Therapeutic regimens and methods for reducing body weight in subjects with fatty liver disease using GLP-1R and GCGR agonists

Abstract

The present disclosure relates to a once weekly dosing regimen of a dual GLP-1R and GCGR agonist, formulations, and methods of using the same to induce weight loss in humans suffering from fatty liver disease, where the humans may or may not suffer from other complications, such as type 2 diabetes and / or cardiovascular disease / disorder. The dual agonist of the GLP-1R and the GCGR comprises a peptide product of SEQ ID NO. 1 (pemvidude), and a preparation method of the GLP-1R and GCGR dual agonist.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 406,681 filed on September 14, 2022; U.S. Provisional Application Serial No. 63 / 422,981 filed on November 5, 2022; U.S. Provisional Application Serial No. 63 / 476,370 filed on December 20, 2022, and U.S. Provisional Application Serial No. 63 / 490,465 filed on March 15, 2023, each of which is hereby incorporated into this application in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on September 14, 2022 is named MED012PRV_ST25.TXT and is 1050 bytes in size.

[0005] Areas of public content

[0006] The present disclosure relates to the use of the GLP-1R and GCGR agonist pemvidutide (ALT-801, a composition comprising SEQ ID NO.: 1) in certain dosing regimens for treating obesity with certain comorbidities (e.g., fatty liver disease, such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH); type 2 diabetes).

[0007] Background of Public Content

[0008] The increasing prevalence of metabolic disorders including obesity, diabetes (e.g., type 2 diabetes), non-alcoholic fatty liver disease (NAFLD) and its advanced form, non-alcoholic steatohepatitis (NASH), is a world health crisis of epidemic proportions, a major contributor to patient morbidity and mortality, and a significant economic burden. Obesity is an important risk factor for type 2 diabetes and NASH, and approximately 90% of patients with type 2 diabetes are overweight or obese. Obesity is a rapidly increasing problem worldwide, and currently more than 65% of adults in the United States are overweight, and the number of obese people doubles every year.

[0009] In the United States (US), NASH has become the leading cause of end-stage liver disease or liver transplantation. Obesity is a core driver of NASH, and weight loss leads to reduced liver fat and improved NASH. More than 80% of individuals with NASH are overweight or obese, and there are currently no available US Food and Drug Administration (FDA)-approved pharmacological options for inducing weight loss, and therapy is mainly based on lifestyle interventions aimed at achieving weight loss. However, it is difficult to achieve and maintain long-term weight loss through lifestyle changes alone.

[0010] Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are associated with modest weight loss at approved doses, and these agents have become a treatment option for patients with NASH. In a recent clinical trial, liraglutide, a GLP-1RA taken daily, was associated with regression of NASH, with a trend toward improved liver fibrosis. However, patients lost only 5.5% of their body weight. In one study, optimal NASH regression required a 10% or greater weight loss. Higher levels of weight loss were also associated with a lower incidence of cardiovascular disease and non-hepatic malignancies, which represent the most serious comorbidities faced by NASH patients.

[0011] GLP-1RAs exert central effects on appetite and food intake, while GCGR agonists (GCGRAs) drive increased energy expenditure in animal models and humans. GCGRAs and GLP-1RAs have been shown to act synergistically in driving greater weight loss than GLP-1RAs alone. GCGRAs also enhance lipolysis and inhibit hepatic fat synthesis, providing an additional pathway for hepatic fat reduction and NASH resolution.

[0012] Dual agonists combine a GCGRA with a GLP-1RA in the same molecule. In obese non-human primates, chronic administration of a GLP-1R / GCGR dual agonist reduced body weight and improved glucose tolerance to a greater extent than a GLP-1RA single agonist. Clinical studies of cotadutide, a GLP-1 / GCGR dual agonist with a 5:1 bias of GLP-1 to glucagon activity, showed an impressive 39% reduction in liver fat content in just 6 weeks, and a greater reduction in NASH-associated alanine aminotransferase (ALT) than liraglutide alone. However, the extent of weight loss with cotadutide administration over 26 weeks was comparable to liraglutide (5.4% vs. 5.5%), suggesting that a 5:1 ratio is acceptable for liver fat reduction but suboptimal for weight loss. Balanced (1:1) agonism has been shown to be associated with greater weight loss and metabolic effects than a biased ratio that favors one agonist over another. A recent study of the balanced dual agonist JNJ 64565111 achieved an impressive 8% weight loss in just 12 weeks (NCT03586830).

[0013] Unfortunately, GLP-1RA and dual receptor agonists based on GLP-1R and GLP-1 that are biased towards GLP-1 are associated with a high rate of nausea, vomiting and diarrhea. These agents must also be titrated over a long period of time to reduce side effects, so agents with improved tolerability and dosing regimens are needed. Therefore, there is still a need for convenient administration (e.g., once a week instead of once a day) of a therapeutic dose that does not require a long period of time (e.g., more than 4 weeks) to reach a therapeutic level to reduce the weight of subjects with fatty liver disease (unrelated to other comorbidities such as type 2 diabetes) without gastrointestinal side effects.

[0014] Overview of public content

[0015] Described herein are dual agonist peptides and products (e.g., formulations) thereof and their use for treating metabolic disorders associated with the function of the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), including use for treating obesity and related co-morbidities, such as fatty liver disease.

[0016] In certain embodiments, a method for reducing the body weight of a human with fatty liver disease is provided, wherein the method comprises administering pemvidutide once a week to a human in need thereof in an amount of at least 1.8 mg up to 2.4 mg; and wherein the human may suffer from type 2 diabetes, and wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In embodiments, after the 12th week and / or the 24th week of weekly administration, the body weight of the human is reduced by at least 3% or at least 4% relative to baseline. In embodiments, pemvidutide is administered once a week in an amount of 1.8 mg, or once a week in an amount of 2.4 mg. In certain embodiments, a steady-state dose is reached after a dose escalation phase having a duration of about 2 weeks, about 3 weeks, or about 4 weeks. In certain embodiments, the human suffers from type 2 diabetes. In alternative embodiments, the human does not suffer from type 2 diabetes. In embodiments, the human has a body mass index (BMI kg / m 2 ), or a body mass index (BMI kg / m 2 In certain embodiments, the human has a liver fat level of 10% or greater as measured by MRI-PDFF (magnetic resonance imaging - proton density fat fraction).

[0017] In certain embodiments, the methods provided herein induce an absolute reduction of about 8% to about 15% or more in liver fat measured by MRI-PDFF after 12 weeks of weekly dosing and / or after 24 weeks of dosing. In certain other embodiments, the methods provided herein induce a relative reduction of about 40% to about 70% in liver fat measured by MRI-PDFF relative to baseline after 12 weeks of weekly dosing and / or after 24 weeks of dosing. .

[0018] Other aspects of the present disclosure are also contemplated as will be appreciated by one of ordinary skill in the art from this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Four treatment groups are shown, wherein subjects (overweight / obesity and NAFLD) are randomly assigned to one of the four groups, and subjects are stratified according to the presence or absence of type 2 diabetes (T2D). Two treatment groups (1.2mg and 1.8mg dosage) do not have dose escalation, while the 2.4mg dosage group has a rapid 4-week dose titration. The study does not carry out calorie restriction or lifestyle intervention on subjects.

[0021] Figure 2 Shown is the reduction in liver fat content at week 12 as both an absolute and relative reduction as determined by MRI-PDFF.

[0022] Figure 3 The reduction in liver fat content according to MRI-PDFF at Week 12 is shown (responder analysis), with data expressed as the proportion of those patients in the groups showing 30% liver fat reduction, 50% liver fat reduction, and normalization.

[0023] Figure 4 Weight loss at week 12 is shown for non-diabetic and diabetic (type 2 diabetes) subjects.

[0024] Figure 5 ALT reduction at week 12 is shown.

[0025] Figure 6 Iron-corrected T1 (cT1) responder analysis at week 12 is shown

[0026] Figure 7 Adverse events (AEs) recorded in the study are shown, with very low incidence for all three doses. The 2.4 mg dose is compared to 2.4 mg*, which are adverse events recorded from an earlier study in which no dose titration was given. Pemvidutide was well tolerated in the study and in each treatment group with reduced AEs compared to the 2.4 mg dose without titration. See WO 2022 / 125598.

[0027] Figure 8 Schematic representation of lipid metabolite extraction, UHPLC-MS analysis, and data processing.

[0028] Fig. 9 Lipoprotein changes in obese / overweight subjects treated with pemvidutide 1.2 mg (n=6), 1.8 mg (n=9), 2.4 mg (n=9), or placebo (n=10) are shown. Day 43 relative to Day -1, Day 84 relative to Day -1. Color codes represent log 2 (robust fold change), blue indicates a decrease in lipoprotein (negative fold change), and red indicates an increase in lipoprotein (positive fold change).

[0029] Fig.10 The lipidomic profiles of obese / overweight subjects treated with pemvidutide 1.2 mg (n=6), 1.8 mg (n=9), 2.4 mg (n=9) or placebo (n=10) are shown comparing the changes on day 84 relative to day -1. The results are expressed as log 2Paired fold change, blue indicates a decrease in metabolite (negative fold change), and red indicates an increase in metabolite (positive fold change). Grey / black bars indicate significant p-values ​​from the Wilcoxon test (light grey, p<0.05; dark grey, p<0.01; black, p<0.001). (Definition: PE = phosphatidylethanolamine; PC = phosphatidylcholine; PI = phosphatidylinositol).

[0030] Fig.11 Figure 2. Volcano plot of changes in known atherogenic lipid species after treatment with 1.8 mg pemvidutide. The Y axis is the Log-squared value based on the Student’s T test. 10 Statistical change, where the horizontal dashed line corresponds to the p-value threshold of 0.01. The X-axis represents Log 2 Fold change, where the dashed line corresponds to an arbitrary change threshold of ±0.75.

[0031] Fig. 12A The graph shows that pemvidutide administration induced significant weight loss compared to placebo.

[0032] Fig. 12B The graph shows that pemvidutide administration induced greater than or equal to 5% or 10% body weight loss compared to placebo.

[0033] Fig.13 Extended study treatment as described in Example 3 is shown.

[0034] Fig.14 The baseline characteristics of the participants in the study of Example 3 are described in detail.

[0035] Fig.15 Robust reductions in liver fat content as determined by MRI-PDFF at Week 24 were shown for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and Week 0 baseline, in terms of both absolute and relative % reductions in liver fat.

[0036] Fig.16 Robust reductions in liver fat content as determined by MRI-PDFF in responders at Week 24 were shown, with all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) resulting in significant reductions in liver fat content in responders compared to placebo and Week 0 baseline (p<0.001 or p<0.0001 as shown therein).

[0037] Fig.17Shown (pemvidutide, 1.8 mg dose) is a significant defatting of the liver as determined by MRI-PDFF at week 24. In this scan of an exemplary subject taking pemvidutide, the reduction in liver volume was dramatic (1.7% liver fat at week 24 compared to 32.3% liver fat at baseline).

[0038] Fig.18 Robust reductions in liver volume determined by MRI-PDFF, measured as both absolute and relative reductions, were shown at Week 24. As shown therein, for example, significant reductions in liver fat content were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and Week 0 baseline (p<0.05 or p<0.001 as shown therein).

[0039] Fig.19 Robust reductions in ALT levels, a biomarker of liver inflammation, were shown in all subjects and subjects with a baseline of ALT ≥ 30 IU / L at Week 24. As shown therein, for example, significant reductions in ALT were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and Week 0 baseline (p<0.001).

[0040] Fig. 20 A high CT1 response rate at week 24 was shown, where a response was defined as a decrease of 80MS in CT1 compared to baseline (week 0), which was associated with a two (2) point decrease in the NASH activity score (NAS) (Dennis A., Front. Endocrinol., 2021). As shown therein, for example, a significant decrease in cT1 response was observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and week 0 baseline (p<0.05 or p<0.005 as shown therein).

[0041] Fig.21 Shown that sustained weight loss was observed in all subjects (non-diabetic and diabetic) at week 24 for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and week 0 baseline, with significant weight loss (p<0.005 or p<0.001 as indicated therein). This shows weight loss in humans with fatty liver disease (non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) in both diabetic and non-diabetic subjects.

[0042] Fig. 22Improvements in serum lipids at week 24 compared to baseline (week 0) are shown for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week).

[0043] Fig.23 It was shown that all doses of pemvidutide tested induced improvements in blood pressure at week 24 compared with placebo and baseline (week 0) without clinically meaningful increases in heart rate, and the reduction in systolic blood pressure was significant (p<0.05) at the 2.4 mg weekly dose.

[0044] Fig.24 An overview of the safety profile of the trial, including adverse events over 24 weeks, is shown.

[0045] Fig.25 Improvements in glycemic control in diabetic patients at Week 24 were shown for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and Week 0 baseline.

[0046] Fig.26 Baseline characteristics of study participants in the Phase II study are shown. See Example 4.

[0047] Fig. 27 Shown are the mean weight losses expressed as percent change from baseline body weight achieved over 24 weeks of treatment with 1.2 mg, 1.8 mg, and 2.4 mg doses of pemvidutide and placebo in all evaluated subjects.

[0048] Fig.28 Shown are the weight losses expressed as mean percent change from baseline body weight achieved over 24 weeks of treatment with 1.2 mg, 1.8 mg, and 2.4 mg doses of pemvidutide and placebo in a subgroup of subjects with a baseline body weight of ≤115 kg.

[0049] Fig.29 Shown are the percentages of subjects in each group who achieved >5%, >10%, or >15% weight loss (expressed as a percentage of baseline body weight) after 24 weeks of treatment with 1.2 mg, 1.8 mg, and 2.4 mg doses of pemvidutide and placebo.

[0050] Fig.30 Shown are the mean weight losses expressed as percent change from baseline weight achieved by Hispanic subjects relative to non-Hispanic subjects after 24 weeks of treatment with 1.2 mg, 1.8 mg, and 2.4 mg doses of pemvidutide and placebo.

[0051] Fig.31Shown are systolic and diastolic blood pressure (A) and heart rate (B) expressed as mean percent change from baseline after 24 weeks of treatment with 1.2 mg, 1.8 mg, and 2.4 mg doses of pemvidutide and placebo.

[0052] Fig.32 Shown are serum lipids at Week 24 compared to baseline for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg).

[0053] Fig.33 All doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) showed a significant reduction in waist circumference at week 24. See Example 4.

[0054] Detailed description of the public content

[0055] The present disclosure relates to one or more dual agonist peptides and pharmaceutical dosage formulations comprising the one or more dual agonist peptides and methods of using the one or more dual agonist peptides. The dual agonist peptide has affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), and in preferred embodiments has approximately equal affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), which can be determined using cell assays. In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to induce weight loss in humans with fatty liver disease. In some embodiments, humans may or may not suffer from type 2 diabetes. In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to reduce pathogenic plasma lipid mediators. As used herein, "pathogenic" serum lipid mediators include, but are not limited to, the following reactive lipid species: malondialdehyde (MDA), isolevuglandins (IsoLG), methylglyoxal (MGO), 4-oxononenal (ONE), and 4-hydroxynonenal (HNE). Oxidized phospholipids include 1-palmitoyl-2-oxopentanoyl-sn-glycerol-3-phosphocholine (POVPC), 1-O-alkyl-2-azelayl-sn-glycerol-3-phosphocholine (azPAF), 1-(palmitoyl)-2-(5-keto-6-octen-diacyl)phosphatidylcholine (KOdiA-PC), 1-palmitoyl-2-F2-isoprostane-sn-glycerol-3-phosphocholine (F2IsoP-PC), and 1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycerol-3-phosphocholine (PEIPC).

[0056] In some embodiments, the present disclosure provides a pharmaceutical dosage formulation configured to induce weight loss (including for long-term weight management and treatment of related comorbidities). In some embodiments, the present disclosure provides a pharmaceutical dosage formulation configured to induce weight loss and reduce pathogenic serum lipid mediators for the treatment of obesity (e.g., long-term weight management) and / or treatment of cardiovascular (CV) related risk factors. In some embodiments, the present disclosure provides a peptide-based GLP-1 / glucagon receptor dual agonist intended for the treatment of potential metabolic dysfunction leading to non-alcoholic steatohepatitis (NASH).

[0057] As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and preventive measures, wherein the goal is to slow down (mitigate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, relieving symptoms; reducing the extent of the condition, disorder, or disease; stabilizing the state of the condition, disorder, or disease (i.e., not worsening); delaying the onset of the condition, disorder, or disease or slowing the progression of the condition, disorder, or disease; improving the condition, disorder, or disease state or alleviation (whether partially or completely), whether detectable or undetectable; improving at least one measurable physical parameter (not necessarily discernible by the patient); or enhancing or ameliorating the condition, disorder, or disease. Treatment includes causing a clinically significant response without excessive levels of side effects. Treatment also includes prolonged survival relative to expected survival if treatment is not received. Thus, "treatment of obesity" refers to activities that reduce or ameliorate any primary phenomenon or secondary symptoms associated with obesity or other conditions described herein, including but not limited to fatty liver disease (FDL) (e.g., NASH and NAFLD) and type 2 diabetes.

[0058] In an embodiment, a method for reducing the body weight of a human with fatty liver disease is provided herein, wherein the method comprises administering pemvidutide once a week to a human in need thereof in an amount of at least 1.8 mg up to 2.4 mg; and, wherein the human may suffer from type 2 diabetes, and wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Pemvidutide, also referred to herein as ALT-801, is a composition comprising a synthetic peptide (SEQ ID NO: 1) containing naturally occurring amino acids, and is a chimeric analog of two natural hormones GLP-1 and glucagon, having primarily a glucagon residue at the N-terminus and a GLP-1 residue at the C-terminus. See U.S. Patent No. 9,856,306, which is incorporated herein by reference. ALT-801 also incorporates a non-proteinogenic amino acid 2-aminoisobutyric acid, an amino acid side chain amide bond (lactam bridge), and a surfactant side chain comprising glucuronic acid connected to an octadecanoic acid side chain. The surfactant side chains show slow entry into the circulation and can form micelles after subcutaneous (SC) injection. The lower maximum concentration (Cmax) associated with slower entry can lead to fewer GI side effects and better tolerance. This latter feature also enhances binding to plasma proteins and improves metabolic stability, extending the half-life (t 1 / 2 ). The design of ALT-801 provides a co-agonist with equivalent (1:1) activity (approximately 40 pM and 100% activity) for both receptors. Compositions comprising SEQ ID NO: 1 have been administered to humans at various doses using standard techniques and have been found not to induce side effects such as nausea, vomiting, diarrhea, abdominal pain, and / or constipation. See U.S. Patent Publication No. 2021 / 0290732 and PCT Publication No. WO 2022 / 125598, each of which is incorporated herein by reference.

[0059] ALT-801 (SEQ ID NO: 1 (also referred to herein as pemvidutide)) has the following amino acid sequence:

[0060] 1 His- 2 Aib- 3 Gln- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Tyr- 11 Ser- 12 Lys- 13 Tyr- 14 Leu-15 Asp- 16 Glu*- 17 Lys #-18 Ala- 19 Ala- 20 Lys*- 21 Glu- 22 Phe- 23 Ile- 24 Gln- 25 Trp- 26 Leu- 27 Leu- 28 Gln- 29 Thr-NH 2 ,

[0061] in * indicating that a lactam bridge is formed between Glu16 and Lys 20, and 17 Lys # Indication Glucuronic Acid C-18 * (Z17COOH) attachment site. Stated differently, SEQ ID NO: 1 (i.e. ALT-801 or pemvidutide) is composed of 29 amino acid residues and attached to 17 Lys glucuronic acid / C 18 A peptide amide composed of a diacid moiety, wherein 16 Glu and 20 The side chain of Lys forms an intramolecular ring as shown below:

[0062]

[0063] In some embodiments, provided herein is a pharmaceutical formulation of SEQ ID NO: 1 in an aqueous buffer solution, referred to herein as ALT-801 (and pemvidutide). The dual agonist peptide products herein (including SEQ ID NO: 1) comprise an amino acid side chain amide bond (lactam bridge) and a surfactant side chain comprising glucuronic acid connected to a fatty acid side chain. The side chain (surfactant comprising a hydrophilic sugar group and a hydrophobic alkyl chain portion covalently attached to the peptide via a linker amino acid). The synthesis of SEQ ID NO. 1 is described in U.S. Pat. No. 11,541,028B2, which is incorporated by reference in its entirety into the present disclosure. In some embodiments, the dual agonist peptide may comprise one or more conservatively substituted amino acids as described herein. In preferred embodiments, SEQ ID NO: 1 may comprise one or more conservatively substituted amino acids, but preferably not at amino acid residues 16, 17 or 20.

[0064] "Peptide" (e.g., dual agonist peptide) comprises two or more natural or / and non-natural amino acid residues, usually linked via peptide bonds. Such amino acids may include naturally occurring structural variants, naturally occurring non-proteinogenic amino acids or / and non-naturally occurring analogs of synthetic natural amino acids. The terms "peptide" and "polypeptide" are used interchangeably herein. Peptides include short peptides (about 2-20 amino acids), medium-length peptides (about 21-50 amino acids), and long peptides (> about 50 amino acids, also referred to as "proteins"). In some embodiments, the peptide product comprises a surfactant portion covalently and stably attached to a peptide having no more than about 50, 40 or 30 amino acids. For example, synthetic peptides can be synthesized using an automated peptide synthesizer. Peptides can also be recombinantly produced in cells expressing a nucleic acid sequence encoding the peptide. Conventional symbols are used herein to describe peptide sequences: the left-hand end of the peptide sequence is the amino (N) end, and the right-hand end of the peptide sequence is the carboxyl (C) end. Standard single-letter and three-letter abbreviations of common amino acids are used herein. Although the abbreviations used in the amino acid sequences disclosed herein represent L-amino acids, unless otherwise specified as D-amino acids or DL-amino acids or the amino acid is achiral, the corresponding D-isomers can generally be used at any position (e.g., to resist proteolytic degradation). Other abbreviations for amino acids used herein include: Aib = α-aminoisobutyric acid (or 2-methylalanine or Cα-methylalanine); Xaa: any amino acid, usually explicitly defined in the formula.Other abbreviations for amino acids that may be used as described herein include: Ac3c = 1-aminocyclopropane-1-carboxylic acid; Ac4c = 1-aminocyclobutane-1-carboxylic acid; Ac5c = 1-aminocyclopentane-1-carboxylic acid; Ac6c = 1-aminocyclohexane-1-carboxylic acid; Aib = α-aminoisobutyric acid (or 2-methylalanine or Cα-methylalanine); Bip = 3-(biphenyl-4-yl)alanine; Bip2Et = 3-(2'-ethylbiphenyl-4-yl)alanine; Bip2EtMeO = 3-(2'-ethyl-4'-methoxybiphenyl-4-yl)alanine; Cit = citrulline; Deg = 2,2-diethylglycine; Dmt = (2,6-dimethyl)tyrosine; 2FPhe = (2-fluorophenyl)alanine; 2FMePhe or 2 FaMePhe = Cα-methyl-(2-fluorophenyl)alanine; hArg = homoarginine; MeLys or aMeLys = Cα-methyllysine; MePhe or aMePhe = Cα-methylphenylalanine; MePro or aMePro = Cα-methylproline; Nall or Nal(l) = 3-(l-naphthyl)alanine; Nal2 or Nal(2) = 3-(2-naphthyl)alanine; Nle = norleucine; Om = ornithine; and Tmp = (2,4,6-trimethylphenyl)alanine; l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and the Tic-Phe dipeptide portion having reduced amide bonds between the residues (designated Tic-Ψ[CFl2-NFl]-Ψ-Phe) has the following structure:.

[0065]

[0066] Unless otherwise specifically stated or the context clearly indicates otherwise, the present disclosure encompasses any and all forms of dual agonist peptides that can be produced, whether the dual agonist peptide is produced synthetically (e.g., using a peptide synthesizer) or produced by a cell (e.g., by recombinant production). Such forms of dual agonist peptides can include one or more modifications that can be performed during the synthesis or cell production process of the peptide, such as one or more post-translational modifications, whether or not the one or more modifications are intentional. Dual agonist peptides can have the same type of modification at two or more different positions, or / and can have two or more different types of modifications. Modifications that can be performed during the synthesis or cell production process of dual agonist peptides include chemical and post-translational modifications, including but not limited to glycosylation (e.g., N-linked glycosylation and O-linked glycosylation), lipidation, phosphorylation, sulfation, acetylation (e.g., acetylation of the N-terminus), amidation (e.g., C-terminal amidation), hydroxylation, methylation, formation of intramolecular or intermolecular disulfide bonds, formation of lactams between two side chains, formation of pyroglutamic acid, and ubiquitination. The dual agonist peptide may have one or more modifications anywhere, such as the N-terminus, the C-terminus, one or more amino acid side chains, or the dual agonist peptide backbone, or any combination thereof. In some embodiments, the dual agonist peptide is acetylated at the N-terminus and / or has a carboxamide (-CONH 2 ) group, which can increase the stability of the dual agonist peptide.

[0067] Possible modifications of dual agonist peptides also include the deletion of one or more amino acids, the addition / insertion of one or more natural or / and non-natural amino acids, or substitution with one or more natural or / and non-natural amino acids, or any combination or all thereof. Substitution may be conservative or non-conservative. Such modifications may be intentional, such as via site-directed mutagenesis or in the chemical synthesis of dual agonist peptides, or may be accidental, such as via mutations occurring in host cells producing dual agonist peptides or via errors caused by PCR amplification. Non-natural amino acids may have the same chemical structure as the counterpart natural amino acids, but with D stereochemistry, or it may have different chemical structures and D stereochemistry or L stereochemistry. Non-natural amino acids may be used, for example, to promote α-helix formation or / and increase the stability of dual agonist peptides (e.g., resist proteolytic degradation). Dual agonist peptides with one or more modifications relative to a reference dual agonist peptide may be referred to as "analogs" or "variants" of the reference dual agonist peptide, as appropriate. "Analogs" generally retain one or more basic properties of the reference dual agonist peptide (e.g., receptor binding, activation of a receptor or enzyme, inhibition of a receptor or enzyme, or other biological activity). A "variant" may or may not retain the biological activity of the reference dual agonist peptide, or / and may have a different biological activity. Preferably, such a variant maintains its ability to act as an agonist of GLP-1R and GCGR, and in a more preferred embodiment, has about equal affinity for GLP-1R and GCGR. In some embodiments, an analog or variant of a reference peptide has an amino acid sequence that is different from the reference dual agonist peptide.

[0068] The term "conservative substitution" refers to the substitution of an amino acid in a dual agonist peptide with a natural or non-natural amino acid that is functionally, structurally or chemically similar. In certain embodiments, the following groups each comprise natural amino acids that are conservative substitutions for each other: 1) glycine (Gly / G), alanine (Ala / A); 2) isoleucine (Ile / I), leucine (Leu / L), methionine (Met / M), valine (Val / V); 3) phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W); 4) serine (Ser / S), threonine (Thr / T), cysteine ​​(Cys / C); 5) asparagine (Asn / N), glutamine (Gln / Q); 6) aspartic acid (Asp / D), glutamic acid (Glu / E); and 7) arginine (Arg / R), lysine (Lys / K), histidine (His / H). In another embodiment, the following groups each comprise natural amino acids that are conservative substitutions for each other: 1) non-polar: Ala, Val, Leu, Ile, Met, Pro (proline / P), Phe, Trp; 2) hydrophobic: Val, Leu, Ile, Phe, Trp; 3) aliphatic: Ala, Val, Leu, Ile; 4) aromatic: Phe, Tyr, Trp, His; 5) uncharged polar or hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln, Tyr; 6) aliphatic containing hydroxyl or thiol: Ser, Thr, Cys; 7) amide containing: Asn, Gln; 8) acidic: Asp, Glu; 9) basic: Lys, Arg, His; and, 10) small: Gly, Ala, Ser, Cys. In other embodiments, the amino acids can be grouped into conservative substitutions as listed below: 1) hydrophobic: Val, Leu, Ile, Met, Phe, Trp; 2) aromatic: Phe, Tyr, Trp, His; 3) neutral hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln; 4) acidic: Asp, Glu; 5) basic: Lys, Arg, His; and 6) residues affecting main chain orientation: Pro.

[0069] Examples of non-natural or non-proteinogenic amino acids include, but are not limited to, alanine analogs (e.g., α-ethyl Gly [α-aminobutyric acid or Abu], α-n-propyl Gly [norvaline or Nva], α-tert-butyl Gly [Tbg], α-vinyl Gly [Vg or Vlg], α-allyl Gly [Alg], α-propargyl Gly [Prg], 3-cyclopropyl Ala [Cpa], and Aib), leucine analogs (e.g., norleucine, Nle), proline analogs (e.g., α-MePro), phenylalanine analogs (e.g., Phe(2-F), Phe(2-Me), Tmp, Bip, Bip(2'-Et-4'-OMe), Nall, NaI2, Tic, α-MePhe, α-MePhe(2- F) and α-MePhe (2-Me)), tyrosine analogs (e.g., Dmt and α-MeTyr), serine analogs (e.g., homoserine [isothreonine or hSer]), glutamine analogs (e.g., Cit), arginine analogs (e.g., hArg, N,N'-g-dialkyl-hArg), lysine analogs (e.g., homolysine [hLys], Orn and α-MeLys), α,α-disubstituted amino acids (e.g., Aib, α,α-diethylGly [Deg], α-cyclohexylAla [2-Cha], Ac3c, Ac4c, Ac5c and Ac6c), and other unnatural amino acids disclosed in A. Santoprete et al., Pept. Sci., 17: 270-280 (2011). α,α-disubstituted amino acids can provide conformational constraints and / or α-helix stabilization. A reduced amide bond between two residues (such as in, e.g., Tic-Ψ[CF12-NF1]-Ψ-Phe) increases protease resistance and may also, for example, alter receptor binding. The present disclosure encompasses all pharmaceutically acceptable salts of dual agonist peptides, including salts with a positive net charge, salts with a negative net charge, and salts with no net charge.

[0070] "Alkyl" groups refer to aliphatic hydrocarbon groups. Alkyl groups can be saturated or unsaturated, and can be straight-chain (linear), branched or cyclic. In some embodiments, the alkyl group is not cyclic. In some embodiments, the alkyl group contains 1-30, 6-30, 6-20, or 8-20 carbon atoms. "Substituted" alkyl groups are substituted with one or more substituents. In some embodiments, one or more substituents are independently selected from halogen, nitro, cyano, oxo, hydroxyl, alkoxy, haloalkoxy, aryloxy, thiol, alkylthio, arylthio, alkyl sulfoxide, aryl sulfoxide, alkyl sulfone, aryl sulfone, amino, alkylamino, dialkylamino, arylamino, alkylacyl, carboxyl, carboxylate, ester, amide, carbonate, carbamate, urea, alkyl, haloalkyl, fluoroalkyl, aromatic alkyl, alkyl chain containing acyl group, heteroalkyl, heteroalicyclic, aryl, alkyloxyaryl, heteroaryl, hydrophobic natural compound (e.g., steroid), etc. In some embodiments, the alkyl group as a substituent is a linear or branched C 1 -C 6 Hydrocarbyl, which may be referred to as "lower hydrocarbon". Non-limiting examples of lower hydrocarbon groups include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including all isomeric forms, such as n-butyl, isobutyl, sec-butyl and / or tert-butyl), pentyl (including all isomeric forms, such as n-pentyl) and hexyl (including all isomeric forms, such as n-hexyl). In some embodiments, the hydrocarbon group is attached to the Na atom of the peptide residue (e.g., Tyr or Dmt). In certain embodiments, the N-hydrocarbyl group is a straight or branched C 1 -C 10 Hydrocarbyl or aryl substituted hydrocarbyl such as benzyl, phenethyl, etc. One or two hydrocarbyl groups can be attached to the Na atom of the N-terminal residue. In some embodiments, the hydrocarbyl group is a 1-hydrocarbyl group attached to the Cl position of a sugar (e.g., glucose) via a glycosidic bond (e.g., O-glycosidic bond, S-glycosidic bond, N-glycosidic bond, or C-glycosidic bond). In some embodiments, such a 1-hydrocarbyl group is unsubstituted or substituted C 1 -C 30 , C 6 -C 30 , C 6 -C 20 or C 8 -C 20 In some embodiments, the hydrocarbyl group (e.g., 1-hydrocarbyl group) is substituted with one or more (e.g., 2 or 3) groups independently selected from aryl, -OH, -OR 1 , -SH, -SR 1 、-NH2 、-NHR 1 、-N(R 1 ) 2 , Oxo(=O), -C(=O)R 2 , carboxyl (-CO 2 H), carboxylate (-CO 2 -), -C(=O)OR 1 、-OC(=O)R 3 、-C(=O)N(R 1 ) 2 、-NR 4 C(=O)R 3 、-OC(=O)OR 5 、-OC(=O)N(R 1 ) 2 、-NR 4 C(=O)OR 5 and -NR4C(=O)N(R 1 ) 2 , where: R 1 is independently hydrogen, hydrocarbyl or aryl at each occurrence, or R 1 The two occurrences of and the nitrogen atom to which they are attached form a heterocyclyl ring or a heteroaryl ring; R 2 R is independently at each occurrence a hydrocarbyl, heterocyclyl, aryl or heteroaryl group; 3 R is independently at each occurrence hydrogen, hydrocarbyl, heterocyclyl, aryl or heteroaryl; 4 is independently hydrogen or hydrocarbyl at each occurrence; and, R 5In each occurrence, it is independently a hydrocarbon group or an aryl group. In some embodiments, a hydrocarbon group (e.g., a 1-hydrocarbon group) is replaced by a carboxyl group / carboxylate group, an aryl group, or an -O-aryl group in the interior or / and at the end. In certain embodiments, a hydrocarbon group (e.g., a 1-hydrocarbon group) is replaced by a carboxyl group or a carboxylate group at the distal end of the hydrocarbon group. In other embodiments, a hydrocarbon group (e.g., a 1-hydrocarbon group) is replaced by an aryl group at the distal end of the hydrocarbon group. In other embodiments, a hydrocarbon group (e.g., a 1-hydrocarbon group) is replaced by an -O-aryl group at the distal end of the hydrocarbon group. The terms "halogen", "halide", and "halo" refer to fluoride, chloride, bromide, and iodide. The term "acyl" refers to -C(=O)R, wherein R is an aliphatic group that can be saturated or unsaturated and can be linear, branched, or cyclic. In certain embodiments, R comprises 1-20, 1-10 or 1-6 carbon atoms.Acyl groups may be optionally substituted with one or more groups, such as halogen, oxo, hydroxyl, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cycloalkyl, aryl, acyl, carboxyl, ester, amide, hydrophobic natural compounds (e.g., steroids), etc. The terms "heterocyclyl" and "heterocycle" refer to monocyclic non-aromatic groups or polycyclic groups comprising at least one non-aromatic ring, wherein at least one non-aromatic ring comprises one or more heteroatoms independently selected from O, N and S. Non-aromatic rings comprising one or more heteroatoms may be attached or combined to one or more saturated, partially unsaturated or aromatic rings. In certain embodiments, heterocyclyl or heterocyclic groups have 3 to 15, or 3 to 12, or 3 to 10, or 3 to 8, or 3 to 6 ring atoms. Heterocyclic radical or heterocyclic group include but are not limited to aziridine base, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, azepanyl, azocanyl, oxirane base, oxetane base, tetrahydrofuranyl (oxolanyl), tetrahydropyranyl, oxepanyl and oxocanyl. The term "aryl" refers to a monocyclic aromatic hydrocarbon group or a polycyclic group comprising at least one aromatic hydrocarbon ring. In certain embodiments, the aryl group has 6 to 15, or 6 to 12, or 6 to 10 ring atoms. The aryl group includes but is not limited to phenyl, naphthyl (naphthalenyl) (naphthyl), fluorenyl, azulenyl (azulenyl), anthracenyl, phenanthrenyl, biphenyl and terphenyl. The aromatic hydrocarbon ring of the aryl group may be attached to or incorporated into one or more saturated, partially unsaturated or aromatic rings, for example dihydronaphthyl, indenyl, indanyl and tetrahydronaphthyl (tetaraphthyl).The aryl group may be optionally substituted with one or more (e.g., 2 or 3) substituents independently selected from the group consisting of halogen (including -F and -Cl), cyano, nitro, hydroxy, alkoxy, thiol, alkylthio, alkyl sulfoxide, alkyl sulfone, amino, alkylamino, dialkylamino, alkyl, halogenated alkyl (including fluoroalkyl such as trifluoromethyl), acyl, carboxyl, ester, amide, and the like. The term "heteroaryl" refers to a monocyclic aromatic group or a polycyclic group comprising at least one aromatic ring, wherein at least one aromatic ring comprises one or more heteroatoms independently selected from O, N, and S. The heteroaromatic ring may be attached or incorporated to one or more saturated, partially unsaturated, or aromatic rings that may comprise only carbon atoms or may comprise one or more heteroatoms. In certain embodiments, the heteroaryl group has 5 to 15, or 5 to 12, or 5 to 10 ring atoms. Monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl (thiophenyl), oxadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridonyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridazinonyl, and triazinyl. Non-limiting examples of bicyclic heteroaryl groups include indolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzothiophenyl (benzothiophenyl), quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzotriazolyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinazolinyl, quinoxalinyl, indazolyl, naphthyridinyl, phthalazinyl, quinazolinyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl.

[0071] In some embodiments, for example, the dual agonist peptide can be associated with a sugar, such as in a pharmaceutically acceptable composition or lyophilizate. Sugars include monosaccharides, disaccharides, and oligosaccharides (e.g., trisaccharides, tetrasaccharides, etc.). Reducing sugars exist in a balanced form of rings and open chains, usually with a preference for rings. The functionalized sugar of the surfactant portion has a functional group suitable for forming a stable covalent bond with the amino acids of the dual agonist peptide.

[0072] The term "pharmaceutically acceptable" refers to a substance (e.g., an active ingredient or excipient) that is suitable for use in contact with tissues and organs of a subject without undue irritation, allergic response, immunogenicity, and toxicity, commensurate with a reasonable benefit / risk ratio, and effective for its intended use. A "pharmaceutically acceptable" excipient or carrier for a pharmaceutical composition is also compatible with the other ingredients of the composition. In one embodiment, a pharmaceutically acceptable composition in which a dual agonist peptide can be formulated comprises polysorbate 20 (e.g., about 0.050% (w / w)) in distilled (DI) water; optionally, methyl paraben (e.g., about 0.300% (w / w)); arginine (e.g., about 0.348% (w / w)) and mannitol (e.g., about 4.260% (w / w)).

[0073] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to prevent, reduce the risk of developing, delay the onset of, slow the progression of, or cause regression of the medical condition being treated, or to alleviate to some extent the medical condition or one or more symptoms or complications of the condition, at least in a portion of the subjects taking the compound. The term "therapeutically effective amount" also refers to an amount of a compound that is sufficient to elicit a biological or medical response in a cell, tissue, organ, or human being that is sought by a physician or clinician.

[0074] The terms "treat," "treating," and "treatment" include alleviating, ameliorating, inhibiting the progression of a medical condition or one or more symptoms or complications associated with the condition, reversing or eliminating a medical condition or one or more symptoms or complications associated with the condition, and alleviating, ameliorating, or eradicating one or more causes of the condition. Reference to "treatment" of a medical condition includes prevention of the condition. The terms "prevent," "preventing," and "prevention" include eliminating, reducing the risk of development of a medical condition or one or more symptoms or complications associated with the condition, and delaying the onset of a medical condition or one or more symptoms or complications associated with the condition. The term "medical condition" (or "condition" for brevity) includes diseases and disorders. The terms "disease" and "disorder" are used interchangeably herein.

[0075] The present disclosure also provides a pharmaceutical composition comprising a dual agonist peptide product described herein or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition comprises a therapeutically effective amount of a peptide product or its appropriate fraction. The composition may optionally comprise an additional therapeutic agent. In some embodiments, the peptide product is at least about 90%, 95% or 98% pure. Pharmaceutically acceptable excipients and carriers include pharmaceutically acceptable substances, materials and vehicles. Non-limiting examples of excipient types include liquid and solid fillers, diluents, adhesives, lubricants, glidants, surfactants, dispersants, disintegrants, emulsifiers, wetting agents, suspending agents, thickeners, solvents, isotonic agents, buffers, pH regulators, absorption delay agents, stabilizers, antioxidants, preservatives, antimicrobials, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweeteners, flavoring agents, coloring agents, encapsulating materials and coating materials. The use of such excipients in pharmaceutical preparations is known in the art. For example, conventional vehicles and carriers include, but are not limited to, oils (e.g., vegetable oils such as olive oil and sesame oil), aqueous solvents (e.g., saline, buffered saline (e.g., phosphate buffered saline [PBS]), and isotonic solutions (e.g., Ringer's solution)), and organic solvents (e.g., dimethyl sulfoxide and alcohols [e.g., ethanol, glycerol, and propylene glycol]). Unless any conventional excipient or carrier is incompatible with the peptide product, the present disclosure encompasses the use of conventional excipients and carriers in formulations comprising the peptide product. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania) (2005); Handbook of Pharmaceutical Excipients, 5th ed., Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd ed., Ash and Ash, eds., Gower Publishing Co. (2007); and Pharmaceutical Pre-formulation and Formulation, Gibson, ed., CRC Press (Boca Raton, Florida) (2004).

[0076] In an embodiment, the pharmaceutical formulation comprises a peptide product and about 0.02%-0.075% (w / w) polysorbate 20, about 0.2%-0.5% (w / w) arginine, about 3%-6% (w / w) mannitol (pH 7.7±0.1) in deionized water; optionally about 0.050% (w / w) polysorbate 20, about 0.348% (w / w) arginine, about 4.260% (w / w) mannitol (pH 7.7±0.1) in deionized water. In certain embodiments, the pharmaceutical formulation of the present invention comprises SEQ ID NO: 1 and about 0.050% (w / w) polysorbate 20, about 0.348% (w / w) arginine, about 4.260% (w / w) mannitol (pH 7.7±0.1) in deionized water. In certain embodiments, the pharmaceutical formulation of the present invention comprises SEQ ID NO: 1 and about 0.020% (w / w) polysorbate 20, about 0.348% (w / w) arginine, about 4.260% (w / w) mannitol in deionized water (pH 7.7±0.1). In certain embodiments, the pharmaceutical formulation comprises SEQ ID NO: 1 (ALT-801, pemvidutide) and is configured for subcutaneous (SC) administration of a once-weekly therapeutic dose.

[0077] Appropriate or suitable formulations may depend on various factors, such as the route of administration selected. Possible routes of administration of pharmaceutical compositions comprising peptide products include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intracavitary and topical) and topical (including transdermal, transmucosal, intranasal (e.g., by nasal spray or drops), ocular (e.g., by eye drops), pulmonary (e.g., by oral inhalation or nasal inhalation (oral ornasal inhalation)), buccal, sublingual, rectal (e.g., by suppository) and vaginal (e.g., by suppository)). In certain embodiments, the dual agonist peptide products of the present invention are administered parenterally (e.g., subcutaneously, intravenously or intramuscularly). In other embodiments, the peptide products are administered by oral inhalation or nasal inhalation or insufflation. In some embodiments, the carrier is a water-based carrier, such as in a parenteral (e.g., subcutaneous, intravenous or intramuscular) formulation. In other embodiments, the carrier is a nonaqueous-based carrier. In certain embodiments, the non-aqueous based carrier is a hydrofluoroalkane (HFA) or HFA-like solvent which may include submicron anhydrous alpha-lactose and / or other excipients, such as in formulations for administration by oral inhalation or nasal inhalation or insufflation.

[0078] In some embodiments, the peptide product is administered parenterally (e.g., subcutaneously, intravenously, or intramuscularly) by injection. Parenteral administration bypasses the highly acidic environment of the stomach, gastrointestinal (GI) absorption, and first-pass metabolism. Excipients and carriers that can be used to prepare parenteral formulations include, but are not limited to, solvents (e.g., aqueous solvents such as water, saline, physiological saline, buffered saline [e.g., PBS], balanced salt solutions [e.g., Ringer's BSS], and aqueous dextrose solutions), isotonic / iso-osmotic agents (e.g., salts [e.g., NaCl, KCl, and CaCl 2 ] and sugars [e.g., sucrose]), buffers and pH adjusters (e.g., sodium dihydrogen phosphate, monobasic sodium phosphate] / di sodium hydrogen phosphate, dibasic sodium phosphate, citric acid / sodium citrate and L-histidine / L-histidine HCl), and emulsifiers (e.g., nonionic surfactants such as polysorbates [e.g., polysorbate 20 and polysorbate 80] and poloxamers [e.g., poloxamer 188]). Peptide formulations and delivery systems are discussed in, e.g., AJ Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Edition, CRC Press (Boca Raton, Florida) (2015). Excipients may optionally include one or more substances that increase peptide stability, increase peptide solubility, inhibit peptide aggregation, or reduce solution viscosity, or any combination or all thereof. Such substances include, but are not limited to, hydrophilic amino acids (e.g., arginine and histidine), polyols (e.g., inositol, mannitol, and sorbitol), sugars (e.g., glucose (including D-glucose [dextrose]), lactose, sucrose, and trehalose), osmotic agents (e.g., trehalose, taurine, amino acids [e.g., glycine, sarcosine, alanine, proline, serine, b-alanine, and g-aminobutyric acid], and betaines [e.g., trimethylglycine and trimethylamine N-oxide]), and nonionic surfactants (e.g., alkyl polysaccharides, Hydrocarbyl sugars (e.g., monosaccharides [e.g., glucose] or disaccharides [e.g., maltose or sucrose] coupled to long-chain fatty acids or corresponding long-chain alcohols) and polypropylene glycol / polyethylene glycol block copolymers (e.g., poloxamers [e.g., Pluronic TM F-68] and PF-10 and its variants). Because such substances increase the solubility of the peptide, they can be used to increase the peptide concentration in the formulation. Higher peptide concentrations in the formulation are particularly advantageous for subcutaneous administration, which has a limited bolus administration volume (e.g., < about 1.5 mL). In addition, such substances can be used to stabilize the peptide during preparation, storage, and reconstitution of lyophilized peptides. Exemplary parenteral formulations include peptide products, mannitol, methionine, sodium thioglycolate, polysorbate 20, pH regulators (e.g., NaOH or / and HCl), and deionized water. Excipients suitable for parenteral formulations used with the dual agonist peptides described herein (e.g., various combinations of excipients including NaCl, etc.) are well known and available to those of ordinary skill in the art.

[0079] For parenteral (for example, subcutaneous, intravenous or intramuscular) use, the peptide product sterile solution or suspension in the aqueous solvent comprising one or more excipients can be prepared in advance, and can be provided in the pre-filled syringe of for example single-use pen or the pen with dose counter.Alternatively, the peptide product can be dissolved or suspended in the aqueous solvent that can optionally comprise one or more excipients before lyophilization (freeze drying).Soon before parenteral administration, the lyophilized peptide product stored in the suitable container (for example, vial) can be reconstructed with the sterile water that can optionally comprise one or more excipients.In other embodiments, the agonist peptide product is administered intranasally.Nasal mucosa provides large surface area, porous endothelium, high vascular epithelial sublayer and high absorption rate, and therefore allows high bioavailability. Intranasal formulations can include peptide products and excipients, such as solubility enhancers (e.g., propylene glycol), wetting agents (e.g., mannitol or sorbitol), buffers and water, and optionally preservatives (e.g., benzalkonium chloride), mucoadhesives (e.g., hydroxyethylcellulose) or / and penetration enhancers. Intranasal solutions or suspension formulations can be administered to the nasal cavity by any suitable device, including but not limited to droppers, pipettes, or sprayers using, for example, metered atomizing spray pumps. Table 2 shows exemplary excipients for nasal spray formulations.

[0080] Table 1

[0081] Exemplary Excipients and Carriers for Nasal and Pulmonary Formulations

[0082]

[0083] In another embodiment, the peptide product is administered via a pulmonary route, such as by oral inhalation or nasal inhalation. Pulmonary administration of the drug can treat pulmonary disorders or / and systemic disorders because the lungs are used as the entrance to the systemic circulation. The advantages of pulmonary drug delivery include, for example: 1) avoiding first-pass metabolism; 2) rapid drug action; 3) large surface area of ​​the alveolar region for absorption, high permeability of the lungs (thin air-blood barrier) and rich vascular system of the airways; and 4) due to the large alveolar surface area, the extracellular enzyme level reduced compared to the GI tract. The advantages of oral inhalation over nasal inhalation include that the drug penetrates / deposits deeper into the lungs, although nasal inhalation can deliver the drug to the systemic circulation through the mucosa in the nasal cavity and the lungs. Oral inhalation or nasal inhalation can be achieved by, for example, a metered dose inhaler (MDI), a nebulizer or a dry powder inhaler (DPI). For example, a peptide product can be formulated for aerosol administration to the respiratory tract by oral inhalation or nasal inhalation. Drug is delivered with small particle size (for example, between about 0.5 micron and about 5 microns), and the small particle size can be obtained by micronization, to improve the stability of drug deposition and drug suspension in lung, for example. Drug can be provided with pressurized packaging with suitable propellant, such as hydrofluoroalkane (HFA, for example, 1,1,1,2-tetrafluoroethane [HFA-134a]), chlorofluorocarbon (CFC, for example, dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane) or suitable gas (for example, oxygen, compressed air or carbon dioxide). Drug in aerosol preparation is dissolved in propellant, or more commonly suspended in propellant, for delivery to lung. Aerosol can include excipient, such as surfactant (it strengthens the penetration into lung by reducing the high surface tension at air-water interface in alveolar, can also make drug emulsification, dissolve or / and stabilize, and can be for example phospholipid, such as lecithin) or / and stabilizer, although the surfactant part of peptide product can perform the function of surfactant. For example, an MDI formulation can include a peptide product, a propellant (e.g., an HFA such as 1,1,1,2-tetrafluoroethane) and a cosolvent (e.g., an alcohol such as ethanol), and optionally a surfactant (e.g., a fatty acid such as oleic acid). The MDI formulation can optionally include a dissolved gas (e.g., CO 2 ). After the device is activated, the CO in the sprayed aerosol droplets 2 The burst of bubbles breaks the droplets into smaller droplets, thereby increasing the respirable portion of the drug. As another example, the nebulizer formulation can contain a peptide product, a chelating agent or preservative (e.g., edetate disodium), an isotonic agent (e.g., NaCl), a pH buffer (e.g., citric acid / sodium citrate), and water, and optionally a surfactant (e.g., Such as polysorbate 80). The drug can be delivered by, for example, a nebulizer or an MDI, with or without a spacer, and the dose of drug delivered can be controlled by a metering chamber (nebulizer) or a metering valve (MDI).

[0084] Table 1 shows exemplary MDI, nebulizer and DPI formulations. Metered dose inhalers (also known as pressurized metered dose inhalers [pMDIs]) are the most widely used inhalation devices. Each time the device is actuated, a metering valve delivers a precise amount of aerosol (e.g., about 20 pL-100 pL). MDIs typically generate aerosols faster than the user can inhale, which can cause much of the aerosol to be deposited in the mouth and throat. The problem of poor coordination between device actuation and inhalation can be solved by using, for example, a breath-actuated MDI or a coordination device. In breath-actuated MDIs (e.g., Easi ) senses the user's inhalation, the device is activated and releases the drug dose in response. The inhalation flow is coordinated by the actuator, and the user has time to reliably actuate the device during inhalation. In the coordination device, a spacer (or a holding chamber with a valve) of a tube attached to the mouthpiece end of the inhaler is used as a reservoir or chamber to hold the drug ejected by the inhaler, and reduces the speed of the aerosol entering the mouth, thereby allowing the propellant to evaporate from larger droplets. The spacer simplifies the use of the inhaler and increases the amount of drug deposited in the lungs rather than in the upper respiratory tract. The spacer can be made of an antistatic polymer to minimize the electrostatic adhesion of the ejected drug particles to the inner wall of the spacer. The nebulizer produces aerosol droplets of about 1 micron to 5 microns. They do not require the user to coordinate between device actuation and inhalation, which may significantly affect the amount of drug deposited in the lungs. Compared with MDI and DPI, nebulizers can deliver larger doses of drugs, but the application time is longer. Examples of nebulizers include, but are not limited to, manual nebulizers, jet nebulizers (e.g., II BAN [breath-actuated], CompAIR TM NE-C80l[virtual valve], PARI Plus [Breathe Enhanced] and SideStream Plus [Breathe Enhanced]), ultrasonic nebulizers, and vibrating mesh nebulizers (e.g., Apixneb, I-neb AAD system with metering chamber, NE-U22, Omron U22 and PARI Rapid). As an example, a pulse ultrasonic nebulizer can atomize a fixed amount of medicine per pulse, and can include a photoacoustic trigger that allows the user to synchronize each breath with each pulse. For oral inhalation or nasal inhalation using a dry powder inhaler (DPI), a peptide product can be provided in the form of a dry micronized powder, wherein the drug particles have a specific small size (e.g., between about 0.5 microns and about 5 microns) to improve, for example, the aerodynamic properties of the dispersed powder and the deposition of the drug in the lungs. Particles between about 0.5 microns and about 5 microns are deposited in the terminal bronchioles and alveolar regions by sedimentation. In contrast, most larger particles (>5 microns) do not follow the airflow into the many bifurcations of the airway, but are deposited in the upper airway by impact, including the oropharyngeal region of the throat. The DPI formulation can include separate drug particles or powders mixed with a suitable larger base / carrier (such as lactose, starch, starch derivatives (e.g., hydroxypropyl methylcellulose) or polyvinyl pyrrolidine). Carrier particles enhance flow, reduce aggregation, improve dose uniformity, and contribute to the dispersion of drug particles. The DPI formulation may optionally contain excipients, such as magnesium stearate or / and leucine, which improve the performance of the formulation by interfering with interparticle binding (through anti-adhesion). The powder formulation may be provided in unit dosage form, such as capsules (e.g., gelatin capsules) or cartridges in blister packages that can be manually loaded or preloaded in an inhaler. The drug particles may be inhaled into the lungs as follows: by placing the mouthpiece or nozzle of the inhaler into the mouth or nose, taking a deep breath to produce turbulent airflow, and holding the breath for a period of time (e.g., about 5 seconds to 10 seconds) to allow the drug particles to settle in the bronchiolar and alveolar regions. When the user actuates the DPI and inhales, the airflow through the device generates shear and turbulence, the inhaled air is introduced into the powder bed, and the static powder mixture is fluidized and enters the airway of the user. There, the drug particles are separated from the carrier particles due to turbulence and are carried to the deep lungs, while the larger carrier particles impact the oropharyngeal surface and are cleared. Thus, the user's inspiratory airflow achieves powder deagglomeration and air ionization and determines drug deposition in the lungs. (While passive DPIs require rapid inspiratory airflow to deagglomerate drug particles, rapid inhalation is not recommended for MDIs or nebulizers because it produces turbulent airflow and rapid velocity, which increases drug deposition in the upper airways through impaction.) DPIs (including breath-activated DPIs) may be able to deliver larger doses of drug and larger sized drugs (e.g., macromolecules) to the lungs compared to MDIs.

[0085] Lactose (e.g., α-lactose monohydrate) is the most commonly used carrier in DPI formulations. Examples of grades / types of lactose monohydrate used in DPI formulations include, but are not limited to, DCL 11, 100. 230, 300, SD 250 (spray dried lactose), SV003 and 400. DPI formulations may contain a single lactose grade or a combination of different lactose grades. For example, a fine lactose grade such as 300 or 400 may not be a suitable DPI carrier and may need to be combined with a crude lactose grade such as DCL 11, 100. 230 or SV003 is mixed (eg, about 1:9 ratio of fine lactose to coarse lactose) to improve flowability.

[0086] Tables 2 and 3 show non-limiting examples of lactose grades / types that can be used in DPI formulations. The distribution of carrier particle sizes affects the fine particle fraction / dose (FPF or FPD) of the drug, and drug delivery to the lungs requires a high FPF. FPF / FPD is the respirable fraction / dose mass of aerodynamic particles <5 microns in the inhaled air that exit the DPI device. High FPF and therefore good DPI performance can be obtained, for example, from a fine lactose having a ratio of about 1:9 (e.g., 300) and crude lactose (e.g. SV003) ratio and about 20% w / w excess DPI formulation to avoid drug deposition in the capsule shell or DPI device and to deliver substantially all of the drug to the airways.

[0087] Table 2

[0088]

[0089] Table 3

[0090]

[0091] Other carriers used in DPI formulations include, but are not limited to, glucose, mannitol (e.g., crystalline mannitol [Pearlitol 110C] and spray-dried mannitol [Pearlitol 100SD]), maltitol (e.g., crystalline maltitol [Maltisorb P90]), sorbitol, and xylitol. Most DPIs are breath-activated ("passive"), relying on the user's inhalation to generate an aerosol. Examples of passive DPIs include, but are not limited to and Otsuka DPI (compact cake). Air Classifier Technology (ACT) is an effective passive powder dispersion mechanism employed in DPIs. In ACT, more than one supply channel generates a tangential airflow that creates a cyclone within the device during inhalation. There are also power-assisted ("active") DPIs (based on, for example, pneumatics, impact forces, or vibrations) that use energy to help, for example, particle deagglomeration. For example, The active mechanism of the inhaler utilizes mechanical energy stored in a spring or compressed air chamber. Examples of active DPIs include, but are not limited to (single unit dose), (Multiple doses), (single unit dose), (multi-unit dose and electronic activation), (single unit dose), Pfeiffer DPI (single unit dose), and (Multiple unit doses). The peptide product can also be administered by other routes, such as oral administration. Oral formulations can include peptide products and conventional excipients known in the art, and optionally absorption enhancers, such as sodium V-[8-(2-hydroxybenzoyl)aminocaprylate] (SNAC). SNAC prevents enzymatic degradation via local buffering, and enhances GI absorption. Oral dosage forms (e.g., tablets, capsules, or pills) can optionally have an enteric coating to protect its contents from the strong acid and proteolytic enzymes of the stomach. In some embodiments, the peptide product is delivered from a sustained release composition. As used herein, the term "sustained release composition" encompasses sustained release (sustained-release), long-term release (prolonged-release), extended release (extended-release), delayed release, slow release, and controlled release compositions, systems, and devices. In some embodiments, sustained release compositions deliver peptide products in at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or longer time periods. In some embodiments, sustained release compositions are formulated as nanoparticles or microparticles comprising biodegradable polymers and incorporating peptide products. In certain embodiments, the biodegradable polymer comprises lactic acid or / and glycolic acid [e.g., copolymers based on L-lactic acid, such as poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)]. In other embodiments, the sustained release composition is in the form of a depot produced when a mixture of the peptide product and the polymer is injected intramuscularly or subcutaneously into a subject. In certain embodiments, the polymer is or comprises PEG, polylactic acid (PLA) or polyglycolic acid (PGA), or a copolymer thereof (e.g., PLGA or PLA-PEG).

[0092] The pharmaceutical composition can be presented in a unit dosage form as a single dose, wherein all active ingredients and inactive ingredients are combined in a suitable system, and the components do not need to be mixed to form the composition to be administered. The unit dosage form usually contains a therapeutically effective dose of the drug, but may also contain an appropriate proportion thereof so that taking multiple unit dosage forms achieves a therapeutically effective dose. Examples of unit dosage forms include tablets, capsules or pills for oral intake; solutions in pre-filled syringes of single-use pens or pens with dose counters for parenteral (e.g., intravenous, subcutaneous or intramuscular) injections; and capsules, cartridges or blisters pre-loaded or manually loaded into an inhaler. Alternatively, the pharmaceutical composition can be presented as a kit, wherein active ingredients, excipients and carriers (e.g., solvents) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles or syringes), and need to be combined to form the composition to be administered. The kit may include instructions for storing, preparing and administering the composition (e.g., a solution to be injected parenterally). The kit may include all active ingredients and inactive ingredients of a unit dosage form, or include active ingredients and inactive ingredients in two or more separate containers, and may include instructions for applying or using a pharmaceutical composition to treat a medical condition disclosed herein. The kit may also include a device for delivering a composition, such as an injection pen or an inhaler. In some embodiments, the kit includes a peptide product or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the peptide product or a pharmaceutically acceptable salt thereof, and instructions for applying or using the peptide product or the composition to treat a medical condition disclosed herein, such as insulin resistance, diabetes, metabolic syndrome, cardiovascular disease, obesity (including "chronic obesity", which means to continue for more than one year or cause obesity-related conditions such as, but not limited to, insulin resistance, diabetes, metabolic syndrome and / or cardiovascular disease), or conditions associated therewith (e.g., NASH or NAFLD). In certain embodiments, the kit also includes a device for delivering a peptide product or composition, such as an injection pen or an inhaler.

[0093] Treatment

[0094] The present disclosure also provides for the use of the dual agonist peptide products described herein for preventing and / or treating conditions associated with GLP1R and / or GCGR, such as, but not limited to, insulin resistance, diabetes, obesity, metabolic syndrome, and cardiovascular disease, as well as conditions related thereto, such as NASH and PCOS. In some embodiments, the dual agonist peptide products can be used to treat hyperglycemia, insulin resistance, hyperinsulinemia, prediabetes, diabetes (including type 1 diabetes and type 2 diabetes, gestational diabetes, and juvenile diabetes), diabetic complications, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, elevated blood levels of free fatty acids, obesity, metabolic syndrome, syndrome X, cardiovascular disease (including coronary artery disease), atherosclerosis, acute cardiovascular syndrome, ischemia (including myocardial ischemia and cerebral ischemia / stroke), ischemia-reperfusion injury (including myocardial and cerebral IRI), infarction (including myocardial and cerebral infarction), angina pectoris, heart failure (e.g., congestive heart failure), peripheral vascular disease, thrombosis (e.g., deep vein thrombosis), embolism (e.g., pulmonary embolism), systemic inflammation (e.g., inflammation characterized by elevated blood levels of C-reactive protein), and hypertension. Dual agonist peptide products can achieve their therapeutic effects through various mechanisms, including stimulation of blood sugar-dependent insulin secretion, increased insulin sensitivity, stimulation of fat burning and weight loss. Dual agonist peptide products can also promote, for example, pancreatic β cell protection, cardioprotection and / or wound healing.

[0095] In certain embodiments, the method includes treating obesity and / or one or more symptoms or complications thereof in a subject in need thereof with a dual agonist peptide product of the present disclosure. In embodiments, symptoms and / or complications are one or more fatty liver diseases (FLD), wherein FLD is selected from NASH and NAFLD. In certain embodiments, symptoms and / or complications of obesity include type 2 diabetes. Nonalcoholic fatty liver disease (NAFLD) is a condition in which fat accumulates in the liver of people who drink little or no alcohol. Nonalcoholic steatohepatitis (NASH) is a type of NAFLD that involves inflammation and liver damage, as well as fat in your liver. The symptoms of NASH are usually not obvious, but some common symptoms include fatigue and right upper abdominal pain. Therefore, "treatment of obesity" means an activity that reduces or improves any primary phenomenon or secondary symptoms associated with obesity or other conditions described herein.

[0096] Non-alcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in the United States. NAFLD is associated with metabolic disorders, such as type 2 diabetes, hypertension, dyslipidemia and obesity. The potential pathophysiological mechanism of NAFLD development is mainly the change of glucose and lipid metabolism, insulin resistance (IR) and insulin secretion, which explains the close relationship between NAFLD and T2D. In addition, patients with both NAFLD and T2D usually share comorbidities associated with metabolic syndrome, i.e. fasting hyperglycemia, hypertension, hypertriglyceridemia, low high-density lipoprotein cholesterol and / or abdominal fat accumulation. It has been fully determined that individuals with NAFLD have more insulin resistance than individuals without NAFLD, even if they are thin and do not have diabetes. 7 Both longitudinal and cross-sectional studies show that increased IR is the earliest detectable abnormality in prediabetes and overt T2D. The pancreas responds to increased IR by secreting more insulin, and the liver reduces insulin clearance to increase peripheral insulin concentrations and prevent the development of diabetes. In NAFLD, IR is present in muscle, liver, and adipose tissue. Therefore, hepatic glucose production and adipose tissue lipolysis are only partially inhibited by insulin, resulting in higher fasting blood glucose and free fatty acid (FFA) concentrations, increasing the risk of T2D in these patients. Therefore, there is a need for treating obesity in subjects with NAFLD or NASH.

[0097] The prevalence of NAFLD in the United States is estimated to be between 20% and 30%, and it is also expected to be the leading cause of liver transplantation in the coming decade. NAFLD exists on a spectrum from simple steatosis to steatohepatitis (nonalcoholic steatohepatitis [NASH]), which is marked by lobular inflammation and ballooning. Throughout the disease spectrum, fibrosis progression can lead to the development of cirrhosis, although fibrosis progression is generally more common and more rapid with NASH than with simple steatosis.

[0098] Provided herein are methods for treating the symptoms and potential conditions (eg, pathogenesis) of obesity, including NAFLD and NASH.

[0099] NAFLD can be diagnosed by evidence of liver steatosis on imaging or histology and the lack of secondary causes of liver fat accumulation (alcoholic steatosis, drugs or genetic diseases). Non-alcoholic fatty liver (NAFL) is defined as the presence of ≥5% liver steatosis without evidence of liver cell damage in the form of hepatocellular ballooning. Although most patients tend to remain in the benign NAFL stage, some patients progress to NASH, which is characterized by the presence of ≥5% steatosis and inflammation with hepatocellular damage, with or without fibrosis. The established scoring system for assessing the histology of NAFLD is the NAFLD activity score (NAS). NAS is quantified using the following features: steatosis (0-3), lobular inflammation (0-3) and hepatocellular ballooning (0-2), which are added together to obtain a final score (0-8). In embodiments, provided herein is a method for treating obesity in a subject with NASH. In certain embodiments, the method provides an improvement in the NAS score of the subject.

[0100] The peptide products of the present disclosure are dual agonist GLP-1 / glucagon peptide products that act in part to help restore normal blood sugar and improve the risk of CVD or chronic kidney disease. In certain embodiments, SEQ ID NO: 1 promotes satiety and weight loss through direct effects on the central nervous system, reverses abnormal insulin and glucagon secretion in T2D, and has other beneficial metabolic effects associated with the pathophysiology of NAFLD. In vitro and in vivo studies have shown that GLP-1RA induces hepatic gene expression of pathways that improve autophagy / endoplasmic reticulum stress, macrophage recruitment and enhance mitochondrial function and fatty acid oxidation in hepatocytes, leading to a reduction in steatosis and inflammation.

[0101] The histological endpoints for evaluating the response to therapeutic intervention can be defined as the improvement of NAS, the regression of NASH, or the improvement of liver fibrosis. For the regression of NASH, it is defined as the complete regression of hepatocyte ballooning, the inflammation score is 0 or 1, and the fibrosis is not deteriorated. Hepatocyte ballooning is an important parameter of NASH and has been shown to be associated with disease progression and fibrosis. Fat accumulation in ballooning hepatocytes causes oxidative damage, endoplasmic reticulum dysfunction and cytoskeletal abnormalities, manifested as Mallory-Denk bodies. In an embodiment, biomarkers can also be used as markers of therapeutic efficacy. For the assessment of hepatic steatosis, controlled attenuation parameters as part of vibration-controlled transient elastography (FibroScan) and MRI proton density fat fraction (MRI-PDFF) and multi-parameter MRI (LiverMultiScan) are used. For liver inflammation and ballooning, liver enzymes ALT and aspartate aminotransferase (AST) can be used. LiverMultiScan is used for inflammation assessment and has received attention, but confirmation data is required. Transient elastography, LiverMultiScan, MRI, or MR elastography can be used to analyze fibrosis. Biomarkers can also be used for accurate hepatocellular fibrosis assessment, including Pro-C3, FIB-4, NAFLD fibrosis score, and enhanced liver fibrosis score are commonly studied non-invasive tools for fibrosis assessment.

[0102] The peptide products described herein can be used to treat other conditions associated with insulin resistance or / and obesity. Other conditions associated with insulin resistance or / and obesity include, but are not limited to, arthritis (e.g., osteoarthritis), low back pain, respiratory disorders (e.g., asthma, obesity hypoventilation syndrome [Pickwickian syndrome], and obstructive sleep apnea), dermatological disorders (e.g., diabetic ulcers, acanthosis nigricans, cellulitis, hirsutism, intertrigo, and lymphedema), gastroenterological disorders (e.g., cholelithiasis [gallstones], gastroesophageal reflux disease [GERD], and gastroparesis), gout, hypercortisolism (e.g., Cushing's syndrome), renal disorders (e.g., chronic kidney disease), liver disorders (e.g., fatty liver disease [FLD], including alcohol and non-alcoholic FLD), neurological disorders (e.g., carpal tunnel syndrome, dementia [e.g., Alzheimer's disease and vascular dementia], meralgia paresthesia, migraine, and multiple sclerosis), urinary disorders (e.g., erectile dysfunction, hypogonadism, and urinary incontinence), polycystic ovary syndrome, infertility, menstrual disorders, mood disorders (e.g., depression), and cancer (e.g., endometrial cancer, esophageal cancer, colorectal cancer, gallbladder cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, and skin cancer (e.g., melanoma] and leukemia). In certain embodiments, the dual agonist peptide products described herein are used to treat polycystic ovary syndrome (PCOS ). In other embodiments, the peptide products are used to treat chronic kidney disease (CKD), also known as chronic kidney / renal failure (CKF / CRF). The most common causes of CKD are diabetes and long-term uncontrolled hypertension. In additional embodiments, the dual agonist peptide products described herein are used to treat fatty liver disease (FLD). In some embodiments, FLD is non-alcoholic fatty liver disease (NAFLD), which should also be understood to include metabolic fatty liver disease (MFLD). In certain embodiments, NAFLD is non-alcoholic steatohepatitis (NASH). FLD, also known as hepatic steatosis, Characterized by excessive fat accumulation in the liver. FLD includes alcoholic fatty liver disease (AFLD) and NAFLD. Chronic alcoholism causes fatty liver due to the production of toxic metabolites such as aldehydes during alcohol metabolism in the liver. NAFLD is described as follows. FLD is associated with diabetes, obesity and metabolic syndrome. Fatty liver may develop into cirrhosis or liver cancer (e.g., hepatocellular carcinoma [HCC]). Less than about 10% of people with cirrhotic AFLD develop HCC, but up to about 45% of people with non-cirrhotic NASH may develop HCC. HCC is the most common type of primary liver cancer in adults and occurs in the context of chronic liver inflammation.NAFLD is characterized by fatty liver, which occurs when fat, especially free fatty acids and triglycerides, accumulate in hepatocytes (hepatic steatosis) due to reasons other than excessive alcohol consumption, such as nutritional overload, high caloric intake, and metabolic dysfunction (e.g., dyslipidemia and impaired glycemic control). The liver can remain fatty without interfering with liver function, but fatty liver can progress to NASH, a condition in which steatosis is accompanied by inflammation, ballooning of hepatocytes, and cell damage with or without liver fibrosis. Fibrosis is the strongest predictor of mortality caused by NASH. NAFLD can be characterized by: steatosis alone; steatosis with lobular or portal inflammation but without ballooning; steatosis with ballooning but without inflammation; or steatosis with inflammation and ballooning. NASH is the most extreme form of NAFLD. NASH is a progressive disease, with about 20% of patients developing cirrhosis and about 10% dying from liver disease, such as cirrhosis or liver cancer (e.g., HCC). NAFLD is the most common liver disorder in developed countries, and it is expected that by 2020, NASH will replace hepatitis C as the main cause of liver transplantation in the United States. About 12%-25% of Americans suffer from NAFLD, of which NASH affects about 2%-5% of Americans. NAFLD, including NASH, is associated with insulin resistance, obesity and metabolic syndrome. For example, insulin resistance contributes to the progression of fatty liver to liver inflammation and fibrosis and thus NASH. In addition, obesity drives and exacerbates NASH, and weight loss can alleviate NASH. Therefore, the peptide products described herein, including GLP-1 receptor (GLP1R) agonists, glucagon receptor (GCGR) agonists and GLP1R / GCGR dual agonists, can be used to treat NAFLD, including NASH. In some embodiments, the dual agonist peptide products disclosed herein for treating conditions associated with insulin resistance or / and obesity (such as fatty liver disease, including NAFLD and NASH) are pemvidutide and / or its derivatives and pharmaceutically acceptable salts thereof.

[0103] In some embodiments, one or more dual agonist peptides of the present invention can be used to control blood sugar while reducing one or more adverse events (i.e., unexpected events that negatively affect patient and / or animal welfare). Exemplary non-limiting adverse events may include nausea, vomiting, diarrhea, abdominal pain and / or constipation. Adverse events may also include any event known to those of ordinary skill in the art, such as listed in industry resources and / or events otherwise known to those of ordinary skill in the art (see, e.g., Medical Dictionary for Regulatory Activities (MedDRA) (Pharm., Med. Transl. Med. 2018) and / or Clark, MJ Biomed. Inf., 54, April 2015, pp. 167-173). Such adverse events can be determined in humans using standard techniques (e.g., doctor visits, surveys / questionnaires) commonly used in clinical trials. The dual agonist peptides of the present disclosure (e.g., any one of SEQ ID NO.1 or its derivatives) can reduce the frequency and / or severity of such adverse events by, for example, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90% or more (up to 100%) compared to the frequency and / or severity of such adverse events that occur when an agonist with unbalanced affinity for GLP-1R and GCGR is administered to a subject (e.g., semaglutide). In some embodiments, the dual agonist peptides of the present disclosure (e.g., pemvidutide) do not cause any adverse events.

[0104] The dual agonist peptide products of the present invention can be administered by any suitable route for treating the conditions disclosed herein. Possible routes of administration of the peptide products include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intracavitary and surface) and surface (including transdermal, transmucosal, intranasal (e.g., by nasal spray or drops), ocular (e.g., by eye drops), pulmonary (e.g., by oral inhalation or nasal inhalation), buccal, sublingual, rectal (e.g., by suppository) and vaginal (e.g., by suppository)). In some embodiments, the peptide products are administered parenterally, such as subcutaneously, intravenously or intramuscularly. In other embodiments, the peptide products are administered by oral inhalation or nasal inhalation or insufflation. The therapeutically effective amount and frequency of administration of the peptide products for treating the conditions disclosed herein and the length of time for treatment with the peptide products can depend on various factors, including the nature and severity of the condition, the efficacy of the compound, the route of administration, the age, weight, general health, sex and diet of the subject and the response of the subject to treatment, and can be determined by the treating physician. In some embodiments, the peptide product is administered parenterally (e.g., subcutaneously (sc), intravenously (iv), or intramuscularly (im)) at a dose of about 0.01 mg to about 0.1 mg, 1 mg, 5 mg, or 10 mg, or about 0.1 mg-1 mg, or 1 mg-10 mg, over a period of about one week for the treatment of conditions disclosed herein (e.g., conditions associated with insulin resistance or / and obesity, such as NASH or NAFLD). In additional embodiments, the peptide product is administered parenterally (e.g., sc, iv, or im) at a dose of about 0.1 mg-0.5 mg, 0.5 mg-1 mg, 1 mg-5 mg, or 5 mg-10 mg over a period of about one week. In certain embodiments, the peptide product is administered parenterally (e.g., subcutaneously (SC), intravenously (IV), or intramuscularly (IM)) at a dose of about 0.1 mg-1 mg, or about 0.1 mg-0.5 mg, or 0.5 mg-1 mg over a period of about one week. Those skilled in the art will appreciate that effective doses in mice or other preclinical animal models can be scaled for humans. Thus, doses for larger animals can be inferred from doses for mice by allometric scaling (also referred to as bioscaling) to obtain equivalent doses based on animal body weight or body surface area.

[0105] The peptide product can be administered at any suitable frequency for treating conditions disclosed herein (e.g., conditions associated with insulin resistance or / and obesity, such as NASH or NAFLD). In some embodiments, the dual agonist peptide product is administered, for example, once a day, once every two days, once every three days, twice a week, once a week, or once every two weeks sc or iv. In certain embodiments, the peptide product is administered, for example, once a week SC, IV, or IM. The dual agonist peptide product can be administered at any time of the day that is convenient for the patient. The dual agonist peptide product can be taken substantially with food (e.g., with a meal or within about 1 hour or 30 minutes before or after a meal) or substantially without food (e.g., at least about 1 hour or 2 hours before or after a meal). The length of time for treating a medical condition with a dual agonist peptide product can be based on, for example, the nature and severity of the condition and the subject's response to treatment, and can be determined by the treating physician. In some embodiments, the dual agonist peptide product is administered chronically to treat the conditions disclosed herein, such as at least about 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 5 years, 10 years or longer. The dual agonist peptide product can also be taken as needed (as needed) until the clinical manifestations of the condition disappear or a clinical goal is achieved, such as blood sugar level, blood pressure, blood lipid level, body weight or body mass index, waist-to-hip ratio or body fat percentage or any combination thereof. If the clinical manifestations of the condition reoccur or the clinical goal is not maintained, the administration of the dual agonist peptide product can be resumed. The present disclosure provides a method for treating the medical conditions described herein, comprising administering a therapeutically effective amount of a peptide product described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the peptide product or a pharmaceutically acceptable salt thereof to a subject in need of treatment. The present disclosure also provides a peptide product described herein or a pharmaceutically acceptable salt thereof or a composition comprising the peptide product or a pharmaceutically acceptable salt thereof for use as a medicament. In addition, the present disclosure provides the use of the peptide product described herein or a pharmaceutically acceptable salt thereof in the preparation of a medicament. The medicament comprising the peptide product can be used to treat any medical condition described herein. The peptide products may optionally be used in combination with one or more additional therapeutic agents.

[0106] The dual agonist peptide products described herein can be administered as the sole active agent, or optionally used in combination with one or more other dual agonist peptide products and / or additional therapeutic agents to treat any disorder disclosed herein, such as insulin resistance, diabetes, obesity, metabolic syndrome or cardiovascular disease, or any condition associated therewith, such as NASH or NAFLD. In some embodiments, one or more additional therapeutic agents are selected from antidiabetic agents, antiobesity agents (including lipid-lowering agents and satiety-promoting agents), antiatherosclerotic agents, anti-inflammatory agents, antioxidants, antifibrotic agents, antihypertensive agents, and combinations thereof. Antidiabetic agents include, but are not limited to, AMP-activated protein kinase (AMPK) agonists, including biguanides (e.g., buformin and metformin); peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, including thiazolidinediones (e.g., balaglitazone, ciglitazone, darglitazone, englitazone, lobeglitazone, neglitazone, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), MSDC-0602K, and saroglitazar (PPAR-α / γ dual agonist); glucagon-like peptide-1 (GLP-1) receptor agonists, including glucagon-4, albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, tasiglutide, CNT0736, CNT03649, HM11260C (LAP S-Exendin), NN9926 (OG9S7GT), TT401, and ZYOG1; dipeptidyl peptidase 4 (DPP-4) inhibitors, including alogliptin, anagliptin, dugliptin, evogliptin, gemagliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, septagliptin, sitagliptin, teneligliptin, trelagliptin, and vildagliptin; sodium-glucose transporter 2 (SGLT2) inhibitors, including canagliflozin (also inhibits SGLT1), dapagliflozin, empagliflozin, erpagliflozin, ipragliptin, rugliflozin, remogliflozin, and efogliptin. etabonate), sogliflozin (also inhibits SGLT1), and togliflozin; ATP-dependent K + (KA TP) channel blockers, including meglitinides (e.g., mitiglinide, nateglinide, and repaglinide) and sulfonylureas {including first generation (e.g., acetohexamide, amoxicillin, chlorpropamide, glycicloamide [toluamide], metahexamide, tolazamide, and tolbutamide) and second generation (e.g., glibenclamide, glyburide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisipide, and glypyramide); insulin and its analogs, including rapid-acting insulins (e.g., insulin aspart, insulin glulisine, and insulin lispro), intermediate-acting insulins (e.g., NPH insulin), and long-acting insulins (e.g., insulin degludec); In some embodiments, the antidiabetic agent is or includes a biguanide (e.g., metformin), a thiazolidinedione (e.g., pioglitazone or rosiglitazone), or an SGLT2 inhibitor (e.g., empagliflozin or togliflozin), or any combination thereof. Anti-obesity agents include, but are not limited to, appetite suppressants (anorectics), including amphetamine, dextroamphetamine, amfepramone, clobenzylex, mazindol, phentermine (with or without topiramate), and lorcaserin; satiety promoters, including ciliary neurotrophic factor (e.g., axokine) and long-acting analogs of amylin, calcitonin, cholecystokinin (CCK), GLP-1, leptin, oxyntomodulin, pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY); lipase inhibitors, including caulerpenyne, cetilistat, ebelactone A and ebelactone B, esterastin, lipstatin, orlistat, percyquinin, panclicin AE, valilactone and vibralactone; antihyperlipidemic agents; and analogs, derivatives and salts thereof. Antihyperlipidemic agents include, but are not limited to, HMG-CoA reductase inhibitors, including statins {e.g., atorvastatin, cerivastatin, fluvastatin, mevastatin, monacolins {e.g., monacolin K (lovastatin), pitavastatin, pravastatin, rosuvastatin and simvastatin} and flavanones (e.g., naringenin);Squalene synthase inhibitors, including lapaquistat, zaragozic acid, and RPR-107393; acetyl CoA carboxylase (ACC) inhibitors, including anthocyanidins, avenaciolides, chloroacetylated biotins, cyclodim, diclofop, haloxyfop, soraphens (e.g., soraphen Ala), 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), CP-640186, GS-0976, NDI-010976; 7-(4-propoxy-phenylethynyl)-3,3-dimethyl-3,4-dihydro-2H-benzo[b][l,4]dioxoheptane; N-ethyl-N'-(3-{[4-(3,3-dimethyl-l-oxo-2-oxa-7-azaspiro[4.5]dodecan-7-yl)piperidin-l-yl]-carbonyl}-l-benzothiophene -2-yl) urea; 5-(3-acetylaminobut-1-ynyl)-2-(4-propoxyphenoxy)thiazole; and 1-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dodecan-7-yl)piperidin-1-yl]-carbonyl}-5-(pyridin-2-yl)-2-thienyl)-3-ethylurea; PPAR-α agonists, including fibrates (e.g., bezafibrate, ciprofibrate, clinofibrate, clofibric acid, clofibrate, aluminum clofibrate, clofibrate [alfibrate], clofibride, etofibrate, fenofibrate acid, fenofibrate, gemfibrozil, cloniafibrate, and sibibrate), isoflavones (e.g., daidzein and genistein), and perfluoroalkanoic acids (e.g., perfluorooctanoic acid and perfluorononanoic acid); PPAR-delta agonists, including elafibranor (PPAR-alpha / gamma dual agonist), GFT505 (PPAR-alpha / gamma dual agonist), GW0742, GW501516 (PPAR-beta / delta dual agonist), solafiltazar (GW677954), MBX-8025, and isoflavones (e.g., daidzein and genistein);PPAR-γ agonists, including thiazolidinediones {as above), saroglitazar (dual PPAR-α / γ agonist), 4-oxo-2-thiothiazolines (e.g., rhodanine), berberine, honokiol, perfluorononanoic acid, cyclopentenone prostaglandins ( prostaglandins (e.g., cyclopentenone 15-deoxy-A-prostaglandin J2 [15d-PGJ2]) and isoflavonoids (e.g., daidzein and genistein); liver X receptor (LXR) agonists, including endogenous ligands (e.g., oxysterols such as 22(S)-hydroxycholesterol, 24(R)-hydroxycholesterol, 27-hydroxycholesterol and cholesteryl acid) and synthetic agonists (e.g., acetyl-podocarpic acid dimer, hypocholamide, A(X-dimethyl-3b-hydroxy-cholenamide, DMHCA], GW3965 and T0901317); retinoid X receptor (RXR) agonists, including endogenous ligands (e.g., 9-cis-retinoic acid) and synthetic agonists (e.g., bexarotene, AGN 191659, AGN191701, AGN 192849, BMS649, LG100268, LG100754, and LGD346); inhibitors of acyl-CoA cholesterol acyltransferase (ACAT, also known as sterol G-acyltransferase [SOAT], including ACAT1 [SOAT1] and ACAT2 [SOAT2]), including avasimibe, pactimibe, pellitorine, terpendole C and flavanones (e.g., naringenin); inhibitors of stearoyl CoA desaturase-1 (SCD-1, also known as stearoyl CoA delta-9 desaturase) activity or expression, including aramchol, CAY-10566, CVT-11127, SAR-224, SAR-707, XEN-103; 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide and 4-ethylamino 1'-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4'-piperidine]; 5-fluoro-1'-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-3,4-dihydrospiro[chromene-2,4'-piperidine];6-[5-(cyclopropylmethyl)-4,5-dihydro-1'H,3H-spiro[1,5-benzoxazepine-2,4'-piperidin]-1'-yl]-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-carboxamide; 6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxylic acid (2-hydroxy-2-pyridin-3-ylethyl)amide; 4-(2-chlorophenoxy)-N-[3-(methylcarbamoyl)phenyl]piperidine-1-carboxamide; cis-9, trans-11 isomers and trans-10, cis-12 isomers of conjugated linoleic acid, WO2009 / 129625 Substituted heteroaryl compounds disclosed in A1, antisense polynucleotides and peptide nucleic acids (PNA) targeting mRNA of SCD-1, and siRNA targeting SCD-1; cholesterol ester transfer protein (CETP) inhibitors, including anacetrapib, dalcetrapib, evacetrapib, torcetrapib and AMG 899 (TA-8995); inhibitors of microsomal triglyceride transfer protein (MTTP) activity or expression, including implitapide, lomitapide, dirlotapide, mitratapide, CP-346086, JTT-130, SLx-4090, antisense polynucleotides and PNAs targeting mRNA of MTTP, microRNAs targeting MTTP (e.g., miRNA-30c), and siRNAs targeting MTTP; GLP-1 receptor agonists; fibroblast growth factor 21 (FGF21) and its analogs and derivatives, including BMS-986036 (PEGylated FGF21); proprotein convertase subtilisin / kexin inhibitors of PCSK9 activity or expression, including berberine (which reduces PC8K9 levels), annexin A2 (which inhibits PCSK9 activity), anti-PCSK9 antibodies (e.g., alirocumab, bococizumab, evolocumab, LGT-209, LY3015014, and RG7652), peptides that mimic the epidermal growth factor A (EGF-A) domain of the LDL receptor that binds to PCSK9, adnectins that bind to PCSK9 (e.g., BMS-962476), antisense polynucleotides and PNAs that target PCSK9 mRNA, and siRNAs that target PCSK9 (e.g., inclisiran [ALN-PCS] and ALN-PCS02);Apolipoprotein mimetic peptides, including apoA-I mimetics (e.g., 2F, 3F, 3F-1, 3F-2, 3F-14, 4F, 4F-P-4F, 4F-IHS-4F, 4F2, 5F, 6F, 7F, 18F, 5A, 5A-C1, 5A-CH1, 5A-CH2, 5A-H1, 18A, 37pA [18A-P-18A], ELK, ELK-1A, ELK-1F, ELK-1K1A1E, ELK-1L1K, ELK-1 W, ELK-2A, ELK-2A2K2E, ELK-2E2K, ELK-2F, ELK-3E3EK, ELK-3E3K3A, ELK-3E3LK, ELK-PA, ELK-P2A, ELKA, ELKA-CH2, ATI-5261, CS-6253, ETC-642, FAMP, FREL and KRES, and apoE mimetics (e.g., Ac-hE18A-NH2, AEM-28, Ac-[R]hE1 8A-NH2, AEM-28-14, EpK, hEp, mR18L, COG-112, COG-133 and COG-1410); ω-3 fatty acids, including docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), fish oil (which contains, for example, DHA and EPA), and esters thereof (e.g., glycerides and ethyl esters); and analogs, derivatives and salts thereof. In certain embodiments, the anti-obesity agent is or includes a lipase inhibitor (e.g., orlistat) or / and an anti-hyperlipidemic agent (e.g., statins such as atropine) Antihypertensive agents include, but are not limited to, antagonists of the renin-angiotensin-aldosterone system (RAAS), including renin inhibitors (e.g., aliskiren), angiotensin converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandolapril), angiotensin II receptor type 1 (ATIII) antagonists (e.g., azilsartan, candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan medoxomil, olmesartan, telmisartan and valsartan) and aldosterone receptor antagonists (e.g., eplerenone and spironolactone);Diuretics, including loop diuretics (eg, bumetanide, ethacrynic acid, furosemide, torsemide), thiazide diuretics (eg, bendrofluazide, chlorothiazide, hydrochlorothiazide, epithiazide, methylclothiazide, and polythiazide), thiazide-like diuretics (eg, chlorthalidone, indapamide, and metolazone), cilclotanide (early distal tubular diuretics), diuretics), potassium-sparing diuretics (e.g., amiloride, eplerenone, spironolactone, and triamterene), and theobromine; calcium channel blockers, including dihydropyridines (e.g., amlodipine, levamlodipine, cilnidipine, clevidipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine) and nondihydropyridines (e.g., diltiazem and verapamil); alpha2-adrenoceptor agonists, including clonidine, guanabenz, guanfacine, methyldopa, and moxonidine; alpha1-adrenoceptor antagonists (alpha blockers), including doxazosin, indolaamine, nicergoline, phenoxybenzamine , phentolamine, prazosin, terazosin, and tolazoline; beta-adrenergic receptor (beta1 or / and beta2) antagonists (beta blockers), including atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, and timolol; mixed alpha / beta blockers, including bucindolol, carvedilol, and labetalol; endothelin receptor antagonists, including selective ETA receptor antagonists (e.g., ambrisentan, atresentan, edonantan, sitaxentan, zilpotentan, and BQ-123) and dual ETA / ETB antagonists (e.g., bosentan, macitentan, and tezosentan);Other vasodilators, including hydralazine, minoxidil, theobromine, sodium nitroprusside, organic nitrates (e.g., isosorbide mononitrate, isosorbide dinitrate, and nitroglycerin, which are converted to nitric oxide in the body), endothelial nitric oxide synthase (eNOS) stimulators (e.g., cilostanine), soluble guanylate cyclase activators (e.g., cinaciguat and riociguat), phosphodiesterase type 5 (PDE5) inhibitors (e.g., avanafil, benzamide fil), daxantafil, dynafil, lodenafil, mironafil, sildenafil, tadalafil, udenafil, vardenafil, dipyridamole, papaverine, propentofylline, zaprinast, and T-1032), prostaglandin Ei (alprostadil) and its analogs (e.g., limaprost and misoprostol), prostacyclin and its analogs (e.g., ataprost, beraprost [e.g., esuberaprost]), 5,6,7-triazine-4 ,8-m-w-phenylene-9-fluoro-PGl2, carbacyclin, isocarbacyclin, clinprostil, ciprostene, eptaprost, cicaprost, iloprost, pimidoprost, SM-10906 (desmethylpimidoprost), naxaprostene, taprostene, treprostinil, CS-570, OP-2507 and TY-11223), non-prostaglandin prostacyclin receptor agonists (e.g. (e.g., l-phthalazinol, ralinepag, selexipag, ACT-333679 [MRE-269, the active metabolite of selexipag], and TRA-418), phospholipase C (PLC) inhibitors and protein kinase C (PKC) inhibitors (e.g., BIM-1, BIM-2, BIM-3, BIM-8, chelerythrine, cilostanine, gossypol, miyabenol C, myricitrin, ruboxistaurin, and verbascoside); minerals, including magnesium and magnesium sulfate;And its analogs, derivatives and salts.In certain embodiments, antihypertensive agent is or includes thiazide or thiazide diuretic (for example, hydrochlorothiazide or chlorthalidone), calcium channel blocker (for example, amlodipine or nifedipine), ACE inhibitor (for example, benazepril, captopril or perindopril) or angiotensin II receptor antagonist (for example, Olmesartan Medoxomil, Olmesartan, Telmisartan or Valsartan), or any combination thereof.In some embodiments, peptide products described herein are used in combination with one or more other therapeutic agents to treat NAFLD, such as NASH.In some embodiments, one or more other therapeutic agents are selected from antidiabetic agents, antiobesity agents, anti-inflammatory agents, antifibrotic agents, antioxidants, antihypertensive agents and combinations thereof.In some embodiments, one or more other therapeutic agents are selected from antidiabetic agents, antiobesity agents, anti-inflammatory agents, antifibrotic agents, antioxidants, antihypertensive agents and combinations thereof. Therapeutic agents that can be used to treat NAFLD (e.g., NASH) include, but are not limited to: PPAR agonists, including PPAR-δ agonists (e.g., MBX-8025, elafibranor [PPAR-α / δ dual agonist] and GW501516 [PPAR-β / δ dual agonist]) and PPAR-γ agonists (e.g., thiazolidinediones, such as pioglitazone and saroglitazar [PPAR-α / γ dual agonist])—PPAR-δ and PPAR-γ agonism increase insulin sensitivity, PPAR-α agonism reduces hepatic steatosis, and PPAR-δ agonists inhibit activation of macrophages and Kupffer cells; farnesoid X receptor (FXR) agonists, such as levofloxacin and nonsteroidal FXR agonists, such as GS-9674, reduce hepatic gluconeogenesis, lipogenesis, steatosis and fibrosis; fibroblast growth factor 19 (FGF19) and its analogs fibroblast growth factor 21 (FGF21) and its analogs and derivatives, such as BMS-986036 (PEGylated FGF21) - FGF21 analogs reduce hepatic steatosis, cell damage and fibrosis; HMG-CoA reductase inhibitors, including statins (e.g., rosuvastatin) - statins reduce steatohepatitis and fibrosis; ACC inhibitors, such as NDI-010976 (liver-targeted) and GS-0976 - ACC inhibitors reduce de novo lipogenesis and hepatic steatosis; SCD-1 inhibitors, such as aramchol - SCD-1 inhibitors reduce hepatic steatosis and increase insulin sensitivity; SGLT2 inhibitors, such as canagliflozin, ipragliflozin and rupagliflozin - SGLT2 inhibitors reduce body weight, hepatic ALT levels and fibrosis;Antagonists of CCR2 or / and CCR5, such as cenicriviroc - antagonists of CCR2 (binding to CCL2 [MCP1]) and CCR5 (binding to CCL5 [RANTES]) inhibit activation and migration of inflammatory cells (e.g., macrophages) to the liver and reduce liver fibrosis; apoptosis inhibitors, including apoptosis signal-regulating kinase 1 (ASK1) inhibitors (e.g., selonsertib) and caspase inhibitors (e.g., emricasan [pan-caspase inhibitor]) - apoptosis inhibitors reduce liver steatosis and fibrosis; lysyl oxidase-like 2 (LOXL) 2) Inhibitors, such as simtuzumab—LOXL2 is a key matrix enzyme in collagen formation and is highly expressed in the liver; galectin-3 inhibitors, such as GR-MD-02 and TD139—galectin-3 is critical for the development of liver fibrosis; antioxidants, including vitamin E (e.g., α-tocopherol) and scavengers of reactive oxygen species (ROS) and free radicals (e.g., cysteamine, glutathione, melatonin, and pentoxifylline [also anti-inflammatory via inhibition of TNF-α and phosphodiesterase])—vitamin E reduces hepatic steatosis, hepatocellular ballooning, and lobular inflammation; and analogs, derivatives, and salts thereof. In some embodiments, the peptide products described herein are used in combination with a PPAR agonist (e.g., a PPAR-δ agonist such as elafibranor or / and a PPAR-γ agonist such as pioglitazone), an HMG-CoA reductase inhibitor (e.g., a statin such as rosuvastatin), an FXR agonist (e.g., obeticholic acid), or an antioxidant (e.g., vitamin E), or any combination thereof, to treat NAFLD (e.g., NASH). In certain embodiments, one or more additional therapeutic agents used to treat NAFLD (e.g., NASH) is or includes vitamin E or / and pioglitazone. Other combinations may also be used, as will be appreciated by those of ordinary skill in the art. ;

[0107] Pharmacokinetic (“PK”) parameters can be measured using Version 8.1 or higher (Certara USA, Inc., Princeton, New Jersey) is used for estimation. Non-compartmental methods consistent with the extravascular route of administration can be used for parameter estimation. Individual plasma concentration-time data can be used for pharmacokinetic calculations. In addition to parameter estimation for individual animals, descriptive statistics (e.g., mean, standard deviation, coefficient of variation, median, minimum, maximum) can be determined as appropriate. For the determination of descriptive statistics and pharmacokinetic analysis, concentration values ​​below the limit of quantification can be considered zero. Embedded concentration values ​​below the limit of quantification can be excluded from pharmacokinetic analysis. All parameters can be generated from the concentration of a single dual agonist peptide (or its derivatives and / or metabolites) in the plasma of the test article treatment group from the day of administration (Day 1). Parameters can be estimated using nominal dose levels unless an analysis result of a dose formulation exceeding the specification is obtained, in which case the actual dose level can be used. Parameters can be estimated using nominal sampling times; if a bioanalysis sample collection deviation is recorded, the actual sampling time can be used at the affected time point. Bioanalytical data may be used for pharmacokinetic analysis as received and may be presented in tables and figures in the units provided. Pharmacokinetic parameters may be calculated and presented in the units provided by the analytical laboratory (orders of magnitude may be adjusted appropriately for presentation in the report, e.g. h * ng / mL to h * μg / mL). Descriptive statistics (e.g., mean, standard deviation, coefficient of variation, median, minimum, maximum) and pharmacokinetic parameters may be determined to three significant figures as appropriate. Additional data processing items may be recorded as needed. Where data permit, the PK parameters to be determined may include, but are not limited to, the following: C max : Maximum observed concentration; DN C max : Dose-normalized maximum concentration, calculated as C max / dose; T max : time of maximum observed concentration; AUC 0-t : Area under the curve from time 0 to the time of the last measurable concentration, calculated using the linear trapezoidal rule; AUC 0-96 : Area under the curve from time 0 to 96 hours, calculated using the linear trapezoidal rule; DN AUC 0-96 : Dose-normalized AUC 0-96 , calculated as AUC 0-96 / dose; AUC 0-inf : Area under the curve from time 0 to infinity (day 1 only), calculated as AUC 0-inf =AUC 0-t +Ct / λ z , where C t is the last observed quantifiable concentration and λ z is the elimination rate constant; t 1 / 2 : Elimination half-life, calculated as ln(2) / λ z Additional parameters and comparisons (eg, gender ratio, proportionality ratio, etc.) may also be determined, as will be appreciated by one of ordinary skill in the art.

[0108] In some embodiments, the present disclosure provides one or more pharmaceutical dosage formulations comprising pemvidutide, wherein the peptide product is modified with a hydrophobic surfactant; the dosage is configured to induce weight loss while reducing one or more adverse events after administration to a mammal, wherein the subject has fatty liver disease and may also suffer from type 2 diabetes, wherein the adverse event is selected from nausea, vomiting, diarrhea, abdominal pain, and constipation.

[0109] "Reducing" or "reduction" of an adverse effect or event refers to a reduction in the extent, duration and / or frequency of the adverse effect experienced by a subject, as well as a reduction in the incidence in a group of subjects, after administration of an agonist with approximately balanced affinity for GLP1R and GCGR. Such a reduction includes preventing some adverse effects that a subject would otherwise experience when responding to an agonist with unbalanced affinity for GLP1R and GCGR. Such a reduction also includes the elimination of an adverse effect that a subject previously experienced after administration of an agonist with unbalanced affinity for GLP1R and GCGR. In some embodiments, "reducing" an adverse effect or a "reduction of" an adverse effect includes a reduction in gastrointestinal side effects, wherein the adverse event is reduced to zero or undetectable levels. In other embodiments, the adverse effect is reduced to a level equivalent to that of an untreated subject, but is not completely eliminated. Furthermore, administration of analogs with unbalanced affinities for GLP-1R or GCGR to a mammal may result in excessively high doses being required to maximally activate the receptor with the less sensitive ligand, thereby leading to the potential for exceeding the biologically effective dose level of the other ligand and causing dose-related undesirable side effects.

[0110] In preferred embodiments, the present disclosure provides a method for reducing the body weight of a human with fatty liver disease, wherein the method comprises administering pemvidutide once a week to a human in need thereof in an amount of at least 1.8 mg up to 2.4 mg; and wherein the human may suffer from type 2 diabetes, and wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some preferred embodiments of such methods, the body weight of the human is reduced by at least 3% relative to baseline at week 12. In some preferred embodiments of such methods, the body weight of the human is reduced by at least 4% relative to baseline at week 12. In some preferred embodiments of such methods, pemvidutide is administered once a week in an amount of 1.8 mg. In some preferred embodiments of such methods, pemvidutide is administered once a week in an amount of 2.4 mg.

[0111] In some preferred embodiments, the present disclosure provides a method of reducing liver fat content as determined by MRI-PDFF in a human with fatty liver disease, the method comprising administering pemvidutide to the human once weekly for at least 12 weeks in an amount of at least about 1.2 mg up to about 2.4 mg, wherein the human: has been diagnosed with fatty liver disease as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH); has a body mass index (BMI kg / m2) greater than about 27; 2); and having a liver fat content of at least about 10% as measured by MRI-PDFF. In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces an absolute reduction in liver fat content of at least about 7%, optionally at least about 9%, in a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces an absolute reduction in liver fat content of at least about 12.5%, optionally at least about 14%, in a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces an absolute reduction in liver fat content of at least about 10%, optionally at least about 11%, in a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces a relative decrease in liver fat content of at least about 40%, optionally at least about 45%, in a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces a relative decrease in liver fat content of at least about 60%, optionally at least about 65%, in a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces a relative decrease in liver fat content of at least about 50%, optionally at least about 55%, in a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces a relative decrease in liver fat content of at least about 30% in at least about 55%, optionally at least about 60% of a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces at least about 30% reduction in liver fat content in at least about 80%, optionally at least about 85% of a human population at week 12, which is significant (p<0.001) compared to placebo. In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces at least about 30% reduction in liver fat in at least about 75%, optionally at least about 80% of a human population. In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces at least about 50% reduction in liver fat content in at least about 35%, optionally at least about 38% of a human population at week 12, which is significant (p<0.001) compared to placebo.In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces a decrease in liver fat content by about 50% in at least about 55%, optionally at least about 60% of a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces a decrease in liver fat content by at least about 50% in at least about 60%, optionally at least about 65% of a population of humans at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content in at least about 20% of a population of humans at week 12, which is significant compared to placebo (p<0.05). In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content in at least about 50%, optionally at least about 55% of a human population at week 12, which is significant (p<0.0001) compared to placebo. In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content in at least about 50% of a human population at week 12, which is significant (p<0.001) compared to placebo.

[0112] In some preferred embodiments, the present disclosure provides a method of inducing weight loss in a human with fatty liver disease, the method comprising administering pemvidutide to the human once weekly for at least 12 weeks in an amount of at least about 1.2 mg up to about 2.4 mg, wherein the human: has been diagnosed with fatty liver disease that is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH); has a body mass index (BMI kg / m2) greater than about 27; 2); and having a liver fat content of at least about 10% as measured by MRI-PDFF. In some preferred embodiments of such methods, the human does not have diabetes and a dose of about 1.2 mg per week reduces the human's body weight by at least about 2.5%, optionally at least about 3%, at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, the human does not have diabetes and a dose of about 1.8 mg per week reduces the human's body weight by at least about 4%, optionally at least about 5%, at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, the human does not have diabetes and a dose of about 2.4 mg per week reduces the human's body weight by at least about 2.5%, optionally at least about 3.5%, at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, the human has type 2 diabetes and a dose of about 1.2 mg per week reduces the human's body weight by at least about 2%, optionally at least about 3%, at week 12. In some preferred embodiments of such methods, the human has type 2 diabetes and a dose of about 1.8 mg per week reduces the human's body weight by about 2.5%, optionally at least about 3.5%, at week 12, which is significant compared to placebo (p<0.05). In some preferred embodiments of such methods, the human has type 2 diabetes and a dose of about 2.4 mg per week reduces the human's body weight by about 3%, optionally at least about 4%, at week 12, which is significant compared to placebo (p<0.05). In some preferred embodiments of such methods, a dose of about 1.2 mg per week reduces the body weight of the human population by at least about 3% at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dose of about 1.8 mg per week reduces the body weight of the human population by at least about 4% at week 12, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a dosage of about 2.4 mg per week reduces body weight in a human population by at least about 3%, optionally at least about 3.5%, at week 12, which is significant compared to placebo (p<0.001).

[0113] In some preferred embodiments of the methods herein, a dose of about 1.2 mg per week induces at least about 11% reduction in ALT in a population of humans at week 12. In some preferred embodiments of the methods herein, a dose of about 1.8 mg per week induces at least about 13% reduction in ALT in a population of humans at week 12, which is significant compared to placebo (p<0.05). In some preferred embodiments of the methods herein, a dose of about 2.4 mg per week induces at least about 13% reduction in ALT in a population of humans at week 12, which is significant compared to placebo (p<0.05). In some preferred embodiments of the methods herein, a dose of about 1.2 mg per week induces at least about 19% reduction in ALT in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L at week 12. In some preferred embodiments of the methods herein, a dose of about 1.8 mg per week induces at least about 20% reduction in ALT in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L at week 12. In some preferred embodiments of the methods herein, a dose of about 2.4 mg per week induces at least about 20%, optionally at least about 25% reduction in ALT at week 12 in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L, which is significant (p<0.005) compared to placebo.

[0114] In some preferred embodiments, pemvidutide is administered by parenteral injection. In some preferred embodiments, pemvidutide is administered by subcutaneous injection. In some preferred embodiments, the human has a body mass index (BMI kg / m 2 In some preferred embodiments, the human has a body mass index (BMI kg / m 2). In some preferred embodiments, the human has a liver fat level of 10% or greater measured by MRI-PDFF before treatment. In some preferred embodiments, after 12 weeks of treatment, the absolute reduction in liver fat determined using MRI-PDFF is about 8%, 10%, 12%, or preferably about 15%. In some preferred embodiments, after 12 weeks of treatment, the relative reduction in liver fat determined using MRI-PDFF relative to baseline is greater than about 40%, 50%, or 60%. In some preferred embodiments, a steady-state dose is reached after a dose escalation phase with a duration of 2 to 4 weeks or about 6, 8, 10, 12, or 16 weeks. In some preferred embodiments, pemvidutide is administered from a liquid containing at least about 2.5 mg / ml pemvidutide. In some embodiments, pemvidutide is administered from a pharmaceutical dosage form as an aqueous formulation containing one or more of polysorbate 20, arginine, or mannitol.

[0115] In some embodiments, the disclosure provides a method for reducing the body weight of a human with fatty liver disease, the method comprising administering pemvidutide to a human in need thereof once a week in an amount of at least about 1.2 to about 2.4 mg (preferably about 1.2 mg, about 1.8 mg, or about 2.4 mg) for at least about 24 weeks; wherein the human optionally has type 2 diabetes and / or optionally wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some embodiments, pemvidutide is administered once a week in an amount of 1.2 mg for 24 weeks. In some such embodiments, the disclosure provides a method for administering pemvidutide once a week at a dosage of about 1.2 mg, 1.8 mg or 2.4 mg, wherein after 24 weeks of pemvidutide administration once a week, the relative reduction of liver fat measured by MRI-PDFF compared to baseline is about 30% to about 50%, wherein the relative reduction is statistically significant, defined as p<0.001 or p<0.0001. In some embodiments, 1.8 mg and 2.4 mg pemvidutide are administered once a week for 24 weeks to induce liver fat reduction of at least 40%, wherein the reduction is significant compared to placebo, defined as p<0.001 or p<0.01. In some embodiments, compared to the baseline in humans, after 24 weeks of administration of about 1.8 mg pevidutide once a week, the delipidation of the liver measured by MRI-PDFF is about 30% compared to the baseline. In some embodiments, after 24 weeks of weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pevidutide, liver volume measured by MRI-PDFF is reduced compared to baseline compared to placebo. In some embodiments, after 24 weeks of weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg, alanine aminotransferase (ALT) is reduced compared to placebo, optionally wherein humans have a baseline ALT of> 30 IU / L before dosing. In some embodiments, after 24 weeks of weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide, the reduction of iron-corrected T1 in about 80% of subjects is better than 80 ms, optionally wherein the reduction is significant, defined as p<0.05 or p<0.005. In some embodiments, once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide per week for 24 weeks results in weight loss in non-diabetic and / or diabetic patients compared to placebo. In some embodiments, once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide per week for 24 weeks results in reduced serum lipid levels compared to placebo.In some embodiments, once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide per week for 24 weeks reduces blood pressure without significantly increasing heart rate, optionally wherein systolic blood pressure is significantly reduced by 2.4 mg once weekly dosing, defined as p<0.05. In some embodiments, once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide per week for 24 weeks improves glycemic parameters, optionally wherein the glycemic parameters are reductions in fasting blood glucose and / or HbA1c levels.

[0116] In some preferred embodiments, the present disclosure provides the following aspects:

[0117] 1. A method of reducing body weight in a human with fatty liver disease, the method comprising administering pemvidutide once weekly to the human in need thereof in an amount of at least about 1.2 mg to about 2.4 mg, optionally about 1.8 mg up to about 2.4 mg, or about 1.2 mg, about 1.8 mg or about 2.4 mg, for at least about 12 weeks and / or up to and including at least about 24 weeks; wherein the human optionally has type 2 diabetes and / or optionally wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0118] 2. The method of aspect 1, wherein the human's body weight is reduced by at least 3% relative to baseline at week 12.

[0119] 3. The method of aspect 1, wherein the human's body weight is reduced by at least 4% relative to baseline at week 12.

[0120] 4. The method according to aspect 1, wherein the pemvidutide is administered in an amount of 1.2 mg once a week for 24 weeks.

[0121] 5. The method according to aspect 1, wherein the pemvidutide is administered in an amount of 1.8 mg once a week.

[0122] 6. The method according to any preceding aspect, wherein the pemvidutide is administered in an amount of 2.4 mg once a week.

[0123] 7. The method according to aspect 1, wherein the steady-state dose is reached after a dose escalation phase having a duration of about 2 weeks, about 3 weeks or about 4 weeks.

[0124] 8. The method of aspect 1, wherein the human suffers from type 2 diabetes.

[0125] 9. The method of aspect 1, wherein the human does not suffer from type 2 diabetes.

[0126] 10. The method of aspect 1, wherein the human has a body mass index (BMI kg / m 2 ).

[0127] 11. The method according to aspect 1, wherein the human has a body mass index (BMI kg / m 2 ).

[0128] 12. The method of aspect 1, wherein the human has a liver fat level of 10% or greater as measured by MRI-PDFF.

[0129] 13. The method of aspect 1, wherein after once weekly dosing for 12 weeks, the absolute reduction in liver fat as measured by MRI-PDFF is about 8% to about 15% reduction.

[0130] 14. The method of aspect 1, wherein after 12 weeks of weekly dosing, the relative reduction in liver fat as measured by MRI-PDFF compared to baseline is about 40% to about 70%.

[0131] 15. The method according to aspect 1, wherein after 24 weeks of once weekly dosing, the relative reduction in liver fat as measured by MRI-PDFF compared to baseline is about 30% to about 50%, wherein the relative reduction is statistically significant, defined as p<0.001 or p<0.0001.

[0132] 16. The method according to aspect 1, wherein the once weekly doses of 1.8 mg and 2.4 mg induce a reduction in liver fat of at least 40% after 24 weeks, wherein the reduction is significant compared to placebo, defined as p<0.001 or p<0.01.

[0133] 17. The method of aspect 1, wherein after once weekly dosing of about 1.8 mg pemvidutide for 24 weeks, liver fat loss as measured by MRI-PDFF is about 30% compared to baseline in said human.

[0134] 18. The method of aspect 1, wherein after weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide for 24 weeks, liver volume as measured by MRI-PDFF is reduced compared to baseline compared to placebo.

[0135] 19. The method of aspect 1, wherein alanine aminotransferase (ALT) is reduced compared to placebo following weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg for 24 weeks, optionally wherein the human has a baseline ALT of >30 IU / L prior to dosing.

[0136] 20. The method of aspect 1, wherein the reduction in iron-corrected T1 is better than 80 ms in about 80% of subjects, optionally wherein the reduction is significant, defined as p<0.05 or p<0.005.

[0137] 21. The method of aspect 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks results in weight loss in non-diabetic and / or diabetic patients compared to placebo.

[0138] 22. The method of aspect 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks results in a reduction in serum lipid levels compared to placebo.

[0139] 23. The method of aspect 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg or about 2.4 mg for 24 weeks reduces blood pressure without significantly increasing heart rate, optionally wherein systolic blood pressure is significantly reduced by once weekly dosing of 2.4 mg, defined as p<0.05.

[0140] 24. The method of aspect 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg or about 2.4 mg per week for 24 weeks improves glycemic parameters, optionally wherein the glycemic parameters are reductions in fasting blood glucose and / or HbA1c levels.

[0141] 25. A method of reducing liver fat content as determined by MRI-PDFF in a human with fatty liver disease, the method comprising administering pemvidutide in an amount of at least about 1.2 mg up to about 2.4 mg to the human once weekly for at least 12 weeks, wherein the human:

[0142] Have been diagnosed with fatty liver disease, either non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH);

[0143] Have a body mass index (BMI kg / m 2 );and,

[0144] Having a liver fat content of at least about 10% as measured by MRI-PDFF.

[0145] 26. The method according to any aspect herein, wherein a dose of about 1.2 mg per week induces an absolute reduction in liver fat content of at least about 7%, optionally at least about 8%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

[0146] 27. A method according to any aspect of the present invention, wherein a dose of about 1.8 mg per week induces an absolute reduction in liver fat content of at least about 12.5%, optionally at least about 14%, in a human population at week 12, which is significant compared to placebo (p<0.001).

[0147] 28. A method according to any aspect herein, wherein a dose of about 2.4 mg per week induces an absolute reduction in liver fat content of at least about 10%, optionally at least about 11%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

[0148] 29. A method according to any aspect of the present invention, wherein a dose of about 1.2 mg per week induces a relative reduction in liver fat content of at least about 40%, optionally at least about 45%, in a human population at week 12, which is significant compared to placebo (p<0.001).

[0149] 30. The method according to any aspect herein, wherein a dose of about 1.8 mg per week induces a relative reduction in liver fat content of at least about 60%, optionally at least about 65%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

[0150] 31. A method according to any aspect herein, wherein a dose of about 2.4 mg per week induces a relative reduction in liver fat content of at least about 50%, optionally at least about 55%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

[0151] 32. A method according to any aspect of the present invention, wherein a dose of about 1.2 mg per week induces at least about 30% reduction in liver fat content in at least about 55%, optionally at least about 60% of a human population at week 12, which is significant compared to placebo (p<0.001).

[0152] 33. A method according to any aspect of the present invention, wherein a dose of about 1.8 mg per week induces at least about 30% reduction in liver fat content at week 12 in at least about 80%, optionally at least about 90% of a human population, which is significant compared to placebo (p<0.001).

[0153] 34. A method according to any aspect of the present invention, wherein a dose of about 2.4 mg per week induces at least about a 30% reduction in liver fat content at week 12 in at least about 75%, optionally at least about 85% of a human population, which is significant compared to placebo (p<0.001).

[0154] 35. A method according to any aspect of the present invention, wherein a dose of about 1.2 mg per week induces at least about 50% reduction in liver fat content in at least about 35%, optionally at least about 40% of a human population at week 12, which is significant compared to placebo (p<0.001).

[0155] 36. A method according to any aspect of the present invention, wherein a dose of about 1.8 mg per week induces about a 50% reduction in liver fat content at week 12 in at least about 60%, optionally at least about 70% of a human population, which is significant compared to placebo (p<0.001).

[0156] 37. A method according to any aspect of the present invention, wherein a dose of about 2.4 mg per week induces at least about 50% reduction in liver fat content in at least about 60%, optionally at least about 70% of a human population at week 12, which is significant compared to placebo (p<0.001).

[0157] 38. A method according to any aspect of the present invention, wherein a dose of about 1.2 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content at week 12 in at least about 20% of a human population, which is significant compared to placebo (p<0.05).

[0158] 39. A method according to any aspect of the present invention, wherein a dose of about 1.8 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content at week 12 in at least about 50%, optionally at least about 55% of a human population, which is significant compared to placebo (p<0.0001).

[0159] 40. The method according to any aspect of the present invention, wherein a dosage of about 2.4 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content at week 12 in at least about 50% of a human population, which is significant compared to placebo (p<0.001).

[0160] 41. A method of inducing weight loss in a human with fatty liver disease, the method comprising administering pemvidutide in an amount of at least about 1.2 mg up to about 2.4 mg to the human once weekly for at least 12 weeks, wherein the human:

[0161] Have been diagnosed with fatty liver disease, either non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH);

[0162] Have a body mass index (BMI kg / m 2 );

[0163] Optionally, has been diagnosed with type 2 diabetes; and,

[0164] Having a liver fat content of at least about 10% as measured by MRI-PDFF.

[0165] 42. The method of aspect 41, wherein the human has not been diagnosed with type 2 diabetes, and a dose of about 1.2 mg per week reduces the human's body weight by at least about 2.5%, optionally at least about 3%, at week 12, which is significant compared to placebo (p<0.001).

[0166] 43. A method according to any aspect of the present invention, wherein the human has not been diagnosed with type 2 diabetes, and a dose of about 1.8 mg per week reduces the human's body weight by at least about 4%, optionally about 5%, at week 12, which is significant compared to placebo (p<0.001).

[0167] 44. A method according to any aspect of the present invention, wherein the human has not been diagnosed with type 2 diabetes, and a dosage of about 2.4 mg per week reduces the human's body weight by at least about 2.5%, optionally about 3.5%, at week 12, which is significant compared to placebo (p<0.001).

[0168] 45. A method according to any aspect of the present invention, wherein the human has been diagnosed with type 2 diabetes and a dose of about 1.2 mg per week reduces the human's body weight by at least about 2%, optionally at least about 3%, at week 12, which is significant (p<0.05) compared to placebo at week 12.

[0169] 46. ​​A method according to any aspect of the present invention, wherein the human has been diagnosed with type 2 diabetes and a dose of about 1.8 mg per week reduces the human's body weight by about 2.5%, optionally at least about 3.5%, at week 12, which is significant compared to placebo (p<0.005).

[0170] 47. A method according to any aspect of the present invention, wherein the human has been diagnosed with type 2 diabetes and a dose of about 2.4 mg per week reduces the human's body weight by about 3%, optionally at least about 4%, at week 12, which is significant compared to placebo (p<0.001).

[0171] 48. The method according to any aspect herein, wherein a dosage of about 1.2 mg per week reduces body weight in a human population by at least about 3% at week 12, which is significant compared to placebo (p<0.001).

[0172] 49. The method according to any aspect herein, wherein a dosage of about 1.8 mg per week reduces body weight in a human population by at least about 4% at week 12, which is significant compared to placebo (p<0.001).

[0173] 50. The method according to any aspect herein, wherein a dose of about 2.4 mg per week reduces body weight in a human population by at least about 3%, optionally at least about 3.5% at week 12, which is significant compared to placebo (p<0.001).

[0174] 51. The method according to any one of the aspects herein, wherein a dosage of about 1.2 mg per week induces at least about an 11% reduction in ALT at week 12 in a population of humans.

[0175] 52. The method according to any aspect herein, wherein a dose of about 1.8 mg per week induces at least about a 13% reduction in ALT at week 12 in a population of humans, which is significant (p<0.05) compared to placebo.

[0176] 53. The method according to any aspect herein, wherein a dose of about 2.4 mg per week induces at least about a 13% reduction in ALT at week 12 in a population of humans, which is significant (p<0.05) compared to placebo.

[0177] 54. The method according to any aspect herein, wherein a dosage of about 1.2 mg per week induces at least about an 18% reduction in ALT at week 12 in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L.

[0178] 55. The method according to any aspect herein, wherein a dosage of about 1.8 mg per week induces at least about a 20% reduction in ALT at week 12 in a population of humans having a baseline ALT of greater than or equal to about 30 IU / L.

[0179] 56. A method according to any aspect herein, wherein a dose of about 2.4 mg per week induces at least about 20%, optionally at least about 25% reduction in ALT at week 12 in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L, which is significant (p<0.005) compared to placebo.

[0180] 57. The method according to any one of the aspects herein, wherein the pemvidutide is administered by parenteral injection.

[0181] 58. The method according to any one of the aspects herein, wherein the pemvidutide is administered by subcutaneous injection.

[0182] 59. A method according to any aspect of the present invention, wherein the human has a body mass index (BMI kg / m 2 ).

[0183] 60. A method according to any aspect of the present invention, wherein the human has a body mass index (BMI kg / m 2 ).

[0184] 61. The method of any preceding aspect, wherein the human has a liver fat level of 10% or greater as measured by MRI-PDFF prior to the once weekly dosing.

[0185] 62. The method according to any one of the aspects herein, wherein after once weekly dosing for 12 weeks, the absolute reduction in liver fat as determined using MRI-PDFF is from about 8% to about 15%.

[0186] 63. The method of any aspect herein, wherein after 12 weeks of once weekly dosing, the liver fat content normalized to less than or equal to 5% is greater than 20% to about 55% of the population.

[0187] 64. The method according to any aspect herein, wherein after 12 weeks of weekly dosing, the relative reduction from baseline in liver fat as determined using MRI-PDFF is greater than about 40% to about 60%.

[0188] 65. The method according to any one of the aspects herein, wherein after 12 weeks of once weekly dosing, ALT is reduced by greater than about 13% to about 25% of the population.

[0189] 66. The method according to any aspect herein, wherein a steady-state dose is reached after a dose escalation phase having a duration of two to four weeks.

[0190] 67. The method according to any aspect herein, wherein the pemvidutide is administered from a liquid formulation comprising at least about 2.5 mg / ml pemvidutide.

[0191] 68. The method according to any one of the aspects herein, wherein the administration of pemvidutide induces a significant reduction in serum lipids and / or concentrations of atherogenic small and medium-sized LDL particles.

[0192] 69. A method according to any aspect of the present invention, wherein the concentration of atherogenic small to intermediate-sized LDL particles is reduced by at least -0.2 log relative to placebo after 43 days and / or 84 days of administration of pemvidutide. 2 Change multiple.

[0193] 70. A method according to any aspect of the present invention, wherein after 43 days and / or 84 days of administration of pemvidutide, the subject's total serum triglyceride concentration is reduced by at least -0.2 log relative to placebo when measured by 2D-NMR. 2 Change multiple.

[0194] 71. A method according to any aspect of the present invention, wherein the serum lipids are selected from glycerides, sterols, glycerophospholipids and sphingolipids, optionally wherein the reduction is at least -0.2 log 2 Change multiple.

[0195] 72. The method according to any aspect herein, wherein serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine and / or lysophosphatidylcholine are reduced following administration of Pemvidutide.

[0196] 73. A liquid pharmaceutical formulation comprising: SEQ ID NO: 1 and about 0.020% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in deionized water (pH 7.7 ± 0.1).

[0197] 74. The formulation according to aspect 73, comprising 1.8 mg of SEQ ID NO: 1 as a therapeutic dose.

[0198] 75. The formulation of aspect 74, wherein the therapeutic dose induces weight loss in a subject in need thereof.

[0199] Other aspects of the disclosure are also contemplated, as will be appreciated by one of ordinary skill in the art.

[0200] As will be understood by those of ordinary skill in the art, other aspects of the present disclosure are also contemplated, unless otherwise defined or otherwise clearly indicated by its use in this article, all technical and scientific terms used in this article have the same meanings commonly understood by those of ordinary skill in the art to which this application belongs. As used in the specification and the appended claims, the words "a" or "an" mean one or more. As used herein, the word "another" means a second or more. The acronym "aka" means also known as. The term "exemplary" as used herein means "used as an example, instance or illustration". Any embodiment or feature characterized as "exemplary" herein is not necessarily interpreted as being preferred or advantageous over other embodiments or features. In some embodiments, the term "about" or "approximately" means within ±10% or 5% of a specified value. Whenever the term "about" or "approximately" precedes the first value in a series of two or more numerical values ​​or a series of two or more numerical ranges, the term "about" or "approximately" applies to each numerical value in the series of numerical values ​​or the series of numerical ranges. The range can be expressed in this article as from about one specific value, and / or to about another specific value. The range can be expressed in this article as from about one specific value, and / or to about another specific value. When such a range is expressed, another aspect includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation, by using the antecedent about or approximately, it can be understood that the specific value forms another aspect. It will be further understood that the endpoints of each range are both associated with and independent of the other endpoint. It is meaningful to include the range itself and each independent value in the range, as if each value is listed separately. Optional or optional means that the event or situation described later may or may not occur, and the description includes instances where the event or situation occurs and instances where the event or situation does not occur. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0201] Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way. Example

[0202] Example 1. A Phase 1, 12-week, randomized, double-blind, placebo-controlled study of ALT-801 (Pemvidutide) in diabetic and non-diabetic overweight and obese subjects with non-alcoholic fatty liver disease (NAFLD)

[0203] The following disclosure is provided herein: A phase I, multicenter, randomized, double-blind, placebo-controlled study to evaluate the efficacy of NAFLD in diabetic and non-diabetic (type 2 diabetes) overweight and obese (BMI 28.0 kg / m 2 ) subjects, the safety of Pemvidutide and the effects on liver fat fraction, anthropometric parameters, lipid metabolism, inflammatory markers and fibrosis markers. The study aims to evaluate the changes in liver fat fraction in diabetic and non-diabetic overweight and obese subjects with NAFLD after 12 weeks of ALT-801 treatment by MRI-PDFF (magnetic resonance imaging-proton density fat fraction). The trial was conducted without auxiliary diet and exercise intervention. The study evaluated changes in body weight, lipid metabolism, metabolic markers and inflammatory markers after 12 weeks of treatment and the safety and tolerability of ALT-801. Subjects were stratified for the presence or absence of diabetes at baseline. Fibroscan and cT1 assessments evaluated changes in liver inflammatory and fibrotic activity at the end of 12 weeks of treatment.

[0204] The study included patients with a BMI ≥ 28 kg / m 2 Overweight and obese volunteers were included, as this is a typical group of individuals with NAFLD and these subjects are better able to tolerate the predicted pharmacodynamic (PD) effects of weight loss and may even benefit from treatment. For the evaluation of liver fat fraction in subjects with NAFLD, a liver fat fraction of 10% is typical. In other words, individuals with a liver fat fraction of 10% or greater are usually diagnosed with NAFLD. The study excluded diabetic subjects who required insulin, sulfonylureas, or DDP-4 inhibitors to control their diabetes. Exclusions were made for those that might otherwise affect the safety of the effects of ALT-801 or the accurate assessment of PD. The upper BMI limit was set at 45 kg / m 2 , because subjects above this BMI are unlikely to fit into the MRI scanner.

[0205] Fibroscan values ​​of <10 kPa sufficiently exclude subjects with advanced fibrosis who are not considered appropriate candidates for this clinical trial.

[0206] The study described in this example was conducted in 94 subjects (overweight and obese (body mass index [BMI] ≥ 28.0 kg / m 2), type 2 diabetic and non-diabetic subjects with non-alcoholic fatty liver disease (NAFLD), were treated with ALT-801 (a composition comprising SEQ ID NO.: 1, the "study drug") or placebo administered by subcutaneous (SC) injection once weekly for up to twelve (12) doses for approximately 4.5 months, including a screening phase of up to 35 days, a treatment phase of 85 days, and a follow-up phase of 25 days.

[0207] The baseline characteristics of the subjects included in this study are shown in Table 5 below:

[0208] Table 5

[0209]

[0210]

[0211] The safety objective of the study was to evaluate the efficacy of ALT-801 in patients aged 18-65 years with NAFLD (NAFLD without significant fibrosis, defined as Safety and tolerability in subjects with a controlled attenuation parameter (CAP) ≥ 280 dB / m, and liver stiffness measurement (LSM) < 10 kPa (i.e., indicating the absence of significant fibrosis), magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF) ≥ 10%), hemoglobin A1c (HbA1c) less than 9.5%, and alanine aminotransferase (ALT) or aspartate aminotransferase (AST) values ​​≤ 75 IU / ml). The pharmacodynamic (PD) objectives of the study are to evaluate the effects of ALT-801 on liver fat fraction, anthropometric parameters, lipid metabolism, metabolic markers, inflammatory markers, fibrosis markers, and lipotoxicity markers. The pharmacokinetic (PK) objectives of the study are to evaluate the effects of ALT-801 and metformin exposure (drug interactions) on subjects (e.g., changes in ALT-801 and metformin concentrations in the blood over time).

[0212] After providing informed consent, subjects underwent a screening phase of up to 35 days. Subjects were instructed on how to maintain their normal diet, alcohol, and physical activity at all times during their participation in the study and not to start any new diet, supplement, or exercise program. Diet and exercise advice was provided at the Day 1 visit and reinforced at subsequent visits. Study subjects were randomized 1:1:1:1 to one of the following treatment groups: 1) ALT-801 1.2 mg SC once weekly for 12 weeks; 2) ALT-801 1.8 mg SC once weekly for 12 weeks; 3) ALT-801 0.6 mg SC at Week 1, 1.2 mg SC at Week 2, 1.8 mg SC once weekly for 2 weeks (Weeks 3 and 4), and 2.4 mg SC once weekly for Weeks 5 to 12; or 4) placebo SC once weekly for 12 weeks. See Figure 1 Subjects were stratified by the presence or absence of diabetes at baseline. Subjects in the treatment group with a final treatment dose of 2.4 mg underwent a 4-week rapid dose escalation to improve tolerability and reduce the potential for adverse events. Previous first-in-human studies of pemvidutide showed that the 2.4 mg dose had a relatively high incidence of certain adverse events. See Figure 7 , the second 2.4 mg column is compared to the first 2.4 mg column (this study). See WO 2022 / 125598. Therefore, a rapid dose escalation regimen of pemvidutide is provided herein, with an initial weekly dose of 25% of the total therapeutic dose; the second week dose is 50% of the total therapeutic dose; and the third and fourth week doses are each 75% of the total therapeutic dose. In the fifth week and subsequent weeks, 100% of the total therapeutic dose is administered to the subject. In certain embodiments, for a final therapeutic dose of 2.4 mg, the dose escalation regimen is 0.6 mg, 1.2 mg, and 1.8 mg.

[0213] Subjects received the first dose of study drug on day 1 ("baseline"). Subsequent visits were conducted weekly in the clinic, at home or at work until day 85 or early termination. Subjects returned for a safety follow-up visit on day 110. Researchers followed decision criteria for the time and method of intervention for subjects with abnormal worsening of liver function tests during the 12-week treatment phase. Fasting blood glucose levels were measured by a blood glucose meter at baseline and before each dose and recorded by researchers. On non-visit days, subjects were monitored to record fasting blood glucose every morning, and if the reading was >240mg / dL or <70mg / dL, the research center was contacted. Subjects also received education on the symptoms and treatment of hypoglycemia, and if they experienced blood glucose <70mg / mL or symptoms suggesting hypoglycemia, a blood glucose meter reading would be obtained. Subjects recorded any symptoms of hypoglycemia experienced at home in a log, and researchers reviewed them at each visit starting from day 8. Subjects were advised by investigators on how to keep their fasting glucose within limits, including repeated dietary advice, and followed decision criteria for timing and method of intervention for subjects with persistent hyperglycemia during the 12-week treatment phase. Certain subjects who demonstrated significant reductions in fasting glucose (<50 mg / dL) were observed repeatedly. Small blood samples were collected for ALT-801 PK to be combined with data from other studies in population PK and PK-PD modeling, as well as data for metformin PK, to assess changes in metformin concentrations over time in the presence of ALT-801. Blood samples were also collected to assess immunogenicity.

[0214] The power and sample size assumptions used in this study were that the sample size was considered adequate for safety assessments in a Phase I study. Based on the treatment effects observed in previous NAFLD studies, the study was also adequately powered to detect a meaningful difference in change from baseline in liver fat fraction (based on MRI-PDFF) at the 0.05 significance level (two-sided) in subjects receiving ALT-801 SC injections compared to those receiving placebo.

[0215] For statistical analysis, all randomized subjects who received at least one dose of study drug (safety population) were included in the safety analysis. Secondary endpoints and PD endpoints were assessed in the PD population, which consisted of all randomized subjects who received at least one dose of study drug and had baseline and at least one post-baseline PD assessment results.

[0216] Two interim analyses were performed: 1) when all subjects completed the Day 43 visit; and 2) when all subjects completed the Day 85 visit. For these analyses, unblinding was limited to the treatment group level, and the study team was blinded to individual treatment assignments. Analyses included safety, weight loss, and MRI-PDFF since the start of treatment, as well as available PK data. Summary data by study part, dose level, treatment group (active or placebo), and day (where applicable) were reported. Continuous safety data were summarized by dose level and treatment (active or placebo) using descriptive statistics (arithmetic mean, standard deviation [SD], median, minimum, and maximum). Categorical safety data were summarized by dose level, treatment group, and day (where applicable) using frequency counts and percentages. AEs were coded using the latest version of the Medical Dictionary for Regulatory Activities (MedDRA). A list of AE data by subject is provided, including verbatim terms, preferred terms, system organ class (SOC), treatment, severity, and relationship to study drug. The number of subjects experiencing treatment-emergent AEs (TEAEs) and the number of individual TEAEs and injection site reactions are summarized by treatment group, SOC, and preferred term. TEAEs are also summarized by severity and by relationship to study drug. Laboratory evaluations, including liver function tests and fasting glucose, vital signs (including RPP calculations), and ECG assessments are summarized by treatment group, dose level, and protocol-specified collection time points. A summary of changes from baseline at each protocol-specified time point is also determined by treatment group. Physical examination changes for each subject will be listed. Concomitant medications are listed by subject and coded using the latest version of the World Health Organization (WHO) Drug Dictionary. Medical history is coded using the latest MedDRA version and will be listed by subject.

[0217] For pharmacodynamic determinations, descriptive statistics, including the number and percentage of categorical variables and the number, mean, SD, median, minimum, and maximum of continuous variables, were provided by dose level and treatment (ALT-801 or placebo) and, when applicable, by day. Changes from baseline in liver fat fraction, anthropometric parameters, lipid metabolism, metabolic markers, lipotoxicity markers, and inflammatory markers were summarized by treatment group and stratification using descriptive statistics (sample size [N], arithmetic mean, SD, median, minimum, maximum, geometric mean, and geometric coefficient of variation [CV%]). The effect of baseline BMI on PD parameters was evaluated by covariate analysis. Inferential statistics were performed where applicable. All analyses were described in the Statistical Analysis Plan (SAP). Changes in liver fat fraction according to MRI-PDFF, body composition according to MRI and Fibroscan, and hepatic inflammatory and fibrotic markers, as well as other continuous variables, were compared between the ALT-801 group and the placebo group using analysis of covariance (ANCOVA) tests, with treatment group as a factor and stratification by the presence or absence of diabetes or corresponding baseline demographic characteristics (sex, race, BMI) as covariates. For secondary endpoints of categorical nature, the Cochrane Mantel Haenszel test will be applied, taking into account the stratification by the presence or absence of diabetes, with a one-sided significance level of 0.025.

[0218] Quality of life was also measured as two summary scores for physical health and mental health, as well as eight domain scores of the SF-36 and the IWQoL-Lite composite score for CT. Changes from baseline will be listed and summarized by treatment group using descriptive statistics (N, arithmetic mean, SD, median, minimum, maximum, geometric mean, and geometric CV%). Inferential statistics applicable to continuous endpoints were applied as described above.

[0219] For pharmacokinetic (PK) determinations, individual ALT-801 and metformin concentration data are listed and summarized by treatment group and time point using descriptive statistics (N, arithmetic mean, SD, CV%, median, minimum and maximum). Individual and mean ± SD ALT-801 concentration-time profiles for each cohort are also presented graphically. Changes relative to baseline metformin concentrations are also listed and summarized by treatment group and time point using descriptive statistics (N, arithmetic mean, SD, CV%, median, minimum and maximum). A population PK model was developed to be able to predict individual subject ALT-801 plasma concentration-time profiles and related PK parameters. Covariates, including sex, age, weight, BMI, and concomitant medications, were explored as much as possible, and efficacy and safety endpoints of exposure-response relationships were explored as much as possible. This analysis combines data from multiple studies and will be conducted and reported as a separate study.

[0220] like Figure 2 As shown in , at week 12, ALT-801 induced significant absolute reductions in liver fat of approximately 10% or more at all non-placebo doses (placebo: 0.2%; 1.2 mg: 8.9%; 1.8 mg dose: 14.7%; and 2.4 mg dose: 11.3% (all doses relative to placebo, p<.001,)), and induced significant relative reductions in liver fat of at least about 40% or more at all non-placebo doses (placebo: 4.4%; 1.2 mg: 46.6%; 1.8 mg dose: 68.5%; and 2.4 mg dose: 57.1% (relative to placebo, p<.001,)).

[0221] like Figure 3 As shown in Figure 2, ALT-801 achieved at least a 30% reduction in liver fat content as measured by MRI-PDFF at Week 12 at all doses in a significant proportion of subjects who completed 12 weeks of treatment (p<.0001 relative to placebo), with the 1.2 mg dose: 65% of patients; 1.8 mg: 94.4% of patients; and 2.4 mg dose: 85% of patients at Week 12, compared to placebo (4.2% of patients).

[0222] Figure 3 It was also shown that ALT-801 achieved at least a 50% reduction in liver fat content as measured by MRI-PDFF at Week 12 at all doses in a significant proportion of subjects who completed 12 weeks of treatment (1.2 mg dose: in approximately 40% of patients (p<.001); 1.8 mg dose: at 1.8 mg, 72.2% of patients (p<.0001); and 2.4 mg dose; 70% (p<.0001)) compared to placebo (0% of patients) at Week 12.

[0223] Figure 3 It was also shown that ALT-801 achieved normalization of liver fat content (equal to or less than 5%) as measured by MRI-PDFF at all doses in a significant proportion of subjects who completed 12 weeks of treatment: at Week 12, 1.2 mg dose: 20% of patients (p<.05); 1.8 mg dose: 55.6% of patients (p<.0001); 2.4 mg dose: 50.0% (p<.001), compared to placebo (0%).

[0224] Figure 4It was shown that in non-diabetic subjects, ALT-801 induced significant weight loss at all doses, 3.4% at the 1.2 mg dose (p<.001); 4.9% at the 1.8 mg dose (p<.001); and 3.5% at the 2.4 mg dose (p<.001) at week 12, compared to placebo (0.2%).

[0225] Figure 4 It was also shown that in diabetic subjects, ALT-801 induced weight loss at all doses, 3.3% at the 1.2 mg dose (p<.005); 3.8% at the 1.8 mg dose (p<.05); and 4.4% at the 2.4 mg dose (p<.001) at week 12, compared to placebo (0.5%).

[0226] Figure 4 It was also shown that ALT-801 induced significant weight loss in all subjects at all doses, 3.4% at the 1.2 mg dose (p<.001); 4.3% at the 1.8 mg dose (p<.001); and 3.7% at the 2.4 mg dose (p<.001) compared to placebo (0.2%) at week 12.

[0227] Figure 5 It was shown that ALT-801 induced at least 10% reduction in ALT at week 12 in all subjects at the 1.2 mg dose (-11.2%); at the 1.8 mg dose (-13.8%, p<0.05); and at the 2.4 mg dose (-13.6%, p<0.05), compared to placebo (-6.2%).

[0228] Figure 5 ALT-801 was also shown to induce significant ALT reductions in subjects with baseline ALT greater than or equal to 30 IU / L after Week 12 in all subjects (n=83) at the 1.2 mg dose (-17.8%); at the 1.8 mg dose (-20.8%); and at the 2.4 mg dose (-27%, p<0.005), compared to placebo (-12.6%).

[0229] Figure 6 The proportions of subjects showing ALT-801-induced iron-corrected T1 (cT1) ≥80 ms are shown: after Week 12, at the 1.2 mg dose (87.5%); at the 1.8 mg dose (83.3%), and at the 2.4 mg dose (85.7%), compared to placebo (0%).

[0230] Therefore, a method for using pemvidutide to reduce the weight of a human with fatty liver disease is provided herein, wherein the method comprises administering pemvidutide once a week to a human in need thereof in an amount of at least 1.8 mg up to 2.4 mg; and wherein the human may suffer from (or may not suffer from) type 2 diabetes, and wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In certain embodiments, a method for using pemvidutide to reduce the weight of a human (e.g., an overweight or obese subject) with NAFLD or NASH is provided herein, wherein the method comprises administering pemvidutide once a week to a human in need thereof in an amount of at least 1.8 mg up to 2.4 mg; and wherein the person does not suffer from type 2 diabetes (e.g., non-diabetic).

[0231] There were no significant changes in serum lipids in subjects after 12 weeks of ALT-801 administration. Even without dose titration, the symptoms experienced by subjects after 12 weeks of treatment with ALT-801 were mainly mild and transient in nature, consistent with the known GLP-1 class effects. No severe adverse events (AEs) or serious AEs were observed after 12 weeks of treatment with ALT-801. In addition, after 12 weeks of continuous administration of ALT-801, a very low rate of AEs leading to treatment discontinuation was observed. In addition, the mean serum alanine aminotransferase (ALT) levels of all subjects decreased, and in subjects with baseline serum ALT above 30 IU / L, the mean serum alanine aminotransferase (ALT) levels decreased by more than 17 IU / L at all dose levels, and the mean serum alanine aminotransferase (ALT) levels decreased by 27.0 IU / L in the 2.4 mg dose cohort. No clinically significant ALT elevations (defined as increases to 3 times or more of the upper limit of normal) were observed for 12 weeks. Glycemic control was not affected, with no clinically meaningful changes in HbA1c or fasting glucose over 12 weeks.

[0232] At the conclusion of this 12-week clinical study, this example shows that ALT-801 administration resulted in a robust (>60%) relative reduction in liver fat, superior to the effects of other GLP-1 agonists and leading NASH candidates. Regarding weight loss, this example shows that placebo-adjusted weight loss (4.7%) at 12 weeks in non-diabetic subjects was superior to semaglutide (Wegovy; GLP-1 agonist), and placebo-adjusted weight loss (4.5%) at 12 weeks in diabetic subjects was superior to tirzepatide (GIP / GLP-1 agonist). The trial was conducted without adjunctive diet and exercise intervention, which is standard for obesity trials. In addition, no serious or severe adverse events ( Figure 7 ); no 3-fold or greater ALT elevations were observed; the rate of AEs leading to treatment discontinuation was very low, including 1 case (4.3%) in the 1.8 mg ALT-801 group and 1 case (4.2%) in the 2.4 mg ALT-801 group, both secondary to gastrointestinal intolerance; and only increases in heart rate (HR) of 1-3 beats per minute (bpm) were observed, which is within the range of other GLP-1-based agents that have been used in humans.

[0233] Example 2. Effects of the GLP-1 / glucagon dual receptor agonist Pemvidutide (ALT-801) on pathogenic lipid mediators

[0234] There is increasing interest in the potential to reduce cardiovascular (CV) risk through incretin-based therapies. Pemvidutide is a long-acting GLP-1 / glucagon (1:1) dual receptor agonist being developed for the treatment of NASH and obesity. Pemvidutide combines the anorectic effects of GLP-1 receptor agonism (RA) with the energy expenditure-increasing and lipid-lowering effects of glucagon RA. Plasma lipids have multiple functions in biological systems, such as energy storage, metabolic regulation, signaling, proliferation, and apoptosis. The plasma lipidome can be analyzed using nuclear magnetic resonance (NMR) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS).

[0235] To test the lipid-lowering effect of pemvidutide, data from a phase I clinical trial (NCT0456124) were analyzed. 2) Subjects were randomized at 1 study center in Australia (NCT0456124). Subjects were randomized to a 4:1 pemvidutide:placebo group with placebo pooled. Pemvidutide doses were 1.2 mg, 1.8 mg, and 2.4 mg, administered once weekly for twelve (12) weeks without dose titration or adjunctive lifestyle intervention (no diet or exercise intervention). Pemvidutide was well tolerated at all dose levels without the use of dose titration. All AEs in these groups were mild or moderate in severity, therefore no grade 3 (severe) AEs were observed here, and no SAEs or AEs leading to treatment discontinuation were reported. Lipoprotein and glycoprotein profiling covering 33 lipoprotein-related parameters was performed by 1 H-NMR was performed on fasting plasma samples obtained on day -1 (baseline), day 43, and day 84 from 34 subjects who completed NCT0456124. Lipidomic profiling covering 600 lipid classes was performed by ultra-high performance liquid chromatography-mass spectrometry on fasting plasma samples obtained on day -1 (baseline), day 43, and day 84 from 34 subjects who completed NCT0456124. For lipid profiling, plasma fractionation was performed using methanol to extract fatty acyl groups, bile acids, steroids, and lysoglycerophospholipids, or using chloroform / methanol mixtures to extract glycerolipids, cholesterol esters, sphingolipids, and glycerophospholipids. Lipid classification followed the classification system proposed by Fahy et al. (J. Lipid Res. 2005; 46: 839-861) and the LIPID MAPS initiative (http: / / www.lipidmaps.org) (see Figure 8 ).

[0236] Consistent with the data presented in Example 1, the data presented here show that pemvidutide has a favorable effect on weight loss, body mass index (BMI), blood pressure, total cholesterol, LDL cholesterol, triglycerides, and apolipoprotein B, as summarized in Table 6:

[0237] Table 6

[0238] Key pharmacodynamic endpoints (change from baseline)

[0239]

[0240] Compared with placebo, * p<0.05, ** p<0.01, *** p<0.001

[0241] Pemvidutide also caused overall consistent and favorable changes in lipoprotein particle subspecies, as determined by 2D-NMR analysis (see Fig. 9 ). The color code indicates log 2 (robust fold change), blue indicates a decrease in lipoprotein (negative fold change), and red indicates an increase in lipoprotein (positive fold change). Fig. 9 It was shown that the number of VLDL and LDL particles as well as the number of smaller HDL particles tended to decrease after treatment with pemvidutide.

[0242] Given the observed changes in total cholesterol, triglycerides, and lipoproteins, serum lipid composition covering 600 lipid classes was evaluated at Day -1 (baseline), Day 43, and Day 84 from 34 subjects who completed NCT0456124. Results are expressed as log 2 Pairwise fold changes are presented in Fig.10 In the Figure 2, blue indicates a decrease in lipids relative to baseline, and red indicates an increase. Grey / black bars indicate significant p-values ​​from the Wilcoxon test. Within 12 weeks of treatment, pemvidutide significantly reduced lipid levels, particularly glycerolipids (diglycerides and triglycerides), glycerophospholipids (phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine), and sphingolipids (ceramide and sphingomyelin), which are associated with reduced cardiovascular (CV) risk and reduced insulin resistance (see Fig.10 ). Thus, at day 85, large and highly statistically significant reductions in multiple bioactive lipid classes were observed compared to placebo.

[0243] Based on the lipidomics data, a volcano plot was generated, such as Fig.11 The horizontal dashed line indicates the lower limit of significance, while the vertical line shows the log 2 The fold change from baseline is ± 0.75 on the scale. Responses in the upper left quadrant indicate particularly interesting changes in atherogenic lipids. Pemvidutide treatment significantly reduced atherogenic lytic PC levels (see Fig.11 ), suggesting the possibility of a reduction in oxidized LDL (Law et al. J. Mol. Sci. 2019; 20(5): 1149). Pemvidutide treatment also significantly reduced levels of atherogenic phosphatidylethanolamine (PE) glycerophospholipids. Based on these findings, pemvidutide shows promise as an agent for reducing cardiovascular (CV) risk.

[0244] In this trial, pemvidutide induced significant weight loss at week 12, as Fig. 12A and Fig. 12BAt the 1.8 mg dose level, a mean weight loss of up to 10.3% (placebo adjusted 8.7%) was observed, with high statistical significance after only 12 weeks of weekly treatment. Additionally, in this treatment group, all subjects lost at least 5% of their body weight, and more than half achieved at least 10% weight loss.

[0245] In a Phase 1a trial, pemvidutide was shown to be safe and well tolerated without the need for dose titration. In this study, mean body weight loss of up to 10.3% was observed, and statistically significant reductions in multiple atherogenic lipid classes as well as in the size of atherogenic small and medium lipoprotein particle concentrations were observed. Based on these findings, pemvidutide shows promise as an agent for inducing weight loss (independent of a diagnosis of type 2 diabetes) while simultaneously improving obesity comorbidities, including reductions in CV risk factors (e.g., pathogenic serum lipid mediators).

[0246] Example 3. Phase 1b, 24-week, randomized, double-blind, placebo-controlled study of ALT-801 (Pemvidutide) in diabetic and non-diabetic overweight and obese subjects with non-alcoholic fatty liver disease (NAFLD)

[0247] Provided herein is a disclosure of a 12-week extension study of the study provided in Example 1, which provides a 24-week Phase 1b, randomized, double-blind, placebo-controlled study to evaluate the efficacy of lipopolysaccharide in patients with NAFLD (defined as liver fat content (LFC) ≥ 10% by MRI-PDFF; the absence of significant fibrosis (defined as LSM <10 kPa), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) laboratory values ​​≤75 IU / L, and HbA1c <9.5) in diabetic and non-diabetic (type 2 diabetes) overweight and obesity (BMI > 28.0kg / m 2) subjects, the safety of pemvidutide, and the effects on weight loss, liver fat fraction, anthropometric parameters, lipid metabolism, and inflammatory markers, fibrosis markers, and other markers. Males and females aged 18-65 years were studied. Diabetic subjects were defined as subjects who had undergone stable doses (≥3 months) of metformin or SLGT-2 treatment and were not treated with insulin, sulfonylureas, DPP-4, GLP-1; Ninety-four (94) subjects were included in the initial 12-week Phase 1b NAFLD trial, and 83 completing subjects were invited to receive additional blinded treatment for a total of 12 weeks. Of these subjects, 66 agreed to an extension (rollover), of which 64 were eligible to participate. Study dispositions are shown in Fig.13 middle.

[0248] The primary endpoint of the study was a reduction in liver fat content (LFC) based on MRI-PDFF at week 24 compared to week 0 (zero). Key secondary endpoints included percent (%) weight loss at week 24 compared to week 0 (zero), and liver inflammation as measured by alanine aminotransferase (ALT) levels and corrected T1 (cT1) imaging at week 24 compared to week 0 (zero). Adverse events (AEs), including severe and serious AEs, AEs leading to discontinuation, GI tolerability, vital signs, and glycemic control, including fasting glucose and HbA1c levels, were also measured. Detailed baseline characteristics of the trial participants are shown in Fig.14 middle.

[0249] like Fig.15 As shown in, robust reductions in liver fat content were observed at week 24, as determined by MRI-PDFF. As shown therein, significant (p<0.001) reductions in liver fat content were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo in both absolute and relative values. In terms of absolute reduction, the 1.2 mg, 1.8 mg, and 2.4 mg doses showed an average absolute reduction of 11.2%, 17%, and 15.6% in liver fat, respectively (p<0.001). In terms of relative reduction, the 1.2 mg, 1.8 mg, and 2.4 mg doses showed an average relative reduction of 56.3%, 75.2%, and 76.4% in liver fat, respectively (p<0.001).

[0250] Fig.16Further, the percentage of subjects who achieved at least 30% and 50% reduction in liver fat content and normalization of liver fat content (≤5%) at week 24 is also shown. As shown therein, for all doses of pemvidutide administered (1.2 mg, 1.8 mg and 2.4 mg per week), the percentage of subjects who achieved at least 30% and 50% reduction in liver fat content was highly significant compared to placebo (as shown therein, p<0.001 or p<0.0001). In addition, in a high proportion of subjects, especially at 1.8 mg dose (53.8%) and 2.4 mg dose (45.3%), normalization of liver fat content was achieved, and 1.8 mg and 2.4 mg doses were highly significant compared to placebo (p<0.001 or p<0.01).

[0251] Fig.17 Shown (pemvidutide, 1.8 mg dose) was a significant defatting of the liver as determined by MRI-PDFF at week 24. The reduction in liver volume in this scan of the exemplary subject taking pemvidutide was dramatic (32.3% at baseline vs. 1.7% at week 24).

[0252] Fig.18 Robust reductions in liver volume determined by MRI-PDFF, measured as both absolute and relative reductions, were shown at week 24. As shown therein, significant reductions in liver fat content were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo in both absolute and relative values ​​(p<0.05 or p<0.001 as indicated therein).

[0253] Fig.19 ALT levels (a biomarker of liver inflammation) were significantly reduced in all subjects at week 24, which was significantly higher in subjects with baseline ALT levels. > As shown therein, for example, significant reductions in ALT were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo in all subjects and in subjects with baseline ALT ≥ 30 IU / L (p<0.001).

[0254] Fig. 20A high cT1 response ratio at week 24 is shown. The response threshold is defined as a decrease of 80 milliseconds (ms) in CT1 compared to baseline. As shown therein, for example, all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) were observed to have a significantly reduced cT1 response compared to placebo (as indicated therein, p<0.05 or p<0.005). It is noted that a decrease of about 80 ms in cT1 is associated with a 2-point decrease in the NASH activity score (NAS) (Dennis, A., Front. Endocrinology, 2021), and increased cT1 levels are associated with an increased risk of major adverse cardiac events (MACE) and major adverse liver outcomes (MALO) (Jayaswal, A. Liver Int., 2020; Roca-Fernandez A., MedRxiv, 2022).

[0255] Fig.21 Sustained weight loss at week 24 compared to week 12 is shown. This distinguishes pemvidutide from other NASH drugs that have comparable reductions in liver fat levels. As shown therein, significant reductions in body weight were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo in non-diabetic patients as well as in all subjects (p<0.005 or p<0.001 as indicated therein). Mean weight loss was also observed in diabetic patients (see Fig.21 ).

[0256] Fig. 22 Improvements in serum lipids are shown at week 24. As shown therein, for example, improvements in serum lipids were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week).

[0257] Fig.23 It was shown that all doses of pemvidutide tested induced improvements in blood pressure at week 24 without clinically significant increases in heart rate. As shown therein, for example, such improvements in blood pressure were observed for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week). It was noted that the reduction in systolic blood pressure was significant (p<0.05) at the 2.4 mg dose per week.

[0258] Fig.24The safety overview of the trial is shown. In the treatment group, the number of severe or serious adverse events was low and comparable to the placebo group. Severe and serious AEs were the same events and included: 1) chest pain after selective coronary stenting (placebo), 2) Salmonella infection (pemvidutide, 1.2 mg), and 3) hypertension > 3 weeks after the last dose of study drug (pemvidutide, 1.8 mg), all of which were unrelated to the study drug, with only Salmonella infection leading to treatment discontinuation. The two (2) gastrointestinal (GI) AEs that led to treatment discontinuation were mild (grade 1) abdominal pain in two (2) subjects. ALT was not reported to be significantly elevated. In addition, across the treatment group, the number of adverse events leading to treatment discontinuation and gastrointestinal-related adverse events leading to treatment discontinuation was low. Gastrointestinal-related adverse events such as nausea, vomiting, diarrhea, and constipation were mild to moderate.

[0259] Fig.25 Improvements in glycemic parameters (fasting blood glucose and HbA1c) in diabetic patients and maintenance of glycemic control in non-diabetic patients at week 24 were shown. As shown therein, for example, such improvements in glycemic control, including reductions in fasting blood glucose and HbA1c, were observed in diabetic patients for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week). These clinical effects support the anti-hyperglycemic effect of pemvidutide, presumably related to its GLP-1 activity that counteracts the hyperglycemic effects of its glucagon activity.

[0260] The study showed that pemvidutide induced liver fat reduction, including a relative liver fat reduction of greater than 75% at 24 weeks (better than or equal to the effect of other leading NASH candidates), and significant reductions in cT1 and serum ALT, indicating effective effects in NASH clinical trials. The study also showed that pemvidutide induced weight loss in non-diabetic subjects (sustained weight loss, reaching 7.2% at week 24) and diabetic subjects (5.3% weight loss at week 24). The study also showed that pemvidutide was safe and tolerable (e.g., low AE rate leading to treatment discontinuation, no severe / serious AEs related to pemvidutide; well tolerated, no dose titration required, consistent with previous experience; no clinically significant ALT elevations; and glycemic control was maintained, with reductions in fasting blood glucose and HbA1c in diabetic patients).

[0261] Example 4: Efficacy Interim Analysis of a Phase II, 24-week, Randomized, Double-blind, Placebo-controlled Study of ALT-801 (Pemvidutide) in Non-diabetic Obese and Overweight Subjects at Higher Risk for NAFLD and NASH.

[0262] The results presented in this example correspond to a 24-week interim analysis obtained from a placebo-controlled Phase II study evaluating pemvidutide (ALT-801) in obese and overweight subjects (ClinicalTrials.gov Identifier: NCT05295875). It is estimated that 70% to 75% of subjects in this group have NAFLD, while 34% may have NASH (Quek J. et al. Global prevalence of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in the overweight and obese population: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol. 2023 Jan; 8 (1): 20-30). The key eligibility criteria are: (1) male and female, aged 18-75 years; (2) at least one unsuccessful weight loss attempt as judged by the investigator; (3) body mass index (BMI) ≥ 30 kg / m 2 or BMI ≥ 27 kg / m 2 , with at least one obesity-related comorbidity (cardiovascular disease, hypertension, dyslipidemia, prediabetes, or history of obstructive sleep apnea), and (4) non-diabetes (HbA1c ≤ 6.5%, fasting glucose ≤ 125 mg / dL). Eligible subjects were randomized 1:1:1:1 to one of the following treatment groups: Group 1 (39 subjects): pemvidutide 1.2 mg SC once weekly for 24 weeks; Group 2 (40 subjects): pemvidutide 1.8 mg SC once weekly for 24 weeks; Group 3 (40 subjects): pemvidutide 0.6 mg SC for 1 week, 1.2 mg SC for 1 week, 1.8 mg SC once weekly for 2 weeks, then 2.4 mg SC once weekly for an additional 20 weeks (sequentially); and Group 4 (41 subjects): placebo SC once weekly for 24 weeks. According to gender and baseline body mass index (BMI < 35 kg / m 2 and ≥35kg / m 2) randomization of subjects was stratified. Minimum 25% of randomized subjects were male. At follow-up visits during the screening and treatment phases, all subjects received qualified health care professionals' advice on a reduced calorie diet of 1200-1500 calories (for individuals <250lb (113.6kg)) and 1500-1800 calories (for individuals ≥250lb (113.6kg)) and gradually increased physical activity (the goal is 150min physical activity per week). Subjects were instructed to record their food intake and physical activity every day, and researchers will regularly assess compliance with lifestyle interventions. Subject subgroups received MRI-PDFF to evaluate liver fat fractions and components to measure total body adipose tissue (AT) and fat-free tissue mass (ATFM).

[0263] Baseline characteristics of the study participants are presented in Fig.26 middle. Fig. 27 Mean percentage weight loss over time for all evaluated subjects over 24 weeks is presented for the three pemvidutide doses and placebo. Results are presented as efficacy estimates assuming that subjects remained on treatment for the entire duration, with missing values ​​handled by a mixed model for repeated measures. Fig. 27 Gradual, dose-dependent weight loss over time was shown with pemvidutide compared to placebo. Fig.28 The mean percentage weight loss achieved over 24 weeks with pemvidutide and placebo in the subgroup of subjects with a baseline body weight ≤ 115 kg is presented (presented as efficacy estimates as described above). Fig. 27 The results presented in Fig.28 A gradual, dose-dependent weight loss over time was demonstrated with pemvidutide compared with placebo. Fig. 27 When compared, Fig.28 The results presented in indicate that each of the three doses of pemvidutide achieved higher levels of weight loss in subjects with lower baseline body weight. This phenomenon may be related to lower drug exposure levels in subjects with higher body weight and would support the possibility of increasing dose levels in this population to maximize efficacy. Fig.29 A weight loss responder analysis based on calculation of the percentage of subjects achieving ≥5%, ≥10%, and ≥15% weight loss for the three pemvidutide doses and placebo, respectively, is presented. Fig.30 The percentage of weight loss achieved by Hispanic subjects and non-Hispanic subjects in the four groups is presented. The results show that overall, subjects of Hispanic descent tended to lose less weight after treatment with pemvidutide, indicating a possible influence of genetic background. Fig.31A shows that treatment with pemvidutide for 24 weeks improved both systolic and diastolic blood pressure in a dose-dependent manner compared to placebo. This result is important considering that obesity represents a major cause of hypertension. Fig.31 B shows that treatment with pemvidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg for 24 weeks did not induce significant changes in heart rate. Fig.32 All doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg) showed a reduction in serum lipids at week 24 compared to baseline. Fig.32 Improvements in serum lipids at week 24 are shown for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week). Fig.33 All doses of pemvidutide administered (1.2 mg, 1.8 mg and 2.4 mg per week) showed a significant reduction in waist circumference at week 24. Waist circumference is an index of central or abdominal obesity recommended by the WHO for assessing the risk of metabolic diseases such as NAFLD and NASH and cardiovascular diseases.

[0264] Thus, provided herein are methods of using pemvidutide to reduce body weight in a human at high risk for fatty liver disease, wherein the method comprises administering pemvidutide once weekly to a human in need thereof in an amount of at least 1.8 mg up to 2.4 mg; and, wherein the human does not suffer from type 2 diabetes, and wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0265] As will be appreciated by those of ordinary skill in the art, other advantages of reagents and methods using the reagents are also provided herein. Although certain embodiments have been described according to preferred embodiments, it should be understood that variations and modifications will occur to those skilled in the art. Therefore, the appended claims are intended to cover all such equivalent variations that fall within the scope of the following claims.

Claims

1. A method of reducing body weight in a human with fatty liver disease, the method comprising administering pemvidutide once weekly to the human in need thereof in an amount of at least about 1.2 mg to about 2.4 mg, optionally about 1.8 mg up to about 2.4 mg, or about 1.2 mg, about 1.8 mg or about 2.4 mg, for at least about 12 weeks and / or up to and including at least about 24 weeks; wherein the human optionally has type 2 diabetes and / or optionally wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

2. The method of claim 1, wherein the human's body weight is reduced by at least 3% from baseline at week 12.

3. The method of claim 1, wherein the human's body weight is reduced by at least 4% from baseline at week 12.

4. The method according to any preceding claim, wherein the pemvidutide is administered in an amount of 1.2 mg once a week for 24 weeks.

5. The method according to any preceding claim, wherein the pemvidutide is administered in an amount of 1.8 mg once weekly.

6. The method according to any preceding claim, wherein the pemvidutide is administered in an amount of 2.4 mg once weekly.

7. The method of claim 1, wherein a steady-state dose is reached after a dose escalation phase having a duration of about 2 weeks, about 3 weeks, or about 4 weeks.

8. The method of claim 1, wherein the human suffers from type 2 diabetes.

9. The method of claim 1, wherein the human does not suffer from type 2 diabetes.

10. The method of claim 1, wherein the human has a body mass index (BMI kg / m 2 ).

11. The method of claim 1, wherein the human has a body mass index (BMI kg / m 2 ).

12. The method of claim 1, wherein the human has a liver fat level of 10% or greater as measured by MRI-PDFF.

13. The method of claim 1, wherein the absolute reduction in liver fat as measured by MRI-PDFF is about 8% to about 15% reduction after once weekly dosing for 12 weeks.

14. The method of claim 1, wherein after 12 weeks of weekly dosing, the relative reduction in liver fat as measured by MRI-PDFF compared to baseline is about 40% to about 70%.

15. The method of claim 1, wherein after 24 weeks of weekly dosing, the relative reduction in liver fat as measured by MRI-PDFF compared to baseline is about 30% to about 50%, wherein the relative reduction is statistically significant, defined as p<0.001 or p<0.0001.

16. The method of claim 14, wherein the once weekly doses of 1.8 mg and 2.4 mg induce a reduction in liver fat of at least 40% after 24 weeks, wherein the reduction is significant compared to placebo, defined as p<0.001 or p<0.

01.

17. The method of claim 1, wherein after once weekly dosing of about 1.8 mg pemvidutide for 24 weeks, liver fat loss as measured by MRI-PDFF is about 30% compared to baseline in said human.

18. The method of claim 1, wherein liver volume as measured by MRI-PDFF is reduced from baseline following weekly administration of about 1.2 mg, about 1.8 mg, or about 2.4 mg pemvidutide for 24 weeks compared to placebo.

19. The method of claim 1, wherein alanine aminotransferase (ALT) is reduced compared to placebo following weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg for 24 weeks, optionally wherein the human has a baseline ALT of >30 IU / L prior to dosing.

20. The method of claim 1, wherein the reduction in iron-corrected T1 is better than 80 ms in about 80% of subjects, optionally wherein the reduction is significant, defined as p<0.05 or p<0.

005.

21. The method of claim 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks results in weight loss in non-diabetic and / or diabetic patients compared to placebo.

22. The method of claim 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks results in a reduction in serum lipid levels compared to placebo.

23. The method of claim 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks reduces blood pressure without significantly increasing heart rate, optionally wherein systolic blood pressure is significantly reduced by the 2.4 mg once weekly dosing, defined as p<0.

05.

24. The method of claim 1, wherein once weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks improves a glycemic parameter, optionally wherein the glycemic parameter is a reduction in fasting blood glucose and / or HbA1c levels.

25. A method of reducing liver fat content as determined by MRI-PDFF in a human with fatty liver disease, the method comprising administering pemvidutide in an amount of at least about 1.2 mg up to about 2.4 mg to the human once weekly for at least 12 weeks, wherein the human: Have been diagnosed with fatty liver disease, either non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH); Have a body mass index (BMI kg / m 2 );and, Having a liver fat content of at least about 10% as measured by MRI-PDFF.

26. The method of claim 15, wherein a dose of about 1.2 mg per week induces an absolute reduction in liver fat content of at least about 7%, optionally at least about 8%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

27. The method of claim 15, wherein a dose of about 1.8 mg per week induces an absolute reduction in liver fat content of at least about 12.5%, optionally at least about 14%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

28. The method of claim 15, wherein a dose of about 2.4 mg per week induces an absolute reduction in liver fat content of at least about 10%, optionally at least about 11%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

29. The method of claim 15, wherein a dose of about 1.2 mg per week induces a relative reduction in liver fat content of at least about 40%, optionally at least about 45%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

30. The method of claim 15, wherein a dose of about 1.8 mg per week induces a relative reduction in liver fat content of at least about 60%, optionally at least about 65%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

31. The method of claim 15, wherein a dose of about 2.4 mg per week induces a relative reduction in liver fat content of at least about 50%, optionally at least about 55%, in a population of humans at week 12, which is significant compared to placebo (p<0.001).

32. The method of claim 15, wherein a dose of about 1.2 mg per week induces at least about a 30% reduction in liver fat content at week 12 in at least about 55%, optionally at least about 60% of a population of humans, which is significant compared to placebo (p<0.001).

33. The method of claim 15, wherein a dose of about 1.8 mg per week induces at least about a 30% reduction in liver fat content at week 12 in at least about 80%, optionally at least about 90% of a population of humans, which is significant compared to placebo (p<0.001).

34. The method of claim 15, wherein a dose of about 2.4 mg per week induces at least about a 30% reduction in liver fat content at week 12 in at least about 75%, optionally at least about 85% of a human population, which is significant (p<0.001) compared to placebo.

35. The method of claim 15, wherein a dose of about 1.2 mg per week induces at least about a 50% reduction in liver fat content at week 12 in a population of at least about 35%, optionally at least about 40%, of humans, which is significant compared to placebo (p<0.001).

36. The method of claim 14, wherein a dose of about 1.8 mg per week induces about a 50% reduction in liver fat content at week 12 in a population of at least about 60%, optionally at least about 70%, of humans, which is significant compared to placebo (p<0.001).

37. The method of claim 15, wherein a dose of about 2.4 mg per week induces at least about a 50% reduction in liver fat content at week 12 in at least about 60%, optionally at least about 70% of a human population, which is significant compared to placebo (p<0.001).

38. The method of claim 15, wherein a dosage of about 1.2 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content at week 12 in at least about 20% of a human population, which is significant (p<0.05) compared to placebo.

39. The method of claim 15, wherein a dose of about 1.8 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content at week 12 in at least about 50%, optionally at least about 55% of a population of humans, which is significant compared to placebo (p<0.0001).

40. The method of claim 15, wherein a dose of about 2.4 mg per week induces normalization of liver fat content to less than or equal to about 5% liver fat content at week 12 in at least about 50% of a human population, which is significant compared to placebo (p<0.001).

41. A method of inducing weight loss in a human with fatty liver disease, the method comprising administering pemvidutide in an amount of at least about 1.2 mg up to about 2.4 mg to the human once weekly for at least 12 weeks, wherein the human: Have been diagnosed with fatty liver disease, either non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH); Have a body mass index (BMI kg / m 2 ); Optionally, has been diagnosed with type 2 diabetes; and, Having a liver fat content of at least about 10% as measured by MRI-PDFF.

42. The method of claim 31, wherein the human has not been diagnosed with type 2 diabetes, and a dose of about 1.2 mg per week reduces the human's body weight by at least about 2.5%, optionally at least about 3%, at week 12, which is significant compared to placebo (p<0.001).

43. The method of claim 31, wherein the human has not been diagnosed with type 2 diabetes, and a dose of about 1.8 mg per week reduces the human's body weight by at least about 4%, optionally about 5%, at week 12, which is significant compared to placebo (p<0.001).

44. The method of claim 31, wherein the human has not been diagnosed with type 2 diabetes, and a dosage of about 2.4 mg per week reduces the human's body weight by at least about 2.5%, optionally about 3.5%, at week 12, which is significant (p<0.001) compared to placebo.

45. The method of claim 31, wherein the human has been diagnosed with type 2 diabetes and a dose of about 1.2 mg per week reduces the human's body weight by at least about 2%, optionally at least about 3%, at week 12, which is significant (p<0.05) compared to placebo at week 12.

46. ​​The method of claim 31, wherein the human has been diagnosed with type 2 diabetes and a dose of about 1.8 mg per week reduces the human's body weight by about 2.5%, optionally at least about 3.5%, at week 12, which is significant compared to placebo (p<0.005).

47. The method of claim 31, wherein the human has been diagnosed with type 2 diabetes and a dose of about 2.4 mg per week reduces the human's body weight by about 3%, optionally at least about 4%, at week 12, which is significant compared to placebo (p<0.001).

48. The method of claim 31, wherein a dosage of about 1.2 mg per week reduces body weight in a human population by at least about 3% at week 12, which is significant compared to placebo (p<0.001).

49. The method of claim 31, wherein a dosage of about 1.8 mg per week reduces body weight in a human population by at least about 4% at week 12, which is significant compared to placebo (p<0.001).

50. The method of claim 31, wherein a dose of about 2.4 mg per week reduces body weight in a human population by at least about 3%, optionally at least about 3.5%, at week 12, which is significant compared to placebo (p<0.001).

51. The method of any preceding claim, wherein a dosage of about 1.2 mg per week induces at least about an 11% reduction in ALT at week 12 in a human population.

52. The method of any preceding claim, wherein a dose of about 1.8 mg per week induces at least about a 13% reduction in ALT at week 12 in a population of humans that is significant compared to placebo (p<0.05).

53. The method of any preceding claim, wherein a dose of about 2.4 mg per week induces at least about a 13% reduction in ALT at week 12 in a population of humans that is significant (p<0.05) compared to placebo.

54. The method of any preceding claim, wherein a dosage of about 1.2 mg per week induces at least about an 18% reduction in ALT at week 12 in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L.

55. The method of any preceding claim, wherein a dosage of about 1.8 mg per week induces at least about a 20% reduction in ALT at week 12 in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L.

56. The method of any preceding claim, wherein a dose of about 2.4 mg per week induces at least about 20%, optionally at least about 25%, reduction in ALT at week 12 in a population of humans with a baseline ALT of greater than or equal to about 30 IU / L, which is significant (p<0.005) compared to placebo.

57. The method of any preceding claim, wherein the pemvidutide is administered by parenteral injection.

58. The method of any preceding claim, wherein the pemvidutide is administered by subcutaneous injection.

59. The method of any preceding claim, wherein the human has a body mass index (BMI kg / m 2 ).

60. The method of any preceding claim, wherein the human has a body mass index (BMI kg / m 2 ).

61. The method of any preceding claim, wherein the human has a liver fat level of 10% or greater as measured by MRI-PDFF prior to the once weekly dosing.

62. The method of any preceding claim, wherein after 12 weeks of once weekly dosing, the absolute reduction in liver fat as determined using MRI-PDFF is about 8% to about 15%.

63. The method of any preceding claim, wherein the liver fat content normalized to less than or equal to 5% is greater than 20% to about 55% of the population after 12 weeks of once weekly dosing.

64. The method of any preceding claim, wherein the relative reduction from baseline in liver fat as determined using MRI-PDFF is greater than about 40% to about 60% after 12 weeks of weekly dosing.

65. The method of any preceding claim, wherein after 12 weeks of once weekly dosing, ALT is reduced by greater than about 13% to about 25% of the population.

66. The method of any preceding claim, wherein a steady-state dose is reached after a dose escalation phase having a duration of two to four weeks.

67. The method of any preceding claim, wherein the pemvidutide is administered from a liquid formulation comprising at least about 2.5 mg / ml pemvidutide.

68. The method according to any preceding claim, wherein administration of pemvidutide induces a significant reduction in serum lipids and / or concentrations of atherogenic small and medium-sized LDL particles.

69. The method of claim 58, wherein the concentration of atherogenic small to intermediate sized LDL particles is reduced by at least -0.2 log relative to placebo after 43 days and / or 84 days of administration of pemvidutide. 2 Change multiple.

70. The method of any preceding claim, wherein after 43 days and / or 84 days of administration of pemvidutide, the subject's total serum triglyceride concentration is reduced by at least -0.2 log relative to placebo when measured by 2D-NMR. 2 Change multiple.

71. The method of any one of claims 58-60, wherein the serum lipids are selected from glycerides, sterols, glycerophospholipids and sphingolipids, optionally wherein the reduction is at least -0.2 log 2 Change multiple.

72. The method of any preceding claim, wherein serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine and / or lysophosphatidylcholine is reduced following administration with Pemvidutide.

73. A liquid pharmaceutical formulation comprising: SEQ ID NO: 1 and about 0.020% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in deionized water (pH 7.7 ± 0.1).

74. The formulation of claim 63, comprising 1.8 mg of SEQ ID NO: 1 as a therapeutic dose.

75. The formulation of claim 64, wherein the therapeutic dose induces weight loss in a subject in need thereof.

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