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

Abstract

The present disclosure relates to weekly dosing regimens, formulations, and methods of using dual GLP-1R and GCGR agonists to induce weight loss in humans with fatty liver, who may or may not have other comorbidities such as type 2 diabetes and / or cardiovascular disease / disorder. The dual GLP-1R and GCGR agonist comprises the peptide product of SEQ ID NO: 1 (pembidutide).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 406,681, filed September 14, 2022, U.S. Provisional Patent Application No. 63 / 422,981, filed November 5, 2022, U.S. Provisional Patent Application No. 63 / 476,370, filed December 20, 2022, and U.S. Provisional Patent Application No. 63 / 490,465, filed March 15, 2023, each of which is incorporated herein in its entirety.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically via EFS-Web in ASCII format and is incorporated herein in its entirety. The ASCII copy, created on September 14, 2022, is entitled MED012PRV_ST25.TXT and is 1050 bytes in size.

[0003] Field of the Disclosure The present disclosure relates to the use of the GLP-1R and GCGR agonist pembidutide (ALT-801), compositions comprising SEQ ID NO: 1 in certain dosing regimens for the treatment of obesity with certain comorbidities (e.g., fatty liver such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH); type 2 diabetes). [Background technology]

[0004] The high prevalence of metabolic disorders, including obesity, diabetes (e.g., type 2 diabetes), nonalcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), is a global health crisis of epidemic proportions that is a major contributor to patient morbidity and mortality and a major economic burden. Obesity is a significant 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 growing problem worldwide; currently, more than 65% of adults in the United States are overweight, and the number of obese people is doubling annually.

[0005] In the United States (US), NASH is the leading cause of end-stage liver disease or liver transplantation. Obesity is a core driver of NASH, and weight loss leads to a reduction in liver fat and amelioration of NASH. Over 80% of individuals with NASH are overweight or obese, and with US Food and Drug Administration (FDA)-approved pharmacological options currently available to induce weight loss, therapy is largely based on lifestyle interventions aimed at achieving weight loss. However, achieving and maintaining long-term weight loss through lifestyle changes alone is difficult.

[0006] Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are associated with modest weight loss at approved doses, and these agents have emerged as a treatment option for patients with NASH. In a recent clinical trial, liraglutide, a daily GLP-1RA, was associated with resolution of NASH, along with a trend toward improvement in liver fibrosis. However, patients only lost 5.5% of their body weight. In one study, a weight loss of 10% or more was required for optimal NASH resolution. Higher levels of weight loss are also associated with lower rates of cardiovascular disease and non-hepatic malignancies, which represent the most severe comorbidities faced by NASH patients.

[0007] While GLP-1RA exerts central effects on appetite and food intake, GCGR agonists (GCGRAs) drive increased energy expenditure in animal models and humans. The actions of GCGRAs and GLP-1RAs have been shown to be synergistic in driving greater weight loss compared to GLP-1RAs alone. GCGRAs also promote lipolysis and inhibit hepatic lipogenesis, providing an additional pathway for the reduction of liver fat and resolution of NASH.

[0008] Dual agonists combine GCGRA with GLP-1RA in the same molecule. In obese nonhuman primates, long-term administration of a GLP-1R / GCGR dual agonist reduced body weight and improved glucose tolerance to a greater extent than a GLP-1RA monoagonist. Clinical studies of cotadutide, a GLP-1 / GCGR dual agonist with a 5:1 bias of GLP-1 to glucagon activity, demonstrated an impressive 39% reduction in liver fat content after just 6 weeks and a greater improvement in reducing NASH-related alanine aminotransferase (ALT) activity than liraglutide alone. However, the extent of weight loss over 26 weeks of cotadutide treatment was comparable to that of liraglutide (5.4% vs. 5.5%), suggesting that the 5:1 ratio was acceptable for reducing liver fat but suboptimal for weight loss. Balanced (1:1) antagonism has been shown to be associated with greater weight loss and metabolic effects than a skewed ratio favoring one agonist over the other. A recent study with JNJ 64565111, a balanced dual agonist, achieved an impressive 8% reduction in body weight in just 12 weeks (NCT03586830). Summary of the Invention [Problem to be solved by the invention]

[0009] Unfortunately, GLP-1Ra and dual receptor agonists based on GLP-1R and GLP-1 with a bias toward GLP-1 are associated with high rates of nausea, vomiting, and diarrhea. These drugs also need to be titrated over a long period of time to reduce side effects, and drugs with improved tolerability and dosing regimens are needed. Therefore, there remains a need for convenient dosing (e.g., once a week instead of once daily) at therapeutic doses that reduce weight in subjects with fatty liver, independent of other comorbidities such as type 2 diabetes, and that do not require long-term (e.g., more than 4 weeks) titration to reach therapeutic levels without gastrointestinal side effects. [Means for solving the problem]

[0010] Described herein are dual agonist peptides and their products (e.g., formulations), 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 the treatment of obesity, and associated with common areas such as fatty liver.

[0011] In certain embodiments, a method of reducing body weight in a human with fatty liver is provided, comprising administering pembidutide to a human in need thereof in an amount of at least 1.8 mg to 2.4 mg once a week, wherein the human may have type 2 diabetes, and the fatty liver is nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In several embodiments, the human's body weight is reduced by at least 3%, or at least 4%, from baseline at 12 weeks and / or after 24 weekly doses. In several embodiments, pembidutide 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 achieved after a dose-escalation phase having a duration of about 2 weeks, about 3 weeks, or about 4 weeks. In certain embodiments, the human has type 2 diabetes. In alternative embodiments, the human does not have type 2 diabetes. In several embodiments, the human has a body mass index (BMI, kg / m) of at least 27. 2 ) or a body mass index (BMI, kg / m ) of 30 or more 2 In certain embodiments, the human has a level of liver fat as measured by MRI-PDFF (Magnetic Resonance Imaging-Proton Density Fat Fraction) of 10% or greater.

[0012] In certain embodiments, the methods provided herein induce an absolute reduction in liver fat as measured by MRI-PDFF of about 8% to about 15% or more after 12 weeks of weekly dosing and / or after 24 weekly dosing. In certain other embodiments, the methods provided herein induce a relative reduction in liver fat as measured by MRI-PDFF compared to baseline of about 40% to about 70% after 12 weeks of weekly dosing and / or after 24 weekly dosing.

[0013] Other aspects of the present disclosure are also contemplated as will be appreciated therefrom by those skilled in the art. [Brief explanation of the drawings]

[0014] [Figure 1] Subjects (overweight / obese and NAFLD) were randomized to one of four arms, and subjects were stratified by the presence or absence of type 2 diabetes (T2D). The four treatment arms are shown. Two treatment arms (1.2 mg and 1.8 mg doses) had no dose escalation, and the 2.4 mg dose arm underwent rapid 4-week dose titration. The study did not involve calorie restriction or lifestyle intervention in subjects. [Figure 2] Figure 1 shows reductions in liver fat content as determined by MRI-PDFF at 12 weeks, both as absolute and relative reductions. [Figure 3] MRI-PDFF at 12 weeks showed a reduction in liver fat content (responder analysis) and data are expressed as the percentage of patients showing a 30% liver fat reduction, a 50% liver fat reduction and a normalized group. [Figure 4] Weight loss at 12 weeks in non-diabetic and diabetic (type 2 diabetes) subjects is shown. [Figure 5] ALT reduction at 12 weeks is shown. [Figure 6] Iron-corrected T1 (cT1) responder analysis at 12 weeks is shown. [Figure 7] Adverse events (AEs) recorded in this study are shown with very low incidence for all three doses. The 2.4 mg dose compared to 2.4 mg*, which was an adverse event recorded from an earlier study, and dose titration was not given in this study. Pembidutide in this study and in each treatment arm was well tolerated with a reduction in AEs for the 2.4 mg dose compared to no dose titration. See WO2022 / 125598. [Figure 8] Lipid metabolite extraction, UHPLC-MS analysis, and data processing are shown. [Figure 9]Figure 1 shows lipoprotein changes in obese / overweight subjects treated with pembidutide 1.2 mg (n=6), 1.8 mg (n=9), 2.4 mg (n=9) or placebo (n=10). Day 43 vs. Day -1, Day 84 vs. Day -1. Color code represents log2 (robust fold change), with blue indicating decreased lipoproteins (negative fold change) and red indicating increased lipoproteins (positive fold change). [Figure 10] Lipidomic signatures in obese / overweight subjects treated with pembidutide 1.2 mg (n=6), 1.8 mg (n=9), 2.4 mg (n=9), or placebo (n=10) compared to changes at day 84 vs. day -1. Results are expressed as log2 vs. fold change, with blue indicating decreased metabolites (negative fold change) and red indicating increased metabolites (positive fold change). Gray / black bars indicate significant p-values ​​from the Wilcoxon test (light gray, p<0.05; dark gray, p<0.01; black, p<0.001). (Definitions: PE = phosphatidylethanolamine; PC = phosphatidylcholine; PI = phosphatidylinositol). [Figure 11] 1 shows a volcano plot of changes in known atherogenic lipid species following treatment with 1.8 mg of pemvidutide. The Y-axis is the Log10 statistical change based on a Student's T-test, with a horizontal dashed line corresponding to a threshold for p-value of 0.01. The X-axis represents the Log2 fold change, with a horizontal dashed line corresponding to a threshold for arbitrary change of ±0.75. [Figure 12A] 1 shows significant weight loss induced by pembidutide administration compared to placebo. [Figure 12B] A weight loss of 5% or more or 10% induced by pembidutide administration compared to placebo. [Figure 13] 1 shows the design of the extension study described in Example 3. [Figure 14] 1 details the baseline characteristics of participants in the study of Example 3. [Figure 15]Both absolute and relative % reductions in liver fat demonstrate robust reductions in liver fat content as determined by MRI-PDFF at week 24 for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and baseline at week 0. [Figure 16] Showing a robust reduction in liver fat content in responders at week 24 as determined by MRI-PDFF, with significant (p<0.001 or p<0.0001 as shown) reduction in liver fat content in responders for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and baseline at week 0. [Figure 17] Shows significant delipidation of the liver as determined by MRI-PDFF at week 24 (pembidutide, 1.8 mg dose). The reduction in liver volume in this scan for the exemplary subject receiving pembidutide was significant (32.3% liver fat at baseline compared to 1.7% liver fat at week 24). [Figure 18] The figures show robust reductions in MRI-PDFF determined liver volume at week 24, measured as both absolute and relative reductions. As shown here, for example, significant (p<0.05 or p<0.001 as shown) reductions in liver fat content were observed for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and baseline at week 0. [Figure 19] All subjects and subjects with a baseline ALT of 30 IU / L or greater demonstrate robust reductions in ALT levels, a biomarker of liver inflammation, at week 24. As shown here, for example, significant (p<0.001) reductions in ALT were observed for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and baseline at week 0. [Figure 20]High rates of cT1 response at week 24 were observed, with response defined as an 80MS reduction in CT1 compared to baseline (week 0) and associated with a 2-point reduction in the NASH Activity Score (NAS) (Dennis A., Front. Endocrinol., 2021). As shown here, for example, significant (p<0.05 or p<0.005 as shown) cT1 responses were observed for all pembidutide doses administered (1.2mg, 1.8mg, and 2.4mg per week) compared to placebo and baseline at week 0. [Figure 21] Sustained weight loss was observed in all subjects (non-diabetic and diabetic) at week 24, with significant (p<0.005 or p<0.001 as shown) reductions in body weight for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and baseline at week 0. This demonstrates weight loss in people with fatty liver (non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) in both diabetic and non-diabetic subjects. [Figure 22] Figure 1 shows the improvement in serum lipids at week 24 for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to baseline (week 0). [Figure 23] All doses of pembidutide tested induced improvements in blood pressure without clinically meaningful increases in heart rate at week 24 compared to placebo and baseline (week 0), with the reduction in systolic blood pressure being significant (p<0.05) at the 2.4 mg per week dose. [Figure 24] A safety overview of this study with adverse events over 24 weeks is presented. [Figure 25] Demonstrates improvement in glycemic control in diabetic patients at week 24 for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo and baseline at week 0. [Figure 26]

[0023] Figure 1 shows baseline characteristics of study participants in a Phase II study. See Example 4. [Figure 27] 1 shows weight loss, expressed as the mean percentage change from baseline body weight, achieved over 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg and placebo in all evaluated subjects. [Figure 28] 1 shows the weight loss, expressed as the mean percentage change in baseline body weight, achieved over 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg and placebo in the subgroup of subjects with a baseline body weight of 115 kg or less. [Figure 29] The percentage of subjects in each group achieving a weight loss of 5% or more, 10% or more, or 15% or more, expressed as a percentage of baseline weight, after 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg, and placebo, is shown. [Figure 30] 1 shows the weight loss, expressed as the mean percentage change from baseline body weight, achieved in Hispanic versus non-Hispanic subjects after 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg, and placebo. [Figure 31] Systolic and diastolic blood pressure (A) expressed as mean percentage change from baseline and heart rate (B) expressed as mean percentage change from baseline after 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg and placebo. [Figure 32] Serum lipids at week 24 are shown for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg) compared to baseline. [Figure 33] 1 shows a significant reduction in waist circumference at week 24 for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week). See Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure relates to dual agonist peptides, pharmaceutical dosage formulations comprising the same, and methods for using the same. The dual agonist peptides have affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), and in preferred embodiments, approximately equal affinity, as determined using a cellular assay. In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to induce weight loss in humans with fatty liver. In some embodiments, the humans may or may not have 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), isolevuglandin (IsoLG), methyglyoxal (MGO), 4-oxononenal (ONE), and 4-hydroxynonenal (HNE). Oxidized phospholipids include 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-O-alkyl-2-azelaoyl-sn-glycero-3-phosphorylcholine (azPAF), 1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine (KOdiA-PC), 1-palmitoyl-2-F2-isoprostane-sn-glycero-3-phosphocholine (F2IsoP-PC), and 1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC).

[0016] In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to induce weight loss, including for long-term weight management and treatment of associated comorbidities. In some embodiments, the present disclosure provides pharmaceutical dosage formulations 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 for cardiovascular (CV)-related risk factors. In some embodiments, the present disclosure provides peptide-based dual GLP-1 / glucagon receptor agonists designed to treat the underlying metabolic dysfunction that leads to nonalcoholic steatohepatitis (NASH).

[0017] As used herein, the terms "treat," "treated," or "treating" refer to both therapeutic treatment and prophylactic measures in which the goal is to slow (delay) 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, alleviation of symptoms, a decrease in the severity of the condition, disorder, or disease, a stabilized (i.e., not worsening) state of the condition, disorder, or disease, a delay or slowing of the onset of the progression of the condition, disorder, or disease, an improvement or remission (partial or total) of the state of the condition, disorder, or disease, detectable or undetectable, an improvement in at least one measurable physical parameter, not necessarily discernible by the patient, or promotion or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival in the absence of treatment. Thus, "treatment of obesity" refers to activities that alleviate or ameliorate either the 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.

[0018] In several embodiments, provided herein is a method for reducing weight in a human with fatty liver, comprising administering pembidutide to a human in need thereof in an amount of at least 1.8 mg to 2.4 mg once weekly, wherein the human may have type 2 diabetes, and the fatty liver is nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). Pembidutide, also referred to herein as ALT-801, is a composition comprising a synthetic peptide (SEQ ID NO: 1) composed of natural amino acids and is a chimeric analog of two natural hormones, GLP-1 and glucagon, with a predominantly N-terminal glucagon residue and a C-terminal GLP-1 residue. See U.S. Patent No. 9,856,306, incorporated herein by reference. ALT-801 also incorporates one nonproteinogenic amino acid, 2-aminoisobutyric acid, an amino acid side chain amide bond (lactam bridge), and a surfactant side chain composed of glucuronic acid attached to an octadecanoic fatty acid side chain. The surfactant side chains appear to enter the circulation slowly and can form micelles after subcutaneous (SC) injection. This slower entry is associated with a lower maximum concentration (C max ) may result in lower GI side effects and better tolerability. This latter feature also improves binding to plasma proteins, improves metabolic stability, and reduces half-life (t 1 / 2 ). The design of ALT-801 results in a co-agonist with equipotent (1:1) activity at both receptors at approximately 40 pM and 100% activity. Compositions containing SEQ ID NO:1 have been administered to humans at various doses and found not to induce side effects such as nausea, vomiting, diarrhea, abdominal pain, and / or constipation using standard techniques. See U.S. Patent Publication No. 2021 / 0290732 and PCT Publication No. WO2022 / 125598, each incorporated herein by reference.

[0019] ALT-801 (SEQ ID NO: 1, also referred to herein as pembidutide) has the following amino acid sequence: 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-NH2 * indicates that a lactam bridge is formed between Glu16 and Lys20, and 17Lys# indicates that the glucuronic acid C-18 * As shown differently, SEQ ID NO: 1 (i.e., ALT-801 or pemvidutide) has 29 amino acid residues and 17 Glucuronic acid / C bound to Lys 18 is a peptide amide consisting of a diacid moiety, 16 Glu and 20 The side chains of Lys form an intramolecular cycle as shown below.

[0020] [ka]

[0021] In several embodiments, provided herein is a pharmaceutical formulation of SEQ ID NO: 1 in an aqueous buffer, referred to herein as ALT-801 (and pemvidutide). The dual agonist peptide products herein comprising SEQ ID NO: 1 comprise an amino acid side chain amide bond (lactam bridge) and a surfactant side chain composed of glucuronic acid linked to a fatty acid side chain. The surfactant side chain, composed of a hydrophilic saccharide group, is covalently attached to the peptide via a linker amino acid and a hydrophobic alkyl chain moiety. The synthesis of SEQ ID NO: 1 is described in U.S. Pat. No. 11,541,028 B2, which is incorporated by reference in its entirety into this disclosure. In some embodiments, the dual agonist peptide may comprise one or more conservatively substituted amino acids as described herein. In a preferred embodiment, SEQ ID NO: 1 comprises one or more conservatively substituted amino acids, preferably excluding amino acid residues 16, 17, or 20.

[0022] A "peptide" (e.g., a dual agonist peptide) comprises two or more naturally occurring and / or unnatural amino acid residues typically joined via a peptide bond. Such amino acids may include natural structural variants, naturally occurring non-proteinogenic amino acids, or / and synthetic non-natural analogs of naturally occurring 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 (more than about 50 amino acids, also referred to as "proteins"). In some embodiments, peptide products include a surfactant moiety covalently and stably attached to a peptide of about 50, 40, or 30 amino acids or less. Synthetic peptides can be synthesized, for example, using an automated peptide synthesizer. Peptides can also be produced recombinantly in cells expressing a nucleic acid sequence encoding the peptide. Conventional symbols are used herein to represent peptide sequences. The left-hand end of a peptide sequence is the amino (N)-terminus, and the right-hand end of a peptide sequence is the carboxyl (C)-terminus. Standard one-letter and three-letter abbreviations for common amino acids are used herein. Abbreviations used in the amino acid sequences disclosed herein represent L-amino acids unless otherwise specified as D or DL, or the amino acid is achiral, although the corresponding D isomer can generally be used at any position (e.g., is resistant to proteolysis). Other amino acid abbreviations used herein include: Aib = α-aminoisobutyric acid (or 2-methylalanine or Ca-methylalanine); Xaa = any amino acid, typically specifically defined within the formula.Other amino acid abbreviations that can be used as described herein are: 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 = alpha-aminoisobutyric acid (or 2-methylalanine or Calpha-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. lysine; 2FMePhe or 2FaMePhe = Ca-methyl-(2-fluorophenyl)alanine; hArg = homoarginine; MeLys or aMeLys = Ca-methyllysine; MePhe or aMePhe = Ca-methylphenylalanine; MePro or aMePro = Ca-methylproline; Na1 or Na1(1) = 3-(1-naphthyl)alanine; Na1 or Na1(2) = 3-(2-naphthyl)alanine; Nle = norleucine; Om = ornithine; and Tmp = (2,4,6-trimethylphenyl)alanine, and the Tic-Phe dipeptide moiety (referred to as Tic-Ψ[CF12-NF1]-Ψ-Phe) containing 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) and a reduced amide bond between the residues has the following structure:

[0023] [ka] It has.

[0024] Unless otherwise specifically stated or clearly indicated by the context, the present disclosure encompasses any and all forms of dual agonist peptides that can be produced, whether they are produced synthetically (e.g., using a peptide synthesizer) or cellularly (e.g., by recombinant production). Such forms of dual agonist peptides can include one or more modifications, such as one or more post-translational modifications, that may occur during the synthetic or cellular production pathway of the peptide, regardless of whether one or more modifications are contemplated. Dual agonist peptides can have the same type of modification at two or more different positions, or / and two or more different types of modifications. Modifications that may occur during the synthesis or cellular production of dual agonist peptides, including chemical and post-translational modifications, include, but are not limited to, glycosylation (e.g., N-linked glycosylation and O-linked glycosylation), lipidation, phosphorylation, sulfation, acetylation (e.g., N-terminal acetylation), amidation (e.g., C-terminal amidation), hydroxylation, methylation, intramolecular or intermolecular disulfide bond formation, lactam formation between two side chains, pyroglutamate formation, and ubiquitination. Dual agonist peptides can have one or more modifications anywhere, for example, at the N-terminus, 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 can be acetylated at the N-terminus or / and have a carboxamide (-CONH) group at the C-terminus to increase the stability of the dual agonist peptide.

[0025] Possible modifications of the dual agonist peptide also include the deletion of one or more amino acids, the addition / insertion of one or more natural and / or unnatural amino acids, or the substitution of one or more natural and / or unnatural amino acids, or any combination thereof. Substitutions can be conservative or non-conservative. Such modifications can be contemplated, for example, through site-directed mutagenesis or during chemical synthesis of the dual agonist peptide, or can be accidental, for example, through mutations occurring in host cells producing the dual agonist peptide or through errors in PCR amplification. Unnatural amino acids can have the same chemical structure as the corresponding natural amino acid but with D-stereochemistry, or can have a different chemical structure and D- or L-stereochemistry. Unnatural amino acids can be used, for example, to promote helix formation and / or increase the stability of the dual agonist peptide (e.g., to resist proteolysis). A dual agonist peptide having one or more modifications relative to a reference dual agonist peptide can be referred to as an "analog" or "variant" of the reference dual agonist peptide, as appropriate. An "analog" typically retains one or more essential properties of the reference dual agonist peptide (e.g., receptor binding, receptor or enzyme activation, receptor or enzyme inhibition, 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 more preferred embodiments, has approximately equal affinity for GLP-1R and GCGR. In some embodiments, an analog or variant of a reference peptide has an amino acid sequence that differs from that of the reference dual agonist peptide.

[0026] The term "conservative substitution" refers to the substitution of an amino acid in a dual agonist peptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In certain embodiments, the following groups each contain natural amino acids that are conservative substitutions for one another: 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 further embodiments, the following groups each contain natural amino acids that are conservative substitutions for one another: 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 hydroxyl or sulfhydryl containing: 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, amino acids can be grouped into conservative substitutions as follows: 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 that affect backbone orientation: Pro.

[0027] Examples of non-naturally occurring 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., nor-leucine, 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, Nall, 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). The α,α-disubstituted amino acids may confer conformational constraint or / and stabilization of the α-helix. A reduced amide bond between two residues (e.g., Tic-Ψ[CF12-NF1]-Ψ-Phe) may enhance protease resistance and, for example, alter receptor binding. The present disclosure encompasses all pharmaceutically acceptable salts of the dual agonist peptides, including those with a positive net charge, a negative net charge, and no net charge.

[0028] An "alkyl" group refers to an aliphatic hydrocarbon group. An alkyl group can be saturated or unsaturated and can be straight-chain (linear), branched-chain, or cyclic. In some embodiments, an alkyl group is not cyclic. In some embodiments, an alkyl group contains 1 to 30, 6 to 30, 6 to 20, or 8 to 20 carbon atoms. A "substituted" alkyl group is substituted with one or more substituents. In some embodiments, the one or more substituents are independently selected from halogen, nitro, cyano, oxo, hydroxy, alkoxy, haloalkoxy, aryloxy, thiol, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, amino, alkylamino, dialkylamino, arylamino, alkoyl, carboxyl, carboxylate, ester, amide, carbonate, carbamate, urea, alkyl, haloalkyl, fluoroalkyl, aralkyl, alkyl chain containing acyl groups, heteroalkyl, heteroalicyclic, aryl, alkoxyaryl, heteroaryl, hydrophobic natural compounds (e.g., steroids), and the like. In some embodiments, an alkyl group as a substituent is a linear or branched C1-C6 alkyl, which can be referred to as a "lower alkyl." Non-limiting examples of lower alkyl groups include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including all isomeric forms, e.g., n-butyl, isobutyl, sec-butyl, and / er / -butyl), pentyl (including all isomeric forms, e.g., n-pentyl), and hexyl (including all isomeric forms, e.g., n-hexyl). In some embodiments, the alkyl group is attached to the Na atom of the residue of the peptide (e.g., Tyr or Dmt). In certain embodiments, the N-alkyl group is a linear or branched C1-C6 alkyl. 10The alkyl groups are alkyl or alkyl substituted with aryl, such as benzyl, phenylethyl, etc. One or two alkyl groups can be attached to the Na atom of the N-terminal residue. In some embodiments, the alkyl group is a 1-alkyl group attached to the C-1 position of a saccharide (e.g., glucose) via a glycosidic bond (e.g., an O-, S-, N-, or C-glycosidic bond). In some embodiments, such 1-alkyl groups are unsubstituted or substituted C1-C30, C6-C30, C6-C 20 Or C8~C 20 In some embodiments, the alkyl group (e.g., 1-alkyl group) is an aryl, —OH, —OR 1 , -SH, -SR 1 , -NH2, -NHR 1 , -N(R 1 )2, oxo(=O), -C(=O)R 2 , carboxyl (-CO2H), carboxylate (-CO2-), -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 -NR 4 C(=O)N(R 1 )2; and R 1 is independently at each occurrence hydrogen, alkyl, or aryl, or R 1 and the connecting nitrogen atom together form a heterocyclyl or heteroaryl ring, R 2 is independently at each occurrence alkyl, heterocyclyl, aryl, or heteroaryl, and R 3 is, independently at each occurrence, hydrogen, alkyl, heterocyclyl, aryl, or heteroaryl; R 4 is, independently at each occurrence, hydrogen or alkyl; R5is independently at each occurrence alkyl or aryl. In some embodiments, an alkyl group (e.g., an 1-alkyl group) is internally and / or terminally substituted with a carboxyl / carboxylate group, an aryl group, or an -O-aryl group. In certain embodiments, an alkyl group (e.g., an 1-alkyl group) is substituted with a carboxyl or carboxylate group at the distal end of the alkyl group. In further embodiments, an alkyl group (e.g., an 1-alkyl group) is substituted with an aryl group at the distal end of the alkyl group. In other embodiments, an alkyl group (e.g., an 1-alkyl group) is substituted with an -O-aryl group at the distal end of the alkyl group. The terms "halogen," "halide," and "halo" refer to fluoride, chloride, bromide, and iodide. The term "acyl" refers to -C(=O)R, where R is an aliphatic group that may be saturated or unsaturated and may be linear, branched, or cyclic. In certain embodiments, R contains 1 to 20, 1 to 10, or 1 to 6 carbon atoms. The acyl group can 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 "heterocyclic" refer to a monocyclic non-aromatic group or a polycyclic group containing at least one non-aromatic ring, wherein at least one non-aromatic ring contains one or more heteroatoms independently selected from O, N, and S. The non-aromatic ring containing one or more heteroatoms can be attached to or fused to one or more saturated, partially unsaturated, or aromatic rings. In certain embodiments, the heterocyclyl or heterocyclic group has 3 to 15, 3 to 12, 3 to 10, 3 to 8, or 3 to 6 ring atoms. Heterocyclyl or heterocyclic groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, azepanyl, azocanyl, oxiranyl, oxetanyl, tetrahydrofuranyl (oxolanyl), tetrahydropyranyl, oxepanyl, and oxocanyl.The term "aryl" refers to a monocyclic aromatic hydrocarbon group or a polycyclic group containing at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group has 6 to 15, or 6 to 12, or 6 to 10 ring atoms. Aryl groups include, but are not limited to, phenyl, naphthalenyl (naphthyl), fluorenyl, azulenyl, anthryl, phenanthryl, biphenyl, and terphenyl. The aromatic hydrocarbon ring of an aryl group can be bonded to or fused with one or more saturated, partially unsaturated, or aromatic rings, such as dihydronaphthyl, indenyl, indanyl, and tetrahydronaphthyl (tetralinyl). Aryl groups can be optionally substituted with one or more (e.g., two or three) substituents independently selected from halogen (including F and Cl), cyano, nitro, hydroxyl, alkoxy, thiol, alkylthio, alkylsulfoxide, alkylsulfone, amino, alkylamino, dialkylamino, alkyl, haloalkyl (including fluoroalkyl, e.g., trifluoromethyl), acyl, carboxyl, ester, amide, and the like. The term "heteroaryl" refers to a monocyclic aromatic or polycyclic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, N, and S. The heteroaromatic ring can contain only carbon atoms or can be attached to or fused to one or more saturated, partially unsaturated, or aromatic rings, which may contain one or more heteroatoms. In certain embodiments, heteroaryl groups have 5 to 15, 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, benzothienyl (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.

[0029] In some embodiments, for example, the dual agonist peptide can be associated with a saccharide, e.g., in a pharmaceutically acceptable composition or lyophilizate. Saccharides include monosaccharides, disaccharides, and oligosaccharides (e.g., trisaccharides, tetrasaccharides, etc.). Reducing saccharides exist in equilibrium in cyclic and open-chain forms, with cyclic forms generally being preferred. The functionalized saccharide of the surfactant moiety has a functional group suitable for forming a stable covalent bond with an amino acid of the dual agonist peptide.

[0030] The term "pharmaceutically acceptable" refers to a substance (e.g., an active ingredient or excipient) that is suitable for use in contact with the tissues and organs of a subject without excessive irritation, allergic response, immunogenicity, or toxicity, and that is effective for its intended use, commensurate with a reasonable benefit / risk ratio. A "pharmaceutically acceptable" excipient or carrier of a pharmaceutical composition is also compatible with the other components of the composition. In one embodiment, a pharmaceutically acceptable composition into which the dual agonist peptide can be formulated includes polysorbate 20 (e.g., about 0.050% (w / w)), optionally methylparaben (e.g., about 0.300% (w / w)), arginine (about 0.348% (w / w)), and mannitol (e.g., about 4.260% (w / w)) in distilled (DI) water.

[0031] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to prevent the medical condition being treated, reduce the risk of developing the medical condition, delay the onset of the medical condition, slow the progression of the medical condition, or cause regression of the medical condition, or to alleviate to some extent the medical condition or one or more symptoms or complications of the condition, in at least some fraction of the subjects who receive the compound. The term "therapeutically effective amount" also refers to an amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, organ, or human that is desired by a physician or clinician.

[0032] The terms "treat," "treating," and "treatment" include alleviating, ameliorating, inhibiting progression, reversing, or arresting a medical condition or one or more symptoms or complications associated with a 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, or delaying the onset of a medical condition or one or more symptoms or complications associated with a condition. The term "medical condition" (or simply "condition") includes diseases and disorders. The terms "disease" and "disorder" are used interchangeably herein.

[0033] The present disclosure also provides pharmaceutical compositions 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 contains a therapeutically effective amount of the peptide product or a suitable fraction thereof. The composition may optionally contain 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 types of excipients include liquid and solid fillers, diluents, binders, lubricants, glidants, surfactants, dispersing agents, disintegrating agents, emulsifiers, wetting agents, suspending agents, thickening agents, solvents, isotonicity agents, buffers, pH adjusters, absorption delaying agents, stabilizers, antioxidants, preservatives, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweeteners, flavoring agents, coloring agents, encapsulating materials, and coating materials. The use of such excipients in pharmaceutical formulations 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 containing peptide products.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).

[0034] In some embodiments, the pharmaceutical formulation comprises the peptide product and about 0.02-0.075% (w / w) polysorbate 20, about 0.2-0.5% (w / w) arginine, and about 3-6% (w / w) mannitol in deionized water (pH 7.7±0.1), optionally about 0.050% (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). In certain embodiments, the pharmaceutical formulation comprises SEQ ID NO: 1 and about 0.050% (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). In certain embodiments, the pharmaceutical formulation comprises 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). In certain embodiments, the pharmaceutical formulation comprises SEQ ID NO:1 (ALT-801, pembidutide) and is configured for subcutaneous (SC) administration of a once-weekly therapeutic dose.

[0035] Suitable or preferred formulations may depend on various factors, such as the route of administration chosen. Potential routes of administration for pharmaceutical compositions containing peptide products include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarteriolar, intraperitoneal, intracavity, and topical), topical (including transdermal and transmucosal), intranasal (e.g., nasal spray or drops), ophthalmic (e.g., eye drops), pulmonary (e.g., oral or nasal inhalation), buccal, sublingual, rectal (e.g., suppository), and vaginal (e.g., suppository). In certain embodiments, the dual agonist peptide product is administered parenterally (e.g., subcutaneously, intravenously, or intramuscularly). In other embodiments, the peptide product is administered by oral or nasal inhalation or insufflation. In some embodiments, the carrier is an aqueous carrier, for example, in parenteral (e.g., subcutaneous, intravenous, or intramuscular) formulations. In other embodiments, the carrier is a non-aqueous carrier. In certain embodiments, the non-aqueous carrier is a hydrofluoroalkane (HFA) or HFA-like solvent, which may contain submicron anhydrous α-lactose or / and other excipients in formulations for administration, for example, by oral or nasal inhalation or insufflation.

[0036] In some embodiments, the peptide product is administered parenterally by injection (e.g., subcutaneously, intravenously, or intramuscularly), which 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 solution), isotonic / iso-osmotic agents (e.g., salts [e.g., NaCl, KCl, and CaCl] and sugars [e.g., sucrose]), buffering agents and pH adjusting agents (e.g., sodium dihydrogen phosphate [monobasic sodium phosphate] / disodium hydrogen phosphate [dibasic sodium phosphate], citric acid / sodium citrate, and L-histidine / L-histidine HCl), and emulsifiers (e.g., non-ionic surfactants, such as polysorbates [e.g., polysorbate 20 and 80] and poloxamers [e.g., poloxamer 188]). Peptide formulations and delivery systems are discussed, for example, in A. J. Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd ed., CRC Press (Boca Raton, Florida) (2015). Excipients can 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., myo-inositol, mannitol, and sorbitol), saccharides (e.g., glucose (including D-glucose [dextrose]), lactose, sucrose, and trehalose), osmolytes (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 non-ionic surfactants (e.g., alkyl polyglycosides, ProTek® alkyl saccharides (e.g., monosaccharides [e.g., glucose] or disaccharides [e.g., maltose or sucrose] linked to long-chain fatty acids or corresponding long-chain alcohols), and polypropylene glycol / polyethylene glycol block copolymers (e.g., poly Examples of suitable parenteral formulations include oxamers (e.g., Pluronic™ F-68, and Genapol® PF-10 and its variants). Because such agents increase peptide solubility, they can be used to increase peptide concentration in formulations. Higher peptide concentrations in formulations are particularly advantageous for subcutaneous administration, with limited bolus volumes (e.g., less than about 1.5 mL). Additionally, such agents can be used to stabilize peptides during preparation, storage, and reconstitution of lyophilized peptides. An exemplary parenteral formulation includes the peptide product, mannitol, methionine, sodium thioglycolate, polysorbate 20, a pH adjuster (e.g., NaOH or / and HCl), and deionized water. Parenteral formulation excipients suitable for use with the dual agonist peptides described herein (e.g., various combinations of excipients, including NaCl, etc.) are well known and available to those skilled in the art.

[0037] For parenteral (e.g., subcutaneous, intravenous, or intramuscular) administration, a sterile solution or suspension of the peptide product in an aqueous solvent containing one or more excipients can be prepared in advance, e.g., in a single-use pen or a pre-filled syringe of a pen containing a dose counter. Alternatively, the peptide product can be dissolved or suspended in an aqueous solvent, optionally containing one or more excipients, before lyophilization (freeze-drying). Immediately prior to parenteral administration, the lyophilized peptide product stored in a suitable container (e.g., a vial), can be reconstituted, e.g., with sterile water, optionally containing one or more excipients. In other embodiments, the agonist peptide product is administered intranasally. The nasal mucosa provides a large surface area, a porous endothelium, a highly vascular subepithelial layer, and a high absorption rate, allowing for high bioavailability. Intranasal formulations may contain excipients such as a peptide product with a solubility enhancer (e.g., propylene glycol), a humectant (e.g., mannitol or sorbitol), a buffer, and water, and optionally a preservative (e.g., benzalkonium chloride), a mucoadhesive agent (e.g., hydroxyethylcellulose), or / and a penetration enhancer. Intranasal solution or suspension formulations can be administered to the nasal cavity by any suitable means, including, but not limited to, a dropper, a pipette, or a spray, for example, using a metered atomizing spray pump. Table 2 shows exemplary excipients for nasal spray formulations.

[0038] [Table 1]

[0039] In a further embodiment, the peptide product is administered via a pulmonary route, such as oral or nasal inhalation. Pulmonary administration of drugs can treat pulmonary or / and systemic disorders because the lungs serve as the gateway to the systemic circulation. The advantages of pulmonary drug delivery include, for example, 1) avoidance of first-pass metabolism, 2) rapid drug action, 3) a large surface area for absorption in the alveolar region, high permeability of the lungs (thin air-blood barrier), and the abundant vasculature of the airways, and 4) lower extracellular enzyme levels compared to the GI tract due to the large alveolar surface area. The advantages of oral inhalation over nasal inhalation include deeper penetration / deposition of drugs into the lungs, while nasal inhalation can deliver drugs to the systemic circulation via the nasal cavity and lungs. Oral or nasal inhalation can be achieved, for example, by a metered dose inhaler (MDI), nebulizer, or dry powder inhaler (DPI). For example, peptide products can be formulated for aerosol administration to the respiratory tract via oral or nasal inhalation. Drugs are delivered in small particle sizes (e.g., about 0.5 microns to about 5 microns), obtainable by micronization, to improve, for example, drug deposition in the lungs and drug suspension stability. Drugs can be provided in pressurized packs containing a suitable propellant, such as a hydrofluoroalkane (HFA, e.g., 1,1,1,2-tetrafluoroethane [HFA-134a]), a chlorofluorocarbon (CFC, e.g., dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane), or a suitable gas (e.g., oxygen, compressed air, or carbon dioxide). In aerosol formulations, the drug is dissolved, or more often suspended, in the propellant for pulmonary delivery. Aerosols can contain excipients, such as surfactants (which promote lung penetration by reducing the high surface tension at the air-water interface within the pits and can also emulsify, solubilize, and / or stabilize the drug, e.g., phospholipids such as lecithin), or / and stabilizers, although the surfactant portion of the peptide product can perform the surfactant function.For example, an MDI formulation may contain 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 may optionally contain a dissolved gas (e.g., CO). After the device is actuated, the bursting of CO2 bubbles within the emitted aerosol droplets breaks them into smaller droplets, thereby increasing the respirable fraction of the drug. As another example, a nebulizer formulation may contain a peptide product, a chelating agent or preservative (e.g., edetate disodium), a tonicity agent (e.g., NaCl), a pH buffer (e.g., citric acid / sodium citrate), and water, and optionally a surfactant (e.g., Tween®, such as polysorbate 80). The drug can be delivered, for example, by a nebulizer or MDI, with or without a spacer, and the drug dose delivered can be controlled by a metering chamber (nebulizer) or a metering valve (MDI).

[0040] Table 1 shows exemplary MDI, nebulizer, and DPI formulations. Metered-dose inhalers (also called compressed metered-dose inhalers [pMDIs]) are the most widely used inhalation devices. A metering valve delivers a precise amount of aerosol (e.g., approximately 20-100 pL) each time the device is actuated. MDIs typically generate aerosol more rapidly than the user can inhale, which can result in significant aerosol deposition in the mouth and throat. The problem of poor coordination between device actuation and inhalation can be addressed, for example, by using breath-actuated MDIs or regulating devices. Breath-actuated MDIs (e.g., Easibreathe®) operate when the device senses the user's inhalation and releases a drug dose in response. The inhalation flow rate is regulated through an actuator, allowing the user time to reliably actuate the device during inhalation. In metered devices, the spacer (or valved holding chamber), a tube attached to the mouthpiece end of the inhaler, acts as a reservoir or chamber to hold the medication being aerosolized by the inhaler, slowing the rate at which the aerosol enters the mouth and allowing the propellant to evaporate from the larger droplets. Spacers simplify inhaler use and increase the amount of medication deposited in the lungs instead of the upper airways. Spacers can be made from antistatic polymers that minimize the electrostatic adhesion of released drug particles to the spacer's inner walls. Nebulizers produce aerosol droplets approximately 1 to 5 microns in size. These do not require user coordination between device actuation and inhalation and can significantly affect the amount of medication deposited in the lungs. Compared to MDIs and DPIs, nebulizers can deliver larger doses of medication, even with longer administration times.Examples of nebulizers include, but are not limited to, manual nebulizers, jet nebulizers (e.g., AeroEclipse® II BAN [breath-actuated], CompAIR™ NE-C801 [substantial valve], PARI LC® Plus [breath-enhanced], and SideStream Plus [breath-enhanced]), ultrasonic nebulizers, and mesh vibratory nebulizers (e.g., Akita2® Apixneb, I-neb AAD system with metering chamber, MicroAir® NE-U22, Omron U22, and PARI eFlow® Rapid). By way of example, pulsed ultrasonic nebulizers can aerosolize a fixed amount of medication per pulse and may include an acousto-optic trigger that allows the user to synchronize each breath to each pulse. For oral or nasal inhalation using a dry powder inhaler (DPI), the peptide product can be provided in the form of a dry, micronized powder, with the drug molecules being of a certain small size (e.g., about 0.5 microns to about 5 microns) that improves the aerodynamic properties of the dispersed powder and drug deposition in the lungs. Particles of about 0.5 microns to about 5 microns are deposited by precipitation in the terminal bronchioles and alveolar regions. In contrast, the majority of larger particles (greater than 5 microns) are not transported by airflow to the many branching points of the respiratory tract, but rather are deposited by impaction in the upper respiratory tract, including the oropharyngeal region of the pharynx. DPI formulations can contain drug particles alone or blended with a suitable larger base / carrier powder, such as lactose, starch, starch derivatives (e.g., hydroxypropylmethylcellulose), or polyvinylpyrrolidine. Carrier particles promote flowability, reduce clumping, improve dose uniformity, and aid in the dispersion of drug particles. DPI formulations can optionally contain excipients such as magnesium stearate or / and leucine, which improve the performance of the formulation by interfering with interparticle bonding (anti-adhesion). The powder formulations can be presented in unit dosage form, such as capsules (e.g., gelatin capsules) or cartridges in blister packs, which can be manually filled into the inhaler or can be pre-filled.Drug particles can be inhaled into the lungs by placing the inhaler's mouthpiece or nosepiece over the mouth or nose, inhaling sharply and deeply, and holding the breath for a period of time (e.g., approximately 5–10 seconds) that creates turbulence and allows drug particles to settle in the bronchiolar and alveolar regions. When the user activates the DPI and inhales, the airflow through the device creates shear and turbulence, directing the inhaled air into the powder bed and fluidizing the electrostatic powder blend, which then enters the user's airway. Here, drug particles separate from carrier particles due to the turbulence and are transported deep into the lungs, while larger carrier particles impact the oropharyngeal surfaces and are expelled. Thus, the user's inspiratory airflow achieves powder deagglomeration and air ionization, determining drug deposition in the lungs. (Passive DPIs require rapid inspiratory airflow to deagglomerate drug particles, but rapid inspiratory airflow is not recommended for MDIs or nebulizers because of the turbulence and rapid velocity that increase drug deposition due to impaction in the upper airway.) Compared to MDIs, DPIs (including breath-actuated DPIs) can deliver higher doses of drugs and larger drugs (eg, macromolecules) to the lungs.

[0041] Lactose (e.g., alpha-lactose monohydrate) is most commonly used as a carrier in DPI formulations. Examples of lactose monohydrate grades / types for DPI formulations include, but are not limited to, DCL 11, Flowlac® 100, Inhalac® 230, Lactohale® 300, Lactopress® SD 250 (spray-dried lactose), Respitose® SV003, and Sorbolac® 400. DPI formulations can contain a single lactose grade or a combination of different lactose grades. For example, fine lactose grades such as Lactohale® 300 or Sorbolac® 400 may not be suitable DPI carriers and need to be blended with coarse lactose grades such as DCL 11, Flowlac® 100, Inhalac® 230 or Respitose® SV003 to improve flowability (e.g., approximately a 1:9 ratio of fine to coarse lactose).

[0042] Tables 2 and 3 show non-limiting examples of lactose grades / types that can be used in DPI formulations. The particle size distribution of the carrier affects the fine particle fraction / dose (FPF or FPD) of the drug, with a high FPF being desirable for pulmonary drug delivery. The FPF / FPD is the respirable fraction / dosage mass from a DPI device with an aerodynamic particle size of less than 5 microns in inspired air. A high FPF, and therefore good DPI performance, can be obtained, for example, from a DPI formulation with an approximately 1:9 ratio of fine lactose (e.g., Lactohale® 300) to coarse lactose (e.g., Respitose® SV003) and an excess of about 20% w / w, avoiding drug deposition in the capsule shell or DPI device and delivering essentially all of the drug to the respiratory tract.

[0043] [Table 2]

[0044]

Table 3

[0045] Other carriers for DPI formulations include, but are not limited to, glucose, mannitol (e.g., crystallized mannitol [Pearlitol 110 C] and spray-dried mannitol [Pearlitol 100 SD]), maltitol (e.g., crystallized maltitol [Maltisorb P90]), sorbitol, and xylitol. Many DPIs are breath-actuated ("passive"), relying on the user's inhalation for aerosol generation. Examples of passive DPIs include, but are not limited to, Airmax®, Novolizer®, and Otsuka DPI (dense cake). Air classification technology (ACT) is an effective passive powder dispersion mechanism utilized in DPIs. In ACT, multiple feed channels generate tangential airflow that creates a cyclone within the device upon inhalation. Power-assisted ("active") DPIs also exist (e.g., based on aerodynamics, impact force, or vibration) that use energy to assist in particle deagglomeration, for example. For example, the active mechanism of the Exubera® inhaler utilizes mechanical energy stored in a spring or compressed air chamber. Examples of active DPIs include, but are not limited to, Actispire® (single-unit dose), Aspirair® (multiple dose), Exubera® (single-unit dose), MicroDose® (multiple-unit dose and electronically activated), Omnihaler® (single-unit dose), Pfeiffer DPI (single-unit dose), and Spiros® (multiple-unit dose). Peptide products can also be administered by other routes, such as orally. Oral formulations can contain the peptide product and conventional excipients known in the art, and optionally, an absorption enhancer such as sodium V-[8-(2-hydroxybenzoyl)aminocaprylate] (SNAC). SNAC protects against enzymatic degradation through local buffering and promotes GI absorption. Oral dosage forms (e.g., tablets, capsules, or pills) may optionally have an enteric coating to protect their contents from the strong acids 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, extended-release, prolonged-release, delayed-release, slow-release, and controlled-release compositions, systems, and devices. In some embodiments, the sustained-release composition delivers a peptide product for a period of at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, the sustained-release composition is comprised of a biodegradable polymer and formulated as nanoparticles or microparticles incorporated into the peptide product. In certain embodiments, the biodegradable polymer comprises lactic acid and / or glycolic acid (e.g., an L-lactic acid-based copolymer, such as poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)). In further embodiments, the sustained-release composition is in the form of a depot created when a mixture of the peptide product and polymer is injected intramuscularly or subcutaneously into a subject. In certain embodiments, the polymer is or includes PEG, polylactic acid (PLA) or polyglycolic acid (PGA), or copolymers thereof (eg, PLGA or PLA-PEG).

[0046] Although a pharmaceutical composition may be present in a unit dosage form as a single dose, all active and inactive ingredients need not be combined in a suitable system and the ingredients need not be mixed to form the administered composition. A unit dosage form generally contains a therapeutically effective dose of a drug, but can contain an appropriate fraction thereof so that ingestion of multiple unit dosage forms achieves a therapeutically effective dose. Examples of unit dosage forms include tablets, capsules, or pills for oral ingestion, solutions in pre-filled syringes of single-use pens or pens containing dose counters for parenteral (e.g., intravenous, subcutaneous, or intramuscular) injection, and capsules, cartridges, or blisters that are pre-filled or manually filled into an inhaler. Alternatively, a pharmaceutical composition may be provided as a kit in which the active ingredient, excipients, and carriers (e.g., solvents) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles, or syringes) that must be combined to form the administered composition. The kit may contain instructions for storing, preparing, and administering the composition (e.g., a solution to be parenterally injected). The kit can contain all active and inactive ingredients in a unit dosage form or in two or more separate containers, and can contain instructions for administering or using the pharmaceutical composition to treat a medical condition disclosed herein. The kit can further contain a device for delivering the composition, such as an injection pen or an inhaler. In some embodiments, the kit contains a peptide product or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, and instructions for administering or using the peptide product or composition to treat a medical condition disclosed herein, such as insulin resistance, diabetes, metabolic syndrome, cardiovascular disease, obesity (including "chronic obesity" meaning obesity that lasts for more than one year or that leads to obesity-related conditions such as, but not limited to, insulin resistance, diabetes, metabolic syndrome, and / or cardiovascular disease), or a condition related thereto (e.g., NASH or NAFLD). In certain embodiments, the kit further contains a device for delivering the peptide product or composition, such as an injection pen or an inhaler.

[0047] Treatment method The present disclosure further provides uses of the dual agonist peptide products described herein for preventing and / or treating conditions associated with GLP1R and / or GCGR, including, but not limited to, insulin resistance, diabetes, obesity, metabolic syndrome and cardiovascular disease, and 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 and type 2, gestational and juvenile diabetes), diabetic complications, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, high 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., characterized by high blood levels of C-reactive protein), and hypertension. Dual agonist peptide products can achieve their therapeutic effects through a variety of mechanisms, including stimulating blood glucose-dependent insulin secretion, increasing insulin sensitivity, stimulating fat burning, and reducing body weight. Dual agonist peptide products can also promote, for example, pancreatic beta cell protection, cardioprotection, and / or wound healing.

[0048] 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 several embodiments, the symptom and / or complication is fatty liver disease (FLD), and the FLD is selected from nonalcoholic steatohepatitis (NASH) and NAFLD. In certain embodiments, the symptom and / or complication of obesity includes 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 accompanied by liver fat. Although symptoms of NASH are often subtle, some common symptoms include fatigue and right upper quadrant pain. Thus, "treating obesity" refers to activities that alleviate or improve either the primary phenomenon or secondary symptoms associated with obesity or other conditions described herein.

[0049] Nonalcoholic fatty liver disease (NAFLD) is 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 pathophysiological mechanisms underlying the development of NAFLD are primarily alterations in glucose and lipid metabolism, insulin resistance (IR), and insulin secretion, which explain the close association between NAFLD and T2D. Furthermore, patients with both NAFLD and T2D often share comorbidities associated with metabolic syndrome, namely, fasting hyperglycemia, hypertension, hypertriglyceridemia, low high-density lipoprotein cholesterol, and / or abdominal fat accumulation. It is well established that individuals with NAFLD, even when lean and without diabetes, have higher insulin resistance than individuals without NAFLD. 7Both longitudinal and cross-sectional studies have shown that elevated IR is the earliest detectable abnormality in both prediabetes and overt T2D. The pancreas responds to elevated IR by secreting more insulin, and the liver increases peripheral insulin concentrations and reduces insulin clearance to prevent the development of diabetes. In NAFLD, IR is present in muscle, liver, and adipose tissue. As a result, hepatic glucose production and adipose tissue lipolysis are only partially suppressed by insulin, leading to higher fasting glucose and free fatty acid (FFA) concentrations, increasing the risk of T2D in these patients. Therefore, there is a need to treat obesity in subjects with NAFLD or NASH.

[0050] The prevalence of NAFLD is estimated at 20%-30% in the United States and is expected to become the leading cause of liver transplantation within the next decade. NAFLD exists on a spectrum ranging from simple steatosis to steatohepatitis (nonalcoholic steatohepatitis [NASH]) and is characterized by lobular inflammation and ballooning. Across the disease spectrum, progression of fibrosis can lead to the development of cirrhosis, but fibrosis progression is typically more common and more rapid in NASH as opposed to simple steatosis.

[0051] Provided herein are methods for treating the symptoms and underlying conditions (eg, pathogenesis) of obesity, including NAFLD and NASH.

[0052] NAFLD can be diagnosed by imaging or histology evidence of hepatic steatosis and the absence of secondary causes of hepatic fat accumulation (alcoholic steatosis, medications, or genetic disorders). Nonalcoholic fatty liver disease (NAFL) is defined as the presence of 5% or more hepatic steatosis in the form of hepatocyte ballooning, without evidence of hepatocellular injury. While most patients tend to remain in the benign NAFL stage, some progress to NASH, which is characterized by the presence of 5% or more steatosis and inflammation accompanied by hepatocellular injury, with or without fibrosis. An established scoring system for assessing NAFLD histology is the NAFLD Activity Score (NAS). NAS is quantified using the following features: steatosis (0-3), lobular inflammation (0-3), and hepatocyte ballooning (0-2), which are added together to form a final score (0-8). In several embodiments, methods for treating obesity in subjects with NASH are provided herein. In certain embodiments, the methods provide an improvement in the subject's NAS score.

[0053] The peptide products of the present disclosure are dual agonist GLP-1 / glucagon peptide products, which act in part to restore normal blood glucose levels and help 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 related to the pathophysiology of NAFLD. In vitro and in vivo studies suggest that GLP-1RA improves autolysis / endoplasmic reticulum stress, macrophage recruitment, and induces hepatic gene expression in pathways that promote mitochondrial function and hepatocyte fatty acid oxidation, resulting in reduced steatosis and inflammation.

[0054] Histological endpoints assessing response to therapeutic intervention can be defined as improvement in NAS, resolution of NASH, or improvement in liver fibrosis. Resolution of NASH is defined as complete resolution of hepatocyte ballooning with an inflammation score of 0 or 1 and no worsening of fibrosis. Hepatocyte ballooning is an important parameter of NASH and has been shown to correlate with progressive disease and fibrosis. Fat accumulation in ballooned hepatocytes leads to oxidative damage, endoplasmic reticulum dysfunction, and cytoskeletal abnormalities, as evidenced by Mallory-Denk bodies. In several embodiments, biomarkers can also be used as markers of treatment efficacy. For hepatic steatosis assessment, controlled attenuation parameters are used as part of vibration-controlled transient elastography (FibroScan), as well as MRI proton density fat fraction (MRI-PDFF) and multiparameter MRI (LiverMultiScan). For liver inflammation and ballooning, the liver enzymes ALT and aspartate aminotransferase (AST) can be used. LiverMultiScan focuses on inflammation assessment, but confirmatory data are needed. Transient elastography, LiverMultiScan, MRI, or MR elastography can be used to analyze fibrosis. Biomarkers are also available for accurate hepatocellular fibrosis assessment, including Pro-C3, FIB-4, and NAFLD fibrosis scores, with improved liver fibrosis scores being commonly studied noninvasive tools for fibrosis assessment.

[0055] 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), 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), gastrointestinal disorders (e.g., cholelithiasis [gallstones], gastroesophageal reflux disease [GERD], and gastroparesis), gout, hypercortisolism (e.g., Cushing's syndrome), kidney disorders (e.g., chronic kidney disease), liver disorders (e.g., liver cirrhosis, liver cirrhosis, liver ulcers ... These include fatty liver disease (FLD), including alcoholic and non-alcoholic FLD, neurological disorders (e.g., carpal tunnel syndrome, dementia [e.g., Alzheimer's disease and vascular dementia], dysesthesias, femoral neuralgia, 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 cancers (e.g., endometrial, esophageal, colorectal, gallbladder, kidney, liver [e.g., hepatocellular carcinoma], pancreatic, and skin [e.g., melanoma] cancers, 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 further 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 is understood to also include metabolic fatty liver disease (MFLD). In certain embodiments, NAFLD is non-alcoholic steatohepatitis (NASH). FLD, also known as hepatic steatosis, is 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 the metabolism of alcohol in the liver. NAFLD is described below. FLD is associated with diabetes, obesity, and metabolic syndrome. Fatty liver can develop into cirrhosis or liver cancer (e.g., hepatocellular carcinoma [HCC]). Fewer than 10% of individuals with cirrhotic AFLD develop HCC, whereas up to 45% of individuals with NASH without cirrhosis may develop HCC. HCC is the most common type of primary liver cancer in adults and occurs in the setting of chronic inflammation of the liver. NAFLD is characterized by fatty liver, which occurs when fat, particularly free fatty acids and triglycerides, accumulate in hepatocytes (hepatic steatosis) due to causes other than excessive alcohol consumption, such as nutrient overload, high calorie intake, and metabolic dysfunction (e.g., dyslipidemia and impaired glycemic control). Although the liver can retain fat without interfering with liver function, fatty liver disease can progress to NASH, a condition in which steatosis, with or without hepatic fibrosis, is associated with inflammation, hepatocellular ballooning, and cellular injury. Fibrosis is the strongest predictor of mortality in NASH. NAFLD can be characterized by steatosis alone, steatosis with lobular or portular 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 approximately 20% of patients developing cirrhosis of the liver and approximately 10% succumbing to liver disease such as cirrhosis or liver cancer (e.g., HCC). NAFLD is the most common liver disorder in developed countries, and NASH is expected to replace hepatitis C as the leading cause of liver transplantation in the United States by 2020. Approximately 12-25% of people in the United States have NAFLD, and NASH affects approximately 2-5% of people in the United States. NAFLD, including NASH, is associated with insulin resistance, obesity, and metabolic syndrome. For example, insulin resistance contributes to liver inflammation and fibrosis, thus contributing to the progression of fatty liver to NASH. Furthermore, obesity contributes to the development and exacerbation of NASH, whereas weight loss can improve NASH.Thus, the peptide products described herein, including GLP-1 receptor (GLP1R) agonists, glucagon receptor (GCGR) agonists, and dual GLP1R / GCGR agonists, can be used to treat NAFLD, including NASH. In some embodiments, the dual agonist peptide product disclosed herein for use in treating conditions associated with insulin resistance or / and obesity, such as fatty liver, including NAFLD and NASH, is pembidutide, and / or its derivatives, and pharmaceutically acceptable salts thereof.

[0056] In some embodiments, the dual agonist peptides can be used to control blood glucose with a reduction in one or more adverse events (i.e., unexpected events that negatively impact patient and / or animal welfare). Exemplary, non-limiting adverse events can include nausea, vomiting, diarrhea, abdominal pain, and / or constipation. Adverse events can also include any known to those skilled in the art, such as those listed in industry resources and / or others known to those skilled in the art (see, e.g., Medical Dictionary for Regulatory Activities (MedDRA) (Pharm., Med. Transl. Med. 2018) and / or Clark, M.J. Biomed. Inf., 54, April 2015, pp. 167-173). Such adverse events can be determined in humans using standard techniques typically used in clinical trials (e.g., physician visits, surveys / questionnaires). Compared to the frequency and / or severity of such adverse events that occur upon administration of an agonist with unequal affinity for GLP-1R and GCGR (e.g., semaglutide) to a subject, a dual agonist peptide of the present disclosure (e.g., SEQ ID NO: 1, or any of its derivatives) can reduce such frequency and / or severity by, for example, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90% or more (up to 100%). In some embodiments, a dual agonist peptide of the present disclosure (e.g., pembidutide) does not cause any adverse events.

[0057] The dual agonist peptide product can be administered by any route suitable for treating the conditions disclosed herein. Possible routes of administration of the peptide product include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarteriolar, intraperitoneal, intracavity, and topical), topical (including transdermal and transmucosal), intranasal (e.g., nasal spray or drops), ophthalmic (e.g., eye drops), intrapulmonary (e.g., oral or nasal inhalation), buccal, sublingual, rectal (e.g., suppository), and vaginal (e.g., suppository). In some embodiments, the peptide product is administered parenterally, for example, subcutaneously, intravenously, or intramuscularly. In other embodiments, the peptide product is administered by oral or nasal inhalation or insufflation. The therapeutically effective amount and frequency of administration of a peptide product for treating a condition disclosed herein, as well as the length of treatment, can depend on various factors, including the nature and severity of the condition, the potency of the compound, the route of administration, the subject's age, weight, general health, sex, and diet, and the subject's response 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, 1, 5, or 10 mg, or about 0.1-1 mg or 1-10 mg, for about one week to treat a condition disclosed herein (e.g., a condition associated with insulin resistance and / or obesity, e.g., NASH or NAFLD). In further embodiments, the peptide product is administered parenterally (e.g., sc, iv, or im) at a dose of about 0.1-0.5 mg, 0.5-1 mg, 1-5 mg, or 5-10 mg, for 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 0.1-1 mg, or about 0.1-0.5 mg, or 0.5-1 mg, for about one week. One skilled in the art will appreciate that effective doses in mice or other preclinical animal models can be scaled to humans. Thus, through relative scaling (also called biological scaling), doses for larger animals can be extrapolated from mouse doses to obtain equivalent amounts based on the animal's body weight or body surface area.

[0058] The peptide product can be administered at any suitable frequency for the treatment of a condition disclosed herein (e.g., a condition associated with insulin resistance or / and obesity, e.g., NASH or NAFLD). In some embodiments, the dual agonist peptide product is administered once daily, once every two days, once every three days, twice weekly, once weekly, or once every two weeks, e.g., sc or iv. In certain embodiments, the peptide product is administered once weekly, e.g., SC, IV, or IM. The dual agonist peptide product can be administered at any time 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 or 2 hours before or after a meal). The length of treatment of a medical condition with the dual agonist peptide product can be based, for example, on 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 a condition disclosed herein, for example, for 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 until clinical symptoms of the condition no longer appear, or until a clinical target, such as blood glucose level, blood pressure, blood lipid level, body weight or body mass index, waist-to-hip ratio, or body fat percentage, or any combination thereof, is achieved. If clinical symptoms of the condition reappear or the clinical target is not maintained, administration of the dual agonist peptide product can be resumed. The present disclosure provides a method of treating a medical condition 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 same, to a subject in need of treatment. The present disclosure further provides a peptide product described herein, or a pharmaceutically acceptable salt thereof, or a composition comprising the same, for use as a pharmaceutical.In addition, the present disclosure provides the use of the peptide product or its pharmaceutically acceptable salt described herein in the preparation of a medicament.The medicament containing the peptide product can be used to treat any medical condition described herein.The peptide product can optionally be used in combination with one or more additional therapeutic agents.

[0059] The dual agonist peptide products described herein can be administered as the sole active agent or, optionally, can be used in combination with one or more other dual agonist peptide products and / or additional therapeutic agents treating any of the disorders disclosed herein, such as insulin resistance, diabetes, obesity, metabolic syndrome, or cardiovascular disease, or any condition related thereto, such as NASH or NAFLD. In some embodiments, the one or more additional therapeutic agents are selected from antidiabetic agents, antiobesity agents (including lipid-lowering agents and pro-satiety 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), thiazolidinediones (e.g., balaglitazone, ciglitazone, darglitazone, englitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, including MSDC-0602K and saroglitazar (a dual PPAR-α / γ agonist). Glucagon-like peptide-1 (GLP-1) receptor agonists, including exendin-4, albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, taspoglutide, CNT0736, CNT03649, HM11260C (LAPS-Exendin), NN9926 (OG9S7GT), TT401, and ZYOGl, alogliptin, anagliptin, dutogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, septagliptin, and septagliptin. dipeptidyl peptidase 4 (DPP-4) inhibitors, including gliptin, sitagliptin, teneligliptin, trelagliptin, and vildagliptin; sodium-glucose transport protein 2 (SGLT2) inhibitors, including canagliflozin (which also inhibits SGLT1), dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, remogliflozin etabonate, sotagliflozin (which also inhibits SGLT1), and tofogliflozin; meglitinides (e.g., ATP-dependent K+ upregulation in pancreatic beta cells, including 1st generation (e.g., mitiglinide, nateglinide, and repagiinide) and sulfonylureas (including first generation (e.g., acetohexamide, carbutamide, chlorpropamide, giycyclamide [tolhexamide], metahexamide, tolazamide, and tolbutamide) and second generation (e.g., glibenclamide, glyburide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, and glyclopyramide)). + (KA TP) channel blockers, insulin and its analogs, including fast-acting insulins (e.g., insulin asparig, insulin glulisine, and insulin lispro), intermediate-acting insulins (e.g., NPH insulin), and long-acting insulins (e.g., insulin degludec, insulin detemir, and insulin glargine), and / or analogs, derivatives, and salts thereof. In certain 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 tofogliflozin), or any combination thereof. Anti-obesity agents include, but are not limited to, appetite suppressants (appetite suppressants) including amphetamine, dexamphetamine, amfepramone, clobenzorex, mazindol, phentermine (with or without topiramate) and lorcaserin; satiety promoters including ciliary neurotrophic factor (e.g., axoxoquin) 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 caulerpenine, cetilistat, ebelactone A and B, estellastin, lipstatin, orlistat, percyquinin, panclicin A-E, valilactone, and vibralactone; antihyperlipidemic agents, and analogs, derivatives, and salts thereof. Antihyperlipidemic agents include, but are not limited to, statins {e.g., atorvastatin, cerivastatin, fluvastatin, mevastatin, monacolins (e.g., monacolin K (lovastatin), pitavastatin, pravastatin, rosuvastatin, and simvastatin} and HMG-CoA reductase inhibitors, including flavanones (e.g., naringenin), squalene synthase inhibitors, including lapaquistat, zaragozic acid, and RPR-107393, anthocyanins, avenaciolide, chloroacetylated biotin, cyclodim, diclofop, haloxyfop, soraphen (e.g., soraphen A la), 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), acetyl-CoA carboxylase (ACC) inhibitors, including CP-640186, GS-0976, and NDI-010976, 7-(4-propyloxy-phenylethynyl)-3,3-dimethyl-3,4 dihydro-2H-benzo[b][1,4]dioxepin, N-ethyl-N'-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dec-7-yl)piperidin-1-yl]-carbonyl}-1-benzothien-2-yl)urea, 5-(3 -acetamidobut-1-ynyl)-2-(4-propyloxyphenoxy)thiazole, and 1-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dec-7-yl)piperidin-1-yl]-carbonyl}-5-(pyridin-2-yl)-2-thienyl)-3-ethylurea, fibrates (e.g., bezafibrate, ciprofibrate, clinofibrate, clofibric acid, clofibrate, aluminum clofibrate [alfibrate], clofibrate, etofibrate, fenofibrate, PPAR-α agonists, including benzodiazepines (e.g., benzoyl perfluorooctanoic acid, fenofibrate, gemfibrozil, lonifibrate, and simfibrate), isoflavones (e.g., daidzein and genistein), and perfluoroalkanoic acids (e.g., perfluorooctanoic acid and perfluorononanoic acid), elafibranor (dual PPAR-α / γ agonist), GFT505 (dual PPAR-α / γ agonist), GW0742, GW501516 (dual PPAR-β / δ agonist), soderglitazar (GW677954), MBX-802 PPAR-δ agonists, including 5, and isoflavones (e.g., daidzein and genistein), PPAR-γ agonists, including thiazolidinediones (described above), saroglitazar (a dual PPAR-α / γ agonist), 4-oxo-2-thioxothiazolinone (e.g., rhodanine), berberine, honokiol, perfluorononanoic acid, cyclopentenone prostaglandins (e.g., cyclopentenone 15-deoxy-α-prostaglandin J2 [15d-PGJ2]), and isoflavones (e.g., daidzein and genistein),Liver X receptor (LXR) agonists, including endogenous ligands (e.g., oxysterols, such as 22(i?)-hydroxycholesterol, 24(A)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid) and synthetic agonists (e.g., acetyl-podocarpic acid dimer, hypocholesterolamide, A(X-dimethyl-3b-hydroxy-cholenamide [DMHCA], GW3965, and T0901317), endogenous ligands (e.g., 9-cis-retinoic acid) and synthetic agonists (e.g., bexarotene, AGN 191659, AGN 191701, AGN Retinoid X receptor (RXR) agonists, including 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, pellitorin, terpendol C, and flavanones (e.g., naringenin); inhibitors of stearoyl-CoA desaturase-1 (SCD-1, also known as stearoyl-CoA delta-9 desaturase), including aramchol, CAY-10566, CVT-11127, SAR-224, SAR-707, and XEN-103; inhibitors, 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide and 4-ethylamino-3-(2-hydroxyethoxy)-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide, 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)-N-methyl]pyridazine-3-carboxylic acid (2-hydroxy-2-pyridin-3-ylethyl)amide

[0010] cis-9, trans-11 and trans-10, cis-12 isomers of conjugated linoleic acid, substituted heteroaromatic compounds disclosed in WO2009 / 129625A1, antisense polynucleotides and peptide-nucleic acids (PNAs) targeting mRNA for SCD-1, and SCD-1-targeting siRNA, anacetrapib, dalcetrapib, evacetrapib, torcetrapib, and AMG 899 (TA-8995), cholesteryl ester transfer protein (CETP) inhibitors, including implitapide, lomitapide, dirlotapide, mitratapide, CP-346086, JTT-130, SLx-4090, inhibitors of the activity or expression of microsomal triglyceride transfer protein (MTTP), including antisense polynucleotides and PNAs targeting mRNA for MTTP, MTTP-targeting microRNAs (e.g., miRNA-30c), and MTTP-targeting siRNAs, and GLP-1 receptor fibroblast growth factor 21 (FGF21) and its analogs and derivatives, including the agonist BMS-986036 (pegylated FGF21), 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, PCSK9-binding adnectins (e.g., BMS-9, 62476), antisense polynucleotides and PNAs targeting mRNA for PCSK9, and PCSK9-targeting siRNA (e.g., inclisiran [ALN-PCS] and ALN-PCS02), inhibitors of the activity or expression of proprotein convertase (eonvertase) subtilisin / kexin type 9 (PCSK9), 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-C H2, 5A-H1, 18A, 37pA [18A-P-18A], ELK, ELK-1A, ELK-1F, ELK-1K1A1E, ELK-1L1K, ELK-1W, ELK-2A, ELK-2A2K2E, ELK-2E2K, ELK-2F, ELK-3E3EK, ELK-3E3K3 A, 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-hEl8A-NH2, AEM-28, Ac-[R]hEl 8 Apolipoprotein-mimetic peptides, including A-NH2, AEM-28-14, EpK, hEp, mR18L, COG-112, COG-133, and COG-1410; omega-3 fatty acids, including docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), fish oils (e.g., containing DHA and EPA), and esters thereof (e.g., glyceryl and ethyl esters), and analogs, derivatives, and salts thereof. In certain embodiments, anti-obesity agents may be used. The stimulating agent is or includes a lipase inhibitor (e.g., orlistat) or / and an antihyperlipidemic agent (e.g., a statin such as atorvastatin, or / and a fibrate such as fenofibrate). The antihypertensive agent includes, but is not limited to, a renin inhibitor (e.g., aliskiren), angiotensin-converting enzyme (ACE) inhibitor (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril),Antagonists of the renin-angiotensin-aldosterone system (RAAS), including angiotensin II receptor type 1 (ATII1) antagonists (e.g., azilsartan, candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan medoxomil, olmesartan, telmisartan, and valsartan) and aldosterone receptor antagonists (e.g., eplerenone and spironolactone), loop diuretics (e.g., bumetanide, ethacrynic acid, furosemide, and torasemide), thiazolinone, ... Diuretics, dihydropyridines (e.g., amlodipine, levamlodipine, cilnidipine, clevidipine, felodipine, isradipine, levodipine), including diazide diuretics (e.g., bendroflumethiazide, chlorothiazide, hydrochlorothiazide, epitizide, methyclothiazide, and polythiazide), thiazide-like diuretics (e.g., chlorthalidone, indapamide, and metolazone), cicletanine (an early distal tubular diuretic), potassium-sparing diuretics (e.g., amiloride, eplerenone, spironolactone, and triamterene), and theobromine calcium channel blockers, including nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine) and non-dihydropyridines (e.g., diltiazem and verapamil); alpha 2-adrenergic receptor agonists, including clonidine, guanabenz, guanfacine, methyldopa, and moxonidine; alpha 1-adrenergic receptor antagonists (alpha blockers), including doxazosin, indoramin, nicergoline, phenoxybenzamine, phentolamine, prazosin, terazosin, and tolazoline; beta-adrenergic receptor (beta1 or / and beta2) antagonists (beta-blockers), including bucindolol, carvedilol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, and timolol; mixed alpha / beta-blockers, including bucindolol, carvedilol, and labetalol; selective ETA receptor antagonists (e.g., ambrisentan, atrasentan, edonentan, sitaxsentan,zibotentan and BQ-123) and dual ET, A / ET BEndothelin receptor antagonists, including antagonists (e.g., bosentan, macitentan, and tezosentan), 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., cicletanine), activators of soluble guanylate cyclase (e.g., cinaciguat and riociguat), phosphodiesterase type 5 (PDE5) inhibitors (e.g., avanafil, benzamidenafil, dasantafil, dinafil, lodenafil, mirodenafil, 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., esveraprost], 5,6 , 7-trinor-4,8-inter-w-phenylene-9-fluoro-PGl2, carbacycline, isocarbacycline, clinprost, ciprostene, eptaloprost, cicaprost, iloprost, pimilprost, SM-10906 (des-methylpimilprost), naxaprostene, taprosten, treprostinil, CS-570, OP-2507, and TY-11223), non-prostanoid prostacyclin receptor agonists (e.g., 1-phthalazinol, larinepag, selexipag, ACT-333679 [MRE-269, 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, other vasodilators, including chelerythrine, cicletanine, gossypol, myabenol C, myricitrin, ruboxistaurin, and verbascoside, minerals, including magnesium sulfate and magnesium sulfate, and analogs, derivatives, and salts thereof. In certain embodiments, the antihypertensive agent is a thiazide or thiazide-like diuretic (e.g.,hydrochlorothiazide or chlorthalidone), calcium channel blockers (e.g., amlodipine or nifedipine), ACE inhibitors (e.g., benazepril, captopril, or perindopril), or angiotensin II receptor antagonists (e.g., olmesartan medoxomil, olmesartan, telmisartan, or valsartan), or any combination thereof. In some embodiments, the peptide products described herein are used in combination with one or more additional therapeutic agents to treat NAFLD, such as NASH. In some embodiments, the one or more additional 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-δ agonists (e.g., MBX-8025, elafibranor [dual PPAR-α / δ agonist], and GW501516 [dual PPAR-β / δ agonist]) and PPAR-γ agonists (e.g., thiazolidinediones such as pioglitazone and saroglitazar [dual PPAR-α / γ agonist]) (PPAR-δ and -γ antagonism increases insulin sensitivity, PPAR-α antagonism reduces hepatic steatosis, and PPAR-δ antagonism inhibits macrophage and Kupffer cell activation), farnesoid X receptor agonists, such as obeticholic acid. receptor (FXR) agonists (non-steroidal FXR agonists like GS-9674 reduce hepatic gluconeogenesis, lipogenesis, steatosis, and fibrosis), fibroblast growth factor 19 (FGF19) and its analogs and derivatives, such as NGM-282 (FGF19 analogs reduce hepatic gluconeogenesis and steatosis), fibroblast growth factor 21 (FGF21) and its analogs and derivatives, such as BMS-986036 (pegylated FGF21) (FGF21 analogs reduce hepatic steatosis, cytotoxicity, and fibrosis), HMG-CoA reductase inhibitors, including statins (e.g., rosuvastatin), (statins reduce steatohepatitis and fibrosis), such as NDI-010976 (liver-targeted) and GS-0976.ACC inhibitors (ACC inhibitors reduce de novo lipogenesis and hepatic steatosis), SCD-1 inhibitors (SCD-1 inhibitors reduce hepatic steatosis and increase insulin sensitivity), such as aramchol, SGLT2 inhibitors (SGLT2 inhibitors reduce body weight, hepatic ALT levels, and fibrosis), such as canagliflozin, ipragliflozin, and luseogliflozin, CCR2 and / or CCR5 antagonists (antagonists of CCR2 (binding to CCL2 [MCP1]) and CCR5 (binding to CCL5 [RANTES]) inhibit the activation and migration of inflammatory cells (e.g., macrophages) to the liver and reduce liver fibrosis), apoptosis signal-regulating kinase 1 (ASK1) inhibitors (e.g., selonsertib), and caspase inhibitors (e.g., emricasan [a pan-caspase inhibitor] ]) (apoptosis inhibitors reduce hepatic steatosis and fibrosis), lysyl oxidase-like 2 (LOXL2) 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 in the development of hepatic 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 [which also has anti-inflammatory properties via inhibition of TNF-α and phosphodiesterase]) (vitamin E reduces hepatic steatosis, hepatocyte 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).The one or more additional therapeutic agents for treatment of NASH (non-alcoholic steatohepatitis) are or include vitamin E and / or pioglitazone. Other combinations may also be used, as will be appreciated by those skilled in the art.

[0060] Pharmacokinetic ("PK") parameters can be estimated using Phoenix® WinNonlin® version 8.1 or higher (Certara USA, Inc., Princeton, New Jersey). A non-compartmental approach consistent with the extravascular route of administration can be used in parameter estimation. Individual plasma concentration-time data can be used to calculate pharmacokinetics. 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. Concentration values ​​below the limit of quantification can be treated as zero for the determination of descriptive statistics and pharmacokinetic analysis. Embedded concentration values ​​below the limit of quantification can be excluded from the pharmacokinetic analysis. All parameters can be generated from the plasma concentrations of individual dual agonist peptides (or their derivatives and / or metabolites) from the test article treatment groups on the day of dosing (Day 1). Parameters can be estimated using nominal dosing levels unless analytical results are available for an out-of-specification dose formulation, in which case the actual dose level can be used. Parameters can be estimated using nominal sampling times; if bioanalytical sampling variances are recorded, actual sampling times can be used at the affected time points. Bioanalytical data can be used for pharmacokinetic analysis and presented in tables and figures in the units presented. Pharmacokinetic parameters can be calculated and presented in the units presented by the laboratory (the digits can be adjusted appropriately for reporting, e.g., h*ng / mL converted to h*μg / mL). Descriptive statistics (e.g., mean, standard deviation, coefficient of variation, median, minimum, maximum) and pharmacokinetic parameters can be determined to three significant figures, as appropriate. Additional data entries can be recorded as needed. PK parameters to be determined, where data are available, include, but are not limited to, the following: max : Maximum observed concentration; DN C max : Dose-normalized maximum concentration, C max Calculated as / dose; Tmax : 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 method; AUC 0-96 : Area under the curve from time 0 to time 96, calculated using the linear trapezoidal method; DN AUC 0-96 : Dose-normalized AUC 0-96 , AUC 0-96 Calculated as / dose; AUC 0-inf : Area under the curve from time 0 to infinity (day 1 only), AUC 0-inf =AUC 0-t +C t / λ z Calculated as: t is the last observed quantifiable concentration, and λ z is the elimination rate constant; t 1 / 2 : elimination half-life, ln(2) / λ z Additional parameters and comparisons (e.g., gender ratio, dose proportionality ratio, etc.) can also be determined as will be understood by one of skill in the art.

[0061] In some embodiments, the present disclosure provides a pharmaceutical dosage formulation comprising pembidutide, wherein the peptide product is modified with a hydrophobic surfactant, and the dosage is configured to induce weight loss accompanied by reduction in one or more adverse events in a subject having fatty liver and possibly also suffering from type 2 diabetes, wherein the adverse events are selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to a mammal.

[0062] "Reducing" adverse effects or events or "reducing" these refers to reducing the severity, duration, and / or frequency of adverse effects experienced by a subject following administration of an agonist with approximately balanced affinity for GLP1R and GCGR, as well as the incidence of morbidity in a group of subjects. Such reduction includes the prevention of some adverse effects that a subject would otherwise experience in response to an agonist with disproportionate affinity for GLP1R and GCGR. Such reduction also includes the elimination of adverse effects previously experienced by a subject following administration of an agonist with disproportionate affinity for GLP1R and GCGR. In some embodiments, "reducing" adverse effects or "reducing" these include the reduction of gastrointestinal side effects, and adverse events are reduced to zero or undetectable levels. In other embodiments, adverse effects are reduced to a level comparable to that of untreated subjects, but are not completely eliminated. Furthermore, administration to mammals of analogs with disproportionate affinities for GLP-1R or GCGR may result in the need for excessively high doses to maximally activate receptors that are less sensitive to the ligand, thus potentially exceeding biologically effective dose levels for other ligands and causing dose-related undesirable side effects.

[0063] In preferred embodiments, the present disclosure provides methods for reducing body weight in a human with fatty liver, comprising administering pembidutide to a human in need thereof once weekly in an amount of at least 1.8 mg to 2.4 mg, wherein the human may have type 2 diabetes, and the fatty liver is nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In some preferred embodiments of such methods, the human's body weight is reduced by at least 3% from baseline at week 12. In some preferred embodiments of such methods, the human's body weight is reduced by at least 4% from baseline at week 12. In some preferred embodiments of such methods, pembidutide is administered once weekly in an amount of 1.8 mg. In some preferred embodiments of such methods, pembidutide is administered once weekly in an amount of 2.4 mg.

[0064] In some preferred embodiments, the disclosure provides a method for reducing liver fat content, as determined by MRI-PDFF, in a human having hepatic steatosis, comprising administering pembidutide to the human once weekly for at least 12 consecutive weeks in an amount of at least about 1.2 mg to about 2.4 mg, wherein the human has been diagnosed with hepatic steatosis, which is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), and has a body mass index (BMI, kg / m) greater than about 27. 2) and have 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 human population at 12 weeks that 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 an absolute reduction in liver fat content of at least about 12.5%, optionally at least about 14%, in a human population at 12 weeks that 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 an absolute reduction in liver fat content of at least about 10%, optionally at least about 11%, in a human population at 12 weeks that is significant (p<0.001) compared to placebo. In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces a relative reduction in liver fat content in a human population of at least about 40%, optionally at least about 45%, which is significant (p<0.001) compared to placebo at week 12. In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces a relative reduction in liver fat content in a human population of at least about 60%, optionally at least about 65%, which is significant (p<0.001) compared to placebo at week 12. In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces a relative reduction in liver fat content in a human population of at least about 50%, optionally at least about 55%, which is significant (p<0.001) compared to placebo at week 12. In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces at least about a 30% reduction in liver fat content in at least about 55%, optionally at least about 60% of a human population, which is significant compared to placebo at week 12 (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% of a human population, optionally at least about 85%, 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% of a human population, optionally at least about 80%. 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% of a human population, optionally at least about 38%, 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 at least a 50% reduction in liver fat content in at least about 55% of a human population, optionally at least about 60%, 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 a 50% reduction in liver fat content in at least about 60% of a human population, optionally at least about 65%, at week 12, which is significant (p<0.001) compared to placebo. In some preferred embodiments of such methods, a dose of about 1.2 mg per week induces a normalization of liver fat content to about 5% or less in at least about 20% of a human population, which is significant (p<0.05) compared to placebo. In some preferred embodiments of such methods, a dose of about 1.8 mg per week induces a normalization of liver fat content to about 5% or less in at least about 50%, optionally at least about 55%, of a human population that is significant (p<0.0001) compared to placebo at week 12. In some preferred embodiments of such methods, a dose of about 2.4 mg per week induces a normalization of liver fat content to about 5% or less in at least about 50% of a human population that is significant (p<0.001) compared to placebo at week 12.

[0065] In some preferred embodiments, the disclosure provides a method of inducing weight loss in a human having fatty liver, comprising administering pembidutide to the human once weekly for at least 12 consecutive weeks in an amount of at least about 1.2 mg to about 2.4 mg, wherein the human has been diagnosed with fatty liver disease, which is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), and has a body mass index (BMI, kg / m) greater than about 27. 2) and have a liver fat content of at least about 10% as measured by MRI-PDFF. In some preferred embodiments of such methods, the human has not been diagnosed with 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 12 weeks, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, the human has not been diagnosed with diabetes, and a dose of about 1.8 mg per week reduces the human's body weight by at least about 4%, optionally at about 5%, at 12 weeks, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, the human has not been diagnosed with 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 about 3.5%, at 12 weeks, which is significant compared to placebo (p<0.001). In some preferred embodiments of such methods, a 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. In some preferred embodiments of such methods, a 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 (p<0.05) compared to placebo. In some preferred embodiments of such methods, a 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 (p<0.05) compared to placebo. In some preferred embodiments of such methods, a dose of about 1.2 mg per week reduces the human's body weight by at least about 3%, 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 reduces body weight by at least about 4% in a human population, which is significant compared to placebo at 12 weeks (p<0.001).In some preferred embodiments of such methods, a dose of about 2.4 mg per week significantly reduces body weight in a human population by at least about 3%, optionally by at least about 3.5%, compared to placebo at week 12 (p<0.001).

[0066] In some preferred embodiments of the methods herein, a dose of about 1.2 mg per week induces an ALT reduction of at least about 11% in a human population at week 12. In some preferred embodiments of the methods herein, a dose of about 1.8 mg per week induces an ALT reduction of at least about 13% in a human population that is significant (p<0.05) compared to placebo at week 12. In some preferred embodiments of the methods herein, a dose of about 2.4 mg per week induces an ALT reduction of at least about 13% in a human population that is significant (p<0.05) compared to placebo at week 12. In some preferred embodiments of the methods herein, a dose of about 1.2 mg per week induces an ALT reduction of at least about 19% at week 12 in a human population with a baseline ALT of about 30 IU / L or greater. In some preferred embodiments of the methods herein, a dose of about 1.8 mg per week induces an ALT reduction of at least about 20% at week 12 in a population of humans with a baseline ALT of about 30 IU / L or greater. In some preferred embodiments of the methods herein, a dose of about 2.4 mg per week induces an ALT reduction of at least about 20%, optionally at least about 25%, at week 12 that is significant (p<0.005) compared to placebo in a population of humans with a baseline ALT of about 30 IU / L or greater.

[0067] In some preferred embodiments, pembidutide is administered by parenteral injection. In some preferred embodiments, pembidutide is administered by subcutaneous injection. In some preferred embodiments, the human has a body mass index (BMI, kg / m) of at least 27. 2 In some preferred embodiments, the human has a body mass index (BMI, kg / m) of 30 or greater. 2In some preferred embodiments, the human has a liver fat level measured by MRI-PDFF of 10% or greater before treatment. In some preferred embodiments, the absolute reduction in liver fat as determined using MRI-PDFF is about 8%, 10%, 12%, or preferably about 15% after 12 weeks of treatment. In some preferred embodiments, the relative reduction in liver fat as determined using MRI-PDFF relative to baseline is greater than approximately 40%, 50%, or 60% after 12 weeks of treatment. In some preferred embodiments, the steady-state dose is achieved after a dose-escalation phase having a duration of 2-4 weeks, or about 6, 8, 10, 12, or 16 weeks. In some preferred embodiments, pembidutide is administered from a liquid containing at least about 2.5 mg / ml of pembidutide. In some embodiments, pembidutide is administered from a pharmaceutical dosage form as an aqueous formulation containing one or more of polysorbate 20, arginine, or mannitol.

[0068] In some embodiments, the disclosure provides a method for reducing body weight in a human having fatty liver, comprising administering pembidutide 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) once a week for at least about 24 weeks to a human in need thereof, wherein the human optionally has type 2 diabetes and / or optionally the fatty liver is nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In some embodiments, pembidutide is administered in an amount of 1.2 mg once a week for 24 weeks. In some such embodiments, the disclosure provides methods for administering pembidutide once weekly in an amount of about 1.2 mg, 1.8 mg, or 2.4 mg, wherein the relative reduction in liver fat compared to baseline as measured by MRI-PDFF is about 30% to about 50% after 24 weeks of weekly dosing with pembidutide, where the relative reduction is statistically significant, defined as p<0.001 or p<0.0001. In some embodiments, weekly dosing with 1.8 mg and 2.4 mg of pembidutide induces at least a 40% reduction in liver fat after 24 weeks, where the reduction is significant, defined as p<0.001 or p<0.01, compared to placebo. In some embodiments, liver delipidation as measured by MRI-PDFF compared to baseline is about 30% after 24 weeks of weekly dosing with about 1.8 mg of pemvidutide compared to baseline in a human. In some embodiments, liver volume as measured by MRI-PDFF compared to baseline is reduced after 24 weeks of weekly dosing with about 1.2 mg, about 1.8 mg, or about 2.4 mg of pemvidutide compared to placebo. In some embodiments, alanine aminotransferase (ALT) is reduced after 24 weeks of weekly dosing with about 1.2 mg, about 1.8 mg, or about 2.4 mg compared to placebo, optionally with a baseline ALT of greater than 30 IU / L prior to dosing.In some embodiments, the reduction in iron-corrected T1 is greater than 80 ms in about 80% of subjects, optionally with the reduction being significant, defined as p<0.05 or p<0.005, following 24 weeks of weekly dosing with about 1.2 mg, about 1.8 mg, or about 2.4 mg of pembidutide. In some embodiments, weekly dosing with about 1.2 mg, about 1.8 mg, or about 2.4 mg of pembidutide for 24 weeks results in weight loss in non-diabetic and / or diabetic patients compared to placebo. In some embodiments, weekly dosing with about 1.2 mg, about 1.8 mg, or about 2.4 mg of pembidutide for 24 weeks results in lower serum lipid levels compared to placebo. In some embodiments, weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg of pembidutide per week for 24 weeks reduces blood pressure without significantly increasing heart rate, and optionally systolic blood pressure is significantly reduced as defined by p<0.05 with weekly dosing of 2.4 mg. In some embodiments, weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg of pembidutide per week for 24 weeks improves glycemic parameters, and optionally the glycemic parameters are reduced fasting glucose and / or HbA1c levels.

[0069] In some preferred embodiments, the present disclosure provides the following aspects: 1. A method for reducing weight in a human having fatty liver, comprising administering pembidutide in an amount of at least about 1.2 to about 2.4 mg, optionally about 1.8 mg to about 2.4 mg, or about 1.2 mg, about 1.8 mg, or about 2.4 mg, once a week to a human in need thereof for at least about 12 weeks and / or up to at least about 24 weeks, wherein the human optionally has type 2 diabetes, and / or optionally the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). 2. The method of embodiment 1, wherein the human loses at least 3% of their body weight from baseline at week 12. 3. The method of embodiment 1, wherein the human loses at least 4% of their body weight from baseline at week 12. 4. The method of embodiment 1, wherein pembidutide is administered in an amount of 1.2 mg once a week for 24 weeks. 5. The method of embodiment 1, wherein pembidutide is administered once a week in an amount of 1.8 mg. 6. The method of any one of aspects 1 to 5, wherein pembidutide is administered once a week in an amount of 2.4 mg. 7. The method of embodiment 1, wherein the steady-state dose is achieved after a dose-escalation phase having a duration of about 2 weeks, about 3 weeks, or about 4 weeks. 8. The method of embodiment 1, wherein the human has type 2 diabetes. 9. The method of embodiment 1, wherein the human does not have type 2 diabetes. 10. Humans must have a body mass index (BMI, kg / m) of at least 28. 2 2. The method of embodiment 1, comprising: 11. A person has a body mass index (BMI, kg / m) of 30 or greater. 2 2. The method of embodiment 1, comprising: 12. The method of embodiment 1, wherein the human has a liver fat level as measured by MRI-PDFF of 10% or greater. 13. The method of embodiment 1, wherein the absolute reduction in liver fat as measured by MRI-PDFF is about 8% to about 15% after 12 weeks of weekly dosing. 14. The method of embodiment 1, wherein the relative reduction in liver fat as measured by MRI-PDFF compared to baseline is about 40% to about 70% after 12 weeks of weekly dosing. 15. The method of embodiment 1, wherein the relative reduction in liver fat as measured by MRI-PDFF compared to baseline is about 30% to about 50% after 24 weeks of weekly dosing, wherein the relative reduction is statistically significant, defined as p<0.001 or p<0.0001. 16. The method of embodiment 1, wherein weekly dosing at 1.8 mg and 2.4 mg induces at least a 40% reduction in liver fat after 24 weeks, with the reduction being significant defined as p<0.001 or p<0.01 compared to placebo. 17. The method of embodiment 1, wherein liver delipidation as measured by MRI-PDFF compared to baseline is about 30% after 24 weeks of weekly dosing of about 1.8 mg of pemvidutide compared to baseline in a human. 18. The method of embodiment 1, wherein liver volume as measured by MRI-PDFF compared to baseline is reduced after 24 weeks of weekly dosing of about 1.2 mg, about 1.8, or about 2.4 mg of pembidutide compared to placebo. 19. The method of embodiment 1, wherein alanine aminotransferase (ALT) is reduced after 24 weeks of weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg relative to placebo, and optionally wherein the human has a baseline ALT of greater than 30 IU / L prior to dosing. 20. The method of embodiment 1, wherein the decrease in iron-corrected T1 is greater than 80 ms in about 80% of subjects, optionally with the decrease being significant, defined as p<0.05 or p<0.005. 21. The method of embodiment 1, wherein 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 embodiment 1, wherein weekly dosing of about 1.2 mg, about 1.8 mg, or about 2.4 mg per week for 24 weeks results in serum lipid levels compared to placebo. 23. The method of embodiment 1, wherein 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, and optionally systolic blood pressure is significantly reduced with weekly dosing of 2.4 mg, as defined as p<0.05. 24. The method of embodiment 1, wherein 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 the glycemic parameter being a reduction in fasting glucose and / or HbA1c levels. 25. A method for reducing liver fat content as determined by MRI-PDFF in a human having fatty liver, comprising administering pembidutide to the human once weekly for at least 12 consecutive weeks in an amount of at least about 1.2 mg to about 2.4 mg, wherein the human: Have been diagnosed with fatty liver disease, either nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), A body mass index (BMI) higher than approximately 28 kg / m 2 ) and have a liver fat content of at least about 10% as measured by MRI-PDFF, method. 26. The method of any embodiment 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 human population, at week 12 that is significant compared to placebo (p<0.001). 27. The method of any embodiment herein, 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 that is significant compared to placebo (p<0.001). 28. The method of any embodiment 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 human population, at week 12 that is significant compared to placebo (p<0.001). 29. The method of any embodiment herein, 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, which is significant compared to placebo at week 12 (p<0.001). 30. The method of any embodiment 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 human population, which is significant compared to placebo at week 12 (p<0.001). 31. The method of any embodiment 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 human population, at week 12 that is significant compared to placebo (p<0.001). 32. The method of any embodiment herein, wherein a dose of about 1.2 mg per week induces a significant (p<0.001) reduction in liver fat content of at least about 30% in at least about 55%, optionally at least about 60% of a human population, compared to placebo at week 12. 33. The method of any embodiment herein, wherein a dose of about 1.8 mg per week induces a significant (p<0.001) reduction in liver fat content of at least about 30% in at least about 80%, optionally at least about 90% of a human population, compared to placebo at week 12. 34. The method of any embodiment herein, wherein a dose of about 2.4 mg per week induces a significant (p<0.001) reduction in liver fat content of at least about 30% in at least about 75%, optionally at least about 85% of a human population, compared to placebo at week 12. 35. The method of any embodiment herein, wherein a dose of about 1.2 mg per week induces at least about a 50% reduction in liver fat content in at least about 35%, optionally at least about 40% of a human population, which is significant compared to placebo at week 12 (p<0.001). 36. The method of any embodiment herein, wherein a dose of about 1.8 mg per week induces a significant (p<0.001) reduction in liver fat content of about 50% in at least about 60%, optionally at least about 70% of a human population, compared to placebo at week 12. 37. The method of any embodiment herein, wherein a dose of about 2.4 mg per week induces at least about a 50% reduction in liver fat content in at least about 60%, optionally at least about 70% of a human population, which is significant compared to placebo at week 12 (p<0.001). 38. The method of any embodiment herein, wherein a dose of about 1.2 mg per week induces a normalization of liver fat content to about 5% or less in at least about 20% of a human population that is significant compared to placebo at week 12 (p<0.05). 39. The method of any embodiment herein, wherein a dose of about 1.8 mg per week induces a normalization of liver fat content to about 5% or less in at least about 50%, optionally at least about 55%, of a human population that is significant compared to placebo at week 12 (p<0.0001). 40. The method of any embodiment herein, wherein a dose of about 2.4 mg per week induces a normalization of liver fat content to about 5% or less in at least about 50% of a human population that is significant compared to placebo at week 12 (p<0.001). 41. A method for inducing weight loss in a human having fatty liver, comprising administering pembidutide to the human once weekly for at least 12 consecutive weeks in an amount of at least about 1.2 mg to about 2.4 mg, wherein the human: Have been diagnosed with fatty liver disease, either nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), A body mass index (BMI) higher than approximately 28 kg / m 2 ) and Any diagnosed type 2 diabetes, have a liver fat content of at least about 10% as measured by MRI-PDFF, method. 42. The method of embodiment 31, wherein the human has not been diagnosed with type 2 diabetes, and wherein a dose of about 1.2 mg per week significantly reduces the human's body weight by at least about 2.5%, optionally at least about 3%, compared to placebo at week 12 (p<0.001). 43. The method of any embodiment herein, wherein the human has not been diagnosed with type 2 diabetes, and wherein a dose of about 1.8 mg per week reduces the human's body weight by at least about 4%, optionally about 5%, significantly compared to placebo at week 12 (p<0.001). 44. The method of any embodiment herein, wherein the human has not been diagnosed with type 2 diabetes, and wherein a dose of about 2.4 mg per week significantly reduces the human's body weight by at least about 2.5%, optionally about 3.5%, compared to placebo at week 12 (p<0.001). 45. The method of any embodiment herein, wherein the human has been diagnosed with type 2 diabetes, and wherein a dose of about 1.2 mg per week significantly reduces the human's body weight by at least about 2%, optionally at least about 3%, at week 12 compared to placebo (p<0.05) at week 12. 46. ​​The method of any embodiment herein, wherein the human has been diagnosed with type 2 diabetes, and wherein a dose of about 1.8 mg per week significantly reduces the human's body weight by about 2.5%, optionally at least about 3.5%, compared to placebo at week 12 (p<0.005). 47. The method of any embodiment herein, wherein the human has been diagnosed with type 2 diabetes, and wherein a dose of about 2.4 mg per week significantly reduces the human's body weight by about 3%, optionally at least about 4%, compared to placebo at week 12 (p<0.001). 48. The method described in any embodiment herein, wherein a dose of about 1.2 mg per week significantly reduces body weight in a human population by at least about 3% compared to placebo at week 12 (p<0.001). 49. The method of any embodiment herein, wherein a dose of about 1.8 mg per week reduces body weight in a human population by at least about 4%, which is significant compared to placebo at week 12 (p<0.001). 50. The method of any embodiment herein, wherein a dose of about 2.4 mg per week significantly reduces body weight in a human population by at least about 3%, optionally at least about 3.5%, compared to placebo at week 12 (p<0.001). 51. The method described in any embodiment herein, wherein a dose of about 1.2 mg per week induces an ALT decrease of at least about 11% in a human population at 12 weeks. 52. The method described in any embodiment herein, wherein a dose of about 1.8 mg per week induces an ALT reduction of at least about 13% in a human population, which is significant compared to placebo at week 12 (p<0.05). 53. The method described in any embodiment herein, wherein a dose of about 2.4 mg per week induces an ALT reduction of at least about 13% in a human population that is significant compared to placebo at week 12 (p<0.05). 54. The method of any embodiment herein, wherein a dose of about 1.2 mg per week induces an ALT reduction of at least about 18% at week 12 in a population of humans with a baseline ALT of about 30 IU / L or greater. 55. The method of any embodiment herein, wherein a dose of about 1.8 mg per week induces an ALT reduction of at least about 20% at week 12 in a population of humans with a baseline ALT of about 30 IU / L or greater. 56. The method of any embodiment herein, wherein a dose of about 2.4 mg per week induces an ALT reduction of at least about 20%, optionally at least about 25%, at week 12 that is significant (p<0.005) compared to placebo in a population of humans with a baseline ALT of about 30 IU / L or greater. 57. The method of any one of the aspects herein, wherein pembidutide is administered by parenteral injection. 58. The method of any one of the aspects herein, wherein pembidutide is administered by subcutaneous injection. 59. Humans must have a body mass index (BMI, kg / m) of at least 28. 2 The method of any aspect herein, comprising: 60. A person with a body mass index (BMI) of 30 or greater (kg / m 2 The method of any aspect herein, comprising: 61. The method of any one of the embodiments herein, wherein the human has a liver fat level as measured by MRI-PDFF of 10% or greater prior to the weekly dosing. 62. The method of any one of the embodiments herein, wherein the absolute reduction in liver fat as determined using MRI-PDFF is about 8% to about 15% after 12 weeks of weekly dosing. 63. The method of any one of the embodiments herein, wherein normalization of liver fat content to 5% or less is greater than 20% to about 55% of the population after 12 weeks of weekly dosing. 64. The method of any embodiment herein, wherein the relative reduction in liver fat as determined using MRI-PDFF relative to baseline is greater than about 40% to about 60% after 12 weeks of weekly dosing. 65. The method of any one of the embodiments herein, wherein the ALT reduction is greater than about 13% to about 25% of the population after 12 weeks of weekly dosing. 66. The method of any one of the embodiments herein, wherein the steady-state dose is achieved after a dose-escalation phase having a duration of 2 to 4 weeks. 67. The method of any one of the embodiments herein, wherein the pembidutide is administered from a liquid formulation comprising at least about 2.5 mg / ml of pembidutide. 68. The method of any one of the embodiments herein, wherein administration of pembidutide induces a significant reduction in serum lipids and / or atherogenic small and intermediate LDL particle concentrations. 69. A method according to any one of the embodiments herein, wherein atherogenic small and intermediate LDL particle concentrations are reduced by at least a -0.2 log2-fold change relative to placebo following 43 and / or 84 days of administration of pembidutide. 70. A method according to any embodiment herein, wherein the subject's total serum triglyceride concentration is reduced by at least a -0.2 log2-fold change relative to placebo following 43 and / or 84 days of administration of pembidutide, as measured by 2D-NMR. 71. The method of any one of the aspects herein, wherein the serum lipid is selected from a glycerolipid, a sterol, a glycerophospholipid, and a sphingolipid, and optionally the reduction is at least a -0.2 log2 fold change. 72. The method of any one of the aspects herein, wherein a decrease in serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, and / or lysophosphatidylcholine follows administration of 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 aspect 73, comprising 1.8 mg of SEQ ID NO:1 as a therapeutic dose. 75. The formulation of embodiment 74, wherein a therapeutic dose induces weight loss in a subject in need thereof.

[0070] Other aspects of the present disclosure are also contemplated, as will be appreciated by those skilled in the art.

[0071] Unless otherwise defined or otherwise clearly indicated by the use of this specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used in this application and the appended claims, the word "a" or "an" means one or more. As used herein, the word "another" means second or more. The acronym "aka" means "also known as." The term "exemplary," as used herein, means "serving as an example, instance, or illustration." Any embodiment or feature characterized herein as "exemplary" is not necessarily construed as preferred or advantageous over other embodiments or features. In some embodiments, the term "about" or "approximately" means within ±10% or 5% of a given value. Whenever the term "about" or "approximately" precedes the first number in a series of two or more numbers or in a series of two or more numerical ranges, the term "about" or "approximately" applies to each of the numbers in the series or numerical range. Ranges can be expressed herein from about one particular value and / or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that by using the antecedent "about" or "approximately," the particular value forms another embodiment. It is further understood that each endpoint of a range is significant both relative to the other endpoint and independently of the other endpoint. A range (e.g., 90-100%) is meant to include the range itself and each individual value within the range, as if each value were individually recited. Optionally or optionally means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur. All publications, patents, and patent applications mentioned in this application are incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

[0072] Certain embodiments are further described in the following examples, which are offered by way of example only and are not intended to limit the scope of the claims in any way. [Example]

[0073] Example 1 A Phase 1, 12-Week, Randomized, Double-Blind, Placebo-Controlled Study of ALT-801 (Pembidutide) in Diabetic and Nondiabetic Overweight and Obese Subjects With Nonalcoholic Fatty Liver Disease (NAFLD) Overweight and obese patients (BMI, 28.0 kg / m ) with and without diabetes (type 2 diabetes) with NAFLD 2 Disclosure is provided herein of a Phase 1, multicenter, randomized, double-blind, placebo-controlled study evaluating the effects of pembidutide (also referred to herein as ALT-801), the safety of pembidutide, and the effects on hepatic fat fraction, anthropometric parameters, lipid metabolism, inflammatory markers, and fibrosis markers in subjects with NAFLD. The study was designed to evaluate changes in hepatic fat fraction by MRI-PDFF (magnetic resonance imaging-proton density fat fraction) in diabetic and non-diabetic overweight and obese subjects with NAFLD after 12 weeks of ALT-801 treatment. The study was conducted without supplemental dietary and exercise intervention. The study evaluated changes in body weight, lipid metabolism, metabolic markers, and inflammatory markers after 12 weeks of treatment, as well as the safety and tolerability of ALT-801. Subjects were stratified by the presence or absence of diabetes at baseline. Fibroscan and cT1 assessments evaluated changes in hepatic inflammation and fibrotic activity at the end of 12 weeks of treatment.

[0074] 28kg / m 2Overweight and obese volunteers with a BMI above 10% were tested because this represents a representative population of individuals with NAFLD, and these subjects may be better able to tolerate the expected pharmacodynamic (PD) effects of weight loss and benefit from treatment. A liver fat fraction of 10% is typical for assessing liver fat fraction in subjects with NAFLD. In other words, individuals with a liver fat fraction of 10% or higher are typically diagnosed with NAFLD. Diabetic subjects who require insulin, sulfonylureas, or DDP-4 inhibitors for diabetes control were excluded from this study. Exclusions were made that may otherwise affect the safety or accurate assessment of the effects of ALT-801 on PD. The upper BMI limit was 45 kg / m 2 , because subjects above this BMI may not fit into the MRI scanner.

[0075] Fibroscan values ​​below 10 kPa adequately exclude subjects with advanced fibrosis who would not be suitable candidates for this clinical trial.

[0076] The study described in this embodiment involves treatment with ALT-801 (a composition comprising SEQ ID NO: 1, "Test Drug") or placebo administered by subcutaneous (SC) injection once weekly for up to 12 doses, over a period of approximately 4.5 months, including a screening period of up to 35 days, and an 85-day treatment period and a 25-day follow-up period, in 94 subjects with overweight and obesity (28.0 kg / m) associated with nonalcoholic fatty liver disease (NAFLD). 2 The study was conducted in type 2 diabetic and non-diabetic subjects with a body mass index (BMI) of ≥ 1.

[0077] The baseline characteristics of the subjects included in this study are shown in Table 5 below.

[0078] [Table 4]

[0079] The safety objective of this study was to evaluate the safety and tolerability of ALT-801 in subjects aged 18 to 65 years with NAFLD (NAFLD without significant fibrosis, defined as a Fibroscan®-controlled attenuation parameter (CAP) of 280 dB / m or greater and a liver stiffness measurement (LSM) of less than 10 kPa (i.e., indicating the absence of significant fibrosis), a magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF) of 10% or greater), a hemoglobin A1c (HbA1c) of less than 9.5%, and an alanine aminotransferase (ALT) or aspartate aminotransferase (AST) level of 75 IU / ml or less. The pharmacodynamic (PD) objective of this study was to evaluate the effects of ALT-801 on hepatic fat fraction, anthropometric parameters, lipid metabolism, metabolic markers, inflammatory markers, fibrosis markers, and lipotoxicity markers. The pharmacokinetic (PK) objective of this study was to evaluate the effect of ALT-801 and metformin exposure (drug interactions) in subjects (eg, ALT-801 and metformin concentrations in the blood over time).

[0080] After providing informed consent, subjects underwent a screening period of up to 35 days. Subjects were instructed on how to maintain their normal diet, alcohol intake, and physical activity during their study participation and not to start any new dietary, nutritional supplement, or exercise programs at any time. Counseling regarding diet and exercise 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 arms: 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 once weekly in week 1, 1.2 mg SC once weekly in week 2, 1.8 mg SC once weekly for 2 weeks (Weeks 3 and 4), and 2.4 mg SC once weekly from 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 arm with a final therapeutic dose of 2.4 mg received a rapid 4-week dose escalation, which improved tolerability and reduced the likelihood of adverse events. Prior studies with pembidutide in first-in-human trials showed that the 2.4 mg dose had a relatively high incidence of certain adverse events. See Figure 7, second 2.4 mg column compared to the first 2.4 mg column (this study). See WO2022 / 125598. Thus, a rapid dose escalation regimen of pembidutide is provided herein, with an initial week 1 dose of 25% of the total therapeutic dose, a week 2 dose of 50% of the total therapeutic dose, and week 3 and week 4 doses of 75% of the total therapeutic dose. From week 5 onwards, subjects receive 100% of the total therapeutic dose. In certain embodiments, the dose escalation regimen is 0.6 mg, 1.2 mg, and 1.8 mg for a final therapeutic dose of 2.4 mg.

[0081] Subjects received their first dose of study medication on Day 1 ("Baseline"). Subsequent visits occurred weekly at the clinic, home, or workplace until Day 85 or early termination. Subjects returned for a safety follow-up visit on Day 110. Investigators followed timing criteria and intervention methods for subjects who developed abnormal liver function test deterioration during the 12-week treatment period. Fasting glucose levels were measured with a glucometer at baseline and before each dose and recorded by study staff. Outside of clinic visits, subjects monitored and recorded their fasting glucose every morning and contacted the study site if their reading was greater than 240 mg / dL or less than 70 mg / dL. Subjects also received education on hypoglycemic symptoms and treatment and had their glucometer read if they experienced a glucose reading less than 70 mg / mL or symptoms suggestive of hypoglycemia. Subjects also recorded any hypoglycemic symptoms they experienced at home in a log, which was reviewed by investigators at each clinic visit beginning on Day 8. Investigators advised subjects on how to maintain fasting glucose within limits, including repeated dietary counseling, and followed study decision criteria and intervention methods for timing in subjects who maintained hyperglycemia during the 12-week treatment period. Certain subjects who exhibited significant reductions in fasting glucose (<50 mg / dL) were repeatedly observed. Sparse blood samples were collected for ALT-801 PK to be combined with data from other studies in population PK and PK-PD modeling, and for metformin PK to evaluate changes in metformin concentrations over time in the presence of ALT-801. Blood samples were also collected to assess immunogenicity.

[0082] The power and sample size assumptions used in this study indicated that the sample size was adequate to meet the safety assessments of a Phase 1 trial. Based on the treatment effects observed in previous studies of NAFLD, the study was also adequately powered to detect meaningful differences in change in liver fat fraction by MRI-PDFF compared to baseline in subjects receiving ALT-801 compared to subjects receiving placebo SC injections at the 0.05 significance level (two-sided).

[0083] For statistical analysis, all randomized subjects receiving at least one dose of study drug (safety population) were included in the safety analysis. Secondary and PD endpoint assessments were performed in the PD population, which consisted of all randomized subjects receiving at least one dose of study drug and available from baseline and at least one post-baseline PD assessment.

[0084] Two interim analyses were conducted: 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 remained blinded to individual treatment assignments. Analyses included safety, weight loss, and MRI-PDFF since the start of treatment and available PK data. Summary data were reported by study arm, dose level, treatment group (active or placebo), and date, if applicable. Ongoing safety data were summarized using descriptive statistics (arithmetic mean, standard deviation [SD], median, minimum, and maximum) by dose level and treatment (active or placebo). Categorical safety data were summarized as frequency counts and percentages by dose level, treatment group, and date, if applicable. AEs were coded using the latest version of the Medical Dictionary for Regulatory Affairs (MedDRA). Itemized AE data listings were presented, 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, as well as injection site reactions, were summarized by treatment group, SOC, and preferred term. TEAEs were also summarized by severity and relationship to study drug. Laboratory assessments, including liver function tests and fasting glucose, vital signs (including RPP calculations), and ECG assessments, were summarized by treatment group, dose level, and protocol-specified collection time points. Summary changes from baseline at each protocol-specified time point by treatment group were also determined. Physical examination changes were listed for each subject. Concomitant medications were listed by subject and coded using the latest version of the World Health Organization (WHO) Drug Dictionary. Medical history was coded using the latest version of MedDRA and listed by subject.

[0085] For pharmacodynamic determinations, descriptive statistics, including numbers and percentages for categorical variables and means, SDs, medians, minimums, and maximums for continuous variables, were provided by dose level and treatment (ALT-801 or placebo), and date, if applicable. Changes from baseline in liver fat fraction, anthropometric parameters, lipid metabolism, metabolic markers, lipotoxicity markers, and inflammatory markers were summarized by stratification by treatment group and 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 assessed in covariate analyses. Inferential statistics were performed when applicable. All analyses were described in the statistical analysis plan (SAP). Changes in liver fat fraction by MRI-PDFF, body composition, and liver inflammation, as well as fibrotic markers by MRI and fibroscan, and other continuous variables were compared between the ALT-801 and placebo groups using analysis of covariance (ANCOVA) tests, with stratification by treatment arm and diabetes status as factors or corresponding baseline demographic characteristics (gender, race, BMI) as covariates. The Cochran-Mantel-Haenszel test was applied to secondary endpoints that were categorical in nature, taking into account the stratification by diabetes status at a one-sided significance level of 0.025.

[0086] Quality of life was also measured as the change from baseline in two summary scores for physical health and mental health, the eight domain scores for the SF-36, and the composite score for the IWQoL-Lite for CT, 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.

[0087] For pharmacokinetic (PK) determinations, individual ALT-801 and metformin concentration data were listed and summarized by treatment group and time point with descriptive statistics (N, arithmetic mean, SD, CV%, median, minimum, and maximum). Individual and mean ± SD ALT-801 concentration-time profiles for each cohort were also presented graphically. Changes from baseline metformin concentrations were also listed and summarized by treatment group and time point with descriptive statistics (N, arithmetic mean, SD, CV%, median, minimum, and maximum). A population PK model was developed to enable prediction of individual subject ALT-801 plasma concentration-time curves and associated PK parameters. Covariates, including sex, age, weight, BMI, and concomitant medications, were considered, and exposure-response relationships, where possible, were examined for efficacy and safety endpoints. This analysis combines data from multiple studies and is performed as a separate study and reported.

[0088] As shown in Figure 2, ALT-801 induced a significant absolute reduction in liver fat of about 10% or more across 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, p<0.001 vs. placebo)), and a significant relative reduction in liver fat of at least about 40% or more across all non-placebo doses at week 12 (placebo: 4.4%; 1.2 mg: 46.6%; 1.8 mg dose: 68.5%; and 2.4 mg dose: 57.1% (p<0.001 vs. placebo)).

[0089] As shown in Figure 3 , ALT-801 achieved at least a 30% reduction in liver fat content as measured by MRI-PDFF at week 12 compared with placebo (4.2% of patients) at all doses: 1.2 mg dose: 65% of patients; 1.8 mg: 94.4% of patients; and 2.4 mg dose: 85% of patients, in a significant proportion of subjects who completed 12 weeks of treatment (p<.0001 vs. placebo).

[0090] Figure 3 also shows that ALT-801 achieved at least a 50% reduction in liver fat content as measured by MRI-PDFF at week 12 in a significant proportion of subjects who completed 12 weeks of treatment at all doses (1.2 mg dose: approximately 40% of patients (p<0.001); 1.8 mg dose: 72.2% of patients at 1.8 mg (p<0.0001); and 2.4 mg dose: 70% (p<0.0001)) compared to placebo (0% of patients) at week 12.

[0091] Figure 3 also shows that ALT-801 achieved normalization of liver fat content (<5%) as measured by MRI-PDFF in a significant proportion of subjects who completed 12 weeks of treatment compared to placebo (0%) at week 12: 1.2 mg dose: 20% of patients (p<0.05); 1.8 mg dose: 55.6% of patients (p<0.0001); 2.4 mg dose: 50.0% (p<0.001).

[0092] Figure 4 shows that ALT-801 induced significant weight loss at all doses in non-diabetic subjects compared to placebo (0.2%) at week 12: 3.4% for the 1.2 mg dose (p<0.001); 4.9% for the 1.8 mg dose (p<0.001); and 3.5% for the 2.4 mg dose (p<0.001).

[0093] Figure 4 also shows that ALT-801 induced weight loss at all doses in diabetic subjects at week 12 compared to placebo (0.5%): 3.3% for the 1.2 mg dose (p<0.005); 3.8% for the 1.8 mg dose (p<0.05); and 4.4% for the 2.4 mg dose (p<0.001).

[0094] Figure 4 also shows that ALT-801 induced significant weight loss at all doses in all subjects compared to placebo (0.2%) at week 12: 3.4% for the 1.2 mg dose (p<0.001); 4.3% for the 1.8 mg dose (p<0.001); and 3.7% for the 2.4 mg dose (p<0.001).

[0095] Figure 5 shows that ALT-801 induced an ALT reduction of at least 10% in all subjects at the 1.2 mg dose (-11.2%); 1.8 mg dose (-13.8%, p<0.05); and 1.8 mg dose (-13.6%, p<0.05) compared to placebo (-6.2%) at week 12.

[0096] Figure 5 also shows that ALT-801 induced significant ALT reductions in subjects with baseline ALT of 30 IU / L or greater at the 1.2 mg dose (-17.8%); 1.8 mg dose (-20.8%); and 2.4 mg dose (-27%, p<0.005) compared to placebo (-12.6%) after 12 weeks in all subjects (n=83).

[0097] Figure 6 shows that ALT-801 induces a proportion of subjects with an iron-corrected T1 (cT1) of 80 ms or greater at the 1.2 mg dose (87.5%); 1.8 mg dose (83.3%), and 2.4 mg dose (85.7%) compared to placebo (0%) after 12 weeks.

[0098] Thus, provided herein is a method of using pembidutide to reduce weight in a human having fatty liver, comprising administering pembidutide in an amount of at least 1.8 mg to 2.4 mg once a week to a human in need thereof, wherein the human may (or may not) have type 2 diabetes, and the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In certain embodiments, provided herein is a method of using pembidutide to reduce weight in a human having NAFLD or NASH (e.g., an overweight or obese subject), comprising administering pembidutide in an amount of at least 1.8 mg to 2.4 mg once a week to a human in need thereof, wherein the human does not have type 2 diabetes (e.g., is non-diabetic).

[0099] Serum lipids were not significantly altered in subjects following ALT-801 administration after 12 weeks. Without dose titration, symptoms experienced by subjects after 12 weeks of treatment with ALT-801 were primarily mild and transient in nature, consistent with known GLP-1 class effects. No serious adverse events (AEs) were observed after 12 weeks of treatment with ALT-801. Furthermore, AEs leading to treatment discontinuation were observed in very low rates following 12 weeks of ALT-801 administration. Additionally, mean serum alanine aminotransferase (ALT) levels decreased in all subjects; in subjects with baseline serum ALT levels greater than 30 IU / L, levels decreased by more than 17 IU / L at all dose levels and by 27.0 IU / L in the 2.4 mg dose cohort. No clinically significant ALT elevations (defined as an elevation greater than 3x the upper limit of normal) were observed at 12 weeks. Glycemic control was unaffected with no clinically meaningful changes in HbA1c or fasting glucose at 12 weeks.

[0100] At the end of this 12-week clinical trial, 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 superior placebo-adjusted weight loss (4.7%) to semaglutide (Wegovy; a GLP-1 agonist) over 12 weeks in non-diabetic subjects, and superior placebo-adjusted weight loss (4.5%) to tirzepatide (a GIP / GLP-1 agonist) over 12 weeks in diabetic subjects. This study was conducted without supplemental diet and exercise interventions, which are standard in obesity trials. Additionally, no severe or serious AEs were observed (Figure 7), no ALT elevations of 3-fold or greater were observed, very few AEs resulted in treatment discontinuation, including 1 (4.3%) in the 1.8 mg ALT-801 arm and 1 (4.2%) in the 2.4 mg ALT-801 arm (both secondary to gastrointestinal intolerance), and only 1-3 beats per minute (bpm) increases in heart rate (HR) were observed, which is within the range of other GLP-1 agents used in humans.

[0101] Example 2 Effects of pembidutide (ALT-801), a GLP-1 / glucagon dual receptor agonist, on pathogenic lipid mediators The potential for cardiovascular (CV) risk reduction through incretin-based therapies is receiving increasing attention. Pembidutide is a long-acting GLP-1 / glucagon (1:1) dual receptor agonist under development for the treatment of NASH and obesity. Pembidutide combines the appetite-suppressing effects of GLP-1 receptor antagonism (RA), which is associated with high energy expenditure, with the lipid-lowering effects of glucagon RA. Plasma lipids perform numerous functions in biological systems, such as energy storage, metabolic regulation, signal transduction, proliferation, and apoptosis. The plasma lipidome can be analyzed using nuclear magnetic resonance (NMR) and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS).

[0102] To examine the lipid-lowering effects of pembidutide, we performed an analysis of data from a phase I clinical trial (NCT0456124). 2 ) subjects were randomized at a single site in Australia (NCT0456124). Subjects were randomized 4:1 to pembidutide:placebo with pooled placebo. Pembidutide doses were 1.2 mg, 1.8 mg, and 2.4 mg, administered weekly for 12 weeks without dose titration or supportive lifestyle intervention (no diet or exercise intervention). Pembidutide was well tolerated at all dose levels without dose titration. All AEs in these groups were mild or moderate in severity; no grade 3 (severe) AEs were observed, and no SAEs or AEs leading to treatment discontinuation were reported. Lipoprotein and glycoprotein profiling, covering 33 lipoprotein-related parameters, was performed on fasting plasma samples obtained on days -1 (baseline), 43, and 84 from 34 subjects who completed NCT0456124. 1Lipid profiling, covering 600 lipid species, was performed by ultra-performance liquid chromatography-mass spectrometry on fasting plasma samples obtained on days -1 (baseline), 43, and 84 from 34 subjects who completed NCT0456124. For lipid profiling, plasma fractionation was performed either with methanol, which extracts fatty acyls, bile acids, steroids, and lyso-glycerophospholipids, or with a mixture of chloroform and methanol, which extracts glycerolipids, cholesteryl 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).

[0103] Consistent with the data presented in Example 1, the data presented here show that pembidutide has favorable effects on weight loss, body mass index (BMI), blood pressure, total cholesterol, LDL cholesterol, triglycerides, and apoprotein B, as summarized in Table 6.

[0104] [Table 5]

[0105] Pembidutide also generally induces consistent, favorable changes in lipoprotein particle subpopulations as determined by 2D-NMR analysis (see Figure 9). The color code represents log2 (robust fold change), with blue indicating decreased lipoproteins (negative fold change) and red indicating increased lipoproteins (positive fold change). Figure 9 shows that the number of VLDL and LDL particles, as well as the number of smaller HDL particles, tend to decrease following treatment with pembidutide.

[0106] From the changes observed in total cholesterol, triglycerides, and lipoproteins, an assessment of serum lipid composition encompassing 600 lipid species was performed on days -1 (baseline), 43, and 84 from 34 subjects who completed NCT0456124. Results are presented in Figure 10 as log2 vs. fold change relative to baseline, with blue indicating lipid reduction and red indicating increase. Gray / black bars indicate significant p-values ​​from the Wilcoxon test. Within 12 weeks of treatment, pembidutide significantly reduced serum lipid levels, particularly glycerolipids (diglycerides and triglycerides), glycerophospholipids (phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine), and sphingolipids (ceramides and sphingomyelin), which are associated with reduced cardiovascular (CV) risk and reduced insulin resistance (see Figure 10). Thus, at day 85, substantial and highly statistically significant reductions across multiple bioactive lipid classes were observed compared to placebo.

[0107] Based on the lipodomics data, a volcano plot was generated, as shown in Figure 11, to highlight changes in atherogenic lipids following 1.8 mg pemvidutide treatment. The horizontal dashed line represents the lower limit of significance, and the vertical lines indicate fold changes from baseline of ±0.75 on a log2 scale. The upper left quadrant shows particularly meaningful changes in atherogenic lipids. Pemvidutide treatment significantly reduced proatherogenic lyso-PC levels (see Figure 11), suggesting a potential reduction in oxidized LDL (Law et al., Int. J. Mol. Sci. 2019;20(5):1149). Pemvidutide treatment also significantly reduced atherogenic plaque forming phosphatidyl-ethanolamine (PE) glycerophospholipid levels. Based on these findings, pemvidutide shows promise as an agent for reducing cardiovascular (CV) risk.

[0108] In this study, pembidutide induced substantial weight loss at 12 weeks, as shown in Figures 12A and 12B. At the 1.8 mg dose level, a maximum average weight loss of 10.3% (8.7% adjusted placebo) was observed, with a high degree of statistical significance after only 12 weeks of weekly treatment. In addition, within this treatment group, all subjects experienced a weight loss of at least 5%, and more than half of the subjects achieved a weight loss of at least 10%.

[0109] Pembidutide was shown to be safe and well-tolerated in a Phase 1a study without dose titration. In this study, mean weight loss of up to 10.3% was observed, accompanied by statistically significant reductions across multiple atherogenic lipid classes and particle concentration sizes in atherogenic small and intermediate lipoproteins. Based on these findings, pembidutide shows promise as an agent for inducing weight loss independent of type 2 diabetes diagnosis and simultaneously improving obesity comorbidities, including lowering CV risk factors (e.g., pathogenic serum lipid mediators).

[0110] Example 3 A Phase 1b, 24-Week, Randomized, Double-Blind, Placebo-Controlled Study of ALT-801 (Pembidutide) in Diabetic and Non-Diabetic Overweight and Obese Subjects With Nonalcoholic Fatty Liver Disease (NAFLD) NAFLD (defined as liver fat content (LFC) by MRI-PDFF ≥ 10%; absence of significant fibrosis, defined as FibroScan® LSM < 10 kPa), diabetic and non-diabetic (type 2 diabetes) overweight and obese (28.0 kg / m ) with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) laboratory values ​​≤ 75 IU / L and HbA1c < 9.5. 2Provided herein is a disclosure of a 12-week extension study of the study shown in Example 1, providing a 24-week Phase 1b, randomized, double-blind, placebo-controlled study evaluating the efficacy of pembidutide (also referred to herein as ALT-801) in subjects with a BMI of ≥ 18 years, the safety and efficacy of pembidutide in weight loss, liver fat fraction, anthropometric parameters, lipid metabolism, and markers of inflammation, fibrosis, and other conditions. Men and women aged 18 to 65 years were studied. Diabetic subjects were defined as those receiving stable doses (≥ 3 months) of metformin or SLGT-2 therapy and not using insulin, sulfonylurea, DPP-4, or GLP-1 treatment. Ninety-four subjects were enrolled in the initial 12-week Phase 1b NAFLD study, and 83 subjects who completed were invited to continue blinded treatment for a total of 12 additional weeks. Of these subjects, 66 consented to rollover, and 64 were eligible to participate. The study design is shown in Figure 13.

[0111] The primary endpoint of this study was a reduction in liver fat content (LFC) by MRI-PDFF at week 24 compared to week 0. Key secondary endpoints included percent weight loss at week 24 compared to week 0 and liver inflammation by alanine aminotransferase (ALT) levels and corrected T1 (cT1) imaging at week 24 compared to week 0. Adverse events (AEs), including serious and severe 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 participants in this study are shown in Figure 14.

[0112] As shown in Figure 15, we observed robust reductions in liver fat content as determined by MRI-PDFF at week 24. As shown here, significant (p<0.001) reductions in liver fat content were observed for all pembidutide doses administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo, both in absolute and relative terms. In terms of absolute reductions, the 1.2, 1.8, and 2.4 mg doses demonstrated mean absolute reductions in liver fat of 11.2%, 17%, and 15.6%, respectively (p<0.001). In terms of relative reductions, the 1.2, 1.8, and 2.4 mg doses demonstrated mean relative reductions in liver fat of 56.3%, 75.2%, and 76.4%, respectively (p<0.001).

[0113] Figure 16 also shows the percentage of subjects who achieved at least 30% and 50% reduction in liver fat content and normalized liver fat content (5% or less) at 24 weeks. As shown here, the percentage of subjects who achieved at least 30% and 50% reduction in liver fat content was highly significant for all administered doses of pembidutide (1.2 mg, 1.8 mg, and 2.4 mg per week) compared with placebo (p<0.001 or p<0.0001 as shown here). In addition, normalization of liver fat content was achieved in a high proportion of subjects, especially in the 1.8 mg dose (53.8%) and 2.4 mg dose (45.3%), and was highly significant for the 1.8 mg and 2.4 mg doses compared with placebo (p<0.001 or p<0.01).

[0114] Figure 17 shows significant liver delipidation as determined by MRI-PDFF at week 24 (pembidutide, 1.8 mg dose). The reduction in liver volume in this scan for an exemplary subject receiving pembidutide was significant (32.3% at baseline compared to 1.7% at week 24).

[0115] Figure 18 shows the robust reduction in liver volume as determined by MRI-PDFF at week 24, measured as both absolute and relative reductions. As shown, significant (p<0.05 or p<0.001 as shown) reductions in liver fat content were observed for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo, both in absolute and relative terms.

[0116] Figure 19 shows a robust reduction in ALT levels, a biomarker of liver inflammation, in all subjects at week 24, with even more pronounced reductions in subjects with baseline ALT of 30 IU / L or greater. As shown here, for example, a significant (p<0.001) reduction in ALT was observed for all administered pembidutide doses (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo in all subjects and in subjects with baseline ALT of 30 IU / L or greater.

[0117] Figure 20 shows a high rate of cT1 response at 24 weeks. The response threshold is defined as an 80 millisecond (ms) decrease in CT1 compared to baseline. As shown here, for example, significant (p<0.05 or p<0.005, as shown here) cT1 responses were observed for all administered doses of pembidutide (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo. It is noted that an 80 ms decrease in cT1 is associated with an approximate 2-point decrease in the NASH Activity Score (NAS) (Dennis, A., Front. Endocrinology, 2021), and elevated 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).

[0118] Figure 21 shows the sustained weight loss at week 24 compared to week 12, distinguishing pembidutide from other NASH medications with comparable levels of liver fat reduction. As shown here, a significant (p<0.005 or p<0.001 as shown here) reduction in body weight for all administered doses of pembidutide (1.2 mg, 1.8 mg, and 2.4 mg per week) compared to placebo was observed in non-diabetic patients as well as all subjects. Mean weight loss was also observed in diabetic patients (see the "Diabetes" panel in Figure 21).

[0119] Figure 22 shows improvements in serum lipids at week 24. As shown there, for example, improvements in serum lipids were observed for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week).

[0120] Figure 23 shows that all doses of pembidutide tested induced improvements in blood pressure without a clinically meaningful increase in heart rate at 24 weeks. As shown here, for example, such improvements in blood pressure were observed for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week). It is noted that the reduction in systolic blood pressure was significant (p<0.05) at the 2.4 mg per week dose.

[0121] Figure 24 shows a safety summary of the study. The number of serious or severe adverse events in the treatment groups was low and comparable to that in the placebo group. Serious and severe AEs were the same events and included 1) chest pain after elective coronary stent placement (placebo), 2) Salmonella infection (pembidutide, 1.2 mg), and 3) hypertension 3 weeks or more after the last dose of study drug (pembidutide, 1.8 mg). All of these were unrelated to study drug; only the Salmonella infection led to treatment discontinuation. The two gastrointestinal (GI) AEs leading to treatment discontinuation were mild (Grade 1) abdominal pain in two subjects. No significant ALT elevations were reported. Additionally, the number of adverse events leading to treatment discontinuation and the number of gastrointestinal-related adverse events leading to treatment discontinuation were low across treatment groups. Gastrointestinal-related adverse events, such as nausea, vomiting, diarrhea, and constipation, were mild to moderate in severity.

[0122] Figure 25 shows improvement in glycemic parameters (fasting glucose and HbA1c) in diabetic patients and maintenance of glycemic control in non-diabetic patients at 24 weeks. As shown here, for example, such improvement in glycemic control, including reduction in fasting glucose and HbA1c, was observed for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg per week) in diabetic patients. These clinical effects support the antihyperglycemic role of pembidutide, likely related to its GLP-1 activity, which offsets the hyperglycemic effects of its glucagon activity.

[0123] This study demonstrates that pembidutide induces liver fat reduction, including a relative liver fat reduction of more than 75% at 24 weeks (better or comparable to the effects of other leading NASH candidates), as well as significant reductions in cT1 and serum ALT scores, leading to robust efficacy in NASH clinical trials. This study also demonstrates that pembidutide induces weight loss in non-diabetic subjects (sustained weight loss, reaching 7.2% at 24 weeks) and in diabetic subjects (5.3% weight loss at 24 weeks). This study also demonstrates that pembidutide is safe and tolerable (e.g., low rates of treatment discontinuation, no serious / severe AEs associated with pembidutide; well tolerated without the need for dose titration, consistent with prior experience; no clinically significant elevations in ALT; and glycemic control was maintained, with diabetes achieving reduced fasting glucose and HbA1c).

[0124] Example 4 Interim Efficacy Analysis of a Phase II, 24-Week, Randomized, Double-Blind, Placebo-Controlled Study of ALT-801 (Pembidutide) in Non-Diabetic Obese and Overweight Subjects at Higher Risk for NAFLD and NASH The results presented in this example represent a 24-week interim analysis from a placebo-controlled Phase II trial (ClinicalTrials.gov Identifier: NCT05295875) evaluating pembidutide (ALT-801) in obese and overweight subjects. 70-75% of subjects in this population 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). Key eligibility criteria were: (1) men and women, 18–75 years of age; (2) at least one unsuccessful weight loss event per investigator judgment; (3) a body mass index (BMI) of 30 kg / m2 or greater or a BMI of 27 kg / m2 or greater with at least one obesity-related comorbidity (cardiovascular disease, hypertension, dyslipidemia, history of prediabetes or obstructive sleep apnea), and (4) non-diabetic (HbA1c ≤ 6.5% and fasting glucose ≤ 125 mg / dL). Eligible subjects were randomized 1:1:1:1 to one of the following treatment arms: Group 1 (39 subjects): pembidutide 1.2 mg SC once weekly for 24 weeks; Group 2 (40 subjects): pembidutide 1.8 mg SC once weekly for 24 weeks; Group 3 (40 subjects): pembidutide 0.6 mg SC for 1 week, 1.2 mg SC for 1 week, 1.8 mg SC once weekly for 2 weeks, followed by 2.4 mg SC once weekly for an additional 20 weeks (in that order); and Group 4 (41 subjects): placebo SC once weekly for 24 weeks. Randomization was stratified based on sex and baseline body mass index (BMI <35 kg / m² vs. BMI ≥35 kg / m²). At least 25% of randomized subjects were male.All subjects received counseling from qualified health care providers at screening and subsequent visits during the treatment period regarding a reduced-calorie diet of 1200-1500 calories for individuals weighing less than 250 lbs (113.6 kg) and 1500-1800 calories for individuals weighing 250 lbs (113.6 kg) and gradually increasing physical activity (targeting 150 minutes of physical activity per week). Subjects were instructed to record their daily food intake and physical activity, and compliance with the lifestyle intervention was periodically assessed by the investigator. A subgroup of subjects underwent MRI-PDFF to assess liver fat fraction and composition to measure total body adipose tissue (AT) and lean tissue mass (ATFM).

[0125] Baseline characteristics of study participants are shown in Figure 26. Figure 27 shows the mean percentage weight loss over time for all evaluated subjects across the three pemvidutide doses and placebo over 24 weeks. Results are presented as efficacy estimands assuming subjects remain on treatment for the entire period, including missing values ​​handled with mixed models using repeated measures. Figure 27 shows the progressive dose-dependent weight loss achieved with pemvidutide relative to placebo over time. Figure 28 shows the mean percentage weight loss (shown as the efficacy estimand, as described above) achieved over 24 weeks with pemvidutide and placebo in the subgroup of subjects with a baseline weight of 115 kg or less. For the results shown in Figure 27, Figure 28 shows the progressive dose-dependent weight loss achieved with pemvidutide relative to placebo over time. When compared to Figure 27, the results shown in Figure 28 indicate that higher levels of weight loss are achieved with each of the three pemvidutide doses in subjects with low baseline weight. This phenomenon may be related to lower levels of drug exposure in heavier subjects, supporting the possibility of increasing dose levels in this population to maximize efficacy. Figure 29 shows a weight loss responder analysis based on calculation of the percentage of subjects achieving 5% or greater, 10% or greater, and 15% or greater weight loss across the three pemvidutide doses and placebo, respectively. Figure 30 shows the percentage weight loss achieved across the four groups comparing Hispanic subjects to non-Hispanic subjects. Overall, the results indicated a significant trend for Hispanic subjects to lose weight following treatment with pemvidutide, indicating a possible influence of genetic background. Figure 31A shows that 24 weeks of treatment with pemvidutide improves 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. Figure 31B shows that 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg did not induce significant changes in heart rate. Figure 32 shows that serum lipids at 24 weeks were reduced for all doses of pembidutide administered (1.2 mg, 1.8 mg, and 2.4 mg) compared to baseline.Thus, Figure 32 shows the improvement in serum lipids at week 24 for all administered doses of pembidutide (1.2 mg, 1.8 mg, and 2.4 mg per week). Figure 33 shows a significant reduction in waist circumference at week 24 for all administered doses of pembidutide (1.2 mg, 1.8 mg, and 2.4 mg per week). Waist circumference is an index of central or abdominal obesity recommended by the WHO to assess the risk of metabolic diseases such as NAFLD and NASH, as well as cardiovascular disease.

[0126] Thus, provided herein is a method of using pembidutide to reduce weight in a human having high-risk fatty liver, comprising administering pembidutide in an amount of at least 1.8 mg to 2.4 mg once a week to a human in need thereof, wherein the human does not have type 2 diabetes, and the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0127] Other advantages of the reagent and methods of using same will be appreciated by those skilled in the art and are provided herein. While certain embodiments are described as preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. It is therefore intended that the appended claims cover all such equivalent variations that fall within the scope of the following claims.

Claims

1. Use of a peptide product or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical for the treatment of reducing the body weight of a person having fatty liver, wherein the peptide product comprises SEQ ID NO: 1 (pemvidutide) in a dose for administration once a week in an amount of at least about 1.2 mg to about 2.4 mg over a treatment period of at least 12 weeks, wherein the fatty liver is non-alcoholic fatty liver (NAFLD) or non-alcoholic steatohepatitis (NASH).

2. The use according to claim 1, wherein the person's body weight decreases by at least 3% from baseline at 12 weeks.

3. The use according to claim 1, wherein the person's body weight decreases by at least 4% from baseline at 12 weeks.

4. The use according to claim 1, wherein the treatment comprises administering pembidutide at a dose of 1.2 mg once weekly for 24 weeks.

5. The use according to claim 1, wherein the treatment comprises administering pembidutide at a dose of 1.8 mg once a week.

6. The use according to claim 1, wherein the treatment comprises administering pembidutide at a dose of 2.4 mg once a week.

7. The use according to claim 1, wherein the treatment comprises achieving a steady-state dose after a dose-escalating phase having a period of about two weeks, about three weeks, or about four weeks.

8. The use according to claim 1, wherein the person has a body mass index (BMI, kg / m²) of at least 28.

9. The use according to claim 1, wherein the person has a body mass index (BMI, kg / m²) of 30 or more.

10. The use according to claim 1, wherein the human has a level of liver fat measured by MRI-PDFF of 10% or more.

11. The use according to claim 1, wherein the absolute reduction in liver fat, as measured by MRI-PDFF, is about 8% to about 15% after 12 weeks of weekly administration.

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

13. The use according to claim 1, wherein the relative reduction in liver fat, as measured by MRI-PDFF, compared to baseline is from about 30% to about 50% after 24 weeks of once-weekly administration, and the relative reduction is defined as p < 0.001 or p < 0.0001 and is statistically significant.

14. The use according to claim 12, wherein weekly dosing of 1.8 mg and 2.4 mg induces a reduction of at least 40% of liver fat after 24 weeks, the reduction being statistically significant, defined as p < 0.001 or p < 0.01 compared to placebo.

15. The use according to claim 1, wherein liver fat reduction as measured by MRI-PDFF compared to baseline is approximately 30% in the human after 24 weeks of weekly administration of approximately 1.8 mg of pemvidutide compared to baseline.

16. The use according to claim 1, wherein liver volume, as measured by MRI-PDFF compared to baseline, is reduced after 24 weeks of weekly administration of approximately 1.2 mg, approximately 1.8 mg, or approximately 2.4 mg of pembidutide compared to placebo.

17. The use according to claim 1, wherein alanine aminotransferase (ALT) is reduced after weekly administration of approximately 1.2 mg, approximately 1.8 mg, or approximately 2.4 mg for 24 weeks compared to placebo, and optionally the person has a baseline ALT greater than 30 IU / L prior to administration.

18. The use according to claim 1, wherein the reduction in iron-corrected T1 is better than 80 ms in about 80% of the subjects, and the reduction is optionally defined as p < 0.05 or p < 0.005 and is significant.

19. The use according to claim 1, wherein a once-weekly dose of approximately 1.2 mg, approximately 1.8 mg, or approximately 2.4 mg per week for 24 weeks results in weight loss in non-diabetic and / or diabetic patients compared to placebo.

20. The use according to claim 1, wherein a weekly dose of approximately 1.2 mg, approximately 1.8 mg, or approximately 2.4 mg per week for 24 weeks results in a reduction in serum lipid levels compared to placebo.

21. The use according to claim 1, wherein weekly administration of approximately 1.2 mg, approximately 1.8 mg, or approximately 2.4 mg per week for 24 weeks lowers blood pressure without significantly increasing heart rate, and optionally, weekly administration of 2.4 mg significantly lowers systolic blood pressure as defined by p < 0.

05.

22. The use according to claim 1, wherein a once-weekly dose of approximately 1.2 mg, approximately 1.8 mg, or approximately 2.4 mg per week for 24 weeks improves a blood glucose parameter, optionally the blood glucose parameter being a decrease in fasting glucose and / or HbA1c levels.

23. Use of a peptide product or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical for a treatment to reduce the hepatic fat content determined by MRI-PDFF in a person having fatty liver, wherein the peptide product comprises SEQ ID NO: 1 (pembidutide) in a dose for administration to the person once a week in an amount of at least about 1.2 mg to about 2.4 mg for a continuous treatment period of at least 12 weeks, wherein the person If you have been diagnosed with fatty liver disease, specifically non-alcoholic fatty liver (NAFLD) or non-alcoholic steatohepatitis (NASH), Having a body mass index (BMI, kg / m²) higher than approximately 28, Use of a liver with a fat content of at least approximately 10%, as measured by MRI-PDFF.

24. The use according to claim 23, 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 human population, which is significant compared to placebo at 12 weeks (p < 0.001).

25. The use according to claim 23, 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, which is significant compared to placebo at 12 weeks (p < 0.001).

26. The use according to claim 23, 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 human population, which is significant compared to placebo at 12 weeks (p < 0.001).

27. ​​The use according to claim 23, 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, which is significant compared to placebo at 12 weeks (p < 0.001).

28. The use according to claim 23, wherein a dose of approximately 1.8 mg per week induces a relative reduction in liver fat content of at least approximately 60%, optionally at least approximately 65%, in a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

29. The use according to claim 23, wherein a dose of approximately 2.4 mg per week induces a relative reduction in liver fat content of at least approximately 50%, optionally at least approximately 55%, in a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

30. The use according to claim 23, wherein a dose of about 1.2 mg per week induces a reduction of at least about 30% in liver fat content in at least about 55%, optionally at least about 60%, of a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

31. The use according to claim 23, wherein a dose of about 1.8 mg per week induces a reduction of at least about 30% in liver fat content in at least about 80%, optionally at least about 90%, of a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

32. The use according to claim 23, wherein a dose of approximately 2.4 mg per week induces a reduction of at least approximately 30% in liver fat content in at least approximately 75%, optionally at least approximately 85%, of a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

33. The use according to claim 23, wherein a dose of about 1.2 mg per week induces a reduction of at least about 50% in liver fat content in at least about 35%, optionally at least about 40%, of a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

34. The use according to claim 23, wherein a dose of approximately 1.8 mg per week induces a reduction of approximately 50% in liver fat content in at least approximately 60%, optionally at least approximately 70%, of a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

35. The use according to claim 23, wherein a dose of approximately 2.4 mg per week induces a reduction of at least approximately 50% in liver fat content in at least approximately 60%, optionally at least approximately 70%, of a human population, which is significant compared to placebo at 12 weeks (p < 0.001).

36. The use according to claim 23, wherein a dose of about 1.2 mg per week induces normalization of liver fat content to about 5% or less in at least about 20% of a human population, and is significant compared to placebo at 12 weeks (p < 0.05).

37. The use according to claim 23, wherein a dose of approximately 1.8 mg per week induces normalization of liver fat content to approximately 5% or less in at least approximately 50%, optionally at least approximately 55%, of a human population, and is significant compared to placebo at 12 weeks (p < 0.0001).

38. The use according to claim 23, wherein a dose of approximately 2.4 mg per week induces normalization of liver fat content to approximately 5% or less in at least approximately 50% of a human population, and is significant compared to placebo at 12 weeks (p < 0.001).

39. Use of a peptide product or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical for a treatment to induce weight loss in a person having fatty liver, wherein the peptide product comprises SEQ ID NO: 1 (pembidutide) in a dose for administration to the person once a week in an amount of at least about 1.2 mg to about 2.4 mg for a continuous treatment period of at least 12 weeks, wherein the person If you have been diagnosed with fatty liver disease, specifically non-alcoholic fatty liver (NAFLD) or non-alcoholic steatohepatitis (NASH), Having a body mass index (BMI, kg / m²) higher than approximately 28, He has been voluntarily diagnosed with type 2 diabetes. Use of a liver with a fat content of at least approximately 10%, as measured by MRI-PDFF.

40. The use according to claim 39, wherein the person has not been diagnosed with type 2 diabetes, and a dose of about 1.2 mg per week reduces the person's body weight by at least about 2.5%, optionally at least about 3%, which is significant compared to placebo at 12 weeks (p < 0.001).

41. The use according to claim 39, wherein the person has not been diagnosed with type 2 diabetes, and a dose of about 1.8 mg per week reduces the person's body weight by at least about 4%, optionally about 5%, and is significant compared to placebo at 12 weeks (p < 0.001).

42. The use according to claim 39, wherein the person has not been diagnosed with type 2 diabetes, and a dose of about 2.4 mg per week reduces the person's body weight by at least about 2.5%, optionally about 3.5%, which is significant compared to placebo at 12 weeks (p < 0.001).

43. The use according to claim 39, wherein the person has been diagnosed with type 2 diabetes, and a dose of about 1.2 mg per week reduces the person's body weight by at least about 2%, optionally at least about 3%, at 12 weeks, which is significant compared to placebo (p < 0.05).

44. The use according to claim 39, wherein the person has been diagnosed with type 2 diabetes, and a dose of about 1.8 mg per week reduces the person's body weight by about 2.5%, optionally at least about 3.5%, which is significant compared to placebo at 12 weeks (p < 0.005).

45. The use according to claim 39, wherein the person has been diagnosed with type 2 diabetes, and a dose of about 2.4 mg per week reduces the person's body weight by about 3%, optionally at least about 4%, and is statistically significant compared to placebo at 12 weeks (p < 0.001).

46. The use according to claim 39, wherein a dose of approximately 1.2 mg per week reduces the body weight of a human population by at least approximately 3%, and is significant compared to placebo at 12 weeks (p < 0.001).

47. The use according to claim 39, wherein a dose of approximately 1.8 mg per week reduces the body weight of a human population by at least approximately 4%, and is significant compared to placebo at 12 weeks (p < 0.001).

48. The use according to claim 39, wherein a dose of approximately 2.4 mg per week reduces the body weight of a human population by at least approximately 3%, optionally at least approximately 3.5%, and is significant compared to placebo at 12 weeks (p < 0.001).

49. The use according to any one of claims 1 to 48, wherein a dose of about 1.2 mg per week induces at least about 11% ALT reduction in a human population at 12 weeks.

50. The use according to any one of claims 1 to 48, wherein a dose of about 1.8 mg per week induces at least about 13% ALT reduction in a human population, which is significant compared to placebo at 12 weeks (p < 0.05).

51. The use according to any one of claims 1 to 48, wherein a dose of approximately 2.4 mg per week induces at least approximately 13% ALT reduction in a human population, which is significant compared to placebo at 12 weeks (p < 0.05).

52. The use according to any one of claims 1 to 48, wherein a dose of about 1.2 mg per week induces at least about 18% ALT reduction at week 12 in a population of humans having a baseline ALT of about 30 IU / L or higher.

53. The use according to any one of claims 1 to 48, wherein a dose of about 1.8 mg per week induces at least about 20% ALT reduction at week 12 in a population of humans having a baseline ALT of about 30 IU / L or higher.

54. The use according to any one of claims 1 to 48, wherein a dose of about 2.4 mg per week induces at least about 20%, optionally at least about 25%, of ALT levels at 12 weeks in a population of humans having a baseline ALT of about 30 IU / L or higher, which is statistically significant compared to placebo (p < 0.005).

55. The use according to any one of claims 1 to 48, wherein the treatment comprises the administration of pembidutide by parenteral injection.

56. The use according to any one of claims 1 to 48, wherein the treatment includes administration of pemvidutide by subcutaneous injection.

57. The use according to any one of claims 1 to 48, wherein the person has a body mass index (BMI, kg / m²) of at least 28.

58. The use according to any one of claims 1 to 48, wherein the person has a body mass index (BMI, kg / m²) of 30 or more.

59. The use according to any one of claims 1 to 48, wherein the human has a level of liver fat measured by MRI-PDFF of 10% or more before weekly administration of the drug.

60. The use according to any one of claims 1 to 48, wherein the absolute reduction in liver fat, as determined using MRI-PDFF, is about 8% to about 15% after 12 weeks of weekly administration.

61. The use according to any one of claims 1 to 48, wherein the normalization of liver fat content to 5% or less is from 20% to more than 55% of the population after 12 weeks of weekly administration.

62. The use according to any one of claims 1 to 48, wherein the relative reduction in liver fat, as determined using MRI-PDFF relative to baseline, is from about 40% to more than about 60% after 12 weeks of weekly administration.

63. The use according to any one of claims 1 to 48, wherein the ALT reduction is in more than 13% to about 25% of the population after 12 weeks of once-weekly administration.

64. The use according to any one of claims 1 to 48, wherein the treatment comprises achieving a steady-state dose after a dose-escalating phase having a period of 2 to 4 weeks.

65. The use according to any one of claims 1 to 48, wherein the treatment comprises administering the pembidutide from a liquid formulation containing at least about 2.5 mg / ml of pembidutide.

66. The use according to any one of claims 1 to 48, wherein administration of pembidutide induces a significant decrease in serum lipids and / or small to medium LDL particle concentrations that lead to atheroma.

67. The use according to claim 66, wherein the small to medium-sized LDL particle concentrations associated with atheroma development decrease by at least -0.2 log 2 times compared to placebo following administration of pembidutide for 43 days and / or 84 days.

68. The use according to any one of claims 1 to 48, wherein the total serum triglyceride concentration of the subject, as measured by 2D-NMR, decreases by at least -0.2 log 2 times compared to placebo following administration of pembidutide for 43 days and / or 84 days.

69. The use according to claim 66, wherein the serum lipid is selected from glycerolipids, sterols, glycerophospholipids, and sphingolipids, and optionally the decrease is at least a -0.2 log 2-fold change.

70. The use according to any one of claims 1 to 48, wherein a decrease in serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, and / or lysophosphatidylcholine follows administration of pembidutide.

71. Use of a peptide product or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of fatty liver in overweight or obese humans, wherein the peptide product comprises SEQ ID NO: 1 (pemvidutide) in a dose for administration to the human at an amount of at least about 1.2 mg to about 2.4 mg once weekly for a treatment period of at least 12 consecutive weeks, wherein the fatty liver is non-alcoholic fatty liver (NAFLD) or non-alcoholic steatohepatitis (NASH).

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