Therapeutic regimens and methods for the treatment of cardiovascular risk factors using GLP-1R and GCGR agonists - Patent Application 20070122999

Abstract

The present disclosure relates to once-weekly dosing regimens, formulations, and methods of using dual agonists of GLP-1R and GCGR to treat cardiovascular diseases / disorders and / or reduce their risk factors by inducing a reduction in pathogenic serum lipid mediators in humans, who may or may not have other comorbidities such as obesity and / or type 2 diabetes. The dual agonist of GLP-1R and GCGR includes the peptide product of SEQ ID NO: 1 (pembidutide).

Description

[Technical Field]

[0001] Related Applications This application claims priority to Ser. No. 63 / 422,983, filed November 5, 2022, and Ser. No. 63 / 490,491, filed March 15, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format via the USPTO Patent Center and is incorporated herein by reference in its entirety. The ASCII copy, created on November 5, 2023, is named MED013US1-PCT_ST26.xml and is 4,453 bytes in size.

[0003] Field of the Disclosure The present disclosure relates to the use of a composition comprising SEQ ID NO: 1, the GLP-1R and GCGR agonist pembidutide (ALT-801), in specific dosing regimens for the treatment of obesity comorbidities, including cardiovascular (CV) disease / risk factors, by reducing pathogenic plasma lipid mediators. [Background technology]

[0004] The increasing prevalence of cardiovascular (CV) disease and CV disease mortality in various human populations, including overweight and / or obese individuals, is a global health crisis of epidemic proportions that is a major contributor to patient morbidity and mortality as well as a major economic burden. For example, obesity (e.g., a body mass index (BMI, BMI kg / m) greater than 25 (overweight) or 30 (obese)) is a leading cause of death. 2Obesity is a rapidly growing problem worldwide, with overweight now accounting for over 65% of adults in the United States. Furthermore, over 80% of patients with heart failure with preserved ejection fraction (HFpEF) are overweight or obese (Kitzman et al., Effect of caloric restriction or aerobic exercise training on peak oxygen consumption and quality of life in obese older patients with heart failure with preserved ejection fraction: A randomized clinical trial. JAMA. 2016;315(1):36-46). In a study of 100 elderly patients (67+ / -5 years) with obesity and clinically stable HFpEF, a 7% weight loss was associated with increased exercise tolerance and improved other measures of diastolic cardiac function (Id.; https: / / www.rethinkobesity.com / disease-progression / comorbidities-of-obesity.html). The majority of individuals considered overweight or obese have limited available U.S. Food and Drug Administration (FDA)-approved pharmacological drug options for inducing weight loss, and treatment 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.

[0005] 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 overweight patients. While GLP-1RAs exert central effects on appetite and food intake, GCGR agonists (GCGRAs) drive increased energy expenditure and lipid oxidation in animal models and humans. The actions of GCGRAs and GLP-1RAs have been shown to be synergistic in driving greater weight loss compared with GLP-1RAs alone. GCGRAs also promote lipolysis and inhibit hepatic lipogenesis, providing additional pathways for obesity-related conditions such as hepatic fat reduction and / or resolution or amelioration of nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), both of which are associated with overweight, particularly obesity. However, such agonists have not yet been shown to have any effect on CV risk factors, such as serum lipid levels or ratios.

[0006] 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 improved NASH-related alanine aminotransferase (ALT) reduction compared with 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 liver fat reduction but suboptimal for weight loss. Balanced (1:1) antagonism has been shown to be associated with greater weight loss and metabolic benefits than a skewed ratio favoring one agonist over the other. A recent study with JNJ 64565111, a balanced dual agonist, achieved an impressive 8% weight loss in just 12 weeks (NCT03586830). However, it is unclear whether the observed weight loss is related to any CV disease-related factors (e.g., elevated cholesterol, triglyceride, LDL, and VLDL particle concentrations and / or diameters, and other relevant parameters).

[0007] Unfortunately, GLP-1Ra and dual receptor agonists based on GLP-1R and GLP-1 with a bias towards 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 to prevent and / or treat conditions related to CV disease that do not require long-term (e.g., more than 4 weeks) titration to reach therapeutic levels without gastrointestinal side effects.The compounds and methods presented herein provide a solution to this problem, and have been shown to have beneficial effects on factors related to CV disease, in particular.

[0008] Summary of the Disclosure Disclosed herein are dual agonist peptides and their products (e.g., formulations), and their uses for treating cardiovascular (CV)-related diseases (e.g., disorders) related to the function of the glucagon-like peptide 1 receptor (GLP-1R) and glucagon receptor (GCGR) to induce beneficial effects on factors associated with CV disease, such as elevated cholesterol, triglyceride, LDL, and / or VLDL particle concentration and / or diameter, and / or other related parameters (e.g., serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine). In certain embodiments, the method reduces, prevents, and / or normalizes factors associated with CV disease, and comprises administering pembidutide to a human in need thereof once weekly in an amount ranging from a minimum of 1.2 mg to a maximum of 2.4 mg. In several embodiments, a person's body weight is reduced by at least 3%, or at least 4% from baseline at 12 weeks, and conditions associated with CV disease, such as elevated cholesterol, triglycerides, LDL, and / or VLDL particle concentrations and / or diameters, and / or other related parameters, are also reduced or normalized (e.g., preferably reduced toward lower and / or normal levels).

[0009] In certain embodiments, the human is overweight, obese, and / or 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 25, 27, 30, or more. 2). In certain embodiments, administration of pemvidutide disclosed herein results in weight loss and / or a reduction in waist circumference measurement. In preferred embodiments, conditions associated with CV disease, including but not limited to elevated cholesterol, triglyceride, LDL, and / or VLDL particle concentrations and / or diameters, weight loss, and / or a reduction in waist circumference measurement, when measured, are significant relative to untreated or control (e.g., vehicle-treated) individuals and / or populations. In several embodiments, pemvidutide is administered once weekly in an amount of 1.2 mg or once weekly 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. Some preferred embodiments are summarized in the aspects presented below. As will be understood by one of ordinary skill in the art, other aspects of the present disclosure are also contemplated. [Brief explanation of the drawings]

[0010] [Figure 1] Metabolite extraction, UHPLC-MS analysis, and data processing are described. [Figure 2] Figure 1 shows changes in serum lipoproteins analyzed by 2D-MDR 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). See Example 1. [Figure 3]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) are shown, comparing 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). Pembidutide treatment significantly reduced serum lipid levels, particularly glycerolipids, glycerophospholipids, and sphingolipids, within 12 weeks of treatment. [Figure 4] Volcano plots of changes in atherogenic lipid species following pemvidutide treatment at a 1.8 mg dose are shown. The Y-axis represents the Log10 statistical change based on a Student's T-test, with a horizontal dashed line corresponding to a threshold for a 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. Pemvidutide treatment significantly reduced proatherogenic lyso-PC levels, suggesting a possible reduction in oxidized LDL. Pemvidutide treatment significantly reduced lyso-phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which form atherosclerotic plaques. [Figure 5A] Describe the significant weight loss induced by weekly pembidutide administration for 12 weeks compared to placebo. [Figure 5B] Describe a weight loss of 5% or more or 10% or more induced by weekly pembidutide administration for 12 weeks compared to placebo. [Figure 6]

[0023] Figure 1 shows baseline characteristics of study participants in a Phase II study. See Example 2. [Figure 7]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 8] Systolic and diastolic blood pressure expressed as mean percentage change from baseline (FIG. 8A) and heart rate expressed as mean percentage change from baseline (FIG. 8B) are shown after 24 weeks of treatment with pembidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg and placebo. [Figure 9] 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 10] Figure 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 2. [Figure 11] The effects of pembidutide and vehicle on % weight loss (FIG. 11A), total cholesterol (FIG. 11B), LDL (FIG. 11C), HDL (FIG. 11D), and triglycerides (FIG. 11E) measured in diet-induced obese hamsters are shown. See Example 3. [Figure 12] The effects of pembidutide and vehicle on liver weight (FIG. 12A), liver fatty acids (FIG. 12B), liver triglycerides (FIG. 12C), and liver cholesterol (FIG. 12D) measured in diet-induced obese hamsters 72 hours after injection of labeled macrophages are shown. See Example 3. [Figure 13]The effects of pembidutide and vehicle on fecal weight measured in grams (FIG. 13A), % of injected dose in fecal cholesterol per gram of feces (FIG. 13B), % of injected dose in fecal bile acids per gram of feces (FIG. 13C), % of injected dose in fecal cholesterol per total feces (FIG. 13D), and % of injected dose in fecal bile acids per total feces (FIG. 13E) measured in diet-induced obese hamsters treated after injection of labeled macrophages are shown. See Example 3. [Figure 14] 1 shows the effect of pembidutide and vehicle on % of injected dose in plasma cholesterol in diet-induced obese hamsters 72 hours after injection of labeled macrophages. See Example 3. [Figure 15]

[0023] Figure 1 shows the effect of pembidutide on hepatic expression of CYP7A, ABCA1, ABCG1, ABCG5, ABCG8, SRB1, LDL-R or SREBP1c compared to semaglutide or vehicle. See Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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 thereto, as can be determined using a cellular assay. In preferred embodiments, the disclosure provides methods for using pembidutide (e.g., a pharmaceutical dosage formulation preferably comprising SEQ ID NO: 1) to prevent and / or treat cardiovascular (CV) disease and / or CV disease-related conditions, symptoms, causes, and / or risks when administered to a human. In some embodiments, the human is overweight or obese (e.g., has a body mass index of at least about 25), and may or may not have type 2 diabetes. In preferred embodiments, administration of pembidutide disclosed herein results in weight loss and / or a decrease in waist circumference ("waist circumference") measurement. In preferred embodiments, CV disease-related conditions, including but not limited to elevated cholesterol, triglyceride, LDL, and / or VLDL particle concentrations and / or diameters, weight loss, and / or waist circumference measurements, as measured, are significant relative to untreated or control (e.g., vehicle-treated) individuals and / or populations. In several embodiments, pembidutide is administered once weekly in an amount of 1.2 mg or once weekly in an amount of 2.4 mg. In certain embodiments, 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. In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to prevent and / or reduce conditions and / or parameters associated with CV disease-related conditions, including, but not limited to, reducing and / or normalizing serum cholesterol, triglycerides, VLDL / LDL particle concentrations, and / or other parameters (e.g., serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine) in humans.In some preferred embodiments, the method comprises inducing weight loss in a human. In some embodiments, the CV disease includes, but is not limited to, heart failure with preserved ejection fraction (HFpEF), coronary heart disease, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, and / or deep vein thrombosis and pulmonary embolism. In preferred embodiments, the method comprises administering pembidutide to a human in need thereof once weekly in an amount of at least about 1.2 mg, 1.8 mg, or 2.4 mg pembidutide. In certain embodiments, the method comprises administering pembidutide to a human in need thereof once weekly in an amount of 1.8 mg for the treatment of CV disease / disorder by inducing a reduction in pathogenic serum 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).

[0012] Pemvidutide, 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 the two natural hormones GLP-1 and glucagon, primarily with an 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 non-proteinogenic 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 octadecane fatty acid side chain. The surfactant side chain appears to enter the circulation slowly and can form micelles after subcutaneous (SC) injection. The slower entry associated with a lower maximum concentration ( Cmax ) 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 are administered to humans at various doses 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.

[0013] ALT-801 (SEQ ID NO: 1) 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 the attachment site for glucuronic acid C-18* (Z17CO2H) ("1-(17-carboxylheptadecyloxy)-beta-D-glucuronyl"). As indicated differently, SEQ ID NO: 1 consists of 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.

[0014] [ka]

[0015] 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. 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.

[0016] 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 natural amino acids. The terms "peptide" and "polypeptide" are used interchangeably herein. Peptides include short peptides (about 2-20 amino acids), medium-length peptides (about 21-50 amino acids), and long peptides (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:

[0017] [ka] It has.

[0018] 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 peptide's synthetic or cellular production pathway, 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, such as 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.

[0019] 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 chemical synthesis of the dual agonist peptide, or can be accidental, for example, through mutations occurring in the host cell 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 they can have a different chemical structure and D- or L-stereochemistry. Unnatural amino acids can be utilized, for example, to promote helix formation and / or increase the stability of the dual agonist peptide (e.g., resistance to proteolysis). Dual agonist peptides having one or more modifications relative to a reference dual agonist peptide can be referred to as "analogs" or "variants" 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. It is preferred that such variants retain their ability to act as agonists of GLP-1R and GCGR, and in more preferred embodiments, have approximately equal affinity for GLP-1R and GCGR. In some embodiments, an analog or variant of a reference peptide has a different amino acid sequence than the reference dual agonist peptide.

[0020] 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.

[0021] 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.

[0022] 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 Cl 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 4is, independently at each occurrence, hydrogen or alkyl; R 5is 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. Heteroaromatic rings 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.

[0023] In some embodiments, for example, the dual agonist peptide can be associated with a saccharide, for example, 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.

[0024] 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 undue 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 comprises 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.

[0025] 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 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.

[0026] The terms "treat," "treating," and "treatment" include alleviating, ameliorating, inhibiting the progression of, 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.

[0027] 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).

[0028] 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 sterile 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 sterile 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 sterile water (pH 7.7±0.1). In certain embodiments, the pharmaceutical formulation comprises SEQ ID NO:1 and about 0.20% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in sterile water (pH 7.7±0.1). In certain embodiments, the pharmaceutical formulation comprises SEQ ID NO:1 and is configured for subcutaneous (SC) administration of a once-weekly therapeutic dose.

[0029] 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.

[0030] 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., nonionic 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 roxamers (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.

[0031] 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 include excipients such as 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 peptide product with 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 using, for example, a metered atomizing spray pump. Table 2 shows exemplary excipients for nasal spray formulations.

[0032] [Table 1]

[0033] 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 this function.For example, an MDI formulation may include a peptide product, a propellant (e.g., an HFA such as 1,1,1,2-tetrafluoroethane) and a cosolvent (e.g., an alcohol such as ethanol), and optionally a surfactant (e.g., a fatty acid such as oleic acid). The MDI formulation 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 include 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).

[0034] 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.

[0035] 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 must 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).

[0036] 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.

[0037] [Table 2]

[0038] Table 3

[0039] 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 (e.g., based on aerodynamics, impact force, or vibration) also exist, which 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). The peptide product 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 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).

[0040] 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 carrier (e.g., solvent) 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 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," which means 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 (for example) a condition related thereto. In certain embodiments, the kit further contains a device for delivering the peptide product or composition, such as an injection pen or an inhaler.

[0041] 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 various mechanisms, including stimulation of blood glucose-dependent insulin secretion, increased insulin sensitivity, stimulation of fat burning, and weight loss. Dual agonist peptide products can also promote, for example, pancreatic beta cell protection, cardioprotection, and wound healing.

[0042] The peptide products described herein can be used to treat other conditions associated with insulin resistance and / or obesity, including, but 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), and liver diseases (e.g., liver cirrhosis). The therapeutic effects of the dual agonist peptide products described herein 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 cancer (e.g., endometrial, esophageal, colorectal, gallbladder, kidney, liver [e.g., hepatocellular carcinoma], pancreatic, and skin [e.g., melanoma] cancer, 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 liver inflammation. 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.

[0043] 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.

[0044] 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 understand that effective doses in mice or other preclinical animal models can be extended 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 weight or body surface area.

[0045] The peptide product can be administered at any suitable frequency for the prevention and / or treatment of a CV disease-related condition disclosed herein (e.g., elevated cholesterol, triglyceride, LDL, and / or VLDL particle concentration and / or diameter, and / or other related parameters (e.g., serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine)), e.g., heart failure with preserved ejection fraction (HFpEF). 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 on a day that is convenient for the patient. The dual agonist peptide product can be taken substantially with food (e.g., with a meal or within about 1 hour or 30 minutes before or after a meal) or substantially without food (e.g., at least about 1 or 2 hours before or after a meal). The length of treatment for 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 for 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 medicament. In addition, the present disclosure provides the use of a peptide product described herein or a pharmaceutically acceptable salt thereof in the preparation of a medicament. A 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.

[0046] 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, e.g., insulin resistance, diabetes, obesity, metabolic syndrome, or CV disease (e.g., heart failure with preserved ejection fraction (HFpEF) or any condition related thereto, e.g., elevated cholesterol, triglycerides, LDL, and / or VLDL particle concentration and / or diameter, and / or other related parameters (e.g., serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine). 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), anti-atherosclerotic agents, anti-inflammatory agents, antioxidants, anti-fibrotic agents, anti-hypertensive 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][l,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 avasimibe, pactimibe, pellitorinin, terpendol C, and flavanones (e.g., naringenin), acyl-CoA cholesterol acyltransferase (ACAT, also known as sterol G-acyltransferase [SOAT], ACAT1 [SOAT1]), including 192849, BMS649, LG100268, LG100754, and LGD346; and ACAT2 [SOAT2]), inhibitors of stearoyl-CoA desaturase-1 (SCD-1, also known as stearoyl-CoA delta-9 desaturase) activity or expression, including aramchol, CAY-10566, CVT-11127, SAR-224, SAR-707, and XEN-103, 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], Drospiro[chromene-2,4'-piperidine], 6-[5-(cyclopropylmethyl)-4,5-dihydro-1'H,3H-spiro[1,5-benzoxazepine-2,4'-piperidine]-1'-yl]-N-(2-hydroxy-2-pyridin-3-ylethyl)pyridazine-3-carboxamide, 6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxylic acid (2-hydroxy-2-pyridin-3-ylethyl)amide, 4-(2-chlorophenoxy)- N-[3-(methylcarbamoyl)phenyl]piperidine-1-carboxamide, cis-9, trans-11 and trans-10, cis-12 isomers of conjugated linoleic acid, substituted heteroaromatic compounds as disclosed in WO2009 / 129625A1, antisense polynucleotides and peptide-nucleic acids (PNAs) targeting mRNA for SCD-1, and SCD-1-targeting siRNA, cholesteryl ester transfer protein (CETP) inhibitors, including anacetrapib, dalcetrapib, evacetrapib, torcetrapib, and AMG 899 (TA-8995), implitapide, lomitapide, dirlotapide, mitratapide, CP-346086, JTT-130, SLx-4090,inhibitors of microsomal triglyceride transfer protein (MTTP) activity or expression, including antisense polynucleotides and PNAs targeting mRNA for MTTP, MTTP-targeting microRNAs (e.g., miRNA-30c), and MTTP-targeting siRNA; GLP-1 receptor agonists; fibroblast growth factor 21 (FGF21) and its analogs and derivatives, including BMS-986036 (pegylated FGF21); berberine (decreases PC8K9 levels); Annexin A2 (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-962476), antisense polynucleotides and PNAs that target mRNA for PCSK9, and PCSK9-targeting siRNA (e.g., inhibitors of proprotein convertase subtilisin / kexin type 9 (PCSK9) activity or expression, including inclisiran [ALN-PCS] and ALN-PCS02), apoA-I mimetics (e.g., 2F, 3F, 3F-1, 3F-2, 3F-14, 4F, 4F-P-4F, 4F-IHS-4F, 4F2, 5F, 6F, 7F, 18F, 5A, 5A-C1, 5A-CH1, 5A-CH2, 5A-H1, 18A, 37pA [18A-P-18A], ELK, EL K-1A, ELK-1F, ELK-1K1A1E, ELK-1L1K, ELK-1W, ELK-2A, ELK-2A2K2E, ELK-2E2K, ELK-2F, ELK-3E3EK, ELK-3E3K3A, ELK-3E3LK, ELK-P A, 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 Apolipoprotein-mimetic peptides, including 8 A-NH2, AEM-28-14, EpK, hEp, mR18L, COG-112, COG-133, and COG-1410), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA),Omega-3 fatty acids, including eicosapentaenoic acid (EPA), α-linolenic acid (ALA), fish oils (e.g., containing DHA and EPA), and esters thereof (e.g., glyceryl and ethyl esters), as well as analogs, derivatives, and salts thereof. In certain embodiments, the anti-obesity 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). Antihypertensive agents include, but are not limited to, antagonists of the renin-angiotensin-aldosterone system (RAAS), including renin inhibitors (e.g., aliskiren), angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril), angiotensin II receptor type 1 (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, including azide 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 dihydropyridines (e.g., amlodipine, levamlodipine, cilnidipine, clevidipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine) and non-dihydropyridines (e.g., diltiazem and verapamil), including clonidine, guanabenz, guanfacine, methyldopa, and moxonidine;α1-adrenergic receptor antagonists (alpha blockers), including α2-adrenergic receptor agonists, doxazosin, indoramin, nicergoline, phenoxybenzamine, phentolamine, prazosin, terazosin, and tolazoline; α1-adrenergic receptor antagonists (alpha blockers), including atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, and timolol; β-adrenergic receptor (β1 or / and β2) antagonists (beta blockers), including bucindolol, carvedilol, and labetalol; mixed alpha / beta blockers, including selective ETA receptor antagonists (e.g., ambrisentan, atrasentan, edonentan, sitaxsentan, zibotentan, and BQ-123) and dual ETA receptor antagonists; 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 (PDE) inhibitors (e.g., thiazolinone ... 5) 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, prostanoid prostacyclin receptor agonists (e.g., 1-phthalazinol, larine phosphate, benzodiazepines, benzocaine, benzodiazepines, benzocaine, benzodiazepines), benzocaine, ... Other vasodilators, including vasodilators such as benzodiazepines, benzodiazepines, benzodiazepines, benzodiazepines (e.g., benzodiazepines), ...In certain embodiments, the antihypertensive agent is or includes a thiazide or thiazide-like diuretic (e.g., hydrochlorothiazide or chlorthalidone), a calcium channel blocker (e.g., amlodipine or nifedipine), an ACE inhibitor (e.g., benazepril, captopril, or perindopril), or an angiotensin II receptor antagonist (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 that prevent and / or treat conditions associated with CV disease, such as elevated cholesterol, triglycerides, LDL, and / or VLDL particle concentration and / or diameter, and / or other related parameters (e.g., serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine). 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, and PPAR-α antagonism reduces hepatic steatosis). PPAR agonists, including PPAR-δ antagonism (which reduces hepatic gluconeogenesis, lipogenesis, steatosis, and fibrosis), such as obeticholic acid, farnesoid X receptor (FXR) agonists (nonsteroidal 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), and BMS-986036 (pegylated FGF21). fibroblast growth factor 21 (FGF21) and its analogs and derivatives (FGF21 analogs reduce hepatic steatosis, cellular injury, and fibrosis), HMG-CoA reductase inhibitors, including statins (e.g., rosuvastatin), (statins reduce steatohepatitis and fibrosis), ACC inhibitors, such as NDI-010976 (liver-targeted) and GS-0976, (ACC inhibitors reduce de novo lipogenesis and hepatic steatosis), SCD-1 inhibitors, such as aramchol (SCD-1 inhibitors reduce hepatic steatosis and insulin resistance), and steroid hormone receptor 1 (SCD-1 inhibitors reduce hepatic steatosis and insulin resistance). These include: SGLT2 inhibitors, such as canagliflozin, ipragliflozin, and luseogliflozin (SGLT2 inhibitors reduce body weight, hepatic ALT levels, and fibrosis); CCR2 and / or CCR5 antagonists, such as cenicriviroc (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, thereby reducing liver fibrosis); and apoptosis signal-regulating kinase 1 (A apoptosis inhibitors, including SK1 inhibitors (e.g., selonsertib), and caspase inhibitors (e.g., emricasan [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 for the development of liver fibrosis), vitamin E (e.g.,and antioxidants, including 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 through inhibition of TNF-α and phosphodiesterase] (vitamin E reduces hepatic steatosis, hepatocyte ballooning, and lobular inflammation), as well as analogs, derivatives, and salts thereof. In some embodiments, the peptide products described herein are used in combination with a PPAR agonist (e.g., a PPAR-δ agonist such as elafibranor or / and a PPAR-γ agonist such as pioglitazone), an HMG-CoA reductase inhibitor (e.g., a statin such as rosuvastatin), an FXR agonist (e.g., obeticholic acid), or an antioxidant (e.g., vitamin E), or any combination thereof, to treat NAFLD (e.g., NASH). In certain embodiments, the one or more additional therapeutic agents for treating NAFLD (e.g., NASH) are or include vitamin E and / or pioglitazone. Other combinations may also be used, as will be appreciated by those skilled in the art.

[0047] 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.

[0048] In some embodiments, the 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 in a subject at risk for and / or suffering from a CV disease-related condition (and / or CV disease), who may be overweight, obese, and / or who may also suffer from type 2 diabetes, with the adverse events selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to a mammal. In certain embodiments, the formulation comprises 1.8 mg of pembidutide as a therapeutic dose.

[0049] "Reducing" adverse effects or events or their "reduction" refers to the reduction in 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 reduction in the prevalence of 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 their "reduction" includes 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.

[0050] In preferred embodiments, the present disclosure provides a method for reducing body weight in a human with fatty liver, comprising administering pembidutide in an amount of at least 1.8 mg to 2.4 mg once weekly to a human in need thereof, wherein the human exhibits a CV disease-related condition (and / or CV disease), is overweight, may be obese, and / or may suffer from type 2 diabetes. 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. In some preferred embodiments, the disclosure provides a method for administering about 1.2 mg or about 1.8 mg of pembidutide to a population of humans for at least 12 weeks, where at week 12, at least about 65% of the population experience a body weight reduction from baseline of about 5% or more; about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, where at week 12, at least about 80% of the population experience a body weight reduction from baseline of about 5% or more; about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, where at week 12, at least about 30% of the population experience a body weight reduction from baseline of about 10% or more; or about 1 The present invention provides methods in which about 0.8 mg or about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 45% of the population experience a body weight reduction of about 10% or more from baseline; about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 5% of the population experience a body weight reduction of about 15% or more from baseline; and / or about 1.8 mg or about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 20% of the population experience a body weight reduction of about 15% or more from baseline. See Figure 7. In some preferred embodiments, the mean change in blood pressure from baseline in the population is reduced. See Figure 8.In some preferred embodiments, the mean change in blood total cholesterol from baseline for the population is reduced by at least about 10%. See Figure 9. In some preferred embodiments, the mean change in blood low-density lipoprotein (LDL) from baseline for the population is reduced by at least about 5%. In some preferred embodiments, the mean change in blood high-density lipoprotein (HDL) from baseline for the population is reduced by at least about 15%. In some preferred embodiments, the mean change in blood triglycerides from baseline for the population is reduced by at least about 15%. See Figure 9. In some preferred embodiments, the mean change in waist circumference for the population is reduced by at least about 8%. See Figure 10. In some preferred embodiments, administration of pembidutide induces a significant increase in ABCG5 (p<0.005) and ABCG8 (p<0.05) gene expression compared to semaglutide.

[0051] In some preferred embodiments, the disclosure provides a method of reducing the risk of cardiovascular (CV) disease in a human, the method comprising: administering pembidutide to the human once weekly in an amount of at least 1.2 mg to at most 2.4 mg, wherein at least one risk factor associated with CV disease is reduced and / or eliminated, wherein the at least one risk factor is selected from the group consisting of excess body weight, high serum lipids, cholesterol, triglycerides, LDL and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine in the human's serum, and optionally, the human is overweight, obese, and has a body mass index (BMI kg / m) of 25 or greater. 2) and / or type 2 diabetes. In some preferred embodiments of this method, pemvidutide is administered once weekly for 5 to 12 weeks. In some preferred embodiments of this method, pemvidutide is administered once weekly in an amount of about 1.8 mg, or once weekly in an amount of about 2.4 mg. In some preferred embodiments of this method, 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, or a steady-state dose is achieved after a dose-escalation phase having a duration of 2 to 4 weeks. In preferred embodiments of this method, pemvidutide is administered by parenteral injection, or pemvidutide is administered by subcutaneous injection. In preferred embodiments of this method, pemvidutide is administered from a liquid formulation comprising at least about 1.8 mg / ml of pemvidutide.

[0052] In some preferred embodiments of this method, administration of pemvidutide induces a significant decrease in atherogenic lipid and / or lipoprotein levels compared to baseline. In some preferred embodiments of this method, the atherogenic lipoprotein levels are reduced by at least -0.2 log2 fold change compared to baseline after 43 and / or 84 days of pemvidutide administration, and the subject's total serum triglyceride levels are reduced by at least -0.2 log2 fold change compared to placebo after 43 and / or 84 days of pemvidutide administration, and the serum lipids are selected from glycerolipids, sterols, glycerophospholipids, and sphingolipids, and optionally the decrease is at least -0.2 log2. In some preferred embodiments of this method, decreases in serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine are induced.

[0053] In certain preferred embodiments of this method, the human has a weight loss of at least 3% or at least 4% from baseline at week 12. In certain preferred embodiments of this method, the human has type 2 diabetes and / or the human has a body mass index (BMI kg / m) of at least 25, at least about 28, or at least about 30. 2 )

[0054] 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 is overweight, obese, and / or 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. 2 In some preferred embodiments, the human has a level of a parameter associated with a CV disease-related condition of 10% or greater prior to treatment (e.g., at baseline). In some preferred embodiments, the absolute reduction in the CV disease-related condition is about 8%, 10%, 12%, or preferably about 15% after 12 weeks of treatment compared to the baseline measurement. In some preferred embodiments, the relative reduction in the CV disease-related condition compared to baseline is about 40%, 50%, or 60% or greater 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, 10, 12, or 16 weeks. In some preferred embodiments, pembidutide is administered from a liquid containing at least about 1.8 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.

[0055] In preferred embodiments, the present disclosure relates to the following aspects:

[0056] Embodiment 1. A method of reducing the risk of cardiovascular (CV) disease in a human, comprising the step of administering pembidutide to the human once weekly in an amount of at least 1.2 mg to at most 2.4 mg, wherein at least one risk factor associated with CV disease is reduced and / or eliminated, wherein the at least one risk factor is selected from the group consisting of excess body weight, high serum lipids, cholesterol, triglycerides, LDL, and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine in the human's serum, and optionally, the human is overweight, obese, and has a body mass index (BMI kg / m) of 25 or greater. 2 ) and / or have type 2 diabetes.

[0057] Embodiment 2 The method of embodiment 1, wherein pembidutide is administered once weekly in an amount of about 1.2 mg, about 1.8 mg, or about 2.4 mg.

[0058] Embodiment 3. The method of embodiment 1 or 2, wherein pembidutide is administered once a week for 5 to 12 weeks.

[0059] Embodiment 4. The method of any one of embodiments 1-3, wherein administration of pembidutide induces a significant decrease in the number of atherogenic lipids and / or lipoproteins compared to baseline.

[0060] Embodiment 5. The method of any one of embodiments 1-4, wherein the concentration of atherogenic lipoprotein number is reduced by a fold change of at least -0.2 log2 compared to baseline at 43 and / or 84 days after administration of pembidutide.

[0061] Embodiment 6. The method of any one of embodiments 1-5, wherein the subject's total serum triglyceride concentration is reduced by a fold change of at least -0.2 log2 relative to placebo at 43 and / or 84 days after administration of pembidutide.

[0062] Embodiment 7. The method of any one of embodiments 1-6, wherein the serum lipid is selected from a glycerolipid, a sterol, a glycerophospholipid, and a sphingolipid, and optionally, the reduction is a fold change of −0.2 log2.

[0063] Embodiment 8. The method of any one of embodiments 1 to 7, wherein a decrease in serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine is induced.

[0064] Embodiment 9 The method of embodiment 2, wherein the human has a body weight reduction of at least 3% or at least 4% from baseline at week 12.

[0065] Embodiment 10. About 1.2 mg or about 1.8 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 65% of the population experiences a body weight reduction from baseline of about 5% or more; about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 80% of the population experiences a body weight reduction from baseline of about 5% or more; about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 30% of the population experiences a body weight reduction from baseline of about 10% or more; about 1.8 mg or about 2.4 mg The method of any one of aspects 1 to 9, wherein about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 45% of the population experience a body weight reduction of about 10% or more from baseline; about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 5% of the population experience a body weight reduction of about 15% or more from baseline; and / or about 1.8 mg or about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 20% of the population experience a body weight reduction of about 15% or more from baseline.

[0066] Embodiment 11 The method of embodiment 10, wherein the mean change in blood pressure from baseline for the population is reduced.

[0067] Embodiment 12 The method of embodiment 10 or 11, wherein the mean change in blood total cholesterol from baseline for the population is reduced by at least about 10%.

[0068] Embodiment 13. The method of any one of embodiments 10-12, wherein the mean change in blood low-density lipoprotein (LDL) from baseline for the population is reduced by at least about 5%.

[0069] Embodiment 14. The method of any one of embodiments 10 to 13, wherein the mean change in blood high density lipoprotein (HDL) from baseline for the population is reduced by at least about 15%.

[0070] Embodiment 15. The method of any one of embodiments 10-14, wherein the mean change in blood triglycerides from baseline for the population is reduced by at least about 15%.

[0071] Embodiment 16 The method of any one of embodiments 10 to 15, wherein the mean change in waist circumference of the population is reduced by at least about 8%.

[0072] Embodiment 17. The method of any one of embodiments 1 to 16, wherein pembidutide induces a significant increase in ABCG5 (p<0.005) and ABCG8 (p<0.05) gene expression compared to baseline.

[0073] Embodiment 18 The method of any one of embodiments 1 to 17, wherein pembidutide is administered once a week in an amount of about 1.8 mg.

[0074] Embodiment 19 The method of any one of embodiments 1 to 18, wherein pembidutide is administered once a week in an amount of about 2.4 mg.

[0075] Embodiment 20. The method of any one of embodiments 1-19, 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.

[0076] Embodiment 21. The method of any one of embodiments 1 to 20, wherein the human has type 2 diabetes and / or the cardiovascular disease is atherosclerotic cardiovascular disease.

[0077] Embodiment 22. A human has a body mass index (BMI kg / m) of at least 25, at least about 28, or at least about 30. 2 22. The method of any one of aspects 1 to 21, wherein

[0078] Embodiment 23 The method of any one of embodiments 1 to 22, wherein pembidutide is administered by parenteral injection.

[0079] Embodiment 24 The method of any one of embodiments 1 to 22, wherein pembidutide is administered by subcutaneous injection.

[0080] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the steady-state dose is achieved after a dose-escalation phase having a duration of 2 to 4 weeks.

[0081] Embodiment 26 The method of any one of embodiments 1 to 25, wherein pembidutide is administered from a liquid formulation comprising at least about 1.8 mg / ml pembidutide.

[0082] Embodiment 27. The method of any one of embodiments 1 to 26, wherein pembidutide is administered as a liquid pharmaceutical formulation comprising SEQ ID NO: 1 and about 0.20% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in sterile water (pH 7.7±0.1).

[0083] Embodiment 28. A method of reducing the risk of cardiovascular (CV) disease in a human, comprising the step of administering pembidutide to the human once weekly in an amount of at least 1.2 mg to at most 2.4 mg, wherein at least one risk factor associated with CV disease is reduced and / or eliminated, wherein the at least one risk factor is selected from the group consisting of excess body weight, high serum lipids, cholesterol, triglycerides, LDL, and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine in the human's serum, and optionally, the human is overweight, obese, and has a body mass index (BMI kg / m) of 25 or greater. 2 ) and / or have type 2 diabetes.

[0084] Embodiment 29. A method of treating a cardiovascular (CV) disease risk factor, comprising administering to a patient in need thereof a pharmaceutical composition comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, wherein the patient is overweight, obese, and has a body mass index (BMI kg / m) of 25 or greater. 2 ), and / or have type 2 diabetes, and the method reduces at least one CV disease risk factor selected from the group consisting of body weight, waist circumference, blood pressure, hyperglycemia, serum lipids, total cholesterol, triglycerides, HDL, LDL, and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine compared to baseline.

[0085] Embodiment 30 The method of embodiment 29, wherein the patient in need of treatment is overweight.

[0086] Embodiment 31 The method of embodiment 29, wherein the patient in need of treatment is obese.

[0087] Aspect 32. The patient in need of treatment has a body mass index (BMI kg / 230. The method of embodiment 29, wherein

[0088] Embodiment 33 The method of embodiment 29, wherein the patient in need of treatment has type 2 diabetes.

[0089] Embodiment 34 The method of embodiment 29, wherein the pharmaceutical composition is administered once a week in an amount of about 1.2 mg, about 1.8 mg, or about 2.4 mg.

[0090] Embodiment 35 The method of embodiment 29, wherein the pharmaceutical composition is administered in an amount of about 1.8 mg about once a week.

[0091] Embodiment 36 The method of embodiment 29, wherein the pharmaceutical composition is administered in an amount of about 2.4 mg about once a week.

[0092] Embodiment 37. The method of embodiment 29, 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.

[0093] Embodiment 38 The method of embodiment 29, wherein the pharmaceutical composition is administered once a week for about 5 to 12 weeks.

[0094] Embodiment 39 The method of embodiment 29, wherein the pharmaceutical composition is administered by parenteral injection.

[0095] Embodiment 40. The method of embodiment 29, wherein the pharmaceutical composition is administered by subcutaneous injection.

[0096] Embodiment 41 The method of embodiment 29, wherein the pharmaceutical composition is administered from a liquid formulation comprising at least about 1.8 mg / ml of pembidutide.

[0097] Embodiment 42. The method of any one of embodiments 29 to 41, wherein the pharmaceutical composition is administered as a liquid pharmaceutical formulation comprising SEQ ID NO:1 and about 0.20% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in sterile water (pH 7.7±0.1).

[0098] Embodiment 43. A method of treating a cardiovascular (CV) disease risk factor, comprising administering to a patient in need of treatment a pharmaceutical composition comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, wherein the patient is overweight, obese, and has a body mass index (BMI kg / m) of 25 or greater. 2 ), and / or have diabetes, and wherein the method reduces at least one of plasma triglyceride, total plasma cholesterol, plasma LDL-cholesterol, or plasma HDL-cholesterol levels by about 3%, 5%, 7%, or preferably 10% from baseline.

[0099] Embodiment 44. The method of embodiment 43, wherein plasma triglycerides are reduced from baseline.

[0100] Embodiment 45 The method of embodiment 55, wherein total plasma cholesterol is reduced from baseline.

[0101] Embodiment 46 The method of embodiment 43, wherein plasma LDL-cholesterol is reduced from baseline.

[0102] Embodiment 47 The method of embodiment 43, wherein plasma HDL-cholesterol is reduced from baseline.

[0103] Embodiment 48. A method of reducing plasma lipids in a patient in need thereof, comprising the step of administering to the patient in need thereof a pharmaceutical composition comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof, wherein the patient is suffering from cardiovascular disease, and wherein the method reduces at least one of plasma triglyceride, total plasma cholesterol, plasma LDL-cholesterol, or plasma HDL-cholesterol levels by 3%, 5%, 7%, or preferably 10% from baseline.

[0104] Embodiment 49. The method of embodiment 48, wherein one or more pathogenic serum lipid mediators are reduced from baseline.

[0105] As will be appreciated by those skilled in the art, other aspects of the present disclosure are also contemplated.

[0106] Unless otherwise defined or clearly indicated otherwise by their usage herein, 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 specification and the appended claims, the terms "a" or "an" mean one or more. As used herein, the term "another" means second or more. The acronym "aka" means also known. As used herein, the term "exemplary" means "serving as an example, instance, or illustration." Any embodiment or feature characterized herein as "exemplary" should not necessarily be construed as preferred or advantageous over other embodiments or features. In some embodiments, the term "about" or "approximately" means within ±10% or ±5% of the specified value. Whenever the term "about" or "approximately" precedes a series of two or more numerical values ​​or the first numerical value in a series of two or more numerical ranges, the term "about" or "approximately" applies to each of the numerical values ​​in the series or numerical range. Ranges may be expressed herein as 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, by use of the antecedent "approximately" or "around," it will be understood that the particular value forms another embodiment. It will be further understood that each endpoint of a range is significant both in relation 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. Optional or optionally means that the subsequently described event or circumstance may or may not occur, and the description includes instances when the event or circumstance occurs and instances when it does not occur.All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0107] Certain embodiments are further described in the following examples, which are provided by way of example only and are not intended to limit the scope of the claims in any way. [Example]

[0108] Example 1. Effects of pembidutide (ALT-801), a GLP-1 / glucagon dual receptor agonist, on pathogenic lipid mediators

[0109] The potential for CV risk reduction with incretin-based therapies has attracted 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).

[0110] 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. 1 Lipid profiling was performed by H-NMR. Lipidomic profiling, encompassing 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 1.

[0111] 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 4.

[0112] [Table 4]

[0113] Pembidutide also generally causes consistent changes in lipoprotein particle subpopulations as determined by 2D-NMR analysis. See Figure 2. 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 2 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.

[0114] Based on changes observed in total cholesterol, triglycerides, and lipoproteins, serum lipid composition encompassing 600 lipid species was assessed on days -1 (baseline), 43, and 84 from 34 subjects who completed NCT0456124. Results are presented in Figure 3 as log2 versus 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 3. Thus, at day 85, substantial and highly statistically significant reductions across multiple bioactive lipid classes were observed compared to placebo.

[0115] Based on the lipodomics data, a volcano plot was generated, as shown in Figure 4, 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 4), suggesting a possible reduction in oxidized LDL (Law et al., Int. J. Mol. Sci. 2019;20(5):1149). Pemvidutide treatment also significantly reduced atherogenic plaque forming phosphatidylethanolamine (PE) glycerophospholipid levels. Based on these findings, pemvidutide shows promise as an agent for reducing cardiovascular (CV) risk.

[0116] In this study, pembidutide induced substantial weight loss at 12 weeks, as shown in Figures 5A and 5B. A maximum mean weight loss of 10.3% (8.7% after placebo subtraction) was observed at the 1.8 mg dose level, with a high degree of statistical significance after only 12 weeks of weekly treatment. Additionally, within this treatment group, all subjects experienced at least a 5% weight loss, with over half achieving at least a 10% weight loss.

[0117] Pembidutide was shown to be safe and well-tolerated without dose titration in a Phase 1a study. A mean weight loss of up to 10.3% was observed in the study, accompanied by statistically significant reductions across multiple atherogenic lipid classes and in atherogenic lipoprotein particles. Based on these findings, pembidutide shows promise as an agent for reducing the risk of CV disease.

[0118] Example 2: Efficacy of ALT-801 (pembidutide) on Pathogenic Lipid Mediators in a Phase II, 24-Week, Randomized, Double-Blind, Placebo-Controlled Study in Non-Diabetic Obese and Overweight Subjects

[0119] The results presented in this example represent a 24-week interim analysis from a placebo-controlled Phase II trial evaluating pembidutide (ALT-801) in obese and overweight subjects (ClinicalTrials.gov Identifier: NCT05295875). Key eligibility criteria were: (1) men and women, 18-75 years of age; (2) at least one unsuccessful weight loss trial 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).

[0120] Baseline characteristics of study participants are shown in Figure 6. Figure 7 shows a weight loss responder analysis based on calculation of the percentage of subjects achieving ≥5%, ≥10%, and ≥15% weight loss across the three pemvidutide doses and placebo, respectively. Figure 8A 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 8B shows that 24 weeks of treatment with pemvidutide at doses of 1.2 mg, 1.8 mg, and 2.4 mg did not induce significant changes in heart rate. Figure 9 shows serum lipids at 24 weeks for all doses of pemvidutide administered (1.2 mg, 1.8 mg, and 2.4 mg) compared to baseline. Figure 9 shows improvements in serum lipids at 24 weeks. As shown therein, for example, improvements in serum lipids were observed for all administered doses of pembidutide (1.2 mg, 1.8 mg, and 2.4 mg per week). Figure 10 shows a significant reduction in waist circumference at 24 weeks 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 for assessing the risk of metabolic diseases such as NAFLD and NASH, as well as cardiovascular disease.

[0121] Example 3: Effect of pembidutide on reverse cholesterol transport from macrophages to feces in diet-induced obese hamsters

[0122] The study was conducted in male Golden Syrian hamsters fed a free-choice diet (chow and regular tap water or a high-fat / cholesterol diet (Safe Diets, 40.8% fat, 14.8% protein, 44.4% carbohydrate, and 0.5% cholesterol, 4444.2 kcal / kg diet) and 10% fructose-enriched tap water (0.4 kcal / L)) for a minimum of 20 weeks prior to the start of the study and until the end of the experiment. After randomization based on body weight and plasma cholesterol levels, hamsters were assigned to two groups of 10 animals each that received vehicle or pemvidutide (10 nmol / kg) subcutaneously daily for 35 days and a 2-hour macrophage-to-fecal reverse cholesterol transport (RCT) experiment. Body weights were measured three times weekly. On day 35, hamsters were fasted for 6 hours, and blood (150 μL / heparin) was collected by retro-orbital bleeding under isoflurane anesthesia to isolate plasma and measure fasting plasma triglyceride, total cholesterol, LDL-cholesterol, and HDL-cholesterol levels. On day 36, hamsters were intraperitoneally injected with oxidized LDL-loaded / [H]-cholesterol-labeled J774 macrophages for a 72-hour macrophage-to-feces RCT experiment, as previously described in Castro-Perez et al. (Castro-Perez et al. Anacetrapibs promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian Olden Hamsters. J Lipid Res. 2011;52:1965-73). Blood was collected into heparin tubes under slight isoflurane anesthesia at 24, 48, and 72 hours after injection of labeled J774 cells to measure plasma radioactivity. Feces were collected continuously during the 72-hour experiment and then weighed before measuring fecal [H]-cholesterol and [H]-bile acids after chemical extraction from fecal homogenates. After 72 hours, hamsters were sacrificed and livers were harvested for liver lipid measurements after chemical extraction from liver homogenates.

[0123] Figure 11A shows the significant effect of pemvidutide treatment on the percent change in body weight from baseline (p<0.0001) compared to the vehicle group at day 35. Figures 11B-E show the significant effect of pemvidutide treatment on lowering plasma lipids, including LDL-c (p<0.05), HDL-c (p<0.005), and triglycerides (p<0.0005), compared to vehicle at day 35. Lower plasma cholesterol was also observed as a result from the day of pemvidutide treatment compared to vehicle, but this difference was not considered statistically significant. The reductions in both body weight and plasma lipids are consistent with pemvidutide's intended mechanism of action and broadly replicate the drug's activity observed in the clinical trials presented in Examples 1 and 2.

[0124] Figure 12A shows the significant effect of pembidutide treatment on the reduction of liver weight compared to the vehicle group (p<0.005). Additionally, Figures 12B, C, and D show the effect of 35 days of pembidutide treatment on the reduction of cholesterol (p<0.05), triglycerides, and fatty acids, respectively, compared to the vehicle.

[0125] After injection of [H]-cholesterol-labeled J774 macrophages, [H]-cholesterol and / or [H]-bile acids (resulting from enzymatic conversion of [H]-cholesterol in the liver) were measured in plasma, liver, and fecal samples at 72 hours and expressed as a percentage of the [H]-cholesterol injected dose. Taking into account the equivalent amounts of feces excreted across the two groups (Figure 13A), Figures 13B-E show that pembidutide promoted a significant increase (p<0.05) in fecal [H]-cholesterol and a trend toward increased [H]-bile acids compared to vehicle, expressed as % of the injected dose per gram of feces or total fecal weight. Related to this result, Figure 14 shows the effect of pembidutide treatment on lowering plasma [H]-cholesterol compared to the vehicle group, likely reflecting more efficient cholesterol transport from peripherally labeled macrophages to the liver.

[0126] Taken together, these results support the beneficial effect of pembidutide on reverse cholesterol transport (RCT), a mechanism associated with increased cholesterol removal from peripheral macrophages and excretion in the feces. This result may also be related to the reduction in plasma HDL-c levels observed with pembidutide, possibly reflecting a higher metabolic turnover of these lipid particles known to mediate RCT. In conclusion, pembidutide promotes RCT, an anti-atherosclerotic mechanism associated with cardiovascular benefits in addition to weight loss.

[0127] Example 4: Effect of pembidutide on gene expression involved in reverse cholesterol transport in a diet-induced obese hamster model

[0128] The study was conducted in male Golden Syrian hamsters that were fed a free-choice diet (chow and regular tap water, or a high-fat / cholesterol diet (Safe Diets, 40.8% fat, 14.8% protein, 44.4% carbohydrate, and 0.5% cholesterol, 4444.2 kcal / kg diet) and 10% fructose-enriched tap water (0.4 kcal / L)) for a minimum of 20 weeks before the start of the study and until the end of the experiment. After randomization based on weight, the hamsters were assigned to three groups of five animals each that received vehicle, pembidutide (10 nmol / kg), or semaglutide (10 nmol / kg) subcutaneously daily for 21 days. After 21 days, the hamsters were sacrificed and their livers were harvested. Total RNA was extracted from frozen liver tissue, and then gene expression was measured by RT-PCR across a range of markers including CYP7A, ABCA1, ABCG1, ABCG5, ABCG8, SREBP1c, SRB1, and LDL-R.

[0129] Figure 15 shows the effect of pembidutide on hepatic expression of CYP7A, ABCA1, ABCG1, ABCG5, ABCG8, ACAT2, SREBP1c, SRB1, or LDL-R compared with semaglutide or vehicle. Results are expressed as fold changes compared with vehicle. Surprisingly, pembidutide, in contrast to semaglutide and vehicle, induces a significant increase in ABCG5 and ABCG8 gene expression. This result supports the effect of pembidutide on ABCG5 and ABCG8 genes as a result of glucagon activity in the liver, an organ that expresses glucagon receptors but not GLP-1 receptors. Both hepatic ATP-binding cassette (ABC) transporters G5 (ABCG5) and G8 (ABCG8) are known to promote biliary secretion of cholesterol and phytosterols into the intestine. Mutations in either of these two genes cause sitosterolemia, a condition in which cholesterol and plant sterols accumulate in the circulation, leading to premature cardiovascular disease (Tada et al., Adv Clin Chem. 2022; 110:145-169). Overexpression of ABCG5 and ABCG8 in mice delays diet-induced atherosclerosis due to reduced circulating and hepatic cholesterol (Wilund et al., High-level expression of ABCG5 and ABCG8 attenuates diet-induced hypercholesterolemia and atherosclerosis in Ldlr- / - mice. J Lipid Res 45: 1429-1436, 2004). Overall, these results suggest that pembidutide may exert an anti-atherosclerotic role through overexpression of ABCG5 and ABCG8 in the liver as a mechanism supporting reverse cholesterol transport.

[0130] Thus, provided herein is a method of using pembidutide to reduce the risk of cardiovascular (CV) disease in a non-diabetic overweight or obese human, comprising administering pembidutide to the human once a week in an amount of at least 1.2 mg to 2.4 mg, wherein at least one risk factor associated with CV disease selected from the group consisting of excess body weight, cholesterol, triglycerides, HDL, and LDL in the human's serum is reduced and / or eliminated.

[0131] Other advantages of the reagents and methods of using them are also provided herein, as will be appreciated by those skilled in the art. While certain embodiments have been described with reference to 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. 1. A method of reducing the risk of cardiovascular (CV) disease in a human, comprising the step of administering pembidutide to the human once weekly in an amount of at least 1.2 mg to at most 2.4 mg, at least one risk factor associated with CV disease is reduced and / or eliminated, said at least one risk factor being selected from the group consisting of excess body weight, high serum lipids, cholesterol, triglycerides, LDL and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine in said human's serum; Optionally, the human is overweight, obese, and has a body mass index (BMI kg / m) of 25 or greater. ２ ) and / or have type 2 diabetes.

2. 10. The method of claim 1, wherein the pembidutide is administered once weekly in an amount of about 1.2 mg, about 1.8 mg, or about 2.4 mg.

3. 3. The method of claim 1 or 2, wherein the pembidutide is administered once a week for 5 to 12 weeks.

4. The method of any one of claims 1 to 3, wherein administration of pembidutide induces a significant decrease in the number of atherogenic lipids and / or lipoproteins compared to baseline.

5. A decrease in atherogenic lipoprotein levels of at least -0.2 log compared to baseline at 43 and / or 84 days after administration of pembidutide. ２ The method of any one of claims 1 to 4, wherein the expression level is reduced by a fold change of

6. The subject's total serum triglyceride concentration is at least -0.2 log lower than placebo at 43 and / or 84 days after administration of pembidutide. ２ The method of any one of claims 1 to 5, wherein the expression level is reduced by a fold change of

7. the serum lipids are selected from glycerolipids, sterols, glycerophospholipids, and sphingolipids, and optionally, the reduction is greater than or equal to -0.2 log ２ The method of any one of claims 1 to 6, wherein the fold change is

8. The method of any one of claims 1 to 7, wherein a decrease in serum phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine is induced.

9. 3. The method of claim 2, wherein the human's body weight is reduced by at least 3% or at least 4% from baseline at 12 weeks.

10. about 1.2 mg or about 1.8 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 65% of the population experiences a body weight reduction of about 5% or more from baseline; about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 80% of the population experiences a body weight reduction of about 5% or more from baseline; about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 30% of the population experiences a body weight reduction of about 10% or more from baseline; about 1.8 or about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 45% of the population experiences a body weight reduction of about 10% or more from baseline; about 1.2 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 5% of the population experiences a weight loss of about 15% or more from baseline; and / or about 1.8 or about 2.4 mg of pembidutide is administered to a population of humans for at least 12 weeks, and at week 12, at least about 20% of the population experiences a body weight reduction of about 15% or more from baseline; The method according to any one of claims 1 to 3.

11. 11. The method of claim 10, wherein the mean change in blood pressure from baseline for the population is reduced.

12. 12. The method of claim 10 or 11, wherein the mean change in blood total cholesterol from baseline for said population is reduced by at least about 10%.

13. 13. The method of any one of claims 10-12, wherein the mean change in blood low density lipoprotein (LDL) from baseline for the population is reduced by at least about 5%.

14. 14. The method of any one of claims 10-13, wherein the mean change in blood high density lipoprotein (HDL) from baseline for the population is reduced by at least about 15%.

15. 15. The method of any one of claims 10-14, wherein the mean change in blood triglycerides from baseline for the population is reduced by at least about 15%.

16. 16. The method of any one of claims 10 to 15, wherein the mean change in waist circumference of the population is reduced by at least about 8%.

17. 17. The method of any one of claims 1 to 16, wherein the pembidutide induces a significant increase in ABCG5 (p<0.005) and ABCG8 (p<0.05) gene expression compared to baseline.

18. 18. The method of any one of claims 1-17, wherein the pembidutide is administered once weekly in an amount of about 1.8 mg.

19. 19. The method of any one of claims 1-18, wherein the pembidutide is administered once weekly in an amount of about 2.4 mg.

20. 20. The method of any one of claims 1-19, 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.

21. 21. The method of any one of claims 1 to 20, wherein the human has type 2 diabetes and / or the cardiovascular disease is atherosclerotic cardiovascular disease.

22. The human has a body mass index (BMI kg / m) of at least 25, at least about 28, or at least about 30. ２ 22. The method according to claim 1, wherein

23. 23. The method of any one of claims 1 to 22, wherein the pembidutide is administered by parenteral injection.

24. 24. The method of any one of claims 1 to 23, wherein the pembidutide is administered by subcutaneous injection.

25. 25. The method of any one of claims 1 to 24, wherein the steady state dose is achieved after a dose escalation phase having a duration of 2 to 4 weeks.

26. 26. The method of any one of claims 1 to 25, wherein the pembidutide is administered from a liquid formulation comprising at least about 1.8 mg / ml of pembidutide.

27. 27. The method of any one of claims 1 to 26, wherein the pembidutide is administered as a liquid pharmaceutical formulation comprising SEQ ID NO: 1 and about 0.20% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in sterile water (pH 7.7±0.1).

28. 1. A method of reducing the risk of cardiovascular (CV) disease in a human, comprising the step of administering pembidutide to the human once weekly in an amount of at least 1.2 mg to at most 2.4 mg, at least one risk factor associated with CV disease is reduced and / or eliminated, said at least one risk factor being selected from the group consisting of excess body weight, high serum lipids, cholesterol, triglycerides, LDL and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine in said human's serum; Optionally, the human is overweight, obese, has a body mass index (BMI) of 25 or greater (kg / m ２ ) and / or have type 2 diabetes.

29. 1. A method of treating a cardiovascular (CV) disease risk factor, comprising administering to a patient in need thereof a pharmaceutical composition comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, The patient is overweight, obese, and has a body mass index (BMI) of 25 or greater. ２ ), and / or have type 2 diabetes, wherein the method reduces at least one CV disease risk factor selected from the group consisting of body weight, waist circumference, blood pressure, hyperglycemia, serum lipids, total cholesterol, triglycerides, HDL, LDL, and / or VLDL particle concentration and / or diameter, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, sphingolipids, and / or lysophosphatidylcholine compared to baseline.

30. 30. The method of claim 29, wherein the patient in need of treatment is overweight.

31. 30. The method of claim 29, wherein the patient in need of treatment is obese.

32. The patient in need of said treatment has a body mass index (BMI kg / kg) of 25 or greater. ２ 30. The method of claim 29, wherein

33. 30. The method of claim 29, wherein the patient in need of treatment has type 2 diabetes.

34. 30. The method of claim 29, wherein the pharmaceutical composition is administered once weekly in an amount of about 1.2 mg, about 1.8 mg, or about 2.4 mg.

35. 30. The method of claim 29, wherein the pharmaceutical composition is administered about once weekly in an amount of about 1.8 mg.

36. 30. The method of claim 29, wherein the pharmaceutical composition is administered about once weekly in an amount of about 2.4 mg.

37. 30. The method of claim 29, 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.

38. 30. The method of claim 29, wherein the pharmaceutical composition is administered once a week for about 5 to 12 weeks.

39. 30. The method of claim 29, wherein the pharmaceutical composition is administered by parenteral injection.

40. 30. The method of claim 29, wherein the pharmaceutical composition is administered by subcutaneous injection.

41. 30. The method of claim 29, wherein the pharmaceutical composition is administered from a liquid formulation comprising at least about 1.8 mg / ml of pembidutide.

42. 42. The method of any one of claims 29-41, wherein the pharmaceutical composition is administered as a liquid pharmaceutical formulation comprising SEQ ID NO:1 and about 0.20% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in sterile water (pH 7.7±0.1).

43. 1. A method of treating a cardiovascular (CV) disease risk factor, comprising administering to a patient in need thereof a pharmaceutical composition comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, The patient is overweight, obese, and has a body mass index (BMI) of 25 or greater. ２ ), and / or have type 2 diabetes, wherein the method reduces at least one of plasma triglyceride, total plasma cholesterol, plasma LDL-cholesterol, or plasma HDL-cholesterol levels by about any of 3%, 5%, 7%, or 10% from baseline.

44. 44. The method of claim 43, wherein the plasma triglycerides are reduced from baseline.

45. 44. The method of claim 43, wherein the total plasma cholesterol is reduced from baseline.

46. 44. The method of claim 43, wherein the plasma LDL-cholesterol is reduced from baseline.

47. 44. The method of claim 43, wherein the plasma HDL-cholesterol is reduced from baseline.

48. 1. A method of reducing plasma lipids in a patient in need thereof, comprising the step of administering to said patient a pharmaceutical composition comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof, wherein said patient is suffering from cardiovascular disease, and said method reduces at least one of plasma triglyceride, total plasma cholesterol, plasma LDL-cholesterol, or plasma HDL-cholesterol levels by 3%, 5%, 7%, or 10% from baseline.

49. 49. The method of claim 48, wherein one or more pathogenic serum lipid mediators are reduced from baseline.

50. 1. A method of reducing the risk of cardiovascular death and / or hospitalization, comprising administering to a patient in need thereof a pharmaceutical composition comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, The patient is overweight, obese, and has a body mass index (BMI) of 25 or greater. ２ ), and / or have type 2 diabetes, wherein the method reduces at least one of plasma triglyceride, total plasma cholesterol, plasma LDL-cholesterol, or plasma HDL-cholesterol levels by about any of 3%, 5%, 7%, or 10% from baseline.

51. 51. The method of claim 50, wherein the plasma triglycerides are reduced from baseline.

52. 51. The method of claim 50, wherein the total plasma cholesterol is reduced from baseline.

53. 51. The method of claim 50, wherein the plasma LDL-cholesterol is reduced from baseline.

54. 51. The method of claim 50, wherein the plasma HDL-cholesterol is reduced from baseline.

55. 1. A method of reducing major adverse cardiac events (MACEs), comprising administering a pharmaceutical composition comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof to a patient in need thereof, wherein the patient is overweight, obese, and has a body mass index (BMI) of 25 or greater (kg / m). ２ ), and / or have type 2 diabetes, wherein the method reduces at least one of plasma triglyceride, total plasma cholesterol, plasma LDL-cholesterol, or plasma HDL-cholesterol levels by about any of 3%, 5%, 7%, or 10% from baseline.

56. 56. The method of claim 55, wherein the plasma triglycerides are reduced from baseline.

57. 56. The method of claim 55, wherein the total plasma cholesterol is reduced from baseline.

58. 56. The method of claim 55, wherein the plasma LDL-cholesterol is reduced from baseline.

59. 56. The method of claim 55, wherein the plasma HDL-cholesterol is reduced from baseline.

60. 56. The method of claim 55, wherein one or more of acute myocardial infarction (AMI), stroke, or cardiovascular mortality is reduced following administration of the pharmaceutical composition.

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