Glp-1r and gcgr agonists, formulations, and methods of use

Abstract

To provide a simple method of dosing at a therapeutic dose to control blood glucose levels and / or induce weight loss without the need for titration to reach a therapeutic level in the absence of gastrointestinal side effects.SOLUTION: A liquid pharmaceutical dosage formulation is provided comprising an agonist peptide product selected from any one of the specific sequence groups, wherein the peptide is modified with a non-ionic glycolipid surfactant, and at least 0.66 milligrams of polysorbate 20 per milligram of peptide in the liquid pharmaceutical dosage formulation.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application Nos. 62 / 980,093, filed February 21, 2020, 63 / 122,108, filed December 7, 2020, and 63 / 133,540, filed January 4, 2021, each of which is incorporated herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. This ASCII copy, created on February 19, 2021, is named MED007PCT_ST25.TXT and is 24,576 bytes in size.

[0003] Field of Disclosure The present disclosure relates to the field of GLP-1R and GCGR agonists, formulations, and methods of use thereof. [Background technology]

[0004] Background to the disclosure The increasing prevalence of obesity, diabetes mellitus, nonalcoholic fatty liver disease (NAFLD), and its advanced form, nonalcoholic steatohepatitis (NASH), constitutes part of a global health epidemic, which is a major cause of patient morbidity and mortality and a major economic burden. Obesity is a significant risk factor for type 2 diabetes and NASH, and approximately 90% of type 2 diabetes patients are overweight or obese. Obesity is a rapidly growing problem worldwide, with more than 65% of U.S. adults currently overweight (Hedley, AA, et al. (2004) JAMA 291:2847-2850). NASH is expected to become the leading cause of liver transplants in the near future. There is a need for the development of safe and effective pharmaceutical treatments for obesity and diabetes mellitus. The present disclosure provides improved peptide pharmaceuticals for the treatment of obesity- and / or diabetes-related disorders, such as nonalcoholic steatohepatitis (NASH) and polycystic ovary syndrome (PCOS).

[0005] In the United States (US), NASH is the leading cause of end-stage liver disease or liver transplantation. Obesity is a central driver of NASH, and weight loss results in reduction of liver fat and amelioration of NASH. Because more than 80% of NASH patients are overweight or obese and there are no currently available US Food and Drug Administration (FDA)-approved pharmacological options for inducing weight loss, treatment has primarily been based on lifestyle interventions aimed at achieving weight loss. However, achieving and sustaining long-term weight loss through lifestyle changes alone is difficult.

[0006] Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are associated with modest weight loss at approved doses, and these agents have emerged as treatment options for patients with NASH. A recent clinical trial showed that daily administration of the GLP-1RA liraglutide was associated with resolution of NASH and a trend toward improvement in liver fibrosis. However, patients only lost 5.5% of their body weight. One study found that optimal resolution of NASH required a weight loss of 10% or more. Higher levels of weight loss have also been associated with reduced incidence of cardiovascular disease and nonhepatic malignancies, which represent the most serious comorbidities faced by patients with NASH.

[0007] While GLP-1RA exerts a central effect on appetite and food intake, GCR agonists promote increased energy expenditure in animal models and humans. The effects of GCR agonists and GLP-1RA have been shown to be synergistic in promoting greater weight loss compared with GLP-1RA alone. GCR also enhances lipolysis and suppresses hepatic lipogenesis, providing an additional pathway for liver fat reduction and resolution of NASH.

[0008] Dual agonists combine GLP-1R and GLP-1RA in the same molecule. In obese non-human primates, long-term administration of a GLP-1R / GCR dual agonist reduced body weight and significantly improved glucose tolerance compared with a GLP-1RA monoagonist. Clinical studies of cotadutide, a GLP-1 / GCR dual agonist with a 5:1 bias ratio of GLP-1 to glucagon activity, demonstrated an impressive 39% reduction in liver fat in just 6 weeks and a greater reduction in NASH-related alanine aminotransferase (ALT) activity than liraglutide alone. However, the extent of weight loss with cotadutide treatment over 26 weeks was comparable to that of liraglutide (5.4% vs. 5.5%), suggesting that the 5:1 ratio, while acceptable for liver fat reduction, was suboptimal for weight loss. Balanced (1:1) agonism has been shown to be associated with greater weight loss and metabolic benefits than skewed ratios favoring one agonist over the other. A recent study using the balanced dual agonist JNJ 64565111 resulted in an 8% weight loss in just 12 weeks (NCT03586830).

[0009] Unfortunately, GLP-1RAs are associated with high rates of nausea, vomiting, and diarrhea. These drugs must be titrated over time to reduce side effects, and there is a need for drugs with improved tolerability and dosing regimens. Therefore, there remains a need for convenient dosing (e.g., weekly rather than daily) at therapeutic doses to control blood glucose levels and / or induce weight loss without the need for titration to reach therapeutic levels in the absence of gastrointestinal side effects. Summary of the Invention

[0010] Disclosure Overview Described herein are dual agonist peptides and their products (e.g., formulations), as well as their uses in treating disorders associated with glucagon-like peptide 1 receptor (GLP-1R) and glucagon receptor (GCGR) function, such as type 2 diabetes, metabolic syndrome, cardiovascular disease (including coronary artery disease such as atherosclerosis and myocardial infarction), hypertension, NASH, chronic kidney disease, PCOS, and conditions associated with such disorders, including, but not limited to, insulin resistance and / or obesity. Such dual agonist peptides have affinity for both GLP-1R and GCGR, as can be determined, for example, by the cellular assays described herein or using a separate assay for making such a determination. In some embodiments, the dual agonist peptide is any of SEQ ID NOS: 1-10 or 12-27, or derivatives thereof, e.g., conservatively substituted derivatives thereof, and / or combinations thereof. In some embodiments, the dual agonist peptide exhibits approximately equal affinity for GLP-1R and GCGR, as can be determined using the cellular assay described above, which in a preferred embodiment is SEQ ID NO: 1 or a derivative thereof.

[0011] In some embodiments, the present disclosure provides pharmaceutical dosage formulations of such dual agonist peptides configured to improve glycemic control by reducing one or more adverse events compared to agonists (e.g., semaglutide) that have disproportionate affinities for GLP-1R and GCGR or that have excessively high maximum blood concentrations (Cmax) after administration. In some embodiments, the present disclosure provides pharmaceutical dosage formulations of such dual agonist peptides configured to induce weight loss by reducing one or more adverse events compared to agonists that have disproportionate affinities for GLP-1R and GCGR. In some embodiments, the adverse events are selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to a mammal. These adverse events are typically observed when the (dual) agonist enters the circulation rapidly after administration, resulting in an excessively high Cmax. In contrast, the pharmaceutical dosage formulations of the present invention provide a therapeutic dose for controlling blood glucose levels and / or treating obesity by inducing weight loss, while reducing or eliminating dose-related adverse events, such as gastrointestinal (GI) adverse events. In some embodiments, administration of the dual agonist peptide(s) disclosed herein (e.g., SEQ ID NOs: 1-10 or 12-27 or derivatives thereof) may result in other results (e.g., weight loss, fat loss, lipid reduction) and / or improved pharmacokinetic (PK) parameters when compared with agonists with imbalanced affinity for GLP-1R and GCGR (e.g., semaglutide). Other aspects of the present disclosure are also contemplated, as will be understood by those skilled in the art from this disclosure. [Brief explanation of the drawings]

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and examples sections, serve to explain the principles and implementations of the present disclosure. [Figure 1] Glycemic response to subcutaneous (SC) injection of semaglutide or SEQ ID NO: 1 (db / db mice). [Figure 2]Blood glucose response to semaglutide or SEQ ID NO: 1 (diet-induced obese (DIO) mice). [Figure 3] Blood glucose levels IPGTT Semaglutide or SEQ ID NO: 1 (DIO mice). [Figure 4] Body weight response (% of day 0), SC injection of semaglutide or SEQ ID NO: 1 (db / db mice, leptin receptor-deficient mice). [Figure 5] Feeding response to subcutaneous (SC) injection of semaglutide or SEQ ID NO: 1 (db / db mice). [Figure 6A] Body weight response (% of day 0) (Figure 6A) and body weight response (g of day 0) (Figure 6B). Subcutaneous (SC) injection of semaglutide or SEQ ID NO: 1 (17) (DIO mice). [Figure 6B] Body weight response (% of day 0) (Figure 6A) and body weight response (g of day 0) (Figure 6B). Subcutaneous (SC) injection of semaglutide or SEQ ID NO: 1 (17) (DIO mice). [Figure 7] Delta Fat Mass and Delta Lean Mass after administration of semaglutide or SEQ ID NO: 1. [Figure 8] Semaglutide and SEQ ID NO: 1 ligand concentrations measured over 120 hours for a single dose administered subcutaneously (SC) in DIO mice. [Figure 9] Semaglutide and SEQ ID NO: 1 (ALT-801) ligand concentrations measured over 96 hours for a single dose administered subcutaneously (SC) to C57BL / 6J mice. [Figure 10] Semaglutide and SEQ ID NO: 1 ligand concentrations measured over 144 hours for a single dose in rats. [Figure 11] Ligand concentrations of SEQ ID NO: 1 measured over 360 hours for a single dose administered intravenously (IV) or subcutaneously (SC) to Yucatan miniature pigs. [Figure 12A]Plasma ligand concentrations (ng / mL) of SEQ ID NO: 1 measured over 192 hours after three doses (10 nmol / kg (FIG. 12B), 20 nmol / kg (FIG. 12C), 40 nmol / kg (FIG. 12D)) administered subcutaneously (SC) to cynomolgus monkeys (FIG. 12A). [Figure 12B] Plasma ligand concentrations (ng / mL) of SEQ ID NO: 1 measured over 192 hours after three doses (10 nmol / kg (FIG. 12B), 20 nmol / kg (FIG. 12C), 40 nmol / kg (FIG. 12D)) administered subcutaneously (SC) to cynomolgus monkeys (FIG. 12A). [Figure 12C] Plasma ligand concentrations (ng / mL) of SEQ ID NO: 1 measured over 192 hours after three doses (10 nmol / kg (FIG. 12B), 20 nmol / kg (FIG. 12C), 40 nmol / kg (FIG. 12D)) administered subcutaneously (SC) to cynomolgus monkeys (FIG. 12A). [Figure 12D] Plasma ligand concentrations (ng / mL) of SEQ ID NO: 1 measured over 192 hours after three doses (10 nmol / kg (FIG. 12B), 20 nmol / kg (FIG. 12C), 40 nmol / kg (FIG. 12D)) administered subcutaneously (SC) to cynomolgus monkeys (FIG. 12A). [Figure 13] Body weight change in male cynomolgus monkeys treated with SEQ ID NO: 1 (0.03 mg / kg-0.25 mg / kg). [Figure 14] Body weight change in female cynomolgus monkeys treated with SEQ ID NO: 1 (0.03 mg / kg-0.25 mg / kg). [Figure 15] Body weight of treatment groups (NASH mice) with SEQ ID NO: 1 (ALT-801) compared to semaglutide and elafibranor. [Figure 16] Change in NAFLD activity score under treatment with SEQ ID NO: 1 (ALT-801) compared to semaglutide and elafibranor. [Figure 17] Treatment with SEQ ID NO: 1 (ALT-801) improved liver morphology, liver weight, NAS, and fibrosis compared to semaglutide and elafibranor. [Figure 18]Mean terminal liver TG, liver TC, and plasma ALT with SEQ ID NO: 1 (ALT-801) compared to semaglutide and elafibranor. [Figure 19] Regulation of gene expression by ALT-801 (SEQ ID NO: 1). [Figure 20] Regulation of genes affecting fat utilization and transport after treatment with SEQ ID NO: 1 (ALT-801) and semaglutide. [Figure 21] Regulation of profibrotic, cell death, and inflammatory genes in hepatic stellate cell pathways after treatment with SEQ ID NO: 1 (ALT-801) and semaglutide. [Figure 22] In vitro stability in human plasma. See Table 14. [Figure 23] In vivo pharmacokinetic behavior of the compound after subcutaneous administration to Göttingen minipigs. [Figure 24] In vivo PK behaviour of SEQ ID NO: 1 and semaglutide after subcutaneous (sc) administration. [Figure 25] In vivo pharmacokinetic behaviour of SEQ ID NO: 1 after a single subcutaneous (sc) and intravenous (iv) administration at 20 nmol / kg to male minipigs (n=4, weighing approximately 75 kg). [Figure 26] In vivo dose response behavior of 17 (SEQ ID NO: 1) and the literature standard semaglutide after single dose subcutaneous (sc) administration in male db / db mice (n=9). [Figure 27] Body weights of DIO rats (n=9) during 28 days of treatment (followed by recovery) with vehicle, literature standard semaglutide (12 nmol / kg), SEQ ID NO: 1 (6 and 12 nmol / kg), and paired experimental groups fed to the amount of food consumed by animals in the 12 nmol / kg semaglutide and SEQ ID NO: 1 groups. [Figure 28] Cumulative food consumption by DIO rats during 27 days of treatment with vehicle, literature standard semaglutide (12 nmol / kg), SEQ ID NO: 1 (6 and 12 nmol / kg), and paired experimental groups fed up to the amount of food consumed by animals in the 12 nmol / kg semaglutide group or SEQ ID NO: 1 group. [Figure 29]DIO rats during 27 days of treatment in response to daily subcutaneous (sc) doses of vehicle, literature standard semaglutide (12 nmol / kg), SEQ ID NO: 1 (6 and 12 nmol / kg), and daily food consumption by paired experimental groups fed up to the amount of food consumed by animals treated with daily sc 12 nmol / kg semaglutide or SEQ ID NO: 1 groups. [Figure 30] Surface tension data for ALT-801 in pure water. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description of Disclosure The present disclosure relates to dual agonist peptides, as well as 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), as can be determined using a cellular assay, and in preferred embodiments, have approximately equal affinity. In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to control blood glucose levels. In some embodiments, blood glucose levels are better controlled (e.g., lowered and stabilized) after administration of the dual agonist peptide compared to selective (e.g., semaglutide) and / or unbalanced agonists. In some embodiments, the present disclosure provides pharmaceutical dosage formulations configured to induce weight loss. In some embodiments, weight loss is improved (e.g., lowered and / or stabilized) after administration of the dual agonist peptide compared to selective (e.g., semaglutide) and / or unbalanced agonists. In some embodiments, such pharmaceutical dosage formulations exhibit reduced adverse events compared to agonists with selective and / or disproportionate affinity for GLP-1R and GCGR (e.g., semaglutide). In some embodiments, adverse events may include nausea, vomiting, diarrhea, abdominal pain, and / or constipation, which are typically observed after administration of agonists with disproportionate affinity for GLP-1R and GCGR (e.g., semaglutide) to mammals. In some embodiments, the present disclosure provides novel peptide-based dual GLP1 / glucagon receptor agonists designed to treat the underlying metabolic dysfunction that leads to nonalcoholic steatohepatitis (NASH).

[0014] In some embodiments, the dual agonist peptide is any one of SEQ ID NOs: 1-10 or 12-27, or a derivative thereof.

[0015] In a preferred embodiment, the dual agonist peptide is EU-A1873 (SEQ ID NO: 1), EU-A1588 (SEQ ID NO: 2), EU-A1871 (SEQ ID NO: 3), or EU-A1872 (SEQ ID NO: 4), as shown in Table 1.

[0016] [Table 1]

[0017] In Table 1, the numbers 1, 5, 10, 15, 20, 25, 30 at the top refer to the amino acid residue numbers (a total of 29 amino acid residues are present in each of SEQ ID NOS: 1-5). Semaglutide shown in Table 1 is SEQ ID NOS: 11 (31 amino acid residues). As shown in Table 1, SEQ ID NOS: 1 (EU-A1873 in Table 1, where ALT-801 is the active pharmaceutical ingredient (API) present in the disclosed pharmaceutical formulation, and the API is represented by SEQ ID NOS: 1) has the following amino acid sequence conjugated to a non-ionic glycolipid surfactant at amino acid position 17 (aa17): [ka] where: * indicates the formation of a lactam bridge between Glu16 and Lys20, and 17Lys # indicates the binding site of glucuronic acid C-18 (EuPort, Z17CO2H, also referred to herein as GC18c). As another example, SEQ ID NO: 1 contains 29 amino acid residues and 17 Glucuronic acid / C linked to Lys 18 A peptide amide consisting of a diacid moiety, wherein: 16 Glu and 20 The side chains of Lys form an intramolecular cycle as shown below. [ka]

[0018] In some embodiments, the dual agonist peptide can be any of the following: [ka] or a derivative thereof, wherein Xaa1 is any amino acid, preferably Aib (α-aminoisobutyric acid (or 2-methylalanine or C alpha-methylalanine)), Xaa2 is Lys (N-omega(1-(17-carboxyl-heptadecyloxy)beta-D-glucuronyl)) or Lys(Z17CO2H), wherein Z17CO2H(EuPort) is (beta-D-glucuron-1-yl)-1-oxa)17-carboxyheptadecane, and Glu16 and Lys20 are cyclized to each other through their respective side chains to form a lactam bond; [ka] or a derivative thereof, wherein Xaa1 is any amino acid, preferably Aib (α-aminoisobutyric acid (or 2-methylalanine or Cα-methylalanine)), Xaa2 is Me17CO2H, which is beta-D-merobiuranyl-1-yl)-1-oxa)17-carboxyheptadecane, and Glu16 and Lys20 are cyclized to each other through their respective side chains to form a lactam bond; [ka] or a derivative thereof, wherein Xaa1 is any amino acid, preferably Aib (α-aminoisobutyric acid (or 2-methylalanine or C alpha-methylalanine)), Glu16 and Lys20 are cyclized to each other through their respective side chains to form a lactam bond, and Xaa3 is Lys(Z15CO2H), wherein Z15CO2H is (beta-D-glucuron-1-yl)-1-oxa)15-carboxyheptadecane; [ka] or a derivative thereof, wherein Xaa1 is any amino acid, preferably Aib (α-aminoisobutyric acid (or 2-methylalanine or C alpha-methylalanine)), Glu16 and Lys20 are cyclized to each other through their respective side chains to form a lactam bond, and Xaa4 is Lys(Z17CO2H), wherein Z17CO2H is (beta-D-glucuron-1-yl)-1-oxa)17-carboxyheptadecane, or [ka] or a derivative thereof, wherein Xaa1 is any amino acid, preferably Aib (α-aminoisobutyric acid (or 2-methylalanine or C alpha-methylalanine)), Xaa2 is Lys(N-omega(1-(17-carboxyl-heptadecyloxy)beta-D-glucuronyl)), wherein Lys(ZCOH) is (beta-D-glucuron-1-yl)-1-oxa)17-carboxyheptadecane, and Xaa5 is Arg, and Glu16 and Lys20 cyclize to each other via their respective side chains to form a lactam bond.

[0019] In some embodiments, the dual agonist peptide is selected from the group consisting of SEQ ID NOs: 1 and 12-27 shown below. [ka] Analogs marked with an asterisk have a side chain lactam from Glu16 to Lys20; the bracketed G, M, and Me refer to D-glucosidic, D-maltosidic, and D-melibiosidic bonds, respectively, and S1 and S2 refer to α-Lys or γ-Glu residues, respectively. Cn refers to an n-carbon methylene chain, and c refers to the carboxylate at the end of the chain. X in semaglutide refers to a Lys residue acylated with the γGlu-2×OEG (see Reference 27) elongation modifier, which contains octadecanoic acid on the γGlu / short PEG spacer. Compound #33 in Reference 8 refers to compound #32 alkylated on Cys24 with 40 kDa PEG via a maleimide linker.

[0020] In a preferred embodiment, the dual agonist peptide is a peptide having the amino acid sequence of any one of SEQ ID NOS: 1-10 or 12-27, or a derivative thereof. In a preferred embodiment, the dual agonist peptide is SEQ ID NO: 1. In some embodiments, the dual agonist peptide is formulated as an injectable solution containing pharmaceutically acceptable excipients, such as a tonicity adjuster or salt, a buffer, a stabilizer and / or surfactant, a pH adjuster, and a solvent. In some embodiments, the tonicity adjuster is mannitol, sorbitol, glycerol, glycine, propylene glycol, or sodium chloride. In some embodiments, the buffer is histidine, arginine, lysine, phosphate, acetate, carbonate, bicarbonate, citrate, meglumine, or Tris. In some embodiments, the stabilizer is histidine, arginine, or lysine. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the pH adjuster is hydrochloric acid and / or sodium hydroxide. In a preferred embodiment, the tonicity adjusting agent is mannitol, the buffering and stabilizing agent is arginine, and the surfactant is polysorbate 20. In some embodiments, the dual agonist peptide is in a pharmaceutical dosage formulation comprising 0.025-0.15% (w / w) polysorbate 20, about 0.2-0.5% (w / w) arginine, and about 3-6% (w / w) mannitol in deionized water (pH 7.7±1.0). In some embodiments, the pharmaceutical dosage formulation comprises "ALT-801" represented by SEQ ID NO: 1 in a formulation comprising, consisting essentially of, or consisting of about 0.050% (w / w) polysorbate 20, about 0.35% (w / w) arginine, and about 4.3% (w / w) mannitol in deionized water (pH 7.7±1). As used herein, the test article formulation is also referred to as the F58 formulation. See Example 4.In a preferred embodiment, the pharmaceutical dosage formulation of "ALT-801" comprises SEQ ID NO: 1 in a formulation comprising, consisting essentially of, or consisting of about 0.35% (w / w) arginine and about 4.3% (w / w) mannitol, 0.6-1.0 mg of polysorbate 20 per mg of "ALT-801" (SEQ ID NO: 1), or 1.0-1.5 mg of polysorbate 80 per mg of "ALT-801" (SEQ ID NO: 1). See Example 8. In some embodiments, the pharmaceutical dosage formulation comprises "ALT-801" at a concentration ranging from 0.05 mg / ml to 20 mg / ml, preferably from 0.1 mg / ml to 10 mg / ml, or more preferably from 0.5 mg / ml to 10 mg / ml. In some embodiments, the pH of the pharmaceutical dosage formulation comprising "ALT-801" is 6-10, more preferably 6-8.

[0021] The synthesis of dual agonist peptides containing nonionic glycolipid surfactants (e.g., SEQ ID NOS: 1-10 or 12-27, or derivatives thereof) is described herein (e.g., Example 1) and in U.S. Pat. No. 9,856,306 B2, the entire contents of which are incorporated by reference herein. In some embodiments, the dual agonist peptides can contain one or more conservatively substituted amino acids as described herein. In a preferred embodiment, SEQ ID NOS: 1 can contain one or more conservatively substituted amino acids, but preferably not at amino acid residues 16, 17, or 20. In a preferred embodiment, SEQ ID NOS: 2 can contain one or more conservatively substituted amino acids, but preferably not at amino acid residues 16, 17, or 20. In a preferred embodiment, SEQ ID NOS: 3 can contain one or more conservatively substituted amino acids, but preferably not at amino acid residues 16, 20, or 24. In preferred embodiments, SEQ ID NO: 4 may contain one or more conservatively substituted amino acids, but preferably not at amino acid residues 16, 20, or 24, and SEQ ID NO: 5 may contain one or more conservatively substituted amino acids, but preferably not at amino acid residues 12, 16, 17, or 20.

[0022] The peptides of SEQ ID NOs: 1-10 or 12-27 can be collectively referred to herein as "dual agonist peptides" (or individually as "dual agonist peptides") because they are agonists of the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), respectively. In some embodiments, the peptides are dual agonists of GLP-1R and GCGR, as determined by a cellular assay such as that described in Example 2 herein. Briefly, in some embodiments, the cellular assay can be performed by measuring cAMP stimulation or arrestin activation in CHO cells in which human GLP-1R or GCGR is expressed (LeadHunter assay (DiscoveRx)). Because the dual agonist peptides of SEQ ID NOs: 1-10 or 12-27 can bind very strongly to serum albumin (>99%), potentially skewing results, such assays are preferably performed in the presence of 0.1% ovalbumin, as compared to 0.1% bovine serum albumin (BSA), which may be typical (see, e.g., Example 2 herein). In some embodiments, as determined using such assays, the dual agonist peptides can have affinity for both GLP-1R and GCGR, and in preferred embodiments, can have approximately equal affinity for GLP-1R and GCGR. "Approximately equal affinity" means that the dual agonist peptides have an affinity for either GLP-1R or GCGR that is no more than about 2-3 times, and preferably no more than 2-fold, their affinity for the other, as determined by such cellular assays. For example, as shown in the Examples herein, the dual agonist peptide SEQ ID NO: 1 (EU-A1873) was surprisingly found to be a dual agonist peptide with approximately equal affinity for GLP-1R and GCGR (e.g., an EC50 of approximately 39 pm for GLP-1R (115% intrinsic activity) and 44 pm for GCGR (115% intrinsic activity)).This is different from GLP-1 "specific" compounds, including semaglutide and exendin-4, which exhibit a strongly biased affinity toward GLP-1R or only toward GLP-1R, or the hormone glucagon, which does not exhibit high or nearly equal affinity to both GLP-1R and GCGR, and is strongly biased toward GCGR.The natural hormone oxyntomodulin has agonistic action on both GLP-1R and GDGR, but this action is not strong and unbalanced.Those skilled in the art will understand that affinity for GLP-1R and GCGR can be determined by methods and / or assays other than those described herein, and that such methods and / or assays for determining affinity are contemplated herein (for example, the determination of nearly equal affinity can be performed by such other methods and / or assays).

[0023] In embodiments, as used herein, "a dual agonist peptide having approximately equal affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR)" means that the dual agonist peptide has an affinity for GLP-1R or GCGR that is no more than about 2-fold greater than its affinity for the other, as determined by such cellular assays. In embodiments, the binding affinity of the dual agonist peptide for one receptor is no more than 1.9, 1.8, 1.6, 1.5, 1.4, or 1.2-fold greater than its affinity for the other receptor, as determined by known cellular assays. In embodiments, "an agonist with disproportionate affinity for GLP-1R and GCGR" as used herein means an agonist peptide having an affinity for GLP-1R or GCGR that is at least about 1.5-fold greater than its affinity for the other, as determined by known cellular assays. In embodiments, the binding affinity of the agonist with disproportionate affinity for GLP-1R and GCGR is at least 1.6, 1.8, 2, 2.5, 3, 5, 7.5, 10, 20 fold, or more, as can be determined by known cellular assays.

[0024] A "peptide" (e.g., a dual agonist peptide) comprises two or more naturally occurring and / or unnatural amino acid residues, usually linked via a peptide bond. Such amino acids may include naturally occurring structural variants, naturally occurring non-proteinogenic amino acids, or / and synthetic non-naturally occurring analogs of natural amino acids. The terms "peptide" and "polypeptide" are used synonymously. Peptides include short peptides (about 2-20 amino acids), intermediate-length peptides (about 21-50 amino acids), and long peptides (more than about 50 amino acids, sometimes also referred to as "proteins"). In some embodiments, the peptide product comprises a surfactant moiety covalently and stably attached to a peptide of about 50, 40, or 30 or fewer amino acids. 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 notation is used herein to depict 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. Unless otherwise specified as D- or DL-, abbreviations used in the amino acid sequences disclosed herein represent L-amino acids, or amino acids are achiral, although the equivalent D-isomers can generally be used at any position (e.g., to resist proteolysis). Other amino acid abbreviations used herein include: Aib = α-aminoisobutyric acid (or 2-methylalanine or Ca-methylalanine); Xaa: any amino acid, generally as specifically defined within the formula. Other amino acid abbreviations that can be used as described herein include: Aib = α-aminoisobutyric acid (or 2-methylalanine or Ca-methylalanine); Xaa = any amino acid, generally as specifically defined within the formula.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; 2FMePhe or 2Fα MePhe = Cα-methyl-(2-fluorophenyl)alanine; hArg = homoarginine; MeLys or αMeLys = Ca-methyllysine; MePhe or aMePhe = Ca-methylphenylalanine; MePro or αMePro = Cα-methylproline; Na1 or Nal(1) = 3-(1-naphthyl)alanine; Na1 or Nal(2) = 3-(2-naphthyl)alanine; N1e = norleucine; Orn = ornithine; and Tmp = (2,4,6-trimethylphenyl)alanine; 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and the Tic-Phe dipeptide moiety with a reduced interresidue amide bond (designated as Tic-Ψ[CF12-NF1]-Ψ-Phe) have the following structures: [ka]

[0025] Unless specifically stated otherwise or unless the context clearly dictates otherwise, the present disclosure encompasses any and all forms of dual agonist peptides that may be produced, whether the dual agonist peptides are produced synthetically (e.g., using a peptide synthesizer) or cellularly (e.g., by recombinant production). Such forms of dual agonist peptides may include one or more modifications that may occur during the synthetic or cellular production of the peptide, such as one or more post-translational modifications, whether or not the one or more modifications are intentional. Dual agonist peptides can have the same type of modification in two or more different locations, or / and two or more different types of modification. Modifications that may occur during synthetic 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), lipid modification, 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 at the N-terminus, C-terminus, one or more amino acid side chains, or the dual agonist peptide backbone, or any combination thereof. In some embodiments, the dual agonist peptide is acetylated at the N-terminus and / or has a carboxamide (-CONH) group at the C-terminus, which can increase the stability of the dual agonist peptide.

[0026] 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 non-natural amino acids, or the substitution of one or more natural and / or non-natural amino acids, or any or all combinations thereof. Substitutions may be conservative or non-conservative. Such modifications may be intentional, such as through site-directed mutagenesis or during chemical synthesis of the dual agonist peptide, or may be accidental, such as through mutations occurring in the host cell producing the dual agonist peptide or errors during PCR amplification. The non-natural amino acid may have the same chemical structure as the corresponding natural amino acid but with a D-stereochemical configuration, or may have a different chemical structure and D- or L-stereochemical configuration. Non-natural amino acids can be used, for example, to promote α-helix formation and / or increase the stability of the dual agonist peptide (e.g., to resist proteolysis). Peptides with one or more modifications relative to a reference dual agonist peptide may be referred to as "analogs" or "variants" of the reference peptide, as appropriate. An "analog" typically retains one or more essential properties of the reference 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, and / or may have a different biological activity. Such variants preferably retain the 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 an amino acid sequence that differs from the reference dual agonist peptide.

[0027] The term "conservative substitution" refers to the substitution of an amino acid in a peptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In certain embodiments, each of the following groups comprises 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, each of the following groups comprises 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 classified 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.

[0028] Examples of unnatural or non-proteinogenic amino acids include, but are not limited to, alanine analogs (e.g., α-ethyl Gly [α-aminobutyric acid or Abu], α-n-propyl Gly [norvaline or Nva], α-tert-butyl Gly [Tbg], α-vinyl Gly [Vg or Vlg], α-allyl Gly [Alg], α-propargyl Gly [Prg], 3-cyclopropyl Ala [Cpa], and Aib), leucine analogs (e.g., norleucine, Nle), proline analogs (e.g., α-MePro), phenylalanine analogs {e.g., Phe(2-F), Phe(2-Me), Tmp, Bip, Bip(2'-Et-4'-OMe), NaIl, , Nal2, 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., J. Pept. Sci., 17:270-280 (2011). The α,α-disubstituted amino acids can constrain conformation and / or stabilize the α-helix. Reducing the amide bond between two residues (e.g., in Tic-Ψ[CH2-NH]-Ψ-Phe) can increase protease resistance and, for example, modify receptor binding. The present disclosure includes all pharmaceutically acceptable salts of the dual agonist peptides, including those with a positive overall charge, a negative overall charge, and no overall charge.

[0029] An "alkyl" group refers to an aliphatic hydrocarbon group. Alkyl groups can be saturated or unsaturated and can be straight-chained (linear), branched-chained, or cyclic. In some embodiments, an alkyl group is not cyclic. In some embodiments, an alkyl group contains 1-30, 6-30, 6-20, or 8-20 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, 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, the alkyl group as a substituent is a linear or branched C1-C6 alkyl, 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 such as n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl (including all isomeric forms such as n-pentyl), and hexyl (including all isomeric forms such as n-hexyl). In some embodiments, the alkyl group is attached to the Nα-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 group may be an alkyl or an aryl-substituted alkyl, such as benzyl or phenylethyl. One or two alkyl groups may be attached to the N-terminal Nα-atom. In some embodiments, the alkyl group is a 1-alkyl group attached to the C-1 position of a sugar (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 C-C 30 , C6-C 30 , 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, wherein R 1 is independently at each occurrence hydrogen, alkyl, or aryl, and R 1 and the nitrogen atom to which they are attached together form a heterocyclyl or heteroaryl ring; R 2 is independently at each occurrence alkyl, heterocyclyl, aryl, or heteroaryl; R 3 is independently at each occurrence hydrogen, alkyl, heterocyclyl, aryl, or heteroaryl; R 4is, independently at each occurrence, hydrogen or alkyl; and R 5is independently alkyl or aryl at each occurrence. In some embodiments, an alkyl group (e.g., an 1-alkyl group) is internally or / and 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 can be saturated or unsaturated and can be linear, branched, or cyclic. In certain embodiments, R contains 1-20, 1-10, or 1-6 carbon atoms. The acyl group may be optionally substituted with one or more groups such as halogen, 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 may be bonded to or fused to one or more saturated, partially unsaturated, or aromatic rings. In certain embodiments, the heterocyclyl or heterocyclic group has 3-15, 3-12, 3-10, 3-8, or 3-6 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 6-15, 6-12, or 6-10 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 may 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 such as trifluoromethyl), acyl, carboxyl, ester, amide, etc. The term "heteroaryl" refers to a monocyclic aromatic or polycyclic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, N, and S. The heteroaromatic ring can be bonded to or fused to one or more saturated, partially unsaturated, or aromatic rings, which may contain only carbon atoms or which may contain one or more heteroatoms. In certain embodiments, heteroaryl groups have 5-15, 5-12, or 5-10 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, pyrrole pyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl.

[0030] In some embodiments, for example, the dual agonist peptide can be associated with a sugar, such as in a pharmaceutically acceptable composition or lyophilizate. Sugars include monosaccharides, disaccharides, and oligosaccharides (e.g., trisaccharides, tetrasaccharides, etc.). Reducing sugars exist in equilibrium in cyclic and open-chain forms, with the cyclic form generally predominating. The functionalized sugar of the surfactant moiety has a functional group suitable for forming a stable covalent bond with an amino acid of the dual agonist peptide.

[0031] 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, is commensurate with a reasonable benefit-risk ratio, and is effective for its intended use. 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 in which a dual agonist peptide can be formulated contains polysorbate 20 (e.g., about 0.050% (w / w)), optionally methylparaben (e.g., about 0.300% (w / w)), arginine (e.g., about 0.348% (w / w)), and mannitol (e.g., about 4.260% (w / w)) in distilled water (DI).

[0032] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to prevent, reduce the risk of development of, delay the onset of, slow the progression of, or cause regression of the medical condition being treated, or to alleviate to some extent the medical condition or one or more symptoms or complications thereof, in at least some of the subjects receiving the compound. The term "therapeutically effective amount" also refers to an amount of a compound sufficient to elicit the biological or medical response of a cell, tissue, organ, or human that is desired by a physician or clinician.

[0033] The terms "treat," "treating," and "treatment" include alleviating, ameliorating, inhibiting the progression of, reversing, or suppressing a medical disease, or one or more symptoms or complications associated therewith, and alleviating, ameliorating, or eradicating one or more causes of a medical disease. A reference to "treatment" of a medical disease includes prevention thereof. The terms "prevent," "preventing," and "prevention" include eliminating, reducing the risk of progression, and delaying the onset of a medical disease, or one or more symptoms or complications associated therewith. The term "medical condition" (or "condition" for brevity) includes diseases and disorders. The terms "disease" and "disorder" are used synonymously herein.

[0034] 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 can optionally contain an additional therapeutic agent. In some embodiments, the purity of the peptide product is at least about 90%, 95%, or 98%. 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, antimicrobial agents, 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]), isotonic solutions (e.g., Ringer's solution)), and organic solvents (e.g., dimethyl sulfoxide, and alcohols (e.g., ethanol, glycerol, and propylene glycol)). Except where any conventional excipient or carrier is incompatible with a peptide product, the present disclosure encompasses the use of conventional excipients and carriers in formulations containing peptide products.See, for example, 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).

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

[0036] In some embodiments, peptide products are administered parenterally by injection (e.g., subcutaneously, intravenously, or intramuscularly), which avoids the strongly 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 salt solution, buffered saline [e.g., PBS], balanced salt solutions [e.g., Ringer's BSS], and aqueous dextrose), isotonic / iso-osmotic agents (e.g., salts [e.g., NaCl, KCl, and CaCl2] and sugars [e.g., sucrose]), buffers, and pH adjusters (e.g., sodium phosphate dibasic [monobasic sodium phosphate] / disodium phosphate dibasic [dibasic sodium phosphate], citric acid / sodium citrate, and L-histidine / L-histidine HCl), and emulsifiers (e.g., non-ionic surfactants such as polysorbates [e.g., polysorbate 20 and 80] and poloxamers [e.g., poloxamer 188]). Peptide formulations and delivery systems are discussed, for example, in A. J. Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Ed., CRC Press (Boca Raton, Florida) (2015). Excipients may optionally include one or more substances that increase peptide stability, increase peptide solubility, inhibit peptide aggregation, reduce solution viscosity, or any or all combinations 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), sugars {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, β-alanine, and γ-aminobutyric acid], and betaines [e.g., trimethylglycine and trimethylamine]). N-oxides]), and non-ionic surfactants (e.g., alkyl polyglycosides, ProTek® alkyl saccharides (e.g., monosaccharides [e.g., glucose] or disaccharides [e.g., maltose or sucrose] conjugated to long-chain fatty acids or corresponding long-chain alcohols), and polypropylene glycol / polyethylene glycol block copolymers (e.g., poloxamers [e.g., Pluronic® F-68] and Genapol® PF-10, and their variants)). Such substances increase peptide solubility and can be used to increase peptide concentration in the formulation. Higher peptide concentrations in the formulation are particularly effective for subcutaneous administration, where bolus volumes are limited (e.g., approximately 1.5 mL or greater). In addition, such substances can be used to stabilize peptides during preparation, storage, and reconstitution of lyophilized peptides. An exemplary parenteral formulation includes the peptide product, mannitol, methionine, sodium thioglycolate, polysorbate 20, a pH adjuster (e.g., NaOH or / and HCl), and deionized water. Parenteral formulation excipients suitable for use with the dual agonist peptides described herein (e.g., various combinations of excipients including NaCl, etc.) are well known and available to those skilled in the art.

[0037] For parenteral (e.g., subcutaneous, intravenous, or intramuscular) administration, a sterile solution or suspension of the peptide product in an aqueous solvent containing one or more excipients can be prepared in advance and provided, for example, in a pre-filled syringe of a disposable pen or a pen with 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 before parenteral administration, the lyophilized peptide product stored in a suitable container (e.g., a vial) can be reconstituted, for example, with sterile water, optionally containing one or more excipients. In another embodiment, the peptide product is administered intranasally. The nasal mucosa offers a large surface area, a porous endothelium, a highly vascular epithelial layer, and a high absorption rate, thus enabling a high level of bioavailability. Intranasal formulations may include the peptide product along with 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 (e.g., hydroxyethylcellulose), or / and a penetration enhancer. Intranasal solution or suspension formulations can be administered to the nasal cavity by any suitable means, including, but not limited to, a dropper, pipette, or spray, for example, using a metered atomizing spray pump. Table 2 lists typical excipients for nasal spray formulations.

[0038] [Table 2]

[0039] In further embodiments, the peptide product is administered via the pulmonary route, such as by oral or nasal inhalation. Pulmonary administration of drugs can treat lung diseases or / and systemic disorders because the lungs serve as a gateway to the systemic circulation. Advantages of pulmonary drug delivery include, for example: 1) avoidance of first-pass metabolism; 2) rapid drug action; 3) a large surface area in the alveolar region for absorption, high lung permeability (thin air-blood barrier), and ample vasculature in the airways; and 4) lower extracellular enzyme levels compared to the GI tract due to the large alveolar surface area. Advantages of oral administration over nasal inhalation include deeper penetration / deposition of drugs in the lungs, while nasal inhalation can deliver drugs via the oral mucosa into the nasal cavity and lungs and into the systemic circulation. 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 may be formulated for aerosol administration to the respiratory system via oral or nasal inhalation. Drugs can be delivered in small particle sizes (e.g., about 0.5 microns to about 5 microns) obtainable through micronization, which can improve, for example, drug deposition in the lungs and the stability of drug suspensions. Drugs may be provided in pressurized packs with a suitable propellant, such as hydrofluoroalkanes (HFAs, e.g., 1,1,1,2-tetrafluoroethane [HFA-134a]), chlorofluorocarbons (CFCs, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane), or a suitable gas (e.g., oxygen, compressed air, or carbon dioxide). In aerosol formulations, the drug is dissolved, or often suspended, in the propellant for pulmonary delivery. The aerosol contains excipients such as surfactants (which enhance pulmonary penetration by reducing the high surface tension at the air-water interface within the alveoli and which can emulsify, solubilize, or / and stabilize the drug, and which may be, for example, a phospholipid such as lecithin) or / and stabilizers, although the surfactant portion of the peptide product can perform the surfactant function.For example, an MDI formulation may contain a peptide product, a propellant (e.g., an HFA such as 1,1,1,2-tetrafluoroethane), and a cosolvent (e.g., an alcohol such as ethanol), and optionally, a surfactant (e.g., a fatty acid such as oleic acid). The MDI formulation may optionally contain a dissolved gas (e.g., CO). After activation of the device, the bursting of CO2 bubbles within the released aerosol droplets causes the droplets to break down into smaller droplets, thereby increasing the respirable fraction of the drug. As another example, a nebulizer formulation may contain a peptide product, a chelating agent or preservative (e.g., edetate disodium), an isotonic 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). Drugs can be delivered, for example, by a nebulizer or an MDI with or without a spacer, and the drug dose delivered can be controlled by a metering chamber (nebulizer) or a metering valve (MDI).

[0040] Table 2 shows exemplary MDI, nebulizer, and DPI formulations. Metered-dose inhalers (also called pressurized 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 μL) per actuation of the device. MDIs typically generate aerosol faster 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 by the use of breath-actuated MDIs or coordinated devices. Breath-actuated MDIs (e.g., Easi breathe®) are activated when the device senses the user's inhalation and expel a drug dose accordingly. The inhalation flow rate is regulated via an actuator, allowing the user time to reliably actuate the device during inhalation. In a linked device, the spacer (or valved holding chamber) is a tube attached to the mouthpiece end of the inhaler. It acts as a reservoir or chamber to hold the medication being sprayed by the inhaler and slows the rate at which the aerosol enters the mouth, allowing for evaporation of the propellant from larger droplets. The spacer simplifies inhaler use and increases the amount of medication deposited in the lungs instead of the upper airways. Spacers are sometimes made of antistatic polymers to minimize electrostatic adhesion of released medication particles to the inner walls of the spacer. Nebulizers produce aerosol droplets of approximately 1-5 microns. Nebulizers do not require user coordination between device activation and inhalation, which can significantly affect the amount of medication deposited in the lungs. Compared to MDIs and DPIs, nebulizers can deliver larger amounts of medication despite 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 [virtual valve], PARI LC® Plus [breath-augmented], and SideStream Plus [breath-augmented]), ultrasonic nebulizers, and vibrating mesh 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 a photoacoustic trigger, allowing the user to synchronize each breath to each pulse. For oral or nasal inhalation using a powder inhaler (DPI), the peptide product can be provided in the form of a micronized dry powder, where the drug particles are of a small size (e.g., about 0.5 microns to about 5 microns) to improve, for example, 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 sedimentation in the terminal bronchioles and alveolar regions. In contrast, the majority of larger particles (>5 microns) do not follow airflow to the many branches of the respiratory tract and are deposited by impaction in the upper respiratory tract, including the oropharyngeal region of the throat. DPI formulations can contain drug particles alone or mixed with larger powders of a suitable base / carrier, such as lactose, starch, starch derivatives (e.g., hydroxypropylmethylcellulose), or polyvinylpyrrolidine. Carrier particles enhance flow, reduce aggregation, improve dose uniformity, and aid in the dispersion of drug particles. DPI formulations may optionally contain excipients such as magnesium stearate or / and leucine, which improve formulation performance by preventing interparticle bonding (due to their anti-adhesion effect). The powder formulations may be presented in unit dosage forms such as capsules (e.g., gelatin capsules) or cartridges in blister packs that can be manually filled or pre-filled into the inhaler.Drug particles can be drawn into the lungs by placing the inhaler's mouthpiece or nosepiece over the mouth or nose, inhaling forcefully and deeply to create turbulence, and maintaining the breath for a period of time (e.g., about 5-10 seconds), allowing the drug particles to settle in the bronchiolar and alveolar regions. When a user activates the DPI and inhales, the airflow through the device creates shear and turbulence, directing the inhaled air into the powder bed, causing the electrostatic powder mixture to become fluid and enter the user's airways. There, the drug particles are separated from the carrier particles by the turbulence and carried deep into the lungs, while the larger carrier particles impact the surface of the oropharynx and are dislodged. Thus, the user's inspiratory airflow achieves powder deagglomeration and air ionization, determining drug deposition in the lungs. (Passive DPIs require rapid inspiration to deagglomerate drug particles, but rapid inspiration is not recommended with MDIs or nebulizers because it creates turbulence and high velocities that increase drug deposition due to impaction on the upper airway.) Compared with MDIs, DPIs (including breath-actuated DPIs) can deliver larger doses of drug and larger size drugs (e.g., macromolecules) to the lungs.

[0041] Lactose (e.g., alpha-lactose monohydrate) is the most commonly used 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 may 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 may need to be mixed with coarse lactose such as DCL 11, Flowlac® 100, Inhalac® 230, or Respitose® SV003 to improve flow (e.g., in a ratio of about 1:9 fine to coarse lactose).

[0042] Tables 3 and 4 show non-limiting examples of lactose grades / types that can be used in DPI formulations. The carrier particle size distribution influences the fine particle fraction / dose (FPF or FPD) of a drug, with a high FPF being desirable for pulmonary drug delivery. The FPF / FPD is the respirable fraction / dose mass from a DPI device with an aerodynamic particle diameter of ≦5 microns in inspired air. A high FPF, and therefore good DPI performance, can be achieved, for example, from a DPI formulation comprising fine lactose (e.g., Lactohale® 300) and coarse lactose (e.g., Respitose® SV003) in a ratio of about 1:9 and with an overage of about 20% w / w, which avoids drug deposition in the capsule shell or DPI device and allows nearly all of the drug to be delivered to the respiratory tract.

[0043] [Table 3]

[0044] Table 4

[0045] Other carriers for DPI formulations include, but are not limited to, glucose, mannitol (e.g., crystallized mannitol [Pearlitol 110C] and spray-dried mannitol [Pearlitol 100 SD]), maltitol (e.g., crystallized maltitol [Maltisorb P90]), sorbitol, and xylitol. Most DPIs are breath-activated ("passive"), relying on the user's inhalation to generate an aerosol. Examples of passive DPIs include, but are not limited to, Airmax®, Novolizer®, and Otsuka DPI (compact cake). Air classifier technology (ACT) is an efficient passive powder dispersion mechanism utilized in DPIs. In ACT, multiple feed channels generate tangential airflow, which creates a cyclone within the device during inhalation. Power-assisted ("active") DPIs (e.g., based on aerodynamics, impact force, or vibration) also exist, which use energy to assist, for example, in particle deagglomeration. 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 doses), Exubera® (single unit dose), MicroDose® (multiple unit doses and electronically activated), Omnihaler® (single unit dose), Pfeiffer DPI (single unit dose), and Spiros® (multiple unit doses). Peptide products can also be administered by other routes, such as orally. Oral formulations may contain the peptide product, conventional excipients known in the art, and optionally, an absorption enhancer such as sodium N-[8-(2-hydroxybenzoyl)aminocaprylate] (SNAC). SNAC protects against enzymatic degradation through local buffering and enhances GI absorption. Oral dosage forms (eg, tablets, capsules, or pills) can optionally be enteric coated to protect the contents thereof 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" includes sustained-release, extended-release, extended-release, delayed-release, slow-release, and controlled-release compositions, systems, and devices. In some embodiments, the sustained-release composition delivers the peptide product for a period of at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or more. In some embodiments, the sustained-release composition is formulated as nanoparticles or microparticles configured with a biodegradable polymer and incorporating 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 product is in the form of a depot that is generated 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), polyglycolic acid (PGA), or copolymers thereof (eg, PLGA or PLA-PEG).

[0046] Pharmaceutical compositions can be provided in unit dosage forms as single doses, where all active and inactive ingredients are combined in a suitable system, and the components do not need to be mixed to form the administered composition. Unit dosage forms generally contain a therapeutically effective dose of a drug, but can contain an appropriate fraction thereof so that a therapeutically effective dose can be achieved by providing multiple unit dosage forms. Examples of unit dosage forms include tablets, capsules, or pills for oral ingestion; solutions in pre-filled syringes for parenteral (e.g., intravenous, subcutaneous, intramuscular) injection, disposable pens, or pens equipped with dose counters; and capsules, cartridges, or blisters pre-filled or manually filled into inhalers. Alternatively, pharmaceutical compositions can be provided as kits, in which the active ingredient, excipients, and carriers (e.g., solvents) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles, or syringes) and must be combined to form the administered composition. The kit may include instructions for storing, preparing, and administering the composition (e.g., a solution to be parenterally injected). The kit may include all active and inactive ingredients in unit dosage form, or the active and inactive ingredients in two or more separate containers, and may include instructions for administering or using the pharmaceutical composition to treat a medical condition disclosed herein. The kit may further include a device for delivering the composition, such as an injection pen or inhaler. In some embodiments, the kit includes 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, obesity, metabolic syndrome, or cardiovascular disease, or a condition related thereto (e.g., NASH or PCOS). In certain embodiments, the kit further includes a device for delivering the peptide product or composition, such as an injection pen or inhaler.

[0047] The present disclosure further provides uses of the peptide products described herein for treating, for example, insulin resistance, diabetes, obesity, metabolic syndrome, and cardiovascular disease, and diseases related thereto, such as NASH and PCOS. In some embodiments, the dual agonist peptide products are used to treat hyperglycemia, insulin resistance, hyperinsulinemia, prediabetes, diabetes (including type 1 and type 2, fetal and juvenile diabetes), diabetic complications, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, elevated blood levels of free fatty acids, obesity, metabolic syndrome, syndrome X, cardiovascular disease (including coronary artery disease), atherosclerosis, acute cardiovascular syndrome, ischemia (including cardiac ischemia and cerebral ischemia / stroke), ischemic reperfusion injury (including myocardial and cerebral IRI), infarction (including myocardial infarction 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 elevated blood levels of C-reactive protein), and hypertension. Dual agonist peptide products can achieve therapeutic effects through various mechanisms, including stimulation of blood glucose-dependent insulin secretion, increasing insulin sensitivity, stimulating fat burning, and weight loss. Dual agonist peptide products can also promote, for example, pancreatic beta cell protection, cardioprotection, and wound healing.

[0048] The peptide products described herein can be used to treat other diseases 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), gastroenterological disorders (e.g., cholelithiasis [gallstones], gastroesophageal reflux disease [GERD], and gastroparesis), gout, hypercortisolism (e.g., Cushing's syndrome), renal disorders (e.g., chronic kidney disease), and liver disorders (e.g., alcoholism). These include fatty liver disease (FLD), including alcoholic and non-alcoholic FLD, neurological disorders (e.g., carpal tunnel syndrome, dementia (e.g., Alzheimer's disease and vascular dementia), paresthesia, migraine, and multiple sclerosis), urinary tract disorders (e.g., erectile dysfunction, hypogonadism, and urinary incontinence), polycystic ovary syndrome, infertility, menstrual irregularities, mood disorders (e.g., depression), and cancer (e.g., endometrial cancer, esophageal cancer, colorectal cancer, gallbladder cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, and skin cancer (e.g., melanoma), and leukemia). In one embodiment, the dual agonist peptide products described herein are used to treat polycystic ovary syndrome (PCOS). In another embodiment, 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 product described herein is used to treat fatty liver disease (FLD).In some embodiments, FLD is non-alcoholic fatty liver disease (NAFLD).In some embodiments, NAFLD is non-alcoholic steatohepatitis (NASH).FLD, also known as fatty liver disease, is characterized by excessive fat accumulation in the liver.FLD includes alcoholic fatty liver disease (AFLD) and NAFLD.Chronic alcoholism leads to fatty liver due to the production of toxic metabolites, such as aldehydes, during the liver's metabolism of alcohol. NAFLD is discussed below. FLD is associated with diabetes, obesity, and metabolic syndrome. Fatty liver can progress to 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 develop HCC. HCC is the most common type of primary liver cancer in adults and occurs in conditions of chronic hepatitis. NAFLD is characterized by fatty liver, which occurs when fat, particularly free fatty acids and triglycerides, accumulates in hepatocytes (fatty liver) due to causes other than excessive alcohol consumption, such as nutrient overload, high calorie intake, and metabolic dysfunction (e.g., dyslipidemia and impaired glucose control). Although the liver may retain fat without interfering with liver function, fatty liver disease can progress to nonalcoholic steatohepatitis (NASH), a condition characterized by steatosis accompanied by inflammation, hepatocyte ballooning, and cellular damage with or without liver fibrosis. Fibrosis is the strongest predictor of death from NASH. NAFLD is characterized by steatosis alone; steatosis with lobular or portal venous 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 progressing to cirrhosis, and approximately 10% dying from liver disease, such as cirrhosis or liver cancer (e.g., HCC). NAFLD is the most common liver disorder in developed countries, and NASH is expected to replace hepatitis C as the leading cause of liver transplants 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 the progression of fatty liver to liver inflammation and fibrosis, and thus to NASH. Furthermore, obesity induces and exacerbates NASH, and weight loss can alleviate NASH.Therefore, the peptide products described herein, including GLP-1 receptor (GLP1R) agonists, glucagon receptor (GCGR) agonists, and dual GLP1R / GCGR agonists, can be used to treat NAFLD, including NASH. In some embodiments, the dual agonist peptide products used to treat diseases associated with insulin resistance and / or obesity disclosed herein, such as NAFLD (e.g., NASH) or PCOS, are selected from the peptide products of SEQ ID NOs: 1-10 or 12-27, and / or derivatives thereof, and pharmaceutically acceptable salts thereof.

[0049] In some embodiments, the dual agonist peptides of the present invention can be used to control blood glucose levels by reducing one or more adverse events (i.e., unexpected events that adversely affect the welfare of patients and / or animals). Agonists with unbalanced affinities for GLP-1R and GCGR (e.g., semaglutide) are used. Exemplary, non-limiting adverse events may include nausea, vomiting, diarrhea, abdominal pain, and / or constipation. Adverse events may include any known to those skilled in the art, such as those listed in industry resources and / or otherwise known to those skilled in the art (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 procedures (physician examination, surveys / questionnaires, etc.) typically used in clinical trials. Compared to the frequency and / or severity of such adverse events that occur when an agonist with disproportionate affinity for GLP-1R and GCGR (e.g., semaglutide) is administered to a subject, a dual agonist peptide of the present disclosure (e.g., any of SEQ ID NOs: 1-10 or 12-27, or a derivative thereof) can reduce such frequency and / or severity by, for example, 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., any of SEQ ID NOs: 1-10 or 12-27, or a derivative thereof) does not cause any adverse events.

[0050] The dual agonist peptide products of the present invention can be administered by any route suitable for the treatment of the diseases disclosed herein. Possible routes of administration of peptide products include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intracavity, and topical), and topical (transdermal, oral intramucosal, intranasal [e.g., via nasal spray or drops], ophthalmic [e.g., via eye drops], pulmonary [e.g., via oral or nasal inhalation], buccal, sublingual, rectal [e.g., via suppository], and vaginal [e.g., via suppository]. In some embodiments, the peptide product is administered parenterally, such as subcutaneously, intravenously, or intramuscularly. In other embodiments, the peptide product is administered by oral or nasal inhalation, or by insufflation. The therapeutically effective amount of a peptide product, the frequency of administration, and the length of treatment therewith for treating a disease disclosed herein may depend on various factors, including the nature and severity of the disease, the potency of the compound, the route of administration, the age, weight, health, sex, and diet of the subject, 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 dosage of about 0.1 mg to about 1, 5, 10 mg, or about 0.1-1 mg or 1-10 mg for a period of about one week for the treatment of a disease disclosed herein (e.g., one associated with insulin resistance and / or obesity, such as NASH or PCOS). In further embodiments, the peptide product is administered parenterally (e.g., subcutaneously [sc], intravenously [iv], or intramuscularly [im]) at a dosage of about 0.1 mg to about 1, 5, 10 mg, or about 0.1-1 mg or 1-10 mg for a period of about one week. 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. 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, 0.1-0.5 mg, or 0.5-1 mg over a period of about one week. Those skilled in the art will understand that effective doses in mice or other preclinical animal models can be scaled to humans.As such, through allometric scaling (also called biological scaling), doses for larger animals can be extrapolated from mouse doses to obtain equivalent doses based on animal weight or body surface area.

[0051] The peptide product can be administered at a frequency suitable for treating a disease disclosed herein (e.g., one associated with insulin resistance and / or obesity, such as NASH or PCOS). In some embodiments, the dual agonist peptide product is administered once daily, once every two days, once every three days, twice a week, once a week, or once every two weeks, e.g., sc or iv. In certain embodiments, the peptide product is administered once a week, e.g., SC, IV, or IM. The dual agonist peptide product can be administered at any time convenient for the patient. The dual agonist peptide product can be taken substantially with food (e.g., with a meal or within about 1 hour or 30 minutes before or after a meal) or substantially without food (e.g., at least about 1 hour or 2 hours before or after a meal). The length of treatment of a medical disease with the dual agonist peptide product can be determined by the treating physician based, for example, on the nature and severity of the disease and the subject's response to treatment. In some embodiments, the peptide product is administered to treat a disease disclosed herein chronically, such as for at least about 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 5 years, 10 years, or more. The dual agonist peptide product can also be taken ad libitum (as needed) until clinical symptoms of the disease disappear or a clinical target, such as blood glucose level, blood pressure, blood lipid level, body weight or body mass index, waist-to-hip ratio, body fat percentage, or any combination thereof, is achieved. If clinical symptoms of the disease reappear or a clinical target is not maintained, administration of the dual agonist peptide product can be resumed. The present disclosure provides a method of treating a medical disease disclosed herein, comprising administering to a subject a therapeutically effective amount of a peptide product described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. The present disclosure further provides a peptide product described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical 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. The medicament containing the peptide product can be used to treat any of the medical diseases described herein. The peptide product can optionally be used in combination with one or more additional therapeutic agents.

[0052] The dual agonist peptide products described herein can be administered as the sole active agent or can optionally be used in combination with one or more other dual agonist peptide products and / or in combination with additional therapeutic agents to treat any disorder disclosed herein, such as insulin resistance, diabetes, obesity, metabolic syndrome or cardiovascular disease, or any disease related thereto, NASH or PCOS, etc. In some embodiments, the one or more additional therapeutic agents are selected from diabetes agents, anti-obesity 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 drugs include, but are not limited to, AMP-activated protein kinase (AMPK) agonists, including biguanides (e.g., buformin and metformin); peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, including thiazolidinediones (e.g., balaglitazone, ciglitazone, daraglitazone, englitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone) and saroglitazar (a dual PPAR-α / γ agonist); exendin-4, albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, taspoglutide, CNTO736, CNTO Glucagon-like peptide-1 (GLP-1) receptor agonists, including 3649, HM11260C (LAPS-Exendin), NN9926 (OG9S7GT), TT401, and ZY0G1; dipeptidyl peptidase-4 (DPP-4) inhibitors, including alogliptin, anagliptin, dutogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, septagliptin, sitagliptin, teneligliptin, trelagliptin, and vildagliptin; canagliflozin (which also inhibits SGLT1), dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, and remogliflozin Sodium-glucose transport protein 2 (SGLT2) inhibitors, including etabonate, sotagliflozin (which also inhibits SGLT1), and tofogliflozin; meglitinides (e.g., mitiglinide, nateglinide, and repaglinide), and sulfonylureas {including first-generation (e.g., acetohexamide, carbutamide, chlorpropamide, glycilamide [tolhexamide], metahexamide, tolazamide, and tolbutamide), and second-generation (e.g., glibenclamide [glyburide], glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, and glyclopyramide)} that inhibit ATP-dependent K+ on pancreatic beta cells. + (K ATP) channel blockers; insulin and its analogs, including fast-acting insulins (e.g., insulin aspart, insulin glulisine, and insulin slipro), intermediate-acting insulins (e.g., NPH insulin), and long-acting insulins (e.g., insulin degludec, insulin detemir, and insulin glargine); and analogs, derivatives, and salts thereof. In certain embodiments, the diabetes drug 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 reducers), including amphetamine, dextroamphetamine, amfepramone, clobenzorex, matindole, phentermine (with or without topiramate), and lorcaserin; satiety promoters, including ciliary neurotrophic factor (e.g., axoxoquin) and longer-acting analogs of amylin, calcitonin, cholecystokinin (CCK), GLP-1, leptin, oxyntomodulin, pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY); lipase inhibitors, including caulerpinine, cetilistat, ebelactone A and B, estellastin, lipstatin, orlistat, persikinin, panclicin AE, valilactone, and vibralactone; antihyperlipidemic agents; and analogs, derivatives, and salts thereof. Antihyperlipidemic agents include, but are not limited to, HMG-CoA reductase inhibitors, including statins {(e.g., atorvastatin, cerivastatin, fluvastatin, mevastatin, monacolins (e.g., monacolin K [lovastatin]), pitavastatin, pravastatin, rosuvastatin, and simvastatin} and flavanones (e.g., naringenin); squalene synthase inhibitors, including lapaquistat, zaragozic acid, and RPR-107393; anthocyanins, avenaciolide, chloroacetylated biotin, cyclodium, diclofop, haloxyfop, soraphen (e.g., soraphen A 1α), 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), CP-640186, GS-0976, NDI-010976; 7-(4-propyloxy-phenylethynyl)-3,3-dimethyl-3,4-dihydro-2H-benzo[b][1,4]dioxepin; N-ethyl-N'-(3-{[4-(3,3-dimethyl-1-oxo-2-oxa-7-azaspiro[4.5]dec-7-yl)piperidin-1-yl]-carbonyl}-1-benzothien-2-yl)urea; 5-(3-acetamidobut-1-ynyl)-2-(4-propyloxy) acetyl-CoA carboxylase (ACC) inhibitors, including 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 perfluoroalkanoic acids (e.g., 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 / δ agonist), GFT505 (dual PPAR-α / δ agonist), GW0742, GW501516 (dual PPAR-β / δ agonist), soderglitazar (GW677954), MBX-80 PPAR-δ agonists, including 25, 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-Δ-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(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid), and synthetic agonists (e.g., acetylpodocarpic acid dimer, hypocholamide, N,N-dimethyl-3β-hydroxy-cholenamide [DMHCA], GW3965, and T0901317); endogenous ligands (e.g., 9-cis-retinoic acid) and synthetic agonists (e.g., bexarotene, Retinoid X receptor (RXR) agonists, including AGN191659, AGN191701, AGN192849, BMS649, LG100268, LG100754, and LGD346; inhibitors of acyl-CoA cholesterol acyltransferase (ACAT, also known as sterol O-acyltransferase [SOAT], including ACAT1 [SOAT1] and ACAT2 [SOAT2]), including avasimide, pactimibe, pellitorin, terpendol C, and flavanones (e.g., naringenin); Aramco ol, CAY-10566, CVT-11127, SAR-224, SAR-707, XEN-103; 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide and 4-ethylamino-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)pyridazin-3-yl}- 5-Fluoro-1'-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-3,4-dihydrospiro[chromene-2,4'-piperidine]; 6-[5-(cyclopropylmethyl)-4,5-dihydro-1'H,3H-spiro[1,5-benzoxazepine-2,4'-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; inhibitors of stearoyl-CoA desaturase-1 (SCD-1, also known as stearoyl-CoA delta-9 desaturase) activity or expression, including the cis-9, trans-11 isomer and the trans-10, cis-12 isomer, of conjugated linoleic acid, substituted heteroaromatic compounds disclosed in WO2009 / 129625A1, antisense polynucleotides and peptide nucleic acids (PNAs) targeting mRNA for SCD-1, and SCD-1-targeting siRNA; anacetrapib, dalcetrapib, evacetrapib, torcetrapib, and AMG cholesteryl ester transfer protein (CETP) inhibitors, including 899 (TA-8995); inhibitors of microsomal triglyceride transfer protein (MTTP) activity or expression, including implitapide, lomitapide, dirlotapide, mitratapide, CP-346086, JTT-130, SLx-4090, antisense polynucleotides and PNAs targeting mRNA for MTTP, MTTP-targeting microRNAs (e.g., miRNA-30c), and MTTP-targeting siRNA; GLP-1 receptor agonists (as above); BMS- Fibroblast growth factor 21 (FGF21) and its analogs and derivatives, including 986036 (pegylated FGF21); berberine (reducing PCSK9 levels), annexin A2 (inhibiting 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), and mRNAs against PCSK9; inhibitors of proprotein convertase subtilisin / kexin type 9 (PCSK9) activity or expression, including antisense polynucleotides and PNAs targeting A, PCSK9-targeting siRNA (e.g., inclisiran [ALN-PCS] and ALN-PCS02); apoA-I mimetics (e.g., 2F, 3F, 3F-1, 3F-2, 3F-14, 4F, 4F-P-4F, 4F-I HS-4F, 4F2, 5F, 6F, 7F, 18F, 5A, 5A-C1, 5A-CH1, 5A-CH2, 5A-H1, 18A, 37pA [18AP-18A], ELK [name], ELK-1A, E LK-1F, ELK-1K1A1E, ELK-1L1K, ELK-1W, ELK-2A, ELK-2A2K2E, ELK-2E2K, ELK-2F, ELK-3E3EK, ELK-3E3K3A , ELK-3E3LK, ELK-PA, ELK-P2A, ELKA [name], ELKA-CH2, ATI-5261, CS-6253, ETC-642, FAMP [name], FREL [name], and KRES [name]), and apoE mimetics (e.g., Ac-hE18A-NH2[AEM-28], Ac-[R]hE18A-NH2, AEM-28-14, EpK, hEp, mR18 apolipoprotein mimetic peptides, including COG-L, COG-112, COG-133, and COG-1410; omega-3 fatty acids, including docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA), fish oils (e.g., containing DHA and EPA), and their esters (e.g., glyceryl and ethyl esters);and analogs, derivatives, and salts thereof. In certain embodiments, the antiobesity 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, 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, eprosa), and / or antihyperlipidemic agents (e.g., acetaminophen, benzodiazepines ... antagonists of the renin-angiotensin-aldosterone system (RAAS), including aldosterone receptor antagonists (e.g., valsartan, 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 torsemide), thiazide diuretics (e.g., diuretics, including (e.g., bendroflumethiazide, chlorothiazide, hydrochlorothiazide, epithizide, 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; dihydropyridines calcium channel blockers, including (e.g., amlodipine, levamlodipine, cilnidipine, clevidipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine), and non-dihydropyridines (e.g., diltiazem and verapamil); alpha2-adrenergic receptor agonists, including clonidine, guanabenz, guanfacine, methyldopa, and moxonidine;alpha 1-adrenergic receptor antagonists (alpha blockers), including doxazosin, indoramin, nicergoline, phenoxybenzamine, phentolamine, prazosin, terazosin, and tolazoline; beta-adrenergic receptor antagonists, including atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, and timolol endothelin receptor (β1 or / and β2) antagonists (β-blockers); mixed α / β-blockers, including bucindolol, carvedilol, and labetalol; endothelin receptor antagonists, including selective ETA receptor antagonists (e.g., ambrisentan, atrasentan, edonentan, sitaxsentan, zibotentan, and BQ-123), and dual ETA / ETB antagonists (e.g., bosentan, macitentan, and tezosentan);Hydralazine, minoxidil, theobromine, sodium nitroprusside, organic nitrates (e.g., isosorbide mononitrate, isosorbide dinitrate, and nitroglycerin, which are converted to nitric oxide in the body), endothelial nitric oxide synthase (eNOS) stimulators (e.g., cicletanine), activators of soluble guanylate cyclase (e.g., cinaciguat and riociguat), phosphodiesterase type 5 (PDE5) inhibitors (e.g., avanafil, benzamidonafil, dasatinib ... prostaglandin E1 (alprostadil) and its analogs (e.g., limaprost and misoprostol), prostacyclin and its analogs (e.g., ataprost, beraprost [e.g., esveraprost], 5,6,7-triazoloside ... 4,8-inter-m-phenylene-9-fluoro-PGI2, carbacyclin, isocarbacyclin, clinprost, ciprostene, eptaloprost, cicaprost, iloprost, pimilprost, SM-10906 (des-methylpimilprost), naxaprostene, taprostenol, treprostinil, CS-570, OP-2507, and TY-11223), and non-prostanoid prostacyclin receptor agonists (e.g., 1-phthalazinol , larinepag, selexipag, ACT-333679 [MRE-269, the active metabolite of selexipag], and TRA-418), phospholipase C (PLC) inhibitors, and protein kinase C (PKC) inhibitors (e.g., BIM-1, BIM-2, BIM-3, BIM-8, chelerythrine, cicletanine, gossypol, myabenol C, myricitrin, rubaxistaurin, and verbascoside); minerals, including magnesium and magnesium sulfate;and analogs, derivatives, and salts thereof. 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 to treat NAFLD, such as NASH. In some embodiments, the one or more additional therapeutic agents are selected from diabetes drugs, antiobesity agents, anti-inflammatory drugs, antifibrotic drugs, antioxidants, antihypertensive drugs, and combinations thereof. Therapeutic agents that can be used to treat NAFLD (e.g., NASH) include, but are not limited to: PPAR agonists (PPAR-δ and -γ agonism increases insulin sensitivity, PPAR-α agonism reduces hepatic steatosis, and PPAR-δ agonism reduces macrophages and Kupffer cells), including 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]), have been shown to be effective in reducing hepatic steatosis. inhibits hepatic steatosis, cell damage, and fibrosis; farnesoid X receptor (FXR) agonists, such as obeticholic acid and GS-9674 (FXR agonists reduce hepatic gluconeogenesis, lipogenesis, adiposity, and fibrosis); fibroblast growth factor 19 (FGF19), its analogs, and derivatives, such as NGM-282 (FGF19 analogs reduce hepatic gluconeogenesis and adiposity); fibroblast growth factor 21 (FGF21), its analogs, and derivatives, such as BMS-986036 (pegylated FGF21) (FGF21 reduces hepatic steatosis, cell damage, and fibrosis) HMG-CoA reductase inhibitors, including statins (e.g., rosuvastatin) (statins reduce steatohepatitis and fibrosis); ACC inhibitors, such as NDI-010976 (liver-targeted) and GS-0976 (ACC inhibitors reduce de novo lipogenesis and hepatic steatosis); SCD-1 inhibitors, such as aramchol (SCD-1 inhibitors reduce hepatic steatosis and increase insulin sensitivity); SGLT2 inhibitors, such as canagliflozin, ipragliflozin, and luseogliflozin (SGLT2 inhibitors reduce body weight, liver ALT levels, and fiber CCR2 and / or CCR5 antagonists, such as cenicriviroc (CCR2 (binding to CCL2 [MCP1]) and CCR5 (binding to CCL5 [RANTES]) inhibit the activation and migration of inflammatory cells (e.g., macrophages) to the liver and reduce liver fibrosis); apoptosis inhibitors, including apoptosis signal-regulating kinase 1 (ASK1) 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 inhibitors are important in the progression of liver fibrosis); antioxidants (vitamin E reduces hepatic steatosis, hepatocyte ballooning, and lobular inflammation), including vitamin E (e.g., α-tocopherol) and scavengers of reactive oxygen species (ROS) and free radicals (e.g., cysteamine, glutathione, melatonin, and pentoxifylline [also anti-inflammatory via inhibition of TNF-α and phosphodiesterase]); and their analogs, derivatives, and salts. 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 the treatment of NAFLD (e.g., NASH) are or include vitamin E and / or pioglitazone. As will be appreciated by those skilled in the art, other combinations may also be used.

[0053] 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 to estimate parameters. Individual plasma concentration-time data can be used for pharmacokinetic calculations. In addition to parameter estimates for individual animals, descriptive statistics (e.g., mean, standard deviation, coefficient of variation, median, minimum, maximum) can be determined as needed. Concentration values ​​below the limit of quantification can be treated as zero for the purpose of determining descriptive statistics and pharmacokinetic analysis. Embedded concentration values ​​below the limit of quantification can be excluded from pharmacokinetic analysis. All parameters can be generated from the individual dual agonist peptide (or its derivatives and / or metabolites) concentrations in the plasma of the test article treatment group on the day of dosing (Day 1). Parameters can be estimated using the nominal dose level unless out-of-specification dose formulation analysis results are obtained. In this case, the actual dose level can be used. Parameters can be estimated using the nominal sampling time. If deviations in bioanalytical sample collection are documented, actual sampling times can be used for the affected time points. Bioanalytical data can be used as received for pharmacokinetic analysis and can be presented in tables and figures in the units provided. Pharmacokinetic parameters can be calculated and displayed in the units provided by the analytical laboratory (orders of magnitude can be adjusted appropriately for presentation in reports, e.g., h * ng / mL to h * (These parameters may be converted to μg / mL.) Descriptive statistics (e.g., mean, standard deviation, coefficient of variation, median, minimum, maximum) and pharmacokinetic parameters may be determined to three significant figures as needed. Additional data processing aspects may be documented as needed. When data permit, PK parameters to be determined may include, but are not limited to: C max : Maximum observed concentration, DN C max : Dose-normalized maximum concentration, calculated as Cmax / dose; T max : Time of maximum observed concentration, AUC 0-t: Area under the curve from time 0 to the time of the last measurable concentration, AUC, calculated using the linear trapezoidal rule 0-96 : Area under the curve from 0 to 96 hours, DN AUC, calculated using the linear trapezoidal rule 0-96 :AUC 0-96 Dose-normalized AUC calculated as / dose 0-96 AUC 0-inf :AUC 0-inf =AUC 0-t +C t / λ z is the area under the curve from time 0 to infinity (day 1 only), calculated as t is the last observed quantifiable concentration, and λ z is the elimination rate constant; t 1 / 2 : elimination half-life calculated as ln(2) / λz. As will be appreciated by those skilled in the art, additional parameters and comparisons (e.g., sex ratio, dose proportionality ratio, etc.) can also be determined.

[0054] In some embodiments, the present disclosure provides a pharmaceutical dosage formulation comprising at least one dual agonist peptide having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), wherein the peptide is modified with a hydrophobic surfactant, and the dosage is designed to control blood glucose levels and / or induce weight loss, along with a reduction in one or more adverse events selected from nausea, vomiting, diarrhea, abdominal pain, and constipation, compared to an agonist having disproportionate affinity for GLP-1R and GCGR. In some embodiments, the dual agonist peptide is any one of SEQ ID NOS: 1-10 or 12-27, or a derivative thereof, or a combination thereof. In some embodiments, the dual agonist peptide has approximately equal affinity for GLP-1R and GCGR, and in an even more preferred embodiment, is SEQ ID NOS: 1. In some embodiments, administration of the dual agonist peptide to a mammal results in a reduction in blood glucose levels at about 48 or 96 hours after administration (optionally a reduction by at least about any of 10, 20, 30, 40, or 50%, preferably at least about 50%), a reduction in blood glucose levels at about 72 hours after administration (optionally a reduction by at least about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, preferably at least about 100%), and / or a reduction in blood glucose levels at about 120 hours after administration, compared to administration of an approximately equimolar dose of semaglutide. In some embodiments, administration of the dual agonist peptide to a mammal induces total body weight loss and / or induces liver weight loss, compared to administration of an approximately equimolar dose of semaglutide.In some embodiments, administration of the dual agonist peptide to a mammal exhibits a lower Cmax (optionally at least about a 10, 20, 30, 40, or 50% decrease, preferably at least about a 50% decrease), an approximately equal or greater Tmax (optionally at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater, preferably at least about 100% greater), and a similar AUC compared to administration of an approximately equimolar dose of semaglutide. (0-inf) (optionally at least about 50, 60, 70, 80, 90, 95, 100% thereof, preferably at least about 80-90% thereof, e.g., about 85-93% thereof), and exhibits about the same or greater T 1 / 2(時間)and / or when administered to treat NASH, exhibits increased whole body weight loss (optionally at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, preferably at least about 50 or 75%, e.g., about 50-75% thereof), exhibits prolonged MRT (time) (optionally at least about 10, 20, 30, 40, or 50% prolongation, preferably at least about 25% prolongation), exhibits a prolonged PK / PD profile, exhibits comparable or greater glucoregulatory efficacy, optionally induces about twice as much greater whole body weight loss (optionally at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, preferably at least about 100% lower body fat mass loss), and / or when administered to treat NASH, increases whole body weight loss, liver weight loss, improved NAS score, improved liver steatosis, improved ballooning, improved col1A1 staining, In some embodiments, administration of the dual agonist peptide to a mammal results in a greater loss of body weight (optionally at least about 10, 20, 30, 40 or 50% greater, preferably at least about 15% greater) by about 14 days after administration of the dosage formulation compared to administration of an approximately equimolar dose of semaglutide, and / or results in a greater loss of body weight (optionally at least about 10, 20, 30, 40 or 50% greater, preferably at least about 25% greater) by about 20-28 days after administration of the dosage formulation compared to administration of an approximately equimolar dose of semaglutide. In some embodiments, administration of the dual agonist peptide to a mammal results in a weight loss in the obese mammal sufficient to return the mammal to the normal weight range for lean, normal mammals, compared to administration of an approximately equimolar dose of semaglutide.

[0055] "Reduction" or "decrease" of adverse effects or events refers to the reduction in the severity, duration, and / or frequency of adverse effects experienced by a subject after administration of an agonist with balanced affinity for GLP1R and GCGR, as well as the reduction in the incidence or severity of adverse effects in a group of subjects, compared with an agonist with disproportionate affinity for GLP1R and GCGR. 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 side effects previously experienced by a subject after administration of an agonist with disproportionate affinity for GLP1R and GCGR. In some embodiments, "reduction" or "reduction" of side effects includes the reduction of gastrointestinal side effects, where adverse events are reduced to zero or to undetectable levels. In other embodiments, side effects are reduced to levels comparable to those of untreated subjects, but are not completely eliminated. Furthermore, administration of analogs with disproportionate affinities for GLP-1R or GCGR to mammals may result in the need for excessively high doses to maximally activate receptors with lower sensitivity to the ligand, thus potentially exceeding the biologically effective dose for other ligands and causing dose-related undesirable side effects.

[0056] The present disclosure also provides a method for lowering and / or stabilizing blood glucose levels in a mammal, the method comprising administering to the mammal a pharmaceutical dosage formulation comprising a dual agonist peptide of SEQ ID NO: 1-10 or 12-27 (or a derivative thereof), preferably a dual agonist peptide having approximately equal affinity for GLP-1R and GCGR (preferably SEQ ID NO: 1), wherein the method reduces the incidence or severity of one or more adverse events compared to an agonist with imbalanced affinity for GLP-1R and GCGR (e.g., semaglutide), the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain and constipation upon administration to the mammal. In some embodiments, such methods result in lower blood glucose levels (10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% lower, preferably at least about 50% lower) at about 48 hours or 96 hours after administration, and preferably lower (optionally at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% lower, preferably at least about 50% lower) at about 72 hours after administration compared to methods in which an approximately equimolar dose of semaglutide is administered. and / or result in lower blood glucose levels at about 120 hours after administration (optionally at least about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% lower, preferably at least about 100% lower), induce whole body weight loss and / or liver weight loss, lower Cmax (optionally about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% lower, preferably about 40-50% lower), T max about equal to or greater than (optionally about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% lower, preferably at least about 100% higher than) T max ), and similar AUC (0-inf) (optionally at least about 50, 60, 70, 80, 90, 95, 100%, preferably at least about 80-90%, e.g., about 85-93%, thereof), about the same as or greater than T 1 / 2(時間)and / or a combination of the above, wherein the administration of the dosage formulation results in a greater overall body weight loss (optionally about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% lower), preferably at least about 50% or 75%, or about 50-75% thereof, and / or ... and / or, if the method is for treating NASH, results in increased total body weight loss, liver weight loss, improved NAS score, improved hepatic steatosis, improved ballooning, improved col1A1 staining, improved ALT, improved liver TG / TC, and improved plasma triglycerides (TG) / total cholesterol (TC), by about 20-28 days after administration of the dosage formulation (optionally, about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, preferably at least about 25%). .

[0057] In some embodiments, the present disclosure provides an agonist peptide product (preferably SEQ ID NO: 1) and about 0.025-0.075% (w / w) polysorbate 20 (PS-20, Tween 20), about 0.2-0.5% (w / w) arginine, and about 3-6% (w / w) mannitol in deionized water (pH 7.7±0.1). In a preferred embodiment, the pharmaceutical dosage formulation is ALT-801, comprising SEQ ID NO: 1, about 0.050% (w / w) polysorbate 20, about 0.348% (w / w) arginine, and about 4.260% (w / w) mannitol in deionized water (pH 7.7±0.1).

[0058] In some embodiments, the F58 formulation (i.e., a pharmaceutical formulation containing ALT-801 as the API) can be modified to include a high concentration of a surfactant, such as polysorbate 20 (PS-20), to maintain micelle formation within the formulation. See Example 8. These results identify the minimum concentration of PS-20 to be used across a range of ALT-801 concentrations to achieve its critical micelle concentration (CMC). The concentration of PS-20 in the F58 formulation (i.e., 0.5 mg / ml) can be increased to achieve the CMC and avoid the hazy appearance of the solution (indicative of large aggregates that precipitate from solution) when stored at +2-8°C. As shown in Example 8 herein, this can be achieved by modifying the F58 formulation to include at least about 0.66 mg of PS-20 per mg of peptide (preferably SEQ ID NO: 1) to achieve the CMC. In some embodiments, the F58 formulation can be modified to replace PS-20 with polysorbate 80 (PS-80, Tween 80) in an amount of at least about 1.03 mg of polysorbate 80 (PS-80, Tween 80) per mg of peptide (preferably SEQ ID NO: 1) to achieve a CMC.

[0059] In some embodiments, the pharmaceutical dosage formulation comprises a preservative, which in certain embodiments can be selected from methylparaben, ethylparaben, propylparaben, butylparaben, benzyl alcohol, chlorobutanol, phenol, metacresol, chlorocresol, benzoic acid, sorbic acid, thiomersal, phenylmercuric nitrate, bronopol, propylene glycol, benzylconium chloride, or benzethonium chloride.

[0060] In some embodiments, the disclosure provides a pharmaceutical dosage formulation configured to administer an agonist peptide product (e.g., SEQ ID NO: 1) to a mammal at less than about 0.72 mg / kg / dose, optionally about 0.001-0.72 mg / kg / dose. In some embodiments, the pharmaceutical dosage formulation is configured to administer less than 0.36 mg / kg / dose of the agonist peptide product to a mammal. In some embodiments, the methods of the invention include administering between 0.001-0.3 mg / kg / dose, optionally about 0.007 mg / kg, or about 0.014 mg / kg, or about 0.03 mg / kg, or about 0.07 mg / kg, or about 0.18 mg / kg, or about 0.25 mg / kg / dose. In some embodiments, the pharmaceutical dosage formulation can be configured to administer between about 0.05 and about 20 mg per week, optionally between 0.1 and about 10 mg per week, or optionally between about 1 and about 7 mg per week, or optionally between about 1 and 5 mg per week. In some embodiments, the pharmaceutical dosage formulation is configured to be administered to a mammal once per week for up to 6 weeks. In some embodiments, the present disclosure provides a pharmaceutical dosage formulation configured to achieve a therapeutic dose in about 4 weeks or less. In some embodiments, the therapeutic dose is a C of about 10 to about 2000 ng / ml. max , T from about 10 hours to about 168 hours max , and / or approximately 1,000-100,000h * AUC in ng / mL 0-168 In some embodiments, ALT-801 may be administered repeatedly to achieve a plasma concentration of about 5-1000 ng / ml, or about 50 ng / ml, or about 150 ng / ml, or about 250 ng / ml, or about 500 ng / ml.

[0061] In some embodiments, the present disclosure provides methods described herein that include administering to a mammal less than about 0.72 mg / kg / dose, optionally from about 0.001 mg / kg / dose to less than about 0.36 mg / kg / dose, or optionally about 0.36 mg / kg / dose, of an agonist peptide product. In preferred embodiments of such methods, less than about 0.36 mg / kg / dose is administered to the mammal. In some embodiments, each dose is administered about once per week or once every two weeks, optionally for at least one month, and optionally each dose contains approximately the same amount of agonist peptide product. In some embodiments, such methods include administering a single dose of about 0.72 mg / kg / dose, followed by one or more subsequent doses of about 0.001 mg / kg / dose to about 0.36 mg / kg / dose. In some embodiments, the method comprises administering between 0.001-0.30 mg / kg / dose, optionally about 0.007 mg / kg, or about 0.014 mg / kg, or about 0.03 mg / kg, or about 0.07 mg / kg, or about 0.18 mg / kg, or about 0.25 mg / kg / dose. In some embodiments, the pharmaceutical dosage formulation can be configured to administer between about 0.05 and about 20 mg per week, optionally between 0.1 and about 10 mg per week, or optionally between about 1 and about 7 mg per week, or optionally between about 1 and 5 mg per week.

[0062] In preferred embodiments, such methods include subcutaneously administering a pharmaceutical dosage formulation. In some embodiments, such methods include administering a pharmaceutical dosage formulation to a mammal at about 0.03-0.25 mg / kg / dose, and provide a C of about 50-1000 ng / ml. max , T from about 10 hours to about 96 hours max , and / or approximately 5,000-80,000h * AUC in ng / mL 0-168 In some such methods, the time to reach a therapeutic dose is about 4 weeks or less. In some embodiments, the therapeutic dose is a C of about 50 to about 700 ng / ml. max, T from about 10 hours to about 72 hours max , and / or approximately 6,000-70,000h * AUC in ng / mL 0-168 Shows.

[0063] In some embodiments, the methods disclosed herein do not include a treatment initiation phase. In other words, the first administered dose is therapeutic without the need for titration to avoid adverse gastrointestinal side effects. For example, in some embodiments, the method involves administering an initial dose or doses (treatment initiation phase) of a peptide of the present disclosure, such as SEQ ID NO: 1, followed by one or more second doses, each of which is administered for one or more weeks. In some embodiments, the first and second doses can be followed by one or more third doses, which may be higher than the second dose. Switching from the first, second, and third doses can be done on a weekly basis. For example, if the first dose does not appear to induce a reduction in blood glucose levels and / or weight loss after one or more weeks, a second higher dose can be administered for one or more weeks, and then the effects of the second dose can be analyzed. If a beneficial effect is observed (e.g., a reduction in blood glucose levels and / or weight), the second dose can be continued. If a beneficial effect is not observed, a third dose can be administered for one or more weeks, after which the beneficial effect can be measured. This cycle of administration and analysis can be repeated as necessary, so long as no adverse events are observed with each administration. In some embodiments, if glycemic control (e.g., reduction in blood glucose levels) is not achieved about four weeks after the initial one or more administrations, a subsequent second or more doses of the peptide can be administered. In some embodiments, the initial one or more doses can be administered without any intention of producing a therapeutic effect (e.g., reduction in blood glucose levels and / or weight loss). However, in some embodiments, the method can be practiced without a treatment initiation step.

[0064] In some embodiments, the method may be the first indication for glycemic control and / or weight loss in a human, meaning that it is the first and only active agent administered to a patient for the purpose of controlling blood glucose levels and / or inducing weight loss in a human. In some embodiments, the methods disclosed herein may include supplemental diet and / or exercise therapy. In such embodiments, a pharmaceutical dosage may be administered to a human, and dietary and / or exercise instructions may be provided that can enhance the beneficial effects of the pharmaceutical dosage. In some embodiments, the human to whom the pharmaceutical dosage is administered has type 2 diabetes mellitus. In some embodiments, the human may exhibit established cardiovascular disease, with or without type 2 diabetes mellitus.

[0065] In some embodiments, the pharmaceutical dosage is administered approximately weekly. In some embodiments, the pharmaceutical dosage is administered to a human approximately weekly for about 2 weeks to about 8 weeks or more. In some embodiments, a pharmaceutical dosage administered to a human as a weekly dose for about 4 to about 8 weeks, optionally about 6 weeks, results in greater total body weight loss at about week 1, about week 2, about week 3, and about week 4 compared to administration of an approximately equimolar dose of semaglutide. In some embodiments, the pharmaceutical dosage is administered on about days 1, 8, 15, 22, 29, and 36. In some embodiments, the method may include administration to a human of a single dose, which results in lower blood glucose levels at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide. In some embodiments, the methods may involve administering to a human a weekly dose for about 4 to about 8 weeks, optionally for 6 weeks, which results in greater weight loss at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or about 7 weeks after administration compared to administration of an approximately equimolar dose of semaglutide. In some embodiments, the methods may involve administering to a human a single dose, which results in a lower C at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide. maxIn some embodiments, the method can include administering a pharmaceutical dosage to an adult at about 0.5 mg / dose, about 1.0 mg / dose, about 1.5 mg / dose, about 2.0 mg / dose, about 2.5 mg / dose, about 3.0 mg / dose, about 3.5 mg / dose, about 4.0 mg / dose, about 4.5 mg / dose, about 5.0 mg / dose, or about 5.5 mg / dose. In some embodiments, the pharmaceutical dosage can be administered about once per week or once every two weeks, optionally for at least one month, and optionally, each dose contains approximately the same amount of agonist peptide product. In some embodiments, the pharmaceutical dosage can be administered subcutaneously. In some embodiments, one or more doses can be administered via a first route (e.g., subcutaneously) and then by a different route (e.g., orally). In some embodiments, the time to reach a therapeutic dose is about four weeks or less. In some embodiments, administration of the pharmaceutical dosage formulation provides a C of about 400 to about 1300 ng / ml. max , T from about 10 hours to about 36 hours max , and / or approximately 15,000-45,000h * AUC in ng / mL 0-48In preferred embodiments, the human experiences a weight loss of at least 5%, at least 10%, or about 1% to about 20%; or about 5% to about 10% (w / w). In some embodiments, administration to a mammal results in a weight loss in an obese mammal sufficient to return the human to the normal weight range for a lean, normal human. In some embodiments, administration to a human with a body mass index (BMI) indicative of obesity (e.g., about 30 or greater) exhibits a weight loss of about 5-20%, such as about 15%, over a suitable period of time (e.g., after about 2, 4, 8, 10, 20, or 30-100 weeks, such as about 50, 60, or 70 weeks). In preferred embodiments, such a human experiences a significant weight loss (e.g., P<0.001, 95% confidence interval (CI)). In some preferred embodiments, within about 4 weeks, administration to a human results in a weight loss of at least about 2-5%, which in some embodiments continues and / or stabilizes until administration is discontinued. In some embodiments, in addition to weight loss, administration may also improve cardiovascular risk factors, including significant reductions in waist circumference, BMI, systolic and diastolic blood pressure, HbA1c, fasting plasma glucose, C-reactive protein, and / or fasting lipid levels, as well as, in some embodiments, physical function scores and quality of life. In some embodiments, the pharmaceutical dosage formulation is an aqueous formulation comprising one or more of polysorbate 20, arginine, or mannitol.

[0066] CERTAIN EMBODIMENTS OF THE DISCLOSURE Preferred aspects of the present disclosure include: A pharmaceutical dosage formulation comprising an agonist peptide product having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), the peptide being modified with a non-ionic glycolipid surfactant, the dosage being designed to improve blood glucose control upon administration to a mammal compared to an agonist having disproportionate affinity for GLP-1R and GCGR with a reduction in one or more adverse events, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain, and constipation.

[0067] 1. A pharmaceutical dosage formulation comprising an agonist peptide having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), the peptide being modified with a non-ionic glycolipid surfactant, the dosage being designed to induce weight loss with reduced one or more adverse events upon administration to a mammal compared to an agonist having disproportionate affinity for the GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain, and constipation.

[0068]

[0023] 2. The pharmaceutical dosage formulation of any preceding embodiment, wherein said weight loss is at least 5%, at least 10%, or from about 1% to about 20%, or from about 5% to about 10% (w / w).

[0069]

[0023] The pharmaceutical dosage formulation of any preceding aspect, wherein the dosage is configured as a once-weekly dosage form, optionally configured for administration for about 2 weeks to about 8 weeks.

[0070]

[0023] The pharmaceutical dosage formulation of any preceding aspect, wherein administration of a single dose to the mammal results in a reduction in blood glucose levels at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide.

[0071]

[0023] The pharmaceutical dosage formulation of any preceding embodiment, wherein administration to a mammal for about 4 to about 8 weeks, optionally about 6 weeks, results in greater total body weight loss at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks after administration compared to administration of an approximately equimolar dose of semaglutide.

[0072] administration of a single dose to the mammal results in a lower C at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide. max 10. The pharmaceutical dosage formulation of any preceding aspect, wherein

[0073]

[0023] The pharmaceutical dosage formulation of any preceding embodiment, wherein the dual agonist peptide is any one of SEQ ID NOs: 1-10 or 12-27.

[0074]

[0023] The pharmaceutical dosage formulation of any preceding embodiment, wherein the dual agonist peptide has approximately equal affinity for GLP-1R and GCGR, and optionally wherein the dual agonist peptide is SEQ ID NO:1.

[0075]

[0023] A pharmaceutical dosage formulation according to any preceding aspect, wherein the surfactant is a surfactant of the 1-alkylglycoside class.

[0076]

[0023] The pharmaceutical dosage formulation of any preceding aspect, wherein the pharmaceutical dosage formulation is present as an aqueous formulation comprising one or more of polysorbate 20, arginine, or mannitol.

[0077] 2. The method of claim 1, wherein administration of a pharmaceutical dosage formulation to a mammal compared to administration of an approximately equimolar dose of semaglutide: a reduction in blood glucose levels at about 48 hours or 96 hours after administration, optionally resulting in a reduction in said blood glucose levels by about 50%; a reduction in blood glucose levels at about 72 hours after administration, optionally with a reduction in blood glucose levels of about 100%; and / or Blood sugar levels decrease approximately 120 hours after administration 10. The pharmaceutical dosage formulation of any preceding aspect, wherein

[0078] 10. The pharmaceutical dosage formulation of any preceding aspect, comprising: a) administering to a mammal the dosage formulation, Induce generalized weight loss, and / or Induces liver weight loss, and / or b) administration of the dosage formulation to a mammal compared to semaglutide administered at approximately equimolar doses; exhibit a lower Cmax, optionally about a 50% lower Cmax; exhibiting about the same or greater Tmax, optionally exhibiting about 100% longer Tmax; Similar AUC (0-inf) and optionally about 85-93% thereof; exhibiting a T1 / 2 (time) that is approximately equal to or longer, optionally about 25-75% thereof; exhibiting an extended MRT (time), optionally at least about 25% higher; demonstrated a long-term PK / PD profile, It shows almost the same or better glucose regulating effect, induce greater total body weight loss, optionally approximately double the weight loss, Induce a loss of body fat mass, optionally about 50-100% lower; and / or When administered to treat NASH, it induces whole body weight loss, liver weight loss, improvement in NAS score, improvement in hepatic steatosis, improvement in ballooning, improvement in col1A1 staining, improvement in ALT, improvement in liver TG / TC, and improvement in plasma TG / TC. 10. The pharmaceutical dosage formulation of any preceding aspect. .

[0079] The pharmaceutical dosage formulation of the preceding embodiment, wherein administration to a mammal results in a significant reduction in body weight, optionally about 15% greater, by about 14 days after administration of the dosage formulation, and / or a significant reduction in body weight, optionally about 25% greater, by about 20-28 days after administration of the dosage formulation, compared to semaglutide administered at an approximately equimolar dose.

[0080] 10. The pharmaceutical dosage formulation of any preceding aspect, wherein administration to said mammal results in weight loss in an obese mammal sufficient to return said mammal to a normal weight range for lean, normal mammals.

[0081] 10. The pharmaceutical dosage formulation of any preceding aspect, comprising one or more pharmaceutically acceptable excipients selected from a buffering agent or an osmolality adjusting agent.

[0082] 10. The pharmaceutical dosage formulation of any preceding aspect, further comprising a surfactant.

[0083] 10. The pharmaceutical dosage formulation of any preceding embodiment, wherein the concentration of the dual peptide agonist is 0.05-20 mg / ml.

[0084] 10. The pharmaceutical dosage formulation of any preceding embodiment, wherein the concentration of the dual peptide agonist is 0.1-10 mg / ml.

[0085] 10. The pharmaceutical dosage formulation of any preceding aspect, wherein the dual peptide agonist has a pH of 6-10.

[0086] 10. The pharmaceutical dosage formulation of any preceding embodiment, comprising about 0.025-0.15% (w / w) polysorbate 20 or polysorbate 80, about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol in water (pH 7.7±1.0), optionally about 0.050% (w / w) polysorbate 20, about 0.35% (w / w) arginine, about 4.3% (w / w) mannitol in water (pH 7.7±1.0).

[0087]

[0023] The pharmaceutical dosage formulation of any preceding embodiment, comprising about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol, and 0.6-1.0 mg of polysorbate 20 or 1.0-1.5 mg of polysorbate 80 in water (pH 7.7±1.0) per mg of ALT-801 (SEQ ID NO: 1).

[0088]

[0033] Aspect 1. The pharmaceutical dosage formulation of any preceding aspect, adapted to be administered to said mammal, wherein said agonist peptide product is less than about 0.25 mg / kg / dose, optionally greater than about 0.001 mg / kg / dose and less than about 0.15 mg / kg / dose.

[0089] 10. The pharmaceutical dosage formulation of the preceding embodiment, configured to administer less than 0.25 mg / kg / dose of the agonist peptide product to said mammal.

[0090] The pharmaceutical dosage formulation of the preceding embodiment, configured to administer between 0.001-0.15 mg / kg / dose, optionally about 0.03 mg / kg / dose or about 0.10 mg / kg / dose.

[0091] The pharmaceutical dosage formulation of the preceding embodiment, configured for administration to a human of between about 0.1 and about 15 mg per week, optionally between about 1-about 7 mg per week, or optionally between about 1-5 mg per week.

[0092] The pharmaceutical dosage formulation of the preceding embodiment, configured to be administered to the mammal once a week for at least six weeks, or for up to six weeks.

[0093]

[0023] The pharmaceutical dosage formulation of any preceding aspect, wherein the time to reach a therapeutic dose is about 4 weeks or less.

[0094] C with a therapeutic dose of about 10 to about 300 ng / ml max , T from about 10 hours to about 36 hours max , and / or approximately 1,000-100,000h * AUC in ng / mL 0-168 10. The pharmaceutical dosage formulation of the preceding aspect, wherein:

[0095] 1. A method for lowering blood glucose levels in a mammal, the method comprising administering to the mammal a pharmaceutical dosage formulation according to any preceding aspect, the method comprising: a) upon administration to a mammal, reduces the incidence of one or more adverse events selected from nausea, vomiting, diarrhea, abdominal pain and constipation compared to an agonist having disproportionate affinity for GLP-1R and GCGR; b) produces about 50% lower blood glucose levels at about 48 hours or 96 hours after administration, about 100% lower blood glucose levels at about 72 hours after administration, and / or a reduction in blood glucose levels at about 120 hours after administration compared to administering an approximately equimolar dose of semaglutide; c) induces total body weight loss and / or induces liver weight loss; d) compared to a regimen in which approximately equimolar doses of semaglutide are administered; a lower Cmax or optionally about a 50% lower Cmax; a Tmax that is approximately equal to or greater than, or optionally about 100% greater than, Similar AUC(0-inf) or optionally approximately 85-93% AUC (0-inf) , Approximately equal to or lower T (hours), or optionally about 50-75% of T 1 / 2 (time), an extended MRT (hours), or optionally at least about 25% higher MRT (hours); an extended PK / PD profile demonstrating comparable or even greater glucoregulatory efficacy; greater total body weight loss, or optionally approximately double the total body weight loss; Lower body fat mass, optionally reduced by about 100%; and / or When the method is for treating NASH, it results in increased total body weight loss, decreased liver weight, improved NAS score, improved hepatic steatosis, improved ballooning, improved col1A1 staining, improved ALT, improved liver TG / TC, and improved plasma TG / TC; e) resulting in a greater weight loss by about 14 days after administration of the dosage formulation, optionally about a 15% greater weight loss, compared to semaglutide administered at an approximately equimolar dose, and / or resulting in a greater weight loss by about 20-28 days after administration of the dosage formulation, optionally about a 25% greater weight loss; and / or f) A method which results in weight loss in an obese mammal sufficient to restore said mammal's weight to the normal weight range for a lean, normal mammal. .

[0096] 10. A method for inducing weight loss in a mammal, the method comprising administering to the mammal the pharmaceutical dosage formulation of any preceding aspect, wherein the method reduces the incidence of one or more adverse events compared to an agonist with disproportionate affinity for GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to the mammal.

[0097] 10. The method of the preceding embodiment, wherein the dual agonist peptide is any one of SEQ ID NOs: 1-10 or 12-27.

[0098] The method of the preceding embodiment, wherein the dual agonist peptide has approximately equal affinity for GLP-1R and GCGR, and optionally said dual agonist peptide is SEQ ID NO:1.

[0099] The method of the preceding aspect, wherein the pharmaceutical dosage is administered about weekly.

[0100]

[0023] Aspect 11. The method of any preceding aspect, wherein the pharmaceutical dose is administered subcutaneously.

[0101]

[0023] The method of any preceding embodiment, wherein the pharmaceutical dosage is administered about weekly for about 2 weeks to about 8 weeks, or longer.

[0102] The method of any preceding aspect, wherein administering the pharmaceutical dosage to the mammal as a weekly dose for about 4 to about 8 weeks, optionally about 6 weeks, compared to administration of an approximately equimolar dose of semaglutide, results in a greater reduction in total body weight at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks after administration to the mammal.

[0103]

[0039] Aspect 11. The method of any preceding aspect, comprising administering to the mammal the agonist peptide product at less than about 0.25 mg / kg / dose, optionally from greater than about 0.001 mg / kg / dose to less than about 0.15 mg / kg / dose.

[0104] 10. The method of the preceding embodiment, wherein said mammal is administered at less than about 0.25 mg / kg / dose.

[0105]

[0033] Aspect 11. The method of any preceding aspect, wherein the agonist peptide product is administered at 0.001-0.15 mg / kg / dose, optionally at about 0.03 mg / kg / dose or about 0.10 mg / kg / dose.

[0106]

[0023] Aspect 11. The method of any preceding aspect, wherein each dose is administered about once a week or once every two weeks, optionally for at least one month, and optionally each dose comprises approximately the same amount of agonist peptide product.

[0107]

[0042] The method of any preceding embodiment, comprising administering a single dose of less than about 0.25 mg / kg / dose, followed by one or more subsequent doses of about 0.03 mg / kg / dose to about 0.10 mg / kg / dose.

[0108]

[0039] Aspect 11. The method of any preceding aspect, comprising administering between 0.001-0.15 mg / kg / dose of the agonist peptide product.

[0109]

[0039] The method of any preceding embodiment, wherein the pharmaceutical dosage formulation comprises about 0.025-0.15% (w / w) polysorbate 20 or polysorbate 80, about 0.2-0.5% (w / w) arginine, about 3-6% mannitol in water (pH 7.7±1.0), optionally about 0.050% (w / w) polysorbate 20, about 0.35% (w / w) arginine, about 4.3% (w / w) mannitol in water (pH 7.7±1.0), and optionally wherein the dual agonist peptide is SEQ ID NO: 1.

[0110]

[0039] The method of any preceding embodiment, wherein the formulation comprises about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol, and 0.6-1.0 mg of polysorbate 20 or 1.0-1.5 mg of polysorbate 80 in water (pH 7.7±1.0) per mg of ALT-801 (SEQ ID NO: 1).

[0111]

[0023] The method of any preceding embodiment, wherein administration of said pharmaceutical dosage formulation is configured for administration to a human at between about 0.1 and about 15 mg per week, optionally at about 1-7 mg per week, or optionally at about 1-5 mg per week.

[0112]

[0013] The method of any preceding embodiment, wherein the time to reach a therapeutic dose is about 4 weeks or less.

[0113] 1. A pharmaceutical dosage formulation configured for subcutaneous administration comprising an agonist peptide product having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), wherein the peptide product is represented by SEQ ID NO: 1, and wherein the dosage is configured to improve blood glucose control by reducing one or more adverse events compared to an agonist having disproportionate affinity for GLP-1R and GCGR, wherein the adverse events are selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to a mammal.

[0114] 1. A pharmaceutical dosage formulation configured for subcutaneous administration comprising an agonist peptide having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), wherein the peptide product is represented by SEQ ID NO: 1, and wherein the dosage is configured to induce weight loss with reduced one or more adverse events when administered to a mammal, compared to an agonist having disproportionate affinity for GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain, and constipation.

[0115] 10. The pharmaceutical dosage formulation of the preceding embodiment, wherein said weight loss is at least 5%, at least 10%, or about 1% to about 20%, or about 5% to about 10% (w / w).

[0116]

[0023] The pharmaceutical dosage formulation of any preceding embodiment, wherein the dosage is formulated as a once-weekly dosage form, optionally for administration for about 2 weeks to about 8 weeks.

[0117]

[0039] The pharmaceutical dosage formulation of any preceding embodiment, wherein the formulation comprises, per mg of ALT-801 (SEQ ID NO: 1), about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol, and 0.6-1.0 mg of polysorbate 20 or 1.0-1.5 mg of polysorbate 80 in water (pH 7.7±1.0).

[0118] Administration of a single dose to a mammal results in a lower C at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide. max 2. The pharmaceutical dosage formulation of the preceding aspect, wherein

[0119]

[0023] The pharmaceutical dosage formulation of any preceding embodiment, wherein the dosage is configured for administration to a human at between about 0.1 mg and about 15 mg per week, optionally about 1-7 mg per week, or optionally about 1-5 mg per week.

[0120]

[0023] The pharmaceutical dosage formulation of any preceding aspect, wherein the pharmaceutical dosage formulation is adapted to be administered to a mammal once a week for at least 6 weeks, or for up to 6 weeks.

[0121]

[0023] 2. The pharmaceutical dosage formulation of any preceding embodiment, wherein the dosage is configured to reach a therapeutic dose within about 4 weeks after the initial weekly administration.

[0122] The therapeutic dose is about 10 to about 300 ng / ml of C max , optionally C<200ng / ml max , T from about 10 hours to about 36 hours max , and / or approximately 1,000-100,000h * AUC in ng / mL 0-168 2. The pharmaceutical dosage formulation of the preceding aspect, wherein

[0123] 10. A method of inducing weight loss in a mammal, comprising administering to the mammal the pharmaceutical dosage formulation of any preceding aspect, wherein the method reduces the incidence of one or more adverse events compared to an agonist with disproportionate affinity for GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to the mammal at a therapeutic dose.

[0124]

[0023] A method according to the preceding aspect, wherein said pharmaceutical dosage is administered about weekly, and wherein the initial dose is a therapeutic dose.

[0125]

[0023] The method of the preceding embodiment, wherein said pharmaceutical dosage is administered about weekly for about 2 weeks to about 8 weeks, or longer.

[0126] As will be appreciated by those skilled in the art, other aspects of the present disclosure are also contemplated.

[0127] Unless otherwise defined or clearly indicated otherwise by 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. 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" appears before the first number in a series of two or more numerical values, or in a range of two or more numerical values, the term "about" or "approximately" applies to each and every numerical value in that series or range of numerical values. 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 aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedents "about" or "approximately," it is understood that the particular value forms another aspect. It is 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 not only the range itself, but each individual value within the range, as if each value were listed individually. Optionally or optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs or 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.

[0128] Certain embodiments are further described in the following examples, which are offered by way of example only and are not intended to limit the scope of the claims in any way. [Example]

[0129] Example 1. Peptide synthesis There are many standard protecting groups and coupling agents that can be successfully used in a typical N-α-Fmoc-based peptide synthesis. Typical examples are listed in U.S. Pat. No. 9,856,306 B2, which is incorporated by reference in its entirety into this disclosure. Further examples can be found in many reviews and protocols, such as those published by Novabiochem and regularly updated online, as well as more specialized reviews (e.g., Behrendt, R., et al. (2015) JP Peptide Sci 22:4-27 and references therein). A typical commercial protocol used by many contract peptide synthesis companies was used in the synthesis herein. A more specialized protocol is provided below.

[0130] Preparation of C-terminal amide analogue—SEQ ID NO:1. Boc-His(Trt)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(tBu)-Glu * -Lys(ivDDE)-Ala-Ala-Lys *A sample of -Glu(tBu)Phe-Ile-Gln(Trt)-Trp(Boc)-Leu-Leu-Gln(Trt)-Thr(tBu)-Rink amide resin (SEQ ID NO: 1) was prepared by sequential addition of N-α-Fmoc-protected amino acids using standard coupling protocols, such as diisopropylcarbodiimide (DIC) / hydroxybenztriazole (HBT) coupling, followed by standard deprotection with piperazine and subsequent coupling (Glu * and Lys * The side-chain cyclic lactam linkage was achieved by deprotection of the allyl-based side-chain protection using Pd(PPh3)4 / 1,3-dimethylbarbituric acid catalyst, washing with DIPEA and 0.5% sodium diethyldithiocarbamate trihydrate in NMP, and then coupling with DIC / Oxyma. The ivDDE group at the Lys-N-ε position of residue 17 was deprotected by incubation with 2% or more hydrazine hydrate in DMF, followed by washing with DMF / CHCl2. The Lys side chain was acylated with tert-butyl 18-([beta-D-glucuron-1-yl]oxy)octadecanoate in DMF / CHCl2 using DIC / HBt or other coupling agents. Completion of the coupling was checked with ninhydrin, and the product was thoroughly washed with CHCl2.

[0131] The product resin underwent final deprotection and cleavage from the resin by treatment with a cleavage cocktail (94% TFA:2% EDT; 2% HO; 2% TIS) for 240 min at room temperature. The mixture was treated with EtO to precipitate the product, which, after extensive washing with EtO and drying in vacuo, gave the crude title peptide.

[0132] Purification was performed in batches by reverse-phase (C18) HPLC. The crude peptide was loaded onto a 4.1 x 25 cm HPLC column at a flow rate of approximately 15 mL / min (CHCN organic modifier in 0.1% aqueous trifluoroacetic acid, Buffer A; CHCN with 0.1% TFA, Buffer B) and eluted with a gradient from 40-70% Buffer B. The product fractions were lyophilized to give the title product peptide (SEQ ID NO: 1) with a purity of >94% by analytical HPLC (10.5 min; 40-70% CHCN in 0.1% TFA) / mass spectrometry (M+1 peak = 1937.44; observed molecular weight 3872.88). Other analogs of the invention were prepared in a similar manner using glucuronic acid or melibiuronic acid prepared as shown in the Examples.

[0133] The analytical data are shown in Table A: TIFF2026021305000015.tif32161

[0134] Compounds were analyzed by HPLC / MS to provide purity and identity data (molecular ion detection). The HPLC technique utilized an analytical column packed with the listed material of the listed particle size, and data are reported here as k' values ​​(k' = (Tr - To) / To), which are largely independent of system configuration and dead volume, but depend on the gradient and packing material. All compounds were reported to be approximately 95% pure.

[0135] The corresponding 1-methyl and 1-octyl analogs of the title compound were prepared in a similar manner, but using D-glucuronic acid and 1'-octyl β-D-glucuronic acid (Carbosynth). 1-Decyl, 1-dodecyl, 1-tetradecyl, 1-hexadecyl, 1-octadecyl, and 1-eicosyl analogs, as well as larger analogs, are prepared using the corresponding mono- and disaccharide uronic acids prepared as described above. Alternatively, 1-alkylglucuronyl or other uronic acid acylated analogs can be prepared by first purifying a deprotected or partially deprotected peptide, followed by acylation with the desired uronic acid reagent. Alternatively, 1-alkylglucuronyl or other uronic acid acylated analogs can be prepared by first purifying a recombinantly prepared peptide, followed by selective acylation of the side chain amino function with the desired uronic acid reagent.

[0136] A. 1-Alkyl β-d-glucuronic acid. Common oxidation method. To a solution of 1-dodecyl β-d-glucopyranoside [2.0 g, 5.74 mmol] in 20 mL of acetonitrile and 20 mL of deionized water, (diacetoxyiodo)benzene [4.4 g, 13.7 mmol] and TEMPO [0.18 g, 1.15 mmol] were added. The resulting mixture was stirred at room temperature until the reaction was complete (up to 20 h). The reaction mixture was diluted with water and lyophilized to give the crude product as a white powder (1.52 g, 73%) of sufficient purity for direct coupling to peptide Lys side chains. In a similar manner, other 1-alkyl β-d-glucuronic acids or melibiuronic acids were prepared for use in acylation of other peptide products described herein. The corresponding mono-substituted glucosides or melivosides were prepared using the procedures of these examples, but substituting the appropriate chain length dicarboxylic acid starting material to produce the desired chain length, e.g., hexadecanedioic acid, dodecanedioic acid, etc., from the synthetic procedures of the examples in place of octadecanedioic acid.

[0137] B. 18-(tert-butoxy)-18-oxooctadecanoic acid A suspension of octadecanedioic acid (40 g, 127 mmol) in toluene (500 mL) was heated at 95 °C under nitrogen. To the resulting solution, N,N-dimethylformamide di-tert-butyl acetal (98 g, 434 mmol) was added dropwise over 3–4 h. The reaction was stirred overnight at the same temperature, concentrated to dryness in vacuo, and placed under high vacuum overnight. The resulting solid was suspended in CHCl (200 mL) with heating and sonication, filtered at room temperature, and washed with CHCl. ​​The filtrate (2) was concentrated to give the product as a solid (45 g, 86%), which was used without further purification.

[0138] C. tert-Butyl 18-hydroxyoctadecanoate A solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (45 g, 121 mmol) in THF was cooled in an ice bath under nitrogen and treated dropwise with borane dimethyl sulfide complex (16 mL, 158 mmol). Vigorous gas evolution occurred over the first few milliliters of addition. After the addition, the mixture was allowed to warm slowly to room temperature and stirred overnight. The reaction was cooled in an ice bath, quenched with saturated sodium carbonate solution, diluted with ethyl acetate, and washed with saturated sodium carbonate solution. The organic layer was concentrated in vacuo and placed under high vacuum overnight. The residue was dissolved in warm toluene (200 mL) and allowed to stand at room temperature for several hours. The precipitated diol was removed by filtration through Celite, and the cake was washed with toluene. The toluene solution was applied directly to a silica gel column and eluted with 10% ethyl acetate / hexane, then 20% ethyl acetate / hexane, then 30% ethyl acetate / hexane. Concentration afforded the product (24 g, 51%) as an oil that solidified upon standing. 1 HNMR(500MHz,d4-MeOH):δ=3.64(m,2H),2.21(t,2H,J=9),1.44(s,9H)1.50-1.62(m,4H),1.20-1.40(m,27H)

[0139] D. tert-Butyl 18-([1-beta-D-glucos-1-yl]oxy)octadecanoate tert-Butyl 18-hydroxyoctadecanoate (46 g, 129 mmol) was dissolved in toluene (400 mL), concentrated under reduced pressure to approximately 250 mL, and allowed to warm to room temperature under nitrogen. To this solution, HgO (yellow) (22.3 g, 103 mmol), HgBr2 (37 g, 103 mmol), and acetobromoglucose were added with vigorous stirring. Stirring was continued overnight until the alcohol was consumed, and the mixture was filtered through Celite. The filtrate was treated with copper(II) triflate (1 g) and stirred for 1 hour until the orthoester (product spotted above by TLC) was decomposed. The reaction was then washed with water, and the organic layer was concentrated in vacuo. The residue was dissolved in methanol (500 mL) and treated with 0.5 mL portions of sodium methoxide (5.4 M in methanol) to a pH of 9 (direct spot on pH paper). The pH was checked every 0.5 hours, and further sodium methoxide was added as needed to maintain the pH at 9. The reaction was complete in 4 hours. Acetic acid was added dropwise to bring the pH to 7, and the mixture was concentrated under reduced pressure. The residue was loaded onto silica gel and purified by silica gel chromatography eluting with 5% methanol / CHCl followed by 10% methanol / CHCl to give the product as a white solid (55 g, 82%). 1 HNMR(400MHz,d4-MeOH):δ=4.30(d,1H,J=7.6),3.84(m,1H),3.77(d,1H,J=9.6),3.45-3.60(m,2H),3.36(t,1 H,J=9.2),3.21(t,1H,J=8.4),2.20(t,2H,J=7.2),1.50-1.67(m,4H),1.43(s,9H),1.43-1.33(m,2H),1.28(b rs,24H)

[0140] E. tert-Butyl 18-([β-D-glucuron-1-yl]oxy)octadecanoate tert-Butyl 18-([1-beta-D-glucos-1-yl]oxy)octadecanoate (50 g, 96 mmol) was dissolved in dioxane (800 mL) with mechanical stirring in a 2000 mL three-neck flask and cooled to 10°C. To this solution were added 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) (150 mg, 0.96 mmol) and KBr (1.14 g, 9.6 mmol). A dropping funnel containing saturated Na2CO3 solution (300 mL) and 13% NaOCl solution (120 mL) was attached to the flask. The carbonate solution was added rapidly, followed by slower NaOCl addition (approximately 1 drop / sec). After the addition of 100 mL of carbonic acid, the pH was checked and more added as needed to maintain a pH of approximately 10. The temperature was maintained between 10°C and 15°C throughout. After 3 hours, Starting material remained, so additional NaOCl (10 ml) was added quickly. After 0.5 h, the reaction was quenched with methanol (10 ml). The mixture was poured into a 4000 ml Erlenmeyer flask, submerged in an ice bath, and adjusted to pH 3 with 6 N HCl. The mixture was diluted with ethyl acetate, washed with 1 N HCl, and washed twice with distilled water, with the layers separated after the final wash. The organic layer was concentrated in vacuo to give the product as a white foam (38 g, 74%).

[0141] Quantitative analysis using 2,3,4,5-tetrachloronitrobenzene (TCNB) internal standard for the anomeric CH 1 H NMR (500 MHz, d4-MeOH) gives 94.8% of the expected weight. Purity >95% by TLC (staining using 20% ​​MeOH / DCM / 2 drops HOAc, 20% H2SO4 / EtOH + heat). 1 HNMR(500MHz,d4-MeOH):δ=4.30(d,1H,J=9.5),3.85(m,1H),3.77(d,1H,J=7.5),3.48-3.56(m,2H),3.37(t,1H,J= 11.5), 3.21(t,1H,J=9.5),2.20(t,2H,J=9.5),1.52-1.66(m,4H),1.44(s,9H),1.34-1.42(br,2H),1.28(s,25H).

[0142] Example 2. Dual Agonist Peptides - In Vitro Assay Cellular assays were performed using standard cell assays (DiscoveRx, LeadHunter assays) that use cAMP stimulation or arrestin activation as a readout. Compounds were accurately weighed in approximately 1 mg amounts and shipped to DiscoverX (Fremont, CA) for dilution and assay. The assays used involved glucagon (human, cloned into CHO cells) and GLP-1 (human, cloned into CHO cells) receptors in cellular assays. Assays were performed in the presence of 0.1% ovalbumin. Historically, such assays have been performed in the presence of 0.1% BSA, but for these compounds that bind very strongly to serum albumin (>99%), this can skew the results and significantly reduce the compound's potency. Using 0.1% ovalbumin circumvents this issue. The improvement seen with the use of ovalbumin can be seen as an indicator of the relative strength of serum albumin binding to the peptide.

[0143] [Table 5]

[0144] EU-A1588 = SEQ ID NO: 2; EU-A1871 = SEQ ID NO: 3; EU-A1872 = SEQ ID NO: 4; EU-A1873 = SEQ ID NO: 1; Semaglutide = SEQ ID NO: 11.

[0145] Assays were performed in the presence of 0.1% ovalbumin. Historically, such assays have been performed in the presence of 0.1% BSA, but for those compounds that bind very strongly to serum albumin (>99%), this can skew the results and significantly reduce the potency of the compound. Using 0.1% ovalbumin circumvents this issue. The improvement seen with the use of ovalbumin can be seen as an indication of the relative strength of serum albumin binding to the peptide. See table below.

[0146] [Table 6]

[0147] Here, it can be seen that a very tight serum albumin binder (with COH-containing substituents, mimicking a fatty acid head group) shows a substantial fold improvement when BSA is replaced with ovalbumin, which does not appreciably bind the fatty acid mimic. The degree of fold improvement provides a readout of the strength of binding to the fatty acid binding site on BSA. Thus, semaglutide shows a 12-fold improvement (tight binding) and EU-A1873 shows a 30- to 40-fold improvement, representing a significant increase in serum albumin binding. This degree of serum albumin binding would be expected to result in a reduced Cmax and extended duration of action, as seen in the bioassay of SEQ ID NO: 1.

[0148] The data shown in Tables 5 and 6 above demonstrate that the test compound is an agonist of both GLP-1R and GCGR (a "dual agonist"), unlike semaglutide, which shows a biased high affinity for GLP-1. The data also show that SEQ ID NO: 1 is a dual agonist peptide with approximately equal affinity for GLP-1R and GCGR.

[0149] Example 3. In vivo effects on glucose, body weight, and fat loss A. In vivo assay using db / db mice. Approximately 75 BKS.Cg-m+ / + Leprdb / J (Jackson Labs stock number 000642) male ("db / db") mice, 7-9 weeks old, were used in these studies and maintained using standard animal care procedures. The study began after a one-week acclimation to facility conditions. On the morning of study day 0, mice were weighed and fasted for 4 hours. Blood glucose levels were measured by glucometer using standard procedures. At least 54 mice were selected based on weight, and mice with blood glucose levels ≥ 300 mg / dL (i.e., diabetic) were randomly assigned to six groups (n = 9). The groups were as follows: Group 1, vehicle; Group 2, semaglutide 3 nmol / kg; Group 3, semaglutide 10 nmol / kg; Group 4, SEQ ID NO: 1, 1 nmol / kg; Group 5, SEQ ID NO: 1, 3 nmol / kg; Group 6, SEQ ID NO: 1, 10 nmol / kg. Clinical observations were performed at the time of receipt prior to randomization and daily from days 1 to 5. Body weights were measured and recorded at the time of receipt prior to randomization and daily from days 1 to 5. Food consumption was measured and recorded daily from days 1 to 5. Blood samples for glucose analysis were taken pre-study (day -3) and at 0, 1, 4, 8, 24, 48, 72, 96 and 120 hours after a single dose of the indicated compound (e.g., SEQ ID NO: 1) on day 1.

[0150] B. In vivo assay using "DIOJAX" mice. Eighty-one 18-week-old male C57BL / 6J mice, fed a high-fat diet (Research Diets D12492) from 6 weeks of age, were transferred to the Jackson in vivo research laboratory (Sacramento, CA). Mice were ear-notched for identification and housed individually in actively ventilated polycarbonate cages with HEPA-filtered air at a maximum density of three mice per cage. Cages were changed every two weeks. The animal room was illuminated entirely with artificial fluorescent light and controlled on a 12-h light / dark cycle (lights on from 6:00 AM to 6:00 PM). The normal temperature and relative humidity ranges in the animal room were 22 ± 4°C and 50 ± 15%, respectively. The animal room was set to allow 15 air changes per hour. All mice continued on the high-fat diet (60% kcal; D12492) and were allowed to acclimate for 4 weeks before the start of the study. On the morning of Study Day 1, baseline body composition of each mouse was determined by NMR analysis. Sixty-three mice were divided into seven groups (n = 9). The remaining ungrouped mice were euthanized. Subcutaneous administration of the compound was performed every other day. On the morning of Study Day 0, pre-dose blood glucose levels were measured using a glucometer. The mice were administered the compound according to Table 7 below, and the administration time was recorded. Blood glucose levels were measured 1, 2, 4, 8, 10, and 24 hours after administration. After Study Day 1, pre-dose blood glucose levels were measured on Days 4, 7, 9, 11, 13, 17, 21, and 25. Body weight and clinical observations were recorded every two days. After administration, food intake was measured daily for all groups. The first food intake measurement was performed on Study Day 1. Group 4 was pair-fed with Group 3, and Group 7 was pair-fed with Group 6. The amount of food consumed by Groups 4 and 7 was determined by the average amount of food consumed by Groups 3 and 6 in the previous 24-hour window, respectively. Food intake in groups 1, 2, 3, 5, and 6 was measured ad libitum and daily. On study day 27, mice were fasted for 5 hours and then subjected to a glucose tolerance test (GTT). All mice received a bolus of glucose (2 g / kg) intraperitoneally, and blood glucose levels were assessed before and 15, 30, 60, 90, and 120 minutes after administration.All blood glucose values ​​were entered into a GTT blood glucose log.

[0151] [Table 7] Note 1: Mice in groups 5 and 6 were dosed at 3 nmol / kg and 6 nmol / kg on days 0, 2, and 4. Starting on day 8, mice in these groups were dosed at 6 nmol / kg and 12 nmol / kg, respectively, as shown in Table 1. Note 2: SEQ ID NO: 1 is referred to as MD-1373 in Table 7.

[0152] C. Glucose Regulation and Tolerance Using the db / db mouse model, high doses of semaglutide suppressed glucose levels for 24 hours and returned to pre-treatment levels by 48 hours, whereas SEQ ID NO: 1 began suppressing blood glucose levels at 4 hours and continued for at least 96 hours, and even up to 120 hours (Figure 1). Thus, SEQ ID NO: 1 was found to exhibit an increased blood glucose response and a prolonged duration of action compared to equimolar amounts of semaglutide in db / db mice. Those skilled in the art will appreciate that the onset of action of SEQ ID NO: 1 indicates a potential reduction in acute gastrointestinal (GI) side effects observed compared to when semaglutide is used. Those skilled in the art will also appreciate that the onset of action of SEQ ID NO: 1 indicates a potential reduction in acute gastrointestinal (GI) side effects observed at lower doses compared to when semaglutide is used.

[0153] The DIOJAX mouse study also showed that blood glucose levels for low (6 nmol / kg) and high (12 nmol / kg) doses of semaglutide decreased to the normoglycemic range 2 hours after administration, then remained suppressed in the normoglycemic range until day 1 after administration, but returned to hyperglycemic levels by day 2 after administration. Low and high doses (6 nmol / kg and 12 nmol / kg, respectively) of SEQ ID NO: 1 ("MD-1373") suppressed blood glucose levels to the normoglycemic range by 4 hours after administration, with the low dose remaining suppressed until day 2 after administration, only returning to the hyperglycemic range by day 4 after administration. Blood glucose levels in animals administered the high dose (12 nmol / kg) of SEQ ID NO: 1 were suppressed to the normoglycemic range from day 7 (7) until the last measurement on day 26 (Figure 2). In the other groups, there was a slight decrease, but blood glucose levels remained in the hyperglycemic range throughout the remainder of the assay. This data indicates that lower doses of SEQ ID NO: 1 (compared to agonists with disproportionate affinity for GLP1R / GCGR) achieve the desired biological effect with reduced adverse events following administration to mammals.

[0154] Furthermore, DIOJAX mice showed large glucose excursions in response to a 2 g / kg IP glucose challenge (intraperitoneal glucose tolerance test (IPGTT)). Both the low-dose and high-dose SEQ ID NO: 1 groups showed blunted glucose excursions, indicating good glucoregulatory effects. For example, as shown in Figure 3, glucose tolerance was found to be similar between SEQ ID NO: 1 and semaglutide using an IPGTT in the DIOJAX mouse model. As shown there, IPGTT assays on day 27 showed similar results for high-dose SEQ ID NO: 1 and semaglutide.

[0155] D. Weight and fat loss SEQ ID NO: 1 was found to result in greater weight loss than semaglutide in BKS.Cg-m+ / +Leprdb / J (Jackson Labs stock number 000642) (db / db) mice. Significant weight changes relative to vehicle were observed for semaglutide and SEQ ID NO: 1 on day 1 after dosing, and for the medium and high doses of SEQ ID NO: 1 on days 2 to 4 (Figure 4). Analysis of food intake showed that the high dose of semaglutide significantly suppressed food intake only on day 1 after dosing, whereas SEQ ID NO: 1 suppressed food intake between days 1 and 4 (Figure 5).

[0156] Glucagon coagonism with SEQ ID NO:1 was found to induce highly potent and stable weight loss of over 25% (12 nmol / kg dose) in DIOJAX mice, more than double that observed after semaglutide administration (e.g., 8-10%), despite similar food intake between the groups (Figure 6). Surprisingly, this data suggests that SEQ ID NO:1 acts via a second mechanism of action (e.g., acting on both sides of the "energy equation," inducing both a decrease in food intake and an increase in energy output). Note that on day 8, the SEQ ID NO:1 group of DIOJAX mice was switched from a 6 to a 12 nmol / kg regimen to compensate for pharmacodynamic (PD) differences between this group of DIOJAX mice and the db / db mice for which previous dose findings had been determined.

[0157] Furthermore, as shown in Figure 7, SEQ ID NO: 1 almost doubled the fat loss observed after semaglutide administration (51% vs. 28% respectively (-6% in the vehicle control group). The observed loss in lean mass was approximately 12% with SEQ ID NO: 1 compared to 6% with semaglutide (approximately 3% in the vehicle control group).

[0158] Example 4. Pharmacokinetics A. Mouse Research The survival phase of the study was conducted at the Jackson Laboratory (Sacramento, CA) with 67 C57BL6 / J male mice (7-9 weeks old) (Diet-Induced Obese (DIO) JAX mice). Mice were ear-notched for identification and housed individually in actively ventilated polycarbonate cages with HEPA-filtered air at a maximum density of four mice per cage. The animal room was illuminated entirely with artificial fluorescent light and controlled on a 12-h light-dark cycle (lights on from 6:00 AM to 6:00 PM). The normal temperature and relative humidity ranges in the animal room were 22 ± 4°C and 50 ± 15%, respectively. The animal room was set to allow a minimum of 15 air changes per hour. Filtered tap water acidified to pH 2.5-3.0 and standard rodent chow were provided ad libitum.

[0159] Both SEQ ID NO: 1 and semaglutide were formulated at 0.02 mg / mL in 50 mM phosphate buffer containing 0.05% Tween 80, at a pH of approximately 8. Dosage volumes were 1.9365 and 5.8095 mL / kg at 10 and 30 nmol / kg for SEQ ID NO: 1, respectively, and 2.057 mL / kg for 10 nmol / kg semaglutide. Three mice in Group 1, who received no medication, were bled only at time 0. For Group 2 (semaglutide; 10 nmol / kg SC), Group 3 (SEQ ID NO: 1; 10 nmol / kg SC), Group 4 (SEQ ID NO: 1; 10 nmol / kg IV), and Group 5 (SEQ ID NO: 1; 30 nmol / kg SC), blood samples were collected up to 120 hours post-dose (n=4 per time point). Plasma concentrations of SEQ ID NO: 1 and semaglutide were determined using LC-MS / MS and pharmacokinetic parameters were determined by non-compartmental analysis using WinNonlin.

[0160] Blood samples were collected at 1, 4, 8, 24, 48, 72, 96, and 120 hours post-dose. For groups 2–5, four mice were bled at two time points, with the second time point designated terminal. At each time point, a minimum of ∼200 μL of whole blood was collected by retroorbital bleeding or cardiac puncture. Blood samples were collected in K2EDTA anticoagulant and centrifuged. Plasma (minimum 100 μL) was transferred to tubes and stored frozen until shipped to a bioanalytical laboratory for analysis by LCMS / MS.

[0161] Determination of SEQ ID NO:1 and semaglutide concentrations in plasma was performed at Climax Laboratories (San Jose, CA). A 100 μL aliquot of plasma was mixed with 10 μL of internal standard (20 μg / mL standard in phosphate-buffered saline) and then mixed with 300 μL of acetonitrile. The sample was vortexed and centrifuged. The supernatant was transferred to a clean 96-well plate for LC-MS / MS analysis. The data are presented in visual format in Figure 8 and in tabular format in Table 8.

[0162] [Table 8]

[0163] After SC administration, as shown in Figure 9, plasma levels of SEQ ID NO: 1 peaked later than semaglutide, with T max At 10 nmol / kg, the AUC of SEQ ID NO: 1 was comparable to that of semaglutide, and the C of SEQ ID NO: 1 max was 54% of that of semaglutide. The reduced Cmax with similar AUC shown by SEQ ID NO: 1 is considered a more favourable profile as it indicates higher than therapeutic blood levels and a minimised peak to trough concentration ratio, thus reducing the potential for side effects.

[0164] Overall, SEQ ID NO: 1 had a slightly longer MRT than semaglutide, 18.3-22.2 hours and 15.5 hours, respectively. After SC administration, plasma concentrations of SEQ ID NO: 1 increased approximately dose-proportionally with a 3-fold increase in dose, with C max and AUC increased 3.2-fold and 2.8-fold, respectively. After IV administration, plasma concentrations of SEQ ID NO: 1 increased over time, with T max was 8 hours after administration. The bioavailability of SEQ ID NO: 1 after SC injection was not calculated because the plasma concentration-time profile suggested that the IV dose may have been delivered perivascularly rather than by the intended intravascular injection.

[0165] A similar study was conducted at The Jackson Laboratory-JAX West (Sacramento, CA) using male C57BL6 / J mice. Pharmacokinetic (PK) parameters were evaluated after a single subcutaneous (sc) dose of ALT801 (containing SEQ ID NO: 1) or semaglutide (both at 10 nmol / kg). Both compounds were formulated at 0.02 mg / mL in 50 mM phosphate buffer, 0.05% Tween 80, pH approximately 8. The dose volume was approximately 2 mL / kg. Blood samples (approximately 200 μL) were collected at 1, 4, 8, 24, 48, 72, 96, and 120 hours post-dose (n=4 per time point). Each mouse was bled at two time points, with the second time point being the terminal bleed. Plasma concentrations of ALT-801 and semaglutide were determined using liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) with limits of quantification of 1.00 ng / mL and 2.00 ng / mL for semaglutide and ALT-801, respectively. Non-compartmental PK analysis using WinNonlin was performed using the mean concentrations at each sampling time point and the maximum concentration (C max ), C max The time at which the max ), the area under the plasma concentration curve from time zero to the last measurable concentration (AUC 0-t), the plasma concentration-time curve from time zero to infinity (AUC), the terminal elimination half-life (T), and the mean residence time (MRT) were reported. The PK parameters observed for ALT-801 and semaglutide administered by the subcutaneous route at a dose of 10 nmol / kg are shown in Figure 9 (T max = 8 and 4 hours, C max = 92 and 182 ng / mL, MRT = 22 and 16 hours), C in mice treated with ALT-801 compared with semaglutide max suggests a more cautious and delayed approach to ALT-801's C max was 50% of the literature standard semaglutide value, while the AUC was >86%. PK parameters for elafibranor were not evaluated because oral administration was required and therefore could not be compared with ALT-801 or semaglutide administered by the subcutaneous injection route.

[0166] B. Miniature pig research The test animals were four single-housed, untreated male Yucatan miniature pigs (Susscrofa). They weighed 73-75 kg. The housing room was set to maintain a room temperature of 16-27°C (61-81°F). Relative humidity was recorded. A 12-hour light / 12-hour dark photoperiod was maintained. Room lights may be on during the dark cycle to facilitate sample collection and other life activities. The animals were fed a maintenance dose of Purina S-9 swine chow. Clean, fresh water from an on-site deep-water well was available ad libitum. General cage observations were conducted at least twice daily (morning and evening) throughout the study period to assess general health, moribundity, or mortality.

[0167] After a 22-day acclimation period, each minipig was treated subcutaneously (behind the cheek jaw) with 20 nmol / kg of SEQ ID NO: 1, and pharmacokinetic blood samples were collected at -0.25, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 168, 192, 216, 264, 312, and 360 hours after administration. After a 2-week washout period, the same animals were intravenously administered SEQ ID NO: 1, and pharmacokinetic blood samples were collected at -0.25, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 168, 192, 216, 264, 312, and 360 hours after administration. The administration concentration was 5.5 mg / mL (dose 0.015 mL / kg) for both treatments.

[0168] Whole blood samples (~3 mL / time point) for pharmacokinetic analysis in tubes containing K2EDTA were collected via the vascular access port (VAP). Samples were kept on wet ice until processing within ~30 minutes of collection. All samples were centrifuged at ~3000 rpm at ~4°C for approximately 15 minutes. The resulting plasma was transferred equally into two cryovials (primary and backup) and placed on dry ice. Plasma samples were stored frozen at approximately -70°C until the primary samples were shipped for analysis.

[0169] No abnormal clinical observations were observed during the study. The concentrations of the test substances are shown in Figure 11.

[0170] After SC administration of SEQ ID NO: 1, plasma levels of SEQ ID NO: 1 increased with an MRT of 86 hours, followed by T max It was also observed that the Cmax increased to 887 ng / mL at 52 hours. In contrast, the MRT of semaglutide has been reported to be 64 hours in minipigs (Lau, J., et al. (2015) J Med Chem 58:7370-80). This lower Cmax and prolonged MRT again indicates a longer duration of action compared to semaglutide, indicating a longer PD profile for SEQ ID NO: 1.

[0171] C. Rat studies 1. Single-dose protocol Sixteen (plus two spares) male CRL:CD(SD) rats, weighing approximately 250-300 g at the start of the study, were received from a standing colony maintained at Charles River Labs. Animals were maintained on a standard diet (Lab Diet C504). Food intake was monitored on study days -1 through 7 by weighing the food and hopper together. Food and drinking water were available ad libitum throughout the study, except for an overnight fasting period prior to dosing on study day -1. All animals were assigned to groups upon receipt.

[0172] On study day 1, all animals received a group-dependent bolus dose of test article (TA) via interscapular (mid) subcutaneous injection. Individual animal weights were recorded beginning on day -1. Animals were observed for any clinically relevant abnormalities during dosing and at all sample collection time points. The study activities are detailed in Table 9.

[0173] [Table 9]

[0174] After TA administration on study day 1, 300 μL whole blood samples were collected into K2EDTA tubes via an indwelling jugular vein catheter (JVC) at the listed time points. The maximum available amount of blood was collected via cardiac puncture for the final time point (144 hours post-dose) after CO2 euthanasia. Whole blood samples were stored on ice for no more than 30 minutes before centrifugation at 2200 × g for 10 minutes at 5°C ± 3°C. The resulting plasma was then pipetted into polypropylene tubes and stored nominally in a freezer set to maintain a temperature of -80°C until transfer to Climax Laboratories (San Jose, CA) for pharmacokinetic analysis. SEQ ID NO: 1 was administered in formulation buffer (0.050% (w / w) polysorbate 20, 0.300% (w / w) methylparaben, 0.348% (w / w) arginine, 4.260% (w / w) mannitol in deionized water) at target dose levels of 0.03 mg / kg, 0.1 mg / kg, or 0.2 mg / kg.

[0175] After SC administration, plasma levels of SEQ ID NO: 1 and semaglutide rose rapidly in rats, as shown in Figure 10. Semaglutide increased the T max On the other hand, the concentration of SEQ ID NO: 1 was still increasing at 8 hours, indicating that the true T max By the next time point, 24 hours, this peaks and decreases somewhat, but is still higher than semaglutide. At 10 nmol / kg, the AUC of SEQ ID NO: 1 (2350 ng.hr / mL) is comparable (93%) to semaglutide (2530 ng.hr / mL), and the C of SEQ ID NO: 1 max was 54% of that of semaglutide. The reduced Cmax with similar AUC exhibited by SEQ ID NO: 1 is considered a very favourable profile as it indicates the potential for reduced side effects due to higher than therapeutic blood levels and a minimised peak-to-trough concentration ratio. Overall, SEQ ID NO: 1 had a longer MRT than semaglutide, 20.6 hours compared to 15.4 hours for semaglutide, respectively.

[0176] 2. Repeated administration protocol in rats The objectives of this study were to evaluate the toxicity and toxicokinetics of the test article ALT-801 when administered daily via subcutaneous injection to rats for at least 6 weeks, and to assess the reversibility, persistence, or delayed onset of any effects after a 4-week recovery period. Animals administered 0.03 mg / kg / day of ALT-801 were treated without incident throughout the entire study period. In contrast, animals treated with doses ≥ 0.09 mg / kg / dose were placed on significant drug holiday during the first 3 weeks of the study due to significant ALT-801 dose-related food intake and associated weight suppression. Dose formulation analysis revealed significant out-of-specification results for all ALT-801 dose formulations as the plausible underlying cause of the exaggerated effects observed during the first 3 weeks of the study in Groups 3 and 4. The dose formulation analysis issues were resolved by the end of week 3, and treatment was resumed for Groups 3 and 4 animals on Day 22. The study period was then extended for an additional 2 weeks of treatment (final necropsy on Day 57). Treatment resumed on week 3, and for animals in groups 3 and 4, on day 22, after which the study period was extended for another 2 weeks (final necropsy on day 57). Group 3 animals were treated with 0.03 mg / kg / day ALT-801 on days 22 and 23, followed by a target dose of 0.09 mg / kg / dose every other day (Q2D) for the remainder of the study. Group 4 animals were treated with 0.09 mg / kg / day on days 22 and 23, followed by a target dose of 0.15 mg / kg / dose, 3 days on / 4 days off, for the remainder of the study. Thus, ALT-801 was administered overall at 0.03 mg / kg / day daily for 8 consecutive weeks (Group 2), 0.09 mg / kg / dose once every other day (Q2D) for 5 consecutive weeks (Group 3), or 0.15 mg / kg / dose 3 days on / 4 days off for 5 consecutive weeks.

[0177] [Table 10] aGroup 1 received the vehicle control article only. b Animals in Group 4 were administered 0.15 mg / kg / dose. Starting on Day 14, animals in Group 4 were administered 0.09 mg / kg / dose. Starting on Day 16, animals in Group 4 were dose escalated to 0.15 mg / kg / dose. Starting on Day 22 of the dosing phase, animals in Group 4 were administered 0.09 mg / kg / dose. Starting on Day 24 of the dosing phase, animals in Group 4 were dose escalated to 0.15 mg / kg / dose until the end of the dosing phase. c Animals in Group 3 were dosed at 0.09 mg / kg / dose. Starting on Day 22 of the dosing phase, animals in Group 3 were dosed at 0.03 mg / kg / dose. Starting on Day 24 of the dosing phase, animals in Group 3 were dosed up to 0.09 mg / kg / dose until Day 35 of the dosing phase. Animals in Group 3 were not dosed on Day 36 of the dosing phase. d Starting on day 32 of the dosing phase, animals in Group 4 were dosed for 3 days (dosing days 32-34), followed by 4 days off dosing. This dosing regimen continued throughout the remainder of the dosing phase (dosing days 39-41, 46-48, and 53-55). e Starting on day 37 of the dosing phase, animals in Group 3 received 0.09 mg / kg / dose once every other day throughout the dosing phase (days 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55).

[0178] Blood samples were collected from three toxicokinetic animals / sex / group / time point at approximately 1.5, 3, 6, 12, 24, 48 (Day 55 and 56 only), and 72 (Day 56 only) hours post-dose in Groups 2 through 4 on Day 1, Groups 3 and 4 on Day 55, and Group 2 pre-dose on Day 56. Blood samples were also collected from three toxicokinetic animals / sex / group / time point in the vehicle control group pre-dose on Days 1 and 56 and approximately 3, 12, 24 (Day 1 only), and 48 (Day 56 only) hours post-dose. Blood samples were processed to plasma and analyzed for ALT-801 at Covance-Madison, and the results were used in generating this toxicokinetic report.

[0179] [Table 11] The NR excretion step cannot be characterized and therefore has not been reported. NR b No measurable concentrations were obtained 72 hours after administration and therefore not reported. NR c Not reported due to lack of measurable concentrations at 168 hours post-dose. Note: AUC 0-168 were calculated using extrapolation and should be interpreted with caution. Combined male and female (MF) parameters were calculated by combining concentration data for all animals (males and females) at each dose level for each interval and using these data as separate composite profiles for TK analysis. These parameters are not the average of values ​​calculated separately for males and females. aAnimals were dosed once daily for at least 8 weeks (the dosing phase). Group 3 animals were not dosed on day 36. Starting on day 37, Group 3 animals were dosed every other day throughout the dosing phase (dosing on days 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55). Starting on day 32 of the dosing phase, Group 4 animals were dosed for 3 days (dosing on days 32-34), followed by a 4-day dosing holiday. This dosing regimen continued throughout the remainder of the dosing phase (dosing on days 39-41, 46-48, and 53-55).

[0180] ALT-801C max , AUC 0-24 , AUC 0-72 , or AUC 0-168 The sex difference in values ​​was less than two-fold. ma x and AUC 0-24 Exposure, as assessed by C values, increased with increasing dose level from 0.03 to 0.15 mg / kg / dose on Day 1. max and AUC 0-24 Increases in values ​​were generally proportional to dose on day 1. Potential accumulation of ALT-801 was observed after multiple doses in rats.

[0181] D. Single-Dose Cynomolgus Monkey Study The purpose of this study was to determine the pharmacokinetics of SEQ ID NO: 1 after a single subcutaneous administration to cynomolgus monkeys (3 monkeys per dose group). No significant adverse events were observed in the animals during the study.

[0182] As shown in Table 12 below and Figure 12, increasing doses of SEQ ID NO: 1 in formulation buffer (0.050% (w / w) polysorbate 20, 0.300% (w / w) methylparaben, 0.348% (w / w) arginine, 4.260% (w / w) mannitol in deionized water) when tested using the cynomolgus monkey model (subcutaneous administration) exhibit the pharmacokinetic parameters shown in Table 12 as measured over a 192 hour period post-dose.

[0183] [Table 12]

[0184] Figure 12B illustrates the plasma concentrations of SEQ ID NO: 1 in animals (labeled 1215, 1216, and 1217 in Figure 12B) administered 10 nmol / kg SEQ ID NO: 1 (as ALT-801) 9 days after administration of ALT-801. Animal 1215 was found to have slightly unformed stool 9 days after treatment (thus unlikely related to ALT-801) and exhibited a Cmax of 126 ng / mL (33 nM) compared to an average of 80 ng / mL for the other two animals in the study (1216 and 1217). This data indicates that the biologically effective level of ALT-801 is likely less than 5 nM SEQ ID NO: 1. This low-dose group (10 nmol / kg) showed no evidence of vomiting (0 / 3), making it unclear whether the "unformed stool, scanty" was compound-related. The Cmax for animals with no formed stool is 158% of the average for the other two animals. All animals exhibit blood levels above 5 nM through 120 hours.

[0185] Figure 12C illustrates the concentration of SEQ ID NO: 1 in animals (labeled 2215, 2216, and 2217 in Figure 12C) administered 20 nmol / kg of SEQ ID NO: 1 (as ALT-801) 9 days after administration of ALT-801. Animal 2217 showed some vomiting on day 2 after administration and had a Cmax of 225 ng / mL (58 nM), compared to an average of 147 ng / mL for the other two animals in this study. This data also indicates that the biologically effective level of ALT-801 is likely less than 5 nM. This mid-dose group (20 nmol / kg) shows little evidence of vomiting (1 / 3). The Cmax of this animal's vomiting is 153% of the average of the other two animals. All animals show blood levels greater than 5 nM throughout 192 hours.

[0186] Figure 12D shows the concentration of SEQ ID NO: 1 (as ALT-801) in animals (labeled 3215, 3216, and 3217 in Figure 12D) administered 40 nmol / kg of SEQ ID NO: 1 (as ALT-801) 9 days after administration of ALT-801. All three animals exhibited vomiting, which may be related to ALT-801 and Cmax. The mean Cmax for this group was 467 ng / mL (121 nM). This data also indicates that the biologically effective level of ALT-801 is likely less than 5 nM. This high-dose group (40 nmol / kg) shows strong evidence of emesis (3 / 3). The Cmax for this relatively homogenous group is 467 ng / mL (121 nM). All animals exhibit blood concentrations greater than 10 nM throughout the assay (192 hours).

[0187] Evidence of GI side effects supports our suggestion that they are Cmax-related, at least in NHPs (non-human primates). If biologically effective blood concentrations are less than 5 nM, 10 nmol / kg may be a higher dose than necessary. Dose accumulation is expected with ALT-801 treatment. In embodiments, a pharmaceutical formulation is provided containing ALT-801 as an API designed for subcutaneous administration to provide a Cmax of 150-200 ng / ml, where adverse GI side effects are reduced or eliminated, yet ALT-801 is effective in lowering blood glucose levels and / or treating obesity.

[0188] E. Multiple-Dose Cynomolgus Monkey Study 1. Six-week repeated administration study in cynomolgus monkeys Research purpose The objectives of this study were to evaluate the toxicity and toxicokinetics of ALT-801 (comprising SEQ ID NO: 1) when administered via subcutaneous injection to cynomolgus monkeys once weekly for at least 6 weeks (6 doses total), and to evaluate the reversibility, persistence, or delayed onset of effects after a 4-week recovery phase. The study was conducted by Covance.

[0189] animal Male and female cynomolgus monkeys (28 animals / sex; Macaca fascicularis) of Asian origin were received from Envigo Global Services Inc. (formerly Covance Research Products) of Alice, Texas. Animals were acclimated to the testing facility for at least 30 days prior to initiation.

[0190] At the start of treatment, the animals were 31-54 months old. On the day before treatment began, the body weight was 2.2-4.2 kg for males and 2.2-3.2 kg for females.

[0191] research design Male and female cynomolgus monkeys were assigned to five groups and administered the doses shown in the table below. Animals were dosed by subcutaneous injection in the back at a volume of 2.0 mL / kg on days 1, 8, 15, 22, 29, and 36 of the dosing phase. The vehicle control article was F58 formulation buffer, consisting of 0.050% (w / w) polysorbate 20, 0.348% (w / w) arginine, 4.260% (w / w) mannitol in deionized water (pH 7.7±0.1).

[0192] [Table 13] (a) Control = vehicle control article only. (b) Two animals designated for recovery evaluation underwent 4 weeks of recovery after completion of the dosing phase. D = dosing; E = evaluation.

[0193] Toxicity assessment was based on mortality, clinical observations, body weight, qualitative food consumption, ophthalmological observations, electrocardiogram (ECG) measurements, neurological examination, qualitative respiratory rate, and clinical and anatomic pathology. Blood samples were collected for toxicokinetic evaluation. Test item description Test item Storage lot Retest date Purity a Frozen (-10 to -30°C) Protect from light and with desiccant November 19, 2020 JPEG2026021305000025.jpg27170 a Purity was determined by high performance liquid chromatography on an anhydrous basis. A correction factor of 1.192 was assigned. b Allocated as per Covance SOP as 365 days from receipt.

[0194] Description of Vehicle Control Article The vehicle control article was F58 formulation buffer, which consisted of 0.050% (w / w) polysorbate 20, 0.348% (w / w) arginine, 4.260% (w / w) mannitol in deionized water (pH 7.7±0.1).

[0195] Test product formulation Test article formulations were prepared in the vehicle control article at least once weekly according to the compounding procedure and dispensed for use. Dose concentrations were corrected for lot-specific purity using a correction factor of 1.192. The pH of each test article formulation was adjusted to pH 7.7 ± 0.1 using dilute hydrochloric acid or sodium hydroxide, as needed. Prepared test article formulations were sterile filtered using 0.2 μm polyvinylidene fluoride (PVDF) filters. Handling after filtration was performed using aseptic technique.

[0196] Formulation of Vehicle Control Article Vehicle control article formulations were prepared at least weekly by Covance according to the compounding procedure and dispensed for use. The prepared vehicle control article formulations were sterile filtered using 0.2 μm PVDF. Post-filtration processing was performed using aseptic technique, and the filtered solution was dispensed into dosing aliquots for Group 1. All ALT-801 concentration values ​​in the vehicle control group were below the lower limit of quantitation (<4.00 ng / mL).

[0197] dosage The dosing sites were in the dorsal scapular region of each animal. Dose volumes were rotated between sites. Dosing sites were as follows: Site A: upper left scapular region, Site B: upper right scapular region, Site C: lower left scapular region, and Site D: lower right scapular region. The following animals were not dosed on the days listed in the following table due to weight loss, body condition score, and veterinary recommendation: JPEG2026021305000026.jpg64170

[0198] Toxicokinetic analysis The toxicokinetic analysis included the parameters listed in the table below. JPEG2026021305000027.jpg51166

[0199] A summary of the mean ALT-801 toxicokinetic parameters in monkey plasma is shown in the table below. All concentration values ​​of ALT-801 in the vehicle control group were below the lower limit of quantitation (<4.00 ng / mL). JPEG2026021305000028.jpg144170

[0200] Veterinary Treatment and Examination No ALT-801-related veterinary health issues were observed. No notable ophthalmological observations were noted during the dosing period. Based on these results, no ophthalmological examinations were performed during the recovery phase. No notable neurological observations were noted during the dosing or recovery phases. Electrocardiograms indicate that no ALT-801-related changes in PR interval, QRS duration, QT interval, QTc interval, or heart rate were observed approximately 24 hours post-dose on Day 1 or Day 36 of the dosing phase. No abnormal electrocardiogram waveforms or arrhythmias were observed during qualitative evaluation of the electrocardiograms.

[0201] Clinical laboratory evaluation No ALT-801-related findings were observed in hematology, coagulation, clinical chemistry, or urinalysis results. No ALT-801-related changes in organ weights were noted at terminal or recovery necropsy. No ALT-801-related macroscopic findings were observed at terminal or recovery sacrifice. No ALT-801-related microscopic findings were observed in animals at terminal sacrifice or recovery sacrifice.

[0202] Weight change Animal weights were recorded four times during the pre-dose phase, on Day -1 of the dosing phase (the day before dosing began), and weekly thereafter (based on Day -1) until Day 14 of the dosing phase. Starting on Day 14 of the dosing phase, weights were collected twice weekly (based on Day 14) until the end of the dosing phase. Body weights were collected on Days 1, 8, 15, 22, and 28 of the recovery phase. Data shown in Figures 13 and 14 represent weight change as % of Day -1 for males and females, respectively. At the two highest doses of ALT-801 (0.18 mg / kg and 0.25 mg / kg), significant weight loss of up to 10% was observed in both males and / or females during the dosing period.

[0203] Clinical observations No ALT-801-related mortality or effects on neurological observations, ECG, clinical pathology, organ weights, or gross or microscopic examinations occurred during the dosing or recovery phases.

[0204] ALT-801-related clinical observations in females administered ≥0.03 mg / kg / dose included reduced food consumption. No ALT-801-related clinical observations were observed in males administered ≥0.03 mg / kg / dose. An ALT-801-related decrease in food consumption was observed in females administered ≥0.03 mg / kg / dose or higher. No ALT-801-related changes in food consumption were observed in males administered ≥0.03 mg / kg / dose. Decreased food consumption was observed in females administered ≥0.03 mg / kg / dose on days 19 and 36 of the treatment phase, with a dose-dependent increase in incidence.

[0205] One episode of vomiting was observed on Day 2 of the dosing phase in one female (animal P0701) receiving 0.18 mg / kg / dose and one female (animal P0901) receiving 0.25 mg / kg / dose. This observation was not sustained and did not show a dose-responsive increase in incidence. Because there was no dose-responsive increase in the incidence of vomiting and these observations were not sustained, this was not considered an ALT-801-related clinical observation.

[0206] No ALT-801-related clinical observations were observed during the recovery phase.

[0207] One female (animal P0604) administered 0.03 mg / kg was sacrificed at an unscheduled interval on day 26 of the recovery phase. Clinical observations noted in this animal included decreased activity, hunched back, thin mucous membranes, a rough hair coat; pale appearance; dark, dry feces on the tail, and no liquid feces in the pan. Because animal P0604 was in the recovery phase and the clinical observations observed in this animal were not observed in other animals administered ALT-801, this unscheduled sacrifice was not related to ALT-801.

[0208] Other clinical observations included swollen tails, scabs, abnormal skin color, liquid / non-formed feces, abnormally colored fur, thinning fur, and red discharge from the vulva. These were fairly infrequent and either transient or occurred with incidences similar to those in controls. Therefore, they were not considered ALT-801-related.

[0209] conclusion In conclusion, male and female monkeys were administered a vehicle control or 0.03, 0.06, 0.18, or 0.25 mg / kg / dose of ALT-801 by subcutaneous injection once weekly.

[0210] As shown in Figures 13 and 14, the two highest doses of ALT-801 tested in the study (0.18 mg / kg and 0.25 mg / kg) resulted in significant weight loss of up to 10% during the dosing period in both males and / or females. This effect was not associated with mortality or gastrointestinal events considered treatment-related at any dose tested.

[0211] No adverse ALT-801-related findings occurred during the dosing or recovery phases, with a no-observed-adverse-effect level (NOAEL) of 0.25 mg / kg / dose. This dose level was associated with a 562 ng / mL and 62300 h * These corresponded to mean peak concentration (Cmax) and area under the concentration-time curve (AUC) values ​​in ng / mL, respectively.

[0212] F. Summary of rat and monkey data from Example 4: These multiple-dose studies did not reveal any significant adverse events (AEs) in rats or cynomolgus monkeys. Decreased food intake and weight loss, expected pharmacological properties of ALT-801, were observed at the mid- and high-dose levels, but no ALT-801-related emesis was observed. High doses of 0.45 mg / kg / week in rats and 0.25 mg / kg / week in monkeys have been established as the no-observed-adverse-effect levels (NOAELs), respectively. Safety pharmacology evaluations incorporated into the general toxicity studies did not reveal any neurological, cardiac, or respiratory findings. As previously mentioned, the observed decreased food intake and weight loss were expected to be on-target effects of GLP-1 and glucagon agonists. These effects were more pronounced in rats compared to monkeys, likely related to the more frequent dosing cycle (first QD), corresponding to the shorter t1 / 2 in rats. On an exposure basis, both Cmax and area under the plasma concentration-time curve (AUC0-168h) (i.e., over the entire dosing interval) were remarkably similar in rats given 0.15 mg / kg for 3 days and off for 4 days weekly (weekly dose of 0.45 mg / kg / week) and monkeys given 0.25 mg / kg / week once weekly. In rats, Cmax and AUC 0-168 are approximately 500ng / mL and 42,600ng, respectively.* Similarly, monkey exposures were 5560 ng / mL and 54,400 ng / mL, respectively. * h / mL.

[0213] Example 5. Murine non-alcoholic steatohepatitis (NASH) In the DIO-NASH mouse study, five DIO-NASH groups (n=12) of male C57BL / 6J mice were fed an Amylin high-fat diet containing 40% fat (including trans fat), 18% fructose, and 2% cholesterol for 29+ weeks. All mice were biopsied and stratified based on liver biopsy (only animals with fibrosis ≥1 and steatosis ≥2 were included). Animals were also stratified into groups based on Col1a1 immunostaining. For a total of 12 weeks, the QD-treated groups were: 1) vehicle; 2) SEQ ID NO: 1, 5 nmol / kg (SC, QD); 3) SEQ ID NO: 1, 10 nmol / kg (SC, QD); 4) elafibranor, 78 μmol / kg (PO, QD); and 5) semaglutide, 10 nmol / kg (SC, QD). Body weight (BW) was measured daily throughout the study period, and food intake was measured daily for the first 14 days and then weekly until study termination. Terminal plasma was measured for ALT, AST, TG, and TC levels. Livers were removed and sampled for pre- and post-NAFLD activity scores (NAS; HE staining), including fibrosis stage (Picro Sirius Red, PSR). Terminal histology was performed for steatosis, Col1a1, and galectin-3 quantification. Terminal liver workup included TG and TC (extraction and measurement). Terminal liver biopsies were set up: 1) 4% PFA for histology, 2) fresh-frozen liver for biochemistry, and 3) fresh-frozen liver for RNA extraction and RNA sequencing.

[0214] Treatment with ALT-801 (a pharmaceutical formulation containing SEQ ID NO: 1) was shown to reduce body weight in a NASH mouse model. Treatment with ALT-801 and semaglutide rapidly and dose-responsively reduced body weight, which stabilized for the remainder of the study (Figure 15). Treatment with ALT-801 (5 nmol / kg and 10 nmol / kg), as well as elafibranor (78 μmol / kg) and semaglutide (10 nmol / kg), resulted in statistically significant weight loss compared to NASH controls (p≦0.001). The weight loss achieved in ALT-801-treated animals was dose-dependent, reaching -25% within 4 weeks of administration. This is approximately twice the weight loss induced by an equimolar dose of semaglutide. Importantly, ALT-801 (10 nmol / kg) reduced the body weight of this group to the lean body mass range (~30 g) for this mouse strain and maintained this range thereafter. On day 63 (week 9 of treatment), a single accidental dose of 10 nmol / kg ALT-801 was administered to the vehicle group, resulting in a rapid loss of body weight, which then recovered to the vehicle trend line over approximately 10 days.

[0215] SEQ ID NO:1 was also shown to exhibit superior reductions in NAFLD activity score (NAS) compared to elafibranor and semaglutide. See Figure 16. As shown there, compared to the start of treatment (day 0), 5 nmol / kg SEQ ID NO:1 exhibited a 32% reduction, and 10 nmol / kg SEQ ID NO:1 exhibited a 61% reduction, compared to 42% for elafibranor and 18% for semaglutide. A 6% increase was observed in the control group. NAS scores improved in all treatment groups at the end of the treatment period (Figure 15). The percent change in NAS score achieved by the elafibranor and semaglutide treatment groups was significantly lower than that achieved by the ALT-801 10 nmol / kg group (both p<0.0001). All animals in the ALT-801 10 nmol / kg group achieved an NAS score of ≤3.

[0216] As shown herein, at the end of the treatment period, the liver fat content of mice treated with low and high doses of ALT-801 was subsequently reduced to the lean normal range of fat content (Figure 17). Low- and high-dose treatment with ALT-801 resulted in a significant reduction in liver weight compared to the NASH vehicle control, semaglutide, and elafibranor (p<0.01; Figure 17). The mean liver weights of mice treated with elafibranor and semaglutide were statistically significantly higher than those of mice treated with high dose (10 nmol / kg) ALT-801 (p<0.0001 and p<0.01, respectively). The liver weights of both ALT-801-treated groups were similar to those of lean, normal mice fed a chow diet. .

[0217] Treatment with ALT-801 (a pharmaceutical formulation containing SEQ ID NO: 1) was also found to have a greater beneficial effect on fibrosis, as measured by liver Col1A1 and galectin-3 content, compared with elafibranor, semaglutide, or NASH vehicle controls. Low- and high-dose treatment with ALT-801 significantly reduced liver end-stage Col1A1 and galectin-3 levels compared with NASH vehicle controls, elafibranor, and semaglutide (p<0.0001; Figure 17). The mean liver Col1A1 levels in mice treated with elafibranor were statistically significantly higher than those in mice treated with high-dose (10 nmol / kg) ALT-801 (p<0.0001). The mean liver galectin-3 levels in mice treated with elafibranor and semaglutide were statistically significantly higher than liver galectin-3 in mice treated with high dose (10 nmol / kg) ALT-801 (both p<0.0001).

[0218] Treatment with ALT-801 (a pharmaceutical formulation containing SEQ ID NO: 1) was also found to normalize hepatic triglycerides (TG), total cholesterol (TC), and plasma ALT. Low- and high-dose treatment with ALT-801 significantly reduced hepatic TG (p<0.01) and TC (p<0.0001) levels compared with NASH vehicle control, semaglutide, and elafibranor (FIG. 18). The mean hepatic TG levels of mice treated with elafibranor and semaglutide were statistically significantly higher than those of mice treated with high-dose (10 nmol / kg) ALT-801 (p≦0.01 and p≦0.0001, respectively; one-way ANOVA with Dunnett's adjustment for multiplicity). Similarly, mean liver TC levels in mice treated with elafibranor and semaglutide were statistically significantly higher than liver TC in high-dose (10 nmol / kg) ALT-801-treated mice (both p<0.0001).

[0219] Low- and high-dose treatment with ALT-801 resulted in significantly lower terminal plasma AST levels compared with NASH vehicle controls (p<0.001), and significantly lower terminal plasma ALT levels compared with NASH vehicle controls, elafibranor, and semaglutide (p<0.01; Figure 18). Mean liver ALT levels in mice treated with elafibranor and semaglutide were statistically significantly higher than plasma ALT in mice treated with high-dose (10 nmol / kg) ALT-801 (p<0.0001 and p<0.01, respectively), which was within the normal range for this strain.

[0220] RNA sequencing showed that treatment with SEQ ID NO: 1 was superior to treatment with elafibranor or semaglutide, resulting in significant suppression of inflammatory and pro-fibrotic gene expression, particularly in astrocytic pathways involved in the development of fibrotic lesions.

[0221] The high-dose ALT-801 (pharmaceutical formulation containing SEQ ID NO: 1) treatment group showed the greatest number of differentially expressed genes (~8000) compared to elafibranor (~5800) or semaglutide (~2800) (Figure 19). Principal component analysis of the 500 most variable liver genes resulted in a clear treatment-related clustering of samples (Figure 19). PC1 explained 52% of the variability, and PC2 explained 21% of the variability.

[0222] Treatment of NASH mice with 10 nmol / kg ALT-801 resulted in modulation of genes affecting fat utilization and transport, including statistically significant increases in the expression levels of carnitine palmitoyltransferase 1a (CPT-1) (p<0.05), glycerol-3-phosphate acyltransferase 4 (GPAT-4) (p<0.001), and sterol regulatory element-binding transcription factor 1 (SREBTF-1) (p<0.05) compared with NASH vehicle controls after correction for multiple testing per gene (Figure 20). Treatment of NASH mice with a low dose of ALT-801 (5 nmol / kg) also increased the expression of CPT-1 (p<0.05) and GPAT-4 (p<0.001) (Figure 18). Expression of fatty acid synthase (FASN) (p<0.05), glycerol-3-phosphate acyltransferase 2 (GPAT2) (p<0.001), stearoyl-coenzyme A desaturase 1 (SCT-1) (p<0.05), and CD36 antigen (CD36) (p<0.001) was decreased in mice treated with ALT-801 at 10 nmol / kg compared to NASH vehicle controls after correction for multiple testing per gene (Figure 20). CD36 expression was also significantly lower in mice treated with ALT-801 at 5 nmol / kg (p<0.05) (Figure 20). The gene expression changes observed in mice after semaglutide treatment were not statistically significant. However, the elafibranor group had significantly lower GPAT2 (p<0.001) and GPAT4 (p<0.001) compared to NASH vehicle controls.

[0223] Treatment of NASH mice with ALT-801 resulted in the suppression of profibrotic genes in the astrocyte pathway. Markers of myofibroblast proliferation and astrocytes, A-SMA (ACTA2), platelet-derived growth factor (PDGFB), and transforming growth factor-beta (TGFB1) (Figure 20), were statistically significantly reduced in treatment groups receiving low or high doses of ALT-801 compared to NASH vehicle controls (all p<0.01 after correction for multiple testing per gene). Expression of A-SMA (p<0.001) and TGFB1 (p<0.05) was also statistically significantly reduced in NASH mice treated with semaglutide, whereas expression of PDGF (p<0.01) was statistically significantly reduced in NASH mice treated with elafibranor.

[0224] Treatment of NASH mice with ALT-801 resulted in the suppression of cell death genes. Hepatocyte cell death and pyroptosis markers absent in melanoma (AIM2), ICE protease activating factor (IPAF), and receptor-interacting kinase 3 (RIPK3) (Figure 20) were statistically significantly reduced in treatment groups receiving low or high doses of ALT-801 compared to NASH vehicle controls (all p<0.01 after correction for multiple testing per gene). AIM2 expression (p<0.01) was also statistically significantly reduced in NASH mice treated with semaglutide. No statistical differences in cell death genes were observed when treated with elafibranor.

[0225] Treatment of NASH mice with ALT-801 resulted in suppression of liver inflammatory genes. The proinflammatory signaling markers c-Jun (JUN), c-FOS (FOSB), and Toll-like receptor 4 (TLR4) (Figure 20) were statistically significantly reduced in treatment groups receiving low or high doses of ALT-801 compared with NASH vehicle controls, except for c-FOS in the low-dose ALT-801 group (all p<0.01 after correction for multiple testing per gene). Expression of TLR4 (p<0.01) was also statistically significantly reduced in NASH mice treated with semaglutide. No statistically significant changes were observed in the FOSB, JUN, or TLR4 genes in NASH mice treated with elafibranor.

[0226] Example 6. Pharmacodynamic (PD) and Pharmacokinetic (PK) Profiles and Weekly Dosing This example relates to a series of peptide analogs with an altered balance of receptor agonist activity at human GLP-1R and GCGR, and analogs with a duration of action suggesting suitability for once-weekly (QW) administration to patients, including but not limited to SEQ ID NO: 1 as in ALT-801. A comparison of certain peptide analogs of the present disclosure with GLP-1 and glucagon is provided below. JPEG2026021305000029.jpg38170

[0227] Unnatural amino acids are underlined and italicized. * and K. * All analogs of Glu 16 and Lys 20 and Z1 and Z2 represents a Lys residue conjugated by acylation to various glycolipid surfactant-derived duration-of-action modifiers (i.e., surfactants discussed below). Z1 or Z2 If neither of these is present in the analog, it is replaced by Q (Gln). The peptide analogs studied in this example are shown in Table 14 below.

[0228] [Table 14] Analogs marked with an asterisk have a side chain lactam from Glu16 to Lys20; the bracketed G, M, and Me refer to D-glucosidic, D-maltosidic, and D-melibiosidic bonds, respectively, and S1 and S2 refer to α-Lys or γ-Glu residues, respectively. Cn refers to an n-carbon methylene chain, and c refers to the carboxylate at the end of the chain. X in semaglutide refers to a Lys residue acylated with the γGlu-2×OEG (see Reference 27) elongation modifier, which contains octadecanoic acid on the γGlu / short PEG spacer. Compound #33 in Reference 8 refers to compound #32 alkylated on Cys24 with 40 kDa PEG via a maleimide linker. In Table 1, "Compound #" refers to analogs 1-17.

[0229] The structures of exemplary glycolipid surfactant-based reagents used herein are shown below: 1-O-alkyl β-D-glucopyranosiduronic acid, 1'-O-alkyl[β-(α-D-galactopyranosiduronic acid-(1→6')]-D-glucoside, or 1-O-alkyl β-[β-D-glucopyranosiduronic acid-(1→4)]-D-glucopyranosiduronic acid, respectively. [ka]

[0230] The reagents are prepared from the corresponding 1-O-alkyl β-D-glucosides, 1-O-alkyl β-D-melibiosides, or 1-O-alkyl β-D-mannosides by chemoselective oxidation of the primary OH group. (Seconds) The R1 alkyl group can be linear, branched, saturated, unsaturated, normal, or functionally modified. The physical and micellar properties of surfactants are known to depend on the specific head-to-tail group combination. In this study, the length of the R1 alkyl chain ranged from C8 to C18. Attachment of glycolipid modifiers is via the 6- or 6'-(distal) carboxylic acid, usually by amide formation with the ε-amino function of a peptide Lys residue. For example, such surfactant reagents are typically obtained at CS Bio Co. (Menlo Park, CA) from commercially available nonionic surfactants (Anatrace, Maumee, OH) by chemoselectively oxidizing the primary alcohol groups of such surfactants in the presence of water with [bis(acetoxy)-iodo]benzene (BAIB) as the oxidant using 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-mediated oxidation. This reaction is completed with high chemoselectivity, producing the desired primary carboxylic acid in virtually quantitative yield, with HOAc and Ph-I as the only volatile byproducts. Simple lyophilization of the pH 3 aqueous solution affords the desired free carboxylic acid, ready for activation and coupling to the free amino group as the penultimate solid-phase synthesis step before cleavage. If necessary, additional purification by trituration with EtO to remove traces of TEMPO can be applied, but is not required for the solid-phase synthesis procedure used here. For large-scale oxidation, a different stoichiometric oxidizing agent (e.g., sodium hypochlorite) can be used. Coupling with EuPort reagents proceeds more slowly than typical amino acid couplings, typically requiring over 8 hours to complete at low molar excess. Additional glycolipid surfactants are prepared by König's-Knorr / Helferich glycosylation reactions with appropriate alkyl alcohols and protected glycosyl bromides.

[0231] The solid-phase peptide synthesis used in generating the peptide analogs of this example was a standard Nα-Fmoc protocol (t-butyloxycarbonyl and N-trityl side chain protection; further Arg(Pbf); Nα-Boc-His(Trt) on Rink amide resin from CS Bio Co (Menlo Park, CA)), Glu 16 and Lys 20 Orthogonal protection of the N- and N-allyloxycarbonyl positions (allyl ester and N-allyloxycarbonyl, respectively) was used. The Lys position to be modified by EuPort conjugation was protected with N-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (iv-Dde), selectively deprotected using 4% hydrazine in DMF as the penultimate step, and then coupled with the appropriate EuPort reagent (as the carboxylic acid) using DIC and HBT (or other coupling additives as needed). The final peptide was cleaved and deprotected using trifluoroacetic acid (TFA) / water / triisopropylsilane (95:2.5:2.5), precipitated with ether, washed with ether, dried, and purified by appropriate reverse-phase (C-18) HPLC chromatography using acetonitrile in a TFA (0.1%) buffer gradient. The compounds were characterized by analytical HPLC / mass spectrometry using similar buffers on the analytical column, and all tested analogs had a purity of 95% or greater (Table 15).

[0232] [Table 15] a Purity is estimated by integration of the anomalous peak after injection. b k' is an HPLC system-independent measure of retention: k' = (tr - t0) / t0. The analysis was performed on a Phenomenex Luna 5μC-18 250 × 4.6 mm column at 1 mL / min. *16 was similarly run on a Polymer Labs PLRP-S100A 8μ 250 × 4.6 mm column. The elution gradient was (min) from low to high %B (B = % CH3CN in 0.1% TFA): a = 35-65% for 20 min; b = 40-70% for 20 min; c = 45-75% for 20 min; d = 50-80 for 20 min; e = 30-90 for 20 min.

[0233] A. In Vitro Receptor Activation Assay Receptor activation assays were performed at DiscoverX Laboratories (Fremont, CA) using human GLP-1R and GCGR cloned into Chinese hamster ovary (CHO) cells (LeadHunter Discovery Services; Assay Product 86-0007D cAMP Hunter™, using huGLP1R and huGCGR; Whole-cell cAMP accumulation assay; the cell lines used were cAMP Hunter™ CHO-K1 GCGR Gs Cell Line, catalog 95-0042C2 and cAMP Hunter™ CHO-K1 GLP1 Gs Cell Line, catalog 95-0062C2; accumulated cAMP was readout using the Readout Hit Hunter cAMP XS+ Assay). Cell lines were maintained at DiscoverX and incubated with test drugs for 30 minutes at 37°C for cAMP accumulation. Results were evaluated in DiscoverX using in-house parameters and literature standards (exendin-4 and glucagon for GLP-1R and GCGR, respectively) for reportability. The results described herein are from a single assay performed in duplicate on cells, and data are from pEC 50Data were replotted in Prism 5 to provide (SE) data. Cytotoxicity observations were reported for all assays. Most assays were performed in the presence of 0.1% BSA to minimize nonspecific binding, but assays 15-17 were also tested in the presence of 0.1% chicken ovalbumin (OVA). These compounds bind very strongly to BSA (>99%; data not shown), and its presence can skew results and significantly reduce compound efficacy.

[0234] B. In vitro stable plasma Stability studies were performed at Climax Laboratories, Inc. (San Jose, CA). Test article samples (approximately 0.5 mg, GLP-17-36 amide, Bachem, Analog 3, Analog 5) were dissolved in pooled human plasma (Bioreclamation LLC, Lot BRH392992) at concentrations of 1–10 μM, and the compound levels remaining at given time points were quantified as described in Bioanalytical Methods (2.54). The time / concentration course (Figure 22) shows that GLP-17-36 amide was rapidly destroyed to below the limit of quantitation (BQL; approximately 2 ng / mL) after 4 hours of incubation, whereas the amounts of Analogs 3 and 5 remained unchanged for 8 hours, indicating excellent intact stability in the presence of pooled human plasma.

[0235] The stability of analog 17 (SEQ ID NO: 1, similar to ALT-801) in plasma was also studied, particularly its binding to the plasma protein albumin. Such noncovalent binding to albumin is expected to delay peptide degradation in plasma and result in reduced renal clearance. The binding of ALT-801 (15,000 ng / mL) to plasma proteins in rats, dogs, monkeys, and humans was assessed by 6-hour ultracentrifugation. Pooled plasma was obtained from at least three Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. Pooled human plasma was obtained from three human males who reportedly had not taken any medications in the past 7 days prior to collection. K2EDTA was used as an anticoagulant. If necessary, the pH of each plasma pool was adjusted to approximately pH 7.4 with hydrochloric acid or sodium hydroxide. To achieve separation of PUC (supernatant) from plasma proteins, ultracentrifugation was performed using polycarbonate ultracentrifuge tubes placed in an S80AT2 rotor at 357,000 x g for 6 hours at 37°C. After centrifugation, PUC was analyzed by LCMS and protein binding was calculated. Protein binding was estimated as percent unbound = (Cu / Co) x 100 and percent bound = 100 - percent unbound, where Co is the concentration of the test substance in plasma before ultracentrifugation (ng / mL) and Cu is the concentration of the test substance in plasma at ultracentrifugation (ng / mL). The results are shown in Table 16.

[0236] [Table 16] The average protein binding rates of ALT-801 were 99.8% in rat plasma, 99.8% in dog plasma, 100% in monkey plasma, and 99.8% in human plasma. These results indicated that ALT-801 has extensive protein binding (≥99.8%) in rat, dog, monkey, and human plasma.

[0237] C. Pharmacokinetics PK and PD assays were performed according to standard protocols in rats at Charles River Laboratories (Shrewsbury, MA) and db / db mice at JAX Laboratories (Sacramento, CA). PK studies were also performed in Göttingen or Yucatan minipigs at MPI Research (Mattawan, MI). No compound-related injection site reactions were observed for any of the compounds tested. Bioanalytical analysis by LC / MS / MS was performed at Climax Laboratories, Inc. (San Jose, CA) or, for the Yucatan minipig study, at Frontage Laboratories, Inc. (Exton, PA).

[0238] D. Pharmacokinetics in Rats The pharmacokinetic profiles of 17 (as ALT-801) and semaglutide after a single subcutaneous dose of 10 nmol / kg were evaluated in male CRL:CD(SD) rats (250-300 g) at Charles River Laboratories. Both ALT-801 and semaglutide were formulated at 0.1 mg / mL in 50 mM phosphate buffer (pH ∼8) containing 0.05% Tween 80. Blood samples (∼300 μL) were collected into ice-cooled K2EDTA tubes at 2, 4, 8, 24, 48, 72, 96, 120, and 144 hours post-dose (n = 4 per time point) and stored on ice until processed for plasma by centrifugation at 2200 rpm for 10 minutes at 5°C. Plasma concentrations of ALT-801 and semaglutide were determined as outlined in the bioanalytical methods below (2.5.3).

[0239] 1. Pharmacokinetics in Göttingen minipigs This study used cassette-style dosing to minimize the use of large animals, but subcutaneous injections were administered at separate sites to prevent each compound from affecting the uptake of the other. Two male Göttingen minipigs were assigned to the study. The animals were housed in pairs in a raised-floor cage enclosure. Animal weights were approximately 11-15 kg at the time of implantation and approximately 5-8 months of age. After a minimum one-week washout period, the same animals were to be used across multiple phases. To facilitate administration and ensure the animals' safety during the administration procedure, animals were sedated with Telazol (IM, 4-6 mg / kg) prior to administration. Dosing was administered subcutaneously via bolus injection between the skin and the underlying tissue layer in the ventral region of the animal. A total of three to four sites were used in each phase, with a different compound administered at each of the four sites. The compounds were formulated in saline containing 0.2% BSA (approximately 0.4 mg / mL) at pH 3.5. Each stock solution was diluted with normal saline (pH 7.4) to the required final concentration and sterile filtered. The dose was 20 nmol / kg. Blood samples were collected pre-dose and at 2, 4, 6, 8, 12, 24, 36, 48, 72, and 96 hours post-dose. At each time point, 1 mL samples were collected from the jugular vein on ice into K2EDTA tubes and then processed to plasma by centrifugation. Plasma samples containing the four test compounds were sent to Climax Labs for separation and quantification by LC-MS / MS (2.5.3).

[0240] 2. Pharmacokinetics in Yucatan Minipigs The study animals were four single-housed, untreated male Yucatan minipigs (Susscrofa; body weight 73-81 kg). Animals were fed a maintenance dose of Purina S-9 pig chow. General cage observations were performed at least twice daily (morning and evening) throughout the study to assess general health, moribundity, or mortality.

[0241] After a 22-day acclimation period, each minipig received 17 subcutaneously (behind the cheek jaw) at 20 nmol / kg (0.2 mL / kg), and PK blood samples were collected at -0.25, 0.25, 4, 6, 8, 12, 24, 48, 72, 96, 120, 168, 192, 216, 264, 312, and 360 h post-dose. After a 2-week washout period, the same animals received 17 i.v., and PK blood samples were collected at -0.25, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 168, 192, 216, 264, 312, and 360 h post-dose. The administration concentration was 5.5 mg / mL (dose 0.015 mL / kg) for both treatments. Whole blood samples (approximately 3 mL / time point) for pharmacokinetic analysis were collected via the vascular access port into tubes containing K2EDTA. Samples were kept on wet ice until processing within 30 minutes of collection. All samples were centrifuged at approximately 3000 rpm at approximately 4°C for approximately 15 minutes. Plasma samples were stored frozen at -70°C until primary samples were shipped to Frontage Laboratories (Exton, PA) for bioanalysis by LC-MS / MS, as outlined below. No abnormal clinical observations were noted during the conduct of the study.

[0242] E. Pharmacodynamics 1. Effects on Blood Glucose—db / db Mice Approximately 75 BKS.Cg-m+ / +Leprdb / J (Jackson Labs strain no. 000642) male ("db / db") mice, 7-9 weeks of age, were used. These studies were maintained using standard animal care procedures. The study began after one week of acclimation to facility conditions. On the morning of study day 0, mice were weighed and fasted for four hours. Blood glucose levels were measured using a glucometer using standard procedures. At least 54 mice were selected based on body weight, and mice with blood glucose levels above 300 mg / dL (i.e., diabetic) were randomly assigned to six groups (n=9). The groups were as follows: Group 1, vehicle; Group 2, semaglutide 3 nmol / kg; Group 3, semaglutide 10 nmol / kg; Group 4, 17, 1 nmol / kg; Group 5, 17, 3 nmol / kg; and Group 6, 17, 10 nmol / kg. Body weights were measured and recorded at the time of receipt, prior to randomization, and daily on days 1 through 5. 0, 2, 4, 8, 24, 48, 72, 96, and 120 hours after a single dose of the indicated compound.

[0243] 2. Body weight - "DIOCRL:CD(SD)" rats Fifty-four male DIOCRL:CD rats, approximately 14-15 weeks old at the start of the study, were enrolled in the study at Charles River Laboratories (Shrewsbury, MA). Animals were maintained on a high-fat diet (ResearchDiets 12492, 60% kcal, % fat) for 11 weeks prior to arriving at the study facility. After arrival, animals were maintained on the high-fat diet for 7 days during acclimation and throughout the study. Food consumption was monitored by weighing the food and hopper together from study day 1 until study day 27 (main study) or 41 (recovery). The mean food intake of group 2 determined the amount of food available to group 3 in subsequent feeding sessions. Similarly, the mean food consumed by group 5 determined the amount of food available to group 6 in subsequent feeding sessions. Food and drinking water were available ad libitum throughout the study, except for 5-hour fasting periods occurring on study days 1, 28, and 42. Data collected on study day -1. On study days 1–27 (main study) or 42 (recovery), all animals received a bolus dose of vehicle, semaglutide standard (12 nmol / kg), or 17 (6, 12 nmol / kg) via interscapular injection. Group-dependent total doses (mL / kg) were based on the most recently recorded body weight. Individual animal weights were recorded starting on day -1. Animals were observed for clinically relevant abnormalities during dosing and at all sample collection time points. On study days -1, 1, 3–27, 29, and 36, 3 μL of whole blood was collected via tail snip to assess blood glucose levels using a handheld glucometer (AlphaTrak2, Abbott). Blood glucose readings were taken at approximately the same time each day, except on day 1, when blood glucose readings were taken pre-dose and 2, 4, 8, and 24 hours post-dose. Additionally, on study day 28, following a 5-hour fast, animals received a 10 mL / kg dose of glucose (2 g / kg) via intraperitoneal injection. A 3 μL blood sample was collected by tail snip and analyzed for glucose levels at the following time points (relative to glucose administration): 0, 15, 30, 60, 90, 120, and 180 minutes post-administration. Glucose samples were read using a handheld glucometer.

[0244] F. Bioanalytical method Analysis was performed at Climax Laboratories (San Jose, CA) using an API-4000 mass spectrometer, ESI positive, MRM scan. Samples were loaded onto a Shimadzu HPLC / CTC Autosampler equipped with an ACEC4 column (2.1 x 50 mm, 5 μm). Elution was with a gradient from aqueous 0.5% formic acid, 5 mM NHOAc, to 0.5% formic acid in CHCN / HO (9:1). Plasma samples (100 μL) were placed in a 96-well plate, and 30 μL of internal peptide standard (10 μg / mL in PBS) was added. A 300 μL aliquot of CHCN was added, and the sample was vortexed and centrifuged to precipitate plasma proteins. After transfer to a 96-well plate, 40 μL of sample was injected, and individual compound peaks were quantified using a standard curve. Noncompartmental pharmacokinetic analysis using WinNonlin was performed using the mean concentration at each sampling time point to report the maximum concentration (Cmax), the time at which Cmax was observed (Tmax), the area under the plasma concentration curve from time zero to the last measurable concentration point (AUC0-t), the plasma concentration-time curve from time zero to infinity (AUC0-∞), the terminal elimination half-life (t1 / 2), and the MRT. Depending on the analog structure, the limit of quantitation is 1-2 ng / mL.

[0245] G. Statistical analysis In vitro data are presented as pEC50 (SE) determined in Prism5 by nonlinear regression analysis of raw fluorescence data normalized by the corresponding response to the internal standard (see Supplementary Information for data plots). For assays where statistical significance was quoted, statistical data analysis was performed using GraphPadPrism software (version 5) by performing an analysis of variance (ANOVA type 2 with multiple measures) followed by a Bonferroni test with p<0.05 as the minimum level of significance.

[0246] H. Peptide elongation Our approach to extending the serum half-life of peptide GLP-1R / GCGR dual agonists focused on a novel approach: the use of covalently attached glycolipid surfactant-derived modifiers. The reagents were primarily derived from commercial nonionic surfactants widely used in the cosmetic and pharmaceutical industries and generally recognized as safe, such as 1-octyl β-D-glucose and 1-dodecyl β-D-maltose (Anatrace, Maumee, OH). Additional surfactant structures can be obtained by König's-Knorr / Helfferich glycosylation of acetobromoglucose (or similar activated carbohydrates) with the appropriate alcohol (e.g., HgO(yellow) / HgBr2 catalysis) and deprotection using NaOMe / MeOH to yield the free surfactant. The desired reagents are readily accessible by chemoselective TEMPO-mediated oxidation of the primary alcohol groups of such surfactants in the presence of water. Thus, a typical structure contains 1-O-alkyl β-D-glucopyranosiduronic acid (also known as 1-O-alkyl β-D-glucuronic acid adduct), a type of structure frequently formed in the liver for solubilization (Phase II metabolism) / detoxification of hydrophobic molecules, in this case acylation at Lys residues. Solid-phase peptide synthesis of the target peptides used a standard Fmoc protocol with orthogonal protection of the Glu16 and Lys20 positions (allyl ester and alloc, respectively), allowing for the formation of side-chain lactams and modification of the Lys position with N-ε-ivDde. Glycolipid-detergent linkage. Peptides were obtained in good yields with high purity (>95%, analytical RP-HPLC).

[0247] I. Pharmacokinetic behavior The primary objective of these studies was to investigate the effect of the novel glycolipid-surfactant conjugation approach on increasing the peptide's duration of action, stability, potency, and bioavailability. Preliminary in vitro stability studies in pooled human plasma showed rapid degradation of GLP-17-36 amide (4 h), whereas concentrations of analogs 3 and 8 remained completely unchanged at 8 h. This demonstrates the excellent stability of these representative surfactant-conjugated analogs in the presence of plasma.

[0248] The duration of action of the analogs was evaluated in rodent and minipig models. Compound series (analogs) 1 through 6 (Table 15) were designed to examine the effect on potency and duration of action of increasing alkyl chain length and hydrophobicity (from octyl to hexadecyl) at the 1-position of analogs. These O-alkyl β-D-glucopyranosiduronic acid modifiers were used. As seen in Figure 23, the relationship between chain length and duration of action in pharmacokinetic studies in Göttingen minipigs was not strictly proportional. As chain length increases, multiple variables can be envisioned that may affect the measured PK and PD. For example, with increasing chain length, depot formation (increases), solubility (decreases), affinity to SA (increases), affinity to hormone receptors (increases, then decreases), and potency of receptor activation (increases, then decreases). Within this group, the Cmax and PK profiles appear optimal for analogs 4 (C14) and 5 (C16), likely due to their optimal solubility and SA binding resulting in favorable distribution. The behavior of liraglutide as a standard in this assay (C16 acylated with palmitic acid on the γGlu spacer) best matched that of analog 3 (C12), which contains a shorter side chain. In vivo pharmacokinetic behavior of the compounds after subcutaneous administration to Göttingen minipigs at 20 nmol / kg for each analog. All data from a single assay, except for 4 and 5, obtained from parallel assays in Göttingen minipigs, were also profiled against liraglutide as a literature standard. Significantly higher plasma levels are measured at 4 hours for analog 2 (**); at 2 and 4 hours (**), 6 and 8 hours (***), and 12 hours (*); and at 2 and 12 hours (***), and 24 hours (*) for analog 5. All compared with liraglutide: *, P<0.05; **, P<0.01; ***, P<0.001.

[0249] J. In Vitro Structure-Activity Analysis We sought highly potent analogs with equally balanced agonist activity at both GLP-1R and GCGR, with good in vivo bioavailability and a significantly extended duration of action. Another goal was to understand the effect of novel glycolipid surfactant modifications on potency and duration of action. Therefore, the peptide structures were identical for most of the analogs studied. Initial SAR studies were directed toward evaluating the efficacy of the analogs for activating the cloned human receptor in vitro (Table 17). The EC50 values ​​of compounds 1–4 (side chain modifications from 1-O-octyl β-D-glucopyranosiduronyl to 1-O-tetradecyl β-D-glucopyranosiduronyl) demonstrated highly potent and variably balanced activation, with EC50 values ​​in the 10–30 pM range and selectivity ratios of 2–3 (SR = GCGREC50 / GLP-1EC50). 163 to 884 pM), resulting in an increased bias toward GLP-1R activation (SR = 4x and 17x, respectively). Although detailed optimization of the assay for these hydrophobic analogs was not performed, the EC50 values ​​for GLP-1R did not increase as rapidly.

[0250] [Table 17] [a] All structures have a Glu16 to Lys20 side chain lactam. G, M, and Me denote D-glucosidic, D-maltosidic, and D-melibiosidic linkages, respectively. S1 and S2 denote the spacer α-Lys or γ-Glu residues between Lys and the surfactant, respectively. Cn denotes a methylene chain of carbon number n. c denotes the carboxylate at the end of the chain. [b] All screening data generated with DiscoverX from the accumulated cAMP responses in CHO cells (duplicates) expressing hCCGR and hGLP-1R were analyzed using nonlinear regression analysis with R2 typically >90%. Data were replotted and analyzed in Prism 5. pEC50(SE) values ​​are reported, and curves are displayed in the Supporting Information. [c] Selectivity ratio (SR = GCGREC50 / GLP-1EC50) generated from EC50 data in pM. [d] Data for compounds 15, 16, and 17 were obtained in the presence of 0.1% OVA-containing buffer. For all others, 0.1% BSA-containing buffer was used.

[0251] The physical properties of such surfactant-modified peptides can be expected to be widely varied and tunable through the use of various alkyl chains (various hydrophobicity, solubility, SA affinity, CMC, and micelle size) and through the use of different carbohydrate head groups, such as disaccharides (various solubility, micelle size, and hydrophilic-lipophilic balance) in glycolipid surfactant precursors. Thus, "dodecyl maltoside" is a widely used commercial surfactant, and its use here yields 7, a highly potent but favorable dual agonist for GCGR. This surfactant is not as convenient as glucose in that it possesses two primary OH groups and therefore generates two carboxyl functional groups upon oxidation.

[0252] As a disaccharide head group, melibiose is more useful, as it has only one glycosylation site and only one primary OH functionality for oxidation to a uronic acid. The use of melibiose affords 1'-O-alkyl[β-(α-D-galactopyranosiduronic acid-(1→6'))]-D-glucoside intermediates (MeC12-MeC18), which give analogs 8-12. This disaccharide series includes highly potent (7) and well-balanced (8) dual agonists, although evidence suggests steric hindrance to GCGR activation. (9-11)

[0253] The l-O-dodecyl β-D-maltoside-derived modification favored GCGR activation (7; SR 0.3), whereas the l-dodecyl β-D-melibioside-derived analog 8 possessed a nearly balanced receptor-selective potency (SR -1). Further increase in the size of the melibioside-based modifications (C14, C16, C18; 9-11) rapidly decreased GCGR potency (SR 7, 28, 14, respectively). Increasing the size (or hydrophobicity) of the ligand side chain appears to hinder GCGR activation.

[0254] All of the aforementioned modifications were placed at residue 24, toward the C-terminal side of the side chain lactam bond (Glu16 to Lys20). Side chain modifications within the lactam ring were also investigated by placing a Lys(Me14) residue at position 17 (compound 12), and high potency was found with only a slight bias toward GLP-1R activation (SR2). In contrast, the same modification at position 24 showed a strong bias toward GLP-1R activation (SR7). Perhaps the conformation of the 12 binding region within the lactam ring disfavors GLP-1R activation for this headgroup and chain length combination.

[0255] Because intermediate-length glycolipid surfactant modifications yielded highly potent and relatively well-balanced analogs, we next investigated the effect of spacer modifications on hydrophobic side chains, as seen in liraglutide, semaglutide, and other similar compounds. The attachment of such linkers proved crucial to potency in the semaglutide drug design story, and 15 linkers were explored with a wide range of potency variations before settling on the γGlu-shortPEG sequence linker. Thus, compound 14, which possesses a Glu(γCO) linked to the Lys24 position with a 1-O-tetradecyl β-D-glucopyranosiduronyl modification linked to a Glu(α-NH2) function (S2GC14), significantly weakened GCGR activation potency (vs. 4). Using a Lys(α-CO) linkage to Lys24 as a spacer and attaching a 1-O-tetradecyl β-D-glucopyranosiduronyl modification to the ε-amino function of the spacer yielded 13, a molecule highly unfavorable for GCGR interactions (SR5). In addition to added bulk, the Glu(γCO) linker adds a negative charge at the attachment site, while the Lys(α-CO) linkage adds a positive charge to this side chain linker. Importantly, our glycolipid surfactant modification does not appear to require a spacer or spacer-receptor interaction, as seen with other side chain modifiers, to generate a highly potent molecule.

[0256] While we have previously primarily investigated the substitution of hydrophobic amino acids into peptide sequences as a route to enhanced HSA binding, here, structures 15–17 are analogs designed to test the effect of mimicking fatty acid head groups by incorporating carboxylic acids. Similar to those used in semaglutide, these function at the terminus of the surfactant alkyl chain. Thus, 15 incorporates 1-O-[(15-carboxypentadecyl)oxy]β-D-glucopyranosiduronic acid via an amide bond to the ε-NH group of Lys24 (Lys24GC16c), while 16 contains 1-O-[(17-carboxyheptadecyl)oxy]β-D-glucopyranosiduronic acid similarly linked to Lys24 (Lys24GC18c). Similarly, 17 contains 1-O-[(17-carboxyheptadecyl)oxy]β-D-glucopyranosiduronic acid. However, the glycolipid surfactant is attached to Lys17 (Lys17GC18c) similarly to 12, thereby conjugating within a lactam ring formed between the side chains of Glu16 and Lys20. Analog 17 exhibited high potency, strong evidence of very high serum albumin (SA) binding, and evenly balanced dual receptor activation potency (SR = approximately 1; Table 16). Analog 17 was therefore selected for more detailed characterization studies.

[0257] It is well known that strong HSA binding can result in reduced potency in vitro and in vivo. This was documented for semaglutide binding to GLP-1R, where the ratio of binding in the presence of 2% HSA compared with binding in the absence of HSA resulted in a 940-fold decrease in measured affinity. Nevertheless, engineering peptides in solution without the presence of any protein to block nonspecific binding can result in a decrease in apparent potency due to loss of ligand. The use of OVA, which has not evolved into a fatty acid carrier protein and has minimal fatty acid-binding properties, is a useful alternative. Comparison of EC50 data for activation of human GLP-1R and GCGR cloned in Chinese hamster ovary (CHO) cells (DiscoverX) by compounds 15-17 and semaglutide. The ratio of EC50 values ​​measured in the presence of BSA versus OVA can be used as a qualitative measure of BSA affinity. Here, we see that substituting low concentrations of BSA (0.1%) with OVA (0.1%) produced negligible improvements for the assay standards exendin-4 and glucagon, while the effect of the C16 side chain of analogue 15 was modest (4-9x fold improvement). In contrast, for 16 or 17, which have C18 alkyl chains, the effect of substituting BSA with OVA was greater (29-47x improvement). The improvement for 16 and 17 was even greater than that seen with semaglutide (13x), suggesting that tighter binding and a longer duration of action can be expected with 17 than with semaglutide. This data is shown in Table 18.

[0258] [Table 18] a Fold improvement = (EC in the presence of BSA) 50 ECs in the presence of / OVA 50 ), which is hypothesized to indicate the degree of binding to BSA, since the replacement of BSA with unconjugated OVA increases the observed potency (EC 50As discussed herein, very tight BSA binding distorts the actual receptor activation potency of semaglutide and these analogues.

[0259] K. In vivo characterization The PK profile of compound 17 was first determined compared to semaglutide in rats after subcutaneous administration at 10 nmol / kg. The measured Tmax for 17 and semaglutide is 8 hours (Figure 24). However, plasma levels of 17 still appeared to rise rapidly, indicating a true Tmax of >8 hours. The Cmax of 17 was 62% of that of semaglutide (76 vs. 122 ng / mL), while the AUCs were comparable (2,350 vs. 2,530 ng·h / mL, respectively). Overall, the MRT for 17 was slightly longer than that for semaglutide, at 21 and 15 hours, respectively. After subcutaneous administration, plasma concentrations of 17 increased dose-proportionally, with a 3-fold increase in dose (30 nmol / kg) resulting in a 2.8-fold and 3-fold increase in Cmax and AUC, respectively (data not shown). This profile, with its lower and delayed Cmax, was also observed in mice (data not shown), providing a lower peak-to-trough ratio than semaglutide and potentially reducing side effects. The 10 nmol / kg intravenous dose (data not shown) had a t of 10 hours, and the same dose administered subcutaneously demonstrated 29% bioavailability (F% in minipigs), albeit limited by apparent imprecision in Tmax and AUC. Figure 24 shows the in vivo PK behavior of 17 and the literature reference semaglutide after subcutaneous administration of 10 nmol / kg to CRL:CD(SD) rats. Analog 17 exhibited significantly lower plasma concentrations (***t = 2 and 4 hours; *t = 8 hours) and subsequent PK profiles in this and other assays. This may lead to a reduced peak-to-trough ratio. Comparison with semaglutide: *, P<0.05; ***, P<0.001.

[0260] The PK behavior of 17 in larger animals was investigated by iv and sc injection of a single dose of 20 nmol / kg in Yucatan minipigs (Figure 25). A very long PK curve was observed (SC, t = 52 h, MRT = 84 h), and the Cmax was low (890 ng / mL). The bioavailability of subcutaneous 17 administration relative to intravenous (iv) administration was 73%. The PK behavior of 17 was similar to published reports of semaglutide (sc, MRT = 64 h) in Göttingen minipigs, and similarly, 17 is expected to be suitable for QW (once weekly) administration in patients. There were no reported clinical observations (e.g., evidence of nausea, vomiting, or decreased appetite) during this study in adult minipigs. Figure 25 shows the in vivo pharmacokinetic behaviour of 17 following single subcutaneous and intravenous (iv) administration at 20 nmol / kg to male minipigs (n=4; weighing approximately 75 kg). Analogue 17 exhibited a very long pK profile, somewhat longer than that reported for semaglutide (MRT 86 h vs 64 h, respectively), indicating that 17 is suitable for QW dosing in patients.

[0261] The glucose-lowering efficacy of 17 was first investigated in a dose-finding study in db / db mice against the literature standard semaglutide (Figure 26). Semaglutide was not fully effective at 3 nmol / kg, but at 10 nmol / kg it rapidly reduced blood glucose levels (105 mg / dL), slightly below the baseline level (126 mg / dL) in normal C57BL / 6J mice at 8 hours. With high-dose semaglutide, blood glucose levels remained in a near-normalized range by 24 hours and returned to elevated levels (280 mg / dL) by 48 hours. Thus, 10 nmol / kg appears to be a sufficiently effective dose of QD semaglutide in this mouse model. The effects of 3 and 10 nmol / kg of 17 were similar acutely, lowering blood glucose levels to 129 mg / dL, close to the normal range in mice, with the maximum effect seen at 24 hours. High doses of 17 (10 nmol / kg) maintain blood glucose levels in the reduced range (153 and 187 mg / dL) at 48 and 72 hours after administration. Blood glucose levels were significantly higher than those of semaglutide at 2 and 4 hours (p<0.0001 and <0.02, respectively) and lower than those of semaglutide at 48, 72, and 96 hours (p<0.01, respectively). Thus, in this dose-finding assay in db / db mice, 17 appears to be more potent and long-acting than semaglutide in terms of glucoregulatory effects, while approaching maximum glucose lowering in a more gradual manner. Figure 25 shows the in vivo dose-response behavior of 17 and the literature standard semaglutide after single subcutaneous administration in male db / db mice (n=9). Analogue 17 appears to have a more potent, more measured, and longer-lasting PD effect compared to semaglutide, which causes a rapid drop in blood glucose to levels lower than those seen in normal C57BL / 6J mice. At equimolar doses of 17 (10 nmol / kg) and semaglutide (10 nmol / kg), blood glucose levels were significantly different at t = 2, 48, 72, and 96 hours. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0262] The pharmacodynamic profile of 17 was investigated in a 28-day diet-induced obesity (DIO) rat model compared with semaglutide as the literature standard (Figure 27). Groups of DIOCD:SD (Sprague-Dawley) rats (n = 9) were treated subcutaneously with vehicle, 12 nmol / kg semaglutide, or 6 or 12 nmol / kg 17 QDs. Groups were pair-fed with the same amount of chow consumed by the 12 nmol / kg semaglutide or 17 groups. Groups treated with either compound rapidly reached a stable, reduced body weight throughout the assay. Importantly, analog 17 treatment dose-dependently restored animals to the lean body mass range typically observed with moderate to severe food restriction (approximately 350 to 500 g, indicating longer survival), and they subsequently maintained that weight. Ad libitum-fed SD rats are known to develop diabetes, resulting in a shortened lifespan and making them unsuitable for long-term studies (spontaneous tumors, degenerative diseases). However, a restricted diet leads to a loss of total body weight and consistently longer survival times. No hyperglycemia was observed, and all animals survived to completion. During the 2-week recovery phase, animals in all treatment groups (4 per group) rapidly regained the weight lost during treatment. Figure 27 shows the amount of food consumed by animals in the vehicle, literature standard semaglutide (12 nmol / kg), Analog 17 (6 and 12 nmol / kg), and 12 nmol / kg semaglutide and 17 groups pair-fed. Treatment groups rapidly reached and maintained a stable weight, then rapidly regained weight during recovery (n=4). Analog 17 treatment achieved greater weight loss (-24% and -40%, respectively; 6 and 12 nmol / kg) than semaglutide treatment (-13%). The 17 animals treated with the low dose (6 nmol / kg) showed significantly lower body weights compared to semaglutide (12 nmol / kg) on ​​days 14-17 (*), 23-25 ​​(*), and 26-28 (**). The 17 animals treated with the equimolar dose (12 nmol / kg) showed significantly lower body weights compared to semaglutide on days 9 (*), 10 (**), and 11-28 (***). *=p<0.05, **=p<0.01, ***=p<0.001.

[0263] As can be seen in Figure 28, animals treated with the low dose of 17 (6 nmol / kg) showed very similar food intake suppression to animals treated with twice the molar equivalent of semaglutide, but lost approximately twice as much weight (-24% vs. -13%, 17 vs. semaglutide, respectively). This difference indicates a second mechanism of action that promotes weight loss: GCGR activation. Whereas animals treated with semaglutide showed significant but transient food intake suppression, animals treated with equimolar 17 showed more sustained food intake suppression throughout the majority of the assay (Figure 29), resulting in significantly greater weight loss (-40% vs. -13%, 17 vs. semaglutide, respectively). Figure 28 shows cumulative food intake by DIO rats during 27 days of treatment (followed by recovery) in groups fed with vehicle, the literature standard semaglutide (12 nmol / kg), analogue 17 (6 and 12 nmol / kg), and pair-fed rats. Amount of food consumed by animals in the 12 nmol / kg semaglutide or 17 groups. Note that the lower doses of 17 and semaglutide achieve similar levels of food intake suppression early on. Meanwhile, 17 at an equimolar dose (12 nmol / kg) to semaglutide exhibits food intake suppression throughout most of the assay. Both 17-treated groups achieved significantly greater weight loss compared to semaglutide (Figure 27). Compared to vehicle, all treatment groups experienced significant reductions in food intake from day 8 onwards, with semaglutide (12 nmol / kg) significantly reducing food intake from day 7 onwards and 17 (12 nmol / kg) significantly reducing food intake by day 6. Equimolar 17 and semaglutide (12 nmol / kg) showed that 17 induced a reduction in food intake relative to semaglutide on days 14 (p<0.05), 15 (p<0.01), and 16–28 (p<0.001). The semaglutide / 17 paired group showed a roughly consistent reduction in food intake, whereas the corresponding treatment groups showed greater weight loss (−6% vs. −13% and −18% vs. −40%; paired feeding vs. treatment, semaglutide vs. 17), again confirming the additional mechanisms of action of both semaglutide and analogue 17. The additive effect on weight loss was modest for the GLP-1 analogue semaglutide and very substantial for analogue 17, a dual GLP-1R / GCGR agonist.Studies with other GLP-1 / GCGR analogs have shown that increased metabolic rate, browning of white adipose tissue, and thermogenesis contribute to the increased weight loss seen with such analogs, but the results of such studies have been mixed and have not been performed.

[0264] Because obesity is thought to drive hepatic enlargement, steatosis, and inflammation in the NAFLD / NASH disease spectrum, an important aspect when evaluating the DIO rat model is its effect on liver weight. In this study, liver weights (and % of body weight) at day 28 were significantly different in the following rats: vehicle (18.6 g, 2.9%), semaglutide (14.9 g, 2.8%), pair-fed semaglutide (16.5 g, 2.9%), low-dose 17 (11.5 g, 2.5%), high-dose 17 (8.9 g, 2.4%), and pair-fed high-dose 17 (14.3 g, 2.8%). The reduction in liver weight in the 12 nmol / kg 17 group was statistically different from both the vehicle and equimolar semaglutide groups (p<0.01). Given the significant reduction in liver weight with 17, it is intriguing that studies using carefully validated antibodies have demonstrated the presence of GCGR in the liver, while GLP-1R is not. The beneficial effects of GCGR agonists on the liver are likely direct, whereas the beneficial effects of GLP-1R agonists on liver weight and histology are likely due to indirect effects on body weight and lipid levels.

[0265] L. Conclusions from Example 6 The rapidly increasing global obesity epidemic is driving a series of metabolic syndrome-related diseases, exemplified by type 2 diabetes and NASH. Existing drugs, including GLP-1 analogs and previously studied GLP-1 / GCGR dual agonists, do not adequately address the need for very significant weight loss (>10%) at approved doses, prompting the inventors to search for substantially more effective and tolerable agents. These drugs have the potential for QW delivery in humans. Based on previous studies demonstrating a significant extension of the peptide's duration of action through transient binding to human serum albumin (HSA), we utilized a novel approach to modify a relatively evenly balanced GLP-1R / GCGR dual agonist peptide framework using functionalized nonionic glycolipid surfactants called EuPort reagents. It was interesting to compare the potency and selectivity of selected peptide frameworks to explore the structure-activity behavior of this new class of peptide modifiers. The peptide sequence (compound 32 in Day, et al., A new glucose and GLP-1 co-agonist removes obesity in rodents. Nat Chem Biol 2009, 5, 749-757) has been successfully synthesized using the widely used polyethylene glycol (40 kDa; PEGylation) approach to generate a long-acting PEGylated molecule (compound 33). However, PEGylation typically causes a very substantial loss of potency (a 12-fold loss in GCGR potency and a 5-fold loss in GLP-1 potency in 33 compared to 32), resulting in a loss of selectivity balance (the potency ratio in 33 decreases from 32 to 0.45-0.17, thus favoring the GLP-1R and is no longer balanced). The studies presented herein also identified the sensitivity of GCGR activation to steric bulk (analogs 9-11). PEGylation also poses challenges regarding characterization (enveloping molecules with various molecular weights) and concerns regarding the immunogenicity and delayed clearance of PEG. In contrast, conjugation with glycolipid surfactants, here and in the PTH series, resulted in an extended and tunable duration of action with high potency and selectivity without the need for an additional linker.Thus, the relatively rigid presentation to the solvent of lipid tails on carbohydrate ring systems appears to be the preferred new approach in at least two hormone analogue series. A detailed evaluation of the physical properties of peptides similarly modified with glycolipid surfactants would be of great interest.

[0266] Seeking a duration of action suitable for QW delivery to patients, we developed analog 17, which demonstrated the desired exceptionally high and evenly balanced potency for in vitro activation of cloned human GLP-1R and GCGR, and reversion of DIO rodents in models of dietary restriction, chow, lean body mass, and very high SA binding. The latter aspect resulted in an exceptionally long duration of action in rodents and minipigs (t = 52 h; MRT = 84 h), a profile suggesting suitability for QW administration in humans. Benchmarking against the literature standard, the QW GLP-1R agonist semaglutide, demonstrated that dual agonist 17 was more potent, longer acting, and more effective in causing weight loss in DIO rodent models, reverting them to a lean body phenotype. Therefore, 17 (formulated as ALT-801 and formerly known as SP-1373) is currently completing studies to evaluate its therapeutic potential in the treatment of metabolic diseases such as obesity and NASH.

[0267] Example 7. Clinical Trial to Determine the Safety and Tolerability of Single and Repeat SC Doses of ALT-801 in Healthy Overweight and Obese Subjects and to Characterize the Effective Dose Range Based on PK-PD Relationships This study is designed to evaluate the safety and tolerability of single and repeated SC doses of ALT-801 in healthy overweight and obese subjects (BMI 25.0-40.0 kg / m²) and characterize the effective dose range based on pharmacokinetic-pharmacodynamic (PK-PD) relationships. Healthy overweight and obese volunteers are being studied because PK in such subjects may differ from that in normal-weight individuals. Furthermore, these subjects may be better able to tolerate the predicted weight-loss PD effects and may even benefit from treatment. Appropriate contraceptive measures are being taken to minimize the possibility of pregnancy, and precautions are being taken to rule out pre-existing conditions that may put them at risk from treatment with GLP-1 or glucagon analogs. Diabetic patients are being excluded until the effects of ALT-801 on glucose homeostasis have been better characterized in nondiabetic populations. Because overweight and obese subjects are expected to have varying levels of insulin resistance, observations made in these studies, combined with data from other compounds in this class, should be predictive of effects observed when diabetic subjects are studied. Exclusions have been established that may affect the accurate assessment of ALT-801's effects on safety, PK, or PD. Analyses will be performed to assess the effect of the BMI range employed in this study on PK and PD parameters. This study demonstrates the effect of ALT-801 on body weight, supporting its use as a first-line treatment for obesity.

[0268] The primary objective of this study is to evaluate the safety and tolerability of ALT-801 in healthy overweight and obese subjects after single and multiple ascending subcutaneous (SC) doses by assessing adverse events (AEs), vital signs, clinical symptoms, safety laboratories, urinalysis, physical examination, and injection site reactions; glucose homeostasis; blood pressure; electrocardiogram (ECG), Holter monitoring, etc. Secondary objectives of this study are to evaluate: 1) the PK of ALT-801 after single and multiple ascending SC doses; and 2) the PD effect of ALT-801 after single and multiple doses. Exploratory objectives of this study include evaluating: 1) the extended PD effect of ALT-801 after multiple doses; and 2) the effect of ALT-801 on heart rate-corrected QT interval (QTc) prolongation. Study assessments, including liver fat content by MRI-PDFF, body weight, body composition by whole-body MRI, insulin resistance, systemic inflammation, and GLP-1 and glucagon target engagement, are based on ALT-801's expected PD characteristics. These include weight loss and changes in body composition. Measurement of glucose homeostasis is based on the potential effects of GLP-1 and glucagon analogs on glucose control. Because GLP-1 and glucagon agonists are associated with clinically insignificant changes in blood pressure and heart rate, ambulatory blood pressure monitoring (ABPM) and Holter monitoring are included. Holter monitoring is also included to provide information on ALT-801's potential effects on QT interval prolongation. Based on experience with the pharmacology and safety of GLP-1 and GLP-1 / glucagon dual agonists, a dose-related incidence of gastrointestinal AEs, including nausea and vomiting, may occur. Glucose homeostasis, including the incidence and severity of hyperglycemia and hypoglycemia, will also be assessed. Because weight loss is a desirable attribute of this compound, efficacy will be monitored rather than safety. However, if weight loss is deemed excessive, doses may be adjusted in subsequent cohorts. If the level of weight loss is deemed dangerous or excessive, the study drug may be suspended or discontinued for an individual subject. Subjects will also be monitored for drug-induced liver injury.Blood samples will be collected for biobanking in subjects who provide separate consent, pre-dose and after the final dose of study drug. These samples will be used to discover and / or validate biomarkers for NASH and related diseases, including potential genetic analysis.

[0269] This study, described herein, is a first-in-human (FIH), phase 1, randomized, double-blind, placebo-controlled, two-part study of ALT-801 in healthy overweight and obese subjects. Overweight to obese subjects (body mass index [BMI] 25.0-40.0 kg / m²) will be enrolled. In Part 1, the single ascending dose (SAD) phase, subjects will undergo a screening period of up to 28 days. Overweight to obese subjects who meet the inclusion criteria and no exclusion criteria will be randomized 3:1 in cohorts of eight subjects, with six subjects receiving ALT-801 and two subjects receiving placebo. The study drug (SEQ ID NO: 1 formulated as ALT-801 for subcutaneous (SC) administration) will be administered subcutaneously (SC) in the abdominal site for all SAD cohorts. Subjects will be admitted to the research unit approximately 1 day prior to study drug administration (Day -1) and discharged on Day 8. Subjects will receive a single SC dose of ALT-801 or placebo on Day 1. Two additional optional cohorts for Part 1 are planned. The following dose levels are planned: 0.4, 1.2, 2.4, 4.8, 7.2, and 9.4 mg as weekly doses administered once weekly (QW) based on a 60 kg human. These doses may be modified based on clinical observations or, if available, pharmacokinetic (PK) data. The first two subjects (one ALT-801 and one placebo) in each SAD cohort will be dosed in a sentinel fashion at least 48 hours before the remaining subjects. Prior to assessments on Days 1 through 5 and on Day 8, subjects will fast overnight for at least 10 hours, and diet will be standardized. Subjects will undergo study evaluations to assess safety, including ECG, CGM, and ABPM, and blood samples will be collected for PK as described in the schedule of assessments below. After discharge from the study unit, subjects will return for outpatient visits for PK and safety assessments every 3 days until Day 26, and for follow-up visits on Day 35 or at least 5 half-lives as determined over the course of dosing. If the predicted effective dose and exposure based on pharmacological modeling is not achieved and / or a single-dose maximum tolerated dose (MTD) is not identified after completing the six planned cohorts, up to two additional single-dose cohorts will be enrolled in the study.1. Upon completion of Day 8 of Part 2, SAD Cohort 3, and evaluation of safety in that cohort, the Multiple Ascending Dose (MAD) phase will begin. The starting dose in Part 2 will be half the dose in SAD Cohort 3.

[0270] After providing informed consent, overweight to obese subjects undergo a screening period of up to 28 days. Subjects are instructed to maintain their usual diet and activity levels during screening and not to start any new dietary, supplement, or exercise programs at any time while participating in the study. Subjects are admitted to the research unit approximately 4 days before study drug administration (Day -4) and undergo a diet and exercise run-in period during which they are provided with a standardized diet. The standardized diet is provided with individualized daily calories using predicted BMR × 1.5 to account for differences between subjects based on weight, height, age, and sex. Study participants' activity levels are also standardized. Subjects who meet the inclusion criteria but not the exclusion criteria are randomized on Day 1 in a 5:1 ratio in cohorts of 12 subjects, with 10 subjects receiving ALT-801 QW and 2 subjects receiving placebo QW for 6 weeks. In all MAD cohorts, the study drug is administered subcutaneously (SC) in the abdominal region.

[0271] Subjects will receive their first dose of study medication on Day 1 and remain on the study unit until receiving their second dose on Day 8. Subjects will then return for three outpatient dosing visits, spaced weekly (Days 15, 22, and 29) and be readmitted between Days 32 and 43. Subjects will receive their last dose of study medication on Day 36, whichever occurs first. Subjects will undergo several study assessments to evaluate the safety, PD, and PK of ALT-801, as described herein. Safety assessments will include ECG, CGM, and ABPM. PD assessments will include anthropomorphic measurements, dietary assessments, imaging, and blood sampling for biomarkers. The Patient Assessment of Gastrointestinal Disorders Symptom Severity Index (PAGI-SYM) will be performed to evaluate the effect of treatment on gastrointestinal symptoms. Blood samples will be collected for PK and immunogenicity. Subjects will fast overnight for at least 10 hours on days -1 through 5, and before days 7, 8, 36, 37, 42, and 43. In addition, subjects will receive a standard breakfast on days -1, 7, and 42 for mixed meal tolerance testing.

[0272] The MAD dose will be selected based on clinical data and, if available, PK data from previously completed SAD and MAD cohorts. Three MAD cohorts are planned, with up to two optional additional cohorts planned as needed to achieve the predicted effective dose and exposure based on pharmacometric modeling, expand previously studied dose levels, and continue dose escalation if the MTD for this phase is not identified. Alternatively, dose titration schemes will be investigated if gastrointestinal intolerance is observed before the maximum effective dose based on pharmacological modeling is reached.

[0273] The maximum recommended starting dose (MRSD) for Part 1 was based on one-tenth the human equivalent dose (HED) at the NOAEL determined in animals (rats and monkeys) in pivotal Good Laboratory Practice toxicity studies. While both rats and monkeys were expected to have similar clinical responses to ALT-801 (see Example 4), the exposure at the NOAEL was slightly lower in rats, resulting in a more conservative human starting dose. The rat NOAEL was a high dose of 0.45 mg / kg / week, which corresponds to 0.44 mg / week in a 60 kg human based on body surface area scaling. Notably, the monkey NOAEL was also a high dose of 0.25 mg / kg, which corresponds to 0.49 mg / week in a 60 kg human based on body surface area scaling. A 10-fold scaling factor was used for safety, resulting in a human starting dose of 0.40 mg / week for a 60 kg human. Furthermore, human exposures extrapolated at the maximum recommended starting dose (MRSD) are significantly lower than those at the NOAEL in monkeys and are particularly comparable to those at the NOAEL in rats. This is particularly important because monkeys, while not the most sensitive species, are biologically associated with the most clinically relevant toxicities (i.e., reduced food intake and vomiting). Clinical observations and PK in Part 1 will ultimately guide dosing considerations in Part 2.

[0274] The primary finding of ALT-801 in rat and monkey studies was weight loss (see, e.g., Example 4). Changing the dosing schedule in rats from daily to three days per week reduced ALT-801's effects on food intake and weight loss, improving tolerability consistent with its mechanism of action (see Example 4). The toxicity of GLP-1 and glucagon agonists has also been well characterized in human studies. Preclinical safety findings support a three-fold dose escalation increase to SAD cohort 2. Subsequent escalations will not exceed two-fold in any part of the study. If tolerability needs to be improved, a dose escalation scheme can be considered. In addition to the reliability of these predictions, the dose-exposure relationship in humans is predicted to be linear based on population PK models of several preclinical species (mice, rats, minipigs, and monkeys), as described in Example 4. As studies are ongoing, the model will be updated with human data. The predicted t1 / 2 of ALT-801 in humans is in the range of 100 hours, an assumption that will also be confirmed in Part 1. Based on once-weekly (QW) dosing, estimated accumulation with repeated dosing at steady state is less than two-fold. To ensure that multiple exposures fall within the range of single exposures, the starting dose in Part 2 is planned to be half the dose in Part 1 Cohort 3. However, subsequent Part 2 cohorts may be adjusted based on safety and pharmacokinetic (PK) data. The decision to escalate to each successive dose level is based on safety and tolerability assessments through Day 8 of Part 1 (7 days after the single dose) and Day 15 of Part 2 (7 days after the second dose). Escalation after completion of Week 2 is based on observations from previous GLP-1 and GLP-1 / glucagon dual agonist studies that AEs, expected to be primarily nausea or vomiting, occur during the first 2 weeks of dosing. Furthermore, Cmax and AUCtau during the final week of dosing are not expected to exceed Cmax or AUCinf of the dose in the previously completed and safety-evaluated SAD cohort. The target dose for maximum efficacy in adults, corresponding to the ED80 to ED90, is estimated to be between 1 and 5 mg, with target plasma concentrations between 50 and 100 ng / ml. Modeling of animal PK parameters to predict human PK.Therefore, the estimated starting dose is approximately 2.5-fold lower than the predicted lowest effective dose and is expected to be inactive.

[0275] To maximize safety, single ascending dose (SAD) and multiple ascending dose (MAD) escalations are planned to not exceed the NOAEL exposure in rats. However, if PD and tolerability suggest that overweight and obese subjects would benefit from doses expected to exceed the NOAEL exposure in rats, supportive safety and efficacy data will be presented to the IEC and consensus will be reached before continuing with SAD and MAD escalation.

[0276] Dose escalation requires a minimum of six subjects in Part 1 and eight subjects in Part 2, with at least one subject receiving placebo in each cohort. If observations suggest that dose escalation exceeds the MTD, the recommended next dose level may be adjusted downward based on an evaluation of safety and tolerability data observed in previous treatment cohorts. Dosing may continue until the MTD, which is determined individually for each part of the study, is identified. Available pharmacokinetic data can be used to guide decision-making and will be explicitly considered if exposure is expected to exceed the NOAEL in rats. To maximize safety, planned SAD and MAD escalations will not exceed exposure at the NOAEL in rats.

[0277] After completing screening activities, subjects who meet all inclusion criteria (e.g., none of the exclusion criteria are randomized via the interactive web response system (IWRS)) will be randomly assigned in Part 1 to either the ALT-801 or placebo treatment group for sentinel administration. The remaining 6 subjects in each cohort of 18 subjects will be randomly assigned to either the ALT-801 or placebo treatment group, 5 to the ALT-801 group, and 1 to the placebo group, for an overall 3:1 ratio of ALT-801 to placebo in each cohort. In Part 2, cohorts of 12 subjects will be randomly assigned to either the ALT-801 or placebo treatment group in a 5:1 ratio, 10 to the ALT-801 group and 2 to the placebo group.

[0278] ALT-801 will be formulated in glass vials in sterile, buffered aqueous solution to a final concentration of 2.5 mg / mL and a total fill volume of 1.2 mL and administered as a subcutaneous (SC) injection. In Part 1, a single dose of study drug will be administered on Day 1. The first two subjects in each SAD cohort (one ALT-801 and one placebo) will be dosed in a sentinel fashion at least 48 hours before the remaining subjects. In Part 2, study drug will be administered QW for 6 weeks. Doses will be administered on Days 1, 8, 15, 22, 29, and 36. The starting dose in Part 1 will be 0.40 mg, which corresponds to one-tenth the human equivalent dose at the no-observed-adverse-effect level (NOAEL) in rats. Dose escalation will follow a modified Fibonacci approach, with no more than threefold increases at planned dose levels of 0.40, 1.2, 2.4, 4.8, 7.2, and 9.4 mg (rounded down for safety) every seven days. The starting dose in Part 2 is planned to be half the dose in Part 1 Cohort 3; however, subsequent Part 2 cohorts may be adjusted based on safety and pharmacokinetic (PK) data. The decision to escalate to each successive dose level will be based on safety and tolerability assessments through Day 8 of Part 1 (7 days after the single dose) and Day 15 of Part 2 (7 days after the second dose). Modified dose titration schemes will be implemented as needed or as described herein. Each dose of ALT-801 or placebo will be administered as an SC injection into the abdomen by appropriately trained clinical staff. Dosage is based on the selected dose and the final formulation concentration of 2.5 mg / mL. The saline placebo is dose-matched based on the dose and amount of ALT-801 administered in that cohort. Because weight loss is a desirable characteristic of this compound, efficacy is monitored rather than safety. However, if weight loss is deemed excessive, the dose may be adjusted in subsequent cohorts. If the level of weight loss is deemed excessive, the study drug may be suspended or discontinued for an individual subject. If the level of gastrointestinal adverse events is deemed excessive and intolerable despite antiemetic treatment (e.g., severe gastrointestinal adverse events lasting more than 24 hours), the study drug may be suspended or discontinued for an individual subject. If vomiting persists, the subject may be administered an antiemetic.In this situation, a 5HT3 receptor antagonist (e.g., ondansetron) is preferred. If observations suggest that dose escalation exceeds the MTD, the recommended dose level can be adjusted downward based on evaluation of safety and tolerability data observed in previous treatment cohorts. Dosing can continue until the MTD is identified, which is determined individually for each portion of the study. Available PK data can be used to guide decision-making.

[0279] Blood samples for PK assessment are collected at hours 0, 1, 4, 6, 8, 12, and 16 on days -1, 1, 2, 3, 4, 5, 8, 11, 14, 17, 20, 23, and 26 in Part 1, and at hours 0, 1, 4, 6, 8, 12, and 16 on days -1, 1, 2, 3, 4, 5, 8, 15, 22, 29, and 36–38 in Part 2. The remaining plasma from the PK samples can be stored frozen without time restrictions and used for ALT-801 bioanalytical method development and ALT-801 metabolite discovery. ECG readings are time-matched to the PK sample times. If multiple activities occur at the same time, the ECG should be collected first, and PK blood draws should be performed at the nominal time. PD assessments are performed only in Part 2.

[0280] Height will be measured in centimeters using a wall-mounted stadiometer or a stadiometer attached to a balance beam scale, whichever is available. Subjects are required to wear socks or be barefoot. Except for the screening visit, weight will be measured in kilograms using a calibrated scale at approximately the same time at each nominal time point. Measurements should be taken with subjects wearing a gown (or other standard clothing provided by the clinical research unit), underwear, and socks (without shoes), both fasting and after the subject is asked to urinate (i.e., empty their bladder). Waist circumference should be taken with the subject wearing a gown. Measurements should be performed midway between the superior aspect of the iliac crest and the lower lateral margin of the ribs. Measurements do not need to be taken at umbilical level. The measuring tape should be held horizontally. Height, weight, and waist circumference will be measured according to the schedules in Part 1 and Part 2, and BMI will be calculated and recorded. Height measurement is required only at screening. Waist circumference will only be measured for Part 2 subjects.

[0281] FibroScan® is an ultrasound-like instrument capable of simultaneously measuring liver stiffness and steatosis via vibration contrast transient elastography (VCTE) and CAP, respectively. For Part 2 subjects, FibroScan® CAP is measured during screening after at least a 10-hour overnight fast. FibroScan® CAP is measured prior to MRI-PDFF. MRI-PDFF is a quantitative imaging biomarker that allows for accurate, repeatable, and reproducible quantitative assessment of liver fat throughout the liver. For Part 2 subjects, MRI-PDFF is measured during screening (occurring only if CAP is ≥ 300 dB / m) and at the EOS visit after at least a 10-hour fast. Liver fat percentage is corrected for total liver volume, which is measured simultaneously with liver fat mass. Whole-body MRI is an established imaging technique used to measure body composition, including lean body mass. For Part 2 subjects, whole-body MRI is performed during screening and the EOS visit in conjunction with MRI-PDFF.

[0282] In Parts 1 and 2, subjects will be provided with a standardized diet during their stay in the research unit. Daily calories will be individualized using the predicted BMR formula multiplied by an activity factor of 1.5, and macronutrient composition will be standardized at 40-50% carbohydrate, 15-25% protein, and 30-40% fat. In Part 2, the same standardized diet will be provided on days 4-2 and 39-41, prior to PD assessments on days 1 and 42. MRI, PDFF, and MMTT assessments will be performed as described in the corresponding manuals.

[0283] Food intake and appetite are assessed using an ad libitum eating test and a VAS questionnaire. VAS questionnaires are a standard method for appetite studies, recording hunger, satiety, fullness, and desire for specific flavors (e.g., sweet, salty, savory, and fatty foods) [Flint 2000]. Subjects complete VAS questionnaires before and after an ad libitum meal on days specified in the assessment schedule. The size of the ad libitum meal exceeds the expected intake of healthy overweight and obese volunteers. During the test meal, subjects are isolated, and environmental cues are minimized (i.e., no television, cell phones, computers, etc.). Subjects are instructed to eat as much as they want within 30 minutes and until they feel full. Weight is recorded before and after the meal to capture food intake and determine calorie expenditure.

[0284] Basal metabolic rate (BMR) and resting energy exposure (REE) are assessed in the morning under fasting conditions, after a fasting period of at least 10 hours. Resting energy expenditure is assessed on days 1 and 42. BMR and REE are determined using the ventilated hood method (indirect calorimetry). Because BMR is typically the primary setting factor for daily energy expenditure, changes in BMR may be clinically relevant within the context of metabolic drug development programs targeting energy expenditure.

[0285] Following a minimum 10-hour fast, subjects consume a standardized liquid meal (6 fluid ounces of Ensure Plus [700 kcal], with fat, carbohydrate, and protein components consistent with a standard MMTT) within 5 minutes. The t=0 sample (i.e., before the standardized liquid meal) is the final HOMAIR2 blood sample (see above). Hormonal markers include glucose, insulin, and C-peptide. Samples are collected at 5-minute intervals for the first 15 minutes, then for 30 minutes until 240 minutes after ingestion of the standardized liquid meal (no additional food intake during this time). The MMTT procedure is performed on the day specified in the assessment schedule. To standardize the test and reduce variability, each test is preceded by a 3-day run-in period of standardized diet and standardized physical activity following admission to the clinical research unit.

[0286] After subjects have fasted overnight for at least 10 hours, blood samples for ketone assessment will be collected one day before the first and second doses and six days after the final dose. Blood samples for FGF-21 and adiponectin assessment will be collected after subjects have fasted overnight for at least 10 days. After a minimum 10-hour fast, blood will be collected for lipid assessment, including cholesterol (total, HDL, LDL), ApoA, B, lipoprotein(a), TG, and tripalmitin, as shown in Table 4, before the first dose and six days after the final dose of study drug. hs-CRP, leptin, MCP-1, and IL-6 will be collected before the first dose and six days after the final dose of study drug, as shown in Table 4. Glucose homeostasis will be assessed during the time periods indicated in Parts 1 and 2 by 24-hour CGM using a Dexcom G6 CGM.

[0287] The safety population includes all randomized subjects who receive at least one dose of study drug. Subjects will be analyzed according to the treatment they receive. The PK population includes all randomized subjects who received at least one dose of ALT-801 and have sufficient PK data for analysis. The QT population includes all subjects in the PK population who had at least one time-matched ECG at baseline and a corresponding time-matched PK-ECG after dosing. The PD population includes all randomized subjects who received at least one dose of study drug and had results from baseline and at least one post-baseline PD assessment.

[0288] Statistical methods used included descriptive statistics to assess differences in demographics and baseline characteristics. Medical histories were coded using the Current Medical Dictionary for Regulatory Activities (MedDRA) version and listed by subject. Ongoing safety data were summarized with descriptive statistics (arithmetic mean, standard deviation [SD], median, minimum, and maximum) by dose level and treatment (active or placebo). Categorical safety data were summarized with frequency counts and percentages by study part, dose level, treatment, and day, where applicable.

[0289] AEs are coded using the latest MedDRA version. A per-subject AE data listing is provided, including verbatim term, preferred term, SOC, treatment, severity, and relationship to study drug. The number of subjects experiencing treatment-emergent AEs (TEAEs) and the number of individual TEAEs and injection site reactions are summarized by treatment group, SOC, and preferred term. TEAEs are also summarized by severity (grade 1 to 4) and relationship to study drug (unlikely, likely, or probably). Relevance for stopping rules is defined as possibly related or likely related. Clinical laboratory assessments, vital sign assessments, continuous cardiac monitoring, ECG parameters (excluding Holter monitoring), CGM measurements, ABPM measurements, and food tolerance test parameters are summarized by study portion, treatment group, dose level, and protocol-specified collection timepoints. A summary of changes from baseline at each protocol-specified timepoint by treatment group is also presented. Physical examination changes are described by subject. PAGI-SYM analysis is detailed in the statistical analysis plan (SAP). Concomitant medications are listed by subject and coded using the most recent WHO drug dictionary.

[0290] Pharmacokinetics data include individual ALT-801 concentration data listed and summarized by cohort along with descriptive statistics (sample size [N], arithmetic mean, SD, coefficient of variation [CV%], median, minimum, and maximum). Individual and mean ± SD ALT-801 concentration-time profiles for each cohort are also displayed graphically. Plasma ALT-801 noncompartmental PK parameters Cmax, time to maximum plasma concentration (Tmax), AUC0-t, AUC0-inf, elimination rate constant (Kel), t1 / 2, apparent total body clearance (CL / F), and apparent terminal volume of distribution (Vz / F) (if sufficient data are available for parameter determination) are estimated for the SAD portion. For the MAD portion, the PK parameters Tmax, Cmax, and AUCtau are estimated after the first and last doses (Weeks 1 and 6). If data permit, Kel, t1 / 2, apparent total body clearance at steady state (CLSS / F), and apparent volume of distribution at steady state (VSS / F) will be estimated after the 6th dose. Pharmacokinetic parameters will be listed for each individual and summarized by cohort using descriptive statistics (N, arithmetic mean, SD, CV%, median, minimum, maximum, geometric mean, and geometric CV%). The effect of baseline BMI on PK parameters will be assessed by correlation analysis. Dose proportionality will be assessed using a power model approach, if appropriate. Accumulation will be assessed as the ratio of Cmax and AUC0-tau from Week 6 to Week 1. Steady state will be assessed by comparing trough concentrations from the first dose to the last dose.

[0291] ECGs extracted from Holter monitors are analyzed by a central ECG laboratory with a selected group of experienced readers blinded to the subject, visit, treatment, and nominal time point. One reader reviews each subject's ECG unless a second review is required based on quality control or availability. All ECGs are analyzed using the same lead for each subject. The primary analytical lead is Lead II. Unless analysis is unavailable, V2 or V5 is used for the entire dataset for each subject.

[0292] The primary analysis will be the placebo-corrected mean change from baseline post-dose time points using the Fridericia-corrected QT interval (ΔΔQTcF) and one-sided upper 95% confidence limit. Other correction methods, such as Bazett's (QTcB), individual correction (QTcI), and population correction (QTcP), can be considered and compared. At a minimum, the Fridericia and Bazett corrections will be analyzed and presented. Secondary analyses will include the relationship between time-matched plasma concentrations and ΔΔQTcF using linear mixed-effects modeling. The immunogenicity of repeat-dose ALT-801 will be assessed by evaluating serum samples collected at the final visit of the MAD period using an ELISA-based assay. If end-of-study samples are positive, mid-study samples will also be analyzed. Immunogenicity may be correlated with safety and PK, if applicable.

[0293] Pharmacodynamic studies will include changes in liver fat content, anthropometric parameters, GLP-1 involvement and insulin resistance, glucagon involvement, and lipid and inflammatory markers, and will be listed and summarized by treatment group using descriptive statistics (sample size [N], arithmetic mean, SD, median, minimum, maximum, geometric mean, and geometric CV%). Inferential statistics will be applied as appropriate. The impact of baseline BMI on PD parameters will be assessed by covariate analysis.

[0294] An interim analysis may be conducted after completion of two or more doses in MAD Cohort 3. The purpose of this analysis is to enable dose selection for subsequent studies. In this analysis, the study remains blinded, and subject-level safety, PD, and available PK data are anonymized for analysis. Summary data by study part, dose level, treatment group (active or placebo), and day, if applicable, are reported. The conduct of the interim analysis is detailed in the SAP.

[0295] Example 8. Formulation Studies To support clinical development and future commercialization, a formulation of ALT-801 needs to be developed to achieve long-term stability, ideally at or above +2-8°C. Additionally, the formulation of ALT-801 can be optimized to improve pharmacokinetic parameters.

[0296] The initial formulation of ALT-801 (F58), disclosed above, contains 2.5 mg / mL ALT-801 as the API, 3.48 mg / mL arginine, 0.5 mg / mL polysorbate 20 (PS-20), and 42.6 mg / mL mannitol adjusted to a pH of -7.75 with hydrochloric acid, and was developed to support early clinical development. F58 is stored at -20°C. The F58 formulation exhibited turbidity at +2-8°C, indicating that larger aggregates had precipitated from solution. Analysis by RP-HPLC revealed no change in ALT-801 purity or content, supporting the theory that this hazy appearance is related to the physical instability of the supramolecular structure formed by ALT-801 in solution. ALT-801 is a peptide amphiphile formed by the covalent attachment of the hydrophobic alkyl chain of EuPort (e.g., a functionalized nonionic glycolipid surfactant) to a hydrophilic peptide moiety. Therefore, ALT-801 is intended to self-assemble into supramolecular structures such as micelles. ALT-801 in water was demonstrated to form micelles at concentrations above the critical micelle concentration (CMC) of 1.33 mg / ml as measured by a surface tensiometer (see Figure 30). The CMC for ALT-801 is expected to be the same in F58 buffer (without PS-20).

[0297] To improve the formulation of ALT-801 for subcutaneous administration, critical micelle concentration (CMC) experiments were performed by surface tension measurements on both polysorbate 20 and polysorbate 80 in F58 buffer prepared at pH 7.7 under four conditions: alone, with 2.5 mg / ml added ALT-801, with 5.0 mg / ml added ALT-801, and with 10.0 mg / ml added ALT-801. The CMC values, and the resulting shifts due to ALT-801, and the extent of interaction between polysorbate 20 or polysorbate 80 and ALT-801 were determined as shown in Tables 19 and 20, respectively. The CMC shift is simply calculated as the CMC in the presence of ALT-801 minus the CMC of the surfactant alone in solution. The extent of interaction on a mass or molar basis is calculated as the CMC shift divided by the concentration of ALT-801 causing the shift.

[0298] [Table 19]

[0299] [Table 20]

[0300] These results identify the minimum concentrations of PS-20 or PS-80 to be used across a range of ALT-801 concentrations to achieve that CMC. They also demonstrate that the concentration of PS-20 in the F58 formulation (0.5 mg / ml) is too low to achieve the CMC, potentially explaining the cloudy appearance of the solution when stored at +2-8°C. The results indicate that at least 0.66 mg of PS-20 per 1 mg of ALT-801 is required to achieve the CMC. Similarly, at least 1.03 mg of PS-80 per 1 mg of ALT-801 is required to achieve the CMC.

[0301] As will be appreciated by those skilled in the art, other advantages of the reagents and methods of using same are also provided herein. 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. Accordingly, it is intended that the appended claims cover all such.

Claims

1. 1. A pharmaceutical dosage formulation comprising an agonist peptide product having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), wherein the peptide is modified with a non-ionic glycolipid surfactant, and wherein the dosage is designed to improve blood glucose control by reducing one or more adverse events when administered to a mammal compared to an agonist having disproportionate affinity for GLP-1R and GCGR, wherein the adverse events are selected from nausea, vomiting, diarrhea, abdominal pain, and constipation.

2. 1. A pharmaceutical dosage formulation comprising an agonist peptide having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), the peptide being modified with a non-ionic glycolipid surfactant, the dosage being designed to induce weight loss with reduced one or more adverse events upon administration to a mammal compared to an agonist having disproportionate affinity for GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain, and constipation.

3. 3. The pharmaceutical dosage formulation of claim 2, wherein the weight loss is at least 5%, at least 10%, or from about 1% to about 20%, or from about 5% to about 10% (w / w).

4. 4. The pharmaceutical dosage formulation of any one of claims 1-3, wherein the dosage is designed as a once-weekly dosage form, optionally designed for administration for about 2 weeks to about 8 weeks.

5. 5. The pharmaceutical dosage formulation of claim 4, wherein administration of a single dose to a mammal results in a reduction in blood glucose levels about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide.

6. 5. The pharmaceutical dosage formulation of claim 4, wherein administration to a mammal for about 4 to about 8 weeks, optionally about 6 weeks, results in greater total body weight loss at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks after administration compared to administration of an approximately equimolar dose of semaglutide.

7. Administration of a single dose to a mammal results in a lower C at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide. ｍａｘ 5. The pharmaceutical dosage formulation of claim 4, wherein:

8. 8. The pharmaceutical dosage formulation of any one of claims 1-7, wherein the dual agonist peptide is any one of SEQ ID NOs: 1-10 or 12-27.

9. 9. The pharmaceutical dosage formulation of any one of claims 1-8, wherein the dual agonist peptide has approximately equal affinity for GLP-1R and GCGR, and optionally said dual agonist peptide is SEQ ID NO:

1.

10. 10. The pharmaceutical dosage formulation of any one of claims 1-9, wherein the surfactant is a surfactant of the 1-alkyl glycoside class.

11. 11. The pharmaceutical dosage formulation of any one of claims 1-10, which is present as an aqueous formulation comprising one or more of polysorbate 20, arginine, or mannitol.

12. 2. The method of claim 1, wherein administration of a pharmaceutical dosage formulation to a mammal compared to administration of an approximately equimolar dose of semaglutide: a reduction in blood glucose levels at about 48 hours or 96 hours after administration, optionally wherein the blood glucose levels are reduced by about 50%; a reduction in blood glucose levels about 72 hours after administration, optionally a reduction in blood glucose levels of about 100%; and / or Blood sugar levels decrease approximately 120 hours after administration 12. The pharmaceutical dosage formulation of any one of claims 1-11, which produces

13. 13. The pharmaceutical dosage formulation according to any one of claims 1 to 12, c) administering said dosage formulation to a mammal, induces generalized weight loss, and / or Induces liver weight loss, and / or d) administration of said dosage formulation to a mammal compared to semaglutide administered at an approximately equimolar dose; exhibit a lower Cmax, optionally about a 50% lower Cmax; exhibiting a Tmax that is about equal to or greater than about 100%; optionally exhibiting a Tmax that is about 100% longer; Similar AUC （０－ｉｎｆ） and optionally about 85-93% thereof; exhibiting a T½ (time) that is approximately equal to or longer, optionally about 25-75% thereof; exhibiting a prolonged MRT (time), optionally at least about 25% higher; demonstrated a long-term PK / PD profile, It shows almost the same or better glucose regulating effect, induce greater total body weight loss, optionally about twice as much; Induce a loss of body fat mass, optionally about 50-100% lower; and / or When administered to treat NASH, it induces increased whole body weight loss, liver weight loss, improved NAS score, improved hepatic steatosis, improved ballooning, improved col1A1 staining, improved ALT, improved liver TG / TC, and improved plasma TG / TC. A pharmaceutical dosage formulation according to any one of claims 1-12.

14. Administration to mammals compared to semaglutide administered at approximately equimolar doses and / or, by about 14 days after administration of the dosage formulation, a significant reduction in body weight, optionally resulting in a reduction of about 15%.

14. The pharmaceutical dosage formulation of claim 13, wherein the dosage formulation results in a significant weight loss, optionally about a 25% weight loss, by about 20-28 days after administration of the dosage formulation.

15. 15. The pharmaceutical dosage formulation of any one of claims 1-14, wherein administration to the mammal results in weight loss in the obese mammal sufficient to return the mammal to a normal weight range for lean, normal mammals.

16. 16. The pharmaceutical dosage formulation of any one of claims 1-15, comprising one or more pharmaceutically acceptable excipients selected from a buffering agent or an osmolality adjusting agent.

17. 17. The pharmaceutical dosage formulation of any one of claims 1-16, further comprising a surfactant.

18. 18. The pharmaceutical dosage formulation of any one of claims 1-17, wherein the concentration of the dual peptide agonist is 0.05-20 mg / ml.

19. 19. The pharmaceutical dosage formulation of any one of claims 1-18, wherein the concentration of the dual peptide agonist is 0.1-10 mg / ml.

20. 20. The pharmaceutical dosage formulation of any one of claims 1-19, wherein the dual peptide agonist has a pH between 6-10.

21. 21. The pharmaceutical dosage formulation of any one of claims 1-20, comprising about 0.025-0.15% (w / w) polysorbate 20 or polysorbate 80, about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol in water (pH 7.7±1.0), optionally about 0.050% (w / w) polysorbate 20, about 0.35% (w / w) arginine, about 4.3% (w / w) mannitol in water (pH 7.7±1.0).

22. 21. The pharmaceutical dosage formulation of claims 1-20, comprising about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol, and 0.6-1.0 mg polysorbate 20 or 1.0-1.5 mg polysorbate 80 in water (pH 7.7±1.0) per mg of ALT-801 (SEQ ID NO: 1).

23. 23. The pharmaceutical dosage formulation of any one of claims 1-22, configured to be administered to the mammal, wherein the agonist peptide product is less than about 0.25 mg / kg / dose, optionally greater than about 0.001 mg / kg / dose and less than about 0.15 mg / kg / dose.

24. 24. The pharmaceutical dosage formulation of claim 23, configured to administer less than 0.25 mg / kg / dose of the agonist peptide product to the mammal.

25. 24. The pharmaceutical dosage formulation of claim 23, configured to administer between 0.001-0.15 mg / kg / dose, optionally about 0.03 mg / kg / dose or about 0.10 mg / kg / dose.

26. 26. The pharmaceutical dosage formulation of any one of claims 1-25, configured for administration to a human of between about 0.1 and about 15 mg per week, optionally between about 1-about 7 mg per week, or optionally between about 1-5 mg per week.

27. 27. The pharmaceutical dosage formulation of any one of claims 1-26, adapted to be administered to the mammal once a week for at least 6 weeks, or for up to 6 weeks.

28. 28. The pharmaceutical dosage formulation of any one of claims 1-27, wherein the time to reach a therapeutic dose is about 4 weeks or less.

29. The therapeutic dose is about 10 to about 300 ng / ml of C ｍａｘ , T of about 10 hours to about 36 hours ｍａｘ , and / or approximately 1,000-100,000 h ＊ AUC in ng / mL ０－１６８ 29. The pharmaceutical dosage formulation of claim 28, wherein:

30. 30. A method for lowering blood glucose levels in a mammal, said method comprising administering to the mammal a pharmaceutical dosage formulation according to any one of claims 1-29, said method comprising: g) reducing the incidence of one or more adverse events selected from nausea, vomiting, diarrhea, abdominal pain and constipation when administered to a mammal compared to an agonist with disproportionate affinity for GLP-1R and GCGR; h) produces about 50% lower blood glucose levels at about 48 hours or 96 hours after administration, about 100% lower blood glucose levels at about 72 hours after administration, and / or a reduction in blood glucose levels at about 120 hours after administration compared to administering an approximately equimolar dose of semaglutide; i) induces total body weight loss and / or induces liver weight loss; j) compared to a method in which an approximately equimolar dose of semaglutide is administered, a lower Cmax or optionally about 50% lower Cmax; a Tmax that is about equal to or greater than, or optionally about 100% greater than, Similar AUC(0-inf) or optionally about 85-93% AUC （０－ｉｎｆ） , Approximately equal to or lower T (hours), or optionally about 50-75% of T １／２ (time), Prolonged MRT (hours), or optionally at least about 25% higher MRT (hours), an extended PK / PD profile showing comparable or greater glucoregulatory efficacy; greater total body weight loss, or optionally about twice the total body weight loss; lower body fat mass, optionally a reduction of about 100% body fat mass; and / or when the method is for treating NASH, it results in increased total body weight loss, decreased liver weight, improved NAS score, improved hepatic steatosis, improved ballooning, improved col1A1 staining, improved ALT, improved liver TG / TC, and improved plasma TG / TC; k) resulting in a greater weight loss by about 14 days after administration of the dosage formulation, optionally about 15% greater weight loss, compared to semaglutide administered at an approximately equimolar dose, and / or resulting in a greater weight loss by about 20-28 days after administration of the dosage formulation, optionally about 25% greater weight loss; and / or l) A method which results in weight loss in an obese mammal sufficient to restore said mammal's weight to the normal weight range for a lean, normal mammal.

31. 31. A method for inducing weight loss in a mammal, the method comprising administering to the mammal a pharmaceutical dosage formulation according to any one of claims 1-30, the method reducing the incidence of one or more adverse events compared to an agonist with disproportionate affinity for GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain and constipation upon administration to the mammal.

32. 32. The method of claim 30 or 31, wherein the dual agonist peptide is any one of SEQ ID NOs: 1-10 or 12-27.

33. 32. The method of claim 30 or 31, wherein the dual agonist peptide has approximately equal affinity for GLP-1R and GCGR, and optionally said dual agonist peptide is SEQ ID NO:

1.

34. 32. The method of claim 30 or 31, wherein the pharmaceutical dosage is administered approximately weekly.

35. 35. The method of any one of claims 30-34, wherein the pharmaceutical dose is administered subcutaneously.

36. 36. The method of any one of claims 30 to 35, wherein the pharmaceutical dosage is administered approximately weekly for about 2 weeks to about 8 weeks or more.

37. 37. The method of any one of claims 30-36, wherein administering the pharmaceutical dosage to the mammal as a weekly dose for about 4 to about 8 weeks, optionally about 6 weeks, compared to administration of an approximately equimolar dose of semaglutide, results in a greater reduction in total body weight at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks after administration to the mammal.

38. 38. The method of any one of claims 30-37, comprising administering the agonist peptide product to the mammal at less than about 0.25 mg / kg / dose, optionally greater than about 0.001 mg / kg / dose to less than about 0.15 mg / kg / dose.

39. 39. The method of claim 38, wherein the mammal is administered less than about 0.25 mg / kg / dose.

40. 40. The method of claims 30-39, wherein the agonist peptide product is administered at between 0.001-0.15 mg / kg / dose, optionally at about 0.03 mg / kg / dose or about 0.10 mg / kg / dose.

41. 41. The method of any one of claims 30-40, wherein each dose is administered about once a week or once every two weeks, optionally for at least a month, and optionally each dose contains approximately the same amount of agonist peptide product.

42. 42. The method of any one of claims 30-41, comprising administering a single dose of less than about 0.25 mg / kg / dose, followed by one or more subsequent doses of from about 0.03 mg / kg / dose to about 0.10 mg / kg / dose.

43. 43. The method of any one of claims 30-42, comprising administering between 0.001-0.15 mg / kg / dose of the agonist peptide product.

44. 44. The method of any one of claims 30-43, wherein the pharmaceutical dosage formulation comprises about 0.025-0.15% (w / w) polysorbate 20 or polysorbate 80, about 0.2-0.5% (w / w) arginine, and about 3-6% (w / w) mannitol in water (pH 7.7±1.0), optionally about 0.050% (w / w) polysorbate 20, about 0.35% (w / w) arginine, and about 4.3% (w / w) mannitol in water (pH 7.7±1.0), and optionally wherein the dual agonist peptide is SEQ ID NO:

1.

45. 45. The method of any one of claims 30-44, wherein the formulation comprises about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol, and 0.6-1.0 mg polysorbate 20 or 1.0-1.5 mg polysorbate 80 in water (pH 7.7±1.0) per mg of ALT-801 (SEQ ID NO: 1).

46. 46. ​​The method of any one of claims 30-45, wherein the administration of the pharmaceutical dosage formulation is configured for administration to a human at between about 0.1 to about 15 mg per week, optionally at about 1 to about 7 mg per week, or optionally at about 1 to 5 mg per week.

47. 47. The method of any one of claims 30-46, wherein the time to reach a therapeutic dose is about 4 weeks or less.

48. 1. A pharmaceutical dosage formulation configured for subcutaneous administration comprising an agonist peptide product having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), said peptide product being represented as SEQ ID NO: 1, said dosage being configured to improve glycemic control with a reduction in one or more adverse events compared to an agonist having disproportionate affinity for GLP-1R and GCGR, said adverse events being selected from nausea, vomiting, diarrhea, abdominal pain and constipation upon administration to a mammal.

49. 1. A pharmaceutical dosage formulation configured for subcutaneous administration comprising an agonist peptide having affinity for the glucagon-like peptide 1 receptor (GLP-1R) and the glucagon receptor (GCGR), wherein the peptide product is represented by SEQ ID NO: 1, and wherein the dosage is configured to induce weight loss with reduced one or more adverse events compared to an agonist having disproportionate affinity for GLP-1R and GCGR, wherein the adverse events are selected from nausea, vomiting, diarrhea, abdominal pain, and constipation upon administration to a mammal.

50. 50. The pharmaceutical dosage formulation of claim 49, wherein the weight loss is at least 5%, at least 10%, or about 1% to about 20%, or about 5% to about 10% (w / w).

51. 51. The pharmaceutical dosage formulation of any one of claims 48-50, wherein the dosage is administered as a once-weekly dosage, optionally for about 2 weeks to about 8 weeks of administration.

52. 52. The pharmaceutical dosage formulation of any one of claims 48-51, wherein the formulation comprises about 0.2-0.5% (w / w) arginine, about 3-6% (w / w) mannitol, and 0.6-1.0 mg polysorbate 20 or 1.0-1.5 mg polysorbate 80 in water (pH 7.7±1.0) per mg of ALT-801 (SEQ ID NO: 1).

53. Administration of a single dose to a mammal results in a lower C at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration compared to administration of an approximately equimolar dose of semaglutide. ｍａｘ 52. The pharmaceutical dosage formulation of claim 51, wherein:

54. 54. The pharmaceutical dosage formulation of any one of claims 48-53, wherein the dosage is designed for administration to a human at between about 0.1 mg and about 15 mg per week, optionally about 1 to about 7 mg per week, or optionally about 1 to 5 mg per week.

55. 55. The pharmaceutical dosage formulation of any one of claims 48-54, adapted to be administered to the mammal once a week for at least 6 weeks, or for up to 6 weeks.

56. 56. The pharmaceutical dosage formulation of any one of claims 48-55, wherein the dosage is designed to reach a therapeutic dose within about 4 weeks after the first weekly administration.

57. The therapeutic dose is from about 10 to about 300 ng / ml of C ｍａｘ , optionally a C of less than 200 ng / ml ｍａｘ , T from about 10 hours to about 36 hours ｍａｘ , and / or approximately 1,000-100,000 h ＊ AUC in ng / mL ０－１６８ 57. The pharmaceutical dosage formulation of claim 56, wherein:

58. 58. A method of inducing weight loss in a mammal, comprising administering to the mammal the pharmaceutical dosage formulation of any one of claims 48-57, wherein the method reduces the incidence of one or more adverse events compared to an agonist with disproportionate affinity for GLP-1R and GCGR, the adverse events being selected from nausea, vomiting, diarrhea, abdominal pain and constipation upon administration to the mammal at a therapeutic dose.

59. 59. The method of claim 58, wherein the pharmaceutical dosage is administered approximately weekly and the initial dose is a therapeutic dose.

60. 60. The method of any one of claims 58 or 59, wherein the pharmaceutical dosage is administered about weekly for about 2 weeks to about 8 weeks or longer.

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