Peptide conjugates and methods of use

Abstract

Disclosed herein are peptide conjugates comprising a peptide selected from a peptide that modulates a PYY receptor, a peptide that modulates both a GLP-1 receptor and a GCG receptor, a peptide that modulates both a GLP-1 receptor and a GIP receptor, and a peptide that modulates a GLP-1 receptor, and a staple attached to the peptide at the first and second amino acids. Also provided are peptide conjugates comprising prolactin-releasing peptides. The peptide conjugates can be used to treat diseases such as obesity. Additionally, stapled prolactin-releasing peptides are provided.

Description

[Technical Field]

[0001] cross reference This application claims the benefits under U.S. Provisional Patent Application No. 62 / 943,667 filed on 4 December 2019 and U.S. Provisional Patent Application No. 62 / 994,791 filed on 25 March 2020, which are incorporated herein by reference in their entirety. [Background technology]

[0002] The development of therapeutic agents is often hindered by short half-lives. The biological half-life of a drug is the time it takes for the drug to lose half of its pharmacological, physiological, or radiological activity. As a result, patients are often administered higher doses of the agent more frequently, which can lead to decreased compliance, increased costs, and a higher risk of side effects. Therefore, there is a need to develop therapeutic agents with extended half-lives. [Overview of the project]

[0003] This specification includes, a) A peptide selected from a peptide that modulates the PYY receptor, a peptide that modulates both the GLP-1 receptor and the GCG receptor, a peptide that modulates both the GLP-1 receptor and the GIP receptor, and a peptide that modulates the GLP-1 receptor, b) Staples that are bound to the peptide by a first sulfhydryl-containing amino acid and a second sulfhydryl-containing amino acid A peptide conjugate containing, The staple is given by formula (I):

[0004] [ka] It is, During the ceremony, A is -N-, X 1 and X 2is a single bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene-, -alkylene-C(=O)NR 3 -, -alkylene-NR 3 C(=O)-, -C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-, -alkylene-C(=O)NR 3 -alkylene-, or -alkylene-NR 3 is C(=O)-alkylene-, where X 1 is bonded to the sulfhydryl-containing amino acid of the peptide, X 2 is bonded to the sulfhydryl-containing amino acid of the peptide, X 1 and X 2 are the same, R is hydrogen or -(L) s -Y, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-, -NR 3 C(=O)NR 3 -, -NR 3 C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-NR 3 -, -alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)-, or -NR 3 is C(=O)-alkylene-, v is 2 to 20, R 1 or R2 These are hydrogen, halogen, -CN, and -OR, respectively, independently. a , -SR a -S(=O)R b -NO2, -NR c R d -S(=O)2R d , -NR a S(=O)2R d -S(=O)2NR c R d -C(=O)R b -OC(=O)R b , -CO2R a , -OCO2R a -C(=O)NR c R d -OC(=O)NR c R d , -NR a C(=O)NR c R d , -NR a C(=O)R b , -NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , or -NR c R d It is replaced by one, two, or three of the following, or R 1 and R 2 These combine to form a C1-C6 cycloalkyl or C1-C6 heterocycloalkyl group. Each R 3 These are hydrogen and -S(=O)R, which are independent of each other. b -S(=O)2R a-S(=O)2NR c R d -C(=O)R b , -CO2R a -C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , or -NR c R d It is replaced by one, two, or three of the following, Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl). s is between 0 and 20. R a is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. R bis a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogens, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogens, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2, and R c and R d Each is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. Or R c and R d A peptide conjugate is disclosed in which these molecules, together with the nitrogen atom to which they are bonded, form a heterocycloalkyl or heteroaryl molecule, where the heterocycloalkyl and heteroaryl molecules are optionally substituted with one, two, or three of the following: halogens, C1-C6 alkyl groups, C1-C6 haloalkyl groups, -OH groups, -OMe groups, or -NH2 groups.

[0005] This specification also includes, a) A peptide that modulates the PYY receptor, comprising a peptide sequence having at least approximately 95% identity with one of sequence numbers 3, 5, 6, 8, 14-30, 36, or 37, b) Staples that are bound to the peptide at the first and second amino acids Peptide conjugates containing the same are also disclosed.

[0006] This specification also includes, a) A peptide that modulates both the GLP-1 receptor and the GCGR receptor, comprising a peptide sequence having at least approximately 95% identity with any one of sequence numbers 50-59, b) Staples that are bound to the peptide at the first and second amino acids Peptide conjugates containing the same are also disclosed.

[0007] This specification also includes, a) A peptide that modulates both the GLP-1 receptor and the GIP receptor, comprising a peptide sequence having at least approximately 95% identity with any one of sequence numbers 62-71, b) Staples that are bound to the peptide at the first and second amino acids Peptide conjugates containing the same are also disclosed.

[0008] This specification also includes, a) A peptide that modulates the GLP-1 receptor, comprising peptide sequences having at least approximately 95% identity with SEQ ID NOs. 74 and 79, b) Staples that are bound to the peptide at the first and second amino acids Peptide conjugates containing the same are also disclosed.

[0009] This specification also provides pharmaceutical compositions comprising the peptide conjugate described herein and pharmaceutically acceptable excipients.

[0010] Furthermore, this specification also provides a method for treating a target disease or illness, comprising the step of administering a composition containing a therapeutically effective amount of the peptide conjugate described herein to the target. [Brief explanation of the drawing]

[0011] [Figure 1A] This figure shows the dose-response curves for PYY analogs symmetrically stapled in the absence of fetal bovine serum. [Figure 1B] This figure shows the dose-response curves for PYY analogs symmetrically stapled in the presence of fetal bovine serum (10%). [Figure 2] This figure illustrates the pharmacokinetics of PYY2, conjugate 40, and conjugate 62 in rats. [Figure 3A] This figure illustrates the changes in food intake over 24 hours in mice treated with a single dose of conjugate 187, conjugate 40, a combination of conjugate 187 and 40, or the vehicle alone. [Figure 3B] This figure illustrates the 24-hour body weight change in mice treated with a single dose of conjugate 187, conjugate 40, a combination of conjugate 187 and 40, or the vehicle alone. [Figure 4A] This figure illustrates the changes in food consumption on day 1 in mice with diet-induced obesity treated with a single dose of conjugate 187, conjugate 40, a combination of conjugate 187 and 40, or vehicle alone. [Figure 4B] This figure illustrates the changes in food consumption on day 5 in diet-induced obese mice treated with a single dose of conjugate 187, conjugate 40, a combination of conjugate 187 and 40, or vehicle alone. [Figure 4C] This figure illustrates the 2-week weight change in diet-induced obesity mice treated with conjugate 187, conjugate 40, a combination of conjugate 187 and 40, or a vehicle. [Figure 4D] This figure illustrates blood glucose levels 14 days after oral glucose tolerance testing in diet-induced obese mice treated with conjugate 187, conjugate 40, a combination of conjugate 187 and 40, or vehicle alone. [Figure 4E]Figure showing the area under the curve 14 days after treatment in an oral glucose tolerance test performed on diet-induced obese mice treated with conjugate 187, conjugate 40, a combination of conjugates 187 and 40, or vehicle alone. [Figure 4F] Figure showing fasting blood glucose 14 days after treatment in diet-induced obese mice treated with conjugate 187, conjugate 40, a combination of conjugates 187 and 40, or vehicle. [Figure 5A] Figure representing the dose-response curves of glucagon, semaglutide, conjugate 122, and conjugate 135 for GLP-1R. [Figure 5B] Figure representing the dose-response curves of glucagon, semaglutide, conjugate 12 and conjugate 135 for GCGR. [Figure 5C] Figure showing the stability of conjugates 122 and 135 over 50 hours in 2% plasma. [Figure 6A] Figure showing the pharmacokinetics of conjugate 135 when administered intravenously and subcutaneously. [Figure 6B] Figure showing the pharmacokinetics of conjugate 122 when administered intravenously and subcutaneously. [Figure 6C] Figure showing the pharmacokinetics of peptide 14 when administered intravenously and subcutaneously to mice. <000090> [Figure 6D] Figure showing the pharmacokinetics of peptide 183 when administered intravenously and subcutaneously to mice. [Figure 7A] Figure showing the time-dependent effect on blood glucose levels 6 hours after administration of the compound in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: cotadutide, E: semaglutide. [Figure 7B] Figure showing the time-dependent effect on blood glucose levels 48 hours after administration of the compound in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: cotadutide, E: semaglutide. [Figure 7C]This figure shows the time course effect of compounds on blood glucose levels 96 hours after administration in an oral glucose tolerance test. A: 122 and E: semaglutide. [Figure 7D] This figure shows the treatment effect on blood glucose levels measured by the area under the curve (AUC) 6 hours after administration of a compound in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: Cotadutide, E: Semaglutide. [Figure 7E] This figure shows the treatment effect on blood glucose levels, measured by the area under the curve (AUC) 48 hours after administration of a compound in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: Cotadutide, E: Semaglutide. [Figure 7F] This figure shows the treatment effect on blood glucose levels, measured by the area under the curve (AUC) 96 hours after administration of a compound in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: Cotadutide, E: Semaglutide. [Figure 7G] This figure shows the effect of compounds on fasting blood glucose levels 6 hours after administration in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: Cotadutide, E: Semaglutide. [Figure 7H] This figure shows the effect of compounds on fasting blood glucose levels 48 hours after administration in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: Cotadutide, E: Semaglutide. [Figure 7I] This figure shows the effect of compounds on fasting blood glucose levels 96 hours after administration in an oral glucose tolerance test. A: 122, B: 135, C: 138, D: Cotadutide, E: Semaglutide. [Figure 8A] This figure shows the results of GLP1 receptor activation reporter assays for tilzepatide, NNC0090-2746, conjugates 142, 141, and 171. [Figure 8B] This figure shows the results of GIP receptor activation reporter assays for tilzepatide, NNC0090-2746, conjugates 142, 141, and 171. [Figure 9A]This figure shows the time course of treatment effects at 2 hours after administration of compounds in an oral glucose tolerance test, compared to a vehicle control. A:141, B:171, C:142, D:Cotadutide, E:Tilzepatide. [Figure 9B] This figure compares the time course of treatment effects 72 hours after administration of compounds in an oral glucose tolerance test with those of a vehicle control. A:141, B:171, C:142, D:Cotadutide, E:Tilzepatide. [Figure 9C] This figure shows the time course of treatment effects 96 hours after administration of compounds in an oral glucose tolerance test, compared to the vehicle control. A:141, B:171, C:142, D:Cotadutide, E:Tilzepatide. [Figure 9D] This figure shows the time course of treatment effects 144 hours after administration of compounds in an oral glucose tolerance test, compared to the vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9E] This figure compares the treatment effect of compounds on blood glucose levels, measured by the area under the curve (AUC) 2 hours after administration in an oral glucose tolerance test, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9F] This figure compares the treatment effect of compounds on blood glucose levels, measured by the area under the curve (AUC) 72 hours after administration in an oral glucose tolerance test, with that of a vehicle control. In the entire figure, A:141, B:171, C:142, D:cotadutide, E:chirzepatide. [Figure 9G] This figure compares the treatment effect of compounds on blood glucose levels, measured by the area under the curve (AUC) 96 hours after administration in an oral glucose tolerance test, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9H] This figure compares the treatment effect on blood glucose levels, measured by the area under the curve (AUC) 144 hours after administration of a compound in an oral glucose tolerance test, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9I]This figure compares the treatment effect on fasting blood glucose, measured by the area under the curve (AUC) at 2 hours after administration of each compound, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9J] This figure compares the treatment effect on fasting blood glucose, measured by the area under the curve (AUC) 72 hours after administration of each compound, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9K] This figure compares the treatment effect on fasting blood glucose, measured by the area under the curve (AUC) 96 hours after administration of each compound, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 9L] This figure compares the treatment effect on fasting blood glucose, measured by area under the curve (AUC) 144 hours after administration of each compound, with that of a vehicle control. A: 141, B: 171, C: 142, D: Cotadutide, E: Chilzepatide. [Figure 10A] This figure shows the change in body weight of mice administered a peptide or vehicle control subcutaneously once or twice daily. Gray arrows indicate the number of days with once-daily administration of the compound, and black arrows indicate the number of days with twice-weekly administration. A: 142 (7x / wk), B: 142 (2x / wk), C: Tilzepatide (2x / wk), D: Semaglutide (2x / wk). [Figure 10B] This figure shows the percentage change in body weight of mice administered a peptide or vehicle control subcutaneously once or twice daily. Gray arrows indicate the number of days with once-daily administration of the compound, and black arrows indicate the number of days with twice-weekly administration. A: 142 (7x / wk), B: 142 (2x / wk), C: Tilzepatide (2x / wk), D: Semaglutide (2x / wk). [Figure 10C]This is a graph showing the incremental food intake of mice over 7 days after subcutaneous administration of a peptide or vehicle control. The gray arrow indicates the number of days of once-daily administration of the compound, and the black arrow indicates the number of days of twice-weekly administration. A: 142 (7x / wk), B: 142 (2x / wk), C: Tirzepatide (2x / wk), D: Semaglutide (2x / wk). [Figure 10D] This is a graph showing the results of the compound on blood glucose levels over time in an oral glucose tolerance test (OGTT). A: 142 (7x / wk), B: 142 (2x / wk), C: Tirzepatide (2x / wk), D: Semaglutide (2x / wk). [Figure 10E] This is a graph showing the results of the compound on blood glucose levels measured by the area under the curve (AUC) in an oral glucose tolerance test (OGTT). A: 142 (7x / wk), B: 142 (2x / wk), C: Tirzepatide (2x / wk), D: Semaglutide (2x / wk). [Figure 10F] This is a graph showing the results of the compound on fasting blood glucose on day 8 in an oral glucose tolerance test (OGTT). A: 142 (7x / wk), B: 142 (2x / wk), C: Tirzepatide (2x / wk), D: Semaglutide (2x / wk). [Figure 11A] This is a graph showing the incremental food intake of mice administered a peptide or vehicle daily. A: 122, B: 142, C: Semaglutide, D: Cotadutide. [Figure 11B] This is a graph showing the change in body weight of mice treated with a peptide or vehicle control over 21 days. A: 122, B: 142, C: Semaglutide, D: Cotadutide. [Figure 11C] This is a graph showing the rate of change in body weight of mice treated with a peptide or vehicle control over 21 days. A: 122, B: 142, C: Semaglutide, D: Cotadutide. [Figure 11D] This is a graph comparing the effect of the compound on the feeding plasma glucose excursion on day 20 with the vehicle control. A: 122, B: 142, C: Semaglutide, D: Cotadutide. [Figure 11E]This figure compares the effect of compounds on fasting plasma glucose excursion on day 20 with that of a vehicle control. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11F] This figure compares the treatment effects of the compounds in the oral glucose tolerance test (OGTT) on day 21 with those of the vehicle control. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11G] This graph shows blood glucose levels determined by measuring the area under the curve (AUC). A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11H] This figure shows the effect of compound treatment on plasma levels of aspartate aminotransferase (AST). A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11I] This figure shows the effect of compound treatment on plasma levels of alanine aminotransferase (ALT). A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11J] This figure shows the effect of compound treatment on plasma alkaline phosphatase (ALP) levels. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11K] This diagram illustrates the effect of various compounds on plasma cholesterol levels. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11L] This diagram shows the therapeutic effects of various compounds on plasma triglyceride levels. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11M] This figure shows the effect of each compound and vehicle on the ratio of liver to body weight. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11N] This figure shows the effect of the compound and vehicle control on fat mass. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11O]This figure shows the effect of each compound and vehicle on liver weight. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11P] This figure shows the therapeutic effects of compounds and vehicle controls on liver triglyceride levels. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 11Q] This figure shows the therapeutic effects of compounds and vehicle controls on hyperlipidemia grades. A: 122, B: 142, C: semaglutide, D: cotadutide. [Figure 12] This figure shows the in vitro plasma stability of PrRP31, conjugate 255 (97-L3), and conjugate 263 (97-L5). [Figure 13] This figure shows the plasma concentration of conjugate 263(97-L5) after a single subcutaneous injection of 1 mg / kg into mice. [Figure 14] This figure shows the plasma concentration of conjugate 263(97-L5) after a single subcutaneous injection of 5 mg / kg into female CD-1 mice (n=4). [Figure 15] This figure shows a 12-day weight test of a diet-induced obesity (DIO) mouse model (n=8 per group) that received daily subcutaneous administration of conjugate 263(97-L5). [Figure 16] This figure shows the weight loss over 12 days in DIO model mice (n=8 per group) that were administered conjugate 263(97-L5) subcutaneously daily (0.5 and 5 mg / kg). [Figure 17] This figure shows blood glucose levels in diet-induced obesity (DIO) model mice that were fasted on day 14 of the in vivo weight study, after oral administration of PrRP31 and conjugate 263(97-L5). [Figure 18] This figure shows the AUC after oral administration of PrRP31 and conjugate 263(97-L5) to diet-induced obesity (DIO) model mice that were fasted on day 14 of the in vivo body weight study. [Modes for carrying out the invention]

[0012] Peptide YY (PYY) and glucagon-like peptide (GLP)-1 are peptides secreted from intestinal L cells in response to food. Increased plasma levels of each peptide have been shown to reduce appetite and inhibit food intake. In rodents administered PYY and GLP-1 compounds, an additive effect on feeding inhibition was observed in individuals administered either compound. Such additive feeding inhibition was also observed in genetically obese models, ob / ob mice and db / db mice. Additional studies in healthy human volunteers also demonstrated an additive effect of PYY and GLP-1 compounds on reducing energy intake at meals (at a buffet) by 27%. This reduction in energy intake was greater in individuals administered in combination with PYY or GLP-1 than in individuals administered with either compound alone. Therefore, PYY, when randomly combined with GLP-1 or a similar compound, is a promising therapeutic agent for treating weight loss-related diseases.

[0013] The neuropeptide Y family modulates signaling between the brain and gut via neuropeptide Y receptors and includes peptides PYY, NPY (neuropeptide Y), and PP (pancreatic polypeptide). PYY is spontaneously secreted and is a 36-amino acid peptide PYY(1-36) that is cleaved into PYY(3-36). However, PYY(3-36) is rapidly eliminated, and its half-life in pigs has been reported to be less than 30 minutes. Therefore, the pharmacokinetic properties of spontaneously occurring PYY compounds are suboptimal for therapeutic use.

[0014] G protein-binding receptors (GPCRs) are membrane-bound proteins with seven transmembrane domains linked by three intracellular loops and three extracellular loops. Their ligand-binding sites are highly specific, ensuring that each receptor responds only to a limited range of chemicals with high affinity. Examples of GPCR ligands include peptides, proteins, lipid-derived molecules, small organic compounds, and ions. GPCRs have long been of interest as drug targets due to their involvement in excessive pathophysiological processes, including neuronal excitability, metabolism, replication, hormonal homeostasis, and behavior. It is estimated that approximately 34% of the U.S. Food and Drug Administration (FDA) has approved 108 drug targets from the GPCR family. GPCRs are generally classified into several superfamilies. Family B GPCRs, the so-called secretin receptor family, represent a small but structurally and functionally diverse set of receptors. These proteins are essential for many physiological functions and serve as important drug targets in various human diseases, including type 2 diabetes mellitus (T2DM), migraines, osteoporosis, depression, and anxiety disorders. Members of this family include polypeptide hormone receptors ranging in length from 27 to 141 residues. Nine of these receptors are targeted by structurally related ligands, including glucagon-like peptides (GLP-1 and GLP-2), glucagon, glucose-dependent insulin-secreting polypeptide (GIP), vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating polypeptide (PACAP), and growth hormone-releasing hormone (GHRH).

[0015] Glucagon-like peptide-1 (GLP-1) is a naturally occurring incretin hormone released into circulation by L cells in the intestines in response to nutrient intake. By binding to its homologous receptor (GLP-1R), GLP-1 can stimulate insulin secretion while suppressing glucagon secretion. However, only when blood glucose levels are elevated, this can potentially lower plasma glucose levels and reduce the risk of hypoglycemia. Furthermore, GLP-1 can lead to weight loss by slowing gastric emptying and suppressing appetite.

[0016] GLP-1 receptor agonists (GLP-1RAs) represent a unique approach to the treatment of diabetes, with benefits exceeding glycemic control and including favorable effects on body weight, blood pressure, cholesterol levels, and β-cell function. Two short-acting GLP-1RAs (exenatide and liraglutide, administered once or twice daily) and three long-acting GLP-1RAs (albiglutide, dulaglutide, and exenatide LAR, administered weekly) are currently approved in the United States. In particular, exenatide, a GLP-1 analog initially isolated from the saliva of the Gila monster lizard, has a half-life of 30 minutes after intravenous administration in humans, while exenatide has a half-life of 2-3 hours. These drugs mimic the effects of the naturally occurring incretin hormone GLP-1 by activating GLP-1 receptors in the pancreas, thereby resulting in glucose-dependent increased insulin release and decreased glucagon release. As a result, the risk of hypoglycemia is low. The effects of these GLP-1RAs on GLP-1 receptors in the CNS and gastrointestinal tract result in decreased appetite accompanied by weight loss and delayed glucose absorption. Considering their limited oral bioavailability, these GLP-1RAs a It is currently administered by subcutaneous injection. In some embodiments, the Specified Provisions provide GLP-1RAs that are coupled to fatty acid-derived side chain staples to increase their half-life.

[0017] Incretin peptides are effective therapeutic agents for treating type 2 diabetes mellitus (T2DM). Oxintomodulin (OXM), i.e., a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose-lowering effects compared to GLP-1R agonists alone. To overcome the short half-life and rapid renal clearance that limit OXM's potential as a therapeutic agent, OXM analogs modified with both lipids and PEG have been reported. However, these methods often result in reduced potency or PEG-related toxicity. In one embodiment, this specification provides a GLP-1R and GCGR dual agonist in which activation of both GLP-1R and GCGR increases plasma stability and enhances potency.

[0018] GIP is also characterized as an incretin that stimulates insulin secretion in a glucose-dependent manner. Dual agonists of GIP and the GLP-1 receptor have been shown to reduce fasting serum glucose and decrease body weight compared to placebo. This dual agonist, namely LY3298176, is administered subcutaneously once weekly. In some embodiments, the specification further provides GIPR and GLP-1R dual agonists that include features stapled to increase serum stability and half-life.

[0019] Prolactin-releasing peptide (PrRP) was initially discovered in the hypothalamus as a novel peptide that stimulates prolactin secretion in anterior pituitary cells via activation of the orphan G protein-binding receptor human taste receptor 3 (Gr3) and its rat ortholog, the previously unknown hypothalamic receptor-1 (UHR-1). However, later reports have shown that PrRP does not stimulate the secretion of prolactin or other pituitary hormones, but rather acts mostly as a neuromodulator, potentially playing a crucial role in regulating energy balance through activation of the prolactin-releasing peptide receptor, also known as G protein-binding receptor 10 (GPR10, identical to hGr3). PrRP reduces body weight and food intake and regulates body temperature upon central administration, suggesting a role in energy homeostasis. The anorexia-inducing effect of PrRP is mediated by the corticotropin-releasing hormone (CRH) receptor and also interacts with leptin to reduce food intake and body weight. PrRP-deficient mice exhibit delayed-onset obesity and steatosis, suggesting that PrRP relays satiety signals in the brain. Disruption of PrRP receptor signaling may lead to obesity and metabolic abnormalities. Therefore, PrRP may offer potential as a therapeutic agent for diabetes and obesity, utilizing its appetite-stimulating properties in reducing food intake and weight loss.

[0020] However, central administration of PrRP significantly increases myocardial contractility, heart rate, and blood pressure. PrRP belongs to the RF amide peptide family and, in addition to activating GPR10, exhibits high affinity for NPFF2R (neuropeptide FF receptor 2 or GPR74). NPFF2R signaling exerts an additional anorexic effect that may enhance those mediated by GPR10, but NPFF2R is associated with increased arterial blood pressure and may contribute to PrRP-induced cardiovascular effects. PrRP causes increases in arterial blood pressure and heart rate, which may be abolished by co-administration of RF9, a specific NPFF2R antagonist, rather than neuropeptide Y, a putative GPR10 antagonist. Direct conjugation of palmitic acid to the N-terminus of PrRP via the Lys side chain at position 11 significantly extends the half-life and in vivo anorexia-inducing effect, reducing food intake, body weight, and glucose tolerance abnormalities in obese rat and mouse models. Regardless of the central nervous system benefits after peripheral administration, palmitoylated PrRP analogs are thought to demonstrate increased activity against NPFF2R. Therefore, there is a need to express a GPR10-selective PrRP analog that reduces their activity against NPFF2R jaw syndrome and its associated cardiovascular risks while retaining anorexia and anti-diabetic effects.

[0021] This specification provides peptide conjugates, including therapeutic peptides stapled to molecules, such as half-life extension molecules.

[0022] In some embodiments, the stapled peptide comprises an incretin peptide or an incretin peptide mimetic. Since incretin peptides generally bind to their homologous receptors in an α-helical configuration, some embodiments herein provide modifications that stabilize the α-helix, which may in some cases increase their binding affinity to their receptors. Furthermore, proteolytic stability may also be enhanced in the helical configuration rather than the expanded configuration. In some embodiments, conjugated peptides with increased circulating half-life and potency to their homologous receptors are provided herein.

[0023] In some embodiments, this specification describes peptide manipulation strategies used to produce stapled, long-acting peptide analogs with potency comparable to natural peptides and significantly enhanced pharmacokinetic properties.

[0024] Peptides that regulate the PYY receptor In one embodiment, the Specified Information provides peptide conjugates comprising peptides specific to derivatives of neuropeptide Y family receptors (the NPY family of biologically active peptides, NPY, peptide YY (PYY), and pancreatic polypeptide (PP)). In one embodiment, the Specified Information provides peptide conjugates comprising peptides that modulate the PYY receptor. In some embodiments, the peptides that modulate the PYY receptor are PYY receptor agonists.

[0025] The binding affinity of the peptide conjugate described herein may be within approximately 5% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 10% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 15% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 20% of the binding affinity of the unmodified peptide.

[0026] In some cases, NPY derivatives represent PYY derivatives. In some cases, NPY derivatives represent PYY derivatives that have an amino acid sequence of 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 10 amino acids, unlike PYY having SEQ ID NO: 1 or 2.

[0027] An NPY derivative, for example a PYY derivative, may contain one or more sulfhydryl-containing amino acid residues. One or more sulfhydryl-containing amino acid residues may be used to staple to the PYY. One or more sulfhydryl-containing amino acid residues may be used to staple to the NPY derivative. One or more sulfhydryl-containing amino acid residues may occur spontaneously in the NPY derivative. One or more sulfhydryl-containing amino acid residues may be inserted into the PYY derivative. One or more sulfhydryl-containing amino acid residues may replace one or more amino acid residues in the PYY derivative. Methods of amino acid substitution and / or insertion are known in the art.

[0028] NPY derivatives, such as PYY derivatives, may contain one or more amine-containing residues. Non-limiting examples of amine-containing residues include lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine. One or more amine-containing residues may be used to connect staples to the PYY derivative. One or more amine-containing residues may be used to connect HEMs to the PYY derivative. One or more amine-containing residues may occur spontaneously in the PYY derivative. One or more amine-containing residues may be inserted into the PYY derivative. One or more amine-containing residues may replace one or more amino acid residues in the PYY derivative.

[0029] NPY derivatives, such as PYY derivatives, may contain at least a portion of a wild-type peptide that includes one or more amino acid mutations. These one or more amino acid mutations may include deletions, substitutions, additions, or combinations thereof. One or more amino acid mutations may include the addition of one or more amino acid residues to the wild-type peptide. One or more amino acid mutations may include the deletion of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the substitution of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more cysteine, lysine, or other sulfhydryl or amine-containing residues. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more D-amino acid residues. One or more amino acid residues from the wild-type peptide may include one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0030] NPY derivatives, such as PYY derivatives, may be modified by, for example, acetylation, phosphorylation, and methylation. Peptide modifications may include chemical modifications. Peptide modifications may occur at the N-terminus of a peptide. Peptide modifications may include acetylation of an amino group at the N-terminus of a peptide. Alternatively or additionally, peptide modifications may occur at the C-terminus of a peptide. Peptide modifications may occur at one or more internal amino acids of a peptide. Peptide modifications may include replacement of a carboxyl group at the C-terminus of a peptide. Peptide modifications may include modification of a carboxyl group at the C-terminus of a peptide. The carboxyl group at the C-terminus of a peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of a peptide may be modified to produce an amine group.

[0031] In some embodiments, the peptide derivative may be a PYY modified with D-serine instead of L-serine. In some embodiments, the peptide derivative may be a PYY modified with aminoisobutyric acid [Aib] instead of L-serine. In some embodiments, the peptide derivative may be a PYY modified with norleucine [Nle] instead of leucine (Leu).

[0032] In some embodiments, the peptide modulating the PYY receptor comprises a truncated wild-type 36-amino acid PYY peptide. In some embodiments, the N-terminus is truncated by 1, 2, 3, or 4 residues. In some embodiments, the N-terminus is truncated by 2 residues. The PYY derivative may contain a peptide sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1 or 2. The peptide derivative may have an amino acid sequence that is at least 80% identical to either SEQ ID NO: 1 or 2. The peptide modulating the PYY receptor may contain a peptide sequence that is at least about 90% identical to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 91% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 92% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 93% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 94% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 95% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 96% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 97% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 98% identical peptide sequences to SEQ ID NO: 1 or 2. In some cases, the peptide that modulates the PYY receptor contains at least approximately 99% identical peptide sequences to SEQ ID NO: 1 or 2.

[0033] In some embodiments, the peptide modulating the PYY receptor contains one of the peptide sequences of SEQ ID NOs: 3-45. In some cases, the peptide modulating the PYY receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs: 3-45. In some cases, the peptide modulating the PYY receptor contains at least about 90% identical peptide sequences to one of SEQ ID NOs: 3-45. In some cases, the peptide modulating the PYY receptor contains at least about 95% identical peptide sequences to one of SEQ ID NOs: 3-45. In some cases, the peptide modulating the PYY receptor contains at least about 99% identical peptide sequences to one of SEQ ID NOs: 3-45. In some cases, the peptides that modulate the PYY receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 3-45.

[0034] In some embodiments, the peptide that modulates the PYY receptor contains the peptide sequence that is SEQ ID NO: 6. In some cases, the peptide that modulates the PYY receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to SEQ ID NO: 6. In some cases, the peptide that modulates the PYY receptor contains at least about 90% identical peptide sequence to SEQ ID NO: 6. In some cases, the peptide that modulates the PYY receptor contains at least about 95% identical peptide sequence to SEQ ID NO: 6. In some cases, the peptide that modulates the PYY receptor contains at least about 99% identical peptide sequence to SEQ ID NO: 6. In some cases, the peptides that modulate the PYY receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 6.

[0035] In some embodiments, the peptide that modulates the PYY receptor comprises the peptide sequence that is SEQ ID NO: 10. In some cases, the peptide that modulates the PYY receptor comprises at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to SEQ ID NO: 10. In some cases, the peptide that modulates the PYY receptor comprises at least about 90% identical peptide sequence to SEQ ID NO: 10. In some cases, the peptide that modulates the PYY receptor comprises at least about 95% identical peptide sequence to SEQ ID NO: 10. In some cases, the peptide that modulates the PYY receptor comprises at least about 99% identical peptide sequence to SEQ ID NO: 10. In some cases, the peptides that modulate the PYY receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 10.

[0036] In some embodiments, the peptide that modulates the PYY receptor includes the peptide sequence that is SEQ ID NO: 20. In some cases, the peptide that modulates the PYY receptor includes at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to SEQ ID NO: 20. In some cases, the peptide that modulates the PYY receptor includes at least about 90% identical peptide sequence to SEQ ID NO: 20. In some cases, the peptide that modulates the PYY receptor includes at least about 95% identical peptide sequence to SEQ ID NO: 20. In some cases, the peptide that modulates the PYY receptor includes at least about 99% identical peptide sequence to SEQ ID NO: 20. In some cases, the peptides that modulate the PYY receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 20.

[0037] In some cases, the PYY derivative is numbered as position 36, along with the last amino acid in the sequence.

[0038] Table 1 shows non-restrictive examples of peptide derivatives.

[0039] [Table 1-1]

[0040] [Table 1-2]

[0041] Peptides that regulate both the GLP-1 receptor and the GCG receptor. In one embodiment, the Specified Reference Indicators provides peptide conjugates comprising peptides that modulate the GLP-1 receptor and / or the GCG receptor. In some embodiments, the peptide modulates both the GLP-1 receptor and the GCG receptor. In some embodiments, the peptide that modulates the GLP-1 receptor is a GLP-1 receptor agonist. In some embodiments, the peptide that modulates the GCG receptor is a GCG receptor agonist.

[0042] The binding affinity of the peptide conjugate described herein may be within approximately 5% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 10% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 15% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 20% of the binding affinity of the unmodified peptide.

[0043] Peptides that modulate both the GLP-1 receptor and the GCG receptor may contain one or more sulfhydryl-containing amino acid residues. One or more sulfhydryl-containing amino acid residues may be used to connect staples. One or more sulfhydryl-containing amino acid residues may be used to connect HEMs. One or more sulfhydryl-containing amino acid residues may occur spontaneously in peptides that modulate both the GLP-1 receptor and the GCG receptor. One or more sulfhydryl-containing amino acid residues may be inserted into peptides that modulate both the GLP-1 receptor and the GCG receptor. One or more sulfhydryl-containing amino acid residues may replace one or more amino acid residues in peptides that modulate both the GLP-1 receptor and the GCG receptor. Methods of amino acid substitution and / or insertion are known in the art.

[0044] Peptides that modulate both the GLP-1 receptor and the GCG receptor may contain one or more amine-containing residues. Non-limiting examples of amine-containing residues include lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine. One or more amine-containing residues may be used to connect staples. One or more amine-containing residues may be used to connect HEMs. One or more amine-containing residues may occur spontaneously in peptides that modulate both the GLP-1 receptor and the GCG receptor. One or more amine-containing residues may be inserted into peptides that modulate both the GLP-1 receptor and the GCG receptor. One or more amine-containing residues may replace one or more amino acid residues in peptides that modulate both the GLP-1 receptor and the GCG receptor.

[0045] Peptides that modulate both the GLP-1 receptor and the GCG receptor may contain at least a portion of the wild-type peptide containing one or more amino acid mutations. These one or more amino acid mutations may include deletions, substitutions, additions, or combinations thereof. One or more amino acid mutations may include the addition of one or more amino acid residues to the wild-type peptide. One or more amino acid mutations may include the deletion of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the substitution of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more cysteine, lysine, or other sulfhydryl or amine-containing residues. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more D-amino acid residues. One or more amino acid residues from the wild-type peptide may include one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0046] Peptides that modulate both GLP-1 receptors and GCG receptors may be modified, for example, by acetylation, phosphorylation, or methylation. Peptide modifications may include chemical modifications. Peptide modifications may occur at the N-terminus of the peptide. Peptide modifications may include acetylation of an amino group at the N-terminus of the peptide. Alternatively or additionally, peptide modifications may occur at the C-terminus of the peptide. Peptide modifications may occur at one or more internal amino acids of the peptide. Peptide modifications may include substitution of a carboxyl group at the C-terminus of the peptide. Peptide modifications may include modification of a carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide may be modified to produce an amine group.

[0047] In some embodiments, the peptide that modulates both the GLP-1 receptor and the GCG receptor may be a peptide modified with D-serine instead of L-serine. In some embodiments, the peptide that modulates both the GLP-1 receptor and the GCG receptor may be modified with aminoisobutyric acid [Aib] instead of L-serine. In some embodiments, the peptide that modulates both the GLP-1 receptor and the GCG receptor may be a peptide modified with norleucine [Nle] instead of leucine (Leu).

[0048] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. 48-61 or 80-82. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 95% identical peptide sequences to either SEQ ID NOs. 48-61 or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to either SEQ ID NOs. 48-61 or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences with up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to either SEQ ID NOs. 48-61 or 80-82.

[0049] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. 108-114. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 95% identical peptide sequences to any one of SEQ ID NOs. 108-114. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 108-114. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 108-114.

[0050] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 90% identical peptide sequences to one of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 80–82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 80–82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 80–82.

[0051] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. 48–59. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 90% identical peptide sequences to one of SEQ ID NOs. 48–59. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 48–59. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 48–59. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 48–59.

[0052] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. 108-110. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 95% identical peptide sequences to any one of SEQ ID NOs. 108-110. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 108-110. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 108-110.

[0053] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. 60-61 or 80-82. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 90% identical peptide sequences to one of SEQ ID NOs. 60-61 or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 95% identical peptide sequences to either SEQ ID NOs. 60-61 or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to either SEQ ID NOs. 60-61 or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences with up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to either SEQ ID NOs. 60-61 or 80-82.

[0054] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor comprises the peptide sequence that is SEQ ID NO: 111. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor comprises at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to SEQ ID NO: 111. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor comprises at least about 90% identical peptide sequence to SEQ ID NO: 111. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor comprises at least about 95% identical peptide sequence to SEQ ID NO: 111. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to SEQ ID NO: 111. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 111.

[0055] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains one of the peptide sequences of SEQ ID NOs. 48-52, 55-61, or 80-82. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of the SEQ ID NOs. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 95% identical peptide sequences to one of SEQ ID NOs. 48-52, 55-61, or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to one of SEQ ID NOs. 48-52, 55-61, or 80-82. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences with up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to one of SEQ ID NOs. 48-52, 55-61, or 80-82.

[0056] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains the peptide sequence that is Sequence ID No. 48. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to Sequence ID No. 48. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 90% identical peptide sequence to Sequence ID No. 48. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 95% identical peptide sequence to Sequence ID No. 48. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to SEQ ID NO: 48. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 48.

[0057] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains the peptide sequence that is Sequence ID No. 60. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to Sequence ID No. 60. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 90% identical peptide sequence to Sequence ID No. 60. In some cases, a peptide that modulates both the GLP-1 receptor and the GCG receptor contains at least about 95% identical peptide sequence to Sequence ID No. 60. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain at least approximately 99% identical peptide sequences to SEQ ID NO: 60. In some cases, peptides that modulate both the GLP-1 receptor and the GCG receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 60.

[0058] Table 2 shows non-restrictive examples of peptide derivatives.

[0059] [Table 2]

[0060] Peptides that regulate both the GLP-1 receptor and the GIP receptor. In one embodiment, the Specified Reference Indicators provides peptide conjugates comprising peptides that modulate the GLP-1 receptor and / or the GIP receptor. In some embodiments, the peptide modulates both the GLP-1 receptor and the GIP receptor. In some embodiments, the peptide that modulates the GLP-1 receptor is a GLP-1 receptor agonist. In some embodiments, the peptide that modulates the GIP receptor is a GIP receptor agonist.

[0061] The binding affinity of the peptide conjugate described herein may be within approximately 5% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 10% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 15% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 20% of the binding affinity of the unmodified peptide.

[0062] Peptides that modulate both the GLP-1 receptor and the GIP receptor may contain one or more sulfhydryl-containing amino acid residues. One or more sulfhydryl-containing amino acid residues may be used to connect staples. One or more sulfhydryl-containing amino acid residues may be used to connect HEMs. One or more sulfhydryl-containing amino acid residues may occur spontaneously in peptides that modulate both the GLP-1 receptor and the GIP receptor. One or more sulfhydryl-containing amino acid residues may be inserted into peptides that modulate both the GLP-1 receptor and the GIP receptor. One or more sulfhydryl-containing amino acid residues may replace one or more amino acid residues in peptides that modulate both the GLP-1 receptor and the GIP receptor. Methods of amino acid substitution and / or insertion are known in the art.

[0063] Peptides that modulate both the GLP-1 receptor and the GIP receptor may contain one or more amine-containing residues. Non-limiting examples of amine-containing residues include lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine. One or more amine-containing residues may be used to connect staples. One or more amine-containing residues may be used to connect HEMs. One or more amine-containing residues may occur spontaneously in peptides that modulate both the GLP-1 receptor and the GIP receptor. One or more amine-containing residues may be inserted into peptides that modulate both the GLP-1 receptor and the GIP receptor. One or more amine-containing residues may replace one or more amino acid residues in peptides that modulate both the GLP-1 receptor and the GIP receptor.

[0064] Peptides that modulate both the GLP-1 receptor and the GIP receptor may contain at least a portion of the wild-type peptide containing one or more amino acid mutations. These one or more amino acid mutations may include deletions, substitutions, additions, or combinations thereof. One or more amino acid mutations may include the addition of one or more amino acid residues to the wild-type peptide. One or more amino acid mutations may include the deletion of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the substitution of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more cysteine, lysine, or other sulfhydryl or amine-containing residues. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more D-amino acid residues. One or more amino acid residues from the wild-type peptide may include one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0065] Peptides that modulate both GLP-1 and GIP receptors may be modified, for example, by acetylation, phosphorylation, or methylation. Peptide modifications may include chemical modifications. Peptide modifications may occur at the N-terminus of the peptide. Peptide modifications may include acetylation of an amino group at the N-terminus of the peptide. Alternatively or additionally, peptide modifications may occur at the C-terminus of the peptide. Peptide modifications may occur at one or more internal amino acids of the peptide. Peptide modifications may include substitution of a carboxyl group at the C-terminus of the peptide. Peptide modifications may include modification of a carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide may be modified to produce an amine group.

[0066] In some embodiments, the peptide that modulates both the GLP-1 receptor and the GIP receptor may be a peptide modified with D-serine instead of L-serine. In some embodiments, the peptide that modulates both the GLP-1 receptor and the GIP receptor may be modified with aminoisobutyric acid [Aib] instead of L-serine. In some embodiments, the peptide that modulates both the GLP-1 receptor and the GIP receptor may be a peptide modified with norleucine [Nle] instead of leucine (Leu).

[0067] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains one of the peptide sequences of SEQ ID NOs. 62–71. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 90% identical peptide sequences to one of SEQ ID NOs. 62–71. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 62–71. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 62–71. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 62–71.

[0068] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains the peptide sequence of either SEQ ID NO: 62 or 65. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to either SEQ ID NO: 62 or 65. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 90% identical peptide sequence to either SEQ ID NO: 62 or 65. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 95% identical peptide sequence to either SEQ ID NO: 62 or 65. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 99% identical peptide sequences to either SEQ ID NO: 62 or 65. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to either SEQ ID NO: 62 or 65.

[0069] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains one of the peptide sequences of SEQ ID NOs. 114–120. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 95% identical peptide sequences to any one of SEQ ID NOs. 114–120. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 114–120. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 114–120.

[0070] In some embodiments, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains one of the peptide sequences of SEQ ID NOs. 62–68. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 90% identical peptide sequences to one of SEQ ID NOs. 62–68. In some cases, a peptide that modulates both the GLP-1 receptor and the GIP receptor contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 62–68. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 62–68. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 62–68. In some embodiments, peptides that modulate both the GLP-1 receptor and the GIP receptor contain any one of SEQ ID NOs. 69–71. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical peptide sequences to any one of SEQ ID NOs. 69–71. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 90% identical peptide sequences to any one of SEQ ID NOs. 69–71. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 95% identical peptide sequences to any one of SEQ ID NOs. 69–71.In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 69–71. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 69–71. In some embodiments, peptides that modulate both the GLP-1 receptor and the GIP receptor contain the peptide sequence that is SEQ ID NO. 63. In some cases, peptides that modulate both GLP-1 receptors and GIP receptors contain at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical peptide sequences to SEQ ID NO: 63. In some cases, peptides that modulate both GLP-1 receptors and GIP receptors contain at least approximately 90% identical peptide sequences to SEQ ID NO: 63. In some cases, peptides that modulate both GLP-1 receptors and GIP receptors contain at least approximately 95% identical peptide sequences to SEQ ID NO: 63. In some cases, peptides that modulate both GLP-1 receptors and GIP receptors contain at least approximately 99% identical peptide sequences to SEQ ID NO: 63. In some cases, peptides that modulate both the GLP-1 receptor and the GIP receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 63.

[0071] Table 3 shows non-limiting examples of peptide derivatives.

[0072] [Table 3]

[0073] Peptides that regulate the GLP-1 receptor In one embodiment, the Specified Reference Indicators provides peptide conjugates comprising peptides that modulate the GLP-1 receptor. In some embodiments, the peptides that modulate the GLP-1 receptor are GLP-1 receptor agonists.

[0074] The binding affinity of the peptide conjugate described herein may be within approximately 5% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 10% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 15% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 20% of the binding affinity of the unmodified peptide.

[0075] A peptide that modulates the GLP-1 receptor may contain one or more sulfhydryl-containing amino acid residues. One or more sulfhydryl-containing amino acid residues may be used to connect staples. One or more sulfhydryl-containing amino acid residues may be used to connect HEMs. One or more sulfhydryl-containing amino acid residues may occur spontaneously in a peptide that modulates the GLP-1 receptor. One or more sulfhydryl-containing amino acid residues may be inserted into a peptide that modulates the GLP-1 receptor. One or more sulfhydryl-containing amino acid residues may replace one or more amino acid residues in a peptide that modulates the GLP-1 receptor. Methods of amino acid substitution and / or insertion are known in the art.

[0076] Peptides that modulate the GLP-1 receptor may contain one or more amine-containing residues. Non-limiting examples of amine-containing residues include lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine. One or more amine-containing residues may be used to connect staples. One or more amine-containing residues may be used to connect HEMs. One or more amine-containing residues may occur spontaneously in peptides that modulate the GLP-1 receptor. One or more amine-containing residues may be inserted into peptides that modulate the GLP-1 receptor. One or more amine-containing residues may replace one or more amino acid residues in peptides that modulate the GLP-1 receptor.

[0077] Peptides that modulate the GLP-1 receptor may contain at least a portion of the wild-type peptide containing one or more amino acid mutations. These one or more amino acid mutations may include deletions, substitutions, additions, or combinations thereof. One or more amino acid mutations may include the addition of one or more amino acid residues to the wild-type peptide. One or more amino acid mutations may include the deletion of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the substitution of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more cysteine, lysine, or other sulfhydryl or amine-containing residues. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more D-amino acid residues. One or more amino acid residues from the wild-type peptide may include one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0078] Peptides that modulate the GLP-1 receptor may be modified, for example, by acetylation, phosphorylation, or methylation. Peptide modifications may include chemical modifications. Peptide modifications may occur at the N-terminus of the peptide. Peptide modifications may include acetylation of an amino group at the N-terminus of the peptide. Alternatively or additionally, peptide modifications may occur at the C-terminus of the peptide. Peptide modifications may occur at one or more internal amino acids of the peptide. Peptide modifications may include replacement of a carboxyl group at the C-terminus of the peptide. Peptide modifications may include modification of a carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide may be modified to produce an amine group.

[0079] In some embodiments, the peptide modulating the GLP-1 receptor may be a peptide modified with D-serine instead of L-serine. In some embodiments, the peptide modulating the GLP-1 receptor may be modified with aminoisobutyric acid [Aib] instead of L-serine. In some embodiments, the peptide modulating the GLP-1 receptor may be a peptide modified with norleucine [Nle] instead of leucine (Leu).

[0080] In some embodiments, the GLP-1 receptor modulating peptide comprises one of the peptide sequences of SEQ ID NOs. 48-82 or 108-120. In some cases, the GLP-1 receptor modulating peptide comprises at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least approximately 95% identical peptide sequences to one of SEQ ID NOs. 48-82 or 108-120. In some cases, the GLP-1 receptor modulating peptide contains at least approximately 99% identical peptide sequences to one of SEQ ID NOs. 48-82 or 108-120. In some cases, the GLP-1 receptor modulating peptide contains amino acid sequences with up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to one of SEQ ID NOs. 48-82 or 108-120.

[0081] In some embodiments, the GLP-1 receptor modulating peptide contains one of the peptide sequences of SEQ ID NOs. 72-79. In some cases, the GLP-1 receptor modulating peptide contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least about 90% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 72-79. In some cases, the GLP-1 receptor modulating peptide contains at least approximately 99% identical peptide sequences to one of SEQ ID NOs. 72–79. In some cases, the GLP-1 receptor modulating peptide contains amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to one of SEQ ID NOs. 72–79.

[0082] In some embodiments, the GLP-1 receptor modulating peptide contains one of the peptide sequences of SEQ ID NOs. 72-75. In some cases, the GLP-1 receptor modulating peptide contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least about 90% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 72-75. In some cases, the GLP-1 receptor modulating peptide contains at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 72–75. In some cases, the GLP-1 receptor modulating peptide contains amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 72–75.

[0083] In some embodiments, the GLP-1 receptor modulating peptide contains one of the peptide sequences of SEQ ID NOs. 76-79. In some cases, the GLP-1 receptor modulating peptide contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least about 90% identical peptide sequences to one of SEQ ID NOs. In some cases, the GLP-1 receptor modulating peptide contains at least about 95% identical peptide sequences to one of SEQ ID NOs. 76-79. In some cases, the peptides that modulate the GLP-1 receptor contain at least approximately 99% identical peptide sequences to any one of SEQ ID NOs. 76–79. In some cases, the peptides that modulate the GLP-1 receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs. 76–79.

[0084] In some embodiments, the GLP-1 receptor modulating peptide includes the peptide sequence of SEQ ID NO: 76. In some cases, the GLP-1 receptor modulating peptide includes at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical peptide sequence to SEQ ID NO: 76. In some cases, the GLP-1 receptor modulating peptide includes at least about 90% identical peptide sequence to SEQ ID NO: 76. In some cases, the GLP-1 receptor modulating peptide includes at least about 95% identical peptide sequence to SEQ ID NO: 76. In some cases, the GLP-1 receptor modulating peptide includes at least about 99% identical peptide sequence to SEQ ID NO: 76. In some cases, peptides that modulate the GLP-1 receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 76.

[0085] In some embodiments, the GLP-1 receptor modulating peptide comprises the peptide sequence of SEQ ID NO: 77. In some cases, the GLP-1 receptor modulating peptide comprises a peptide sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 77. In some cases, the GLP-1 receptor modulating peptide comprises a peptide sequence that is at least about 90% identical to SEQ ID NO: 77. In some cases, the GLP-1 receptor modulating peptide comprises a peptide sequence that is at least about 95% identical to SEQ ID NO: 77. In some cases, the GLP-1 receptor modulating peptide comprises a peptide sequence that is at least about 99% identical to SEQ ID NO: 77. In some cases, peptides that modulate the GLP-1 receptor contain amino acid sequences that have up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 77.

[0086] Table 4 shows non-restrictive examples of peptide derivatives.

[0087] [Table 4]

[0088] Prolactin-releasing peptide (PrRP) In one embodiment, the Specified Information provides a peptide conjugate comprising a prolactin-releasing peptide (PrRP).

[0089] The binding affinity of the peptide conjugate described herein may be within approximately 5% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 10% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 15% of the binding affinity of the unmodified peptide. The binding affinity of the peptide conjugate described herein may be within approximately 20% of the binding affinity of the unmodified peptide.

[0090] A prolactin-releasing peptide (PrRP) may contain one or more sulfhydryl-containing amino acid residues. One or more sulfhydryl-containing amino acid residues may be used to staple the prolactin-releasing peptide (PrRP). One or more sulfhydryl-containing amino acid residues may occur spontaneously in a prolactin-releasing peptide (PrRP). One or more sulfhydryl-containing amino acid residues may be inserted into a prolactin-releasing peptide (PrRP). One or more sulfhydryl-containing amino acid residues may replace one or more amino acid residues in a prolactin-releasing peptide (PrRP). Methods of amino acid substitution and / or insertion are known in the art.

[0091] Prolactin-releasing peptides (PrRPs) may contain at least a portion of a wild-type peptide that includes one or more amino acid mutations. These one or more amino acid mutations may include deletions, substitutions, additions, or combinations thereof. One or more amino acid mutations may include the addition of one or more amino acid residues to the wild-type peptide. One or more amino acid mutations may include the deletion of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the substitution of one or more amino acid residues from the wild-type peptide. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more cysteine, lysine, or other sulfhydryl or amine-containing residues. One or more amino acid mutations may include the replacement of one or more amino acid residues from the wild-type peptide with one or more D-amino acid residues. One or more amino acid residues from the wild-type peptide may include one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0092] Prolactin-releasing peptides (PrRPs) may be modified, for example, by acetylation, phosphorylation, and methylation. Peptide modifications may include chemical modifications. Peptide modifications may occur at the N-terminus of the peptide. Peptide modifications may include acetylation of an amino group at the N-terminus of the peptide. Alternatively or additionally, peptide modifications may occur at the C-terminus of the peptide. Peptide modifications may occur at one or more internal amino acids of the peptide. Peptide modifications may include replacement of a carboxyl group at the C-terminus of the peptide. Peptide modifications may include modification of a carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide may be modified to produce an amine group.

[0093] In some embodiments, the peptide derivative may be a prolactin-releasing peptide (PrRP) modified with hArg instead of Arg. In some embodiments, the peptide derivative may be a prolactin-releasing peptide (PrRP) modified with β-hArg instead of Arg. In some embodiments, the peptide derivative may be a prolactin-releasing peptide (PrRP) modified with NMe-Arg instead of Arg. In some embodiments, the peptide derivative may be a prolactin-releasing peptide (PrRP) modified with Nle instead of Met.

[0094] Table 5 shows non-specific examples of prolactin-releasing peptides (PrRPs).

[0095] In some cases, prolactin-releasing peptides (PrRPs) have an amino acid sequence that is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs. In some cases, prolactin-releasing peptides (PrRPs) have an amino acid sequence that is at least approximately 90% identical to one of SEQ ID NOs. 83-105. In some cases, prolactin-releasing peptides (PrRPs) have an amino acid sequence that is at least approximately 95% identical to one of SEQ ID NOs. 83-105. In some cases, prolactin-releasing peptides (PrRPs) have an amino acid sequence that is at least approximately 99% identical to one of SEQ ID NOs. 83-105. In some cases, PrRPs contain an amino acid sequence that has up to approximately 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to one of SEQ ID NOs. 83-105.

[0096] [Table 5-1]

[0097] [Table 5-2]

[0098] staples This specification discloses peptide conjugates containing staples.

[0099] In some embodiments, the staple that binds to the peptide has the formula (I):

[0100] [Chemical formula] where in the formula A is optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted -NR 3 -alkylene-NR 3 -, or -N- X 1 and X 2 are independently a single bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene-, -alkylene-C(=O)NR 3 -, -alkylene-NR 3 C(=O)-, -C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-, -alkylene-C(=O)NR 3 -alkylene-, or -alkylene-NR 3 C(=O)-alkylene-, where X 1 is bonded to the first amino acid of the peptide, and X 2 is bonded to the second amino acid of the peptide R is hydrogen or -(L) s -Y each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-, -NR 3 C(=O)NR3 -, -NR 3 C(=O)NR 3 -Alkilen-, -NR 3 C(=O)-alkylene-NR 3 -,-alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)-, or -NR 3 It is C(=O)-alkylene-, v is between 2 and 20. R 1 or R 2 These are hydrogen, halogen, -CN, and -OR, respectively, independently. a , -SR a -S(=O)R b -NO2, -NR c R d -S(=O)2R d , -NR a S(=O)2R d -S(=O)2NR c R d -C(=O)R b -OC(=O)R b , -CO2R a , -OCO2R a -C(=O)NR c R d -OC(=O)NR c R d , -NR a C(=O)NR c R d , -NR a C(=O)R b , -NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R dThey are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , -NR c R d It is replaced by one, two, or three of the following, or R 1 and R 2 These combine to form a C1-C6 cycloalkyl or C1-C6 heterocycloalkyl group. Each R 3 These are hydrogen and -S(=O)R, which are independent of each other. b -S(=O)2R a -S(=O)2NR c R d -C(=O)R b , -CO2R a -C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , or -NR c R d It is replaced by one, two, or three of the following, Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl). Furthermore, s is between 0 and 20. R ais hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. R b The elements are C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogens, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogens, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. R c and R d Each is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. Or R c and R dThese atoms, together with the nitrogen atom to which they are bonded, form a heterocycloalkyl or heteroaryl group, where the heterocycloalkyl and heteroaryl groups are optionally substituted with one, two, or three of the following: halogens, C1-C6 alkyl groups, C1-C6 haloalkyl groups, -OH groups, -OMe groups, or -NH2 groups.

[0101] In some embodiments, the staple bound to the peptide is of formula (I):

[0102] [ka] It is, During the ceremony, A is -N-, X 1 and X 2 These are single bonds, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene-, and -alkylene-C(=O)NR 3 -, -alkylene-NR 3 C(=O)-, -C(=O)NR 3 -Alkilen-, -NR 3 C(=O)-alkylene-,-alkylene-C(=O)NR 3 -alkylene-, or -alkylene-NR 3 C(=O)-alkylene-, where X 1 It binds to the first amino acid of the peptide, X 2 It is bound to the second amino acid of the peptide, X 1 and X 2 They are identical, R is hydrogen or -(L) s -Y is, Each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3-, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-, -NR 3 C(=O)NR 3 -, -NR 3 C(=O)NR 3 -Alkilen-, -NR 3 C(=O)-alkylene-NR 3 -,-alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)-, or -NR 3 It is C(=O)-alkylene-, v is between 2 and 20. R 1 or R 2 These are hydrogen, halogen, -CN, and -OR, respectively, independently. a , -SR a -S(=O)R b -NO2, -NR c R d -S(=O)2R d , -NR a S(=O)2R d -S(=O)2NR c R d -C(=O)R b -OC(=O)R b , -CO2R a , -OCO2R a -C(=O)NR c R d -OC(=O)NR c R d , -NR a C(=O)NR c R d , -NR a C(=O)R b , -NR a C(=O)OR a, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , or -NR c R d It is replaced by one, two, or three of the following, or R 1 and R 2 These combine to form a C1-C6 cycloalkyl or C1-C6 heterocycloalkyl group. Each R 3 These are hydrogen and -S(=O)R, which are independent of each other. b -S(=O)2R a -S(=O)2NR c R d -C(=O)R b , -CO2R a -C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , or -NR c R d It is replaced by one, two, or three of the following, Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl). s is between 0 and 20. R a is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. R b is a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogens, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogens, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2, and R c and R dEach is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. Or R c and R d These atoms, together with the nitrogen atom to which they are bonded, form a heterocycloalkyl or heteroaryl group, where the heterocycloalkyl and heteroaryl groups are optionally substituted with one, two, or three of the following: halogens, C1-C6 alkyl groups, C1-C6 haloalkyl groups, -OH groups, -OMe groups, or -NH2 groups.

[0103] In some embodiments, A is an optionally substituted alkylene. In some embodiments, A is -(CH2) t -where t is 1 to 12. In some embodiments, A is -(CH2) t -where t is 1 to 10. In some embodiments, A is -(CH2) t -where t is 1 to 8. In some embodiments, A is -(CH2) t -where t is 1 to 6. In some embodiments, A is -(CH2) t - and here t is 1 to 4.

[0104] In some embodiments, A is an optionally substituted arylene. In some embodiments, A is an arylene optionally substituted with a halogen, alkyl, or haloalkyl. In some embodiments, A is an unsubstituted arylene.

[0105] In some embodiments, A is -NR 3 -Alkilen-NR 3 -. In some embodiments, A is -N-.

[0106] In some embodiments, X 1 and X 2 They are identical. In some embodiments, X 1 and X 2 They are different.

[0107] In some embodiments, X 1 and X 2 is -C(=O)-. In some embodiments, X 1 and X 2 These are independently -alkylene-C(=O)- or -C(=O)alkylene-. In some embodiments, X 1 and X 2 These are independently -CH2-C(=O)- or -C(=O)-CH2-. In some embodiments, X 1 and X 2 These are independently -alkylene-C(=O)NR 3 -or -C(=O)NR 3 -Alkylene-. In some embodiments, X 1 and X 2 Independently, -CH2-C(=O)NR 3 -or -C(=O)NR 3 -CH2-. In some embodiments, X 1 and X 2 These are independently -alkylene-C(=O)NR 3 -alkylene- or -alkylene-NR 3 It is C(=O)-alkylene-. In some embodiments, X 1 and X 2 Independently, -CH2-C(=O)NR 3 -CH2CH2- or -CH2-NR 3 In some embodiments, X 1 and X 2These are independently -CH2-C(=O)NH-CH2CH2- or -CH2-NHC(=O)-CH2CH2-.

[0108] Several embodiments, each R 3 R is independently hydrogen or a C1-C6 alkyl group. In some embodiments, each R 3 It is hydrogen.

[0109] In some embodiments, >AR has the following structure:

[0110] [ka] It has, in the formula, R 1 and R 2 Each of these values ​​is independently between 0 and 4.

[0111] In some embodiments, R 1 and R 2 Each of these is independently between 0 and 2. In some embodiments, R 1 and R 2 These are each 0. In some embodiments, R 1 and R 2 Each of these is 1. In some embodiments, R 1 and R 2 These are 3, respectively. In some embodiments, R 1 and R 2 These are both 2.

[0112] In some embodiments, >AR has the following structure:

[0113] [ka] It has.

[0114] In some embodiments, >AR has the following structure:

[0115] [ka] The formula has the following properties, where p1 is between 1 and 5.

[0116] In some embodiments, p1 is 1 to 3. In some embodiments, p1 is 1 to 2. In some embodiments, p1 is 1. In some embodiments, p1 is 2. In some embodiments, p1 is 3. In some embodiments, p1 is 4. In some embodiments, p1 is 5.

[0117] In some embodiments, >AR has the following structure:

[0118] [ka] It has.

[0119] In some embodiments, >AR has the following structure:

[0120] [ka] It has.

[0121] In some embodiments, s is 1 to 15. In some embodiments, s is 1 to 10. In some embodiments, s is 5 to 15. In some embodiments, s is 5 to 10. In some embodiments, s is 5 to 20.

[0122] In some embodiments, Y is hydrogen or -CO2H. In some embodiments, Y is hydrogen. In some embodiments, Y is -CO2H.

[0123] In some embodiments, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3-, or -alkylene-NR 3 C(=O)- and v is between 2 and 20.

[0124] In some embodiments, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)- and v is between 2 and 16.

[0125] In some embodiments, v is 2 to 16. In some embodiments, v is 2 to 5. In some embodiments, v is 5 to 16. In some embodiments, v is 5 or 16. In some embodiments, v is 2 or 16.

[0126] In some embodiments, R 1 or R 2 These are hydrogen, halogen, -CN, and -OR, respectively, independently. a , -NR c R d -C(=O)R b , -CO2R a -C(=O)NR c R d , or C1-C6 alkyl.

[0127] In some embodiments, R 1 or R 2 These are hydrogen, halogen, and -CO2R, respectively, independently. a -C(=O)NR c R d , or C1-C6 alkyl. In some embodiments, R 1 or R 2 These are hydrogen and -CO2R, respectively, independently. a , or -C(=O)NR c R d In some embodiments, R1 or R 2 These are, independently, hydrogen or -CO2R a That is the case.

[0128] In some embodiments, staples are

[0129] [ka] That is the case.

[0130] In some embodiments, the staples bound to the peptide are

[0131] [ka] And each L 1 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s1 is between 1 and 15.

[0132] In some embodiments, the staples bound to the peptide are

[0133] [ka] And each L 2 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s2 is between 1 and 15.

[0134] In some embodiments, the staples bound to the peptide are

[0135] [ka] And each L 3 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s3 is between 1 and 15.

[0136] In some embodiments, the staples bound to the peptide are

[0137] [ka] And each L 4 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s4 is between 1 and 15.

[0138] In some embodiments, the staples bound to the peptide are

[0139] [ka] And each L 5 (CR) is independent. 1 R 2 )v -, -C(=)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s5 is between 1 and 10.

[0140] In some embodiments, the staples bound to the peptide are

[0141] [ka] And each L 6 (CR) is independent. 1 R 2 ) v -, -C(=)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s6 is between 1 and 5.

[0142] In some embodiments, the staples bound to the peptide are

[0143] [ka] And each L 7 (CR) is independent. 1 R 2 ) v -, -C(=)NR 3 -, or -NR 3 C(=O)-, v is between 2 and 20, and s7 is between 1 and 5.

[0144] In some embodiments, the staples bound to the peptide are

[0145] [ka] And each L8 Ha-(CR 1 R 2 ) v - and v is between 10 and 20.

[0146] In some embodiments, the staples bound to the peptide are

[0147] [ka] And each L 9 (CR) is independent. 1 R 2 ) v -, -C(=)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s9 is between 1 and 5.

[0148] In some embodiments, the staples bound to the peptide are

[0149] [ka] And each L 10 Ha-(CR 1 R 2 ) v - and v is between 10 and 20.

[0150] In some embodiments, the staples bound to the peptide are

[0151] [ka] That is the case.

[0152] In some embodiments, the staples bound to the peptide are

[0153] [ka] And each L 11 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s11 is between 1 and 15.

[0154] In some embodiments, the staples bound to the peptide are

[0155] [ka] And each L 12 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s12 is between 1 and 15.

[0156] In some embodiments, the staples bound to the peptide are

[0157] [ka] And each L 13 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3C(=O)-, v is between 2 and 20, and s13 is between 1 and 15.

[0158] In some embodiments, the staples bound to the peptide are

[0159] [ka] Each L 14 (CR) is independent. 1 R 2 ) v -, -alkylene-O-, -O-alkylene, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O-), v is between 2 and 20, and s14 is between 1 and 15.

[0160] In some embodiments, the staples bound to the peptide are

[0161] [ka] And each L 15 (CR) is independent. 1 R 2 ) v -, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is between 2 and 20, and s15 is between 1 and 10.

[0162] In some embodiments, the staples bound to the peptide are

[0163] [ka] And each L 16 (CR) is independent. 1 R2 ) v -, -C(=O)NR 3 -, or -NR 3 C(=O)-, v is between 2 and 20, and s16 is between 1 and 5.

[0164] In some embodiments, the staples bound to the peptide are

[0165] [ka] And each L 17 (CR) is independent. 1 R 2 ) v -, -C(=O)NR 3 -, or -NR 3 C(=O)-, v is between 2 and 20, and s17 is between 1 and 5.

[0166] In some embodiments, the staples bound to the peptide are

[0167] [ka] And each L 18 Ha-(CR 1 R 2 ) v - and v is between 10 and 20.

[0168] In some embodiments, the staples bound to the peptide are

[0169] [ka] And each L 19 (CR) is independent. 1 R 2 ) v -, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3C(=O)-, v is between 2 and 20, and s19 is between 1 and 5.

[0170] In some embodiments, the staples bound to the peptide are

[0171] [ka] And each L 20 Ha-(CR 1 R 2 ) v - and v is between 10 and 20.

[0172] In some embodiments, the staples bound to the peptide are

[0173] [ka]

[0174] [ka]

[0175] [ka] And,

[0176] [ka] This is the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue portion.

[0177] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0178] In some embodiments, the staples bound to the peptide are

[0179] [ka] In the formula, n is between 1 and 4, and m is between 6 and 20.

[0180] [ka] This is the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue portion.

[0181] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0182] In some embodiments, the staples bound to the peptide are

[0183] [ka] And,

[0184] [ka] This is the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue portion.

[0185] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0186] In some embodiments, the staples bound to the peptide are

[0187] [ka] And,

[0188] [ka] This is the portion of the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0189] In some embodiments, the staples bound to the peptide are

[0190] [ka] And,

[0191] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0192] In some embodiments, the staples bound to the peptide are

[0193] [ka] And,

[0194] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0195] In some embodiments, the staples bound to the peptide are

[0196] [ka] And,

[0197] [ka] This is the portion of the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0198] In some embodiments, the staples bound to the peptide are

[0199] [ka] And,

[0200] [ka] This is the portion of the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0201] In some embodiments, the staples bound to the peptide are

[0202] [ka] And,

[0203] [ka] This is the portion of the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0204] In some embodiments, the staples bound to the peptide are

[0205] [ka] And,

[0206] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0207] In some embodiments, the staples bound to the peptide are

[0208] [ka] And,

[0209] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0210] In some embodiments, the staples bound to the peptide are

[0211] [ka] And,

[0212] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0213] Half-life extension portion (HEM) This specification discloses peptide conjugates containing HEM.

[0214] In some embodiments, the HEM bound to the peptide is given by formula (II): -X 3 -(L) s -Y formula (II) It is, During the ceremony, X3 These are single bonds, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene-, and -alkylene-C(=O)NR 3 -, -alkylene-NR 3 C(=O)-, -C(=O)NR 3 -Alkilen-, -NR 3 C(=O)-alkylene-,-alkylene-C(=O)NR 3 -alkylene-, or -alkylene-NR 3 It is C(=O)-alkylene-, Here, X 3 It binds to the first amino acid of the peptide, Each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-, -NR 3 C(=O)NR 3 -, -NR 3 C(=O)NR 3 -Alkilen-, -NR 3 C(=O)-alkylene-NR 3 -,-alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)-, or -NR 3 It is C(=O)-alkylene-, v is between 2 and 20. R 1 or R 2 These are hydrogen, halogen, -CN, and -OR, respectively, independently. a , -SR a -S(=O)R b -NO2, -NRc R d -S(=O)2R d , -NR a S(=O)2R d -S(=O)2NR c R d -C(=O)R b -OC(=O)R b , -CO2R a , -OCO2R a -C(=O)NR c R d -OC(=O)NR c R d , -NR a C(=O)NR c R d , -NR a C(=O)R b , -NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , -NR c R d It is replaced by one, two, or three of the following, or R 1 and R 2 These combine to form a C1-C6 cycloalkyl or C1-C6 heterocycloalkyl group. Each R 3 These are hydrogen and -S(=O)R, which are independent of each other. b -S(=O)2R a -S(=O)2NR c R d -C(=O)R b , -CO2R a -C(=O)NR c Rd , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR a , or -NR c R d They are optionally substituted with 1, 2, or 3 of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a , or -NR c R d It is replaced by one, two, or three of the following, Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl), and s is between 0 and 20. R a is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. R bThe elements are C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogens, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogens, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. R c and R d Each is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one, two, or three of halogen, -OH, -OMe, or -NH2, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one, two, or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe, or -NH2. Or R c and R d These atoms, together with the nitrogen atom to which they are bonded, form a heterocycloalkyl or heteroaryl group, where the heterocycloalkyl and heteroaryl groups are optionally substituted with one, two, or three of the following: halogens, C1-C6 alkyl groups, C1-C6 haloalkyl groups, -OH groups, -OMe groups, or -NH2 groups.

[0215] In some embodiments, X 3 It is a single bond.

[0216] In some embodiments, X 3 is -alkylene-C(=O)- or -C(=O)alkylene-. In some embodiments, X 3is -CH2-C(=O)- or -C(=O)-CH2-. In some embodiments, X 3 is -alkylene-C(=O)NR 3 -or -C(=O)NR 3 -Alkylene-. In some embodiments, X 3 -CH2-C(=O)NR 3 -or -C(=O)NR 3 -CH2-. In some embodiments, X 3 is -alkylene-C(=O)NR 3 -alkylene- or -alkylene-NR 3 It is C(=O)-alkylene-. In some embodiments, X 3 -CH2-C(=O)NR 3 -CH2CH2- or -CH2-NR 3 In some embodiments, X 3 This is -CH2-C(=O)NH-CH2CH2- or -CH2-NHC(=O)-CH2CH2-.

[0217] Several embodiments, each R 3 R is independently hydrogen or a C1-C6 alkyl group. In some embodiments, each R 3 It is hydrogen.

[0218] In some embodiments, s is 1 to 15. In some embodiments, s is 1 to 10. In some embodiments, s is 5 to 15. In some embodiments, s is 5 to 10. In some embodiments, s is 5 to 20.

[0219] In some embodiments, Y is hydrogen or -CO2H. In some embodiments, Y is hydrogen. In some embodiments, Y is -CO2H.

[0220] In some embodiments, each L is independently -(CR 1 R 2 ) v-, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)- and v is between 2 and 20.

[0221] In some embodiments, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -, -NR 3 C(=O)-,-alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)- and v is between 2 and 16.

[0222] In some embodiments, v is 2 to 16. In some embodiments, v is 2 to 5. In some embodiments, v is 5 to 16. In some embodiments, v is 5 or 16. In some embodiments, v is 2 or 16.

[0223] In some embodiments, R 1 or R 2 These are hydrogen, halogen, -CN, and -OR, respectively, independently. a , -NR c R d -C(=O)R b , -CO2R a -C(=O)NR c R d , or C1-C6 alkyl.

[0224] In some embodiments, R 1 or R 2 These are hydrogen, halogen, and -CO2R, respectively, independently. a -C(=O)NR c R d , or C1-C6 alkyl. In some embodiments, R 1 or R 2These are hydrogen and -CO2R, respectively, independently. a , or -C(=O)NR c R d In some embodiments, R 1 or R 2 These are, independently, hydrogen or -CO2R a That is the case.

[0225] In some embodiments, the HEM bound to the peptide is

[0226] [ka] And,

[0227] [ka] This is the portion of the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0228] In some embodiments, the HEM bound to the peptide is

[0229] [ka] In the formula, n is between 1 and 4, and m is between 6 and 20.

[0230] [ka] This is the portion of the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0231] Peptide conjugate with staples In one embodiment, the Specified herein discloses a peptide conjugate comprising (a) a peptide selected from a peptide that modulates the PYY receptor, a peptide that modulates both the GLP-1 receptor and the GCG receptor, a peptide that modulates both the GLP-1 receptor and the GIP receptor, and a peptide that modulates the GLP-1 receptor, and (b) a staple that is bound to the peptide at a first amino acid and a second amino acid.

[0232] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates the PYY receptor, and (b) a staple that is bound to the peptide at a first amino acid and a second amino acid.

[0233] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates both the GLP-1 receptor and the GCG receptor, and (b) a staple that is bound to the peptide at a first amino acid and a second amino acid.

[0234] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates both the GLP-1 receptor and the GIP receptor, and (b) a staple that is bound to the peptide at a first amino acid and a second amino acid.

[0235] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates the GLP-1 receptor, and (b) a staple that is bound to the peptide at a first amino acid and a second amino acid.

[0236] Non-limiting examples of amino acids for use in conjugation include cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, 2-amino-6-mercaptohexanoic acid, lysine, ornithine, diaminobutyric acid, diaminopropionic acid, homolysine, other sulfhydryl-containing amino acids, or other amine-containing amino acids. In some embodiments, the two amino acids linked by staples are separated by approximately or at least approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more amino acid units. For example, the first amino acid has position i, and the second amino acid has position i+7, i+11, i+13, i+15, or i+16. For example, the first amino acid has position i in the peptide, and the second amino acid has position i+n in the peptide, where n is 4 to 16. In some embodiments, the first amino acid is located at position 14 of the peptide, and the second amino acid is located at position 21 of the peptide. In some embodiments, the first amino acid is located at position 17 of the peptide, and the second amino acid is located at position 24 of the peptide.

[0237] For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+4. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+5. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+6. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+7. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+8. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+9. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+10. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+11. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+12. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+13 in the peptide. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+14 in the peptide. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+15 in the peptide. For example, the first amino acid occupies position i in the peptide, and the second amino acid occupies position i+16 in the peptide.

[0238] In some embodiments, the first amino acid and the second amino acid are independently selected from the group consisting of amine-containing amino acids and sulfhydryl-containing amino acids.

[0239] In some embodiments, the first and second amino acids are independently selected from cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, and 2-amino-6-mercaptohexanoic acid. In some embodiments, the first and second amino acids are cysteine.

[0240] In some embodiments, the first and second amino acids are independently selected from lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine.

[0241] In some embodiments, the first and second amino acids are lysine.

[0242] In some embodiments, the first and second amino acids are ornithine.

[0243] In some embodiments, the peptide conjugate further comprises a half-life-extending molecule that binds to a sulfhydryl-containing amino acid or amine-containing amino acid residue in the peptide.

[0244] In some embodiments, the amine-containing amino acid is selected from lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine.

[0245] In some embodiments, the amine-containing amino acid is lysine.

[0246] In some embodiments, the sulfhydryl-containing amino acid is selected from cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, and 2-amino-6-mercaptohexanoic acid.

[0247] In some embodiments, the sulfhydryl-containing amino acid is cysteine.

[0248] Peptide conjugate with half-life extension portion In one embodiment, the Specified herein discloses a peptide conjugate comprising (a) a peptide selected from a peptide that modulates the PYY receptor, a peptide that modulates both the GLP-1 receptor and the GCG receptor, a peptide that modulates both the GLP-1 receptor and the GIP receptor, and a peptide that modulates the GLP-1 receptor, and (b) a half-life extension moiety (HEM) that is bound to the peptide at a first amino acid.

[0249] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates the PYY receptor, and (b) a half-life extension moiety (HEM) that is bound to the peptide at a first amino acid.

[0250] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates both the GLP-1 receptor and the GCG receptor, and (b) a half-life extension moiety (HEM) that is bound to the peptide at a first amino acid.

[0251] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates both the GLP-1 receptor and the GIP receptor, and (b) a half-life extension moiety (HEM) that is bound to the peptide at a first amino acid.

[0252] In some embodiments, the peptide conjugate comprises (a) a peptide that modulates the GLP-1 receptor, and (b) a half-life extension moiety (HEM) that is bound to the peptide at a first amino acid.

[0253] Non-limiting examples of amino acids for use in conjugation include cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, 2-amino-6-mercaptohexanoic acid, lysine, ornithine, diaminobutyric acid, diaminopropionic acid, homolysine, other sulfhydryl-containing amino acids, or other amine-containing amino acids. In some embodiments, the first amino acid is selected from the group consisting of amine-containing amino acids and sulfhydryl-containing amino acids. In some embodiments, the first amino acid is selected from cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, and 2-amino-6-mercaptohexanoic acid. In some embodiments, the first amino acid is cysteine. In some embodiments, the first amino acid is selected from lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine. In some embodiments, the first amino acid is lysine. In some embodiments, the first amino acid is ornithine. In some embodiments, the peptide conjugate further comprises a second half-life extension moiety that binds to a sulfhydryl-containing amino acid or amine-containing amino acid residue in the peptide. In some embodiments, the amine-containing amino acid is selected from lysine, ornithine, diaminobutyric acid, diaminopropionic acid, and homolysine. In some embodiments, the amine-containing amino acid is lysine. In some embodiments, the sulfhydryl-containing amino acid is selected from cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, and 2-amino-6-mercaptohexanoic acid. In some embodiments, the sulfhydryl-containing amino acid is cysteine.

[0254] In some embodiments, the peptide conjugate is a) A peptide that modulates the PYY receptor, comprising a peptide sequence having at least approximately 95% identity with one of sequence numbers 3, 5, 6, 8, 14-30, 36, or 37, b) Staples that are bound to the peptide at the first and second amino acids Includes.

[0255] In some embodiments, the peptide that modulates the PYY receptor comprises a peptide sequence having at least about 99% identity to any one of SEQ ID NOs: 3, 5, 6, 8, 14-30, 36, or 37.

[0256] In some embodiments, the peptide that modulates the PYY receptor includes the peptide sequence SEQ ID NOs. 3, 5, 6, 8, 14-30, 36, or 37.

[0257] In some embodiments, the peptide that modulates the PYY receptor includes a sequence that is at least about 99% identical to SEQ ID NO: 6.

[0258] In some embodiments, the peptide that modulates the PYY receptor includes the sequence SEQ ID NO: 6.

[0259] In some embodiments, the peptide conjugate is a) A peptide that modulates both the GLP-1 receptor and the GCGR receptor, comprising a peptide sequence having at least approximately 95% identity with any one of sequence numbers 50-59, b) Staples that are bound to the peptide at the first and second amino acids Includes.

[0260] In some embodiments, the peptides that modulate both the GLP-1 receptor and the GCG receptor include a peptide sequence having at least about 99% identity with any one of SEQ ID NOs. 50-59.

[0261] In some embodiments, the peptide that modulates both the GLP-1 receptor and the GCG receptor comprises a peptide sequence selected from SEQ ID NOs. 50-59.

[0262] In some embodiments, the peptide conjugate is a) A peptide that modulates both the GLP-1 receptor and the GIP receptor, comprising a peptide sequence having at least approximately 95% identity with any one of sequence numbers 62-71, b) Staples that are bound to the peptide at the first and second amino acids Includes.

[0263] In some embodiments, the peptides that modulate both the GLP-1 receptor and the GIP receptor include a peptide sequence having at least about 99% identity with any one of SEQ ID NOs. 62-71.

[0264] In some embodiments, the peptide that modulates both the GLP-1 receptor and the GIP receptor comprises a peptide sequence selected from SEQ ID NOs. 62-71.

[0265] In some embodiments, the peptides that modulate both the GLP-1 receptor and the GIP receptor include a sequence that has at least about 99% identity with SEQ ID NO: 63.

[0266] In some embodiments, the peptide that modulates both the GLP-1 receptor and the GIP receptor includes the sequence SEQ ID NO: 63.

[0267] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-1 receptor, comprising a peptide sequence having at least approximately 95% identity with either SEQ ID NO: 74 or 79, b) Staples that are bound to the peptide at the first and second amino acids Includes.

[0268] In some embodiments, the peptide that modulates the GLP-1 receptor comprises a peptide sequence having at least about 99% identity with either SEQ ID NO: 74 or 79.

[0269] In some embodiments, the peptide that modulates the GLP-1 receptor comprises a peptide sequence selected from SEQ ID NO: 74 or 79.

[0270] In some embodiments, the peptide conjugate is a) A peptide that modulates the PYY receptor, comprising the peptide sequence of SEQ ID NO: 6, b) A staple bound to the peptide by a first cysteine ​​and a second cysteine, having the following structure:

[0271] [ka] (

[0272] [ka] Staples that have (which are part of the cysteine ​​residue) Includes.

[0273] In some embodiments, the peptide conjugate is a) A peptide that modulates the PYY receptor, comprising the peptide sequence of SEQ ID NO: 10, b) A HEM bound to the peptide by a first cysteine, the following structure

[0274] [ka] (

[0275] [ka] HEM has (which is part of the cysteine ​​residue) Includes.

[0276] In some embodiments, the peptide conjugate is a) A peptide comprising the peptide sequence of Sequence ID No. 48, which modulates both the GLP-1 receptor and the GCG receptor, b) A staple bound to the peptide by a first lysine and a second lysine, having the following structure:

[0277] [ka] (

[0278] [ka] Staples that have (which are part of the lysine residue) Includes.

[0279] In some embodiments, the peptide conjugate is a) A peptide comprising the peptide sequence of SEQ ID NO: 60 that modulates both the GLP-1 receptor and the GCG receptor, b) A staple bound to the peptide by a first cysteine ​​and a second cysteine, having the following structure:

[0280] [ka] (

[0281] [ka] Staples that have (which are part of the cysteine ​​residue) Includes.

[0282] In some embodiments, the peptide conjugate is a) A peptide comprising the peptide sequence of Sequence ID No. 63 that modulates both the GLP-1 receptor and the GIP receptor, b) A staple bound to the peptide by a first lysine and a second lysine, having the following structure:

[0283] [ka] (

[0284] [ka] Staples that have (which are part of the lysine residue) Includes.

[0285] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-1 receptor, comprising the peptide sequence of SEQ ID NO: 76, b) A staple bound to the peptide by a first cysteine ​​and a second cysteine, having the following structure:

[0286] [ka] (

[0287] [ka] Staples that have (which are part of the cysteine ​​residue) Includes.

[0288] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-1 receptor, comprising the peptide sequence of SEQ ID NO: 77, b) A staple bound to the peptide by a first cysteine ​​and a second cysteine, having the following structure:

[0289] [ka] (

[0290] [ka] Staples that have (which are part of the cysteine ​​residue) Includes.

[0291] Prolactin-releasing peptide (PrRP) peptide conjugate In one embodiment, the Specified herein discloses a peptide conjugate comprising a prolactin-releasing peptide (PrRP). In an exemplary example, the prolactin-releasing peptide (PrRP) comprises two amino acids linked by a staple. Non-limiting examples of amino acids used in the conjugation include cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, 2-amino-6-mercaptohexanoic acid, or other sulfhydryl-containing amino acids. In a prolactin-releasing peptide (PrRP) comprising two amino acids linked by a staple, the two amino acids are separated by approximately, or at least approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acid units. For example, the first amino acid occupies position i, and the second amino acid occupies positions i+7, i+11, i+13, i+15, or i+16. For example, the first amino acid occupies the i position of the peptide, and the second amino acid occupies the i+n position, where n is between 4 and 16. For example, the first amino acid occupies the i position of the peptide, and the second amino acid occupies the i+7 position. For example, the first amino acid occupies the i position of the peptide, and the second amino acid occupies the i+11 position. For example, the first amino acid occupies the i position of the peptide, and the second amino acid occupies the i+15 position. For example, the first amino acid occupies the i position of the peptide, and the second amino acid occupies the i+16 position.

[0292] This specification includes, a) Prolactin-releasing peptide (PrRP), b) A half-life extension molecule bound to a staple, wherein the staple is bound to a peptide at a first amino acid and a second amino acid, and A peptide conjugate containing is disclosed.

[0293] In some embodiments, the first amino acid and the second amino acid are independently selected from sulfhydryl-containing amino acids.

[0294] In some embodiments, the first and second amino acids are independently selected from cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, and 2-amino-6-mercaptohexanoic acid. In some embodiments, the first and second amino acids are cysteine.

[0295] In some embodiments of a prolactin-releasing peptide (PrRP) and a half-life-extending molecule bound to a staple, the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) A half-life extension molecule bound to a staple, wherein the staple is bound to the peptide at a first cysteine ​​and a second cysteine, and The half-life extension molecule contains the following structure:

[0296] [ka]

[0297] [ka] (

[0298] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0299] In some embodiments of a prolactin-releasing peptide (PrRP) and a half-life-extending molecule bound to a staple, the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) A half-life extension molecule bound to a staple, wherein the staple is bound to the peptide at a first cysteine ​​and a second cysteine, and The half-life extension molecule contains the following structure:

[0300] [ka]

[0301] [ka] (

[0302] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0303] In some embodiments of a prolactin-releasing peptide (PrRP) and a half-life-extending molecule bound to a staple, the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) A half-life extension molecule bound to a staple, wherein the staple is bound to the peptide at a first cysteine ​​and a second cysteine, and The half-life extension molecule contains the following structure:

[0304] [ka] (

[0305] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0306] In some embodiments of a prolactin-releasing peptide (PrRP) and a half-life-extending molecule bound to a staple, the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) A half-life extension molecule bound to a staple, wherein the staple is bound to the peptide at a first cysteine ​​and a second cysteine, and The half-life extension molecule contains the following structure:

[0307] [ka] (

[0308] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0309] In some embodiments of a prolactin-releasing peptide (PrRP) and a half-life-extending molecule bound to a staple, the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) A half-life extension molecule bound to a staple, wherein the staple is bound to the peptide at a first cysteine ​​and a second cysteine, and The half-life extension molecule contains the following structure:

[0310] [ka] (

[0311] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0312] In some embodiments of a prolactin-releasing peptide (PrRP) and a half-life-extending molecule bound to a staple, the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) A half-life extension molecule bound to a staple, wherein the staple is bound to the peptide at a first cysteine ​​and a second cysteine, and The half-life extension molecule contains the following structure:

[0313] [ka] (

[0314] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0315] In some embodiments of prolactin-releasing peptide (PrRP), the peptide conjugate is a) A prolactin-releasing peptide (PrRP) containing a peptide sequence selected from sequence numbers 83-105, b) Staples bound to the peptide at the first and second cysteine The half-life extension molecule contains the following structure:

[0316] [ka] (

[0317] [ka] It is bound to a staple that has (a part of the cysteine ​​residue).

[0318] Pharmacokinetics The mechanisms by which peptide conjugates have a beneficial effect on pharmacokinetic or pharmacodynamic behavior include, but are not limited to, (i) preventing or mitigating in vivo proteolytic degradation or chemical modifications that reduce the activity of other therapeutic agents; (ii) improving half-life or pharmacokinetic properties by reducing renal filtration, reducing receptor-mediated clearance, or increasing bioavailability; (iii) reducing toxicity; (iv) improving solubility; and / or (v) increasing the biological activity and / or target selectivity of unconjugated therapeutic agents. Therapeutic agents may include agents that modulate PYY receptor modulators, GLP-1 receptor modulators, GCG receptor modulators, GIP receptor modulators, or combinations thereof, such as peptides.

[0319] Peptide conjugates, when bound to a therapeutic agent, can enhance one or more of the therapeutic agent's pharmacokinetic properties. The peptide conjugates disclosed herein, when measured by pharmacodynamic methods, can enhance one or more of the therapeutic agent's pharmacokinetic properties by at least about 200% compared to the therapeutic agent or the unmodified therapeutic peptide alone. The peptide conjugates disclosed herein, when measured by pharmacodynamic methods, can enhance one or more of the therapeutic agent's pharmacokinetic properties by at least about 300%, 400%, 500%, 600%, 700%, 800%, 900%, and 1000% compared to the therapeutic agent or the unmodified therapeutic peptide alone.

[0320] Pharmacokinetic properties may include half-life. The half-life of a peptide conjugate may be at least about twice as long as the half-life of the unmodified peptide alone. The half-life of peptide conjugates disclosed herein may be at least about three, four, five, or ten times longer than the half-life of the therapeutic agent or the unmodified therapeutic agent alone. The half-life of peptide conjugates disclosed herein may be at least about six, seven, eight, nine, ten, fifteen, twenty, twenty-five, thirty, thirty-five, forty-five, forty-five, or fifty times longer than the half-life of the unmodified peptide alone.

[0321] In some embodiments, the half-life of the peptide conjugate is at least about twice the half-life of the unmodified peptide. In some embodiments, the half-life of the peptide conjugate is at least about five times the half-life of the unmodified peptide. In some embodiments, the half-life of the peptide conjugate is at least about ten times the half-life of the unmodified peptide.

[0322] In addition, the peptide conjugates described herein may have a positive effect in that they increase the productivity of the peptide and / or decrease the immunogenicity of the peptide compared to the unconjugated form of the unmodified therapeutic peptide.

[0323] therapeutic use In one embodiment, the peptide conjugates disclosed herein are useful for treating, mitigating, inhibiting, and / or preventing one or more diseases and / or illnesses. The diseases and / or illnesses may be chronic diseases or illnesses. Alternatively, the diseases and / or illnesses may be acute diseases or illnesses. The diseases or illnesses may be relapsing, refractory, progressive, or in remission. The diseases or illnesses may affect one or more cell types. The one or more diseases and / or illnesses may be autoimmune diseases, inflammatory diseases, or metabolic diseases.

[0324] This specification discloses a method for treating a target disease or illness of interest, comprising the step of administering a peptide conjugate described herein to a target. The disease or illness may be diabetes or obesity, or a medical condition associated with diabetes or obesity. The disease or illness may be non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or a cardiovascular disease. The disease or illness may be an autoimmune disorder. The disease or illness may be Crohn's disease or ulcerative colitis. The disease or illness may be short bowel syndrome (SBS). The disease or illness may be inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or psoriasis. The disease or illness may be Alzheimer's disease, Parkinson's disease, or Huntington's disease. PLC may be administered together with one or more additional therapeutic agents. This specification discloses a method for treating a target disease or illness of interest, comprising the step of administering a composition disclosed herein, comprising one or more peptide conjugates to a target.

[0325] This specification provides a method for preventing or treating a metabolic disorder or disease of interest, comprising the step of administering a peptide conjugate described herein to the target. The metabolic disorder or disease may be diabetes mellitus. The metabolic disorder or disease may be obesity. The metabolic disorder or disease may be glycogen storage disorder, phenylketonuria, maple syrup urine disease, glutaricemia type 1, carbamoyl phosphate synthetase I deficiency, alkaptonuria, medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipid-like congenital adrenal hyperplasia, congenital adrenal hyperplasia, POMPC deficiency, LEPR deficiency, Valde-Biedl syndrome, Alström syndrome, Prader-Willi syndrome, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher disease, or Niemann-Pick disease.

[0326] This specification provides a method for preventing or treating a target NAFLD, NASH, or cardiovascular disease, comprising the step of administering a peptide conjugate described herein.

[0327] This specification provides a method for preventing or treating short bowel syndrome (SBS) in a target area, comprising the step of administering a peptide conjugate described herein.

[0328] This specification provides a method for preventing or treating an inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or psoriasis of interest, comprising the step of administering a peptide conjugate described herein to an interest.

[0329] This specification provides a method for preventing or treating Crohn's disease or ulcerative colitis of a desired target, comprising the step of administering a peptide conjugate described herein to the target.

[0330] This specification provides methods for preventing or treating sleep disorders.

[0331] This specification provides methods for preventing or treating absence seizures. This specification provides methods for preventing or treating chronic kidney disease (e.g., complications of diabetes). This specification provides methods for preventing or treating diabetic heart disease. This specification provides methods for preventing or treating cardiovascular events.

[0332] This specification provides a method for preventing or treating Alzheimer's disease, Parkinson's disease, or Huntington's disease of a desired target, comprising the step of administering a peptide conjugate described herein to the target.

[0333] This specification provides methods for preventing or treating gastrointestinal disorders, including the treatment of neonates with impaired bowel function, osteoporosis, and DPP-IV (dipeptidyl peptidase-IV)-mediated diseases. Examples of gastrointestinal disorders include ulcers, gastritis, digestive disorders, malabsorption syndromes, short bowel syndrome, blind bag syndrome, inflammatory bowel disease, celiac disease (e.g., resulting from gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemia sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, irritable bowel syndrome with diarrhea, small bowel injury, and short bowel syndrome.

[0334] This specification provides methods for preventing or treating radiation enteritis, infectious or post-infectious enteritis, and small bowel damage caused by toxic or other chemotherapeutic agents. These methods may require the administration of peptide conjugates before, concurrently with, or after chemotherapy or radiotherapy to reduce chemotherapy side effects such as diarrhea, abdominal cramps, and vomiting, and to reduce structural and functional damage to the intestinal epithelium resulting from chemotherapy or radiotherapy.

[0335] This specification provides methods for preventing or treating malnutrition, such as diseases like wasting syndrome cachexia and anorexia nervosa.

[0336] This specification provides a method for preventing or treating a disease or illness that would benefit from a PYY receptor modulator in a target subject, the method comprising the step of administering the peptide conjugate described herein to the target subject.

[0337] This specification provides a method for preventing or treating a disease or illness that would benefit from a GLP-1 receptor modulator in a target subject, the method comprising the step of administering the peptide conjugate described herein to the target subject.

[0338] This specification provides a method for preventing or treating a disease or illness that would benefit from a GLP-1 / GIP receptor modulator in a target subject, the method comprising the step of administering the peptide conjugate described herein to the target subject.

[0339] This specification provides a method for preventing or treating a disease or illness that would benefit from a GLP-1 / GCG receptor modulator in a target subject, the method comprising the step of administering the peptide conjugate described herein to the target subject.

[0340] This specification provides a method for preventing or treating a disease or illness that would benefit from a prolactin-releasing peptide (PrRP) receptor modulator in a target of interest, the method comprising the step of administering the peptide conjugate described herein to the target.

[0341] combination This specification provides pharmaceutical compositions comprising the peptide conjugate described herein and one or more additional therapeutic agents.

[0342] Additional therapeutic agents may include one or more other antidiabetic drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic agents, insulin secretagogues, TZD drugs, insulin and insulin analogs, FGF21 and its analogs, leptin or leptin analogs, amylin and amylin analogs, anti-inflammatory drugs, cyclosporine A or FK506, 5-ASA, or statins, or any combination thereof. Additional therapeutic agents may include aspirin.

[0343] Additional therapeutic agents may include therapeutic incretins or their derivatives. Non-exclusive examples of incretins or their derivatives include GLP-1, glucagon, oxytomodulin, exendin-4, GLP-2, GIP, and combinations thereof.

[0344] In some embodiments, the combination therapy demonstrates superior glycemic control, reduced food intake, and weight loss compared to monotherapy. In some embodiments, the combination therapy mimics the beneficial effects of bariatric surgery for obese patients.

[0345] In some embodiments, the PYY receptor modulator is administered together with the GLP-1 receptor modulator.

[0346] In some embodiments, the PYY receptor modulator is administered together with the GLP-1 / GIP receptor modulator.

[0347] In some embodiments, the PYY receptor modulator is administered together with the GLP-1 / GCG receptor modulator.

[0348] In some embodiments, the GLP-1 / GIP receptor modulator is administered together with the GLP-1 receptor modulator.

[0349] In some embodiments, the GLP-1 / GIP receptor modulator is administered together with the GLP-1 / GCG receptor modulator.

[0350] In some embodiments, the GLP-1 / GCG receptor modulator is administered together with the GLP-1 receptor modulator.

[0351] In some embodiments, the combination includes a plurality of peptide conjugates described herein. In some embodiments, the additional therapeutic agent is conjugate 187.

[0352] composition This specification discloses pharmaceutical compositions comprising peptide conjugates described herein and pharmaceutically acceptable excipients or vehicles. Examples of pharmaceutically acceptable excipients or vehicles include carriers, excipients, diluents, antioxidants, preservatives, colorants, fragrances, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobial agents, and surfactants.

[0353] Neutral buffered saline or saline mixed with serum albumin is an exemplary and suitable carrier. Examples of pharmaceutical compositions include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; monosaccharides and disaccharides, and other carbohydrates including glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronic®, or polyethylene glycol (PEG). Further examples of suitable isotonic enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, and sorbitol. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, and sorbic acid. Hydrogen peroxide can also be used as a preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, and tyroxapole. Buffers may be acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl or its conventional buffers. The pH of acetate buffers may be approximately 4-5.5, and the pH of Tris buffer may be approximately 7-8.5. Further pharmaceuticals are specified in Remington's Pharmaceutical Sciences, 18th Edition, ARGennaro, ed., Mack Publishing Company, 1990.

[0354] The composition may be in liquid or lyophilized or freeze-dried form and may contain one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives, and / or fillers. In one embodiment, the lyoprotectant is a non-reducing sugar such as sucrose, lactose, or trehalose. The amount of lyoprotectant typically included is such that the formulation obtained upon reconstitution is isotonic, although hypertonic or slightly hypotonic formulations are also appropriate. In addition, the amount of lyoprotectant must be sufficient to prevent unacceptable levels of protein degradation and / or aggregation during lyophilization. Typical lyoprotectant concentrations of sugars (e.g., sucrose, lactose, trehalose) in pre-lyophilized formulations are about 10 mM to about 400 mM. In another embodiment, the surfactant may be, for example, a nonionic surfactant or an ionic surfactant such as polysorbate (e.g., polysorbate 20, polysorbate 80); poloxamer (e.g., poloxamer 188); poly(ethylene glycol)phenyl ether (e.g., Triton); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl Examples include sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristoamidopropyl-, palmidropropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristoamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate- or disodium methyl ophylline-taurate; MONAQUAT® series (Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic®, PF68, etc.).Exemplary amounts of surfactants that may be present in pre-freeze-dried formulations may be around 0.001-0.5%. Examples of high molecular weight structural additives (e.g., fillers, binders) include acacia, albumin, alginic acid, calcium phosphate (dibase), cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrate, sucrose, tyrose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugars, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives are 0.1% to 10% by weight. In other embodiments, bulking agents (e.g., mannitol, glycine) may be included.

[0355] The compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals via any route available to those skilled in the art, such as intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, intraocular, intraarterial, or intrainjury routes. Parenteral formulations may generally be sterile, pyrogen-free, isotonic aqueous solutions and may optionally contain pharmaceutically acceptable preservatives.

[0356] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions containing saline media or buffering media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactoringer, and fixative oils. Intravenous vehicles include fluid and nutrient supplements, and electrolyte replacement drugs such as ringer's dextrose-based solutions. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. For general information, see Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

[0357] The pharmaceutical compositions described herein may be formulated for controlled or sustained delivery in the form of local concentrations of products (e.g., high doses, storage effects) and / or increased stability or half-life in specific local environments. The compositions may include formulations of peptide conjugates, polypeptides, nucleic acids, or vectors disclosed herein, along with microparticle preparations of polymer compounds such as polylactic acid and polyglycolic acid, as well as implantable delivery devices that provide controlled or sustained release of active agents that can subsequently be delivered as depot injections. Techniques for formulating such sustained or controlled delivery means are known, and various polymers have been developed and used for controlled release and delivery of drugs. Such polymers are generally biodegradable and biocompatible. Polymeric hydrogels, including those formed by the complex formation of enantiomerized polymers or polypeptide segments, and hydrogels with temperature or pH-sensitive properties, may be desirable to provide drug depot effects due to the mild aqueous conditions involved in the capture of bioactive protein agents (e.g., peptide conjugates).

[0358] An appropriate and / or preferred pharmaceutical formulation can be determined based on the intended route of administration, mode of delivery, and desired dose, taking into account the present disclosure and general knowledge of pharmaceutical technology. Regardless of the method of administration, an effective dose can be calculated based on the patient's body weight, body surface area, or organ size. Further improvements to the calculations for determining the appropriate dose for each formulation described herein for the treatment requiring it are customary in the art and are within the scope of work customary in the art. The appropriate dose can be verified using appropriate dose-response data.

[0359] definition When used herein and in the appended claims, the singular forms “a,” “an,” and “the” are to include the plural unless the context makes it clear. Thus, for example, a reference to “drug” includes multiple such drugs, and a reference to “cell” includes one or more cells (or more cells) and their equivalents known to those skilled in the art. When ranges relating to physical properties such as molecular weight or chemical properties such as chemical formulas are used herein, all combinations of ranges and specific embodiments within them, and subcombinations, are intended to be inclusive. When the term “about” refers to a number or range, it means that the number or range referred to is an approximation within the range of experimental variability (or statistical experimental error), and therefore, in some examples, varies by 1% to 15% of the number or range described. The term “comprising” (and related terms such as “comprise,” “comprises,” “having,” or “including”) is not intended to exclude, in other specific embodiments, any embodiment of any material composition, composition, method, or process described herein from “consist of” or “consist essentially of” the described feature.

[0360] As used herein and in the appended claims, unless otherwise specified to the contrary, the following terms have the meanings set forth below:

[0361] "Alkyl" refers to a linear or branched hydrocarbon monoradical having 1 to 10 carbon atoms, or 1 to 6 carbon atoms, which may be fully saturated or unsaturated. The sp3-hybridized carbon of the alkyl residue is attached to the rest of the molecule by a single bond. Examples of saturated hydrocarbon monoradicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, hexyl, heptyl, and octyl, which are long alkyl groups. Wherever it appears herein, a numerical range such as “C1-C6 alkyl” means that the alkyl group consists of one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, or six carbon atoms; however, where no numerical range is given, this definition also encompasses the occurrence of the term “alkyl.” In some embodiments, alkyl is C1-C 10Alkyls are C1-C9 alkyls, C1-C8 alkyls, C1-C7 alkyls, C1-C6 alkyls, C1-C5 alkyls, C1-C4 alkyls, C1-C3 alkyls, C1-C2 alkyls, or C1 alkyls. When alkyls refer to unsaturated linear or branched hydrocarbon monoradicals, they are known as "alkenyls" or "alkynyls." Alkenyls should be understood as being either cis or trans in configuration with respect to the double bond and including both isomers. Examples of alkenyls include, but are not limited to, ethenyl (-CH=CH2), 1-propenyl (-CH2CH=CH2), isopropenyl [-C(CH3)=CH2], butenyl, and 1,3-butadienyl. Wherever it appears herein, numerical ranges such as “C2-C6 alkenyl” mean that the alkenyl group consists of two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, or six carbon atoms; however, where no numerical range is given, this definition also encompasses the occurrence of the term “alkenyl.” In some embodiments, the alkenyl is C2-C 10 Alkenyls are C2-C9 alkenyls, C2-C8 alkenyls, C2-C7 alkenyls, C2-C6 alkenyls, C2-C5 alkenyls, C2-C4 alkenyls, C2-C3 alkenyls, or C2 alkenyls. Examples of alkynyls include, but are not limited to, ethynyl, 2-propynyl, and 2-. Wherever it appears herein, numerical ranges such as "C2-C6 alkynyl" mean that the alkynyl group consists of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms; however, where no numerical range is given, this definition also includes the occurrence of the term "alkynyl". In some embodiments, the alkynyl is C2-C 10These are alkynyl, C2-C9 alkynyl, C2-C8 alkynyl, C2-C7 alkynyl, C2-C6 alkynyl, C2-C5 alkynyl, C2-C4 alkynyl, C2-C3 alkynyl, or C2 alkynyl. Unless otherwise specified herein, alkyl groups are optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc., as described below. In some embodiments, alkyl groups are optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, alkyl groups are optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, alkyl groups are optionally substituted with halogens.

[0362] "Alkylene" refers to a straight-chain or branched-chain divalent hydrocarbon chain. Wherever it appears herein, numerical ranges such as "C1-C6 alkyl" mean that the alkylene consists of one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, or six carbon atoms; however, where no numerical range is given, this definition also encompasses the occurrence of the term "alkylene." In some embodiments, the alkylene is C1-C 10The alkylene is C1-C9 alkylene, C1-C8 alkylene, C1-C7 alkylene, C1-C6 alkylene, C1-C5 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, or C1 alkylene. Unless otherwise specified herein, the alkylene group is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc., as described below. In some embodiments, the alkylene is optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkylene is optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, the alkylene is optionally substituted with halogen.

[0363] "alkoxy" is the formula -OR a R represents the radical, a is an alkyl radical as defined. Unless otherwise specified herein, the alkoxy group may be optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc., as described below. In some embodiments, the alkoxy may be optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkoxy may be optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, the alkoxy may be optionally substituted with halogen.

[0364] "Aryl" represents a radical derived from a hydrocarbon ring system containing hydrogen, 6 to 30 carbon atoms, and at least one aromatic ring. Aryl radicals may be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include condensed ring systems (where the aryl is bonded via aromatic ring atoms when condensed with a cycloalkyl or heterocycloalkyl ring) or bridging ring systems. In some embodiments, the aryl is a 6- to 10-membered aryl. In some embodiments, the aryl is a 6-membered aryl. Examples of aryl radicals derived from the hydrocarbon ring systems of anthreene, naphthylene, phenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indan, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene include, but are not limited to, aryl radicals derived from these hydrocarbon ring systems. In some embodiments, the aryl is phenyl. Unless otherwise specified herein, aryls are optionally substituted with, for example, halogens, aminos, nitriles, nitros, hydroxyls, alkyls, alkenyls, alkynyls, haloalkyls, alkoxys, aryls, cycloalkyls, heterocycloalkyls, heteroaryls, etc. In some embodiments, aryls are optionally substituted with halogens, methyls, ethyls, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, aryls are optionally substituted with halogens, methyls, ethyls, -CN, -CF3, -OH, or -OMe. In some embodiments, aryls are optionally substituted with halogens.

[0365] A "cycloalkyl" refers to a stable, partially or fully saturated monocyclic or polycyclic cyclic carbon atom, which may include fused ring systems (when fused with an aryl or heteroaryl ring, the cycloalkyl is bonded via a non-aromatic ring atom) or bridging ring systems. Typical cycloalkyls include those with 3 to 15 carbon atoms (C3-C3). 15 Cycloalkyl), 3-10 carbon atoms (C3-C 10Examples of cycloalkyls include, but are not limited to, cycloalkyls having 3 to 8 carbon atoms (C3-C8 cycloalkyl), 3 to 6 carbon atoms (C3-C6 cycloalkyl), 3 to 5 carbon atoms (C3-C5 cycloalkyl), or 3 to 4 carbon atoms (C3-C4 cycloalkyl). In some embodiments, the cycloalkyl is a 3 to 6-membered cycloalkyl. In some embodiments, the cycloalkyl is a 5 to 6-membered cycloalkyl. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl or carbocyclic compounds include adamantyl, norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.2]decane, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Examples of partially saturated cycloalkyls include cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Unless otherwise specified herein, cycloalkyls are optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, cycloalkyls are optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, cycloalkyls are optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -OH, or -OMe. In some embodiments, cycloalkyls are optionally substituted with halogens.

[0366] "Halo" or "halogen" refers to bromo, chloro, fluoro, or iodine. In some embodiments, the halogen is fluoro or chloro. In some embodiments, the halogen is fluoro.

[0367] "Haloalkyl" represents an alkyl radical as defined above, which is substituted by one or more halo radicals as defined above, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc.

[0368] "Hypercycloalkyl" refers to a stable 3-24 membered, partially or fully saturated ring radical containing 2-23 carbon atoms and 1-8 heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. Typical heterocycloalkyls include those with 2-15 carbon atoms (C2-C2). 15 Heterocycloalkyl), 2 to 10 carbon atoms (C2-C 10Examples of heterocycloalkyls include, but are not limited to, heterocycloalkyls having 2 to 8 carbon atoms (C2-C8 heterocycloalkyls), 2 to 6 carbon atoms (C2-C6 heterocycloalkyls), 2 to 5 carbon atoms (C2-C5 heterocycloalkyls), or 2 to 4 carbon atoms (C2-C4 heterocycloalkyls). In some embodiments, the heterocycloalkyl is a 3 to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 5 to 6-membered heterocycloalkyl. Unless otherwise specified herein, the heterocycloalkyl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused ring system (where the heterocycloalkyl is bonded via a non-aromatic ring atom when fused with an aryl or heteroaryl ring) or a bridging ring system, and the nitrogen, carbon, or sulfur atoms in the heterocycloalkyl radical may be optionally oxidized, and the nitrogen atom may be optionally quaternized. Examples of such heterocycloalkyl radicals include azilidinyl, azetidinyl, dioxolanil, thienyl[1,3]dithianil, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperadinyl, 4-piperidonyl, pyrrolidinyl Examples include, but are not limited to, yl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianil, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, 1,3-dihydroisobenzofuran-1-yl, 3-oxo-1,3-dihydroisobenzofuran-1-yl, methyl-2-oxo-1,3-dioxol-4-yl, and 2-oxo-1,3-dioxol-4-yl. The term "heterocycloalkyl" also includes, but is not limited to, all cyclic forms of carbohydrates, including monosaccharides, disaccharides, and oligosaccharides.Unless otherwise specified, heterocycloalkyls have 2 to 10 carbon atoms in the ring. It is understood that the number of carbon atoms in a heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) (skeletal atoms of the heterocycloalkyl ring) that make up the heterocycloalkyl. Examples of partially saturated heterocycloalkyls include dihydropyrrolyl and tetrahydropyricine. Unless otherwise specified herein, heterocycloalkyls are optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, heterocycloalkyls are optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, heterocycloalkyls are optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe. In some embodiments, the heterocycloalkyl group is optionally substituted with a halogen.

[0369] "Heteroalkyl" refers to an alkyl group in which one or more of the alkyl backbone atoms are atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or a combination thereof. The heteroalkyl group is bonded to the remainder of the molecule at the carbon atoms of the heteroalkyl group. In one embodiment, the heteroalkyl group is a C1-C6 heteroalkyl group, where the heteroalkyl group consists of 1 to 6 carbon atoms and one or more atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or a combination thereof, and the heteroalkyl group is bonded to the remainder of the molecule at the carbon atoms of the heteroalkyl group. Unless otherwise specified herein, heteroalkyl groups may be optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the heteroalkyl group is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the heteroalkyl group is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, OH, or -OMe. In some embodiments, the heteroalkyl group is optionally substituted with halogen.

[0370] A "heteroaryl" refers to a 5- to 14-membered cyclic radical comprising a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur, and at least one aromatic ring. Heteroaryl radicals may be monocyclic, bicyclic, tricyclic, or tetracyclic, and may include fused cyclic systems (where the heteroaryl is bonded via aromatic ring atoms when fused with a cycloalkyl or heterocycloalkyl ring) or bridging cyclic systems. The nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized, and the nitrogen atom may be optionally quaternized. In some embodiments, the heteroaryl is a 5- to 10-membered heteroaryl. In some embodiments, the heteroaryl is a 5- to 6-membered heteroaryl. In some embodiments, the heteroaryl is a 5-membered heteroaryl. In some embodiments, the heteroaryl is a 6-membered heteroaryl.For example, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranil, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanil, benzonaphthofuranil, benzoxazolyl, benzodioxolyl, benzodioxynil, benzopyranil, benzopyranonil, benzofuranil, benzofuranonil, benzothienyl, benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, sinnolinil, dibenzofuranil, dibenzothiophenyl, furanil, furanonil, isothiazolyl, imidazolyl, indazolyl, indazolyl, isoindolyl, indri Examples include, but are not limited to, nyl, isoindolinyl, isoquinolyl, indolidinyl, isoxazolyl, naphthilidinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxyranil, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyradinyl, 1-oxidepyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxadinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridadinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless otherwise specified herein, heteroaryls are optionally substituted with, for example, halogens, aminos, nitriles, nitros, hydroxyls, alkyls, alkenyls, alkynyls, haloalkyls, alkoxys, aryls, cycloalkyls, heterocycloalkyls, heteroaryls, etc. In some embodiments, heteroaryls are optionally substituted with halogens, methyls, ethyls, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, heteroaryls are optionally substituted with halogens, methyls, ethyls, -CN, -CF3, -OH, or -OMe.In some embodiments, the heteroaryl is optionally replaced with a halogen.

[0371] The term "percent identity" refers to a comparison between two nucleic acid or amino acid sequences. Such comparisons are performed using any number of alignment methods known in the art, including but not limited to global (e.g., Needleman-Wunsch algorithm) or local (e.g., Smith-Waterman, Sellers, or other algorithms). Percent identity often represents the proportion of positions in adjacent sections where the two sequences coincide, and the two sequences are aligned to maximize the coincidences and minimize the discrepancies. In some examples, alignment is performed with no discrepancy between the two sequences. In some examples, the resulting discrepancy is less than 5%, less than 3%, or less than 1%. Further sequence comparison or alignment methods are also consistent with this disclosure.

[0372] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after achieving maximum percentage sequence identity by aligning the sequences and introducing gaps as necessary, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved using various known methods, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning the sequences can be determined, and algorithms required to achieve maximum alignment over the entire length of the sequences being compared are also available. However, for the purposes of this specification, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted to the U.S. Copyright Office, Washington DC, 20559 in user documentation and is registered under copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program must be compiled for use on UNIX® operating systems, including Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain constant.When ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, to an amino acid sequence B, or to an amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a specific % amino acid sequence identity to, with, or to, a given amino acid sequence B) is calculated as 100 × fraction X / Y, where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0373] "Pharmacologically acceptable" means that it is approved or eligible for approval by a federal or state regulatory authority, or that it is listed in the United States Pharmacopeia or any other generally accepted pharmacopoeia for use in animals, including humans.

[0374] A "pharmaceutically acceptable salt" refers to a salt of a compound that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.

[0375] "Pharmacologically acceptable excipients, carriers, or adjuvants" refers to excipients, carriers, or adjuvants that can be administered to a subject together with at least one antibody of the present disclosure, which do not disrupt the pharmacological activity and are non-toxic when administered in a dose sufficient to deliver a therapeutic dose of the compound.

[0376] "Pharmacologically acceptable vehicle" means a diluent, adjuvant, excipient, or carrier to which at least one of the antibodies of this disclosure is administered together.

[0377] Terms such as “treating,” “treatment,” “to treat,” “alleviating,” or “to alleviate” may refer to 1) therapeutic means that cure, delay, reduce the symptoms of, and / or halt the progression of a diagnosed condition or disorder, and / or 2) preventive or relapse-preventive means that prevent and / or delay the onset of a target condition or disorder. “Treatment” refers to a clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed for preventive purposes or during the course of clinicopathology. Desired treatment effects include prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or relief of the condition, and remission or improved prognosis. For this reason, people who need treatment may include those who already have a disorder, those who are prone to developing a disorder, and those who should be prevented from developing a disorder.

[0378] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs or amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, such as hydrogen, a carboxyl group, an amino group, and an alpha carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but can retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes are chemical compounds that have a structure different from the general chemical structure of amino acids but function similarly to naturally occurring amino acids.

[0379] "Disorder" or "disease" means a condition for which one benefits from treatment by the substance / molecule (e.g., the peptide conjugate disclosed herein) or method disclosed herein. This includes chronic and acute disorders or diseases, including conditions that cause mammals to suffer from the disorder of interest.

[0380] For treatment purposes, “mammal” means humans, rodents (e.g., mice and rats), and monkeys; domestic animals and livestock; and any animal classified as a mammal, including dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and other animals used in zoos, sports, laboratories, or as pets. In some embodiments, the mammal is selected from humans, rodents, or monkeys.

[0381] "Modifying" refers to the ability of a peptide to bind to a protein receptor. In some embodiments, the modulator is the ligand for the receptor. In some embodiments, the modulator is an agonist. In some embodiments, the modulator is an antagonist. For example, a peptide that modulates the GLP-1 receptor binds to the GLP-1 receptor (GLP-1R). For example, a peptide that modulates the GCG receptor binds to the GCG receptor (GCGR). For example, a peptide that modulates the GIP receptor binds to the GIP receptor (GIPR). For example, a peptide that modulates the PYY receptor binds to the PYY receptor (PYYR). As a non-limiting example, a peptide that modulates the GLP-1 receptor is a GLP-1R agonist. As a non-limiting example, a peptide that modulates both the GLP-1 receptor and the GCG receptor is a dual GLP-1R / GCGR agonist. As a non-limiting example, a peptide that modulates both the GLP-1 receptor and the GIP receptor is a dual GLP-1R / GIPR agonist. As a non-limiting example, peptides that modulate the PYY receptor are PYYR agonists.

[0382] An "unmodified peptide" refers to either an unmodified sequence (wild-type peptide) or a modified sequence without staples. [Examples]

[0383] Peptides were synthesized using standard solid-phase peptide synthesis (SPPS) techniques and purified via HPLC (as described).

[0384] Unless otherwise specified, all reagents were purchased from suppliers (Sigma Aldrich, Fisher, Oakwood) and used without further purification. All reactions involving air or humidity-sensitive reagents or intermediates were carried out under an inert atmosphere of nitrogen or argon. All solvents used were HPLC grade. Reactions were monitored by LC-MS or thin-layer chromatography (TLC) on Merck 50 × 100 mm silica gel 60 aluminum sheets stained with KMnO4 aqueous solution.

[0385] Flash chromatography purification was performed on a CombiFlash® Rf (Teledyne Isco) on a silica gel packed column (40 μm, Teledyne Isco's RediSep® Rf). The purified final compound eluted as single and symmetrical peaks (confirming a purity of 95% or higher). Half-slice chromatography was performed on a Shimadzu HPLC with a Phenomenex Luna column (C18, 100A pore size, 10 μm particle size, 250 × 10.0 mm, flow: 4 mL / min) or an Agilent 1200 HPLC with a Phenomenex Luna column (C18, 100A pore size, 5 μm particle size, 150 × 21.2 mm, flow: 20 mL / min).

[0386] 1 H and 13¹³C NMR spectra were recorded on a Bruker 400 system for d6-DMSO, CDCl3, or CD3OD. Chemical shifts are provided in parts per million (ppm) using tetramethylsilane as an internal standard. Abbreviations are used as follows: s=singlet, d=doublet, t=triplet, q=quadruplet, p=quintlet, m=multiplet, dd=doublet of doublets, br=broad. Binding constants (J values) are provided in Hertz (Hz). Low-resolution mass spectra were recorded on a Waters Acquity UPLC with a hemomenex Luna Omega C18 column (C18, 100A pore size, 1.6 μm particle size, 50 × 2.1 mm, flow: 0.4 mL / min). Solvents: A- H2O + 0.1% formic acid, B- MeCN + 0.1% formic acid, gradient: 0-1 min 10-90% B, 1-1.6 min 90% B, 1.6-1.7 min 90-10% B, 1.7-2 min 10% B.

[0387] High-resolution mass spectra (HRMS) were recorded using an Agilent 1200 Series Accurate Mass Time-of-Flight (TOF) with an Aeris Widepore column (XB-C8, 3.6 μm particle size, 150 × 2.1 mm, flow: 0.5 mL / min). Solvents: A- H2O + 0.1% formic acid, B- MeCN + 0.1% formic acid, gradient: 0-2 min 5% B, 2-12 min 5-60% B, 12-13 min 60-80% B, 13-14 min 80-20% B, 14-15 min 20-80% B, 15-16 min 80-20% B, 16-17 min 20-95% B, 17-20 min 95% B, 20-21 min 95-5% B.

[0388] General protocol A for loading chlorotrityl chloride resin Fmoc-Lys(ivDde)-OH (60 mg, 100 μmol) was bound to 2-chlorotrityl chloride resin (Novabiochem) (100 mg, 80 μmol) by mixing it with amino acids, resin, and DIEA (70 μL, 400 μmol) in 5 mL of DMF and stirring for 30 minutes. The resin was then washed with DMF (3 times) and DCM (3 times), and the unreacted trityl chloride moieties were capped by treatment with CH3OH / DCM / DIEA (8:1:1) for 10 minutes. The resin was dried under vacuum and stored in a desiccator.

[0389] General Protocol B for Deprotection of Fmoc Protectors Pipericin dissolved in DMF (20%) was added to the resin. The mixture was shaken for 5 minutes and then drained. A fresh 20% pipericin was added, and this time the mixture was shaken for 15 minutes. Ninhydrin and / or TNBS tests were positive. The resin was then washed with DMF (3 times) and DCM (3 times).

[0390] General protocol C for deprotecting the ivDde protecting group After washing with DMF and DCM, the resin was treated with 2% hydrazine dissolved in DMF (5 mL, 15 minutes twice). Ninhydrin and / or TNBS tests were positive. The resin was then washed with DMF (3 times) and DCM (3 times).

[0391] General Protocol D for Peptide Coupling The resin was treated with carboxylic acid derivatives (3 eq) identified using coupling reagents HATU (3.3 eq) and DIEA (3.3 eq) in DMF (5 mL), or this was repeated until the ninhydrin and / or TNBS test was negative. The resin was then washed with DMF (3 times) and DCM (3 times).

[0392] General protocol E for bromoacetylation of resins Next, the resin was treated with anhydrous bromoacetic acid (2.4 eq) and DIEA (2.6 eq) in 200 mL of DCM for 30 minutes.

[0393] General protocol F for peptide cleavage from chlorotrityl resin The resin was treated with DCM (3 times), and the product was cleaved from the resin in DCM containing 10% H2O and 10% triisopropylsilane for 1 hour using 5 mL of 10% TFA. [Examples]

[0394] Synthesis of fatty acid conjugation reagent (FA2)

[0395] [ka]

[0396] Intermediate FA2a. Myristic acid (0.46 g, 2 mmol) was dissolved in 5 mL of DMF. HATU (0.8 g, 2.1 mmol) and DIEA (0.4 mL, 2.2 mmol) were added, followed by Boc-NH-PEG2-COOH (0.5 g, 2 mmol). The reaction mixture was then stirred for 6 hours, and the solvent was removed. The product was extracted with ELISA (3 times with 15 mL). The organic layer was sequentially washed with saturated NaHCO3, chilled HCl (1 M), and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel yielded 0.81 g of tert-butyl(2-(2-tetradecanamideethoxy)ethoxy)ethyl)carbamate as a white solid with a 90% product yield. MS(ES) + )m / z 459.6([M+H] + ), calcd MW 458.4.

[0397] FA2. A solution of FA2a (0.23 g, 0.5 mmol) dissolved in DCM (10 mL) was treated with TFA (2 mL) for 2 hours. The mixture was concentrated, and then bromoacetic anhydride (0.14 g, 0.55 mmol) and DIEA (0.17 mL, 1 mmol) were added to 10 mL of DCM at 0°C. The reaction mixture was then stirred for 2 hours, and the solvent was removed. The product was extracted with ELISA (3 times with 15 mL). The organic layer was sequentially washed with saturated NaHCO3, chilled HCl (1 M), and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel yielded 0.2 g of FA2 as a white solid with 83% product purity. MS(ES) + )m / z 480.4([M+H] + ), calcd MW 479.5. [Examples]

[0398] L1 synthesis

[0399] [ka]

[0400] To a solution of 1,4-diaminobutane (80 μL, 0.795 mmol, 1 eq) dissolved in DCM (10 mL) at 0°C, DIEA (276 μL, 1.59 mmol, 2 eqs) was added, followed by bromoacetic anhydride (413 g, 1.59 mmol, 2 eqs) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification by flash column chromatography on silica gel yielded 1 L as a white solid (162 mg, 0.49 mmol, 61%). MS(ES) + )m / z 331.0([M+H] + ). 1 H NMR (400MHz, methanol-d4) δ 3.94 (s, 4H), 3.40-3.30 (m, 4H), 1.68 (p, J = 3.5Hz, 4H). [Examples]

[0401] L1B synthesis

[0402] [ka]

[0403] To a solution of 1,2-ethylenediamine (30 μL, 0.448 mmol, 1 eq) dissolved in DCM (5 mL) at 0°C, DIEA (172 μL, 0.985 mmol, 2.2 eqs) was added, followed by bromoacetic anhydride (233 mg, 0.897 mmol, 2 eqs) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification on silica gel by flash column chromatography yielded L1B as a white solid (43.9 mg, 0.145 mmol, 32%). MS(ES) + )m / z 302.55([M+H] + ),304.54([M+H] + ). 1 ¹H NMR (400 MHz, methanol-d4) δ 2.49 (s, 4H), 2.06 (s, 4H). [Examples]

[0404] L1C synthesis

[0405] [ka]

[0406] To a solution of 1,3-diaminopropane (30 μL, 0.359 mmol, 1 eq) dissolved in DCM (5 mL) at 0°C, DIEA (138 μL, 0.789 mmol, 2.2 eqs) was added, followed by bromoacetic anhydride (186 mg, 0.718 mmol, 2 eqs) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification by flash column chromatography on silica gel yielded L1C as a white solid (60.8 mg, 0.19 mmol, 53%). MS(ES) +)m / z 316.32([M+H] + ),318.6([M+H] + ). 1 H NMR (400MHz, methanol-d4) δ 3.86 (s, 4H), 3.27 (t, J = 6.8Hz, 4H), 1.74 (p, J = 6.8Hz, 2H). [Examples]

[0407] L1D synthesis

[0408] [ka]

[0409] To a solution of 1,7-diaminohexane (65 μL mg, 0.499 mmol, 1 eq) dissolved in DCM (15 mL) at 0°C, DIEA (208 μL, 1.197 mmol, 2.4 eq) was added, followed by bromoacetic anhydride (259 mg, 0.998 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L1D was obtained as a white solid (120 mg, 0.322 mmol, 64%) by purification on silica gel using flash column chromatography. MS(ES) + )m / z 372.71([M+H] + ),374.70([M+3H] + ). 1 H NMR (400MHz, chloroform-d) δ 6.55(s,2H),3.91(s,4H),3.30(q,J=7.1Hz,4H),1.56(p,J=7.1Hz,4H),1.45-1.29(m,6H). [Examples]

[0410] L1E synthesis

[0411] [ka]

[0412] To a solution of 1,1-diaminoundecane (48 μL mg, 0.257 mmol, 1 eq) dissolved in DCM (10 mL) at 0°C, DIEA (108 μL, 0.616 mmol, 2.4 eq) was added, followed by bromoacetic anhydride (134 mg, 0.515 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification on silica gel by flash column chromatography yielded L1E as a white solid (62.3 mg, 0.145 mmol, 56%). MS(ES) + )m / z 428.33([M+H] + ). 1 H NMR (400MHz, chloroform-d) δ 6.53(s,2H),3.91(s,4H),3.30(q,J=6.8Hz,4H),1.57(q,J=7.2Hz,4H),1.42-1.20(m,14H). [Examples]

[0413] L1F synthesis

[0414] [ka]

[0415] To a solution of cadaverine (48 mg, 0.257 mmol, 1 eq) dissolved in DCM (20 mL) at 0°C, DIEA (284 μL, 1.63 mmol, 2.4 eq) was added, followed by bromoacetic anhydride (353 mg, 1.36 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification by flash column chromatography on silica gel yielded L1F as a white solid (156 mg, 0.453 mmol, 66%). MS(ES) + )m / z 344.65([M+H] + ),346.64([M+H] + ). 1H NMR (400MHz, methanol-d4) δ 3.83(s,4H),3.23(q,J=6.8Hz,4H),1.57(p,J=7.2Hz,4H),1.44-1.33(m,2H). [Examples]

[0416] L1G synthesis

[0417] [ka]

[0418] Intermediate L1Ga To a solution of tert-butylbis(2-aminoethyl)carbamate (167 mg, 0.82 mmol, 1 eq) dissolved in DCM (20 mL), DIEA (342 μL, 11.96 mmol, 2.4 eq) was added, followed by bromoacetic anhydride (426 mg, 1.64 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L1Ga was obtained as a white solid (289 mg, 0.65 mmol, 79%) by purification on silica gel using flash column chromatography. MS(ES) + )m / z 445.71([M+H] + ),447.7([M+H] + ). 1 ¹H NMR (400 MHz, methanol-d4) δ 3.85 (s, 4H), 3.39 (s, 9H), 1.50 (s, 10H).

[0419] L1G Compound L1Ga (20 mg) was dissolved in TFA / DCM (1:1, v / v, 2 mL), stirred at room temperature for 30 minutes, and evaporated (co-evaporation with hexane) to obtain compound L1G as oil. This product was used directly in the previous step. MS(ES + )m / z 345.2([M+H] + ). [Examples]

[0420] L3 synthesis

[0421] [ka]

[0422] Intermediate L3a Myristic acid (184 mg, 0.805 mmol, 1 eq) was dissolved in 4 mL of DMF. HATU (321 mg, 0.845 mmol, 1.1 eq) and DIEA (154 μL, 0.885 mmol, 1.1 eq), followed by Boc-NH-PEG2-COOH (200 mg, 0.805 mmol, 1 eq), were added. The reaction mixture was then stirred for 1.5 hours, and the solvent was removed. The product was dissolved in toluene. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L3a as a white solid (254 mg, 0.55 mmol, 69%). 1 H NMR(400MHz,chloroform-d)δ 3.66-3.54(m,8H),3.49(q,J=5.2Hz,2H),3.35(d,J=6.1Hz,2H),2.20(t,J=7.7Hz ,2H),1.63-1.58(m,2H),1.47(s,8H),1.33-1.24(m,21H),0.90(t,J=6.9Hz,3H). t R = 2.21 minutes (Agilent). MS(ES + )m / z 459.6([M+H] + )

[0423] Intermediate L3b A solution of compound L3a (242 mg, 0.527 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (224 mg, 0.59 mmol, 1.1 eq) was added to a solution of BocNH-PEG2-CO2H (146 mg, 0.527 mol, 1 eq) in DMF (5 mL). Deprotected compound L3a dissolved in DMF and DIEA (183 μL, 1.05 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L3b was obtained as an oil (129 mg, 0.209 mmol, 40%) by purification on silica gel using flash column chromatography. 1 H NMR(400MHz,chloroform-d)δ 6.76(s,1H),6.19(s,1H),5.29(s,1H),3.76(t,J=5.8Hz,2H),3.69-3.62(m,8H),3.57(dt,J=12.3,5.0Hz,6H),3.48(dt,J=10.4,5.5Hz,4 H),3.33(s,2H),2.51(t,J=5.8Hz,2H),2.20(t,J=7.0Hz,2H),1.90-1.75(m,4H),1.64(p,J=7.3Hz,2H),1.46(s,9H),1.33-1.22(m,17H).

[0424] Intermediate L3c Compound L3b (129 mg, 0.209 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. Boc-Orn(Boc)-OH (69 mg, 0.209 mmol, 1 eq) was dissolved in DMF (5 mL) and HATU (88 mg, 0.23 mmol, 1.1 eq) was added. Deprotected compound L3b dissolved in DMF and DIEA (73 μL, 0.419 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L3c was obtained as an oil (137 mg, 0.164 mmol, 78%) by purification on silica gel using flash column chromatography. R = 4.07 minutes (Agilent). MS(ES + )m / z 832.9([M+H] + ). 1 H NMR(400MHz,chloroform-d)δ 7.12(s,1H),6.80(s,1H),6.30(s,1H),4.87(s,1H),3.85-3.73(m,2H),3.68-3.61(m,7H),3.58(p,J=6.1,5.5Hz,7H),3.53-3.36(m,6H), 3.29-3.00(m,2H),2.51(t,J=5.8Hz,2H),2.20(t,J=7.7Hz,2H),2.00-1.74(m,6H),1.71-1.51(m,5H),1.45(s,18H),1.35-1.22(m,21H).

[0425] L3 Compound L3c (137 mg, 0.165 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 10 mL of DCM, and cooled to 0°C. DIEA (115 μL, 0.66 mmol, 4 eq) was added, followed by bromoacetic anhydride (85.8 g, 0.33 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification by flash column chromatography on silica gel yielded L3 as a white solid (56 mg, 0.064 mmol, 39%). R = 3.4 minutes (Agilent). MS(ES + )m / z 872.4([M+H] + ),874.3([M+H] + ). [Examples]

[0426] L4 Synthesis

[0427] [ka]

[0428] Intermediate L4a To a solution of Boc-Orn(Boc)-OH (595 mg, 1.79 mmol, 1 eq) dissolved in DMF (5 mL), HATU (750 mg, 1.79 mmol, 1.1 eq), DIEA (343 μL, 1.97 mmol, 1.1 eq), and amine-PEG3-N3 (391 mg, 1.79 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 16 hours. The product was diluted with ethyl acetate. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L4a as oil (558 mg, 1.05 mmol, 58%). MS(ES) + )m / z 533.13([M+H] +). 1 ¹H NMR (400MHz, chloroform-d)δ 6.82 (s, 1H), 5.25 (d, J=8.3Hz, 1H), 4.75 (s, 1H), 4.19 (s, 1H), 3.76-3.60 (m, 10H), 3.57 (t, J=5.1Hz, 2H), 3.43 (t, J=4.6Hz, 2H), 3.30-3.19 (m, 1H), 3.18-3.03 (m, 1H), 1.85 (s, 4H), 1.68-1.49 (m, 2H), 1.45 (s, 18H).

[0429] Intermediate L4b Compound L4a (548 mg, 1.02 mmol, 1 eq) was dissolved in anhydrous MeOH (10 mL) under argon, and Pd / C (10.9 mg, 0.102 mmol, 0.1 eq) was added, replacing the argon with H2. The reaction mixture was stirred at room temperature for 6 hours, filtered on Celite, and evaporated to obtain compound L4b as oil (516 mg, 1.02 mmol, quantified). This product was used without further purification.

[0430] Intermediate L4c To a solution of octadecanediocate monotert-butyl ester (370 mg, 1.02 mmol, 1 eq) dissolved in DMF (5 mL), HATU (387 mg, 1.02 mmol, 1.1 eq), DIEA (186 μL, 1.07 mmol, 2 eq), and compound L4b (516 mg, 1.02 mmol, 1 eq), dissolved in 1 mL of DMF, were added. The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L4c as oil (697 mg, 0.81 mmol, 79%). 1H NMR(400MHz,chloroform-d)δ 6.94(s,1H),6.42(s,1H),4.81(s,1H),4.20(s,1H),3.65(d,J=6.7Hz,8H),3.59(dt,J=9.7,5.1Hz,4H),3.51-3.35(m,4H) ,3.31-3.18(m,1H),3.17-3.06(m,1H),2.20(q,J=8.0Hz,4H),1.87(s,4H),1.71-1.53(m,6H),1.45(s,26H),1.26(s,24H).

[0431] L4 A solution of L4c (422 mg, 0.49 mmol, 1 eq) dissolved in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0°C. DIEA (327 μL, 1.96 mmol, 4 eq) was added, followed by bromoacetic anhydride (254 mg, 0.98 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. Purification on silica gel by flash column chromatography yielded L4 as a white solid (53 mg, 0.063 mmol, 12%). MS(ES) + )m / z 845.08([M+H] + ),847.07([M+H] + ) 1 H NMR(400MHz, methanol-d4)δ 3.68-3.60(m,8H),3.54(td,J=5.4,3.4Hz,4H),3.43-3.35(m,4H),3.30-3.16(m,2H),2.27(t, J=7.5Hz,2H),2.17(t,J=7.6Hz,2H),1.86-1.73(m,1H),1.72-1.45(m,8H),1.37-1.19(m,28H). [Examples]

[0432] L4A synthesis

[0433] [ka]

[0434] Intermediate L4Aa To a solution of tert-butylbis(2-aminoethyl)carbamate (500 mg, 2.45 mmol, 1 eq) and DIEA (1.02 mL, 5.88 mmol, 2 eq) dissolved in DCM (20 mL) at 0°C, bromoacetic anhydride (1.31 g, 5.04 mmol, 2.05 eq in 1 mL of DCM) was added dropwise. The reaction mixture was stirred at 0°C for 30 minutes and at room temperature for 6 hours, then evaporated under vacuum. The product was purified by flash chromatography and obtained as oil (883 mg, 81%). 1 ¹H NMR (400 MHz, methanol-d4) δ 1.50 (s, 9H), 3.39 (s, 8H), 3.85 (s, 4H). R = 1.04 minutes. MS(ES + )m / z 445.71 / 447.70([M+H] + ).

[0435] Intermediate L4Ab Compound L4Aa (1 eq) was dissolved in DCM / TFA (1:1, v / v), and the solution was stirred at room temperature for 30 minutes and concentrated under vacuum (co-evaporated with heptane). Compound L4Ab was used directly in the next step without purification. R = 0.58 minutes. MS(ES + )m / z 345.65 / 347.67([M+H] + ).

[0436] Intermediate L4Ac HATU (1.05 eq) was added to a solution of mono-tert-butyl succinate (1.05 eq) dissolved in DMF. The reaction mixture was stirred at room temperature for 5 minutes. Compounds L4Ab and DIEA (4 eq) were dissolved in DMF (1 mL) and added to the reaction mixture. The reaction mixture was stirred at room temperature overnight and diluted with AcOEt. The organic phase was washed with HCl 1N and saturated NaHCO3 solution, dried over MgSO4, and evaporated. The product was purified by flash chromatography and obtained as oil. R = 1.07 minutes. MS(ES +)m / z 501.52 / 503.80([M+H] + ).

[0437] Intermediate L4Ad A solution of compound L4Ac (1 eq) dissolved in DCM / TFA (1:1, v / v) was stirred at room temperature for 30 minutes and concentrated under vacuum (co-evaporated with heptane). Compound L4Ac was used directly in the next step without purification. R = 0.57 minutes. MS(ES + )m / z 445.71 / 447.73([M+H] + ).

[0438] Intermediate L4Ae Octadecanedioic acid mono-tert-butyl ester (200 mg, 0.54 mmol, 1 eq) was dissolved in 5 mL of DMF. HATU (225 mg, 0.59 mmol, 1.1 eq) and DIEA (103 μL, 0.59 mmol, 1.1 eq), followed by Boc-NH-PEG3-NH2 (157.8 g, 0.54 mmol, 1 eq), were added. The reaction mixture was then stirred for 3 hours to remove the solvent. The product was dissolved in ethyl acetate. The organic layer was sequentially washed with saturated NaHCO3, 1 M HCl, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired product L4Ae as a white solid (281 mg, 0.43 mmol, 81%). MS(ES) + )m / z 645.5([M+H] + ). 1 H NMR(400MHz,chloroform-d)δ 3.76-3.61(m,8H),3.63-3.54(m,4H),3.48(q,J=5.1Hz,2H),3.34(s,2H),2.20(dt,J=9 .8,7.6Hz,4H),1.67-1.55(m,4H),1.49-1.44(m,17H),1.30(s,6H),1.30-1.24(m,19H).

[0439] L4A A solution of compound L4Ae dissolved in DCM was treated with TFA for 30 minutes. The mixture was concentrated, co-evaporated with heptane, dissolved in DMF, and added to a solution of compounds L4Ad, HATU, and DIEA dissolved in DMF. The reaction mixture was stirred for 3 hours, and half of the sample was purified by HPLC to obtain the desired product L4A. [Examples]

[0440] L5 Synthesis

[0441] [ka]

[0442] General protocols A, B, D (octadecane diacitate mono-tert-butyl ester), C, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0443] Product L5 was obtained as a white solid (73 mg, 0.065 mmol, 11%) by purifying the crude product by half-separation HPLC with mass detection. 1 H NMR(400MHz, methanol-d4)δ 4.36(td,J=8.9,5.1Hz,2H),3.89(q,J=11.4Hz,2H),3.82(s,2H),3.74(t, J=6.2Hz,2H),3.60(s,4H),3.54(t,J=5.5Hz,2H),3.37(q,J=5.2Hz,2H),3 .29-3.11(m,5H),2.44(t,J=6.2Hz,2H),2.26(dt,J=12.3,7.5Hz,4H),1.8 9-1.77(m,2H),1.76-1.49(m,10H),1.48-1.38(m,2H),1.37-1.25(m,25H). [Examples]

[0444] L5A synthesis

[0445] [ka]

[0446] Intermediate L5Aa A solution of Fmoc-OSu (131 g, 388 mmol) dissolved in DCM (200 mL) was added dropwise to a solution of diethylenetriamine (20 g, 194 mmol) dissolved in DCM (200 mL) under N2 at -40°C, and the mixture was stirred for 2 hours. Completion of the reaction was confirmed by LC-MS. The crude product in the solution was not purified and was used directly in the next step. 1 H NMR(400MHz,DMSO-d6)δ 7.88(d,J=7.6Hz,4H),7.68(d,J=7.6Hz,4H),7.43-7.24(m,10H),4.30(d,J=6 .4Hz,4H),4.21(d,J=6.4Hz,2H),3.06(d,J=5.6Hz,4H),2.57(d,J=7.6Hz,4H). MS(ES) + )m / z 548.2([M+H] + ).

[0447] Intermediate L5Ab Compound L5Aa (106 g, 194 mmol) was dissolved in DCM (400 mL), to which DMAP (4.74 g, 38.8 mmol) and tetrahydrofuran-2,5-dione (67.9 g, 678 mmol) were added, and the mixture was stirred at 25°C for 14 hours. Completion of the reaction was confirmed by LC-MS. 1N HCl was added to the reaction mixture until the pH reached 5-6, and the mixture was stirred for 15 minutes to separate the organic phase. The organic phase was then washed with water and saturated NaCl (500 mL), and the aqueous phase was extracted twice with DCM (500 mL). The combined DCM was dried over anhydrous Na2SO4 and concentrated under vacuum. Using DCM / MeOH (80:0-5:1) as the eluate, the crude product was purified by column chromatography on silica gel to obtain compound L5Ab (57.6 g, 45% yield) as a white solid powder. 1H NMR(400MHz,DMSO-d6)δ 12.09(s,1H),7.87(d,J=7.5Hz,4H),7.66(d,J=7.0Hz,4H),7.23-7.48(m,10H),4.24 -4.33(m,4H),4.14-4.22(m,2H),3.27(s,4H),2.95-3.19(m,4H),2.37-2.44(m,4H). MS(ES) + )m / z 648.2([M+H] + ).

[0448] L5A General protocols A, B, D (octadecane diacitate mono-tert-butyl ester), C, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-PEG2-propionic acid), B, D (compound L5Ab), B, E, F.

[0449] The crude product was purified by HPLC to obtain product L5A as a white solid (5.2 g, 11% yield). MS(ES) + )m / z 1188.5([M+H] + ). [Examples]

[0450] L6 Synthesis

[0451] [ka]

[0452] Intermediate L6a Palmitic acid (235 mg, 0.919 mmol, 1.05 eq) was dissolved in 4 mL of DMF. HATU (349 mg, 0.919 mmol, 1.1 eq) and DIEA (167 μL, 0.963 mmol, 1.05 eq), followed by Boc-NH-PEG2-NH2 (200 mg, 0.875 mmol, 1 eq), were added. The reaction mixture was then stirred for 2 hours, and the solvent was removed. The product was dissolved in RINKAN. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, HCl, and brine, dried over Na2SO4, filtered, and concentrated to obtain the desired compound L6a as a white solid (412 mg, 0.84 mmol, 97%). 1 H NMR(400MHz,chloroform-d)δ 6.17(s,1H),5.07(s,1H),3.58(s,4H),3.53(t,J=5.0Hz,3H),3.43(q,J=5.3Hz,2H),3.36-3.2 1(m,2H),2.15(t,J=7.5Hz,2H),1.66-1.54(m,2H),1.32-1.15(m,26H),0.84(t,J=6.6Hz,3H).

[0453] Intermediate L6b A solution of compound L6a (412 mg, 0.84 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (353 mg, 0.931 mmol, 1.1 eq) was added to a solution of BocNH-PEG2-CO2H (258 mg, 0.931 mmol, 1.1 eq) in DMF (5 mL). Deprotected compound L6a dissolved in DMF and DIEA (294 μL, 1.69 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, HCl, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L6b was obtained as an oil (329 mg, 0.51 mmol, 60%) by purification on silica gel using flash column chromatography. 1H NMR(400MHz,chloroform-d)δ 6.79(s,1H),6.28(s,1H),5.28(s,1H),3.68(t,J=5.8Hz,2H),3.61-3.44(m,14H),3.38(p,J=5.6Hz,4H),3.24(q,J=5.5Hz,2H ),2.42(t,J=5.8Hz,2H),2.11(t,J=7.9Hz,2H),1.55(p,J=7.2Hz,2H),1.38(s,9H),1.32-1.10(m,24H),0.81(t,J=6.7Hz,3H).

[0454] Intermediate L6c A solution of compound L6b (329 mg, 0.51 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (213 mg, 0.56 mmol, 1.1 eq) was added to a solution of Boc-Orn(Boc)-OH (186 mg, 0.56 mmol, 1.1 eq) in DMF (5 mL). Deprotected compound L6b dissolved in DMF and DIEA (177 μL, 1.02 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with ELISA. The organic layer was sequentially washed with saturated NaHCO3, 1 M HCl, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L6c was obtained as an oil (326 mg, 0.37 mmol, 94%) by purification on silica gel using flash column chromatography. 1 1H NMR (400 MHz, chloroform-d)δ 7.18(s,1H),6.92(s,1H),6.48(s,1H),5.61(d,J=8.4Hz,1H),5.08(t,J=5.9Hz ,1H),4.13(s,1H),3.73-3.65(m,2H),3.59-3.44(m,14H),3.42-3.29(m,8H),3 .19-2.86(m,2H),2.42(t,J=5.9Hz,2H),2.10(d,J=7.3Hz,2H),1.78-1.63(m,1 H),1.60-1.40(m,5H),1.35(s,18H),1.26-1.09(m,22H),0.80(t,J=6.7Hz,3H).

[0455] L6 Compound L6c (100 mg, 0.116 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 10 mL of DCM, and cooled to 0°C. DIEA (80.8 μL, 0.46 mmol, 4 eq) was added, followed by bromoacetic anhydride (61.9 mg, 0.238 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L6 was obtained as a white solid (50.1 mg, 0.055 mmol, 40%) by purification on silica gel using flash column chromatography. 1 1H NMR (400 MHz, methanol-d4) δ 4.39(dd,J=8.4,5.5Hz,1H),3.91(q,J=11.4Hz,2H),3.84(s,2H),3.76(t,J= 6.2Hz,2H),3.63(d,J=7.1Hz,8H),3.57(q,J=5.5Hz,6H),3.43-3.36(m,6H),3 .25(t,J=13.9,6.8Hz,2H),2.49(t,J=6.2Hz,2H),2.21(t,J=7.5Hz,2H),1.9 1-1.79(m,1H),1.75-1.53(m,5H),1.42-1.25(m,24H),0.92(t,J=6.7Hz,3H). [Examples]

[0456] L7 Synthesis

[0457] [ka]

[0458] Intermediate L7a Stearic acid (261 mg, 0.919 mmol, 1.05 eq) was dissolved in 4 mL of DMF. HATU (349 mg, 0.919 mmol, 1.1 eq) and DIEA (167 μL, 0.963 mmol, 1.05 eq), followed by Boc-NH-PEG2-NH2 (200 mg, 0.875 mmol, 1 eq), were added. The reaction mixture was then stirred for 2 hours, and the solvent was removed. The product was dissolved in RINKAN. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated to obtain the desired compound L7a as a white solid (430 mg, 0.83 mmol, 95%). 1 H NMR(400MHz,chloroform-d)δ 3.69-3.59(m,4H),3.56(t,J=5.1Hz,4H),3.46(q,J=5.2Hz,2H),3.40-3.23(m,2H),2.18(t, J=7.6Hz,2H), 1.62(t,J=7.3Hz,2H),1.45(s,9H),1.35-1.19(m,30H),0.88(t,J=6.7Hz,4H).

[0459] Intermediate L7b A solution of compound L7a (426 mg, 0.87 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (366 mg, 0.96 mmol, 1.1 eq) was added to a solution of BocNH-PEG2-CO2H (266 mg, 0.96 mmol, 1.1 eq) in DMF (5 mL). Deprotected compound L7a dissolved in DMF and DIEA (304 μL, 1.75 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L7b was obtained as an oil (360 mg, 0.53 mmol, 61%) by purification on silica gel using flash column chromatography. 1H NMR(400MHz,chloroform-d)δ 6.75(s,1H),6.18(s,1H),5.26(s,1H),3.75(t,J=5.8Hz,2H),3.69-3.52(m,14H),3.47(p,J=5.4Hz,4H),3.33(q,J=5.5Hz,2H),2.50 (t,J=5.8Hz,2H),2.19(t,J=7.5Hz,2H),2.07(s,1H),1.63(p,J=7.3Hz,2H),1.46(s,9H),1.37-1.19(m,29H),0.89(t,J=6.7Hz,3H).

[0460] Intermediate L7c A solution of compound L7b (360 mg, 0.53 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (223 mg, 0.58 mmol, 1.1 eq) was added to a solution of Boc-Orn(Boc)-OH (195 mg, 0.58 mmol, 1.1 eq) in DMF (5 mL). Deprotected compound L7b dissolved in DMF and DIEA (186 μL, 1.07 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L7c was obtained as an oil (373 mg, 0.42 mmol, 78%) by purification on silica gel using flash column chromatography. 1 H NMR(400MHz,chloroform-d)δ 7.14(s,1H),6.84(s,1H),6.35(s,1H),5.53(d,J=8.2Hz,1H),5.05-4.88 (m,1H),4.20(s,1H),3.82-3.69(m,2H),3.65-3.31(m,22H),3.23-3.00(m ,2H),2.48(t,J=5.8Hz,2H),2.17(t,J=7.8Hz,2H),1.87-1.72(m,1H),1. 67-1.48(m,5H),1.42(s,18H),1.34-1.14(m,29H),0.87(t,J=6.9Hz,3H).

[0461] L7 Compound L7c (100 mg, 0.112 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 10 mL of DCM, and cooled to 0°C. DIEA (78 μL, 0.44 mmol, 4 eq), followed by bromoacetic anhydride (62 mg, 0.24 mmol, 2.05 eq) dissolved in 1 mL of DCM, was added. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. The product was dissolved in ethyl acetate. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. L7 was obtained as a white solid (95 mg, 0.10 mmol, 91%) by purification on silica gel by flash column chromatography. MS(ES) + )m / z 931.31([M+H] + ),933.25([M+H] + ). 1 H NMR(400MHz, methanol-d4)δ 4.39(dd,J=8.5,5.4Hz,1H),3.91(q,J=11.3Hz,2H),3.84(s,2H),3.76(t, J=6.2Hz,2H),3.63(d,J=7.0Hz,8H),3.57(t,J=5.5Hz,6H),3.42-3.35(m,6 H),3.31-3.13(m,4H),2.49(t,J=6.2Hz,2H),2.20(t,J=7.4Hz,2H),1.91- 1.79(m,1H),1.75-1.56(m,6H),1.39-1.26(m,26H),0.92(t,J=6.3Hz,3H). [Examples]

[0462] L8 synthesis

[0463] [ka]

[0464] General protocols A, B, D (hexadecanedioic acid mono-tert-butyl ester), C, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0465] Product L8 was obtained as a white solid (42.6 mg, 0.038 mmol, 22%) by purifying the crude product by half-separation HPLC with mass detection. 1 1H NMR (400 MHz, methanol-d4) δ 4.38(td,J=8.6,5.1Hz,2H),3.91(q,J=11.3Hz,2H),3.84(s,2H),3.76(q,J=6 .1Hz,4H),3.65-3.59(m,8H),3.56(td,J=5.5,1.7Hz,4H),3.43-3.37(m,4H),3 .31-3.16(m,4H),2.48(dt,J=15.7,6.2Hz,4H),2.28(dt,J=12.6,7.5Hz,4H), 1.95-1.79 (m, 1H), 1.77-1.51 (m, 10H), 1.49-1.41 (m, 2H), 1.40-1.26 (m, 31H). [Examples]

[0466] L9 Synthesis

[0467] [ka]

[0468] General protocols A, B, D (heptadecanedioic acid mono-tert-butyl ester), C, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0469] Product L9 was obtained as a white solid (49 mg, 0.089 mmol, 9%) by purifying the crude product by half-separation HPLC with mass detection. 11H NMR (400 MHz, methanol-d4) δ 4.45-4.33(m,2H),3.92(t,J=10.9Hz,2H),3.85(d,J=1.1Hz,2H),3.77(q,J=6. 0Hz,4H),3.63(s,8H),3.57(t,J=5.6Hz,4H),3.40(t,J=5.5Hz,4H),3.25(dq,J =22.7,6.7Hz,4H),2.48(dt,J=15.6,6.2Hz,4H),2.29(dt,J=13.2,7.4Hz,4H), 1.95-1.79(m,2H),1.80-1.50(m,10H),1.51-1.41(m,2H),1.40-1.27(m,20H). [Examples]

[0470] L12 Synthesis

[0471] [ka]

[0472] General protocols A, B, D (octadecane dioic acid), C, D (Fmoc-PEG2-propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0473] Product L12 was obtained as a white solid (51.7 mg, 0.054 mmol, 3%) by purifying the crude product by half-separation HPLC with mass detection. 1 1H NMR (400 MHz, methanol-d4) δ 4.39(td,J=9.2,5.1Hz,2H),3.92(qd,J=11.4,1.2Hz,2H),3.85(s,2H),3.7 6(t,J=6.2Hz,2H),3.63(s,4H),3.57(t,J=5.5Hz,2H),3.40(q,J=5.1Hz,2H) ,3.30-3.12(m,6H),2.47(t,J=6.1Hz,2H),2.29(dt,J=12.1,7.4Hz,4H),1. 95-1.77 (m, 2H), 1.78-1.50 (m, 10H), 1.48-1.40 (m, 2H), 1.39-1.26 (m, 22H). [Examples]

[0474] L14 Synthesis

[0475] [ka]

[0476] Intermediate L14a To a solution of hexadecanedioic acid monotert-butyl ester (102 mg, 0.3 mmol, 1 eq) dissolved in DMF (5 mL), HATU (125 mg, 0.33 mmol, 1.1 eq), DIEA (51 μL, 0.33 mmol, 1.1 eq), and compound L4b (151.9 mg, 0.30 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L14a as oil (147 mg, 0.176 mmol, 59%). 1 H NMR(400MHz,chloroform-d)δ 6.87(s,1H),6.40(s,1H),5.32(s,2H),4.79(s,1H),4.20(s,1H),3.66 (d,J=7.0Hz,8H),3.60(dt,J=10.0,5.1Hz,4H),3.49-3.45(m,3H),3.3 1-3.18(m,1H),3.13-3.06(m,1H),2.21(td,J=7.8,6.0Hz,4H),1.88-1 .78(m,1H),1.66-1.53(m,7H),1.51-1.42(m,27H),1.36-1.19(m,20H).

[0477] L14 Compound L14a (40 mg, 0.048 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0°C. DIEA (34 μL, 0.1924 mmol, 4 eq), followed by bromoacetic anhydride (23.63 mg, 0.098 mmol, 2.05 eq) dissolved in 1 mL of DCM, was added. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L14 was obtained as a white solid (18.3 mg, 0.022 mmol, 46%) by purification on silica gel using flash column chromatography. MS(ES) + )m / z 817.1([M+H] + ),819.09([M+H] + ). 1 H NMR(400MHz, methanol-d4)δ 4.38(dd,J=8.4,5.5Hz,1H),3.92(q,J=11.2,10.6Hz,2H),3.84(s,2H),3.69-3.61(m,8H),3.56(td,J=5.5,2.6Hz,4H),3.44-3.36(m,4H),3 .30-3.14(m,2H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.91-1 .78(m,1H),1.76-1.67(m,1H),1.67-1.54(m,6H),1.40-1.29(m,20H). [Examples]

[0478] L15 Synthesis

[0479] [ka]

[0480] Intermediate L15a To a solution of 20-(tert-butoxy)-20-oxoicosanoic acid (360 mg, 0.90 mmol, 1.05 eq) dissolved in DMF (5 mL), HATU (343 mg, 0.90 mmol, 1.05 eq), DIEA (300 μL, 1.71 mmol, 2 eq), and compound L4b (435 mg, 0.858 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with ethyl acetate. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L15a as oil (555 mg, 0.625 mmol, 72%). 1 H NMR(400MHz,chloroform-d)δ 6.87(s,1H),6.40(s,1H),4.79(s,1H),4.21(s,1H),3.76-3.53(m,15H),3.47(s,5H),3.32-3.05(m,3 H),2.29-2.17(m,4H),1.90-1.76(m,4H),1.69-1.53(m,2H),1.52-1.41(m,33H),1.36-1.20(m,29H).

[0481] L15 Compound L15a (100 mg, 0.112 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0°C. DIEA (79 μL, 0.45 mmol, 4 eq) was added, followed by bromoacetic anhydride (60 mg, 0.231 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L15 was obtained as a white solid (17.5 mg, 0.02 mmol, 18%) by purification on silica gel using flash column chromatography. MS(ES) + )m / z 873.21([M+H] + ),875.20([M+H] + ) 1H NMR(400MHz, methanol-d4)δ 4.38(dd,J=8.4,5.5Hz,1H),3.91(q,J=11.4Hz,2H),3.84(s,2H),3.72-3.61(m,8H),3.56(td,J=5.5,2.7Hz,4H),3.44-3.35(m, 5H),3.30-3.17(m,2H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.92-1.77(m,1H),1.75-1.53(m,7H),1.40-1.27(m,27H). [Examples]

[0482] L16 Synthesis

[0483] [ka]

[0484] Intermediate L16a To a solution of Boc-Orn(Boc)-OH (400 mg, 1.2 mmol, 1 eq) dissolved in DMF (10 mL), HATU (504 mg, 1.32 mmol, 1.1 eq), DIEA (230 μL, 1.32 mmol, 1.1 eq), and amine-PEG2-N3 (210 mg, 1.20 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 4 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. The desired compound L16a was obtained as oil (471 mg, 0.96 mmol, 80%) by purification on silica gel using flash column chromatography. 1 ¹H NMR (400MHz, methanol-d4) δ 4.01 (t,J=6.6Hz,1H), 3.71-3.60 (m,6H), 3.55 (t,J=5.5Hz,2H), 3.41-3.37 (m,3H), 3.04 (t,J=6.2Hz,2H), 1.78-1.66 (m,1H), 1.62-1.48 (m,3H), 1.48-1.39 (m,18H).

[0485] Intermediate L16b Compound L16a (471 mg, 0.9 mmol, 1 eq) was dissolved in anhydrous MeOH (10 mL) under argon. Pd / C (10.2 mg, 0.09 mmol, 0.1 eq) was added to the solution, and the argon was replaced with H2. The reaction mixture was stirred at room temperature for 6 hours, filtered on Celite, and evaporated to obtain compound L16b as oil (295.5 mg, 0.64 mmol, 71%). This product was used without further purification. MS(ES) + )m / z 462.51([M+H] + ).

[0486] Intermediate L16c To a solution of octadecanediocate monotert-butyl ester (281 mg, 0.76 mmol, 1 eq) dissolved in DMF (5 mL), HATU (288 mg, 0.76 mmol, 1 eq), DIEA (132 μL, 0.76 mmol, 1 eq), and compound L16b (351 mg, 0.76 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with dimethyl phosphate. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L16c as oil (351 mg, 0.43 mmol, 57%). 1 H NMR(400MHz, methanol-d4)δ 3.61(s,4H),3.54(td,J=5.6,2.3Hz,4H),3.40-3.34(m,4H),3.04(t,J=6.6Hz,2H),2.20(td,J= 7.6,5.9Hz,4H),1.77-1.68(m,2H),1.64-1.48(m,2H),1.48-1.42(m,28H),1.35-1.26(m,26H).

[0487] L16 A solution of compound L16c (31 mg, 0.038 mmol, 1 eq) dissolved in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0°C. DIEA (27 μL, 0.152 mmol, 4 eq) was added, followed by bromoacetic anhydride (21 mg, 0.078 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L16 was obtained as a white solid (12.6 mg, 0.015 mmol, 41%) by purification on silica gel using flash column chromatography. MS(ES) + )m / z 801.13([M+H] + ),803.12([M+H] + ). 1 H NMR(400MHz, methanol-d4)δ 4.37(dd,J=8.5,5.4Hz,1H),3.91(q,J=11.3Hz,2H),3.84(s,2H),3.63(s,4H),3.57(td,J=5.6,2.6Hz,4H),3.43-3.36(m,4H) ),3.31-3.17(m,1H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.90-1.79(m,1H),1.76-1.54(m,7H),1.41-1.30(m,26H). [Examples]

[0488] L17 Synthesis

[0489] [ka]

[0490] Intermediate L17a To a solution of Boc-Orn(Boc)-OH (400 mg, 1.2 mmol, 1 eq) dissolved in DMF (10 mL), HATU (504 mg, 1.32 mmol, 1.1 eq), DIEA (230 μL, 1.32 mmol, 1.1 eq), and amine-PEG2-N3 (316 mg, 1.20 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 4 hours. The product was diluted with ELISA. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L17a as oil (454 mg, 0.78 mmol, 66%). 1 ¹H NMR (400MHz, methanol-d4) δ 4.04-3.97 (m, 1H), 3.71-3.58 (m, 14H), 3.54 (t, J=5.4Hz, 2H), 3.37 (t, J=5.0Hz, 4H), 3.04 (t, J=6.6Hz, 2H), 1.75-1.67 (m, 1H), 1.62-1.48 (m, 3H), 1.48-1.41 (m, 18H).

[0491] Intermediate L17b Compound L17a (454 mg, 0.9 mmol, 1 eq) was dissolved in anhydrous MeOH (10 mL) under argon, and Pd / C (8.3 mg, 0.078 mmol, 0.1 eq) was added, replacing the argon with H2. The reaction mixture was stirred at room temperature for 6 hours, filtered on Celite, and evaporated to obtain compound L17b as oil (192 mg, 0.35 mmol, 45%). This product was used without further purification.

[0492] Intermediate L17c To a solution of octadecanediocate monotert-butyl ester (225 mg, 0.61 mmol, 1 eq) dissolved in DMF (5 mL), HATU (231 mg, 0.61 mmol, 1 eq), DIEA (106 μL, 0.61 mmol, 1 eq), and compound L17b (335 mg, 0.61 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with dimethyl ethyl acetate. The organic layer was sequentially washed with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification on silica gel by flash column chromatography yielded the desired compound L17c as oil (178 mg, 0.20 mmol, 32%). 1 H NMR(400MHz,chloroform-d)δ 5.32(s,2H),3.74-3.63(m,11H),3.59(dt,J=10.9,5.0Hz,4H),3.52-3.43(m,4H),3.27- 3.08(m,2H),2.22(d,J=7.6Hz,4H),1.69-1.52(m,6H),1.51-1.42(m,27H),1.27(s,26H).

[0493] L17 Compound L17c (45.6 mg, 0.05 mmol, 1 eq) was dissolved in DCM (2 mL) and treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0°C. DIEA (36 μL, 0.202 mmol, 4 eq) was added, followed by bromoacetic anhydride (27 mg, 0.103 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0°C for 30 minutes and at room temperature for 1.5 hours to remove the solvent. L17 was obtained as a white solid (14.9 mg, 0.017 mmol, 33%) by purification on silica gel using flash column chromatography. MS(ES) + )m / z 889.18([M+H] + ),891.17([M+H] + ) 1H NMR(400MHz, methanol-d4)δ 4.38(dd,J=8.3,5.5Hz,1H),3.92(q,J=11.3Hz,2H),3.84(s,2H),3.67-3.60(m,7H),3.56(td,J=5.5,3.5Hz,4H),3.45-3.35(m, 5H),3.32-3.15(m,3H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.90-1.76(m,1H),1.74-1.57(m,7H),1.41-1.26(m,25H). [Examples]

[0494] L18 Synthesis

[0495] [ka]

[0496] General protocols A, B, D (Octadecanedioic acid mono-), C, D (Fmoc-PEG2-propionic acid), B (Fmoc-PEG2-propionic acid), B, (Fmoc-PEG2-propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0497] Product L18 was obtained as a white solid (47 mg, 0.036 mmol, 10%) by purifying the crude product by half-separation HPLC with mass detection. + )m / z 1276.39([M+H] + ),1278.37([M+H] + ).

[0498] General procedure for bromoacetyl peptide stapling / conjugation The peptide was dissolved at a concentration of 2 mM in 1:3 (v / v) MeCN / 30 mM NH4HCO3 buffer (pH 8.5) using 1.5 eq of bromoacetyl staples. The pH of the reaction mixture was readjusted with ammonium hydroxide to correct the pH drop caused by the peptide TFA counterion. More MeCN was added, especially for the insoluble peptide. The reaction mixture was stirred at room temperature for 2-4 hours, and then acidified by adding acetic acid dropwise to a pH of 5. The resulting solution was lyophilized and purified by reverse-phase HPLC.

[0499] General solid-phase protocol for lactam stapling The orthogonal protection (Dde / Mmt) of the amine side chain holding the peptide-resin at each stapling position was swollen in DMF for 1 hour. The Dde protecting group was removed from the first side chain by treatment with 2% hydrazine solution in DMF (twice for 15 minutes). TNBS testing was positive. The linker construction blocks specified below were coupled as described, and TNBS testing was negative. The solvent was changed to DCM, and the Mmt group was removed from the second side chain by treatment with 1% TFA in DCM containing 5% TIPS (five times for 2 minutes). The resin was washed with DCM, 10% DIEA in DMF, and DMF, and TNBS testing was positive. The linker was cyclized, and the PEG-fatty acid portion of the staple (where applicable) was extended as described below. The fully stapled peptide was cleaved from the resin over 3 hours using 95% TFA, 2.5% TIPS, and 2.5% H2O. The peptide cleavage mixture was evaporated to form an oil, which was then triturated, washed with diethyl ether, and purified by reverse-phase HPLC. The Dde / Alloc protection scheme can also be used in this method, but this requires the addition of allyl alcohol as a scavenger to the Dde deprotection cocktail to prevent the co-occurrence of the Alloc allyl moiety.

[0500] Synthesis of K(Fmoc) linker

[0501] [ka]

[0502] Intermediate Ka Fmoc-β-Ala-OH (1.00 g, 3.21 mmol) and di-tert-butyliminodiaacetate (0.461 g, 2.68 mmol) were suspended in 100 mL of DCM. HATU (1.02 g, 2.68 mmol) and DIEA (3.32 mL, 12.8 mmol) were added, and the reaction mixture was stirred at room temperature for 3.5 hours. The solvent was evaporated, and the residue was dissolved in MeOH. The product was purified by flash column chromatography on silica gel (hexane / siRNA) to obtain a white solid (0.802 g, 56%). 1 H NMR(400MHz,chloroform-d)δ 7.78(d,J=7.4Hz,2H),7.62(d,J=7.4Hz,2H),7.42(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),5.66(t,J=5.7Hz,1H),4.35(d, J=7.3Hz,2H),4.23(t,J=7.3Hz,1H),4.10(s,2H),4.02(s,2H),3.56(q,J=5.7Hz,2H),2.55(t,J=5.7Hz,2H),1.49(s,18H).

[0503] K(Fmoc) Linker Compound Ka was treated with 20 mL of 1:1 TFA / DCM for 2 hours. After evaporating the solvent and triturating the residue, the K(Fmoc) linker was obtained as a white solid (0.371 g, 58%) by washing with diethyl ether. MS(ES) + )m / z 427.15([M+H] + ).

[0504] Synthesis of A(Fmoc) linker

[0505] [ka]

[0506] A solution of 5-aminoisophthalic acid (1.00 g, 5.5 mmol) dissolved in 10 mL of dioxane was added to a degassed solution of Na2CO3 (1.46 g, 5.5 mmol) dissolved in 15 mL of water. The solution was then cooled on ice, and a solution of Fmoc chloride (1.42 g, 5.5 mmol) dissolved in 10 mL of dioxane was added dropwise over 15 minutes with stirring. The reaction mixture was then stirred for 1 hour, and then at room temperature for 24 hours. The dioxane was removed under vacuum, and the remaining aqueous solution was acidified with 1 M HCl. The resulting solid precipitate was then washed with diethyl ether (4 times with 10 mL), dissolved again in ethyl ether, filtered, washed with brine, dried over Na2SO4, filtered, and concentrated to obtain A(Fmoc) linker as a white solid (119 mg, 5%). 1 H NMR(500MHz,DMSO-d6)δ 13.24(s,2H),10.12(s,1H),8.33(d,J=1.5Hz,2H),8.12(t,J=1.5Hz,1H),7.91(d,J=7.6Hz,2H),7.76(dd,J=7. 6,1.2Hz,2H),7.43(t,J=7.6Hz,2H),7.36(td,J=7.6,1.2Hz,2H),4.50(d,J=6.8Hz,2H),4.33(t,J=6.8Hz,1H).

[0507] General Protocol G for Simple Lactam Staples of the "A1" and "K1" Series In the linker coupling, a suitable diacid construction block (2 eq) was attached using HATU (4 eq) and DIEA (4 eq) in DMF (2 hours, once). The cyclization step was achieved using HATU (1 eq) and DIEA (2 eq) in DMF (2 hours, once).

[0508] General Protocol H for Trifunctional Lactam Staples of "K" PEG Fatty Acids In linker coupling, intramolecular symmetric anhydride coupling of the constructed block K(Fmoc) linker (2eq) was performed at room temperature for 10 minutes using DIC (2eq) in dry DCM and catalyst DMAP. The peptide-resin solvent was replaced with DCM, and then the anhydride was added and stirred overnight. The resin was drained and washed with DCM and DMF. The linker was cyclized overnight by treatment with DIC (1eq) and HOBt or HOAt (1eq) in DMF, and the TNBS test was negative. The remaining uncyclized linker was capped by treatment with 10% acetic anhydride in DMF (30 minutes). The linker Fmoc group was deprotected by treatment with 20% pipericin in DMF (twice for 10 minutes). The TNBS test was positive. For the deprotection cycle (5+10 mins, room temperature), 20% pipericin in DMF was used, and the subsequent stapled PEG and fatty acid construction blocks were successively attached to a linker-free amine via the standard coupling chemistry method: construction blocks (3 eq), HATU (3 eq), and DIEA (6 eq) in DMF (1 hour at room temperature).

[0509] General Protocol I for the Trifunctional Lactam Staple of "A"PEG Fatty Acids In linker coupling, the constructed block A (Fmoc) linker (2eq) was attached using HATU (4eq) and DIEA (4eq) in DMF (2 hours, once). Cyclization was achieved using HATU (1eq) and DIEA (2eq) in DMF (2 hours, once). The remaining uncyclized linker was capped by treatment with 10% acetic anhydride in DMF (30 minutes). The Fmoc linker group was deprotected by treatment with 20% pipericin in DMF (10 minutes, twice). Positive TNBS testing was not observed with aniline nitrogen. Fmoc-β-Ala-OH (3eq) was coupled using HATU (3eq) and DIEA (6eq) in DMF (1 hour, four times, room temperature), or as a symmetric anhydride using DIC / DMAP in DCM (2 hours, room temperature). For the deprotection cycle (5+10 mins, room temperature), 20% pipericin in DMF was used, and the subsequent stapled PEG and fatty acid construction blocks were successively attached to a linker-free amine via the standard coupling chemistry method: construction blocks (3 eq), HATU (3 eq), and DIEA (6 eq) in DMF (1 hour at room temperature).

[0510] In some embodiments, the peptide conjugates described herein include half-life extension portions or staples as shown in Table 6.

[0511] [Table 6-1]

[0512] [Table 6-2]

[0513] [Table 6-3]

[0514] [Table 6-4]

[0515] [Table 6-5]

[0516] [ka] This is the cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue portion.

[0517] [ka] This refers to the lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0518] In some embodiments, the PYY peptide conjugates described herein are as shown in Table 7.

[0519] [Table 7-1]

[0520] [Table 7-2]

[0521] [Table 7-3]

[0522] [Table 7-4]

[0523] [Table 7-5]

[0524] [Table 7-6]

[0525] In some embodiments, the GLP-1R / GCGR dual agonist peptide conjugates described herein are as shown in Table 8.

[0526] [Table 8-1]

[0527] [Table 8-2]

[0528] [Table 8-3]

[0529] [Table 8-4]

[0530] In some embodiments, the GLP-1R / GIPR dual agonist peptide conjugates described herein are as shown in Table 9.

[0531] [Table 9-1]

[0532] [Table 9-2]

[0533] [Table 9-3]

[0534] In some embodiments, the GLP-1R peptide conjugates described herein are as shown in Table 10.

[0535] [Table 10]

[0536] In some embodiments, the peptide conjugates described herein are as shown in Tables 11 and 12.

[0537] [Table 11]

[0538] [Table 12-1]

[0539] [Table 12-2]

[0540] [Table 12-3]

[0541] Bioassay protocol A β-arrestin supplementation assay for the GPR10-activated PathHunter CHO-K1 GPR101 β-arrestin orphan GPCR cell line was purchased from DiscoverX. Briefly, cells (20 μL of 5000 cells per well) were seeded into a 384-well white solid plate, covered with a metal lid, and incubated overnight. On day 2, the culture medium was replaced with fresh medium (FBS-free in the 0% FBS group). Cells were treated in three ways at 37°C, 5% CO2 for 90 minutes with 5 μL of 12-fold diluted PrRP31 as a positive control and sample peptide (starting at a concentration of 400 nM and a 1:3 serial dilution) in protein dilution buffer (0.1% BSA) (from the PathHunter® detection kit). The PathHunter® detection kit purchased from DiscoverX was used for detection. 12.5 μl of diluted standard detection solution was added to each well and incubated at room temperature in the dark for 1 hour. The emission signal was measured using ViewLux (PerkinElmer). EC was used with prism software. 50 The value was retrieved.

[0542] cAMP assay for NPFF2R activation Human NPFF2R-stably overexpressing CHO cells (20 μL, 5000 cells per well; obtained from Christopher McCurdy's lab at the University of Florida College of Pharmacy) were seeded in 384-well white solid plates, covered with a metal lid, and incubated overnight. On day 2, the culture medium was replaced with fresh medium (without FBS in the 0% FBS group). Cells were treated with 5 μL of PrRP31 or an analog (starting at a concentration of 20 μM and a 1:3 serial dilution) in a 12-point dose-response assay, 20 μM forskolin in culture medium as a positive control, and 0.5 mM IBMX (3-isobutyl-1-methylxanthine) to inhibit cAMP degradation. The assay was performed in three runs at 37°C and 5% CO2 for 30 minutes. cAMP levels were detected using the Cisbio cAMP dynamic 2 kit. Briefly, 25 μL of cAMP detection reagent (containing cAMP-d2, cryptotate conjugate, and lysis buffer in a 1:1:38 ratio) was added and stirred at room temperature for 1 hour. In the negative control wells of the cells, cAMP detection reagent without d2 was added. The plates were then read at Ex 320 nm, Em-1 665 nm, and Em-2 615 nm. Graphs were plotted as ratios or delta-F using Prism software, and EC 50 Obtained. Ratio = A 665nm / B 620nm X10^4. %Delta F = (Standard or sample ratio - Ratio) neg ) / Ratio neg x100.

[0543] Peptide stability in plasma 12 μL of 1 mM peptide stock solution (in DMSO) was added to 300 μL of mouse plasma (final concentration 20 μM). The sample was incubated at 37°C for 48 hours. At specific time intervals (0, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours), 25 μL of plasma was withdrawn and added to 150 μL of cold acetonitrile / H2O (9:1, v / v) + 0.1% TFA to precipitate plasma proteins. The sample was incubated at 0°C for 30 minutes and centrifuged at 17 rpm for 10 minutes (4°C). The sample was analyzed using LC-MS (QTOF).

[0544] In vivo pharmacokinetic studies In vivo PK was performed by WuXi AppTec Co., Ltd. in accordance with the IACUC guidelines, which comply with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals, the WuXi IACUC standard animal treatment, applicable WuXi standard operating procedures, and generally recognized good laboratory practices. Unfed male C57 mice (7-9 weeks old) obtained from SLAC Laboratory Animal Co. Ltd. or SIPPR / BK Laboratory Animal Co. Ltd. (Shanghai, China) were acclimated for at least 3 days, and then administered subcutaneously (sc) at a dose of 5 mL per kg of body weight of 0.2 mg / mL of compound 60 (18-S4) (1 mg / kg dose) dissolved in normal physiological saline (0.9% NaCl). Blood samples (70 μL) were collected from the posterior orbital or saphenous vein at the following time points: 0.25, 0.5, 1, 3, 7, 24, 48, and 72 hours (n=3 per group, 3 groups). During acclimatization, animals were housed in groups and individually during the experiment. The animal housing environment was controlled (18–26°C, 30–70% relative humidity, 12 hours of artificial light and 12 hours of darkness). All animals were given free access to Certified Rodent Diet (SLAC Laboratory Animal Co. Ltd) and water. All blood samples were transferred to microcentrifuge tubes with 2 μL of 0.5 M K2 EDTA anticoagulant and placed on wet ice until centrifugation, which was performed at 3000 g for 15 minutes (4°C) within 30 minutes of collection. Plasma was stored in polypropylene tubes, rapidly frozen on dry ice, and stored at -70°C until LC-MS / MS analysis.

[0545] LC-MS analysis of plasma samples obtained from pharmacokinetic studies WuXi AppTec performed PK bioanalysis. An aliquot of 8 μL plasma sample was added to 8 μL of 4% H3PO4, and plasma proteins were precipitated using 160 μL of methanol with 100 ng / mL of Glybride as an internal standard. The mixture was thoroughly stirred and centrifuged at 3220 g for 15 minutes (4°C). 10 μL of the supernatant was analyzed using a SCIEX Triple Quad® 6500. + LC-MS / MS system (ES + The compound was injected into an ACQUITY UPLCR HSS T3 column (1.8 μm, 2.1 × 50 mm) according to the specified procedure. A solvent gradient of 10–60% B over 1 minute was used for analysis, where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile (flow rate 0.6 mL / min, column temperature 60°C). The retention time for compound 60(18-S4) was 0.96 minutes. LC-MS data were analyzed using Analyst 1.6.3 software. Calibration curves were generated using eight non-zero calibration standards consisting of high, medium, and low concentrations, including those at the limit of quantification (LLOQ) of 1–3 ng / mL. Test sample analysis was performed simultaneously with one set of calibration standards and two sets of samples using the calibration curves. Plasma concentration versus time data were analyzed using a non-compartmental method with Phoenix WinNonlin 6.3 software. Due to limitations in mouse volume / sampling, sparse sampling was used. Therefore, a single PK profile was obtained by combining concentrations from various animals, and the PK parameter estimates were averaged.

[0546] Example A: cAMP HTRP assay (PYY) To measure the effect of peptide-induced NPY2R-mediated inhibition of cAMP production, a cAMP HTRF assay was performed according to the manufacturer's instructions (cAMP-Gs Dynamic kit, Cisbio). Briefly, cAMP Hunter CHO cells expressing NPY2R (DiscoveRx) were seeded overnight in 20 μl of F12 medium at 37°C and 5% CO2 in a white 384-well plate at a cell density of 5,000 cells per well. The following day, the medium was removed and replaced with 20 μl of Opti-MEM (Gibco) in or without 10% FBS. Various concentrations of peptide (prepared as 5-fold Opti-MEM solution) and forskolin (final concentration 10 μM, direct activator of adenylate cyclase enzyme) were added and incubated at 37°C for 30 minutes. Detection reagents were added and incubated for a further 60 minutes at room temperature, and read on a compatible HTRF reader (PHERAstar). Concentration-response curves were obtained using nonlinear regression analysis with Prism software (GraphPad Software Inc.).

[0547] [Table 13]

[0548] [Table 14-1]

[0549] [Table 14-2]

[0550] [Table 14-3]

[0551] Example B: In vivo study Intravenous injection The compound was dissolved in sterile saline and administered intravenously at a final dose of 0.033 mg / kg to male, non-fasted Sprague Dolly rats (n=3 per group) via femoral vein cannula over 1 hour. The formulation was administered at a rate of 1.67 mL / kg / h. Blood samples (approximately 250 μL) were collected via jugular vein cannula at 0.25, 0.5, 0.75, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8, 24, 30, and 48 hours after the start of injection for pharmacokinetic analysis. These samples were transferred to microtainer tubes containing 25 μL of K2EDTA as an anticoagulant and a protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at -80°C until analysis.

[0552] Preparation of plasma samples Aliquots of each plasma sample were placed in a 96-well plate. Tween-20 was added to each well to a final concentration of 0.05%. The plate was then vortexed, and 3 volumes of 0.1% TFA in a 2:1 ethanol:acetonitrile mixture with a suitable internal standard were added to each well. The plate was vortex-mixed again, and then centrifuged at 2844xg for 10 minutes. The supernatant was placed in a clean 96-well plate and evaporated under a nitrogen stream at 45°C. The residue was reconstituted in 20% acetonitrile (aq) with 0.1% formic acid.

[0553] LC / MS quantification of peptides in plasma All calibration standards were prepared in control rat plasma with K2EDTA and a protease inhibitor cocktail. Samples and standards were analyzed by TurboIonSpray® ULC-MS / MS using a system consisting of a CTC HTS PAL autoinjector (Leap, Carrboro, NC), an Agilent Infinity 1290 system with a column oven (Palo Alto, CA), a Valco switching valve (Houston, TX), and an AB Sciex API 5600 TripleTOF® or Sciex API 4000QTrap mass spectrometer (Framingham, MA). Samples were injected into a 2.1 × 50 mm reversed-phase C18 analytical column, typically Waters ACQUITY UPLC HSS T3, 1.8 μm (Waters Corporation, Milford, MA) or similar. Chromatographic separation was achieved by a gradient method using water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) as mobile phases. The initial conditions consisted of 95% A and 5% B. Depending on the peptide, the organic component B was increased to 95% over a period of 3-4 minutes. The typical flow rate was 600 μL / min. The column temperature was kept constant at 40 or 45°C. Peptides were quantified by monitoring ions, which were one or more products produced from diversely charged parent ions.

[0554] [Table 15]

[0555] [Table 16]

[0556] Example C: Optimization of staple length and position in PYY analog By examining the structure of homologous neuropeptide Y (NPY) that binds to the human G protein-binding neuropeptide Y receptor Y2 (NPY2R), we induced the selection of stapling sites on PYY. To minimize disruption of critical peptide receptor interactions, we avoided residues on the surfaces interacting with receptors (Y20, L24, Y27, and I28) during site selection for covalent modification. Rapid cleavage of the N-terminus of PYY by post-secretion dipeptidyl peptidase-4 (DPP-4) resulted in the truncated peptide PYY, which is the dominant form in circulation. 3-36 (PYY1: Sequence ID 1) was obtained. Because PYY1 exhibited higher specificity for the Y2 receptor subtype than PYY, it was decided to use this truncated form for development. A pair of PYY1 analogs were synthesized by incorporating di-cysteine ​​mutations at selected stapling sites representing a scan of the entire sequence. Cysteine ​​substitutions were selected to enable bromoacetyl-functionalized staple binding using the aforementioned solution-phase chemistry method. 40

[0557] We first screened the i,i+7diCys mutant and found the optimal position of the sequence in stapling using 10-atom staples L1.

[0558] To measure peptide-induced NPY2R-mediated inhibition of cAMP production, a cAMP HTRF (cyclic adenosine monophosphate homogeneous time-degraded fluorescence) assay was performed according to the manufacturer's instructions (cAMP-Gs Dynamic kit, Cisbio). Briefly, cAMP Hunter CHO cells expressing NPY2R (DiscoveRx) were seeded overnight in 20 μl of F12 medium at a cell density of 5,000 cells per well in a white 384-well plate at 37°C and 5% CO2. The following day, the medium was removed and replaced with 20 μl of Opti-MEM (Gibco) in or without 10% FBS. Various concentrations of peptides (prepared as 5X solutions in Opti-MEM) and forskolin (a direct activator of adenylate cyclase enzyme, final concentration 10 μM) were added and incubated at 37°C for 30 minutes. The detection reagent was added and incubated at room temperature for a further 60 minutes, and the results were read on a compatible HTRF reader (PHERAstar). Concentration-response curves were obtained by nonlinear regression analysis using Prism software (GraphPad Software Inc).

[0559] Numerous diCys substitution sites showed resistance to unstapled PYY1 (2-9, 10-17, 20-27, and 23-30), and stapling at positions 23-30 by L1 resulted in sub-nanomolecular potency similar to the natural sequence (Table 17). Furthermore, longer stapling at positions i,i+11 and i,i+15 may enhance the proteolytic stability of the peptide, but stapling at length-matched L1D and L1E is expected to negatively affect their own activity. In addition, mutations were incorporated into the PYY1 sequence to enhance the potency of the natural peptide (sequence "PYY2" or SEQ ID NO: 2). PYY2 analogs stapled at positions 10-17 and 23-30 were also found to be powerful NPY2R agonists. Staples L1F and L1G (slightly longer than L1) also showed resistance at positions 10-17. Therefore, the PYY1 and PYY2 sequences that were stapled at positions 10-17 and 23-30 were taken forward for fatty acid conjugation to improve serum binding.

[0560] [Table 17]

[0561] Example D: Fatty acid conjugation enhances serum protein binding and extends the half-life. A library of staples was synthesized by incorporating various PEG linkers and fatty acid types to facilitate rapid screening of conjugates. To measure peptide-induced NPY2R-mediated inhibition of cAMP production, a cAMP HTRF (cyclic adenosine monophosphate homogeneous time-degraded fluorescence) assay was performed according to the manufacturer's instructions (cAMP-Gs Dynamic kit, Cisbio). Briefly, cAMP Hunter CHO cells expressing NPY2R (DiscoveRx) were seeded overnight in 20 μl of F12 medium at a cell density of 5,000 cells per well in a white 384-well plate at 37°C and 5% CO2. The following day, the medium was removed and replaced with 20 μl of Opti-MEM (Gibco) in the presence or absence of 10% FBS. Peptides of various concentrations (prepared as 5X solutions in Opti-MEM) and forskolin (a direct activator of adenylate cyclase enzyme, final concentration 10 μM) were added and incubated at 37°C for 30 minutes. Detection reagents were added and incubated at room temperature for a further 60 minutes, and the results were read on a compatible HTRF reader (PHERAstar). Concentration-response curves were obtained by nonlinear regression analysis using Prism software (GraphPad Software Inc).

[0562] The results of this assay are confirmed in Table 18. Overall, a significant change was observed between the activity determined in the presence and absence of serum in staples L4 and L5. For example, the EC of conjugates with staples L4 and L5 was compared to that of PYY1 conjugates tested under conditions with 10% FBS. 50 The EC2 levels were 250 nM and 310 nM at positions 10-17, respectively, and 150 nM and 340 nM at positions 23-30, respectively. In contrast, the EC2 levels of unstapled PYY1 tested under the same conditions were... 50 The EC of the conjugate with staples L4 and L5 was 0.97 nM. Similarly, the EC of the conjugate with staples L4 and L5 compared to the PYY2 conjugate at 10% FBS. 50The values ​​for positions 10-17 were 21 and 15 respectively, and for positions 23-30 were 9.2 and 170 respectively, in contrast to the EC of unstapled PYY1 tested under the same conditions. 50 The value was 0.45 nM.

[0563] [Table 18]

[0564] Example E: Symmetrically stapled conjugates are promising for NPY2R. A "symmetrical" staple L5A was incorporated to avoid the formation of positional isomers, which can occur during stapling with "asymmetrical" L5. To measure peptide-induced NPY2R-mediated inhibition of cAMP production, a cAMP HTRF (cyclic adenosine monophosphate homogeneous time-resolved fluorescence) assay was performed according to the manufacturer's instructions (cAMP-Gs Dynamic kit, Cisbio). Briefly, cAMP Hunter CHO cells expressing NPY2R (DiscoveRx) were seeded overnight in 20 μl of F12 medium at a cell density of 5,000 cells per well in a white 384-well plate at 37°C and 5% CO2. The following day, the medium was removed and replaced with 20 μl of Opti-MEM (Gibco) in the presence or absence of 10% FBS. Peptides of various concentrations (prepared as 5X solutions in Opti-MEM) and forskolin (a direct activator of adenylate cyclase enzyme, final concentration 10 μM) were added and incubated at 37°C for 30 minutes. Detection reagents were added and incubated at room temperature for a further 60 minutes, and the results were read on a compatible HTRF reader (PHERAstar). Concentration-response curves were obtained by nonlinear regression analysis using Prism software (GraphPad Software Inc).

[0565] Table 19 shows the activity of conjugates stapled with L5A. The EC of symmetrically stapled conjugates is shown below. 50In 10% FBS, the EC was 160 nM in the 23rd and 30th staples of PYY1. In PYY2, the EC of the symmetrically stapled conjugates was 160 nM. 50 The levels were 14 nM and 36 nM in the 10-17th and 23-30th staples, respectively, within 10% FBS.

[0566] [Table 19]

[0567] In addition, simple lipid modification using FA2 conjugates (without stapling) was found to result in remarkably potent NPY2R agonists. As shown in the dose-response curves in and out of serum, clear serum transitions were observed for stapling (diCys mutant 40) or lipid-modified fatty acid conjugate PYY analogs at a single Cys conjugation site (62), suggesting enhanced serum binding and further extension of the in vivo half-life.

[0568] Example F: The half-life of the PYY conjugate is extended. To determine the effect of extending the half-life, the pharmacokinetic properties of the conjugate were assessed in vivo. The conjugate was dissolved in sterile saline and administered intravenously at a final dose of 0.033 mg / kg to male non-fasted Sprague Dolly rats (n=3 per group) via femoral vein cannula over 1 hour. The formulation was administered at a rate of 1.67 mL / kg / h. Blood samples (approximately 250 μL) were collected via jugular vein cannula at 0.25, 0.5, 0.75, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8, 24, 30, and 48 hours after the start of injection for pharmacokinetic analysis and transferred to a microtainer tube containing 25 μL of K2EDTA as an anticoagulant and a protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at -80°C until analysis.

[0569] Aliquots of each plasma sample were placed in a 96-well plate. Tween-20 was added to each well to a final concentration of 0.05%. The plate was then vortex-mixed, and 3 volumes of 0.1% TFA in a 2:1 ethanol:acetonitrile mixture with a suitable internal standard were added to each well. The plate was vortex-mixed again, and then centrifuged at 2844xg for 10 minutes. The supernatant was placed in a clean 96-well plate and evaporated under nitrogen at 45°C. The residue was reconstituted in 20% acetonitrile (aq) with 0.1% formic acid.

[0570] All calibration standards were prepared in control rat plasma with K2EDTA and a protease inhibitor cocktail. Samples and standards were analyzed by TurboIonSpray® ULC-MS / MS using a system consisting of a CTC HTS PAL autoinjector (Leap, Carrboro, NC), an Agilent Infinity 1290 system with a column oven (Palo Alto, CA), a Valco switching valve (Houston, TX), and an AB Sciex API 5600 TripleTOF® or Sciex API 4000QTrap mass spectrometer (Framingham, MA). Samples were injected into a 2.1 × 50 mm reversed-phase C18 analytical column, typically Waters ACQUITY UPLC HSS T3, 1.8 μm (Waters Corporation, Milford, MA) or similar. Chromatographic separation was achieved by a gradient method using water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) as mobile phases. The initial conditions consisted of 95% A and 5% B. Depending on the conjugate, the organic component B was increased to 95% over a period of 3-4 minutes. The typical flow rate was 600 μL / min. The column temperature was kept constant at 40°C or 45°C. The conjugate was quantified by monitoring one or more ions, which were products produced from diversely charged parent ions.

[0571] As shown in Table 20, PYY1 analogs stapled at positions 10–17 with L4 and L5 showed half-lives of 0.45 hours and 5.4 hours in rats, respectively. PYY1 analogs stapled at positions 23 and 30 with L4 and L5 showed half-lives of 2.4 hours and 3.9 hours in rats, respectively. PYY2 analogs with staples at positions 10–17 retained activity with half-lives of 1.9 hours and 2.0 hours, respectively. As shown in Figure 2 and Table 21, PYY2 stapled at positions 23–30 exhibited even better long-acting effects, with a maximum half-life of 15 hours in rats. Lipid modification at position 10 also extended the half-life to a maximum of 12 hours. As observed with other commercially available lipid-modified conjugate therapeutics such as semaglutide, this effect corresponds to a large in vitro serum shift and is presumed to be due to favorable interaction between the conjugate and serum albumin. Staples L4, L5, and L5A, as well as lipid FA2 (unstapled), all incorporated fatty acid moieties with carboxylic acid groups, and were found to yield the most favorable pharmacokinetic properties. L5, FA2, and L5A, which were adorned with "internal" carboxylates on the lysine linker, showed even better results. Serum transitions were found to show enhanced albumin binding, but the predicted in vivo extension of half-life was not observed with analogs stapled at positions 10–17, likely due to the fact that stapling at this position does not prevent proteolysis.

[0572] [Table 20]

[0573] Detailed pharmacokinetic profiles of the long-acting analogs 40 and 62 are shown in Table 21. Both conjugates exhibited a more than 10-fold increase in half-life and a significant decrease in clearance compared to SEQ ID NO: 2. This result is similar to that of semaglutide, a fatty acid conjugated GLP-1R agonist administered once weekly to humans.

[0574] [Table 21]

[0575] Example G: The predicted half-life of the stapled PYY conjugate in human plasma is long. The conjugate serum albumin binding affinity was directly measured using the Biacore surface plasmon resonance (SPR) assay. This affinity was used to determine the unbound fraction (f) of each conjugate. u The steady-state distributed volume (V) was calculated. ss Using ) and clearance (CL), the following formula:

[0576]

number

[0577]

number

[0578]

number

[0579] Table 19 shows the predicted human half-lives based on the relative growth rate corrected for albumin binding, along with parameters obtained experimentally for both rat serum albumin (RSA) and human serum albumin (HSA). Comparing all compounds with semaglutide, the affinity of all compounds was relatively high for both RSA and HSA. In addition, no significant species differences were observed.

[0580] [Table 22]

[0581] In particular, conjugate 40 demonstrated a remarkable extension of half-life by 14 hours in rats, compared to a planned human half-life of approximately 4.5 days. Furthermore, in vivo studies revealed highly favorable effects of food intake restriction and significant weight loss in a chronic efficacy study when combined with the previously discovered long-acting GLP-1R agonist conjugate 187. Comparison with the approved peptide therapy semaglutide supports the likelihood that the observed rodent half-life translates to a planned pharmacokinetic profile in humans suitable for once-weekly administration.

[0582] Example H: The PYY analog showed high specificity for NPY2R. The specificity of PYY analogs to NPY2R was assessed using a luciferase assay. HEK293 cells were infected with a lentivirus encoding the firefly luciferase gene under the control of a cAMP-reactive region (CRE) promoter (Qiagen, Netherlands), and then selected for 1 week using 1 μg / mL puromycin (Life Technologies, Carlsbad). Viable cells (referred to as CRE-HEK293) were expanded and then transfected with G418 selective mammalian expression plasmids encoding human NPY1R, NPY2R, NPY4R, and NPY5R. The plasmids were transfected into CRE-HEK293 cells using Lipofectamine2000 and selected with 400 μg / mL genethecin (Life Technologies, Carlsbad, CA). Stable single-colony cell lines overexpressing both CRE luciferase and NPY receptors were then established for in vitro activity assays of each NPY receptor. These cells were seeded at a density of 5000 cells / well in 384-well plates and cultured for 18 hours at 37°C and 5% CO2 in DMEM containing 10% FBS. Cells were treated with conjugates for 24 hours, and receptor activation was reported by luminescence intensity using One-Glo(Promega,WI) luciferase reagent according to the manufacturer's instructions. EC of each conjugate was measured using GraphPad Prism 6 software (GraphPad, San Diego, CA). 50 They sought it.

[0583] As shown in Table 23, SEQ ID NO: 1, SEQ ID NO: 2, and conjugate 21 showed high specificity for NPY2R compared to other NPY receptors. Unstapled PYY1 analogs showed high specificity for EC 50 The EC2 levels for NPY1R, NPY4R, and NPY5R were 1900 nM, 6700 nM, and 410 nM, respectively, but for NPY2R they were EC2 levels. 50 It was 0.49 nM. In the unstapled PYY2 analog, EC 50For NPY1R and NPY4R, the EC was over 10,000 nM, for NPY5R it was 1,200 nM, but for NPY2R it was 2.2 nM. For conjugate 21, i.e., the PYY2 analog with staple L5 at positions 10-17, EC 50 The levels were 0.39 for NPY2R and over 10,000 nM for all other NPY receptors tested.

[0584] [Table 23]

[0585] Example I: The PYY analog reduced food intake in mice. Considering the well-established anorexia-inducing effect of PYY administration, food intake studies were conducted in C57BL / 6 wild-type mice using conjugate 40 administered by subcutaneous injection (SC) at doses of 0.04 and 0.2 mg / kg. Conjugate 40 was tested in combination with conjugate 187, a previously published long-acting GLP-1R agonist.

[0586] C57BL / 6 wild-type male mice (15 weeks old, Jackson Labs, Bar Harbor, ME) that had been regularly fed solid feed were adapted to a reverse light cycle and administered a single dose of conjugate (5 mL / kg) subcutaneously (n=6, 2 mice per cage). Food intake was monitored at 0 (start of dark cycle), 3, 6, 12, and 24 hours after administration, and body weight was monitored at 0 and 48 hours after administration. As shown in Figure 3A, a significant decrease in food intake was observed in all groups, and administration of conjugate 40 alone showed a dose-dependent decrease in food consumption in the wild-type model. Conjugate 187 administered at 0.01 mg / kg showed a similar decrease in food intake as conjugate 40 administered at 0.04 mg / kg, but the most significant decrease in food intake was observed when conjugate 187 was used in combination with conjugate 40 at both 0.04 and 0.2 mg / kg. As a result, cumulative food intake over 24 hours decreased by 64% and 90%, respectively. As shown in Figure 3B, a single dose of the combined conjugates 187 and 40 resulted in a significant weight loss (-5%) 48 hours after administration, demonstrating a long-lasting effect.

[0587] Example J: Administration of the PYY analog resulted in weight loss in mice. A two-week chronic study was conducted in a diet-induced obesity (DIO) mouse model to investigate the effects of daily administration of Conjugate 40 on body weight and glucose homeostasis.

[0588] Male mice (18 weeks old, Taconic Biosciences) of a diet-induced obesity (DIO) model were continuously fed a high-fat diet (D12492, 60% fat diet) and administered a conjugate subcutaneously daily for up to 13 days (n=6, 2 mice per cage, regular light cycle). The mean body weight at the start of the experiment was 50g. Mouse body weight was measured on days 0, 2, 4, 6, 8, 10, 12, and 13. Mice were fasted overnight, and an oral glucose tolerance test (OGTT) was performed on day 14, followed by administration of the conjugate. Six hours later, 1g of glucose solution per kg of body weight was orally administered, and tail glucose levels were measured before (0 hours) and after the 2-hour glucose load. Data were compared using independent Student's t-tests. Where appropriate, measurements were repeated or one-way ANOVA was used, followed by Student-Niemann-Coyrs post-hoc studies for comparison of data.

[0589] Both administrations of the PYY analog (40) alone demonstrated a dose-dependent reduction in food intake on day 1 (Figure 4A), but this effect appeared to diminish over time (day 5, Figure 4B). As shown in Figure 4C, administration of conjugate 40 alone at a high dose resulted in a significant reduction in body weight compared to the vehicle control. As observed in the acute food intake test, conjugate 187 alone showed some efficacy, but the group receiving both demonstrated better weight loss. When 0.01 mg / kg of conjugate 187 was administered in combination with a high dose (0.2 mg / kg) of conjugate 40, body weight decreased by nearly 25% after 13 days. Furthermore, the weight loss in the combination group substantially exceeded the profile predicted based on the additive effect alone (plotted expected addictiveness), thus supporting a synergistic enhancement of efficacy with concomitant administration. Similarly, as shown in Figure 4B, the combination therapy demonstrated a superior inhibitory effect on food consumption on day 5 after administration compared to the administration of GLP-1R agonist conjugate 187 alone.

[0590] Blood glucose homeostasis on day 14 was evaluated via oral glucose tolerance tests (OGTT, Figures 4D-4F). Treatment with PYY analog 40 alone did not show a significant effect on OGTT results or fasting blood glucose. Significant improvement was observed in the GLP-1R agonist (conjugate 187) group, but as expected, the combination therapy group showed slightly better glycemic control. The demonstrated relatively moderate glycemic control effect was not unexpected, and this is probably due to the somewhat mild hyperglycemia observed in the prediabetic DIO model. The clear difference between the conjugate 187 group and the group without conjugate 187 indicates that the glycemic control effect in this study is due to the GLP-1R agonist. However, as shown in Figure 4F, the combination therapy group showed a better effect on fasting blood glucose levels, suggesting that the combination therapy may have a somewhat sustained additive effect on blood glucose management.

[0591] Example K: Generation of a CRE-Luc stable cell line overexpressing GLP-1R or GCGR. Under the control of a cAMP-reactive region (CRE) promoter (Qiagen, The Netherlands), HEK293 cells were infected with a lentivirus encoding the firefly luciferase gene and then selected for 1 week using 1 μg / mL puromycin (Life Technologies, Carlsbad). Viable cells (referred to as CRE-HEK293) were expanded and then transfected with G418 selective mammalian expression plasmids encoding human GLP-1R or GCGR. In short, GLP-1R or GCGR plasmids were transfected into CRE-HEK293 cells using Lipofectamine 2000 and selected with 400 μg / mL genethecin (Life Technologies, Carlsbad, CA). Stable single-colony cell lines overexpressing CRE-luciferase and GLP1R or GCGR (HEK293-GLP-1R-CRE or HEK293-GCGR-CRE) were then established for in vitro activity assays.

[0592] Example L: In vitro receptor activation reporter assay (receptor-mediated cAMP synthesis) HEK293-GLP-1R-CRE or HEK293-GCGR-CRE cells were seeded at a density of 5000 cells / well in 384-well plates and cultured for 18 hours at 37°C and 5% CO2 in DMEM containing 10% FBS. Cells were treated with peptides in a dose-dependent manner for 24 hours, and receptor activation was observed by luminescence intensity using One-Glo(Promega,WI) luciferase reagent according to the manufacturer's instructions. EC of each peptide was measured using GraphPad Prism 6 software (GraphPad, San Diego, CA). 50 They sought it.

[0593] [Table 24]

[0594] Example M: ​​cAMP assay CHOK1 cells stably overexpressing human GLP-1R or GCGR (20 μL of 5000 cells per well) were seeded in a 384-well white solid plate covered with a metal lid and incubated overnight. On day 2, the culture medium was replaced with fresh medium without FBS (0% FBS group). Cells were treated in three ways using 5 μL of peptide in a 12-point dose-response system in culture medium with 0.5 mM IBMX at 37°C and 5% CO2 for 30 minutes. cAMP levels were detected using the Cisbio cAMP dynamic 2 kit. Briefly, 25 μL of cAMP detection reagent (containing cAMP-d2, cryptotate conjugate, and lysis buffer in a 1:1:38 ratio) was added and mixed at room temperature for 1 hour. In the negative control wells of the cells, a cAMP detection reagent without d2 was added. Next, the plate was read at Ex320nm, Em-1 665nm, and Em-2 615nm. Prism software was used to plot the graphs as ratio or delta-F, and EC 50 Obtained. Ratio = A 665nm / B 620nmX10^4. %Delta F = (Standard or sample ratio - Ratio) neg ) / Ratio neg 100. The results are confirmed in Figures 5A to 5C.

[0595] Example N: PK test Female CD-1 mice (n=3 or 4 per group) obtained from the Charles River Laboratory were fasted overnight, and 100 μL of each peptide in phosphate-buffered saline (pH=8.2) was administered intravenously (iv) or subcutaneously (sc). Three hours after blood collection, the mice were fed. Blood was collected, transferred to heparin tubes, and centrifuged at 3000xg for 15 minutes. The resulting plasma was then stored at -80°C for peptide concentration determination. Peptide concentrations in the plasma at each time point were determined by in vitro cell-based activity assays. Briefly, HEK293-GLP-1R-CRE cells were treated with plasma samples at various time points (5 dose-response points, with dilutions starting at 1:10 and progressing to 1:100 for each plasma sample), incubated in DMEM containing 10% FBS for 16 hours at 37°C and 5% CO2, and then firefly luciferase activity was measured. Simultaneously, the same peptide was used to perform Bottom, Top, and EC assays. 50 Standard curves and parameters were obtained for the hill slope. Using the relative luciferase unit (RLU) (RLU) in each plasma sample, a standard curve (RLU = Bottom + (Top - Bottom) / (1 + 10^(log EC)) was obtained. 50 -Conc) * The peptide concentration (nmol / L) in plasma was calculated using parameters derived from the hill slope. The in vivo half-life of each peptide was obtained by acquiring the peptide concentration in plasma using WinNonLin Phoenix software (Pharsight Corp, St. Louis, MO) and plotting it against time. The results for peptides 122 and 135 are shown in Figures 6A-6B and Tables 25a and 25b.

[0596] [Table 25a]

[0597] [Table 25b]

[0598] The plasma concentrations of peptide 142 over time are shown in Figure 6C. Pharmacokinetic parameters are listed in Tables 26a and 26b. The mean half-life of peptide 142 was 10.71 hours after intravenous administration and 11.56 hours after subcutaneous administration.

[0599] [Table 26a]

[0600] [Table 26b]

[0601] The plasma concentrations of peptide 183 over time are shown in Figure 6D. Pharmacokinetic parameters are listed in Tables 26c and 26d. The mean half-life of peptide 142 was 6.335 hours after intravenous administration and 7.87 hours after subcutaneous administration.

[0602] [Table 26c]

[0603] [Table 26d]

[0604] Example O: Efficacy in vivo C57BL / 6J mice (n=6 / group) between 10 and 12 weeks of age were fasted overnight, and then 5 mL / kg of each peptide in PBS (pH=8.2) was administered subcutaneously. Six hours later, the mice were orally or intraperitoneally administered 2 g of glucose solution per kg of body weight, and tail blood glucose levels were measured before (0 min) and after a 2-hour glucose load. In the same mice that had been fasted overnight, observational oral glucose tolerance tests (OGTT) were also performed at 48 and 96 hours after the initial administration.

[0605] Next, the efficacy of the GLP-1R / GCGR dual agonist 135 was evaluated in an oral glucose tolerance test (OGTT) in wild-type mice. As positive controls, semaglutide, a single GLP-1R agonist administered once weekly, and cotadutide, a once-daily dual GLP-1R / GCGR agonist currently in a Phase II trial by AstraZeneca, were used.

[0606] Figures 7A to 7C show the effects of the compounds on oral glucose tolerance tests (OGTT) at 6, 48, and 96 hours after administration, compared to the vehicle control (PBS pH 8.2). Figures 7A to 7C show the time-dependent effects of the compounds on blood glucose. Figures 7D to 7F show the effects of the compounds on blood glucose levels as measured by the area under the curve. Figures 7G to 7I show the effects of the compounds on fasting blood glucose. In all figures, A: 122 4, B: 135, C: 138, D: cotadutide, E: semaglutide. All peptides significantly reduced blood glucose levels to similar values ​​to the vehicle 6 hours after administration. Similar results were observed for fasting blood glucose with all peptides. However, significant differences in blood glucose levels were observed after peptide administration. Cotadutide showed no improvement compared to the vehicle at 48 hours, consistent with its suitability as a once-daily injection for human subjects. On the other hand, mice treated with conjugate 135 showed a significant improvement in blood glucose management after 48 hours compared to mice treated with semaglutide. Furthermore, conjugate 135 was able to significantly reduce fasting blood glucose levels, while the remaining peptides did not show any improvement in efficacy. The increased in vivo efficacy of conjugate 135 observed herein is likely due to both higher dual agonist activity and an extended in vivo half-life. Assuming a direct relationship between the pharmacokinetics, the results of this experiment suggest that peptide conjugate 135, exhibiting a longer half-life than semaglutide, could be developed with appropriate formulations for weekly or bi-weekly administration.

[0607] Example P: Generation of a CRE-Luc stable cell line overexpressing GLP-1R or GCGR. Under the control of a cAMP-reactive region (CRE) promoter (Qiagen, The Netherlands), HEK293 cells were infected with a lentivirus encoding the firefly luciferase gene and then selected for 1 week using 1 μg / mL puromycin (Life Technologies, Carlsbad). Viable cells (referred to as CRE-HEK293) were expanded and then transfected with G418 selective mammalian expression plasmids encoding human GLP-1R or GCGR. In short, GLP-1R or GCGR plasmids were transfected into CRE-HEK293 cells using Lipofectamine 2000 and selected with 400 μg / mL genethecin (Life Technologies, Carlsbad, CA). Stable single-colony cell lines overexpressing CRE-luciferase and GLP1R or GIPR (HEK293-GLP-1R-CRE or HEK293-GIPR-CRE) were then established for in vitro activity assays.

[0608] Example Q: In vitro receptor activation reporter assay (receptor-mediated cAMP synthesis) HEK293 GLP-1R-CRE or HEK293-GIPR-CRE cells were seeded at a density of 5000 cells / well in 384-well plates and cultured for 18 hours at 37°C and 5% CO2 in DMEM containing 10% FBS. Cells were treated with peptides in a dose-dependent manner for 24 hours, and receptor activation was observed by luminescence intensity using One-Glo(Promega,WI) luciferase reagent according to the manufacturer's instructions. EC of each peptide was measured using GraphPad Prism 6 software (GraphPad, San Diego, CA). 50 We calculated the value. The results are shown in Figures 8A and 8B.

[0609] [Table 27]

[0610] Example R: cAMP assay CHOK1 cells stably overexpressing human GLP-1R or GIPR (20 μL of 5000 cells per well) were seeded in a 384-well white solid plate covered with a metal lid and incubated overnight. On day 2, the culture medium was replaced with fresh medium without FBS (0% FBS group). Cells were treated in three ways using 5 μL of peptide in a 12-point dose-response system in culture medium with 0.5 mM IBMX at 37°C and 5% CO2 for 30 minutes. cAMP levels were detected using the Cisbio cAMP dynamic 2 kit. Briefly, 25 μL of cAMP detection reagent (containing cAMP-d2, cryptotate conjugate, and lysis buffer in a 1:1:38 ratio) was added and mixed at room temperature for 1 hour. In the negative control wells of the cells, cAMP detection reagent without d2 was added. Next, the plate was read at Ex320nm, Em-1 665nm, and Em-2 615nm. Prism software was used to plot the graphs as ratio or delta-F, and EC 50 Obtained. Ratio = A 665nm / B 620nm X10^4. %Delta F = (Standard or sample ratio - Ratio) neg ) / Ratio neg x100. The results are confirmed in Figures 5A to 5C.

[0611] [Table 28]

[0612] Example S: Oral glucose tolerance test (OGTT) C57BL / 6J mice (n=6 / group) between 10 and 12 weeks of age were fasted overnight, and then 5 mL / kg of each peptide in PBS (pH=8.2) was administered subcutaneously. Six hours later, the mice were orally or intraperitoneally administered 2 g of glucose solution per kg of body weight, and tail glucose levels were measured before (0 min) and after the 2-hour glucose load. In the same mice that had been fasted overnight, follow-up oral glucose tolerance tests (OGTTs) were also performed at 72, 96, and 144 hours after the initial administration. In all figures, A: 141 4, B: 171, C: Tirzepatide, D: Cotadutide, E: Semaglutide. As shown in Figures 9A to 9D, oral glucose tolerance tests (OGTTs) were performed at 2, 72, 96, and 144 hours after administration. As shown in Figure 9E, in the 2-hour test, AUC measurements showed a significant decrease in blood glucose levels in mice treated with compound 141, compound 171, compound 142, semaglutide, and tilzepatide compared to mice treated with vehicle alone. As shown in Figure 9F, in the OGTT performed 72 hours after administration, AUC measurements showed a significant decrease in blood glucose levels in mice treated with compound 141, compound 171, compound 142, and semaglutide compared to mice treated with vehicle alone. As shown in Figure 9G, in the OGTT performed 96 hours after administration, AUC measurements showed a significant decrease in blood glucose levels in mice treated with compound 141, compound 142, and semaglutide compared to mice treated with vehicle alone. As shown in Figure 9F, in an OGTT performed 144 hours after administration, AUC measurements showed a significant decrease in blood glucose levels in mice treated with compound 141, compound 142, and semaglutide compared to mice treated with vehicle alone. As shown in Figure 9I, treatment with compound 141, compound 171, compound 142, semaglutide, and tilzepatide resulted in a significant decrease in fasting blood glucose levels 2 hours after treatment compared to treatment with vehicle alone. As shown in Figure 9J, treatment with compound 141, compound 171, and semaglutide resulted in a significant decrease in fasting blood glucose levels 72 hours after treatment compared to treatment with vehicle alone.As shown in Figures 9K-9L, treatment with compound 141, compound 142, and semaglutide resulted in a significant decrease in fasting blood glucose levels at 96 and 144 hours post-treatment compared to treatment with vehicle alone.

[0613] Example T: DIO mouse test Results were expressed as mean ± SE, and data were compared using independent Student t-tests. Where appropriate, measurements were repeated or one-way ANOVA was used, followed by comparisons using the Student-Niemann-Coyrs post-hoc study. Area under the incremental curve (AUC) analysis for plasma glucose was calculated using GraphPad Prism 6. Data groups were identified as significantly different when p was less than 0.01.

[0614] Measurement of body weight, food intake, and visceral fat mass. DIO mice (C57BL / 6, male, 37 weeks old) were randomized according to body weight and administered either peptide or vehicle subcutaneously daily or twice weekly (n=6 / group). Body weight and food intake were monitored daily throughout the study.

[0615] As shown in Figures 10A and 10B, mice treated with compound 142 or tilzepatide showed a decrease in total body weight and body weight percentage over time compared to mice treated with vehicle alone. Furthermore, as shown in Figure 10C, mice treated with compound 142 or tilzepatide showed a decrease in cumulative food intake compared to mice treated with vehicle alone. As shown in Figure 10D, in oral glucose tolerance tests (OGTT), blood glucose levels over time were lower in mice treated with compound 142 or tilzepatide compared to mice treated with vehicle alone. Furthermore, when measured by area under the curve (AUC), a significant decrease in total blood glucose levels was observed in mice treated with compound 142 or tilzepatide compared to mice treated with vehicle alone. Mice treated with these compounds also showed a decrease in blood glucose levels after overnight fasting on day 8 compared to mice treated with vehicle alone. In mice treated with compound 142 seven times a week, blood glucose levels decreased by 53%. In mice treated with compound 142 twice a week, blood glucose levels decreased by 42%. In mice treated with tilzepatide, blood glucose levels decreased by 30%.

[0616] Animal and statistical analysis All treatment and experimental procedures on animals were approved by the Animal Experimentation Committee (IACUC) of the California Institute for Biomedical Research (Calibr) and strictly adhered to NIH guidelines for painless treatment of animals. Results were expressed as mean ± SE, and data were compared using independent Student t-tests. Where appropriate, measurements were repeated or one-way ANOVA was used, followed by Student-Niemann-Coyrs post-hoc studies for data comparison. Area under the curve (AUC) analysis of plasma glucose increments was calculated using GraphPad Prism 6. Data groups were identified as significantly different when p < 0.01.

[0617] Measurement of body weight, food intake, and visceral fat mass. DIO mice (C57BL / 6, male, 28 weeks old) were randomized according to body weight and administered either peptide or vehicle subcutaneously twice a week daily (n=7 / group). Body weight and food intake were monitored daily throughout the study. Mice were sacrificed at the end of the experiment and visceral fat mass was measured. Collected plasma was used to determine cholesterol levels according to the manufacturer's guidelines (cholesterol assay kit, Abcam, Cambridge, England), and triglyceride levels were determined using a triglyceride colorimetric assay kit (Cayman Chemical, Ann Arbor, Michigan).

[0618] Cholesterol level determination Cholesterol levels were determined using collected plasma according to the manufacturer's guidelines (Cholesterol Assay Kit, Abcam, Cambridge, England). In short, plasma was diluted with cholesterol assay buffer and then reacted with the same volume of reaction mixture containing cholesterol assay buffer, cholesterol probe, enzyme mixture, and cholesterol esterase. After incubation at 37°C for 1 hour, absorption was measured at 560 nm using an Envision multi-label plate reader (PerkinElmer, Waltham, MA). Plasma cholesterol concentrations were subsequently calculated according to a standard curve.

[0619] Measurement of triglyceride levels Triglyceride levels were determined using a triglyceride colorimetric assay kit (Cayman Chemical, Ann Arbor, Michigan) with collected plasma. 5 μL of plasma sample or standard was seeded into a 384-well plate, followed by the addition of 75 μL of diluted enzyme buffer to each well. The mixture was incubated at room temperature for 15 minutes, and the absorption was read at 560 nm using an Envision plate reader (PerkinElmer, Waltham, MA). Plasma triglyceride concentrations were calculated using a standard curve.

[0620] Biochemical and histological analysis Terminal serum analytes, including total cholesterol, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), were analyzed using an Alfa Wassermann Vet Axcel® clinical analyzer. Liver triglycerides were measured from liver homogenates produced using a colorimetric quantitative triglyceride kit (Cayman Chemical). Paraformaldehyde-fixed livers were paraffin-embedded, dissected, and stained with hematoxylin-eosin and picro-sirius red by HistoTox Labs (Boulder, CO). All histological evaluations (scoring for hyperlipidemia and fibrosis) were performed by certified histopathologists (HistoTox Labs) who were blinded to the procedure, based on the classification outlined in Kleiner et al.3.

[0621] result The results are shown in Figures 11A to 11Q. In all figures, A: 122, B: 142, C: semaglutide, and D: cotadutide. The vehicle control was PBS at pH 8.2. As shown in Figure 11A, mice treated with compound 122, compound 142, or semaglutide showed a decrease in food intake over 20 days compared to mice treated with vehicle alone. Furthermore, as shown in Figures 11B to 11C, mice treated with compound 122, compound 142, or semaglutide showed a decrease in both total body weight and initial body weight change rate over 21 days compared to mice treated with vehicle alone. Treatment with these compounds also affected blood glucose levels. As shown in Figures 11D-11E, mice treated with compound 122, compound 142, or semaglutide showed a significant decrease in plasma glucose excursion on day 20 (feeding state) and day 21 (fasting state) compared with mice treated with vehicle alone. Only mice treated with cotadutide showed a significant decrease in plasma glucose excursion in the fasting state. As shown in Figures 11F-11G, in the oral glucose tolerance test (OGTT) performed on day 21, mice treated with compound 122, compound 142, or semaglutide showed a significant decrease in blood glucose levels over time, as measured by the area under the curve, compared with mice treated with vehicle alone.

[0622] Treatment with these compounds affects plasma levels of liver function markers. As shown in Figures 11J-11L, treatment with compound 122 or semaglutide significantly reduced ALT, ALP, cholesterol, and triglyceride levels compared to treatment with vehicle alone, while treatment with compound 142 significantly reduced AST, ALT, ALP, cholesterol, and triglyceride levels compared to treatment with vehicle alone. As shown in Figures 11M, 11O, and 11P, liver-to-body weight ratio, liver weight, and liver triglyceride levels were significantly lower in mice treated with compound 122, compound 142, semaglutide, or cotadutide compared to mice treated with vehicle alone. As shown in Figure 11N, treatment with compound 122, compound 142, or semaglutide significantly reduced fat mass compared to treatment with vehicle alone. The grade of hyperlipidemia was lower in mice treated with compound 122, compound 142, or semaglutide compared to mice treated with vehicle alone.

[0623] Example U: cAMP assay (GLP-1R single agonist) Under the control of a cAMP-reactive region (CRE) promoter (Qiagen, Netherlands), HEK293 cells were infected with a lentivirus encoding the firefly luciferase gene and then selected for 1 week using 1 μg / mL puromycin (Life Technologies, Carlsbad). Viable cells (referred to as CRE-HEK293) were expanded and then transfected with the G418 selective mammalian expression plasmid encoding human GLP-1R. In short, GLP-1R plasmid was transfected into CRE-HEK293 cells using Lipofectamine 2000 and selected with 400 μg / mL genethecin (Life Technologies, Carlsbad, CA). A stable single-colony cell line overexpressing both CRE-luciferase and GLP-1R (HEK293-GLP-1R-CRE) was then established for in vitro activity assays.

[0624] HEK293-GLP-1R-CRE cells were seeded at a density of 5000 cells / well in 384-well plates and cultured for 18 hours at 37°C and 5% CO2 in DMEM containing 10% FBS. Cells were treated with peptides in a dose-dependent manner for 24 hours, and receptor activation was observed by luminescence intensity using One-Glo(Promega,WI) luciferase reagent according to the manufacturer's instructions. EC of each peptide was measured using GraphPad Prism 6 software (GraphPad, San Diego, CA). 50 They sought it.

[0625] [Table 29]

[0626] Example A: β-arrestin recruitment assay The results are shown in Tables 30 to 32.

[0627] [Table 30]

[0628] [Table 31]

[0629] [Table 32]

[0630] Example B: Plasma Stability To investigate the stability of the conjugates in plasma, PrRP31, conjugate 255 (97-L3), and conjugate 263 (97-L5) were incubated in mouse plasma for up to 24 hours (Figure 12). The remaining intact peptide levels were quantified by LC-MS (QTOF) after serum protein precipitation. PrRP31 degraded rapidly in mouse plasma, with a half-life of approximately 11 minutes and complete elimination after 1 hour. Stapling at L3 between positions 6 and 13 improved stability, extending the half-life to 30-60 minutes. Conjugation to staple L5 further extended the half-life to approximately 3 hours.

[0631] Peptides were incubated in mouse plasma at various time points, followed by plasma protein precipitation in methanol and quantification via LC-MS. Plasma stability tests were then performed on the resulting single replicas.

[0632] Example C: Pharmacokinetic study The pharmacokinetic profile of conjugate 263(97-L5) in male C57 mice administered 1 mg / kg subcutaneously was evaluated (Figure 13). Plasma peptide concentrations were determined at various time points (0.25, 0.5, 1, 3, 7, 24, 48, and 72 hours) using LC-MS. max The concentration reached approximately 1.67 μg / mL about 3 hours after administration, and the elimination half-life was 8 hours, which was similar to that of semaglutide in rodents.

[0633] Mouse PK studies were conducted by subcutaneously injecting mice with 1 mg / kg of conjugate 263(97-L5). Plasma samples were collected at different time points and quantified by LC-MS. PK parameters were calculated by data fitting using WinNonlin. Due to limitations in mouse volume / sampling, sparse sampling was used. Therefore, a single PK profile was obtained by combining concentrations from various animals, and the estimated PK parameters were averaged. Consequently, SEM results are not reported.

[0634] Additional PK studies were performed with a 5 mg / kg dose, and plasma concentrations were determined using cell-based functional assays, resulting in a similar pharmacokinetic profile (Figure 14).

[0635] [Table 33]

[0636] Example D: In vivo efficacy assay To demonstrate the translation of the extended half-life of conjugate 263(97-L5) into in vivo efficacy, a 12-day weight study (Figure 15) was performed on a diet-induced obesity (DIO) mouse model (n=8 per group) administered daily subcutaneously. A significant weight-reducing effect was observed with conjugate 263(97-L5) at 0.5 mg / kg. Furthermore, daily injection of a higher dose of compound 263(97-L5) at 5 mg / kg yielded similar efficacy to the 0.5 mg / kg administration (Figure 16), indicating that the ED50 of conjugate 263(97-L5) is less than 0.5 mg / kg. While this selectivity is thought to result in reduced appetite-inducing effects, conjugate 263(97-L5) is expected to exhibit a more favorable safety profile in terms of undesirable cardiovascular side effects associated with NPFF2R agonism. 24-hour plasma exposure in PK studies at 5 mg / kg and 1 mg / kg was equivalent to the EC of conjugate 263(97-L5). 50 This is significantly higher than the previous dose, which may indicate that lower doses are required to exhibit a dose-response effect. Detailed dose-response and efficacy studies for more chronic obesity and metabolic disease models are currently underway.

[0637] Efficacy studies were conducted on eight diet-induced obesity (DIO) mice (n=8) that received either 0.5 mg / kg of conjugate 263(97-L5) or vehicle subcutaneously daily for 12 days. Body weight decreased significantly more in mice treated with conjugate than in those treated with vehicle. **** =p≦0.0001, *** =p≦0.001, **= p ≤ 0.01.

[0638] Example E: In vivo body weight study and oral glucose tolerance test (OGTT) All treatment and experimental procedures for animals were approved by the Animal Experimentation Committee (IACUC) of the California Institute for Biomedical Research (Calibr) and strictly adhered to NIH guidelines for painless treatment of animals. Male mice (24 weeks old, Jackson Labs, Bar Harbor, ME) of the Charles River diet-induced obesity (DIO) model were fed a high-fat diet (D12492, 60% fat diet) for 18 weeks and administered 0.5 or 5 mg / kg of peptide subcutaneously daily for up to 12 days (two mice per cage). The mean body weight at the start of the experiment was 50 g. Body weight of the mice was monitored daily throughout the study period, and food intake was monitored on days 1, 2, 6, and 9. After fasting overnight, the mice underwent an oral glucose tolerance test (OGTT) on day 14 and were administered the peptide. Six hours later, 1 g of glucose solution per kg of body weight was orally administered, and tail glucose levels were measured before (0 hours) and after the 2-hour glucose load. Data were compared using an independent Student's tt test. Where appropriate, measurements were repeated or one-way ANOVA was used, followed by comparison of data using the Student-Niemann-Coirs post-hoc study. Blood glucose levels and AUC after oral administration of PrRP31 or conjugate 263(97-L5) are shown in Figures 17 and 18.

Claims

1. A peptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, or an amino acid sequence that has one amino acid addition, deletion, or substitution compared to SEQ ID NO: 6, A peptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 76, or an amino acid sequence that has one amino acid addition, deletion, or substitution compared to SEQ ID NO: 76, or A peptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 63, or an amino acid sequence having one amino acid addition, deletion, or substitution compared to SEQ ID NO: 63, A staple bonded to the peptide by a first amino acid and a second amino acid, wherein the first amino acid and the second amino acid are independently sulfhydryl or amine-containing amino acids, and the staple is of formula (I): 【Chemistry 1】 It is, During the ceremony, A is a substituted or unsubstituted alkylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, or a substituted or unsubstituted -NR ３ -Alkilen-NR ３ -, or -N-, X １ and X ２ is a single bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene-, -alkylene-C(=O)NR ３ -, -alkylene-NR ３ C(=O)-, -C(=O)NR ３ -alkylene-, -NR ３ C(=O)-alkylene-, -alkylene-C(=O)NR ３ -alkylene-, or -alkylene-NR ３ C(=O)-alkylene- and is Here, the X １ The first amino acid of the peptide is bound to the X ２ It binds to the second amino acid of the peptide, R is hydrogen or -(L) ｓ -Y is, Each L is independently - (CR １ R ２ ) ｖ -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR ３ -Alkylene-, -Alkylene-NR ３ -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O) ２ -alkylene, -alkylene-S (=O) ２ -, -C(=O)-, -C(=O)NR ３ -, -NR ３ C(=O)-, -NR ３ C(=O)NR ３ -, -NR ３ C(=O)NR ３ -Alkilen-, -NR ３ C(=O)-alkylene-NR ３ -,-alkylene-C(=O)NR ３ -, -C(=O)NR ３ -Alkylene-, -Alkylene-NR ３ C(=O)-, or -NR ３ It is C(=O)-alkylene-, v is between 2 and 20. R １ or R ２ These are hydrogen, halogen, -CN, and -OR, respectively, independently. ａ ,-SR ａ , -S(=O)R ｂ , -NO ２ , -NR ｃ R ｄ , -S (=O) ２ R ｄ , -NR ａ S (=O) ２ R ｄ , -S (=O) ２ NR ｃ R ｄ , -C(=O)R ｂ -OC(=O)R ｂ , -CO ２ R ａ , -OCO ２ R ａ , -C(=O)NR ｃ R ｄ , -OC(=O)NR ｃ R ｄ , -NR ａ C(=O)NR ｃ R ｄ , -NR ａ C(=O)R ｂ , -NR ａ C (=O) OR ａ , C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C １ -C ６ Heteroalkyl, C ３ -C ８ Cycloalkyl, C ２ -C ８ Heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OR ａ , or -NR ｃ R ｄ They are substituted or unsubstituted by one, two, or three of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogens, C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, -OR ａ , or -NR ｃ R ｄ It is either substituted or not substituted by one, two, or three of the following: Alternatively, the said R １ and the said R ２ are combined to form C １ -C ６ cycloalkyl or C １ -C ６ heterocycloalkyl, Each R ３ is independently hydrogen, -S(=O)R ｂ , -S(=O) ２ R ａ , -S(=O) ２ NR ｃ R ｄ , -C(=O)R ｂ , -CO ２ R ａ , -C(=O)NR ｃ R ｄ , C １ -C ６ alkyl, C ２ -C ６ alkenyl, C ２ -C ６ alkynyl, C １ -C ６ heteroalkyl, C[[ID=4I]] ３ -C ８ cycloalkyl, C ２ -C ８ heterocycloalkyl, aryl, or heteroaryl, where alkyl, alkenyl, alkynyl, and heteroalkyl are substituted or unsubstituted by one, two, or three of halogen, -OR ａ , or -NR ｃ R ｄ , and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are substituted or unsubstituted by one, two, or three of halogen, C １ -C ６ alkyl, C １ -C ６ haloalkyl, -OR ａ , or -NR ｃ R ｄ , and Y is hydrogen, C １ -C ６ Alkyl, -CO ２ H, -CO ２ (C １ -C ６ Alkyl), -CO ２ NH ２ , -CO ２ N (alkyl) ２ , or -CO ２ It is NH (alkyl), s is between 0 and 20. R ａ is hydrogen, C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C １ -C ６ Heteroalkyl, C ３ -C ８ Cycloalkyl, C ２ -C ８ A heterocycloalkyl, aryl, or heteroaryl group, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OH, -OMe, or -NH ２ They are substituted or unsubstituted by one, two, or three of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogens, C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, -OH, -OMe, or -NH ２ It is either substituted or not substituted by one, two, or three of the following: R ｂ C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C １ -C ６ Heteroalkyl, C ３ -C ８ Cycloalkyl, C ２ -C ８ A heterocycloalkyl, aryl, or heteroaryl group, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OH, -OMe, or -NH ２ They are substituted or unsubstituted by one, two, or three of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogens, C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, -OH, -OMe, or -NH ２ It is either substituted or not substituted by one, two, or three of the following, and R ｃ and R ｄ These are hydrogen and C, respectively, independently. １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C １ -C ６ Heteroalkyl, C ３ -C ８ Cycloalkyl, C ２ -C ８ A heterocycloalkyl, aryl, or heteroaryl group, where alkyl, alkenyl, alkynyl, and heteroalkyl are halogens, -OH, -OMe, or -NH ２ They are substituted or unsubstituted by one, two, or three of the following, and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are halogens, C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, -OH, -OMe, or -NH ２ It is either substituted or not substituted by one, two, or three of the following: Or R ｃ and R ｄ These, together with the nitrogen atom to which they are bonded, form a heterocycloalkyl or heteroaryl, where the heterocycloalkyl and heteroaryl are halogens, C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, -OH, -OMe, or -NH ２ The staples are substituted or not substituted by one, two, or three of the following: A peptide conjugate containing this compound.

2. The peptide conjugate according to claim 1, wherein the first amino acid and the second amino acid are independently cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid.

3. The peptide conjugate according to claim 1, wherein the first amino acid and the second amino acid are independently lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine.

4. The peptide conjugate according to any one of claims 1 to 3, wherein the first amino acid has the i position in the peptide, and the second amino acid has the i+n position in the peptide, where n is 4 to 16.

5. The peptide conjugate according to any one of claims 1 to 3, wherein the first amino acid has the i position in the peptide and the second amino acid has the i+7 position in the peptide.

6. The peptide conjugate according to any one of claims 1 to 5, wherein the half-life of the peptide conjugate is at least twice the half-life of the unmodified form of the peptide.

7. The peptide conjugate according to any one of claims 1 to 6, wherein the binding affinity of the peptide conjugate is within 20% of the binding affinity of the unmodified form of the peptide.

8. The aforementioned X １ and the aforementioned X ２ However, -C(=O)-, -alkylene-C(=O)-, -C(=O)alkylene-, -CH ２ -C(=O)-, -C(=O)-CH ２ -,-alkylene-C(=O)NR ３ -, -C(=O)NR ３ -Alkylene-, -CH ２ -C(=O)NR ３ -, -C(=O)NR ３ -CH ２ -,-alkylene-C(=O)NR ３ -Alkylene-, -Alkylene-NR ３ C(=O)-alkylene-, -CH ２ -C(=O)NR ３ -CH ２ CH ２ -ien-CH ２ -NR ３ C(=O)-CH ２ CH ２ -ien-CH ２ -C(=O)NH-CH ２ CH ２ -, or -CH ２ -NHC(=O)-CH ２ CH ２ - The peptide conjugate according to any one of claims 1 to 7.

9. > A-R has the following structure: 【Chemistry 2】 A peptide conjugate according to any one of claims 1 to 8, wherein s is 5 to 15 in the formula.

10. The peptide conjugate according to any one of claims 1 to 8, wherein s is 1 to 15.

11. The peptide conjugate according to any one of claims 1 to 8, wherein s is 1 to 10.

12. > A-R has the following structure: 【Transformation 3】 The peptide conjugate according to any one of claims 1 to 8, wherein r1 and r2 are each independently 0 to 4 and s is 5 to 15.

13. The aforementioned Y is hydrogen or -CO ２ A peptide conjugate according to any one of claims 1 to 12, wherein H.

14. Each of the above L independently - (CR １ R ２ ) ｖ -, -alkylene-O-, -C(=O)-, -C(=O)NR ３ -, -NR ３ C(=O)-,-alkylene-C(=O)NR ３ - or -alkylene-NR ３ A peptide conjugate according to any one of claims 1 to 13, wherein it is C(=O)- and v is 2 to 20.

15. The staple has the following structure 【Chemistry 4】 Each has 【Transformation 5】 The peptide conjugate according to claim 1, wherein represents the sulfur atom of the cysteine ​​of the peptide.

16. The staple has the following structure 【Transformation 6】 Each has 【Transformation 7】 The peptide conjugate according to claim 1, wherein represents the sulfur atom of the cysteine ​​of the peptide.

17. The staple has the following structure 【Transformation 8】 Each has 【Chemistry 9】 The peptide conjugate according to claim 1, wherein represents the sulfur atom of the cysteine ​​of the peptide.

18. The staple has the following structure 【Chemistry 10】 Each has 【Chemistry 11】 The peptide conjugate according to claim 1, wherein represents the sulfur atom of the cysteine ​​of the peptide.

19. a) A peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, b) The following structure 【Chemistry 12】 A staple to which the peptide having each 【Chemistry 13】 The staple and represent the sulfur atom of the cysteine ​​in the peptide. A peptide conjugate containing this compound.

20. a) A peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 60, b) The following structure 【Chemistry 14】 A staple to which the peptide having each 【Chemistry 15】 The staple and represent the sulfur atom of the cysteine ​​in the peptide. A peptide conjugate containing this compound.

21. a) A peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 76, b) The following structure 【Chemistry 16】 A staple to which the peptide having each 【Chemistry 17】 The staple and represent the sulfur atom of the cysteine ​​in the peptide. A peptide conjugate containing this compound.

22. a) A peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 116, b) The following structure [Chemistry 18] A staple to which the peptide having each 【Chemistry 19】 The staple and represent the sulfur atom of the cysteine ​​in the peptide. A peptide conjugate containing this compound.

23. a) A peptide consisting of a peptide sequence that is at least 95% identical to SEQ ID NO: 6 or 63, b) Staples that are bound to the peptide at the first and second amino acids of the peptide A peptide conjugate containing this peptide.

24. The peptide conjugate according to claim 23, wherein the first amino acid has the i position in the peptide, and the second amino acid has the i+n position in the peptide, where n is 4 to 16.

25. The peptide conjugate according to claim 23 or 24, wherein the first amino acid has the i position in the peptide and the second amino acid has the i+7 position in the peptide.

26. The peptide conjugate according to any one of claims 23 to 25, wherein the half-life of the peptide conjugate is at least twice the half-life of the unmodified form of the peptide.

27. The peptide conjugate according to any one of claims 23 to 25, wherein the binding affinity of the peptide conjugate is within 20% of the binding affinity of the unmodified form of the peptide.

28. The staple has the following structure 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 Each has 【Chemistry 24】 Each represents the sulfur atom of the cysteine ​​in the peptide, 【Chemistry 25】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the nitrogen atom of the lysine of the peptide.

29. The staple has the following structure 【Chemistry 26】 【Chemistry 27】 Each has 【Chemistry 28】 Each represents the sulfur atom of the cysteine ​​in the peptide, 【Chemistry 29】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the nitrogen atom of the lysine of the peptide.

30. The staple has the following structure 【Transformation 30】 Each has 【Chemistry 31】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the sulfur atom of the cysteine ​​of the peptide.

31. The staple has the following structure 【Chemistry 32】 Each has 【Transformation 33】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the sulfur atom of the cysteine ​​of the peptide.

32. The staple has the following structure 【Transformation 34】 Each has 【Chemistry 35】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the sulfur atom of the cysteine ​​of the peptide.

33. The staple has the following structure 【Transformation 36】 Each has 【Chemistry 37】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the sulfur atom of the cysteine ​​of the peptide. 【Request Item 34】 【Chemistry 38】 Includes each 【Chemistry 39】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the nitrogen atom of the lysine of the peptide.

35. The staple has the following structure 【Chemistry 40】 Each has 【Chemistry 41】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the nitrogen atom of the lysine of the peptide.

36. The staple has the following structure 【Chemistry 42】 Each has 【Chemistry 43】 The peptide conjugate according to any one of claims 23 to 27, wherein represents the nitrogen atom of the lysine of the peptide.

37. a) A peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 63, b) A staple that is bound to the peptide by the first lysine of SEQ ID NO: 63 and the third lysine of SEQ ID NO: 63, having the following structure 【Chemistry 44】 Each has 【Chemistry 45】 A staple and a lysine molecule that represents the nitrogen atom of the first or third lysine of the peptide. A peptide conjugate containing this compound.

38. A pharmaceutical composition comprising a peptide conjugate according to any one of claims 1 to 37 and a pharmaceutically acceptable excipient.

39. Use of a composition comprising a therapeutically effective amount of a peptide conjugate according to any one of claims 1 to 37 in the manufacture of a drug for use in a method of treating a target disease or illness, wherein the method comprises the step of administering the composition to the target.

40. The use according to claim 39, wherein the disease or illness is diabetes or obesity.

41. The use according to claim 39, wherein the disease or illness is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or a cardiovascular disease.

42. The use according to claim 39, wherein the disease or illness is short bowel syndrome (SBS).

43. The use according to claim 39, wherein the disease or illness is inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or psoriasis.

44. The use according to claim 39, wherein the disease or illness is Crohn's disease or ulcerative colitis.

45. The use according to claim 39, wherein the disease or illness is Alzheimer's disease, Parkinson's disease, or Huntington's disease.

46. The use according to any one of claims 39 to 45, further comprising the step of administering one or more additional therapeutic agents to the subject.

47. A composition comprising a peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO:

6.

48. A composition comprising a peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 63.

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