Dual GLP-1 and glucagon agonist peptides with improved biostability

Abstract

Provided herein are dual agonists of GLP-1 and glucagon with improved biological stability (e.g., proteolytic stability) and duration of action. These peptides can be administered, for example, once a week.

Description

[Technical field]

[0001] The incidence of obesity and diabetes is rising like an epidemic. Diabetes is characterized by hyperglycemia resulting from defects in insulin production, insulin action, or both. Type 2 diabetes mellitus (T2DM) accounts for approximately 90-95% of all diagnosed cases of diabetes, and the risk of T2DM increases with increasing body weight. The prevalence of T2DM is 3-7 times higher in obese than in normal weight adults, and is associated with a body mass index (BMI) of 35 kg / m 2 In larger individuals, the risk is 20 times higher. However, weight loss can improve the control of type 2 diabetes or even reverse it. [Background technology]

[0002] Glucagon and glucagon-like peptide-1 (GLP-1) are derived from a 158 amino acid precursor polypeptide, preproglucagon, which is processed in different tissues to form many different proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM), which are involved in various physiological functions including glucose homeostasis, insulin secretion, gastric emptying, intestinal growth, and food intake regulation. Glucagon is produced as a 29 amino acid peptide corresponding to amino acids 33 to 61 of proglucagon (53 to 81 of preproglucagon), and GLP-1 is produced as a 37 amino acid peptide corresponding to amino acids 72 to 108 of proglucagon (92 to 128 of preproglucagon). GLP-1(7-36)amide or GLP-1(7-37)acid are the biologically active forms of GLP-1 and exhibit essentially equivalent activity at the GLP-1 receptor.

[0003] Glucagon is produced in the pancreas and interacts with the glucagon receptor ("glucR"). Glucagon acts in the liver to increase blood glucose through gluconeogenesis and glycogenolysis. When blood glucose begins to fall, glucagon signals the liver to break down glycogen and release glucose, raising blood glucose levels back to normal.

[0004] GLP-1 has distinct biological activities compared to glucagon. GLP-1 is secreted from the L-cells of the intestine and binds to the GLP-1 receptor. Its activities include stimulating insulin synthesis and secretion, inhibiting glucagon secretion, and inhibiting food intake.

[0005] Both glucagon and GLP-1 act as agonists at their respective receptors and have been shown to be effective in weight loss. Certain GLP-1 analogues are marketed or in development for the treatment of obesity, including, for example, liraglutide (Novo Nordisk's Saxenda®) and semaglutide (Novo Nordisk's Ugovi®). Glucagon / GLP-1 dual agonist peptides such as cotadutide are also known and are in clinical development for the treatment of diabetes, obesity, and nonalcoholic steatohepatitis (NASH). However, both of these proposed treatments involve chronic self-medication, which requires long-term patient compliance with medication. Other peptides, such as amylin analogues, are also being investigated for the treatment of obesity, excessive food intake, and diabetes (WO2018 / 046719). Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need for therapeutic agents that can agonize the functions of both GLP-1 and glucagon, for example, to improve glycemic control, reduce weight, treat type 2 diabetes mellitus (T2DM), and / or treat NASH, while minimizing the burden associated with administration and improving patient compliance and quality of life. [Means for solving the problem]

[0007] Provided herein is a GLP-1 and glucagon dual agonist with improved biological stability (e.g., proteolytic stability) and duration of action.Provided herein is a GLP-1 and glucagon dual agonist peptide, for example, compared with semaglutide (H(Aib)EGTFTSDVSSYLEGQAAX20EFIAWLVRG-acid, where X20 is Lys[O2Oc-O2Oc-γE-C18 diacid], (SEQ ID NO: 539)) and / or cotadutin (HSQGTFTSDX10SEYLDSERARDFVAWLEAGG-acid, where X10=Lys[ε-γE-palmitoyl] (SEQ ID NO: 538)).Therefore, provided herein is a GLP-1 and glucagon dual agonist peptide, which can be administered once a week.

[0008] Certain embodiments of the present disclosure are directed to a peptide comprising the sequence: H-X2-X3-G-X5-X6-TSD-X10-S-X12-α-methyl-phenylalanine (αMePhe)-L-X15-X16-X17-X18-A-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-Z, where X2 is aminoisobutyric acid (Aib), S or A, X3 is Q, H or E, X5 is T or S, X6 is F or αMePhe, X10 is V, K or Y, X12 is K, E or S, and X15 is D or or E, X16 is T, S or G, X17 is K, R, E or Q, X18 is R or A, X20 is R, K or Q, X21 is D or E, X22 is αMePhe or F, X23 is V or I, X24 is Q or A, X25 is Aib or W, X26 is L or I, X27 is L, A, E, V or M, X28 is E, N, A, R or K, X29 is Aib, T or G, X30 is G, R or absent, X31 is G or absent, and Z is an amide or acid (SEQ ID NO: 540).

[0009] In some embodiments, X2 is Aib. In some embodiments, X3 is Q. In some embodiments, X3 is H. In some embodiments, X5 is T. In some embodiments, X5 is S. In some embodiments, X6 is F. In some embodiments, X6 is αMePhe. In some embodiments, X10 is V. In some embodiments, X12 is K. In some embodiments, X15 is D. In some embodiments, X16 is T. In some embodiments, X16 is S. In some embodiments, X17 is K. In some embodiments, X17 is R. In some embodiments, X18 is R. In some embodiments, X18 is A. In some embodiments, X20 is R. In some embodiments, X20 is K. In some embodiments, X21 is D. In some embodiments, X22 is F. In some embodiments, X22 is αMePhe. In some embodiments, X23 is V. In some embodiments, X24 is Q. In some embodiments, X25 is W. In some embodiments, X25 is Aib. In some embodiments, X26 is L. In some embodiments, X26 is I. In some embodiments, X27 is L. In some embodiments, X27 is A. In some embodiments, X28 is E. In some embodiments, X28 is N. In some embodiments, X29 is Aib. In some embodiments, X29 is T. In some embodiments, X30 is G. In some embodiments, X30 is absent. In some embodiments, X31 is absent. In some embodiments, Z is amide. In some embodiments, Z is acid.

[0010] In some embodiments, X2 is Aib, X12 is K, and X24 is Q. In some embodiments, X16 is T, X17 is K, X27 is L, X28 is E, and X29 is Aib. In some embodiments, X3 is Q, X5 is T, X6 is F, X10 is V, X12 is K, X15 is D, X16 is T, X17 is K, X18 is R, X20 is R, X21 is D, X22 is F, X23 is V, X24 is Q, X25 is W, X26 is L, X27 is L, X28 is E, X29 is Aib, X30 is G, X31 is absent, and Z is an acid. In some embodiments, X3 is H, X5 is S, X6 is αMePhe, X10 is V, X12 is K, X15 is D, X16 is S, X17 is R, X18 is A, X20 is K, X21 is D, X22 is αMePhe, X23 is V, X24 is Q, X25 is Aib, X26 is I, X27 is A, X28 is N, X29 is T, X30 is absent, X31 is absent, and Z is amide.

[0011] In some embodiments, one or more lysine residues are acylated. In some embodiments, the lysine at position 17 is acylated. In some embodiments, the lysine at position 20 is acylated.

[0012] In some embodiments, one or more lysines are lipidated. In some embodiments, the lysine at position 17 is lipidated. In some embodiments, the lysine at position 20 is lipidated.

[0013] In some embodiments, the lipid is selected from the group consisting of octadecanedioic acid (C18 diacid) and icosane diacid (C20 diacid). In some embodiments, the lipid is octadecanedioic acid (C18 diacid). In some embodiments, the lipid is icosane diacid (C20 diacid). In some embodiments, the lipid is linked to the epsilon amino group of lysine at position 17 or 20 via a linker.

[0014] In some embodiments, the linker is, from the C-terminus to the N-terminus, ((O2Oc)-(O2Oc)-γE) or ((O2Oc)-(O2Oc)-γE-γE). In some embodiments, the linker is, from the C-terminus to the N-terminus, ((O2Oc)-(O2Oc)-γE). In some embodiments, the linker is, from the C-terminus to the N-terminus, ((O2Oc)-(O2Oc)-γE-γE). In some embodiments, the linker is, from the C-terminus to the N-terminus, ((O2Oc)-(O2Oc)-γE-γE). In some embodiments, the linker is linked to the epsilon amino group of the residue at position 17 or 20.

[0015] Certain aspects of the present disclosure are directed to a peptide comprising the sequence H-Aib-QGTFTSDVSK-αMePhe-LDTKRARDFVQWLLE-Aib-G-acid (SEQ ID NO:541).

[0016] In some embodiments, the lysine at position 17 is acylated and lipidated, and the lipid is linked to the acylated lysine via its epsilon amino group and a linker that is ((O2Oc)-(O2Oc)-γE) in the C-terminal to N-terminal direction, and the lipid is octadecanedioic acid (C18 diacid). In some embodiments, the lysine at position 17 is acylated and lipidated, and the lipid is linked to the acylated lysine via its epsilon amino group and a linker that is ((O2Oc)-(O2Oc)-γE) in the C-terminal to N-terminal direction, and the lipid is icosane diacid (C20 diacid).

[0017] Certain aspects of the present disclosure are directed to a peptide comprising the sequence H-Aib-HGS-αMePhe-TSDVSK-αMePhe-LDSRAAK(ε-(O2Oc)-(O2Oc)-γE-C18 diacid) 20-D-αMePhe-VQ-Aib-IANT-amide (SEQ ID NO: 228).

[0018] Certain aspects of the present disclosure are directed to a peptide comprising the sequence H-Aib-HGS-αMePhe-TSDVSK-αMePhe-LDSRAAK(ε-(O2Oc)-(O2Oc)-γE-γE-C20 diacid) 20-D-αMePhe-VQ-Aib-IANT-amide (SEQ ID NO: 233).

[0019] In some embodiments, the peptide binds to the GLP-1 receptor (GLP-1R), binds to the glucagon receptor (GCGR), or binds to both the GLP-1 receptor and the glucagon receptor. In some embodiments, the GLP-1R is human GLP-1R. In some embodiments, the GCGR is human GCGR.

[0020] In some embodiments, the peptide is an agonist of GLP-1 activity, an agonist of glucagon activity, or an agonist of both GLP-1 and glucagon activity. In some embodiments, the peptide has increased resistance to proteolysis compared to the natural ligands of GLP-1R and / or GCGR.

[0021] In some embodiments, the peptide is isolated.

[0022] In some embodiments, the peptides have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% intact peptide remaining after incubation with the protease for 5 minutes, 10 minutes, 15 minutes, 30 minutes, 2 hours, 4 hours, or 24 hours at 37° C. In some embodiments, the protease is selected from the group consisting of neprilysin, pepsin, pancreatin, simulated gastric fluid with pepsin, and simulated intestinal fluid with pancreatin.

[0023] In some embodiments, following intravenous administration, the peptide has a half-life in cynomolgus monkeys of at least 45 hours, at least 50 hours, at least 60 hours, at least 70 hours, at least 80 hours, at least 90 hours, at least 100 hours, at least 110 hours, at least 120 hours, or about 130 hours. In some embodiments, the peptide has a subcutaneous bioavaiablity in cynomolgus monkeys of at least 75%, at least 80%, at least 90%, or about 95%.

[0024] In some aspects, provided herein is a pharmaceutical composition comprising the peptide. In some aspects, the composition is a solid composition. In some aspects, the composition is a liquid composition.

[0025] In some embodiments, the present invention provides a method for treating or preventing the disease or condition caused or characterized by excess weight, comprising administering to the subject in need of treatment an effective amount of any peptide or composition provided herein.In some embodiments, the disease or condition is obesity.In some embodiments, the disease or condition is type 2 diabetes.

[0026] In some aspects, provided herein are methods for treating or preventing non-alcoholic steatohepatitis (NASH), comprising administering to a subject in need of treatment an effective amount of any of the peptides or compositions provided herein.

[0027] In some embodiments of the methods provided herein, the administration is by injection. In some embodiments of the methods provided herein, the administration is by oral. In some embodiments of the methods provided herein, the administration reduces the subject's body weight, increases the subject's insulin secretion, delays gastric emptying in the subject, reduces food intake in the subject, increases mitochondrial function in the subject, inhibits de novo lipogenesis in the subject, reduces HbA1c in the subject, enhances fat oxidation in the subject, reduces hepatic mitochondrial oxidative stress in the subject, reduces steatosis in the subject, reduces fibrosis in the subject, reduces glycogen synthesis in the subject, increases gluconeogenesis in the subject, stops disease progression in the subject, reverses fibrosis in the subject, and / or reduces the risk of death due to liver cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in the subject. In some embodiments of the methods provided herein, the subject is a human. In some embodiments of the methods provided herein, the peptide is administered about once a week. [Brief description of the drawings]

[0028] [Figure 1A] 1A-1G are diagrams depicting exemplary lipids icosane diacid (C20 diacid) (FIG. 1A), octadecanedioic acid (C18 diacid) (FIG. 1B), stearoyl (FIG. 1C), margalloyl (FIG. 1D), palmitoyl (FIG. 1E), myristoyl (FIG. 1F), and lauryl (FIG. 1G). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 2A]2A-2F show exemplary linkers attached to the lipids depicted in FIGS. 1A-1G, including 2-(2-(2-aminoethoxy)ethoxy)acetic acid (O2Oc) (FIG. 2A), (O2Oc)-(O2Oc) (FIG. 2B), (O2Oc)-γE-(O2Oc) (FIG. 2C), (PEG)2-(PEG)2-γE-γE (FIG. 2D), (PEG)2-γE-(PEG)2-γE (FIG. 2E), gamma glutamic acid (γE) (FIG. 2F), γE-(O2Oc) (FIG. 2G), γE-(O2Oc)-(O2Oc) (FIG. 2H), γE-(O2Oc)-γE-(O2Oc) (FIG. 2I), and γE-(PEG)2-(PEG)2 (FIG. 2J). 2K), γE-(PEG)4 (FIG. 2L), γE-γE (FIG. 2M), γE-γE-(O2Oc) (FIG. 2N), γE-γE-(O2Oc)-(O2Oc) (FIG. 2O), γE-γE-(PEG)12 (FIG. 2P), γE-γE-(PEG)2-(PEG)2 (FIG. 2Q), γE-γE-(PEG)2-γE-γE (FIG. 2R), γE-γE-(PEG)4 (FIG. 2S), γE-γE-(PEG)8 (FIG. 2T), γE-γE-(O2Oc)-(O2Oc)-γE-γE (FIG. 2U), and (PEG)2-(PEG)2-γE (FIG. 2V). All linkers are shown with the N-terminus on the left and the C-terminus on the right. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above. [Figure 2M] Same as above. [Figure 2N] Same as above. [Figure 2O] Same as above. [Figure 2P] Same as above. [Figure 2Q] Same as above. [Figure 2R] Same as above. [Figure 2S] Same as above. [Figure 2T] Same as above. [Figure 2U] Same as above. [Figure 2V] Same as above. [Figure 3A] 3A-3P show exemplary unnatural amino acids, 2-amino-2-methylpropanoic acid (Aib) ( FIG. 3A ), (S)-2-amino-2-methyl-3-phenylpropanoic acid (αMePhe) ( FIG. 3B ), (S)-2-amino-3-hydroxy-2-methylpropanoic acid (αMeSer) ( FIG. 3C ), D-glutamine (dGln) ( FIG. 3D ), β-dimethylglutamine (β-dimethylGln) ( (Figure 3E), (S)-2,5-diamino-2-methyl-5-oxopentanoic acid (αMeGln) (Figure 3F), D-serine (dSer) (Figure 3G), methyl-L-glutamine (N-MeGln) (Figure 3H), ((1H-imidazol-4-yl)methyl)glycine (NHis) (Figure 3I), 1-aminocyclopropane-1-carboxylic acid (Acpr) (Figure 3J), 1-amino Nonocyclobutane-1-carboxylic acid (Acbu) (Figure 3K), N6-acetyl-L-lysine (Ac-Lys) (Figure 3L), (S)-2-amino-5-guanidino-3,3-dimethylpentanoic acid (β-dimethylArg) (Figure 3M), (S)-2-amino-3,3-diphenylpropanoic acid (Dip) (Figure 3N), (S)-2-amino-3-cyclohexylpropanoic acid (Cha) ( FIG. 3O), (S)-2-aminohexanoic acid (Nle) (FIG. 3P), (S)-3-([1,1′-biphenyl]-4-yl)-2-aminopropanoic acid (Bip) (FIG. 3Q), 1-methyl-L-tryptophan (1-methyl-Trp) (FIG. 3R), and (S)-2-amino-3-(5-bromo-1H-indol-3-yl)propanoic acid (5-Br-Trp) (FIG. 3S). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 3J] Same as above. [Figure 3K] Same as above. [Figure 3L] Same as above. [Figure 3M] Same as above. [Figure 3N] Same as above. [Figure 3O] Same as above. [Figure 3P] Same as above. [Figure 3Q] Same as above. [Figure 3R] Same as above. [Figure 3S] Same as above. [Figure 4A] 4A-4D are drawings depicting the structures of exemplary peptides: peptide 224 (FIG. 4A), peptide 229 (FIG. 4B), peptide 188 (FIG. 4C), and peptide 195 (FIG. 4D). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0030] Units, prefixes, and symbols are written in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not limitations of the various aspects of the disclosure, but can be understood by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0031] Throughout this disclosure, the terms "a" or "an" refer to one or more of that entity, e.g., "a polynucleotide" is understood to refer to one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0032] Furthermore, "and / or" as used herein is deemed to specifically disclose each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A (alone)", and "B (alone)". Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0033] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate a deviation of 10% above and below the numerical value or range while remaining within the intended meaning of the stated numerical value or range. When embodiments are described herein with a numerical value or range of the word "about" or "approximately," it is understood that other similar embodiments that refer to the particular numerical value or range are also provided.

[0034] When an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided. A peptide "comprising" a particular amino acid sequence refers to a peptide containing that amino acid sequence, which may or may not contain additional amino acids or other modifications to that amino acid sequence. A peptide "consisting of" a particular amino acid sequence refers to a peptide containing only that amino acid sequence, without additional amino acids or other modifications to that amino acid sequence. A peptide "comprising" an amino acid sequence "consisting of" a particular amino acid sequence refers to a peptide containing that amino acid sequence and no additional amino acids, although the peptide may contain other modifications to the amino acid sequence (e.g., acyl or palmitoyl moieties).

[0035] As used herein, the term "amino acid" refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bonded with hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.

[0036] "Unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrrolysine or selenocysteine; other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated versions thereof. The term "unnatural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids, or pyrrolysine or selenocysteine) but that are not themselves incorporated into a growing polypeptide chain by the translation complex.

[0037] Examples of naturally occurring amino acids that are not naturally encoded include ((1H-imidazol-4-yl)methyl)glycine (NHis), S, 1-aminocyclobutane-1-carboxylic acid (1-aminocyclobutane-1-carboxylic acid; Acbu), 1-aminocyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carboxylic acid; Acpr), aminoisobutyric acid (2-amino-2-methylpropanoic acid; Aib), D-serine (d Ser), α-methyl-serine ((S)-2-amino-3-hydroxy-2-methylpropanoic acid (αMeSer); αMeSer), methyl-L-glutamine (N-MeGln), α-methyl-glutamine ((S)-2,5-diamino-2-methyl-5-oxopentanoic acid; αMeGln), β-dimethylGln, α-methyl-phenylalanine ((S)-2-amino-2-methyl-3-phenylpropanoic acid; αMePhe), acetylated lysine N 6 These include, but are not limited to, -acetyl-L-lysine (Ac-Lys), diphenylalanine ((S)-2-amino-3,3-diphenylpropanoic acid; Dip), β-dimethylarganine ((S)-2-amino-5-guanidino-3,3-dimethylpentanoic acid; β-dimethylArg), β-cyclohexyl-L-alanine ((S)-2-amino-3-cyclohexylpropanoic acid; Cha), norleucine ((S)-2-aminohexanoic acid; Nle), D-glutamine (dGln), (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid (Bip), 1-methyl-L-tryptophan (1-methyl-Trp), and (S)-2-amino-3-(5-bromo-1H-indol-3-yl)propanoic acid (5-Br-Trp).

[0038] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes.

[0039] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides" and includes any chain or chains of two or more amino acids. Thus, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," or other terms used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," even though each of these terms may have a more specific meaning. The term "polypeptide" may be used in place of or interchangeably with these terms. The term further includes polypeptides that have been post-translationally or post-synthetically modified, such as the attachment of palmitoyl groups, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids.

[0040] More specifically, the term "peptide" as used herein includes full-length peptides and fragments, variants or derivatives thereof, such as GLP-1 / glucagon agonist peptides (e.g., 29, 30 or 31 amino acids long). The "peptides" disclosed herein, e.g., GLP-1 / glucagon agonist peptides, may be part of a fusion polypeptide that includes additional components, such as, for example, an Fc domain or an albumin domain, to increase half-life. The peptides described herein can also be derivatized in many different ways. The peptides described herein can include modifications including, for example, the attachment of lipids, including palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, octadecanedioic acid (C18 diacid), or icosandioic acid (C20 diacid). Exemplary lipids are shown in Figures 1A-1G.

[0041] The peptides described herein may include modifications including, for example, the attachment of linkers including 2-(2-(2-aminoethoxy)ethoxy)acetic acid (O2Oc), PEG, and / or gamma glutamic acid (γE). In some embodiments, the linker is selected from the group consisting of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), 3-(2-(2-aminoethoxy)ethoxy)propanoic acid) ((PEG)2), 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid ((PEG)4), 1-amino-3,6,9,12,15,18,21,14-octaoxaheptacosane-27-oic acid ((PEG)8), 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid ((PEG)12), gamma glutamic acid (γE) , (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2O c)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γE-(PEG)2-γE-(PEG)2, γE-(PEG)4, γE-γE, γE-γE-(O2O c), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, γE-γE-(PEG)8, γE-γE-(O2Oc)-(O2Oc)-γE-γE, and (PEG)2-(PEG)2-γE. Exemplary linkers are shown in Figures 2A-2V.

[0042] The term "isolated" refers to the peptide or nucleic acid being in a state generally in accordance with the present disclosure. Isolated peptides and isolated nucleic acids are free or substantially free of the substances with which they are naturally associated, such as other peptides or nucleic acids, when they are found in their natural environment, or when they are found in the environment in which they are prepared (e.g., cell culture) when preparation is by recombinant DNA technology carried out in vitro or in vivo. Peptides and nucleic acids can be formulated with diluents or adjuvants and still be practically isolated, for example, peptides are usually mixed with gelatin or other carriers when used to coat microtiter plates for use in immunoassays, or mixed with pharma-ceutically acceptable carriers or diluents when used for diagnosis or treatment.

[0043] "Recombinant" peptide refers to a peptide produced by recombinant DNA technology. Recombinantly produced peptides expressed in a host cell are considered isolated for purposes of this disclosure as native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0044] The terms "fragment," "analog," "derivative," or "variant," when referring to GLP-1 / glucagon agonist peptides, include any peptide that retains at least some desired activity, e.g., binding to the glucagon receptor and / or the GLP-1 receptor. Fragments of the GLP-1 / glucagon agonist peptides provided herein include proteolytic fragments, deletion fragments that exhibit desirable properties during expression, purification, and / or administration to a subject.

[0045] The term "variant" as used herein refers to a peptide that differs from the referenced peptide by amino acid substitution, deletion, insertion, and / or modification. Variants can be made using mutagenesis techniques known in the art. Variants may alternatively contain other modifications. Peptides may be bound or linked, e.g., fused, to heterologous amino acid sequences or other moieties, e.g., to increase half-life, solubility, or stability. Examples of moieties that may be bound or linked to the peptides provided herein include, but are not limited to, albumin, immunoglobulin Fc region, polyethylene glycol (PEG), and the like. Peptides may also be produced bound or linked to linkers or other sequences to facilitate peptide synthesis, purification, or identification (e.g., 6-His), or to enhance binding of the polypeptide to a solid support.

[0046] The term "composition" or "pharmaceutical composition" refers to a composition containing a GLP-1 / glucagon agonist peptide provided herein and, e.g., a pharma- ceutical acceptable carrier, excipient, or diluent, for administration to a subject in need of treatment, e.g., a human subject in need of improved glycemic control, weight loss, treatment of type 2 diabetes (Mellitis), and / or treatment of NASH.

[0047] The term "pharmacologically acceptable" refers to a composition that is suitable, within the scope of sound medical judgment, for contact with the tissues of human beings and animals without undue toxicity or other complications commensurate with a reasonable benefit / risk ratio.

[0048] An "effective amount" is an amount of an agent provided herein (e.g., a GLP-1 / glucagon agonist peptide) whose administration to a subject, either as a single dose or as part of a series of doses, is effective for treatment, e.g., improved glycemic control, weight loss, treatment of type 2 diabetes, and / or treatment of NASH.

[0049] As used herein, the terms "subject" and "patient" are used interchangeably. The subject may be an animal. In some embodiments of the present disclosure, the subject is a mammal, such as a non-human animal (e.g., a cow, a pig, a horse, a cat, a dog, a rat, a mouse, a monkey or other primate, etc.). In some embodiments of the present disclosure, the subject is a cynomolgus monkey. In some embodiments of the present disclosure, the subject is a human.

[0050] As used herein, "subject in need thereof" or "patient in need thereof" refers to an individual for whom treatment is desirable, e.g., a subject in need of improved glycemic control, weight loss, treatment of type 2 diabetes, and / or treatment of NASH.

[0051] Terms such as "treat" or "treatment" or "to treat" refer to therapeutic measures that cure and / or halt the progression of a diagnosed pathological condition or disorder. Terms such as "prevent" refer to prophylactic or preventative measures that prevent and / or delay the onset of the pathological condition or disorder of interest. Thus, patients in need of treatment include those already with the disease or condition. Those in need of prevention include those susceptible to the disease or condition as well as those in whom the disease or condition is to be prevented.

[0052] "Reducing the severity" and like terms refer to therapeutic measures that slow or alleviate the symptoms of a diagnosed pathological condition or disorder.

[0053] As used herein, a "GLP-1 agonist peptide" is a peptide that is not native GLP-1 but exhibits at least about 1% or more activity at the GLP-1 receptor compared to native GLP-1 under cAMP assay conditions (see Example 2). In some embodiments, the GLP-1 agonist peptide exhibits at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity at the GLP-1 receptor compared to native GLP-1 under cAMP assay conditions (see Example 2).

[0054] As used herein, a "glucagon agonist peptide" is a peptide that is not native glucagon, but that exhibits at least 1% or more activity at the glucagon receptor compared to native glucagon under cAMP assay conditions (see Example 2). In some embodiments, the glucagon agonist peptide exhibits at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity at the glucagon receptor compared to native GLP-1 under cAMP assay conditions (see Example 2).

[0055] As used herein, a "GLP-1 / glucagon agonist peptide," "GLP-1 / glucagon co-agonist peptide," "GLP-1 and glucagon dual agonist peptide," or "GLP-1 and glucagon dual co-agonist peptide" is a peptide that is neither native GLP-1 nor native glucagon, and that exhibits at least about 1% or more activity at the glucagon receptor compared to native glucagon and at least about 1% or more activity at the GLP-1 receptor compared to native GLP-1 under cAMP assay conditions (see Example 2). In some embodiments, the "GLP-1 / glucagon agonist peptides" or "GLP-1 and glucagon dual agonist peptides" exhibit at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity at the glucagon receptor compared to native glucagon, and at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity at the GLP-1 receptor compared to native GLP-1 under cAMP assay conditions (see Example 2).

[0056] As used herein, "relative potency ratio" refers to the % GLP-1R activity for GLP-1 / % GlucR activity for glucagon.

[0057] As used herein, the term "native glucagon" refers to naturally occurring glucagon, e.g., human glucagon contained in the sequence of SEQ ID NO:1. The term "native GLP-1" refers to naturally occurring GLP-1, e.g., human GLP-1, and is a generic term encompassing, e.g., GLP-1(7-36)amide (SEQ ID NO:2), GLP-1(7-37)acid (SEQ ID NO:3), or a mixture of these two compounds. As used herein, a general reference to "glucagon" or "GLP-1" without further specification is intended to mean native human glucagon or native human GLP-1, respectively. Unless otherwise specified, "glucagon" refers to human glucagon and "GLP-1" refers to human GLP-1.

[0058] As used herein, the term "sequence identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. If a position in a sequence is occupied by the same nucleic acid base or amino acid as the corresponding position in the compared sequence, the sequences are said to be "identical" at that position. The percentage of "sequence identity" is calculated by determining the number of positions where the same nucleic acid base or amino acid occurs in both sequences to obtain the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to calculate the percentage of "sequence identity". The percentage of "sequence identity" is determined by comparing two sequences that are optimally aligned over the comparison window. To optimally align sequences for comparison, portions of the polynucleotide or polypeptide sequences within the comparison window can include additions or deletions, called gaps, while keeping the reference sequence constant. An optimal alignment is one that produces as many "identical" positions as possible between the reference and comparison sequences, even with gaps. The percentage of "sequence identity" between two sequences can be determined using the version of the program "BLAST2 Sequences" available from the National Center for Biotechnology Information as of September 1, 2004, which incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST2 Sequences", the default parameters as of September 1, 2004 can be used for other required parameters, including but not limited to word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expectation value (10), and matrix options. Glucagon (SEQ ID NO: 1) HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-acid GLP-1(7-36)amide (SEQ ID NO:2) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Amide GLP-1(7-37) acid (SEQ ID NO:3) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-acid

[0059] II. GLP-1 / Glucagon Agonist Peptides Provided herein is a peptide that binds to both glucagon receptor and GLP-1 receptor.In some embodiments, provided herein is a peptide that is a co-agonist (dual agonist) of glucagon and GLP-1 activity.Such peptide is referred to herein as GLP-1 / glucagon agonist peptide.In some embodiments, provided herein is a GLP-1 / glucagon agonist peptide that is active at human GLP1 receptor and human glucagon receptor.

[0060] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein exhibit an in vitro potency at the GLP-1 receptor, represented by EC50 in a cAMP assay (see Example 2), of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

[0061] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein exhibit an in vitro potency at the glucagon receptor, represented by EC50 in a cAMP assay (see Example 2), of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

[0062] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 1 to about 25. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 1 to about 20. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 1 to about 15. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 1 to about 10.

[0063] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 2 to about 25. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 2 to about 20. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 2 to about 15. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have a hGLP-1R / hGCGR relative potency ratio of about 2 to about 10.

[0064] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein, when administered to mice at 10 nmol / kg (as performed in Example 3), reduce 24-hour food intake by at least 10% compared to mice treated with a vehicle control. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein, when administered to mice at 10 nmol / kg (as performed in Example 3), reduce 24-hour food intake by at least 20% compared to mice treated with a vehicle control. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein, when administered to mice at 10 nmol / kg (as performed in Example 3), reduce 24-hour food intake by at least 30% compared to mice treated with a vehicle control.

[0065] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein, when administered to mice at 10 nmol / kg (as performed in Example 3), reduce 24-hour food intake by 10-70% compared to mice treated with a vehicle control. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein, when administered to mice at 10 nmol / kg (as performed in Example 3), reduce 24-hour food intake by at least 20% or 20-70% compared to mice treated with a vehicle control. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein, when administered to mice at 10 nmol / kg (as performed in Example 3), reduce 24-hour food intake by at least 30% or 30-70% compared to mice treated with a vehicle control.

[0066] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable.For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSGF (fasting state simulated gastric fluid), and after 30 minutes of incubation with FasSSGF, at least 25% of the GLP-1 / glucagon agonist peptides remain intact (as carried out in Example 4).In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSGF, and after 30 minutes of incubation with FasSSGF, at least 40% of the GLP-1 / glucagon agonist peptides remain intact (as carried out in Example 4). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSGF, where at least 50% of the GLP-1 / glucagon agonist peptide remains intact after 30 minutes of incubation with FasSSGF (as performed in Example 4). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSGF, where at least 60% of the GLP-1 / glucagon agonist peptide remains intact after 30 minutes of incubation with FasSSGF (as performed in Example 4). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSGF, where at least 70% of the GLP-1 / glucagon agonist peptide remains intact after 30 minutes of incubation with FasSSGF (as performed in Example 4).

[0067] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, and at least 55% of the GLP-1 / glucagon agonist peptides remain intact after 24 hours of incubation with neprilysin (as performed in Example 5). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, and at least 60% of the GLP-1 / glucagon agonist peptides remain intact after 24 hours of incubation with neprilysin (as performed in Example 5). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, and at least 65% of the GLP-1 / glucagon agonist peptides remain intact after 24 hours of incubation with neprilysin (as performed in Example 5). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, and at least 70% of the GLP-1 / glucagon agonist peptides remain intact after 24 hours of incubation with neprilysin (as carried out in Example 5). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, and at least 75% of the GLP-1 / glucagon agonist peptides remain intact after 24 hours of incubation with neprilysin (as carried out in Example 5). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, and at least 80% of the GLP-1 / glucagon agonist peptides remain intact after 24 hours of incubation with neprilysin (as carried out in Example 5). In certain embodiments, the GLP-1 / Glucagon agonist peptides provided herein are stable in the presence of neprilysin, with at least 85% of the GLP-1 / Glucagon agonist peptide remaining intact after 24 hours of incubation with neprilysin (as performed in Example 5).In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, such that at least 90% of the GLP-1 / glucagon agonist peptide remains intact after 24 hours of incubation with neprilysin (as performed in Example 5). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of neprilysin, such that at least 95% of the GLP-1 / glucagon agonist peptide remains intact after 24 hours of incubation with neprilysin (as performed in Example 5).

[0068] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable.For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSIF (fasted state simulated intestinal fluid) / pancreatin, and at least 10% of the GLP-1 / glucagon agonist peptides remain intact after 30 minutes of incubation with FasSSIF / pancreatin (as carried out in Example 6).In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable.For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSIF / pancreatin, and at least 20% of the GLP-1 / glucagon agonist peptides remain intact after 30 minutes of incubation with FasSSIF / pancreatin (as carried out in Example 6). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable. For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSIF / pancreatin, and at least 25% of the GLP-1 / glucagon agonist peptides remain intact after 30 minutes of incubation with FasSSIF / pancreatin (as carried out in Example 6). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable. For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FaSSSIF / pancreatin, and at least 50% of the GLP-1 / glucagon agonist peptides remain intact after 30 minutes of incubation with FasSSSIF / pancreatin (as carried out in Example 6). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable.For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSIF / pancreatin, and at least 75% of the GLP-1 / glucagon agonist peptides remain intact after 30 minutes of incubation with FasSSIF / pancreatin (as carried out in Example 6). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable. For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSIF / pancreatin, and at least 80% of the GLP-1 / glucagon agonist peptides remain intact after 30 minutes of incubation with FasSSIF / pancreatin (as carried out in Example 6). In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable. For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stable in the presence of FasSSIF / pancreatin, with at least 90% of the GLP-1 / glucagon agonist peptide remaining intact after a 30 minute incubation with FasSSIF / pancreatin (as performed in Example 6).

[0069] The GLP-1 / glucagon agonist peptides disclosed herein can include a heterologous moiety, for example to extend half-life. The heterologous moiety can be a protein, peptide, protein domain, linker, organic polymer, inorganic polymer, polyethylene glycol (PEG), biotin, albumin, human serum albumin (HSA), FcRn binding portion of HSA, antibody, antibody domain, antibody fragment, single chain antibody, domain antibody, albumin binding domain, enzyme, ligand, receptor, binding peptide, non-FnIII scaffold, epitope tag, recombinant polypeptide polymer, cytokine, and combinations of two or more of such moieties.

[0070] In some embodiments, the GLP-1 / glucagon agonist peptides disclosed herein bind to the GLP-1 receptor (GLP-1R), bind to the glucagon receptor (GCGR), or bind to both GLP-1R and GCGR. In some embodiments, the GLP-1R is human GLP-1R. In some embodiments, the GCGR is human GCGR. In some embodiments, the peptides are agonists of GLP-1 activity, agonists of glucagon activity, or agonists of both GLP-1 and glucagon activity.

[0071] In some embodiments, the GLP-1 / glucagon agonist peptides as disclosed herein have increased proteolytic resistance compared to the natural ligands of GLP-1R and / or GCGR. In some embodiments, the GLP-1 / glucagon agonist peptides as disclosed herein have increased proteolytic resistance compared to cotadutide (SEQ ID NO: 358). In some embodiments, the GLP-1 / glucagon agonist peptides as disclosed herein have increased proteolytic resistance compared to semaglutide (SEQ ID NO: 359). In some embodiments, the GLP-1 / glucagon agonist peptides as disclosed herein have increased proteolytic resistance compared to cotadutide (SEQ ID NO: 358) and semaglutide (SEQ ID NO: 359).

[0072] In some embodiments, the GLP-1 / glucagon agonist peptides provided herein comprise the sequence: X1-X2-X3-G-X5-X6-TSD-X10-S-X12-X13-L-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-Z, where X1 is H or NHis and X2 is S, 1-aminocyclobutane-1-carboxylic acid (Acbu), 1-aminocyclopropane-1-carboxylic acid (Acpr), aminoisobutyric acid (Aib), D-selenoic acid (Dss), 1-aminocyclopropane-1-carboxylic acid (Ab ... X3 is Q, H, I, D-glutamine (dGln), methyl-L-glutamine (N-MeGln), α-methyl-glutamine (αMeGln), or β-dimethylglutamine (β-dimethylGln); X5 is T or S; X6 is F or α-methyl-phenylalanine (αMePhe); X10 is Y, K, or V, where K may contain an acyl moiety and / or may be lipidated; X12 is K, acetylated lysine (Ac-Lys), acetylated lysine (Ac-Lys), or acetylated lysine (Ac-Lys), X4 is H, I, D-glutamine (dGln), methyl-L-glutamine (N-MeGln), α-methyl-glutamine (αMeGln), or β-dimethylglutamine (β-dimethylGln); X5 is T or S; X6 is F or α-methyl-phenylalanine (αMePhe); X15 is D or E, X16 is S, Aib, E, T, R, A, K, L, or V, and X17 is R, E, K, Q, or β-dimethylarganine (β-dimethylArg), where K may comprise an acyl moiety and / or may be lipidated. X18 is R, A, Aib, Q, S, or β-dimethylArg, X19 is A or V, X20 is Q, Aib, E, K, L, or R, where K may comprise an acyl moiety and / or may be lipidated, X21 is D, E, or L, X22 is F, I, or αMePhe, X23 is V or I, X24 is Q, A, E, K, L, or R, where K may comprise an acyl moiety and / or may be lipidated, X25 is W, Aib, Dip, H, I, S,X27 is M, A, E, I, L, norleucine (Nle), S, K, or V; X28 is N, (PEG)4, A, Aib, E, G, R, S, or absent; X29 is L, beta-cyclohexyl-L-alanine (Cha), I, or V; X28 is N, (PEG)4, A, Aib, E, G, R, S, or absent; X29 is T, Aib, E, G, A, R, or absent, X30 is absent, E, A, Aib, K, T, or G, X31 is absent, I, or G, and Z is an amide or acid (SEQ ID NO:4), where the peptide does not comprise SEQ ID NO:1, and HSQGTFTSDX10SEYLDSERARDFVAWLEAGG-acid, where X10=Lys[ε-γE-palmitoyl] (SEQ ID NO:538). In some embodiments, X2 is Aib, and / or X10 is V. In some embodiments, X3 is Q, X15 is D, X18 is R, X20 is R, X21 is D, X23 is V, and / or X30 is G. In some embodiments, X13 is αMePhe, X16 is T, X17 is K, X27 is L, X28 is E, and / or X29 is Aib.

[0073] In some embodiments, the residues at positions 10, 13, 17, 20, or 24 are acylated. In some embodiments, the residues at positions 10, 13, 17, 20, or 24 are lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, arachidoyl, octadecanedioic acid (a C18 diacid), and icosane diacid (a C20 diacid).

[0074] In some embodiments, the lipid attached to residues 10, 13, 17, 20, or 24 is attached via a linker. In some embodiments, the linker is selected from the group consisting of (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γE-(PEG)2-γE-(PEG)2, γE -(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, (PEG)2-(PEG)2-γE, γE-γE-(O2Oc)-(O2Oc)-γE-γE, and γE-γE-(PEG)8.

[0075] In some embodiments, the linker is linked to the epsilon amino group of residues 10, 13, 17, 20, and / or 24.

[0076] In some embodiments, the peptide comprises any one of SEQ ID NOs: 6-411 and 418-537. In some embodiments, the peptide comprises SEQ ID NO: 99. In some embodiments, the peptide comprises SEQ ID NO: 106. In some embodiments, the peptide comprises SEQ ID NO: 228. In some embodiments, the peptide comprises SEQ ID NO: 233.

[0077] In some embodiments, the peptide comprises the sequence: H-X2-X3-G-X5-X6-TSD-X10-S-X12-X13-L-X15-X16-X17-X18-A-X20-D-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-Z, where X2 is S, aminoisobutyric acid (Aib), or alpha methyl-serine (alpha MeSer), X3 is Q or H, X5 is T or S, and X6 is F or alpha methyl-phenyl. X10 is Y or V, X12 is K or acetylated lysine (Ac-Lys), X13 is Y, αMePhe, Aib, diphenylalanine (Dip), or I, X15 is D or E, X16 is S, T, A, E, K, L, R, or V, X17 is R or K, where K may contain an acyl moiety and / or be lipidated, X18 is R, A, Q, or β- X20 is Q, R, Aib, L, or E, X22 is F, I, or αMePhe, X23 is V or I, X24 is Q, E, A, L, or R, X25 is W, Aib, S, Dip, I, H, biphenyl-alanine (Bip), 1-methyltryptophan (1-methyl-Trp), 5-bromotryptophan (5-BrTrp), or αMePhe, and X26 is L, I, or Be X27 is M, A, L, E, V, I, K, norleucine (Nle), or S, X28 is N, Aib, E, (PEG)4, A, S, or G, X29 is T, absent, Aib, G, A, R, or E, X30 is absent, G, A, Aib, K, or E, X31 is absent, and Z is amide or acid (SEQ ID NO:5), wherein the peptide does not include SEQ ID NO:1.

[0078] In some embodiments, the residue at position 17 is acylated. In some embodiments, the residue at position 17 is lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, arachidoyl, octadecanedioic acid (a C18 diacid), and icosane diacid (a C20 diacid).

[0079] In some embodiments, the lipid attached to residue 17 is attached via a linker. In some embodiments, the linker is selected from the group consisting of (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γ Selected from the group consisting of E-(PEG)2-γE-(PEG)2, γE-(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, and γE-γE-(PEG)8. In some embodiments, the linker is selected from the group consisting of γE-γE-(O2Oc)-(O2Oc)-γE-γE, (O2Oc)-(O2Oc), γE, γE-(O2Oc)-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)4, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)8, γE-γE-(PEG)12, (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE-(PEG)2-γE-(PEG)2, γE-(PEG)2-(PEG)2, (PEG)2-(PEG)2-γE, and γE-(PEG)4.

[0080] In some embodiments, the linker is attached to the epsilon amino group of the 17th residue.

[0081] In some embodiments, the peptide comprises the sequence: H-aminoisobutyric acid (Aib)-QGT-X6-TSDVSK-alpha methyl-phenylalanine (alpha MePhe)-L-X15-X16-K-X18-A-X20-X21-X22-X23-X24-W-X26-X27-X28-X29-X30-X31-Z, where X6 is F or alpha methyl-phenylalanine (alpha MePhe), X15 is E or D, X16 is T, S, K, E, A, L, or R, X18 is R or A, X20 is R, Q, or L, X21 is D or E, X22 is F or alpha MePhe, and X23 is V. or I, X24 is R, A, Q, or L, X25 is W, αMePhe, biphenyl-alanine (Bip), 1-methyltryptophan (1-methyl-Trp), 5-bromotryptophan (5-BrTrp), or Aib, X26 is L, I, or V, X27 is L, A, E, V, I, or K, X28 is E, S, A, Aib, absent, or R, X29 is G, Aib, R, T, E, A, or absent, X30 is G, Aib, E, A, K, or absent, X31 is I or absent, and / or G, and Z is an amide or acid (SEQ ID NO: 412).

[0082] In some embodiments, the lysine at position 17 is acylated. In some embodiments, the lysine at position 17 is lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margaroyl, arachidoyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid). In some embodiments, the lipid is selected from the group consisting of octadecanedioic acid (C18 diacid) and icosane diacid (C20 diacid). In some embodiments, the lipid is octadecanedioic acid (C18 diacid). In some embodiments, the lipid is icosane diacid (C20 diacid).

[0083] In some embodiments, the lipid is linked to lysine 17 via a linker. In some embodiments, the linker is (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG), γE-(PEG)2-γE-(PEG)2 , γE-(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, (PEG)2-(PEG)2-γE, γE-γE-(O2Oc)-(O2Oc), and γE-γE-(PEG)8. In some embodiments, the peptide of claim X, wherein the linker is selected from the group consisting of (PEG)2-(PEG)2-γE, (O2Oc)-(O2Oc), γE, γE-(O2Oc)-(O2Oc), γE-γE-(O2Oc)-(O2Oc), and γE-γE(O2Oc)-(O2Oc)-γE-γE. In some embodiments, the linker is γE-(O2Oc)-(O2Oc).

[0084] In some embodiments, the linker is attached to the epsilon amino group of the 17th residue.

[0085] In certain embodiments, the GLP-1 / glucagon agonist peptides as disclosed have desirable potencies at the glucagon and GLP-1 receptors and desirable relative potencies for promoting weight loss.

[0086] In some embodiments, the peptide has any one of SEQ ID NOs: 6-206 and 418-531. In some embodiments, the peptide comprises SEQ ID NO: 99. In some embodiments, the peptide comprises SEQ ID NO: 106.

[0087] In some embodiments, the peptide has the structure of any one of the structures depicted in Figures 4C-4D. In some embodiments, the peptide has the structure of Figure 4C. In some embodiments, the peptide has the structure of Figure 4D.

[0088] In some embodiments, the peptide is any one of the peptides in Table 1.

[0089] Table 1. Peptides modified at position 17 [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

[0090] In some embodiments, the peptide comprises the sequence: H-X2-X3-G-X5-X6-TSD-X10-S-X12-X13-L-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-Z, where X2 is S, aminoisobutyric acid (Aib), alpha methyl-serine (alphaMeS), D-serine (dSer), 1-aminocyclopropane-1-carboxylic acid (Acpr), or is S, 1-aminocyclobutane-1-carboxylic acid (Acbu), X3 is Q, H, α-methyl-glutamine (αMeGln), N-methyl-glutamine (N-MeGln), D-glutamine (dGln) or β-dimethylglutamine (β-dimethylGln), X5 is T or S, X6 is F or α-methyl-phenylalanine (αMePhe), X10 is Y or V, X12 is K, E or R, X13 is Y, αMePhe, or Aib, X15 is D or E, X16 is S, T, E, or Aib, X17 is R, Q, or E, X18 is R, A, Aib, or S, X19 is A or V, X20 is Q or K, where K may contain an acyl moiety and / or be lipidated, X21 is D or L, X22 is F or α-methyl-phenylalanine (αMePhe), X23 is V or I, and X X24 is Q, E, A, or R, X25 is W, Aib, or S, X26 is L or I, X27 is M, A, L, E, I, or V, X28 is N, E, (PEG)4, Aib, S, or A, X29 is T, absent, E, or G, X30 is absent, E, T, or G, X31 is absent or G, and Z is amide or acid (SEQ ID NO:413), wherein the peptide does not include SEQ ID NO:1.

[0091] In some embodiments, the residue at position 20 is acylated. In some embodiments, the residue at position 20 is lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid). In some embodiments, the lipid is selected from the group consisting of stearoyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid).

[0092] In some embodiments, the lipid attached to residue 20 is attached via a linker. In some embodiments, the linker is selected from the group consisting of (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γ Selected from the group consisting of E-(PEG)2-γE-(PEG)2, γE-(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, and γE-γE-(PEG)8. In some embodiments, the linker is selected from the group consisting of γE, γE-γE, γE-γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-(O2Oc), γE-γE-(PEG)2-(PEG)2, γE-(O2Oc)-γE-(O2Oc), γE-(PEG)4, γE-γE-(PEG)4, (O2Oc)-γE-(O2Oc), (O2Oc)-(O2Oc) and (O2Oc).

[0093] In some embodiments, the peptide comprises the sequence: H-aminoisobutyric acid (Aib)-QGTFTSD-X10-S-X12-alpha methyl-phenylalanine (alpha MePhe)-LD-X16-X17-X18-AKDFV-X24-W-X26-X27-X28-X29-X30-Z, where X10 is V or Y, X12 is K or E, X16 is S or Aib, X17 is R or E, X18 is R or A, X24 is A, R, or Q, X26 is L or I, X27 is E, L, A, or I, X28 is A, E, Aib, S, or N, X29 is G, Aib, T, or E, X30 is G, E, T, or absent, and Z is an amide or acid (SEQ ID NO: 414).

[0094] In some embodiments, the lysine at position 20 is acylated. In some embodiments, the lysine at position 20 is lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid). In some embodiments, the lipid is octadecanedioic acid (C18 diacid).

[0095] In some embodiments, the lipid is linked via a linker to the lysine at position 20. In some embodiments, the linker is (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γ The linker is selected from the group consisting of E-(PEG)2-γE-(PEG)2, γE-(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, and γE-γE-(PEG)8. In some embodiments, the linker is selected from the group consisting of γE, γE-(O2Oc)-(O2Oc), and γE-γE(O2Oc)-(O2Oc)-γE-γE.

[0096] In some embodiments, the peptide comprises any one of SEQ ID NOs: 207-347 and 532-537. In some embodiments, the peptide comprises SEQ ID NO: 228. In some embodiments, the peptide comprises SEQ ID NO: 233.

[0097] In some embodiments, the peptide has the structure of any one of the structures depicted in Figures 4A-4B. In some embodiments, the peptide has the structure of Figure 4A. In some embodiments, the peptide has the structure of Figure 4B.

[0098] In some embodiments, the peptide is any one of the peptides in Table 2.

[0099] Table 2. Peptides modified at position 20 [Table 2-1] [Table 2-2] [Table 2-3]

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

[0100] In some embodiments, the peptide comprises the sequence: X1-X2-X3-G-X5-X6-TSD-X10-SK-X13-L-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-Z, where X1 is H or ((1H-imidazol-4-yl)methyl)glycine (N X2 is S or aminoisobutyric acid (Aib), X3 is Q, H or I, X5 is T or S, X6 is F or α-methyl-phenylalanine (αMePhe), X10 is Y or V, X13 is Y, αMePhe, Aib or diphenylalanine (Dip), X15 is D or E, and X16 is S, E or L. X17 is R, Q, or E, X18 is R, A, or Aib, X19 is A or V, X20 is Q or R, X21 is D or L, X22 is F or αMePhe, X23 is V or I, X24 is Q or K, where K may contain an acyl moiety and / or be lipidated, X25 is W, Aib, or S, X26 is L or I, X27 is M, V, L, or A, X28 is N, E, or Aib, X29 is T, Aib, G or absent, X30 is absent, Aib, or G, X31 is absent or G, and Z is an amide or acid (SEQ ID NO:415), wherein the peptide does not comprise SEQ ID NO:1.

[0101] In some embodiments, the residue at position 24 is acylated. In some embodiments, the residue at position 24 is lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid). In some embodiments, the lipid is selected from the group consisting of octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid).

[0102] In some embodiments, the lipid attached to residue 24 is attached via a linker. In some embodiments, the linker is selected from the group consisting of (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γ The linker is selected from the group consisting of E-(PEG)2-γE-(PEG), γE-(PEG), γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG), γE-γE-(PEG), γE-γE-(PEG), γE-γE-(PEG), γE-γE-(PEG), γE-γE-(PEG), γE-γE-(PEG). In some embodiments, the linker is selected from the group consisting of γE, γE-(O2Oc)-(O2Oc), γE-γE-(PEG)2-(PEG), γE-γE-(PEG)2-γE-γE, and γE-γE-(O2Oc)-(O2Oc).

[0103] In some embodiments, the peptide comprises the sequence: H-aminoisobutyric acid (Aib)-QGTFTSDVSK-alpha methyl-phenylalanine (alpha MePhe)-L-X15-X16-RRAQDFVKWL-X27-X28-X29-X30-Z, where X15 is D or E, X16 is S or L, X27 is V or L, X28 is E or Aib, X29 is T, Aib or G, X30 is G or Aib or absent, and Z is amide or acid (SEQ ID NO: 416).

[0104] In some embodiments, the lysine at position 24 is acylated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid). In some embodiments, the lysine at position 24 is lipidated. In some embodiments, the lipid is octadecanedioic acid (C18 diacid).

[0105] In some embodiments, the lipid is linked via a linker to lysine 24. In some embodiments, the linker is (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γ The linker is selected from the group consisting of E-(PEG)2-γE-(PEG)2, γE-(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, and γE-γE-(PEG)8. In some embodiments, the linker is γE-(O2Oc)-(O2Oc).

[0106] In certain embodiments, the GLP-1 / glucagon agonist peptides as disclosed have desirable potency at the glucagon receptor and the GLP-1 receptor.

[0107] In some embodiments, the peptide comprises any one of SEQ ID NOs:348-395.

[0108] In some embodiments, the peptide is any one of the peptides in Table 3.

[0109] Table 3. Peptides modified at position 24 [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0110] In some embodiments, the peptide comprises the sequence: H-X2-X3-G-X5-X6-TSD-X10-SK-X13-LDS-X17-X18-AQD-X22-V-X24-X25-X26-X27-NTX(30)-X(31)-Z, where X2 is S or aminoisobutyric acid (Aib), X3 is Q or H, X5 is T or S, X6 is F or α-methylphenylalanine (αMePhe), X10 is Y, V, or K, where K may comprise an acyl moiety and / or be lipidated, X13 is Y, αMePhe, X17 is R, Q, or β-dimethylarginine (β-diMeArg), X18 is R or A, X22 is F or αMePhe, X24 is Q or E, X25 is W, Aib or H, X26 is L or I, X27 is M or A, X(30) is absent, X(31) is absent, and Z is amide or acid (SEQ ID NO:417), wherein the peptide does not include SEQ ID NO:1.

[0111] In some embodiments, the residue at position 10 or 13 is acylated. In some embodiments, the residue at position 10 or 13 is lipidated. In some embodiments, the lipid is selected from the group consisting of palmitoyl, stearoyl, lauryl, myristoyl, margalloyl, octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid). In some embodiments, the lipid is selected from the group consisting of octadecanedioic acid (C18 diacid), and icosane diacid (C20 diacid), stearoyl, and palmitoyl.

[0112] In some embodiments, the lipid attached to residue 10 or 13 is attached via a linker. In some embodiments, the linker is selected from the group consisting of (O2Oc), (O2Oc)-(O2Oc), (O2Oc)-γE-(O2Oc), (PEG)2-(PEG)2-γE-γE, (PEG)2-γE-(PEG)2-γE, γE, γE-(O2Oc), γE-(O2Oc)-(O2Oc), γE-(O2Oc)-γE-(O2Oc), γE-(PEG)2-(PEG)2, γ The linker is selected from the group consisting of E-(PEG)2-γE-(PEG)2, γE-(PEG)4, γE-γE, γE-γE-(O2Oc), γE-γE-(O2Oc)-(O2Oc), γE-γE-(PEG)12, γE-γE-(PEG)2-(PEG)2, γE-γE-(PEG)2-γE-γE, γE-γE-(PEG)4, and γE-γE-(PEG)8. In some embodiments, the linker is selected from the group consisting of γE-γE-(O2Oc)-(O2Oc), γE-(O2Oc)-(O2Oc), γE-γE-(PEG)2-(PEG)2, and γE-(PEG)2-(PEG)2.

[0113] In some embodiments, the peptide comprises any one of SEQ ID NOs:396-411.

[0114] In some embodiments, the peptide is any one of the peptides in Table 4 or Table 5.

[0115] Table 4. Peptides modified at position 10 [Table 4-1] [Table 4-2]

[0116] Table 5. Peptides modified at position 13 [Table 5-1] [Table 5-2]

[0117] In some embodiments, the peptide comprises the sequence: H-X2-X3-G-X5-X6-TSD-X10-S-X12-α-methyl-phenylalanine (αMePhe)-L-X15-X16-X17-X18-A-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-Z, where X2 is aminoisobutyric acid (Aib), S, or A, X3 is Q, H, or E, X5 is T or S, X6 is F or αMePhe, X10 is V, K, or Y, X11 is S, X12 is K, E, or S, and X15 is D or or E, X16 is T, S, or G, X17 is K, R, E, or Q, X18 is R or A, X20 is R, K, or Q, X21 is D or E, X22 is αMePhe or F, X23 is V or I, X24 is Q or A, X25 is Aib or W, X26 is L or I, X27 is L, A, E, V, or M, X28 is E, N, A, R, or K, X29 is Aib, T, or G, X30 is G, R, or absent, X31 is G, or absent, and Z is an amide or acid (SEQ ID NO:540).

[0118] In some embodiments, X2 is Aib. In some embodiments, X3 is Q. In some embodiments, X3 is H. In some embodiments, X5 is T. In some embodiments, X5 is S. In some embodiments, X6 is F. In some embodiments, X6 is αMePhe. In some embodiments, X10 is V. In some embodiments, X12 is K. In some embodiments, X15 is D. In some embodiments, X16 is T. In some embodiments, X16 is S. In some embodiments, X17 is K. In some embodiments, X17 is R. In some embodiments, X18 is R. In some embodiments, X18 is A. In some embodiments, X20 is R. In some embodiments, X20 is K. In some embodiments, X21 is D. In some embodiments, X22 is F. In some embodiments, X22 is αMePhe. In some embodiments, X23 is V. In some embodiments, X24 is Q. In some embodiments, X25 is W. In some embodiments, X25 is Aib. In some embodiments, X26 is L. In some embodiments, X26 is I. In some embodiments, X27 is L. In some embodiments, X27 is A. In some embodiments, X28 is E. In some embodiments, X28 is N. In some embodiments, X29 is Aib. In some embodiments, X29 is T. In some embodiments, X30 is G. In some embodiments, X30 is absent. In some embodiments, X31 is absent. In some embodiments, Z is amide. In some embodiments, Z is acid.

[0119] In some embodiments, X2 is Aib, X12 is K, and X24 is Q. In some embodiments, X16 is T, X17 is K, X27 is L, X28 is E, and X29 is Aib.

[0120] In some embodiments, X3 is Q, X5 is T, X6 is F, X10 is V, X12 is K, X15 is D, X16 is T, X17 is K, X18 is R, X20 is R, X21 is D, X22 is F, X23 is V, X24 is Q, X25 is W, X26 is L, X27 is L, X28 is E, X29 is Aib, X30 is G, and X31 is absent.

[0121] In some embodiments, X3 is H, X5 is S, X6 is αMePhe, X10 is V, X12 is K, X15 is D, X16 is S, X17 is R, X18 is A, X20 is K, X21 is D, X22 is αMePhe, X23 is V, X24 is Q, X25 is Aib, X26 is I, X27 is A, X28 is N, X29 is T, X30 is absent, and X31 is absent.

[0122] In some embodiments, one or more lysine residues are acylated. In some embodiments, the lysine at position 17 is acylated. In some embodiments, the lysine at position 20 is acylated.

[0123] In some embodiments, one or more lysine residues are lipidated. In some embodiments, the lysine at position 17 is lipidated. In some embodiments, the lysine at position 20 is lipidated.

[0124] In some embodiments, the lipid is selected from the group consisting of octadecanedioic acid (C18 diacid) and icosane diacid (C20 diacid). In some embodiments, the lipid is octadecanedioic acid (C18 diacid). In some embodiments, the lipid is icosane diacid (C20 diacid).

[0125] In some embodiments, the lipid is linked to the residue at position 17 or 20 via a linker. In some embodiments, the linker is γE-(O2Oc)-(O2Oc) or γE-γE-(O2Oc)-(O2Oc). In some embodiments, the linker is γE-(O2Oc)-(O2Oc). In some embodiments, the linker is γE-γE-(O2Oc)-(O2Oc). In some embodiments, the linker is γE-γE-(O2Oc)-(O2Oc). In some embodiments, the linker is linked to the epsilon amino group of the residue at position 17 or 20.

[0126] Certain aspects of the present disclosure are directed to a peptide comprising the sequence H-Aib-QGTFTSDVSK-αMePhe-LDTKRARDFVQWLLE-Aib-G-acid (SEQ ID NO:541).

[0127] In some embodiments, the lysine at position 17 is acylated and lipidated, and the lipid is linked to the acylated lysine via (ε-(O2Oc)-(O2Oc)-γE-C18 diacid).

[0128] In some embodiments, the lysine at position 17 is acylated and lipidated, and the lipid is linked to the acylated lysine at position 17 via (ε-(O2Oc)-(O2Oc)-γE-C20 diacid).

[0129] Certain aspects of the present disclosure are directed to a peptide comprising the sequence H-Aib-HGS-αMePhe-TSDVSK-αMePhe-LDSRAAK(ε-(O2Oc)-γE-C18 diacid)20-D-αMePhe-VQ-Aib-IANT-amide (SEQ ID NO: 228).

[0130] Certain aspects of the present disclosure are directed to a peptide comprising the sequence H-Aib-HGS-αMePhe-TSDVSK-αMePhe-LDSRAAK(ε-(O2Oc)-γE-γE-C20 diacid)20-D-αMePhe-VQ-Aib-IANT-amide (SEQ ID NO: 233).

[0131] In some embodiments, the peptide binds to the GLP-1 receptor (GLP-1R), binds to the glucagon receptor (GCGR), or binds to both the GLP-1 receptor and the glucagon receptor. In some embodiments, the GLP-1R is human GLP-1R. In some embodiments, the GCGR is human GCGR. In some embodiments, the peptide is an agonist of GLP-1 activity, an agonist of glucagon activity, or an agonist of both GLP-1 and glucagon activity.

[0132] In some embodiments, the peptide has increased resistance to proteolysis compared to the natural ligands of GLP-1R and / or GCGR.

[0133] In some embodiments, the peptide is isolated.

[0134] In some embodiments, the peptides have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% intact peptide remaining after incubation with the protease for 5 minutes, 10 minutes, 15 minutes, 30 minutes, 2 hours, 4 hours or 24 hours at 37° C. In some embodiments, the protease is selected from the group consisting of neprilysin, pepsin, pancreatin, simulated gastric fluid with pepsin, and simulated intestinal fluid with pancreatin.

[0135] In some embodiments, the peptide has a half-life in cynomolgus monkeys following intravenous administration of at least 45 hours, at least 50 hours, at least 60 hours, at least 70 hours, at least 80 hours, at least 90 hours, at least 100 hours, at least 110 hours, at least 120 hours, or about 130 hours.

[0136] In some embodiments, the peptide has a subcutaneous bioavailability in cynomolgus monkeys of at least 75%, at least 80%, at least 90%, or about 95%.

[0137] III. Methods of Making GLP-1 / Glucagon Agonist Peptides The GLP-1 / glucagon agonist peptide for use provided herein can be produced by any suitable method.For example, in some embodiments provided herein, the GLP-1 / glucagon agonist peptide for use provided herein is chemically synthesized by methods well known to those skilled in the art, for example, by solid-phase synthesis as described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154).Solid-phase peptide synthesis can be achieved using standard reagents, for example, by using an automated synthesizer, as described in Example 1 of WO2014 / 091316, which is incorporated herein by reference in its entirety.

[0138] Alternatively, the GLP-1 / glucagon agonist peptides for use as provided herein can be produced recombinantly using a convenient vector / host cell combination, as known to those skilled in the art. A variety of methods are available for recombinantly producing GLP-1 / glucagon agonist peptides. In general, the polynucleotide sequence encoding the GLP-1 / glucagon agonist peptide is inserted into a suitable expression vehicle, such as a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. The nucleic acid encoding the GLP-1 / glucagon agonist peptide is inserted into the vector in the appropriate reading frame. The expression vector is transfected into a suitable host cell that expresses the GLP-1 / glucagon agonist peptide. Suitable host cells include, but are not limited to, bacteria, yeast, or mammalian cells. A variety of commercially available host expression vector systems can be utilized to express the GLP-1 / glucagon agonist peptides described herein.

[0139] Pharmaceutical Compositions Further provided are compositions, e.g., pharmaceutical compositions, formulated for the treatment of metabolic diseases, e.g., obesity, type 2 diabetes, and / or NASH, comprising an effective amount of a GLP-1 / glucagon agonist peptide as provided herein.

[0140] The composition of the present disclosure can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, AR Gennaro, ed., Mack Publishing Co., Easton, PA (1995), the entirety of which is incorporated herein by reference. The composition can be in various forms, including, but not limited to, aqueous solutions, emulsions, gels, suspensions, lyophilized forms, or any other forms known in the art. In addition, the composition can contain pharma- ceutically acceptable additives, including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, the composition of the present invention can be administered directly to a subject.

[0141] In some embodiments, the pharmaceutical composition is a solid composition. In some embodiments, the pharmaceutical composition is a liquid composition.

[0142] IV. METHODS OF USE OF GLP-1 / GLUCAGON AGONIST PEPTIDES As provided herein, GLP-1 / glucagon agonist peptides can be used to improve glycemic control, weight loss, type 2 diabetes mellitus (T2DM), and / or treat or prevent nonalcoholic steatohepatitis (NASH).

[0143] In some embodiments, administration of a GLP-1 / glucagon agonist peptide reduces weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo adipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

[0144] As provided herein, a method for improving glycemic control or reducing weight in a human subject suffering from T2DM and / or NASH can include administering a GLP-1 / glucagon agonist peptide to the subject.

[0145] The present disclosure also provides a GLP-1 / glucagon agonist peptide for use in the manufacture of a medicament for improving glycemic control or reducing weight in a human subject suffering from T2DM and / or NASH.

[0146] In some embodiments, the peptide is administered about once a week.

[0147] In some embodiments, the GLP-1 / glucagon agonist peptide is administered to treat or prevent a disease or condition that is caused or characterized by excess weight.In some embodiments, the disease or condition is obesity.In some embodiments, the disease or condition is type 2 diabetes.

[0148] In some embodiments, the GLP-1 / glucagon agonist peptide is administered to treat or prevent non-alcoholic steatohepatitis (NASH). In some embodiments, the GLP-1 / glucagon agonist peptide is administered by injection. In some embodiments, the GLP-1 / glucagon agonist peptide is administered orally. In some embodiments, administration of GLP-1 / glucagon agonist peptide reduces the subject's weight, increases the subject's insulin secretion, delays gastric emptying in the subject, reduces the subject's food intake, increases mitochondrial function in the subject, inhibits de novo lipogenesis in the subject, reduces HbA1c in the subject, enhances fat oxidation in the subject, reduces hepatic mitochondrial oxidative stress in the subject, reduces steatosis in the subject, reduces fibrosis in the subject, reduces glycogen synthesis in the subject, increases gluconeogenesis in the subject, stops disease progression in the subject, reverses fibrosis in the subject, and / or reduces the risk of death due to liver cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in the subject.In some embodiments, the subject is a human.In some embodiments, the peptide is administered about once a week. EXAMPLES

[0149] [Example 1]

[0150] Preparation of lipidated GLP-1 / glucagon dual agonist peptide analogs Lipidated GLP-1R / GCGR dual agonist peptides were synthesized as C-terminal carboxamides or carboxylic acids using Rink amide MBHA resin (100-200 mesh) or Wang resin (100-200 mesh). All peptides were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) / tert-butyl ( tThe peptides were prepared by automated synthesis on a PTI Prelude solid-phase peptide synthesizer using the N,N-dimethylformamide (DMF) Fmoc (Fmoc) protocol. The manufacturer's protocols were applied for coupling of the amino acids in N,N-dimethylformamide (DMF) and deprotection of the Fmoc protecting group using piperidine in DMF (20% v / v). Asparagine (Asn), glutamine (Gln) and histidine (His) were incorporated as their side chain triphenylmethyl, trityl (Trt) derivatives. Lysine (Lys) was incorporated as its side chain tert-butyloxycarbonyl (Boc) derivative. Serine (Ser), threonine (Thr) and tyrosine (Tyr) were incorporated as their side chain triphenylmethyl, trityl (Trt) derivatives. t Aspartic acid (Asp) and glutamic acid (Glu) are incorporated as Bu ethers, and their side chains are t Arginine (Arg) was incorporated as the side chain 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) derivative; other amino acids were incorporated with appropriate side chain protection.

[0151] If subsequent chemical modification of the lysine side chain was required, Lys(Mmt) was incorporated. After completion of peptide chain elongation, Mmt side chain protection was removed by treating the resin with a selective deprotection cocktail (1% trifluoroacetic acid (TFA), 5% TIPS in dichloromethane (DCM)) at 100 mL / mmol for 1 min, which was repeated at least 10 times until deprotection of the Mmt group was complete. The reaction was quenched with 10% N,N-diisopropylethylamine (DIPEA) / NMP. Subsequent coupling of albumin binding moieties, such as lipids and linkers, was performed manually in the presence of DIPEA using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) as a coupling reagent.

[0152] The peptide was cleaved from the solid support by treatment with a mixture of TFA:triisopropylsilane (TIS):water (92.5:5:2.5 v / v) for 4 h at room temperature with stirring. The cleavage mixture was then filtered, concentrated in vacuo, precipitated, washed with diethyl ether, and the solid was isolated by centrifugation. The crude peptide was dried under a stream of nitrogen, dissolved in 20% acetonitrile (MeCN) / water (v / v), and filtered. The crude peptide was purified using a preparative reversed-phase high-performance liquid chromatography (RP-HPLC) Xbridge C18-A stationary phase (19.0 × 250 mm, 5 micron) column on a Varian SD-1 Prep Star binary pump system, eluting with a linear solvent gradient of 25–70% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) over 25 min while monitoring ultraviolet (UV) absorption at 210 nm. The purified fractions were pooled, frozen, and lyophilized.

[0153] Liquid chromatography / mass spectrometry (LC / MS) characterization of purified peptides was performed on a Waters MassLynx 3100 platform using an XBridge C18 stationary phase (4.6 x 100 mm, 3 microns) by elution with a linear binary gradient of 10-90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) at 1.5 mL / min over 10 min at ambient temperature. Analytes were detected by both UV absorption at 210 nm and ionization using a Waters 3100 mass detector (electrospray ionization (ESI)+ mode). Analytical RP-HPLC characterization was performed on an Agilent 1260 Infinity system using an Agilent Polaris C8-A stationary phase (4.6 × 100 mm, 3 micron) eluted with a linear binary gradient of 10–90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) over 15 min at 1.5 mL / min at 40°C. [Example 2]

[0154] In vitro functional assays of GLP-1 receptor and glucagon receptor CHO cell lines stably recombinantly expressing the human GLP-1 receptor (hGLP-1R) or the human glucagon receptor (hGCGR) were used to test the functional activities of lipidated-GLP-1R / GCGR peptides, such as cAMP production.

[0155] Cryopreserved cell stocks were quickly thawed in a water bath and suspended in assay buffer (HBSS (Sigma, Cat. No. H8264) containing 0.1% BSA (Sigma, Cat. No. A3059), 0.5 mM IBMX (Sigma, Cat. No. 17018) in 25 mM HEPES, pH 7.4) and centrifuged at 240 × g for 5 min. The cells were incubated at a batch-dependent optimized concentration (usually 1 × 10 hGLP-1R cells). 5 cells / mL, hGCGR cells were 2 × 10 5 Cells were resuspended in assay buffer at 100x the concentration (cells / mL).

[0156] Test peptide stocks were prepared in DMSO, serially diluted in assay buffer, and 11-point concentration-response curves were generated in duplicate in 384-well low-volume microtiter assay plates (Corning, Cat. No. 4514) using a non-contact liquid dispenser (ECHO™, LabCyte). Cells were added to the assay plates using a multi-drop dispenser and incubated at room temperature for 30 minutes before measuring cAMP levels using the cAMP dynamic2 homogeneous time resolved Fluorescence (HTRF) kit (Cisbio Bioassays, Cat. No. 62AM4PEJ) following the manufacturer's recommended two-step protocol. Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were made separately, diluted 1 in 20 in the conjugate and lysis buffers provided with the kit. Anti-cAMP cryptate was added to all wells of the assay plate, followed by cAMP-d2 in all wells except the non-specific binding (NSB) wells (where conjugate and lysis buffer were added). Plates were incubated for 1 hour at room temperature and then read on an Envision (Perkin Elmer) using an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm.

[0157] Data were converted to % delta F as described in the manufacturer's guidelines and EC 50 The peptides were analyzed by a four-parameter logistic fit to determine the selectivity ratio for hGLP-1R versus hGCGR, which is defined as follows: %Relative Potency Ratio = %GLP-1R activity for GLP-1 / %GlucR activity for glucagon. Data are the geometric mean EC 50 Shown as (pM).

[0158] The relative potency ratios of the peptides are listed in Tables 6-10.

[0159] Table 6. Potency of peptides with modifications at amino acid position 17. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0160] Table 7. Potency of peptides with modifications at amino acid position 20. [Table 7-1] [Table 7-2] [Table 7-3]

[0161] Table 8. Potency of peptides with modifications at amino acid position 24. [Table 8]

[0162] Table 9. Potency of peptides with modifications at amino acid position 10 [Table 9]

[0163] Table 10. Potency of peptides with modifications at amino acid position 13. [Table 10] [Example 3]

[0164] Acute food intake test in mice Male C57Bl / 6 mice, 8–9 weeks of age, obtained from Jackson Laboratories or Charles River, were housed one per cage in BioDaq (Research Diets) cages. Mice were placed on Alpha Dri bedding, standard chow chow (Envigo, 2018), and provided with water pouches. Mice were allowed to acclimate for 1–2 weeks. Weight was measured to confirm proper acclimatization. After acclimatization, mice were administered a placebo a minimum of two times before food intake testing began. On the day of testing, mice were placed in clean cages, weighed, and fasted for 6–8 h. Mice were assigned to groups based on 24-h average food intake data and body weight. Vehicle or test peptide was administered subcutaneously to mice at 5 mL / kg in the appropriate vehicle 1–2 h before lights out. Mice were left undisturbed with food and water available for 48 h. During this time, automated food intake was monitored with the BioDaq system. Individual food intake data was exported into MS Excel, from which cumulative food intake data was generated and analyzed. The results of the comparison of 24 hour food intake to vehicle food intake (percent) are shown in Table 11.

[0165] Table 11. Food intake in mice administered lipidated peptides [Table 11] [Example 4]

[0166] Evaluation of proteolytic resistance of lipidated peptides in fasted-state simulated gastric fluid (FasSSGF) containing porcine pancreatic pepsin Lyophilized porcine pancreatic pepsin (Sigma: P7012) was reconstituted in freshly prepared FasSSGF (Biorelevant media) to 0.5 mg / mL (~2500 units / mL) to obtain the enzyme stock solution. Peptide stock solutions were prepared in FasSSGF to a concentration of 250 μM (~1.0 mg / mL). 200 μL (10 μg, ~250 units) of pepsin stock solution was added to 200 μL of peptide solution (1.0 mg / mL, ~100 μg peptide, ~25 nmoles) and the mixture was incubated together for the duration of the experiment in a temperature-controlled incubator at 37°C. Aliquots of 30 μL of peptide-enzyme mixture were withdrawn periodically (t=0, 5, 10, 15, 30 min) and immediately quenched by the addition of 80 μL of 0.1 M ammonium bicarbonate solution in water / acetonitrile (4:1, pH 8) to stop the proteolytic activity. Aliquots of 30 μL were analyzed by analytical RP-HPLC. Analytical RP-HPLC method: An Agilent Polaris C8-A column (4.6×100 mm, 3 micron) was used to elute with a linear binary gradient of 10-90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) at 1.5 mL min−1 over 10 or 15 min at 40° C. with detection by UV absorbance at 210 nm. Manual integration (AUC) allowed an estimate of intact peptide remaining over the time course of the experiment. Peptide stability data is shown in Table 12.

[0167] Table 12. Stability of lipidated peptides incubated with FasSSGF [Table 12] [Example 5]

[0168] Evaluation of peptide proteolytic resistance to neprilysin 10.0 μg (approximately 10 units) of recombinant neprilysin (R&D Systems: 1182-ZNC-010) was reconstituted to 100 μL (100 μg / mL, approximately 100 units / mL) in assay buffer (50 mM Tris, 50 mM NaCl, 50 mM NaHCO3, adjusted to pH 8.3) to obtain the enzyme stock solution. Peptide stocks were prepared to a concentration of approximately 250 μM (approximately 1.0 mg / mL of 4 kDa peptide) in assay buffer. 100 μL (10 μg, approximately 10 units) of neprilysin stock solution was added to 100 μL of peptide stock solution (1.0 mg / mL, approximately 100 μg of peptide) and the mixture was incubated together for the duration of the experiment in a 37°C temperature-controlled incubator. Aliquots of 25 μL (approximately 12.5 μg of initial peptide) of the peptide-enzyme mixture were periodically withdrawn (t = 0 min, 30 min, 1 h, 2 h, 4 h, and 24 h) and immediately quenched by the addition of an equal volume (75 μL) of 10% TFA (v / v) in 1:1 water / acetonitrile to stop proteolytic activity. The quenched aliquots were centrifuged at 7800 rpm, and 30 μL of the supernatant was analyzed by analytical RP-HPLC as follows: Analytical RP-HPLC method: An Agilent Polaris C8-A column (4.6 × 100 mm, 3 micron) was used to perform a linear binary gradient of 10 to 90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) over either 10 or 15 min, 1.5 mL min. -1 The peptides were eluted at 0.5% CO at 40° C. with detection by UV absorbance at 210 nm. Manual integration (AUC) allowed an estimate of intact peptide remaining over the time course of the experiment. Peptide stability data is shown in Table 13.

[0169] Table 13. Stability of lipidated peptides incubated with neprilysin [Table 13] [Example 6]

[0170] Evaluation of proteolytic resistance of mono-lipidated peptides in fasted-state simulated intestinal fluid (FasSSIF / pancreatin) A fresh suspension of FasSSIF / P (fasted state simulated intestinal fluid + USP Pancreatin®) was prepared according to the requirements of Galia, Nicolaides, Hoerter, Loebenberg, Reppas and Dressman: Pharm.Res. 15 (1998) 698-705, and USP XL. The preparation of FasSSIF / P is shown in Table 14. The resulting preparation was used immediately without storage.

[0171] Table 14. Preparation of FasSSIF / pancreatin [Table 14]

[0172] The peptide to be evaluated (1.0 mg) was dissolved in pre-warmed FasSSIF without pancreatin® (200 μL). To this was added fresh FasSSIF / pancreatin® (100 μL) to initiate potential proteolysis. After momentary vortexing of the reaction tube, the mixture was incubated in a 37°C water bath for the duration of the experiment. Aliquots of 25 μL of the co-incubated peptide-enzyme mixture were periodically removed (t=0, 5, 10, 15 and 30 min) and immediately quenched with 75 μL of 10% TFA in 1:1 water / acetonitrile to stop proteolytic activity. The quenched samples were centrifuged (7800 RPM, 3 min) to pellet the solids and a 10 μL aliquot of the supernatant was analyzed using analytical RP-HPLC as follows: Analytical RP-HPLC method: A linear binary gradient of 10–90% MeCN (0.1% TFA v / v) in water (0.1% TFA v / v) was run over either 10 or 15 min on an Agilent Polaris C8-A column (4.6×100 mm, 3 micron) in 1.5 mL min. -1 The peptides were eluted at 0.5 °C at 40 °C with detection by UV absorbance at 210 nm. Manual integration (AUC) allowed an estimate of intact peptide remaining over the time course of the experiment. Peptide stability data are shown in Table 15.

[0173] Table 15. Stability of lipidated peptides incubated with FasSSIF [Table 15] [Example 7]

[0174] Glucose levels in GLP-1R knockout mice treated with GLP-1 / glucagon peptide GLP-1R knockout (KO) and wild-type (WT) mice, 8–14 weeks of age, were housed singly on a standard chow diet (Envigo, 2918) with automatic water supply. Mice were acclimated for a minimum of 1–2 weeks and divided into groups (n=4–8 / group) based on body weight. On the day of study, mice were fasted for a short 2-hour period before the start of the study. In some studies, mice were pretreated with octreotide (BaChem, 10 mg / kg) 30 minutes prior to peptide injection. Mice were dosed by subcutaneous injection, and peptide doses are listed in Tables 16 and 17. Glucose was measured by glucometer at various time points including -30 minutes (before octreotide, if used in the study), 0 minutes (before peptide administration), 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, and 360 minutes. Because these studies were acute in nature, mice were reused in multiple studies (not more than three times), always with a minimum washout period of one week. Glucose changes expressed as percent glucose change from 0 minutes (60 minutes or 120 minutes) are shown in Tables 16 and 17.

[0175] Table 16. Glucose changes in GLP-1R KO and WT mice treated with GLP-1 / glucagon peptides [Table 16-1] [Table 16-2]

[0176] Table 17. Glucose changes in GLP-1R KO and WT mice treated with GLP-1 / glucagon peptides. [Table 17] [Example 8]

[0177] Pharmacokinetic profiling in preclinical species Selected test peptides (n=8) were profiled for pharmacokinetic (PK) properties in mice following a single intravenous (iv) or subcutaneous (sc) dose. A subset (n=4) that met the acceptance criteria were then profiled in dogs (single iv dose) and non-human primates (single iv and sc doses). Blood was collected at selected time points following dosing, up to 48 hours in mice, 120 hours in dogs, and 168 hours in non-human primates, to accurately determine the complete pharmacokinetic profile in each species. Low-binding plastic containers were used during sample preparation and handling to avoid non-specific binding.

[0178] Mice: PK was studied in lean male mice C57BL / 6 (peptide 140, peptide 188, peptide 195, peptide 420, peptide 477 and peptide 472) or male mice C57BL / 6 DIO (peptide 224, peptide 229) prefed on a high-fat diet. After dosing, PK samples were collected from the dorsal metatarsal vein. Blood from each sample was transferred to a plastic microcentrifuge tube containing EDTA-K2 and placed on wet ice after inverting several times to properly mix the contents. Blood samples were centrifuged at 4°C to obtain plasma, which was stored at -75°C prior to analysis.

[0179] Dogs: PK was studied in non-naive male beagle dogs. After dosing, PK samples were collected from the jugular vein. Blood from each sample was transferred to a plastic microcentrifuge tube containing EDTA-K2, inverted several times to adequately mix the contents, and then placed on wet ice. Plasma was separated by centrifugation, placed in matrix tubes, and stored frozen at a temperature set to maintain -20°C until analysis.

[0180] Non-human primates: PK was studied in non-naive male cynomolgus monkeys. After dosing, PK samples were collected from the femoral vein. Blood from each sample was transferred to a plastic microcentrifuge tube containing EDTA-K2, inverted several times to adequately mix the contents, and then placed on wet ice. Blood samples were centrifuged under refrigerated (2°C to 8°C) conditions within 30 minutes of sample collection to obtain plasma, which was stored at -60°C to -90°C prior to analysis.

[0181] Formulation: Test articles were formulated in 20 mM sodium phosphate, 220 mM sorbitol pH 7.5 (Peptide 224, Peptide 229, Peptide 140, Peptide 188, Peptide 195) or 20 mM sodium acetate, 220 mM mannitol pH 4.5 (Peptide 420, Peptide 477, Peptide 472).

[0182] Sample Analysis: Samples were analyzed by LC-MS / MS. Calibration standards were injected at the beginning and end of each batch, and calibration curves were generated using the determined concentrations of each prepared standard. Concentrations in plasma and administered aliquots were determined from the plasma calibration curve.

[0183] Data Analysis: Plasma concentration data were analyzed by noncompartmental analysis (NCA) using Phoenix Winnonlin v.8.3.3.33. The linear trapezoidal rule was used for increasing values ​​and the logarithmic trapezoidal rule for decreasing values, with uniform weighting applied to calculate lambda_z. Subcutaneous bioavailability was calculated as the ratio, AUC(subcutaneous): 0-inf / AUC(intravenous) 0-inf The reported values ​​(Table 18) represent the average of the individual estimates.

[0184] Results: The half-lives of the test peptides after intravenous administration and the absolute bioavailability after subcutaneous administration calculated by NCA are reported in Table 18. In general, the test peptides showed long circulating half-lives in all preclinical species studied, with an additional significant prolongation in higher species compared to rodents. Estimated half-lives ranged from 2.9 to 19 hours in mice, 86 to 71 hours in dogs, and 49 to 130 hours in non-human primates. Bioavailability after subcutaneous administration was generally greater than 50% and was comparable in mice and non-human primates. All test peptides evaluated in non-human primates showed high bioavailability, greater than 75%.

[0185] Table 18. Preclinical PK. Half-life and absolute subcutaneous bioavailability following intravenous administration at specific doses of profiled test peptides in mice, dogs and non-human primates. [Table 18] nd: Not determined.

Claims

Claim 1: A peptide comprising the sequence H-Aib-Q-GT-F-T-S-D-V-S-K-αMePhe-L-D-T-K(O2Oc)-(O2Oc)-γE-C18 diacid)17-R-A-R-D-F-V-Q-W-L-L-E-Aib-G-acid.

2. A peptide comprising the sequence H-Aib-Q-GT-F-T-S-D-V-S-K-αMePhe-L-D-T-K(O2Oc)-(O2Oc)-γE-C20 diacid)17-R-A-R-D-F-V-Q-W-L-L-E-Aib-G-acid.

3. A peptide containing the sequence H-Aib-H-G-S-αMePhe-T-S-D-V-S-K-αMePhe-L-D-S-R-A-A-K(ε-(O2Oc)-(O2Oc)-γE-C18 diacid)20-D-αMePhe-V-Q-Aib-I-A-N-T-amide.

4. A peptide containing the sequence H-Aib-H-G-S-αMePhe-T-S-D-V-S-K-αMePhe-L-D-S-R-A-A-K(ε-(O2Oc)-(O2Oc)-γE-γE-C20 diacid)20-D-αMePhe-V-Q-Aib-I-A-N-T-amide.

5. 5. The peptide of any one of claims 1 to 4, wherein the peptide binds to the GLP-1 receptor (GLP-1R), binds to the glucagon receptor (GCGR), or binds to both the GLP-1 receptor and the glucagon receptor, and optionally (i) the GLP-1R is human GLP-1R; and / or (ii) the GCGR is human GCGR.

6. 6. The peptide of claim 5, wherein the peptide is an agonist of GLP-1 activity, an agonist of glucagon activity, or an agonist of both GLP-1 and glucagon activity.

7. 6. The peptide of claim 5, wherein the peptide (i) has increased resistance to proteolysis compared to the natural ligands of GLP-1R and / or GCGR; and / or (ii) is isolated. (i) after incubation with a protease at 37°C for 5 minutes, 10 minutes, 15 minutes, 30 minutes, 2 hours, 4 hours or 24 hours, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the intact peptides remain, and optionally the protease is selected from the group consisting of neprilysin, pepsin, pancreatin, simulated gastric fluid with pepsin, and simulated intestinal fluid with pancreatin; (ii) has a half-life in cynomolgus monkeys after intravenous administration of at least 45 hours, at least 50 hours, at least 60 hours, at least 70 hours, at least 80 hours, at least 90 hours, at least 100 hours, at least 110 hours, at least 120 hours, or about 130 hours; and / or (iii) has a subcutaneous bioavailability in cynomolgus monkeys of at least 75%, at least 80%, at least 90%, or about 95%; A peptide according to any one of claims 1 to 4.

9. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 4, optionally in the form of a solid or liquid composition.

10. The pharmaceutical composition of claim 9, for treating or preventing a disease or condition caused by or characterized by excess weight in a subject.

11. The pharmaceutical composition described in claim 10, wherein the disease or condition is obesity or type 2 diabetes.

12. The pharmaceutical composition of claim 9, for treating or preventing nonalcoholic steatohepatitis (NASH) in a subject.

13. The pharmaceutical composition of claim 10, wherein administration of the pharmaceutical composition reduces body weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo lipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

14. The pharmaceutical composition of claim 12, wherein administration of the pharmaceutical composition reduces body weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo lipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

15. The pharmaceutical composition of claim 9, for improving glycemic control or reducing weight in a human subject with type 2 diabetes and / or NASH.

16. (i) The pharmaceutical composition is administered to a subject orally or by injection; (ii) the pharmaceutical composition is administered about once a week; and / or (iii) the subject is a human; The pharmaceutical composition of claim 10.

17. (i) The pharmaceutical composition is administered to a subject orally or by injection; (ii) the pharmaceutical composition is administered about once a week; and / or (iii) the subject is a human; The pharmaceutical composition of claim 12.

18. (i) the pharmaceutical composition is administered orally or by injection to a human subject; and / or (ii) the pharmaceutical composition is administered about once a week; 16. The pharmaceutical composition of claim 15.

19. A peptide described in any one of claims 1 to 4 for treating or preventing a disease or condition caused by or characterized by excess weight in a subject.

20. The peptide described in claim 19, wherein the disease or condition is obesity or type 2 diabetes.

21. A peptide described in any one of claims 1 to 4 for treating or preventing non-alcoholic steatohepatitis (NASH) in a subject.

22. The peptide of claim 19, wherein administration of the peptide reduces body weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo lipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

23. The peptide of claim 21, wherein administration of the peptide reduces body weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo lipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

24. A peptide described in any one of claims 1 to 4 for improving glycemic control or reducing weight in a human subject with type 2 diabetes and / or NASH.

25. (i) The peptide is administered to the subject orally or by injection; (ii) the peptide is administered about once a week; and / or (iii) the subject is a human; 20. The peptide of claim 19.

26. (i) The peptide is administered to the subject orally or by injection; (ii) the peptide is administered about once a week; and / or (iii) the subject is a human; 22. The peptide of claim 21.

27. (i) the peptide is administered orally or by injection to a human subject; and / or (ii) the peptide is administered about once a week; 25. The peptide of claim 24.

28. Use of a peptide described in any one of claims 1 to 4 in the manufacture of a medicament for treating or preventing a disease or condition caused by or characterized by excess weight in a subject.

29. The use described in claim 28, wherein the disease or condition is obesity or type 2 diabetes.

30. Use of a peptide described in any one of claims 1 to 4 in the manufacture of a medicament for treating or preventing non-alcoholic steatohepatitis (NASH) in a subject.

31. The use of claim 28, wherein administration of the pharmaceutical reduces body weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo lipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

32. The use of claim 30, wherein administration of the pharmaceutical reduces body weight in a subject, increases insulin secretion in a subject, delays gastric emptying in a subject, reduces food intake in a subject, increases mitochondrial function in a subject, inhibits de novo lipogenesis in a subject, reduces HbA1c in a subject, enhances fat oxidation in a subject, reduces hepatic mitochondrial oxidative stress in a subject, reduces steatosis in a subject, reduces fibrosis in a subject, reduces glycogen synthesis in a subject, increases gluconeogenesis in a subject, halts disease progression in a subject, reverses fibrosis in a subject, and / or reduces the risk of death due to cirrhosis, hepatocellular carcinoma, and / or cardiorenal disease in a subject.

33. Use of a peptide according to any one of claims 1 to 4 in the manufacture of a medicament for improving glycemic control or reducing weight in a human subject with type 2 diabetes and / or NASH.

34. (i) The pharmaceutical agent is administered to the subject orally or by injection; (ii) the medication is administered about once a week; and / or (iii) the subject is a human; 29. The use according to claim 28.

35. (i) The pharmaceutical agent is administered to the subject orally or by injection; (ii) the medication is administered about once a week; and / or (iii) the subject is a human; 31. The use according to claim 30.

36. (i) The pharmaceutical is administered orally or by injection to a human subject; and / or (ii) the medication is administered about once a week; 34. The use according to claim 33.

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