GIP / GLP1 / GCG tri-receptor agonists and uses thereof

Abstract

A polypeptide that is active against each of a GIP, a GLP-1, and a glucagon receptor is provided. The polypeptides have structural features that result in each of the receptors having activity and extended action time. Also provided are methods for treating diseases and / or conditions such as obesity, long term weight management, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-associated sleep apnea (OSA), and polycystic ovarian syndrome (PCOS).

Description

TECHNICAL FIELD

[0001] The present disclosure relates to polypeptides having activity at each of the glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and glucagon (GCG) receptors. The polypeptides described herein have structural features that can provide appropriate levels of activity at each receptor and extended duration of action. In addition, the present invention relates to compounds that can be administered orally or subcutaneously. Such polypeptides can be used to treat diseases or conditions such as obesity, long-term weight management, type 2 diabetes mellitus (T2DM), dyslipidemia, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and / or polycystic ovary syndrome (PCOS). BACKGROUND

[0002] Over the past several decades, the prevalence of diabetes has continued to rise. T2DM is the most common type of diabetes, accounting for approximately 90% of all diabetes. T2D is characterized by high blood glucose levels resulting from insulin resistance. Current standard of care for T2DM includes diet and exercise, as well as treatment with oral and injected glucose-lowering medications, including incretin-based therapies, such as GLP-1 receptor agonists. Several GLP-1 analogs are currently available for the treatment of T2DM, including dulaglutide, exenatide, and liraglutide. However, many of the currently marketed GLP-1 receptor agonists are dose-limited due to gastrointestinal side effects, such as nausea and vomiting. Subcutaneous injection is the typical route of administration for existing GLP-1 receptor agonists. Insulin can be considered when existing oral and incretin-based therapies fail to provide adequate glycemic control. Despite the availability of multiple treatment options, a significant number of individuals receiving approved therapies fail to achieve glycemic control targets (see, e.g., Casagrande et al., (2013) Diabetes Care 36:2271-2279). Uncontrolled diabetes can lead to one or more conditions that affect morbidity and mortality in such individuals.

[0003] One of the major risk factors for T2DM is obesity, and the majority of individuals with T2DM (~90%) are overweight or obese. Obesity is a complex medical disorder that results in an excessive accumulation of adipose tissue mass. Today, obesity has become a global public health issue associated with adverse health outcomes and morbidity. The ideal treatment for patients with obesity is to reduce excess body weight, improve obesity-related comorbidities, and maintain long-term weight loss. Existing treatments for obesity are particularly unsatisfactory for patients with severe obesity. Thus, alternative treatment options are needed to facilitate therapeutic weight loss in patients in need of such treatment.

[0004] In view of this, new therapies under investigation include compounds that are not only active at the GLP-1 receptor but also at one or more other receptors such as the GIP and / or the glucagon receptor.

[0005] For example, International Patent Application Publication Nos. WO 2013 / 164483 and WO 2016 / 111971 describe polypeptides that are purported to have GLP-1 and GIP receptor activity. WO 2011 / 075393, WO 2012 / 177444, and WO 2016 / 209707 describe polypeptides that are purported to have GCG and GIP receptor activity.

[0006] In addition, certain compounds have been described as having triple agonist activity (i.e., activity at the GIP, GLP-1, and glucagon receptors). For example, WO 2015 / 067716 describes glucagon analogs having triple agonist activity. Likewise, WO 2016 / 198624 describes analogs of exendin-4 (which is itself a GLP-1 analog) having triple agonist activity. Likewise, WO 2014 / 049610 and WO 2017 / 116204 describe, respectively, a variety of analogs having triple agonist activity. In addition, International Patent Application No. WO 2017 / 153375 describes glucagon and GLP-1 co-agonists that are purported to also have GIP activity. Furthermore, WO 2019 / 125938, WO 2019 / 125929, and WO 2021 / 126695 each describe a variety of polypeptides having triple agonist activity.

[0007] Nonetheless, there remains a need for compounds that can provide effective glucose control, weight loss benefits, and favorable side effect profiles. In addition, there remains a need for alternative treatment options to provide therapeutic weight loss or long-term weight management for patients in need of such treatment. There also remains a need for therapeutic agents that can be used to extend the duration of action sufficiently to allow for infrequent dosing once daily, three times per week, twice per week, or once per week. In addition, it is also desirable and needed for compounds to be suitable for convenient modes of administration (such as subcutaneous or oral routes). In particular, it is desirable for the compounds to exhibit sufficient efficacy, have favorable side effect profiles, and / or stability and bioavailability so that they can be administered orally.

[0008] SUMMARY

[0009] The polypeptides described herein are intended to meet one or more of the above needs. Accordingly, the present disclosure describes polypeptides that are active at each of the GIP, GLP-1, and glucagon receptors. The polypeptides described herein allow for administration at a dose that provides sufficient activity at each receptor to provide the benefits of agonizing that receptor, while avoiding adverse side effects associated with too much activity. In addition, the polypeptides described herein have an extended duration of action at the GIP, GLP-1, and glucagon receptors, allowing for infrequent dosing once daily, three times per week, twice per week, or once per week. In this way, these polypeptides result in enhanced glycemic control, metabolic benefits such as weight loss and / or body composition improvement, lipid benefits, and / or other benefits such as increased bone mass or bone formation or reduced bone resorption. In addition, the polypeptides described herein are suitable for subcutaneous or oral administration. The present disclosure also describes effective treatment methods for disorders or conditions including obesity, long-term weight management, type 2 diabetes, NAFLD, NASH, dyslipidemia, metabolic disorders, CKD, OA, OSA, and PCOS.

[0010] In one embodiment, a polypeptide comprising Formula I (SEQ ID NO: 4) is provided:

[0011] X1X2QGTX6TSDX 10 X 11 X 12 X 13 LDX 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X38X 39 X 40 X 41 X 42 wherein:

[0012] X1is Y, NMeY, or H,

[0013] X2is Aib,

[0014] X6is F, aMeF, or aMeF(2F),

[0015] X 10is F, 4-Pal, F(4CN), 3-Pal, F(4N02) or Y,

[0016] X 11 is S or aMeS,

[0017] X 12 is Orn, K, R, Q, Dap, Dab, S, E or I,

[0018] X 13 is aMeL, I or L,

[0019] X 16 is K, Orn, A or E,

[0020] X 17 is any amino acid with a functional group available for conjugation to a fatty acid, A, I or Q,

[0021] X 19 is Q or A,

[0022] X 20 is any amino acid with a functional group available for conjugation to a fatty acid, Aib, aMe-4-Pal, Q or R,

[0023] X 21 is A, Q, Orn, Aad, Aib, S, N, E or T,

[0024] X 23 is I or V,

[0025] X 24 is any amino acid with a functional group available for conjugation to a fatty acid, E, Q, D-Glu or N,

[0026] X 25 is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0027] X 27 is L, I, E, V, A, Q or S,

[0028] X 28 is any amino acid with a functional group available for conjugation to a fatty acid, E or A,

[0029] X 29 is G, D-Ala, Aib, T or A,

[0030] X 30 is A or G,

[0031] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp (hydroxyproline),

[0032] X 34 is G or Aib,

[0033] X 35 is A, Aib, E, H or 4-Pal,

[0034] X 36 is P or Hyp,

[0035] X 37 is P, Hyp or E,

[0036] X 38 is P or Hyp,

[0037] X 39 is E, S, G, T, H, 4-Pal, yE or A,

[0038] X 40 is absent or is G, E, S, A or T,

[0039] wherein if X 40 is G, E, S, A or T, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q or H,

[0040] wherein if X 41 is E, S, T, 4-Pal, D, G, Q or H, then X 42 is absent or is G, E or yE,

[0041] wherein if X 40 is absent, then X 41 is also absent, 42

[0042] wherein if X 41 is absent, then X 42 is also absent,

[0043] wherein at least one of X 17 , X 20 , X 24 and X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0044] wherein the C-terminal amino acid is optionally amidated;

[0045] or a pharmaceutically acceptable salt thereof.

[0046] In some embodiments, position X 17 , X 20 , X 24 or X 28 ​The amino acid at position X having a functional group available for conjugation to a fatty acid is K, C, E, or D. In some embodiments, X 17 The amino acid at position X having a functional group available for conjugation to a fatty acid is K, C, E, or D. In some embodiments, X 20 The amino acid at position X having a functional group available for conjugation to a fatty acid is K, C, E, or D. In some embodiments, X 24 The amino acid at position X having a functional group available for conjugation to a fatty acid is K, C, E, or D. In some embodiments, X 28 The amino acid at position X having a functional group available for conjugation to a fatty acid is K, C, E, or D. In some embodiments, X 10 is F or 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q.

[0047] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid.

[0048] In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid by a direct bond or a linker between the amino acid and the fatty acid. In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid by a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is C 16 -C 22 fatty acid.

[0049] In one embodiment, a polypeptide comprising Formula I’ (SEQ ID NO: 1243) is provided:

[0050] X1X2QX4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 SX 34 X 35 X36X 37 X38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 wherein:

[0051] X1is Y, NMeY or H,

[0052] X2is Aib,

[0053] X4is G or D-Ala,

[0054] X6is F, aMeF or aMeF(2F),

[0055] X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4N02) or Y,

[0056] X 11 is S or aMeS,

[0057] X 12 is Orn, K, R, Q, Dap, Dab, S, E or I,

[0058] X 13 is aMeL, I or L,

[0059] X 15 is D or E,

[0060] X 16 is K, Orn, A or E,

[0061] X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, Q or Orn,

[0062] X 19 is Q or A,

[0063] X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, aMe-4-Pal, Q, R or L-Iva,

[0064] X 21 is A, Q, Orn, Aad, Aib, S, N, E or T,

[0065] X 23 is I or V,

[0066] X 24is any amino acid having a functional group available for conjugation with a fatty acid, E, Q, D-Glu, N, or D-Gln,

[0067] X 25 is W, Y, F, 4-Pal, aMeY, or aMe-4-Pal,

[0068] X 27 is L, I, E, V, A, Q, S, T, or Aad,

[0069] X 28 is any amino acid having a functional group available for conjugation with a fatty acid, E, Q, or A,

[0070] X 29 is G, D-Ala, Aib, T, or A,

[0071] X 30 is A, S, or G,

[0072] X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp (hydroxyproline),

[0073] X 32 is S or P,

[0074] X 34 is G or Aib,

[0075] X 35 is A, Aib, E, D, H, or 4-Pal or Orn,

[0076] X 36 is P or Hyp,

[0077] X 37 is P, Hyp, or E,

[0078] X 38 is P or Hyp,

[0079] X 39 is E, S, G, T, H, 4-Pal, yE, or A or Orn,

[0080] X 40 is absent or is G, E, S, A, T, or D-Glu,

[0081] wherein if X 40 is G, E, S, A, T, or D-Glu, then X 41 is absent or is A, E, S, T, 4-Pal, D, G, yE, D-Glu(e), Q, or H,

[0082] wherein if X 41If X is A, E, S, T, 4-Pal, D, G, γE, D-Glu, Q, or H, then X 42 The deficiency may be due to G, E, γE, D-Glu, or AEEA.

[0083] Where X 42 If it is G, E, γE, D-Glu(e), or AEEA, then X 43 The deficiency may be due to E, γE, or D-Glu.

[0084] Where X 43 If it is E, γE, or D-Glu, then X 44 Missing or E, where if X 44 If it is E, then X 45 Missing or E, where if X 45 If it is E, then X 46 Missing or E,

[0085] Where X 40 If X is missing, then 41 To X 46 Also missing,

[0086] Where X 41 If X is missing, then 42 To X 46 Also missing,

[0087] Where X 42 If X is missing, then 43 To X 46 Also missing,

[0088] Where X 43 If X is missing, then 44 To X 46 Also missing, where X is missing. 44 If X is missing, then 45 and X 46 Also missing; where X is missing. 45 If X is missing, then 46 Also missing,

[0089] Where X 17 X 20 X 24 and X 28 At least one of them is an amino acid having a functional group that can be used for conjugation with fatty acids.

[0090] The C-terminal amino acid is optionally amidated;

[0091] Or its pharmaceutically acceptable salt.

[0092] In some implementations, location X 17 X20 , X 24 or X 28 at position X 17 , X 20 , X 24 or X 28 is K. In some embodiments, position X 17 , X 20 , X 24 or X 28 is Orn. In some embodiments, X 10 is F or 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q.

[0093] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid.

[0094] In some embodiments, one of X 17 , X 20 , X 24 or X 28 is conjugated to a fatty acid through a direct bond or a linker between the amino acid and the fatty acid. In some embodiments, one of X 17 , X 20 , X 24 or X 28 is conjugated to a fatty acid through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is C 16 -C 22 fatty acid.

[0095] In some embodiments, none of X 17 , X 20 , X 24 or X 28 is conjugated to a fatty acid.

[0096] In one embodiment, a polypeptide comprising Formula II (SEQ ID NO: 5) is provided:

[0097] YX2QGTFTSDX 10 SX 12 X 13 LDX 16 X 17 AQX 20X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X 38 X 39 X40X 41 X 42 wherein

[0098] X2 is Aib,

[0099] X 10 is F, 4-Pal or F(4CN),

[0100] X 12 is Orn, K, R, Q, Dap or Dab,

[0101] X 13 is aMeL,

[0102] X 16 is K or Orn,

[0103] X 17 is any amino acid having a functional group available for conjugation to a fatty acid,

[0104] X 20 is Aib, aMe-4-Pal or Q,

[0105] X 21 is A, Q or Orn,

[0106] X 24 is E or Q,

[0107] X 25 is W, Y, 4-Pal, aMeY or aMe-4-Pal,

[0108] X 27 is L or I,

[0109] X 28 is E or A,

[0110] X 29 is G, D-Ala or Aib,

[0111] X 30 is A or G,

[0112] X 31is P, H, S, 4-Pal, E, T, K(Ac), or Hyp,

[0113] X 34 is G or Aib,

[0114] X 35 is A, Aib, E, H, or 4-Pal,

[0115] X 36 is P or Hyp,

[0116] X 37 is P or Hyp,

[0117] X 38 is P or Hyp,

[0118] X 39 is E, S, G, T, H, 4-Pal, or yE,

[0119] X 40 is absent or is G, E, or S,

[0120] wherein if X 40 is G, E, or S, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q, or H,

[0121] wherein if X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or yE,

[0122] wherein if X 40 is absent, then X 41 and X 42 are also absent,

[0123] wherein if X 41 is absent, then X 42 is also absent,

[0124] wherein the C-terminal amino acid is optionally amidated;

[0125] or a pharmaceutically acceptable salt thereof.

[0126] In some embodiments of the polypeptide comprising Formula II, X 17 is K, C, E, or D. In some embodiments, X 17 is K. In some embodiments, X 10 is F or 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q.

[0127] In some embodiments, X 17It is K, and it is conjugated to fatty acids via a direct bond or linker between amino acids and fatty acids. In some embodiments, X 17 It is K, and it is conjugated to fatty acids via a linker between amino acids and fatty acids. In some embodiments, the fatty acid is C. 16 -C 22 fatty acid.

[0128] In some implementation schemes, X 17 It is K, and through X 17 amino acids and C 16 -C 22 The linker between fatty acids and C 16 -C 22 Fatty acid conjugation.

[0129] In one embodiment, a polypeptide comprising formula II' (SEQ ID NO: 1244) is provided:

[0130] X1X2QGTFTSDX 10 SX 12 X 13 LDX 16 X 17 AQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X 38 X 39 X40X 41 X 42 X 43 X 44 ,in

[0131] X1 is Y or NMeY.

[0132] X2 is Aib.

[0133] X 10 It is F, 4-Pal, or F(4CN).

[0134] X 12 It is Orn, K, R, I, Q, Dap, or Dab.

[0135] X 13 It is αMeL.

[0136] X 16is K or Orn,

[0137] X 17 is any amino acid having a functional group available for conjugation to a fatty acid,

[0138] X 20 is Aib, aMe-4-Pal, Q or L-Iva,

[0139] X 21 is A, Q or Orn,

[0140] X 24 is E or Q,

[0141] X 25 is W, Y, 4-Pal, aMeY or aMe-4-Pal,

[0142] X 27 is L or I,

[0143] X 28 is E, Q or A,

[0144] X 29 is G, D-Ala or Aib,

[0145] X 30 is A, S or G,

[0146] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp,

[0147] X 34 is G or Aib,

[0148] X 35 is A, Aib, E, H or 4-Pal,

[0149] X 36 is P or Hyp,

[0150] X 37 is P or Hyp,

[0151] X 38 is P or Hyp,

[0152] X 39 is E, S, G, T, H, 4-Pal or yE,

[0153] X 40 is absent or is G, E, S or D-Glu,

[0154] wherein if X 40 is G, E, S or D-Glu, then X 41is absent or is E, S, T, 4-Pal, D, G, Q, or H,

[0155] wherein if X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or yE,

[0156] wherein if X 42 is G, E, or yE, then X 43 is absent or is E,

[0157] wherein if X 43 is E, then X 44 is absent or is E,

[0158] wherein if X 40 is absent, then X 41 , X 42 , X 43 , and X 44 are also absent,

[0159] wherein if X 41 is absent, then X 42 , X 43 , and X 44 are also absent,

[0160] wherein if X 43 is absent, then X 44 is also absent,

[0161] wherein the C-terminal amino acid is optionally amidated;

[0162] or a pharmaceutically acceptable salt thereof.

[0163] In some embodiments of the polypeptide comprising Formula II’, X 17 is K, C, E, or D. In some embodiments, X 17 is K. In some embodiments, X 10 is F or 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q.

[0164] In some embodiments, X 17 is K and is conjugated to a fatty acid by a direct bond between the amino acid and the fatty acid or a linker. In some embodiments, X 17 is K and is conjugated to a fatty acid by a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0165] In some embodiments, X 17is K, and is conjugated to X 17 amino acid and C 16 -C 22 linker between the fatty acid and C 16 -C 22 fatty acid conjugation.

[0166] In some embodiments, X 17 , X 20 , X 24 or X 28 is not conjugated to a fatty acid.

[0167] In one embodiment, a polypeptide comprising Formula III (SEQ ID NO: 6) is provided:

[0168] X1X2QGTX6TSDX 10 X 11 X 12 X 13 LDX 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 GX 31 SSX 34 X 35 X 36 X 37 X38X 39 X 40 X 41 wherein

[0169] X1is Y, NMeY, or H,

[0170] X2is Aib,

[0171] X6is F, aMeF, or aMeF(2F),

[0172] X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4N02), or Y,

[0173] X 11 is S or aMeS,

[0174] X 12 is Orn, K, R, Q, Dap, S, E, or I,

[0175] X 13 is aMeL, I, or L,

[0176] X 16 is K, Orn, A, or E,

[0177] X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q,

[0178] X 19 is A or Q,

[0179] X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, Q, R, or aMe-4-Pal,

[0180] X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn,

[0181] X 23 is I or V,

[0182] X 24 is any amino acid having a functional group available for conjugation to a fatty acid, E, D-Glu, Q, or N,

[0183] X 25 is W, Y, F, 4-Pal, aMeY, or aMe-4-Pal,

[0184] X 27 is L, I, E, V, A, Q, or S,

[0185] X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A,

[0186] X 29 is G, Aib, T, D-Ala, or A,

[0187] X 31 is P or E,

[0188] X 34 is G or Aib,

[0189] X 35 is A or E,

[0190] X 36 is P,

[0191] X 37 is P or E,

[0192] X 38 is P,

[0193] X 39 is E, S, G, or A,

[0194] X 40 is absent or is G, E, S, A, or T,

[0195] wherein if X 40 is G, E, S, A, or T, then X 41 is absent or is E, S, D, or G,

[0196] wherein if X 41 is E, S, D, or G, then X 42 is absent or is G, E, or γE,

[0197] wherein if X 40 is absent, then X 41 and X 42 are also absent,

[0198] wherein if X 41 is absent, then X 42 is also absent,

[0199] wherein if X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), then X 12 is I,

[0200] wherein at least one of X 17 , X 20 , X 24 , or X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0201] wherein the C-terminal amino acid is optionally amidated;

[0202] or a pharmaceutically acceptable salt thereof.

[0203] In some embodiments of Formula III, the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K, C, E, or D. In some embodiments, the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K. In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), and X 12 is I. In some embodiments, X 10 is Y, and X 12 is Orn, K, R, Q, Dap, S, E, or I.

[0204] In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is an amino acid having a functional group available for conjugation to a fatty acid.

[0205] In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid via a direct bond or a linker between the amino acid and the fatty acid. In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid via a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0206] In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is K and is conjugated to a C 16 -C 22 fatty acid via a linker between the amino acid and the C 16 -C 22 fatty acid.

[0207] In one embodiment, a polypeptide comprising Formula III’ (SEQ ID NO: 1245) is provided:

[0208] X1X2QX4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 GX 31 X 32 SX 34 X 35 PX 37 PX 39 X 40 X41 X 42 X 43 X 44 X 45 X 46 wherein

[0209] X1is Y, NMeY or H,

[0210] X2is Aib,

[0211] X4is G or D-Ala,

[0212] X6is F, aMeF or aMeF(2F),

[0213] X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4N02) or Y,

[0214] X 11 is S or aMeS,

[0215] X 12 is Orn, K, R, Q, Dap, S, E or I,

[0216] X 13 is aMeL, I or L,

[0217] X 15 is D or E,

[0218] X 16 is K, Orn, A or E,

[0219] X 17 is any amino acid with a functional group available for conjugation to a fatty acid, A, I or Q or Orn,

[0220] X 19 is A or Q,

[0221] X 20 is any amino acid with a functional group available for conjugation to a fatty acid, Aib, Q, R or aMe-4-Pal,

[0222] X 21 is A, Aad, Aib, S, N, Q, E, T or Orn,

[0223] X 23 is I or V,

[0224] X 24 is any amino acid with a functional group available for conjugation to a fatty acid, E, D-Glu, Q, N or D-Gln,

[0225] X 25is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0226] X 27 is L, I, E, V, A, Aad, T, Q or S,

[0227] X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E or A,

[0228] X 29 is G, Aib, T, D-Ala or A,

[0229] X 31 is P or E,

[0230] X 32 is S or P,

[0231] X 34 is G or Aib,

[0232] X 35 is A, D or E,

[0233] X 37 is P or E,

[0234] X 39 is E, S, G, A, T or Orn,

[0235] X 40 is absent or is G, E, S, A or T,

[0236] wherein if X 40 is G, E, S, A, T or D-Glu, then X 41 is absent or is E, S, D, G, Q, T, A, yE or D-Glu,

[0237] wherein if X 41 is E, S, D, G, Q, T, A, yE or D-Glu, then X 42 is absent or is G, E, D-Glu or yE,

[0238] wherein if X 42 is G, E, D-Glu or yE, then X 43 is absent or is E, yE or D-Glu,

[0239] wherein if X 43 is E, yE or D-Glu, then X 44 is absent or is E,

[0240] wherein if X 44 is E, then X 45 is absent or is E,

[0241] wherein if X 45 is E, then X 46 is absent or E,

[0242] wherein if X 40 is absent, then X 41 is absent, 46

[0243] wherein if X 41 is absent, then X 42 is absent, 46

[0244] wherein if X 42 is absent, then X 43 is absent, 46

[0245] wherein if X 43 is absent, then X 44 is absent, 46

[0246] wherein if X 44 is absent, then X 45 and X 46 are also absent,

[0247] wherein if X 45 is absent, then X 46 is also absent,

[0248] wherein the polypeptide comprises at least one of: X6is aMeF or aMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02); X 11 is aMeS; X 13 is aMeL; X 24 is D-Glu; and / or X 25 is aMeY,

[0249] wherein if X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02), then X 12 is I,

[0250] and wherein at least one of X 17 , X 20 , X 24 , or X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0251] wherein the C-terminal amino acid is optionally amidated;

[0252] ​​​​or a pharmaceutically acceptable salt thereof.

[0253] In some embodiments of III’, position X 17 , X 20 , X 24 , or X 28 is an amino acid having a functional group available for conjugation to a fatty acid. In some embodiments, position X 17 , X 20 , X 24 , or X 28 is K. In some embodiments, position X 17 , X 20 , X 24 , or X 28 is Orn. In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02), and X 12 is I. In some embodiments, X 10 is Y, and X 12 is Orn, K, R, Q, Dap, S, E, or I.

[0254] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid.

[0255] In some embodiments, one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid by a direct bond between the amino acid and the fatty acid or a linker. In some embodiments, one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid by a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0256] In some embodiments, only one of X 17 , X 20 , X 24 , or X 28 is K, and is conjugated to a C 16 -C 22 fatty acid by a linker between the amino acid and the C16 -C 22 fatty acid conjugation. In some embodiments, X 17 , X 20 , X 24 , or X 28 are not conjugated to a fatty acid.

[0257] In another embodiment, provided herein is a pharmaceutical composition comprising a polypeptide described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0258] In another embodiment, provided is a method of treating a disease or disorder, including obesity, long-term weight management, type 2 diabetes, NAFLD, NASH, dyslipidemia, metabolic disorder, CKD, OA, OSA, and PCOS. Another embodiment provides a method of non-therapeutic weight loss, comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof. Such a method can comprise at least one step of administering to an individual in need thereof an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0259] In another embodiment, a polypeptide described herein is provided for use in therapy. For example, a polypeptide described herein is provided for use in treating a disease or disorder, including obesity, long-term weight management, type 2 diabetes, NAFLD, NASH, dyslipidemia, metabolic disorder, CKD, OA, OSA, and / or PCOS.

[0260] In another embodiment, provided is use of a polypeptide described herein in the manufacture of a medicament for treating a disease or disorder, including obesity, long-term weight management, type 2 diabetes, NAFLD, NASH, dyslipidemia, metabolic disorder, CKD, OA, OSA, and / or PCOS.

[0261] DETAILED DESCRIPTION

[0262] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the polypeptides, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0263] Further, the indefinite articles "a" or "an," as used in this application, are each generally defined as meaning one or more, unless the context clearly dictates otherwise. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to one or at least one), unless the context clearly dictates otherwise. Thus, use of the singular herein will also cover the plural unless the context clearly dictates otherwise.

[0264] GIP is a 42-amino acid peptide (SEQ ID NO: 1) that is an incretin that plays a physiological role in glucose homeostasis by stimulating pancreatic beta cells to secrete insulin in the presence of glucose.

[0265] GLP-1 is a 36-amino acid peptide that is also an incretin that stimulates glucose-dependent insulin secretion and has been shown to prevent hyperglycemia in diabetic patients. The major biologically active fragment of GLP-1 is generated as a 30-amino acid, C-terminally amidated peptide (GLP-1 7-36 )(SEQ ID NO: 2).

[0266] Glucagon is a 29-amino acid peptide (SEQ ID NO: 3) that helps maintain blood glucose levels by binding to and activating glucagon receptors on liver cells, causing the liver to release glucose stored in the form of glycogen through a process called glycogenolysis.

[0267] In addition to T2DM, incretins and analogs thereof having activity at one or more GIP, GLP-1, and / or glucagon receptors have been described as having potential therapeutic value in a number of other conditions, diseases, or disorders, including, for example, obesity, NAFLD and NASH, dyslipidemia, metabolic syndrome, bone-related disorders, and neurodegenerative and / or cognitive disorders such as Alzheimer’s disease and Parkinson’s disease. See, e.g., Jall et al. (2017) Mol. Metab. 6:440-446; Carbone et al. (2016) J. Gastroenterol. Hepatol. 31 :23-31; Finan et al. (2016) Trends Mol. Med. 22:359-376; Choi et al. (2017) Potent body weight loss and efficacy in a NASH animal model by a novel long-acting GLP-1 / Glucagon / GIP triple-agonist (HM15211), ADA Poster 1139-P; Ding (2008) J. Bone Miner. Res. 23:536-543; Tai et al. (2018) Brain Res. 1678:64-74; Muller et al. (2017) Physiol. Rev. 97:721-766; Finan et al. (2013) Sci. Transl. Med. 5:209; (2014) Biochem. Soc. Trans. 42:593-600.

[0268] As used herein, “about” refers to a range of statistical significance within one or more numerical values, e.g., a specified concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such numerical values or ranges can be within one order of magnitude, typically within 20% of a given numerical value or range, more typically within 10% of a given numerical value or range, and even more typically within 5% of a given numerical value or range. The permissible variation encompassed by “about” will depend on the particular system under study, as will be readily appreciated by one of skill in the art.

[0269] As used herein, with respect to one or more GIP, GLP-1, or glucagon receptors, “activity,” “activate,” “activation,” and the like refer to the ability of a compound, e.g., a polypeptide as described herein, to bind to one or more receptors and induce a response at the receptor, as measured using assays known in the art, e.g., in vitro assays as described below.

[0270] As used herein, "amino acid having a functional group available for conjugation" refers to any natural (encoded) or unnatural (non-encoded) amino acid having a functional group that can be conjugated to a fatty acid, either directly or through, for example, a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo, and thiol. Examples of natural amino acids that include such functional groups include K (amino), C (thiol), E (carboxyl), and D (carboxyl).

[0271] As used herein, "conservative amino acid substitution" refers to the substitution of one amino acid for another amino acid with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) and minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution of functionally similar amino acids is well known in the art and need not be described in detail herein.

[0272] As used herein, "C 16 -C 22 Fatty acid" refers to a carboxylic acid having 16 to 22 carbon atoms. Suitable C 16 -C 22 The fatty acid can be a saturated monoacid or a saturated diacid. As used herein, "saturated" refers to a fatty acid that does not contain carbon-carbon double or triple bonds.

[0273] As used herein, "effective amount" refers to an amount, concentration, or dosage of one or more polypeptides described herein, or a pharmaceutically acceptable salt thereof, which, upon single or multiple dose administration to an individual in need thereof, provides the intended effect in that diagnosed or treated individual. An effective amount can be readily determined by a person skilled in the art by using known techniques and by observing results obtained under analogous circumstances. In determining an effective amount, a variety of factors are considered, including but not limited to: the species of mammal; its size, age, and general health condition; the specific disease or disorder involved; the degree, extent, or severity of the disease or disorder; the response of the individual patient; the particular polypeptide administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dosage regimen desired; the use of concomitant medication; and other relevant circumstances.

[0274] As used herein, "extended duration of action" refers to a longer duration of binding affinity and activity of the polypeptide than native human GIP, GLP-1, and glucagon peptides, allowing for less frequent dosing of at least once daily, even three times per week, twice per week, or once per week. The time course of the polypeptide can be measured using known pharmacokinetic test methods, such as those used in the following examples.

[0275] As used herein, "polypeptide" or "peptide" refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids as well as to those modified naturally occurring amino acids, and to those containing one or more non-naturally occurring amino acids.

[0276] As used herein, "individual in need thereof refers to a mammal, such as a human, who has a condition, disease, disorder, or symptom for which treatment or therapy is desired, including, for example, those listed herein.

[0277] As used herein, "treat" and the like, refer to the inhibition, slowing, stopping, or reversal of the progression or severity of an existing condition, disease, disorder, or symptom.

[0278] As used herein, with respect to polypeptides, "triple agonist activity" refers to a polypeptide that has activity at each of the GIP, GLP-1, and glucagon receptors, and in particular, to a polypeptide that has sufficient activity at each receptor to provide the benefit of agonizing that receptor while avoiding the adverse side effects associated with too much activity. A polypeptide having triple agonist activity (also referred to herein as a "GGG polypeptide") has a prolonged duration of action at the GIP, GLP-1, and glucagon receptors, which advantageously allows for infrequent dosing once daily, three times per week, twice per week, or once per week.

[0279] As used herein, the term "sequence identity" refers to the extent to which two sequences are similar. The extent of sequence identity between two polypeptides can be expressed as a percentage, calculated as follows:

[0280] % sequence identity = 100% * (number of identical amino acids) / (length of the shortest common sequence).

[0281] The structural features of the polypeptides described herein allow for appropriate activity at each of the GIP, GLP-1, and glucagon receptors to achieve a beneficial active effect at each receptor (i.e., triple agonist activity), but not so much activity at any one receptor as to overwhelm the activity at the other two receptors or cause adverse side effects when administered at a dose sufficient to produce activity at all three receptors. In some embodiments, the polypeptides described herein are partial agonists of the GLP-1 receptor, showing 80% or less agonism compared to native GLP-1 7-36 (SEQ ID NO: 2), as evidenced by the HEK293 cell GLP-1 receptor internalization assay described herein. In other embodiments, the polypeptides described herein are full agonists of the GLP-1 receptor, showing 7-36 (SEQ ID NO: 2), as evidenced by the HEK293 cell GLP-1 receptor internalization assay described herein. In other embodiments, the polypeptides described herein are full agonists of the GLP-1 receptor, showing >80%, as evidenced by the HEK293 cell GLP-1 receptor internalization assay described herein. In some embodiments, the polypeptides described herein have a higher potency for each of the glucagon, GIP, and GLP-1 receptors compared to native glucagon (SEQ ID NO: 3), GIP (SEQ ID NO: 1), and GLP-1 (SEQ ID NO: 2). 7-36 The structural features of the polypeptides described herein also result in the polypeptides having a number of other beneficial attributes relevant to their developability as therapeutic treatment methods, including improving solubility of the analog in aqueous solutions at near neutral pH, improving chemical and physical formulation stability, improving peptide membrane permeability in the presence of permeation enhancers, prolonging pharmacokinetic properties, and minimizing the potential for injection site reactions or immunogenicity.

[0282] The structural features of the polypeptides described herein also result in the polypeptides having a number of other beneficial attributes relevant to their developability as therapeutic treatment methods, including improving solubility of the analog in aqueous solutions at near neutral pH, improving chemical and physical formulation stability, improving peptide membrane permeability in the presence of permeation enhancers, prolonging pharmacokinetic properties, and minimizing the potential for injection site reactions or immunogenicity.

[0283] It should be noted that the combination of beneficial attributes of the exemplary analogs described herein is not the result of any single modification in isolation, but rather is achieved through the novel combination of structural features described herein.

[0284] Polypeptide sequences

[0285] In one embodiment, provided herein is a polypeptide comprising Formula I (SEQ ID NO: 4):

[0286] X1X2QGTX6TSDX 10 X 11 X 12 X 13 LDX 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X38X 39 X 40 X 41 X 42 wherein

[0287] X1is Y, NMeY, or H,

[0288] X2is Aib,

[0289] X6is F, aMeF or aMeF(2F),

[0290] X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4N02) or Y,

[0291] X 11 is S or aMeS,

[0292] X 12 is Orn, K, R, Q, Dap, Dab, S, E or I,

[0293] X 13 is aMeL, I or L,

[0294] X 16 is K, Orn, A or E,

[0295] X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I or Q,

[0296] X 19 is Q or A,

[0297] X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, aMe-4-Pal, Q or R,

[0298] X 21 is A, Q, Orn, Aad, Aib, S, N, E or T,

[0299] X 23 is I or V,

[0300] X 24 is any amino acid having a functional group available for conjugation to a fatty acid, E, Q, D-Glu or N,

[0301] X 25 is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0302] X 27 is L, I, E, V, A, Q or S,

[0303] X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E or A,

[0304] X 29 is G, D-Ala, Aib, T or A,

[0305] X 30 is A or G,

[0306] X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp,

[0307] X 34 is G or Aib,

[0308] X 35 is A, Aib, E, H, or 4-Pal,

[0309] X 36 is P or Hyp,

[0310] X 37 is P, Hyp, or E,

[0311] X 38 is P or Hyp,

[0312] X 39 is E, S, G, T, H, 4-Pal, yE, or A,

[0313] X 40 is absent or is G, E, S, A, or T,

[0314] wherein if X 40 is G, E, S, A, or T, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q, or H,

[0315] wherein if X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or yE,

[0316] wherein if X 40 is absent, then X 41 and X 42 are also absent,

[0317] wherein if X 41 is absent, then X 42 is also absent,

[0318] wherein at least one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0319] and wherein the C-terminal amino acid is optionally amidated;

[0320] or a pharmaceutically acceptable salt thereof.

[0321] if X 40 is absent, then X 41 and X42 is also deleted, and the polypeptide comprises a 39 amino acid sequence. If X 41 is deleted, then X 42 is also deleted, and the polypeptide comprises a 40 amino acid sequence. If X 42 is deleted, then the polypeptide comprises a 41 amino acid sequence. If X 40 , X 41 , and X 42 are all not deleted (in other words, X 40 , X 41 , and X 42 are all present), then the polypeptide comprises a 42 amino acid sequence.

[0322] In one embodiment, X 40 is G, E, S, A, or T, X 41 is deleted, and X 42 is deleted. In such embodiments, the polypeptide comprises a 40 amino acid sequence. In one embodiment, X 40 is G, E, S, A, or T, X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is deleted. In such embodiments, the polypeptide comprises a 41 amino acid sequence. In one embodiment, X 40 is G, E, S, A, or T, X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is G, E, or γE. In such embodiments, the polypeptide comprises a 42 amino acid sequence.

[0323] In some embodiments, the polypeptides of the application include, at position X 17 , X 20 , X 24 , or X 28 , any amino acid having a functional group available for conjugation to a fatty acid (natural or unnatural). In certain embodiments, the amino acid having a functional group available for conjugation to a fatty acid is K, C, E, or D. In particularly preferred embodiments, the amino acid is K, and the conjugation is to the epsilon-amino group of the K side chain.

[0324] Thus, in some embodiments, the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K, C, E, or D. In some embodiments, the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28The amino acid having a functional group available for conjugation with a fatty acid is K.

[0325] In some embodiments, X 10 is F or 4-Pal. In some embodiments, X 10 is F. In some embodiments, X 10 is 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q. In some embodiments, X 12 is Orn. In some embodiments, X 12 is K. In some embodiments, X 12 is R. In some embodiments, X 12 is Q.

[0326] In one embodiment, only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation with a fatty acid. In one embodiment, the conjugation is acylation.

[0327] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid. In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is optionally conjugated to a fatty acid through a linker between the amino acid and the fatty acid. Thus, in some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a direct bond between the amino acid and the fatty acid or through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0328] Thus, in one embodiment, X 17 is K and is conjugated to a C 16 -C 22 fatty acid through a direct bond between the amino acid and the C 16 -C 22 fatty acid or a linker. In one embodiment, X 17 is K and is conjugated to a C 16 -C 22 fatty acid through a linker between the amino acid and the C 16-C 22 fatty acid conjugation. In such embodiments, X 20 is Aib, aMe-4-pal, Q, or R, X 24 is E, Q, D-Glu, or N, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0329] In one embodiment, X 20 is K, and is conjugated to C 16 -C 22 fatty acid via a direct bond or linker between the amino acid and C 16 -C 22 fatty acid conjugation. In one embodiment, X 20 is K, and is conjugated to C 16 -C 22 fatty acid via a linker between the amino acid and C 16 -C 22 fatty acid conjugation. In such embodiments, X 17 is A, I, or Q, X 24 is E, Q, D-Glu, or N, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0330] In one embodiment, X 24 is K, and is conjugated to C 16 -C 22 fatty acid via a direct bond or linker between the amino acid and C 16 -C 22 fatty acid conjugation. In one embodiment, X 24 is K, and is conjugated to C 16 -C 22 fatty acid via a linker between the amino acid and C 16 -C 22 fatty acid conjugation. In such embodiments, X 17 is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0331] In one embodiment, X 28 is K, and is conjugated to C 16 -C 22 fatty acid via a direct bond or linker between the amino acid and C 16 -C 22 fatty acid conjugation. In one embodiment, X 28is K, and conjugated through an amino acid and C 16 - C 22 Linker between fatty acids and C 16 - C 22 Fatty acid conjugation. In such embodiments, X 17 is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0332] In one embodiment, provided herein is a polypeptide comprising Formula I’ (SEQ ID NO: 1243):

[0333] X1X2QX4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 SX 34 X 35 X36X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 wherein:

[0334] X1is Y, NMeY, or H,

[0335] X2is Aib,

[0336] X4is G or D-Ala,

[0337] X6is F, aMeF, or aMeF(2F),

[0338] X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4N02), or Y,

[0339] X 11 is S or aMeS,

[0340] X 12 is Orn, K, R, Q, Dap, Dab, S, E or I,

[0341] X 13 is aMeL, I or L,

[0342] X 15 is D or E,

[0343] X 16 is K, Orn, A or E,

[0344] X 17 is any amino acid with a functional group available for conjugation to a fatty acid, A, I, Q or Orn,

[0345] X 19 is Q or A,

[0346] X 20 is any amino acid with a functional group available for conjugation to a fatty acid, Aib, aMe-4-Pal, Q, R or L-Iva,

[0347] X 21 is A, Q, Orn, Aad, Aib, S, N, E or T,

[0348] X 23 is I or V,

[0349] X 24 is any amino acid with a functional group available for conjugation to a fatty acid, E, Q, D-Glu, N or D-Gln,

[0350] X 25 is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0351] X27is L, I, E, V, A, Q, S, T or Aad,

[0352] X 28 is any amino acid with a functional group available for conjugation to a fatty acid, E, Q or A,

[0353] X 29 is G, D-Ala, Aib, T or A,

[0354] X 30 is A, S or G,

[0355] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp (hydroxyproline),

[0356] X 32 is S or P,

[0357] X 34 is G or Aib,

[0358] X 35 is A, Aib, E, D, H or 4-Pal or Orn,

[0359] X 36 is P or Hyp,

[0360] X 37 is P, Hyp or E,

[0361] X 38 is P or Hyp,

[0362] X 39 is E, S, G, T, H, 4-Pal, yE or A or Orn,

[0363] X 40 is absent or is G, E, S, A, T or D-Glu,

[0364] wherein if X 40 is G, E, S, A or T, then X 41 is absent or is A, E, S, T, 4-Pal, D, G, yE, D-Glu, Q or H,

[0365] wherein if X 41 is A, E, S, T, 4-Pal, D, G, yE, D-Glu, Q or H, then X 42 is absent or is G, E, yE, D-Glu or AEEA,

[0366] wherein if X 42 is G, E, yE, D-Glu(e) or AEEA, then X 43 is absent or is E, yE or D-Glu,

[0367] wherein if X 43 is E, yE or D-Glu, then X 44 is absent or is E, wherein if X 44 is E, then X 45 is absent or is E, wherein if X 45 is E, then X 46 is absent or is E,

[0368] wherein if X 40 is absent, then X 41 is absent to X 46 is also absent,

[0369] wherein if X 41 is absent, X 42 is absent, 46 also is absent,

[0370] wherein if X 42 is absent, X 43 is absent, 46 also is absent,

[0371] wherein if X 43 is absent, X 44 is absent, 46 also is absent, wherein if X 44 is absent, X 45 and X 46 are also absent; wherein if X 45 is absent, X 46 is also absent,

[0372] wherein at least one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0373] wherein the C-terminal amino acid is optionally amidated;

[0374] or a pharmaceutically acceptable salt thereof.

[0375] if X 40 is absent, X 41 is absent, 46 and the polypeptide comprises a 39 amino acid sequence backbone. If X 41 is absent, X 42 is absent, 46 and the polypeptide comprises a 40 amino acid sequence backbone. If X 42 is absent, the polypeptide comprises a 41 amino acid sequence backbone. If X 43 is absent, the polypeptide comprises a 42 amino acid sequence backbone. If X 44 is absent, the polypeptide comprises a 43 amino acid sequence backbone. If X 45 is absent, the polypeptide comprises a 44 amino acid sequence backbone. If X 46 is absent, the polypeptide comprises a 45 amino acid sequence backbone. If X 40 is absent, X 46 is absent, (in other words, X 40 is present, X 46 is present), the polypeptide comprises a 46 amino acid sequence backbone.

[0376] In one embodiment, X40 is G, E, S, A, or T, X 41 is absent, and X 42 is absent. In such embodiments, the polypeptide comprises a 40 amino acid sequence. In one embodiment, X 40 is G, E, S, A, or T, X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is absent. In such embodiments, the polypeptide comprises a 41 amino acid sequence. In one embodiment, X 40 is G, E, S, A, or T, X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is G, E, or γE. In such embodiments, the polypeptide comprises a 42 amino acid sequence.

[0377] In some embodiments, the polypeptides of the application include any amino acid (natural or non-natural) having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 In certain embodiments, the amino acid having a functional group available for conjugation to a fatty acid is K, C, E, or D. In certain embodiments, the amino acid is K, and the fatty acid is conjugated to the epsilon-amino group of the K side chain.

[0378] Thus, in some embodiments, the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K, C, E, or D. In some embodiments, the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K.

[0379] In some embodiments, X 10 is F or 4-Pal. In some embodiments, X 10 is F. In some embodiments, X 10 is 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q. In some embodiments, X 12 is Orn. In some embodiments, X 12 is K. In some embodiments, X 12 is R. In some embodiments, X 12 is Q.

[0380] In one embodiment, X 17 , X 20 , X 24 , and X 28 only one of which is an amino acid having a functional group available for conjugation to a fatty acid. In one embodiment, the conjugation is acylation.

[0381] In some embodiments, X 17 , X 20 , X 24 , and X 28 only one of which is conjugated to a fatty acid. In some embodiments, X 17 , X 20 , X 24 , and X 28 only one of which is conjugated to a fatty acid, optionally through a linker between the amino acid and the fatty acid. Thus, in some embodiments, X 17 , X 20 , X 24 , and X 28 only one of which is conjugated to a fatty acid, either through a direct bond between the amino acid and the fatty acid or through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0382] Thus, in one embodiment, X 17 is K, and is conjugated to a C 16 -C 22 fatty acid, either through a direct bond between the amino acid and the C 16 -C 22 fatty acid or through a linker between the amino acid and the C 17 -C 16 fatty acid. In one embodiment, X 22 is K, and is conjugated to a C 16 -C 22 fatty acid through a linker between the amino acid and the C 20 -C 24 fatty acid. In such embodiments, X 28 is Aib, alphaMe-4-pal, Q, or R, X 20 is E, Q, D-Glu, or N, and X 16 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0383] In one embodiment, X 22 is K, and is conjugated to a C 16 -C 22Fatty acid conjugation. In one embodiment, X 20 is K, and the conjugation is to the epsilon-amino group of the side chain of K via an amino acid and C 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 17 is A, I, or Q, X 24 is E, Q, D-Glu, or N, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K.

[0384] In one embodiment, X 24 is K, and the conjugation is to the epsilon-amino group of the side chain of K via an amino acid and C 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 24 is K, and the conjugation is to the epsilon-amino group of the side chain of K via an amino acid and C 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 17 is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K.

[0385] In one embodiment, X 28 is K, and the conjugation is to the epsilon-amino group of the side chain of K via an amino acid and C 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 28 is K, and the conjugation is to the epsilon-amino group of the side chain of K via an amino acid and C 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K. 17 is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K.

[0386] In some embodiments, X 17 , X 20 , X24 and X 28 none of which are conjugated to a fatty acid.

[0387] In one embodiment, provided herein is a polypeptide comprising Formula II (SEQ ID NO: 5):

[0388] YX2QGTFTSDX 10 SX 12 X 13 LDX 16 X 17 AQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X 38 X 39 X40X 41 X 42 wherein:

[0389] X2 is Aib,

[0390] X 10 is F, 4-Pal, F(4CN),

[0391] X 12 is Orn, K, R, Q, Dap or Dab,

[0392] X 13 is aMeL,

[0393] X 16 is K or Orn,

[0394] X 17 is any amino acid having a functional group available for conjugation to a fatty acid,

[0395] X 20 is Aib, aMe-4-pal or Q,

[0396] X 21 is A, Q or Orn,

[0397] X 24 is E or Q,

[0398] X 25 is W, Y, 4-Pal, aMeY or aMe-4-Pal,

[0399] X 27 is L or I,

[0400] X 28 is E or A,

[0401] X 29 is G, D-Ala or Aib,

[0402] X 30 is A or G,

[0403] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp,

[0404] X 34 is G or Aib,

[0405] X 35 is A, Aib, E, H or 4-Pal,

[0406] X 36 is P or Hyp,

[0407] X 37 is P or Hyp,

[0408] X 38 is P or Hyp,

[0409] X 39 is E, S, G, T, H, 4-Pal or γE,

[0410] X 40 is absent or is G, E or S,

[0411] wherein if X 40 is G, E or S, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q or H,

[0412] wherein if X 41 is E, S, T, 4-Pal, D, G, Q or H, then X 42 is absent or is G, E or γE,

[0413] wherein if X 40 is absent, then X 41 and X 42 are also absent,

[0414] wherein if X 41 is absent, then X 42 is also absent,

[0415] wherein the C-terminal amino acid is optionally amidated;

[0416] Or its pharmaceutically acceptable salt.

[0417] As mentioned earlier, if X 40 If X is missing, then 41 and X 42 Also missing, and the polypeptide contains a 39-amino acid sequence. If X 41 If X is missing, then 42 Also missing, and the polypeptide contains a 40-amino acid sequence. If X 42 If X is missing, the polypeptide contains a 41-amino acid sequence. 40 X 41 and X 42 None are missing (in other words, X) 40 X 41 and X 42 If all of them are present, then the polypeptide contains a 42-amino acid sequence.

[0418] In one implementation, X 40 Is it G, E, or S, X 41 Missing, and X 42 Missing. In such embodiments, the polypeptide comprises a 40-amino acid sequence. In one embodiment, X 40 Is it G, E, or S, X 41 It is E, S, T, 4-Pal, D, G, Q, or H, and X 42 Missing. In such embodiments, the polypeptide comprises a 41-amino acid sequence. In one embodiment, X 40 Is it G, E, or S, X 41 It is E, S, T, 4-Pal, D, G, Q, or H, and X 42 It is G, E, or γE. In such embodiments, the polypeptide comprises a 42-amino acid sequence.

[0419] In some embodiments of the polypeptide of formula II, position X 17 The amino acid at position X that has a functional group that can be used for conjugation with fatty acids is K, C, E, or D. In a preferred embodiment, position X... 17 The amino acid at the site that has a functional group that can be used for conjugation with fatty acids is K, and the conjugation is an ε-amino conjugation with the side chain of K.

[0420] In some embodiments of the polypeptide of formula II, X 10 It is either F or 4-Pal. In some implementations, X 10 It is F. In some implementations, X 10 It is 4-Pal. In some implementations, X 12 It is Orn, K, R, or Q. In some implementations, X 12is Orn. In some embodiments, X 12 is K. In some embodiments, X 12 is R. In some embodiments, X 12 is Q. In some embodiments, X 10 is F or 4-Pal, and X 12 is Orn, K, R or Q. In some embodiments, X 10 is F, and X 12 is Q. In some embodiments, X 10 is F, and X 12 is Orn. In some embodiments, X 10 is F, and X 12 is K. In some embodiments, X 10 is F, and X 12 is R. In some embodiments, X 10 is 4-Pal, and X 12 is Q. In some embodiments, X 10 is 4-Pal, and X 12 is Orn. In some embodiments, X 10 is 4-Pal, and X 12 is K. In some embodiments, X 10 is 4-Pal, and X 12 is R.

[0421] In further embodiments, X 10 is selected from F or 4-Pal. In some embodiments, X 12 is selected from Orn, K, R or Q. In some embodiments, X 16 is K. In some embodiments, X 17 is K. In some embodiments, X 20 is selected from Aib or aMe-4-pal. In some embodiments, X 24 is E. In some embodiments, X 28 is selected from E or A. In some embodiments, X 31 is selected from P, H, S, 4-Pal, T or E. In some embodiments, X 35 is selected from A, Aib or E. In some embodiments, X 36 is P. In some embodiments, X 37 is P. In some embodiments, X 38 is P. In some embodiments, X 39 is selected from E, S or G. In some embodiments, X 40 is selected from G, E or S. In some embodiments, X 41selected from E, S, T, 4-Pal, or H.

[0422] In some embodiments, X is selected from F and 4-Pal. 10 In some embodiments, X is selected from F and 4-Pal. 12 In some embodiments, X is selected from Orn, K, R, and Q. 16 In some embodiments, X is K. 17 In some embodiments, X is K. 20 In some embodiments, X is selected from Aib and aMe-4-pal. 24 In some embodiments, X is E. 28 In some embodiments, X is selected from E and A. 31 In some embodiments, X is selected from P, H, S, 4-Pal, T, and E. 35 In some embodiments, X is selected from A, Aib, and E. 36 In some embodiments, X is P. 37 In some embodiments, X is P. 38 In some embodiments, X is P. 39 In some embodiments, X is selected from E, S, and G. 40 In some embodiments, X is selected from G, E, and S. 41 In some embodiments, X is selected from E, S, T, 4-Pal, and H.

[0423] In some embodiments, X is selected from F and 4-Pal. 10 In some embodiments, X is selected from F or 4-Pal; X 12 In some embodiments, X is Orn, K, R, or Q; X 16 In some embodiments, X is K; X 17 In some embodiments, X is K; X 20 In some embodiments, X is selected from Aib or aMe-4-pal; X 24 In some embodiments, X is E; X 28 In some embodiments, X is selected from E or A; X 31 In some embodiments, X is selected from P, H, S, 4-Pal, T, or E; X 35 In some embodiments, X is selected from A, Aib, or E; X 36 In some embodiments, X is P; X 37 In some embodiments, X is P; X 38 In some embodiments, X is P; X 39 In some embodiments, X is selected from E, S, or G; X 40 In some embodiments, X is selected from G, E, or S; and X 41 In some embodiments, X is selected from E, S, T, 4-Pal, or H.

[0424] In some embodiments, X is selected from F and 4-Pal. 10 In some embodiments, X is selected from F and 4-Pal. 12 In some embodiments, X is selected from Orn, K, R, and Q. 16 In some embodiments, X is K. 17 In some embodiments, X is K.20 is selected from Aib and aMe-4-pal; X 24 is E; X 28 is selected from E and A; X 31 is selected from P, H, S, 4-Pal, T, and E; X 35 is selected from A, Aib, and E; X 36 is P; X 37 is P; X 38 is P; X 39 is selected from E, S, and G; X 40 is selected from G, E, and S; and X 41 is selected from E, S, T, 4-Pal, and H.

[0425] In some embodiments, the amino acid X 17 is conjugated to a fatty acid. In some embodiments, the amino acid X 17 is optionally conjugated to a fatty acid through a linker between the amino acid and the fatty acid. Thus, in some embodiments, the amino acid X 17 is conjugated to a fatty acid through a direct bond between the amino acid and the fatty acid or through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is C 16 -C 22 fatty acid.

[0426] Thus, in some embodiments, X 17 is K and is conjugated to a C 16 -C 22 fatty acid through a direct bond between the amino acid and the C 16 -C 22 fatty acid or through a linker between the amino acid and the C 16 -C 22 fatty acid. In one embodiment, X 17 K is conjugated to a C 16 -C 22 fatty acid through a linker between the amino acid and the C 16 -C 22 fatty acid. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of X 17 K.

[0427] In one embodiment, provided herein is a polypeptide comprising Formula II’ (SEQ ID NO: 1244):

[0428] X1X2QGTFTSDX 10 SX 12 X 13 LDX 16 X 17 AQX 20 X 21FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X 38 X 39 X40X 41 X 42 X 43 X 44 wherein

[0429] X1is Y or NMeY,

[0430] X2is Aib,

[0431] X 10 is F, 4-Pal or F(4CN),

[0432] X 12 is Orn, K, R, I, Q, Dap or Dab,

[0433] X 13 is aMeL,

[0434] X 16 is K or Orn,

[0435] X 17 is any amino acid having a functional group available for conjugation to a fatty acid,

[0436] X 20 is Aib, aMe-4-Pal, Q or L-Iva,

[0437] X 21 is A, Q or Orn,

[0438] X 24 is E or Q,

[0439] X 25 is W, Y, 4-Pal, aMeY or aMe-4-Pal,

[0440] X 27 is L or I,

[0441] X 28 is E, Q or A,

[0442] X 29 is G, D-Ala or Aib,

[0443] X 30is A, S or G,

[0444] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp,

[0445] X 34 is G or Aib,

[0446] X 35 is A, Aib, E, H or 4-Pal,

[0447] X 36 is P or Hyp,

[0448] X 37 is P or Hyp,

[0449] X 38 is P or Hyp,

[0450] X 39 is E, S, G, T, H, 4-Pal or γE,

[0451] X 40 is absent or is G, E, S or D-Glu,

[0452] wherein if X 40 is G, E, S or D-Glu, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q or H,

[0453] wherein if X 41 is E, S, T, 4-Pal, D, G, Q or H, then X 42 is absent or is G, E or γE,

[0454] wherein if X 42 is G, E or γE, then X 43 is absent or is E,

[0455] wherein if X 43 is E, then X 44 is absent or is E,

[0456] wherein if X 40 is absent, then X 41 , X 42 , X 43 and X 44 are also absent,

[0457] wherein if X 41 is absent, then X 42 , X 43 and X 44 are also absent,

[0458] Where X 43 If X is missing, then 44 Also missing,

[0459] The C-terminal amino acid is optionally amidated;

[0460] Or its pharmaceutically acceptable salt.

[0461] As mentioned before, if X 40 If X is missing, then 41 To X 44 Also missing, and the polypeptide contains a 39-amino acid sequence backbone. If X 41 If X is missing, then 42 To X 44 Also missing, and the polypeptide contains a 40-amino acid sequence backbone. If X 42 If X is missing, then 43 To X 44 Also missing, and the polypeptide contains a 41-amino acid sequence backbone. If X 43 If X is missing, then 44 Also missing, and the polypeptide contains a 42-amino acid sequence backbone. If X 40 To X 44 None are missing (in other words, X) 40 To X 44 (All of them exist), the polypeptide contains a 44-amino acid sequence backbone.

[0462] In one implementation, X 40 Is it G, E, or S, X 41 Missing, and X 42 Missing. In such embodiments, the polypeptide comprises a 40-amino acid sequence. In one embodiment, X 40 Is it G, E, or S, X 41 It is E, S, T, 4-Pal, D, G, Q, or H, and X 42 Missing. In such embodiments, the polypeptide comprises a 41-amino acid sequence. In one embodiment, X 40 Is it G, E, or S, X 41 It is E, S, T, 4-Pal, D, G, Q, or H, and X 42 It is G, E, or γE. In such embodiments, the polypeptide comprises a 42-amino acid sequence backbone.

[0463] In some embodiments of the polypeptide including formula II', position X 17 The amino acid at position X that has a functional group that can be used for conjugation with fatty acids is K, C, E, or D. In some embodiments, position X... 17The amino acid having a functional group available for conjugation with a fatty acid in the context of the polypeptide of Formula II is K, and the conjugation is with the epsilon-amino group of the K side chain.

[0464] In some embodiments of the polypeptide of Formula II, X 10 is F or 4-Pal. In some embodiments, X 10 is F. In some embodiments, X 10 is 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q. In some embodiments, X 12 is Orn. In some embodiments, X 12 is K. In some embodiments, X 12 is R. In some embodiments, X 12 is Q. In some embodiments, X 10 is F or 4-Pal, and X 12 is Orn, K, R, or Q. In some embodiments, X 10 is F, and X 12 is Q. In some embodiments, X 10 is F, and X 12 is Orn. In some embodiments, X 10 is F, and X 12 is K. In some embodiments, X 10 is F, and X 12 is R. In some embodiments, X 10 is 4-Pal, and X 12 is Q. In some embodiments, X 10 is 4-Pal, and X 12 is Orn. In some embodiments, X 10 is 4-Pal, and X 12 is K. In some embodiments, X 10 is 4-Pal, and X 12 is R.

[0465] In further embodiments, X 10 is selected from F or 4-Pal. In some embodiments, X 12 is selected from Orn, K, R, or Q. In some embodiments, X 16 is K. In some embodiments, X 17 is K. In some embodiments, X 20 is selected from Aib or alphaMe-4-pal. In some embodiments, X 24 is E. In some embodiments, X 28 is selected from E or A. In some embodiments, X 31Selected from P, H, S, 4-Pal, T, or E. In some implementations, X 35 Selected from A, Aib, or E. In some implementations, X 36 It is P. In some implementations, X 37 It is P. In some implementations, X 38 It is P. In some implementations, X 39 Selected from E, S, or G. In some implementations, X 40 Selected from G, E, or S. In some implementations, X 41 Selected from E, S, T, 4-Pal or H.

[0466] In some implementation schemes, X 10 Selected from F and 4-Pal. In some implementations, X 12 Selected from Orn, K, R, and Q. In some implementations, X 16 It is K. In some implementations, X 17 It is K. In some implementations, X 20 Selected from Aib and αMe-4-pal. In some implementations, X 24 It is E. In some implementations, X 28 Selected from E and A. In some implementations, X 31 Selected from P, H, S, 4-Pal, T, and E. In some implementations, X 35 Selected from A, Aib, and E. In some implementations, X 36 It is P. In some implementations, X 37 It is P. In some implementations, X 38 It is P. In some implementations, X 39 Selected from E, S, and G. In some implementations, X 40 Selected from G, E, and S. In some implementations, X 41 Selected from E, S, T, 4-Pal and H.

[0467] In some implementation schemes, X 10 Selected from F or 4-Pal;X 12 Is it Orn, K, R, or Q; X 16 It is K; X 17 It is K; X 20 Selected from Aib or αMe-4-pal; X 24 It is E; X 28 Selected from E or A; X 31 Selected from P, H, S, 4-Pal, T, or E; X 35 Selected from A, Aib, or E; X 36 It is P; X 37 It is P; X38 is P; X 39 is selected from E, S, or G; X 40 is selected from G, E, or S; and X 41 is selected from E, S, T, 4-Pal, or H.

[0468] In some embodiments, X 10 is selected from F and 4-Pal; X 12 is selected from Orn, K, R, and Q; X 16 is K; X 17 is K; X 20 is selected from Aib and aMe-4-pal; X 24 is E; X 28 is selected from E and A; X 31 is selected from P, H, S, 4-Pal, T, and E; X 35 is selected from A, Aib, and E; X 36 is P; X 37 is P; X 38 is P; X 39 is selected from E, S, and G; X 40 is selected from G, E, and S; and X 41 is selected from E, S, T, 4-Pal, and H.

[0469] In some embodiments, amino acid X 17 is conjugated to a fatty acid. In some embodiments, amino acid X 17 is optionally conjugated to a fatty acid through a linker between the amino acid and the fatty acid. Thus, in some embodiments, amino acid X 17 is conjugated to a fatty acid through a direct bond between the amino acid and the fatty acid or through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is C 16 -C 22 fatty acid.

[0470] Thus, in some embodiments, X 17 is K and is conjugated to C 16 -C 22 fatty acid through a direct bond between the amino acid and C 16 -C 22 fatty acid through a linker between the amino acid and C 16 -C 22 fatty acid. In one embodiment, X 17 K is conjugated to C 16 -C 22 fatty acid through a linker between the amino acid and C 16 -C 22 fatty acid. In one embodiment, the conjugation is to X 17epsilon-amino group of the K side chain.

[0471] In one embodiment, provided herein is a polypeptide comprising Formula III (SEQ ID NO: 6):

[0472] YX2QGTFTSDX 10 SX 12 X 13 LDX 16 X 17 AQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X 38 X 39 X40X 41 wherein

[0473] X1is Y, NMeY, or H,

[0474] X2is Aib,

[0475] X6is F, aMeF, or aMeF(2F),

[0476] X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4N02), or Y,

[0477] X 11 is S or aMeS,

[0478] X 12 is Orn, K, R, Q, Dap, S, E, or I,

[0479] X 13 is aMeL, I, or L,

[0480] X 16 is K, Orn, A, or E,

[0481] X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q,

[0482] X 19 is A or Q,

[0483] X 20is any amino acid having a functional group available for conjugation with a fatty acid, Aib, Q, R, or aMe-4-pal,

[0484] X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn,

[0485] X 23 is I or V,

[0486] X 24 is any amino acid having a functional group available for conjugation with a fatty acid, E, D-Glu, Q, or N,

[0487] X 25 is W, Y, F, 4-Pal, aMeY, or aMe-4-pal,

[0488] X 27 is L, I, E, V, A, Q, or S,

[0489] X 28 is any amino acid having a functional group available for conjugation with a fatty acid, E, or A,

[0490] X 29 is G, Aib, T, D-Ala, or A,

[0491] X 31 is P or E,

[0492] X 34 is G or Aib,

[0493] X 35 is A or E,

[0494] X 36 is P,

[0495] X 37 is P or E,

[0496] X 38 is P,

[0497] X 39 is E, S, G, or A,

[0498] X 40 is absent or G, E, S, A, or T,

[0499] wherein if X 40 is G, E, S, A, or T, then X 41 is absent or E, S, D, or G,

[0500] wherein if X 41 is E, S, D, or G, then X 42The deficiency could be G, E, or γE.

[0501] Where X 40 If X is missing, then 41 and X 42 Also missing,

[0502] Where X 41 If X is missing, then 42 Also missing, where X 10 If the derivatives are F, 3-Pal, 4-Pal, F(4CN), and F(4NO2), then X 12 It is I, and

[0503] Where X 17 X 20 X 24 or X 28 At least one of them is available for use with C 16 -C 22 Amino acids with functional groups conjugated with fatty acids.

[0504] The C-terminal amino acid is optionally amidated;

[0505] Or its pharmaceutically acceptable salt.

[0506] As mentioned earlier, if X 40 If X is missing, then 41 and X 42 Also missing, and the polypeptide contains a 39-amino acid sequence. If X 41 If X is missing, then 42 Also missing, and the polypeptide contains a 40-amino acid sequence. If X 42 If X is missing, the polypeptide contains a 41-amino acid sequence. 40 X 41 and X 42 None are missing (in other words, X) 40 X 41 and X 42 If all of them are present, then the polypeptide contains a 42-amino acid sequence.

[0507] In one implementation, X 40 It is G, E, S, A, or T, and X 41 Missing. In this embodiment, the polypeptide comprises a 40-amino acid sequence. In one embodiment, X 40 It is G, E, S, A or T, X 41 It is E, S, D, or G. In this embodiment, the polypeptide comprises a 41-amino acid sequence. In one embodiment, X 40 It is G, E, S, A or T, X 41is E, S, D, or G, and X 42 is G, E, or γE. In such embodiments, the polypeptide comprises a 42 amino acid sequence.

[0508] In some embodiments of the polypeptide of Formula III, the amino acid at position X 17 , X 20 , X 24 , or X 28 having a functional group available for conjugation to a fatty acid is K, C, E, or D. In some embodiments, the amino acid at position X 17 , X 20 , X 24 , or X 28 having a functional group available for conjugation to a fatty acid is K, and the conjugation is to the epsilon-amino group of the K side chain.

[0509] In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02), and X 12 is I. In some embodiments of the polypeptide of Formula III, X 10 is Y, and X 12 is Orn, K, R, Q, Dap, S, E, or I.

[0510] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a C 16 -C 22 fatty acid.

[0511] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is optionally conjugated to a fatty acid through a linker between the amino acid and the fatty acid. Thus, in some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a direct bond between the amino acid and the fatty acid or through a linker between the amino acid and the fatty acid. In one embodiment, only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0512] Thus, in one embodiment, X is K, and conjugated to C 17 -C 16 -C 22 -C 16 -C 22 -C 17 -C 16 -C 22 -C 16 -C 22 -C 20 A, I, or Q, X is E, Q, D-Glu, or N, and X is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain. 24 28

[0513] Thus, in one embodiment, X is K, and conjugated to C 20 -C 16 -C 22 -C 16 -C 22 -C 20 -C 16 -C 22 -C 16 A, I, or Q, X is E, Q, D-Glu, or N, and X is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain. 22 17 24 28

[0514] Thus, in one embodiment, X is K, and conjugated to C 24 -C 16 -C 22 -C 16 -C 22 -C 24 -C 16 -C 22 -C 16 -C 22 A, I, or Q, X is E, Q, D-Glu, or N, and X is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain. 17 20 ​​​​​​​is Aib, aMe-4-pal, Q, or R, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0515] In one embodiment, X 28 is K, and conjugated through an amino acid and C 16 -C 22 a direct bond or a linker between the fatty acid and C 16 -C 22 a fatty acid conjugation. In one embodiment, X 28 is K, and conjugated through an amino acid and C 16 -C 22 a direct bond or a linker between the fatty acid and C 16 -C 22 a fatty acid conjugation. In such embodiments, X 17 is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0516] In one embodiment, provided herein is a polypeptide comprising Formula III’ (SEQ ID NO: 1245):

[0517] X1X2QX4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 GX 31 X 32 SX 34 X 35 PX 37 PX 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 wherein

[0518] X1is Y, NMeY, or H,

[0519] X2 is Aib,

[0520] X4 is G or D-Ala,

[0521] X6 is F, aMeF or aMeF(2F),

[0522] X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4N02) or Y,

[0523] X 11 is S or aMeS,

[0524] X 12 is Orn, K, R, Q, Dap, S, E or I,

[0525] X 13 is aMeL, I or L,

[0526] X 15 is D or E,

[0527] X 16 is K, Orn, A or E,

[0528] X 17 is any amino acid with a functional group available for conjugation to a fatty acid, A, I or Q or Orn,

[0529] X 19 is A or Q,

[0530] X 20 is any amino acid with a functional group available for conjugation to a fatty acid, Aib, Q, R or aMe-4-Pal,

[0531] X 21 is A, Aad, Aib, S, N, Q, E, T or Orn,

[0532] X 23 is I or V,

[0533] X 24 is any amino acid with a functional group available for conjugation to a fatty acid, E, D-Glu, Q, N or D-Gln,

[0534] X 25 is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0535] X 27 is L, I, E, V, A, Aad, T, Q or S,

[0536] X 28is any amino acid having a functional group available for conjugation with a fatty acid, E or A,

[0537] X 29 is G, Aib, T, D-Ala or A,

[0538] X 31 is P or E,

[0539] X 32 is S or P,

[0540] X 34 is G or Aib,

[0541] X 35 is A, D or E,

[0542] X 37 is P or E,

[0543] X 39 is E, S, G, A, T or Orn,

[0544] X 40 is absent or G, E, S, A or T,

[0545] wherein if X 40 is G, E, S, A, T or D-Glu, then X 41 is absent or E, S, D, G, Q, T, A, γE or D-Glu,

[0546] wherein if X 41 is E, S, D, G, Q, T, A, γE or D-Glu, then X 42 is absent or G, E, D-Glu or γE,

[0547] wherein if X 42 is G, E, D-Glu or γE, then X 43 is absent or E, γE or D-Glu,

[0548] wherein if X 43 is E, γE or D-Glu, then X 44 is absent or E,

[0549] wherein if X 44 is E, then X 45 is absent or E, wherein if X 45 is E, then X 46 is absent or E,

[0550] wherein if X 40 is absent, then X 41 is absent to X 46 is also absent,

[0551] wherein if X 41 is absent, X 42 is absent, 46 also is absent,

[0552] wherein if X 42 is absent, X 43 is absent, 46 also is absent,

[0553] wherein if X 43 is absent, X 44 is absent, 46 also is absent,

[0554] wherein if X 44 is absent, X 45 and X 46 also is absent,

[0555] wherein if X 45 is absent, X 46 also is absent,

[0556] wherein the polypeptide comprises at least one of: X6is aMeF or aMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02); X 11 is aMeS; X 13 is aMeL; X 24 is D-Glu; and / or X 25 is aMeY,

[0557] wherein if X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02), X 12 is I,

[0558] and wherein at least one of X 17 , X 20 , X 24 , or X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0559] wherein the C-terminal amino acid is optionally amidated;

[0560] or a pharmaceutically acceptable salt thereof.

[0561] As previously described, if X 40 is absent, X 41 is absent, 46 also is absent, and the polypeptide comprises a 39 amino acid sequence backbone. If X 41 is absent, X 42 is absent, 46Also missing, and the polypeptide contains a 40-amino acid sequence backbone. If X 42 If X is missing, the polypeptide contains a 41-amino acid sequence backbone. 43 If X is missing, the polypeptide comprises a 42-amino acid sequence backbone. 44 If X is missing, the polypeptide comprises a 43-amino acid sequence backbone. 45 If X is missing, the polypeptide comprises a 44-amino acid sequence backbone. 46 If X is missing, the polypeptide comprises a 45-amino acid sequence backbone. 40 To X 46 All are missing (in other words, X) 40 To X 46 If all of them are present, then the polypeptide contains a 46-amino acid sequence backbone.

[0562] In one implementation, X 40 It is G, E, S, A, or T, and X 41 Missing. In this embodiment, the polypeptide comprises a 40-amino acid sequence. In one embodiment, X 40 It is G, E, S, A or T, X 41 It is E, S, D, or G. In this embodiment, the polypeptide comprises a 41-amino acid sequence. In one embodiment, X 40 It is G, E, S, A or T, X 41 It is E, S, D or G, and X 42 It is G, E, or γE. In this embodiment, the polypeptide comprises a 42-amino acid sequence.

[0563] In some embodiments of the polypeptide of formula III', position X 17 X 20 X 24 or X 28 The amino acid at position X that has a functional group that can be used for conjugation with fatty acids is K, C, E, or D. In some embodiments, position X... 17 X 20 X 24 or X 28 The amino acid at the site that has a functional group that can be used for conjugation with fatty acids is K, and the conjugation is an ε-amino conjugation with the side chain of K.

[0564] In some implementation schemes, X 10 It is F, 3-Pal, 4-Pal, F(4CN) or F(4NO2), and X 12 It is I. In some embodiments of the polypeptide of formula III, X 10 It is Y, and X 12is Orn, K, R, Q, Dap, S, E, or I.

[0565] In some embodiments, the polypeptide comprises at least three of: X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02); X 11 is aMeS; X 13 is aMeL; X 16 is Orn; X 24 is D-Glu; and / or X 25 is aMeY.

[0566] In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02).

[0567] In some embodiments, X1is Y, X2is Aib, X4is G, X6is aMeF(2F), X 10 is 4-Pal, X 12 is I, X 13 is aMeL, X 15 is D, X 16 is Orn, X 19 is Q, X 20 is aMe-4-Pal, X 21 is E or Orn, X 23 is I, X 24 is D-Glu, X 25 is aMeY, X 27 is I or V, X 28 is E, X 29 is G, X 31 is P, X 34 is G, X 35 is A or E, X 37 is P, X 39 is E or S, X 40 is G or T, X 41 is E, S or G, X 42 is absent, X 43 is absent, and X 44 is absent.

[0568] In some embodiments, X 11 is S, X 21 is Orn, X 27 is I, X 35 is E, X 39 is E, X 40 is T, and X 41 is E.

[0569] In some embodiments, X 11 is a MeS, X 21 is E, X 27 is V, X 35 is A, X 39 is S, X 40 is G, and X 41 is S.

[0570] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a C 16 -C 22 fatty acid.

[0571] In some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is optionally conjugated to a fatty acid through a linker between the amino acid and the fatty acid. Thus, in some embodiments, only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a direct bond between the amino acid and the fatty acid or through a linker between the amino acid and the fatty acid. In one embodiment, only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is a C 16 -C 22 fatty acid.

[0572] Thus, in one embodiment, X 17 is K and is conjugated to a C 16 -C 22 fatty acid through a direct bond between the amino acid and the C 16 -C 22 fatty acid or through a linker. In one embodiment, X 17 is K and is conjugated to a C 16 -C 22 fatty acid through a linker between the amino acid and the C 16 -C 22 fatty acid. In such embodiments, X 20 is Aib, a Me-4-pal, Q, or R, X 24 is E, Q, D-Glu, or N, and X 28is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0573] In one embodiment, X 20 is K, and is conjugated to C 16 -C 22 through a direct bond or a linker between the amino acid and C 16 -C 22 a fatty acid. In one embodiment, X 20 is K, and is conjugated to C 16 -C 22 through a linker between the amino acid and C 16 -C 22 a fatty acid. In such embodiments, X 17 is A, I, or Q, X 24 is E, Q, D-Glu, or N, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0574] In one embodiment, X 24 is K, and is conjugated to C 16 -C 22 through a direct bond or a linker between the amino acid and C 16 -C 22 a fatty acid. In one embodiment, X 24 is K, and is conjugated to C 16 -C 22 through a linker between the amino acid and C 16 -C 22 a fatty acid. In such embodiments, X 17 is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 28 is E or A. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain.

[0575] In one embodiment, X 28 is K, and is conjugated to C 16 -C 22 through a direct bond or a linker between the amino acid and C 16 -C 22 a fatty acid. In one embodiment, X 28 is K, and is conjugated to C 16 -C 22 through a linker between the amino acid and C 16 -C 22 a fatty acid. In such embodiments, X 17is A, I, or Q, X 20 is Aib, aMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, the conjugation is to the epsilon-amino group of the side chain of K.

[0576] The amino acid sequences of the polypeptides described herein include naturally occurring amino acids, typically described herein using the standard one-letter code (e.g., L = leucine), as well as residues of alpha-methyl substitution of natural amino acids (e.g., alpha-methyl leucine (aMeL)), and certain other non-natural amino acids (such as alpha aminoisobutyric acid (Aib)). The structures of these amino acids are shown below:

[0577]

[0578]

[0579] As used herein, “Orn” means L-ornithine. As used herein, “4-Pal” or “4Pal” means 3-(4-pyridyl)-L-alanine or (S)-2-amino-3-(pyridin-4-yl)propanoic acid. As used herein, “3-Pal” or “3Pal” means 3-(3-pyridyl)-L-alanine or (S)-2-amino-3-(pyridin-3-yl)propanoic acid. As used herein, “aMe-4-Pal” or “aMe4Pal” means alpha-methyl-3-(4-pyridyl)-L-alanine. As used herein, “aMeY” means alpha-methyl-L-tyrosine. As used herein, “aMeL” means alpha-methyl-leucine. As used herein, “D-Ala” and “a” each mean D-alanine. As used herein, “D-Glu” and “e” each mean D-glutamic acid. As used herein, “Aib” means 2-aminoisobutyric acid. As used herein, “NMeY” means N-methyl-tyrosine. As used herein, “Dap” means (S)-2,3-diaminopropionic acid. As used herein, “Dab” means (S)-2,4-diaminobutyric acid. As used herein, “Hyp” means hydroxy-L-proline. As used herein, “K(Ac)” means N 6- acetyl-L-lysine. As used herein, "yGlu" refers to y-L-glutamic acid. As used herein, "Aad" refers to (S)-2-aminoadipic acid. As used herein, "F(4CN)" refers to 4-cyano-L-phenylalanine or (S)-2-amino-3-(4-cyanophenyl)propanoic acid. As used herein, "F(4N02)" refers to 4-nitro-L-phenylalanine or (S)-2-amino-3-(4-nitrophenyl)propanoic acid. As used herein, "aMeS" refers to a-methyl-L-serine. As used herein, "aMeF" refers to a-methyl-L-phenylalanine. As used herein, "aMeF(2F)" refers to a-methyl-2-fluoro-L-phenylalanine or (S)-2-amino-3-(2-fluorophenyl)-2-methylpropanoic acid. As used herein, "L-Iva" and "Iva" refer to L-isovaline. As used herein, "D-Gln" and "q" each refer to D-glutamine.

[0580] As previously described, in some embodiments, the polypeptides described herein include a fatty acid moiety conjugated to a natural or non-natural amino acid having a functional group available for conjugation, for example, by a direct bond or a linker. Such conjugation is sometimes referred to as acylation. In certain instances, the amino acid having a functional group available for conjugation can be K, C, E, and D. In particular instances, the amino acid having a functional group available for conjugation is K, wherein the conjugation is to the epsilon-amino group of the K side chain.

[0581] Acylation of the polypeptides described herein is at position X 17 , X 20 , X 24 or X 28 in SEQ ID NO: 4 or 6, or position X 17 in SEQ ID NO: 5. The fatty acid, and in certain embodiments, the linker and / or amino acid sequence backbone, can act as an albumin binder and provide the potential to create a long-acting compound.

[0582] In some embodiments, the polypeptides described herein utilize C 16 -C 22 The fatty acid is chemically conjugated to the amino acid functional group by a direct bond or by a linker. The length and composition of the fatty acid affects the half-life of the polypeptide, its efficacy in in vivo animal models, and its solubility and stability. Conjugation with C 16 -C 22 Saturated fatty mono- or di-acids produce polypeptides with desirable half-life, desirable efficacy in in vivo animal models, and desirable solubility and stability characteristics.

[0583] For use herein, saturated C 16 -C 22Examples of fatty acids include, but are not limited to, palmitic acid (hexadecanoic acid) (C 16 monoacid), hexadecanedioic acid (C 16 diacid ) , margaric acid (heptadecanoic acid) (C 17 monoacid), heptadecanedioic acid (C 17 diacid), stearic acid (octadecanoic acid) (C 18 monoacid), octadecanedioic acid (C 18 diacid), nonadecanoic acid (nonadecanoic acid) (C 19 monoacid), nonadecanedioic acid (C 19 diacid), arachidic acid (eicosanoic acid) (C 20 monoacid), eicosanedioic acid (C 20 diacid), heneicosanoic acid (heneicosanoic acid) (C 21 monoacid), heneicosanedioic acid (C 21 diacid), behenic acid (docosanoic acid) (C 22 monoacid), docosanedioic acid (C 22 diacid), including branched and substituted derivatives thereof.

[0584] In certain instances, the C 16 -C 22 fatty acid can be a saturated C 18 monoacid, a saturated C 18 diacid, a saturated C 19 monoacid, a saturated C 19 diacid, a saturated C 20 monoacid, a saturated C 20 diacid, and branched and substituted derivatives thereof. In more particular instances, the C 16 -C 22 fatty acid can be octadecanedioic acid (C 18 diacid) or eicosanedioic acid (C 20 diacid).

[0585] In certain instances, the linker can have one or more (2-[2-(2-amino- ethoxy)-ethoxy]-acetyl) moieties or εK, optionally in combination with one to four amino acids.

[0586] Where the linker includes at least one amino acid, the amino acid can be one to five Glu or γGlu amino acid residues. In certain instances, the linker can include one or two or three or four or five Glu or γGlu amino acid residues, including the D form thereof. For example, the linker can include one or two or three or four γGlu amino acid residues. Alternatively, the linker can include one to five amino acid residues (e.g., Glu or γGlu amino acids) in combination with one to five (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ("AEEA") or one to five εK moieties. Specifically, the linker can be one to five Glu or γGlu amino acids in combination with one to five (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties or one to five Glu or γGlu amino acids in combination with one to five εK moieties. In some instances, the linker can be one or two or three γGlu amino acids in combination with one or two (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) or εK moieties. ε K moieties. In some instances, the linker can be one or two or three γGlu amino acids in combination with one or two (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) or εK moieties.

[0587] For example, in some embodiments, the polypeptides described herein have a linker and a fatty acid component of the following formula:

[0588] (γGlu) a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2) p -CO2H, where a is 0, 1, or 2; b is 0, 1, or 2; c is 0, 1, 2, or 3; and p is an integer between 14 and 20.

[0589] In some preferred embodiments, a is 0 or 1; b is 0, 1, or 2; c is 1, 2, or 3; and p is an integer between 14 and 20.

[0590] In some embodiments, a is 0, b is 1, c is 1 or 2, and p is 16 or 18.

[0591] For example, in some embodiments, a is 0, b is 1, c is 1, and p is 16, which has the following structure:

[0592]

[0593] For example, in some embodiments, a is 0, b is 1, c is 1, and p is 18, which has the following structure:

[0594]

[0595] In some embodiments, a is 0, b is 1, c is 2, and p is 16, which structure is shown below.

[0596]

[0597] In some embodiments, a is 0, b is 1, c is 2, and p is 18, which structure is shown below.

[0598]

[0599] In some embodiments, a is 0, b is 2, c is 1, and p is 16 or 18.

[0600] For example, in some embodiments, a is 0, b is 2, c is 1, and p is 16, which structure is shown below.

[0601]

[0602] In some embodiments, a is 0, b is 2, c is 1, and p is 18, which structure is shown below.

[0603]

[0604] In some embodiments, a is 0, b is 0, c is 2, and p is 16 or 18.

[0605] For example, in some embodiments, a is 0, b is 0, c is 2, and p is 16, which structure is shown below.

[0606]

[0607] In some embodiments, a is 0, b is 0, c is 2, and p is 18, which structure is shown below.

[0608]

[0609] In some embodiments, a is 0, b is 0, c is 3, and p is 16 or 18.

[0610] For example, in some embodiments, a is 0, b is 0, c is 3, and p is 16, which structure is shown below.

[0611]

[0612] In some embodiments, a is 0, b is 0, c is 3, and p is 18, which structure is shown below.

[0613]

[0614] In some embodiments, a is 1, b is 1, c is 1, and p is 16 or 18.

[0615] For example, in some embodiments, a is 1, b is 1, c is 1, and p is 16, which has the structure shown below.

[0616]

[0617] For example, in some embodiments, a is 1, b is 1, c is 1, and p is 18, which has the structure shown below.

[0618]

[0619] In some embodiments, the polypeptides described herein have a linker and fatty acid component of the following formula structure:

[0620] (γGlu) d -(εK) e -(γGlu) f -CO-(CH2) q -CO2H, where d is 0, 1, or 2; e is 0, 1, or 2; f is 0, 1, 2, or 3; and q is an integer between 14 and 20.

[0621] For example, in one embodiment, d is 0; e is 2; f is 1; and q is an integer between 14 and 20. In some embodiments, d is 0; e is 2; f is 1; and q is 16 or 18.

[0622] For example, in some embodiments, d is 0; e is 2; f is 1; and q is 16, which has the structure shown below.

[0623]

[0624] For example, in some embodiments, d is 0; e is 2; f is 1; and q is 18, which has the structure shown below.

[0625]

[0626] As shown in the chemical structures of Examples 1-1229 below, the linker-fatty acid moiety described above can be attached to an amino acid present at position 17, 20, 24, or 28. In some embodiments, the linker-fatty acid moiety described above is attached or conjugated to an amino acid present at position 17, e.g., to the ε-amino group of the side chain of a lysine (K) present at position 17. In some embodiments, the linker-fatty acid moiety described above is attached or conjugated to an amino acid present at position 20, e.g., to the ε-amino group of the side chain of a lysine (K) present at position 20. In some embodiments, the linker-fatty acid moiety described above is attached or conjugated to an amino acid present at position 24, e.g., to the ε-amino group of the side chain of a lysine (K) present at position 24. In some embodiments, the linker-fatty acid moiety described above is attached or conjugated to an amino acid present at position 28, e.g., to the ε-amino group of the side chain of a lysine (K) present at position 28.

[0627] In some embodiments, the polypeptides described herein comprise a sequence selected from any one of SEQ ID NOs: 7-1242 (as described in Examples 1-1236 below). In some embodiments, the polypeptides described herein consist of a sequence selected from any one of SEQ ID NOs: 7-1242 (as described in Examples 1-1236 below).

[0628] In some embodiments, the polypeptides described herein are amidated. In some embodiments, the polypeptides described herein have a modification of the C-terminal group, wherein the modification is NH2or is absent. In some embodiments, the polypeptides described herein have an OH group at the C-terminus.

[0629] In addition to the sequences described herein, the polypeptides described herein can include one or more conservative amino acid substitutions, provided that the polypeptide is still capable of binding and activating GIP, GLP-1, and glucagon receptors.

[0630] In certain embodiments of any of the polypeptides of the general formulae described herein, the polypeptide is an isotopic derivative of any one of the polypeptides described herein, or a pharmaceutically acceptable salt thereof. It will be appreciated that the isotopic derivatives can be prepared using a variety of art-recognized techniques. For example, isotopic derivatives can generally be prepared by carrying out the methods disclosed in the examples described herein using isotopically-labeled reagents in place of non-isotopically-labeled reagents. In embodiments of any of the polypeptides of the general formulae described herein, or a pharmaceutically acceptable salt thereof, the polypeptide is a deuterated derivative of any one of the polypeptides described herein.

[0631] In the polypeptides of the application, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise indicated, when an atom is specifically designated as "H" or "hydrogen," it is understood to have its natural abundance isotopic composition of hydrogen. Further, unless otherwise indicated, when an atom is specifically designated as "D" or "deuterium," it is understood to have a deuterium abundance significantly higher than the natural abundance of deuterium (0.015%).

[0632] The affinities of the polypeptides described herein for the GIP, GLP-1, and glucagon receptors can be measured using techniques known in the art to measure levels of receptor binding, typically expressed as an inhibition constant (Ki) value. The activities of the polypeptides described herein at each of the receptors can also be measured using techniques known in the art (e.g., the in vitro activity assays described below), typically expressed as an effective concentration 50 (EC 50 ) value, which is the concentration of the compound that elicits a half-maximal stimulatory effect in a dose response curve.

[0633] The polypeptides described herein can be reacted with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts, and common techniques for preparing them are well known in the art (see, e.g., Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2 nd Revised Edition (Wiley-VCH, 2011)). As used herein, pharmaceutically acceptable salts include sodium, potassium, trifluoroacetate, hydrochloride, and / or acetate salts. Accordingly, in some embodiments, provided herein are pharmaceutically acceptable salt forms of the GGG polypeptides. In some embodiments, the pharmaceutically acceptable form is selected from a sodium salt or a potassium salt. In some embodiments, the pharmaceutically acceptable form is selected from a sodium salt, a potassium salt. In some preferred embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0634] The polypeptides described herein are suitable for administration by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal) or oral routes (e.g., tablets, capsules). In some preferred embodiments, the polypeptides described herein are suitable for oral administration. The in vitro permeability (P app ) assays and in vivo ileal absorption assays are useful tools for assessing the potential of a polypeptide for oral delivery.

[0635] In another embodiment, provided herein are pharmaceutical compositions comprising a polypeptide described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. Some pharmaceutical compositions and techniques for their preparation are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (Troy, Ed., 21st edition, Lippincott, Williams & Wilkins, 2006). st

[0636] In some embodiments, the pharmaceutical composition is suitable for administration by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). In some embodiments, the pharmaceutical composition is administered orally (e.g., tablet, capsule). In some embodiments, the pharmaceutical composition is administered parenterally. In some embodiments, the pharmaceutical composition is administered orally.

[0637] In addition to the anatomical and physiological features of the gastrointestinal tract, the physicochemical properties of the polypeptide can pose a challenge to the effective oral delivery of the peptide. In one embodiment, the pharmaceutical composition for oral administration comprises a polypeptide described herein, or a pharmaceutically acceptable salt thereof, and a penetration enhancer. In one embodiment, the pharmaceutical composition for oral administration comprises a polypeptide described herein, or a pharmaceutically acceptable salt thereof, a penetration enhancer, and a protease inhibitor.

[0638] As used herein, the term “penetration enhancer” refers to a penetration enhancer that enhances the oral absorption of the polypeptide of the application. As used herein, penetration enhancer refers to penetration enhancers such as sodium caprate (C10), sodium taurodeoxycholate (NaTDC), lauroyl carnitine (LC), dodecyl maltoside, dodecyl phosphatidyl choline, SNAC, rhamnolipid (Thamnolipid), and penetration enhancers reported in the literature such as PIP-250, PIP-640, phosphatase penetration inhibitor. See Pharmaceutics. 2019 Jan; 11(1): 41, (see Biomaterials. 2012; 33: 3464-3474), ZOT (zonula occludens toxin), AG (ZOT fragment) (see Int. J. Pharm. 2009; 365, 121-130). In one embodiment, the penetration enhancer is selected from the group consisting of sodium caprate, sodium taurodeoxycholate, and lauroyl carnitine. In one embodiment, the penetration enhancer is selected from the group consisting of C10, LC, or NaTDC. In one embodiment, the penetration enhancer is selected from the group consisting of sodium caprate, sodium taurodeoxycholate, and lauroyl carnitine. In one embodiment, the penetration enhancer is selected from the group consisting of C10, LC, and NaTDC.​

[0639] As used herein, the term "protease inhibitor" refers to a protease inhibitor that can be selected from the group consisting of proteins, peptides, and small molecules. Protease inhibitors are well known and can include, for example, soybean trypsin inhibitor ("SBTI"), soybean trypsin-chymotrypsin inhibitor ("SBTCI"), ecotin, sunflower trypsin inhibitor ("SFTI"), leupeptin, citric acid, ethylenediaminetetraacetic acid ("EDTA"), sodium glycocholate, and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride ("AEBSF"). In one embodiment, the protease inhibitor is selected from the group consisting of SBTI, SBTCI, and SFTI. In one embodiment, the protease inhibitor is SBTI.

[0640] The present disclosure also provides and thus contemplates novel intermediates and methods of synthesizing the polypeptides described herein, or pharmaceutically acceptable salts thereof. The intermediates and polypeptides described herein can be prepared by a variety of techniques known in the art. For example, one chemical synthesis method, or one biological expression method, is set forth in the Examples below. The specific synthesis steps of each approach can be combined in different ways to prepare the polypeptides described herein. Reagents and starting materials are readily available to one of skill in the art.

[0641] With respect to chemical synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are available from commercial sources such as CEM (Charlotte, North Carolina), Csbio (Menlo Park, California), and Gyros Protein Technologies Inc. (Tucson, AZ). Solid-phase synthesis reagents are readily available from commercial sources. Solid-phase synthesis instruments can be used according to the manufacturer's instructions to block interfering groups, protect amino acids during reactions, perform coupling, deprotection, and capping of unreacted amino acids.

[0642] With respect to biological expression, a polynucleotide having a nucleic acid sequence encoding all or part of the amino acid sequence of a polypeptide can be constructed using standard recombinant techniques, integrated into a recombinant expression vector, and the vector introduced into a host cell (e.g., bacterial, yeast, and mammalian cells) to produce the polypeptide. See, e.g., Green & Sambrook, “Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, 4th ed. 2012). Polypeptides can be readily produced in mammalian cells (e.g., CHO, NSO, 20HEK293, BHK, or COS cells); bacterial cells (e.g., E. coli, Bacillus subtilis, or Pseudomonas fluorescens); insect cells; fungal or yeast cells (grown using techniques known in the art). Vectors containing the polynucleotide sequence of interest can be transferred into the host cell by well-known methods, which depend on the type of cellular host. A variety of protein purification methods can be employed and such methods are known in the art.

[0643] The polypeptides described herein can be used to treat a variety of conditions, disorders, diseases, or symptoms. In particular, methods for treating obesity in an individual are provided, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0644] Further, methods for long-term weight management in an individual are provided, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0645] Further, methods of treating type 2 diabetes (T2DM) in an individual are provided, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0646] Further, methods of treating nonalcoholic fatty liver disease (NAFLD) in an individual are provided, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0647] Further, methods of treating nonalcoholic steatohepatitis (NASH) in an individual are provided, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0648] Further provided are methods of treating dyslipidemia in an individual, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0649] Further provided are methods of treating metabolic syndrome in an individual, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0650] Further provided are methods of treating osteoarthritis (OA) in an individual, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0651] Further provided are methods of treating obesity-related sleep apnea (OSA) in an individual, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0652] Further provided are methods of treating polycystic ovary syndrome (PCOS) in an individual, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0653] Further provided are methods of inducing non-therapeutic weight loss in an individual, wherein such methods comprise at least one step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein, or a pharmaceutically acceptable salt thereof.

[0654] In these methods, the effectiveness of the polypeptide can be assessed by, for example, observing a significant decrease in blood glucose, observing a significant increase in insulin, observing a significant decrease in HbAlc, and / or observing a significant decrease in body weight.

[0655] Alternatively, the polypeptides described herein, or a pharmaceutically acceptable salt thereof, can be used to improve bone strength in an individual in need thereof. In certain cases, the individual in need is suffering from hypo-ostosis or hypo-osteoidosis, or is recovering from a bone fracture, orthopedic surgery, prosthetic implantation, tooth implantation, and / or spinal fusion. The polypeptides described herein can also be used to treat other disorders, such as Parkinson's disease or Alzheimer's disease.

[0656] Further provided are polypeptides described herein, or pharmaceutically acceptable salts thereof, for use in therapy. In some embodiments, provided herein are polypeptides described herein, or pharmaceutically acceptable salts thereof, for use in the treatment of obesity, long-term weight management, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS). Also provided are uses of polypeptides described herein, or pharmaceutically acceptable salts thereof, for inducing non-therapeutic weight loss.

[0657] Further provided are uses of polypeptides described herein, or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for the treatment of obesity, long-term weight management, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS). Also provided are uses of polypeptides described herein, or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inducing non-therapeutic weight loss.

[0658] A polypeptide or pharmaceutical composition described herein can be provided as part of a kit. In certain instances, the kit comprises a device for administering at least one polypeptide (and optionally at least one additional therapeutic agent) to an individual, such as a syringe, auto-injector, or pump.

[0659] Additional non-limiting embodiments are as follows:

[0660] 1. A polypeptide comprising:

[0661] X1X2QGTX6TSDX 10 X 11 X 12 X 13 LDX 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X38X 39 X 40 X 41 X 42 , wherein:

[0662] X1is Y, NMeY or H,

[0663] X2is Aib,

[0664] X6is F, aMeF or aMeF(2F),

[0665] X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4N02) or Y,

[0666] X 11 is S or aMeS,

[0667] X 12 is Orn, K, R, Q, Dap, Dab, S, E or I,

[0668] X 13 is aMeL, I or L,

[0669] X 16 is K, Orn, A or E,

[0670] X 17 is any amino acid with a functional group available for conjugation to a fatty acid, A, I or Q,

[0671] X 19 is Q or A,

[0672] X 20 is any amino acid with a functional group available for conjugation to a fatty acid, Aib, aMe-4-Pal, Q or R,

[0673] X 21 is A, Q, Orn, Aad, Aib, S, N, E or T,

[0674] X 23 is I or V,

[0675] X 24 is any amino acid with a functional group available for conjugation to a fatty acid, E, Q, D-Glu or N,

[0676] X 25 is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0677] X 27 is L, I, E, V, A, Q or S,

[0678] X 28 is any amino acid with a functional group available for conjugation to a fatty acid, E or A,

[0679] X29 is G, D-Ala, Aib, T or A,

[0680] X 30 is A or G,

[0681] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp,

[0682] X 34 is G or Aib,

[0683] X 35 is A, Aib, E, H or 4-Pal,

[0684] X 36 is P or Hyp,

[0685] X 37 is P, Hyp or E,

[0686] X 38 is P or Hyp,

[0687] X 39 is E, S, G, T, H, 4-Pal, yE or A,

[0688] X 40 is absent or is G, E, S, A or T,

[0689] wherein if X 40 is G, E, S, A or T, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q or H, wherein if X 41 is E, S, T, 4-Pal, D, G, Q or H, then X 42 is absent or is G, E or yE,

[0690] wherein if X 40 is absent, then X 41 and X 42 are also absent,

[0691] wherein if X 41 is absent, then X 42 is also absent, wherein at least one of X 17 , X 20 , X 24 and X 28 is an amino acid having a functional group available for conjugation to a fatty acid,

[0692] wherein the C-terminal amino acid is optionally amidated;

[0693] or a pharmaceutically acceptable salt thereof.

[0694] 2. The polypeptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein

[0695] X 40 is G, E, S, A, or T,

[0696] X 41 is absent, and

[0697] X 42 is absent.

[0698] 3. The polypeptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein

[0699] X 40 is G, E, S, A, or T,

[0700] X 41 is E, S, T, 4-Pal, D, G, Q, or H, and

[0701] X 42 is absent.

[0702] 4. The polypeptide of embodiment 1, wherein

[0703] X 40 is G, E, S, A, or T,

[0704] X 41 is E, S, T, 4-Pal, D, G, Q, or H, and

[0705] X 42 is G, E, or γE.

[0706] 5. The polypeptide of any one of embodiments 1 to 4, wherein the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K, C, E, or D.

[0707] 6. The polypeptide of embodiment 5, wherein the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K.

[0708] 7. The polypeptide of any one of embodiments 1 to 6, or a pharmaceutically acceptable salt thereof, wherein X 10 is F or 4-Pal.

[0709] 8. The polypeptide of any one of embodiments 1 to 7, or a pharmaceutically acceptable salt thereof, wherein X 12 is Orn, K, R, or Q.

[0710] 9. The polypeptide of any one of embodiments 1 to 8, or a pharmaceutically acceptable salt thereof, wherein only one of X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid.

[0711] 10. The polypeptide of any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof, wherein only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a direct bond or a linker between the amino acid and the fatty acid.

[0712] 11. The polypeptide of embodiment 10, or a pharmaceutically acceptable salt thereof, wherein only one of X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid through a linker between the amino acid and the fatty acid.

[0713] 12. The polypeptide of any one of embodiments 1 to 11, wherein the fatty acid is a C 16 -C 22 fatty acid.

[0714] 13. The polypeptide of embodiment 12, or a pharmaceutically acceptable salt thereof, wherein

[0715] X 17 is K and is conjugated to the C 16 -C 22 fatty acid through a direct bond or a linker between the amino acid and the C 16 -C 22 fatty acid,

[0716] X 20 is Aib, aMe-4-Pal, Q, or R,

[0717] X 24 is E, Q, D-Glu, or N, and

[0718] X 28 is E or A.

[0719] 14. The polypeptide of embodiment 12, or a pharmaceutically acceptable salt thereof, wherein

[0720] X 17 is A, I, or Q,

[0721] X 20 is K and is conjugated to the C 16 -C 22 fatty acid through a direct bond or a linker between the amino acid and the C 16 -C 22 fatty acid.16 - C 22 a direct bond or linker between the fatty acids and C 16 - C 22 the fatty acid is conjugated to,

[0722] X 24 is E, Q, D-Glu, or N, and

[0723] X 28 is E or A.

[0724] 15. The polypeptide of embodiment 12, or a pharmaceutically acceptable salt thereof, wherein

[0725] X 17 is A, I, or Q,

[0726] X 20 is Aib, aMe-4-Pal, Q, or R,

[0727] X 24 is K, and is conjugated to, through an amino acid and C 16 - C 22 a direct bond or linker between the fatty acids and C 16 - C 22 the fatty acid is conjugated to, and X 28 is E or A.

[0728] 16. The polypeptide of embodiment 12, or a pharmaceutically acceptable salt thereof, wherein

[0729] X 17 is A, I, or Q,

[0730] X 20 is Aib, aMe-4-Pal, Q, or R,

[0731] X 24 is E, Q, D-Glu, or N, and

[0732] X 28 is K, and is conjugated to, through an amino acid and C 16 - C 22 a direct bond or linker between the fatty acids and C 16 - C 22 the fatty acid is conjugated.

[0733] 17. The polypeptide of embodiment 1, comprising:

[0734] YX2QGTFTSDX 10 SX 12 X 13 LDX 16 X 17 AQX 20 X 21FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 SSX 34 X 35 X 36 X 37 X 38 X 39 X40X 41 X 42 wherein

[0735] X2is Aib,

[0736] X 10 is F, 4-Pal or F(4CN),

[0737] X 12 is Orn, K, R, Q, Dap or Dab,

[0738] X 13 is aMeL,

[0739] X 16 is K or Orn,

[0740] X 17 is any amino acid having a functional group available for conjugation to a fatty acid,

[0741] X 20 is Aib, aMe-4-Pal or Q,

[0742] X 21 is A, Q or Orn,

[0743] X 24 is E or Q,

[0744] X 25 is W, Y, 4-Pal, aMeY or aMe-4-Pal,

[0745] X 27 is L or I,

[0746] X 28 is E or A,

[0747] X 29 is G, D-Ala or Aib,

[0748] X 30 is A or G,

[0749] X 31 is P, H, S, 4-Pal, E, T, K(Ac) or Hyp,

[0750] X 34 is G or Aib,

[0751] X 35 is A, Aib, E, H or 4-Pal,

[0752] X 36 is P or Hyp,

[0753] X 37 is P or Hyp,

[0754] X 38 is P or Hyp,

[0755] X 39 is E, S, G, T, H, 4-Pal or γΕ,

[0756] X 40 is absent or is G, E or S,

[0757] wherein if X 40 is G, E or S, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q or H,

[0758] wherein if X 41 is E, S, T, 4-Pal, D, G, Q or H, then X 42 is absent or is G, E or γΕ,

[0759] wherein the C-terminal amino acid is optionally amidated;

[0760] or a pharmaceutically acceptable salt thereof.

[0761] 18. The polypeptide of embodiment 17, or a pharmaceutically acceptable salt thereof, wherein

[0762] X 40 is G, E or S,

[0763] X 41 is absent, and

[0764] X 42 is absent.

[0765] 19. The polypeptide of embodiment 17, or a pharmaceutically acceptable salt thereof, wherein

[0766] X 40 is G, E or S,

[0767] X 41 is E, S, T, 4-Pal, D, G, Q or H, and

[0768] X 42Deletion.

[0769] 20. The polypeptide of embodiment 17, or a pharmaceutically acceptable salt thereof, wherein X

[0770] X 40 is G, E, or S,

[0771] X 41 is E, S, T, 4-Pal, D, G, Q, or H, and

[0772] X 42 is G, E, or γE.

[0773] 21. The polypeptide of any one of embodiments 17 to 20, wherein X 17 is K, C, E, or D.

[0774] 22. The polypeptide of embodiment 21, wherein X 17 is K.

[0775] 23. The polypeptide of any one of embodiments 17 to 22, or a pharmaceutically acceptable salt thereof, wherein X 10 is F or 4-Pal.

[0776] 24. The polypeptide of any one of embodiments 17 to 23, or a pharmaceutically acceptable salt thereof, wherein X 12 is Orn, K, R, or Q.

[0777] 25. The polypeptide of any one of embodiments 17 to 24, or a pharmaceutically acceptable salt thereof, wherein X 17 is K, and is conjugated to a fatty acid through a direct bond or a linker between the amino acid and the fatty acid.

[0778] 26. The polypeptide of embodiment 25, or a pharmaceutically acceptable salt thereof, wherein X 17 is K, and is conjugated to a fatty acid through a linker between the amino acid and the fatty acid.

[0779] 27. The polypeptide of any one of embodiments 17 to 26, wherein the fatty acid is C 16 -C 22 fatty acid.

[0780] 28. The polypeptide of embodiment 27, or a pharmaceutically acceptable salt thereof, wherein X 17 is K, and is conjugated to a C 16 -C 22 fatty acid through a direct bond or a linker between the amino acid and the C 16 -C 22 fatty acid.

[0781] 29. The polypeptide of embodiment 1, comprising:

[0782] X1X2QGTX6TSDX 10 X 11 X 12 X 13 LDX 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 GX 31 SSX 34 X 35 X 36 X 37 X38X 39 X 40 X 41 ,

[0783] wherein

[0784] X1is Y, NMeY or H,

[0785] X2is Aib,

[0786] X6is F, aMeF or aMeF(2F),

[0787] X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4N02) or Y,

[0788] X 11 is S or aMeS,

[0789] X 12 is Orn, K, R, Q, Dap, S, E or I,

[0790] X 13 is aMeL, I or L,

[0791] X 16 is K, Orn, A or E,

[0792] X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I or Q,

[0793] X 19 is A or Q,

[0794] X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, Q, R or aMe-4-Pal,

[0795] X 21 is A, Aad, Aib, S, N, Q, E, T or Orn,

[0796] X 23 is I or V,

[0797] X 24 is any amino acid with a functional group available for conjugation to a fatty acid, E, D-Glu, Q or N,

[0798] X 25 is W, Y, F, 4-Pal, aMeY or aMe-4-Pal,

[0799] X 27 is L, I, E, V, A, Q or S,

[0800] X 28 is any amino acid with a functional group available for conjugation to a fatty acid, E or A,

[0801] X 29 is G, Aib, T, D-Ala or A,

[0802] X 31 is P or E,

[0803] X 34 is G or Aib,

[0804] X 35 is A or E,

[0805] X 36 is P,

[0806] X 37 is P or E,

[0807] X 38 is P,

[0808] X 39 is E, S, G or A,

[0809] X 40 is absent or is G, E, S, A or T,

[0810] wherein if X 40 is G, E, S, A or T, X 41 is absent or is E, S, D or G,

[0811] wherein if X 41 is E, S, D or G, X 42 is absent or is G, E or γE,

[0812] wherein if X 10is F, 3-Pal, 4-Pal, F(4CN), F(4N02), then X 12 is I,

[0813] wherein X 17 , X 20 , X 24 or X 28 at least one of X

[0814] is optionally amidated;

[0815] or a pharmaceutically acceptable salt thereof.

[0816] 30. The polypeptide of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein:

[0817] X 40 is G, E, S, A, or T,

[0818] X 41 is absent, and

[0819] X 42 is absent.

[0820] 31. The polypeptide of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein:

[0821] X 40 is G, E, S, A, or T,

[0822] X 41 is E, S, D, or G, and

[0823] X 42 is absent.

[0824] 32. The polypeptide of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein:

[0825] X 40 is G, E, S, A, or T,

[0826] X 41 is E, S, D, or G, and

[0827] X 42 is G, E, or γE.

[0828] 33. The polypeptide of any one of embodiments 29 to 32, wherein the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 or X 28 is K, C, E, or D.

[0829] 34. The polypeptide of embodiment 5, wherein the amino acid having a functional group available for conjugation to a fatty acid at position X 17 , X 20 , X 24 , or X 28 is K.

[0830] 35. The polypeptide of any one of embodiments 29 to 34, or a pharmaceutically acceptable salt thereof, wherein X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4N02), and X 12 is I.

[0831] 36. The polypeptide of any one of embodiments 29 to 34, or a pharmaceutically acceptable salt thereof, wherein X 10 is Y, and X 12 is Orn, K, R, Q, Dap, S, E, or I.

[0832] 37. The polypeptide of any one of embodiments 29 to 36, or a pharmaceutically acceptable salt thereof, wherein only one of X 17 , X 20 , X 24 , or X 28 is an amino acid having a functional group available for conjugation to a fatty acid.

[0833] 38. The polypeptide of any one of embodiments 29 to 37, or a pharmaceutically acceptable salt thereof, wherein only one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid by a direct bond or a linker between the amino acid and the fatty acid.

[0834] 39. The polypeptide of embodiment 38, or a pharmaceutically acceptable salt thereof, wherein only one of X 17 , X 20 , X 24 , or X 28 is conjugated to a fatty acid by a linker between the amino acid and the fatty acid.

[0835] 40. The polypeptide of any one of embodiments 29 to 39, wherein the fatty acid is a C 16 -C 22 fatty acid.

[0836] 41. The polypeptide of embodiment 40, or a pharmaceutically acceptable salt thereof, wherein

[0837] X 17 is K, and is conjugated to the C 16 -C 22 fatty acid by a direct bond or a linker between the amino acid and the C 16-C 22 fatty acid conjugation,

[0838] X 20 is Aib, aMe-4-Pal, Q, or R,

[0839] X 24 is E, Q, D-Glu, or N, and

[0840] X 28 is E or A.

[0841] 42. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 40, wherein:

[0842] X 17 is A, I, or Q,

[0843] X 20 is K, and is conjugated to C 16 -C 22 fatty acid via a direct bond or linker between the amino acid and C 16 -C 22 fatty acid conjugation,

[0844] X 24 is E, Q, D-Glu, or N, and

[0845] X 28 is E or A.

[0846] 43. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 40, wherein:

[0847] X 17 is A, I, or Q,

[0848] X 20 is Aib, aMe-4-Pal, Q, or R,

[0849] X 24 is K, and is conjugated to C 16 -C 22 fatty acid via a direct bond or linker between the amino acid and C 16 -C 22 fatty acid conjugation, and

[0850] X 28 is E or A.

[0851] 44. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 40, wherein:

[0852] X 17 is A, I, or Q,

[0853] X 20 is Aib, aMe-4-Pal, Q, or R,

[0854] X 24 is E, Q, D-Glu, or N, and

[0855] X 28 is K, and is conjugated to C 16 -C 22 through a direct bond or a linker between an amino acid and C 16 -C 22 a fatty acid.

[0856] 45. The polypeptide or a pharmaceutically acceptable salt thereof of any one of embodiments 10, 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 38, 39, 40, 41, 42, 43, and 44, wherein the linker comprises one to four amino acids.

[0857] 46. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 45, wherein the amino acid is Glu, γGlu, or a combination thereof.

[0858] 47. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 45 or 46, wherein the linker comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) or εK moieties.

[0859] 48. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 47, wherein the linker comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

[0860] 49. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 45, wherein the linker comprises a (γGlu) a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2) p -CO2H structure, wherein a is 0 or 1, b is 0, 1, or 2, c is 1, 2, or 3, and p is an integer between 14 and 20.

[0861] 50. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 49, wherein a is 0.

[0862] 51. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 49, wherein a is 1.

[0863] 52. The polypeptide or a pharmaceutically acceptable salt thereof of any one of embodiments 49 to 51, wherein b is 0.

[0864] 53. The polypeptide or a pharmaceutically acceptable salt thereof of any one of embodiments 49 to 51, wherein b is 1.

[0865] 54. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 49-51, wherein b is 2.

[0866] 55. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 49-54, wherein c is 1.

[0867] 56. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 49-54, wherein c is 2.

[0868] 57. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 49-54, wherein c is 3.

[0869] 58. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 49-57, wherein p is 16.

[0870] 59. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 49-57, wherein p is 18.

[0871] 60. The polypeptide or pharmaceutically acceptable salt thereof selected from the group consisting of SEQ ID NOs: 7-504.

[0872] 61. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 1-60, wherein the C-terminus is amidated.

[0873] 62. The polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 1-61, wherein the pharmaceutically acceptable salt is selected from the group consisting of a sodium salt, a potassium salt, a trifluoroacetate salt, a hydrochloride salt, or an acetate salt.

[0874] 63. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 62, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium and potassium.

[0875] 64. A pharmaceutical composition comprising the polypeptide or pharmaceutically acceptable salt thereof of any one of embodiments 1-63 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0876] 65. The pharmaceutical composition of embodiment 64, wherein the composition is formulated for oral administration.

[0877] 66. The pharmaceutical composition of embodiment 64, wherein the composition is formulated for subcutaneous administration.

[0878] 67. A method of treating a disease or condition selected from the group consisting of diabetes, obesity, long-term weight management, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS), the method comprising the step of administering to an individual in need thereof an effective amount of the polypeptide of any one of embodiments 1 to 63, or a pharmaceutically acceptable salt thereof.

[0879] 68. The polypeptide of any one of embodiments 1 to 63, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0880] 69. The polypeptide of any one of embodiments 1 to 63, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease or condition selected from the group consisting of diabetes, obesity, long-term weight management, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS).

[0881] 70. Use of the polypeptide of any one of embodiments 1 to 63, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease or condition selected from the group consisting of diabetes, obesity, long-term weight management, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS).

[0882] 71. A polypeptide comprising a sequence having more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% sequence identity to any one of SEQ ID NOs: 7-1242.

[0883] 72. The polypeptide of embodiment 71, comprising a sequence selected from the group consisting of SEQ ID NOs. 7-1242.

[0884] 73. A polypeptide or a pharmaceutically acceptable salt thereof, comprising a sequence having more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% sequence identity to any one of SEQ ID NOs. 294 to 775 or 1146 to 1240.

[0885] 74. A polypeptide or a pharmaceutically acceptable salt thereof, selected from the group consisting of SEQ ID NOs: 294 to 775 or 1146 to 1240.

[0886] 75. The polypeptide or a pharmaceutically acceptable salt thereof of embodiment 74, which is selected from the group consisting of SEQ ID NO: 692, 700, 702, 705, 706, 716, 718, 743, 747, 749, 767.

[0887] 76. The polypeptide of any of the above embodiments, wherein the polypeptide has a higher potency for each of the glucagon, GIP, and GLP-1 receptors compared to native glucagon (SEQ ID NO: 3), GIP (SEQ ID NO: 1), and GLP-1 7-36 (SEQ ID NO: 2).

[0888] 77. The polypeptide or a pharmaceutically acceptable salt thereof of any of the above embodiments, wherein one or more hydrogen atoms are replaced by deuterium.

[0889] The present application is further illustrated by the following examples, which should not be construed as limiting.

[0890] Peptide synthesis

[0891] Example 1:

[0892] Example 1 is the compound represented by the following description:

[0893] Y-Aib-QGTFTSDFSK-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γ-Glu)-CO-(CH2) 18 -CO2H)AQ-Aib-AFIEYLLAGGPSSGEPPPSEG-NH2(SEQ ID NO: 7).

[0894] The following is a description of the structure of Example 1 using standard one-letter amino acid code, except for residues Aib2, αMeL13, and Aib20, where the structure of these amino acid residues has been expanded:

[0895]

[0896] The peptide backbone of Example 1 was synthesized using fluorenylmethyloxycarbonyl (Fmoc) / tert-butyl (t-Bu) chemistry on a Symphony 12-channel multiplexed peptide synthesizer (Protein Technologies, Inc. Tucson, AZ).

[0897] The resin consisted of 1% DVB cross-linked polystyrene (Fmoc-Rink-MBHA low loading resin, 100-200 mesh, EMD Millipore) with a substitution of 0.3-0.4 meq / g. Standard side chain protecting groups were used. Lysine at position 17 was used Fmoc-Lys(Mtt)-OH and tyrosine at position 1 was used Boc-Tyr(tBu)-OH. Prior to each coupling step (2x7 min) the Fmoc group was removed using 20% piperidine in DMF. All standard amino acid couplings were performed in equimolar ratios of Fmoc amino acid (0.3 M), diisopropylcarbodiimide (0.9 M) and Oxyma (0.9 M) in a 9-fold molar excess relative to the theoretical peptide loading, with coupling for 1 hour for primary amines and 3 hours for secondary amines. The exception was coupling with the Ca-methylated amino acid, which was coupled for 3 hours. After completion of the A peptide backbone synthesis, the resin was washed thoroughly 6 times with DCM to remove residual DMF. The Mtt protecting group on the lysine at position 17 was selectively removed from the peptide resin using 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM for two treatments (2x 40 min treatments).

[0898] The subsequent fatty acid-linker moiety was attached by coupling of 2-[2-(2-Fmoc-amino- ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid a-tert-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.), mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China). A 3-fold excess of reagents (AA:PyAOP:DIPEA = 1:1:1 mol / mol) was used for each coupling, with a coupling time of 1 hour.

[0899] After synthesis, the peptide resin was washed with DCM and then thoroughly air-dried. The dried resin was treated with 10 mL of a lysis mixture (trifluoroacetic acid:water:triisopropylsilane, 95:2.5:2.5 v / v) at room temperature for 2 hours. The resin was filtered off, washed twice with 2 mL of pure TFA each time, and the combined filtrates were treated with 5 times (v / v) of cold diethyl ether (-20°C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 3500 rpm for 2 min to form a solid precipitate. The supernatant was decanted, and the solid precipitate was ground twice with ether and dried under vacuum. The crude peptide was dissolved in 20% acetonitrile / 20% acetic acid / 60% water and purified by RP-HPLC on a Luna 5 μm phenyl-hexyl preparative column (21 x 250 mm, Phenomenex) using a linear gradient of 100% acetonitrile and 0.1% TFA / water buffer (30%–50% acetonitrile, 60 min). The purity of the peptide was assessed using analytical RP-HPLC, with pooling criteria >95%. The purity of the main pool in Example 1 was found to be 98.8%. The final main product pool was then lyophilized to obtain the lyophilized peptide TFA salt. The molecular weight was determined by LC-MS (measured value: M+4H). + / 4 = 1226.8; Calculated value: M + 4H + / 4=1226.9).

[0900] Examples 2 to 1236

[0901] The polypeptides according to Examples 2 (SEQ ID NO: 8) to 1236 (SEQ ID NO: 1242) were prepared essentially according to the method described in Example 1. These polypeptides are listed in Table 1 below. Additional descriptions of some examples are provided after Table 1.

[0902] Table 1

[0903]

[0904]

[0905]

[0906]

[0907]

[0908]

[0909]

[0910]

[0911]

[0912]

[0913]

[0914]

[0915]

[0916]

[0917]

[0918]

[0919]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925]

[0926]

[0927]

[0928]

[0929]

[0930]

[0931]

[0932]

[0933]

[0934]

[0935]

[0936]

[0937]

[0938]

[0939]

[0940]

[0941]

[0942]

[0943]

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950]

[0951]

[0952]

[0953]

[0954]

[0955]

[0956]

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974]

[0975]

[0976]

[0977]

[0978]

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

[0992]

[0993]

[0994]

[0995]

[0996]

[0997]

[0998]

[0999]

[1000]

[1001] Structural descriptions of certain embodiments are provided as follows:

[1002]

[1003]

[1004]

[1005]

[1006]

[1007] In vitro function

[1008] Functional activity assay:

[1009] Functional activity was determined in HEK-293 clonal cell lines expressing GIP-R, GLP-1R and GcgR. Peptides were used (20-point concentration response curves prepared by Labcyte Echo acoustic liquid handler, using 2.75-fold serial dilutions) in 1X GlutaMAX I supplemented with 10% FBS, 1% Pen / Strep, 1% NEAA, 1% HEPES, 1% L-Glutamine, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids, 1% Sodium Bicarbonate, 1% Pluronic F-127, 1% MEM NEAA, 1% MEM Non-Essential Amino Acids, 1% MEM Vitamin Solution, 1% MEM Sodium Pyruvate, 1% MEM Sodium TMEach recipient cell line was treated in a 20 pl assay volume in DMEM (Gibco Cat#31053) containing 0.1% L-alanyl-L-glutamine dipeptide (Gibco Cat#35050), 0.1% casein (Sigma Cat#C4765), 1% HSA (Human Serum Albumin, Sigma Cat#A3782), 500 mM IBMX (3-isobutyl-l-methylxanthine) and 20 mM HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid).

[1010] After 30 minutes incubation at 37°C, the resulting increase in intracellular cAMP was quantitatively determined using the CisBio cAMP Dynamic 2 HTRF assay kit (62AM4PEJ). Briefly, intracellular cAMP levels were detected by adding a cell lysis buffer containing cAMP-d2 conjugate followed by the addition of an antibody anti-cAMP-Eu 3+ Cryptate, also in cell lysis buffer. The resulting competition assay was incubated at room temperature for at least 60 min before being detected using a Pherastar instrument (BMG Labtech) with an excitation wavelength of 320 nm and emission wavelengths of 665 nm and 620 nm. Raw data values (emission wavelength 665 nm / 620 nm*10,000) were inversely proportional to the amount of cAMP present and converted to cAMP (nM) per well using a cAMP standard curve.

[1011] The amount of cAMP (nM) produced per well was converted to the percentage of the maximal response observed for human GLP-l(7-36)NH2, human glucagon (Gcg) or human GIP 1-42 NH2. Relative EC 50 values were derived by non-linear regression analysis, fitting to a four parameter logistic equation using maximal response percentage vs. added peptide concentration.

[1012] Geometric mean values of the relative EC 50 data for exemplary analogs vs. hGIP(l-42)NH2, hGLP-l(7-36)NH2 and hGcg are shown in Table 2 below.

[1013] Table 2. Functional cAMP potency (relative EC 50 )

[1014]

[1015]

[1016]

[1017]

[1018]

[1019]

[1020]

[1021]

[1022]

[1023]

[1024]

[1025]

[1026]

[1027]

[1028]

[1029]

[1030]

[1031]

[1032]

[1033]

[1034]

[1035]

[1036]

[1037]

[1038]

[1039]

[1040]

[1041]

[1042]

[1043]

[1044] Note: EC of human GLP-1 (7-36) NH2 on human GLP-1 R, human Gcg on human GcgR and human GIP (1-42) NH2 on human GIP-R 50 Assay: Peptide concentration range 346 pM to 9950 nM. EC of exemplary analogs on human GLP-1 R, human GcgR and human GIP-R 50 Assay: Peptide concentration range 6.87 pM to 9.95 μΜ.

[1045] As shown in Table 2, exemplary analogs have agonist activity in the presence of HSA, as determined by the human GIP-R, GLP-1 R and GcgR cAMP assays, which is lower than the native ligand.

[1046] To determine the intrinsic potency of exemplary analogs and control molecules, the above cAMP assays were performed in the presence of 0.1% casein only (without human serum albumin). Casein was used as a non-specific blocker in both cAMP assays and does not interact with the fatty acid moiety of the analyzed molecules.

[1047] Intracellular cAMP levels were determined using standard curve extrapolation. Dose response curves of compounds were plotted as percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) and analyzed using four parameter non-linear regression fit with variable slope. EC 50 refers to the concentration of a compound that elicits a half-maximal stimulatory effect in a dose response curve. Relative EC 50 values for each calculation of the geometric mean were determined by curve fitting.

[1048] Data are shown in Table 3 below.

[1049] Table 3. Functional activation of hGLP-1 R, hGIPR, hGcgR in the presence of 0.1% casein

[1050]

[1051]

[1052]

[1053]

[1054]

[1055]

[1056]

[1057]

[1058]

[1059]

[1060]

[1061]

[1062]

[1063]

[1064]

[1065]

[1066]

[1067]

[1068]

[1069]

[1070]

[1071]

[1072]

[1073]

[1074]

[1075]

[1076]

[1077]

[1078]

[1079]

[1080] As shown in Table 3, exemplary analogs stimulated cAMP in human GIP, GLP-1, and glucagon receptors in the presence of 0.1% casein.

[1081] In vivo studies

[1082] Pharmacokinetics in male Sprague Dawley rats:

[1083] The pharmacokinetics of exemplary analogs were evaluated following a single subcutaneous (SC) administration of 10 nmol / kg in 40 mM Tris pH 8 solution or a single jejunal (IJ) administration of 4 mg / kg mixed with 40 mM Tris pH 8 solution of 250 mM sodium decanoate / C10 to male Sprague Dawley rats. Blood samples were collected 96 hours post-SC administration and 72 hours post-IJ administration, respectively, and the pharmacokinetic parameters were calculated using the resulting individual plasma concentrations. Peptide plasma (K3EDTA) concentrations were determined using a qualified LC / MS method that measures the intact mass of the analog. Each peptide and an analog as an internal standard were extracted from rat plasma using methanol. LC / MS detection was performed using a high resolution instrument. The mean pharmacokinetic parameters are shown in Table 4 and Table 5.

[1084] Table 4. Mean pharmacokinetic parameters of peptides following a single subcutaneous administration of 10 nmol / kg to male Sprague Dawley rats.

[1085]

[1086] Abbreviations: T 1 / 2 = half-life, CL / F = apparent clearance

[1087] Note: Data are means, where n = 3 / group.

[1088] Table 5. Mean pharmacokinetic parameters of peptides following a single IJ administration of 4 mg / kg to male Sprague Dawley rats.

[1089]

[1090] Abbreviations: Cmax / D = dose normalized maximum plasma concentration; T 1 / 2 = half-life, CL / F = apparent clearance.

[1091] Note: Data are means, where n = 4 / group.

[1092] The results of this study for the test examples are consistent with the extended pharmacokinetic properties.

[1093] Pharmacokinetics in cynomolgus monkeys

[1094] This study was designed to evaluate the oral bioavailability of the example polypeptides in cynomolgus monkeys. High resolution liquid chromatography / mass spectrometry (HR-LC / MS) was used to determine the concentration of example 510, 548, 558, 694, 699, 686, 712, 741, 883, and 1027 in cynomolgus monkey plasma. Standards and controls were prepared in cynomolgus monkey plasma and diluted in control cynomolgus monkey plasma as needed to bring the samples into the quantitation range. To control for assay variability, an internal standard (IS) was added to all standards and samples. Isopropanol and methanol (50:50 v / v) were used for protein precipitation to extract the examples and internal standard from 100% monkey plasma (50 μί). The samples were then centrifuged (3000 rpm, 10 minutes) and the supernatant was transferred to a Siricco protein precipitation plate. The samples were loaded onto a Sep-Pak tC18 SPE microplate that had been treated with 2% formic acid in acetonitrile and water. The compounds were then washed with 2% formic acid in water, eluted with 2% formic acid in acetonitrile into a plate containing 5x Invitrosol and 1% formic acid in water, and then aliquots (10 μί) were injected onto an Xselect CSH C18 (3.5 μιη, 2.1 x 20 mm) for LC / MS analysis.

[1095] Plasma pharmacokinetics (PK) of example 510, 548, 558, 694, 699, 686, 712, 741, 883, and 1027 following a single intravenous (IV) administration dose (10 nmol / kg) was evaluated in male and female cynomolgus monkeys. Blood samples were collected over 504 hours. Plasma was collected from the blood samples by centrifugation and stored frozen (-70 °C) until analysis. The plasma concentration of the molecules was detected using the bioanalytical method described above.

[1096] Table 6: Pharmacokinetic parameters (mean ± SD) following a single IV administration of example polypeptides (10 nmol / kg) dose in male and female cynomolgus monkeys (n=3).

[1097]

[1098] Abbreviations: AUC 0-inf = Area under the curve from time 0 hours to infinity; CL = clearance; T 1 / 2 = half-life.

[1099] PK parameters for TG-2474, TG-2728, TG-2565, TG-2698, TG-2708, TG-3329, TG-3270, TG-3169, TG-3211, and TG-3120 were determined following a single 10 mg oral dose in male and female cynomolgus monkeys. Blood samples were collected over 504 hours post-dose. Plasma was collected from blood samples by centrifugation and stored frozen (-70 °C) until analysis. Plasma concentrations of molecules were determined using the bioanalytical method described above.

[1100] Table 7. Pharmacokinetic parameters (mean ± SD) following a single oral dose of example polypeptides (10 mg / animal) and sodium salcaprozate (N-[8-(2-hydroxybenzoyl)amino]octanoate) (SNAC), 300 mg, in male and female cynomolgus monkeys (n = 4).

[1101]

[1102]

[1103] Abbreviations: AUC 0-inf = area under the curve from time 0 hours to infinity; CL / F = apparent clearance; Cmax= maximum concentration; T max = time to reach maximum concentration; T max = time to reach maximum concentration; T 1 / 2 = half-life; F = bioavailability.

[1104] The percent F results in Table 7 indicate the relative bioavailability of the example polypeptides when administered orally compared to IV administration.

[1105] Studies in diet-induced obese C57BL / 6 mice:

[1106] To investigate the effect of the polypeptides described herein on weight loss, exemplary polypeptides were administered to C57BL / 6 diet-induced obese (DIO) mice.

[1107] Specifically, DIO male C57BL / 6 mice (Taconic, Germantown, NY) were used in the following studies that were maintained on a high caloric diet. Mice were individually housed in a temperature-controlled (24 °C) facility with a 12-hour light / dark cycle (lights on at 22:00) with free access to food (TD95217) and water. After at least 2 weeks to acclimate to the facility, mice were randomly grouped according to their body weight so that each group of experimental animals had a similar starting body weight. Body weight ranged from 41 to 50 g.

[1108] All groups contained 5 mice. Mice were treated with vehicle (40 mM Tris-HCl, pH 8.0) or 10 nmol / kg of the example polypeptides. Treatments were administered by subcutaneous (SC) injection (10 mL / kg) to DIO mice fed ad libitum once daily (QD) or every 3 days (Q3D) for 9-16 days, 30 to 90 minutes prior to the start of the dark cycle. Body weight and food intake were measured daily throughout the study. Body weight is expressed as a percentage of the starting body weight. Vehicle-treated mice (control group) maintained a body weight between 98.00 ± 0.84% and 101.45 ± 2.39% throughout the study.

[1109] Data are shown in Table 7 below as the mean ± SEM of 5 animals per group. Statistical analysis was performed using repeated measures ANOVA followed by comparison tests by Dunnett’s method.

[1110] Table 8. % Body weight after treatment with example polypeptides

[1111]

[1112]

[1113] *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparison test compared to control group

[1114] GLP1-R internalization assay

[1115] The potency of the peptide to stimulate ligand-induced GLP-1R internalization was determined using HEK293 cells expressing human GLP1-R.

[1116] The day before transfection, HEK293 cells were seeded in white 384-well plates at a density of 20,000 cells / well. Cells were transfected with Lipofectamine 2000 (Invitrogen) for SNAP-GLP-1R transfection. The next day, the medium was removed and the tagged receptors were labeled with 100 nM Tag-Lite SNAP-Lumi4-Tb (donor, Cisbio) in OptiMEM medium for 75 min at 37 °C. After that, cells were washed with internalization buffer (HBSS supplemented with 1 mM CaCl2, 2.5 mM MgCl2, 20 mM HEPES, and 0.1% Pluronic F-68, pH 7.4) and then 100 μΜ pre-warmed fluorescein-O' acetic acid (acceptor, Sigma-Aldrich) was added. The plates were placed in a 37 °C incubator for 5 min and then the ligand was added to adjust the temperature. Cells were then stimulated with ligand pre-warmed at 37 °C and the internalization of GLP1-R was measured every 3 min for 60 min at 37 °C using an EnVision plate reader. Data were normalized to GLP-1 maximum concentration (100%) and no ligand (0%) and plotted using GraphPad Prism 7 software.

[1117] Table 9 reports the potency of exemplary polypeptide to stimulate ligand-induced GLP-1R internalization. The assay results determine whether the polypeptide is a partial agonist of GLP-1R with respect to GLP-1R internalization.

[1118]

[1119]

[1120]

[1121]

[1122]

[1123]

[1124] GLP-1R CHO Cell β-arrestin recruitment assay

[1125] Activated G protein coupled receptors can interact with the family of β-arrestin signaling proteins. The potency of a peptide to induce arrestin recruitment by GLP-1R can be determined by the PathHunter enzyme fragment complementation assay, essentially as described in the literature (von Degenfeld et al., FASEB J., 2007 (14): 3819-26 and Hamdouchi et al., J. Med Chem., 2016 59(24): 10891-10916).

[1126] CHO-K1 cells expressing Pro-Link labeled human GLP-1R and enzyme receptor labeled β-arrestin-2 were obtained from DiscoveRx and prepared as frozen cells for use in the assay. Test peptides were dissolved in DMSO and serially diluted using an Echo ultrasonic liquid handler (LabCyte). Assay medium was PathHunter Cell Assay Buffer (DiscoveRx) containing 0.1% w / v hydrolyzed casein (Sigma). 100 nl of peptide was dispensed into 10 μΐ of assay medium in a 384 well plate, followed by the addition of 10 μΐ of cell containing assay medium to give 5000 cells per well. The plates were incubated in a 37°C / 5% CO2 incubator for 90 minutes, followed by the addition of 10 μΐ of PathHunter detection reagent (DiscoveRx) and incubation of the plates at room temperature for 60 minutes. Luminescence signals were measured. Peptide concentration-response curves were fitted to a four parameter logistic model to give EC50 values. 50 Potency was calculated. Data were normalized as % stimulation with DMSO and GLP-1 (7-36) as minimum and maximum controls (Campbell et al., Assay Guidance Manual 2017).

[1127] Table 10 reports the potency of sample peptides to stimulate GLP-1R induced β-arrestin recruitment. The assay results determine whether the peptides are partial and biased agonists of GLP-1R in terms of β-arrestin-2 recruitment.

[1128] Table 10.

[1129]

[1130]

[1131] Cell-based in vitro permeability (Papp) assay

[1132] Intestinal organoids from Gottingen minipig jejunum tissue and their two-dimensional monolayer model were generated using the protocol described previously (van der Hee, B.; Loonen, L. M. P.; Taverne, N.; Taverne-Thiele, J. J.; Smidt, H.; Wells, J. M., Optimized procedures for generating an enhanced, near-physiological 2D culture system from porcine intestinal organoids. Stem Cell Res 2018, 28, 165-171). After 7 days of culture, organoids were dissociated into single cells with TrypLE (Gibco) and the single-cell suspension was added to 24-well transparent transwell chambers (0.3 cm 2 When transepithelial resistance reached > 600 Ω·cm 2 , peptide permeability was tested in the presence of 10 or 20 mM C10. In the presence of C10, basal chamber samples were collected at 20 min. Peptide concentrations in apical and basal chambers were measured by LC / MS and apparent permeability coefficients were calculated as described previously (Twarog, C.; Liu, K.; O'Brien, P. J.; Dawson, K. A.; Fattal, E.; Illel, B.; Brayden, D. J., A head-to-head Caco-2 assay comparison of the mechanisms of action of the intestinal permeation enhancers: SNAC and sodium caprate (C10). Eur J Pharm Biopharm 2020, 152, 95-107).

[1133] Table 11 reports the P app values of exemplary polypeptides.

[1134] Table 11.

[1135]

[1136]

[1137]

[1138] In vivo ileum (distal loop) absorption assay

[1139] Example polypeptides were evaluated for ileal absorption in the presence of C10 in rats using a previously reported closed loop model of the intestine (Lawrence, S. A.; Blankenship, R.; Brown, R.; Estwick, S.; Ellis, B.; Thangaraju, A.; Datta-Mannan, A., Influence of FcRn binding properties on the gastrointestinal absorption and exposure profile of Fc molecules. Bioorg Med Chem 2021, 32, 115942).

[1140] Sprague Dawley rats weighing between 250-280 grams, fasted overnight, were anesthetized by inhalation of isoflurane and placed on a warm surgical table maintained at 37°C with a circulating water bath. The surgical area was shaved and the skin was disinfected with betadine scrub followed by 70% isopropyl alcohol. A 2 cm incision was made at the midline to expose the intestine and a 10 cm segment of ileum was ligated. Peptides were formulated in 100 mM C10 Tris buffer (pH 8.0, 50 mM) to a final concentration of 300 µM. Formulations were prepared as follows: Formulations were administered directly into the ileal loop. Blood samples (0.2 mL) were collected from the tail vein prior to peptide administration and at 10, 20, 40, 60 minutes post administration. Blood samples were collected into tubes containing K3EDTA (5%) and processed into plasma for subsequent analysis. Peptide concentrations in plasma were determined by LC / MS.

[1141] Table 12.

[1142]

[1143] Proteolytic stability

[1144] Pepsin stability assays were used to determine the relative stability of the example polypeptides in a simulated pepsin proteolytic environment. Pepsin A was dissolved in simulated gastric fluid (SGF) with the example polypeptides or controls. Samples were taken and quenched at TO, 15, 30, and 60 minutes. Controls were semaglutide and Example Compound Number 4 (“Cmpd4”) from U.S. Patent Application 2020 / 0024322. The relative content of intact peptide was quantified by mass spectrometry (MS) method.

[1145] Example polypeptides were assayed for proteolytic stability relative to controls in simulated gastric fluid (SGF, 2 g / L NaCl, pH 1.2). Examples were dissolved in 50 mM Tris buffer (pH 8.0) at a concentration of 10 mg / mL as a stock solution. Pepsin A was reconstituted at 10 mg / mL in SGF. Reaction systems were prepared by diluting the peptides in SGF and spiking in pepsin A to a final concentration of 0.4 mg / mL for the example polypeptides and 1 mg / mL for pepsin. The example peptide and pepsin solutions were then incubated in a shaking incubator set to 37 °C at 100 rpm. Samples (25 μL) were taken at various time intervals and quenched with 100 mM ammonium bicarbonate (pH 9) (50 μL) to stop proteolytic activity. All samples were centrifuged and the supernatant was analyzed by LC / MS to determine the remaining intact peptide.

[1146] Results are provided in Tables 12-14 below:

[1147] Table 12. Stability in 0.1 mg / mL pepsin A solution.

[1148]

[1149]

[1150] Table 13. Stability in 1 mg / mL pepsin A solution.

[1151]

[1152] Table 14. Stability in 1 mg / mL pepsin A solution.

[1153]

[1154] The data in Tables 12-14 support improved stability of certain example polypeptides.

[1155] Sequences

[1156] SEQ ID NO: 1 - Human GIP amide

[1157] YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-NH2

[1158] SEQ ID NO: 2 - Human GLP-1 (7-36) amide

[1159] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2

[1160] SEQ ID NO: 3 - Human glucagon

[1161] HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

[1162] SEQ ID NO: 4 - Polypeptide of Formula I

[1163] SEQ ID NO: 5 - Polypeptide of Formula II

[1164] SEQ ID NO: 6 - Polypeptide of Formula III

[1165] SEQ ID NO: 7-1242 - Polypeptides of Examples 1-1236

[1166] SEQ ID NO: 1243 - Polypeptide of Formula I'

[1167] SEQ ID NO: 1244 - Polypeptide of Formula II'

[1168] SEQ ID NO: 1245 - Polypeptide of Formula III'

Claims

1. Polypeptides, which include: X1X2QX4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 X 17 AX 19 X 20 X 21 FX 23 X 24 X 25 LX 27 X 28 X 29 GX 31 X 32 SX 34 X 35 PX 37 PX 39 X40X 41 X 42 X 43 X 44 X 45 X 46 , in X1 is Y, NMeY, or H. X2 is Aib. X4 is either G or D-Ala. X6 is F, αMeF, or αMeF(2F). X 10 It is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), or Y. X 11 Is it S or αMeS? X 12 It is Orn, K, R, Q, Dap, S, E, or I. X 13 Is it αMeL, I or L, X 15 Is it D or E? X 16 Is it K, Orn, A, or E? X 17 It is any amino acid, A, I, or Q, or Orn, that has a functional group that can be used to conjugate with fatty acids. X 19 Is it A or Q? X 20 It is any amino acid, Aib, Q, R, or αMe-4-Pal that has a functional group that can be used to conjugate with fatty acids. X 21 It is A, Aad, Aib, S, N, Q, E, T, or Orn. X 23 Is it I or V? X 24 It is any amino acid, E, D-Glu, Q, N, or D-Gln that has a functional group that can be used to conjugate with fatty acids. X 25 It is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal. X 27 is L, I, E, V, A, Aad, T, Q, or S, X 28 It is any amino acid, E or A, that has a functional group that can be used to conjugate with fatty acids. X 29 It is G, Aib, T, D-Ala, or A. X 31 Is it P or E? X 32 Is it S or P? X 34 Is it G or Aib? X 35 It is A, D, or E. X 37 Is it P or E? X 39 Is it E, S, G, A, T, or Orn? X 40 The missing digits are G, E, S, A, or T. Where X 40 If it is G, E, S, A, T, or D-Glu, then X 41 The deficiency may be due to E, S, D, G, Q, T, A, γE, or D-Glu. Where X 41 If it is E, S, D, G, Q, T, A, γE, or D-Glu, then X 42 The deficiency may be due to G, E, D-Glu, or γE. Where X 42 If it is G, E, D-Glu, or γE, then X 43 The deficiency may be due to E, γE, or D-Glu. Where X 43 If it is E, γE, or D-Glu, then X 44 Missing or E, Where X 44 If it is E, then X 45 Missing or E, Where X 45 If it is E, then X 46 Missing or E, Where X 40 If X is missing, then 41 To X 46 Also missing, Where X 41 If X is missing, then 42 To X 46 Also missing, Where X 42 If X is missing, then 43 To X 46 Also missing, Where X 43 If X is missing, then 44 To X 46 Also missing, Where X 44 If X is missing, then 45 and X 46 Also missing, Where X 45 If X is missing, then 46 Also missing, The polypeptide comprises at least one of the following: X6 is αMeF or αMeF(2F); X 10 It is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2); X 11 It is αMeS; X 13 It is αMeL; X 24 It is D-Glu; and / or X 25 It is αMeY; Where X 10 If it is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), then X 12 It's I, And X among them 17 X 20 X 24 or X 28 At least one of them is an amino acid having a functional group that can be used for conjugation with fatty acids. The C-terminal amino acid is optionally amidated; Or its pharmaceutically acceptable salt.

2. The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least two of the following: X 10 It is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2); X 11 It is αMeS; X 13 It is αMeL; X 16 It is Orn; X 24 It is D-Glu; and / or X 25 It is αMeY.

3. The polypeptide of any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least three of the following: X 10 It is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2); X 11 It is αMeS; X 13 It is αMeL; X 16 It is Orn; X 24 It is D-Glu; and / or X 25 It is αMeY.

4. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein: X 10 It is F, 3-Pal, 4-Pal, F(4CN) or F(4NO2).

5. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein: X1 is Y; X4 is G; X6 is αMeF(2F); X 10 It is 4-Pal;X 12 It is I; X 13 It is αMeL; X 15 It is D; X 16 It is Orn; X 19 It is Q; X 20 It is αMe-4-Pal;X 21 Is it E or Orn; X 23 It is I; X 24 It is D-Glu;X 25 It is αMeY; X 27 Is it I or V; X 28 It is E; X 29 It is G; X 31 It is P; X 34 It is G; X 35 It is A or E; X 37 It is P; X 39 Is it E or S; X 40 Is it G or T; X 41 It is E, S, or G; and X 42 Missing.

6. The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, wherein: X 11 It is S, X 21 It is Orn, X 27 It is I, X 35 It is E, X 39 It is E, X 40 It is T, and X 41 It is E.

7. The polypeptide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein: X 11 It is αMeS, X 21 It is E, X 27 It is V, X 35 It is A, X 39 It is S, X 40 It is G, and X 41 It is S.

8. The polypeptide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein: X 11 It is αMeS, X 21 It is E, X 27 It is I, X 35 It is A, X 39 It is S, X 40 It is G, and X 41 It is S.

9. The polypeptide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein: X 11 It is αMeS, X 21 It is E, X 27 It is I, X 35 It is A, X 39 It is S, X 40 It is G, and X 41 It's G.

10. The polypeptide of any one of claims 1-9 or a pharmaceutically acceptable salt thereof, wherein: X 17 It is K, and through amino acids and C 16 -C 22 The linker between fatty acids and C 16 -C 22 Fatty acid conjugation.

11. The polypeptide of claim 10 or a pharmaceutically acceptable salt thereof, wherein the linker comprises one to four amino acids.

12. The polypeptide of claim 11 or a pharmaceutically acceptable salt thereof, wherein the amino acid contained in the linker is Glu, γGlu, or a combination thereof.

13. The polypeptide of any one of claims 10-12 or a pharmaceutically acceptable salt thereof, wherein the linker comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

14. The polypeptide of claim 13 or a pharmaceutically acceptable salt thereof, wherein the linker comprises (γGlu). a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2) p The structure is -CO2H, where a is 0 or 1, b is 0, 1 or 2, c is 1, 2 or 3, and p is an integer between 14 and 20.

15. The polypeptide of claim 14 or a pharmaceutically acceptable salt thereof, wherein a is 0, b is 1, and c is 1.

16. A polypeptide or a pharmaceutically acceptable salt thereof, selected from SEQ ID NO:294-775, 1146-1240.

17. A polypeptide comprising more than 90% sequence identity with any one of SEQ ID NO: 692, 700, 702, 705, 706, 716, 718, 743, 747, 749, 767.

18. The polypeptide of claim 17 or a pharmaceutically acceptable salt thereof, selected from: SEQ ID NO: 692, 700, 705, 718 and 747.

19. The polypeptide of claim 18 or a pharmaceutically acceptable salt thereof, comprising SEQ ID NO.

692.

20. The polypeptide of claim 18 or a pharmaceutically acceptable salt thereof, comprising SEQ ID NO.

700.

21. The polypeptide of claim 18 or a pharmaceutically acceptable salt thereof, comprising SEQ ID NO.

705.

22. The polypeptide of claim 18 or a pharmaceutically acceptable salt thereof, comprising SEQ ID NO.

718.

23. The polypeptide of claim 18 or a pharmaceutically acceptable salt thereof, comprising SEQ ID NO.

747.

24. The polypeptide of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein the C-terminus is amidated.

25. The polypeptide of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from sodium salts, potassium salts, trifluoroacetates, hydrochlorides, or acetates.

26. The polypeptide of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein one or more hydrogen atoms are replaced by deuterium.

27. The polypeptide of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein the polypeptide is associated with natural glucagon (SEQ ID NO:3), GIP (SEQ ID NO:1), and GLP-1. 7-36 Compared to (SEQ ID NO:2), the polypeptide has higher potency against each of the glucagon, GIP and GLP-1 receptors.

28. A pharmaceutical composition comprising a polypeptide of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

29. The pharmaceutical composition of claim 28, wherein the composition is formulated for oral administration.

30. The pharmaceutical composition of claim 28, wherein the composition is formulated for subcutaneous administration.

31. A method for treating a disease or condition selected from diabetes, obesity, long-term weight management, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), polycystic ovary syndrome (PCOS), Parkinson's disease, and Alzheimer's disease, said method comprising the following steps: Administer to an individual in need an effective amount of any one of claims 1 to 23 of the polypeptide or a pharmaceutically acceptable salt thereof.

32. The polypeptide or a pharmaceutically acceptable salt thereof claimed in any one of claims 1 to 23, for use in treatment.

33. The polypeptide or a pharmaceutically acceptable salt thereof claimed in any one of claims 1 to 23, for the treatment of a disease or condition selected from diabetes, obesity, long-term weight management, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS).

34. Use of the polypeptide or a pharmaceutically acceptable salt thereof claimed in any one of claims 1 to 23 in the preparation of a medicament for treating a disease or condition selected from diabetes, obesity, long-term weight management, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS).

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