GIP / GLP1 / GCG triple receptor agonists and their uses

Abstract

Polypeptides are provided that have activity at each of the GIP, GLP-1, and glucagon receptors. The polypeptides have structural features that result in activity at each of these receptors and a prolonged duration of action. Also provided are methods for treating diseases and / or conditions, such as obesity, chronic weight management, type 2 diabetes mellitus, 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).

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 provide appropriate levels of activity and extended duration of action at each of these receptors. Additionally, the present invention relates to compounds that can be administered orally or subcutaneously. Such polypeptides may be useful for treating diseases or conditions such as obesity, chronic weight management, type 2 diabetes mellitus (T2DM), dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), and / or polycystic ovary syndrome (PCOS). [Background technology]

[0002] The prevalence of diabetes has been increasing over the past few decades. T2DM is the most common form of diabetes, accounting for approximately 90% of all diabetes cases. T2DM is characterized by high blood glucose levels caused by insulin resistance. The current standard of care for T2DM includes dietary restriction and exercise, as well as treatment with oral medications and injectable hypoglycemic drugs, including incretin-based therapies such as GLP-1 receptor agonists. Currently, various GLP-1 analogs, including dulaglutide, exenatide, and liraglutide, are available for treating T2DM. However, many currently available GLP-1 receptor agonists are dose-limited by gastrointestinal side effects such as nausea and vomiting. Subcutaneous injection is the most common route of administration for available GLP-1 receptor agonists. Insulin is considered when available oral medications and incretin-based therapies are insufficient. Despite available treatment options, a significant number of individuals receiving approved therapy do not achieve glycemic control goals (see, e.g., Casagrande et al. (2013) Diabetes Care 36:2271-2279). Uncontrolled diabetes can lead to one or more conditions that affect the morbidity and mortality of such individuals.

[0003] One of the major risk factors for T2DM is obesity, and the majority of individuals with T2DM (approximately 90%) are overweight or obese. Obesity is a complex medical disorder that leads to excessive accumulation of adipose tissue mass. Today, obesity is a global public health concern associated with unfavorable health outcomes and morbidity. Desirable treatments for patients suffering from obesity aim to reduce excess weight, improve obesity-related comorbidities, and maintain long-term weight loss. Available obesity treatments are particularly inadequate for patients with severe obesity. Alternative treatment options are needed to induce therapeutic weight loss in patients in need of such treatment.

[0004] In that regard, new therapies being investigated include compounds that have activity not only at the GLP-1 receptor, but also at one or more other receptors, such as the GIP and / or glucagon receptors.

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

[0006] Furthermore, certain compounds have been described as having triple agonist activity (i.e., activity at each of the GIP, GLP-1, and glucagon receptors). For example, International Publication No. WO 2015 / 067716 describes glucagon analogs with triple agonist activity. Similarly, International Publication No. WO 2016 / 198624 describes exendin-4 analogs that are themselves GLP-1 analogs and have triple agonist activity. Similarly, International Publication Nos. WO 2014 / 049610 and WO 2017 / 116204 each describe various analogs with triple agonist activity. International Patent Application Publication No. WO 2017 / 153375 also describes glucagon and GLP-1 co-agonists that are said to have GIP activity. Additionally, WO 2019 / 125938, WO 2019 / 125929, and WO 2021 / 126695 each describe various polypeptides with triple agonist activity.

[0007] However, there remains a need for compounds that can provide effective glucose control with weight loss benefits and a favorable side effect profile. There is also a need for alternative treatment options to provide therapeutic weight loss or chronic weight management in patients in need of such treatment. There is also a need for therapeutic agents that are available for use with a sufficiently extended duration of action to allow for less frequent dosing, such as once daily, three times weekly, twice weekly, or once weekly. Furthermore, there is a desire and need for compounds that are suitable for convenient administration modes, such as subcutaneous or oral routes. In particular, there is a desire for compounds that exhibit sufficient efficacy with a favorable side effect profile, and / or stability and bioavailability, so that they can be administered orally. Summary of the Invention

[0008] The polypeptides described herein aim to meet one or more of the above needs. Accordingly, the present disclosure describes polypeptides having activity at each of the GIP, GLP-1, and glucagon receptors. The polypeptides described herein provide sufficient activity at each receptor, allowing for administration of a dose that provides the benefits of agonism at that receptor while avoiding undesirable side effects associated with excessive activity. Furthermore, the polypeptides described herein have an extended duration of action at the GIP, GLP-1, and glucagon receptors, allowing for less frequent dosing, such as once daily, three times weekly, twice weekly, or once weekly. In this manner, the polypeptides provide enhanced glucose control, metabolic benefits, such as weight loss and / or improved body composition, lipid benefits, and / or other benefits, such as an increase in bone mass or bone formation, or a decrease in bone resorption. Furthermore, the polypeptides described herein are suitable for subcutaneous or oral administration. The present disclosure also describes effective treatments for disorders or conditions including obesity, chronic weight management, type 2 diabetes mellitus, NAFLD, NASH, dyslipidemia, metabolic disorders, CKD, OA, OSA, and PCOS.

[0009] In one embodiment, the amino acid sequence of Formula I (SEQ ID NO: 4): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X6 is F, αMeF, or αMeF(2F); X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, Dab, S, E, or I; X 13 is αMeL, I, or L; X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q; X 19 is Q or A, X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, αMe-4-Pal, Q, or R; X 21 is A, Q, Orn, Aad, Aib, S, N, E, or T; X 23 is I or V, X 24 is a fatty acid, E, Q, D-Glu, or any amino acid with a functional group available for conjugation to N; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L, I, E, V, A, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, D-Ala, Aib, T, or A; X 30 is A or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp (hydroxyproline); X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P, Hyp, or E; X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, γE, or A; X 40 is absent or is G, E, S, A, or T; 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; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41 and X 42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, X 17 , X 20 , X 24 , and X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; A polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the C-terminal amino acid is optionally amidated.

[0010] In some embodiments, positions X have functional groups available for conjugation to fatty acids. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid in 10 is F or 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q.

[0011] In some embodiments, X 17 , X 20 , X 24 , and X 28 Only one of the amino acids has a functional group available for conjugation to a fatty acid.

[0012] In some embodiments, X 17 , X 20 , X 24 , or X 28 In some embodiments, only one of X is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid. 17 , X 20 , X 24 , or X 28 In some embodiments, only one of the amino acids is conjugated to the fatty acid via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0013] In one embodiment, the compound of Formula I' (SEQ ID NO: 1243): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X4 is G or D-Ala, X6 is F, αMeF, or αMeF(2F); X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, Dab, S, E, or I; X 13 is αMeL, I, or L; X 15 is D or E, X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, Q, or Orn; X 19 is Q or A, X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, αMe-4-Pal, Q, R, or L-Iva; X 21 is A, Q, Orn, Aad, Aib, S, N, E, or T; X 23 is I or V, X 24 is any amino acid having a functional group available for conjugation to a fatty acid, E, Q, D-Glu, N, or D-Gln; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L, I, E, V, A, Q, S, T, or Aad; X 28is any amino acid having a functional group available for conjugation to a fatty acid, E, Q, or A; X 29 is G, D-Ala, Aib, T, or A; X 30 is A, S, or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp (hydroxyproline); X 32 is S or P, X 34 is G or Aib, X 35 is A, Aib, E, D, H, or 4-Pal, or Orn; X 36 is P or Hyp, X 37 is P, Hyp, or E; X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, γE, or A, or Orn; X 40 is absent or is G, E, S, A, T, or D-Glu; 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, γE, D-Glu(e), Q, or H; X 41 is A, E, S, T, 4-Pal, D, G, γE, D-Glu, Q, or H, then X 42 is absent or is G, E, γE, D-Glu, or AEEA; X 42 is G, E, γE, D-Glu(e), or AEEA, then X 43 is absent or is E, γE, or D-Glu; X 43 is E, γE, or D-Glu, then X 44 is non-existent or E, and X44 If E, then X 45 is non-existent or E, and X 45 If E, then X 46 is absent or E, X 40 If X is non-existent, 41 ~X 46 is also non-existent, X 41 If X is non-existent, 42 ~X 46 is also non-existent, X 42 If X is non-existent, 43 ~X 46 is also non-existent, X 43 If X is non-existent, 44 ~X 46 is also non-existent, and X 44 If X is non-existent, 45 and X 46 is also non-existent, and X 45 If X is non-existent, 46 is also non-existent, X 17 , X 20 , X 24 , and X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; A polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the C-terminal amino acid is optionally amidated.

[0014] In some embodiments, positions X have functional groups available for conjugation to fatty acids. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 In some embodiments, the amino acid in 10 is F or 4-Pal. In some embodiments, X 12 is Orn, K, R, or Q.

[0015] In some embodiments, X 17 , X 20 , X 24 , and X 28 Only one of the amino acids has a functional group available for conjugation to a fatty acid.

[0016] In some embodiments, X 17 , X 20 , X 24 , or X 28 In some embodiments, one of X is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid. 17 , X 20 , X 24 , or X 28 In some embodiments, one of the amino acids is conjugated to the fatty acid via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0017] In some embodiments, X 17 , X 20 , X 24 , or X 28 None of the amino acids are conjugated to a fatty acid.

[0018] In one embodiment, the amino acid sequence of Formula II (SEQ ID NO: 5): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X2 is Aib, X 10 is F, 4-Pal, or F(4CN), X 12 is Orn, K, R, Q, Dap, or Dab; X 13 is αMeL, X 16 is K or Orn, X 17 is any amino acid having a functional group available for conjugation to a fatty acid; X 20 is Aib, αMe-4-Pal, or Q; X 21 is A, Q, or Orn, X 24 is E or Q, X 25 is W, Y, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L or I, X 28 is E or A, X 29 is G, D-Ala, or Aib, X 30 is A or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P or Hyp, X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, or γE; X 40is absent or is G, E, or S; X 40 is G, E, or S, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q, or H; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41 and X 42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, A polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the C-terminal amino acid is optionally amidated.

[0019] In some embodiments of the polypeptide of 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.

[0020] In some embodiments, X 17 is K and is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid. 17 is K and is conjugated to a fatty acid via a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is C 16 ~C 22 It is a fatty acid.

[0021] In some embodiments, X 17 is K and C 16 ~C 22 Fatty acids, X 17Amino acids and C 16 ~C 22 The fatty acid is conjugated via a linker between the fatty acid and the carboxyl group.

[0022] In one embodiment, the compound of Formula II' (SEQ ID NO: 1244): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y or NMeY; X2 is Aib, X 10 is F, 4-Pal, or F(4CN), X 12 is Orn, K, R, I, Q, Dap, or Dab; X 13 is αMeL, X 16 is K or Orn, X 17 is any amino acid having a functional group available for conjugation to a fatty acid; X 20 is Aib, αMe-4-Pal, Q, or L-Iva; X 21 is A, Q, or Orn, X 24 is E or Q, X 25 is W, Y, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L or I, X 28 is E, Q, or A, X 29 is G, D-Ala, or Aib, X 30 is A, S, or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P or Hyp, X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, or γE; X 40 is absent or is G, E, S, or D-Glu; 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; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 42 If is G, E, or γE, then X 43 is absent or E, X 43 If E, then X 44 is absent or E, X 40 If X is non-existent, 41 , X 42 , X 43 , and X 44 is also non-existent, X 41 If X is non-existent, 42 , X 43 , and X 44 is also non-existent, X 43 If X is non-existent, 44 is also non-existent, A polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the C-terminal amino acid is optionally amidated.

[0023] In some embodiments of the polypeptide of 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.

[0024] In some embodiments, X 17 is K and is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid. 17 is K and is conjugated to a fatty acid via a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is C 16 ~C 22 It is a fatty acid.

[0025] In some embodiments, X 17 is K and C 16 ~C 22 Fatty acids, X 17 Amino acids and C 16 ~C 22 The fatty acid is conjugated via a linker between the fatty acid and the carboxyl group.

[0026] In some embodiments, X 17 , X 20 , X 24 , or X 28 None of the amino acids are conjugated to a fatty acid.

[0027] In one embodiment, the amino acid sequence of Formula III (SEQ ID NO: 6): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X6 is F, αMeF, or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, S, E, or I; X 13 is αMeL, I, or L; X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q; X 19 is A or Q, X 20 is any amino acid having a functional group available for conjugation to the fatty acid Aib, Q, R, or αMe-4-Pal; X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn; X 23 is I or V, X 24 is any amino acid having a functional group available for conjugation to the fatty acid E, D-Glu, Q, or N; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L, I, E, V, A, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, Aib, T, D-Ala, or A; X 31 is P or E, X 34 is G or Aib, X 35 is A or E, X 36 is P, X 37 is P or E, X 38 is P, X 39 is E, S, G, or A; X 40 is absent or is G, E, S, A, or T; X 40 is G, E, S, A, or T, then X 41 is absent or is E, S, D, or G; X 41 If is E, S, D, or G, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41 and X 42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, X 10 If is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), then X 12 But I, X 17 , X 20 , X 24 , or X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; A polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the C-terminal amino acid is optionally amidated.

[0028] In some embodiments of Formula III, position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X20 , X 24 , or X 28 The amino acid in 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.

[0029] In some embodiments, X 17 , X 20 , X 24 , and X 28 Only one of the amino acids has a functional group available for conjugation to a fatty acid.

[0030] In some embodiments, X 17 , X 20 , X 24 , or X 28 In some embodiments, only one of X is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid. 17 , X 20 , X 24 , or X 28 In some embodiments, only one of the amino acids is conjugated to the fatty acid via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0031] In some embodiments, X 17 , X 20 , X 24 , or X 28 Only one of the is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 The fatty acid is conjugated via a linker between the fatty acid and the carboxyl group.

[0032] In one embodiment, the compound of Formula III' (SEQ ID NO: 1245): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X4 is G or D-Ala, X6 is F, αMeF, or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, S, E, or I; X 13 is αMeL, I, or L; X 15 is D or E, X 16 is K, Orn, A, or E; X 17 is a fatty acid, A, I, or Q, or any amino acid having a functional group available for conjugation to Orn; X 19 is A or Q, X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, Q, R, or αMe-4-Pal; X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn; X 23 is I or V, and X 24 is any amino acid having a functional group available for conjugation to the fatty acid E, D-Glu, Q, N, or D-Gln; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X27 is L, I, E, V, A, Aad, T, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, Aib, T, D-Ala, or A; X 31 is P or E, X 32 is S or P, X 34 is G or Aib, X 35 is A, D, or E, X 37 is P or E, X 39 is E, S, G, A, T, or Orn; X 40 is absent or is G, E, S, A, or T; 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, γE, or D-Glu; X 41 is E, S, D, G, Q, T, A, γE, or D-Glu, then X 42 is absent or is G, E, D-Glu, or γE; X 42 is G, E, D-Glu, or γE, then X 43 is absent or is E, γE, or D-Glu; X 43 is E, γE, or D-Glu, then X 44 is absent or E, X 44 If E, then X 45 is absent or E, X 45 If E, then X 46 is absent or E, X 40If X is non-existent, 41 ~X 46 is also non-existent, X 41 If X is non-existent, 42 ~X 46 is also non-existent, X 42 If X is non-existent, 43 ~X 46 is also non-existent, X 43 If X is non-existent, 44 ~X 46 is also non-existent, X 44 If X is non-existent, 45 and X 46 is also non-existent, X 45 If X is non-existent, 46 is also non-existent, The polypeptide is as follows: X6 is αMeF or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), X 11 is αMeS, X 13 is αMeL, X 24 is D-Glu, and / or X 25 is αMeY; X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), then X 12 But I, X 17 , X 20 , X 24 , or X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; A polypeptide or a pharmaceutically acceptable salt thereof is provided, wherein the C-terminal amino acid is optionally amidated.

[0033] In some embodiments of Formula III', position X has a functional group available for conjugation to a fatty acid.17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 In some embodiments, the amino acid in 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.

[0034] In some embodiments, X 17 , X 20 , X 24 , and X 28 Only one of the amino acids has a functional group available for conjugation to a fatty acid.

[0035] In some embodiments, X 17 , X 20 , X 24 , or X 28 In some embodiments, one of X is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid. 17 , X 20 , X 24 , or X 28 In some embodiments, one of the amino acids is conjugated to the fatty acid via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0036] In some embodiments, X 17 , X 20 , X 24 , or X 28 Only one of the is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In some embodiments, X is conjugated via a linker between the fatty acid and the 17 , X 20 , X 24 , or X 28 None of the amino acids are conjugated to a fatty acid.

[0037] 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.

[0038] In another embodiment, a method for treating a disease or disorder, including obesity, chronic weight management, type 2 diabetes mellitus, NAFLD, NASH, dyslipidemia, metabolic disorders, CKD, OA, OSA, and PCOS, is provided. Another embodiment provides a method for providing non-therapeutic weight loss, comprising administering to a subject in need thereof an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof. Such a method can at least comprise the step of administering to an individual in need thereof an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof.

[0039] In another embodiment, there is provided a polypeptide described herein for use in therapy, for example, for use in the treatment of a disease or disorder, including obesity, chronic weight management, type 2 diabetes mellitus, NAFLD, NASH, dyslipidemia, metabolic disorders, CKD, OA, OSA, and / or PCOS.

[0040] In another embodiment, there is provided a use of a polypeptide described herein in the manufacture of a medicament for treating a disease or disorder, including obesity, chronic weight management, type 2 diabetes mellitus, NAFLD, NASH, dyslipidemia, metabolic disorders, CKD, OA, OSA, and / or PCOS. DETAILED DESCRIPTION OF THE INVENTION

[0041] 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.

[0042] Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0043] GIP is a 42 amino acid peptide (SEQ ID NO: 1) and an incretin, which plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose.

[0044] GLP-1 is a 36-amino acid peptide and incretin that has been shown to stimulate glucose-dependent insulin secretion and prevent hyperglycemia in diabetic patients. The primary biologically active fragment of GLP-1 is a 30-amino acid C-terminal amidated peptide (GLP-1 7~36 ) (SEQ ID NO: 2).

[0045] Glucagon is a 29 amino acid peptide (SEQ ID NO: 3) that helps maintain blood glucose by binding to and activating glucagon receptors in liver cells, causing the liver to release glucose stored in the form of glycogen through a process called glycogenolysis.

[0046] In addition to T2DM, incretins and their analogs with activity at one or more of the GIP, GLP-1, and / or glucagon receptors have been described as having potential therapeutic value in several other conditions, diseases, or disorders, including, for example, obesity, NAFLD and NASH, dyslipidemia, metabolic syndrome, bone-related disorders, and neurodegenerative and / or cognitive disorders, e.g., Alzheimer's disease and Parkinson's disease. For example, 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. al.(2017)Physiol.Rev.97:721-766, Finan et al. See al. (2013) Sci. Transl. Med. 5:209, Holscher (2014) Biochem. Soc. Trans. 42:593-600.

[0047] As used herein, "about" means within a statistically significant range of a value, such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0048] As used herein, "activity," "activate," "activating," and the like, in reference to one or more of the GIP, GLP-1, or glucagon receptors, refer to the ability of a compound, e.g., a polypeptide described herein, to bind to the receptor and elicit a response in the receptor, as measured using assays known in the art, e.g., the in vitro assays described below.

[0049] As used herein, "amino acid having a functional group available for conjugation" means any natural (encoded) or unnatural (non-encoded) amino acid having a functional group that can be conjugated to a fatty acid directly or, for example, via a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo, and thiol groups. Examples of natural amino acids containing such functional groups include K (amino), C (thiol), E (carboxyl), and D (carboxyl).

[0050] As used herein, "conservative amino acid substitution" refers to the substitution of an amino acid with an amino acid that has similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, rigidity, etc.) and that has 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 exhaustively herein.

[0051] As used herein, "C 16 ~C 22 "Fatty acid" means a carboxylic acid having 16 to 22 carbon atoms. 16 ~C 22 Fatty acids can be saturated monoacids or saturated diacids. As used herein, "saturated" means that the fatty acid does not contain any carbon-carbon double or triple bonds.

[0052] As used herein, "effective amount" refers to an amount, concentration, or dosage of one or more polypeptides described herein or pharmaceutically acceptable salts thereof that, after single or multiple administration to an individual in need thereof, provides a desired effect in such an individual under diagnosis or treatment. An effective amount can be readily determined by one skilled in the art by using known techniques and observing results obtained under similar circumstances. In determining an effective amount for an individual, several factors are taken into consideration, including, but not limited to, the species of mammal; its size, age, and general health; the specific disease or disorder involved; the extent or involvement or severity of the disease or disorder; the individual patient's response; the specific polypeptide administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances.

[0053] As used herein, "extended duration of action" means that the binding affinity and activity of the polypeptide continues for a longer period than native human GIP, GLP-1, and glucagon peptides, allowing for less frequent dosing, such as at least once daily, or even three times weekly, twice weekly, or once weekly. The time-action profile of the polypeptide may be measured using known pharmacokinetic testing methods, such as those utilized in the Examples below.

[0054] As used herein, "polypeptide" or "peptide" refers to a polymer of amino acid residues. The term applies to polymers containing naturally occurring amino acids and to polymers containing one or more non-naturally occurring amino acids.

[0055] As used herein, "individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein.

[0056] As used herein, "treat," "treatment," "treating," and the like mean to inhibit, slow, arrest, or reverse the progression or severity of an existing condition, disease, disorder, or symptom.

[0057] As used herein, "triple agonist activity" in reference to a polypeptide means that the polypeptide has activity at each of the GIP, GLP-1, and glucagon receptors, and in particular, that the polypeptide has sufficient activity at each receptor to provide the benefits of agonism at that receptor while avoiding the undesirable side effects associated with excessive activity. Polypeptides with triple agonist activity (also referred to herein as "GGG polypeptides") have an extended duration of action at the GIP, GLP-1, and glucagon receptors, which advantageously allows for less frequent dosing, such as once daily, three times weekly, twice weekly, or once weekly.

[0058] As used herein, the term "sequence identity" refers to the degree of similarity between two sequences. The degree of sequence identity between two polypeptides can be expressed as a percentage, calculated as follows: % sequence identity = 100% x (number of identical amino acids) / (length of shortest consensus sequence).

[0059] The structural features of the polypeptides described herein result in the polypeptides having adequate activity at each of the GIP, GLP-1, and glucagon receptors to obtain the advantageous effects of activity at each receptor (i.e., triple agonist activity), but not so much that the activity at any one receptor either overwhelms the activity at the other two receptors or results in undesirable side effects when administered at a dose sufficient to provide activity at all three receptors. In some embodiments, the polypeptides described herein are partial agonists at the GLP-1 receptor, and exhibit a similar activity to native GLP-1 receptors as demonstrated by the HEK293 cell GLP-1 receptor internalization assay described herein. 7~36In other embodiments, the polypeptides described herein are full agonists at the GLP-1 receptor, exhibiting 80% or less agonism compared to native GLP-1 (SEQ ID NO: 2), as demonstrated by the HEK293 cell GLP-1 receptor internalization assay described herein. 7~36 In some embodiments, the polypeptides described herein are full agonists, exhibiting 80% or greater agonism compared to native glucagon (SEQ ID NO: 3), GIP (SEQ ID NO: 1), and GLP-1 (SEQ ID NO: 2). 7~36 (SEQ ID NO: 2) has greater potency at each of the glucagon, GIP, and GLP-1 receptors.

[0060] The structural features of the polypeptides described herein also result in polypeptides with many other beneficial attributes related to the potential development of the polypeptides as therapeutic treatments, including improved solubility of the analogs in aqueous solutions at near-neutral pH, improved chemical and physical formulation stability, improved peptide membrane permeability in the presence of permeation enhancers, an extended pharmacokinetic profile, and minimized potential for injection site reactions or immunogenicity.

[0061] It should be noted that the combination of beneficial features of the exemplary analogs described herein is not the result of any single modification in isolation, but instead is achieved through novel combinations of the structural features described herein.

[0062] Polypeptide Sequence In one embodiment, the amino acid sequence of Formula I (SEQ ID NO: 4): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X6 is F, αMeF, or αMeF(2F); X10 is F, 4-Pal, F(4CN), 3-Pal, F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, Dab, S, E, or I; X 13 is αMeL, I, or L; X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q; X 19 is Q or A, X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, αMe-4-pal, Q, or R; X 21 is A, Q, Orn, Aad, Aib, S, N, E, or T; X 23 is I or V, X 24 is a fatty acid, E, Q, D-Glu, or any amino acid with a functional group available for conjugation to N; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-pal; X 27 is L, I, E, V, A, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, D-Ala, Aib, T, or A; X 30 is A or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P, Hyp, or E; X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, γE, or A; X 40 is absent or is G, E, S, A, or T; 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; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41 and X 42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, X 17 , X 20 , X 24 , and X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; Provided herein are polypeptides, or pharmaceutically acceptable salts thereof, wherein the C-terminal amino acid is optionally amidated.

[0063] X 40 If absent, X 41 and X 42 is also absent, and the polypeptide contains a 39 amino acid sequence. 41 If absent, X 42 is also absent, and the polypeptide comprises a 40 amino acid sequence. 42If X is absent, the polypeptide contains a 41 amino acid sequence. 40 , X 41 , and X 42 None of the X 40 , X 41 , and X 42 are present), the polypeptide comprises a 42 amino acid sequence.

[0064] In one embodiment, X 40 is G, E, S, A, or T, and X 41 is non-existent, and X 42 is absent. In one such embodiment, the polypeptide comprises a 40 amino acid sequence. 40 is G, E, S, A, or T, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is absent. In one such embodiment, the polypeptide comprises a 41 amino acid sequence. 40 is G, E, S, A, or T, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is G, E, or γE. In one such embodiment, the polypeptide comprises a 42 amino acid sequence.

[0065] In some embodiments, the polypeptide of the invention has a nucleotide sequence at position X 17 , X 20 , X 24 , or X 28 In the formula (I), the amino acid includes any amino acid (natural or non-natural) having a functional group available for conjugation to a fatty acid. 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.

[0066] Thus, in some embodiments, position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid in is K.

[0067] 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 In some embodiments, X is Orn. 12 is K. In some embodiments, X 12 is R. In some embodiments, X 12 is Q.

[0068] In one embodiment, X 17 , X 20 , X 24 , and X 28 is an amino acid having a functional group available for conjugation to a fatty acid. In one embodiment, the conjugation is acylation.

[0069] In some embodiments, X 17 , X 20 , X 24 , and X 28 In some embodiments, only one of X is conjugated to a fatty acid. 17 , X 20 , X 24 , and X 28is conjugated to a fatty acid, optionally via a linker between the amino acid and the fatty acid. Thus, in some embodiments, X 17 , X 20 , X 24 , and X 28 In some embodiments, only one of the amino acids is conjugated to the fatty acid via a direct bond between the amino acid and the fatty acid or via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0070] Thus, in one embodiment, X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 17 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 20 is Aib, αMe-4-pal, Q, or R, and 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.

[0071] In one embodiment, X 20 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 20 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and 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.

[0072] In one embodiment, X 24 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 24 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-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.

[0073] In one embodiment, X 28 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 28 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20is Aib, αMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, conjugation is to the epsilon-amino group of the K side chain.

[0074] In one embodiment, the compound of Formula I' (SEQ ID NO: 1243): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X4 is G or D-Ala, X6 is F, αMeF, or αMeF(2F); X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, Dab, S, E, or I; X 13 is αMeL, I, or L; X 15 is D or E, X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, Q, or Orn; X 19 is Q or A, X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, αMe-4-Pal, Q, R, or L-Iva; X 21 is A, Q, Orn, Aad, Aib, S, N, E, or T; X 23 is I or V, X 24 is any amino acid having a functional group available for conjugation to a fatty acid, E, Q, D-Glu, N, or D-Gln; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L, I, E, V, A, Q, S, T, or Aad; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, Q, or A; X 29 is G, D-Ala, Aib, T, or A; X 30 is A, S, or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp (hydroxyproline); X 32 is S or P, X 34 is G or Aib, X 35 is A, Aib, E, D, H, or 4-Pal, or Orn; X 36 is P or Hyp, X 37 is P, Hyp, or E; X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, γE, or A, or Orn; X 40 is absent or is G, E, S, A, T, or D-Glu; X 40 is G, E, S, A, or T, then X 41 is absent or is A, E, S, T, 4-Pal, D, G, γE, D-Glu, Q, or H; X 41 is A, E, S, T, 4-Pal, D, G, γE, D-Glu, Q, or H, then X 42is absent or is G, E, γE, D-Glu, or AEEA; X 42 is G, E, γE, D-Glu(e), or AEEA, then X 43 is absent or is E, γE, or D-Glu; X 43 is E, γE, or D-Glu, then X 44 is non-existent or E, and X 44 If E, then X 45 is non-existent or E, and X 45 If E, then X 46 is absent or E, X 40 If X is non-existent, 41 ~X 46 is also non-existent, X 41 If X is non-existent, 42 ~X 46 is also non-existent, X 42 If X is non-existent, 43 ~X 46 is also non-existent, X 43 If X is non-existent, 44 ~X 46 is also non-existent, and X 44 If X is non-existent, 45 and X 46 is also non-existent, and X 45 If X is non-existent, 46 is also non-existent, X 17 , X 20 , X 24 , and X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; Provided herein are polypeptides, or pharmaceutically acceptable salts thereof, wherein the C-terminal amino acid is optionally amidated.

[0075] X 40 If absent, X 41~X 46 is also absent, and the polypeptide contains a 39 amino acid backbone. 41 If absent, X 42 ~X 46 is also absent, and the polypeptide comprises a 40 amino acid backbone. 42 If X is absent, the polypeptide contains a 41 amino acid backbone. 43 If X is absent, the polypeptide contains a 42 amino acid backbone. 44 If X is absent, the polypeptide contains a 43 amino acid backbone. 45 If X is absent, the polypeptide contains a 44 amino acid backbone. 46 If X is absent, the polypeptide contains a 45 amino acid backbone. 40 ~X 46 None of the X 40 ~X 46 are present), the polypeptide comprises a 46 amino acid sequence backbone.

[0076] In one embodiment, X 40 is G, E, S, A, or T, and X 41 is non-existent, and X 42 is absent. In one such embodiment, the polypeptide comprises a 40 amino acid sequence. 40 is G, E, S, A, or T, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is absent. In one such embodiment, the polypeptide comprises a 41 amino acid sequence. 40 is G, E, S, A, or T, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is G, E, or γE. In one such embodiment, the polypeptide comprises a 42 amino acid sequence.

[0077] In some embodiments, the polypeptide of the invention has a nucleotide sequence 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.

[0078] Thus, in some embodiments, position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid in is K.

[0079] 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 In some embodiments, X is Orn. 12 is K. In some embodiments, X 12 is R. In some embodiments, X 12 is Q.

[0080] In one embodiment, X 17 , X 20 , X 24 , and X 28is an amino acid having a functional group available for conjugation to a fatty acid. In one embodiment, the conjugation is acylation.

[0081] In some embodiments, X 17 , X 20 , X 24 , and X 28 In some embodiments, only one of X is conjugated to a fatty acid. 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid, optionally via a linker between the amino acid and the fatty acid. Thus, in some embodiments, X 17 , X 20 , X 24 , and X 28 In some embodiments, only one of the amino acids is conjugated to the fatty acid via a direct bond between the amino acid and the fatty acid or via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0082] Thus, in one embodiment, X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 17 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 20 is Aib, αMe-4-pal, Q, or R, and 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.

[0083] In one embodiment, X 20 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 20 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and 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.

[0084] In one embodiment, X 24 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 24 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-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.

[0085] In one embodiment, X 28 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 28 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, conjugation is to the epsilon-amino group of the K side chain.

[0086] In some embodiments, X 17 , X 20 , X 24 , and X 28 None of the amino acids are conjugated to a fatty acid.

[0087] In one embodiment, the amino acid sequence of Formula II (SEQ ID NO: 5): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X2 is Aib, X 10 are F, 4-Pal, and F(4CN), X 12 is Orn, K, R, Q, Dap, or Dab; X 13 is αMeL, X 16 is K or Orn, X 17is any amino acid having a functional group available for conjugation to a fatty acid; X 20 is Aib, αMe-4-pal, or Q; X 21 is A, Q, or Orn, X 24 is E or Q, X 25 is W, Y, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L or I, X 28 is E or A, X 29 is G, D-Ala, or Aib, X 30 is A or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P or Hyp, X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, or γE; X 40 is absent or is G, E, or S; X 40 is G, E, or S, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q, or H; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41 and X42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, Provided herein are polypeptides, or pharmaceutically acceptable salts thereof, wherein the C-terminal amino acid is optionally amidated.

[0088] As mentioned above, X 40 If absent, X 41 and X 42 is also absent, and the polypeptide contains a 39 amino acid sequence. 41 If absent, X 42 is also absent, and the polypeptide comprises a 40 amino acid sequence. 42 If X is absent, the polypeptide contains a 41 amino acid sequence. 40 , X 41 , and X 42 None of the X 40 , X 41 , and X 42 are present), the polypeptide comprises a 42 amino acid sequence.

[0089] In one embodiment, X 40 is G, E, or S, and X 41 is non-existent, and X 42 is absent. In one such embodiment, the polypeptide comprises a 40 amino acid sequence. 40 is G, E, or S, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is absent. In one such embodiment, the polypeptide comprises a 41 amino acid sequence. 40 is G, E, or S, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is G, E, or γE. In one such embodiment, the polypeptide comprises a 42 amino acid sequence.

[0090] In some embodiments of the polypeptide of Formula II, position X has a functional group available for conjugation to a fatty acid. 17 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 The amino acid in is K and the conjugation is to the epsilon-amino group of the K side chain.

[0091] 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 In some embodiments, X is Orn. 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 In some embodiments, X is Orn. 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 In some embodiments, X is Orn.10 is 4-Pal and X 12 is K. In some embodiments, X 10 is 4-Pal and X 12 is R.

[0092] In a further embodiment, X 10 is selected from F or 4-Pal. 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 αMe-4-pal. 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 41 is selected from E, S, T, 4-Pal, or H.

[0093] In some embodiments, X 10 is selected from the group consisting of F and 4-Pal. 12 is selected from the group consisting of Orn, K, R, and Q. In some embodiments, X 16 is K. In some embodiments, X 17is K. In some embodiments, X 20 is selected from the group consisting of Aib and αMe-4-pal. 24 is E. In some embodiments, X 28 is selected from the group consisting of E and A. In some embodiments, X 31 is selected from the group consisting of P, H, S, 4-Pal, T, and E. In some embodiments, X 35 is selected from the group consisting of A, Aib, and 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 the group consisting of E, S, and G. In some embodiments, X 40 is selected from the group consisting of G, E, and S. In some embodiments, X 41 is selected from the group consisting of E, S, T, 4-Pal, and H.

[0094] In some embodiments, X 10 is selected from F or 4-Pal, and X 12 is Orn, K, R, or Q, and X 16 is K and X 17 is K and X 20 is selected from Aib or αMe-4-pal, and X 24 But E and X 28 is selected from E or A, and X 31 is selected from P, H, S, 4-Pal, T, or E, and X 35 is selected from A, Aib, or E, and X 36 is P and X 37 is P and X 38 is P and X 39 is selected from E, S, or G, and X 40 is selected from G, E, or S, and X 41 is selected from E, S, T, 4-Pal, or H.

[0095] In some embodiments, X 10 is selected from the group consisting of F and 4-Pal; X 12 is selected from the group consisting of Orn, K, R, and Q; and X 16 is K and X 17 is K and X 20 is selected from the group consisting of Aib and αMe-4-pal, and X 24 But E and X 28 is selected from the group consisting of E and A, and X 31 is selected from the group consisting of P, H, S, 4-Pal, T, and E; and X 35 is selected from the group consisting of A, Aib, and E, and X 36 is P and X 37 is P and X 38 is P and X 39 is selected from the group consisting of E, S, and G; and X 40 is selected from the group consisting of G, E, and S; and X 41 is selected from the group consisting of E, S, T, 4-Pal, and H.

[0096] In some embodiments, the amino acid X 17 is conjugated to a fatty acid. 17 is conjugated to a fatty acid, optionally via a linker between the amino acid and the fatty acid. Thus, in some embodiments, the amino acid X 17 is conjugated to the fatty acid via a direct bond between the amino acid and the fatty acid or via a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is 16 ~C 22 It is a fatty acid.

[0097] Thus, in some embodiments, X 17 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22via a direct bond between the fatty acid or the amino acid and C 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 17 K is C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In one embodiment, the conjugation is via a linker between the X 17 K is to the epsilon-amino group of the side chain.

[0098] In one embodiment, the compound of Formula II' (SEQ ID NO: 1244): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y or NMeY; X2 is Aib, X 10 is F, 4-Pal, or F(4CN), X 12 is Orn, K, R, I, Q, Dap, or Dab; X 13 is αMeL, X 16 is K or Orn, X 17 is any amino acid having a functional group available for conjugation to a fatty acid; X 20 is Aib, αMe-4-Pal, Q, or L-Iva; X 21 is A, Q, or Orn, X 24 is E or Q, X 25 is W, Y, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L or I, X 28is E, Q, or A, X 29 is G, D-Ala, or Aib, X 30 is A, S, or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P or Hyp, X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, or γE; X 40 is absent or is G, E, S, or D-Glu; 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; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 42 If is G, E, or γE, then X 43 is absent or E, X 43 If E, then X 44 is absent or E, X 40 If X is non-existent, 41 , X 42 , X 43 , and X 44 is also non-existent, X 41 If X is non-existent, 42 , X 43 , and X 44is also non-existent, X 43 If X is non-existent, 44 is also non-existent, Provided herein are polypeptides, or pharmaceutically acceptable salts thereof, wherein the C-terminal amino acid is optionally amidated.

[0099] As mentioned above, X 40 If absent, X 41 ~X 44 is also absent, and the polypeptide contains a 39 amino acid backbone. 41 If absent, X 42 ~X 44 is also absent, and the polypeptide comprises a 40 amino acid backbone. 42 If X is non-existent, 43 ~X 44 is also absent, and the polypeptide contains a 41 amino acid backbone. 43 If X is non-existent, 44 is also absent, and the polypeptide contains a 42 amino acid backbone. 40 ~X 44 None of the X 40 ~X 44 are present), the polypeptide comprises a 44 amino acid sequence backbone.

[0100] In one embodiment, X 40 is G, E, or S, and X 41 is non-existent, and X 42 is absent. In one such embodiment, the polypeptide comprises a 40 amino acid sequence. 40 is G, E, or S, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X 42 is absent. In one such embodiment, the polypeptide comprises a 41 amino acid sequence. 40 is G, E, or S, and X 41 is E, S, T, 4-Pal, D, G, Q, or H, and X42 is G, E, or γE. In such an embodiment, the polypeptide comprises a 42 amino acid sequence backbone.

[0101] In some embodiments of the polypeptide of Formula II', position X has a functional group available for conjugation to a fatty acid. 17 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 The amino acid in is K and the conjugation is to the epsilon-amino group of the K side chain.

[0102] 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 In some embodiments, X is Orn. 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 In some embodiments, X is Orn. 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 X12 is Q. In some embodiments, X 10 is 4-Pal and X 12 In some embodiments, X is Orn. 10 is 4-Pal and X 12 is K. In some embodiments, X 10 is 4-Pal and X 12 is R.

[0103] In a further embodiment, X 10 is selected from F or 4-Pal. 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 αMe-4-pal. 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 41 is selected from E, S, T, 4-Pal, or H.

[0104] In some embodiments, X 10 is selected from the group consisting of F and 4-Pal. 12is selected from the group consisting of Orn, K, R, and Q. In some embodiments, X 16 is K. In some embodiments, X 17 is K. In some embodiments, X 20 is selected from the group consisting of Aib and αMe-4-pal. 24 is E. In some embodiments, X 28 is selected from the group consisting of E and A. In some embodiments, X 31 is selected from the group consisting of P, H, S, 4-Pal, T, and E. In some embodiments, X 35 is selected from the group consisting of A, Aib, and 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 the group consisting of E, S, and G. In some embodiments, X 40 is selected from the group consisting of G, E, and S. In some embodiments, X 41 is selected from the group consisting of E, S, T, 4-Pal, and H.

[0105] In some embodiments, X 10 is selected from F or 4-Pal, and X 12 is Orn, K, R, or Q, and X 16 is K and X 17 is K and X 20 is selected from Aib or αMe-4-pal, and X 24 But E and X 28 is selected from E or A, and X 31 is selected from P, H, S, 4-Pal, T, or E, and X 35 is selected from A, Aib, or E, and X 36 is P and X 37 is P and X 38 is P and X 39is selected from E, S, or G, and X 40 is selected from G, E, or S, and X 41 is selected from E, S, T, 4-Pal, or H.

[0106] In some embodiments, X 10 is selected from the group consisting of F and 4-Pal; X 12 is selected from the group consisting of Orn, K, R, and Q; and X 16 is K and X 17 is K and X 20 is selected from the group consisting of Aib and αMe-4-pal, and X 24 But E and X 28 is selected from the group consisting of E and A, and X 31 is selected from the group consisting of P, H, S, 4-Pal, T, and E; and X 35 is selected from the group consisting of A, Aib, and E, and X 36 is P and X 37 is P and X 38 is P and X 39 is selected from the group consisting of E, S, and G; and X 40 is selected from the group consisting of G, E, and S; and X 41 is selected from the group consisting of E, S, T, 4-Pal, and H.

[0107] In some embodiments, the amino acid X 17 is conjugated to a fatty acid. 17 is conjugated to a fatty acid, optionally via a linker between the amino acid and the fatty acid. Thus, in some embodiments, the amino acid X 17 is conjugated to the fatty acid via a direct bond between the amino acid and the fatty acid or via a linker between the amino acid and the fatty acid. In some embodiments, the fatty acid is 16 ~C 22 It is a fatty acid.

[0108] Thus, in some embodiments, X 17is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 via a direct bond between the fatty acid or the amino acid and C 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 17 K is C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In one embodiment, the conjugation is via a linker between the X 17 K is to the epsilon-amino group of the side chain.

[0109] In one embodiment, the amino acid sequence of Formula III (SEQ ID NO: 6): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X6 is F, αMeF, or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, S, E, or I; X 13 is αMeL, I, or L; X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q; X 19 is A or Q, X 20is any amino acid having a functional group available for conjugation to the fatty acid Aib, Q, R, or αMe-4-pal; X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn; X 23 is I or V, X 24 is any amino acid having a functional group available for conjugation to the fatty acid E, D-Glu, Q, or N; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-pal; X 27 is L, I, E, V, A, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, Aib, T, D-Ala, or A; X 31 is P or E, X 34 is G or Aib, X 35 is A or E, X 36 is P, X 37 is P or E, X 38 is P, X 39 is E, S, G, or A; X 40 is absent or is G, E, S, A, or T; X 40 is G, E, S, A, or T, then X 41 is absent or is E, S, D, or G; X 41 If is E, S, D, or G, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41and X 42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, and X 10 If is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), then X 12 But I, X 17 , X 20 , X 24 , or X 28 At least one of the 16 ~C 22 any amino acid having a functional group available for conjugation to a fatty acid; Provided herein are polypeptides, or pharmaceutically acceptable salts thereof, wherein the C-terminal amino acid is optionally amidated.

[0110] As mentioned above, X 40 If absent, X 41 and X 42 is also absent, and the polypeptide contains a 39 amino acid sequence. 41 If absent, X 42 is also absent, and the polypeptide comprises a 40 amino acid sequence. 42 If X is absent, the polypeptide contains a 41 amino acid sequence. 40 , X 41 , and X 42 None of the X 40 , X 41 , and X 42 are present), the polypeptide comprises a 42 amino acid sequence.

[0111] In one embodiment, X 40 is G, E, S, A, or T, and X 41 is absent. In one such embodiment, the polypeptide comprises a 40 amino acid sequence. 40 is G, E, S, A, or T, and X 41is E, S, D, or G. In one such embodiment, the polypeptide comprises a 41 amino acid sequence. 40 is G, E, S, A, or T, and X 41 is E, S, D, or G, and X 42 is G, E, or γE. In one such embodiment, the polypeptide comprises a 42 amino acid sequence.

[0112] In some embodiments of the polypeptide of Formula III, position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid in is K and the conjugation is to the epsilon-amino group of the K side chain.

[0113] In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), 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.

[0114] In some embodiments, X 17 , X 20 , X 24 , and X 28 Only one of them is C 16 ~C 22 It is an amino acid that has a functional group available for conjugation to a fatty acid.

[0115] In some embodiments, X 17 , X 20 , X24 , and X 28 is conjugated to a fatty acid, optionally via a linker between the amino acid and the fatty acid. Thus, in some embodiments, X 17 , X 20 , X 24 , and X 28 In one embodiment, only one of X is conjugated to the fatty acid via a direct bond between the amino acid and the fatty acid or via a linker between the amino acid and the fatty acid. 17 , X 20 , X 24 , and X 28 In some embodiments, only one of the amino acids is conjugated to the fatty acid via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0116] Thus, in one embodiment, X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 17 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 20 is Aib, αMe-4-pal, Q, or R, and 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.

[0117] In one embodiment, X 20 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids,16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 20 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and 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.

[0118] In one embodiment, X 24 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 24 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-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.

[0119] In one embodiment, X 28 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 28 is K and C 16 ~C22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, conjugation is to the epsilon-amino group of the K side chain.

[0120] In one embodiment, the compound of Formula III' (SEQ ID NO: 1245): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X4 is G or D-Ala, X6 is F, αMeF, or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, S, E, or I; X 13 is αMeL, I, or L; X 15 is D or E, X 16 is K, Orn, A, or E; X 17 is a fatty acid, A, I, or Q, or any amino acid having a functional group available for conjugation to Orn; X 19 is A or Q, X 20is any amino acid having a functional group available for conjugation to a fatty acid, Aib, Q, R, or αMe-4-Pal; X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn; X 23 is I or V, X 24 is any amino acid having a functional group available for conjugation to the fatty acid E, D-Glu, Q, N, or D-Gln; X 25 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 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, Aib, T, D-Ala, or A; X 31 is P or E, X 32 is S or P, X 34 is G or Aib, X 35 is A, D, or E, X 37 is P or E, X 39 is E, S, G, A, T, or Orn; X 40 is absent or is G, E, S, A, or T; 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, γE, or D-Glu; X 41 is E, S, D, G, Q, T, A, γE, or D-Glu, then X 42 is absent or is G, E, D-Glu, or γE; X42 is G, E, D-Glu, or γE, then X 43 is absent or is E, γE, or D-Glu; X 43 is E, γE, or D-Glu, then X 44 is absent or E, X 44 If E, then X 45 is non-existent or E, and X 45 If E, then X 46 is absent or E, X 40 If X is non-existent, 41 ~X 46 is also non-existent, X 41 If X is non-existent, 42 ~X 46 is also non-existent, X 42 If X is non-existent, 43 ~X 46 is also non-existent, X 43 If X is non-existent, 44 ~X 46 is also non-existent, X 44 If X is non-existent, 45 and X 46 is also non-existent, X 45 If X is non-existent, 46 is also non-existent, The polypeptide is as follows: X6 is αMeF or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), X 11 is αMeS, X 13 is αMeL, X 24 is D-Glu, and / or X 25 is αMeY; X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), then X12 But I, X 17 , X 20 , X 24 , or X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; Provided herein are polypeptides, or pharmaceutically acceptable salts thereof, wherein the C-terminal amino acid is optionally amidated.

[0121] As mentioned above, X 40 If absent, X 41 ~X 46 is also absent, and the polypeptide contains a 39 amino acid backbone. 41 If does not exist, then X 42 ~X 46 is also absent, and the polypeptide comprises a 40 amino acid backbone. 42 If X is absent, the polypeptide contains a 41 amino acid backbone. 43 If X is absent, the polypeptide contains a 42 amino acid backbone. 44 If X is absent, the polypeptide contains a 43 amino acid backbone. 45 If X is absent, the polypeptide contains a 44 amino acid backbone. 46 If X is absent, the polypeptide contains a 45 amino acid backbone. 40 ~X 46 (in other words, X 40 ~X 46 are present), the polypeptide comprises a 46 amino acid sequence backbone.

[0122] In one embodiment, X 40 is G, E, S, A, or T, and X 41 is absent. In one such embodiment, the polypeptide comprises a 40 amino acid sequence. 40 is G, E, S, A, or T, and X 41is E, S, D, or G. In one such embodiment, the polypeptide comprises a 41 amino acid sequence. 40 is G, E, S, A, or T, and X 41 is E, S, D, or G, and X 42 is G, E, or γE. In one such embodiment, the polypeptide comprises a 42 amino acid sequence.

[0123] In some embodiments of the polypeptide of Formula III', position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid at position X has a functional group available for conjugation to a fatty acid. 17 , X 20 , X 24 , or X 28 The amino acid in is K and the conjugation is to the epsilon-amino group of the K side chain.

[0124] In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), 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.

[0125] In some embodiments, the polypeptide is 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), X 11 is αMeS, X 13 is αMeL, X 16 is Orn, X 24 is D-Glu, and / or X 25 and αMeY.

[0126] In some embodiments, X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2).

[0127] In some embodiments, X1 is Y, X2 is Aib, X4 is G, X6 is αMeF(2F), and X 10 is 4-Pal and X 12 is I and X 13 is αMeL and X 15 is D and X 16 is Orn and X 19 is Q and X 20 is αMe-4-Pal, and X 21 is E or Orn, and X 23 is I and X 24 is D-Glu, and X 25 is αMeY and X 27 is I or V, and X 28 is E and X 29 is G and X 31 is P and X 34 is G and X 35 is A or E, and X 37 is P and X 39 is E or S, and X 40 is G or T, and X 41 is E, S, or G, and X 42 is non-existent, and X 43 is non-existent, and X 44 is non-existent.

[0128] In some embodiments, X 11 is S and X 21 is Orn and X 27 is I and X 35 is E and X 39 is E and X 40 is T and X 41 is E.

[0129] In some embodiments, X11 is αMeS, and X 21 is E and X 27 is V and X 35 is A and X 39 is S and X 40 is G and X 41 is S.

[0130] In some embodiments, X 17 , X 20 , X 24 , and X 28 Only one of them is C 16 ~C 22 It is an amino acid that has a functional group available for conjugation to a fatty acid.

[0131] In some embodiments, X 17 , X 20 , X 24 , and X 28 is conjugated to a fatty acid, optionally via a linker between the amino acid and the fatty acid. Thus, in some embodiments, X 17 , X 20 , X 24 , and X 28 In one embodiment, only one of X is conjugated to the fatty acid via a direct bond between the amino acid and the fatty acid or via a linker between the amino acid and the fatty acid. 17 , X 20 , X 24 , and X 28 In some embodiments, only one of the amino acids is conjugated to the fatty acid via a linker between the amino acid and the fatty acid. 16 ~C 22 It is a fatty acid.

[0132] Thus, in one embodiment, X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22In one embodiment, X is conjugated via a linker between the fatty acid and 17 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 20 is Aib, αMe-4-pal, Q, or R, and 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.

[0133] In one embodiment, X 20 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 20 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and 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.

[0134] In one embodiment, X 24 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 24 is K and C 16 ~C 22Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-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.

[0135] In one embodiment, X 28 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 In one embodiment, X is conjugated via a linker between the fatty acid and 28 is K and C 16 ~C 22 Fatty acids, amino acids and C 16 ~C 22 In such embodiments, X is conjugated via a linker between the fatty acid and the 17 is A, I, or Q, and X 20 is Aib, αMe-4-pal, Q, or R, and X 24 is E, Q, D-Glu, or N. In one embodiment, conjugation is to the epsilon-amino group of the K side chain.

[0136] The amino acid sequences of the polypeptides described herein incorporate naturally occurring amino acids, which are typically depicted herein using the standard single-letter code (e.g., L=leucine), as well as alpha-methyl substituted residues of natural amino acids (e.g., α-methyl leucine (αMeL)), and certain other unnatural amino acids, such as alpha amino isobutyric acid (Aib). The structures of these amino acids are shown below. [ka] [ka] [ka] [ka] [ka]

[0137] As used herein, "Orn" refers to L-ornithine. As used herein, "4-Pal" or "4Pal" refers to 3-(4-pyridyl)-L-alanine or (S)-2-amino-3-(pyridin-4-yl)propanoic acid. As used herein, "3-Pal" or "3Pal" refers to 3-(3-pyridyl)-L-alanine or (S)-2-amino-3-(pyridin-3-yl)propanoic acid. As used herein, "αMe-4-Pal" or "αMe4Pal" refers to alpha-methyl-3-(4-pyridyl)-L-alanine. As used herein, "αMeY" refers to alpha-methyl-L-tyrosine. As used herein, "αMeL" refers to alpha-methyl-leucine. As used herein, "D-Ala" and "α" each refer to D-alanine. As used herein, "D-Glu" and "e" each refer to D-glutamic acid. As used herein, "Aib" refers to 2-aminoisobutyric acid. As used herein, "NMeY" refers to N-methyl-tyrosine. As used herein, "Dap" refers to (S)-2,3-diaminopropanoic acid. As used herein, "Dab" refers to (S)-2,4-diaminobutanoic acid. As used herein, "Hyp" refers to hydroxy-L-proline. As used herein, "K(Ac)" refers to N 6As used herein, "γGlu" means gamma-L-glutamic acid. As used herein, "Aad" means (S)-2-aminohexanedioic acid. As used herein, "F(4CN)" means 4-cyano-L-phenylalanine or (S)-2-amino-3-(4-cyanophenyl)propanoic acid. As used herein, "F(4NO2)" means 4-nitro-L-phenylalanine or (S)-2-amino-3-(4-nitrophenyl)propanoic acid. As used herein, "αMeS" means alpha-methyl-L-serine. As used herein, "αMeF" means alpha-methyl-L-phenylalanine. As used herein, "αMeF(2F)" means alpha-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.

[0138] As noted above, in some embodiments, the polypeptides described herein comprise a fatty acid moiety conjugated, e.g., by a direct bond or a linker, to a natural or unnatural amino acid having a functional group available for conjugation. Such conjugation is sometimes referred to as acylation. In certain examples, the amino acid having a functional group available for conjugation can be K, C, E, and D. In particular examples, the amino acid having a functional group available for conjugation is K, and the conjugation is to the epsilon-amino group of the K side chain.

[0139] Acylation of the polypeptides described herein can be achieved by acylation at position X in SEQ ID NO: 4 or 6. 17 or X 20 or X 24 or X 28 or at position X in SEQ ID NO: 517 The fatty acid, and in certain embodiments, the linker and / or amino acid sequence backbone, may act as an albumin binder, offering the potential to generate long-acting compounds.

[0140] In some embodiments, the polypeptides described herein comprise a C 1 -amino acid that is chemically conjugated either directly to a functional group of an amino acid or via a linker. 16 ~C 22 Fatty acids are utilized. The length and composition of the fatty acids influence the half-life of polypeptides, their efficacy in in vivo animal models, and their solubility and stability. 16 ~C 22 Conjugation to saturated fatty mono- or diacids results in polypeptides that exhibit desirable half-lives, desirable efficacy in in vivo animal models, and desirable solubility and stability characteristics.

[0141] Saturated C for use herein 16 ~C 22 Examples 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 (C 18 monoacid), octadecanedioic acid (C 18 diacid), nonadecylic acid (nonadecanoic acid) (C 19 monoacid), nonadecanedioic acid (C 19 diacid), alachadic acid (eicosanoic acid) (C 20 monoacid), eicosanedioic acid (C 20 diacid), heneicosylic acid (heneicosanoic acid) (C 21 monoacid), heneicosanedioic acid (C 21 diacid), behenic acid (docosanoic acid) (C 22 monoacid), docosanedioic acid (C 22 diacids), including branched and substituted derivatives thereof.

[0142] In certain cases, C 16 ~C 22 Fatty acids are saturated C 18 monoacid, saturated C 18 Diacid, saturated C 19 monoacid, saturated C 19 Diacid, saturated C 20 monoacid, saturated C 20 In certain instances, C 16 ~C 22 The fatty acid is octadecanedioic acid (C 18 diacid) or eicosanedioic acid (C 20 diacid).

[0143] In certain cases, the linker can have one or more (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties or εK, optionally in combination with 1 to 4 amino acids.

[0144] In cases where the linker includes at least one amino acid, the amino acid can be one to five Glu or γGlu amino acid residues. In some cases, the linker can include one, two, three, four, or five Glu or γGlu amino acid residues, including the D-forms of the Glu or γGlu amino acid residues. For example, the linker can include one, two, three, or four γGlu amino acid residues. Alternatively, the linker can include one to five (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl, "AEEA") or one to five amino acid residues (e.g., Glu or γGlu amino acids) used in combination with one to five εK moieties. Specifically, the linker can include 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. εIn some cases, the linker can be a combination of one, two, or three γGlu amino acids and one or two (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) or εK moieties.

[0145] For example, in some embodiments, the polypeptides described herein have the following formula: (γGlu) a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2) p It has a linker with the structure —CO2H and a fatty acid component, 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 from 14 to 20.

[0146] 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 from 14 to 20.

[0147] In some embodiments, a is 0, b is 1, c is 1 or 2, and p is 16 or 18.

[0148] For example, in some embodiments, a is 0, b is 1, c is 1, and p is 16, the structure of which is shown below. [ka]

[0149] For example, in some embodiments, a is 0, b is 1, c is 1, and p is 18, the structure of which is shown below. [ka]

[0150] In some embodiments, a is 0, b is 1, c is 2, and p is 16, the structure of which is shown below. [ka]

[0151] In some embodiments, a is 0, b is 1, c is 2, and p is 18, the structure of which is shown below. [ka]

[0152] In some embodiments, a is 0, b is 2, c is 1, and p is 16 or 18.

[0153] For example, in some embodiments, a is 0, b is 2, c is 1, and p is 16, the structure of which is shown below. [ka]

[0154] In some embodiments, a is 0, b is 2, c is 1, and p is 18, the structure of which is shown below. [ka]

[0155] In some embodiments, a is 0, b is 0, c is 2, and p is 16 or 18.

[0156] For example, in some embodiments, a is 0, b is 0, c is 2, and p is 16, the structure of which is shown below. [ka]

[0157] In some embodiments, a is 0, b is 0, c is 2, and p is 18, the structure of which is shown below. [ka]

[0158] In some embodiments, a is 0, b is 0, c is 3, and p is 16 or 18.

[0159] For example, in some embodiments, a is 0, b is 0, c is 3, and p is 16, the structure of which is shown below. [ka]

[0160] In some embodiments, a is 0, b is 0, c is 3, and p is 18, the structure of which is shown below. [ka]

[0161] In some embodiments, a is 1, b is 1, c is 1, and p is 16 or 18.

[0162] For example, in some embodiments, a is 1, b is 1, c is 1, and p is 16, the structure of which is shown below. [ka]

[0163] For example, in some embodiments, a is 1, b is 1, c is 1, and p is 18, the structure of which is shown below. [ka]

[0164] In some embodiments, the polypeptides described herein have the following formula: (γGlu) d -(εK) e -(γGlu) f -CO-(CH2) q It has a linker with the structure —CO2H and a fatty acid component, 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 from 14 to 20.

[0165] For example, in one embodiment, d is 0, e is 2, f is 1, and q is an integer from 14 to 20. In some embodiments, d is 0, e is 2, f is 1, and q is 16 or 18.

[0166] For example, in some embodiments, d is 0, e is 2, f is 1, and q is 16, the structure of which is shown below. [ka]

[0167] For example, in some embodiments, d is 0, e is 2, f is 1, and q is 18, the structure of which is shown below. [ka]

[0168] As shown in the chemical structures of Examples 1-1229 below, the linker-fatty acid moiety can be linked to an amino group present at positions 17, 20, 24, or 28. In some embodiments, the linker-fatty acid moiety is linked to or conjugated to an amino acid present at position 17, e.g., the epsilon (ε)-amino group of the lysine (K) side chain present at position 17. In some embodiments, the linker-fatty acid moiety is linked to or conjugated to an amino acid present at position 20, e.g., the epsilon (ε)-amino group of the lysine (K) side chain present at position 20. In some embodiments, the linker-fatty acid moiety is linked to or conjugated to an amino acid present at position 24, e.g., the epsilon (ε)-amino group of the lysine (K) side chain present at position 24. In some embodiments, the linker-fatty acid moiety is linked to or conjugated to the amino acid present at position 28, e.g., the epsilon (ε)-amino group of the lysine (K) side chain present at position 28.

[0169] In some embodiments, the polypeptides described herein comprise a sequence selected from any one of SEQ ID NOs: 7-1242 (described below in Examples 1-1236). In some embodiments, the polypeptides described herein consist of a sequence selected from any one of SEQ ID NOs: 7-1242 (described below in Examples 1-1236).

[0170] In some embodiments, the polypeptides described herein are amidated. In some embodiments, the polypeptides described herein have a C-terminal group modification, where the modification is NH or absent. In some embodiments, the polypeptides described herein have an OH group at the C-terminus.

[0171] In addition to the sequences described herein, the polypeptides described herein may include one or more conservative amino acid substitutions, provided, however, that the polypeptide is still capable of binding to and activating GIP, GLP-1, and glucagon receptors.

[0172] In certain embodiments of a polypeptide of any of the formulas described herein, the polypeptide is an isotopic derivative of any one of the polypeptides described herein or a pharmaceutically acceptable salt thereof. It is understood that isotopic derivatives can be prepared using any of a variety of art-recognized techniques. For example, isotopic derivatives can generally be prepared by performing the procedures disclosed in the examples described herein, substituting an isotopically labeled reagent for a non-isotopically labeled reagent. In one embodiment of a polypeptide of any of the formulas described herein or a pharmaceutically acceptable salt thereof, the polypeptide is a deuterated derivative of any one of the polypeptides described herein.

[0173] In the polypeptides of the present invention, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise specified, when an atom is specifically designated as "H" or "hydrogen," the atom is understood to have hydrogen at its natural abundance isotopic composition. Also, unless otherwise specified, when an atom is specifically designated as "D" or "deuterium," the atom is understood to have deuterium at an abundance substantially greater than the natural abundance of deuterium, which is 0.015%.

[0174] The affinity of the polypeptides described herein for each of the GIP, GLP-1, and glucagon receptors can be measured using techniques known in the art for measuring receptor binding levels, including, for example, those described in the Examples below, and is generally expressed as an inhibition constant (Ki) value. The activity of the polypeptides described herein at each of the receptors can also be measured using techniques known in the art, including, for example, the in vitro activity assays described below, and is generally expressed as an effective concentration 50 (EC 50 ) value, which is the concentration of compound that causes half-maximal simulation of the dose-response curve.

[0175] The polypeptides described herein can react with any of several inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and general techniques for preparing them are well known in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Revised 2nd Edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use herein include sodium salts, potassium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts. Thus, in some embodiments, pharmaceutically acceptable salt forms of GGG polypeptides are provided herein. In some embodiments, the pharmaceutically acceptable form is selected from the sodium or potassium salt. In some embodiments, the pharmaceutically acceptable form is selected from the group consisting of sodium and potassium salts. In some preferred embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0176] 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) assays described herein are also suitable for administration by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal) or oral routes (e.g., tablets, capsules). app ) assay and in vivo ileal absorption assay are useful tools for assessing the potential for oral delivery of polypeptides.

[0177] In another embodiment, provided herein is a pharmaceutical composition 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, for example, Remington: The Science and Practice of Pharmacy (Troy, ed., 21st ed., Lippincott, Williams & Wilkins, 2006).

[0178] 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 suitable for oral administration (e.g., tablet, capsule). In some embodiments, the pharmaceutical composition is administered parenterally. In some embodiments, the pharmaceutical composition is administered orally.

[0179] In addition to the anatomical and physiological characteristics of the gastrointestinal tract, the physiochemical properties of peptides can make efficient oral delivery of peptides difficult. In one embodiment, a pharmaceutical composition for oral administration comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof and a permeation enhancer. In one embodiment, a pharmaceutical composition for oral administration comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof, a permeation enhancer, and a protease inhibitor.

[0180] As used herein, the term "permeation enhancer" refers to a permeation enhancer that promotes oral absorption of the polypeptide of the present invention. As used herein, permeation enhancer refers to permeation enhancers such as sodium decanoate (C10), sodium taurodeoxycholate (NaTDC), lauroyl carnitine (LC), dodecyl maltoside, dodecyl phosphatidylcholine (SNAC), rhamnolipid, and permeation enhancers reported in the literature, such as phosphatase permeation inhibitors PIP-250 and PIP-640. See Pharmaceutics. 2019 Jan;11(1):41, (see Biomaterials. 2012;33:3464-3474), ZOT (zonula occludens toxin), ΔG (fragment of ZOT) (see Int. J. Pharm. 2009;365,121-130). In one embodiment, the permeation enhancer is selected from sodium decanoate, sodium taurodeoxycholate, and lauroylcarnitine. In one embodiment, the permeation enhancer is selected from the group consisting of C10, LC, or NaTDC. In one embodiment, the permeation enhancer is selected from the group consisting of sodium decanoate, sodium taurodeoxycholate, and lauroylcarnitine. In one embodiment, the permeation enhancer is selected from the group consisting of C10, LC, and NaTDC.

[0181] As used herein, the term "protease inhibitor" refers to a protease inhibitor that may be selected from the group consisting of protein-based, peptide-based, and small molecule-based. Protease inhibitors are well known and may include 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"), among others. In one embodiment, the protease inhibitor is selected from the group consisting of SBTI, SBTCI, and SFTI. In one embodiment, the protease inhibitor is an SBTI.

[0182] The present disclosure also provides and therefore encompasses novel intermediates and methods for synthesizing the polypeptides described herein or pharmaceutically acceptable salts thereof. The intermediates and polypeptides described herein can be prepared by various techniques known in the art. For example, methods using chemical synthesis are illustrated in the examples below, or using biological expression. The specific synthetic steps for each of the described pathways can be combined in different ways to prepare the polypeptides described herein. The reagents and starting materials are readily available to those skilled in the art.

[0183] For chemical synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, for example, CEM (Charlotte, North Carolina), CSBio (Menlo Park, California), and Gyros Protein Technologies Inc. (Tucson, Arizona). Reagents for solid-phase synthesis are readily available from commercial suppliers. Solid-phase synthesis equipment can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during the reaction, coupling, deprotecting, and capping unreacted amino acids.

[0184] For biological expression, standard recombinant techniques can be used to construct a polynucleotide having a nucleic acid sequence encoding the amino acid sequence of all or a portion of a polypeptide, incorporate the polynucleotide into a recombinant expression vector, and introduce the vector into host cells, such as bacteria, yeast, and mammalian cells, to produce the polypeptide. See, for example, Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4th ed. 2012). Polypeptides can be readily produced in mammalian cells, such as CHO, NSO20, HEK293, BHK, or COS cells; bacterial cells, such as E. coli, Bacillus subtilis, or Pseudomonas fluorescens; insect cells; or fungal or yeast cells, which are cultured using techniques known in the art. Vectors containing the polynucleotide sequence of interest can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. Various methods of protein purification can be used, and such methods are known in the art.

[0185] The polypeptides described herein can be used to treat various conditions, disorders, diseases, or symptoms. Specifically, methods are provided for treating obesity in an individual, comprising at least the 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.

[0186] Additionally, methods for chronic weight management in an individual are provided, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a polypeptide as described herein or a pharmaceutically acceptable salt thereof.

[0187] Additionally, methods are provided for treating type 2 diabetes mellitus (T2DM) in an individual, such methods comprising at least the 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.

[0188] Additionally, methods are provided for treating non-alcoholic fatty liver disease (NAFLD) in an individual, such methods comprising at least the 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.

[0189] Additionally, methods are provided for treating non-alcoholic steatohepatitis (NASH) in an individual, such methods comprising at least the 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.

[0190] Additionally, methods for treating dyslipidemia in an individual are provided, such methods comprising at least the 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.

[0191] Additionally, methods for treating metabolic syndrome in an individual are provided, such methods comprising at least the 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.

[0192] Additionally, methods for treating osteoarthritis (OA) in an individual are provided, which comprise at least the 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.

[0193] Additionally, methods are provided for treating obesity-related sleep apnea (OSA) in an individual, such methods comprising at least the 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.

[0194] Additionally, methods are provided for treating polycystic ovary syndrome (PCOS) in an individual, such methods comprising at least the 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.

[0195] Additionally, methods are provided for inducing non-therapeutic weight loss in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a polypeptide as described herein, or a pharmaceutically acceptable salt thereof.

[0196] In these methods, the effectiveness of the composition can be assessed, for example, by observing a significant reduction in blood glucose, observing a significant increase in insulin, observing a significant reduction in HbA1c, and / or observing a significant reduction in body weight.

[0197] Alternatively, the polypeptides described herein or their pharmaceutically acceptable salts can be used to improve bone strength in individuals in need thereof. In some instances, the individuals in need thereof have hypoostosis or osteoid hypoplasia, or are recovering from a fracture, joint muscle restoration surgery, prosthetic implants, dental implants, 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.

[0198] Additionally, provided herein are polypeptides or pharmaceutically acceptable salts thereof described herein for use in therapy. In some embodiments, provided herein are polypeptides or pharmaceutically acceptable salts thereof described herein for use in the treatment of obesity, chronic weight management, type 2 diabetes mellitus, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS). Also provided are uses of polypeptides or pharmaceutically acceptable salts thereof described herein for inducing non-therapeutic weight loss.

[0199] Additionally provided is the use of a polypeptide or a pharmaceutically acceptable salt thereof described herein in the manufacture of a medicament for treating obesity, chronic weight management, type 2 diabetes mellitus, 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 is the use of a polypeptide or a pharmaceutically acceptable salt thereof described herein in the manufacture of a medicament for inducing non-therapeutic weight loss.

[0200] The polypeptides or pharmaceutical compositions described herein may be provided as part of a kit. In some cases, the kit includes a device, such as a syringe, auto-injector, or pump, for administering at least one polypeptide (and optionally at least one additional therapeutic agent) to an individual.

[0201] Additional non-limiting embodiments are described below.

[0202] 1. [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X6 is F, αMeF, or αMeF(2F); X 10 is F, 4-Pal, F(4CN), 3-Pal, F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, Dab, S, E, or I; X 13 is αMeL, I, or L; X 16 is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q; X 19 is Q or A, X 20 is any amino acid having a functional group available for conjugation to a fatty acid, Aib, αMe-4-Pal, Q, or R; X 21 is A, Q, Orn, Aad, Aib, S, N, E, or T; X 23 is I or V, X 24 is a fatty acid, E, Q, D-Glu, or any amino acid with a functional group available for conjugation to N; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L, I, E, V, A, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, D-Ala, Aib, T, or A; X 30 is A or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P, Hyp, or E; X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, γE, or A; X 40 is absent or is G, E, S, A, or T; 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; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; X 40 If X is non-existent, 41 and X 42 is also non-existent, X 41 If X is non-existent, 42 is also non-existent, X 17 , X 20 , X 24 , and X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; A polypeptide or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is optionally amidated.

[0203] 2. X 40 is G, E, S, A, or T; X 41 But it is non-existent, X 42 2. The polypeptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein:

[0204] 3. X 40 is G, E, S, A, or T; X 41 is E, S, T, 4-Pal, D, G, Q, or H; X 42 2. The polypeptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein:

[0205] 4. X 40 is G, E, S, A, or T; X 41 is E, S, T, 4-Pal, D, G, Q, or H; X 42 is G, E, or γE.

[0206] 5. Position X, bearing a functional group available for conjugation to a fatty acid 17 , X 20 , X 24 , or X 285. The polypeptide according to any one of embodiments 1 to 4, wherein the amino acid in is K, C, E, or D.

[0207] 6. Position X, bearing a functional group available for conjugation to a fatty acid 17 , X 20 , X 24 , or X 28 6. The polypeptide of embodiment 5, wherein the amino acid in is K.

[0208] 7.X 10 7. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 6, wherein is F or 4-Pal.

[0209] 8.X 12 is Orn, K, R, or Q, or a pharmaceutically acceptable salt thereof.

[0210] 9.X 17 , X 20 , X 24 , and X 28 9. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 8, wherein only one of the amino acids is an amino acid having a functional group available for conjugation to a fatty acid.

[0211] 10.X 17 , X 20 , X 24 , and X 28 10. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 9, wherein only one of the amino acids is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid.

[0212] 11.X 17 , X 20 , X 24 , and X 28 11. The polypeptide of embodiment 10, or a pharmaceutically acceptable salt thereof, wherein only one of the amino acid residues is conjugated to the fatty acid via a linker between the amino acid and the fatty acid.

[0213] 12. Fatty acids are C 16 ~C 22 12. The polypeptide of any one of embodiments 1 to 11, which is a fatty acid.

[0214] 13. X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 conjugated via a linker between the fatty acid, X 20 is Aib, αMe-4-Pal, Q, or R; X 24 is E, Q, D-Glu, or N; X 28 is E or A.

[0215] 14. X 17 is A, I, or Q, X 20 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 conjugated via a linker between the fatty acid, X 24 is E, Q, D-Glu, or N; X 28 is E or A.

[0216] 15. X 17 is A, I, or Q, X 20 is Aib, αMe-4-Pal, Q, or R; X 24 is K and C 16 ~C 22to fatty acids, via direct bonds or with amino acids, 16 ~C 22 conjugated via a linker between the fatty acid, X 28 is E or A.

[0217] 16. X 17 is A, I, or Q, X 20 is Aib, αMe-4-Pal, Q, or R; X 24 is E, Q, D-Glu, or N; X 28 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 13. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 12, wherein the polypeptide is conjugated via a linker between the fatty acid and the polypeptide.

[0218] 17. [ka] Including, During the ceremony, X2 is Aib, X 10 is F, 4-Pal, or F(4CN), X 12 is Orn, K, R, Q, Dap, or Dab; X 13 is αMeL, X 16 is K or Orn, X 17 is any amino acid having a functional group available for conjugation to a fatty acid; X 20 is Aib, αMe-4-Pal, or Q; X 21 is A, Q, or Orn, X24 is E or Q, X 25 is W, Y, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L or I, X 28 is E or A, X 29 is G, D-Ala, or Aib, X 30 is A or G, X 31 is P, H, S, 4-Pal, E, T, K(Ac), or Hyp; X 34 is G or Aib, X 35 is A, Aib, E, H, or 4-Pal; X 36 is P or Hyp, X 37 is P or Hyp, X 38 is P or Hyp, X 39 is E, S, G, T, H, 4-Pal, or γE; X 40 is absent or is G, E, or S; X 40 is G, E, or S, then X 41 is absent or is E, S, T, 4-Pal, D, G, Q, or H; X 41 is E, S, T, 4-Pal, D, G, Q, or H, then X 42 is absent or is G, E, or γE; 2. The polypeptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is optionally amidated.

[0219] 18. X 40 is G, E, or S; X 41 But it is non-existent, X42 18. The polypeptide of embodiment 17, or a pharmaceutically acceptable salt thereof, wherein

[0220] 19. X 40 is G, E, or S; X 41 is E, S, T, 4-Pal, D, G, Q, or H; X 42 18. The polypeptide of embodiment 17, or a pharmaceutically acceptable salt thereof, wherein

[0221] 20. X 40 is G, E, or S; X 41 is E, S, T, 4-Pal, D, G, Q, or H; X 42 18. The polypeptide or a pharmaceutically acceptable salt thereof according to embodiment 17, wherein is G, E, or γE.

[0222] 21.X 17 is K, C, E, or D.

[0223] 22.X 17 22. The polypeptide of embodiment 21, wherein

[0224] 23.X 10 23. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 17 to 22, wherein is F or 4-Pal.

[0225] 24.X 12 is Orn, K, R, or Q, or a pharmaceutically acceptable salt thereof.

[0226] 25.X 17is K and is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid, or a pharmaceutically acceptable salt thereof.

[0227] 26.X 17 is K and is conjugated to the fatty acid via a linker between the amino acid and the fatty acid, or a pharmaceutically acceptable salt thereof.

[0228] 27. Fatty acids are C 16 ~C 22 27. The polypeptide of any one of embodiments 17 to 26, which is a fatty acid.

[0229] 28.X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 28. The polypeptide of embodiment 27, or a pharmaceutically acceptable salt thereof, conjugated via a linker between the polypeptide and the fatty acid.

[0230] 29. [ka] Including, During the ceremony, X1 is Y, NMeY, or H; X2 is Aib, X6 is F, αMeF, or αMeF(2F); X 10 is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), or Y; X 11 is S or αMeS, X 12 is Orn, K, R, Q, Dap, S, E, or I; X 13 is αMeL, I, or L; X 16is K, Orn, A, or E; X 17 is any amino acid having a functional group available for conjugation to a fatty acid, A, I, or Q; X 19 is A or Q, X 20 is any amino acid having a functional group available for conjugation to the fatty acid Aib, Q, R, or αMe-4-Pal; X 21 is A, Aad, Aib, S, N, Q, E, T, or Orn; X 23 is I or V, X 24 is any amino acid having a functional group available for conjugation to a fatty acid, E, D-Glu, Q, or N; X 25 is W, Y, F, 4-Pal, αMeY, or αMe-4-Pal; X 27 is L, I, E, V, A, Q, or S; X 28 is any amino acid having a functional group available for conjugation to a fatty acid, E, or A; X 29 is G, Aib, T, D-Ala, or A; X 31 is P or E, X 34 is G or Aib, X 35 is A or E, X 36 is P, X 37 is P or E, X 38 is P, X 39 is E, S, G, or A; X 40 is absent or is G, E, S, A, or T; X 40 is G, E, S, A, or T, then X 41is absent or is E, S, D, or G; X 41 If is E, S, D, or G, then X 42 is absent or is G, E, or γE; X 10 If is F, 3-Pal, 4-Pal, F(4CN), F(4NO2), then X 12 But I, X 17 , X 20 , X 24 , or X 28 at least one of which is an amino acid having a functional group available for conjugation to a fatty acid; 2. The polypeptide of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is optionally amidated.

[0231] 30. X 40 is G, E, S, A, or T; X 41 But it is non-existent, X 42 30. The polypeptide of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein

[0232] 31. X 40 is G, E, S, A, or T; X 41 is E, S, D, or G, X 42 30. The polypeptide of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein

[0233] 32. X 40 is G, E, S, A, or T; X 41 is E, S, D, or G, X 42 is G, E, or γE, or a pharmaceutically acceptable salt thereof.

[0234] 33. Position X, having a functional group available for conjugation to a fatty acid 17 , X 20 , X 24 , or X 28 33. The polypeptide of any one of embodiments 29 to 32, wherein the amino acid in is K, C, E, or D.

[0235] 34. Position X, with a functional group available for conjugation to a fatty acid 17 , X 20 , X 24 , or X 28 6. The polypeptide of embodiment 5, wherein the amino acid in is K.

[0236] 35.X 10 is F, 3-Pal, 4-Pal, F(4CN), or F(4NO2), and X 12 or a pharmaceutically acceptable salt thereof according to any one of embodiments 29 to 34, wherein

[0237] 36.X 10 is Y and X 12 is Orn, K, R, Q, Dap, S, E, or I, or a pharmaceutically acceptable salt thereof.

[0238] 37.X 17 , X 20 , X 24 , or X 28 37. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 29 to 36, wherein only one of the amino acids is an amino acid having a functional group available for conjugation to a fatty acid.

[0239] 38.X 17 , X 20 , X 24 , or X 2838. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 29 to 37, wherein only one of the amino acids is conjugated to the fatty acid via a direct bond or via a linker between the amino acid and the fatty acid.

[0240] 39.X 17 , X 20 , X 24 , or X 28 39. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 38, wherein only one of the amino acid residues is conjugated to the fatty acid via a linker between the amino acid and the fatty acid.

[0241] 40. Fatty acids are C 16 ~C 22 40. The polypeptide of any one of embodiments 29 to 39, which is a fatty acid.

[0242] 41. X 17 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 conjugated via a linker between the fatty acid, X 20 is Aib, αMe-4-Pal, Q, or R; X 24 is E, Q, D-Glu, or N; X 28 is E or A.

[0243] 42. X 17 is A, I, or Q, X 20 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 conjugated via a linker between the fatty acid, X 24is E, Q, D-Glu, or N; X 28 is E or A.

[0244] 43. X 17 is A, I, or Q, X 20 is Aib, αMe-4-Pal, Q, or R; X 24 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 conjugated via a linker between the fatty acid, X 28 is E or A.

[0245] 44. X 17 is A, I, or Q, X 20 is Aib, αMe-4-Pal, Q, or R; X 24 is E, Q, D-Glu, or N; X 28 is K and C 16 ~C 22 to fatty acids, via direct bonds or with amino acids, 16 ~C 22 41. The polypeptide or pharmaceutically acceptable salt thereof of embodiment 40, which is conjugated via a linker between the fatty acid and the polypeptide.

[0246] 45. The polypeptide or a pharmaceutically acceptable salt thereof according to 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 1 to 4 amino acids.

[0247] 46. ​​The polypeptide or a pharmaceutically acceptable salt thereof according to embodiment 45, wherein the amino acid is Glu, γGlu, or a combination thereof.

[0248] 47. The polypeptide or a pharmaceutically acceptable salt thereof according to embodiment 45 or 46, wherein the linker comprises 1 to 4 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties or eK moieties.

[0249] 48. The polypeptide of embodiment 47, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 4 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

[0250] 49. The linker is (γGlu) a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2) p 46. ​​The polypeptide or a pharmaceutically acceptable salt thereof according to embodiment 45, comprising the structure -CO2H, wherein a is 0 or 1, b is 0, 1, or 2, c is 1, 2, or 3, and p is an integer of 14 to 20.

[0251] 50. The polypeptide or a pharmaceutically acceptable salt thereof according to embodiment 49, wherein a is 0.

[0252] 51. The polypeptide of embodiment 49, or a pharmaceutically acceptable salt thereof, wherein a is 1.

[0253] 52. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 51, wherein b is 0.

[0254] 53. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 51, wherein b is 1.

[0255] 54. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 51, wherein b is 2.

[0256] 55. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 54, wherein c is 1.

[0257] 56. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 54, wherein c is 2.

[0258] 57. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 54, wherein c is 3.

[0259] 58. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 57, wherein p is 16.

[0260] 59. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 49 to 57, wherein p is 18.

[0261] 60. A polypeptide selected from the group consisting of SEQ ID NOs: 7 to 504, or a pharmaceutically acceptable salt thereof.

[0262] 61. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 60, wherein the C-terminus is amidated.

[0263] 62. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-61, wherein the pharmaceutically acceptable salt is selected from sodium, potassium, trifluoroacetate, hydrochloride, or acetate.

[0264] 63. The polypeptide or a pharmaceutically acceptable salt thereof according to embodiment 62, wherein the pharmaceutically acceptable salt is selected from sodium and potassium.

[0265] 64. A pharmaceutical composition comprising a polypeptide according to any one of embodiments 1 to 63 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0266] 65. The pharmaceutical composition of embodiment 64, wherein the composition is formulated for oral administration.

[0267] 66. The pharmaceutical composition of embodiment 64, wherein the composition is formulated for subcutaneous administration.

[0268] 67. A method for treating a disease or condition selected from the group consisting of diabetes mellitus, obesity, chronic 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), the method comprising: 64. A 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.

[0269] 68. A polypeptide or a pharmaceutically acceptable salt thereof described by any one of embodiments 1-63, for use in therapy.

[0270] 69. The polypeptide or pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 63, for use in the treatment of a disease or condition selected from the group consisting of diabetes mellitus, obesity, chronic 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).

[0271] 70. Use of the polypeptide or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 63 in the manufacture of a medicament for treating a disease or condition selected from the group consisting of diabetes mellitus, obesity, chronic 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).

[0272] 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 of SEQ ID NOs: 7-1242.

[0273] 72. The polypeptide of embodiment 71, comprising a sequence selected from the group consisting of SEQ ID NOs: 7-1242.

[0274] 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 of SEQ ID NOs: 294-775 or 1146-1240.

[0275] 74. A polypeptide selected from the group consisting of SEQ ID NOs: 294 to 775 or 1146 to 1240, or a pharmaceutically acceptable salt thereof.

[0276] 75. The polypeptide of embodiment 74, selected from the group consisting of SEQ ID NOs: 692, 700, 702, 705, 706, 716, 718, 743, 747, 749, 767, or a pharmaceutically acceptable salt thereof.

[0277] 76. The polypeptide is selected from the group consisting of natural glucagon (SEQ ID NO: 3), GIP (SEQ ID NO: 1), and GLP-1 7~36 2. The polypeptide of any of the preceding embodiments, which has greater potency at each of the glucagon, GIP, and GLP-1 receptors compared to (SEQ ID NO: 2).

[0278] 77. The polypeptide or a pharmaceutically acceptable salt thereof according to any of the above embodiments, wherein one or more hydrogen atoms are replaced by deuterium.

[0279] This invention is further illustrated by the following examples which should not be construed as limiting.

[0280] Peptide synthesis Example 1: Example 1 is a compound represented by the following description: Y-Aib-QGTFTSDFSK-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γ-Glu)-CO—(CH2) 18 —COH)AQ-Aib-AFIEYLLAGGPSSGEPPPSEG-NH (SEQ ID NO: 7).

[0281] Below is a depiction of the structure of Example 1 using standard single letter amino acid codes, except for residues Aib2, αMeL13, and Aib20, the structures of these amino acid residues have been expanded. [ka]

[0282] The peptide backbone of Example 1 is synthesized on a Symphony 12-channel multiplex peptide synthesizer (Protein Technologies, Inc. Tucson, AZ) using Fluorenylmethyloxycarbonyl (Fmoc) / tert-Butyl (t-Bu) chemistry.

[0283] The resin consists 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 are used. Fmoc-Lys(Mtt)-OH is used for lysine at position 17, and Boc-Tyr(tBu)-OH is used for tyrosine at position 1. The Fmoc group is removed using 20% ​​piperidine in DMF before each coupling step (2 × 7 min). All standard amino acid couplings are performed using equimolar ratios of Fmoc amino acid (0.3 M), diisopropylcarbodiimide (0.9 M), and Oxyma (0.9 M) at a 9-fold molar excess over theoretical peptide loading, for 1 h for primary amines and 3 h for secondary amines. The exception is coupling to Ca-methylated amino acids, which are coupled for 3 h. After peptide backbone synthesis is complete, the resin is thoroughly washed six times with DCM to remove residual DMF. The Mtt protecting group at lysine 17 is selectively removed from the peptide resin by two 40-minute treatments with 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM.

[0284] Subsequent attachment of the fatty acid-linker moiety is achieved by coupling 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid at-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.), and mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China). A three-fold excess of reagents (AA:PyAOP:DIPEA = 1:1:1 mol / mol) is used for each coupling, which is 1 hour long.

[0285] Upon completion of the synthesis, the peptide resin is washed with DCM and air-dried thoroughly. The dried resin is treated with 10 mL of cleavage cocktail (trifluoroacetic acid:water:triisopropylsilane, 95:2.5:2.5 v / v) for 2 hours at room temperature. The resin is filtered off and washed twice with 2 mL of undiluted TFA each time, and the combined filtrate is treated with 5 volumes (by volume) of cold diethyl ether (-20°C) to precipitate the crude peptide. The peptide / ether suspension is then centrifuged at 3500 rpm for 2 minutes to form a solid pellet. The supernatant is decanted, and the solid pellet is triturated twice more with ether and dried in vacuo. The crude peptide is solubilized in 20% acetonitrile / 20% acetic acid / 60% water and purified by RP-HPLC on a Luna 5 μm phenyl-hexyl preparative column (21 × 250 mm, Phenomenex) with a linear gradient of 100% acetonitrile and 0.1% TFA / water buffer system (30 to 50% acetonitrile in 60 min). Peptide purity is assessed using analytical RP-HPLC, with a pooling standard of >95%. The main pool purity of Example 1 is found to be 98.8%. Subsequent lyophilization of the final main product pool yields the lyophilized peptide TFA salt. Molecular weights are determined by LC-MS (observed: M + 4H). + / 4=1226.8, calculated value M+4H + / 4=1226.9).

[0286] Examples 2 to 1236 Polypeptides according to Example 2 (SEQ ID NO: 8) through Example 1236 (SEQ ID NO: 1242) are prepared substantially as described by the procedure of Example 1. These are listed below in Table 1. Additional descriptions of certain examples are provided after Table 1.

[0287] Table 1 [Table 1-1] TIFF2026505071000037.tif66170

[0288] (Continued from Table 1) [Table 1-2] TIFF2026505071000039.tif183170

[0289] (Continued from Table 1) [Table 1-3] TIFF2026505071000041.tif172170

[0290] (Continued from Table 1) [Table 1-4] TIFF2026505071000043.tif167170

[0291] (Continued from Table 1) [Table 1-5] TIFF2026505071000045.tif178170

[0292] (Continued from Table 1) [Table 1-6] TIFF2026505071000047.tif172170

[0293] (Continued from Table 1) [Table 1-7] TIFF2026505071000049.tif193170

[0294] (Continued from Table 1) [Table 1-8] TIFF2026505071000051.tif172170

[0295] (Continued from Table 1) [Table 1-9] TIFF2026505071000053.tif178170

[0296] (Continued from Table 1) [Table 1-10] TIFF2026505071000055.tif220170

[0297] (Continued from Table 1) [Table 1-11] TIFF2026505071000057.tif231170

[0298] (Continued from Table 1) [Table 1-12] TIFF2026505071000059.tif199170

[0299] (Continued from Table 1) [Table 1-13] TIFF2026505071000061.tif236170

[0300] (Continued from Table 1) [Table 1-14] TIFF2026505071000063.tif215170

[0301] (Continued from Table 1) [Table 1-15] TIFF2026505071000065.tif220170

[0302] (Continued from Table 1) [Table 1-16] TIFF2026505071000067.tif225170

[0303] (Continued from Table 1) [Table 1-17] TIFF2026505071000069.tif172170

[0304] (Continued from Table 1) [Table 1-18] TIFF2026505071000071.tif167170

[0305] (Continued from Table 1) [Table 1-19] TIFF2026505071000073.tif172170

[0306] (Continued from Table 1) [Table 1-20] TIFF2026505071000075.tif220170

[0307] (Continued from Table 1) [Table 1-21] TIFF2026505071000077.tif215170

[0308] (Continued from Table 1) [Table 1-22] TIFF2026505071000079.tif199170

[0309] (Continued from Table 1) [Table 1-23] TIFF2026505071000081.tif215170

[0310] (Continued from Table 1) [Table 1-24] TIFF2026505071000083.tif209170

[0311] (Continued from Table 1) [Table 1-25] TIFF2026505071000085.tif231170

[0312] (Continued from Table 1) [Table 1-26] TIFF2026505071000087.tif241170

[0313] (Continued from Table 1) [Table 1-27] TIFF2026505071000089.tif230170

[0314] (Continued from Table 1) [Table 1-28] TIFF2026505071000091.tif215170

[0315] (Continued from Table 1) [Table 1-29] TIFF2026505071000093.tif193170

[0316] (Continued from Table 1) [Table 1-30] TIFF2026505071000095.tif215170

[0317] (Continued from Table 1) [Table 1-31] TIFF2026505071000097.tif215170

[0318] (Continued from Table 1) [Table 1-32] TIFF2026505071000099.tif193170

[0319] (Continued from Table 1) [Table 1-33] TIFF2026505071000101.tif255169 TIFF2026505071000102.tif66170

[0320] (Continued from Table 1) [Table 1-34] TIFF2026505071000104.tif204170

[0321] (Continued from Table 1) [Table 1-35] TIFF2026505071000106.tif171113

[0322] (Continued from Table 1) [Table 1-36] TIFF2026505071000108.tif231170

[0323] (Continued from Table 1) [Table 1-37] TIFF2026505071000110.tif230170

[0324] (Continued from Table 1) [Table 1-38] TIFF2026505071000112.tif241170

[0325] (Continued from Table 1) [Table 1-39] TIFF2026505071000114.tif193170

[0326] (Continued from Table 1) [Table 1-40] TIFF2026505071000116.tif204170

[0327] (Continued from Table 1) [Table 1-41] TIFF2026505071000118.tif172170

[0328] (Continued from Table 1) [Table 1-42] TIFF2026505071000120.tif204170

[0329] (Continued from Table 1) [Table 1-43] TIFF2026505071000122.tif252170

[0330] (Continued from Table 1) [Table 1-44] TIFF2026505071000124.tif241170

[0331] (Continued from Table 1) [Table 1-45] TIFF2026505071000126.tif241170 TIFF2026505071000127.tif29170

[0332] (Continued from Table 1) [Table 1-46] TIFF2026505071000129.tif255169

[0333] (Continued from Table 1) [Table 1-47] TIFF2026505071000131.tif252170 TIFF2026505071000132.tif50170

[0334] (Continued from Table 1) [Table 1-48] TIFF2026505071000134.tif255169 TIFF2026505071000135.tif23170

[0335] (Continued from Table 1) [Table 1-49] TIFF2026505071000137.tif247170 TIFF2026505071000138.tif66170

[0336] (Continued from Table 1) [Table 1-50] TIFF2026505071000140.tif252170 TIFF2026505071000141.tif34170

[0337] (Continued from Table 1) [Table 1-51] TIFF2026505071000143.tif247170 TIFF2026505071000144.tif23170

[0338] (Continued from Table 1) [Table 1-52] TIFF2026505071000146.tif255169

[0339] (Continued from Table 1) [Table 1-53] TIFF2026505071000148.tif255169 TIFF2026505071000149.tif7170

[0340] (Continued from Table 1) [Table 1-54] TIFF2026505071000151.tif210170

[0341] (Continued from Table 1) [Table 1-55] TIFF2026505071000153.tif172170

[0342] (Continued from Table 1) [Table 1-56] TIFF2026505071000155.tif236170

[0343] (Continued from Table 1) [Table 1-57] TIFF2026505071000157.tif220170

[0344] (Continued from Table 1) [Table 1-58] TIFF2026505071000159.tif172170

[0345] (Continued from Table 1) [Table 1-59] TIFF2026505071000161.tif151170

[0346] (Continued from Table 1) [Table 1-60] TIFF2026505071000163.tif172170

[0347] (Continued from Table 1) [Table 1-61] TIFF2026505071000165.tif172170

[0348] (Continued from Table 1) [Table 1-62] TIFF2026505071000167.tif187170

[0349] (Continued from Table 1) [Table 1-63] TIFF2026505071000169.tif182170

[0350] (Continued from Table 1) [Table 1-64] TIFF2026505071000171.tif236170

[0351] A structural description of one particular embodiment is provided below.

[0352] Example 694 (SEQ ID NO: 700) [ka]

[0353] Example 699 (SEQ ID NO: 705) [ka]

[0354] Example 686 (SEQ ID NO: 692) [ka]

[0355] Example 712 (SEQ ID NO: 718) [ka]

[0356] Example 741 (SEQ ID NO: 747) [ka]

[0357] Example 510 (SEQ ID NO: 516) [ka]

[0358] Example 883 (SEQ ID NO: 889) [ka]

[0359] Example 548 (SEQ ID NO: 554) [ka]

[0360] Example 558 (SEQ ID NO: 564) [ka]

[0361] Example 1027 (SEQ ID NO: 1033) [ka]

[0362] In vitro function Functional activity: Functional activity was determined in HEK-293 clonal cell lines expressing GIP-R, GLP-1R, and GcgR. Each receptor cell line was treated with peptides (20-point concentration-response curves with 2.75-fold serial dilutions prepared using a Labcyte Echo acoustic liquid handler) in a 20 μl assay volume in DMEM (Gibco catalog number 31053) supplemented with 1× GlutaMAX™ (L-alanyl-L-glutamine dipeptide, Gibco catalog number 35050), 0.1% casein (Sigma catalog number C4765), 1% HSA (human serum albumin, Sigma catalog number A3782), 500 μM IBMX (3-isobutyl-1-methylxanthine), and 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0363] After 30 minutes of incubation at 37°C, the resulting increase in intracellular cAMP is quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit (62AM4PEJ). Briefly, cAMP-d2 conjugate in cell lysis buffer followed by antibody, anti-cAMP-cAMP-Eu, also in cell lysis buffer, is used. 3+ -cryptate to detect intracellular cAMP levels. The resulting competitive assay is incubated at room temperature for at least 60 minutes and then detected using a Pherastar instrument (BMG Labtech) with excitation at 320 nm and emission at 665 nm and 620 nm. The raw data value (emission at 665 nm / 620 nm x 10,000) is inversely proportional to the amount of cAMP present. The raw data value was converted to cAMP (nM) per well using a cAMP standard curve.

[0364] The amount of cAMP (nM) produced in each well was measured using human GLP-1(7-36)NH2, human glucagon (Gcg), or human GIP ( 1~42) converted to percent of the maximal response observed with either NH2. Relative EC2 values ​​were calculated by nonlinear regression analysis using percent maximal response versus added peptide concentration fitted to a four-parameter logistic equation. 50 Find the value.

[0365] Exemplary analogs and relative EC values ​​for hGIP(1-42)NH2, hGLP-1(7-36)NH2, and hGcg 50 The geometric means of the data are shown in Table 2 below. Table 2. Functional cAMP potency (relative EC for exemplary analogs and comparators in the presence of 1% HSA 50 ). [Table 2-1] TIFF2026505071000183.tif57125

[0366] (Continued from Table 2) [Table 2-2] TIFF2026505071000185.tif69125

[0367] (Continued from Table 2) [Table 2-3] TIFF2026505071000187.tif69125

[0368] (Continued from Table 2) [Table 2-4] TIFF2026505071000189.tif69125

[0369] (Continued from Table 2) [Table 2-5] TIFF2026505071000191.tif69125

[0370] (Continued from Table 2) [Table 2-6] TIFF2026505071000193.tif69125

[0371] (Continued from Table 2) [Table 2-7] TIFF2026505071000195.tif69125

[0372] (Continued from Table 2) [Table 2-8] TIFF2026505071000197.tif69125

[0373] (Continued from Table 2) [Table 2-9] TIFF2026505071000199.tif69125

[0374] (Continued from Table 2) [Table 2-10] TIFF2026505071000201.tif69125

[0375] (Continued from Table 2) [Table 2-11] TIFF2026505071000203.tif69125

[0376] (Continued from Table 2) [Table 2-12] TIFF2026505071000205.tif69125

[0377] (Continued from Table 2) [Table 2-13] TIFF2026505071000207.tif69125

[0378] (Continued from Table 2) [Table 2-14] TIFF2026505071000209.tif69125

[0379] (Continued from Table 2) [Table 2-15] TIFF2026505071000211.tif69125

[0380] (Continued from Table 2) [Table 2-16] TIFF2026505071000213.tif69125

[0381] (Continued from Table 2) [Table 2-17] TIFF2026505071000215.tif69125

[0382] (Continued from Table 2) [Table 2-18] TIFF2026505071000217.tif69125

[0383] (Continued from Table 2) [Table 2-19] TIFF2026505071000219.tif69125

[0384] (Continued from Table 2) [Table 2-20] TIFF2026505071000221.tif69125

[0385] (Continued from Table 2) [Table 2-21] TIFF2026505071000223.tif69125

[0386] (Continued from Table 2) [Table 2-22] TIFF2026505071000225.tif69125

[0387] (Continued from Table 2) [Table 2-23] Note: EC2 of human GLP-1(7-36)NH2 at human GLP-1R, human Gcg at human GcgR, and human GIP(1-42)NH2 at human GIP-R 50Determination: Peptide concentration range was 346 pM to 9950 nM. EC of the examples in human GLP-1R, human GcgR, and human GIP-R 50 Determination: The peptide concentration range was 6.87 pM to 9.95 μM.

[0388] As shown in Table 2, in the presence of HSA, the exemplary analogs have lower agonist activity than the native ligands as determined by human GIP-R, GLP-1R, and GcgR cAMP assays.

[0389] To determine the intrinsic potency of exemplary analogs and comparative molecules, the cAMP assays described above were performed in the presence of 0.1% casein only (without human serum albumin). Casein was used in both cAMP assays as a nonspecific blocker; it does not interact with the fatty acid moieties of the molecules analyzed.

[0390] Intracellular cAMP levels are determined by extrapolation using a standard curve. Compound dose-response curves are plotted as percentage of stimulation normalized to the minimum (buffer only) and maximum (maximum concentration of each control ligand) and analyzed using a four-parameter nonlinear regression fit with variable slope (Genedata Screener 13). EC 50 is the concentration of compound that produces half-maximal simulation of the dose-response curve. Each relative EC50 value of the geometric mean calculation is determined from the curve fitting.

[0391] The data is provided below in Table 3. Table 3. Functional activation of hGLP-1R, hGIPR, and hGcgR in the presence of 0.1% casein. [Table 3-1]

[0392] (Continued from Table 3) [Table 3-2] TIFF2026505071000229.tif101125

[0393] (Continued from Table 3) [Table 3-3] TIFF2026505071000231.tif69125

[0394] (Continued from Table 3) [Table 3-4] TIFF2026505071000233.tif117125

[0395] (Continued from Table 3) [Table 3-5] TIFF2026505071000235.tif139125

[0396] (Continued from Table 3) [Table 3-6] TIFF2026505071000237.tif123125

[0397] (Continued from Table 3) [Table 3-7] TIFF2026505071000239.tif128125

[0398] (Continued from Table 3) [Table 3-8] TIFF2026505071000241.tif252125 TIFF2026505071000242.tif67125

[0399] (Continued from Table 3) [Table 3-9] TIFF2026505071000244.tif251125 TIFF2026505071000245.tif72125

[0400] (Continued from Table 3) [Table 3-10] TIFF2026505071000247.tif255124 TIFF2026505071000248.tif45125

[0401] (Continued from Table 3) [Table 3-11] TIFF2026505071000250.tif253125 TIFF2026505071000251.tif13125

[0402] (Continued from Table 3) [Table 3-12] TIFF2026505071000253.tif17183 TIFF2026505071000254.tif34125

[0403] (Continued from Table 3) [Table 3-13] TIFF2026505071000256.tif255121 TIFF2026505071000257.tif78125

[0404] (Continued from Table 3) [Table 3-14] TIFF2026505071000259.tif252125 TIFF2026505071000260.tif67125

[0405] (Continued from Table 3) [Table 3-15] TIFF2026505071000262.tif221125

[0406] (Continued from Table 3) [Table 3-16] TIFF2026505071000264.tif96125

[0407] (Continued from Table 3) [Table 3-17] TIFF2026505071000266.tif128125

[0408] (Continued from Table 3) [Table 3-18] TIFF2026505071000268.tif101125

[0409] (Continued from Table 3) [Table 3-19] TIFF2026505071000270.tif79125

[0410] (Continued from Table 3) [Table 3-20] TIFF2026505071000272.tif95125

[0411] (Continued from Table 3) [Table 3-21] TIFF2026505071000274.tif231125

[0412] (Continued from Table 3) [Table 3-22] TIFF2026505071000276.tif255124 TIFF2026505071000277.tif89125

[0413] (Continued from Table 3) [Table 3-23] TIFF2026505071000279.tif186125

[0414] As shown in Table 3, exemplary analogs stimulate cAMP from human GIP, GLP-1, and glucagon receptors in the presence of 0.1% casein.

[0415] In vivo studies Pharmacokinetics in male Sprague Dawley rats: The pharmacokinetics of exemplary analogs are evaluated following a single subcutaneous (SC) dose of 10 nmol / kg (dissolved in 40 mM Tris, pH 8) or a single intrajejunal (IJ) dose of 4 mg / kg (mixed with 250 mM sodium decanoate / ClO in 40 mM Tris, pH 8) to male Sprague Dawley rats. Blood samples are collected over 96 hours after SC administration and over 72 hours after IJ dosing, and the resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K3EDTA) concentrations are determined using a qualified LC / MS method that measures the intact mass of the analogs. Each peptide and analog as an internal standard is extracted from rat plasma using methanol. LC / MS detection was performed using a high-resolution instrument. Mean pharmacokinetic parameters are shown in Tables 4 and 5.

[0416] Table 4. Mean pharmacokinetic parameters of peptides after a single subcutaneous administration of 10 nmol / kg to male Sprague Dawley rats. [Table 4] Abbreviation: T 1 / 2 = half-life, CL / F = apparent clearance Note: Data are mean values, n=3 / group.

[0417] Table 5. Mean pharmacokinetic parameters of peptides following a single IJ administration of 4 mg / kg to male Sprague Dawley rats. [Table 5] Abbreviation: Cmax / D=dose normalized maximum plasma concentration, T 1 / 2 = half-life, CL / F = apparent clearance. Note: Data are means, n=4 / group.

[0418] The results of this study for the tested examples are consistent with an extended pharmacokinetic profile.

[0419] Pharmacokinetics in cynomolgus monkeys The study is designed to evaluate the oral bioavailability of example polypeptides in cynomolgus monkeys. High-resolution liquid chromatography / mass spectrometry (HR-LC / MS) is used to measure the concentrations of Examples 510, 548, 558, 694, 699, 686, 712, 741, 883, and 1027 in cynomolgus monkey plasma. Standards and controls are prepared in cynomolgus monkey plasma, and any dilutions required to bring samples into the quantification range are performed in control cynomolgus monkey plasma. To control assay variability, an internal standard (IS) is added to all standards and samples. Examples and IS are extracted from 100% monkey plasma (50 μL) by protein precipitation using isopropyl alcohol and methanol (50:50 v / v). The sample is then centrifuged (3000 rpm for 10 minutes) and the supernatant is transferred to a Siricco protein precipitation plate. The sample is loaded onto a Sep-Pak tC18 SPE microelution plate conditioned with acetonitrile and 2% formic acid in water. The compound is then washed with 2% formic acid in water and eluted using 2% formic acid in acetonitrile into a plate containing 5x Invitrosol and 1% formic acid in water, after which an aliquot (10 μL) is injected into an Xselect CSH C18, 3.5 μm, 2.1 × 20 mm column for LC / MS analysis.

[0420] The plasma pharmacokinetics (PK) of Examples 510, 548, 558, 694, 699, 686, 712, 741, 883, and 1027 are evaluated in male and female cynomolgus monkeys after a single intravenous (IV) dose (10 nmol / kg). Blood samples are collected over 504 hours. Plasma is collected from the blood samples by centrifugation and stored frozen (-70°C) until analysis. The plasma concentration of the molecule is detected using the bioanalytical method described above.

[0421] Table 6: Pharmacokinetic parameters (mean±SD) following a single IV administration of example polypeptides (10 nmol / kg) to male and female cynomolgus monkeys (n=3). [Table 6] Abbreviation: AUC 0-無限大 = area under the curve from time 0 to infinity. CL = clearance, T 1 / 2 = Half-life.

[0422] The PK parameters of TG-2474, TG-2728, TG-2565, TG-2698, TG-2708, TG-3329, TG-3270, TG-3169, TG-3211, and TG-3120 are determined after a single 10 mg oral dose to male and female cynomolgus monkeys. Blood samples are collected up to 504 hours after administration. Plasma is collected from the blood samples by centrifugation and stored frozen (-70°C) until analysis. Plasma concentrations of the molecules are detected using the bioanalytical methods described above.

[0423] Table 7: Pharmacokinetic parameters (mean ± SD) following a single oral administration of the example polypeptide (10 mg per animal) and sodium salcaprozate ((N-[8-(2-hydroxybenzoyl)amino]caprylate) (SNAC), 300 mg) to male and female cynomolgus monkeys (n=4). [Table 7] Abbreviation: AUC0-無限大 = area under the curve from time 0 to infinity. CL / F = apparent clearance, C max = maximum concentration, T max = time to maximum concentration, T 1 / 2 = half-life, F = bioavailability.

[0424] The percent F results in Table 7 demonstrate the relative bioavailability of the example polypeptides when administered orally compared to IV administration.

[0425] Studies in diet-induced obese C57BL / 6 mice: To investigate the effect of the polypeptides described herein on weight loss, exemplary polypeptides are administered to C57BL / 6 Diet-Induced Obese (DIO) mice.

[0426] Specifically, DIO male C57BL / 6 mice (Taconic, Germantown, NY) maintained on a calorie-rich diet are used in the following study. Mice are individually housed in a temperature-controlled (24°C) facility with a 12-hour light / dark cycle (lights on at 22:00) and free access to food (TD95217) and water. After a minimum of two weeks of acclimation to the facility, mice are randomized according to their body weight, so that each experimental group of animals has a similar starting weight. Body weights range from 41 to 50 g.

[0427] All groups contain five mice. Mice are treated with vehicle (40 mM Tris-HCl at pH 8.0) or example polypeptides at 10 nmol / kg. Treatments are administered to ad libitum-fed DIO mice by subcutaneous (SC) injection (10 mL / kg) 30-90 minutes before the onset of the dark cycle, either once daily (QD) or once every three days (Q3D) for 9-16 days. Body weight and food intake are measured daily throughout the study. Body weights are presented as a percentage of starting body weight. Vehicle-treated mice (control group) maintain body weights ranging from 98.00±0.84% ​​to 101.45±2.39% throughout the study.

[0428] Data are presented below in Table 7 as the mean ± SEM of 5 animals per group. Statistical analysis is performed using repeated measures ANOVA followed by Dunnett's method comparison test.

[0429] Table 8. % Body Weight After Treatment with Exemplary Polypeptides [Table 8] TIFF2026505071000285.tif145137 * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the control group, one-way ANOVA, Dunnett's multiple comparison test

[0430] GLP1-R internalization assay The potency of the peptides to stimulate ligand-induced internalization of the GLP-1R is determined using HEK293 cells expressing the human GLP1-R.

[0431] HEK293 cells were seeded in white 384-well plates at a density of 20,000 cells / well the day before transfection. Cells were transfected with Lipofectamine 2000 (Invitrogen) for SNAP-GLP-1R. The next day, the medium was removed, and the tagged receptor was labeled with 100 nM Tag-Lite SNAP-Lumi4-Tb (donor, Cisbio) in OptiMEM for 75 minutes at 37°C. Afterwards, the cells were washed with internalization buffer (HBBS at pH 7.4 supplemented with 1 mM CaCl, 2.5 mM MgCl, 20 mM HEPES, and 0.1% Pluronic F-68), followed by the addition of 100 μM preheated fluorescein-O'-acetate (acceptor, Sigma-Aldrich). The plate was placed in a 37°C incubator for 5 minutes, after which the ligand was added and the temperature was adjusted. The cells were then stimulated with the ligand preheated at 37°C, and GLP1-R internalization was measured every 3 minutes for 60 minutes at 37°C using an EnVision plate reader. Data were normalized to the maximum concentration of GLP-1 (100%) and no ligand (0%) and plotted using GraphPad Prism 7 software.

[0432] The potency of exemplary polypeptides to stimulate ligand-induced internalization of GLP-1R is reported in Table 9. The assay results identify whether the polypeptides are partial agonists for GLP-1R with respect to GLP-1R internalization.

[0433] Table 9. [Table 9-1] TIFF2026505071000287.tif52147

[0434] (Continued from Table 9) [Table 9-2] TIFF2026505071000289.tif63147

[0435] (Continued from Table 9) [Table 9-3] TIFF2026505071000291.tif63147

[0436] (Continued from Table 9) [Table 9-4]

[0437] GLP-1R CHO cell β-arrestin recruitment assay Activated G protein-coupled receptors can interact with the β-arrestin family of signaling proteins. The potency of peptides for GLP-1R-induced arrestin recruitment is determined using the PathHunter Enzyme Fragment Complementation method, essentially as described (von Degenfeld et al., FASEB J., 2007(14):3819-26, and Hamdouchi et al., J. Med Chem., 2016 59(24):10891-10916).

[0438] CHO-K1 cells expressing Pro-Link-tagged human GLP-1R and enzyme acceptor-tagged β-arrestin-2 are available from DiscoveRx and can be prepared as frozen cells for the assay. Test peptides are dissolved in DMSO, and serial dilutions are performed using an Echo acoustic dispenser (LabCyte). The assay medium is PathHunter Cell Assay Buffer (DiscoveRx) containing 0.1% w / v hydrolyzed casein (Sigma). 100 nl of peptide is dispensed into 10 μl of assay medium in a 384-well plate, followed by the addition of 10 μl of cells in assay medium to achieve 5000 cells per well. The plate is incubated for 90 minutes in a 37°C / 5% CO2 incubator, 10 μl of PathHunter detection reagent (DiscoveRx) is added, and the plate is incubated for 60 minutes at room temperature. The luminescence signal is measured. Peptide concentration-response curves were fitted to a four-parameter logistic model to determine EC 50 Calculate potency at % stimulation. Data normalization to % stimulation is performed using DMSO and GLP-1(7-36) as min and max controls (Campbell et al., Assay Guidance Manual 2017).

[0439] The potency of sample peptides to stimulate GLP-1R-induced β-arrestin recruitment is reported in Table 10. The assay results identify whether the peptides are partial and biased agonists to the GLP-1R with respect to β-arrestin-2 recruitment.

[0440] Table 10. [Table 10]

[0441] Cell-based in vitro permeability (Papp) assay Intestinal organoids and their 2D monolayers from Göttingen minipig jejunal tissue were generated using a previously described protocol (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 by TrypLE (GIbco), and the single-cell suspension was then transferred to 24-well transparent transwell inserts (0.3 cm). 2 (Falcon, BD) to achieve a transepithelial electrical resistance of 600 Ω cm 2 When the apical permeability of the intestinal permeation enhancers (SNAC and sodium caprate (C10)) reaches 1000kJ / mL, peptide permeability is examined in the presence of 10 or 20 mM C10. Samples from the basolateral compartment are collected at 20 min in the presence of C10. Peptide concentrations in the apical and basolateral compartments are measured by LC / MS, and apparent permeability coefficients (Papp) are calculated as previously described (Twarog, C.; Liu, K.; O'Brien, PJ; Dawson, KA; Fattal, E.; Illel, B.; Brayden, DJ, 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).

[0442] P for exemplary polypeptides app The values ​​are reported in Table 11.

[0443] Table 11. [Table 11-1] TIFF2026505071000295.tif30145

[0444] (Continued from Table 11) [Table 11-2]

[0445] In vivo ileal (distal loop) absorption assay Ileal absorption of exemplary polypeptides in the presence of C10 in rats is assessed using a previously reported closed intestinal loop model (Lawrence, SA; 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).

[0446] Overnight-fasted Sprague-Dawley rats weighing 250-280 grams were anesthetized by inhalation of isoflurane and placed on a heated operating table maintained at 37°C by water circulation. The surgical area was shaved, and the skin was sterilized with a betadine scrub followed by 70% isopropyl alcohol. An approximately 2 cm ventral midline incision was made to expose the intestine, and a 10 cm ileal segment was ligated. Peptides were formulated with 100 mM C10 in Tris buffer (pH 8.0, 50 mM) at a final concentration of 300 μM. The formulation was administered directly into the ileal loop. Blood samples (0.2 mL) were collected from the tail vein before and 10, 20, 40, and 60 minutes after peptide administration. Blood samples were collected into tubes containing K3EDTA (5%) and processed to plasma for subsequent analysis. Peptide concentrations in plasma are determined by LC / MS.

[0447] Table 12. [Table 12]

[0448] Proteolytic stability A pepsin stability assay is utilized to determine the relative stability of example polypeptides in a simulated gastric proteolytic environment. Pepsin A is dissolved in simulated gastric fluid (SGF) with example polypeptides or comparisons, and the solutions are sampled and quenched at TO, 15, 30, and 60 minutes. The comparisons are semaglutide and Example Compound No. 4 ("Cmpd 4") from U.S. Patent Application No. 2020 / 0024322. The relative amounts of intact peptides are quantified by mass spectrometry (MS).

[0449] The proteolytic stability of the example polypeptides relative to the controls was performed in simulated gastric fluid (SGF 2 g / L NaCl, pH 1.2). The examples were dissolved as stock solutions at a concentration of 10 mg / mL in 50 mM Tris buffer at pH 8.0. Pepsin A was reconstituted at 10 mg / mL in SGF. Reactions were prepared by diluting the peptides in SGF and adding pepsin A to have final concentrations of the example polypeptides at 0.4 mg / mL and pepsin at 1 mg / mL. The example peptide and pepsin solutions were then incubated at 37°C in a shaking incubator set at 100 rpm. Samples (25 μL) were removed at different time intervals and quenched with 100 mM ammonium bicarbonate (50 μL) at pH 9 to stop proteolytic activity. All samples were centrifuged, and the supernatants were analyzed by LC / MS to determine the remaining intact peptide.

[0450] The results are shown in Tables 12 to 14 below. Table 12. Stability in 0.1 mg / mL pepsin A solution. [Table 13]

[0451] Table 13. Stability in 1 mg / mL pepsin A solution. [Table 14]

[0452] Table 14. Stability in 1 mg / mL pepsin A solution. [Table 15]

[0453] The data in Tables 12-14 support the improvements in stability for certain example polypeptides.

[0454] array SEQ ID NO:1 - Human GIPamide YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-NH2 SEQ ID NO:2 - Human GLP-1(7-36)amide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 SEQ ID NO:3 - Human glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO:4 - Polypeptide of Formula I SEQ ID NO:5 - Polypeptide of Formula II SEQ ID NO: 6 - Polypeptide of Formula III SEQ ID NOs: 7-1242 - Polypeptides of Examples 1-1236 SEQ ID NO: 1243 - Polypeptide of Formula I' SEQ ID NO: 1244 - Polypeptide of Formula II' SEQ ID NO: 1245 - Polypeptide of Formula III'

Claims

【Request Item 1】 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, During the ceremony, X １ is Y, NMeY, or H; X ２ is Aib, X ４ is G or D-Ala, X ６ is F, αMeF, or αMeF(2F), X １０ F, 3-Pal, 4-Pal, F(4CN), F(4NO ２ ), or Y; X １１ is S or αMeS, X １２ is Orn, K, R, Q, Dap, S, E, or I; X １３ is αMeL, I, or L; X １５ is D or E, X １６ is K, Orn, A, or E; X １７ is a fatty acid, A, I, or Q, or any amino acid with a functional group available for conjugation to Orn; X １９ is A or Q, X ２０ is a fatty acid, Aib, Q, R, or any amino acid with a functional group available for conjugation to αMe-4-Pal; X ２１ is A, Aad, Aib, S, N, Q, E, T, or Orn; X ２３ is I or V, X ２４ is any amino acid having a functional group available for conjugation to a fatty acid E, D-Glu, Q, N, or D-Gln; X ２５ 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 is any amino acid with a functional group available for conjugation to a fatty acid, E, or A; X ２９ is G, Aib, T, D-Ala, or A; X ３１ is P or E, X ３２ is S or P, X ３４ is G or Aib, X ３５ is A, D, or E; X ３７ is P or E, X ３９ is E, S, G, A, T, or Orn; X ４０ is absent or is G, E, S, A, or T; 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, γE, or D-Glu; X ４１ is E, S, D, G, Q, T, A, γE, or D-Glu, then X ４２ is absent or is G, E, D-Glu, or γE; X ４２ is G, E, D-Glu, or γE, then X ４３ is absent or is E, γE, or D-Glu; X ４３ is E, γE, or D-Glu, then X ４４ is absent or E, X ４４ If E, then X ４５ is absent or E, X ４５ If E, then X ４６ is absent or E, X ４０ If X is absent, ４１ ~X ４６ is also non-existent, X ４１ If X is absent, ４２ ~X ４６ is also non-existent, X ４２ If X is absent, ４３ ~X ４６ is also non-existent, X ４３ If X is absent, ４４ ~X ４６ is also non-existent, X ４４ If X is absent, ４５ and X ４６ is also non-existent, X ４５ If X is absent, ４６ is also non-existent, The polypeptide is hereinafter referred to as X ６ is αMeF or αMeF(2F), X １０ is F, 3-Pal, 4-Pal, F(4CN), or F(4NO ２ ) and X １１ is αMeS, X １３ is αMeL, X ２４ is D-Glu, and / or X ２５ is αMeY; X １０ is F, 3-Pal, 4-Pal, F(4CN), or F(4NO ２ ), then X １２ But I, X １７ , X ２０ , X ２４ , or X ２８ is an amino acid having a functional group available for conjugation to a fatty acid; A polypeptide or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is optionally amidated.

2. The polypeptide is hereinafter referred to as X １０ is F, 3-Pal, 4-Pal, F(4CN), or F(4NO ２ ) and X １１ is αMeS, X １３ is αMeL, X １６ is Orn, X ２４ is D-Glu, and / or X ２５ The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, comprising at least two of the following:

3. The polypeptide is hereinafter referred to as X １０ is F, 3-Pal, 4-Pal, F(4CN), or F(4NO ２ ) and X １１ is αMeS, X １３ is αMeL, X １６ is Orn, X ２４ is D-Glu, and / or X ２５ 3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, comprising at least three of the following:

4. X １０ is F, 3-Pal, 4-Pal, F(4CN), or F(4NO ２ 4. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein

5. X １ is Y and X ４ is G and X ６ is αMeF(2F), and X １０ is 4-Pal, and X １２ is I and X １３ is αMeL, and X １５ is D and X １６ is Orn, and X １９ is Q and X ２０ is αMe-4-Pal, and X ２１ is E or Orn, and X ２３ is I and X ２４ is D-Glu, and X ２５ is αMeY, and X ２７ is I or V, and X ２８ is E and X ２９ is G and X ３１ is P and X ３４ is G and X ３５ is A or E, and X ３７ is P and X ３９ is E or S, and X ４０ is G or T, and X ４１ is E, S, or G, and X ４２ The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein is absent.

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

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

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

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

10. X １７ is K and C １６ ~C ２２ The fatty acid contains the amino acid and the C １６ ~C ２２ The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, which is conjugated via a linker between the polypeptide and the fatty acid.

11. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 10, wherein the linker comprises 1 to 4 amino acids.

12. 12. The polypeptide or pharmaceutically acceptable salt thereof of claim 11, wherein the amino acids contained within the linker are Glu, γGlu, or a combination thereof.

13. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 10 to 12, wherein the linker comprises 1 to 4 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

14. The linker is (γGlu) ａ -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ｂ -(γGlu) ｃ -CO-(CH ２ ) ｐ -CO ２ H, wherein a is 0 or 1, b is 0, 1, or 2, c is 1, 2, or 3, and p is an integer of 14 to 20.

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

16. A polypeptide selected from the group consisting of SEQ ID NOs: 294-775, 1146-1240, or a pharmaceutically acceptable salt thereof.

17. A polypeptide comprising greater than 90% sequence identity to any of the polypeptides in SEQ ID NOs: 692, 700, 702, 705, 706, 716, 718, 743, 747, 749, 767.

18. 18. The polypeptide of claim 17, selected from the group consisting of SEQ ID NOs: 692, 700, 705, 718, and 747, or a pharmaceutically acceptable salt thereof.

19. 19. The polypeptide of claim 18, consisting of SEQ ID NO: 692, or a pharmaceutically acceptable salt thereof.

20. 19. The polypeptide of claim 18, consisting of SEQ ID NO: 700, or a pharmaceutically acceptable salt thereof.

21. 19. The polypeptide of claim 18, consisting of SEQ ID NO: 705, or a pharmaceutically acceptable salt thereof.

22. 19. The polypeptide of claim 18, consisting of SEQ ID NO: 718, or a pharmaceutically acceptable salt thereof.

23. 19. The polypeptide of claim 18, consisting of SEQ ID NO: 747, or a pharmaceutically acceptable salt thereof.

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

25. 24. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, wherein the pharmaceutically acceptable salt is selected from sodium, potassium, trifluoroacetate, hydrochloride, or acetate.

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

27. The polypeptides are selected from the group consisting of native glucagon (SEQ ID NO: 3), GIP (SEQ ID NO: 1), and GLP-1 ７～３６ 24. The polypeptide of any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof, having greater potency at each of the glucagon, GIP, and GLP-1 receptors compared to (SEQ ID NO: 2).

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

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

31. 24. A method for treating a disease or condition selected from the group consisting of diabetes mellitus, obesity, chronic 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 step of administering to an individual in need thereof an effective amount of the polypeptide of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof.

32. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23 for use in therapy.

33. 24. The polypeptide of any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease or condition selected from the group consisting of diabetes mellitus, obesity, chronic 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 pharmaceutically acceptable salt thereof according to any one of claims 1 to 23 in the manufacture of a medicament for treating a disease or condition selected from the group consisting of diabetes mellitus, obesity, chronic 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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