Long-acting amyrin receptor agonist and use of the same

Abstract

To provide a medicine that can be used for treating diabetes mellitus, obesity, NASH and / or dyslipidemia.SOLUTION: A medicine contains a compound including a specific sequence or pharmaceutically acceptable salt thereof, which is used simultaneously or continuously and in combination separately from incretin or incretin analog for treating type 2 diabetes mellitus, obesity, dyslipidemia or NASH. The incretin or incretin analog is selected from the group consisting of GIP / GLP-1 dual agonist, glucagon, GIP, and triagonist of GLP-1, analog of oxyntomodulin, and GLP-1 agonist of oxyntomodulin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to the field of medicine. In particular, the present disclosure is in the field of the treatment of diabetes, obesity and / or chronic weight management, dyslipidemia and / or non-alcoholic steatohepatitis (NASH). The present disclosure relates to compounds that can stimulate amylin receptors and thus reduce food intake, body weight, glucose HbA1c (glycated hemoglobin), and / or triglycerides, and can be used to treat diabetes, obesity and / or chronic weight management, dyslipidemia, and / or NASH. The present disclosure also includes pharmaceutical compositions containing such compounds, as well as the therapeutic uses of such compounds and pharmaceutical compositions.

Background Art

[0002] Over the past few decades, the prevalence of diabetes has been continuously increasing. Type 2 diabetes mellitus ("T2DM") is the most common form of diabetes, accounting for 90% of all diabetes. T2DM is characterized by hyperglycemia and is mainly related to insulin resistance. The desired treatment for diabetic patients should be to lower blood glucose levels and stabilize HbA1c.

[0003] Obesity is a complex medical disorder that leads to excessive accumulation of adipose tissue mass. Today, obesity is a global public health concern that is often associated with undesirable health outcomes and morbidity. In the desired treatment of obese patients, excessive body weight should be reduced, comorbidities associated with obesity should be improved, and / or long-term weight loss should be maintained.

[0004] Dyslipidemia refers to abnormal levels of cholesterol and other lipids (also called fats) in the blood. Dyslipidemia increases the chance of clogged arteries (atherosclerosis) and heart attacks, strokes, or other cardiovascular problems, especially in smoking individuals.

[0005] NASH represents non-alcoholic steatohepatitis. This is an expression in the liver of metabolic disorders and is the most severe form of non-alcoholic fatty liver disease (NAFLD). NASH is closely related to the coexistence of diabetes and obesity.

[0006] Amylin is a 37-amino acid peptide hormone that is co-secreted with insulin from pancreatic β-cells and is deficient in people with diabetes. It suppresses the secretion of glucagon, delays the emptying of the contents of the stomach, and acts as a satiety agent. As a result, amylin helps regulate the amount of glucose in the body after a meal. However, the physicochemical properties of human amylin make its use as a medicine difficult. For example, amylin is chemically unstable and precipitates at physiological pH, making formulation difficult and requiring formulation in an acidic solution. Furthermore, the half-life of this drug is less than 1 hour, and since this drug is used at mealtime, patients will need to take it multiple times a day to use this drug for therapy.

[0007] Pramlintide is a commercially available amylin agonist peptide used in the treatment of diabetes as an adjunct to insulin. Pramlintide is chemically unstable at neutral pH due to the presence of disulfide bonds (-S-S-) and deamidation. Therefore, it is provided in an acidic formulation. Compared to human amylin, the amino acids at positions 25, 28, and 29 of pramlintide are replaced with prolines. These modifications reduce the tendency of the peptide towards fibril formation. However, pramlintide still has a very short plasma half-life and thus has to be injected 2 to 3 times a day, which is inconvenient and makes it impossible to adequately adhere to the dosing schedule. As a result, the effectiveness of the drug can be incomplete or suboptimal. In addition, its low pH formulation is not compatible with the neutral pH formulations used for insulin and GLP-1 analogs, thereby complicating its co-administration with such compounds, which in itself can be synergistic and thus have improved clinical effectiveness.

[0008] Human amylin binds to two different receptor complexes. These two complexes also include the calcitonin receptor and the receptor activity-modifying proteins, RAMP1 or RAMP3. The calcitonin receptor is found in many tissues throughout the body and is thought to be involved in the regulation of bone metabolism. However, little is known about the physiological function of the calcitonin receptor in humans outside of bone regulation, and thus there is an increased risk of off-target toxicity with molecules that have a high affinity for the calcitonin receptor. Therefore, amylin-based polypeptides with increased selectivity for the amylin receptor compared to the calcitonin receptor are thought to provide advantageous pharmacokinetic and pharmacological profiles.

[0009] Due to the close relationship between the calcitonin receptor and the amylin receptor, some cross-reactivity to the calcitonin receptor can be expected for amylin receptor agonists. As an example, pramlintide, an amylin agonist peptide, is 14-fold more potent for the amylin receptor but has some affinity for the calcitonin receptor.

[0010] Polypeptides are disclosed that include human amylin agonist peptides and have albumin-binding moieties. See International Publication Nos. WO 2010 / 046357, WO 2009 / 034119, and WO 2009 / 034119. These polypeptides having albumin-binding moieties exhibit improved pharmacokinetic (PK) properties or pharmacodynamic (PD) properties compared to pramlintide, but they can still exhibit insufficient physical stability under certain conditions. Further, the polypeptides generally do not show selectivity for the amylin receptor over the calcitonin receptor. U.S. Patent Application Publication No. US 2014 / 0087995 relates to polypeptides that include amylin agonist peptides having selectivity for the amylin receptor over the calcitonin receptor. However, there is still a need for amylin agonist peptide drug candidates having higher potency, lower development risk, lower immunogenicity risk, improved chemical stability, and compatibility with formulations at neutral pH.

[0011] There is a need for compounds (e.g., peptides) that show selectivity for amylin receptor agonism over the calcitonin receptor. Further, there is a need for amylin receptor agonists having an extended duration of action and maintained potency. Therapeutically desirable compounds will stimulate the amylin receptor and provide one or more advantageous properties such as a decrease in food intake, a decrease in body weight, a decrease in blood glucose level, a decrease in HbA1c, a decrease in triglycerides, and / or a decrease in insulin level. Further, therapeutically desirable compounds can have one or more additional advantageous properties such as a long-lasting action with maintained or improved potency to stimulate the amylin receptor, a low risk of immunogenic response, and / or a low risk of fibrosis.

[0012] Furthermore, to provide treatment for diabetes, obesity, NASH, and / or dyslipidemia, a combination of the amylin receptor agonists of the present disclosure, optionally in combination with an incretin or an incretin analog, is desirable. Such combinations will also preferably be more effective than either molecule alone. For example, such treatment with such a combination may allow for the use of lower doses of either or both molecules compared to each molecule alone, potentially resulting in lower side effects (or a shorter duration of one or the other therapy) while maintaining efficacy. The novel combination(s) provided herein are believed to be effective treatments for diabetes, obesity, NASH, and / or dyslipidemia.

Summary of the Invention

[0013] Accordingly, the present disclosure provides a method of treating diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of an amylin receptor agonist of the present invention and an effective amount of an additional agent.

[0014] Accordingly, the present disclosure describes novel compounds (peptides) that stimulate the amylin receptor and have one or more of the following properties: (1) effective reduction of food intake and body weight; (2) reduction of glucose and insulin; (3) reduced immunogenic risk and lower risk of fibrosis compared to pramlintide; (4) significantly extended half-life compared to pramlintide. The extended half-life of the compounds disclosed herein allows these compounds to be dosed only once a week when used in therapy.

Detailed Description of the Invention

[0015] One embodiment of the present disclosure is Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -Xaa 12 -AE-Xaa 15 -LVRSS-Xaa21 -Xaa 22 -FGP-Xaa 26 -LPPTEVGSNTY-NH2 (In the sequence, Xaa1 is K or γE, Xaa3 is E, N, or G, Xaa 10 is G or Q, Xaa 11 is Orn or K, Xaa 12 is L or αMeL, Xaa 15 is αMeF or F, Xaa 21 is N or H, Xaa 22 is NMeD, NMeN, or N, Xaa 26 is I or K). A compound (SEQ ID NO: 14) containing the same or a pharmaceutically acceptable salt thereof. Optionally, the compound containing SEQ ID NO: 14 further includes additional elements for extending the time-action profile of this compound. These additional elements include, regardless of the presence or absence of a linker, at any suitable position in the sequence, the Fc portion of an immunoglobulin, a fragment of the Fc portion of an immunoglobulin, human serum albumin (HSA), a variant of VHH (variable domain of a heavy-chain antibody), a nanobody, a fragment of human serum albumin, C 20 monoacid, C 20 dioic acid, and a polyethylene glycol (PEG) moiety, or other types of high-molecular-weight polymers. Preferably, the additional element is optionally bound to a lysine on the compound using a linker. Further, in any of the embodiments disclosed herein, an amylin agonist peptide and a compound containing an amylin agonist peptide may further include an element as described above, in addition to or as an alternative to any element already present that binds or is linked to the peptide sequence to extend the time-action profile.

[0016] In some embodiments of the compound containing SEQ ID NO: 14, Xaa 26 is lysine. In some embodiments of the compound containing SEQ ID NO: 14, when the lysine at position 26 is present, the lysine has the formula: (γE)2-CO-(CH2) 18It is attached to the fatty acid via a linker represented by -CO2H. In some embodiments of the compound comprising SEQ ID NO: 14, when lysine at position 26 is present, that lysine has the formula: AEEA2-γE-CO-(CH2) 18 It is attached to the fatty acid via a linker represented by -CO2H. In some embodiments of the compound comprising SEQ ID NO: 14, when lysine at position 26 is present, that lysine has the formula: γE-AEEA2-CO-(CH2) 18 It is attached to the fatty acid via a linker represented by -CO2H. In some embodiments of the compound comprising SEQ ID NO: 14, when lysine at position 26 is present, that lysine has the formula: (γE)2-AEEA-CO-(CH2) 18 It is attached to the fatty acid via a linker represented by -CO2H. In other embodiments of the compound comprising SEQ ID NO: 14, when lysine at position 26 is present, that lysine is attached to the fatty acid via another linker known in the art. Further, in any of the embodiments disclosed herein, the amylin agonist peptide and the compound comprising the amylin agonist peptide may include another linker for attaching the fatty acid, represented by the formulas herein.

[0017] In some embodiments of the compound comprising SEQ ID NO: 14, Xaa1 is lysine. In some embodiments of the compound comprising SEQ ID NO: 14, when lysine at position 1 is present, that lysine has the formula: (γE)2-CO-(CH2) 18 It is attached to the fatty acid via a linker represented by -CO2H. In other embodiments of the compound comprising SEQ ID NO: 14, when lysine at position 1 is present, that lysine is attached to the fatty acid via another linker known in the art.

[0018] In some embodiments of the compound comprising SEQ ID NO: 14, a disulfide bridge is present between cysteine at position 2 and cysteine at position 7. In a preferred embodiment of the compound comprising SEQ ID NO: 14, a thioacetal bridge is present between cysteine at position 2 and cysteine at position 7.

[0019] One of the embodiments herein includes the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 (SEQ ID NO: 1). An alternative embodiment herein consists of the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 (SEQ ID NO: 1). The following depicts Compound I using the standard one-letter amino acid code, provided that for glutamic acid (γE) at position 1 (the peptide bond is formed using the carboxylic acid group of the side chain at the γ-position instead of the typical α-position), cysteine at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeN at position 22, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0020] [Chemical formula] Compound I; SEQ ID NO: 1

[0021] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 1. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 1.

[0022] In some embodiments of the compound comprising SEQ ID NO: 1, lysine at position 26 is linked to a fatty acid via a linker. In a preferred embodiment of the compound comprising SEQ ID NO: 1, lysine at position 26 is linked to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H.

[0023] One of the embodiments herein includes the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is linked to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H (SEQ ID NO: 2)). An alternative embodiment herein consists of the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is linked to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H (SEQ ID NO: 2)). The following depicts Compound II using the standard one-letter amino acid code, provided that for the glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-side chain carboxylic acid instead of the typical α-position), the cysteines at positions 2 and 7, the Orn at position 11, the αMeF at position 15, the NMeN at position 22, the lysine at position 26, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0024]

Chemical Structure

[0025] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 2. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 2.

[0026] One embodiment herein includes the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 (SEQ ID NO: 3). An alternative embodiment herein consists of the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 (SEQ ID NO: 3). The following depicts Compound III using the standard one-letter amino acid code, except that for the glutamic acid at position 1 (γE) (the peptide bond is formed using the carboxylic acid group of the side chain at the γ-position instead of the typical α-position), the cysteines at positions 2 and 7, the Orn at position 11, the αMeF at position 15, the NMeD at position 22, and the tyrosine at position 37, the structures of these amino acid residues are expanded:

[0027]

Chemical Structure

[0028] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 3. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to Compound III (SEQ ID NO: 3).

[0029] In some embodiments of the compounds containing SEQ ID NO: 3, the lysine at position 26 is attached to a fatty acid via a linker. In a preferred embodiment of the compounds containing SEQ ID NO: 3, the lysine at position 26 is attached to the fatty acid linker moiety of the formula: (γE)2-CO-(CH2) 18 -CO2H.

[0030] One of the embodiments herein includes the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is of the formula: (γE)2-CO-(CH2) 18-CO2H (SEQ ID NO: 4) is attached to the fatty acid linker moiety. An alternative embodiment herein is the following sequence: γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 has the formula: (γE)2-CO-(CH2) 18 -CO2H (SEQ ID NO: 4) is attached to the fatty acid linker moiety). The following depicts Compound IV using the standard one-letter amino acid code, provided that for glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-side chain carboxylic acid group instead of the typical α-position), cysteine at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, lysine at position 26, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0031]

Chemical formula

[0032] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 4. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 4.

[0033] One embodiment of the present specification includes the following sequence: KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 (SEQ ID NO: 5). An alternative embodiment herein consists of the following sequence: KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 (SEQ ID NO: 5). The following depicts Compound V using the standard one-letter amino acid code, provided that for the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0034] [Chemical Formula] Compound V; SEQ ID NO: 5

[0035] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 5. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 5.

[0036] In some embodiments of the compound comprising SEQ ID NO: 5, the lysine at position 1 is linked to a fatty acid via a linker. In a preferred embodiment of the compound comprising SEQ ID NO: 5, the lysine at position 1 is linked to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H.

[0037] One embodiment herein includes the following sequence: KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 1 is of the formula: (γE)2-CO-(CH2) 18-CO2H (linked to the fatty acid linker moiety represented by SEQ ID NO: 6). One alternative embodiment herein consists of the following sequence: KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 1 has the formula: (γE)2-CO-(CH2) 18 -CO2H (linked to the fatty acid linker moiety represented by SEQ ID NO: 6). The following depicts Compound VI using the standard one-letter amino acid code, except that for the lysine at position 1, the cysteines at positions 2 and 7, the Orn at position 11, the αMeF at position 15, the NMeD at position 22, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0038]

Chemical Structure

[0039] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 6. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to Compound VI (SEQ ID NO: 6).

[0040] One embodiment herein includes the following sequence: KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 (SEQ ID NO: 7). One alternative embodiment herein consists of the following sequence: KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 (SEQ ID NO: 7). The following depicts Compound VII, except that for the cysteines at positions 2 and 7, the Orn at position 11, the αMeL at position 12, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0041]

Chemical Structure

[0042] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 7. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 7.

[0043] In some embodiments of the compound comprising SEQ ID NO: 7, the lysine at position 1 is attached to a fatty acid linker moiety. In a preferred embodiment of the compound comprising SEQ ID NO: 7, the lysine at position 1 is attached to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H.

[0044] One embodiment herein comprises the following sequence: KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 1 is attached to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H (SEQ ID NO: 8)). An alternative embodiment herein consists of the following sequence: KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 1 is attached to a fatty acid via a linker represented by the formula: (γE)2-CO-(CH2) 18 -CO2H (SEQ ID NO: 8)). The following depicts Compound VIII using the standard one-letter amino acid code, except that for the lysine at position 1, Orn at positions 2 and 7 which are cysteines, αMeL at position 12, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0045]

Chemical Structure

[0046] Alternatively, the present compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 8. Alternatively, the present compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 8.

[0047] One embodiment herein includes the following sequence: γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is bound to a fatty acid via a linker represented by the formula: AEEA2-γE-CO-(CH2) 18 -CO2H (SEQ ID NO: 9)). An alternative embodiment herein consists of the following sequence: γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is bound to a fatty acid via a linker represented by the formula: AEEA2-γE-CO-(CH2) 18 -CO2H (SEQ ID NO: 9)). The following depicts Compound IX using the standard one-letter amino acid code, provided that for the glutamic acid at position 1 (γE) (where the peptide bond is formed using the γ-position side chain carboxylic acid group instead of the typical α-position), the cysteines at positions 2 and 7, the lysine at position 26, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0048]

Chemical Structure

[0049] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO:9. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:9.

[0050] One embodiment herein includes the following sequence: γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is bound to a fatty acid via a linker represented by the formula: γE-AEEA2-CO-(CH2) 18 -CO2H (SEQ ID NO:10)). An alternative embodiment herein consists of the following sequence: γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is bound to a fatty acid via a linker represented by the formula: γE-AEEA2-CO-(CH2) 18 -CO2H (SEQ ID NO:10)). The following depicts Compound X using the standard single-letter amino acid code, provided that for the glutamic acid at position 1 (γE) (the peptide bond is formed using the carboxylic acid group in the γ-position side chain rather than the typical α-position), the cysteines at positions 2 and 7, the lysine at position 26, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0051]

Chemical Structure

[0052] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO:10. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:10.

[0053] One of the embodiments described herein includes the following sequence: γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is bound to a fatty acid linker moiety represented by the formula: (γE)2-AEEA-CO-(CH2) 18 -CO2H (SEQ ID NO: 11)). An alternative embodiment herein consists of the following sequence: γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is bound to a fatty acid linker moiety represented by the formula: (γE)2-AEEA-CO-(CH2) 18 -CO2H (SEQ ID NO: 11)). The following depicts Compound XI using the standard one-letter amino acid code, provided that for the glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-position side chain carboxylic acid group instead of the typical α-position), the cysteines at positions 2 and 7, the lysine at position 26, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0054]

Chemical Structure

[0055] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 11. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 11.

[0056] One of the embodiments described herein includes the following sequence: γE-CNTATCATQ-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is linked to a fatty acid via a linker represented by the formula: AEEA2-γE-CO-(CH2) 18 -CO2H (SEQ ID NO: 12)). An alternative embodiment consists of the following sequence: γE-CNTATCATQ-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is linked to a fatty acid via a linker represented by the formula: AEEA2-γE-CO-(CH2) 18 -CO2H (SEQ ID NO: 12)). The following depicts compound XII using the standard one-letter amino acid code, provided that for the glutamic acid (γE) at position 1 (the peptide bond is formed using the carboxylic acid group of the side chain at the γ-position instead of the typical α-position), the cysteines at positions 2 and 7, the ornithine at position 11, the lysine at position 26, and the tyrosine at position 37, the structures of those amino acid residues are expanded:

[0057]

Chemical Structure

[0058] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 12. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 12.

[0059] One of the embodiments herein includes the following sequence: γE-CGTATCATG-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 is linked to a fatty acid via a linker represented by the formula: γE2-CO-(CH2) 18-CO2H (linked to the fatty acid linker moiety represented by SEQ ID NO: 13). An alternative embodiment herein consists of the following sequence: γE-CGTATCATG-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 (wherein there is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7, and the lysine at position 26 has the formula: γE2-CO-(CH2) 18 -CO2H (linked to the fatty acid linker moiety represented by SEQ ID NO: 13). The following depicts Compound XIII using the standard one-letter amino acid code, provided that for glutamic acid (γE) at position 1 (the peptide bond is formed using the carboxylic acid group of the side chain at the γ-position rather than the typical α-position), cysteine at positions 2 and 7, ornithine at position 11, lysine at position 26, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0060]

Chemical Structure

[0061] Alternatively, the compound may have at least about 90% to about 99% sequence similarity to SEQ ID NO: 13. Alternatively, the compound may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 13.

[0062] The present disclosure provides a compound consisting of SEQ ID NO: 1, a compound consisting of SEQ ID NO: 2, a compound consisting of SEQ ID NO: 3, a compound consisting of SEQ ID NO: 4, a compound consisting of SEQ ID NO: 5, a compound consisting of SEQ ID NO: 6, a compound consisting of SEQ ID NO: 7, a compound consisting of SEQ ID NO: 8, a compound consisting of SEQ ID NO: 9, a compound consisting of SEQ ID NO: 10, a compound consisting of SEQ ID NO: 11, a compound consisting of SEQ ID NO: 12, and / or a compound consisting of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof. The present disclosure also provides a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The present disclosure also provides a pharmaceutical composition comprising a compound consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0063] The present disclosure further provides a compound comprising SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a pharmaceutically acceptable salt thereof. In another embodiment, the present disclosure provides a compound consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a pharmaceutically acceptable salt thereof. Preferably, the compounds of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 comprise a disulfide or thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. More preferably, the compound comprising SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 has a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7 and is attached to a fatty acid linker moiety. Preferably, the fatty acid moiety is attached to lysine.

[0064] The present disclosure provides a method for treating diabetes, obesity, dyslipidemia, and / or NASH in a patient, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. The present disclosure further provides a method for reducing food intake, reducing body weight, reducing blood glucose level, reducing HbA1c, and / or reducing triglyceride, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. Preferably, the present disclosure provides a method for treating diabetes, obesity, dyslipidemia, and / or NASH in a patient, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutical composition comprising the same. More preferably, the present disclosure provides a method for treating diabetes in a patient, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutical composition comprising the same. Alternatively, the present disclosure provides a method for treating obesity in a patient, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutical composition comprising the same.

[0065] This application provides a compound comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in therapy. This application further provides a compound comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment of diabetes, obesity, dyslipidemia, and / or NASH. This application also provides a compound comprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the therapy or treatment of diabetes, obesity, dyslipidemia, and / or NASH. More preferably, this application provides a compound comprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment of diabetes. Alternatively, this application provides a compound comprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment of obesity. Alternatively, this application provides a compound comprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment of dyslipidemia. Alternatively, this application provides a compound comprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment of NASH.

[0066] The present application provides a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, or a composition comprising the same, for use in reducing food intake, reducing body weight, reducing blood glucose level, reducing HbA1c, and / or reducing triglyceride. More preferably, the present application provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, or a composition comprising the same, for use in reducing food intake, reducing body weight, reducing blood glucose level, reducing HbA1c, and / or reducing triglyceride.

[0067] The present disclosure further provides the use of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of diabetes, obesity, dyslipidemia, and / or NASH. Preferably, the present disclosure further provides the use of a compound comprising SEQ ID NO: 2 in the manufacture of a medicament for the treatment of diabetes, obesity, dyslipidemia, and / or NASH.

[0068] The present disclosure further provides the use of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing food intake, reducing body weight, reducing blood glucose level, reducing HbA1c, and / or reducing triglyceride. Preferably, the present invention further provides the use of a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing food intake, reducing body weight, reducing blood glucose level, reducing HbA1c, and / or reducing triglyceride.

[0069] 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 amylin agonist peptides, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0070] Also, references to an element by the indefinite article “a” or “an” do not exclude the possibility of there being more than one element, unless the context clearly requires that there be one and only one element. Thus, the indefinite articles “a” or “an” typically mean “at least one.”

[0071] As used herein, “about” means within a statistically meaningful range of a value (singular or plural), such as, for example, a concentration, length, molecular weight, pH, percent sequence identity, time frame, temperature, volume, etc., as specified. Such a value or range can be within a magnitude typically within 20% of a given value or range, more typically within 10% of the given value or range, and even more typically within 5% of the given value or range. The allowable variation encompassed by “about” depends on the particular system in the study and can be readily understood by one of ordinary skill in the art.

[0072] As used herein, with respect to one or more receptors, “activity,” “activate,” “activating,” etc. mean the ability of a compound such as a peptide herein to bind to a receptor and induce a response at the receptor, as measured using assays known in the art such as the in vitro assays described below.

[0073] As used herein, the term "amino acid" means both naturally occurring amino acids and non-coded amino acids. As is known in the art, "amino acids" are molecules that, from a chemical perspective, are characterized by the presence of one or more amine groups and one or more carboxylic acid groups, and may contain other functional groups. Further, amino acids are typically denoted using the standard one-letter code (e.g., L is leucine), and α-methyl substituted residues of natural amino acids (e.g., α-methyl leucine or αMeL, and α-methyl phenylalanine or αMeF), as well as other specific non-coded amino acids such as "γE", "NMeN", "NMeD", "Orn", etc. The structures of the non-coded amino acids of the present disclosure are shown below:

[0074] [Chemical formula]

[0075] As used herein, "γE" means γ-glutamic acid (wherein the peptide bond is formed using the side-chain carboxylic acid group at the γ-position rather than the typical α-position). As used herein, "Orn" means L-ornithine. As used herein, "αMeL" means α-methyl-L-leucine. As used herein, "αMeF" means α-methyl-L-phenylalanine. As used herein, "NMeN" means N-methyl-asparagine, and "NMeD" means N-methyl-aspartic acid.

[0076] As used herein, "AEEA" means 2-[2-(2-amino-ethoxy)-ethoxy]acetic acid. The structure is as follows:

[0077] [Chemical formula]

[0078] As used herein, "amylin agonist peptide" means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and elicits at least one in vivo or in vitro effect induced by the natural agonist of that receptor.

[0079] As used herein, "chronic weight management" means a method of maintaining weight loss in humans characterized as currently or previously obese, as an adjunct to a low calorie diet and increased physical activity.

[0080] The term "diabetes" refers to a disease in which the body's ability to produce or respond to the hormone insulin is impaired, resulting in abnormal metabolism of carbohydrates and elevated levels of glucose in the blood and urine. As used herein, the term "diabetes" can refer to a chronic condition that affects the way the body processes blood sugar or glucose, such as type 2 diabetes mellitus (T2DM), a chronic condition in which the pancreas produces little or no insulin, such as type 1 diabetes mellitus (T1DM), a condition in which blood sugar levels are high but not as high as in type 2 diabetes, such as prediabetes, a form of hyperglycemia that affects pregnant women, such as gestational diabetes.

[0081] The term "dyslipidemia" refers to a disorder of lipoprotein metabolism, including overproduction or deficiency of lipoproteins. Dyslipidemia can manifest as elevated levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides, and / or decreased levels of high-density lipoprotein (HDL) cholesterol in the blood. Dyslipidemia may or may not be associated with diabetes.

[0082] As used herein, "fatty acid" consists of a straight chain of an even number of carbon atoms, the straight chain having hydrogen atoms along the length of the chain and having a carboxyl group (-COOH) at one end (monoacid) or both ends (diacid) of the chain. In a preferred embodiment, the "fatty acid" moiety is C 20 is a diacid.

[0083]

Chem.

[0084] As used herein, "thioacetal crosslinking" refers to the organic synthesis of thioacetal groups for the functional re-crosslinking of disulfide bond(s) in a peptide structure. The thioacetal crosslink (-S-CH2-S-) is shown below together with the methylene group inserted between two sulfur atoms.

[0085]

Chem.

[0086] As used herein, the term "treating" or "treatment" refers to the management and care of a patient having a condition for which administration of an amylin receptor peptide agonist is indicated, with the purpose of counteracting or alleviating the symptoms and complications of the condition. Treatment includes administering to a patient in need thereof one of the compounds disclosed herein or a pharmaceutical composition containing the same, in order to prevent the onset of symptoms or complications, to alleviate symptoms or complications, or to eliminate a disease, condition or disorder. Preferably, treatment includes administering a compound of the present disclosure or a pharmaceutical composition containing the compound of the present disclosure to a patient in need thereof, resulting in a net decrease in body weight, a decrease in food intake, a decrease in blood glucose level, a decrease in HbA1c, and / or a decrease in triglyceride level. The patient to be treated is a mammal, preferably a human.

[0087] As used herein, the term "effective amount" means one or more amounts or dosages of a compound described herein that, upon single or multiple administration to an individual in need thereof, provides a desired effect in such an individual during diagnosis or treatment (i.e., causes a clinically measurable difference in the condition of the individual, such as, for example, a net decrease in body weight, a decrease in food intake, a decrease in blood glucose level, a decrease in HbA1c, and / or a decrease in triglyceride level). An effective amount can be readily demonstrated by one of ordinary skill in the art using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for an individual, a number of factors are considered, including, but not limited to, the mammalian species, its size, age, and general health, the particular disease or disorder involved, the degree or severity of the disease or disorder involvement, the individual's response, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the selected regimen, the use of concomitant medications, and other related circumstances. Preferably, the effective amount of a compound of the present disclosure or a pharmaceutical composition containing a compound of the present disclosure administered to a patient in need thereof will result in a net decrease in body weight, a decrease in food intake, a decrease in blood glucose level, a decrease in HbA1c, and / or a decrease in triglyceride level. The dosage can include a higher initial loading dose followed by a lesser dose thereafter. The effective dosage of the compounds provided herein can be from 0.05 μg / kg to 5000 μg / kg, or from 0.01 nmol / kg to 1000 nmol / kg.

[0088] As used herein, "half-life" or "t 1 / 2 " means the time it takes for half of the amount of a compound, such as a peptide herein, to be removed from a fluid, such as the serum or plasma of an individual, or other physiological space, by a biological process. Alternatively, t 1 / 2 can also mean the time it takes for the amount of such a peptide to lose half of its pharmacological, physiological, or radiological activity.

[0089] As used herein, "half maximal effect concentration" or "EC 50 " means the concentration of a compound that produces 50% activation / stimulation of an assay endpoint such as a dose-response curve.

[0090] As used herein, "long-acting" means that the binding affinity and activity of the compositions herein persist for a longer period than a native peptide or protein, such that it is possible to administer the drug at a frequency of at least once a day or even three times a week, twice a week, once a week, or once a month. The time-action profile of the compounds herein may be measured using known pharmacokinetic testing methods such as those described in the following examples.

[0091] The term "NASH" refers to non-alcoholic steatohepatitis, also known as fatty liver disease. "NASH" also refers to inflammation and damage of the liver caused by the accumulation of fat in the liver. "NASH" also refers to a subtype of non-alcoholic fatty liver disease ("NAFLD"). In some embodiments, "NASH" may be synonymous with "NAFLD".

[0092] As used herein, the term "obesity" refers to a disorder with excessive body fat that increases the risk of health problems. The term "obesity" also refers to a weight that is heavier than what is considered a healthy weight for a given height. The term "obesity" also refers to a BMI of greater than 30.0, or a BMI of 27.0 or more (overweight) accompanied by at least one weight-related co-morbidity (e.g., hypertension, type 2 diabetes mellitus, or dyslipidemia).

[0093] As used herein, the body mass index (BMI) refers to a person's weight in kilograms divided by the square of their height in meters.

[0094] As used herein, "patient" and "individual" are used interchangeably and mean a mammal, preferably a human. In certain embodiments, the patient, preferably a human, is further characterized by a disease, disorder, or condition that may benefit from administration of a compound that stimulates both amylin receptors and calcitonin receptors.

[0095] In some embodiments, a pharmaceutical composition comprising a compound disclosed herein can be administered orally to a patient in need of such treatment. A pharmaceutical composition comprising a compound disclosed herein can be administered parenterally to a patient in need of such treatment. Parenteral administration can be effected by subcutaneous, intramuscular, or intravenous injection using a syringe, optionally a pen-like syringe, or a mechanically-driven syringe. Alternatively, parenteral administration can be effected using an infusion pump. Embodiments of the compounds disclosed herein provide a pharmaceutical composition, which is suitable for administration to a patient, comprising a therapeutically effective amount of a compound disclosed herein and one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can be prepared by any of a variety of techniques using conventional excipients for pharmaceutical products well known in the art. (Remington’s Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Philadelphia, PA, USA (2006)).

[0096] As used herein, "sequence similarity" refers to a quantitative property of two or more nucleic acid sequences or amino acid sequences of biological compounds, such as, for example, identity over the full length or a comparison window of two or more sequences. Sequence similarity can be measured by (1) percent identity or (2) percent similarity. Percent identity measures the percentage of identical residues between two biological compounds divided by the length of the shortest sequence, while percent similarity measures identity and, in addition, includes sequence gaps and residue similarity in the evaluation. Methods and algorithms for determining sequence similarity are well known in the art and need not be described in detail herein. A particular percentage of identical nucleotide or amino acid positions is at least about 75%, 80%, 85%, 86, 76, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more.

[0097] The compounds disclosed herein can be used simultaneously, separately, or sequentially in combination with one or more additional therapeutic agents useful for the induction of weight loss, the treatment of diabetes, diabetes-related conditions, obesity and / or chronic weight management, dyslipidemia, and / or NASH. Non-limiting examples of additional therapeutic agents that can be combined with the compounds recited in the claims include insulin or insulin analogs; biguanides; sulfonylureas; thiazolidinediones; dipeptidyl peptidase-4 (“DPP-4”) inhibitors; sodium-dependent glucose transporter (SGLT2) inhibitors; incretin compounds such as glucagon-like peptide-1 (GLP-1) or GLP-1 analogs, gastric inhibitory polypeptide (GIP) or GIP analogs, oxyntomodulin or oxyntomodulin analogs; growth differentiation factor 15 (GDF15) agonist compounds; peptide YY (PYY) analogs; GIP / GLP-1 dual agonists; Gcg / GIP / GLP-1 triple agonists (agonists of glucagon, GIP, and GLP-1); or any combination of the above agents. The compounds recited in the claims and the additional therapeutic agent(s) can be co-administered via the same delivery route and device, such as a single pill, capsule, tablet, or injectable formulation, or can be administered separately simultaneously via separate delivery devices or routes, or sequentially.

[0098] Another embodiment of the present disclosure is a method for treating a condition selected from the group consisting of diabetes, obesity, NASH, and / or dyslipidemia in a patient in need thereof, the method comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an incretin or an incretin analog. Preferably, one embodiment of the present disclosure is a method for treating a condition selected from the group consisting of diabetes, obesity, NASH, and / or dyslipidemia in a patient in need thereof, the method comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an incretin or an incretin analog.

[0099] A particular embodiment of the present disclosure is a method of treating a condition selected from the group consisting of diabetes, obesity, NASH, and / or dyslipidemia in a patient in need of such treatment, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with a GLP-1 agonist. A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, in combination with a CLP-1 agonist comprising Compound XVII (SEQ ID NO: 18). Another particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof, in combination with a CLP-1 agonist comprising Compound XVII (SEQ ID NO: 18).

[0100] A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with an analog of oxyntomodulin. A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with an analog of oxyntomodulin comprising compound XVIII (SEQ ID NO: 19). Another particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof, in combination with an analog of oxyntomodulin comprising compound XVIII (SEQ ID NO: 19).

[0101] A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with an agonist of glucagon, GIP, and GLP-1. A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with an agonist of glucagon, GIP, and GLP-1 comprising compound XIX (SEQ ID NO: 20). Another particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof, in combination with an agonist of glucagon, GIP, and GLP-1 comprising compound XIX (SEQ ID NO: 20).

[0102] A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with a GIP / GLP-1 dual agonist. A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, in combination with a GIP / GLP-1 dual agonist comprising SEQ ID NO: 21. Another particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, the method comprising administering to a patient in need thereof an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof, in combination with a GIP / GLP-1 dual agonist comprising SEQ ID NO: 21.

[0103] Another embodiment of the present disclosure is a compound comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, for use separately, simultaneously, or sequentially in combination with an incretin or an incretin analog for the treatment of diabetes, obesity, NASH, and / or dyslipidemia. Preferably, an embodiment of the present disclosure is a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use separately, simultaneously, or sequentially in combination with an incretin or an incretin analog for the treatment of diabetes, obesity, NASH, and / or dyslipidemia. More preferably, the incretin or incretin analog is a GIP / GLP-1 dual agonist comprising SEQ ID NO: 21.

[0104] The specific abbreviations used in this specification are defined as follows: "ACN" refers to acetonitrile, "AMY1R" refers to amylin receptor 1, "cAMP" refers to cyclic adenosine monophosphate, "CT" refers to calcitonin, "DCM" refers to dichloromethane, "DIEA" refers to diisopropylethylamine, "DMF" refers to N,N-dimethylformamide, "DMSO" refers to dimethyl sulfoxide, "DODT" refers to 2,2'-(ethylenedioxy)diethanethiol, "FBS" refers to fetal bovine serum, "Fmoc" refers to fluorenylmethyloxycarbonyl, "GDF15" refers to growth differentiation factor 15, "GPCR" refers to G protein-coupled receptor, "HEPES" refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, "HTRF" refers to homogeneous time-resolved fluorescence, "IBMX" refers to 1-methyl-3-isobutylxanthine, "MEM" refers to minimum essential medium, "Mtt" refers to 4-methyltrityl, "NASH" refers to non-alcoholic steatohepatitis, "NEAA" refers to non-essential amino acids, "PyAOP" refers to (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, "RP-HPLC" refers to reverse-phase high performance liquid chromatography, "TCEP" refers to tris(2-carboxyethyl)phosphine hydrochloride, "TFA" refers to trifluoroacetic acid, and "TRIS" refers to tris(hydroxymethyl)aminomethane.

Example

[0105] Example 1: Preparation and Purification of Compound I and Compound II Compound I and Compound II are produced according to the following steps. First, on a Symphony 12-channel multiple peptide synthesizer (manufactured by Protein Technologies, Inc. (Tucson, Arizona)), Compound I (SEQ ID NO: 1) is synthesized using fluorenylmethyloxycarbonyl (Fmoc) / tert-butyl (t-Bu) chemistry.

[0106] Polystyrene Rink Amide MBHA resin LL resin (Novabiochem, sub: 0.35 meq / g, 100 - 200 mesh, catalog number 855045) is used for synthesis on a 0.13 mmol scale. Standard side-chain protecting groups are used. Boc-Glu-OtBu is used at the 1-position. Fmoc-Lys(Mtt)-OH is used for the lysine at the 26-position. Prior to each coupling step (2 times × 7 minutes), the Fmoc group is removed using 20% piperidine in DMF. All amino acid couplings are carried out at 60 °C for 30 minutes with a 7.7-fold molar excess relative to the theoretical peptide loading using an equimolar ratio of Fmoc amino acid (0.3 M), diisopropylcarbodiimide (0.9 M), and Oxyma (0.9 M). The amino acid couplings following αMeF at the 15-position and NMeN at the 22-position are carried out at 60 °C for 3 hours and 6 hours, respectively. For compound I (SEQ ID NO: 1), at this point, the resin is treated with the cleavage cocktail (conditions are described after the procedure for adding the fatty acid-linker moiety to produce compound II). The following is a schematic depiction of compound I (SEQ ID NO: 1) using the standard one-letter amino acid code, however, for glutamic acid (γE) at the 1-position (the peptide bond is formed using the carboxylic acid group of the side chain at the γ-position rather than the typical α-position), cysteine at the 2- and 7-positions, Orn at the 11-position, αMeF at the 15-position, NMeN at the 22-position, and tyrosine at the 37-position, the structures of those amino acid residues are expanded:

[0107]

Chemical Structure

[0108] Next, the resin is carefully washed six times with DCM to remove residual DMF. The Mtt protecting group of lysine at position 26 is selectively removed from the peptide resin using two treatments (2 treatments × 40 minutes each) with 30% hexafluoroisopropanol (manufactured by Oakwood Chemical) in DCM. The subsequent attachment of the fatty acid linker moiety is achieved by coupling Fmoc-glutamic acid α-t-butyl ester (Fmoc-Glu-OtBu (manufactured by Ark Pharm, Inc.)) and mono-OtBu-eicosanoic acid (manufactured by WuXi AppTec (Shanghai, China)). A three-fold excess of reagents (AA:PyAOP:DIEA = 1:1:1 mol / mol) is used for each coupling with a length of 1 hour.

[0109] Once the synthesis is complete, the peptide resin is washed with DCM and air-dried completely. The dried resin is treated with 10 mL of cleavage cocktail (TFA:DODT:TIS:H2O = 89:3:3:5 v / v) for 2 hours at room temperature. The resin is filtered off, washed twice with 2 mL of neat TFA each, and the combined filtrate is treated with 4-fold cold diethyl ether (-20 °C) to precipitate the crude peptide. Subsequently, the peptide / ether suspension is centrifuged at 3500 rpm for 2 minutes to form a solid pellet, the supernatant is decanted, and the solid pellet is triturated with ether two more times. To create the thioacetal bridge, the air-dried pellet is dissolved in 5 mL of 20 mM potassium phosphate buffer and 3 mL of ACN. Once completely dissolved, while stirring well, TCEP (30 μM, 5 equivalents), diiodomethane (8 equivalents), and triethylamine (10 equivalents) in H2O are added and stirred for 5 - 30 minutes. The reaction is monitored using LCMS. This usually takes 5 minutes to complete. After the reaction is complete, 1 mL of H2O with 0.1% TFA is added to the mixture along with 6 mL of acetic acid. If the solution is turbid, additional ACN is added as necessary.

[0110] The crude peptide is purified by RP-HPLC on a Phenomenex PhenylHexyl column (5 μm, 100 Å, 250×21.2 mm, part number: 00G-4257-P0-AX) using a linear gradient with 100% acetonitrile and 0.1% TFA / water buffer system. The purity of the peptide is evaluated using analytical RP-HPLC with a Waters Symmetry Shield RP18 column (3.5 μm, 6×100 mm, part number: 186000179), and the pooling criterion is over 95%. The major pool purity of Compound II (SEQ ID NO: 2) has been found to be over 98.4%. Subsequent lyophilization of the final major product pool yields the lyophilized peptide TFA salt. The molecular weight of Compound I (SEQ ID NO: 1) is determined by LC / MS (found: 3+ [M+3H] 3+ = 1315.2; calculated [M+3H] 3+ = 1315.5; MW (average) found = 3942.6; MW (average) calculated = 3943.4). The molecular weight of Compound II (SEQ ID NO: 2) is determined by LC / MS (found: 3+ [M+3H]

[0111]

Chemical Structure

[0112] Using methods similar to those described above and known to those skilled in the art, the peptide backbone can be synthesized, the fatty acid-linker moiety can be conjugated, the purity can be examined, and the molecular weight of the compounds of the present invention described herein can be confirmed.

[0113] Example 2: Preparation and Purification of Compounds III and IV Compounds III and IV are produced according to the process outlined in Example 1.

[0114] The following is a schematic depiction of Compound III (SEQ ID NO: 3) using the standard one-letter amino acid code, except that for Glutamic acid (γE) at position 1 (the peptide bond is formed using the side-chain carboxylic acid group at the γ-position rather than the typical α-position), Cysteine at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and Tyrosine at position 37, the structures of those amino acid residues are expanded:

[0115]

Chemical Structure

[0116] The molecular weight of Compound III (SEQ ID NO: 3) is determined by LC / MS (measured value: [M + 3H] 3+ = 1314.9; calculated value [M + 3H] 3+ = 1315.8; MW (average) measured value = 3941.7; MW (average) calculated value = 3944.4).

[0117] The following is a schematic depiction of Compound IV (SEQ ID NO: 4) using the standard one-letter amino acid code, except that for Glutamic acid (γE) at position 1 (the peptide bond is formed using the side-chain carboxylic acid group at the γ-position rather than the typical α-position), Cysteine at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, Lysine at position 26, and Tyrosine at position 37, the structures of those amino acid residues are expanded:

[0118]

Chemical Structure

[0119] The molecular weight was determined by LC / MS (found: [M+3H] 3+ = 1509.7; calcd [M+3H] 3+ = 1510.0; MW (average) found = 4526.1; MW (average) calcd = 4527.1).

[0120] Example 3: Preparation and Purification of Compound V and Compound VI Compound V and Compound VI are produced according to the process outlined in Example 1.

[0121] The following is a schematic depiction of Compound V (SEQ ID NO: 5) using the standard one-letter amino acid code, except that for the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0122]

Chemical Structure

[0123] The molecular weight of Compound V (SEQ ID NO: 5) was determined by LC / MS (found: [M+3H] 3+ = 1314.9; calcd [M+3H] 3+ = 1315.5; MW (average) found = 3941.7; MW (average) calcd = 3943.4).

[0124] The following is a schematic depiction of Compound VI (SEQ ID NO: 6) using the standard one-letter amino acid code, except that for the lysine at position 1, cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0125]

Chemical Structure

[0126] The molecular weight was determined by LC / MS (found: [M+3H] 3+ = 1509.4; calcd [M+3H] 3+ = 1509.7; MW (average) found = 4525.2; MW (average) calcd = 4526.1).

[0127] Example 4: Preparation and Purification of Compounds VII and VIII Compounds VII and VIII are prepared according to the method outlined in Example 1.

[0128] The following is a schematic depiction of Compound VII (SEQ ID NO: 7) using the standard one-letter amino acid code, except that for the cysteines at positions 2 and 7, Orn at position 11, αMeL at position 12, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0129]

Chem.

[0130] The molecular weight of Compound VII (SEQ ID NO: 7) was determined by LC / MS (found: [M+3H] 3+ = 1317.9; calcd [M+3H] 3+ = 1318.1; MW (average) found = 3950.7; MW (average) calcd = 3951.4).

[0131] The following is a schematic depiction of Compound VIII (SEQ ID NO: 8) using the standard one-letter amino acid code, except that for the lysine at position 1, cysteines at positions 2 and 7, Orn at position 11, αMeL at position 12, and tyrosine at position 37, the structures of those amino acid residues are expanded:

[0132]

Chem.

[0133] The molecular weight is determined by LC / MS (measured value: [M+3H] 3+ = 1512.2; calculated value [M+3H] 3+ = 1512.4; MW (average) measured value = 4533.6; MW (average) calculated value = 4534.2).

[0134] Example 5: Preparation and Purification of Compound IX Compound IX is produced according to the process outlined in Example 1.

[0135] The following is a schematic depiction of Compound IX (SEQ ID NO: 9) using the standard one-letter amino acid code, except that for Glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-position side chain carboxylic acid group instead of the typical α-position), Cysteine at positions 2 and 7, Lysine at position 26, and Tyrosine at position 37, the structures of those amino acid residues are expanded:

[0136]

Chemical Structure

[0137] The molecular weight is determined by LC / MS (measured value: [M+3H] 3+ = 1558.3; calculated value [M+3H] 3+ = 1558.8; MW (average) measured value = 4671.9; MW (average) calculated value = 4673.3).

[0138] Example 6: Preparation and Purification of Compound X Compound X is produced according to the process outlined in Example 1.

[0139] The following is a schematic depiction of compound X (SEQ ID NO: 10) using the standard one-letter amino acid code, except that for glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-side chain carboxylic acid group rather than the typical α-position), cysteine at positions 2 and 7, lysine at position 26, and tyrosine at position 37, the structures of those amino acid residues are enlarged:

[0140]

Chem.

[0141] The molecular weight is determined by LC / MS (found: [M + 3H] 3+ = 1558.5; calcd [M + 3H] 3+ = 1558.8; MW (average) found = 4672.5; MW (average) calcd = 4673.3).

[0142] Example 7: Preparation and Purification of Compound XI Compound XI is produced according to the process outlined in Example 1.

[0143] The following is a schematic depiction of compound XI (SEQ ID NO: 11) using the standard one-letter amino acid code, except that for glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-side chain carboxylic acid group rather than the typical α-position), cysteine at positions 2 and 7, lysine at position 26, and tyrosine at position 37, the structures of those amino acid residues are enlarged:

[0144]

Chem.

[0145] The molecular weight is determined by LC / MS (found: [M + 3H] 3+ = 1553.2; calcd [M + 3H] 3+ = 1553.4; MW (average) found = 4656.6; MW (average) calcd = 4657.2).

[0146] Example 8: Preparation and Purification of Compound XII Compound XII is produced according to the process outlined in Example 1.

[0147] The following is a schematic depiction of Compound XII (SEQ ID NO: 12) using the standard one-letter amino acid code, except that for Glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-side chain carboxylic acid group rather than the typical α-position), Cysteine at positions 2 and 7, Orn at position 11, Lysine at position 26, and Tyrosine at position 37, the structures of those amino acid residues are expanded:

[0148]

Chemical Structure

[0149] The molecular weight is determined by LC / MS (measured value: [M + 3H] 3+ = 1577.4; calculated value [M + 3H] 3+ = 1577.8; MW (average) measured value = 4729.2; MW (average) calculated value = 4730.3).

[0150] Example 9: Preparation and Purification of Compound XIII Compound XIII is produced according to the process outlined in Example 1.

[0151] The following is a schematic depiction of Compound XIII (SEQ ID NO: 13) using the standard one-letter amino acid code, except that for Glutamic acid (γE) at position 1 (the peptide bond is formed using the γ-side chain carboxylic acid group rather than the typical α-position), Cysteine at positions 2 and 7, Orn at position 11, Lysine at position 26, and Tyrosine at position 37, the structures of those amino acid residues are expanded:

[0152]

Chemical Structure

[0153] The molecular weight was determined by LC / MS (found: [M + 3H] 3+ = 1481.2; calculated [M + 3H] 3+ = 1481.3; MW (average) found = 1440.6; MW (average) calculated = 4441.0).

[0154] Preparation of disulfide bond crosslinking To create a disulfide bond, an oxidation solution is prepared by adding 10 drops of iodine solution (2% iodine in AcOH) to 40 mL of 20% MeCN / 20% AcOH / 60% water in a flask. Separately, a peptide solution is made by dissolving the air-dried peptide pellet in 5 mL of AcOH. With stirring, the peptide solution is added dropwise to the oxidation solution. Additional iodine solution (2% iodine in AcOH) is added as needed to maintain a pale yellow solution in the flask. After all of the peptide solution has been added, a light brown / yellow color is maintained for 5 minutes. Excess iodine is neutralized by adding 1 drop of saturated ascorbic acid to the oxidation solution. The solution is filtered through a 0.45 μm filter, and the peptide is ready for purification.

[0155] Example 10: In Vitro Functional Activity of Amylin Agonist Peptides The AMY1 receptor and the CT receptor are GPCRs that are functionally coupled to the Gαs protein. Stimulation of these receptors increases the production of intracellular cAMP, which can be detected using standard in vitro techniques. The in vitro activity of the peptide is measured by the amount of cAMP formed in cells overexpressing human AMY1R and CTR.

[0156] The human CT receptor is stably expressed in human bladder cells (UM-UC-3 or UMUC3) under the control of a pcDNA expression vector. The UMUC3 cell line is cultured in MEM 1X (Meditech Inc., 17-305-CV) supplemented with 10% FBS, 1% antibiotic / antifungal agent solution, 1 mM sodium pyruvate, 1X MEM NEAA, and 1X GlutaMAX-I. The plasmid of human CTa-pcDNA3.1 Hygro(+)(T2616) DNA is transfected into UMUC3 cells using LipofectAMINE 2000 transfection reagent (Invitrogen, 11668-019). Twenty days later under selection, the mRNA levels from different clones are measured to confirm the expression of hCTR. To determine the function of the overexpressed hCTR cells, the intracellular cAMP levels in response to salmon calcitonin are measured and compared with the expression of hCTR mRNA in each clone.

[0157] The human AMY1R stable cell line is generated by further transfecting human RAMP1-pCMVpuroPB (T14213) into the hCTR clone cells of UMUC3. After selection, the mRNA levels of human RAMP1 from different clones are measured to confirm the expression of human AMY1R.

[0158] hAMY1R cells are cultured in MEM 1X (manufactured by Corning) supplemented with 10% FBS, 1% antibiotic / antifungal solution, 1 mM sodium pyruvate, 1X MEM NEAA, 1X GlutaMAX-I, 200 μg / mL hygromycin B, and 0.4 μg / mL puromycin. hCTR cells are cultured in the same medium lacking puromycin. The cultured cells are grown to a confluence density of 70%, and then incubated overnight in fresh medium.

[0159] On the assay day, 10 μL of assay buffer (MEM without phenol red (Corning, catalog number 17 - 305 - CV), 0.1% casein, 0.5 mM IBMX, 5 mM HEPES, pH 7.4) is dispensed into each well of a white poly - D - lysine - coated 384 - well plate (Corning, catalog number 354661). Peptides diluted in DMSO are added in a 1:3 dilution series (200 nL / well) using an ECHO acoustic liquid handler (Beckman). Cultured cells are detached with TrypLE Express (Gibco) and resuspended in assay buffer, and 10 μL containing 1200 cells / well (hCTR) or 1500 cells / well (hAMY1R) is dispensed into each well. The plate is incubated at room temperature for 1 hour.

[0160] The HTRF technology (homogeneous time - resolved fluorescence, Cisbio) is used to quantify the amount of intracellular cAMP according to the vendor's instructions. Briefly, 10 μL of cAMP - d2 conjugate and 10 μL of anti - cAMP - cryptate conjugate in lysis buffer are incubated with the treated cells at room temperature for 60 minutes. The HTRF signal is immediately detected using an Envision plate reader (Perkin - Elmer), and the ratio of fluorescence at 665 nm to 620 nm is calculated. The raw data is converted to the amount of cAMP (pmol / well) using the cAMP standard curve generated for each experiment. The relative EC 50 values are calculated from the upper and lower ranges of the concentration - response curve defined using a four - parameter logistic curve - fitting program (Genedata Screener® v12.0.4), with 1 nM salmon CT (Bachem) as the maximum value and buffer alone as the minimum value. The compounds of the present application show selective activity at the amylin receptor versus the calcitonin receptor, as shown in Table 1.

[0161] Table 1. Comparison of functional activity data at the amylin receptor and the calcitonin receptor [Table 1]

[0162] Example 11: In Vitro Binding Affinity of Amylin Agonist Peptides for hAMY1 Receptor and hCT Receptor Membranes from human AMY1R- and CTR-overexpressing cells (described in Example 10) were isolated by standard methods and used in the binding assay. The equilibrium dissociation constants (Kd) for various receptor / radioactive ligand interactions were determined from saturation binding analyses using the same reagents and buffers as described below for the compound testing. The Kd values measured for the receptor preparations used in this study were as follows: hAMY1R: 0.067 nM; human calcitonin receptor (hCTR): 0.046 nM.

[0163] hAMY1R Binding Protocol The receptor binding affinities (Ki) of rAMY, hCT, and hAMY agonist peptides are determined from a competitive radioligand binding filter assay. The assay buffer consists of 50 mM HEPES, pH 7.1, 5 mM MgCl2, 5 mM KCl, 0.2% (w / v) bacitracin, and 0.003% (w / v) saponin and is used to dilute the radioligand and membrane preparations. The binding reaction is carried out in a polystyrene 96-deep well assay block with a total reaction volume of 0.1 mL. Iodinated rat amylin (custom synthesis by ViTrax; 2200 Ci / mmol; 125I-rAMY) is first diluted to a concentration of approximately 50 pM in assay buffer. Test compounds and non-specific binding (NSB: defined as 300 nM rAMY) are added to aliquots of radioligand buffer. Briefly, test compounds are diluted to an initial concentration of 200 nM and serially diluted in 4-fold steps in 125I-rAMY, then 0.05 mL of diluted test compound, NSB, or total binding (defined as neat 125I-rAMY) is transferred to a 96-well polystyrene assay block. The binding reaction is initiated by adding 0.05 mL of 200 μg / mL hAMY1R diluted in assay buffer to the radioligand buffer. The assay block is gently vortexed, sealed with parafilm, and incubated at room temperature for approximately 20 hours. Thirty minutes before the incubation is complete, a filter mat (Perkin Elmer printed filter mat, catalog number 1450-421) is immersed in a solution consisting of assay buffer without saponin but supplemented with 0.1% (w / v) fatty acid-free bovine serum albumin (FAF-BSA) and 0.5% (v / v) polyethyleneimine (PEI). At the end of the incubation, the bound ligand is separated from the free ligand by the addition of ice-cold quench buffer consisting of 100 mM NaCl, 50 mM Tris-HCl (pH 7.1) and immediately recovered onto the filter mat by vacuum filtration using a TomTec 96-well filter harvester. The following filter protocol is used: dry time, 7; cycle 3 repeats, 0; wash time, 1; soak time, 1; first aspiration time, 4; wash / aspiration time, 7; second aspiration time, 4; air pressure, 2 PSI.In the response curve, the final assay concentration range of the tested peptide is 100 nM to 0.00038 nM.

[0164] hCTR binding protocol Determine the receptor binding affinity (Ki) of rAMY, hCT, and hAMY agonist peptides on hCTR membranes from a competitive radioligand binding filter assay. The assay procedure is the same as the hAMY1R binding assay, except that 125I-hCT (custom synthesis by ViTrax; 2200 Ci / mmol) is used as the hot ligand. The binding reaction is carried out in a total reaction volume of 0.2 mL using 14 pM of 125I-hCT and 20 μg / mL of hCTR membranes and incubated for 20 hours. In the response curve, the final assay concentration range of the tested peptide is 2500 nM to 0.00128 nM. The compounds of the present application show selective activity at the amylin receptor versus the calcitonin receptor, as shown in Table 2.

[0165] Table 2. In Vitro Binding (Ki) at Human Amylin and Calcitonin Receptors

Table 2

[0166] Example 12: In Vivo Effects on Food Intake and Body Weight of Normal Rats Male Sprague Dawley rats obtained from Envigo RMS (Indianapolis, Indiana) were housed individually in a temperature-controlled facility (72.0°F; 22.2°C) on a solid diet (2014; Teklad Global (Envigo RMS, Indianapolis, Indiana)) and allowed free access to food and water on a 12:12 hour reverse light cycle (lights off at 10:00 am). At 10 weeks of age, the non-fasted body weight and initial food weight were recorded, and the animals were given a single subcutaneous injection (SC) of vehicle or acylated peptide (1 mL / kg). Thereafter, body weight and food intake were measured daily for 4 days after administration. Area under the curve (AUC) analysis was calculated for both body weight and food intake relative to vehicle.

[0167] Table 3: Changes in body weight and food intake over 4 days in Sprague Dawley rats after a single administration of a long-acting amylin agonist peptide [Table 3] * Change in mean body weight at 96 hours after administration compared to the initial body weight of untreated animals. ** Cumulative food intake of peptide-treated animals at 96 hours after administration vs. cumulative food intake of vehicle-treated animals at 96 hours after administration.

[0168] Example 13: In Vivo Effects on Food Intake and Body Weight in Diet-Induced Obese Rats Male 14-week-old Long Evans rats obtained from Envigo RMS (Indianapolis, Indiana) are fed a high-fat diet (40% of the calories consumed are from fat, TD.95217 (manufactured by Envigo RMS, Indianapolis, Indiana)) from 35 to 45 weeks of age. The animals are individually housed in a temperature-controlled facility (75.0°F; 23.9°C) on a 12-hour reverse light / dark cycle (lights out at 10:00 AM) with free access to food and water. Body weights are recorded, and body composition (fat mass) is determined using quantitative nuclear magnetic resonance analysis (ECHO MRI, 3-1 Composition Analyzer; manufactured by Echo Medical Systems, Houston, Texas), and then randomized into experimental groups (n = 5). Rats are injected subcutaneously with vehicle or peptide (1 mL / kg) every 3 days (days 1, 4, 7, 10, and 13). Daily body weights are recorded, and the change at the end of treatment (day 14) is calculated as a percentage of the body weight before treatment (day 1). Body composition is measured on day 14, and the changes in fat mass and fat-free mass (body weight - fat mass) are calculated as gram changes from the pre-treatment values.

[0169] Table 4: Changes in body weight in a 2-week study in diet-induced obese rats using an amylin agonist peptide [Table 4] * Change in mean body weight or fat mass or fat-free mass at week 2 compared to the initial body weight of untreated animals.

[0170] Example 14: Pharmacokinetics in male Sprague Dawley rats The pharmacokinetics of Compound II (SEQ ID NO: 2), Compound IV (SEQ ID NO: 4), Compound VI (SEQ ID NO: 6), and Compound VIII (SEQ ID NO: 8) are evaluated after a single subcutaneous administration of 30 nmol / kg to male Sprague Dawley rats. Blood samples are collected at 1, 3, 6, 12, 24, 48, 72, and 96 hours after SC administration. Pharmacokinetic parameters are calculated using the obtained individual plasma concentrations. The peptide plasma (K3EDTA) concentration is determined using a qualified LC / MS method that measures the intact mass of the peptide. Each peptide and analog is extracted from plasma using an internal standard. A high-resolution Thermo Q-Exactive is used for LC / MS detection. The mean pharmacokinetic parameters are shown in Table 5.

[0171] Table 5. Mean pharmacokinetic parameters of the peptides after a single subcutaneous administration of 30 nmol / kg to male Sprague Dawley rats.

Table 5

[0172] Example 15: Pharmacokinetics in Male Cynomolgus Monkeys The pharmacokinetics of Compound II (SEQ ID NO: 2), Compound IV (SEQ ID NO: 4), Compound VI (SEQ ID NO: 6), and Compound VIII (SEQ ID NO: 8) are evaluated after a single subcutaneous administration of 20 nmol / kg to male cynomolgus monkeys. Blood samples are collected at the following time points: 1, 3, 6, 12, 24, 48, 72, 120, 168, 240, 336, 408, and 504 hours after SC administration. Using the obtained individual plasma concentrations, pharmacokinetic parameters are calculated. The peptide plasma (K3EDTA) concentration is determined using a qualified LC / MS method that measures the intact mass of the peptide. Each peptide and analog is extracted from plasma using an internal standard. A high-resolution Thermo Q-Exactive is used for LC / MS detection. The average pharmacokinetic parameters are shown in Table 6.

[0173] Table 6. Average pharmacokinetic parameters of the peptides after a single subcutaneous administration of 20 nmol / kg to cynomolgus monkeys.

Table 6

[0174] Example 16: Immunogenicity Risk Assessment Dendritic Cell (DC) Internalization Assay This assay evaluates the ability of human DCs to internalize the test antibody. CD14+ cells are cultured and differentiated into immature DCs using IL-4 and GM-CSF. The test antibody, isotype control, or positive control is pre-incubated with the detection agent (Fab-QSY7-TAMRA) at a 1:1 ratio to form a complex, which is then added to the culture. The cells are incubated for 1 day. At the time of internalization and cleavage, the positive TAMRA signal is detected by flow cytometry, and the normalized internalization index is calculated using the IgG1-EN isotype control and the anti-CXCR antibody.

[0175] MAPPS assay (MHC-associated peptide proteomics) MAPPS reveals the characteristics of MHC-II presented peptides on human dendritic cells treated with the test molecule. CD14+ cells isolated from PBMC of normal human donors are cultured and differentiated into immature DCs by incubation with IL-4 and GM-CSF. On day 4, the medium is replaced with fresh medium containing the test molecule. On day 5, LPS is added to convert the cells into mature DCs. On day 6, the cells are lysed in RIPA buffer containing protease inhibitors. Immunoprecipitation of the MHC-II complex is performed using a biotinylated anti-MHC-II antibody conjugated to streptavidin beads. The bound complex is eluted and filtered. The isolated MHC-II peptides are analyzed by a mass spectrometer. Peptide identification is generated by an in-house proteomics pipeline using an enzyme-free search algorithm and a bovine / human database containing the test sequences added to the database. The KNIME workflow is used to process the identification files from the samples. The peptides identified from the test articles are aligned against the parental sequences of the test molecules. The output is used to determine the percentage of donors presenting MHC-II peptides from the region of the test molecule. Compound II, Compound IV, Compound VI, Compound VIII, the compound represented by US Patent No. 9,023,789 (hereinafter referred to as "Compound '789"), and plumricotide are tested in the MAPPs assay. Compound II, Compound IV, Compound VI, and Compound VIII do not show peptides presented on the MHC-II complex in the assay. Compound '789 presents a peptide cluster spanning residues 8-23 on the MHC-II complex. Plumricotide shows two peptide clusters, both of which span residues 1-34 presented on the MHC-II complex.

[0176] In-silico TCEM (T cell exposure motif) analysis This analysis assesses the potential of specific peptide clusters identified by MAPPS to activate CD4+ T cells. Peptide sequences identified by MAPPS that contain non-germline residues are entered into the MHCII Binding Prediction page of the ImmunoEpitope Database (IEDB) analysis resource. The prediction method recommended by IEDB is selected. In the prediction, the 27 most frequent HLA-DR, -DP, and -DQ alleles are considered to cover a substantial portion of the population. Each input sequence of a length of 15 residues or more is split into overlapping 15-mers (1 amino acid offset) across the entire sequence. For each peptide, a percentile rank is generated by comparing the score of the peptide to the scores of 5 million random 15-mers selected from the SWISSPROT database. Amino acids located at the putative P-1, P2, P3, P5, P7, and P8 positions of the register generate the TCEM, and the risk is defined based on the presence of non-germline residues at these positions. The potential for non-germline residues and core binding to multiple alleles is reported in a graphic rendering and considered for immunogenic risk assessment.

[0177] MS serum binding This assay evaluates off-target binding to human serum proteins of test candidates. The test antibody is coated on Immulon 4 HBX microplates. After blocking, human serum is added and incubated overnight. The plates are washed, and the bound proteins are eluted, reduced, alkylated, and digested. The peptides are analyzed by mass spectrometry. Peptide identification is generated by an in-house proteomics pipeline using a search algorithm based on trypsin enzyme specificity and a human database augmented with the sequence of the test molecule. Ions are quantified by in-house proteomics tools (Chrom-Alignment, Metaconsense, Quant) and analyzed in JMP using the one-way ANOVA / each pair, Student's t-test platform. Analysis of log2auc of ions using JMP: approximation of Y by X for each ion / comparison of means / all pairs, Tukey HSD.

[0178] T Cell Proliferation Assay This assay evaluates the ability of a test antibody or test MAPPS peptide to activate CD4+ T cells by inducing cell proliferation. PBMCs depleted of CD8+ T cells are prepared and labeled with CFSE. Each sample is tested with a media control, keyhole limpet hemocyanin (KLH; positive clinical benchmark control), the test antibody, or the test MAPPS peptide. The cultures are incubated for 7 days. 。 On day 7, the samples are analyzed by flow cytometry.

[0179] Existing Reactivity (ACE Assay Format) This assay evaluates the presence of existing antibodies (ADA) against the test molecule in untreated normal human serum (NHS). Diluted NHS is incubated overnight on a Pierce Streptavidin plate coated with the biotinylated test molecule. The next day, the captured binding proteins are acid eluted, hard coated onto a Mesoscale (MSD) plate, and detected with a combination of biotin-labeled molecules and ruthenium-labeled streptavidin. If anti-drug antibodies are present, they bind to the labeled drug, and the resulting signal is called the Tier 1 signal (represented as electrochemiluminescence). This signal is confirmed in Tier 2 by adding an excess of unlabeled test molecule in the detection step (which suppresses the Tier 1 signal). The presence of existing anti-drug antibodies is represented as the magnitude of the 90th percentile of Tier 2 inhibition. The 90th percentile of Tier 2 inhibition is a statistical tool for evaluating the magnitude of the specificity of the Tier 1 reactivity. This 90th percentile is used to rank molecules with respect to ADA risk.

[0180] Table 7. Summary of Immunogenicity Risk Assessment

Table 7

[0181] Example 17: In Vivo Efficacy of Compound II in Combination with Other Incretin Compounds in Diet Induced Obese (DIO) Rats This study is conducted to investigate the effect of Compound II on diabetes and / or obesity in diet-induced obesity (DIO) rats when administered in combination with GLP-1 agonist (Compound XVII), oxyntomodulin analog (Compound XVIII), and other incretin compounds including a triagonist of glucagon, GLP-1, and GIP (Compound XIX). Male Long Evans rats (obtained from Envigo) with diet-induced obesity (DIO) that have been fed a high-calorie diet (TD95217 (manufactured by Teklad, Madison, Wisconsin)) since arrival at Lilly are used in the following study. The animals are individually housed in a temperature-controlled (24°C) facility with a 12-hour light / dark cycle (lights on at 22:00) and have free access to diet (TD95217) and water.

[0182] The rats are randomized according to their body weight so that each experimental group of animals has a similar body weight. The body weight ranges from 529 to 823 grams.

[0183] Each group contains 5 rats. Vehicle and Compound II (1 nmol / kg) are dissolved in vehicle (20 mM Tris-HCl, pH 8 + 0.02% PS80) and administered by subcutaneous (SC) injection (1 mL / kg) to DIO rats with free access to food 30 - 90 minutes before the start of the dark cycle every 3 days for 14 days. SC injections are performed on days 1, 4, 7, 10, and 13. Body weight and food intake are measured daily throughout the study. The absolute change in body weight is calculated by subtracting the body weight of the same animal before the first injection of the molecule.

[0184] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured using an AccuChek glucose meter (manufactured by Roche, Indianapolis, Indiana). Insulin is measured by ELISA (manufactured by MSD, Rockville, Maryland).

[0185] All data are presented as mean ± SEM of five animals per group. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison test to compare the treatment groups with the vehicle group or with each other. Significant differences were identified with p-values less than 0.05.

[0186] Table 8: Effects of Compound II with and without combination with Compound XVII, Compound XVIII, or Compound XIX on body weight and cumulative food intake. [Table 8] * Treatment was administered subcutaneously every three days on days 1, 4, 7, 10, and 13. ** Body weight measurements were taken daily. The change in body weight was expressed as the difference in grams from day 1 to day 14. *** Cumulative food intake was the total food consumed over the 14-day treatment period. Statistical analysis was performed by one-way ANOVA followed by Tukey's test. * Compared to the vehicle group, p < 0.05; # Compared to any of the Compound XVII, Compound XVIII, or Compound XIX groups, p < 0.05; + Compared to Compound II, p < 0.05.

[0187] In the combination of Compound II with Compound XVII, Compound XVIII, or Compound XIX, there was greater weight loss than with Compound II alone.

[0188] SEQ ID NO: 1: Compound I γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7.

[0189] SEQ ID NO: 2: Compound II γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is linked to the fatty acid linker moiety represented by the formula (γE)2-CO-(CH2) 18 -CO2H.

[0190] SEQ ID NO: 3: Compound III γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7.

[0191] SEQ ID NO: 4: Compound IV γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is linked to the fatty acid linker moiety represented by the formula (γE)2-CO-(CH2) 18 -CO2H.

[0192] SEQ ID NO: 5: Compound V KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7.

[0193] SEQ ID NO: 6: Compound VI KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 1 is linked to the fatty acid linker moiety represented by the formula (γE)2-CO-(CH2) 18It is bound to a fatty acid linker moiety represented by -CO2H.

[0194] SEQ ID NO: 7: Compound VII KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7.

[0195] SEQ ID NO: 8: Compound VIII KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 1 is bound to a fatty acid linker moiety represented by the formula (γE)2-CO-(CH2) 18 -CO2H.

[0196] SEQ ID NO: 9: Compound IX γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is bound to a fatty acid linker moiety represented by the formula AEEA2-γE-CO-(CH2) 18 -CO2H.

[0197] SEQ ID NO: 10: Compound X γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is bound to a fatty acid linker moiety represented by the formula γE-AEEA2-CO-(CH2) 18 -CO2H.

[0198] SEQ ID NO: 11: Compound XI γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is linked to the fatty acid linker moiety represented by the formula (γE)2-AEEA-CO-(CH2) 18 -CO2H.

[0199] SEQ ID NO: 12: Compound XII γE-CNTATCATQ-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is linked to the fatty acid linker moiety represented by the formula AEEA2-γE-CO-(CH2) 18 -CO2H.

[0200] SEQ ID NO: 13: Compound XIII γE-CGTATCATG-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH2 There is a thioacetal bridge between the cysteine at position 2 and the cysteine at position 7. The lysine at position 26 is linked to the fatty acid linker moiety represented by the formula γE2-CO-(CH2) 18 -CO2H.

[0201] SEQ ID NO: 14: Compound XIV Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -Xaa 12 -AE-Xaa 15 -LVRSS-Xaa 21 -Xaa 22 -FGP-Xaa 26 -LPPTEVGSNTY-NH2 (In the sequence, Xaa1 is K or γE Xaa3 is E, N, or G Xaa 10 is G or Q, Xaa 11 is Orn or K, Xaa 12 is L or αMeL Xaa 15 is αMeF or F Xaa 21 is N or H Xaa 22 is NMeD, NMeN, or N Xaa 26 is I or K.)

[0202] SEQ ID NO: 15: Compound XV; Plumrintide KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH2 There is a disulfide bridge between the cysteine at position 2 and the cysteine at position 7.

[0203] SEQ ID NO: 16: Compound XVI; hCT CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH2 There is a disulfide bridge between the cysteine at position 1 and the cysteine at position 7.

[0204] SEQ ID NO: 17: hAMY KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2 There is a disulfide bridge between the cysteine at position 2 and the cysteine at position 7.

[0205] SEQ ID NO: 18: Compound XVII H-Aib-EGTFTSDVSSYLEGQAAK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 16 -CO2H)EFIAWLVRGRG

[0206] SEQ ID NO: 19: Compound XVIII H-Aib-QGTFTSDYSKYLDEKKAK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO-(CH2) 18-CO2H)EFVEWLLEGGPSSG-NH2

[0207] SEQ ID NO: 20: Compound XIX Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2

[0208] SEQ ID NO: 21 YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS X1 is Aib, X2 is Aib, K at position 20 is (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 18 -CO2H, chemically modified through conjugation to the epsilon-amino group of the K side chain, and the C-terminal amino acid is amidated as a C-terminal primary amide.

[0209] SEQ ID NO: 22: Compound I backbone γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN-FGPKLPPTEVGSNTY-NH2

[0210] SEQ ID NO: 23: Compound III backbone γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPKLPPTEVGSNTY-NH2

[0211] SEQ ID NO: 24: Compound V backbone KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD-FGPILPPTEVGSNTY-NH2

[0212] SEQ ID NO: 25: Compound VII backbone KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH2

Claims

1. ︁ １ _______________ ３ _____________________________ １０ ︁︁ １１ ︁︁ １２ _____________________ １５ ______________________________ ２１ ︁︁ ２２ P!︁ ２６ __________________________________________ [In the sequence, Xaa １ is K or γE, Xaa ３ is E, N, or G; Xaa １０ is G or Q, Xaa １１ is Orn or K, Xaa １２ is L or αMeL, Xaa １５ is αMeF or F, Xaa ２１ is N or H, Xaa ２２ is NMeD, NMeN, or N; Xaa ２６ is I or K. or a pharma- ceutically acceptable salt thereof.

2. Xaa １ is γE, Xaa ３ is N, Xaa １０ is G, Xaa １１ is Orn, Xaa １２ is L, Xaa １５ is αMeF, Xaa ２１ is N, Xaa ２２ is NMeN, Xaa ２６ is K (SEQ ID NO: 22), or a pharma- ceutically acceptable salt thereof.

3. Xaa １ is γE, Xaa ３ is N, Xaa １０ is G, Xaa １１ is Orn, Xaa １２ is L, Xaa １５ is αMeF, Xaa ２１ is N, Xaa ２２ is NMeD, Xaa ２６ is K (SEQ ID NO: 23), or a pharma- ceutically acceptable salt thereof.

4. Xaa ２６ The lysine in the formula: (γE) ２ -CO-(CH ２ ) １８ -CO ２ 4. The compound of claim 2 or 3, attached to a fatty acid linker moiety designated H.

5. Xaa １ is K, Xaa ３ is E, Xaa １０ is G, Xaa １１ is Orn, Xaa １２ is L, Xaa １５ is αMeF, Xaa ２１ is N, Xaa ２２ is NMeD, Xaa ２６ The compound of claim 1 , wherein is I (SEQ ID NO: 24), or a pharma- ceutically acceptable salt thereof.

6. Xaa １ is K, Xaa ３ is E, Xaa １０ is G, Xaa １１ is Orn, Xaa １２ is αMeL, Xaa １５ is F, Xaa ２１ is H, Xaa ２２ is N, Xaa ２６ is I (SEQ ID NO: 25), or a pharma- ceutically acceptable salt thereof.

7. Xaa １ The lysine in the formula: (γE) ２ -CO-(CH ２ ) １８ -CO ２ 7. The compound of claim 5 or 6, which is attached to a fatty acid linker moiety designated H.

8. The compound according to any one of claims 1 to 7, wherein a thioacetal bridge is present between the cysteine ​​at position 2 and the cysteine ​​at position 7.

9. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the compound is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

10. A compound consisting of SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof.

11. A compound consisting of SEQ ID NO: 4 or a pharma- ceutically acceptable salt thereof.

12. A compound consisting of SEQ ID NO:6 or a pharma- ceutically acceptable salt thereof.

13. A compound consisting of SEQ ID NO: 8 or a pharma- ceutically acceptable salt thereof.

14. 14. A method of treating type 2 diabetes in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

15. 17. A method of treating obesity in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

16. 14. A method of treating dyslipidemia in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

17. 14. A method of treating NASH in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

18. 14. A method of reducing food intake in a patient in need thereof, comprising administering to said patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

19. 14. A method of reducing body weight in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

20. 14. A method of lowering blood glucose levels in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

21. 14. A method of lowering triglycerides in a patient in need thereof comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

22. The method of any one of claims 13 to 21, wherein the compound is administered once a week.

23. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

24. A method for treating a condition in a patient in need of treatment selected from the group consisting of clinical or preclinical diabetes, obesity, NASH, and dyslipidemia, comprising administering to the patient an effective amount of a compound described in claim 1 in combination with an effective amount of an incretin or incretin analog.

25. 25. The method of treatment of claim 24, wherein the compound is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, or a pharmaceutical salt thereof, and the incretin or incretin analog is SEQ ID NO:

21.

26. 14. A compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof, for use in therapy.

27. 24. A pharmaceutical composition according to claim 23 for use in therapy.

28. 14. A compound according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition selected from the group consisting of type 2 diabetes, obesity, dyslipidemia, and NASH.

29. 24. The pharmaceutical composition of claim 23 for use in the treatment of a condition selected from the group consisting of type 2 diabetes, obesity, dyslipidemia, and NASH.

30. 14. Use of a compound according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment of a condition selected from the group consisting of type 2 diabetes, obesity, dyslipidemia and NASH.

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