Dual amylin and calcitonin receptor agonists and uses thereof

Abstract

To provide a compound that stimulates both calcitonin and amylin receptors and can reduce food intake, body weight, glucose, and / or triglycerides, and therefore can be used to treat diabetes, obesity, and / or dyslipidemia, and to provide a pharmaceutical composition containing such a compound.SOLUTION: There is provided a pharmaceutical composition comprising a compound comprising a specific sequence or a pharmaceutically acceptable salt thereof for the treatment of a disease selected from the group consisting of diabetes, obesity, non-alcoholic steatohepatitis (NASH), and dyslipidemia, wherein the compound or pharmaceutically acceptable salt thereof is used to be administered in combination with a GLP-1 agonist.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the field of medicine. More particularly, the present disclosure is in the field of treating diabetes, obesity, non-alcoholic steatohepatitis (NASH), and / or dyslipidemia. The present disclosure relates to compounds that stimulate both amylin and calcitonin receptors and thus can reduce food intake, body weight, glucose, and / or triglycerides and can be used to treat diabetes, obesity, NASH, and / or dyslipidemia. The present disclosure also includes pharmaceutical compositions containing such compounds, as well as therapeutic uses of such compounds and pharmaceutical compositions. [Background technology]

[0002] Amylin is a peptide hormone co-secreted with insulin by pancreatic beta cells and is deficient in people with diabetes. It suppresses glucagon secretion, slows gastric emptying, and acts as a satiety agent. Pramlintide, an amylin analog, lowers blood glucose levels and can be used to treat insulin-dependent diabetes patients. Because the drug has an elimination half-life of less than one hour and is administered with meals, patients require multiple daily doses to use the drug therapeutically.

[0003] Calcitonin is a hormone produced by the thyroid gland that regulates calcium and phosphate levels in the blood. Salmon calcitonin can be used to treat conditions with high blood calcium levels, such as hypercalcemia. Because salmon calcitonin has a short half-life of less than two hours, patients must take the peptide once or multiple times a day to use it therapeutically.

[0004] Compounds that stimulate both amylin and calcitonin receptors have been shown to have beneficial effects, such as lowering blood glucose levels and inducing weight loss. See WO2016 / 034604, WO2015 / 071229, and WO2010 / 085700. Known amylin and calcitonin receptor agonists have short natural half-lives, making long-term action desirable. However, chemical modifications intended to extend activity have so far shown reduced efficacy. Furthermore, amylin and calcitonin receptor agonists have stability issues due to their tendency to fibrillate and the presence of unstable disulfide bonds at neutral pH.

[0005] There is a need for alternative compounds that stimulate both amylin and calcitonin receptors. Additionally, there is a need for amylin and calcitonin receptor agonists with extended duration of action and retained efficacy. A therapeutically desirable compound would stimulate both amylin and calcitonin receptors and provide one or more advantageous properties, such as reduced food intake, reduced body weight, reduced blood glucose levels, reduced triglycerides, and / or reduced insulin levels. Furthermore, a therapeutically desirable compound may have one or more additional advantageous properties, such as a long duration of action with retained or improved efficacy in stimulating both amylin and calcitonin receptors, a low risk of immunogenic response, chemical stability at neutral pH, and / or a low risk of fibrillation.

[0006] Furthermore, combinations of the dual amylin and calcitonin receptor agonists of the present disclosure, optionally combined with an incretin or incretin analog, are desirable for providing treatment for diabetes, obesity, NASH, and / or dyslipidemia. Such combinations will also preferably be more effective than either molecule alone. For example, treatment with such combinations may allow for the use of lower doses of either or both molecules compared to each molecule alone, potentially resulting in fewer side effects (or a shorter duration of one or the other therapy) while maintaining efficacy. The novel combinations provided herein are believed to be effective treatments for diabetes, obesity, NASH, and / or dyslipidemia. Summary of the Invention

[0007] Accordingly, the present disclosure provides methods for treating diabetes, obesity, NASH, and / or dyslipidemia, comprising administering to a patient in need of such treatment an effective amount of a dual amylin and calcitonin receptor agonist with an effective amount of an incretin or incretin analog. The present disclosure further provides methods for treating clinical or preclinical diabetes, obesity, NASH, and / or dyslipidemia, comprising administering to a patient in need of such treatment an effective amount of a dual amylin and calcitonin receptor agonist in combination with an effective amount of an incretin or incretin analog.

[0008] Certain compounds disclosed herein that stimulate amylin and calcitonin receptors exhibit effective reductions in food intake and body weight, as well as glucose and insulin lowering. Furthermore, certain compounds disclosed herein exhibit reduced risk of immunogenicity and low levels of fibrillation. The pharmacokinetic properties of certain compounds disclosed herein exhibit significantly extended half-lives, thereby enabling once-weekly dosing of these compounds for use in therapy. Other embodiments of the present disclosure are useful for preparing such compounds for use in therapy. Methods for combining the compounds disclosed herein with incretins or incretin analogs are also disclosed. DETAILED DESCRIPTION OF THE INVENTION

[0009] One of the compounds of the present disclosure, Compound I, comprises the following sequence: Acetyl-ASHLSTAVLGKLS-Aib-ELHKLEDYPRTDVGAESP-NH2 (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof. Below is a depiction of the structure of Compound I using standard single-letter amino acid codes, except for residues acetyl-A1, Aib14, and P32-NH2, where the structures of these amino acid residues have been extended: [ka]

[0010] Thus, one of the compounds of the present disclosure is [ka] or a pharmaceutically acceptable salt thereof.

[0011] The compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof may further comprise one or more additional elements to extend the half-life of the compound. Elements that can be added to the compound comprising SEQ ID NO: 1 for this purpose, with or without a linker and at any suitable position in the sequence, include the Fc portion of an immunoglobulin, a fragment of the Fc portion of an immunoglobulin, human serum albumin (HSA), VHH, a variant of a VHH (variable domain of a heavy chain antibody) nanobody, a fragment of human serum albumin, C 16 ~C 20 Monoacid, C 16 ~C 20 Other elements that can be added to compounds containing SEQ ID NO: 1 for this purpose include C, C-C, C-D, C-E, C-F, C-H ... 16 ~C 22 Monoacid or C 16 ~C 22 diacid, C 18 ~C 20 Monoacid, or C 18 ~C 20 Diacids may also be included.

[0012] One embodiment of the compounds disclosed herein is a compound comprising SEQ ID NO: 1, which further comprises a fatty acid moiety with or without a linker.

[0013] In another embodiment, the compounds disclosed herein have an acetyl group (CHCO) on the alanine amino acid residue at the N-terminus of the peptide. This is indicated herein by the word "acetyl" and a dash before the N-terminal amino acid residue in descriptions of the peptide. The compounds disclosed herein also have an amide group (NH) on the C-terminal amino acid residue, proline. This is indicated herein by the formula "NH" and a dash after the C-terminal amino acid residue in descriptions of the peptide.

[0014] In another embodiment, the compound comprising SEQ ID NO: 1 is acylated at any suitable amino acid in the sequence, resulting in an extended half-life. In another embodiment, the compound comprising SEQ ID NO: 1 is acylated at the epsilon amino group on the lysine side chain, resulting in an extended half-life. In a particular ... 18 It is acylated at the epsilon amino group on the lysine side chain with -COH. Thus, one of the compounds of the present disclosure is Compound II, which comprises the following sequence: Acetyl-ASHLSTAVLGK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2) 18 —CO₂H)LS-Aib-ELHKLEDYPRTDVGAESP-NH₂ (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof.

[0015] In certain embodiments, the compound consisting of SEQ ID NO: 1 has the formula 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH 18 It is acylated at the epsilon amino group on the lysine side chain with -COH. Thus, one of the compounds of the present disclosure is Compound II, which consists of the following sequence: Acetyl-ASHLSTAVLGK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2) 18 -COH)LS-Aib-ELHKLEDYPRTDVGAESP-NH (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound consisting essentially of SEQ ID NO: 1 has the formula 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH) 18 The epsilon amino group on the lysine side chain is acylated with -COH. Thus, one of the compounds of the present disclosure is Compound II, which consists essentially of the following sequence: Acetyl-ASHLSTAVLGK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2) 18 -COH)LS-Aib-ELHKLEDYPRTDVGAESP-NH (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof. The following is a depiction of the structure of Compound II using standard single-letter amino acid codes, except for residues acetyl-A1, K11, Aib14, and P32-NH2, the structures of these amino acid residues being elongated: [ka]

[0016] Thus, one of the compounds of the present disclosure is [ka] or a pharmaceutically acceptable salt thereof.

[0017] Another embodiment of the present disclosure provides a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. Another embodiment of the present disclosure provides a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0018] Another embodiment of the present disclosure provides a method for treating diabetes in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method for treating diabetes in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. A preferred embodiment of the present disclosure provides a method for treating type 2 diabetes in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another preferred embodiment of the present disclosure provides a method for treating type 2 diabetes in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method for treating diabetes in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of treating diabetes in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2. A preferred embodiment of the present disclosure provides a method of treating type 2 diabetes in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another preferred embodiment of the present disclosure provides a method of treating type 2 diabetes in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0019] Another embodiment of the present disclosure provides a method of treating obesity in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating obesity in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating obesity in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of treating obesity in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0020] Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0021] Another embodiment of the present disclosure provides a method of treating NASH in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating NASH in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating NASH in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of treating NASH in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0022] Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0023] Another embodiment of the present disclosure provides a method of reducing weight in a patient in need of weight loss, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of reducing weight in a patient in need of weight loss, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of reducing weight in a patient in need of weight loss, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of reducing weight in a patient in need of weight loss, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0024] Another embodiment of the present disclosure provides a method of lowering blood glucose in a patient in need of lowering blood glucose, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of lowering blood glucose in a patient in need of lowering blood glucose, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of lowering blood glucose in a patient in need of lowering blood glucose, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of lowering blood glucose in a patient in need of lowering blood glucose, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0025] Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 1. Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

[0026] Another embodiment of the present disclosure provides a method of treating diabetes in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of treating diabetes in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0027] Another embodiment of the present disclosure provides a method of treating type 2 diabetes in a patient in need of such treatment, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of treating type 2 diabetes in a patient in need of such treatment, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0028] Another embodiment of the present disclosure provides a method of treating obesity in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of treating obesity in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0029] Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of such treatment, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of such treatment, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0030] Another embodiment of the present disclosure provides a method of treating NASH in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of treating NASH in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0031] Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0032] Another embodiment of the present disclosure provides a method of reducing weight in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of reducing weight in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0033] Another embodiment of the present disclosure provides a method of lowering blood glucose in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of lowering blood glucose in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0034] Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need thereof, wherein a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need thereof, wherein a compound comprising SEQ ID NO: 2 is administered once weekly.

[0035] Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, for use in therapy. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 1, for use in therapy. Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use in therapy. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 2, for use in therapy.

[0036] Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of diabetes. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 1, for use in the treatment of diabetes. Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of diabetes. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 2, for use in the treatment of diabetes.

[0037] Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of type 2 diabetes. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 1, for use in the treatment of type 2 diabetes. Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of type 2 diabetes. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 2, for use in the treatment of type 2 diabetes.

[0038] Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of obesity. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 1, for use in the treatment of obesity. Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of obesity. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 2, for use in the treatment of obesity.

[0039] Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of dyslipidemia. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 1, for use in the treatment of dyslipidemia. Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of dyslipidemia. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 2, for use in the treatment of dyslipidemia.

[0040] Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of NASH. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 1, for use in the treatment of NASH. Another embodiment of the compounds disclosed herein provides a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of NASH. Another embodiment of the disclosure provides a pharmaceutical composition comprising SEQ ID NO: 2, for use in the treatment of NASH.

[0041] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diabetes. Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diabetes. A preferred embodiment of the compound of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of type 2 diabetes. Another preferred embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diabetes.

[0042] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of obesity.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of obesity.

[0043] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of dyslipidemia.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of dyslipidemia.

[0044] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of NASH.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of NASH.

[0045] Another embodiment of the compounds disclosed herein is a compound having a half-life (t 1 / 2 ) is the compound of SEQ ID NO: 1 in which the amino acid sequence is extended.

[0046] Another 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, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an incretin or incretin analog.Another 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, 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 incretin analog.

[0047] 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, comprising administering to the 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 GLP-1 agonist. A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, comprising administering to the 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 GLP-1 agonist selected from the group consisting of compounds comprising Compound III (SEQ ID NO: 6), Compound VIII (SEQ ID NO: 12), or Compound IX (SEQ ID NO: 13). A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, comprising administering to the 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 GLP-1 agonist. A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, 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 GLP-1 agonist selected from the group consisting of compounds comprising Compound III (SEQ ID NO: 6), Compound VIII (SEQ ID NO: 12), or Compound IX (SEQ ID NO: 13).

[0048] A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 GIP / GLP-1 dual agonist. A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 GIP / GLP-1 dual agonist selected from the group consisting of compounds comprising Compound VI (SEQ ID NO: 9) and Compound VII (SEQ ID NO: 10). A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 2 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, 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 GIP / GLP-1 dual agonist selected from the group consisting of compounds comprising Compound VI (SEQ ID NO: 9) and Compound VII (SEQ ID NO: 10).

[0049] A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 triagonist of glucagon, GIP, and GLP-1. A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 triagonist of glucagon, GIP, and GLP-1. A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 triagonist of glucagon, GIP, and GLP-1, including compound V (SEQ ID NO: 8). A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, 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 triagonist of glucagon, GIP, and GLP-1, including compound V (SEQ ID NO: 8).

[0050] A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 an analog of oxyntomodulin. A particular embodiment of the compounds disclosed herein is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, comprising administering to a patient in need of such treatment an effective amount of a compound comprising SEQ ID NO: 2 in combination with an analog of oxyntomodulin. A particular embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 an analog of oxyntomodulin, including compound IV (SEQ ID NO: 7). A particular embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, 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 an analog of oxyntomodulin, including compound IV (SEQ ID NO: 7).

[0051] Another embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 an incretin or incretin analog, wherein the compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof and the incretin or incretin analog are administered simultaneously. Another embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 an incretin or incretin analog, wherein the compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof and the incretin or incretin analog are administered simultaneously.

[0052] Another embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 an incretin or incretin analog, wherein the compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof is administered prior to administration of the incretin or incretin analog. Another embodiment of the present disclosure is a method of treating a patient with diabetes, obesity, NASH, and / or dyslipidemia, 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 an incretin or incretin analog, wherein the compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof is administered prior to administration of the incretin or incretin analog.

[0053] Another embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, 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 an incretin or an incretin analog, wherein the incretin or incretin analog is administered prior to administration of the compound comprising SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating a patient having diabetes, obesity, NASH, and / or dyslipidemia, 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 an incretin or an incretin analog, wherein the incretin or incretin analog is administered prior to administration of the compound comprising SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof.

[0054] Another embodiment of the present disclosure is a compound comprising SEQ ID NO: 1 for use in separate, simultaneous, or sequential combination with an incretin or incretin analog for the treatment of diabetes, obesity, NASH, and / or dyslipidemia. Another embodiment of the present disclosure is a compound comprising SEQ ID NO: 2 for use in separate, simultaneous, or sequential combination with an incretin or incretin analog for the treatment of diabetes, obesity, NASH, and / or dyslipidemia.

[0055] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 in the manufacture of a medicament for the treatment of diabetes.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 in the manufacture of a medicament for the treatment of diabetes.

[0056] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 in the manufacture of a medicament for the treatment of type 2 diabetes. Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 in the manufacture of a medicament for the treatment of type 2 diabetes.

[0057] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 in the manufacture of a medicament for the treatment of obesity.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 in the manufacture of a medicament for the treatment of obesity.

[0058] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 in the manufacture of a medicament for the treatment of dyslipidemia.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 in the manufacture of a medicament for the treatment of dyslipidemia.

[0059] Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 1 in the manufacture of a medicament for the treatment of NASH.Another embodiment of the present disclosure provides the use of a compound comprising SEQ ID NO: 2 in the manufacture of a medicament for the treatment of diabetes.

[0060] 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 dual amylin and calcitonin receptor agonists, pharmaceutical compositions, and pharmaceutical combinations, the preferred methods and materials are described herein.

[0061] Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0062] The term "analog" refers to 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 elicited by a natural agonist for that receptor.

[0063] As used herein, the term "combination" means that the compounds disclosed herein can be used simultaneously, separately, or sequentially in combination with one or more additional therapeutic agents useful for inducing weight loss, treating diabetes, conditions related to diabetes, obesity, NASH, and / or dyslipidemia. Non-limiting examples of additional therapeutic agents that can be combined with the claimed compounds include insulin or insulin analogs; incretin compounds or incretin analogs, such as glucagon-like peptide-1 (GLP-1) or GLP-1 analogs, gastric inhibitory polypeptide (GIP) or GIP analogs, oxyntomodulin or oxyntomodulin analogs; GIP / GLP-1 dual agonists; Gcg / GIP / GLP-1 triagonists (triagonists of glucagon, GIP, and GLP-1); or any combination of the foregoing agents. The claimed compound and additional therapeutic agent may be co-administered via the same delivery route and device, such as a single pill, capsule, tablet, or injectable formulation, or may be administered separately either simultaneously in separate delivery devices or routes, or may be administered sequentially.

[0064] 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 carbohydrate metabolism and elevated blood and urine glucose levels.As used herein, the term diabetes can refer to a chronic condition that affects the body's blood sugar or glucose processing process, 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 is high but not high enough to result in type 2 diabetes, such as prediabetes; a form of hyperglycemia that affects pregnant women, such as gestational diabetes.

[0065] As used herein, the term "dyslipidemia" refers to a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. Dyslipidemia can manifest as elevated total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride levels in the blood, and / or decreased high-density lipoprotein (HDL) levels. Dyslipidemia may or may not occur in association with diabetes.

[0066] As used herein, the term "incretin" refers to a group of endogenous metabolic hormones excreted from enteroendocrine cells in the stomach and pancreas to stimulate a lowering of blood glucose, typically through regulation of the amount of insulin secreted after a meal. As used herein, the term "incretin analog" refers to a group of synthetic incretin mimetics that are physiologically similar to incretins. Furthermore, "incretin analogs" may have favorable pharmacological properties compared to endogenous incretins.

[0067] As used herein, the term "treatment" or "treating" refers to the management and care of a patient having a condition for which administration of an amylin and calcitonin receptor peptide agonist is indicated for the purpose of combating or alleviating the symptoms and complications of the condition. Treatment involves administering one of the compounds disclosed herein or a pharmaceutical composition containing the same to a patient in need thereof to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate a disease, condition, or disorder. Preferably, treatment involves administering one of the compounds disclosed herein or a pharmaceutical composition containing the same to a patient in need thereof to result in a net loss of body weight, reduced food intake, reduced blood glucose levels, and / or reduced triglyceride levels. The patient to be treated is an animal, preferably a human.

[0068] As used herein, the term "effective amount" refers to an amount or dose of one of the compounds disclosed herein or a pharmaceutical composition containing a compound disclosed herein that, when administered to a patient in single or multiple doses, elicits the biological or medical response or desired therapeutic effect sought by a medical professional. Preferably, an effective amount of one of the compounds disclosed herein or a pharmaceutical composition containing the same administered to a patient in need thereof will result in a net loss of body weight, reduced food intake, reduced blood glucose levels, and / or reduced triglyceride levels. Dosages can include a higher initial loading dose, followed by smaller doses.

[0069] As used herein, the term "patient" refers to an animal, preferably a human. In certain embodiments, the patient, preferably a human, is further characterized by a disease, disorder, or condition that may benefit from the administration of a compound that stimulates both amylin and calcitonin receptors. Pharmaceutical compositions comprising the compounds disclosed herein can be administered orally or parenterally to a patient in need of such treatment. Parenteral administration can be performed by subcutaneous, intramuscular, or intravenous injection using a syringe, optionally a pen-like syringe, or a mechanically driven injector. Alternatively, parenteral administration can be performed using an infusion pump. Embodiments of the compounds disclosed herein provide pharmaceutical compositions suitable for administration to a patient, comprising administering to a patient in need thereof 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, as are well known in the art. (Remington's Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Philadelphia, PA, USA (2006)).

[0070] As used herein, the term "NASH" refers to non-alcoholic steatohepatitis, also known as fatty liver disease. "NASH" also refers to inflammation and damage to 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" can be synonymous with "NAFLD."

[0071] As used herein, the term "obesity" refers to a disorder involving excess body fat, which increases the risk of health problems. The term "obesity" also refers to a weight that is higher than what is considered healthy for a given height. The term "obesity" also refers to a BMI greater than 30.0. As used herein, body mass index (BMI) refers to a person's weight in kilograms divided by the square of their height in meters.

[0072] Certain abbreviations are defined as follows: "Aib" refers to 2-aminoisobutyric acid, "AMY1" refers to amylin receptor 1, "cAMP" refers to cyclic adenosine monophosphate, "CT" refers to calcitonin, "DCM" refers to dichloromethane, "DIEA" refers to diisopropylethylamine, "DIO" refers to diet-induced obesity, "DMF" refers to dimethylformamide, "FBS" refers to fetal bovine serum, "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, "Mtt" refers to 4-methyltrityl, "PBMC" refers to peripheral blood mononuclear cells, "PyAOP" refers to (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, "RP-HPLC" refers to reverse-phase high-pressure liquid chromatography, and "TFA" refers to trifluoroacetic acid. [Example]

[0073] Example 1: Preparation and purification of Compound I and Compound II Compound I and Compound II are prepared according to the following steps: First, Compound I is synthesized using fluorenylmethyloxycarbonyl (Fmoc) / tert-butyl (t-Bu) chemistry on a Symphony 12-Channel Multiplex Peptide Synthesizer (Protein Technologies, Inc. Tucson, AZ).

[0074] Polystyrene Rink Amide AM LL resin (Novabiochem, sub: 0.33 meq / g, 100-200 mesh, catalog number 855045) is used for the synthesis on a 0.13 mmol scale. Standard side chain protecting groups are used. Fmoc-Lys(Mtt)-OH) is used at lysine 11. The Fmoc group is removed using 20% ​​piperidine in DMF before each coupling step (2 x 7 min). All amino acid couplings are performed at 50 °C for 30 min at a 7.7-fold molar excess over theoretical peptide loading using equimolar ratios of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0.9 mM), and Oxyma (0.9 mM). The N-terminus is acetylated using 5% acetic anhydride, 5% DIEA in DMF for 30 min. Below is a schematic diagram of compound I (SEQ ID NO: 1). [ka] The resin is then thoroughly washed six times with DCM to remove residual DMF. The Mtt protecting group on lysine 11 is selectively removed from the peptide resin using two treatments of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (2 x 40 min treatments).

[0075] The synthesis of Compound II is carried out according to the following steps. The subsequent attachment of the fatty acid linker moiety is achieved by coupling 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid α-t-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.), and mono-OtBu-eicosanoic acid (WuXi AppTec, Shanghai, China). A three-fold excess of reagents (AA:PyAOP:DIEA = 1:1:1 mol / mol) is used for each coupling, which lasts for 1 hour.

[0076] Upon completion of the synthesis, the peptide resin is washed with DCM and air-dried thoroughly. The dried resin is treated with 10 mL of cleavage cocktail (TFA:water:triisopropylsilane, 95:2.5:2.5 v / v) for 2 h at room temperature. The resin is filtered off and washed twice with 2 mL of neat TFA each time. The combined filtrates are treated with four portions of cold diethyl ether (-20 °C) to precipitate the crude peptide. The peptide / ether suspension is then centrifuged at 3500 rpm for 2 min to form a solid pellet. The supernatant is decanted, and the solid pellet is triturated twice more with ether and dried in vacuo. The crude peptide was solubilized in 20% acetonitrile / 20% acetic acid / 60% water and purified by RP-HPLC on a Waters Xselect Peptide CSH C18 prep column 130A 5µm 19x150mm PN 186007021 with a linear gradient using 100% acetonitrile and 0.1% TFA / water buffer system (20-40% acetonitrile in 60 min). Peptide purity was assessed using analytical RP-HPLC, with pooling criteria exceeding 95%. The purity of the main pool of compound I was found to be greater than 99.0%. Subsequent lyophilization of the final main product pool yielded the lyophilized peptide TFA salt. The molecular weight was determined by LC / MS. Average MW = 3447.8 Da. Expected mass: (M+3H): 1150.3, found: 1150.0.

[0077] The purity of the main pool of Compound II was found to be greater than 98.0%. Subsequent lyophilization of the final main product pool resulted in the lyophilized peptide TFA salt. The molecular weight was determined by LC / MS. Average MW=4191.8 Da. Expected mass: (M+3H): 1398.2, Found 1398.2. Below is a schematic diagram of Compound II (SEQ ID NO: 2). [ka]

[0078] Methods similar to those described above and known to those skilled in the art can be used to synthesize the peptide backbone, conjugate the fatty acid-linker moiety, check the purity, and confirm the molecular weight of the compounds of the invention described herein.

[0079] Example 2: In vitro functional activity AMY1 and CT receptors are GPCRs functionally coupled to Gαs proteins. Stimulation of these receptors increases intracellular cAMP production, which can be detected using standard in vitro techniques. Human AMY1 or CT receptors are stably expressed in human bladder cells (UM-UC-3) under the control of the pcDNA(CALCR) or pCMVpiggybac(RAMP1) expression vectors. AMY1 cells are cultured in MEM 1X (Corning) supplemented with 10% FBS, 1% antibiotic / antimycotic solution, 1 mM sodium pyruvate, 1X MEM NEAA, 1X GlutaMAX-I, 200 μg / mL hygromycin B, and 0.4 μg / mL puromycin. CT cells are cultured in the same medium, except lacking puromycin. Cultured cells are grown to 70% confluency and then incubated overnight in fresh medium.

[0080] On the day of the assay, 10 μL of assay buffer (phenol red-free MEM (Corning, catalog no. 17-305-CV), 0.1% casein, 0.5 mM IBMX, 5 mM HEPES, pH 7.4) was dispensed into each well of a white poly-D-lysine-coated 384-well plate (Corning, catalog no. 354661). Peptides diluted in DMSO were added (200 nL / well) in a 1:3 dilution series using an ECHO acoustic liquid handler (Beckman). Cultured cells were detached with TrypLE Express (Gibco) and resuspended in assay buffer. 10 μL containing 1200 cells / well (hCT) or 1500 cells / well (hAMY1) was dispensed into each well. The plate was incubated at room temperature for 1 hour.

[0081] The amount of intracellular cAMP is quantified using HTRF technology (Homogeneous Time-Resolved Fluorescence, Cisbio) according to the manufacturer'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 for 60 minutes at room temperature. The HTRF signal is immediately detected using an Envision plate reader (Perkin-Elmer), and the fluorescence ratio is calculated at 665-620 nm. The raw data are converted to cAMP amounts (pmol / well) using a cAMP standard curve generated for each experiment. The relative EC 50 Values ​​are calculated from the upper and lower ranges of concentration-response curves defined with a four-parameter logistic curve fitting program (Genedata Screener® v12.0.4) using 1 nM salmon CT (Bachem) as the maximum and buffer alone as the minimum. Compounds of the present application exhibit activity at amylin and calcitonin receptors, as shown in Table 1. [Table 1]

[0082] Example 3: In vivo efficacy in a normal rat model Male Sprague Dawley (SD) rats weighing approximately 300 grams from Envigo Laboratories are used to evaluate the effects of Compound II on acute reduction in food intake and body weight loss in vivo during a 96-hour evaluation study. Rats are maintained under Lilly Research Laboratories' approved Animal Care and Use protocols and housed individually in a 12-hour reverse light cycle from 10 PM to 10 AM. On the morning of the study, baseline animal body and food weights are measured, and different doses of vehicle (20 mM Tris pH 8, 50 mg / mL D-mannitol, 0.02% PS80) or Compound II (SEQ ID NO: 2) alone in vehicle (20 mM Tris pH 8, 50 mg / mL D-mannitol, 0.02% PS80) are subcutaneously administered.

[0083] 24 hours, 48 ​​hours, 72 hours, and 96 hours after administration, the weight of the animal's body and food are measured. Daily food intake and percent weight loss are calculated. These results are listed in Tables 2 and 3 below. All data are shown as average daily food intake and percent weight loss. As shown by the results, Compound II has the effect of reducing food consumption and reducing body weight rapidly and dose-dependently. Also, even at very low doses, it shows the effect of reducing food intake and body weight. [Table 2] [Table 3]

[0084] Example 4: In vivo efficacy in diet-induced obese (DIO) rats This study was conducted to investigate the effects of Compound II on diabetic and / or obese conditions in DIO rats. Male, 24-30 week-old, diet-induced obese (DIO) Long Evans (Envigo) rats maintained on a high-calorie diet since arrival at Lilly (TD95217; Teklad, Madison, WI) were used in the following study. Animals were housed individually in a temperature-controlled (24°C) facility with a 12-hour light / dark cycle (lights on at 22:00) and free access to food (TD95217) and water.

[0085] Randomize the rats according to their weight so that each experimental group of animals has a similar weight, ranging from 514 to 710 grams.

[0086] Each group contained five rats. Vehicle and Compound II (0.1 and 100 nmol / kg) dissolved in vehicle (Tris pH 8, (50 mg / mL D-mannitol) + 0.02% PS80) were administered by subcutaneous (SC) injection (1 mL / kg) to ad libitum-fed DIO rats 30–60 min before the onset of the dark cycle every 3 days for 15 days. SC injections were administered on days 1, 4, 7, 10, and 13. Body weight and food intake were measured daily throughout the study. Absolute changes in body weight were calculated by subtracting the weight of the same animal before the first injection of the molecule. Body composition was assessed by quantitative nuclear magnetic resonance (QMNR, EchoMRI LLC, Houston, TX) on days -1 (1 day before treatment) and 15.

[0087] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured by AccuChek blood glucose meter (Roche, Indianapolis, IN). Insulin is measured by ELISA (MSD, Rockville, MD).

[0088] All data are presented as the mean ± SEM of 5 animals per group. Statistical analysis was performed using repeated measures ANOVA followed by Dunnett's method for multiple comparisons. Significant differences were defined at p<0.05.

[0089] Compound II dose-dependently reduces body weight and food intake in male DIO rats. The weight loss is believed to be primarily due to a decrease in fat mass. As shown in Tables 4 and 5, treatment with Compound II resulted in a substantial weight loss as well as a dose-dependent decrease in serum glucose and insulin.

[0090] Body composition measurements are taken on days 0 and 14. Changes from the first measurement are presented as the measurement on day 0 to the measurement on day 14. All data are presented as the mean ± SEM of 5 animals per group from day 14. Statistical analysis is performed using repeated measures ANOVA followed by Dunnett's method for multiple comparisons. Significant differences are defined at p<0.05. [Table 4] [Table 5]

[0091] Example 5: Pharmacokinetic behavior of Compound II Plasma concentrations of Compound II are determined by a certified liquid chromatography-mass spectrometry (LC / MS) method at Q Squared Solutions BioSciences LLC, Ithaca, NY. Compound II and analogs as internal standards are extracted from 100% species-specific plasma using protein precipitation followed by solid-phase extraction. The intact mass of Compound II, including the peptide and acyl chains, is detected by a Q-Exactive Orbitrap™ mass spectrometer.

[0092] In the monkey pharmacokinetic study, male and female cynomolgus monkeys were administered a single subcutaneous dose of Compound II at 20 nmol / kg (0.084 mg / kg) in a 0.2 mL / kg volume of Tris-mannitol buffer (pH 8.0). Blood samples were collected pre-dose and 1, 3, 6, 12, 24, 48, 72, 96, 120, 144, 168, 240, 336, 408, and 504 hours post-dose for pharmacokinetic characterization. The results are shown in Table 6. [Table 6]

[0093] In the rat pharmacokinetic study, male Sprague Dawley rats were administered a single subcutaneous dose of Compound II at 20 nmol / kg (0.084 mg / kg) in a volume of 0.2 mL / kg Tris-mannitol buffer (pH 8.0). Blood samples were collected at 1, 3, 6, 12, 24, 48, 72, 96, 120, and 144 hours after dosing for pharmacokinetic characterization. The results are shown in Table 7. [Table 7]

[0094] The extended half-life exhibited by Compound II in cynomolgus monkeys indicates that treatment with a once-weekly dose of Compound II is feasible in patients.

[0095] Example 6: In vivo efficacy of Compound II in combination with other incretin compounds in diet-induced obese (DIO) rats This study was conducted to investigate the effects of Compound II on diabetic and / or obese conditions in DIO rats when administered in combination with other incretin compounds, including a GLP-1 agonist (Compound III), an oxyntomodulin analog (Compound IV), and triagonists of glucagon, GLP-1, and GIP (Compound V). Diet-induced obese (DIO) male Long Evans (Envigo) rats maintained on a high-calorie diet upon arrival at Lilly (TD95217; Teklad, Madison, WI) were used in the following study. Animals were housed individually in a temperature-controlled (24°C) facility with a 12-hour light / dark cycle (lights on at 2200) and free access to food (TD95217) and water.

[0096] Randomize the rats according to their weight so that each experimental group of animals has a similar weight, ranging from 529 to 823 grams.

[0097] Each group contained five rats. Vehicle and Compound II (1 nmol / kg) were dissolved in vehicle (40 mM Tris-HCl pH 8 + 0.02% PS80) and administered by subcutaneous (SC) injection (1 mL / kg) to ad libitum-fed DIO rats 30-90 min before the onset of the dark cycle every 3 days for 14 days. SC injections were administered on days 1, 4, 7, 10, and 13. Body weight and food intake were measured daily throughout the study. Absolute changes in body weight were calculated by subtracting the weight of the same animal before the first injection of the molecule.

[0098] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured by AccuChek blood glucose meter (Roche, Indianapolis, IN). Insulin is measured by ELISA (MSD, Rockville, MD).

[0099] All data are presented as the mean ± SEM of 5 animals per group. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test to compare treatment groups with the vehicle group or each other. Significant differences were defined at p<0.05. [Table 8]

[0100] Example 7: Analogs of Compound II in combination with agonists of GIP-GLP This study is conducted to investigate the effects of an analog of Compound II on diabetic and / or obese conditions in DIO rats when administered with Compound VI (SEQ ID NO: 9), a dual agonist of GIP and GLP-1. Diet-induced obese (DIO) male Long Evans (Envigo) rats maintained on a high-calorie diet upon arrival at Lilly (TD95217; Teklad, Madison, WI) are used in the following study. Animals are housed individually in a temperature-controlled (24°C) facility with a 12-hour light / dark cycle (lights on at 2200) and free access to food (TD95217) and water.

[0101] Randomize the rats according to their weight so that each experimental group of animals has a similar weight, ranging from 549 to 683 grams.

[0102] Each group contained five rats. Vehicle and Compound II (1 nmol / kg) dissolved in vehicle (10 mM Tris-HCl pH 7.5, (50 mg / mL D-mannitol) + 0.02% PS80) were administered by subcutaneous (SC) injection (1 mL / kg) to ad libitum-fed DIO rats 30–90 min before the onset of the dark cycle every 3 days for 14 days. SC injections were administered on days 1, 4, 7, 10, and 13. Body weight and food intake were measured daily throughout the study. Absolute changes in body weight were calculated by subtracting the weight of the same animal before the first injection of the molecule.

[0103] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured by AccuChek blood glucose meter (Roche, Indianapolis, IN). Insulin is measured by ELISA (MSD, Rockville, MD).

[0104] All data are presented as the mean ± SEM of 5 animals per group. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test to compare treatment groups with the vehicle group or each other. Significant differences were defined at p<0.05. [Table 9]

[0105] The analog of Compound II in combination with Compound VI caused greater weight loss than either treatment individually, which is likely due primarily to the significant reduction in cumulative food intake induced by the combined treatment.

[0106] Example 8: Immunogenicity Risk Assessment MAPPs assay (MHC-associated peptide proteomics) Primary human dendritic cells from 10 normal human donors were prepared from buffy coats by isolation of CD14+ cells as described and differentiated into immature dendritic cells by incubation with 20 ng / mL IL-4 and 40 ng / mL GM-CSF in complete RPMI medium containing 5% serum replacement (Thermo Fisher Scientific, Cat. No. A2596101) at 37°C and 5% CO2 for 3 days (Knierman et al., "The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein", Cell Reports, 33, 108454 (2020)). On day 4, 3 micromolar test antibodies were added to approximately 5 x 10 6Fresh medium containing 5 μg / mL LPS was added to the cells and replaced after 5 hours of incubation to transform the cells into mature dendritic cells. The following day, mature cells were lysed in 1 mL of RIPA buffer containing protease inhibitors and DNAse. Lysates were stored at -80°C until sample analysis.

[0107] Using an automated liquid handling system, HLA-II molecules were isolated from thawed lysates using a biotinylated anti-pan-HLA class II antibody (clone Tu39). Bound receptor-peptide complexes were eluted with 5% acetic acid, 0.1% TFA. The eluted MHC-II peptides were passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. The isolated MHC-II peptides were analyzed by nanoLC / MS using a Thermo Easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. This separation was performed using a 75 μm x 7 cm YMC-ODS C18 column with a flow rate of 250 nL / min, using a 65-minute gradient of 0.1% formic acid in water as solvent A and 80% acetonitrile containing 0.1% formic acid as solvent B. Mass spectrometry was performed in full scan mode at a resolution of 240,000, followed by a 3 second data-dependent MS / MS cycle consisting of an ion trap rapid scan with HCD and EThcD fragmentation.

[0108] Peptide identification was generated by an internal proteomics pipeline (Higgs et al., "Label-free LC-MS method for the identification of biomarkers," Methods in Molecular Biology, 428, 209-230 (2008)) using multiple search algorithms without enzyme search parameters against bovine and human databases containing test antibody sequences. The KNIME workflow was used to process the sample identification files. Peptides identified from the test article were aligned to the parent sequence. A summary of all donors was generated, annotating the percentage of donors displaying non-germline residues, the number of distinct regions displaying peptides containing non-germline residues, and the depth of peptide display in each region containing non-germline residues. An increased degree of non-germline peptide display is associated with an increased risk of immunogenicity. Results for Compound II are shown in Table 9 and suggest a low level of immunogenicity risk associated with Compound II. [Table 10]

[0109] T cell proliferation assay This assay evaluates the ability of test candidates to activate CD4+ T cells by inducing cell proliferation as described (Walsh et al., "Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms," mAbs, 12, 1764829 (2020)). Cryopreserved PBMCs from 10 healthy donors were used. CD8+ T cells were depleted from PBMCs and labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). PBMCs were cultured at 4 × 10 in AIM-V medium (Life Technologies, Cat. No. 12055-083) containing 5% CTS™ Immune Cell SR (Gibco, Cat. No. A2596101).6 Cells were seeded at 1000 cells / ml / well and tested in triplicate in 2.0 mL containing various test substances, medium control, keyhole limpet hemocyanin (KLH; positive control), lixisenatide (assay positive control), or Compound II. Cells were cultured and incubated at 37°C with 5% CO for 7 days. On day 7, samples were stained with the following cell surface markers for viability detection by flow cytometry using a BD LSRFortessa™ equipped with a high-throughput sampler (HTS). Data were analyzed using FlowJo® software (FlowJo, LLC, TreeStar) to calculate the mitotic index (CDI). Briefly, the CDI for each test candidate MAPPs-derived peptide cluster was calculated by dividing the number of proliferating CFSE clusters from peptide-stimulated wells by the number of CFSE clusters. dim The percentage of CD4+ T cells was calculated by multiplying the number of proliferating CFSE in unstimulated wells. dim The CD4+ T cell frequency was calculated by dividing by the percentage of CD4+ T cells. A CDI of greater than 2.5 was considered to represent a positive response. The donor frequency percentage across all donors was assessed. All donors demonstrated a positive T cell response to KLH (100%). The clinical immunogenicity positive control, lixisenatide, induced a positive T cell response in 50% of the cohort in this study, which is within the expected range for this assay (40-90% positive donor frequency). The results for Compound II are shown in Table 10 and suggest a low level of immunogenicity risk for Compound II. [Table 11]

[0110] array Compound I (SEQ ID NO: 1) Acetyl-ASHLSTAVLGKLS-Aib-ELHKLEDYPRTDVGAESP-NH2 Compound II (SEQ ID NO: 2) Acetyl-ASHLSTAVLGK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2) 18 -CO2H)LS-Aib-ELHKLEDYPRTDVGAESP-NH2 Pramlintide (SEQ ID NO: 3) KCNTATCATQRLANFLVHSSNNFGPILPTNVGSNTY-NH2 Contains a disulfide bond between Cys2 and Cys7 Human calcitonin (SEQ ID NO: 4) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH2 Contains a disulfide bond between Cys1 and Cys7 Human amylin (SEQ ID NO: 5) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2 Contains a disulfide bond between Cys2 and Cys7 Compound III (SEQ ID NO: 6) H-Aib-EGTFTSDVSSYLEGQAAK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO—(CH2) 16 -CO2H)EFIAWLVRGRG Compound IV (SEQ ID NO: 7) H-Aib-QGTFTSDYSKYLDEKKAK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)-CO—(CH2) 18 -CO2H)EFVEWLLEGGPSSG-NH2 Compound V (SEQ ID NO: 8) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO—(CH2) 18 -CO2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 Compound VI (SEQ ID NO: 9) Y-Aib-EGTFTSDYSI-Aib-LDKIAQK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO—(CH2) 18 -CO2H)A-(1Nal)-VQWLIAGGPSSGAPPPS-NH2 Compound VII (SEQ ID NO: 10) YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS In the formula, X1 is Aib, X2 is Aib, and K at position 20 is (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO—(CH2) 18 It is chemically modified through conjugation of the K side chain with -CO2H to the epsilon-amino group, and the C-terminal amino acid is amidated as a C-terminal primary amide. CAS registration number: 2023788-19-2 Lixisenatide (SEQ ID NO: 11) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 CAS registration number: 320367-13-3 Compound VIII (SEQ ID NO: 12) HAEGTFTSDVSSYLEGQAAK(γGlu-CO-(CH2) 14 -CH3)EFIAWLVRGRG CAS registration number: 204656-20-2 Compound IX (SEQ ID NO: 13) Dulaglutide is a human GLP-1 receptor agonist, comprising a dimer of a GLP-1 analog fused at its C-terminus via a peptide linker to the N-terminus of an analog of the Fc portion of an immunoglobulin, and is identified as CAS Registry Number 923950-08-7 and provided with the following chemical name: 7-37-Glucagon-Like Peptide I [8-Glycine, 22-Glutamic Acid, 36-Glycine] (Synthetic Human) Fusion Protein, Dimer, with Peptide (Synthetic 16-Amino Acid Linker) Fusion Protein, with Immunoglobulin G4 (Synthetic Human Fc Fragment). Each monomer of dulaglutide has the amino acid sequence set forth in SEQ ID NO: 13: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

Claims

1. Acetyl-ASHLSTAVLGK ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ２ -(γ-Glu)-CO-(CH ２ ) １８ -CO ２ H) LS-Aib-ELHKLEDYPRTDVGAESP-NH ２ (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof.

2. Acetyl-ASHLSTAVLGK ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ２ -(γ-Glu)-CO-(CH ２ ) １８ -CO ２ H) LS-Aib-ELHKLEDYPRTDVGAESP-NH ２ 2. The compound of claim 1, consisting of: (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof.

3. 10. A method for treating type 2 diabetes in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. 10. A method of treating obesity in a patient in need thereof, comprising administering to said patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

5. 10. A method for treating dyslipidemia in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

6. 10. A method of treating NASH in a patient in need thereof, comprising administering to said patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

7. 10. A method for reducing food intake in a patient in need thereof, comprising administering to said patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

8. 10. A method for reducing weight in a patient in need thereof, comprising administering to said patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

9. 10. A method for lowering blood glucose in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

10. 10. A method of lowering triglycerides in a patient in need thereof, comprising administering to said patient an effective amount of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

11. The method of any one of claims 3 to 10, wherein the compound of claim 1 or 2 is administered once a week.

12. 10. A pharmaceutical composition comprising a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

13. 13. A method of treating type 2 diabetes in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

14. 13. A method of treating obesity in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

15. 13. A method for treating dyslipidemia in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

16. 13. A method of treating NASH in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

17. 13. A method for reducing food intake in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

18. 13. A method for reducing weight in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

19. 13. A method of lowering blood glucose in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

20. 13. A method of lowering triglycerides in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 12.

21. 13. A method for treating a condition selected from the group consisting of clinical or preclinical diabetes, obesity, NASH, and dyslipidemia in a patient in need of such treatment, comprising administering to the patient an effective amount of the pharmaceutical composition of claim 12 in combination with an effective amount of an incretin or incretin analog.

22. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use in therapy.

23. 13. A pharmaceutical composition according to claim 12 for use in therapy.

24. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of type 2 diabetes.

25. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of obesity.

26. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of dyslipidemia.

27. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of NASH.

28. 13. The pharmaceutical composition of claim 12 for use in the treatment of a condition selected from the group consisting of type 2 diabetes, obesity, dyslipidemia, and NASH.

29. 10. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for the treatment of type 2 diabetes.

30. 10. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for the treatment of obesity.

31. 10. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for the treatment of dyslipidemia.

32. 10. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for the treatment of NASH.

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