Dual amylin and calcitonin receptor agonists and uses thereof

Abstract

To provide compounds that stimulate both the calcitonin and amylin receptors and can lower food intake, body weight, glucose and / or triglycerides, making them suitable for treating diabetes, obesity and / or dyslipidemia.SOLUTION: The present disclosure provides compounds with specific sequences, serving as dual amylin and calcitonin receptor agonists. The present disclosure also includes pharmaceutical compositions containing the compounds and therapeutic uses of the compounds and compositions.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 the treatment of 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 the beta cells of the pancreas and is deficient in people with diabetes. It inhibits glucagon secretion, slows gastric emptying, and acts as a satiety agent. Pramlintide, an amylin analog, lowers blood glucose levels and is available to treat diabetes patients using insulin. The elimination half-life of this drug is less than one hour, and because the drug is used at mealtimes, patients require multiple doses per day to use the drug as a therapy.

[0003] Calcitonin is a hormone produced by the thyroid gland that plays a role in regulating calcium and phosphate levels in the blood. Salmon calcitonin can be used to treat conditions in which there is high calcium in the blood, such as hypercalcemia. Salmon calcitonin has a short half-life of less than 2 hours, so patients need to take the peptide once a day or multiple times a day for therapeutic use.

[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 a short natural half-life, so a long duration of action is desirable.However, chemical modifications intended to extend the duration of activity have also shown reduced efficacy so far.In addition, amylin and calcitonin receptor agonists have stability problems due to their tendency to fibrillate and the presence of labile 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. Additionally, 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, low risk of immunogenic response, chemical stability at neutral pH, and / or low risk of fibrillation.

[0006] Furthermore, a combination of the dual amylin and calcitonin receptor agonist of the present disclosure, optionally combined with an incretin or incretin analog, is desirable to provide treatment for diabetes, obesity, NASH, and / or dyslipidemia. Such a combination would also preferably be more effective than either molecule alone. For example, 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 fewer side effects (or shorter duration of one or the other therapy) while maintaining efficacy. It is believed that the novel combinations provided herein will be effective treatments for diabetes, obesity, NASH, and / or dyslipidemia. Summary of the Invention

[0007] Thus, the present disclosure provides methods of 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 analogue. The present disclosure further provides methods of 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 analogue.

[0008] Certain compounds disclosed herein that stimulate amylin and calcitonin receptors show effective reduction in food intake and body weight, as well as glucose and insulin lowering. Additionally, certain compounds disclosed herein show reduced risk of immunogenicity and low fibrillation levels. The pharmacokinetic properties of certain compounds disclosed herein show significantly extended half-life, which allows these compounds to be dosed once a week for use in therapy. Other embodiments of the present disclosure are useful for making such compounds for use in therapy. Methods are also disclosed for combining the compounds disclosed herein with incretins or incretin analogs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 pharma- ceutically acceptable salt thereof. Below is a depiction of the structure of Compound I using standard one-letter amino acid codes, except for residues Acetyl-A1, Aib14, and P32-NH2, in which the structures of these amino acid residues have been elongated: [ka]

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

[0011] The compound comprising SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof may further comprise one or more additional elements to extend the half-life of the compound. Elements that may 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-D, C-D-D-E, C ... 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 (CH3CO) on the alanine amino acid residue at the N-terminus of the peptide, which is indicated herein by the word "acetyl" and a dash before the N-terminal amino acid residue in the description of the peptide. The compounds disclosed herein also have an amide group (NH2) on the C-terminal amino acid residue, proline, which is indicated herein by the formula "NH2" and a dash after the C-terminal amino acid residue in the description 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 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 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. 18 It is acylated at the epsilon amino group on the lysine side chain with -CO2H. Thus, one of the compounds of the present disclosure is compound II, which includes 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 pharma- ceutical acceptable salt thereof.

[0015] In certain embodiments, the compound 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 -CO2H. 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 pharma- ceutical 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 It is acylated at the epsilon amino group on the lysine side chain with -CO2H. 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 -CO2H)LS-Aib-ELHKLEDYPRTDVGAESP-NH2 (SEQ ID NO: 2) or a pharma- ceutical 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 pharma- ceutically acceptable salt thereof.

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

[0018] Another embodiment of the present disclosure provides a method of treating diabetes in a patient in need of diabetes treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating diabetes in a patient in need of diabetes treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof. A preferred embodiment of the present disclosure provides a method of treating type 2 diabetes in a patient in need of type 2 diabetes treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. Another preferred embodiment of the present disclosure provides a method of treating type 2 diabetes in a patient in need of type 2 diabetes treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating diabetes in a patient in need of diabetes 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 pharma- ceutically 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 pharma- ceutically 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 dyslipidemia treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of dyslipidemia treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating dyslipidemia in a patient in need of dyslipidemia 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 dyslipidemia 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 NASH treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating NASH in a patient in need of NASH treatment, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of treating NASH in a patient in need of NASH 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 NASH 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 of reduced food intake, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need of reduced food intake, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of reducing food intake in a patient in need of reduced food intake, 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 of reduced food intake, 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 of triglyceride lowering, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need of triglyceride lowering, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof. Another embodiment of the present disclosure provides a method of lowering triglycerides in a patient in need of triglyceride lowering, 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 of triglyceride lowering, comprising administering to the patient a pharmaceutical composition comprising a compound comprising SEQ ID NO: 2.

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

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

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

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

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

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

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

[0034] Another embodiment of the disclosure provides a method of lowering triglycerides in a patient in need thereof, in which a compound comprising SEQ ID NO: 1 is administered once weekly.Another embodiment of the disclosure provides a method of lowering triglycerides in a patient in need thereof, in which 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 disclosure provides the use of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diabetes. Another embodiment of the disclosure provides the use of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diabetes. A preferred embodiment of the compound of the disclosure provides the use of a compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for the treatment of type 2 diabetes. Another preferred embodiment of the disclosure provides the use of a compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diabetes.

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

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

[0046] Another embodiment of the 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 pharma- ceutical acceptable salt thereof, in combination with an effective amount of an incretin or incretin analog.Another embodiment of the 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 pharma-ceutical 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 pharma- tically 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 pharma- tically acceptable salt thereof in combination with a GLP-1 agonist selected from the group consisting of a compound 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 pharma- tically 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 pharma- ceutical 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutical 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutical 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 in combination with an analog of oxyntomodulin, or a pharma- ceutically acceptable salt thereof. 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 in combination with an analog of oxyntomodulin, including compound IV (SEQ ID NO: 7), or a pharma-ceutically acceptable salt thereof. 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 pharma- ceutical acceptable salt thereof, in combination with an analog of oxyntomodulin, including compound IV (SEQ ID NO:7).

[0051] Another embodiment of the 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 pharma- ceutically acceptable salt thereof in combination with an incretin or incretin analog, wherein the compound comprising SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof and the incretin or incretin analog are administered simultaneously. Another embodiment of the 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 pharma- ceutically acceptable salt thereof in combination with an incretin or incretin analog, wherein the compound comprising SEQ ID NO:2 or a pharma- ceutically acceptable salt thereof and the incretin or incretin analog are administered simultaneously.

[0052] Another embodiment of the 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 pharma- ceutically acceptable salt thereof in combination with an incretin or incretin analog, where the compound comprising SEQ ID NO:1 or a pharma- ceutically acceptable salt thereof is administered prior to administration of the incretin or incretin analog. Another embodiment of the 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 pharma- ceutically acceptable salt thereof in combination with an incretin or incretin analog, where the compound comprising SEQ ID NO:2 or a pharma- ceutically 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 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 pharma- ceutically acceptable salt thereof in combination with an incretin or an incretin analog, where the incretin or incretin analog is administered prior to administration of the compound comprising SEQ ID NO: 1 or a pharma- ceutically acceptable salt thereof. 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 pharma- ceutically acceptable salt thereof in combination with an incretin or an incretin analog, where the incretin or incretin analog is administered prior to administration of the compound comprising SEQ ID NO: 2 or a pharma- ceutically acceptable salt thereof.

[0054] Another embodiment of the 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 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 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 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 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 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 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 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, pharmaceutical combinations, the preferred methods and materials are described herein.

[0061] Furthermore, 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 may be used in combination, simultaneously, separately, or sequentially, 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 may 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 aforementioned agents. The claimed compounds and the 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 levels of glucose in blood and urine.As used herein, the term diabetes can refer to a chronic condition that affects the body's process of 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 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 overproduction or deficiency of lipoproteins. Dyslipidemia may be manifested in elevated total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and / or decreased high-density lipoprotein (HDL) concentrations in the blood. 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 the lowering of blood glucose, typically via regulating the amount of insulin secreted after a meal. As used herein, the term "incretin analogs" 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 to combat or alleviate the symptoms and complications of the condition. Treatment includes administering one of the compounds disclosed herein or a pharmaceutical composition containing the same to a patient in need of the compound or a pharmaceutical composition containing the same to prevent the onset of the symptoms or complications, to alleviate the symptoms or complications, or to eliminate a disease, condition, or disorder. Preferably, treatment includes administering one of the compounds disclosed herein or a pharmaceutical composition containing the same to a patient in need of the compound or a pharmaceutical composition containing the same to cause a net reduction in 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 induces the biological or medical response or desired therapeutic effect sought by a medical professional when administered to a patient in a single or multiple doses. Preferably, an effective amount of one of the compounds disclosed herein or a pharmaceutical composition containing the compound administered to a patient in need thereof will result in a net reduction in 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 administration of a compound that stimulates both amylin and calcitonin receptors. A pharmaceutical composition comprising a compound disclosed herein may be administered orally or parenterally to a patient in need of such treatment. Parenteral administration may be performed by subcutaneous, intramuscular, or intravenous injection using a syringe, optionally a pen-like syringe, or a mechanically driven injector. Alternatively, parenteral administration may be performed using an infusion pump. An embodiment of the compounds disclosed herein provides a pharmaceutical composition suitable for administration to a patient, comprising administering to a patient in need of said pharmaceutical composition a therapeutically effective amount of a compound disclosed herein and one or more pharma- ceutical acceptable excipients. Such pharmaceutical compositions may 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)).

[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 of the liver caused by fat accumulation in the liver. "NASH" also refers to subtypes 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 that 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 reversed-phase high pressure liquid chromatography, and "TFA" refers to trifluoroacetic acid. EXAMPLES

[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 for lysine at position 11. The Fmoc group is removed using 20% ​​piperidine in DMF before each coupling step (2 x 7 min). All amino acid couplings are carried out 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 with 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 at position 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 alpha-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 with a duration of 1 hour.

[0076] Once synthesis is complete, 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, washed twice with 2 mL each of neat TFA, and the combined filtrates are treated 4 times with 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 vacuum. The crude peptide is solubilized in 20% acetonitrile / 20% acetic acid / 60% water and purified by RP-HPLC on a Waters Xselect Peptide CSH C18 prep column 130A 5um 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 is assessed using analytical RP-HPLC with pooling criteria >95%. The main pool purity of compound I is found to be >99.0%. Subsequent lyophilization of the final main product pool results in the lyophilized peptide TFA salt. Molecular weight is determined by LC / MS. Average MW=3447.8Da. Expected mass: (M+3H): 1150.3, observed 1150.0.

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

[0078] Methods similar to those described above and known to those of skill 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 that are functionally coupled to Gαs proteins. Stimulation of these receptors increases the production of intracellular cAMP, which can be detected using standard in vitro techniques. Human AMY1 or CT receptors are stably expressed in human bladder cells (UM-UC-3) cells under the control of 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 that it lacks 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, Cat. No. 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, Cat. No. 354661). Peptides diluted in DMSO are added (200 nL / well) in a 1:3 dilution series using an ECHO acoustic liquid handler (Beckman). Cultured cells are detached with TrypLE Express (Gibco), resuspended in assay buffer, and 10 μL containing 1200 cells / well (hCT) or 1500 cells / well (hAMY1) is dispensed into each well. Plates are incubated at room temperature for 1 h.

[0081] The amount of intracellular cAMP is quantified using HTRF technology (Homogeneous Time-Resolved Fluorescence, Cisbio) 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 treated cells for 60 min at room temperature. The HTRF signal is immediately detected using an Envision plate reader (Perkin-Elmer) and the ratio of fluorescence at 665-620 nm is calculated. The raw data is converted to cAMP amounts (picomoles / well) using a cAMP standard curve generated for each experiment. 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 effect 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 with a 12-hour reverse light cycle from 10 p.m. to 10 a.m. On the morning of the study, baseline animal body and food weights are measured and different doses of Compound II (SEQ ID NO:2) alone in vehicle (20 mM Tris pH 8, 50 mg / mL D-mannitol, 0.02% PS80) or vehicle (20 mM Tris pH 8, 50 mg / mL D-mannitol, 0.02% PS80) are administered subcutaneously.

[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 daily average 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 efficacy of lowering food intake and body weight. [Table 2] [Table 3]

[0084] Example 4: In vivo efficacy in diet-induced obese (DIO) rats This study is conducted to investigate the effect of Compound II on diabetic and / or obese conditions in DIO rats. Male Long Evans (Envigo) rats, 24-30 weeks of age, with diet-induced obesity (DIO), maintained on a hypercaloric diet since arrival at Lilly (TD95217; Teklad, Madison, WI) are used for the following study. Animals are housed individually in a temperature-controlled (24°C) facility with a 12-h light / dark cycle (lights on at 2200) and free access to food (TD95217) and water.

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

[0086] Each group contains 5 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) are 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 are given on days 1, 4, 7, 10, and 13. Body weight and food intake are measured daily throughout the study. Absolute changes in body weight are calculated by subtracting the weight of the same animal before the first injection of the molecule. On day -1 (1 day before treatment) and day 15, body composition was assessed by quantitative nuclear magnetic resonance (QMNR, EchoMRI LLC, Houston, TX).

[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 is performed using repeated measures ANOVA followed by Dunnett's method for multiple comparisons. Significant differences are identified at p<0.05.

[0089] Compound II dose-dependently reduces body weight and food intake in male DIO rats. The reduction in body weight is believed to be mainly due to the reduction in fat mass. As shown in Tables 4 and 5, treatment with Compound II is observed to reduce serum glucose and insulin in a dose-dependent manner, in addition to substantial weight loss.

[0090] Body composition measurements are taken on days 0 and 14. Changes from the first measurement are presented as day 0 measurement to day 14 measurement. 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 peptides and acyl chains, is detected by a Q-Exactive Orbitrap™ mass spectrometer.

[0092] In the monkey pharmacokinetic study, male and female cynomolgus monkeys are administered a single subcutaneous dose of Compound II at 20 nmol / kg (0.084 mg / kg) in Tris-mannitol buffer (pH 8.0) at a volume of 0.2 mL / kg. Blood is 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 are administered a single subcutaneous dose of Compound II at 20 nmol / kg (0.084 mg / kg) in Tris-mannitol buffer (pH 8.0) at a volume of 0.2 mL / kg. Blood is 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 is conducted to investigate the effect 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) 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.

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

[0097] Each group contains 5 rats. Vehicle and compound II (1 nmol / kg) are 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 are given on days 1, 4, 7, 10, and 13. Body weight and food intake are measured daily throughout the study. Absolute changes in body weight are 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 with 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 effect 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 body weight so that each experimental group of animals has a similar weight, ranging from 549 to 683 grams.

[0102] Each group contains 5 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) are 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 are given on days 1, 4, 7, 10, and 13. Body weight and food intake are measured daily throughout the study. Absolute changes in body weight are 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 with 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 decrease 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 for 3 days at 37°C and 5% CO2 with 20ng / mL IL-4 and 40ng / mL GM-CSF in complete RPMI medium containing 5% serum replacement (Thermo Fisher Scientific, Cat. No. A2596101) (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 × 10 6Fresh medium containing 5 μg / mL LPS is added to the cells and replaced after 5 hours of incubation to transform the cells into mature dendritic cells. The next day, mature cells are lysed in 1 mL of RIPA buffer with protease inhibitors and DNAse. Lysates are stored at -80°C until sample analysis.

[0107] HLA-II molecules are isolated from thawed lysates using a biotinylated anti-pan HLA class II antibody (clone Tu39) using an automated liquid handling system. Bound receptor-peptide complexes are eluted with 5% acetic acid, 0.1% TFA. Eluted MHC-II peptides are passed through a pre-washed 10k MWCO filter to remove high molecular weight proteins. Separated MHC-II peptides are analyzed by nano LC / MS using a Thermo easy 1200 nLC-HPLC system equipped with a Thermo LUMOS mass spectrometer. The separation uses a 75 μm × 7 cm YMC-ODS C18 column with a flow rate of 250 nL / min and a 65 min gradient of 0.1% formic acid in water as solvent A and 80% acetonitrile with 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 a rapid scan in the ion trap with HCD and EThcD fragmentation.

[0108] Peptide identification is 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 / human databases containing test antibody sequences. KNIME workflow is used to process the sample identification files. Peptides identified from the test article are aligned against the parent sequence. A summary of all donors is generated that annotates the percentage of donors displaying non-germline residues, the number of different 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, and 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, Catalog No. 12055-083) containing 5% CTS™ Immune Cell SR (Gibco, Catalog 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% CO2 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 and mitotic index (CDI) was calculated using FlowJo® software (FlowJo, LLC, TreeStar). Briefly, CDI for each test candidate MAPPs-derived peptide cluster was calculated as the ratio of the number of proliferating CFSE from peptide-stimulated wells to the number of proliferating CFSE from peptide-stimulated wells. dim The percentage of CD4+ T cells was calculated by multiplying CFSE in unstimulated wells. dim The CD4+ T cell frequency was calculated by dividing by the percentage of CD4+ T cells. A CDI of >2.5 was considered to represent a positive response. The percentage of donor frequency 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 the 20th position 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 Registry Number: 204656-20-2 Compound IX (SEQ ID NO: 13) Dulaglutide is a human GLP-1 receptor agonist and comprises 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, 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 pharma- ceutically 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, comprising: (SEQ ID NO:2), or a pharma- ceutically acceptable salt thereof.

3. 13. 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 according to claim 1 or 2, or a pharma- ceutical acceptable salt thereof.

4. 13. A method of treating obesity in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound according to claim 1 or 2, or a pharma- ceutical acceptable salt thereof.

5. 13. A method for treating dyslipidemia in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound according to claim 1 or 2, or a pharma- ceutical acceptable salt thereof.

6. 13. A method of treating NASH in a patient in need of such treatment, comprising administering to the patient an effective amount of a compound of claim 1 or 2, or a pharma- ceutical acceptable salt thereof.

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

8. 13. A method of reducing weight in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 or 2, or a pharma- ceutically acceptable salt thereof.

9. 13. A method of lowering blood glucose in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to claim 1 or 2, or a pharma- ceutical acceptable salt thereof.

10. 13. A method of lowering triglycerides in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof.

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

12. 3. A pharmaceutical composition comprising a compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable excipient.

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

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

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

17. 13. A method of 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 of 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. 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 pharma- ceutically 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 pharma- ceutically acceptable salt thereof, for use in the treatment of type 2 diabetes.

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

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

27. 3. A compound according to claim 1 or 2, or a pharma- ceutically 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. 13. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for the treatment of type 2 diabetes.

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

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

32. 13. 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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