Co-agonist of GLP-1 receptor and amylin receptor

Abstract

Provided is a co-agonist of a GLP-1 receptor and an amylin receptor. The provided co-agonist can be used for preventing, alleviating and / or treating diseases related to metabolic, cardiovascular and / or cognitive dysfunction.

Description

Co-agonists of GLP-1 receptor and amylin receptor

[0001] CROSS-REFERENCE

[0002] This application claims the benefit of Chinese Application No. 202411380790.0, filed September 30, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention belongs to the field of biological medicine, and relates to co-agonists of GLP-1 receptor and amylin receptor. BACKGROUND

[0004] Glucagon-like peptide 1 (GLP-1) is a polypeptide hormone synthesized and secreted by endocrine L cells that plays a role in the regulation of blood glucose, mainly by stimulating insulin secretion and suppressing glucagon secretion, and can also inhibit beta cell apoptosis, promote its regeneration, slow gastric emptying, and suppress appetite by acting on the hypothalamus. Endogenous GLP-1 is rapidly degraded, mainly by dipeptidyl peptidase-4 (DPP-4), with a half-life of about 2 minutes. Several marketed products containing GLP-1 receptor agonists as active pharmaceutical ingredients have been approved for use in patients with type 2 diabetes. These include dulaglutide Exenatide Liraglutide Lixisenatide and semaglutide

[0005] Amylin is co-secreted with insulin from pancreatic beta cells, and it exerts its effects through amylin receptors (AMYR1, AMYR2, AMYR3) in several different organ systems, the main physiological functions being slowing gastric emptying and delaying glucose absorption, also reducing hepatic glucose production by suppressing glucagon, reducing appetite, and assisting insulin in regulating blood glucose levels. Other effects of amylin on the cardiovascular system and bone have also been reported. Clinical studies suggest that amylin receptor agonists can be useful in the treatment of overweight, obesity, type 1 diabetes, and / or type 2 diabetes. There is one product on the market which contains an amylin receptor agonist (pramlintide acetate) as an active pharmaceutical ingredient.

[0006] A fixed-dose combination of the amylin receptor agonist cagrilintide and the GLP-1 receptor agonist semaglutide is currently under investigation for the treatment of overweight and obesity (Lancet 2021; 397: 1736-48). The combination of semaglutide and cagrilintide induced greater weight loss in obese patients than the maximum approved dose of semaglutide monotherapy without causing a significant worsening of side effects. However, the formulation conditions of the two molecules are incompatible, and a double-chambered cartridge is required to achieve a one-needle administration, which is more expensive to manufacture. SUMMARY

[0007] The purpose of the present disclosure is to provide a polypeptide or a derivative thereof having a completely new polypeptide sequence, which has a dual agonistic activity for both GLP-1 receptor and amylin receptor, and has the characteristics of strong biological activity and high stability, and can provide enhanced weight loss, and thus can be used as a therapeutic agent for obesity and related diseases.

[0008] In a first aspect, the present disclosure provides a co-agonist of GLP-1 receptor and amylin receptor, comprising or consisting of a polypeptide R1 of Formula I:

[0009] Z1-Z2-Z3 (Formula I),

[0010] characterized in that,

[0011] Z1 is a GLP-1 receptor agonist peptide comprising the amino acid sequence set forth in SEQ ID NO: 17 or comprising at most 3 amino acid modifications relative to the amino acid sequence set forth in SEQ ID NO: 17;

[0012] Z2 is a peptide linker or is absent;

[0013] Z3 is an amylin receptor agonist peptide comprising the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 23, or comprising at most 4 amino acid modifications relative to the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 23;

[0014] The co-agonist of GLP-1 receptor and amylin receptor further comprises a fatty acid side chain attached to a lysine (Lys, K) residue in the polypeptide R1.

[0015] In some embodiments, the lysine (Lys, K) residue can be directly connected or indirectly connected (e.g., through a linker) to the fatty acid side chain.

[0016] In some embodiments, the co-agonist of GLP-1 receptor and amylin receptor described above, the carboxy terminus (C-terminus) of the polypeptide R1 has an NH2 modification.

[0017] In some embodiments, in the above-mentioned co-agonists of GLP-1 receptor and amylin receptor, the polypeptide R1 does not contain disulfide bond.

[0018] In some embodiments, the polypeptide R1 comprises 1-3 lysine (Lys, K) residues linked to fatty acid side chains; more preferably, comprises 1 lysine (Lys, K) residue linked to fatty acid side chains.

[0019] In some embodiments, in the above-mentioned co-agonists of GLP-1 receptor and amylin receptor, the lysine (Lys, K) residues linked to fatty acid side chains are located in the GLP-1 receptor agonist peptide Z1, preferably, the GLP-1 receptor agonist peptide Z1 comprises 0-3 lysine (Lys, K) residues, for example, comprises 0, 1, 2 or 3 lysine (Lys, K) residues.

[0020] In some embodiments, the lysine (Lys, K) residues linked to fatty acid side chains are located in the GLP-1 receptor agonist peptide Z1, preferably, the GLP-1 receptor agonist peptide Z1 comprises 0-3 lysine (Lys, K) residues, for example, comprises 0, 1, 2 or 3 lysine (Lys, K) residues.

[0021] In some embodiments, at least 1 lysine (Lys, K) residue in the GLP-1 receptor agonist peptide Z1 is located at position 17 or 24 of the GLP-1 receptor agonist peptide Z1.

[0022] In some embodiments, in the above-mentioned co-agonists of GLP-1 receptor and amylin receptor, the GLP-1 receptor agonist peptide Z1 is a polypeptide of formula II:

[0023] His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa17-Ala-Ala-Arg-Glu-Phe-Ile-Xaa24-Trp-Leu-Val-Arg-Gly-Arg-Xaa31 (formula II),

[0024] wherein,

[0025] Xaa17 is Gin (Q) or Lys (K);

[0026] Xaa24 is Lys (K) or Ala (A); and / or

[0027] Xaa31 is Gly (G) or absent.

[0028] and at least one Lys (K) residue is present at Xaa17 or Xaa24.

[0029] In some embodiments, the GLP-1 receptor agonist peptide Z1 consists of or comprises a polypeptide according to Formula II:

[0030] His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa17-Ala-Ala-Arg-Glu-Phe-Ile-Xaa24-Trp-Leu-Val-Arg-Gly-Arg-Xaa31 (Formula II),

[0031] wherein,

[0032] Xaa17 is Gin (Q) or Lys (K);

[0033] Xaa24 is Lys (K) or Ala (A); and / or

[0034] Xaa31 is Gly (G) or is absent;

[0035] and at least one Lys (K) residue is present at Xaa17 or Xaa24.

[0036] In some embodiments, the GLP-1 receptor agonist peptide Z1 is a polypeptide according to Formula II:

[0037] His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa17-Ala-Ala-Arg-Glu-Phe-Ile-Xaa24-Trp-Leu-Val-Arg-Gly-Arg-Xaa31 (Formula II),

[0038] wherein,

[0039] Xaa17 is Gin (Q) or Lys (K);

[0040] Xaa24 is Lys (K) or Ala (A); and / or

[0041] Xaa31 is Gly (G) or is absent;

[0042] and at least one Lys (K) residue is present at Xaa17 or Xaa24.

[0043] In some embodiments, in the polypeptide of Formula II, Xaa17 is a Lys (K) residue.

[0044] In some embodiments, Xaa17 is a Lys (K) residue.

[0045] In some embodiments, in the polypeptide of Formula II, Xaa24 is a Lys (K) residue.

[0046] In some embodiments, Xaa24 is a Lys (K) residue.

[0047] In some embodiments, in any of the co-agonists of GLP-1 receptor and amylin receptor described above, the GLP-1 receptor agonist peptide Z1 is a polypeptide of SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 36.

[0048] In some embodiments, the GLP-1 receptor agonist peptide Z1 consists of or comprises a polypeptide of SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 36.

[0049] In some embodiments, the GLP-1 receptor agonist peptide Z1 is a polypeptide of SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 36.

[0050] In some embodiments, in any of the co-agonists of GLP-1 receptor and amylin receptor described above, the peptide linker Z2 is a linking peptide containing 1-12 amino acids; preferably, a linking peptide rich in Gly (G) and / or Ser (S), such as a linking peptide of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40; or,

[0051] In some embodiments, the peptide linker Z2 is a linking peptide containing 1-12 amino acids; preferably, a linking peptide rich in Gly (G) and / or Ser (S), such as a linking peptide of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40; or, the peptide linker Z2 is a linking peptide of SEQ ID NO: 41 or SEQ ID NO: 42.

[0052] In some embodiments, the peptide linker Z2 is a linking peptide containing 1-12 amino acids; preferably, a linking peptide rich in Gly (G) and / or Ser (S), such as a linking peptide of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40; or, the peptide linker Z2 is a linking peptide of SEQ ID NO: 41 or SEQ ID NO: 42.

[0053] In some embodiments, in any of the above described co-agonists of the GLP-1 receptor and the amylin receptor, the peptide linker Z2 is absent.

[0054] In some embodiments, the peptide linker Z2 is absent.

[0055] In some embodiments, in any of the above described co-agonists of the GLP-1 receptor and the amylin receptor, the amylin receptor agonist peptide Z3 is a polypeptide according to Formula III:

[0056] Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu- Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (Formula III),

[0057] wherein,

[0058] Xaa3 is Gin (Q) or Glu (E);

[0059] Xaa8 is Val (V) or Ala (A);

[0060] Xaa9 is Leu (L) or Thr (T);

[0061] Xaa10 is Gly (G) or Gin (Q);

[0062] Xaa13 is Ser (S) or Ala (A);

[0063] Xaa14 is Asp (D), Ala (A) or Aib;

[0064] Xaa15 is Glu (E) or Phe (F);

[0065] Xaa17 is His (H) or Arg (R);

[0066] Xaa18 is Arg (R), Glu (E) or His (H);

[0067] Xaa19 is Leu (L) or Phe (F);

[0068] Xaa20 is Gin (Q), Ala (A) or Thr (T);

[0069] Xaa22 is Tyr (Y) or Arg (R);

[0070] Xaa23 is Pro (P) or Asp (D);

[0071] Xaa25 is Thr (T) or Asp (D);

[0072] Xaa26 is He (I) or is absent;

[0073] Xaa27 is Leu (L) or is absent;

[0074] Xaa28 is Pro (P) or is absent;

[0075] Xaa29 is Pro (P) or is absent;

[0076] Xaa30 is Thr (T) or is absent;

[0077] Xaa31 is Asp (D) or Asn (N);

[0078] Xaa34 is Ser (S) or Ala (A);

[0079] Xaa35 is Gly (G), Glu (E) or Asn (N); and / or

[0080] Xaa36 is Ser (S) or Thr (T).

[0081] In some embodiments, the amylin receptor agonist peptide Z3 consists of or comprises a polypeptide according to Formula III:

[0082] Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu- Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (Formula III),

[0083] wherein,

[0084] Xaa3 is Gin (Q) or Glu (E);

[0085] Xaa8 is Val (V) or Ala (A);

[0086] Xaa9 is Leu (L) or Thr (T);

[0087] Xaa10 is Gly (G) or Gin (Q);

[0088] Xaa13 is Ser (S) or Ala (A);

[0089] Xaa14 is Asp (D), Ala (A), or Aib;

[0090] Xaa15 is Glu (E) or Phe (F);

[0091] Xaa17 is His (H) or Arg (R);

[0092] Xaa18 is Arg (R), Glu (E), or His (H);

[0093] Xaa19 is Leu (L) or Phe (F);

[0094] Xaa20 is Gin (Q), Ala (A), or Thr (T);

[0095] Xaa22 is Tyr (Y) or Arg (R);

[0096] Xaa23 is Pro (P) or Asp (D);

[0097] Xaa25 is Thr (T) or Asp (D);

[0098] Xaa26 is He (I) or is absent;

[0099] Xaa27 is Leu (L) or is absent;

[0100] Xaa28 is Pro (P) or is absent;

[0101] Xaa29 is Pro (P) or is absent;

[0102] Xaa30 is Thr (T) or is absent;

[0103] Xaa31 is Asp (D) or Asn (N);

[0104] Xaa34 is Ser (S) or Ala (A);

[0105] Xaa35 is Gly (G), Glu (E), or Asn (N); and / or

[0106] Xaa36 is Ser (S) or Thr (T).

[0107] In some embodiments, the amylin receptor agonist peptide Z3 is a polypeptide of Formula III:

[0108] Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu- Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (Formula III),

[0109] wherein,

[0110] Xaa3 is Gin (Q) or Glu (E);

[0111] Xaa8 is Val (V) or Ala (A);

[0112] Xaa9 is Leu (L) or Thr (T);

[0113] Xaa10 is Gly (G) or Gin (Q);

[0114] Xaa13 is Ser (S) or Ala (A);

[0115] Xaa14 is Asp (D), Ala (A) or Aib;

[0116] Xaa15 is Glu (E) or Phe (F);

[0117] Xaa17 is His (H) or Arg (R);

[0118] Xaa18 is Arg (R), Glu (E) or His (H);

[0119] Xaa19 is Leu (L) or Phe (F);

[0120] Xaa20 is Gin (Q), Ala (A) or Thr (T);

[0121] Xaa22 is Tyr (Y) or Arg (R);

[0122] Xaa23 is Pro (P) or Asp (D);

[0123] Xaa25 is Thr (T) or Asp (D);

[0124] Xaa26 is He (I) or absent;

[0125] Xaa27 is Leu (L) or absent;

[0126] Xaa28 is Pro (P) or absent;

[0127] Xaa29 is Pro (P) or absent;

[0128] Xaa30 is Thr (T) or absent;

[0129] Xaa31 is Asp (D) or Asn (N);

[0130] Xaa34 is Ser (S) or Ala (A);

[0131] Xaa35 is Gly (G), Glu (E) or Asn (N); and / or

[0132] Xaa36 is Ser (S) or Thr (T).

[0133] In some embodiments, in any of the above described co-agonists of the GLP-1 receptor and the amylin receptor, the amylin receptor agonist peptide Z3 is a polypeptide set forth in SEQ ID NO: 21, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 23.

[0134] In some embodiments, the amylin receptor agonist peptide Z3 consists of or comprises a polypeptide set forth in SEQ ID NO: 21, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 23.

[0135] In some embodiments, the amylin receptor agonist peptide Z3 is a polypeptide set forth in SEQ ID NO: 21, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 23.

[0136] In some embodiments, in any of the above described co-agonists of the GLP-1 receptor and the amylin receptor, the lysine (Lys, K) residue on the polypeptide R1 is linked to the fatty acid side chain via an amide bond; preferably via the epsilon amino group on the lysine (Lys, K) residue to the fatty acid side chain.

[0137] In some embodiments, in any of the above described co-agonists of the GLP-1 receptor and the amylin receptor, the lysine (Lys, K) residue on the polypeptide R1 is linked to the fatty acid side chain via an amide bond; preferably via the epsilon amino group on the lysine (Lys, K) residue to the fatty acid side chain.

[0138] In some embodiments, in any of the above described co-agonists of the GLP-1 receptor and the amylin receptor, the fatty acid side chain is selected from

[0139] Cx diacid:

HOOC(CH2) x-2 CO-

[0140] C X Phosphoric acid:

PO(OH)2(CH2) x-1 CO-

[0141] or

[0142] C X Tetrazole:

CN4H(CH2) x-1 CO-

[0143] wherein x is an arbitrary integer selected from 10-30;

[0144] Preferably, the fatty acid side chain is selected from:

[0145] HOOC(CH2) 14 CO-, HOOC(CH2) 15 CO-, HOOC(CH2) 16 CO-, HOOC(CH2) 17 CO-, HOOC(CH2) 18 CO-, HOOC(CH2) 19 CO-, HOOC(CH2) 20 CO-, HOOC(CH2) 21 CO-, HOOC(CH2) 22 CO-, PO(OH)2(CH2) 15 CO-, PO(OH)2(CH2) 16 CO-, PO(OH)2(CH2) 17CO-, PO(OH)2(CH2) 18 CO-, PO(OH)2(CH2) 19 CO-, PO(OH)2(CH2) 20 CO-, PO(OH)2(CH2) 21 CO-, PO(OH)2(CH2) 22 CO-, PO(OH)2(CH2) 23 CO-, CN4H(CH2) 14 CO-, CN4H(CH2) 15 CO-, CN4H(CH2) 16 CO-, CN4H(CH2) 17 CO-, CN4H(CH2) 18 CO-, CN4H(CH2) 19 CO-, CN4H(CH2) 20 CO-, CN4H(CH2) 21 CO-, CN4H(CH2) 22 one or more of CO-.

[0146] In some embodiments, the fatty acid side chain is selected from

[0147] Cx diacid:

HOOC(CH2) x -2CO-

[0148] C X phosphonic acid:

PO(OH)2(CH2) x -1CO-

[0149] or

[0150] C X tetrazole:

CN4H(CH2) x-1 CO-

[0151] one or more of wherein x is an arbitrary integer selected from 10-30;

[0152] Preferably, the fatty acid side chain is selected from:

[0153] HOOC(CH2) 14 CO-, HOOC(CH2) 15 CO-, HOOC(CH2) 16 CO-, HOOC(CH2) 17 CO-, HOOC(CH2) 18CO-, HOOC(CH2) 19 CO-, HOOC(CH2) 20 CO-, HOOC(CH2) 21 CO-, HOOC(CH2) 22 CO-, PO(OH)2(CH2) 15 CO-, PO(OH)2(CH2) 16 CO-, PO(OH)2(CH2) 18 CO-, PO(OH)2(CH2) 19 CO-, PO(OH)2(CH2) 20 CO-, PO(OH)2(CH2) 21 CO-, PO(OH)2(CH2) 22 CO-, PO(OH)2(CH2) 23 CO-, CN4H(CH2) 14 CO-, CN4H(CH2) 15 CO-, CN4H(CH2) 16 CO-, CN4H(CH2) 17 CO-, CN4H(CH2) 18 CO-, CN4H(CH2) 19 CO-, CN4H(CH2) 20 CO-, CN4H(CH2) 21 CO-, CN4H(CH2) 22 one or more of CO-.

[0154] In some embodiments, in any of the above described co-agonists of GLP-1 receptor and amylin receptor, the fatty acid side chain is connected to the amino acid Lys residue through a linker.

[0155] In some embodiments, the fatty acid side chain is connected to the amino acid Lys residue through a linker.

[0156] In some embodiments, in any of the above described co-agonists of GLP-1 receptor and amylin receptor, the linker is selected from

[0157] n AEEA + m y GIu:

[0158] n * e Lys + m y GIu:

[0159] or

[0160] n AEEA + m b Asp:

[0161] one or more of: wherein m and n are each an integer selected from 0-4;

[0162] Preferably, the linker is selected from:

[0163] 2*AEEA + γGlu:

[0164] γGlu:

[0165] 2AEEA + βAsp:

[0166] 2*εLys + γGlu:

[0167] In some embodiments, the linker is selected from

[0168] nAEEA + mγGlu:

[0169] n*εLys + mγGlu:

[0170] or

[0171] nAEEA + mβAsp:

[0172] one or more of: wherein m and n are each an integer selected from 0-4;

[0173] Preferably, the linker is selected from:

[0174] 2*AEEA + γGlu:

[0175] γGlu:

[0176] 2AEEA + βAsp:

[0177] 2*εLys + γGlu:

[0178] In some embodiments, any of the above described co-agonists of GLP-1 receptor and amylin receptor is any of the polypeptide derivatives shown in Table 1 herein.

[0179] In some embodiments, the co-agonist of GLP-1 receptor and amylin receptor is any of the polypeptide derivatives shown in Table 1 herein.

[0180] In a second aspect, the present disclosure provides a polypeptide having an amino acid sequence according to Formula I above. Preferably, the present disclosure provides a polypeptide of Formula I: Z1-Z2-Z3 (Formula I), characterized in that Z1 is a GLP-1 receptor agonist peptide comprising the amino acid sequence set forth in SEQ ID NO: 17 or up to 3 amino acid modifications relative to SEQ ID NO: 17; Z2 is a peptide linker or is absent; and Z3 is an amylin receptor agonist peptide comprising the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 23, or up to 4 amino acid modifications relative to SEQ ID NO: 21 or SEQ ID NO: 23.

[0181] In a third aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned polypeptide.

[0182] In a fourth aspect, the present disclosure provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0183] In a fifth aspect, the present disclosure provides a recombinant cell comprising the above-mentioned nucleic acid molecule and / or the above-mentioned recombinant vector and capable of expressing and optionally secreting the above-mentioned polypeptide.

[0184] In a sixth aspect, the present disclosure provides a method of manufacturing the above-mentioned co-agonist of GLP-1 receptor and amylin receptor or the above-mentioned polypeptide;

[0185] Preferably, the method of manufacturing comprises the step of manufacturing the co-agonist of GLP-1 receptor and amylin receptor or the polypeptide using chemical and / or biological methods;

[0186] Preferably, the chemical methods comprise liquid phase and / or solid phase polypeptide synthesis.

[0187] In a seventh aspect, the present disclosure provides a pharmaceutical composition comprising the above-mentioned co-agonist of GLP-1 receptor and amylin receptor or the above-mentioned polypeptide, and a pharmaceutically acceptable excipient.

[0188] The pharmaceutical composition of the present disclosure can be administered by any suitable route known in the art, including but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intra-arterial, lymphatic and / or cerebrospinal;

[0189] Preferably, the pharmaceutical composition is in the form of a solid, liquid, semi-solid.

[0190] In some embodiments, the pharmaceutical composition of the present disclosure is formulated as a liquid suitable for administration by injection or infusion.

[0191] In some embodiments, the pharmaceutical composition of the present disclosure further comprises one or more additional pharmaceutically active ingredients that can have beneficial effects on preventing, alleviating, and / or treating metabolic, cardiovascular, and / or cognitive dysfunction related diseases;

[0192] The active ingredients are, for example, pharmaceutically active ingredients for the treatment of obesity, such as GIP, Glucagon, PYY, GDF15, Cannabinoid Receptor-1 blockers, mitochondrial uncouplers, THR-beta agonists, etc.

[0193] In some embodiments, the pharmaceutical composition of the present disclosure can be used in combination with one or more additional pharmaceutical compositions. In some embodiments, the different pharmaceutical compositions can be administered simultaneously, sequentially, or separately to a patient in need thereof. In some embodiments, the different pharmaceutical compositions are administered to a patient in need thereof in a time sequence, for example, within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, 2 months, 3 months, or more, each pharmaceutical composition is administered one, two, three, or more times a day, simultaneously, sequentially, or separately;

[0194] The additional pharmaceutical composition(s) are, for example, weight regulating, anti-obesity, lipid metabolism regulating, blood glucose regulating, hypertension treating, cardiovascular regulating, brain system disease, mental system disease or nervous system regulating pharmaceuticals, etc. The corresponding medical uses are provided for the therapeutic methods disclosed herein. Thus, when medical is referred to herein, this can refer to any of the GLP-1 receptor and amylin receptor co-agonists or polypeptides described above for use in the therapeutic methods disclosed herein. Thus, provided herein are any of the polypeptides or pharmaceutical compositions disclosed herein for use in a method of preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease; preferably, the metabolic related disease comprises diabetes, obesity related disorder, appetite abnormality disease; the cardiovascular related disease comprises myocardial infarction, stroke, angina pectoris, heart failure; the cognitive dysfunction related disease comprises dementia, Alzheimer’s disease. Also provided herein are any of the co-agonists, polypeptides or pharmaceutical compositions disclosed herein for use in a method of preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease, wherein the method comprises the step of administering to a patient in need thereof a therapeutically effective amount of the co-agonist, polypeptide or pharmaceutical composition; preferably, the metabolic related disease comprises diabetes, obesity related disorder, appetite abnormality disease; the cardiovascular related disease comprises myocardial infarction, stroke, angina pectoris, heart failure; the cognitive dysfunction related disease comprises dementia, Alzheimer’s disease. In an eighth aspect, the disclosure provides any of the GLP-1 receptor and amylin receptor co-agonists or polypeptides described above for use in medical. As medical is also provided, the disclosure also provides any of the GLP-1 receptor and amylin receptor co-agonists or polypeptides described above for use in the therapeutic methods disclosed herein.

[0195] In some embodiments, the medical comprises one or more of the following:

[0196] (1) preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease;

[0197] (2) reducing food intake, reducing body weight, suppressing appetite, inducing satiety, reducing gastric motility and / or delaying gastric emptying;

[0198] (3) preventing weight gain, in particular weight maintenance after weight loss;

[0199] (4) delaying or reducing the development of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization for unstable angina pectoris, and hospitalization for heart failure;

[0200] (5) delaying or improving cognitive function decline or improving cognitive function.

[0201] Since medical treatment is also provided herein, the present disclosure also provides any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor or polypeptides for use in a method of treatment comprising one or more of the following:

[0202] (1) preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease;

[0203] (2) reducing food intake, reducing body weight, suppressing appetite, inducing satiety, reducing gastric motility and / or delaying gastric emptying;

[0204] (3) preventing weight gain, especially weight maintenance after weight loss;

[0205] (4) delaying or reducing the development of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization for unstable angina, and hospitalization for heart failure;

[0206] (5) delaying or improving cognitive decline or enhancing cognitive function.

[0207] In a ninth aspect, the present disclosure provides use of any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides or pharmaceutical compositions in the manufacture of a medicament for preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease. Since medical treatment is also provided herein, the present disclosure also provides any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides or pharmaceutical compositions for use in a method of preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease.

[0208] In a tenth aspect, the present disclosure provides a method of preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease, comprising the step of administering to a patient in need thereof a therapeutically effective amount of any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides or pharmaceutical compositions. Since medical treatment is also provided herein, the present disclosure also provides any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides or pharmaceutical compositions for use in a method of preventing, alleviating and / or treating a metabolic, cardiovascular and / or cognitive dysfunction related disease, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides or pharmaceutical compositions.

[0209] In the present disclosure, the metabolic related disease includes diabetes, obesity related disorders, appetite abnormality diseases; the cardiovascular related disease includes myocardial infarction, stroke, angina pectoris, heart failure; the cognitive dysfunction related disease includes dementia, Alzheimer's disease;

[0210] Preferably, the diabetes is all forms of diabetes, such as hyperglycemia, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, maturity onset diabetes of the young (MODY), non-insulin dependent diabetes mellitus, and / or gestational diabetes (for reducing HbAlc);

[0211] Preferably, the obesity-related disease is obesity, overweight, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH).

[0212] Preferably, the appetite disorder disease includes binge eating, eating impulsivity, and bulimia.

[0213] In some embodiments, in any of the methods described above, the GLP-1 receptor and amylin receptor co-agonist, polypeptide, or pharmaceutical composition described above is administered in a cycle of administration of 1 or more times per day, per week, per two weeks, per three weeks, per 1 month, per 2 months, per 3 months, per 4 months, per 5 months, per 6 months, per 7 months, per 8 months, per 9 months, per 10 months, per 11 months, per 12 months, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times per day, per week, per two weeks, per three weeks, per 1 month, per 2 months, per 3 months, per 4 months, per 5 months, per 6 months, per 7 months, per 8 months, per 9 months, per 10 months, per 11 months, per 12 months.

[0214] In some embodiments, the administration cycle of any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides, or pharmaceutical compositions is 1 or more times per day, per week, per two weeks, per three weeks, per 1 month, per 2 months, per 3 months, per 4 months, per 5 months, per 6 months, per 7 months, per 8 months, per 9 months, per 10 months, per 11 months, per 12 months, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times per day, per week, per two weeks, per three weeks, per 1 month, per 2 months, per 3 months, per 4 months, per 5 months, per 6 months, per 7 months, per 8 months, per 9 months, per 10 months, per 11 months, per 12 months.

[0215] In some embodiments, the total number of administrations in any of the aforementioned methods can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0216] The present disclosure also provides a kit comprising any of the aforementioned co-agonists of GLP-1 receptor and amylin receptor, polypeptides, or pharmaceutical compositions and optionally instructions for use. BRIEF DESCRIPTION OF DRAWINGS

[0217] Figure 1 shows the weight loss effect of a single dose (5 nmol / kg) of a polypeptide derivative in SD rats.

[0218] Figure 2 shows the weight loss effect of a single dose (5 nmol / kg) of a polypeptide derivative in SD rats.

[0219] Figure 3 shows the effect of different fatty acid side chains and connecting peptides on efficacy.

[0220] DETAILED DESCRIPTION

[0221] DEFINITIONS

[0222] The terms used herein, unless otherwise indicated, have the following definitions.

[0223] In the present text, the conventional one-letter and three-letter codes for natural amino acids and the generally accepted three-letter code for other amino acids, such as alpha- aminoisobutyric acid (Aib), are used. All amino acid residues in the peptides of the present disclosure are in the L-configuration, unless otherwise specified.

[0224] In the present disclosure, Aib denotes alpha-aminoisobutyric acid, which has the following structural formula:

[0225] The sequences disclosed herein are sequences incorporating an "NH2" moiety at the carboxy terminus (C-terminus) of the sequence, the "NH2" moiety representing an amide group (-CONH2) at the C-terminus.

[0226] The terms "polypeptide" or "peptide" or "protein" can be used interchangeably. A "polypeptide" or "peptide" or "protein" is any chain comprising two or more amino acids, whether post-translationally modified (e.g., glycosylated or phosphorylated) or not, including naturally occurring or non-naturally occurring (e.g., artificially synthesized) amino acids or amino acid analogs, wherein the amino acids in any chain are covalently linked by peptide bonds.

[0227] The term "amino acid modification" as used herein refers to an alteration of an amino acid or amino acid residue at a particular position, including a substitution, modification, deletion of the amino acid or amino acid residue, or an insertion of an amino acid / amino acid residue at that site.

[0228] The terms "comprising", "containing", "having" or "including" are to be construed open- ended, permitting the inclusion of additional components, features, structures, steps, etc.

[0229] The term "agonist" refers to a substance (ligand) that activates the receptor.

[0230] The terms "compound", "co-agonist compound", "co-agonist", "derivative" or "polypeptide derivative" can be used interchangeably herein.

[0231] The terms "patient", "subject" and "individual" can be used interchangeably and include humans and non-human animals, including mammals, such as monkeys, rats, mice, cows, pigs, goats, sheep, dogs, cats.

[0232] "Administering" and "treatment" when used in reference to an animal, human, subject, cell, tissue, organ or biological fluid means contacting the exogenous agent, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid. "Administering" and "treatment" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods and experimental methods. Treating a cell includes contacting the agent with the cell and contacting the agent with a fluid in which the cell is in contact. "Administering" and "treatment" also mean, for example, in vitro and ex vivo treatment of cells by agents, diagnostic agents, binding compositions or by other cells.

[0233] "Preventing" or "treatment" as used herein includes delaying the development of symptoms associated with a disease and / or reducing the severity of such symptoms where the disease is to develop or is expected to develop. The term also includes slowing of existing symptoms, preventing additional symptoms and slowing or preventing the underlying cause of the symptoms. Thus, the term indicates that a beneficial result has been imparted to a vertebrate subject, such as a human, who has a disease.

[0234] The term "therapeutically effective amount" or "effective amount" as used herein means that amount of a co-agonist of GLP-1 receptor and amylin receptor or the polypeptide that, when administered alone or in combination with another therapeutic agent to a cell, tissue or subject, is effective to prevent or slow the disease or condition to be treated. A therapeutically effective dose further refers to that amount of the co-agonist of GLP-1 receptor and amylin receptor or the polypeptide sufficient to result in slowing of symptoms, e.g., treatment, healing, prevention or amelioration of an associated medical condition, or an increase in rate of treatment, healing, prevention or amelioration of the medical condition. An effective amount for a particular subject can vary according to factors such as the disease being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects. A therapeutically effective amount, when administered to an individual, refers to the individual component when administered alone. When administered in combination, a therapeutically effective amount refers to the combined amounts of the active ingredients that result in the therapeutic effect, regardless of whether they are administered together, sequentially or simultaneously. A therapeutically effective amount will reduce symptoms by typically at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

[0235] Co-agonist

[0236] A "co-agonist" refers to a compound having the activity of activating at least two signaling pathways, e.g., a compound comprising two different ligands, each of which binds to a given biological receptor to produce the biological response characteristic of the natural ligand. The co-agonists of the present disclosure are referred to herein as "co-agonists of GLP-1 receptor and amylin receptor."

[0237] Co-agonists of GLP-1 receptor and amylin receptor

[0238] The compounds of the present disclosure are co-agonists of GLP-1 receptor and amylin receptor, having agonist activity at both GLP-1 receptor and amylin receptor. Co-agonists of GLP-1 receptor and amylin receptor comprise a GLP-1 receptor agonist peptide, a peptide linker (or none), and an amylin receptor agonist peptide. The GLP-1 receptor agonist peptide component binds to and activates the GLP-1 receptor, and the amylin receptor agonist peptide component binds to and activates at least the human amylin 3 receptor (AMYR3).

[0239] The GLP-1 receptor agonist peptide is capable of binding to the GLP-1 R and activating signaling through the receptor, for example by producing cAMP or inducing Ca 2+ release. The activity of the GLP-1 receptor agonist peptide is assessed herein by measuring the signal produced by luciferase driven by a cAMP response element.

[0240] Similarly, the amylin receptor agonist peptide is capable of binding to the amylin receptor and activating signaling through the receptor, for example by producing cAMP or inducing Ca 2+ release. The activity of the amylin receptor agonist peptide is assessed herein by measuring the signal produced by luciferase driven by a cAMP response element.

[0241] The present disclosure achieves co-agonist compounds having good activity and stability at both GLP-1 receptor and amylin receptor through polypeptide design, which show significantly better drug effects than single agonists, showing good GLP-1 R / AMYR3 activation synergy.

[0242] The co-agonists of GLP-1 receptor and amylin receptor of the present disclosure comprise or consist of a polypeptide R1 of Formula I:

[0243] Z1-Z2-Z3 (Formula I),

[0244] Z1 is a GLP-1 receptor agonist peptide of SEQ ID NO: 17 or comprising up to 3 amino acid modifications relative to SEQ ID NO: 17;

[0245] Z2 is a peptide linker or is absent;

[0246] Z3 is a amylin receptor agonist peptide of SEQ ID NO: 21 or SEQ ID NO: 23, or comprising up to 4 amino acid modifications relative to SEQ ID NO: 21 or SEQ ID NO: 23;

[0247] The co-agonist further comprises a fatty acid side chain attached to a lysine (Lys, K) residue in the polypeptide R1;

[0248] Preferably, the polypeptide R1 comprises 1-3 lysine (Lys, K) residues to which a fatty acid side chain is attached; more preferably, 1 lysine (Lys, K) residue to which a fatty acid side chain is attached;

[0249] Optionally, the polypeptide R1 has an NH2 modification at the carboxy terminus and / or is free of disulfide bonds.

[0250] The side chain attached to the polypeptide R1 in the present disclosure comprises a fatty acid side chain selected from:

[0251] Cx diacid:

HOOC(CH2) x-2 CO-

[0252] C X Phosphoric acid:

PO(OH)2(CH2) x-1 CO-

[0253] or

[0254] C X Tetrazole:

CN4H(CH2) x-1 CO-

[0255] wherein x is an arbitrary integer selected from 10-30;

[0256] Preferably, the fatty acid side chain is selected from:

[0257] C18 diacid:

HOOC(CH2) 16 CO-

[0258] C20 diacid:

HOOC(CH2) 18 CO-

[0259] C22 diacid:

HOOC(CH2) 20 CO-

[0260] C24 diacid:

HOOC(CH2) 22 CO-

[0261] C17 phosphoric acid:

PO(OH)2CH 16 CO-

[0262] C19 phosphoric acid: [PO(OH)2CH 18 CO-],

[0263] C21 phosphoric acid: [PO(OH)2CH 20 CO-],

[0264] C17 tetrazole: [CN4H(CH2) 16 CO-],

[0265] C19 tetrazole: [CN4H(CH2) 18 CO-],

[0266] C21 tetrazole: [CN4H(CH2) 20 CO-] one or more of.

[0267] The connection of the polypeptide R1 to the fatty acid side chain in the present disclosure can be a direct connection or an indirect connection. Among them, the indirect connection is for example through a linker.

[0268] The linker of the indirect connection is selected from

[0269] selected from

[0270] n AEEA + m yGlu:

[0271] n * eLys + m yGlu:

[0272] or

[0273] n AEEA + m bAsp:

[0274] one or more of, wherein m and n are respectively any integer selected from 0-4;

[0275] Preferably, the linker is selected from:

[0276] 2 * AEEA + yGlu:

[0277] yGlu:

[0278] 2 AEEA + bAsp:

[0279] 2 * eLys + yGlu:

[0280] The polypeptide R1-linked side chains according to the present disclosure are preferably selected from the group consisting of:

[0281] 2 * AEEA + γGlu + C18 diacid:

[0282] 2 * AEEA + γGlu + C20 diacid:

[0283] 2 * AEEA + γGlu + C22 diacid

[0284] 2 * AEEA + γGlu + C17 phosphoric acid:

[0285] 2 * AEEA + γGlu + C17 tetrazole:

[0286] 2 * AEEA + γGlu + C19 phosphoric acid:

[0287] 2 * AEEA + γGlu + C21 phosphoric acid:

[0288] 2 * εLys + γGlu + C18 diacid:

[0289] 2 * εLys + γGlu + C20 diacid:

[0290] 2 * εLys + γGlu + C22 diacid:

[0291] 2 * εLys + γGlu + C17 phosphoric acid:

[0292] 2 * εLys + γGlu + C19 phosphoric acid:

[0293] 2 * εLys + γGlu + C21 phosphoric acid:

[0294] γGlu + C20 diacid:

[0295] 2 * AEEA + βAsp + C18 diacid:

[0296] The co-agonists of GLP-1 receptor and amylin receptor designed according to the present disclosure are shown in Table 1.

[0297] It is understood that the compounds of the present disclosure can also be provided in the form of salts. Pharmaceutically acceptable salts include salts in anionic form and salts in cationic form. Some examples of salts in anionic form include hydrochloride, citrate, chloride, and acetate. Preferably, the salt is acetate. Some examples of salts in cationic form include salts in which the cation is selected from alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium), and the like.

[0298] Additionally, the compounds of the present disclosure can also form coordination complexes with metal ions (e.g., Mn2+and Zn2+) and thus exist in the form of complexes. Since the compounds of the present disclosure have a hydroxyl group or carboxylic acid, the derivative can also react with a suitable carboxylic acid or alcohol to form an ester and thus exist in the form of an ester. The compounds of the present disclosure can also exist in the form of a prodrug, which can be converted to one of the parent compounds in vivo or in vitro. Generally, at least one biological activity of the compound will be reduced in the prodrug form and can be activated by conversion of the prodrug to release the compound or its metabolite. Some examples of prodrugs include the use of protecting groups, which can be removed in situ to release the active compound or to inhibit the clearance of the drug in vivo.

[0299] GLP-1

[0300] The term "GLP-1" refers to human glucagon-like peptide-1.

[0301] GLP-1 receptor agonist peptide

[0302] The GLP-1 receptor agonist peptide of the present disclosure is a polypeptide of SEQ ID NO: 17 or comprising up to 3 amino acid modifications relative to SEQ ID NO: 17, which is a polypeptide of Formula II:

[0303] His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa17-Ala-Ala-Arg-Glu-Phe-Ile-Xaa24-Trp-Leu-Val-Arg-Gly-Arg-Xaa31 (Formula II),

[0304] wherein,

[0305] Xaa17 is Gin (Q) or Lys (K);

[0306] Xaa24 is Lys (K) or Ala (A); and / or

[0307] Xaa31 is Gly (G) or is absent;

[0308] and at least one Lys (K) residue is present at Xaa17 and Xaa24.

[0309] Preferably, in the polypeptide of Formula II, Xaa17 is a Lys (K) residue.

[0310] Preferably, in the polypeptide of Formula II, Xaa24 is a Lys (K) residue.

[0311] More preferably, the GLP-1 receptor agonist peptide Z1 is a polypeptide of SEQ ID NO: 17, SEQ ID NO: 35, or SEQ ID NO: 36.

[0312] Amylin

[0313] The term "amylin" refers to a polypeptide having the same amino acid sequence as an endogenous amylin, such as human amylin.

[0314] Amylin receptor

[0315] The amylin receptor is a heterodimeric complex composed of a calcitonin receptor (CTR) and a receptor activity-modifying protein (RAMP). There are three subtypes of RAMP: RAMP 1, RAMP 2, RAMP 3, and depending on the RAMP species bound to the CTR, three possible amylin receptor subtypes are generated: amylin receptors 1-3 (AMYR1-3). While, as with natural ligands, amylin receptor agonists / ligands can have some cross-reactivity with amylin receptors, unless otherwise specified, "amylin receptor" refers at least to amylin receptor 3 (AMYR3).

[0316] Amylin receptor agonist peptide

[0317] An amylin receptor agonist peptide can activate or agonize a calcitonin receptor (CTR) and / or an amylin receptor (AMYR). Examples of endogenous amylin receptor agonists are human amylin and human calcitonin. Examples of exogenous amylin receptor agonists are pramlintide and carbaglu tide.

[0318] The amylin receptor agonist peptide Z3 of the present disclosure is a polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23, or a polypeptide comprising up to 4 amino acid modifications relative to SEQ ID NO: 21 or SEQ ID NO: 23, which is a polypeptide of Formula III:

[0319] Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu- Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (Formula III),

[0320] wherein,

[0321] Xaa3 is Gin (Q) or Glu (E);

[0322] Xaa8 is Val (V) or Ala (A);

[0323] Xaa9 is Leu (L) or Thr (T);

[0324] Xaa10 is Gly (G) or Gin (Q);

[0325] Xaa13 is Ser (S) or Ala (A);

[0326] Xaa14 is Asp (D), Ala (A) or Aib;

[0327] Xaa15 is Glu (E) or Phe (F);

[0328] Xaa17 is His (H) or Arg (R);

[0329] Xaa18 is Arg (R), Glu (E) or His (H);

[0330] Xaa19 is Leu (L) or Phe (F);

[0331] Xaa20 is Gin (Q), Ala (A) or Thr (T);

[0332] Xaa22 is Tyr (Y) or Arg (R);

[0333] Xaa23 is Pro (P) or Asp (D);

[0334] Xaa25 is Thr (T) or Asp (D);

[0335] Xaa26 is He (I) or absent;

[0336] Xaa27 is Leu (L) or absent;

[0337] Xaa28 is Pro (P) or is absent;

[0338] Xaa29 is Pro (P) or is absent;

[0339] Xaa30 is Thr (T) or is absent;

[0340] Xaa31 is Asp (D) or Asn (N);

[0341] Xaa34 is Ser (S) or Ala (A);

[0342] Xaa35 is Gly (G), Glu (E) or Asn (N); and / or

[0343] Xaa36 is Ser (S) or Thr (T).

[0344] Preferably, the amylin receptor agonist peptide Z3 is a polypeptide as set forth in SEQ ID NO: 21, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 23.

[0345] Peptide linker

[0346] The peptide linker Z2 of the present disclosure is a Gly (G) and / or Ser (S) rich connecting peptide, such as a connecting peptide as set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40; or,

[0347] The peptide linker Z2 is a connecting peptide as set forth in SEQ ID NO: 41 or SEQ ID NO: 42.

[0348] Synthesis of co-agonist compounds or polypeptides

[0349] The co-agonist compounds or polypeptides of the present disclosure can be prepared using chemical and / or biological methods. Preparation using chemical methods is preferred, such as synthesis of the co-agonist compounds or polypeptides of the present disclosure using liquid or solid phase polypeptide synthesis means. The biological methods include molecular biological methods and cell biological methods.

[0350] The method of preparing the co-agonist compounds or polypeptides of the present disclosure can comprise the following steps:

[0351] synthesis of the co-agonist compounds or polypeptides of the present disclosure by liquid or solid phase polypeptide synthesis means according to the polypeptide sequence step by step or by fragment assembly; or

[0352] In some embodiments, the co-agonist compound of the present disclosure is prepared by solid phase polypeptide synthesis on a suitable resin. The solid phase polypeptide synthesis steps are well known in the art, for example, the solid phase synthesis is initiated by attaching an N-terminal protected amino acid and its carboxyl terminal to an inert solid support carrying a cleavable linker. The solid support can be any polymer that allows the initial amino acid coupling, for example, Rink amide AM resin. In some embodiments, starting with Rink amide AM as the starting material, the amino acids with Fmoc-protecting groups are sequentially linked according to the method of solid phase synthesis, and the Fmoc-protecting groups are sequentially removed, and the peptide is cleaved from the resin, and the side chain protecting groups are simultaneously removed to obtain the fatty acid modified polypeptide resin, the peptide is cleaved from the resin, and separated and purified by chromatography column, and then freeze-dried to obtain the polypeptide derivative in powder form.

[0353] In some embodiments, the co-agonist compound of the present disclosure is prepared by solid phase polypeptide synthesis on a suitable resin. The solid phase polypeptide synthesis steps are well known in the art, for example, the solid phase synthesis is initiated by attaching an N-terminal protected amino acid and its carboxyl terminal to an inert solid support carrying a cleavable linker. The solid support can be any polymer that allows the initial amino acid coupling, for example, Rink amide AM resin. In some embodiments, starting with Rink amide AM as the starting material, the amino acids with Fmoc-protecting groups are sequentially linked according to the method of solid phase synthesis, and the Fmoc-protecting groups are sequentially removed, and the peptide is cleaved from the resin, and the side chain protecting groups are simultaneously removed to obtain the fatty acid modified polypeptide resin, the peptide is cleaved from the resin, and separated and purified by chromatography column, and then freeze-dried to obtain the polypeptide derivative in powder form.

[0354] In some embodiments, the co-agonist compound of the present disclosure is prepared by solid phase polypeptide synthesis on a suitable resin. The solid phase polypeptide synthesis steps are well known in the art, for example, the solid phase synthesis is initiated by attaching an N-terminal protected amino acid and its carboxyl terminal to an inert solid support carrying a cleavable linker. The solid support can be any polymer that allows the initial amino acid coupling, for example, Rink amide AM resin. In some embodiments, starting with Rink amide AM as the starting material, the amino acids with Fmoc-protecting groups are sequentially linked according to the method of solid phase synthesis, and the Fmoc-protecting groups are sequentially removed, and the peptide is cleaved from the resin, and the side chain protecting groups are simultaneously removed to obtain the fatty acid modified polypeptide resin, the peptide is cleaved from the resin, and separated and purified by chromatography column, and then freeze-dried to obtain the polypeptide derivative in powder form.

[0355] In some embodiments, the co-agonist compound of the present disclosure is prepared by solid phase polypeptide synthesis on a suitable resin. The solid phase polypeptide synthesis steps are well known in the art, for example, the solid phase synthesis is initiated by attaching an N-terminal protected amino acid and its carboxyl terminal to an inert solid support carrying a cleavable linker. The solid support can be any polymer that allows the initial amino acid coupling, for example, Rink amide AM resin. In some embodiments, starting with Rink amide AM as the starting material, the amino acids with Fmoc-protecting groups are sequentially linked according to the method of solid phase synthesis, and the Fmoc-protecting groups are sequentially removed, and the peptide is cleaved from the resin, and the side chain protecting groups are simultaneously removed to obtain the fatty acid modified polypeptide resin, the peptide is cleaved from the resin, and separated and purified by chromatography column, and then freeze-dried to obtain the polypeptide derivative in powder form.

[0356] In some embodiments, the polypeptide sequence of the co-agonist compound of the present disclosure is prepared using a recombinant technique, in which case the present disclosure also provides a recombinant vector comprising the above-described nucleic acid molecule; the recombinant vector includes a cloning vector for replicating the relevant sequence and an expression vector for expressing the relevant gene. The vector can be any vector generally used in the art, such as a plasmid, a bacteriophage, a cosmid, a minichromosome, or a virus. In addition to the nucleic acid encoding the above polypeptide sequence of the co-agonist compound, the expression vector can include not only a promoter that initiates the transcription of the gene of the polypeptide sequence, but also a signal peptide sequence, a terminator that terminates the transcription of the gene of the polypeptide sequence, and an enhancer sequence.

[0357] The method for constructing the recombinant expression vector can be any known method. The above-described promoter, nucleic acid encoding the polypeptide sequence, and other DNA segments (e.g., terminator, enhancer) if present can be introduced in a predetermined order into a properly selected vector used as a basis. For example, the recombinant vector can be constructed by using restriction enzymes and ligases, etc.

[0358] In some embodiments, the polypeptide sequence of the co-agonist compound of the present disclosure is prepared using a recombinant technique, in which case the present disclosure also provides a recombinant cell comprising the above-described recombinant vector, which expresses the polypeptide sequence of the co-agonist compound of the present disclosure with or without induction. In some embodiments, the method for constructing the recombinant cell includes the following: transforming the recombinant expression vector into an expression host cell, culturing and inducing expression (if necessary) by adding an inducer, to obtain the above-described polypeptide sequence of the co-agonist compound. Further, the expression host cell is a prokaryotic cell or a eukaryotic cell, such as E. coli, yeast, plant cells, animal cells, etc.

[0359] More specifically, the method for constructing the above-described recombinant cell includes the following steps:

[0360] (1) amplification of the gene encoding the polypeptide sequence of the co-agonist compound;

[0361] (2) construction of the recombinant expression vector;

[0362] (3) transformation or transfection of the recombinant expression vector into an expression host cell;

[0363] (4) optional selection of positive clones.

[0364] The polypeptide of the present disclosure can be secreted outside the cell, expressed on the cell surface, or expressed inside the cell.

[0365] In some embodiments, the method for preparing the polypeptide sequence of the co-agonist compound of the present disclosure using a recombinant technique includes the following steps:

[0366] (1) culturing the recombinant cell and adding an inducer to induce (if necessary) expression of the polypeptide sequence of the co-agonist compound of the present disclosure, to obtain a cell culture;

[0367] (2) optionally, isolating, purifying the polypeptide sequence of the co-agonist compound of the present disclosure from the cell culture (e.g. cells, cell culture supernatant).

[0368] Biological activity

[0369] In the present disclosure, EC 50 values are used as a numerical measure of agonist activity for a given receptor (i.e. GLP-1R and AMYR3). An EC 50 value refers to the concentration that elicits 50% of the maximum effect. A compound with a lower EC 50 value can be considered to have higher activity for the receptor.

[0370] The cell activity of the co-agonist compounds of the present disclosure was detected, and it was found that the co-agonist compounds of the present disclosure have good agonist activity for both GLP-1R and AMYR3, and can effectively activate the corresponding downstream pathways of GLP-1R and AMYR3 to exert appropriate agonist effects.

[0371] Further, the weight loss effect was evaluated in a rat model, and the results showed that the co-agonist compounds of the present disclosure all exhibited significantly higher weight loss effect than single agonists and prior art (such as Cagrilintide).

[0372] Pharmaceutical composition

[0373] The co-agonist compounds or polypeptides of the present disclosure can be prepared into a pharmaceutical composition, wherein the co-agonist compound or its pharmaceutically acceptable salt or polypeptide is present in a therapeutically effective amount.

[0374] The pharmaceutical composition described herein comprises, in addition to the active ingredient co-agonist compound or its pharmaceutically acceptable salt or polypeptide, a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable adjuvants are well known to those skilled in the art, such as non-toxic fillers, stabilizers, diluents, carriers, solvents or other formulation adjuvants. For example, diluents, excipients, such as microcrystalline cellulose, mannitol, etc.; fillers, such as starch, sucrose, etc.; binders, such as starch, cellulose derivatives, alginate, gelatin and / or polyvinylpyrrolidone; disintegrants, such as calcium carbonate and / or sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants, such as cetyl alcohol; carriers, solvents, such as water, physiological saline, kaolin, soap clay, etc.; lubricants, such as talc, calcium / magnesium stearate, polyethylene glycol, etc.

[0375] In some embodiments, the co-agonist compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or polypeptides, are formulated into a liquid suitable for administration by injection or infusion.

[0376] The co-agonist compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or polypeptides, can be used alone, or in combination with any active ingredient that is beneficial for the prevention, alleviation and / or treatment of diseases related to metabolic, cardiovascular and / or cognitive dysfunction, which is expected to enhance the beneficial therapeutic effects of the co-agonist compounds or polypeptides of the present disclosure. Such active ingredients are, for example, GIP, Glucagon, PYY, GDF15, Cannabinoid Receptor-1 blockers, Mitochondrial uncouplers, THR-beta agonists.

[0377] The pharmaceutical compositions of the present disclosure can also be used in combination with another drug or drugs, which combination is expected to have a synergistic effect in the prevention, alleviation and / or treatment of diseases related to metabolic, cardiovascular and / or cognitive dysfunction. Such another drug or drugs are, for example, weight regulation, anti-obesity, lipid metabolism regulation, glycemic regulation, hypertension treatment, cardiovascular regulation, brain system disease, mental system disease or nervous system regulation drugs, etc.

[0378] Medical conditions

[0379] By administering an effective amount of the co-agonist compounds or polypeptides as described herein, the formulations of the present disclosure can be used for:

[0380] (1) the prevention, alleviation and / or treatment of diseases related to metabolic, cardiovascular and / or cognitive dysfunction;

[0381] (2) the reduction of food intake, the reduction of body weight, the suppression of appetite, the induction of satiety, the reduction of gastric motility and / or the delay of gastric emptying;

[0382] (3) the prevention of weight gain, especially the maintenance of weight loss after dieting;

[0383] (4) the delay or reduction of the development of major adverse cardiovascular events (MACE) selected from the group consisting of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, revascularization, hospitalization for unstable angina, and hospitalization for heart failure;

[0384] (5) the delay or improvement of cognitive function decline or the enhancement of cognitive function.

[0385] In Table 1, each polypeptide derivative comprises a GLP-1 receptor agonist peptide as shown in Table A.

[0386] Table A

[0387] In Table 1, each polypeptide derivative comprises a peptide linker as shown in Table B.

[0388] Table B

[0389] In Table 1, the amylin receptor agonist peptide comprised by each polypeptide derivative is as shown in Table C.

[0390] Table C

[0391] In Table 1, the linker + fatty acid side chain comprised by each polypeptide derivative is as shown in Table D.

[0392] Table D DETAILED DESCRIPTION

[0393] The experimental methods used in the following examples are conventional unless otherwise specified.

[0394] The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

[0395] The application is further described in conjunction with the following examples. It is to be understood that the following examples are merely illustrative of the application and do not limit the scope of the application.

[0396] Example 1: Preparation of polypeptide derivatives

[0397] The polypeptide derivatives of the application and the control polypeptide derivatives were prepared using a general synthetic method (solid phase synthesis) as follows:

[0398] 1) Swell the starting resin: Load 6.0 g of Rink amide AM resin into a 250 mL solid phase reaction vessel and swell the resin with 60 mL of N,N-dimethylformamide (DMF) for 20 minutes. Wash the resin twice with 60 mL of DMF and stir for 2-3 minutes.

[0399] 2) General Fmoc deprotection procedure: Perform two Fmoc deprotections using 60 mL of a solution of 5 wt% piperidine, 1.25 wt% diphenylurea (DPU) and 1 wt% hydroxybenzotriazole (HOBt) in DMF for 10 minutes and 20 minutes respectively. Then wash the resin once with 60 mL of DMF and stir for 2-3 minutes, wash twice with 60 mL of methyl tert-butyl ether (MTBE) and stir for 2-3 minutes, and wash twice with 60 mL of DMF and stir for 2-3 minutes.

[0400] 3) General Fmoc amino acid coupling procedure: A solution of Fmoc-amino acid (1.5 equivalents) corresponding to the polypeptide sequence and HOBt (3.0 equivalents, 1.62 g) in 28 mL of DMF was added to the resin followed by the addition of N,N'-diisopropylcarbodiimide (DIC) (3 equivalents, 1.9 mL). The reaction was continued for 2 to 24 hours at room temperature and monitored by testing with Ninhydrin reagent. Where the fatty acid side chain was pre-synthesized onto the Fmoc amino acid, the amino acid to which the fatty acid was coupled was added as a Fmoc amino acid unit to the polypeptide sequence.

[0401] 4) Peptide resin drying: After the polypeptide chain assembly was completed, the peptide resin was washed with DMF three times, 60 mL each, and then with MTBE three times, 60 mL each. The peptide resin was then dried under vacuum at 20-30 °C, -0.08 MPa for 23 hours to obtain the peptide resin.

[0402] 5) Cleavage: Trifluoroacetic acid (TFA), dithiothreitol (DTT), water and triisopropylsilane (TIS) were mixed in the ratio of 95 mL: 5 g: 2.5 mL: 2.5 mL (volume / volume / volume / volume ratio, total 360 mL) and cooled to 0-5 °C. The peptide resin was then added to the cleavage solution while maintaining the temperature of the mixture < 15 °C. The reaction mixture was warmed to approximately 25 ± 2 °C and the reaction was continued at room temperature for 3 hours. The resin was then filtered off through a sintered glass funnel and washed with TFA twice, 36 mL each. The filtrates were combined and the peptide was precipitated by the addition of pre-cooled MTBE (0-5 °C, approximately 1 mL of filtrate per 10 mL of MTBE, total 3600 mL). The suspension was stirred for 30 minutes. The solid was collected by filtration and washed with MTBE three times, 300 mL each.

[0403] 6) Crude peptide drying: The crude peptide was dried under vacuum at 20-30 °C, -0.08 MPa to obtain the crude peptide dry powder.

[0404] The polypeptide derivatives of the present application prepared are shown in Table 1 and the control polypeptide derivatives prepared are shown in Table 2.

[0405] In Table 1 and Table 2, each polypeptide derivative represents a derivative obtained by modifying an amino acid sequence with a fatty acid-containing side chain; the modification site and the fatty acid column represent the modification position of the fatty acid side chain on the amino acid sequence and the specific fatty acid side chain connected at the position, respectively.

[0406] For example, 10083, the modification site 17K and the fatty acid 2*AEEA+γGlu+C18 diacid in the table mean that the fatty acid side chain modification is carried out on the 17th Lys in the polypeptide sequence (SEQ ID NO: 1) of 10083, and specifically, the modification is achieved by connecting the carboxyl of the AEEA end in 2*AEEA+γGlu+C18 diacid to the epsilon amino group on the Lys through an amide bond. All fatty acid side chain modifications with 2*AEEA are achieved by connecting the carboxyl of the AEEA end in 2*AEEA+γGlu+C18 diacid, 2*AEEA+γGlu+C20 diacid, 2*AEEA+γGlu+C22 diacid, 2*AEEA+γGlu+C17 phosphonic acid, 2*AEEA+γGlu+C19 phosphonic acid, 2*AEEA+γGlu+C21 phosphonic acid, 2*AEEA+γGlu+C17 tetrazole or 2*AEEA+βAsp+C18 diacid to the epsilon amino group on the specified Lys through an amide bond.

[0407] For example, 10093, the modification site 17K and the fatty acid 2*AEEA+γGlu+C18 diacid in the table mean that the fatty acid side chain modification is carried out on the 17th Lys in the polypeptide sequence (SEQ ID NO: 1) of 10093, and specifically, the modification is achieved by connecting the carboxyl of the AEEA end in 2*AEEA+γGlu+C18 diacid to the epsilon amino group on the Lys through an amide bond. All fatty acid side chain modifications with 2*AEEA are achieved by connecting the carboxyl of the AEEA end in 2*AEEA+γGlu+C18 diacid, 2*AEEA+γGlu+C20 diacid, 2*AEEA+γGlu+C22 diacid, 2*AEEA+γGlu+C17 phosphonic acid, 2*AEEA+γGlu+C19 phosphonic acid, 2*AEEA+γGlu+C21 phosphonic acid, 2*AEEA+γGlu+C17 tetrazole or 2*AEEA+βAsp+C18 diacid to the epsilon amino group on the specified Lys through an amide bond.

[0408] For example, 10093, the modification site 17K and the fatty acid 2*AEEA+γGlu+C18 diacid in the table mean that the fatty acid side chain modification is carried out on the 17th Lys in the polypeptide sequence (SEQ ID NO: 1) of 10093, and specifically, the modification is achieved by connecting the carboxyl of the AEEA end in 2*AEEA+γGlu+C18 diacid to the epsilon amino group on the Lys through an amide bond. All fatty acid side chain modifications with 2*AEEA are achieved by connecting the carboxyl of the AEEA end in 2*AEEA+γGlu+C18 diacid, 2*AEEA+γGlu+C20 diacid, 2*AEEA+γGlu+C22 diacid, 2*AEEA+γGlu+C17 phosphonic acid, 2*AEEA+γGlu+C19 phosphonic acid, 2*AEEA+γGlu+C21 phosphonic acid, 2*AEEA+γGlu+C17 tetrazole or 2*AEEA+βAsp+C18 diacid to the epsilon amino group on the specified Lys through an amide bond.

[0409] The "NH2" at the C-terminus of the polypeptide derivative indicates that the amino acid at the C-terminus has a "NH2" modification.

[0410] The meanings of the abbreviations used in this example are as follows:

[0411] Fmoc: 9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl

[0412] AEEA: 2-(2-(2-Aminoethoxy)ethoxy)acetic acid

[0413] yGlu: y-Glutamic Acid

[0414] C18 diacid: octadecandioic acid

[0415] C20 diacid: icosandioic acid

[0416] C22 diacid: docosandioic acid

[0417] C17 phosphonic acid: 17-phosphonoheptadecanoic acid

[0418] C19 phosphonic acid: 19-phosphonononadecanoic acid

[0419] C21 phosphonic acid: 21-phosphonohenicosanoic acid

[0420] C17 tetrazole: 17-(4H-1,2,3,4-tetraazol-5-yl)heptadecanoic acid

[0421] eLys: e-lysine

[0422] bAsp: b-aspartic acid

[0423] Example 2: Cell activity test of polypeptide derivatives

[0424] The purpose of this example is to test the activity of the polypeptide derivatives of the disclosure on human GLP-1 receptor (GLP-1R), amylin receptor in vitro using luciferase assay.

[0425] Activity on amylin receptor

[0426] Using standard methods, the hygromycin B selection marker of plasmid pGL4.29 [luc2P / CRE / Hygro] Vector (Promega, Cat# E8471) containing a multiple copy cAMP response element (CRE)-driven luciferase expression cassette was replaced with a puromycin selection marker, and the constructed plasmid was used to transfect CHO-K1 / Ga15 / AMYR3 cells (Jinsirui Biotech Co., Ltd.) in which the calcitonin receptor (CTR) and receptor activity modifying protein 3 (RAMP3) have been constructed, and the cells were cultured in Ham’s F12 medium containing Zeocin 200 μg / mL, puromycin 2 μg / mL, hygromycin 100 μg / mL, and G418 400 μg / mL to obtain a stably transfected AMYR3 / CRE-luc cell line.

[0427] The polypeptide dry powder prepared in Example 1 was dissolved in 20 mM phosphate buffer at pH 7.0, and further diluted with growth medium containing 10% FBS in DMEM medium to obtain a polypeptide derivative sample with an initial concentration of 2 nM, and then the polypeptide derivative sample was gradient-diluted with growth medium to obtain 7 samples with concentrations that differ by 5 times in turn. 50 μL of the sample determination solution with the corresponding concentration was added to each well of a white 96-well plate.

[0428] The stably transfected AMYR3 / CRE-luc cell line constructed above was resuspended in growth medium at a certain density, and 50 μL of cell resuspension was added to the white 96-well plate containing the sample determination solution at a certain cell density. After incubation at 37°C and 5% CO2 for 4-5 hours, 100 μL of Luciferase substrate (Promega, Cat# E2650) was added to each well, incubated for 3 minutes, and finally the luminescence was determined on a SPARK (TECAN) enzyme marker with SparkControl Magellan 3.0 software. The standard curve was drawn by the fluorescence value, and the EC 50 .

[0429] Activity of GLP-1 receptor (GLP-1R)

[0430] The HEK293 / CRE-Luc / GLP1R cell line (Jinsirui Biotech Co., Ltd., Cat# M00562) was used for the activity determination of GLP-1 receptor (GLP-1R).

[0431] The polypeptide dry powder prepared in Example 1 was dissolved in 20 mM phosphate buffer at pH 7.0, and further diluted with growth medium containing 10% FBS in F12 medium to obtain a polypeptide derivative sample with an initial concentration of 10 nM, and then the polypeptide derivative sample was gradiently diluted with growth medium to obtain 7 samples with concentrations differing by 4 times in turn, and 50 μL of sample solution with corresponding concentration was added to each well of a white 96-well plate.

[0432] The HEK293 / CRE-Luc / GLP1R cell line was resuspended in growth medium at a certain density, 50 μL of cell resuspension was added to each well of a white 96-well plate with sample solution added, and after incubation at 37°C and 5% CO2 for 20-24 hours, 100 μL of Luciferase substrate (Promega, item number E2650) was added to each well, incubated for 3 minutes, and finally the luminescence was determined on a SPARK (TECAN) microplate reader with SparkControl Magellan 3.0 software, and the EC 50 was calculated by drawing a standard curve with fluorescence values. The results are shown in Tables 3 and 4.

[0433] Table 3 GLP-1R and AMYR3 cell activity results of polypeptide derivatives of the application

[0434] Table 3 shows that the polypeptide derivative molecules of the application as GLP-1R and AMYR3 co-agonists have excellent agonistic activity on both GLP-1R and AMYR3.

[0435] Table 4 Comparison of GLP-1R and AMYR3 cell activity effects of control polypeptide derivatives and polypeptide derivatives of the application

[0436] Table 4 shows that it is extremely difficult to obtain GLP-1R and AMYR3 co-agonists by directly or indirectly connecting effective GLP-1 receptor agonists and effective amylin receptor agonists, and unlike the usual expectation, the molecules obtained by such connection do not necessarily have equally effective agonistic ability on both receptors.

[0437] Example 3: Pharmacodynamic study of polypeptide derivatives in SD rats

[0438] Experimental method

[0439] The in vivo weight loss effects of polypeptide derivatives of the application (divided into three batches) and control polypeptide derivatives were studied respectively to evaluate the pharmacodynamic effect.

[0440] SPF level male SD rats (7-8 weeks, 240-260 g) were used in the experiment after quarantine period. The experimental animal breeding conditions were room temperature 20℃-23℃, relative humidity 40%-50%; during the quarantine period and the experimental process, the feed was used for rat maintenance feed (Kaoxie (Tianjin) Feed Co., Ltd.), and the drinking water was purified water supplied by drinking water bottle, and the rats were free to drink water.

[0441] During the experiment, the initial body weight of the animals was recorded on the day before administration (D0), and the initial feed was added. The body weight of the experimental animals and the remaining food were recorded every day from the first day (D1) to the nth day (Dn), and the body weight change rate and food intake were calculated, and any abnormal conditions were recorded and reported. The rats were in good condition during the experiment, and no abnormal phenomena were observed. The body weight change rate = (BWDn-BWD1) / BWD1*100%, BW represents the body weight value, D1 and Dn represent the administration and n days after administration, respectively. The points in the figure are represented by the mean value ± standard error of the mean (SEM) method. The food intake is the average food intake per animal in each group at two time points after administration. The cumulative food intake is the total food intake of each animal before a certain time point after administration. The concentration of polypeptide derivatives in each administration group is 1 nmol / ml.

[0442] 3.1 Weight loss efficacy of the polypeptide derivatives of the invention

[0443] Batch A: The experimental animals were divided into 5 groups (n=5) according to the principle of equal weight distribution, and were named A1-A5, respectively. The polypeptide derivatives and doses of each administration group are shown in Table 5. Subcutaneous injection was used for single administration, and the experiment lasted for 5 days. The administration day was recorded as D1, and the efficacy comparison of different molecules is shown in Figure 1.

[0444] Table 5

[0445] Batch B: The experimental animals were divided into 3 groups (n=5) according to the principle of equal weight distribution, and were named B1-B3, respectively. The polypeptide derivatives and doses of each administration group are shown in Table 6. Subcutaneous injection was used for single administration, and the experiment lasted for 4 days. The administration day was recorded as D1, and the efficacy comparison of different molecules is shown in Figure 2.

[0446] Table 6

[0447] C batch: the experimental animals were divided into 8 groups (n=5) according to the principle of average grouping by weight, and were named as C1-C8. The polypeptide derivatives and doses of each administration group are shown in Table 7. Subcutaneous injection was used for single administration, and the experiment lasted for 4 days. The administration time was recorded as T0, and the body weight change rate was calculated as (BWTn-BWT0) / BWT0*100%, where BW represents the body weight value, and T0 and Tn represent the administration time and n hours after administration, respectively. The pharmacodynamic comparison of different molecules is shown in Figure 3.

[0448] Table 7

[0449] Result analysis

[0450] The experimental results of Figures 1-3 further show the significant differences in the co-agonistic activities of polypeptide molecules with different structures against GLP-1R and AMYR3.

[0451] The data in Figures 1-3 show that, compared with the original GLP-1R agonist 801111 (Ecnoglutide) from which the polypeptide derivatives of the present application are derived, the polypeptide derivatives of the present application derived from Ecnoglutide and the amylin receptor agonist 801502 all maintain the original or even higher weight loss effect of Ecnoglutide in vivo. Notably, this further illustrates that the combination of GLP-1R and AMYR3 agonists in the composition plays a major and key role in whether the compound itself has co-agonistic activity, while the position of the single fatty acid modification in the compound (batch A experiment, Figure 1, compounds 10083 and 10084), the fatty acid modification and / or the type of connecting sequence (batch C experiment, Figure 3, compounds 10086 and 10087, etc., compounds 10094 and 10095, etc.) have less effect on the co-agonistic activity of the compound. At the same time, the activity of the polypeptide derivatives of the present application also shows a more optimal weight loss effect compared with the existing AMYR agonist (803333, cagrilintide) or GLP-1R / AMYR co-agonist (804444).

[0452] The co-agonist of the present application also shows similar beneficial effects in controlling cumulative food intake as weight loss.

Claims

1. A co-agonist of the GLP-1 receptor and the amylin receptor comprising a polypeptide R1 of formula I: Z1-Z2-Z3 (formula I), characterized in that Z1 is a GLP-1 receptor agonist peptide comprising the amino acid sequence of SEQ ID NO: 17 or at most 3 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 17; Z2 is a peptide linker or is absent; and Z3 is an amylin receptor agonist peptide comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23 or at most 4 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23; the co-agonist further comprises a fatty acid side chain attached to a lysine (Lys, K) residue in the polypeptide R1; preferably, the polypeptide R1 comprises 1-3 lysine (Lys, K) residues to which a fatty acid side chain is attached; more preferably, comprises 1 lysine (Lys, K) residue to which a fatty acid side chain is attached; optionally, the polypeptide R1 has an NH2 modification at its carboxy terminus and / or is free of disulfide bonds.

2. The co-agonist of GLP-1 receptor and amylin receptor according to claim 1, characterized in that: the lysine (Lys, K) residue to which a fatty acid side chain is attached is located in the GLP-1 receptor agonist peptide Z1, preferably at position 17 or 24 of the GLP-1 receptor agonist peptide Z1.

3. The co-agonist of the GLP-1 receptor and the amylin receptor according to claim 1 or 2, characterized in that: the GLP-1 receptor agonist peptide Z1 consists of or comprises a polypeptide of formula II: His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa17-Ala-Ala-Arg-Glu-Phe-Ile-Xaa24-Trp-Leu-Val-Arg-Gly-Arg-Xaa31 (formula II), wherein Xaa17 is Gin (Q) or Lys (K); Xaa24 is Lys (K) or Ala (A); and / or Xaa31 is Gly (G) or is absent; and at least one Lys (K) residue is present at Xaa17 or Xaa24.

4. The co-agonist of GLP-1 receptor and amylin receptor according to claim 1 or 2, characterized in that: the GLP-1 receptor agonist peptide Z1 consists of or comprises a polypeptide of formula II: His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa17-Ala-Ala-Arg-Glu-Phe-Ile-Xaa24-Trp-Leu-Val-Arg-Gly-Arg-Xaa31 (formula II), wherein Xaa17 is Gin (Q) or Lys (K); Xaa24 is Lys (K) or Ala (A); and / or Xaa31 is Gly (G) or is absent; and at least one Lys (K) residue is present at Xaa17 or Xaa24.

5. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 4, characterized in that: The GLP-1 receptor agonist peptide Z1 consists of or comprises a polypeptide as shown in SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO:

36.

6. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 4, characterized in that: The GLP-1 receptor agonist peptide Z1 is a polypeptide as shown in SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO:

36.

7. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 6, characterized in that: The peptide linker Z2 consists of or comprises a Gly (G) and / or Ser (S) rich linker peptide, such as a linker peptide as shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40: or The peptide linker Z2 consists of or comprises a linker peptide as shown in SEQ ID NO: 41 or SEQ ID NO:

42.

8. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 6, characterized in that: The peptide linker Z2 is a Gly (G) and / or Ser (S) rich linker peptide, such as a linker peptide as shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40: or The peptide linker Z2 is a linker peptide as shown in SEQ ID NO: 41 or SEQ ID NO:

42.

9. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 8, characterized in that: The amylin receptor agonist peptide Z3 consists of or comprises a polypeptide as shown in formula III: Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu-Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (formula III), wherein, Xaa3 is Gin (Q) or Glu (E); Xaa8 is Val (V) or Ala (A); Xaa9 is Leu (L) or Thr (T); Xaa10 is Gly (G) or Gin (Q); Xaa13 is Ser (S) or Ala (A); Xaa14 is Asp (D), Ala (A) or Aib; Xaa15 is Glu (E) or Phe (F); Xaa17 is His (H) or Arg (R); Xaa18 is Arg (R), Glu (E) or His (H); Xaa19 is Leu (L) or Phe (F); Xaa20 is Gin (Q), Ala (A) or Thr (T); Xaa22 is Ala (A), Ser (S) or Thr (T); Xaa23 is Ala (A), Ser (S) or Thr (T); Xaa25 is Ala (A), Ser (S) or Thr (T); Xaa26 is Ala (A), Ser (S) or Thr (T); Xaa27 is Ala (A), Ser (S) or Thr (T); Xaa28 is Ala (A), Ser (S) or Thr (T); Xaa29 is Ala (A), Ser (S) or Thr (T); Xaa30 is Ala (A), Ser (S) or Thr (T); Xaa31 is Ala (A), Ser (S) or Thr (T); Xaa34 is Ala (A), Ser (S) or Thr (T); Xaa35 is Ala (A), Ser (S) or Thr (T); Xaa36 is Ala (A), Ser (S) or Thr (T). Xaa22 is Tyr (Y) or Arg (R); Xaa23 is Pro (P) or Asp (D); Xaa25 is Thr (T) or Asp (D); Xaa26 is lie (I) or is absent; Xaa27 is Leu (L) or is absent; Xaa28 is Pro (P) or is absent; Xaa29 is Pro (P) or is absent; Xaa30 is Thr (T) or is absent; Xaa31 is Asp (D) or Asn (N); Xaa34 is Ser (S) or Ala (A); Xaa35 is Gly (G), Glu (E) or Asn (N); and / or Xaa36 is Ser (S) or Thr (T).

10. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 8, characterized in that: The amylin receptor agonist peptide Z3 is a polypeptide according to formula III: Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu- Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (formula III), wherein, Xaa3 is Gin (Q) or Glu (E); Xaa8 is Val (V) or Ala (A); Xaa9 is Leu (L) or Thr (T); Xaa10 is Gly (G) or Gin (Q); Xaa13 is Ser (S) or Ala (A); Xaa14 is Asp (D), Ala (A) or Aib; Xaa15 is Glu (E) or Phe (F); Xaa17 is His (H) or Arg (R); Xaa18 is Arg (R), Glu (E) or His (H); Xaa19 is Leu (L) or Phe (F); Xaa20 is Gin (Q), Ala (A) or Thr (T); Xaa22 is Tyr (Y) or Arg (R); Xaa23 is Pro (P) or Asp (D); Xaa25 is Thr (T) or Asp (D); Xaa26 is lie (I) or is absent; Xaa27 is Leu (L) or is absent; Xaa28 is Pro (P) or is absent; Xaa29 is Pro (P) or is absent; Xaa30 is Thr (T) or is absent; Xaa31 is Asp (D) or Asn (N); Xaa34 is Ser (S) or Ala (A); Xaa35 is Gly (G), Glu (E) or Asn (N); and / or Xaa36 is Ser (S) or Thr (T). The amylin receptor agonist peptide Z3 is a polypeptide according to formula III: Ala-Ser-Xaa3-Leu-Ser-Thr-Ala-Xaa8-Xaa9-Xaa10-Arg-Leu-Xaa13-Xaa14-Xaa15-Leu- Xaa17-Xaa18-Xaa19-Xaa20-Asp-Xaa22-Xaa23-Arg-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Val-Gly-Xaa34-Xaa35-Xaa36-Pro (formula III), wherein, Xaa3 is Gin (Q) or Glu (E); Xaa8 is Val (V) or Ala (A); Xaa9 is Leu (L) or Thr (T); Xaa10 is Gly (G) or Gin (Q); Xaa13 is Ser (S) or Ala (A); Xaa14 is Asp (D), Ala (A) or Aib; Xaa15 is Glu (E) or Phe (F); Xaa17 is His (H) or Arg (R); Xaa18 is Arg (R), Glu (E) or His (H); Xaa19 is Leu (L) or Phe (F); Xaa20 is Gin (Q), Ala (A) or Thr (T); Xaa22 is Tyr (Y) or Arg (R); Xaa23 is Pro (P) or Asp (D); Xaa25 is Thr (T) or Asp (D); Xaa26 is lie (I) or is absent; Xaa27 is Leu (L) or is absent; Xaa28 is Pro (P) or is absent; Xaa29 is Pro (P) or is absent; Xaa30 is Thr (T) or is absent; Xaa31 is Asp (D) or Asn (N); Xaa34 is Ser (S) or Ala (A); Xaa35 is Gly (G), Glu (E) or Asn (N); and / or Xaa36 is Ser (S) or Thr (T).

11. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 10, characterized in that: The amylin receptor agonist peptide Z3 consists of or comprises a polypeptide as set forth in SEQ ID NO: 21, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO:

23.

12. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 10, characterized in that: The amylin receptor agonist peptide Z3 is a polypeptide as set forth in SEQ ID NO: 21, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO:

23.

13. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 12, characterized in that: The lysine (Lys, K) residue on the polypeptide R1 is linked to the fatty acid side chain via an amide bond; preferably via the epsilon amino group on the lysine (Lys, K) residue to the fatty acid side chain.

14. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 13, characterized in that: The fatty acid side chain is selected from one or more of Cx diacids: C X phosphoric acid: or C X tetrazole: wherein x is an arbitrary integer selected from 10-30; HOOC(CH2)CO-, HOOC(CH2)CO-, 14 HOOC(CH2)CO-, HOOC(CH2)CO-, 15 HOOC(CH2)CO-, HOOC(CH2)CO-, 16 HOOC(CH2)CO-, HOOC(CH2)CO-, 17 HOOC(CH2)CO-, HOOC(CH2)CO-, 18 HOOC(CH2)CO-, HOOC(CH2)CO-, 19 HOOC(CH2)CO-, HOOC(CH2)CO-, 20 HOOC(CH2)CO-, HOOC(CH2)CO-, 21 HOOC(CH2)CO-, HOOC(CH2)CO-, 22 HOOC(CH2)CO-, PO(OH)2(CH2) 15 HOOC(CH2)CO-, PO(OH)2(CH2) 16 HOOC(CH2)CO-, PO(OH)2(CH2) 17 HOOC(CH2)CO-, PO(OH)2(CH2) 18 HOOC(CH2)CO-, PO(OH)2(CH2) 19 HOOC(CH2)CO-, PO(OH)2(CH2) 20 HOOC(CH2)CO-, PO(OH)2(CH2) 21 HOOC(CH2)CO-, PO(OH)2(CH2) 22 HOOC(CH2)CO-, PO(OH)2(CH2) 23 HOOC(CH2)CO-, CN4H(CH2) 14 HOOC(CH2)CO-, CN4H(CH2) 15 HOOC(CH2)CO-, CN4H(CH2) 16 HOOC(CH2)CO-, CN4H(CH2) 17 HOOC(CH2)CO-, CN4H(CH2) 18 HOOC(CH2)CO-, CN4H(CH2) 19 HOOC(CH2)CO-, CN4H(CH2) 20 HOOC(CH2)CO-, CN4H(CH2) 21 HOOC(CH2)CO-, CN4H(CH2) 22 one or more of HOOC(CH2)CO-, CN4H(CH2).

15. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 14, characterized in that: The fatty acid side chain is linked to the amino acid Lys residue via a linker.

16. The co-agonist of GLP-1 receptor and amylin receptor according to claim 15, characterized in that: The linker is selected from one or more of nAEEA+mγGlu: n*εLys + m*γGlu: or nAEEA + mβAsp: wherein m and n are each an arbitrary integer selected from 0-4; Preferably, the linker is selected from one or more of 2 * AEEA + γGlu: Yglu: 2 AEEA + βAsp: 2*εLys + γGlu:

17. The co-agonist of the GLP-1 receptor and the amylin receptor according to any one of claims 1 to 16, characterized in that: The co-agonist of GLP-1 receptor and amylin receptor is any one of the polypeptide derivatives as set forth in Table 1 herein.

18. A polypeptide having an amino acid sequence as set forth in formula I in any one of claims 1 to 17, preferably wherein the polypeptide comprises a polypeptide of formula I: Z1-Z2-Z3 (formula I), characterized in that, Z1 is a GLP-1 receptor agonist peptide comprising an amino acid sequence as set forth in SEQ ID NO: 17 or at most 3 amino acid modifications relative to SEQ ID NO: 17; Z2 is a peptide linker or is absent; and Z3 is an amylin receptor agonist peptide comprising an amino acid sequence as set forth in SEQ ID NO: 21 or SEQ ID NO: 23 or at most 4 amino acid modifications relative to SEQ ID NO: 21 or SEQ ID NO:

23.

19. A polypeptide comprising an amino acid sequence as set forth in formula I in any one of claims 1 to 17.

20. A nucleic acid molecule encoding a polypeptide of claim 18 or 19.

21. A recombinant vector comprising a nucleic acid molecule of claim 20.

22. A recombinant cell comprising a nucleic acid molecule of claim 20 and / or a recombinant vector of claim 21 and being capable of expressing and optionally secreting a polypeptide of claim 18 or 19.

23. A method of preparing the co-agonist of GLP-1 receptor and amylin receptor according to any one of claims 1 to 17 or the polypeptide according to claim 18 or 19; Preferably, the method of preparation comprises a step of preparing the co- agonist of GLP-1 receptor and amylin receptor or the polypeptide using chemical and / or biological methods; Preferably, the chemical method comprises liquid phase and / or solid phase polypeptide synthesis.

24. A pharmaceutical composition comprising the co-agonist of GLP-1 receptor and amylin receptor according to any one of claims 1 to 17 or the polypeptide according to claim 18 or 19, and a pharmaceutically acceptable excipient.

25. The co-agonist of GLP-1 receptor and amylin receptor according to any one of claims 1 to 17 or the polypeptide according to claim 18 or 19 for use in medical treatment.

26. Use of the co-agonist of GLP-1 receptor and amylin receptor according to any one of claims 1 to 17, the polypeptide according to claim 18 or 19, or the pharmaceutical composition according to claim 24 in the manufacture of a medicament for preventing, alleviating and / or treating a disease associated with metabolic, cardiovascular and / or cognitive dysfunction; Preferably, the disease associated with metabolism comprises diabetes, obesity related disorders, abnormal appetite diseases; the disease associated with cardiovascular comprises myocardial infarction, stroke, angina pectoris, heart failure; the disease associated with cognitive dysfunction comprises dementia, Alzheimer's disease.

27. A method of preventing, alleviating and / or treating a disease associated with metabolic, cardiovascular and / or cognitive dysfunction, comprising a step of administering to a patient in need a therapeutically effective amount of the co-agonist of GLP-1 receptor and amylin receptor according to any one of claims 1 to 17, the polypeptide according to claim 18 or 19, or the pharmaceutical composition according to claim 24; Preferably, the disease associated with metabolism comprises diabetes, obesity related disorders, abnormal appetite diseases; the disease associated with cardiovascular comprises myocardial infarction, stroke, angina pectoris, heart failure; the disease associated with cognitive dysfunction comprises dementia, Alzheimer's disease.

Images