Polypeptide and derivative thereof, composition, and use thereof

Abstract

The present application provides a polypeptide, and a derivative of the polypeptide or a pharmaceutically acceptable salt thereof. The present application further provides a pharmaceutical composition containing the polypeptide or the derivative or the pharmaceutically acceptable salt thereof of the present application, and a pharmaceutically acceptable adjuvant, and a use of the polypeptide, the derivative of the polypeptide or the pharmaceutically acceptable salt thereof, and the composition in the field of disease prevention / treatment.

Description

Polypeptide and its derivatives, composition and application Technical Field

[0001] The present application relates to the field of polypeptide technology, and in particular to a polypeptide and its derivatives, compositions and applications. Background Art

[0002] Amylin, also known as islet amyloid polypeptide (IAPP, amylin, or hAMY1-37), is a 37-amino acid polypeptide hormone that belongs to the calcitonin protein family (which also includes calcitonin (CT), calcitonin gene-related peptide (CGRP), and adrenomedullin (hAM) and its precursors). Amylin is co-secreted with insulin in pancreatic β-cells and is deficient in diabetics. It exerts its effects in several different organ systems, primarily through amylin receptors 1-3 (AMYR1-3). Amylin inhibits glucagon secretion, delays gastric emptying, signals satiety, and suppresses appetite. Clinical studies have shown that amylin receptor agonists are useful for treating overweight, obesity, type 1 diabetes, and / or type 2 diabetes. However, amylin has several drawbacks, such as a high profibrogenic potential, a short in vivo half-life, and chemical instability at pH 7. Therefore, native amylin is not suitable for use as a pharmaceutical active ingredient.

[0003] Numerous amylin analogs or derivatives are known in the prior art that attempt to address some of the known shortcomings of human amylin. A successful example is the amylin analog pramlintide (trade name Symlin), which has been approved by the FDA for use in type I and type II diabetes. However, pramlintide has a half-life of less than one hour and is taken at mealtimes, requiring patients to take the drug multiple times a day for treatment. Furthermore, pramlintide is formulated at pH 4 because it fibrillates at pH 7 and becomes ineffective due to precipitation. Therefore, there remains a need for new amylin analogs or derivatives with increased chemical stability, increased metabolic stability, and / or reduced fibrillation tendency. In particular, there is a need for amylin analogs or derivatives that are stable over a wider pH range. Furthermore, there is a need for new amylin analogs or derivatives that increase efficacy by increasing potency, improving efficacy, and / or extending half-life to reduce frequent dosing and improve patient compliance. Pramlintide, currently available on the market, has a half-life of less than one hour, requiring multiple daily doses for treatment. It cannot be formulated in a liquid formulation with other drugs under neutral conditions.

[0004] Summary of the Invention

[0005] The technical problem to be solved by the present application is to address the deficiencies in the background technology and to provide a polypeptide and its derivatives, compositions and applications.

[0006] The present application provides a polypeptide derivative or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the following amino acid sequence: ASX3LS TAX8X9X 10 RLADF LRHX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILPPT NVGSN TX 37 -NH2;

[0007] Among them: X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N, Q, E, S, T, A, G, H, K, R, or D; X 22 is N, Q, E, S, NMeAsn, αMeAsn, NMeAsp, or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 is P or trans-Hyp; the derivative comprises a fatty acid-containing side chain attached to the N-terminus of the polypeptide.

[0008] In some embodiments of the present application, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0009] In some embodiments of the present application, X3 is E, X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0010] In some embodiments of the present application, X3 is Q, X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0011] In some embodiments of the present application, X8 is A, X3 is E or Q; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0012] In some embodiments of the present application, X8 is V, X3 is E or Q; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0013] In some embodiments of the present application, X9 is L, X3 is E or Q; X8 is A or V; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0014] In some embodiments of the present application, X9 is T, X3 is E or Q; X8 is A or V; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0015] In some embodiments of the present application, X 10 is G, X3 is E or Q; X8 is A or V; X9 is L or T; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0016] In some embodiments of the present application, X 10 is Q; X3 is E or Q; X8 is A or V; X9 is L or T; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0017] In some embodiments of the present application, X 19 is S, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0018] In some embodiments of the present application, X 19is F; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0019] In some embodiments of the present application, X 20 is S; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0020] In some embodiments of the present application, X 20 is T; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0021] In some embodiments of the present application, X 21 is N, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0022] In some embodiments of the present application, X 21 is D; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0023] In some embodiments of the present application, X 21 is Q; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0024] In some embodiments of the present application, X 21 is E; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0025] In some embodiments of the present application, X 21 is S; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0026] In some embodiments of the present application, X 21 is T; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0027] In some embodiments of the present application, X 21 is A; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0028] In some embodiments of the present application, X 21 is G; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0029] In some embodiments of the present application, X 21 is H; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0030] In some embodiments of the present application, X 21 is K; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X25 P or D; X 37 It is P or trans-Hyp.

[0031] In some embodiments of the present application, X 21 is R; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 22 N or R; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0032] In some embodiments of the present application, X 22 is N, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0033] In some embodiments of the present application, X 22 is R, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0034] In some embodiments of the present application, X 22 is Q, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0035] In some embodiments of the present application, X 22 is E, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0036] In some embodiments of the present application, X 22 is S, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0037] In some embodiments of the present application, X 22 is NMeAsn, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0038] In some embodiments of the present application, X 22 is αMeAsn, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0039] In some embodiments of the present application, X22 is NMeAsp, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 23 L or D; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0040] In some embodiments of the present application, X 23 is L, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0041] In some embodiments of the present application, X 23 is D; X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 24 K or R; X 25 P or D; X 37 It is P or trans-Hyp.

[0042] In some embodiments of the present application, X 24 is K, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 25 P or D; X 37 It is P or trans-Hyp.

[0043] In some embodiments of the present application, X 24 is R; X3 is E or Q; X8 is A or V; X9 is L or T; X 10G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 25 P or D; X 37 It is P or trans-Hyp.

[0044] In some embodiments of the present application, X 25 is P, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 37 It is P or trans-Hyp.

[0045] In some embodiments of the present application, X 25 is D, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 37 It is P or trans-Hyp.

[0046] In some embodiments of the present application, X 37 is P, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X 21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 It is P or D.

[0047] In some embodiments of the present application, X 37 For trans-Hyp, X3 is E or Q; X8 is A or V; X9 is L or T; X 10 G or Q; X 19 S or F; X 20 S or T; X21 N or D; X 22 N or R; X 23 L or D; X 24 K or R; X 25 It is P or D.

[0048] In some embodiments of the present application, X3 is Q; preferably, X8X9X 10 Any of ALG, VLG, VTQ, ATQ, and VLQ; X8X9X 10 More preferably, X 19 F, X 20 T, X 23 D, X 24 R, X 25 D, X 37 is P, further preferably, X 21 D or / and X 22 For R.

[0049] In some embodiments of the present application, X3 is Q; preferably, X8X9X 10 Any of ALG, VLG, VTQ, ATQ, and VLQ; X8X9X 10 More preferably, ATQ; more preferably, X 19 S, X 20 S, X 23 L, X 24 K, X 25 P, X 37 is trans-Hyp, further preferably, X 21 N, Q, E, S, T, A, G, H, K, R or / and X 22 For N, Q, E, S, MeAsn, αMeAsn, NMeAsp.

[0050] In some embodiments of the present application, the polypeptide sequence comprises any one of SEQ ID No. 1 to SEQ ID No. 25 sequences.

[0051] In some embodiments of the present application, the polypeptide sequence is ASELS TAALG RLADF LRHSS NNLKP ILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID No. 1).

[0052] In some embodiments of the present application, the polypeptide sequence is ASQLS TAVLG RLADF LRHSS NNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 2).

[0053] In some embodiments of the present application, the polypeptide sequence is ASELS TAALG RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID No. 3).

[0054] In some embodiments of the present application, the polypeptide sequence is ASQLS TAVLG RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID No. 4).

[0055] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ

[0056] RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID No. 5).

[0057] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID No. 6).

[0058] In some embodiments of the present application, the polypeptide sequence is ASQLS TAVTQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID No. 7).

[0059] In some embodiments of the present application, the polypeptide sequence is ASQLS TAVTQ RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID No. 8).

[0060] In some embodiments of the present application, the polypeptide sequence is ASQLS TAVLQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID No. 9).

[0061] In some embodiments of the present application, the polypeptide sequence is ASQLS TAVLQ RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID No. 10).

[0062] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS QQLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 11).

[0063] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS EELKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 12).

[0064] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS SSLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 13).

[0065] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS N-NMeAsn-LKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 14).

[0066] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS N-NMeAsp-LKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 15).

[0067] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS QNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 16).

[0068] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS ENLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 17).

[0069] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS SNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 18).

[0070] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS TNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 19).

[0071] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS ANLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 20).

[0072] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS GNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 21).

[0073] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS HNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 22).

[0074] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS KNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 23).

[0075] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS RNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 24).

[0076] In some embodiments of the present application, the polypeptide sequence is ASQLS TAATQ RLADF LRHSS N-αMeAsn-LKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID No. 25).

[0077] In some embodiments of the present application, the side chain containing fatty acids is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid; Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist; Z3 is 0-6 AEEA, for example, it can be 0, 1, 2, 3, 4, 5, 6; and Z1, Z2 and Z3 are connected in sequence by amide bonds.

[0078] In some embodiments of the present application, Z1 is a C22 fatty diacid.

[0079] In some embodiments of the present application, Z1 is a C20 fatty diacid.

[0080] In some embodiments of the present application, the Z1 is a C19 fatty diacid.

[0081] In some embodiments of the present application, Z1 is a C18 fatty diacid.

[0082] In some embodiments of the present application, Z1 is a C17 fatty diacid.

[0083] In some embodiments of the present application, Z1 is a C16 fatty diacid.

[0084] In some embodiments of the present application, the Z2 is γGlu.

[0085] In some embodiments of the present application, the Z2 is αGlu.

[0086] In some embodiments of the present application, the Z2 is βAsp.

[0087] In some embodiments of the present application, the Z2 is αAsp.

[0088] In some embodiments of the present application, Z2 is Inp.

[0089] In some embodiments of the present application, Z2 is Trx.

[0090] In some embodiments of the present application, Z2 does not exist.

[0091] In some embodiments of the present application, Z3 does not exist.

[0092] In some embodiments of the present application, the Z3 is 1 AEEA.

[0093] In some embodiments of the present application, the Z3 is 2 AEEAs.

[0094] In some embodiments of the present application, the Z3 is 3 AEEAs.

[0095] In some embodiments of the present application, the Z3 is 4 AEEAs.

[0096] In some embodiments of the present application, the Z3 is 5 AEEAs.

[0097] In some embodiments of the present application, the Z3 is 6 AEEAs.

[0098] In some embodiments of the present application, the polypeptide derivative is M1, or M2, or M3, or M4, or M5, or M6, or M7, or M8, or M9, or M10, or M11, or M12, or M13, or M14, or M15, or M16, or M17, or M18, or M19, or M20, or M21, or M22, or M23, or M24, or M25, or M26, or M 27, or M28, or M29, or M30, or M31, or M32, or M33, or M34, or M35, or M36, or M37, or M38, or M39, or M40, or M41, or M42, or M43, or M44, or M45, or M46, or M47, or M48, or M49, or M50, or M51, or M52, or M53, or M54, or M55.

[0099] The present application provides a polypeptide, wherein the polypeptide comprises the above-mentioned polypeptide sequence.

[0100] In some embodiments of the present application, the polypeptide comprises a polypeptide sequence of any one of SEQ ID No. 1 to SEQ ID No. 25.

[0101] The present application provides a pharmaceutical composition comprising the above-mentioned polypeptide, or the above-mentioned derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0102] In some embodiments of the present application, the pharmaceutical composition further comprises one or more other active pharmaceutical ingredients. The pharmacologically active ingredients include weight regulators, anti-obesity agents, lipid metabolism regulators, blood sugar regulators, hypertension therapeutic agents, cardiovascular regulators, brain system diseases, mental system diseases, or nervous system regulators. Preferably, the pharmacologically active substances are selected from the group consisting of: GLP-1 receptor agonists (including but not limited to GLP-1, GLP-1 analogs, GLP-1 derivatives), GIP receptor agonists (including but not limited to GIP, GIP analogs, GIP derivatives), and insulin receptor agonists (including but not limited to insulin, insulin analogs, and insulin derivatives).

[0103] Use of the above polypeptide or derivative or pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for preventing and / or treating amylin receptor-related metabolic diseases or fat metabolism disorders.

[0104] Use of the above polypeptide or derivative or pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for preventing and / or treating overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain.

[0105] Use of the above polypeptide or derivative or pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for preventing and / or treating reduced food intake. Use of the above polypeptide or derivative or pharmaceutically acceptable salt or composition thereof in reducing food intake.

[0106] The present application provides a method for preventing and / or treating amylin receptor-related metabolic diseases or fat metabolism disorders, comprising administering to a subject a preventive or therapeutically effective amount of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition.

[0107] The present application provides a method for preventing and / or treating overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain, comprising administering to a subject a preventively or therapeutically effective amount of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition.

[0108] The present application provides a method for reducing food intake, comprising administering an effective amount of the above polypeptide or the above derivative or a pharmaceutically acceptable salt thereof or the above pharmaceutical composition to a subject.

[0109] Compared to the prior art, at the same dose, the polypeptides and their derivatives or compositions of the present application have significantly improved advantages or positive effects in terms of drug activity, molecular stability, in vivo biological exposure, etc. Furthermore, compared to traditional amylin analogs or derivatives, the polypeptides and their derivatives described in the present application do not contain disulfide bonds. However, surprisingly, compared to traditional amylin analogs or derivatives, the polypeptides and their derivatives described in the present application have unexpectedly high stability, with a downward trend in fibrillation, and even when formulated at a neutral or non-acidic pH, they still have higher stability. In addition, the polypeptides or their derivatives of the present application have good pharmacokinetic effects and do not require frequent injections like known amylin derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] FIG1 shows the results of thermal acceleration experiments of the polypeptide derivative M6 in Example 3;

[0111] FIG2A shows the results of the weight loss effect of the polypeptide derivatives in Example 4 in the SD rat model over time;

[0112] FIG2B shows the results of the effect of the polypeptide derivatives on food intake in the SD rat model in Example 4 over time;

[0113] Figures 3A and 3C show the results of the weight loss effects of different doses of the polypeptide derivatives in Example 5 in the SD rat model over time;

[0114] Figures 3B and 3D show the effects of different doses of the polypeptide derivatives in Example 5 on food intake in the SD rat model over time;

[0115] FIG4A shows the results of the weight loss effects of different doses of the polypeptide derivatives in Example 6 in the DIO SD rat model over time;

[0116] FIG4B shows the results of the effects of different doses of the polypeptide derivatives in Example 6 on food intake in the DIO SD rat model over time;

[0117] Figures 5A-5B show the results of the weight loss effect of the polypeptide derivatives in Example 7 in the SD rat model over time;

[0118] Figures 5C-5D show the results of the effects of the polypeptide derivatives on food intake in the SD rat model in Example 7 over time;

[0119] FIG6 shows the changes in the concentrations of different molecules in plasma over time in Example 8. DETAILED DESCRIPTION

[0120] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0121] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0122] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may comprise natural amino acids, and it may be interrupted by unnatural amino acids.

[0123] As used herein, "derivatives" refer to products obtained by modifying functional groups (e.g., amino acid residues) of biomacromolecules (e.g., polypeptides and proteins) with certain compounds or molecules. Such modifications include, but are not limited to, acylation, amidation, esterification, and thioesterification.

[0124] As used herein, a pharmaceutical composition may refer to a composition used for the treatment of a disease or for in vitro cell culture experiments. When used for the treatment of a disease, the term pharmaceutical composition generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more adjunct ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid excipient, a finely divided solid excipient, or both.

[0125] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0126] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present application, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of this application.

[0127] As used herein, treatment refers to the use of a method for obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0128] As used herein, "prevent" or "prevent" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (eg, cancer).

[0129] In this article, "amylin receptor-related metabolic diseases" are diseases that can improve the symptoms of subjects by regulating the activation of amylin receptors and thus improving their downstream cellular pathways, including hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, diseases often associated with diabetes and fat metabolism disorders, such as methods for osteoporosis, methods for treating cognitive disorders, neurodegenerative diseases (such as Parkinson's syndrome, Alzheimer's disease), and treatment of gastrointestinal diseases, such as inflammatory bowel disease, malnutrition, and peptic ulcers.

[0130] In some embodiments of the present application, the polypeptide derivative is coupled to the polypeptide via a fatty acid in the form of an amide bond.

[0131] In some embodiments of the present application, the fatty acid is a C1 to C25 fatty acid, such as formic acid, acetic acid, a C3 fatty acid, a C4 fatty acid, a C5 fatty acid, a C6 fatty acid, a C7 fatty acid, a C8 fatty acid, a C9 fatty acid, a C10 fatty acid, a C11 fatty acid, a C12 fatty acid, a C13 fatty acid, a C14 fatty acid, a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a C18 fatty acid, a C19 fatty acid, a C20 fatty acid, a C21 fatty acid, a C22 fatty acid, a C23 fatty acid, a C24 fatty acid, or a C25 fatty acid.

[0132] In the present application, the C1 to C25 fatty acids include straight-chain fatty acids and branched-chain fatty acids.

[0133] In the present application, the C1 to C25 fatty acids include saturated fatty acids and unsaturated fatty acids.

[0134] In some embodiments of the present application, the fatty acid is one or more of caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanedioic acid, stearic acid, nonadecanedioic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosapentaenoic acid or docosahexaenoic acid, 1,8-octanedioic acid, 1,7-heptanedicarboxylic acid, 1,10-decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid or nonadecanedioic acid, eicosanedioic acid, heneicosanedioic acid, docosanedioic acid or tricosanedioic acid.

[0135] The fatty acid portion of the present application can be connected to the above polypeptide via a linker. The linker is selected from: -γGlu-, -αGlu-, -βAsp-, -αAsp-, -Inp-, -Trx-,

[0136] One or more of , wherein m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; and p is any integer from 1 to 8.

[0137] In the present application, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the polypeptide derivatives of the present application and is generally not biologically or otherwise undesirable. In many cases, the polypeptide derivatives of the present application can form acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto.

[0138] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate, and trifluoroacetate.

[0139] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0140] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.

[0141] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0142] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the Periodic Table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0143] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0144] The pharmaceutically acceptable salts of the present application can be synthesized from parent compound, alkaline or acidic moieties by customary chemical methods. Usually, these salts can be prepared by reacting the free acid form of these compounds with a stoichiometric suitable base (for example, hydroxide, carbonate, bicarbonate of Na or K, etc.), or by reacting the free base form of these compounds with a stoichiometric suitable acid. These reactions are typically carried out in water or an organic solvent or a mixture of the two. Usually, where feasible, it is desirable to use a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile.

[0145] The present application provides a pharmaceutical composition, which includes the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition may further include a buffer system, a preservative, a surface tension agent, a chelating agent, a stabilizer and a surfactant. In some embodiments, the buffer is selected from: sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium phosphate and tris (hydroxymethyl) aminomethane, N-bis (hydroxyethyl) glycine, tricine, malic acid, lactic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or its mixture. Each of these specific buffers or their combination constitutes an alternative of the present application. In some embodiments, the medicine or preparation described in the present application is an aqueous medicine or preparation, for example, they can generally be a solution or suspension. In other specific embodiments of the present application, the medicine or preparation is a lyophilized preparation, to which a solvent and / or diluent is added before use.

[0146] The present application provides a use of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating amylin receptor-related metabolic diseases or fat metabolism disorders.

[0147] The present application provides a use of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain.

[0148] The present application provides a use of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating reduced food intake.

[0149] The present application provides a use of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof in reducing food intake.

[0150] The present application also relates to a method for preventing and / or treating amylin receptor-related diseases or fat metabolism disorder-related diseases, comprising administering to a subject a preventive or therapeutically effective amount of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition.

[0151] The present application provides a method for preventing and / or treating overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain, comprising administering to a subject a preventively or therapeutically effective amount of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition.

[0152] The present application provides a method for reducing food intake, comprising administering an effective amount of the above-mentioned polypeptide or the above-mentioned derivative or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition to a subject.

[0153] In some embodiments of the present application, the method comprises administering the above-mentioned polypeptide or derivative or pharmaceutically acceptable salt or pharmaceutical composition thereof to a subject in need thereof.

[0154] In some embodiments of the present application, the polypeptide or the above-mentioned derivative or pharmaceutically acceptable salt thereof, pharmaceutical composition is used in combination with other drugs, pharmaceutical compounds or compositions.

[0155] abbreviation

[0156] The corresponding names or structural forms of some abbreviations used in the examples of this application are as follows:

[0157] AA:Amino Acid

[0158] Boc:t-Butyloxy carbonyl, tert-Butyloxycarbonyl

[0159] DCM: dichloromethane

[0160] DMF:N,N-Dimethyl formamide

[0161] DIEA:N,N-Diisopropylethylamine

[0162] EDT: 1,2-Ethanedithiol

[0163] Fmoc:9-fluorenylmethyloxycarbonyl,9-fluorenylmethyloxycarbonyl

[0164] OtBu: tert-butyl ester group

[0165] Pbf:2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl chloride

[0166] Pip:Piperidine, piperidine

[0167] TBT: O-(Benzotriazol-l-yl)-N,N,N,N,-tetramethyluronium Tetrafluoroborate, O-Benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0168] tBu:tertiary butyl

[0169] TFA: Trifluoroacetic acid

[0170] TIS: Triisopropylsilane, triisopropylsilane

[0171] Trt:Triphenylmethyl, triphenylmethyl

[0172] αGlu: α-glutamate

[0173] γGlu: γ-glutamate

[0174] αAsp: α-aspartic acid

[0175] βAsp: β-aspartic acid

[0176] αMeAsn: α-methylated asparagine

[0177] NMeAsn: N-methylated asparagine

[0178] NMeAsp: N-methylated aspartate

[0179] Example 1 Preparation of polypeptide derivatives

[0180] The polypeptide derivatives prepared in this application are shown in Table 2.

[0181] Solid phase organic synthesis, Fmoc-protected amino acid strategy, SPPS solid phase synthesis technology were used to complete peptide synthesis, cleavage, oxidation, and purification to obtain the target products in Table 2, which were labeled as Cagrilintide, M1-M55, respectively.

[0182] Taking the compound Cagrilintide as an example, the synthesis process is as follows:

[0183] 1.1 Solid-phase synthesis

[0184] Using Fmoc-Linker MBHA Resin S = 0.32 mmol / g and Fmoc / tBu technology, amino acids were condensed and linked in sequence from the C-terminus to the N-terminus (from right to left) according to the above peptide sequence using the method in Table 1:

[0185] Table 1 Peptide synthesis program list

[0186] The following amino acids were coupled sequentially:

[0187] A-01Fmoc-Pro-OH,A-02Fmoc-Thr(tBu)-OH,A-03Fmoc-Asn(Trt)-OH,A-04Fmoc-Ser(tBu)-OH,A-05Fmoc-Gly-OH ,A-06Fmoc-Val-OH,A-07Fmoc-Asn(Trt)-OH,A-08Fmoc-Thr(tBu)-OH,A-09Fmoc-Pro-OH,A-10Fmoc-Pro-OH,A-1 1Fmoc-Leu-OH,A-12Fmoc-Ile-OH,A-13Fmoc-Pro-OH,A-14Fmoc-Gly-OH,A-15Fmoc-Phe-OH,A-16Fmoc-Asn(Trt)-OH,A-17Fmoc-Asn(Trt)-OH,A-18Fmoc-Ser(tBu )-OH,A-19Fmoc-Ser(tBu)-OH,A-20Fmoc-His(Trt)-OH,A-21Fmoc-Arg(Pbf)-OH,A-22Fmoc-Leu-OH,A-23Fmoc-Phe-OH,A-24Fmoc-Glu(OtBu)-OH,A-25Fmoc-Ala-O H,A-26Fmoc-Leu-OH,A-27Fmoc-Arg(Pbf)-OH,A-28Fmoc-Gln(Trt)-OH,A-29Fmoc-Thr(tBu)-OH,A-30Fmoc-Ala-OH,A-31Fmoc-Cys(Trt)-OH,A-32Fmoc-Thr(tBu) -OH,A-33Fmoc-Ala-OH,A-34Fmoc-Thr(tBu)-OH,A-35Fmoc-Asn(Trt)-OH,A-36Fmoc-Cys(Trt)-OH,A-37Fmoc-Lys(Boc)-OH,A-38Fmoc-Glu-OtBu,A-39C20diacid;

[0188] Finally, the peptide derivative resin is formed.

[0189] The polypeptide derivative resin is washed, transferred out, dried to a constant weight, and then cleaved.

[0190] 1.2 Cracking

[0191] Preparation of the cleavage reagent: Calculate the cleavage reagent dosage based on a ratio of 10 mL ± 2 mL per 1 g of peptide derivative resin: TFA:H₂O:EDT:TIS = 95:1:2:2. Place the required cleavage reagents, H₂O, TFA, EDT, and TIS, in the cleavage reaction flask in this order. Control the cleavage reagent temperature between 0 and 10°C. Add the cleavage reagents to the resin with stirring. Once the system temperature stabilizes, continue stirring at 25-30°C for 2.5 hours. Filter the lysate and precipitate with 5 times the liquid volume of glacial ether. Filter the precipitate and wash it three times with 3 times the liquid volume of glacial ether. Dry under reduced pressure at room temperature to obtain a crude solid.

[0192] 1.3 Oxidation

[0193] Grind the crude product into powder, prepare purified water, slowly add the ground crude product under stirring, and at the same time add acetonitrile aqueous solution dropwise. After the crude product is added and completely dissolved, add iodine methanol solution and stir for half an hour.

[0194] 1.4 Purification and freeze-drying

[0195] The oxidized liquid was filtered through a 0.45 μm microporous membrane. The crude product was purified using a C-18 preparative column using an appropriate gradient at room temperature. The target product was collected, analyzed, tested, and classified. Purity was required to be ≥90%. Unqualified target products were collected and further separated and purified using an appropriate gradient. The qualified liquid peak was collected. The qualified liquid sample was freeze-dried under reduced pressure to obtain a powdered, purified polypeptide derivative lyophilized powder.

[0196] The polypeptide derivatives prepared in this application are shown in Table 2 as M1-M55, and the difference lies in the different amino acid sequences or different fatty acid side chains.

[0197] Table 2 Summary of the design of peptides and their derivatives (modifications are all at the N-terminus)

[0198] M1-M55 represent derivatives obtained by modifying an amino acid sequence with a fatty acid-containing side chain in the present application, such as the derivatives obtained by modifying SEQ ID No.1-SEQ ID No.10 with the corresponding fatty acid-containing side chains in Table 2. For example, M1 represents the derivative M1 obtained by modifying SEQ ID No.1 with (C20diacid+γGlu). The fatty acid-containing side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid; Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx, or Z2 is absent; Z3 is 0-6 AEEAs, for example, 0, 1, 2, 3, 4, 5, or 6; and Z1, Z2, and Z3 are sequentially connected by amide bonds. The C16-C22 fatty diacid portion is located outside the fatty acid-containing side chain used for modification relative to the amino acid sequence, and is at the distal end where the side chain is connected to the amino acid sequence.

[0199] Example 2 Cellular activity detection of derivatives

[0200] The purpose of this experiment is to use luciferase assay to detect the efficacy of the polypeptide derivatives of the present application on human amylin receptor in vitro.

[0201] 2.1 Construction of the Amylin Receptor / CRE-luc Cell Line

[0202] Using standard methods, CHO-K1 / Ga15 / AMY3 cells (calcitonin receptor and receptor-activating modified peptide RAMP have been constructed and purchased from GenScript) were transfected with a plasmid containing multiple copies of a cAMP response element (CRE)-driven luciferase expression cassette and cultured in F12 medium containing 200 μg / mL of Zeocin, 2 μg / mL of puromycin, 100 μg / mL of hygromycin, and 400 μg / mL of G418 to obtain a stably transfected amylin receptor / CRE-luc cell line.

[0203] 2.2 Amylin-luciferase assay

[0204] The lyophilized powder obtained in Example 1 was dissolved in 20 mM phosphate buffer at pH 7.0 and diluted with growth medium (F12 medium containing 10% FBS) to obtain a derivative sample with an initial concentration of 10 nM. The derivative sample was then gradiently diluted with growth medium to obtain seven concentrations of samples with a concentration difference of 5 times from 10 nM to 2 nM-0.4 nM. 50 μL of the sample assay solution of the corresponding concentration was added to each well of a white 96-well plate.

[0205] CHO cells stably transfected with amylin / CRE-luc were resuspended in growth medium at a defined density. 50 μL of the cell suspension was added to a white 96-well plate containing the sample assay solution at a density of approximately 20,000 cells / well. After incubation at 37°C and 5% CO₂ for 24 hours, 100 μL of Luciferase substrate was added to each well and incubated for 3 minutes. Luminescence was measured on a SpectraMax L (Molecular Devices) using SoftMax Pro 7.0.3 GxP software. Fluorescence values ​​were used to plot a standard curve and calculate the EC50. Differences between groups of different peptide derivatives were statistically analyzed by calculating relative activity (ratio of cagrilintide to compound EC50), with the relative activity of cagrilintide being 100%. The results are shown in the table below.

[0206] Table 3 Amylin receptor cell activity results

[0207] Example 3 Thermal Accelerated Stability Test

[0208] 3.1 Materials and Equipment

[0209] Stability test chamber (BINDER GmbH), 1 / 100,000 balance (Mettler-Toledo), pH meter (Mettler-Toledo), biological safety cabinet (ESCO), T2G-Ⅱ small electric capping machine (Changsha Zhongya Pharmaceutical Equipment Co., Ltd.), high-speed refrigerated centrifuge (Eppendorf), sterilization cabinet (Xinhua Medical), Agilent 1260 high-performance liquid chromatograph, Sepax Bio-C18 4.6*250mm 3μm Reversed-phase chromatography column, TSKgel G2000SWXL 7.8*300mm 3μm.

[0210] 3.2 Experimental methods and results

[0211] The derivative was mixed with sodium dihydrogen phosphate (1.42 mg / mL) at a concentration of 1 mg / mL and dissolved in ultrapure water. The pH was adjusted to approximately 7.4 using hydrochloric acid / sodium hydroxide. The solution was then filtered through a 0.22 μm sterile filter in a clean bench and transferred to a sterilized vial. The vial was capped and placed in a 40°C stability chamber. Testing was performed on day 7 (7d) and day 28 (28d). During the experiment, changes in the properties of the polypeptide were observed and recorded. The sample was then centrifuged at 10,000 rpm, 4°C, for 3 min. The supernatant was transferred to a liquid phase injection vial and the concentration and purity of the polypeptide were determined using the following liquid chromatography method.

[0212] Reversed-phase chromatography conditions: flow rate: 1.0 mL / min; autosampler temperature: 15°C; column temperature: 25°C; detection wavelength: 214 nm; mobile phase A: 100% H2O + 0.05% TFA, mobile phase B: 100% ACN; elution gradient: see the table below:

[0213] Table 4 Liquid chromatography elution gradient list

[0214] The results are shown in Table 5 below.

[0215] Table 5 Purity changes in thermally accelerated stability

[0216] Note: Change (7d-0d) = purity of 7d - purity of 0d, change (28d-0d) = purity of 28d - purity of 0d.

[0217] The stability data of M6 is shown in Figure 1. As can be seen from the results in Figure 1, after seven days or even one month of thermal acceleration, the inverted spectrum of M6 remains essentially unchanged, and the spectra before and after heating are essentially overlapping.

[0218] The derivative was mixed with sodium dihydrogen phosphate (1.42 mg / mL) at a concentration of 1 mg / mL and dissolved in ultrapure water. The pH was adjusted to approximately 7.4 using hydrochloric acid / sodium hydroxide. The solution was then filtered through a 0.22 μm sterile filter in a clean bench and transferred to a sterile vial. The vial was capped and placed in a 40°C stability chamber. Testing was performed 10 and 20 days after the experiment. During the experiment, changes in the properties of the peptide were observed and recorded. The sample was then centrifuged at 10,000 rpm, 4°C, for 3 minutes. The supernatant was transferred to a liquid chromatography vial and the peptide concentration and purity were determined using the following liquid chromatography method.

[0219] Reversed-phase chromatography conditions

[0220] Flow rate: 1.0 mL / min; autosampler temperature: 25°C; column temperature: 25°C; detection wavelength: 214 nm; mobile phase A: 110 mM phosphate (pH 5.8): ACN = 90:10; mobile phase B: ACN: IPA: H2O = 85:5:10; elution gradient see Table 6:

[0221] Table 6 Liquid chromatography elution gradient list

[0222] The experimental results are shown in Table 7.

[0223] Table 7

[0224] The results of the thermal stability experiment showed that compared with the control Cagriliintide molecule group, the polypeptide molecules described in this application all exhibited significantly better stability.

[0225] Example 4 Animal Experiment

[0226] SPF male SD rats (7-8 weeks, 200-250g) were used for this experimental study after a quarantine period. The experimental animals were kept at room temperature of 20℃~23℃ and relative humidity of 40%~50%; during the animal quarantine period and the experimental process, the feed was Co60 mouse breeding feed 1035, and the drinking water was purified water supplied by drinking bottles, with free water intake. The experimental animals were averaged according to the weight principle. Each derivative test group had 5 rats, and each group of rats was subcutaneously administered with a 30nmol / kg dose of the derivative or solvent (PBS pH7.4). The administration time of each group was recorded. After administration, the rats were returned to their home cages, where they could then access food and water. Food consumption and rat weight changes were recorded every 24 hours by online recording or manual recording. The results of the animal experiments are shown in Figures 2A and 2B.

[0227] The specific experimental results are shown in Figures 2A and 2B. Compared with Cagrilintide, the activity of the candidate molecules of the present application is significantly higher than that of Cagrilintide, among which M6 is the most active molecule, which is significantly higher than Cagrilintide.

[0228] Example 5: Pharmacological efficacy experiment in SD rats - dose-dependence study

[0229] SPF male Sprague-Dawley rats (7-8 weeks, 220-230 g) were used in this study after a quarantine period. Animals were housed at room temperature (20°C-23°C) and relative humidity (40%-50%). During quarantine and the experiment, they were fed Co60 Rat Breeding Diet 1035 and provided with purified water via drinking bottles with free access to water. Animals were divided into 10 groups (designated A1-A10) based on average body weight, with n=5 animals, including vehicle, cagrilintide (high, medium, and low) dose groups, M41 (high, medium, and low) dose groups, and M43 (high, medium, and low) dose groups. The compounds and doses for each treatment group are shown in Table 8. A single subcutaneous injection was used for 6 days, with the day of administration designated as Day 1 / Time 0 (T0). The initial weight of the animals and the initial feed were recorded on Day 0. The weight of the experimental animals and the remaining food were recorded every day from Day 1 to Day 5. The weight change rate and food intake were calculated. Any abnormalities were also recorded and reported. The rats were in good condition during the experiment and no abnormalities were observed. Weight change = (BWTn-BWT0) / BWT0*100%, where BW represents the body weight value, T0 and Tn represents the time of dosing and n hours after dosing, respectively. Each point in the figure is expressed as the mean ± standard error of the mean (SEM). Cumulative food intake is the total amount of food consumed by each animal up to a certain time point after dosing. The dose-effect relationship of the molecule is shown in Figures 3A-3D.

[0230] Table 8 Active ingredients and dosage of test products in dose-dependence study

[0231] As can be seen from the results of Figures 3A-3D, both M41 and M43 reduced the body weight and food intake of animals in a dose-dependent manner; compared with Cagrilintide, M41 and M43 were more effective in reducing the body weight and food intake of animals at the same dose.

[0232] Example 6: DIO SD rats efficacy experiment - dose dependence study

[0233] SPF male DIO Sprague-Dawley rats (36-37 weeks, 680-110 g, fed a high-fat diet starting at 3 weeks of age for 34 weeks) were used in this study. Animals were housed at room temperature (20°C–23°C) and at a relative humidity (40%–50%). During DIO model establishment and the experimental period, animals were fed a high-fat diet (D12492) and provided with purified water via drinking bottles with free access to water. The animals were divided equally into six groups (n=5) based on body weight, designated B1–B6. The active ingredient and dosage of each group are shown in Table 9. Subcutaneous injections were administered once daily for 10 consecutive days. The first day of administration was designated Day 1 (D1). Initial body weight and initial feed addition were recorded on Day 0. Daily weight and remaining food were recorded from Day 1 to Day 11. Weight change and food intake were calculated, and any abnormalities were recorded and reported. The rats were in good condition throughout the experiment, and no abnormalities were observed. Body weight change = (BWDn - BWD1) / BWD1 * 100%, where BW represents body weight, D1 and Dn represent the first and Nth day of dosing, respectively. Graphs represent the mean ± standard error of the mean (SEM). Cumulative food intake is the total amount of food consumed by each group of animals up to a specific time point after dosing. The weight loss and food suppression effects in DIO model rats are shown in Figures 4A and 4B.

[0234] Table 9: DIO SD rats efficacy experiment - dose-dependence study of the active ingredients and dosage of the test products

[0235] As can be seen from the results in Figures 4A and 4B, both M41 and M43 reduced the body weight and food intake of DIO model rats in a dose-dependent manner; compared with Cagrilintide, M41 and M43 were more effective in reducing the body weight and food intake of animals at the same dose.

[0236] Example 7: Drug efficacy experiment in SD rats - single dose screening

[0237] SPF male Sprague-Dawley rats (7-8 weeks, 220-230 g) were used for this study after a quarantine period. Animals were housed at room temperature (20°C-23°C) and relative humidity (40%-50%). During quarantine and the experiment, they were fed Co60 Rat Breeding Diet 1035 and provided with purified water via drinking bottles with free access to water. A single-dose screening study was conducted in two separate trials. Animals were grouped according to mean body weight (n=5) and designated C1-C6 and D1-D9, respectively. The compounds and doses for each treatment group are shown in Table 10. A single subcutaneous injection was used for a total of six days. The day of administration was designated Day 1 / Time 0 (T0). Daily weight and food remaining were recorded from Day 1 to Day 5, and the relative rate of weight change and food intake were calculated. Any abnormalities were recorded and reported. The rats were in good condition throughout the experiment, and no abnormalities were observed. Body weight change = (BWTn-BWT0) / BWT0*100%, BW represents body weight, T0 and T n The values ​​represent the time of administration and the number of hours after administration, respectively. Each point in the figure is expressed as the mean ± standard error of the mean (SEM). Comparison of the efficacy of different molecules is shown in Figures 5A-5D.

[0238] Table 10 Active ingredients and dosages of test products in single-dose screening experiment

[0239] From the results in Figures 5A-5D , it can be seen that compared with Cagrilintide, the efficacy of the polypeptide and its derivatives of the present application is comparable to or improved upon that of Cagrilintide.

[0240] Example 8 Pharmacokinetic Study in SD Rats

[0241] After passing the inspection and quarantine, SPF-grade experimental animals (male, 6-8 weeks, 200-240g) were divided into three groups (n=3) according to their body weight, namely M41 group, M43 group and Cagrilintide group. The experimental animals were subcutaneously injected with the corresponding test sample (1 mg / kg, 5 ml / kg), and the day of administration was recorded as D1. On D1, whole blood was collected from the animals before administration (-10 minutes) and on D1-2 hours, D1-4 hours, D1-6 hours, D2-24 hours, D3-48 hours, D4-72 hours, D5-96 hours, and D6-120 hours after administration, and plasma (EDTA anticoagulation) was prepared. The concentration of drug molecules in plasma was detected by liquid chromatography-mass spectrometry (LC-MS / MS), the drug concentration-time curve was plotted using GraphPad Prism 10, and the pharmacokinetic parameters (Cmax, Tmax, T1 / 2, AUC, MRT) of the drug were calculated using PKSolver. The changes in the concentrations of different molecules in plasma over time are shown in Figure 6. The experimental results are analyzed and listed in Table 11.

[0242] Table 11 Summary of comparison results of different molecules

[0243] As can be seen from the data in Figure 6 and Table 11, compared with Cagrilintide, peptide molecules M41 and M43 showed significantly higher biological exposure.

[0244] Although the present invention has been disclosed above with reference to the embodiments, they are not intended to limit the present invention. Anyone with ordinary knowledge in the art may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the patent application attached hereto.

Claims

1. A polypeptide derivative or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the following amino acid sequence: ASX3LS TAX8X9X 10 RLADF LRHX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILPPT NVGSN TX 37 -NH2 in: X3 is E or Q; X8 is A or V; X9 is L or T; X 10 is G or Q; X 19 is S or F; X 20 is S or T; X 21 is N, Q, E, S, T, A, G, H, K, R or D; X 22 is N, Q, E, S, NMeAsn, αMeAsn, NMeAsp or R; X 23 is L or D; X 24 is K or R; X 25 is P or D; X 37 is P or trans-Hyp; The derivative comprises a fatty acid-containing side chain attached to the N-terminus of the polypeptide.

2. The derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein: X3 is Q; Preferably, X8X9X 10 Select any of ALG, VLG, VTQ, ATQ, VLQ; X8X9X 10 Further preferably, ATQ; More preferably, X 19 For F, X 20 T, X 23 D, X 24 R, X 25 D, X 37 is P; further preferably, X 21 D or / and X 22 is R; or X 19 For S, X 20 For S, X 23 For L, X 24 K, X 25 P, X 37 is trans-Hyp, further preferably, X 21 N, Q, E, S, T, A, G, H, K, R or / and X 22 For N, Q, E, S, NMeAsn, αMeAsn, NMeAsp.

3. The derivative according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein: The polypeptide sequence comprises any one of SEQ ID No. 1 to SEQ ID No.

25.

4. The derivative according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, The side chain containing fatty acids is in the form of Z1+Z2+Z3, in, Z1 is a C16-C22 fatty diacid; Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 is absent; Z3 is 0-6 AEEAs, for example, 0, 1, 2, 3, 4, 5, 6; The Z1, Z2 and Z3 are sequentially connected via amide bonds. 5 . The derivative or pharmaceutically acceptable salt thereof according to claim 4 , wherein the polypeptide derivative is selected from M1 to M55.

6. A polypeptide, wherein The polypeptide comprises the polypeptide sequence involved in any one of claims 1-5.

7. A pharmaceutical composition comprising the derivative according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof or the polypeptide according to claim 6, and a pharmaceutically acceptable excipient.

8. Use of the derivative according to any one of claims 1 to 5 and its pharmaceutically acceptable salt or the polypeptide according to claim 6 in the preparation of a medicament for preventing and / or treating overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain.

9. Use of the derivative according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof or the polypeptide according to claim 6 in reducing food intake.

10. A method for preventing and / or treating overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain, comprising administering to a subject a preventively or therapeutically effective amount of the derivative according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof, or the polypeptide according to claim 6, or the pharmaceutical composition according to claim 7.

11. A method for reducing food intake, comprising administering to a subject a preventively or therapeutically effective amount of the derivative according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof, or the polypeptide according to claim 6, or the pharmaceutical composition according to claim 7.

12. A method for preventing and / or treating amylin receptor-related diseases or dyslipidemia, comprising administering to a subject a preventively or therapeutically effective amount of the derivative according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof, or the polypeptide according to claim 6, or the pharmaceutical composition according to claim 7.