Polypeptides and derivatives thereof, compositions containing same, and uses thereof

Abstract

The present application provides a polypeptide and a polypeptide derivative or a pharmaceutically acceptable salt thereof. The present application also provides a pharmaceutical composition comprising the polypeptide described herein or a derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. The use of the above polypeptide, polypeptide derivative or a pharmaceutically acceptable salt thereof, or composition in the field of disease prevention / treatment is also provided.

Description

[Technical Field]

[0001] The present application relates to the technical field of polypeptides, and in particular to polypeptides and derivatives thereof, compositions containing same, and uses thereof. [Background technology]

[0002] Amylin, also known as islet amyloid polypeptide (IAPP, amylin, or hAMY1-37), is a 37-amino acid polypeptide hormone that functions as a member of the calcitonin protein family, which also includes calcitonin (CT), calcitonin gene-related peptide (CGRP), and human adrenomedullin (hAM), as well as their precursors. Amylin is secreted simultaneously with insulin by pancreatic islet β cells and is deficient in diabetic patients. Amylin acts in several different organ systems, primarily via amylin receptors 1-3 (AMYR1-3). Amylin inhibits glucagon secretion, delays gastric emptying, transmits satiety signals, and suppresses appetite. Clinical trials have shown that amylin receptor agonists can be used to treat overweight, obesity, type 1 diabetes, and / or type 2 diabetes. However, amylin has several drawbacks, including a high tendency to fibrillate, a short in vivo half-life, and chemical instability at pH 7. Therefore, natural amylin is not suitable for use as an active pharmaceutical ingredient.

[0003] Numerous amylin analogs or derivatives are known in the prior art in an attempt to address some of the known shortcomings of human amylin. A successful example is an amylin analog named pramlintide (trade name Symlin), approved by the FDA for use in type 1 and type 2 diabetes. However, pramlintide has a half-life of less than one hour and is administered with meals, requiring patients to take the drug multiple times per day for treatment. Furthermore, pramlintide is formulated at pH 4 because it fibrillates and precipitates at pH 7, rendering it ineffective. Therefore, there remains a need for novel amylin analogs or derivatives that exhibit increased chemical stability, increased metabolic stability, and / or reduced tendency to fibrillate. In particular, there is a need for amylin analogs or derivatives that are stable over a wider pH range. Additionally, there is a need for novel amylin analogs or derivatives that have increased potency, improved therapeutic efficacy, and / or increased half-life to reduce dosing frequency and improve patient compliance, and thus improve efficacy. Currently available pramlintide has a half-life of less than one hour, requires multiple daily doses for treatment, and cannot be formulated with other drugs into a single liquid formulation under neutral conditions. Summary of the Invention

[0004] The technical problem that the present application aims to address is to find a solution to the shortcomings as mentioned in the "Background Art" by providing polypeptides and derivatives thereof, compositions containing same, and uses thereof.

[0005] In this application, ASX3LS TAX8X9X 10 RLADF LRHX 19 X 20 X 21 X 22 X 23 X 24 X 25 ILPPT NVGSN TX 37A derivative of a polypeptide comprising an amino acid sequence of -NH2 or a pharmaceutically acceptable salt thereof is provided, where X3 is E or Q, X8 is A or V, X9 is L or T, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N, Q, E, S, T, A, G, H, K, R, or D, and X 22 is N, Q, E, S, NMeAsn, αMeAsn, NMeAsp, or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp, and the derivative comprises a fatty acid-containing side chain attached to the N-terminus of the polypeptide.

[0006] In some embodiments of the present application, X3 is E or Q, X8 is A or V, X9 is L or T, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0007] In some embodiments of the present application, X3 is E, X8 is A or V, X9 is L or T, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

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

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

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

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

[0013] 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, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37is P or trans-Hyp.

[0014] 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, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0015] 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, and X 10 is G or Q, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

[0017] 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, and X 10 is G or Q, and X 19 is S or F, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0018] 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, and X 10 is G or Q, and X 19 is S or F, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0019] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0020] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0021] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0022] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0023] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0024] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0025] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0026] In some embodiments of the present application, X21 is G, X3 is E or Q, X8 is A or V, X9 is L or T, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0027] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0028] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

[0030] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0031] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

[0033] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0034] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

[0036] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 23 is L or D, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

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

[0039] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 24 is K or R, and X 25 is P or D, and X 37 is P or trans-Hyp.

[0040] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 25 is P or D, and X 37 is P or trans-Hyp.

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

[0042] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 37 is P or trans-Hyp.

[0043] 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, and X 10 is G or Q, and X 19 is S or F, and X 20 is S or T, and X 21 is N or D, and X 22 is N or R, and X 23 is L or D, and X 24 is K or R, and X 37 is P or trans-Hyp.

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

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

[0046] In some embodiments of the present application, X3 is Q, preferably X8X9X 10 is selected from any of ALG, VLG, VTQ, ATQ, and VLQ, and more preferably X8X9X 10 is ATQ, and more preferably, X 19 is F and X 20 is T and X 23 is D and X 24 is R and X 25 is D and X 37 is P, and more preferably, X 21 is D and / or X 22 is R.

[0047] In some embodiments of the present application, X3 is Q, preferably X8X9X 10 is selected from any of ALG, VLG, VTQ, ATQ, and VLQ, and more preferably X8X9X 10 is ATQ, and more preferably, X 19 is S and X 20 is S and X 23 is L and X 24 is K and X 25 is P and X 37 is trans-Hyp, and more preferably, X 21 is N, Q, E, S, T, A, G, H, K, or R, and / or X 22 is N, Q, E, S, NMeAsn, αMeAsn, or NMeAsp.

[0048] In some embodiments of the present application, the sequence of the polypeptide comprises any one of the sequences of SEQ ID NO:1 to SEQ ID NO:25.

[0049] In some embodiments of the present application, the sequence of the polypeptide is ASELS TAALG RLADF LRHSS NNLKP ILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID NO: 1).

[0050] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAVLG RLADF LRHSS NNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 2).

[0051] In some embodiments of the present application, the sequence of the polypeptide is ASELS TAALG RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID NO: 3).

[0052] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAVLG RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID NO: 4).

[0053] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID NO: 5).

[0054] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID NO: 6).

[0055] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAVTQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID NO: 7).

[0056] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAVTQ RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID NO: 8).

[0057] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAVLQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 (SEQ ID NO: 9).

[0058] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAVLQ RLADF LRHFT DRDRD ILPPT NVGSN TP-NH2 (SEQ ID NO: 10).

[0059] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS QQLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 11).

[0060] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS EELKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 12).

[0061] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS SSLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 13).

[0062] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS N-NMeAsn-LKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 14).

[0063] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS N-NMeAsp-LKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 15).

[0064] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS QNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 16).

[0065] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS ENLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 17).

[0066] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS SNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 18).

[0067] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS TNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 19).

[0068] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS ANLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 20).

[0069] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS GNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 21).

[0070] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS HNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 22).

[0071] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS KNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 23).

[0072] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS RNLKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 24).

[0073] In some embodiments of the present application, the sequence of the polypeptide is ASQLS TAATQ RLADF LRHSS N-αMeAsn-LKP ILPPT NVGSN T-trans-Hyp-NH2 (SEQ ID NO: 25).

[0074] In some embodiments of the present application, the side chain comprising a fatty acid is of the form Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty diacid, Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent, and Z3 is 0-6 AEEA, e.g., 0, 1, 2, 3, 4, 5, or 6 AEEA, and Z1, Z2, and Z3 are linked via an amide bond.

[0075] In some embodiments of the present application, Z1 is a C22 fatty diacid.

[0076] In some embodiments of the present application, Z1 is a C20 fatty diacid.

[0077] In some embodiments of the present application, Z1 is a C19 fatty diacid.

[0078] In some embodiments of the present application, Z1 is a C18 fatty diacid.

[0079] In some embodiments of the present application, Z1 is a C17 fatty diacid.

[0080] In some embodiments of the present application, Z1 is a C16 fatty diacid.

[0081] In some embodiments of the present application, Z2 is γGlu.

[0082] In some embodiments of the present application, Z2 is αGlu.

[0083] In some embodiments of the present application, Z2 is βAsp.

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

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

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

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

[0088] In some embodiments of the present application, Z3 is absent.

[0089] In some embodiments of the present application, Z3 is one AEEA.

[0090] In some embodiments of the present application, Z3 is two AEEA.

[0091] In some embodiments of the present application, Z3 is 3 AEEA.

[0092] In some embodiments of the present application, Z3 is 4 AEEA.

[0093] In some embodiments of the present application, Z3 is 5 AEEA.

[0094] In some embodiments of the present application, Z3 is 6 AEEA.

[0095] In some embodiments of the present application, the derivative of the polypeptide 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 M27, 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.

[0096] The present application provides a polypeptide comprising the above polypeptide sequence.

[0097] In some embodiments of the present application, the polypeptide sequence comprised in the polypeptide is any one of SEQ ID NO: 1 to SEQ ID NO: 25.

[0098] The present application provides a pharmaceutical composition comprising the above polypeptide, or the above derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0099] 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 control agents, anti-obesity agents, lipid metabolism control agents, blood glucose control agents, antihypertensive agents, cardiovascular control agents, and brain-based disease control agents, psychiatric disease control agents, or nervous system control agents. Preferably, the pharmacologically active substance is selected from GLP-1 receptor agonists, including but not limited to GLP-1, GLP-1 analogs, and GLP-1 derivatives; GIP receptor agonists, including but not limited to GIP, GIP analogs, and GIP derivatives; and insulin receptor agonists, including but not limited to insulin, insulin analogs, and insulin derivatives.

[0100] The above polypeptide, the above derivative or a pharmaceutically acceptable salt thereof, or the composition is for use in the manufacture of a medicament for preventing and / or treating an amylin receptor-associated metabolic disease or lipid metabolism disorder.

[0101] The above polypeptide, the above derivative or a pharmaceutically acceptable salt thereof, or the composition is for use in the manufacture of a medicament for preventing and / or treating overweight, and / or obesity, and / or type I diabetes or type II diabetes, and / or osteoporosis, and / or neuropathic pain.

[0102] The polypeptide, the derivative or a pharmaceutically acceptable salt thereof, or the composition is for use in the manufacture of a medicament for reducing food intake. The polypeptide, the derivative or a pharmaceutically acceptable salt thereof, or the composition is for use in reducing food intake.

[0103] The present application provides a method for preventing and / or treating an amylin receptor-associated metabolic disease or lipid metabolism disorder, the method comprising administering to a subject a prophylactically or therapeutically effective amount of the above-mentioned polypeptide, the above-mentioned derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0104] The present application provides a method for preventing and / or treating overweight, and / or obesity, and / or type I diabetes or type II diabetes, and / or osteoporosis, and / or neuropathic pain, the method comprising administering to a subject a prophylactically or therapeutically effective amount of the above-mentioned polypeptide, the above-mentioned derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0105] The present application provides a method for reducing food intake, the method comprising administering to a subject an effective amount of the above-mentioned polypeptide, the above-mentioned derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0106] The polypeptides and their derivatives or compositions of the present application have significantly improved advantages or positive effects in terms of in vivo pharmaceutical activity, molecular stability, and bioavailability compared to equivalent dosages of the prior art polypeptides and their derivatives or compositions. Furthermore, in contrast to conventional amylin analogs or derivatives, the polypeptides and their derivatives described in the present application, although not containing disulfide bonds, surprisingly and unexpectedly exhibit high stability, show reduced tendency to fibrillate, and still have relatively high stability when formulated at neutral or non-acidic pH. Furthermore, because the polypeptides or their derivatives of the present application have desirable pharmacokinetic effects, they do not need to be injected as frequently as known amylin derivatives. [Brief explanation of the drawings]

[0107] [Figure 1] 1 shows the results of a thermal acceleration assay of polypeptide derivative M6 in Example 3. [Figure 2A] 1 shows the effect of a polypeptide derivative on weight loss over time in an SD rat model in Example 4. [Figure 2B] 1 shows the effect of a polypeptide derivative on food intake over time in an SD rat model in Example 4. [Figure 3A] 1 shows the effect of various doses of polypeptide derivatives on weight loss over time in an SD rat model in Example 5. [Figure 3B] 1 shows the effect of various doses of polypeptide derivatives on food intake over time in an SD rat model in Example 5. [Figure 3C] 1 shows the effect of various doses of polypeptide derivatives on weight loss over time in an SD rat model in Example 5. [Figure 3D] 1 shows the effect of various doses of polypeptide derivatives on food intake over time in an SD rat model in Example 5. [Figure 4A] 1 shows the effect of various doses of polypeptide derivatives over time on weight loss in a DIO SD rat model in Example 6. [Figure 4B] 1 shows the effect of various doses of polypeptide derivatives over time on food intake in a DIO SD rat model in Example 6. [Figure 5A] 1 shows the effect of a polypeptide derivative on weight loss over time in an SD rat model in Example 7. [Figure 5B] 1 shows the effect of a polypeptide derivative on weight loss over time in an SD rat model in Example 7. [Figure 5C] 1 shows the effect of a polypeptide derivative on food intake over time in an SD rat model in Example 7. [Figure 5D] 1 shows the effect of a polypeptide derivative on food intake over time in an SD rat model in Example 7. [Figure 6] 1 is a plot showing the concentration of various molecules in plasma over time in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0108] The present invention will now be further described with reference to the following examples, which are intended to further illustrate and explain the present application and are not intended to limit the present application.

[0109] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The following describes methods and materials, but methods and materials similar to or identical to those described herein may be used in assays or practical applications. In the event of any discrepancy, the definitions contained herein shall prevail. Furthermore, materials, methods, and examples are merely illustrative and not limiting. The following provides a further explanation of the present application in conjunction with specific examples, but this is not intended to limit the scope of the present application.

[0110] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, which may be linear or branched, and which may comprise naturally occurring amino acids, interspersed with non-naturally occurring amino acids.

[0111] As used herein, the term "derivative" refers to a product produced by modifying a functional group (e.g., an amino acid residue) in a biological polymer (e.g., a polypeptide or protein) with a compound or molecule. Modifications include, but are not limited to, acylation, amidation, esterification, and thioesterification.

[0112] The pharmaceutical composition herein may be used for the treatment of disease or in vitro cell culture assay. When used for the treatment of disease, the term "pharmaceutical composition" generally refers to a unit dosage form and may be prepared by any method well known in the pharmaceutical field. The method used includes mixing an active ingredient with one or more auxiliary ingredients, i.e., excipients. Usually, the composition is prepared by uniformly and appropriately mixing the active compound with a liquid excipient, a finely divided solid excipient, or both.

[0113] As used herein, "pharmaceutically acceptable" refers to the property of a substance or composition that must be chemically and / or toxicologically compatible with other ingredients contained in the formulation and / or the mammal being treated therewith.

[0114] The term "pharmaceutically acceptable excipient" herein may include any solvent, solid excipient, diluent, or other liquid excipient, etc., that is suitable for a particular target dosage form. The use of any conventional excipients is also within the scope contemplated in this application, except to the extent that they are incompatible with the compounds of the present application, e.g., produce any undesired biological effect, or interact in a deleterious manner with any other component of the pharmaceutically acceptable composition.

[0115] As used herein, treatment refers to the achievement of a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects that may result from the disease are completely or partially cured. As used herein, "treatment" encompasses mammalian, particularly human, disease, and includes (a) preventing the occurrence of a disease or condition in an individual who is susceptible to but has not been diagnosed as having the disease; (b) inhibiting the disease, e.g., preventing the progression of the disease; or (c) alleviating the disease, e.g., reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit the individual's disease, including, but not limited to, administering a drug, including a compound described herein, to an individual in need thereof.

[0116] As used herein, "prevention" refers to reducing the likelihood of occurrence (or recurrence) of a disease, illness, or disorder, or related condition (eg, cancer).

[0117] As used herein, "amylin receptor-associated metabolic diseases" refer to diseases whose symptoms can be improved in a subject by regulating the activation of amylin receptors to improve the status of downstream cellular pathways, including hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, diabetes, and diseases that usually involve lipid metabolism disorders, such as osteoporosis, cognitive impairment, neurodegenerative diseases (e.g., Parkinson's syndrome and Alzheimer's disease), and gastrointestinal diseases, e.g., inflammatory bowel disease, dystrophies, or gastrointestinal ulcers.

[0118] In some embodiments of the present application, the polypeptide is linked to the fatty acid by an amide bond in the polypeptide derivative.

[0119] In some embodiments of the present application, the fatty acid is a C1-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.

[0120] The C1 to C25 fatty acids in the present application may include straight-chain fatty acids and branched fatty acids.

[0121] The C1 to C25 fatty acids in the present application may include saturated fatty acids and unsaturated fatty acids.

[0122] In some embodiments of the present application, the fatty acid is selected from the group consisting of octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, isocitric acid, eicosanoic acid, and the like. and one or more fatty acids selected from the group consisting of sapentaenoic acid, 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, nonadecanedioic acid, eicosanediocic acid, heneicosanediocic acid, docosanediocic acid, and tricosanedioic acid.

[0123] The fatty acid moiety of the present application may be attached to the above polypeptide via a linker selected from one or more of -γGlu-, -αGlu-, -βAsp-, -αAsp-, -Inp-, -Trx-.

[0124] [ka] Here, 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.

[0125] In this application, the term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the polypeptide derivatives of this application and that are generally not biologically or otherwise undesirable. In many cases, the polypeptide derivatives of this application are capable of forming acid and / or base addition salts by virtue of the presence of amino and / or carboxyl groups or similar groups.

[0126] Inorganic and organic acids can be used to form pharmaceutically acceptable acid addition salts, such as acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorotheophylline, citrate, ethanedisulfonate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactate. These include bionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, toluenesulfonate, and trifluoroacetate.

[0127] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.

[0128] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, hydroxyacetic 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, and sulfosalicylic acid.

[0129] Pharmaceutically acceptable addition salts can be formed with inorganic and organic bases.

[0130] 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, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly suitable salts including ammonium, potassium, sodium, calcium, and magnesium salts.

[0131] Organic bases from which salts can be derived can include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Certain organic bases include isopropylamine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0132] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds or basic or acidic moieties by conventional chemical methods.Generally, these salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (for example, hydroxide, carbonate, bicarbonate, etc. of Na or K), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid.This reaction is usually carried out in water, an organic solvent, or a mixture of the two.Generally, if possible, it is desirable to use non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile.

[0133] The present application provides a pharmaceutical composition comprising the above-described polypeptide, or a derivative or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In preferred embodiments, the pharmaceutical composition may further comprise a buffer system, a preservative, a surfactant, 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, disodium hydrogen phosphate, sodium phosphate, tri(hydroxymethyl)-aminomethane, N-di(hydroxyethyl)glycine, tricine, malic acid, lactic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or a mixture thereof. Each of these specific buffers, or combinations thereof, constitutes an alternative of the present application. In some embodiments, the agent or formulation described in the present application is, for example, a water-containing agent or formulation that may normally be a solution or suspension. In some other specific embodiments of the present application, the agent or formulation is a lyophilized formulation to which a solvent and / or diluent is added prior to use.

[0134] The present application provides use of the above-mentioned polypeptide, or the above-mentioned derivative or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for preventing and / or treating an amylin receptor-associated metabolic disease or lipid metabolism disorder.

[0135] The present application provides the use of the above-mentioned polypeptide, or the above-mentioned derivative or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for preventing and / or treating overweight, and / or obesity, and / or type I diabetes or type II diabetes, and / or osteoporosis, and / or neuropathic pain.

[0136] The present application provides the use of the above polypeptide, or the above derivative or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing food intake.

[0137] The present application provides the use of the above polypeptide, or the above derivative or a pharmaceutically acceptable salt thereof, in reducing food intake.

[0138] The present application also relates to a method for preventing and / or treating an amylin receptor-related disease or a lipid metabolism disorder-related disease, the method comprising administering to a subject a prophylactically or therapeutically effective amount of the above-mentioned polypeptide, the above-mentioned derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0139] The present application provides a method for preventing and / or treating overweight, and / or obesity, and / or type I diabetes or type II diabetes, and / or osteoporosis, and / or neuropathic pain, the method comprising administering to a subject a prophylactically or therapeutically effective amount of the above-mentioned polypeptide, the above-mentioned derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0140] The present application provides a method for reducing food intake, the method comprising administering to a subject an effective amount of the above-mentioned polypeptide, the above-mentioned derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0141] Some embodiments of the present application include administering to a subject in need thereof the above-described polypeptide, the above-described derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

[0142] In some embodiments of the present application, the described polypeptides, the derivatives or pharmaceutically acceptable salts thereof, or pharmaceutical compositions are used in combination with other drugs, pharmaceutical compounds, or compositions.

[0143] Abbreviation The names or structures corresponding to some of the abbreviations used in the examples of this application are as follows:

[0144] [Table 1] AA: amino acid Boc: t-butyloxycarbonyl DCM: dichloromethane DMF: N,N-dimethylformamide DIEA: N,N-diisopropylethylamine EDT: 1,2-ethanedithiol Fmoc: 9-fluorenylmethyloxycarbonyl OtBu: t-butyl ester Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Pip: piperidine TBT: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate tBu: tertiary butyl TFA: Trifluoroacetic acid TIS: Triisopropylsilane Trt: triphenylmethyl αGlu: α-glutamic acid γGlu: γ-glutamic acid αAsp: α-aspartic acid βAsp: β-aspartic acid αMeAsn: α-methylated asparagine NMeAsn: N-methyl asparagine NMeAsp: N-methylated aspartic acid [Example]

[0145] Example 1 Preparation of Polypeptide Derivatives

[0146] The polypeptides and their derivatives prepared in this application are shown in Table 2.

[0147] Polypeptides were synthesized by solid-phase organic synthesis, specifically solid-phase peptide synthesis (SPPS) using Fmoc-protected amino acids, and then cleaved, oxidized, and purified to obtain the target compounds listed in Table 2, which are Cagrilintide and M1 to M55, respectively.

[0148] Taking the compound caglilintide as an example, the synthesis process was as follows:

[0149] 1.1 Solid phase synthesis

[0150] Using Fmoc-Linker MBHA resin S = 0.32 mmol / g, amino acids were sequentially condensed from the C-terminus to the N-terminus (right to left) according to the peptide sequence using Fmoc / tBu treatment according to the method shown in Table 1.

[0151] [Table 2]

[0152] The following amino acids were coupled in order:

[0153] A-01 Fmoc-Pro-OH, A-02 Fmoc-Thr(tBu)-OH, A-03 Fmoc-Asn(Trt)-OH, A-04 Fmoc-Ser(tBu)-OH, A-05 Fmoc-Gly-OH, A-06 Fmoc-Val-OH, A-07 Fmoc-Asn(Trt)-OH, A-08 Fmoc-Thr(tBu)-OH, A-09 Fmoc-Pro-OH, A-10 Fmoc-Pro-OH, A-11 Fmoc-Leu-OH, A-12 Fmoc-Ile-OH, A-13 Fmoc-Pro-OH, A-14 Fmoc-Gly-OH, A-15 Fmoc-Phe-OH, A-16 Fmoc-Asn(Trt)-OH, A-17 Fmoc-Asn(Trt)-OH, A-18 Fmoc-Ser(tBu)-OH, A-19 Fmoc-Ser(tBu)-OH, A-20 Fmoc-His(Trt)-OH, A-21 Fmoc-Arg(Pbf)-OH, A-22 Fmoc-Leu-OH, A-23 Fmoc-Phe-OH, A-24 Fmoc-Glu(OtBu)-OH, A-25 Fmoc-Ala-OH, A-26 Fmoc-Leu-OH, A-27 Fmoc-Arg(Pbf)-OH, A-28 Fmoc-Gln(Trt)-OH, A-29 Fmoc-Thr(tBu)-OH, A-30 Fmoc-Ala-OH, A-31 Fmoc-Cys(Trt)-OH, A-32 Fmoc-Thr(tBu)-OH, A-33 Fmoc-Ala-OH, A-34 Fmoc-Thr(tBu)-OH, A-35 Fmoc-Asn(Trt)-OH, A-36 Fmoc-Cys(Trt)-OH, A-37 Fmoc-Lys(Boc)-OH, A-38 Fmoc-Glu-OtBu, and A-39 C20 diacid.

[0154] Finally, the polypeptide derivative was formed on the resin.

[0155] The polypeptide derivative on the resin was washed, excised, and dried to constant weight for cleavage.

[0156] 1.2 Cutting

[0157] Cleavage reagent formulation: The cleavage reagent was TFA:HO:EDT:TIS = 95:1:2:2, with a volume of 10 mL ± 2 mL per gram of polypeptide derivative on the resin. The necessary cleavage reagent components, HO, TFA, EDT, and TIS, were added sequentially to a cleavage reaction flask, and the temperature was controlled at 0-10°C. The cleavage reagent was added to the resin under stirring. After the system temperature stabilized, the reaction mixture was stirred for an additional 2.5 hours at a controlled temperature of 25-30°C. The cleavage solution was filtered and precipitated using 5 times the volume of the liquid, using ice-diethyl ether. The precipitate was filtered, washed three times with 3 times the volume of the liquid, and then dried under reduced pressure at room temperature to obtain a solid crude product.

[0158] 1.3 Oxidation

[0159] The crude product was pulverized and slowly added to purified water with stirring while adding dropwise an acetonitrile solution. After the crude product was added and completely dissolved, a methanol solution of iodine was added and stirred for 30 minutes.

[0160] 1.4 Purification and lyophilization

[0161] The oxidized liquid was filtered through a 0.45 μm microporous filter film. The crude product was separated and purified using a column prepared with C-18 column packing at ambient temperature with an appropriate gradient. The target substance was then recovered, detected, analyzed, and selected. Purity of 90% or more was required. Target substances that did not meet the standard were recovered, separated with an appropriate gradient, and purified again to achieve a suitable liquid peak. The suitable liquid sample was freeze-dried under reduced pressure to obtain a freeze-dried powder of the purified polypeptide derivative.

[0162] The polypeptide derivatives prepared in this application are designated M1 to M55 in Table 2 and are distinguished from one another by their amino acid sequences or fatty acid side chains.

[0163] [Table 3-1]

[0164] [Table 3-2]

[0165] [Table 3-3]

[0166] M1 to M55 represent derivatives resulting from modifying an amino acid sequence with a fatty acid-containing side chain in the present application, e.g., those derived from SEQ ID NOs: 1 to 10 after modification with the corresponding fatty acid-containing side chain in Table 2. For example, M1 represents a derivative M1 resulting from modifying SEQ ID NO: 1 with a C20 diacid plus γGlu. The fatty acid-containing side chain is in the form Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty diacid, Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent, and Z3 is 0 to 6, e.g., 0, 1, 2, 3, 4, 5, or 6 AEEAs, and Z1, Z2, and Z3 are linked via an amide bond. The C16-C22 fatty diacid portion is located outside the fatty acid-containing side chain due to the modification to the amino acid sequence, and is therefore located distal to the bond between the side chain d and the amino acid sequence.

[0167] Example 2 Activity test of derivatives in cells

[0168] The purpose of this assay is to test the efficacy of derivatives of the polypeptides of the present application on human amylin receptors in vitro using a luciferase assay.

[0169] 2.1 Construction of amylin receptor / CRE-luc cell line

[0170] Using standard protocols, CHO-K1 / Ga15 / AMY3 cells (purchased from GenScript, in which calcitonin receptor and receptor activity-modifying peptide (RAMP) had already been constructed) were transfected with a plasmid containing a luciferase expression cassette driven by multiple copies of the cAMP response element (CRE). Cells were cultured in F12 medium containing 200 μg / mL Zeocin®, 2 μg / mL puromycin, 100 μg / mL hygromycin, and 400 μg / mL G418 to obtain a stably transfected amylin receptor / CRE-luc cell line.

[0171] 2.2 Amylin luciferase assay

[0172] The lyophilized powder of Example 1 was dissolved in 20 mM phosphate buffer (pH 7.0) and diluted in growth medium (F12 medium containing 10% FBS) to obtain a derivative sample with an initial concentration of 10 nM, which was then serially diluted 5-fold with growth medium to obtain seven samples with concentrations ranging from 2 nM to 0.4 nM. 50 μL of the test sample solution with the corresponding concentration was added to each well of a white 96-well plate.

[0173] Stably transfected amylin receptor / CRE-luc CHO cells were resuspended at a specific density in growth medium, and 50 μL of the cell suspension was added to a white 96-well plate containing the test sample solution at a density of approximately 20,000 cells / well. After 24 hours of incubation at 37°C and 5% CO2, 100 μL of luciferase substrate was added to each well, incubated for 3 minutes, and finally, luminescence was tested using a SpectraMax L (Molecular Devices) with the software SoftMax Pro 7.0.3 GxP. Fluorescence intensities were used to plot a standard curve, from which EC50 values ​​were calculated. Variability among various polypeptide derivatives was summarized by calculating the relative activity (the ratio of the EC50 of caglilintide to the compound), with the relative activity of caglilintide set at 100%. The results are shown in the table below.

[0174] [Table 4]

[0175] Example 3 Heat-Accelerated Stability Assay

[0176] 3.1 Materials and Equipment

[0177] A stability test chamber (BINDER GmbH), a 0.01 mg readout scale (METTLER TOLEDO), a pH meter (METTLER TOLEDO), a biosafety cabinet (ESCO), a T2G-II small motor-driven capping machine (Changsha Zhongya Pharmaceutical Equipment Co. LTD), a high-speed refrigerated centrifuge (Eppendorf), a sterilization cabinet (Shinva Medical), an Agilent 1260 high-performance liquid chromatography system with a Sepax Bio-C18 4.6*250mm 3μm 200Å reversed-phase column, and a TSKgel G2000SWXL 7.8*300mm 3μm column were used.

[0178] 3.2 Assay Methods and Results

[0179] The derivative (1 mg / mL) was mixed with sodium dihydrogen phosphate (1.42 mg / mL) and dissolved in ultrapure water. After adjusting the pH to approximately 7.4 with hydrochloric acid / sodium hydroxide, the solution was filtered into a sterile vial using a 0.22 μm sterile filter in a clean bench. The vial was then capped and placed in a stability test chamber at 40°C. Detection was performed on days 7 (7d) and 28 (28d), and changes in the characteristics and properties of the polypeptide were observed and recorded during the assay. The sample was then centrifuged at 10,000 rpm for 3 minutes at 4°C, and the supernatant was transferred to a liquid-phase sample vial. The concentration and purity of the polypeptide were detected using liquid-phase chromatography as described below.

[0180] The conditions for reverse phase chromatography were: flow rate: 1.0 mL / min, autosampler temperature: 15°C, column temperature: 25°C, detection wavelength: 214 nm, mobile phase A: 100% HO + 0.05% TFA, mobile phase B: 100% ACN, and the elution gradient is listed in Table 4.

[0181] [Table 5]

[0182] The results are shown in Table 5 below.

[0183] [Table 6]

[0184] The stability data for M6 is specifically shown in Figure 1, which shows that after 7 days or even 1 month of thermal acceleration, the reversed-phase chromatographic spectrum of M6 showed little change, and the pre- and post-heating spectra were virtually overlapping.

[0185] The derivative (1 mg / mL) was mixed with sodium dihydrogen phosphate (1.42 mg / mL) and dissolved in ultrapure water. After adjusting the pH to approximately 7.4 with hydrochloric acid / sodium hydroxide, the solution was filtered into a sterile vial in a clean bench using a 0.22 μm sterile filter. The vial was then capped and placed in a stability test chamber at 40°C. Detection was performed at 10 and 20 days, and changes in the characteristics and properties of the polypeptide were observed and recorded during the assay. The sample was then centrifuged at 10,000 rpm at 4°C for 3 minutes, and the supernatant was transferred to a liquid-phase sample vial. The concentration and purity of the polypeptide were detected using liquid-phase chromatography as described below.

[0186] Reversed-phase chromatography conditions

[0187] Flow rate: 1.0 mL / min, autosampler temperature: 25°C, column temperature: 25°C, detection wavelength: 214 nm, mobile phase A: 100 mM phosphate buffer (pH 5.8):ACN = 90:10, mobile phase B: ACN:IPA:HO = 85:5:10, and the elution gradient is listed in Table 6.

[0188] [Table 7]

[0189] The results are shown in Table 7 below.

[0190] [Table 8]

[0191] The results of the thermal stability assay revealed that the polypeptide molecules of the present application exhibited significantly superior stability compared to the control caglilintide molecule.

[0192] Example 4 Animal Testing

[0193] SPF male SD rats (7-8 weeks old, 200-250 g) were used in this study after undergoing a sanitary inspection and quarantine. The experimental animals were maintained at a room temperature of 20-23°C and a relative humidity of 40-50%. They were fed Co60 mouse breeding diet 1035 throughout the sanitary inspection, quarantine, and study. Purified water was provided via a water bottle and was available ad libitum. Five rats were assigned to each derivative test group based on their average body weight. The derivative or vehicle (PBS pH 7.4) was administered subcutaneously to the rats at a dose of 30 nmol / kg, and the administration time was recorded for each group. After administration, the rats were returned to their cages, where they were kept alive and allowed access to food and water. The rats' food intake and weight changes were recorded online or manually every 24 hours. The results of the animal tests are shown in Figure 2A and Figure 2B.

[0194] The specific results shown in Figures 2A and 2B indicate that the candidate molecules of the present application have significantly higher activity than caglilintide, and among them, M6 is the most active molecule, having significantly higher activity than caglilintide.

[0195] Example 5 Efficacy Assay in SD Rats - Dose-Dependent Study

[0196] SPF male SD rats (7-8 weeks old, 220-230 g) were used in this study after undergoing a sanitary inspection and quarantine. The experimental animals were maintained at a room temperature of 20-23°C and a relative humidity of 40-50%. They were fed Co60 mouse breeding diet 1035 throughout the sanitary inspection and quarantine period and throughout the study. Purified water was provided by water bottle and was available ad libitum. Based on average body weight, animals were assigned to 10 groups (designated A1-A10) with n=5 each: vehicle group, various doses of caglilintide groups (high, medium, and low doses), various doses of M41 group (high, medium, and low doses), and various doses of M43 group (high, medium, and low doses). The compounds and doses used in the treatment groups for this study are listed in Table 8. The study lasted a total of 6 days using a single dose administered by subcutaneous injection, and the administration day was represented as Day 1 / 0 time (T0). On Day 0, the animals' initial body weight was recorded and the first meal was introduced. From Day 1 to Day 5, the body weight and remaining food of the test animals were recorded daily to calculate the percentage of body weight change and food intake, and any abnormalities during that time were recorded and reported. The rats were in good condition throughout the study, and no abnormalities were observed. Body weight change = (BWT n -BWT0) / BWT0*100%, where BW represents the body weight value, and T0 and T n The dosing time and n hours after dosing were represented, respectively. Points in the graph were represented using the mean ± standard error of the mean (SEM). Cumulative food intake was the total amount of food intake for each animal prior to a specific time point after dosing. The dose-response relationship of the molecules is shown in Figures 3A-3D.

[0197] [Table 9]

[0198] The results in Figures 3A-3D showed that both M41 and M43 dose-dependently reduced the body weight and food intake of animals, and performed better in these respects than comparable doses of caglilintide.

[0199] Example 6 Efficacy Assay in DIO SD Rats - Dose Dependence Study

[0200] This study used SPF male DIO SD rats (36-37 weeks old, 680-110 g, fed a high-fat diet for 34 weeks from 3 weeks of age). The experimental animals were maintained at room temperature of 20-23°C and relative humidity of 40-50%. They were fed the high-fat diet D12492 throughout the construction and testing of the DIO animal model, with purified water provided ad libitum via water bottle. Based on their average body weight, the animals were assigned to six groups (n = 5), designated B1 to B6. The active ingredients and dosages of the compounds administered to the various groups in this study are listed in Table 9. Subcutaneous injections were administered once daily for 10 consecutive days. The first day of administration was designated Day 1 (D1). On Day 0, the animals' initial body weights were recorded and the first meal was introduced. From Day 1 to Day 11, the body weights and remaining food were recorded daily to calculate the rate of body weight change and food intake. Any abnormalities during this period were recorded and reported. During the test, the rats were in good condition and no abnormalities were observed. Weight change = (BWD n -BWD1) / BWD1*100%, where BW represents the body weight value, D1 and D n The dots represent the first and Nth days of administration, respectively. The points in the figure are represented using the mean ± standard error of the mean (SEM). The cumulative food intake was the total amount of food intake per animal in each group prior to a specific time point after administration. The effects on weight loss and food intake inhibition in the DIO rat model are shown in Figure 4A and Figure 4B.

[0201] [Table 10]

[0202] The results in Figures 4A and 4B showed that in the DIO rat model, both M41 and M43 dose-dependently reduced animal body weight and food intake, and performed better in these respects than comparable doses of caglilintide.

[0203] Example 7 Efficacy Assay in SD Rats - Single Dose Screening

[0204] SPF male SD rats (7-8 weeks old, 220-230 g) were used in this study after undergoing a sanitary inspection and quarantine. The experimental animals were maintained at a room temperature of 20-23°C and a relative humidity of 40-50%. They were fed Co60 mouse breeding diet 1035 throughout the sanitary inspection and quarantine period and throughout the study. Purified water was provided by water bottle and was available ad libitum. Two consecutive single-dose screening assays were performed, and the animals were assigned to groups of n = 5, designated C1-C6 and D1-D9, based on their average body weight. The compounds and doses used for the treatment groups in this study are listed in Table 10. The study lasted a total of 6 days using a single dose administered by subcutaneous injection. The day of administration was designated Day 1 / Time 0 (T0). The body weight and remaining food of the test animals were recorded daily from Day 1 to Day 5 to calculate the relative body weight change and food intake. Any abnormalities during this period were recorded and reported. During the test, the rats were in good condition and no abnormalities were observed. Body weight change = (BWT n -BWT0) / BWT0*100%, where BW represents the body weight value, and T0 and T n The dots represent the time of administration and n hours after administration, respectively. Points in the figures are represented as mean ± standard error of the mean (SEM). A comparison of the efficacy of various molecules is shown in Figures 5A-5D.

[0205] [Table 11]

[0206] The results in Figures 5A to 5D reveal that the efficacy of the polypeptide of the present application and its derivatives is comparable to or superior to that of caglirintide.

[0207] Example 8 Pharmacokinetic study in SD rats

[0208] SPF experimental animals (male, 6-8 weeks old, 200-240 g) that passed sanitary inspection and quarantine were assigned to three groups (n=3) based on their average body weight: M41, M43, and caglilintide. To conduct the study, animals were administered a subcutaneous injection of the corresponding compound (1 mg / kg and 5 ml / kg). The administration day was designated D1. Whole blood was collected from the animals on D1 (-10 min) before administration and on D1-2 h, D1-4 h, D1-6 h, D2-24 h, D3-48 h, D4-72 h, D5-96 h, and D6-120 h after administration. Plasma was then prepared using EDTA for anticoagulation. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to detect the concentrations of drug molecules in plasma, and GraphPad Prism 10 was used to plot the drug concentration-time curves. Then, PKSolver was used to calculate its pharmacokinetic parameters, including C, T, T, AUC, and MRT. The concentrations of various molecules in plasma over time are shown in Figure 6. A summary of the experimental results is listed in Table 11.

[0209] [Table 12]

[0210] The data from Figure 6 and Table 11 demonstrate that the polypeptide molecules M41 and M43 exhibited significantly higher bioavailability than caglilintide.

[0211] Although the present invention has been disclosed by way of examples as above, these examples are not intended to limit the present invention. Those skilled in the art will be able to make appropriate changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention is to be defined by the claims.

Claims

1. ASX ３ LS TAX ８ X ９ X １０ RLADF LRHX １９ X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ ILPPT NVGSN TX ３７ -NH ２ A polypeptide derivative or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence of where: X ３ is E or Q, X ８ is A or V, X ９ 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, or a pharmaceutically acceptable salt thereof.

2. X ３ is Q, Preferably, X ８ X ９ X １０ is selected from the group consisting of ALG, VLG, VTQ, ATQ, and VLQ, and more preferably X ８ X ９ X １０ is ATQ, More preferably, X １９ is F and X ２０ is T and X ２３ is D and X ２４ is R and X ２５ is D and X ３７ is P, and more preferably, X ２１ is D, and / or X ２２ is R, Or, X １９ is S and X ２０ is S and X ２３ is L and X ２４ is K and X ２５ is P and X ３７ is trans-Hyp, and more preferably, X ２１ is N, Q, E, S, T, A, G, H, K, or R, and / or X ２２ The derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein is N, Q, E, S, NMeAsn, αMeAsn, or NMeAsp.

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

25.

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

5. The derivative of the polypeptide or a pharmaceutically acceptable salt thereof according to claim 4, wherein the derivative is selected from M1 to M55.

6. A polypeptide comprising the amino acid sequence involved in any one of claims 1 to 5.

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

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

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