Polypeptide derivative for oral delivery

Abstract

The use of a fatty acid-containing side chain in improving the activity, activity selectivity and oral availability of a polypeptide derivative, a method for achieving the use, and a polypeptide derivative modified by the side chain.

Description

A polypeptide derivative for oral delivery Technical Field

[0001] The present application relates to the field of biomedicine or biopharmaceutical technology, and more specifically to a polypeptide or its derivatives that can be used for oral delivery, a composition for oral delivery of the polypeptide or its derivatives, and their use in treating and / or preventing metabolism-related diseases. Background Art

[0002] Peptides / proteins are not only essential building blocks of cells but also crucial players in signaling between cells and tissues, playing a vital role in maintaining normal physiological activity. Since the early 21st century, when bovine and porcine insulin were first used to treat diabetes, the continuous advancement of modern molecular biology, organic chemistry, and other fields has led to the production and application of an increasing number of peptides / proteins in human medicine. Compared to other types of drugs, peptide / protein drugs offer significant advantages in the treatment of cancer, diabetes, cardiovascular and cerebrovascular diseases, autoimmune diseases, and metabolic disorders, due to their high target specificity, favorable safety profile, and tolerability. However, due to the limitations of the human body's absorption mechanism, the vast majority of therapeutic peptide / protein drugs lack good bioavailability via oral administration, requiring injections and other methods for treatment. This has limited the development of peptide / protein drugs and, at the same time, has brought additional pain and inconvenience to patients.

[0003] In order to solve the problem of oral administration of peptides, people have tried many ways to increase the oral efficacy of peptide drugs. For example, Fiona McCartney et al. (Journal of Controlled Release, Volume 310, 28 September 2019, Pages 115-126) disclosed a non-ionic surfactant excipient. ALF It can be used to enhance the utilization of insulin in the intestine; P. Uhl et al. (Nanomedicine: Nanotechnology, Biology and Medicine, Volume 24, February 2020, 102132) used arginine-rich cyclic cell-penetrating peptides to coat PLA nanoparticles to enable oral administration of liraglutide; Vivek Gupta et al. (Journal of Controlled Release, Volume 172, Issue 3, 28 December 2013, Pages 753-762) disclosed the preparation of a mucosal adhesive device by compressing a polymer matrix containing carbomer, pectin and sodium carboxymethylcellulose (1:1:2), and demonstrated its enhanced effect on the delivery of salmon calcitonin (sCT) in vivo.

[0004] However, to date, peptide drugs still have problems such as poor oral efficacy, low bioavailability, unpredictable delivery effects of corresponding oral administration regimens for peptide molecules, and poor versatility.

[0005] Summary of the Invention

[0006] Therefore, the present application provides a polypeptide derivative with higher activity and stability, better oral effect and better bioavailability, which can effectively treat and / or prevent metabolic-related diseases. Specifically, the present application adopts the following technical solutions:

[0007] 1. A use of a fatty acid side chain in improving the activity selectivity of a polypeptide derivative, wherein the polypeptide derivative is obtained by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0008] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0009] Z3 is n AEEAs, where n≥0.

[0010] 2. A use of a fatty acid side chain in improving the activity of a polypeptide derivative, wherein the polypeptide derivative is obtained by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0011] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0012] Z3 is n AEEAs, where n≥0.

[0013] 3. A use of a fatty acid side chain in improving the oral availability of a polypeptide derivative, wherein the polypeptide derivative is obtained by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0014] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0015] Z3 is n AEEAs, where n≥0.

[0016] 4. A method for improving the activity selectivity of a polypeptide derivative, comprising modifying a polypeptide molecule with a fatty acid side chain to obtain a polypeptide derivative, wherein the fatty acid side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0017] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0018] Z3 is n AEEAs, where n≥0.

[0019] 5. A method for improving the activity of a polypeptide derivative, comprising modifying a polypeptide molecule with a fatty acid side chain to obtain a polypeptide derivative, wherein the fatty acid side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0020] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0021] Z3 is n AEEAs, where n≥0.

[0022] 6. A method for improving the oral availability of a polypeptide derivative, the method comprising modifying a polypeptide molecule with a fatty acid side chain to obtain a polypeptide derivative, wherein the fatty acid side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0023] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0024] Z3 is n AEEAs, where n≥0.

[0025] 7. The use according to any one of items 1 to 3 or the method according to items 4 to 6, wherein Z1 is a C16, C17, C18, C19 or C20 fatty diacid.

[0026] 8. The use or method according to any one of items 1 to 7, wherein Z2 is selected from any one of γGlu, βAsp, γGlu, Inp, and Trx, preferably selected from βAsp or γGlu.

[0027] 9. The use or method according to any one of items 1 to 8, wherein n is greater than 0, preferably 1-10, more preferably 2-8, and further preferably n=2.

[0028] 10. The use or method according to any one of items 1 to 9, wherein Z1 is a C16, C18 or C20 fatty diacid.

[0029] 11. The use or method according to any one of items 1 to 10, wherein Z2 is Inp or Trx; and n is greater than 0.

[0030] 12. The use or method according to any one of items 1-11, wherein Z1 is a C16, C17, C18, C19 or C20 fatty diacid; Z2 is γGlu; ​​and n=2. 13. The use or method according to any one of items 1-12, wherein Z1, Z2 and Z3 are sequentially connected via amide bonds.

[0031] 14. The use or method according to any one of items 1 to 13, wherein the side chain modification is on 1 to 10 amino acids, preferably on 1 to 3 amino acids, preferably on 1 amino acid of the polypeptide.

[0032] 15. The use or method according to any one of items 1 to 14, wherein the side chain modification is at the N-terminus or any intermediate amino acid of the polypeptide sequence; the amino acid is preferably selected from glutamic acid, aspartic acid, lysine, glutamine or asparagine, and is further preferably selected from lysine.

[0033] 16. The use or method according to any one of items 1 to 15, wherein the polypeptide is a natural polypeptide or an artificially synthesized polypeptide molecule, which can be an agonist or antagonist; preferably, selected from: GLP-1 receptor agonist, GIP receptor antagonist, insulin receptor agonist, heparin receptor agonist, growth hormone, interferon receptor agonist, interleukin receptor agonist, follicle-stimulating hormone receptor agonist, gonadotropin receptor agonist, erythropoietin receptor agonist, PYY (peptide tyrosine kinase) receptor agonist, oxygenated regulatory factor receptor agonist, GLP-2 (glucose agonist), The invention relates to an agonist of the present invention, wherein the agonist comprises a leptin-like peptide-2 (LSP-2) receptor agonist, a calcitonin receptor agonist, a PTH (parathyroid hormone) receptor agonist, a ghrelin receptor agonist, an endocannabinoid receptor agonist, a leptin receptor agonist, a serotonin receptor agonist, a fibroblast growth factor 21 (FGF21) receptor agonist, a cholecystokinin (CCK) receptor agonist, an oxyntomodulin receptor agonist or a glucagon receptor agonist; preferably, an amylin receptor agonist; further preferably, an amylin analog or a calcitonin analog.

[0034] 17. The use or method according to any one of items 1 to 16, wherein the polypeptide comprises the following amino acid sequence:

[0035] ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2 or KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 or

[0036] KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2.

[0037] Preferably, the polypeptide derivative is selected from any one or more of N1 or N2 or M1-M30.

[0038] Preferably, the polypeptide derivative is selected from any one or more of N1, N2, N3 or M1-M30.

[0039] 18. The use according to item 1 or items 7-17, or the method according to item 4 or items 7-17, wherein the selective enhancement of the activity of the polypeptide derivative is achieved by preferentially enhancing the activity of the polypeptide derivative at the amylin receptor over the activity of the polypeptide derivative at the calcitonin receptor. Preferably, the selective enhancement of the biological activity refers to enhancing the ratio of the activity of the polypeptide derivative at the amylin receptor to the activity of the polypeptide derivative at the calcitonin receptor; further preferably, the activity of the polypeptide derivative at the amylin receptor is also enhanced.

[0040] 19. The use according to item 1 or items 7-18 or the method according to item 4 or items 7-18, wherein the improvement of the activity selectivity of the polypeptide derivative is that the ratio of the cellular activity at the amylin receptor to the calcitonin receptor is increased by more than 1.5 times, preferably, the multiple is more than 2 times; further preferably, the multiple is more than 5 times; more preferably, the multiple is more than 10 times.

[0041] 20. The use or method according to item 18 or 19, wherein the activity is agonistic activity.

[0042] 21. The use according to item 1 or items 7-20 or the method according to item 4 or items 7-20, wherein the polypeptide derivative is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin derivative; more preferably, the polypeptide derivative comprises the amino acid sequence: ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2 or more preferably, the polypeptide derivative comprises the amino acid sequence: KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2.

[0043] 22. The use according to item 2 or items 7 to 17 or the method according to item 5 or items 7 to 17, wherein the activity of the polypeptide derivative is its activity in reducing weight and / or reducing food intake.

[0044] 23. The use according to item 2 or items 7 to 17 or the method according to item 5 or items 7 to 17, wherein said increasing the activity of the polypeptide derivative is increasing the cellular activity of the polypeptide derivative on amylin receptor and calcitonin receptor.

[0045] 24. The use according to item 2 or items 7 to 17 or items 22 to 23, or the method according to item 5 or items 7 to 17 or items 22 to 23, wherein the polypeptide derivative is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin derivative; more preferably, the polypeptide derivative comprises the following amino acid sequence:

[0046] KCNTATCATQ RLADFLRHS NNLKPILPPT NVGSNT-trans-Hyp-NH2, or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2.

[0047] 25. The use according to item 3 or items 7-17 or the method according to item 6 or items 7-17, wherein the polypeptide derivative containing fatty acid side chains is co-administered with one or more oral delivery agents; preferably, the oral delivery agent is N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC) salt; more preferably PNAC.

[0048] 26. The use according to item 3 or items 7 to 17 or the method according to item 6 or items 7 to 17, wherein the improved oral availability of the polypeptide derivative is higher than that of the Cagrilintide polypeptide molecule.

[0049] 27. The use according to item 3 or items 7 to 17 or the method according to item 6 or items 7 to 17, wherein the polypeptide derivative is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin derivative; more preferably, the polypeptide derivative comprises the following amino acid sequence:

[0050] KCNTATCATQ RLADFLRHS NNLKPILPPT NVGSNT-trans-Hyp-NH2, or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2.

[0051] 28. A polypeptide derivative comprising a polypeptide and a side chain connected to the polypeptide, wherein the side chain is in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty diacid;

[0052] Z2 is selected from any one of γGlu, αGlu, βAsp, αAsp, Inp, Trx or Z2 does not exist;

[0053] Z3 is n AEEAs, where n≥0.

[0054] 29. The derivative according to item 28, wherein Z1 is a C16, C17, C18, C19 or C20 fatty diacid.

[0055] 30. The derivative according to item 28 or 29, wherein Z2 is selected from any one of γGlu, Inp, Trx or Z2 is absent.

[0056] 31. The derivative according to any one of items 28-30, wherein n is greater than 0.

[0057] 32. The derivative according to any one of items 28-31, wherein Z2 is Inp or Trx; and n is greater than 0.

[0058] 33. The derivative according to any one of items 28-32, wherein Z2 is absent; and n is greater than 0.

[0059] 34. The derivative according to any one of items 28 to 33, wherein Z2 is γGlu; ​​and n=2.

[0060] 35. The derivative according to any one of items 28 to 34, wherein the polypeptide is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin-like organism; more preferably, the polypeptide comprises the following amino acid sequence:

[0061] ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2 or KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 or

[0062] KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2,

[0063] Preferably, the polypeptide derivative is selected from any one or more of N1 or N2 or M1-M30.

[0064] Preferably, the polypeptide derivative is selected from any one or more of N1, N2, N3 or M1-M30.

[0065] 36. A pharmaceutical composition comprising a derivative or a pharmaceutically acceptable salt thereof according to any one of items 28-35, and a pharmaceutically acceptable excipient. Preferably, the composition comprises one or more oral delivery agents; more preferably, the oral delivery agent is N-[8-(2-hydroxybenzoyl)amino]octanoic acid (NAC) salt; further preferably PNAC.

[0066] 37. Use of the derivative or pharmaceutically acceptable salt thereof described in any one of items 28 to 35 or the composition described in item 36 in the preparation of a polypeptide drug; preferably, the polypeptide drug is used to prevent and / or treat overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain.

[0067] 38. The use according to item 37, wherein the polypeptide drug is an oral drug; preferably, the oral drug comprises one or more oral delivery agents; more preferably, the oral delivery agent is N-[8-(2-hydroxybenzoyl)amino]octanoic acid (NAC) salt; further preferably, it is PNAC.

[0068] 39. Use of the derivative according to any one of items 28 to 35 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to item 36, for reducing food intake.

[0069] 40. 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 or pharmaceutically acceptable salt thereof according to any one of items 28 to 35 or the pharmaceutical composition according to item 36.

[0070] 41. A method for reducing food intake, comprising administering to a subject an effective amount of the derivative or pharmaceutically acceptable salt thereof according to any one of items 28 to 35 or the pharmaceutical composition according to item 36.

[0071] Effects of the Invention

[0072] The polypeptide derivatives of the present application have high cell activity, high activity or efficacy on amylin receptors and calcitonin receptors, stable properties, and little change in concentration and purity when stored for a long time.

[0073] The polypeptide derivatives of the present application have outstanding effects in reducing weight and reducing food intake. As oral drugs, they have better effects and higher bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The accompanying drawings are provided to facilitate a better understanding of the present application and do not constitute an undue limitation on the present application.

[0075] FIG1 is a trend diagram showing the effects of various polypeptide derivatives on changes in rat body weight;

[0076] FIG2 is a trend diagram showing the effects of various polypeptide derivatives on the cumulative food intake of rats;

[0077] FIG3 is a trend diagram showing the effects of some polypeptide derivatives on rat body weight changes;

[0078] FIG4 is a trend diagram showing the effects of some polypeptide derivatives on the cumulative food intake of rats;

[0079] Figure 5 shows the drug content of D1 and N2 polypeptide derivatives in plasma;

[0080] Figure 6 shows the drug content in plasma of some polypeptide derivative oral tablets. DETAILED DESCRIPTION

[0081] The following description of exemplary embodiments of the present application includes various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Therefore, it should be recognized by those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, unless otherwise defined, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description, and the relevant technical and scientific terms in this specification have the same meanings as those commonly understood by those skilled in the art.

[0082] 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.

[0083] The term "N-terminus" refers to the presence of a free, unbonded α-amino (-NH2) and free α-carboxyl (-COOH) group at each end of a linear polypeptide formed from multiple amino acids via peptide bonds, unless further modified. The end containing the unbonded free α-NH2 is the N-terminus or amino terminus of the polypeptide. A modification site located at or at the N-terminus indicates that the amino acid at the N-terminus of the polypeptide has been modified.

[0084] 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.

[0085] In this article, the "activity selectivity" of a polypeptide or polypeptide derivative refers to the polypeptide or polypeptide derivative having a stronger effect on the receptor it acts on than on another receptor it acts on. In some embodiments of the present application, the effect refers to agonist activity.

[0086] In some embodiments of the present application, the activity selectivity may further indicate a preference for enhancing the activity of the polypeptide derivative at the amylin receptor over enhancing the activity of the polypeptide derivative at the calcitonin receptor. Preferably, the biological activity selectivity refers to the ratio of the activity of the polypeptide derivative at the amylin receptor to the activity of the polypeptide derivative at the calcitonin receptor.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] As used herein, "treatment" refers to the use of drugs or compounds to obtain the desired pharmacological and / or physiological effects. The effect can be preventive in terms of completely or partially preventing a disease or its symptoms, and / or can 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, especially humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the development 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.

[0091] 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).

[0092] abbreviation

[0093] The corresponding names or structural forms of some abbreviations used in the examples of this application are as follows:

[0094] Trx:

[0095] Inp:

[0096] AEEA:

[0097] AA: Amino Acid; Boc: t-Butyloxy carbonyl; DCM: dichloromethane; DMF: N,N-Dimethyl formamide; DIEA: N,N-Diisopropylethylamine; EDT: 1,2-Ethanedithiol; Fmoc: 9-fluorenylmethyloxycarbonyl; OtBu: tert-butyl ester; Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl chloride; Pip: Piperidine; TBTU: O-(Benzotriazol-l-yl)-N,N,N,N,-tetramethyluroniumtetrafluorobor ate, O-benzotriazole-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;

[0098] Example

[0099] Example 1 Preparation method of polypeptide derivatives

[0100] The polypeptide derivatives prepared in this application are shown in Table 2.

[0101] Solid phase organic synthesis was used, utilizing Fmoc-protected amino acid strategy and SPPS solid phase synthesis technology to complete synthesis, cleavage, oxidation and purification to obtain the target products shown in Table 2.

[0102] Taking compound D1 (Cagrilintide) as an example, its structural formula is as follows:

[0103] The synthesis process is as follows:

[0104] 1.1 Solid-phase synthesis

[0105] 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 peptide sequence using the method in Table 1:

[0106] Table 1 Synthesis procedures

[0107] The following amino acids were coupled sequentially:

[0108] A-01Fmoc-Pro-OH,A-02Fmoc-Thr(tBu)-OH,A-03Fmoc-Asn(Trt)-OH,A-04Fmoc-Ser(tBu)-OH,A-05Fmoc-Gl y-OH,A-06Fmoc-Val-OH,A-07Fmoc-Asn(Trt)-OH,A-08Fmoc-Thr(tBu)-OH,A-09Fmoc-Pro-OH,A-10Fmoc-Pr o-OH,A-11Fmoc-Leu-OH,A-12Fmoc-Ile-OH,A-13Fmoc-Pro-OH,A-14Fmoc-Gly-OH,A-15Fmoc-Phe-OH,A-16F moc-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-25Fm oc-Ala-OH,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-39C20 diacid;

[0109] Finally, the polypeptide derivative resin is formed.

[0110] The peptide derivative resin is washed, transferred out, dried to constant weight, and then cleaved.

[0111] 1.2 Cracking

[0112] Calculate the amount of cleavage reagent based on 10 mL ± 2 mL of cleavage reagent per gram of peptide derivative resin. For cleavage, add the required cleavage reagents, H₂O, TFA, EDT, and TIS, to the cleavage reaction flask in the following order: TFA:H₂O:EDT:TIS = 95:1:2:2. Maintain the cleavage reagent temperature at 0-10°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 cleavage solution and precipitate it 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.

[0113] 1.3 Oxidation

[0114] Grind the crude product into powder, prepare purified water, slowly add the ground crude product while 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.

[0115] 1.4 Purification and freeze-drying

[0116] 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.

[0117] The polypeptide derivatives prepared in this application are shown in Table 2 and can be prepared by using a similar method to that of this example, except that different amino acid sequences and / or different fatty acid side chains are used in solid phase synthesis.

[0118] Table 2 Amino acid sequences and modifications of peptides and their derivatives

[0119] N1-N3 or M1-M30 represent derivatives obtained by modifying the amino acids shown in SEQ ID No. 1 to SEQ ID No. 5 with the corresponding fatty acid-containing side chains in Table 2 (if no fatty acid modification is present, the derivatives are polypeptides). The fatty acid-containing side chains are 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 0-8 AEEAs, for example, 0, 1, 2, 3, 4, 5, or 6; Z1, Z2, and Z3 are sequentially connected by amide bonds, and the C16-C22 fatty diacid portion is located outside the fatty acid-containing side chain for modification relative to the amino acid sequence, at the distal end where the side chain is connected to the amino acid sequence. For example, M1 represents a derivative obtained by modifying the amino acid shown in SEQ ID No. 1 with a fatty acid-containing side chain "C18 diacid+γGlu", wherein the γGlu end of the side chain is connected to the amino acid through a peptide bond, and the C18 fatty diacid end is at the distal end where the side chain is connected to the amino acid sequence.

[0120] Example 2 In vitro activity detection of polypeptide derivatives

[0121] The purpose of this experiment is to use luciferase assay to detect the efficacy of the polypeptide derivatives of the present application on amylin receptor and calcitonin receptor in vitro.

[0122] 2.1. Amylin receptor agonism assay

[0123] The amylin receptor / CRE-luc cell line was constructed using standard methods. CHO-K1 / Ga15 / AMY3 cells (calcitonin receptor and receptor-activating modified peptide RAMP were 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.

[0124] In the amylin luciferase assay, the lyophilized powder obtained according to the method described 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-fold 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.

[0125] 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 7.0.3GxP software. A standard curve was plotted based on the fluorescence values, and the EC50 was calculated.

[0126] Compounds N2 and N3 were assayed using the same method as above, except that the assay solution and CHO cells were incubated at 37° C. and 5% CO 2 for 4 hours before adding 100 μL of Luciferase substrate to each well.

[0127] The results are shown in Table 3.

[0128] Table 3 Amylin receptor cell activity results

[0129] It can be seen from the data in Table 3 that the polypeptide derivatives in the present application have high cell activity and have good agonist activity on amylin receptors.

[0130] 2.2 Calcitonin receptor agonism

[0131] 2.2.1 Construction of calcitonin receptor / CRE-luc cell line

[0132] Using standard methods, CHO-K1 / CALCR / Gα15 cells (calcitonin receptor has 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, and 100 μg / mL of hygromycin to obtain a stably transfected calcitonin receptor / CRE-luc cell line.

[0133] 2.2.2 Calcitonin luciferase assay

[0134] The lyophilized powder obtained in step 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 samples with concentrations ranging from 10 nM to 2 nM-0.4 nM, each with a 5-fold difference in concentration. 50 μL of the sample assay solution of the corresponding concentration was added to each well of a white 96-well plate.

[0135] CHO cells stably transfected with calcitonin / 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. A standard curve was constructed using the fluorescence values, and the EC50 was calculated. The results are shown in the table below.

[0136] Table 4 Calcitonin receptor cell activity results

[0137] It can be seen from the data in Table 4 that the polypeptide derivatives in the present application have high cell activity and have agonist activity on the calcitonin receptor.

[0138] A comprehensive analysis of the cell activity data of some polypeptide derivatives in the present application on the amylin receptor (hAMY3R) and calcitonin receptor (hCTR) is shown in Table 5. Surprisingly, taking the cell activity of D1 molecule on hAMY3R and hCTR as a benchmark, compared with the polypeptide derivatives of the present application containing fatty acid side chains in the form of Z1+Z2 (i.e., fatty diacid + amino acid form), when the side chains of fatty acids contained are in the form of Z1+Z2+Z3 (i.e., fatty diacid + amino acid + AEEA form), the molecule has a significant difference in cell activity against the amylin receptor and calcitonin receptor, and shows a higher hAMY3R activity / relative activity than the activation ability of hCTR, especially when Z2 is Trx or Inp, the cell activity is more significantly improved; for example, compared with M17 with SEQ ID NO. 4 modified with C18 diacid+Inp, M17 with SEQ ID NO. 4 modified with C18 diacid+Inp+2AEEA has a significantly higher cell activity / relative activity than hCTR, and especially when Z2 is Trx or Inp, the cell activity is more significantly improved; for example, compared with M17 with SEQ ID NO. 4 modified with C18 diacid+Inp, M17 with SEQ ID NO. NO.4 M30, the amylin receptor agonist activity increased by nearly 70 times, and relative to the calcitonin receptor agonist activity, the amylin receptor activity also increased from the original 1.7 times to about 42.5 times, showing a higher amylin receptor agonist tendency.

[0139] Similarly, in the derivative N3 containing another group of amino acid sequences (SEQ ID NO. 2) modified with fatty acid side chains in the form of Z1+Z2+Z3 (i.e., fatty diacid + amino acid + AEEA), high agonist activity on the amylin receptor was also exhibited.

[0140] Table 5 Cellular activity data of amylin receptor and calcitonin receptor

[0141] Example 3 Study on the stability of polypeptide derivatives

[0142] 3.1 Materials and Equipment

[0143] 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 columns.

[0144] Reagents and consumables: ultrapure water (18.2 MΩ self-made ultrapure water), acetonitrile (HPLC grade), trifluoroacetic acid (HPLC grade), sodium dihydrogen phosphate (pharmaceutical grade / Hunan Jiudian Hongyang), hydrochloric acid (pharmaceutical grade / Hunan Erkang), sodium hydroxide (analytical grade / National Pharmaceutical Shanghai Testing), vials, rubber stoppers, disposable syringes, sterile filters.

[0145] 3.2 Experimental methods

[0146] The lyophilized powder of the peptide derivative prepared according to Example 1 was mixed with sodium dihydrogen phosphate (1.42 mg / ml) at a mass-volume ratio 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 on the 7th day (7d) and the 26th day (26d). During the experiment, changes in the peptide properties were observed and recorded. The samples were then centrifuged at 10,000 rpm, 4°C, for 3 min. The supernatant was transferred to a liquid chromatography vial and the peptide concentration and purity were determined using the following liquid chromatography method. Peptide concentration = peak area / sample load / extinction coefficient / 60 × flow rate. Peptide purity = target peak area / total peak area × 100%.

[0147] Reversed-phase chromatography conditions: flow rate: 1.0 ml / min; autosampler temperature: 15°C; column temperature: 25°C; detection wavelengths: 280 nm and 214 nm; mobile phase A: 100% H2O + 0.05% TFA; mobile phase B: 100% ACN; elution gradient: see Table 6 below:

[0148] Table 6 Liquid chromatography elution gradient list

[0149] 3.3 Experimental Results

[0150] In the thermal acceleration stability experiment, it was observed that the solutions of the polypeptide derivatives of the present application remained clear on the 7th day of thermal acceleration. Compared with the D1 molecule solution which showed obvious visible foreign matter on the 26th day of thermal acceleration, the solutions of the polypeptide derivatives of the present application maintained a more obvious high clarity.

[0151] As can be seen from Table 7, the concentration changes of the polypeptide derivatives provided by the present application under neutral conditions of thermal acceleration stability are not obvious. Compared with the molecule D2, the concentration change reached -40% on the 7th day of the accelerated experiment. The polypeptide derivatives of the present application maintained a very low concentration change. Even some polypeptide derivatives such as M6, M19, M22, M23, M24, M27, M29, M30, etc. have concentration changes of less than 1% (marked as no change). Even on the 26th day of thermal acceleration, the concentration change in the solution of the polypeptide derivatives of the present application was still maintained below 10%, and the concentration change of some solution derivatives was even less than 3%.

[0152] Table 7 Concentration changes of thermally accelerated stability

[0153] Note: Concentration change = (concentration at 7 days or 26 days - concentration at 0 days) / concentration at 0 days × 100%

[0154] Purity was determined by reverse-phase chromatography. As can be seen from Table 8, the peptide derivatives provided by this application showed little change in purity during heat-accelerated stability under neutral conditions. On day 7 of the accelerated experiment, the reverse-phase purity change for most peptide solutions was less than 1%, with no concentration reduction observed. Compared to the prior art molecule D1, which showed a 12.73% decrease in purity after 7 days of heat acceleration, the purity of each peptide derivative in this application remained at a very high level. Furthermore, during the experiment, it was found that the purity of molecules such as M7, M18, M19, and M23 showed almost no significant change on day 26 of the accelerated experiment, indicating excellent purity stability.

[0155] Table 8 Purity Changes in Thermally Accelerated Stability (Reversed Phase Purity - 214nm)

[0156] Note: Purity change = 7d or 26d purity - 0d purity

[0157] Example 4 Animal Experiment Results

[0158] SD (Sprague Dawley) rats weighing 200-250g were selected for this experimental study. Before the experiment began, the rats arrived for at least 10-14 days to acclimate to the experimental environment. During this period, the rats were raised for two weeks under a reversed light / dark phase (meaning that the light was turned off during the day and turned on at night) after arrival, and were raised individually in the first week of arrival to ensure data accuracy and high test sensitivity. Rats had free access to food and water during the entire adaptation period and experimental period. Each derivative test group had 5-8 rats, and each group of rats was subcutaneously administered a 10nmol / kg dose of the derivative or solvent (20mM phosphate buffer solution, PB, pH 7.0). The dosing time of each group was recorded. After administration, the rats were returned to their home cages, where they could then obtain food and water. Food consumption and rat weight changes were recorded every 24 hours by online recording or manual recording. Body weight change rate = (BWTn-BWT0) / BWT0*100%, BW represents body weight value, T0 and Tn represent the time of administration and n hours after administration, respectively. Each point in the figure is expressed as the mean ± standard error of the mean (SEM).

[0159] As shown in Figures 1-4, compared with the control group (solvent or D1 group), the polypeptide derivatives described in the present application showed significantly better effects in reducing the body weight and cumulative food intake of SD rats. More importantly, compared with molecules with a single amino acid as a linker, under the same polypeptide structure, the addition of AEEA in the linker has significantly further improved the effect of reducing the body weight and cumulative food intake of SD rats, such as polypeptide derivatives M18, M23, M25, M27, and M29.

[0160] Example 5: Pharmacological efficacy experiment in SD rats - dose-dependence study

[0161] SPF male Sprague-Dawley rats (7-8 weeks, 220-230 g) were used in this study after a quarantine period. The animals were housed at a room temperature of 20°C to 23°C and a relative humidity of 40% to 50%. During the quarantine period and the experimental period, they were fed Co60 Rat Breeding Diet 1035 and provided with purified water via drinking bottles, allowing free access to water.

[0162] In the comparison of the pharmacological effects of compounds D1 and N3, the experimental animals were divided into 5 groups according to the average body weight, with n=5 animals in each group. The experiments were conducted according to the solvent group (20 mM phosphate buffer solution, PB, pH 7.0), D1 compound 30 nmol / kg and 100 nmol / kg dosage groups, and N3 compound 30 nmol / kg and 100 nmol / kg dosage groups.

[0163] In the comparison of the pharmacological effects of compounds N2 and N3, the experimental animals were divided into 7 groups according to the average body weight, with n=5 animals in each group. The experiments were conducted according to the solvent group (20 mM phosphate buffer solution, PB, pH 7.0), N2 compound 5nmol / kg, 25nmol / kg, 125nmol / kg dosage groups, and N3 compound 5nmol / kg, 25nmol / kg, 125nmol / kg dosage groups.

[0164] During the experiment, subcutaneous injection was used for single administration. The day of administration was recorded as Day1 / Time0 (T0). Day0 recorded the initial body weight of the animals and the addition of initial feed. The body weight and remaining food of the experimental animals were recorded every day, and the body weight change rate and food intake were calculated. At the same time, any abnormal conditions were recorded and reported. The rats were in good condition during the experiment and no abnormal phenomena were observed in the rats. Body weight change rate = (BWTn-BWT0) / BWT0*100%, BW represents body weight value, T0 and Tn represent the time of administration and n hours after administration, respectively. Each point in the figure is expressed as the mean ± standard error (SEM). Food intake is the amount of food consumed by each animal per day.

[0165] Table 9

[0166] Table 10

[0167] Compared to D1, compound N2 has a significantly better weight-reducing effect (see Example 3 of CN 202310614769.1). As can be seen from Table 9, N3 also showed a significant dose-dependent improvement in reducing the body weight and cumulative food intake of SD rats compared to D1. Both N2 and N3 are derivatives of another amino acid sequence (SEQ ID NO. 2) containing a fatty acid with a side chain form of Z1+Z2+Z3 (i.e., a fatty diacid + amino acid + AEEA). Surprisingly, compared to N2, N3 showed a stronger effect in reducing animal weight and food intake (Table 10).

[0168] Example 6 Study on PK properties of amylin derivative tablets

[0169] The amylin derivatives were made into oral tablets containing the same amount of the oral delivery agent PNAC. The structure of the PNAC described in this application is shown below:

[0170] N-[8-(2-Hydroxybenzoyl)amino]octanoic acid (NAC) was prepared according to the method described in Example 1 of International Patent Application WO2008 / 028859. Isopropyl alcohol (22070.0 ml, 4.0 vol) was added to a 50 L reactor. Stirring was initiated, and NAC (5518 g, 1.0 eq) was added. The system was heated to 50°C, and a 50% potassium hydroxide solution (1304.0 g, 1.0 eq) was added dropwise. The solution became a clear, transparent yellow solution, which was then maintained at 50°C for 1 h. The reaction solution was concentrated in batches at 40°C to yield a light orange-yellow crude product.

[0171] The crude product was slurried in isopropanol (19310.0 ml, 3.5 vol) for 1 hour. The mixture was filtered, and the filter cake was rinsed with isopropanol (2760.0 ml, 0.5 vol). The filter cake was transferred to a vacuum drying oven, dried at 60°C under nitrogen pressure for 16 hours, and then transferred to a vacuum drying oven at 100°C for 24 hours. After drying, 4.52 kg of the product (72.8% yield) was obtained as an off-white powdery solid, namely PNAC.

[0172] The contents of some main components in the polypeptide derivative tablet containing fatty acid side chains are shown in Table 9:

[0173] Table 11: Examples of polypeptide derivative tablets prepared in this application

[0174] The preparation method of the polypeptide derivative tablet containing fatty acid side chains is as follows: the polypeptide derivative and PNAC are sieved, mixed evenly with excipients, and directly compressed into tablets. Male Beagle dogs (9-12 kg) aged 10-15 months were orally administered amylin derivative (D1, N2, respectively) at a dose of 7 mg / dog once daily for 5 consecutive days (n=5), with the first oral administration of the drug protein molecule being designated as Day 1 and the last oral administration of the drug protein molecule being designated as Day 5. On Day 1, whole blood was collected from the animals before administration (-10 minutes) and 2 hours, 4 hours, and 8 hours after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). On Days 2-4, whole blood was collected from the animals before administration (-10 minutes) and 2 hours and 4 hours after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). On Day 5, whole blood was collected from the animals before administration (-10 minutes) and 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). Plasma was stored at −80°C and used for subsequent analysis. Drug content in plasma was analyzed using LC-MS / MS (Waters ACQUITY I Class Premier UPLC tandem with Sciex 6500+QQQ). The ion-pair mass-to-charge ratio used for D1 was 1102.9 / 1074.5, and the ion-pair mass-to-charge ratio used for N2 was 945.6 / 919.8. Experimental data were plotted using GraphPad Prism 9.3.1.

[0175] The results are shown in FIG5 . Compared with the tablet group containing compound D1, the compound N2 molecule in the tablet containing the delivery agent PNAC showed a special synergistic effect, exhibiting extremely high oral bioavailability.

[0176] In the comparison of oral bioavailability of D1 and N3, N3 also showed a synergistic effect of significantly improved bioavailability in tablets of delivery agent PNAC.

[0177] Example 7 Effect of Side Chain Modification on the PK Properties of Oral Tablets of Peptide Derivatives

[0178] The effects of side chain modification on the oral administration of polypeptide derivatives were further verified by using the sequence of Amylin polypeptide analogs. The PK properties of oral tablets of polypeptide derivatives D1, M2, and M3, which differed only in side chains as shown in Table 1, were tested using an experimental method similar to that of Example 6.

[0179] The results, as shown in Figure 6, show that compared to the D1 molecule, the peptide derivatives M2 and M3 exhibit significantly higher oral absorption. This is similar to the results of Example 6, further demonstrating that the side chain modifications provided herein can effectively enhance the oral absorption of peptide derivatives, and that M3, containing C18 fatty acids, exhibits better oral absorption than M2, containing C20 fatty acids.

[0180] Although the embodiments of the present application are described above, the present application is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are merely illustrative and instructive, and not restrictive. A person of ordinary skill in the art, under the guidance of this specification and without departing from the scope of protection of the claims of this application, can also make many forms, all of which fall within the scope of protection of this application.

Claims

1. A use of a fatty acid side chain in improving the activity selectivity of a polypeptide derivative, wherein the polypeptide derivative is obtained by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid 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 n AEEAs, where n≥0.

2. A use of a fatty acid side chain in improving the activity of a polypeptide derivative, wherein the polypeptide derivative is obtained by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid 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 n AEEAs, where n≥0.

3. A use of a fatty acid side chain in improving the oral availability of a polypeptide derivative, wherein the polypeptide derivative is obtained by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid 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 n AEEAs, where n≥0.

4. A method for improving the activity selectivity of a polypeptide derivative, the method comprising obtaining a polypeptide derivative by modifying a polypeptide molecule with a fatty acid side chain, wherein the fatty acid 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 n AEEAs, where n≥0.

5. A method for improving the activity of a polypeptide derivative, the method comprising obtaining a polypeptide derivative by modifying a polypeptide molecule with a fatty acid side chain, wherein: 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 n AEEAs, where n≥0.

6. A method for improving the oral availability of a polypeptide derivative, the method comprising obtaining a polypeptide derivative by modifying a polypeptide molecule with a fatty acid side chain, wherein: 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 n AEEAs, where n≥0.

7. The use according to any one of claims 1 to 3 or the method according to claims 4 to 6, wherein: The Z1 is C16, C17, C18, C19 or C20 fatty diacid.

8. The use or method according to any one of claims 1 to 7, wherein The Z2 is selected from any one of γGlu, βAsp, γGlu, Inp, and Trx, preferably selected from βAsp or γGlu.

9. The use or method according to any one of claims 1 to 8, wherein The n is greater than 0, preferably 1-10, more preferably 2-8, and further preferably n=2.

10. The use or method according to any one of claims 1 to 9, wherein The Z1 is C16, C18 or C20 fatty diacid.

11. The use or method according to any one of claims 1 to 10, wherein: The Z2 is Inp or Trx; and the n is greater than 0.

12. The use or method according to any one of claims 1 to 11, wherein The Z1 is C16, C17, C18, C19 or C20 fatty diacid; the Z2 is γGlu; ​​and n=2.

13. The use or method according to any one of claims 1 to 12, wherein: The Z1, Z2 and Z3 are sequentially connected via amide bonds.

14. The use or method according to any one of claims 1 to 13, wherein The side chain modification is on 1-10 amino acids of the polypeptide, preferably on 1-3 amino acids, and preferably on 1 amino acid.

15. The use or method according to any one of claims 1 to 14, wherein The side chain modification is at the N-terminus of the polypeptide sequence, or an amino acid at any intermediate position; the amino acid is preferably selected from glutamic acid, aspartic acid, lysine, glutamine or asparagine, and is further preferably selected from lysine.

16. The use or method according to any one of claims 1 to 15, wherein the polypeptide is a natural polypeptide or an artificially synthesized polypeptide molecule, and can be an agonist or an antagonist; preferably, the protein or polypeptide drug is selected from: GLP-1 receptor agonist, GIP receptor antagonist, insulin receptor agonist, heparin receptor agonist, growth hormone, interferon receptor agonist, interleukin receptor agonist, follicle stimulating hormone receptor agonist, gonadotropin receptor agonist, erythropoietin receptor agonist, PYY (peptide tyrosine) receptor agonist, oxygenation regulator receptor agonist, GLP -2 (glucagon-like peptide-2) receptor agonist, calcitonin receptor agonist, PTH (parathyroid hormone) receptor agonist, ghrelin receptor agonist, endocannabinoid receptor agonist, leptin receptor agonist, serotonin receptor agonist, fibroblast growth factor 21 (FGF21) receptor agonist, cholecystokinin (CCK) receptor agonist, oxyntomodulin receptor agonist or glucagon receptor agonist; preferably an amylin receptor agonist; further preferably, an amylin analog or a calcitonin analog.

17. The use or method according to any one of claims 1 to 16, wherein The polypeptide comprises the following amino acid sequence: ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2 or KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2, preferably, the polypeptide derivative is selected from: any one or more of N1, N2, N3 or M1-M30.

18. The use according to claim 1 or claims 7-17 or the method according to claim 4 or claims 7-17, wherein The selectivity of improving the activity of the polypeptide derivative is that, compared with improving the activity of the polypeptide derivative on the calcitonin receptor, it is more inclined to select improving the activity of the polypeptide derivative on the amylin receptor. Preferably, the biological activity selectivity refers to improving the ratio of the activity of the polypeptide derivative on the amylin receptor to the activity of the calcitonin receptor; further preferably, it also includes improving the activity of the polypeptide derivative on the amylin receptor.

19. The use according to claim 1 or claims 7-18 or the method according to claim 4 or claims 7-18, wherein The method for improving the activity selectivity of the polypeptide derivative is to increase the ratio of the cellular activity of the amylin receptor to the calcitonin receptor by more than 1.5 times, preferably, more than 2 times; further preferably, more than 5 times; more preferably, more than 10 times.

20. The use or method of claim 18 or claim 19, wherein the activity is agonist activity.

21. The use according to claim 1 or claims 7-20 or the method according to claim 4 or claims 7-20, wherein The polypeptide derivative is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin derivative; more preferably, the polypeptide derivative comprises the following amino acid sequence: ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2 or KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2.

22. The use according to claim 2 or the method according to claim 5, wherein The activity of the polypeptide derivative is its activity in reducing weight and / or reducing food intake.

23. The use according to claim 2 or claims 7-17 or the method according to claim 5 or claims 7-17, wherein The method of improving the activity of the polypeptide derivatives is to improve the cell activity of the polypeptide derivatives on amylin receptors and calcitonin receptors.

24. The use according to claim 2 or claims 7-17 or claims 22-23 or the method according to claim 5 or claims 7-17 or claims 22-23, wherein The polypeptide derivative is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin derivative; more preferably, the polypeptide derivative comprises the following amino acid sequence: KCNTATCATQ RLADFLRHS NNLKPILPPT NVGSNT-trans-Hyp-NH2, or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2.

25. The use according to claim 3 or claims 7-17 or the method according to claim 6 or claims 7-17, wherein The polypeptide derivative containing fatty acid side chains is co-administered with one or more oral delivery agents; preferably, the oral delivery agent is N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC) salt; more preferably, PNAC.

26. The use according to claim 3 or claims 7-17 or the method according to claim 6 or claims 7-17, wherein The improved oral availability of the polypeptide derivative is higher than that of the Cagrilintide polypeptide molecule.

27. The use according to claim 3 or claims 7-17 or the method according to claim 6 or claims 7-17, wherein The polypeptide derivative is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin derivative; more preferably, the polypeptide derivative comprises the following amino acid sequence: KCNTATCATQ RLADFLRHS NNLKPILPPT NVGSNT-trans-Hyp-NH2, or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2.

28. A polypeptide derivative, comprising a polypeptide and a side chain connected to the polypeptide, wherein the 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 n AEEAs, where n≥0.

29. The derivative according to claim 28, wherein The Z1 is C16, C17, C18, C19 or C20 fatty diacid.

30. The derivative according to claim 28 or 29, wherein The Z2 is selected from any one of γGlu, Inp, Trx or Z2 does not exist.

31. The derivative according to any one of claims 28 to 30, wherein The n is greater than 0.

32. The derivative according to any one of claims 28 to 31, wherein The Z2 is Inp or Trx; and the n is greater than 0.

33. The derivative according to any one of claims 28 to 32, wherein The Z2 does not exist; the n is greater than 0.

34. The derivative according to any one of claims 28 to 33, wherein The Z2 is γGlu; ​​and n=2.

35. The derivative according to any one of claims 28 to 34, wherein The polypeptide is an amylin receptor agonist; preferably, the polypeptide derivative is an amylin or calcitonin-like organism; more preferably, the polypeptide comprises the following amino acid sequence: ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2 or KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2, preferably, the polypeptide derivative is selected from: any one or more of N1, N2, N3 or M1-M30.

36. A pharmaceutical composition comprising a derivative or a pharmaceutically acceptable salt thereof according to any one of claims 18 to 25, and a pharmaceutically acceptable excipient, preferably, the composition comprises one or more oral delivery agents; more preferably, the oral delivery agent is N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC) salt; further preferably PNAC.

37. Use of the derivative according to any one of claims 28 to 35 or a pharmaceutically acceptable salt thereof or the composition according to claim 36 in the preparation of a polypeptide drug; preferably, the polypeptide drug is used to prevent and / or treat overweight, obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain.

38. The use according to claim 37, wherein the polypeptide drug is an oral drug; preferably, the oral drug comprises one or more oral delivery agents; more preferably, the oral delivery agent is N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC) salt; further preferably PNAC.

39. Use of the derivative according to any one of claims 28 to 35 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 36 in reducing food intake.

40. 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 or pharmaceutically acceptable salt thereof according to any one of claims 28 to 35 or the pharmaceutical composition according to claim 36.

41. A method for reducing food intake, comprising administering to a subject an effective amount of the derivative or pharmaceutically acceptable salt thereof according to any one of claims 28 to 35 or the pharmaceutical composition according to claim 36.