Human amylin analog, and derivative and medical use thereof

Abstract

The present invention relates to a human amylin analog, and a derivative and a medical use thereof. The present invention introduces unnatural amino acids into amylin, and a human amylin polypeptide analog and a derivative thereof are obtained by means of process technologies such as amino acid substitution, deletion, addition, insertion, and chemical fragment modification. The human amylin polypeptide analog and derivative thereof have improved physicochemical properties and / or pharmacological efficacy characteristics, and can be used for the treatment of metabolic-related diseases such as diabetes and obesity.

Description

A human amylin analogue and its derivatives and their medicinal uses Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a human pancreatic amylin analogue and its derivatives, and their medical uses. Background Technology

[0002] Diabetes and obesity are chronic metabolic diseases affecting global health, and along with cardiovascular disease and malignant tumors, they have become major global health problems. Obesity leads to insulin resistance and hyperglycemia, a major risk factor for type 2 diabetes, with nearly 80% of type 2 diabetes patients being obese or overweight. Conversely, hyperglycemia inhibits fat breakdown, causing fat, especially visceral fat, to accumulate in the body, further worsening insulin resistance and metabolic disorders. A vicious cycle exists between diabetes and obesity, mutually promoting each other's development. This poses a significant threat to human health, increasing the risk of various chronic diseases such as cardiovascular disease, cancer, fatty liver, and kidney disease. Although the growing number of people with diabetes and obesity will impact public health and the economy, there are currently no effective measures to curb the rapid increase in diabetes and obesity.

[0003] The amylin receptor (AMYR) is a specialized protein heterodimer composed of a calcitonin receptor and a receptor activity modifier (RAMP). There are three subtypes: AMY1R, AMY2R, and AMY3R. Amylin receptors respond to peptides such as amylin and calcitonin, regulating appetite, energy metabolism, and blood glucose levels, making them important drug targets for the treatment of diabetes and obesity.

[0004] Human amylin (hIAPP) is a peptide composed of 37 amino acids. It regulates blood sugar control through mechanisms such as slowing gastric emptying, modulating postprandial glucagon, and reducing food intake. However, natural amylin is chemically unstable and precipitates at physiological pH levels due to its tendency to form fibrosis in solution, making it difficult to formulate as a drug.

[0005] Amylin Pharmaceuticals developed pramlintide through a mutation of amylin, which was approved by the FDA in 2005 for the treatment of diabetes. Compared to natural amylin, pramlintide has proline substitutions at positions 25, 28, and 29. This mutation reduces the tendency for peptide fibrillation; however, pramlintide has a plasma half-life of only 48 minutes, requiring 2-3 injections daily, thus limiting clinical convenience. Furthermore, due to disulfide bonds and deamidation, pramlintide is chemically unstable at neutral pH and can only be formulated into acidic preparations. These acidic preparations are incompatible with neutral preparations used for GLP-1 analogs and insulin, preventing the formulation of combination drugs with these compounds.

[0006] CN201280028554.1 reports an improved amylin analogue, wherein the human amylin polypeptide has an albumin-binding moiety and significantly prolongs the half-life compared to pramlintide. One preferred compound disclosed is Cagrilintide (also known as AM833), which is under clinical development. Its chemical structure is “N-α-[(S)-4-carboxy-4-(19-carboxynonadecanylamino)butyryl]-[Glu14,Arg17,Pro37]-pramlintide” analogue. The amino acid sequence of the polypeptide is as follows: KC()NTATC()ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2, where the -NH2 at the C-terminus indicates that the C-terminal carboxyl group is in amide form; and the parentheses () connecting the two cysteine ​​(C, Cys) residues at positions 2 and 7 indicate the existence of an intramolecular disulfide bridge between the two Cys residues involved.

[0007] In addition, WO2015 / 040182, WO2016 / 146739, WO2018 / 046719, WO2022 / 187305, WO2022 / 187305, WO2023 / 227133, WO2024 / 164776 and WO2024 / 022465 reported different modified forms of amylin analogs and their derivatives, with the aim of extending the half-life, improving physical / chemical stability or drug activity.

[0008] However, there is still a need to develop long-acting human amylin analogues or derivatives with higher potency, improved physicochemical stability, and / or better hypoglycemic / weight-loss effects, thereby improving patient adherence.

[0009] Invention Overview

[0010] On one hand, the present invention relates to human amylin polypeptide analogs, pharmaceutically acceptable salts or solvates thereof, said amylin polypeptide analogs comprising the following amino acid sequence:

[0011] Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Equation I), where:

[0012] Xaa1 is selected independently from K, R, Har, Orn, Dab, hLys, or is missing;

[0013] Xaa3 is independently selected from N or G;

[0014] Xaa 10 Independently selected from Q, G, E, N, or D;

[0015] Xaa 11 Independently selected from: R, K, or Orn;

[0016] Xaa 14 Choose independently from: E, D, or Aad;

[0017] Xaa 17 Selected independently from: R, K, or Q;

[0018] Xaa 18 Independently selected from: H or R;

[0019] Xaa 21 Selected independently from: N, S, P, H, or missing;

[0020] Xaa 22 Selected independently from: N, S, P, MeAsn or missing;

[0021] Xaa 24 Independently selected from: G or MeGly;

[0022] Xaa 25 Independently selected from: P or A;

[0023] Xaa 26 Independently selected from: I, K, or MeIle;

[0024] Xaa28 Independently selected from: S, T, or P;

[0025] Xaa 29 Independently selected from: S, T, or P;

[0026] Xaa 31 Choose independently from: E, D, N, A, or Q;

[0027] Xaa 37 Independently selected from: Y, S, T, P, (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, 4,4-difluoro-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro;

[0028] Among them, there is disulfide bond cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

[0029] In some preferred embodiments, the amylin polypeptide analogue comprises the amino acid sequences described in numbers 4-80 of Table 1.

[0030] In a second aspect, the present invention provides a human amylase analog derivative having the structural formula AB, wherein the chemically modified fragment A has the structural formula XY. n -L m (Formula III), where X is the albumin-binding group, Y is the hydrophilic spacer group, and L is the linker; B is an amylase polypeptide analog whose amino acid sequence includes: Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Equation I), where:

[0031] Xaa1 is K, R, Har, Orn, Dab, hLys, or missing;

[0032] Xaa3 is N or G;

[0033] Xaa 10 It is Q, G, E, N, or D;

[0034] Xaa 11 It is R, K, or Orn;

[0035] Xaa 14 It is E, D, or Aad;

[0036] Xaa 17 It is R, K, or Q;

[0037] Xaa 18 It is H or R;

[0038] Xaa 21 It is N, S, P, H, or missing;

[0039] Xaa 22 It is N, S, P, MeAsn, or missing;

[0040] Xaa 24 It is G or MeGly;

[0041] Xaa25 is either P or A;

[0042] Xaa 26 Is it I, K, or MeIle?

[0043] Xaa 28 Is it S, T, or P;

[0044] Xaa 29 Is it S, T, or P;

[0045] Xaa 31 It is E, D, N, A, or Q;

[0046] Xaa 37 Is Y, S, T, P, (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, 4,4-difluoro-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro;

[0047] Among them, there is disulfide bond cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

[0048] In another embodiment, the present invention relates to human amylin analog derivatives, wherein the linking site of the chemically modified fragment A is the α-amino or Epsilon-amino group of the N-terminal (Xaa1) amino acid in the human amylin analog polypeptide B, or the linking site of the chemical fragment A is the K amino group in the polypeptide B. 26The Epsilon-amino group of amino acids.

[0049] In some preferred embodiments, the chemical modification fragment A in the amylin analogue derivative comprises the chemical fragments described in Tables 1-28.

[0050] In some preferred embodiments, the amylin analogue derivative comprises the polypeptide compounds described in Table 3, 4-213.

[0051] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide analog or a derivative thereof, and a pharmaceutically acceptable carrier.

[0052] In another embodiment, the present invention provides the use of the human amylin polypeptide analogue or its derivatives for the treatment of obesity and obesity-related diseases, including but not limited to overweight, morbid obesity, preoperative obesity, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea and breathing problems, cartilage degeneration, osteoarthritis, non-alcoholic fatty liver disease (NAFLD), and reproductive health complications of obesity or overweight (e.g., infertility). The subjects may be affected by obesity accompanied by at least one weight-related comorbidity (e.g., diabetes, hypertension, dyslipidemia, sleep apnea, and cardiovascular disease).

[0053] In another embodiment, the present invention provides the use of the human amylin polypeptide analogue or its derivatives for the prevention or treatment of diabetes and related diseases, including type 1 diabetes, type 2 diabetes, prediabetes, insulin resistance syndrome, non-alcoholic fatty liver disease (NAFLD), impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, and diabetes-related complications, including but not limited to cardiovascular and cerebrovascular diseases (atherosclerosis, large vessel disease, microvascular disease, coronary heart disease, peripheral artery disease, or stroke), lower extremity vascular disease (diabetic foot ulcers), eye diseases, peripheral neuropathy, diabetic cardiomyopathy, and nephropathy, or combinations thereof.

[0054] In another embodiment, the present invention provides the use of combining the human amylin polypeptide analogue or its derivative with one or more targeted drugs for the prevention or treatment of obesity or diabetes and related diseases. The targeted drugs include, but are not limited to, diabetes medications, obesity medications, and hypertension medications, such as GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, GLP-1 / GIP / GCGR triple agonists, FGF21 derivatives, insulin, metformin, sulfonylureas, meglitinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc., with more specific drug types selected from exenatide, lixinatide, liraglutide, smegglutide, dulaglutide, abiglutide, leptin, neuropeptide Y, tirzepatide, retatrutide, mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, etc. Attached Figure Description

[0055] Figure 1 shows the pharmacodynamic evaluation, illustrating the rate of weight change in DIO rats after subcutaneous administration of different human amylin analogue derivatives.

[0056] Figure 2 shows the pharmacodynamic evaluation, illustrating the changes in food intake in DIO rats after subcutaneous administration of different human amylin analogue derivatives.

[0057] Figure 3 shows the pharmacokinetic evaluation, illustrating the drug metabolism patterns of different human amylin analogues after subcutaneous / intravenous administration.

[0058] Figure 4. Effects of a single subcutaneous injection of JY4 on OGTT blood glucose levels and AUC in SD rats. (0～180min) The impact (N=6, Mean±SEM).

[0059] Figure 5. Random blood glucose levels and glycosylated hemoglobin at 8 weeks in ZDF(fa / fa) type II diabetic rats.

[0060] Figure 6. Detection of renal function biochemical indicators in ZDF(fa / fa) type II diabetic rats after 8 weeks of administration.

[0061] Figure 7. Kidney HE staining and PAS staining pathological scores of ZDF(fa / fa) type II diabetic rats after 8 weeks of administration.

[0062] Figure 8 Random blood glucose monitoring in BKS-db / db mice 24 hours after the first administration.

[0063] Figure 9. Monitoring of fasting blood glucose levels in BKS-db / db mice.

[0064] Figure 10 Monitoring of fasting blood glucose levels in DIO mice.

[0065] Figure 11 Random blood glucose monitoring in DIO mice 24 hours after the first and last administration.

[0066] Figure 12 Effects of repeated subcutaneous injection of JY4 on body weight and food intake in DIO rats (N=8, Mean±SEM).

[0067] Figure 13 Effects of repeated subcutaneous administration of JY4 injection combined with semaglutide on body weight and food intake in DIO rats (N=8, Mean±SEM).

[0068] Figure 14 Effects of repeated subcutaneous administration of JY4 injection combined with Tirzepatide on body weight and food intake in DIO rats (N=6, Mean±SEM).

[0069] Invention Details

[0070] To facilitate understanding of this invention, certain terms are first defined. Unless otherwise stated, scientific and technical terms used herein should have the meanings commonly understood by one of ordinary skill in the art. Other definitions will be clarified throughout the detailed description.

[0071] Unless otherwise stated, the present invention will be carried out using conventional techniques of chemistry, molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, immunology, pharmacology, and protein and nucleic acid chemistry, all of which are within the scope of the art and are fully explained in the technical literature and general textbooks in the field, such as Molecular Cloning: A Laboratory Manual.

[0072] The term "amino acid" as used in this article is defined as both natural and non-natural amino acids. Natural amino acids include, but are not limited to, alanine (Ala), arginine (Arg), asparagine (Asn), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).

[0073] "Non-natural amino acids" refer to amino acids that are not encoded by the existing 64 genetic codons. The non-natural amino acids referred to in this article include, but are not limited to, L-ornithine (L-Orn), L-diaminobutyric acid, dimethylalanine (Aib), L-homogeneous acid, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine (D-Orn), D-diaminobutyric acid, D-homogeneous acid, D-citrulline, D-diaminopropionic acid, (2S,4R)-4-hydroxyproline, (2S,4S)-4-hydroxyproline, (2S,4R)-4-fluoroproline, (2S,4S)-4-fluoroproline, 4,4-difluoroproline, (2S,3R)-3-hydroxyproline, (2S,3S)-3-hydroxyproline, and 4-oxoproline.

[0074] The term "human amylin polypeptide" as used in this article refers to the polypeptide having the sequence described in SEQ ID No:1. Human amylin polypeptide (hIAPP) is a polypeptide hormone composed of 37 amino acid residues. After being synthesized intracellularly, it is stored co-located with insulin in insulin-secreting vesicles within pancreatic β-cells and is secreted co-located by pancreatic β-cells in response to external stimuli. In this article, SEQ ID No:1 and human amylin polypeptide are used interchangeably. The amino acid sequence and structure of human amylin polypeptide (hIAPP) are shown below:

[0075] Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID NO: 1)

[0076] As used herein, "pralinin peptide" refers to a synthetic polypeptide having the sequence described in SEQ ID No:2. In this document, SEQ ID No:2 and pralinin peptide are used interchangeably. The amino acid sequence and structure of pralinin peptide as defined herein are shown below:

[0077] Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID NO: 2)

[0078] As used herein, “Cagrilintide” refers to a synthetic polypeptide having the sequence described in SEQ ID No:3. In this document, SEQ ID No:3 and Cagrilintide / AM833 are used interchangeably. The amino acid sequence of Cagrilintide as defined herein is shown below, wherein an intramolecular disulfide bond exists between the two Cys residues at positions 2 and 7, and the C-terminus of the polypeptide is an amide:

[0079] KCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP(SEQ ID No:3) or Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Glu Phe Leu Arg His Ser Ser Asn Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val Gly Ser Asn Thr Pro(SEQ ID No:3)

[0080] The sequence “variant” used in this article refers to a sequence that differs from the parent sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0081] The terms "amylin analogue" or "amylin polypeptide analogue" used herein are interchangeable and are defined as: one or more amino acid substitutions and / or one or more deletions and / or one or more additions / insertions of an amylin polypeptide as described above. Amino acid substitution modification refers to the replacement of an amino acid residue with an amino acid residue having similar side chains or physicochemical characteristics. The amino acid can be natural or non-natural. Such amino acid substitution modifications include, but are not limited to, substitutions of lysine-arginine, aspartic acid-glutamic acid, valine-arginine, and tyrosine-proline in the peptide chain. The number of amino acid insertions, additions, deletions, or substitutions can be at least one, but can be up to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen amino acid insertions, additions, deletions, or substitutions. Substitutions or additions can be any natural or non-natural amino acid, synthetic amino acid, peptide-like compound, or other compound.

[0082] The term "amylin analogue derivative" as used herein is defined as: a product obtained by chemically modifying the aforementioned amylin polypeptide or analogue. Chemical modification includes, but is not limited to, amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. Amylin analogue derivatives may contain one or more substituents on one or more amino acid residues of the polypeptide.

[0083] The term "disulfide cyclization" as used in this article refers to the organic synthesis of disulfide groups to functionally rebridge disulfide bonds within the peptide structure. A disulfide bond (-SS-) is shown below, with an asterisk (*) indicating that other groups or atoms can be attached at that position (and so on):

[0084] The term "thioacetal cyclization" used in this article refers to the organic synthesis of thioacetal groups to functionally rebridge disulfide bonds within a peptide structure. The thioacetal bridge (-S-CH2-S-) is shown below, with a methylene group inserted between two sulfur atoms:

[0085] As used herein, the term "medicinal salt" is intended to mean a salt that is harmless to a patient or subject when administered to them. It may suitably be a salt selected, for example, from acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrates, and acetates. Examples of basic salts include salts in which the cation is selected from: alkali metal cations (e.g., sodium or potassium ions), alkaline earth metal cations (e.g., calcium or magnesium ions), and substituted ammonium ions. Further examples of medicinal salts are described in Remington's Pharmaceutical Sciences, 17th edition, edited by Alfonso R. Gennaro, Mark Publishing Company, Easton, PA, USA, 1985 (and its latest edition), Encyclopaedia of Pharmaceutical Technology, 3rd edition, edited by James Swarbrick, Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66:2 (1977).

[0086] In the context of this invention, the term "solvate" refers to a stoichiometric complex formed between a solute (in this case, a peptide or its pharmaceutically acceptable salt according to the invention) and a solvent. In this respect, the solvent can be, for example, water, ethanol, or other pharmaceutically acceptable (typically small molecule) organic substances, such as, but not limited to, acetic acid or lactic acid. When the solvent involved is water, such sovates are generally referred to as hydrates.

[0087] As used herein, the term "substituent" refers to any suitable portion that bonds (especially covalently) to an amino acid residue, particularly to any available position on the amino acid residue. Typically, the suitable portion is the chemical part. Substituents are attached to native or non-native amino acids of the polypeptide. In some embodiments, the derivative has a substituent on an amino acid residue that is an N-terminal amino acid residue. In some embodiments, the derivative has a substitution on an N-terminal amino acid residue that is homoarginine.

[0088] As used herein, the term "albumin-binding group" refers to a group that is non-covalently bound to human serum albumin. Albumin-binding residues linked to human amylin polypeptide analogs typically have a binding affinity to human serum albumin of less than about 10 μM or even less than about 1 μM. "Albumin-binding affinity" can be determined by several methods known in the art, and the EC50 value used for competition is a measure of the affinity of the compound. Various albumin-binding residues are known, including linear and branched lipophilic moieties containing 12-40 carbon atoms, compounds with a cyclopentanophenylene backbone, and / or peptides with 10-45 amino acid residues. Albumin binding properties can be measured by surface plasmon resonance as described in the following reference: J. Biol. Chem. 277(38), 35035-35042, (2002). The method for determining albumin-binding residues and affinity is described in detail in CN201280028554.1 and CN201180015252.6, both of which are incorporated herein by reference. In one embodiment, the albumin-binding residue is preferably C20 diacid.

[0089] As used herein, the term "hydrophilic spacer" refers to any suitable portion that connects the albumin-binding group and the linker. The hydrophilic spacer may be used together with a substituent portion (e.g., the albumin-binding group and / or the linker) to form a new substituent. The hydrophilic spacer may be present independently or absent.

[0090] As used herein, the term "linker" refers to any suitable portion that links an albumin-binding group and / or a hydrophilic spacer to a human amylin polypeptide analog. The linker may be a novel substituent together with the substituent portion (e.g., the albumin-binding group and / or the hydrophilic spacer). The linker may be present independently or absent. The linker may contain one or more amino acids; in one embodiment, the linker is preferably Glu. The linker binds to the albumin-binding portion at one end and to an amino group on a non-natural amino acid at the N-terminus of the human amylin analog at the other end.

[0091] As used herein, the term "fibrillation" refers to the physical interactions between polypeptide molecules that result in the formation of oligomers, which can remain dissolved or precipitate from solution as large, visible aggregates. The degree of polypeptide fibrillation can be assessed by visual inspection, chromatographic methods, ThT fibrillation assays (sometimes called ThT fibrillation formation assays), and / or turbidity measurements, the relevant methods of which are described in detail in CN201280028554.1, etc., and are incorporated herein by reference in their entirety.

[0092] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective dose also means a dose sufficient to enable or facilitate diagnosis. The effective dose for a specific patient can vary depending on various factors, such as the disease being treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0093] "Pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, etc. Preferably, the carrier for a composition containing a polypeptide or a derivative thereof is suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0094] The terms "subject" or "patient" include both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, mice, rats, cats, cattle, horses, chickens, amphibians, and reptiles.

[0095] Human pancreatic amylin polypeptide analogs and their derivatives

[0096] This invention relates to human amylin polypeptide analogs and their derivatives. Human amylin polypeptide analogs and their derivatives are obtained through processes such as amino acid substitution, deletion, addition, insertion, and chemical fragment modification. These analogs possess improved physicochemical properties and / or pharmacological and pharmacodynamic characteristics and can be used for the treatment of metabolic-related diseases such as diabetes and obesity.

[0097] On one hand, the present invention relates to human amylin polypeptide analogs, pharmaceutically acceptable salts or solvates thereof, said amylin analogs comprising the following amino acid sequence:

[0098] Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22-F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Equation I), where:

[0099] Xaa1 is selected independently from K, R, Har, Orn, Dab, hLys, or is missing;

[0100] Xaa3 is independently selected from N or G;

[0101] Xaa 10 Independently selected from Q, G, E, N, or D;

[0102] Xaa 11 Independently selected from: R, K, or Orn;

[0103] Xaa 14 Choose independently from: E, D, or Aad;

[0104] Xaa 17 Selected independently from: R, K, or Q;

[0105] Xaa 18 Independently selected from: H or R;

[0106] Xaa 21 Selected independently from: N, S, P, H, or missing;

[0107] Xaa 22 Selected independently from: N, S, P, MeAsn or missing;

[0108] Xaa 24 Independently selected from: G or MeGly;

[0109] Xaa 25 Independently selected from: P or A;

[0110] Xaa 26 Independently selected from: I, K, or MeIle;

[0111] Xaa 28 Independently selected from: S, T, or P;

[0112] Xaa 29 Independently selected from: S, T, or P;

[0113] Xaa 31 Choose independently from: E, D, N, A, or Q;

[0114] Xaa37 Independently selected from: Y, S, T, P, (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, 4,4-difluoro-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro;

[0115] Among them, there is disulfide bond cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

[0116] In some preferred embodiments, the amyloid analogue comprises Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 The amino acid sequence shown in Formula I, wherein:

[0117] Xaa1 is independently selected from: R, Har, Orn, or K;

[0118] Xaa3 is independently selected from N or G;

[0119] Xaa 10 Independently selected from Q, E, or G;

[0120] Xaa 11 Independently selected from R, K, or Orn;

[0121] Xaa 14 Independently selected from: E or Aad;

[0122] Xaa 17 Independently selected from R or Q;

[0123] Xaa 18 Independently selected from H or R;

[0124] Xaa 21 Selected independently from N or missing;

[0125] Xaa 22 Selected independently from N or missing;

[0126] Xaa 24 Independently selected from G or MeGly;

[0127] Xaa 25 Independently selected from P or A;

[0128] Xaa 26 Independently selected from I, K, or MeIle;

[0129] Xaa 28 Independently selected from S or P;

[0130] Xaa 29 Independently selected from S or P;

[0131] Xaa 31 Independently selected from E or N;

[0132] Xaa 37 Independently selected from (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro;

[0133] The amino acids at positions 2 and 7 exhibit disulfide cyclization or thioacetal bridging, and the C-terminus of the polypeptides are all amides.

[0134] In some preferred embodiments, the amyloid analogue comprises Har-C-Xaa3-TATCAT-Xaa 10 -RLA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 The amino acid sequence described in (Formula II) wherein:

[0135] Xaa3 is independently selected from N or G;

[0136] Xaa 10 Independently selected from Q or E;

[0137] Xaa 17 Independently selected from R or Q;

[0138] Xaa 18 Independently selected from H or R;

[0139] Xaa 21 Selected independently from N or missing;

[0140] Xaa 22 Selected independently from N or missing;

[0141] Xaa 24 Independently selected from G or MeGly;

[0142] Xaa 25 Independently selected from P or A;

[0143] Xaa 26 Independently selected from I, K, or MeIle;

[0144] Xaa 28 Independently selected from S or P;

[0145] Xaa 29 Independently selected from S or P;

[0146] Xaa 31 Independently selected from E or N;

[0147] Xaa 37 The polypeptide is independently selected from (2S,4R)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp, or 4-Oxo-Pro; wherein there is disulfide cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

[0148] In some embodiments, preferred amylin polypeptide analogs comprise the amino acid sequences described in numbers 4-80 of Table 1.

[0149] Table 1. Amino acid sequence list of pancreatin analogues Note: In the polypeptide sequence 4-80, there is disulfide bond cyclization or thioacetal bridging between amino acids at positions 2 and 7, and the C-terminus is amide.

[0150] In a second aspect, the present invention provides a human amylase analog derivative modified with an albumin-binding group, having the structural formula AB, wherein the chemically modified fragment A in the derivative has the following structural formula XY. n -L m(Formula III), where X is the albumin-binding group, Y is the hydrophilic spacer group, and L is the linker; B in the derivative is an amylin polypeptide analog, whose amino acid sequence includes:

[0151] Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Equation I), where:

[0152] Xaa1 is K, R, Har, Orn, Dab, hLys, or missing;

[0153] Xaa3 is N or G;

[0154] Xaa 10 It is Q, G, E, N, or D;

[0155] Xaa 11 It is R, K, or Orn;

[0156] Xaa 14 It is E, D, or Aad;

[0157] Xaa 17 It is R, K, or Q;

[0158] Xaa 18 It is H or R;

[0159] Xaa 21 It is N, S, P, H, or missing;

[0160] Xaa 22 It is N, S, P, MeAsn, or missing;

[0161] Xaa 24 It is G or MeGly;

[0162] Xaa25 is either P or A;

[0163] Xaa 26 Is it I, K, or MeIle?

[0164] Xaa 28Is it S, T, or P;

[0165] Xaa 29 Is it S, T, or P;

[0166] Xaa 31 It is E, D, N, A, or Q;

[0167] Xaa 37 Is Y, S, T, P, (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, 4,4-difluoro-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro;

[0168] Among them, there is disulfide bond cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

[0169] In some embodiments, the human amylin analogue derivative of the present invention, wherein the amylin polypeptide analogue B comprises the amino acid sequence shown in Formula I, wherein:

[0170] Xaa1 is independently selected from: R, Har, Orn, or K;

[0171] Xaa3 is independently selected from N or G;

[0172] Xaa 10 Independently selected from Q, E, or G;

[0173] Xaa 11 Independently selected from R, K, or Orn;

[0174] Xaa 14 Independently selected from: E or Aad;

[0175] Xaa 17 Independently selected from R or Q;

[0176] Xaa 18 Independently selected from H or R;

[0177] Xaa 21 Selected independently from N or missing;

[0178] Xaa 22 Selected independently from N or missing;

[0179] Xaa 24 Independently selected from G or MeGly;

[0180] Xaa 25 Independently selected from P or A;

[0181] Xaa 26 Independently selected from I, K, or MeIle;

[0182] Xaa 28 Independently selected from S or P;

[0183] Xaa 29 Independently selected from S or P;

[0184] Xaa 31 Independently selected from E or N;

[0185] Xaa 37 Independently selected from (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro;

[0186] The amino acids at positions 2 and 7 exhibit disulfide cyclization or thioacetal bridging, and the C-terminus of the polypeptides are all amides.

[0187] In some embodiments, the human amylin analogue derivative of the present invention, wherein the amylin polypeptide analogue B comprises the amino acid sequence of Formula II, wherein:

[0188] Har-C-Xaa3-TATCAT-Xaa 10 -RLA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Formula II)

[0189] Xaa3 is independently selected from N or G;

[0190] Xaa 10 Independently selected from Q or E;

[0191] Xaa 17 Independently selected from R or Q;

[0192] Xaa 18 Independently selected from H or R;

[0193] Xaa21 Selected independently from N or missing;

[0194] Xaa 22 Selected independently from N or missing;

[0195] Xaa 24 Independently selected from G or MeGly;

[0196] Xaa 25 Independently selected from P or A;

[0197] Xaa 26 Independently selected from I, K, or MeIle;

[0198] Xaa 28 Independently selected from S or P;

[0199] Xaa 29 Independently selected from S or P;

[0200] Xaa 31 Independently selected from E or N;

[0201] Xaa 37 The polypeptide is independently selected from (2S,4R)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp, or 4-Oxo-Pro; wherein there is disulfide cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

[0202] In some embodiments, the human amylin analogue derivatives of the present invention, wherein the amylin polypeptide analogue B comprises the amino acid sequences shown in SEQ ID NO:4-SEQ ID NO:80.

[0203] In some embodiments, the albumin-binding group in the human amylase analog derivative of the present invention may have the following structure:

[0204] HOOC(CH2) O CO-, where o is an integer between 14 and 22;

[0205] (HO)2PO(CH2) p CO-, where p is an integer from 14 to 22;

[0206] HO3S(CH2) q CO-, where q is an integer between 14 and 22;

[0207] Where r is an integer between 14 and 22;

[0208] Where s is an integer between 14 and 22;

[0209] Where t is an integer between 13 and 21;

[0210] Where u is an integer between 14 and 22;

[0211] or Where v is an integer between 14 and 22.

[0212] In another preferred embodiment, the albumin-binding group is selected from HOOC(CH2). o CO-, where o is an integer between 16 and 22, and more preferably selected from HOOC(CH2). 16 CO-, HOOC(CH2) 18 CO-, HOOC(CH2) 20 CO- or HOOC(CH2) 22 CO-.

[0213] In another preferred embodiment, the albumin-binding group is selected from (HO)₂PO(CH₂). P CO-, where p is an integer from 16 to 22, and more preferably selected from (HO)2PO(CH2). 16 CO-, (HO)2PO(CH2) 18 CO-, (HO)2PO(CH2) 20 CO- or (HO)2PO(CH2) 22 CO-.

[0214] In another preferred embodiment, the albumin-binding group is selected from -CO(CH2). r CN4H(tetrazole), where r is an integer from 16 to 22, and more preferably selected from -CO(CH2). 16 CN4H(tetrazole), -CO(CH2) 18 CN4H(tetrazole), -CO(CH2) 20 CN4H (tetrazole) or -CO (CH2) 22 CN4H (tetrazole);

[0215] In some embodiments, the hydrophilic spacer group Y in the human pancreatic amylin analogue derivative of the present invention n The hydrophilic spacer group may be absent, or it may consist of one or more hydrophilic spacer groups, where n is an integer from 0 to 3. The structure of the hydrophilic spacer group Y is selected from the following structures:

[0216] In another preferred embodiment, the hydrophilic spacer group is absent.

[0217] In another preferred embodiment, the hydrophilic spacer group may be AEEA, with the structure shown below:

[0218] In another preferred embodiment, the hydrophilic spacer group may be AEEA-AEEA-Glu, with the structure shown below:

[0219] In some embodiments, in the human pancreatic amylin analogue derivatives of the present invention, the connector L m The connector may be absent, or it may consist of one or more connectors, where m is an integer from 0 to 2. The connector L is selected from the following structures:

[0220] In another preferred embodiment, the connector is not present.

[0221] In another preferred embodiment, the connector can be γGlu, with the structure shown below:

[0222] In some embodiments, the amylin analogue derivatives of the present invention contain chemical modification fragment A comprising the chemical fragments described in Tables 1-28.

[0223] Table 2 List of Structural Formulas of Chemically Modified Fragments

[0224] *In the table, Glu refers to γGlu.

[0225] In another preferred embodiment, the chemically modified fragment may be Glu-C20 diacid, with the structure shown below:

[0226] In another preferred embodiment, the chemically modified fragment may be Glu-CO(CH2). 18 PO3H2 has the following structure:

[0227] In another preferred embodiment, the chemically modified fragment may be Glu-CO(CH2). 18 CN4H (tetrazole) has the following structure:

[0228] In another preferred embodiment, the chemically modified fragment may be AEEA-AEEA-Glu-C20diacid, with the following structure:

[0229] In another preferred embodiment, the chemically modified fragment may be AEEA-AEEA-Glu-CO(CH2). 18 PO3H2 has the following structure:

[0230] This invention relates to human amylin analogue derivatives, wherein the linking site of the chemically modified fragment A is the α-amino or Epsilon-amino group of the N-terminal (Xaa1) amino acid in the human amylin analogue polypeptide B, or the linking site of the chemical fragment A is the K amino group in the polypeptide B. 26 The epsilon-amino group of an amino acid. Preferably, the chemical fragment A is linked to the N-terminus of polypeptide B; more preferably, the chemical fragment A is linked to the α-amino group at the N-terminus of polypeptide B. More preferably, the chemical fragment A is linked to the α-amino group of the N-terminal homoarginine in polypeptide B.

[0231] In some embodiments, the amylin analogue derivatives of the present invention preferably comprise the compounds described in Table 3, 4-213.

[0232] Table 3 List of compounds that are analogues of amylin Note: Unless otherwise specified, the side chain of fatty acids is attached to the α-amino group of amino acids; Glu in the table refers to γGlu.

[0233] In some embodiments, the amylin analogue derivatives of the present invention are preferably selected from the compounds shown in Table 5, including JY4, JY5, JY6, JY11, JY13, JY14, JY17, JY18, JY19, JY22, JY26, JY28, JY29, JY49, JY50, JY131, JY132, JY133, JY134, JY135, JY136, JY137, JY138, JY193, JY201, JY209, JY210, JY204, JY211, JY212, or JY213.

[0234] In another preferred embodiment, the amylin analogue derivative of the present invention is compound JY4, the structure of which is shown below:

[0235] The C-terminal Hyp is (2S-4R)-Hyp and forms an amide (-NH2), and the others are similar.

[0236] In another preferred embodiment, the amylin analogue derivative of the present invention is JY26, the structure of which is shown below:

[0237] In another preferred embodiment, the amylin analogue derivative of the present invention is JY14, the structure of which is shown below:

[0238] In another preferred embodiment, the amylin analogue derivative of the present invention is JY17, the structure of which is shown below:

[0239] In another preferred embodiment, the amylin analogue derivative of the present invention is JY49, the structure of which is shown below:

[0240] In another preferred embodiment, the amylin analogue derivative of the present invention is JY209, the structure of which is shown below:

[0241] In another preferred embodiment, the amylin analogue derivative of the present invention is JY210, the structure of which is shown below:

[0242] In another preferred embodiment, the amylin analogue derivative of the present invention is JY204, the structure of which is shown below:

[0243] In another preferred embodiment, the amylin analogue derivative of the present invention is JY193, the structure of which is shown below:

[0244] In another preferred embodiment, the amylin analogue derivative of the present invention is JY211, the structure of which is shown below:

[0245] In another preferred embodiment, the amylin analogue derivative of the present invention is JY201, the structure of which is shown below:

[0246] In another preferred embodiment, the amylin analogue derivative of the present invention is preferably JY213, the structure of which is shown below:

[0247] This invention introduces non-natural amino acids into amylin, which can effectively improve amylin activity; effectively prevent enzyme degradation and prolong half-life; and, when a non-natural basic amino acid is preferably introduced at the N-terminus, the isoelectric point of the peptide can be effectively increased, resulting in improved solubility and / or physical stability. Compared with reported human amylin analogs or derivatives thereof, it exhibits improved performance in at least one aspect, such as physicochemical properties, pharmacodynamics, or pharmacokinetic parameters.

[0248] This invention introduces fatty acid chemically modified fragments into amylin, which can effectively prevent polypeptides from being degraded by enzymes and reduce glomerular filtration clearance, thus prolonging the half-life.

[0249] Human amylin and most amylin analogues (such as pramlintide and cagrilintide) have low chemical stability under neutral conditions. This invention removes N21 and N22 and / or replaces certain amino acids at different positions, which can effectively improve the chemical stability of amylin analogues.

[0250] Human amylin is prone to fibrosis in aqueous solution. The present invention can effectively inhibit the fibrosis of amylin analogues by replacing certain amino acids at different positions (such as replacing them with N-methylated amino acids or proline).

[0251] In some embodiments, the amylin analogue derivatives provided by the present invention have comparable or better solubility, comparable or better chemical stability, and comparable or better physical stability compared to Cagrilintide.

[0252] In some embodiments, the amylin analogue derivatives provided by the present invention have comparable or higher in vitro biological activity compared to Cagrilintide.

[0253] In some embodiments, the amylin analogue derivatives provided by the present invention have similar albumin-binding capacity to Cagrilintide.

[0254] In some embodiments, the amylin analogues provided by this invention exhibit comparable or better appetite-suppressing and weight-loss effects compared to Cagrilintide, Eloralintide, or Petrelintide. When the amylin analogues provided by this invention are injected in combination with Semaglutide, the weight-loss effect in DIO rats is significantly enhanced compared to the single-component administration, and the weight loss is greater than that in the CagriSema combination group, demonstrating a good synergistic effect.

[0255] In some embodiments, the amylin analogues provided by this invention have comparable or better appetite-suppressing and weight-loss effects compared to Eloralintide. The amylin analogues provided by this invention, whether used alone or in combination with Tirzepatide, exhibit greater weight changes compared to Eloralintide or Eloralintide & Tirzepatide, demonstrating a better synergistic weight-loss effect.

[0256] In some embodiments, the amylin analogues provided by this invention have comparable or better hypoglycemic effects compared to Cagrilintide. In some embodiments, the human amylin analogues provided by this invention, alone or in combination with semaglutide, significantly reduced 4-hour random blood glucose (RBG) levels in ZDF(fa / fa) type II diabetic rats after a single subcutaneous injection.

[0257] In another embodiment, the present invention provides human amylin analogues that have good effects on improving blood glucose and protecting against diabetic nephropathy. When used in combination with semaglutide or tirzepatide, they exhibit a synergistic effect, suggesting that the human amylin analogues of the present invention can be used to treat diabetic nephropathy.

[0258] Synthesis of amylin analogues and their derivatives

[0259] This invention also relates to a method for preparing the human amylin polypeptide analogue and its derivatives, the method comprising steps of stepwise or fragment assembly via solid-phase and / or liquid-phase methods, and optionally the separation and / or purification of the final product. The method further comprises the step of forming a disulfide bond between the cysteine ​​side chain thiol groups at positions 2 and 7 via oxidative cyclization or by forming a thioacetal bridged ring via nucleophilic substitution. The method further comprises the step of forming a C-terminal amidation via recombinant expression, purification, and induction using non-synthetic methods.

[0260] The prepared human amylin polypeptide analogue can be further obtained by condensation reaction of fatty acid side chains with polypeptides to yield human amylin polypeptide derivatives, which can then be optionally separated and / or purified to obtain the final product. Similar synthetic and purification processes are described in detail in the prior art WO2006 / 105527, CN201180015252.6, CN201280028554.1, WO2012 / 168431, WO2013 / 156594, WO2015 / 040182, WO2016 / 146739, WO2018 / 046719, WO2022 / 187305, WO2022 / 187305, WO2023 / 227133 and WO2024 / 022465, all of which are incorporated herein by reference.

[0261] pharmaceutical preparations

[0262] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide analog or a derivative thereof, and a pharmaceutically acceptable carrier.

[0263] On one hand, the present invention provides a pharmaceutical composition comprising a human amylin polypeptide analog or derivative thereof as described above, formulated together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all physiologically compatible carriers such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0264] Pharmaceutical compositions must generally be sterile and stable under manufacturing and storage conditions. The compositions can be formulated into dosage forms such as solutions, microemulsions, liposomes, or lyophilized powders for injection. In one embodiment, the pharmaceutical formulation is a liquid formulation, which can be formulated as a solution or suspension. In one embodiment, the concentration of the human amylin polypeptide analog or its derivative present in the formulation is from about 0.1 mg / ml to about 25 mg / ml, more preferably from about 1 mg / ml to about 10 mg / ml. The drug can be administered directly in unit dose form, for example, via an injection pen containing the pharmaceutical formulation. Furthermore, it can be administered parenterally, such as subcutaneously, intramuscularly, intravenously, or percutaneously.

[0265] In another embodiment, the pharmaceutical preparation is a lyophilized preparation to which a doctor, nurse, or patient adds solvents and / or diluents before use.

[0266] The present invention also relates to pharmaceutical formulations comprising the aforementioned human pancreatic amylin polypeptide analogue or derivative thereof. The pharmaceutical formulation may include a pharmaceutical carrier, excipient, or protein protectant. Preferred routes of administration for the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal / spinal cord, or other parenteral routes, such as by injection or infusion.

[0267] The actual dose level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and administration method. The "therapeuticly effective amount" of the present invention's human amylin polypeptide analogue or its derivative preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability caused by the disease. Those skilled in the art will be able to determine such amounts based on factors such as the subject's body size, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0268] Drug Use

[0269] The present invention also relates to the pharmaceutical use of the aforementioned human amylin polypeptide analogue or its derivatives. In another embodiment, the present invention provides a method of treating a disease, the method comprising administering a therapeutically effective amount of the human amylin polypeptide analogue or its derivative to a subject in need of treatment.

[0270] The invention provides a method for treating a disease, the method comprising administering a therapeutically effective amount of the human amylin polypeptide analog or a derivative thereof of the present invention to a subject requiring treatment. The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, mice, rats, cats, cattle, horses, chickens, amphibians, and reptiles. In another embodiment, the recipient or individual of the human amylin polypeptide analog or a derivative thereof is a mammal, such as a mouse, monkey, dog, cattle, horse, or human, preferably a human.

[0271] In another embodiment, the present invention provides the use of the human amylin analogue or its derivatives for the prevention or treatment of obesity and obesity-related diseases, including but not limited to overweight, morbid obesity, preoperative obesity, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea and breathing problems, cartilage degeneration, osteoarthritis, metabolic dysfunction-associated steatohepatitis (MASH), and reproductive health complications of obesity or overweight (e.g., infertility). The subjects may be affected by obesity accompanied by at least one weight-related comorbidity (e.g., diabetes, hypertension, dyslipidemia, sleep apnea, and cardiovascular disease).

[0272] In another embodiment, the present invention provides a method for using the human amylin polypeptide analogue or its derivatives to prevent or treat, inhibit or reduce weight gain, promote weight loss, and / or reduce excess weight. For example, treatment can be achieved by controlling appetite, food intake, food consumption, calorie intake, and / or energy expenditure.

[0273] In another embodiment, the present invention provides the use of the human amylin analogue or its derivatives for the prevention or treatment of diabetes and related diseases, including type 1 diabetes, type 2 diabetes, prediabetes, insulin resistance syndrome, metabolic dysfunction-associated steatohepatitis (MASH), impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, and diabetes-related complications, including but not limited to cardiovascular and cerebrovascular diseases (atherosclerosis, macrovascular disease, microvascular disease, coronary artery disease, peripheral artery disease, or stroke) caused by diabetes, lower extremity vascular disease (diabetic foot ulcers), eye diseases, peripheral neuropathy, diabetic cardiomyopathy, and nephropathy, or combinations thereof.

[0274] Combination therapy

[0275] In another embodiment, the present invention provides the use of combining the human amylin polypeptide analogue or its derivative with one or more targeted drugs for the prevention or treatment of obesity or diabetes and related diseases, wherein the targeted drugs include, but are not limited to, diabetes drugs, obesity drugs and hypertension drugs, such as GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, GLP-1 / GIP / GCGR triple agonists, FGF21 derivatives, insulin, metformin, sulfonylureas, meglitinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc., wherein more specific drug types are selected from exenatide, lixinatide, liraglutide, smegglutide, dulaglutide, abiglutide, leptin, neuropeptide Y, tirzepatide, retatrutide, mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, etc. In some embodiments, the amylin analogue derivative preferably comprises the compounds described in Table 3, 4-213.

[0276] When the human amylin polypeptide analogue or its derivatives described in this invention are administered in combination with GLP-1 receptor agonists for the prevention or treatment of diabetes or obesity-related diseases, such as in combination with GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, or GLP-1 / GIP / GCGR triple agonists for the prevention or treatment of diabetes or obesity-related diseases, they exhibit a synergistic hypoglycemic or weight-loss effect. In another preferred embodiment, this invention provides the use of the human amylin polypeptide analogue or its derivatives in combination with smegglutide, tirzepatide, or retatrutide for the prevention or treatment of obesity and obesity-related diseases. These uses include, but are not limited to, overweight, morbid obesity, pre-operative obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea and breathing problems, cartilage degeneration, osteoarthritis, metabolic dysfunction-associated steatohepatitis (MASH), and reproductive health complications of obesity or overweight (e.g., infertility). The preferred human amylin polypeptide analogue is... The structural formulas of starch polypeptide analogs or their derivatives are shown in Table 5, including compounds JY4, JY5, JY6, JY11, JY13, JY14, JY17, JY18, JY19, JY22, JY26, JY28, JY29, JY49, JY50, JY131, JY132, JY133, JY134, JY135, JY136, JY137, JY138, JY193, JY201, JY209, JY210, JY204, JY211, JY212 or JY213, with JY4 being a more preferred compound.

[0277] In another preferred embodiment, the present invention provides the use of co-administering the human amylin polypeptide analog or its derivative with smegglutide, tirzepatide, or retatrutide for the prevention or treatment of diabetes-related diseases, wherein preferred human amylin polypeptide analogs or their derivatives have the structural formulas shown in Table 5, and a more preferred compound is JY4. The diabetes-related diseases include type 1 diabetes, type 2 diabetes, prediabetes, insulin resistance syndrome, metabolic dysfunction-associated steatohepatitis (MASH), impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, and diabetes-related complications, including but not limited to cardiovascular and cerebrovascular diseases, lower extremity vascular diseases, eye diseases, peripheral neuropathy, and nephropathy caused by diabetes, such as diabetic retinopathy, diabetic nephropathy, diabetic foot ulcers, diabetic atherosclerosis, diabetic polyneuropathy, and diabetic fatty liver disease.

[0278] In another embodiment, the present invention provides an efficacy study of human amylin analogue derivatives JY1 (Cagrilintide) and JY4 injection combined with semaglutide on weight loss in DIO rats. When JY1 (Cagrilintide) or JY4 was injected in combination with semaglutide, the weight loss in DIO rats was significantly enhanced compared to the individual compounds used alone, demonstrating a good synergistic effect; the weight loss effect of JY4 combined with semaglutide was superior to that of JY1 (Cagrilintide) combined with semaglutide.

[0279] In another embodiment, the present invention provides a repeated-dose weight-loss test of human amylin analogue derivative JY4 injection combined with Tirzepatide in DIO rats. Compared with the model control group, the body weight of both the single-dose group and the combined-dose group of the test substance JY4 and the reference drug Eloralintide was significantly reduced, with highly significant differences compared with the model control group; JY4 combined with the positive control drug Tirzepatide showed a synergistic effect in inhibiting food intake. The body weight change was greater in both the single-dose group and the JY4 & Tirzepatide combination group compared with the combination of Eloralintide or Eloralintide & Tirzepatide, indicating that the weight-loss effect of JY4 injection is superior to that of the reference drug Eloralintide.

[0280] In another embodiment, the present invention provides a hypoglycemic efficacy study of human amylin analogue derivatives alone or in combination with semaglutide in rats. SD rats treated with different doses (3, 10, 30 nmol / kg) of JY4 injection showed reduced peak blood glucose levels and area under the glucose tolerance curve (AUC). Subcutaneous single injections of JY1, JY4, and semaglutide into ZDF(fa / fa) type II diabetic rats significantly reduced random blood glucose (RBG) levels. When JY4 was combined with semaglutide, the hypoglycemic effect in ZDF(fa / fa) type II diabetic rats was significantly enhanced, exhibiting a synergistic effect.

[0281] In another embodiment, the present invention provides an efficacy test of human amylin analogue derivative JY4 injection combined with semaglutide on hypoglycemic and renal protective effects in ZDF rats. Results showed that random blood glucose (RBG) and glycated hemoglobin (HbA1c) levels in ZDF (fa / fa) type II diabetic rats were significantly reduced after administration. JY4 injection significantly reduced urine output, urinary microalbumin, and serum cystatin C in diabetic animals, and the combined use of semaglutide improved urine output and all renal biochemical indicators. HE and PAS staining pathological results further verified the renal protective effect of JY4 injection. JY4 injection showed good effects in improving blood glucose and protecting against diabetic nephropathy, exhibiting a synergistic effect when used in combination with semaglutide.

[0282] The optimal dose of the human amylin polypeptide analog or its derivative, used alone or in combination with other drugs, will depend on the disease being treated, the severity of the disease, and the presence or absence of side effects. The optimal dose can be determined through routine experiments. For parenteral administration, doses of 1 μg / kg to 5 mg / kg, or 5 μg / kg to 1000 μg / kg, or 10 μg / kg to 500 μg / kg, or 20 μg / kg to 100 μg / kg, or 30 μg / kg to 80 μg / kg of the human amylin polypeptide analog or its derivative are given. Exemplary treatment regimens may include once daily, once weekly, once every two weeks, once every three weeks, or once every four weeks.

[0283] This invention provides a human amylin polypeptide analog and its derivatives, and discloses their use in the preparation of medicaments for treating metabolic diseases such as obesity and diabetes. The technical solutions disclosed in this invention are summarized below: Detailed Implementation

[0284] The following examples are provided to fully disclose and describe how to prepare, screen, identify, and use the present invention. These examples are not intended to limit the scope of the invention in any way, nor do they represent all or only the experiments conducted. The inventors guarantee the objectivity and accuracy of the experimental data, but a certain degree of experimental error and bias should be allowed.

[0285] Table 4 Abbreviations

[0286] Example 1: Preparation of albumin binding base

[0287] Synthesis of albumin-binding base 1 (A01):

[0288] 7-Bromoheptanoate methyl ester was reacted with triphenylphosphine to form the corresponding phosphonium salt (intermediate 3), and 12-bromo-1-dodecanoate was oxidized by PCC to obtain the corresponding 12-bromo-1-dodecanoate (intermediate 4). The phosphonium salt (intermediate 3) was reacted with NaHMDS to prepare the corresponding ylide, which was then reacted with 12-bromo-1-dodecanoate (intermediate 4) via a Witting reaction to prepare a bromoolefin (intermediate 5). The bromoolefin (intermediate 5) was reduced by Pd / C hydrogenation to give 19-bromo-1-nonadecanoate methyl ester (intermediate 6). 19-bromo-1-nonadecanoate methyl ester (intermediate 6) was reacted with dibenzyl phosphite under alkaline conditions to prepare the corresponding phosphate ester (intermediate 7). The phosphate ester (intermediate 7) was hydrolyzed in the presence of lithium hydroxide to obtain the desired albumin-binding group (A01).

[0289] 1 H NMR(400MHz,Chloroform-d)δ5.40–5.25(m,2H),3.66(s,3H),3.41(t,J=6.8Hz,2H ),2.31(t,J=7.6Hz,2H),2.10–1.80(m,2H),1.60–1.50(m,2H),1.45–1.25(m,24H).

[0290] GCMS(M) + 388.2, Calculated value (M) + :388.2.

[0291] 1 H NMR (400MHz, Chloroform-d)3.67(s,3H),3.41(t,J=6.8Hz,2H),2.30(t,J=7.6Hz,2H),1.90–1.80(m,2H),1.65–1.51(m,2H),1.45–1.24(m,28H).

[0292] GCMS(M) + 390.2, calculated value (M) + 390.2.

[0293] 1 H NMR (400MHz, Chloroform-d) δ7.39–7.30(m,10H),5.10–4.90(m,4H),3.66(s,3H),2.25(t,J=7.8,2H),1.92–1.50(m,6H),1.30–1.20(m,28H).

[0294] LCMS(M+H) + 573.4, calculated value (M+H) + : 573.4.

[0295] 1 H NMR (400MHz, Chloroform-d) δ7.39–7.30(m,10H),5.10–4.90(m,4H),2.34(t,J=7.8,2H),1.85–1.50(m,6H),1.37–1.20(m,28H).

[0296] LCMS(M+H) + 559.3, calculated value (M+H) + : 559.3.

[0297] Synthesis of albumin-binding base 2 (AO2):

[0298] 19-Bromo-nonadecanoate tert-butyl ester was de-tert-butylated in the presence of trifluoroacetic acid to yield the corresponding 19-bromo-nonadecanoic acid (intermediate 2). 19-Bromo-nonadecanoic acid (intermediate 2) was reacted with sodium cyanide to prepare the corresponding 19-cyano-nonadecanoic acid (intermediate 3). 19-Cyano-nonadecanoic acid (intermediate 3) was reacted with sodium azide to prepare the desired albumin-binding group (AO2).

[0299] 1 H NMR (400MHz, CDCl3) δ2.45 (t, J = 7Hz, 2H), 2.18 (t, J = 7Hz, 2H), 1.70–1.40 (m, 4H), δ1.30–1.20 (m, 28H).

[0300] 1 H NMR (400MHz, DMSO-d6)2.85(t,J=7Hz,2H),2.18(t,J=7Hz,2H),1.70–1.60(m,2H),1.48–1.40(m,2H), δ1.30–1.20(m,28H).

[0301] LCMS(MH) - 365.3, calculated value (MH) - 365.3.

[0302] Synthesis of albumin-binding base 3 (A03):

[0303] 19-Bromo-nonadecanoate tert-butyl ester reacted with 5-mercapto-1,2,3-triazole monosodium salt to prepare the corresponding intermediate 2. Intermediate 2 was de-tert-butylated in the presence of trifluoroacetic acid to obtain the desired albumin-binding group (A03).

[0304] LCMS(MH) - 368.3, calculated value (MH) - 368.2.

[0305] Synthesis of albumin-binding base 4 (A04):

[0306] 19-Bromo-nonadecanoate tert-butyl ester reacts with 1-H-1,2,3-triazole to prepare the corresponding intermediate 2. Intermediate 2 is then detert-butylated in the presence of trifluoroacetic acid to obtain the desired albumin-binding group (A04).

[0307] LCMS(MH) - 364.3, calculated value (MH) - 364.3.

[0308] Synthesis of albumin-binding base 5 (A05):

[0309] 19-Bromo-nonadecanoate tert-butyl ester reacts with 1-H-1,2,3-triazole to prepare the corresponding intermediate 2. Intermediate 2 is then de-tert-butylated in the presence of trifluoroacetic acid to obtain the desired albumin-binding group (A05).

[0310] LCMS(MH) - 364.3, calculated value (MH) - 364.3.

[0311] Synthesis of albumin-binding base 6 (A06):

[0312] 19-Bromo-nonadecanoate tert-butyl ester reacts with imidazole to prepare the corresponding intermediate 2. Intermediate 2 is then de-tert-butylated in the presence of trifluoroacetic acid to obtain the desired albumin-binding group (A06).

[0313] 1 H NMR (400MHz, Chloroform-d) δ7.49(s,1H),7.09(s,1H),6.94(s,1H),3.95(t,J=7.2Hz,2H),2.34(t,J=7.5Hz,2H),1.87–1.50(m,4H),1.42–1.10(m,28H).

[0314] LCMS(MH) - 363.3, calculated value (MH) - 363.3.

[0315] Synthesis of albumin-binding base 7 (A07):

[0316] The corresponding albumin-binding group (A07) can be prepared by reacting methyl 16-bromo-hexadecanoate with sodium sulfite.

[0317] 1 H NMR (400MHz, DMSO-d6) δ2.47–2.30(m,2H),2.18(t,J=7.5Hz,2H),1.60–1.40(m,4H),1.42–1.10(m,22H).

[0318] Synthesis of albumin-binding base 8 (A08):

[0319] 19-Bromononadecanoate tert-butyl ester reacts with trimethylsilylacetylenide lithium to form the corresponding alkynyl compound (intermediate 2). Intermediate 2 reacts with p-toluenesulfonyl azide to form the corresponding intermediate 3. Intermediate 3 is deprotected by potassium carbonate to obtain intermediate 4. Intermediate 4 is deprotected by trifluoroacetic acid to obtain the desired albumin-binding group (A08).

[0320] LCMS(MH) - 364.3, calculated value (MH) - 364.3.

[0321] Synthesis of albumin-binding 9 (A09) eicosanoic acid monotert-butyl ester:

[0322] Eicosanoic acid (50 g, 146.0 mmol) was suspended in acetic anhydride (200 ml) and reacted at 140 °C for 10 hours. The acetic anhydride was then removed by vacuum distillation. Toluene (120 ml), tert-butanol (50 g), and DMAP (4.1 g) were added, and the mixture was reacted at 85 °C for 8 hours. The solvent was then removed by vacuum distillation. Dichloromethane (500 ml) and concentrated hydrochloric acid (3 ml) were added, and the mixture was stirred at 25 °C for 30 min. The mixture was filtered, and the organic phase was collected. The organic phase was extracted three times with 5% hydrochloric acid aqueous solution (200 ml * 3), and dried with anhydrous sodium sulfate (20 g) for 30 min. The mixture was filtered, and the filtrate was collected. The solvent was then removed by vacuum distillation. Hexane (500 ml) was added and heated to dissolve the filtrate. The mixture was then crystallized at -20 °C for 4 hours. The precipitate was filtered, collected, and dried under vacuum to constant weight to give a white solid product, tert-butyl eicosanoic acid, yield: 18.7 g (46.7 mmol, 32.0%).

[0323] 1 H NMR (400MHz, CDCl3)2.31(t,J=7.6Hz,2H),2.17(t,J=7.6Hz,2H),1.50-1.65(m,4),1.41(s,9),1.20-1.35(m,28).

[0324] Example 2: Synthesis of human pancreatic amylin analogues and their derivatives

[0325] Using solid-phase synthesis, employing the Fmoc-protected amino acid strategy and SPPS solid-phase synthesis technology, the peptide was completed through steps such as coupling, deprotection, cyclization, and cleavage. After purification, the target peptide product, an amylin analog, was obtained.

[0326] Amylin analogues and their derivatives were prepared according to the general method for polypeptide synthesis described below (Tables 1 and 3 (JY1-JY166)), and JY167-JY213 in Table 3 were prepared according to the thioacetal cyclization synthesis steps.

[0327] Couplet:

[0328] CEM Liberty Blue Peptide Synthesizer: Add Fmoc-amino acids from DMF, along with DIC / DMF and Oxyma / DMF, to the resin in the CEM Discover microwave unit. Heat the coupling mixture to 90°C for 2 minutes while bubbling nitrogen through it, then wash the resin with DMF. Alternatively, coupling can be performed without heating, in which case the reaction time is extended to 60 minutes.

[0329] In cases where coupling is difficult (e.g., N-methylated amino acid residues or other sterically hindered amino acid residues as recognized by those skilled in the art), the coupling may be repeated once or more, or the coupling reaction time may be extended.

[0330] Go to protect:

[0331] Piperidine / DMF (V / V = 1:4) was added to the resin for initial deprotection, and the mixture was heated by microwave (40°C; 30 seconds). The reaction vessel was drained, and a second portion of piperidine / DMF (V / V = 1:4) was added and heated again (75°C; 3 minutes), followed by washing the resin with DMF.

[0332] Oxidative cyclization:

[0333] The Acm deprotection and disulfide formation steps are performed simultaneously using thallium(III) trifluoroacetate [Tl(TFA)3] in NMP, with intramolecular ring formation (disulfide bridge formation) between Cys residues at positions 2 and 7 (initially coupled as Acm-protected cysteine ​​residues) of the peptide still attached to the resin. (Alternative method: Add 10 eq iodine to a 50 mM solution of the resin-bound peptide in acetic acid and stir for 18 to 24 hours).

[0334] Cutting:

[0335] The resin was washed with ethanol and methyl tert-butyl ether and dried under vacuum at room temperature (rt) to constant weight. The crude peptide was cleaved from the resin by treatment with TFA / TIS / H2O (90:5:5) at room temperature for 2 h, or alternatively with TFA / EDT / TIS / H2O (94:2:2:2) at room temperature for 2 h. Most of the trifluoroacetic acid was removed by rotary evaporation, and the crude peptide was precipitated with methyl tert-butyl ether. The crude peptide was then washed three times with methyl tert-butyl ether and dried under vacuum at room temperature to constant weight.

[0336] Thioacetal cyclization:

[0337] To prepare the thioacetal bridge, the dried crude peptide was dissolved in 20 mM potassium phosphate buffer and acetonitrile. Once completely dissolved, TCEP hydrochloride (5 eq), diiodomethane (10 eq), and DIEA (10 eq) were added with vigorous stirring for 5–30 minutes. The reaction was monitored by HPLC and LCMS. After the reaction was complete, formic acid was added to the mixture. If the solution became clear, reverse-phase purification was performed directly. If complete dissolution was not achieved, NMP was added to aid dissolution, followed by reverse-phase purification.

[0338] Purification and characterization:

[0339] Crude peptides were purified by semi-preparative HPLC on a 10*250 mm C8 column packed with 10-100 μL of acetonitrile. The modified peptide B sample was dissolved in 5-10% acetonitrile / water, filtered through a 0.45 μm microporous membrane, and then injected onto the column. The crude peptide was purified by RP-HPLC at a column temperature of 30 °C and a flow rate of 2 mL / min using a linear gradient of acetonitrile and 0.1% formic acid / water buffer. Fractions containing peptides were collected. The collected fractions were analyzed by HPLC and LC-MS, and the relevant fractions were combined and lyophilized. The final product was characterized by HPLC and MS.

[0340] Taking the synthesis of compound JY4 as an example, the preparation methods of the other compounds in Table 3 are similar.

[0341] Using polystyrene Rink Amide MBHA resin (degree of substitution = 0.4 mmol / g), with standard side chain protecting groups, a 0.25 mmol scale synthesis was carried out following a common coupling, deprotection, and oxidative cyclization procedure. Amino acids, C20 alkyl dicits, and other chemical fragments are sequentially coupled onto the resin in the following order: A01(2S,4R)-4-OH-Fmoc-Pro(tBu)-OH, A02 Fmoc-Thr(tBu)-OH, A03 Fmoc-Asn(Trt)-OH, A04 Fmoc-Ser(tBu)-OH, A05 Fmoc-Gly-OH, A06 Fmoc-Val-OH, A07 Fmoc-Asn(Trt)-OH, A08 Fmoc-Thr(tBu)-OH, A09 Fmoc-Pro-OH, A10 Fmoc-Pro-OH, A11 Fmoc-Leu-OH, A12 Fmoc-Ile-OH, A13 Fmoc-Pro-OH, A14 Fmoc-Gly-OH, A15 Fmoc-Phe-OH, A16 Fmoc-Asn(Trt)-OH, A17 Fmoc-Asn(Trt)-OH, A18 Fmoc-Ser(tBu)-OH, A19 Fmoc-Ser(tBu)-OH, A20 Fmoc-His(Trt)-OH, A21 Fmoc-Arg(Pbf)-OH, A22 Fmoc-Leu-OH, A23 Fmoc-Phe-OH, A24 Fmoc-Glu(OtBu)-OH, A25 Fmoc-Ala-OH, A26 Fmoc-Leu-OH, A27 Fmoc-Arg(Pbf)-OH, A28 Fmoc-Gln(Trt)-OH, A29 Fmoc-Thr(tBu)-OH, A30 Fmoc-Ala-OH, A31 Fmoc-Cys(Acm)-OH,A32 Fmoc-Thr(tBu)-OH, A33 Fmoc-Ala-OH, A34 Fmoc-Thr(tBu)-OH, A35 Fmoc-Asn(Trt)-OH, A36 Fmoc-Cys(Acm)-OH, A37 Fmoc-Har(Pbf)-OH, A38 Fmoc-γGlu(OtBu)-OH, A39 Eicosanoic acid monotert-butyl ester; finally, a resin containing polypeptide derivatives was obtained; all amino acid coupling conditions were that Fmoc-amino acid\DIC\Oxyma were coupled with 5 times the molar amount; the coupling conditions for eicosanoic acid monotert-butyl ester were that eicosanoic acid monotert-butyl ester\PyBop\DIEA\HOBT were coupled with 5 times the molar amount.

[0342] After synthesis, the peptide resin was washed with ethanol (3 x 10 ml) and methyl tert-butyl ether (3 x 10 ml), and dried under vacuum at room temperature (rt) to constant weight. The crude peptide was cleaved from the resin for 2 h with 30 ml of TFA / TIS / H2O (90:5:5) cleavage buffer at room temperature. The cleavage buffer was filtered, collected, and most of the trifluoroacetic acid was removed by rotary evaporation. The cleavage buffer was then added to 10 times its volume of methyl tert-butyl ether (-20 °C) for precipitation. The peptide / ether suspension was then centrifuged at 10,000 rpm for 10 minutes to form a solid precipitate. The supernatant was discarded, and the precipitate was washed with methyl tert-butyl ether and centrifuged twice to obtain the crude peptide, which was then dried under vacuum at room temperature to constant weight.

[0343] Crude peptides were purified by semi-preparative HPLC on a 10*250 mm C8 column packed with 10-100 μL of acetonitrile. The modified peptide B sample was dissolved in 20 mL of 5-10% acetonitrile / water, filtered through a 0.45 μm microporous membrane, and then injected onto the column. The crude peptide was purified by RP-HPLC at a column temperature of 30 °C and a flow rate of 2 mL / min using a linear gradient of acetonitrile and 0.1% formic acid / water buffer. Fractions containing peptides were collected. The collected fractions were analyzed by HPLC and LCMS, and the relevant fractions (purity >95%) were combined and lyophilized. The final product had an HPLC purity of 96.7% and an LCMS purity of (M+H) of [missing value]. + =4465.28; Calculated value (M+H) + =4465.28.

[0344] Example 3: Solubility Measurement Experiment

[0345] Weigh the amylin analogue derivative into a suitable vial, and add 1 ml of the corresponding aqueous solution (acetate buffer pH = 4.0, ultrapure water pH = 7.0). Shake the vial thoroughly at 25 degrees Celsius to ensure complete dissolution of the peptide, and visually assess the degree of dissolution.

[0346] The above solution was filtered through a 0.45 μm filter and eluted using a trifluoroacetic acid / acetonitrile / water elution system on a C18 column. The area of ​​the main peak was determined by UV spectroscopy at 210 nm, and the concentration of the sample (mg / ml) was calculated by the external standard method. The relevant results are shown in Table 6.

[0347] Example 4 Freeze-thaw test

[0348] Weigh 1-1.5 mg of the amylin analogue derivative into a suitable vial, add 1 ml of ultrapure water (pH = 7.0) to the vial, and shake the vial thoroughly at 25°C to ensure the peptide is fully dissolved (if the peptide has poor solubility, discard the peptide molecule and do not proceed with subsequent experiments). Observe and record the state of the solution. Perform gradient elution on a C18 column using a trifluoroacetic acid / acetonitrile / water elution system. Determine the area of ​​the main peak at 210 nm using UV spectroscopy. Calculate the sample concentration (mg / ml) using the external standard method and record the concentration.

[0349] The dissolved sample was stored at -20°C for 16 hours, then removed and allowed to thaw at room temperature. The state of the solution was observed and recorded after thawing. Gradient elution was performed on a C18 column using a trifluoroacetic acid / acetonitrile / water elution system. The area of ​​the main peak was determined by UV spectroscopy at 210 nm. The concentration (mg / ml) of the thawed sample was calculated using the external standard method, and the concentration was recorded.

[0350] Calculate the freeze-thaw yield: Freeze-thaw yield = concentration of peptide after freeze-thaw / concentration of peptide before freeze-thaw. The relevant results are shown in Table 6.

[0351] Example 5 Chemical Stability Experiment

[0352] The amylin analogue derivative sample was dissolved in acetate buffer at pH 4 to prepare a concentration of 1 mg / mL. The sample was placed in a glass vial and incubated at 25°C for 7 days. Gradient elution was performed on a C18 column using a trifluoroacetic acid / acetonitrile / water elution system. The normalized concentration of the main peak was determined by UV spectroscopy at 214 nm. The relevant results are shown in Table 6.

[0353] The amylin analogue derivative sample was dissolved in acetate buffer (pH 4) to a concentration of 1 mg / mL. The sample was placed in a glass vial and incubated at 25°C for 7 days. After filtration through a 0.45 μm filter, gradient elution was performed on a C18 column using a trifluoroacetic acid / acetonitrile / water elution system. The peak area of ​​the main peak was determined by UV spectroscopy at 214 nm. The yield was calculated as: Yield = Peptide concentration after 7 days of incubation / Peptide concentration before incubation. The relevant results are shown in Table 6.

[0354] Example 6 Physical Stability (Fiberization) Experiment

[0355] It can generally be measured by visual inspection of the sample, or it can be detected by the amyloid protein-specific dye thioflavin T (ThT). Thioflavin T has a unique fluorescent signal when it binds to fibrils, and this dye can be used in experiments to show the presence of fibrils in the solution.

[0356] Amylin analogue derivative samples were dissolved in acetate buffer (pH 4) at 25°C to a concentration of 1 mg / mL. Thiamine T was added to the sample solution to a final concentration of 1 μM. The solutions were then packaged in triplicate into 96-well black fluorescent plates (transparent bottom). After incubation at 37°C for 96 hours, the samples were visually inspected and their fluorescence intensity was measured using a fluorescence meter. The results are shown in Table 6.

[0357] Amylin analogue derivative samples were dissolved in acetate buffer solution at pH 4 at 25°C to prepare a concentration of 1 mg / ml. After being placed at 37°C for 96 hours, the samples were removed and visually inspected to determine the relevant fibrosis status in the solution. The relevant results are shown in Table 6.

[0358] Table 6. Solubility, freeze-thaw state and yield, chemical stability, and physical stability data. A: Represents a normalized content greater than or equal to 90%; B: Represents a normalized content between 80% and 90%; C: Represents a normalized content between 70% and 80%; D: Represents a normalized content less than or equal to 70%.

[0359] Example 7: In vitro activity assay (hCTR, rCTR, rAMY3R, hAMY3R)

[0360] CHOK1 cells stably transfected with CRE-Luc were transfected with hCTR, rCTR, or rCTR+rRAMP3 (rAMY3R) to construct reporter gene stable cell lines or mini pools for activity assays. Cells reaching 90% confluence were trypsinized, collected, and counted. Cells were resuspended to 20 w / mL in F12K+0.1% BSA or F12K+1% BSA viability assay medium, and 100 μL of cell resuspension was added to each well of a 96-well plate and incubated overnight at 37°C with 5% CO2. Amylin analogue derivatives were serially diluted 5-fold and added to 50 μL per well of the cell culture plate and incubated at 37°C with 5% CO2 for 6 h. 50 μL of Bio-glo luciferase substrate was added to each well and incubated at room temperature for 10 min. The plates were read using a Molecular Devicer SpectraMax M5e. Data analysis was performed using GraphPad Prism 9, with JY1 (Cagrilintide) as the 100% standard for percentage analysis.

[0361] BHK-21 cells were transfected with hCTR+hRAMP3 (hAMY3R) and CRE-Luc to construct a reporter gene stable cell line for activity assay. Cells reaching 90% confluence were collected after trypsin digestion and counted. Cells were resuspended in complete medium MEM + 10% FBS + 1% Sodium Pyruvate + 1% GlutaMAX to 10 w / mL. 100 μL of the cell resuspended medium was added to each well of a 96-well plate and incubated overnight at 37°C in a 5% CO2 incubator. The medium was carefully discarded, and amylin analogue derivatives were serially diluted 5-fold with either MEM + 0.1% BSA + 1% Sodium Pyruvate + 1% GlutaMAX or MEM + 4% BSA + 1% Sodium Pyruvate + 1% GlutaMAX. 100 μL of each dilution was added to each well of the cell culture plate and incubated at 37°C in a 5% CO2 incubator for 6 h. Add 50 μL of Bio-glo luciferase substrate to each well and incubate at room temperature for 10 min. Read the plate using a Molecular Devicer SpectraMax M5e. Perform data analysis using a GraphPad Prism 9, with JY1 (Cagrilintide) as the 100% standard for percentage analysis.

[0362] Table 7-1 Relative Activity Data ND: Not determined, indicating that a definite EC could not be calculated for this derivative after activity testing. 50 The value is too low to calculate the activity.

[0363] A higher percentage indicates better activity. The activity of derivatives modified with side-chain phosphorylation of JY49 and JY50 decreased significantly. Under both 0.1% BSA and 1% BSA testing conditions, JY4 showed significantly higher activity than JY1 (Cagrilintide).

[0364] Table 7-2 Relative Activity Data

[0365] Cagrilintide is disclosed in WO 2012 / 168430 and has the following sequence;

[0366] KCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2

[0367] Petrelintide is disclosed in WO 2018 / 046719 and has the following sequence;

[0368] RDGTATKATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH2

[0369] PD069 is disclosed in WO 2023 / 227133 and has the following sequence;

[0370] KCNTAT CATQRL ADFLRH SSNN LGPIL PPTNVGSNT-trans-Hyp-NH2

[0371] PD071 is disclosed in WO 2023 / 227133 and has the following sequence;

[0372] KCNTAT CATQRL ADFLRH SSNN LKPIL PPTNVGSNT-trans-Hyp-NH2

[0373] According to WO2023 / 227133, the structure of trans-Hyp is as follows:

[0374] A higher percentage indicates better activity. JY4 showed significantly higher activity than Cagrilintide, Petrelintide, PD069, and PD071 under both 0.1% BSA and 4% BSA testing conditions.

[0375] Example 8: Kinetic analysis of binding to human serum albumin (octet)

[0376] Biotin-labeled human serum albumin (abcam, ab8033) was diluted to 50 μg / mL and immobilized with an SA probe for 30 min using a macromolecular interaction analyzer (Fortebio, Octet RED96). Simultaneously, amylin analogues were serially diluted with 0.02% DPBST in black polypropylene 96-well plates (Greiner, 655209). The immobilized probe was associated with the serially diluted amylin analogues for 60 s, followed by disassociation in 0.02% DPBST for 60 s. This process was repeated with unimmobilized probes. Steady-state analysis was performed using Octet Analysis Studio 12.2 software after double subtraction to obtain the corresponding KD values ​​and R0. 2 .

[0377] Table 8 Albumin-binding activity data

[0378] A lower KD value indicates a better binding affinity between the amylin analogue derivative and human serum albumin. Except for JY133, JY134, and JY136, the binding affinity of amylin analogue derivatives to human serum albumin is no weaker than that of Cagrilintide.

[0379] Example 9: Detection of binding to human calcitonin receptor (CTR) and amylin receptor 3 (AMY3R) membrane proteins.

[0380] 293F cells were transfected with PEI (PolyScience, 23966-100) with either human CTR plus empty vector plasmid (hCTR group) or human CTR plus human RAMP3 plasmid (hAMY3R group) using pcDNA3.1 as the vector. After 18 hours, 10% (v / v) F01 supplemented medium (PD Biosciences, PDF01-1000) was added to the transfected cells. Two days later, 5 weeks of cells were collected for flow cytometry identification. If the identification results showed >50% positive expression, 1*10t cells from each of the hCTR and hAMY3R groups were collected. 8 Membrane proteins were extracted using Pierce™ GPCR extraction and stabilization reagent (Thermo Scientific, A43436).

[0381] Take a black, low-absorption 96-well plate, add 50 μL of membrane protein, 20 nM sCT(8-32):5-FAM fluorescently labeled peptide, serially diluted amylin analogue derivative, and buffer HBSS (Gibco, 14025-092) + 10 mM HEPES (Gibco, 15630080) + 0.1% BSA to each well, for a total volume of 100 μL. Detection is performed using a ELISA reader (Molecular Devices, SpectraMax M5e) with fluorescence polarization module, Ex / Em at 485 / 525 nm. The group without peptides serves as a blank control. After subtracting the S / P value of the blank control group, calculate mP = ABS[1000*(SP) / (S+P)], the result of which is automatically obtained using SoftMax Pro software. Plot mP value on the ordinate and concentration on the x-axis, and fit the results using GraphPad Prism 9 to obtain EC. 50 The values ​​are shown in the table below.

[0382] Table 9 Receptor binding affinity data

[0383] EC 50The lower the value, the higher the affinity of the amylin analogue derivative for the corresponding receptor. The affinities of JY4 for hAMY3R and hCTR are both higher than those of JY1 (Cagrilintide).

[0384] Example 10 Single-dose Feeding Inhibition Experiment on SD Rats

[0385] Male SD rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License No.: SCXK(Zhe)2024-0001, Animal Quarantine Certificate No.: 20240701Aazz0619999373). After arrival, the animals were adaptively fed for one week. The SD rats were randomly grouped according to body weight, and the groups were: normal control group (Control), 10 nmol / kg amylin analogue derivative group, with 6 rats in each group. Subsequently, the corresponding samples were given to each group by subcutaneous injection in the neck, with a single dose. After the dosing period, the body weight and food intake of the test animals were monitored at 0, 24, 48, and 72 h. The relevant data are shown in Tables 10 and 11.

[0386] Results: In this experiment, the weight loss effect of JY4 on SD rats was better than that of the control molecule JY1 (Cagrilintide); the feeding inhibition effect of JY4 on SD rats was also significantly better than that of the control molecule JY1 (Cagrilintide). Based on the above results, it shows that JY4 exhibits the best drug efficacy in the single-dose subcutaneous injection experiment on SD rats. The weight loss and feeding inhibition effects of the JY29 molecule are basically equivalent to those of JY1 (Cagrilintide).

[0387] Table 10 Changes in Body Weight

[0388] Table 11 Changes in Food Intake

[0389] Example 11 Repeated-dose Weight Loss Experiment on Obese Animals

[0390] Male Sprague-Dawley (SD) rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License No.: SCXK(Zhe)2024-0001, Animal Quarantine Certificate No.: 20240219Aazz0619999827). During the adaptation period, induction, and experiment, they were fed with the XTHF60 high-fat diet provided by Synergy Biosciences. Additionally, 6 SD rats were randomly selected and fed with a normal diet as the normal control group (Control). After 20 weeks of high-fat diet feeding, animals with a body weight exceeding 20% of the average body weight of normal SD rats were selected as the diet-induced obesity (DIO) rat model. The DIO rats were randomly divided into 4 groups according to body weight: the model control group (Vehicle), and the injection groups of amylin analog derivatives at 10 nmol / kg, with 6 rats in each group. Subsequently, each group was administered the test sample by subcutaneous injection, 3 times a week for 4 consecutive weeks. During the administration period, the body weight and food intake of the test animals were monitored (2 times a week). The relevant test results are shown in Figure 1 and Figure 2.

[0391] Results: In this experiment, the DIO rats were subcutaneously injected continuously for 4 weeks (3 times a week). JY1 (Cagrilintide), JY3, and JY4 all had a significant weight loss effect on DIO rats, with JY4 showing the best weight loss effect. The weight loss amplitude of JY4 alone was significantly higher than that of JY3 and the control molecule JY1 (Cagrilintide). During the monitoring of daily food intake, the food intake inhibition effect in the test group of the control molecule JY1 (Cagrilintide) was weak and the food intake recovery rate was fast; the initial food intake inhibition effects of the test animals in JY3 and JY4 were basically equivalent, but the food intake recovery rate in the JY3 group was significantly faster than that in JY4. In summary, the JY4 molecule showed the best food intake inhibition effect.

[0392] Example 12 Pharmacokinetic Experiment in SD Rats

[0393] Male and female SD rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License No.: SCXK(Zhe)2024-0001, Animal Quarantine Certificate No.: 20240708Aazz0619999452). After the animals arrived, they were adaptively fed for one week. The rats were randomly grouped according to their body weight, and the groups were: normal control group (Control), and the test peptide injection group at 30 nmol / kg, with 6 rats in each group (3♂&3♀). Subsequently, the corresponding test samples were given to each group, administered subcutaneously in the neck / tail vein injection, once. Blood samples were collected from all groups at the time points of 30 min, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after administration. Note to stop bleeding in time after each blood collection. All the above blood samples were collected from the posterior orbital venous plexus of the rats, with the single blood collection volume controlled at 180 - 200 μL. The collected blood samples were collected in EDTA-K2 anticoagulant tubes and temporarily stored on ice, and centrifuged at 6000 rpm for 5 min within 1 hour, and the supernatant plasma was transferred as soon as possible (within 30 min) and frozen in an -80°C refrigerator. The plasma samples were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS), and the average plasma concentration was used to calculate the pharmacokinetic parameters. The relevant data are shown in Table 12 and Figure 3.

[0394] Results: In this experiment, SD rats were administered a single subcutaneous or tail vein injection. Figure 3 shows that the drug action patterns of JY1 (Cagrilintide) and JY4 were basically the same. Table 12 shows that there were no significant differences observed in the control molecules JY1 (Cagrilintide) and JY4 in terms of pharmacokinetic parameters (T 1 / 2 、T max 、C max and AUC Inf ), etc.

[0395] Table 12 Pharmacokinetic Parameters

[0396] Example 13: Oral Glucose Tolerance Test of JY4 Injection in SD Rats

[0397] Male SD rats were all purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License Number: SCXK(Zhe)2024-0001, Animal Quarantine Certificate Number: 20240912Aazz0619999498). The SD rats were randomly divided into 6 groups according to body weight: blank control group (Vehicle), 1, 3, 10, 30 nmol / kg JY4 injection groups, and 10 nmol / kg reference molecule JY1 injection group, with 6 rats in each group. All the test animals were fasted for 16 h without water deprivation before the oral glucose tolerance test (OGTT), and were administered by single subcutaneous injection in the neck at the same time. On the experimental day, a 2 g / kg glucose solution was intragastrically administered. Before giving glucose (0 minute) and at 15, 30, 60, 120, and 180 minutes after giving glucose, rat blood samples were collected by tail tip puncture, and the whole blood glucose value was detected using a Roche Accu-Chek Aviva blood glucose meter and blood glucose test strips held by hand.

[0398] Results: As shown in Figure 4, the blood glucose values of the control group (Vehicle), JY1, and 1 nmol / kg JY4 group reached the peak at 15 min after glucose loading, while the 3, 10, 30 nmol / kg JY4 groups reached the peak at 30 min (10, 30 nmol / kg) or even 60 min (3 nmol / kg) after glucose loading. When the dosing concentration of JY4 reached 3 nmol / kg, the animal blood glucose level and AUC 0-180min were significantly lower than those of JY1. As the dose of JY4 continued to increase, the animal blood glucose level and AUC 0-180min no longer decreased significantly.

[0399] Compared with the control group, the decline rate of AUC 0-180min of 10 nmol / kg JY1 was only 11.0%, while the decline rate of AUC 0-180min of 1 nmol / kg JY4 reached 15.1%. The decline rate of AUC 0-180min in the JY4 dose groups above 3 nmol / kg reached more than 22.0%, which was significantly better than the blood glucose regulation effect of the reference molecule JY1.

[0400] Example 14: Hypoglycemic and Renal Protection Pharmacodynamic Test of JY4 Injection Combined with Semaglutide in ZDF Rats

[0401] Male ZDF (fa / fa) type II diabetic rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License No.: SCXK (E) 2022-0030, Animal Quarantine Certificate No.: 422023600013416, 422023600013415). ZDF (fa / fa) type II diabetic rats were randomly divided into 6 groups according to body weight and glycated hemoglobin HbA1c: model control group (ZDF), 10 nmol / kg Semaglutide injection group, 10 nmol / kg JY1, 10 nmol / kg JY4, 10&10 nmol / kg JY1 combined with Semaglutide dose group, 10&10 nmol / kg JY4 combined with Semaglutide dose group, with 8 rats in each group; another 8 ZDF (fa / +) normal rats were purchased as blank control group (Control), and the animals were adaptively fed for two weeks. After grouping, the food intake of rats before drug administration was monitored for 2-3 days to collect the initial food intake data. Subsequently, according to the drug administration design, the corresponding drugs were injected subcutaneously (S.C.) into the neck of each group (Note: the two drugs in the combined group need to be mixed immediately before use), three times a week (TIW) for 8 consecutive weeks. The whole blood glucose value was detected with a Roche Accu-Chek Aviva blood glucose meter, and the glycated hemoglobin value was detected with a Botech BOTANGPING glycated hemoglobin detector of Wuxi Biosciences. Animal serum and urine were collected in the experiment, and relevant indicators were detected with a Beke BK-400 biochemical analyzer. Kidney tissues were collected for HE staining and PAS staining analysis.

[0402] Results: As shown in Figure 5, the random blood glucose value (RBG) and glycated hemoglobin value of ZDF (fa / fa) type II diabetic rats decreased significantly after drug administration. The reduction amplitude of JY4 injection alone and in combination was basically the same as that of JY1 injection, but the significant effect of JY4 combined with Semaglutide was better than that of Semaglutide or JY4 alone, showing a synergistic effect. The study on the renal protective effect of JY4 injection on ZDF type II diabetic rats is shown in Figures 6 and 7. JY4 injection significantly reduced the urine volume, urinary microalbumin and serum cystatin C of diabetic animals, and showed an improvement effect on urine volume and all renal function biochemical indexes after combining with Semaglutide. The pathological results of HE staining and PAS staining verified again the renal protective effect of JY4 injection.

[0403] In summary, JY4 injection has good effects on blood glucose improvement and protection against diabetic nephropathy, and shows a drug synergistic effect after combining with Semaglutide.

[0404] Example 15: Study on the blood glucose regulation of JY4 injection combined with Semaglutide in BKS-db / db mice

[0405] Male BKS-db / db mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Animal Production License No.: SCXK(Su)2023-0009, Animal Quarantine Certificate No.: B202411270225). After arrival, the animals were acclimatized for one week. Data on glycated hemoglobin and random blood glucose were used as the primary indicators, with body weight as a secondary indicator. The mice were divided into six groups: a model control group (60% HFD), a 3 nmol / kg Semaglutide injection group, a 3 nmol / kg JY1 group, a 3 nmol / kg JY4 group, a 3 & 3 nmol / kg JY1 combined with Semaglutide group, and a 3 & 3 nmol / kg JY4 combined with Semaglutide group, with eight mice in each group. Eight normal BKS mice were also purchased as a blank control group. The animals were acclimatized for two weeks. Subsequently, each group received a subcutaneous (SC) injection of the corresponding drug in the neck (Note: the two drugs in the combination group must be mixed immediately before use), once daily (QD) for six consecutive weeks. After weekly administration, whole blood fasting blood glucose levels were measured using a Roche Vitality blood glucose meter. Random blood glucose levels were measured within 24 hours of free feeding after the first administration (measurement time points: 1h, 2h, 4h, 8h, 24h).

[0406] Results: Figure 8 shows that after the first administration, amylin analogs JY1 and JY4 improved blood glucose levels in the test animals, and the combined use of semaglutide significantly enhanced the efficacy; moreover, the improvement level in the JY4 & semaglutide group was better than that in the JY1 & semaglutide group. Figure 9 shows the changes in fasting blood glucose levels in BKS-db / db mice after 6 weeks of administration. The most significant decrease in blood glucose levels was observed in the first week of administration, after which blood glucose levels gradually returned to their initial levels; this was mainly due to the resistance to semaglutide developed in BKS-db / db mice. Although blood glucose levels were not effectively suppressed in the later stages of the experiment, it was clearly observed throughout the experiment that the hypoglycemic effect of the combination of amylin analogs and semaglutide was significantly better than that of single-drug treatment. Furthermore, the therapeutic effect of JY4 & semaglutide remained superior to that of JY1 & semaglutide during the 6-week administration period.

[0407] Example 16: Study on the regulation of blood glucose in DIO mice (30 weeks) by JY4 injection combined with semaglutide

[0408] Male DIO mice (60% HFD induced for 30 weeks) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Animal Production License No.: SCXK(Su)2023-0009, Animal Quarantine Certificate No.: B202503240551). After arrival, the animals were acclimatized for one week. Based primarily on random blood glucose data and secondarily on body weight data, they were divided into 6 groups: a model control group (60% HFD), a 3 nmol / kg Semaglutide injection group, a 3 nmol / kg JY1 group, a 3 nmol / kg JY4 group, a 3 & 3 nmol / kg JY1 combined with Semaglutide group, and a 3 & 3 nmol / kg JY4 combined with Semaglutide group, with 8 mice in each group. Eight normal C57BL / 6 mice were also purchased as a blank control group. The animals were acclimatized for two weeks. Subsequently, each group received a subcutaneous (SC) injection of the corresponding drug in the neck (Note: the two drugs in the combination group must be mixed immediately before use), once daily (QD) for 3 consecutive weeks. After weekly administration, whole blood fasting blood glucose levels were measured using a Roche Vitality blood glucose meter. Random blood glucose levels were measured within 24 hours of free feeding after the first and last administration (measurement time points: 1h, 2h, 4h, 8h, 24h).

[0409] Results: The changes in fasting blood glucose levels in DIO mice after 3 weeks of administration are shown in Figure 10. The most significant decrease in blood glucose levels was observed in the first week of administration, followed by a gradual, slight increase, remaining lower than the model control group until the end of the experiment. This difference in efficacy was mainly due to resistance to semaglutide in DIO mice. However, throughout the experiment, the hypoglycemic effect of the combination of amylin analogues and semaglutide was significantly better than that of single-drug treatment. Furthermore, the therapeutic effect of JY4 & semaglutide was consistently superior to JY1 & semaglutide during the 3-week administration period. Figure 11 shows a significant difference in the rate of blood glucose recovery between the first and last 24 hours after administration, further demonstrating that resistance to semaglutide in DIO mice affected blood glucose regulation in the later stages of the experiment. In addition, the combination of amylin analogues (JY1 and JY4) and semaglutide significantly enhanced efficacy after both the first and last administrations; moreover, the improvement in the JY4 & semaglutide group was superior to that in the JY1 & semaglutide group.

[0410] Example 17: Repeated-dose weight loss test of JY4 injection in DIO rats

[0411] Male SD rats and DIO rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. DIO rats were randomly divided into 6 groups based on body weight: a model control group (60% HFD), a group receiving 10 nmol / kg of the reference molecule Cagrilintide, Eloralintide, Petrelintide injection, and a group receiving the test molecule JY4 injection, with 8 rats in each group; 8 normal SD rats were also purchased as a blank control group. Animals were acclimatized for one week. After grouping, food intake was monitored for 2-3 days before drug administration to collect initial food intake data. Subsequently, each group received subcutaneous (SC) injections of the corresponding drugs in the neck three times a week (TIW) for four consecutive weeks, according to the drug administration design. Animal body weight and food intake were monitored twice a week (BIW) after the start of the experiment.

[0412] result:

[0413] Figure 12A shows that during the administration period, the test molecule JY4 injection and the reference drugs Cagrilintide, Eloralintide, and Petrelintide all reduced the body weight of DIO rats, while the body weight of rats in the model control group and normal control group increased slowly during the experiment. Towards the later stage of the experiment, the body weight of animals in both the JY4 injection group and the reference drug group rebounded to some extent, but throughout the entire experiment, the decrease in body weight in the JY4 group at the same dose was consistently greater than that in the reference drug groups Cagrilintide, Eloralintide, and Petrelintide. On Day 28, the body weight reduction of DIO rats in the JY4 injection group relative to the model control group was 19.34%, significantly better than the 14.24% reduction in the Cagrilintide group, 12.45% reduction in the Eloralintide group, and 14.24% reduction in the Petrelintide group.

[0414] During the drug administration period, the average daily food intake of both the model control group (DIO rats) and the normal control group fluctuated little. All doses of the test substance JY4 and the reference drugs Cagrilintide, Eloralintide, and Petrelintide reduced the average daily food intake of DIO rats in all groups, showing a consistent trend of "first decreasing and then increasing." Specifically, the food intake of the drug-treated DIO rats was lowest upon the first administration, then gradually increased, remaining significantly lower than that of the model control group until the end of the administration period. Furthermore, Figure 12B shows that the food intake of the JY4-treated rats was significantly less than that of the reference drug groups (especially during Day 0-Day 12); while the food intake of the reference drug groups (Cagrilintide, Eloralintide, and Petrelintide) showed little difference during the experiment.

[0415] Example 18: Repeated-dose weight loss test of JY4 injection in DIO rats

[0416] Male SD rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. During acclimatization, induction, and the experimental period, they were fed a high-fat diet containing XTHF60 from Synergistic Biotech. Six SD rats were randomly selected and fed a normal diet as a control group. After 14 weeks of high-fat diet feeding, animals exceeding the average body weight of normal SD rats by 20% were selected as DIO rat models. DIO rats were randomly divided into six groups according to body weight: a model control group (Vehicle), a 10 nmol / kg Semaglutide injection group, a 10 nmol / kg JY4 injection group, and a 10 & 10 nmol / kg dose of the test peptide combined with Semaglutide, with six rats in each group. Subsequently, each group received the test sample via subcutaneous injection three times per week (TIW) for seven consecutive weeks. During the administration period, the weight and food intake of the test animals were monitored (twice per week, BIW).

[0417] result:

[0418] As shown in Figure 13A, the results of the body weight change rate are recorded as Day 0. During the dosing period, compared with the test drug JY4 and the reference drug Cagrilintide single-drug group, the JY4 & Semaglutide and CagriSema combination groups showed greater body weight changes and greater decreases, demonstrating a good synergistic effect. Moreover, compared with the reference drug Cagrilintide single-drug or combination therapy, the JY4 test group showed a greater decrease in body weight.

[0419] As shown in Figure 13B, during the drug administration period, the average daily food intake of the model control group (DIO rats) and the normal control group fluctuated within a certain range without significant changes. All drug administration groups significantly reduced the average daily food intake of DIO rats, and the trend was generally consistent, showing a "decrease followed by increase" trend. Specifically, the food intake of DIO rats dropped to its lowest point after the first administration, and subsequently, the food intake of each group gradually increased. During the drug administration period, the food intake of each combination drug group remained lower than that of each single drug group, and the food intake of each drug administration group remained lower than that of the model control group. The food intake results suggest that JY4 may produce a weight-loss effect by reducing the average daily food intake of DIO rats. Furthermore, the food intake of the animals in the test groups treated with JY4 injection, whether as a single drug or in combination, was basically consistent with that of the reference drug Cagrilintide.

[0420] Example 19: Repeated-dose weight loss test of JY4 injection in DIO rats

[0421] Male SD rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Upon delivery, six SD rats were randomly selected and fed a standard SPF maintenance diet as normal controls, while 60 rats were fed a 60% high-fat diet (60% kcal High-Fat Die, HFD) to induce diet-induced obesity (DIO). The obesity induction period was approximately 20 weeks, and rats whose body weight exceeded that of control rats by approximately 20% were considered to have met the DIO model criteria. After induction, all rats were grouped according to body weight. Six SD rats fed a maintenance diet under standard SPF conditions served as the control group. DIO rats were randomly divided into six groups according to body weight: model control group (60% HFD), 10 nmol / kg Tirzepatide positive control group, 10 nmol / kg Eloralintide reference group, 10 nmol / kg JY4 group, 10 nmol / kg Tirzepatide & 10 nmol / kg Eloralintide combination group, and 10 nmol / kg Tirzepatide & 10 nmol / kg JY4 combination group. Subsequently, each group was administered the test sample subcutaneously three times per week (TIW) for six consecutive weeks. During the administration period, the body weight and food intake of the test animals were monitored (twice per week, BIW).

[0422] result:

[0423] The day of the first administration is designated as Day 0. Figure 14A shows the weight change rate, indicating that at the end of the administration period, compared with the model control group, the weight of both the single-use group of the test substance JY4 and the combination group of the reference drug Eloralintide was significantly reduced, with highly significant differences compared to the model control group. The weight change of the test substance JY4 injection, whether used alone or in combination with Tirzepatide, was greater than that of Eloralintide or Eloralintide & Tirzepatide, indicating that the weight-loss effect of JY4 injection is superior to that of the reference drug Eloralintide.

[0424] Figure 14B shows that during the administration period, all treatment groups reduced the average daily food intake of DIO rats, and the effect on food intake was basically consistent, showing a "decrease followed by increase" trend. Specifically, the food intake of rats dropped to the lowest level after the first administration, and then gradually recovered. By the end of the administration period, the food intake of each treatment group was still lower than that of the model control group. During the administration period, the food intake of the JY4 and Eloralintide single-drug groups was basically the same; the food intake of the JY4 & Tirzepatide combination group and the Eloralintide & Tirzepatide combination group was also basically the same, but both were significantly lower than those of the JY4 or reference drug single-drug groups. This phenomenon reveals that the combination of JY4 and the positive control drug Tirzepatide showed a synergistic inhibitory effect on food intake.

Claims

1. A human amylin polypeptide analog, a pharmaceutically acceptable salt or solvate thereof, said amylin polypeptide analog comprising the following amino acid sequence: Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Formula I), wherein: Xaa1 is selected independently from K, R, Har, Orn, Dab, hLys, or is missing; Xaa3 is independently selected from N or G; Xaa 10 Independently selected from Q, G, E, N, or D; Xaa 11 Independently selected from: R, K, or Orn; Xaa 14 Choose independently from: E, D, or Aad; Xaa 17 Selected independently from: R, K, or Q; Xaa 18 Independently selected from: H or R; Xaa 21 Selected independently from: N, S, P, H, or missing; Xaa 22 Selected independently from: N, S, P, MeAsn or missing; Xaa 24 Independently selected from: G or MeGly; Xaa 25 Independently selected from: P or A; Xaa 26 Independently selected from: I, K, or MeIle; Xaa 28 Independently selected from: S, T, or P; Xaa 29 Independently selected from: S, T, or P; Xaa 31 Choose independently from: E, D, N, A, or Q; Xaa 37 Independently selected from: Y, S, T, P, (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, 4,4-difluoro-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro; Among them, there is disulfide bond cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

2. The human pancreatic amylin polypeptide analogue according to claim 1, and its pharmaceutically acceptable salt or solvate, characterized in that, The amylase polypeptide analogue comprises the amino acid sequence shown in Formula I, wherein: Xaa1 is independently selected from: R, Har, Orn, or K; Xaa3 is independently selected from N or G; Xaa 10 Independently selected from Q, E, or G; Xaa 11 Independently selected from R, K, or Orn; Xaa 14 Independently selected from: E or Aad; Xaa 17 Independently selected from R or Q; Xaa 18 Independently selected from H or R; Xaa 21 Selected independently from N or missing; Xaa 22 Selected independently from N or missing; Xaa 24 Independently selected from G or MeGly; Xaa 25 Independently selected from P or A; Xaa 26 Independently selected from I, K, or MeIle; Xaa 28 Independently selected from S or P; Xaa 29 Independently selected from S or P; Xaa 31 Independently selected from E or N; Xaa 37 Independently selected from (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro; The amino acids at positions 2 and 7 exhibit disulfide cyclization or thioacetal bridging, and the C-terminus of the polypeptides are all amides.

3. The human pancreatic amylin polypeptide analogue according to claim 1, and its pharmaceutically acceptable salt or solvate, characterized in that, The amylase polypeptide analogue comprises the amino acid sequence described in Formula II, Har-C-Xaa3-TATCAT-Xaa 10 -RLA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Formula II) in: Xaa3 is independently selected from N or G; Xaa 10 Independently selected from Q or E; Xaa 17 Independently selected from R or Q; Xaa 18 Independently selected from H or R; Xaa 21 Selected independently from N or missing; Xaa 22 Selected independently from N or missing; Xaa 24 Independently selected from G or MeGly; Xaa 25 Independently selected from P or A; Xaa 26 Independently selected from I, K, or MeIle; Xaa 28 Independently selected from S or P; Xaa 29 Independently selected from S or P; Xaa 31 Independently selected from E or N; Xaa 37 The polypeptide is independently selected from (2S,4R)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp, or 4-Oxo-Pro; wherein there is disulfide cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

4. The human pancreatic amylin polypeptide analogue according to claim 1, and its pharmaceutically acceptable salt or solvate, characterized in that, The amylin polypeptide analogue comprises the amino acid sequences shown in SEQ ID NO:4-SEQ ID NO:

80.

5. A human amylin analogue derivative, with the structural formula: AB, wherein: The chemically modified fragment A in the derivative has the following structural formula XY n -L m (Formula III), where X is the albumin-binding group, Y is the hydrophilic spacer group, and L is the linker; B in the derivative is an amylin polypeptide analog, whose amino acid sequence includes: Xaa1-C-Xaa3-TATCAT-Xaa 10 -Xaa 11 -LA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa[[ID=**23**]] 29 -T-Xaa 31 -VGSNT-Xaa 37 (Formula I), wherein: It should be noted that there may be some inaccuracies in the translation as the original text seems to be a rather specialized and potentially incomplete or unclear chemical / biological formula description. If possible, more context or clarification of the "Xaa" and other notations would be beneficial for a more precise translation. Also, it seems there might be a formatting issue in the original text where the tag 29 is not properly separated in the text editor. I've assumed it's just a continuous text flow for the purpose of translation. Xaa1 is K, R, Har, Orn, Dab, hLys, or missing; Xaa3 is N or G; Xaa 10 It is Q, G, E, N, or D; Xaa 11 It is R, K, or Orn; Xaa 14 It is E, D, or Aad; Xaa 17 It is R, K, or Q; Xaa 18 It is H or R; Xaa 21 It is N, S, P, H, or missing; Xaa 22 It is N, S, P, MeAsn, or missing; Xaa 24 It is G or MeGly; Xaa25 is either P or A; Xaa 26 Is it I, K, or MeIle? Xaa 28 Is it S, T, or P; Xaa 29 Is it S, T, or P; Xaa 31 It is E, D, N, A, or Q; Xaa 37 is Y, S, T, P, (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, 4,4-difluoro-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro; Among them, there is disulfide bond cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

6. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The derivative, amylin polypeptide analog B, comprises the amino acid sequence shown in Formula I, wherein: Xaa1 is independently selected from: R, Har, Orn, or K; Xaa3 is independently selected from N or G; Xaa 10 Independently selected from Q, E, or G; Xaa 11 Independently selected from R, K, or Orn; Xaa 14 Independently selected from: E or Aad; Xaa 17 Independently selected from R or Q; Xaa 18 Independently selected from H or R; Xaa 21 Selected independently from N or missing; Xaa 22 Selected independently from N or missing; Xaa 24 Independently selected from G or MeGly; Xaa 25 Independently selected from P or A; Xaa 26 Independently selected from I, K, or MeIle; Xaa 28 Independently selected from S or P; Xaa 29 Independently selected from S or P; Xaa 31 Independently selected from E or N; Xaa 37 Independently selected from (2S,4R)-Hyp, (2S,4S)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp or 4-Oxo-Pro; The amino acids at positions 2 and 7 exhibit disulfide cyclization or thioacetal bridging, and the C-terminus of the polypeptides are all amides.

7. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The derivative, amylin polypeptide analog B, comprises the amino acid sequence described in Formula II, wherein: Har-C-Xaa3-TATCAT-Xaa 10 -RLA-Xaa 14 -FL-Xaa 17 -Xaa 18 -SS-Xaa 21 -Xaa 22 -F-Xaa 24 -Xaa 25 -Xaa 26 -L-Xaa 28 -Xaa 29 -T-Xaa 31 -VGSNT-Xaa 37 (Formula II) Xaa3 is independently selected from N or G; Xaa 10 Independently selected from Q or E; Xaa 17 Independently selected from R or Q; Xaa 18 Independently selected from H or R; Xaa 21 Selected independently from N or missing; Xaa 22 Selected independently from N or missing; Xaa 24 Independently selected from G or MeGly; Xaa 25 Independently selected from P or A; Xaa 26 Independently selected from I, K, or MeIle; Xaa 28 Independently selected from S or P; Xaa 29 Independently selected from S or P; Xaa 31 Independently selected from E or N; Xaa 37 The polypeptide is independently selected from (2S,4R)-Hyp, (2S,4R)-4-F-Pro, (2S,4S)-4-F-Pro, (2S,3R)-Hyp, (2S,3S)-Hyp, or 4-Oxo-Pro; wherein there is disulfide cyclization or thioacetal bridging between the amino acids at positions 2 and 7, and the C-terminus of the polypeptide is an amide.

8. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The derivative, amylin polypeptide analog B, contains the amino acid sequences shown in SEQ ID NO:4-SEQ ID NO:

80.

9. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The albumin-binding group in the derivative has the following structure: HOOC(CH2) O CO-, where o is an integer between 14 and 22; (HO)2PO(CH2) p CO-, where p is an integer from 14 to 22; HO3S(CH2) q CO-, where q is an integer between 14 and 22; Where r is an integer between 14 and 22; Where s is an integer between 14 and 22; Where t is an integer between 13 and 21; Where u is an integer between 14 and 22; or Where v is an integer between 14 and 22.

10. The human pancreatic amylin analogue derivative according to claim 9, characterized in that, The albumin-binding group in the derivative is selected from HOOC(CH2). o CO-, where o is an integer between 16 and 22, selected from HOOC (CH2). 16 CO-, HOOC(CH2) 18 CO-, HOOC(CH2) 20 CO- or HOOC(CH2) 22 CO-.

11. The human pancreatic amylin analogue derivative according to claim 9, characterized in that, The albumin-binding group in the derivative is selected from (HO)₂PO(CH₂). P CO-, where p is an integer between 16 and 22, selected from (HO)2PO(CH2). 16 CO-, (HO)2PO(CH2) 18 CO-, (HO)2PO(CH2) 20 CO- or (HO)2PO(CH2) 22 CO-.

12. The human pancreatic amylin analogue derivative according to claim 9, characterized in that, The albumin-binding group in the derivative is selected from -CO(CH2). r CN4H(tetrazole), where r is an integer between 16 and 22, selected from -CO(CH2). 16 CN4H(tetrazole), -CO(CH2) 18 CN4H(tetrazole), -CO(CH2) 20 CN4H (tetrazole) or -CO (CH2) 22 CN4H (tetrazole).

13. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The hydrophilic spacer group Y in the derivative n The hydrophilic spacer group may be absent, or it may consist of one or more hydrophilic spacer groups, where n is an integer from 0 to 3.

14. The human pancreatic amylin analogue derivative according to claim 13, characterized in that, The hydrophilic spacer Y in the derivative is selected from the following structures:

15. The human pancreatic amylin analogue derivative according to claim 13, characterized in that, The hydrophilic spacer group is absent in the derivative.

16. The human pancreatic amylin analogue derivative according to claim 13, characterized in that, The hydrophilic spacer group in the derivative is AEEA, and its structure is shown below:

17. The human pancreatic amylin analogue derivative according to claim 13, characterized in that, The hydrophilic spacer group in the derivative is AEEA-AEEA-Glu, and its structure is shown below:

18. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The connector L in the derivative m The connector may be absent, or it may consist of one or more connectors, where m is an integer from 0 to 2.

19. The human pancreatic amylin analogue derivative according to claim 18, characterized in that, The connector L in the derivative is selected from the following structures:

20. The human pancreatic amylin analogue derivative according to claim 18, characterized in that, The connector is absent in the derivative.

21. The human pancreatic amylin analogue derivative according to claim 18, characterized in that, The linker in the derivative is Glu, and its structure is shown below:

22. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The structure of chemically modified fragment A in the derivative is shown below:

23. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The chemically modified fragment in the derivative is Glu-C20diacid, whose structure is shown below:

24. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The chemical modification fragment in the derivative is Glu-CO(CH2). 18 PO3H2 has the following structure:

25. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The chemical modification fragment in the derivative is Glu-CO(CH2). 20 CN4H (tetrazole) has the following structure:

26. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The chemically modified fragment in the derivative is AEEA-AEEA-Glu-C20diacid, and its structure is shown below:

27. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The chemically modified fragment in the derivative is AEEA-AEEA-Glu-CO(CH2). 18 PO3H2 has the following structure:

28. The human pancreatic amylin analogue derivative according to any one of claims 5-27, characterized in that, The linking site of the chemically modified fragment A in the derivative is either the α-amino group or the epsilon-amino group of the N-terminal (Xaa1) amino acid in the human amylin analog peptide B, or the linking site of the chemical fragment A is the K amino group in peptide B. 26 The Epsilon-amino group of amino acids.

29. The human pancreatic amylin analogue derivative according to claim 28, characterized in that, Chemical fragment A in the derivative is linked to the α-amino group of the N-terminal high arginine in polypeptide B.

30. The human pancreatic amylin analogue derivative according to claim 5, characterized in that, The amylin analogue derivatives mentioned above are selected from the compounds described in JY4-JY213.

31. The human pancreatic amylin analogue derivative according to claim 30, characterized in that, The amylin analogue derivatives mentioned above are selected from the following compounds:

32. A pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide analogue or a derivative thereof as described in any one of claims 1-31, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition according to claim 32, characterized in that... The pharmaceutical composition is formulated into dosage forms such as solutions, microemulsions, liposomes, or lyophilized powder injections.

34. The pharmaceutical composition according to claim 33, characterized in that... The concentration of the human amylin polypeptide analog or its derivative in the pharmaceutical composition is 0.1 mg / ml to 25 mg / ml.

35. The pharmaceutical composition according to claim 34, characterized in that... The concentration of the human amylin polypeptide analog or its derivative in the pharmaceutical composition is 1 mg / ml to 10 mg / ml.

36. The pharmaceutical composition according to claim 33, characterized in that... The pharmaceutical composition is a lyophilized preparation, and a solvent and / or diluent are added before use.

37. The pharmaceutical composition according to claim 32, characterized in that... The routes of administration of the pharmaceutical composition include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal / spinal cord, or other parenteral routes.

38. Use of the human amylin polypeptide analog or derivative thereof according to any one of claims 1-31 in the preparation of a medicament.

39. The use according to claim 38, characterized in that: The stated purpose is to prevent or treat obesity and obesity-related diseases.

40. The use according to claim 39, characterized in that: The diseases mentioned include, but are not limited to, overweight, morbid obesity, preoperative obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea and breathing problems, cartilage degeneration, osteoarthritis, metabolic dysfunction-associated steatohepatitis (MASH), and reproductive health complications of obesity or overweight such as infertility.

41. The use according to claim 40, characterized in that: The disease subjects may be affected by obesity accompanied by at least one weight-related comorbidity, such as diabetes, hypertension, dyslipidemia, sleep apnea, and cardiovascular disease.

42. The use according to claim 40, characterized in that: The intended use is for the prevention or treatment of, inhibition or reduction of weight gain, promotion of weight loss and / or reduction of excess weight.

43. The use according to claim 42, characterized in that: The stated purpose is achieved by controlling appetite, eating, food intake, calorie intake and / or energy expenditure.

44. The use according to claim 38, characterized in that: The stated purpose is for the prevention or treatment of diabetes and related diseases.

45. The use according to claim 44, characterized in that: The diseases mentioned include type 1 diabetes, type 2 diabetes, prediabetes, insulin resistance syndrome, metabolic dysfunction-associated steatohepatitis (MASH), impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, and diabetes-related complications.

46. ​​The use according to claim 45, characterized in that: The diabetes-related complications include, but are not limited to, cardiovascular and cerebrovascular diseases caused by diabetes, such as atherosclerosis, macrovascular disease, microvascular disease, coronary heart disease, peripheral artery disease or stroke, lower extremity vascular diseases (such as diabetic foot ulcers), eye diseases, peripheral neuropathy, diabetic cardiomyopathy and nephropathy, or combinations thereof.

47. The use of the human amylin polypeptide analogue or its derivative as described in any one of claims 1-31 in combination with one or more targeted drugs for the prevention or treatment of obesity or diabetes and related diseases, wherein, The targeted drugs mentioned include, but are not limited to, drugs for diabetes, obesity, and hypertension.

48. The use according to claim 47, characterized in that: The targeted drugs are selected from GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, GLP-1 / GIP / GCGR triple agonists, FGF21 derivatives, insulin, metformin, sulfonylureas, meglitinides, glitazones, DPP-IV inhibitors, and AGLT2 inhibitors.

49. The use according to claim 48, characterized in that: The drug is selected from exenatide, lixinatide, liraglutide, smegglutide, dulaglutide, abiglutide, leptin, neuropeptide Y, tirzepatide, retatrutide, mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, or BIO89-100.

50. The use according to claim 47, characterized in that: When the aforementioned human amylin polypeptide analogue or its derivative is administered in combination with GLP-1 receptor agonist drugs for the prevention or treatment of diabetes or obesity-related diseases, such as in combination with GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, or GLP-1 / GIP / GCGR triple agonists for the prevention or treatment of diabetes or obesity-related diseases, it exhibits a synergistic hypoglycemic or weight-loss effect.

51. The use according to claim 47, characterized in that: The stated uses refer to the administration of human amylin polypeptide analogues or their derivatives in combination with smegglutinin, tirzepatide or retatrutide for the prevention or treatment of obesity and obesity-related diseases.

52. The use according to claim 51, characterized in that: The diseases mentioned include overweight, morbid obesity, preoperative obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea and breathing problems, cartilage degeneration, osteoarthritis, metabolic dysfunction-associated steatohepatitis (MASH), and reproductive health complications of obesity or overweight (such as infertility).

53. The use according to claim 47, characterized in that: The stated uses refer to the administration of human amylin polypeptide analogues or their derivatives in combination with smegglutinin, tirzepatide, or retatrutide for the prevention or treatment of diabetes-related diseases.

54. The use according to claim 53, characterized in that: The diabetes-related diseases mentioned include type 1 diabetes, type 2 diabetes, prediabetes, insulin resistance syndrome, metabolic dysfunction-associated steatohepatitis (MASH), impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia, and diabetes-related complications.

55. The use according to claim 54, characterized in that: The diabetes-related complications mentioned include cardiovascular and cerebrovascular diseases, lower extremity vascular diseases, eye diseases, peripheral neuropathy, and kidney diseases caused by diabetes.

56. The use according to claim 55, characterized in that: The diabetes-related complications mentioned include diabetic retinopathy, diabetic nephropathy, diabetic foot ulcers, diabetic atherosclerosis, diabetic polyneuropathy, and diabetic fatty liver disease.

57. The use according to any one of claims 38-56, characterized in that: The human amylin analogue derivatives mentioned are selected from compounds JY4, JY5, JY6, JY11, JY13, JY14, JY17, JY18, JY19, JY22, JY26, JY28, JY29, JY49, JY50, JY131, JY132, JY133, JY134, JY135, JY136, JY137, JY138, JY193, JY201, JY209, JY210, JY204, JY211, JY212, or JY213.

58. The use according to claim 57, characterized in that: The human pancreatic amylin analogue derivative mentioned above is JY4, and its structural formula is as follows: The C-terminus is (2S,4R)-Hyp-NH2.

59. The use according to any one of claims 38-56, characterized in that: When the human amylin polypeptide analog or its derivative is administered parenterally, the dosage is 1 μg / kg-5 mg / kg, or 5 μg / kg-1000 μg / kg, or 10 μg / kg-500 μg / kg, or 20 μg / kg-100 μg / kg, or 30 μg / kg-80 μg / kg.

60. The use according to claim 59, characterized in that: The treatment regimen of the human amylin polypeptide analogue or its derivative is once daily, once weekly, once every two weeks, once every three weeks, or once every four weeks.

61. A method for preparing the human amylin polypeptide analog or derivative thereof according to any one of claims 1-31, wherein the method may be carried out by solid-phase and / or liquid-phase methods, stepwise or by fragment assembly, and optionally by separating and / or purifying the final product.

62. The preparation method according to claim 61, characterized in that: The method further includes the step of forming a disulfide bond between the cysteine ​​side chain thiol groups at positions 2 and 7 by oxidative cyclization or by forming a thioacetal bridged ring through a nucleophilic substitution reaction.

63. The preparation method according to claim 61, characterized in that: The method also includes a step of forming C-terminal amidation by non-synthetic methods through recombinant expression, purification, and induction.

64. The preparation method according to claim 61, characterized in that: The prepared human amylin polypeptide analogue can be further obtained by condensation reaction of fatty acid side chains with polypeptides to obtain human amylin polypeptide derivatives, and optionally separated and / or purified to obtain the final product.

Images