Therapeutic application of human amylin polypeptide derivative

Abstract

The invention provides a human amylin polypeptide analogue containing non-natural amino acid and application of a derivative of the human amylin polypeptide analogue to treatment of obesity and diabetes related diseases. The human amylin polypeptide derivative obtained by introducing non-natural amino acid into the N terminal of human amylin and modifying by albumin binding residues can effectively prevent enzymatic degradation, prolong the half-life period, participate in delay of gastric emptying and inhibition of postprandial glucagon release, can influence the rising speed of postprandial blood sugar through various mechanisms, and can be used for preparing the anti-hyperglycemia human amylin polypeptide derivative. The compound can be used for preventing or treating obesity, excessive ingestion, diabetes mellitus and other related metabolic diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a human amylin polypeptide derivative and its medical use. Background Art

[0002] The global prevalence of diabetes and obesity is becoming increasingly severe.

[0003] The World Obesity Atlas 2023 report shows that among people aged ≥5 years worldwide, overweight (BMI ≥ 25 kg / m 2 ) / obesity rate (BMI≥30kg / m 2 ) will rapidly increase from 38% in 2020 to 51% in 2035, and the number of people will climb from 2.6 billion in 2020 to more than 4 billion in 2035; the obesity rate will rise from 14% in 2020 to 24% in 2035, and the number of people will reach nearly 2 billion.

[0004] With rapid economic growth and rapidly changing lifestyles, the prevalence of diabetes and obesity continues to rise. Diabetes and obesity have become the most significant economic burdens and public health issues for countries, societies, and individuals. Without effective interventions, obesity- and diabetes-related complications will emerge globally, including in my country. These complications include cardiovascular disease, ophthalmological disorders, hepatobiliary diseases, stroke, and chronic kidney disease, posing a serious challenge to public health. Therefore, it is necessary to develop more effective innovative anti-diabetic and anti-obesity drugs and further investigate novel therapeutic mechanisms.

[0005] Islet amyloid polypeptide (IAPP), also known as amylin, is a glucose-regulating pancreatic hormone secreted alongside insulin. Amylin is involved in delaying gastric emptying and inhibiting postprandial glucagon release. It can influence the rate of postprandial blood glucose rise through various mechanisms, thereby lowering postprandial blood glucose. IAPP is also a satiety factor that, by activating receptors in the hindbrain and nucleus tractus solitarius, reduces energy intake and participates in regulating appetite and satiety. It can also enhance hepatocyte and plasma triglyceride metabolism, reducing hepatic adipose tissue and lipid deposition and plasma triglyceride fluctuations. Therefore, amylin has potential for the treatment of obesity and diabetes-related diseases.

[0006] Human amylin (hIAPP, SEQ ID No: 1) is a polypeptide hormone composed of 37 amino acid residues, with short peptide modifications at both ends. The terminal amide group and intramolecular disulfide bond are important for the integrity of its molecular biological function. hIAPP binds to two different receptor complexes, which contain calcitonin receptors and receptor activity-modifying proteins RAMP1 or RAMP3. hIAPP can play a role in regulating food selection and preference through the hypothalamus, ventral tegmental area and lateral tegmental nucleus. Whether in mice or humans, hIAPP binds to hIAPP receptors on the cell membranes of brain neurons, and receptor-mediated signal transduction affects the feeding center of the hypothalamus, delays gastric emptying, produces a satiety effect, and ultimately reduces food intake, slows down the absorption of glucose in the small intestine, prolongs the time of absorption into the blood, and lowers postprandial blood sugar. Therefore, some have proposed using hIAPP-modified proteins targeting the amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) as adjunctive therapies for diabetes and weight management, potentially complementing other treatments such as GLP-1 and leptin. However, hIAPP's amyloidogenicity (fibrosis), chemical instability, and solubility make it unsuitable as a therapeutic agent.

[0007] Pramlintide (SEQ ID No: 2) is a peptide peptide developed by Amylin Pharmaceuticals. A marketed adjunctive treatment for diabetes (types 1 and 2) with limited affinity for the calcitonin receptor and high-affinity binding to the amylin polypeptide receptor. Compared to hIAPP, pramlintide features amino acid substitutions at A25P, S28P, and S29P, overcoming the persistent proliferation and insolubility of hIAPP. This mitigates the effects of self-replication, amyloid deposition, and B cell apoptosis, while retaining the glucose-lowering effects of amylin. Despite its advantages, pramlintide has a shorter half-life, requiring two to three daily injections. Like hIAPP, it also exhibits poor solubility at physiological pH.

[0008] WO2006105527, WO2012168431, CN201280028554.1, and WO2013156594 disclose hIAPP analogs and / or derivatives with improved pharmacokinetic or pharmacodynamic (PK / PD) properties. However, most currently disclosed hIAPP analogs and derivatives suffer from susceptibility to fibrillation, poor solubility, short half-lives, and poor efficacy. Compared to other disclosed hIAPP analogs and / or derivatives, the human amylin polypeptide analog disclosed in CN201280028554.1 exhibits superior efficacy and drugability. The human amylin polypeptide has an albumin-binding moiety, significantly extending its half-life compared to pramlintide. The "N-α-[(S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]-[Glu14,Arg17,Pro37]-pramlintide" analogue described in this patent is currently under clinical development and is referred to as Cagrilintide (also known as AM833). The partial amino acid sequence of its polypeptide is shown in SEQ ID No: 3. However, there remains a need to identify hIAPP polypeptides with superior performance in at least one of the following aspects: physicochemical properties, pharmacodynamics, or pharmacokinetic parameters. SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention relates to a human amylin polypeptide analog containing unnatural amino acids, the sequence of which is shown below:

[0010] X CNTATCATQ RLAEFLRHS NNFGPILPPT NVGSNTP(SEQ ID No:4)

[0011] wherein X is an unnatural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid;

[0012] There is an intramolecular disulfide bond between the two Cys residues at positions 2 and 7 in the polypeptide sequence; and the C-terminus of the polypeptide is an amide.

[0013] In another embodiment, the inventors of the present application unexpectedly discovered that when a basic amino acid is present at the N-terminus of a human amylin polypeptide, the isoelectric point of the polypeptide can be effectively increased, resulting in improved performance in at least one of the following aspects: physicochemical properties, pharmacodynamics, or pharmacokinetic parameters. Therefore, in a preferred embodiment, the inventors preferably introduce a non-natural basic amino acid into the N-terminus of human amylin, such as L-ornithine (L-Orn), L-diaminobutyric acid, L-homoarginine, L-diaminopropionic acid, diaminoacetic acid, D-ornithine (D-Orn), D-diaminobutyric acid, D-homoarginine, or D-diaminopropionic acid.

[0014] In another preferred embodiment, the non-natural basic amino acid introduced in the present invention is L-ornithine (L-Orn) or D-ornithine (D-Orn).

[0015] In another embodiment, the present invention provides a human amylin polypeptide derivative modified with an albumin binding residue, the structural formula of which is shown below:

[0016] YLZ (I)

[0017] Wherein, Y is an albumin binding residue; L is a linker; Z is a human amylin polypeptide analog, the sequence of which is shown in SEQ ID No: 4, wherein an intramolecular disulfide bond exists between the two Cys residues at positions 2 and 7 in the sequence, and the C-terminus of the polypeptide is an amide;

[0018] wherein X in SEQ ID No: 4 is a non-natural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid;

[0019] The albumin binding residue is connected to the non-natural amino acid at the N-terminus of the human amylin polypeptide via a linker.

[0020] In another embodiment, in the human amylin polypeptide derivative modified with an albumin-binding residue, L is a linker, which may be present independently or absent. The linker may comprise one or more amino acids, which is bound to the albumin-binding moiety at one end and to the amino group of the non-natural amino acid at the N-terminus of the human amylin polypeptide at the other end.

[0021] In another embodiment, in the human amylin polypeptide derivative modified with an albumin binding residue, wherein Y is an albumin binding residue, the term "albumin binding residue" as used herein means a residue that non-covalently binds to human serum albumin. The albumin binding residue attached to the human amylin polypeptide analog typically has a binding affinity for human serum albumin of less than about 10 μM or even less than about 1 μM.

[0022] In another embodiment, in the human amylin polypeptide derivative represented by structural formula (I), the albumin binding residue (Y) and the linker (L) moiety can be collectively referred to as a substituent. In another embodiment, the human amylin polypeptide derivative represented by structural formula (I) can comprise at least one substituent, such as one, two, or three substituents. In another embodiment, the human amylin polypeptide derivative represented by structural formula (I) preferably comprises one substituent.

[0023] In another embodiment the albumin binding residue has 6-40 carbon atoms, 8-26 carbon atoms or 14-22 carbon atoms, such as 16, 17, 18, 19, 20 carbon atoms.

[0024] In another embodiment, the preferred albumin binding residue comprises a group that is negatively charged at pH 7.4.

[0025] In another embodiment, the albumin binding residue is an acyl group selected from:

[0026] c) CH3(CH2) r CO-*, where r is an integer from 12 to 20;

[0027] d)HOOC(CH2) s CO-*, where s is an integer from 12 to 22.

[0028] The present invention introduces non-natural amino acids into the N-terminus of human amylin, which can effectively prevent enzymatic degradation and prolong the half-life; at the same time, when non-natural basic amino acids are preferably introduced into the N-terminus, the isoelectric point of the polypeptide can be effectively increased, so that it has improved performance.

[0029] In another embodiment, the human amylin polypeptide derivatives provided by the present invention have improved performance in at least one aspect, such as physicochemical properties, pharmacodynamics, or pharmacokinetic parameters, compared to previously reported hIAPP polypeptide substances.

[0030] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide analog or derivative thereof and a pharmaceutically acceptable carrier.

[0031] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or derivative thereof for preventing or treating obesity and obesity-related diseases, including but not limited to obesity, non-alcoholic steatohepatitis (NASH), hypertension, and cardiovascular disease. In another embodiment, the pharmaceutical use includes reducing food intake, reducing appetite, and / or promoting weight loss.

[0032] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or its derivative for preventing or treating diabetes-related diseases, including type 1 diabetes, type 2 diabetes and diabetes-related complications, including but not limited to cardiovascular and cerebrovascular diseases, lower limb vascular diseases, eye diseases, peripheral neuropathy and kidney disease caused by diabetes.

[0033] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or its derivative in combination with one or more targeted drugs for treating 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, insulins, metformin, sulfonylureas, glinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Liquid phase diagram of compound A. The liquid phase results showed that the liquid phase purity of compound A was 97%.

[0035] Figure 2 Mass spectrum of compound A. The mass spectrometry results confirmed that the molecular weight of compound A was 3824.85.

[0036] Figure 3 Liquid phase diagram of compound 1. The liquid phase results showed that the liquid phase purity of compound 1 was 98.6%.

[0037] Figure 4 Mass spectrum of compound 1. The mass spectrometry results confirmed that the molecular weight of compound 1 was 4392.24.

[0038] Figure 5 Liquid phase diagram of compound 4. The liquid phase results showed that the liquid phase purity of compound 4 was 95.63%.

[0039] Figure 6 Mass spectrum of compound 4. The mass spectrometry results confirmed that the molecular weight of compound 4 was 4392.24.

[0040] Figure 7 Biological activity assay. The results of the calcitonin receptor and β-galactosidase reporter gene assays for compound 1 and AM833 showed that the β-galactosidase signals generated by both compounds after stimulating the calcitonin receptor were basically the same, and the EC 50 The EC values of compound 1 were 6.308 nM and 6.720 nM, respectively. The two compounds have similar biological activities. 50 The value is about 6% higher than that of AM833.

[0041] Figure 8 Kinetic analysis of the binding of compound 1 and AM833 to human serum albumin.

[0042] Figure 9 Binding curves of compound 1, AM833, compound 4 and compound 12 to human membrane protein.

[0043] Figure 10 Binding curves of compound 1, AM833, compound 4 and compound 12 to rat membrane protein.

[0044] Figure 11 Activity curves of compound 1, AM833, compound 4, and compound 12 against hCTR and hAMY3R overexpressing cells.

[0045] Figure 12 cAMP agonist curves of compound 1, AM833h and compound 4 stimulating rCTR and rAMY3R overexpressing cells.

[0046] Figure 13 Pharmacokinetic evaluation: Compound 1 has a longer half-life and higher bioavailability than AM833, showing pharmacokinetic advantages.

[0047] Figure 14 Pharmacodynamic evaluation: Compound 1 had an appetite suppressant and weight-reducing effect comparable to that of AM833.

[0048] Figure 15 The weight loss efficacy of AM833, Compound 1, and Compound 4 injections combined with Semaglutide was tested on DIO rats. When AM833, Compound 1, or Compound 4 were injected together with Semaglutide, the weight loss effect on DIO rats was significantly enhanced compared to the individual compounds. The weight loss effect of Compound 1 combined with Semaglutide was comparable to that of the AM833 combined with Semaglutide group, while the weight loss effect of Compound 4 combined with Semaglutide was superior to that of either AM833 or Compound 1 combined with Semaglutide.

[0049] Figure 16 Oral glucose tolerance test of SD rats with injection of compound 1.

[0050] Figure 17 Compound 1 injection combined with semaglutide demonstrated a synergistic effect in ZDF rats, demonstrating a significant synergistic effect in the blood glucose lowering effect of compound 1 in combination with semaglutide, as shown in the 4-hour randomized blood glucose level changes in ZDF (fa / fa) type 2 diabetic rats. Detailed Description of the Invention

[0052] To make the present invention easier to understand, certain terms are first defined. Unless otherwise indicated, the scientific and technical terms used herein should have the meanings commonly understood by those of ordinary skill in the art. Additional definitions will be set forth throughout the detailed description.

[0053] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art and are fully explained in the technical literature and general textbooks in the art, such as Molecular Cloning: A Laboratory Manual.

[0054] As used herein, "amino acid" is defined as natural amino acids 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).

[0055] "Non-natural amino acids" refer to amino acids that are not encoded by the existing 64 genetic codons. Non-natural amino acids referred to herein include, but are not limited to, L-ornithine (L-Orn), L-diaminobutyric acid, dimethylalanine (Aib), L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine (D-Orn), D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid. Its structural formula is as follows:

[0056]

[0057] As used herein, "human amylin polypeptide" refers to a 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 intracellular synthesis, it is stored together with insulin in insulin-secreting vesicles within pancreatic β cells and is secreted together with insulin 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:

[0058] 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)

[0059]

[0060] As used herein, "pramlintide" refers to a synthetic polypeptide having the sequence set forth in SEQ ID No: 2. SEQ ID No: 2 and pramlintide are used interchangeably herein. The amino acid sequence and structure of pramlintide, as defined herein, are as follows: 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)

[0061]

[0062] As used herein, "Cagrilintide" refers to a synthetic polypeptide having the sequence set forth in SEQ ID No: 3. SEQ ID No: 3 and Cagrilintide / AM833 are used interchangeably herein. 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:

[0063] KCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP(SEQ ID No:3) or

[0064] Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Glu Phe Leu ArgHis Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val Gly Ser Asn ThrPro(SEQ ID No:3)

[0065]

[0066] As used herein, a sequence "variant" refers to a sequence that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0067] As used herein, "polypeptide analogs" are defined as polypeptides described above with one or more amino acid substitutions and / or one or more deletions and / or one or more additions / insertions. Amino acid substitution modification refers to the replacement of an amino acid residue by an amino acid residue having similar side chains or physicochemical characteristics, wherein the amino acid may be a natural or non-natural amino acid, and the amino acid substitution modification includes but is not limited to lysine-ornithine replacement, aspartic acid-glutamic acid replacement, valine-arginine replacement, and tyrosine-proline replacement in the peptide chain. The number of amino acid insertions, additions, deletions, or substitutions may be at least one, but there may be up to 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, additions, deletions, or substitutions. Any natural or non-natural amino acid, synthetic amino acid, peptidomimetic, or other compound may be used for substitution or addition. Addition or deletion of amino acid residues may occur at the N-terminus and / or the C-terminus of the peptide.

[0068] As used herein, a "human amylin polypeptide derivative" is defined as a product obtained by chemically modifying the above-described polypeptide or analog from its amino terminus (N-terminus) to its carboxyl terminus (C-terminus). Chemical modifications include, but are not limited to, amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation, and cyclization. A human amylin polypeptide derivative may include one or more substituents on one or more of the amino acid residues in the polypeptide.

[0069] As used herein, the term "substituent" refers to any suitable moiety that is bonded (particularly covalently bonded) to an amino acid residue, particularly to any available position on the amino acid residue. Typically, a suitable moiety is a chemical moiety. The substituent is attached to a natural or non-natural amino acid in a polypeptide. In some embodiments, the derivative has a substituent on one amino acid, the amino acid residue being the amino acid residue at the N-terminal residue. In some embodiments, the derivative has a substitution at the N-terminal amino acid residue, the amino acid residue being ornithine.

[0070] As used herein, the term "albumin binding residue" means a residue that non-covalently binds to human serum albumin. Albumin binding residues linked to human amylin polypeptide analogs typically have a binding affinity for 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 for competition is a measure of the affinity of a compound. Various albumin binding residues are known, including linear and branched lipophilic moieties containing 12-40 carbon atoms, compounds having a cyclopentaphenanthrene backbone, and / or peptides having 10-45 amino acid residues. Albumin binding properties can be measured by surface plasmon resonance as described in J. Biol. Chem. 277 (38), 35035-35042, (2002). Albumin binding residues and affinity determination methods are described in detail in CN201280028554.1 and CN201180015252.6, which are incorporated herein by reference in their entirety.

[0071] The term "fibrillation" as used herein refers to the physical interaction between polypeptide molecules that results in the formation of oligomers, which can remain dissolved or precipitate from solution as large, visible aggregates. The degree of fibrillation of a polypeptide can be measured by visual inspection, chromatographic methods, ThT fibrillation assay (sometimes referred to as ThT fibrillogenesis assay), and / or turbidity. Related methods are described in detail in CN201280028554.1, etc., which are incorporated herein by reference in their entirety.

[0072] The term "linker" as used herein refers to any suitable portion that connects a substituent to a human amylin polypeptide or an analog thereof. The linker can form a new substituent together with a substituent portion (e.g., an albumin binding portion). The linker can exist independently or not. The linker can comprise one or more amino acids, or a combination of at least one amino acid and an amine. In one embodiment, the amine is preferably the group OEG. The linker is bound to the albumin binding portion at one end and to the amino group on the non-natural amino acid at the N-terminus of the human amylin polypeptide at the other end.

[0073] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also means an amount sufficient to allow for or facilitate diagnosis. The effective amount for a particular subject may vary depending on a variety of factors, such as the condition being treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0074] "Pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, carriers for compositions containing polypeptides or derivatives thereof are suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0075] The term "subject" or "patient" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, mice, rats, cats, cows, horses, chickens, amphibians, and reptiles.

[0076] Human amylin polypeptide analogs and derivatives

[0077] The present invention relates to human amylin polypeptide analogs and polypeptide derivatives thereof. By substituting non-natural amino acids for pramlintide (SEQ ID No: 2) and modifying the N-terminal fatty acid, the human amylin polypeptide derivatives have improved properties in at least one aspect, such as physicochemical properties, pharmacodynamics, or pharmacokinetic parameters, and can be used for the treatment of obesity and diabetes-related diseases.

[0078] In one aspect, the present invention relates to a human amylin polypeptide analog containing unnatural amino acids, the sequence of which is shown below:

[0079] X CNTATCATQ RLAEFLRHS NNFGPILPPT NVGSNTP(SEQ ID No:4)

[0080] wherein X is an unnatural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid;

[0081] There is an intramolecular disulfide bond between the two Cys residues at positions 2 and 7 in the polypeptide sequence; and the C-terminus of the polypeptide is an amide.

[0082] The inventors introduced non-natural amino acids into the N-terminus of human amylin, which can effectively prevent enzymatic degradation and prolong half-life. In another embodiment, the inventors of the present application unexpectedly discovered that when the N-terminus of the human amylin polypeptide is a basic amino acid, the isoelectric point of the polypeptide can be effectively improved, so that it has improved physicochemical properties. Therefore, in a preferred embodiment, the inventors preferably introduce non-natural basic amino acids into the N-terminus of human amylin, such as L-ornithine (L-Orn), L-diaminobutyric acid, L-homoarginine, L-diaminopropionic acid, diaminoacetic acid, D-ornithine (D-Orn), D-diaminobutyric acid, D-homoarginine and D-diaminopropionic acid. In another preferred embodiment, the non-natural basic amino acids introduced by the present invention are L-ornithine (L-Orn) or D-ornithine (D-Orn). The basic amino acid structural formula is as follows:

[0083]

[0084] In another embodiment, the present invention provides a human amylin polypeptide derivative modified with an albumin binding residue, the structural formula of which is shown below:

[0085] YLZ(I)

[0086] Wherein, Y is an albumin binding residue; L is a linker; Z is a human amylin polypeptide analog, the sequence of which is shown in SEQ ID No: 4, wherein an intramolecular disulfide bond exists between the two Cys residues at positions 2 and 7 in the sequence, and the C-terminus of the polypeptide is an amide;

[0087] wherein X in SEQ ID No: 4 is a non-natural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid;

[0088] The albumin binding residue is connected to the non-natural amino acid at the N-terminus of the human amylin polypeptide via a linker.

[0089] In another embodiment, in the human amylin polypeptide derivative, the sequence of Z is as shown in SEQ ID No: 4, wherein X is a non-natural basic amino acid, which may be selected from the amino acids shown in the following table. More preferably, X as shown in SEQ ID No: 4 is L-ornithine or D-ornithine.

[0090]

[0091]

[0092] In another embodiment, in the human amylin polypeptide derivative modified with an albumin-binding residue, L is a linker, which may be present independently or absent. The linker may comprise one or more amino acids, which is bound to the albumin-binding moiety at one end and to the amino group of the non-natural amino acid at the N-terminus of the human amylin polypeptide at the other end.

[0093] For some embodiments, preferred linkers are selected from γGlu, γGlu-γGlu, γGlu-γGlu-γGlu, γGlu-γGlu-γGlu-γGlu, Glu, Glu-Glu, Glu-γGlu, Glu-Arg, Glu-G lu-Arg, His, His-His, His-γGlu, His-His-γGlu, Gly, Gly-γGlu, Ser, Ser-γGlu, D-Arg-D-Arg, Arg, Arg-Arg, Arg-Arg-γ Glu,Ser-Ser,-Gly-Ser-Ser,Ser-Ser,-Gly-Ser-Ser-γGlu,Ser-Ser-Gly-Ser-Ser-Gly,Ser-Ser-Gly-Ser-Ser-Gly- γGlu, γGlu-OEG, γGlu-2xOEG and OEG, preferably the linker is selected from γGlu, γGlu-γGlu, γGlu-OEG, γGlu-2xOEG, OEG and -NHC(O)CH2CH2CH2S(O)2NH-, etc.

[0094] For example, the linker term "γGlu" refers to an amino acid residue having the following structure:

[0095]

[0096] Another example of a spacer linker is a combination of at least one amino acid and an amine. In one embodiment, it is preferred that the amine is a group OEG, wherein the structural formula of OEG is as follows:

[0097]

[0098] By use of the term "γGlu-OEG" is meant a moiety having the following structure:

[0099]

[0100] By use of the term "γGlu-OEG-OEG" is meant a moiety having the following structure:

[0101]

[0102] In another embodiment, more preferably the linker can be selected from the following groups:

[0103] a)

[0104] b)

[0105] In another embodiment, in the human amylin polypeptide derivative modified with an albumin binding residue, Y is an albumin binding residue. As used herein, the term "albumin binding residue" means a residue that non-covalently binds to human serum albumin. The albumin binding residue attached to the human amylin polypeptide analog typically has a binding affinity for human serum albumin of less than about 10 μM or even less than about 1 μM. Various albumin binding residues are known, including linear and branched lipophilic moieties containing 12-40 carbon atoms, compounds having a cyclopentaphenanthrene backbone, and / or peptides having 10-45 amino acid residues. Albumin binding properties can be measured by surface plasmon resonance as described in the following literature: J. Biol. Chem. 277 (38), 35035-35042, (2002).

[0106] In one embodiment the albumin binding residue has 6-40 carbon atoms, 8-26 carbon atoms or 12-22 carbon atoms, such as 16, 17, 18, 19, 20 carbon atoms.

[0107] In another embodiment, the preferred albumin binding residue comprises a group that is negatively charged at pH 7.4.

[0108] In another embodiment, the albumin binding residue is an acyl group selected from:

[0109] c) CH3(CH2) r CO-*, where r is an integer from 12 to 20;

[0110] d)HOOC(CH2) s CO-*, where s is an integer from 12 to 22.

[0111] In some embodiments, the albumin binding residue is selected from CH3(CH2) r CO-acyl, wherein r is an integer from 12 to 20, more preferably selected from CH3(CH2) 12 CO-, CH3(CH2) 14 CO-, CH3(CH2) 16 CO-, CH3(CH2) 18 CO-, CH3(CH2) 20 CO- and CH3(CH2) 22 CO-;

[0112] In some embodiments, the albumin binding residue further comprises a carboxylic acid group, such as HOOC(CH2) s CO-, wherein s is an integer from 12 to 22. More preferably selected from HOOC(CH2) 14 CO-, HOOC(CH2) 16 CO-, HOOC(CH2) 18 CO- or HOOC(CH2) 20 CO-.

[0113] In another embodiment, in the human amylin polypeptide derivative represented by structural formula (I), the albumin binding residue (Y) and the linker (L) moiety can be collectively referred to as a substituent. In another embodiment, the human amylin polypeptide derivative represented by structural formula (I) can comprise at least one substituent, such as one, two, or three substituents. In another embodiment, the human amylin polypeptide derivative represented by structural formula (I) preferably comprises one substituent.

[0114] In an embodiment of the present invention, preferred substituents have a binding affinity for human serum albumin of less than about 10 μM or less than about 1 μM. Preferred substituents include groups that can be negatively charged at pH 7.4. In another embodiment, preferred substituents are:

[0115]

[0116] In another embodiment, the present invention provides a human amylin polypeptide derivative, wherein:

[0117] (1) The derivative has a structure as shown in formula (I), wherein Z is a human amylin polypeptide analog as shown in SEQ ID No: 4, wherein X at the N-terminus is ornithine;

[0118] (2) The ornithine residue is connected to the albumin binding residue via a linker.

[0119] In another embodiment, the present invention provides a human amylin polypeptide derivative, wherein:

[0120] (1) The derivative has a structure as shown in formula (I), wherein Z is a human amylin polypeptide analog as shown in SEQ ID No: 4, wherein X at the N-terminus is L-ornithine or D-ornithine;

[0121] (2) the ornithine residue is connected to the albumin binding residue via a linker;

[0122] (3) The derivative has a solubility of about 200 μM or higher at pH 7.0.

[0123] In one embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0124]

[0125] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0126]

[0127] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0128]

[0129] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0130] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0131]

[0132] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0133]

[0134] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0135]

[0136] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0137]

[0138] wherein r is an integer from 12 to 20, and s is an integer from 12 to 22.

[0139] In another preferred embodiment, the structures of the human amylin polypeptide derivatives provided by the present invention are shown in Compounds 1-33 in Table 2 of Example 3.

[0140] In a particularly preferred embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0141]

[0142]

[0143] The present invention introduces non-natural amino acids into the N-terminus of human amylin, which can effectively prevent enzymatic degradation and prolong the half-life. At the same time, when the N-terminus is a basic amino acid, the isoelectric point of the polypeptide can be more effectively increased, and compared with previously reported hIAPP polypeptide substances, at least one aspect of the physicochemical properties, pharmacodynamics or pharmacokinetic parameters is improved.

[0144] As demonstrated in the Examples of the present invention, the human amylin polypeptide derivatives provided herein have a solubility of approximately 200 μM or higher at pH 7.0. For example, under the conditions of Example 4 of the present invention, human amylin polypeptide derivatives Compounds 1, 4, and AM833 all have a solubility of >1 g / ml in water and >50 mg / ml in acetate-phosphate buffer (pH 2.0-8.0) (20°C).

[0145] In another embodiment, the present invention provides human amylin polypeptide derivatives that bind to human calcitonin receptor (CTR) and amylin receptor 3 (AMY3R) membrane proteins. Overall, these derivatives exhibit comparable in vitro biological activity to Cagrilintide (AM833). Compounds 1 and 4 exhibit similar affinities to AM833 for human membrane proteins, while compound 12 exhibits slightly weaker affinity than AM833. Compound 4 exhibits a stronger affinity for AMY3R than CTR, while compound 1 and AM833 exhibit comparable affinities for CTR and AMY3R.

[0146] In another embodiment, the present invention provides assays for the activity of human amylin peptide derivatives against human CTR and AMY3R cells. Compared to Cagrilintide (also known as AM833), Compound 1 exhibited comparable agonist activity against human CTR and AMY3R cells, Compound 4 exhibited approximately three times greater agonist activity than AM833, and Compound 12 exhibited slightly weaker agonist activity than AM833.

[0147] In another embodiment, the human amylin polypeptide derivative provided by the present invention has a comparable or longer half-life and higher bioavailability than Cagrilintide (also known as AM833). In another embodiment, Compound 1 and AM833 provided by the present invention have similar binding abilities to human serum albumin.

[0148] In another embodiment, the human amylin polypeptide derivatives provided by the present invention have comparable or better appetite suppressant and weight reduction effects than Cagrilintide (also known as AM833). When co-injected with Semaglutide, the weight reduction effect in DIO rats was significantly enhanced compared to each compound used alone.

[0149] In another embodiment, the present invention provides a human amylin polypeptide derivative, which, alone or in combination with Semaglutide, is administered subcutaneously to ZDF (fa / fa) type II diabetic rats. The 4-hour random blood glucose (RBG) value of the ZDF (fa / fa) type II diabetic rats is significantly reduced.

[0150] Synthesis of Human Amylin Peptide Analogs and Derivatives

[0151] The present invention also relates to a preparation method for synthesizing the human amylin polypeptide analogs and derivatives thereof, which can be assembled stepwise or by fragmentation using solid-phase and / or liquid-phase methods, and optionally includes the steps of isolating and / or purifying the final product. The method also includes the step of forming a disulfide bond between the cysteine side chain sulfhydryl groups at positions 2 and 7 by oxidative cyclization. The method also includes the step of forming a C-terminal amidation using non-synthetic methods through recombinant expression, purification, and induction.

[0152] The prepared human amylin polypeptide analog can be further subjected to a condensation reaction between a fatty acid side chain and the polypeptide to obtain a human amylin polypeptide derivative, which can then be optionally isolated and / or purified to obtain the final product. Similar synthesis and purification processes are described in detail in the prior art WO2006105527, CN201180015252.6, CN201280028554.1, WO2012168431, and WO2013156594, all of which are incorporated herein by reference.

[0153] pharmaceutical preparations

[0154] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide analog or derivative thereof and a pharmaceutically acceptable carrier.

[0155] In one aspect, 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 solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other physiologically compatible carriers. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0156] The pharmaceutical composition must generally be sterile and stable under the conditions of production and storage. The composition can be formulated into dosage forms such as solutions, microemulsions, liposomes, or freeze-dried powder injections. In one embodiment, the pharmaceutical preparation is a liquid preparation that can be formulated as a solution or a suspension. In one embodiment, the concentration of the human amylin polypeptide analog or its derivative present in the preparation is about 0.1 mg / ml to about 25 mg / ml, more preferably about 1 mg / ml to about 10 mg / ml. The drug can be provided directly in a unit dosage form, such as an injection pen containing the pharmaceutical preparation. Furthermore, it can be administered parenterally, such as subcutaneously, intramuscularly, intravenously, transdermally, etc.

[0157] In another embodiment, the pharmaceutical preparation is a lyophilized preparation, to which a solvent and / or diluent is added by a doctor, nurse, or patient before use.

[0158] The present invention also relates to pharmaceutical preparations comprising the human amylin polypeptide analogs or derivatives thereof. The pharmaceutical preparations may include pharmaceutically acceptable carriers, excipients, or protein protectants. Preferred routes of administration of the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal / spinal, or other parenteral administration routes, such as by injection or infusion.

[0159] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, while being non-toxic to the patient. A "therapeutically effective amount" of the human amylin polypeptide analog or derivative of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of damage or disability caused by the disease. One of ordinary skill in the art will be able to determine such an amount based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0160] Drug uses

[0161] The present invention also relates to the pharmaceutical use of the human amylin polypeptide analog or its derivative. In another embodiment, the present invention provides a method for treating a disease, comprising administering a therapeutically effective amount of the human amylin polypeptide analog or its derivative to a subject in need of treatment.

[0162] The invention provides a method for treating a disease, comprising administering a therapeutically effective amount of a human amylin polypeptide analog or derivative thereof according to the present invention to a subject in need of treatment. The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, mice, rats, cats, cows, horses, chickens, amphibians, and reptiles. In another embodiment, the subject or individual to whom the human amylin polypeptide analog or derivative thereof is administered is a mammal, e.g., a mouse, monkey, dog, cow, horse, or human, preferably a human.

[0163] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or derivative thereof for preventing or treating obesity and obesity-related diseases, including but not limited to overweight, morbid obesity, pre-operative 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 subject may be affected by obesity accompanied by at least one weight-related comorbidity (e.g., diabetes, hypertension, dyslipidemia, sleep apnea, and cardiovascular disease, etc.).

[0164] In another embodiment, the present invention provides a method for preventing or treating, inhibiting or reducing weight gain, promoting weight loss and / or reducing excess body weight using the human amylin polypeptide analog or derivative thereof. For example, treatment can be achieved by controlling appetite, food intake, food intake, calorie intake and / or energy expenditure.

[0165] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or its derivative for preventing or treating diabetes-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 caused by diabetes (atherosclerosis, large vessel disease, microvascular disease, coronary heart disease, peripheral arterial disease or stroke, etc.), lower limb vascular lesions (diabetic foot ulcers), eye lesions, peripheral neuropathy, diabetic heart disease and kidney disease, or a combination thereof.

[0166] Combination therapy

[0167] In another embodiment, the present invention provides a use of the human amylin polypeptide analog or its derivative for preventing or treating obesity or diabetes and related diseases, wherein the human amylin polypeptide analog or its derivative can also be used in combination with one or more targeted drugs, the targeted drugs including but 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 class, metformin, sulfonylureas, glinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc., wherein more specific drug types are selected from exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, leptin, neuropeptide Y, Tirzepatide, retatrutide, Mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, etc.

[0168] When the human amylin polypeptide analog or its derivative described in the present invention is administered in combination with a GLP-1 receptor agonist drug for the prevention or treatment of diabetes or obesity-related diseases, for example, when it is administered in combination with a GLP-1 derivative, a GLP-1R / GCGR dual agonist, a GLP-1 / GIP dual agonist, or a GLP-1 / GIP / GCGR triple agonist for the prevention or treatment of diabetes or obesity-related diseases, it exhibits a synergistic hypoglycemic or weight-reducing effect.

[0169] In another preferred embodiment, the present invention provides a use of the human amylin polypeptide analog or its derivative in combination with semaglutide, Tirzepatide or retatrutide for the prevention or treatment of obesity and obesity-related diseases, including but not limited to overweight, morbid obesity, pre-operative 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 (such as infertility), etc., wherein the structural formula of the human amylin polypeptide analog or its derivative is as shown in Compound 1, 4 or 12.

[0170] In another preferred embodiment, the present invention provides a use of the human amylin polypeptide analog or derivative thereof in combination with semaglutide, Tirzepatide or retatrutide for preventing or treating diabetes-related diseases, wherein the human amylin polypeptide analog or derivative thereof has a structural formula as shown in Compound 1, 4 or 12. The diabetes-related diseases include 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, lower limb vascular diseases, eye diseases, peripheral neuropathy and kidney diseases caused by diabetes, such as diabetic retinopathy, diabetic nephropathy, diabetic foot ulcers, diabetic atherosclerosis, diabetic polyneuropathy and diabetic fatty liver disease.

[0171] In another embodiment, the present invention provides a weight loss efficacy test for human amylin polypeptide derivatives Compound 1 and Compound 4, combined with Semaglutide, in DIO rats. When AM833, Compound 1, or Compound 4 were injected together with Semaglutide, the weight loss effect in DIO rats was significantly enhanced compared to each compound alone. The weight loss effect of Compound 1 combined with Semaglutide was comparable to that of the AM833 combined with Semaglutide group, while the weight loss effect of Compound 4 combined with Semaglutide was superior to that of AM833 or Compound 1 combined with Semaglutide.

[0172] In another embodiment, the present invention provides a hypoglycemic efficacy test of a human amylin polypeptide derivative alone or in combination with Semaglutide on rats. After SD rats were intervened with different doses (10, 30, 50 nmol / kg) of compound 1 injection, the peak blood glucose and the area under the glucose tolerance curve (AUC) were significantly reduced. After a single subcutaneous injection of compound 1 and Semaglutide injection in ZDF (fa / fa) type Ⅱ diabetic rats, the 4h random blood glucose (RBG) values of ZDF (fa / fa) type Ⅱ diabetic rats were significantly reduced, but the reduction of compound 1 was slightly lower than that of Semaglutide. When compound 1 was injected in combination with Semaglutide, the blood glucose-lowering effect on ZDF (fa / fa) type Ⅱ diabetic rats was significantly enhanced, showing a synergistic effect.

[0173] The optimal dosage of the human amylin polypeptide analog or its derivative of the present invention, when 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 dosage can be determined by routine experiments. For parenteral administration, a dosage of 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 of human amylin polypeptide analog or its derivative is administered. Exemplary treatment regimens can be administered once a day, once a week, once every two weeks, once every three weeks, or once every four weeks.

[0174] The present invention provides a use of a human amylin polypeptide analog and its derivatives in the preparation of a medicament for preventing or treating obesity, excessive food intake, diabetes, and related diseases. The technical solutions disclosed in the present invention are summarized as follows:

[0175] 1. Use of a human amylin polypeptide analog containing unnatural amino acids in the preparation of a medicament for preventing or treating obesity, excessive food intake, diabetes, and related diseases, wherein the sequence of the human amylin polypeptide analog is as follows:

[0176] X CNTATCATQ RLAEFLRHS NNFGPILPPT NVGSNTP(SEQ ID No:4)

[0177] wherein X is an unnatural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, and D-diaminopropionic acid;

[0178] There is an intramolecular disulfide bond between the two Cys residues at positions 2 and 7 in the polypeptide sequence; and the C-terminus of the polypeptide is an amide.

[0179] 2. The use according to technical solution 1, characterized in that the first X in the sequence SEQ ID No: 4 is a non-natural basic amino acid L-ornithine (L-Orn) or D-ornithine (D-Orn).

[0180] 3. Use of a human amylin polypeptide derivative modified with an albumin-binding residue in the preparation of a medicament for preventing or treating obesity, excessive food intake, diabetes, and related diseases, wherein the structural formula of the human amylin polypeptide derivative is as follows:

[0181] YLZ(I)

[0182] Wherein, Y is an albumin binding residue; L is a linker; Z is a human amylin polypeptide analog, the sequence of which is shown in SEQ ID No: 4, wherein an intramolecular disulfide bond exists between the two Cys residues at positions 2 and 7 in the sequence, and the C-terminus of the polypeptide is an amide;

[0183] wherein X in SEQ ID No: 4 is a non-natural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid;

[0184] The albumin binding residue is connected to the non-natural amino acid at the N-terminus of the human amylin polypeptide via a linker.

[0185] 4. The use according to technical solution 3, characterized in that: X in the sequence SEQ ID No: 4 is a non-natural basic amino acid selected from L-ornithine (L-Orn), L-diaminobutyric acid, L-homoarginine, L-diaminopropionic acid, diaminoacetic acid, D-ornithine (D-Orn), D-diaminobutyric acid, D-homoarginine and D-diaminopropionic acid.

[0186] 5. The use according to technical solution 4, characterized in that: X in the sequence SEQ ID No: 4 is L-ornithine (L-Orn) or D-ornithine (D-Orn).

[0187] 6. The use according to technical solution 3 is characterized in that: in the human amylin polypeptide derivative, L is a linker, which can exist independently or not.

[0188] 7. The use according to technical solution 6 is characterized in that: the linker may contain one or more amino acids, which are bound to the albumin binding portion at one end and to the amino group on the non-natural amino acid at the N-terminus of human amylin polypeptide at the other end.

[0189] 8. The use according to technical solution 7, characterized in that: the linker is selected from γGlu, γGlu-γGlu, γGlu-γGlu-γGlu, γGlu-γGlu-γGlu-γGlu, Glu, Glu-Glu, Glu-γGlu, Glu-Arg, Glu-Glu-Arg, His, His-His, His-γGlu, His-His-γGlu, Gly, Gly-γGlu, Ser, Ser-γGlu, D-Ar g-D-Arg, Arg, Arg-Arg, Arg-Arg-γGlu, Ser-Ser, -Gly-Ser-Ser, Ser-Ser, -Gly-Ser-Ser-γGlu, Ser-Ser-Gly -Ser-Ser-Gly, γGlu-2xOEG, OEG, Ser-Ser-Gly-Ser-Ser-Gly-γGlu, γGlu-OEG and -NHC(O)CH2CH2CH2S(O)2NH-.

[0190] 9. The use according to technical solution 8, characterized in that the linker is selected from the following groups:

[0191] a)

[0192] b)

[0193] c)

[0194]

[0195] d)

[0196] or

[0197] e)

[0198]

[0199] 10. The use according to technical solution 3, characterized in that the albumin binding residue has 6-40 carbon atoms, 8-26 carbon atoms or 12-22 carbon atoms.

[0200] 11. The use according to technical solution 3, characterized in that the albumin binding residue has 16, 17, 18, 19, 20, 21 or 22 carbon atoms.

[0201] 12. The use according to technical solution 3 is characterized in that the albumin binding residue contains a group that can be negatively charged at pH 7.4.

[0202] 13. The use according to technical solution 3, characterized in that the albumin binding residue is an acyl group selected from the following:

[0203] c) CH3(CH2) r CO-*, where r is an integer from 12 to 20;

[0204] d)HOOC(CH2) s CO-*, where s is an integer from 12 to 22.

[0205] 14. The use according to technical solution 13, characterized in that: the albumin binding residue is selected from CH3(CH2) r CO-, wherein r is an integer from 12 to 20 and is selected from CH3(CH2)6CO-, CH3(CH2)8CO-, CH3(CH2) 10 CO-, CH3(CH2) 12 CO-, CH3(CH2) 14 CO-, CH3(CH2) 16 CO-, CH3(CH2) 18 CO-, CH3(CH2) 20 CO- and CH3(CH2) 22 CO-;

[0206] 15. The use according to technical solution 13, characterized in that: the albumin binding residue is a HOOC(CH2)-containing carboxylic acid group s CO-acyl, wherein s is an integer from 12 to 22 selected from HOOC(CH2) 12 CO-, HOOC(CH2) 14 CO-, HOOC(CH2) 16 CO-, HOOC(CH2) 18 CO- or HOOC(CH2) 20 CO-.

[0207] 16. The use according to technical solution 13 is characterized in that the human amylin polypeptide derivative shown in structural formula (I) may contain at least one substituent, such as 1, 2 or 3.

[0208] 17. The use according to technical solution 16 is characterized in that the human amylin polypeptide derivative shown in structural formula (I) preferably contains 1 substituent.

[0209] 18. The use according to technical solution 16, characterized in that: the substituent is:

[0210]

[0211] 19. The use according to any one of technical solutions 3 to 18, wherein:

[0212] (1) The derivative has a structure as shown in formula (I), wherein Z is a human amylin polypeptide analog as shown in SEQ ID No: 4, wherein X at the N-terminus is L-ornithine (L-Orn) or D-ornithine (D-Orn);

[0213] (2) The ornithine residue is connected to the albumin binding residue via a linker.

[0214] 20. The use according to technical solution 19 is characterized in that the derivative has a solubility of about 200 μM or higher at pH 7.0.

[0215] 21. The use according to technical solution 19, characterized in that the structure of the human amylin polypeptide derivative is:

[0216] Here, r is an integer from 12 to 20.

[0217] 22. The use according to technical solution 19, characterized in that the structure of the human amylin polypeptide derivative is:

[0218]

[0219] Here, r is an integer from 12 to 20.

[0220] 23. The use according to technical solution 19, characterized in that the structure of the human amylin polypeptide derivative is:

[0221]

[0222] Here, s is an integer from 12 to 22.

[0223] 24. The use according to technical solution 19, characterized in that the structure of the human amylin polypeptide derivative is:

[0224]

[0225] Here, s is an integer from 12 to 22.

[0226] 25. The use according to any one of technical solutions 3 to 18, characterized in that the structure of the human amylin polypeptide derivative is:

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] 26. A human amylin polypeptide derivative, having the structure:

[0236]

[0237]

[0238] Here, r is an integer from 12 to 20.

[0239] 27. A human amylin polypeptide derivative, having the structure:

[0240]

[0241] Here, s is an integer from 12 to 22.

[0242] 28. A pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide derivative as described in any one of technical solutions 26-27 and a pharmaceutically acceptable carrier.

[0243] 29. The pharmaceutical composition according to technical solution 28 is characterized in that the composition is formulated into a dosage form such as solution, microemulsion, liposome or freeze-dried powder injection.

[0244] 30. The pharmaceutical composition according to technical solution 29 is characterized in that the composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.

[0245] 31. The use according to any one of technical solutions 1 to 25, characterized in that:

[0246] The sequence of the human amylin polypeptide analog is shown in SEQ ID No: 4, wherein the X at the N-terminus is D-ornithine (D-Orn);

[0247] The structural formula of the human amylin polypeptide derivative is:

[0248]

[0249] Here, s is an integer from 12 to 22.

[0250] 32. The use according to any one of technical solutions 1-25 or 31 is characterized in that the use for preventing or treating obesity, excessive food intake and related diseases includes overweight, morbid obesity, pre-operative 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.

[0251] 33. The use according to technical solution 32 is characterized in that the drug use includes reducing food intake, reducing appetite and / or promoting weight loss.

[0252] 34. The use according to technical solution 32 is characterized in that the human amylin polypeptide analog or its derivative can also be used in combination with one or more targeted drugs, and the targeted drugs include but are not limited to diabetes drugs, obesity drugs and hypertension drugs.

[0253] 35. The use according to technical solution 34 is characterized in that the diabetes drugs, obesity drugs and hypertension 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, insulins, metformin, sulfonylureas, glinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc.

[0254] 36. The use according to technical solution 35 is characterized in that the diabetes drugs, obesity drugs and hypertension drugs are selected from exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, Tirzepatide, retatrutide, Mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, leptin, neuropeptide Y, etc.

[0255] 37. The use according to technical solution 31 is characterized in that: the human amylin polypeptide analog or its derivative is administered in combination with semaglutide, tirzepatide or retatrutide for the prevention or treatment of obesity and obesity-related diseases, including but not limited to overweight, morbid obesity, pre-operative 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, wherein the structural formula of the human amylin polypeptide analog or its derivative is as shown in Compound 4.

[0256] 38. The use according to any one of technical solutions 1-25 or 31 is characterized in that the use for preventing or treating diabetes and its related diseases includes 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 sugar levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia and diabetes-related complications.

[0257] 39. The use according to technical solution 38 is characterized in that the diabetes-related complications include cardiovascular and cerebrovascular diseases, lower limb vascular diseases, eye diseases, peripheral neuropathy and kidney disease caused by diabetes.

[0258] 40. The use according to technical solution 39 is characterized in that the diabetes-related complications include atherosclerosis, large vessel disease, microvascular disease, coronary heart disease, peripheral arterial disease, stroke, diabetic retinopathy, diabetic nephropathy, diabetic foot ulcer, diabetic atherosclerosis, diabetic polyneuropathy and diabetic fatty liver disease, etc.

[0259] 41. The use according to technical solution 38 is characterized in that the human amylin polypeptide analog or its derivative is used in combination with one or more targeted drugs, and the targeted drugs include but are not limited to diabetes drugs, obesity drugs and hypertension drugs.

[0260] 42. The use according to technical solution 41 is characterized in that the human amylin polypeptide analog or its derivative is used in combination with GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, GLP-1 / GIP / GCGR triple agonists, FGF21 derivatives, insulins, metformin, sulfonylureas, glinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc.

[0261] 43. The use according to technical solution 42 is characterized in that the human amylin polypeptide analog or its derivative is used in combination with exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, leptin, neuropeptide Y, Tirzepatide, retatrutide, Mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, etc.

[0262] 44. The use according to technical solution 38 is characterized in that the human amylin polypeptide analog or its derivative is administered in combination with semaglutide, tirzepatide or retatrutide for the prevention or treatment of obesity and diabetes-related diseases, wherein the human amylin polypeptide analog or its derivative has a structural formula as shown in Compound 1, 4 or 12.

[0263] 45. The use according to any one of technical solutions 1-25 is characterized in that the drug 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.

[0264] 46. The use according to technical solution 45 is characterized in that the drug treatment regimen can be administered once a day, once a week, once every two weeks, once every three weeks or once every four weeks. DETAILED DESCRIPTION

[0265] 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 present invention in any way, nor do they represent that the experiments described below are all or the only experiments performed. The inventors guarantee the objectivity and accuracy of the experimental data, but allow for certain experimental errors and deviations.

[0266] Abbreviations:

[0267]

[0268]

[0269] Fmoc:9H-fluoren-9-ylmethoxycarbonyl

[0270] tBu: tert-butyl

[0271] Trt: triphenylmethyl

[0272] Pbf: Fluorenylmethoxycarbonyl

[0273] Boc: tert-Butyloxycarbonyl

[0274] HCTU: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate

[0275] HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0276] DIEA: N,N-diisopropylethylamine

[0277] DMF: N,N-dimethylformamide

[0278] NMP: N-methylpyrrolidone

[0279] DCM: dichloromethane

[0280] TFA: trifluoroacetic acid

[0281] TIPS: Triisopropylsilane

[0282] NHS: N-hydroxysuccinimide

[0283] DCC: dicyclohexylcarboimide

[0284] AEEA: Hydroxyethylethylenediamine

[0285] Example 1: Preparation of the polypeptide sequence CNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (ss ring) (SEQ ID No: 5)

[0286] The peptide was synthesized using solid-phase Fmoc chemistry, Wang resin, HCTU / DIEA as coupling reagent, DMF as reaction solvent, and ninhydrin detection method as reaction monitoring. After synthesis, the resin was washed with DMF and DCM and dried, and the peptide was treated with TFA / TIPS / water (95 / 2.5 / 2.5) for 2 hours to cleave from the resin, and then precipitated with 10 times the volume of ice methyl tert-ether, washed, and dried to obtain a crude peptide. The crude peptide was subjected to SS cyclization reaction with iodine, and then filtered with a filter membrane to obtain a crude product. After purification of the crude product, the target product was obtained, and the liquid phase test showed that the target product was 1.147kJ / 1.3kJ / 1.3kJ. Figure 1 The purity can be as high as 97%. The structure of the peptide was confirmed by LCMS. Figure 2 :MS(ESI):3828.3[M+H] + ,957.8[M+4H] 4+ .

[0287] Purification Method: The crude peptide was purified by semi-preparative HPLC on a 10 x 250 mm column packed with 10-100 C8 filler. The crude product was dissolved in 30 ml of 10% to 20% acetonitrile / water and injected onto the column. Elution was then performed using a gradient of 30% to 60% acetonitrile in 0.2% to 2% formic acid over 60 minutes at a flow rate of 2 ml / min at a column temperature of 30°C. Fractions containing the peptide were collected, and the purified sample was diluted and lyophilized.

[0288] The synthesis of the single sequence of SEQ ID No: 4 can refer to the synthesis steps of SEQ ID No: 5, except that an additional non-natural amino acid X is added to the N-terminus. X can be L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid.

[0289] Example 2: Preparation of Albumin Binding Residue Derivatives

[0290] The albumin binding residue derivatives are shown in Table 1 below. The preparation methods used are similar, and the preparation process takes the synthesis steps of Sample 1 as an example.

[0291]

[0292]

[0293]

[0294]

[0295] Sample 1 preparation process:

[0296] 1) Preparation of mono-tert-butyl eicosanedioate

[0297]

[0298] Eicosanedioic acid (50 g, 146.0 mmol) was suspended in acetic anhydride (200 ml) and heated at 140°C for 10 hours. The acetic anhydride was then removed under reduced pressure. Toluene (120 ml), tert-butyl alcohol (50 g), and DMAP (4.1 g) were added and the reaction was continued at 85°C for 8 hours. The solvent was then removed under reduced pressure. Dichloromethane (500 ml) and 3 ml of concentrated hydrochloric acid were added, and the mixture was stirred at 25°C for 30 minutes. The mixture was filtered and the organic phase was collected. The mixture was extracted three times with 5% aqueous hydrochloric acid (200 ml x 3). The organic phase was collected and dried over anhydrous sodium sulfate (20 g) for 30 minutes. The filtrate was filtered and the solvent was evaporated under reduced pressure. The mixture was dissolved in n-hexane (500 ml) and heated to -20°C for 4 hours. The precipitate was filtered, collected, and dried under vacuum to constant weight to obtain a white solid product. Yield: 18.7 g (46.7 mmol, 32.0%).

[0299] 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).

[0300] 2) Preparation of tert-butyl succinimidyl eicosanedioate

[0301]

[0302] Dissolve mono-tert-butyl eicosanedioate (10.03 g, 25.2 mmol) in dichloromethane (204 ml), add N-hydroxysuccinimide (3.03 g) and dicyclohexylcarboimidine (5.70 g) at 0°C, stir and react for 2 hours, return to 25°C and stir for 12 hours, filter, evaporate the solvent under reduced pressure, add isopropanol (90 ml), filter and collect the filter cake, wash the filter cake with n-hexane (34 ml), collect the filter cake, and dry it under reduced pressure to constant weight to obtain a white solid product with a yield of 10.28 g (20.7 mmol, 82.1%).

[0303] 1 H NMR (400MHz, CDCl3)2.81(s,4H),2.58(t,J=7.6Hz,2H),2.17(t,J=7.6Hz,2H),1.64-1.76(m,2),1.50-1.58(m,2),1.42(s,9),1.15-1.41(m,38).

[0304] 3) Preparation of tert-butyleicosandioyl-L-Glu(OtBu)-OH

[0305]

[0306] Dissolve tert-butyl succinimidyl eicosanedioate (10.28 g, 20.7 mmol) in N,N-dimethylformamide (213 ml). Add L-Glu(OtBu)-OH (4.13 g) and DIEA (5.3 ml) at 25°C, then heat to 30°C and react for 24 hours. DMF and DIEA are evaporated to dryness under reduced pressure. After cooling, the product is dissolved in ethyl acetate (250 ml) and extracted and washed sequentially with 0.2 mol / L hydrochloric acid solution (100 ml), purified water (100 ml), and saturated brine (100 ml). The ethyl acetate phase is collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and dried in vacuo to constant weight to obtain a viscous oil (yield: 12.98 g (crude product)).

[0307] 1 H NMR(400MHz, CDCl3)6.37(d,J=8.0Hz,1),4.45-4.55(m,1),2.25-2.45(m,2),2.15-2.25 (m,5),1.80-1.95(m,1),1.45-1.65(m,4),1.43(s,9H),1.40(s,9H),1.20-1.25(m,28).

[0308] 4) Preparation of tert-butyleicosandioyl-L-Glu(OtBu)-NHS

[0309]

[0310] Tert-butyleicosanedioyl-L-Glu(OtBu)-OH (crude product, 12.98 g, 20.7 mmol) was dissolved in dichloromethane (250 ml), and N-hydroxysuccinimide (2.68 g) and dicyclohexylcarboimidine (5.04 g) were added at 0°C, stirred for 2 hours, returned to 25°C and stirred for 12 hours, filtered, and the solvent was evaporated under reduced pressure. Dichloromethane (10 ml) and n-hexane (375 ml) were added and stirred for 6 hours. Filtered, the filter cake was collected, and the filter cake was washed with n-heptane (100 ml). The filter cake was collected and dried under reduced pressure to constant weight to obtain a white solid with a yield of 12.80 g (18.8 mmol, 90.8%).

[0311] 1H NMR(400MHz, CDCl3)6.22(d,J=7.6Hz,1),4.50-4.60(m,1),2.81(s,4),2.65-2.75(m,1),2.30-2.50(m,1),2.24 -2.35(m,1),2.13-2.22(m,4),1.98-2.10(m,1),1.45-1.70(m,4),1.45(s,9H),1.41(s,9H),1.17-1.30(m,28).

[0312] 5) Preparation of tert-butyleicosandioyl-L-Glu(OtBu)-Orn-OH

[0313]

[0314] Dissolve tert-butyl succinimidyl eicosandioate (4.5 g, 6.6 mmol) in N,N-dimethylformamide (60 ml). Add L-Orn(Boc)-OH (1.76 g) and DIEA (1.78 ml) at 25°C. Heat to 60°C and react for 12 hours. DMF and DIEA are evaporated to dryness under reduced pressure. After cooling, the product is dissolved in ethyl acetate (250 ml) and extracted and washed sequentially with purified water (100 ml), purified water (100 ml), and saturated brine (100 ml). The ethyl acetate phase is collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and filtered through a column chromatography column to obtain a viscous oil (yield: 4.1 g (5.1 mmol, 77.3%).

[0315] 1 H NMR(400MHz, CDCl3)7.16(d,J=8.0Hz,1),6.57(d,J=8.0Hz,1),5.00(s,1),4.50-4.60(m,1),4.37-4 .46(m,1),3.05-3.15(m,2),2.30-2.45(m,2),2.10-2.23(m,5),1.60-2.00(m,4),1.38-1.60(m,60).

[0316] Example 3: Preparation of human amylin polypeptide derivatives (ss ring formation)

[0317] The human amylin polypeptide derivatives are shown in Table 2 below. The preparation processes of the human amylin polypeptide derivatives are similar, and the synthesis steps of Compound 1 are taken as an example.

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] Preparation process of compound 1:

[0324]

[0325] Process 1:

[0326]

[0327] According to Scheme 1, tert-butyleicosanedioyl-L-Glu(OtBu)-Orn-OH (19 mg) was dissolved in NMP (8 ml). HATU (8 mg) and DIEA (18 μl) were added at 25°C, and the mixture was stirred for 0.5 hours to activate the tert-butyleicosanedioyl-L-Glu(OtBu)-Orn-OH. CNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (ss cyclization) peptide A (50 mg) and NMP (2 ml) were added, and the reaction was stirred at 27°C for 20 hours. The reaction solution was purified to obtain modified peptide B in a yield of 25 mg.

[0328] Take modified polypeptide B (25 mg), add 1 ml of cutting solution (TFA:TIS:H20=95:2.5:2.5), stir and react at 25°C for 0.5 hour, and concentrate under reduced pressure at 25°C to obtain a crude product. The crude product was purified to obtain the target compound 1, yield: 17 mg.

[0329] Purification method:

[0330] Purification of Modified Polypeptide B: Purify the crude polypeptide by semi-preparative HPLC on a 10 x 250 mm column packed with C8 or polymeric fillers. Dissolve the modified polypeptide B sample in 30 ml of 10% to 20% acetonitrile / water and inject it onto the column. Elute with a gradient of 35% to 65% acetonitrile in 0.1% formic acid over 60 minutes at a flow rate of 2 ml / min at 30°C. Collect the fractions containing the polypeptide, dilute the purified sample, and lyophilize or pump dry.

[0331] Purification of Compound 1: The crude peptide was purified by semi-preparative HPLC on a 10x250 mm column packed with 10-100 C8 filler. A sample of modified peptide B was dissolved in 30 ml of 10% to 20% acetonitrile / water and injected onto the column. The column was then eluted with a gradient of 30% to 60% acetonitrile in 0.2% to 2% formic acid over 60 minutes at a column temperature of 30°C and a flow rate of 2 ml / min. The fractions containing the peptide were collected, the purified sample was diluted, and lyophilized or pumped dry to obtain the desired product. Liquid chromatography analysis showed that the product was eluted with a 0.2% to 2% formic acid gradient of 30% to 60% acetonitrile. Figure 3 The purity can be as high as 97.9%. The structure of the peptide was confirmed by LCMS. Figure 4 :MS(ESI):1099.07[M+4H] 4+ The confirmed molecular weight of compound 1 is 4392.24.

[0332] The solid phase synthesis of compound 1 can also refer to the synthesis steps of SEQ ID No: 5, except that ornithine, γ-glutamic acid and eicosanedioic acid are added sequentially to the N-terminus.

[0333] The preparation process of compound 4 was similar to that of compound 1, and the purity after purification reached 95.63% ( Figure 5 The structure of the peptide was confirmed by LCMS. Figure 6 :MS(ESI):1099.56[M+4H] 4+ The molecular weight of compound 4 was confirmed to be 4392.24.

[0334] The preparation processes of the remaining compounds were similar to that of compound 1.

[0335] The confirmed molecular weight test results of the human amylin polypeptide derivatives are shown in Table 3.

[0336] Table 3 Molecular weight of human amylin polypeptide derivatives

[0337]

[0338]

[0339] Example 4 Solubility Test of Human Amylin Polypeptide Derivatives

[0340] The solubility of the human amylin polypeptide derivative at different pH values is shown in Table 4. The solubility test was performed by dissolving an appropriate amount of the API in a solution and visually detecting the solubility limit in the solution.

[0341] Table 4 Solubility of human amylin polypeptide derivatives at different pH

[0342]

[0343] Example 5 Determination of biological activity of human amylin polypeptide derivatives

[0344] (1) Overview of β-galactosidase assay

[0345] Calcitonin Receptor-β-Galactosidase ProLink TM Upon activation of β-Arrestin (PK), β-galactosidase EA can be recruited into the cell. When two functionally complementary proteins, β-galactosidase ProLink (PK) and β-galactosidase EA, come into proximity, active β-galactosidase is produced. Therefore, it is possible to assay the biological activity of human amylin polypeptide derivatives using a β-galactosidase reporter gene assay introduced into CHO-K1 cells (which also express the calcitonin receptor).

[0346] (2) β-galactosidase detection kit

[0347] Using Eurofins DiscoverX eXpress CALCR-RAMP3(AMY3) CHO-K1β-Arrestin GPCR Assay Kit was used for detection.

[0348] (3) Determination of β-galactosidase

[0349] For activity assay, CALCR-RAMP3 (AMY3) CHO-K1β-Arrestin cells were cultured at a rate of approximately 1×10 4 The density of cells / well was inoculated into a white 96-well culture plate and the cells were placed in 100 μl of detection medium. After incubation in a 37°C, 5% CO2 incubator for 48 hours, 10 μl / well of gradient diluted sample solution was added. After incubation in a 37°C, 5% CO2 incubator for 1.5 hours, 55 μl / well detection reagent was added and incubated in the dark for 1 hour at room temperature. Finally, the Lum module was used for detection on a microplate reader. GraphPad was used to perform four-parameter fitting of different drug concentrations (nM) and their response values. The results are shown in Tables 5 and 6 below. Figure 7 .

[0350] Table 5 Relative biological activity determination

[0351] name <![CDATA[Human Calcitonin Polypeptide Receptor EC 50 (nM)]]> Relative activity AM833 6.720 100％ Compound 1 6.308 106％ Compound A (terminal amidation) 7.693 87％ Compound A-terminal carboxyl group 417.018 1.6％ Compound 4 3.594 187％ Compound 5 12.000 56％ Compound 7 6.222 108％ Compound 12 9.081 74％

[0352] The results of the calcitonin receptor and β-galactosidase reporter gene assays for compound 1 and AM833 showed that the β-galactosidase signals generated by the two compounds after stimulating the calcitonin receptor were basically the same. 50 The EC values of compound 1 were 6.308 nM and 6.720 nM, respectively. The two compounds have similar biological activities. 50The value is about 6% higher than that of AM833.

[0353] The results of the calcitonin receptor and β-galactosidase reporter gene assays for compound A (terminal amidation) and compound A terminal carboxyl group showed that EC 50 The biological activities of the two peptides were 7.693 nM and 417.018 nM, respectively. The difference in biological activity was nearly 54 times, indicating that the amidation of the C-terminus of the peptide was very important for the biological activity of the human amylin peptide derivative.

[0354] Example 6 Kinetic Analysis of Binding to Human Serum Albumin (octet)

[0355] Biotin-labeled human serum albumin (abcam, ab8033) was diluted to 50 μg / mL and immobilized with SA probe for 30 min using a macromolecular interaction analyzer (Fortebio, Octet RED96). At the same time, the peptide was gradient diluted with NaCl in a black polypropylene 96-well plate (Greiner, 655209). The immobilized probe was associated with the gradient diluted peptide for 60 s, followed by disassociation in NaCl for 60 s. The unimmobilized probe was repeated. Double subtraction and steady-state analysis were performed using Octet Analysis Studio 12.2 software to obtain the binding curve of the peptide and human serum albumin and the steady-state fitting curve ( Figure 8 , Table 6).

[0356] Table 6 KD values of compound 1 and AM833 binding to human serum albumin

[0357]

[0358] Conclusion: Compound 1 and AM833 have similar binding abilities to human serum albumin.

[0359] Example 7 Detection of binding to human calcitonin receptor (CTR) and amylin receptor 3 (AMY3R) membrane proteins

[0360] 293F cells were transfected with pcDNA3.1-based human CTR plus an empty plasmid (hCTR group) or human CTR plus human RAMP3 plasmid (hAMY3R group) using PEI (PolyScience, 23966-100). 18 hours later, 10% (v / v) F01 feed medium (PD Biosciences, PDF01-1000) was added to the transfected cells. Two days later, 5 weeks of cells were collected for flow cytometry analysis. If the expression was >50%, 1 x 10 cells were collected for hCTR and 1 x 10 cells were collected for hAMY3R. 8 By Pierce TMMembrane proteins were extracted using GPCR extraction and stabilization reagent (Thermo Scientific, A43436).

[0361] A black low-adhesion 96-well plate was added to each well with 50 μL of membrane protein, 20 nM sCT(8-32):5-FAM fluorescently labeled peptide, and serially diluted peptide molecules in HBSS (Gibco, 14025-092) + 10 mM HEPES (Gibco, 15630080) + 0.1% BSA buffer, for a total volume of 100 μL. Detection was performed using a fluorescence polarization module on a microplate reader (Molecular Devices, SpectraMax M5e) at an Ex / Em ratio of 485 / 525 nm. A blank control was used, and after deducting the S / P value of the blank control, mP was calculated as ABS [1000*(SP) / (S+P)]. Results were automatically generated by SoftMax Pro software.

[0362] Table 7 IC binding of compound 1, AM833, compound 4 and compound 12 to human membrane protein 50

[0363]

[0364] Conclusion: Overall, the affinity of compound 1 and compound 4 for human membrane proteins is similar to that of AM833, and the affinity of compound 12 for human membrane proteins is slightly weaker than that of AM833. Among them, compound 4 prefers to bind to AMY3R between CTR and AMY3R, while compound 1 and AM833 have similar affinities for CTR and AMY3R ( Figure 9 ).

[0365] Example 8 Binding Detection to Rat Calcitonin Receptor (CTR) and Amylin Receptor 3 (AMY3R) Membrane Proteins

[0366] 293F cells were transfected with pcDNA3.1-based rat CTR plus empty plasmid (rCTR group) or rat CTR plus rat RAMP3 plasmid (rAMY3R group) using PEI (PolyScience, 23966-100). 18 hours later, 10% (v / v) F01 feed medium (PD Biosciences, PDF01-1000) was added to the transfected cells. Two days later, 5 weeks of cells were collected for flow cytometry analysis. If the results showed >50% positive expression, 1*10 cells were collected for rCTR and 1*10 cells for rAMY3R. 8 By Pierce TMMembrane proteins were extracted using GPCR extraction and stabilization reagent (Thermo Scientific, A43436).

[0367] A black low-adhesion 96-well plate was added to each well with 50 μL of membrane protein, 20 nM sCT(8-32):5-FAM fluorescently labeled peptide, and serially diluted peptide molecules in HBSS (Gibco, 14025-092) + 10 mM HEPES (Gibco, 15630080) + 0.1% BSA buffer, for a total volume of 100 μL. Detection was performed using a fluorescence polarization module on a microplate reader (Molecular Devices, SpectraMax M5e) at an Ex / Em ratio of 485 / 525 nm. A blank control was used, and after deducting the S / P value of the blank control, mP was calculated as ABS [1000*(SP) / (S+P)]. Results were automatically generated by SoftMax Pro software.

[0368] Table 8 IC binding of compound 1, AM833, compound 4 and compound 12 to rat membrane protein 50

[0369]

[0370] Conclusion: Overall, the affinity of compounds 1, 4, and 12 for rat AMY3R is comparable to that of AM833. Among them, compound 4 is more inclined to bind to rat AMY3R, while compounds 1, 12, and AM833 are more inclined to bind to rat CTR ( Figure 10 ).

[0371] Example 9 Activity detection of human CTR and AMY3R cells

[0372] CHOK1 cells stably transfected with CRE-Luc and hCTR were plated overnight in 6-well plates and then transiently transfected with either an empty pcDNA3.1 plasmid (hCTR group) or a human RAMP3 plasmid (hAMY3R group) using 3.75 μL Lipofectamin 3000 (Invitrogen, L3000008). Two days later, cells were resuspended in F12K (Gibco, 25200-056) + 0.1% BSA (Shanghai Yuanpei, S476T7) activation medium. Two weeks of cell per well were plated in white 96-well plates overnight. Peptide drugs were serially diluted in activation medium, and 50 μL of drug was added to each well. The cells were then incubated at 37°C for 3 hours. 50 μL of Luciferase assay substrate (Promega, G7940) was added to each well and incubated in the dark for 10 min. LUM was detected using a microplate reader (Molecular Devices, SpectraMax M5e) and graphed using Graphpad Prism 9.0.

[0373] Table 9 EC activity of compound 1, AM833, compound 4 and compound 12 against hCTR and hAMY3R overexpressing cells 50

[0374]

[0375] Conclusion: Compound 1 and AM833 have comparable agonist activity against human CTR and AMY3R. Compound 4 has approximately 3-fold higher agonist activity than AM833, while compound 12 is slightly weaker than AM833 ( Figure 11 ).

[0376] Example 10 cAMP agonist activity on rat CTR and AMY3R cells

[0377] After plating CHOK1 cells overnight in 6-well plates, cells were transfected with either rat CTR (rCTR) or rat CTR plus a rat RAMP3 plasmid (rAMY3R) using 3.75 μL of Lipofectamin 3000 (Invitrogen, L3000008) containing pcDNA3.1. Two days later, cells were resuspended in F12K (Gibco, 25200-056) medium supplemented with 0.1% BSA (Shanghai Yuanpei, S476T7) and plated at 3,000 cells per well in white 384-well plates overnight. cAMP levels following peptide stimulation were measured using the cAMP HiRange Kit (cisbio, 62AM6PEB). Briefly, the F12K medium was aspirated, and 10 μL of peptide diluted in Stimulation Buffer 1 (containing 0.5 mM IBMX (Sigma, I5879)) was added to each well. After incubation at 37°C for 30 minutes, 5 μL of d2 working solution diluted in Lysis Buffer and 5 μL of Eu working solution diluted in Lysis Buffer were added, and the cells were incubated at room temperature for 1 hour. HTRF results were analyzed using a microplate reader (Molecular Devices, SpectraMax M5e) using the Eu Optimized Assay plate. Ratio = (Signal 665nm / Signal 620nm) * 10000 was calculated, and graphed using Graphpad Prism 9.0. Forskolin was used as a positive control.

[0378] Table 10 Compound 1, AM833h and Compound 4 stimulate cAMP activation EC in rCTR and rAMY3R overexpressing cells 50

[0379]

[0380] Conclusion: Compound 1 has slightly weaker agonist activity on rat CTR and AMY3R than AM833, while compound 4 has significantly stronger agonist activity than AM833 ( Figure 12 ).

[0381] Example 11 Pharmacokinetic Study of Human Amylin Polypeptide Derivatives in SD Rats

[0382] After adaptive feeding, male SD rats were divided into two groups, with 6 rats in each group. The corresponding drugs were given subcutaneously. Blood was collected before and 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h, and 96h after administration. The serum was separated and the drug concentration in the serum was detected by LC-MS.

[0383] Table 11 Main pharmacokinetic parameters

[0384]

[0385] Conclusion: After subcutaneous injection, compound 1 (T 1 / 2 ) half-life and exposure (C max and AUC 0-t ) were slightly larger than the positive control AM833 ( Figure 13 ).

[0386] Example 12: Human Amylin Polypeptide Derivatives Feeding Suppression Test in SD Rats

[0387] After five days of adaptive feeding, male SD rats were divided into eight groups, each containing eight rats, one rat per cage. Each group received the corresponding drug via subcutaneous injection before the light-dark cycle on the day of grouping. Following administration, a certain amount of feed and water were added, and the animals' food intake and body weight were monitored regularly.

[0388] Table 12-1 Statistics of food intake of SD rats at different stages (g)

[0389]

[0390] Table 12-2 SD rat weight statistics

[0391]

[0392]

[0393] Conclusion: After a single subcutaneous injection, the food intake of SD rats was inhibited, and the body weight decreased sharply, and then slowly recovered, showing a certain dose-effect relationship. The same dose of compound 1 and the positive control AM833 had basically the same effect on the food intake and body weight of rats (Table 12, Figure 14 ).

[0394] Example 13: Weight loss efficacy test of compound 1 and compound 4 injection combined with semaglutide in DIO rats

[0395] Male Sprague-Dawley rats were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. (Animal Production License No.: SCXK(Zhejiang)2019-0001, Animal Quarantine Certificate No.: 20230922Aazz0619999597). They were fed a Synergistic Biological XTHF60 high-fat diet for adaptive feeding, induction, and the experimental period. Six Sprague-Dawley rats were randomly selected and fed a standard diet to serve as a control group. After 10 weeks of high-fat diet feeding, animals whose weight exceeded 20% of the average weight of normal Sprague-Dawley rats were selected to establish the DIO rat model. DIO rats were randomly divided into 8 groups according to body weight: model control group (Vehicle), 10 nmol / kg Semaglutide injection group, 10 nmol / kg AM833, 10 nmol / kg Compound 1, 10 nmol / kg Compound 4, 10 & 10 nmol / kg AM833 combined with Semaglutide group, 10 & 10 nmol / kg Compound 1 combined with Semaglutide group, and 10 & 10 nmol / kg Compound 4 combined with Semaglutide group, with 6 rats in each group. Subsequently, each group should be given the test sample by subcutaneous injection, 3 times / week for 4 consecutive weeks. The body weight of the test animals was monitored during the administration period (2 times / week).

[0396] Results: In this experiment, DIO rats were administered subcutaneous injections for 3 weeks (3 times / week). AM833, compound 1, compound 4 and semaglutide all had a significant weight loss effect on DIO rats, especially semaglutide, which had the best weight loss effect. Compound 4 alone had a higher weight loss rate than compound 1. When AM833, compound 1 or compound 4 were combined with semaglutide, the weight loss effect of DIO rats was significantly enhanced compared with the single compound. The weight loss effect of compound 1 combined with semaglutide was comparable to that of the AM833 combined with semaglutide group, while the weight loss effect of compound 4 combined with semaglutide was better than that of AM833 or compound 1 combined with semaglutide ( Figure 15 ).

[0397] Example 14: Oral glucose tolerance test of SD rats with injection of compound 1

[0398] Male and female SD rats were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd. (Animal Production License No.: SCXK (Zhejiang) 2019-0001, Animal Quarantine Certificate No.: 20230828Aazz0619999343, 20230828Aazz0619999266). SD rats were randomly divided into four groups according to sex and body weight: blank control group (Vehicle), 10, 30, and 50 nmol / kg compound 1 injection groups, with 6 rats in each group (half male and half female). All test animals were fasted but not water for 16 hours before the oral glucose tolerance test (OGTT) and were simultaneously given a single subcutaneous injection in the neck. On the day of the experiment, 2 g / kg glucose solution was orally gavaged. Blood samples were collected from the rats by tail tip acupuncture before (0 minute) and 15, 30, 60, 120, and 180 minutes after glucose administration. Whole blood glucose levels were measured using a Roche Active Blood Glucose Meter and blood glucose test strips.

[0399] Results: In this experiment, after SD rats were treated with different doses (10, 30, 50 nmol / kg) of compound 1 injection, the peak blood glucose level and the area under the glucose tolerance curve (AUC) were significantly reduced. When the compound 1 injection dose was 10-30 nmol / kg, the AUC showed a dose-effect relationship ( Figure 16 ).

[0400] Example 15: Hypoglycemic efficacy test of Compound 1 injection combined with Semaglutide in ZDF rats

[0401] Male ZDF (fa / fa) type 2 diabetic rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Animal Production License No.: SCXK (Beijing) 2021-0011, Animal Quarantine Certificate No.: 110011231111771753). ZDF (fa / fa) type 2 diabetic rats were randomly divided into four groups based on body weight: a model control group (Vehicle), a 10 nmol / kg semaglutide injection group, a 10 nmol / kg compound 1 injection group, and a 10 & 10 nmol / kg compound 1 combined with semaglutide injection group, with eight rats in each group. Eight ZDF (fa / +) normal rats were also purchased as a blank control group (Control), and the animals were acclimated for one week. At the start of the experiment, all animals consumed a normal diet and water intake. A single subcutaneous injection of the drug was administered subcutaneously in the neck. Blood samples were collected by tail puncture before (0 h) and 4 h after administration. Whole blood glucose levels were measured using a Roche Vitality blood glucose meter and blood glucose test strips.

[0402] Results: In this experiment, after a single subcutaneous injection of Compound 1 and Semaglutide injection in ZDF (fa / fa) type II diabetic rats, the 4-hour random blood glucose (RBG) values of ZDF (fa / fa) type II diabetic rats were significantly reduced, but the reduction of Compound 1 was slightly lower than that of Semaglutide. When Compound 1 was combined with Semaglutide, the blood glucose-lowering effect in ZDF (fa / fa) type II diabetic rats was significantly enhanced, showing a synergistic effect ( Figure 17 ).

[0403] The foregoing describes the subject matter, preferred embodiments, and modes of implementation of the present invention. However, the present invention should not be construed as being limited to the specific embodiments described above. Rather, the embodiments described herein should be considered illustrative rather than restrictive, and modifications and variations made by others without departing from the scope of the present invention should be considered within the scope of the technical solutions of the present invention.

Claims

1. Use of a human amylin polypeptide analog containing unnatural amino acids in the preparation of a medicament for preventing or treating obesity, excessive food intake, diabetes and related diseases, wherein: The sequence of the human amylin polypeptide analog is as follows: X CNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP(SEQ ID No:4) wherein X is an unnatural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, and D-diaminopropionic acid; wherein an intramolecular disulfide bond exists between the two Cys residues at positions 2 and 7 in the polypeptide sequence; and The C-terminus of the polypeptide is an amide.

2. Use of a human amylin polypeptide derivative modified with an albumin binding residue in the preparation of a drug for preventing or treating obesity, excessive food intake, diabetes, and related diseases, wherein: The structural formula of the human amylin polypeptide derivative is shown below: YLZ(I) Wherein, Y is an albumin binding residue; L is a linker; Z is a human amylin polypeptide analog, the sequence of which is shown in SEQ ID No: 4, wherein an intramolecular disulfide bond exists between the two Cys residues at positions 2 and 7 in the sequence, and the C-terminus of the polypeptide is an amide; wherein X in SEQ ID No: 4 is a non-natural amino acid independently selected from L-ornithine, L-diaminobutyric acid, dimethylalanine Aib, L-homoarginine, L-citrulline, L-diaminopropionic acid, diaminoacetic acid, D-ornithine, D-diaminobutyric acid, D-homoarginine, D-citrulline, or D-diaminopropionic acid; The albumin binding residue is connected to the non-natural amino acid at the N-terminus of the human amylin polypeptide via a linker.

3. The use according to claim 2, characterized in that: The albumin binding residue is an acyl group selected from the following: c) CH3(CH2) r CO-*, where r is an integer from 12 to 20; d)HOOC(CH2) s CO-*, where s is an integer from 12 to 22.

4. The use according to any one of claims 2 to 3, wherein: (1) The derivative has a structure as shown in formula (I), wherein Z is a human amylin polypeptide analog as shown in SEQ ID No: 4, wherein X at the N-terminus is L-ornithine (L-Orn) or D-ornithine (D-Orn); (2) The ornithine residue is connected to the albumin binding residue via a linker.

5. The use according to any one of claims 2 to 3, characterized in that: The structure of the human amylin polypeptide derivative is:

6. The use according to any one of claims 1-2, characterized in that: The use of preventing or treating obesity, excessive food intake and related diseases includes overweight, morbid obesity, pre-operative obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea and breathing problems, cartilage degeneration, osteoarthritis, non-alcoholic fatty liver disease (NAFLD) and reproductive health complications of obesity or overweight.

7. The use according to any one of claims 1-2, characterized in that: The use of the present invention for preventing or treating diabetes and its related diseases includes 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 sugar levels, metabolic diseases including metabolic syndrome, hyperglycemia, hypertension, atherogenic dyslipidemia and diabetes-related complications.

8. The use according to claim 6 or 7, characterized in that: The human amylin polypeptide analog or its derivative is used in combination with GLP-1 derivatives, GLP-1R / GCGR dual agonists, GLP-1 / GIP dual agonists, GLP-1 / GIP / GCGR triple agonists, FGF21 derivatives, insulins, metformin, sulfonylureas, glinides, glitazones, DPP-IV inhibitors, AGLT2 inhibitors, etc.

9. The use according to claim 8, characterized in that: The human amylin polypeptide analog or derivative thereof is used in combination with exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, leptin, neuropeptide Y, Tirzepatide, retatrutide, Mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, etc.

10. The use according to claim 9, characterized in that: The human amylin polypeptide analog or its derivative is used for preventing or treating obesity and diabetes-related diseases by combined administration with semaglutide, tirzepatide or retatrutide, wherein the structural formula of the human amylin polypeptide analog or its derivative is shown as compound 1, 4 or 12.

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