Human amylin polypeptide derivative and application thereof

Abstract

The invention provides a human amylin polypeptide analogue containing non-natural amino acid and a derivative of the human amylin polypeptide analogue. According to the present invention, the human amylin polypeptide derivative obtained by introducing the non-natural amino acid to the N-terminal of the human amylin and modifying with the albumin binding residue can effectively prevent enzymatic degradation and prolong the half-life period, and has characteristics of improved solubility and / or physical stability and / or bioavailability and / or pharmacokinetic properties, the compound can be used for treating obesity and obesity-related diseases.

Description

A human amylin polypeptide derivative and its use 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 obesity epidemic is becoming increasingly severe, with 1.24 billion people suffering from obesity. With rapid economic growth and changing lifestyles, the prevalence continues to rise, making obesity a major economic burden and a significant public health issue for countries, societies, and individuals. Obesity carries a high risk of life-threatening comorbidities and carries significant social costs, including complications such as an increased risk of diabetes, cardiovascular disease, sleep apnea, and osteoarthritis. Furthermore, in the context of the global COVID-19 pandemic, research evidence indicates that patients with obesity and diabetes experience significantly increased rates of severe illness and mortality from COVID-19, further highlighting the urgency and importance of weight management.

[0003] Despite the high incidence of obesity worldwide and the heavy burden of related diseases, few drugs have been approved for the treatment of obesity. Currently, the FDA has only approved six long-term obesity treatment drugs, and most of them have limited efficacy, only promoting a weight loss of about 3%-15%, which may not be enough to improve some obesity-related complications. It is necessary to develop more effective anti-obesity drugs and further study new treatment mechanisms.

[0004] Islet amyloid polypeptide (IAPP), also known as amylin, is a glucose-regulating pancreatic hormone secreted alongside insulin. It is involved in delaying gastric emptying and inhibiting postprandial glucagon release. IAPP is also a satiety factor that reduces energy intake and regulates appetite and satiety by activating receptors in the hindbrain and nucleus of the solitary tract. It also enhances hepatocyte and plasma triglyceride metabolism, reducing hepatic adipose tissue and lipid deposition and plasma triglyceride fluctuations.

[0005] Human amylin (hIAPP, SEQ ID No: 1) is a 37-amino acid polypeptide hormone with short peptide modifications at both ends. Terminal amide groups and intramolecular disulfide bonds are crucial for the integrity of its molecular biological functions. hIAPP binds to two distinct receptor complexes: the calcitonin receptor and the receptor activity-modifying proteins RAMP1 or RAMP3. hIAPP may regulate food choice and preference through the hypothalamus, ventral tegmental area, and lateral tegmental nucleus. Therefore, IAPP-modified proteins targeting the amylin receptor (AMYR) and the calcitonin G protein-coupled receptor (CTR) have been proposed as adjunctive therapies for weight management, potentially complementing other treatments such as GLP-1 and leptin. However, hIAPP's amyloidogenicity (fibrosis), chemical instability, and solubility have hindered its use as a therapeutic agent.

[0006] 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.

[0007] 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 patented analog of "N-α-[(S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]-[Glu14,Arg17,Pro37]-pramlintide" is under clinical development and is called Cagrilintide (also known as AM833). The partial amino acid sequence of its polypeptide is shown in SEQ ID No: 3. However, Cagrilintide may still exhibit poor physical stability under certain conditions, and there is still a need to find hIAPP polypeptide substances that possess native hIAPP activity, a prolonged in vivo action time, good stability, higher solubility, and resistance to fibrosis.

[0008] 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] 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;

[0011] 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.

[0012] In another embodiment, the inventors of the present application unexpectedly discovered that when the N-terminus of a human amylin polypeptide is a basic amino acid, the isoelectric point of the polypeptide can be effectively increased, resulting in improved solubility and / or physical stability. 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.

[0013] In another preferred embodiment, the non-natural basic amino acid introduced by the present invention is L-ornithine (L-Orn).

[0014] 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:

[0015] 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;

[0016] 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;

[0017] The albumin binding residue is connected to the non-natural amino acid at the N-terminus of the human amylin polypeptide via a linker.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In another embodiment, the preferred albumin binding residue comprises a group that is negatively charged at pH 7.4.

[0023] In another embodiment, the albumin binding residue is an acyl group selected from:

[0024] c) CH3(CH2) r CO-*, where r is an integer from 12 to 20;

[0025] d)HOOC(CH2) s CO-*, where s is an integer from 12 to 22.

[0026] 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, thereby improving its solubility and / or physical stability.

[0027] In another embodiment, the human amylin polypeptide derivatives provided by the present invention exhibit improved solubility, good stability in solution, and are less susceptible to fibrosis. Furthermore, biological activity assays have shown that the human amylin polypeptide derivatives provided by the present invention have comparable or higher in vitro biological activity than cagrilintide.

[0028] 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.

[0029] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or derivative thereof for treating obesity and obesity-related diseases, including but not limited to obesity, diabetes, 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.

[0030] The human amylin polypeptide analogs or derivatives thereof of the present invention may also be used in combination with one or more targeted drugs, including but not limited to diabetes drugs, obesity drugs and hypertension drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the liquid phase diagram of Compound A. The liquid phase results show that the liquid phase purity of Compound A is 97%.

[0032] Figure 2 Mass spectrum of compound A. The mass spectrometry results confirmed that the molecular weight of compound A was 3824.85.

[0033] Figure 3 is a liquid phase diagram of compound 1. The liquid phase results show that the liquid phase purity of compound 1 is 98.6%.

[0034] Figure 4 Mass spectrum of compound 1. The mass spectrometry results confirmed that the molecular weight of compound 1 was 4392.24.

[0035] Figure 5 Biological activity assay. The results of the calcitonin receptor and β-galactosidase reporter gene assay 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.

[0036] Figure 6 Kinetic analysis of the binding of compound 1 and AM833 to human serum albumin.

[0037] Figure 7 Concentration-response binding curves of compound 1 and AM833 to CTR / AMY1R / AMY2R / AMY3R transiently transfected COS-1 cells.

[0038] Figure 8 shows the concentration response curves of compound 1 and AM833 on calcium ion stimulation in CTR / AMY1R / AMY2R / AMY3R transiently transfected COS-1 cells.

[0039] Figure 9 Pharmacokinetic evaluation. Compound 1 has a longer half-life and higher bioavailability than AM833, indicating pharmacokinetic advantages.

[0040] Figure 10 Pharmacodynamic evaluation. Compound 1 has comparable appetite suppressant and weight loss effects to AM833.

[0041] Figure 11 shows the weight loss efficacy of Compound 1 combined with Semaglutide in DIO mice. When Compound 1 and Semaglutide were injected together, the weight loss effect on DIO mice was significantly stronger than that of either drug alone, indicating that the combined administration had a synergistic weight loss effect.

[0042] Detailed Description of the Invention

[0043] 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.

[0044] 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.

[0045] 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).

[0046] "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:

[0047] 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:

[0048] 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)

[0049] 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:

[0050] 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)

[0051] 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 as follows, 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: KCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP (SEQ ID No: 3) or Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Glu Phe Leu Arg His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val Gly Ser Asn Thr Pro (SEQ ID No: 3)

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] "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).

[0061] 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.

[0062] Human amylin polypeptide analogs and derivatives

[0063] The present invention relates to human amylin polypeptide analogs and polypeptide derivatives thereof. These derivatives, obtained by substituting non-natural amino acids for pramlintide (SEQ ID No. 2) and modifying its N-terminal fatty acid, significantly reduce the fibrogenic potential of these human amylin polypeptide derivatives, significantly increase their solubility in water, and significantly enhance the stability of the compounds. Furthermore, these human amylin polypeptide derivatives exhibit significantly prolonged pharmacokinetic half-lives and favorable pharmacodynamic properties, possessing comparable or higher biological activity than AM833, and are therefore useful in the treatment of obesity and obesity-related diseases.

[0064] In one aspect, the present invention relates to a human amylin polypeptide analog containing unnatural amino acids, the sequence of which is shown below:

[0065] 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;

[0066] 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.

[0067] The inventors introduced non-natural amino acids into the N-terminus of human amylin, which can effectively prevent enzymatic degradation and extend the 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 solubility and / or physical stability. 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 acid introduced by the present invention is L-ornithine (L-Orn). The basic amino acid structural formula is as follows:

[0068] 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:

[0069] 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;

[0070] 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;

[0071] The albumin binding residue is connected to the non-natural amino acid at the N-terminus of the human amylin polypeptide via a linker.

[0072] 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.

[0073] 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.

[0074] 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-Glu-Arg, His, His-His, His-γGl u, His-His-γGlu, Gly, Gly-γGlu, Ser, Ser-γGlu, D-Arg-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, 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.

[0075] For example, the linker term "γGlu" refers to an amino acid residue having the following structure:

[0076] 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:

[0077] By use of the term "γGlu-OEG" is meant a moiety having the following structure:

[0078] By use of the term "γGlu-OEG-OEG" is meant a moiety having the following structure:

[0079] In another embodiment, more preferably the linker can be selected from the following groups:

[0080] 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).

[0081] 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.

[0082] In another embodiment, the preferred albumin binding residue comprises a group that is negatively charged at pH 7.4.

[0083] In another embodiment, the albumin binding residue is an acyl group selected from:

[0084] c) CH3(CH2) r CO-*, where r is an integer from 12 to 20;

[0085] d)HOOC(CH2) s CO-*, where s is an integer from 12 to 22.

[0086] 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-;

[0087] 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) 16CO-, HOOC(CH2) 18 CO- or HOOC(CH2) 20 CO-.

[0088] 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.

[0089] 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:

[0090] In another embodiment, the present invention provides a human amylin polypeptide derivative, wherein:

[0091] (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;

[0092] (2) The ornithine residue is connected to the albumin binding residue via a linker.

[0093] In another embodiment, the present invention provides a human amylin polypeptide derivative, wherein:

[0094] (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;

[0095] (2) the ornithine residue is connected to the albumin binding residue via a linker;

[0096] (3) The derivative has a solubility of about 200 μM or higher at pH 7.0.

[0097] In one embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0098] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0099] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0100] In another embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0101] wherein r is an integer from 12 to 20, and s is an integer from 12 to 22.

[0102] 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.

[0103] In a particularly preferred embodiment, the structure of the human amylin polypeptide derivative provided by the present invention is:

[0104] 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, resulting in improved solubility and / or physical stability.

[0105] As demonstrated in the Examples herein, the human amylin polypeptide derivatives provided herein have a solubility of approximately 200 μM or higher at pH 7.0. For example, the human amylin polypeptide derivatives shown under the conditions of Example 4 herein have a solubility of >60 mg / ml and exhibit a delayed fibrosis tendency compared to Cagrilintide (also known as AM833), which clearly exceeded expectations.

[0106] In another embodiment, the human amylin polypeptide derivatives provided herein exhibit improved physical stability, good stability in solution, and are less susceptible to fibrosis. Furthermore, biological activity assays have shown that the human amylin polypeptide derivatives provided herein have comparable or higher in vitro biological activity than cagrilintide.

[0107] In another example, the human amylin polypeptide derivative Compound 1 provided herein showed no calcium ion agonism in CTR transiently transduced cells, but exhibited stronger calcium ion agonism than AM833 in AMYR transiently transduced cells, and slightly stronger binding to AMY2R than AM833. Compound 1 and AM833 had similar binding abilities to human serum albumin.

[0108] In another embodiment, the human amylin polypeptide derivatives provided by the present invention have comparable or longer half-life and higher bioavailability than Cagrilintide (also known as AM833).

[0109] In another embodiment, the human amylin polypeptide derivative provided by the present invention has comparable or better appetite suppressant and weight loss effects than Cagrilintide (also known as AM833).

[0110] Synthesis of Human Amylin Peptide Analogs and Derivatives

[0111] 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.

[0112] 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.

[0113] pharmaceutical preparations

[0114] 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.

[0115] 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).

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] Drug uses

[0121] 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.

[0122] 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.

[0123] In another embodiment, the present invention provides the use of the human amylin polypeptide analog or derivative thereof for treating obesity and obesity-related diseases, including but not limited to obesity, diabetes, non-alcoholic steatohepatitis (NASH), hypertension, and cardiovascular disease. In another embodiment, the pharmaceutical use includes reducing food intake, reducing appetite, and promoting weight loss.

[0124] The human amylin polypeptide analog or derivative thereof of the present invention can also be used in combination with one or more 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, insulins, 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.

[0125] In another preferred embodiment, the present invention provides a method for treating obesity and obesity-related diseases by administering the human amylin polypeptide analog or its derivative in combination with semaglutide, tirzepatide or retatrutide, including but not limited to obesity, diabetes, non-alcoholic steatohepatitis (NASH), hypertension and cardiovascular disease, wherein the human amylin polypeptide analog or its derivative has a structural formula as shown in Compound 1.

[0126] The optimal dosage of the human amylin polypeptide analog or derivative thereof of the present invention 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 derivative thereof is administered. Exemplary treatment regimens may be administered once a day, once a week, once every two weeks, once every three weeks, or once every four weeks. DETAILED DESCRIPTION

[0127] Abbreviations: Fmoc: 9H-fluoren-9-ylmethoxycarbonyl tBu: tert-butyl Trt: triphenylmethyl Pbf: fluorenylmethoxycarbonyl Boc: tert-butyloxycarbonyl HCTU: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate DIEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide NMP: N-methylpyrrolidone DCM: dichloromethane TFA: trifluoroacetic acid TIPS: triisopropylsilane NHS: N-hydroxysuccinimide DCC: dicyclohexylcarboimide AEEA: hydroxyethylethylenediamine

[0128] Example 1: Preparation of the polypeptide sequence CNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (ss ring) (SEQ ID No: 5)

[0129] The peptide was synthesized using solid-phase Fmoc chemistry, Wang resin, HCTU / DIEA as coupling reagent, DMF as reaction solvent, and ninhydrin detection method for reaction monitoring. After synthesis, the resin was washed with DMF and DCM and dried. The peptide was treated with TFA / TIPS / water (95 / 2.5 / 2.5) for 2 hours to cleave from the resin. The peptide was then precipitated with 10 volumes of icy tertiary methyl ether, washed, and dried to obtain a crude peptide. The crude peptide was subjected to SS cyclization reaction with iodine and then filtered through a filter membrane to obtain a crude product. After purification of the crude product, the target product was obtained. Liquid chromatography test, as shown in Figure 1, showed a purity of up to 97%. The structure of the peptide was confirmed by LCMS, as shown in Figure 2: MS (ESI): 3828.3 [M+H] + ,957.8[M+4H] 4+ .

[0130] 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.

[0131] 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.

[0132] Example 2: Preparation of Albumin Binding Residue Derivatives

[0133] 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.

[0134] Sample 1 preparation process:

[0135] 1) Preparation of mono-tert-butyl eicosanedioate

[0136] 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%).

[0137] 1H 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).

[0138] 2) Preparation of tert-butyl succinimidyl eicosanedioate

[0139] 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%).

[0140] 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).

[0141] 3) Preparation of tert-butyleicosandioyl-L-Glu(OtBu)-OH

[0142] 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)).

[0143] 1H 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).

[0144] 4) Preparation of tert-butyleicosandioyl-L-Glu(OtBu)-NHS

[0145] 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%).

[0146] 1 H 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).

[0147] 5) Preparation of tert-butyleicosandioyl-L-Glu(OtBu)-Orn-OH

[0148] 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%).

[0149] 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).

[0150] Example 3: Preparation of human amylin polypeptide derivatives (ss ring formation)

[0151] 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.

[0152] Preparation process of compound 1:

[0153] Process 1:

[0154] 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.

[0155] 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.

[0156] Purification method:

[0157] 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.

[0158] 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%-20% acetonitrile / water and injected onto the column. Elution was then performed over 60 minutes using a gradient of 30%-60% acetonitrile in 0.2%-2% formic acid at a column temperature of 30°C and a flow rate of 2 ml / min. Fractions containing the peptide were collected, and the purified sample was diluted and lyophilized or pumped dry to obtain the desired product. Liquid chromatography analysis, as shown in Figure 3, showed a purity of up to 97.9%. The peptide structure was confirmed by LCMS, as shown in Figure 4: MS (ESI): 1099.07 [M+4H]. 4+ The confirmed molecular weight of compound 1 is 4392.24.

[0159] 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.

[0160] The preparation processes of the remaining compounds were similar to that of compound 1.

[0161] The detection results of the human amylin polypeptide derivatives are shown in Table 3.

[0162] Example 4 Solubility and fibrosis testing of human amylin polypeptide derivatives

[0163] The solubility and fibrillation of the human amylin polypeptide derivatives at different pH values ​​are shown in Table 4.

[0164] Compound 1 exhibited a solubility exceeding 60 mg / ml at pH 7.0 and 20°C, with no noticeable fibrillation after 56 hours. AM833 exhibited a solubility exceeding 50 mg / ml at pH 7.0 and 20°C, with no noticeable fibrillation after 48 hours. This experiment demonstrates that human amylin peptide derivatives incorporating unnatural amino acids exhibit similar or superior solubility and reduced fibrillation to AM833.

[0165] Example 5 Biological Activity Determination of Human Amylin Polypeptide Derivatives

[0166] (1) Overview of β-galactosidase assay

[0167] 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).

[0168] (2) β-galactosidase detection kit

[0169] Using Eurofins DiscoverX Detection was performed using the CALCR-RAMP3(AMY3) CHO-K1 β-Arrestin GPCR Assay Kit.

[0170] (3) Determination of β-galactosidase

[0171] For activity assay, CALCR-RAMP3 (AMY3) CHO-K1 β-Arrestin cells were cultured at a density of approximately 1×10 4 Cells were seeded into a white 96-well culture plate at a density of 100 μl per well in 100 μl of assay medium. After incubation for 48 hours in a 37°C, 5% CO2 incubator, 10 μl per well of the sample solution was added. After incubation for 1.5 hours in a 37°C, 5% CO2 incubator, 55 μl per well of assay reagent was added and incubated in the dark for 1 hour at room temperature. Finally, the assay was performed on a microplate reader using the Lum module. Four-parameter fitting of different drug concentrations (nM) and their response values ​​was performed using GraphPad. The results are shown in Table 5 and Figure 5 below.

[0172] 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. 50 The value is about 6% higher than that of AM833.

[0173] 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.

[0174] Example 6 Kinetic Analysis of Binding to Human Serum Albumin (octet)

[0175] Biotin-labeled human serum albumin (abcam, ab8033) was diluted to 50 μg / mL and immobilized with a SA probe for 30 minutes using a macromolecular interaction analyzer (Fortebio, Octet RED96). Simultaneously, the peptide was diluted with a NaCl gradient in a black polypropylene 96-well plate (Greiner, 655209). The immobilized probe was associated with the peptide in the gradient dilution for 60 seconds, followed by disassociation in NaCl for 60 seconds. This procedure was repeated with the unimmobilized probe. Double-subtraction analysis was performed using Octet Analysis Studio 12.2 software, and steady-state analysis was performed to generate binding curves and steady-state fitting curves for the peptide and human serum albumin (Figure 6, Table 6).

[0176] Table 6 KD values ​​of compound 1 and AM833 binding to human serum albumin

[0177] Conclusion: Compound 1 and AM833 have similar binding abilities to human serum albumin.

[0178] Example 7 Binding Assay to Human CTR and AMYR (FACS)

[0179] (1) Construction of COS-1 transiently transfected cells

[0180] COS-1 cells were seeded overnight at a density of 80%-90% and then transfected with hCTR and empty pcDNA3.1, hRAMP1, hRAMP2, or hRAMP3 (1:1 mass ratio) using Lipofectamine 3000 (Invitrogen, L3000008) and incubated for 2 days. Transfection steps (using one T75 flask as an example): 1. Vortex and mix 750 μL of Opti-MEM with 29.6 μL of Lipofectamine 3000. 2. Mix another 750 μL of Opti-MEM with 20 μg of plasmid and 40 μL of P3000. 3. Mix the two premixes and incubate at room temperature for 15 minutes. 4. Add the lipofectamine-DNA mixture to the culture flask and incubate for 2 days.

[0181] (2) FACS detection of binding to human receptors

[0182] The transfected cells were collected and counted, and 5-10w cells were plated on a low-adsorption 96-well plate per well. After washing, the supernatant was removed by centrifugation at 300g for 5 minutes. A gradient dilution of peptide was added to each well. At the same time, each well contained 10nM fluorescent peptide sCT8-32:5-FAM and 0.5% w / v BSA. The binding ability of the sample was determined by competitive binding with the fluorescent peptide. Non-specific binding was defined by the results of 30μM sCT and 10nM fluorescent peptide. After the peptide and cells were incubated in the dark for 10 minutes, the flow cytometer was used to detect the FITC channel. The calculation formula is as follows: Binding (%) = 1-[MFI (peptide) - MFI (30μM sCT)] / MFI (10nM fluorescent peptide). GraphPad Prism 9 was used to plot and calculate IC 50 (Figure 7, Table 7).

[0183] Table 7 IC values ​​of compound 1 and AM833 binding to CTR / AMY1R / AMY2R / AMY3R transiently transfected COS-1 cells 50

[0184] Conclusion: Compound 1 and AM833 have similar binding abilities to CTR and the three AMYRs, and are concentration-responsive. Compound 1 has slightly stronger binding ability to AMY2R than AM833, and slightly weaker binding ability to AMY3R at high concentrations. In addition, in this example, compound 1 has not yet reached a high plateau at AMY1R and AMY3R, so there is no effective IC 50 .

[0185] Example 8 Calcium ion stimulation assay

[0186] COS-1 transiently transduced cells were constructed using reference Example 7. 3-4 weeks of cells were plated per well in a 96-well, black-walled, clear-bottom ViewPlate (Corning, 3904). After overnight culture, the culture medium was discarded and the cells were stained using the Fluo-4NW kit (Invitrogen, F36206). 0.5% BSA was added to the working staining solution and incubated at 37°C for 30 min. Peptides were serially diluted in Greiner black polypropylene 96-well plates (Greiner, 655209), and 10 μM ATP was used as a positive control. Calcium flux was measured using an MD FlexStation 3, with a total reading of 2 min 30 s, an average of 0.9 s per reading, including a 30 s baseline reading and a 30 s peptide injection. Peak values ​​for each well were subtracted from the baseline and vehicle values ​​and normalized to 10 μM ATP as 100%. Results were plotted and calculated using GraphPad Prism 9 (Figure 8, Table 8).

[0187] Table 8 IC of compound 1 and AM833 on calcium ion agonism in CTR / AMY1R / AMY2R / AMY3R transiently transfected COS-1 cells 50

[0188] Conclusion: In this example, compound 1 did not exhibit calcium ion stimulatory ability on CTR transiently transfected cells, but its calcium ion stimulatory ability on AMYR transiently transfected cells was stronger than that of AM833.

[0189] Example 9 Pharmacokinetic Study of Human Amylin Polypeptide Derivatives in SD Rats

[0190] After adaptive feeding, male SD rats were divided into two groups, with 6 rats in each group. The corresponding drugs were injected subcutaneously. Blood was collected before and 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after administration. The serum was separated and the drug concentration in the serum was detected by LC-MS.

[0191] Table 9 Main pharmacokinetic parameters

[0192] Conclusion: After subcutaneous injection, compound 1 (T 1 / 2 ) half-life and exposure (C max and AUC 0-t ) were slightly greater than the positive control AM833 (Table 9).

[0193] Example 10 Human Amylin Polypeptide Derivatives Feeding Suppression Test in SD Rats

[0194] 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.

[0195] Table 10-1 Statistics of food intake of SD rats at different stages (g)

[0196] Table 10-2 SD rat body weight statistics

[0197] 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 effects on the food intake and body weight of rats (Table 10, Figure 10).

[0198] Example 11: Weight loss efficacy test of compound 1 combined with semaglutide in DIO mice

[0199] DIO mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. (Animal Production License No.: SCXK(Su)2021-0005, Animal Quarantine Certificate No.: 320726230100211516) and continued to be fed a D12492 high-fat diet during adaptive feeding and the experimental period. DIO mice were randomly divided into four groups based on body weight: a vehicle control group, a 1 nmol / kg semaglutide injection group, a 10 nmol / kg compound 1 group, and a 10 nmol / kg compound 1 combined with 1 nmol / kg semaglutide group, with 8 mice in each group. Eight C57BL / 6 mice were purchased (Biocytogen, Animal Production License No.: SCXK(Su)2021-0005, Animal Quarantine Certificate No.: 320726230100211613) and continued to be fed a control diet during adaptive feeding and the experimental period as a normal control group. Each group was then given the drug solution by subcutaneous injection once a day for two consecutive weeks. Body weight was monitored twice a week during the drug administration period.

[0200] Results: In this experiment, DIO mice were administered subcutaneously for two weeks (once daily). Both 10 nmol / kg of Compound 1 and 1 nmol / kg of Semaglutide significantly reduced body weight in DIO mice. When Compound 1 was combined with Semaglutide, the weight-reducing effect on DIO mice was significantly enhanced, demonstrating a synergistic weight-reducing effect of combined administration (Figure 11).

[0201] 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.

[0202] Sequence Listing Information:

Claims

1. A human amylin polypeptide analog containing unnatural amino acids, the sequence of which is as follows: XCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP (SEQ ID No: 4) in, 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; 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.

2. The human amylin polypeptide analog according to claim 1, characterized in that: The first X in the sequence SEQ ID No: 4 is the non-natural basic amino acid L-ornithine (L-Orn).

3. A human amylin polypeptide derivative modified with an albumin binding residue, the structural formula of which is shown below: YLZ (I) in, 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.

4. The human amylin polypeptide derivative according to claim 3, characterized in that: In the sequence SEQ ID No: 4, X 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.

5. The human amylin polypeptide derivative according to claim 4, characterized in that: In the sequence SEQ ID No: 4, X is L-ornithine (L-Orn).

6. The human amylin polypeptide derivative according to claim 3, characterized in that: In the human amylin polypeptide derivative, L is a linker, which may exist independently or not.

7. The human amylin polypeptide derivative according to claim 6, characterized in that: The linker may comprise one or more amino acids which bind to the albumin binding moiety at one end and to the amino group on the N-terminal unnatural amino acid of the human amylin polypeptide at the other end.

8. The human amylin polypeptide derivative according to claim 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-Arg-D-Arg, Ar g, 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-.

9. The human amylin polypeptide derivative according to claim 8, characterized in that: The linker is selected from the following groups: a) b) c) d) e) 10. The human amylin polypeptide derivative according to claim 3, characterized in that: The albumin binding residue has 6-40 carbon atoms, 8-26 carbon atoms or 12-22 carbon atoms.

11. The human amylin polypeptide derivative according to claim 3, characterized in that: The albumin binding residue has 16, 17, 18, 19, 20, 21 or 22 carbon atoms.

12. The human amylin polypeptide derivative according to claim 3, characterized in that: The albumin binding residue comprises a group that is negatively charged at pH 7.

4.

13. The human amylin polypeptide derivative according to claim 3, 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.

14. The human amylin polypeptide derivative according to claim 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-; 15. The human amylin polypeptide derivative according to claim 13, characterized in that: The albumin binding residue is a carboxylic acid group-containing structure such as HOOC(CH2) 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-.

16. The human amylin polypeptide derivative according to claim 13, characterized in that: The human amylin polypeptide derivative represented by structural formula (I) may comprise at least one substituent, for example, 1, 2 or 3 substituents.

17. The human amylin polypeptide derivative according to claim 16, characterized in that: The human amylin polypeptide derivative represented by structural formula (I) preferably contains one substituent.

18. The human amylin polypeptide derivative according to claim 16, characterized in that: The substituents are:

19. The human amylin polypeptide derivative according to any one of claims 3 to 18, 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 ornithine; (2) The ornithine residue is connected to the albumin binding residue via a linker.

20. The human amylin polypeptide derivative according to claim 19, characterized in that: The derivatives have a solubility of about 200 μM or higher at pH 7.

0.

21. The human amylin polypeptide derivative according to claim 19, characterized in that: The structure of the human amylin polypeptide derivative is: Here, r is an integer from 12 to 20.

22. The human amylin polypeptide derivative according to claim 19, characterized in that: The structure of the human amylin polypeptide derivative is: Here, r is an integer from 12 to 20.

23. The human amylin polypeptide derivative according to claim 19, characterized in that: The structure of the human amylin polypeptide derivative is: Here, s is an integer from 12 to 22.

24. The human amylin polypeptide derivative according to claim 19, characterized in that: The structure of the human amylin polypeptide derivative is: Here, s is an integer from 12 to 22.

25. The human amylin polypeptide derivative according to any one of claims 3 to 18, characterized in that: The structure of the human amylin polypeptide derivative is:

26. A pharmaceutical composition comprising a therapeutically effective amount of the human amylin polypeptide analog according to any one of claims 1 to 2 or the human amylin polypeptide derivative according to any one of claims 3 to 25 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition according to claim 26, characterized in that: The composition is prepared into dosage forms such as solution, microemulsion, liposome or freeze-dried powder injection.

28. The pharmaceutical composition according to claim 27, characterized in that: The composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.

29. Use of the human amylin polypeptide analog according to any one of claims 1 to 2 or the human amylin polypeptide derivative according to any one of claims 3 to 25 in treating obesity and obesity-related diseases.

30. The use according to claim 29, characterized in that: Diseases of the use include, but are not limited to, obesity, diabetes, non-alcoholic steatohepatitis (NASH), hypertension, and cardiovascular disease.

31. The use according to claim 29, characterized in that: Such pharmaceutical uses include reducing food intake, decreasing appetite and / or promoting weight loss.

32. The use according to claim 29, characterized in that: The human amylin polypeptide analog or derivative thereof can also be used in combination with one or more targeted drugs, including but not limited to diabetes drugs, obesity drugs and hypertension drugs.

33. The use according to claim 32, 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.

34. The use according to claim 33, characterized in that: The diabetes drug, obesity drug and hypertension drug are selected from exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, Tirzepatide, retatrutide, Mazdutide, BI-456906, pemvidutide, cotadutide, SAR425899, efruxifermin, BIO89-100, leptin, neuropeptide Y, etc.

35. The use of a human amylin polypeptide analog or its derivative in combination with semaglutide, tirzepatide or retatrutide for the treatment of obesity and obesity-related diseases, including but not limited to obesity, diabetes, non-alcoholic steatohepatitis (NASH), hypertension and cardiovascular disease, wherein the human amylin polypeptide analog or its derivative has the structural formula shown in Compound 1.

36. The use according to claim 29, 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.

37. The use according to claim 29, characterized in that: The drug treatment regimen may be administered once daily, once weekly, once every two weeks, once every three weeks, or once every four weeks.

Images