Long-acting amylin receptor agonist and use thereof

Abstract

Provided are a long-acting amylin receptor agonist and the use thereof. By means of modifying an amylin receptor agonist, the isoelectric point of the amylin receptor agonist is reduced, and the water solubility and stability of the amylin receptor agonist in a neutral formulation solution are improved. By means of fatty acid chain modification, the half-life of the molecule is prolonged, and a good drug effect is achieved in animal models.

Description

A long-acting amyloid receptor agonist and its uses

[0001] This application claims priority to an earlier application filed on November 15, 2024, with patent application number 202411637253X, entitled "A Long-Acting Amylin Receptor Agonist and Its Use Thereof," which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention belongs to the field of biomedicine and relates to a long-acting amylin receptor agonist and its uses. Background Technology

[0003] Pancreatic amyloid polypeptide (IAPP), also known as amylin, is a polypeptide hormone synthesized and secreted by pancreatic β-cells. Amylin acts on two fronts: firstly, it acts on the feeding center in the hypothalamus, producing a feeling of satiety and inhibiting gastrointestinal motility and gastric emptying, thus prolonging the feeling of fullness after eating and reducing food intake; secondly, it inhibits the secretion of glucagon, slowing down the absorption of glucose in the small intestine, thereby lowering postprandial blood glucose. The secretion concentration of amylin in a fasting state is 3-5 pM, while the postprandial secretion concentration is 15-25 pM. Normally, amylin and insulin are secreted together in a molar ratio of approximately 1:100 (amyloid:insulin), and their synergistic effect allows for more precise regulation of blood glucose levels. Within pancreatic β cells, the 89-residue precursor of amylin, PreproIAPP, is first synthesized. PreproIAPP is then hydrolyzed in the endoplasmic reticulum by signal peptidase to form ProIAPP, containing 67 residues. ProIAPP then enters the secretory vesicles within the Golgi apparatus after enzymatic cleavage by prohormone convertases PC1 / 3 and PC2. Further action by carboxypeptidase E and peptidylglycine α-amiditase PAM leads to the formation of mature amylin, composed of 37 amino acids. Amylin possesses a pair of intramolecular disulfide bonds and a C-terminal amidation modification; both post-translational modifications significantly influence amylin activity and are indispensable.

[0004] Amyrin receptors (AMYRs) belong to the large family of cell surface G protein-coupled receptors (GPCRs). They are a special type of protein heterodimer formed by the polymerization of a calcitonin receptor (CTR) with three homologous receptor-modifying proteins (RAMP 1, RAMP 2, and RAMP 3), resulting in three subtypes (AMY1R, AMY2R, and AMY3R). AMYRs have become important potential disease targets, and selective AMYR agonists and AMYR / CTR dual agonists are being developed for obesity treatment. However, it remains unclear whether amyrin-templated agonists and calcitonin-templated agonists activate the target receptors through similar or different molecular mechanisms.

[0005] Normal human pancreatic islet tissue contains a comparable number of amylin-positive cells to insulin-positive cells, and a certain amount of amylin is also present in the blood, with its content closely related to blood insulin levels. However, amylin is almost undetectable in the pancreatic islet tissue and blood of type 1 diabetic patients, while the levels of amylin in the pancreatic tissue and blood of type 2 diabetic patients who require insulin to control blood sugar are also significantly lower than normal. Studies have found that amyloid deposits occur in the pancreatic islet tissue of most type 2 diabetic patients; these substances are misfolded amylin. Monomeric amylin easily aggregates and misfolds into cytotoxic oligomers under physiological conditions. These oligomers further fold, homogeneously arrange, and form β-sheet structures, ultimately constituting a rigid, unbranched, fibrous network with a diameter of 8–10 nm—an insoluble amyloid deposit. The amyloid protein fibers formed by amylin deposition in the islets further damage islet cells, accelerating islet cell dysfunction. Therefore, addressing the tendency of human amylin to form amyloid deposits is key to amylin drug development.

[0006] In 2005, a modified amylin, pramlintide (developed by Amylin Pharmaceuticals), was approved by the FDA for use in combination with insulin to treat type 1 and type 2 diabetes. Pramlintide replaces amino acids 25, 28, and 29 of human amylin with proline, altering the protein's primary structure and conformation. While retaining the physiological effects of amylin, it changes the physical properties of human amylin, such as its tendency to aggregate and misfold, preventing amyloid deposition and avoiding inducing pancreatic β-cell apoptosis. Animal studies have shown that pramlintide, whether used alone or in combination with leptin, can sustainably reduce the weight of test animals. Amylin is primarily cleared by the kidneys, with a half-life of only 13 minutes in rats, while pramlintide's half-life in humans is only 20-45 minutes. This short half-life severely limits the efficacy of amylin. Clinically, pramlintide requires three subcutaneous injections daily, and its formulation has a pH of 4.0, leading to poor patient adherence. Similar to GLP-1 inhibitors, pramlintide can cause side effects such as nausea and vomiting at the start of treatment.

[0007] Canagliptin, developed by Novo Nordisk, is a non-selective dual agonist of amylin receptors (AMYRs) and calcitonin receptors (CTRs) (DACRA). It is the first long-acting amylin analog studied for weight management, overcoming the short half-life of pramlinide, which requires multiple daily doses. Canagliptin further incorporates mutations (N14E, V17R, Y37P) and fatty acid chain modifications to pramlinide, enhancing its activation and stability against calcitonin receptors and significantly prolonging its half-life. In a phase II clinical trial for weight loss, after 32 weeks of treatment, subjects in the semaglutide group experienced an average weight loss of approximately 5.07%, the canagliptin group an average weight loss of 8.09%, and the combination of canagliptin and semaglutide an average weight loss of 15.59%. Therefore, canagliflozin alone has a slightly better weight-loss effect than smegglutinin, and the two can work synergistically to exhibit a more significant efficacy. Furthermore, clinical studies have shown that the combined use of canagliflozin and smegglutinin not only reduces weight but also effectively controls blood sugar. Common side effects of canagliflozin include nausea, diarrhea, constipation, fatigue, and injection site reactions, indicating a high safety profile. Besides canagliflozin, Calcitonin (sCT) from salmon is also a natural, non-selective dual agonist of amylin receptors (AMYRs) and calcitonin receptors (CTRs) (DACRA). However, due to its short half-life, natural sCT cannot be directly developed as a weight-loss and blood sugar-lowering drug. Additionally, the high isoelectric point of natural sCT makes it unstable in neutral formulation solutions, hindering its potential for drug development.

[0008] Amylin is unstable and prone to aggregation and misfolding to form insoluble amylin fibers, making the development of amylin-based drugs challenging. Therefore, there are currently few drugs targeting amylin, with only Novo Nordisk's canagliflozin entering Phase III clinical trials. Furthermore, due to their molecular properties, both pramlintide and canagliflozin formulations are acidic solutions with a pH of around 4.0, significantly different from smegglutide formulations (pH around 7.4). Consequently, pramlintide and canagliflozin cannot currently be directly combined with smegglutide to form compound formulations, leading to inconvenience in use. Given the increasing demand for weight control, the development of safe and effective amylin-based drugs has a vast market potential. Summary of the Invention

[0009] To improve the above-mentioned technical problems, the present invention provides an amylin receptor agonist comprising the following polypeptide: X1CX2TATCVLGX3LSQELHX4LX5TYPX6TX7TGSGTX8-NH2.

[0010] Where X1 is independently selected from K, R, or does not exist;

[0011] X2 is independently selected from N, Q, or E;

[0012] X3 can be independently selected from L, V, K, R, or M;

[0013] X4 is independently selected from K, E, or R;

[0014] X5 can be independently selected from K, Q, or E;

[0015] X6 can be independently selected from R, E, A, K, or Q;

[0016] X7 can be independently selected from R, N, or E;

[0017] X8 can be independently selected from F, Y, P, or E;

[0018] The C-terminus of the polypeptide may optionally be an amide or its derivative;

[0019] The α-amino group of X1 can be optionally connected to an N-terminal extension via a connector, wherein the N-terminus is a fatty acid.

[0020] In one embodiment, the amylin receptor agonist has at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with any one of SEQ ID NO:3-11, 17-22.

[0021] In one embodiment, the amylin receptor agonist has an amino acid sequence as shown in any one of SEQ ID NO:3-11, 17-22.

[0022] In one embodiment, the amino acid residue X1, X3, or X5 may be lysine K, and the ε-amino group in the lysine residue may be linked to a fatty acid via one or more linkers I;

[0023] The fatty acid may have the structure described in Formula I, wherein the fatty acid contains at least one carboxyl group at its terminal, wherein n may be 5, 6, 7, 8, 9, 10, 11 or 12, and R1 is a chemical group to which the fatty acid is attached to one or more linkers I, wherein R1 may be a carboxyl group or a succinimide group.

[0024] (Fatty acid, formula I);

[0025] Furthermore, the R1 group in the fatty acid can be linked to the R2 group in linker I (Formula II), and the aforementioned amylin receptor agonist is linked to the R3 group in linker I. The fatty acid and one or more linkers I form a fatty acid chain, wherein linker I has the structure described in Formula II, wherein the R2 group is an amino group (-NH2), and the R3 group can be a carboxyl group or a succinimide group. The R3 group of the terminal linker I in the aforementioned fatty acid chain can be linked to the α-amino group at the N-terminus of the aforementioned amylin receptor agonist or the ε-amino group in the side chain of the N-terminal lysine K.

[0026] (Connector I, Equation II).

[0027] In one embodiment, the R3 group of linker I in the aforementioned fatty acid chain can be further linked to one or more linkers II (Formula III) or linker III (Formula IV), wherein the R4 group of linker II or linker III is linked to the R3 group of linker I, thereby forming a longer fatty acid chain. In the fatty acid chain, the R5 group of linker II or linker III can be further linked to the aforementioned amylin receptor agonist, wherein the R4 group is an amino group (-NH2), and the R5 group can optionally be a carboxyl group or a succinimide group. The R5 group of the terminal linker II or linker III in the aforementioned fatty acid chain can be linked to the α-amino group at the N-terminus of the aforementioned amylin receptor agonist or the ε-amino group in the K side chain of lysine.

[0028] (Connector II, Formula III) or

[0029] (Connector III, Formula IV).

[0030] In one embodiment, the fatty acid chain comprises the structure described in formula V, VI, VII, or VIII:

[0031] (Fatty acid chain 1, formula V);

[0032] (Fatty acid chain 2, formula VI);

[0033] (Fatty acid chain 3, formula VII);

[0034] (Fat acid chain 4, formula VIII);

[0035] The R3 or R5 group can be a carboxyl group or a succinimide group, and n can be 5, 6, 7, 8, 9, 10, 11 or 12, preferably 8, 9 or 10.

[0036] On the other hand, the present invention provides the use of the aforementioned amylin receptor agonist in the preparation of medicaments for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, and / or for reducing food intake, reducing β-cell apoptosis, improving β-cell function and β-cell quality, and / or for restoring β-cell glucose sensitivity.

[0037] On the other hand, this disclosure provides a pharmaceutical composition comprising the aforementioned amylin receptor agonist and a pharmaceutically acceptable excipient.

[0038] In one embodiment, the aforementioned pharmaceutical composition further comprises one or more additional active agents;

[0039] Preferably, the active agent is selected from GLP-1 receptor agonists, GIP / GLP-1 or GLP-1 / GCG dual receptor agonists, and insulin;

[0040] More preferably, the GLP-1 receptor agonist is selected from smegglutide, liraglutide, and benaglutide; the GIP / GLP-1 or GLP-1 / GCG dual receptor agonist is selected from telposide and mascara.

[0041] In one embodiment of the present invention, the present invention provides the aforementioned amylin receptor agonist or pharmaceutical composition for treating or preventing hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcer. Beneficial effects

[0042] This invention modifies the amylin receptor agonist, lowering its isoelectric point and improving its water solubility and stability in neutral formulation solutions. Furthermore, fatty acid chain modification prolongs the molecule's half-life and achieves good efficacy in animal models. Attached Figure Description

[0043] Figure 1 shows the RMSD comparison of AsCT-1 and sCT peptides after molecular dynamics simulation.

[0044] Figure 2 shows the changes in food intake in the DIO model of SD rats after injection of amylin receptor agonists.

[0045] Figure 3 shows the weight changes of the DIO model in SD rats after injection of amylin receptor agonists. Detailed Implementation

[0046] definition

[0047] As used herein, the term "amylin receptor agonist" refers to a wild-type or modified polypeptide molecule capable of activating the human amylin receptor AMY3R. In one embodiment, examples of said amylin receptor agonist include polypeptides comprising the amino acid sequences shown in any one of SEQ ID NO: 3-11, 17-22. Further examples of amylin receptor agonists can be found in CA2555877C or CN116669753A, which are incorporated herein by reference in their entirety.

[0048] For example, the amylin receptor agonist includes, but is not limited to, substitutions and / or deletions and / or additions of any amino acid residue of mature salmon Calcitonin (sCT) to any natural or non-natural amino acid, synthetic amino acid, or peptide mimicry, and / or the linking of a substituent to any natural or non-natural amino acid, synthetic amino acid, or peptide mimicry at any available position. In the amylin receptor agonist defined above, the amylin receptor agonist obtained where a substituent is linked to any natural or non-natural amino acid, synthetic amino acid, or peptide mimicry at any available position may also be referred to herein as an "amylin receptor agonist".

[0049] The term "sequence identity" refers to the degree of association between two amino acid sequences or two nucleotide sequences, described by the parameter "sequence identity". For the purposes of this invention, the sequence identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later). Parameters used may include, for example, a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The output of "longest identity" marked by Nieder (obtained using the non-simplified (-nobrief) option) is used as the identity percentage and calculated as follows:

[0050] (Same residue x 100) / (Alignment length - total number of vacancies in alignment).

[0051] The term "natural amino acid" as used herein is selected from the following amino acids (their conventional three-letter and one-letter codes are in parentheses): glycine (Gly and G), proline (Pro and P), alanine (Ala and A), valine (Val and V), leucine (Leu and L), isoleucine (Ile and I), methionine (Met and M), cysteine ​​(Cys and C), phenylalanine (Phe and F), tyrosine (Tyr and Y), tryptophan (Trp and W), histidine (His and H), lysine (Lys and K), arginine (Arg and R), glutamine (Gln and Q), asparagine (Asn and N), glutamic acid (Glu and E), aspartic acid (Asp and D), serine (Ser and S), and threonine (Thr and T). If there is a discrepancy with the commonly used codes due to typos, the commonly used codes shall apply. The amino acids present in the polypeptides of this invention are preferably amino acids that can be encoded by nucleic acids.

[0052] As used herein, the term "fatty acid chain" includes residues that can non-covalently bind to human serum albumin in this invention. These residues are linked to the human amylin receptor agonist described in this invention and can also bind to human serum albumin non-covalently. The fatty acid chain described in this invention includes, but is not limited to, linear or branched fatty acid chain structures containing 12-24 carbon atoms, and may have carboxyl (-COOH), amino (-NH2), or other groups and specific linkers. One possible fatty acid chain in this invention includes structural formulas V, VI, VII, and VIII.

[0053] As used herein, the term "linker" includes suitable substituents that link a portion (e.g., a fatty acid chain) to a polypeptide (e.g., a polypeptide backbone). Thus, the linker and the chemical portion together become substituents. The portion linked to the linker can be any suitable portion. Examples of linkers include structures as described in any one of formulas II-IV, wherein the R2 group is an amino group (-NH2), and the R3 group is optionally a carboxyl group (-COOH) or a succinimide group. The R4 group is an amino group (-NH2), and the R5 group can be a carboxyl group (-COOH) or a succinimide group.

[0054] The term "fatty acid" as used in this article refers to a long aliphatic hydrocarbon chain organic compound containing a carboxyl group at one end, with the general formula C(n)H(2n+1)COOH. Based on carbon chain length, it can be classified into four categories: short-chain (containing 4–6 carbon atoms) fatty acids; medium-chain (containing 8–14 carbon atoms) fatty acids; long-chain (containing 16–20 carbon atoms) fatty acids; and very long-chain (containing 20 or more carbon atoms) fatty acids.

[0055] The terms “pharmaceutically acceptable carrier” and “excipient” used in this article are found in all pharmacopoeias known to medicinal chemists: Remington’s Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), and in particular Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol.

[0056] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0057] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0058] Example 1: Design and structural simulation of salmon Calcitonin-human amylin heterozygous sequence

[0059] Mature salmon Calcitonin (sCT) and human amylin have similar polypeptide structures, both containing a disulfide bond at the N-terminus and an amidation modification at the C-terminus. However, sCT is only 32 amino acids long (its amino acid sequence is shown in SEQ ID NO:1), while human amylin contains 37 amino acids (its amino acid sequence is shown in SEQ ID NO:2). According to literature reports, the additional 5 amino acids in human amylin are mainly related to its formation of amyloid fibrils, thus sCT is more stable than human amylin. Furthermore, sCT and human amylin also have different N-terminal disulfide bond structures. The N-terminal disulfide bond sequence of sCT is CSNLSTC (SEQ ID NO:13), where the first cysteine ​​C forms a disulfide bond with the second cysteine ​​C, while the N-terminal disulfide bond sequence of human amylin is KCNTATC (SEQ ID NO:14), where the first cysteine ​​C forms a disulfide bond with the second cysteine ​​C. To assess the impact of two different sequences containing disulfide bonds on protein stability, the inventors transplanted the N-terminal disulfide bond sequence of human amylin onto the corresponding sequence of sCT, generating the heterozygous sequence AsCT-1 (SEQ ID NO:3). The stability of AsCT-1 and sCT was compared using computer simulation, as detailed below.

[0060] First, the target molecules AsCT-1 and sCT were pretreated separately. The molecules were then placed in a cubic box with the boundary 1.0 nm away from the molecule to ensure no interaction with the periodic boundary. Water molecules were then filled into the box using the SPC216 water model to ensure a suitable solution environment. Na was then added. + and Cl -The system was neutralized and electroneutrality was ensured. Subsequently, energy minimization was used to reduce unreasonable overlap or high-energy conformations in the system. The system was equilibrated at fixed volume and temperature, and further equilibrated at fixed pressure and temperature (allowing volume variation) for 100 ps each. Finally, a final molecular dynamics simulation was performed to record the system's evolution under the specified conditions. The Leap-Frog algorithm was used for integration, with a simulation duration of 300 ns, a time step of 2 fs, and a reference temperature of 300 K. Three-dimensional periodic boundary conditions were used in all three directions, and coordinates, velocity, energy, and logs were recorded approximately every 10 ps. The simulated tracks were processed and analyzed as follows: periodic boundary conditions (PBCs) were removed to generate trajectory files without PBCs. Trajectories were extracted every 100 frames to reduce data volume. The root mean square deviation (RMSD) of the simulated trajectories was compared with that of the reference structure to evaluate the difference in peptide stability. The RMSD comparison results of AsCT-1 and sCT peptides after molecular dynamics simulation are shown in Figure 1.

[0061] Comparative analysis of the results revealed that the AsCT-1 sequence converged quickly in molecular dynamics simulations and maintained a stable RMSD state, indicating that the sequence could maintain good stability under simulation conditions. In contrast, sCT failed to converge within 300 ns of simulation time, exhibiting significant conformational fluctuations. This suggests that sCT has greater molecular flexibility than AsCT-1, indicating that AsCT-1 has better stability under simulation conditions. The only difference between AsCT-1 and sCT is the N-terminal disulfide bond sequence, suggesting that the disulfide bond sequence derived from human amylin is more conducive to improving peptide stability.

[0062] Example 2: Synthesis and Modification of Amylin Receptor Agonists

[0063] Mutation at certain sites in AsCT-1 yielded amylin receptor agonists AsCT-2, AsCT-3, AsCT-4, AsCT-5, AsCT-6, and AsCT-10, as well as their corresponding fatty acid chain-modified derivatives AsCT-4F, AsCT-4F1, AsCT-4F2, AsCT-5F, AsCT-6F, AsCT-6F1, AsCT-6F2, and AsCT-10F. All peptides involved in this invention were synthesized using a common solid-phase peptide synthesis method (Fmoc Chemistry). The related work was completed by commissioned research institutions (CROs) (Shanghai ChuTai Biotechnology Co., Ltd. and Nanjing Genscript Biotech Co., Ltd.). The synthesized peptides were initially peptide sequences without fatty acid chain modifications but with C-terminal amidation modifications, and the N-terminal disulfide bonds were completely closed. If the peptide sequence requires further fatty acid chain modification, the specific steps are as follows: Weigh the peptide powder and dissolve it in 50mM borate buffer (pH 10.0) to a concentration of 3 mg / ml. Simultaneously, dissolve the fatty acid chain stock solution in isopropanol to prepare a 10 mg / ml solution. Mix the peptide solution and fatty acid chain solution at a molar ratio of 1:2, maintaining the isopropanol concentration in the final mixture at 30%. Stir rapidly until homogeneous, and react for 15–20 min. Detect the coupling efficiency using HPLC reverse-phase chromatography and purify using the same method. Collect the correctly modified product, ensuring the final fatty acid chain-modified peptide purity exceeds 90%. The structure of the fatty acid chain used is shown below.

[0064] Fatty acid chain-1: (Formula V)

[0065] Fatty acid chain-2: (Formula VI)

[0066] Fatty acid chain-3: (Formula VII)

[0067] Fatty acid chain-4: (Formula VIII);

[0068] Table 1. Polypeptide sequences involved in this invention

[0069] Example 3: Stability test of amylin receptor agonist

[0070] To further test the stability differences among different amylin receptor agonists, we tested the stability of some amylin receptor agonists and their derivatives listed in Table 1 in a neutral formulation solution. This neutral formulation solution contained 10 mM Na-phosphate, pH 7.4, 14 mg / ml propylene glycol, and 32 mM phenol. The specific method was as follows: the lyophilized powder of the amylin receptor agonist and its derivatives to be tested was dissolved in the above neutral solution to a final concentration of 2.5 mg / ml. After thorough mixing, the mixture was placed in a 37°C incubator for 48 hours, then filtered through a 0.22 μm filter membrane to obtain the test sample. The test sample was analyzed using an ACQUITY UPLC Peptide BEH C18 column (SKU: 186003687), a Waters Acquity UPLC HClass-plus liquid chromatography system, and a Waters Empower3 web-based liquid chromatography workstation. The flow rate used in the experiment was 0.3 ml / min, the column temperature was 50℃, the detection UV was 280 nm, mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. First, the column was washed with 5% mobile phase B for 2.5 minutes. At 3 minutes, the proportion of mobile phase B increased to 10%, and the target protein was eluted with a linear gradient of 10%-90% mobile phase B over 15 minutes, and the corresponding peak areas were calculated. Subsequently, within 0.5 minutes, mobile phase B linearly increased to 95% and was maintained for 2 minutes of washing. Then, the initial equilibrium ratio was returned, and washing was maintained for 4 minutes. Table 2 lists the recovery rates of different amylin receptor agonists, calculated using the formula: Recovery rate = Peak area after standing at 37℃ for 48 hours / Peak area before standing * 100%

[0071] To further investigate the stability of co-formulations of the aforementioned different amylin receptor agonists with GLP-1 receptor agonists (such as semaglutide), we mixed solutions of the aforementioned different amylin receptor agonists (AsCT-4F1, AsCT-6F, or AsCT-6F1) at a concentration of 5 mg / ml with a semaglutide solution at a 1:1 volume ratio. Both solutions contained 10 mM Na-phosphate, pH 7.4, 14 mg / ml propylene glycol, and 32 mM phenol. The resulting mixed solution contained 2.5 mg / ml of the amylin receptor agonist (AsCT-4F1, AsCT-6F, or AsCT-6F1), 2.5 mg / ml of semaglutide, 10 mM Na-phosphate, pH 7.4, 14 mg / ml propylene glycol, and 32 mM phenol. Following the method described in the first paragraph of this embodiment, after thorough mixing, the mixture was placed in a 37°C constant temperature chamber for 48 hours and then removed. The same method was used for analysis, and the results are shown in Table 2.

[0072] The results in Table 2 show that after the N-terminal disulfide bond was replaced, the stability of AsCT-1 in neutral formulation solutions was improved compared to wild-type sCT, which is similar to the results of computer simulations. However, after 48 hours of incubation at 37°C, the recovery rate was only about 50%. By lowering the isoelectric point from 9.6 to 5.0 through point mutations (K18E, R24E, N26E), the recovery rate increased to over 90%. This indicates that the stability of this amylin receptor agonist is affected by multiple factors, including the N-terminal disulfide bond and the isoelectric point, and a lower isoelectric point is beneficial for improved stability. Furthermore, a comparison between AsCT-6F and AsCT-6F1 shows that introducing fatty acid chains of different lengths at the K20 position, such as those containing 18 or 20 carbon atoms, has little impact on the molecular stability of the amylin receptor agonist. Meanwhile, compared to canagliflozin, which has an isoelectric point as high as 9.4, some of the amylin receptor agonist molecules involved in this invention, such as AsCT-4F1, AsCT-6F and AsCT-6F1, have lower isoelectric points that are close to those of smegglutinin, and have also shown good stability in co-formulation studies with smegglutinin.

[0073] Table 2. Comparison of recovery rates of different amylin receptor agonists

[0074] Example 4: Detection of cellular activity of amylin receptor agonists and their derivatives

[0075] The method for detecting amylin receptor agonists and their receptor activity is the cAMP assay.

[0076] COS-7 cells were transfected using lentiviral transfection to detect the hCT receptor, hRAMP3, and cAMP response element-mediated firefly luciferase reporter gene (CREB-Luc). COS-7 cell lines were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. hCTR cells transfected with hCT receptor and CREB-Luc were selected using hygromycin B and puromycin pressure, while hAMY3R cells transfected with hCT receptor, hRAMP3, and CREB-Luc were selected using hygromycin B, blastomycin, and puromycin pressure. After selection, hCT receptor expression was detected by flow cytometry. hAMY3R expression was confirmed by detecting hRAMP3 mRNA levels in the cells.

[0077] hAMY3R cells were cultured in DMEM medium containing 10% FBS, 1% penicillin and streptomycin, 1 μg / mL puromycin, 200 μg / mL hygromycin B, and 10 μg / mL blastcin. hCTR cells were cultured in the same medium without blastcin. For assays, cells were digested and resuspended in medium without selection antibiotics, and 20 μL per well (7000 cells) was added to 384-well plates. The plates were incubated overnight at 37°C with 5% CO2.

[0078] On the day of assay, the positive control and sample were diluted to the specified concentration using culture medium without screening antibiotics, followed by a 5-fold serial dilution to 10 concentration points. 20 μL of the diluted protein was added to each well of a 384-well plate, with two replicates. The plate was incubated at 37°C for 3 hours. After incubation, 20 μL of GMOne-Step assay reagent was added to each well, and the plate was incubated at room temperature for 30 minutes. The chemiluminescence values ​​were read using a microplate reader. The EC50 value was obtained by plotting a dose-response curve using Prism8 software.

[0079] The results are shown in Table 3. Using canagliflozin (its amino acid sequence is shown in SEQ ID NO:12) as a positive control, the cell activities corresponding to different amino acid substitution strategies varied. Specifically, comparing wild-type sCT (SEQ ID NO:1), AsCT-1 (SEQ ID NO:3), and AsCT-3 (SEQ ID NO:5), it was found that replacing the N-terminal disulfide bond structure with a more stable one and introducing negatively charged amino acids at specific positions (K18E, R24E, N26E) to lower the isoelectric point did not significantly affect the activity of the amylin receptor agonist. However, replacing the last amino acid residue Pro at the C-terminus with Tyr (SEQ ID NO:4) resulted in an approximately 100-fold decrease in activity. Meanwhile, the fatty acid chain modification sites were also crucial. Of the three modification sites used in this invention, K1 and K20 had relatively small effects on activity, while K11 (SEQ ID NO:9) had a significant impact.

[0080] Table 3. Assay of amylin receptor agonist activity

[0081] Example 5: Animal activity test of amylin receptor agonist in SD rats

[0082] To further test the efficacy of the aforementioned amylin receptor agonists in animals, we used SD rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.) fed for 5-6 weeks to conduct efficacy tests on some molecules. The specific experimental steps are as follows: All male SD rats were weighed before grouping and randomly divided into groups based on their body weight. After grouping, they were housed individually. Before administration, the weight of the feed and the weight of the rat were weighed. After the first administration, the weight of the rat and the weight of the feed were measured and recorded every 12 hours. The feed intake at the current time period was calculated by subtracting the previous feed weight from the current feed weight. Measurements were taken twice a day for 5 consecutive days. The specific administration regimen is shown in Table 4 below.

[0083] Table 4. Dosing regimens for SD rats

[0084] Note: The dosing frequency Q3D*2 means dosing twice every 3 days.

[0085] Table 5. Activity assays of amyloid receptor agonists in SD rats

[0086] The experimental results are shown in Table 5. SD rats injected with the amylin receptor agonist provided in this invention showed further reductions in food intake and body weight compared to the control group or canagliflozin. The long-acting amylin receptor agonist effectively prolongs the efficacy of amylin and reduces the frequency of administration. The novel amylin receptor agonist designed in this invention maintains excellent cellular and animal activity while also effectively improving stability in neutral formulation solutions.

[0087] Example 6: Animal activity test of amylin receptor agonist in a DIO model of SD rats

[0088] To further test the efficacy of the above-mentioned amylin receptor agonists in animals, we selected 4-week-old male SD rats and fed them a high-fat diet (D12451, SPAF Biotechnology Co., Ltd.) for 4 weeks. The rats were randomly divided into 4 groups of 6 rats each, housed individually, and given the same weight of feed. The following treatments were administered: (1) solvent control (PBS), subcutaneously injected, Q3D six times; (2) AsCT-6F 10 nmol / kg, subcutaneously injected, Q3D six times; (3) Semaglutide, subcutaneously injected, Q3D six times; (4) AsCT-6F 10 nmol / kg, subcutaneously injected, Q3D six times, combined with Semaglutide, subcutaneously injected, Q3D six times. During the administration period, the rats' weight and feed weight were measured three times per week. The feed intake for that period was calculated by subtracting the current feed weight from the previous feed weight. At the end of the experiment, the changes in the rats' weight and feed intake during the administration period were statistically analyzed, as shown in Figures 2 and 3.

[0089] Experiments using it in combination with semaglutide showed that the amylin receptor agonist in this invention has a synergistic effect with GLP-1 receptor agonists, which can better enhance the weight loss effect of the drug.

[0090] Example 7: Pharmacokinetics of amylin receptor agonists in rats

[0091] To further verify the in vivo half-life of the fatty acid chain-modified molecules in this invention, we selected canglitide, AsCT-6F, AsCT-6F1, AsCT-6F2, and AsCT-10F, which have the same C20 fatty acid modification, for in vivo pharmacokinetic testing in rats. Taking AsCT-10F as an example, we randomly divided 6-week-old male SD rats into two groups of 4 rats each according to their body weight. They were given: (1) canglitide 10 nmol / kg, subcutaneously, as a single dose; and (2) AsCT-10F 10 nmol / kg, subcutaneously, as a single dose. Subsequently, blood was collected from the inner canthus of each rat at 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after administration, and serum was separated and collected. After processing, the serum was quantified and pharmacokinetic parameters were calculated using mass spectrometry. The specific values ​​are shown in Table 6 below.

[0092] Table 6. Pharmacokinetics of some amylin receptor agonists in rats

[0093] The comparison of canagliflozin in the table above reveals that AsCT-10F, which has similar fatty acid chain modifications, as well as AsCT-6F, AsCT-6F1, and AsCT-6F2, all have relatively long half-lives. Among them, AsCT-10F and AsCT-6F2 have better half-lives than canagliflozin, indicating that the optimized amylin receptor agonist can improve the pharmacokinetic performance of the molecule.

[0094] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An amylin receptor agonist comprising the following polypeptide: X1CX2TATCVLGX3LSQELHX4LX5TYPX6TX7TGSGTX8-NH2; Where X1 is independently selected from K, R, or does not exist; X2 is independently selected from N, Q, or E; X3 can be independently selected from L, V, K, R, or M; X4 is independently selected from K, E, or R; X5 can be independently selected from K, Q, or E; X6 is independently selected from R, E, A, K, or Q; X7 can be independently selected from R, N, or E; X8 can be independently selected from F, Y, P, or E; The C-terminus of the polypeptide may optionally be an amide or its derivative; The α-amino group of X1 can be optionally connected to an N-terminal extension via a linker, wherein the N-terminus extends into a fatty acid chain.

2. The amylin receptor agonist according to claim 1, wherein the amylin receptor agonist has at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with any one of SEQ ID NO:3-11, 17-22.

3. The amylin receptor agonist according to claim 1 or 2, wherein the amylin receptor agonist has an amino acid sequence as shown in any one of SEQ ID NO: 3-11, 17-22.

4. The amylin receptor agonist according to any one of claims 1-3, wherein the amino acid residue X1, X3 or X5 may be lysine K, and the ε-amino group of the lysine residue may be linked to a fatty acid via one or more linkers I; The fatty acid may have the structure described in Formula I, wherein the fatty acid contains at least one carboxyl group at its terminal, wherein n may be 5, 6, 7, 8, 9, 10, 11 or 12, and R1 is a chemical group to which the fatty acid is attached to one or more linkers I, wherein R1 may be a carboxyl group or a succinimide group. Furthermore, the R1 group in the fatty acid can be linked to the R2 group in linker I (Formula II), and the amylin receptor agonist is linked to the R3 group in linker I. The fatty acid and one or more linkers I form a fatty acid chain, and linker I has the structure described in Formula II, wherein the R2 group is an amino group (-NH2), and the R3 group can be a carboxyl group or a succinimide group. The R3 group of the terminal linker I in the fatty acid chain can be linked to the α-amino group at the N-terminus of the amylin receptor agonist or the ε-amino group in the side chain of lysine K.

5. The amylin receptor agonist according to any one of claims 1-4, wherein the R3 group of linker I in the fatty acid chain may be further linked to one or more linkers II (Formula III) or linker III (Formula IV), wherein the R4 group of linker II or linker III is linked to the R3 group in linker I, thereby forming a longer fatty acid chain, wherein the R5 group of linker II or linker III in the fatty acid chain may be further linked to the amylin receptor agonist, wherein the R4 group is an amino group (-NH2), and the R5 group may be a carboxyl group or a succinimide group, wherein the R5 group of the terminal linker II or linker III in the fatty acid chain may be linked to the α-amino group at the N-terminus of the amylin receptor agonist or the ε-amino group in the K side chain of lysine.

6. The amylin receptor agonist according to any one of claims 1-5, said fatty acid chain comprising a structure according to formula V, VI, VII or VIII: The R3 or R5 group can be a carboxyl group or a succinimide group, and n can be 5, 6, 7, 8, 9, 10, 11 or 12, preferably 8, 9 or 10.

7. A pharmaceutical composition comprising the amylin receptor agonist of any one of claims 1-6 and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, further comprising one or more additional active agents; Preferably, the active agent is selected from GLP-1 receptor agonists, GIP / GLP-1, GCG / GLP-1 dual receptor agonists, and insulin; More preferably, the GLP-1 receptor agonist is selected from smegglutide, liraglutide, and benaglutide; the GIP / GLP-1 or GCG / GLP-1 dual receptor agonist is selected from telposide and mascara.

9. The amylin receptor agonist of any one of claims 1-6 or the pharmaceutical composition of claim 7 or 8 for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcer.

10. Use of the amylin receptor agonist of any one of claims 1 to 6 for the manufacture of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, and / or for reducing food intake, reducing beta-cell apoptosis, increasing beta-cell function and beta-cell mass, and / or for restoring glucose sensitivity of beta-cells.

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