Incretin analogs and their use

Abstract

Incretin analogs and their use. It contains a triple-acting agent protein for human glucose-dependent insulinotropic polypeptide (GIP) receptor, glucagon-like peptide-1 (GLP-1) receptor and glucagon (GCG) receptor, and is useful for the treatment of type 2 diabetes (T2D), obesity, non-alcoholic fatty liver disease (NAFLD) and other carbohydrate and lipid metabolism disorders.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals. Specifically, this invention relates to incretin analogs and their use. [Background technology]

[0002] Diabetes and obesity are diseases caused by abnormalities in carbohydrate and lipid metabolism and are associated with many other diseases, including cardiovascular disease (CVD), peripheral artery disease, microvascular complications, and osteoarthritis. Therefore, the development of new therapies and treatments for obesity, diabetes, and their complications is of great importance to improving human health. Currently, incretins have excellent clinical efficacy in the treatment of type 2 diabetes and obesity. Glucagon-like peptide-1 (GLP-1) is a 37-amino acid incretin that stimulates insulin secretion, protects pancreatic β-cells, and suppresses glucagon secretion, gastric emptying, and food intake, leading to weight loss. Liraglutide and semaglutide are GLP-1 receptor agonists and are already approved for use in type 2 diabetes and obesity. However, many patients with type 2 diabetes and obesity are still not adequately controlled, and currently available incretin analogs or dipeptidyl peptidase IV (DPP-IV) inhibitors are used to control blood glucose and weight through only a single established mechanism.

[0003] Currently, research into new-generation metabolic drugs for diabetes and other conditions is primarily focused on dual-effect or multi-effect incretin receptor agonists, such as GLP-1R / GCGR, GLP-1R / GIPR dual-effect agonists, and GLP-1R / GCGR / GIPR triple-effect agonists. For example, LY3437943, developed by Eli Lilly, is a polypeptide modified with a fatty acid chain possessing GLP-1 / GCG / GIP activity, and it can significantly reduce the body weight of DIO mice.

[0004] In the prior art, incretin analogs with GLP-1R / GIPR / GCGR triagonist activity having different amino acid sequences have been developed, but there are still many deficiencies. Therefore, the research and development of incretin analogs with GLP-1R / GIPR / GCGR triagonist activity that have novel structures, higher activity, better efficacy, higher stability and lower production costs can provide safer and effective treatment methods for obesity, diabetes and their complications, and is an important problem that is urgently needed to be solved in this field. Summary of the Invention Problem to be Solved by the Invention

[0005] An object of the present invention is to provide an incretin analog and use thereof. Means for Solving the Problem

[0006] In a first aspect of the present invention, there is provided an incretin analog or a pharmaceutically acceptable salt thereof, wherein the incretin analog is X1X2QGTFTSDYSILLDX 16 X 17 AAQAFIEX 25 LX 27 X 28 GGPSSGAPPPS (SEQ ID NO: 7) (provided that X1 is His or Tyr, X2 is Aib, X 16 is Lys or modified Lys, X 17 is Ile or Gln or Lys or modified Lys, X 25 is Trp or Tyr, X 27 is Ile or Leu, X 28 is Ala or Glu.) comprises, and The incretin analog simultaneously has the activity to bind to and activate the class B G protein-coupled receptor GLP-1R and the activity to bind to the glucagon (GCG) receptor. To provide something.

[0007] In another preferred example, the incretin analog further has the activity to bind to and activate human glucose-dependent insulinotropic polypeptide (GIP) receptors. In another preferred example, X 16 This is Lys modified with fatty acid chains. In another preferred example, X 17 This is Lys modified with fatty acid chains.

[0008] In another preferred example, the incretin analog is HX2QGTFTSDYSILLDX 16 IAAQAFIEX 25 LX 27 X 28 GGPSSGAPPPS (Sequence ID 13) (however, X2 is Aib, X 16 is Lys or modified Lys, X 25 is Trp or Tyr, X 27 is Ile or Leu, X 28 (It is either Ala or Glu.) including and The incretin analog simultaneously has the activity to bind to and activate the class B G protein-coupled receptor GLP-1R, and the activity to bind to the glucagon (GCG) receptor.

[0009] In another preferred example, the incretin analog further has the activity to bind to and activate human glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0010] In another preferred example, X 16 or X 17 These are K molecules that have been chemically modified by independently compounding with the ε-amino group of the K side chain via the following structures. (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γE)b-CO-(CH2)c-CO2H (where a is 0, 1, or 2, b is 1 or 2, and c is an integer between 16 and 20.)

[0011] In another preferred example, a is 1, b is 1, and c is 18. In another preferred example, the fatty acid chain is ((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2)18-CO2H).

[0012] In another preferred example, the modification with the fatty acid chain includes compounding (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2)18-CO2H) with the ε-amino group of the 16th or 17th Lys side chain of the incretin analog.

[0013] In another preferred example, the incretin analog is selected from the following group: (1) Polypeptides having the amino acid sequence shown in SEQ ID NOs. 8, 9, 10, 11, or 12; (2) an amino acid sequence having at least 80%, preferably at least 85% or 90%, more preferably at least 95%, more preferably at least 98%, and more preferably at least 99% homology with the sequence shown in SEQ ID NOs: 8, 9, 10, 11, or 12, and having the activity to bind to and activate the class B G protein-coupled receptor GLP-1R and the activity to bind to the glucagon (GCG) receptor.

[0014] In another preferred example, the incretin analog further has the activity to bind to and activate human glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0015] In another preferred example, the incretin analog has an amino acid sequence selected from the following group: (1) Polypeptides having the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, or 6; (2) A polypeptide derived from (1) having the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, or 6, which is obtained by substitution, deletion, or addition of 1-2 amino acid residues, and which simultaneously has the activity to bind to and activate the class B G protein-coupled receptor GLP-1R and the activity to bind to the glucagon (GCG) receptor.

[0016] In another preferred example, the incretin analog further has the activity to bind to and activate human glucose-dependent insulinotropic polypeptide (GIP) receptors. In another preferred example, the amino acids include natural or non-natural amino acids.

[0017] In a second aspect of the present invention, a drug composition, (I) Incretin analogs according to the first aspect of the present invention, and (II) Pharmaceutically acceptable carriers The present invention provides a drug composition containing the following:

[0018] In another preferred example, component (a) accounts for 0.1 to 99.9 wt%, preferably 10 to 99.9 wt%, and more preferably 70 to 99.9 wt%, of the total weight of the drug composition.

[0019] In another preferred example, the drug composition is a liquid, a solid, or a semi-solid. In another preferred example, the dosage form of the drug composition is an oral dosage form, an injection, a spray, an aerosol, or a topical drug form.

[0020] In another preferred example, the dosage form of the drug composition includes tablets, granules, capsules, oral solutions, or injections. In another preferred example, the drug composition is a liquid composition. In another preferred example, the drug composition is an orally administered formulation. In another preferred example, the dosage form of the drug composition is a subcutaneous injection or an intramuscular injection.

[0021] In another preferred example, the pharmaceutically acceptable carrier is selected from the group consisting of carriers for intravenous solutions and / or carriers for injectable solutions, and preferably the carrier is one or more carriers selected from the group consisting of physiological saline, glucose saline, or a combination thereof. In another preferred example, the pharmaceutically acceptable carrier may be a carrier containing nanomaterials.

[0022] In another preferred example, the drug composition is in the form of a sustained-release formulation. In another preferred example, the dosage form of the drug composition is a freeze-dried powder. In another preferred example, the freeze-dried powder includes a freeze-drying protective agent. In another preferred example, the freeze-drying protective agent is selected from the group consisting of glucose, sucrose, mannitol, or combinations thereof.

[0023] In another preferred example, the drug composition is for the prevention and / or treatment of diseases related to abnormalities in carbohydrate and lipid metabolism. In another preferred example, the drug composition further contains other drugs that can be used for the prevention and / or treatment of diseases related to abnormalities in carbohydrate and lipid metabolism.

[0024] In another preferred example, other drugs that can be used for the prevention and / or treatment of diseases associated with the abnormalities of carbohydrate and lipid metabolism include, but are not limited to, metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase IV inhibitors, and sodium-glucose cotransporters.

[0025] In another preferred example, the diseases associated with the aforementioned abnormalities in carbohydrate and lipid metabolism include type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

[0026] A third aspect of the present invention provides the use of the incretin analog described in the first aspect of the present invention for the manufacture of a drug used for the following: (a) Lower the blood glucose level of the target organism as needed; (b) Lower the blood lipid glucose content of the target individual; (c) To reduce the weight or body fat percentage of the subject as required; and / or (d) To improve liver function; and / or (e) To prevent and / or treat diseases associated with abnormalities in carbohydrate and lipid metabolism.

[0027] A fourth aspect of the present invention provides the use of an incretin analog according to the first aspect of the present invention, or a drug composition according to the second aspect of the present invention, in the manufacture of a drug for treating a disease selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

[0028] A fifth aspect of the present invention provides a method for treating diseases related to abnormalities in carbohydrate and lipid metabolism, comprising administering an effective amount of the incretin analog described in the first aspect of the present invention or a drug composition containing the same to a required subject.

[0029] Of course, within the scope of the present invention, it is understood that the above-mentioned technical features of the present invention and the technical features specifically described below (for example, in the examples) can be combined to form new or preferred technical solutions. Due to space limitations, each will not be explained in detail here. [Modes for carrying out the invention]

[0030] Through extensive and in-depth research, the inventors have unexpectedly discovered that, for the first time, altering the position of the fatty acid chain modification and specific amino acids in the GLP-1 / GCG / GIP tripartite activity polypeptide of the present invention significantly improves the biological activity and stability of the polypeptide. Compared to compounds with existing structures, the polypeptide of the present invention exhibits significantly improved biological activity in the human blood albumin system, higher stability in serum, fewer non-natural amino acids used simultaneously, and lower immunogenicity and production costs. Specifically, the polypeptide obtained in the present invention possesses GLP-1, GCG, and GIP tripartite activity, significantly lowers body weight and body fat ratio in DIO mice, with a greater reduction than that of the control tirzepatide, which also possesses GLP-1 and GIP activity, and simultaneously significantly lowers blood glucose, plasma total cholesterol, and low-density lipoprotein cholesterol, improving liver function. It can be used to treat metabolic diseases such as type 2 diabetes, obesity, and hyperlipidemia. Based on this, the present invention was completed.

[0031] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which this invention pertains.

[0032] The term "approximately" refers to a value or composition that falls within the permissible margin of error of a particular value or composition as determined by those skilled in the art, thereby partially determining how the value or composition is measured or measured.

[0033] As used herein, the terms “contain” or “include” may be open, semi-closed, or closed. In other words, the terms also include “basically composed of…” or “composed of…”.

[0034] Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-1 (GLP-1) is a 37-amino acid incretin that stimulates insulin secretion, protects pancreatic β-cells, and suppresses glucagon secretion, gastric emptying, and food intake, leading to weight loss. Liraglutide and semaglutide are GLP-1 receptor agonists and are already approved for use in type 2 diabetes and obesity. However, many patients with type 2 diabetes and obesity are still not adequately controlled, and currently available incretin analogs or dipeptidyl peptidase IV (DPP-IV) inhibitors are used to control blood glucose and weight through a single established mechanism.

[0035] Glucagon (GCG) Glucagon (GCG), similarly converted from proglucagon, binds to and activates the glucagon receptor, thereby activating the corresponding signaling pathway and regulating gluconeogenesis and glycogenolysis to raise blood glucose levels and maintain blood glucose levels. Studies have already shown that glucagon suppresses appetite, reduces food intake, and simultaneously has fat-breaking and weight-reducing effects. Pocai et al. (Obesity 2012;20:1566-1571; Diabetes 2009, 58, 2258) and Day et al. (Nat Chem Biol 2009;5:749) described the therapeutic principle by which dual agonists of the GLP-1 receptor and glucagon receptor combine the actions of GLP-1 and glucagon within a single molecule, resulting in anti-diabetic effects and significant weight-reducing effects.

[0036] Human glucose-dependent insulin-stimulating polypeptide (GIP) Human glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that protects pancreatic β-cells by stimulating insulin secretion from them in the presence of glucose, thereby performing physiological functions in a stable glucose environment. The published patent CN201680005007.X describes compound LY3298176, a GLP-1 / GIP receptor dual agonist based on a natural GIP polypeptide sequence, as having a beneficial effect in lowering blood glucose and body weight. This product has already been approved by the FDA for use in the treatment of type 2 diabetes, and its generic name is tirzepatide.

[0037] Both naturally occurring GIP and GLP-1 are rapidly inactivated by the commonly present protease DPPIV, making them unsuitable for short-term metabolic control. Existing technologies generally improve the half-life of polypeptides in the body by linking fatty acid chains to polypeptides and adding non-standard amino acids. For example, LY3298176 (Tilzepatide) is modified at the 20th lysine position by a drug modification of GLP-1, and the 2nd and 13th mutations are non-standard amino acids Aib.

[0038] The incretin analog of the present invention As used herein, "incretin analog of the present invention," "polypeptide having GLP-1 / GCG / GIP triple-acting activity of the present invention," "incretin analog having GLP-1R / GIPR / GCGR triple-acting activity," and "polypeptide of the present invention" are interchangeable and all refer to the polypeptide described in the first aspect of the present invention.

[0039] Specifically, the present invention obtained polypeptides having triple-acting activity on GLP-1 receptors, GIP receptors, and GCG receptors through screening. As used herein, “triple-acting activity” refers to an incretin analog that is active on any of the GLP-1, GIP, and glucagon receptors. In particular, because it has balanced activity on each receptor, the administered dose provides sufficient activity to deliver the agonizing effect on each receptor, while simultaneously avoiding analogs with excessively high levels of unwanted side effects associated with the activity. Furthermore, incretin analogs with triple-acting activity have extended durations of action on any of the GIP, GLP-1, and glucagon receptors, which is advantageous for low-frequency administration such as once daily, three times a week, twice a week, or once a week.

[0040] The structural features of the incretin analogs described herein result in analogs that exhibit sufficient activity at all three receptors: GIP, GLP-1, and glucagon receptors. This provides the advantageous effect of activity at each receptor (i.e., triple agonist activity), without the activity at any one receptor being excessively high compared to the other two, nor does it lead to unnecessary side effects when administered at a dose sufficient to activate all three receptors.

[0041] The structural features of the incretin analogs described herein include further fatty acid modification of their side chains, enhanced optimal binding and efficacy at each receptor, improved stability, and reduced immunogenicity.

[0042] Preferably, this publication first describes an incretin analog or a pharmaceutically acceptable salt thereof, wherein the incretin analog comprises the following base amino acid sequence. X1X2QGTFTSDYSILLDX 16 X 17 AAQAFIEX 25 LX 27 X 28 GGPSSGAPPPS (Sequence ID 7) (However, X1 is His or Tyr, X2 is Aib, X 16 Lys or compound Lys, X 17 is Ile or Gln or Lys or modified Lys, X 25 is Trp or Tyr, X 27 is Ile or Leu, X 28 (It is either Ala or Glu.)

[0043] Preferably, X1 is H, X2 is Aib, and X 16 is K, X 17 is K, X 25 is W, X 27 is I, X 28 A is the incretin analog, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKIAAQAFIEWLIAGGPSSGAPPPS(Sequence ID 8)

[0044] Preferably, X1 is H, X2 is Aib, and X 16 is K, X 17 is K, X 25 is W, X 27 is I, X 28 A is the incretin analog, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKKAAQAFIEWLIAGGPSSGAPPPS(Sequence ID 9)

[0045] Preferably, X1 is H, X2 is Aib, and X 16 is K, X 17 Q is X 25 is W, X 27 is I, X 28 A is the incretin analog, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKQAAQAFIEWLIAGGPSSGAPPPS(Sequence ID 10)

[0046] Preferably, X1 is Y, X2 is Aib, and X 16 is K, X 17 is I, X 25 is W, X 27 is I, X 28 A is the incretin analog, and the incretin analog has the following amino acid sequence. Y-Aib-QGTFTSDYSILLDKIAAQAFIEWLIAGGPSSGAPPPS(Sequence ID 11)

[0047] Preferably, X1 is H, X2 is Aib, and X 16 is K, X 17 is I, X 25 is Y, X 27 is L, X 28 E is present, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKIAAQAFIEYLLEGGPSSGAPPPS(Sequence ID 12) Among these, the incretin analogs described in the present invention include naturally occurring amino acids and unnatural amino acids, such as α-aminoisobutyric acid (Aib).

[0048] The incretin analogs described in this invention have structural similarities to natural human peptides, but also many structural differences. For example, modifications at positions 2, 16, or 17 include Aib at position 2, K modified with a fatty acid chain at position 16, or K modified with a fatty acid chain at position 17.

[0049] As described above, the incretin analogs described herein include a fatty acid moiety compounded with a natural or unnatural amino acid having a functional group that can be compounded via a linker, for example. Such compounding is sometimes called acylation. In some cases, the amino acid having the functional group that can be compounded may be K, C, E, and D. In specific cases, the amino acid having the functional group that can be compounded is K, in which case it compoundes with the ε-amino group of the K side chain. In which case the linker may be 1 to 4 amino acids, or aminopolyethylenecarboxylic acid, or a mixture thereof. If the linker is at least one amino acid, the amino acid may be one or more E or γE amino acid residues. The length and composition of the fatty acid moiety affect the half-life of the incretin analog, the potency of the incretin analog in in vivo animal models, and the solubility and stability of the incretin analog, for example, C 16 -C 22 fatty acid portion (for example, C 16 -C 22 Examples include saturated monounsaturated or diunsaturated fatty acids.

[0050] As used herein, "modified Lys" or "lysine modified with a fatty acid chain" means that the fatty acid portion is compounded with the ε-amino group of the K side chain of lysine via a linker.

[0051] Apart from the modifications described herein, the incretin analogs described herein may further include one or more other amino acid modifications, provided that the analog is still capable of binding to and activating any of the GIP, GLP-1, and glucagon receptors.

[0052] In one specific case, the incretin analog described herein contains a linker and a fatty acid component having the structure of the following formula. (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γGlu)b-CO-(CH2)c-CO2H (where a is 0, 1, or 2, b is 1 or 2, and c is 16 or 18.)

[0053] The present invention further includes active fragments, derivatives, and analogues of the polypeptide of the present invention. As used herein, the terms “fragment,” “derivative,” and “analog” essentially refer to polypeptides that maintain the triple-acting activity of GLP-1R / GIPR / GCGR. Fragments, derivatives, and analogues of the polypeptide of the present invention may be (i) polypeptides substituted with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues), or (ii) polypeptides with substituents on one or more amino acid residues, or (iii) polypeptides obtained by fusing the polypeptide of the present invention with another compound (for example, a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides obtained by fusing an additional amino acid sequence to this polypeptide (subsequent proteins obtained by fusing with a leader sequence, secretory sequence, or tag sequence such as 6His). Based on the disclosure herein, these fragments, derivatives, and analogues are known to those skilled in the art.

[0054] Some suitable active derivatives refer to polypeptides in which, compared to the amino acid sequence of formula I or formula II, five or fewer, preferably three or fewer, more preferably two or fewer, and most preferably one amino acid is replaced with an amino acid having similar or similar properties. These conservative mutant polypeptides are preferably produced by amino acid substitution as shown in Table A. [Table A]

[0055] Manufacturing method The polypeptide of the present invention may be chemically synthesized. Alternatively, the polypeptide of the present invention may be artificially synthesized by conventional methods.

[0056] One preferred method is to use liquid-phase synthesis or solid-phase synthesis, such as the Boc solid-phase method, the Fmoc solid-phase method, or a combination of both. Solid-phase synthesis allows for rapid sample acquisition, and an appropriate resin support and synthesis system can be used depending on the characteristics of the target peptide sequence. For example, a suitable solid support in the Fmoc system, such as Wang resin with linked C-terminal amino acids in the peptide, has a polystyrene structure with 4-alkoxybenzyl alcohol as the arm between the amino acids. The Fmoc protecting group is removed by treatment with 25% hexahydropyridine / dimethylformamide at room temperature for 20 minutes, and the peptide is extended from the C-terminus to the N-terminus along the given amino acid sequence. After the synthesis is complete, the proinsulin-related peptide synthesized with trifluoroacetic acid containing 4% p-cresol is cleaved from the resin to remove the protecting group, the resin is removed by filtration, and then precipitated and separated with ethyl ether to obtain the crude peptide. After lyophilization of the resulting product solution, the required peptide is purified by gel filtration and reverse-phase high-pressure liquid-phase chromatography. When performing solid-phase synthesis using the Boc system, a suitable resin is a PAM resin in which the C-terminal amino acid of the peptide is linked. The structure of the PAM resin is polystyrene, and the arm between the amino acid is 4-hydroxymethylphenylacetamide. In the Boc synthesis system, the deprotection, neutralization, and coupling cycle involves removing the protecting group Boc with TFA / dichloromethane (DCM) and neutralizing with diisopropylethylamine (DIEA) / dichloromethane. After the condensation of the peptide chain is complete, the mixture is treated with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0°C for 1 hour to cleave the peptide chain from the resin and simultaneously remove the protecting group. The mixture is extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), the solution is freeze-dried, and then further separated and purified using a molecular sieve Sephadex G10 or Tsk-40f. Further purification by high-pressure liquid phase is then performed to obtain the required peptide.Each amino acid residue can be coupled using various coupling agents and methods known in the field of peptide chemistry, for example, by direct coupling with diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBt), or 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). The purity and structure of the synthesized short-chain peptides can be confirmed by reverse-phase high-performance liquid spectrometry and mass spectrometry.

[0057] In one preferred example, the polypeptide of the present invention is prepared by solid-phase synthesis depending on its sequence, purified by high-performance liquid-phase chromatography to obtain a lyophilized powder of the target peptide of high purity, and stored at -20°C.

[0058] Drug composition Furthermore, the present invention provides a drug composition containing an effective amount (for example, 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70-99.9 wt%) of the polypeptides of the present invention (particularly polypeptide 1 and polypeptide 3), as well as a pharmaceutically acceptable carrier.

[0059] Typically, the polypeptides of the present invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous medium, where the pH is usually about 5-8, preferably about 6-8.

[0060] As used herein, the term “effective dose” or “effective dosage” means the amount that is functional or active in humans and / or animals and is available to humans and / or animals.

[0061] As used herein, a “pharmaceutically acceptable” ingredient is a substance that, when applied to humans and / or mammals, does not cause excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio. The term “pharmaceutically acceptable carrier” refers to a carrier for the delivery of a therapeutic agent, including various excipients and diluents.

[0062] The drug composition of the present invention contains a safe and effective amount of the polypeptide of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline solution, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. Since drug formulations should generally be adapted to the mode of administration, the drug composition of the present invention may also be an injectable preparation and can be prepared by conventional methods using, for example, physiological saline solution or an aqueous solution containing glucose and other adjuvants. The drug composition is preferably prepared under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The drug formulation of the present invention may also be a sustained-release formulation.

[0063] In another preferred example, the dosage form of the drug composition is a spray, an injection (for example, an intramuscular or intravenous injection).

[0064] The effective dose of the protein described in this invention can be modified depending on the mode of administration and the severity of the disease to be treated. A suitable effective dose can be determined by a person skilled in the art based on various factors (e.g., by clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters such as bioavailability, metabolism, and half-life, the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. Typically, a satisfactory effect can be obtained by administering the polypeptide of this invention at a dose of 0.1-1 mg / kg animal body weight (preferably 0.3-0.6 mg / kg animal body weight) daily. For example, depending on the treatment situation, the dose can be divided into several doses daily, or the dose can be reduced proportionally.

[0065] The incretin analogs described herein may react with any of several inorganic and / or organic acids / bases to form pharmaceutically acceptable acid / base addition salts (pharmaceutically acceptable salts). The pharmaceutically acceptable salts and their common preparation techniques are known in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)). The pharmaceutically acceptable salts used herein include, but are not limited to, sodium salts, trifluoroacetate salts, hydrochloride salts, and acetate salts.

[0066] The main advantages of this invention include the following: The present invention offers the following advantages compared to compounds described in the existing technology: (1) Higher in vitro bioactivity; (2) Higher stability in serum; (3) Fewer non-natural amino acids are used, production costs are lower, and immunogenicity in the body is lower; (4) Compounds with GLP-1 / GIP dual activity have a better effect in lowering body weight and body fat percentage.

[0067] The present invention will be further described below, along with specific examples. These examples are used solely to illustrate the present invention and are not intended to limit its scope. Experimental methods in the following examples where detailed conditions are not given typically follow standard conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are calculated by weight.

[0068] Example 1 Synthesis of polybutylenes This invention involves producing polypeptides and their modification sites by a solid-phase polypeptide synthesis method, using standard side-chain protecting groups. The solid-phase polypeptide synthesis method is as follows: 1.1 Sufficient swelling of the resin 0.22 g of Fmoc-linker-MBHA resin containing 0.451 mmol / g of substitution was weighed into a clean, dry reaction tube, 5 mL of DMF was added, and the tube was immersed at room temperature for 2 hours.

[0069] 1.2 Removal of Fmoc protecting group The solvent from the previous step was removed by suction filtration, 5 mL of 20% piperidine DMF solution was added to the resin, nitrogen gas was blown over it for 30 minutes, the solution was removed by filtration, and the resin was washed five times with 5 mL of DMF.

[0070] 1.3 Detection of elimination effect by ninhydrin A small amount of resin was placed in a detector tube, washed twice with ethanol, and two drops each of ethanol solutions A: 20% ethanol + 80% phenol, B: redistilled pyridine, and C: 5% ninhydrin were added. The mixture was heated at 110°C for 2 minutes, and the color of the resin was observed. A deep blue color indicates complete elimination, meaning the next reaction can proceed. If the resin is colorless, it indicates that the protecting group has not been completely removed, and the deprotection procedure described above needs to be repeated.

[0071] 1.4 Linking of the first amino acid At room temperature, the solvent from the previous step was removed by suction filtration using a sand core. Three times the amount of the first amino acid at the C-terminus (an amino acid protected with Fmoc), three times the amount of HOBT, and three times the amount of DIC were added, and the reaction was allowed to proceed at room temperature for 1-2 hours using DMF as the solvent. A small amount of resin was taken and detected with ninhydrin detection reagent. If it was detected as clear, it indicated that the reaction was complete. After suction filtration of the solvent, the mixture was washed three times with 5 mL of DMF.

[0072] 1.5 Sequential addition of amino acids to the main chain Similarly, steps 1.2-1.4 were repeated to synthesize the polypeptide up to the last amino acid at the N-terminus, following the polypeptide sequence, to obtain compound 1.

[0073] 1.6 De-Dde protection 5 mL of a 3% hydrazine hydrate / DMF solution was placed in the resin, nitrogen gas was blown through for 10 minutes, the solution was removed by filtration, and the procedure was repeated twice. Then, the resin was washed five times with 5 mL of DMF to obtain compound 2.

[0074] 1.7 Sequential addition of side chain amino acids Similarly, following the above procedure, steps 1.2-1.44 were repeated to synthesize compound 3 according to the polypeptide sequence, up to the last amino acid, eicosanedioic acid mono-t-butyl ester, and then vacuum-dried.

[0075] 1.8 Extraction of resin and separation and detection of refined products Finally, the compound was cut with trifluoroacetic acid cleavage solution (95% TFA:2.5% TIS:2.5% H2O) for 3 hours, filtered by suction, and the filtrate was collected and placed in methyl t-butyl ether. The solid was collected by centrifugation, and the precipitated crude product was washed three times with methyl t-butyl ether. After vacuum drying, the crude product was obtained. The product was desalted and purified by HPLC, and after freeze-drying, the purified product was obtained. The molecular weight was measured by LC-MS. The sequences of the chemically synthesized polypeptides are shown in Table 1. [Table 1]

[0076] Example 2 Detection of in vitro activity In vitro activity of the polypeptide GLP-1 was detected using CHO-K1 cells expressing human GLP-1 receptor and CRE-luciferase. The cells were inoculated into 96-well plates and cultured for 1 hour at 37°C and 5% CO2 at a density of 30,000 cells / well / 100 μL. For each test protein sample, the protein solution was diluted nine times in a 4-fold gradient from a constant concentration with PBS containing 0.25% FBS / 0.01% BSA. A total of 10 different dilutions were prepared, and 50 μL of each was taken and placed in the 96-well plates inoculated with cells. The plates were cultured for 5 hours at 37°C and 5% CO2. The 96-well plates were removed from the incubator and allowed to equilibrate at room temperature for 10 minutes. 100 μL of reaction solution was added to each well, and after shaking at 200 times / min for 10 minutes, fluorescence readings were detected using a multifunctional plate reader. The half-effective concentration (EC50) for each sample was calculated by blotting, with the sample concentration as the x-coordinate and the fluorescence reading as the y-coordinate. GLP-1 was used as a positive control.

[0077] Using CHO-K1 cells containing reporter genes expressing the GCG receptor and GIP receptor, respectively, the GCG and GIP activity of polypeptides was detected. The procedure was the same as described above, with glucagon and GIP used as positive controls. [Table 2] Among these, polypeptides 1, 3, and 6 showed a significant increase in GCG activity, and corresponding changes in GIP activity.

[0078] Example 3: Bioactivity in different buffer systems Since different buffer systems affect the degree of binding between polypeptides and buffer components during the detection process, GLP-1, GCG, and GIP biological activities of different polypeptide species were detected in 0.01% casein, 2% mouse serum albumin, and 2% human serum albumin (HSA) systems, respectively, in order to better predict the in vivo and clinical activities of the polypeptides. The procedure was the same as in Example 2. In the human serum albumin, mouse serum albumin, and casein systems, polypeptide 1 showed superior GLP-1R activity compared to the control LY3437943. In the human serum albumin system, polypeptide 1 (SEQ ID NO: 1) showed superior GLP-1, GCG, and GIP activity compared to the control LY3437943 (Table 3), indicating better activity in actual clinical applications. [Table 3]

[0079] Example 4: Stability of polypeptides in serum Polypeptide 1 (SEQ ID NO: 1), Polypeptide 3 (SEQ ID NO: 3), and control LY3437943 were diluted to 100 μg / ml with cynomolgus monkey serum, and each was divided into three vials of 200 μl each. One vial was placed in a -80°C refrigerator as a zero-point control, while the other two samples were placed in a 37°C incubator. After 24 hours and 72 hours, they were removed and placed in a -80°C refrigerator. The GIP activity of the above samples was detected in human GIPR-expressing CHO cells (CHOK1-GIPR), and the changes in activity after different samples were placed for different periods of time were compared.

[0080] In particular, polypeptide 1 (SEQ ID NO: 1) showed higher stability in serum than polypeptide 3 (SEQ ID NO: 3), suggesting that the linking site of the fatty acid chain in the polypeptide affects polypeptide stability. LY3437943 contains three non-natural amino acids, which primarily improve the serum stability of the polypeptide and avoid enzymatic cleavage by endopeptidases. However, introducing a relatively large number of non-natural amino acids increases immunogenicity in the body and simultaneously leads to higher production costs.

[0081] Surprisingly, polypeptide 1 can significantly improve polypeptide stability even in screening of amino acids at different sites of the fatty acid chain. Compared to the control, the polypeptide of the present invention is more stable. Furthermore, because the polypeptide of the present invention has only one non-natural amino acid introduced, it has lower immunogenicity and production costs. [Table 4]

[0082] Example 10: Experiment on weight loss in DIO mice by multiple subcutaneous injections. The purpose of this embodiment is to study the effects of subcutaneous injection of polypeptides on indicators such as body weight in DIO mice, and to simultaneously compare its efficacy with that of tirzepatide.

[0083] A model was constructed using 7-week-old C57BL / 6Nju mice, fed a high-fat diet (approximately 20% protein and 60% fat) for 12 weeks. The feeding conditions were: lighting switched every 12 hours / 12 hours daily, free feeding, temperature of 20-25°C, relative humidity of 40-70%, and ventilation rate of 10-15 times / hour. The day before administration, the mice were randomly divided into groups based on body weight, water intake, and food intake. The groups were designed as follows: tilzepatide group (30 nmol / kg), polypeptide 1 (30 nmol / kg), normal control group, and model control group, with 6 mice in each group. The treatment was administered by subcutaneous injection, twice a week for 5 consecutive weeks, for a total of 10 injections. After administration, the mice's body weight, OGTT, body fat ratio, four blood lipid parameters, and liver function were detected. All data were displayed as (X±s), and statistical analysis was performed on the data using one-way ANOVA.

[0084] [Table 5]

[0085] [Table 6]

[0086] The results show that polypeptide 1 significantly reduced body weight and body fat percentage in DIO mice, with a greater reduction than the control group tilzepatide. Simultaneously, it significantly reduced blood glucose, plasma total cholesterol, and low-density lipoprotein cholesterol, and improved liver function.

[0087] All documents relating to the present invention are cited herein by reference, so that each document may be cited independently. Furthermore, after reading the above, those skilled in the art will understand that various variations and modifications of the present invention may be made, and that equivalent forms thereof are included within the scope of the claims of the present invention.

Claims

1. An incretin analog or a pharmaceutically acceptable salt thereof, wherein the incretin analog is X １ X ２ QGTFTSDYSILLDX １６ X １７ AAQAFIEX ２５ LX ２７ X ２８ GGPSGAPPPS (Sequence ID 7) (however, X １ His is X ２ is Aib, X １６ is a modified Lys, X １７ はLys, X ２５ is Trp, X ２７ It is Ile, X ２８ (It is Ala.) including and The incretin analog simultaneously has the activity to bind to and activate the class B G protein-coupled receptor GLP-1R and the activity to bind to the glucagon (GCG) receptor. The incretin analog further has the activity to bind to and activate human glucose-dependent insulin-stimulating polypeptide (GIP) receptors. The incretin analog has a polypeptide having the amino acid sequence shown in SEQ ID NO:

1. The incretin analog or a pharmaceutically acceptable salt thereof, characterized by the above.

2. The incretin analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the modified Lys is compounded with the ε-amino group of the K side chain of lysine via a linker.

3. X16 has the following structure: (2-[2-(2-aminoethoxy)-ethoxy]acetyl)-(γGlu)-CO-(CH2)18-CO2H) The incretin analog according to claim 1, wherein K is chemically modified by compounding with the ε-amino group of the K side chain.

4. A drug composition, (I) The incretin analog according to claim 1, and (II) Pharmaceutically acceptable carriers The drug composition characterized by containing the following:

5. The drug composition according to claim 4, characterized in that the drug composition is for the prevention and / or treatment of diseases related to abnormalities in carbohydrate and lipid metabolism.

6. The drug composition according to claim 5, further characterized in that it contains other drugs that can be used for the prevention and / or treatment of diseases related to abnormalities in carbohydrate and lipid metabolism.

7. The drug composition according to claim 6, characterized in that other drugs that can be used for the prevention and / or treatment of diseases related to the aforementioned abnormalities in carbohydrate and lipid metabolism include, but are not limited to, metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase IV inhibitors, and sodium-glucose cotransporters.

8. The drug composition according to claim 5, characterized in that the diseases related to the abnormality of carbohydrate and lipid metabolism mentioned above include type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

9. Use of the incretin analog according to claim 1 or the drug composition according to claim 4 in the manufacture of a drug for treating a disease selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

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