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] The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to incretin analogs and their use.

Background Art

[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 arterial disease, microvascular complications, and osteoarthritis. Therefore, the development of new therapies and therapeutic agents for obesity, diabetes, and their complications is of great significance for improving human health. Currently, incretin has excellent clinical effects 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 that have already been approved for use in type 2 diabetes and obesity. However, many type 2 diabetes patients and obese patients are still not well controlled, and currently marketed incretin analogs or dipeptidyl peptidase-IV (DPP-IV) inhibitors are used for blood glucose and weight control with only a single established mechanism.

[0003] Currently, next-generation metabolic drugs such as diabetes mainly focus on the research of 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 having GLP-1 / GCG / GIP activity, which can significantly reduce the body weight of DIO mice.

[0004] In the prior art, incretin analogs with GLP-1R / GIPR / GCGR triple agonistic activity of 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 triple agonistic activity, which have a novel structure, higher activity, better efficacy, higher stability and lower production cost, is an important issue that is urgently desired to be solved in this field to provide a safer and more effective treatment method for obesity, diabetes and their complications.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] An object of the present invention is to provide an incretin analog and its use.

MEANS FOR SOLVING THE PROBLEMS

[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.) and includes The incretin analog has the activity of simultaneously binding to and activating the class B G protein-coupled receptor GLP-1R and the activity of binding to the glucagon (GCG) receptor. provided.

[0007] In another preferred example, the incretin analog further has the activity of binding to and activating the human glucose-dependent insulinotropic polypeptide (GIP) receptor. In another preferred example, X 16 is Lys modified with a fatty acid chain. In another preferred example, X 17 is Lys modified with a fatty acid chain.

[0008] In another preferred example, the incretin analog HX2QGTFTSDYSILLDX 16 IAAQAFIEX 25 LX 27 X 28 GGPSSGAPPPS (SEQ ID NO: 13) (where X2 is Aib, X 16 is Lys or modified Lys, X 25 is Trp or Tyr, X 27 is Ile or Leu, X 28 is Ala or Glu). and the incretin analog has the activity of simultaneously binding to and activating the class B G protein-coupled receptor GLP-1R and the activity of binding to the glucagon (GCG) receptor.

[0009] In another preferred example, the incretin analog further has the activity of binding to and activating the human glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0010] In another preferred example, X 16 or X 17 is K that is chemically modified by binding to the ε-amino group of the K side chain via the following structure, respectively. (2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)a-(γE)b-CO-(CH2)c-CO2H (wherein a is 0, 1 or 2, b is 1 or 2, and c is an integer of 16 to 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 by the fatty acid chain includes binding (2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2)18-CO2H) to 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) A polypeptide having the amino acid sequence shown by SEQ ID NO: 8, 9, 10, 11 or 12; (2) An amino acid sequence having at least 80% homology with that shown by SEQ ID NO: 8, 9, 10, 11 or 12, preferably at least 85% or 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and simultaneously having the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the activity of binding to the glucagon (GCG) receptor.

[0014] In another preferred example, the incretin analog further has the activity of binding to and activating the human glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0015] In another preferred example, the incretin analog has an amino acid sequence selected from the following group: (1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6; (2) A polypeptide derived from (1) in which the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6 has undergone substitution, deletion or addition of 1 to 2 amino acid residues, and which simultaneously has the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the activity of binding to the glucagon (GCG) receptor.

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

[0017] In a second aspect of the present invention, there is provided a pharmaceutical composition comprising: (I) The incretin analog according to the first aspect of the present invention, and (II) a pharmaceutically acceptable carrier .

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

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

[0020] In another preferred example, the dosage form of the drug composition includes tablets, granules, capsules, oral administration solutions, or injections. In another preferred example, the drug composition is a liquid composition. In another preferred example, the drug composition is an oral administration preparation. 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 infusion solutions and / or carriers for injections. Preferably, the carrier is one or more carriers selected from the group consisting of physiological saline, glucose saline, or combinations thereof. In another preferred example, the pharmaceutically acceptable carrier may be a carrier containing a nanomaterial.

[0022] In another preferred example, the drug composition is in a sustained release dosage form. In another preferred example, the dosage form of the drug composition is a lyophilized powder. In another preferred example, the lyophilized powder contains a lyoprotectant. In another preferred example, the lyoprotectant 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 disorders of 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 disorders of carbohydrate and lipid metabolism.

[0024] In another preferred example, other drugs that can be used for the prevention and / or treatment of diseases related to abnormal 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 related to the abnormal carbohydrate and lipid metabolism include type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

[0026] In a third aspect of the present invention, there is provided the use of an incretin analog described in the first aspect of the present invention for the manufacture of a drug used for the following: (a) Lowering the blood glucose content of a subject in need; (b) Lowering the blood fat sugar content of a subject in need; (c) Lowering the body weight or body fat percentage of a subject in need; and / or (d) Improving liver function; and / or (e) Preventing and / or treating diseases related to abnormal carbohydrate and lipid metabolism.

[0027] In a fourth aspect of the present invention, there is provided the use of an incretin analog described in the first aspect of the present invention or a drug composition described in 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] In a fifth aspect of the present invention, there is provided a method for treating a disease related to abnormal carbohydrate and lipid metabolism, the method comprising administering to a subject in need an effective amount of an incretin analog described in the first aspect of the present invention or a drug composition containing the same.

[0029] Of course, within the scope of the present invention, it is understood that each of the above technical features of the present invention and each of the specifically described technical features below (for example, in the examples) can be combined with each other to form a new or preferred technical solution. Due to limited space, not all combinations will be described herein one by one.

Mode for Carrying Out the Invention

[0030] Through extensive and in-depth research, the inventor of the present invention unexpectedly found for the first time that by changing the position of the fatty acid chain modification and the amino acids at specific positions of the polypeptide having GLP-1 / GCG / GIP triple agonistic activity of the present invention, the biological activity and stability of the polypeptide can be significantly improved. Compared with compounds of existing structures, the polypeptide of the present invention has significantly improved biological activity in the human blood albumin system, higher stability in serum, fewer unnatural amino acids used at the same time, and lower immunogenicity and production costs. Specifically, the polypeptide obtained in the present invention has GLP-1, GCG, and GIP triple activities, significantly reduces the body weight and body fat ratio of DIO mice, and the reduction range is larger than that of the control tirzepatide having GLP-1 and GIP activities. At the same time, it can significantly reduce blood glucose, total plasma cholesterol, and low-density lipoprotein cholesterol, and improve liver function. It can be used for the treatment of metabolic diseases such as type 2 diabetes, obesity, and hyperlipidemia. Based on this, the present invention has been completed.

[0031] Term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] The term "about" refers to a value or composition within an acceptable error range of a specific value or composition determined by one of ordinary skill in the art, thereby determining in part how the value or composition is measured or determined.

[0033] As used herein, the terms "comprising" or "including" may be open-ended, semi-closed or closed. In other words, the said terms also include "consisting essentially of...", or "consisting 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 that have already been 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 marketed incretin analogs or dipeptidyl peptidase-IV (DPP-IV) inhibitors are used to control blood glucose and body weight through only a single established mechanism.

[0035] Glucagon (GCG) Glucagon (GCG) is similarly converted from proglucagon and binds to and activates the glucagon receptor to activate the corresponding signaling pathway, regulate gluconeogenesis and glycogenolysis to increase blood glucose, and maintain glucose levels in the blood. Glucagon has been shown in some studies to suppress appetite, reduce food intake, and at the same time has the effects of decomposing fat and reducing body weight. 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 that dual agonists of the GLP-1 receptor and the glucagon receptor produce anti-diabetic effects and significant weight loss effects by combining the actions of GLP-1 and glucagon within a single molecule.

[0036] Human Glucose-dependent Insulinotropic Polypeptide (GIP) Human Glucose-dependent Insulinotropic Polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic β-cells in the presence of glucose, protects pancreatic β-cells, and plays a physiological role in glucose homeostasis. The published patent CN201680005007.X shows that the compound LY3298176, a GLP-1 / GIP receptor dual agonist based on the natural GIP polypeptide sequence, has good effects on reducing blood glucose and body weight. Currently, 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 natural GIP and GLP-1 are rapidly inactivated by the commonly existing protease DPPIV, so they can only be used for short-term metabolic control. Existing technologies generally improve the half-life of polypeptides in the body by means of linking fatty acid chains to polypeptides and adding non-standard amino acids. For example, LY3298176 (Tirzepatide) is modified at the 20th lysine by a drug modification means of GLP-1, and the 2nd and 13th mutations are non-standard amino acid Aib.

[0038] The Incretin Analogue of the Present Invention As used herein, "the incretin analogue of the present invention", "the polypeptide having GLP-1 / GCG / GIP triple agonistic activity of the present invention", "the incretin analogue having GLP-1R / GIPR / GCGR triple agonistic activity", and "the polypeptide of the present invention" can be used interchangeably, and all refer to the polypeptide described in the first aspect of the present invention.

[0039] Specifically, the present invention obtained a polypeptide having triple agonistic activity for GLP-1 receptor, GIP receptor, and GCG receptor by screening. As used herein, "triple agonistic activity" refers to an incretin analog having activity for any of GLP-1, GIP, and glucagon receptors. In particular, because it has balanced activity in each receptor, the administered dose provided provides sufficient activity to provide the agonistic effect of the receptor in each receptor, and at the same time can avoid analogs with overly high unwanted side effects related to the activity. In addition, the incretin analog having triple agonist activity has an extended duration of action for any of GIP, GLP-1, and glucagon receptors, which is advantageous for low-frequency administration such as once a day, three times a week, twice a week, or once a week.

[0040] The structural features of the incretin analogs described herein result in analogs having sufficient activity for any of GIP, GLP-1, and glucagon receptors, obtaining the advantageous effect of activity (i.e., triple agonist activity) in each receptor, but the activity in any one receptor is not so high as to overwhelm the activity in the other two receptors, and when administered at a dose sufficient to produce activity in all three receptors, it does not lead to unwanted side effects.

[0041] The structural features of the incretin analogs described herein include further side-chain fatty acid modifications, promotion of optimal binding and potency in each receptor, improvement of its stability, reduction of its immunogenicity, and the like.

[0042] Preferably, the present disclosure first described an incretin analog or a pharmaceutically acceptable salt thereof, wherein the incretin analog comprises the following basic amino acid sequence. X1X2QGTFTSDYSILLDX 16 X 17 AAQAFIEX 25 LX 27 X 28 GGPSSGAPPPS (SEQ ID NO: 7) (However, X1 is His or Tyr, X2 is Aib, X 16 is Lys or complexed 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.)

[0043] Preferably, X1 is H, X2 is Aib, X 16 is K, X 17 is K, X 25 is W, X 27 is I, X 28 is A, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKIAAQAFIEWLIAGGPSSGAPPPS (SEQ ID NO: 8)

[0044] Preferably, X1 is H, X2 is Aib, X 16 is K, X 17 is K, X 25 is W, X 27 is I, X 28 is A, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKKAAQAFIEWLIAGGPSSGAPPPS (SEQ ID NO: 9)

[0045] Preferably, X1 is H, X2 is Aib, X 16 is K, X 17 is Q, X 25 is W, X 27 is I, X 28 is A, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKQAAQAFIEWLIAGGPSSGAPPPS (SEQ ID NO: 10)

[0046] Preferably, X1 is Y, X2 is Aib, and X 16 is K, and X 17 is I, and X 25 is W, and X 27 is I, and X 28 is A, and the incretin analog has the following amino acid sequence. Y-Aib-QGTFTSDYSILLDKIAAQAFIEWLIAGGPSSGAPPPS (SEQ ID NO: 11)

[0047] Preferably, X1 is H, X2 is Aib, and X 16 is K, and X 17 is I, and X 25 is Y, and X 27 is L, and X 28 is E, and the incretin analog has the following amino acid sequence. H-Aib-QGTFTSDYSILLDKIAAQAFIEYLLEGGPSSGAPPPS (SEQ ID NO: 12) Among them, the incretin analogs described in the present invention contain natural amino acids and unnatural amino acids, such as α-aminoisobutyric acid (Aib).

[0048] The incretin analogs described in the present invention have structural similarities with natural human peptides, and at the same time, there are also many structural differences. For example, modifications at position 2, position 16 or position 17: 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, for example, a fatty acid moiety conjugated to a natural or unnatural amino acid having a functional group capable of conjugation via a linker. Such conjugation is sometimes referred to as acylation. In some cases, the amino acid having a functional group capable of conjugation may be K, C, E, and D. In certain cases, the amino acid having a functional group capable of conjugation is K, and among them, it conjugates to the ε-amino group of the K side chain. Among them, the linker may be 1 to 4 amino acids, or aminopolyethylene carboxylic acid or a mixture thereof. When the linker is at least 1 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 in an in vivo animal model of the incretin analog, and the solubility and stability of the incretin analog. For example, C 16 -C 22 fatty acid moiety (e.g., C 16 -C 22 saturated monovalent or divalent fatty acid) can be mentioned.

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

[0051] In addition to 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 analogs described herein contain 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 analogs of the polypeptide of the present invention. As used herein, the terms "fragment", "derivative", and "analog" basically refer to polypeptides that maintain the triple agonistic activity of GLP-1R / GIPR / GCGR. Fragments, derivatives, and analogs of the polypeptide of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides in which one or more amino acid residues have substituents, or (iii) polypeptides formed 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 formed by fusing an additional amino acid sequence to this polypeptide (a polypeptide formed by fusing with a leader sequence, a secretion sequence, or a tag sequence such as 6His). Based on the disclosure herein, these fragments, derivatives, and analogs are within the scope known to those skilled in the art.

[0054] Some preferred active derivatives refer to polypeptides in which, when compared with the amino acid sequence of Formula I or Formula II, 5 or fewer, preferably 3 or fewer, more preferably 2 or fewer, and most preferably 1 amino acid is replaced by an amino acid having similar or close properties. These polypeptides with conservative mutations are preferably generated by performing amino acid substitutions as shown in Table A.

Table A

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

[0056] One preferred method is to use liquid-phase synthesis technology or solid-phase synthesis technology, such as the Boc solid-phase method, the Fmoc solid-phase method, or a combination of the two methods. Solid-phase synthesis enables rapid sample acquisition, and an appropriate resin support and synthesis system can be used according to the characteristics of the target peptide sequence. For example, in the Fmoc system, a suitable solid-phase support, such as Wang resin linked with the C-terminal amino acid in the peptide, has a polystyrene structure, an arm between the amino acid and 4-alkoxybenzyl alcohol, and the Fmoc protecting group is removed by treating with 25% hexahydropyridine / dimethylformamide at room temperature for 20 minutes, and then extended from the C-terminal to the N-terminal along the predetermined amino acid sequence. After the synthesis is completed, the proinsulin-related peptide synthesized with trifluoroacetic acid containing 4% p-cresol is cleaved from the resin to remove the protecting group. After removing the resin by filtration, precipitation and separation with ethyl ether can obtain the crude peptide. After freeze-drying the solution of the obtained product, the required peptide is purified by gel filtration and reverse-phase high-pressure liquid chromatography. When performing solid-phase synthesis in the Boc system, a suitable resin is PAM resin linked with the C-terminal amino acid in the peptide. The structure of PAM resin is polystyrene, and the arm between the amino acid and 4-hydroxymethylphenylacetamide. In the Boc synthesis system, in the cycle of deprotection, neutralization, and coupling, the protecting group Boc is removed with TFA / dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA) / dichloromethane. After the condensation of the peptide chain is completed, hydrogen fluoride (HF) containing p-cresol (5-10%) is used to treat at 0°C for 1 hour to cleave the peptide chain from the resin and simultaneously remove the protecting group. Extract with 50-80% acetic acid (containing a small amount of mercaptoethanol), freeze-dry the solution, and then separate and purify with molecular sieve Sephadex G10 or Tsk-40f, and further purify by high-pressure liquid phase to obtain the required peptide.Each amino acid residue can be coupled by various coupling agents and coupling methods known in the field of peptide chemistry. For example, it may be directly coupled with diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBt), or 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). For the short-chain peptides obtained by synthesis, their purity and structure can be confirmed by reverse-phase high-performance liquid chromatography and mass spectrometry.

[0057] In one preferred example, the polypeptide of the present invention is produced by the method of solid-phase synthesis according to its sequence, purified by high-performance liquid chromatography, and the freeze-dried powder of the target peptide with high purity is obtained and stored at -20°C.

[0058] Drug composition In addition, 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 polypeptide of the present invention (especially polypeptide 1, polypeptide 3), as well as a pharmaceutically acceptable carrier.

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

[0060] As used herein, the term "effective amount" or "effective dosage" refers to an amount that has a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0061] As used herein, a "pharmaceutically acceptable" component has no excessive adverse side effects (such as toxicity, irritation, and allergic reactions) when applied to humans and / or mammals, that is, a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent and includes various excipients and diluents.

[0062] The pharmaceutical 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, buffer solutions, glucose, water, glycerin, ethanol, and combinations thereof. Usually, since the pharmaceutical formulation should depend on the mode of administration, the pharmaceutical composition of the present invention may be an injection, and for example, it can be produced by a conventional method using an aqueous solution containing physiological saline or glucose and other adjuvants. The above-mentioned pharmaceutical composition is preferably produced under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulation of the present invention may be a sustained-release formulation.

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

[0064] The effective amount of the protein described in the present invention can be changed depending on the mode of administration, the severity of the disease to be treated, etc. The selection of a suitable effective amount can be determined by those skilled in the art based on various factors (for example, by clinical trials). The above-mentioned factors include, but are not limited to, the pharmacokinetic parameters, such as bioavailability, metabolism, half-life, etc., the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Usually, when the polypeptide of the present invention is administered at a dosage of 0.1-1 mg / kg body weight of the animal per day (preferably 0.3-0.6 mg / kg body weight of the animal), a satisfactory effect can be obtained. For example, depending on the urgency of the treatment situation, it can be administered in divided doses several times a day, or at a proportionally reduced dosage.

[0065] The incretin analogs in this specification may react with any of several inorganic and / or organic acids / bases to form medicinal acid / base addition salts (pharmaceutically acceptable salts). Pharmaceutically acceptable salts and their usual manufacturing 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)). 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 the present invention include the following. The present invention has the following advantages compared to the compounds described in the prior art: (1) Higher in vitro biological activity; (2) Higher stability in serum; (3) Fewer unnatural amino acids are used, the production cost is lower, and the immunogenicity in vivo is lower; (4) It has a better effect of reducing body weight and body fat percentage for compounds with GLP-1 / GIP dual activity.

[0067] Hereinafter, specific examples will be combined to further describe the present invention. It is understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which detailed conditions are not shown in the following examples usually follow normal conditions such as those described in, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or the recommended conditions of the manufacturer. Unless otherwise specified, % and parts are calculated by weight.

[0068] Example 1 Synthesis of Polypeptide The present invention manufactures polypeptides and their modified sites by a solid-phase polypeptide synthesis method and uses 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 with a substitution of 0.451 mmol / g was weighed into a clean and dry reaction tube, 5 mL of DMF was added, and it was immersed at room temperature for 2 h.

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

[0070] 1.3 Detection of the deprotection effect by ninhydrin A small amount of the resin was taken into a detection tube, washed twice with ethanol, 2 drops each of A: 20% ethanol + 80% phenol, B: redistilled pyridine, and C: 5% ninhydrin ethanol solution were added, heated at 110 °C for 2 min, and the color of the resin was observed. When it turned dark blue, it indicated complete deprotection, that is, the reaction of the next step could be carried out. When it was colorless, it indicated that the protecting group was not completely deprotected, and it was necessary to repeat the above deprotection operation.

[0071] 1.4 Coupling of the first amino acid At room temperature, the solvent from the previous step was removed by suction filtration through a sintered glass funnel. 3 equivalents of the first amino acid at the C-terminus (Fmoc-protected amino acid), 3 equivalents of HOBT, and 3 equivalents of DIC were added, and reacted at room temperature for 1 - 2 h using DMF as the solvent. A small amount of the resin was taken and detected with the ninhydrin detection reagent. When it was detected as transparent, it meant that the reaction was complete. After suction filtration of the solvent, it was washed 3 times with 5 mL of DMF.

[0072] 1.5 Sequential addition of the main-chain amino acids Similarly according to the above, the steps of 1.2 - 1.4 were repeated, and according to the polypeptide sequence, it was synthesized up to the last amino acid at the N-terminus to obtain Compound 1.

[0073] 1.6 Removal of the Dde protection A 5 mL of 3% hydrazine hydrate / DMF solution was added to the resin. After purging with nitrogen gas for 10 min, the solution was filtered off and the operation was repeated twice. Then, it was washed five times with 5 mL of DMF to obtain Compound 2.

[0074] 1.7 Sequential addition of side-chain amino acids Similarly according to the above, Steps 1.2 - 1.44 were repeated. According to the polypeptide sequence, synthesis was carried out up to the last amino acid, eicosanedioic acid mono-t-butyl ester, to obtain Compound 3. After suction drying, it was washed successively with 5 mL of DMF, 5 mL of DCM, and 5 mL of methanol. After vacuum drying, a crude resin product was obtained.

[0075] 1.8 Removal of resin and separation / detection of purified product Finally, cleavage was carried out with a trifluoroacetic acid cleavage solution (95% TFA: 2.5% TIS: 2.5% H2O) for 3 h. After suction filtration, the cleavage filtrate was collected and placed in methyl-t-butyl ether. The precipitate was obtained by centrifugation and the solid was collected. The precipitated crude product was washed three times with methyl-t-butyl ether. After vacuum drying, a crude product was obtained. It was desalted and purified by HPLC, and after lyophilization, a purified product was obtained. The molecular weight was measured by LC-MS. Here, the sequence of the chemically synthesized polypeptide is shown in Table 1.

Table 1

[0076] Example 2 Detection of in vitro activity The in vitro activity of GLP-1 of the polypeptide was detected using CHO-K1 cells expressing human GLP-1 receptor and CRE-luciferase. The cells were inoculated into a 96-well plate at a density of 30,000 cells / well / 100 μL and cultured at 37 °C under 5% CO2 for 1 hour. For each test protein sample, the protein solution was diluted 9 times from a certain concentration in a 4-fold gradient with PBS containing 0.25% FBS / 0.01% BSA. A total of 10 different concentrations of dilutions were taken from each sample, 50 μL each, and placed into the 96-well plate inoculated with cells, and cultured at 37 °C under 5% CO2 for 5 hours. The 96-well plate was taken out of the incubator and equilibrated at room temperature for 10 minutes. 100 μL of the reaction solution was added to each well, shaken at 200 rpm for 10 minutes, and then the fluorescence reading value was detected using a multifunctional plate reader. The concentration of the sample was plotted on the abscissa and the fluorescence reading value was plotted on the ordinate, and the half-maximal effective concentration (EC50) of each sample was calculated. GLP-1 was used as the positive control.

[0077] Reporter gene-containing CHO-K1 cells expressing GCG receptor and GIP receptor respectively were used to detect the GCG and GIP activities of the polypeptide. The operation process was the same as above, and glucagon and GIP were used as the positive controls respectively.

Table 2

[0078] Example 3 Biological Activities in Different Buffer Systems In the detection process, different buffer systems affect the binding degree between polypeptides and buffer components. Therefore, in order to more fully predict the in vivo and clinical activities of different species of polypeptides, the GLP-1, GCG, and GIP bioactivities of polypeptides in 0.01% casein, 2% mouse serum albumin, and 2% human serum albumin (HSA) systems were detected respectively. The operation process is the same as that in Example 2. Among them, in the human serum albumin system, mouse serum albumin, and casein system, polypeptide 1 had better GLP-1R activity than the control LY3437943. In the human serum albumin system, polypeptide 1 (SEQ ID NO: 1) was superior to the control LY3437943 in terms of GLP-1, GCG, and GIP activities (Table 3), and had 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 the control LY3437943 were diluted to 100 μg / ml with cynomolgus monkey serum respectively, and divided into three tubes each, 200 μl per tube. Among them, one sample was placed in an -80°C refrigerator as a zero control, and the other two samples were placed in a 37°C incubator. After 24 h and 72 h respectively, they were taken out and placed in an -80°C refrigerator. The GIP activity of the above samples was detected using CHO cells (CHOK1-GIPR) expressing human GIPR, and the activity changes of different samples after being placed for different times were compared.

[0080] Among them, since the stability of polypeptide 1 (SEQ ID NO: 1) in serum was higher than that of polypeptide 3 (SEQ ID NO: 3), it was suggested that the linkage site of the fatty acid chain in the polypeptide affects the stability of the polypeptide. LY3437943 has three non-natural amino acids, which mainly improve the stability of the polypeptide in serum and avoid enzymatic cleavage by endopeptidases. However, introducing a relatively large number of non-natural amino acids will increase the immunogenicity in vivo and at the same time lead to a higher production cost.

[0081] Surprisingly, Polypeptide 1 can significantly improve the stability of the polypeptide even in the screening of the amino acids at the fatty acid chain linkage site and different sites. In contrast, the polypeptide of the present invention has higher stability. In addition, since only one non-natural amino acid is introduced into the polypeptide of the present invention, the immunogenicity and production cost are lower. [Table 4]

[0082] Experiment on weight loss in DIO mice by multiple subcutaneous injections in Example 10 The purpose of this example is to study the effect of the polypeptide on indicators such as the body weight of DIO mice by subcutaneous injection, and at the same time to compare the drug efficacy with tirzepatide.

[0083] 7-week-old C57BL / 6Nju mice were used and fed a high-fat diet (protein accounted for about 20% and fat accounted for about 60%) for 12 weeks to construct a model. The feeding conditions were that the lighting was switched every 12h / 12h, free access to food, the temperature was 20-25°C, the relative humidity was 40-70%, and the ventilation rate was 10-15 times / hour. On the day before administration, the mice were randomly grouped according to body weight, water intake and food intake. The group design was the tirzepatide group (30 nmol / kg), the polypeptide 1 (30 nmol / kg), the normal control group, and the model control group, with 6 mice in each group. They were administered by subcutaneous injection, injected twice a week for 5 consecutive weeks, for a total of 10 administrations. After the administration, the body weight, OGTT, body fat ratio, blood fat, and liver function of the mice were detected. All the data were expressed as (X±s), and statistical analysis was performed on the data by one-way analysis of variance (One-Way ANOVA).

[0084] [Table 5]

[0085]

Table 6

[0086] From the results, polypeptide 1 can significantly reduce the body weight and body fat percentage of DIO mice, and the range of reduction is larger than that of the control tilzepatide. At the same time, it can significantly reduce blood glucose, total plasma cholesterol and low-density lipoprotein cholesterol, and improve liver function.

[0087] All documents related to the present invention are cited herein for reference as if each document were cited separately. After reading the above content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and it should be understood that those equivalent forms are included in 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 QGFTTSDYSILLDX １６ X １７ AAQAFIEX ２５ LX ２７ X ２８ GGPPSSGAPPPPS (SEQ ID NO: 7) (provided that X1 is His or Tyr, X ２ 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.) including the incretin analog is characterized by having an activity of simultaneously binding to and activating the class B G protein-coupled receptor GLP-1R and an activity of binding to the glucagon (GCG) receptor, the incretin analog or a pharmaceutically acceptable salt thereof.

2. The incretin analog according to claim 1, wherein the incretin analog further has an activity of binding to and activating the human glucose-dependent insulinotropic polypeptide (GIP) receptor.

3. X １７ The incretin analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein X is Lys modified with a fatty acid chain.

4. The incretin analog is HX ２ QGTTFTSDYSILLDX １６ IAAQAFIEX ２５ LX ２７ X ２８ GGPPSSGAPPPS (SEQ ID NO: 13) (provided that X ２ is Aib, X １６ is Lys or modified Lys, X ２５ is Trp or Tyr, X ２７ is Ile or Leu, X ２８ is Ala or Glu.) including the incretin analog has an activity of simultaneously binding to and activating the class B G protein-coupled receptor GLP-1R and an activity of binding to the glucagon (GCG) receptor, the incretin analog or a pharmaceutically acceptable salt thereof according to claim 1.

5. X １６ or X １７ each independently has the following structure: (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γE)b-CO-(CH2)c-CO2H (wherein a is 0, 1 or 2, b is 1 or 2, and c is an integer from 16 to 20). K which is chemically modified by complexing with the ε-amino group of the K side chain via, the retin analog according to claim 1.

6. The incretin analog is selected from the following group: (1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 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%, more preferably at least 99% homology with the sequence shown in SEQ ID NO: 8, 9, 10, 11 or 12, and having an activity of simultaneously binding to and activating the class B G protein-coupled receptor GLP-1R and an activity of binding to the glucagon (GCG) receptor The incretin analog according to claim 1, characterized by being selected from the group consisting of.

7. The incretin analog is selected from the following group: (1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6; A polypeptide derived from (1), wherein the amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, 5, or 6 has undergone substitution, deletion, or addition of 1-2 amino acid residues, and simultaneously has the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the activity of binding to the glucagon (GCG) receptor The incretin analog according to claim 1, characterized by having an amino acid sequence selected from the group consisting of

8. The incretin analog according to claim 1, characterized in that the incretin analog further has the activity of binding to and activating the human glucose-dependent insulinotropic polypeptide (GIP) receptor

9. A pharmaceutical composition comprising (I) the incretin analog according to claim 1, and (II) a pharmaceutically acceptable carrier The pharmaceutical composition, characterized by comprising

10. The pharmaceutical composition according to claim 9, characterized in that the pharmaceutical composition is for the prevention and / or treatment of diseases related to abnormal carbohydrate and lipid metabolism

11. The pharmaceutical composition according to claim 10, characterized in that the pharmaceutical composition further contains other drugs that can be used for the prevention and / or treatment of diseases related to abnormal carbohydrate and lipid metabolism

13. The other drugs that can be used for the prevention and / or treatment of diseases related to abnormal carbohydrate and lipid metabolism include, but are not limited to, metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase IV inhibitors, and sodium-glucose cotransporters, according to the pharmaceutical composition of claim 11

15. The diseases related to abnormal carbohydrate and lipid metabolism include type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia, according to the pharmaceutical composition of claim 10

17. Use of the incretin analog according to claim 1, comprising the following (a) lowering the blood glucose content of a subject in need (b) lowering the blood fat sugar content of a subject in need (c) lowering the body weight or body fat percentage of a subject in need; and / or (d) improving liver function; and / or (e) preventing and / or treating diseases related to abnormal carbohydrate and lipid metabolism The use, characterized by being used for

15. Use of the incretin analog according to claim 1 or the pharmaceutical composition according to claim 7 in the manufacture of a medicament 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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