GLP-1GIP analog, FGF21 protein, trifunctional protein, and use thereof

Abstract

Provided are a GLP-1GIP analog, an FGF21 protein, a trifunctional protein, and use thereof. The trifunctional protein comprises the GLP-1 / GIP analog and the FGF21 protein. Further provided is use of the GLP-1 / GIP analog, the FGF21 protein, and the trifunctional protein in the preparation of a drug for preventing and / or treating diseases related to GLP-1, GIP, and / or FGF21. The provided GLP-1 / GIP analog, FGF21 protein, and trifunctional protein can synergistically regulate blood glucose and lipid levels in vivo, meet multiple needs in patients with type 2 diabetes, including lowering blood glucose, alleviating hepatic steatosis, promoting weight loss, ameliorating circulating lipid metabolism disorder, etc., and have better biological safety and tolerability and longer in vivo half-lives.

Description

GLP-1 GIP analogue, FGF21 protein, trifunctional protein and application thereof

[0001] This application claims priority to Chinese patent application 2024104694852 with the filing date of 2024 / 4 / 18. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine. Specifically, the present application relates to a GLP-1 GIP analogue, FGF21 protein, trifunctional protein and application thereof, and also relates to the use of the trifunctional protein in the preparation of a medicament for treating type 2 diabetes, obesity, hyperlipidemia, fatty liver disease and / or metabolic syndrome. BACKGROUND

[0003] Glucagon-like peptide-1 (GLP-1) is a 36-amino acid incretin secreted by L cells in the mammalian intestine, which stimulates insulin secretion from pancreatic beta cells, suppresses glucagon release from pancreatic alpha cells, and exerts biological effects on glycemic control in a glucose-dependent manner by binding to and activating GLP-1 receptor (GLP-1R), and exhibits biological effects on inhibiting gastrointestinal motility and controlling appetite (Knudsen LB, J Med Chem, 2004, 4128-4134). Native human GLP-1 is easily inactivated by dipeptidyl peptidase IV (DDP-IV) in vivo, resulting in a short half-life. Exendin-4 is extracted from the saliva of the South African garter snake, has 39 amino acids, 53% homology with the human GLP-1 amino acid sequence, similar biological activity, but the second amino terminal of Exendin-4 is replaced by Gly instead of Ala in human GLP-1, which resists enzyme degradation by DPP-IV to some extent, thereby prolonging the circulating half-life in vivo. The carboxy terminal of Exendin-4 has a special Trap-cage structure, which makes its binding affinity to GLP-1 receptor significantly higher than that of human GLP-1 (Neidigh JW et al., Biochemistry, 2001, 40:13188-13200), and Exendin-4 exhibits stronger effects on promoting insulin release from pancreatic beta cells at equimolar concentrations. The two slightly different blood glucose metabolism regulators have been marketed, and the most representative ones are Novo Nordisk's Liraglutide and Novo Nordisk's Exenatide, which can effectively control the blood glucose level of type 2 diabetes patients by injecting 2-3 times a day. However, the high injection frequency leads to a substantial increase in patient treatment costs and low clinical compliance. In order to prolong the in vivo half-life and bioavailability of GLP-1 and Exendin-4 analogs, Fc fragment or HSA fusion technology has been applied to the development of such long-acting drugs. The currently marketed product is Liraglutide (Dulaglutide) from Lilly. The most prominent clinical performance is Dulaglutide, a GLP-1 hIgG4Fc fusion protein (Dulaglutide), which has a mean half-life of up to 90 hours (Chinese patent CN1802386B), and its clinical indications are type 2 diabetes, and the recommended use is subcutaneous injection once a week. Clinical studies have shown that Dulaglutide can effectively control postprandial blood glucose and glycosylated hemoglobin in diabetic patients, and reduce the body weight of obese patients by suppressing appetite, but there are varying degrees of gastrointestinal adverse reactions. Epidemiological surveys have shown that most type 2 diabetes patients are accompanied by non-alcoholic fatty liver disease and symptoms of lipid metabolism disorder (Radaelli MG et al., J Endocrinol Invest, 2017, s40618).Current clinical research results do not support GLP-1 or Exendin-4 analogues to have therapeutic effects on fatty liver and hyperlipidemia independent of weight loss (Petit JM, Diabetes Metab, 2017, 43, 2S28-2S33), so this class of products cannot fully meet the overall treatment needs of patients with type 2 diabetes.

[0004] Gut incretins are important hormones connecting the intestine with the brain and pancreas, and play a regulatory role in energy metabolism. Glucose-dependent insulinotropic polypeptide (GIP) is an important component of gut incretins, and cooperates with another member of gut incretins, glucagon-like peptide-1 (GLP-1), to achieve the regulation of energy metabolism. The half-life of GIP is <2 min in rodents, and about 7 and 5 min in healthy subjects and patients with type 2 diabetes mellitus (T2DM), respectively. The inactivation of GIP is mediated by proteolysis catalyzed by dipeptidyl peptidase-4, and is cleared by the kidney. Unlike GLP-1 receptors, GIP receptors are expressed in white adipose tissue, and play a regulatory role in the regulation of circulating lipids by white adipose tissue, which is equally important as the regulation of blood glucose in vivo. Current research suggests that GIP may regulate body weight by regulating the distribution and volume of adipose tissue. At the same time, GIP can directly promote the esterification of free fatty acids and the storage of triglycerides in adipose tissue, to achieve rapid clearance of triglycerides in circulation. The regulation of GIP on lipid storage may be achieved by affecting the activity of enzymes related to lipid metabolism. The addition of GIP in cultured preadipocytes in vitro can enhance lipoproteinase activity, and the clearance of triglyceride-rich proteins and chylomicrons from the circulation is helpful for lipid storage. In addition, GIP can also improve adipose tissue inflammation. After injection of GIP in diet-induced obese mice, the mRNA levels of chemokines and inflammatory cytokines can be significantly reduced. The synergistic effect of GIP and GLP-1 makes the treatment of GIP as a target combined with GLP-1 receptor agonists an important direction for drug research and development, including the combination of GLP-1 receptor agonists with GIP receptor agonists or antagonists. Tirzepatide is a diabetes treatment drug developed by Eli Lilly. It is a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor. Activation of GLP-1 receptor can reduce hunger, thereby reducing food and calorie intake, while activation of GIP receptor can reduce food and calorie intake and increase energy expenditure.

[0005] The fibroblast growth factors (FGFs) family has 22 members, 7 subfamilies, among which the FGF19 subfamily exerts physiological activity in an endocrine manner, participates in the regulation of energy and bile acid homeostasis, glucose and lipid metabolism, phosphate and vitamin D homeostasis (Moore DD et al., Science, 2007, 316: 1436-1438 and Beenken et al., Nature Reviews Drug Discover, 2009, 8: 235). FGF21 is one of the members of the FGF19 subfamily, with 182 amino acids. In vivo, the carboxy terminus of FGF21 first binds to the auxiliary factor β-Klotho transmembrane protein, and then the amino terminus binds to FGFR, forming a stable FGF21 / β-Klotho / FGFR complex, which activates downstream related signaling molecules (Yie J et al., FEBS Lett, 2009, 583(1): 19-24 and Micanovic R et al., J Cell Physiol, 2009, 219(2): 227-234). The physiological activity of FGF21 is manifested as insulin-independent promotion of glucose utilization function (Kharitonenkov A et al., J Clin Invest, 2005, 115(6): 1627-1635), insulin sensitization (Duthchak PA et al., Cell, 2012, 148, 387-393), inhibition of liver lipid neogenesis, promotion of liver fatty acid β oxidation, and reduction of serum triglyceride levels (Xu J et al., Diabetes, 2009, 58, 250-259); by inhibiting liver SREBP-2 synthesis, reducing serum total cholesterol and low-density lipoprotein content, thereby relieving hypercholesterolemia (Lin Z et al., Circulation, 2015, 131, 1861-1871).

[0006] In summary, FGF21 shows beneficial metabolic regulation for metabolic diseases such as obesity, type 2 diabetes, non-alcoholic fatty liver and hyperlipidemia. At the same time, FGF21 is the only cytokine in the FGF family that has not been found to have mitogenic effect, greatly reducing the possible carcinogenicity in clinical use (Wu X et al., Proc Natl Acad Sci USA, 2010, 170: 14158-14163). However, due to its own physicochemical properties defects, it is difficult to develop natural FGF21 into a therapeutic biological agent, the main reasons including: 1. FGF21 protein stability is poor, easy to be hydrolyzed by protease; 2. FGF21 conformation is unstable, easy to aggregate, increasing the difficulty of large-scale production of FGF21; 3. The half-life of natural FGF21 is short, the half-life of human FGF21 in mice is 0.5-1 hour, and the half-life in cynomolgus monkeys is 2-3 hours (Kharitonenkov A et al., J Clin Invest, 2005, 115: 1627-1635). At present, a variety of protein long-acting technologies have been applied to prolong the in vivo half-life of recombinant FGF21. For example, FGF21 is linked with PEG molecules to increase the molecular weight, reduce the glomerular filtration rate, and prolong the in vivo retention time (see patents WO2005 / 091944, WO2006 / 050247, WO2008 / 121563 and WO2012 / 066075); FGF21 is fused with long-chain fatty acids (which can bind to serum albumin) (see WO2010 / 084169 and WO2012 / 010553); or prepare agonist antibodies that can specifically bind to FGFR or FGFR / β-klotho complex to mimic the mechanism of action of FGF21 to activate the FGF / FGFR signaling pathway (see WO2011 / 071783, WO2011 / 130417, WO2012 / 158704 and WO2012 / 170438); or by fusion with Fc fragment can also improve the half-life of FGF21 (see WO2004 / 110472, WO2005 / 113606, WO2009 / 149171, WO2010 / 042747, WO2010 / 129503, WO2010 / 129600, WO2013 / 049247, WO2013 / 188181 and WO2016 / 114633). There is no FGF21 long-acting protein on the market at present, but there are three long-acting FGF21 proteins LY2405319 of Lilly, PF-05231023 of Pfizer and BMS986036 of Bristol-Myers Squibb in the clinical trial stage.LY2405319 and PF-05231023 only show weight loss and serum TG reduction in type 2 diabetes patients, and do not show positive therapeutic effects on glycemic control (Gaich G et al., Cell Metab, 2013, 18:333-340 and Dong JQ et al., Br J Clin Pharmacol, 2015, 80-1051-1063). BMS986036 shows good therapeutic effects in a clinical study on non-alcoholic fatty liver, but it has not been tested for glycemic control in type 2 diabetes patients. The above results show that the use of FGF21 long-acting protein alone can show multiple pharmacodynamic activities such as weight loss, treatment of non-alcoholic fatty liver and hyperlipidemia, but cannot meet the most critical requirement of glycemic control in the treatment of type 2 diabetes patients.

[0007] Recent studies have reported that the combination of GLP-1 and FGF21 has a synergistic effect on glycemic control. For example, CN102802657A discloses that the combination of GLP-1 and FGF21 can synergistically reduce the blood glucose level of db / db mice. However, the combination of drugs not only increases the frequency of drug administration for patients and reduces the compliance of patients to treatment, but also greatly increases the cost of treatment. In addition, there have been reports of bifunctional proteins prepared by fusing GLP-1 and FGF21. In order to solve the problem of easy degradation of FGF21 in vivo, researchers have introduced corresponding mutations into the natural FGF21 molecule, but this will inevitably increase the potential immunogenicity of the bifunctional protein (WO2017 / 074123 and CN104024273B). In addition, the observed synergistic effect of GLP-1 and FGF21 is mainly reflected in the control of blood glucose, and there is a lack of comparative study of the therapeutic effects of GLP-1 and FGF21 on other metabolic diseases such as obesity, non-alcoholic fatty liver and lipid metabolism disorders. There is no report on the combination of GLP-1 / GIP and FGF21.

[0008] The lack of the above studies may include the following reasons: (1) natural GLP-1, GIP or FGF21 is not stable in vivo, and any one of the three molecules cannot maintain structural integrity and stability in vivo, and cannot produce a synergistic effect in function; (2) in the process of fusing GLP-1 / GIP and FGF21 into a single protein, it is necessary to maintain the three-dimensional conformation of each other to the greatest extent without interfering with each other, so as to realize the functional synergy, which must be carefully treated at the molecular design level; (3) the functions of GLP-1 / GIP and FGF21 depend on the binding of their respective receptors, and it is necessary to verify a large number of in vitro and in vivo tests under what conditions the dynamic balance is achieved, and there is no related description in the published patents or other non-patent literature.

[0009] In summary, if the GLP-1 / GIP-FGF21 and its variant synergistic three-function protein drugs with enhanced stability can be developed in the art, the effects of prolonged half-life and low immunogenicity can also meet the multiple needs of many type 2 diabetes patients to solve the problems of blood glucose reduction, alleviation of liver steatosis, weight loss and improvement of circulating lipid metabolism disorders. SUMMARY

[0010] In order to solve the defects of unstable structure, short in vivo half-life and the like of natural GLP-1, GIP and FGF21 in the prior art, retain the physiological effects of GLP-1 / GIP on strong blood glucose reduction and FGF21 on insulin sensitization, weight loss, fatty liver and hypercholesterolemia treatment, and to some extent alleviate the technical problem of gastrointestinal adverse reactions caused by GLP-1, the present application provides a GLP-1 GIP analogue, FGF21 protein, three-function protein and application thereof. The present application aims to provide a three-function protein comprising human GLP-1 / GIP analogue and FGF21 and its variant, which has synergistic effects in blood glucose and lipid regulation, a preparation method and use thereof.

[0011] To solve the above technical problems, one of the technical solutions provided by the present application is a GLP-1 / GIP analogue, which comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0012] In a specific embodiment of the present application, the amino acid sequence of the GLP-1 / GIP analogue is as shown in SEQ ID NO: 1.

[0013] In a specific embodiment of the present application, the GLP-1 / GIP analogue is fused with a polypeptide stabilizing fragment, preferably the polypeptide stabilizing fragment is fused to the C-terminal end of the GLP-1 / GIP analogue.

[0014] In a specific embodiment of the present application, the polypeptide stabilizing fragment is an Fc fragment, preferably a human Fc fragment.

[0015] In a specific embodiment of the present application, the Fc fragment is from immunoglobulin IgG, IgM or IgA or their variants, preferably from IgG1, IgG2, IgG3 or IgG4 or their variants.

[0016] In the present application, the "variant" of the Fc fragment of immunoglobulin refers to some Fc variants known in the art by mutating or modifying at least one amino acid in the wild-type Fc fragment of immunoglobulin, and the formed Fc variant has no cleavage and shows minimal Fc-mediated adverse side effects (ADCC and CDC effects) and / or enhanced binding affinity to FcRn receptor. For example, in a specific embodiment of the present application, the Fc fragment comprises the amino acid sequence as shown in SEQ ID NO: 9 (which belongs to an IgG Fc variant, IgG2 / 4Fc).

[0017] In a specific embodiment of the present application, the GLP-1 / GIP analogue is connected to the polypeptide stabilizing fragment by a first connecting peptide.

[0018] In the present application, the first connecting peptide is preferably non-immunogenic and generates sufficient distance between the GLP-1 / GIP analogue and the polypeptide stabilizing fragment to minimize the steric effect between each other so as not to affect or seriously affect the correct folding and spatial conformation of the GLP-1 / GIP analogue and the polypeptide stabilizing fragment. The connecting peptide can be designed by a person skilled in the art according to the conventional method in the art.

[0019] In a specific embodiment of the present application, the first connecting peptide is a flexible peptide and is selected from the following amino acids: Gly (G), Ser (S), Ala (A) and Thr (T); preferably, the first connecting peptide comprises G and S residues.

[0020] In a specific embodiment of the present application, the first connecting peptide is preferably a flexible peptide containing two or more than two amino acids, for example, 5-30 amino acids; the first connecting peptide more preferably comprises the amino acid sequence as shown in SEQ ID NO: 2.

[0021] To solve the above technical problems, the second technical solution provided by the present application is an FGF21 protein, which comprises the amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0022] In a specific embodiment of the present application, the amino acid sequence of the FGF21 protein is as shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0023] To solve the above technical problems, the third technical solution provided by the present application is a trifunctional protein, which comprises the GLP-1 / GIP analogue and the FGF21 protein as described in any one of the technical solutions of the present application.

[0024] In a specific embodiment of the present application, the trifunctional protein comprises a GLP-1 / GIP analogue as described in one of the technical solutions of the present application, a polypeptide stabilizing fragment as defined in the GLP-1 / GIP analogue as described in one of the technical solutions of the present application (the defined range can include the relevant description of the connection order of the polypeptide stabilizing fragment and the GLP-1 / GIP analogue, the type of the polypeptide stabilizing fragment and / or the first connection peptide between the polypeptide stabilizing fragment and the GLP-1 / GIP analogue in one of the technical solutions of the present application), and an FGF21 protein.

[0025] In a specific embodiment of the present application, the trifunctional protein comprises a GLP-1 / GIP analogue as described in one of the technical solutions of the present application, a polypeptide stabilizing fragment as defined in the GLP-1 / GIP analogue as described in one of the technical solutions of the present application (the defined range can include the relevant description of the connection order of the polypeptide stabilizing fragment and the GLP-1 / GIP analogue, the type of the polypeptide stabilizing fragment and / or the first connection peptide between the polypeptide stabilizing fragment and the GLP-1 / GIP analogue in one of the technical solutions of the present application), and an FGF21 protein.

[0026] In a specific embodiment of the present application, the polypeptide stabilizing fragment and the FGF21 protein are connected by a second connection peptide.

[0027] In the present application, the "first connection peptide" and the "second connection peptide" are only convenient to understand to distinguish them as different connection peptides, and do not impose any additional restrictions on the two connection peptides themselves.

[0028] In the present application, the second connection peptide is preferably non-immunogenic, and generates sufficient distance between the polypeptide stabilizing fragment and the FGF21 protein to minimize the steric hindrance effect between them, so as not to affect or seriously affect the correct folding and spatial conformation of the polypeptide stabilizing fragment and the FGF21 protein. The skilled person can design the connection peptide according to the conventional method in the art.

[0029] In a specific embodiment of the present application, the second connection peptide is a flexible peptide, and is selected from the following amino acids: Gly (G), Ser (S), Ala (A) and Thr (T); preferably, the second connection peptide comprises G and S residues.

[0030] In a specific embodiment of the present application, the second connection peptide is preferably a flexible peptide containing two or more than two amino acids, for example, 5-30 amino acids; the second connection peptide more preferably comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0031] In a specific embodiment of the present application, the FGF21 protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 3-7.

[0032] In a specific embodiment of the present application, the trifunctional protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 10-14.

[0033] In a specific embodiment of the present application, the trifunctional protein comprises a glycosylation modification. A person skilled in the art can achieve glycosylation by expressing in a mammalian cell, preferably by expressing in a Chinese hamster ovary cell, by a routine method.

[0034] To solve the above technical problem, the sixth technical solution of the present application provides a transformant comprising the nucleic acid according to the fourth technical solution of the present application or the recombinant expression vector according to the fifth technical solution of the present application.

[0035] According to the amino acid sequence of the present application, a person skilled in the art can easily prepare the coding nucleic acid of the present application by various known methods, such as but not limited to PCR, DNA artificial synthesis, etc. The specific method can be found in J. Sambrook, Molecular Cloning: A Laboratory Manual. As an embodiment of the present application, the coding nucleic acid sequence of the present application can be constructed by the method of synthesizing nucleotide sequence in segments and then performing overlap extension PCR.

[0036] To solve the above technical problem, the sixth technical solution of the present application provides a transformant comprising the nucleic acid according to the fourth technical solution of the present application or the recombinant expression vector according to the fifth technical solution of the present application.

[0037] The recombinant expression vector of the present application comprises the nucleic acid according to the fourth technical solution of the present application and an expression control sequence operatively linked thereto. The "operatively linked" refers to a condition that some parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, if a promoter control sequence controls transcription, it is operatively linked to the coding sequence.

[0038] In a specific embodiment of the present application, the backbone of the recombinant expression vector is pDNA3.4, pIRES, pDR or pUC18. The backbones of these recombinant expression vectors are commercially available, and a person skilled in the art can select a suitable recombinant expression vector according to the host cell.

[0039] According to the known restriction map of the empty expression vector (backbone), a person skilled in the art can insert the nucleic acid according to the fourth technical solution of the present application into a suitable restriction site by restriction enzyme cleavage and splicing according to a routine method, to prepare the recombinant expression vector of the present application.

[0040] To solve the above technical problem, the sixth technical solution of the present application provides a transformant comprising the nucleic acid according to the fourth technical solution of the present application or the recombinant expression vector according to the fifth technical solution of the present application.

[0041] In a specific embodiment of the present application, the host cell of the transformant is a eukaryotic cell.

[0042] In a specific embodiment of the present application, the eukaryotic cell is a mammalian cell, preferably a CHO cell such as CHO K1 cell, COS cell, 293 cell or RSF cell, more preferably the CHO cell is deficient in DHFR enzyme.

[0043] The introduction of the nucleic acid or the recombinant expression vector into the host cell can be performed using a variety of known techniques in the art, such as but not limited to: calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, retroviral transduction and alkali metal ion method.

[0044] To solve the above technical problems, the seventh technical solution of the present application provides a method for preparing the GLP-1 / GIP analogue according to the first technical solution of the present application, the FGF21 protein according to the second technical solution of the present application or the trifunctional protein according to the third technical solution of the present application, which comprises culturing the transformant according to the sixth technical solution of the present application, and obtaining the target product from the culture.

[0045] The target product according to the present application is generally prepared by a biosynthetic method. The culture and expression of the host cell can refer to Olander RM Dev Biol Stand, 1996, 86: 338. The cells and residues in the suspension can be removed by centrifugation, and the supernatant can be collected. The identification can be performed by agarose gel electrophoresis technology.

[0046] The target product can be purified to be substantially uniform in nature, such as a single band on SDS-PAGE electrophoresis. For example, when the target protein is expressed by secretion, a commercial ultrafiltration membrane can be used to separate the protein, such as the products of Millipore, Pellicon and the like, and the expression supernatant is first concentrated. The concentrated solution can be further purified by gel chromatography or purified by ion exchange chromatography. For example, anion exchange chromatography (DEAE, etc.) or cation exchange chromatography. The gel matrix can be agarose, dextran, polyamide and the like commonly used for protein purification. Q- or SP- groups are more ideal ion exchange groups. Finally, the above purified product can be further refined and purified by hydroxyapatite adsorption chromatography, metal chelation chromatography, hydrophobic interaction chromatography and reverse phase high performance liquid chromatography (RP-HPLC) and the like. All the above purification steps can be combined in different combinations, and finally the purity of the protein reaches substantially uniform.

[0047] The expressed product of interest can be purified using an affinity column containing a specific antibody, receptor or ligand for the product of interest. Depending on the nature of the affinity column used, the product of interest bound to the affinity column can be eluted using conventional methods, such as high salt buffer, change of pH, etc. Alternatively, the amino or carboxyl terminus of the product of interest can also contain one or more polypeptide fragments as a protein tag. Any suitable tag can be used in the present application. For example, the tag can be FLAG, HA, HA1, c-Myc, 6-His or 8-His, etc. These tags can be used for purification of the product of interest.

[0048] To solve the above technical problem, the eighth technical solution of the present application provides a pharmaceutical composition comprising the GLP-1 / GIP analogue according to the first technical solution of the present application, the FGF21 protein according to the second technical solution of the present application, or the trifunctional protein according to the third technical solution of the present application, and a pharmaceutically acceptable carrier such as an excipient and / or a diluent.

[0049] To solve the above technical problem, the ninth technical solution of the present application provides use of the GLP-1 / GIP analogue according to the first technical solution of the present application, the FGF21 protein according to the second technical solution of the present application, the trifunctional protein according to the third technical solution of the present application, the nucleic acid according to the fourth technical solution of the present application, the recombinant expression vector according to the fifth technical solution of the present application, the transformant according to the sixth technical solution of the present application, or the pharmaceutical composition according to the eighth technical solution of the present application in the preparation of a medicament for preventing and / or treating a disease related to GLP-1, GIP and / or FGF21.

[0050] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0051] In specific embodiments of the present application, the disease includes metabolic syndrome, endocrine abnormality and cardiovascular and cerebrovascular disease.

[0052] In specific embodiments of the present application, the disease includes hyperglycemia, type 1 or type 2 diabetes, hyperlipidemia, obesity, metabolic syndrome caused by obesity, non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis or cirrhosis induced by liver steatosis, hypercholesterolemia and hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis induced by the same, and pancreatitis, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, acute myocardial infarction, peripheral arterial disease, stroke, heart failure, kidney disease, diabetic complications, neuropathy, gastroparesis, and a condition associated with a severe inactivating mutation of the insulin receptor.

[0053] To solve the above technical problems, the tenth technical solution of the present application provides a method for preventing and / or treating a disease related to GLP-1, GIP and / or FGF21, which comprises administering to a subject in need a therapeutically effective amount of the GLP-1 / GIP analogue according to any one of the technical solutions of the present application, the FGF21 protein according to the second technical solution of the present application, the trifunctional protein according to the third technical solution of the present application, or the pharmaceutical composition according to the eighth technical solution of the present application.

[0054] In the present application, the term "effective amount" refers to the amount of a drug or a pharmaceutical agent that elicits the biological or medicinal response of a tissue, system, animal or human that is being sought by a researcher or clinician. Furthermore, the term "therapeutically effective amount" refers to the amount that causes an improved treatment, cure, prevention or lessening of a disease, disorder or side effect, or a rate of progression of a disease or condition to be reduced, as compared to a corresponding subject who does not receive the amount. The term also includes within its scope an amount effective to enhance normal physiological function.

[0055] In specific embodiments of the present application, the disease includes metabolic syndrome, endocrine abnormality and cardiovascular and cerebrovascular diseases.

[0056] In specific embodiments of the present application, the disease includes hyperglycemia, type 1 or type 2 diabetes, hyperlipidemia, obesity, metabolic syndrome caused by obesity, non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis or cirrhosis induced by liver steatosis, hypercholesterolemia and hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis induced thereby, and pancreatitis, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, acute myocardial infarction, peripheral arterial disease, stroke, heart failure, nephropathy, diabetic complications, neuropathy, gastroparesis and a condition associated with a severe inactivating mutation of the insulin receptor.

[0057] To solve the above technical problems, the eleventh technical solution of the present application provides the GLP-1 / GIP analogue according to any one of the technical solutions of the present application, the FGF21 protein according to the second technical solution of the present application, the trifunctional protein according to the third technical solution of the present application, or the pharmaceutical composition according to the eighth technical solution of the present application for use in preventing and / or treating a disease related to GLP-1, GIP and / or FGF21.

[0058] In specific embodiments of the present application, the disease includes metabolic syndrome, endocrine abnormality and cardiovascular and cerebrovascular diseases.

[0059] In specific embodiments of the present application, the diseases include hyperglycemia, type 1 or 2 diabetes, hyperlipidemia, obesity, metabolic syndrome caused by obesity, non-alcoholic fatty liver induced by liver steatosis, non-alcoholic steatohepatitis, liver fibrosis or cirrhosis, hypercholesterolemia and hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis induced thereby, and pancreatitis, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, acute myocardial infarction, peripheral arterial disease, stroke, heart failure, kidney disease, diabetic complications, neuropathy, gastroparesis, and conditions associated with severe inactivation mutations of the insulin receptor.

[0060] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining preferred embodiments of the present application.

[0061] The reagents and raw materials used in the present application are commercially available.

[0062] The positive progress effect of the present application is that:

[0063] 1. The GLP-1 / GIP analog of the present application has the functions of reducing fat synthesis and promoting fat metabolism and utilization in vivo, and can be applied to the treatment of obesity and metabolic syndrome caused by obesity, and is superior to Tirzepatide in the prior art.

[0064] 2. Compared with the existing products, the synergistic trifunctional protein of the present application, such as HT-201 (G1-G5), has the following outstanding advantages:

[0065] ①It shows good therapeutic effect on diseases related to GLP-1, GIP and / or FGF21, such as type 2 diabetes, obesity, hyperlipidemia, fatty liver disease and / or metabolic syndrome, etc.

[0066] ②Better biological safety and tolerance. Tirzepatide administration can induce severe gastrointestinal adverse reactions. In high-fat diet-induced obese mice, the food intake of mice administered with HT-201 is significantly higher than that of mice administered with Tirzepatide, indicating that the synergistic trifunctional protein HT-201 can effectively alleviate the anorexia symptoms induced by gastrointestinal adverse reactions.

[0067] ③Prolonged half-life in vivo, maintaining therapeutic activity for a longer period of time. The trifunctional protein provided in the present application fuses a polypeptide stabilizing fragment such as an Fc fragment, which not only has a significantly prolonged circulating half-life in vivo, but also has a synergistic effect on blood glucose and lipid regulation, i.e., the trifunctional protein constructed in the present application well maintains the functions of active molecules and has strong stability. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 shows a schematic diagram of the protein structure of the trifunctional protein HT-201 of the present application, which is composed of GLP-1 / GIP analogue, L1, Fc fragment, L2 and FGF21, and the trifunctional protein HT-201 can form stable dimers through disulfide bond linkage of Fc hinge region.

[0069] Figure 2a is a reduced SDS-PAGE electropherogram of GLP-1 / GIP-FGF21 trifunctional protein HT-201 (wherein M lane is Marker, R lane is protein reduction, and N-R lane is non-protein reduction).

[0070] Figure 2b is a SEC-HPLC profile of GLP-1 / GIP-FGF21 trifunctional protein HT-201.

[0071] Figure 3 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on cumulative food intake of high-fat diet-induced obese mice (means ± SEM, n = 8).

[0072] Figure 4 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on glycosylated hemoglobin value of high-fat diet-induced obese mice (means ± SEM, n = 8).

[0073] Figure 5 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on body weight of high-fat diet-induced obese mice (means ± SEM, n = 8).

[0074] Figure 6 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on liver weight of high-fat diet-induced obese mice (means ± SEM, n = 8); statistical difference marked notes: compared with the tirzepatide group, *P < 0.05, **P < 0.01.

[0075] Figure 7 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on liver triglyceride and total cholesterol content of high-fat diet-induced obese mice (means ± SEM, n = 8); statistical difference marked notes: compared with the tirzepatide group, **P < 0.05, **P < 0.01.

[0076] Figure 8 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on serum ALT and AST content of high-fat diet-induced obese mice (means ± SEM, n = 8); statistical difference marked notes: compared with the tirzepatide group, *P < 0.05, **P < 0.01.

[0077] Figure 9 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on the total cholesterol content in serum of high-fat diet-induced obese mice (means ± SEM, n = 8); statistical difference marked notes: compared with the tirzepatide group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0078] Figure 10 is the effect of GLP-1 / GIP-FGF21 trifunctional protein HT-201 on the total cholesterol content of HDL and LDL in serum of high-fat diet-induced obese mice (means ± SEM, n = 8); statistical difference marked notes: compared with the tirzepatide group, *P < 0.05, **P < 0.01.

[0079] Figure 11 is the effect of GLP-1 / GIP analogues of the present application on the body weight of high-fat diet-induced obese mice (means ± SEM, n = 8); compared with the tirzepatide group, the body weight level of obese mice in the GLP-1 / GIP analogue group was significantly reduced (P < 0.01). DETAILED DESCRIPTION

[0080] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. And, the molecular genetic, nucleic acid chemistry, chemical, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc. The operation steps used herein are conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows:

[0081] GLP-1 / GIP analogue

[0082] The term "GLP-1 / GIP analogue" used herein, i.e. the analogue of glucagon-like peptide-1 and enterogastrone, refers to the analogue, fusion peptide and derivative of wild-type human GLP-1 / GIP which can maintain the activity of human GLP-1 / GIP obtained by substitution, deletion or addition of one or more amino acid residues. For example, the GLP-1 / GIP analogue includes but is not limited to the amino acid sequence as shown in SEQ ID NO: 1 in the sequence listing of the present application.

[0083] FGF21 and its variants

[0084] The term "FGF21 and variants thereof" as used herein refers to variants, fusion peptides and derivatives of the amino acid sequence of wild-type human FGF21 (human fibroblast growth factor 21) obtained by substitution, deletion or addition of one or several amino acid residues, which are capable of maintaining the activity of human FGF21. For example, the FGF21 and variants thereof include, but are not limited to, the amino acid sequences as shown in SEQ ID NOs: 3 to 7 in the sequence listing of the present application.

[0085] Disease associated with GLP-1, GIP and / or FGF21

[0086] The term "disease associated with GLP-1, GIP and / or FGF21" refers to a disease or condition caused by a deficiency of GLP-1, GIP and / or FGF21 in the body of a patient or a short half-life of GLP-1, GIP and / or FGF21, or an abnormality in the metabolic regulation pathway mediated by the receptors of GLP-1, GIP and / or FGF21, GLP-1R, GIPR and / or FGFR, including but not limited to obesity, type 1 and type 2 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, nephropathy, diabetic complications, neuropathy, gastroparesis, conditions associated with severe inactivating mutations of the insulin receptor, and the like.

[0087] "Conditions associated with severe inactivating mutations of the insulin receptor" describes conditions in subjects with mutations in the insulin receptor (or a protein directly downstream of it) that result in severe insulin resistance, but typically without the obesity that is common in type 2 diabetes. In many aspects, subjects with these conditions exhibit a combination of symptoms of type 1 and type 2 diabetes. Thus, affected subjects are generally classified into several categories in increasing order of severity, including: diabetes mellitus resistant type A, insulin resistance type C (AKA HAIR-AN syndrome), Rabson-Mendenhall syndrome, Donohue's syndrome or Leprechaunism. These conditions are associated with very high endogenous insulin levels, leading to elevated blood glucose levels. Thus, affected subjects also exhibit a variety of clinical features associated with "insulin toxicity", including hyperandrogenism, polycystic ovary syndrome (PCOS), hirsutism and acanthosis nigricans (excessive growth of skin creases and pigmentation).

[0088] "Diabetic complications" are dysfunction in other parts of the body caused by chronic high blood glucose, such as diabetic nephropathy, diabetic neuropathy, diabetic foot (foot ulcers and poor circulation), and ocular disorders (retinopathy). Diabetes also increases the risk of heart disease, as well as bone and joint disorders. Other long-term complications of diabetes include skin, digestive, sexual, and dental-gum problems.

[0089] "Metabolic syndrome" (MS) is a pathological state of clustering of multiple metabolic abnormalities, including: (1) abdominal obesity or overweight; (2) atherogenic dyslipidemia, such as hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C); (3) hypertension; (4) insulin resistance and / or glucose intolerance. Some criteria also include microalbuminuria, hyperuricemia, and increased proinflammatory (C-reactive protein) and prothrombotic (fibrinogen and plasminogen activator inhibitor-1) states.

[0090] "Dyslipidemia" is a disorder of lipoprotein metabolism, including overproduction or defects. Dyslipidemia can be manifested as elevated concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides in the blood, and decreased concentrations of high-density lipoprotein (HDL) cholesterol.

[0091] "Non-alcoholic fatty liver disease" (NAFLD) is a liver disease not associated with alcohol consumption, characterized by hepatocellular steatosis.

[0092] "Non-alcoholic steatohepatitis" (NASH) is a liver disease not associated with alcohol consumption, characterized by hepatocellular steatosis, accompanied by inflammation and fibrosis within the lobule.

[0093] "Atherosclerosis" is a vascular disease characterized by irregular deposits of lipids in the intima of large and medium-sized arteries, leading to narrowing of the arterial lumen, and eventually to fibrosis and calcification.

[0094] The present application is further described in connection with the following specific examples. It should be understood that these examples are intended to illustrate the application and are not intended to limit the scope of the application. The experimental methods in the following examples, where not otherwise specified, were generally performed according to routine conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0095] Example 1: Construction of Synergistic Tri-functional Protein Expression Plasmid

[0096] The synergistic trifunctional protein of the present application comprises, from N-terminus to C-terminus, GLP-1 / GIP analogue, L1, Fc fragment, L2 and FGF21, and its structure is shown in Figure 1.

[0097] wherein the gene sequences of GLP-1 / GIP analogue (SEQ ID NO: 1), L1 (SEQ ID NO: 2), FGF21 and its variants (SEQ ID NO: 3-7), L2 (SEQ ID NO: 8) and Fc fragment (human IgG Fc variant, SEQ ID NO: 9) are all artificially optimized CHO cell preferred codons, and the full-length sequences are obtained by chemical synthesis method.

[0098] GLP-1 / GIP analogue (SEQ ID NO: 1)

[0099] L1 (SEQ ID NO: 2)

[0100] FGF21-G1 (SEQ ID NO: 3)

[0101] FGF21-G2 (SEQ ID NO: 4)

[0102] FGF21-G3 (SEQ ID NO: 5)

[0103] FGF21-G4 (SEQ ID NO: 6)

[0104] FGF21-G5 (SEQ ID NO: 7)

[0105] L2 (SEQ ID NO: 8)

[0106] Human IgG Fc variant (SEQ ID NO: 9)

[0107] The present application constructs a plurality of recombinant expression vector plasmids of synergistic trifunctional proteins containing GLP-1 / GIP and FGF21 (the backbone of the recombinant expression vector is pDNA3.4, which is a conventional commercially available product in the art), and five of them, HT-201 (G1-G5), are exemplarily illustrated, and their amino acid compositions are as follows:

[0108] G1 (SEQ ID NO: 10):

[0109] G2 (SEQ ID NO: 11):

[0110] G3 (SEQ ID NO: 12):

[0111] G4 (SEQ ID NO: 13):

[0112] G5 (SEQ ID NO: 14):

[0113] wherein the GLP-1 / GIP analog is in bold, the Linker (L1 and L2) is in italics, and the FGF21 is underlined.

[0114] Example 2: Expression of the synergistic tri-functional protein in transfected cell lines

[0115] The recombinant expression vector plasmids constructed in Example 1 were transfected into a mammalian host cell line to express the synergistic tri-functional protein. To achieve stable high level expression, the preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Patent No. 4,818,679), and in this example the host cell is the CHO K1 cell line (CHO K1 cell line provided by BBI, source ECACC global sub-license). One preferred method of transfection is electroporation, but other methods can be used, including calcium phosphate co-precipitation, lipofection. In electroporation, 50 μg of highly purified expression plasmid was added to 5 x 10 7 cells in a cuvette with a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, CA) set at 300 V electric field and 1500 μFd capacitance. Two days after transfection, the culture medium was changed to growth medium containing 0.6 mg / mL G418. Transfectants resistant to the selection drug were screened by anti-human IgG Fc ELISA assay. Anti-human FGF21 or anti-human GLP-1 / GIP ELISA can also be used to quantify the expression level of the synergistic tri-functional protein. The wells producing high level of the synergistic tri-functional protein were subcloned by limiting dilution 96-well plates.

[0116] To achieve a high level of expression of the synergistic trifunctional protein, it is appropriate to use the DHFR gene inhibited by MTX for co-amplification. The synergistic trifunctional protein gene transfected by co-amplification of the DHFR gene is used in a growth medium containing an increasing concentration of MTX. The subclone positive for DHFR expression is subjected to extreme dilution, gradually increased pressure and screening of transfected cells capable of growing in a medium containing up to 6 μM MTX, and determination of the secretion rate, and screening of cell lines with high expression of foreign proteins. Cell lines with a secretion rate of more than about 10 (preferably about 20) μg / 10 6 (i.e. million) cells / 24 hours are subjected to adaptive suspension culture in serum-free medium, and then the synergistic trifunctional protein is purified using conditioned medium.

[0117] Example 3: Purification and qualification of the synergistic trifunctional protein

[0118] In this example, the purification and qualification method of HT-201 is described. The cell culture supernatant is subjected to clarification treatment such as high-speed low-temperature centrifugation and 0.22 μm sterilization filtration, and then purified by protein A affinity, anion exchange and hydrophobic three-step chromatography. The specific method is as follows: the first step of capture uses protein A affinity chromatography, the equilibrium liquid is PBS buffer, and the eluent is citrate buffer at pH 3.5, and the eluted target protein is then neutralized with 1M Tris solution. The intermediate purification selects high-resolution anion exchange filler Q Sepharose HP (GE Company) to remove residual impurity proteins. The binding mode is used, and the elution is performed using 20 mM Tris-HCl, 0.2M NaCl, pH 7.5 solution. The elution is performed using 20 mM Tris-HCl, 0.3M NaCl, pH 7.5 solution. The final purification step selects Butyl Sepharose FF (GE Company) to remove polymers. The hydrophobic properties of HT-201 monomers and polymers are different, the monomers with weak hydrophobicity directly flow through, and the polymers with strong hydrophobicity are bound to the medium. The flow-through mode of hydrophobic chromatography is selected, and the equilibrium liquid is PBS buffer.

[0119] The results of the qualitative analysis of the target protein product are shown in Figures 2a and 2b. The theoretical molecular weight of HT-201 single chain is about 53KD. Under reducing conditions, SDS-PAGE electrophoresis shows that the actual size of HT-201 single chain molecule is about 70KD due to the glycosylation site. The same method is used to obtain the target protein.

[0120] Example 4: Experimental study on the therapeutic effect of the synergistic trifunctional protein on obesity and fatty liver and lipid metabolism disorder induced by high-fat diet in obese mice

[0121] 8-week-old C57BL / 6 mice were purchased from Shanghai Slac Laboratory Animal Co. Ltd. and raised in an environment with a temperature of 22-25°C, a relative humidity of 45-65%, and a lighting time of 12 h / d. After 1 week of adaptive feeding, the mice were fed with high-fat feed (D12450B, Research diets). After 40 weeks, the obese mice were adaptively fed for 1 week, three per cage, and then randomly divided into the following groups according to the body weight: a vehicle group, an FGF21 group (Efruxifermin, which is an Fc-FGF21 fusion protein, CAS No. 2375240-92-7), a tirzepatide group, and HT-201 groups (G1-G5) (n=8). The mice in the vehicle group were subcutaneously injected with a PBS buffer solution, and the mice in each administration group were subcutaneously injected with 10 nmol / kg of a corresponding drug solution, twice a week, for a total of 12 administrations. The body weight, food intake, and blood glucose of each mouse were recorded before and after the experiment. After the last administration period, the mice in each group were fasted for 16 hours, and whole blood was taken from the eye orbit, centrifuged at 2000 x g for 15 min, and serum was separated. Serum biochemistry was detected by an automatic biochemical analyzer. The liver tissue was separated, washed with normal saline, and dried with filter paper, and the weight was measured. About 50 mg of liver tissue was taken from the same part, and the triglyceride content in the liver tissue was detected by the Folch method. The total cholesterol content in the liver tissue was detected by using a cholesterol (TC) content detection kit (Solebao: BC1985). The data were expressed in the form of means ± standard error (means ± SEM), and the data were analyzed by using SPSS 18.0 statistical software. For normal distribution, the difference between the means of multiple groups was analyzed by one-way analysis of variance, the LSD test was used for variance homogeneity, the Dunnet T3 test was used for variance heterogeneity; for non-normal distribution, a non-parametric test was used, and P<0.05 indicated a statistically significant difference.

[0122] As shown in FIG. 3, for high-fat feed-induced obese mice, the cumulative food intake of the mice was significantly reduced after treatment with tirzepatide (P<0.01). This indicates that tirzepatide can cause severe gastrointestinal adverse reactions and has a certain central nervous system appetite-inhibiting effect, thereby greatly reducing the food intake of animals. The results in this example show that the weight loss, liver steatosis relief, and lipid metabolism disorder regulation effects of tirzepatide are basically dependent on its inhibition of food intake, and do not have other independent treatment mechanisms.

[0123] As shown in FIG. 4, compared with the tirzepatide group, the glycosylated hemoglobin value of the obese mice in the HT-201 group was significantly reduced (P<0.01), indicating that HT-201 has a blood glucose-lowering function and can be applied to the treatment of hyperglycemia, type 1 or type 2 diabetes.

[0124] As shown in Figure 5, compared with the tirzepatide group, the body weight of the obese mice in the HT-201 group was significantly reduced (P<0.01), indicating that HT-201 has the function of reducing fat synthesis and promoting fat metabolism and utilization in vivo, and can be applied to the treatment of hyperlipidemia, obesity and metabolic syndrome caused by obesity.

[0125] As shown in Figures 6-8, compared with the tirzepatide group, the liver weight (Figure 6) and the contents of triglyceride and cholesterol in the liver (Figure 7) and the contents of AST and ALT in the serum (Figure 8) of the mice in the HT-201 group were significantly reduced (P<0.01 or P<0.05), indicating that HT-201 can effectively reduce the excessive deposition of fat in liver tissue, restore liver function, and suggest that HT-201 can be applied to the treatment of various liver diseases such as non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis induced by liver steatosis.

[0126] As shown in Figures 9-10, compared with the tirzepatide group, the contents of total cholesterol (Figure 9) and low-density lipoprotein (Figure 10) in the serum of the mice in the HT-201 group were significantly reduced (P<0.01 or P<0.05), suggesting that HT-201 can be applied to the treatment of various cardiovascular and cerebrovascular diseases such as hypercholesterolemia, hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis induced by hypercholesterolemia.

[0127] The above research results show that HT-201 can treat obesity, fatty liver disease and lipid metabolism disorder through the physiological activity of GLP-1 / GIP and FGF21 synergistic effect, not completely dependent on the food intake regulation effect of GLP-1 / GIP analogs, and the therapeutic effect of HT-201 is the most prominent, which is significantly better than tirzepatide, and can fill the gap in the clinical treatment of tirzepatide. In summary, the therapeutic targets of HT-201 are more abundant than tirzepatide, and more in line with the needs of clinical diversified treatment.

[0128] Example 5: The GLP-1 / GIP analog of the present application has a better therapeutic effect on obesity, fatty liver and lipid metabolism disorder in high-fat diet-induced obese mice than tirzepatide

[0129] 8-week-old C57BL / 6 mice were purchased from Shanghai Slac Laboratory Animal Co. Ltd. and were raised in an environment with a temperature of 22-25℃, a relative humidity of 45-65%, and a lighting time of 12h / d. After 1 week of adaptive feeding, the mice were fed with the same amount of high-fat feed (D12450B, Research diets). After 40 weeks, the obese mice were adaptively fed for 1 week, three per cage, and then randomly divided into groups according to body weight: a vehicle group, a tirzepatide group, and a GLP-1 / GIP analogue (SEQ ID NO: 1) group (n=8). The vehicle group was subcutaneously injected with a PBS buffer solution, and each administration group was subcutaneously injected with 10 nmol / kg of a corresponding drug solution. The administration was performed once every 7 days, and a total of 6 administrations were performed. The body weight and food intake of each mouse before and after the experiment were recorded. The data were expressed as means ± SEM, and the data were analyzed using SPSS 18.0 statistical software. For normally distributed data, the differences between the means of multiple groups were analyzed using one-way ANOVA, the LSD test was used for variance homogeneity, and the Dunnet T3 test was used for variance inhomogeneity. For non-normally distributed data, non-parametric tests were used. P<0.05 was considered to be statistically significant.

[0130] As shown in FIG. 11, compared with the tirzepatide group, the body weight level of the obese mice in the GLP-1 / GIP analogue group was significantly reduced (P<0.01), indicating that the GLP-1 / GIP analogue has the functions of reducing fat synthesis and promoting fat metabolism and utilization in vivo, and can be applied to the treatment of obesity and metabolic syndrome caused by obesity, and is superior to the tirzepatide group.

[0131] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. In addition, it should be understood that various modifications and changes can be made to the present application by those skilled in the art upon reading the above description of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. A GLP-1 / GIP analogue, characterized in that, The amino acid sequence of the GLP-1 / GIP analogue is shown as SEQ ID NO: 1; Preferably, the GLP-1 / GIP analogue is fused with a polypeptide stabilizing fragment, preferably the polypeptide stabilizing fragment is fused to the C-terminal end of the GLP-1 / GIP analogue; More preferably, the polypeptide stabilizing fragment is an Fc fragment, preferably a human Fc fragment; the Fc fragment is preferably from immunoglobulin IgG, IgM or IgA or their variants, more preferably from IgG1, IgG2, IgG3 or IgG4 or their variants; further more preferably, the Fc fragment comprises an amino acid sequence shown as SEQ ID NO: 9; Further more preferably, the GLP-1 / GIP analogue is connected with the polypeptide stabilizing fragment by a first connecting peptide; the first connecting peptide is preferably a flexible peptide containing two or more than two amino acids; the first connecting peptide more preferably comprises an amino acid sequence shown as SEQ ID NO:

2.

2. An FGF21 protein, characterized in that, The amino acid sequence of the FGF21 protein is shown as SEQ ID NO: 6 or SEQ ID NO:

7.

3. A tri-functional protein, characterized in that, The trifunctional protein comprises the GLP-1 / GIP analogue as claimed in claim 1, and the FGF21 protein; preferably further comprises the polypeptide stabilizing fragment as defined in the GLP-1 / GIP analogue as claimed in claim 1; Preferably, the trifunctional protein comprises, from N-terminal to C-terminal, the GLP-1 / GIP analogue, the polypeptide stabilizing fragment and the FGF21 protein in sequence; More preferably, the polypeptide stabilizing fragment and the FGF21 protein are connected by a second connecting peptide; the second connecting peptide is preferably a flexible peptide containing two or more than two amino acids; the second connecting peptide more preferably comprises an amino acid sequence shown as SEQ ID NO:

8.

4. The tri-functional protein of claim 3, wherein, The FGF21 protein comprises an amino acid sequence shown as any one of SEQ ID NO: 3-7; Preferably, the trifunctional protein comprises an amino acid sequence shown as any one of SEQ ID NO: 10-14; More preferably, the trifunctional protein comprises a glycosylation modification.

5. An isolated nucleic acid, comprising, The nucleic acid encodes the GLP-1 / GIP analogue as claimed in claim 1, the FGF21 protein as claimed in claim 2, or the trifunctional protein as claimed in claim 3 or 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as claimed in claim 5; preferably, the backbone of the recombinant expression vector is pDNA3.4, pIRES, pDR or pUC18.

7. A transformant characterized in that, The transformant comprises the nucleic acid as claimed in claim 5 or the recombinant expression vector as claimed in claim 6; Preferably, the host cell of the transformant is a eukaryotic cell; More preferably, the eukaryotic cell is a mammalian cell, preferably a CHO cell such as CHO K1 cell, COS cell, 293 cell or RSF cell, more preferably the CHO cell is deficient in DHFR enzyme.

8. A method of preparing a GLP-1 / GIP analog of claim 1, an FGF21 protein of claim 2, or a trifunctional protein of claim 3 or 4, characterized in that, The method comprises culturing the transformant as claimed in claim 7, and obtaining the target product from the culture.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the GLP-1 / GIP analogue of claim 1, the FGF21 protein of claim 2, or the trifunctional protein of claim 3 or 4, and a pharmaceutically acceptable carrier.

10. Use of the GLP-1 / GIP analogue of claim 1, the FGF21 protein of claim 2, the trifunctional protein of claim 3 or 4, the nucleic acid of claim 5, the recombinant expression vector of claim 6, the transformant of claim 7, or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing and / or treating a disease associated with GLP-1, GIP and / or FGF21. Preferably, the disease comprises metabolic syndrome, endocrine abnormality and cardiovascular and cerebrovascular diseases. More preferably, the disease comprises hyperglycemia, type 1 or 2 diabetes, hyperlipidemia, obesity, obesity-induced metabolic syndrome, liver steatosis-induced non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis or cirrhosis, and hypercholesterolemia and hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis induced thereby. Preferably, the disease comprises metabolic syndrome, endocrine abnormality and cardiovascular and cerebrovascular diseases. More preferably, the disease comprises hyperglycemia, type 1 or 2 diabetes, hyperlipidemia, obesity, obesity-induced metabolic syndrome, liver steatosis-induced non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis or cirrhosis, and hypercholesterolemia and hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis induced thereby.

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