Polypeptide compounds and their use in preventing or treating diabetes or diabetic complications

Abstract

The present invention discloses a polypeptide compound and its application in preventing or treating diabetes or diabetic complications. The polypeptide compound shows good long-term hypoglycemic effect and therapeutic effect of diabetic nephropathy; at the same time, it has the effects of high enzymatic stability, high biological activity, no adverse reactions, etc., and can be used to prepare drugs for treating overeating, obesity and overweight, elevated cholesterol, diabetes and diabetic nephropathy.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical technology, and in particular, relates to a class of polypeptide compounds and their application in preventing or treating diabetes or diabetic complications. Background Art

[0002] In recent years, with the rapid development of the economy, changes in dietary structure and lifestyle, and the acceleration of population aging, the prevalence of diabetes in the world has also shown a rapid increase. According to statistics, nearly 9% of adults worldwide suffer from type II diabetes. According to the International Diabetes Federation (IDF), the global prevalence of diabetes will increase from 415 million people in 2015 to 642 million people in 2040 (Unnikrishnan R, Pradeepa R, Joshi SR, Mohan V. Type 2 Diabetes: Demystifying the Global Epidemic. Diabetes, 2017. 66: 1432-1442). Diabetes has become another important chronic non-communicable disease that seriously endangers human health after cardiovascular and cerebrovascular diseases and tumors.

[0003] Many surveys in recent years have shown that the control of diabetes is not optimistic, whether in developed countries in Europe and America or developing countries such as China. One of the most serious consequences of diabetes is the development of diabetic vascular disease, which clinically manifests as microvascular and macrovascular complications. Diabetic nephropathy is a microvascular complication and the most common cause of end-stage renal disease in developed countries.

[0004] About 40% of patients with type I diabetes will develop diabetic nephropathy. Although the number of cases of type II diabetic nephropathy developing into end-stage renal disease is relatively small, the rapid increase in the number of patients with type II diabetes means that if there is a lack of effective measures for diabetes management, the incidence of diabetic nephropathy will also increase significantly. Therefore, effective and long-acting hypoglycemic drugs are urgently needed for the treatment of diabetic nephropathy. About 40% of patients with type I diabetes develop diabetic nephropathy. Due to the considerable racial variability in the incidence of diabetic nephropathy, only about one-third of patients with type I diabetes develop diabetic nephropathy, which is greatly related to genetic factors. Although the number of cases of type 2 diabetic nephropathy developing into end-stage renal disease is relatively small, the number of type 2 diabetes patients is increasing rapidly, which means that if there is a lack of effective measures for diabetes management, the incidence of diabetic nephropathy will also increase significantly. Therefore, there is an urgent need for effective and long-acting hypoglycemic drugs for the treatment of diabetic nephropathy (Chen L, Magliano DJ, Zimmet PZ. The worldwide epidemiology of type 2 diabetes mellitus—present and future perspectives. Nat Rev Endocrinol, 2011, 8(4): 228-236).

[0005] With the in-depth study of diabetes and its treatment, new drug targets for the treatment of type II diabetes are constantly being discovered. The GLP-1 analogs that have been on the market since 2005 (Meier JJ. GLP-1 Receptor Agonists for Individualized Treatment of Type-2 Diabetes Mellitus. Nat. Rev. Endocrinol, 2012. 8 (12): 728) have different targets from previous drugs, providing new treatment options for the treatment of diabetes. Currently, the GLP-1 analogs that have been approved for the treatment of type II diabetes include exenatide, liraglutide and lixisenatide, as well as the subsequent albiglutide and dulaglutide. In addition, Novo Nordisk's once-weekly long-acting GLP-1 analog semaglutide has also been marketed for the treatment of type II diabetes.

[0006] Compared with single-target agonists of homologous ligands, GLP-1R / GCGR dual agonists have synergistic effects of lowering blood sugar, lowering lipids, and reducing body weight. In addition, the synergistic effect of GLP-1R / GCGR dual agonists, GCGR and GLP-1, can enhance the function of pancreatic β-cells, while potentially minimizing or preventing the progression of type II diabetes. These innovations increase the established effects of GLP-1, and the second activity may add more efficacy, such as: weight reduction, cardiovascular benefits, and treatment of diabetic nephropathy.

[0007] For example, Chinese patent CN201711194175.0 discloses a glucagon analog for treating metabolic diseases. However, studies have found that the effect of the glucagon analog in improving glucose tolerance is comparable to that of liraglutide, but is inferior to that of semaglutide in improving glucose tolerance. The glucagon analog also cannot meet the requirements for clinical use.

[0008] In summary, although there are many drugs for the treatment of diabetes, the treatment of type II diabetes and related complications of kidney disease is still challenging, and curbing the global spread of diabetic nephropathy is destined to be a long and arduous task. The development of new dual-target or multi-target agonist peptide drugs is the main direction for the treatment of diabetes and its complications. Summary of the invention

[0009] The purpose of the present invention is to provide a new type of polypeptide compound. The inventors of the present invention have proved through a large number of experimental studies that the new type of polypeptide compound has a longer half-life, has insulinotropic activity, has no adverse reactions, and can be used for the prevention or treatment of diabetic complications such as diabetes and diabetic nephropathy.

[0010] Another object of the present invention is to provide the use of the novel polypeptide compound, which can potentially be used as a new generation of drugs for preventing or treating diabetes and diabetic nephropathy and other diabetic complications, and can also be used for lowering blood sugar or reducing body weight.

[0011] To achieve the above object, the technical solution of the present invention is as follows:

[0012] The present invention provides a polypeptide compound, which contains a parent peptide represented by the following amino acid sequence:

[0013] His-Xaa2-Gln-Gly-Thr 5 -Phe-Thr-Ser-Asp-Lys 10 -Ser-Lys-Tyr-Leu-Xaa15 15 -Xaa16-Xaa17-Ala-Ala-Gln 20 -Xaa21-Phe-Xaa23-Xaa24-Trp25 -Leu-Xaa27-Xaa28-Gly-Gly 30 -Pro-Ser-Ser-Gly-Xaa35 35 -Pro-Pro-Pro-Ser,

[0014] in:

[0015] Xaa2=Aib, Ser or D-Ser;

[0016] Xaa15 = Asp or Glu;

[0017] Xaa16 = Aib or Glu;

[0018] Xaa17 = Lys or Arg;

[0019] Xaa21 = Asp or Glu;

[0020] Xaa23=Val or Iva;

[0021] Xaa24=Glu or Gln;

[0022] Xaa27 = Leu or Lys;

[0023] Xaa28 = Asp, Glu or Ala;

[0024] Xaa35 = Ala or Aib.

[0025] Preferably, in the amino acid sequence of the parent peptide, Xaa2 = Aib or D-Ser.

[0026] Preferably, in the amino acid sequence of the parent peptide, Xaa21 = Asp.

[0027] Preferably, the carboxyl terminus of the amino acid sequence of the parent peptide is not modified or is modified with the amino group to form a -CONH2 group.

[0028] Furthermore, preferably, the side chain of the 10th or 12th Lys in the amino acid sequence of the parent peptide is connected to the lipophilic substituent via a bridging group, and the bridging group is (PEG) m , (PEG) m -γGlu, (PEG) m -Asp, (Gly) x -(Gly-Ser) y -(Gly) z -, (Gly) x -(Gly-Ser) y -(Gly) z -γGlu and (Gly)x -(Gly-Ser) y -(Gly) z The connection method is: the side chain amino group of the 10th or 12th Lys and the carboxyl group of the glycine residue at one end of the bridging group or (PEG) m The terminal modified carboxyl group forms an amide bond to connect to the parent peptide; at the same time, the lipophilic substituent is connected to the amino group of the bridging group at the other end by forming an amide bond with its carboxyl group. The lipophilic substituent is CH3(CH2) n C(O)- or HOOC(CH2) n C(O)- and its acyl group forms an amide bond with the amino group in the bridging group.

[0029] In the bridging group described in the present invention, m is an integer of 2-10; x is an integer of 0-5; y is an integer of 1-5; z is an integer of 1-5; in the lipophilic substituent described in the present invention, n is an integer of 14-20.

[0030] Alternatively, Lys at position 10 or 12 in the amino acid sequence of the parent peptide may be replaced by HomoLys, Orn, Dap or Dab.

[0031] Further preferably, in the amino acid sequence of the parent peptide:

[0032] Xaa2=Aib or D-Ser;

[0033] Xaa15 = Glu;

[0034] Xaa16 = Aib;

[0035] Xaa17 = Lys;

[0036] Xaa21 = Asp;

[0037] Xaa23 = Val;

[0038] Xaa24 = Glu;

[0039] Xaa27 = Lys;

[0040] Xaa28 = Ala;

[0041] Xaa35=Ala;

[0042] or,

[0043] Xaa2=Aib or D-Ser;

[0044] Xaa15=Asp;

[0045] Xaa16 = Glu;

[0046] Xaa17=Arg;

[0047] Xaa21 = Asp;

[0048] Xaa23 = Iva;

[0049] Xaa24=Gln;

[0050] Xaa27=Leu;

[0051] Xaa28 = Asp or Glu;

[0052] Xaa35 = Ala or Aib.

[0053] According to a specific embodiment of the present invention, the amino acid sequence of the parent peptide is selected from the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16.

[0054] According to a specific embodiment of the present invention, the structure formed by connecting the 10th or 12th Lys, preferably the 10th Lys, in the amino acid sequence of the parent peptide to the lipophilic substituent via a bridging group is:

[0055]

[0056]

[0057] According to a specific embodiment of the present invention, the present invention provides any of the following polypeptide compounds:

[0058] Compound 1:

[0059] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLE-Aib-KAAQDFVEWLKAGGGPSSGAPPPS-NH2

[0060] Compound 2:

[0061] HsQGTFTSDK(PEG2-PEG2-CO(CH2) 18CO2H)HOLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0062] compound 3:

[0063] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CO2H)HOLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0064] Compound 4:

[0065] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 16 CO2H)HOLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0066] Compound 5:

[0067] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CH3)HOLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0068] compound 6:

[0069] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0070] Compound 7:

[0071] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CO2H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH2

[0072] Compound 8:

[0073] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0074] Compound 9:

[0075] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 16 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0076] compound 10:

[0077] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CH3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0078] compound 11:

[0079] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CH3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0080] compound 12:

[0081] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2 18 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0082] compound 13:

[0083] HsQGTFTSDK(GGSGSG-γGlu-CO(CH2 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS

[0084] compound 14:

[0085] HsQGTFTSDK(GGSGSG-γGlu-CO(CH2 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0086] compound 15:

[0087] H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH2 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS

[0088] Compound 16:

[0089] H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH2) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGPSSG-Aib-PPPS-NH2.

[0090] The present invention also provides a composition comprising the polypeptide compound of the present invention.

[0091] Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier or excipient.

[0092] The present invention further provides a use of the polypeptide compound or the composition in the preparation of a drug for preventing or treating diabetes and / or diabetic complications; wherein preferably, the diabetic complication is diabetic nephropathy.

[0093] The present invention further provides an application of the polypeptide compound or the composition in preparing a health product or medicine for reducing body weight.

[0094] The technical solution of the present invention is described in detail as follows.

[0095] The first aspect of the present invention is to provide a novel agonist polypeptide compound, which contains a parent peptide represented by the following amino acid sequence:

[0096] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys-Ser-Lys-Tyr-Leu-Xaa15-Xaa16-Xaa17-Ala-Ala-Gln-Xa a21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly-Xaa35-Pro-Pro-Pro-Ser-COR1

[0097] Wherein, R1=-NH2 or -OH;

[0098] Xaa2=Aib, Ser or D-Ser;

[0099] Xaa15 = Asp or Glu;

[0100] Xaa16 = Aib or Glu;

[0101] Xaa17 = Lys or Arg;

[0102] Xaa21 = Asp or Glu;

[0103] Xaa23=Val or Iva;

[0104] Xaa24=Glu or Gln;

[0105] Xaa27 = Leu or Lys;

[0106] Xaa28 = Asp, Glu or Ala;

[0107] Xaa35 = Ala or Aib.

[0108] In the amino acid sequence of the parent peptide, the side chain of the Lys at position 10 or 12 is connected to the lipophilic substituent via a bridging group.

[0109] The bridging group comprises (PEG) m , or (PEG) m -γGlu or (PEG) m -Asp; may also contain (Gly) x -(Gly-Ser) y -(Gly) z -, or (Gly) x -(Gly-Ser) y -(Gly) z -γGlu or (Gly) x -(Gly-Ser) y -(Gly) z The connection method is: the side chain amino group of the 10th or 12th Lys and the carboxyl group of the glycine residue at one end of the bridging group or (PEG) m The terminal modified carboxyl group forms an amide bond to connect to the parent peptide; at the same time, the lipophilic substituent is connected to the amino group of the bridging group at the other end by forming an amide bond. The 10th or 12th Lys connected to the lipophilic substituent can be replaced by HomoLys, Orn, Dap or Dab. Wherein m is an integer of 2-10; x is an integer of 0-5; y is an integer of 1-5; z is an integer of 1-5. The lipophilic substituent is selected from CH3(CH2) n CO- or HOOC(CH2) n CO-, where n is an integer from 14 to 20. For details on the connection method, see Figure 6 and Figure 7 , Figure 6 is an exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention, Figure 7 is another exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention.

[0110] Throughout the specification of this application, conventional three-letter codes are used to represent natural amino acids, and recognized three-letter codes are used to represent other amino acids, such as Aib (aminoisobutyric acid), Orn (ornithine). The compounds of the present invention are based on the theory that the intramolecular bridge can stabilize the helical structure of the molecule, thereby improving the efficacy and / or selectivity for the glucagon-like peptide 1 (GLP-1R) receptor and the glucagon (GCGR) receptor.

[0111] The compounds of the present invention are based on the theory that the lipophilic substituents can bind to albumin in the blood, thereby protecting the compounds of the present invention from enzymatic degradation, thereby increasing the half-life of the compounds.

[0112] Another aspect of the present invention is to provide a pharmaceutical composition containing the novel agonist polypeptide compound of the present invention, wherein the pharmaceutical composition is prepared by adding the novel agonist polypeptide compound as an active ingredient to a pharmaceutically acceptable carrier and / or excipient.

[0113] Another aspect of the present invention is to provide the medical use of the novel agonist polypeptide compound of the present invention. Cell and animal experiments show that the novel agonist polypeptide compound of the present invention has a long-lasting hypoglycemic effect and can be used as a drug for treating diabetes.

[0114] Another aspect of the present invention is to provide other medical uses of the novel agonist polypeptide compound of the present invention. Cell and animal experiments show that the novel agonist polypeptide compound of the present invention has a pharmacodynamic effect in treating diabetic complications such as diabetic nephropathy. Animal experiments show that the therapeutic effect is better than that of the marketed GLP-1 analog semaglutide, and can be used as a drug for treating diabetic complications such as diabetic nephropathy.

[0115] The novel agonist polypeptide compound of the present invention can also reduce body weight and has potential use as a drug for treating obesity.

[0116] The parent peptide in the novel agonist polypeptide compound mentioned in the present invention is a homologous polypeptide. The homologous polypeptide in the present invention means that the polypeptide originally has the amino acid sequence of oxyntomodulin (OXM), glucagon-like peptide (GLP-1), exenatide (Exenatide) or glucagon (Glucagon), but one or more amino acid residues therein have been replaced by different amino acid residues, these amino acid residues are conserved with each other, and the obtained polypeptide can be used to implement the present invention.

[0117] The polypeptide compounds of the present invention can also be used to prevent weight gain or promote weight loss. The polypeptide compounds can cause a decrease in food intake and / or an increase in energy expenditure, so that an observable effect on body weight is produced. Therefore, the polypeptide compounds of the present invention can be used to directly or indirectly treat any condition caused by or characterized by overweight, such as treating and / or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, and sleep apnea caused by obesity. The effect of the polypeptide compounds of the present invention in these conditions may be caused by or related to the effect of the polypeptide compounds on body weight, or may be independent of its effect on body weight.

[0118] Those skilled in the art will appreciate that the pharmaceutical composition of the present invention is suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. According to the mode of administration adopted, the pharmaceutical composition of the polypeptide compound of the present invention can be prepared into various suitable dosage forms, which contain at least one effective dose of the polypeptide compound of the present invention and at least one pharmaceutically acceptable pharmaceutical carrier. Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions that can be used for injection.

[0119] The pharmaceutical composition containing the polypeptide compound of the present invention can be prepared into a solution or lyophilized powder for parenteral administration. Before use, an appropriate solvent or other pharmaceutically acceptable carrier can be added to reconstitute the powder. Liquid formulas are generally buffer solutions, isotonic solutions and aqueous solutions.

[0120] The dosage of the pharmaceutical composition of the present invention can vary within a wide range, and those skilled in the art can easily determine it based on objective factors such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, etc.

[0121] Compared with the prior art, the present invention has the following advantages:

[0122] 1) Compared with natural OXM at the same mass number, the polypeptide compound of the present invention has better biological activity;

[0123] 2) Compared with GLP-1 analogs at the same mass number, the polypeptide compound of the present invention has better pharmacodynamic effect in treating diabetic complications such as diabetic nephropathy;

[0124] 3) The drug showed significantly prolonged half-life and stability in pharmacokinetic experiments;

[0125] 4) The synthesis yield is high, the stability is good, it is easy to scale up the production, and the cost is low.

[0126] In a specific embodiment, the present invention relates to the following novel agonist polypeptide compounds, which have the following sequence and side chain modification results:

[0127] Compound 1 (SEQ ID NO.1):

[0128] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0129] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLE-Aib-KAAQDFVEWLKAGGGPSSGAPPPS-NH2

[0130] Compound 2 (SEQ ID NO. 2):

[0131] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0132] HsQGTFTSDK(PEG2-PEG2-CO(CH2) 18 CO2H)SKYLE-Aib-KAAQDFVEWLKAGGGPS SGAPPPS-NH2

[0133] Compound 3 (SEQ ID NO.3):

[0134] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0135] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0136] Compound 4 (SEQ ID NO.4):

[0137] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0138] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0139] Compound 5 (SEQ ID NO.5):

[0140] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CH3)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0141] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18(CH3)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0142] Compound 6 (SEQ ID NO.6):

[0143] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0144] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPS SG-Aib-PPPS-NH2

[0145] Compound 7 (SEQ ID NO.7):

[0146] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0147] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH2

[0148] Compound 8 (SEQ ID NO.8):

[0149] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0150] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0151] Compound 9 (SEQ ID NO.9):

[0152] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0153] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0154] Compound 10 (SEQ ID NO.10):

[0155] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CH3)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0156] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18CH3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0157] Compound 11 (SEQ ID NO.11):

[0158] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2) 18 CH3)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0159] H-Aib-QGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CH3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0160] Compound 12 (SEQ ID NO.12):

[0161] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0162] HsQGTFTSDK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH2

[0163] Compound 13 (SEQ ID NO.13):

[0164] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2) 18-COOH)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser

[0165] HsQGTFTSDK(GGSGSG-γGlu-CO(CH2) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS

[0166] Compound 14 (SEQ ID NO.14):

[0167] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2) 18 -COOH)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0168] HsQGTFTSDK(GGSGSG-γGlu-CO(CH2) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH2

[0169] Compound 15 (SEQ ID NO.15):

[0170] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2) 18 -COOH)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser

[0171] H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH2) 18-COOH)SKYLDERAAQDF-Iva-QWLLDGPSSG-Aib-PPPS

[0172] Compound 16 (SEQ ID NO.16):

[0173] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2) 18 -COOH)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH2

[0174] H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH2) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGPSSG-Aib-PPPS-NH2.

[0175] In the above sequence, the modification of Lys can be one of the following structures:

[0176]

[0177]

[0178] The above Lys group connected to the lipophilic substituent can be replaced by:

[0179]

[0180] The specific meanings of the abbreviations used in the present invention are as follows:

[0181] Boc is tert-butyloxycarbonyl, Fmoc is fluorenylmethoxycarbonyl, t-Bu is tert-butyl, ivDDe is 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl, resin is resin, TFA is trifluoroacetic acid, EDT is 1,2-ethanedithiol, Phenol is phenol, FBS is fetal bovine serum, BSA is bovine serum albumin, HPLC is high performance liquid chromatography, GLP-1R is glucagon-like peptide 1 receptor, GCGR is glucagon receptor, GLP-1 is glucagon-like peptide, and mPEG is Monomethoxypolyethylene glycol, His is histidine, Ser is serine, D-Ser is D-serine, Gln is glutamine, Gly is glycine, Glu is glutamic acid, Ala is alanine, Thr is threonine, Lys is lysine, Arg is arginine, Tyr is tyrosine, Asp is aspartic acid, Trp is tryptophan, Phe is phenylalanine, IIe is isoleucine, Leu is leucine, Cys is cysteine, Pro is proline, Val is valine, Met is methionine, Asn is asparagine. HomoLys is homolysine, Orn is ornithine, Dap is diaminopimelic acid, Dab is 2,4-diaminobutyric acid, Aib is 2-aminoisobutyric acid, and Iva is isovaline. BRIEF DESCRIPTION OF THE DRAWINGS

[0182] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0183] Figure 1 The area under the curve (AUC) is calculated based on the initial blood glucose level of the experimental animals 4 hours after administration and the 24-hour blood glucose change after oral administration of glucose at different time points in Example 2, p<0.05.

[0184] Figure 2A This is the gel electrophoresis diagram of the FN expression of different groups detected by Western blot in Example 3, in which MCS mesangial cells and NRK renal epithelial cells were induced by high glucose to construct an in vitro renal fibrosis cell model.

[0185] Figure 2B The in vitro renal fibrosis cell model was constructed by inducing MCS mesangial cells and NRK renal epithelial cells by high glucose in Example 3, and the FN expression in different groups was detected in the bar chart, p<0.05; *: indicates that it is within 95% confidence level compared with the control (p<0.05); **: indicates that it is within 99% confidence level compared with the control (p<0.01).

[0186] Figure 3AThe 8-week-old db / db diabetic mice in Example 4 were subcutaneously injected with normal saline, compound 3 (80 and 120 μg / kg, 2 groups in total), compound 8 (80 and 120 μg / kg, 2 groups in total), compound 13 (80 and 120 μg / kg, 2 groups in total), compound 15 (80 and 120 μg / kg, 2 groups in total) and semaglutide (80 and 120 μg / kg, 2 groups in total) every day or every other day. The blood glucose and body weight of the mice were measured after fasting for 6 hours every other day after daily administration, and p<0.05.

[0187] Figure 3B The 8-week-old db / db diabetic mice in Example 4 were subcutaneously injected with normal saline, compound 3 (80 and 120 μg / kg, 2 groups in total), compound 8 (80 and 120 μg / kg, 2 groups in total), compound 13 (80 and 120 μg / kg, 2 groups in total), compound 15 (80 and 120 μg / kg, 2 groups in total) and semaglutide (80 and 120 μg / kg, 2 groups in total) every day or every other day. The blood glucose and body weight of the mice were measured after administration every other day and fasting for 6 hours every other day, and p<0.05.

[0188] Figure 4A The 12-week-old db / db diabetic mice in Example 4 were subcutaneously injected with normal saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, and MED C18-acid for 6 consecutive weeks, and the blood glucose changes of the mice were measured every week.

[0189] Figure 4B The 12-week-old db / db diabetic mice in Example 4 were subcutaneously injected with normal saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, and MED C18-acid, respectively. The OGTT results and corresponding AUC values ​​were measured at the 4th week.

[0190] Figure 4C The 12-week-old db / db diabetic mice in Example 4 were subcutaneously injected with normal saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, and MED C18-acid, respectively. The ITT results and corresponding AUC values ​​were measured at week 5.

[0191] Figure 4D The 12-week-old db / db diabetic mice in Example 4 were subcutaneously injected with normal saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, and MED C18-acid, respectively. The results of ALT and AST in mouse serum were p<0.05.

[0192] Figure 5AFrom left to right are the urine protein content, urine sugar content in urine, and glycosylated hemoglobin content, urea nitrogen content and blood creatinine content in serum of mice in Example 4 after 8 weeks of drug treatment.

[0193] Figure 5B The H&E staining results of the kidneys of mice in Example 4 after 8 weeks of drug treatment.

[0194] Figure 5C The PAS staining results and statistical graphs of the kidneys of mice in Example 4 after 8 weeks of drug treatment.

[0195] Figure 5D The Sirius red staining results and statistical graphs of the kidneys of mice treated with the drug for 8 weeks in Example 4 are shown.

[0196] Figure 5E The FN staining results and statistical graphs of the kidneys of mice in Example 4 after 8 weeks of drug treatment.

[0197] Fig. 5F The α-SMA staining results and statistical graphs of the kidneys of mice treated with the drug for 8 weeks in Example 4, p<0.05.

[0198] Figure 6 is an exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention.

[0199] Figure 7 is another exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention. DETAILED DESCRIPTION

[0200] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0201] Example 1 Synthesis of peptide compounds

[0202] The polypeptide compounds 1-16, positive control polypeptide compounds 17-20 (derived from compounds in Chinese Patent Publication No. CN104926934A), positive control polypeptide compounds 21-26 (derived from patent CN201711194175.0), SAR425899 and MED C18-acid involved in the present invention are all synthesized independently, among which SAR425899 and MED C18-acid are compounds reported in the prior art.

[0203] The amino acid sequences of the above-mentioned positive control peptide compounds 17-26, SAR425899 and MED C18-acid compounds are as follows:

[0204] Compound 17 (SEQ ID NO.17):

[0205] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0206] HsQGTFTSDYSKYLDK(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)RRAQDFVQWLMNTGGPSSGAPPPS-NH2

[0207] Compound 18 (SEQ ID NO.18):

[0208] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0209] HsQGTFTSDYSKYLDK(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)RRAQDFVQWLLNTGGPSSGAPPPS-NH2

[0210] Compound 19 (SEQ ID NO.19):

[0211] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-CO(CH2) 16CO2H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0212] HsQGTFTSDYSK(PEG2-PEG2-CO(CH2) 16 CO2H)KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2

[0213] Compound 20 (SEQ ID NO.20):

[0214] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0215] H-(d-S)-QGTFTSDYSKYLDK(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)RRAQDFVQWLMNTGGPSSGAPPPS-NH2

[0216] Compound 21 (SEQ ID NO.21):

[0217] His-Xaa-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0218] HXQGTFTSDK*SKYLDEQAAQDFVQWLLDGPSSGAPPPS-NH2

[0219] Compound 22 (SEQ ID NO.22):

[0220] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Ser-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0221] HSQGTFTSDK*SKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH2

[0222] Compound 23 (SEQ ID NO.23):

[0223] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Glu-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0224] HSQGTFTSDK * SKYLDEEAAQDFVQWLMNGGPSSGAPPPS-NH2

[0225] Compound 24 (SEQ ID NO.24):

[0226] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0227] HSQGTFTSDK * SKYLDEQAAQDFVQWLMNGGPSSGAPPPS-NH2

[0228] Compound 25 (SEQ ID NO.25):

[0229] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OHHSQGTFTSDK * SKYLDERAAQDFVQWLMNTGPSSGAPPPS-OH

[0230] Compound 26 (SEQ ID NO.26):

[0231] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OHHSQGTFTSDK * SKYLDSRAAQDFVQWLMNTGPSSGAPPPS-OH

[0232] Note: X in compound 21 in the table is aminoisobutyric acid, and K in compounds 21-25 is * This indicates that there is a lysine residue at this position and it is covalently linked to a fatty acid, the structure of which is shown below:

[0233]

[0234] MED C18-acid (SEQ ID NO.27):

[0235] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Lys(PEG2-PEG2-γGlu-(CH2) 17 COOH)-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Val-Ala-Gly-Gly-NH2

[0236] HsQGTFTSDYSKK(PEG2-PEG2-γGlu-CO(CH2) 16CO2H)LDSERARDFVAWLVAGG-NH2

[0237] SAR425899 (SEQ ID NO. 28):

[0238] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Lys(CO(CH2) 14 CH3)-Glu-Ser-Lys-Ala-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0239] HsQGTFTSDLSKQK(CO(CH2) 14 CH3)ESKAAQDFIEWLKAGGGPSSGAPPPS-NH2

[0240] To facilitate the description of the preparation method of the above polypeptide compound, the present invention takes polypeptide compound 3 as an example, and the specific operation steps are as follows:

[0241] 1. Materials:

[0242] All amino acids were purchased from NovaBiochem. All other reagents were analytical grade and purchased from Sigma unless otherwise specified. CS336X peptide synthesizer (CS Bio, USA). Phenomenex Luna C18 preparative column (46 mm x 250 mm) was used to purify peptides. High performance liquid chromatography was a product of Waters. Mass spectrometry was performed using an Agilent mass spectrometer.

[0243] (II) Methods:

[0244] 1. Synthesis of polypeptide compound 3:

[0245] Structural sequence:

[0246] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0247] 1a) Main peptide chain assembly:

[0248] The following peptides were synthesized on a CS336X peptide synthesizer (CS Bio, USA) at a scale of 0.25 mol according to the Fmoc / t-Bu strategy:

[0249] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gl u(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin

[0250] (1) Step 1: 0.75 g of Rink amide resin was swollen in dichloromethane and the resin was washed three times with N,N-dimethylformamide;

[0251] (2) The second step: using Rink amide resin as a carrier, a mixture of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine in a molar ratio (molar ratio) of 1:1:1 as a coupling agent, and N,N-dimethylformamide as a solvent, a program reaction is carried out, and condensation reactions are carried out in sequence to connect Fmoc-Ser(t-Bu)-OH, Fmoc-Pro-OH(3x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(t-Bu)-OH(2x), Fmoc-Pro-OH, Fmoc-Gly-OH(2x), Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc -Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH(2x), Fmoc-Lys(Boc )-OH, Fmoc-Aib-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Phe-OH, Fmoc- Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Ser(t-Bu)-OH, Boc-His(Boc)-OH, get:

[0252] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG2-PEG2-γGlu-CO(CH2) 18The invention relates to a novel condensation-enhanced polyol (CO2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rinkamide resin, wherein the molar ratio (molar ratio) of the amount of the Fmoc-protected amino acid to the amount of the resin in each condensation reaction is 1:1 to 6:1, and the molar ratio (molar ratio) of the amount of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate to the amount of the Fmoc-protected amino acid in each condensation reaction is 3:1.

[0253] 1b)Lys(PEG2-PEG2-γGlu-CO(CH2) 18 Removal of CO2H) and introduction of lipophilic substituents:

[0254] In a mixed solution of N-methylpyrrolidone: dichloromethane = 1:1 (volume ratio), the protected peptidyl resin synthesized in 1a) was washed twice, and a freshly prepared 2.0% hydrazine hydrate N-methylpyrrolidone solution was added. The reaction mixture was shaken at room temperature for 12.0 minutes and then filtered. The hydrazine treatment step was repeated twice to obtain:

[0255] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. The resin was then washed thoroughly with dichloromethane and N-methylpyrrolidone. Add FmocNH-PEG2-OH, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, diisopropylethylamine in N-methylpyrrolidone mixed coupling solution, shake for 3.0 hours, filter, wash and repeat the hydrazine treatment step twice to obtain:

[0256] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG2-OH)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Ly s(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin, piperidine / N,N-dimethylformamide solution to remove the Fmoc group, repeat the Fmoc-PEG2-OH coupling reaction once to obtain:

[0257] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp( OtBu)-Lys(PEG2-PEG2-OH)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys( Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rinkamide resin. The Fmoc group was removed by piperidine / N,N-dimethylformamide solution, and then Fmoc-γGlu-OtBu, tBu mono-protected twenty-carbon fatty acid were coupled in sequence under conventional conditions to obtain:

[0258] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG2-PEG2-γGlu-CO(CH2) 18CO2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gl u(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin.

[0259] 1c) Removal of full protection of polypeptide:

[0260] Will

[0261] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin was added to a round-bottom flask. Under ice bath conditions, cutting solution TFA / EDT / Phenol / H2O (88 / 2 / 5 / 5, volume ratio) was added to raise the temperature. The temperature of the cleavage solution was controlled at 25°C and the reaction was carried out for 120 minutes. Filter, wash the filter cake three times with a small amount of trifluoroacetic acid, and combine the filtrate. The filtrate was slowly poured into ice ether under stirring. Let it stand for more than 1.0 hour until the precipitation is complete. Pour off the supernatant, centrifuge the precipitate, and wash it with ice ether 6 times to obtain the crude compound:

[0262] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0263] 1d) Purification of polypeptide compounds:

[0264] The crude product obtained in 1c was dissolved in a solution of 5.0% acetic acid in acetonitrile:H2O=1:1 (volume ratio) and purified by two semi-preparative HPLCs on a 50mm x 250mm column packed with reverse phase C18. The column was eluted with a gradient of 30-60% acetonitrile-0.1% trifluoroacetic acid / H2O at 40mL / min for 45.0 minutes, and the peptide-containing fractions were collected, concentrated to remove acetonitrile, and then freeze-dried. A pure product with an HPLC purity of more than 95% was obtained. The separated product was analyzed by LC-MS, and the m / z value of the protonated molecular ion peak was found to be: 4860.9, and the theoretical value was 4863.5.

[0265] Table 1. Polypeptide compounds of the present invention

[0266]

[0267]

[0268]

[0269]

[0270] Example 2 Effects of peptide compounds 1-16, positive control peptide compounds 17-26, liraglutide and semaglutide on oral glucose tolerance (OGTT)

[0271] Semaglutide in this example was purchased from Zhejiang Paipei Biotechnology Co., Ltd. (CAS No.: 910463-68-2), and liraglutide was purchased from Shenzhen Jianyuan Pharmaceutical Technology Co., Ltd. (CAS No.: 204656-20-2).

[0272] Eight-week-old male C57BL / 6J mice (Model Animal Research Center, Nanjing University) were randomly divided into groups with similar blood glucose (assessed from blood samples obtained from the tail tip), with 6 mice in each group.

[0273] The animals were dosed after fasting (6 hours). The polypeptide compounds 1-16, positive control polypeptide compounds 17-26, liraglutide, semaglutide, SAR425899 and MED C 18-acid in the present invention were subcutaneously administered at a dose of 100ug / kg, and the control group was given PBS. After about 4 hours, an initial blood sample was obtained (fasting blood glucose level). Subsequently, an oral dose of glucose (2g / kg) was administered, and the animals were returned to their cages (t=0). Blood glucose was measured at t=15 minutes, t=30 minutes, t=60 minutes and t=120 minutes. Then, after 8h, 12h and 24h of administration, oral doses of glucose were repeated, and blood glucose changes were detected and tracked until 24 hours. The data were processed using the software GraphPadPrism to make a blood glucose change line graph, and the area under the curve was calculated to obtain the AUC graph. The results are shown in Figure 1 shown.

[0274] Compared with the vehicle (control group PBS), the AUC of liraglutide and compound 17-26 in the first OGTT curve period (0-120min) can be significantly reduced (P<0.05), and in the subsequent three OGTT curve periods (4-22h), the AUC began to increase. In contrast, the AUC of semaglutide in the four OGTT curve periods was significantly reduced (P<0.05), indicating that it has a long-term hypoglycemic effect. Compared with the vehicle (control group PBS), the polypeptide compounds of the present invention all have the effect of improving the glucose tolerance of mice to varying degrees, among which polypeptide compounds 1-16 showed a better and more significant effect of improving glucose tolerance in the four OGTT curve periods (0-22h). At the same time, the results also show that polypeptide compounds 1-16 have a better and more significant long-term hypoglycemic effect than polypeptide compounds 17-26, liraglutide, SAR425899 and MED C18-acid. Compared with semaglutide, polypeptide compounds 1, 2, 3, 8, 13, 14 and 15 showed better and more significant long-term hypoglycemic effects in four OGTT curve periods (0-22h).

[0275] Example 3 In the high glucose-induced renal fibrosis model, peptide compounds 1, 2, 3, 8, 13, 14, and 15 improve renal fibrosis

[0276] Hyperglycemia and glycosylation end products will cause mesangial cell hyperplasia and hypertrophy after the increase of extracellular matrix (ECM), morphological changes such as mesangial expansion, and ultimately lead to the loss of its physiological function. In addition, renal tubular epithelial cell transdifferentiation is also an important pathological basis of renal fibrosis. Studies have found that high concentrations of glucose further induce glomerular fibrosis by activating the RAS system. Therefore, the present embodiment uses high sugar to induce MCS mesangial cells and NRK renal epithelial cells, and constructs an in vitro renal fibrosis cell model to screen drugs that can be used to treat diabetic nephropathy.

[0277] The renal mesangial cell line GMC and glomerular epithelial cell mTEC used in this example are cells preserved in the applicant's laboratory (both are primary cells extracted from mouse kidneys) and cultured in a low-glucose medium containing 10% fetal bovine serum. When the cells grew to a confluence of 70%, they were starved with serum-free culture medium for 24 h, and the cells were divided into the following 9 groups by adjusting the glucose concentration as follows: ① Low group (5.5 mmol / L glucose), ② High group (30 mmol / L glucose), ③ High glucose + compound 1 group (30 mmol / L glucose + NO.1 10 uM), ④ High glucose + compound 2 group (30 mmol / L glucose + NO.2 10 uM), ⑤ High glucose + compound 3 group (30 mmol / L glucose + NO.3 10 uM), ⑥ High glucose + compound 8 group (30 mmol / L glucose + NO.8 10 uM), ⑦ High glucose + compound 13 group (30 mmol / L glucose + NO.13 10 uM), ⑧ High glucose + compound 14 group (30 mmol / L glucose + NO.14 10 uM) and ⑨ High glucose + compound 15 group (30 mmol / L glucose + NO.15 10uM), cultured at 37℃5%CO2 for 48h, and FN protein (fibronectin) was detected by Western blot. Figure 2A As shown in FIG. 1 , in this example, MCS mesangial cells and NRK renal epithelial cells were induced by high glucose to construct an in vitro renal fibrosis cell model, and the FN expression gel electrophoresis of different groups detected by Western blot was shown; Figure 2B As shown, in Example 3, an in vitro renal fibrosis cell model was constructed by inducing MCS mesangial cells and NRK renal epithelial cells with high glucose, and the FN expression in different groups was detected in the bar chart.

[0278] According to previous experimental studies and previous literature, 30mmol / L glucose can induce hyperplasia of glomerular mesangial cells and transdifferentiation of renal epithelial cells. Western blot test results showed that under high glucose stimulation, the expression level of FN protein in the High group was higher than that in the control Low group; and the drug-treated groups were able to downregulate the expression of FN protein, indicating that the polypeptide compounds of the present invention can significantly reduce the increase of ECM caused by high glucose. Among them, polypeptide compounds 3, 8, 13, 14 and 15 have a more significant effect in inhibiting FN expression.

[0279] Example 4 The therapeutic effects of peptide compounds 3, 8, 13, 14, 15 and 16, semaglutide, SAR425899 and MED C18-acid on diabetic mice

[0280] (I) Drug concentration determination experiments of peptide compounds 3, 8, 13 and 15

[0281] The db / db mouse diabetic model was obtained (purchased from Nanjing University-Nanjing Institute of Biomedicine, about 8 weeks old, and blood glucose and body weight were measured to ensure the smooth progress of subsequent experiments). The model mice were randomly divided into 12 groups (including polypeptide compounds 3, 8, 13, 15, semaglutide and saline groups), 6 mice in each group, and there was no difference in basal body weight and blood glucose. Each group of mice was subcutaneously injected with compound 3 (80 and 120 μg / kg, 2 groups in total), compound 8 (80 and 120 μg / kg, 2 groups in total), compound 13 (80 and 120 μg / kg, 2 groups in total), compound 15 (80 and 120 μg / kg, 2 groups in total), semaglutide (80 and 120 μg / kg, 2 groups in total) and saline every day or every other day (DN group: normal mice born in the same litter; DC group: db / db mouse control group).

[0282] After the mice were dosed daily, the blood glucose and body weight of the mice were measured after fasting for 6 h every other day.

[0283] Figure 3AIt was shown that compared with the saline group and DC group, the semaglutide group (120 μg / kg) showed a better hypoglycemic effect, and then maintained a relatively stable normal blood sugar level, but its weight reduction effect was not significant. In the drug administration group, polypeptide compounds 3, 8, 13 and 15 had a significant hypoglycemic effect at a dose of 80ug / kg, and polypeptide compounds 3 and 8 had a significant hypoglycemic effect, and polypeptide compounds 13 and 15 had a significant hypoglycemic effect and were long-lasting; at a dose of 120ug / kg, polypeptide compounds 3, 8, 13 and 15 had achieved a good hypoglycemic effect in 3-5 days, and blood sugar was stably maintained at a normal level. At the same time, polypeptide compounds 3, 8, 13 and 15 showed a better weight reduction effect, and the effects of polypeptide compounds 13 and 15 were better than those of polypeptide compounds 3 and 8. Therefore, the drug concentration was set at 120ug / kg.

[0284] After the mice were given the drug every other day, they were fasted for 6 hours to measure their blood sugar and body weight.

[0285] Figure 3B It was shown that compared with the saline group and DC group, after administration every other day, the 80μg / kg and 120μg / kg groups of Semaglutide had hypoglycemic effects, but the hypoglycemic effect was not obvious, and the weight reduction effect was not significant. In the administration group, polypeptide compounds 3 and 8, at a dose of 80ug / kg, the blood sugar of polypeptide compounds 3 and 8 first decreased and then increased, and the overall hypoglycemic effect was obvious, and polypeptide compounds 3 and 8 had the effect of reducing weight. While polypeptide compounds 13 and 15 had a significant hypoglycemic effect on the third day, but the subsequent blood sugar level did not change much and tended to be stable; at a dose of 120ug / kg, the blood sugar of the Semaglutide group rebounded on the third day, and its long-term effect was not enough. Polypeptide compounds 3, 8, 13 and 15 have achieved a good hypoglycemic effect in 3-5 days, and the blood sugar did not change much after polypeptide compounds 3 and 8. Polypeptide compounds 13 and 15 have maintained a good hypoglycemic effect, proving that polypeptide compounds 13 and 15 have good long-term hypoglycemic effect. At the same time, peptide compounds 3, 8, 13 and 15 showed better weight loss effects, and the effects of peptide compounds 13 and 15 were better than those of peptide compounds 3 and 8. Therefore, the drug concentration was set at 120ug / kg.

[0286] (II) Effects of peptide compounds 3, 8, 13, 14, 15, 16, semaglutide, SAR425899 and MED C18-acid on diabetes mellitus in diabetic mice

[0287] The db / db mouse diabetes model was obtained (purchased from Nanjing University-Nanjing Institute of Biomedicine, mice of about 12 weeks old, and blood glucose and body weight were measured to ensure the smooth progress of subsequent experiments). The model mice were randomly divided into 10 groups (peptide compounds 3, 8, 13, 14, 15, 16, semaglutide, SAR425899, MED C18-acid and saline groups), 6 mice in each group, and there was no difference in the basal body weight and blood glucose of each mouse. Each group of mice was subcutaneously injected with peptide compounds 3, 8, 13, 14, 15, 16 (120 μg / kg), semaglutide (120 μg / kg), SAR425899 (120 μg / kg), MED C18-acid (120 μg / kg), and saline every day (DN group: normal mice born in the same litter; DC group: db / db mouse control group). After the mice were given the drug every day, the blood glucose and body weight of the mice were measured after fasting for 6 hours every other day, and the water intake and food intake were measured once a week. OGTT was measured in the 4th week, insulin tolerance (ITT) was measured in the 5th week, mouse metabolism was detected using mouse metabolic cages in the 6th and 7th weeks, and samples were collected in the 8th week to test various serological and pathological indicators.

[0288] 1. Effects of the drug on lowering blood sugar, reducing body weight and improving insulin resistance in diabetic mice

[0289] The type II diabetes model is characterized by obesity, insulin resistance, hyperglycemia, dyslipidemia and liver fat vacuolar degeneration. When the diabetic mice were treated with peptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, and MED C18-acid for 4 and 5 weeks, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed, respectively. The results are as follows: Figures 4A-4D shown.

[0290] Figures 4A to 4C The results showed that compared with the DN group, peptide compounds 3, 8, 13, 14, 15, 16, semaglutide, SAR425899 and MED C18-acid all had significant hypoglycemic effects, and blood sugar had dropped to normal levels in the first week, and blood sugar was relatively stable thereafter. However, peptide compounds 3, 8, 13, 14, 15, and 16 performed better in improving oral glucose tolerance (OGTT) and insulin resistance (ITT) in diabetic mice.

[0291] also, Figure 4D The results showed that polypeptide compounds 3, 8, 13, 14, 15, and 16 had better liver protection function.

[0292] 2. Therapeutic effect of drugs on diabetic nephropathy in diabetic mice

[0293] After 6 weeks of drug treatment, urine was collected from mice using metabolic cages, and the protein content in urine was detected using relevant kits. After 7 weeks of drug administration, blood was collected and used to detect serological indicators such as blood creatinine and urea nitrogen. Figures 5A to 5F shown.

[0294] Depend on Figure 5A It can be seen that the proteinuria and urine sugar content of the 20-week-old db / db diabetic mice DC group were significantly increased compared with the control DN group. In addition, the glycated hemoglobin content, blood creatinine and urea nitrogen content also increased significantly, indicating that the diabetic nephropathy of the mice was already very serious. After administration, the proteinuria content, urine sugar content, glycated hemoglobin content, blood creatinine and urea nitrogen content of the mice improved to varying degrees.

[0295] Mouse kidneys were taken and embedded in paraffin for subsequent staining and immunohistochemical studies. Figure 5B The results of H&E staining of mouse kidneys showed that the mouse kidney tissue had local inflammation and glomerular adhesion. After administration, the glomerular adhesion phenomenon was alleviated. Compared with the positive control drugs semaglutide, SAR425899 and MED C18-acid group, the improvement and therapeutic effects of peptide compounds 3, 8, 13, 14, 15 and 16 were more significant. In general, the observation of renal lesions is mostly concentrated in the cortex, mainly including glomeruli and renal interstitium, and the observation of glomeruli is concentrated in the mesangial area. By PAS staining ( Figure 5C ) It can be observed that compared with the DN group, the DC group had a wider glomerular mesangial area, proliferated mesangial cells and mesangial matrix, and thickened basement membrane. After administration, the glomerular vascular loops were thin and clear, the number of endothelial cells and mesangial cells was normal, and the surrounding renal tubules were also normal. The improvement effect of polypeptide compounds 3, 8, 13, 14, 15 and 16 was more significant. Figures 5D to 5F Sirius red, FN and α-SMA staining showed that compared with the DN group, the collagen deposition in the DC group was significantly increased, and the drug group could significantly reduce the collagen content, among which polypeptide compounds 3, 8, 13, 14, 15 and 16 had more significant improvement effects, and their therapeutic effects were better than those of the positive control semaglutide, SAR425899 and MED C18-acid groups.

[0296] In summary, through further optimization and design of the amino acid sequence and modification of the side chain technology, the compounds reported in the present invention are superior to previously reported compounds in terms of reducing blood sugar, reducing body weight, improving and treating diabetic complications, and have more advantages in clinical application.

[0297] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention. Sequence Listing <110> Sun Yat-sen University <120> Polypeptide compound and its application in preventing or treating diabetes or diabetic complications <130> GD1899-21P125434 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 1 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 2 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> The side chain of Lys is connected to PEG2-PEG2-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 3 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 3 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 4 <211> 39 <212> PRT <213> Artificial Sequence <220> <221> MISC_FEATURE <222> (2)..(2) <223> X = D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> The side chain of Lys is linked to -PEG2-PEG2-γGlu-CO(CH2)16CO2H <220> <221> MISC_FEATURE <222> (16)..(16) <223> X = Aib <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 5 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CH3 <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 5 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 6 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 6 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 7 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <400> 7 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 8 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 8 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 9 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)16CO2H <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 9 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 10 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CH3 <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 10 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 11 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-CO(CH2)18CH3 <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 11 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 12 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)18CO2H <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 12 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 13 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18-COOH <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 13 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 14 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18-COOH <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 14 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa 1 5 10 15 Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 15 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18-COOH <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 15 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 16 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=Aib <220> <221> MISC_FEATURE <222> (10)..(10) <223> Lys side chain connection -Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18-COOH <220> <221> MISC_FEATURE <222> (23)..(23) <223> X=Iva <220> <221> MISC_FEATURE <222> (35)..(35) <223> X=Aib <400> 16 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Xaa Pro Pro Pro Ser 35 <210> 17 <211> 40 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (16) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)14CH3 <400> 17 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser 35 40 <210> 18 <211> 40 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (16) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)14CH3 <400> 18 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser 35 40 <210> 19 <211> 40 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (12)..(12) <223> Lys side chain connection -PEG2-PEG2-CO(CH2)16CO2H <220> <221> MISC_FEATURE <222> (16) <223> X=Aib <400> 19 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser 35 40 <210> 20 <211> 40 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (16) <223> The side chain of Lys is connected to -(PEG2-PEG2-γGlu-CO(CH2)14CH3 <400> 20 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser 35 40 <210> twenty one <211> 38 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> Xaa = aminoisobutyric acid <400> twenty one His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Pro Ser Ser 20 25 30 Gly Ala Pro Pro Pro Ser 35 <210> 22 <211> 39 <212> PRT <213> Artificial Sequence <400> 22 His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser 1 5 10 15 Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 23 <211> 39 <212> PRT <213> Artificial Sequence <400> 23 His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 24 <211> 39 <212> PRT <213> Artificial sequence <400> 24 His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 25 <211> 39 <212> PRT <213> Artificial sequence <400> 25 His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 26 <211> 39 <212> PRT <213> Artificial sequence <400> 26 His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 27 <211> 30 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (13) <223> Lys side chain connection -PEG2-PEG2-γGlu-CO(CH2)17COOH <400> 27 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Ser 1 5 10 15 Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Val Ala Gly Gly 20 25 30 <210> 28 <211> 39 <212> PRT <213> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> X=D-Ser <220> <221> MISC_FEATURE <222> (14)..(14) <223> The side chain of Lys is connected to -CO(CH2)14CH3 <400> 28 His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Glu Ser 1 5 10 15 Lys Ala Ala Gln Asp Phe How Much Glu Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Gly Ala Pro Pro Pro Cheese 35

Claims

1. A polypeptide compound, which is any one of the following compounds: Compound 1: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 2: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2) 18 CO2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2。 2. A composition comprising the polypeptide compound according to claim 1.

3. The composition according to claim 2, characterized in that The composition is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier or excipient.

4. Use of the polypeptide compound according to claim 1 or the composition according to any one of claims 2 to 3 in the preparation of a medicament for preventing or treating diabetes and / or diabetic complications; the diabetic complication is diabetic nephropathy.

5. Use of the polypeptide compound according to claim 1 or the composition according to any one of claims 2 to 3 in the preparation of a health product or medicine for reducing body weight.

Images