Intestinal insulinotropic polypeptide analogue and application thereof

Abstract

The invention provides an insulinotropic polypeptide analogue and application thereof, and relates to the technical field of polypeptide preparation and application thereof. The intestinal insulinotropic polypeptide analogue comprises a co-agonist, the co-agonist is GLP-1, GIP and GCG three-receptor co-agonist polypeptide which is in acylation connection with a fatty acid side chain, and the amino acid sequence of the polypeptide or a salt thereof or a solvate thereof is shown as the following formula: Y (Aib) QGTFTSDX10SIX13LDX16X17AQ (Aib) X21FIX24X25LX27EGGPSSGAPPPS-NH2. In the present invention, it provides an enteric insulinotropic analogue having activity in each of a glucose-dependent insulinotropic polypeptide, a glucagon-like peptide-1, and a glucagon receptor, the polypeptide analogue can be used for treating and preventing metabolic disorders and other diseases including diabetes mellitus, obesity, metabolic syndromes and related diseases through various administration modes, and the polypeptide analogue can be used for treating and preventing metabolic disorders including diabetes mellitus, obesity, metabolic syndromes and related diseases through various administration modes.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide preparation and its applications, and specifically to an incretin polypeptide analog and its applications. Background Art

[0002] Over the past few decades, the prevalence of diabetes has been continuously increasing. Type II diabetes patients are the most common form of diabetes, accounting for nearly 90% of diabetes cases, and are characterized by high blood glucose levels caused by insulin resistance. Overweight or obesity usually also causes insulin resistance and is prone to metabolic diseases such as non-alcoholic fatty liver disease. Type II diabetes patients with overweight or obesity are the largest group among Type II diabetes patients, and abnormal insulin resistance index is also considered an important indicator for pre-diabetes. Therefore, for Type II diabetes patients, therapies with dual effects of controlling blood glucose and weight loss are usually required.

[0003] In healthy individuals, oral glucose intake causes an increase in insulin secretion (also known as the incretin effect), which is due to the action of peptide substances produced by gastrointestinal neuroendocrine cells. The two most important peptide substances are glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic peptide (GIP), which are secreted by L cells in the small intestine or colon to produce proglucagon and by K cells in the small intestinal mucosa, respectively.

[0004] Glucagon (GCG) is derived from the secretion of pancreatic alpha cells and proglucagon secreted by intestinal L cells. After its receptor GCGR (glucagon receptor) is activated, it reduces glycogen accumulation by promoting glycogenolysis and accelerates fat metabolism, increasing energy consumption, and releasing glucose from liver glycogen to prevent hypoglycemia. Gastric inhibitory polypeptide (Oxyntomodulin), which is also derived from proglucagon like GCG, consists of non-enzymatically cleaved GCG and intervening peptide 1. It has the ability to activate GLP-1R and GCGR, can inhibit appetite, increase energy consumption and induce weight loss in obese individuals.

[0005] Therefore, those skilled in the art have provided an incretin polypeptide analog and its applications to solve the problems raised in the above background art. Summary of the Invention

[0006] (1) Technical Problems to be Solved

[0007] In view of the deficiencies of the prior art, the present invention provides an incretin polypeptide analog and its applications. This polypeptide analog can be used for treating and preventing metabolic disorders including diabetes, obesity, metabolic syndrome and related diseases through various administration routes.

[0008] (2) Technical Solutions

[0009] To achieve the above object, the present invention is realized by the following technical solutions:

[0010] An incretin polypeptide analogue, comprising a co-agonist, wherein the co-agonist is a GLP-1, GIP and GCG triple receptor co-agonist polypeptide acylated with a fatty acid side chain, and the amino acid sequence of the polypeptide or its salt or its solvate is shown as follows:

[0011] Y(Aib)QGT(α-meF)TSDX 10 SIX 13 LDX 16 X 17 AQ(Aib)X 21 X 22 IX 24 X 25 LX 27 EGGPSSGAPPPS-NH2;

[0012] Wherein:

[0013] X 10 is selected from Y or V;

[0014] X 13 is selected from Y, α-meF or α-meY;

[0015] X 16 is selected from Orn;

[0016] X 17 is selected from K or any amino acid having a functional group available for conjugation, and the ε-amino group or other functional group is conjugated to C through a linker 16 -C 22 fatty acid;

[0017] X 21 is selected from E or Aib;

[0018] X 22 is selected from F, α-meF;

[0019] X 24 is selected from E or D-Glu;

[0020] X 25 is selected from W, α-meW or Y;

[0021] X 27 is selected from I or V;

[0022] The Aib is α-aminoisobutyric acid; α-meF is α-methylphenylalanine; α-meY is α-methyltyrosine; Orn is ornithine; α-meW is α-methyltryptophan; D-Glu is D-glutamic acid, and the above structures are as shown in the appendix Figure 7 as shown.

[0023] Further, the amino acid sequence of the polypeptide is selected from any one of the following:

[0024] Compound 01: Y(Aib)QGT(α-meF)TSDYSI(α-meF)LD(Orn)(Lys(AEEA - AEEA - γGlu - C20diacid))AQ(Aib)EFIEYLIEGGPSSGAPPPS - NH2;

[0025] Compound 02: Y(Aib)QGT(α-meF)TSDYSIYLD(Orn)(Lys(AEEA - γGlu - C20diacid))AQ(Aib)EFIEYLIEGGPSSGAPPPS - NH2;

[0026] Compound 03: Y(Aib)QGT(α-meF)TSDYSI(α-meY)LD(Orn)(Lys(AEEA - γGlu - C20diacid))AQ(Aib)EFIEYLVEGGPSSGAPPPS - NH2;

[0027] Compound 04: Y(Aib)QGT(α-meF)TSDYSIYLD(Orn)(Lys(εK - εK - γGlu - C20diacid))AQ(Aib)EFI(D - Glu)WLIEGGPSSGAPPPS - NH2;

[0028] Compound 05: Y(Aib)QGT(α-meF)TSDYSI(α-meY)LD(Orn)(Lys(εK - εK - γGlu - C20diacid))AQ(Aib)EFIEWLIEGGPSSGAPPPS - NH2;

[0029] Compound 06: Y(Aib)QGT(α-meF)TSDVSI(α-meF)LD(Orn)(Lys(AEEA - AEEA - γGlu - C18diacid))AQ(Aib)E(α-meF)IE(α-meW)LVEGGPSSGAPPPS - NH2;

[0030] Compound 07: Y(Aib)QGT(α-meF)TSDYSI(α-meF)LD(Orn)(Lys(AEEA - AEEA - γGlu - C20diacid))AQ(Aib)(Aib)FIEWLVEGGPSSGAPPPS - NH2.

[0031] Furthermore, the co-agonist is linked to the fatty acid side chain through the ε-amino group on the amino acid K residue at position 17 of the polypeptide and through a linker.

[0032] Furthermore, the fatty acid side chain is selected from any one of the following:

[0033] HOOC(CH2) 16 CO-, HOOC(CH2) 17 CO-, HOOC(CH2) 18 CO-, HOOC(CH2) 19 CO-, HOOC(CH2) 20 CO-, HOOC(CH2) 21 CO-, HOOC(CH2) 22 CO-, (HO)3P(CH2) 16 CO-, (HO)3P(CH2) 18 CO- or (HO)3P(CH2) 20 CO-.

[0034] Furthermore, the fatty acid side chain is linked to the amino acid K through a linker.

[0035] Furthermore, the linker is selected from any one of -(AEEA)-(AEEA)-γE-, -εK-εK-γE- or -(AEEA)-γE-.

[0036] Furthermore, it further includes a fatty acid component having a linker and a structure of the following formula:

[0037] -(εK) a -γE-CO(CH2) c -COOH or -(AEEA) b -γE-CO(CH2) c -COOH

[0038] where a is 1 or 2, b is 1 or 2, and c is 16 or 18.

[0039] Furthermore, a method for preparing an incretin polypeptide analog, the preparation method comprising the following steps:

[0040] Step S1. Swell the resin

[0041] Add 0.4 g of Rink-Ser resin (SD = 0.35) to the reactor, add 6 ml of DCM and shake to swell for 30 min;

[0042] Step S2. Deprotect the resin

[0043] Vacuum dry the swelling reagent, add 6 mL of 20% Pip / DMF and shake for 5 min. After the reaction ends, drain the reaction solution, and then add 6 mL of 20% Pip / DMF again and continue the reaction for 5 min;

[0044] Step S3. Deprotection and washing

[0045] Vacuum dry the deprotection reagent, wash 6 times with 6 mL of DMF, wash for 1 min each time, and then dry for 1 min;

[0046] Step S4. Deprotection detection

[0047] Take 20 resin beads and put them into a test tube for detection, then add 1 ml of ninhydrin detection reagent. Place the test tube in a 100 °C metal bath for 2 min, take it out and observe the color of the resin. If the color of the resin becomes darker, it is positive, indicating that deprotection is successful;

[0048] Step S5. Condense the second amino acid

[0049] Take 2 mL (3 eq) of a pre-prepared 0.2 M Fmoc-Pro-O solution containing Oxyma, and then add 3 eq of DIC and shake for 1 h;

[0050] Step S6. Reaction detection and washing

[0051] Take 20 resin beads and put them into a test tube for detection, then add 1 mL of ninhydrin detection reagent. Place the test tube in a 100 °C metal bath for 2 min, take it out and observe the color of the resin. If there is no obvious change in the color of the resin, it proves that the condensation is successful. After successful condensation, wash the reaction solution, wash 4 times with 6 mL of DMF each time, wash for 1 min each time, and then dry for 1 min;

[0052] Step S7. Peptide chain extension

[0053] Repeat steps 2 to 6 to condense the amino acids in the sequence from right to left successively. Among them, Q at position 19, Orn at position 16, D at position 15, I at position 12, α-meF, α-meL or α-meY at positions 13 and 6, and T at positions 5 and 7. After condensing with Oxyma / DIC for 30 min, withdraw the reaction solution, add 3 eq of amino acid solution, 3 eq of HATU, and 6 eq of DIEA again, and continue the reaction for 30 min. After the reaction is completed, perform resin detection; after double charging of T at position 5, there is still color detected. Add 6 mL of capping reagent (0.5 mL of acetic anhydride, 1 mL of DIEA solution, and 4.5 mL of DMF), and continue the capping reaction for 30 min; among them, Fmoc-L-Lys[C20-OtBu-Glu-(OtBu)-AEEA-AEEA]-OH or Fmoc-L-Lys[C20-OtBu-Glu-(OtBu)-εK(Boc)-εK(Boc)]-OH at position 17. After condensing with Oxyma / DIC for 1 h, withdraw the reaction solution, add 3 eq of Fmoc-L-Lys[C20-OtBu-Glu-(OtBu)-AEEA-AEEA]-OH or Fmoc-L-Lys[C20-OtBu-Glu-(OtBu)-εK(Boc)-εK(Boc)]-OH, and 3 eq of Oxyma / DIC again, and continue the reaction for 1 h. After the reaction is completed, perform resin detection;

[0054] Step S8. Resin drying

[0055] After the deprotection and washing of the last amino acid are completed, wash the resin 3 times with 6 mL of methanol, and dry it for cleavage;

[0056] Step S9. Resin cleavage

[0057] Add 5 mL of cleavage solution, cleave at room temperature for 3 h, filter out 1 tube of the reaction solution, add it to 40 mL of ice-cold diethyl ether, centrifuge to precipitate, and wash with ice-cold diethyl ether 3 times. The centrifuge is set at a rotation speed of 3000 R / min and a centrifugation time of 2 min to obtain the crude polypeptide solid for purification;

[0058] Step S10. Intermediate control detection of the crude product

[0059] Perform high performance liquid chromatography (HPLC) analysis:

[0060] The chromatographic column is a C18 reversed-phase silica gel column; column temperature: 40 °C; detection wavelength: 220 nm and 254 nm; flow rate: 1.0 mL / min; injection volume of the automatic injection needle: 5 μL; mobile phase A: aqueous solution of 0.065% TFA; mobile phase B: acetonitrile solution of 0.05% TFA;

[0061] Step S11. Purification of the crude product

[0062] Dissolution of crude peptide: Take the crude peptide, add an acetonitrile or pure water mixed reagent and dissolve it by ultrasonic treatment. After the sample becomes clear and transparent, filter it with a vacuum filter and then load the sample. Perform C18 gradient elution according to the settings, and judge the peak shape at a wavelength of 220 nm. If there is an obvious inflection, change the tube to collect the liquid; if there is no obvious inflection, change the tube appropriately according to the situation.

[0063] Step S12. Sample salt conversion

[0064] Freeze-dry the fraction, weigh it to be 13 mg, and completely dissolve 15 mg of the sample with acetonitrile and water.

[0065] Step S13. Freeze-dry and subpackage

[0066] Load the qualified fraction after purification into a freeze-drying tray, cover it, and then put it into a freeze dryer for freeze-drying. After the freeze-drying is completed, take out the freeze-drying tray, weigh and subpackage the polypeptide sample, and store it at -20 °C.

[0067] Step S14. QC / QA inspection and release

[0068] Take a small sample and send it to QC for various index detections according to the order requirements. After the detection is qualified, QC / QA releases it according to the standard process, and the qualified sample is sent to the warehouse for shipment.

[0069] Furthermore, an exendin polypeptide analog drug composition and application, comprising the co-agonist or its salt or its solvate according to any one of claims 1-7, and a pharmaceutically acceptable excipient, and its application in drugs for treating diabetes or weight loss.

[0070] (III) Beneficial effects

[0071] The present invention provides an exendin polypeptide analog and its application. It has the following beneficial effects:

[0072] 1. The present invention provides an exendin polypeptide analog and its application, which provides an exendin analog having activity in each of glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, and glucagon receptor. This polypeptide analog can be used for treating and preventing metabolic disorders such as diabetes, obesity, and metabolic syndrome and related diseases through various administration methods. Description of the drawings

[0073] Figure 1 It is a graph showing the hypoglycemic effect of oral administration of compound 01 of the present invention and Rybelsus;

[0074] Figure 2 It is a graph showing the hypoglycemic effect of oral administration of compound 05 of the present invention and Rybelsus;

[0075] Figure 3 This is the graph showing the hypoglycemic effect of oral administration of Compound 07 of the present invention and Rybelsus;

[0076] Figure 4 This is the graph showing the hypoglycemic effect of oral administration of Compound 1, Compound 2 of the present invention and Semaglutide;

[0077] Figure 5 This is the graph showing the hypoglycemic effect of subcutaneous administration of Compound 1 of the present invention and Semaglutide;

[0078] Figure 6 This is the graph showing the hypoglycemic effect of subcutaneous administration of Compound 2 of the present invention and Semaglutide;

[0079] Figure 7 This is the schematic diagram of the amino acid structure of the present invention. Detailed implementation mode

[0080] Next, the technical solutions in the specific implementation modes of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the specific implementation modes of the present invention. Obviously, the described specific implementation modes are only a part of the specific implementation modes of the present invention, rather than all of the specific implementation modes. Based on the specific implementation modes of the present invention, all other specific implementation modes obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0081] Example 1:

[0082] The amino acid sequence of the polypeptide in the specific example of the present invention is selected from:

[0083] Compound 01: Y(Aib)QGT(α-meF)TSDYSI(α-meF)LD(Orn)(Lys(AEEA - AEEA - γGlu - C20diacid))AQ(Aib)EFIEYLIEGGPSSGAPPPS - NH2.

[0084] The co - agonist is connected to the fatty acid side chain through the ε - amino group on the amino acid K residue at the 17th position of the polypeptide and through a linker.

[0085] The fatty acid side chain is selected from HOOC(CH2) 16 CO-.

[0086] The fatty acid side chain is connected to the amino acid K through a linker, and the linker is selected from -(AEEA)-(AEEA)-γE-.

[0087] The co - agonist also includes a fatty acid component having a linker and having the following formula:

[0088] -(εK)a -γE-CO(CH2) c -COOH or -(AEEA) b -γE-CO(CH2) c -COOH

[0089] Where a is 1, b is 2, and c is 16.

[0090] The method for preparing the incretin polypeptide analogue comprises the following steps:

[0091] Step S1. Swelling the resin

[0092] Add 0.4 g of Rink-Ser resin (SD = 0.35) into a reactor, add 6 ml of DCM and shake to swell for 30 min;

[0093] Step S2. Deprotecting the resin

[0094] Vacuum-dry the swelling reagent, add 6 mL of 20% Pip / DMF and shake for 5 min. After the reaction is completed, draw off the reaction solution, and add 6 mL of 20% Pip / DMF again and continue the reaction for 5 min;

[0095] Step S3. Washing after deprotection

[0096] Vacuum-dry the deprotection reagent, wash with 6 ml of DMF 6 times, wash for 1 min each time, and then dry for 1 min;

[0097] Step S4. Detecting deprotection

[0098] Take 20 resin beads and put them into a test tube for detection, then add 1 ml of ninhydrin detection reagent. Place the test tube in a 100 °C metal bath for 2 min, take it out and observe the color of the resin. If the color of the resin becomes darker, it is positive, indicating that deprotection is successful;

[0099] Step S5. Condensing the second amino acid

[0100] Take 2 mL (3 eq) of a pre-prepared 0.2 M Fmoc-Pro-O solution containing Oxyma, and then add 3 eq of DIC and shake to react for 1 h;

[0101] Step S6. Detecting the reaction and washing

[0102] Take 20 resin beads and put them into a test tube for detection, then add 1 ml of ninhydrin detection reagent. Place the test tube in a 100 °C metal bath for 2 min, take it out and observe the color of the resin. If there is no obvious change in the color of the resin, it proves that condensation is successful. After successful condensation, wash the reaction solution, wash 4 times with 6 ml of DMF each time, wash for 1 min each time, and then dry for 1 min;

[0103] Step S7. Peptide chain elongation

[0104] Repeat the operations in steps 2 to 6, and sequentially condense the amino acids in the sequence from right to left. Among them, for Q at position 19, Orn at position 16, D at position 15, I at position 12, α-meF at positions 13 and 6, and T at positions 5 and 7, after condensing with Oxyma / DIC for 30 min, withdraw the reaction solution, add 3 eq of amino acid solution, 3 eq of HATU, and 6 eq of DIEA again, and continue the reaction for 30 min. After the reaction ends, conduct resin detection; after double charging of T at position 5, there is still color detected. Add 6 mL of capping reagent (0.5 mL of acetic anhydride, 1 mL of DIEA solution, and 4.5 mL of DMF), and continue the capping reaction for 30 min; for Fmoc-L-Lys[C20-OtBu-Glu-(OtBu)-AEEA-AEEA]-OH at position 17, after condensing with Oxyma / DIC for 1 h, withdraw the reaction solution, add 3 eq of Fmoc-L-Lys[C20-OtBu-Glu-(OtBu)-AEEA-AEEA]-OH and 3 eq of Oxyma / DIC again, and continue the reaction for 1 h. After the reaction ends, conduct resin detection;

[0105] Step S8. Drying the resin

[0106] After the deprotection and washing of the last amino acid are completed, wash the resin 3 times with 6 ml of methanol, and dry it for cleavage;

[0107] Step S9. Cleavage of the resin

[0108] Add 5 mL of cleavage solution, cleave at room temperature for 3 h, filter out 1 tube of the reaction solution, add it to 40 ml of ice-cold diethyl ether, centrifuge to precipitate, and wash 3 times with ice-cold diethyl ether. Set the centrifuge speed to 3000 R / min and the centrifugation time to 2 min to obtain the crude polypeptide solid for purification;

[0109] Step S10. In-process control detection of the crude product

[0110] Perform high performance liquid chromatography (HPLC) analysis:

[0111] The chromatographic column is a C18 reversed-phase silica gel column; column temperature: 40 °C; detection wavelengths: 220 nm and 254 nm; flow rate: 1.0 mL / min; injection volume of the automatic injection needle: 5 μL; mobile phase A: aqueous solution of 0.065% TFA; mobile phase B: acetonitrile solution of 0.05% TFA;

[0112] High performance liquid chromatography elution gradient:

[0113]

[0114]

[0115] Step S11. Crude product purification

[0116] Dissolution of crude peptide: Take the crude peptide, add an acetonitrile or pure water mixed reagent and dissolve it by ultrasonic treatment. After the sample becomes clear and transparent, filter it with a vacuum filter and then load the sample. Perform a C18 gradient elution according to the settings. Judge the peak shape at a wavelength of 220 nm. If there is an obvious inflection, change the tube to collect the liquid. If there is no obvious inflection, change the tube appropriately according to the situation;

[0117] First purification A: Ammonia mobile phase (pH = 10.4) B: Pure acetonitrile

[0118] Gradient: 5 - 45 min / B: 10% - 40%

[0119] Second purification: 0.1% TFA / water, 0.1% TFA / acetonitrile

[0120] Gradient: 5 - 45 min / B: 35% - 55%

[0121] Step S12. Sample salt conversion

[0122] Lyophilize the fraction, weigh it to be 13 mg, and completely dissolve 15 mg of the sample with acetonitrile and water;

[0123] Step S13. Lyophilization and sub-packaging

[0124] Load the qualified fraction after purification into a lyophilization tray, cover it, and then put it into a freeze dryer for lyophilization. After the lyophilization is completed, take out the lyophilization tray, weigh and sub-package the polypeptide sample, and store it at -20°C;

[0125] Step S14. QC / QA inspection and release

[0126] Take a small sample and send it to QC for various index detections according to the order requirements. After the detection is qualified, QC / QA releases it according to the standard process, and the qualified sample is sent to the warehouse for shipment.

[0127] An incretin polypeptide analog drug composition and its application, including a co-agonist or its salt or its solvate, and a pharmaceutically acceptable excipient, and its application in drugs for treating diabetes or weight loss.

[0128] Example 2: Detection of the activity of cells overexpressing GLP-1 receptor, GIP receptor or GCG receptor in vitro

[0129] 1) First, construct HEK-293 cells overexpressing human GLP-1, GIP or GCG receptor by lentiviral transfection respectively, and then determine the agonist activity of each compound on the corresponding receptor by measuring the cAMP signal response level of the above cells. The intracellular cAMP content is measured using a kit based on HTRF (homogeneous time-resolved fluorescence) technology. Adjust the cell density to 2.0×10 with DMEM blank medium5 Inoculate at a density of 100 - 150 μL / well into a 96-well cell culture plate and culture at 37 °C under 5% CO₂ for 12 to 20 hours;

[0130] 2) Dilute the test article to 8 concentrations and add 100 - 200 μL / well, with duplicate wells for each dilution. Incubate in the dark at room temperature for 2 - 5 h or at 4 °C for 12 - 18 h;

[0131] 3) Add 5 μL of cAMP-d2 working solution to each well and 5 μL of cAMP Eu-Cryptate antibody working solution to each well. Cover the 96-well plate with a sealing film and incubate at room temperature for 60 min. Process using four-parameter regression fitting to obtain the EC50 value of the test sample;

[0132] 4) Use natural human ligands GLP-1(7 - 37), GIP, and GCG as positive controls for the agonist effects of the test compounds on the receptors. The EC50 values and relative natural human ligands for the determination results of the agonist effects of the in vitro GLP-1 receptor, in vitro GIP receptor, and in vitro GCG receptor are shown in Table 1 below:

[0133] Table 1

[0134]

[0135]

[0136] As can be seen from Table 1 above, the co-agonist prepared in the present invention has GLP-1 receptor, GIP receptor, and GCG receptor agonist activities.

[0137] Example 3: Pharmacokinetics study in male beagle dogs

[0138] 1) Select healthy male beagle dogs with free access to food, weighing 9 - 12 kg. Dissolve Compound 01, Compound 02, Compound 06, Compound 07, or Retatrutide in phosphate buffer (pH = 8.0) and administer 0.05 mL / kg subcutaneously to the hind limb once. Collect plasma (anticoagulated with EDTA-K2) before and after administration up to 168 or 192 hours with at least 12 sampling points, and store the separated plasma in a -60 °C refrigerator;

[0139] 2) Add a polypeptide analogue as an internal standard, extract the test peptide and internal standard with a solvent containing methanol:acetonitrile = 1:2, and use LC / MS-MS to measure the responses of the fragments of the test peptide and internal standard. Convert to the concentration of the test peptide through a standard curve. Among them, Retatrutide is a drug developed by Eli Lilly and Company that can treat obesity and type 2 diabetes simultaneously. This drug is an incretin polypeptide analogue and has a co-activation effect on the GLP-1 receptor, GIP receptor, and GCG receptor;

[0140] The pharmacokinetic parameters are shown in Table 2 below:

[0141] Compound Dosage <![CDATA[T 1 / 2 (hour)]]> <![CDATA[AUC 0-t (ng*h / mL)]]> Retatrutide 0.05 mg / kg 69.5 24891 01 0.05 mg / kg 121.8 29882 02 0.05 mg / kg 57.1 21330 07 0.05 mg / kg 71.6 16263

[0142] As can be seen from Table 2 above, for the incretin mimetics prepared by the present invention, in the pharmacokinetic experiment in beagle dogs, compared with Retatrutide: the half-lives of Compound 02 and Compound 07 are close to or similar to that of Retatrutide; the half-life of Compound 01 is significantly longer than the former, having the potential to be applicable to a longer interval dosing strategy.

[0143] Example 4: OGTT hypoglycemic study in mice, using oral administration

[0144] 1) Select SPF-grade male mice, weighing 30 - 35 g;

[0145] 2) Compound 01, Compound 02, Retatrutide, Semaglutide or blank excipients dissolved in phosphate buffer (PH = 8.0) were formulated into suspensions and orally administered to male healthy mice that had fasted for 4 hours. Blood glucose was measured 30 minutes later, and glucose (2 g / kg) was orally administered. Blood glucose of the mice was measured at 30 minutes, 60 minutes, 90 minutes, and 120 minutes subsequently.

[0146] Oral glucose tolerance test:

[0147] ① Experimental materials

[0148] Experimental animals: C57 / BL6J mice, 20 in number, 8 weeks old, male;

[0149] Experimental drug preparations: PBS buffer, 200 mg / mL glucose saline solution, Compound 01, Compound 07 polypeptide, Rybelsus or blank excipients;

[0150] ② Experimental methods

[0151] Grouping: control group and experimental group;

[0152] Fasting: Fast in a clean cage for 12 h, and keep normal drinking water during this period;

[0153] Fasting basal blood glucose measurement: Gently pick up the mouse, cut off the end 1 - 2 mm of the mouse's tail, gather the blood into a drop, discard the first drop, and use a blood glucose meter and test strips to detect the blood glucose value of the second drop of blood in the same way, which is regarded as the blood glucose value at 0 min;

[0154] Glucose gavage: The mice were allowed to stabilize for a moment, and then glucose was gavaged at a dose of 2 g / kg. The mice were gently grasped, and according to the standard gavage procedure, a 1 mL syringe was connected to an 8-gauge gavage needle to administer the glucose solution. Timing started after the injection was completed, and the operation time for each mouse was controlled within 1 - 2 minutes;

[0155] Drug administration: Oral gavage of Compound 01, Compound 02, Compound 05, Compound 07 polypeptide, Semaglutide, Retatrutide, or blank excipient;

[0156] Data collection: Using the above-mentioned tail tip blood sampling method, the blood glucose values of the mice were measured at 30, 60, 90, and 120 minutes respectively, and the detected data are as Figures 1-4 shown;

[0157] According to the appendix Figures 1-4 It can be seen that the incretin analog prepared by the present invention can have a significant hypoglycemic effect through oral administration. In the comparative study, it was also confirmed that Compound 01 has a hypoglycemic effect comparable to that of Rybelsus and semaglutide.

[0158] Example 5: OGTT hypoglycemic study in mice, using subcutaneous administration

[0159] 1) Select SPF-grade male mice, weighing 30 - 35 g;

[0160] 2) Compound 01, Compound 02, semaglutide, or blank solvent dissolved in phosphate buffer (PH = 8.0) was administered to healthy male mice freely fed by intraperitoneal injection. Immediately after that, the blood glucose was measured after fasting for 4 hours, and glucose (2 g / kg) was orally administered. The blood glucose of the mice was measured at 30 minutes, 60 minutes, 90 minutes, and 120 minutes subsequently;

[0161] Oral glucose tolerance test:

[0162] ① Experimental materials

[0163] Experimental animals: C57 / BL6J mice, 20 in number, 8 weeks old, male;

[0164] Experimental drug preparations: PBS buffer, 200 mg / mL glucose saline solution, Compound 01, Compound 07 polypeptide, Rybelsus, or blank excipient;

[0165] ② Experimental method

[0166] Grouping: Control group and experimental group;

[0167] Fasting: Fast in a clean cage for 12 h, and keep normal drinking water during this period;

[0168] Fasting basal blood glucose measurement: Gently pick up the mouse, cut off the end 1 - 2 mm of the mouse's tail, gather the blood into a drop, discard the first drop, and use a blood glucose meter and test strips to detect the blood glucose value of the second drop of blood in the same way, which is regarded as the blood glucose value at 0 min;

[0169] Glucose gavage: Let the mouse stabilize for a moment, then gavage glucose at a dose of 2 g / kg. Gently hold the mouse and, according to the standard gavage procedure, connect an 8 - gauge gavage needle to a 1 - mL syringe to administer the glucose solution. Start timing after the injection is completed, and control the operation time for each mouse within 1 - 2 minutes;

[0170] Administration: Subcutaneously inject compound 01, compound 02 polypeptide, Semaglutide, or blank excipient;

[0171] Data collection: Use the above - mentioned method of taking blood from the tail tip to detect the blood glucose values of the mice at 30, 60, 90, and 120 min respectively. The detected data are as Figures 5-6 shown;

[0172] According to the appendix Figures 5-6 As can be seen, the incretin analog prepared by the present invention can have a significant hypoglycemic effect through oral administration. In the comparative study, it is also confirmed that its compound 01 has a hypoglycemic effect comparable to that of semaglutide.

[0173] Although the specific embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these specific embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An incretin polypeptide analog, including a co-agonist, characterized in that: The co-agonist is a GLP-1, GIP and GCG three-receptor co-agonist polypeptide acylated and connected to a fatty acid side chain. The amino acid sequence of the polypeptide or its salt or solvate is shown in the following formula: Y(Aib)QGT(α-meF)TSDX 10 SIX 13 LDX 16 X 17 AQ(Aib)X 21 X 22 IX 24 X 25 LX 27 EGGPSSGAPPPS-NH2; in: X 10 Selected from Y or V; X 13 is selected from Y, α-meF or α-meY; X 16 Selected from Orn; X 17 The ε-amino group or other functional groups are conjugated to C through a linker. 16 -C 22 fatty acid; X 21 Selected from E or Aib; X 22 Selected from F, α-meF; X 24 Selected from E or DG lu; X 25 is selected from W, α-meW or Y; X 27 Select from I or V. The Aib is α-aminoisobutyric acid; α-meF is α-methylphenylalanine; α-meY is α-methyltyrosine; Orn is ornithine; α-meW is α-methyltryptophan; and D-Glu is D-glutamic acid.

2. The co-agonist according to claim 1, characterized in that The amino acid sequence of the polypeptide is selected from any one of the following: Compound 01: Y(Aib)QGT(α-meF)TSDYSI(α-meF)LD(Orn)(Lys(AEEA-AEEA-γGlu-C20diacid))AQ(Aib)EFIEYLIEGGPSSGAPPPS-NH2; Compound 02: Y(Aib)QGT(α-meF)TSDYSI(α-meY)LD(Orn)(Lys(AEEA-AEEA-γGlu-C20diacid))AQ(Aib)(Aib)FIEYLIEGGPSSGAPPPS-NH2; Compound 03: Y(Aib)QGT(α-meF)TSDYSI(α-meY)LD(Orn)(Lys(AEEA-γGlu-C20diacid))AQ(Aib)EFIEYLVEGGPSSGAPPPS-NH2; Compound 04: Y(Aib)QGT(α-meF)TSDYSIYLD(Orn)(Lys(εK-εK-γGlu-C20diacid))AQ(Aib)EFI(D-Glu)WLIEGGPSSGAPPPS-NH2; Compound 05: Y(Aib)QGT(α-meF)TSDYSI(α-meY)LD(Orn)(Lys(εK-εK-γGlu-C20diacid))AQ(Aib)EFIEWLIEGGPSSGAPPPS-NH2; Compound 06: Y(Aib)QGT(α-meF)TSDVSI(α-meF)LD(Orn)(Lys(AEEA-AEEA-γGlu-C18diacid))AQ(Aib)E(α-meF)IE(α-meW)LVEGGPSSGAPPPS-NH2; Compound 07: Y(Aib)QGT(α-meF)TSDYSI(α-meF)LD(Orn)(Lys(AEEA-AEEA-γGlu-C20diacid))AQ(Aib)(Aib)FIEWLVEGGPSSGAPPPS-NH2.

3. The co-agonist according to claim 1, characterized in that The co-agonist is linked to the fatty acid side chain via the epsilon amino group on the amino acid K residue at position 17 of the polypeptide and via a linker.

4. The co-agonist according to any one of claims 1 to 3, characterized in that The fatty acid side chain is selected from any one of the following: HOOC(CH2) 16 CO-, HOOC(CH2) 17 CO-, HOOC(CH2) 18 CO-, HOOC(CH2) 19 CO-, HOOC(CH2) 20 CO-, HOOC(CH2) 21 CO-, HOOC(CH2) 22 CO-, (HO)3P(CH2) 16 CO-, (HO)3P(CH2) 18 CO- or (HO)3P(CH2) 20 CO-.

5. The co-agonist according to any one of claims 1 to 4, characterized in that The fatty acid side chain is connected to the amino acid K via a linker.

6. The co-agonist according to any one of claims 1 to 5, characterized in that The linker is selected from any one of -(AEEA)-(AEEA)-γE-, -εK-εK-γE- or -(AEEA)-γE-.

7. The co-agonist according to any one of claims 1 to 4, characterized in that Also included are fatty acid components having a linker and a structure having the formula: -(εK) a -γE-CO(CH2) c -COOH or-(AEEA) b -γE-CO(CH2) c -COOH Where a is 1 or 2, b is 1 or 2, and c is 16 or 18.

8. A pharmaceutical composition of incretin polypeptide analogs and its application, characterized in that: It comprises the co-agonist or its salt or solvate as described in any one of claims 1 to 7, and a pharmaceutically acceptable excipient, and its use in a drug for treating diabetes or weight loss.

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