GLP-1 AGONIST POLYPEPTIDE COMPOUND AND SALT THEREOF, SYNTHESIS METHOD THEREFOR AND USE THEREOF

Abstract

The disclosure provides a GLP-1 agonist polypeptide compound having an amino acid sequence: H Xaa.sup.1EGTFTSDVSSYLE Xaa.sup.2QAA Xaa.sup.3EFIAWLVRGRG (SEQ ID NO: 1). The C-terminal amino acid of the polypeptide compound is a carboxyl or the carboxyl is amidated. Xaa.sup.1 and Xaa.sup.2 are the same or different; R.sub.1 is: a straight-chain or branched-chain alkyl containing 2-6 carbon atoms, R.sub.2 is: H or CH.sub.3, X is: O, S or N—CH.sub.3, in the formula, R.sub.1 and R.sub.2 alkyl is optionally substituted by 1-6 halogen atoms.

Claims

1. A GLP-1 agonist polypeptide compound, having an amino acid sequence: H Xaa.sup.1EGTFTSDVSSYLE Xaa.sup.2QAA Xaa.sup.3EFIAWLVRGRG (SEQ ID NO: 1), wherein a C-terminal amino acid of the polypeptide compound is a carboxyl or the carboxyl is amidated; wherein: Xaa.sup.1 is: ##STR00010## Xaa.sup.2 is: G, ##STR00011## Xaa.sup.1 and Xaa.sup.2 are the same or different; R.sub.1 is: a straight-chain or branched-chain alkyl containing 2-6 carbon atoms, R.sub.2 is: H or CH.sub.3, X is: O, S or N—CH.sub.3, in the formula, R.sub.1 and R.sub.2 alkyl is optionally substituted by 1-6 halogen atoms; Xaa.sup.3 is: ##STR00012##

2. The GLP-1 agonist polypeptide compound of claim 1, wherein n is a natural number ranging from 12 to 20; and m is a natural number ranging from 0 to 3.

3. The GLP-1 agonist polypeptide compound of claim 1, wherein the amino acid sequence of the polypeptide compound is one of the following sequences: TABLE-US-00033 (1) NO: 1 HX.sub.2EGTFTSDVSSYLEX.sub.2QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (2) NO: 2 HX.sub.2EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (3) NO: 3 HX.sub.3EGTFTSDVSSYLEX.sub.3QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (4) NO: 4 HX.sub.3EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (5) NO: 5 HX.sub.4EGTFTSDVSSYLEX.sub.4QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (6) NO: 6 HX.sub.4EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (7) NO: 7 HX.sub.5EGTFTSDVSSYLEX.sub.5QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (8) NO: 8 HX.sub.5EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (9) NO: 9 HX.sub.6EGTFTSDVSSYLEX.sub.6QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (10) NO: 10 HX.sub.6EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (11) NO: 11 HX.sub.7EGTFTSDVSSYLEX.sub.7QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (12) NO: 12 HX.sub.7EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (13) NO: 13 HX.sub.8EGTFTSDVSSYLEX.sub.8QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (14) NO: 14 HX.sub.8EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (15) NO: 15 HX.sub.9EGTFTSDVSSYLEX.sub.9QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (16) NO: 16 HX.sub.9EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (17) NO: 17 HX.sub.10EGTFTSDVSSYLEX.sub.10QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu- AEEA-AEEA)EFIAWLVRGRG (18) NO: 18 HX.sub.10EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (19) NO: 19 HX.sub.11EGTFTSDVSSYLEX.sub.11QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu- AEEA-AEEA)EFIAWLVRGRG (20) NO: 20 HX.sub.11EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (21) NO: 21 HX.sub.12EGTFTSDVSSYLEX.sub.12QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG (22) NO: 22 HX.sub.12EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG wherein: ##STR00013##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a diagram showing the effect of each test drug on the random blood sugar of db/db mice in the experiment;

[0066] FIG. 2 is a diagram showing the rate of change of each test drug to random blood sugar in db/db mice in the experiment;

[0067] FIG. 3 is a diagram showing the effect of each test drug on the body weight of ob/ob mice in the experiment;

[0068] FIG. 4 is a diagram showing the body weight change rate of each test drug to ob/ob mice in the experiment.

DETAILED DESCRIPTION

[0069] The disclosure is described with the following embodiments, but these embodiments do not constitute any limitation to the rights of the disclosure.

[0070] In the formula:

##STR00009##

EXAMPLE 1

[0071] Synthesis of Polypeptide Compound of NO: 1:

TABLE-US-00003 HX.sub.2EGTFTSDVSSYLEX.sub.2QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0072] 1.1 Swelling of Resin

[0073] 10 g of Wang-Resin (with the substitution degree of 0.53 mmol/g) was weighed and swelled for 30 min with 100 mL of DCM, suction filtration was conducted to remove DCM, swelling was conducted for 30 min with 100 mL of DMF, and the resin was washed with 100 mL of DMF and 100 mL of DCM respectively.

[0074] 1.2 Synthesis of Fmoc-Gly-Wang-Resin

[0075] Fmoc-Gly-OH (15 mmol), HOBt (18 mmol) and DIC (18 mmol) were dissolved in 100 mL of DMF, then the solution was added into resin obtained in the previous step to react for 2 hours, after reaction ended, the reaction solution was filtered out, and the resin was washed 3 times with 100 mL of DCM and 100 mL of DMF respectively.

[0076] 1.3 Removal of Fmoc Protecting Group

[0077] A 25% piperidine/DMF (V/V) solution containing 0.1 M of HOBt was added into the washed resin to remove Fmoc, and after the reaction ended, the resin was washed 3 times with 100 mL of DCM and 100 mL of DMF respectively.

[0078] 1.4 Extension of Peptide Chain

[0079] According to the sequence, the steps of deprotection and coupling were repeated to sequentially bind corresponding amino acids, and the corresponding amino acids were sequentially bound until peptide chain synthesis was completed, to obtain the peptide resin. The specific coupling protected amino acids were as follows:

[0080] Fmoc-Arg.sup.30(Pbf)-OH, Fmoc-Gly.sup.29-OH, Fmoc-Arg.sup.28(Pbf)-OH, Fmoc-Val.sup.27-OH, Fmoc-Leu.sup.26-OH, Fmoc-Trp.sup.25(Boc)-OH, Fmoc-Ala.sup.24-OH, Fmoc-Ile.sup.23-OH, Fmoc-Phe.sup.22-OH, Fmoc-Glu.sup.21(OtBu)-OH, Fmoc-Lys.sup.20(Xaa.sup.3)-OH, Fmoc-Ala.sup.19-OH, Fmoc-Ala.sup.18-OH, Fmoc-Gln.sup.17(Trt)-OH, Fmoc-X.sub.2-OH, Fmoc-Glu.sup.15(OtBu)-OH, Fmoc-Leu.sup.14-OH, Fmoc-Tyr.sup.13(tBu)-OH, Fmoc-Ser.sup.12(tBu)-OH, Fmoc-Ser.sup.11(tBu)-OH, Fmoc-Val.sup.10-OH, Fmoc-Asp.sup.9(OtBu)-OH, Fmoc-Ser.sup.8(tBu)-OH, Fmoc-Thr.sup.7(tBu)-OH, Fmoc-Phe.sup.6-OH, Fmoc-Thr.sup.5(tBu)-OH, Boc-His.sup.1(Trt)-X.sub.2-Glu.sup.3(OtBu)-Gly.sup.4-OH were coupled to Fmoc-Gly.sup.31-Wang one by one by using a solid-phase synthesis method;

[0081] 1.5 Cleavage of Peptide Resin

[0082] Trifluoroacetic acid was measured to a reactor and cooled to −10° C. to 0° C., triisopropylsilane, 1,2-dithioglycol and purified water (TFA:TIS:EDT:H.sub.2O=95:2:2:1) were added and stirred to be uniformly mixed. Peptide resin was slowly added, heated to 20-30° C., and a cracking reaction was conducted for 115-125 min. After the reaction ended, the resin was filtered out, the filtered resin was washed with M×8×20% ml of TFA, the filtered solution and the washing solution were completely transferred into M×8×1.2×4 ml of diethyl ether, stirred for 5-10 min, kept standing to precipitate for 15 min or more. The precipitated turbid liquid was added into a centrifugal machine, and solids were centrifuged and collected; the solids were washed with diethyl ether six times, and the amount of diethyl ether used each time was not less than 5 L. The solids were subjected to vacuum drying for 6-10 hat the temperature of 20-35° C., and 10.90 g of crude peptide was obtained.

[0083] 2. Purification of NO: 1 Polypeptide Compound

[0084] Purification was conducted through preparative liquid chromatography, and the chromatographic conditions were that a C18 column (100 mm×250 mm, 10 μm) was adopted; a mobile phase A was 0.1% H.sub.3PO.sub.4/water (V/V), and a mobile phase B was 0.1% H.sub.3PO.sub.4/acetonitrile (V/V); the mobile phase gradient was 20%-60% of the mobile phase B, and the time was 60 min; the flow velocity was 200 mL/min, the detection wavelength was 214 nm, fractions with the purity larger than 98.0% were collected, and 3.10 g of samples were obtained through freeze drying after rotary evaporation and concentration.

[0085] The synthesis methods of polypeptide compounds of NO: 2-30 were the same as that of Example 1:

EXAMPLE 2

[0086] Synthesis of Polypeptide Compound of NO: 2:

TABLE-US-00004 HX.sub.2EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0087] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.93 g.

EXAMPLE 3

[0088] Synthesis of Polypeptide Compound of NO: 3:

TABLE-US-00005 HX.sub.3EGTFTSDVSSYLEX.sub.3QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0089] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.87 g.

EXAMPLE 4

[0090] Synthesis of Polypeptide Compound of NO: 4:

TABLE-US-00006 HX.sub.3EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0091] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.55 g.

EXAMPLE 5

[0092] Synthesis of Polypeptide Compound of NO: 5

TABLE-US-00007 HX.sub.4EGTFTSDVSSYLEX.sub.4QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0093] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.60 g.

EXAMPLE 6

[0094] Synthesis of Polypeptide Compound of NO: 6

TABLE-US-00008 HX.sub.4EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0095] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.61 g.

EXAMPLE 7

[0096] Synthesis of Polypeptide Compound of NO: 7

TABLE-US-00009 HX.sub.5EGTFTSDVSSYLEX.sub.5QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0097] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.1 g.

EXAMPLE 8

[0098] Synthesis of Polypeptide Compound of NO: 8

TABLE-US-00010 HX.sub.5EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0099] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.35 g.

EXAMPLE 9

[0100] Synthesis of Polypeptide Compound of NO: 9

TABLE-US-00011 HX.sub.6EGTFTSDVSSYLEX.sub.6QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0101] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.70 g.

EXAMPLE 10

[0102] Synthesis of Polypeptide Compound of NO: 10

TABLE-US-00012 HX.sub.6EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEQ)-EFIAWLVRGRG

[0103] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.90 g.

EXAMPLE 11

[0104] Synthesis of Polypeptide Compound of NO: 11

TABLE-US-00013 HX.sub.7EGTFTSDVSSYLEX.sub.7QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0105] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.74 g.

EXAMPLE 12

[0106] Synthesis of Polypeptide Compound of NO: 12

TABLE-US-00014 HX.sub.7EGTFTSDVSSYLEX.sub.7QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0107] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.22 g.

EXAMPLE 13

[0108] Synthesis of Polypeptide Compound of NO: 13

TABLE-US-00015 HX.sub.8EGTFTSDVSSYLEX.sub.8QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0109] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.48 g.

EXAMPLE 14

[0110] Synthesis of Polypeptide Compound of NO: 14

TABLE-US-00016 HX.sub.8EGTFTSDVSSYLEX.sub.8QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0111] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 5.30 g.

EXAMPLE 15

[0112] Synthesis of Polypeptide Compound of NO: 15

TABLE-US-00017 HX.sub.9EGTFTSDVSSYLEX.sub.9QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0113] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.70 g.

EXAMPLE 16

[0114] Synthesis of Polypeptide Compound of NO: 16

TABLE-US-00018 HX.sub.9EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0115] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.67 g.

EXAMPLE 17

[0116] Synthesis of Polypeptide Compound of NO: 17

TABLE-US-00019 HX.sub.10EGTFTSDVSSYLEX.sub.10QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0117] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.33 g.

EXAMPLE 18

[0118] Synthesis of Polypeptide Compound of NO: 18

TABLE-US-00020 HX.sub.10EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0119] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.440 g.

EXAMPLE 19

[0120] Synthesis of Polypeptide Compound of NO: 19

TABLE-US-00021 HX.sub.11EGTFTSDVSSYLEX.sub.11QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0121] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 2.70 g.

EXAMPLE 20

[0122] Synthesis of Polypeptide Compound of NO: 20

TABLE-US-00022 HX.sub.11EGTFTSDVSSYLEX.sub.1QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0123] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.92 g.

EXAMPLE 21

[0124] Synthesis of Polypeptide Compound of NO: 21

TABLE-US-00023 HX.sub.12EGTFTSDVSSYLEX.sub.12QAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0125] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.62 g.

EXAMPLE 22

[0126] Synthesis of Polypeptide Compound of NO: 22

TABLE-US-00024 HX.sub.12EGTFTSDVSSYLEXQAAK(HOOC-(CH.sub.2).sub.16-CO-γ-Glu-AEEA- AEEA)EFIAWLVRGRG

[0127] The synthesis procedures were the same as those in Example, and the purified sample solution was collected and freeze-dried to obtain a purified product in a mass of 3.80 g.

[0128] The relevant pharmacological experimental methods and results of a GLP-1 agonist polypeptide compound (hereinafter referred to as a polypeptide compound):

[0129] 1. GLP-1 Receptor Agonist Activity of Polypeptide Compound

[0130] A glucagon-like peptide-1 receptor (GLP-1R) belongs to a B-type G protein coupling receptor, and plays an important role in the conditions of blood glucose and weight and is recognized as an important target of antidiabetic drugs. A Chinese hamster ovary cell line (CHO) capable of stably expressing human GLP-1R was used for measuring the induction activity of a polypeptide compound sample and a control compound to a GLP-1R downstream cAMP signal.

[0131] A wild human GLP-1R (NM_002062.5) was subjected to transient transfection and screened for two weeks by 600 μg/ml of hygromycin-B to obtain a cell line which was recombined and integrated into an FlpInCHO (Invitrogen) cell stable expression system. The culture condition of the FlpInCHO cell was as follows: 10% of heat-inactivated fetal calf serum was added to a DMEM culture solution, and the culture was performed in a 5% carbon dioxide cell incubator.

[0132] In a downstream cAMP signal detection test, cells were placed in a 6-well cell culture plate for overnight culture, then transferred into a 384-well plate according to the concentration of 8,000 cells in each well and continuously cultured for 24 h under the conditions of 37° C. and 5% of CO.sub.2. In the test, an LANCE cAMP detection kit was used for measuring the cAMP signal intensity. After the cells were incubated for 30 min, the LANCE cAMP detection kit was used, a microplate reader was used for measuring fluorescence value, a standard curve was established to convert the fluorescence value into corresponding cAMP value, and the EC.sub.50 value of the compound was calculated by nonlinear regression of Graphpad Prism 7.0 software.

[0133] cAMP Signal Analysis of GLP-1R

TABLE-US-00025 Peptides GLP-1R EC.sub.50 (pM) Semaglutide 645.17 ± 48.37  GLP-1(7-36) 121.64 ± 87.78  NO: 1 91733.33 ± 9692.46  NO: 2 21410.0 ± 1320.27 NO: 3 2842.33 ± 512.87  NO: 4 2024.33 ± 276.5  NO: 5 914.63 ± 126.1  NO: 6 623.22 ± 83.5  NO: 7 8795.33 ± 1407   NO: 8 7924.72 ± 1062   NO: 9 675.33 ± 67    NO: 10 524.72 ± 52    NO: 11 8095.65 ± 507    NO: 12 8924.22 ± 310    NO: 13 5590.19 ± 575    NO: 14 5400.39 ± 364.7  NO: 15 2901.56 ± 390    NO: 16 2508.81 ± 307    NO: 17 3324.82 ± 677.2  NO: 18 3124.17 ± 511.8  NO: 19 791.33 ± 63.5  NO: 20 692.89 ± 55.7  NO: 21 652.07 ± 41.7  NO: 22 630.43 ± 51.9 

[0134] An in-vivo animal test was carried out for polypeptide compounds NO: 3 and NO: 4, a detailed comparison experiment was carried out in the aspects of hypoglycemic effect and weight loss, and the experiment scheme and results were as follows.

[0135] Experiments on Glucose Lowering of Polypeptide Compound and Effect on Weight

[0136] After DB/DB mice with high blood glucose were acclimated for 5-7 days, a non-fasting random blood glucose level was determined; after fasting for 12 h, a fasting blood glucose level was determined by a rapid blood glucose meter. With comprehensive consideration on the body weight, and fasting blood glucose 12 hours after fasting and random blood glucose results, the animals were randomly grouped (random block design) and were divided into a solvent control group, a polypeptide compound group (selected test drugs: NO: 3 and NO: 4), a positive control Semaglutide group and a known control group SPN013 according to the random weight, the random blood glucose and the fasting blood glucose. Mice in each test drug group and each control group received single subcutaneous injection of each test drug or a control solution respectively, and the mice in the model control group received single subcutaneous injection of PBS buffer solution; and the experiment grouping and dosage setting conditions were shown in Table 1:

TABLE-US-00026 TABLE 1 Grouping of experimental animals before administration Dose Volume parameter Route of (nmol/ of administration administra- Frequency of Group kg) (μL/g) tion administration n Solvent — 5 SC Single dose 8 control (PBS) Semaglutide 30 5 SC Single dose 8 NO: 3 30 5 SC Single dose 8 NO: 4 30 5 SC Single dose 8 SPNO13 30 5 SC Single dose 8

[0137] Note: n: the number of the mice in each group; dosing volume: 5 μl/g according to the body weight of the mice.

[0138] Experimental instrument: fast blood glucose meter (Johnson & Johnson, One Touch UltraEasy; instrument serial number: MGC23B4ER, MGC23B5ER)

[0139] Experimental Observation:

[0140] Clinical symptoms: the formulation of this experimental protocol and any modification would be implemented after evaluation and approval by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica. Animal health and death were monitored daily.

[0141] Body weight: after grouping the animals, the body weight was measured once a day at a fixed time period

[0142] Experimental Indicators:

[0143] Blood glucose level: the blood was taken from the tail tip of the mice to measure the blood glucose level with a fast blood glucose meter and blood glucose test strips.

[0144] After grouping, different test articles were subcutaneously administered once, and blood glucose levels were measured in 0 h, 2 h, 4 h, 6 h, 10 h, 24 h, 34 h, 48 h, 58 h, 72 h, 82 h, and 96 h following the administration, respectively.

[0145] The glucose lowering effect of the test article was evaluated by BG (mmol/L) or AUC (mmol/L.Math.min).

[0146] Calculation of AUC:

AUC=[(BG.sub.0+BG.sub.2)×2+(BG.sub.2+BG.sub.4)×2+(BG.sub.4+BG.sub.6)×2+(BG.sub.6+BG.sub.10)×4+(BG.sub.10+BG.sub.24)×14+(BG.sub.24+BG.sub.34)×10+(BG.sub.34+BG.sub.48)×14+(BG.sub.48+BG.sub.58)×10+(BG.sub.58+BG.sub.72)×14+(BG.sub.72+BG.sub.82)×10+(BG.sub.82+BG.sub.96)×14]/2.

[0147] Data Analysis:

[0148] T test was used for comparison between two groups. One-way ANOVA was used for comparison among three or more groups. If there was a significant difference in F value, multiple comparisons should be performed after ANOVA analysis. All data analyses were performed with SPSS 17.0. If p<0.05, it was considered a significant difference.

[0149] As shown in Tables 2 to 4 and FIGS. 1 to 2, the results of blood glucose lowering experiment data and charts showed that when the administration concentration of the polypeptide compound of the disclosure was 30 nmol/kg, the glucose lowering effect was almost consistent with that of Semaglutide; the positive control group and the polypeptide compound group were both significantly different from the solvent control group. As shown in Tables 5-6 and FIGS. 3-4, the solvent control group had no obvious effect on the random weight and the variation of weight of the db/db mice after single subcutaneous injection. The positive control group and the polypeptide compound group were both significantly different from the solvent control group; particularly, the difference was most significant in 24 h following administration. Meanwhile, the experiment results showed that the polypeptide compounds NO: 3, NO: 4 and SPN013 could significantly reduce the random weight variation of the db/db mice in 24 h, 48 h and 72 h following the single subcutaneous injection, which was superior to the Semaglutide group.

[0150] Therefore, the single subcutaneous injection of the GLP-1 agonist compound could significantly reduce the random blood glucose of the ob/ob mice with type 2 diabetes, wherein the effects of NO: 3 and NO: 4 were comparable to that of Semaglutide with the same dosage. The NO: 3 and NO: 4 polypeptide compounds showed a better weight control effect.

[0151] 3. Pharmacokinetic Experiments of Peptide Compounds

[0152] Pharmacokinetic comparison experiment of Semaglutide, SPN009 (NO: 3) and SPN010 (NO: 4): 9 male rats, 3 in each group, were respectively given Semaglutide, SPN009 and SPN010. A single subcutaneous injection was performed with a dose of 0.02 mg/kg; blood collection was performed by tail clipping, and the blood collection time points were: 0 h, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 48 h and 72 h. The compound concentration in plasma was determined by LC-MS/MS, all data were collected and exported by Unifi 1.9.3 software, and Excel software was used for data calculation. A non-compartmental model of DAS 3.0 software was used to calculate the pharmacokinetic parameters of rats after administration. The results were shown in Table 7.

[0153] As shown in Table 7, the main pharmacokinetic parameters t.sub.1/2 and Tmax of NO: 3 and NO: 4 were comprehensively superior to those of Semaglutide when the rats received a single subcutaneous administration at a concentration of 0.02 mg/kg.

TABLE-US-00027 TABLE 2 Effect of each test drug on random blood glucose in db/db mice in experiment (X ± s, n = 8) Random blood Dosage(nmol/ glucose Blood glucose at different time after administration (mmol/L) Groups kg) (mmol/L) 0 h 2 h 4 h Solvent — 25.7 ± 3.1 29.3 ± 2 28.9 ± 3.5 29.7 ± 2.sup.  control (PBS) Semaglutide 30 25.8 ± 3.9 .sup. 26.8 ± 3.2 14.9 ± 4.1 13.2 ± 3.2 NO: 3 30 25.6 ± 2.7 27.3 ± 2 15.9 ± 4.sup.  14.3 ± 3.5 NO: 4 30 25.5 ± 2.sup.  .sup. 26.9 ± 2.7 14.4 ± 3.5 12.5 ± 2.2 SPNO13 30 25.6 ± 2.7 29.6 ± 2 15.3 ± 3.3 18.2 ± 4.4 Blood glucose at different time after administration (mmol/L) Groups 6 h 10 h 24 h 36 h 48 h 56 h 72 h Solvent 27.4 ± 2.3 33.3 ± 0 31.1 ± 2.2 30.6 ± 2.2 29.3 ± 2.8 31.1 ± 2.2 33.3 ± 0.sup.  control (PBS) Semaglutide 13.3 ± 3.7 .sup. 11.5 ± 3.7 12.6 ± 5.6 21.1 ± 4.1 21.3 ± 5.6 28.3 ± 4.8 32.3 ± 1.9 NO: 3 14.1 ± 5.sup.  .sup. 11.8 ± 3.4 11.1 ± 2.4 16.7 ± 6.8 .sup. 21 ± 4.1 28.9 ± 3.4 31.7 ± 1.9 NO: 4 .sup. 13 ± 4.6 11.9 ± 4 11.1 ± 4.6 18.5 ± 8.sup.  19.6 ± 7.1 .sup. 28 ± 6.1 32.5 ± 1.5 SPNO13 16.9 ± 4.2 17.8 ± 6 .sup. 16 ± 4.5 22.7 ± 6.3 23.7 ± 3.9 31.5 ± 1.5 32.4 ± 1.2

TABLE-US-00028 TABLE 3 Rate of change of random blood sugar of db/db mice with each test drug in experiment Random Dosage(nmol/ blood Ratio(%) Groups kg) glucose 0 h 2 h 4 h 6 h 10 h 24 h 36 h 48 h 56 h 72 h Solvent — 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 control (PBS) Semaglutide 30 100.7 91.4 51.4 44.5 48.3 34.6 40.6 68.9 72.7 91.1 96.8 NO: 3 30 99.6 93.0 55.1 48.3 51.4 35.5 35.7 54.5 71.7 92.9 95.1 NO: 4 30 99.2 91.6 49.8 42.1 47.5 35.8 35.6 60.4 66.9 90.1 97.6 SPNO13 30 99.8 100.9 53.0 61.2 61.5 53.3 51.5 74.1 81.0 101.2 97.3

TABLE-US-00029 TABLE 4 T test of random blood sugar of db/db mice with each test drug in experiment Random Dosage(nmol/ blood T-Test P value Groups kg) glucose 0 h 2 h 4 h 6 h 10 h 24 h 36 h 48 h 56 h 72 h Solvent — — — — — — — — — — — — control (PBS) Semaglutide 30 0.92 0.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.16 0.14 NO: 3 30 0.95 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.14 0.03 NO: 4 30 0.88 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.20 0.16 SPNO13 30 0.97 0.80 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.70 0.05

TABLE-US-00030 TABLE 5 Random weight of db/db mice with each test drug in experiment (X ± s, n = 8) Dosage (nmol/ Random body weight at different time after administration (mmol/L) Groups kg) Day −2 Day −1 Day 0 Day 1 Day 2 Day 3 Solvent — 40.70 ± 1.40 39.64 ± 1.32 40.92 ± 1.35 40.80 ± 1.19 41.21 ± 1.44 41.82 ± 1.70 control (PBS) Semaglutide 30 41.37 ± 1.64 40.24 ± 1.44 41.62 ± 1.56 38.86 ± 1.37 40.56 ± 1.51 41.03 ± 1.72 NO: 3 30 40.38 ± 0.84 39.43 ± 0.83 40.55 ± 0.77 38.09 ± 0.58 39.41 ± 0.57 40.06 ± 0.50 NO: 4 30 40.14 ± 1.62 39.21 ± 1.44 40.49 ± 1.73 37.62 ± 1.48 39.26 ± 1.79 39.90 ± 1.60 SPNO13 30 40.22 ± 1.65 39.23 ± 1.65 40.21 ± 1.65 37.98 ± 2.11 39.04 ± 2.10 39.75 ± 2.13

TABLE-US-00031 TABLE 6 T test of random weight of db/db mice with each test drug in experiment T-Test P value Groups Dosage Day −2 Day −1 Day 0 Day 1 Day 2 Day 3 Solvent — — — — — — — control (PBS) Semaglutide 30 0.39 0.40 0.35 0.01 0.39 0.37 NO: 3 30 0.58 0.71 0.51 0.00 0.01 0.01 NO: 4 30 0.47 0.55 0.59 0.00 0.03 0.04 SPNO13 30 0.54 0.59 0.36 0.01 0.03 0.05

TABLE-US-00032 TABLE 7 Comparison of pharmacokinetic parameters between Semaglutide and drug candidates Semaglutide SPN009 SPN010 Parameter Unit Mean SD RSD/% Mean SD RSD/% Mean SD RSD/% AUC(0-t) μg/L*h 2244.71 293.93 13.10 2630.55 679.80 25.80 1969.77 164.97 8.40 AUC(0-∞) μg/L*h 2320.02 302.12 13.00 2807.29 691.63 24.60 2064.93 123.42 6.00 R_AUC(t/∞) % 96.77 0.09 0.90 93.53 0.18 1.90 95.30 0.26 2.70 AUMC(0-t) h*h*μg/L 54463.48 7571.79 13.90 73156.82 15271.94 20.90 47513.48 2032.32 4.30 AUMC(0-∞) h*h*μg/L 61264.52 9552.95 15.60 89827.62 16348.27 18.20 56436.94 4262.31 7.60 t½z h 12.50 0.88 7.10 15.38 1.43 9.30 14.25 2.63 18.40 Tmax h 18.00 10.39 57.70 24.00 0.00 0.00 18.67 9.24 49.50 Vz/F L/kg 0.16 0.02 14.60 0.17 0.05 29.50 0.20 0.05 24.40 CLz/F L/h/kg 0.009 0.001 11.100 0.008 0.002 25.000 0.009 0.001 11.100 Cmax μg/L 58.30 7.07 12.10 66.80 16.86 25.20 47.17 5.44 11.50