Acylated GLP-1 derivative

Abstract

Provided are a GLP-1(7-37) polypeptide analogue, a fatty acid-modified derivative of the analogue, and a medicament comprising the derivative. Further, also provided are a preparation method of the derivative, and use of the same in the preparation of a medicament.

Claims

1. A derivative of a GLP-1(7-37) analogue or a pharmaceutically acceptable salt thereof, wherein the derivative is: N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34 GLP-1(7-37)) peptide (M2), or N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M4).

2. A method for preparing the derivative or a pharmaceutically acceptable salt thereof, comprising: (1) mixing a solution in which the GLP-1 analogue according to claim 1 is dissolved with a solution in which the extension portion is dissolved; (2) adjusting the pH to 4-5 to quench the reaction, standing until a precipitate is generated, and then collecting the precipitate; and (3) adding TFA to the precipitate, and adjusting the pH to 7.5-8.5 to quench the reaction.

3. The method according to claim 2, further comprising: adding triethylamine to a solution in which the GLP-1 analogue is dissolved, followed by mixing with a solution in which the extension portion is dissolved.

4. The method according to claim 2, wherein the solution of the extension portion is dissolved by acetonitrile.

5. A pharmaceutical composition comprising the derivative or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

6. A method for treating diabetes or diabetic complications, comprising: administering a prophylactically or therapeutically effective amount of the derivative or the pharmaceutically acceptable salt thereof according to claim 1 to a subject.

7. The method according to claim 6, wherein the diabetic complication is diabetic nephropathy.

8. A method for reducing blood glucose, increasing glucose tolerance, reducing islet β-cell apoptosis, enhancing islet β-cell function, increasing islet β-cell number, and/or restoring islet β-cell glucose sensitivity, comprising: administering a therapeutically effective amount of the derivative or the pharmaceutically acceptable salt thereof according to claim 6 to a subject.

9. The method according to claim 8, wherein said reducing blood glucose includes reducing fasting blood glucose and/or postprandial blood glucose.

10. A kit comprising: a container in which the pharmaceutical composition according to claim 5 is contained, and a package insert, wherein the package insert contains instructions for use of the pharmaceutical composition.

11. The kit according to claim 10, further comprising a container containing one or more other medicaments.

12. The kit according to claim 11, wherein the one or more other medicaments are other medicaments for treating diabetes or diabetic complications.

13. The derivative of a GLP-1(7-37) analogue or pharmaceutically acceptable salt thereof according to claim 1 wherein the derivative is N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (M2).

14. The derivative of a GLP-1(7-37) analogue or pharmaceutically acceptable salt thereof according to claim 1 wherein the derivative is N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M4).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the glucose-lowering effects of different acylated GLP-1 derivative molecules on type II diabetic db/db mice.

(2) FIG. 2 shows a trend graph of the effects of different doses of M0, M4, and Semaglutide on fasting blood glucose in diabetic mice.

(3) FIG. 3 shows the effects of different doses of M0, M4 and Semaglutide on random blood glucose in diabetic mice.

(4) FIG. 4 shows the effects of different doses of M0, M4 and Semaglutide on the blood glucose area under the curve of diabetic mice.

(5) FIG. 5 shows the trend graph of M4 and Semaglutide molecules against pepsin degradation.

(6) FIG. 6 shows the trend graph of M4 and Semaglutide molecules against trypsin degradation.

DETAILED DESCRIPTION OF THE INVENTION

Examples

(7) Hereinafter, the invention will be described through specific examples. Unless otherwise specified, it may be implemented according to the methods listed in the experimental manuals such as “Molecular Cloning: A Laboratory Manual” and “Cells: A Laboratory Manual” familiar to those skilled in the art, as well as CFDA's experimental guidelines. Among them, the reagent raw materials used are all commercially available products, which may be purchased through public channels.

Example 1: Construction of the Expression Plasmids of GLP-1 Analogues

(8) TABLE-US-00001 Construction of DNA of Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37)

(9) The 6-His tag, SUMO tag, and Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37) encoding gene sequence (SEQ ID NO:7) are fused successively in series, and the gene fragment (SEQ ID NO:18) is obtained by chemical synthesis. Through the BamHI and XhoI sites, the above fragment is inserted into the prokaryotic expression plasmid pET-24(+) and verified by sequencing. The resulting expression plasmid for transformation assay is named

(10) TABLE-US-00002 pET-24(+)-His-SUMO-Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37).

(11) According to the above method, the corresponding expression plasmids of the following peptides are successively constructed:

(12) TABLE-US-00003 (the encoding gene is SEQ ID NO: 3) Val.sup.8Glu.sup.22Lys.sup.26Arg.sup.34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 11) Val.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 5) Val.sup.8Glu.sup.22Lys.sup.19Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 9) Val.sup.8Glu.sup.22Lys.sup.27Arg.sup.26, .sup.34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 13) Val.sup.8Glu.sup.22Lys.sup.34Arg.sup.26-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 15) Val.sup.8Glu.sup.22Arg.sup.26, 34Lys.sup.36-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 17) Val.sup.8Glu.sup.22Arg.sup.26, 34Lys.sup.37-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 20) Thr.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 22) Ile.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 24) Leu.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 26) Gly.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37),  (the encoding Gene is SEQ ID NO: 28) Ser.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 30) Thr.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34-GLP- 1(7-37),  (the encoding gene is SEQ ID NO: 32) Ile.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 34) Leu.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34-GLP- 1(7-37),  (the encoding gene is SEQ ID NO: 36) Gly.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34-GLP-1(7-37),  (the encoding gene is SEQ ID NO: 38) Ser.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34-GLP-1(7-37). 

Example 2: Expression of the Fusion Proteins

(13) The DNA construct described in Example 1 is transformed into BL21 host cells (TrabsGenBiotech, catalog #CD601) for producing the target protein of the present invention. 50 μl of BL21 competent cells are put in an ice bath to melt, then adding the DNA of interest and gently shaking gently, and placing in the ice bath for 30 min. Then heat shock in water bath at 42° C. for 30 seconds, followed by quickly transferring the centrifuge tube to ice bath for 2 min, and do not shake the centrifuge tube during this process. 500 μl of sterile LB medium (without antibiotics) is added to the centrifuge tube, then mixing and culturing at 37° C., 180 rpm for 1 hour to recover the bacteria. 200 μl of transformed competent cells are pipetted and added onto LB agar medium plate containing kanamycin resistance, spreading the cells evenly; placing the plate at 37° C. until the liquid is absorbed, then inverting the plate and incubating at 37° C. overnight. The next day, the monoclonal colonies in the transformation dish is picked by using inoculation ring to inoculate in 15 ml of sterile LB medium (containing antibiotics), then culturing at 30° C. overnight.

Example 3: Fermentation of the Recombinant GLP-1 Analogues

(14) 50 μl of bacterial suspension (GLP-1 expressing bacterial suspension) is added to 50 ml of LB medium, adding 50 μl of kanamycin at the same time, mixing and putting in 30° C. constant temperature shaker, then inoculating overnight. 10 ml of the bacterial suspension inoculated overnight is added to 1000 ml of LB medium, adding 1000 μl of kanamycin at the same time; then shaking and placing it in a 37° C. shaker at 200 rpm. After inoculation for 4 hours, IPTG with a final concentration of 0.1 mol/L is added into the medium, then shaking and placing it in 30° C. shaker at 180 rpm to induce expression overnight. The bacterial suspension expressed overnight is centrifuged at 13000 g for 60 min. The bacteria yield is about 4 g bacteria/L fermentation broth, and the expression of the protein of interest determined by SDS-PAGE is about up to 40%.

Example 4: Purification of the Recombinant GLP-1 Analogues

(15) 100 g of cell slurry is weighed and re-suspended in 500 ml of 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, then sonicating in an ultrasonic cell mill for 30 min to break up the cells. The homogenate is centrifuged at 13000 g for 60 min at 4° C. After centrifugation the supernatant is collected as Ni column chromatography sample.

(16) The obtained supernatant is concentrated by Chelating Sepharose FF equilibrated with 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole (equilibrium liquid 1) in advance; after rinsing with the equilibrium liquid 1, it is eluted with 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 0.3 M imidazole (eluent). According to SDS-PAGE analysis, the purity of the intermediate product of GLP-1 produced by the above purification process is higher than 70%.

(17) Excising the Sumo tag sequence by using ULP enzyme: adding 20 mM PB, pH7.4 buffer to the intermediate product to make a three-fold dilution, adding ULP enzyme to mix and digest overnight at the condition of 4° C. at a 1:150 ratio of ULP enzyme:the intermediate product. The digestion rate is nearly 100% according to SDS-PAGE analysis.

(18) Purification of GLP-1 analogue: the product obtained after digestion is concentrated by using Tosoh Butyl 550C medium equilibrated with 20 mM Na.sub.2HPO.sub.4, 0.7M NaCl (equilibrium liquid 2) in advance, after rinsing with the equilibrium liquid 2, it is eluted with 20% ethanol, and the purity is about 90% according to SDS-PAGE analysis.

(19) 0.2M Na.sub.2HPO.sub.4 is added to the eluted sample to make a final concentration of 20 mM Na.sub.2HPO.sub.4, then adjusting the pH to 4.8-5.0 with 1M citric acid for acid precipitation at 4° C. overnight. The yield is over 90% according to SDS-PAGE assay. Centrifuging at 13000 g for 30 min at 4° C., then the precipitate is collected and stored at −20° C.

Example 5: Preparation of the Derivatives of GLP-1 Analogues

(20) Preparation of the derivative of GLP-1 analogue as shown below, N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (abbreviated as M2)

(21) ##STR00010##

(22) 1. Fatty acid modification: water is added to the precipitate of Val.sup.8Gu.sup.22Lys.sup.23Arg.sup.26, 34-GLP-1(7-37) prepared and collected in the above example to prepare 4-6 mg/ml solution, then adding 1M sodium hydroxide to adjust the pH to 11.0-11.5, shaking to make the protein completely dissolved, and the concentration of the peptide is quantified by HPLC. Fatty acid powder is weighed and dissolved in acetonitrile at a 1:4 molar ratio of the peptide to fatty acid (the structure is as follows). Two thousandths of triethylamine is added to this polypeptide solution, mixing with the fatty acid solution, and letting the mixture stand at 4° C. for one hour.

(23) ##STR00011##

(24) The sample is diluted 5 times with water, then adjusting to pH 4.8 with 1M citric acid (or 10% acetic acid) to quench the reaction, standing at 4° C. for acid precipitation for 10 min, centrifuging at 13000 g after acid precipitation and centrifuging at 4° C. for 30 min, and then the precipitate is stored at −80° C.

(25) 2. Deprotection of fatty acid and purification: TFA is added to the acid precipitation sample to a final peptide concentration of about 10 mg/ml, then shaking to dissolve the precipitate, letting it stand at room temperature for deprotection for 30 min, and dropping 4M NaOH into the reaction solution to adjust the pH to 7.5-8.5 to quench the reaction.

(26) By using a preparative HPLC (Shimadzu LC-8A), the reaction liquid after quenching is pumped into UniSil 10-120 C18 (purchased from Suzhou Nanomicro Technology Co., Ltd.) equilibrated with 10 mM ammonium acetate, 20% ethanol (equilibrium liquid 3) in advance for concentration. After rinsing with the equilibrium liquid 3, a gradient of 0-100% eluent (10 mM ammonium acetate, 80% ethanol) is used for elution. The elution peak is collected, and the purity is about 90% according to RP-HPLC analysis.

(27) The elution peak is diluted 3 times with water, then adjusting the pH of acid precipitation to 4.80, and acid precipitation is performed at 4° C. for 30 min. After centrifugation, PBST buffer (pH7.0) is added to the pellet to reconstitute it, then freezing and storing at −80° C.

(28) The following peptides are prepared successively according to the above method, N-ε.sup.26-[2-(2-[2-(2-[2-(2[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]Val.sup.8Gu.sup.22Lys.sup.26Arg.sup.34-GLP-1(7-37) peptide (M0), N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl])(Val.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M4), N-ε.sup.19-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Lys.sup.19Gu.sup.22Arg.sup.26,34-GLP-1(7-37)) peptide (M1), N-ε.sup.27-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Lys.sup.27Arg.sup.26,34-GLP-1(7-37)) peptide (M3), N-ε.sup.34-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Gu.sup.22Arg.sup.26Lys.sup.34-GLP-1(7-37)) peptide (M5), N-ε.sup.36-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Arg.sup.26,34Lys.sup.36-GLP-1(7-37)) peptide (M6), N-ε.sup.37-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val.sup.8Glu.sup.22Arg.sup.26,34Lys.sup.37-GLP-1(7-37)) peptide (M7); N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Thr.sup.8Gu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (M8), N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ile.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (M9), N-ε.sup.23-(2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Leu.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (M10), N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Gly.sup.8Glu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (M11), N-ε.sup.23-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl)](Ser.sup.8Gu.sup.22Lys.sup.23Arg.sup.26,34-GLP-1(7-37)) peptide (M12); N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxylbutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl])(Thr.sup.8Gu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M13), N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ile.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M14), N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Leu.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M5), N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Gly.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M6), N-ε.sup.30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4 (s)-carboxybutyryl amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ser.sup.8Glu.sup.22Lys.sup.30Arg.sup.26,34-GLP-1(7-37)) peptide (M7).

(29) TABLE-US-00004 TABLE 1  Comparison table of GLP-1(7-37) analogues and their corresponding derivatives Abbreviations of the Abbreviations of the Amino acid sequences of derivatives GLP-1 analogues the GLP-1 analogues GLP-1(7-37) HAEGTFTSDVSSYLEGQAAKEFIA WLVKGRG (SEQ ID NO: 1) Semaglutide Aib.sup.8Lys.sup.26Arg.sup.34-GLP-1 H-Aib-EGTFTSDVSSYLEGQAAKE (Aib) (7-37) FIAWLVRGRG (SEQ ID NO: 40) M0  Val.sup.8Glu.sup.22Lys.sup.26Arg.sup.34-GLP-1 HVEGTFTSDVSSYLEEQAAKEFIA (7-37) WLVRGRG (SEQ ID NO: 2) M1  Val.sup.8Lys.sup.19Glu.sup.22Arg.sup.26, 34- HVEGTFTSDVSSKLEEQAAREFIA GLP-1 (7-37) WLVRGRG (SEQ ID NO: 4) M2  Val.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34- HVEGTFTSDVSSYLEEKAAREFIA GLP-1 (7-37) WLVRGRG (SEQ ID NO: 6) M3  Val.sup.8Glu.sup.22Lys.sup.27Arg.sup.26, 34- HVEGTFTSDVSSYLEEQAARKFIA GLP-1 (7-37) WLVRGRG (SEQ ID NO: 8) M4  Val.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34- HVEGTFTSDVSSYLEEQAAREFIK GLP-1 (7-37) WLVRGRG (SEQ ID NO: 10) M5  Val.sup.8Glu.sup.22Arg.sup.26Lys.sup.34-GLP-1 HVEGTFTSDVSSYLEEQAAREFIA (7-37) WLVKGRG (SEQ ID NO: 12) M6  Val.sup.8Glu.sup.22Arg.sup.26, 34 HVEGTFTSDVSSYLEEQAAREFIA Lys.sup.36-GLP-1(7-37) WLVRGKG (SEQ ID NO: 14) M7  Val.sup.8Glu22Arg.sup.26, 34 HVEGTFTSDVSSYLEEQAAREFIA Lys.sup.37-GLP-1(7-37) WLVRGRK (SEQ ID NO: 16) M8  Thr.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34- HTEGTFTSDVSSYLEEKAAREFIA GLP-1(7-37) WLVRGRG (SEQ ID NO: 19) M9  Ile.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34- HIEGTFTSDVSSYLEEKAAREFIA GLP-1(7-37) WLVRGRG (SEQ ID NO: 21) M10 Leu.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34- HLEGTFTSDVSSYLEEKAAREFIA GLP-1(7-37) WLVRGRG (SEQ ID NO: 23) M11 Gly.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34- HGEGTFTSDVSSYLEEKAAREFIA GLP-1(7-37) WLVRGRG (SEQ ID NO: 25) M12 Ser.sup.8Glu.sup.22Lys.sup.23Arg.sup.26, 34- HSEGTFTSDVSSYLEEKAAREFIA GLP-1(7-37) WLVRGRG (SEQ ID NO: 27) M13 Thr.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34- HTEGTFTSDVSSYLEEQAAREFIK GLP-1(7-37) WLVRGRG (SEQ ID NO: 29) M14 Ile.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34- HIEGTFTSDVSSYLEEQAAREFIK GLP-1(7-37) WLVRGRG (SEQ ID NO: 31) M15 Leu.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34- HLEGTFTSDVSSYLEEQAAREFIK GLP-1(7-37) WLVRGRG (SEQ ID NO: 33) M16 Gly.sup.8Glu.sup.22Lys.sup.30Arg.sup.26, 34- HGEGTFTSDVSSYLEEQAAREFIK GLP-1(7-37) WLVRGRG (SEQ ID NO: 35) M17 Ser.sup.8Glu.sup.22Lys.sup.30Ar.sup.g26, 34- HSEGTFTSDVSSYLEEQAAREFIK GLP-1(7-37) WLVRGRG (SEQ ID NO: 37)

Example 6: In Vitro Activity Determination of Derivatives of GLP-1 Analogues in RIN-m5F Cells

(30) RIN-m5F cells with good culturing status are selected. Cells are then collected, counted, and prepared into a cell suspension of 1×10.sup.5 cells/ml with RPMI1640 basal medium. The cell suspension is inoculated in a 96-well cell culture plate, 100 μl per well, then incubating overnight at 37° C. and 5% CO.sub.2. The in vitro activity of the derivatives of GLP-1 analogs is measured by using cAMP assay kit (Promega): preparing diluted samples (Aib, M0, M1, M2, M3, M4, M5, M6, and M7) to 300 ng/ml with the assay medium, then performing a 3-fold gradient dilution in 96-well plates, a total of 8 concentrations, and making 2 duplicate wells for each dilution, wherein M0, M1, M2, M3, M4, M5, M6, and M7 are prepared as described above, and Aib is:

(31) N-ε.sup.26-[2-(2-[2-(2-[2-(2[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy) ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib.sup.8, Arg.sup.34]GLP-1-(7-37) peptide (see CN101133082B, Example 4), the trade name is Semaglutide, and it is prepared according to the method disclosed in patent CN101133082B.

(32) The prepared cell plate is taken out, then discarding the medium, and blotting it dry on the filter paper. The sample solutions are transferred correspondingly into the cell plate, 40 μl/well; treating with the lid open for 15 min at 37° C. and 5% CO.sub.2. The cell culture plate is taken out from the incubator, then adding 10 μl of CD solution (cAMP assay kit, Promega) to each well, keeping the cell plate at 22-25° C., and shaking horizontally at 500 rpm for 20 min. 50 μl of KG solution (cAMP assay kit, Promega) is added to each well, then shaking horizontally at 22° C.-25° C., 500 rpm and avoiding light for 10 min. The chemiluminescence value is read by using the Molecular Devices SpectraMax L chemiluminescence apparatus, completing the measurement in 30 min. EC50 of a sample is calculated by using the four-parameter regression in softmax Pro software.

(33) TABLE-US-00005 TABLE 2 Results of In vitro Activity Assay Samples Aib M0 M1 M2 M3 M4 M5 M6 M7 EC50 2.437 10.68 5.386 1.996 5.387 2.322 3.043 7.650 3.208

(34) The in vitro pharmacodynamics of RIN-m5F cells shows that the in vitro activities of Semaglutide, M2, M4, M5, and M7 are comparable, and generally they are slightly higher than those of M0, M1, M3, and M6.

Example 7: In Vitro Activity Determination of the Derivatives of GLP-1 Analogues in HEK293/CRE-Luc/GLP1R Cells

(35) Based on the fact that GLP-1 may bind to the receptor on the cell membrane, the HEK293/CRE-Luc/GLP1R cell line is constructed, the cAMP response elements (CRE) are activated through a series of signal transduction, the expression of downstream luciferase is initiated, and the amount of its expression is positively correlated with the biological activity of GLP-1. After adding the luciferase substrate, the chemiluminescence assay is performed to determine the luminous intensity, thereby determining the biological activity of GLP-1.

(36) Experimental Materials

(37) 96-well cell culture plate (white and opaque), DMEM medium (GIBCO), 0.05% TRYPSIN-EDTA (GIBCO), fetal bovine serum (GIBCO), G418, hygromycin B, Bright-Glo™ Luciferase Assay System Kit (Promega), and HEK293/CRE-luc/GLP1R cells.

(38) Experimental Operations

(39) (1) Cell preparation: the cells are cultured until they grow vigorously and reach a sufficient quantity, discarding the culture medium in the culture bottle, adding 3 ml of Versene solution and shaking once; then adding 2 ml of 0.05% TRYPSIN-EDTA digestion solution, covering the bottle and standing for 1 minute, and then adding 6 ml of the assay medium to quench the digestion; after centrifugation at 1000r/min for 3 min, the supernatant is discarded, and the cells are resuspended in 5 ml of assay medium and counted on a hemacytometer. The cell density is adjusted to an appropriate range for later use by using DMEM assay medium.

(40) (2) Sample preparation: the derivatives of different GLP-1 analogues in Table 1 are diluted to 20 ng/ml with the assay medium, then gradient diluted into 8 concentrations in 96-well plates, and using the assay medium instead of the sample as the cell blank control. 2 duplicate wells are made for each dilution concentration.

(41) (3) Culturing with addition of the samples: the prepared solutions of the control and test samples are transferred to a 96-well cell culture plate (white board), adding 50 μl of the solution to each well; then adding the prepared cell suspension, adding 50 μl of the suspension to each well; and then incubating for a certain period of time under the conditions of 37° C. and 5% CO.sub.2.

(42) (4) Chemiluminescence assay: substrate is added, then taking out the 96-well cell culture plate, adding 100 μl of Bright Glo reagent to each well, and leaving in the dark for 3 min.

(43) (5) Reading: determination is performed with a chemiluminescence microplate reader SpectraMax L, then reading the plate within 30 min, and recording the determined results.

(44) TABLE-US-00006 TABLE 3 Experimental results of in vitro activity of HEK293/CRE-Luc/GLP1R cells Experimental plate 1 Experimental plate 2 Experimental plate 3 Samples EC50 Samples EC50 Samples EC50 Semaglutide 0.14 Semaglutide 0.138 Semaglutide 0.111 Liraglutide 0.206 Liraglutide 0.211 Liraglutide 0.142 M2 0.134 M4 0.184 M4 0.137 M9 0.177 M13 0.454 M16 0.13 M11 0.183 M14 0.232 M17 0.15

(45) The pharmacodynamics of HEK293/CRE-Luc/GLP1R cells shows that the in vitro activities of Semaglutide, M2, M4, M9, M11, M14, M16, and M17 are comparable, and generally they are slightly higher than that of M13.

Example 8: Research on Glucose-Lowing Effect of Fatty Acid Modified Derivatives of GLP-1 Analogues in Normal Mice

(46) Twenty-eight healthy female CD-1 mice aged 4-6 weeks are selected and divided into 4 groups, and they are injected subcutaneously and respectively with M2, M4, M0 and Semaglutide (Aib) at a dose of 0.15 mg/kg body weight. 20% glucose is intragastrically administered pre-administration, and after intervals of 6h, 1 day, 2 days, 3 days, and 4 days from administration, at a dose of 2 g/kg body weight, and fasting for 6 hours before giving the glucose; then blood is respectively collected from the tip of the tail at 0h, 0.5h, 1h and 2h after giving the glucose and measured for blood glucose value in real time by using Roche blood glucose test paper; and the blood glucose AUC (area under the curve of blood glucose˜time) within 0-120 min is calculated, and the blood glucose inhibition rate is obtained (Table 4).
Blood glucose inhibition rate=[(Blood glucose AUC of mice before administration−Blood glucose AUC of mice after administration)/Blood glucose AUC of mice before administration]×100%

(47) TABLE-US-00007 TABLE 4 Comparison of the glucose-lowing effects in normal mice 6 h 30 h 54 h 78 h 102 h Semaglutide Inhibition rate 35.95% 30.87% 21.00% 1.68% 0.15 mg/kg P 0.0000 0.0000 0.0010 0.7728 M2 Inhibition rate 34.29% 29.51% 27.23% 21.97% 10.15% 0.15 mg/kg P 0.0000 0.0000 0.0000 0.0002 0.0408 M4 Inhibition rate 36.11% 34.19% 31.51% 24.82% 15.00% 0.15 mg/kg P 0.0000 0.0000 0.0000 0.0001 0.0064 M0 Inhibition rate 38.47% 33.79% 25.18% 13.43% −1.05% 0.15 mg/kg P 0.0000 0.0000 0.0002 0.0180 0.7909 P value: compared with blood glucose before administration

(48) It may be seen from Table 4 that, the glucose-lowing effect of Semaglutide in normal mice lasts about 2 days, and the glucose-lowing effect of M0 in normal mice lasts about 3 days; however, the glucose-lowing effect of M2 and M4 in normal mice are still obvious on day 4, and their duration of sustained glucose-lowing effect in the body is significantly longer than that of Semaglutide or M0, and at various time points after the 3rd day of administration the glucose-lowing effects of both M2 and M4 are also significantly stronger than that of Semaglutide or M0.

(49) Twenty-eight healthy female CD-1 mice aged 4-6 weeks are selected and divided into 4 groups, and they are injected subcutaneously with M4, M5, M7 and M0 at a dose of 0.15 mg/kg body weight. 20% glucose is intragastrically administered pre-administration and after intervals of 6h, 1 day, 2 days, 3 days, and 4 days from administration, at a dose of 2 g/kg body weight, and fasting for 6 hours before intragastrically administering the 20% glucose; and blood is respectively collected from the tip of the tail at 0h, 0.5h, 1h and 2h after giving the glucose, then measuring the blood glucose value in real time by using Roche blood glucose test paper, and the blood glucose AUC (area under the curve of blood glucose-time) within 0-120 min is calculated, the blood glucose inhibition rate (Table 5) is obtained.
Blood glucose inhibition rate=[(Blood glucose AUC of mice before administration−Blood glucose AUC of mice after administration)/Blood glucose AUC of mice before administration]×100%

(50) TABLE-US-00008 TABLE 5 Comparison of glucose-lowing effects in normal mice 6 h 30 h 54 h 78 h 102 h M0 Inhibition rate 26.71% 33.57% 17.32% 8.97% −12.89% 0.15 mg/kg P 0.0018 0.0004 0.0210 0.2329 0.2792 M4 Inhibition rate 26.26% 32.92% 22.22% 24.07% 16.55% 0.15 mg/kg P 0.0045 0.0004 0.0066 0.0049 0.0706 M5 Inhibition rate 29.59% 39.47% 30.11% 27.07% 15.38% 0.15 mg/kg P 0.0041 0.0006 0.0046 0.0156 0.1144 M7 Inhibition rate 27.22% 38.82% 22.56% 22.48% 11.60% 0.15 mg/kg P 0.0049 0.0006 0.0142 0.0084 0.1389

(51) From the results of Table 4 and Table 5, the glucose-lowing effects of M2 and M4 are better than those of M0 and Semaglutide; and the glucose-lowing effects of M2, M4, M5 and M7 are comparable, and there is no significant difference between them.

Example 9: Research on the Glucose-Lowing Effect by Using ICR Mice

(52) OGTT test of ICR mice: 30 ICR mice aged 4-6 weeks are selected and divided into 6 groups, 5 mice per group, and they are injected subcutaneously with M0, Semaglutide, M2, M4, M5 and M7 respectively at a dose of 0.15 mg/kg body weight by single administration. 20% glucose is intragastrically administered every day according to the time schedule of 4h, 1d, 2d, 3d, 4d, and 5d, at a dose of 2 g/kg body weight, and fasting for 6 hours before administering the glucose; and blood is respectively collected from the tip of the tail at 0h, 0.5h, 1h and 2h after giving the 20% glucose, then measuring the blood glucose value in real time by using Roche blood glucose test paper. Blood is collected from the tip of the tail, the blood glucose value is measured in real time by using Roche blood glucose test paper, and the blood glucose AUC (area under the curve of blood glucose-time) within 0-120 min is calculated, and the blood glucose inhibition rate (Table 6) is obtained.
Blood glucose inhibition rate=[(Blood glucose AUC of mice before administration−Blood glucose AUC of mice after administration)/Blood glucose AUC of mice before administration]×100%

(53) TABLE-US-00009 TABLE 6 Comparison of glucose-lowing effects in ICR mice Groups 0 h 4 h 1 d 2 d 3 d 4 d 5 d M0 G-AUC 18.0 ± 1.9 11.1 ± 1.2 11.9 ± 0.7 13.5 ± 1.1 15.6 ± 1.3 18.2 ± 1.1 / 0.15 mg/kg Inhibition rate / 38.47% 33.79% 25.18% 13.43%  0.60% / Semaglutide G-AUC 18.3 ± 2.1 11.7 ± 1.2 12.7 ± 0.7 14.5 ± 1.1 18.0 ± 1.8 / / 0.15 mg/kg Inhibition rate / 35.95% 30.87% 21.00%  1.68% / / M2 G-AUC 17.8 ± 1.9 11.7 ± 0.6 12.5 ± 0.6 13.0 ± 0.7 13.9 ± 0.6 16.0 ± 1.0 18.3 ± 0.6 0.15 mg/kg Inhibition rate / 34.29% 29.51% 27.23% 21.97% 12.61% 0.08% M4 G-AUC 18.0 ± 1.8 11.5 ± 1.0 11.8 ± 0.9 12.3 ± 0.9 13.5 ± 0.9 15.3 ± 1.1 17.3 ± 1.0 0.15 mg/kg Inhibition rate / 36.11% 34.19% 31.51% 24.82% 16.39% 5.68% M5 G-AUC 16.3 ± 2.5 11.5 ± 1.0  9.9 ± 0.7 11.4 ± 1.3 11.9 ± 2.0 13.8 ± 1.9 14.4 ± 2.2 0.15 mg/kg Inhibition rate / 29.59% 39.47% 30.11% 27.07% 15.38% 11.66% M7 G-AUC 16.3 ± 2.7 11.8 ± 1.4 10.0 ± 1.6 12.6 ± 1.5 12.6 ± 0.7 14.4 ± 1.1 15.2 ± 1.0 0.15 mg/kg Inhibition rate / 27.22% 38.82% 22.56% 22.48% 11.60% 6.76%

(54) It may be seen from the results in Table 6 that, the glucose level is maintenance constantly: the glucose-lowing effects of M4, M5, M2, and M7 may all be maintained for at least 4 days, which are much better than those of M0 (only maintained for 3 days) and Semaglutide (only maintained for 2 days), and all of which are statistically significant.

Example 10: Glucose-Lowing Pharmacokinetic Test for Type II Diabetic db/db Mice

(55) Fifty 8-9 week-old female db/db mice are evenly divided into 10 groups based on body weight and fasting blood glucose value (FBG) before administration, 5 mice per group; and they are respectively administered with a single subcutaneous injection of vehicle, M2, M4, Semaglutide, M9, M11, M13, M14, M16 and M17 at 10 ml/kg. The dosage is 0.05 mg/kg for each, and the administration time is set as 0h. Fasting blood glucose is measured after fasting for 6-8h every day, and the fasting blood glucose after administration is measured every day until the fasting blood glucose value of each animal of the test group recovers to the value measured before administration. The blood glucose value measured before administration is called the basal blood glucose value, and is set as 0.
Change of the fasting blood glucose (Δ: delta)=Blood glucose value after administration−Basal blood glucose value before administration.

(56) The results are shown in FIG. 1, it may be seen from day 4 and day 5 that the glucose-lowing effects of M9, M13, and M14 are better than that of Semaglutide, and are also not lower than that of M2; however, the glucose-lowing effects of M11, M16 and M17 are lower than that of Semaglutide on day 2.

Example 11: Glucose-Lowing Effect of Different Doses of Semaglutide, M0 and M4 for Type II Diabetic db/db Mice

(57) Thirty-five 8-9 week-old female db/db mice are evenly divided into 7 groups based on body weight and blood glucose area under the curve (G-AUC) before administration, 5 mice per group; and they are respectively administered with a single subcutaneous injection of vehicle, M4 (0.15, 0.015 mg/kg), Semaglutide (0.15, 0.015 mg/kg), and M0 (0.15, 0.015 mg/kg) at 10 ml/kg. The administration time is set as 0h, and the fasting blood glucose and OGTT (oral glucose tolerance assay) are determined after fasting for 7-8h every day, then 10% glucose is intragastrically administered at 1 g/kg body weight, and then blood is collected from the tip of the tail to measure the blood glucose in real time at 0, 0.5, 1, and 2h after glucose load. After administration, the blood glucose is measured every day before fasting as random blood glucose, until the fasting blood glucose value of each animal of the test group recovers to the value before administration. All of the basal blood glucose value, random blood glucose value, and the blood glucose area under the curve (G-AUC) value determined before administration are bases for evaluating the efficacy of the medicament, and they are set as 0.

(58) Change of the blood glucose (Δ: delta)=Blood glucose value after administration−Basal blood glucose value before administration.

(59) Change of the blood glucose area under the curve (Δ: delta)=blood glucose area under the curve after administration−blood glucose area under the curve before administration.

(60) The results are shown in Tables 7, 8 and 9, and FIGS. 2, 3 and 4.

(61) TABLE-US-00010 TABLE 7 Fasting blood glucose changes of mice in each test group Average changes of fasting blood glucose after administration Administration (mM) Groups dosage (mg/kg) −21 h 3 h 27 h 51 h 75 h 99 h 123 h 147 h Vehicle — 0.0 3.8 4.2 6.1 3.4 6.8 8.9 8.7 control M4 0.15 0.0 −4.2 −4.5 −3.8 −2.5 −2.0 0.0 1.1 0.015 0.0 −4.4 −3.3 −3.2 −2.6 0.4 0.6 2.3 Semaglutide 0.15 0.0 −3.6 −3.3 −3.0 3.0 3.6 3.0 4.7 0.015 0.0 −4.2 −2.0 0.0 1.0 3.9 / / M0 0.15 0.0 −4.1 −3.0 −3.2 −0.7 1.4 4.4 6.7 0.015 0.0 −3.5 −2.7 −2.4 2.5 3.9 3.7 9.8 Note: “−21 h” represents the fasting blood glucose base before administration.

(62) TABLE-US-00011 TABLE 8 Average changes of random blood glucose of mice in each test group Average changes of random blood glucose after administration Administration (mM) Groups dosage (mg/kg) −5 h 6 h 19 h 43 h 67 h 91 h 115 h 139 h Vehicle — 0 −1.7 −0.8 0.9 1.9 3.7 5.2 4.8 control M4 0.15 0 −17.3 −17.5 −17.0 −16.8 −13.5 −1.8 2.8 0.015 0 −16.6 −15.4 −14.1 −12.9 −9.2 −1.3 3.0 Semaglutide 0.15 0 −17.6 −17.5 −16.1 −10.0 0.4 5.2 3.6 0.015 0 −17.9 −17.5 −14.6 −11.3 −0.6 / / M0 0.15 0 −15.7 −14.8 −14.1 −7.1 1.7 4.5 7.8 0.015 0 −15.7 −13.5 −10.1 −3.1 −0.1 4.9 5.9 Note: “−5 h” represents the random blood glucose base at 5 h before administration.

(63) TABLE-US-00012 TABLE 9 Changes of the blood glucose area under the curve (G-AUC) of mice in each test group Average changes of the blood glucose area under the curve after Administration administration (mmol/L .Math. h) Groups dosage (mg/kg) −21 h 3 h 27 h 51 h 75 h 99 h 123 h 147 h Vehicle — 0 13.1 23.1 24.9 16.6 18.7 22.9 22.2 control M4 0.15 0 −9.6 −8.0 −8.6 −7.7 −4.3 2.4 −0.8 0.015 0 −12.2 −10.6 −8.4 −9.6 −3.9 −0.4 7.5 Semaglutide 0.15 0 −10.4 −10.6 −8.4 1.3 7.7 6.8 12.8 0.015 0 −9.1 −1.3 −2.6 10.0 18.6 / / M0 0.15 0 −10.3 −10.2 −7.1 1.5 3.9 9.9 16.0 0.015 0 −9.0 −5.2 1.1 8.2 7.9 13.0 21.9 Note: “−21 h” represents the base of blood glucose area under the curve before administration.

(64) The results in Tables 7-9 and FIGS. 2-4 indicate:

(65) Fasting blood glucose: as for M4, at 123h after administration that of the 0.15 mg/kg dosage group returns to the basal blood glucose base before administration, and at 99h after administration that of the 0.015 mg/kg dosage group returns to the basal blood glucose base before administration; as for Semaglutide, at 51h after administration that of the 0.15 mg/kg dosage group returns to the basal blood glucose base before administration, and at 27h after administration that of the 0.015 mg/kg dosage group returns to the basal blood glucose base before administration; as for M0, at 75h after administration that the 0.15 mg/kg dosage group returns to the basal blood glucose base before administration, and at 51h after administration that of the 0.015 mg/kg dosage group returns to the basal blood glucose base before administration; wherein all the reduction values of fasting blood glucose in the 0.015 mg/kg dosage group of M4 at each time point for measurement are not lower than those in the 0.15 mg/kg dosage group of Semaglutide or M0.

(66) Random blood glucose: as for M4, at 115h after administration that of the 0.15 mg/kg dosage group returns to the random blood glucose base before administration, and at 115h after administration that of the 0.015 mg/kg dosage group returns to the random blood glucose base before administration; as for Semaglutide, at 67h after administration that of the 0.15 mg/kg dosage group returns to the random blood glucose base before administration, and at 67h after administration that of the 0.015 mg/kg dosage group returns to the random blood glucose base before administration; as for M0, at 67h after administration that of the 0.15 mg/kg dosage group returns to the random blood glucose base before administration, and at 67h after administration that of the 0.015 mg/kg dosage group returns to the random blood glucose base before administration; wherein all the inhibitory effects on random blood glucose in the 0.015 mg/kg dosage group of M4 at each time point for measurement are not lower than those in the 0.15 mg/kg dosage group of Semaglutide or M0.

(67) Blood glucose area under the curve (G-AUC): as for M4, at 99h after administration that of the 0.15 mg/kg dosage group returns to the base of blood glucose area under the curve before administration, and at 99h after administration that of the 0.015 mg/kg dosage group returns to the base of blood glucose area under the curve before administration; as for Semaglutide, at 51h after administration that of the 0.15 mg/kg dosage group returns to the base of blood glucose area under the curve before administration, and at 51 h after administration that of the 0.015 mg/kg dosage group returns to the base of blood glucose area under the curve before administration; as for M0, at 51h after administration that of the 0.15 mg/kg dosage group returns to the base of blood glucose area under the curve before administration, and at 27h after administration that of the 0.015 mg/kg dosage group returns to the base of blood glucose area under the curve before administration; wherein all the values of blood glucose area under the curve in the 0.015 mg/kg dosage group of M4 at each time point for measurement are not lower than those in the 0.15 mg/kg dosage group of Semaglutide or M0.

(68) These glucose-lowing results indicate that: after a single subcutaneous injection of M4, or Semaglutide, or M0, each group shows a significant glucose-lowing effect, however, M4 has the best glucose-lowing effect. The glucose-lowing effect of the 0.015 mg/kg dosage of M4 is comparable to that of the 0.15 mg/kg dosage of Semaglutide, or that of the 0.15 mg/kg dosage of M0.

Example 12: Research on the Stability of M4 and Semaglutide Against Enzymatic Degradation

(69) Pepsin (3200-4500U/mg protein, from Sigma, catalog number: P6887), trypsin (approximately 10000AEE U/mg protein, from Sigma, catalog number: T8003).

(70) (1) Reaction Solution

(71) A: Reaction buffer of pepsin: three 20 mM citric acid-phosphate buffers with different pH (2.6, 4.0, and 7.4) are prepared, then adding 0.005% Tween 20 and 0.001% BSA to prepare a reaction buffer of pepsin.

(72) B: Reaction buffer of trypsin: three 20 mM citric acid-phosphate buffers with different pH (4.0, 6.8, and 8.0) are prepared, then adding 0.005% Tween 20 and 0.001% BSA to prepare a reaction buffer of trypsin.

(73) C: Simulated gastric fluid containing pepsin (SGF): obtained by taking 5 ml of 0.1M hydrochloric acid and adding and dissolving 0.019 g of pepsin.

(74) D: Simulated intestinal fluid containing trypsin (SIF): obtained by taking 0.0684 g of potassium dihydrogen phosphate, adding 2.5 ml of water to dissolve it, adding 0.77 ml of 0.2M sodium hydroxide solution and 5 ml of water, and then adding and dissolving 0.1001 g of trypsin; the pH is measured as 6.82, then diluting by adding water to 10 ml.

(75) (2) Sample Preparation

(76) M4 and Semaglutide samples are taken and respectively diluted to 1.33 mg/ml with PB buffer at pH 7.4 as the stock solutions of the test samples.

(77) (3) Pepsin Degradation Experiments

(78) An appropriate amount of the stock solution of each test samples is taken respectively, then diluted to 0.06 mg/ml with reaction buffers of pepsin with different pH; reaction solution of each group is divided into 1 ml/tube, a total of 7 tubes, then mixing well and incubating in a 37° C. water bath for 30 min. 1 tube without SGF is taken out as the 0 point of enzyme-free reaction (recorded as −5 min point), and then other 6 tubes are taken out and added with SGF separately and mixed well, one tube of them is immediately added with an appropriate volume of 1M NaOH to quench the reaction, acting as the 0 point after adding enzyme (recorded as 0 min point), and the remaining 5 tubes are placed at 37° C. continuously for reaction; and one group is taken out at 5 min, 10 min, 20 min, 35 min and 50 min respectively and added with an appropriate volume of 1M NaOH respectively to quench the reaction. All tubes in all experimental groups are ensured that the total volume after termination of the reaction is the same.

(79) (4) Trypsin Degradation Experiments

(80) An appropriate amount of the stock solution of the test sample is taken respectively, then diluting it to 0.06 mg/ml with reaction buffers of trypsin with different pH; each group of reaction solution is divided into 1 ml/tube, a total of 7 tubes, then mixing well and incubating in a 37° C. water bath for 30 min. 1 tube without SIF is taken out as the 0 point of enzyme-free reaction (recorded as −5 min point), and then other 6 tubes are taken out to add SIF separately and mix well, among them one tube is immediately added an appropriate volume of 6M HCl to quench the reaction, as the 0 point after adding enzyme (recorded as 0 min point), and the remaining 5 tubes are placed at 37° C. continuously for reaction; and one group is respectively taken out at 5 min, 10 min, 20 min, 35 min and 50 min to add an appropriate volume of 6M HCl respectively to quench the reaction. All tubes in all experimental groups are ensured that the total volume after termination of the reaction is the same.

(81) HPLC assay is performed with samples from the enzyme degradation experiment. The peak area of the main peak of the sample without enzyme reaction at 0 point (recorded as −5 min) is used as the basal peak area, and the remaining percentage of the peak area of the main peak at different time points after enzyme addition is calculated.

(82) Experimental data of pepsin degradation (n=3) shows (FIG. 5) that, the degradation rates of M4 and Semaglutide molecules under acidic condition (pH 2.6) are comparable, which is due to the highest pepsin activity at this pH; at neutral pH 7.4, both molecules are basically not degraded, and at this time the activity of gastric protein is the lowest; while at pH 4.0, the degradation rate of Semaglutide is significantly higher than that of M4, the t1/2 of the former is about 10 min, and the t1/2 of the latter is about 45 min, indicating that the ability of M4 against pepsin degradation is significantly better than that of Semaglutide.

(83) Experimental data of trypsin degradation (n=4) shows (FIG. 6) that, the degradation rates of the two are basically the same under the conditions of pH6.8 and 8.0, because trypsin has the highest activity in this pH range; M4 and Semaglutide also show the ability against trypsin degradation under the condition of pH4.0, and there is basically no difference between the two.