Polypeptide Compounds and Use Thereof in the Prevention or Treatment of Diabetes or Diabetic Complications

Abstract

The present invention discloses polypeptide compounds and use thereof in the prevention or treatment of diabetes or diabetic complications, which shows good long-acting hypoglycemic effect and therapeutic effect of diabetic nephropathy; it also has the effect of high enzymatic stability, high biological activity, and no occurrence of adverse effects, and can be used in the preparation of medicaments for the treatment of hyperphagia, obesity and overweight, elevated cholesterol, diabetes and diabetic nephropathy.

Claims

1. A polypeptide compound comprising a parent peptide represented by the following amino acid sequence: TABLE-US-00010 His-Xaa2-Gln-Gly-Thr.sup.5-Phe-Thr-Ser-Asp-Lys.sup.10-Ser-Lys- Tyr-Leu-Xaa15.sup.15-Xaa16-Xaa17-Ala-Ala-Gln20-Xaa21-Phe- Xaa23-Xaa24-Trp.sup.25-Leu-Xaa27-Xaa28-Gly-Gly.sup.30-Pro-Ser- Ser-Gly-Xaa35.sup.35-Pro-Pro-Pro-Ser, wherein, Xaa2=Aib, Ser or D-Ser; Xaa15=Asp or Glu; Xaa16=Aib or Glu; Xaa17=Lys or Arg; Xaa21=Asp or Glu; Xaa23=Val or Iva; Xaa24=Glu or Gln; Xaa27=Leu or Lys; Xaa28=Asp, Glu or Ala; Xaa35=Ala or Aib.

2. The polypeptide compound according to claim 1, wherein Xaa2=Aib or D-Ser.

3. The polypeptide compound according to claim 2, wherein Xaa21=Asp.

4. The polypeptide compound according to claim 3, wherein a carboxyl terminal of the amino acid sequence of the parent peptide is not modified or is modified by an amino group to form a —CONH.sub.2 group.

5. The polypeptide compound according to claim 1, wherein a side chain of Lys at position 10 or 12 in the amino acid sequence of the parent peptide is linked to a lipophilic substituent via a bridging group; the bridging group is one selected from (PEG).sub.m, (PEG).sub.m-γGlu, (PEG).sub.m-Asp, (Gly).sub.x-(Gly-Ser).sub.y-(Gly).sub.z-, (Gly).sub.x-(Gly-Ser).sub.y-(Gly).sub.z-γGlu and (Gly).sub.x-(Gly-Ser).sub.y-(Gly).sub.z-Asp; the linkage is a formation of an amide bond by an amino group of the Lys at position 10 or 12 with a carboxyl group of a glycine residue of the bridging group or a carboxyl group of a (PEG).sub.m terminal modification at one end of the bridging group, and the lipophilic substituent is attached to the amino group of the bridging group at the other end thereof by forming an amide bond with its carboxyl group; the lipophilic substituent is CH.sub.3(CH.sub.2).sub.nC(O)— or HOOC(CH.sub.2).sub.nC(O)— and its acyl group forms an amide bond with the amino group in the bridging group; wherein m is an integer of 2 to 10; n is an integer of 14 to 20; x is an integer of 0 to 5; y is an integer of 1-5; z is an integer of 1-5.

6. The polypeptide compound according to claim 1, wherein the Lys at position 10 or 12 in the amino acid sequence of the parent peptide is replaced with HomoLys, Orn, Dap or Dab.

7. The polypeptide compound according to claim 1, wherein the parent peptide has in its amino acid sequence: Xaa2=Aib or D-Ser; Xaa15=Glu; Xaa16=Aib; Xaa17=Lys; Xaa21=Asp; Xaa23=Val; Xaa24=Glu; Xaa27=Lys; Xaa28=Ala; Xaa35=Ala.

8. The polypeptide compound according to claim 1, wherein the parent peptide has in its amino acid sequence: Xaa2=Aib or D-Ser; Xaa15=Asp; Xaa16=Glu; Xaa17=Arg; Xaa21=Asp; Xaa23=Iva; Xaa24=Gln; Xaa27=Leu; Xaa28=Asp or Glu; Xaa35=Ala or Aib.

9. The polypeptide compound according to claim 1, wherein the amino acid sequence of the parent peptide is selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16.

10. The polypeptide compound according to claim 5, wherein the Lys at position 10 or 12 in the amino acid sequence of the parent peptide is linked via the bridging group to the lipophilic substituent to form any one of the following structures: ##STR00007## ##STR00008##

11. The polypeptide compound according to claim 1, wherein the polypeptide compound is any one of the following compounds: TABLE-US-00011 Compound 1: H-Aib-QGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS-NH.sub.2 Compound 2: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.18CO.sub.2H)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS-NH.sub.2 Compound 3: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS-NH.sub.2 Compound 4: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.16CO.sub.2H)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS-NH.sub.2 Compound 5: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2)18CH.sub.3)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS-NH.sub.2 Compound 6: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)SKYLDERAAQDF- Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2 Compound 7: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)SKYLDERAAQDF- Iva-QWLLEGGPSSGAPPPS-NH) Compound 8: H-Aib-QGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2 Compound 9: H-Aib-QGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.16CO.sub.2H) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2 Compound 10: H-Aib-QGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CH.sub.3) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2 H-Aib-QGTFTSDK(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.18CH.sub.3) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2 Compound 12: HsQGTFTSDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)SKYLDERAAQDF- Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2 Compound 13: HsQGTFTSDK(GGSGSG-γGlu-CO(CH.sub.2).sub.18-COOH)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS Compound 14: HsQGTFTSDK(GGSGSG-γGlu-CO(CH.sub.2).sub.18-COOH)SKYLE-Aib- KAAQDFVEWLKAGGPSSGAPPPS-NH.sub.2 Compound 15: H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH.sub.2).sub.18-COOH) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS Compound 16: H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH.sub.2).sub.18-COOH) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH.sub.2

12. A composition comprising the polypeptide compound of claim 1.

13. The composition according to claim 12, wherein the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carriers or excipients.

14. A method of preventing or treating diabetes and/or diabetic complications, or reducing body weight; wherein the method comprises the polypeptide compound of claim 1, and the diabetic complication is diabetic nephropathy.

15. (canceled)

Description

DESCRIPTION OF THE DRAWINGS

[0096] Hereinafter, embodiments of the present invention are described in detail in conjunction with the accompanying drawings, in which:

[0097] FIG. 1 shows the area under the curve (AUC) calculated in Example 2, based on the initial blood glucose level of the experimental animal at 4 hours after administration versus the 24-hour traced blood glucose changes at different time points when oral glucose was administered again, p<0.05.

[0098] FIG. 2A shows gel electrophoresis plots of FN expression in different groups detected using Western blot in the in vitro renal fibrosis cell model constructed by high glucose induction of MCS mesangial cells and NRK renal epithelial cells in Example 3.

[0099] FIG. 2B shows bar graphs of FN expression in different groups detected by high glucose induction of MCS mesangial cells and NRK renal epithelial cells in Example 3 to construct an in vitro renal fibrosis cell model, p<0.05; *: indicates a confidence level within 95% compared to control (p<0.05); **: indicates a confidence level within 99% compared to control (p<0.01).

[0100] FIG. 3A shows the blood glucose and body weight of mice measured 8-week-old db/db diabetic mice in Example 4, subcutaneously injected daily or every other day with saline, compound 3 (80 and 120 μg/kg, total 2 groups), compound 8 (80 and 120 μg/kg, total 2 groups), compound 13 (80 and 120 μg/kg, total 2 groups), compound 15 (80 and 120 μg/kg, total 2 groups) and semaglutide (80 and 120 μg/kg, total 2 groups) followed by administration and 6 h fasting on alternate days, p<0.05.

[0101] FIG. 3B shows the blood glucose and body weight of mice measured on 8-week-old db/db diabetic mice in Example 4, subcutaneously injected daily or every other day with saline, compound 3 (80 and 120 μg/kg, 2 groups), compound 8 (80 and 120 μg/kg, 2 groups), compound 13 (80 and 120 μg/kg, 2 groups), compound 15 (80 and 120 μg/kg, 2 groups) and semaglutide (80 and 120 μg/kg, total 2 groups) followed by administration and 6 h fasting on alternate days, p<0.05.

[0102] FIG. 4A shows the weekly blood glucose changes in 12-week-old db/db diabetic mice in Example 4, subcutaneously injected with saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, MED C18-acid, for 6 weeks, respectively.

[0103] FIG. 4B shows the OGTT results and corresponding AUC values measured at week 4 in 12-week-old db/db diabetic mice in Example 4, subcutaneously injected with saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, MED C18-acid, respectively.

[0104] FIG. 4C shows the ITT results and corresponding AUC values measured at week 5 in 12-week-old db/db diabetic mice of Example 4, subcutaneously injected with saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, MED C18-acid, respectively.

[0105] FIG. 4D shows the results of ALT and AST in the serum of 12-week-old db/db diabetic mice in Example 4, subcutaneously injected with saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, MED C18-acid, respectively, p<0.05.

[0106] FIG. 5A shows, from left to right, the urinary protein content, urine glucose content, and serum glycated hemoglobin content, urea nitrogen content, and blood creatinine content in the urine of mice in Example 4 after 8 weeks of treatment.

[0107] FIG. 5B shows the H&E staining results of the kidneys of the mice in Example 4 after 8 weeks of medical treatment.

[0108] FIG. 5C shows the PAS staining results and statistics of the kidneys of mice in Example 4 after 8 weeks of medical treatment.

[0109] FIG. 5D shows the results and the statistical graph of Sirius red staining of the kidneys of mice in Example 4 after 8 weeks of medical treatment.

[0110] FIG. 5E shows the results and the statistical graph of FN staining in the kidneys of mice in Example 4after 8 weeks of medical treatment.

[0111] FIG. 5F shows the results and statistical graph of α-SMA staining of the kidneys of mice in Example 4 after 8 weeks of medical treatment, p<0.05.

[0112] FIG. 6 shows an exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention.

[0113] FIG. 7 shows another exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0114] Embodiments of the invention will be described in detail hereafter in connection with the examples, it will be understood by those skilled in the art that the following examples are only intended to illustrate the invention and should not be considered as limiting the scope of the invention. Where specific conditions are not indicated in the examples, they are carried out according to conventional conditions or those recommended by the manufacturer. Where the manufacturer is not specified for the reagents or apparatus used, they are conventional products available commercially.

EXAMPLE 1

Synthesis of Polypeptide Compounds

[0115] Polypeptide compounds 1-16, positive control polypeptide compounds 17-20 (from compounds in Chinese Patent Publication No. CN104926934A), positive control polypeptide compounds 21-26 (from patent CN201711194175.0), SAR425899 and MED C18-acid involved in the present invention were synthesized by the applicant, where SAR425899 and MED C18-acid are both compounds reported in the prior art.

[0116] The amino acid sequences of the above positive control polypeptide compounds 17-26, SAR425899 and MED C18-acid compounds are as follows.

TABLE-US-00005 Compound 17 (SEQ ID NO. 17): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu- Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.14CH.sub.3)-Arg-Arg-Ala-Gln-Asp-Phe- Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 HsQGTFTSDYSKYLDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.14CH.sub.3) RRAQDFVQWLMNTGGPSSGAPPPS-NH.sub.2 Compound 18 (SEQ ID NO. 18): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu- Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.14CH.sub.3)-Arg-Arg-Ala-Gln-Asp-Phe- Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 HsQGTFTSDYSKYLDK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.14CH.sub.3) RRAQDFVQWLLNTGGPSSGAPPPS-NH.sub.2 Compound 19 (SEQ ID NO. 19): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG.sub.2- PEG.sub.2-CO(CH.sub.2).sub.16CO.sub.2H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe- Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 HsQGTFTSDYSK(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.16CO.sub.2H)KLD-Aib- RRAQDFVQWLMNTGGPSSGAPPPS-NH.sub.2 Compound 20 (SEQ ID NO. 20): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu- Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.14CH.sub.3)-Arg-Arg-Ala-Gln-Asp-Phe- Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 H-(d-S)-QGTFTSDYSKYLDK(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.14CH.sub.3)RRAQDFVQWLMNTGGPSSGAPPPS-NH.sub.2 Compound 21 (SEQ ID NO. 21): His-Xaa-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys*-Ser-Lys-Tyr-Leu-Asp- Glu-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Pro-Ser- Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2 HXQGTFTSDK*SKYLDEQAAQDFVQWLLDGPSSGAPPPS-NH.sub.2 Compound 22 (SEQ ID NO. 22): His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys*-Ser-Lys-Tyr-Leu-Asp- Ser-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2 HSQGTFTSDK*SKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH.sub.2 Compound 23 (SEQ ID NO. 23): His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys*-Ser-Lys-Tyr-Leu-Asp- Glu-Glu-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2 HSQGTFTSDK*SKYLDEEAAQDFVQWLMNGGPSSGAPPPS-NH.sub.2 Compound 24 (SEQ ID NO. 24): His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys*-Ser-Lys-Tyr-Leu-Asp- Glu-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2 HSQGTFTSDK*SKYLDEQAAQDFVQWLMNGGPSSGAPPPS-NH.sub.2 Compound 25 (SEQ ID NO. 25): His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys*-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH HSQGTFTSDK*SKYLDERAAQDFVQWLMNTGPSSGAPPPS-OH Compound 26 (SEQ ID NO. 26): His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys*-Ser-Lys-Tyr-Leu-Asp- Ser-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH HSQGTFTSDK*SKYLDSRAAQDFVQWLMNTGPSSGAPPPS-OH

[0117] Note: X in Compound 21 in the table is aminoisobutyric acid, and K* in Compound 21-25 indicates that this position is a lysine residue and is covalently linked to a fatty acid whose structure is shown in the following formula:

##STR00006##

TABLE-US-00006 MED C18-acid (SEQ ID NO. 27): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Lys(PEG.sub.2- PEG.sub.2-γGlu-(CH.sub.2).sub.17COOH)-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe- Val-Ala-Trp-Leu-Val-Ala-Gly-Gly-NH.sub.2 HsQGTFTSDYSKK(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.16CO.sub.2H) LDSERARDFVAWLVAGG-NH.sub.2 SAR425899 (SEQ ID NO. 28): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln- Lys(CO(CH.sub.2).sub.14CH.sub.3)-Glu-Ser-Lys-Ala-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2 HsQGTFTSDLSKQK(CO(CH.sub.2).sub.14CH.sub.3)ESKAAQDFIEWLKAGGPS SGAPPPS-NH.sub.2

[0118] To facilitate the demonstration of the preparation method of the above polypeptide compounds, the present invention takes polypeptide compound 3 as an example, and the specific operation steps are as follows.

[0119] (i) Materials:

[0120] All amino acids were purchased from NovaBiochem Company. Unless otherwise specified, other reagents were all analytically pure and purchased from Sigma Company. CS336X peptide synthesizer (CS Bio American Company). Phenomenex Luna C18 preparative column (46 mm×250 mm) was used to purify the peptides. The high performance liquid chromatograph was manufactured by Waters Company. Mass analysis was determined using Agilent mass spectrometer.

[0121] (ii) Methods:

[0122] 1. Synthesis of Polypeptide Compound 3.

[0123] Structural Sequence:

TABLE-US-00007 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Glu-Aib- Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala- Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2

[0124] 1a) Main Peptide Chain Assembly:

[0125] The following peptides were synthesized on a 0.25 mol scale on a CS336X peptide synthesizer (CS Bio American Company) according to the Fmoc/t-Bu strategy:

TABLE-US-00008 Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)- Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)-Ser(t-Bu)- Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)- Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly- Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Ser(t-Bu)-rink amide resin

[0126] (1) Step 1: 0.75 g of Rink amide resin was dissolved in dichloromethane and the resin was washed with N,N-dimethylformamide for three times.

[0127] (2) Step 2: The procedure reaction was performed using Rink amide resin as carrier, the mixture of benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine in the ratio of the amount of substance (molar ratio) of 1:1:1 was used as the coupling agent, and N,N-dimethylformamide was used as the solvent, the procedure reaction was carried out and the condensation reaction was performed sequentially to couple Fmoc-Ser(t-Bu)-OH, Fmoc-Pro-OH (3×), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(t-Bu)-OH (2×), Fmoc-Pro-OH, Fmoc-Gly-OH (2×), Fmoc-Ala-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH (2×), Fmoc-Lys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)—OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Phe-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Ser(t-Bu)-OH, Boc-His(Boc)-OH to obtain:

[0128] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin, wherein the ratio of the amount of substance (molar ratio) of the amount of the Fmoc-protected amino acids fed to the amount of resin used in each condensation reaction was 1:1 to 6:1, and the amount of Fmoc-protected amino acids fed to the amount of resin used in each condensation reaction was determined by the ratio of the amount of substance (molar ratio) of the amount of benzotriazole-N,N,N′,N′-tetramethylformamidiniumhexafluorophosphatefed to the amount of Fmoc-protected amino acids used in each condensation reaction was 3:1.

[0129] 1b) Removal of Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H) and Introduction of Lipophilic Substituents:

[0130] The protected peptide-based resin synthesized in 1a) was washed twice in a mixture of N-methylpyrrolidone:/dichloromethane (DCM)=1:1 (v/v), freshly prepared 2.0% solution of hydrazine hydrate N-methylpyrrolidone was added, and the reaction mixture was shaken for 12.0 min at room temperature and then filtered. The hydrazine treatment step was repeated twice to obtain:

[0131] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Thereafter the resin was washed well with dichloromethane and N-methylpyrrolidone. A mixed coupling solution of FmocNH-PEG.sub.2-OH, benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N-methylpyrrolidone with diisopropylethylamine was added thereto and shaken for 3.0 h, filtered and washed, the hydrazine treatment step was repeated twice to yield: Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-OH)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. The Fmoc group was removed from the piperidine/N,N-dimethylformamide solution and the Fmoc-PEG.sub.2-OH coupling reaction was repeated once to yield: Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-OH)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. The Fmoc group was removed from piperidine/N,N-dimethylformamide solution, and then the Fmoc-γGlu-OtBu, tBu monoprotected eicosanoic fatty acids were sequentially coupled according to conventional conditions to yield: Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin.

[0132] 1c) Removal of Total Peptide Protection:

[0133] Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Ala-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin was added to a round bottom flask, and the reaction was carried out under ice bath conditions by adding cutting solution TFA/EDT/Phenol/H.sub.2O (88/2/5/5, v/v) to raise the temperature and the temperature of the lysate was controlled at 25° C. for 120 min. Filtered, the filter cake was washed 3 times with a small amount of trifluoroacetic acid and the filtrate was combined. The filtrate was slowly poured into ice ether under stirring, and was allowed to stand for more than 1.0 hour till complete precipitation. The supernatant was decanted off and the precipitate was centrifuged and washed 6 times with ice ether to obtain the crude compound: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2

[0134] 1d) Refinement and Purification of Polypeptide Compounds:

[0135] The crude product obtained in 1c was dissolved in 5.0% acetic acid in a solution of acetonitrile:H.sub.2O=1:1 (v/v) and purified by semi-preparative HPLC on a reversed-phase C18 packed 50 mm×250 mm column for 2 times. The column was eluted with 30-60% acetonitrile-0.1% trifluoroacetic acid/H.sub.2O gradient at 40 mL/min for 45.0 min. The fraction containing peptides was collected, concentrated to remove acetonitrile and then lyophilized. The pure product with >95% purity by HPLC was obtained. The separated product was analyzed by liquid-mass spectrometry, and the m/z value of the ion peak of the protonated molecule was found to be: 4860.9, while the theoretical value was 4863.5.

TABLE-US-00009 TABLE 1 Polypeptide compounds of the present invention Theo- retical Measured Compound Parent peptide Structure value value  1 SEQ ID NO. 1 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 4861.5 4858.5 Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Glu- Aib-Lys-Ala-Ala-Gln- Asp-Phe-Val-Glu- Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2  2 SEQ ID NO. 2 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4734.4 4731.5 Asp-Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.18CO.sub.2H)- Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala- Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala- Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2  3 SEQ ID NO. 3 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4863.5 4860.9 Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Glu- Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu- Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2  4 SEQ ID NO. 4 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4835.5 4832.5 Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.16CO.sub.2H)-Ser-Lys-Tyr-Leu-Glu- Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu- Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2  5 SEQ ID NO. 5 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4833.5 4830.5 Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CH.sub.3)-Ser-Lys-Tyr-Leu-Glu- Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu- Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2  6 SEQ ID NO. 6 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4963.5 4960.5 Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln- Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser- Gly-Aib-Pro-Pro-Pro-Ser-NH.sub.2  7 SEQ ID NO. 7 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4963.5 4960.9 Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln- Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2  8 SEQ ID NO. 8 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 4961.6 4959.3 Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln- Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser- Gly-Aib-Pro-Pro-Pro-Ser-NH.sub.2  9 SEQ ID NO. 9 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 4933.5 4930.4 Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.16CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln- Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser- Gly-Aib-Pro-Pro-Pro-Ser-NH.sub.2 10 SEQ ID NO. 10 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 4931.6 4928.6 Lys(PEG.sub.2-PEG.sub.2-γGlu-CO(CH.sub.2).sub.18CH.sub.3)- Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala- Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp- Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro- Pro-Ser-NH.sub.2 11 SEQ ID NO. 11 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 4802.5 4799.5 Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.18CH.sub.3)-Ser- Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln- Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly- Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro- Ser-NH.sub.2 12 SEQ ID NO. 12 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4963.5 4960.6 Asp-Lys(PEG.sub.2-PEG.sub.2-γGlu- CO(CH.sub.2).sub.18CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln- Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser- Gly-Aib-Pro-Pro-Pro-Ser-NH.sub.2 13 SEQ ID NO. 13 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4948.5 4948.8 Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly- γGlu-CO(CH.sub.2).sub.18-COOH)-Ser-Lys-Tyr- Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe- Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser 14 SEQ ID NO. 14 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- 4963.5 4962.6 Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly- γGlu-CO(CH.sub.2).sub.18-COOH)-Ser-Lys-Tyr- Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe- Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2 15 SEQ ID NO. 15 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 5046. 4 5046. 8 Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu- CO(CH.sub.2).sub.18-COOH)-Ser-Lys-Tyr-Leu- Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva- Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser- Ser-Gly-Aib-Pro-Pro-Pro-Ser 16 SEQ ID NO. 16 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- 5061.4 5060.6 Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu- CO(CH.sub.2).sub.18-COOH)-Ser-Lys-Tyr-Leu- Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva- Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser- Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH.sub.2

EXAMPLE 2

Effects of Polypeptide Compounds 1-16, Positive Control Polypeptide Compounds 17-26, Liraglutide and Semaglutide on Oral Glucose Tolerance (OGTT)

[0136] Semaglutide was purchased from Zhejiang Paitai Biological Co., Ltd (CAS No.: 910463-68-2) and Liraglutide was purchased from Shenzhen Jianyuan Pharmaceutical Technology Co., Ltd. (CAS No.: 204656-20-2).

[0137] Eight-week-old male C57BL/6J mice (Model Animal Research Center, Nanjing University) were randomly grouped according to similar blood glucose (assessed by blood samples obtained from the tail tip), with six mice in each group.

[0138] After fasting (6 h), the animals were administered with polypeptide compounds 1-16, positive control polypeptide compounds 17-26, liraglutide, semaglutide, SAR425899 and MED C18-acid in the present invention at a dose of 100 μg/kg, subcutaneously, while the control group was administered with PBS. The initial blood samples (fasted blood glucose levels) were obtained after about 4 h. Subsequently, an oral dose of glucose (2 g/kg) was administered and the animals were placed back in their holding cages (t=0). Blood glucose was measured at t=15 min, t=30 min, t=60 min and t=120 min. The oral dose of glucose was then repeated at 8 h of administration, 12 h of administration and 24 h of administration, and blood glucose changes were detected and followed until 24 hours. The data were processed using the software GraphPadPrism to make a line graph of blood glucose changes, and the area under the curve was calculated to obtain an AUC graph, and the results are shown in FIG. 1.

[0139] Compared to the carrier agent (control PBS), liraglutide and compounds 17-26 could have a significantly lower AUC (p<0.05) in the first OGTT curve period (0-120 min), while in the next three OGTT curve periods (4-22 h), their AUC started to elevate. In contrast, the AUC of semaglutide was significantly reduced (P<0.05) in all four OGTT curves, indicating its long-lasting hypoglycemic effect. And compared with the carrier agent (control PBS), the polypeptide compounds of the present invention all showed different degrees of improvement in glucose tolerance in mice, with polypeptide compounds 1-16 exhibiting a more excellent and significant improvement in glucose tolerance at four OGTT curve periods (0-22h), while the results also indicate that polypeptide compounds 1-16, compared with polypeptide compounds 17-26, liraglutide SAR425899 and MED C18-acid have more excellent and significant long-lasting hypoglycemic effects. Among them, polypeptide compounds 1, 2, 3, 8, 13, 14 and 15 exhibited superior and significant long-acting hypoglycemic effects compared to semaglutide at four OGTT curves (0-22 h).

EXAMPLE 3

Amelioration of Renal Fibrosis by Polypeptide Compounds 1, 2, 3, 8, 13, 14, and 15 in a High Glucose-Induced Renal Fibrosis Model

[0140] Hyperglycemia and increased production of glycosylation end products will cause morphological changes such as proliferation and hypertrophy of glomerular mesangial cells, increase in extracellular matrix (ECM) and mesangial expansion, which will eventually lead to the loss of its physiological function. In addition, trans-differentiation of renal tubular epithelial cell is also an important pathological basis of renal fibrosis, and it has been found that high concentration of glucose further induces glomerular fibrosis by activating the RAS system. Therefore, this example used high glucose to induce MCS mesangial cells and NRK renal epithelial cells to construct an in vitro renal fibrosis cell model for screening medicaments useful for the treatment of diabetic nephropathy.

[0141] The renal mesangial cell strain GMC and glomerular epithelial cells mTEC used in this example were both cells kept in the applicant's laboratory (both were extracted from rat renal primary cells) and cultured in low sugar medium containing 10% fetal bovine serum. The cells were starved with serum-free culture medium for 24 h when grew to 70% confluence. The cells were divided into the following 9 groups by adjusting the glucose concentration as follows: (i) Low group (5.5 mmol/L glucose), (ii) High group (30 mmol/L glucose), (iii) High glucose+compound 1 group (30 mmol/L glucose+NO. 1 10 μM), (iv) High glucose+compound 2 group (30 mmol/L glucose+NO. 2 10 μM), (v) high sugar+compound 3 group (30 mmol/L glucose+NO.3 10 μM), (vi) high sugar+compound 8 group (30 mmol/L glucose+NO.8 10 μM), (vii) high sugar+compound 13 group (30 mmol/L glucose+NO.13 10 μM), (viii) high sugar+compound 14 group (30 mmol/L glucose+NO.14 10 μM), and (ix) high sugar+compound 15 group (30 mmol/L glucose+NO.15 10 μM) were incubated at 37° C. 5% CO.sub.2 for 48 h for Western blot assay of FN protein (fibronectin). As shown in FIG. 2A, the gel electrophoresis graphs of FN expression in different groups detected by Western blot was shown in an in vitro renal fibroblast cell model constructed by high glucose induction of MCS mesangial cells and NRK renal epithelial cells in this example; as shown in FIG. 2B, the bar graphs of FN expression detected in different groups was shown in the in vitro renal fibroblast cell model constructed by high glucose induction of MCS mesangial cells and NRK renal epithelial cells in this example 3.

[0142] According to the previous experimental study and previous literature, 30 mmol/L glucose can induce the hypertrophy of glomerular mesangial cells and the trans-differentiation of renal epithelial cells. Western blot assay results showed that the expression level of FN protein was higher in the High group than the control Low group under high glucose stimulation; while all the administered groups can down-regulate the expression of FN protein, suggesting that the polypeptide compound of the present invention can significantly reduce the increase of ECM caused by high glucose. Among them, polypeptide compounds 3, 8, 13, 14 and 15 inhibited FN expression more significantly.

EXAMPLE 4

Therapeutic Effects of Polypeptide Compounds 3, 8, 13, 14, 15 and 16, Semaglutide, SAR425899, MED C18-Acid on Diabetic Mice

[0143] (i) Experiments for the Determination of Different Concentrations of Medicaments of Polypeptide Compounds 3, 8, 13 and 15

[0144] The db/db mice diabetic model (purchased from Nanjing University-Nanjing Institute of Biomedical Research, about 8 weeks, and blood glucose and body weight were measured to ensure the smooth subsequent experiments) was obtained, and the model mice were randomly divided into 12 groups (including polypeptide compounds 3, 8, 13, 15, semaglutide and physiological saline groups), with 6 mice in each group and no difference in basal body weight and blood glucose. Each group was injected subcutaneously with compound 3 (80 and 120 μg/kg, 2 groups), compound 8 (80 and 120 μg/kg, 2 groups), compound 13 (80 and 120 μg/kg, 2 groups), compound 15 (80 and 120 μg/kg, 2 groups), semaglutide (80 and 120 μg/kg, 2 groups) and physiological saline (DN group: normal mice born in the same litter; DC group: db/db mice control group) every day or every other day.

[0145] Blood glucose and body weight of mice were measured after daily administration and 6 h fasting on alternate days.

[0146] FIG. 3A shows that compared with the physiological saline group DC group, the semaglutide group (120 μg/kg) exhibited a better hypoglycemic effect, after which it was maintained at a more stable normoglycemic level, but its hypoglycemic effect was not significant. In the administration group, polypeptide compounds 3, 8, 13 and 15 at a dose of 80 μg/kg, polypeptide compounds 3 and 8 showed significant glucose-lowering effect, and polypeptide compounds 13 and 15 showed significant glucose-lowering effect with long-lasting effect; at a dose of 120 μg/kg, polypeptide compounds 3, 8, 13 and 15 already achieved good glucose-lowering effect in 3-5 days, and their blood glucose was stably maintained at normal level, besides, polypeptide compounds 3, 8, 13 and 15 showed better weight-lowering effect, and the effect of compounds 13 and 15 was superior to that of compounds 3 and 8. Therefore, the medicament concentration was set at 120 μg/kg.

[0147] Blood glucose and body weight of mice were measured after daily administration and 6 h fasting on alternate days.

[0148] FIG. 3B shows that compared with the physiological saline group DC group, the semaglutide group had a hypoglycemic effect at the dose of 80 μg/kg and 120 μg/kg every other day, but the hypoglycemic effect was not significant, and its hypoglycemic effect was not significant. In the administration group of polypeptide compounds 3 and 8, at the dose of 80 μg/kg, the blood glucose of polypeptide compounds 3 and 8 showed a first decrease and then increase, and the overall hypoglycemic effect was significant, and polypeptide compounds 3 and 8 had a hypoglycemic effect. While polypeptide compounds 13 and 15 had significant hypoglycemic effect on day 3, but the subsequent blood glucose level did not change much and tended to be stable; at the dose of 120 μg/kg, the blood glucose of semaglutide group rebounded on day 3, and its long-lasting effect was not enough. Polypeptide compounds 3, 8, 13 and 15 had achieved good hypoglycemic effect on days 3-5, and after then, the blood glucose did not change much for polypeptide compounds 3 and 8. Polypeptide compounds 13 and 15 kept good hypoglycemic effect, which proved that polypeptide compounds 13 and 15 have good hypoglycemic long-lasting effect. Meanwhile, polypeptide compounds 3, 8, 13 and 15 showed more excellent hypoglycemic effect, and the effect of polypeptide compounds 13 and 15 was better than that of polypeptide compounds 3 and 8. Therefore, the medicament concentration was set at 120 μg/kg.

[0149] (II) Ameliorating Effects of Polypeptide Compounds 3, 8, 13, 14, 15, 16, Semaglutide, SAR425899 and MED C18-Acid on Diabetes in Diabetic Mice

[0150] A db/db mouse model of diabetes (purchased from Nanjing University-Nanjing Institute of Biomedical Research, mice at about 12 weeks, and blood glucose and body weight were measured to ensure that the subsequent experiments were carried out smoothly) was obtained, and the model mice were randomly divided into 10 groups (polypeptide compounds 3, 8, 13, 14, 15, 16, semaglutide, SAR425899, MED C18-acid and saline group), with 6 mice in each group, and no difference in basal body weight and blood glucose in each mouse, each group was injected subcutaneously daily with polypeptide compounds 3, 8, 13, 14, 15, and 16 (120 μg/kg), semaglutide (120 μg/kg), SAR425899 (120 μg/kg), MED C18-acid (120 μg/kg), and saline (DN group. Normal mice born in the same litter; DC group: db/db mice control group). Blood glucose and body weight were measured on alternate days after daily administration of the medicament, and water intake and food intake were measured once a week. OGTT was measured at week 4, insulin tolerance (ITT) was measured at week 5, metabolism of mice was examined using mouse metabolic cages at weeks 6-7, and various serological and pathological indices were taken at week 8.

[0151] 1. The Effect of the Medicaments on the Glucose Lowering Weight and Improvement of Insulin Resistance in Diabetic Mice

[0152] Type 2-diabetes mice were characterized by obesity, insulin resistance, hyperglycemia, dyslipidemia and hepatic fat vacuole-like degeneration. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed in diabetic mice treated with polypeptide compounds 3, 8, 13, 14, 15, 16 and semaglutide, SAR425899, MED C18-acid at 4 and 5 weeks, respectively, and the results are shown in FIGS. 4A-4D.

[0153] The results in FIGS. 4A to 4C showed that polypeptide compounds 3, 8, 13, 14, 15, 16, semaglutide, SAR425899 and MED C18-acid all had significant hypoglycemic effects compared to the DN group, with blood glucose already falling to normal levels at week 1 and relatively stable thereafter. However, the polypeptide compounds 3, 8, 13, 14, 15 and 16 performed more excellent in improving oral glucose tolerance (OGTT) and insulin resistance (ITT) in diabetic mice.

[0154] In addition, the results in FIG. 4D showed that polypeptide compounds 3, 8, 13, 14, 15, and 16 had more excellent hepatoprotective functions.

[0155] 2. Therapeutic Effect of the Medicaments on Diabetic Nephropathy in Diabetic Mice

[0156] After 6 weeks of medical treatment, the mice was collected for urine using metabolic cages, and the protein content in the urine was detected using relevant kits. After 7 weeks of medical treatment, the material was taken and blood was used to detect serological indicators such as blood creatinine and urea nitrogen. The results are shown in FIGS. 5A to 5F.

[0157] As shown in FIG. 5A, the 20-week-old db/db diabetic mice in the DC group showed a significant increase in urine protein content as well as urine glucose content compared to the control DN group. In addition, glycosylated hemoglobin content, blood creatinine, and urea nitrogen content were also significantly increased, indicating that the diabetic nephropathy in the mice was already severe. After the administration of the medicament, the urine protein content, urine glucose content, glycosylated hemoglobin content, blood creatinine and urea nitrogen content of the mice were all improved to different degrees.

[0158] The kidneys of mice were taken for paraffin embedding and used for subsequent staining and immunohistochemical studies. According to the results of H&E staining of mouse kidneys in FIG. 5B, local inflammation and glomerular adhesions were observed in the kidney tissue. After administration, the glomerular adhesion phenomenon was alleviated, and the ameliorative and therapeutic effects of polypeptide compounds 3, 8, 13, 14, 15 and 16 were more significant compared to the positive control medicament semaglutide, SAR425899 and MED C18-acid groups. In general, renal lesions were observed mostly in the cortex, mainly including glomeruli and interstitium, and glomeruli were observed in the mesangial region. As observed by PAS staining (FIG. 5C), compared with the DN group, the glomerular mesangial region was widened, the mesangial cells and mesangial matrix were proliferated, and the basement membrane was thickened in the DC group. After administration of the medicament, the glomerular vascular collaterals were thin and clear, the number of endothelial and mesangial cells was normal, and the surrounding tubules were normal, and the ameliorative effects of polypeptide compounds 3, 8, 13, 14, 15 and 16 were more significant. As shown by the staining of Sirius novel red as well as FN and α-SMA in FIGS. 5D to 5F, compared with the DN group, the collagen deposition was significantly increased in the DC group, and the medicament group could significantly reduce the collagen content, in which the polypeptide compounds 3, 8, 13, 14, 15 and 16 improved more significantly, and their therapeutic effect was better than the positive control semaglutide, SAR425899 and MED C18-acid groups.

[0159] In conclusion, by further optimization and design of the amino acid sequence, along with modification of the side chain technology, the compounds reported in the present invention are superior to the previously reported compounds in terms of hypoglycemia, weight reduction, improvement and treatment of diabetic complications, and are more advantageous in clinical applications.

[0160] The above description of specific embodiments of the present invention does not limit the present invention, and various changes or variations can be made by those skilled in the art according to the present invention, which shall fall within the scope of the appended claims of the present invention as long as they do not depart from the spirit of the present invention.