GLP-1 (7-37) glycosylated derivative as well as pharmaceutical composition and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a GLP-1 (7-37) glycosylated derivative as well as a pharmaceutical composition and application thereof. The GLP-1 (7-37) glycosylated derivative disclosed by the invention is formed by connecting sugar with oxygen of side chains of 11-site threonine, 13-site threonine or 18-site serine of GLP-1 (7-37). Compared with the GLP-1 (7-37), the GLP-1 (7-37) glycosylated derivative disclosed by the invention has higher DPP-4 enzymolysis stability, also maintains the hypoglycemic activity equivalent to that of the GLP-1 (7-37), and has the potential of being further developed into a medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a GLP-1(7-37) glycosylation derivative, a pharmaceutical composition thereof, and applications thereof. Background Art

[0002] Glucagon-like peptide-1 (GLP-1(7-37)) is a multifunctional hormone containing 31 amino acids. Research has shown that GLP-1(7-37) can reduce blood glucose concentration through pathways such as glucose concentration-dependent insulin secretion stimulation, inhibition of gastric emptying, and reduction of food intake. In addition, GLP-1(7-37) also has cardioprotective and neuroprotective effects, reducing inflammation and apoptosis. These unique properties have inspired extensive efforts to use GLP-1(7-37) in clinical applications. However, GLP-1(7-37) is easily degraded and inactivated by the protease dipeptidyl peptidase-4 (DPP-4) widely present in the body, resulting in a half-life in vivo of only about 2 minutes. To extend its half-life, some pharmaceutical companies have chosen to use the exendin-4 peptide (a natural peptide obtained from the venom of the Heloderma lizard, which has 53% homology with human GLP-1(7-37)) to replace GLP-1(7-37), as its half-life in vivo reaches about 30 minutes. This difference stems from a difference in one amino acid at the 2nd position of the N-terminus: GLP-1(7-37) has an alanine (Ala) at this position, while exendin-4 has a glycine (Gly). Early studies have shown that introducing alternative amino acids at position 8 can reduce the instability of GLP-1(7-37) to DPP-4. Currently, the most commonly used Ala substitute at position 8 is amino isobutyric acid (Aib). Among them, the effect of replacing Ala with Gly is not obvious enough, while replacing it with Aib cannot be directly obtained by recombinant expression methods because it is a non-natural amino acid, and means such as chemical fragment ligation need to be combined for preparation, thus increasing the preparation cost. Generally speaking, current strategies for inhibiting the degradation of GLP-1(7-37) by DPP-4 are relatively limited, and more strategies need to be developed by scientists.

[0003] Glycosylation, as one of the most common post-translational modifications, widely affects the inherent properties of proteins, including properties such as folding, stability, intracellular transport, and pharmacokinetics. Among them, glycosylation often plays an important role in the enzymatic stability of proteins. Summary of the Invention

[0004] In order to inhibit the degradation of GLP-1(7-37) by DPP-4, the present invention provides a GLP-1(7-37) glycosylation derivative, its pharmaceutical composition and application, providing more alternative technical means for this field. Specifically, the present invention provides a GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof, characterized in that the GLP-1(7-37) glycosylation derivative is formed by connecting a sugar to the oxygen of the side chain of threonine at position 11, threonine at position 13 or serine at position 18 of GLP-1(7-37), and the sequence of GLP-1(7-37) is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, and the sugar is selected from G1, G2, G3, G4, G5, G6 or G7;

[0005] The structural formulas of G1, G2, G3, G4, G5, G6 and G7 are as follows:

[0006] Structural formula G1:

[0007] Structural formula G2:

[0008] Structural formula G3:

[0009] Structural formula G4:

[0010] Structural formula G5:

[0011] Structural formula G6:

[0012] Structural formula G7:

[0013] When the GLP-1(7-37) glycosylation derivative contains the G1, G2 or G3 sugar structure, its synthesis method is briefly described as follows:

[0014] 1. Solid-phase synthesis: Using sugar amino acids as raw materials, a glycosylated GLP-1(7-37) polypeptide chain is prepared by solid-phase peptide synthesis.

[0015] 2. Removal of acetyl group on the sugar: The synthesized glycosylated GLP-1(7-37) polypeptide chain is dissolved in a methanol / water (1 / 1, v / v) solution of 40 mM NaOH for reaction.

[0016] 3. Purification: The reaction solution obtained in step 2 above is purified to obtain a glycosylated GLP-1(7-37) polypeptide.

[0017] When the GLP-1(7-37) glycosylation derivative contains a G4, G5, G6 or G7 sugar structure, its synthesis method is briefly described as follows:

[0018] 1. Solid-phase synthesis: Using sugar amino acids as raw materials, a glycosylated GLP-1(7-37) polypeptide chain is prepared by solid-phase peptide synthesis.

[0019] 2. Hydrolysis of methyl ester on the sugar and partial deacetylation: The synthesized glycosylated GLP-1(7-37) polypeptide chain is dissolved in an aqueous solution of 40 mM NaOH for reaction, a termination solution is added, and then freeze-dried.

[0020] 3. Deacetylation of the sugar: An aqueous solution of 10% NH2·NH2 is added to the freeze-dried powder obtained in step 2 for reaction, and a termination solution is added;

[0021] 4. Purification: The reaction solution obtained in step 3 above is purified to obtain a glycosylated GLP-1(7-37) polypeptide.

[0022] Preferably, the GLP-1(7-37) glycosylation derivative is selected from GLP1, GLP2, GLP3, GLP4, GLP5, GLP6, GLP7, GLP8, GLP9, GLP10, GLP11, GLP12, GLP13, GLP14, GLP15, GLP16, GLP17, GLP18, GLP19, GLP20 or GLP21;

[0023] The structural formulas of GLP1, GLP2, GLP3, GLP4, GLP5, GLP6, GLP7, GLP8, GLP9, GLP10, GLP11, GLP12, GLP13, GLP14, GLP15, GLP16, GLP17, GLP18, GLP19, GLP20, GLP21 are respectively:

[0024] Structural formula of GLP1:

[0025] Structural formula of GLP2:

[0026] Structural formula of GLP3:

[0027] Structural formula of GLP4:

[0028]

[0029] Structural formula of GLP5:

[0030]

[0031] Structural formula of GLP6:

[0032]

[0033] Structural formula GLP7:

[0034]

[0035] Structural formula GLP8:

[0036] Structural formula GLP9:

[0037] Structural formula GLP10:

[0038] Structural formula GLP11:

[0039]

[0040] Structural formula GLP12:

[0041]

[0042] Structural formula GLP13:

[0043]

[0044] Structural formula GLP14:

[0045]

[0046] Structural formula GLP15:

[0047] Structural formula GLP16:

[0048] Structural formula GLP17:

[0049] Structural formula GLP18:

[0050] Structural formula GLP19:

[0051] Structural formula GLP20:

[0052]

[0053] Structural formula GLP21:

[0054]

[0055] Among them, the sugar in GLP1 to GLP7 is covalently linked to the oxygen of the side chain of threonine at position 11 of GLP-1(7-37); the sugar in GLP8 to GLP14 is covalently linked to the oxygen of the side chain of threonine at position 13 of GLP-1(7-37), and the sugar in GLP15 to GLP21 is covalently linked to the oxygen of the side chain of serine at position 18 of GLP-1(7-37).

[0056] The present invention also relates to a pharmaceutical composition comprising a biologically active amount of at least one of the above-mentioned GLP-1(7-37) glycosylation derivatives or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0057] The term "pharmaceutically acceptable carrier" broadly refers to any component other than the active therapeutic ingredient. The excipient can be an inert substance, an inactive substance, and / or a non-pharmaceutically active substance. The formulation of pharmaceutically active ingredients with various excipients is known in the art.

[0058] Preferably, the pharmaceutical composition may further include one or more other active ingredients, such as those selected from rapid-acting insulin, long-acting insulin, glucagon-like peptide-1 receptor agonists, glucagon-like peptide-1 / glucagon receptor co-agonists, glucagon-like peptide-1 / glucose-dependent insulinotropic polypeptide receptor co-agonists, or glucagon-like peptide-1 / glucose-dependent insulinotropic polypeptide / glucagon receptor co-agonists.

[0059] Preferably, the long-acting insulin in the pharmaceutical composition of the present invention can be selected from one or more of insulin glargine, insulin detemir, insulin degludec, and insulin icodec.

[0060] Preferably, the glucagon-like peptide-1 receptor agonist can be selected from one or more of liraglutide, semaglutide, dulaglutide, and albiglutide.

[0061] Preferably, the dosage form of the pharmaceutical composition is an injection dosage form or an oral dosage form. The injectable composition containing the GLP-1(7-37) glycosylation derivative of the present invention can be prepared by conventional techniques in the pharmaceutical industry, which include appropriately dissolving and mixing the components to obtain the desired final product. Therefore, the GLP-1(7-37) glycosylation derivative of the present invention can be dissolved in a suitable buffer at an appropriate pH to minimize or avoid precipitation. For example, the injectable composition is made sterile by filtration sterilization.

[0062] The present invention also relates to the use of the above-mentioned GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in the preparation of a drug for the treatment and / or prevention of type II diabetes.

[0063] The present invention also relates to the use of the above-mentioned GLP-1(7-37) glycosylation derivative or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing at least one of the following diseases: impaired glucose tolerance, hyperglycemia, perioperative hyperglycemia, metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0064] The present invention also relates to the use of the above-mentioned GLP-1(7-37) glycosylation derivative or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing neurodegenerative diseases (including Alzheimer's disease, Parkinson's disease, etc.).

[0065] The present invention also relates to the use of the above-mentioned GLP-1(7-37) glycosylation derivative or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing at least one of the following diseases: non-alcoholic steatohepatitis, non-alcoholic fatty liver disease.

[0066] The present invention also relates to the use of the above-mentioned GLP-1(7-37) glycosylation derivative or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing at least one of the following diseases: obesity, atherosclerosis.

[0067] The technical solution provided by the present invention has the following advantages compared with the prior art:

[0068] By glycosylation modification of threonine at position 11, threonine at position 13 or serine at position 18 of GLP-1(7-37), the present invention improves the stability of GLP-1(7-37) against DPP-4 enzyme degradation, providing another technical means for enhancing the DPP-4 enzymatic stability. Among them, the DPP-4 enzymatic stability of the GLP-1(7-37) glycosylation derivative GLP7 is 632 times that of GLP-1(7-37). This fully proves the effectiveness and superiority of this technology. Finally, the mouse activity experiment shows that the GLP-1(7-37) glycosylation derivative retains considerable hypoglycemic activity and has the potential for further development into a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments.

[0070] Figure 1 . Structure, UPLC chart and MS chart of the product GLP1. Molecular formula: C 159 H 241 N 41 O 52 ; Theoretical value: [M+3H] 3+m / z = 1186.59, [M+4H] 4+ m / z = 890.19, [M+5H] 5+ m / z = 712.36; Observed values: 1186.59, 890.20, 712.35. Theoretical value of sugar loss in mass spectrometry: [M+4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0071] Figure 2 . Structure, UPLC chromatogram and MS spectrum of product GLP2. Molecular formula: C 165 H 251 N 41 O 57 ; Theoretical value: [M+3H] 3+ m / z = 1240.61, [M+4H] 4+ m / z = 930.71, [M+5H] 5+ m / z = 744.77; Observed values: 1240.61, 930.71, 744.77. Theoretical value of losing 1 sugar in mass spectrometry to become GLP1: [M+5H] 5+ m / z = 712.36; Observed value: 712.33 (marked with *). Theoretical value of losing 2 sugars in mass spectrometry: [M+4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0072] Figure 3 . Structure, UPLC chromatogram and MS spectrum of product GLP3. Molecular formula: C 173 H 264 N 42 O 62 ; Theoretical value: [M+3H] 3+ m / z = 1308.30, [M+4H] 4+ m / z = 981.48, [M+5H] 5+ m / z = 785.38; Observed values: 1308.30, 981.48, 785.38. Theoretical value of losing 1 sugar in mass spectrometry to become GLP2: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0073] Figure 4 . Structure, UPLC chromatogram and MS spectrum of product GLP4. Molecular formula: C 170 H 258 N 42 O 60 ; Theoretical value: [M+3H] 3+ m / z = 1283.62, [M+4H] 4+m / z = 962.97, [M+5H] 5+ m / z = 770.58; Observed values: 1283.62, 962.97, 770.58. Theoretical value for losing one sugar in the mass spectrum to become GLP1: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0074] Figure 5 . Structure, UPLC chromatogram and MS spectrum of product GLP5. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M+3H] 3+ m / z = 1337.64, [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value for losing one sugar in the mass spectrum to become GLP2: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0075] Figure 6 . Structure, UPLC chromatogram and MS spectrum of product GLP6. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M+3H] 3+ m / z = 1337.64, [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value for losing one sugar in the mass spectrum to become GLP2: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value for losing two sugars in the mass spectrum to become GLP1: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0076] Figure 7 . Structure, UPLC chromatogram and MS spectrum of product GLP7. Molecular formula: C 187 H 285 N 43 O 73 ; Theoretical value: [M+3H] 3+ m / z = 1434.67, [M+4H] 4+m / z = 1076.26, [M+5H] 5+ m / z = 861.21; Observed values: 1434.67, 1076.26, 861.22. Theoretical value for losing 1 sugar in the mass spectrum to become GLP5 / GLP6: [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1003.49, 802.99.

[0077] Figure 8 . Structure, UPLC chromatogram and MS spectrum of product GLP8. Molecular formula: C 159 H 241 N 41 O 52 ; Theoretical value: [M+3H] 3+ m / z = 1186.59, [M+4H] 4+ m / z = 890.19, [M+5H] 5+ m / z = 712.36; Observed values: 1186.59, 890.20, 712.36.

[0078] Figure 9 . Structure, UPLC chromatogram and MS spectrum of product GLP9. Molecular formula: C 165 H 251 N 41 O 57 ; Theoretical value: [M+3H] 3+ m / z = 1240.61, [M+4H] 4+ m / z = 930.71, [M+5H] 5+ m / z = 744.77; Observed values: 1240.61, 930.71, 744.77. Theoretical value for losing 1 sugar in the mass spectrum to become GLP8: [M+5H] 5+ m / z = 712.36; Observed value: 712.33 (marked with *). Theoretical value for losing 2 sugars in the mass spectrum: [M+4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0079] Figure 10 . Structure, UPLC chromatogram and MS spectrum of product GLP10. Molecular formula: C 173 H 264 N 42 O 62 ; Theoretical value: [M+3H] 3+ m / z = 1308.30, [M+4H] 4+ m / z = 981.48, [M+5H] 5+m / z = 785.38; Observed values: 1308.30, 981.48, 785.38. Theoretical value of losing one sugar in the mass spectrum to become GLP9: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0080] Figure 11 . Structure, UPLC chromatogram and MS spectrum of product GLP11. Molecular formula: C 170 H 258 N 42 O 60 ; Theoretical value: [M+3H] 3+ m / z = 1283.62, [M+4H] 4+ m / z = 962.97, [M+5H] 5+ m / z = 770.58; Observed values: 1283.62, 962.97, 770.58. Theoretical value of losing one sugar in the mass spectrum to become GLP8: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0081] Figure 12 . Structure, UPLC chromatogram and MS spectrum of product GLP12. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M+3H] 3+ m / z = 1337.64, [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing one sugar in the mass spectrum to become GLP9: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0082] Figure 13 . Structure, UPLC chromatogram and MS spectrum of product GLP13. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M+3H] 3+ m / z = 1337.64, [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing one sugar in the mass spectrum to become GLP9: [M+4H] 4+m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value after losing 2 sugars in the mass spectrum to become GLP8: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0083] Figure 14 . Structure, UPLC chromatogram and MS spectrum of product GLP14. Molecular formula: C 187 H 285 N 43 O 73 ; Theoretical value: [M+3H] 3+ m / z = 1434.67, [M+4H] 4+ m / z = 1076.26, [M+5H] 5+ m / z = 861.21; Observed values: 1434.68, 1076.26, 861.21. Theoretical value after losing 1 sugar in the mass spectrum to become GLP12 / GLP13: [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1003.49, 802.99.

[0084] Figure 15 . Structure, UPLC chromatogram and MS spectrum of product GLP15. Molecular formula: C 159 H 241 N 41 O 52 ; Theoretical value: [M+3H] 3+ m / z = 1186.59, [M+4H] 4+ m / z = 890.19, [M+5H] 5+ m / z = 712.36; Observed values: 1186.59, 890.20, 712.36. Theoretical value after losing sugars in the mass spectrum: [M+4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0085] Figure 16 . Structure, UPLC chromatogram and MS spectrum of product GLP16. Molecular formula: C 165 H 251 N 41 O 57 ; Theoretical value: [M+3H] 3+ m / z = 1240.61, [M+4H] 4+ m / z = 930.71, [M+5H] 5+m / z = 744.77; Observed values: 1240.61, 930.71, 744.77. Theoretical value for GLP15 with one sugar removed in the mass spectrum: [M + 5H] 5+ m / z = 712.36; Observed value: 712.34 (marked with *). Theoretical value for GLP15 with two sugars removed in the mass spectrum: [M + 4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0086] Figure 17 . Structure, UPLC chromatogram and MS spectrum of product GLP17. Molecular formula: C 173 H 264 N 42 O 62 ; Theoretical value: [M + 3H] 3+ m / z = 1308.30, [M + 4H] 4+ m / z = 981.48, [M + 5H] 5+ m / z = 785.38; Observed values: 1308.30, 981.48, 785.39. Theoretical value for GLP16 with one sugar removed in the mass spectrum: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value for GLP15 with two sugars removed in the mass spectrum: [M + 4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0087] Figure 18 . Structure, UPLC chromatogram and MS spectrum of product GLP18. Molecular formula: C 170 H 258 N 42 O 60 ; Theoretical value: [M + 3H] 3+ m / z = 1283.62, [M + 4H] 4+ m / z = 962.97, [M + 5H] 5+ m / z = 770.58; Observed values: 1283.62, 962.97, 770.58.

[0088] Figure 19 . Structure, UPLC chromatogram and MS spectrum of product GLP19. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M + 3H] 3+ m / z = 1337.64, [M + 4H] 4+ m / z = 1003.48, [M + 5H] 5+m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing one sugar in the mass spectrum to become GLP16: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0089] Figure 20 . Structure, UPLC chromatogram and MS spectrum of product GLP20. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M+3H] 3+ m / z = 1337.64, [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing one sugar in the mass spectrum to become GLP16: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value of losing two sugars in the mass spectrum to become GLP15: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0090] Figure 21 . Structure, UPLC chromatogram and MS spectrum of product GLP21. Molecular formula: C 187 H 285 N 43 O 73 ; Theoretical value: [M+3H] 3+ m / z = 1434.67, [M+4H] 4+ m / z = 1076.26; Observed values: 1434.67, 1076.26. Theoretical value of losing one sugar in the mass spectrum to become GLP19 / GLP20: [M+4H] 4+ m / z = 1003.48; Observed value: 1003.49 (marked with *). Theoretical value of losing two sugars in the mass spectrum to become GLP18: [M+4H] 4+ m / z = 962.97; Observed value: 962.97 (marked with *). Theoretical value of losing two sugars in the mass spectrum to become GLP16: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0091] In the above figures: AU is the absorbance unit; Time (min) is the retention time in UPLC and its unit is minute; m / z is the mass-to-charge ratio. Detailed implementation manners

[0092] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention, and is not used to limit the present invention as described in detail in the claims. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0093] The partial English abbreviations used in the present invention and their meanings are shown in Table l as follows:

[0094] Table 1:

[0095] Fmoc 9-Fluorenylmethyloxycarbonyl DMF N,N-Dimethylformamide DCM Dichloromethane DIC 1,3-Diisopropylcarbodiimide Oxyma Ethyl 2-cyano-2-oxoacetimidate TFA Trifluoroacetic acid TIPS Triisopropylsilane DODT 3,6-Dioxa-1,8-octanedithiol <![CDATA[NH2·NH2]]> Hydrazine NaOH Sodium hydroxide

[0096] The sugar amino acids involved in the present invention can be synthesized with reference to the literature or directly purchased, and other reagents can be directly purchased. The structures of the sugar amino acids involved are as follows:

[0097] F1:

[0098] F2:

[0099] F3:

[0100] F4:

[0101] F5:

[0102] F6:

[0103] F7:

[0104] Among them, R = CH3 or H.

[0105] When the synthesis site is threonine, the sugar amino acid with R = CH3 is used; when the synthesis site is serine, the sugar amino acid with R = H is used.

[0106] Example 1. Preparation of GLP-1.

[0107] Solid-phase peptide synthesis (SPPS) of glycosylated GLP-1(7-37) polypeptide GLP1 was carried out using a CEM Liberty Blue automatic microwave peptide synthesizer. The specific steps are as follows: First, weigh 0.05 mmol of Fmoc-Gly-2-ClTrt resin, add it to the reactor, and add 10 mL of DMF to swell for 5 min. After removing the DMF solution, add 3 mL of 4-methylpiperidine / DMF solution (20 / 80, v / v), and microwave heat at 50 °C for 5 min to remove the Fmoc protecting group. Wash the resin four times with DMF (4 mL × 4). After removing the DMF solution, add 0.2 M Fmoc-protected amino acid dissolved in DMF (5.0 equivalents), 0.5 M DIC dissolved in DMF (10 equivalents), and 0.25 M Oxyma dissolved in DMF (5.0 equivalents), and microwave heat at 50 °C for 10 min for the coupling reaction. For the amino acids (Gly10, Phe12, Asp15, Phe28, Leu32, Gly35) after the amino acids with β-side chain branching (Thr, Ile, Val) and amino acids with large steric hindrance side chain protecting groups (Arg), two coupling reactions are required. The coupling reaction of Arg needs to be carried out at room temperature for 25 min, and a total of two rounds are carried out. In addition, the coupling of sugar amino acid F1 (0.2 M concentration, 4.5 equivalents) can be completed by microwave heating at 50 °C for 20 min. After completing the coupling of amino acid or sugar amino acid F1, wash the resin four times with DMF (4 mL × 4). Repeat the above coupling-deprotection cycle until the synthesis of the GLP1 glycopeptide chain is completed.

[0108] After solid-phase synthesis, transfer the resin from the reactor to a peptide synthesis reaction tube, and wash it three times alternately with DMF and DCM (5 mL each time). Then add 3.5 mL of TFA / TIPS / H2O / DODT (90 / 5 / 2.5 / 2.5, v / v / v / v) cleavage solution, and stir at 38 °C for 40 min. Collect the cleavage solution with a 50 mL centrifuge tube, and then add 40 mL of methyl tert-butyl ether at 4 °C to precipitate the polypeptide, and centrifuge (9000 g, 7 min, 4 °C). After discarding the supernatant, dissolve the precipitate in 10 mL of MeCN / H2O (1 / 1, v / v), and obtain 104 mg of crude peptide after freeze-drying.

[0109] Weigh 6.03 mg of the above crude peptide, add 3 mL of methanol / water (1 / 1, v / v) solution of 40 mM NaOH, stir at room temperature until all acetyl groups are removed (2 - 3 h), and then add 5% acetic acid for neutralization. After passing the reaction solution through a 0.2 μm filter membrane, purify it by HPLC using a C18 column (10x 250mm, 5μm, ), mobile phase A is water containing 0.05% TFA, mobile phase B is acetonitrile containing 0.05% TFA, linearly eluted with 20% to 40% (B%) for 40 minutes, flow rate is 4 mL / min, detection wavelength is 214 nm. During the purification process, LC-MS (Waters SQD2-ACQUITY UPLC, mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, gradient is 20% to 40% (B%), flow rate is 0.3 mL / min, detection wavelength is 214 nm) is used to identify the collected eluate. The eluates containing the product are combined and after lyophilization, 2.08 mg of product GLP1 is obtained, and the yield is 20%. The product GLP1 is characterized by LC-MS, and its structure is correct. The results are as shown in the attached Figure 1 as follows.

[0110] Example 2. Preparation of GLP2, GLP3, GLP8 - GLP10 and GLP15 - GLP17.

[0111] The preparation of GLP2, GLP3, GLP8 - GLP10 and GLP15 - GLP17 is basically the same as that in Example 1, except that the glycoamino acids they use are different. The corresponding relationships between the synthesized glycopeptides and glycoamino acids are listed in the following table:

[0112] Table 2: Corresponding relationships between glycopeptides and the glycoamino acids used in their synthesis.

[0113] Glycopeptide Glycoamino acid GLP8, GLP15 F1 GLP2, GLP9, GLP16 F2 GLP3, GLP10, GLP17 F3

[0114] The structures of the synthesized GLP2, GLP3, GLP8 - GLP10 and GLP15 - GLP17 are all correct. Their LC-MS characterization results are as shown in the attached Figure 2 , 3 , 8 - 10 and 15 - 17. Their synthesis yields are shown in the following table:

[0115] Table 3: Statistical results of the synthesis yields of GLP1 - GLP3, GLP8 - GLP10 and GLP15 - GLP17.

[0116] Yield Yield Yield GLP1 20％ GLP8 21％ GLP15 26％ GLP2 27％ GLP9 26％ GLP16 23％ GLP3 19％ GLP10 20％ GLP17 19％

[0117] Example 3. Preparation of GLP4.

[0118] Solid-phase peptide synthesis (SPPS) of glycosylated GLP-1(7-37) polypeptide GLP4 was carried out using a CEM Liberty Blue automatic microwave peptide synthesizer. The specific steps are as follows: First, weigh 0.05 mmol of Fmoc-Gly-2-ClTrt resin, add it to the reactor, and add 10 mL of DMF to swell for 5 min. After removing the DMF solution, add 3 mL of 4-methylpiperidine / DMF solution (20 / 80, v / v), and microwave heat at 50 °C for 5 min to remove the Fmoc protecting group. Wash the resin four times with DMF (4 mL×4). After removing the DMF solution, add 0.2 M Fmoc-protected amino acid (5.0 equivalents) dissolved in DMF, 0.5 M DIC (10 equivalents) dissolved in DMF, and 0.25 M Oxyma (5.0 equivalents) dissolved in DMF, and microwave heat at 50 °C for 10 min for the coupling reaction. For the amino acids (Gly10, Phe12, Asp15, Phe28, Leu32, Gly35) after the amino acids with β-side chain branching (Thr, Ile, Val) and amino acids with large steric hindrance side chain protecting groups (Arg), two coupling reactions are required. The coupling reaction of Arg needs to be carried out at room temperature for 25 min, and a total of two rounds are carried out. In addition, the coupling of the sugar amino acid F4 (0.2 M concentration, 4.5 equivalents) can be completely reacted by microwave heating at 50 °C for 20 min. After the coupling of the amino acid or the sugar amino acid F4 is completed, wash the resin four times with DMF (4 mL×4). Repeat the above coupling-deprotection cycle until the synthesis of the GLP4 glycopeptide chain is completed.

[0119] After solid-phase synthesis, transfer the resin from the reactor to a peptide synthesis reaction tube, and wash it three times alternately with DMF and DCM (5 mL each time). Then add 3.5 mL of TFA / TIPS / H2O / DODT (90 / 5 / 2.5 / 2.5, v / v / v / v) cleavage solution, and stir at 38 °C for 40 min. Collect the cleavage solution with a 50 mL centrifuge tube, and then add 40 mL of methyl tert-butyl ether at 4 °C to precipitate the polypeptide, and centrifuge (9000 g, 7 min, 4 °C). After discarding the supernatant, dissolve the precipitate in 10 mL of MeCN / H2O (1 / 1, v / v), and obtain 96 mg of crude peptide after freeze-drying.

[0120] Weigh 5.19 mg of the above crude peptide, add 2.5 mL of an aqueous solution of 40 mM NaOH, stir at room temperature until most of the acetyl groups and all the methyl esters are removed (0.5 h), and then add 5% acetic acid for neutralization. After freeze-drying the reaction solution, continue to add 1.6 mL of an aqueous solution of 10% NH2·NH2, stir at room temperature for 2 h to remove the remaining acetyl groups. After the reaction is completed, add 5% acetic acid for neutralization. After passing the reaction solution through a 0.2 μm filter membrane, purify it by HPLC using a C18 column (10x 250 mm, 5 μm, ) The mobile phase A is water containing 0.05% TFA, and the mobile phase B is acetonitrile containing 0.05% TFA. Linear elution is performed at 20% to 40% (B%) for 40 minutes at a flow rate of 4 mL / min, and the detection wavelength is 214 nm. During the purification process, LC-MS (Waters SQD2-ACQUITY UPLC, the mobile phase A is water containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, the gradient is 20% to 40% (B%), the flow rate is 0.3 mL / min, and the detection wavelength is 214 nm) is used to identify the collected eluate. The eluates containing the product are combined and lyophilized to obtain 1.54 mg of the product GLP4 with a yield of 15%. The product GLP4 is characterized by LC-MS, and its structure is correct. The results are as shown in the appendix Figure 4 as follows.

[0121] Example 4. Preparation of GLP5 - GLP7, GLP11 - GLP14, and GLP18 - GLP21.

[0122] The preparation of GLP5 - GLP7, GLP11 - GLP14, and GLP18 - GLP21 is basically the same as that in Example 3, except that different glycoamino acids are used. The corresponding relationships between the synthesized glycopeptides and glycoamino acids are listed in the following table:

[0123] Table 4: Corresponding relationships between glycopeptides and the glycoamino acids used in their synthesis.

[0124] Glycopeptide Glycoamino acid GLP11, GLP18 F4 GLP5, GLP12, GLP19 F5 GLP6, GLP13, GLP20 F6 GLP7, GLP14, GLP21 F7

[0125] The structures of the synthesized GLP4 - GLP7, GLP11 - GLP14, and GLP18 - GLP21 are all correct. Their LC-MS characterization results are as shown in the appendix Figures 5 - 7 as well as those shown in 11 - 14 and 18 - 21. Their synthesis yields are shown in the following table:

[0126] Table 5: Statistical results of the synthesis yields of GLP4 - GLP7, GLP11 - GLP14, and GLP18 - GLP21.

[0127] Yield Yield Yield GLP4 15％ GLP11 16％ GLP18 26％ GLP5 13％ GLP12 13％ GLP19 16％ GLP6 14％ GLP13 14％ GLP20 14％ GLP7 13％ GLP14 14％ GLP21 13％

[0128] Example 5. Effect of glycosylation modification on the DPP-4 enzymatic stability of GLP-1(7 - 37).

[0129] Dissolve 50 μg of GLP-1(7-37) or its glycosylated derivative in 100 μL of 100 mM HEPES buffer (pH 7.4). After vortexing, take 80 μL and place it in a 0.6 mL centrifuge tube, and incubate it in a metal bath at 37 °C for 15 minutes. Take out 4 μL from it and add it to a mass spectrometry sample vial containing 36 μL of 0.2% TFA. Add 4 μL (0.2 μg) of DPP-4 enzyme to the remaining sample, and take 4 μL samples at different time points (0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 180, 240, 300, 360 min), and add them to mass spectrometry sample vials containing 36 μL of 0.2% TFA. Analyze the above samples using UPLC with an elution gradient of 20-50% (B%), which can separate the full-length glycopeptide and the glycopeptide after enzymatic cleavage, and use the UV peak integration in the LC of the full-length glycopeptide to represent its remaining amount. The above experiments for each compound were carried out in three parallel operations.

[0130] Plot the remaining amount of the full-length glycopeptide against the sampling time point in Origin, and use the formula y = A*e∧(-x / t1)+y0 for fitting, where y represents the UV peak integration and x represents the degradation time, so as to obtain the enzymatic hydrolysis half-life H = t1*ln2. The obtained results are shown in the following table:

[0131] Table 6: DPP-4 enzymatic hydrolysis half-lives of GLP-1(7-37) and its glycosylated derivatives.

[0132] Half-life Half-life Half-life GLP-1(7-37) 5.50 min GLP1 4.56 min GLP8 3.93 min GLP15 13.1 min GLP2 9.10 min GLP9 3.45 min GLP16 4.67 min GLP3 57.6 min GLP10 11.8 min GLP17 9.31 min GLP4 24.1 min GLP11 7.90 min GLP18 4.95 min GLP5 53.8 min GLP12 18.3 min GLP19 16.7 min GLP6 88.0 min GLP13 13.1 min GLP20 16.0 min GLP7 3478 min GLP14 40.2 min GLP21 15.3 min

[0133] The results show that most of the glycosylated derivatives of GLP-1(7-37) have higher DPP-4 enzymatic hydrolysis stability than GLP-1(7-37). Among these derivatives, glycosylation at the 11th position has a greater improvement in enzymatic hydrolysis stability than glycosylation at the 13th and 18th positions. Among glycosylation at the 11th and 13th positions, the tetrasaccharide has a much greater improvement in enzymatic hydrolysis stability than other sugars. In particular, the DPP-4 enzymatic hydrolysis half-life of the glycosylated derivative GLP7 is 632 times that of GLP-1(7-37).

[0134] Example 6. Effect of glycosylation modification on the hypoglycemic activity of GLP-1(7-37)

[0135] We characterized the hypoglycemic activities of GLP-1(7-37) and its glycosylated derivatives using db / db diabetic model mice. For representativeness, we selected derivatives GLP8, GLP11, GLP13, and GLP15 with different sizes of sugars (monosaccharides, disaccharides, trisaccharides), different types of sugars (with or without sialic acid), and different sites (13th and 18th positions, which are relatively far apart) for study.

[0136] SPF-grade 6-week-old male db / db mice (30 - 40 g) were housed in an environment with constant temperature (23 ± 1°C) and humidity (55 - 65%), under a 12:12-hour light-dark cycle (lights on from 7 am to 7 pm), and fed standard feed and water ad libitum. The mice were intraperitoneally injected with a dose of 75 nmol / kg of GLP-1(7-37) or its glycosylated derivative (7.5 nmol / mL) dissolved in PBS buffer. The blood glucose level was continuously monitored for 6 hours. The obtained experimental data were imported into Origin for further analysis, and the results were expressed as the mean of 6 repeated experiments in each group. The results are shown in the following table.

[0137] Table 7: Statistical results of normalized blood glucose concentrations in the hypoglycemic experiments of GLP-1(7-37) and its glycosylated derivatives.

[0138] Time GLP-1(7-37) GLP8 GLP11 GLP13 GLP15 0h 100％ 100％ 100％ 100％ 100％ 10 min 109％ 20 min 82％ 30 min 74% (minimum) 93％ 118％ 121％ 79％ 40 min 75％ 50 min 76％ 1h 85％ 65％ 131％ 128％ 78% (minimum) 1.5h 114％ 2h 117％ 62% (minimum) 84％ 85％ 85％ 3h 78％ 71% (minimum) 66% (minimum) 99％ 4h 90％ 88％ 5.5h 81％ 116％ 6h 99％

[0139] The results showed that all GLP-1(7-37) glycosylated derivatives exhibited hypoglycemic activities comparable to or lower than those of GLP-1(7-37). Notably, the blood glucose levels of GLP-1(7-37) and some derivatives increased at the beginning. It is speculated based on experience that this might be due to the stimulation brought by the injection preparation (PBS buffer) and the influence of the absorption of the preparation and the drug. The percentages of the lowest blood glucose concentrations of GLP-1(7-37), GLP8, GLP11, GLP13, and GLP15 were 74%, 62%, 71%, 66%, and 78% respectively. In terms of the time to reach the lowest value, GLP-1(7-37) was the shortest, approximately 30 minutes. For the other four derivatives, GLP8, GLP11, GLP13, and GLP15 reached the lowest values at 2 hours, 3 hours, 3 hours, and 1 hour respectively. This is related to the extended half-life of these compounds in vivo, probably because the sugar modification reduced renal clearance and inhibited DPP-4 enzymatic hydrolysis in vivo.

Claims

1. A GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof, characterized in that, The GLP-1(7-37) glycosylation derivative is formed by the oxygen linkage of a sugar to the side-chain oxygen of threonine at position 11, threonine at position 13, or serine at position 18 of GLP-1(7-37). The sequence of GLP-1(7-37) is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, and the sugar is selected from G1, G2, G3, G4, G5, G6, or G7; The structural formulas of G1, G2, G3, G4, G5, G6, and G7 are respectively: Structural formula G1: Structural formula G2: Structural formula G3: Structural formula G4: Structural formula G5: Structural formula G6: Structural formula G7:

2. The GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein The GLP-1(7-37) glycosylation derivative is selected from GLP1, GLP2, GLP3, GLP4, GLP5, GLP6, GLP7, GLP8, GLP9, GLP10, GLP11, GLP12, GLP13, GLP14, GLP15, GLP16, GLP17, GLP18, GLP19, GLP20, or GLP21; The structural formulas of GLP1, GLP2, GLP3, GLP4, GLP5, GLP6, GLP7, GLP8, GLP9, GLP10, GLP11, GLP12, GLP13, GLP14, GLP15, GLP16, GLP17, GLP18, GLP19, GLP20, and GLP21 are respectively: Structural formula GLP-1: Structural formula GLP2: Structural formula GLP3: Structural formula GLP-4: Structural formula of GLP5: Structural formula of GLP6: Structural formula of GLP7: Structural formula GLP8: Structural formula of GLP9: Structural formula GLP10: Structural formula of GLP11: Structural formula of GLP12: Structural formula of GLP13: Structural formula of GLP14: Structural formula GLP15: Structural formula GLP16: Structural formula GLP17: Structural formula GLP18: Structural formula GLP19: Structural formula of GLP20: Structural formula of GLP21: Among them, the sugar in GLP1 to GLP7 is covalently linked to the side-chain oxygen of threonine at position 11 of GLP-1(7-37); the sugar in GLP8 to GLP14 is covalently linked to the side-chain oxygen of threonine at position 13 of GLP-1(7-37), and the sugar in GLP15 to GLP21 is covalently linked to the side-chain oxygen of serine at position 18 of GLP-1(7-37).

3. A pharmaceutical composition comprising at least one of the GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to claim 1 in a biologically active amount and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, wherein The pharmaceutical combination further includes one or more other active ingredients, and the active ingredients are selected from rapid-acting insulin, long-acting insulin, glucagon-like peptide-1 receptor agonist, glucagon-like peptide-1 / glucagon receptor co-agonist, glucagon-like peptide-1 / glucose-dependent insulinotropic polypeptide receptor co-agonist, or glucagon-like peptide-1 / glucose-dependent insulinotropic polypeptide / glucagon receptor co-agonist.

5. The pharmaceutical composition according to any one of claims 3 to 4, characterized in that, The dosage form of the pharmaceutical composition is an injection dosage form or an oral dosage form.

6. Use of the GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 4 in the preparation of a drug for the treatment and / or prevention of type II diabetes.

7. Use of the GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, or the pharmaceutical composition according to any one of claims 3 to 4, in the preparation of a medicament for the treatment and / or prevention of at least one of the following diseases: impaired glucose tolerance, hyperglycemia, perioperative hyperglycemia, metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

8. Use of the GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, or the pharmaceutical composition according to any one of claims 3 to 4, in the preparation of a medicament for the treatment and / or prevention of neurodegenerative diseases (including Alzheimer's disease, Parkinson's disease, etc.).

9. Use of the GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, or the pharmaceutical composition according to any one of claims 3 to 4, in the preparation of a medicament for the treatment and / or prevention of at least one of the following diseases: non-alcoholic steatohepatitis, non-alcoholic fatty liver disease.

10. Use of the GLP-1(7-37) glycosylation derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, or the pharmaceutical composition according to any one of claims 3 to 4, in the preparation of a medicament for the treatment and / or prevention of at least one of the following diseases: obesity, atherosclerosis.

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