Glucagon glycosylated derivative and pharmaceutical composition and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a glucagon glycosylation derivative as well as a pharmaceutical composition and application thereof. The glucagon glycosylation derivative disclosed by the invention is formed by connecting sugar with oxygen of side chains of 7-site threonine, 8-site serine or 11-site serine of glucagon, so that the solubility of glucagon in a neutral water phase is remarkably improved, and fibrosis of glucagon in the neutral water phase is inhibited. Another technical means is provided for improving the solubility and the stability of the glucagon in a neutral water phase. Rat activity experiments show that the glucagon glycosylation derivative has good blood sugar increasing activity and has the potential of being further developed into drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a glucagon glycosylation derivative, a pharmaceutical composition thereof, and an application thereof. Background Art

[0002] Human glucagon is a 29 - amino - acid linear peptide hormone secreted by pancreatic alpha cells, and its physiological function is mediated by the glucagon receptor mainly expressed in the liver. Glucagon stimulates hepatic glycogenolysis during hypoglycemia, prolonged fasting, and protein - rich food intake, counteracting the hypoglycemic effect of insulin. Clinically, glucagon is mainly used as a basic first - aid drug for severe diabetic hypoglycemia.

[0003] The earliest glucagon preparation (in 1991) was available as a lyophilized powder and needed to be immediately redissolved with the provided acidic diluent before use. This involves a multi - step injection - mixing - aspiration procedure, which can be very challenging in hypoglycemic emergencies and often leads to confusion and misuse by those with limited experience. In cases where a lower dose is required (e.g., in children), the remaining solution must be discarded, resulting in significant waste. These inconveniences are attributed to the physical and chemical instability of glucagon in aqueous solutions. At neutral pH, glucagon has poor water solubility. Under alkaline conditions, glucagon has better solubility but undergoes chemical degradation. In an acidic environment, glucagon has good solubility but spontaneously aggregates into tightly packed fibers, which may be cytotoxic. The fibrillation process can also be accelerated by elevated temperature and physical agitation.

[0004] Subsequently, a variety of glucagon drugs have been marketed. Nasal powder glucagon: Trade name Baqsimi, developed by Eli Lilly and approved for marketing in the United States, Canada, and Europe in 2019. Nasal powder administration greatly reduces the difficulty of administration and significantly improves the success rate of patient emergency treatment. However, its bioavailability is low, and a three - fold dose is required, resulting in more adverse reactions. Ready - to - use glucagon: Trade name Gvoke, developed by Xeris Pharmaceuticals and approved by the US FDA in 2020, uses a non - aqueous solvent DMSO in which glucagon can be dissolved and stabilized at room temperature. However, its absorption is delayed, and there is obvious discomfort at the injection site. Aqueous ready - to - use glucagon: Trade name Zegalogue, developed by Zealand Pharma and approved by the US FDA in 2021. This drug is formed by mutating 7 amino acids of glucagon and can be dissolved and stably exist in neutral aqueous phase.

[0005] Glycosylation has always been a well-established method for enhancing the water solubility of peptides / proteins and reducing fibrosis. Recent research (Bioconjugate Chem. 2021, 32, 2148 - 2153.) has shown that replacing the tryptophan at position 25 of glucagon with cysteine and attaching an undecasaccharide to the side chain of cysteine can reduce the fibrillation tendency of glucagon. The above-mentioned glycosylated glucagon molecule uses a relatively large sugar, namely undecasaccharide, which is costly. This sugar belongs to the N-glycoside type and is usually linked to the nitrogen of the asparagine side chain in the protein sequence in vivo. In addition, the solubility of this molecule in neutral aqueous phase was not tested in this study. Summary of the Invention

[0006] In order to improve the solubility of glucagon in neutral aqueous phase and its anti-fibrillation tendency, the present invention provides an insulin glycosylation derivative, its pharmaceutical composition and application. The sugar in this patent will use O-glycoside, which is usually linked to the oxygen of the serine and threonine side chains in the protein sequence in vivo. The present invention provides more alternative technical means for this field. Specifically, the present invention provides a glucagon glycosylation derivative or a pharmaceutically acceptable salt thereof, characterized in that the glucagon glycosylation derivative is formed by linking a sugar to the oxygen of the side chain of threonine at position 7, serine at position 8 or serine at position 11 of glucagon, and the sequence of glucagon is HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, and the sugar is selected from G1, G2, G3, G4, G5 or G6;

[0007] The structural formulas of G1, G2, G3, G4, G5, and G6 are as follows:

[0008] Structural formula G1:

[0009] Structural formula G2:

[0010] Structural formula G3:

[0011] Structural formula G4:

[0012] Structural formula G5:

[0013] Structural formula G6:

[0014] When the glucagon glycosylation derivative contains the G1 or G2 sugar structure, its synthesis method is briefly described as follows:

[0015] 1. Solid-phase synthesis: Using sugar amino acids as raw materials, the glycosylated glucagon polypeptide chain is prepared by solid-phase peptide synthesis method.

[0016] 2. Removal of acetyl groups on the sugar: The synthesized glycosylated glucagon polypeptide chain was dissolved in an aqueous solution of 40 mM NaOH for reaction.

[0017] 3. Purification: The reaction solution obtained in step 2 above was purified to obtain glycosylated glucagon.

[0018] When the glucagon glycosylation derivative contains G3, G4, G5 or G6 sugar structures, its synthesis method is briefly described as follows:

[0019] 1. Solid-phase synthesis: Using sugar amino acids as raw materials, a glycosylated glucagon polypeptide chain was prepared by solid-phase peptide synthesis.

[0020] 2. Hydrolysis of methyl ester on the sugar and partial removal of acetyl groups: The synthesized glycosylated glucagon polypeptide chain was dissolved in an aqueous solution of 40 mM NaOH for reaction, a termination solution was added, and then freeze-dried.

[0021] 3. Removal of acetyl groups on the sugar: An aqueous solution of 10% NH2·NH2 was added to the freeze-dried powder obtained in step 2 for reaction, and a termination solution was added;

[0022] 4. Purification: The reaction solution obtained in step 3 above was purified to obtain glycosylated glucagon.

[0023] Preferably, the glucagon glycosylation derivative is selected from GCG1, GCG2, GCG3, GCG4, GCG5, GCG6, GCG7, GCG8, GCG9, GCG10, GCG11, GCG12, GCG13, GCG14, GCG15, CCG16, GCG17 or GCG18;

[0024] The structural formulas of GCG1, GCG2, GCG3, GCG4, GCG5, GCG6, GCG7, GCG8, GCG9, GCG10, GCG11, GCG12, GCG13, GCG14, GCG15, CCG16, GCG17, GCG18 are respectively:

[0025] Structural formula GCG1:

[0026] Structural formula GCG2:

[0027] Structural formula GCG3: Structural formula GCG4: Structural formula GCG5:

[0028]

[0029] Structural formula GCG6:

[0030]

[0031] Structural formula GCG7: Structural formula GCG8: Structural formula GCG9: Structural formula GCG10: Structural formula GCG11:

[0032]

[0033] Structural formula GCG12:

[0034]

[0035] Structural formula GCG13: Structural formula GCG14: Structural formula GCG15: Structural formula GCG16: Structural formula GCG17:

[0036]

[0037] Structural formula GCG18:

[0038]

[0039] Among them, the sugars in GCG1 - GCG6 are covalently linked to the oxygen of the 7 - threonine of glucagon; the sugars in GCG7 - GCG12 are covalently linked to the oxygen of the 8 - serine of glucagon; the sugars in GCG13 - GCG18 are covalently linked to the oxygen of the 11 - serine of glucagon.

[0040] The present invention also provides a pharmaceutical composition, which comprises at least one of a glucagon glycosylation derivative as claimed in claim 1 in a biologically active amount or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0041] The term "pharmaceutically acceptable carrier" broadly refers to any component other than the active therapeutic ingredient. Excipients can be inert substances, inactive substances, and / or non - pharmaceutically active substances. The formulation of pharmaceutically active ingredients with various excipients is known in the art.

[0042] Preferably, the pharmaceutical composition further comprises one or more other active ingredients, and the active ingredients are selected from insulin, glucagon - like peptide - 1 receptor agonists, and glucose - dependent insulinotropic polypeptide receptor agonists.

[0043] Preferably, the dosage form of the pharmaceutical composition is an injectable dosage form. Injectable compositions comprising the glucagon glycosylation derivatives of the present invention can be prepared using conventional techniques of the pharmaceutical industry, which include appropriately dissolving and mixing the ingredients to obtain the desired final product. Thus, the glucagon glycosylation derivatives 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.

[0044] The present invention also provides the use of any one of the above glucagon glycosylation derivatives or pharmaceutically acceptable salts thereof and the above pharmaceutical compositions in the preparation of a medicament for treating or preventing hypoglycemia.

[0045] Preferably, the hypoglycemia is selected from one or more of congenital hyperinsulinemic hypoglycemia, diabetic hypoglycemia, non-diabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, gestational hypoglycemia, drug-induced hypoglycemia, surgery-induced hypoglycemia, and tumor-induced hypoglycemia.

[0046] The technical solution provided by the present invention has the following advantages compared with the prior art:

[0047] By glycosylation modification at threonine at position 7, serine at position 8 or serine at position 11 of glucagon, the solubility of glucagon in neutral aqueous phase is significantly increased, and the fibrillation of glucagon in neutral aqueous phase is inhibited. This provides another technical means for improving the solubility and stability of glucagon in neutral aqueous phase. This fully demonstrates the effectiveness and superiority of this technology. Finally, the rat activity experiment shows that the glucagon glycosylation derivative has good blood glucose-raising activity and has the potential for further development into a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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.

[0049] Figure 1 . Structure, UPLC chromatogram and MS chromatogram of product GCG1. Molecular formula: C 167 H 248 N 44 O 59 S; Theoretical value: [M + 3H] 3+ m / z = 1282.92, [M + 4H] 4+ m / z = 962.44, [M + 5H] 5+ m / z = 770.16; Observed values: 1282.93, 962.45, 770.16.

[0050] Figure 2. Structure, UPLC chromatogram and MS spectrum of product GCG2. Molecular formula: C 175 H 261 N 45 O 64 S; Theoretical value: [M + 3H] 3+ m / z = 1350.62, [M + 4H] 4+ m / z = 1013.21, [M + 5H] 5+ m / z = 810.77; Observed values: 1350.62, 1013.22, 810.78.

[0051] Figure 3 . Structure, UPLC chromatogram and MS spectrum of product GCG3. Molecular formula: C 172 H 255 N 45 O 62 S; Theoretical value: [M + 3H] 3+ m / z = 1325.94, [M + 4H] 4+ m / z = 994.70, [M + 5H] 5+ m / z = 795.97; Observed values: 1325.94, 994.71, 795.96. Theoretical value of losing 1 sugar in the mass spectrum: [M + 3H] 3+ m / z = 1228.91, [M + 4H] 4+ m / z = 921.93; Observed values: 1228.90, 921.93.

[0052] Figure 4 . Structure, UPLC chromatogram and MS spectrum of product GCG4. Molecular formula: C 178 H 265 N 45 O 67 S; Theoretical value: [M + 3H] 3+ m / z = 1379.96, [M + 4H] 4+ m / z = 1035.22, [M + 5H] 5+ m / z = 828.38; Observed values: 1379.96, 1035.22, 828.37.

[0053] Figure 5 . Structure, UPLC chromatogram and MS spectrum of product GCG5. Molecular formula: C 178 H 265 N 45 O 67 S; Theoretical value: [M + 3H] 3+ m / z = 1379.96, [M + 4H] 4+ m / z = 1035.22, [M + 5H] 5+m / z = 828.38; Observed values: 1379.96, 1035.22, 828.37. Theoretical value of sugar loss in mass spectrometry to become GCG1: [M+4H] 4+ m / z = 962.44; Observed value: 962.44 (marked with *).

[0054] Figure 6 . Structure, UPLC chromatogram and MS spectrum of product GCG6. Molecular formula: C 189 H 282 N 46 O 75 S; Theoretical value: [M+3H] 3+ m / z = 1476.99, [M+4H] 4+ m / z = 1107.99, [M+5H] 5+ m / z = 886.60; Observed values: 1477.00, 1108.00, 886.60.

[0055] Figure 7 . Structure, UPLC chromatogram and MS spectrum of product GCG7. Molecular formula: C 167 H 248 N 44 O 59 S; Theoretical value: [M+3H] 3+ m / z = 1282.92, [M+4H] 4+ m / z = 962.44; Observed values: 1282.93, 962.45.

[0056] Figure 8 . Structure, UPLC chromatogram and MS spectrum of product GCG8. Molecular formula: C 175 H 261 N 45 O 64 S; Theoretical value: [M+3H] 3+ m / z = 1350.62, [M+4H] 4+ m / z = 1013.21, [M+5H] 5+ m / z = 810.77; Observed values: 1350.62, 1013.22, 810.78.

[0057] Figure 9 . Structure, UPLC chromatogram and MS spectrum of product GCG9. Molecular formula: C 172 H 255 N 45 O 62 S; Theoretical value: [M+3H] 3+ m / z = 1325.94, [M+4H] 4+ m / z = 994.70, [M+5H] 5+m / z = 795.97; Observed values: 1325.94, 994.71, 795.96. Theoretical value for loss of one sugar in the mass spectrum: [M+4H] 4+ m / z = 921.93; Observed value: 921.93 (marked with *).

[0058] Figure 10 . Structure, UPLC chromatogram and MS spectrum of product GCG10. Molecular formula: C 178 H 265 N 45 O 67 S; Theoretical value: [M+3H] 3+ m / z = 1379.96, [M+4H] 4+ m / z = 1035.22, [M+5H] 5+ m / z = 828.38; Observed values: 1379.96, 1035.22, 828.37. Theoretical value for loss of sugar to form GCG7 in the mass spectrum: [M+4H] 4+ m / z = 962.44; Observed value: 962.45 (marked with *).

[0059] Figure 11 . Structure, UPLC chromatogram and MS spectrum of product GCG11. Molecular formula: C 178 H 265 N 45 O 67 S; Theoretical value: [M+3H] 3+ m / z = 1379.96, [M+4H] 4+ m / z = 1035.22, [M+5H] 5+ m / z = 828.38; Observed values: 1379.96, 1035.22, 828.37. Theoretical value for loss of sugar to form GCG7 in the mass spectrum: [M+4H] 4+ m / z = 962.44; Observed value: 962.44 (marked with *).

[0060] Figure 12 . Structure, UPLC chromatogram and MS spectrum of product GCG12. Molecular formula: C 189 H 282 N 46 O 75 S; Theoretical value: [M+3H] 3+ m / z = 1476.99, [M+4H] 4+ m / z = 1107.99, [M+5H] 5+ m / z = 886.60; Observed values: 1477.00, 1108.00, 886.60. Theoretical value for loss of sugar to form GCG10 / GCG11 in the mass spectrum: [M+4H] 4+m / z = 1035.22; Observed value: 1035.22 (marked with *).

[0061] Figure 13 . Structure, UPLC chromatogram and MS spectrum of product GCG13. Molecular formula: C 167 H 248 N 44 O 59 S; Theoretical value: [M + 3H] 3+ m / z = 1282.92, [M + 4H] 4+ m / z = 962.44, [M + 5H] 5+ m / z = 770.16; Observed values: 1282.93, 962.45, 770.16.

[0062] Figure 14 . Structure, UPLC chromatogram and MS spectrum of product GCG14. Molecular formula: C 175 H 261 N 45 O 64 S; Theoretical value: [M + 3H] 3+ m / z = 1350.62, [M + 4H] 4+ m / z = 1013.21, [M + 5H] 5+ m / z = 810.77; Observed values: 1350.62, 1013.22, 810.78.

[0063] Figure 15 . Structure, UPLC chromatogram and MS spectrum of product GCG15. Molecular formula: C 172 H 255 N 45 O 62 S; Theoretical value: [M + 3H] 3+ m / z = 1325.94, [M + 4H] 4+ m / z = 994.70; Observed values: 1325.94, 994.71.

[0064] Figure 16 . Structure, UPLC chromatogram and MS spectrum of product GCG16. Molecular formula: C 178 H 265 N 45 O 67 S; Theoretical value: [M + 3H] 3+ m / z = 1379.96, [M + 4H] 4+ m / z = 1035.22, [M + 5H] 5+ m / z = 828.38; Observed values: 1379.96, 1035.22, 828.37. Theoretical value of sugar loss in the mass spectrum to become GCG13: [M + 4H]4+ m / z = 962.44; Observed value: 962.45 (marked with *).

[0065] Figure 17 . Structure, UPLC chromatogram and MS spectrum of product GCG17. Molecular formula: C 178 H 265 N 45 O 67 S; Theoretical value: [M + 3H] 3+ m / z = 1379.96, [M + 4H] 4+ m / z = 1035.22, [M + 5H] 5+ m / z = 828.38; Observed values: 1379.96, 1035.23, 828.38.

[0066] Figure 18 . Structure, UPLC chromatogram and MS spectrum of product GCG18. Molecular formula: C 189 H 282 N 46 O 75 S; Theoretical value: [M + 3H] 3+ m / z = 1476.99, [M + 4H] 4+ m / z = 1107.99, [M + 5H] 5+ m / z = 886.60; Observed values: 1477.00, 1108.00, 886.60.

[0067] Figure 19 . Plot of fluorescence intensity vs. time in the fibrillation test of glucagon or its glycosylated derivatives.

[0068] In the above figures: mAU is the absorbance unit; Time (min) is the retention time in UPLC and its unit is minutes; m / z is the mass-to-charge ratio. Detailed implementation manners

[0069] The following is a further detailed description of the present invention 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 does not 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.

[0070] The explanations of some English abbreviations used in the present invention and their meanings are shown in Table 1 below:

[0071] Table 1:

[0072]

[0073]

[0074] The glycoamino acids involved in the present invention can be synthesized according to the references or purchased directly, and other reagents can be purchased directly. The structures of the glycoamino acids involved are as follows:

[0075] F1:

[0076] F2:

[0077] F3:

[0078] F4:

[0079] F5:

[0080] F6:

[0081] Among them, R = CH3 or H.

[0082] When the synthesis site is threonine, the glycoamino acid with R = CH3 is used; when the synthesis site is serine, the glycoamino acid with R = H is used.

[0083] Example 1. The solid-phase synthesis, post-treatment, purification and identification process of glucagon.

[0084] The solid-phase peptide synthesis (SPPS) of glycosylated glucagon was completed using a CEM Liberty Blue automatic microwave peptide synthesizer.

[0085] Swelling: 0.05 mmol of resin was added to the solid-phase synthesis reactor, and the resin was washed three times with DMF (3 mL × 3), and then 10 mL of DMF was added to swell for 5 min.

[0086] Deprotection: 3 mL of 20% 4-methylpiperidine dissolved in DMF was added. The temperature of the mixture was raised to 50 °C using microwave heating and maintained at 50 °C. Nitrogen was introduced from the bottom of the container (usually open for 2 s, closed for 3 s) for mixing. After reacting for 10 min, the resin was washed four times with DMF (4 mL × 4).

[0087] Coupling: Add Fmoc-protected amino acid (0.2M in DMF, 1.25 mL, 5 eq.), DIEA (0.5M in DMF, 1 mL, 10 eq.), and HATU (0.25M in DMF, 1 mL, 5 eq.) to the reaction vessel in sequence. After reacting at room temperature for 2 min, raise the temperature of the reaction mixture to 50 °C and maintain it at 50 °C with nitrogen mixing for 8 min. Among them, for amino acids with β-branches in the sequence (Ile, Thr, Val), Pro, the residue following Fmoc-Arg(Pbf)-OH with a bulky protecting group, and Arg itself, perform two couplings to ensure complete reaction.

[0088] Peptide chain elongation: Repeat the above deprotection and coupling steps until the sequence is completed.

[0089] Cleavage: Transfer the obtained resin to a glass peptide synthesis reaction tube and wash the resin three times (5 mL each time) alternately with DMF and DCM. Add 4 mL of freshly prepared TFA / TIPS / H2O (90 / 5 / 2.5 / 2.5, v / v / v) cleavage solution and gently stir at room temperature for 2 h. Filter and collect the cleavage solution with a 50 mL centrifuge tube.

[0090] Collection: Concentrate the cleavage solution to 0.5 mL by blowing with nitrogen, 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). After stirring at 38 °C for 1 h, lyophilize to obtain the crude peptide.

[0091] High-performance liquid chromatography (HPLC) purification: Use a C18, 10×250 mm, 5 μm, separation column, mobile phase A is water containing 0.05% TFA, and mobile phase B is acetonitrile containing 0.05% TFA. Elute the column with 95% (B%) for 15 min and equilibrate the column with 5% (B%) for 15 min. After loading the sample, elute with a linear gradient of 20% - 40% (B%) for 40 min at a flow rate of 4 mL / min, and the detection wavelengths are 214 nm and 275 nm. During the purification process, use LC-MS to identify the collected eluates, combine the eluates containing the product, and obtain the pure product after lyophilization.

[0092] LC-MS analysis: Chromatographic separation was performed using a Waters Acquity ultra-high performance liquid chromatography (UPLC) system with an Acquity UPLC BEH 300C4, 1.7 μm column. The flow rate was 0.3 mL / min, and the detection wavelength was 214 nm. Phase A was H2O containing 0.1% formic acid, and phase B was MeCN containing 0.1% formic acid, eluting with a linear gradient of 20% - 40% (B%) for a total of 6 min. After each run, the column was washed with 95% (B%) for 2 min and re-equilibrated with 5% (B%) for 2 min. Mass spectrometry analysis was performed using an Exactive Plus Orbitrap mass spectrometer. The electrospray ionization (ESI) source was used in the positive ion mode, with a scanning range of m / z 300 - 4500 and a spray voltage of 3 kV. Depending on the concentration, the injection volume ranged from 1 μL to 10 μL.

[0093] Example 2. Preparation of GCG2.

[0094] Solid-phase synthesis and post-treatment: Fmoc-Thr(tBu)-2-Cl-Trityl resin was used. Among them, the coupling of sugar amino acids was carried out as follows: Sugar amino acid F2 (0.1 M in DMF, 1 mL, 2 eq.), DIEA (0.5 M in DMF, 0.4 mL, 4 eq.), and HATU (0.25 M in DMF, 0.4 mL, 2 eq.) were added to the reaction vessel in sequence. After reacting at room temperature for 2 min, the temperature of the reaction mixture was raised to 50 °C and maintained at 50 °C under nitrogen mixing for 18 min. Two couplings were performed to ensure complete reaction. Other steps were the same as in Example 1.

[0095] Removal of protecting groups on sugar: 1 mg of the crude peptide was dissolved in 0.5 mL of 40 mM aqueous NaOH solution. After stirring the reaction mixture for 1 h, 25 μL of 5% aqueous AcOH solution was added to stop the reaction. Subsequently, it was diluted with 5 mL of water and filtered through a 0.22 μm membrane. The resulting solution was purified by HPLC to obtain GCG2 with a yield of 27%. The product GCG2 was characterized by LC-MS, and its structure was correct, as shown in the appendix Figure 2 as follows.

[0096] Example 3. Preparation of GCG1, GCG7, GCG8, GCG13, and GCG14.

[0097] The preparation of GCG1, GCG7, GCG8, GCG13, and GCG14 was basically the same as in Example 2, except that the sugar amino acids they used were different. The following table lists the corresponding relationship between the synthesized glycopeptides and sugar amino acids:

[0098] Table 2: Corresponding relationship between glycopeptides and the glycoamino acids used in their synthesis.

[0099] Glycopeptide Sugar amino acid GCG1, GCG7, GCG13 F1 GCG8, GCG14 F2

[0100] The structures of the synthesized GCG1, GCG7, GCG8, GCG13, and GCG14 are all correct, and their LC-MS characterization results are as shown in Appendices Figure 1 、 7 、8, 13, and 14. Their synthesis yields are shown in the following table:

[0101] Table 3: Statistical results of the synthesis yields of GCG1, GCG2, GCG7, GCG8, GCG13, and GCG14.

[0102] Yield Yield Yield GCG1 28％ GCG7 27％ GCG13 30％ GCG2 27％ GCG8 25％ GCG14 28％

[0103] Example 4. Preparation of GCG17.

[0104] Solid-phase synthesis and post-treatment: Use Fmoc-Thr(tBu)-2-Cl-Trityl resin. Among them, the coupling of glycoamino acids is carried out in the following steps: sequentially add glycoamino acid F5 (0.1M in DMF, 1mL, 2eq.), DIC (0.5M in DMF, 0.4mL, 4eq.), and Oxyma (0.25M in DMF, 0.4mL, 2eq.) into the reaction vessel. After reacting at room temperature for 2 min, raise the temperature of the reaction mixture to 50 °C and maintain it at 50 °C with nitrogen mixing for 18 min. Carry out two couplings to ensure complete reaction. Other steps are the same as those in Example 1.

[0105] Removal of the protecting group on the sugar: Dissolve 1 mg of the crude peptide in 0.5 mL of 40 mM NaOH aqueous solution. After stirring the reaction mixture for 1 h, add 25 μL of 5% AcOH aqueous solution to quench the reaction, and then lyophilize. Add 200 μL of 10% NH2·NH2 aqueous solution to the lyophilized powder and stir for 2 h. Add 1 mL of 5% AcOH aqueous solution to quench the reaction. Subsequently, dilute with 5 mL of water and filter through a 0.22 μm membrane. The resulting solution is purified by HPLC to obtain GCG17 with a yield of 22%. The product GCG17 is characterized by LC-MS, and the results are as shown in Appendices Figure 17 shown.

[0106] Example 5. Preparation of GCG3 - GCG6, GCG9 - GCG12, GCG15, GCG16, and GCG18.

[0107] The preparation of GCG3 - GCG6, GCG9 - GCG12, GCG15, GCG16 and GCG18 is basically the same as that in Example 4, except that different sugar amino acids are used. The following table lists the corresponding relationships between the synthesized glycopeptides and sugar amino acids:

[0108] Table 4: Corresponding relationships between glycopeptides and the sugar amino acids used in their synthesis.

[0109] Glycopeptide Sugar amino acid GCG3, GCG9, GCG15 F3 GCG4, GCG10, GCG16 F4 GCG5, GCG11 F5 GCG6, GCG12, GCG18 F6

[0110] The structures of the synthesized GCG3 - GCG6, GCG9 - GCG12, GCG15, GCG16 and GCG18 are all correct, and their LC-MS characterization results are as shown in Appendices Figures 3 - 6 9 - 12, 15, 16 and 18. Their synthesis yields are shown in the following table:

[0111] Table 5: Statistical results of the synthesis yields of GCG3 - GCG6, GCG9 - GCG12 and GCG15 - GCG18.

[0112]

[0113]

[0114] Example 6. Effect of glycosylation modification on the solubility of glucagon.

[0115] Dissolve 200 μg of glucagon (GCG) or glucagon glycosylation derivative in 100 μL of pure water, add 1 μL of 0.1 M NaOH to adjust the solution to neutral pH, and then perform lyophilization. Add 5 μL of PBS, and vortex and sonicate for 5 min to promote protein dissolution. Let the mixture stand at room temperature (25 °C) for 1 h. Then, centrifuge at 10,000 rpm (relative centrifugal force = 9,600 g) for 10 min, and observe whether precipitation occurs. If precipitation occurs, take the supernatant for dilution and perform LC-MS analysis, and calculate the solubility of the protein according to the area of the absorption peak at 214 nm. If the protein is completely dissolved under this condition, the following tests are carried out.

[0116] Dissolve 1 mg of the glucagon glycosylation derivative in 300 μL of pure water, add 14 μL of 0.1 M NaOH to adjust the protein solution to neutral pH, and then perform lyophilization. Subsequently, add 5 μL of PBS, and vortex and sonicate for 5 min to promote protein dissolution. Let the mixture stand at room temperature (25 °C) for 1 h. Then, centrifuge at 10,000 rpm (relative centrifugal force = 9,600 g) for 10 min to observe whether precipitation occurs. If precipitation occurs, take the supernatant for dilution and perform LC-MS analysis, and calculate the protein solubility based on the area of the absorption peak at 214 nm. If the protein remains completely dissolved under these conditions, it indicates that its solubility exceeds the upper limit of the test.

[0117] The above experiment was repeated three times. The experimental results are shown in the following table:

[0118] Table 2: Solubility of glucagon and its glycosylation derivatives.

[0119] Solubility (g / L) Solubility (g / L) Solubility (g / L) GCG 0.229 GCG1 23.7 GCG7 9.97 GCG13 1.10 GCG2 182 GCG8 >200 GCG14 26.3 GCG3 >200 GCG9 >200 GCG15 27.7 GCG4 >200 GCG10 >200 GCG16 8.61 GCG5 >200 GCG11 >200 GCG17 78.0 GCG6 >200 GCG12 >200 GCG18 >200

[0120] The results show that all glucagon glycosylation derivatives have higher solubility than glucagon. Glycosylation significantly enhances the solubility of glucagon, and even more than half of the glycosylation derivatives have a solubility exceeding the upper limit of the test (200 g / L). Specifically, from the perspective of glycosylation sites, there are significant differences in the degree of enhancement of glucagon solubility at different glycosylation sites. By comparing the glycoisomers with the same glycan type at different glycosylation sites, we can find that the degree of solubility enhancement is 7th, 8th > 11th, and the degree of enhancement of glucagon solubility at the first two glycosylation sites is significantly higher than that at the third glycosylation site. From the perspective of glycan types, there are also obvious differences in the degree of enhancement of solubility. Glycan types containing sialic acid often have better ability to enhance solubility.

[0121] Example 7. Effect of glycosylation modification on the fibrillation tendency of glucagon.

[0122] Buffer: Prepare a buffer of 50 mM Glycine, 40 mM Na2SO4, pH 2.0.

[0123] Dissolve glucagon or its glycosylated derivatives in a buffer to obtain a sample stock solution with a concentration of 4 mg / mL. Similarly, dissolve thioflavin T (ThT) in a buffer and filter it through a 0.22-μm membrane to obtain a ThT stock solution with a concentration of 1 mg / mL. Next, sequentially add the above sample stock solution, buffer, and ThT stock solution into a black flat-bottom 96-well microplate so that the total volume of the reaction solution reaches 50 μL, the final concentration of glucagon or its glycosylated derivatives reaches 2 mg / mL, and the ThT concentration reaches 300 μM. To prevent the evaporation of the reaction solution from affecting the data stability, the 96-well microplate is covered with a fluorescence quantitative pressure-sensitive film. In addition, a buffer containing 300 μM ThT is used as an optical control.

[0124] Perform fluorescence kinetics experiments using a Molecular Devices FlexStation 3 microplate reader, with an excitation wavelength of 450 nm and an emission wavelength of 485 nm. The experiment is carried out at 30 °C with rapid shaking. During the experiment, select the top reading mode, with a reading duration of 12 h and readings taken every 10 min. Subsequently, import the fluorescence intensity data into Origin for further analysis, and the results are expressed as the mean of three repeated experiments per group. The results are as Figure 19 shown.

[0125] The appearance of the fluorescence signal represents the occurrence of fibrosis. It should be noted that in some cases, the fluorescence signal first increases and then decreases, which is due to the change in the fiber configuration generated, resulting in a configuration with a weakened signal when interacting with ThT, rather than the result of fiber depolymerization. The experimental results show that all glucagon glycosylated derivatives have a lower tendency to fibrosis than glucagon. Among them, sialic acid significantly inhibits fibrosis. Under our test conditions, only one derivative GCG9 containing sialic acid undergoes fibrosis, and other sialic acid-containing derivatives (GCG3 - GCG6, GCG10 - GCG12, GCG15 - GCG18) do not undergo fibrosis. Among the derivatives without sialic acid, only GCG14 containing a trisaccharide at position 11 does not undergo fibrosis. From the perspective of anti-fibrosis, among these derivatives without sialic acid, GCG1 and GCG13 exhibit strong anti-fibrosis capabilities because their fibrosis occurs relatively late.

[0126] Example 8. Effect of Glucagon Glycosylation Modification on Blood Glucose-Raising Activity.

[0127] We characterized the blood glucose-raising activity of glucagon glycosylated derivatives using a Wistar rat. For representativeness, we selected two derivatives, GCG6 and GCG12, with the largest sugar (tetrasaccharide) and different sites for research.

[0128] SPF-grade 8-week-old male Wistar rats (295 ± 25 g) were housed in an environment with a constant temperature of (25 ± 1) °C and a constant humidity of (55 - 65)%, under a 12:12 h light-dark cycle (lights on from 7 am to 7 pm), and were allowed free access to standard feed and water. Hyperglycemia was measured and confirmed. On the day of the experiment, after the rats were fasted for 2 h, a glucagon glycosylation derivative dissolved in PBS (40 nmol / mL) was subcutaneously injected at a dose of 40 nmol / kg. Blood glucose was collected from the tail vein every 15 min after injection, and the blood glucose level was monitored using a handheld blood glucose meter for 1 h. The results are expressed as the mean of 6 repeated experiments per group, as shown in the following table:

[0129] Table 3: Statistical results of normalized blood glucose concentration in the blood glucose elevation experiment of glucagon glycosylation derivatives. The results showed that all glucagon glycosylation derivatives exhibited significant blood glucose elevation activity. It should be noted that due to the stimulation caused by the injection preparation (PBS buffer) and the influence of the absorption of the preparation and the drug, the basal blood glucose increased (see PBS data). Therefore, referring to the PBS data for the end time of blood glucose monitoring, GCG12 basically restored a blood glucose level similar to that of PBS at 60 min. After injecting GCG6, the blood glucose reached the highest point during the period from 15 to 45 min, while the blood glucose after injecting GCG6 reached the highest point within 30 min.

Claims

1. A glucagon glycosylation derivative or a pharmaceutically acceptable salt thereof, characterized in that, The glucagon glycosylation derivative is formed by the oxygen linkage of a sugar to the side-chain oxygen of threonine at position 7, serine at position 8, or serine at position 11 of glucagon. The sequence of glucagon is HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, and the sugar is selected from G1, G2, G3, G4, G5, or G6; The structural formulas of G1, G2, G3, G4, G5, and G6 are respectively: Structural formula G1: Structural formula G2: Structural formula G3: Structural formula G4: Structural formula G5: Structural formula G6:

2. The glucagon glycosylation derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein The glucagon glycosylation derivative is selected from GCG1, GCG2, GCG3, GCG4, GCG5, GCG6, GCG7, GCG8, GCG9, GCG10, GCG11, GCG12, GCG13, GCG14, GCG15, CCG16, GCG17, or GCG18; The structural formulas of GCG1, GCG2, GCG3, GCG4, GCG5, GCG6, GCG7, GCG8, GCG9, GCG10, GCG11, GCG12, GCG13, GCG14, GCG15, CCG16, GCG17, and GCG18 are respectively: Structural formula GCG1: Structural formula GCG2: Structural formula GCG3: Structural formula GCG4: Structural formula GCG5: Structural formula GCG6: Structural formula GCG7: Structural formula GCG8: Structural formula GCG9: Structural formula GCG10: Structural formula GCG11: Structural formula GCG12: Structural formula GCG13: Structural formula GCG14: Structural formula GCG15: Structural formula GCG16: Structural formula GCG17: Structural formula GCG18: Among them, the sugar in GCG1 - GCG6 is covalently linked to the oxygen of threonine at position 7 of glucagon; the sugar in GCG7 - GCG12 is covalently linked to the oxygen of serine at position 8 of glucagon; the sugar in GCG13 - GCG18 is covalently linked to the oxygen of serine at position 11 of glucagon.

3. A pharmaceutical composition, which comprises at least one of the glucagon glycosylation derivative or its pharmaceutically acceptable salt according to claim 1 in a biologically active amount and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical combination further includes one or more other active ingredients, and the active ingredients are selected from insulin, glucagon-like peptide-1 receptor agonists, and glucose-dependent insulinotropic polypeptide receptor agonists.

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.

6. Use of the glucagon glycosylation derivative or its pharmaceutically acceptable salt according to any one of claims 1 - 2 or the pharmaceutical composition according to any one of claims 3 - 4 in the preparation of a drug for treating or preventing hypoglycemia.

7. The use according to claim 6, wherein, The hypoglycemia is selected from one or more of congenital hyperinsulinemic hypoglycemia, diabetic hypoglycemia, non-diabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, gestational hypoglycemia, drug-induced hypoglycemia, surgery-induced hypoglycemia, and tumor-induced hypoglycemia.

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