Insulin glycosylation derivative as well as pharmaceutical composition and application thereof

Abstract

The invention relates to the technical field of insulin derivatives, in particular to an insulin glycosylation derivative as well as a pharmaceutical composition and application thereof. The insulin glycosylation derivative disclosed by the invention is formed by connecting sugar with hydroxyl of human insulin B chain 27-site threonine. The insulin glycosylation derivative disclosed by the invention has higher anti-auto-agglutination tendency and solubility than insulin, also maintains the hypoglycemic activity equivalent to that of insulin, and has the potential of being developed into quick-acting insulin which takes effect more quickly.

Description

Technical Field

[0001] The present invention relates to the technical field of insulin derivatives, and particularly relates to an insulin glycosylation derivative, its pharmaceutical composition and application. Background Art

[0002] Insulin is one of the most important hormones for maintaining blood glucose homeostasis in the human body. Since its discovery 100 years ago, it has played a crucial role in the treatment of diabetes. The use of insulin drugs saves millions of lives globally every year.

[0003] The use of insulin drugs has gone through three important stages. In the initial stage, insulin was mainly extracted from the internal organs of pigs and cows obtained from slaughterhouses. The development and use of such insulin drugs got the treatment of diabetes out of the predicament of having no available drugs. However, due to the difference in the amino acid sequences between animal insulin and human insulin, the use of this drug would bring some side effects including immune reactions. With the development of gene recombination and protein expression technologies, the research and development of insulin drugs entered the second stage. In this stage, the use of human insulin drugs produced by expression helped to overcome the problems brought by the use of animal insulin. In order to better improve the treatment effect of diabetes, in the third stage of the research and development of such drugs, multiple amino acids of the insulin molecule were mutated or their side chain structures were modified and changed. Through these changes, the performance of the insulin molecule was optimized and improved to some extent. These improvements further improved the lives of diabetic patients. Among them, in order to better control the blood glucose fluctuations caused by meals, scientists developed rapid-acting insulin. The currently commonly used rapid-acting insulins in clinical practice are insulin aspart and insulin lispro, which are produced by two different companies respectively. The principle is to mutate amino acids at the C-terminus of the B-chain of insulin, increasing the electrostatic repulsion during the formation of dimers, thereby enhancing the anti-self-aggregation tendency of insulin. Through these changes, the insulin molecule can dissociate, be absorbed and enter the bloodstream rapidly after entering the subcutaneous tissue.

[0004] Currently, the research and development of insulin drugs are gradually entering a new stage, and it is necessary to further improve the anti-self-aggregation tendency of insulin to obtain a faster onset time. However, the existing technologies cannot meet the market's demand for a higher onset speed of rapid-acting insulin. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an insulin glycosylation derivative, its pharmaceutical composition and application. Specifically, the present invention provides an insulin glycosylation derivative or a pharmaceutically acceptable salt thereof, and the insulin glycosylation derivative is formed by connecting a sugar to the hydroxyl group of threonine at position 27 of the B-chain of human insulin. The sugar is selected from G1, G2, G3, G4, G5 or G6.

[0006] The structural formulas of G1, G2, G3, G4, G5, and G6 are respectively as follows:

[0007] Structural formula G1:

[0008] Structural formula G2:

[0009] Structural formula G3:

[0010] Structural formula G4:

[0011] Structural formula G5:

[0012] Structural formula G6:

[0013] In the present invention, the term "insulin glycosylation derivative" refers to an insulin derivative modified with sugar, wherein the sugar is covalently linked to the hydroxyl group of the threonine side chain at position 27 of the B chain of human insulin.

[0014] The term "human insulin" used herein means the natural human insulin hormone without structural changes, the structure and properties of which are well known. Human insulin has two polypeptide chains, named the A chain and the B chain. The A chain is a peptide of 21 amino acids, and the B chain is a peptide of 30 amino acids. These two chains are linked by the following disulfide bridges: the first bridge between cysteine at position 7 of the A chain and cysteine at position 7 of the B chain, and the second bridge between cysteine at position 20 of the A chain and cysteine at position 19 of the B chain. The third bridge exists between cysteine at positions 6 and 11 of the A chain.

[0015] The insulin A chain has the following amino acid sequence:

[0016] GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1),

[0017] The insulin B chain has the following amino acid sequence:

[0018] FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2).

[0019] Preferably, the insulin glycosylation derivative is selected from Ins1, Ins2, Ins3, Ins4, Ins5, or Ins6. The structural formulas of Ins1, Ins2, Ins3, Ins4, Ins5, and Ins6 are respectively as follows:

[0020] Structural formula Ins1:

[0021] Structural formula Ins2:

[0022] Structural formula Ins3:

[0023] Structural formula Ins4:

[0024] Structural formula Ins5:

[0025] Structural formula Ins6:

[0026] Among them, the sugar is covalently linked to the hydroxyl group of threonine at position 27 of the insulin B chain.

[0027] The above insulin glycosylation derivatives can be prepared by the methods reported in the literature (ACS.Chem.Biol.2018,13,73 - 81.).

[0028] The present invention also relates to a pharmaceutical composition comprising at least one of a biologically active amount of the above insulin glycosylation derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0029] 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.

[0030] Preferably, the pharmaceutical composition may further include one or more other active ingredients, such as those selected from 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.

[0031] 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.

[0032] Preferably, the glucagon - like peptide - 1 receptor agonist can be selected from one or more of liraglutide, semaglutide, dulaglutide, and albiglutide.

[0033] Preferably, the dosage form of the pharmaceutical composition is an injection dosage form. Injectable compositions containing the insulin glycosylation derivatives of the present invention can be prepared using conventional techniques in the pharmaceutical industry, which include appropriately dissolving and mixing the ingredients to obtain the desired final product. Thus, the insulin 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.

[0034] The present invention also relates to the use of the above insulin glycosylation derivatives or their pharmaceutically acceptable salts or the above pharmaceutical compositions in the preparation of drugs for the treatment or prevention of diabetes.

[0035] The present invention also relates to the use of the above insulin glycosylation derivatives or their pharmaceutically acceptable salts or the above pharmaceutical compositions in the preparation of drugs for the treatment or prevention of type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, perioperative hyperglycemia, or metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0036] The technical solution provided by the present invention has the following advantages compared with the prior art:

[0037] By glycosylating the threonine at position 27 of the human insulin B chain, the present invention increases the anti-self-aggregation tendency of insulin and unexpectedly improves the solubility of insulin, making it more suitable for the treatment of sugar metabolism diseases. Among them, the insulin glycosylation derivatives Ins3, Ins4, Ins5, and Ins6 have a higher anti-self-aggregation tendency than the rapid-acting insulins such as aspart insulin and lispro insulin on the market, and have the potential to be developed into rapid-acting insulins with a faster onset of action. In addition, the insulin glycosylation derivatives Ins2, Ins3, Ins4, Ins5, and Ins6 have a higher solubility than the rapid-acting insulins such as aspart insulin and lispro insulin on the market. Finally, mouse activity experiments show that the insulin glycosylation derivatives maintain a hypoglycemic activity comparable to that of insulin. Brief Description of the Drawings

[0038] 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 use in the description of the specific embodiments.

[0039] Figure 1 . Schematic diagram of the synthesis of insulin glycosylation derivatives.

[0040] Figure 2 . UPLC and HRMS spectra of the product Ins1. Theoretical values: [M + 4H] 4+ m / z = 1543.20, [M + 5H] 5+ m / z = 1234.76, [M + 6H]6+ m / z = 1029.14; Observed values: 1543.21, 1234.77, 1029.14. Theoretical value for loss of one sugar in the mass spectrum: [M+6H] 6+ m / z = 1002.13; Observed value: 1002.14.

[0041] Figure 3 . UPLC and HRMS spectra of product Ins2. Theoretical value: [M+4H] 4+ m / z = 1575.46, [M+5H] 5+ m / z = 1260.57, [M+6H] 6+ m / z = 1050.64; Observed values: 1575.47, 1260.58, 1050.65.

[0042] Figure 4 . UPLC and HRMS spectra of product Ins3. Theoretical value: [M+4H] 4+ m / z = 1593.97, [M+5H] 5+ m / z = 1275.38, [M+6H] 6+ m / z = 1062.98; Observed values: 1593.98, 1275.38, 1062.99.

[0043] Figure 5 . UPLC and HRMS spectra of product Ins4. Theoretical value: [M+4H] 4+ m / z = 1615.97, [M+5H] 5+ m / z = 1292.98, [M+6H] 5+ m / z = 1077.65; Observed values: 1615.98, 1292.99, 1077.66.

[0044] Figure 6 . UPLC and HRMS spectra of product Ins5. Theoretical value: [M+4H] 4+ m / z = 1615.97, [M+5H] 5+ m / z = 1292.98, [M+6H] 6+ m / z = 1077.65; Observed values: 1615.98, 1292.99, 1077.66. Theoretical value for loss of one sugar in the mass spectrum: [M+5H] 5+ m / z = 1234.76, [M+6H] 6+ m / z = 1029.14; Observed values: 1234.77, 1029.14.

[0045] Figure 7UPLC and HRMS spectra of product Ins6. Theoretical values: [M+4H] 4+ m / z = 1688.75, [M+5H] 5+ m / z = 1351.20, [M+6H] 6+ m / z = 1126.17; Observed values: 1688.74, 1351.20, 1126.17. Theoretical value of losing one sugar in mass spectrometry: [M+5H] 5+ m / z = 1292.98, [M+6H] 6+ m / z = 1077.65; Observed values: 1292.98, 1077.65. Detailed implementation manners

[0046] The following combines specific embodiments to further elaborate on the present invention. It should be noted that the following detailed descriptions are all exemplary, aiming to provide further explanations for the present invention and are not used to limit the present invention described in detail in the claims. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0047] Example 1. Insulin solid-phase peptide synthesis (SPPS), purification and identification process:

[0048] Swelling: Add 0.05 mmol of resin into the solid-phase synthesis reactor, wash the resin three times with DMF (3 mL×3), and add 10 mL of DMF to swell for 5 minutes.

[0049] Deprotection: Add 3 mL of 20% 4-methylpiperidine dissolved in DMF. Use microwave heating to raise the temperature of the mixture to 50 °C and maintain it at 50 °C. Nitrogen is introduced from the bottom of the container (usually open for 2 seconds and closed for 3 seconds) for mixing. After reacting for 10 min, wash the resin four times with DMF (4 mL×4).

[0050] Condensation: Sequentially add Fmoc-protected amino acid (0.2 M in DMF, 1.25 mL, 5 eq.), DIEA (0.5 M in DMF, 1 mL, 10 eq.) and HATU (0.25 M in DMF, 1 mL, 5 eq.) into the reaction vessel. After reacting at room temperature for 2 minutes, raise the temperature of the reaction mixture to 50 °C and maintain it at 50 °C with nitrogen mixing for 8 minutes. Among them, for amino acids containing β-branches (Ile, Thr and Val), Pro in the sequence and residues after Fmoc-Arg(Pbf)-OH with large protecting groups, two condensations are carried out to ensure complete reaction.

[0051] Peptide chain elongation: Repeat the above deprotection and condensation steps until the sequence is complete. The Fmoc group on the last amino acid is removed through the deprotection step.

[0052] Cleavage: Transfer the obtained resin to a glass polypeptide synthesis reactor, and wash the resin three times (5 mL each time) alternately with DMF and DCM. Add freshly prepared cleavage solution (4 mL), and gently stir at room temperature for 2 hours. Filter, and collect the cleavage solution using a 50 mL centrifuge tube.

[0053] Collection: Add 40 mL of ice-cold diethyl ether to precipitate the crude peptide. Centrifuge (5800 g, 4 °C, 5 min) to collect the precipitate, and then carefully pour out the liquid from the centrifuge tube. Dissolve the residue in 15 mL of MeCN / H 2 O (1 / 1, v / v).

[0054] UPLC-HRMS 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 H 2 O containing 0.1% formic acid, and phase B was MeCN containing 0.1% formic acid, eluted with a linear gradient (20% - 40%) for a total of 6 minutes. After each run, wash the column with 95% (B%) for 2 min and re-equilibrate the column with 5% (B%) for 2 min. Mass spectrometry analysis was performed using an Exactive Plus Orbitrap mass spectrometer. An electrospray ionization (ESI) source was used, in positive ion mode, with a scan 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.

[0055] High performance liquid chromatography (HPLC) purification: Use a C18, 10×250 mm, 5 μm, separation column. Elute the column with 95% (B%) for 15 min and equilibrate the column with 5% (B%) for 15 min. Inject the filtered solution into the column and elute with a linear gradient. Monitor the eluate at wavelengths of 214 nm and 275 nm, collect the desired product according to the UPLC-MS analysis results, and obtain the pure product through lyophilization.

[0056] Example 2. Synthesis of insulin glycosylation derivatives

[0057] The synthesis of the insulin glycosylation derivatives in this patent was achieved by referring to the method in the reference (ACS.Chem.Biol.2018, 13, 73 - 81.).

[0058] As attachedFigure 1 As shown, Fmoc-Asn-NovaSyn TGT resin was used as the solid-phase carrier, and the insulin A chain was synthesized by solid-phase peptide synthesis (SPPS) technology. Except that Fmoc-Cys(Acm)-OH was used for CysA6, CysA11 and CysA20, the remaining steps were the same as those described in Example 1. After SPPS was completed, freshly prepared TFA / TIPS / H 2 O (92.5 / 5 / 2.5, v / v / v, 5 mL) cleavage solution was added to the resin, and the mixture was stirred at room temperature for 2 hours. The solution was filtered and collected into a 50 mL centrifuge tube, and 40 mL of cold Et 2 O (+4 °C) was added to form a white precipitate. After centrifugation (5800 g, 4 °C, 5 min), the liquid was carefully decanted. The crude peptide was dissolved in 10 mL of MeCN / H 2 O (1 / 1, v / v), and lyophilized to obtain the crude peptide. The crude peptide was dissolved in MeCN / H 2 O (2 / 8, v / v) to a concentration of 1 mg / mL and filtered through a 0.22 μm membrane. In each operation, 5 mg (5 mL) of the sample was loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted with a 20% to 40% gradient for 40 min. The target fraction was collected and lyophilized to obtain the desired A chain with a yield of 10%.

[0059] Fmoc-Thr-NovaSyn TGT resin was used as the solid-phase carrier, and the insulin B chain was synthesized by SPPS technology. Among them, a sugar group was introduced into the B chain using a Fmoc-protected glycoamino acid (FPGA). For the condensation reaction of the Fmoc-protected glycoamino acid, a DMF solution containing 0.75 mL of 0.1 M FPGA (1.5 eq.), 0.3 mL of 0.5 M DIEA (3 eq.) and 0.3 mL of 0.25 M HATU (1.5 eq.) was added to the reaction vessel in sequence. The reaction was carried out at room temperature for 2 minutes, then the temperature was raised to 50 °C, and the reaction was continued at 50 °C for 18 minutes while mixing was carried out by bubbling nitrogen. This condensation reaction was carried out 2 times in total. In addition, except that Fmoc-Cys(Acm)-OH was used for CysB19, the remaining steps were the same as those described in Example 1. After SPPS, the resin was placed in a peptide synthesis reaction tube, and a TFA / TIPS / H 2 O (92.5 / 5 / 2.5, v / v / v, 5 mL) cleavage solution containing 20 equivalents of 2,2′-dithiobis(5-nitropyridine) was added, and the mixture was stirred at room temperature for 2 h. The solution was filtered and collected into a 50 mL centrifuge tube, and 40 mL of cold Et 2At O(+4 °C), a white precipitate is formed. After centrifugation (5800 g, 4 °C, 5 min), the liquid is carefully decanted. The crude polypeptide is dissolved in 10 mL of MeCN / H 2 O (1 / 1, v / v), and freeze-dried to obtain the crude polypeptide. The crude polypeptide is dissolved in MeCN / H 2 O (2 / 8, v / v) to a concentration of 4 mg / mL and filtered through a 0.22 μm membrane. In each operation, 8 mg (2 mL) of the sample is loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted with a 30% to 50% gradient for 40 min. The target fraction is collected and freeze-dried to obtain the B chain, with a yield of 20 - 25%.

[0060] The A chain (4 mg, 1.63 μmol, 1 eq.) and the B chain (1.96 μmol, 1.2 eq.) are dissolved in 1 mL of a buffer solution with pH 8.0 containing 8.0 M Gn·HCl and 0.1 M Tris·HCl, and reacted at room temperature for 5 minutes. 8 mL of AcOH / H 2 O (4 / 1, v / v) is added for dilution, and then 1.9 mL of a methanol solution containing I 2 (20.6 mg, 81.6 μmol, 50 eq.) is added, and the reaction is carried out at room temperature for 15 min. An appropriate amount of 1 M ascorbic acid is added until the color of iodine disappears, and the solution is filtered through a 0.45 μm membrane, loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted with a 20% to 50% gradient for 40 min. The target fraction is freeze-dried to obtain the insulin glycosylation derivative intermediate (sugar with protecting group), with yields of 10% - 15% respectively.

[0061] Synthesis of Ins1 and Ins3: 3 mg of the intermediate is dissolved in 3 mL of 5% NH 2 NH 2 solution, and stirred for 0.5 h. The pH of the reaction mixture is adjusted to 5 with 10% acetic acid and filtered through a 0.45 μm membrane, loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted with a 20% to 50% gradient for 40 min. The target fraction is freeze-dried to obtain Ins1 and Ins3, with yields of 62% and 58% respectively.

[0062] Synthesis of Ins2, Ins4, Ins5 and Ins6: 3 mg of the intermediate is dissolved in 3 mL of 100 mM aqueous NaOH solution, and stirred for 10 h. The pH of the reaction mixture is adjusted to 5 with 10% acetic acid and filtered through a 0.45 μm membrane, loaded onto a C18 column (10 × 250 mm, 5 μm, ) Elute with a gradient of 20% to 50% for 40 min. The target components were freeze-dried to obtain Ins2, Ins4, Ins5, and Ins6, with yields of 25%, 22%, 18%, and 19% respectively.

[0063] The LC-MS of Ins1 to Ins6 is shown in detail in Figures 2 - 7 .

[0064] Example 3. Effect of Insulin Glycosylation Modification on the Tendency of Self-Aggregation

[0065] Insulin, aspart insulin, lispro insulin, or insulin glycosylation derivatives were respectively dissolved in a buffer solution (pH 7.47) containing phenol (1.5 mg / mL), m-cresol (1.72 mg / mL), zinc ions (19.6 μg / mL), disodium phosphate dihydrate (1.25 mg / mL), and sodium chloride (0.58 mg / mL) to prepare samples with a concentration of 60 μM.

[0066] Analytical ultracentrifugation experiments were carried out using a Beckman Coulter Optima analytical ultracentrifuge equipped with an An-60 Ti rotor. The samples were loaded into a standard double-sector aluminum centerpiece with a diameter of 12 mm, and sapphire windows were connected above and below the centerpiece. The buffer solution was used as the optical reference. The experiments were carried out at 20 °C and a rotational speed of 60 k rpm, and data were collected using Rayleigh interference detection with a 1024x1088 CCD camera. The obtained data were analyzed using the software SEDFIT, and the results are shown in Table 1:

[0067] Table 1: Proportions of monomers, dimers, and hexamers of insulin and its derivatives.

[0068] Monomer ratio Dimer ratio Hexamer ratio Insulin 14.9％ 3.3％ 81.8％ Insulin aspart 33.8％ 11.7％ 54.5％ Insulin lispro 24.1％ 6.8％ 69.1％ Ins1 25.6％ 4.3％ 70.1％ Ins2 20.2％ 3.4％ 76.4％ Ins3 43.9％ 0.0％ 56.1％ Ins4 78.9％ 7.2％ 13.9％ Ins5 87.1％ 7.2％ 5.7％ Ins6 77.7％ 16.8％ 5.5％

[0069] The results show that all insulin glycosylation derivatives have a higher anti-self-aggregation tendency than insulin. Among them, Ins3, Ins4, Ins5, and Ins6 all have a higher anti-self-aggregation tendency than the commercial rapid-acting insulins aspart insulin and lispro insulin, and have the potential to be developed into rapid-acting insulins with a faster onset of action.

[0070] Example 4. Effect of Insulin Glycosylation Modification on Solubility

[0071] 40 μL of sodium phosphate buffer (pH 7.3) was added to 400 μg of insulin, insulin aspart, insulin lispro or insulin glycosylation derivative, and vortexed for 2 minutes. Turbidity appeared, indicating supersaturation. After centrifugation at 2680 g for 2 minutes, 20 μL of the supernatant was transferred to another 0.6 mL centrifuge tube. The sample was diluted with water to an appropriate concentration and then quantitatively analyzed by UPLC. For Ins6, when 750 μg of Ins6 was added to 40 μL of sodium phosphate buffer, a gel-like mixture was formed. Therefore, the determined concentration for Ins6 was not its true solubility, and the actual solubility should be higher than the obtained value. The solubility was calculated by substituting the UV integration into the calibration curve, and the results are shown in Table 2.

[0072] Table 2: Solubility of insulin and its derivatives.

[0073] Solubility (g / L) Insulin 0.10 Insulin aspart 0.25 Insulin lispro 0.25 Ins1 0.15 Ins2 0.83 Ins3 0.33 Ins4 0.51 Ins5 3.53 Ins6 >63.83

[0074] The results showed that all insulin glycosylation derivatives had higher solubility than insulin. Among them, insulin glycosylation derivatives Ins2, Ins3, Ins4, Ins5 and Ins6 had higher solubility than rapid-acting insulins such as insulin aspart and insulin lispro on the market. The solubility of derivative Ins6 with a tetrasaccharide structure was at least 638 times that of insulin and at least 255 times that of insulin aspart or insulin lispro.

[0075] Example 5. Effect of insulin glycosylation modification on hypoglycemic activity

[0076] We characterized the hypoglycemic activities of insulin and insulin glycosylation derivatives using a type 1 diabetic mouse model.

[0077] SPF-grade 5-week-old male C57BL / 6N mice were housed in an environment with a constant temperature of (25 ± 1°C) and a constant humidity of (55 - 65%), and a 12:12-hour light-dark cycle (lights on from 7 am to 7 pm) was adopted. They had free access to standard feed and water. After a 6-hour fast, the mice were intraperitoneally injected with streptozotocin (18 mg / mL) dissolved in 0.1 M sodium citrate buffer (pH 4.5) at a dose of 180 mg / kg. Food was restored 2 hours after injection, and then they were raised for 7 days. Blood glucose was collected from the tail vein and measured using a handheld blood glucose meter to confirm hyperglycemia. Only mice with a fasting blood glucose concentration of at least 16.7 mmol / L were included in the study. On the day of the experiment, after a 3-hour fast, the mice (weighing 21.4 ± 2 g) were subcutaneously injected with insulin or insulin glycosylation derivative (0.36 U / mL) dissolved in a buffer (pH 7.47) containing phenol (1.5 mg / mL), m-cresol (1.72 mg / mL), zinc ions (19.6 μg / mL), disodium hydrogen phosphate dihydrate (1.25 mg / mL), and sodium chloride (0.58 mg / mL) at a dose of 3.6 U / kg. The blood glucose level was monitored every 30 minutes for 6.5 hours after injection. Subsequently, the in vivo experimental data obtained were imported into GraphPad for further analysis, and the results were expressed as the mean of 8 repeated experiments in each group. The results are shown in Table 3.

[0078] Table 3: Statistical results of normalized blood glucose concentration in the hypoglycemic experiments of insulin and its derivatives.

[0079] Time Insulin Ins1 Ins2 Ins3 Ins4 Ins5 Ins6 0h 100％ 100％ 100％ 100％ 100％ 100％ 100％ 0.5h 39.8％ 52.7％ 69.9％ 70.7％ 58.6％ 65.9％ 68.3％ 1h 20.6％ 26.3％ 32.6％ 34.5％ 27.7％ 32.4％ 31.2％ 1.5h 20.4％ 22.0％ 23.3％ 27.4％ 26.4％ 25.3％ 26.4％ 2h 19.8％ 22.9％ 20.6％ 23.7％ 24.8％ 20.7％ 22.2％ 2.5h 21.1％ 24.2％ 19.2％ 27.9％ 29.0％ 21.2％ 24.9％ 3h 28.9％ 29.5％ 22.5％ 35.8％ 32.9％ 21.8％ 28.5％ 3.5h 39.1％ 39.5％ 28.1％ 45.5％ 40.2％ 30.8％ 34.9％ 4h 45.6％ 48.1％ 34.7％ 56.5％ 51.3％ 39.2％ 44.8％ 4.5h 69.2％ 68.6％ 47.8％ 70.7％ 62.2％ 49.7％ 56.7％ 5h 76.8％ 75.4％ 58.4％ 83.5％ 65.7％ 61.1％ 65.9％ 5.5h 82.3％ 79.8％ 69.5％ 89.3％ 69.4％ 71.5％ 71.4％ 6h 84.6％ 79.9％ 77.5％ 90.2％ 75.3％ 82.4％ 76.2％ 6.5h 83.5％ 80.7％ 83.6％ 93.7％ 78.8％ 94.1％ 87.8％

[0080] The results showed that all glycosylated insulin molecules exhibited hypoglycemic activity comparable to that of insulin.

Claims

1. An insulin glycosylation derivative or a pharmaceutically acceptable salt thereof, characterized in that, the insulin glycosylation derivative is formed by connecting a sugar to the hydroxyl group of threonine at position 27 of the B chain of human insulin, 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 insulin glycosylation derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, the insulin glycosylation isoform is selected from Ins1, Ins2, Ins3, Ins4, Ins5 or Ins6; the structural formulas of Ins1, Ins2, Ins3, Ins4, Ins5, and Ins6 are respectively: Structural formula Ins1: Structural formula Ins2: Structural formula Ins3: Structural formula Ins4: Structural formula Ins5: Structural formula Ins6: wherein, the sugar is covalently linked to the hydroxyl group of threonine at position 27 of the B chain of human insulin.

3. A pharmaceutical composition comprising a bioactive amount of at least one of the insulin glycosylation derivatives or pharmaceutically acceptable salts thereof according to claim 1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, the pharmaceutical composition further comprises one or more other active ingredients, and the active ingredients are selected from 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.

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

7. Use of the insulin glycosylation derivative or a pharmaceutically acceptable salt thereof according to claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 4 in the preparation of a drug for treating or preventing type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, perioperative hyperglycemia or metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

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