Zwitterionic polymer-insulin conjugate and preparation method and application thereof

Abstract

The application discloses an amphoteric ion polymer-insulin conjugate and a preparation method and application thereof. 2-bromoisobutyric acid N-hydroxysuccinimide ester is reacted with INS to introduce an ATRP reaction site at the Lys B29 position of the INS, so that INS-Br is obtained; the INS-Br, MPC and a catalyst are mixed, deoxygenated, CuBr is further mixed, and full polymerization reaction is carried out, so that CuBr and PMDETA are used as a catalytic system, INS-Br is used to initiate polymerization of the amphoteric ion monomer MPC, and through dialysis and cation chromatographic column purification technology, INS-PMPC is obtained. Compared with INS and INS-PEG, the INS-PMPC has a longer blood glucose control time under the same dose, and can significantly reduce the risk of hypoglycemia, has high stability under room temperature and mechanical stirring, is resistant to storage, is suitable for areas where cold chain facilities are lacking, and is a long-acting insulin drug with higher safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a zwitterionic polymer-insulin conjugate, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by high blood sugar, which can lead to serious complications such as kidney disease, cardiovascular disease, and foot injuries. As a global public health epidemic, diabetes affects more than 400 million people worldwide. Insulin is the primary drug for treating type 1 diabetes, and its discovery has fundamentally improved the treatment and prognosis of diabetes, transforming it from an incurable disease into a manageable chronic condition. However, like many protein drugs, insulin also has some problems, such as a short half-life and poor stability during storage and transportation. To achieve blood sugar control, diabetic patients need to inject insulin multiple times a day, which severely impacts their quality of life. Furthermore, insulin needs to be stored and transported at low temperatures, as high temperatures can cause it to aggregate and degrade, which not only impairs its biological activity but may also trigger unexpected immune responses. Additionally, the demanding cold storage requirements of insulin limit its use in areas lacking cold chain facilities, such as disaster areas and rural clinics in developing countries.

[0003] To address these two shortcomings of insulin, significant effort and resources have been invested for decades in developing long-acting insulin with high stability. Genetic engineering is one method for producing long-acting insulin analogs. For example, insulin glargine (Sanofi-Aventis) has a half-life extended to approximately 13 hours. Precise manipulation of certain amino acid sequences and structures alters the isoelectric point, causing precipitation at the subcutaneous injection site, thus delaying dissolution and absorption. However, genetic engineering mutagenesis procedures are highly complex. More importantly, degradation of insulin glargine at the injection site reduces its bioavailability and can induce unintended immune responses. Besides mutagenesis strategies, conjugation with water-soluble polymers has become a common method for improving protein circulation time and shelf-life stability. To date, relatively few polymers have been used for insulin conjugation, primarily including polyethylene glycol (PEG) and glycopolymers. While conjugation with these polymers can improve the pharmacokinetics and stability of insulin, it often affects its activity. PEG is also readily oxidatively degraded when applied to biological media. Furthermore, although PEGylation is generally touted as reducing the immunogenicity of proteins, there are increasing reports of PEG antibodies following injection of PEG-protein conjugates. PEG antibodies not only impair the efficacy of PEG-protein conjugates but can also cause severe allergic reactions. These drawbacks hinder the further development of PEG. Therefore, there is a need to develop novel insulin-polymer conjugates that do not affect insulin activity while improving its pharmacokinetic characteristics and storage stability. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide an amphoteric polymer-insulin conjugate, its preparation method, and its application.

[0005] The first objective of this invention is to provide a method for preparing an insulin conjugate.

[0006] A second objective of this invention is to provide an insulin conjugate.

[0007] A third object of the present invention is to provide the use of the insulin conjugate in the preparation of a medicament for treating diabetes.

[0008] A fourth objective of this invention is to provide a medicament for treating diabetes.

[0009] To achieve the above objectives, the present invention is implemented through the following solution:

[0010] A method for preparing an amphoteric polymer-insulin conjugate, comprising the following specific steps:

[0011] S1: 2-Bromoisobutyric acid N-hydroxysuccinimide ester reacts with insulin to give the large insulin initiator;

[0012] S2: Mix the insulin initiator, 2-methacryloyloxyethylphosphorylcholine and catalyst from step S1, deoxygenate, then mix with cuprous bromide and fully polymerize to obtain the zwitterionic polymer-insulin conjugate.

[0013] The catalyst is 1,1,4,7,10,10-hexamethyltriethylenetetramine, 2,2'-bipyridine, or N,N,N',N",N"-pentamethyldiethylenetriamine.

[0014] Preferably, a Na2CO3 buffer solution containing insulin and a DMSO solution containing N-hydroxysuccinimide ester of 2-bromoisobutyric acid are mixed and stirred to obtain a large insulin initiator.

[0015] Preferably, the molar ratio of insulin to N-hydroxysuccinimide 2-bromoisobutyric acid is 1:1 to 1.5.

[0016] More preferably, the molar ratio of insulin to N-hydroxysuccinimide 2-bromoisobutyric acid is 1:1.2.

[0017] Preferably, the Na2CO3 buffer solution is 0.1M, the insulin concentration in the Na2CO3 buffer solution containing insulin is 40 mg / mL, and the concentration of 2-bromoisobutyric acid N-hydroxysuccinimide ester in the DMSO solution containing 2-bromoisobutyric acid N-hydroxysuccinimide ester is 22 mg / mL.

[0018] Preferably, the catalyst is N,N,N',N",N"-pentamethyldiethylenetriamine.

[0019] More preferably, the molar ratio of the insulin-initiating agent to 2-methylacryloyloxyethylphosphorylcholine is 1:20 to 1:200.

[0020] More preferably, the molar ratio of the insulin-initiating agent to 2-methylacryloyloxyethylphosphocholine is 1:40.

[0021] More preferably, the molar ratio of the insulin-initiating agent: cuprous bromide: N,N,N',N",N"-pentamethyldiethylenetriamine is 0.25 to 10:4:4.

[0022] More preferably, the insulin initiator, 2-methacryloyloxyethylphosphonic choline and N,N,N',N'",N'"-pentamethyldiethylenetriamine are dissolved in Tris buffer in a ratio of 30 mg:30 mg:1.73 mg:4-10 mL.

[0023] Preferably, the deoxygenation method in step S2 is nitrogen gas purging and bubbling deoxygenation.

[0024] More preferably, in step S2, the deoxygenation method is to pass nitrogen gas and bubble deoxygenate for 10 minutes.

[0025] Preferably, the polymerization reaction time in step S2 is 7 to 14 hours.

[0026] Preferably, after the polymerization reaction is fully completed in step S2, purification is performed;

[0027] The reaction solution after polymerization in step S2 was dialyzed with deionized water to remove copper ions and other impurities by removing a molecular weight cutoff of 3500, yielding crude INS-PMPC. This crude INS-PMPC was then freeze-dried to obtain freeze-dried crude INS-PMPC.

[0028] Preferably, the crude INS-PMPC is purified using an AKAT protein purification system equipped with a HiTrap SP HP cation exchange column.

[0029] More preferably, the freeze-dried crude INS-PMPC is dissolved in acetate buffer to obtain a crude INS-PMPC solution. The crude INS-PMPC solution is loaded onto a HiTrap SP HP cation exchange column, and an acetate buffer containing NaCl is used as the elution buffer. The elution time is 100 min, and the elution buffer is continuously detected at 280 nm. The purified INS-PMPC is collected.

[0030] The acetate buffer solution has a concentration of 10 mM and a pH of 4.0.

[0031] The NaCl gradient concentration in the eluent is 0–0.25 M.

[0032] An insulin conjugate is prepared by the method described above.

[0033] The application of the insulin conjugate in the preparation of diabetes treatment drugs.

[0034] Preferably, the drug is a drug that reduces the risk of hypoglycemia during diabetes treatment.

[0035] A medication for treating diabetes, the medication containing the insulin conjugate.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention discloses a zwitterionic polymer-insulin conjugate, its preparation method, and its applications. 2-Bromoisobutyric acid N-hydroxysuccinimide ester reacts with insulin (INS) to specifically introduce an ATRP reaction site at the Lys B29 position of INS, yielding an insulin macroinitiator (INS-Br). INS-Br, 2-methacryloyloxyethylphosphorylcholine (MPC), and a catalyst are mixed, deoxygenated, and then mixed with cuprous bromide (CuBr) for a complete polymerization reaction. Using CuBr and PMDETA as a catalytic system, the zwitterionic monomer MPC is polymerized using INS-Br as the initiator. The resulting product is purified by dialysis and cation exchange chromatography to obtain the zwitterionic polymer-insulin conjugate (INS-PMPC). Compared to INS and polyethylene glycol-insulin conjugate (INS-PEG), at the same dosage, the INS-PMPC of this invention exhibits a longer glycemic control time while significantly reducing the risk of hypoglycemia. It also demonstrates high stability at room temperature and under mechanical stirring, good storage resistance, and suitability for areas lacking cold chain facilities, making it a safer long-acting insulin drug. Attached Figure Description

[0038] Figure 1 This is a synthetic route diagram for INS-PMPC.

[0039] Figure 2 The molecular weights of INS-Br and INS-PEG reduced with dithiothreitol (DTT) and untreated INS-Br and INS-PEG were analyzed using MALDI-TOF. In the MALDI-TOF mass spectra, A represents INS-Br with and without DTT treatment, B represents INS-PEG, and C represents INS-PEG treated with DTT.

[0040] Figure 3 The hydrodynamic dimensions of INS-Br, INS, INS-PEG, and INS-PMPC, and the secondary structure of INS-Br are shown. A is the dynamic light scattering (DLS) pattern of INS-Br, B is the circular dichroism (CD) pattern of INS-Br, and C is the DLS pattern of INS, INS-PEG, and INS-PMPC.

[0041] Figure 4 This is a non-denaturing PAGE electrophoresis image. In lane A, lane 1 is INS, lane 2 is INS-Br, lane 3 is crude INS-PMPC, and lane 4 is purified INS-PMPC; in lane B, lane 5 is INS, lane 6 is crude INS-PEG, and lane 7 is purified INS-PEG.

[0042] Figure 5 Gel chromatograms (GPC) of INS-PMPC and INS.

[0043] Figure 6 This is the MALDI-TOF mass spectrum of INS-PMPC.

[0044] Figure 7 The glycemic control capabilities of INS, INS-PEG, and INS-PMPC are shown. A represents the glycemic status of diabetic mice (inducible by streptozotocin) after subcutaneous injection of INS, INS-PEG, and INS-PMPC, and the glycemic status of mice in five glucose tolerance tests; the arrows represent the time of each glucose tolerance test. B represents the number of diabetic mice suffering from hypoglycemia within 4 hours after subcutaneous injection of INS, INS-PEG, and INS-PMPC. C represents... Figure 7 The area under the curve of the first glucose tolerance test in A. Results are expressed as mean ± SD (n = 6).

[0045] Figure 8 The relationship between blood fluorescence intensity and time in mice after intravenous injection of Cy7 fluorescent dyes INS, INS-PEG, and INS-PMPC was investigated. The results are expressed as mean ± SD (n = 5).

[0046] Figure 9 The hypoglycemic index in mice in response to INS, INS-PEG, and INS-PMPC is given. A represents the blood glucose change in mice injected with INS, INS-PEG, and INS-PMPC within 6 hours, and results are expressed as mean ± SD (n = 6). B represents... Figure 9 The area above curve A and below the hypoglycemia indicator line.

[0047] Figure 10 The stability of INS, INS-PEG, and INS-PMPC was evaluated. A represents the changes in blood glucose levels of INS and INS-PMPC in diabetic mice before and after storage, with results expressed as mean ± SD (n = 6). B represents the stability of INS, INS-PEG, and INS-PMPC based on... Figure 10 A shows the area under the curve (AUC) calculated. C shows the changes in the transmittance of INS, INS-PEG, and INS-PMPC solutions under shaking at pH 7.4 and 37℃. D shows the dynamic light scattering (DLS) plots of INS, INS-PEG, and INS-PMPC after the thermal aggregation experiment. E shows the circular dichroism (CD) spectra of INS, INS-PEG, and INS-PMPC. F shows the results based on... Figure 10 The proportions of secondary structures of INS, INS-PEG, and INS-PMPC were calculated from the CD spectrum of E. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1: Preparation of insulin-poly(2-methacryloyloxyethylphosphorylcholine) conjugate (i.e., zwitterionic polymer-insulin conjugate, INS-PMPC) and insulin-PEG conjugate.

[0050] I. Experimental Methods

[0051] like Figure 1 The diagram shows the synthetic route for INS-PMPC. INS-PMPC was synthesized using a grafting-from strategy combined with atom transfer radical polymerization (ATRP) technology.

[0052] 1. Synthetic insulin major initiator (INS-Br)

[0053] Based on the pKa differences of the three residual amino groups in insulin (Gly A1 amino, Phe B1 amino, and Lys B29 amino groups have pKa values ​​of 8.6, 6.8, and 11.2, respectively), an ATRP reaction site was specifically introduced at the Lys B29 position of insulin by controlling the reaction time and the pH of the solution. The insulin Lys B29 is the 29th lysine residue in the insulin B chain.

[0054] 2-Bromoisobutyric acid N-hydroxysuccinimide ester was synthesized as an ATRP initiator according to the method described in the literature (Lecolley F, Lei T, Mantovani G, et al. A new approach to bioconjugates for proteins and peptides (pegylation) utilizing living radical polymerisation[J]. Chimica oggi: international journal of chemistry and biotechnology, 2005(1):23.).

[0055] 200 mg of insulin (INS) was dissolved in 5 mL of 0.1 M Na₂CO₃ buffer to obtain an insulin (INS) solution. 11 mg of 2-bromoisobutyric acid N-hydroxysuccinimide ester was dissolved in 0.5 mL of DMSO buffer to obtain a 2-bromoisobutyric acid N-hydroxysuccinimide ester solution.

[0056] The above INS solution and 2-bromoisobutyric acid N-hydroxysuccinimide ester solution were mixed and stirred at 4°C for 1 h to obtain crude INS-Br. The crude INS-Br was desalted using a PD-10 desalting column, freeze-dried, and stored at -20°C.

[0057] 2. Synthesis of INS-PMPC

[0058] (1) Dissolve 30 mg INS-Br, 30 mg MPC monomer (2-methacryloyloxyethylphosphorylcholine) and 1.73 mg PMDETA (N,N,N',N",N”-pentamethyldiethylenetriamine) in 4 mL Tris buffer (50 mmol, 150 mmol NaCl, pH 7.4) to obtain a mixture solution.

[0059] (2) Purge the mixture solution from step (1) with nitrogen gas and bubble for 10 min to remove oxygen. Then, quickly add 1.43 mg of CuBr to the deoxygenated mixture solution and polymerize overnight (7–14 h). The molar ratio of INS-Br: MPC: PMDETA: CuBr is 1:40:4:4.

[0060] Using CuBr and PMDETA as catalysts, INS-Br was used to initiate the polymerization of zwitterionic monomers MPC to obtain INS-PMPC.

[0061] (3) Dialyze the reaction solution after step (2) with deionized water to remove copper ions and other impurities, and obtain crude INS-PMPC, which is then freeze-dried.

[0062] (4) Purify INS-PMPC using an AKAT protein purification system equipped with a HiTrap SP HP cation exchange column. Dissolve the freeze-dried crude INS-PMPC from step (3) in acetate buffer (10 mM, pH 4.0) and load it onto a HiTrap SP HP cation exchange column. Elute the column with acetate buffer containing a NaCl gradient (0 to 0.25 M) over 100 min. Continuously monitor the eluent at 280 nm and collect the target product, INS-PMPC.

[0063] (5) Dialyze the purified product INS-PMPC from step (4) with deionized water and freeze-dry it.

[0064] 3. Preparation of polyethylene glycol-insulin conjugate (INS-PEG)

[0065] The preparation and purification of the insulin-PEG conjugate were performed according to previously published methods with minor modifications (Wang Yuanpeng, Fu Mian, Wang Zuwei, Zhu XX, Guan Ying, Zhang Yongjun. Assustained zero-order release carrier for long-acting, peakless basal insulin therapy.[J]. Journal of materials chemistry.B,2020,8(9).).

[0066] (1) Dissolve 75 mg of PEG-NHS (methoxy polyethylene glycol carboxymethyl succinimide ester) in DMSO solution to obtain PEG-NHS solution.

[0067] (2) Dissolve 58 mg of insulin in a mixed solvent containing 0.8 mg DMSO and 1.7 mL carbonate buffer (0.1 M, pH 11) to obtain an insulin mixed solution.

[0068] (3) Immediately inject 0.5 mL of the PEG-NHS solution from step (1) into the insulin mixture from step (2), stir at room temperature (25°C) for 1 h, add 12 mL of H2O to end the reaction, and obtain crude INS-PEG, which is then freeze-dried.

[0069] (4) is the same as steps (4) and (5) in the synthesis of INS-PMPC.

[0070] Example 2: Synthesis of INS-PMPC

[0071] In Experimental Group 1 and Example 1, the insulin initiator (INS-Br) was 60 mg, and the molar ratio of INS-Br to MPC monomer (2-methacryloyloxyethyl phosphorylcholine) was 1:20. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0072] In Experimental Group 2 and Example 1, the MPC monomer was 15 mg, and the molar ratio of INS-Br to MPC monomer was 1:20. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0073] In Experimental Group 2 and Example 1, the MPC monomer was 150 mg, and the molar ratio of INS-Br to MPC monomer was 1:200. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0074] In Experimental Group 3 and Example 1, the CuBr content was 0.7 mg, and the molar ratio of CuBr to INS-Br was 1:1. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0075] In Experimental Group 4 and Example 1, the CuBr content was 7 mg, and the molar ratio of CuBr to INS-Br was 1:10. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0076] In Experimental Group 5, the amount of PMDEAT (N,N,N',N",N"-pentamethyldiethylenetriamine) in Example 1 was 0.8 mg, and the molar ratio of PMDEAT to INS-Br was 1:1. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0077] In Experimental Group 6 and Example 1, the amount of PMDEAT was 1.7 mg, and the molar ratio of PMDEAT to INS-Br was 1:10. Other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0078] Experimental Group 7: The volume of Tris buffer in Example 1 was 6 mL, and other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0079] Experimental Group 8: The volume of Tris buffer in Example 1 was 10 mL, and other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0080] Experimental Group 9: The PMDETA in Example 1 was replaced with 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTEMA), and the other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0081] Experimental Group 10: The PMDETA in Example 1 was replaced with 2,2'-bipyridine (BPY), and the other parameters for the synthesis of INS-PMPC were the same as in Example 1.

[0082] Example 2 analyzes the physical properties and in vitro cell activity of insulin (INS), insulin macroinitiator (INS-Br), polyethylene glycol-insulin conjugate (INS-PEG), and insulin-poly(2-methacryloyloxyethylphosphorylcholine) conjugate (INS-PMPC).

[0083] I. Experimental Methods

[0084] 1. The molecular weights of INS-Br and INS-PEG reduced with dithiothreitol (DTT) and untreated INS-Br and INS-PEG were analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF).

[0085] 2. The hydrodynamic dimensions of INS-Br, INS, INS-PEG, and INS-PMPC were analyzed using dynamic light scattering (DLS). The secondary structures of insulin and INS-Br were analyzed using circular dichroism spectroscopy.

[0086] 3. Non-denaturing PAGE gel electrophoresis was performed on insulin, INS-Br, crude INS-PMPC, purified INS-PMPC, crude INS-PEG, and purified INS-PEG.

[0087] 4. Analyze insulin and INS-PMPC using gel permeation chromatography (GPC).

[0088] 5. Analyze the molecular weight of INS-PMPC using MALDI-TOF.

[0089] II. Experimental Results

[0090] 1. For example Figure 2 As shown in Figure A, in addition to the original peak of insulin at m / z = 5808, a new peak at m / z = 5957 appeared, indicating that INS-Br synthesis was successful. An increase in molecular weight (from 3431 to 3580) was observed only on the insulin B chain.

[0091] like Figure 2 As shown in Figure B, the peak at 10949 (m / z) represents INS-PEG, and the difference between the peaks (44 Da) is consistent with the molecular weight of the repeating unit of the PEG chain.

[0092] like Figure 2 As shown in C, INS-PEG was treated with DTT to verify the coupling site. Two peaks were observed in MALDI-TOF mass spectrometry: one at 2384 m / z and the other at 8525 m / z. The peak at 2384 m / z corresponds to the A chain of insulin, and the peak at 8525 m / z matches the sum of the molecular weights of the B chain of insulin and 5000 Da PEG.

[0093] 2. For example Figure 3 Figure A shows the dynamic light scattering (DLS) pattern of INS-Br, as follows: Figure 3 Figure B shows the circular dichroism (CD) spectra of insulin and INS-Br, indicating that ATRP initiator coupling did not significantly alter the hydrodynamic size and secondary structure of insulin.

[0094] like Figure 3 As shown in Figure C, the hydrodynamic dimensions of INS, INS-PEG, and INS-PMPC are 2.4±0.4, 5.8±0.5, and 6.3±0.2 nm, respectively. The hydrodynamics of INS-PEG and INS-PMPC are similar, ensuring fairness in subsequent comparative experiments.

[0095] Small-sized insulin particles are rapidly eliminated from the body through kidney filtration, resulting in a very short blood half-life (approximately 9 minutes). Due to their larger particle size, INS-PEG reduces renal clearance, thus prolonging its circulation time in the body. INS-PMPCs are also larger than insulin particles, and their half-life in the body is also prolonged.

[0096] 3. For example Figure 4 As shown in Figure A, insulin migrates as a single band, but due to the change in net charge between insulin and INS-Br, crude INS-Br migrates as two bands. After polymerization, the INS-Br band almost disappears, and a high molecular weight band of the INS-PMPC conjugate appears. After purification with an ion-exchange column, only the INS-PMPC band is retained.

[0097] like Figure 4 As shown in Figure B, after the coupling reaction, a new INS-PEG band and an unreacted insulin band were observed. Purification retained only the INS-PEG band.

[0098] 4. For example Figure 5 As shown, the retention time of INS-PMPC (24.5 min) is shorter than that of insulin (25.8 min), indicating that INS-PMPC has a larger molecular weight than insulin.

[0099] 5. For example Figure 6 As shown, the peak interval in the MALDI-TOF spectrum is 296 (m / z), which is exactly consistent with the molecular weight of the MPC monomer.

[0100] Example 3: Effect of INS-PEG on in vivo blood glucose control

[0101] I. Experimental Methods

[0102] The glycemic control ability of the insulin-polymer conjugate prepared in Example 1 was evaluated using streptozotocin (STZ)-induced type 1 diabetic mice.

[0103] Blood glucose levels were measured in type 1 diabetic mice after a single subcutaneous (sc) injection of insulin, INS-PEG, INS-PMPC, and PBS buffer (negative control). Each group consisted of 6 mice, and the injection dose was 5 U / kg.

[0104] Different doses of glucose were used to mimic the changes in a person's daily food intake. Mice underwent an intraperitoneal glucose tolerance test (IPGTT) five times, at 4.5h, 8.5h, 11.5h, 14.5h, and 17.5h after subcutaneous injection of the drug, to test the INS-PMPC's response to glucose. The glucose injection doses for the first to last IPGTT tests were 3, 2, 1, 2, and 2 g / kg, respectively.

[0105] To quantify the glycemic regulation capabilities of INS-PMPC, insulin, and INS-PEG, the area under the curve (AUC) for each group in the first IPGTT experiment was calculated using Graphad Prism 7.0 software.

[0106] II. Experimental Results

[0107] 1. For example Figure 7 As shown in Figure A, the blood glucose levels of all three groups of mice rapidly decreased to the normal range within 30 minutes, with blood glucose levels <11.1 mmol / L. The blood glucose levels of the mice in the negative control group remained high. Compared with insulin and INS-PEG, INS-PMPC exhibits gentler blood glucose regulation, a slower rate of glucose reduction, and is less likely to cause hypoglycemia.

[0108] 2. For example Figure 7 As shown in Figure B, within the first 4 hours after glucose injection, 5 mice in the insulin and INS-PEG groups experienced hypoglycemia (mice <3.3 mmol / L), while only 1 mouse in the INS-PMPC group experienced hypoglycemia. Figure 7 As shown in Figure A, the blood glucose levels of mice in the INS-PMPC group experiencing hypoglycemia, such as 2.9 mmol / L and 3.2 mmol / L, were very close to the critical value of 3.3 mmol / L. The blood glucose levels of mice in the insulin and INS-PEG groups, such as 1.1 mmol / L, 1.5 mmol / L, and 2.0 mmol / L, were far from the critical value of 3.3 mmol / L in a hypoglycemic state. Typical symptoms of hypoglycemia, such as hypothermia and even seizures, were observed in mice treated with insulin and INS-PEG. The intraperitoneal glucose tolerance test showed that after a rapid spike in blood glucose, INS-PMPC restored blood glucose to normal levels (<11.1 mmol / L) within 1.5 hours. Insulin did not show a hypoglycemic effect after glucose injection.

[0109] 3. For example Figure 7 As shown in Figure C, INS-PMPC has the lowest AUC compared to insulin and INS-PEG. Figure 7As shown in Figure A, after a second IPGTT 8.5 hours post-injection, INS-PMPC again reduced elevated blood glucose to normal levels, while INS-PEG showed no blood glucose-lowering effect in the second IPGTT. Even after a third IPGTT, INS-PMPC continued to demonstrate its ability to regulate blood glucose back to normal levels. The blood glucose control time of INS-PMPC reached 20 hours, indicating that once-daily administration of INS-PMPC holds great potential in the treatment of diabetes.

[0110] Example 4: INS-PMPC Extends Half-Life

[0111] I. Experimental Methods

[0112] To demonstrate the long-term efficacy of INS-PMPC in glycemic control, preliminary pharmacokinetic studies were conducted on INS-PMPC, INS-PEG, and unmodified insulin (INS) from Example 1. INS-PMPC, INS-PEG, and INS, labeled with 0.1 mg / kg Cy 7 fluorescent dye, were subcutaneously injected (sc) into mice. Blood samples were collected from mice at predetermined time intervals, and fluorescence intensity was measured.

[0113] II. Experimental Results

[0114] like Figure 8 As shown, after drug injection, the fluorescence intensity of blood samples from the INS-PMPC, INS-PEG, and INS groups increased very rapidly, reaching its maximum value within 2 or 3 hours. The maximum blood fluorescence intensity of the INS-PMPC group was the lowest, about one-third lower than that of the INS and INS-PEG groups. Furthermore, the blood fluorescence intensity of the INS and INS-PEG groups rapidly decreased to baseline values ​​comparable to those before injection. Fluorescent signals were still detectable in blood samples from the INS-PMPC group 24 hours after injection, indicating that the circulation time of INS-PMPC was prolonged.

[0115] Example 5: INS-PMPC completely inhibits the occurrence of hypoglycemia.

[0116] I. Experimental Methods

[0117] To assess the risk of hypoglycemia induced by INS-PMPC in Example 1, healthy mice were injected with insulin, INS-PEG, and INS-PMPC from the example, respectively, at a dose of 5 U / kg, and blood glucose changes in the mice were monitored at preset time points.

[0118] II. Experimental Results

[0119] like Figure 9As shown in Figure A, INS-PMPC exhibited a slower rate of blood glucose reduction in the initial stage. Mice treated with INS and INS-PEG showed hypoglycemia starting at 0.5 h and 1 h, respectively. Hypoglycemia was defined as a glucose concentration below 3.3 mmol / L. Figure 9 As shown by the dashed line in A, the blood glucose level in the INS-PMPC group remained above the hypoglycemia indicator line throughout the experiment.

[0120] like Figure 9 As shown in B, calculate Figure 9 The area above the curve and below the dashed line in Figure A quantifies the blood glucose lowering effect. INS-PMPC completely prevented hypoglycemia, while there was no significant difference in hypoglycemia risk between the INS group and the INS-PEG group.

[0121] Example 6: Stability of INS-PMPC

[0122] Similar to other therapeutic proteins, insulin is prone to aggregation and degradation if not stored and transported under proper conditions. For example, at temperatures above 25°C, insulin degradation increases tenfold or more for every 10°C increase. This instability not only leads to the loss of its biological activity but can also trigger unexpected immune responses. High temperatures are a major factor impairing insulin stability during storage and transport, a problem exacerbated by rising diabetes rates in developing countries where cold chain facilities are often inadequate. In addition to high temperatures, proteins are frequently subjected to mechanical agitation during transport, leading to unfolding and irreversible aggregation.

[0123] I. Experimental Methods

[0124] After storing INS, INS-PEG, and INS-PMPC from Example 1 at 37°C for 10 days, streptozotocin (STZ)-induced type 1 diabetic mice were injected subcutaneously (sc) with INS and INS-PMPC before and after storage, respectively, according to the method in Example 3. The hypoglycemic efficacy within 6 hours was tested to evaluate the effect of high temperature on the bioactivity of the drugs.

[0125] Aggregation experiments were conducted on INS, INS-PEG, and INS-PMPC from Example 1 under physiological pH and temperature (pH 7.4 and 37°C) with continuous stirring for 60 h. The optical transmittance of the drugs at 540 nm was measured to evaluate the kinetics of drug aggregation; aggregation time was defined as a transmittance change greater than 10% of the initial transmittance value. Size changes of different insulin samples before and after reheat stability experiments were measured using a nanoparticle size analyzer.

[0126] To understand the reason for the good stability of INS-PMPC, the secondary structures of INS, INS-PEG and INS-PMPC were analyzed using a circular dichroism spectroscopy instrument.

[0127] II. Experimental Methods

[0128] like Figure 10 A and Figure 10 As shown in Figure B, the blood glucose curves of INS-PMPC stored at 37℃ for 10 days were basically the same as those before storage. The blood glucose lowering efficacy of INS stored at 37℃ for 10 days was significantly reduced compared to before storage.

[0129] like Figure 10 As shown in Figure C, INS aggregated after 10 hours of continuous stirring, while the transmittance of INS-PEG and INS-PMPC changed by no more than 3% throughout the 60-hour continuous stirring experiment.

[0130] like Figure 10 As shown in Figure D, the size of INS-PEG increased from 5.8±0.5 nm before the aggregation experiment to 52.3±5.8 nm after the experiment, while the size of INS-PMPC remained at a similar level before and after the aggregation experiment (6.3±0.2 nm and 6.1±0.2 nm). This indicates that INS-PEG exhibited some aggregation in the aggregation experiment, while INS-PMPC did not.

[0131] like Figure 10 E and Figure 10 As shown in Figure F, the secondary structure parameters of INS and INS-PEG remained essentially unchanged, except for the change in random coil structure (31% for INS and 29% for INS-PEG). The percentage of α-helix structure in INS-PMPC was 47% (significantly higher than 37% for both INS and INS-PEG), while the percentage of random coil structure in INS-PMPC was 22% (lower than 31% for INS and 29% for INS-PEG). This indicates that PMPC conjugation has a good stabilizing effect on the secondary structure of insulin, which is crucial for insulin storage and transport.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of an insulin conjugate in the preparation of a diabetes treatment drug, characterized in that, The drug mentioned is one that reduces the risk of hypoglycemia during diabetes treatment; The method for preparing the insulin conjugate includes the following steps: S1: 2-Bromoisobutyric acid N-hydroxysuccinimide ester reacts with insulin to specifically introduce the ATRP reaction site at the Lys B29 position of insulin, thus obtaining the large insulin initiator; S2: Dissolve insulin initiator, 2-methacryloyloxyethylphosphonic choline and N,N,N',N",N”-pentamethyldiethylenetriamine in Tris buffer, deoxygenate, then mix with cuprous bromide and polymerize to obtain zwitterionic polymer-insulin conjugate. The ratio of the insulin primer, 2-methacryloyloxyethylphosphorylcholine, N,N,N',N",N"-pentamethyldiethylenetriamine, Tris buffer, and cuprous bromide is 30 mg: 30 mg: 1.73 mg: 4 mL: 1.43 mg.

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