CN-115845076-B - Zwitterionic polymer-insulin conjugate and preparation method and application thereof

Abstract

The invention discloses a zwitterionic polymer-insulin conjugate and a preparation method and application thereof. The preparation method comprises the steps of reacting 2-bromoisobutyric acid N-hydroxysuccinimide ester with INS, introducing an ATRP reaction site at the position of INS Lys B29 to obtain INS-Br, mixing INS-Br, MPC and a catalyst, deoxidizing, mixing CuBr, fully polymerizing, using CuBr and PMDETA as a catalytic system, initiating zwitterionic monomer MPC polymerization by using INS-Br, and obtaining INS-PMPC by dialysis and cation chromatography column purification technology. Compared with INS and INS-PEG, the INS-PMPC provided by the invention has longer blood sugar control time under the same dosage, meanwhile, the risk of hypoglycemia is obviously reduced, the stability under room temperature and mechanical stirring is high, the storage is durable, the INS-PMPC is suitable for areas lacking cold chain facilities, and the INS-PMPC is a long-acting insulin medicament with higher safety.

Inventors

• ZENG ZHIPENG
• CHEN YONGMING

Assignees

• 中山大学

Dates

Publication Date

20260508

Application Date

20221122

Claims (1)

1. 1. An application of an insulin conjugate in preparing a medicament for treating diabetes, which is characterized in that the medicament is a medicament for reducing the risk of hypoglycemia in the treatment process of the diabetes; the preparation method of the insulin conjugate comprises the following steps: s1, reacting N-hydroxysuccinimide ester of 2-bromoisobutyric acid with insulin, and specifically introducing an ATRP reaction site at the position of insulin Lys B29 to obtain an insulin macroinitiator; S2, dissolving an insulin macroinitiator, 2-methacryloyloxyethyl phosphorylcholine and N, N, N' -pentamethyl diethylenetriamine in a Tris buffer solution, deoxidizing, mixing cuprous bromide, and fully polymerizing to obtain a zwitterionic polymer-insulin conjugate; The ratio of the insulin macroinitiator, the 2-methacryloyloxyethyl phosphorylcholine, the N, N, N ', the N' -pentamethyldiethylenetriamine, the Tris buffer and the cuprous bromide is 30mg:30mg:1.73mg:4mL:1.43mg.

Description

Zwitterionic polymer-insulin conjugate and preparation method and application thereof Technical Field The invention relates to the technical field of biological medicine, in particular to a zwitterionic polymer-insulin conjugate and a preparation method and application thereof. Background Diabetes is a chronic metabolic disease characterized by hyperglycemia, which can lead to serious complications such as kidney disease, cardiovascular disease, foot injury, etc. Diabetes affects more than 4 million people worldwide as a global public health epidemic. Insulin is the main drug for treating type 1 diabetes, and its discovery radically improves the treatment and prognosis of diabetes, changing it from an incurable disease to a controllable chronic disease. However, like many protein drugs, insulin has problems such as short half-life and poor stability in storage and transport. To achieve the hypoglycemic goal, diabetics need to inject insulin multiple times per day, which severely affects their quality of life. In addition, insulin needs to be stored and transported under low temperature conditions, since high temperature causes aggregation and degradation thereof, which not only impairs its biological activity, but may also cause unexpected immune response. In addition, the need for low temperature storage of insulin, the demanding transportation requirements limit its use in areas where cold chain facilities are lacking, such as disaster areas and rural clinics in developing countries. To ameliorate the deficiencies of insulin, a great deal of effort and resources have been devoted to developing long acting insulin with high stability for decades. Genetic engineering is a method of producing long acting insulin analogues. For example, the half-life of insulin glargine (sirofine-amont) is extended to about 13 hours. Precise manipulation of part of the amino acid sequence and structure changes the isoelectric point, causing precipitation of such insulin analogues at the site of subcutaneous injection, thereby delaying dissolution and absorption. However, the engineering mutagenesis procedure is very complex. More importantly, degradation of insulin glargine at the injection site reduces its bioavailability and induces an unexpected immune response. In addition to mutagenesis strategies, coupling to water-soluble polymers has become a popular approach to improve protein circulation time and shelf life stability in vivo. To date, relatively few polymers have been used for insulin coupling, including mainly polyethylene glycol (PEG) and sugar-containing polymers. While coupling with these polymers can improve the pharmacokinetics and stability of insulin, this tends to affect its activity. PEG is also easily degraded by oxidation when applied to biological media. Furthermore, while PEGylation is generally stated to reduce the immunogenicity of proteins, more and more recent reports have shown that PEG antibodies appear after injection of PEG-protein conjugates. PEG antibodies not only impair the efficacy of PEG-proteins, but may also cause severe allergic reactions. These drawbacks hamper the further development of PEG. There is therefore a need to develop new insulin-polymer conjugates that do not affect insulin activity but improve their pharmacokinetic profile and storage stability. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides a zwitterionic polymer-insulin conjugate, and a preparation method and application thereof. The first object of the present invention is to provide a method for preparing an insulin conjugate. A second object of the present invention is to provide an insulin conjugate. A third object of the present invention is to provide an application of the insulin conjugate in preparing a medicament for treating diabetes. A fourth object of the present invention is to provide a medicament for treating diabetes. In order to achieve the above object, the present invention is realized by the following means: A preparation method of a zwitterionic polymer-insulin conjugate comprises the following specific steps: S1, reacting N-hydroxysuccinimide ester of 2-bromoisobutyric acid with insulin to obtain an insulin macroinitiator; S2, mixing the insulin macroinitiator, the 2-methacryloyloxyethyl phosphorylcholine and the catalyst in the step S1, deoxidizing, mixing cuprous bromide, and fully polymerizing to obtain the zwitterionic polymer-insulin conjugate; The catalyst is 1,1,4,7,10,10-hexamethyltriethylenetetramine, 2 '-bipyridine or N, N, N' -pentamethyldiethylenetriamine. Preferably, na 2CO3 buffer containing insulin and DMSO solution containing N-hydroxysuccinimide ester of 2-bromoisobutyric acid are mixed and stirred for reaction to obtain the insulin macroinitiator. Preferably, the molar ratio of insulin to N-hydroxysuccinimide ester of 2-bromoisobutyric acid is 1:1 to 1.5. More preferably, the molar ratio of insulin to N-hydroxysuccinimide ester of