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New monomeric insulin, its medicinal composition and prepn process

一种胰岛素、组合物的技术，应用在医学和药物学领域，能够解决难大规模生产、工艺流程复杂、化学副反应等问题

Inactive Publication Date: 2006-03-29
SHANGHAI C A S SHENGLONGDA BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process is difficult to produce on a large scale due to the following main disadvantages: (1) small peptides such as GFFY(But)Obut need to be chemically synthesized; (2) when trypsin enzymatically transpeptides, the yield is within 80%; (3) ) After enzymatic transpeptidation, the intermediate needs to be treated with strong acid, such as trifluoroacetic acid (TFA) to remove the protecting group; (4) The process is complicated and there are chemical side reactions

Method used

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  • New monomeric insulin, its medicinal composition and prepn process
  • New monomeric insulin, its medicinal composition and prepn process
  • New monomeric insulin, its medicinal composition and prepn process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Preparation of DOI

[0077] 613 mg of dezincified insulin was dissolved in 127 ml (5 mg / ml) of 0.05mol / L borax solution (containing 0.001M CaCl 2 ), adjust pH=8.9. Add 24.5 mg of crystalline trypsin (the mass ratio of enzyme to substrate is 1: 25), stir and react in a water bath at 37°C for 3 hours, purify through a DEAE-Sephadex A25 ion exchange column, and collect the main peak ( figure 1 ) was dialyzed against water, freeze-dried, and then desalted with Sephadex G25 to obtain DOI.

Embodiment 2

[0078] The preparation of embodiment 2 GFFYK (Boc) Obut

[0079] 2.1 Preparation of Boc-Phe-Osu

[0080] Dissolve 3.18g Boc-Phe (12mmol) and an equimolar amount of HOsu in 15ml THF, and place the reaction bottle in a -5°C salt ice bath for 10 minutes; another equimolar amount of DCCI was dissolved in 2ml THF, and slowly added dropwise to the reaction In the bottle, stir at -5°C at the same time; after adding liquid, continue to stir at -5°C for 2 hours, and white DCU precipitates appear. The reaction bottle was sealed and placed at 4°C overnight, filtered through three layers of filter paper, and the filtrate turned into a white solid after rotary evaporation. 15ml of isopropanol was added to the solid and heated to 90°C to form a yellow solution, which was recrystallized at room temperature. After suction filtration, washing and drying, the white crystals obtained 2.5 g of the product, with a melting point of 138-140° C., and thin-layer chromatography showed homogeneity, si...

Embodiment 3

[0095] Example 3B 27 Preparation of K-DTrI

[0096] Take 0.237g (0.172mmol) Gly-Phe-Phe-Tyr-Lys(Boc)Obut, add 0.26ml DMSO, heat below 50°C until completely dissolved, and place in a 37°C water bath to keep warm; take another DOI 86mg (0.0172mmol), Slowly add to the reactor to dissolve all, then add 1.82ml of 1-4 butanediol preheated to 37°C, the reaction liquid is slightly muddy, then add deionized water preheated to 37°C, the reaction liquid becomes clear, add 5 microliters of N-methylmorpholine was used to adjust the pH to an accurate value of 6.5 (determined with a pH meter). After adding 7.8mg of TPCK-Trypsin, the temperature of the water bath was slowly lowered to 30°C, and 4.5g of TPCK-Trypsin were added after 2 hours and 4 hours, and the enzyme amount was one-fifth of the DOI amount. After 20 hours, the reaction solution was moderately turbid, and 1.1 ml of glacial acetic acid and a small amount of 1N HCl were added to adjust the pH to 3 to terminate the enzymatic rea...

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Abstract

The present invention provides new insulin strand B, monomeric insulin containing strand B, medicinal composition containing the monomeric insulin and their preparation process and use. The insulin analog B27K-DTrI sample of high purity is obtained via enzymatic semi-synthesis process and is proved to have the property of monomeric insulin and overall insulin activity 80 % of that of normal insulin. The monomeric insulin precursor is secreted and expressed in yeast expressing system and enzyme cut to obtain B27K-DTrI, and the enzymatic peptide converting step is omitted to raise the final yield and suit for industrial production.

Description

technical field [0001] The present invention relates to the fields of medicine and pharmacy. More specifically, the present invention relates to a novel insulin B chain, a monomeric insulin containing the B chain, a pharmaceutical composition containing the monomeric insulin, and its preparation method and use. Background technique [0002] The incidence of diabetes is increasing year by year, and it can cause death from cardiovascular disease. It is one of the major diseases threatening human health today. Hyperglycemia is the cause of various diabetic complications, and strictly controlling the blood sugar concentration within the normal range 24 hours a day is the most critical factor to prevent and delay the complications of late diabetes. [0003] Insulin has been an irreplaceable drug since it was used to treat diabetes in the 1920s. However, the secretion of insulin in a normal human body is regulated by the blood sugar concentration. Generally, the blood insulin co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/62A61K38/28C12N15/63C12N15/17C12P21/02A61P3/10
CPCC07K14/62A61P3/10
Inventor 张友尚丁金国崔大敷石嘉豪
Owner SHANGHAI C A S SHENGLONGDA BIOTECH GRP
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