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PEG-modified medicinal kininogenase and preparation method and application thereof

A kininogenase and reaction technology, applied in the field of polyethylene glycol modification, can solve the problems of repeated administration, short half-life, redness and swelling at the injection site, and achieve high biological activity and efficacy, reduced immunogenicity, The effect of half-life extension

Active Publication Date: 2018-03-06
ZONHON BIOPHARMA INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the commercially available porcine pancreatic kininogenase injection is used, the injection site often has symptoms of redness, swelling, and pain, and the applicant also found that the rat died after the drug was administered in the rat acute ischemic stroke experiment. Therefore, the applicant speculates that there are ingredients with relatively large side effects in existing marketed products
In addition, both of these two types have problems of poor biological stability, short half-life, repeated administration, and immunogenicity.
[0004] In addition, in order to overcome the common disadvantages of poor biological stability, short half-life in vivo and immunogenicity of such pharmaceutical proteins and polypeptides, genetic engineering and chemical modification are usually used to modify them to improve the quality of pharmaceutical proteins and peptides. In vivo biological stability of peptides, prolonging half-life, reducing or eliminating immunogenicity
However, in view of the structural specificity of each protein, there is a big gap in the application of various methods and their actual effects.

Method used

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  • PEG-modified medicinal kininogenase and preparation method and application thereof
  • PEG-modified medicinal kininogenase and preparation method and application thereof
  • PEG-modified medicinal kininogenase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Separation and purification of porcine pancreatic kininogenase single components KLK1b, KLK1a and KLK1c

preparation example 1

[0082] Porcine pancreatic kininogenase (from Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd.) was diluted to 6 mg / mL with liquid A, and purified by ion-exchange chromatography. Purified chromatography conditions: ion exchange medium (QFF), A solution: 50mM Tris-HCl (pH9.0), B solution: 50mM Tris-HCl (pH9.0) containing 1M NaCl; flow rate 10mL / min, The detection wavelength is 280nm.

[0083] Loading: the above dilution of porcine pancreatic kininogenase was bound to a QFF ion exchange column.

[0084] Equilibration: Flush 5 column volumes with solution A.

[0085] Elution: The mobile phase ratio is 12% B, 88% A, after elution of 10 column volumes, gradient elution is performed. Gradient conditions were 12% B to 30% B, 30 column volumes.

[0086] Collection: collect part of the eluted product before peak 1 as KLK1b, such as Figure 1a Shown; Partial elution product after peak 2 was collected as KLK1a, as Figure 2a As shown, the partially eluted product after peak 3 w...

Embodiment 2

[0098] Example 2 Detection of the purity of isolated products of porcine pancreatic kininogenase single components KLK1b, KLK1a and KLK1c

[0099] SDS-PAGE detection

[0100] (1) Electrophoresis: After preparing a 12% polyacrylamide gel, load and run. Run at 80V for 30min, and change to 150V for 30min after the bromophenol blue indicator runs out of the stacking gel.

[0101] (2) Coomassie Brilliant Blue staining: After electrophoresis, the gel was peeled off and placed in Coomassie Brilliant Blue staining solution for 30 minutes of staining.

[0102] (3) Decolorization: after dyeing, put the glue in the decolorization solution and decolorize overnight.

[0103] Analysis results such as Figure 4 shown. In terms of yield, the yield of KLK1b can reach more than 85%, the yield of KLK1a can reach 60%, and the yield of KLK1c is lower, but after separation and purification, the purity of each component of KLK1 can reach 95%. Therefore, when the purification method in this exam...

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Abstract

The invention relates to PEG-modified medicinal kininogenase and a preparation method and application thereof. The kininogenase does not contain lowly-glycosylated KLK1, and the lowly-glycosylated KLK1 is a stripe with the lowest molecular weight in three stripes during SDS-PAGE protein electrophoresis of porcine pancreas-derived KLK1; PEG adopts a structural general formula shown as a formula (1)or a formula (2). According to the pegylated kininogenase, on one hand, component nonuniformity caused by different glycosylation modifications of the kininogenase is eliminated, so that the purity,the stability, the bioactivity and the medicinal efficacy are improved; on the other hand, after PEG modification of the kininogenase, the half-life period is significantly prolonged, the immunogenicity is significantly reduced, and side effects of a raw medicine are effectively reduced.

Description

technical field [0001] The invention relates to the polyethylene glycol modification of protein drugs, especially the polyethylene glycol modification of kininogenase with more definite components, its preparation method and its application in drug preparation. Background technique [0002] Kininogenase, also known as kallikrein (kininogenase) or kallidinogenase (kallidinogenase), it is a kind of serine protease, exists in various tissues and biological fluids, and catalyzes macromolecular precursors (kinin Original, Kininogen) releases biologically active peptides (kinin, Kinin). The physiological effects of kininogenase include capillary and arterial vasodilation and increased permeability, increasing the blood supply of coronary arteries, brain, and retina. , vasospasm, thromboangiitis obliterans, frostbite and trauma embolism also have therapeutic effect. At the same time, kininogenase can also be used as an activating factor to activate plasminogen into plasmin, hydro...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N9/96A61K38/48A61K47/60A61P13/12
CPCA61K38/00C12N9/6424C12N9/96
Inventor 马永王俊
Owner ZONHON BIOPHARMA INST
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