Pharmaceutical composition containing human urine kininogenase for treating cerebral infarction

A technique for urokininogenase and composition, which is applied in the fields of biochemistry and medicinal chemistry, can solve the problems such as unreported research on human urokininogenase, and achieve the effects of less side effects, prevention of cerebral infarction, and reduced side effects

Active Publication Date: 2012-01-11
GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] It can be found that there are two bands of human urinary kallikreinase in SDS-PAGE electrophoresis, and the two substances can be effectively separated to obtain a single band of human urinary kallikrein in SDS-PAGE electrophoresis.

Method used

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  • Pharmaceutical composition containing human urine kininogenase for treating cerebral infarction
  • Pharmaceutical composition containing human urine kininogenase for treating cerebral infarction
  • Pharmaceutical composition containing human urine kininogenase for treating cerebral infarction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 SDS-PAGE electrophoresis assay shows the preparation of the human urinary kininogenase of single band

[0043] It is prepared by the following steps (benzaprimidine affinity chromatography):

[0044] 1. Pump 10 tons of fresh male urine into the stirring tank, adjust the pH to 4.5-5.5, add 300kg of deacetylated chitin for adsorption, and adjust the pH to 7.0-9.0 with 10% ammonium sulfate solution to elute; the eluent Use diatomaceous earth as the filter medium, perform suction filtration, drain the salting-out solution completely, and 1 kg of the drained product is the crude product of human urinary kallikreinase;

[0045] 2. Pour 150kg of crude human urinary kininogenase into a stirring tank, add 750L of 0.15M EDTA solution to dissolve, adjust the pH to 9.0, and stir until the crude product is completely dissolved; centrifuge at 3000 rpm for 20 minutes, take the Clear, filter the membrane of 0.45 μm, ultrafiltration obtains ultrafiltrate 45L, therefrom get...

Embodiment 2

[0058] Example 2 Determination of the structure of human urinary kininogenase

[0059] Amino Acid Composition Analysis of Human Urinary Kallikrein:

[0060] 1. Instrument name and model: Hitachi 835-50 high-speed amino acid analyzer

[0061] 2. Sample preparation:

[0062] Accurately measure the sample into the hydrolysis tube, add 6mol / L hydrochloric acid, hydrolyze at 110°C for 24 hours, cool to constant volume, filter, evaporate to remove excess hydrochloric acid, constant volume with 0.02mol / L, and analyze on the machine.

[0063] 3. Determination conditions:

[0064] Ion exchange column: 2.6×150mm; resin specification: No.2619; column temperature: 53°C; pump flow rate: 0.225ml / min; pump pressure: 90kg / cm2, eluent IPH—1, 2, 3, 4; analysis Time: 72min; injection volume: 50μl.

[0065] 4. Measurement results: basically consistent with the theoretically derived values ​​(Table 3).

[0066] Table 3 Analysis results of amino acid composition

[0067]

[0068]

[006...

Embodiment 3

[0071] Example 3 Study on Enzymatic Properties of Human Urinary Kinikreinogenase

[0072] Take 2 small test tubes (2×20cm), add 0.2ml of human urinary kininogenase solution to each, add 4.0ml of 0.2mol / L Tris buffer solution (pH8.0), mix well, and incubate at 37°C for 5 minutes. Add 0.4ml of 50% acetic acid solution to the first tube, add 0.4ml of substrate solution (S-2266 (HD-Val-Leu-Arg-PnA·2HCl), 1.5mM) to the second tube, mix well, and React in a water bath at ±0.5°C for 15 minutes, then add 0.4ml of substrate solution to the first tube, add 0.4ml of 50% acetic acid solution to the second tube, measure at a wavelength of 405nm, and use the first tube as a blank to measure the absorption value A, A value should be controlled between 0.1 and 0.2. Substitute the value of A into the following calculation:

[0073] PNA U / ml=173.6×A×T / 1000 (T is the dilution factor)

[0074] 1PNA U (p-nitroaniline unit): equivalent to human urinary kininogenase when H-D-Val-Leu-Arg-p-nitroni...

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Abstract

The present invention relates to the application of human urokininogenase with single SDS-PAGE electrophoresis strip in serving as active component of medicine composition for treating acute cerebral infarction. The human urokininogenase with single SDS-PAGE electrophoresis strip separated from human urea for the first time has obvious effect of treating acute cerebral infarction. The human urokininogenase of the present invention is normally in the form of medicine composition, such as freeze dried powder for injection or liquid injection.

Description

technical field [0001] The present invention relates to the fields of biochemistry and medicinal chemistry. Specifically, the present invention relates to the preparation and enzymatic properties of a single-component human urokininogenase (Human Urinary Kallidinogenase), a pharmaceutical composition with a single-component human urokininogenase as an active component and the Application research of pharmaceutical composition in the treatment of acute cerebral infarction. Background technique [0002] In 1909, Abelous and Bardier observed that a water-insoluble component of human urine was injected into anesthetized dogs, and their blood pressure decreased. In 1929, Frey and Kraut isolated the substance from urine and found it in the pancreas. There is a higher concentration, and it is considered that the pancreas is its main synthesis site, and it is named "kallikrein". The protease function of kallikrein was discovered in 1937, and it was considered as a new protein, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/49A61P9/10C12N9/72
Inventor 傅和亮苗丕渠王晓岩许文勤郑少亮
Owner GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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