Anti-platelet thrombolysin and preparation method thereof

An anti-platelet and anti-platelet aggregation technology, applied in the field of biomedicine, can solve the problems of short plasma half-life and bleeding, and achieve the effects of small bleeding tendency, high yield and obvious effect.

Active Publication Date: 2010-09-22
ZHAOKE PHARMA HEFEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the anticoagulant drugs are thrombin inhibitors. Due to their inability to directly dissolve thrombosis and the non-specificity of the anticoagulant effect, they are prone to bleeding side effects or other toxicity; the thrombolytic effect of thrombolytic drugs is rapid and direct, and the disadvantage lies in the plasma half-life Short, long-term use often causes bleeding, and the probability of reinfarction after thrombolysis is higher

Method used

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  • Anti-platelet thrombolysin and preparation method thereof
  • Anti-platelet thrombolysin and preparation method thereof
  • Anti-platelet thrombolysin and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Embodiment 1: Preparation of high-purity anti-platelet thrombolysin

[0060] 1. Treatment of snake venom

[0061] Accurately weigh 5 g of Agkistrodon halys venom purchased from Huizhou Venom Research Institute in Huangshan City, and dissolve it in a refrigerator at 4°C with 0.02mol / L Tris-HCl buffer solution at pH 8.0 for 6-12 hours, and then dissolve it at 4000rr / min centrifugation, centrifugation twice, each centrifugation 15min, the supernatant was collected for later use.

[0062] 2. Preliminary purification to obtain antigens for the preparation of monoclonal antibodies

[0063] (1) Anion exchange chromatography

[0064] Take the supernatant obtained from dissolving and centrifuging the crude poison in step 1 and load it on the equilibrated anion exchange chromatography DEAE column at a flow rate of 3.0ml / min. Miscellaneous proteins that cannot be adsorbed by the chromatographic medium, the elution time is 360min, and the elution flow rate is 3ml / min; then add ...

Embodiment 2

[0079] Example 2: Drug efficacy evaluation of the anti-platelet thrombolysin of the present invention

[0080] 1. Activity determination

[0081] One unit of activity of anti-thrombolysin is defined as: the amount of anti-thrombolysin that can inhibit the percentage of platelet aggregation induced by ristocetin to 100±5.

[0082] The potency determination method adopts the platelet aggregation method, as follows:

[0083] (1) Sources of platelet-rich plasma PRP and platelet-poor plasma PPP: Anticoagulant blood from blood donors with normal platelet count was taken. The anticoagulant was 3.8% sodium citrate, and the ratio of the anticoagulant to whole blood was 1:9. Centrifuge at 800r / min for 5min, absorb the supernatant to obtain platelet-rich plasma (PRP), then centrifuge at 3500r / min for 10min, absorb the supernatant to obtain platelet-poor plasma (PPP), and adjust the platelet count of PRP to (150 ~200)×10 9 .

[0084] (2) Blank tube measurement: Take a siliconized turb...

Embodiment 3

[0096] Embodiment 3: High-purity anti-platelet thrombolysin crude drug content detection method

[0097] In order to detect the content of anti-thrombolysin in serum so as to be used in pharmacokinetic tests, a set of methods for detecting the content of anti-thrombolysin raw materials by double-antibody sandwich method was established. The detection sensitivity of this method can reach 1ng / ml .

[0098] The specific detection method is as follows:

[0099] 1. Coating a monoclonal antibody: properly dilute the monoclonal antibody prepared in Example 1 of the present invention with 0.05M pH9.6 carbonate buffer, add 0.1 ml, overnight at 4°C or for 18-24 hours. The next day, the solution in the well was discarded, and washed 3 times with washing buffer for 3 minutes each time (abbreviated as washing, the same below).

[0100] 2. Adding samples: add the sample to be tested or a properly diluted standard reference product into the reaction wells, and simultaneously make blank we...

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Abstract

The invention relates to the field of medicaments, and in particular provides anti-platelet thrombolysin. The anti-platelet thrombolysin of the invention consists of two peptide chains, namely an alpha chain and a beta chain, wherein an amino acid sequence of the alpha chain is shown as SEQ ID NO.1; the amino acid sequence of the beta chain is shown as SEQ ID NO.2; and the specific activity of the anti-platelet thrombolysin is greater than 1,000 activity units in each milligram of protein. Platelet membrane glycoprotein GPIb protein of a key component is aggregated mainly by mediated platelets; and the combination of the platelet membrane glycoprotein GPIb protein and blood plasma vWF is interrupted so as to prevent platelets from adhering to a vascular endothelial cell wall so that the aggregation of the platelets is inhibited. The invention also provides a method for purifying the anti-platelet thrombolysin, which has the characteristics of high specificity, high yield and short period. Pharmacology, toxicology, potency, pharmacokinetic experiments show that the anti-platelet thrombolysin of the invention is a safe and high-efficiency medicament for inhibiting the aggregation of the platelets and has a broad clinical application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an antithrombolysin and a preparation method thereof. Background technique [0002] With the increase of risk factors such as population aging and poor eating habits, the morbidity and mortality of cardiovascular and cerebrovascular diseases are increasing day by day. Human life expectancy and quality of life. [0003] The prevention and treatment of cardiovascular and cerebrovascular diseases has always been a research hotspot in medicine and pharmacy. Antithrombotic drugs are generally divided into three types: anticoagulant drugs, thrombolytic drugs, and anti-platelet aggregation drugs. Anticoagulant drugs are mostly thrombin inhibitors, which can easily cause bleeding side effects or other toxicity due to their inability to directly dissolve thrombosis and non-specific anticoagulant effects; the thrombolytic effect of thrombolytic drugs is rapid and direct, and the di...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K1/22C07K1/18A61K38/17A61P7/02A61P9/10
Inventor 李小羿戴向荣杨中强许贞玉张国辉钱芳方丽
Owner ZHAOKE PHARMA HEFEI
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