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New anti-embolism medicine using small peptide arginine-glycine-aspartic acid (RGD) as leading compound

A compound and leading technology, applied in the field of antithrombotic linear small peptide molecules, to achieve the effect of small molecular weight, strong platelet aggregation activity, and no immunogenicity

Inactive Publication Date: 2004-11-17
牛勃
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the RGD sequence on fibrinogen plays an important role in organisms, as a drug, only the natural RGD sequence cannot achieve appropriate strength and selectivity

Method used

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  • New anti-embolism medicine using small peptide arginine-glycine-aspartic acid (RGD) as leading compound
  • New anti-embolism medicine using small peptide arginine-glycine-aspartic acid (RGD) as leading compound
  • New anti-embolism medicine using small peptide arginine-glycine-aspartic acid (RGD) as leading compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: H 2 N(CH 2 ) 7 The structure of -CO-Gly-Asp-Trp ( figure 1 ), its synthesis, purification, identification are as follows:

[0017] Using the 9-fluorenylmethyloxycarbonyl (Fmoc) protocol, Wang resin was used initially, and the peptide chain was extended from the C-terminal to the N-terminal according to the tetrapeptide sequence, and BOP / HOBt (1-hydroxyphenylacryltriazole) was added to each condensation step ) as a condensing agent, the Fmoc protecting group is removed with 20% hexahydropyridine in DMF (dimethylformamide) solution after condensation. After the peptide chain was synthesized, 95% TFA (trifluoroacetic acid) was used to stir at room temperature to cleave the peptide chain from the resin while removing the side chain protecting group, and the ninhydrin reagent was used to detect the completeness of deprotection and condensation. The synthetic peptide was purified by RP-HPLC (reverse-phase high-pressure liquid chromatography) C18 reverse-phase c...

Embodiment 2

[0018] Embodiment 2: Activity and stability experiment of antithrombotic effect

[0019] Blood was collected through the cubital vein on an empty stomach in the early morning, anticoagulated with 3.8% sodium citrate (the volume ratio of blood to anticoagulant was 9:1), centrifuged at 1000r / min for 8min at room temperature, and the upper plasma was taken as platelet-rich Plasma (PRP platelet-rich plasma), isolated PRP. The remaining blood was centrifuged at 3000r / min for 10min, and the upper plasma was collected to obtain platelet-poor plasma (PPP platelet-poor plasma). PRP was adjusted with PPP so that the number of platelets was about 250,000 / μL. Take 400 μL of PRP, add 0.9% NaCL (negative control) and different concentrations of RGDS (100, 80, 60, 30uM positive control) / small peptide (see Example 1) (10, 5, 2, 1uM final concentration) 50ul respectively, The platelet aggregation rate was measured by turbidimetry (the measuring instrument was Packs-4 platelet aggregation inst...

Embodiment 3

[0020] Embodiment 3: the specific research of antithrombotic effect

[0021] (1) Under aseptic conditions, take the umbilical cord of a fetus with normal delivery or caesarean section, squeeze out the blood in the umbilical cord vessel, put it into cold sterile D-hank's solution, and culture the umbilical cord vein endothelial cells within 3 hours. (2) Primary culture of human umbilical vein endothelial cells (HUVEC). (3) Cell de-adhesion experiment: HUVEC (7.5×10 4 / well) 1 mL was inoculated in a 24-well culture plate and cultured for 2 days to form a monolayer. Rinse twice with serum-free medium, discard the medium, add (see Example 1) (2.3, 5.5, 11, 22mM) 200uL into a 24-well culture plate, incubate together at 37°C for 4h, use serum-free The culture medium was rinsed 3 times to wash away the detached cells. (4) MTT method: Add 20uL of MTT to each well, and place the 24-well culture plate in CO 2 Incubator (37°C, 5% CO 2 ) for 4 h, discard the MTT supernatant, add 300 ...

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Abstract

The present invention relates to a new kind anti-embolism linear small-peptide molecule developed based on the protein structure and function research. It has the amino acid sequence of X-Gly-Asp-Y, powerful activity of inhibiting thrombocyte aggregation, high stability, good specificity and selectivity, small molecular weight, to immunogenicity, simple structure, low cost and easy synthesis. Owing to the effective binding with thrombocyte GPIIbIIIa and competitive inhibition of the combination between fibrinogen and thrombocyte, the small peptide may be used as thrombocyte inhibitor in basic research and clinical application.

Description

Technical field: [0001] The invention relates to a novel high-efficiency, stable and specific antithrombotic linear small peptide molecule designed and synthesized on the basis of protein structure and function research, which can be used in blood biology, pharmacodynamics and clinical treatment. Background technique: [0002] Thrombotic diseases such as myocardial infarction and cerebral infarction have become the first cause of death in my country, but the existing antiplatelet drugs such as aspirin and ticlopidine only affect a certain link of platelet activation, while other methods still Can cause thrombosis. Therefore, it is not difficult to understand that many patients taking existing antiplatelet drugs can still develop thromboembolism and develop serious complications. Finding new efficient and complete antiplatelet drugs has become an important issue. The RGD sequence on the α-chain of fibrinogen specifically binds to platelet glycoprotein (GP) IIb / IIIa receptors...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/06A61P7/02C07K5/08
Inventor 牛勃王晓霞杨琦常冰梅杨涛李乐工韩锐阎占清郭勇
Owner 牛勃
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