Anticoagulation agent and uses thereof

An anticoagulant, a technology for inhibiting coagulation, applied in the branch of hemostasis and in the field of hematology, which can solve the problem of wrong targeting of anticoagulants

Inactive Publication Date: 2008-08-06
贝克医药研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fibrin or fibrin degradation products can circulate in the blood, leading to mistargeting of anticoagulants in the circulation

Method used

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  • Anticoagulation agent and uses thereof
  • Anticoagulation agent and uses thereof
  • Anticoagulation agent and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1: Generation of single-chain antibody scFv 抗-LIBS and fusion construct scFv 抗-LIBS -TAP

[0117] Past literature has described the generation and functional characterization of hybridoma cell lines expressing monoclonal antibodies directed against LIBS epitopes on GPIIb / IIIa (Schwarz et al., JPET 2004;308:1002). Briefly, GPIIb / IIIa purified and eluted with RGD peptides was used as immunogen for producing hybridomas. Clones were screened with activated platelets and with immobilized GPIIb / IIIa saturated with RGD peptides. One of these clones, the monoclonal antibody (mAb) clone 145, was shown to bind to ADP-activated platelets as well as with RGD peptides (GRGDSP, BIOMOL Research Laboratories, Plymouth Meeting, PA), eptifibatide (Integrilin  , Essex Pharma, Muenchen, Germany), tirofiban (tirofiban) (Aggrastat  , MSD, Whitehouse Station, NJ) and abciximab (abciximab) (ReoPro  , Eli Lilly & Co, Indianapolis, IN) increased binding of pre-incubated platelets...

Embodiment 2

[0119] Example 2: Expression and purification of scFv constructs in E. coli

[0120] Escherichia coli (TG1) cells were transformed with the above pHOG21 plasmid and single colonies from streaked agar plates were grown at 37°C in 500 mL flasks in LB medium containing 100 μg / mL ampicillin and 100 mM glucose. The culture was shaken at 200 rpm for approximately 4-6 hours until an OD (600 nm) of -0.8 was reached. Bacteria were pelleted by centrifugation at 5000 rpm for 10 min at 4°C, and resuspended with LB medium containing 100 μg / ml ampicillin and 0.4 M sucrose. IPTG was added to a final concentration of 0.25 mM to induce scFv production and incubated at room temperature (22-24° C.) at 200 rpm for 16-20 hours. For purification of soluble proteins from whole cell extracts, bacteria were harvested by centrifugation at 5000 rpm for 10 min at 4°C. Pellets of bacteria were resuspended in 5mL 1X BugBuster  (Novagen, Madison, USA) solution / g bacterial mass, and incubated at room te...

Embodiment 3

[0121] Example 3: In vitro functional properties of scFv anti-LIBS-TAP

[0122] blood products

[0123] Human blood was collected from healthy volunteers by venipuncture using a 21-gauge butterfly needle and anticoagulated with citrate. Platelet rich plasma was obtained by centrifugation at 100 xg for 10 min in a plastic tube at room temperature in a centrifuge (GS-6R centrifuge, Beckmann Coulter, Gladesville, NSW, Australia).

[0124] Mouse blood was collected from C57BL / 6 mice by intracardiac puncture using a 27-gauge needle and anticoagulated with unfractionated heparin (20 U / mL). In a volume of 50 μl, use 1 mL of modified Tyrode's buffer (150 mM NaCl, 2.5 mM KCl, 1.2 mM NaHCO 3 , 2mM MgCl 2 , 2mM CaCl 2 , 0.1% BSA, 0.1% glucose) were resuspended and centrifuged at 1300×g for 5 min. The supernatant was discarded, and the pellet was resuspended with 1 mL of modified Tyrode's buffer.

[0125] Flow Cytometry

[0126] Citrate-containing human whole blood was diluted 1 / ...

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Abstract

The present invention provides an anticoagulant agent including a first element capable of inhibiting coagulation and a second element capable of targeting an activated platelet wherein upon administration of the agent to a subject the second element directs the first element to the activated platelet. Also provided is a probe for detecting a blood vessel abnormality including (a) a binding element capable of targeting an activated platelet and (b) a label. Applicant has shown that agents and probes directed to activated platelets are useful in the diagnosis and therapy of coagulation disorders.

Description

technical field [0001] The present invention relates to the field of hematology and in particular to the sub-field of hemostasis. More specifically, the present invention relates to: 1) agents that inhibit blood coagulation in mammals, and the use of these agents in the treatment and prevention of diseases such as stroke, myocardial infarction, and deep vein thrombosis; 2) in the following clinical situations ( Probes such as thrombosis, thrombotic emboli, and unstable plaques) can diagnose and characterize activated platelets. Background technique [0002] The body's ability to control blood flow after vascular injury is critical to continued survival. The process of blood clotting and the subsequent dissolution of the clot following the repair of damaged tissue is known as hemostasis. Hemostasis consists of a series of events that occur in a fixed order following loss of vascular integrity. [0003] The initial stage of this process is the narrowing of blood vessels, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K39/395A61P7/02
Inventor K・彼得
Owner 贝克医药研究所
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