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Anticoagulation agent and uses thereof

a technology of anticoagulation agent and anticoagulation agent, which is applied in the field of haemostasis, can solve the problems of reducing the rate of fibrin clot dissolution, affecting the activation of plasminogen, and the inability of the body to control the circulating level, so as to prevent or treat coagulation disorders and achieve higher affinity binding

Inactive Publication Date: 2009-12-24
BAKER IDI HEART & DIABETES INST HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides an anticoagulant agent that targets activated platelets to inhibit coagulation without excessive bleeding. The agent includes a first element that inhibits coagulation and a second element that targets activated platelets. The second element is a single-chain antibody that recognizes a specific clot-specific target, ligand-induced binding sites (LIBS). The first element is a highly potent direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). The invention also provides pharmaceutical compositions and methods for treating or preventing coagulation disorders, as well as diagnostic methods for screening compounds and identifying the presence of thrombosis and unstable plaques."

Problems solved by technology

This leads to an impairment of plasminogen activation, thereby reducing the rate of fibrin clot dissolution (i.e. fibrinolysis).
The inability of the body to control the circulating level of active thrombin would lead to dire consequences.
Thus, from the above it can be seen that the physiological mechanisms involved in coagulation are exceedingly complex, and it will be appreciated that great difficulty exists in designing or identifying agents that are capable of safely modulating the many inter-related pathways in coagulation.
Clotting must be very strictly regulated because even one inappropriate clot can have fatal consequences.
Indeed, blood clots are the leading cause of strokes and heart attack, the two major causes of human death.
Heparin is relatively non-toxic, however heparin overdose or hypersensitivity may result in excessive bleeding.
Coumarins and 1,3-indandiones (see infra) have a further disadvantage in that they interact with certain drugs.
The chief disadvantage of indandiones is their side effects.
Despite the overall benefits achieved, the currently used therapeutic anticoagulants are also a major source of mortality and morbidity, caused by limitations in efficacy and even more so by bleeding complications.
However, it appears that therapeutic anticoagulation inevitably comes with the inherent problem that increased efficiency is only achieved by an increase in bleeding complications.
However fibrin or fibrin degradation products may circulate in the blood leading to mis-targeting of anticoagulants in the circulation.
A further problem in the art relates to the diagnosis of clotting disorders.

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

example 1

Generation of the Single-Chain Antibody scFvanti-LIBS and the Fusion Construct scFvanti-LIBS-TAP

[0110] The generation of the hybridoma cell line expressing a monoclonal antibody against a LIBS epitope on GPIIb / IIIa and its functional characterisation has been described earlier (Schwarz et al. JPET 2004; 308: 1002). Briefly, GPIIb / IIIa purified and eluted with RGD peptides was used as immunogen for hybridoma production. Clones were screened with activated platelets as well as with immobilized GPIIb / IIIa saturated with RGD peptides. One of these clones, monoclonal antibody (mAb) clone 145 demonstrated increased binding to ADP-activated platelets and to platelets pre-incubated with RGD peptides (GRGDSP, BIOMOL Research Laboratories, Plymouth Meeting, Pa.), eptifibatide (Integrilin®, Essex Pharma, Muenchen, Germany), tirofiban (Aggrastat®, MSD, Whitehouse Station, N.J.), and abciximab (ReoPro®, Eli Lilly & Co, Indianapolis, Ind.). The hybridoma was maintained in RPMI, 10% fetal calf se...

example 2

Expression and Purification of scFv Constructs in E. coli

[0112]E. coli (TG1) cells were transformed with the pHOG21 plasmids described above and individual colonies from a freshly streaked agar plate were grown in LB media containing 100 μg / mL ampicillin and 100 mM glucose at 37° C. in 500 mL flasks. Cultures were shaken at 200 rpm for approximate 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 media containing 100 μg / ml ampicillin and 0.4 M sucrose. IPTG was added to a final concentration of 0.25 mM for induction of scFv production and incubated at room temperature (22-24° C.) with 200 rpm for 16-20 hours. For purification of soluble protein from whole cell extract, bacteria were harvested by centrifugation at 5000 rpm for 10 min at 4° C. Pelleted bacteria were resuspended in 5 mL 1× BugBuster® (Novagen, Madison, USA) solution / g pellet and incubated for 15 min at room temperature w...

example 3

In Vitro Functional Characterization of the scFv Anti-LIBS-TAP

Blood Preparation

[0113] Human blood was collected by venipuncture with a 21-gauge butterfly needle from healthy volunteers and anticoagulated with citric acid. Platelet-rich plasma was obtained by centrifugation (GS-6R centrifuge, Beckmann Coulter, Gladesville, NSW, Australia) at 100×g in plastic tubes at room temperature for 10 min in a centrifuge.

[0114] Mouse Blood was collected by intracardial puncture with a 27-gauge needle from C57BL / 6 mice and anticoagulated with unfractionated heparin (20 U / mL). A volume of 50 μl was resuspended with 1 mL modified Tyrode's buffer (150 mM NaCl, 2.5 mM KCl, 1.2 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 0.1% BSA, 0.1% Glucose) and centrifuged at 1300×g for 5 min. The supernatant was discarded and the pellet was resuspended with 1 mL modified Tyrode's buffer.

Flow Cytometry

[0115] Human citrated whole blood was diluted 1 / 50 in modified Tyrode's buffer, either activated by addition of 20 ...

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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

FIELD OF THE INVENTION [0001] The present invention relates to the field of haematology and particularly the sub-speciality of haemostasis. More specifically, the present invention relates to 1) agents that inhibit coagulation in mammalian blood, and uses for these agents in the treatment and prevention of diseases such as stroke, myocardial infarction, and deep vein thrombosis and 2) probes that allow diagnosis and identification of activated platelets in clinical settings such as thrombosis, thrombotic emboli as well as unstable plaques. BACKGROUND TO THE INVENTION [0002] The ability of the body to control the flow of blood following vascular injury is paramount to continued survival. The process of blood clotting and then the subsequent dissolution of the clot, following repair of the injured tissue, is termed hemostasis. Hemostasis is composed of a number of events that occur in a set order following the loss of vascular integrity: [0003] The initial phase of the process is vasc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K49/00C07K16/18A61K39/395C12N15/11G01N33/53
CPCA61K47/48561A61K49/16A61K49/1818A61K51/1027A61K2039/505C07K14/811C07K2316/96C07K2317/622C07K2319/00C07K2317/34A61K49/04C07K16/2848A61K47/6849A61P7/02A61P9/00A61P9/10A61P11/00C07K2317/76A61K38/16A61K38/17A61K39/395
Inventor PETER, KARLHEINZ
Owner BAKER IDI HEART & DIABETES INST HLDG LTD
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