Abstract WP261: In Vitro Characterization, Pharmacokinetics and Reversal of Supratherapeutic Doses of Dabigatran-Induced Bleeding in Rats by a Specific Antibody Fragment Antidote to Dabigatran
One feature all new oral anticoagulants share is the lack of a specific antidote in cases of emergency. A fully humanized monoclonal antibody fragment (Fab) against dabigatran is currently in development for use in the clinic.
Fab binding to dabigatran was tested as dissociation constant (KD), on- and off-rate. Inhibition of glucuronidated dabigatran was tested using diluted thrombin time assay. Any prothrombotic activity of the Fab was tested for the ability to induce coagulation in endogenous thrombin potential (ETP, 5 pM tissue factor) and ecarin chromogenic assay (ECA). Pharmacokinetics (PK) was determined by measuring functional and total dabigatran and total Fab in the rat. Dabigatran etexilate (DE), 30 mg/kg, was given to rats every 8 hrs to induce bleeding. Increasing Fab doses were given to test for bleeding reversal in this rat tail bleeding model.
The Fab bound to dabigatran with a Kd of 2 pM, with a rapid kon (1.5x106M-1s-1) and slow koff (3x10-6s-1). Dabigatran (7nM) inhibition by Fab was 3.5 nM (IC50) and metabolite inhibition was 2nM (IC50) in human plasma. There was no effect on thrombin generation with 3 mg/mL Fab added to plasma (ETP, 0.89-fold of control), in contrast, FEIBA (0.8 U/mL), resulted in 2.06 increase of ETP. ECA was also not elevated vs control. The PK of the Fab in rats showed a short initial phase t1/2 (0.24 h) and longer terminal phase t1/2 (5.8 h). Clearance was low (1.95 mL/min/kg) and steady-state volume of distribution small (0.0688 L/kg). PK of the Fab was not affected by dabigatran. DE (30 mg/kg p.o.) given in 8 hr intervals achieved steady state levels in the rat, plasma levels of 1500 nM (~750 ng/mL), and resulted in ~2-fold increase of tail cut bleeding time. There was a rapid, dose-dependent decrease in blood loss after i.v. injection of Fab. Anticoagulation (clotting ex vivo) was also reversed. Treating rats with warfarin (0.5 mg/kg p.o.) over 3 days resulted in a 2-fold elevation of blood loss. Similar doses of Fab had no effect on reversing warfarin induced blood loss.
Thus this Fab is specific and selective for dabigatran and reversed DE-induced blood loss, correlating with reversal of ex vivo clotting tests. This agent holds promise for reversing dabigatran-induced anticoagulation and bleeding effects and is currently under development.
- © 2012 by American Heart Association, Inc.