In Vitro Characterization, Pharmacokinetics and Reversal of Supratherapeutic Doses of Dabigatran-Induced Bleeding in Rats by a Specific Antibody Fragment Antidote to Dabigatran

The new oral anticoagulants have demonstrated efficacy and safety in preventing stroke in patients with atrial fibrillation; however, one feature they all share is the lack of a specific antidote in cases of emergency, such as excess bleeding or emergency surgical interventions. A fully humanized monoclonal antibody fragment (Fab) against dabigatran is currently in development for use in the clinic.

The Fab was characterised for binding to dabigatran using Kinexa technology. The dissociation constant (K_D) and on-rate (k_on) were determined experimentally; the off-rate (k_off) was calculated. Inhibition of glucuronidated dabigatran metabolites was tested using a diluted thrombin time assay. Any prothrombotic activity of the Fab was determined by testing the ability to induce coagulation in various assays in human plasma including endogenous thrombin potential (ETP, 5 pM tissue factor) and ecarin chromogenic assay (ECA). Animal studies were approved by local ethics committee and followed principles of laboratory animal care. Pharmacokinetics (PK) of Fab with dabigatran was determined by measuring functional dabigatran, total dabigatran and total Fab in the rat. Dabigatran etexilate (DE) was given to rats every 8 hrs to achieve supratherapeutic levels of dabigatran and induce bleeding. Increasing Fab doses were given to test for bleeding reversal in this rat tail bleeding model. Associated ex vivo clotting and plasma levels of dabigatran and Fab were measured.

The Fab has a very tight binding affinity to dabigatran, with a Kd of 2 pM, ∼350-fold more potent than the 0.7 nM Kd of dabigatran binding to thrombin. This binding has a rapid k_on (1.5×10⁶ M⁻¹s⁻¹) and a slow k_off (3×10⁻⁶s⁻¹), resulting in a calculated complex half life (t½) of ∼64 hrs. IC₅₀ of Fab inhibition of 7nM dabigatran was 3.5 nM. Acylglucuronidated dabigatran (7nM) was inhibited by an IC50 of 2nM Fab. There was no effect on thrombin generation when concentrations up to 3 mg/mL Fab were added to plasma (ETP, 0.89-fold of control), in contrast an activated prothrombinase complex concentrate, FEIBA, resulted in 2.06 increase of ETP with 0.8 U/mL. ECA was also not elevated vs control.

The PK of the Fab in rats showed a short initial phase t_1/2 (0.24 h) and a longer terminal phase t_1/2 (5.8 h). Clearance was low (1.95 mL/min/kg), and the steady-state volume of distribution small (0.0688 L/kg; only slightly larger than plasma volume). The 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, with dabigatran plasma levels of 1500 nM (∼750 ng/mL), and resulted in an ∼2-fold prolongation of tail cut bleeding time. There was a rapid, dose-dependent decrease in blood loss after i.v. injection of Fab, which was maintained for 6 hrs after the highest dose 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. As expected, similar doses of Fab had no effect on reversing this elevated blood loss.

These data show the specificity and selectivity of the antibody fragment for dabigatran. Administration of the Fab after DE resulted in a safe and rapid reversal of blood loss, which correlated with reversal of ex vivo clotting tests. Thus this agent holds promise for reversing dabigatran-induced anticoagulation and bleeding effects. It is currently under development for clinical use.

van Ryn:Boehringer Ingelheim: Employment. Litzenburger:Boehringer Ingelheim: Employment. Gan:Boehringer Ingelheim: Employment. Coble:Boehringer Ingelheim: Employment. Schurer:Boehringer Ingelheim: Employment.