In this animal model, bleeding induced by high doses of dabigatran can be reduced by CFCs. However, routine coagulation assays do not predict this effect.

2316 Background and Aims: Dabigatran etexilate (DE) is a prodrug, which is rapidly converted in vivo to the active moiety dabigatran, a potent, direct thrombin inhibitor. It is approved as an alternative to oral vitamin K antagonists for sustained prevention of ischemic and hemorrhagic strokes in patients with atrial fibrillation. However, as with any anticoagulant, bleeding risks are associated with its use. Thus, the effects of different coagulation factor concentrates, prothrombinase complex concentrates, activated prothrombinase complex concentrates and recombinant factor VIIa were tested for their ability to reverse bleeding of high dose oral dabigatran etexilate treatment and the corresponding systemic anticoagulation was measured. Methods: Rats (Hann:Wistar, 220 g BW) were given 30 mg/kg DE or vehicle by gavage and 20 min later were anesthetized for surgical preparation. Catheters for i.v. administration of reversal agent and blood sampling were placed and 45 min after oral DE a reversal agent (Beriplex®, Octaplex®, Feiba® and NovoSeven®) or vehicle was administered intravenously at clinically recommended doses. Bleeding was tested at 5, 15, 30 and 120 min (n=3-6/group) post i.v. dosing. Bleeding was induced using a rat tail cut model, where standardized incisions are made in the rat tail and the time to hemostasis recorded. Blood samples were taken at the same time points as bleeding above to allow correlation between ex vivo clotting and bleeding times. Clotting was performed using aPTT, TT, PT, ECT and diluted TT on a laboratory coagulometer and dabigatran plasma levels were determined by Hemoclot. Data shown as mean±SE. Results: Control bleeding times in the absence of treatment were 171±10s over the 2hr experiment. Oral treatment with 30 mg/kg DE resulted in an ∼3-fold elevation in time to hemostasis (495±65 s) just prior to reversal agent administration, this correlated with supratherapeutic dabigatran plasma levels between 800–1000 ng/ml. Beriplex® 35 U/kg, Octaplex® 40 U/kg, Feiba® 100 U/kg and NovoSeven® 0.5 μg/kg all completely reversed the DE-induced prolonged bleeding time to baseline levels within 5 min of i.v. administration. This effect was maintained over the 2 hr measurement period. All clotting tests were prolonged 3–5-fold baseline with the administered dose of DE, indicating the supratherapeutic dose used. The TT, aPTT and ECT all remained prolonged after administration of the coagulation factor concentrates, despite reversal of bleeding time. In contrast the PT was reversed to baseline levels with all coagulation factor concentrates, regardless of whether bleeding time was reversed or not. Thus all these parameters did not correlate with the bleeding outcome measurement. Conclusions: This study illustrates that high doses of dabigtran that induce bleeding can be reversed by coagulation factor concentrates at clinically recommended doses in this animal model. However, the systemic coagulation parameters routinely measured clinically do not predict this reversal. This may be due to lack of predictiveness of systemic clotting on local hemostasis at the site of injury for DTIs, for instance by not taking the role of local tissue factor generation and other surface-dependent processes into account. Disclosures: van Ryn: Boehringer Ingelheim: Employment. Schurer:Boehringer Ingelheim: Employment. Kink-Eiband:Boehringer Ingelheim: Employment. Clemens:Boehringer Ingelheim Pharma GmbH & Co. KG: Employment.

1065 Poster Board I-87 Dabigatran etexilate is a prodrug, which is rapidly converted in vivo to the active moiety dabigatran, a potent, direct thrombin inhibitor. This drug has been evaluated as an alternative to oral vitamin K antagonists for sustained prevention of ischemic and hemorrhagic strokes in patients with atrial fibrillation. As with any anticoagulant, there are bleeding risks with its use. Thus, the adsorption of drug to activated charcoal to allow for potential neutralization in emergency situations was tested in vitro. Binding of dabigatran etexilate to activated charcoal in water simulates recent ingestion (2-3 hrs) of large amounts of dabigatran etexilate in the stomach fluid. Binding of dabigatran to activated charcoal in plasma simulates situations where dabigatran was absorbed after ingestion and present in high concentrations in plasma. The contents of 5, 10 and 20 capsules (150 mg/capsule) of dabigatran etexilate were suspended in 100 ml water (approx. stomach volume, pH 2.7, 2.5 and 2.4, respectively). Each suspension was divided in half. To one portion a freshly prepared activated charcoal suspension (125 mg/ml, Ultracarbon®, Merck) was added. Both suspensions (with and without charcoal) were filtered and dabigatran etexilate concentrations were assessed via HPLC. Levels of 8.3, 15.6 and 29.6 mg/ml dabigatran etexilate were recovered by HPLC in the untreated suspensions in water. In the charcoal-treated suspensions, the dabigatran etexilate peak was no longer detectable, this indicated that >99.9 % of dabigatran etexilate was adsorbed by the activated charcoal for all three tested concentrations. In the plasma experiments, dabigatran was added to a human plasma pool at concentrations of 470 and 940 ng/ml. The sample was then split, and active charcoal was added to half at the manufacturer's specified concentration (125 mg/ml), or 1:11 dilution of this. Dabigatran plasma levels were measured by a validated LC-MS/MS method. After adding dabigatran active substance to plasma, values of 394 ± 19.4 and 824 ± 39.3 ng/ml (mean ± SD) were obtained in untreated plasma. Addition of activated charcoal at both concentrations reduced levels of dabigatran to <1.01 ng/ml (i.e. close to or below lower limit of quantification of the assay). These preliminary in vitro studies demonstrate that the prodrug dabigatran etexilate, and the active moiety, dabigatran, bind activated charcoal and can be removed from water or plasma. Clinical implications may be that oral activated charcoal could be used to neutralise recent ingestion of overdose quantities of dabigatran etexilate before it is absorbed in the gut. In addition, dabigatran could theoretically be adsorbed from plasma via hemoperfusion over a charcoal filter. However, further testing in more clinically relevant models, is required before this can be recommended in patients. Disclosures: van Ryn: Boehringer Ingelheim Pharma GmbH & Co KG: Employment. Sieger:Boehringer Ingelheim Pharma GmbH & Co KG: Employment. Kink-Eiband:Boehringer Ingelheim Pharma GmbH & Co KG: Employment. Gansser:Boehringer Ingelheim Pharma GmbH & Co K: Employment. Clemens:Boehringer Ingelheim GmbH: Employment.

This study determined if meloxicam, a selective cyclooxygenase (COX)-2 inhibitor, interferes with the antiplatelet effect of aspirin using platelet aggregation and thromboxane (Tx) B(2) endpoints in healthy volunteers. Eight male and 8 female volunteers participated in this open-label, randomized, two-treatment, two-way crossover trial. Treatment 1 was meloxicam (15 mg qd) over 4 days, and then aspirin (100 mg qd) was ingested 2 hours after meloxicam for an additional 7 days. Blood samples were taken 2, 6, and 24 hours after the last dose. Treatment 2 consisted of only aspirin (100 mg) over 2 days. Samples were taken at the same time points. Each subject received both treatments with a 2-week washout between the treatment periods. Treatments were safe and well tolerated. The initial 4-day treatment with meloxicam had no effect on platelet aggregation but reduced serum TxB(2) by 64% +/- 19%. Addition of aspirin (100 mg qd) for 7 days resulted in complete inhibition of aggregation and TxB(2) for 24 hours. Two-day treatment with only 100 mg aspirin also resulted in complete inhibition of platelet aggregation and TxB(2). These results indicate that meloxicam does not affect the ability of aspirin to inhibit COX-1 in platelets, thereby allowing aspirin to effectively prevent platelet aggregation and reduce TxB(2) levels, and that meloxicam is selective for COX-2.

Direct thrombin inhibitors (DTIs) have been shown to be very potent inhibitors of platelet function when platelets are activated with thrombin. This action does not occur by direct binding of the DTI to the platelet PAR-1/-4 receptor, but indirectly, by reducing thrombin concentrations and thereby reducing the interactions of thrombin with its receptor on the platelet. It was hypothesized that both thrombin and factor Xa inhibitors could inhibit platelet aggregation, if the stimulus to initiate aggregation was higher in the cascade than factor Xa, such as tissue factor. Thus, dabigatran, a DTI, and the direct factor Xa inhibitors, rivaroxaban and apixaban were tested. Free flowing whole blood (60 ml) was obtained from an antecubital vein using an 18 gauge needle from healthy human volunteers. Blood was collected in tubes containing 3.13% sodium citrate (1 in 10 dilution with whole blood). Blood was centrifuged at 200x g to obtain platelet rich plasma (PRP). Samples (300 μL PRP) were placed in a 6-channel aggregometer, equilibrated for 5 min at 37°C and calibrated with PPP from same individual (0–1 Volts). Photometric tracings were continuously digitally recorded over 5 min following the addition of tissue factor and curves were evaluated as AUC over this time interval. Each PRP sample was incubated with 2 mg/ml Pefabloc®FG (Gly-Pro-Arg-Pro) to prevent fibrin polymerisation, 5 mM CaCl2 and increasing concentrations of dabigatran or factor Xa inhibitor. Tissue factor stimulus (range, 5–27 μl of 10 ml Innovin solution) was tailored for each individual, so that the minimum concentration that resulted in maximum aggregation was used. As positive controls, aggregation was also performed after stimulating with ADP (10 μM), collagen (2 μg/ml), TRAP (20 μM) or ecarin (0.1 U/ml). All substances inhibited tissue factor-induced platelet aggregation in a concentration-dependent manner. Dabigatran was the most potent inhibitor of platelet aggregation among the substances tested, with an IC50 of 35 nM, rivaroxaban and then apixaban followed, with IC50s of 312 and 817 nM, respectively. All substances had no effect on platelet aggregation induced by ADP, collagen and TRAP. Dabigatran was a potent inhibitor of ecarin-induced platelet aggregation, while the factor Xa inhibitors had no effect, as expected from their mechanism of action. Thus, these studies demonstrate that both direct thrombin inhibitors (by inhibiting thrombin) and direct factor Xa inhibitors (by preventing thrombin generation) indirectly inhibit platelet aggregation, though dabigatran was more potent than rivaroxaban and apixaban under these experimental conditions. Thus, these substances may not only be effective in venous/stasis thrombotic episodes where fibrin formation plays an important role, but may also be effective in more platelet dominant, arterial thrombosis settings.