Abstract
Coagulation factor Xa (fXa) is a validated target for antithrombotic therapy and there are several on-going clinical trials testing direct fXa inhibitors. Current measures for monitoring coagulation status [activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), activated clotting time (ACT), anti fXa units] have been developed for existing anticoagulants (i.e., heparins and warfarin). The available tests are not sensitive enough to evaluate therapeutic concentrations of direct fXa inhibitors. Since, the true target of fXa inhibitors is the membrane associated prothrombinase complex, we hypothesized that an assay measuring thrombin generation would be a superior measure of the level of anti-coagulation achieved in patients dosed with direct fXa inhibitors. PRT54021 (PRT021), an orally bioavailable fXa inhibitor in advanced stages of clinical development (Phase II), was used to validate this hypothesis. PRT021 is an active site directed, competitive inhibitor of human fXa (Ki=117pM ) and exhibits a >86,000 fold specificity against related proteases such as thrombin, factor VIIa, factor IXa, activated protein C, tissue plasminogen activator, plasmin and trypsin. PRT021 is a potent inhibitor of the purified prothrombinase complex (Ki=801pM). Evaluation in a whole blood prothrombinase assay was carried out to compare PRT021 to fondaparinux, an indirect inhibitor of fXa. Like fondaparinux, PRT021 dose-dependently inhibited platelet mediated prothrombinase activity in this test system. To measure prothrombinase inhibition, we developed a new bioassay which used a fluorogenic thrombin substrate to quantitate the amount of thrombin produced upon addition of tissue factor to human plasma. In order to determine if this new bioassay predicts in vivo antithrombotic activity, we tested PRT021 in a baboon model of arteriovenous shunt thrombosis. 111In labeled platelet and 125I fibrinogen deposition on the thrombogenic device were used as indicators of thrombotic activity. Dose-dependent inhibition of venous thrombosis was observed at four doses (0.05, 0.12, 0.21 and 0.49mg/kg) of PRT021. Ex vivo measurements of plasma thrombin generation, aPTT, PT, ACT, and anti fXa units were performed during the time course. In contrast to the observed antithrombotic activity (30 to 90% inhibition of platelet deposition, 0 to 87% inhibition of fibrin deposition), there were minimal extension of clotting parameters upon PRT021 treatment. Anti fXa units were below the limit of quantitation for the three lower doses and 0.31 Units/ml at the highest dose. Template bleeding times were not perturbed for any of the PRT021 treated animals. The only ex vivo parameter that correlated to the antithrombotic activity was the dose proportional inhibition (correlation coefficient R2=0.99) of plasma thrombin generation; which ranged from 13% inhibition at the lowest dose to 72% at the highest dose. Thus we have established that this new prothrombinase bioassay predicts in vivo antithrombotic activity. PRT021 is currently in clinical development for prevention and treatment of venous thromboembolic diseases. On-going work with plasma from PRT021 treated patients will verify if the bioassay correlates with clinical endpoints.
Synthesis and Thrombin, Factor Xa and U46619 Inhibitory Effects of Non-amidino and Amidino N2-Thiophenecarbonyl- and N2-Tosylanthranilamides
