Effect of AR-H067637, the Active Metabolite of the Oral Anticoagulant AZD0837, on Thrombus Formation Using Human Whole Blood in an In Vitro Flow Chamber Model

Abstract

AR-H067637 is a direct thrombin inhibitor derived in vivo from the orally available prodrug AZD0837. AR-H067637 is a reversible and selective direct thrombin inhibitor, which has an inhibition constant, Ki, of 2–4 nmol/L against human alpha-thrombin. During early development of a new anticoagulant drug, data on antithrombotic doses from animal studies may help guide selection of the dose to use in initial human studies. In the present study, using a flow chamber model developed by Badimon and colleagues (Badimon L, et al. J Lab Clin Med 1987; 110:706–718), the antithrombotic effect of AR-H067637 was evaluated using pig aorta and human whole blood. Following collection of informed consent, blood was taken from 11 healthy subjects into citrate-containing (10%, 0.109 M) tubes. Subjects were not permitted to receive medication for 7 days prior to blood collection. Blood was also collected in EDTA-containing tubes for cell counting. Denuded pig aorta pieces were used as the thrombogenic surface in the flow chamber. AR-H067637 was added to the blood at final blood concentrations ranging from 0.01 to 10 μmol/L, corresponding to plasma concentrations of 0.02 to 16 μmol/L. The blood was drawn for 5 minutes through the flow chamber with a shear of 220−s which is comparable with venous flow rate. The thrombus formed inside the chamber was degraded by plasmin, and platelets attached to the thrombus were lysed. The degradation product of fibrin, D-dimer, and the expression of the platelet cell adhesion molecule P-selectin were used as indirect measures of fibrin and platelet content in the thrombus, respectively. The anticoagulant effect of AR-H067637 was determined using the activated partial thromboplastin time (APTT) and prothrombin time (PT) assays. When using D-dimer levels as a measure of thrombus size, 25%, 50% and 75% thrombus inhibition was estimated to occur at AR-H067637 plasma concentrations of 0.21, 0.48 and 1.32 μmol/L, respectively. A significant inhibition of P-selectin expression by AR-H067637 was seen only at the highest concentration. APTT and PT were shown to be prolonged in a concentration-dependent manner; 50% inhibition of thrombus formation on the pig aorta was obtained at 1.8 and 1.2 times prolongations of APTT and PT, respectively. Hematological parameters such as WBC, RBC, HCT and platelets were all within the normal range. In conclusion, this study demonstrates that AR-H067637, the active metabolite of the oral prodrug AZD0837, has antithrombotic effects, causing concentration-dependent inhibition of thrombus formation measured as fibrin degradation products on the denuded pig aorta. Only a small effect at the highest concentration was observed on inhibition of platelet content in the thrombus, measured by P-selectin. This is in accordance with thrombin being a very potent platelet agonist. Therefore, higher concentrations of a thrombin inhibitor are needed to totally prevent platelet activation and aggregation, compared to those needed to prevent fibrin formation. APTT and PT prolongation correlated with the antithrombotic effect of AR-H067637 with >75% inhibition of fibrin formation at APTT and PT prolongations of 2.4 and 1.7, respectively.