AR-H067637, the Active Metabolite of the Oral Anticoagulant AZD0837, with and without Aspirin or Aspirin and Clopidogrel in Experimental Models of Venous and Arterial Thrombosis and Bleeding in Anaesthetized Rats

AR-H067637 is a direct thrombin inhibitor derived in vivo from the orally available prodrug AZD0837. AZD0837 is in development for use in thromboembolic disorders. This study investigated the additional effects of aspirin (ASA) alone and in combination with clopidogrel (Clop) on AR-H067637 in animal models of venous thrombosis (VT) and arterial thrombosis (AT). Anaesthetized rats (9–11 per treatment group) were treated with vehicle or 3 increasing (low, middle and high) doses of AR-H067637 (0.1, 0.3 and 1.0 μmol/kg/h in VT model and 0.5, 1.5 and 4.5 μmol/kg/h in AT model [predicted to obtain 25%, 50% and 75% antithrombotic effect]) given intravenously (iv) as continuous infusions. Rats were also treated with saline, ASA (10mg/kg) or ASA (10mg/kg) + Clop (25mg/kg) as iv bolus injections at the start of the experiment. In the AT model the thrombotic stimulus was ferric chloride administered to the carotid artery, while in the VT model the stimulus was ferric chloride and partial stasis of the caval vein. Thrombus size (TS) was assessed as wet weight (both models) and as protein content (AT model only). Bleeding time (BT) and blood loss (BL) were investigated by tail incision (TI) and muscle transection (MT). Activated partial thromboplastin time (APTT; AT model), thrombin coagulation time (TcT; AT model), thrombin generation (determined using the calibrated automatic thrombogram [CAT] assay variables; both models), lag time (LT), time to peak concentration (ttPeak), peak concentration (peak) and endogenous thrombin potential (ETP) were investigated. Arachidonic acid (AA) and ADP-induced platelet aggregation were measured by the whole blood impedance method in a subset of rats (n=6) to verify antiplatelet effects of ASA and Clop, respectively. Results showed that ARH067637 dose-dependently decreased TS in both models (Table). ASA+Clop, decreased TS in both models. In the VT model, ASA+Clop had no additional effect to that seen with AR-H067637 alone, but in the AT model further decreased TS with total inhibition obtained at the highest dose of AR-H067637. AR-H067637 dose-dependently prolonged TI BT and BL and MT BL (up to 2.4, 1.4 & 2.1 times the vehicle group). ASA in addition to AR-H067637 did not potentiate TI BT or MT BL but potentiated TI BL in addition to the highest dose of AR-H067637. ASA+Clop potentiated TI BT, BL and MT BL (2.6, 5.4 & 2.4 times the saline group). Only high-dose AR-H067637 reinforced this bleeding (TI BT and BL and MT BL 3.4, 17 and 9.2 times the saline group). AR-H067637 concentration-dependently prolonged TcT (2–7 times), APTT (1.3–3 times), CAT LT (up to 2.6 times), CAT ttPeak (up to 2 times), and the highest plasma concentration totally abolished CAT peak and CAT ETP. ASA or ASA+Clop had no influence on these variables. AA and ADP-induced platelet aggregation was inhibited by 96% in ASA-treated and 29% in Clop-treated animals, respectively. This investigation shows that ASA with or without ARH067637 had a small effect on TS, without increasing bleeding. ASA+Clop decreased TS: effects were more marked in the AT model, but increased bleeding was seen, especially at high AR-H067637 plasma concentrations. Pharmacodynamic markers indicate dose-dependant, increased anticoagulation with rising concentrations of AR-H067637; these markers were not influenced by ASA or ASA+Clop.

Thrombus size (TS) in VT and AT models