ALBERT

Description: 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 Med1987;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.

Description: The direct thrombin inhibitor melagatran (Exanta™, Astra Zeneca, Sweden) has proven efficient for the prevention and treatment of thromboembolism. Major bleeding complications are rare and may often be managed by discontinuation of the drug; however, in some cases of acute serious bleeding, an effective and instant haemostatic intervention may be needed. Potential haemostatic agents may include recombinant factor VIIa (rFVIIa - NovoSeven®, Novo Nordisk, Denmark) or activated prothrombin complex concentrate (APCC - Feiba®, Baxter, Austria). We hypothesized that melagatran induces abnormal whole blood (WB) clotting profiles and rFVIIa as well as APCC may improve the deteriorated clotting profiles. This study aimed to investigate the effect of ex vivo addition of melagatran to WB from healthy males and explore the haemostatic potential of rFVIIa and APCC. Following informed consent, 15 healthy males with an average age of 34 years were enrolled for blood sampling. Continuous WB coagulation profiles were recorded by ROTEG® thrombelastography employing activation with minute amounts of tissue factor (Innovin® final dilution 1:17,000 ~0.35pM). The initiation phase of WB clot formation was defined by the clotting time (CT - sec). Coagulation raw data were processed to provide dynamic parameters that concur with the propagation of WB coagulation such as maximum velocity (MaxVel - mm*100/sec) and time to maximum velocity (t, MaxVel - sec). Titration experiments (n=10) with ex vivo addition of melagatran to WB corresponding plasma concentrations ranging from 0 to 5.0 μM (12 steps) showed a significant and dose dependent prolongation of the CT and t, MaxVel. The MaxVel of WB clot formation was initially reduced from average 13.8 mm*100/sec (12.2-15.4, 95 % CI) to a plateau level of average 9.6 (7.5–12.2) at concentrations of melagatran ranging from 0.125 μM to 0.50 μM. A further and progressive decline in MaxVel was observed at concentrations of melagatran exceeding 1.0μM. Intervention studies (n=10) were performed ex vivo on WB spiked with melagatran at 0.25, 0.50, 1.0, and 2.0 μM followed by ex vivo addition of rFVIIa at concentrations of 25, 50, 100, and 200 nM or APCC at concentration of 0.5, 1.0, 2.0, and 4.0 U/mL. In all tested concentrations of melagatran, rFVIIa significantly shortened the CT and t, MaxVel, while the reduced MaxVel was not accelerated. No dose-response effect of rFVIIa was detected. In contrast, at all concentration of melagatran, APCC significantly and dose dependently shortened the CT, the t, MaxVel as well as increased the MaxVel. As compared to rFVIIa, the effect of APCC was statistically more potent. At melagatran 0.25 μM, APCC at 1.0, 2.0, and 4.0 U/mL normalized the MaxVel. In all other experimental settings, rFVIIa or APCC did not normalize the dynamic WB coagulation parameters following anticoagulation with melagatran. In conclusion, melagatran induces unique changes of dynamic WB clot formation as illustrated by the prolonged initiation and plateau interval of MaxVel in clot propagation. rFVIIa as well as APCC significantly improved the WB clot formation, although reversal of melagatran anticoagulation was not obtained. The more pronounced effect of APCC may be caused by addition of prothrombin and activated coagulation factors. However, this intervention may be less safe than use of rFVIIa.

Description: 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 TS in VT (%) TS in AT (%) AR-H=AR-H067637; LD=low dose; MD=middle dose; HD=high dose Controls 100±8 100±9 Saline + ASA 89±9 71±10 Saline + ASA/Clop 73±7 64±11 Low dose (LD) LD AR-H 62±11 82±9 LD AR-H + ASA 72±11 77±8 LD AR-H + ASA/Clop 67±9 36±8 Middle dose (MD) MD AR-H 49±6 64±9 MD AR-H + ASA 46±10 51±6 MD AR-H + ASA/Clop 32±9 34±6 High-dose (HD) HD AR-H 12±3 23±5 HD AR-H + ASA 12±2 27±6 HD AR-H + ASA/Clop 10±1 1±1