Early assessment of efficacy and safety of novel anticoagulants as exemplified by direct thrombin inhibitors. Non-clinical pharmacodynamic studies in vitro and in vivo

The drawbacks of today's anticoagulants are parenteral use (heparins), a narrow interval between efficacy and bleeding, and the high variability of effect with a consequent need for frequent monitoring (coumarins). The aim of the current studies was to evaluate properties important for efficacy and safety of novel anticoagulants, as exemplified by direct thrombin inhibitors, with non-clinical methods. The focus was on the direct thrombin inhibitors inogatran and melagatran. The efficacy, selectivity and safety of the anticoagulants were investigated by using in vivo models of venous and arterial thrombosis, exogenous thrombolysis, endogenous fibrinolysis and bleeding time measurements. The findings were compared with the results obtained in biochemical assays ex vivo and in vitro. Efficacy. Based on similar prolongations of activated partial thromboplastin time (APTT) inogatran, argatroban and heparin were equipotent in a venous thrombosis model. A decrease in the enzyme inhibition constant (Ki) obtained in vitro for melagatran compared with inogatran, led to an increased potency for melagatran in the arterial thrombosis model in vivo, while a similar antithrombotic effect was obtained for melagatran and hirudin despite a large difference in the Ki-values. For thrombin inhibitors, a relationship was found between steep dose-response curves and low association rate constants between thrombin and the inhibitor. Warfarin was also found to have a steep dose-response curve. Selectivity. In rats exogenous fibrinolysis, obtained by administration of recombinant tissue plasminogen activator (rt-PA), or endogenous fibrinolysis was either facilitated or inhibited depending on the degree of selectivity against the fibrinolytic system. The results suggest that the ratio of the Ki-values of fibrinolytic enzymes to the required plasma concentration needed in vivo must be used to differentiate thrombin inhibitors rather than the selectivity against thrombin. Safety. Inogatran and melagatran seemed to have a wider separation of antithrombotic effect and bleeding time compared with warfarin. Feiba , a coagulation factor concentrate containing a mixture of coagulation factors, was found to be effective, without being prothrombotic, in reversing the anticoagulant effect of melagatran.Conclusion: For direct thrombin inhibitors to be effective and safe, potent, rapid and selective inhibition of thrombin is needed. Excessive thrombin inhibition can be reversed by prothrombin complex concentrates

Expression and characterization of recombinant ecarin.

Item does not contain fulltextThe snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 degrees C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K (m) value of 0.4 muM prethrombin-2 was determined but only a rough estimate could be made of the K (m) for prothrombin of 0.9 muM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.1 juni 201