In-vitro profile and ex-vivo anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate

Summary

Dabigatran is a reversible and selective, direct thrombin inhibitor (DTI) undergoing advanced clinical development as its orally active prodrug, dabigatran etexilate.This study set out to determine the molecular potency and anticoagulant efficacy of dabigatran and its prodrug dabigatran etexilate.This was achieved through enzyme inhibition and selectivity analyses, surface plasmon resonance studies, platelet aggregation, thrombin generation and clotting assays in vitro and ex vivo.These studies demonstrated that dabigatran selectively and reversibly inhibited human thrombin (Ki: 4.5 nM) as well as thrombin-induced platelet aggregation (IC₅₀: 10 nM), while showing no inhibitory effect on other platelet-stimulating agents.Thrombin generation in platelet-poor plasma (PPP), measured as the endogenous thrombin potential (ETP) was inhibited concentration-dependently (IC₅₀: 0.56 μM). Dabigatran demonstrated concentration-dependent anticoagulant effects in various species in vitro, doubling the activated partial thromboplastin time (aPTT), prothrombin time (PT) and ecarin clotting time (ECT) in human PPP at concentrations of 0.23, 0.83 and 0.18 μM, respectively. In vivo, dabigatran prolonged the aPTT dose-dependently after intravenous administration in rats (0.3, 1 and 3 mg/kg) and rhesus monkeys (0.15, 0.3 and 0.6 mg/kg). Dose- and time-dependent anticoagulant effects were observed with dabigatran etexilate administered orally to conscious rats (10, 20 and 50 mg/kg) or rhesus monkeys (1, 2.5 or 5 mg/kg), with maximum effects observed between 30 and 120 min after administration, respectively. These data suggest that dabigatran is a potent, selective thrombin inhibitor and an orally active anticoagulant as the prodrug, dabigatran etexilate.

Footnote: Parts of this study were presented at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, July 2001. Thromb Haemost 2001; 86 (Suppl): Abstracts P755, P763.

Institution where work was carried out: Boehringer Ingelheim Pharma GmbH &Co KG, 88397 Biberach, Germany.

Keywords

Activated partial thromboplastin time - dabigatran etexilate - direct thrombin inhibitor - ecarin clotting time - oral anticoagulant

• References

• 1 Lind SE. The hemostatic system. Blood: Principles and Practice of Hematology. Philadelphia: Lippincott; 1995. pp. 949-973.
• 2 Hauptmann J, Stuerzebecher J. Synthetic inhibitors of thrombin and factor Xa: from bench to bedside. Thromb Res 1999; 93: 203-241.
• 3 Weitz JI. Emerging anicoagulants for the treatment of venous thromboembolism. Thromb Haemost 2006; 96: 274-284.
• 4 Becattini C. et al. Initial treatment of venous thromboembolism. Thromb Haemost 2006; 96: 242-250.
• 5 Hirsh J. et al. Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 2001; 119 (Suppl. 01) 8S-21S.
• 6 Hirsh J. et al. Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest 2001; 119 (Suppl. 01) 64S-94S.
• 7 Wittkowsky AK. Warfarin and other coumarin derivatives: pharmacokinetics, pharmacodynamics, and drug interactions. Semin Vasc Med 2003; 3: 221-230.
• 8 Hursting MJ, Jang IK. When heparins promote thrombosis: review of heparin-induced thrombocytopenia. Circulation 2005; 111: 2671-2683.
• 9 Di Nisio M. et al. Direct thrombin inhibitors. N Engl J Med 2005; 353: 1028-1040. Erratum in: N Engl J Med 2005; 353: 2827.
• 10 Hauel N. et al. Stucture-based design of novel potent nonpeptide thrombin inhibitors. J Med Chem 2002; 45: 1757-1766.
• 11 Ries UJ, Wienen W. Serine proteases as targets for antithrombotic therapy. Drugs Future 2003; 28: 355-370.
• 12 Born GVR, Cross MJ. The aggregation of blood platelets. J Physiol(London) 1963; 168: 178-195.
• 13 Hemker HC. et al. Calibrated automated thrombin generation measurement in clottingplasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
• 14 Gustafsson D. et al. Effects of melagatran, a new low-molecular-weight thrombin inhibitor, on thrombin and fibrinolytic enzymes. Thromb Haemost 1998; 79: 110-118.
• 15 Berry CN. et al. SSR182289A, a novel, orally active thrombininhibitor: in vitro profile and ex vivo anticoagulant activity. J Pharmacol Exp Ther 2002; 303: 1189-1198.
• 16 Nilsson T. et al. The mechanism of binding of low-molecular-weight active site inhibitors to human alpha-thrombin. J Enzyme Inhib 1998; 13: 11-29.
• 17 Jochum M. et al. The role of phagocyte proteinases and proteinase inhibitors in multiple organ failure. Am J Resp Crit Care Med 1994; 150: S123-130.
• 18 Elg M. et al. The importance of enzyme inhibition kinetics for the effect of thrombin inhibitors in a rat model of arterial thrombosis. Thromb Haemost 1997; 78: 1286-1292.
• 19 Kahn ML. et al. Protease-activatedreceptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 879-887.
• 20 Boström SL. et al. Effects of melagatran, theactive form of the oral direct thrombin inhibitor ximelagatran, and dalteparin on theendogenous thrombinpotentialin venousblood from healthymale subjects. Blood Coagul Fibrinolysis 2003; 14: 457-462.
• 21 Baglin T. The measurement and application of thrombin generation. Br J Haematol 2005; 130: 653-661.
• 22 Carlson SC. et al. A review of the effects of the oral direct thrombin inhibitor ximelagatran on coagulation assays. Thromb Res 2005; 115: 9-18.
• 23 Okayama T. et al. Anticoagulant activity of the novel thrombin inhibitor 1-butyl-3-(6,7-dimethoxy-2-naphtylsufonyl)amino-3-(2-guanidinopropyl)-2-pyrrolidinone hydrochloride. Drug Res 1997; 47: 1023-1026.
• 24 Mann KG. Thrombinformation. Chest 2003; 124: 4-10.
• 25 Eriksson BI. et al. Dose escalating safety study of a new oral direct thrombin inhibitor, dabigatran etexilate, in patientsundergoing totalhip replacement: BISTRO I. J Thromb Haemost 2004; 2: 1573-1580.
• 26 Eriksson BI. et al. A neworal directthrombininhibitor, dabigatran etexilate, compared with enoxaparin for prevention of thromboembolic events following total hip or knee replacement: the BISTRO II randomized trial. J Thromb Haemost 2005; 3: 103-111.