Biochemical and Pharmacological Characterization of YM-60828, a Newly Synthesized and Orally Active Inhibitor of Human Factor Xa

Summary

YM-60828 was found to potently inhibit human factor Xa following oral administration. YM-60828 showed high affinity for factor Xa (K_i = 1.3 μM), but did not affect thrombin (K_i > 100 μM). YM-60828 doubled factor Xa clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT) at 0.10, 0.21, 0.24 μM, respectively. Importantly, it did not prolong thrombin time at 100 μM. YM-60828 also inhibited factor Xa in the prothrombinase complex with an IC₅₀ value of 7.7 nM. In addition to its anticoagulant activity, YM-60828 inhibited platelet aggregation induced by various agonists (IC₅₀ = 3 to 23 μM). Squirrel monkeys were used to study the ex vivo anticoagulant activity and pharmacokinetic properties of YM-60828. One hour after oral administration at 3 mg/kg, YM-60828 strongly prolonged PT and APTT by 4.8- and 1.9-fold, respectively, and plasma concentration reached 788 ± 167 ng/ml. Bioavailability was calculated to be 20.3%. These results strongly suggest that YM-60828 will be a valuable orally active and potent anticoagulant agent showing potential antithrombotic activity.

• References

• 1 Klement P, Borm A, Hirsh J, Maraganore J, Wilson G, Weitz J. The effect of thrombin inhibitors on tissue plasminogen activator induced thrombolysis in a rat model. Thromb Haemost 1992; 68: 64-8.
• 2 Lynch Jr JJ, Sitko GR, Lehman ED, Vlasuk GP. Primary prevention of coronary arterial thrombosis with the factor Xa inhibitor rTAP in a canine electrolytic injury model. Thromb Haemost 1995; 74: 640-5.
• 3 Kelly AG, Marzec UM, Krupski W, Bass A, Cadroy Y, Hanson SR, Harker LA. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood 1991; 77: 1006-12.
• 4 Berry CN, Girardot C, Lecoffre C, Lunven C. Effects of the synthetic thrombin inhibitor argatroban on fibrin- or clot-incorporated thrombin: comparison with heparin and recombinant hirudin. Thromb Haemost 1994; 72: 381-6.
• 5 Maffrand JP. Direct thrombin inhibitors. Nouv Rev Fr Hematol 1992; 34: 405-19.
• 6 Tapparelli C, Metternich R, Ehrhardt C, Cook NS. Synthetic low-molecular weight thrombin inhibitors: molecular design and pharmacological profile. Trends in Pharmacol Sci 1993; 14: 366-76.
• 7 Davie EW, Fujikawa K. Basic mechanisms in blood coagulation. Anu Rev Biochem 1975; 44: 799-829.
• 8 Radcliffe R, Nemerson Y. Activation and control of factor VII by activated factor X and thrombin. J Biol Chem 1975; 250: 388-95.
• 9 Prager NA, Abendschein DR, McKenzie CR, Eisenberg PR. Role of thrombin compared with factor Xa in the procoagulant activity of whole blood clots. Circulation 1995; 92: 962-7.
• 10 Schaffer LW, Davidson JT, Vlasuk GP, Siegl PK. Antithrombotic efficacy of recombinant tick anticoagulant peptide: a potent inhibitor of coagulation factor Xa in a primate model of arterial thrombosis. Circulation 1991; 84: 1741-8.
• 11 Hara T, Yokoyama A, Tanabe K, Ishihara H, Iwamoto M. DX-9065a, an orally active, specific inhibitor of factor Xa, inhibits thrombosis without affecting bleeding time in rats. Thromb Haemost 1995; 74: 635-9.
• 12 Tuszynski GP, Gasic TB, Gasic GJ. Isolation and characterization of antistatin. J Biol Chem 1987; 262: 9718-23.
• 13 Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 1990; 248: 593-6.
• 14 Neeper MP, Waxman L, Smith DE, Schulman CA, Sardana M, Ellis RW, Schaffer LW, Siegel PKS, Vlasuk GP. Characterization of recombinant tick anticoagulant peptide: A highly selective inhibitor of blood coagulation factor XA. J Biol Chem 1990; 265: 17746-52.
• 15 Vlasuk GP, Ramjit D, Fujita T, Dunwiddie CT, Nutt Em, Smith DE, Shebuski RJ. Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistatin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis. Thromb Haemost 1991; 65: 257-62.
• 16 Sitko GR, Ramjit DR, Stabilito II, Lehman D, Lynch JJ, Vlasuk FP. Conjunctive enhancement of enzymatic thrombolysis and prevention of thrombotic reocclusion with the selective factor Xa inhibitor, tick anticoagulant peptide: comparison to hirudin and heparin in a canine model of acute coronary artery thrombosis. Circulation 1992; 85: 805-15.
• 17 Hara T, Yokoyama A, Ishihara H, Yokoyama Y, Nagahara T, Iwamoto M. DX-9065a, a new synthetic, potent anticoagulant and selective inhibitor for factor Xa. Thromb Haemost 1993; 71: 314-9.
• 18 Yamazaki M, Asakura H, Aoshima K, Saito M, Jokaji H, Uotani C, Kumabashiri I, Morishita E, Ikeda T, Matsuda T. Effects of DX-9065a, an orally active, newly synthesized and specific inhibitor of factor Xa, against experimental disseminated intravascular coagulation in rats. Thromb Haemost 1994; 72: 393-6.
• 19 Herbert JM, Bernat A, Dol F, Hérault JP, Crépon B, Lormeau JC. DX-9065a, a novel, synthetic, selective and orally active inhibitor of factor Xa: In vitro and in vivo studies. J Pharmacol Exper Ther 1996; 276: 1030-8.
• 20 Yamashita T, Tsuji T, Matsuoka A, Giddings JC, Yamamoto J. The antithrombotic effect of synthetic low molecular weight human factor Xa inhibitor, DX-9065a, on the He-Ne laser-induced thrombosis in rat mesenteric microvessels. Thromb Res 1997; 85: 45-51.
• 21 Lineweaver H, Burk D. Determination of enzyme dissociation constants. J Am Chem Soc 1934; 56: 658-66.
• 22 Dixon M. The determination of enzyme dissociation constants. Biochem J 1953; 55: 170-1.
• 23 Hackeng TM, van’t Veer C, Meijers JCM, Bouma BN. Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factors Va and Xa. J Biol Chem 1994; 269: 21051-8.
• 24 Katagiri Y, Hayashi Y, Yamamoto K, Tanoue K, Kosaki G, Yamazaki H. Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib. Thromb Haemost 1990; 63: 122-6.
• 25 Dunwiddie C, Thronberry NA, Bull HG, Sardana M, Friedman PA, Jacobs JW, Simpson E. Antistatin, a leech-derived inhibitor of factor Xa. J Biol Chem 1989; 264: 16694-9.
• 26 Nutt EM, Jain D, Lenny AB, Schaffer R, Siegl PK. Purification and characterization of recombinant antistatin: A leech-derived inhibitor of coagulation of factor Xa. Arch Biochem Biophys 1991; 285: 37-44.
• 27 Hara T, Yokoyama A, Morishima Y, Kunitada S. Species differences in anticoagulant and anti-Xa activity of DX-9065a, a highly selective factor Xa inhibitor. Thromb Res 1995; 80: 99-104.
• 28 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-62.
• 29 Eisenberg PR, Siegel JE, Abendschein DR, Miletich JP. Importance of factor Xa in determining the procoagulant activity of whole-blood clots. J Clin Invest 1993; 91: 1877-83.
• 30 Jackson CV, Crowe VG, Frank JD, Wilson HC, Coffman WJ, Utterback BG, Jakubowski JA, Smith F. Pharmacological assessment of the anti-thrombotic activity of the peptide thrombin inhibitor, D-methylphenylalanyl-prolyl-arginal (GYKI-14766), in a canine model of coronary artery thrombosis. J Pharmacol Exp Ther 1992; 261: 546-52.
• 31 Yokoyama T, Kelly AB, Marzec UM, Hanson SR, Kunitada S, Harker LA. Antithrombotic effect of orally active synthetic antagonist of activated factor X in nonhuman primates. Circulation 1995; 92: 485-91.