A Family of Arginal Thrombin Inhibitors Related to Efegatran

Abstract

Three new tripeptide arginal thrombin inhibitors were shown to have potent anticoagulant and antithrombotic activity: D-MePhg-Pro-Arg-H (LY287045), D-1-Tiq-Pro-Arg-H (LY294291), and D-MePhe-Pro-Arg-H (Efegatran). Efegatran and the related arginals differ mechanistically from old and from new anticoagulant agents. As illustrated with x-ray diffraction analysis of crystals of the LY294291 complex with human thrombin, the family of arginals binds thrombin with the P3, P2, and P1 residues interacting with the putative S3, S2, and S1 fibrinogen-binding sites. A hemi-acetal bond at Ser 195 was shown to contribute to the tight-binding reversible competitive thrombin inhibition properties observed with the arginal family. Tight-binding Kass values from thrombin inhibition studies correlated with thrombin clottin inhibition potency. The thrombin time (TT) assay was prolonged twofold with 33 nM Efegatran, which demonstrated an apparent Kass value of 0.8 x 10(8) L/mol (for comparison, 17 nM hirudin was required to prolong the TT assay two-fold). There are empirical anticoagulant selectivity differences between Efegatran and hirudin, manifested by large activated partial thromboplastin time (aPTT)/TT effect ratios (30 to 55) found with the arginals, as compared to the small aPTT/TT effect ratio (2 to 3) found with hirudin. The underlying anticoagulant mechanism differences between the arginals and hirudin appear to be confined to the aPTT pathway and, therefore, might involve different effects toward thrombin feedback activation of factor VIII. The arginals did not substantially inhibit other coagulation factor serine proteases. Antithrombotic effects of Efegatran and the arginal family occur at low infusion doses in dogs and appear to correlate with effects on TT without requiring perturbation of the aPTT. Selectivity properties regarding the fibrinolytic enzymes were shown to be important for successful use of the arginals in vivo as adjunctive agents during tissue plasminogen activator (t-PA) thrombolysis. The data suggest that LY287045, LY294291, and Efegatran should be expected to be useful as antithrombotic adjuncts to thrombolytic therapy with t-PA, urokinase, or streptokinase and should be expected to spare endogenous fibrinolysis. Efegatran has been evaluated in phase I clinical studies and is currently under clinical investigation in phase II protocols as a new cardiovascular anticoagulant.

The crystal structure of human α‐thrombin complexed with LY178550, a nonpeptidyl, active site‐directed inhibitor

“…The chemical formula of LY178550 is shown in Figure 1. The binding affinity of the inhibitor to human thrombin (and other serine proteases) was determined as apparent association constant (Kass) derived from inhibition kinetics, as described previously (Schacht et al, 1995;Smith et al, 1996). Enzyme inhibition kinetics were performed in 96-well polystyrene plates and rates of reaction were determined from hydrolysis rates of appropriate p-nitroanilide substrates at 405 nm using a Thermomax plate reader from Molecular Devices (San Francisco, California).…”

The Minimum Significant Ratio: A Statistical Parameter to Characterize the Reproducibility of Potency Estimates from Concentration-Response Assays and Estimation by Replicate-Experiment Studies

“…The apparent K ass values were obtained in a high-volume testing protocol [13][14][15] using automated dilutions of inhibitors (performed in triplicate at 4-8 inhibitor concentrations) into 96-well plates and chromogenic substrate hydrolysis rates determined at 405 nm using a Thermomax plate reader from Molecular Devices (San Francisco, CA). The assay protocol was 50 µl buffer (0.06 M tris, 0.3 M NaCl, pH 7.4), 25 µl inhibitor test solution (in MeOH), 25 µl human fXa (32 nM in 0.03 M tris, 0.15 M NaCl, 1 mg/ml HSA), and, finally, 150 µl substrate (0.3 mM in water) added within 2 min to start hydrolysis.…”

Direct Thrombin Inhibitors for the Prevention of Venous Thromboembolism after Major Orthopaedic Surgery

“…Argatroban (argidipine, MD805), a derivative of arginine with a molecular weight of 526 daltons [15], napsagatran (RO 46-6240), a cyclopropyl derivative with a molecular weight of 559 daltons [15,22], inogatran, (H 314/27) is a synthetic dipeptide with a molecular weight of 439 daltons [17,23] and efegatran sulfate (GYKI 14766, LY294468), a tripeptide aldehyde containing arginine [18,23] are direct, selective and reversible inhibitors of the active site of thrombin. The antithrombotic effect of direct antithrombins in the experimental model is reviewed in details elsewhere [24].…”