Abstract

The pharmacokinetics of ethoxycoumarin have been characterized using steady-state plasma concentrations achieved after administration of this compound, at a series of infusion rates, into the hepatic portal vein of rats. The clearance of ethoxycoumarin could be described by a one-site Michaelis-Menten kinetic model withV_max and unboundK_M values of 495 nmol/min/standard rat weight (SRW) and 3.6 μM, respectively, and an intrinsic clearance (CL_int,V_max/K_M ratio) of 137 ml/min/SRW (where SRW is 250 g). Urinary excretion experiments, using both ethoxycoumarin and hydroxycoumarin, demonstrated that 7-hydroxycoumarin, the metabolite frequently measured in in vitro studies, accounted for 26% of the metabolism of ethoxycoumarin. In vitro studies with hepatic microsomes and isolated hepatocytes were undertaken to characterize the kinetics of both hydroxycoumarin formation and ethoxycoumarin depletion and to compare the utility of these methods for predicting in vivoclearance. In both in vitro systems, hydroxycoumarin formation displayed biphasic kinetics, with a high-affinity/low-capacity component (withV_max, K_M , and CL₁ terms) and a low-affinity/high-capacity component (with aCL₂ term) that was not saturated over the substrate concentration range studied (0.5–100 μM). The use of scaling factors to relate in vitro and in vivodata showed that, although microsomal and hepatocyteV_max values were comparable (26 and 17 nmol/min/SRW, respectively), both were substantially lower than thein vivo value. However, scaling of the in vitro CL_int values, by taking into account the fraction of ethoxycoumarin metabolized to hydroxycoumarin, yieldedin vivo predictions of 127 and 122 ml/min/SRW (representing 93 and 89% of the observed CL_int value) for microsomes and hepatocytes, respectively. The depletion of ethoxycoumarin (1–1.5 μM) with time in both microsomes and hepatocytes displayed a monoexponential decline and predicted in vivo CL_int values of 53 and 117 ml/min/SRW (representing 39 and 85% of the observed value), respectively. Therefore, both in vitro systems can accurately predict ethoxycoumarin CL_intvalues using hydroxycoumarin formation rates, providing the importance of this pathway in total clearance is taken into account. Moreover, these results demonstrate that, even when the complete metabolic fate of the compound under investigation is unknown, isolated hepatocytes can be successfully used to predict in vivo CL_int values by measurement of substrate depletion with time.