In vivo clearance of ethoxycoumarin and its prediction from In vitro systems. Use Of drug depletion and metabolite formation methods in hepatic microsomes and isolated hepatocytes.

摘要

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 with Vmax and unbound KM values of 495 nmol/min/standard rat weight (SRW) and 3.6 microM, respectively, and an intrinsic clearance (CLint, Vmax/KM 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 vivo clearance. In both in vitro systems, hydroxycoumarin formation displayed biphasic kinetics, with a high-affinity/low-capacity component (with Vmax, KM, and CL1 terms) and a low-affinity/high-capacity component (with a CL2 term) that was not saturated over the substrate concentration range studied (0.5-100 microM). The use of scaling factors to relate in vitro and in vivo data showed that, although microsomal and hepatocyte Vmax values were comparable (26 and 17 nmol/min/SRW, respectively), both were substantially lower than the in vivo value. However, scaling of the in vitro CLint values, by taking into account the fraction of ethoxycoumarin metabolized to hydroxycoumarin, yielded in vivo predictions of 127 and 122 ml/min/SRW (representing 93 and 89% of the observed CLint value) for microsomes and hepatocytes, respectively. The depletion of ethoxycoumarin (1-1.5 microM) with time in both microsomes and hepatocytes displayed a monoexponential decline and predicted in vivo CLint 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 CLint values 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 CLint values by measurement of substrate depletion with time.