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

The pharmacokinetics of ethoxycoumarin have been characterized using s teady-state plasma concentrations achieved after administration of thi s compound, at a series of infusion rates, into the hepatic portal vei n of rats. The clearance of ethoxycoumarin could be described by a one -site Michaelis-Menten kinetic model with V-max and unbound K-M values of 495 nmol/min/standard rat weight (SRW) and 3.6 mu M, respectively, and an intrinsic clearance (CLint, V-max/K-M ratio) of 137 ml/min/SRW (where SRW is 250 g). Urinary excretion experiments, using both ethox ycoumarin and hydroxycoumarin, demonstrated that 7-hydroxycoumarin, th e metabolite frequently measured in in vitro studies, accounted for 26 % of the metabolism of ethoxycoumarin. In vitro studies with hepatic m icrosomes and isolated hepatocytes were undertaken to characterize the kinetics of both hydroxycoumarin formation and ethoxycoumarin depleti on and to compare the utility of these methods for predicting in vivo clearance. In both in vitro systems, hydroxycoumarin formation display ed biphasic kinetics, with a high-affinity/low-capacity component (wit h V-max, K-M, and CL1 terms) and a low-affinity/high-capacity componen t (with a CL2 term) that was not saturated over the substrate concentr ation range studied (0.5-100 mu M). The use of scaling factors to rela te in vitro and in vivo data showed that, although microsomal and hepa tocyte V-max values were comparable (26 and 17 nmol/min/SRW, respectiv ely), both were substantially lower than the in vivo value. However, s caling of the In vitro CLint values, by taking into account the fracti on of ethoxycoumarin metabolized to hydroxycoumarin, yielded in vivo p redictions of 127 and 122 ml/min/SRW (representing 93 and 89% of the o bserved CLint value) for microsomes and hepatocytes, respectively. The depletion of ethoxycoumarin (1-1.5 mu M) 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 c an accurately predict ethoxycoumarin CLint, values using hydroxycoumar in formation rates, providing the importance of this pathway in total clearance is taken into account. Moreover, these results demonstrate t hat, even when the complete metabolic fate of the compound under inves tigation is unknown, isolated hepatocytes can be successfully used to predict in vivo CLint values by measurement of substrate depletion wit h time.