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
Dj. Carlile et al., 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, Drug metabolism and disposition, 26(3), 1998, pp. 216-221
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.