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Vol. 25, Issue 12, 1359-1369, 1997
Department of Drug Metabolism, Pfizer Central Research
The nonspecific, noncovalent binding of three drugs, imipramine,
warfarin, and propranolol, to pooled human and animal liver microsomes
has been determined using equilibrium dialysis in conditions where no
cofactor (NADPH) was included in the incubation. The binding of
warfarin was dependent upon both protein and drug concentration, whereas the binding of propranolol and imipramine was also dependent upon protein concentration but generally independent of drug
concentration. At a microsomal protein concentration of 1.0 mg/ml and a
warfarin concentration of 10 µM, the free fraction
(fu(mic)) was 0.85. The corresponding values
for propranolol and imipramine were 0.41 and 0.16, respectively. Thus,
although all three drugs exhibit high binding in plasma
(fu<0.1) the acidic drug warfarin differs from
the basic drugs propranolol and imipramine in the extent to which each
binds to microsomal protein. The binding of all three drugs to liver
microsomes obtained from commonly studied animal species (rat, dog, and
monkey) was almost identical to that observed in human. Additionally,
the binding of warfarin and propranolol to microsomes obtained from
insect cells used in baculovirus cytochrome P450 expression systems was
similar to that exhibited in liver microsomes, when equal protein
concentrations were compared. The enzyme kinetics of propranolol,
imipramine, and warfarin oxidative metabolism were determined in pooled
human liver microsomes, and the intrinsic clearance values obtained were used in scaling up to project human in vivo clearance.
The values obtained by incorporating microsomal binding were compared with those in which this factor is ignored. The findings suggest that
the parameter fu(mic) is important to obtain
when attempting to relate in vitro intrinsic clearance to
in vivo clearance. Also, this value is important to
consider when comparing substrates with respect to enzyme specificity,
since measured apparent KM values should
be converted to true "free KM" values
by correcting for the free fraction in the in vitro
incubation. Furthermore, the extent of nonspecific binding to
microsomes is likely an important parameter to consider when attempting
to relate Ki values measured in
vitro to observations of drug-drug interactions (or the lack thereof) in vivo.
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