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Vol. 291, Issue 2, 749-759, November 1999
Department of Drug Metabolism, Merck Research Laboratories, West
Point, Pennsylvania
Cytochrome P-450 (CYP) 3A4 is an inordinately important CYP enzyme that
catalyzes the metabolism of a vast array of clinically used drugs.
Microsomal proteins of Spodoptera frugiperda (Sf21) insect cells infected with recombinant baculoviruses encoding CYP3A4
cDNA were used to immunize mice and to develop a monoclonal antibody
(mAb3A4a) specific to CYP3A4 through the use of hybridoma technology. The mAb is both a potent inhibitor and a strong binder of
CYP3A4. One and 5 µl (0.5 and 2.5 µM IgG2a) of the mAb
mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6
-hydroxylation by 95 and 99%,
respectively, and, to a lesser extent, cross-inhibited CYP3A5 and
CYP3A7 activity. mAb3A4a exhibited no cross-reactivity with
any of the other recombinant human CYP isoforms (CYP1A1, CYP1A2,
CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) in the
course of CYP reaction phenotyping and Western immunoblot analyses. The
potency of mAb-induced inhibition is insensitive to substrate
concentration in human liver microsomes. Therefore, mAb3A4a
was used to assess the quantitative role of CYP3A4/5 to the metabolism
of testosterone and diazepam in five human liver microsomes. The
results showed that CYP3A4 and CYP3A5 contribute >95% to both
testosterone 6-hydroxylation and diazepam 3-hydroxylation and 52 to
73% to diazepam N-demethylation, respectively. In
addition, mAb3A4a significantly inhibited testosterone
6-hydroxylase activity in rhesus monkey liver microsomes to a degree
equal to that observed with CYP3A4 in human liver microsomes. By
comparison, no inhibition of testosterone 6-hydroxylase activity was
observed in the presence of dog, rat, and mouse liver microsomes. The
selectivity of ketoconazole, a chemical inhibitor of CYP3A4, was probed
with mAb3A4a and was shown to be highly concentration
dependent in the diazepam N-demethylation by human liver
microsomes. The results demonstrate that inhibitory and immunoblotting
mAb3A4a can offer a precise and useful tool for
quantitative identification of CYP3A4/5 in the metabolism of drugs in
clinical use and drugs in development.
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