Role of a potent inhibitory monoclonal antibody to cytochrome P-450 3A4 inassessment of human drug metabolism

Cytochrome P-450 (CYP) 3A4 is an inordinately important CYP enzyme that cat alyzes the metabolism of a vast array of clinically used drugs. Microsomal proteins of Spodoptera frugiperda (Sf21) insect cells infected with recombi nant baculoviruses encoding CYP3A4 cDNA were used to immunize mice and to d evelop a monoclonal antibody (mAb(3A4a)) specific to CYP3A4 through the use of hybridoma technology. The mAb is both a potent inhibitor and a strong b inder of CYP3A4. One and 5 mu l (0.5 and 2.5 mu M IgG(2a)) of the mAb mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6 beta-hydroxylation by 95 and 99%, respectively, and, to a lesser extent, cross-inhibited CYP3A5 and CYP3A7 activity. mAb3A4a exhib ited no cross-reactivity with any of the other recombinant human CYP isofor ms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CY P2E1) in the course of CYP reaction phenotyping and Western immunoblot anal yses. The potency of mAb-induced inhibition is insensitive to substrate con centration in human liver microsomes. Therefore, mAb3A4a was used to assess the quantitative role of CYP3A4/5 to the metabolism of testosterone and di azepam in five human liver microsomes. The results showed that CYP3A4 and C YP3A5 contribute >95% to both testosterone 6 beta-hydroxylation and diazepa m 3-hydroxylation and 52 to 73% to diazepam N-demethylation, respectively. In addition, mAb(3A4a) significantly inhibited testosterone 6 beta-hydroxyl ase activity in rhesus monkey liver microsomes to a degree equal to that ob served with CYP3A4 in human liver microsomes. By comparison, no inhibition of testosterone 6 beta-hydroxylase activity was observed in the presence of dog, rat, and mouse liver microsomes. The selectivity of ketoconazole, a c hemical inhibitor of CYP3A4, was probed with mAb3A4a and was shown to be hi ghly concentration dependent in the diazepam N-demethylation by human liver microsomes. The results demonstrate that inhibitory and immunoblotting mAb 3A4a can offer a precise and useful tool for quantitative identification of CYP3A4/5 in the metabolism of drugs in clinical use and drugs in developme nt.