Role of a potent inhibitory monoclonal antibody to cytochrome P-450 3A4 in assessment of human drug metabolism.
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 (mAb(3A4a)) 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 microl (0.5 and 2.5 microM IgG(2a)) of the mAb mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6beta-hydroxylation by 95 and 99%, respectively, and, to a lesser extent, cross-inhibited CYP3A5 and CYP3A7 activity. mAb(3A4a) 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, mAb(3A4a) 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 6beta-hydroxylation and diazepam 3-hydroxylation and 52 to 73% to diazepam N-demethylation, respectively. In addition, mAb(3A4a) significantly inhibited testosterone 6beta-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 6beta-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 mAb(3A4a) and was shown to be highly 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 development.
Mei
Q
Q
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. qin_mei@merck.com
Tang
C
C
Assang
C
C
Lin
Y
Y
Slaughter
D
D
Rodrigues
A D
AD
Baillie
T A
TA
Rushmore
T H
TH
Shou
M
M
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003429:Cross Reactions; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000276:immunology*; D018076:DNA, Complementary / Q000502:physiology; D003975:Diazepam / Q000378:metabolism; D004285:Dogs; D005260:Female; D000882:Haplorhini; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D008875:Middle Aged; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D051381:Rats; D013045:Species Specificity; D018411:Spodoptera / Q000821:virology; D013739:Testosterone / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D004364:Pharmaceutical Preparations; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003975:Diazepam; D007654:Ketoconazole