INHIBITION AND KINETICS OF CYTOCHROME P4503A ACTIVITY IN MICROSOMES FROM RAT, HUMAN, AND CDNA-EXPRESSED HUMAN CYTOCHROME-P450

Midazolam (MDZ) is metabolized in human liver microsomes by the cytoch rome P450 (CYP) 3A subfamily to 1'-hydroxy (1'-OH) and 4-hydroxy (4-OH ) metabolites. MDZ is metabolized in the rat primarily to 4-OH MDZ, 1' -OH MDZ, and 1',4-dihydroxy (1'4-diOH) MDZ. The kinetics of 4-OH and 1 '-OH metabolite formation were determined using hepatic microsomes fro m control, Ro 23-7637 and dexamethasone-treated male rats. K-M values for the major metabolite, 4-OH MDZ, were 24.5, 43.1, and 32.8 mu M, an d the corresponding V-max values were 5.9, 28.9, and 13 nmol/mg/min fo r the control, DEX, and Ro 23-7637-treated animals, respectively. K-M values for 1'-hydroxylation of MDZ (the major metabolite) after incuba tion with human liver microsomes from three individuals were 5.57, 2.5 0, and 3.56 mu M, and the corresponding V-max values were 4.38, 0.49, and 0.19 nmol/mg/min, respectively in parallel studies using cDNA-expr essed human CYP3A4 microsomes, the K-M for 1'-OH formation was 1.56 mu M, and the corresponding V-max was 0.16 nmol/mg/min. MDZ was not meta bolized by cDNA-expressed human CYP2D6, CYP2E1, or CYP1A2, thus confir ming that these isoforms were not responsible for its biotransformatio n. The formation of 1',4-diOH metabolite in rat and 1'-OH formation in cDNA-expressed human CYP3A4 microsomes showed a decrease in velocity at high substrate concentrations. Inhibition studies showed that MDZ h ydroxylation was strongly inhibited by ketoconazole and Ro 23-7637 in rat, human, and cDNA-expressed human CYP3A4 microsomes. alpha-Naphthof lavone stimulated 1'-OH metabolite formation in human and cDNA-express ed human CYP3A4 microsomes at low concentration (10 mu M). Naringenin, a flavonoid present in grapefruit juice, also inhibited MDZ metabolis m in human liver microsomes. Immunoinhibition studies revealed that po lyclonal anti-rat CYP3A2 antibody inhibited MDZ metabolism 80-90% in r at, human, and cDNA-expressed human CYP3A4 microsomes, thus suggesting that members of the CYP3A4 subfamily were involved in the metabolism.