Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cDNA-expressed human cytochrome P450

Abstract

Midazolam (MDZ) is metabolized in human liver microsomes by the cytochrome 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 from control, Ro 23-7637 and dexamethasone-treated male rata. K(M) values for the major metabolite, 4-OH MDZ, were 24.5, 43.1, and 32.8 μM, and the corresponding V(max) values were 5.9, 28.9, and 13 nmol/mg/min for the control, DEX, and Ro 23-7637-treated animals, respectively. K(M) values for l'-hydroxylation of MDZ (the major metabolite) after incubation with human liver microsomes from three individuals were 5.57, 2.50, and 3.56/μM, and the corresponding V(max) values were 4.38, 0.49, and 0.19 nmol/mg/min, respectively. In parallel studies using cDNA-expressed human CYP3A4 microsomes, the K(M) for 1'-OH formation was 1.56/μM, and the corresponding V(max) was 0.16 nmol/mg/min. MDZ was not metabolized by cDNA-expressed human CYP2D6, CYP2E1, or CYP1A2, thus confirming that these isoforms were not responsible for its biotransformation. The formation of l',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 hydroxylation was strongly inhibited by ketoconazole and Ro 23-7637 in rat, human, and cDNA-expressed human CYP3A4 microsomes. α-Naphthoflavone stimulated 1'-OH metabolite formation in human and cDNA-expressed human CYP3A4 microsomes at low concentration (10/αM). Naringenin, a flavonoid present in grapefruit juice, also inhibited MDZ metabolism in human liver microsomes. Immunoinhibition studies revealed that polyclonal anti-rat CYP3A2 antibody inhibited MDZ metabolism 80-90% in rat, human, and cDNA-expressed human CYP3A4 microsomes, thus suggesting that members of the CYP3A4 subfamily were involved in the metabolism.