title: 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 rats. KM values for the major metabolite, 4-OH MDZ, were 24.5, 43.1, and 32.8 microM, and the corresponding Vmax values were 5.9, 28.9, and 13 nmol/mg/min for the control, DEX, and Ro 23-7637-treated animals, respectively KM values for 1'-hydroxylation of MDZ (the major metabolite) after incubation with human liver microsomes from three individuals were 5.57, 2.50, and 3.56 microM, and the corresponding Vmax values were 4.38, 0.49, and 0.19 nmol/mg/min, respectively. In parallel studies using cDNA-expressed human CYP3A4 microsomes, the KM for 1'-OH formation was 1.56 microM, and the corresponding Vmax 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 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 hydroxylation was strongly inhibited by ketoconazole and Ro 23-7637 in rat, human, and cDNA-expressed human CYP3A4 microsomes. alpha-Naphthoflavone stimulated 1'-OH metabolite formation in human and cDNA-expressed human CYP3A4 microsomes at low concentration (10 microM). 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.
authors.affiliation: Hoffmann-La Roche, Inc., Nutley, NJ 07110-1199, USA.
authors.forename: A
authors.initials: A
authors.lastname: Ghosal
authors.forename: H
authors.initials: H
authors.lastname: Satoh
authors.forename: P E
authors.initials: PE
authors.lastname: Thomas
authors.forename: E
authors.initials: E
authors.lastname: Bush
authors.forename: D
authors.initials: D
authors.lastname: Moore
mesh_terms: D018686:Anesthetics, Intravenous / Q000378:metabolism*; D000818:Animals; D014151:Anti-Anxiety Agents / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008874:Midazolam / Q000031:analogs & derivatives / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D018686:Anesthetics, Intravenous; D014151:Anti-Anxiety Agents; D000906:Antibodies; D065607:Cytochrome P-450 Enzyme Inhibitors; D010880:Piperidines; D011725:Pyridines; D011994:Recombinant Proteins; C083287:Ro 23-7637; C042940:4-hydroxymidazolam; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; C040081:1-hydroxymethylmidazolam; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam