title: Diclofenac-induced inactivation of CYP3A4 and its stimulation by quinidine.
abstract: Incubation of human liver microsomes with diclofenac in the presence of NADPH resulted in a decrease in testosterone 6 beta-hydroxylation activity. The decrease in the activity followed time- and concentration-dependent kinetics, required oxidative metabolism, and was resistant to reduced glutathione, suggesting that diclofenac causes a mechanism-based inactivation of cytochrome p450 (p450) 3A4 (CYP3A4). The inactivation was reproduced by using microsomes from B-lymphoblastoid cell lines expressing CYP3A4 instead of human liver microsomes. No other monooxygenase activities measured as indexes of p450 enzymes; CYP2C8, CYP2C9, or CYP2C19 was inactivated by the same incubation procedure. Quinidine, a stimulant of CYP3A4-mediated diclofenac 5-hydroxylation, did not affect the inactivation of CYP3A4 assessed by testosterone 6 beta-hydroxylation activity but accelerated the inactivation assessed by diazepam 3-hydroxylation activity. These results supported the idea that diclofenac 5-hydroxylation is involved in the inactivation of CYP3A4 and described for the first time a stimulation of mechanism-based inactivation attributable to CYP3A4 heterotropic cooperativity. Preincubation of human liver microsomes with 5-hydroxydiclofenac instead of diclofenac did not cause the inactivation of CYP3A4, suggesting that 5-hydroxydiclofenac is not a precursor of a postulated reactive metabolite that inactivates CYP3A4, and thus 5-hydroxylation step is critical to inactivation of CYP3A4.
authors.affiliation: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Masubuchi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ose
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Horie
mesh_terms: D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004008:Diclofenac / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D020536:Enzyme Activators / Q000737:chemistry / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D011802:Quinidine / Q000494:pharmacology*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D011802:Quinidine