Contribution of human CYP3A subfamily members to the 6-hydroxylation of chlorzoxazone.
1. The capability of human CYPs other than 2E1 to catalyse the formation of 6-hydroxychlorzoxazone (6OHCHZ) was examined in vitro using human liver microsomes. 2. 4-Methylpyrazole, diethyldithiocarbamate (DDC), and rabbit anti-human CYP2E1 antibodies reduced chlorzoxazone 6-hydroxylase activity by 60, 60 and 50% respectively. The rate of formation of 6OHCHZ by DDC-treated microsomes was reduced further by the 3A inhibitors midazolam, troleandomycin and gestodene and increased by alpha-naphtholavone, a 3A4 stimulator. 3. Following preincubation with DDC there were significant correlations (p < 0.05) between the residual CHZ 6-hydroxylase activity and immunoquantified CYP3A levels, and corresponding activities (e.g. midazolam 1'-hydroxylation). Rabbit anti-human CYP3A antibodies alone and in combination with DDC reduced the formation of 6OHCHZ by 47 and 62", respectively. 4. cDNA expressed CYP3A4, 2E1 and 2D6 exhibited comparable CHZ 6-hydroxylase activity. CHZ modulated 3A4 activity as reflected by midazolam 1'-hydroxylase and 4-hydroxylase activities. 5. CYP3A may make a significant contribution to CHZ 6-hydroxylation and therefore caution should be exercized when chlorzoxazone is employed as a specific 2E1 probe in vitro and in vivo.
Gorski
J C
JC
Department of Medicine, Indiana University, Indianapolis 46202, USA.
Jones
D R
DR
Wrighton
S A
SA
Hall
S D
SD
D001189:Aryl Hydrocarbon Hydroxylases; D002753:Chlorzoxazone / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D004791:Enzyme Inhibitors; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D009125:Muscle Relaxants, Central / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009125:Muscle Relaxants, Central; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D002753:Chlorzoxazone