CONTRIBUTION OF HUMAN CYP3A SUBFAMILY MEMBERS TO THE 6-HYDROXYLATION OF CHLORZOXAZONE

1. The capability of human CYPs other than 2E1 to catalyse the formati on of 6-hydroxychlorzoxazone (6OHCHZ) was examined in vitro using huma n liver microsomes. 2. 4-Methylpyrazole, diethyldithiocarbamate (DDC), and rabbit anti-human CYP2E1 antibodies reduced chlorzoxazone 6-hydro xylase activity by 60, 60 and 50 %, respectively. The rate of formatio n of 6OHCHZ by DDC-treated microsomes was reduced further by the 3A in hibitors midazolam, troleandomycin and gestodene and increased by alph a-naphthoflavone, a 3A4 stimulator. 3. Following preincubation with DD C there were significant correlations (p < 0.05) between the residual CHZ 6-hydroxylase activity and immuno-quantified CYP3A levels, and cor responding activities (e.g. midazolam 1'-hydroxylation). Rabbit anti-h uman CYP3A antibodies alone and in combination with DDC reduced the fo rmation of 6OHCHZ by 47 and 62% respectively. 4. cDNA expressed CYP3A4 , 2E1 and 2D6 exhibited comparable CHZ 6-hydroxylase activity. CHZ mod ulated 3A4 activity as reflected by midazolam 1'-hydroxylase and 4-hyd roxylase activities. 5. CYP3A may make a significant contribution to C HZ 6-hydroxylation and therefore caution should be exercized when chlo rzoxazone is employed as a specific 2E1 probe in vitro and in vivo.