O-DEMETHYLATION OF EPIPODOPHYLLOTOXINS IS CATALYZED BY HUMAN CYTOCHROME-P450 3A4

We previously demonstrated that O-demethylation of the pendant dimetho xyphenol ring of epipodophyllotoxins to produce their respective catec hol metabolites is catalyzed by cytochrome(s) P450 in human liver micr osomes. Our objective was to identify the specific human cytochrome(s) P450 responsible for catechol formation. Using a panel of prototypica l substrates and inhibitors for specific cytochromes P450, we identifi ed substrates for CYP3A4 (midazolam, erythromycin, cyclosporin, and de xamethasone) as inhibitors of catechol formation from both etoposide a nd teniposide. Dexamethasone inhibition was competitive, with K-i valu es of 60 and 45 mu M for etoposide and teniposide, respectively. In 58 human livers, the correlation coefficients for teniposide catechol fo rmation versus 1'- and 4-hydroxymidazolam formation were 80% and 85%, respectively; for etoposide catechol formation versus 1'- and 4-hydrox ymidazolam formation r(2) was 83% and 79%, respectively. Teniposide an d etoposide catechol formation rates were also significantly correlate d with immunodetectable CYP3A (r(2) = 49% and 51%, respectively) and n ot with immunodetectable CYP1A2, 2E1, or 2C8. Finally, cDNAs for human CYP3A4, 3A5, 2A6, 2B6, 2C8, and 2C9 were functionally expressed in He pG2 cells, using a vaccinia viral vector. Teniposide and etoposide cat echol formation was catalyzed primarily by 3A4 (15.4 and 40.9 pmol/ pm ol/hr, respectively) and to a lesser degree by 3A5 (1.94 and 11.3 pmol /pmol/hr, respectively), whereas there was no detectable O-demethylati on of epipodophyllotoxins by 2A6, 2B6, 2C8, 2C9, or the control virus alone. Moreover, the relative activities of midazolam hydroxylation, c ompared with O-demethylation of epipodophyllotoxins, were similar for heterologously expressed 3A4 and for human liver microsomes. We conclu de that catechol formation from teniposide and etoposide is primarily mediated by human CYP3A4, making these reactions susceptible to inhibi tion by prototypical 3A substrates and inhibitors.