CHARACTERIZATION OF DEXTROMETHORPHAN N-DEMETHYLATION BY HUMAN LIVER-MICROSOMES - CONTRIBUTION OF THE CYTOCHROME-P450 3A (CYP3A) SUBFAMILY

In an effort to identify the human cytochromes P450 involved in the N- demethylation of dextromethorphan, the kinetics of 3-methoxymorphinan formation were studied in microsomal enzyme systems. Under initial rat e conditions, 3-methoxymorphinan formation demonstrated single enzyme Michaelis-Menten kinetics using microsomes obtained from three human l ivers (K-m: 0.52-0.71mM; V-max: 375-812 pmol/mg protein/min). B-lympho blastoid cells expressing CYP3A4 incubated with 0.4mM dextromethorphan catalyzed the formation of 3-methoxymorphinan at a rate of 22 pmol pr oduct/mg protein/min. Midazolam, a prototypic substrate for CYP3A4 and CYP3A5, competitively inhibited dextromethorphan N-demethylation by t wo human liver microsomal samples with K-i values of 46 +/- 10 and 63 +/- 8 mu M. At a dextromethorphan concentration of 0.4 mM, gestodene ( 100 mu M) inhibited 3-methoxymorphinan formation by approximately 50%. Immunoinhibition of dextromethorphan N-demethylation using rabbit ant i-CYP3A4 antibodies resulted in a 60% decrease in 3-methoxymorphinan f ormation at a dextromethorphan concentration of 0.4 mM. Additional inh ibition studies using furafylline, coumarin, sulfaphenazole, mephenyto in, quinidine, and diethyldithiocarbamic acid, which are selective inh ibitors of CYP1A2, CYP2A6, CYP2C8/9, CYP2Cmp, CYP2D6, and CYP2E1, resp ectively, demonstrated no substantial inhibition of dextromethorphan N -demethylation. Correlation analysis was performed using the rate of 3 -methoxymorphinan formation at a concentration of 1 mM dextromethorpha n and immunoquantified levels of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C 19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 and their associated characteri stic catalytic activities. A significant correlation was observed betw een dextromethorphan N-demethylase activity and midazolam 1'- and 4-hy droxylase activity (r(2) = 0.77 and 0.69 respectively, N = 19, P < 0.0 1); the exclusion of those samples containing both CYP3A4 and CYP3A5 i ncreased the correlation significantly (r(2)= 0.87 and 0.91 respective ly, N = 12, P < 0.01). In the absence of CYP3A5, a significant correla tion was observed between 3-methoxymorphinan formation and the sample' s erythromycin N-demethylase activity (r(2) = 0.94, N = 12, P < 0.01), testosterone 6 beta-hydroxylase activity (r(2) = 0.96, N = 7, P < 0.0 1) and relative immunoquantified levels of CYP3A4 (r(2) = 0.96, N = 12 , P < 0.01). Inclusion of those samples expressing CYP3A5 in addition to CYP3A4 reduced the magnitude of the observed correlation. No signif icant correlation between 3-methoxymorphinan formation and the sample' s relative immunoquantified levels of or form-selective activity assoc iated with CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19 (or CYP2Cmp), CYP2D 6, and CYP2E1 was observed. In conclusion, dextromethorphan N-demethyl ation appears to be catalyzed primarily by CYP3A4 and to a lesser exte nt by CYP3A5 in vitro in humans. Thus, the administration of dextromet horphan to human volunteers may provide a means of simultaneously phen otyping the in vivo activity of CYP2D6 and CYP3A.