Comparative contribution to dextromethorphan metabolism by cytochrome P450isoforms in vitro: Can dextromethorphan be used as a dual probe for both CYP2D6 and CYP3A activities?

Dextromethorphan (DXM) is a widely used probe drug for human CYP2D6 activit y both in vitro and in vivo. In humans, DXM is metabolized to dextrorphan ( DXO), as well as 3-methoxymorphinan [MEM) and 3-hydroxymorphinan (HYM). The formation of MEM has been attributed primarily to CYP3A4, and the use of D XM has been debated as a simultaneous probe for CYP3A4 and CYP2D6 activitie s. Recently, we found that highly purified CYP2D6 has significant DXM N-dem ethylase activity in addition to its well known DXM O-demethylase activity. Therefore, we desired to further compare the contribution to DXM metabolis m by individual human cDNA-expressed cytochromes P450, including 2C8, 2C9, 2C18, 2C19, 2D6, 2B6, and 3A4. Metabolites were quantified following separa tion by high-pressure liquid chromatography and apparent Michaelis-Menten c onstants determined for the appearance of DXO and MEM. Intrinsic clearance values were estimated for each P450 and normalized using the average percen tage content and relative activity factor approaches for comparison. Simpli fied kinetic models (when [S] << K-m, V-max/K-m = V-o/[S]) were used at fix ed DXM concentrations of 20 (for DXM N-demethylation) and 0.2 muM (for DXM O-demethylation), as well as 2 muM to mimic plasma DXM concentrations in hu man extensive metabolizers. The results confirm that CYP2D6 contributes at least 80% to the formation of DXO, and CYP3A4 contributes more than 90% to the formation of MEM. All of our in vitro results are consistent and indica te that DXM as a marker for monitoring both CYP2D6 and CYP3A activities is practical in an average human or human liver microsomal preparation.