In vitro analysis of the activity of the major human hepatic CYP enzyme (CYP3A4) using [N-methyl-14C]-erythromycin.

Although the N-demethylation of erythromycin has found widespread use in a noninvasive assay with which to phenotype hepatic CYP3A function, currently, the routine in vitro analysis of erythromycin N-demethylase activity relies on the quantitation of liberated formaldehyde by relatively labor-intensive and insensitive colorimetric or fluorimetric detection. This report describes the development of a rapid, sensitive, and reproducible radioassay for human CYP3A4 using solid-phase extraction (SPE). The kinetics of erythromycin N-demethylation were best described by a one-site Michaelis-Menten model with autoinhibition and the apparent kinetic parameters for pooled human liver microsomes (HLM; Km=88 microM, Vmax=345 pmol/min/mg) and expressed CYP3A4 (Km=33 microM, Vmax=130 pmol/min/ mg) were in good agreement. Erythromycin N-demethylase activity was found to vary 14-fold in HLM and correlated with the rate of testosterone 6beta-hydroxylation (r2=0.92, p < 0.001; N=9). Ketoconazole was a potent inhibitor of the N-demethylation of erythromycin, and the estimated IC50 value (104+/-23 nM) agreed well with that obtained using testosterone as a probe for CYP3A (71+/-4 nM). The addition of this radioassay to those established for human CYP1A2, 2C9, 2D6, and 2E1 and its subsequent automation should enable the routine use of this methodology in the analysis of CYP-dependent reactions.