Identification and characterization of the cytochrome P450 enzymes involved in N-dealkylation of propafenone: molecular base for interaction potential and variable disposition of active metabolites.

The activity of metabolizing enzymes determines plasma concentrations and hence effects of drugs. Identification of these enzymes may allow the prediction of both the interaction potential of drugs and the variability deriving from certain pathways. The antiarrhythmic propafenone is extensively biotransformed to the active metabolites 5-hydroxypropafenone and N-desalkylpropafenone. Whereas 5-hydroxylation is catalyzed by CYP2D6, the enzyme involved in N-dealkylation has not been identified. We, therefore, characterized the enzyme involved in the formation of N-desalkylpropafenone by using both in vitro [human liver microsomes, specific antibodies or inhibitors, and stably expressed cytochrome P450 (P450) enzymes] and in vivo (formation of N-desalkylpropafenone in patients under conditions of chronic therapy) approaches. Formation of N-desalkylpropafenone can be described by Michaelis-Menten kinetics. A strong correlation was observed between maximum rate of formation (Vmax) of N-desalkylpropafenone and the amount of CYP1A2 (r = 0.83, p < 0.001) and CYP3A (r = 0.54, p < 0.05) in the microsomal fraction of 20 human livers. In vitro intrinsic clearances (derived from Vmax/Km) indicated a wide interindividual variability in seven human livers (from 0.01 to 0.1 ml/hr/mg of protein). Antibodies directed against CYP3A and CYP1A2 inhibited formation of N-desalkylpropafenone by 54 +/- 10% and 24 +/- 16%, respectively. The CYP2D6-mediated formation of 5-hydroxypropafenone was unaffected by these antibodies. Verapamil (substrate of CYP3A4 and CYP1A2) and midazolam (substrate of CYP3A4) were competitive inhibitors of N-desalkylpropafenone formation (Ki = 70 microM and 25 microM for verapamil and midazolam, respectively). Coding sequences for CYP1A2 and CYP3A4 were inserted in a yeast expression vector and introduced into Saccharomyces cerevisiae strain W(R). Both CYP1A2 and CYP3A4 catalyzed N-dealkylation of propafenone, with specific activities of 0.32 pmol/min/pmol of P450 and 0.16 pmol/min/pmol of P450, respectively. Our data indicate that N-dealkylation of propafenone is mediated via CYP3A4 and CYP1A2. From experiments on the molecular level interactions of propafenone with other drugs that are metabolized by CYP3A4 and CYP1A2 can be predicted. Such interactions have been reported for cyclosporin, rifampicin, warfarin, and theophylline. Moreover, in vitro intrinsic clearances showed a wide interindividual variability. Therefore, variable plasma concentrations of the active metabolite N-desalkylpropafenone are expected in vivo. We tested this hypothesis in 14 patients (dose of 150 mg of propafenone three times per day) during chronic oral therapy and observed steady state plasma concentrations of N-desalkylpropafenone ranging from 4 to 293 ng/ml.(ABSTRACT TRUNCATED AT 400 WORDS)