ABSTRACT:Several in vitro criteria were used to assess whether three methylenedioxyphenyl (MDP) compounds, the isoquinoline alkaloids bulbocapnine, canadine, and protopine, are mechanism-based inactivators of CYP2C19. The recently reported fluorometric CYP2C19 progress curve analysis approach was applied first to determine whether these alkaloids demonstrate time-dependent inhibition. In this experiment, bulbocapnine, canadine, and proto- , respectively. Additional studies were performed to determine whether other specific criteria for mechanism-based inactivation were fulfilled: NADPH dependence, irreversibility, and involvement of a catalytic step in the enzyme inactivation. CYP2C19 activity was not significantly restored by dialysis when it had been inactivated by the alkaloids in the presence of a NADPH-regenerating system, and a metabolic-intermediate complex-associated increase in absorbance at approximately 455 nm was observed. In conclusion, the CYP2C19 progress curve analysis method revealed time-dependent inhibition by these alkaloids, and additional experiments confirmed its quasi-irreversible nature. This study revealed that the CYP2C19 progress curve analysis method is useful for identifying novel mechanism-based inactivators and yields a wealth of information in one run. The alkaloids bulbocapnine, canadine, and protopine, present in herbal medicines, are new mechanism-based inactivators and the first MDP compounds exhibiting quasi-irreversible inactivation of CYP2C19.
ABSTRACT:Many clinically relevant drug interactions involving cytochrome P450 inhibition are mediated by mechanism-based inactivation (MBI). Time-dependent inhibition is one of the major features distinguishing between reversible inhibition and MBI. It thus provides a useful screening approach for early drug interaction risk assessment. Accordingly, we developed an easy and informative fluorometric method for the assessment of CYP2C19 enzyme inactivation kinetics. Dibenzylfluorescein (DBF) is widely used as a profluorescent probe substrate for P450 activity and inhibition assays, but its use has been considered to be limited to traditional endpoint assays. We monitored CYP2C19-catalyzed metabolism of DBF using , respectively. Tranylcypromine did not display any time-dependent inhibition, which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization.
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