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 protopine displayed time dependence and saturation in their inactivation kinetics with KI and kinact values of 72.4 ± 14.7 μM and 0.38 ± 0.036 min−1, 2.1 ± 0.63 μM and 0.18 ± 0.015 min−1, and 7.1 ± 2.3 μM and 0.24 ± 0.021 min−1, 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.
Footnotes
This work was supported by the Finnish Graduate School in Toxicology.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.041319.
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ABBREVIATIONS:
- P450
- cytochrome P450
- MBI
- mechanism-based inactivation
- MIC
- metabolic-intermediate complex
- TDI
- time-dependent inhibition
- FDA
- U.S. Food and Drug Administration
- MDP
- methylenedioxyphenyl
- DBF
- dibenzylfluorescein.
- Received June 22, 2011.
- Accepted September 9, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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