Fluorescence Kinetic Modeling for Mechanism Based CYP450 1A2 Enzyme Inactivation by Furafylline

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Authors
Jonovich, Matthew R.
Issue Date
2000
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Thesis
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en_US
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Abstract
The enzyme P4501A2 is known to play a significant role in the metabolic clearance of drugs and the activation of environmental contaminants and drugs to toxic or carcinogenic species in the body through biotransformation reactions. It has been discovered that furafylline is a potent and selective inhibitor of P4501A2 activity in uman liver microsomes (Sesardic, D., Boobis, A., Murray, S., Segura, J., De La Torre, ., and Davies, D. 1990). The study of P450 inactivation by furafylline inhibition has been an area of increasing interest as it may provide insight as to the role of CYP450 1A2 in the overall metabolic process (Kunze, et al. 1993). Previous study of the proposed mechanism based inhibition of CYP1A2 by furafylline followed the classic Kitz and Wilson method of determination of kinact and K; of the inhibition (Kunze 1993). This procedure, although effective, requires an extensive series of timed assay dilutions and lengthy analysis by HPLC. This procedure provides extensive opportunity for experimental error, requires a great time investment for the determination of kinact, and only allows for examination of the reaction progress at spaced time increments. In this study a method for a continuous fluorescence kinetic assay for the modeling of CYP450 1A2 enzyme inactivation by furafylline was developed. The substrates methoxyresorufm and 3-cyano-7-ethoxy coumarin were selected and employed in this study. The metabolized products, resorufin and 3-cyano-7-hydroxy of each provide fluorescence at 590nm and 460nm respectively. After the development of the fluorescence kinetic assay for the modeling of CYP450 1 A2 enzyme inactivation this procedure was used to determine kinact and K; of 0.07 +/- 0.002min' and 7.18 +/- 0.74µM for the inhibition of methoxyresorufm o-dealkylation and .047 +1- 0.006min' and 2.31 +/- 1.0241.tM for the 3-cyano-7-ethoxycoumarin deethylation by CYP450 1A2.
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