Abstract
We identify here for the first time the low-affinity cytochrome P450 (P450) isoforms that metabolize paroxetine, using cDNA-expressed human P450s measuring substrate depletion and paroxetine-catechol (product) formation by liquid chromatography-tandem mass spectrometry. CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were identified as paroxetine-catechol-forming P450 isoforms, and CYP2C19 and CYP2D6 were identified as metabolizing P450 isoforms by substrate depletion. Michaelis-Menten constants Km and Vmax were determined by product formation and substrate depletion. Using selective inhibitory studies and a relative activity factor approach for pooled and single-donor human liver microsomes, we confirmed involvement of the identified P450 isoforms for paroxetine-catechol formation at 1 and 20 μM paroxetine. In addition, we used the population-based simulator Simcyp to estimate the importance of the identified paroxetine-metabolizing P450 isoforms for human metabolism, taking mechanism-based inhibition into account. The amount of active hepatic CYP2D6 and CYP3A4 (not inactivated by mechanism-based inhibition) was also estimated by Simcyp. For extensive and poor metabolizers of CYP2D6, Simcyp-estimated pharmacokinetic profiles were in good agreement with those reported in published in vivo studies. Considering the kinetic parameters, inhibition results, relative activity factor calculations, and Simcyp simulations, CYP2D6 (high affinity) and CYP3A4 (low affinity) are most likely to be the major contributors to paroxetine metabolism in humans. For some individuals CYP1A2 could be of importance for paroxetine metabolism, whereas the importance of CYP2C19 and CYP3A5 is probably limited.
Footnotes
This work was supported in part by Madsens's Foundation.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030551.
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- PM
- poor metabolizer
- EM
- extensive metabolizer
- SS
- steady state
- P450
- cytochrome P450
- MBI
- mechanism-based inhibition
- IVIVE
- in vitro/in vivo extrapolation
- HLM
- human liver microsome
- RAF
- relative activity factor
- SOD
- superoxide dismutase
- pHLM
- pooled human liver microsome
- sdHLM
- single-donor human liver microsome
- ICM
- baculovirus-infected insect cell microsome
- ICC
- insect cell control microsome
- mAb
- inhibitory monoclonal antibody
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- MM
- Michaelis-Menten
- LOQ
- limit of quantification.
- Received October 1, 2009.
- Accepted December 9, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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