Abstract
CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC50 = 77 nM) and O-demethylation of astemizole (Ki = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC50 values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues.
Footnotes
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant R01-HL096706]; and the National Institutes of Health National Institute of General Medical Sciences [P01-GM32165].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- P450
- cytochrome P450
- RAF
- relative activity factor
- ISEF
- intersystem extrapolation factor
- HLM
- human liver microsomes
- HPLC
- high-performance liquid chromatography
- 1D
- one dimensional
- COSY
- correlation spectroscopy
- TOCSY
- total correlation spectroscopy
- HSQC
- heteronuclear single quantum correlation
- IS
- internal standard
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- Clint
- intrinsic clearance
- DMSO
- dimethyl sulfoxide
- MRM
- multiple reaction monitoring
- CE
- collision energies
- DP
- declustering potentials
- NCE
- new chemical entities
- PF-05218881
- (E)-3-(4-((2S,3S,4S,5R)-5-1-(3-chloro-2,6-difluorobenzyloxyimino)ethyl)-3,4-dihydroxytetrahydrofuran-2-yloxy)-3-hydroxyphenyl)-2-methyl-N(3aS,4R,5R,6S,7R,7aR)-4,6,7-trihydroxyhexahydrobenzo[d][1,3]dioxol-5-yl) acrylamide.
- Received October 25, 2011.
- Accepted February 10, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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