Abstract
In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.
Copyright 2000 John Wiley & Sons, Ltd.
MeSH terms
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Atmospheric Pressure
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Chromatography, High Pressure Liquid / methods*
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Cytochrome P-450 CYP2D6 / analysis*
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Cytochrome P-450 CYP2D6 Inhibitors*
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Cytochrome P-450 CYP3A
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Cytochrome P-450 Enzyme Inhibitors*
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Cytochrome P-450 Enzyme System / analysis*
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Dextrorphan / chemistry
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Dextrorphan / pharmacology
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Drug Design
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Drug Evaluation, Preclinical
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / pharmacology
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Evaluation Studies as Topic
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Humans
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Hydroxytestosterones / chemistry
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Hydroxytestosterones / pharmacology
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In Vitro Techniques
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Mass Spectrometry / methods*
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Microsomes, Liver / enzymology*
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Mixed Function Oxygenases / analysis*
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Mixed Function Oxygenases / antagonists & inhibitors*
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Reproducibility of Results
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Substrate Specificity
Substances
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Cytochrome P-450 CYP2D6 Inhibitors
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Cytochrome P-450 Enzyme Inhibitors
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Enzyme Inhibitors
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Hydroxytestosterones
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Dextrorphan
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6 beta-hydroxytestosterone
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Cytochrome P-450 Enzyme System
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Mixed Function Oxygenases
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CYP3A protein, human
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Cytochrome P-450 CYP2D6
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Cytochrome P-450 CYP3A
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CYP3A4 protein, human