Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes.
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.
Chu
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Department of Drug Metabolism, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Favreau
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Soares
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T
Lin
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Cc
Nomeir
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D001274:Atmospheric Pressure; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis*; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D003917:Dextrorphan / Q000737:chemistry / Q000494:pharmacology; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005069:Evaluation Studies as Topic; D006801:Humans; D006915:Hydroxytestosterones / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006915:Hydroxytestosterones; D003917:Dextrorphan; C025591:6 beta-hydroxytestosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human