High-Throughput, 384-Well, LC-MS/MS CYP Inhibition Assay Using Automation, Cassette-Analysis Technique, and Streamlined Data Analysi
| Autor: | Jason Halladay, Susan Wong, Daniel Tran, Leslie Wang, S. Cyrus Khojasteh, Erlie Marie Delarosa |
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| Rok vydání: | 2011 |
| Předmět: |
Male
Metabolite Clinical Biochemistry Pharmaceutical Science Automation Inhibitory Concentration 50 chemistry.chemical_compound Tandem Mass Spectrometry Lc ms ms medicine Cytochrome P-450 Enzyme Inhibitors Humans Inhibitory concentration 50 Pharmacology (medical) Column switching Enzyme Inhibitors Incubation Chromatography Biochemistry (medical) CYP1A2 Substrate (chemistry) High-Throughput Screening Assays Isoenzymes chemistry Phenacetin Microsomes Liver Female Chromatography Liquid medicine.drug |
| Zdroj: | Drug metabolism letters. |
| ISSN: | 1874-0758 |
| Popis: | Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature. |
| Databáze: | OpenAIRE |
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