A high-throughput method for enzyme kinetic studies.
A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.
Department of Biology and Bioanalytical Chemistry, ArQule, Inc., Woburn, MA 01801, USA. lsaraswat@arqule.com
Lakshmi D
LD
Saraswat
Kimberley A
KA
Caserta
Kathy
K
Laws
Darren
D
Wei
Simon S
SS
Jones
Adedayo
A
Adedoyin
D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism / Q000494:pharmacology; D004008:Diclofenac / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004798:Enzymes / Q000187:drug effects / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008874:Midazolam / Q000378:metabolism / Q000494:pharmacology; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000378:metabolism / Q000494:pharmacology; D014859:Warfarin / Q000378:metabolism / Q000494:pharmacology
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004798:Enzymes; D004008:Diclofenac; D013739:Testosterone; D014859:Warfarin; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D008874:Midazolam