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Published on: December 18, 2020
An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS
Kuresh A Youdim1, Richard Lyons, Leon Payne
1Pfizer Global Research and Development, Pharmacokinetics Dynamic and Metabolism, Sandwich, Kent, UK CT13 9NJ, United Kingdom. kuresh.youdim@pfizer.com
Journal of Pharmaceutical and Biomedical Analysis
|July 1, 2008
Summary
This study developed an automated IC50 cocktail assay using human liver microsomes and a mix of probe substrates for major CYPs. The assay provides rapid, high-throughput CYP inhibition analysis, crucial for drug discovery and predicting clinical drug-drug interaction risk.
Area of Science:
- Pharmacology
- Biochemistry
- Analytical Chemistry
Background:
- Cytochrome P450 (CYP) enzymes are critical for drug metabolism.
- Assessing CYP inhibition is vital for predicting drug-drug interactions (DDIs).
- Existing methods for measuring CYP inhibition can be time-consuming and low-throughput.
Purpose of the Study:
- To develop a miniaturized, automated IC50 cocktail assay for high-throughput CYP inhibition screening.
- To significantly reduce analytical run times using rapid resolution liquid chromatography-mass spectrometry (LC-MS/MS).
- To validate the assay's performance by comparing IC50 values from cocktail and single-substrate incubations.
Main Methods:
- Development of a 384-well automated assay format.
- Utilized human liver microsomes with a cocktail of five major CYP probe substrates.
- Employed rapid resolution liquid chromatography-mass spectrometry (LC-MS/MS) for analysis with short run times (1 min).
- Determined IC50 values for eight reference compounds in both cocktail and single-substrate assays.
Main Results:
- The automated cocktail assay successfully determined IC50 values for multiple CYP enzymes simultaneously.
- IC50 values from cocktail incubations closely matched those from single-substrate experiments.
- Achieved significantly reduced LC-MS/MS run times (as short as 1 minute) without compromising data integrity.
- Demonstrated increased sample throughput compared to existing cocktail assay methodologies.
Conclusions:
- The developed miniaturized, high-throughput cocktail IC50 assay is a robust and efficient tool for CYP inhibition screening.
- The assay's rapid analytical run times enhance sample throughput for drug discovery libraries.
- The method provides reliable IC50 data, supporting accurate prediction of clinical DDI risk.
