P450 Inhibition

Purpose

Many drug-drug interactions involve cytochrome P450 (CYP) inhibition. In the U.S. FDA Guidance for Industry, it is recommended that the ability of a new drug entity to inhibit CYP isozymes be assessed. Making this evaluation prior to drug candidate selection can reduce the odds of failure during the development process. An accepted method to accomplish this goal is to measure the inhibitory activity of drug leads on individual isozymes of human cDNA expressed CYP.

Assay Protocol

Inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 is measured in separate assays using specific substrates that become fluorescent upon CYP metabolism. Test articles dissolved in DMSO or ACN are serially diluted in a 96-well plate containing incubation/NADPH regenerating buffer. Individual isozymes and CYP-specific substrates are added and incubated at 37°C. The plates are read on a Tecan SpectaFluor Plus and the IC50 (concentration at 50% inhibition) is determined by comparing the activity of enzyme in the presence and in the absence of the test articles. Known positive controls for each isozyme are used each time the assay is run.

Confirmation Data

The results for the positive inhibition controls for each isozyme are tabulated and compared to published results (*).