Readout: % inhibition or IC50 and shifted IC50
Cyp Isoforms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4
Controls: Known isoform-specific inhibitors
Test compounds and control inhibitors (single concentration for single-point; a range of test concentrations, typically 0.1 – 50 µM, for IC50) are separately pre-incubated with human liver microsomes with or without NADPH for 30 min. Following pre-incubation, CYP isoform-specific substrates (see table below) are added to measure the residual enzyme activity and then incubated for a further 20 minutes. Identical incubations are run in parallel lacking the pre-incubation step (or with pre-incubation lacking NADPH). 100% and 0% activity control incubations are run for each part of the experiment. Plotting percent control enzyme activity against log(inhibitor concentration) gives sigmoidal inhibition curves for each set of data. When the inhibition curve is shifted to lower
IC50 value by pre-incubation treatment, this is an indication of TDI. The MS detection is performed by using a Sciex API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC.
The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area).
Single-point: Mean percentage inhibition following pre-incubation
IC50 values are calculated from the substrate conversion rate versus inhibitor concentration plots using the following equation:
R = Rmax / (1 + (I / IC50))
R = substrate conversion rate
Rmax = max. substrate conversion rate
I = initial concentration of inhibitor
A fold shift greater than 1.5 fold is considered to be significant, and the compound is classified as a time-dependent inhibitor.
DDI Drug-drug interaction
HPLC High-performance liquid chromatography
MS Mass spectrometry
NADPH Nicotinamide adenine dinucleotide phosphate
TDI Time-dependent inhibition