Readout: % inhibition at single concentration, IC50, or Ki
Cyp Isoforms: CYP3A4, CYP2C9, CYP2D6, CYP2C19, CYP1A2
Controls: Known isoform-specific inhibitors
CYP isoform-specific substrates (see table below) are incubated with human liver microsomes at a single concentration or at a range of test compound concentrations (typically 0.1 – 50 µM). At the end of the incubation, the amount of parent remaining relative to each substrate is monitored by LC-MS/MS at each of the test compound concentrations. Typical experiments for determining IC50 values involve incubating the substrate at concentrations below its KM. For Ki determinations, both the substrate and inhibitor concentrations are varied to cover ranges above and below the drug’s KM and inhibitor’s Ki.
The MS detection is performed by using a Sciex API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2. C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area).
IC50 or Ki detemination.
Typically compounds can be categorized as follows:
Potent inhibition: IC50 < 1 µM
Moderate inhibition: 1 µM < IC50 < 10 µM
No or weak inhibition: IC50 > 10 µM
DDI Drug-drug interaction
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass Spectrometry