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Cytochrome P450 (CYP) inhibition assay (DDI)

Readout: % inhibition at single concentration, IC50 or Ki

Cyp Isoforms: 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4

Controls: Known isoform-specific inhibitors

CYP Substrate Inhibitor
1A2 Phenacetin Naphthoflavone
2B6 Bupropion Ticlopidine
2C8 Paclitaxel Monteleukast
2C9 Diclofenac Sulfaphenazole
2C19 Omeprazole Tranylcypromine
2D6 Dextromethorphan Quinidine
3A4 Testosterone Ketoconazole

Assay Description:

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).

Data Analysis:

IC50 or Ki determination.

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

DMSO                     Dimethylsulfoxide

HPLC                       High-performance liquid chromatography

LC                             Liquid chromatography

MS                           Mass Spectrometry



  1. Slaughter, R. L.; Edwards, D. J.; “Recent advances: the cytochrome P450 enzymes”,   Pharmacother. 29, 619, (1995).
  2. Fowler, S.; Zhang, H.; “In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug–Drug Interactions”, AAPS J. 10, 410, (2008).
  3. Obach, R. S., et al. “The Utility of in Vitro Cytochrome P450 Inhibition Data in the Prediction of Drug-Drug Interactions”; J. Pharmacol. Exp. Ther. 316, 336, (2006).

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