- Cytochrome P450 CYP) represents a family of more than 50 individual isozymes which play a major role in the metabolism of drugs. The most important enzymes involved in drug metabolism are CYP3A4, CYP2C9, CYP2D6, CYP2C19, and CYP1A2.1
- The human liver microsomal assay is accepted as the ‘gold standard’ for in vitro DDI assessment,2 and in vitro data are generally very predictive of DDI in vivo.3
- Time-dependent inhibition (TDI) of cytochrome P450 can cause clinically relevant DDI and lead to termination of development, withdrawal from the market, or severe restriction on the use of new therapies. Examples of TDI of CYP3A include the HIV protease inhibitors ritonavir and saquinavir, the macrolide antibiotics erythromycin and clarithromycin, and the calcium channel blockers verapamil and diltiazem.
- In general, TDI results from irreversible covalent binding or quasi-irreversible noncovalent tight binding of a chemically reactive intermediate to the enzyme that catalyzes its formation, resulting in loss of enzyme function. In some cases, TDI could result from reversible inhibition from a metabolite(s) generated in situ.
- Recent guidance from the US Food and Drug Administration (FDA) has advocated testing of time-dependent inhibition of cytochrome P450, which can be addressed by assessing whether there is any change in the IC50 value for a test inhibitor with or without inhibitor pre-incubation.4-7
Readout: % inhibition, IC50 value and shifted IC50 value, KI and Kinact
Cyp Isoforms: 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4
Controls: Known isoform-specific time-dependent 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 NADPH for either 0 min or 30 min at 37 °C.
Following pre-incubation, CYP isoform-specific substrates (see table above) are added to measure the residual enzyme activity and the mixtures are incubated for another 20 minutes, after which the reactions are quenched with acetonitrile/methanol (1:1, v/v). 100% and 0% activity control incubations are run for each part of the experiment.
The samples are analyzed by LC-MS/MS.The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Plotting percent control enzyme activity against log(inhibitor concentration) gives sigmoidal inhibition curves for each set of data.
Test compounds which exhibit time dependent inhibition will give a lower IC50 with a 30 min pre-incubation with human liver microsomes in the presence of NADPH compared to a 0 min pre-incubation and an IC50 shift can be calculated.
Single-point: Mean percentage inhibition following pre-incubation
IC50 values are calculated applying the 4 parameter logistic regression model using GraphPad prism.8
IC50 shift is the ratio of IC50 (no pre-incubation) divided by the IC50 (30 min pre-incubation).
IC50 shift = IC50 (no pre-incubation) / IC50 (30 min pre-incubation)
A shift greater than 1.5 fold is considered to be significant, and the compound is classified as a time-dependent inhibitor.
If an IC50 shift is observed, KI and kinact could be determined. Please contact us for further information.
DDI Drug-drug interaction
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass Spectrometry
NADPH Nicotinamide adenine dinucleotide phosphate
TDI Time-dependent inhibition / Time-dependent inhibitor
- Slaughter, R. L.; Edwards, D. J.; “Recent advances: the cytochrome P450 enzymes”; Pharmacother. 29, 619, (1995).
- 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).
- 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).
- FDA Draft Guidance for Industry – Drug Interaction Studies (2012); https://www.fda.gov/downloads/drugs/guidances/ucm292362.pdf
- Berry, L. M.; Zhao, Z.; “An Examination of IC50 and IC50-Shift Experiments in Assessing Time-Dependent Inhibition of CYP3A4, CYP2D6 and CYP2C9 in Human Liver Microsomes”; Drug Metab. Lett., 2, 51, (2008).
- Berry, L. M.; Zhao, Z.; “Dynamic Modeling of Cytochrome P450 Inhibition In Vitro: Impact of Inhibitor Depletion on IC50 Shift”; Drug Metab. Dispos., 41, 1433, (2013).
- Grimm, S. W.; et al. “The Conduct of in Vitro Studies to Address Time-Dependent Inhibition of Drug-Metabolizing Enzymes: A Perspective of the Pharmaceutical Research and Manufacturers of America”; Drug Metab. Dispos., 37, 1355, (2009).
- GraphPad Software, Inc., 7825 Fay Avenue, Suite 230, La Jolla, CA 92037, USA.
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