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Cytochrome P450 (CYP) time-dependent inhibition assay (TDI) (single-point or IC50 shift)

 

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

 

Assay Description:

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.

 

CYP Substrate Inhibitor
1A2 Phenacetin Furafylline
2B6 Bupropion Thiotepa
2C8 Paclitaxel Gemfibrozil glucoronide
2C9 Diclofenac Tienilic acid
2C19 Omeprazole Fluoxetine
2D6 Dextromethorphan Paroxetine
3A4 Midazolam Mifepristone

 

Data Analysis:

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.

 

Abbreviations:

DDI                      Drug-drug interaction

DMSO                  Dimethylsulfoxide

HPLC                   High-performance liquid chromatography

LC                        Liquid chromatography

MS                       Mass Spectrometry

NADPH                Nicotinamide adenine dinucleotide phosphate

TDI                      Time-dependent inhibition / Time-dependent inhibitor

 

Literature:

  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).
  4. FDA Draft Guidance for Industry – Drug Interaction Studies (2012); https://www.fda.gov/downloads/drugs/guidances/ucm292362.pdf
  5. 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).
  6. 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).
  7. 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).
  8. GraphPad Software, Inc., 7825 Fay Avenue, Suite 230, La Jolla, CA 92037, USA.

 

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