Bioduro

中文简体

Let's talk
Contact us

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.

 

CYPSubstrateInhibitor
1A2PhenacetinFurafylline
2B6BupropionThiotepa
2C8PaclitaxelGemfibrozil glucoronide
2C9DiclofenacTienilic acid
2C19OmeprazoleFluoxetine
2D6DextromethorphanParoxetine
3A4MidazolamMifepristone

 

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.