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

 

 

Readout: % inhibition or IC50 and shifted IC50

Cyp Isoforms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4

Controls: Known isoform-specific 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 or without NADPH for 30 min. Following pre-incubation, CYP isoform-specific substrates (see table below) are added to measure the residual enzyme activity and then incubated for a further 20 minutes. Identical incubations are run in parallel lacking the pre-incubation step (or with pre-incubation lacking NADPH). 100% and 0% activity control incubations are run for each part of the experiment. Plotting percent control enzyme activity against log(inhibitor concentration) gives sigmoidal inhibition curves for each set of data. When the inhibition curve is shifted to lower

IC50 value by pre-incubation treatment, this is an indication of TDI. The MS detection is performed by using a Sciex API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC.

The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area).

CYPSubstrateInhibitor
1A2Phenacetin Furafylline
2C9DiclofenacTienilic acid
2C19OmeprazoleFluoxetine
2D6DextromethorphanParoxetine
3A4TestosteroneMifepristone

 

Data Analysis:

Single-point: Mean percentage inhibition following pre-incubation

IC50 values are calculated from the substrate conversion rate versus inhibitor concentration plots using the following equation:

R = Rmax / (1 + (I / IC50))

R = substrate conversion rate

Rmax =  max. substrate conversion rate

I = initial concentration of inhibitor

A fold shift greater than 1.5 fold is considered to be significant, and the compound is classified as a time-dependent inhibitor.

 

Abbreviations:

DDI                          Drug-drug interaction

DMSO                     Dimethylsulfoxide

HPLC                       High-performance liquid chromatography

MS                           Mass spectrometry

NADPH                   Nicotinamide adenine dinucleotide phosphate

TDI                           Time-dependent inhibition

 

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. www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf