Cytochrome P450 Inhibition Information Leaflet
The cytochrome P450 (CYP) enzymes play a critical role in the metabolism of many drug, and alterations to the activity of these enzymes caused by co-administered drugs may lead to metabolic drug-drug interactions (DDI). In vitro tools are available for early detection of potential DDI perpetrators and are an integral part of early safety assessment in the drug discovery process.
Compounds are incubated at increasing concentrations with microsomes in the presence of CYP specific substrates and enzyme co-factor at 37°C. After the incubation, samples are quenched and analysed by LC-MS. IC50 is calculated using GraphPad Prism.
Standard Assay Conditions
- 0.2 mg/mL human liver microsomal protein concentration
- 7 CYP isoforms available:
- CYP1A2
- CYP2B6
- CYP2C8
- CYP3A4 (Midazolam)
- CYP2C9
- CYP2C19
- CYP2D6
- CYP3A4 (Testosterone)
- FDA approved marker inhibitors/substrates
- 7 compound concentrations tested:
- 0.04 — 50.00 µM
- pH 7.4 phosphate buffer
- NADPH co-factor
- 1% organic solvent final in the incubation mixture
- N=2 replicates
- Analysis by LC-MS
Figure 1. IC50 curve of Sulfaphenazole (marker inhibitor compound) with Diclofenac (2C9 substrate) . % inhibition relative to solvent control. Data from three experiments; 2 replicates per experiment.
Figure 2. Geomean of pIC50 values for all isoforms. Data from three experiments.
Compound Requirements
100 µL of a 10 mM stock or 1 mg of solid (accurately weighed to 2 decimal places)
Deliverables
Graphical representation of the inhibition curves and IC550 values in µM for all CYP isoforms tested
