Cytochrome P450 time dependent inhibition: IC50 shift assay

Aim: To determine time-dependent inhibition of test compound for cytochrome P450 in human liver microsomes by the IC50 shift assay.

Background information: Cytochrome P450 (CYP) inhibition is one of the most common types of drug-drug interactions (DDI). The inhibition mechanism can either be a reversible or irreversible (time dependent) interaction and IC50 shift allows to determine it. Irreversible inhibition is undesired since it usually implies the formation of a covalent bond between the enzyme and the metabolite.

The Cytochrome P450 time dependent inhibition assay determines the IC50 value under three different experimental conditions: 0 min pre-incubation and 30 min pre-incubation with/without NADPH. Following the pre-incubation, isoform-specific substrates are incubated individually with human liver microsomes in a range of test compound concentrations. At the end of the incubation, the formation of metabolite is monitored by UPLC-MS/MS at each of the test compound concentrations. A decrease in the formation of the metabolites compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).If the compound is a time-dependent inhibitor a shift to the left (increase in potency) occur between the 30 min pre-incubation without and with NADPH. The ratio of these values gives the IC50 shift.

Compound requirement: Dependent on number of isoforms assessed (2 mg / 1 CYP)

Turnaround time: 1 week / 1 compound / 1 isoform

Experimental design:

  • Test compound concentration: 8 different concentrations available
  • Number of replicates: 2
  • Pre-incubation time: 30 min (+/- NADPH) and 0 min
  • CYP isoforms: CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (other isoforms available)
  • Controls: known isoform specific inhibitors
  • Analytical method: UPLC-MS/MS quantification

Delivered results:

  • IC50 shift for the CYP isoform tested