CYP450 Time Dependent Inhibition (TDI) Assay

It is vital for researchers occupied in drug discovery to evaluate parameters of DDIs for the development of safe and effective drug candidates. Creative Bioarray offers TDI assays, including IC50 shift and Kinact/KI assay. We deliver accurate data and detailed experimental protocols to meet your needs.

CYP450 TDI Assay Introduction

  • Why CYP450 TDI Assay?
    • It is widely accepted that the current therapy of multi-drugs inescapably increases the likelihood of drug-drug interactions (DDI). Serious DDIs are a significant risk for new molecular entities (NCE).
    • A large proportion of adverse drug reactions in the clinic can be attributed to drug-drug interactions. Most cited pharmacokinetic DDIs have involved cytochrome P450s (CYPs), responsible for metabolizing 80% of drugs.
    • The forms of CYP inhibition can be roughly divided into two categories. One of them is the inhibition of CYP activity mediated by the drug itself (direct inhibition, DI). Another form is the inhibition mediated by the metabolites derived from the drug (time-dependent inhibition, TDI). In most cases, the inhibition caused by the time-dependent inhibition is irreversible.
    • The consequences of irreversible inhibition are considered to be more severe than reversible inhibition. IC50 shift assay is possible to distinguish between reversible and irreversible inhibition. The kinact and KI values determined can be used to predict the risk of drug-drug interactions.
  • The relationship between IC50 shift and kinact/ KI

CYP450 time-dependent inhibition IC50 shift assay can identify reversible and time-dependent inhibitors. IC50 values often quantify the potential of enzyme-inhibiting drugs. The IC50 change test is currently the standard method used for the preliminary evaluation of TDI.

However, with IC50 shift assay, problems can arise when dealing with irreversible or mechanism-based inhibitors (MBI). The IC50 value of MBI is time-dependent and can cause serious issues when ranking the inhibitory potential of different compounds. Therefore, most studies and ranking schemes associated with MBI depend on the inhibition constant (KI) and enzyme inactivation rate (kinact) instead of the IC50 value.

The kinact/KI assay determines the Kinetic constant of time-dependent inhibition. kinact is the maximum rate of enzyme inactivation at the saturation concentration of the inhibitor, while KI is the concentration of the inhibitor, which gives half of the maximum rate of inactivation. The experimental conditions are determined based on previously performed assays, such as P450 reversible inhibition and time-dependent inhibition: single point or IC50 shift.

Schematic flow chart for identification and characterization of P450 TDIFigure 1. Schematic flow chart for identification and characterization of P450 TDI (Grimm, Scott W., 2009).

Brief Protocol

  • IC50 Shift
    • The IC50 change determination determined the IC50 values (concentrations that produce 50% inhibition) of the test compound under three different experimental conditions: 0-minute pre-incubation, 30-minute pre-incubation minus NADPH, and 30-minute pre-incubation plus NADPH.
    • After pre-incubation with/without NADPH, the isoform-specific substrate is added to measure the residual enzyme activity. No dilution step is required, and the incubation is run under linear conditions.
    • If the compound is a time-dependent inhibitor, a shift to the left (increased potency) will occur between the 30-minute pre-incubation minus NADPH and the 30-minute pre-incubation plus NADPH. The ratio of these two values gives the IC50 offset.
IC50 Shift Assay
CYP IsoformsCYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4
Test SystemHuman microsomes
Pre-Incubation Time30 min (+/- NADPH) and 0 min
Number of Replicatesn=3 per concentration point
Test Concentration0.1, 0.25, 1, 2.5, 10, 25 μM
Analysis MethodLC-MS/MS
Data DeliveryIC50, Shifted IC50
  • kinact/KI Assay
    • By pre-incubating the selected test compounds concentrations (plus carrier control) with human liver microsomes and pre-incubation times (including 0 minutes) to achieve the scale from no inactivation to maximum inactivation to evaluate the compound NADPH.
    • After the pre-incubation, the pre-incubated aliquot is diluted with a buffer containing the specific cytochrome P450 probe substrate and NADPH for a particular incubation time.
    • After the minor square regression analysis determines the negative slope of the logarithm of the remaining activity percentage and the pre-incubation time, kinact and KI can be calculated by nonlinear regression analysis of the negative gradient of the inhibitor concentration.
kinact and KI Assay
Test Concentration5 concentrations
Substrates and CYP IsoformsPhenacetin (CYP1A2), bupropion (CYP2B6), paclitaxel (CYP2C8), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4)
Test SystemHuman liver microsomes
Pre-Incubation Time7 Pre-incubation times (0, 5, 15, 30, 45, 60, 120 minutes)
Number of ReplicatesN = 3 per time point
Analysis MethodLC-MS/MS
Data Deliverykinact, KI

Quotation and Ordering

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  1. Riley RJ, Wilson CE. Cytochrome P450 time-dependent inhibition and induction: advances in assays, risk analysis and modelling. Expert Opin Drug Metab Toxicol. 2015 Apr;11(4):557-72.
  2. Kozakai K, Taniguchi H. Cocktail-substrate approach-based high-throughput assay for evaluation of direct and time-dependent inhibition of multiple cytochrome P450 isoforms. Drug Metab PharmacoKInet. 2014;29(2):198-207.
  3. Grimm, Scott 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 Metabolism and Disposition 37.7 (2009): 1355-1370.

For research use only. Not for any other purpose.