Time Dependent Inhibition

Time dependent inhibition (TDI) of CYPs refers to a change in enzyme inhibition during an in vitro incubation or dosing period in vivo. Riley, Grime and Weaver, Expert Opin Drug Metab Toxicol. 2007 Feb;3(1):51-66.

TDI can be the cause of serious drug-drug interactions (DDI). TDI is usually measured by comparing potency (IC₅₀) in direct inhibition to potency after a 30 minute pre-incubation in the absence of NADPH. In the case of a TDI the resulting IC₅₀-value will be significantly lower than the IC₅₀-value observed in the direct inhibition experiment.

The cytochrome P450 (CYP450) enzymes play a significant role in the metabolism of both endogenous and exogenous compounds. Within this family, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are predominantly involved in the metabolism of drugs.

The measurement of the inhibition of each of these CYP450 enzymes in human liver microsomes (HLM) helps in predicting the potential for drug-drug interactions with co-administered drugs and in understanding the subsequent clinical consequences.

Sygnature’s Standard Time-Dependent CYP450 Inhibition assay (IC₅₀ Shift) utilises drug-like probe substrates, HLM and the Phase I cofactor, NADPH and two pre-incubation times, 0 and 30 minutes. The assay monitors for the formation of the metabolites of the drug-like probe substrates in the absence and presence of a compound by LC-MS/MS following a pre-incubation period. All assays have two replicates per compound and include a positive control inhibitor. Data output consists of the generation of an inhibition constant or IC₅₀ value.