P450 / CYP Inhibition

Inhibition of cytochrome P450 (CYP450) enzymes is one of the major reasons for drug-drug interactions (DDI), as they play a significant role in the metabolism of both endogenous and exogenous compounds.

Therefore, detailed CYP inhibition profiles are now required for the registration of novel molecular entities. Using either liver microsomes or recombinant CYP preparations like Bactosomes, in combination with specific probe substrates, we can assess inhibition of individual CYPs by your discovery compounds. As a reliable and cost-effective initial screening we recommend assessing inhibition potencies against the 5 most relevant CYP isoforms, i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 using Cypex Bactosomes. 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. Please see Weaver R. et al., 2003, DMD 31:7, p. 955-966 for reference.

By introducing a 30 minute pre-incubation in the absence or presence of NADPH, we can distinguish between direct, time-dependent, and metabolism-dependent inhibition. Our inhibition assays are generally performed using seven concentrations covering four orders of magnitude. Please feel free to inquire about more detailed investigations, e.g. Ki/KI determination.

Direct inhibition

Direct inhibition, sometimes referred to as reversible inhibition, is the most basic form of enzyme inhibition. It is assessed by measurement of an enzyme activity in the presence of increasing concentration of inhibitor without a pre-incubation step. This is the classic in vitro inhibition assay which, if positive, yields an IC50-value as a result.

Sygnature’s Standard Reversible CYP450 Inhibition assay utilises drug-like probe substrates, HLM and the Phase I cofactor, NADPH. 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. 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.

To discuss the availability of other CYP isoform inhibition assays, contact us.