Chapter 32 - CYP Inhibition Methods

Abstract

Cytochrome P450 (CYP) inhibition occurs when two drugs are co-administered and one drug preferentially binds to a CYP metabolizing enzyme that is responsible for appreciable clearance of the second drug. This inhibits clearance of the second drug and can cause toxicity. The binding can be reversible or irreversible. in vitro assays for reversible CYP inhibition co-incubate the test compound with a “probe substrate” that is metabolized by a specific CYP enzyme and, if the metabolic rate of the probe substrate is reduced, inhibition is indicated. Screening is performed at a single test compound concentration. An IC50 is obtained by running a wide range of test compound concentrations. Various methods have been used, which vary in the CYP enzyme source (human recombinant CYP isozymes or human liver microsomes (HLM)), probe substrates (fluorogenic or drugs), and measurement method (fluorescence plate reader or liquid chromatography/mass spectrometry (LC/MS/MS)). The most common reversible method uses HLM, with their full complement of CYP isozymes, a cocktail of probe substrates, each specifically metabolized by a different isozyme, and LC/MS/MS measurement. Definitive reversible methods use single probe substrates and optimized enzymology for highest correlation to human clinical pharmacokinetic effects of CYP inhibition. Irreversible “time dependent inhibition” (TDI) methods pre-incubate the test compound with microsomes and reduced nicotinamide adenine dinucleotide phosphate to allow reactive metabolites to form and react with the enzyme. CYP drug probe substrates are then added to detect the loss of enzyme activity. TDI is indicated if the CYP activity or the IC50 is reduced by pre-incubation. TDI screening uses a single (or limited) test compound concentration and single (or limited) pre-incubation time. More definitive TDI methods use a range of test compound concentrations and multiple pre-incubation times to obtain kinact, the maximum inactivation rate constant, and KI, the inhibitor concentration producing half of the maximum inactivation rate (1/2 kinact).