Cytochrome P450 (CYP450) Profiling

The CYP450 family of enzymes account for the majority of phase I metabolism. Our in vitro ADME services offer phenotyping of test compounds with individual CYP450 isoforms to provide important information regarding metabolism and can be useful as quick screens for lead optimization. In addition, CYP450 inhibition and induction are common causes for drug-drug interactions. The inhibition of a particular CYP450 isoform by a test compound may lead to potentially harmful accumulations of other drugs that are normally metabolized by the inhibited CYP450. The induction of a particular CYP450 isoform by a test compound may lead to the loss of efficacy of co-administered drugs due to increased metabolism by the induced isoforms. Thus, our in vitro ADME services can provide important information to understand and quantify the effect of test compounds on CYP450 activity.

CYP450 Phenotyping

Two types of CYP450 phenotyping studies are provided. Firstly, assessment of test compound metabolism by individual recombinant CYP450 isoforms. Incubation solutions are prepared containing the individual human recombinant CYP450 isoform. The reaction is initiated with the addition of the co-factor NADPH and an aliquot of the solution is taken at pre-determined intervals. The peak area of the test compound at each time point is compared to the peak area of the time zero sample. A control reaction (without co-factor NADPH) is used in order to assess the amount of thermal breakdown, insolubility and non-specific binding that contributes to the overall loss of the test compound.

Secondly, the inhibition of test compound metabolism in liver tissue, typically microsomes, is tested using archetypical inhibitors of each CYP450 isoform.

CYP450 Inhibition

Solutions of specific CYP450 recombinant isoforms and their associated substrate are generated with a range of test compound concentrations to measure the inhibition of the standard CYP450 activity by the test compound. The reaction is initiated with the addition of the co-factor NADPH and following incubation, the reaction is quenched. The dose-response curve for each isoform is generated to determine the extent of CYP450 inhibition, if any, by the test compound.

Figure 1. Typical CYP Inhibition Data Generated at WLI for Standard Inhibitors

As well as using recombinant CYP450 isoforms, liver microsomes can also be used as an alternative tissue source in this work.

CYP450 Induction

The CYP450 induction studies are performed in plated hepatocytes containing a Matrigel ® overlay. The hepatocytes are exposed to vehicle, standard inducer, or test compound for an induction period of 72 hours. Test compounds are typically evaluated at three concentrations spanning two orders of magnitude. The culture media is exchanged every 24 hours to provide fresh exposure to vehicle, standard inducer, or test compound. The major mechanisms of CYP450 induction involve activation of the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), or the pregnane X receptor (PXR).

After the induction period, the metabolic activity of the hepatocytes is assayed by incubating the cells with appropriate substrates and measuring the fold induction as compared to vehicle. Typically, the induction of the following isoforms (and their associated pathways) is evaluated: 1A (AhR), 2B6 (CAR), and 3A (PXR).

Figure 2. Typical CYP Induction Data Generated at WLI for Standard Inducers