CYP Testing

The cytochrome P450 family (CYP) is a large and diverse group of enzymes. The function of most CYP enzymes is to catalyze the oxidation of organic substances, including xenobiotic substances such as drugs. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for about 75% of the total drug metabolism.¹

In September 2006 the FDA released a draft guidance for the drug industry, "Drug Interaction Studies - Study Design, Data Analysis, and Implications for Dosing and Labeling." In this guidance the FDA outlines the assays and methods recommended to test the potential of drug candidates to cause drug-drug interactions. The main assays associated with drug-drug interactions are:

• CYP inhibition
• CYP phenotyping
• CYP induction

Apredica offers CYP assays in both screening and IND-enabling modes. Contact us to learn more our preclinical CRO services.

Drug-Drug Interaction

Drugs may increase or decrease the activity of various CYP isozymes, either by inducing the biosynthesis of an isozyme (CYP induction) or by directly inhibiting the activity of the CYP (CYP inhibition). This is a major source of adverse drug interactions, as changes in CYP enzyme activity may affect the metabolism and clearance of various drugs. For example, if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate within the body to toxic levels, possibly causing an overdose. Early CYP profiling to identify the potential for these drug interactions can guide drug-discovery efforts to compounds less likely to have toxic effects.

CYP Inhibition Assays

• CYP inhibition screen or IC50 testing
• Available CYP enzyme assays:
• CYP1A2
• CYP2B6
• CYP2A6
• CYP2C8
• CYP2C9
• CYP2C19
• CYP2D6
• CYP2E1
• CYP3A4
• CYP3A5
• Both LC/MS/MS and fluorescent assays are available
• CYP Ki determination

CYP Time-Dependent Inhibition (CYP TDI)

CYP time-dependent inhibition (also referred to as mechanism-based inhibition) usually results from irreversible or quasi-irreversible binding of test compound or its metabolite to the active site of a CYP enzyme during the biotransformation process. Thus, each generated metabolite may inactivate the enzyme further, and the percent inhibition increases with the incubation time. Based on this mechanism, CYP time-dependent inhibition can be identified by two sets of simultaneous experiments: one is to pre-incubate enzyme (typically for 30 min) with a test compound in the presence and absence of NADPH. The residual CYP enzyme activity is then analyzed by incubation of diluted pre-incubation mixture with a probe substrate. If the test compound is a time-dependent inhibitor, it would show a higher percent inhibition after a pre-incubation in the presence of NADPH.

Both recombinant enzyme and liver microsomes can be used in the CYP time-dependent inhibition assay, but the liver microsomes are recommended because the metabolite that inactivates a CYP isozyme can also be generated by other CYP isozymes.

CYP Induction Assays

Some chemicals can induce the activity of CYP enzymes. These inducers will increase the metabolism of the co-administered drugs that are the substrates of the induced C YP enzymes, resulting in concomitant drugs' losing efficacy. CYP enzymes such as CYP1A2, CYP2B6, and CYP3A4 are susceptible to induction. Apredica's in vitro induction screening assays allow identification of compounds that may induce the activity of drug metabolizing enzymes, creating the potential for drug-drug interactions.

The following CYP induction assays are available in your choice of fresh or cryo-preserved hepatocytes; or human, rat, or HepaRG cells:

• CYP1A induction
• CYP3A induction
• CYP2B6 induction

Specific activity of the CYPs is measured relevant to controls. The result is expressed as fold induction (by comparing to negative control activity) and/or % of positive control (by comparing net activity changes between test-compound-treated cells and positive-control-treated cells). An induction assay is considered valid if the positive control yields a greater than 2-fold induction. A test compound is considered an in vitro CYP inducer if displays a greater than 40 % of the positive control activity.

CYP Phenotyping (Metabolism) Assays

Profiling of which CYPs are metabolizing your compound is determined using either rCYP enzymes or 10 characterized individual donors.

Contact us to learn more about Apredica's CYP testing services.

References

1. Guengerich FP (January 2008). "Cytochrome p450 and chemical toxicology". Chem. Res. Toxicol. 21 (1): 70-83. doi:10.1021/tx700079z. PMID 18052394.