Background:
Cytochrome P450 (CYP) enzymes represent a heme containing protein superfamily metabolizing a broad variety of xenobiotics, including drugs and toxic chemicals. 11 CYP families are expressed in a human liver and gastrointestinal tract (CYP1A2, CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and CYP3A4/5). Five major isoforms (CYPs 1A2, 3A4, 2C9, 2C19, and 2D6) are involved in about 95% of the known drug metabolism. Recent studies showed the increasing role of CYP2C8 and CYP2B6 in metabolism of numerous drugs and important endogenous compounds. Cytochrome P450s are of critical importance due to the two of the most significant problems in clinical pharmacology: metabolism-mediated drug-drug interactions (DDI) and individual variability in drug metabolism. It is important to evaluate the potential inhibition of a new drug candidate for the most clinically relevant CYP450 enzymes. CYP450 inhibition may potentially lead to elevated in vivo plasma levels of a co-administered drug metabolized by the inhibited enzyme, and, consequently, to adverse drug reactions and toxicity. Assessment of the following CYP450 inhibition by a new drug candidate is recommended by FDA and EMA: CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP1A2, CYP2C8 and CYP2B6.
Advantages of LC-MS/MS method over fluorimetric assay:
- LC-MS/MS analysis method is sensitive and specific, therefore human liver microsomes can be used, the fluorescent probes are not isoform-specific and should be used only with individual recombinant cytochromes
- interference can occur from the test compound exhibiting fluorescence or fluorescence quenching and lead to false results
The service is available both in panel of 5, comprising the most clinically important CYPs (1A2, 3A4, 2C9, 2C19, and 2D6), and expanded panel of 7 (1A2, 3A4, 2C9, 2C19, 2D6, 2B6, and 2C8) formats. Both individual recombinant cytochromes and human liver microsomes can be used. For the rough estimate, single point assays are typically performed for each compound at 10 µM concentration or another concentration stipulated by the customer. All test points are performed in duplicates. If a noticeable inhibition is detected, the IC50 values (test compound concentration which produces 50% inhibition) can be determined upon request. For this purpose, dose-response inhibition curves (8 points, 3-fold serial dilution) of the test compound and reference inhibitor are built starting at 100 µM concentration. The IC50 values are calculated using Microsoft Excel and GraphPad Prism software. Reference inhibitors specific for each CYP enzyme are used to assess inhibition in the control experiments for every batch of tested compounds. Test concentrations of the reference compounds correspond to approximately 5x- fold of IC50 values for the corresponding cytochromes P450, which is expected to produce 80-100% inhibition in the properly performing assay.
Figure 1. Test compounds CYP450 inhibition profiles.
Figure 2. IC50 of Sulphaphenazole
Deliverable:
either single point assay data for each compound at 10 µM concentration or IC50 values for the tested compounds based on 8-point, 3-fold serial dilution dose-response inhibition curves (upon request). Full study report is provided.
Sample Submission:
either single point assay data for each compound at 10 µM concentration or IC50 values for the tested compounds based on 8-point, 3-fold serial dilution dose-response inhibition curves (upon request). Full study report is provided.