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), and 5 of them (CYPs 1A2, 2C9, 2C19, 2D6 and 3A4) are involved in about 95% of the known drug metabolism. Cytochrome P450s are of critical importance due to the two of the most significant issues in clinical pharmacology: drug-drug interactions and individual variability in drug metabolism (CYP450 gene polymorphism). Assessment of potential CYP450 inhibition by a new drug candidate is recommended by FDA and EMA.

Service Details: The potential for CYP inhibition of 5 major cytochromes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 is assessed using LC-MS/MS based assay, in which biotransformations of the CYP450 specific substrates are used as markers to quantify the enzymatic activity, according to FDA recommendations. Quantification of a decrease in the formation of a metabolite in the presence of an inhibitor is used to determine the CYP450 inhibition. In this assay both individual human recombinant cytochromes and human liver microsomes can be used.

Isoform specific CYP450 substrates, metabolites and inhibitors

CYP450 Substrate Metabolite Reference Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
3A4 Testosterone 6β-Hydroxytestosterone Ketoconazole
2C9 Diclofenac 4’-Hydroxydiclofenac Sulphaphenazole
2C19 Omeprazole 5-Hydroxyomeprazole Ticlopidine
2D6 Dextromethorphan Dextrorphan Quinidine

 

For the rough estimate, single point assays are typically performed for each compound at 10 µM concentration or another concentration stipulated by the customer. If a noticeable inhibition is detected, the IC50 values for the tested compounds 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 100µM concentration. All test points are performed in duplicates. The IC50 values are calculated using Microsoft Excel and GraphPad Prism software. Reference inhibitors specific for each CYP enzyme are used to assess CYP inhibition in the control experiments for every batch of tested compounds. Test concentrations of the reference compounds correspond to approximately 4x fold of IC50 values for the corresponding cytochromes P450, which is expected to produce 80-100% inhibition in the properly performing assay.

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: A minimal accurately weighable quantity of dry compound (~1 mg or 2 µmol) or 100 µL of 10-20 mM stock DMSO solution is required for this assay. For multiple assays, lesser amount of compound per assay may be sufficient, which should be discussed for each particular project.