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 problems in clinical pharmacology: drug-drug interactions and individual variability in drug metabolism (CYP450 gene polymorphism). CYP450 reaction phenotyping involves the identification of enzymes responsible for metabolism of the test article, allowing prediction of potential drug-drug interactions. A new drug candidate is preferred to be metabolized by more than one isozyme, so that if one metabolic pathway is blocked by a coadministered inhibiting drug, then clearance can occur by metabolic switching to another uninhibited enzyme. Determination of CYP450 enzyme responsible for the metabolism of new drug candidate is recommended by FDA.

Service Details: In this assay two metabolically active test systems are used: individual human recombinant cytochromes (CYPs 1A2, 2C9, 2C19, 2D6 and 3A4) and human liver microsomes. The test compound is incubated with each CYP450 isoform and the metabolising ability of the enzyme is estimated by disappearance rate of parent drug using HPLC-MS/MS analysis. Reference cytochrome specific substrates are used as controls. In the microsomal assay the isoform specific CYP450 inhibitors are used, and the increase of t1/2 in the presence of the inhibitor indicates which enzyme is responsible for the metabolism of the compound.

Isoform specific substrates and inhibitors for in vitro CYP450 reaction phenotyping

CYP450 Substrate (reference) Inhibitor Inhibitor concentration, uM
1A2 Phenacetin α-Napthoflavone 1
3A4 Testosterone Ketoconazole 1 and 10
2C9 Diclofenac Sulphaphenazole 10
2C19 Omeprazole Ticlopidine 20
2D6 Dextromethorphan Quinidine 1

 

Deliverable: Metabolic stability data of 1 test article and 5 reference compounds (Phenacetin, Testosterone, Diclofenac, Omeprazole, and Dextromethorphan) at the presence of individual human recombinant cytochromes at tree time points over 60 minutes. Metabolic stability data for 1 test article in human liver microsomes at the presence of specific cytochrome inhibitors responsible for its metabolism (identified in the study with recombinant enzymes) at five time points over 60 minutes obtained by HPLC-MS/MS analysis. Data include parent compound percent remaining, half-life and clearance values. 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, smaller amount of compound per assay may be sufficient, which should be discussed for each particular project. We need to know structures of the molecules for metabolite profiling.