Cytochrome P450-Mediated Metabolic Stability Assay in Liver Microsomes

Abstract

In early phase drug discovery, it is important to try to identify compounds that have a high risk of failing as a marketable drug. Extensive metabolism of a drug molecule is one way that a drug candidate can fail. Since most small molecules are metabolized by the liver, the enzymes in the liver need to be heavily accounted for. The majority of these enzymes are cytochrome P450 (CYP), and therefore screening compounds for activity with CYPs is necessary to develop molecules. A simple way of doing this is to incubate the compound with liver microsomes, a subcellular fraction of hepatocytes that contain a high concentration of CYPs from the endoplasmic reticulum. This can be done in vitro through incubation in a potassium phosphate buffer at 37 °C in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH), the cofactor necessary for CYP reactions, and measuring the concentration of the incubation mixture at various time-points through LC/MS/MS analysis. From this procedure, an in vitro metabolism half-life can be measured, and then scaled up to predict the total hepatic clearance of the molecule.