In the human body, the liver plays a major role in the metabolism and clearance of drugs absorbed through the gut. Therefore, in vitro metabolic stability is an important early ADME test for predicting in vivo half-life and clearance of a test compound. Our Metabolic Stability Assay uses microsomes (various species available) and human primary hepatocytes in a sandwich culture. While microsomes contain Phase I enzymes, primary human hepatocytes in sandwich culture contain both the Phase I and the Phase II enzymes. The microsomes and/or primary human hepatocytes in sandwich culture are exposed to the test compound for a number of exposure times and the samples are then assessed by LC-MS/MS to determine intrinsic clearance of a compound.
Microsomes and/or primary human hepatocytes in sandwich culture are exposed to various concentrations of the test compound for a number of exposure times, up to one hour (e.g. 5, 10, 20, 30, 45 and 60 minutes). The samples are centrifuged and the supernatant is analyzed via LC-MS/MS to determine the disappearance of test compound over time (intrinsic clearance or CLint) which is calculated as follows: