The liver accounts for a large amount of endogenous and xenobiotic metabolism. Microsomes are sub-cellular liver structures that contain a variety of enzymes responsible for phase I metabolism. The post-mitochondrial supernatant or S9 fraction of liver preparations contains both phase I and phase II enzymes and provides an alternative to the hepatocyte incubation assay. Liver cells or hepatocytes provide phase I and phase II enzymes in their intact cell environment. As part of our in vitro ADME services we use hepatocytes, liver microsomes, and S9 incubations from humans and animals to determine the potential metabolic stability of test compounds. Test compound is incubated with human and/or animal liver tissue and sample aliquots are removed at pre-defined time intervals. The concentration of the test compound is quantified by LC-MS/MS and the rate of compound disappearance can determine the compound’s metabolic stability and half-life.
We routinely conduct both human and rat microsome studies on test compounds to determine relative stability in both species and correlate the in vivo rat studies with potential expected human stability (in vitro - in vivo correlation or IVIVC). Compounds are incubated at pre-determined concentrations with liver microsomes and NADPH. The peak area of the test compound at each time point is compared to the peak area of the time zero sample. A control reaction (without NADPH co-factor) is used in order to assess the amount of thermal breakdown, insolubility, and non-specific binding that contributes to the overall loss of the test compound.
Intrinsic clearance measurement, an assessment of compound metabolic clearance in vitro, is typically performed with hepatocytes across a range of test compound concentrates to identify the enzyme kinetic constant values of Km and Vmax that describe the reaction kinetics. These values are used to determine intrinsic clearance, which is a useful parameter for assessment of in vitro - in vivo correlation (IVIVC).
The data on liver metabolism and the hepatic clearance are valuable for rational formulation development of orally administered drug products, particularly if one of the goals of formulation development is improvement in oral bioavailability.