Metabolism is a major route of drug elimination. In vitro assays are frequently used to study the metabolic fate of NCEs, to rank-order compounds in terms of metabolic stability, and to predict metabolic pathways in vivo. Using appropriate model systems, both phases of metabolism can be studied in vitro: Phase I, in which the parent compound is converted by hydrolysis or oxidation into a more polar derivative, and Phase II, in which the compound and/or its Phase I metabolites are conjugated to a polar group such as  glucuronic acid to form a charged, more water-soluble derivative that is subsequently excreted in the urine or bile.

Absorption Systems uses several different test systems to evaluate metabolism in vitro. Microsomes are fragments of the endoplasmic reticulum membrane, containing many Phase I enzymes. In the presence of appropriate cofactors, microsomal enzymes will catalyze the conversion of an NCE to one or more metabolites. These reactions can yield a variety of information about potential metabolism of the NCE in vivo. These include prediction of the rate of clearance of the NCE by liver or other tissues, identification of metabolites and species differences in metabolite structures, and production of metabolites for chemical characterization or pharmacological activity assays. Microsomal assays can be performed in a high-throughput mode, where several dozen to several hundred compounds are simply screened for “metabolic stability” by following the disappearance of the parent compound.  Alternatively, they can be used for detailed dissection of the enzymatic pathways responsible for an NCE’s metabolism by carrying out the reactions in the presence and absence of chemical inhibitors or antibodies that selectively delete one or more enzymes from the metabolic scheme.

S9 subcellular fractions contain a full complement of both Phase I and Phase II enzymes. They are the preferred test system for evaluating metabolism by various Phase II enzymatic pathways, or in cases where the metabolic pathway is unknown. Metabolism by Phase II enzymes is evaluated by measuring the rate of disappearance of test compounds from S9 fractions supplemented with appropriate cofactors. One-at-a-time addition of cofactors to the assay can identify which classes of transferases are involved. Specific antibodies to many of these transferases are available and can be used to identify the specific isoform that mediates the reaction. Like microsomal assays, S9 assays can provide information about clearance, metabolite structure and species differences. They can also be used in high-throughput screening, but the protocols are more complicated because of the number of cofactors that have to be evaluated.

Hepatocytes, available from humans as well as various animal species, offer an additional, more physiological, test system for evaluating hepatic clearance of NCEs. Fresh hepatocytes, plated or in suspension, are considered the “gold standard” for in vitro metabolic assays because they express virtually all of the important enzymes and transporters implicated in drug metabolism, at physiological concentrations, along with all necessary cofactors. However, the supply of fresh hepatocytes, particularly from humans, is limited and their availability is unpredictable, which limits the usefulness of this test system. Cryopreserved hepatocytes are an alternative test system, are commercially available from inventory at any time, and retain metabolic activity and viability for years. Individual lots can be pre-characterized for expression of metabolic functions of interest and stored in large quantities for repeated use.  An additional desirable feature of cryopreserved human hepatocytes is that cells from several different donors can be pooled for assay, thereby eliminating the variable of differences in enzyme activity between donors.  In vitro to in vivo correlation studies have demonstrated a good correlation between metabolic clearance predicted from cryopreserved hepatocyte assays and the values observed in in vivo studies.

A typical NCE follows a pathway like this during characterization of its metabolism:

1. Stability in human and animal liver microsomes or S9 fraction
2. Identification of metabolites present in human and/or animal microsomes or S9 fraction
3. Confirmation of metabolite formation using cryopreserved or fresh hepatocytes
4. Dissection of the metabolic pathway (enzyme phenotyping assays)