is to take samples of interest (i.e, microsomal incubations, plasma samples) and
isolate and purify any metabolites present, after which the structure of the
metabolites is determined and each is tested for biological activity. An
alternative approach is to use bioassay-guided methods where biological
samples containing biotransformation products are first evaluated for their
pharmacological activity without any effort to isolate or structurally
characterize the metabolites. The bioassay methods may be based on the
assessment of the pharmacological activity usingin vitro-ligand binding (Lim
et al., 1999; Soldner et al., 1998), cell-based assays orin vivopharmacological
assays (van Heek et al., 1997). Metabolites can be generated by any of thein
vitroandin vivomethods discussed in the following sections. Biological activity
in the sample mixture can then be evaluated as is or after fractionation of the
sample mixture by using chromatographic techniques. The structural identity
of the active metabolite can then be determined and itsin vitroandin vivo
activity confirmed after isolation and/or after further biological or chemical
synthesis.
A systematic approach to profiling active metabolites using a 96-well plate
format was recently described (Shu et al., 2002). The approach is based on a
rapid bioassay-guided metabolite detection and characterization. Drug meta-
bolite mixtures (generated by various methods described below) are separated
and fractions collected into microtiter plates such as 96-well plates. The
fractions are then subjected to one or more relevant activity (e.g., receptor
ligand binding) assays.
8.6.3 Methods for Generation of Metabolites
There are a number ofin vitro and in vivobiotransformation techniques
available to generate metabolites. Thein vitrotechniques include the use of
subcellular fractions prepared from cells that mediate drug metabolism, intact
cell-based systems, intact organs, and isolated expressed enzymes.In vivo
methods involve the use of biological fluids (plasma, bile, urine, etc.) obtained
from laboratory animals or humans dosed with the parent molecule. Microbial
methods and biomimetic systems based on metalloporphyrin chemistry can
also be used as bioreactors to produce metabolites.
8.7 SUMMARY
The past several decades have witnessed an explosion in our knowledge of drug
metabolizing enzymes. It is now possible to fully characterize and predict the
metabolic fate of new chemical entities in humans with reasonable certainty.
The in vitro methods used to do this, that is, human liver microsomes,
expressed enzymes, cryopreserved, or freshly isolated hepatocytes, can be
adapted to be run in medium-throughput fashion and allow the metabolic
properties of new compounds to be optimized during the discovery phase.
SUMMARY 253