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MARÍA JOSÉ GÓMEZ-LECHÓN y MARÍA TERESA DONATO AN. R. ACAD. NAC. FARM.

for a good pharmacokinetic profile as well as a risk for drug-drug
interactions. Metabolic stability is defined as the susceptibility of a
chemical compound to biotransformation, and is expressed as in
vitro half-life (t(1/2)) and intrinsic clearance (CL(int)). Based on these
values, in vivo pharmacokinetic parameters, such as bioavailability
and in vivo half-life, can be calculated when other data concerning
the volume of distribution and fraction absorbed are available (11).
Metabolic stability assays can be easily investigated by incubating
new chemicals with fully competent metabolic models and
performing sensitive chromatographic analysis (e.g. HPLC-MS/MS)
of the incubation mixtures (12). In the early phases of chemical
screening, metabolic rates are estimated by measurement of the
disappearance of the test compound as metabolites are usually
unknown. At advanced stages, formation of metabolites is also
analyzed. Human liver microsomes, hepatocytes, and cDNA-
expressed P450 enzymes are commonly used (Figure 2). A major
disadvantage of using recombinant models expressing a single
enzyme for the study of metabolic stability of a drug is the lack of
other phase I and phase II enzymes. Human liver microsomes
contain high levels of P450s and other drug-metabolizing enzymes
(flavin monooxygenase, UDP-glucuronyltransferases, epoxide
hydrolase). The metabolites identified after a short incubation with
microsomes coincide with those reported as the major metabolites
in human in vivo studies (13). The major limitation of microsomes
is that they lack phase II cytosolic enzymes (glutathione S-
transferases, sulfotransferases, soluble epoxide hydrolases, alcohol
dehydrogenase, xanthine oxidase, etc.). Human hepatocytes represent
a more complete system with physiological levels of cofactors,
natural orientation for linked enzymes and intact membranes to
allow for the modeling of intracellular drug concentrations (14, 15)
(Figure 2). Moreover, the restricted accessibility of suitable human
liver samples has greatly hindered the widespread use of human
hepatocytes for drug metabolism studies. Recently, this scarcity has
been countered by the increasing availability of metabolically
competent cryopreserved human hepatocytes and by the use of
optimized metabolic assays in hepatocytes cultured in multiwell plate
formats (16, 17). Later in non clinical development, the ADME flow
chart focuses on metabolite profiling in different species and
metabolite identification. Significant metabolic interspecies

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