9.5.1 In vitroMetabolite Profiling
In vitro metabolite profiling work is typically done in liver fractions
(microsomes or S-9) or hepatocytes (freshly isolated or cryopreserved). The
latter is often preferred because hepatocytes provide cellular integrity with
respect to enzyme architecture and contain the full complement of drug–
metabolizing enzymes and cofactors (both oxidative and conjugative) (Gomez-
Lechon et al., 2003; Hewitt and Utesch, 2004; McGinnity et al., 2004).
Alternatively, freshly isolated liver slices can be used (Ekins et al., 1996;
Martignoni et al., 2004). These systems tend to complement each other. When
oxidative metabolism is involved, the turnover in hepatocytes are usually lower
compared to subcellular tissue fractions or recombinant enzymes in the
presence of cofactors (de Graaf et al., 2002). Incubation of a drug with
hepatocytes, liver slices, or subcellular tissue fractions followed by chromato-
graphic analysis of the incubation medium by radioactivity detection and LC/
MS/MS is typically conducted for metabolite profiling and identification. The
comparative biotransformation profiles provide an analysis of the similarities
and differences in metabolism across species; information that could be helpful
in the selection of the toxicology species to be evaluated in long-term safety
studies and potentially to help understand toxicity in animal species mediated
through metabolism.
Figure 9.6 shows a comparative profile generated from incubations with
hepatocytes and liver microsomes of rat, monkey, and human for a C-14-
labeled compound. The data shows that metabolism of this compound is
qualitatively similar across species in both microsomes and hepatocyte systems.
Profiles in microsomes showed the oxidative pathways of metabolism (see
Fig. 9.6) whereas hepatocyte profiles, in addition to the oxidative metabolites,
also showed conjugative metabolites (Fig. 9.6, metabolites designated M21,
M30, and M31). This again emphasizes the need to use complementaryin vitro
systems to understand the overall metabolism of a compound.
9.5.2 Identification of Drug-Metabolizing Enzyme(s)
Reaction phenotyping refers to a set of experiments that is conducted to
identify the specific enzymes responsible for the metabolism of a drug (Zhang
et al., 2007). For studies with CYP enzymes, various tools like expressed
enzymes, specific chemical inhibitors, antibodies, and liver banks are available
(Lu et al., 2003; Williams et al., 2003). Identification of drug-metabolizing CYP
enzymes is crucial for predicting the potential for drug–drug interactions in the
clinic. It is well known for drugs that are metabolized mainly by CYP3A4,
there is potential for significant drug–drug interaction when coadministered
with a strong inhibitor like ketoconazole, where changes in AUCs could be
5–10-fold (Dresser et al., 2000; Greenblatt and von Moltke, 2002). It is
obvious that the potential for drug–drug interaction decreases for compounds
that are not primarily eliminated through metabolism, are low clearance
compounds, or are metabolized by multiple enzymes.
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