and/or immunoquantitated levels of individual CYP forms. More integrated
systems, such as primary hepatocytes or precision-cut liver slices, are also used
to identify additional (e.g., Phase II) pathways and enable initial estimates of
fm. As much as possible, a complete CYP reaction phenotyping package should
contain data obtained from cDNA-expressed human CYP systems, human
liver microsomes, and primary human hepatocytes (Rodrigues, 1999).
5.4.2 Data Interpretation and Integration
As discussed above, variousin vitrosystems can yield useful information and
increase one’s understanding of the individual roles each CYP enzyme plays in
drug clearance. Each system has unique properties that must be considered
when analyzing and interpreting data. For example, bridging metabolism data
generated from cDNA-expressed CYP systems to human liver microsomes
requires use of certain scaling factors and at least two approaches (e.g., relative
activity factor, RAF and abundance normalized rates, NR) have been
proposed (Crespi, 1995; Remmel and Burchell, 1993; Rodrigues, 1999;
Venkatakrishnan et al., 2000).
Although cDNA-expressed human CYP enzymes are ‘‘cleaner’’ in vitro
systems for studying CYP-mediated reactions, human liver microsomes are
preferred due to the existence of multiple CYP enzymes and other native
components that allow for consideration of drug–protein interactions and
parallel or sequential metabolism. As such, data obtained from human liver
microsomes are the ‘‘net’’ outcome of CYP-mediated metabolism (Houston
and Kenworthy, 2000). Although complications may arise due to differing
effects on enzyme kinetics at varying substrate concentrations, the role of
CYPs in metabolism of a particular drug can be reasonably assessed with
specific chemical inhibitors, specific inhibitory antibodies, and sensitive
analytical methods (Gelboin et al., 1999; Lu et al., 2003; Walsky and Obach,
2004). As discussed above, activity correlation studies using a panel of human
liver microsomes with specific probe substrates are additional means to
delineate CYP-mediated metabolism (Lu et al., 2003).
Cell- or tissue-basedin vitrosystems possess the advantage of simultaneous
assessment of both Phase I and Phase II drug-metabolizing enzymes. Although
freshly isolated human hepatocytes are the preferred cell-based system to
evaluate drug metabolism, the availability of such cells has limited its broad
utility in the industrial setting. As a result, cryopreserved human hepatocytes
have become a valuable alternative (Gebhardt et al., 2003; Gomez-Lechon
et al., 2004). In addition, precision-cut liver slices have been used to investigate
drug metabolism and liver toxicity (de Kanter et al., 2002). Identification of
non-CYP-mediated metabolism using these integratedin vitro systems has
been critical to understand the overall metabolic clearance of NCEs and a
certain degree of success has been achieved in predictingin vivometabolic
clearance usingin vitrohepatocyte data (Hallifax et al., 2005; Ito et al., 1998a;
Niro et al., 2003).
128 METABOLISM-MEDIATED DRUG–DRUG INTERACTIONS