(Chan and Delucchi, 2000; Desai et al., 2002; Goodwin et al., 1999; He et al.,
1999; Ledirac et al., 2000; Luo et al., 2002, 2003, 2004; Pichard et al., 1999;
Zhao et al., 2002). As a result, overall CYP3A4 activity in cells treated with
these compounds can actually be lower than activity obtained from vehicle
control cells, even though CYP3A4 mRNA levels and protein expression are
significantly induced.
In addition to providing good models for prediction of CYP3A4 induction,
primary human hepatocytes are also an excellent model for evaluating the
potential of a drug to induce other human CYP isoforms, including CYP1A2,
2A6, 2B6, 2C8, 2C9, and 2C19. Positive inducer controls, as well as isoform-
specific substrates, are unique for each different CYP isoform. Table 17.1 lists
preferred and acceptable inducers and model substrates for each inducible
human CYP isoform (Gerbal-Chaloin et al., 2001; Goodwin et al., 2001;
LeCluyse et al., 2000; Meunier et al., 2000; Luo et al., 2002).
Studies using primary human hepatocyte cultures are dependent on the
availability of donor livers. Recently, scientists from Pfizer have developed the
immortalized human hepatocyte cell line Fa2N-4, which may provide a
valuable alternative tool for determining the induction of CYP3A4, as well as
of CYP1A2, 2C9, UGT1A, and MDR1 (PGP). For example, the levels of
induction of CYP3A4 and CYP2C9 by rifampin and phenobarbital, and
CYP1A2 byb-naphthoflavone observed in this model, are very close to those
obtained using primary human hepatocytes (Mills et al., 2004; Ripp et al.,
2006). Cryopreserved human hepatocyte cultures have also been employed for
CYP induction studies, but their use is thus far limited to studying CYP3A4,
1A2, and UDP-glucuronosyl-transferase (Kafert-Kasting et al., 2006; Reinach
et al., 1999; Roymans et al., 2004). The usefulness of cryopreserved human
hepatocytes for induction studies is limited because of the poor attachment of
cells to the culture dish and because their basal CYP activities are reduced
compared to those of fresh human hepatocytes. In addition, HepG2 cells,
derived from human hepatoblastoma, may be useful in combination with RT–
PCR to evaluate CYP induction in some limited cases (Matsuda et al., 2002;
Sumida et al., 1999).
17.2.2.2 PXR Reporter Gene Assay The identification and characterization
of members of the human PXR family have permitted the development of cell-
based reporter gene assays, which provide an accurate measure of the extent to
which putative inducers activate CYP3A4 expression via the PXR signaling
pathway. Figure 17.2 illustrates the basic features of the assay, and how the
interaction of the components can predict the induction potential of a
candidate compound. The basis of the assay is the cotransfection of cells with
two DNA vectors, one coding for expression of human PXR, the other
containing the reporter gene construct. The reporter gene construct contains
PXR responsive elements that activate expression of the reporter gene. A third
vector coding for expression of alkaline phosphatase or another control protein
can also be cotransfected for normalization of results. The expressed human
ASSESSMENTS 557