Drug Metabolism in Drug Design and Development Basic Concepts and Practice

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A eukaryotic expression vector such as pSG5 (Stratagene) or pCMV
(Promega), with a human PXR open reading frame cDNA insert, is employed
to express human PXR. The most critical component is the reporter gene
construct, in which the PXR responsive elements and reporter gene are the key
factors. To represent PXR responsive elements, some laboratories have used
the CYP3A4 proximal promoter alone, usually three copies of ER6 (Bertilsson
et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998); some have used the
entire 1 kb (1087 to57) CYP3A4 5^0 untranslated region (El-Sankary et al.,
2000); and some have used both the proximal promoter and distal enhancer
elements (Goodwin et al., 1999). These approaches differ in their indication of
the maximal fold induction by rifampicin as shown in Table 17.2. In our own
laboratory, we have used several different PXR reporter gene constructs, and
find that constructs where both the CYP3A4 proximal promoter and distal
enhancer regions are present in the reporter gene construct provide the most
facile and sensitive assays (Goodwin et al., 1999; Luo et al., 2002). Among the
commonly employed reporter gene assays, the luciferase assay is simple and
sensitive, whereas the measurement of CAT is much more time-consuming,
and there is a considerably higher background. Finally, an alternative reporter
gene system has been developed, by creating a construct that inserts the LBD
region of PXR into aGAL4 reporter system. In this case, PXR LBD cDNA was
fused withGAL4 in an expression vector, while the reporter gene construct was
either 4USA-luciferase or tk-USA-luciferase (Bertilsson et al., 1998; Blumberg
et al., 1998; Lehmann et al., 1998). This assay employs the same principle as
other PXR reporter gene assays, except that the PXR DBD is replaced with
GAL4 in the expression vector construct, and a PXR responsive element is
substituted for five tandem repeats of the 17-bpGAL4 binding element in the
reporter gene construct. This assay is quite straightforward and the maximal
induction is more than 10-fold. We have studied the correlation between the
prediction of CYP3A4 induction by the PXR reporter gene assay originally
established by Goodwin et al. (Goodwin et al., 1999), and the actual induction
and expression of CYP3A4 in primary cultures of human hepatocytes for 14
clinically used drugs (Luo et al., 2002). This study indicated that the PXR
reporter gene assay generally predicted the degree of CYP3A4 induction
observed with primary cultures of human hepatocytes (Luo et al., 2002). In
some cases (e.g., ritonavir and troleandomycin), compounds that induce
CYP3A4 are also mechanism-based inactivators of CYP3A4in vivo. For these
compounds, the PXR reporter gene assay identified the inducers, even though
the net CYP3A4-dependent metabolism of the model substrates was not
increased in primary human hepatocyte cultures.


17.2.3 Direct Assessment of CYP3A4 InductionIn vivoin Humans


As mentioned above, all the methods currently employed for measuring
CYP3A4 induction, including humanized mice, PXR reporter gene assays, and
primary cultures of human hepatocytes, have limitations. Most importantly,


562 TESTING DRUG CANDIDATES FOR CYP3A4 INDUCTION

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