0.125 M sucrose), and can be stored at 80 oC if they cannot be used
immediately. The total protein concentration is determined by the bicincho-
ninic acid protein assay (BCA-Pierce, Rockford, IL) with bovine serum
albumin (BSA) as a standard (Smith et al., 1985).
Quantitative RT–PCR for mouse cyp3a is performed with the ABI PRISM
79000HT Sequence Detection System (Applied Biosystems), using TaqManR
Universal PCR Master Mix. Detailed instructions are provided in the kit
manual (Prueksaritanont et al., 2005). Fold increase in cyp3a mRNA level
(drug treated group compared to vehicle control) can be used independently to
identify induction of cyp3a gene expression by the test compound, or to
provide confirmatory evidence of induction mediated through enhanced gene
transcription, as is normally the case.
Mouse liver CYP3a activity is measured by suspending liver microsomes
(0.5 mg/mL) in phosphate buffer (50 mM, pH 7.4), and the probe substrates
testosterone (250mM) or midazolam (50mM) is added. The reaction is initiated
by addition of NADPH at a final concentration of 1 mM, and terminated with
an equal volume of acetonitrile after incubation for 10 min (testosterone) or
5 min (midazolam). The rates of generation of the metabolites 6b-OH
testosterone or 1^0 -OH midazolam, which are specifically generated by cyp3a,
are determined (Pearce et al., 1996; Wandel et al., 2000).
Western blot analysis (LeCluyse et al., 2000) can also be employed to
confirm cyp3a induction. However, this method is time consuming and
provides a relatively low throughput.
17.2.2 Assessment of Induction Potential UsingIn vitroModels
The potential of test compounds to induce CYP3A4 can be assessed utilizing
cell-based assays such as primary cultures of hepatocytes or the PXR reporter
gene assay (Goodwin et al., 1999; Jones et al., 2000; LeCluyse, 2001a; Luo et
al., 2002, 2004; Moore and Kliewer, 2000; Zhu et al., 2004). There is a major
advantage of employing such assays early in the drug discovery process. They
provide the capability to screen a large number of test compounds in a
relatively short period of time, thereby detecting the potential for induction by
chemotypes in the early stages of drug discovery so that their potential for
drug–drug interactions can be timely addressed. More importantly, thesein
vitro screening assays generate results that are more reliably predictive of
induction in humans than results obtained from animal studies. In particular,
primary cultures of human hepatocytes are considered by academic, industry,
and regulatory scientists to be the current ‘‘gold standard’’ for CYP3A4
induction studies. In addition, in vitro assays are reproducible and cost-
effective. However, the disadvantages of these assays must also be considered.
All of thein vitroassays are performed under non-physiological conditions, in
an over-simplified system, using cell lines. Exposure of cells in culture is not
equivalent to human ingestion of test compounds. In addition, each of these
assays has its unique disadvantages. For example, the SPA (scintillation
552 TESTING DRUG CANDIDATES FOR CYP3A4 INDUCTION