Our current understanding of the mechanisms of gene regulation offers opportunities to develop assays to evaluate each step of the gene regulation cascade. These include nuclear receptor, mRNA, protein, or enzyme activity assays, and examples of each are either referenced or described below. An enzyme induction assay based on human hepatocyte preparations using enzyme activity as the readout is still considered by many to be the assay of "best practice". An assay based on apoprotein detection is semi-quantitative, and those based on mRNA are open to interpretation because of the potential for post-transcriptional modifications which may result in a lack of correlation with either apoprotein or enzyme activity.
Assays based on nuclear receptors offer the advantage of high throughput in order to develop SAR. The other assays described provide information on specific gene products, such as CYP3A4. Increasingly, however, scientists want to derive a characteristic "signature" gene response which is prototypic of, for instance, a hPXR activator. This is important since, historically, scientists have been "CYP3A4 centric". Nowadays, the totality of gene responses can be considered in the context of potential implications for interactions between co-administered medications. These higher throughput assays also allow the use of human hepatocytes for induction studies to be limited to those compounds of real interest.
The technology of cDNA microarrays allows the investigator to monitor for changes of gene expression on a relatively massive scale; typically 25 - 50 K oligonucleotide probes or cDNA fragments at one time (50, 51). Such experiments are likely to be undertaken in the context of attempting to understand a drug-drug interaction or a specific toxicology. In the context of a drug-drug interaction, once potential associations between selected gene sets and proteins involved in the disposition of the co-administered drugs have been identified, then assays to quantify induction of specific genes is undertaken and will typically be performed using a technique such as quantitative RT-PCR (e.g. TaqMan, Applied Biosystems).
To perform microarray and Taqman studies requires the availability of mRNA from living cells which have been challenged with a drug or test compound (52, 53). The gut and liver can be considered important organs in which to monitor for protein induction since these organs modulate exposure to parent compound via the action of
drug transporters and drug metabolizing proteins. In vitro induction studies are therefore undertaken in fresh and cryopreserved human hepatocytes, and in human liver slices (54, 55). Hepatocytes are often in limited supply, and the additional issue of inter-preparation/individual variability have, in addition, made these assays less attractive as a primary screen.
Preparing primary enterocytes is relatively routine, but reliable protocols for plating them for subsequent drug challenge during the course of undertaking an induction study is not trivial (56). The availability of gut cell lines represent one alternative, but it should be noted that Caco-2 cells, for instance, express the VDR, but not the PXR (57). Induction of the CYP3A4 gene in these cells is therefore controlled by the binding of the VDR-RXR heterodimer to the CYP3A PXR response elements (58). An alternative to using Caco-2 cells is the immortalized human colon carcinoma LS180 cell line which contains both the VDR and PXR (42, 58). Induction of both CYP3A4 and Pgp activity by the hPXR ligand, paclitaxel, in this cell line, has been reported (42).
While there are several in vitro systems in which to evaluate enzyme induction, the main problem lies with interpretation of data in the context of clinical relevance. Factors such as, potency at the nuclear receptor, dose level (exposure), duration of dosing, free versus bound concentrations of drug, the effect of drug transporters on cell concentrations, all potentially confound interpretation of data. Clearly the role of in vitro induction assays is to provide choice; compounds with low potential to induce enzymes being favored over those which do not, all other considerations being equal.
Induction Studies in Human Hepatocytes - Hepatocytes are cultured on an extracellular matrix (e.g. Matrigel or collagen) for a period of 48 - 72 hr and then challenged with drug. It is common, although not essential, to overlay hepatocytes in culture with extracellular matrix in order to recapitulate a cytoarchitecture synonymous with that encountered physiologically. Drug challenge is typically for at least 48 hr if protein levels and enzyme activities are to be assayed. The appropriate controls to run include a positive control (e.g. 10 nM rifampicin), and a vehicle control (e.g. methanol, acetonitrile or dimethylsulfoxide; all < 1%). Induction of enzyme activity is typically performed with midazolam or testosterone for CYP3A4, caffeine, theophylline, or phenacetin for CYP1A, and S-warfarin, diclofenac, or tolbutamide for CYP2C9. For compounds which are inhibitors of enzyme activity, at least in vitro, then assessment of alternative measures of enzyme induction (e.g. mRNA or apoprotein) are recommended.
Nuclear receptor reporter gene assay - A hPXR reporter gene assay based on the transient transfection of HepG2 cells with a full length PXR construct and a reporter plasmid consisting of the CYP3A-5' flanking region linked to the luciferase gene has been developed (59). These workers then evaluated 14 drugs for PXR activation and for their ability to induce CYP3A4 in cultured primary human hepatocytes (using testosterone 63-hydroxylase (T6PH), CYP3A4 mRNA, and protein assays). The overall correlation (not considering the CYP3A4 inhibitors, ritonavir and troleandomycin) between hPXR activation and CYP3A4 activity (T6pH) was 0.864 (P < 0.001). An important component of this analysis was variability of response. For the positive control, rifampicin, the magnitude of the induction over solvent control, based on enzyme activity, was 2- to 10-fold. In contrast, the hPXR reporter gene assay showed less variation, 2 jiM rifampicin providing a 21- to 25-fold increase over control in three separate experiments. In recognizing that hPXR activation was not the only means by which CYP3A4 induction occurs (vide infra), these workers suggested that enzyme induction should be evaluated in more than one experimental system.
hPXR Scintillation proximity assay (60) - In this assay hPXR was immobilized on a scintillant-containing bead and then incubated with the radioligand, [3H]SR12813, a potent activator of hPXR (Kd 41 nM) which has been shown to efficiently induce CYP3A gene expression in human hepatocytes. The binding of non-radioactive ligands was measured as a function of their ability to compete with the radioligand for binding to hPXR. Since this assay does not require separation of bound from free ligand, this assay is amenable to high throughput screening.
CYP3A4 reporter gene assay - One group has investigated the effect of 17 xenobiotics on an approximately 1 kilobase pair fragment of the CYP3A4 proximal promoter, cloned upstream of the secretory placental alkaline phosphatase gene, which was then transfected into the HepG2 cell line (61). They also evaluated a smaller region of the proximal promotor covering the first 301 bp and determined it to be sufficient to act as a basal transcription unit, and to be able to mediate induction of the CYP3A4 reporter gene by compounds previously shown to activate the 1105 bp proximal promotor reporter (35). These workers used a four point concentration response curve (1, 10, 30 and 50 |xM) to calculate maximal induction (lmax) and EC5o values, where the ratio Imax/ECso (the "overall inductive ability" (IA)) was representative of intrinsic transcriptional activation (61). It was concluded that this in vitro model is capable of identifying CYP3A4 transcriptional inducers and yields an IA value allowing the ranking of compounds for their overall ability to induce CYP3A4 transcription. It would be of interest also to determine the effect of free drug concentration on the IA in an expansion of the equation which describes the activation of a reporter gene by a compound that follows the law of mass action for binding of a ligand to its receptor (61):
Effect = lmax * Free Drug Concentration EC5o + Free Drug Concentration
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