The human transcription factor pregnane X receptor (hPXR) is a key regulator of enzyme expression, especially cytochrome P450 3A4 (CYP3A4). Due to the prominence of CYP3A4 in the elimination of many drugs, the development of high throughput in vitro models to predict the effect of drugs on CYP3A4 expression have increased. To better interpret and predict potential drug-drug interactions due to CYP3A4 enzyme induction, we evaluated 170 xenobiotics in a hPXR transactivation assay and compared these results to known clinical drug-drug interactions. Of the 170 xenobiotics tested, 54% of them demonstrated some level of hPXR transactivation. By taking into consideration cell culture conditions (solubility, cytotoxicity, appropriate drug concentration in media), as well as in vivo pharmacokinetics (therapeutic plasma C(max), distribution, route of administration, dosing regimen, liver exposure, potential to inhibit CYP3A4), the risk potential of CYP3A4 enzyme induction for most compounds reduced dramatically. By employing this overall interpretation strategy, the final percentage of compounds predicted to significantly induce CYP3A4 reduced to 5%, all of which are known to cause drug-drug interactions. Also, this is the first report that identifies several potent compounds that have the ability to transactivate hPXR that previously have not been identified, such as terbinafine, diclofenac, sildenafil, glimepiride, montelukast, and ticlopidine.

ABSTRACT:Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (ϳ96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC 50 values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.

From a trial comparing interventions to improve adherence to antiretroviral therapy-directly administered antiretroviral therapy (DAART) or an intensive adherence case management (IACM)-to standard of care (SOC), for HIV-infected participants at public HIV clinics in Los Angeles County, California, we examined the cost of adherence programs and associated health care utilization. We assessed differences between DAART, IACM, and SOC in the rate of hospitalizations, hospital days, and outpatient and emergency department visits during an average of 1.7 years from study enrollment, beginning November 2001. We assigned costs to health care utilization and program delivery. We calculated incremental costs of DAART or IACM v SOC, and compared those costs with savings in health care utilization among participants in the adherence programs. IACM participants experienced fewer hospital days compared with SOC (2.3 versus 6.7 days/1000 person-days, incidence rate ratio [IRR]: 0.34, 97.5% confidence interval [CI]: 0.13-0.87). DAART participants had more outpatient visits than SOC (44.2 versus 31.5/1000 person-days, IRR: 1.4; 97.5% CI: 1.01-1.95). Average per-participant health care utilization costs were $13,127, $8,988, and $14,416 for DAART, IACM, and SOC, respectively. Incremental 6-month program costs were $2,120 and $1,653 for DAART and IACM participants, respectively. Subtracting savings in health care utilization from program costs resulted in an average net program cost of $831 per DAART participant; and savings of $3,775 per IACM participant. IACM was associated with a significant decrease in hospital days compared to SOC and was cost saving when program costs were compared to savings in health care utilization.

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In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.