Protein binding-dependent decreases in hERG channel blocker potency assessed by whole-cell voltage clamp in serum.

In vitro hERG blocking potency is measured in drug discovery as part of an integrated cardiovascular risk assessment. Typically, the concentrations producing 50% inhibition are measured in protein-free saline solutions and compared with calculated free therapeutic in vivo Cmax values to estimate a hERG safety multiple. The free/unbound fraction is believed responsible for activity. We tested the validity of this approach with 12 compounds by determining potencies in voltage clamp studies conducted in the absence and presence of 100% dialyzed fetal bovine serum (FBS). Bath drug concentrations in saline solutions were measured to account for loss of compounds due to solubility, stability, and/or adsorption. Protein binding in dialyzed FBS was measured to enable predictions of serum IC50s based on the unbound fraction and the saline IC50. For 11 of 12 compounds, the measured potency in the presence of dialyzed FBS was within 2-fold of the predicted potency. The predicted IC50 in dialyzed FBS for one highly bound compound, amiodarone, was 9-fold higher than the measured serum IC50. These data suggest that for highly bound compounds, direct measurement of IC50s in the presence of 100% serum may provide a more accurate estimate of in vivo potencies than the approach based on calculated serum shifts.

Temporal evaluation of CYP mRNA in mice administered with prototypical P450 inducers: comparison with conventional protein/enzyme methods.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.

Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system.

In the current drug discovery environment, higher-throughput analytical assays have become essential to keep pace with the screening demands for drug metabolism and pharmacokinetics (DMPK) attributes. This has been dictated by advances primarily in chemical procedures, notably combinatorial and parallel syntheses, which has resulted in many-fold increases in the number of compounds requiring DMPK evaluation. Because of its speed and specificity, liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become the dominant technology for sample analysis in the DMPK screening assays. For higher-throughput assays, analytical speed as well as other factors such as method development, data processing, quality control, and report generation, must be optimized. The four-way multiplexed electrospray interface (MUX), which allows for the analysis of four LC eluents simultaneously, has been adopted to maximize the rate of sample introduction into the mass spectrometer. Generic fast-gradient HPLC methods that are suitable for approximately 80% of the new chemical entities encountered have been developed. In-house-written software programs have been used to streamline information flow within the system, and for quality control by automatically identifying analytical anomalies. By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 permeability has been established.

Generic fast gradient liquid chromatography/tandem mass spectrometry techniques for the assessment of the in vitro permeability across the blood-brain barrier in drug discovery.

Rapid, generic gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays, designed to accelerate sample analyses, have been developed to keep pace with the productivity of advanced synthetic procedures. In this study, LC/MS/MS was combined with an in vitro, cell-based, blood-brain barrier (BBB) model to evaluate the potential of new chemical entities (NCEs) to cross the BBB. This in vitro assay provides the permeability of discovery compounds across a monolayer of a primary culture of bovine brain microvessel endothelial cells in a fraction of the time that is required for in vivo studies (brain/plasma concentrations), using only 2 mg of the compound. The results are consistent with in vivo brain/plasma concentration ratio data.