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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Rosiglitazone and pioglitazone are thiazolidinediones used for treatment of noninsulin-dependent diabetes mellitus. These compounds, along with troglitazone, were evaluated for the ability to induce cytochrome P450 enzymes (P450) in primary human hepatocyte cultures and to inhibit P450 in human microsomes. In induction studies, all three thiazolidinediones caused a dose-dependent increase in CYP3A4 activity and immunoreactive protein.While troglitazone was the most potent, rosiglitazone and pioglitazone generally exceeded troglitazone in absolute CYP3A4 activity achieved at concentrations >10 M. A comparable concentration-dependent increase in CYP2B6 immunoreactive protein was observed with all three thiazolidinediones. Microarray analysis revealed rifampin > troglitazone > pioglitazone > rosiglitazone in terms of CYP3A4 mRNA induction potential with 10 M compound. Inhibition studies conducted for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP2A6, and CYP2E1 showed troglitazone to be the most nonselective and potent inhibitor followed by rosiglitazone and pioglitazone. In vitro, the thiazolidinediones were strong inhibitors of CYP2C8, with K i values between 1.7 and 5.6 M, and of CYP3A4, with K i values between 1.6 and 11.8 M.Troglitazone, in addition, inhibited CYP2C9 (K i 0.6 M). Although the inhibitory effects of the thiazolidinediones have not been demonstrated clinically, our results suggest there is potential for interactions with CYP2C8 substrates. This is the first report of in vitro induction of P450 enzymes by rosiglitazone and pioglitazone. While only the induction of CYP3A4 by troglitazone has been demonstrated in vivo, these results suggest that other thiazolidinediones may have the potential to cause clinically significant drug interactions at sufficiently high doses.

This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.

The metal ion dependence of Escherichia coli ribonuclease H activity has been examined by monitoring the change in absorbance of nucleotide substrate by rapid (stopped-flow) kinetic methods. Kinetic equations that fully account for enzyme activation, and substrate inhibition at high metal concentration, have been derived. Inhibition constants correlate with metal nucleotide binding affinities. Comparison of thermodynamic (Huang, H.-W.; Cowan, J. A Ear. J. Biochem. 1994, 219, 253-260) and kinetic data suggests that there is one essential catalytic metal cofactor required for ribonuclease H activation, rather than a binuclear magnesium site. This conclusion is in accord with recent crystallographic data on the Mg2+-bound enzyme (Katayanagi, K.; Okumura, M.; Morikawa, K. Proteins 1993, 17, 337-346). Similar conclusions are likely to hold for the structurally homologous RNase H domains of retroviral reverse transcriptase.