Metabolic Enzyme Microarray Coupled with Miniaturized Cell-Culture Array Technology for High-Throughput Toxicity Screeni
Due to poor drug candidate safety profiles that are often identified late in the drug development process, the clinical progression of new chemical entities to pharmaceuticals remains hindered, thus resulting in the high cost of drug discovery. To acceler
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troduction The metabolism of chemicals, including drug compounds, in the human body primarily occurs in the liver via a variety of oxidative and conjugative routes. Among the many metabolic enzymes, Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_14, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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cytochrome P450 (CYP450) isoforms, which catalyze first-pass (Phase I) functionalization reactions (e.g., hydroxylation, dealkylation, oxidation, deamination, and dehalogenation), are the most important (1, 2). Subsequent conjugation reactions (e.g., glucuronidation, sulfation, acetylation, and addition of amino acids and peptides) are catalyzed by Phase II metabolic enzymes including UDP-glycosyltransferase (UGT), sulfotransferase (SULT), and glutathione S-transferase (GST), resulting in the formation of more soluble compounds and eventually enhancing excretion (3, 4). Thus, understanding the role of these metabolic enzymes is important in drug metabolism and for human toxicology testing. There have been a number of in vitro approaches developed for human metabolism and toxicology screening, including isolated liver slices, primary hepatocytes, transformed cultured human hepatoma cell lines, purified microsomal preparations, or isolated and purified P450s (5, 6). Two-dimensional (2D) hepatocytes cultures in multi-well plates can be adapted to high-throughput screening, and have been considered as the gold standard of in vitro human metabolism and toxicology for replacement of animal testing (7). However, 2D hepatocyte cultures may not emulate the environment and cellular architecture found in vivo (8, 9). Thus, in vitro results obtained from 2D hepatocyte cultures may not provide toxicity information that correlates with in vivo animal data. To address this limitation, we have developed a metabolic enzyme microarray (the Metabolizing Enzyme Toxicology Assay Chip, or MetaChip) and a miniaturized 3D cell-culture array (the Data Analysis Toxicology Assay Chip, or DataChip) for high-throughput toxicity screening of target compounds and their metabolic enzyme-generated products (10–12). The MetaChip can be prepared by spotting human or rat metabolic enzymes including individual CYP450s, a mixture of CYP450s, individual phase II metabolic enzymes, a mixture of phase II metabolic enzymes, a mixture of all metabolic enzymes, liver microsomes, and s9 fractions, all encapsulated in alginate gels (as small as 15 nL) arrayed on a methyltrimethoxysilane (MTMOS)-coated glass slide. The DataChip can be prepared by printing human or rat cells in alginate gels (as small as 30 nL) onto a poly(styrene-co-maleic anhydride) (PS-MA)-coated glass slide for toxicity screening against multiple target cells. Using the human or rat DataChip coupled with the human or rat MetaChip, the toxicity of parent compounds have been compared to that of their products generated by various metabolic enzymes (Fig. 1). We
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