Screening Tools for the High-Throughput Analysis of CYP450-Mediated Drug Metabolism and CYP450 Inhibition
Sumitra Sukumaran, Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, Moo-Yeal Lee, Solidus Biosciences, 1223 Peoples Avenue, Troy, NY 12180, Benjamin Potsaid, The Center for Automation Technologies and Systems, Rensselaer Polytechnic Institute, Troy, NY 12180, Douglas S. Clark, Chemical Engineering, UC - Berkeley, Dept of Chemical Engineering, Berkeley, CA 94720 - 1462, and Jonathan S. Dordick, Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, NY 12180.
A common in vivo drug-drug interaction is the inhibition of Cytochrome P450 (CYP450) enzymes responsible for drug metabolism, resulting in higher systemic levels of a drug and possible toxicity. Drug-drug interactions represent a safety hurdle and a major expense in drug discovery. Hence it is vital to incorporate in vitro CYP450 inhibition assays early in the drug development process. Previous inhibition assays have been performed in 96-well plates, 1536-well plates, and on microfluidic platforms; however, truly high-throughput assays using nanovolumes of reagents have yet to be explored. We have developed a solid-phase alginate microarray consisting of up to 3,000 isolated CYP450 reactions on a single 25 X 75 mm functionalized glass slide for the high-throughput analysis of CYP450 inhibition. Individual CYP450 isoforms (CYP3A4, CYP2C9, CYP2D6, CYP2E1, and CYP1A2) were encapsulated in 20 nl alginate spots (prepared from a solution of 0.5% alginate and containing a total enzyme content < 50 pg) and used in fluorescence-based inhibition assays. For slide imaging and data extraction, the CYP450 microarrays were interfaced to a novel, wide-field, whole-slide imaging optics system, consisting of a telecentric composite lens coupled with traditional microscopic filters. Individual CYP50 isoforms encapsulated in alginate exhibited comparable and reproducible reactivity and storage stability, and increased linear reaction time, when compared to conventional CYP450 preparations in solution. We show that a single 1,080 or 2,856 spot array on a glass slide can simultaneously provide IC50 values for nine potential inhibitor compounds against several CYP450 isoforms. IC50 values and enzyme kinetics comparable to those in solution were obtained for CYP3A4, CYP2C9, CYP2D6, CYP2E1, and CYP1A2 with a number of compounds, including known inhibitor compounds such as sulfaphenazole, clotrimazole, quinidine, and erythromycin. Thus, a robust, sensitive, and accurate CYP450 inhibition assay has been developed that is high-throughput (nearly 2,000-fold miniaturization when compared to 96-well plate assays). The assay is now being validated by expanding to a broad set of test compounds. These advances represent critical steps toward a fully automated, high-throughput human pharmacokinetics (PK) technology. Potential long-term applications include very early-stage PK analysis in the drug discovery process and ultimately leading to uses that may partially enable personalized medicine.