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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Induction of Drug Metabolism Enzymes and MDR1 Using a Novel Human Hepatocyte Cell Line

Exploratory Medicinal Sciences (J.B.M, S.M.F.de M.), Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut; and Department of Pharmacokinetics, Dynamics, and Metabolism (K.A.R., N.S., J.S.), Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan

Induction of drug-metabolizing enzymes and transporters can cause drug-drug interactions and loss of efficacy. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds. We investigated the use of a novel human hepatocyte clone, the Fa2N-4 cell line, as an alternative reagent, which is readily available and provides a consistent, reproducible system. We used the Invader assay to monitor gene expression in these cells. This assay is a robust, yet simple, high-throughput system for quantification of mRNA transcripts. CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 transcripts were quantified from total RNA extracts from Fa2N-4 cells treated with a panel of known inducers and compared with vehicle controls. In addition, we used enzyme activity assays to monitor the induction of CYP1A2, CYP2C9, and CYP3A4. The Fa2N-4 cells responded in a similar manner as primary human hepatocytes. Treatment with 10 µM rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6--hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4'-hydroxydiclofenac formation, 2-fold). Treatment with 50 µM -naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by -naphthoflavone (2-fold), and MDR1 (P-glycoprotein) mRNA was induced by rifampin (3-fold). These preliminary data using a few prototypical inducers show that Fa2N-4 cells can be a reliable surrogate for primary human hepatocytes, and, when used in conjunction with the Invader technology, could provide a reliable assay for assessment of induction of drug-metabolizing enzymes and transporters.

Address correspondence to: Dr. Sonia M. F. de Morais, Pfizer Global Research and Development, Groton Laboratories, Eastern Point Rd., Groton, CT 06340. E-mail: demoraissm{at}groton.pfizer.com

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