QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: dmd.106.009969v1 34/8/1291    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McGinnity, D. F. Articles by Riley, R. J. Search for Related Content PubMed PubMed Citation Articles by McGinnity, D. F. Articles by Riley, R. J.

EVALUATION OF TIME-DEPENDENT CYTOCHROME P450 INHIBITION USING CULTURED HUMAN HEPATOCYTES

Department of Physical & Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdom

Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters k_inact and K_I, which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4'-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1'-hydroxylation in a time- and concentration-dependent manner. Fluoxetine also inhibited CYP2C19-dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLMs, and recombinant CYP2C19 (K_I 0.4 µM and k_inact 0.5 min^–1). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, a proprietary AstraZeneca compound; K_I 30 µM and k_inact 0.02 min^–1) effectively abolished CYP2C9 activity over 24 h at low (micromolar) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time-dependent inhibitors, which may have dramatic inhibitory effects on P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate.

This article has been cited by other articles:

				O. A. Fahmi, S. Hurst, D. Plowchalk, J. Cook, F. Guo, K. Youdim, M. Dickins, A. Phipps, A. Darekar, R. Hyland, et al. Comparison of Different Algorithms for Predicting Clinical Drug-Drug Interactions, Based on the Use of CYP3A4 in Vitro Data: Predictions of Compounds as Precipitants of Interaction Drug Metab. Dispos., August 1, 2009; 37(8): 1658 - 1666. [Abstract] [Full Text] [PDF]

				D. F. McGinnity, G. Zhang, J. R. Kenny, G. A. Hamilton, S. Otmani, K. R. Stams, S. Haney, P. Brassil, D. M. Stresser, and R. J. Riley Evaluation of Multiple in Vitro Systems for Assessment of CYP3A4 Induction in Drug Discovery: Human Hepatocytes, Pregnane X Receptor Reporter Gene, and Fa2N-4 and HepaRG Cells Drug Metab. Dispos., June 1, 2009; 37(6): 1259 - 1268. [Abstract] [Full Text] [PDF]

				D. M. Stresser, A. K. Mason, E. S. Perloff, T. Ho, C. L. Crespi, A. A. Dandeneau, L. Morgan, and S. S. Dehal Differential Time- and NADPH-Dependent Inhibition of CYP2C19 by Enantiomers of Fluoxetine Drug Metab. Dispos., April 1, 2009; 37(4): 695 - 698. [Abstract] [Full Text] [PDF]

				J. M. Hutzler, L. M. Balogh, M. Zientek, V. Kumar, and T. S. Tracy Mechanism-Based Inactivation of Cytochrome P450 2C9 by Tienilic Acid and ({+/-})-Suprofen: A Comparison of Kinetics and Probe Substrate Selection Drug Metab. Dispos., January 1, 2009; 37(1): 59 - 65. [Abstract] [Full Text] [PDF]

				X. Zhang, D. R. Jones, and S. D. Hall Prediction of the Effect of Erythromycin, Diltiazem, and Their Metabolites, Alone and in Combination, on CYP3A4 Inhibition Drug Metab. Dispos., January 1, 2009; 37(1): 150 - 160. [Abstract] [Full Text] [PDF]

				K. Grime, P. J. H. Webborn, and R. J. Riley Functional Consequences of Active Hepatic Uptake on Cytochrome P450 Inhibition in Rat and Human Hepatocytes Drug Metab. Dispos., August 1, 2008; 36(8): 1670 - 1678. [Abstract] [Full Text] [PDF]

				P. J. Kilford, M. Gertz, J. B. Houston, and A. Galetin Hepatocellular Binding of Drugs: Correction for Unbound Fraction in Hepatocyte Incubations Using Microsomal Binding or Drug Lipophilicity Data Drug Metab. Dispos., July 1, 2008; 36(7): 1194 - 1197. [Abstract] [Full Text] [PDF]

				D. F. McGinnity, N. J. Waters, J. Tucker, and R. J. Riley Integrated in Vitro Analysis for the in Vivo Prediction of Cytochrome P450-Mediated Drug-Drug Interactions Drug Metab. Dispos., June 1, 2008; 36(6): 1126 - 1134. [Abstract] [Full Text] [PDF]

				P. Zhao, C. A. Lee, and K. L. Kunze Sequential Metabolism Is Responsible for Diltiazem-Induced Time-Dependent Loss of CYP3A Drug Metab. Dispos., May 1, 2007; 35(5): 704 - 712. [Abstract] [Full Text] [PDF]