1521-009X/43/1/119–125$25.00 http://dx.doi.org/10.1124/dmd.114.059246 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:119–125, January 2015 Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics HepaRG Cells as Human-Relevant In Vitro Model to Study the Effects of Inflammatory Stimuli on Cytochrome P450 Isoenzymes Katarina Rubin, Annika Janefeldt, Linda Andersson, Zsofia Berke, Ken Grime, and Tommy B. Andersson AstraZeneca R&D, Mölndal, Sweden; and Respiratory, Inflammation and Autoimmunity Innovative Medicines Drug Metabolism and Pharmacokinetics (K.R., K.G.), Cardiovascular and Metabolic Diseases Innovative Medicines (A.J., T.B.A.), Drug Safety and Metabolism Drug Metabolism and Pharmacokinetics (L.A.), Personalized Healthcare and Biomarkers (Z.B.), and Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (T.B.A.) Received May 27, 2014; accepted October 31, 2014 ABSTRACT The suppression of hepatic cytochrome P450 (P450) expression during inflammatory and infectious diseases and the relief of this suppression by successful disease treatment have been previously demonstrated to impact drug disposition. To address this clinically relevant phenomenon preclinically, the effect of proinflammatory cytokines on P450 isoenzymes in human hepatocytes has been examined by several researchers. In the present study we used the human hepatoma cell line (HepaRG) and cryopreserved primary human hepatocytes to investigate the effects of various inflamma- tory stimuli on P450 levels with the aim of further characterizing HepaRG cells as a useful surrogate for primary hepatocytes. In this study, HepaRG cells were exposed to bacterial lipopolysaccharide (LPS), interleukin-6 (IL-6), and interleukin-18 (IL-18) for 48 or 72 hours. The effects on CYP1A2, CYP2B6, and CYP3A4 mRNA and catalytic activity (phenacetin-O-deethylase, bupropion-hydroxylase, and midazolam-19-hydroxylase) were measured. Cryopreserved pooled plateable hepatocytes were also exposed to IL-6 or IL-18 for 48 hours, and the effects on CYP1A2, CYP2B6, and CYP3A4 mRNA levels were measured. The exposure of HepaRG cells to IL-6 and LPS resulted in suppression of CYP1A2, CYP2B6, and CYP3A4 mRNA levels as well as their catalytic activities. However, no suppression of P450 activities or mRNA levels was observed after exposure to IL-18. Similar results on CYP1A2, CYP2B6, and CYP3A4 mRNA levels were observed with primary hepatocytes. The present study indicates that different proinflammatory mediators influence the expression of P450 differentially and that HepaRG cells may be used as an alternative to human hepatocytes for studies on cytokine-mediated suppression of drug-metabolizing enzymes. Introduction Proinflammatory cytokines can impact the transcriptional regulation of cytochrome P450 (P450) enzymes. Thus, owing to elevated levels of some circulating cytokines in chronic inflammatory disease conditions, expression levels and activity of these drug metabolizing enzymes can be affected (Shedlofsky et al., 1994; Morgan, 2001; Kulmatycki and Jamali, 2005). This phenomenon is of importance when accounting for pharmacokinetic differences between healthy and diseased subjects, and when considering the introduction of novel anti-inflammatory therapies. Depending on the type of therapy, there may be a decrease in circulatory proinflammatory cytokine levels and associated increases in expression and activity of certain P450s. This may have a direct effect on the metabolism of other administered drugs, potentially decreasing their exposure and efficacy (Huang et al., 2010). In vitro studies using human hepatocytes can facilitate understanding of the relevance of such events and help predict clinical outcomes. Cytokine-mediated P450 regulation has been studied previously in primary human hepatocytes in vitro, with different cytokines dem- onstrating clear effects measured both at the transcriptional and functional activity level (Sunman et al., 2004; Aitken et al., 2006; Christensen and Hermann, 2012). The mechanisms behind this P450 regulation have been investigated, and the nuclear factor k-light-chain-enhancer of activated B cells–dependent inflammatory response and associated relocalization and dimerization of nuclear receptors such as retinoid X receptor, liver X receptor, farnesoid X receptor, constitutive androstane receptor, pregnane X receptor (PXR), and peroxisome proliferator–activated receptor have been implicated (Jover et al., 2002, Yang et al., 2010; Ghose et al., 2011). However, P450-specific transcriptional regulation has not been fully characterized, and the question of common pathways versus specific receptor-dependent intracellular signaling pathways is still to be elucidated. IL-6 is a primary mediator of the acute-phase response and is a central inflammatory mediator in several chronic inflammatory diseases, such as rheumatoid arthritis, psoriasis, and atherosclerosis (Arican et al., 2005; Rose-John et al., 2007; Barnes et al., 2011). The effect of IL-6 on primary human hepatocytes has been documented, with the expression of several P450s downregulated (Jover et al., 2002; Aitken and Morgan, 2007; Le Vee et al., 2009; Dickmann et al., 2011). In human hepatoma cell line (HepaRG) cells, IL-6 and lipopolysaccharide (LPS) were pre- viously shown to upregulate C-reactive protein (CRP) and downregulate CYP3A4 mRNA (Aninat et al., 2008). Moreover, drug clearance in cancer patients has been shown to inversely correlate with circulating IL-6 levels (Rivory et al., 2002), whereas treatment of rheumatoid arthritis patients with the anti–IL-6 antibody drug tocolizumab has been shown to directly affect CYP3A4 activity (Schmitt et al., 2011). dx.doi.org/10.1124/dmd.114.059246. ABBREVIATIONS: BM, basal medium; CRP, C-reactive protein; HepaRG, human hepatoma cell line; huPO, human acidic ribosomal phosphoprotein; IL, interleukin; LPS, lipopolysaccharide; P450, cytochrome P450; PXR, pregnane X receptor; RT-PCR, reverse transcription–polymerase chain reaction. 119 at ASPET Journals on May 1, 2016 dmd.aspetjournals.org Downloaded from