Regulation of Hepatic Drug Transporters by Pro-inflammatory Cytokines

Abstract

<b>Abstract ID 20942</b> <b>Poster Board 388</b> <i>In vivo</i> studies have shown that the clearance of drugs primarily cleared by cytochrome P450 (CYP) metabolism is reduced during inflammation or infections, potentially resulting in drug toxicity. Inflammation or infectious diseases (e.g. HIV, COVID-19) are associated with increased production of pro-inflammatory cytokines which are known to down-regulate the expression or activity of CYP enzymes. However, their effect on human hepatic drug transporters has been poorly studied, primarily with individual cytokines at supraphysiological concentrations. This makes it difficult to translate these findings to <i>in&nbsp;vivo</i>. Therefore, the goal of this study was to systematically investigate the effect of major cytokines, individually or in combination, on the mRNA expression of hepatic drug transporters in plated primary human hepatocytes. Primary human hepatocytes (lot ZKF, YND, ADR, BioIVT) were treated with interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN- γ), either in combination (each at 0.1 and 1 ng/mL) or individually (0.01, 0.1, 1 and 10 ng/mL) for 72 hours with daily replenishment of media. The cytokine concentrations used encompass the <i>in&nbsp;vivo</i> observed plasma concentrations of cytokines in infectious diseases (e.g. HIV) and autoimmune diseases (rheumatoid arthritis). Then, the total RNA was then isolated, and the mRNA levels of 11 hepatic drug transporters were quantified by real time q-PCR, including organic anion transporting polypeptide 1B1 (OATP1B1), organic anion transporting polypeptide 1B3 (OATP1B3), organic anion transporting polypeptide 2B1 (OATP2B1), organic anion transporter (OAT2), organic cation transporter (OCT1), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1 (MATE1), multi-drug resistance protein 2,3, and 4 (MRP2/3/4). OATP1B1, OATP1B3, OAT2, P-gp, BCRP, and MATE1 mRNA expression was significantly down-regulated by the combination of cytokines in a concentration-dependent manner. OAT2 mRNA was most affected. It was down-regulated by 82% at 0.1 ng/mL and 95% at 1 ng/mL. Conversely, OCT1 mRNA expression was induced by 2-fold and 3-fold at 0.1 ng/mL and 1 ng/mL, respectively. The individual cytokines also modulated transporter mRNA expression in a concentration-dependent manner. The greatest repression (at 10 ng/ml) was of OATP1B1 by TNF-α (by 60%) and IFN- γ (by 60%), OATP1B3 by IL-6 (by 75%) and IFN- γ (by 60%), and OAT2 by TNF-α (by 90%) and IL-1β (by 90%), P-gp by IL-6 (by 50%) and TNF-α (by 50%). While OATP2B1 was down-regulated by IL-1β (by 70%) and induced by IL-6 (by 1.5-fold) at 10 g/ml, this down-regulation was abolished by the combination cytokine treatment. Our data suggested that there is an additive effect of the cytokines on the mRNA expression of most of the hepatic transporters. Similar results were obtained with plated human hepatocytes from three donors. Also, cytokines generally exhibited a greater impact on uptake transporters vs. efflux transporters. Next, we intend to determine if these observed changes in transporter mRNA expression correspond to changes in transporter activity (by transport assays) and/or abundance (by quantitative proteomics). Then, these data will be used to predict changes in transporter-mediated drug PK caused by inflammation or infectious diseases using physiologically based pharmacokinetic modeling and simulations. This work was supported by the NIH Grant R01HD102786.