Detection of Drug-Induced Cholestasis Potential in Sandwich-Cultured Human Hepatocytes
Drug-induced cholestasis poses a major hurdle for the pharmaceutical industry as it is one the primary mechanisms of drug-induced liver injury. Hence, detection of drug-induced cholestasis during the early stages of drug development is of utmost importanc
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induced liver injury (DILI) is a primary cause of both attrition and post-marketing withdrawal of drugs, which leads to considerable losses for the pharmaceutical industry [1]. As DILI presents itself in several predominant clinical forms (e.g., acute hepatitis, cholestasis, and steatosis), the subsequent plethora of possible underlying mechanisms is an immense hurdle for the development of next generation prediction tools [2]. Therefore, a better understanding of these mechanisms is an absolute priority Mathieu Vinken (ed.), Experimental Cholestasis Research, Methods in Molecular Biology, vol. 1981, https://doi.org/10.1007/978-1-4939-9420-5_22, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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for the pharmaceutical industry [3]. During the last decade, it has become more and more apparent that the disturbance of bile acid homeostasis plays a key role in the onset of liver injury, making cholestasis one of the major causes of DILI [4]. However, early detection of drug-induced cholestasis (DIC) remains challenging during the drug development process. Existing in vitro models for detecting compounds that induce cholestatic liver injury predominantly rely on the extent of inhibition of bile salt export pump (BSEP)-mediated taurocholic acid transport, either in sandwichcultured hepatocytes (SCH) or membrane vesicles originating from BSEP-overexpressing cells [5–7]. However, several recent studies have indicated that evaluation of BSEP inhibition alone does not adequately predict DIC [8, 9]. More holistic in vitro tools are therefore needed to accurately identify compounds with cholestatic liability. Sandwich-cultured human hepatocytes (SCHH) represent such a suitable in vitro model, as previous work has indicated that SCHH preserve relevant expression and activity levels of basolateral and canalicular transporters as well as enzymes involved in bile acid homeostasis [10]. We recently developed and validated a SCHH-based in vitro assay to identify compounds that may cause cholestasis in vitro by interfering with bile acid handling in the cultured hepatocytes [9, 11]. The assay relies on determination of drug-induced cholestasis index (DICI) values. DICI values reflect the relative ability of SCHH to maintain urea formation when additionally challenged with bile acids in the presence of a potentially cholestatic compound. The amount of urea produced is determined by the urea assay, a nondestructive assay that is based on the reaction of urea with diacetyl monoxime under strong acidic conditions. The reaction is catalyzed by ferric ions and condenses to a red-colored product in the presence of thiosemicarbazide. The intensity of the red color, which is assessed spectrophotometrically, is proportional to the amount of urea formed. A DICI value of less than 0.8 can be considered as an indication for enhanced risk for disturbed bile acid disposition (or “in vitro cholestasis”). This approach has also been successfully applied to primary human hepatocytes in 3D spheroid cultur
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