Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes

Abstract

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically. Predicting DILI is challenging due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to approximating clinical therapeutic and supra-therapeutic concentrations levels of Nevirapine (11.3 and 175.0 μM) and Ritonavir (3.5 and 62.4 μM) for 48 hours. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain with simultaneous mitochondrial uncoupling supporting clinical reports of a lower propensity for steatosis. This in vitro study offers insights into the disparate direct and immune mediated toxicity mechanisms underlying Nevirapine and Ritonavir toxicity in the clinic.

Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis

“…To mimic the liver microenvironment and multicellularity, primary human hepatocytes were cocultured in a collagen sandwich with primary human MΦs and HSCs, separated by a synthetic membrane ( Figure 1A) under conditions of hemodynamics and transport (7,8,11). In addition to hepatocytes, MΦs and HSCs have been shown to be critical to the signaling and progression of NAFL/NASH; thus, it was important that these additional cell types be present in the system (1,12,13).…”

“…These 3 cell types communicate through direct cell-cell contact via the porous transwell membrane (11) and/or through secreted factors. Under healthy-milieu conditions (i.e., containing physiological levels of hormones) and lacking lipotoxic risk factors, hepatocytes maintain their polarized morphology as previously described (7,8). At the conclusion of each experiment, the hepatocytes were separated and analyzed independently from the nonparenchymal cells (NPCs).…”

“…For our lipotoxic system, we chose to use a larger-scale 3D system (7,8) that utilizes more cells, which enables simultaneous collection of multiple endpoints (e.g., transcriptomics, lipidomics, and imaging) from the same experiment, allowing for interassay comparison within a single experiment. This 3D system design incorporates both perfusion and hemodynamic shear forces while recapitulating elements of sinusoidal spatial architecture.…”

“…Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH. (7,8). Herein, we adapted this system to model human fatty liver disease with lipotoxic stress.…”

Systems biology for organotypic cell cultures

“…Many successful examples of organ/tissue models have been recently reported ( Fig. 1) (Adler et al, 2015;Alépée et al, 2014;Andersen et al, 2014;Dash et al, 2009Dash et al, , 2013Hawkins et al, 2015;Hogberg et al, 2013;Mortensen et al, 2016;Pamies et al, 2014;Sarkar et al, 2015;Sellgren et al, 2014Sellgren et al, , 2015Smirnova et al, 2015;Terelius et al, 2015;Torisawa et al, 2014;Walter et al, 2016;Wikswo, 2014).…”