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

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically 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 concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 µM) and Ritonavir (3.5 and 62.4 µM) for 48 h. 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.

Utility of humanized BLT mice for analysis of dengue virus infection and antiviral drug testing.

Dengue virus (DENV) is the cause of a potentially life-threatening disease that affects millions of people worldwide. The lack of a small animal model that mimics the symptoms of DENV infection in humans has slowed the understanding of viral pathogenesis and the development of therapies and vaccines. Here, we investigated the use of humanized "bone marrow liver thymus" (BLT) mice as a model for immunological studies and assayed their applicability for preclinical testing of antiviral compounds. Human immune system (HIS) BLT-NOD/SCID mice were inoculated intravenously with a low-passage, clinical isolate of DENV-2, and this resulted in sustained viremia and infection of leukocytes in lymphoid and nonlymphoid organs. In addition, DENV infection increased serum cytokine levels and elicited DENV-2-neutralizing human IgM antibodies. Following restimulation with DENV-infected dendritic cells, in vivo-primed T cells became activated and acquired effector function. An adenosine nucleoside inhibitor of DENV decreased the circulating viral RNA when administered simultaneously or 2 days postinfection, simulating a potential treatment protocol for DENV infection in humans. In summary, we demonstrate that BLT mice are susceptible to infection with clinical DENV isolates, mount virus-specific adaptive immune responses, and respond to antiviral drug treatment. Although additional refinements to the model are required, BLT mice are a suitable platform to study aspects of DENV infection and pathogenesis and for preclinical testing of drug and vaccine candidates. IMPORTANCE Infection with dengue virus remains a major medical problem. Progress in our understanding of the disease and development of therapeutics has been hampered by the scarcity of small animal models. Here, we show that humanized mice, i.e., animals engrafted with components of a human immune system, that were infected with a patient-derived dengue virus strain developed clinical symptoms of the disease and mounted virus-specific immune responses. We further show that this mouse model can be used to test preclinically the efficacy of antiviral drugs.