Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.

BACKGROUND AND AIMS: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration. APPROACH AND RESULTS: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy. Using translational noninvasive imaging techniques, CDDO-Me was shown to enhance the rate of restoration of liver volume (MRI) and improve liver function (multispectral optoacoustic imaging of indocyanine green clearance) in wild-type, but not Nrf2 null, mice following partial hepatectomy. Using immunofluorescence imaging and whole transcriptome analysis, these effects were found to be associated with an increase in hepatocyte hypertrophy and proliferation, the suppression of immune and inflammatory signals, and metabolic adaptation in the remnant liver tissue. Similar processes were modulated following exposure of primary human hepatocytes to CDDO-Me, highlighting the potential relevance of our findings to patients. CONCLUSIONS: Our results indicate that pharmacological activation of Nrf2 is a promising strategy for enhancing functional liver regeneration. Such an approach could therefore aid the recovery of patients undergoing liver surgery and support the treatment of acute and chronic liver disease.

Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier-Mediated Process.

The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( Vmax = 3299 pmol/million cells/min, Km = 35.9 µM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331.

A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity.

Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease.

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication.

In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.