human-liver-microphysiological-system-for-assessing-drug-induced

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity <em>In Vitro</em>

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...

CN Bio Innovations Ltd

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models. 

Bioreactor technologies to support liver function in vitro.

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System.

Hepatotoxins cause liver damage via many mechanisms but the formation of reactive metabolites and/or damage to liver mitochondria are commonly implicated. We assess 3D human primary hepatocyte microtissues as a platform for hepatotoxicity studies with reactive metabolite-forming and mitochondria-perturbing compounds. We show that microtissues formed from cryopreserved human hepatocytes had bile canaliculi, transcribed mRNA from genes associated with xenobiotic metabolism and expressed functional cytochrome P450 enzymes. Hierarchical clustering was used to distinguish dose-dependent hepatotoxicity elicited by clozapine, fialuridine and acetaminophen (APAP) from control cultures and less liver-damaging compounds, olanzapine and entecavir. The regio-isomer of acetaminophen, N-acetyl-meta-aminophenol (AMAP) clustered with the hepatotoxic compounds. The principal metabolites of APAP were formed and dose-dependent changes in metabolite profile similar to those seen in patient overdose was observed. The toxicological profile of APAP was indistinguishable from that of AMAP, confirming AMAP as a human hepatotoxin. Tissue oxygen consumption rate was significantly decreased within 2h of exposure to APAP or AMAP, concomitant with glutathione depletion. These data highlight the potential utility of perfused metabolically functional human liver microtissues in drug development and mechanistic toxicology.

Perfused human hepatocyte microtissues identify reactive metabolite-forming and mitochondria-perturbing hepatotoxins.

Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. There is an urgent need for better understanding of the genetic and molecular pathways that underlie NAFLD/NASH, and currently available preclinical models, be they  or , do not fully represent key aspects of the human disease state. We have developed a human  co-culture NASH model using primary human hepatocytes, Kupffer cells and hepatic stellate cells, which are cultured together as microtissues in a perfused three-dimensional microphysiological system (MPS). The microtissues were cultured in medium containing free fatty acids for at least 2 weeks, to induce a NASH-like phenotype. The co-culture microtissues within the MPS display a NASH-like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Addition of lipopolysaccharide resulted in a more pro-inflammatory milieu. In the model, obeticholic acid ameliorated the NASH phenotype. Microtissues were formed from both wild-type and patatin-like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro-inflammatory milieu.  The MPS model displays a phenotype akin to advanced NAFLD or NASH and has utility as a tool for exploring mechanisms underlying the disease. Furthermore, we demonstrate that in co-culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype.

A Microphysiological System for Studying Nonalcoholic Steatohepatitis.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

Non-alcoholic steatohepatitis (NASH) is a common form of chronic liver disease characterised by lipid accumulation, infiltration of immune cells, hepatocellular ballooning, collagen deposition and liver fibrosis. There is a high unmet need to develop treatments for NASH. We have investigated how liver fibrosis and features of advanced clinical disease can be modelled using an in vitro microphysiological system (MPS). The NASH MPS model comprises a co-culture of primary human liver cells, which were cultured in a variety of conditions including+/- excess sugar, fat, exogenous TGFβ or LPS. The transcriptomic, inflammatory and fibrotic phenotype of the model was characterised and compared using a system biology approach to identify conditions that mimic more advanced clinical disease. The transcriptomic profile of the model was shown to closely correlate with the profile of patient samples and the model displayed a quantifiable fibrotic phenotype. The effects of Obeticholic acid and Elafibranor, were evaluated in the model, as wells as the effects of dietary intervention, with all able to significantly reduce inflammatory and fibrosis markers. Overall, we demonstrate how the MPS NASH model can be used to model different aspects of clinical NASH but importantly demonstrate its ability to model advanced disease with a quantifiable fibrosis phenotype.

David Hughes

CN Bio Innovations Ltd

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro