Hepatic 3D spheroids as a model system for drug-induced liver injury and cytochrome P450 induction

Drug-induced liver injury (DILI) is a major cause of post-marketing drug withdrawals and restricted-use warnings. In addition, unexpected adverse hepatic drug reactions occurring in the clinical phases of development are a major reason for drug attrition. Currently, there is an unmet need for reliable in vitro models to faithfully study the impact of drugs on the human liver. In this thesis, we developed and extensively characterized a novel three-dimensional (3D) spheroid culture system comprised of primary human hepatocytes (PHH). We found that the proteomes of PHH in 3D spheroid culture closely resemble those observed in the liver in vivo, whereas in conventional two-dimensional (2D) monolayer cultures PHH rapidly lose their mature phenotype due to dedifferentiation. PHH spheroids retain stable molecular phenotypes and liver-specific functionalities for multiple weeks in culture. These features allow prediction of DILI events, including those that may be delayed in onset, in a more phenotypically adequate system. Moreover, the PHH spheroid system was found suitable to detect the liability of drugs to induce cholestasis and to identify concomitant toxicity mechanisms. Our results also indicate that PHH spheroids can be used to screen drugs for cytochrome P450 3A4 (CYP3A4) induction. Importantly, PHH in 3D spheroid culture could identify a clinically relevant atypical mechanism of CYP3A4 induction that was not possible to detect in the corresponding 2D monolayer cultures. In conclusion, the PHH spheroid system presented here constitutes a versatile in vitro model to study liver biology and to assess the metabolic and toxicological profiles of drugs and drug candidates

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

Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine metabolites of diclofenac and mefenamic acid

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRβ), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFβ1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates

Expression and Function of mARC: Roles in Lipogenesis and Metabolic Activation of Ximelagatran

<div><p>Recently two novel enzymes were identified in the outer mitochondrial membrane, mARC1 and mARC2. These molybdenum containing enzymes can reduce a variety of <i>N</i>-hydroxylated compounds, such as N-hydroxy-guanidines and sulfohydroxamic acids, as well as convert nitrite into nitric oxide (NO). However, their endogenous functions remain unknown. Here we demonstrate a specific developmental pattern of expression of these enzymes. mARC1, but not mARC2, was found to be expressed in fetal human liver, whereas both, in particular mARC2, are abundant in adult liver and also expressed in omental and subcutaneous fat. Caloric diet restriction of obese patients caused a decreased expression of mARC2 in liver, similar to that seen in the livers of starved rats. Knock down of mARC2 expression by siRNA in murine adipocytes had statistically significant effect on the level of diglycerides and on the fatty acid composition of some triglycerides, concomitantly a clear trend toward the reduced formation of most of triglyceride and phospholipid species was observed. The involvement of mARC2 in the metabolism of the hepatotoxic drug ximelagatran was evaluated in hepatocytes and adipocytes. Ximelagatran was shown to cause oxidative stress and knock down of mARC2 in adipocytes prevented ximelagatran induced inhibition of mitochondrial respiration. In conclusion, our data indicate that mARC1 and mARC2 have different developmental expression profiles, and that mARC2 is involved in lipogenesis, is regulated by nutritional status and responsible for activation of ximelagatran into a mitotoxic metabolite(s).</p></div

Clinical characteristics of the obese patients.

<p>Clinical characteristics of the obese patients.</p

Statistical analysis of the mARC2 downregulation effect on specific lipids in the differentiated adipocytes (TG, triglycerides; DG, diglycerides).

<p>Statistical analysis of the mARC2 downregulation effect on specific lipids in the differentiated adipocytes (TG, triglycerides; DG, diglycerides).</p

Nutritional status in both human and rats affects the mARC2 levels.

<p>A. mARC2 protein levels in obese patients that were put on a caloric restriction diet (fasted) prior to surgery (n = 7) and control (non-fasted) individuals (n = 7) (patient data are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138487#pone.0138487.t001" target="_blank">Table 1</a>). Equal amounts of protein from the hepatic mitochondrial fractions were analyzed by western blot for mARC2 and loading control, mitochondrial heat shock protein 70 (mHSP70) (lower panels). B. Both bands were quantified by densitometric analysis and mARC2 levels were normalized by mHSP70. The results represent the mean ±S.D. (n = 7 in each group), **p<0.01. C. mARC2 protein levels and the associated amidoxime reductase activity are decreased in the starvation treated rats. The mitochondrial fractions from the livers from control (n = 3) and starvation treated (n = 3) animals were analyzed for amidoxime reductase activity using benzamidoxime as a substrate (left panel). The results are presented as the mean ± S.D. values. ***, p<0.001. The mitochondrial fractions from these animals were also analyzed by western blot for the presence of mARC2 (right panel). mHSP70, mitochondrial chaperone was used as a loading control.</p

New approach methodologies (NAMs) for human-relevant biokinetics predictions: Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment.

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.</p