Determination of liver specific toxicities in rat hepatocytes by high content imaging during 2-week multiple treatment

AbstractDILI is a major safety issue during drug development and one of the leading causes for market withdrawal. Despite many efforts made in the past, the prediction of DILI using in vitro models remains very unreliable. In the present study, the well-established hepatocyte Collagen I-Matrigel™ sandwich culture was used, mimicking chronic drug treatment after multiple incubations for 14days. Ten drugs associated with different types of specific preclinical and clinical liver injury were evaluated at non-cytotoxic concentrations. Mrp2-mediated transport, intracellular accumulation of neutral lipids and phospholipids were selected as functional endpoints by using Cellomics™ Arrayscan® technology and assessed at five timepoints (day 1, 3, 7, 10, 14). Liver specific functional impairments after drug treatment were enhanced over time and could be monitored by HCI already after few days and before cytotoxicity. Phospholipidosis-inducing drugs Chlorpromazine and Amiodarone displayed the same response as in vivo. Cyclosporin A, Chlorpromazine, and Troglitazone inhibited Mrp2-mediated biliary transport, correlating with in vivo findings. Steatosis remained difficult to be reproduced under the current in vitro testing conditions, resulting into false negative and positive responses. The present results suggest that the repeated long-term treatment of rat hepatocytes in the Collagen I-Matrigel™ sandwich configuration might be a suitable tool for safety profiling of the potential to induce phospholipidosis and impair Mrp2-mediated transport processes, but not to predict steatosis

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: a Comprehensive Validation Using Drugs and Compounds Affecting the Retina

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies

Incorporating microglia‐like cells in human induced pluripotent stem cell‐derived retinal organoids

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina

Etude in vitro et in vivo de l'interaction topoisomerase II-ADN, en fonction de differentes molecules antitumorales sur un ADN viral transformant

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 84742 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Improving the safety assessment of chemicals and drug candidates by the integrating bioinformatics and chemoinformatics

The application of read-across and in silico tools for regulatory decision making has been limited for pharmaceutical compounds to the assessment of genotoxic impurity. In contrast, the broad availability of toxicity data for industrial chemicals has triggered regulatory frameworks for read-across (e.g. ECHA read-across assessment framework), software tools and public databases for an automated process of gap filling for safety assessment framework. This review provides an overview of the currently existing read-across approaches for chemicals and pharmaceutical compounds highlighting particularly the different requirements in the safety assessment of these two fields. The biggest hurdle for establishing preclinical safety databases for pharma compounds are the unwillingness to share proprietary data and lack of published data sets. In a consortial approach thirteen pharmaceutical companies, eleven academic partners and six small to medium size enterprises (SMEs) of the bioinformatics sector joined forces over the last seven years within the European Innovative Medicines Initiative project eTOX ("electronic toxicity") to design and implement a strategy for leveraging these preclinical data and sharing them across project partners. The eTOX database has evolved as the largest preclinical toxicity database for drugs and drug candidates and currently contains more than 1900 different chemical structures and more than 8000 in vivo toxicity study data. Use cases based on chemical or biological similarity are presented. It can be foreseen that the development and application of such databases for drugs or drug candidates will in the future also cross-fertilize the read-across and in silico assessment of industrial or consumer chemicals particularly as soon as human safety data from clinical trials are integrated, too

The eTOX Consortium: To Improve the Safety Assessment of New Drug Candidates

After their incorporation into human risk assessment, reports on preclinical animal studies in most cases get buried in the archives. The wealth of these data is hardly accessible. The European Innovative Medicines Initiative project eTOX ("electronic toxicity") over the last years designed and implemented a strategy for leveraging these preclinical data and sharing them across pharmaceutical companies and academic institutions. The shared toxicological data can now be used by the participating companies to perform early safety assessments of new drug candidates and new pharmacological targets. In addition, the data are used to build in silico predictive tools for specific toxicological endpoint

OntoBrowser: a tool for curating ontologies and code lists by subject matter experts.

Lack of compliance of terminology and ontology usage leads to partial search results and poor inter-operability between databases. The source of the problem is often the difficulty of finding subject matter experts having time, skills and tools to facilitate curation. Existing mapping software are usually not intuitive and the process is time consuming. The primary objective of OntoBrowser is to provide an easy to use online collaborative solution for subject matter experts, to map reported terms to preferred ontologies or controlled terminologies. Additional features include: web service access to data, visualization of ontologies in hierarchical/graph format, advanced search capabilities, peer review/approval workflow

Bile acids in drug induced liver injury: Key players and surrogate markers

Bile acid research has gained great momentum since the role of bile acids as key signaling molecules in the enterohepatic circulation was discovered. Their physiological function in regulating their own homeostasis, as well as energy and lipid metabolism make them interesting targets for the pharmaceutical industry in the context of diseases such as bile acid induced diarrhea, bile acid induced cholestasis or nonalcoholic steatohepatitis. Changes in bile acid homeostasis are also linked to various types of drug-induced liver injury (DILI). However, the key question whether bile acids are surrogate markers for monitoring DILI or key pathogenic players in the onset and progression of DILI is under intense investigation. The purpose of this review is to summarize the different facets of bile acids in the context of normal physiology, hereditary defects of bile acid transport and DILI

Liver enzyme delayed clearance in rat treated by CSF1 receptor specific antagonist Sotuletinib.

Sotuletinib (BLZ945), a CSF1-R specific kinase inhibitor developed for the treatment of Amyotrophic Lateral Sclerosis, induced liver enzyme elevation in absence of hepatic lesions in preclinical rat and monkey studies. The depletion of Kupffer cells through CSF1 pathway inhibition has been hypothesized as responsible for this effect. However, a release of these biomarkers from unseen hepatic lesions or from other organs cannot be excluded. Here we demonstrate a delayed clearance independently of any potential organ lesions by exogenously injecting recombinant his-Tagged ALT1 into rats pretreated with Sotuletinib, excluding a potential undetected cytotoxic effect