Transcriptomic Analysis of Cholestatic Compounds In Vitro.

Drug-induced cholestasis is one of the most severe manifestations of drug-induced liver injury. Drug-induced cholestasis is characterized by an accumulation of endogenous metabolites normally excreted in the bile such as bile salts, cholesterol, bilirubin, or drug metabolites. The possibility to determine early in the drug development process whether a compound presents a risk of inducing drug-induced cholestasis is key information. Since preclinical repeated dose toxicity studies have limited predictive value, large efforts in identifying alternative in vitro models with improved prediction are being made. One of the best current models for in vitro human liver is primary human hepatocytes, and we recently reported that primary human hepatocytes can be kept as long-term cultures in 2D-sandwich configuration when regularly renewing the Matrigel overlay, thereby making the model useful for repeat exposure-related toxicities, as well as for the study of adaptive responses. This primary human hepatocyte culture system combined with transcriptomics carries the future promise to identify individual gene expression profiles predictive of increased drug-induced cholestasis risk.This chapter describes the various steps for culturing and exposing primary human hepatocytes to drugs during long-term 2D-sandwich culture, performing RNA extraction, gene chip assay and selecting hepatotoxic signature using the IPA software and highlighting genes involved in bile acid homeostasis.

Safety differentiation: emerging competitive edge in drug development.

With increasing expectations to provide evidence of drug efficacy, safety, and cost-effectiveness, best-in-class drugs are a major value driver for the pharmaceutical industry. Superior safety is a key differentiation criterion that could be achieved through better risk:benefit profiles, safety margins, fewer contraindications, and improved patient compliance. To accomplish this, comparative safety assessments using innovative and adaptive nonclinical and clinical outcome-based approaches should be undertaken, and continuous strategic adjustments must be made as the risk:benefit profiles evolve. Key success criteria include scientific expertise and integration between all disciplines during the full extent of the drug development process.

Human-Induced Pluripotent Stem Cells Generate Light Responsive Retinal Organoids with Variable and Nutrient-Dependent Efficiency.

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Evaluation of transcriptomic signature as a valuable tool to study drug-induced cholestasis in primary human hepatocytes.

Primary human hepatocyte (PHH) sandwich cultures from five different donors were daily exposed to cyclosporine A (CsA), ibuprofen (IBU), chlorpromazine (CPZ), amiodarone (AMI) and paracetamol (APAP) at their respective Cmax (total) for short-term (1-3 days) and long-term treatment (14 days). Whole genome mRNA profiles (34,693 genes in total) were conducted using an Illumina microarray platform. The impact of compound treatments on gene signatures involved in liver differentiation, cholestasis and in bile acid homeostasis was evaluated. Notably, PHH from the five donors showed a highly comparable phenotype of terminally differentiated hepatocytes. As expected, PHH exposed to 100 µM APAP showed no signs of hepatotoxicity both after short- and long-term treatment. CsA at 0.7 µM, IBU at 100 µM, AMI at 2.5 µM and CPZ at 0.1-0.2 µM presented, in line with their cholestatic syndromes reported at therapeutic doses, transcriptomic signatures of cholestasis in PHH cultures; deregulation of genes involved in bile acid homeostasis further confirmed this finding. The strength of the cholestasis signature obtained after treatment with CsA, IBU and AMI could be directly related to the basal expression of the respective drug metabolizing enzymes in the various PHH cultures from different individuals. Our data show that the PHH model system combined with transcriptomics carries the future promise to identify individual gene expression profiles predictive of increased cholestasis risk. As the present work suggests possible correlation between mRNA levels of ADME relevant genes and a transcriptomic signature of cholestasis, particular focus on this research question could be the emphasis of additional data collection.

Editor's Highlight: Comparative Renal Safety Assessment of the Hepatitis B Drugs, Adefovir, Tenofovir, Telbivudine and Entecavir in Rats.

The aim of this study was to determine the relative safety of 4 antiviral drugs (telbivudine, tenofovir, adefovir, and entecavir) against hepatitis B virus with respect to kidney function and toxicity in male Sprague Dawley rats. The antiviral drugs were administered once daily for 4 weeks by oral gavage at ∼10 and 25-40 times the human equivalent dose. Main assessments included markers of renal toxicity in urine, magnetic resonance imaging (MRI) of kidney function, histopathology, and electron microscopic examination. Administration of adefovir at 11 and 28 mg/kg for 4 weeks caused functional and morphological kidney alterations in a time- and dose-dependent manner, affecting mainly the proximal tubules and suggesting a mechanism of toxicity related to mitochondrial degeneration/depletion. Of note, the observed adefovir-induced reduction of kidney function was not detected by the standard method of glomerular filtration rate (GFR) measurements (clearance rate of the endogenous marker, creatinine), thereby emphasizing the superiority of MRI in terms of sensitive detection of GFR in rats. For the low dose of 300 mg/kg of tenofovir, minor kidney effects such as nuclear enlargement in the tubular epithelium, and hyaline droplets accumulation were detected, which was also observed for the low dose (11 mg/kg) of adefovir. No assessments could be done at the higher dose of 600/1000 mg/kg tenofovir due to gastrointestinal tract toxicity which prevented treatment of the animals for longer than 1 week. Entecavir at 1 and 3 mg/kg and telbivudine at 600 and 1600 mg/kg caused no toxicologically relevant effects on the kidney.

Mechanistic insights into selective killing of OXPHOS-dependent cancer cells by arctigenin.

Arctigenin has previously been identified as a potential anti-tumor treatment for advanced pancreatic cancer. However, the mechanism of how arctigenin kills cancer cells is not fully understood. In the present work we studied the mechanism of toxicity by arctigenin in the human pancreatic cell line, Panc-1, with special emphasis on the mitochondria. A comparison of Panc-1 cells cultured in glucose versus galactose medium was applied, allowing assessments of effects in glycolytic versus oxidative phosphorylation (OXPHOS)-dependent Panc-1 cells. For control purposes, the mitochondrial toxic response to treatment with arctigenin was compared to the anti-cancer drug, sorafenib, which is a tyrosine kinase inhibitor known for mitochondrial toxic off-target effects (Will et al., 2008). In both Panc-1 OXPHOS-dependent and glycolytic cells, arctigenin dissipated the mitochondrial membrane potential, which was demonstrated to be due to inhibition of the mitochondrial complexes II and IV. However, arctigenin selectively killed only the OXPHOS-dependent Panc-1 cells. This selective killing of OXPHOS-dependent Panc-1 cells was accompanied by generation of ER stress, mitochondrial membrane permeabilization and caspase activation leading to apoptosis and aponecrosis.

Antiviral Nucleotide Incorporation by Recombinant Human Mitochondrial RNA Polymerase Is Predictive of Increased In Vivo Mitochondrial Toxicity Risk.

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2'-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 µM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 µM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, ß, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.

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

DILI 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 14 days. 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.

Current approaches and future role of high content imaging in safety sciences and drug discovery.

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems. This allows the identification of signaling pathways underlying cell defense, adaptation, toxicity and death. Therefore, high content imaging is considered a promising technology to address the challenges for the "Toxicity testing in the 21st century" approach. Currently, high content imaging technologies are frequently applied in academia for mechanistic toxicity studies and in pharmaceutical industry for the ranking and selection of lead drug compounds or to identify/confirm mechanisms underlying effects observed in vivo. A recent workshop gathered scientists working on high content imaging in academia, pharmaceutical industry and regulatory bodies with the objective to compile the state-of-the-art of the technology in the different institutions. Together they defined technical and methodological gaps, proposed quality control measures and performance standards, highlighted cell sources and new readouts and discussed future requirements for regulatory implementation. This review summarizes the discussion, proposed solutions and recommendations of the specialists contributing to the workshop.

Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein-dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide sakacin P and a sakacin P variant that is structurally stabilized by an inserted C-terminal disulfide bridge.

The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1-17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel beta-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic alpha-helix (residues 18-33), and this in turn is followed by the C-terminal tail (residues 34-43 for sakacin P and 34-44 for sakP[N24C+44C]) that lacks any apparent common secondary structural motif. In the presence of DPC, the C-terminal tails in both peptides fold back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal halves. The lack of long-range NOEs between the beta-sheet Nu-terminal region and the hairpin-like C-terminal half indicates that there is a flexible hinge between these regions. We discuss which implications such a structural arrangement has on the interaction with the target cell membrane.

Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium.

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.