High‐Content Analysis/Screening for Predictive Toxicology: Application to Hepatotoxicity and Genotoxicity

Abstract

High-content imaging/analysis has emerged as a powerful tool for predictive toxicology as it can be used for identifying and mitigating potential safety risks during drug discovery. By careful selection of end-points, some cellular assays can show better predictivity than routine animal toxicity testing for certain adverse events. Here, we present the perhaps most utilized highcontent screening assays for predictive toxicology in the pharmaceutical industry. Multi-parametric imaging of cell health in simple and cost-effective model systems can be used to predict human hepatotoxicity and elucidate mechanisms of toxicity, and imaging of bile salt transport inhibition in sandwich-cultured hepatocytes can be used to predict cholestasis-inducing compounds. Imaging of micronuclei formation in simple cell models can be used to detect genotoxic potential and elucidate anuegenic or clastogenic mode of actions. The hope is that application of these relatively predictive assays during drug discovery will reduce toxicity and safety-related attrition of drug development programmes at later stages. High-Content Imaging/Analysis for Predictive ToxicologyHigh-content imaging and analysis, often referred to as highcontent screening (HCS), has emerged as a powerful tool for assessing molecular, cellular, and tissue-based toxicity, particularly in the field of predictive toxicology. This field of science aims at predicting adverse events in human beings (and pre-clinical species) associated with drug treatment. It is crucial for the pharmaceutical industry to identify and subsequently mitigate potential safety risks early on in the R&D process, and thereby reduce the toxicity and safety-related attrition of drug development programmes at later stages [1]. Here, we review some of the most commonly used HCS assays for predictive toxicology in the pharmaceutical industry, and at Lundbeck in particular. We discuss the background of the readouts, their predictivity and what conclusions can be drawn from them. Particular emphasis will be placed on endpoints that are not well predicted by routine toxicological studies in animals. The Lundbeck strategy for toxicology assessments during the drug discovery process, including assays performed, is described elsewhere [1,2].Since HCS is based on automatized imaging of cells or tissues, typically making use of fluorescent dyes or antibodies, it offers some key advantages over the biochemical assays traditionally used for in vitro toxicology assessments. The main advantages are that the end-points can be assessed at the single-cell level and that multiple end-points can be assessed simultaneously. This enables the user to customize and multiplex end-points and gate for particular cells of interest, which provides an integrated assessment of cellular or molecular toxicity and its mode of action. The multiplexing of different end-points is only limited by the specificity of the employed dyes and antibodies, and their spectral separation. This allows for highly customized assays where the...

Inhibition of bile canalicular network formation in rat sandwich cultured hepatocytes by drugs associated with risk of severe liver injury

“…Recently, high content imaging analyses have attracted attention on the basis of this concept (Tolosa et al, 2015). These analyses mostly focus on mitochondrial membrane potential, oxidative stress, endoplasmic reticulum stress, membrane integrity, shape of a nucleus, and so on (Donato et al, 2012;Persson et al, 2014;Tolosa et al, 2015). However, to the best of our knowledge, BC network formation has not yet been examined.…”

“…Focusing on these various mechanisms, DILI risk screening assay systems have been extensively proposed (Bale et al, 2014). These assays not only monitor hepatocyte death but also collect data on functional markers including membrane permeability, cell proliferation, mitochondrial activity, endoplasmic reticulum stress, and so on (Donato et al, 2012;Persson et al, 2014;Tolosa et al, 2015). Such multifactorial analyses provide improved predictability and mechanistic information; however, methods to clearly identify drugs at risk of causing severe DILI have not yet been established.…”

Assessment of food toxicology

“…[5,7,34,128]. Consequently, high content as well as highthroughput complex cellular in vitro models focusing on organ-specific toxicity such as hepatotoxicity [127,129,130], nephrotoxicity [131,132], neurotoxicity [133][134][135], cardiotoxicity [59,136], and respiratory toxicity [137,138] are routinely used. An even higher level of complexity is achieved by three-dimensional organotypic models.…”

“…A c c e p t e d M a n u s c r i p t Thanks to significant advances made in biotechnology during the last two decades the majority of toxicity test systems are now analyzed by highthroughput or high content screening technology [4,5,9,25,27,127]. These newly emerged screening systems were originally developed by the pharmaceutical industry to identify bioactive compounds from huge corporate compound libraries [5,184,185].…”

Workshop of Scandinavian Society for Cell Toxicology 25‐27 September 2013 in Denmark

“…Per Artursson also presented weaknesses of the current in vitro methodologies. To overcome these challenges, he presented recent approaches towards more quantitative predictions of drug exposure: for example, models for estimation of free drug concentration, proteomics-informed quantification of drug transport and metabolism, and in vitro predictions of drug-induced liver injury.The scientific programme included presentations of new technologies in cell toxicity testing including efficient and sensitive techniques of high-content screening (HCS) based on in vitro monitoring of biochemical and morphological responses of individual live cells to chemical stress in microtitre plates [2][3][4]. This technique can be further refined and developed for use in preclinical testing, diagnostic and prognostic testing as well as monitoring.…”