High‐Content Analysis in Toxicology: Screening Substances for Human Toxicity Potential, Elucidating Subcellular Mechanisms and In Vivo Use as Translational Safety Biomarkers

Abstract

High-content analysis (HCA) of in vitro biochemical and morphological effects of classic (small molecule) drugs and chemicals is concordant with potential for human toxicity. For hepatotoxicity, concordance is greater for cytotoxic effects assessed by HCA than for conventional cytotoxicity tests and for regulatory animal toxicity studies. Additionally, HCA identifies chronic toxicity potential, and drugs producing idiosyncratic adverse reactions and/or toxic metabolites are also identified by HCA. Mechanistic information on the subcellular basis for the toxicity is frequently identified, including various mitochondrial effects, oxidative stress, calcium dyshomeostasis, phospholipidosis, apoptosis and antiproliferative effects, and a fingerprinting of the sequence and pattern of subcellular events. As these effects are frequently non-specific and affect many cell types, some toxicities may be detected and monitored by HCA of peripheral blood cells, such as for anticancer and anti-infective drugs. Critical methodological and interpretive features are identified that are critical to the effectiveness of the HCA cytotoxicity assessment, including the need for multiple days of exposure of cells to drug, use of a human hepatocyte cell line with metabolic competence, assessment of multiple pre-lethal effects in individual live cells, consideration of hormesis, the need for interpretation of relevance of cytotoxicity concentration compared to efficacy concentration and quality management. Limitations of the HCA include assessment of drugs that act on receptors, transporters or processes not found in hepatocytes. HCA may be used in a) screening lead candidates for potential human toxicity in drug discovery alongside of in vitro assessment of efficacy and pharmacokinetics, b) elucidating mechanisms of toxicity and c) monitoring in vivo toxicity of drugs with known toxicity of known mechanism.Classic (small molecule) drug toxicity is one of the major challenges for the pharmaceutical industry, not only because of the associated loss of human life or health, but because of enormous financial loss of past investment and of future revenue potential. Attrition of drugs is progressively more costly the later it occurs. It costs almost two billion dollars to bring the average drug through discovery and development to registration [1]. Furthermore, typically 10-12 years of investment of time by hundreds of pharmaceutical staff will have been mis-spent.Drug safety has been an important cause of this attrition. Analysis of safety attrition of approved drugs over a 25-year period ending in 1999 indicated an annual average of 0.7 approved drugs was withdrawn and two drugs received black box warnings [2]. From 1994 to 2006, the average safety attrition was even higher with 2.1 drugs per year, with 38 drugs withdrawn from the market by the US Food and Drug Agency More than 80% of market withdrawals due to drug toxicity were due to cardiotoxicity and hepatotoxicity. In 2003, druginduced liver injury was found to be the most fr...

Assessment of food toxicology

“…Xenobiotic metabolism is a commonly encountered problem during development of new drug candidates thus applicable also for potential toxicants in food [4,9,25,28,34,92] M a n u s c r i p t…”

“…Advances of these in silico tools to assess toxicity in food has led to a wealth of mechanistic information of adverse effects of food toxicants and a significant reduction in the number of animals required for toxicological tests for a new active substance [5-8, 27, 33]. Therefore, in silico models are being increasingly recognized as predictive tools to analyze hepatotoxicity, cardiotoxicity and nephrotoxicity [9,33,34,39,172,[181][182][183].…”

High-Content Screening: Understanding and Managing Mechanistic Data to Better Predict Toxicity

“…HCS offers the ability to detect cellular pathways associated with sublethal toxicities allowing a sensitive early indication. Many conventional cytotoxicity assays (e.g., LDH release, trypan blue infl ux, and ATP measurement) demonstrate low sensitivity because the end points are typically only measured at a point very close to or at cell death [ 12 ]. HCS-based assays are much more fl exible, with a huge variety of end points that can be assessed.…”

High-content analysis for drug delivery and nanoparticle applications

“…Its effectiveness in identifying cytotoxicity has now been extended from small molecules to biotech molecules, NPs, polymers, and intestinal permeation enhancers. Applicability is across a range of cell types, including hepatic, intestinal, renal, skeletal and cardiac myocytic, neuronal, lymphocytic, and monocytic [31]. In addition, its effectiveness at identifying specific subcellular toxicities has been improved by the incorporation of fluorescent dyes that detect genetic, mitochondrial, lysosomal, and oxidative stress-associated effects.…”