by Dr A. D. Crawford, Dr N. Mesens, Dr P. A. M. de Witte and Dr C. V. Esguerra
Pharmaceutical companies are under increasing pressure to develop safe and effective drugs, while at the same time controlling R&D expenditures. One partial solution to this challenge may be new technologies capable of generating in vivo data on the safety and efficacy of large numbers of compounds as early as possible in the drug discovery process, thereby reducing the number of drug candidates that fail in clinical development. Zebrafish offer the unique possibility to carry out high-throughput in vivo screens for compounds at the hit to lead stage, to detect both therapeutically relevant bioactivities and to assess potential toxicities. Here, we briefly highlight zebrafish assays for cardiotoxicity and hepatotoxicity.
Regulatory approval rates of new chemical entities (NCEs) are currently lower than at any previous time. Recent data indicate that the average success rate for all therapeutic areas is approximately 11% – implying that only one in nine compounds survives the drug development process – and that the major causes of attrition in the clinic are safety issues [1]. Between 1975-2007, furthermore, 47 drugs were withdrawn from the market due to adverse events. In order to increase the success rate of drug development and to minimise the risk to patients, the prediction of drug safety must improve significantly.
Recent data indicate three toxicities as the most common clinical adverse events: hepatotoxicity (14%), cardiotoxicity (16%) and neurotoxicity (22%). Cardiac and hepatic toxicity also contributed disproportionately to drug withdrawals, with 21 of the 47 drugs withdrawn between 1975-2007 linked to hepatotoxicity and 21 to cardiotoxicity. For these reasons, eliminating potentially toxic compounds as early as possible in the drug discovery process – particularly in the case of hepatotoxicity and cardiotoxicity – would clearly improve the overall efficiency of drug development.
As cell-based toxicity assays address only cell-autonomous toxicity, and in vivo toxicity often involves drug absorption, distribution, metabolism and excretion, in vivo models should be re-considered for early-stage toxicity screening in drug discovery. As large-scale compound screens are not practical in mammalian models due to ethical, financial, and throughput issues, embryos and larvae of the zebrafish (Danio rerio) represent an attractive alternative. Zebrafish have several advantages for screening large numbers of compounds, both for drug discovery and for toxicity applications. Adult zebrafish are small, cost-effective to maintain, and breed easily, producing large numbers of offspring. Zebrafish embryos and larvae, with which most bioassays are performed, are microscopically small – between 1 and 4 mm in length, and therefore compatible with 96-well microtitre plates. Importantly, zebrafish embryos and larvae are optically transparent, readily allowing the visualisation of internal organs such as the liver. The organisation of the genome and the genetic pathways controlling signal transduction and development appear to be highly conserved between zebrafish and humans.
In addition, the cardiovascular, nervous and digestive systems of zebrafish have shown to be similar to their mammalian counterparts at the anatomical, physiological and molecular levels. Finally, histological and transgenesis methods are well-developed for zebrafish applications, allowing the straightforward detection of cells, tissues, and organs via whole-mount in situ hybridisation (WISH) or using transgenic reporters lines. Zebrafish thus offer the opportunity to collect observational data on intact organ systems, of which the physiology closely parallels human organs. Importantly, zebrafish assays offer the possibility to evaluate different organ toxicities simultaneously. Also of interest is the regulatory status of zebrafish – in most jurisdictions, embryos and larvae are not considered to be test animals in the same sense as rodents, thereby reducing the administrative burden for large-scale safety testing.
To date, zebrafish have been extensively used for the toxicological evaluation of environmental contaminants. Along these lines, the zebrafish embryo test was recently introduced in Germany as a standardised ISO assay for water testing, replacing traditional toxicological tests [ISO 15088:2007, Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)].
Zebrafish also offer an attractive platform to analyse off-target effects of drug-like compounds. For example, off-target effects could be identified for acetylcholinesterase inhibitors by comparing the phenotype of an ache -/- mutant with the pharmacological effects of ACHE inhibitors in normal zebrafish. These zebrafish experiments were able to correctly predict that the AChE inhibitor eserine not only blocked AChE activity, but also the acetylcholine receptor (AChR). These results show that zebrafish mutants can function as "phenotypic blueprints" for the ideal bioactivity profile of pharmacological inhibitors, thereby helping to detect off-target effects [2].
With regard to the utility of zebrafish for hepatotoxicity screening, many similarities in liver function between zebrafish and humans have been identified to date, underscoring the suitability of zebrafish as an acceptable model for human disease and toxicity studies. ENU mutagenesis screens have identified mutants with features of liver diseases and a zebrafish model for hepatic steatosis, choleductal cysts and cholestasis has been established. The histopathology of steatosis, cholestasis and neoplasia in zebrafish is quite similar to that in humans. Furthermore, treatment with carcinogens generated liver tumours, and γ-hexachlorocyclohexane, thioacetamide and alcohol can induce hepatic steatosis. Finally, CYP3A4 and CYP2D6, catalysing the majority of known drug-metabolising reactions, have zebrafish orthologues, and functional activity assays based on these enzymes have been performed in zebrafish using human CYP-specific substrates and similar responses have been identified, indicating similar metabolism in the livers of zebrafish and humans.
To develop a more informative in vivo hepatotoxicity assay in developing zebrafish, we are currently examining the utility of a liver-specific molecular marker in zebrafish embryos and larvae – initially via whole-mount in situ hybridisation analysis (WISH). The marker we selected for this purpose was fabp10a, which encodes a liver-specific fatty acid-binding protein. For an initial evaluation of the predictive utility of this assay, we tested a series of compounds with known hepatotoxicity issues in humans: amiodarone, clofibrate, diclofenac, paracetamol, tetracycline and troglitazone, with biotin, ketorolac and rosiglitazone as negative controls. Significant changes in liver size and in liver-specific marker expression were observed for the drugs with known hepatotoxicities in humans, whereas negative controls were negative at the highest concentrations tested [3].
Zebrafish also offer the possibility to investigate various aspects of cardiotoxicity at a very early stage in the drug discovery process. Cardiotoxicity is one of the more common adverse effects seen for many new chemical entities, and the clinical development of numerous drug candidates has been discontinued because of their tendency to induce cardiac arhythmias such QT prolongation and torsades de pointes. One of the primary causes of cardiotoxicity is unforeseen interactions of compounds with the hERG potassium channel. Although interactions of compounds with hERG can be tested in vitro, the predictivity of this assay is not high and examines only one of several possible targets, and a more physiologically relevant assay involving isolated guinea pig hearts requires multi-milligram amounts of each compound to be tested.
With their requirement for only microgram quantities of any given compound to be analysed, combined with the ability to readily observe heart function in transparent larvae, zebrafish are well-suited as a front-line assay for cardiotoxicity. Already in 2003, two laboratories reported an initial validation of zebrafish larvae as a reliable model to test compounds for their ability to induce QT prolongation. These studies showed that 22 out of 23 compounds known to cause QT prolongation in humans induced bradycardia or atrioventricular block in zebrafish. More recently, Scott Mittelstadt and colleagues at Abbott Laboratories further validated zebrafish embryos as an in vivo model for determining the inhibition of hERG, showing that almost all of the drugs known to cause QT prolongation induced a dissociation between atrial and ventricular rates in zebrafish embryos subjected to an acute exposure to these compounds [4]. A number of smaller studies of cardiotoxicity by other groups have found similar results, all falling into the good to excellent score of the guidelines of the European Centre for the Validation of Alternative Methods (ECVAM), which classifies alternative toxicity assays according to their capability to predict adverse effects of chemicals known to be toxic in humans. Together, these findings underscore the suitability of zebrafish to serve as a useful model for prescreening small molecules for potential cardiotoxicity.
In the last few years, pilot studies with new zebrafish toxicity assays in several other areas have been reported – including developmental toxicity, gastrointestinal motility, ototoxicity, neurotoxicity and seizure liability. While the results of these studies were for the most part encouraging, systematic evaluations of zebrafish-based toxicity assays using higher numbers of pharmacologically relevant drugs are still largely missing. Only such studies will enable a more realistic, in-depth and statistically significant view of the predictive power of zebrafish models in toxicity screens. Another important issue that will have to be addressed involves dependence of zebrafish toxicity assays on the absorption, distribution, metabolism and excretion (ADME) properties of compounds [5].
Because of this rapidly growing body of research underscoring the promise of zebrafish-based toxicity studies, together with the growing use of zebrafish in biomedical research and drug discovery, the zebrafish model will likely continue to gain interest from the pharmaceutical industry for use in safety evaluations, as well as increasing interest from cosmetic and food companies for the toxicity analysis of novel ingredients and ingredient combinations.
References
1. Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates? Nature Rev Drug Discov 2004; 3:711-5.
2. Mesens N et al. A strategy combining high-content screening and zebrafish larvae to predict drug-induced hepatotoxicity. 8th World Congress on Alternatives and Animal Use in the Life Sciences. Montréal, Canada, August 21-25, 2011.
3. Mittelstadt SW et al. Evaluation of zebrafish embryos as a model for assessing inhibition of hERG. J Pharmacol Toxicol. Methods 2008; 57:100-5.
4. Sukardi H et al. Zebrafish for drug toxicity screening: bridging in vitro cell-based models and in vivo mammalian models. Expert Opin Drug Metab Toxicol 2011; 7:579-89.
5. Yang L et al. Zebrafish embryos as models for embryotoxic and teratological effects of chemicals. Reprod Toxicol 2009; 28:245-53.
The authors
Alexander D. Crawford1*, Natalie Mesens2, Peter A. M. de Witte1, Camila V. Esguerra1
1 Department of Pharmaceutical Sciences, University of Leuven, Leuven, Belgium
2 Genetic & Exploratory Toxicology, Drug Safety Sciences, Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium
*alexander.crawford@pharm.kuleuven.be
