Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery

Abstract

The zebrafish is a well-established model organism used in developmental biology. In the last decade, this technology has been extended to the generation of high-value knowledge on safety risks of novel drugs. Indeed, the larval zebrafish appear to combine advantages of whole organism phenotypic assays and those (rapid production of results with minimal resource engagement) of in vitro high-throughput screening techniques. Thus, if appropriately evaluated, it can offer undeniable advantages in drug discovery for identification of target and off-target effects. Here, we review some applications of zebrafish to identify potential safety liabilities, particularly before lead/candidate selection. For instance, zebrafish cardiovascular system can be used to reveal decreases in heart rate and atrial-ventricular dissociation, which may signal human ether-a-go-go-related gene (hERG) channel blockade. Another main area of interest is the CNS, where zebrafish behavioural assays have been and are further being developed into screening platforms for assessment of locomotor activity, convulsant and proconvulsant liability, cognitive impairment, drug dependence potential and impaired visual and auditory functions. Zebrafish also offer interesting possibilities for evaluating effects on bone density and gastrointestinal function. Furthermore, available knowledge of the renal system in larval zebrafish can allow identification of potential safety issues of drug candidates on this often neglected area in early development platforms. Although additional validation is certainly needed, the zebrafish is emerging as a versatile in vivo animal model to identify off-target effects that need investigation and further clarification early in the drug discovery process to reduce the current, high degree of attrition in development.

Figure 1

(a) Zebrafish larva at 3 d.p.f. with organs such as the heart clearly visible due to the optical clarity of the larva at this age. Bar, 0.5 mm. (b) Zebrafish larvae at 7 d.p.f in a 96-well plate. Bar 5 mm.

Figure 2

Data analysis of a high-resolution video of a 3 d.p.f zebrafish larvae heart where atrium beat (black line), ventricular beat (grey line) and outflow strength (black bars) are shown for: (a) normal heart rate; (b) 2:1 atrial/ventricular ratio arrhythmia in response to treatment with terfenadine (Berghmans, 2006).

Figure 3

The experimental paradigm for the investigation of conditioned place preference in zebrafish. The fish preferred compartment is assessed (a), the fish is then exposed to the drug in the least preferred compartment (b). Finally the fish is tested for change in preference (c).

Figure 4

(a) Zebrafish larva at 10 d.p.f. has many bones of the head skeleton mineralized and these are stained with Alizarin red. B. Larva exposed to 25 μM prednisolone from 5 to 10 d.p.f. shows a marked reduction in the stained mineralized tissue. Quantification of the staining showed a 50% reduction in the mineralized area with steroid treatment (Barrett et al. (2006). Copyright Wiley-VCH Verlag GmbH & Co. KGaA (reproduced with permission)). Bar, 0.4 mm.