Resumen de Novel tools in drug discovery: optimising the use of zebrafish for assessing drug safety and antitumoral efficacy
The pharmaceutical industry is facing a productivity crisis in which, despite the increasing expenditures in drug R&D, there is no clear correlation between investments and number of new drugs entering the market. The inefficiency of the process leads to extremely high investments costs, which are then reflected in the high prices of the few successful drugs that reach the market. In this context, the productivity crisis has not only negative consequences for the pharmaceutical sector, but it also harms patients that cannot access or afford effective treatments, and society, which has to deal with the elevated costs. The reasons behind this problem are multiple, complex and they involve factors related to the R&D, regulatory and business processes. One major issue is the high drug attrition rate during the clinical and post market phases, which is two to four folds higher in the oncology field than in other therapeutic areas. In this regard, safety and efficacy matters are the two main reasons for promising compounds to be finally discarded. Thus, high drug attrition rate echoes predictivity deficiencies in preclinical models. One solution might be represented by the use of alternative and complementary tools to improve the overall predictive output and so, reduce the posterior drug attrition rate. Zebrafish has emerged as a really promising model since it shows unique biological properties for a vertebrate, in addition to have high genetic and physiologic homology to humans. It is characterized by small size, fast life cycle, large progeny, transparency, ease of maintenance and genetic manipulation, and the ability to absorb molecules from the surrounding water. All these peculiar aspects, allow its utilisation in high throughput studies.
On these bases, during my PhD project, I improved the use of zebrafish for the evaluation of drug safety and antitumoral efficacy. Two different methodologies have been developed and validated: the ZeGlobalTox and the ZeOncoTest.
ZeGlobalTox is a middle-high throughput system in which zebrafish larvae are used to sequentially evaluate the three most important organ toxicities that are the main cause of drug attrition: cardio-, neuro-, and hepato-toxicities. Results have demonstrated high sensitivity, specificity, and accuracy of the model, compared to human data. Furthermore, our assay integrates for the first time the analysis of these three organ-toxicities in the same larvae; reducing animal usage, experimental time and costs, and quantity of compound needed. In a context where animal utilisation in research is being contested, such an alternative fulfilling the 3Rs principles (Replacement, Reduction, and Refinement), is very welcomed.
ZeOncoTest is a methodology for the evaluation of antitumoral drug efficacy through an optimised zebrafish larvae xenograft model. Previously published reports presented contrasting data probably due to differences in incubation times and temperatures, image acquisition and analysis, cell labelling methods prior to transplantation and site of injection. With the ZeOncoTest we to optimised, standardised and automated the zebrafish larvae xenograft assay for anti-cancer drug discovery. Our results demonstrate that with this methodology, human tumour cells are able to engraft, grow and disseminate into zebrafish larvae and that they respond to known drugs as expected. Also, we showed that our system is suitable to investigate drugs mode of action.
Our work helps to validate zebrafish as a promising preclinical model, bridging the gap between high throughput but low predictive in vitro models and more predictive but low throughput in vivo mammalian models. We propose it as a filter of molecules coming from in vitro models and entering in vivo studies, thus improving the rationale of selection. Finally, it is envisioned that zebrafish consolidation in R&D processes will improve drug safety and efficacy predictions during preclinical phases, therefore reducing the high drug attrition rates and ameliorating the productivity crisis.
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