Comparison of effects of anti-angiogenic agents in the zebrafish efficacy–toxicity model for translational anti-angiogenic drug discovery

Abstract

Background

Anti-angiogenic therapy in certain cancers has been associated with improved control of tumor growth and metastasis. Development of anti-angiogenic agents has, however, been saddled with higher attrition rate due to suboptimal efficacy, narrow therapeutic windows, or development of organ-specific toxicities. The aim of this study was to evaluate the translational ability of the zebrafish efficacy–toxicity model to stratify anti-angiogenic agents based on efficacy, therapeutic windows, and off-target effects to streamline the compound selection process in anti-angiogenic discovery.

Methods

The embryonic model of zebrafish was employed for studying angiogenesis and toxicity. The zebrafish were treated with anti-angiogenic compounds to evaluate their effects on angiogenesis and zebrafish-toxicity parameters. Angiogenesis was measured by scoring the development of subintestinal vessels. Toxicity was evaluated by calculating the median lethal concentration, the lowest observed effect concentration, and gross morphological changes. Results of efficacy and toxicity were used to predict the therapeutic window.

Results

In alignment with the clinical outcomes, the zebrafish assays demonstrated that vascular endothelial growth factor receptor (VEGFR) inhibitors are the most potent anti-angiogenic agents, followed by multikinase inhibitors and inhibitors of endothelial cell proliferation. The toxicity assays reported cardiac phenotype in zebrafish treated with VEGFR inhibitors and multikinase inhibitors with VEGFR activity suggestive of cardiotoxic potential of these compounds. Several other pathological features were reported for multikinase inhibitors suggestive of off-target effects. The predicted therapeutic window was translational with the clinical trial outcomes of the anti-angiogenic agents. The zebrafish efficacy–toxicity approach could stratify anti-angiogenic agents based on the mechanism of action and delineate chemical structure-driven biological activity of anti-angiogenic compounds.

Conclusion

The zebrafish efficacy–toxicity approach can be used as a predictive model for translational anti-angiogenic drug discovery to streamline compound selection, resulting in safer and efficacious anti-angiogenic agents entering the clinics.

Keywords:

• angiogenesis
• therapeutic window
• VEGFR inhibitors
• zebrafish toxicity assay

Supplementary materials

Figure S1 Gross morphological changes in the zebrafish larvae following VEGFR inhibitors at 96 hours.

Notes: Treatment with VEGFR inhibitors was associated with development of pericardial edema at their LOEC concentrations. Sunitinib, cabozantinib, axitinib, motesanib, and pazopanib treatments were associated with pericardial edema at and above their LOEC concentrations (arrows). Lack of the cardiac phenotype (pericardial edema) in vandetanib treatment group at NOEC concentration.
Abbreviations: VEGFR, vascular endothelial growth factor receptor; LOEC, lowest observed effect concentration; NOEC, no observed effect concentration.

Figure S2 Gross morphological changes in the zebrafish larvae following non-VEGFR angiogenesis inhibitors at 96 hours post fertilization.

Notes: (A and B) Treatment with non-VEGFR inhibitors like flavopiridol was associated with development of pericardial edema (arrows) and widespread tissue necrosis (*) at LOEC concentrations. (C–F) Treatment with combretastatin was associated with dose- and time-dependent coagulation of embryos at concentrations as low as 5–10 nM (arrows).

Abbreviations: VEGFR, vascular endothelial growth factor receptor; LOEC, lowest observed effect concentration; hpf, hours post-fertilization.

Figure S3 Gross morphological changes in the zebrafish larvae following treatment with thalidomide.

Notes: Thalidomide treatment exhibited 52% reduction in otolith size and 100% absence of fin development. Yellow arrows indicate the fin development and yellow dotted circles indicate otolith boundary.

Author contributions

All authors contributed toward data analysis, drafting and revising the paper and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflict of interest in this work.