In vitro anti-VEGF agent safety profile: apoptosis in ARPE-19 cells treated with ranibizumab, bevacizumab, aflibercept and ziv-aflibercept

Abstract

Purpose : The use of anti-VEGF (anti-vascular endothelial growth factor) therapy has dramatically altered the prognosis of eyes with neovascular age-related macular degeneration. However, chronic use of anti-VEGF medications may have an effect on progression of geographic atrophy. The purpose of this study is to compare the effect of different concentrations of anti-VEGF drugs on apoptosis of cultured retinal pigment epithelium (RPE) cells.

Methods : Human RPE cells (ARPE-19) were plated then treated for 24 hours with either 1/2x, 1x, 2x or 10x of standard clinical concentrations achieved in vitreous of ranibizumab, bevacizumab, aflibercept and ziv-aflibercept. A standard clinical concentration was determined as 0.05ml (50µl) injected in 4ml of vitreous for ranibizumab, bevacizumab and aflibercept; for ziv-aflibercept, a dose of 2.0mg per 0.05ml was used. YO-PRO-1 iodide assay (Molecular Probes – Life Technologies, Eugene, OR) was used to assess apoptosis; under this assay, apoptotic cells fluoresce green. The fluorescent image scanning unit FMBio III (Hitachi Solutions America, San Bruno, CA) was used to detect and quantify fluorescing cells. Experiments were run in quadruplicate and repeated thrice. All results were normalized to untreated controls.

Results : Ranibizumab produced no significant increase in apoptosis levels compared to controls at all tested levels. Aflibercept and bevacizumab showed no increase in apoptosis levels at standard clinical concentrations, but apoptosis significantly increased for both at 2x standard concentration (p=0.0165 and p=0.0003 respectively) and at 10x standard concentration (p<0.0001 and p=0.0014 respectively). Ziv-aflibercept showed significant increase in apoptosis at 1x (p=0.0072), 2x (p<0.0001), and 10x (p<0.0001).

Conclusions : Of the medications studied, only ziv-aflibercept demonstrated significant increase in apoptosis rates at standard clinical concentrations. At 2x standard concentration and higher, aflibercept and bevacizumab produced higher apoptosis rates. Exposure to ranibizumab did not cause a significant increase in apoptosis at all tested concentration levels.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Image 1: Describes the relative rates of apoptosis of each anti-VEGF medication normalized to controls