Reactive Oxygen Species Levels in Human Retinal Pigment Epithelium Cells In vitro to anti-VEGF Agents: Ranibizumab, Bevacizumab, Aflibercept and Ziv-aflibercept

Abstract

 

Purpose

 

The development of anti-VEGF therapy has dramatically changed the prognosis of wet age-related macular degeneration. While patients can often expect stabilization or improvement in vision, chronic use of anti-VEGF medications may also be associated with progression of geographic atrophy. The purpose of this study is to compare the effects of different concentrations of anti-VEGF drugs in cultured retinal pigment epithelium cells by assessing reactive oxygen species (ROS) levels post drug exposure as a measure of oxidative damage.

 

Methods

 

Human retinal pigment epithelium cells (ARPE-19) were cultured in Dulbecco Modified Eagles’ Medium (DMEM) with 10% bovine growth serum (BGS) and then exposed for 24 hours to ranibizumab, bevacizumab, aflibercept and ziv-aflibercept at 1/2x, 1x, 2x, and 10x clinical concentrations. A clinical equivalent dose of 2mg was used for ziv-aflibercept. The cells were then exposed to the fluorescent dye 2', 7’-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and ROS levels were assessed using a fluorescence plate reader.

 

Results

 

There was no statistically significant increase in ROS production at all tested doses of ranibizumab including at 10x the clinical dose. Bevacizumab produced a statistically significant increase in ROS levels at 2x and 10x the clinical dose (p=0.0032 and p=0.0001, respectively). Aflibercept showed significant increases in ROS levels at 10x the clinical dose (p=0.0009), but not in the other studied concentrations. Ziv-aflibercept showed significant increases of ROS at the clinical equivalent dose (p=0.001), 2x (p<0.001) and 10x (p<0.001) the clinical equivalent dose.

 

Conclusions

 

At standard clinical and clinical equivalent doses, only ziv-aflibercept exhibited significant increases in ROS production. At 2x concentration, both bevacizumab and ziv-aflibercept produced significantly increased ROS levels. At 10x the clinical dose, only ranibizumab demonstrated no increase in ROS production. This study suggests that ranibizumab and aflibercept may have the broadest safety profiles of the four studied anti-VEGF drugs when tested on cultured retinal pigment epithelium cells.  

 

Figure 1. Reactive Oxygen Species Assay