Investigation of In vitro ocular toxicity of ranibizumab and aflibercept

Currently, intravitreal injections of anti-VEGF molecules are the gold standard for the treatment of neovascular AMD. Besides this, it is also widely used in other ocular pathologies with neovascular growth and vascular hyperpermeability, especially in RVT, DME, and retinopathy of prematurity. Treatment usually needs to be administered in monthly and repeated doses. Discontinuation of therapy may result in the recurrence of neovascularization. Therefore, in most cases, treatment may need to be continued for several years. Although VEGF antagonists have been shown to be safe, since VEGF is a critical factor in cell survival, several side effects have also been identified. Some studies have reported RPE tears and intraocular inflammation after anti-VEGF therapy. Moreover, the number of intravitreal injections administered was found to be significantly associated with the progression of retinal atrophy. However, it is not possible to distinguish whether these effects are a result of the natural course of the disease or are due to anti-VEGF therapy. In our study, we aimed to evaluate the possible toxic effects of Ranibizumab and Aflibercept, which have been approved by the FDA as a result of numerous randomized clinical studies, on the adult human RPE cell line (ARPE-19), in vitro. In addition, we aimed to elucidate the effects of these drugs on 1) Beclin1 and ATG4 expressions, which are critical in the autophagy pathway, 2) MMP2 and TIMP1, which are enzymes that are of critical importance in the regulation of the extracellular matrix involved in intercellular structural and chemical support. The MTT test was applied to the cells for the evaluation of cytotoxicity. Then RNA isolation was performed. Finally, selected autophagy and matrix metalloproteinase proteins were analyzed by Quantitative Reverse Transcription PCR. In conclusion, we observed that these two anti-VEGF drugs, administered at much higher doses than those used in clinical practice, did not cause significant toxicity to the RPE in vitro. Compared to the control group, it was found that Ranibizumab had no significant effect on Beclin1 expression, while Aflibercept significantly reduced Beclin1 expression. Both anti-VEGFs increased ATG4 gene expression. Compared to the control group, Ranibizumab caused an increase in MMP2 and TIMP1 expression; Aflibercept decreased MMP2 expression and increased TIMP1 expression. When comparing TIMP1 to MMP2, this ratio was higher in Aflibercept.