Anti-VEGF Agents Clearance Through the Aqueous Outflow Pathway in a Rat Model

Sustained increase in intraocular pressure (IOP) following intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) in the treatment of retinal disease has been theoretically attributed to aggregation of anti-VEGF in the iridocorneal angle. However, previous studies by our group showed full clearance of intravitreally injected bevacizumab, aflibercept, and ranibizumab. The objective of this study was to further analyze and compare the clearance of these anti-VEGF agents from the eye after a single injection in a rat model.

Brown Norway rats received an intravitreal injection of 3 µl anti-VEGF at the standard concentration 3 days following induction of choroidal neovascularization. The eyes were processed at 0, 3, 6, 24, and 48 hours thereafter, and immunofluorescence was evaluated with confocal microscopy and 3D reconstruction analysis. The signal concentration was calculated, and the drug clearance rate was measured. The immunohistochemistry process was validated with negative and positive control groups.

The immunofluorescent signal was positive for all anti-VEGF agents at the trabecular meshwork, Schlemm's canal, and episcleral veins. Anti-VEGF immunostaining peaked immediately after injection and then decreased gradually to negligible at 48 hours (P < 0.05). All three agents demonstrated an identical pattern (P > 0.05). The clearance rate of anti-VEGF from the iridocorneal angle ranged between 98.68% and 99.87% at 48 hours.

Bevacizumab, aflibercept, and ranibizumab cleared completely from the iridocorneal angle at 48 hours in the brown Norway rats following a single intravitreal injection and with similar a clearance rate. These findings support our earlier studies refuting the anti-VEGF aggregation hypothesis.