Effects of Anti-VEGF and ALG-1001 on Human Retinal Cells in vitro

Abstract

Purpose : Anti-vascular endothelial growth factor (VEGF) drugs (Lucentis^® , Ranibizumab; Avastin^®, Bevacizumab; Eylea^® , Aflibercept) are used to treat neovascular retinal diseases. ALG-1001 (Luminate^® , Allegro Ophthalmic, Inc.), which is in clinical trials, has anti-angiogenic along with unique neuroprotective and anti-inflammatory properties. This study compares responses of anti-VEGF drugs versus ALG-1001 using in vitro human retinal Müller cells (MIO-M1) cells and pigment epithelial cells (ARPE-19).

Methods : Specific markers defining RPE and Müller cells were expressed at high levels in our ARPE-19 and MIO-M1 cells. ARPE-19 or MIO-M1 cells were treated 24 hrs with Lucentis, Avastin, Eylea or ALG-1001 (1x dose equivalent to intravitreal injection concentrations). Assays for Cell Viability (CV, Trypan Blue exclusion assay), Cellular Metabolic Activity (CMA, tetrazolium MTT dye assay), Mitochondrial Membrane Potential (ΔΨm, JC1 assay) or Reactive Oxygen Species (ROS, H2DCF-DA assay) were performed.

Results : Compared to untreated MIO-M1 cultures (100%), CMA was slightly higher with ALG-1001 (113%, P=0.37) but lower for cultures treated with Lucentis (86%, P<0.001), Avastin (84%, P<0.0001) or Eylea (82%, P<0.0001). ROS level increased with Lucentis (137%, P<0.001) Avastin (124%, p=0.002) and Eylea (129%, P<0.001) while ALG-1001 treated MIO-M1 cells were slightly lower (81%, p=0.13). The ΔΨm levels were decreased in Eylea-treated cultures (85%, P=0.0008) but unchanged (P>0.05) in those treated with ALG-1001 (109%), Lucentis (99%), Avastin (94%).
Untreated ARPE-19 cells (100%) showed similar CV and ROS levels (P>0.05) to ALG-1001 (101%; 93%), Lucentis (98%; 102%), Avastin (97%; 106%) and Eylea (98%; 97%) compared to untreated cultures (100%). The ΔΨm levels decreased in Avastin-treated cultures (86%, P =0.0014) but remained unchanged (P>0.05) in ALG-1001 (102%), Lucentis (97%) and Eylea (95%) compared to controls (100%).

Conclusions : MIO-M1 cells treated with anti-VEGF drugs had significantly decreased CMA and ΔΨm along with higher ROS levels. In contrast, ALG-1001-treated MIO-M1 cells showed more favorable profiles with increased CMA, improved ΔΨm, and lower ROS levels. The ARPE-19 cells showed similar trends but were less sensitive to any medications tested. The damage seen with chronic use of intravitreal anti-VEGF injections may be due to increased susceptibility of Müller cells, while the RPE cells remain more resistant to changes.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.