Novel small molecule with neuroprotective activity

Abstract

Purpose : Embolism and/or high intraocular pressure (IOP) induces oxidative stress in retina via poor retinal circulation and decrease in antioxidant enzymes resulting in RGC death. The goal is to demonstrate if small molecule with both antioxidant and plasminogen activating activity can prevent RGC death via scavenging ROS and improving fibrinolytic activity in ischemic/reperfusion mice model.

Methods : Antioxidant activity: Human Umbilical Vascular Endothelial Cells (HUVECs) were co-treated with or without H₂O₂ (400µM) and test compounds (0.05µM-100µM). After 24 hours of treatment, cytosolic ROS levels were measured using Carboxy-H₂DCFDA following the manufacturer’s protocol. Plasminogen activating inhibitor-1 (PAI-1) activity: primary human trabecular meshwork (hTM) cells were were treated with dexamethasone (100nM) or vehicle (ethanol) in the presence of test compounds (100 µM) for 48hr. Conditioned medium was collected for western blot. Experiments were performed in triplicates (n = 5). Protection of RGC death: Mice (C57Bl6, 12 weeks, n = 5) were anesthetized and the ischemic/reperfusion (I/R) injury was performed on left eye followed by intravitreal injection of 2ul of 2% of compound SA-10 in PBS or only 2ul PBS to left eye (control group) at days 0. At day 7, pattern ERG was performed with the JORVEC System. SD-OCT was done to measure the change in overall thickness of retina layers and flash ERG was done to measure the function of bipolar cells (b-wave) as well as photoreceptor cells (a-wave) after 28 days. Right eye was served as contralateral control.

Results : Compound SA-9 and SA-10 treatment was effective in inhibiting the dexamethasone induced upregulation of PAI-1 in hTM cells and decreased the ROS level significantly in HUVECs (n =4). Intravitreal injection of compound SA-10 restored retina function after 7 days post-I/R. While PERG amplitude was significantly reduced in I/R induced mice eyes (14.8 ± 1.4 µV), SA-10 treated group demonstrated significant improvement (21 ± 1.4 µV, p ≤ 0.001). The retinal thickness and a, b waves in the treated group were similar to control as measured by SD-OCT and ERG where the I/R group had significant thinning of retina.

Conclusions : Our results are consistent with our hypothesis that this novel class of hybrid compound protect RGC against ischemic insult. This class of molecule has high potential to be useful for treatment of ocular stroke and glaucoma.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.