We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm’s canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.

Purpose: Aldose reductase (ALR), the first and rate-limiting enzyme involved in polyol pathway plays a central role in diabetes and its related complications, including diabetic retinopathy (DR). Inhibition of ALR may also be an ideal target for reducing the deleterious effects of DR. Therefore, the purpose of the present study was to investigate the protective effect of epalrestat (EPL), ALR inhibitor on glucose-induced toxicity in ARPE-19 cells. Methods: ARPE-19 cells were challenged with normal glucose (NG, 5 mM) and high glucose (HG1, 25 mM and HG2, 50 mM) in the presence or absence of EPL. ALR and VEGF₁₆₅ expression in retinal pigment epithelial (RPE) cells under experimental conditions were quantified by real-time polymerase chain reaction using SYBR Green chemistry. Vascular endothelial growth factor (VEGF) secretion in the cell supernatant was measured by Sandwich ELISA. Cytotoxicity of EPL was assessed by MTT assay. ALR inhibitory activity, apoptosis, and sorbitol accumulation were also investigated. Results: EPL at studied concentration did not show any toxicity to RPE cells and showed as maximum as 65% ALR inhibition under high glucose condition (HG1). The presence of EPL significantly reduced ALR expression and VEGF levels as induced by high glucose in ARPE-19 cells. Conclusion: Inhibition of ALR appeared to be beneficial in reducing diabetes-related complications in RPE cells under high glucose condition.

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma.

Purpose: Our previous study demonstrated the drug reservoir function of human amniotic membrane (HAM) using stable moxifloxacin as a model drug. The purpose of the present study is to evaluate whether HAM can be used as a drug carrier for extended release of extemporaneous preparation of cefazolin. Methods: HAM Buttons (1 Control, 5 Test) were incubated in a freshly prepared (1 ml) sterile topical solution of cefazolin 5% (w/v) for 3 h and 24 h at two different temperatures. The groups were designated as follows: Group IA: Soaking duration 3 h at 4°C; Group IB: Soaking duration 3 h at room temperature; Group IIA: Soaking duration 24 h at 4°C; and Group IIB: Soaking duration 24 h at room temperature. The release kinetics of cefazolin from different groups of drug-laden HAM was studied for a period of 5 days. Samples were assayed for estimation of cefazolin content at different time intervals by High Performance Liquid Chromatography (HPLC) with Photodiode array (PDA) detector. Results: Three-hour cefazolin treatment with HAM at 4°C caused high drug entrapment (24%) compared to room temperature (11%; P < 0.005); however, the release kinetics was not significantly different between Group IA and IB as well as Group IIA and IIB up to the study period. Increase in drug treatment duration did not show increase in entrapment, but caused two-fold (IA Vs IIA) and 1.6-fold (IB Vs IIB) less drug entrapment at 4°C and room temperature, respectively. Conclusion: The results reveal that HAM may be a suitable drug carrier for extended delivery of fortified formulations without compromising stability.

The purpose of the present study was to assess the differential intraocular pressure response (IOP) to dexamethasone (DEX) treatment at two dose levels (100 or 500 nM) in perfusion cultured Indian cadaveric eyes to investigate glucocorticoid (GC) responsiveness. In a human organ-cultured anterior segment (HOCAS) set-up, the eye pressure was monitored for every 24 h post DEX infusion (100 or 500 nM) or 0.1% ethanol treatment for 7 days after baseline stabilization. The expression of DEX-inducible proteins such as myocilin and fibronectin in HOCAS-TM tissues was assessed by immunostaining. Elevated IOP was observed in 6/16 eyes [Mean ± SEM (mΔIOP): 15.50 ± 1.96 mmHg; 37.5% responders] and 3/15 eyes (Mean ± SEM mΔIOP: 10 ± 0.84 mmHg; 20% responders) in 100 nM and 500 nM dose groups respectively. Elevated IOP in GC responder eyes was substantiated with a significant increase in myocilin (11.8-fold; p = 0.0002) and fibronectin (eightfold; p = 0.04) expression as compared to vehicle-treated eyes by immunofluorescence analysis. This is the first study reporting the GC responsiveness in Indian cadaveric eyes. The observed GC response rate was comparable with the previous studies and hence, this model will enable us to investigate the relationship between differential gene expression and individual GC responsiveness in our population.

Aim: To investigate genes and pathways involved in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. Methods: Using paired human donor eyes, human organ-cultured anterior segment (HOCAS) was established in one eye to characterize GC responsiveness based on intra ocular pressure (IOP) change and, in the other eye, primary HTM cell culture was established. For RNA sequencing, total RNA extracted from GC-responder (GC-R) and non-responder (GC-NR) cells after dexamethasone (DEX) or ethanol (ETH) treatment for 7d was used. Differentially expressed genes (DEGs) were compared among five groups and validated. Results: In total, 616 and 216 genes were identified as significantly dysregulated in Group #1 and #2 (#1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR), respectively. Around 80 genes were commonly dysregulated in Group #3 (overlapping DEGs between #1 and #2), whereas 536 and 136 genes were uniquely expressed in GC-R (#4) and GC-NR HTM (#5) cells, respectively. Pathway analysis revealed that WNT signaling, drug metabolism cytochrome p450, cell adhesion, TGF-β signaling, and MAPK signaling were associated with GC responsiveness. Conclusion: This is the first study reporting distinct gene signatures and their associated pathways for GC-R and GC-NR HTM cells. WNT and MAPK signaling are potential therapeutic targets for the management of GC-induced glaucoma.