Megan E. Cavet

Nitric Oxide (NO): An Emerging Target for the Treatment of Glaucoma

The predominant risk factor for the progression of glaucoma is an increase in IOP, mediated via a reduction in aqueous outflow through the conventional (trabecular meshwork and Schlemms canal) outflow pathway. Current IOP lowering pharmacological strategies target the uveoscleral (nonconventional) outflow pathway or aqueous humor production; however, to date no therapy that primarily targets the conventional pathway exists. Nitric oxide (NO) is an intracellular signaling molecule produced by endogenous NO synthases, well-known for its key role in vasodilation, through its action on smooth muscle cells. Under physiological conditions, NO mediates a multitude of diverse ocular effects, including maintenance of IOP. Nitric oxide donors have been shown to mediate IOP-lowering effects in both preclinical models and clinical studies, primarily through cell volume and contractility changes in the conventional outflow tissues. This review is focused on evaluating the current knowledge of the role and mechanism of action of endogenous NO and NO donors in IOP regulation. Data on key additional functions of NO in glaucoma pathology (i.e., ocular blood flow and effects on optic neuropathy) are also summarized. The potential for future therapeutic application of NO in the treatment of glaucoma is then discussed.

Comparison of the effect of multipurpose contact lens solutions on the viability of cultured corneal epithelial cells

PURPOSE To determine the effect of four marketed multipurpose contact lens solutions (MPSs) on corneal epithelial cell viability. METHODS Comparison of the effect of MPS A (Renu MultiPlus, Bausch & Lomb), MPS B (OPTI-FREE Express, Alcon), MPS C (AQuify, CibaVision), and MPS D (OPTI-FREE RepleniSH, Alcon) on cell viability was performed by quantifying cellular ATP content, resazurin reduction, and lactate dehydrogenase (LDH) release in transformed human corneal epithelial cells (HCEpiC) and primary bovine corneal epithelial cells (BCEpiC). RESULTS Significant reductions in cellular ATP content were observed at 40% solution and above with both MPS B and MPS D, compared to at 100% only for MPS A and MPS C, and similar results were obtained in BCEpiC. Effects on resazurin reduction were also less in HCEpiC exposed to increasing doses of MPS A and MPS C than in cells exposed to MPS B and MPS D. After 15 min, HCEpiC viability measured by both resazurin reduction and cellular ATP levels was significantly lower for cells exposed to MPS B, MPS D, and MPS C, while HCEpiC exposed to MPS A were not affected. MPS B and MPS D reduced cell viability more than MPS A and MPS C over a 2-h time course in both HCEpiC and BCEpiC. CONCLUSIONS Both MPS B and MPS D can cause large decreases in the viability of cultured corneal epithelial cells even with just a 2h exposure at multiple doses. Significant reduction in cell viability is evident at brief 15-30 min exposures. In contrast, MPS A and MPS C have significantly less effect on the cell viability of corneal epithelial cells at multiple doses, after these short exposure times.

Reduced myocilin expression in cultured monkey trabecular meshwork cells induced by a selective glucocorticoid receptor agonist: comparison with steroids.

PURPOSE To assess in vitro myocilin (MYOC) expression in trabecular meshwork (TM) cells exposed to BOL-303242-X, a selective glucocorticoid receptor (GR) agonist (SEGRA), in comparison with dexamethasone (DEX), and prednisolone acetate (PA). METHODS After drug treatment of monkey TM cultures, MYOC protein in conditioned media (CM) was measured by Western blot and densitometry. MYOC mRNA levels were analyzed by qRT-PCR. RU-486 was tested for antagonism of MYOC protein expression induced by DEX and BOL-303242-X. RESULTS Baseline MYOC protein released into CM and MYOC mRNA were detected. DEX or PA elicited dose-dependent increases in MYOC in CM and also in MYOC mRNA. BOL-303242-X effects typified partial agonism, with significantly reduced MYOC protein and mRNA, compared with DEX. Maximum efficacy for BOL-303242-X was 53% of that for DEX. Mean EC(50) across all strains tested was lower, but not significantly different, for BOL-303242-X versus DEX. Compared with DEX, MYOC mRNA levels were significantly lower in BOL-303242-X-treated TM cells at the highest doses tested. EC(50)s for PA were higher than DEX, for both myocilin protein and mRNA. RU-486 displayed a dose-dependent antagonism to drug-induced increases in myocilin levels. CONCLUSIONS In vitro quantitative assays of myocilin expression in TM cells can be used for characterizing anti-inflammatory drugs that are GR ligands. The results suggest that, compared with traditional ocular steroids, therapeutic doses of BOL-303242-X elicit a reduced myocilin expression profile in TM cells by virtue of the partial agonist properties of this compound.

Effect of a novel multipurpose contact lens solution on human corneal epithelial barrier function

PURPOSE To explore the effect of a novel multipurpose contact lens solution (MPS) on the junction protein distribution and barrier function of cultured human corneal epithelial cell monolayers. METHODS Cultured human corneal epithelial cells (HCEpiC) were exposed to a novel MPS (MPS A; Biotrue™ multi-purpose solution, Bausch & Lomb Incorporated) at 50%, 75% and 100% for 10 or 30 min. Four commercially available MPS products, MPS B (AQuify, Ciba Vision), MPS C (COMPLETE MPS Easy Rub, AMO), MPS D (OPTI-FREE Express, Alcon) and MPS E (OPTI-FREE RepleniSH, Alcon) were tested in parallel. Tight junction structure and integrity were evaluated by confocal microscopy using ZO-1 antibody and scanning microscopy (SEM). Quantitative evaluation of MPSs on epithelial barrier function was determined by measuring transepithelial electrical resistance (TEER) across HCEpiC grown on Transwell Clear permeable supports and on electric cell-substrate impedance sensing (ECIS) electrode arrays. RESULTS Overall after exposure to the three concentrations (50%, 75%, and 100%) of MPS A, ZO-1 distribution and fluorescent intensity on the cell surface appeared similar to the media control with continuous tight junctions and clear intercellular junctions. At all measured time points after exposure to MPS A (50% or 75%) there was also no effect on the TEER using both resistance methodologies, and SEM showed that MPS A appeared similar to the Hanks balanced salt solution (HBSS) control. In cells exposed to MPS D there was a dose-dependent change in the distribution of ZO-1, some cell detachment, and a decrease in monolayer resistance at all time points measured. Ultrastructurally, MPS D caused gross changes, including damage to cell junctions and plasma membranes. To a lesser extent, the remaining three commercial MPS products demonstrated some effects on tight junction ZO-1 distribution and/or TEER. CONCLUSIONS Based on the in vitro measurements of tight junction protein expression, monolayer integrity, and transepithelial electrical resistance, the novel multipurpose contact lens solution (MPS A) did not alter corneal barrier function as compared to media, PBS or HBSS control. Clinical significance of the observed differences in epithelial barrier function among the MPSs tested needs further investigation.

Anti-inflammatory effects of besifloxacin, a novel fluoroquinolone, in primary human corneal epithelial cells.

Purpose: The aim of this study was to determine the anti-inflammatory effects of besifloxacin, a novel fluoroquinolone under clinical evaluation for the treatment of ophthalmic infections, in human corneal epithelial cells (HCEpiC). Methods: Cytokine expression in primary HCEpiC was stimulated by interleukin-1ß (IL-1ß), and Luminex technology was used to determine the effect of besifloxacin on IL-1ß-induced cytokine release. Effect of besifloxacin on nuclear factor kappa B (NFκB), and mitogen-activated protein kinase (MAPK) was assessed by measuring inhibitory kappa B protein (IκB) degradation, NFκB nuclear translocation, and MAPK phosphorylation by Western blotting. Moxifloxacin, a marketed fluoroquinolone, was used as the control. Anti-inflammatory efficacy of besifloxacin was also evaluated in rabbits infected with methicillin-resistant Staphylococcus aureus (MRSA). Results: Stimulation of HCEpiC with IL-1ß increased release of 12 of the 29 cytokines measured. Besifloxacin significantly inhibited IL-1ß-induced cytokine release in a dose-dependent manner, with a comparable (IL-8) or better (G-CSF, GM-CSF, IL-6, MCP-1, MIP-1ß, TGF-α, and TNF-α) efficacy compared to moxifloxacin. A significant inhibitory effect of besifloxacin was observed at 1 or 10 μg/ml. Besifloxacin inhibited IκB degradation, NFκB nuclear translocation, and activation of p38 and JNK MAPKs. Based on improvement of clinical score, besifloxacin showed statistically significant anti-inflammatory effect compared to saline treatment. Conclusions: Besifloxacin acts as an anti-inflammatory agent in corneal epithelial cells in vitro, by inhibiting the NFκB and MAPK pathways. Besifloxacin also exhibits anti-inflammatory efficacy in vivo. The anti-inflammatory attribute may enhance its efficacy in the treatment of ocular infections with an inflammatory component and warrants further investigation.

Nonclinical safety evaluation of boric acid and a novel borate-buffered contact lens multi-purpose solution, Biotrue™ multi-purpose solution.

Multipurpose solutions (MPS) often contain low concentrations of boric acid as a buffering agent. Limited published literature has suggested that boric acid and borate-buffered MPS may alter the corneal epithelium; an effect attributed to cytotoxicity induced by boric acid. However, this claim has not been substantiated. We investigated the effect of treating cells with relevant concentrations of boric acid using two cytotoxicity assays, and also assessed the impact of boric acid on corneal epithelial barrier function by measuring TEER and immunostaining for tight junction protein ZO-1 in human corneal epithelial cells. Boric acid was also assessed in an in vivo ocular model when administered for 28 days. Additionally, we evaluated Biotrue multi-purpose solution, a novel borate-buffered MPS, alone and with contact lenses for ocular compatibility in vitro and in vivo. Boric acid passed both cytotoxicity assays and did not alter ZO-1 distribution or corneal TEER. Furthermore, boric acid was well-tolerated on-eye following repeated administration in a rabbit model. Finally, Biotrue multi-purpose solution demonstrated good ocular biocompatibility both in vitro and in vivo. This MPS was not cytotoxic and was compatible with the eye when administered alone and when evaluated with contact lenses. We demonstrate that boric acid and a borate-buffered MPS is compatible with the ocular environment. Our findings provide evidence that ocular effects reported for some borate-buffered MPS may be incorrectly attributed to boric acid and are more likely a function of the unique combination of ingredients in the MPS formulation tested.