Regulation of Endothelin-1-Induced Trabecular Meshwork Cell Contractility by Latanoprostene Bunod.

PURPOSE: Previous in vivo studies demonstrated that latanoprostene bunod (LBN), a nitric oxide (NO)-donating prostaglandin F2α receptor agonist, results in greater intraocular pressure (IOP) lowering than latanoprost. The present series of investigations compared the effects of LBN and latanoprost on primary human trabecular meshwork cell (HTMC) contractility and underlying signaling pathways to determine whether LBN might mediate this additional IOP lowering via the conventional outflow pathway. METHODS: The effect of LBN (1-100 µM) on HTMC cGMP levels was determined by ELISA with or without the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Endothelin-1 (ET-1) was used to induce HTMC contractility. To determine the effect of LBN on myosin light chain-2 (MLC-2) phosphorylation, HTMCs were pretreated with 10 to 60 µM LBN for 1 hour and then ET-1 for 5 minutes. MLC-2 phosphorylation was determined by Western blotting. Effects of LBN (30 and 45 µM) on ET-1-induced filamentous (F)-actin cytoskeletal stress fibers and the focal adhesion associated protein vinculin were determined by confocal microscopy. ET-1-induced HTMC monolayer resistance in the presence of LBN (45 µM) was determined by electrical cell substrate impedance sensing, as an indicator of cell contractility. Latanoprost and SE 175 (an NO donor which releases NO on reductive transformation within the cells) were used as comparators in all studies. RESULTS: LBN (1-100 µM) significantly increased cGMP levels in a dose-dependent manner, with a half maximal effective concentration (EC50) of 1.5 ± 1.3 µM, and with maximal effect similar to that of 100 µM SE 175. In contrast, latanoprost caused a minimal increase in cGMP levels at 100 µM only. The cGMP elevation induced by LBN or SE 175 was abolished by ODQ and was therefore sGC-dependent. The two NO donors SE 175 and LBN elicited a reduction in ET-1-induced MLC-2 phosphorylation that was significantly greater than that mediated by latanoprost in HTMCs. SE 175 (100 µM) and LBN (30 or 45 µM) caused a dramatic reduction in ET-1-induced actin stress fibers and vinculin localization at focal adhesions, whereas 45 µM latanoprost was without observable effect. SE 175 reduced ET-1-induced increases in HTMC resistance in a dose-dependent manner. A synergistic effect on reduction of HTMC resistance was observed when latanoprost and SE 175 doses were given together. LBN significantly reduced ET-1-induced HTMC monolayer resistance increases to a greater extent than latanoprost, indicating a greater reduction in cell contractility with LBN. CONCLUSIONS: LBN, SE 175, and latanoprost caused relaxation of ET-1-contracted HTMCs. The effect on HTMC relaxation observed with LBN was significantly greater in magnitude than that observed with latanoprost or SE 175. Data indicate that the NO-donating moiety of LBN mediates HTMC relaxation through activation of the cGMP signaling pathway and a subsequent reduction in MLC-2 phosphorylation. These findings suggest that increased conventional outflow facility may mediate the additional IOP-lowering effects of LBN over that of latanoprost observed in in vivo studies.

Mapracorat, a selective glucocorticoid receptor agonist, upregulates RelB, an anti-inflammatory nuclear factor-kappaB protein, in human ocular cells.

Selective glucocorticoid receptor agonists (SEGRAs) are a new class of compounds under clinical evaluation for treatment of ocular inflammation. Widely prescribed therapeutics, such as glucocorticoids, are effective at reducing ocular inflammation, but their long term use predisposes to undesirable side effects. The purpose of this study was to investigate a novel SEGRA, mapracorat (BOL-303242-X), and the differences in mapracorat's mechanism of action compared with traditional steroids (i.e. dexamethasone). Keratocytes from three different humans were cultured and treated with mapracorat or dexamethasone, with and without a strong provoking agent, interleukin (IL)-1ß. The effects of mapracorat compared to dexamethasone were determined by measuring protein levels (Western blotting) and DNA binding (ELISA) for two nuclear factor-kappaB (NF-κB) family members, RelA and RelB. Cytokine production (i.e. IL-6, IL-8, prostaglandin E2 (PGE2)) was characterized by immunoassay. Our findings reveal mechanistic differences between mapracorat and traditional steroid therapies. Mapracorat showed partial attenuation of the classical NF-κB pathway, consistent with traditional steroids. However, mapracorat uniquely potentiated a novel anti-inflammatory mechanism through rapid upregulation of RelB, an anti-inflammatory member of the NF-κB alternative pathway. Mapracorat potently inhibits ocular inflammation in vitro and is a promising new treatment for ocular inflammatory disease. Mapracorat acts, in part, by a novel mechanism via upregulation of RelB in the NF-κB alternative pathway.

Safety of repeated topical ocular administration of a polycarbophil-based formulation in several models of ocular surgery in rabbits.

PURPOSE: To evaluate the safety of the polycarbophil-based formulation Durasite in rabbits when administered chronically to intact eyes and acutely to eyes compromised by a corneal epithelial defect, penetrating corneal incision, or laser in situ keratomileusis (LASIK) flap. The rheological properties were evaluated to characterize the behavior of the formulation on the ocular surface. SETTING: Bausch & Lomb, Rochester, New York, USA. DESIGN: Experimental study. METHODS: Intact eyes of albino rabbits received polycarbophil (0.6% or 1.3%) 3 times a day for 1 year. The compromised models using polycarbophil 0.9% were an epithelial defect, penetrating corneal incision, or LASIK flap. Eyes with the epithelial defects were dosed 10 times for 24 hours and then 2 times a day for 2 days, and the defect was monitored with fluorescein. The incision or LASIK eyes were dosed 4 times a day for 11 days starting the day before surgery, with 1 drop just before the surgical procedure. The eyes were examined microscopically. The rheological properties were evaluated using a controlled-stress rheometer with a synthetic tear fluid. RESULTS: No adverse ocular or systemic effects were observed with polycarbophil after chronic administration. In the compromised models, there were no adverse effects of the polycarbophil. There was no evidence of an anterior chamber reaction or qualitative effects on the corneal endothelium. Rheologically, the polycarbophil-based formulation behaved as a sheer-thinning fluid under physical conditions similar to the ocular surface. CONCLUSION: Results suggest that the polycarbophil-based formulation, like other shear-thinning formulations, is safe to use in topical ophthalmic pharmaceutical products indicated for chronic use and for treatment of conditions with compromise of the ocular surface.

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.

Impact of assay selection and study design on the outcome of cytotoxicity testing of medical devices: the case of multi-purpose vision care solutions.

Medical device biocompatibility testing usually includes a cytotoxicity component. Assay selection and protocol design often depend on a specific testing standard rather than on the characteristics of the medical device. To better understand the impact of assay selection on study outcome of unstructured medical devices, we evaluated contact lens multi-purpose solutions (MPS) in the agar diffusion, direct contact and two elution cytotoxicity assays. To simulate the conditions of use, MPS were evaluated alone and in combination with contact lenses. All MPS passed the agar diffusion assay (n=3) and extracts prepared from contact lenses soaked in MPS passed the USP elution assay (n=3). Both the duration of contact and MPS concentration impacted the outcome of a modified elution assay. When tested at 25% strength for 48 h, all MPS evaluated were non-cytotoxic (n>3). Test article movement and mechanical damage were significant issues with the direct contact assay. Movement was effectively controlled by manipulating contact lens orientation while using 0.8 mL culture medium. All MPS passed the USP direct contact cytotoxicity test when evaluated using this optimized methodology (n=3). These data are consistent with MPS results in ocular irritation studies in rabbits (n=3).