Complement factor B is critical for sub-RPE deposit accumulation in a model of Doyne honeycomb retinal dystrophy with features of age-related macular degeneration.

EFEMP1 R345W is a dominant mutation causing Doyne honeycomb retinal dystrophy/malattia leventinese (DHRD/ML), a rare blinding disease with clinical pathology similar to age-related macular degeneration (AMD). Aged Efemp1  R345W/R345W knock-in mice (Efemp1ki/ki) develop microscopic deposits on the basal side of retinal pigment epithelial cells (RPE), an early feature in DHRD/ML and AMD. Here, we assessed the role of alternative complement pathway component factor B (FB) in the formation of these deposits. RNA-seq analysis of the posterior eyecups revealed increased unfolded protein response, decreased mitochondrial function in the neural retina (by 3 months of age) and increased inflammatory pathways in both neural retina and posterior eyecups (at 17 months of age) of Efemp1ki/ki mice compared with wild-type littermate controls. Proteomics analysis of eye lysates confirmed similar dysregulated pathways as detected by RNA-seq. Complement activation was increased in aged Efemp1ki/ki eyes with an approximately 2-fold elevation of complement breakdown products iC3b and Ba (P < 0.05). Deletion of the Cfb gene in female Efemp1ki/ki mice partially normalized the above dysregulated biological pathway changes and oral dosing of a small molecule FB inhibitor from 10 to 12 months of age reduced sub-RPE deposits by 65% (P = 0.029). In contrast, male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females, no elevation of ocular complement activation and no effect of FB inhibition on sub-RPE deposits. The effects of FB deletion or inhibition on Efemp1ki/ki mice supports systemic inhibition of the alternative complement pathway as a potential treatment of dry AMD and DHRD/ML.

Understanding metabolism related differences in ocular efficacy of MGV354.

MGV354 was being developed as a novel ocular therapy for lowering of intraocular pressure, a key modifiable risk factor for glaucoma. MGV354 is an activator of soluble guanylate cyclase, an enzyme known to be involved in the regulation of IOP. MGV354 has been shown to robustly lower IOP over 24 h after a single topical ocular drop in rabbit and monkey pharmacology models. However, MGV354 failed to produce similar results in patients with ocular hypertension or open-angle glaucoma. With an objective of explaining the lack of efficacy in the clinic, we attempted to study whether human metabolism was significantly different from animal metabolism. The present study documents the investigation of metabolism of MGV354 in an effort to understand potential differences in biotransformation pathways of MGV354 in rabbits, monkeys, and humans. Overall twenty-six metabolites, formed via oxidative and conjugative pathways, were identified in vitro and in vivo. In vitro hepatic metabolism was qualitatively similar across species, with minor but distinct differences. There were no observable interspecies differences in the hepatic and ocular metabolism of MGV354. Although ocular metabolism was not as extensive as hepatic, the results do not explain the lack of efficacy of MGV354 in clinical studies.

Application of Cell Impedance as a Screening Tool to Discover Modulators of Intraocular Pressure.

Purpose: To identify new targets and compounds involved in mediating cellular contractility or relaxation in trabecular meshwork (TM) cells and test their efficacy in an ex vivo model measuring outflow facility. Methods: A low-molecular weight compound library composed of 3,957 compounds was screened for cytoskeletal changes using the Acea xCelligence impedance platform in immortalized human NTM5 TM cells. Hits were confirmed by 8-point concentration response and were subsequently evaluated for impedance changes in 2 primary human TM strains, as well as cross-reactivity in bovine primary cells. A recently described bovine whole eye perfusion system was used to evaluate effects of compounds on aqueous outflow facility. Results: The primary screen conducted was robust, with Z' values >0.5. Fifty-two compounds were identified in the primary screen and confirmed to have concentration-dependent effects on impedance in NTM5 cells. Of these, 9 compounds representing distinct drug classes were confirmed to modulate impedance in both human primary TM cells and bovine cells. One of these compounds, wortmannin, an inhibitor of phosphoinositide 3-kinase, increased outflow facility by 11%. Conclusions: A robust phenotypic assay was developed that enabled identification of contractility modulators in immortalized TM cells. The screening hits were translatable to primary TM cells and modulated outflow facility in an ex vivo perfusion assay.

Using Healthcare Databases to Refine Understanding of Exploratory Associations Between Drugs and Progression of Open-Angle Glaucoma.

We sought to refine understanding about associations identified in prior studies between angiotensin-II receptor blockers, metformin, selective serotonin reuptake inhibitors, fibric-acid derivatives, or calcium channel blockers and progression to glaucoma filtration surgery for open-angle glaucoma (OAG). We used new-initiator, active-comparator cohort designs to investigate these drugs in two data sources. We adjusted for confounders using stabilized inverse-probability-of-treatment weights and evaluated results using "intention-to-treat" and "as-treated" follow-up approaches. In both data sources, Kaplan-Meier curves showed trends for more rapid progression to glaucoma filtration surgery in patients taking calcium channel blockers compared with thiazides with as-treated (MarketScan P = 0.15; Medicare P = 0.03) and intention-to-treat follow-up (MarketScan P < 0.01; Medicare P = 0.10). There was suggestion of delayed progression for selective serotonin reuptake inhibitor compared with tricyclic antidepressants in Medicare, which was not observed in MarketScan. Our study provided support for a relationship between calcium channel blockers and OAG progression but not for other investigated drugs.

Glaucoma - Next Generation Therapeutics: Impossible to Possible.

The future of next generation therapeutics for glaucoma is strong. The recent approval of two novel intraocular pressure (IOP)-lowering drugs with distinct mechanisms of action is the first in over 20 years. However, these are still being administered as topical drops. Efforts are underway to increase patient compliance and greater therapeutic benefits with the development of sustained delivery technologies. Furthermore, innovations from biologics- and gene therapy-based therapeutics are being developed in the context of disease modification, which are expected to lead to more permanent therapies for patients. Neuroprotection, including the preservation of retinal ganglion cells (RGCs) and optic nerve is another area that is actively being explored for therapeutic options. With improvements in imaging technologies and determination of new surrogate clinical endpoints, the therapeutic potential for translation of neuroprotectants is coming close to clinical realization. This review summarizes the aforementioned topics and other related aspects.

Mutant myocilin impacts sarcomere ultrastructure in mouse gastrocnemius muscle.

Myocilin (MYOC) is the gene with mutations most common in glaucoma. In the eye, MYOC is in trabecular meshwork, ciliary body, and retina. Other tissues with high MYOC transcript levels are skeletal muscle and heart. To date, the function of wild-type MYOC remains unknown and how mutant MYOC causes high intraocular pressure and glaucoma is ambiguous. By investigating mutant MYOC in a non-ocular tissue we hoped to obtain novel insight into mutant MYOC pathology. For this study, we utilized a transgenic mouse expressing human mutant MYOC Y437H protein and we examined its skeletal (gastrocnemius) muscle phenotype. Electron micrographs showed that sarcomeres in the skeletal muscle of mutant CMV-MYOC-Y437H mice had multiple M-bands. Western blots of soluble muscle lysates from transgenics indicated a decrease in two M-band proteins, myomesin 1 (MYOM1) and muscle creatine kinase (CKM). Immunoprecipitation identified CKM as a MYOC binding partner. Our results suggest that binding of mutant MYOC to CKM is changing sarcomere ultrastructure and this may adversely impact muscle function. We speculate that a person carrying the mutant MYOC mutation will likely have a glaucoma phenotype and may also have undiagnosed muscle ailments or vice versa, both of which will have to be monitored and treated.

Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.

A murine glaucoma model induced by rapid in vivo photopolymerization of hyaluronic acid glycidyl methacrylate.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid in vivo crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.

A Randomized, Controlled Phase I/II Study to Evaluate the Safety and Efficacy of MGV354 for Ocular Hypertension or Glaucoma.

PURPOSE: To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma. DESIGN: Double-masked, randomized, and vehicle-controlled study. METHODS: Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once-daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2) at a single clinical site. Part 3 was a multisite trial that evaluated intraocular pressure (IOP)-lowering efficacy of the MTD administered nightly for 1 week in 50 patients with minimum IOP of 24 mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at day 8 compared to baseline (ClinicalTrials.govNCT02743780). RESULTS: There was no difference in favor of MGV354 for IOP lowering; change from baseline to day 8 in mean diurnal IOP was -0.6 mm Hg for MGV354-treated patients and -1.1 mm Hg for vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common adverse events reported after MGV354 administration were conjunctival and ocular hyperemia. CONCLUSIONS: Overall, MGV354 0.1% demonstrated no statistically significant effect compared to vehicle in lowering IOP based on the study's main outcome measure. MGV354 produced ocular hyperemia consistent with its pharmacology.

A Novel Selective Soluble Guanylate Cyclase Activator, MGV354, Lowers Intraocular Pressure in Preclinical Models, Following Topical Ocular Dosing.

Purpose: The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Methods: Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. Results: sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. Conclusions: MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

The Discovery of ( S)-1-(6-(3-((4-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1 H-inden-4-yl)pyridin-2-yl)-5-methyl-1 H-pyrazole-4-carboxylic Acid, a Soluble Guanylate Cyclase Activator Specifically Designed for Topical Ocular Delivery as a Therapy for Glaucoma.

Soluble guanylate cyclase (sGC), the endogenous receptor for nitric oxide (NO), has been implicated in several diseases associated with oxidative stress. In a pathological oxidative environment, the heme group of sGC can be oxidized becoming unresponsive to NO leading to a loss in the ability to catalyze the production of cGMP. Recently a dysfunctional sGC/NO/cGMP pathway has been implicated in contributing to elevated intraocular pressure associated with glaucoma. Herein we describe the discovery of molecules specifically designed for topical ocular administration, which can activate oxidized sGC restoring the ability to catalyze the production of cGMP. These efforts culminated in the identification of compound (+)-23, which robustly lowers intraocular pressure in a cynomolgus model of elevated intraocular pressure over 24 h after a single topical ocular drop and has been selected for clinical evaluation.

A Compact Whole-Eye Perfusion System to Evaluate Pharmacologic Responses of Outflow Facility.

Purpose: To discover novel therapies that lower IOP by increasing aqueous humor outflow facility, ex vivo ocular perfusion systems provide a valuable tool. However, currently available designs are limited by their throughput. Here we report the development of a compact, scalable perfusion system with improved throughput and its validation using bovine and porcine eyes. Methods: At a fixed IOP of 6 mm Hg, flow rate was measured by flow sensors. We validated the system by measuring the outflow responses to Y-39983 (a Rho kinase inhibitor), endothelin-1 (ET-1), ambrisentan (an antagonist for endothelin receptor A [ETA]), sphigosine-1-phosphate (S1P), JTE-013 (antagonist for S1P receptor 2 [S1P2]), S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor), and 3-Morpholino-sydnonimine (SIN-1, another NO donor). Results: The instrument design enabled simultaneous measurements of 20 eyes with a footprint of 1 m2. Relative to vehicle control, Y-39983 increased outflow by up to 31% in calf eyes. On the contrary, ET-1 decreased outflow by up to 79%, a response that could be blocked by pretreatment with ambrisentan, indicating a role for ETA receptors. Interestingly, the effect of ET-1 was also inhibited by up to 70% to 80% by pretreatment with NO donors, SNAP and SIN-1. In addition to testing in calf eyes, similar effects of ET-1 and ambrisentan were observed in adult bovine and porcine eyes. Conclusions: The compact eye perfusion platform provides an opportunity to efficiently identify compounds that influence outflow facility and may lead to the discovery of new glaucoma therapies.

Effects of Rho Kinase Inhibitors on Intraocular Pressure and Aqueous Humor Dynamics in Nonhuman Primates and Rabbits.

PURPOSE: This study examines the effects of 2 Rho kinase inhibitors on intraocular pressure (IOP) and aqueous humor dynamics. METHODS: IOPs of New Zealand albino rabbits with ocular normotension and cynomolgus macaques (nonhuman primate, NHP) with chronic unilateral laser-induced glaucoma were measured at baseline and periodically after a 9 a.m. dose of H-1152, Y-27632, or vehicle. In a separate group of NHPs, aqueous flow, outflow facility, uveoscleral outflow, and IOP were determined after treatment with Y-27632 or vehicle control. RESULTS: Decreases in IOP were found in rabbits (n = 5) at 6 h after one dose of 2% Y-27632 (29%, P = 0.0002) or 1% H-1152 (35%, P = 0.0001), and in hypertensive eyes of NHPs (n = 7-9) at 3 h after one dose of 2% Y-27632 (35%, P = 0.005) or 1% H-1152 (51%, P = 0.0003). With 2 doses of 1% Y-27632 or vehicle in NHP hypertensive eyes (n = 12), significant drug effects were IOP reduction of 28% (P = 0.05) at 2.5 h after the second dose and increases in aqueous flow (36%; P = 0.013), uveoscleral outflow (59%, P = 0.008), and outflow facility (40%; P = 0.01). In normotensive eyes of the same animals, aqueous flow increased by 21% (P = 0.03). No significant change was found in any of the other parameters. CONCLUSIONS: Y-27632 and H-1152 lower IOP in rabbits and hypertensive eyes of NHPs for at least 6 h after single doses. The Y-27632 effect on IOP in hypertensive NHP eyes is caused by increases in outflow facility and uveoscleral outflow. An increase in aqueous humor formation attenuates but does not prevent an IOP decrease.

Pharmacology of novel intraocular pressure-lowering targets that enhance conventional outflow facility: Pitfalls, promises and what lies ahead?

Intraocular pressure (IOP) lowering drugs that are approved for the treatment of glaucoma and ocular hypertension have limited activity on increasing aqueous humor movement through the trabecular meshwork and Schlemm's canal (TM/SC). The TM/SC complex is considered the conventional outflow pathway and is a primary site of increased resistance to aqueous humor outflow in glaucoma. Novel mechanisms that enhance conventional outflow have shown promise in IOP reduction via modulation of several pathways including Rho kinase, nitric oxide/soluble guanylate cyclase/cGMP, adenosine A1, prostaglandin EP4/cAMP, and potassium channels. The clinical translatability of these pharmacological modulators based on pre-clinical efficacy models is currently being explored. In addition, identification of pathways from GWAS and other studies involving transgenic rodent models with elevated/reduced IOP phenotypes have begun to yield additional insights into IOP regulation and serve as a source for the next generation of IOP lowering targets. Lastly, improvements in drug delivery technologies to enable sustained IOP reduction are also discussed.

Intraocular Pressure-Lowering Activity of NCX 470, a Novel Nitric Oxide-Donating Bimatoprost in Preclinical Models.

PURPOSE: The prostaglandin F2alpha (PGF2α) analogue bimatoprost lowers intraocular pressure (IOP) by increasing uveoscleral outflow at doses shown to elicit redness of the eye. With the aim to enhance the IOP-lowering effect of bimatoprost we studied NCX 470 [(S,E)-1-((1R,2R,3S,5R)-2-((Z)-7-(ethylamino)-7-oxohept-2-enyl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate], a dual-acting compound combining bimatoprost with nitric oxide (NO) known to mainly act via relaxation of trabecular meshwork and Schlemm's canal. METHODS: New Zealand white rabbits with transient hypertonic saline-induced IOP elevation (tOHT-rabbits), cynomolgus monkeys with laser-induced ocular hypertension (OHT-monkeys), and normotensive dogs (ONT-dogs) were used. The levels of NCX 470, bimatoprost, and bimatoprost acid were determined in aqueous humor (AH), cornea (CR), and iris/ciliary body (ICB) by liquid chromatography-mass spectrometry/mass (LC-MS/MS), while cGMP in AH and ICB was monitored using an enzyme immunoassay (EIA) kit in pigmented Dutch Belted rabbits. RESULTS: NCX 470 (0.14%, 30 µL) lowered IOP in tOHT-rabbits with an E(max) of -7.2 ± 2.8 mm Hg at 90 minutes. Bimatoprost at equimolar dose (0.1%, 30 µL) was noneffective in this model. NCX 470 (0.042%, 30 µL) was more effective than equimolar (0.03%, 30 µL) bimatoprost in ONT-dogs (IOP change, -5.4 ± 0.7 and -3.4 ± 0.7 mm Hg, respectively, P < 0.05) and in OHT-monkeys (IOP change, -7.7 ± 1.4 and -4.8 ± 1.7 mm Hg, respectively, P < 0.05) at 18 hours post dosing. NCX 470 (0.042%, 30 µL) or bimatoprost (0.03%, 30 µL) resulted in similar bimatoprost acid exposure in AH, CR, and ICB while cGMP was significantly increased in AH and ICB at 18 and 24 hours after NCX 470 dosing. CONCLUSIONS: NCX 470 lowers IOP more than equimolar bimatoprost in three animal models of glaucoma by activating PGF2α and NO/cGMP signaling pathways.

Effect of PF-04217329 a prodrug of a selective prostaglandin EP(2) agonist on intraocular pressure in preclinical models of glaucoma.

Better control of intraocular pressure (IOP) is the most effective way to preserve visual field function in glaucomatous patients. While prostaglandin FP analogs are leading the therapeutic intervention for glaucoma, new target classes also are being identified with new lead compounds being developed for IOP reduction. One target class currently being investigated includes the prostaglandin EP receptor agonists. Recently PF-04217329 (Taprenepag isopropyl), a prodrug of CP-544326 (active acid metabolite), a potent and selective EP(2) receptor agonist, was successfully evaluated for its ocular hypotensive activity in a clinical study involving patients with primary open angle glaucoma. In the current manuscript, the preclinical attributes of CP-544326 and PF-0421329 have been described. CP-544326 was found to be a potent and selective EP(2) agonist (IC(50) = 10 nM; EC(50) = 2.8 nM) whose corneal permeability and ocular bioavailability were significantly increased when the compound was dosed as the isopropyl ester prodrug, PF-04217329. Topical ocular dosing of PF-04217329 was well tolerated in preclinical species and caused an elevation of cAMP in aqueous humor/iris-ciliary body indicative of in vivo EP(2) target receptor activation. Topical ocular dosing of PF-04217329 resulted in ocular exposure of CP-544326 at levels greater than the EC(50) for the EP(2) receptor. PF-04217329 when dosed once daily caused between 30 and 50% IOP reduction in single day studies in normotensive Dutch-belted rabbits, normotensive dogs, and laser-induced ocular hypertensive cynomolgus monkeys and 20-40% IOP reduction in multiple day studies compared to vehicle-dosed eyes. IOP reduction was sustained from 6 h through 24 h following a single topical dose. In conclusion, preclinical data generated thus far appear to support the clinical development of PF-04217329 as a novel compound for the treatment of glaucoma.

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models.

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 µg) and 0.12% (36 µg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 µg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.

Endothelin, astrocytes and glaucoma.

It has become increasingly clear that astrocytes may play an important role in the genesis of glaucoma. Astrogliosis occurs in response to ocular stress or the presence of noxious stimuli. Agents that appear to stimulate reactive gliosis are becoming increasingly clear. One class of agents that is emerging is the endothelins (ETs; specifically, ET-1). In this review we examine the interactions of ET-1 with astrocytes and provide examples where ET-1 appears to contribute to activation of astrocytes and play a role in the neurodegenerative effects that accompany such reactivation resulting in astrogliosis. These actions are presented in the context of glaucoma although information is also presented with respect to ET-1's role in the central nervous system and brain. While much has been learned with respect to ET-1/astrocyte interactions, there are still a number of questions concerning the potential therapeutic implications of these findings. Hopefully this review will stimulate others to examine this potential.

Thrombin-induced endothelin-1 synthesis and secretion in retinal pigment epithelial cells is rho kinase dependent.

PURPOSE: The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood­retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood­retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1. METHODS: Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels. RESULTS: Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion. CONCLUSIONS: Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.

A novel nitric oxide releasing prostaglandin analog, NCX 125, reduces intraocular pressure in rabbit, dog, and primate models of glaucoma.

PURPOSE: Nitric oxide (NO) is involved in a variety of physiological processes including ocular aqueous humor dynamics by targeting mechanisms that are complementary to those of prostaglandins. Here, we have characterized a newly synthesized compound, NCX 125, comprising latanoprost acid and NO-donating moieties. METHODS: NCX 125 was synthesized and tested in vitro for its ability to release functionally active NO and then compared with core latanoprost for its intraocular pressure (IOP)-lowering effects in rabbit, dog, and nonhuman primate models of glaucoma. RESULTS: NCX 125 elicited cGMP formation (EC(50) = 3.8 + or - 1.0 microM) in PC12 cells and exerted NO-dependent iNOS inhibition (IC(50) = 55 + or - 11 microM) in RAW 264.7 macrophages. NCX 125 lowered IOP to a greater extent compared with equimolar latanoprost in: (a) rabbit model of transient ocular hypertension (0.030% latanoprost, not effective; 0.039% NCX 125, Delta(max) = -10.6 + or - 2.3 mm Hg), (b) ocular hypertensive glaucomatous dogs (0.030% latanoprost, Delta(max)= -6.7 + or - 1.2 mm Hg; 0.039% NCX 125, Delta(max) = -9.1 + or - 3.1 mm Hg), and (c) laser-induced ocular hypertensive non-human primates (0.10% latanoprost, Delta(max) = -11.9 + or - 3.7 mm Hg, 0.13% NCX 125, Delta(max) = -16.7 + or - 2.2 mm Hg). In pharmacokinetic studies, NCX 125 and latanoprost resulted in similar latanoprost-free acid exposure in anterior segment ocular tissues. CONCLUSIONS: NCX 125, a compound targeting 2 different mechanisms, is endowed with potent ocular hypotensive effects. This may lead to potential new perspectives in the treatment of patients at risk of glaucoma.