Safety of intravitreally delivered AAV2 vector-mediated multi-characteristic opsin genetic construct in wild type beagle dogs.

BACKGROUND: A novel adeno-associated virus 2 (AAV2)-carried multi-characteristic opsin (MCO) (MCO-010) is undergoing several clinical trials as a novel therapeutic modality for the treatment of degenerative retinal diseases including retinitis pigmentosa and Stargardt disease. The present study aimed to determine the ocular and systemic safety of MCO-010 and the AAV2 vehicle in adult Beagle dogs following intravitreal (IVT) injection. METHODS: The current safety/toxicology studies spanning 13 weeks described here utilized well-documented techniques to assess the effects of IVT injection of MCO-010 up to 2.2 × 1011 genome copies (gc) per eye, or the AAV2 capsid (vehicle control) on gross behavioral and immunogenic changes, alterations in body weights, blood biochemistry, hematology, blood coagulation, gross necropsy lesions, organ weight changes and histopathology in the dogs (n = 4 per group; two males and two females per group). Immunohistochemical and functional electroretinogram studies were also conducted to determine MCO expression in the retina and determine any retinal toxicity associated with MCO-010. RESULTS: There were no significant deleterious effects of the MCO-010 (or the AAV2 at the tested doses) on any of the examined parameters, including the absence of any severe ocular or systemic adverse events. However, as expected, inflammation after IVT delivery of AAV2 and MCO-010 was observed in the conjunctivae of all groups of animals, although this self-resolved within 1 week post-injection. Quantitative immunohistochemical analyses of MCO-010-associated mCherry revealed successful delivery of the gene therapy within the inner retina. CONCLUSIONS: In summary, MCO-010 demonstrated a favorable safety profile when administered to the eyes of adult Beagle dogs of both sexes at dose levels up to 2.2 × 1011 gc per eye, with no adverse effects observed. This dose was identified as the No Observed Adverse Effect Level (i.e. NOAEL) and guided selection of safe doses for human clinical trials.

Steroid-Induced Ocular Hypertension in Mice Is Differentially Reduced by Selective EP2, EP3, EP4, and IP Prostanoid Receptor Agonists.

We tested five chemically and metabolically stable prostaglandin (PG) receptor agonists in a mouse model of dexamethasone-induced ocular hypertension (OHT). Whilst all compounds significantly (p < 0.05, ANOVA) lowered intraocular pressure (IOP) after twice-daily bilateral topical ocular dosing (5 µg/dose) over three weeks, the time course and magnitude of the responses varied. The onset of action of NS-304 (IP-PG receptor agonist) and rivenprost (EP4-PG receptor agonist) was slower than that of misoprostol (mixed EP2/EP3/EP4-PG receptor agonist), PF-04217329 (EP2-PG receptor agonist), and butaprost (EP2-PG receptor agonist). The rank order of IOP-lowering efficacies aligned with the onset of actions of these compounds. Peak IOP reductions relative to vehicle controls were as follows: misoprostol (74.52%) = PF-04217329 (74.32%) > butaprost (65.2%) > rivenprost (58.4%) > NS-304 (55.3%). A literature survey indicated that few previously evaluated compounds (e.g., latanoprost, timolol, pilocarpine, brimonidine, dorzolamide, cromakalim analog (CKLP1), losartan, tissue plasminogen activator, trans-resveratrol, sodium 4-phenyl acetic acid, etc.) in various animal models of steroid-induced OHT were able to match the effectiveness of misoprostol, PF-04217329 or butaprost. Since a common feature of the latter compounds is their relatively high affinity and potency at the EP2-PG receptor sub-type, which activates the production of intracellular cAMP in target cells, our studies suggest that drugs selective for the EP2-PG receptor may be suited to treat corticosteroid-induced OHT.

Identifying new drugs and targets to treat rapidly elevated intraocular pressure for angle closure and secondary glaucomas to curb visual impairment and prevent blindness.

A multitude of pharmacological compounds have been shown to lower and control intraocular pressure (IOP) in numerous species of animals and human subjects after topical ocular dosing or via other routes of administration. Most researchers have been interested in finding drug candidates that exhibit a relatively long duration of action from a chronic therapeutic use perspective, for example to treat ocular hypertension (OHT), primary open-angle glaucoma and even normotensive glaucoma. However, it is equally important to seek and characterize treatment modalities which offer a rapid onset of action to help provide fast relief from quickly rising IOP that occurs in certain eye diseases. These include acute angle-closure glaucoma, primary angle-closure glaucoma, uveitic and inflammatory glaucoma, medication-induced OHT, and other secondary glaucomas induced by eye injury or infection which can cause partial or complete loss of eyesight. Such fast-acting agents can delay or prevent the need for ocular surgery which is often used to lower the dangerously raised IOP. This research survey was therefore directed at identifying agents from the literature that demonstrated ocular hypotensive activity, normalizing and unifying the data, determining their onset of action and rank ordering them on the basis of rapidity of action starting within 30-60 min and lasting up to at least 3-4 h post topical ocular dosing in different animal species. This research revealed a few health authority-approved drugs and some investigational compounds that appear to meet the necessary criteria of fast onset of action coupled with significant efficacy to reduce elevated IOP (by ≥ 20%, preferably by >30%). However, translation of the novel animal-based findings to the human conditions remains to be demonstrated but represent viable targets, especially EP2-receptor agonists (e.g. omidenepag isopropyl; AL-6598; butaprost), mixed activity serotonin/dopamine receptor agonists (e.g. cabergoline), rho kinase inhibitors (e.g. AMA0076, Y39983), CACNA2D1-gene product inhibitors (e.g. pregabalin), melatonin receptor agonists, and certain K+-channel openers (e.g. nicorandil, pinacidil). Other drug candidates and targets were also identified and will be discussed.

FP and EP2 prostanoid receptor agonist drugs and aqueous humor outflow devices for treating ocular hypertension and glaucoma.

Prostaglandin (PG) receptors represent important druggable targets due to the many diverse actions of PGs in the body. From an ocular perspective, the discovery, development, and health agency approvals of prostaglandin F (FP) receptor agonists (FPAs) have revolutionized the medical treatment of ocular hypertension (OHT) and glaucoma. FPAs, such as latanoprost, travoprost, bimatoprost, and tafluprost, powerfully lower and control intraocular pressure (IOP), and became first-line therapeutics to treat this leading cause of blindness in the late 1990s to early 2000s. More recently, a latanoprost-nitric oxide (NO) donor conjugate, latanoprostene bunod, and a novel FP/EP3 receptor dual agonist, sepetaprost (ONO-9054 or DE-126), have also demonstrated robust IOP-reducing activity. Moreover, a selective non-PG prostanoid EP2 receptor agonist, omidenepag isopropyl (OMDI), was discovered, characterized, and has been approved in the United States, Japan and several other Asian countries for treating OHT/glaucoma. FPAs primarily enhance uveoscleral (UVSC) outflow of aqueous humor (AQH) to reduce IOP, but cause darkening of the iris and periorbital skin, uneven thickening and elongation of eyelashes, and deepening of the upper eyelid sulcus during chronic treatment. In contrast, OMDI lowers and controls IOP by activation of both the UVSC and trabecular meshwork outflow pathways, and it has a lower propensity to induce the aforementioned FPA-induced ocular side effects. Another means to address OHT is to physically promote the drainage of the AQH from the anterior chamber of the eye of patients with OHT/glaucoma. This has successfully been achieved by the recent approval and introduction of miniature devices into the anterior chamber by minimally invasive glaucoma surgeries. This review covers the three major aspects mentioned above to highlight the etiology of OHT/glaucoma, and the pharmacotherapeutics and devices that can be used to combat this blinding ocular disease.

The Role of Autophagy in Chemical Proteasome Inhibition Model of Retinal Degeneration.

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule.

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167-384 nM; Emax = 32-86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g., IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60-84 nM; Emax = 29-44% relative to max. BK-induced effects). FR-190997 (0.3-300 µg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g., 34.5 ± 7.5% decrease; 6 h post-dose of 30 µg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.

FR-190997, a nonpeptide bradykinin B2-receptor partial agonist, is a potent and efficacious intraocular pressure lowering agent in ocular hypertensive cynomolgus monkeys.

Preclinical Research FR-190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnaminoacetyl]-N-methylamino]benzyloxy]-2-methyl-4- (2-pyridylmethoxy) quinoline), a nonpeptide bradykinin (BK) B2-receptor-selective agonist, represents a novel class of ocular hypotensive agents. FR-190997 exhibited a high affinity for the human cloned B2-receptor (Ki = 9.8 nM) and a relatively high potency (EC50 = 155 nM) for mobilizing intracellular Ca(2+) ([Ca(2+)]i) in human ocular cells from nonpigmented ciliary epithelium; trabecular meshwork [h-TM]; ciliary muscle [h-CM] that are involved in regulating intraocular pressure (IOP). Unlike BK, FR-190997 behaved as a partial agonist (Emax = 38-80%) in these cells and its [Ca(2+)]i-mobilizing effects were blocked by the B2-receptor-selective antagonists (HOE-140, Ki = 0.8-7 nM; WIN-64338, Ki = 157-425 nM). FR-190997 stimulated the production of prostaglandins (PGs) in h-CM and h-TM cells (EC50 = 15-19 nM; Emax = 27-33%); an effect that was reduced by the cyclooxygenase-2 inhibitor bromfenac, and by HOE-140. FR-190997 also induced pro-matrix metalloproteinase (MMP)-1 and MMP-3 release from h-CM cells. FR-190997 significantly lowered IOP (37% [P < 0.001] with 30 µg, 24 h post-topical ocular dosing) in ocular hypertensive eyes of conscious Cynomolgus monkeys. This effect was reduced by bromfenac and completely blocked by a B2-antagonist. FR-190997 primarily stimulated uveoslceral outflow (UVSO) of aqueous humor (2.6 to 3.9-fold above baseline). In conclusion, FR-190997 is a B2-receptor selective partial agonist that activates phospholipase C, mobilizes [Ca(2+)]; induces PG and pro-MMP production, and that profoundly lowers IOP by promoting UVSO in ocular hypertensive Cynomolgus monkey eyes.

Human experience and efficacy of omidenepag isopropyl (Eybelis®; Omlonti®): Discovery to approval of the novel non-prostaglandin EP2-receptor-selective agonist ocular hypotensive drug.

More than 75 million people worldwide suffer from ocular hypertension (OHT)-associated retinal and optic nerve degenerative diseases that cause visual impairment and can lead to blindness. In an effort to find novel pharmaceutical therapeutics to combat OHT with reduced side-effect potential, several emerging drug candidates have advanced to human proof-of-concept in recent years. One such compound is a nonprostaglandin (non-PG) EP2-receptor-selective agonist (omidenepag isopropyl ester). Omidenepag (OMD; free acid form) is a novel non-PG that selectively binds to and activates the human EP2-prostglandin receptor (EP2R) with a high affinity (Ki = 3.6 nM) and which potently generates intracellular cAMP in living cells (EC50 = 3.9-8.3 nM). OMD significantly downregulated COL12A1 and COL13A1 mRNAs in human trabecular meshwork (TM) cells, a tissue involved in the pathogenesis of OHT. Omidenepag isopropyl (OMDI) potently and efficaciously lowered intraocular pressure (IOP) in ocular normotensive rabbits, dogs, and monkeys, and also in ocular hypertension (OHT) Cynomolgus monkeys, after a single topical ocular (t.o.) instillation at doses of 0.0001-0.01%. No reduction in IOP-lowering response to OMDI was observed after repeated t.o. dosing with OMDI in dogs and monkeys. Additive IOP reduction to OMDI was noted with brinzolamide, timolol, and brimonidine in rabbits and monkeys. OMDI 0.002% t.o. decreased IOP by stimulating the conventional (TM) and uveoscleral (UVSC) outflow of aqueous humor (AQH) in OHT monkeys. In a Phase-III clinical investigation, 0.002% OMDI (once daily t.o.) reduced IOP by 5-6 mmHg in OHT/primary open-angle glaucoma (POAG) patients (22-34 mmHg baseline IOPs) that was maintained over 12-months. In an additional month-long clinical study, 0.002% OMDI induced IOP-lowering equivalent to that of latanoprost (0.005%), a prostanoid FP-receptor agonist, thus OMDI was noninferior to latanoprost. Additive IOPreduction was also noted in OHT/OAG patients when OMDI (0.002%, once daily t.o.) and timolol (0.05%, twice daily t.o.) were administered. Patients with OHT/POAG who were low responders or nonresponders to latanoprost (0.005%, q.d.; t.o.) experienced significant IOP-lowering (additional approximately 3 mmHg) when they were switched over to OMDI 0.002% (q.d.; t.o.). No systemic or ocular adverse reactions (e.g. iris color changes/deepening of the upper eyelid sulcus/abnormal eyelash growth) were noted after a year-long, once-daily t.o. dosing with 0.002 % OMDI in OHT/POAG patients. However, OMDI caused transient conjunctival hyperemia. These characteristics of OMDI render it a suitable new medication for treating OHT and various types of glaucoma, especially where elevated IOP is implicated.

Assessment of Brain-Derived Neurotrophic Factor on Retinal Structure and Visual Function in Rodent Models of Optic Nerve Crush.

The effects of brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and visual function were assessed in rat and mouse models of optic nerve (ON) crush. ONs were crushed on Day 1, followed by intravitreal injections of a vehicle or BDNF on Days 1 and 8. The spatial frequency threshold was measured using optokinetic tracking on Days 7 and 14. On Day 15, ganglion cell complex (GCC) thickness was quantified using optical coherence tomography. Furthermore, all eyes were enucleated for immunohistochemical analysis of the surviving RGC somas and axons. BDNF significantly reduced the RGC soma in mice and increased GCC thickness in intact eyes, with apparent axonal swelling in both species. It displayed significantly greater RGC soma survival in eyes with ON injury, with moderately thicker axonal bundles in both species and a thicker GCC in rats. Visual function was significantly reduced in all ON-crushed animals, regardless of BDNF treatment. Thus, we obtained a comprehensive analysis of the structural and functional impact of BDNF in intact and ON-crushed eyes in two rodent models. Our results provide a foundation for further BDNF evaluation and the design of preclinical studies on neuroprotectants using BDNF as a reference positive control.

Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B2 receptors in ciliary muscle.

PURPOSE: To examine the bradykinin (BK) B2-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. METHODS: Previously published procedures were used throughout these studies. RESULTS: The human and monkey ciliary bodies expressed high levels of B2-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²âº](i) mobilization in primary h-CM cells (BK EC50=2.4±0.2 nM > Hyp³,ß-(2-thienyl)-Ala5,Tyr(Me)8-(®)-Arg9-BK (RMP-7) > Des-Arg9-BK EC50=4.2 µM [n=3-6]), and this was blocked by B2-selective antagonists, HOE-140 (IC50=1.4±0.1 nM) and WIN-63448 (IC50=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²âº](i) mobilization. Total prostaglandin (primarily PGE2) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B2-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 µM; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg9-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7-10). CONCLUSIONS: These studies have provided evidence of a functional endogenously expressed B2-receptor system in the CM that appears to be involved in modulating IOP.

Neuroaxonal and cellular damage/protection by prostanoid receptor ligands, fatty acid derivatives and associated enzyme inhibitors.

Cellular and mitochondrial membrane phospholipids provide the substrate for synthesis and release of prostaglandins in response to certain chemical, mechanical, noxious and other stimuli. Prostaglandin D2, prostaglandin E2, prostaglandin F2α, prostaglandin I2 and thromboxane-A2 interact with five major receptors (and their sub-types) to elicit specific downstream cellular and tissue actions. In general, prostaglandins have been associated with pain, inflammation, and edema when they are present at high local concentrations and involved on a chronic basis. However, in acute settings, certain endogenous and exogenous prostaglandins have beneficial effects ranging from mediating muscle contraction/relaxation, providing cellular protection, regulating sleep, and enhancing blood flow, to lowering intraocular pressure to prevent the development of glaucoma, a blinding disease. Several classes of prostaglandins are implicated (or are considered beneficial) in certain central nervous system dysfunctions (e.g., Alzheimer's, Parkinson's, and Huntington's diseases; amyotrophic lateral sclerosis and multiple sclerosis; stroke, traumatic brain injuries and pain) and in ocular disorders (e.g., ocular hypertension and glaucoma; allergy and inflammation; edematous retinal disorders). This review endeavors to address the physiological/pathological roles of prostaglandins in the central nervous system and ocular function in health and disease, and provides insights towards the therapeutic utility of some prostaglandin agonists and antagonists, polyunsaturated fatty acids, and cyclooxygenase inhibitors.

Electrical, Electromagnetic, Ultrasound Wave Therapies, and Electronic Implants for Neuronal Rejuvenation, Neuroprotection, Axonal Regeneration, and IOP Reduction.

The peripheral nervous system (PNS) of mammals and nervous systems of lower organisms possess significant regenerative potential. In contrast, although neural plasticity can provide some compensation, the central nervous system (CNS) neurons and nerves of adult mammals generally fail to regenerate after an injury or damage. However, use of diverse electrical, electromagnetic and sonographic energy waves are illuminating novel ways to stimulate neuronal differentiation, proliferation, neurite growth, and axonal elongation/regeneration leading to various levels of functional recovery in animals and humans afflicted with disorders of the CNS, PNS, retina, and optic nerve. Tools such as acupuncture, electroacupuncture, electroshock therapy, electrical stimulation, transcranial magnetic stimulation, red light therapy, and low-intensity pulsed ultrasound therapy are demonstrating efficacy in treating many different maladies. These include wound healing, partial recovery from motor dysfunctions, recovery from ischemic/reperfusion insults and CNS and ocular remyelination, retinal ganglion cell (RGC) rejuvenation, and RGC axonal regeneration. Neural rejuvenation and axonal growth/regeneration processes involve activation or intensifying of the intrinsic bioelectric waves (action potentials) that exist in every neuronal circuit of the body. In addition, reparative factors released at the nerve terminals and via neuronal dendrites (transmitter substances), extracellular vesicles containing microRNAs and neurotrophins, and intercellular communication occurring via nanotubes aid in reestablishing lost or damaged connections between the traumatized tissues and the PNS and CNS. Many other beneficial effects of the aforementioned treatment paradigms are mediated via gene expression alterations such as downregulation of inflammatory and death-signal genes and upregulation of neuroprotective and cytoprotective genes. These varied techniques and technologies will be described and discussed covering cell-based and animal model-based studies. Data from clinical applications and linkage to human ocular diseases will also be discussed where relevant translational research has been reported.

Recently Approved Drugs for Lowering and Controlling Intraocular Pressure to Reduce Vision Loss in Ocular Hypertensive and Glaucoma Patients.

Serious vision loss occurs in patients affected by chronically raised intraocular pressure (IOP), a characteristic of many forms of glaucoma where damage to the optic nerve components causes progressive degeneration of retinal and brain neurons involved in visual perception. While many risk factors abound and have been validated for this glaucomatous optic neuropathy (GON), the major one is ocular hypertension (OHT), which results from the accumulation of excess aqueous humor (AQH) fluid in the anterior chamber of the eye. Millions around the world suffer from this asymptomatic and progressive degenerative eye disease. Since clinical evidence has revealed a strong correlation between the reduction in elevated IOP/OHT and GON progression, many drugs, devices, and surgical techniques have been developed to lower and control IOP. The constant quest for new pharmaceuticals and other modalities with superior therapeutic indices has recently yielded health authority-approved novel drugs with unique pharmacological signatures and mechanism(s) of action and AQH drainage microdevices for effectively and durably treating OHT. A unique nitric oxide-donating conjugate of latanoprost, an FP-receptor prostaglandin (PG; latanoprostene bunod), new rho kinase inhibitors (ripasudil; netarsudil), a novel non-PG EP2-receptor-selective agonist (omidenepag isopropyl), and a form of FP-receptor PG in a slow-release intracameral implant (Durysta) represent the additions to the pharmaceutical toolchest to mitigate the ravages of OHT. Despite these advances, early diagnosis of OHT and glaucoma still lags behind and would benefit from further concerted effort and attention.

Elevated Intraocular Pressure and Glaucomatous Optic Neuropathy: Genes to Disease Mechanisms, Therapeutic Drugs, and Gene Therapies.

This review article focuses on the pathogenesis of and genetic defects linked with chronic ocular hypertension (cOHT) and glaucoma. The latter ocular disease constitutes a group of ocular degenerative diseases whose hallmark features are damage to the optic nerve, apoptotic demise of retinal ganglion cells, disturbances within the brain regions involved in visual perception and considerable visual impairment that can lead to blindness. Even though a number of pharmaceuticals, surgical and device-based treatments already exist addressing cOHT associated with the most prevalent of the glaucoma types, primary open-angle glaucoma (POAG), they can be improved upon in terms of superior efficacy with reduced side-effects and with longer duration of activity. The linkage of disease pathology to certain genes via genome-wide associated studies are illuminating new approaches to finding novel treatment options for the aforementioned ocular disorders. Gene replacement, gene editing via CRISPR-Cas9, and the use of optogenetic technologies may replace traditional drug-based therapies and/or they may augment existing therapeutics for the treatment of cOHT and POAG in the future.

Gene therapies and gene product-based drug candidates for normalizing and preserving tissue functions in animal models of ocular hypertension and glaucoma.

More than 76 million people worldwide are afflicted with the neurodegenerative eye diseases described and grouped together as glaucoma. A common feature amongst the many forms of glaucoma is chronically elevated intraocular pressure (IOP) within the anterior chamber of the eye that physically damages the retina, optic nerve and parts of the brain connected with visual perception. The mediators of the contusing raised IOP responsible for such damage and loss of vision include locally released inflammatory agents, tissue remodeling enzymes and infiltrating immune cells which damage the retinal ganglion cell (RGC) axons and eventually kill a significant number of the RGCs. Additional culprits include genetic defects of the patient that involve aberrations in receptors, enzymes and/or endogenous ligands and possible over- or under-production of the latter. Other genetic abnormalities may include issues with signal transduction machinery within key cells of critical tissues in the front (e.g. trabecular meshwork [TM] and Schlemm's canal [SC]) and back of the eye (e.g. retinal ganglion cells and their axons). Genome-wide associated studies (GWAS) coupled with next generation sequencing have provided powerful linkage of certain gene defects and polymorphic variants to the onset and progression of diseases of the tissues involved in fluid dynamics in the TM and SC, and many retinal elements (lamina cribosa, optic nerve head) at the back of the eye which cause ocular hypertension (OHT) and glaucomatous optic neuropathy (GON), respectively. Despite the availability of some drugs, fluid drainage microshunts and full surgical techniques to lower and control intraocular pressure, the major modifiable biomarker of open-angle and other forms of glaucoma, their side-effect profiles, less than optimum effectiveness and short duration of action present opportunities to clinically manage the glaucomas with next generation of treatments with high therapeutic indices, including gene therapies. Thus, identification, characterization and deployment of genetic data coupled with traditional drug discovery and novel gene replacement, gene editing and genetic engineering technologies may provide some solutions to the aforementioned problems. These aspects will be discussed in this article.

Ocular Treatments Targeting Separate Prostaglandin Receptors in Mice Exhibit Alterations in Intraocular Pressure and Optic Nerve Lipidome.

Background: Prostaglandin (PG) receptor agonists are the first-line eyedrop medication treatment for glaucoma. The pathophysiology of this disease is not completely known, and elevated intraocular pressure (IOP) is the key risk factor. The membranes of the axons (of the retinal ganglion cells) passing through the optic nerve (ON) head experience significant damage. Lipids are an essential component of the cell's membranes, and their profile changes owing to neurodegeneration. In this investigation, three agonists for distinct PG receptors were used to lower IOP and to determine their effect on the ON lipids. We utilized DBA/2J mice as a model of progressive IOP increase and C57BL/6J mice as a model of ON crush. Methods: DBA/2J and C57BL/6J mice were treated daily for 2 weeks with Latanoprost, PF-04217329, or Rivenprost. The IOP was measured every 2 days and pattern electroretinogram was conducted for DBA/2J throughout the study. Lipidomics of ONs were performed for each model and treatment group. Results: Of the tested compounds, Latanoprost and Rivenprost were the most effective agents decreasing IOP in DBA/2J mice. Triglyceride levels increased in the ONs of DBA/2J mouse model, but phosphatidylethanolamine levels underwent highest level changes in the C57BL/6J mouse model when treated with Latanoprost. Conclusions: Topical ocular FP- and EP4-receptor agonists appreciably lowered IOP in the DBA/2J mice representing pigmentary glaucoma. The observed changes in ON lipidomics in the different models of neurodegeneration suggest possible use of such measures in the development of more effective medicines for both IOP reduction and ON protection.

MicroRNA profile of extracellular vesicles released by Müller glial cells.

Introduction: As with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells. Methods: We conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells. Results: Sequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway. Discussion: The results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.

Degeneration of retina-brain components and connections in glaucoma: Disease causation and treatment options for eyesight preservation.

Eyesight is the most important of our sensory systems for optimal daily activities and overall survival. Patients who experience visual impairment due to elevated intraocular pressure (IOP) are often those afflicted with primary open-angle glaucoma (POAG) which slowly robs them of their vision unless treatment is administered soon after diagnosis. The hallmark features of POAG and other forms of glaucoma are damaged optic nerve, retinal ganglion cell (RGC) loss and atrophied RGC axons connecting to various brain regions associated with receipt of visual input from the eyes and eventual decoding and perception of images in the visual cortex. Even though increased IOP is the major risk factor for POAG, the disease is caused by many injurious chemicals and events that progress slowly within all components of the eye-brain visual axis. Lowering of IOP mitigates the damage to some extent with existing drugs, surgical and device implantation therapeutic interventions. However, since multifactorial degenerative processes occur during aging and with glaucomatous optic neuropathy, different forms of neuroprotective, nutraceutical and electroceutical regenerative and revitalizing agents and processes are being considered to combat these eye-brain disorders. These aspects form the basis of this short review article.

PAF-induced inflammatory and immuno-allergic ophthalmic diseases and their mitigation with PAF receptor antagonists: Cell and nuclear effects.

Ocular allergies are becoming more prevalent as more airborne pollutants, irritants and microbes pervade our environment. Inflammatory and allergic mediators released by dendritic and mast cells within the conjunctiva cause allergic conjunctivitis (AC), a prevalent ocular surface disorder that affects >40% of the world's human population on a seasonal or perennial basis. Even though histamine is a major culprit, platelet-activating factor (PAF) also contributes to AC, acting either directly or synergistically with histamine and other mediators. PAF receptor-meditated inflammatory reactions, via cell-membrane-bound and nuclear-membrane-bound and nuclear PAF receptors, are also implicated in the etiology of other eye diseases such as uveitis, diabetic retinopathy, corneal and choroidal neovascularization, and age-related macular degeneration which cause serious visual impairment and can lead to blindness. This review highlights the various deleterious elements implicated in the pathological aspects of ocular allergic reactions and inflammation and provides concepts and treatment options to mitigate these eye disorders with a special focus on PAF and PAF receptor antagonists.

Neuroprotection in neurodegenerations of the brain and eye: Lessons from the past and directions for the future.

Background: Neurological and ophthalmological neurodegenerative diseases in large part share underlying biology and pathophysiology. Despite extensive preclinical research on neuroprotection that in many cases bridges and unifies both fields, only a handful of neuroprotective therapies have succeeded clinically in either. Main body: Understanding the commonalities among brain and neuroretinal neurodegenerations can help develop innovative ways to improve translational success in neuroprotection research and emerging therapies. To do this, analysis of why translational research in neuroprotection fails necessitates addressing roadblocks at basic research and clinical trial levels. These include optimizing translational approaches with respect to biomarkers, therapeutic targets, treatments, animal models, and regulatory pathways. Conclusion: The common features of neurological and ophthalmological neurodegenerations are useful for outlining a path forward that should increase the likelihood of translational success in neuroprotective therapies.