PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Comprehensive Ocular and Systemic Safety Evaluation of Polysialic Acid-Decorated Immune Modulating Therapeutic Nanoparticles (PolySia-NPs) to Support Entry into First-in-Human Clinical Trials.

An inflammation-resolving polysialic acid-decorated PLGA nanoparticle (PolySia-NP) has been developed to treat geographic atrophy/age-related macular degeneration and other conditions caused by macrophage and complement over-activation. While PolySia-NPs have demonstrated pre-clinical efficacy, this study evaluated its systemic and intraocular safety. PolySia-NPs were evaluated in vitro for mutagenic activity using Salmonella strains and E. coli, with and without metabolic activation; cytotoxicity was evaluated based on its interference with normal mitosis. PolySia-NPs were administered intravenously in CD-1 mice and Sprague Dawley rats and assessed for survival and toxicity. Intravitreal (IVT) administration in Dutch Belted rabbits and non-human primates was assessed for ocular or systemic toxicity. In vitro results indicate that PolySia-NPs did not induce mutagenicity or cytotoxicity. Intravenous administration did not show clastogenic activity, effects on survival, or toxicity. A single intravitreal (IVT) injection and two elevated repeat IVT doses of PolySia-NPs separated by 7 days in rabbits showed no signs of systemic or ocular toxicity. A single IVT inoculation of PolySia-NPs in non-human primates demonstrated no adverse clinical or ophthalmological effects. The demonstration of systemic and ocular safety of PolySia-NPs supports its advancement into human clinical trials as a promising therapeutic approach for systemic and retinal degenerative diseases caused by chronic immune activation.

Sialic Acid Mimetic Microglial Sialic Acid-Binding Immunoglobulin-like Lectin Agonism: Potential to Restore Retinal Homeostasis and Regain Visual Function in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), a leading cause of visual loss and dysfunction worldwide, is a disease initiated by genetic polymorphisms that impair the negative regulation of complement. Proteomic investigation points to altered glycosylation and loss of Siglec-mediated glyco-immune checkpoint parainflammatory and inflammatory homeostasis as the main determinant for the vision impairing complications of macular degeneration. The effect of altered glycosylation on microglial maintained retinal para-inflammatory homeostasis and eventual recruitment and polarization of peripheral blood monocyte-derived macrophages (PBMDMs) into the retina can explain the phenotypic variability seen in this clinically heterogenous disease. Restoring glyco-immune checkpoint control with a sialic acid mimetic agonist targeting microglial/macrophage Siglecs to regain retinal para-inflammatory and inflammatory homeostasis is a promising therapeutic that could halt the progression of and improve visual function in all stages of macular degeneration.

Investigational drugs in clinical trials for macular degeneration.

INTRODUCTION: Intravitreal anti-vascular endothelial growth factor (VEGF) injections for exudative age-related macular degeneration (eAMD) are effective and safe but require frequent injections and have nonresponding patients. Geographic atrophy/dry AMD (gaAMD) remains an unmet medical need. New therapies are needed to address this leading cause of blindness in the increasing aged population. AREAS COVERED: This paper reviews the pathogenesis of macular degeneration, current and failed therapeutics, therapies undergoing clinical trials and a rationale for why certain AMD therapies may succeed or fail. EXPERT OPINION: VEGF-inhibitors reduce both vascular leakage and neovascularization. Experimental therapies that only address neovascularization or leakage will unlikely supplant anti-VEGF therapies. The most promising future therapies for eAMD, are those that target, more potently inhibit and have a more sustained effect on the VEGF pathway such as KSI-301, RGX-314, CLS-AX, EYEP-1901, OTX-TKI. GaAMD is a phenotype of phagocytic retinal cell loss. Inhibiting phagocytic activity of retinal microglial/macrophages at the border of geographic atrophy and reducing complement derived activators of microglial/macrophage is the most promising strategy. Complement inhibitors (Pegcetacoplan and Avacincaptad pegol) will likely obtain FDA approval but will serve to pave the way for combined complement and direct phagocytic inhibitors such as AVD-104.

SCORE Study report #11: incidences of neovascular events in eyes with retinal vein occlusion.

PURPOSE: To investigate in The Standard Care versus COrticosteroid for REtinal Vein Occlusion (SCORE) Study: (1) incidences of neovascular events and retinal capillary nonperfusion (abbreviated as "nonperfusion"), and their relationship with treatment groups; (2) neovascular incidences by nonperfusion status; and (3) pertinent baseline factors for their potential risk for neovascular events. DESIGN: Two multicenter, randomized clinical trials, 1 evaluating participants with central retinal vein occlusion (CRVO) and the other evaluating participants with branch retinal vein occlusion (BRVO). PARTICIPANTS: At 36 months, data were available for 81 participants with CRVO and 128 with BRVO. INTERVENTION: Standard care (observation or grid photocoagulation) versus 1 or 4 mg intravitreal triamcinolone. MAIN OUTCOME MEASURES: Neovascularization of the iris (NVI), neovascular glaucoma (NVG), disc or retinal neovascularization (NVD/NVE), preretinal or vitreous hemorrhage (PRH/VH), and nonperfusion. RESULTS: The cumulative 36-month incidences for CRVO and BRVO eyes, respectively, were 8.5% and 2.4% for NVI or NVG; 8.8% and 7.6% for NVD/NVE or PRH/VH. There were no differences in incidences of neovascular events or risk of nonperfusion when comparing the 3 treatment groups within diseases. For CRVO at 36 months, 16.6% of eyes with ≥5.5 disc areas of nonperfusion versus 4.0% of eyes with <5.5 disc areas of nonperfusion developed NVG (P = 0.0003); for BRVO at 36 months, 14.6% versus 2.4% developed NVD/NVE (P<0.0001). Similar results were noted for most other neovascular events. Nonperfusion was the only significant baseline factor for neovascularization in BRVO, with the risk of a neovascular event increasing with greater disc areas of nonperfusion, and the highest risk noted at ≥5.5 disc areas. CONCLUSIONS: In the SCORE Study, triamcinolone treatment was not associated with lower incidences of neovascular events or nonperfusion status compared with observation or grid photocoagulation. Cumulative 36-month incidences for most neovascular events were significantly higher for nonperfused than perfused eyes. Greater baseline disc areas of nonperfusion increased the risk of neovascularization in BRVO but not CRVO eyes, possibly owing to obscuration of retinal capillary details caused by dense hemorrhage at baseline for CRVO eyes. Increased risk of neovascularization was noted below the historical threshold of 10 disc areas of nonperfusion for retinal vein occlusion.

Verteporfin photodynamic therapy combined with intravitreal bevacizumab for neovascular age-related macular degeneration.

PURPOSE: To assess outcomes for patients with choroidal neovascularization (CNV) due to age-related macular degeneration (AMD) treated with verteporfin photodynamic therapy (PDT) and bevacizumab. DESIGN: Retrospective, case series database study (registry). PARTICIPANTS: We included 1196 patients with CNV due to AMD who received > or =1 combination treatment of 1.25 mg intravitreal bevacizumab within 14 days of verteporfin PDT. METHODS: Retrospective analysis of baseline data with ongoing follow-up. Physicians from 45 clinical centers entered patient data at baseline and follow-up examinations, including subsequent treatments, into a secure, Web-accessed database. Snellen visual acuity (VA) was converted to logarithm of the minimum angle of resolution (logMAR) for statistical analyses. MAIN OUTCOME MEASURES: Change from baseline in VA and retreatment rates of any therapy after the initial combination treatment. RESULTS: Of 1196 patients, 1073 patients had > or =6 months of follow-up. For these 1073 patients, mean baseline VA was 0.967 logMAR (approximate Snellen 20/185) and 56.3% of patients (604/1073) were treatment naïve. After their baseline combination treatment, patients received a mean of 0.6 additional verteporfin PDT retreatments and 2.0 bevacizumab retreatments over a mean follow-up period of 15.0 months. By 12 months, 82% of patients (578/701) had stable or improved vision (loss of <3 lines or a gain in VA), 36% (255/701) improved by > or =3 lines, and 17% (121/701) improved by > or =6 lines. By 12 months, patients gained approximately 1.2 lines (6 letters) of VA from baseline. Patients who were treatment naïve gained significantly more VA by month 12 (+8.4 letters) compared with those who had been previously treated (+2.4 letters; P<0.01). Most serious adverse events (26/30) were judged by investigators as not related to any study treatment, although 3 ocular events were judged related to bevacizumab alone, and 1 ocular event was judged related to both bevacizumab and PDT. CONCLUSIONS: Combination therapy with PDT and bevacizumab led to vision benefit for most patients, particularly those who were treatment naïve at baseline. The number of retreatments was lower than published reports with either treatment delivered as monotherapy. Randomized clinical trials are underway to confirm these findings.

Intravitreal bevacizumab for macular edema from idiopathic juxtafoveal retinal telangiectasis.

To assess the potential visual benefit of intravitreal bevacizumab in a patient with idiopathic juxtafoveal retinal telangiectasis refractory to focal laser treatment, an intravitreal injection of bevacizumab (1.25 mg) was given. Within 1 week, visual acuity improved from 20/50 to 20/25 and optical coherence tomography demonstrated complete resolution of macular edema. There was no adverse effect. The macular edema recurred after 3 months, requiring a repeat injection of bevacizumab with subsequent resolution of macular edema. An intravitreal injection of bevacizumab may provide potential short-term visual benefit in patients with macular edema from idiopathic juxtafoveal retinal telangiectasis.

Retinal temperature increase during transpupillary thermotherapy: effects of pigmentation, subretinal blood, and choroidal blood flow.

PURPOSE: To study the risk of adverse events in transpupillary thermotherapy (TTT) for age-related macular degeneration by measuring how laser-induced retinal temperature increase is affected experimentally by subretinal blood, choroidal blood flow, and chorioretinal pigmentation. METHODS: An ultrafine thermocouple technique was developed to measure retinal temperature increase during TTT in albino and pigmented rabbit eyes. TTT was performed with 60-second, 0.78-mm spot size, 810-nm infrared diode laser exposures with power settings ranging from 50 to 950 mW. Intraretinal and subretinal temperature increases were measured in pigmented and albino rabbits, with or without subretinal blood and choroidal blood flow. RESULTS: Threshold power settings for visible lesions in albino and pigmented rabbits were 950 and 90 mW, respectively, corresponding to retinal temperature increases of 11.8 degrees C and 5.28 degrees C, respectively. Power settings required to produce threshold lesions in albino rabbits caused retinal temperature increases in pigmented rabbits that were five times higher than in the albino rabbits. Temperature increases in albino rabbits were 1.5 times higher with subretinal blood than without it. Choroidal blood flow generally did not affect measured retinal temperature increases. CONCLUSIONS: The results confirm prior theoretical recommendations that clinicians should consider decreasing TTT power settings in darkly pigmented eyes and proceed with caution in those with subretinal hemorrhage or pigment clumping.

Systemic rapamycin inhibits retinal and choroidal neovascularization in mice.

PURPOSE: Rapamycin exhibits significant antitumor/antiangiogenic activity that is coupled with a decrease in vascular endothelial growth factor (VEGF) production and a reduction in the response of vascular endothelial cells to stimulation by VEGF. VEGF plays a significant role in neovascular pathologies of the eye, thus we tested the possibility of using rapamycin to inhibit retinal and choroidal neovascularization (CNV). METHODS: CNV was induced in adult mice with laser photocoagulation. Retinal neovascularization was induced using the retinopathy of prematurity (ROP) hyperoxia/hypoxia model. Experimental animals received intraperitoneal (ip) injections of rapamycin (2 mg/kg/day or 4 mg/kg/day) for 1-2 weeks. Controls were not treated or received ip injections of phosphate buffered saline (PBS). Eyes were analyzed histologically for evidence of CNV or retinal neovascularization. ROP eyes were further analyzed for changes in VEGF and VEGF receptor (Flt-1 and Flk-1) protein content following rapamycin treatment. RESULTS: Rapamycin significantly reduced the extent of neovascularization in both the CNV and the ROP model. Immunohistochemical staining of treated and untreated ROP retina did not reveal a significant reduction in levels of VEGF protein or its receptors. Immunostaining for Flt-1 increased, while no obvious changes in Flk-1 were observed. Quantitative analysis of total protein via enzyme linked immunosorbent assay (ELISA) confirmed an increase in Flt-1 and VEGF, following drug treatment, with no effect on Flk-1. CONCLUSIONS: These results suggest rapamycin may provide an effective new treatment for ocular neovascularization.

Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model.

PURPOSE: RNA interference mediated by small interfering RNAs (siRNAs) is a powerful technology allowing the silencing of mamalian genes with great specificity and potency. The purpose of this study was to demonstrate the feasibility of RNA interference mediated by siRNA in retinal cells in vitro and in the murine retina in vivo. METHODS: siRNAs specific for enhanced green fluorescent protein (EGFP) and murine and human vascular endothelial growth factor (VEGF) were designed. In vitro studies in human cell lines entailed modulation of endogenous VEGF levels through chemically induced hypoxia. Effects of siRNA treatment on these levels were measured by ELISA. In vivo studies evaluating effects of siRNA on levels of EGFP and VEGF were performed by co-injecting recombinant viruses carrying EGFP or hVEGF cDNAs along with the appropriate siRNAs subretinally in mice. Additional studies aimed at blocking production of endogenous mVEGF were performed using laser-induced choroidal neovascularization (CNV) in mice. Effects of in vivo treatments were evaluated ophthalmoscopically. Retinal/choroidal flat mounts were evaluated after perfusion with dextran-fluorescein. Alternatively, retinas were evaluated in histological sections or VEGF levels were measured in intact eyes using ELISA. RESULTS: Successful delivery of siRNA to the subretinal space was confirmed by observing significantly reduced levels of EGFP in eyes treated with Ad.CMV.EGFP plus EGFP-directed siRNA. siRNAs directed against hVEGF effectively and specifically inhibit hypoxia-induced VEGF levels in human cell lines and after adenoviral induced hVEGF transgene expression in vivo. In addition, subretinal delivery of siRNA directed against murine Vegf significantly inhibited CNV after laser photocoagulation. CONCLUSIONS: Delivery of siRNA can be used in vitro and in vivo to target specific RNAs and to reduce the levels of the specific protein product in the targeted cells. This work suggests that RNA interference has potential for application to studies of retinal biology and for the treatment of a variety of retinal diseases, including those involving abnormal blood vessel growth.

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops. DESIGN: Experimental animal study. METHODS: Vascular endothelial growth factor 165 was injected into the eyes of normal cynomolgus monkeys at regular intervals. As a control, normal eyes were injected with phosphate buffered saline. Color photography and fluorescein angiography were performed at regular intervals. The retinas were incubated for adenosine diphosphatase (ADPase) activity to visualize retinal vessels. The retinas were flat-embedded and areas of potential preretinal neovascularization were identified en bloc and serially sectioned. RESULTS: Areas of capillary nonperfusion and vessel dilation and tortuousity were seen by angiography. In serial sections, the nonperfused areas were found to be associated with endothelial cell hyperplasia in vessel lumens. Preretinal neovascularization originating only from superficial veins and venules was observed throughout peripheral retina, but was not seen in the posterior pole. Lacunae-like veins were subdivided by the process of intussusception and endothelial cell bridging. Arterioles demonstrated endothelial cell hyperplasia and microaneurysms. CONCLUSION: Intraocular injections of VEGF were sufficient to produce preretinal neovascularization in the nonhuman primate. Most vasculopathic structures were associated with endothelial cell hyperplasia. These results demonstrate that VEGF alone can produce many features of both nonproliferative and proliferative diabetic retinopathy including the previously undescribed development of preretinal neovascularization. This well-characterized VEGF-induced primate model of retinal neovascularization may be useful as a means of testing new treatments for retinal neovascularization.

Central retinal vein occlusion immediately following cardiac surgery.

The relationship between cardiac surgery and central retinal vein occlusion (CRVO) has been previously documented; however, in all of these cases the occlusion occurred months after the cardiac surgery. This is the first report of a patient awakening from cardiac surgery with ophthalmoscopically documented CRVO. This diagnostic case report describes a patient who developed CRVO immediately following extensive cardiac surgery including aortic valve, aortic root, and proximal aortic arch replacements, as well as coronary artery bypass grafts. Ophthalmologists and cardiac surgeons alike should be aware that CRVO is a potential complication of cardiac surgery, especially when cardiopulmonary bypass is necessary.

Current molecular understanding and future treatment strategies for pathologic ocular neovascularization.

The leading cause of blindness in the developed world results from several disorders that have pathologic ocular neovascularization as the common pathway leading to vision loss. These disorders include exudative age related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), retinal vein occlusions (RVO) and ocular tumors. Because neovascularization is the common pathway to blindness in these highly prevalent conditions, the recent understanding of the cascade of angiogenesis has led to clinically available molecular therapeutics that have been proven to restore and preserve vision in patients that suffer from these blinding conditions. This article will summarize the emergence of vascular endothelial growth factor (VEGF) as a validated treatment target for and current understanding of the pathophysiology of ocular neovascularization. The article will then cover promising future strategies and therapeutic targets that are aimed at enhancing the efficacy and/or duration of effect of these clinically available anti-VEGF strategies. In particular molecules that target, VEGF, PDGF, Complement, Inflammation and Integrins that are entering or are currently in clinical trials will be reviewed.

Transmission of retinal laser wavelengths through blood.

PURPOSE: Treatment of retinal and choroidal diseases may be influenced by overlying blood. We compared the penetration through blood of various laser wavelengths used in thermal photocoagulation, photodynamic therapy, and transpupillary thermotherapy. METHODS: Laser light of wavelengths 514 nm, 568 nm, 647 nm, 689 nm, and 810 nm was directed through normal saline (control), whole blood with a hematocrit of 40%, and serial dilutions of whole blood until a steady-state reading of laser power output was obtained. Laser power output was measured with an Orion Laser Power/Energy monitor (Ophir Optronics LTD). Laser power was measured in milliwatts and expressed as a percentage of control. RESULTS: Five hundred fourteen-nanometer and 568-nm laser wavelengths penetrated the least through all dilutions of blood tested (>60% attenuation through the highest dilution tested); 647-nm, 689-nm, and 810-nm laser wavelengths penetrated most effectively through blood but were still significantly attenuated ( approximately 46%, 49.6%, and 47.0% attenuation, respectively, at the highest dilution tested). CONCLUSIONS: The presence of hemorrhage may have a significant effect on the delivery of laser energy to underlying structures/tissue. This may affect parameters used in thermal and nonthermal laser treatment of ocular diseases such as choroidal neovascularization.

Clinical applications of color Doppler imaging in the management of orbital lesions.

PURPOSE: To present the clinical applications of color Doppler imaging (CDI) as an adjunctive study in the diagnosis and treatment of orbital lesions. METHODS: Retrospective noncomparative case series. Medical records of 17 patients with orbital lesions who underwent orbital CDI were reviewed, and their orbital CDI results, diagnosis, and treatment were studied. The main outcome measures were detection of abnormal orbital vascular flow and presence or absence of blood flow within orbital lesions by CDI. RESULTS: Absence of intralesional flow was associated with an orbital lesion considered benign. Tumors in which flow was present were more frequently malignant or had malignant potential. Reversal of flow in the superior ophthalmic vein was observed in patients with carotid-cavernous fistula and orbital varix. CONCLUSIONS: CDI is a useful adjunctive imaging study for evaluating the vascularity of orbital tumors. The absence or presence of intratumoral blood flow as demonstrated by CDI can help determine the nature of the orbital mass and can assist with surgical planning.

Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization.

PURPOSE: To determine the safety and efficacy of small interfering RNA (siRNA) directed against vascular endothelial growth factor (VEGF) in a nonhuman primate model of laser-induced choroidal neovascularization (CNV). METHODS: Each animal received laser rupture of Bruch's membrane to induce CNV in both eyes. Each animal was then randomized to receive 0.05 mL of either vehicle alone or VEGF siRNA at 70 microg, 150 microg, or 350 microg in both eyes by intravitreal injection. Eyes were monitored weekly by ophthalmic examination, color photography, and fluorescein angiography for 36 days after laser injury. Electroretinograms were measured at baseline and at 5 weeks after laser. CNV on fluorescein angiograms were measured for area and graded for clinically significant leakage in a standardized, randomized, and double-masked fashion on days 15, 22, 29, and 36 after laser. RESULTS: VEGF siRNA did not cause any change in electroretinographic, hemorrhage, inflammation, or clinical signs of toxicity. A single administration of VEGF siRNA significantly inhibited growth of CNV and attenuated angiographic leakage in a dose-dependent manner. CONCLUSION: Intravitreal injection of VEGF siRNA is capable of inhibiting the growth and vascular permeability of laser-induced CNV in a nonhuman primate in a dose-dependent manner. This study demonstrates preclinical proof of a principle that supports proceeding to clinical studies of VEGF siRNA in patients with exudative age-related macular degeneration.

Risk factors for choroidal neovascularization and vision loss in the fellow eye study of CNVPT.

PURPOSE: To identify risk factors for the development of choroidal neovascularization (CNV) and vision loss in the Fellow Eye Study of the Choroidal Neovascularization Prevention Trial. METHODS: Retrospective review of 121 patients enrolled in a multicentered, randomized, controlled trial. Patients had neovascular age-related macular degeneration in one eye and more than 10 large drusen in the other eye. Records of patients randomly assigned to laser treatment or observation were reviewed through 4 years of follow-up. Three candidate risk factors for the development of CNV and vision loss were evaluated. RESULTS: Eyes with hyperfluorescent drusen on fluorescein angiography at 3 minutes appeared to have a decreased risk of CNV. Patchy choroidal filling was seen in 14% of patients. Eyes with patchy choroidal perfusion showed a higher risk of developing CNV that was not statistically significant, and the increased risk was present only in treated eyes. Reticular pseudodrusen were present in only three eyes. CONCLUSIONS: Reticular pseudodrusen were rare. Late drusen fluorescence may protect against the development of CNV.