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Neovascular age-related macular degeneration (neoAMD) is the leading cause of blindness in AMD and manifests as choroidal neovascularization (CNV). Anti-vascular endothelial growth factor (VEGF) therapies are the mainstay treatments but with limited efficacy and cause detrimental effects on the retina after long-term application. These disadvantages warrant alternative strategy. Herein, we examined the effect on CNV by intravitreal injection of bortezomib, a reversible proteasome inhibitor, and further dissected the mechanism. Krypton red Laser was used to create CNV model in mice. The angiogenesis volume was assessed in choroidal flat-mount with isolectin GS-IB4 labeling and the leakage was examined with fluorescein fundus angiography. Injection of Bor sub inhibited angiogenesis in the CNV model which was dose-dependent; the injection significantly inhibited leakage as well. Furthermore, Bor sub injection reduced the contents of VEGF-A, macrophage chemotactic factor 1 (MCP-1), and platelet-derived growth factor (PDGF)-D but not PDGF-B, examined by enzyme-linked immunosorbent assay, in choroid/retinal pigment epithelium (RPE) tissue. These injections also reduced phospho-VEGFR-2 and phospho-PDGFRβ in choroid/RPE tissue examined by immunoblotting. Moreover, Bor sub inhibited the recruitment of mural cells or macrophages to laser-injured spots. Injection of Bor sub indicated negative effect on scotopic and photopic responses recorded by electroretinogram. Altogether, intravitreal injection of Bor sub significantly reduced CNV by antagonizing VEGF-A/Flk-1 and PDGF-D/PDGFRβ pathways without impacting electroretinography parameters. Thus, Bor sub may offer an invaluable therapy for the prevention and treatment of neoAMD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Investigate the potential application of corosolic acid (CA) in the treatment of diseases causing retinal neovascularization., Methods: CA cytotoxicity effect was evaluated in ARPE-19 cells by sulforhodamine B colorimetric method, and antiangiogenic activity was studied using chorioallantoic membrane (CAM) assay. An amount of 0.01 mL of CA formulations at 5, 10 and 25 µM was injected in the right eyes of Wistar rats, and the contralateral eyes received the vehicle to verify the safety of ophthalmic use. Electroretinography (ERG) was performed before, 7 and 15 days after CA administration. Animals were killed on the 15th day, and the histological analysis of retina was carried out under light microscopy., Results: CA did not present cytotoxicity at concentrations below 35.5 μM after 48 h of treatment. The antiangiogenic activity was confirmed by CAM assay, since CA (range from 5 to 25 µM) induced a significant reduction in vascularity without any signs of toxicity. ERG recordings and histological evaluation did not show any signs of retinal toxicity., Conclusions: CA was effective in reducing vascularity in a CAM model and was found to be safe for potential ophthalmic use.

Purpose: To assess the retinal toxicity of an intravitreal injection of infliximab, a monoclonal antibody to tumor necrosis factor α, in a rabbit model., Materials and Methods: Two groups of adult albino rabbits (n = 5) received intravitreal injections of infliximab (0.1 ml) in the study eye and balanced salt solution (BSS, 0.1 ml) in the control eye at baseline. Group 1 was administered with 1.5 mg/0.1 ml, and group 2 was injected with 7.5 mg/0.1 ml of infliximab solution. Electroretinography (ERG) was performed at baseline and at 1, 7, 30, and 45 days after the injection. Visual evoked potentials (VEPs) were recorded at 7 and 45 days after the injection. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology and immunhistochemistry for glial fibrillary acidic protein (GFAP) were performed., Results: ERG responses demonstrated no significant deficit in retinal function in eyes injected with infliximab. Mean dark-adapted a-wave and b-wave maximal amplitude and semi-saturation constant values at baseline and throughout the 45 days of follow-up after the injection indicated no remarkable difference in outer retinal function between the control and experimental eyes. VEP responses were similar at each time point (7 and 45 days). No difference was seen in retinal histopathology and immunocytochemistry sections in eyes receiving the 1.5 mg/0.1 ml dose compared to the control eyes. However, increased GFAP labeling in retinal Müller cells was detected in rabbit eyes treated with the 7.5 mg/0.1 ml dose., Conclusions: Intravitreal injection of 1.5 mg/0.1 ml infliximab dose has no toxic effect on the integrity (functional or structural) of the retina in rabbits. A higher dose of 7.5 mg/0.1 ml may be slightly toxic as suggested by positive Müller cell GFAP expression. Additional studies of retinal toxicity at higher doses and after multiple injections are needed to establish the retinal safety of intravitreal infliximab therapy in humans.

Aim: To evaluate the safety of intravitreal ziv-aflibercept (Zaltrap) in the treatment choroidal neovascularization secondary to age-related macular degeneration., Methods: Eligible eyes with choroidal neovascularization secondary to age-related macular degeneration each received a single intravitreal injection of ziv-aflibercept. Comprehensive ophthalmic examinations and detailed systemic evaluations were performed at baseline and Days 1, 7, and 30 after injection, and International Society for Clinical Electrophysiology of Vision standard electroretinography was performed at baseline and Day 30. Primary outcome measures were safety parameters that included signs of clinical and electroretinographic toxicity. Secondary outcome measures included changes in best-corrected visual acuity and central subfield thickness., Results: Twelve eyes of 12 patients were treated. None of the patients complained of blurred vision, ocular pain, or bulbar injection at any of the follow-up visits, nor was intraocular inflammation noted. There were no significant differences in implicit times, "a" and "b" wave amplitudes, or b/a ratios at 1 month when compared with baseline (P = 0.4). None of the patients experienced serious ocular or systemic adverse events. Mean best-corrected visual acuity improved only slightly at 30 days (LogMAR 0.45 ± 0.31 [Snellen equivalent: 20/60]) compared with baseline (LogMAR 0.37 ± 0.24 [Snellen equivalent: 20/50]; P = 0.51)., Conclusion: Single intravitreal injections of ziv-aflibercept into eyes with neovascular age-related macular degeneration appear to be safe through 1 month. Ziv-aflibercept could become a safe, low-cost therapy for macular diseases in developing countries and in those where intravitreal aflibercept (Eylea) is not available.

Retinal diseases characterized by pathologic retinal angiogenesis are the leading causes of blindness worldwide. Although therapies directed toward vascular endothelial growth factor (VEGF) represent a significant step forward in the treatment of proliferative retinopathies, further improvements are needed. In the last few years, an intense research activity has focused around the use of herbal and traditional natural medicines as an alternative for slowing down the progression of proliferative retinopathies. In the present study, we investigated the antiangiogenic effects of acetyl-11-keto-β-boswellic acid (AKBA), one of the active principles derived from the plant Boswellia serrata, used in Ayurvedic systems of medicine. We studied the antiangiogenic properties of AKBA using the mouse model of oxygen-induced retinopathy (OIR), which mimics the neovascular response seen in human retinopathy of prematurity. We first evaluated the effects of subcutaneously administered AKBA on the expression/activity of proteins which are known to play a role in the OIR model. In the retina, AKBA increased expression and activity of Src homology region 2 domain-containing phosphatase 1 and reduced the phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) as well as VEGF expression and VEGF receptor (VEGFR)-2 phosphorylation. Likely as a result of these effects, AKBA significantly reduced retinal neovascularization in OIR mice without affecting retinal cell survival and retinal function. Using retinal explants cultured in hypoxia and an activator of STAT3 phosphorylation, we showed that the AKBA-induced inhibition of VEGFR-2 phosphorylation is likely to be mediated by a mechanism depending on an SHP-1/STAT3/VEGF axis. In the OIR model, neovascularization results from the activation of retinal endothelial cells, therefore we evaluated whether AKBA affected the angiogenic response of human retinal microvascular endothelial cells (HRMECs). We observed that AKBA reduced proliferation, migration and tube formation in HRMECs stimulated with exogenous VEGF, while it reduced migration and tube formation in untreated HRMECs. Taken together, our results demonstrate the antiangiogenic effects of AKBA in a model of pathologic neovascularization, providing a rationale for further investigation of AKBA as a promising therapeutic agent to reduce the impact of proliferative retinopathies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to evaluate the short-term efficacy and safety of repeated intravitreal bevacizumab for myopic choroidal neovascularization., Methods: We performed a prospective, nonrandomized, interventional case series study. Sixteen eyes from 16 patients with myopic choroidal neovascularization secondary to pathologic myopia had a monthly injection of intravitreal bevacizumab 1.25 mg for 3 consecutive months. Best-corrected visual acuity assessment, optical coherence tomography, fluorescein angiography and multifocal electroretinogram were performed before treatment and at 1, 2, 3, and 6 months., Results: The mean logarithm of minimum angle of resolution best-corrected visual acuity was 1.43 before treatment. At 6 months after treatment, the mean best-corrected visual acuity improved significantly (P = 0.02) to 0.7. Fluorescein angiography demonstrated absence of leakage in 14 eyes (87.5%) and persistent but decreased leakage in the remaining 2 eyes (12.5%). Optical coherence tomography showed a highly significant reduction in the foveal central thickness (P < 0.001) at all follow-up visits. The foveal and parafoveal rings showed a significant improvement in the multifocal electroretinogram responses. At the sixth month follow-up, the P1 amplitudes and implicit time P values in the foveal and parafoveal rings were <0.01 and <0.001, respectively. None of the remaining three rings showed any significant change throughout the study period., Conclusion: In 6-month follow-up, repeated injections of intravitreal bevacizumab were effective in treating myopic choroidal neovascularization. No adverse effect was detected on retinal function evaluated by multifocal electroretinogram.

Purpose: To evaluate the in vivo and in vitro toxicity of sunitinib malate, a multikinase inhibitor molecule., Design: Experimental, Prospective, Controlled., Methods: Human retinal pigment epithelial (ARPE-19) and human umbilical vein endothelialcells (HUVECS) were used in a culture toxicity test and exposed to different concentrations of sunitinib malate for 18 hours. The HUVECs also were cultured to evaluate the angiogenesis inhibitory effect of sunitinib malate. Fundus photography and angiographic, electrophysiologic, and histopathologic evaluations with light and electron microscopy were performed in two groups of five rabbits each that received different intravitreal concentrations of the drug. Each rabbit received 0.1 ml of sunitinib malate in the right eye (one group with 12.5 mg/ml, the other group with 25 mg/ml); all animals received 0.1 ml of physiologic saline solution in the left eye. After sacrifice, the eyes were enucleated and fixed with modified Karnovsky solution., Results: No toxicity related to sunitinib malate was observed using an in vitro model with the 12.5 and 25 mg/ml solutions in HUVEC and ARPE cell cultures. No toxicity was observed in the in vivo model with 12.5 mg/ml, but light microscopy showed that the 25 mg/ml solution damaged the photoreceptors layer. No functional changes in the electroretinogram were observed in any group., Conclusions: Sunitinib malate 12.5 mg/ml caused no toxicity in in vivo and in vitro models, but the 25 mg/ml concentration caused retinal changes suggesting toxicity in the in vivo model. Further research with the drug is needed in models of ocular neovascularization.

Purpose: To determine whether intravitreal vasohibin-1 will reduce the grade of the choroidal neovascularization in monkey eyes., Methods: Choroidal neovascularizations were induced in 12 monkey eyes by laser photocoagulation. Three monkeys were evaluated for the safety of the vasohibin-1 injections, 6 monkeys for the effects of a single injection, and 3 monkeys for repeated injections of vasohibin-1. Ophthalmoscopy, fluorescein angiography, focal electroretinograms, and optical coherence tomography were used for the evaluations. The level of vascular endothelial growth factor in the aqueous was determined by enzyme-linked immunosorbent assay. Immunohistochemistry was performed., Results: An intravitreal injection of 10 μg of vasohibin-1 induced mild intraocular inflammation. Eyes with an intravitreal injection of 0.1 μg and 1.0 μg of vasohibin-1 had significant less fluorescein leakage from the choroidal neovascularizations and larger amplitude focal electroretinograms than that of vehicle-injected eyes. Similar results were obtained by repeated injections of 0.1 μg of vasohibin-1. Immunohistochemistry showed that vasohibin-1 was expressed mainly in the endothelial cells within the choroidal neovascularizations. The vascular endothelial growth factor level was not significantly altered by intravitreal vasohibin-1., Conclusion: The reduction of the laser-induced choroidal neovascularizations and preservation of macular function in monkey by intravitreal vasohibin-1 suggest that it should be considered for suppressing choroidal neovascularizations in humans.

Purpose: To determine the short-term safety of intravitreal bevacizumab by multifocal electroretinography testing., Methods: Thirty-one eyes with choroidal neovascularization, proliferative diabetic retinopathy, and retinal vein occlusion received intravitreal bevacizumab (2.5 mg/0.1 mL). All patients underwent best-corrected visual acuity measurement, retinal fluorescein angiography, optical coherence tomography, and multifocal electroretinography at baseline and 1 month after the treatment., Results: Subjects undergoing multifocal electroretinography testing had no statistically significant changes in electrophysiologic responses 1 month after the intravitreal injection of bevacizumab., Conclusion: Multifocal electrophysiologic testing did not demonstrate any short-term cone photoreceptor toxicity after intravitreal bevacizumab.

Purpose: The purpose of this was to study the long-term effects of a single intravitreal dose of bevacizumab injected at different postnatal age on retinal function and development of retinal blood vessels in the adult albino rabbit., Methods: Bevacizumab was injected into the right eye of newborn rabbits, aged 11 days to 25 days, whereas the left eye of each rabbit was injected with identical volume of saline and served as control. The electroretinogram was recorded 1 week and 10 weeks after injection. Visual evoked potentials were recorded 10 weeks after injection. At termination of the follow-up period, the rabbits were killed and the retinas were prepared for histopathologic studies at the light microscopic level, for glial fibrillary acidic protein immunoreactivity, and for reduced form of nicotinamide adenine dinucleotide phosphate diaphorase histochemistry to assess the integrity of the retinal vascular system., Results: The electroretinogram responses demonstrated normal retinal function in adult rabbits injected at postnatal age of 11 days to 25 days. Mean Vmax and σ values calculated for each group of rabbits, 10 weeks after bevacizumab injection, indicated similar retinal function of the experimental and control eyes. Visual evoked potentials recorded by stimulating each eye separately were also similar. The histopathologic studies yielded similar results; no structural retinal damage was observed in the experimental eyes compared with the control eyes of rabbits from all age groups, and no increased glial fibrillary acidic protein immunoreactivity in Müller cells was observed in the experimental eyes. Staining of blood vessels with reduced form of nicotinamide adenine dinucleotide phosphate diaphorase revealed decreased branching of the capillary network in the experimental eyes compared with the control eyes in all age groups., Conclusion: The electroretinographic and morphologic data showed no deleterious effects of a single intravitreal dose of bevacizumab, injected during the first 30 days postnatally, on the structural and functional integrity of the sensory retina in the adult rabbit. Even partial blockage of vascular endothelial growth factor with bevacizumab applied during retinal development seems to interfere with the development of the retinal vascular system in the albino rabbit. However, extrapolation from rabbits to humans should be made with caution.

Purpose: To evaluate changes in the multifocal electroretinogram (mfERG) in patients with neovascular age-related macular degeneration (nAMD) undergoing ranibizumab treatment., Methods: This was an observational, longitudinal, prospective study. Treatment-naive patients with nAMD who met the inclusion and exclusion criteria underwent a course of monthly injections of ranibizumab over 3 months. At baseline and month 3, each subject was evaluated with best corrected visual acuity (BCVA), contrast sensitivity (CS), fluorescein and indocyanine green angiography, optical coherence tomography (OCT), and mfERG. Additional mfERGs were performed at weeks 1 and 4 and BCVA and OCT at weeks 4 and 8., Results: Eighteen patients were enrolled. Between baseline and week 12, median BCVA improved from 59 to 69 ETDRS letters (P = 0.001), median CS improved from 29 to 30 letters (P = 0.05), mean OCT central foveal subfield thickness (CFT) decreased from 294 to 199 μm (P = 0.005), mean P1 amplitude density of the mfERG central zone increased from 35.85 to 51.55 nV/deg(2) (P = 0.009). The mfERG response correlated positively with BCVA (F = 22; P < 0.0001) and negatively with CFT (F = 12.73; P = 0.00078)., Conclusions: Intravitreal ranibizumab therapy appears to induce an increase in mfERGs centrally in patients with nAMD at least in the short term. Longer term studies to investigate the prognostic value of mfERG responses to predict changes in visual acuity in nAMD and other diseases are warranted. (ClinicalTrials.gov number, NCT01023971.).

Aim: To evaluate the in vitro effects of bevacizumab (Avastin®) on the electroretinogram (ERG) in rats using a model of isolated perfused retina ERG recording., Methods: Retinas were isolated from rat eyes and placed in a chamber continuously perfused with a nutrient solution. The ERG was recorded every 3 min. Once the ERG b-wave amplitude was at a steady state, bevacizumab was added at concentrations of 0.25 and 0.5 mg/ml to the perfusion medium for 3 h., Results: We observed no effect on ERG amplitudes or kinetics when bevacizumab was added to the perfusion medium. In addition, we found no significant differences in the survival curves of the b-wave and PIII wave during the application of bevacizumab between bevacizumab-exposed retinas and control retinas., Conclusions: We demonstrate that bevacizumab has no in vitro toxic effects on the ERG of isolated perfused rat retina. Our study supports the retinal safety of bevacizumab with respect to retinal function., (Copyright © 2011 S. Karger AG, Basel.)

Objective: To evaluate the potential toxicity of repeated intravitreal injections of bevacizumab in rabbit eyes., Design: Randomized, placebo-controlled experimental animal study., Participants: Fourteen chinchilla rabbits; 12 assigned to the experimental group and 2 assigned to the normal control group., Methods: Three sequential, biweekly, intravitreal injections of bevacizumab in doses of 2.5 mg/0.1 mL or 5.0 mg/0.2 mL were performed on each rabbit. Evaluations included intraocular pressure (IOP), aqueous flare, B-scan ultrasound, fundus photography, ultrasound biomicroscopy, electroretinography (ERG), and visually evoked potentials (VEPs) performed at baseline and during the follow-up period. The eyes were enucleated at 1 week and 4 weeks after the last intravitreal injection, and underwent light and electron microscopic evaluations, as well as testing for apoptotic activity., Results: After intravitreal injections, no changes were found by regular clinical observation and IOP tests. There was no significant difference in the anterior chamber inflammatory activity evaluated by the laser flare meter. No evidence of retinal toxicity was seen after intravitreal bevacizumab at doses of 2.5 and 5.0 mg by either ERG or flash VEPs. Electron microscopy did show the presence of inflammatory cells and some ultrastructural changes in the photoreceptor cells in the 5.0 mg experimental group 1 week after the third injection. Mild to moderate apoptosis of photoreceptors was detected in the 5.0 mg group at the same time., Conclusions: The biweekly, multiple intravitreal injections of bevacizumab did not result in evidence of toxicity in regular clinical and functional observations at both 2.5 mg and 5.0 mg doses. The 5.0 mg dose may induce transient inflammation, ultrastructural abnormalities, and apoptosis.

Purpose: To evaluate the short-term toxic effects of pegaptanib sodium on retinal function. At present, intraocular anti-vascular endothelial growth factor (VEGF) therapy is the primary choice of treatment for neovascular maculopathy. The isoform VEGF(165) is specifically inhibited by pegaptanib sodium. Therefore, since VEGF(165) has neuroprotective effects against apoptosis of neuronal cells, blockage of VEGF(165) by pegaptanib could induce retinal dysfunction. In the present study, we used an electrophysiological technique for testing retinal toxicity in order to evaluate the short-term toxic effects of pegaptanib sodium on retinal function in a model of isolated perfused vertebrate retina., Methods: Isolated bovine retinas were perfused with an oxygenated, pre-incubated nutrient solution. Electroretinograms (ERGs) were recorded as trans-retinal potentials using Ag/AgCl electrodes. Pegaptanib sodium (0.006, 0.06, or 0.2 mg/ml) and solvent carrier were added to the nutrient solution for 45 min. ERGs were monitored before, during, and after exposure., Results: No significant reductions of b-wave (p = 0.357, p = 0.31, and p = 0.11, respectively) or a-wave (p = 0.189, p = 0.46, and p = 0.23, respectively) amplitudes were detected during application of pegaptanib (0.006, 0.06, or 0.2 mg/ml). The solvent carrier alone had no effect on ERG b- or a-waves (p = 0.98 and p = 0.42, respectively). During washout, ERG amplitudes of all test series remained unchanged., Conclusion: Results suggest that both pegaptanib sodium and its solvent carrier have good safety profiles. Intraocular application of 0.3 mg pegaptanib sodium induced no significant changes in ERGs in our ex vivo model and, thus, appears to be safe.

Purpose: To evaluate the ocular complications of intravitreal injection of high dose bevacizumab in monkey eyes., Methods: Four healthy monkeys (Macaca fuscata) received an intravitreal injection of either 6.25 or 12.5 mg bevacizumab in the right eye; the left control eye received an intravitreal injection of the same volume of saline. The eyes were examined using slit lamp and funduscopy, optical coherence tomography (OCT), electroretinography (ERG), fundus photography (FP), fluorescein angiography (FA), and indocyanine green angiography (ICGA). The eyes were enucleated 28 days after the intravitreal injection and subjected to light microscopy., Results: No pathologic changes were observed by FP, FA, ICGA, OCT, and light microscopy in the eyes injected with either of the two bevacizumab doses. ERG showed no toxic change in the eyes that received the 6.25-mg dose. In two eyes that received the 12.5-mg dose, ERG showed no significant difference between the right and left eyes 4 weeks after injection, although there were transient changes in scotopic responses., Conclusions: No irreversible toxic effects were observed in monkey eyes receiving an intravitreal injection of high dose bevacizumab.

Background: Intraocular ranibizumab (Lucentis, Novartis, Basel Switzerland) is the primary choice in the treatment of neovascular age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is known to be a survival factor for neuronal cells. Therefore, blockage of all VEGF isoforms by ranibizumab could induce retinal dysfunction., Methods: Using isolated bovine retinas, the electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes, while the retinas were perfused with an oxygen preincubated nutrient solution. For 45 min, ranibizumab was applied at a concentration of 0.2 mg/ml and alternatively the solvent carrier without the active agent. The ERG was monitored before, during and after exposure., Results: The concentration of 0.2 mg/ml ranibizumab induced a non-significant b-wave reduction of 22.32% after exposure (p = 0.13). For the a-wave amplitude only a reduction of 4% was detected (p = 0.18). The solvent carrier induced no significant reduction of the a- and b-wave amplitudes (p = 0.30 and p = 0.979, respectively)., Conclusion: In the ex vivo model, the isolated perfused vertebrate retina, ranibizumab has been proven to be a safe compound at the concentrations applied. The stability of the ERG-amplitudes rules out a considerable retinal dysfunction after an injection of up to 1 mg ranibizumab.

Purpose: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an anticancer vascular-disrupting agent, has induced transient visual symptoms in some patients. Exploratory investigations were undertaken to characterize the visual disturbances in two consecutive phase I trials., Methods: Assessments were made in 21 patients before and immediately after a 20-minute IV infusion of DMXAA, including visual acuity, funduscopy, color discrimination, pattern electroretinography (PERG), pattern visual-evoked potentials (VEP), and full-field electroretinography (ERG). Evaluation of late effects was undertaken subsequently in 12 patients before and after 6 weeks of IV DMXAA at one dose per week., Results: Frequency and intensity of transient visual disturbance increased with DMXAA dose, occurring in two thirds of patients at 3000 mg x m(-2). Symptoms included blurring, flickering, fragmentation, alteration of colors, and contrast and mild photosensitivity, starting during the infusion and resolving completely, usually within 60 minutes. Visual acuity was unchanged but color discrimination was perturbed. Dose-dependent increases in PERG P50 implicit time by up to 23 ms returned toward baseline values within 90 minutes. Prominent transient changes on ERG included prolonged scotopic rod and 30-Hz flicker implicit times and reduced 30-Hz flicker amplitude. In the second trial, no clinically significant sustained effects were detected, although an increase in bright flash a-wave implicit time (P = 0.022) was seen on whole-group analysis. In vitro studies showed nonspecific phosphodiesterase inhibition by DMXAA., Conclusions: DMXAA induced acute, transient disturbance of retinal activity consistent with phosphodiesterase inhibition. No clinically significant cumulative effects were noted and most effects occurred at doses higher than those used in ongoing clinical trials (ClinicalTrials.gov numbers, NCT00856336, NCT00863733, and NCT00003697).

Objective: To examine the sensitivity of the multifocal electroretinogram (mf-ERG) at measuring changes in retinal electrical activity in response to Avastin (bevacizumab) treatment for age-related macular degeneration (ARMD)., Methods: Nine subjects with exudative ARMD, not previously treated with bevacizumab in the investigated eye, underwent pretreatment testing with mf-ERG and intravenous fluorescein angiography (IVFA). A second mf-ERG test was conducted post-treatment. The P1 response amplitudes were examined for the hexagons corresponding to areas of pathology on the IVFA. Intertest variability was accounted for by examining areas without pathology. Aggregate responses were also generated for central and lesion-associated responses., Results: Changes in P1 response amplitude correlated with changes in visual acuity (R(2)>0.96). An improvement in Snellen visual acuity correlated with a significant improvement in P1 response amplitude from lesion associated recordings (p<0.03). Changes in P1 response amplitudes were not observed when aggregate responses were generated., Conclusion: This study represents a novel method for assessing an improvement of mf-ERG responses. This is the first study to demonstrate a statistically significant change in retinal electrical activity postbevacizumab in patients with ARMD. This study demonstrates a method for utilising mf-ERG to assess changes in retinal electrical activity and to assess the effectiveness of treatments such as bevacizumab.

Purpose: To investigate the intraocular properties and toxicity of IMS2186, a small molecule developed as an anti-choroidal neovascularization (anti-CNV) drug., Materials and Methods: Cellular toxicity and mechanism of action was tested on cell lines in vitro. Intraocular studies used rabbits for drug dissolution as well as toxicity and rats for the treatment study as well as the toxicity confirmation study. Rabbits' eyes were injected with 2.5 mg of IMS2186 and observed for 36 weeks. Laser-induced CNV in rats was treated with IMS2186, Kenalog, or phosphate-buffered saline (pBS). Fluorescein angiography (FA) and immunohistochemical processing of the globes was performed., Results: The anti-proliferative IC(50) of IMS2186 for human fibroblast cells was 1.0-3.0 microM and 0.3-3.0 microM for human cancer cells; the IC(50) of IMS2186 to inhibit endothelial tube formation was 0.1-0.3 microM. The IC(50) of IMS2186 for inhibiting the production of pro-inflammatory cytokines was 0.3-1 microM. The IC(50) of IMS2186 for inhibiting macrophage migration was 1 micrM. These biological properties were not species specific. IMS2186 can be formulated as a suspension for long-lasting release and when delivered intraocularly, no intraocular toxicity was observed by slit lamp exam, fundus exam, intraocular pressure measurements, or by electroretinography. FA showed a reduction in the leakage in eyes treated with IMS2186 and triamcinolone acetonide; DAPI staining also showed significantly less cellularity in IMS2186-treated lesions as compared to PBS (p = 0.0025)., Conclusion: IMS2186 may be a safe intraocular therapeutic agent for intraocular proliferation and angiogenesis.

Background: Bevacizumab (Avastin) has been used as off-label treatment for the specific inhibition of the vascular endothelial growth factor (VEGF). Although only intravenous administration of the drug is approved in combination therapy of colorectal carcinoma, promising short-term results have been reported about its intravitreal administration. However, VEGF is also known to exhibit neurotrophic capabilities. Therefore, blockage of all VEGF isoforms by bevacizumab could induce toxic effects. Missing randomized controlled studies and unclear long-term risks require further evaluation., Methods: Intensified monitoring of bevacizumab treatment was performed in consecutive patients. In ten patients, the functional field score was calculated after obtaining Goldmann visual fields at baseline and 1 year after injection. The other subgroup was examined by means of EOG, ERG and colour testing at baseline and 4 months following treatment. Naka-Rushton plots were calculated to enable statements about retinal function. Lanthony desaturated D15 test was used for repeated colour testing., Results: Baseline parameters already disclosed predominant cone dysfunction. Drug-related effects caused a significant improvement of visual acuity. There was no sign of clinically relevant retinal toxicity following the bevacizumab injection. No progression of visual field defects was seen within the follow-up of 1 year. Performance in EOG testing was affected by restricted fixation stability, but no parameter indicated deterioration within the 4-month-period., Conclusions: Short-term results underline that intraocular bevacizumab injection promises to be not only a cost-effective, but safe treatment option. Assessed functional parameters as error scores (e.g., Lanthony) corresponded to the impaired retinal function which was presumed to be disease-related. Further long-term results have to confirm the good tolerability in repeated treatment.

Purpose: To evaluate the preclinical safety of intravitreal bevacizumab, which is a full-length humanized monoclonal antibody against the vascular endothelial growth factor (VEGF), in rabbit eyes over a short-term period., Methods: Twenty-four rabbits were divided into two groups, each with two subgroups. The first group (groups 1 and 2) received 1.25 mg (0.05 mL) intravitreal bevacizumab, and the second group (groups 3 and 4) received 3.00 mg (0.12 mL) intravitreal bevacizumab. The right eyes were designated as the study eyes, and the left eyes served as a control and received the same volume of saline intravitreally. Groups 1 and 3 were labeled as early groups and scheduled to be terminated at 14 days. Groups 2 and 4, labeled as late groups, were scheduled to be terminated at 28 days. Besides electroretinography (ERG) and visually evoked potentials (VEP), central corneal thickness, intraocular pressure, fundus photography, and anterior segment imaging were performed at baseline and scheduled time points. Enucleated eyes were preserved for light and electron microscopic investigation., Results: No anterior segment inflammation was observed, except in one eye in group 1 which showed a uveitic reaction. No evidence of retinal toxicity was seen with intravitreal bevacizumab at doses of 1.25 and 3.00 mg, by either ERG or light microscopy. Electron microscopic assessment revealed mitochondrial damage in the inner segments of photoreceptors. Immunohistochemical staining with bax and caspase-3 and -9 showed intensive apoptotic protein expression in all study sections and minimal expression in the control eyes., Conclusions: Although electrophysiologic investigation and light microscopy showed normal retinal function and structure, mitochondrial disruption in the inner segments of photoreceptors was detected by electron microscopy, and apoptotic expression was detected after the injection of intravitreal bevacizumab.

Purpose: To evaluate by MFERG and OCT the macular function before and after intravitreal use of bevacizumab (Avastin) in eyes suffering from CNV due to ARMD., Methods: Eighteen eyes with subfoveal CNV due to ARMD were studied before and after intravitreal use of bevacizumab with MFERG and OCT. The post treatment follow up was three months., Results: Before treatment, OCT shows an increase of the retinal thickening of the fovea and the electrical response densities in the fovea and parafovea were decreased in all patients. Three months after treatment, OCT showed a real resolution of the subretinal fluid. The electrical responses in the fovea and parafovea remained the same or slightly improved in some cases. The intraocular pressure remained normal and no inflammation was observed., Conclusion: The intravitreal use of bevacizumab may provide anatomical correlates that support the concept of disease amelioration but the functional improvement of the macula three months after treatment is not obvious. However the method is promising and needs further evaluation.

Background: Bevacizumab (Avastin) is a recombinant protein that targets vascular endothelial growth factor (VEGF). In vitro, bevacizumab inhibits VEGF induced cell proliferation and tissue factor production. Abnormal angiogenesis involving VEGF is a central event during the development of choroidal neovascularisation (CNV). The present study was designed to evaluate the short term toxic effects of bevacizumab on retinal function for a therapeutic intraocular application., Methods: Isolated bovine retinas were perfused with an oxygen pre-incubated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using silver/silver chloride electrodes. Bevacizumab was added in different concentrations to the nutrient solution for 45 minutes. Thereafter the retina was reperfused for 60 minutes with normal nutrient solution. The percentage of a-wave and b-wave reduction during the application of bevacizumab was calculated and compared to control recordings., Results: During the application of three different concentrations of bevacizumab (0.08 mg/ml, 0.25 mg/ml, 0.8 mg/ml) no significant reduction of the a-wave and b-wave amplitude was observed. During the washout, the ERG amplitudes were unchanged., Conclusion: The present study suggests that an intraocular application of 0.25 mg/ml bevacizumab for the treatment of CNV is reasonable. No significant short term effects of bevacizumab on retinal function were detected, but long term effects cannot be excluded.

To determine the ocular pharmacokinetics, physiological and histological effects of prinomastat (a matrix metalloprotease inhibitor), a total of seventy-seven eyes of New Zealand White rabbits received intravitreous and subtenon injections of prinomastat or of acidified water vehicle as control, Doses of 0.5 mg in 0.05 mL of prinomastat or acidified water were used for intravitreal injection. For the subtenon injections, doses of 5 mg prinomastat in 0.5 mL of acidified water were administered in the superotemporal quadrant. Intraocular pharmacokinetics were determined by analyzing vitreous samples at different postinjection time points using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS). The toxicity was evaluated by biomicroscopy, electroretinography (ERG), pneumatonometry, and histology. No toxicity was found with either administration method. At day 14 after intravitreal injection, levels of prinomastat in the vitreous and choroid were 1.4 ng/mg and 7.8 ng/mg, respectively. The retinal levels of prinomastat were 22 ng/mg at 24 hr and dropped below 1 ng/mg at 48 hr. Prinomastat remained well above minimum effective concentration in the choroid for at least four weeks after a single intravitreal injection, suggesting that local intravitreal injection may have potential in treating choroidal neovascularization.