Phase 1B study of the safety and tolerability of the mineralocorticoid fludrocortisone acetate in patients with geographical atrophy.

OBJECTIVE: To evaluate the safety and tolerability of a mineralocorticoid, in a single-dose intravitreal (IVT) injection of 1 mg/0.1 mL and 2 mg/0.1 mL fludrocortisone acetate (FCA) in subjects with geographical atrophy (GA) secondary to age-related macular degeneration. METHODS AND ANALYSIS: This phase 1b study was a two-part dose-escalation prospective study. Part 1 involved a single participant treated with 1 mg/0.1 mL and monitored up to 28 days before being reviewed by a safety review committee. Two subsequent participants were then dosed with the same dose. Part 2 involved a single participant dosed with 2 mg/0.1 mL and monitored up to 28 days when a further five participants were dosed. All participants were followed up for 6 months after baseline.A full ophthalmic assessment was performed at study visits which included GA area, best-corrected visual acuity (BCVA), low-luminance BCVA (LL-BCVA) and intraocular pressure (IOP). Adverse events (AEs) were reported from the first dose of FCA until the end-of-study visit. RESULTS: There were no serious AEs (ocular or systemic) observed with IVT FCA at either 1 mg/0.1 mL or 2 mg/0.1 mL among nine participants. There was no evidence of increased IOP or cataract development.Neither BCVA or LL-BCVA changed significantly in the study-eye over the follow-up period (p=0.28 and 0.38, respectively). Mean GA area increased in the study (0.5 mm2, p=0.003) and fellow-eyes (0.62 mm2, p=0.02) over 6 months. Differences between eyes were not significant (p=0.64), and at the lower end of population norms. CONCLUSION: IVT FCA is clinically safe and well tolerated and did not increase IOP.

Anti-inflammatory and neuroprotective properties of the corticosteroid fludrocortisone in retinal degeneration.

The pathogenesis of outer retinal degenerations has been linked to the elevation of cytokines that orchestrate pro-inflammatory responses within the retinal milieu, and which are thought to play a role in diseases such as geographic atrophy (GA), an advanced form of AMD. Here we sought investigate the anti-inflammatory and mechanistic properties of fludrocortisone (FA), as well as triamcinolone acetonide (TA), on Müller cell-mediated cytokine expression in response to inflammatory challenge. In addition, we investigated the neuroprotective efficacy of FA and TA in a photo-oxidative damage (PD), a model of outer retinal degeneration. Expression of CCL2, IL-6, and IL-8 with respect to FA and TA were assessed in Müller cells in vitro, following simulation with IL-1ß or TNF-α. The dependency of this effect on mineralocorticoid and glucocorticoid signaling was also interrogated for both TA and TA via co-incubation with steroid receptor antagonists. For the PD model, C57BL/6 mice were intravitreally injected with FA or TA, and changes in retinal pathology were assessed via electroretinogram (ERG) and optical coherence tomography (OCT). FA and TA were found to dramatically reduce the expression of CCL2, IL-6, and IL-8 in Müller glia in vitro after inflammatory challenge with IL-1ß or TNF-α (P < 0.05). Though FA acts as both a mineralocorticoid and glucocorticoid receptor agonist, co-incubation with selective steroid antagonists revealed that the suppressive effect of FA on CCL2, IL-6, and IL-8 expression is mediated by glucocorticoid signaling (P < 0.05). In PD, intravitreal FA was found to ameliorate outer-retinal atrophy as measured by ERG and OCT (P < 0.05), while TA had no significant effect (P > 0.05). Our data indicate potent anti-inflammatory and mechanistic properties of corticosteroids, specifically FA, in suppressing inflammation and neurodegeneration degeneration associated with outer retinal atrophy. Taken together, our findings indicate that corticosteroids such as FA may have value as a potential therapeutic for outer retinal degenerations where such pro-inflammatory factors are implicated, including AMD.

Cone degeneration in aging and age-related macular degeneration.

OBJECTIVE: To examine the morphological features of macular photoreceptors in histologically normal retina from normal donor eyes and eyes with age-related macular degeneration (AMD). METHODS: The macular region was excised from 18 donor eyes (aged 22-96 years) and cryosectioned. Sections were stained with hematoxylin-eosin or double immunolabeled using opsin antibodies or synaptic markers. RESULTS: Three of 8 retinas studied in detail had AMD lesions; the remainder were histologically normal. Immunoreactivity to cone opsin was abnormal in parts of all retinas (3.5%-95.0% of each sample) and was associated with swelling of and altered immunoreactivity in the cone distal axon. In non-AMD retinas, the anomalies were mainly in nonfoveal macular locations. The nature of the anomalies was identical in non-AMD retinas and in parts of AMD retinas adjacent to overt degeneration. CONCLUSION: Redistribution of opsin and anomalies in the distal cone axon are common in the aging human macula and may indicate susceptibility to AMD. CLINICAL RELEVANCE: The findings are consistent with tests of cone function in aging and early AMD, which suggests that integrated cone functions--including contrast sensitivity, color matching, and short wavelength-sensitive cone sensitivity--are the most reliable prognostic indicators of progression in AMD.

Anatomy and development of the macula: specialisation and the vulnerability to macular degeneration.

The central retina in primates is adapted for high acuity vision. The most significant adaptations to neural retina in this respect are: 1. The very high density of cone photoreceptors on the visual axis; 2. The dominance of Midget pathways arising from these cones and 3. The diminishment of retinal blood supply in the macula, and its absence on the visual axis. Restricted blood supply to the part of the retina that has the highest density of neural elements is paradoxical. Inhibition of vascular growth and proliferation is evident during foetal life and results in metabolic stress in ganglion cells and Muller cells, which is resolved during formation of the foveal depression. In this review we argue that at the macula stressed retinal neurons adapt during development to a limited blood supply from the choriocapillaris, which supplies little in excess of metabolic demand of the neural retina under normal conditions. We argue also that while adaptation of the choriocapillaris underlying the foveal region may initially augment the local supply of oxygen and nutrients by diffusion, in the long term these adaptations make the region more vulnerable to age-related changes, including the accumulation of insoluble material in Bruch's membrane and beneath the retinal pigment epithelium. These changes eventually impact on delivery of oxygen and nutrients to the RPE and outer neural retina because of reduced flow in the choriocapillaris and the increasing barriers to effective diffusion. Both the inflammatory response and the sequelae of oxidative stress are predictable outcomes in this scenario.

Safety of an intravitreal injection of triamcinolone: results from a randomized clinical trial.

OBJECTIVE: To determine the safety of a single intravitreal injection of triamcinolone acetonide (4 mg) in patients with subfoveal choroidal neovascularization caused by age-related macular degeneration. METHODS: A double-masked, placebo-controlled, randomized clinical trial was conducted at a public tertiary referral eye hospital. Patients participating had age-related macular degeneration with evidence of choroidal neovascularization, any part of which was classic; age older than 59 years; and best-corrected visual acuity of 20/200 or better. Eyes were assigned to active study treatment or to placebo. Intraocular pressure and cataract grading were performed every 6 months for 3 years. Adverse events, from mild to vision-threatening or life-threatening, were recorded as procedure-related or corticosteroid-related. RESULTS: Seventy-five eyes were assigned to study treatment and 76 eyes to placebo. There were no moderate or severe adverse events related to the surgical procedure in either group. Triamcinolone-treated eyes had a significantly increased risk of developing mild or moderate elevation of the intraocular pressure. Topical glaucoma medication reduced intraocular pressure to acceptable levels in all patients. There was significant progression of cataract in the triamcinolone-treated eyes. CONCLUSION: Despite a significant adverse event profile, intravitreal triamcinolone is generally well tolerated by the human eye as long as patients are carefully followed up by their surgeon and treated appropriately, when necessary.

A randomized clinical trial of a single dose of intravitreal triamcinolone acetonide for neovascular age-related macular degeneration: one-year results.

OBJECTIVE: To determine if a single intravitreal injection of 4 mg of triamcinolone acetonide in patients with classic choroidal neovascularization associated with age-related macular degeneration can safely reduce the risk of severe visual loss. METHODS: A double-masked, placebo-controlled, randomized clinical trial was performed in patients 60 years or older who had choroidal neovascularization with any classic component, a duration of symptoms of less than 1 year, and a visual acuity of 20/200 or better. Best-corrected visual acuity, intraocular pressure, and cataract grading were performed before the injection and then at 3, 6, and 12 months. MAIN OUTCOME MEASURE: The development of severe loss of vision (30 letters) by survival analysis on an intention-to-treat basis. RESULTS: One hundred fifty-one eyes were randomized into the study, and follow-up data were obtained for 73 (97%) of the 75 eyes in the treated group and for 70 (92%) of the 76 eyes in the control group. There was no difference between the 2 groups for the development of severe visual loss during the first year of the study (log-rank chi 2(1) = 0.03, P =.90). In both groups, the 12-month risk of severe visual loss was 35%, with a hazard ratio of 1.05 (95% confidence interval, 0.59-1.86). The change in size of the neovascular membranes, however, was significantly less in eyes receiving triamcinolone than in those receiving placebo 3 months after treatment (P =.01), although no difference was noted after 12 months. After 12 months, treated eyes had a significantly higher risk of an elevated intraocular pressure (31/75 [41%] vs 3/76 [4%]; P<.001), but not of cataract progression (P =.29). CONCLUSIONS: A single dose of intravitreal triamcinolone had no effect on the risk of loss of visual acuity during the first year of the study in eyes with age-related macular degeneration and classic choroidal neovascularization, despite a significant antiangiogenic effect found 3 months after treatment. This biological effect warrants further study.

Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD.

PURPOSE: To demonstrate structural and immunocytochemical changes associated with light-induced degeneration in albino rat retinas and human AMD retinas. METHODS: Retinas from Wistar rats aged 3, 6, or 10 months were examined by immunocytochemistry, with antibodies to neuronal and glial markers. Results were compared with human nonneovascular AMD retinas. RESULTS: In aging rat retinas, many photoreceptors were lost in response to normal ambient light exposure. Photoreceptor loss was preceded by loss of RPE cells. Müller cells extended processes through gaps in Bruch's membrane, into the choroid. immunolabeling for gamma-aminobutyric acid (GABA), the glycine transporter Glyt-1, and the rod bipolar cell marker PKC revealed the presence of numerous neuronal somata and processes that appeared to have migrated into the choroidal region. Processes of presumptive ganglion cells remodeled and stratified in the choroid, where strong labeling for synaptic vesicle antigens was present. Myelination of retinal ganglion cell axons was also observed, especially in the peripheral retina. In AMD retinas, glial rearrangement and displacement of neurons suggestive of their migration were also observed. CONCLUSIONS: In response to loss of RPE and photoreceptor cells, adult retinal neurons migrate out of the retina along remodeled processes of Müller cells. The presence of synaptic vesicle antigens suggests the formation of new synapses between migrating neurons. The myelination is probably due to the ingress of Schwann cells from the sclera. The presence of some similar changes in human AMD retinas suggests that these findings are of broad significance for determining the likely events in transplantation of neurons in the human retina and elsewhere.

Immunoglobulin superfamily expression in primary retinoblastoma and retinoblastoma cell lines.

Retinoblastoma (Rb) is the most common intraocular tumor of childhood. In this study we examined primary Rb specimens and Rb cell lines for the expression of immunoglobulin superfamily (IgSF) antigens: MHC class I and II (MHC-I and MHC-II), neural cell adhesion molecule (NCAM), intercellular adhesion molecule-1 (ICAM-1), and Thy-1, which play an important role in immune system and tumor cell interactions. MHC-I and-II, ICAM-1 (CD54), NCAM (CD56), and Thy-1 (CDw90) immunoreactivity was studied in eight primary Rb biopsy specimens using immunohistochemistry, three using immunoelectron microscopy, and six Rb cell lines using flow cytometry (FCM). Parenchymal and vascular-associated cells, phenotypically similar to retinal microglia, strongly expressed MHC-II immunoreactivity and were distributed throughout primary Rb specimens. However, MHC-II expression on Rb cell lines was similar to nonspecific control levels. Tumor cells in primary Rb specimens displayed high NCAM, moderate Thy-1, and low MHC-I and ICAM-1 immunolabeling. Tumor vasculature expressed low to moderate MHC-I and ICAM-1 immunoreactivity and moderate Thy-1 immunoreactivity. NCAM was not detected on the vasculature of primary Rb specimens. Rb cell lines displayed variable expression of Thy-1, ICAM-1, and MHC-I. NCAM was highly expressed on five of six Rb cell lines. The high levels of constitutive NCAM immunoreactivity on Rb tumor cells confirm the neuroectodermal origins of this tumor. Additionally, the variable expression of Thy-1 may suggest separate neural lineages or differences in the maturational status ofsome Rb tumors. The presence of a population of infiltrating MHC-II-positive cells in primary Rb tumors has implications for immunomodulation of Rb growth.

Modulation of permeability and adhesion molecule expression by human choroidal endothelial cells.

PURPOSE: The therapeutic potential of TA, an anti-inflammatory glucocorticoid, for the treatment of exudative retinopathy has been examined in several independent clinical studies. The modulation of permeability and adhesion molecule expression of an epithelial cell line has been described in vitro, with the use of cytokines and triamcinolone acetonide (TA). In the current study, the influence of proinflammatory cytokines and TA on permeability and adhesion molecule expression in human choroidal endothelial cells (CECs) was investigated. METHODS: Human CEC isolates treated with IFNgamma, TNFalpha, and TA were evaluated by flow cytometry and immunocytochemistry for expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and major histocompatibility complex (MHC)-I and -II. The effects of IFNgamma, TNFalpha, and TA on paracellular permeability of CEC monolayers were assessed in transendothelial cell resistance (TER) assays. RESULTS: Both IFNgamma and TNFalpha significantly upregulated expression of ICAM1 and MHC-I on CECs. Expression of VCAM1 was induced after stimulation with both IFNgamma and TNFalpha, whereas expression of MHC-II was induced only by stimulation with IFNgamma. Cytokine-induced expression of ICAM1, MHC-I, and MHC-II antigen by CECs was significantly downregulated by TA. IFNgamma stimulation also increased permeability of CEC monolayers, whereas subsequent TA treatment decreased permeability of CEC monolayers. CONCLUSIONS: Human CEC isolates provide a useful in vitro model to study choroidal neovascular membrane characteristics and their potential response to pro- and anti-inflammatory agents. In addition, the results indicate that TA has the capacity to reduce adhesion molecule expression and permeability of choroidal vessels in vitro, confirming its potential as a therapeutic agent for treatment of exudative macular degeneration.

Effects of post-mortem delay and storage duration on the expression of GFAP in normal human adult retinae.

Glial fibrillary acidic protein (GFAP) is an established marker of retinal glia and has been shown to be modulated by several cytokines and retinal pathology. The influence of a number of factors, including post-mortem delay, storage duration and retinal pathology, on the distribution and morphology of macroglia and GFAP antigenicity was examined in human retina. The effects of these parameters on GFAP expression were estimated using immunohistochemistry, confocal microscopy and image analysis. Changes in expression of antigenicity were analysed in human retinal cryosections at three levels: constitutive,aberrant and total. The results indicated that short-term and long-term storage duration had no significant effect on GFAP immunoreactivity at all three levels of expression (P > 0.2).However, a significant increase in GFAP immunoreactivity and distribution at all three levels of expression was associated with prolonged post-mortem delay (> 30 h) (P < 0.05). This study highlights the importance of rigorous matching of post-mortem delay between control specimens in histological studies of human retinae. The study further demonstrates the utility of Eye Bank retinae fixed and stored in 2% paraformaldehyde, provided that appropriate controls are applied.