Sustained treatment of retinal vascular diseases with self-aggregating sunitinib microparticles.

Neovascular age-related macular degeneration and diabetic retinopathy are prevalent causes of vision loss requiring frequent intravitreous injections of VEGF-neutralizing proteins, and under-treatment is common and problematic. Here we report incorporation of sunitinib, a tyrosine kinase inhibitor that blocks VEGF receptors, into a non-inflammatory biodegradable polymer to generate sunitinib microparticles specially formulated to self-aggregate into a depot. A single intravitreous injection of sunitinib microparticles potently suppresses choroidal neovascularization in mice for six months and in another model, blocks VEGF-induced leukostasis and retinal nonperfusion, which are associated with diabetic retinopathy progression. After intravitreous injection in rabbits, sunitinib microparticles self-aggregate into a depot that remains localized and maintains therapeutic levels of sunitinib in retinal pigmented epithelium/choroid and retina for more than six months. There is no intraocular inflammation or retinal toxicity. Intravitreous injection of sunitinib microparticles provides a promising approach to achieve sustained suppression of VEGF signaling and improve outcomes in patients with retinal vascular diseases.

Safety and effectiveness of hyaluronic acid dermal filler in correction of moderate-to-severe nasolabial folds in Chinese subjects.

BACKGROUND: Modified sodium hyaluronate gel for injection, Princess® VOLUME (PV), has been on the European market since 2009 to correct deeper wrinkles and folds, increasing or restoring volume of the face, and remodeling facial contours. OBJECTIVE: To evaluate the safety and effectiveness of PV in correction of moderate-to-severe nasolabial folds (NLF) in Chinese subjects. METHODS: In this prospective, split-face, randomized, evaluator and subject-blinded, multicenter, noninferiority trial, 120 subjects were randomized to bilateral NLF treatment with PV administered in one NLF and Restylane® (RL) administered in the other NLF. NLFs were evaluated using the validated 5-point Wrinkle Severity Rating Scale with scores ranging from 1= none (no visible NLF) to 5= very severe (extremely deep and long NLF). Response was defined as ≥1 point improvement at Week 24 assessed by the blinded independent review committee (IRC) and the reduction of NLF severity, assessed by subjects and IRC based on the Global Aesthetic Improvement Scale. RESULTS: Among the 115 subjects who completed the study, median initial and touch-up volumes (mL) were 1.00 for both groups with a maximum dosage per NLF of 2.00 and a minimum of 0.30 for PV and 0.60 for RL. At week 24, the Wrinkle Severity Rating Scale improvement rate, as assessed by the IRC, reached 68.70% for PV and 52.17% for RL. The results indicate that PV is noninferior to RL (p<0.001). Most frequently reported adverse events for both devices were injection site swelling and procedural pain. The severity of the majority of the adverse events was mild. CONCLUSION: This study confirms that PV is a safe and effective treatment for the correction of moderate-to-severe NLFs in Chinese subjects.

Phenotypic characterization of P23H and S334ter rhodopsin transgenic rat models of inherited retinal degeneration.

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

A Controlled, Randomized Double-Blind Study to Evaluate the Safety and Efficacy of Chitosan-N-Acetylcysteine for the Treatment of Dry Eye Syndrome.

PURPOSE: This study was designed to evaluate the effect of chitosan-N-acetylcysteine (C-NAC) eye drops on tear film thickness (TFT) in patients with dry eye syndrome (DES). METHODS: This was a controlled, randomized, double-blind clinical investigation with patients assigned to 2 cohorts. In Cohort I, 21 patients were randomized to receive 1 instillation of C-NAC eye drops in 1 eye and placebo (normal saline solution) in the contralateral eye. In Cohort II, 17 patients were randomized to receive C-NAC eye drops once (QD) or twice (BID) daily for 5 days. TFT was assessed with a custom-built ultrahigh-resolution optical coherence tomography system. RESULTS: In Cohort I, mean TFT increased from 3.9 ± 0.5 µm predose to 4.8 ± 1.1 µm 10 min postdose after treatment with C-NAC. The increase was significantly different from placebo over time (P < 0.0001) and remained stable until 24 h postdose. In Cohort II, TFT increased with QD and BID instillation, with no significant difference between regimens. In both groups, Ocular Surface Disease Index scores improved, fewer patients presented with corneal damage, and symptoms of ocular discomfort/conjunctival redness were reduced. CONCLUSIONS: A single instillation of C-NAC significantly increased mean TFT in patients with DES as early as 10 min after instillation and lasted for 24 h. The magnitude of the increase in TFT following a single instillation was comparable with that after instillation twice daily over 5 days. Corneal damage improved in >60% of patients. C-NAC could be a viable treatment option for DES.

Expression profiling after retinal detachment and reattachment: a possible role for aquaporin-0.

PURPOSE: Retinal detachment (RD) is associated with acute visual loss caused by anatomic displacement of the photoreceptors and with chronic visual loss/disturbance caused by retinal remodeling and photoreceptor cell death, which may occur even after successful reattachment. The P2Y(2) receptor agonist INS37217 improves the rate of retinal reattachment in animal models of induced RD, and has been shown to also significantly enhance the rate of ERG recovery in a mouse model of RD. The identification of genes modulated by INS37217 may allow further drug discovery for treating RD and edema. METHODS: To identify genes involved in RD and subsequent reattachment, a retinal microarray screen was performed using a mouse model of RD in the presence or absence of INS37217. RESULTS: Ninety-two genes were identified as differentially expressed across three time points, most of which were upregulated in the presence of this agonist. Furthermore, it was shown that RD alters the expression of aquaporin-0 (AQP-0), and this modulation is prevented by treatment with INS37217. The presence of AQP-0 in retinal bipolar cells was also demonstrated, whereas it was previously thought to be specific to the lens. Mice lacking functional alleles of AQP-0 had a phototransduction deficit as assessed by electroretinography; however, their photoreceptor structure was normal, indicative of a problem with signal transmission between neurons. CONCLUSIONS: This study establishes the genes involved in RD and reattachment, and also demonstrates for the first time a physiologically significant role for AQP-0 in retinal function.

Effect of 5-MCA-NAT, a putative melatonin MT3 receptor agonist, on intraocular pressure in glaucomatous monkey eyes.

PURPOSE: 5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduced intraocular pressure (IOP) in ocular normotensive rabbit eyes. This study evaluates the effect of topical application of 5-MCA-NAT on IOP in monkey eyes with laser-induced unilateral glaucoma. METHODS: A multiple-dose study was performed in 8 glaucomatous monkey eyes. One 25-microL drop of 5-MCA-NAT (2%) was applied topically to the glaucomatous eye at 9:30 am and 3:30 pm for 5 consecutive days. IOP was measured hourly for 6 hours beginning at 9:30 am for one baseline day, one vehicle-treated day, and treatment days 1, 3, and 5 with 5-MCA-NAT. RESULTS: Compared with vehicle treatment, twice daily administration of 5-MCA-NAT for 5 days reduced (P < 0.05) IOP from 1 hour to 5 hours after the first dose, and the IOP-lowering effects were shown to last at least 18 hours following administration, based on IOP measurements made after the fourth and eighth doses. The ocular hypotensive effect of 5-MCA-NAT was enhanced with repeated dosing. The maximum reduction (P < 0.001) of IOP occurred at 3 hours after each morning dose, and was 4.0 +/- 0.5 (mean +/- SEM) mm Hg (10%) on day 1, 5.6 +/- 0.8 mm Hg (15%) on day 3, and 7.0 +/- 1.1 mm Hg (19%) on day 5. Adverse ocular or systemic side effects were not observed during the 5 days of treatment. CONCLUSIONS: 5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduces IOP in glaucomatous monkey eyes. Melatonin agonists with activity on the putative MT3 receptor may have clinical potential for treating elevated IOP.

Effect of nucleotide P2Y2 receptor agonists on outward active transport of fluorescein across normal blood-retina barrier in rabbit.

BACKGROUND: To evaluate the effect of nucleotide P2Y(2) receptor agonists INS542 and uridine 5'-triphosphate (UTP) on the outward active transport of fluorescein across rabbit blood-retina barrier (BRB) in vivo. METHODS: Injection (0.1 ml) of INS542 (0.1 or 1mM), phosphate buffered solution, or UTP (1 or 10mM) was made in Dutch-belted rabbits. Differential vitreous fluorophotometry (DVF) was performed 3hr later and the fluorescein (F)/fluorescein monoglucuronide (FG) ratio was then calculated. F/FG ratios are inversely proportional to outward active transport of F across BRB at the level of the retinal pigment epithelium (RPE). In another set of experiments, the effect of 0.1 ml vitreous injection of INS542 (1mM) on F/FG ratios was evaluated at different time points ranging from 0.5 to 48hr before conducting DVF. RESULTS: F/FG ratios obtained 3hr after intravitreal injection were as follows (mean+/-standard error): 0.49+/-0.14 (0.1mM INS542), 0.19+/-0.04 (1mM INS542), 0.48+/-0.09 (PBS), 0.40+/-0.08 (1mM UTP) and 0.36+/-0.05 (10mM UTP). The F/FG ratio for 1mM INS542 was significantly lower than in the other groups (P<0.05). In the time course experiments, a significant decrease in the F/FG ratios was observed between 1 and 12hr following administration of INS542 when compared with F/FG ratios obtained in the contralateral (untreated) eye. CONCLUSION: Intravitreal administration of INS542 (but not UTP) enhances outward active transport of F across RPE in intact rabbit eye, indicating that activation of P2Y(2) receptors in vivo directly stimulates RPE active transport.

P2Y(2) receptor agonist INS37217 enhances functional recovery after detachment caused by subretinal injection in normal and rds mice.

PURPOSE: To evaluate the effects of INS37217 on the recovery of retinal function after experimental retinal detachment induced by subretinal injection. METHODS: Subretinal injections of 1 micro L of fluorescent microbeads, saline, or INS37217 (1-200 micro M) were made by the transvitreal method in normal (C57BL/6) mice and in mice heterozygous for the retinal degeneration slow (rds) gene. Control, mock-injected animals underwent corneal puncture without injection. Histologic and ERG evaluations were made at 0 to 1 and 8 hours, and 1, 3, 7, 10, 14, and 60 days post injection (PI). DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL). RESULTS: A single subretinal injection of saline solution containing fluorescent beads caused a histologically evident retinal detachment and distributed the microbeads to almost all the subretinal space. Spontaneous reattachment occurred within 24 hours after injection and was accompanied by evident retinal folding that appeared largely resolved by 6 days later. Relative to controls, injection of saline resulted in approximately 40% recovery of dark-adapted a-wave amplitude at 24 hours PI and gradually improved to approximately 90% of controls at 2 months PI. Subretinal injection of saline containing INS37217 (10 micro M) significantly increased rod and cone ERG of normal and rds(+/-) mice at 1 and 10 days PI, when compared with injection of saline alone. Additionally, INS37217 reduced the number of TUNEL-positive photoreceptors and the enhanced rate of reattachment. CONCLUSIONS: Enhancement of ERG recovery by INS37217 is likely due to reduced retinal folding and cell death associated with detachment. These results support the use of INS37217 to help restore function after therapies that involve subretinal administration of drugs in animal models of retinal diseases.

Localization of ocular P2Y2 receptor gene expression by in situ hybridization.

The objective of this study was to determine the cellular localization of P2Y(2) receptor gene expression in rabbit and primate ocular tissues using the technique of non-isotopic in situ hybridization. Fresh frozen whole eye from a New Zealand White rabbit and whole eye and eyelid from a rhesus macaque were cut into 5 microm thick sections and mounted onto glass slides. In situ hybridization was performed on ocular cryosections using digoxigenin-labeled P2Y(2) receptor riboprobes. Alkaline phosphatase-conjugated anti-digoxigenin antibody was used to localize riboprobe hybridization, which was subsequently visualized by staining with a precipitating alkaline phosphatase substrate. Cytoplasmic staining indicative of antisense riboprobe hybridization to P2Y(2) receptor mRNA was observed in the palpebral and bulbar conjunctival epithelium, including goblet cells, the corneal epithelium, and in meibomian gland sebaceous and ductal cells. Staining was also observed in both layers of the ciliary body epithelium, subcapsular epithelium of the lens, and corneal endothelium. In the posterior eye, staining was observed in various layers of the retina, including ganglion cell, inner nuclear, inner segment and retinal pigment epithelium layers, in the optic nerve head, and in a variety of structures within the choroid. No specific staining of sense riboprobe was seen on any of the ocular structures. These results demonstrate that the P2Y(2) receptor gene is expressed in a variety of ocular cells types and suggest that P2Y(2) receptors are associated with diverse physiological functions throughout the eye.

The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.

PURPOSE: To investigate the effects of INS37217, a synthetic P2Y(2) receptor agonist, on intracellular calcium signaling, electrophysiology, and fluid transport in vitro and on experimentally induced retinal detachment in rat eyes in vivo. METHODS: Freshly isolated monolayers of bovine and human fetal RPE were mounted in Ussing chambers for measurements of cytosolic calcium levels ([Ca(2+)](i)), membrane voltages and resistances, and transepithelial fluid transport. Retinal detachments were experimentally produced in Long-Evans rats by injecting modified phosphate-buffered saline into the subretinal space (SRS). Experimental or vehicle solutions were injected into the vitreous, and the size of blebs in the SRS was scored under masked conditions. RESULTS: Addition of INS37217 to Ringer's solution bathing the apical membrane transiently increased [Ca(2+)](i), altered membrane voltages and resistances and generally produced responses that were similar in magnitude to those of uridine triphosphate (UTP). In fluid transport experiments performed with the capacitance probe technique, INS37217 significantly increased fluid absorption across freshly isolated bovine and fetal human RPE monolayers. All in vitro results were blocked by apical 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which has been shown to block P2Y(2) receptors in the RPE. Intravitreal administration of INS37217, but not UTP, in the rat model of retinal detachment enhanced the removal of SRS fluid and facilitated retinal reattachment when compared with vehicle control. CONCLUSIONS: These findings indicate that INS37217 stimulates the RPE fluid "pump" function in vitro by activating P2Y(2) receptors at the apical membrane. In vivo INS37217 enhances the rates of subretinal fluid reabsorption in experimentally induced retinal detachments in rats and may be therapeutically useful for treating a variety of retinal diseases that result in fluid accumulation in the subretinal space.

Effect of INS37217, a P2Y(2) receptor agonist, on experimental retinal detachment and electroretinogram in adult rabbits.

PURPOSE: To evaluate the effects of subretinal and intravitreal delivery of INS37217, a P2Y(2) receptor agonist, on subretinal fluid reabsorption in experimentally induced retinal detachments in rabbits, and to characterize the effects of INS37217 on electroretinograms (ERG) in rabbits. METHODS: A single retinal detachment was produced in New Zealand White rabbits by injecting approximately 50 micro L of modified phosphate-buffered saline (MPBS) solution into the subretinal space (SRS). In all experiments, one eye served as the INS37217-treated eye and the contralateral eye served as the vehicle control. In the first series of experiments, each rabbit received a SRS injection of MPBS solution, with or without INS37217 (1 mM). In the second series of experiments, each eye received an SRS injection of MPBS solution, followed by an intravitreal injection of MPBS solution, with or without INS37217 (12, 1.4, and 0.15 mM). A masked observer determined the size of blebs by indirect ophthalmoscopy at 30-minute intervals for up to 3 hours after SRS injections. Optical coherence tomography (OCT) was conducted to provide cross-sectional images of the blebs. Cellular expression of P2Y(2) receptor mRNA was localized by nonradioisotopic in situ hybridization in fresh rabbit retina-RPE tissue sections. Bilateral, full-field scotopic and photopic ERGs were made at 1, 7, and 14 days after a single intravitreal injection of 24 mM INS37217. RESULTS: SRS administration of 1 mM INS37217 significantly enhanced subretinal fluid reabsorption when compared with vehicle controls (P < 0.05; repeated measures ANOVA). Intravitreal administration of INS37217 at 12 and 1.4 mM, but not at 0.15 mM, also significantly enhanced subretinal fluid reabsorption (P < 0.05). P2Y(2) receptor mRNA was observed throughout the RPE and in discrete layers of the retina. INS37217 had no adverse effects on scotopic and photopic ERG amplitude and latency parameters at any of the postadministration time points evaluated. CONCLUSIONS: These results demonstrate that INS37217 enhances subretinal fluid reabsorption in experimental retinal detachment in rabbits and support the development of INS37217 for stimulating subretinal fluid reabsorption in conditions that result in retinal detachment or retinal edema.