Retinal Dystrophy Associated With RLBP1 Retinitis Pigmentosa: A Five-Year Prospective Natural History Study.

Purpose: To assess the progression in functional and structural measures over a five-year period in patients with retinal dystrophy caused by RLBP1 gene mutation. Methods: This prospective, noninterventional study included patients with biallelic RLBP1 mutations from two clinical sites in Sweden and Canada. Key assessments included ocular examinations, visual functional measures (best-corrected visual acuity [BCVA], contrast sensitivity [CS], dark-adaptation [DA] kinetics up to six hours for two wavelengths [450 and 632 nm], Humphrey visual fields [HVF], full-field flicker electroretinograms), and structural ocular assessments. Results: Of the 45 patients enrolled, 38 completed the full five years of follow-up. At baseline, patients had BCVA ranging from -0.2 to 1.3 logMAR, poor CS, HVF defects, and prominent thinning in central foveal thickness. All patients had extremely prolonged DA rod recovery of approximately six hours at both wavelengths. The test-retest repeatability was high across all anatomic and functional endpoints. Cross-sectionally, poorer VA was associated with older age (right eye, correlation coefficient [CC]: 0.606; left eye, CC: -0.578; P < 0.001) and HVF MD values decreased with age (right eye, CC: -0.672, left eye, CC: -0.654; P < 0.001). However, no major changes in functional or structural measures were noted longitudinally over the five-year period. Conclusions: This natural history study, which is the first study to monitor patients with RLBP1 RD for five years, showed that severely delayed DA sensitivity recovery, a characteristic feature of this disease, was observed in all patients across all age groups (17-69 years), making it a potentially suitable efficacy assessment for gene therapy treatment in this patient population.

Ocular Pharmacology and Toxicology of TRPV1 Antagonist SAF312 (Libvatrep).

Purpose: To evaluate the pharmacology and toxicology of SAF312, a transient receptor potential vanilloid 1 (TRPV1) antagonist. Methods: TRPV1 expression in human ocular tissues was evaluated with immunohistochemistry. Inhibition of calcium influx in Chinese hamster ovary (CHO) cells expressing human TRPV1 (hTRPV1) and selectivity of SAF312 were assessed by a fluorescent imaging plate reader assay. Ocular tissue and plasma pharmacokinetics (PK) were assessed following a single topical ocular dose of SAF312 (0.5%, 1.0%, 1.5%, 2.5%) in rabbits. Safety and tolerability of SAF312 were evaluated in rabbits and dogs. Effects of SAF312 on corneal wound healing after photorefractive keratectomy (PRK) surgery were assessed in rabbits. Results: TRPV1 expression was noted in human cornea and conjunctiva. SAF312 inhibited calcium influx in CHO-hTRPV1 cells induced by pH 5.5 (2-[N-morpholino] ethanesulfonic acid), N-arachidonoylethanolamine, capsaicin, and N-arachidonoyl dopamine, with IC50 values of 5, 10, 12, and 27 nM, respectively, and inhibition appeared noncompetitive. SAF312 demonstrated high selectivity for TRPV1 (>149-fold) over other TRP channels. PK analysis showed highest concentrations of SAF312 in cornea and conjunctiva. SAF312 was found to be safe and well tolerated in rabbits and dogs up to the highest feasible concentration of 2.5%. No delay in wound healing after PRK was observed. Conclusions: SAF312 is a potent, selective, and noncompetitive antagonist of hTRPV1 with an acceptable preclinical safety profile for use in future clinical trials. Translational Relevance: SAF312, which was safe and well tolerated without causing delay in wound healing after PRK in rabbits, may be a potential therapeutic agent for ocular surface pain.

Topical Ocular TRPV1 Antagonist SAF312 (Libvatrep) for Postoperative Pain After Photorefractive Keratectomy.

Purpose: Evaluation of safety and efficacy of topical ocular SAF312 (Libvatrep) in post-photorefractive keratectomy (PRK) pain. Methods: In this placebo (vehicle)-controlled, participant- and investigator-masked study, 40 participants were randomized (1:1) to two treatment sequences in a bilateral PRK crossover design (SAF312 2.5% followed by vehicle [or vice versa], one eye drop, four times daily for 72 hours after PRK). Primary endpoints were visual analog scale (VAS) pain scores at 6 hours after first drop of study drug and average VAS scores over 0 to 12 hours postoperatively. Secondary endpoints included postoperative oral rescue medication (ORM) use and adverse events (AEs). Results: All 40 participants completed the study. Both primary endpoints were met; mean difference in VAS pain scores between SAF312- and vehicle-treated eyes was -11.13 (P = 0.005, -25%) at 6 hours postoperatively and -8.56 (P = 0.017, -22%) over 0 to 12 hours. Mean VAS pain scores with SAF312 were consistently lower than with vehicle from 1 hour postoperatively up to 30 hours (P ≤ 0.10 observed in 8/11 time points). Less ORM was taken with SAF312 up to 0 to 72 hours postoperatively, with a trend of fewer participants taking ORM at 0 to 24 hours postoperatively with SAF312 versus vehicle. No serious AEs were reported. All ocular AEs were mild and transient, and none were drug related. SAF312-treated eyes showed no delay in wound healing and had a lower grade 4 conjunctival hyperemia 24 hours postoperatively versus vehicle-treated eyes. Conclusions: SAF312 was well tolerated and effective in reducing ocular pain post-PRK. Translational Relevance: Topical SAF312 presents a new therapeutic option for patients undergoing PRK.

Topical Ocular TRPV1 Antagonist SAF312 (Libvatrep) Demonstrates Safety, Low Systemic Exposure, and No Anesthetic Effect in Healthy Participants.

Purpose: This first-in-human (FIH) study evaluated the safety, tolerability, pharmacokinetics, and effect on corneal sensitivity of topical ocular SAF312 in healthy participants. Methods: This double-masked, randomized study comprised single-ascending dose (SAD), multiple-ascending dose (MAD), and esthesiometry parts. In SAD and MAD, 8 participants in each dose cohort were randomized 3:1 to receive SAF312 or vehicle, 1 drop once (SAD), or 1 drop 4 or 8 times daily for 7 days (MAD). Safety and pharmacokinetics were the primary and secondary objectives. Blink rate, tear production, tear film break-up time (TFBUT), and corneal sensitivity were also explored. Results: SAF312 was tolerated in single and multiple doses, including the maximum concentration of 2.5% dosed up to 1 drop 8 times daily for 7 days. Most adverse events (AEs) were mild and similar between SAF312 and vehicle-treated groups. No serious AEs were reported. SAF312 was rapidly absorbed, and had low systemic exposure. After supratherapeutic dosing for 7 days, mean steady-state exposures of SAF312 were low and afforded safety margins of >70-fold compared with no-observed-AE levels following oral dosing in preclinical studies. No clinically relevant changes were observed in blink rate, tear production, and TFBUT. SAF312 showed no undesired anesthetic effect on the cornea. Conclusions: SAF312 was well tolerated, with no ocular or systemic safety concerns; had no anesthetic effect, and demonstrated rapid topical absorption with low systemic exposure. Translational Relevance: This work bridges the gap between basic research and clinical care by providing FIH data of SAF312, supporting the further investigation as a potential treatment for ocular surface pain.

Tear osmolarity and dry eye symptoms in diabetics.

PURPOSE: To assess the relationship between tear osmolarity and dry eye symptoms in patients with diabetes. PATIENTS AND METHODS: Fifty patients with diabetes were enrolled. Demographic information and past medical history were recorded. Symptoms were assessed using the ocular surface disease index (OSDI). Tear osmolarity of each eye was measured with the TearLab® Osmolarity System. RESULTS: The majority of the subjects were female (76%), African American (56%), and/or had a diagnosis of type 2 diabetes (82%). The mean ± standard deviation (SD) for age was 54.6±13.4, and maximum tear osmolarity was 304.6±12.7 mOsm/L. Men had higher osmolarity than women (mean ± standard error (SE) 311.8±4.0 mOsm/L versus 302.3±1.9 mOsm/L, P=0.02). Age, race, use of artificial tears, years of diabetes, and hemoglobin A1c did not have a statistically significant association with tear osmolarity. Longer duration of diabetes was associated with lower (less severe) OSDI scores (r=-0.35, P=0.01). Higher tear osmolarity was associated with lower (less severe) OSDI scores (r=-0.29, P=0.04). CONCLUSION: Approximately half of the diabetic subjects in our study had elevated tear osmolarity, and half of our population also reported symptoms consistent with dry eye disease. However, the two were slightly inversely related in that those with higher osmolarity reported fewer symptoms. Subjects with a longer duration of diabetes also reported fewer dry eye symptoms. Therefore, health care providers should be aware that patients who are most likely to have ocular surface disease, including those with long-standing diabetes, may not experience symptoms and seek care in a timely manner.

Optimal isolation and xeno-free culture conditions for limbal stem cell function.

PURPOSE: To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions. METHODS: Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype. RESULTS: Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2. CONCLUSIONS: The LSC function can be maintained in vitro under appropriate Xeno-free conditions.

Retinal arteriolar spasm during transient monocular visual loss in eosinophilic vasculitis.

A patient with eosinophilic vasculitis and acquired immunodeficiency syndrome (AIDS) developed episodic transient monocular visual loss. During or immediately after two visual loss episodes, we demonstrated narrowed retinal arterioles, delayed arterial filling time, and segmented retinal venous flow in the affected eye on fundus photography and fluorescein angiography (FA). Such findings have only rarely been reported in patients with transient monocular visual loss in other conditions, probably because the episodes have ended before fundus photography and FA could be performed. This is the first report to capture retinal vascular changes associated with transient monocular visual loss in a patient with eosinophilic vasculitis.

Update on phototherapeutic keratectomy.

PURPOSE OF REVIEW: To review all recent publications on the use of phototherapeutic keratectomy (PTK). RECENT FINDINGS: Recent studies confirm the beneficial outcomes of PTK in a variety of anterior corneal disorder: anterior stromal scars, dystrophies of the corneal epithelium and the anterior stroma, and elevated corneal lesions. They also try to evaluate the biomechanical properties of eyes with granular corneal dystrophy undergoing PTK, in an effort to prevent iatrogenic ectasia. The different genotypes in patients with transforming growth factor, beta-induced linked corneal dystrophies have recently been correlated to the surgical outcome after PTK. An extensive review of recurrent corneal erosion syndrome identified PTK as the most effective treatment. In the developing world, the most common indication for PTK is still bullous keratopathy, as PTK can be successfully used while waiting for penetrating keratoplasty. SUMMARY: PTK can successfully treat a variety of conditions of the anterior cornea. New studies may further expand its use.

Ceruloplasmin upregulation in retina of murine and human glaucomatous eyes.

PURPOSE: Ceruloplasmin (Cp) expression is increased locally as a response to many neurodegenerative conditions. The purposes of this study were to confirm findings of Cp upregulation in glaucoma, detect the time course of this upregulation in a glaucoma model, and better localize its expression in the retina. METHODS: mRNA and protein were extracted from the retina and brain of DBA/2 and C57BL/6 mice and were subjected to analysis by RT-PCR and immunoblotting. In addition, eyes from the same mouse strains were subjected to immunohistochemistry using antibodies specific for Cp. Eyes from human subjects with or without glaucoma were also subjected to immunohistochemical analysis for Cp. RESULTS: Cp mRNA and Cp protein were upregulated in the retinas of glaucomatous DBA/2 mice. Upregulation of Cp occurred at approximately the time of extensive retinal ganglion cell (RGC) death and increased with increasing age to 15 months in the retinas but not in the brains of these animals. No age-related Cp upregulation was detected in the reference normal mouse strain (C57BL/6), which can develop significant nonglaucomatous RGC loss toward the end of the same time frame. Cp upregulation was also detected in most eyes from the patients with glaucoma. Cp upregulation was localized to the Müller cells within the retinas and in the area of the inner limiting membrane. CONCLUSIONS: Cp is upregulated in the retina of a commonly used glaucoma model (the DBA/2 mouse) and in most human glaucomatous eyes. The timing of this upregulation suggests that it may represent a reactive change of the retina in response to a noxious stimulus or to RGC death. Such Cp upregulation may represent a protective mechanism within the retina.

Photodynamic treatment in a rabbit model of glaucoma surgery.

PURPOSE: To evaluate the safety and efficacy of postoperative verteporfin photodynamic treatment as an adjunct to glaucoma experimental filtration surgery in rabbits. METHODS: Dutch belted (n = 15) rabbits underwent full thickness sclerectomy in one eye. The experimental group (group 1, n = 7) underwent i.v. injection of verteporfin and subsequent photoactivation at the operative site on postoperative day 1 (POD 1). Control groups of animals received either light exposure (group 2, n = 4) or verteporfin (group 3, n = 2), or no intervention (group 4, n = 2). Intraocular pressure (IOP) was measured prior to the procedure (POD 0) and daily thereafter for the first week after sclerectomy (PODs 1-7) and every other day for the second week (PODs 9, 11, 13, 15). Percentage IOP reductions of operated over contralateral control eyes were compared among the various groups. Success rates (percentage IOP reduction > 15%) were also compared between the experimental and control groups. Eyes were histologically examined for evaluation of fibrosis. RESULTS: Rabbits in the experimental group (group 1) had a mean +/- SEM percentage IOP reduction of 25 +/- 3% during the follow-up period. In contrast, groups 2, 3 and 4 had IOP reductions of 4 +/- 5%, 12 +/- 7% and 4 +/- 6%, respectively (p < 0.005, anova among all four groups). Successful IOP reduction (> or = 15%) over the contralateral eye at POD 15 was achieved in six of seven experimental animals, but only in one of eight control animals (p < 0.02, chi-squared test). Bleb failure occurred significantly earlier in the control eyes compared with eyes receiving PDT (p < 0.003, log rank test). Blebs in the experimental group differed from those in the control groups histologically, lacking significant collagen deposition in the area of the sclerostomy. CONCLUSIONS: Wound healing in glaucoma surgery may be successfully modulated postoperatively using photodynamic therapy with i.v. administered photosensitizer.

Complement component 1Q (C1Q) upregulation in retina of murine, primate, and human glaucomatous eyes.

PURPOSE: Complement has been implicated in the pathogenesis of neurodegenerative diseases. The purpose of this study was to investigate whether complement activation is part of the pathogenesis of retinal ganglion cell (RGC) loss in glaucoma. METHODS: mRNA and protein was extracted from the retina and brain of DBA/2 and C57/BL6 mice and subjected to RT-PCR and immunoblot analysis, respectively. In addition, eyes from the same mouse strains were subjected to immunohistochemistry with antibodies specific to complement component 1q (C1q). Eyes from monkeys with unilateral experimental glaucoma were also subjected to immunohistochemical analysis, as were eyes from human subjects with or without glaucoma. RESULTS: C1q mRNA and C1q protein were found to be upregulated in the retina of glaucomatous DBA/2 mice. Upregulation of C1q preceded the time of extensive RGC death and increased with increasing age to 15 months in the retina, but not in the brain. No age-related C1q upregulation was detected in the reference mouse strain (C57BL/6), which develops significant nonglaucomatous RGC loss toward the end of the same time frame. C1q upregulation was also detected in laser-induced glaucomatous monkey eyes and in some (but not all) eyes of patients with glaucoma. C1q upregulation was localized to the Müller cells within the retina and in the area of the inner limiting membrane. CONCLUSIONS: Complement expression is upregulated in the retina of two commonly used glaucoma models (in the DBA/2 mouse and the monkey) and in some human glaucomatous eyes. The timing of this upregulation suggests that complement activation plays a significant role in the pathogenesis of glaucoma.

Genetically induced retinal degeneration leads to changes in metabotropic glutamate receptor expression.

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [(35)S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

BACKGROUND: The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. METHODS: [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. RESULTS: In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). CONCLUSIONS: The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.