High-dose intravenous pulse steroid therapy for optic disc swelling and subretinal fluid in non-arteritic anterior ischemic optic neuropathy.

Non-arteritic anterior ischemic optic neuropathy (NAION) is a disease with microvascular abnormality that causes acute optic disc swelling (ODS) and, in severe cases, subretinal fluid (SRF) accumulation. ODS causes compartment syndrome and subsequent axonal degeneration and loss of retinal ganglion cells by apoptosis. No treatment modalities have been effective, although some cases improved after the intake of oral systemic steroids. We reported a case of a 72-year-old man who was referred due to a visual defect in the right eye. At first presentation, visual acuity and visual field were disturbed; critical flicker frequency (CFF) was decreased; and optic coherence tomography (OCT) showed ODS and SRF. Microscopic examination revealed parapapillary hemorrhage and fluorescence angiography showed non-filling, temporal-superior choroidal lesion adjacent to the optic disc at an early phase. After high-dose intravenous steroid treatment, SRF and ODS were decreased, and completely resolved after 30 days. Visual acuity and CFF were improved, and visual field was enlarged. High-dose intravenous steroids could possibly resolve SRF and ODS and improve visual function of patients with NAION. Some cases in NAION improved visual acuity and visual function in natural course, more cases were needed to evaluate the efficiency.

Expression of Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells Induced by Amyloid-ß Is Depressed by an Endoplasmic Reticulum Stress Inhibitor.

PURPOSE: Amyloid-ß (Aß) is a 36- to 43-amino-acid peptide that is a constituent of drusen, and it has been demonstrated to upregulate vascular endothelial growth factor (VEGF) expression by retinal pigment epithelial (RPE) cells. This study aimed to determine whether 4-phenylbutyl phosphonylacetate (PBA), a known endoplasmic reticulum (ER) stress inhibitor, can reduce Aß-induced expression of VEGF in RPE cells. METHODS: Aß was added to the medium of regularly cultured or polarized ARPE-19 cells, a human RPE cell line, with or without PBA. The levels of VEGF and ER stress markers, namely GRP78/Bip, cleaved caspases 4 and 12 and GADD153/C-EBP homologous protein, were determined by enzyme-linked immunoassay, immunocytochemistry and Western blotting. RESULTS: Exposure of ARPE-19 cells to Aß induced GRP78/Bip expression and activated caspases 4 and 12; however, their expression was decreased by simultaneous exposure to PBA. Aß increased the expression of VEGF both in regularly cultured and polarized ARPE-19 cells, but it was suppressed by PBA. PBA did not cause RPE cell apoptosis. CONCLUSION: Aß has been suggested to be involved in the development of age-related macular degeneration; therefore, our findings suggest that drugs that target ER stress should be considered for the treatment of age-related macular degeneration.

High osmolarity effect of intravitreal plasmin enzyme on rabbit retina.

We conducted a study of the effect of intravitreal plasmin on retinal thickness in rabbits. Forty rabbit eyes were injected with 0.4, 1, 2, and 5 IU of plasmin (8 eyes/group). The same volume of BSS-plus(®) was injected in control eyes. Four eyes in each group underwent vitrectomy 60 minutes after the injections. The retinal thickness was measured in optical coherence tomographic (OCT) images before, 30 minutes, and 1 week after the injection. To study the effect of hyperosmolarity, 4 eyes received an injection of mannitol solution whose osmolarity was the same as the plasmin solutions. Thirty minutes after the plasmin injection, 4 eyes developed a serous retinal detachment (SRD). The mean retinal thickness including SRD was increased at 30 minutes in a dose-dependent way. The increase in eyes with 5 IU of plasmin was significantly greater than that in eyes with BSS-Plus(®) or 0.4 IU of plasmin (P = 0.0266, P = 0.0371, respectively). One week after the injection, SRD disappeared, and the mean retinal thickness decreased. The eyes injected with mannitol, the same osmolarity of 1, 2, 5 IU of plasmin, developed SRD at 30 minutes, and it disappeared after 1 week in all eyes. The results of this study demonstrated that an intravitreal injection of plasmin increases the retinal thickness in a dose-dependent way in rabbit eyes. The results with mannitol suggest that the increase in retinal thickness following plasmin is most likely caused by the hyperosmolarity of plasmin solution.

A long-term follow-up of patients with retinopathy of prematurity treated with photocoagulation and cryotherapy.

To evaluate the refractive characteristics of adults diagnosed with retinopathy of prematurity (ROP) treated with ablation treatment as children, we measured best corrected visual acuity (BCVA, logMAR), spherical equivalent refraction (SER), axial length (AL), lens thickness (LT), anterior chamber depth (ACD) and the corneal curvature radius (CCR) from 46 eyes, 24 patients (15-30 years old) that were diagnosed with ROP. Patients were divided into two groups dependent on the size of the treated retina at the time of ablation treatment; i.e., 360 degrees group (treatment over the whole circumference of the retina; n = 18) and partial group (treatment over part of the retina; n = 28). The study showed that LT was significantly larger (P < 1x10(-4)) and ACD was significantly shorter (P < 1 x 10(-3)) in 360 degrees group (4.26 +/- 0.40 mm and 2.92 +/- 0.48 mm, respectively) than those in partial group (3.71 +/- 0.34 mm and 3.42 +/- 0.26 mm, respectively). However, there were no differences in SER (-6.52 +/- 3.54 diopter vs. -5.95 +/- 4.12 diopter, P = 0.31), AL (23.9 +/- 1.42 mm vs. 25.0 +/- 21.48 mm, P = 0.08) and CCR (7.59 +/- 0.37 mm vs. 7.59 +/- 0.19 mm, P = 0.86). These results indicated that the eyes in the 360 degrees group had larger LTs but did not have extended ALs compared with the partial group.

Age-dependent alteration of intraocular soluble heparan sulfate levels and its implications for proliferative diabetic retinopathy.

PURPOSE: To assess the relationship between intraocular soluble heparan sulfate (HS) concentration and age in subjects with and without diabetic retinopathy. METHODS: Vitreous from subjects with idiopathic maculopathies (n=17), i.e., macula hole or epiretinal membrane, or nonproliferative diabetic retinopathy (non-PDR; n=5) and aqueous humor from subjects with PDR (n=16), non-PDR (n=7), or cataracts (n=15) was collected. The levels of HS and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay. Concentrations of sulfated glycosaminoglycan were determined through dimethylmethylene blue-based assay. The effect of the vitreal HS level on the binding of exogenous VEGF to surface-bound heparin was determined in vitro. RESULTS: The level of HS in vitreous samples from subjects with idiopathic maculopathies increased concomitantly with age (p=0.020, R²=0.327). Meanwhile, HS levels in aqueous humor were lower in PDR subjects than in non-PDR (p=0.003) and cataract subjects (p=0.007). However, the PDR subjects were significantly younger than the non-PDR subjects (p<0.001) or cataract subjects (p<0.001). When the three groups were controlled for age, the levels of HS glycosaminoglycans were no longer different between the three (p=0.247). The increasing level of HS or sulfated glycosaminoglycan in the vitreous was associated with its increased inhibitory effect on interaction between VEGF and surface heparin in vitro (p=0.014, R²=0.377). CONCLUSIONS: The HS level of the intraocular fluid increased with age. The possible link between low HS in intraocular fluid and increased localization of VEGF at the retinal surface may provide one explanation for the higher susceptibility of younger subjects with diabetes mellitus to developing PDR.

Influence of preoperative intravitreal bevacizumab on visual function in eyes with proliferative diabetic retinopathy.

PURPOSE: Although bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), is effective in treating ocular neovascularization, there are some concerns about whether blocking VEGF might be harmful to retinal neurons. The purpose of this study was to evaluate the effects of preoperative intravitreal bevacizumab (IVB) on the visual function of eyes with proliferative diabetic retinopathy (PDR). METHODS: Thirty eyes of 23 patients (13 men and 10 women) with PDR who were treated at the Nagoya University Hospital from November 2006 to October 2009 were studied. All of the eyes were treated with 1.25 mg/0.05 ml of IVB 2-8 days before the vitrectomy. The protocol was approved by the Institutional Review Board of Nagoya University, and a written informed consent was obtained from each patient. All of the eyes had an active proliferative membrane with vitreous hemorrhage, but the fundus was visible. The mean age of the patients was 41.6 ± 10 years (range, 27-59), and the mean follow-up period was 9.7 ± 8.9 months (range, 1-24) after the vitrectomy. The visual acuity (VA) was measured, the visual fields were determined by Goldmann perimetery, and full-field electroretinograms (ERGs) were recorded before IVB, and before and after the vitrectomy. Fluorescein angiography was also performed before and after IVB. The area of the visual field was measured using a computer software (Scion Image). RESULTS: All eyes showed a regression of the new vessels and a reduction of fluorescent leakage from the new vessels after IVB. In addition, there was less bleeding during the removal of the proliferative membrane. The average VA was improved postoperatively from 20/250 to 20/70. However, there was no significant change in the amplitudes of the a- (from 261.4 to 259.2 µV) and b-waves (from 256.9 to 253.3 µV) of the ERGs, and there was no significant change in the visual field area after the surgery (from 8,322.5 to 7,496.3 degrees(2)). No significant ocular or systemic adverse events were observed. CONCLUSION: IVB-assisted vitrectomy led to an improvement of the VA in eyes with PDR without significant adverse events. There was no change in the visual fields and ERGs. Although only a small number of patients were studied, we conclude that IVB is most likely not harmful to retinal neurons if bevacizumab is washed out in less than 1 week. In addition, preoperative IVB made the surgery much easier by decreasing the activity of new vessels.

Factors Associated with Reclosure of Posterior Capsule Aperture by Flat Opacifications with Pearls after Nd:YAG Laser Posterior Capsulotomy.

In this retrospective case series, we investigated factors associated with posterior capsule aperture (PCA) reclosure following neodymium-yttrium aluminum garnet (Nd:YAG) laser posterior capsulotomy. The study encompassed patients who underwent cataract surgery with intraocular lens (IOL) implantation or a combined vitrectomy, cataract surgery, and IOL implantation between 2009 and 2022. PCA reclosure was observed in 22 eyes of 17 patients: 45% (10 eyes) underwent the triple procedure, and 55% (12 eyes) received cataract surgery with IOL implantation. In our clinic, 14% of patients were given IOLs with a 4% water content, while 73% (13 eyes) of those experiencing PCA reclosure had IOLs with a 4% water content. The mean interval between Nd:YAG capsulotomies was notably shorter than that between the initial cataract surgery and the first Nd:YAG laser capsulotomy. We also identified five stages of PCA reclosure progression. In conclusion, IOL water content may be linked to PCA reclosure, and the time to recurrence is shorter with each successive reclosure. Further research is needed to verify these findings and uncover additional contributing factors.

Association Between Torque Teno Virus and Systemic Immunodeficiency in Patients With Uveitis With a Suspected Infectious Etiology.

PURPOSE: To determine the correlation between the presence of torque teno virus (TTV) in the aqueous humor of patients with uveitis and clinical information, including immunodeficiency history. DESIGN: Multicenter, retrospective, cross-sectional study. METHODS: Fifty-eight patients with uveitis with a suspected infectious etiology and 24 controls with cataract or age-related macular degeneration were included. We used quantitative polymerase chain reaction to test all subjects for TTV and multiplex polymerase chain reaction to test uveitis subjects for common ocular pathogens. When possible, both serum and aqueous humor samples were tested. Ocular TTV positivity was compared with age, sex, and a history of systemic immunodeficiency with logistic analysis. RESULTS: Ocular TTV positivity was found in 23%, 11%, and 0% of patients with herpetic uveitis, nonherpetic uveitis, and controls, respectively. Among patients with herpes infection, positivity for ocular TTV was found in 43%, 8%, 14%, and 50% of patients with cytomegalovirus retinitis, iridocyclitis, acute retinal necrosis, and Epstein-Barr virus-positive uveitis, respectively. Patients with cytomegalovirus retinitis showed a significantly higher rate of ocular TTV infection than controls (P = .008). Serum analysis revealed TTV positivity in 90% of patients with uveitis and in 100% of controls. Age- and gender-adjusted logistic analysis revealed a correlation between ocular TTV positivity and systemic immunodeficiency (P = .01), but no correlations between ocular TTV and age, gender, or viral pathogenic type. CONCLUSIONS: These findings suggest that positivity for ocular TTV was correlated with a clinical history of systemic immunodeficiency.

Findings in pseudophakic eye that developed liquefied aftercataract-like substance one day after vitrectomy.

Purpose: To report our findings in a case that had an accumulation of a translucent fluid between the intraocular lens (IOL) and posterior lens capsule one day after vitrectomy for a vitreous hemorrhage. Observations: A 67-year-old woman was diagnosed with diabetes 20 years before the vitrectomy and was treated with panretinal photocoagulation (PRP) for proliferative diabetic retinopathy (PDR) 14 years earlier. She underwent cataract surgery with an implantation of an IOL 4 years earlier. She was referred to our hospital because of a vitreous hemorrhage, and we performed uneventful vitrectomy. However, the day after the operation, a translucent liquid substance that resembled liquefied aftercataract was observed in the lens capsule bag. With time, the liquid substance became cloudy. The opacification progressed for two years after the vitrectomy, and her visual acuity decreased. We then performed neodymium: YAG (Nd: YAG) laser posterior capsulotomy, and the cloudy liquid dispersed into the vitreous and the visual acuity improved. Conclusions and importance: Our findings indicate that liquified aftercataract-like substance can form after vitrectomy in a pseudophakic eye. We suggest that the aqueous humor might flow into the space behind the IOL during or just after the vitrectomy and was trapped behind the IOL optics. Then, the proliferating lens epithelial cells might be dissolved forming the white liquid substance immediately after the surgery.

BEST1 expression in the retinal pigment epithelium is modulated by OTX family members.

A number of genes preferentially expressed in the retinal pigment epithelium (RPE) are associated with retinal degenerative disease. One of these, BEST1, encodes bestrophin-1, a protein that when mutated causes Best macular dystrophy. As a model for RPE gene regulation, we have been studying the mechanisms that control BEST1 expression, and recently demonstrated that members of the MITF-TFE family modulate BEST1 transcription. The human BEST1 upstream region from -154 to +38 bp is sufficient to direct expression in the RPE, and positive-regulatory elements exist between -154 and -104 bp. Here, we show that the -154 to -104 bp region is necessary for RPE expression in transgenic mice and contains a predicted OTX-binding site (Site 1). Since another non-canonical OTX site (Site 2) is located nearby, we tested the function of these sites using BEST1 promoter/luciferase constructs by in vivo electroporation and found that mutation of both sites reduces promoter activity. Three OTX family proteins - OTX1, OTX2 and CRX - bound to both Sites 1 and 2 in vitro, and all of them increased BEST1 promoter activity. Surprisingly, we found that human and bovine RPE expressed not only OTX2 but also CRX, the CRX genomic region in bovine RPE was hypersensitive to DNase I, consistent with active transcription, and that both OTX2 and CRX bound to the BEST1 proximal promoter in vivo. These results demonstrate for the first time CRX expression in the RPE, and suggest that OTX2 and CRX may act as positive modulators of the BEST1 promoter in the RPE.

Rebound of macular edema after intravitreal bevacizumab therapy in eyes with macular edema secondary to branch retinal vein occlusion.

PURPOSE: To determine the incidence of rebound macular edema after intravitreal bevacizumab in eyes with macular edema secondary to branch retinal vein occlusion and to identify the pretreatment factors that were significantly associated with the rebound. METHODS: The changes in the foveal thickness after the intravitreal bevacizumab (1.25 mg/0.05 mL) were studied in 65 eyes of 65 patients with macular edema secondary to branch retinal vein occlusion. A rebound of macular edema was defined as a ≥110% increase in the foveal thickness or a foveal thickness ratio of ≥110% (foveal thickness at the recurrence/foveal thickness at the baseline × 100). Multivariate logistic regression analyses and subgroup analyses were performed to determine which pretreatment factors were associated with the rebound. RESULTS: Seven of 65 eyes (10.8%) showed a rebound (≥110% of baseline thickness). Subgroup analyses showed that a thinner pretreatment fovea and a shorter interval between symptom onset to the initiation of the intravitreal bevacizumab were significantly associated with a rebound of macular edema (P < 0.01). The interval from symptoms onset to the initiation of treatment was <8 weeks in all 7 eyes with a rebound macular edema. CONCLUSION: These results suggest that a rebound of macular edema in eyes with branch retinal vein occlusion was more likely to occur when the intravitreal bevacizumab therapy is initiated before the macular edema reaches the maximum level. Rebound of macular edema may be effectively avoided by waiting at least 8 weeks after the onset of symptoms to begin the intravitreal bevacizumab.

Generation of immature retinal neurons from proliferating cells in the pars plana after retinal histogenesis in mice with retinal degeneration.

PURPOSE: To study the differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium after retinal histogenesis in mice with inherited or acquired retinal degeneration. METHODS: Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically. The distribution and differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium of mice with inherited (C3H/HeJ) and acquired (C57BL mice injected with 60 mg/kg N-methyl-N-nitrosourea) retinal degeneration were assessed. Proliferating retinal progenitors were labeled with bromodeoxyuridine (BrdU), and they were studied histologically using retinal markers. RESULTS: Many cells of rod and cone photoreceptor lineage were identified within the ciliary epithelium of the pars plana in adult mice with inherited retinal degeneration. Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24). The induction of acquired retinal degeneration in adult wild-type mice (P30) increased the number of BrdU-positve cells by roughly fourfold and recoverin-positive cells by approximately 17-fold in the pars plana. Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana. CONCLUSIONS: In response to retinal damage, an increased number of immature retinal neurons/retinal precursors was observed in the pars plana of mice with acquired and inherited retinal degeneration. Some of these cells differentiated from proliferating cells even after retinal histogenesis.

Intravitreal injection of bevacizumab for macular edema secondary to branch retinal vein occlusion:results after 12 months and multiple regression analysis.

PURPOSE: To evaluate the 12-month follow-up results of intravitreal bevacizumab therapy for macular edema secondary to branch retinal vein occlusion and to identify the pretreatment factors that were associated with an improvement of the final visual outcome. METHODS: Fifty eyes of 50 patients with macular edema secondary to branch retinal vein occlusion received an injection of 1.25 mg/0.05 mL bevacizumab. Additional injections were done when recurrence of macular edema occurred or the treatment was not effective. The best-corrected visual acuity and foveal thickness were measured. Stepwise multiple regression analyses were also performed. RESULTS: The mean logarithm of the minimum angle of resolution visual acuity improved significantly from 0.53 to 0.26, and the mean foveal thickness decreased significantly from 523 to 305 microm during the 12-month follow-up period. The mean number of injections was 2.0 (range, 1-4). Stepwise multiple regression analyses showed that younger patients had both better visual acuity at 12 months and greater improvement of visual acuity during 12 months. In addition, better pretreatment visual acuity was associated with better visual acuity at 12 months but with less improvement of the visual acuity. CONCLUSION: Intravitreal bevacizumab therapy can be a long-term effective treatment for macular edema secondary to branch retinal vein occlusion.

Concentration of vascular endothelial growth factor in aqueous humor of eyes with advanced retinopathy of prematurity before and after intravitreal injection of bevacizumab.

PURPOSE: To determine whether an intravitreal injection of bevacizumab alters the concentration of vascular endothelial growth factor (VEGF) in the aqueous humor of eyes with retinopathy of prematurity. METHODS: Seven Stage 4 and three Stage 5 eyes of eight patients with retinopathy of prematurity were studied. Bevacizumab (0.75 mg/0.03 mL/eye) was injected intravitreally in six eyes of six patients after approval was obtained from the Institutional Review Board of Nagoya University Hospital and an informed consent was signed by the parents. Aqueous humor was collected just before the surgery or before the intravitreal injection of bevacizumab. Aqueous humor was also collected immediately before vitrectomy 4 to 48 days after the injection of bevacizumab. Aqueous humor was also collected from four patients undergoing congenital cataract surgery as controls. The concentration of VEGF was measured by enzyme-linked immunosorbent assay. RESULTS: In the 4 control eyes, the concentration of VEGF in 2 eyes was 156 and 158 pg/mL and was not detectable in the other 2 eyes. The average concentration of VEGF was 1,109 pg/mL in the active Stage 4 eyes and 3,520 pg/mL in the active Stage 5 eyes. After bevacizumab injection, the unbound VEGF concentration was 60, 230, and 290 pg/mL in 3 eyes and not detectable in 1 eye. CONCLUSION: Intravitreal bevacizumab resulted in a marked decrease in the unbound VEGF concentration in eyes with retinopathy of prematurity.

Triamcinolone-assisted removal of internal limiting membrane enhances the effect of vitrectomy for diabetic macular edema.

PURPOSE: To evaluate the efficacy of internal limiting membrane (ILM) peeling during vitrectomy to treat diabetic macular edema (DME). METHODS: In this nonrandomized study, we retrospectively analyzed the medical charts of 107 eyes that had undergone three-port vitrectomy to treat DME. The ILM was peeled in 65 eyes and not peeled in 42 eyes (ILM ON group). Indocyanine green (ICG) was used on 36 eyes (ICG group) and triamcinolone acetonide (TA) on 29 eyes (TA group) to make the ILM more visible. RESULTS: In all groups, the mean foveal thickness was significantly decreased after surgery. Visual acuity (VA) improved gradually after the surgery and was significantly better than the preoperative VA at 6 months in the ICG group and at 3 months in the TA group. Compared with the ILM ON group eyes, the eyes in the TA group had significantly better improvement of VA. CONCLUSION: TA-assisted ILM peeling may improve the VA outcome to treat DME.

[New therapies for age-related macular degeneration].

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly in developed countries. New treatments have been developed, and they can be grouped into those that selectively disrupt new vessels, e.g., photodynamic therapy; and those that target molecules that play an important role in angiogenesis, e.g., anti-vascular endothelial growth factor (VEGF) drugs. Ranibizumab, the anti-VEGF-drug, was the first drug that led to an improvement of visual acuity. However, a disadvantage of this drug is the need of repeated injection, and to overcome this disadvantage, gene therapy and some other methods are being studied. A clinical trial of gene therapy is being performed in the U.S.A. In this review, we describe the new therapies for AMD.

Topical administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema.

Age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi-targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down-stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser-induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de-esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser-induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema.

Development of prodrug 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate (TG100801): a topically administered therapeutic candidate in clinical trials for the treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.

Identification of photoreceptor precursors in the pars plana during ocular development and after retinal injury.

PURPOSE: To study the distribution and differentiation of photoreceptor precursors in the ciliary epithelium in mice. METHODS: Proliferating cells in flat-mount specimens of the ciliary body and retina were studied by bromodeoxyuridine (BRDU; 150 mg/kg) labeling in young C57Bl mice. Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically to assess the distribution and differentiation of photoreceptor progenitors or precursors. Mice injected intraperitoneally with N-methyl-N-nitrosourea (MNU; 60 mg/kg) were also examined. RESULTS: Part of the neuroblast layer composed of BrdU-positive retinal progenitor cells was identified within the ciliary epithelium of the pars plana in continuation of the layer of the peripheral retina during ocular development. In both the ciliary epithelium and the retina, the layer size decreased rapidly and disappeared mostly by postnatal day (P)9. Within the ciliary epithelium of the pars plana, numerous postmitotic rod and cone photoreceptor precursors were identified that rapidly differentiated morphologically and decreased in number with ocular development. Rod precursors were no longer seen in the pars plana at P12, whereas rare presumptive cone precursors persisted even at P120. An increase in the number of presumptive cone precursors (approximately 16-fold) was identified in the pars plana of adult mice with MNU-induced photoreceptor degeneration. CONCLUSIONS: Rod and cone precursors were identified in the ciliary epithelium of the murine pars plana during ocular development but nearly disappeared after the completion of histogenesis. However, in response to retinal injury, an increased number of presumptive cone precursors was found even in the adult pars plana.

Retardation of photoreceptor degeneration in the detached retina of rd1 mouse.

PURPOSE: To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice. METHODS: RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30. RESULTS: The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD. CONCLUSIONS: RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.