Correlation between improvement in visual acuity and QOL after Ranibizumab treatment for age-related macular degeneration patients: QUATRO study.

BACKGROUND: To evaluate the correlation between visual acuity improvement and vision-related QOL after ranibizumab treatment in Japanese patients with AMD. METHODS: In this one-year prospective, interventional, open-label, multicenter study involving four sites, patients with neovascular AMD were enrolled and observed for 12 months. Treatment-naïve patients received 0.5 mg ranibizumab as needed after three initial monthly doses. The best corrected visual acuity (BCVA) and central macular thickness (CMT) were measured at every visit. Evaluations with the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ-25) and patient satisfaction questionnaire were performed at baseline and 3 and 12 months after initial treatment. The primary endpoint was change in BCVA and QOL 3 months after ranibizumab treatment. QOL outcomes were also assessed in the better and poor BVCA subgroups. RESULTS: The study enrolled 100 patients. The mean logMAR BCVA after treatment improved significantly from 0.43 to 0.30 at 3 months (p< 0.0001), and 0.28 at 12 months (p< 0.0001). The mean NEI-VFQ-25 composite scores improved from 79.48 to 84.13 at 3 months (p< 0.0001), and 86.0 at 12 months (p< 0.0001). The 3 and 12-month changes in NEI-VFQ-25 score and BCVA showed significant correlation. In the poor baseline visual acuity group (decimal BCVA ≤0.5), there was a significant correlation between the changes in the NEI-VFQ-25 score and BCVA (p=0.02) but not in the better baseline visual acuity group (decimal BCVA > 0.6, p=0.1) at 3 months. There were no significant differences in the satisfaction questionnaire score from baseline to at 3 months (p=0.54) and 12 months (p=0.23). The average CMT improved significantly from 340 to 264 µm at 3 months (p< 0.0001) and to 268 µm at 12 months (p< 0.0001). CONCLUSIONS: Intravitreal ranibizumab treatment resulted in improvement in visual acuity, anatomical change, and visual function change in Japanese AMD patients. Significant improvement was seen in patient visual function, and this was correlated with changes in VA, except immediately after loading dose treatment in patients with higher baseline VA. The patients' satisfaction with the treatment remained unchanged during the study period. TRIAL REGISTRATION: This study is registered at UMIN Clinical Trials Registry ( UMIN000012013 ). Registered October 10, 2013, as prospective study.

[Comparison of 12 Months Outcome of As-needed Intravitreal Aflibercept or Ranibizumab for the Treatment of Naïve Patients with Age-related Macular Degeneration].

PURPOSE: To compare pro re nata (PRN) intravitreal injections of aflibercept (IVA) and ranibizumab (IVR) in patients with treatment naïve age-related macular degeneration (AMD). MATERIAL AND METHODS: We analyzed 42 eyes that receive 3 times monthly IVA as introduction phase and subsequently received PRN retreatment at the recurrence. As the control, 56 eyes received the same IVR treatments as the IVA criteria. We statistically analyzed chronological changes of VA and the first recurrence following introduction phase by comparing the findings of the 2 groups. RESULTS: There was no difference in the IVA and the IVR in baseline visual acuity (VA) and the mean number of injections during 12 months. Compared to the IVR, the IVA showed better improved VA from baseline at each time point, especially, there was a statistically significant difference in 6 months after introduction (p = 0.041). The IVA proved to have a shorter period until the first recurrence and a lower remission maintenance rate following introduction phase than the IVR. The improvement of VA above 0.2 logMAR was significantly related to cases involving polypoidal choroidal vasculopathy, greatest linear dimension and baseline VA. CONCLUSIONS: The improvement of VA in anti-VEGF therapy for AMD was influenced by the disease type or pathology rather than the choice of therapeutic agents.

[Background factors of recurrence after remission maintenance with photodynamic therapy for age-related macular degeneration].

PURPOSE: To investigate the factors associated with recurrence following remission of at least 12 months after photodynamic therapy in patients with age-related macular degeneration (AMD). SUBJECTS AND METHODS: We analyzed 42 eyes with AMD in 41 patients who did not require additional treatment within 12 months of their photodynamic therapy (PDT). Additional treatment for recurrence beyond 12 months after the last PDT was required in 20 eyes in the recurrent group (n = 20). The remaining eyes (controls; n = 22) showed no recurrence during this period. We statistically analyzed the factors associated with recurrence by comparing the findings of the 2 groups. RESULTS: Compared to the controls, the recurrent group showed significantly improved baseline visual acuity (VA) (p = 0.005), but required significantly more PDT applications to achieve remission (p = 0.010). Age (p = 0.026), multiple PDT sessions (p = 0.001), and high baseline VA (p = 0.003) were factors significantly related to the recurrence following remission maintenance with PDT for AMD (R = 0.63; R2 = 0.39; p < 0.0001). CONCLUSIONS: Age, preoperative VA and number of PDT sessions were associated with a significant risk of recurrence after remission maintenance with PDT.

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.

Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive.

In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Expression of ephrinB2 and its receptors on fibroproliferative membranes in ocular angiogenic diseases.

PURPOSE: To determine whether ephrinB2 plays a role in ocular angiogenesis, we investigated the expression of ephrinB2 and EphB receptors on retinal fibroproliferative membranes. DESIGN: Experimental study of the expression of ephrinB2 and EphB receptors within fibroproliferative membranes in patients with ocular angiogenic diseases collected during vitrectomy. METHODS: Fibroproliferative membranes were obtained at the time of vitrectomy from 20 patients with proliferative diabetic retinopathy (PDR) and from 40 patients who had stage 5 retinopathy of prematurity. Specimens were investigated with immunohistochemistry using polyclonal antibodies directed against ephrinB2 and the EphB2, EphB3, and EphB4 receptors. Immunoreactivity for von Willebrand factor (factor VIII) and alpha-smooth muscle actin (alpha-SMA) was also determined to confirm the identity of the target vascular endothelial cells. RESULTS: Positive staining for ephrinB2 was observed on fibroproliferative membranes that were obtained from patients with PDR (65.0%) and retinopathy of prematurity (25.0%). Specifically, ephrinB2 was found to be present on endothelial cells, as confirmed by its colocalization with factor VIII and alpha-SMA staining. EphB2 and EphB3 expression was observed on fibroproliferative membranes that were harvested from patients with PDR (EphB2, 90.0%; EphB3, 70.0%) and retinopathy of prematurity (EphB2, 35.0%; EphB3, 45.0%). However, EphB4 expression was not observed in any of the membranes derived from patients with PDR or retinopathy of prematurity. The rate of ephrinB2 expression in patients with PDR was significantly higher than that seen in patients with retinopathy of prematurity, which probably reflected differences in the vascular density of their fibroproliferative membranes. CONCLUSION: These data suggest that the ephrinB2-EphB2/B3 system may play an important role in ocular angiogenesis.

Equine infectious anemia viral vector-mediated codelivery of endostatin and angiostatin driven by retinal pigmented epithelium-specific VMD2 promoter inhibits choroidal neovascularization.

Equine infectious anemia virus (EIAV) is a nonprimate lentivirus that does not cause human disease. Subretinal injection into mice of a recombinant EIAV lentiviral vector in which lacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside within the optic nerve. Substitution of the RPE-specific promoter from the vitelliform macular dystrophy (VMD2) gene for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells, albeit reduced 6- to 10-fold compared with the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin [EIAV VMD2 Endo(FLAG)/Angio]; expression was approximately 6-fold lower than with identical vectors in which the CMV promoter drove expression. Compared with murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoter caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruch's membrane. These data support proceeding toward clinical studies with EIAV-based gene therapy for choroidal NV, using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.

Suppression and regression of choroidal neovascularization by systemic administration of an alpha5beta1 integrin antagonist.

Integrin alpha(5)beta(1) plays an important role in developmental angiogenesis, but its role in various types of pathologic neovascularization has not been completely defined. In this study, we found strong up-regulation of alpha(5)beta(1) in choroidal neovascularization. Implantation of an osmotic pump delivering 1.5 or 10 microg/h ( approximately 1.8 or 12 mg/kg/day) of 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid (JSM6427), a selective alpha(5)beta(1) antagonist, caused significant suppression of choroidal neovascularization; the area of neovascularization was reduced by 33 to 40%. When an osmotic pump delivering 10 microg/h of JSM6427 was implanted 7 days after rupture of Bruch's membrane, there was terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in vascular cells within the neovascularization and significant regression of the neovascularization over the next week. JSM6427 also induced apoptosis of cultured vascular endothelial cells. Fibronectin stimulates phosphorylation of extracellular signal-regulated kinase (ERK) in alpha(5)beta(1)-expressing cells that is blocked by JSM6427. These data suggest that alpha(5)beta(1) plays a role in the development and maintenance of choroidal neovascularization and provides a target for therapeutic intervention.

Intraocular injection of an aptamer that binds PDGF-B: a potential treatment for proliferative retinopathies.

Platelet-derived growth factor-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured fibroblasts with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with 20 microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.

Angiopoietin 1 prevents retinal detachment in an aggressive model of proliferative retinopathy, but has no effect on established neovascularization.

Vascular endothelial growth factor (VEGF) plays a central role in vasoproliferative diseases in the retina, however, other gene products modulate its effects. The angiopoietins are particularly important in this regard. Angiopoietin 2 (Ang2) collaborates with VEGF to stimulate neovascularization (NV) in some situations, but in other situations causes regression of NV. Ang2 also causes a transient increase in vascular density during retinal vascular development. In this study, we sought to determine if Ang1 has similar activities. The effects of Ang1 were tested in double transgenic mice with inducible expression of Ang1. Increased expression of Ang1 in the retina during retinal vascular development did not cause a detectable alteration in vascular density. Also, unlike Ang2, increased expression of Ang1 had no effect on established retinal or choroidal NV. However, when Ang1 expression was initiated simultaneously with that of VEGF, it strongly suppressed VEGF-induced NV and prevented retinal detachment. These data indicate that the timing of Ang1 expression is a critical determinate of its effects on VEGF-induced NV in the retina; it effectively blocks the initiation and progression of NV, but cannot reverse established NV or reduce leakage from NV. These data suggest that increased expression of Ang1 may be a good strategy for prophylaxis of retinal NV, but is unlikely to be effective as monotherapy of established NV.

Vascular targeting of ocular neovascularization with a vascular endothelial growth factor121/gelonin chimeric protein.

Tumors provide an extremely abnormal microenvironment that stimulates neovascularization from surrounding vessels and causes altered gene expression within vascular cells. Up-regulation of vascular endothelial growth factor (VEGF) receptors has allowed selective destruction of tumor vessels by administration of a chimeric protein consisting of VEGF121 coupled to the toxin gelonin (VEGF/rGel). We sought to determine whether there is sufficient up-regulation of VEGF receptors in endothelial cells participating in ocular neovascularization to permit a similar strategy. After intravenous injection of 45 mg/kg VEGF/rGel, but not uncoupled recombinant gelonin (rGel), there was immunofluorescent staining for rGel within choroidal neovascularization in mice and regression of the neovascularization occurred, demonstrating successful vascular targeting via the systemic circulation. Intraocular injection of 5 ng of VEGF/rGel also caused significant regression of choroidal neovascularization and regression of retinal neovascularization in two models, transgenic mice with expression of VEGF in photoreceptors and mice with ischemic retinopathy, whereas injection of 5 ng of rGel had no effect. These data suggest that the strategy of vascular targeting can be applied to nonmalignant neovascular diseases and could serve as the basis of a new treatment to reduce established ocular neovascularization.

Colocalization of Tie2, angiopoietin 2 and vascular endothelial growth factor in fibrovascular membrane from patients with retinopathy of prematurity.

PURPOSE: The tyrosine kinase receptor Tie2 and its ligands, the angiopoietins (Angs), play important roles in vascular integrity and neovascularization, modulating vascular endothelial growth factor (VEGF) activity. To elucidate the potential role of Angs and the Tie2 system in retinopathy of prematurity (ROP), we have investigated the expression of Angs, Tie2 and VEGF within fibroproliferative membranes in ROP. METHODS: Fibroproliferative membranes were obtained from 38 cases with stage 5 ROP at the time of vitrectomy. Membranes were fixed in formalin and embedded in paraffin. Each specimen was serially sectioned for immunohistochemistry. Polyclonal antibodies specific for Ang1, Ang2, Tie2 and VEGF were used for immunostaining. Immunoreactivity for von Willebrand factor (factor VIII) was also assessed to confirm the identity of vascular endothelial cells. RESULTS: Positive staining for Tie2 was observed in 23 of 38 specimens (60.5%). Tie2 was localized in vascularized regions of fibrovascular membranes and was co- expressed with VEGF and factor VIII. Ang2 stained positively in 18 of 38 (47.3%) serial sections where Tie2 was present, and was also co-expressed with VEGF and factor VIII. Ang1 was not generally observed in these specimens (3/38). CONCLUSIONS: VEGF and Ang2-Tie2 interactions may play an important role in the pathogenesis of ROP.

Non-paralleled increase of hepatocyte growth factor and vascular endothelial growth factor in the eyes with angiogenic and nonangiogenic fibroproliferation.

Vascular endothelial growth factor (VEGF) has been known as principal angiogenic factor in vasculogenesis, tumor angiogenesis and ocular angiogenesis. Currently, hepatocyte growth factor (HGF) has been reported to play a major role in ocular angiogenesis. We studied distribution of both growth factors in angiogenic and non-angiogenic fibroproliferation to determine the correlation of VEGF and HGF in retinal angiogenesis. Concentrations of VEGF and HGF molecules in vitreous samples from 27 eyes with angiogenic proliferative diabetic retinopathy (PDR) and 9 eyes with non-angiogenic proliferative vitreoretinopathy (PVR) were measured by enzyme-linked immunosorbent assay (ELISA). Vitreous samples with idiopathic macular role (IMH) served as a control. Concentrations of VEGF in the angiogenic PDR were 4.3 +/- 5.8 ng/ml (mean +/- SD), and were significantly higher than in non-angiogenic PVR (0.5 +/- 0.1 ng/ml). No significant differences were observed on VEGF concentrations between PVR to control. On the contrary, HGF concentrations were significantly higher in PVR (22.5 +/- 21.8 ng/ml) than in control (6.9 +/- 5.2 ng/ml), those of PDR (24.0 +/- 16.3 ng/ml) were also significantly higher than control. Among PDR samples, VEGF concentration was significantly higher than in the subgroup with higher angiogenic activity represented by iris neovascularization, although there were no significant differences on HGF concentration between the subgroups. Focal increases in HGF on fibroproliferation in the eye regardless of the involvement of angiogenesis were not in remarkable relation with angiogenic activity, unlike VEGF. These data suggested a more extensive role of HGF than VEGF strictly related to angiogenesis.