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Objective: To evaluate long-term efficacy and safety profiles during 3 years of individualized ranibizumab treatment in patients with visual impairment due to diabetic macular edema (DME). Design: Phase 1Mb, multicenter, 12-month, randomized core study and 24-month open-label extension study. Participants: Of the 303 patients who completed the randomized RESTORE 12-month core study, 240 entered the extension study. Methods: In the extension study, patients were eligible to receive individualized ranibizumab treatment as of month 12 guided by best-corrected visual acuity (BCVA) and disease progression criteria at the investigators' discretion. Concomitant laser treatment was allowed according to the Early Treatment Diabetic Retinopathy Study guidelines. Based on the treatments received in the core study, the extension study groups were referred to as prior ranibizumab, prior ranibizumab + laser, and laser. Main Outcome Measures: Change in BCVA and incidence of ocular and nonocular adverse events (AEs) over 3 years. Results: Overall, 208 patients (86.7%) completed the extension study. In patients treated with ranibizumab during the core study, consecutive individualized ranibizumab treatment during the extension study led to an overall maintenance of BCVA and central retinal subfield thickness (CRST) observed at month 12 over the 2-year extension study (+8.0 letters, -142.1 µm [prior ranibizumab] and +6.7 letters, -145.9 µm [prior ranibizumab + laser] from baseline at month 36) with a median of 6.0 injections (mean, 6.8 injections; prior ranibizumab) and 4.0 (mean, 6.0 injections; prior ranibizumab + laser). In the prior laser group, a progressive BCVA improvement (+6.0 letters) and CRST reduction (-142.7 µm) at month 36 were observed after allowing ranibizumab during the extension study, with a median of 4.0 injections (mean, 6.5 injections) from months 12 to 35. Patients in all 3 treatment groups received a mean of <3 injections in the final year. No cases of endophthalmitis, retinal tear, or retinal detachment were reported. The most frequently reported ocular and nonocular adverse effects over 3 years were cataract (16.3%) and nasopharyngitis (23.3%). Eight deaths were reported during the extension study, but none were suspected to be related to the study drug/procedure. Conclusions: Ranibizumab was effective in improving and maintaining BCVA and CRST outcomes with a progressively declining number of injections over 3 years of individualized dosing. Ranibizumab was generally well tolerated with no new safety concerns over 3 years. [ABSTRACT FROM AUTHOR]

Background: The primary objective of LUTEGA is to determine the long-term effect of a supplementation with fixed combination of lutein, zeaxanthin, omega-3-longchain-polyunsaturated-fatty-acids (O-3-LCPUFAs) and antioxidants on macular pigment optical density (MPOD) in patients with non-exudative age-related macular degeneration (AMD). Methods: The LUTEGA study is a double-blind, placebo-controlled clinical trial. 172 patients with non-exudative AMD were enrolled and randomized to three treatment arms. Supplementation included either once (dosage D1) or twice daily (dosage D2) of 10 mg L / 1 mg Z/ O-3-LCPUFAs (thereof 100 mg DHA, 30 mg EPA)/ antioxidants, or placebo (P). After best-corrected visual acuity (BCVA) test, blood sample was collected and MPOD was measured using the 1-wavelength-reflection method and recording reflection images at 480 nm (modified Visucam NM/FA, Carl Zeiss Meditec, Germany). During 1 year of intervention, AMD patients were followed up after 1, 3, 6 and 12 months. 145 AMD patients (D1 = 50, D2 = 55, P = 40) completed the study. Results: After 12 months of intervention, the MPOD parameters (volume, area, maxOD, meanOD) increased significantly in treatment arms D1 and D2 ( p < 0.001). Volume of MPOD showed the highest within-group difference and increased significantly in D1 and D2, and decreased significantly in P ( p = 0.041). Between-group comparison of absolute changes of all MPOD parameters were significantly different between D1 and P as well as D2 and P with p < 0.001 at end point ( t = 12). BCVA, measured in log MAR, improved in D1 and in D2 ( p < 0.001). After 12 months of intervention, the mean improvement in BCVA was significant in D2 ( p = 0.006) and D1 ( p = 0.038) compared to P. Conclusions: The supplementation of L, Z, O-3-LCPUFAs and antioxidants resulted in considerable increase in MPOD. There was no difference in accumulation of MPOD between both dosages. Thus, we believe that the used supplementation with L and Z seems to reach a saturation level in retinal cell structure. Additionally, the constant supplementation of L, Z, O-3-LCPUFAs and antioxidants in AMD patients seems to be useful, because MPOD reduces without supplementation. We conclude that the supplementation caused an increase of MPOD, which results in an improvement and stabilization in BCVA in AMD patients. Thus, a protective effect on the macula in AMD patients is assumed. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the 2-year safety and efficacy of ranibizumab 0.5 mg in diabetic macular edema (DME). Design: Twenty-four-month, open-label, multicenter, Phase IIIb extension study. Participants: Two hundred forty of 303 patients with visual impairment due to DME who completed the RESTORE core study and entered the extension. Methods: All patients were eligible to receive ranibizumab 0.5 mg pro re nata (PRN) from month 12 (end of core study) to month 36 based on best-corrected visual acuity (BCVA) stability and disease progression retreatment criteria. Patients were also eligible to receive laser PRN according to Early Treatment Diabetic Retinopathy Study guidelines. A preplanned interim analysis was performed at month 24, stratifying by treatment groups as in the RESTORE core study and referred to as prior ranibizumab, ranibizumab plus laser, or laser groups in the extension. Main Outcome Measures: Incidence of ocular and nonocular adverse events (AEs) and mean change in BCVA. Results: Two hundred twenty patients (92%) completed the month 24 visit. Over 2 years, the most frequent ocular serious AE (SAE) and AE were cataract (2.1%) and eye pain (14.6%), respectively. The main nonocular AEs were nasopharyngitis (18.8%) and hypertension (10.4%). There were no cases of endophthalmitis, and the incidences of nonocular SAEs were low. Of the patients entering the extension, 4 deaths were reported in the second year, none of which were related to study drug or procedure. Mean BCVA gain, central retinal thickness (CRT) decrease, and National Eye Institute Visual Functioning Questionnaire-25 (NEI VFQ-25) composite score observed at month 12 were maintained at month 24 (prior ranibizumab: +7.9 letters, –140.6 μm, and 5.6, respectively; prior ranibizumab plus laser: +6.7 letters, –133.0 μm, and 5.8, respectively), with an average of 3.9 (prior ranibizumab) and 3.5 ranibizumab injections (prior ranibizumab plus laser). In patients treated with laser alone in the core study, the mean BCVA, CRT, and NEI VFQ-25 composite score improved from month 12 to month 24 (+5.4 letters, –126.6 μm, and 4.3, respectively), with an average of 4.1 ranibizumab injections. Conclusions: Ranibizumab 0.5 mg administered according to prespecified visual stability and disease progression criteria was well tolerated, with no new safety concerns identified over 2 years. Overall, an average of 3.8 ranibizumab injections was sufficient to maintain (prior ranibizumab) or improve (prior laser) BCVA, CRT, and NEI VFQ-25 outcomes through the second year. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. [Copyright &y& Elsevier]

PURPOSE. Retinal pigment epithelial (RPE) cellproliferation is an important step in the pathogenesis of ocular diseasessuch as proliferative retinopathy. Growth factors play a major role in modulatingthe development and progression of these diseases. The aim of this study wasto examine the effects of single growth factors as compared to growth factorcombinations on the stimulation of RPE cell proliferation. METHODS. Human RPE cells were treated with 10 ng/ml IGF-1, PDGF, bFGF,EGF, VEGF, and TGFβ[sub 2] , or with the combination of each of thegrowth factors. RPE cell proliferation was determined by [[sup 3] H]-thymidineincorporation after growth factor stimulation for 24 hr. RESULTS. RPE cell proliferation was significantly increased when PDGF andbFGF were combined as compared to the single growth factor ( p <0.005). There was a decrease in DNA synthesis for combined growth factorsas compared to the single growth factor for IGF-1 plus TGFβ[sub 2] ( p < 0.05), IGF-1 plus VEGF ( p < 0.005), PDGF plusTGFβ[sub 2] ( p < 0.005), VEGF plus TGFβ[sub 2] ( p < 0.005 as compared to VEGF), and bFGF plus TGFβ[sub 2] ( p = 0.01 as compared to bFGF). The remaining growth factorcombinations showed no significant synergistic effect on human RPE cell proliferation. CONCLUSIONS. These data lead to the implication that DNA synthesis in humanRPE cell culture is synergistically affected by certain growth factor combinations,but not by all of them. This observation may be important for a better understandingof growth factor interactions occuring in proliferative diseases in the eye. [ABSTRACT FROM AUTHOR]

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The main aim of this study was to evaluate the ability of serum biomarkers to predict the worsening of retinal neurodysfunction in subjects with type 2 diabetes. For this purpose, we measured selected molecules (N-epsilon-carboxy methyl lysine (CML), laminin P1 (Lam-P1), and asymmetric dimethylarginine (ADMA)) in the serum of 341 participants of the EUROCONDOR study at baseline, 24, and 48 weeks. Retinal neurodysfunction was assessed by measuring implicit time (IT) using multifocal electroretinography, and structural changes were examined by spectral domain–optical coherence tomography. The values of IT at baseline were directly correlated with baseline serum concentrations of CML (r = 0.135, p = 0.013). Furthermore, in the placebo group, increase in CML concentration throughout follow-up correlated with the IT (r = 0.20; p = 0.03). Baseline serum levels of CML also correlated with macular retinal thickness (RT) (r = 0.231; p < 0.001). Baseline Lam-P1 levels correlated with the increase of the RT at the end of follow-up in the placebo group (r = 0.22; p = 0.016). We provide evidence that CML may be a biomarker of both retinal neurodysfunction and RT, whereas Lam-P1 was associated with RT only. Therefore, circulating levels of these molecules could provide a complementary tool for monitoring the early changes of diabetic retinopathy (DR). [ABSTRACT FROM AUTHOR]

Purpose: Intravitreal injection of the VEGF-binding protein aflibercept is widely used to treat various ocular diseases. In vitro, immortalized bovine retinal endothelial cells (iBREC) take up and transport aflibercept through the cell layer in a serum-dependent manner, likely mediated through the neonatal Fc receptor (FcRn), but degradation of the Fc domain-containing protein might be a competing intracellular process. Therefore, aflibercept's associations with proteins either involved in FcRn-mediated transport or in the lysosomal pathway were studied.Methods: Confluent iBREC pre-cultivated with or without FBS were exposed for 4 h to in vivo achievable 250 μg/ml aflibercept, before cells were harvested for immunofluorescence staining or preparation of protein extracts. Intracellular localization of aflibercept and putative co-localizations with proteins involved in transport of IgG/FcRn complexes, i.e., endosomal Rab4 and Rab11, components of the cytoskeleton, motor proteins, or with marker proteins characteristic of multivesicular bodies or lysosomes were assessed by co-immunofluorescence stainings. Amounts of expressed endogenous proteins and of internalized aflibercept were determined by Western blot analyses.Results: Aflibercept-specific perinuclear staining overlapped with that of the motor protein dynein whereas double staining with an anti-kinesin antibody resulted in a patchy pattern. In addition, aflibercept was typically present close to microtubules and often co-localized with α-tubulin. Rab4 and Rab11 stainings partly overlapped with the perinuclear staining of aflibercept whereas co-localization with Rab7 (in late endosomes/lysosomes) was only rarely seen. Interestingly, aflibercept but not the IgG bevacizumab broadly co-localized with the cation-independent mannose 6-phosphate receptor characteristic of multivesicular endosomes. In accordance with partial degradation beside transcytosis, the amount of intracellular aflibercept increased when cells were treated with protease inhibitors MG-132 or MG-101. Serum-deprived iBREC expressed less Rab11 and dynein but slightly more Rab4.Conclusion: After uptake by iBREC, aflibercept is present in organelles associated with FcRn-mediated transport, but part of the protein is subject to degradation. Transport inhibition of aflibercept during cultivation without FBS is likely a consequence of an attenuated exocytosis due to decreased expression of Rab11. [ABSTRACT FROM AUTHOR]

Background/Aims: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A165), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A165, we investigated if its signaling pathways triggered in REC are redundant. Methods: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A165. Permeability was monitored continuously by measurements of the cell index (Cl) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. Results: After a sharp but transient Cl drop caused by VEGF-A155 early after its addition, further exposure resulted in a continuous Cl decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A165, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. Conclusions: Specific kinase inhibitors differently affect VEGF-A165-triggered processes in distinct phases of its action. VEGF-A165-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown. [ABSTRACT FROM AUTHOR]

Retinal endothelial cells (REC) likely contribute to the clearance of intravitreally injected IgG. Because this is of high relevance to the pharmacokinetic assessment of the widely used therapeutic Fc fusion protein aflibercept, we studied its transport through immortalized bovine REC (iBREC) in detail. For shuttling of IgG or Fc fusion proteins like aflibercept, endothelial cells use the highly conserved neonatal Fc receptor (FcRn) also expressed in iBREC where it is down regulated by serum depletion. Therefore, we focused on studying intracellular localization and transport of aflibercept under conditions affecting its interaction with the FcRn. Intracellular localization of aflibercept was assessed by Western-blot analyses of subcellular protein fractions or by immunofluorescence staining. After uptake in a temperature-dependent process, aflibercept co-localized with early endosomes, which harbor FcRn. Similar amounts of aflibercept were co-extracted with proteins from membranes/organelles irrespectively of the amount of FBS in the culture medium. Lowering the concentration of FBS resulted in a strong, but reversible association with cytoskeletal proteins suggesting a block in intracellular transport. In accordance with this finding, aflibercept's transport through an iBREC monolayer grown on porous membrane inserts was markedly delayed in the absence of FBS in the culture medium indicating that aflibercept is taken up but not exocytosed under these conditions. Transcytosis of aflibercept was also strongly delayed by inhibition of phosphatidylinositol 3-kinase with LY294002, which affects FcRn-mediated IgG transport. A similar inhibition of aflibercept's transport was observed with IgG-binding proteins (i.e. protein A or protein G) that block interaction between FcRn and aflibercept. Interfering with aflibercept's binding to the FcRn with protein A (or protein G) or the inhibitory FcRn-specific monoclonal antibody 1G3 resulted in a reduced amount of intracellular aflibercept. Taken together, our results strongly suggest that FcRn is involved in transport of aflibercept through REC in vitro. [ABSTRACT FROM AUTHOR]

PURPOSE. The retinal pigment epithelium (RPE) isinvolved in the pathogenesis of age-related macular degeneration. The purposeof this study was to assess the effect of thalidomide, octreotide, and prednisoloneon the proliferation and migration of bovine RPE cells in vitro . METHODS. The migration assay was performed in double-chamber-wells separatedby a membrane filter with 8 μm pores. Cells were allowed to migrate verticallyfor 7 hr, afterwards the cells on both filtersides were fixed, stained, andthe migrated cells were counted. To examine RPE proliferation, bovine RPEcells were seeded subconfluently followed by an incubation with octreotide,thalidomide or prednisolone in a concentration gradient for 24 hr. Stimulationor inhibition of DNA synthesis was measured by [[sup 3] H]-thymidine incorporation.Statistical analysis was performed with the paired student's t-test. RESULTS. Statistically significant (p < 0.05) inhibition of RPE cellproliferation was measured for thalidomide at a concentration of 10–50 μg/ml,for octreotide at a concentration of 5 × 10[sup -4] and 5 ×10[sup -5] M, and for prednisolone at a concentration of 250 and 500 μg/mlas compared to the negative control. RPE cell migration was significantly(p < 0.05) inhibited by thalidomide at a concentration of 10 μg/ml,by octreotide at a concentration of 5 × 10[sup -5] M, and alsoby prednisolone at a concentration of 500 μg/ml as compared to the negativecontrol. CONCLUSIONS. Although the main effect of thalidomide, octreotide, and prednisolonewhen treating patients with choroidal neovascular membranes is probably relatedto the inhibition of angiogenesis it should be kept in mind that these substancesmay additionally inhibit RPE proliferation and migration. [ABSTRACT FROM AUTHOR]

Bevacizumab is one of the VEGF-binding proteins that are established in clinical practice to treat various ocular diseases. In view of therapeutic long-term application, potential accumulation of the antibody in retinal cells gave reason for safety concerns. Internalization of considerable amounts of bevacizumab by retinal endothelial (REC) and pigment epithelial cells has been observed which may affect their important functions. Therefore we investigated the transport and intracellular localization of bevacizumab in immortalized bovine REC (iBREC) in detail, considering possible roles of vesicles and receptors mediating uptake and intracellular transport. By performing transcytosis assays with iBREC monolayers cultivated on porous membrane inserts, we demonstrated that bevacizumab was transported efficiently through a tight monolayer from the lower to the upper chamber or vice versa. When added to the lower chamber in excess, the internalized antibody was transported through the cells, but it was also recycled to be set free at the same side of the cell into a bevacizumab-free environment. The rates of both processes strongly depended on the concentration of fetal bovine serum (FBS) in the environment. This observation is important because in vivo REC might be exposed to varying amounts of serum, e.g. in patients with macular edema. FBS also affected the intracellular localization of bevacizumab as shown by analyses of subcellular fractions and direct immunofluorescence staining. When iBREC were cultivated in low-serum medium, most of the antibody was found in the fraction of cytoskeleton proteins and spots of high intensity of bevacizumab-specific staining close to the nuclei were observed. Cultivation in medium with FBS resulted in internalized bevacizumab predominately found in the membrane/organelle fraction in addition to its weaker association with proteins from the cytoskeleton and uniform staining of the cell. Bevacizumab-specific staining close to the cytoskeleton proteins α-tubulin or vimentin was also observed. Accumulation and association of the antibody with the cytoskeleton induced by serum reduction could be reversed by subsequent FBS addition. In uptake and transport of bevacizumab vesicles and binding to a receptor seems to be involved: Internalization was strongly temperature-dependent which ruled out paracellular passage and a fraction of the internalized bevacizumab was associated with early endosomes. Protein A inhibited transcytosis and affected intracellular localization suggesting a key role of the neonatal Fc receptor (FcRn). Interestingly, FcRn expression was decreased when iBREC were cultivated without FBS. Our results suggest this pathway of bevacizumab uptake and transition through iBREC: Independent of serum, bevacizumab is taken up through a nonspecific mechanism. The subsequent sorting into transport vesicles depends on the presence of serum as regulator of FcRn expression. Without sufficient amounts of the receptor being expressed, a likely obstructed exocytosis results in intracellular accumulation and an increased association with cytoskeleton proteins. Interaction of substantial amounts of bevacizumab with the cytoskeleton may be the reason for under these conditions suppressed migration of iBREC. If long-term therapies by intravitreal injection lead to accumulation of bevacizumab in REC in vivo and potentially harmful consequences, will have to be revealed by future investigations. [ABSTRACT FROM AUTHOR]

Abstract: Members of the vascular endothelial growth factor (VEGF) family differently regulate processes in retinal endothelial cells (REC) which are crucially involved in the pathogenesis of diabetic retinopathy: Both, VEGF-A and placenta growth factor (PlGF), stimulate proliferation of primary and immortalized bovine REC ((i)BREC) but only VEGF-A165 stimulates their migration. Diabetic macular edema is most likely a consequence of an elevated permeability of REC which can be induced by VEGF-A, but not by PlGF. Binding of VEGF-A by the antibody fragment ranibizumab is sufficient to completely restore or prevent VEGF-A-induced disturbance of the iBREC barrier or migration of these cells without affecting the basal processes. This was observed even in the presence of other growth factors when surplus proliferation was only partly blocked. The recombinant protein aflibercept (VEGF-trap) not only binds very strongly to VEGF-A, but – in contrast to ranibizumab – also recognizes PlGF. In this study, we investigated whether this additional targeting of PlGF also results in better inhibition of growth factor-induced proliferation and migration, and disturbance of the iBREC barrier. In addition, uptake of aflibercept by iBREC and potential functional consequences were examined. In accordance with its binding specificity, aflibercept strongly and specifically inhibited iBREC proliferation stimulated with VEGF-A, PlGF or a combination of these factors. By treatment with aflibercept at therapeutically achievable concentrations, VEGF-A-stimulated iBREC migration was reduced not only to normal values but driven below the basal level. However, the VEGF-A binding humanized antibody bevacizumab as well as the unrelated control antibody rituximab also inhibited basal or VEGF-A stimulated migration at clinically relevant concentrations, suggesting an effect of high amounts of IgG domain-containing proteins which does not depend on their binding specificity. However, aflibercept specifically blocked VEGF-A stimulated migration at lower concentration without influencing basal processes. Effects on permeability were determined by measuring transendothelial resistance (TER) of iBREC (±VEGF-A165) and their expression of the tight junction protein claudin-1. The VEGF-A-disturbed barrier was completely restored by treatment with ≤25 μg/ml aflibercept of which even much higher concentrations did not interfere with normal barrier function. Uptake of aflibercept by iBREC – analyzed by Western blot – was observed after 1 h of treatment and the amount further increased during prolonged incubation. Most of the internalized aflibercept was present in subcellular fractions of proteins assigned to the membranes and organelles, but it was also detected in the fraction consisting of cytoskeletal proteins. Co-immunofluorescence staining showed aflibercept absorbed by iBREC to be localized in or close to the Golgi apparatus. Aflibercept at high concentrations interferes with an important normal iBREC function, but prevents and restores VEGF-A-induced disturbances at considerably lower concentrations. Therefore, reduction of the doses administered in DR and DME therapy might be considered. [Copyright &y& Elsevier]

Abstract: Purpose: To compare the efficacy and safety of ranibizumab 0.5 mg versus dexamethasone 0.7 mg according to their European labels in macular oedema secondary to branch retinal vein occlusion (BRVO) in a 6‐month, phase IIIb, randomized trial. Methods: Patients received either monthly ranibizumab for 3 months followed by Pro re nata (PRN) treatment (n = 126) or a sustained‐release dexamethasone implant followed by PRN sham injections (n = 118). Main outcomes were mean average change in best‐corrected visual acuity (BCVA) from baseline to month 1 through month 6, mean changes in BCVA and foveal centre point thickness (FCPT), and adverse events (AEs). Results: There was no difference in BCVA gains between the treatments prior to month 3. Best‐corrected visual acuity (BCVA) gain with dexamethasone declined thereafter. From month 3 to month 6, mean BCVA change from baseline was significantly higher with ranibizumab than with dexamethasone [raw means (standard deviation):+16.2 (±11) letters versus +9.3 (±10.1) letters]. At month 6, the difference in BCVA gains from baseline was +17.3 letters in the ranibizumab versus +9.2 letters in the dexamethasone group. Patients in the ranibizumab group received a mean of 2.94 loading injections and 1.74 PRN retreatment injections, while those in the dexamethasone group received a single loading injection. Elevated intraocular pressure (IOP) and AEs were more frequent with dexamethasone than ranibizumab treatment. Conclusion: Ranibizumab PRN resulted in greater visual acuity (VA) gains in macular oedema following BRVO compared with single‐dose dexamethasone over a 6‐month study period, observed from month 3, when administered according to their European label. In clinical practice, retreatment with dexamethasone may be required prior to this point. [ABSTRACT FROM AUTHOR]