Determining vitreous viscosity using fluorescence recovery after photobleaching.

PURPOSE: Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. METHODS: Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. RESULTS: The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. CONCLUSIONS: FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous.

Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart.

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.

Effects of Bevacizumab, Ranibizumab, and Aflibercept on MicroRNA Expression in a Retinal Pigment Epithelium Cell Culture Model of Oxidative Stress.

PURPOSE: This study aimed to evaluate the effects of bevacizumab, ranibizumab, and aflibercept on the microRNA (miRNA) expression in human retinal pigment epithelium cell (ARPE-19) culture model of oxidative stress. METHODS: Control cells were cultured in the hydrogen peroxide (H2O2)-free medium. In H2O2 group ARPE-19 cells were exposed to 600 µM H2O2 alone for 18 h. In study groups, cells were preincubated with bevacizumab, ranibizumab, and aflibercept (1.25-2.5, 0.5 and 2.0 mg/mL, respectively) for 3 h before H2O2 exposure. Another group of ARPE-19 cells were incubated with drugs for 3 h without H2O2 exposure. Cell viability and vascular endothelial growth factor (VEGF) levels were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and enzyme-linked immunosorbent assay. The expression levels of 1,152 miRNAs were determined by quantitative real-time PCR. RESULTS: Incubation with 600 µM H2O2 alone for 18 h decreased cell viability by ∼50%. Cell viability was greater in the anti-VEGF drug groups compared with the H2O2 group, but the differences were not significant (P > 0.05). VEGF levels were significantly lower in the anti-VEGF drug groups compared with the H2O2 group (P < 0.05 for all study groups), with no significant differences between the study groups (P > 0.05). Incubation with anti-VEGF drugs alone had no effect on miRNA expression in ARPE-19 cells. However, preincubation with bevacizumab, ranibizumab, and aflibercept significantly altered the profile of H2O2-modulated miRNA expression. CONCLUSIONS: Preincubation with anti-VEGF drugs can alter the miRNA expression profile in response to H2O2-induced oxidative stress, and these drugs may have epigenetic effects.

Aflibercept Versus Bevacizumab and/or Ranibizumab for Recurrent Macular Edema Secondary to Central Retinal Vein Occlusion.

PURPOSE: To compare functional and anatomic outcomes of treatment with intravitreal aflibercept versus bevacizumab and/or ranibizumab in patients with recurrent macular edema (ME) secondary to central retinal vein occlusion (CRVO). METHODS: Retrospective, comparative case series of patients with recurrent ME in the setting of CRVO. Patients with recurrent ME received treatment with aflibercept (Group 1, G1) or bevacizumab and/or ranibizumab (Group 2, G2). Primary outcome measures were best-corrected visual acuity (BCVA) and central foveal thickness (CFT). RESULTS: Of the 20 eyes (20 patients) with recurrent ME included in the study, 9 received aflibercept (G1) and 11 received bevacizumab and/or ranibizumab (G2). Median BCVA at recurrence of ME and at most recent follow-up was 20/60 (G1) and 20/80 (G2) and 20/40 (G1) and 20/50 (G2, P > 0.05 for all comparisons), respectively. Median CFT at recurrence of ME and at most recent follow-up was 492 µm (G1) and 448 µm (G2) and 291 µm (G1) and 295 µm (G2, P > 0.05 for all comparisons), respectively. Complete resolution of ME for at least 4 months was found in 78% (G1) and 55% (G2) of patients with a median injection free interval of 11 (G1) and 13 (G2) months (P > 0.05). CONCLUSIONS: In patients with recurrent ME secondary to CRVO, there was improvement in BCVA and CFT in all groups, although patients treated with aflibercept showed a trend toward better anatomical outcomes decreased need for recurrent injections.

Long-term release and stability of pharmaceutical proteins delivered from solid lipid implants.

Solid lipid implants (SLIs) prepared by twin-screw (tsc) extrusion represent a promising technology platform for the sustained release of pharmaceutical proteins. In this work, we report on two aspects, long-term release and stability of released protein. First, SLIs were produced by tsc-extrusion containing the low melting triglyceride H12 and the high melting triglyceride Dynasan D118. Two different proteins available in a freeze-dried matrix containing hydroxypropyl-ß-cyclodextrine (HP-ß-CD) were incorporated into the lipid matrix: a monoclonal antibody (mAb) from the IgG1 class and the fab-fragment Ranibizumab (Lucentis®). SLIs, composed of 10% protein lyophilizate and both triglycerides, were extruded at 35°C and 40rpm. Sustained release of both proteins was observed in a sustained manner for approximately 120days. Protein load per implant was increased by three different approaches resulting in a protein load of 3.00mg per implant without affecting the release profiles. The incubation medium containing the released protein was collected, concentrated and analyzed including liquid chromatography (SE-HPLC, IEX, HIC), electrophoresis (SDS-PAGE, on-chip gel electrophoresis) and FT-IR spectroscopy. The mAb showed a monomer loss of up to 7% (SE-HPLC) and IEX analysis revealed the formation of 16% acidic subspecies after 18weeks. FT-IR spectra of mAb indicated the formation of random coil structures towards the end of the release study. Ranibizumab was mainly released in its monomeric form (>95%), and approximately 5% hydrophobic subspecies were formed after 18weeks of release. FT-IR analysis revealed no changes in secondary structure. The release and stability profiles of both proteins underline the potential of SLIs as a delivery system. SLIs provide a promising platform for applications where really long-term release is needed, for example for intraocular delivery of anti-vascular endothelial growth factor (VEGF) drugs for age related macular degeneration (AMD).

Development of a Sustainable Release System for a Ranibizumab Biosimilar Using Poly(lactic-co-glycolic acid) Biodegradable Polymer-Based Microparticles as a Platform.

Ranibizumab is a humanized monoclonal antibody fragment against vascular endothelial growth factor (VEGF)-A and is widely used to treat age-related macular degeneration (AMD) caused by angiogenesis. Ranibizumab has a short half-life in the eye due to its low molecular weight and susceptibility to proteolysis. Monthly intravitreal injection of a large amount of ranibizumab formulation is a burden for both patients and medical staff. We therefore sought to develop a sustainable release system for treating the eye with ranibizumab using a drug carrier. A ranibizumab biosimilar (RB) was incorporated into microparticles of poly(lactic-co-glycolic acid) (PLGA) biodegradable polymer. Ranibizumab was sustainably released from PLGA microparticles (80+% after 3 weeks). Assay of tube formation by endothelial cells indicated that RB released from PLGA microparticles inhibited VEGF-induced tube formation and this tendency was confirmed by a cell proliferation assay. These results indicate that RB-loaded PLGA microparticles are useful for sustainable RB release and suggest the utility of intraocular sustainable release systems for delivering RB site-specifically to AMD patients.

PERMEABILITY AND ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR EFFECTS OF BEVACIZUMAB, RANIBIZUMAB, AND AFLIBERCEPT IN POLARIZED RETINAL PIGMENT EPITHELIAL LAYER IN VITRO.

PURPOSE: To determine the effects of bevacizumab, ranibizumab, and aflibercept on the permeability and the effects of anti-vascular endothelial growth factor (VEGF) on highly polarized retinal pigment epithelial cells (RPECs) in vitro. METHODS: Highly polarized RPECs were cultured in the upper chamber of a Transwell system. Anti-VEGF antibodies were added to the upper chamber, and the concentrations of the drugs in the lower chambers were measured. The permeability rates of the three anti-VEGF drugs through the RPEC layer and the concentration of VEGF in each chamber were determined. RESULTS: The permeability of aflibercept was significantly lower by about 40% than that of bevacizumab through the RPEC layer (P < 0.05). Ranibizumab was significantly more permeable through the RPECs than bevacizumab (180% of bevacizumab, P < 0.05). Although VEGF was almost absent in the upper chamber after exposure to the 3 antibodies, it was decreased more significantly with aflibercept than with bevacizumab in the lower chamber (2.8% vs. 65.8% of control; P < 0.01). Ranibizumab also decreased the VEGF level compared with bevacizumab (31.7% vs. 65.8% of control; P < 0.01). CONCLUSION: The greater reduction of the amount of VEGF in the lower chamber by aflibercept and ranibizumab than bevacizumab may explain why aflibercept and ranibizumab are more effective than bevacizumab against type 1 choroidal neovascularization.

Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions.

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under normal and oxidative stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5 days after treatment. Finally, none of the drugs affected migration. In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.