Aflibercept

CNVs were generated as described previously.⁴⁹ (link) In brief, under deep anesthesia, laser photocoagulation (532 nm, 180 mW, 75 μm, 100 ms; Novus Spectra; Lumenis, Tokyo, Japan) was performed around the optic disc using a slit-lamp delivery system with a cover glass as a contact lens. Four laser spots per eye for immunohistochemistry and six laser spots for mRNA and protein analysis were placed. The formation of a subretinal bubble at the time of laser photocoagulation indicated the rupture of Bruch’s membrane. (P)RR-PshRNA, aflibercept (2.5 μg; Bayer, Leverkusen, Germany), or a mixture of (P)RR-PshRNA and aflibercept in 1 μL PBS per eye was injected into the vitreous cavity of mice immediately after laser injury. An additional injection was given on day 7 for quantification of subretinal fibrosis at day 21 after laser. Controls received control-PshRNA or PBS. The dose of in vivo injection of control or (P)RR-PshRNA was determined to be 100 pM in 1 μL PBS per eye, at which (P)RR-PshRNA significantly suppresses the size of CNV in dose-ranging experiments.

Rabbit RPE cells were isolated and maintained as described by Chang et al.^{12 (link)} After incubating the globes with 2% dispase for 15 minutes, an incision was made 3 mm from the limbus and continued circumferentially. After removal of the cornea and lens, 4 radial incisions were made in the posterior segment, and this part was incubated in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/ F12) medium augmented with 10% fetal bovine serum for 2 hours. Finally, the RPE cells were separated from the neural retina and choroid as a sheet with micropipettes and observed under a stereo microscope (Olympus BX51, Japan). Passage 3 cells were used for the study and drugs were applied to the cultures 24 hours after fresh cell plating.
Ranibizumab (Lucentis, Novartis, Switzerland), a fragment of a human monoclonal antibody against VEGF-A selectively binds all isoforms of VEGF-A (VEGF110, VEGF121, and VEGF165), was applied at a concentration of 0.125 mg/mL. Bevacizumab (Avastin, Genetech/Roche, USA), a monoclonal antibody against VEGF which is used off-label to treat various eye diseases, was added to the cultures at a concentration of 0.3125 mg/mL. Aflibercept (Eylea, Bayer Health Care, Germany), a fusion protein that binds to circulating VEGF (subtypes VEGF-A and VEGF-B) and placental growth factor (PGF), was used at a concentration of 0.5 mg/mL.

Regorafenib, 4‐[4‐ ({[4‐Chloro‐3‐(trifluoromethyl) phenyl]carbamoyl}amino)‐3‐fluorophenoxy]‐N‐methylpyridine‐2‐carboxamide monohydrate, was purchased from Selleck Chemicals Co., Ltd. and Active Biochem, Ltd.. Pazopanib, 5‐[[4‐[(2,3‐dimethylindazol‐6‐yl)‐methylamino]pyrimidin‐2‐yl]amino]‐2‐methylbenzenesulfonamide hydrochloride was purchased from SYNKinase Co., Ltd. Hydroxypropyl cellulose, sodium dihydrogen phosphate, sodium chloride and sodium hydroxide were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Light liquid paraffin and benzalkonium chloride were purchased from NACALAI TESQUE, Inc (Kyoto, Japan). Polysorbate 80 and D‐mannitol were purchased from Junsei Chemical Co., Ltd. Captisol was purchased from Ligand Pharmaceuticals, Inc. Aflibercept (40 mg/mL EYLEA^® Injection For Intravitreal Injection) was purchased from Bayer Yakuhin, Ltd. Mydrin‐P ophthalmic Solution was purchased from Santen Pharmaceutical Co., Ltd.. Scopisol solution was purchased from Senju Pharmaceutical Co., Ltd. (Osaka, Japan). Fluorescite Intravenous Injection 500 mg was purchased from Alcon Japan Ltd. (Tokyo, Japan).