Bevacizumab

Macular edema was defined as a CMT ≥ 300 μm based on the ETDRS protocol, which involved segmentation of the macula into a central circle, inner ring, and outer ring of 1-, 3-, and 6-mm diameters. The CMT was measured by calculating the average retinal thickness in a circle of 1 mm diameter centered on the fovea, and measuring the distance between the first signal from the vitreoretinal interface and the outer border of the retinal pigment epithelium. Recurrence of ME was defined as the reappearance of ME in eyes with previously resolved ME (CMT < 300 μm).
Macular edema was treated with as-needed intravitreal injections of anti-VEGF agents, bevacizumab (1.25 mg; Avastin, Genentech, San Francisco, CA, USA) or ranibizumab (Lucentis, 0.5 mg; Genentech, Inc., San Francisco, CA, USA). If three consecutive monthly anti-VEGF injections failed to resolve ME, the patient was switched to intravitreal dexamethasone implant or triamcinolone acetonide injections.

This was a retrospective multi-center study involving multiple eye clinics in the Australian states of South Australia and Tasmania. All participants were enrolled through the Tasmanian Ophthalmic Biobank Study (University of Tasmania) or the Genetic Risk Factors in Complications of Diabetes Study (Flinders University). The eligibility criteria included T1 and T2 DM patients ≥18 years who had received any intravitreal anti-VEGF injections (Aflibercept, Regeneron; Bevacizumab, Genentech; Ranibizumab, Novartis) between 2013 and 2020 for the treatment of DME. DME cases were defined as those with clinically diagnosed center-involving DME and confirmed by central macular thickness (CMT ≥ 315 microns) measured by optical coherence tomography (OCT). Eyes with cysts in the central 1000 microns or any intraretinal or subretinal fluid were included in this study, independent of the CMT parameter. Exclusion criteria were: (a) any vitreoretinal surgery, systemic anti-VEGF therapy, or intra-ocular steroid in the six months preceding the initiation of anti-VEGF injection, (b) insufficient visibility of the fundus for retinal diagnosis, (c) incomplete follow-up data, and (d) inability to give consent. The better responding eye was included as the study eye for patients receiving bilateral anti-VEGF injections.

Experiments involving human and murine cell lines were performed on 8-12 week old female athymic nude and 4-6 week old female C57/Bl6 mice, respectively, obtained from Taconic Farms (Hudson, NY). All experiments were done in accordance to protocols approved by MD Anderson Institutional Animal Care and Use Committee.
Tumor cells were injected intraperitoneally (1x 10⁶ cells/mouse for IG10, OVCAR432, and OVCAR5) into mice in all groups on day 0. The patient-derived cell line MDA-HGSC-1 (2414) was injected intraperitoneally as ascites, after being grown in NOD-SCID mice. Nude mice were treated with anti-VEGF antibody, bevacizumab, 5 mg/kg, twice weekly, intraperitoneal injection, while C57/Bl6 mice were treated with murine monoclonal VEGF-A and VEGFR-2 antibody, B20, 5 mg/kg, twice weekly, intraperitoneal injections (Genentech Inc, San Francisco, CA). AC708 was given 90 mg/kg, daily oral gavage. 2G2 was administered once weekly via intraperitoneal injection at a dose of 30 mg/kg. Paclitaxel 4 mg/kg once weekly, intraperitoneal injection was given to nude mice. Treatment for therapy experiments began 7 days after injection.

VEGF (specifically, VEGF165 isoform, which shares amino acid sequences between human and canine species [44 (link)]), a key mediator of angiogenesis; the RTK inhibitor SU5416; and the selective COX-2 inhibitor, celecoxib, were obtained from Sigma Aldrich (Madrid, Spain). The anti-VEGF drug, bevacizumab, was kindly supported by Genentech Inc. (San Francisco, USA).
All drugs were dissolved in dimethyl sulfoxide (DMSO), stored at −20 °C and diluted in the corresponding fresh culture medium immediately before use.
Cultured IPC-366 and SUM149 cells were divided into a control group, treated with DMSO (final concentration, <0.1%), and four experimental groups, in which different concentrations of the different drugs tested were added to the culture medium.
To determine the sensitivity of IPC-366 and SUM149 cells to the effects of VEGF, SU5416, bevacizumab and celecoxib, different concentrations were used to determine the critical final concentrations to be used in the MTS assay.
For in vitro treatments, IPC-366 and SUM149 cells were exposed to 0.62, 1.25 and 2.5 uM concentrations of VEGF. SU5416, bevacizumab and celecoxib at 1.5, 3 and 6 uM final concentrations were added to the culture medium.

Poly(d,l-lactide-co-glycolide) (Resomer^® RG 505, Mw = 54,000–69,000 g/mol) 50:50 and poly(l-lactide) (Resomer^® L 207 S, Mw = 200,000–250,000 g/mol) were obtained from Evonik Industries (Essen, Germany), whereas poly(ε-caprolactone) (Mn = 80,000 g/mol), poly(vinyl alcohol) (Mw = 30,000–70,000 g/mol), and a solution of penicillin/streptomycin for cell culture were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Ethyl acetate and chloroform, that were used as solvents to dissolve the polymers, Dulbecco’s modified Eagle medium (high glucose with L-glutamine and sodium pyruvate) (DMEM), fetal bovine serum (FBS), trypsin, and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Carlo Erba (Milan, Italy). Bevacizumab (Avastin^®, Genentech, Inc., South San Francisco, CA, USA) was kindly offered by the pharmacy of Ospedale Policlinico San Martino, Genoa, Italy. The QuantumMicroProtein bicinchoninic acid protein assay kit (Micro BCA) (Euroclone, Pero, Italy) was used in order to quantify BEV for the encapsulation efficiency and for the in vitro release studies. CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS) for cell viability studies was purchased from Promega (Madison, WI, USA).