Research conducted at the University of Kiel, Germany, and funded by Novartis AG (SIX: NOVN; NYSE: NVS), suggests that aflibercept, marketed by Regeneron (NASDAQ: REGN) may impair the wound-healing ability and phagocytic capacity of RPE cells. The study, performed by Prof. Alexa Klettner and colleagues at Kiel’s Department of Ophthalmology is published in the British Journal of Ophthalmology (2014;98:1448–1452. doi:10.1136/bjophthalmol-2014-305105). The research compared the impact of aflibercept, bevacizumab and ranibizumab on cultured porcine retinal pigment epithelium (RPE) cells. The impact of aflibercept on cell viability was assessed with MTT and trypan blue exclusion assays while wound healing was assessed with a standard scratch assay.
The research focused on the three main anti-VEGF compounds – bevacizumab, ranibizumab and aflibercept – used to treat age-related macular degeneration (AMD), diabetic macular edema (DME) and retinal vein occlusion (RVO). The three compounds differ in both molecular structure and VEGF binding affinities: bevacizumab is a humanized murine full-length antibody against VEGF-A molecular weight of 149 kDa; ranibizumab is a high affinity Fab-fragment against VEGF-A, molecular weight of 48 kDa, and; aflibercept is a fusion protein with a molecular weight of 115 kDa, composed of the 2nd immunoglobulin domain of the VEGFR-1 and the 3rd immunoglobulin domain of the VEGFR-2 connected to an Fc-fragment. In addition, the three compounds have three different clinically relevant concentrations: bevacizumab is used at 0.25mg/ml, ranibizumab at 0.125mg/ml and aflibercept at 0.5mg/ml.
The researchers conducted a basic wound healing “scratch assay” designed to measure the migratory ability of the cultured cells. The RPE cells were seeded in 12-well plates and, when confluent, three wounds were scratched in the cell layer with a toothpick following which photographs captured their configuration. The three compounds – aflibercept, ranibizumab, or bevacizumab – were added to the wells at specific concentrations and 24 hours later, further photographs taken at the same coordinates allowed the researchers to analyse the migration of the cells. The assays showed that all three compounds, at a concentration of 0.125mg/ml, showed no statistically significant effect on wound healing however, at their clinically relevant concentrations, both aflibercept (0.5mg/ml) and bevacizumab (0.25mg/ml) showed statistically significant effects on wound healing when compared with control (control 87.3±8.4% closure, aflibercept 500 mg/mL 70.3±10.3% closure, p<0.01; bevacizumab 250 mg/mL 80.7±6.8% closure, p<0.05). Commenting in the result, the researchers stated, “[t]he impairment of wound healing by aflibercept may indeed constitute an adverse effect of this drug which may be of clinical consequences” however, the published work additionally cautioned that, “Whether our in vitro findings are of clinical relevance in vivo, however, remains to be seen, especially as the clinically relevant concentrations refers to the intravitreal concentration immediately after injection, and may not reflect the actual concentration reached at the RPE.”