Benzyl Alcohol–Free Triamcinolone Acetonide: Retinal Toxicity and Modulation of Vascular Endothelial Growth Factor

Abstract

Abstract: : Purpose: Intravitreal triamcinolone acetonide (TA) has been shown to have a beneficial effect in some patients with exudative AMD. One proposed mechanism of action involves modulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). In this study, we evaluated the effects of TA on VEGF levels in cultured retinal cells and in murine eyes. We utilized benzyl alcohol–free TA as commercial preparations of TA have displayed toxicity. Methods: In vitro: Retinal pigment epithelial ARPE–19 cells were treated with 0.01, 0.1, 1, or 6 mg/mL of TA suspended in culture media. Two days later, VEGF protein and mRNA levels were quantified by ELISA and real time RT–PCR. To determine toxicity, cells were examined by microscopy and cytotoxicity evaluated with alamarBlue. In vivo: C57BL mice were injected intravitreally with TA suspended in balanced salt solution (BSS) in one eye and BSS alone in the contralateral eye. One week later, eyes were evaluated for VEGF protein and mRNA levels. Another set of eyes was prepared as plastic sections for histologic analysis of toxicity. Results: ARPE–19 cells treated with TA displayed dose–dependent toxicity. At 1 and 6 mg/mL of TA, percent reduced (a measure of cellular proliferation as determined by alamarBlue) was 52% and 47% respectively, relative to untreated control. There was a dose–dependent increase in VEGF protein and mRNA levels. In murine eyes, intravitreal injection of TA was associated with vacuolization of RPE cells and degeneration of photorecepter outer segments. TA had no effect on VEGF levels in vivo. Conclusions: In the present study, we evaluated the toxicity of benzyl alcohol–free TA and its effects on VEGF levels. Treatment of TA caused dose–dependent toxicity in cultured retinal cells, and at a clinical dose, RPE and photorecepter degeneration in murine eyes. These results are consistent with previous studies of commercial TA preparations containing benzyl alcohol. In terms of VEGF, toxic doses of TA were associated with elevated VEGF levels in cultured cells. This suggests that VEGF production may be stimulated in the presence of TA–induced cytotoxicity. In murine eyes, where only microscopic toxicity was observed, there was no change in VEGF levels. These results suggest that the beneficial effect of TA in AMD is unlikely to be mediated through suppression of VEGF.

Keywords: corticosteroids • retina