Abstract
Purpose: :
To study the effects of Loteprednol Etabonate 0.5% suspension (LE, Lotemax®, Bausch&Lomb, Tampa, FL), its solubilized form, and vehicle on human retinal pigment epithelial cells (ARPE-19) and rat neurosensory retinal cells (R28) in vitro.
Methods: :
ARPE-19 and R28 cells were treated for 2, 6, or 24 hours with LE or DMSO solubilized LE (S-LE) at 250 µg/ml (2X clinical dose), 125 µg/ml (1X ), 62.5 µg/ml (X/2), 31.25 µg/ml (X/4), and 15.62 µg/ml (X/8). Cell viability (CV) was measured using the trypan blue dye-exclusion assay or Calcein Assay. Equivalent concentrations of DMSO were used as controls. The vehicle was obtained as the supernatant after centrifugation of LE. Lactose dehydrogenase (LDH), a measure of necrosis, was quantified using the LDH-Cytotoxicity Assay.
Results: :
In both cell lines LE at 24 hours caused a significant (p<0.01) decrease in CV at all concentrations except at the lowest dose (X/8 clinical dose) in ARPE-19 cells. ARPE-19 cell CV at 24 hrs at LE doses of X, X/2, X/4 and X/8 were 8.2±3.8%, 80.4±0.9%, 80.5±1.5% and 94.4±2.3%. R28 cell 24 hour CV were 22.3±3.1%, 58.8±4.6%, 69.7±8.6% & and 76.1±6.5%. Control CV was normalized to 100 %. S-LE at 24 hours was found to be toxic to ARPE-19 and R 28 cells only at 2X dose (59.6±7.8% for ARPE-19 versus 88.1±4.4% for controls (p=0.01) and 52.3±2.9% for R28 versus 64.9±2.5 % for controls [p=0.03]). LDH was significantly elevated for ARPE-19 cells and R 28 cells treated with 2X S-LE (0.55±0.02 for ARPE-19 versus 0.37±0.01 for control, (p<0.001); 0.14 ± 0.001 for R28 versus 0.13 ± 0.003 for control [p<0.001]). Vehicle decreased CV at 2X and X in ARPE-19 cells (12.6 ± 2.1 and 89.4±3.5 for 2X and X versus 98.2±0.5 for controls (p<0.0001 and p= 0.05 respectively). Vehicle reduced CV of R28 cells only at 2X (53.2±3.8 versus 98.2±0.5 for controls; p<0.001).
Conclusions: :
Loteprednol Etabonate 0.5% suspension was cytotoxic at all doses to both cell lines, except X/8 clinical dose for ARPE-19. Solubilized LE and vehicle were toxic at 2X clinical dose. Based on LDH results, cytotoxicity from LE maybe due to necrosis.
Keywords: corticosteroids • retinal pigment epithelium • inflammation