M. F. Estrago-Franco

Evaluation of the Toxicity of Triamcinolone Acetonide and Dexamethasone Sodium Phosphate on Human Lens Epithelial Cells (HLE B-3)

PURPOSE The purpose of this study was to compare the in vitro effects of triamcinolone acetonide (TA) and dexamethasone sodium phosphate (DEX) on human lens epithelial cells (HLE B-3). METHODS HLE B-3 cells were exposed for 24 h to commercially available TA (c-TA) and dimethylsulfoxide-solubilized TA (s-TA). The cells were treated with 1,000 (clinical dose), 750, 500, 200, and 100 μg/mL concentrations of c-TA, s-TA, and supernatant for 24 h. The cells were also treated with DEX at 2, 1, 0.5, 0.2, 0.1 (clinical dose), and 0.05 mg/mL. Cell viability, caspase-3/7 activity, and DNA fragmentation analyses were performed. RESULTS The mean cell viabilities of HLE B-3 after exposure to c-TA at 1,000, 750, 500, 200, and 100 μg/mL were significantly reduced compared with control untreated cells. The s-TA also significantly reduced cell viability at 1,000, 750, and 500 μg/mL compared with dimethylsulfoxide control. The supernatant did not reduce cell viability. Caspase-3/7 activity significantly increased after treatment with c-TA and s-TA. DNA laddering revealed bands at 200 bp intervals with both c-TA at≥100 μg/mL and s-TA at ≥500 μg/mL. The cell viabilities of HLE B-3 after 24 h exposure to DEX were significantly reduced at 2 and 1 mg/mL but not at lower concentrations tested. Caspase-3/7 activities in HLE B-3 cells were not increased significantly after treatment with DEX at any dose tested. DNA laddering did not reveal any band at any dose tested. CONCLUSION This study showed that TA at its clinical dose (1,000 μg/mL) in both commercial preparation and solubilized forms decrease HLE B-3 cell viability through an apoptotic pathway. DEX at its clinical dose (0.1 mg/mL) does not decrease cell viability or cause any increase of caspase-3/7 activity. This study suggests that for long-term sustained-release devices, DEX may be less damaging to human lens cells than TA.

The Effects of Commercially Available Preservative-Free FDA-Approved Triamcinolone (Triesence®) on Retinal Cells in Culture

PURPOSE To evaluate the effects of Triesence® (TRI), a new preservative-free triamcinolone approved by the U.S. Food and Drug Administration (FDA) for intraocular use, on human retina pigment epithelial (ARPE-19) and rat neurosensory (R28) cells in culture. METHODS ARPE-19 and R28 cell cultures were treated 24 h with 1,000, 500, 200, or 100 μg/mL of crystalline (cTRI) or 1,000, 500, or 200 μg/mL of solubilized (sTRI). TRI was solubilized by centrifuging the drug, discarding the supernatant containing the vehicle and then resuspending the drug pellet in an equivalent amount of Dimethyl sulfoxide to achieve the same concentration as the commercial preparation. Percentage of cell viability (CV) was evaluated by a trypan blue dye-exclusion assay. The mitochondrial membrane potential (ΔΨm) was analyzed with the JC-1 assay. The caspase-3/7 activity was measured by a fluorochrome assay. RESULTS In the ARPE-19 cultures, the cTRI caused a decrease in CV at 1,000 μg/mL (13.03±6.51; P<0.001), 500 μg/mL (28.87±9.3; P<0.001), 200 μg/mL (54.93±5.61; P<0.001), and 100 μg/mL (82.53±0.65; P<0.005) compared with the untreated controls (96.98±0.16). In R28 cultures, the cTRI treatment also reduced CV values significantly (P<0.001) for the 1,000 μg/mL (22.73±2.44), 500 μg/mL (34.63±1.91), 200 μg/mL (58.70±1.39), and 100 μg/m (75.33±2.47) compared with the untreated controls (86.08±3.54). Once the TRI was solubilized (sTRI), the CV and ΔΨm remained similar to the untreated controls for both ARPE-19 and R28 cells. The sTRI treatment with 1,000, 500, and 200 μg/mL increased in caspase-3/7 activity in ARPE-19 cells (P<0.01) and in R28 cells (P<0.05) compared with dimethyl sulfoxide equivalent controls. CONCLUSION The crystalline form of TRI (cTRI) can cause a significant decrease in CV to cultured retinal cells. Once the TRI is solubilized (sTRI), at the same concentrations, the cells remain viable with no decrease in CV or ΔΨm. The sTRI can, however, increase caspase-3/7 activity, thus suggesting some degree of apoptosis.

Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro.

Aim: To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells. Materials and Methods: HTM cells were cultured and treated with 125, 250, 500 and 1000 μg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA-C) (commercial preparation) and solubilized TA (TA-S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II. Results: Mean cell viabilities of HTM cells after 24 h exposure to TA-C 125, 250, 500, and 1000 μg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 μg/mL of TA-S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO-equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO-treated controls of 125, 250, 500, and 1000 μg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase-3/7 activity in cultures treated with either TA-C or TA-S. DNA laddering showed no bands in the TA-C or TA-S treated cultures. There were significantly higher LDH release rates at all concentrations of TA-C compared to TA-S. Conclusions: Results show that the effect of TA-C and TA-S on HTM cells is due to cell death by necrosis at all concentrations except 125 μg/mL of TA-S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA-S over TA-C.