Corticosteroids have been used to treat inflammatory diseases of the eye for several years with success. The side effects of cataract and glaucoma, however, continue to be a concern for physicians who are considering using them to treat retinal disease. We have performed several in vivo studies in our laboratory that have looked at the toxicity of triamcinolone acetonide (TA; Kenalog 40, Bristol-Meyers Squibb) vs dexamethasone. This article will summarize our data. It is important to note that these abstracts have been condensed. For full methods, results, and discussion, please refer to the studies referenced.
TOXICITY STUDY SUMMARIES
Study purpose To evaluate the toxicity of TA to the retinal neurosensory (R28) and retinal pigment epithelial (RPE; ARPE-19) cells.1
Methods: Cells were grown in tissue culture. Cells were then treated with concentrations of 50, 100, and 200 µg/mL TA for 2, 6 and 24 hours (the clinical dose of TA is 1,000 µg). We also treated the cells with triamcinolone with and without vehicle and identified toxicity using trypan blue dyeexclusion and WST-1 mitochondrial dehydrogenase assays.
Conclusions: 200 µg/mL triamcinolone acetonide with or without the vehicle was toxic to both retinal cell lines in vitro using both the cell viability and mitochondrial dehydrogenase assays. Vehicle alone was not toxic to the cells. TA with vehicle caused a greater reduction in cell viability and mitochondrial dehydrogenase activity than triamcinolone without vehicle, in both R28 and ARPE-19 cell lines, although the difference was not statistically significant. We concluded that the clinically used doses of TA are toxic to these cells. The vehicle did not seem to be toxic, but there is a possibility that the vehicle may affect the level of toxicity of TA.
Study Purpose: To evaluate the potential role of solubilized TA in damaging human retinal pigment epithelial (ARPE-19) and neurosensory (R28) cell lines in culture.2
Methods: Commercially available TA was centrifuged and the supernatant containing the vehicle and preservative was discarded. The pellet of TA was dissolved in an equivalent amount of Dimethyl sulfoxide (DMSO) in order to achieve the same concentration of TA as in the commercial suspension. ARPE-19 and R28 cultures were treated with 100, 200, 500 and 1000 µg/ml of TA dissolved in DMSO for 24 hours. Toxicity was evaluated by trypan blue dye-exclusion assay, WST-1 assay and caspase-3/7 activity.
Conclusions: TA can damage retinal cells in vitro cells in part via caspase dependent apoptosis. The toxic effect is not as high as previously assumed since TA particles play an important role in TA toxicity and in clinical settings the vitreous preclude direct cellular contact.
Study purpose: To assess the effect of dexamethasone on mitochondrial function and cell viability in human RPE (ARPE-19) and rat neurosensory (R28) retinal cells in vitro.3
Methods: Cells were treated for 2, 6, and 24 hours with four different concentrations of dexamethasone: 0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, and 1 mg/mL. Cells were also treated with corresponding concentrations of the preservative benzyl alcohol: 0.03125%, 0.0625%, 0.125%, and 0.25%. Trypan-blue dye-exclusion assay was applied to measure cell viability and a WST-1 colorimetric assay to assess mitochondrial function. Absorbance was measured at 490 nm on a multi-well spectrophotometer.
Conclusions: Dexamethasone at concentrations of 0.125, 0.25 and 0.50 mg/mL (up to 5x the clinical dose of free dexamethasone) did not decrease cell viability in the RPE and rat neurosensory cells in vitro. Decreased cell viability was only observed at 10X the clinical dose. The more sensitive WST-1 assay shows that there may be some reduced mitochondrial function in response to the commercial dexamethasone preparation. However, the reduction in cell viability at 10X the clinical dose as well as the reduction of mitochondrial function at the lower doses was primarily due to the preservative in the commercial preparation. The doses of dexamethasone tested, which were at least 500 times the concentration of the dexamethasone used in the intravitreal dexamethasone implant (Ozurdex, Allergan, Inc.) appear to be non-toxic to retinal cells in culture.
Study purpose: To compare the toxicity of TA and dexamethasone sodium phosphate (DEX) on human lens epithelial (HLE B-3) cells.4
Methods: Cell viability assay was performed on cultured HLE cells. Commercially available TA was centrifuged at 5,000 rpm for 1 minute and the supernatant was removed. The pellet was resuspended in equivalent amounts of DMSO to produce solubilized TA. Cells were treated with 100, 200, 500, 750, or 1000 (clinical dose) µg/mL concentrations of commercially available TA and solubilized TA and the supernatant for 24 hours. Other HLE cells were treated with dexamethasone sodium phosphate at 0.05, 0.1 (clinical dose), 0.2, 0.5, 1, and 2 mg/mL. Cell viability was determined by trypan blue dye-exclusion assay.
For caspase detection, HLE cells were grown in 24-well plates in the groups outlined above. Caspase-3/7 was detected with carboxyfluorescein apoptosis detection kits. Caspase- 3/7 activities were measured with a fluorescence image scanning instrument. Apoptosis was quantified as the amount of green fluorescence emitted from FLICA probes bound to caspases. Non-apoptotic cells appeared unstained, whereas cells undergoing apoptosis fluoresced brightly.
A DNA Fragmentation Assay DNA was extracted from groups outlined. Samples were separated by electrophoresis on 3% agarose gels and stained with 5% ethidium bromide. A 100bp marker was used and images were captured with a fluorescence image scanning instrument.
Conclusions: TA at its clinical dose in both commercial preparation and solubilized form decreases human lens epithelial cell viability and that the caspase-3/7 pathway is involved suggestive of an apoptotic pathway. Dexamethasone at its clinical dose does not decrease cell viability nor cause any increase of caspase-3/7 activity.
Study purpose: To study the effects of TA on cultured human trabecular meshwork (HTM) cells.5
Methods: HTM cells were cultured. Cells were treated with 125, 250, 500 and 1000 μg/mL concentration of TA for 24 hours. The cells were treated with both crystalline TA and solubilized TA. Cell viability was measured by a trypan blue dye exclusion test. Activity of caspase-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehyrdrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II.
Conclusions: Commercially available TA and solubilized TA are toxic to HTM cells at all concentrations with the exception of lowest concentration of solubilized TA (125 μg/mL). As there is no caspase-3/7 activity with both TA-C and TA-S and as we could not find any bands on DNA ladder, we concluded 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 solubilized TA over commercially available TA.
Study purpose: To test the effects of varying doses of dexamethasone on human trabecular meshwork cells in vitro.6
Methods: Dexamethasone 2 mg/mL (20 times the clinical dose), 1 mg/mL (10 times [X] the clinical dose), 0.5 mg/mL (5 X), 0.25 mg/mL (2.5 X), 0.1 mg/mL (1 X), or 0.05 mg/mL (0.5 X) was evaluated over 24 hours. Trypanblue exclusion was used to measure cell viability and Fluorochrome assays were applied to evaluate apoptosis via caspase -3/7, -8, -9, and -12 activities. Mitochrondrial dehydrogenase activity/damage was assessed using WST assay.
In addition to comparing treated and untreated cells, some of the human trabecular meshwork cells were pretreated with brimonidine (Alphagan, Allergan, Inc.) to test the neuroprotective effects of the agent.
Conclusions: At the very high doses (20X, 10X, 5X), dexamethasone caused apoptosis via the mitochondrial pathways; however, it is possible that brimonidine could have a partial protective effect when used as pretreatment. The lower doses (2.5 X, 1 X, 0.5 X) of dexamethasone, which more appropriately represent what is used in clinical practice, did not cause any significant reduction in cell viability, apoptotic upregulation, or mitochondrial dysfunction of HTM cells in vitro.
SUMMARY STATEMENT
Steroids can vary not only in their potency but also in their cytotoxicity. TA is the most commonly used intravitreal steroid due to its formulation and duration of action. Despite the clinical benefit seen with TA, it is associated with a high rate of cataract and glaucoma, and may have a hidden or difficult to observe toxic retinal effect that may blunt the magnitude of the clinical benefit. By contrast, dexamethasone is a more potent glucocorticosteroid than TA and may be associated with a lower risk of cataract and glaucoma. Additionally, when tested in vitro on retinal cell lines, there was significantly less cytoxtoxicity than observed with TA. In fact, when TA and dexamethasone were compared with regards to their effect on lens epithelial cells (as a risk factor for cataract formation), trabecular meshwork cells (as a risk factor for glaucoma development), and retinal cells including RPE and neurosensory cells, TA was consistently more toxic than dexamethasone using a variety of assays for cell death, mitochondrial damage, and apoptosis. This was true both when the TA crystals were placed directly on the cells as well as when the TA was dissolved and exposed to the cell lines. This in vitro data corroborates the observation that TA is more likely to cause cataract and glaucoma than dexamethasone. However, it is important to note that dexamethasone has a short half-life, and for effective clinical use it must be employed in the context of an extended release drug delivery system such as the intravitreal dexamethasone implant (Ozurdex, Allergan, Inc.)
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