title: The effects of commercially available preservative-free FDA-approved triamcinolone (Triesence®) on retinal cells in culture.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California-Irvine, CA 92697, USA.
authors.forename: Leandro Cabral
authors.initials: LC
authors.lastname: Zacharias
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Estrago-Franco
authors.forename: Claudio
authors.initials: C
authors.lastname: Ramirez
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Kenney
authors.forename: Walter Y
authors.initials: WY
authors.lastname: Takahashi
authors.forename: Gail M
authors.initials: GM
authors.lastname: Seigel
authors.forename: Baruch D
authors.initials: BD
authors.lastname: Kuppermann
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D051381:Rats; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D014221:Triamcinolone / Q000008:administration & dosage / Q000633:toxicity*
chemical_list: D014221:Triamcinolone; D053148:Caspase 3; D053179:Caspase 7