Korean J Ophthalmol.  2013 Dec;27(6):446-453. 10.3341/kjo.2013.27.6.446.

In Vitro Effects of Preservative-free and Preserved Prostaglandin Analogs on Primary Cultured Human Conjunctival Fibroblast Cells

• Kim EJ
• Kim YH
• Kang SH
• Lee KW
• Park YJ

• KMID: 1792081
• DOI: http://doi.org/10.3341/kjo.2013.27.6.446

Abstract

PURPOSE
Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells.
METHODS
Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis.
RESULTS
BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs.
CONCLUSIONS
This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs formulations has not yet been firmly established. Alternatively preserved or preservative-free glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

Keyword

Benzalkonium compounds; Conjunctiva; Pharmaceutical preservatives; Synthetic prostaglandins

MeSH Terms

Apoptosis
Cell Line
Cell Survival/drug effects
Conjunctiva/drug effects/*pathology
Fibroblasts/drug effects/pathology
Glaucoma/drug therapy/pathology
Humans
Preservatives, Pharmaceutical/*pharmacology
Prostaglandins, Synthetic/*pharmacology
Preservatives, Pharmaceutical
Prostaglandins, Synthetic

Figure

• 

  Fig. 1 The morphological features of cultured human conjunctival fibroblast cells with different concentrations of benzalkonium chloride (BAC) in six-well plates were observed by phase-contrast microscopy (×100). (A) Control, (B) 0.001% BAC, (C) 0.005% BAC, (D) 0.01% BAC, and (E) 0.02% BAC. Cell shirnkage increased dose dependently with BAC concentration. Scale bar, 200 µm.

• 

  Fig. 2 The morphological features of cultured human conjunctival fibroblast cells with different prostaglandin formulations in six-well plates were observed by phase-contrast microscopy. (A) Control, (B) bimatoprost 0.01% (benzalkonium chloride [BAC] 0.02%), (C) latanoprost 0.005% (BAC 0.02%), (D) travoprost 0.004% (0.001% Polyquad), (E) tafloprost 0.0015% (BAC 0.001%), and (F) tafluprost 0.0015% (preservative-free). Compared with the control group, severe cell shrinkage was observed in bimatoprost 0.01% (BAC 0.02%) and latanoprost 0.005% (BAC 0.02%) groups. In the tafluprost 0.0015% group (BAC 0.001%), mild cell shrinkage was visible. In contrast, the travoprost 0.004% (0.001% Polyquad) and preservative-free tafluprost 0.0015% groups showed relatively similar cell morphology to that observed with the control group. Scale bar, 200 µm.

• 

  Fig. 3 Analysis of cell viability by Cell Counting Kit-8 assay. The absorbance was measured at 450 nm. The cells were exposed to the drugs for 15 minutes (A) or 30 minutes (B). *p < 0.05 vs. corresponding value for control by Mann-Whitney U-test. BAC = benzalkonium chloride; PF = preservative-free.

• 

  Fig. 4 Flow cytometric and quantitative analyses of apoptotic cells after culture for 30 minutes. Apoptosis was analyzed as a sub-G1 fraction by fluorescent activated cell sorting analysis (A) and columns (B). *p < 0.05 vs. corresponding value for control by Mann-Whitney U-test. BAC = benzalkonium chloride; PF = preservative-free.

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