Objective: To investigate the cytotoxic effect of three kinds of topical ocular anti-allergic agent, including olopatadine 0.1% (A group), ketotifen fumarate 0.025% (B group) and pemirolast potassium 0.1% (C group), on cultured human corneal epithelial cells in vitro.
Methods: Primary human corneal epithelial cells were cultured with keratinocyte serum-free medium. The cells were exposed to three kinds of topical ocular anti-allergic agent for a period of 10 min, 30 min, 2 h, 6 h, 12 h and 24 h. Toxicity was examined in three ways. MTT assay was used to quantify cytotoxicity. Cell membrane permeability and intracellular esterase activity were analyzed with live-dead viability staining of fluorescent calcein-AM/ethidium homodimer. The morphologic analysis was performed by light and scanning electron microscopy. Statistical methods adopted one-way ANOVA (analysis of variance) and Student-Newman-Keuls q test between each group. The P-value of 0.05 was considered statistically significant.
Results: (1) Morphologic changes: The Findings under the light microscopy were demonstrated that cells became round or edematic and detached from dishes after exposure to topical ocular anti-allergic agent. The cellular damage was more severe with longer exposure time and increasing concentration. Likewise, the electron microscopy examination showed reduced microvilli with longer exposure time and increasing concentration. The cellular changes of 20.0% olopatadine 0.1% were reduced when compared to the other agents. (2) Live/dead viability/cytotoxicity assay: Ethidium homodimeric permeates damaged cell membranes and results in red fluorescence. These results indicated that cell membrane damage caused by 20.0% olopatadine 0.1% at 6, 12, 24 h was less than those of ketotifen fumarate 0.025% and pemirolast potassium 0.1%. The data of A group were (29.7 +/- 2.6)%, (36.9 +/- 3.2)%, (51.2 +/- 4.3)%, B group were (36.5 +/- 3.1)%, (48.5 +/- 4.3)%, (75.5 +/- 3.8)% and C group were (37.1 +/- 2.2)%, (52.7 +/- 3.4)%, (71.1 +/- 5.1)%, respectively. The q values of A to B group and A to C group at 6 h were 3.27, 4.31, respectively (P < 0.05). The green fluorescent staining of calcein-AM indicated intracellular esterase activity was decreased after incubation with increasing concentration and longer exposure time. There was no significantly different result between each group (P > 0.05). The proportion of green staining cell of A, B and C group at 24 h were 100.0% with 50.0% concentration and were (23.2 +/- 4.6)%, (29.5 +/- 5.2)%, (31.1 +/- 5.5)% respectively with 20.0% concentration (F = 1.97, P = 0.377). (3) MTT assay: The results of the three groups revealed cell viability decreased significantly with increasing concentration and longer exposure time at all the concentrations except 0.8%. MTT values for A, B and C group at the concentration of 20.0%, at 6 h were 0.429 +/- 0.028, 0.367 +/- 0.038, 0.379 +/- 0.012 and 4% at 24 h were 0.457 +/- 0.025, 0.401 +/- 0.008, 0.387 +/- 0.012, respectively. The data for olopatadine 0.1% were significantly improved over those of ketotifen fumarate 0.025% and pemirolast potassium 0.1%. The q value of A to B group, A to C group were 3.01, 3.77 (P < 0.05) at the concentration of 20.0%, 6 h and were 3.63, 4.11 (P < 0.05) at the concentration, 24 h. There were no statistical significant results at other concentrations.
Conclusions: The topical ocular anti-allergic agent, olopatadine 0.1%, showed less toxic effects on human corneal epithelial cells compared to ketotifen fumarate 0.025% and pemirolast potassium 0.1%. Olopatadine 0.1% may offer a safer option to the corneal epithelium when used to treat allergic keratoconjunctivitis over an extended period of time.