Pharmaceutical Research Laboratories, Lion Corporation Odawara Research Center, Odawara, Japan.
Department of Ophthalmology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
J Ocul Pharmacol Ther. 2020 Nov;36(9):672-678. doi: 10.1089/jop.2019.0165. Epub 2020 Jun 17.
Benzalkonium Chloride (BAK) is reported to have the potential to damage the cornea. We developed a composition with broad-spectrum antimicrobial activity without preservatives by combining trometamol, boric acid, and ethylenediaminetetraacetic acid (TBE). This study aimed at evaluating the corneal damage caused by TBE and comparing it with that caused by BAK. SV40-immortalized human corneal epithelial cell line (HCE-T) was treated with BAK or TBE, and the cell viability was measured. The exposure time that caused 50% cell death (CDT) was calculated. Transepithelial electrical resistance (TEER) was measured before and after treatment with BAK or TBE. Occludin was detected with immunostaining and Western blotting after treatment with BAK or TBE. The effect of BAK or TBE on membrane-associated mucins was evaluated with rose bengal (RB) staining. In the BAK group, cell viability decreased in a dose-dependent manner. The viability of the TBE group was significantly greater than that of the BAK group. The CDT of the TBE group is greater than that of the BAK groups. In the BAK groups, the recovery of TEER was delayed in a dose-dependent manner, whereas in the TBE group, the recovery occurred earlier. Localization of occludin was disrupted, and the amount of occludin was significantly reduced among the cells exposed to BAK. The area stained with RB in the BAK groups increased, whereas that in the TBE group did not increase. These results suggest that the application of TBE would be useful for developing preservative-free ophthalmic preparations that offer both sufficient safety and antimicrobial activity.
苯扎氯铵（BAK）据报道有损伤角膜的潜力。我们通过组合三羟甲基氨基甲烷、硼酸和乙二胺四乙酸（TBE）开发了一种具有广谱抗菌活性且不含防腐剂的制剂。本研究旨在评估 TBE 引起的角膜损伤，并将其与 BAK 引起的损伤进行比较。
用 BAK 或 TBE 处理 SV40 永生化人角膜上皮细胞系（HCE-T），并测量细胞活力。计算导致 50%细胞死亡（CDT）的暴露时间。用 BAK 或 TBE 处理前后测量跨上皮电阻（TEER）。用免疫染色和 Western blot 检测用 BAK 或 TBE 处理后 occludin 的表达。用玫瑰红（RB）染色评估 BAK 或 TBE 对膜相关粘蛋白的影响。
在 BAK 组中，细胞活力呈剂量依赖性下降。TBE 组的活力明显大于 BAK 组。TBE 组的 CDT 大于 BAK 组。在 BAK 组中，TEER 的恢复呈剂量依赖性延迟，而在 TBE 组中，恢复发生得更早。occludin 的定位被破坏，暴露于 BAK 的细胞中 occludin 的数量明显减少。BAK 组中 RB 染色的面积增加，而 TBE 组中则没有增加。
这些结果表明，应用 TBE 将有助于开发不含防腐剂的眼科制剂，这些制剂既具有足够的安全性又具有抗菌活性。
