Hakkarainen Jenni J, Reinisalo Mika, Ragauskas Symantas, Seppänen Aila, Kaja Simon, Kalesnykas Giedrius
R&D department, Experimentica Ltd., P.O. Box 1199, FI-70211 Kuopio, Finland.
R&D department, Experimentica Ltd., P.O. Box 1199, FI-70211 Kuopio, Finland; School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.
Int J Pharm. 2016 Sep 10;511(1):73-78. doi: 10.1016/j.ijpharm.2016.06.135. Epub 2016 Jun 29.
The purpose of the study was to devise a fast, reliable and sensitive cell viability assay for assessment of acute cytotoxicity on human corneal epithelial cells by using a clinically relevant exposure time. Acute cytotoxic effects of the pharmaceutical excipients benzalkonium chloride (BAC), macrogolglycerol hydroxystearate (MGHS40), polysorbate 80 (PS80) and marketed ophthalmic formulations (Lumigan(®), Monoprost(®), Taflotan(®), Travatan(®), Xalatan(®)) containing these excipients were tested. Human corneal epithelial cell (HCE-T) viability was assessed by measuring the reduction of resazurin to highly fluorescent resorufin. Expression of the tight junction proteins in HCE-T cells were characterized by immunofluorescence staining. Presence of tight junction proteins in HCE-T cells was demonstrated. BAC preserved ophthalmic formulations showed concentration-dependent and time-dependent cytotoxicity to human corneal epithelium. In contrast, no acute cytotoxicity of non-ionic stabilizing/solubilizing excipients (MGSH40 and PS80) or ophthalmic formulation containing these excipients was observed. Marketed ophthalmic formulations used for glaucoma medication show differential toxicity on human corneal epithelial cells. The present study revealed that BAC-preserved ophthalmic formulations were able to induce acute cytotoxic effects even during a clinically relevant exposure time, which was not observed with MGSH40 and PS80 excipients or ophthalmic formulations containing these excipients.
本研究的目的是设计一种快速、可靠且灵敏的细胞活力测定方法，以评估在临床相关暴露时间下对人角膜上皮细胞的急性细胞毒性。测试了药用辅料苯扎氯铵（BAC）、聚乙二醇氢化蓖麻油（MGHS40）、聚山梨酯80（PS80）以及含有这些辅料的市售眼科制剂（卢美根（®）、蒙诺前列素（®）、他氟前列素（®）、曲伏前列素（®）、适利达（®））的急性细胞毒性。通过测量刃天青还原为高荧光的试卤灵来评估人角膜上皮细胞（HCE-T）的活力。通过免疫荧光染色对HCE-T细胞中紧密连接蛋白的表达进行表征。证明了HCE-T细胞中存在紧密连接蛋白。含BAC的眼科制剂对人角膜上皮显示出浓度依赖性和时间依赖性的细胞毒性。相比之下，未观察到非离子型稳定/增溶辅料（MGSH40和PS80）或含有这些辅料的眼科制剂有急性细胞毒性。用于青光眼治疗的市售眼科制剂对人角膜上皮细胞显示出不同的毒性。本研究表明，含BAC的眼科制剂即使在临床相关暴露时间内也能够诱导急性细胞毒性，而MGSH40和PS80辅料或含有这些辅料的眼科制剂则未观察到这种情况。
