Meloni Marisa, Cattaneo Giampiero, De Servi Barbara
VitroScreen In Vitro Research Laboratories, Milan, Italy.
Clin Ophthalmol. 2012;6:1433-40. doi: 10.2147/OPTH.S35057. Epub 2012 Sep 5.
By using a biologically relevant and sensitive three-dimensional model of human corneal epithelium and multiple endpoint analysis, assessment of the potential for eye irritation and long-term compatibility of four registered ophthalmological preparations, ie, Timolabak(®), Timoptol(®), Nyogel(®), and Timogel(®), was performed. This approach enables classification of the potential for irritation, discriminating between mildly irritant and non-irritant ocular substances.
The exposure protocol included two time periods, ie, 24 hours (acute application) and 72 hours (repeated applications twice daily). This approach allows assessment of not only the acute reaction but also possible recovery, as well as mimicking the potential cumulative effects associated with long-term application. Using benzalkonium chloride (BAK) 0.01% as a positive control, the following parameters were quantified: cellular viability by MTT test, histological analysis by hematoxylin and eosin staining, passive release of interleukin-1α by enzyme-linked immunosorbent assay, and OCLN gene expression by quantitative real-time polymerase chain reaction.
Cell viability was reduced to under the 50% cutoff value after acute exposure (24 hours) to BAK 0.01%, and after repeated application (72 hours) of Timoptol and Nyogel. Histological analysis after acute exposure showed signs of superficial damage with all formulations, and severe changes after repeated applications of Timoptol, BAK 0.01%, and Nyogel. Timolabak and Timogel did not significantly alter the morphology of the human corneal epithelial cells after the different exposure times. Interleukin-1α release was greater than that for the negative control (>20 pg/mL) and the positive control (BAK 0.01%), Nyogel, and Timoptol treatments and not different after treatment with Timolabak and Timogel. Expression of OCLN, a sign of epithelial barrier impairment, was only significantly upregulated at 24 hours by BAK 0.01%, suggesting a toxic reaction at the ocular surface. OCLN was also overexpressed after repeated application of Nyogel and Timogel.
OVERALL, THE MULTIPLE ENDPOINT ANALYSIS APPROACH ALLOWS CLASSIFICATION OF THESE PRODUCTS ACCORDING TO DECREASING ORDER OF IRRITATION POTENTIAL AS FOLLOWS: BAK 0.01%, Timoptol, Nyogel, Timogel, and Timolabak.
通过使用具有生物学相关性且敏感的人角膜上皮三维模型以及多终点分析，对四种已注册的眼科制剂（即噻吗洛尔滴眼液（Timolabak®）、噻吗心安滴眼液（Timoptol®）、Nyogel®和Timogel®）的眼刺激潜力和长期相容性进行了评估。这种方法能够对刺激潜力进行分类，区分轻度刺激性和非刺激性眼用物质。
暴露方案包括两个时间段，即24小时（急性应用）和72小时（每日重复应用两次）。这种方法不仅可以评估急性反应，还能评估可能的恢复情况，以及模拟与长期应用相关的潜在累积效应。以0.01%苯扎氯铵（BAK）作为阳性对照，对以下参数进行了量化：通过MTT试验测定细胞活力，通过苏木精和伊红染色进行组织学分析，通过酶联免疫吸附测定法测定白细胞介素-1α的被动释放，以及通过定量实时聚合酶链反应测定OCLN基因表达。
在急性暴露（24小时）于0.01% BAK后，以及在重复应用（72小时）噻吗心安滴眼液和Nyogel后，细胞活力降至50%临界值以下。急性暴露后的组织学分析显示，所有制剂均有浅表损伤迹象，在重复应用噻吗心安滴眼液、0.01% BAK和Nyogel后有严重变化。在不同暴露时间后，噻吗洛尔滴眼液和Timogel对人角膜上皮细胞的形态没有显著改变。白细胞介素-1α的释放量高于阴性对照（>20 pg/mL）以及阳性对照（0.01% BAK）、Nyogel和噻吗心安滴眼液处理组，在用噻吗洛尔滴眼液和Timogel处理后无差异。OCLN（上皮屏障受损的标志）的表达仅在24小时时被0.01% BAK显著上调，表明眼表存在毒性反应。在重复应用Nyogel和Timogel后，OCLN也过表达。
总体而言，多终点分析方法可根据刺激潜力的降序对这些产品进行分类，顺序如下：0.01% BAK、噻吗心安滴眼液、Nyogel、Timogel和噻吗洛尔滴眼液。
