Fujimura Hisako, Dekura Eriha, Kurabe Michie, Shimazu Noriko, Koitabashi Mieko, Toriumi Wataru
Exploratory Toxicology, Exploratory Toxicology & DMPK Research Laboratories, Tanabe Seiyaku Company, Ltd, 2-50, Kawagishi, 2-Chome, Toda, Saitama, Japan.
Exp Toxicol Pathol. 2007 Aug;58(6):375-82. doi: 10.1016/j.etp.2007.01.004. Epub 2007 Apr 6.
To evaluate new-drugs potential for phospholipidosis (PL), we developed a cell-based fluorescence assay using a fluorescent-labeled phospholipid analogue (NBD-PE). CHL/IU cells derived from newborn hamster lung were exposed to positive reference compounds (amiodarone, imipramine, chloroquine, propranolol, chlorpromazine and amantadine) in the presence of NBD-PE, and the level of PL, as indicated by accumulation of fluorescent inclusions in the cytoplasm, was evaluated using fluorescence microscopy and fluorometry. All positive reference compounds induced accumulation of fluorescent inclusions in a concentration-dependent manner with an increase in fluorescence intensity. Fluorescence microscopically, the positive dose of test compound was determined as the concentration with a grade equivalent to or above that of 3.13 microM of amiodarone. Based on this criterion, 8 of 20 test compounds including PL-positive or -negative compounds were judged positive that were concurrent with the pathological results from rat toxicity studies. Furthermore, a positive criterion for fluorometry was decided as equivalent to or above 25% of maximum intensity induced by 1.56-25.0 microM amiodarone. In comparison of fluorometry methods with fluorescence microscopy method, 19 of 20 compounds were judged same. From these findings, we concluded that the assay developed in this study is a rapid and reliable method to predict new-drugs potential for PL at an early stage of drug development.
为评估新药引发磷脂沉积症（PL）的可能性，我们开发了一种基于细胞的荧光检测方法，该方法使用了一种荧光标记的磷脂类似物（NBD-PE）。将新生仓鼠肺来源的CHL/IU细胞在NBD-PE存在的情况下暴露于阳性参考化合物（胺碘酮、丙咪嗪、氯喹、普萘洛尔、氯丙嗪和金刚烷胺），并使用荧光显微镜和荧光测定法评估PL水平，PL水平通过细胞质中荧光包涵体的积累来表示。所有阳性参考化合物均以浓度依赖性方式诱导荧光包涵体的积累，荧光强度增加。在荧光显微镜下，测试化合物的阳性剂量被确定为与3.13 microM胺碘酮相当或更高等级的浓度。基于该标准，在20种测试化合物中，包括PL阳性或阴性化合物在内的8种被判定为阳性，这与大鼠毒性研究的病理结果一致。此外，荧光测定法的阳性标准被确定为相当于或高于由1.56 - 25.0 microM胺碘酮诱导的最大强度的25%。在荧光测定法与荧光显微镜法的比较中，20种化合物中有19种被判定结果相同。从这些发现中，我们得出结论，本研究中开发的检测方法是一种在药物开发早期预测新药引发PL可能性的快速且可靠的方法。
