Cell-based fluorescence assay for evaluation of new-drugs potential for phospholipidosis in an early stage of drug development.
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.
Fujimura
Hisako
H
Exploratory Toxicology, Exploratory Toxicology & DMPK Research Laboratories, Tanabe Seiyaku Company, Ltd, 2-50, Kawagishi, 2-Chome, Toda, Saitama, Japan. h-fuji@tanabe.co.jp
Dekura
Eriha
E
Kurabe
Michie
M
Shimazu
Noriko
N
Koitabashi
Mieko
M
Toriumi
Wataru
W
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions; D005456:Fluorescent Dyes / Q000494:pharmacology*; D002479:Inclusion Bodies / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008064:Lipidoses / Q000139:chemically induced* / Q000378:metabolism; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D004364:Pharmaceutical Preparations / Q000145:classification; D010714:Phosphatidylethanolamines / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence
D004338:Drug Combinations; D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine