title: High content screening analysis of phospholipidosis: validation of a 96-well assay with CHO-K1 and HepG2 cells for the prediction of in vivo based phospholipidosis.
abstract: Drug-induced phospholipidosis is marked by an excessive accumulation of phospholipids in lysosomes which can occur after exposure to cationic amphiphilic drugs. Phospholipidosis is considered as an adverse side effect and may delay or negatively affect registration of drug candidates. Currently, the gold standard method of phospholipidosis detection is electron microscopy on tissue samples. This technique is time consuming and only performed relatively late in drug development. Therefore, in vitro screening methods for phospholipidosis are essential in early drug development. In this study, an in vitro phospholipidosis detection assay is developed with CHO-K1 and HepG2 cells by using the fluorescent marker NBD-PE and high content screening analysis. Lysosomal localization of NBD-PE was demonstrated by colocalization with Lysotracker and lamellar body formation by electron microscopy. Upon drug exposure, lysosomal NBD-PE accumulation can be visualized and quantified. Validation with 56 reference compounds, divided in 25 phospholipidosis inducers and 31 negative compounds, showed that this new in vitro assay has a high sensitivity (CHO-K1=92.0% and HepG2=88.0%) and specificity (CHO-K1=87.1% and HepG2=80.6%) for predicting phospholipidosis in vivo. Thus a selective screening tool has been developed for early selection of drug candidates with low probability for phospholipidosis.
authors.affiliation: Toxicology and Drug Disposition, Merck Sharp and Dohme, P.O. Box 20, 5340 BH Oss, The Netherlands. fm_vdwater@hotmail.com
authors.forename: F M
authors.initials: FM
authors.lastname: van de Water
authors.forename: J
authors.initials: J
authors.lastname: Havinga
authors.forename: W T
authors.initials: WT
authors.lastname: Ravesloot
authors.forename: G J M J
authors.initials: GJ
authors.lastname: Horbach
authors.forename: W G E J
authors.initials: WG
authors.lastname: Schoonen
mesh_terms: D000638:Amiodarone / Q000009:adverse effects; D000639:Amitriptyline / Q000009:adverse effects; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions; D005456:Fluorescent Dyes / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008064:Lipidoses / Q000139:chemically induced* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D000639:Amitriptyline; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; D000638:Amiodarone