title: Evaluation and validation of multiple cell lines and primary mouse macrophages to predict phospholipidosis potential.
abstract: Phospholipidosis (PLD) in preclinical species can lead to regulatory delays thereby creating incentives to screen for PLD during drug discovery. The objective of this work was to compare, optimize, and validate in vitro PLD assays in primary mouse macrophages and hepatocyte- (HepG2, HuH7) or macrophage-derived cells lines (I.13.35, RAW264.7) and to evaluate whether primary cells were better at predicting PLD. Assay precision, determined by a measure of signal to noise window (Z'), within assay variability, and day-to-day variability, using amiodarone, was generally acceptable for all cell types; however, precision limits for HepG2 and HuH7 were slightly below assay acceptance criteria. Up to 66 known PLD inducers and non-inducers were subsequently tested to validate the assays. The concordance for predicting PLD in primary macrophages, I-13.35, RAW264.7, HuH7, and HepG2 cells was 91%, 74%, 73%, 62%, and 62% respectively using a decision limit of EC50≤125 μM as a positive finding. Increasing the number of negative controls tested in RAW264.7 cells and changing the decision limit to ≥4-fold increase in PLD, improved the specificity and overall concordance to 88%. RAW264.7 cells were selected as the primary screen for predicting PLD, and together with the primary macrophages, were integrated into an overall testing paradigm proposed for use in PLD risk identification.
authors.affiliation: Discovery Toxicology, Bristol-Myers Squibb, Princeton, NJ 08543, United States.
authors.forename: Lloyd
authors.initials: L
authors.lastname: LeCureux
authors.forename: Charles S
authors.initials: CS
authors.lastname: Cheng
authors.forename: John
authors.initials: J
authors.lastname: Herbst
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Reilly
authors.forename: Lois
authors.initials: L
authors.lastname: Lehman-McKeeman
authors.forename: Monicah
authors.initials: M
authors.lastname: Otieno
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions; D005456:Fluorescent Dyes / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008064:Lipidoses / Q000139:chemically induced* / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine