Relationship between in vitro phospholipidosis assay using HepG2 cells and 2-week toxicity studies in rats

Abstract

Drug candidates under development by industry frequently show phospholipidosis as a side-effect in pre-clinical toxicity studies. This study sets up a cell-based assay for drug-induced phospholipidosis (PLD) and its performance was evaluated based on the in vivo PLD potential of compounds in 2-week toxicity studies in rats. When HepG2 cells were exposed simultaneously to PLD-inducing chemicals and a phospholipid having a fluorophore, an accumulation of phospholipids was detected as an increasing fluorescent intensity. Amiodarone, amitriptyline, fluoxetine, AY-9944, and perhexiline, which are common PLD-inducing chemicals, increased the fluorescent intensity, but acetaminophen, ampicillin, cimetidine, famotidine, or valproic acid, which are non-PLD-inducing chemicals, did not. The fluorescent intensity showed concordance with the pathological observations of phospholipid lamellar bodies in the cells. Then to confirm the predictive performance of the in vitro PLD assay, the 32 proprietary compounds characterized in 2-week toxicity studies in rats were evaluated with this in vitro assay. Because this in vitro assay was vulnerable to cytotoxicity, the innate PLD potential was calculated for each compound. A statistically significant increase in the in vitro PLD potential was seen for the compounds having in vivo PLD-inducing potential in the rat toxicity studies. The results suggest that the in vitro PLD potential could be appropriate to detect the appearance of PLD as a side effect in pre-clinical toxicity studies in rats.

Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

“…Squares (▪) indicate studies based on human hepatocytes; open square ( □ ) rhesus monkey; triangles (▴), rat hepatocytes; diamonds (♦), mouse hepatocytes; circles (•), spheroids; crosses ( × ), HepG2 cell line; and pluses ( + ), other cell lines. The numbers refer to published references as follows: 1, (Bhandari et al , 2008); 2, (Bort et al , 1998); 3, (Donato et al , 2009); 4, (Dykens et al , 2008); 5, (Flynn and Ferguson, 2008); 6, (Foretz et al , 2010); 7, (Fulgencio et al , 2001); 8, (Golli-Bennour et al , 2012); 9, (Gomez-Lechon et al , 2003); 10, (Jemnitz et al , 2008); 11, (Ji et al , 2010); 12, (Kaufmann et al , 2005); 13, (Kiang et al , 2010); 14, (Kingsley et al , 1983); 15, (Lauer et al , 2009); 16, (Lewerenz et al , 2003); 17, (Lin et al , 2012); 18, (McCarthy et al , 2004); 19, (McGill et al , 2011); 20,(Miyamoto et al , 2009a); 21, (Moon et al , 2010); 22, (Neuman et al , 2001); 23, (Okamoto et al , 2009); 24, (Phillips et al , 2003); 25, (Ponsoda et al , 1995); 26, (Rauchenzauner et al , 2008); 27, (Riches et al , 2009); 28, (Toyoda et al , 2012); 29, (Ubl et al , 1994); 30, (Ullrich et al , 2009); 31, (Waldhauser et al , 2008); 32, (Wang et al , 2002); 33, (Xu et al , 2003a); 34, (Zhou et al , 2001); and 35, data presented in this report.…”

Drug induced phospholipidosis: An acquired lysosomal storage disorder

“…These include conventional pathological examination of tissues from animals used in toxicological studies, bronchoalveolar lavage fluid analysis of macrophages and surfactant, and in vitro cell assays [44, 63–66]. Cell based assays have focused on flow cytometric and morphologically based assays due to the absence of a specifically identified enzymatic target [67].…”

<i>In vitro</i> validation of drug-induced phospholipidosis

“…Cells treated with acetaminophen known to be non-phospholipidogenic that gave controversial results in LipidTox and NBD-PE assays were also observed by TEM, the gold standard method for detection of PLD (Reasor et al, 2006;Miyamoto et al, 2009). Fig.…”

“…Therefore, the accumulation of NBD-PE is used as a surrogate for PLD (Miyamoto et al, 2009;Morelli et al, 2006). We tested whether LipidTox positive results for acetaminophen and menadione can be reproduced in the NBD-PD assay using propranolol as a positive control.…”