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.

Keywords::

• Fluorometry
• screening
• HepG2
• phospholipidosis
• toxicity study

Acknowledgements

We wish to acknowledge particularly Drs Fumio Chatani and Ryo Fukuda for their helpful suggestions. We are grateful to the members of our laboratory for their encouragement and discussions. We especially thank Ms Chihiro Kurozumi and Ms Mika Okai for their excellent technical support. We also thank Dr Ken-ichi Nagatome and Dr Eric Spicer for an advice on the statistical analysis and editing the manuscript, respectively.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.