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

Abstract

-Intracellular accumulation of phospholipids with lamellar bodies is a hallmark of druginduced phospholipidosis (PLD) which is caused by impaired phospholipid metabolism of the lysosome. Although it remains uncertain whether PLD is associated with the adverse effects, sponsors generally terminate the development of a candidate drug when PLD is observed in an organ. For drugs that are used without serious adverse events, there should be labels indicating that the drug can induce PLD. We conducted LipidTox and NBD-PE assays for detecting PLD to compare and validate the methods. In the case of contrary results in both assays, electron microscopy was performed to confirm the data. We selected 12 chemicals and divided them into 4 categories: P+S+, PLD and steatosis positive; P+/S-, PLD positive and steatosis negative; P-S+, PLD negative and steatosis positive; P-/S-, PLD and steatosis negative. In general, results showed very good agreement with the known information with some minor discrepancies. LipidTox assay is proven to be a very sensitive method. Considering the contrary results of acetaminophen and menadione in LipidTox and the NBD-PE assay, the combination of two methods using different phospholipids is advantageous to reduce false positives. The finding that acetaminophen was positive in LipidTos assay and increased the frequency of lamellar body implies that acetaminophen is a weak inducer of PLD.

Comparative analysis of lipotoxicity induced by endocrine, pharmacological, and innate immune stimuli in rat basophilic leukemia cells

“…A second major pathway for induction of PLD, sometimes with accompanying steatosis, is the response to cationic CAD (Halliwell, 1997 Anderson et al, 2006; Park et al, 2012; Shayman et al, 2013). In the present study, whether this drug-induced PLD (diPLD) pathway operated in the basophilic leukemia cell line studied here was assessed.…”

“…Lipotoxicity in tissues such as muscle, pancreas, heart and liver has been described in response to both metabolic overload and exposure to cationic amphiphilic drugs (CAD) (Halliwell, 1997; Anderson et al, 2006; Begriche et al, 2011; Park et al, 2012; Shayman et al, 2013). In cells of the immune system (primarily granulocytes), physiological hyper-accumulation of lipid bodies that resembles steatosis can occur in response to Toll-like receptor (TLR) ligands during infection (Pacheco et al, 2002; Melo et al, 2003), and there is evidence for endocrine regulation of LB numbers in macrophages and mast cells (Greineisen et al, 2012).…”

“…Hyper-accumulation of mast cell LB in response to one of these (hyperinsulinemia) has been documented (Greineisen et al, 2012). However, it remains unclear whether hyperinsulinemia-driven steatotic LB accumulation in mast cells represents a lipotoxic phenotype; whether innate immune stimuli can in fact initiate LB accumulation in mast cells; whether exposure to CAD can drive the steatotic pathway; and whether phospholipidosis accompanies any of these lipotoxic responses (Halliwell, 1997; Anderson et al, 2006; Begriche et al, 2011; Muehlbacheret al, 2012; Park et al, 2012; Shayman et al, 2013). This paper seeks to address these questions.…”

“…PLD is a change in phospholipid metabolism that causes excess accumulation of phospholipids, distinguished by the production of intracellular multi-lammelar lysosomal bodies (Halliwell, 1997; Anderson et al, 2006; Begriche et al, 2011; Muehlbacher et al, 2012; Park et al, 2012; Shayman et al, 2013). A suggested mechanism for PLD involves the formation of a drug-phospholipid complex that inhibits phospholipases.…”

Establishment of an <i>in silico</i> phospholipidosis prediction method using descriptors related to molecular interactions causing phospholipid–compound complex formation

“…Although reports of an in vitro validation study for PLD evaluation (Park et al, 2012) and screening studies exist (Kasahara et al, 2006;Kikkawa et al, 2005), the prediction or screening method for PLDs remains under development. Thus, an in silico method that can predict PLD potential would be a significant tool for planning a study package for assessing PLD.…”