In vitro phospholipidosis

Aim: To assess the phospholipidosis inducing potential in an in vitro experimental system.

Background information: The intracellular accumulation of phospholipids (phospholipids, PLD) is known to be produced by drug accumulation within lysosomes, often triggered by cationic amphiphilic drugs, and can also occur indirectly by altering synthesis and processing of phospholipids. Except for aminoglycoside induced nephrotoxicity, a clear correlation of PLD with toxicity (tissue disfunction) has not been established. However, as several inborn errors of metabolism and some xenobiotic induced disease have been associated with PLD, the observation of such finding in the context of regulatory toxicity studies might raise the need to run additional investigations in order to show that cellular function remains unaltered on tissues where PLD is seen. Early assessment of potential for PLD of individual compounds or lead pharmaceutical series early in the drug discovery process increases the chance of successfully selecting those with better developmental properties.

In this assay, the intracellular accumulation of phospholipids is detected by incubating cells in the presence of phospholipids conjugated to fluorescent dyes, as assessed by means of flow cytometry.

Cell type

  • HepG2 cells
  • Other cell types/lines on demand

Experimental procedure

  • Top concentration: 100 µM, or on demand
  • 48 h treatment; 37ºC, 5% CO2
  • Microscopic morphological changes observation
  • Cytotoxicity assessed by cell count
  • Phospholipids accumulation assessed by flow cytometry

Delivered results

  • Risk for PLD, for concentrations with > 2-fold increase in fluorescence and < 20% cytotoxicity (Messens et al., 2009, 2010):
  • ≤ 10 μM : Strong
  • >10 μM ≤ 50 μM : Weak
  • > 50 μM : Negative

Example of a test item with Weak potential for PLD