Phospholipids and Phospholipid Disorders in Cellular Toxicity

R.G. Ulrich, J. Lin, S.L. Vonderfecht, and G.L. Weber
Phammacia & Upjohn, Inc.
Kaiamazoo, MI

A variety of drugs can induce phospholipidosis, characterized by cytoplasmic phospholipid inclusions (lamellar bodies). Current dogma indicates that lamellar bodies are generated from Iysosomes, primarily through inhibition of Iysosomal phospholipid degradation. Though generally regarded as benign, phospholipidosis is frequently associated with target-organ toxicity. We describe here the approaches used to study the mechanism of cellular phospholipid accumulation by a pyrrolopyrimidine drug candidate (PNU-101033), investigate potential associations with cell injury, and identify chemical structural analogues with reduced toxicity. In preclinical studies, PNU-101033 was found to produce gallbladder toxicity and coincident phospholipidosis in some animal species. Using a fluorescent analog of phosphatidylcholine (NBD-PC) and probes for Iysosomes in cultured hepatocytes, we found that the primary effect of phospholipidosis-inducing agents is on the endosomal compartment, altering membrane recycling dynamics, and that iysosomal involvement is secondary. This alteration may impair a variety of activities including absorptive and secretory processes, and lipid-mediated signalling pathways. In the gallbladder epithelium, impairment of secretory activities may contribute to cell injury. In order to select a follow-up compound without gallbladder toxicity, the in vitro phospholipidosis assay was applied as a moderate through-put screen following validation experiments. A structural analogue, PNU-104067F, was found to have a reduced potential to induce phospholipidosis compared to PNU-101033 in cultured rat and human hepatocytes. In vivo studies with this analogue revealed mild hepatic and pulmonary phospholipidosis with no evidence of gallbladder toxicity. Radiolabel studies in cultured human hepatocytes and in rats showed uptake and accumulation of radiolabel with persistent residual counts after a recovery period. HPLC and mass spectral analysis showed the residue to contain an ethanolamine metabolite that formed a tight association with phospholipid. In vitro studies revealed the metabolite to be an inducer of phospholipidosis with a potency considerably higher than the parent drug, suggesting the phospholipidosis induced by PNU104067 was due to a metabolite. These studies show that a mechanism-based in vitro model can be used to identify potential phospholipidosis induction by drugs, and differentiate the activity of metabolites from each other or parent compound.