Phospholipidosis

Phospholipidosis (PLD) is characterized by concentric-layered multilamellar intracellular lysosomal inclusion bodies that are often composed of complex phospholipids, parent drug, and/or metabolites. Accumulation will often occur in several different cell types of the hepatobiliary, immune, and nervous systems. Typically, hepatocytes, biliary epithelial cells and macrophages of lymph nodes, and pulmonary alveoli and ganglia and nonganglia neuronal cell bodies of the central nervous system can be affected. The evidence to date suggests that phospholipidosis is a structural-related toxicity of cationic amphi-philic compounds irrespective of pharmacologic action. The finding of phos-

pholipidosis may also be associated with inflammation, severe organ damage, and possibly impairment of immune function. Although drugs are marketed that cause phospholipidosis preclinically and clinically, this is an undesirable profile for potentially new candidate drugs, and as such, this liability should be identified and avoided early in the drug discovery process. Therefore, high-throughput in vitro predictive screens can add value, particularly if phospho-lipidosis potency can be ranked and supported by in vivo data.

Several methodologies have been evaluated to assess neutral and phos-pholipid content as an index of phospholipidosis in cells growing in culture. However, accumulation of NBD-PE as a result of cytotoxicity induces false-positive results, particularly at high concentrations. Recently, a high-t hroughput, validated, predictive, sensitive, and selective multichannel fluorescence-based in vitro PLD assay was developed to reduce the false-positive limitation of cytotoxicity. This assay uses I-13.35 adherent mouse spleen macrophages cultured in 96-well plates with fluorescent-tagged phospholipids. Cells with an intact nucleus were differentiated from dead cells using ethidium staining and cell gating that rejects dead cells. Using this improved technique, 26 of 28 positive phospholipidogenic compounds were identified. These findings aided application of this methodology to other techniques, such as flow cytometry, which may be used in preclinical toxicology studies and clinical trials. For example, flow cytometric analysis coupled with Nile Red staining was used to detect neutral and phospholipids in a monocyte cell line, U397. Application of this methodology was utilized in (in vivo) toxicology studies, and this raised the possibility that preclinical toxicology and clinical assessment of phospholipidosis could be done using peripheral blood cells and flow cytometry.

Project Management Made Easy

Project Management Made Easy

What you need to know about… Project Management Made Easy! Project management consists of more than just a large building project and can encompass small projects as well. No matter what the size of your project, you need to have some sort of project management. How you manage your project has everything to do with its outcome.

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