Drug toxicities represent a significant disease burden in the ambulatory and inpatient settings. Many drug toxicities are idiopathic in presentation and severity and often result in significant morbidity and mortality for those affected. Drug-induced phospholipidosis is one such toxicity. Drug-induced phospholipidosis is manifest as the intra-lysosomal accumulation of glycerophospholipids in association with the chronic use of commonly used pharmaceuticals. Specific examples of agents that cause phospholipidosis include, but are not limited to, azithromycin, fluoxetine, amiodarone, chloroquine, imipramine, and ketoconazole. Ascertaining the significance of this complication has been problematic for clinicians, as well as scientists and regulatory agencies overseeing drug discovery and approval. This is due to a limited understanding of the mechanisms responsible for phospholipidosis and the lack of suitable models for its study. Lysosomal phospholipase A2 is a recently discovered and characterized phospholipase that hydrolyzes those phospholipids that accumulate secondary to drug-induced phospholipidosis. In addition, mice engineered to express an inactive form of lysosomal phospholipase A2 develop a phenotype that closely resembles the clinical manifestations of this disorder. Specifically, these mice develop lysosomal phospholipid accumulation most prominently within their alveolar macrophages. When normal alveolar macrophages are exposed to amiodarone and additional phospholipidosis associated drugs, in vitro, lysosomal phospholipase A2 activity is inhibited in concert with phospholipid accumulation. Based on these observations, the primary hypothesis to be tested is that drug-induced phospholipidosis is the consequence of inhibition of lysosomal phospholipase A2. This grant will test this hypothesis by use of a newly developed fluorometric assay for lysosomal phospholipase A2 activity and the use of knockout mice that are partially or totally deficient in lysosomal phospholipase A2 activity. Also included in this project are mechanistic studies on the role of electrostatic charge interactions between the lysosomal phospholipase A2 protein and lipid membranes that contain the substrates for the enzyme. Finally, the association between common mutations in the lysosomal phospholipase A2 gene and susceptibility to drug-induced phospholipidosis will be studied by the expression of the mutated protein in cell lines. If the inhibition of lysosomal phospholipase A2 is demonstrated to be a primary mechanism for drug-induced hospholipidosis, then these findings may serve as both the basis for screening drugs that may cause this complication as well as for the identification of patients who may be uniquely sensitive to this form of drug toxicity.
Among the most frequent health problems for Veterans and the general population are the toxic complications of commonly used drugs. One such complication results from over fifty commonly used drugs and is termed drug-induced phospholipidosis. However, the basis for this toxicity is unknown and therefore, there is limited ability to assess its seriousness and to predic who might be at risk for its development. A newly discovered enzyme that is important in the breakdown of lipids within cells may be the target for drugs that cause phospholipidosis. One commonly used drug, amiodarone, has already been demonstrated to act in this manner. This grant will explore the extent to which other associated drugs inhibit this enzyme and the mechanisms by which this occur. If successful, these studies may provide the basis for preventing this important medical complication.
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