Mechanisms of Drug Induced Phospholipidosis

描述(由申请人提供) 在门诊和住院环境中，药物毒性是一种重大的疾病负担。许多药物毒性在表现和严重程度上都是特发性的，往往会导致患者严重的发病率和死亡率。药物引起的磷脂沉积症就是这样一种毒性。药物诱导的磷脂沉积症表现为甘油磷脂在溶酶体内的积聚，与长期使用常用药物有关。引起磷脂沉积症的药物的具体例子包括但不限于阿奇霉素、氟西汀、胺碘酮、氯喹、丙咪嗪和酮康唑。对于临床医生以及监督药物发现和审批的科学家和监管机构来说，确定这种并发症的重要性一直是个问题。这是由于对磷脂沉积症的机制了解有限，缺乏合适的模型来进行研究。溶酶体磷脂酶A2是一种新近发现的具有特征性的磷脂酶，它能降解药物引起的磷脂沉积所积累的磷脂。此外，被设计为表达非活性溶酶体磷脂酶A2的小鼠，其表型与这种疾病的临床表现非常相似。具体地说，这些小鼠的溶酶体磷脂积聚最显著的是在它们的肺泡巨噬细胞中。当正常肺泡巨噬细胞暴露于胺碘酮和额外的磷脂增多症相关药物时，在体外，溶酶体磷脂酶A2的活性受到抑制，同时磷脂积累也受到抑制。基于这些观察，需要检验的主要假设是药物诱导的磷脂沉积是溶酶体磷脂酶A2抑制的结果。这项资助将通过使用一种新开发的溶酶体磷脂酶A2活性的荧光分析方法以及使用部分或完全缺乏溶酶体磷脂酶A2活性的基因敲除小鼠来验证这一假设。该项目还包括溶酶体磷脂酶A2蛋白和含有该酶底物的脂膜之间静电电荷相互作用的机制研究。最后，将通过突变蛋白在细胞系中的表达来研究溶酶体磷脂酶A2基因的常见突变与药物诱导的磷脂血症易感性之间的关系。如果溶酶体磷脂酶A2的抑制被证明是药物诱导的磷脂沉积症的主要机制，那么这些发现可以作为筛选可能导致这种并发症的药物以及识别可能对这种药物毒性唯一敏感的患者的基础。

DESCRIPTION (provided by applicant) 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.