Mechanisms of Substrate Reduction Therapy for Niemann-Pick C Disease

描述（由申请方提供）：C型尼曼-匹克（NPC）病是一种胆固醇-鞘糖脂（GSL）溶酶体贮积症，最常见的原因是NPC 1缺陷，NPC 1是一种跨膜蛋白，被认为在从溶酶体逆向内吞转运底物中起关键作用。大多数受影响的儿童在出生时表现正常，在早年发展为进行性神经系统疾病，并在第二个十年死亡。我们率先开发了两种治疗这种疾病的化合物。第一种，N-丁基脱氧野尻霉素（NB-DNJ）或麦格司他是一种记录在案的GSL合成抑制剂，而第二种，羟丙基-环糊精（HPBCD）是FDA批准的用于药物溶解的辅料。在NPC 1疾病的小鼠模型中，这两种化合物在延迟神经系统疾病的发作和延长生命（分别延长25%和100%）方面是有效的。然而，这两种药物都没有被理解为其有效性负责的确切机制。对于麦格司他，Npc 1小鼠经口给药后神经节苷脂蓄积持续减少的证据缺乏。类似地，对于HPBCD，虽然在Npc 1小鼠中治疗后胆固醇和GSL储存都大幅减少，但这种益处的机制完全未知，甚至对其穿过血脑屏障的能力仍存在争议。该提案将采用当前和新型试剂和动物模型进行一系列补充性体内和体外研究，以及定量高分辨率成像，生化和遗传评估，每一项都针对NPC疾病的治疗机制。我们的前两个目的是精确定义HPBCD减少神经元中胆固醇/GSL蓄积的作用机制，并严格重新检查和评估麦格司他减少GSL合成的能力，作为其对神经元存活产生有益影响的基础。我们的第三个目标是使用无偏见的基因分析方法来探索每种药物影响的全部代谢途径。利用在这些目标中吸取的经验教训，新的组合治疗策略将在第四个目标中进行测试，作为大幅改善NPC疾病儿童治疗的一种手段。 公共卫生相关性：溶酶体贮积症是一组约60种罕见的致命性遗传疾病，由与内体-溶酶体系统相关的广泛蛋白质缺陷引起。C型尼曼-皮克病（NPC）是一种胆固醇-鞘糖脂（GSL）储存障碍，最常见的是NPC 1缺陷引起的，NPC 1是一种跨膜蛋白，被认为在溶酶体底物的逆向内吞运输中起关键作用。受影响的儿童通常在出生时表现正常，但在4-6岁时开始表现出进行性神经功能衰退，死亡通常发生在生命的第二个十年。NPC疾病的治疗选择非常有限，酶替代，细胞介导和基因疗法几乎没有希望提供益处，因为NPC 1蛋白不可溶并由细胞分泌。这些限制推动了药物的开发，这些药物可以限制大脑和其他器官中有害底物的积累-称为底物减少疗法（SRT）。我们率先研究了两种此类药物，N-丁基脱氧野尻霉素（麦格司他）和2-羟丙基-环糊精（HPBCD），这两种药物均在动物模型中显示出对NPC疾病的疗效。该基金的目的是确定这两种药物在NPC疾病小鼠模型中延迟临床疾病和延长寿命的机制。这一目标现在变得更加及时，因为一项涉及HPBCD治疗儿童NPC疾病的临床试验正在向FDA提出，开始于2012年。入组本试验的许多个体也将接受麦格司他治疗。了解作用机制和可能的相互作用（例如，协同作用）是至关重要的。重要的是，鉴于NPC和其他溶酶体疾病之间的相似性，以及更常见的神经退行性疾病，如阿尔茨海默氏症，这里出现的成功治疗可能会提供远远超出单一罕见疾病的益处。

DESCRIPTION (provided by applicant): Niemann-Pick type C (NPC) disease is a cholesterol-glycosphingolipid (GSL) lysosomal storage disorder caused most commonly by defects in NPC1, a transmembrane protein believed critical in retroendocytic trafficking of substrates from lysosomes. Most affected children appear normal at birth, develop progressive neurological disease in their early years and die in their second decade. We have pioneered the development of two compounds for this disorder. The first, N-butyldeoxynojirimycin (NB-DNJ) or miglustat is a documented inhibitor of GSL synthesis, whereas the second, hydroxypropyl ¿-cyclodextrin (HPBCD), is an FDA-approved excipient used for drug solublization. Both compounds are efficacious in delaying onset of neurological disease and prolonging life (by 25% and 100%, respectively) in the mouse model of NPC1 disease. Yet neither drug is understood in terms of the precise mechanism responsible for its effectiveness. For miglustat, evidence for sustained reductions in ganglioside storage following oral administration to Npc1 mice is lacking. Similarly, for HPBCD, while both cholesterol and GSL storage are substantially reduced following treatment in Npc1 mice, the mechanism underlying this benefit is completely unknown, and indeed controversy continues even over its ability to cross the blood brain barrier. This proposal will carry out a series of complementary in vivo and in vitro studies employing current and novel reagents and animal models, and quantitative high-resolution imaging, biochemical and genetic evaluations, each directed at treatment mechanisms for NPC disease. Our first two aims are to precisely define HPBCD's mechanism of action in reducing cholesterol/GSL storage in neurons and to critically re-examine and assess miglustat's ability to reduce GSL synthesis as a basis for its beneficial impact on neuron survival. Our third aim uses an unbiased gene analysis approach to explore the full range of metabolic pathways impacted by each drug. Capitalizing on lessons learned in these aims, new combinatorial treatment strategies will be tested in the fourth aim as a means to substantially improve therapy for children with NPC disease. PUBLIC HEALTH RELEVANCE: Lysosomal storage disorders are a group of about 60 rare, fatal genetic diseases caused by defects in a wide range of proteins associated with the endosomal-lysosomal system. Niemann-Pick type C (NPC) disease is a cholesterol-glycosphingolipid (GSL) storage disorder caused most commonly by defects in NPC1, a transmembrane protein believed critical in retroendocytic trafficking of substrates from lysosomes. Affected children typically appear normal at birth but exhibit progressive neurological decline beginning at 4-6 years of age with death often occurring in the second decade of life. Therapeutic options for NPC disease are very limited, with enzyme replacement, cell-mediated, and gene therapies providing little hope of benefit since the NPC1 protein is not soluble and secreted by cells. Such limitations have driven development of drugs that can limit the build-up of offending substrates in brain and other organs - known as substrate reduction therapy (SRT). We have pioneered the study of two such agents, N- butyldeoxynojirimycin (miglustat) and 2-hydroxypropyl ¿-cyclodextrin (HPBCD), both of which have shown efficacy for NPC disease in animal models. The purpose of this grant is to determine the mechanisms by which these two agents delay clinical disease and increase longevity in the murine model of NPC disease. This goal has now become all the more timely as a clinical trial involving HPBCD for treatment of children with NPC disease is being proposed to the FDA, to begin in 2012. Many of the individuals enrolling in this trial will also be under treatment with miglustat. Understanding the mechanisms of action and possible interactions (e.g., synergy) of HPBCD and miglustat are of paramount importance. Importantly, given similarities between NPC and other lysosomal diseases, as well as more common neurodegenerative conditions like Alzheimer's, successful treatments emerging here may provide benefit well beyond a single rare disease.