Mechanisms of Substrate Reduction Therapy for Niemann-Pick C Disease

描述(申请人提供)：Niemann-Pick C型(NPC)病是一种胆固醇-鞘糖脂(GSL)溶酶体储存障碍，最常见的原因是NPC1的缺陷，NPC1是一种跨膜蛋白，被认为在溶酶体底物的内吞后转运中起关键作用。大多数受影响的儿童在出生时看起来正常，在早期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。第一种是N-丁基脱氧诺吉霉素(NB-DNJ)或米格列斯特，是已知的GSL合成抑制剂，而第二种是羟丙基-环糊精(HPBCD)，是FDA批准的用于药物溶解的赋形剂。在NPC1病小鼠模型中，这两种化合物都能有效地延缓神经系统疾病的发生，延长寿命(分别为25%和100%)。然而，这两种药物都没有从导致其有效性的确切机制方面得到理解。对于米卢司坦，缺乏口服给药后神经节苷脂存储持续减少的证据。同样，对于HPBCD，尽管在NPC1小鼠中治疗后胆固醇和GSL的存储都显著减少，但这种好处背后的机制完全未知，甚至对于其穿越血脑屏障的能力仍存在争议。这项提议将利用现有的和新的试剂和动物模型，以及定量的高分辨率成像、生化和遗传评估，开展一系列补充的体内和体外研究，每项研究都针对鼻咽癌疾病的治疗机制。我们的前两个目标是准确地确定HPBCD在减少神经元中胆固醇/GSL储存方面的作用机制，并严格地重新检查和评估米卢司汀减少GSL合成的能力，以此作为其对神经元存活的有益影响的基础。我们的第三个目标是使用一种无偏见的基因分析方法来探索每种药物影响的所有代谢途径。根据在这些目标中吸取的经验教训，将在第四个目标中测试新的联合治疗策略，作为大幅改善鼻咽癌儿童治疗的一种手段。 公共卫生相关性：溶酶体储存障碍是一组约60种罕见的、致命的遗传病，由与内酶体-溶酶体系统相关的一系列蛋白质缺陷引起。Niemann-Pick C型(NPC)病是一种胆固醇-鞘糖脂(GSL)储存障碍，最常见的原因是NPC1的缺陷，NPC1是一种跨膜蛋白，被认为在溶酶体底物的内向后转运中起关键作用。受影响的儿童通常在出生时看起来正常，但从4-6岁开始出现进行性神经衰退，死亡通常发生在生命的第二个十年。鼻咽癌的治疗选择非常有限，酶替代、细胞介导和基因治疗几乎没有带来好处的希望，因为NPC1蛋白不能溶解并由细胞分泌。这些限制推动了药物的开发，这种药物可以限制有害底物在大脑和其他器官中的积聚-被称为底物减少疗法(SRT)。我们率先对两种这样的药物进行了研究，N-丁基脱氧诺吉霉素(米卢斯特)和2-羟丙基-环糊精(HPBCD)，这两种药物在动物模型上都显示出对鼻咽癌疾病的疗效。这项资助的目的是确定这两种药物在鼻咽癌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治疗鼻咽癌儿童的临床试验正被提交给FDA，将于2012年开始。参加这项试验的许多人也将接受米卢司坦的治疗。了解HPBCD和米格列斯特的作用机制和可能的相互作用(例如，协同作用)至关重要。重要的是，考虑到鼻咽癌和其他溶酶体疾病之间的相似之处，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方法可能会提供远远超过单一罕见疾病的好处。

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.