Nitazoxanide as a Novel Therapy for Rare Disease Lymphangioleiomyomatosis and Tuberous Sclerosis

摘要 结节性硬化症(TS)，又称结节性硬化症，是一种罕见的多系统遗传性疾病，常为生命性疾病。 一种会导致良性肿瘤在大脑和肾脏等其他重要器官生长的危险疾病， 心脏、眼睛、肺和皮肤。淋巴管肌瘤病(LAM)是一种与TS相关的肿瘤样疾病。两者都以 TSC1或TSC2基因的遗传性或零星突变的后果，其功能为阴性 MTOR途径的调节者。MTORC1活性失控导致肿瘤异常增殖 肺中的平滑肌细胞(LAM细胞)、进行性呼吸急促、复发性气胸和 肺组织结构和功能。在美国，TS影响多达25,000到40,000人， 全球约有100万至200万人。LAM是一种罕见的疾病，影响育龄妇女。第一个和 FDA批准的唯一治疗LAM的药物是免疫抑制剂西罗莫司(Rapamune)，自2015年以来由 辉瑞。它是目前的护理标准，通过抑制mTORC1发挥作用。西罗莫司有几个临床缺点， 包括相当数量的无应答者，由于其免疫抑制特性而产生严重不良事件 和怀孕C类，限制其在育龄妇女中的使用。因此，存在着高度未得到满足的医疗需求 开发LAM和TS的替代和更安全的治疗方案。 我们已经确定了Nitazoxanide(以下简称NTZ；Alinia)，一种FDA批准的安全的抗原虫药物 治疗1岁及以上儿童由蓝氏贾第鞭毛虫或隐孢子虫引起的腹泻 LAM/TS潜在的新疗法。利用捕获化合物质谱学技术，我们发现 NAD(P)H-苯醌氧化还原酶(NQO1)作为硝唑尼特的人体靶标。此外，我们已经表明， NTZ对三种不同LAM亲缘关系的小鼠和人细胞mTORC1相关信号的抑制作用 阐述LAM/TS的潜在病理机制的文章。 我们这笔赠款的目标是研究NTZ治疗LAM/TS的疗效。Aim 1将成为活体证明 在TSC2缺失的小鼠模型中比较三个剂量水平的NTZ的概念。 宾夕法尼亚大学的克里姆斯卡娅。它将评估TSC2缺失病变生长的预防， 免疫组织化学和肺形态改变与小鼠LAM模型动物存活的关系。 AIM 2将包括一项体外研究，调查潜在的细胞对mTOR和相关信号通路的影响。 这项体外研究将研究NTZ在LAM来源的AML(LAMD)细胞和永生化的TSC2缺失细胞中的作用 人类起源的文化，随后是更详细的mTORC1相互作用研究。除了单一的治疗方法 Ntz方法，将探索具有ntz附加物的易剂量毒性雷帕霉素的剂量减少潜力。 该项目旨在为LAM/TS的破坏性疾病患者开发一种新的治疗方法。基于NTZ的 作为一种被批准的抗原虫药物，我们的研究中的阳性疗效结果将使 根据FDA第505(2)(B)条批准途径和孤儿疾病指定加速临床开发。

ABSTRACT Tuberous sclerosis (TS), also called tuberous sclerosis complex, is a rare, multi-systemic genetic and often life- threatening disease that causes benign tumors to grow in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. Lymphangioleiomyomatosis (LAM) is a TS-related tumor-like disorder. Both occur as a consequence of an inherited or sporadic mutation in either the TSC1 or TSC2 gene, which function as negative regulators of the mTOR pathway. Uncontrolled mTORC1 activity leads to the neoplastic proliferation of abnormal smooth muscle cells (LAM cells) in the lungs, progressive shortness of breath, recurrent pneumothoraxes, and loss of pulmonary tissue structure and function. TS affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide. LAM is a rare disease affecting women in childbearing age. The first and only FDA-approved treatment for LAM is the immunosuppressant sirolimus (Rapamune©), marketed since 2015 by Pfizer. It is the current standard-of-care and acts by inhibiting mTORC1. Sirolimus has several clinical disadvantages, including a considerable number of non-responders, severe adverse events due to its immunosuppressive properties and pregnancy category C, limiting its use in women of childbearing age. Thus, there is a high unmet medical need to develop alternative and safer treatment options for LAM and TS. We have identified Nitazoxanide (hereafter NTZ; Alinia©), an antiprotozoal FDA-approved and safe therapy for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum in patients 1 year of age or older, as a potential novel therapy for LAM/TS. Using Capture Compound Mass Spectrometry technology, we discovered NAD(P)H quinone oxidoreductase (NQO1) as the human target of Nitazoxanide. Furthermore, we have shown that NTZ is effective in inhibiting mTORC1 related cellular signaling in three different LAM related murine and human cell lines addressing the underlying pathological mechanism of LAM/TS. Our goal for this grant is the investigation of NTZ’s efficacy for the treatment of LAM/TS. Aim 1 will be the in vivo proof of concept comparing three dose levels of NTZ in a TSC2-null murine model developed at the laboratory of Prof. Krymskaya at the University of Pennsylvania. It will evaluate prevention of TSC2-null lesion growth, immunohistochemical and lung morphology changes and animal survival in a murine LAM model. Aim 2 will cover an in vitro study, investigating underlying cellular effects on mTOR and related signaling pathways. The in vitro study will investigate NTZ's effects in LAM-derived AML (LAMD) cells and immortalized TSC2-null cell cultures of human origin followed by more detailed mTORC1 interactions studies. In addition to the monotherapeutic NTZ approach, dose reduction potential for dose-toxicity prone rapamycine with NTZ add-on will be explored. This project aims to develop a novel therapy for patients with the devastating diseases of LAM/TS. Based on NTZ's excellent safety profile as an approved antiprotozoal drug, positive efficacy results in our studies would enable an accelerated clinical development under FDA section 505 (2)(b) approval pathway and orphan disease designation.