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

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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.