Repurposing Nitazoxanide for Potential Treatment of Rare Disease Lymphangioleiomyomatosis

Abstract

Lymphangioleiomyomatosis (LAM) is a rare genetic lung disease. Unfortunately, treatment with the mTORC1 inhibitor Rapamycin only slows disease progression, and incomplete responses are common. Thus, there remains an urgent need to identify new targets for the development of curative LAM treatments. Nitazoxanide (NTZ) is an orally bioavailable antiprotozoal small molecule drug approved for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum in children and adults, with a demonstrated mTORC1 inhibitory effect in several human cell lines. NTZ's excellent safety profile characterized by its more than 20 years of clinical use makes it a promising candidate for repurposing. Our rationale for this study was to further investigate NTZ's effect using in vitro and in vivo LAM models and to elucidate the underlying molecular mechanism beyond mTORC1 inhibition. For this purpose, we investigated cell proliferation, cell viability, and changes in protein phosphorylation and expression in primary human cell cultures derived from LAM lung samples before translating our results into a syngeneic mouse model utilizing Tsc2-null cells. NTZ reduced cell growth for all tested cell lines at a dose of about 30 µM. Lower doses than that had no effect on cell viability, but doses above 45 µM lowered the viability by about 10 to 15% compared to control. Interestingly, our western blot revealed no inhibition of mTORC1 and only a mild effect on active ß-Catenin. Instead, NTZ had a pronounced effect on reducing pAkt. In the mouse model, prophylactic NTZ treatment via the intraperitoneal and oral routes had some effects on reducing lung lesions and improving body weight retention, but the results remain inconclusive.

Keywords: Nitazoxanide; drug repurposing; lung disease; lymphangioleiomyomatosis; rare disease.

Figure 1

Simplified pathway and targets of Nitazoxanide and Rapamycin involved in LAM/TS. (1) Growth factors regulate mTORC1 activity through the PI3K-AKT pathway by activating PI3K, which leads to the production of PIP3 and the recruitment of AKT to the plasma membrane. (2) At the membrane, AKT is phosphorylated at two sites by PDK1 and mTORC2. (3) Activated Akt phosphorylates and inhibits TSC1/2, a negative regulator of mTORC1. TSC suppressor mutations in LAM lead to constitutively active mTORC1 through the RHEB GTPase, resulting in (4) downstream phosphorylation of S6K and 4E-BP. Rapamycin binds to cytosolic FKBP12 and thereby acts as an allosteric inhibitor of mTORC1, preferentially inhibiting S6K/S6 phosphorylation. mTORC1/S6K and mTORC2 mediate negative feedback of PI3K/AKT [12]. 4E-BP: Eukaryotic translation initiation factor 4E-binding protein 1, AKT: Protein kinase B, eIF4E: Eukaryotic translation initiation factor 4E, FKBP12: Protein FK506-binding protein 12, mTORC: Mammalian target of Rapamycin complex, PDK1: Phosphoinositide-dependent kinase-1, PI3K: Phosphatidylinositol 3-kinase, PIP3: Phosphatidylinositol (3,4,5)-trisphosphate, Raptor: Regulatory-associated protein of mTOR, RHEB: Ras homolog enriched in brain, Rictor: Rapamycin-insensitive companion of mammalian target of rapamycin, RTK: Receptor tyrosine kinase, S6K: P70-S6 Kinase 1, S6: Ribosomal protein S6, TSC1/2: Tuberous sclerosis complex. Red dots indicate activating phosphorylation sites.

Figure 2

NTZ affects cell proliferation and viability in LAM-related cell lines. (a) Percent proliferation for different LAM-related cell lines: murine-derived TTJ cells; human angiomyolipoma cell line 621; primary human-derived LAM cells. Curves were created by applying the four-parameter log-logistic function. (b) Percent viability for the same cells. Trypan blue staining/cell counting was performed at 48 h post-seeding. Assays were performed in triplicate.

Figure 2

NTZ affects cell proliferation and viability in LAM-related cell lines. (a) Percent proliferation for different LAM-related cell lines: murine-derived TTJ cells; human angiomyolipoma cell line 621; primary human-derived LAM cells. Curves were created by applying the four-parameter log-logistic function. (b) Percent viability for the same cells. Trypan blue staining/cell counting was performed at 48 h post-seeding. Assays were performed in triplicate.

Figure 3

Partial inhibition of mTORC1 and Wnt ß-Catenin growth pathways in TTJ Tsc2-null cells by NTZ as determined by immunoblot analysis. (a) Immunoblot bands. (b) Active ß-Catenin is inhibited by NTZ. (c) pAkt is inhibited by NTZ and increased by Rapamycin (* p < 0.05). Original figures can be found in Supplementary Materials.

Figure 4

Tumor volume in the subcutaneous experiment. NTZ given intraperitoneally (n = 10) and orally formulated as chow (n = 10) on day 3 post tumor cell injection were able to significantly reduce tumor growth compared to the untreated control (n = 9). The positive control Rapamycin (n = 10) performed very well. (* p < 0.05, ** p < 0.01, *** p < 0.001). Dots represent outliers of the boxplot.

Figure 5

Representative H&E stainings, percent-lesions, and body weight changes in Tsc2-null lung lesions in a metastatic immunocompromised mouse model of LAM. Mice received 800,000 Tsc2-null TTJ cells injected into the tail vein. The animals received pharmacological treatment beginning 3–7 days after the injection of cells. Animals were euthanized 28 days after injection of the Tsc2-null cells. In two subsequent studies with a similar design, different NTZ and Rapamycin doses and combination were tested. (a) Lungs were inflated and stained with H&E. Scale bars: 4 mm and 500 μm. (b) Percent-lesion formation in lungs of the treated mice at takedown. Shown is an average of all mice in the treatment groups. (c) Development of body weight over the course of the study, presented as percentage change from baseline body weight on day 0. Group sizes were as follows: Vehicle n = 5, Rapa (0.1 mg/kg) i.p. n = 5, NTZ (500 mg/kg) oral n = 10, NTZ (100 mg/kg) i.p. + Rapa (0.1 mg/kg) i.p. n = 5, NTZ (100 mg/kg) i.p. n = 5, NTZ (500 mg/kg) oral + Rapa (0.5 mg/kg) i.p. n = 10, Rapa (0.5 mg/kg) i.p. n = 10. (ns non-significant, ** p < 0.01, **** p < 0.0001).