Preclinical Testing of a Novel Niclosamide Stearate Prodrug Therapeutic (NSPT) Shows Efficacy Against Osteosarcoma.

Gireesh B Reddy,Brian E Brigman,David L Kerr,David S Hsu,William C Eward,Jason A Somarelli,Artak Tovmasyan,David Needham,Ivan Spasojevic

doi:10.1158/1535-7163.mct-19-0689

Gireesh B Reddy, Brian E Brigman + Show 7 more

Open Access

https://doi.org/10.1158/1535-7163.mct-19-0689

Copy DOI

Abstract

Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied in vitro for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; ex vivo pulmonary metastatic assay model; and in vivo pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation in vivo with t 1/2 approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells in vitro at 0.2-2 μmol/L IC50; inhibits recognized growth pathways and induces apoptosis at 20 μmol/L; eliminates metastatic lesions in the ex vivo lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.

Highlights

Osteosarcoma has seen almost no chemotherapeutic advances over the past 3 decadesOsteosarcoma is the most common primary bone malignancy in humans
Materials niclosamide stearate prodrug therapeutic (NSPT) were created using the following materials and manufacturers: niclosamide stearate was synthesized by the Duke Small Molecule Synthesis Facility; niclosamide anhydrous (Sigma Aldrich), chloroform stock solutions (DSPC, cholesterol, and DSPE PEG2000; Avanti Polar Lipids, Inc.), acetone (!99.8%; Sigma Aldrich), absolute ethanol (!99.8%; VWR Chemicals), chloroform (Rathburn Chemicals Ltd.), buffer solution components, acetic acid (Fluka), sodium phosphate monobasic (Sigma Aldrich), and Millipure water
Preparation of NSPTs As described by Walke and colleagues and Hervella and colleagues, the NSPT nanoparticles were made by adapting a rapid solvent exchange method to a new prodrug, niclosamide stearate [41,42,43,44, 56, 57]

Summary

Introduction

Osteosarcoma has seen almost no chemotherapeutic advances over the past 3 decades. Osteosarcoma is the most common primary bone malignancy in humans. It most frequently occurs in adolescence [1]. 15%–20% of patients already have visible pulmonary metastatic disease, and a majority of patients have lung metastases that are not yet detectable [2]. Because most patients without visible metastatic disease at presentation likely have undetectable micrometastasis, systemic therapy, both neoadjuvant and adjuvant therapy, is a critical addition to surgical resection [3]. Osteosarcoma, unlike most other solid tumors, has seen relatively few chemotherapeutic advances over the past 3 decades, with respect to patients presenting with advanced or metastatic disease [4,5,6,7,8].

Objectives

Methods

Results

Conclusion