Toward Repositioning Niclosamide for Antivirulence Therapy of Pseudomonas aeruginosa Lung Infections: Development of Inhalable Formulations through Nanosuspension Technology Gabriella Costabile, † Ivana d’Angelo, ‡ Giordano Rampioni, § Roslen Bondì, § Barbara Pompili, ⊥ Fiorentina Ascenzioni, ⊥ Emma Mitidieri, † Roberta d’Emmanuele di Villa Bianca, † Raffaella Sorrentino, † Agnese Miro, † Fabiana Quaglia, † Francesco Imperi, ⊥ Livia Leoni, § and Francesca Ungaro* ,† † Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Naples, Italy ‡ Di.S.T.A.Bi.F., Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy § Department of Sciences, University Roma Tre, Viale Marconi, 446, 00146 Rome, Italy ⊥ Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Via dei Sardi 70, 00185 Rome, Italy * S Supporting Information ABSTRACT: Inhaled antivirulence drugs are currently consid- ered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of -20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5-10 μM. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e., ≤10 μM). In vivo acute toxicity studies in rats confirmed no observable toxicity of the NCL T80_10 DP formulation upon intratracheal administration at a concentration 100-fold higher than the anti-QS activity concentration. These preliminary results suggest that NCL repurposed in the form of inhalable nanosuspensions has great potential for the local treatment of P. aeruginosa lung infections as in the case of CF patients. KEYWORDS: nanosuspensions, pulmonary delivery, antivirulence drugs, niclosamide, Pseudomonas aeruginosa, cystic fibrosis, quorum sensing, rat 1. INTRODUCTION Cystic fibrosis (CF) is an autosomal disease caused by a defect in a single gene encoding the CF transmembrane conductance regulator (CFTR). 1 This condition predisposes CF patients to recurrent/persistent bacterial lung infections, which are the primary cause of bronchiectasis, respiratory failure, and consequent death in CF patients. 1-3 The dominant pathogen in CF airways is Pseudomonas aeruginosa, though other microorganisms may play a role in lung function decline. 4 Although lung infections can be controlled to some extent by early, aggressive antibiotic treatments, 60-70% of CF patients become chronically infected by P. aeruginosa by the age of 20. Current treatment involves life-long daily inhaled antibiotic therapies. 5 Antivirulence drugs, that is, agents that inhibit the production of disease-causing virulence factors but are neither bacterio- static nor bactericidal, are considered promising therapeutic Special Issue: Advances in Respiratory and Nasal Drug Delivery Received: January 31, 2015 Revised: May 12, 2015 Accepted: May 14, 2015 Article pubs.acs.org/molecularpharmaceutics © XXXX American Chemical Society A DOI: 10.1021/acs.molpharmaceut.5b00098 Mol. Pharmaceutics XXXX, XXX, XXX-XXX