1 3 A novel inhaled multi-pronged attack against respiratory bacteria Q1 4 5 6 Sie Huey Lee a,b Q2 , Jeanette Teo c , Desmond Heng a,⇑ , Yanli Zhao b,d , Wai Kiong Ng a , Hak-Kim Chan e , 7 Tan Li Teng a , Reginald B.H. Tan a,f,⇑ 8 a Institute of Chemical and Engineering Sciences, A / STAR (Agency for Science, Technology and Research), 1, Pesek Road, Jurong Island, Singapore 627833, Singapore 9 b Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore 10 c Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore 11 d School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore 12 e Advanced Drug Delivery Group, Faculty of Pharmacy, A15, The University of Sydney, Sydney, NSW 2006, Australia 13 f Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore 14 15 17 article info 18 Article history: 19 Received 27 October 2014 20 Received in revised form 26 December 2014 21 Accepted 10 January 2015 22 Available online xxxx 23 Keywords: 24 Dry powder inhaler 25 Antiinfectives 26 Mucoactives 27 Combinatorial therapy 28 Synergy 29 Quorum sensing 30 31 abstract 32 Airway mucus hypersecretion is a common clinical feature of many severe respiratory diseases, and when 33 complicated by a recalcitrant bacterial infection, the whole treatment regimen thereby becomes more 34 challenging and protracted. The accumulation of thickened mucus secretions in the lower airways pro- 35 vides a nutrient-rich breeding ground for bacteria that promotes their growth and limits the ease of effec- 36 tive eradication. Unfortunately, no direct-inhaled dry powder formulation to treat these respiratory 37 mucoid infections more effectively is available commercially. This work therefore seeks to develop a 38 highly-efficacious ternary dry powder inhaler (DPI) formulation (ciprofloxacin hydrochloride (CIP), gati- 39 floxacin hydrochloride (GAT) and ambroxol hydrochloride (AMB)) capable of delivering a novel multi- 40 pronged attack (synergy, quorum quenching and mucociliary clearance) on Pseudomonas aeruginosa,a 41 common respiratory bacteria found in mucoid infections. The powders were prepared via spray drying, 42 evaluated on their aerosol performance via a multi-stage liquid impinger and tested for their efficacies 43 in bacteria-spiked artificial sputum medium (ASM). The optimized particles were of respirable-size 44 (d 50 of 1.61 ± 0.03 lm) and slightly corrugated. When dispersed via an Aerolizer Ò inhaler at 60 L/min, 45 the powder showed concomitant in vitro deposition, minimal capsule, device and throat retention, and 46 highly promising and uniform fine particle fractions (of the loaded dose) of 64–69%, which was a vast 47 improvement over the singly-delivered actives. Favourably, when tested on bacteria-spiked ASM, the 48 optimized ternary formulation (with AMB) was more effective at killing bacteria (i.e. faster rate of killing) 49 than just the synergistic antibiotics alone (binary formulation; without AMB). In conclusion, a ternary 50 antibiotic–(non-antibiotic) DPI formulation involving a unique multi-pronged attack mechanism was 51 successfully pioneered and optimized for mucoid infections. 52 Ó 2015 Published by Elsevier B.V. 53 54 55 56 1. Introduction 57 Airway mucus hypersecretion and a recalcitrant bacterial infec- 58 tion are the hallmarks of cystic fibrosis (CF), and as of today, this 59 condition is the most common life-threatening inherited disease 60 afflicting the Caucasian population (O’Sullivan and Freedman, 61 2009). The CF disorder is caused by mutations in a single gene on 62 chromosome 7 that encodes the cystic fibrosis transmembrane 63 conductance regulator (CFTR), a 1480 amino acid polypeptide 64 which regulates the transport of chloride ions across epithelial 65 membranes (Tsui, 1995). Abnormalities of CFTR function result in 66 low volume abnormal secretions leading to mucus plugs, impaired 67 mucociliary clearance and eventually, chronic lung inflammation 68 and infection (Bilton, 2008). 69 As CF patients with Pseudomonas aeruginosa infections typically 70 have more rapid declines in lung function than non-P. aeruginosa 71 infected individuals (Bilton, 2008), anti-pseudomonal therapy for 72 this group of patients should hence be initiated early and aggres- 73 sively upon first isolation of the bacteria, in order to prevent its 74 evolution into a chronic infection (Valerius et al., 1991). Early erad- 75 ication of P. aeruginosa infection is extremely crucial as initial P. http://dx.doi.org/10.1016/j.ejps.2015.01.005 0928-0987/Ó 2015 Published by Elsevier B.V. ⇑ Corresponding Q3 authors at: Institute of Chemical and Engineering Sciences, A / STAR (Agency for Science, Technology and Research), 1, Pesek Road, Jurong Island, Singapore 627833, Singapore. Tel.: +65 67963841 (R.B.H. Tan). Tel.: +65 67963861; fax: +65 63166188 (D. Heng). E-mail addresses: desmond_heng@ices.a-star.edu.sg (D. Heng), reginald_tan@ ices.a-star.edu.sg (R.B.H. Tan). European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps PHASCI 3171 No. of Pages 8, Model 5G 22 January 2015 Please cite this article in press as: Lee, S.H., et al. A novel inhaled multi-pronged attack against respiratory bacteria Q1 . Eur. J. Pharm. Sci. (2015), http:// dx.doi.org/10.1016/j.ejps.2015.01.005