| dc.description.abstract | Chapter 1 Emergence of phage–resistant bacteria has led to repurposing of combination therapy using phage and antibiotics. Inhaled phage and antibiotic combination have the potential to combat respiratory antimicrobial resistant infections. This chapter contains a literature review examining in vitro and in vivo phage and antibiotic combination therapy on respiratory infections. This chapter has been published in Advanced Drug Delivery Reviews, 2018; 133: 80-81, with the title ‘Phage therapy for respiratory infections. Authors: Rachel Yoon Kyung Chang, Martin Wallin, Yu Lin, Sharon Sui Yee Leung, Hui Wang, Sandra Morales, Hak-Kim Chan. Chapter 2 Nebulization is currently used for delivery of antibiotics for respiratory infections. Bacteriophages (or phages) are effective predators of pathogens including Pseudomonas aeruginosa commonly found in the lungs of patients with cystic fibrosis (CF). It is known that phages and antibiotics can potentially show synergistic antimicrobial effect on bacterial killing. In the present study, we investigated synergistic antimicrobial effect of phage PEV20 with five different antibiotics against three P. aeruginosa strains isolated from sputum of CF patients. The antibiotics included ciprofloxacin, tobramycin, colistin, aztreonam and amikacin, which are approved by U.S Food and Drug Administration (FDA) for inhaled administration. Phage and antibiotic synergy was determined by assessing bacterial killing in time-kill studies. Among the different phage-antibiotic combinations, PEV20 and ciprofloxacin exhibited the most synergistic effect. Two phage-ciprofloxacin combinations, containing 1/4 and 1/2 of the minimum inhibitory concentration (MIC) of ciprofloxacin against P. aeruginosa strains FADD1-PA001 and JIP865, respectively were aerosolized using both air-jet and vibrating mesh nebulizers, and the synergistic antibacterial activity was maintained after nebulization. Air-jet nebulizer generated droplets with smaller volume median diameters (3.6-3.7 µm) and slightly larger span (2.3-2.4) than vibrating mesh nebulizers (5.1-5.3 µm; 2.1-2.2), achieving a higher fine particle fraction (FPF) of 70%. In conclusion, the nebulized phage PEV20 and ciprofloxacin combination shows promising antimicrobial and aerosol characteristics for potential treatment of respiratory tract infections caused by drug-resistant P. aeruginosa. This chapter has been published in International Journal of Pharmaceutics, 2018; 551:158-165, with the title ‘Synergy of nebulized phage PEV20 and ciprofloxacin combination against Pseudomonas aeruginosa’. Authors: Yu Lin, Rachel Yoon Kyung Chang, Warwick J. Britton, Sandra Morales, Elizabeth Kutter, Hak-Kim Chan. Chapter 3 Compared to nebulization, dry powders for inhalation may improve patient handling characteristics and compliance. In the present study, we co-spray dried ciprofloxacin and phage PEV20 using L-leucine with or without lactose as excipients. Two formulations were identified for testing in this study. The mass ratios were set at 1:1:1 for ciprofloxacin, lactose and L-leucine (Formulation A) or 2:1 for ciprofloxacin and L-leucine without lactose (Formulation B). Concentrations of PEV20 were set at 108 and 109 PFU/mL for two clinical P. aeruginosa strains FADD1-PA001 and JIP865, respectively. Formulations A and B were characterized as partially crystalline and the powders recrystallized at >40% relative humidity (RH). Both formulations exhibited strong synergistic antimicrobial killing effect on the two strains. Formulations A and B maintained bactericidal synergy after dispersion using both low and high resistance OsmohalerTM inhalers. Powder aerosol performance in the low resistance inhaler at 100 L/min was examined by next generation impactor (NGI) and in the high resistance inhaler at 60 L/min by multi-stage liquid impinger (MSLI). Fine particle fractions (FPF) obtained by NGI were 59.7 ± 2.1 % and 64.3 ± 2.9% for Formulations A and B, respectively. FPF obtained by MSLI were 71.0 ± 3.4% and 73.3 ± 5.0%, respectively. In conclusion, it is feasible to prepare stable and inhalable combination powder formulations of phage PEV20 and ciprofloxacin for potential treatment of respiratory infections caused by multi-drug resistant (MDR) P. aeruginosa. This chapter has been published in European Journal of Pharmaceutics and Biopharmaceutics, 2019; 142:543-552, with the title ‘Inhalable combination powder formulations of phage and ciprofloxacin for P. aeruginosa respiratory infections’. Authors: Yu Lin, Rachel Yoon Kyung Chang, Warwick J. Britton, Sandra Morales, Elizabeth Kutter, Jian Li, Hak-Kim Chan. Chapter 4 In this study, we investigated the storage stability of these powders which comprised phage PEV20, ciprofloxacin, lactose and L-leucine in mass ratios of 1:1:1 (Formulation A) or ciprofloxacin and L-leucine in 2:1 without lactose (Formulation B). These powders were produced by spray drying, collected in polypropylene tubes and packed inside aluminium pouches which were heat-sealed at < 20% relative humidity (RH), then stored at 4°C or 25°C. The phage viability, aerosol performance and solid-state properties of the powders were examined over 12 months. The biological activity and aerosol performance of both formulations showed no significant change over 12 months of storage at 4 °C. However, after four months of storage at 25 °C, a significant titer loss of 2.2 log10 (p<0.01) was observed in Formulation B, but the loss in Formulation A was much less (0.5 log10 (p<0.05)). In contrast, the fine particle fraction (FPF, wt. % particles ≤5 µm) of Formulation A was significantly reduced by 11% (p<0.05) after four months of storage at 25 °C, whereas the aerosol performance of Formulation B remained stable over 12 months. The results showed that ciprofloxacin can sufficiently stabilize phage through vitrification and/or hydrogen bonding at 4 °C. The presence of lactose was necessary with the ciprofloxacin to preserve the phage at 25 °C, while leucine maintained the powder dispersibility. In conclusion, spray dried PEV20-ciprofloxacin combination powders were biologically and physico-chemically stable even without lactose as a stabilising excipient, when stored below 20% RH at 4 °C for 12 months. This chapter has been submitted to International Journal of Pharmaceutics with the title ‘Storage stability of phage-ciprofloxacin combination powders for Pseudomonas aeruginosa respiratory infections’. Authors: Yu Lin, Rachel Yoon Kyung Chang, Warwick J. Britton, Sandra Morales, Elizabeth Kutter, Jian Li, Hak-Kim Chan. Chapter 5 Combination treatment using bacteriophage and antibiotics can be an advanced approach to combatting antimicrobial-resistant bacterial infections. In this chapter, we aimed to assess the in vivo efficacy of the powder using a neutropenic mouse model of acute lung infection. The synergistic activity of PEV20 and ciprofloxacin was investigated by infecting mice with P. aeruginosa, then administering freshly spray-dried single PEV20 (106 PFU/mg), single ciprofloxacin (0.33 mg/mg) or combined PEV20-ciprofloxacin treatment using a dry powder insufflator. Lung tissues were then harvested for colony counting, flow cytometry analysis and histopathological examination at 24 h post-treatment. PEV20 and ciprofloxacin combination powder significantly reduced the bacterial load of clinical P. aeruginosa strain in mouse lungs by 5.9 log10 (P < 0.005). No obvious reduction in bacterial load was observed in the single PEV20 and single ciprofloxacin groups. Assessing immunological responses in the lungs demonstrated that the efficacy of PEV20-ciprofloxacin treatment is associated with reduced inflammation. While all infected mice demonstrated worsened lung pathology, this did not significantly differ between treatment groups. In this pre-clinical study, the co-spray dried combination powder of PEV20 and ciprofloxacin was shown to exhibit synergistic efficacy against P. aeruginosa respiratory infections. Further studies are needed to assess pharmacokinetics as well as long-term safety for potential clinical implementation. This chapter has been submitted to Antimicrobial Agents and Chemotherapy with the title ‘Efficacy of co-spray dried phage and ciprofloxacin combination in an acute neutropenic mouse lung infection model caused by Pseudomonas aeruginosa’. Authors: Yu Lin, Diana Quan, Rachel Yoon Kyung Chang, Micheal Y. T. Chow, Yuncheng Wang, Mengyu Li, Warwick J. Britton, Sandra Morales, Elizabeth Kutter, Jian Li, Hak-Kim Chan. Chapter 6 The major findings from the previous chapters are summarized in this chapter. • Nebulized phage PEV20 and ciprofloxacin combination shows promising antimicrobial and aerosol characteristics for potential treatment of respiratory tract infections caused by multi-drug resistant (MDR) P. aeruginosa. • It is feasible to prepare stable and inhalable combination powder formulations of phage PEV20 and ciprofloxacin for potential treatment of respiratory infections caused by MDR P. aeruginosa. • One-year shelf-life of PEV20-ciprofloxacin combination powders can be achieved by aluminium foil packaging under a dry environment of < 20% RH and storage at a cold temperature of 4°C. • Co-spray dried combination powder of PEV20 and ciprofloxacin was shown to exhibit synergistic efficacy against P. aeruginosa respiratory infections in mice lung. Furthermore, the future projects and challenges are discussed in chapter 6 and the final remarks. | en_AU |
