This study aimed to assess the robustness of using a spray drying approach and formulation design in producing inhalable phage powders. Two types of Pseudomonas phages, PEV2 (Podovirus) and PEV40 (Myovirus) in two formulations containing different amounts of trehalose (70% and 60%) and leucine (30% and 40%) were studied. Most of the surface of the produced powders was found to be covered in crystalline leucine. The powders were stored at 4 °C and 20 °C under vacuum. The phage stability and in vitro aerosol performance of the phage powders were examined on the day of production and after 1, 3 and 12 months of storage. A minor titer loss during production was observed for both phages (0.2-0.8 log10 pfu/ml). The storage stability of the produced phage powders was found to be phage and formulation dependent. No further reduction in titer occurred for PEV2 powders stored at 4 °C across the study. The formulation containing 30% leucine maintained the viability of PEV2 at 20 °C, while the formulation containing 40% leucine gradually lost titer over time with a storage reduction of ∼0.9 log10 pfu/ml measured after 12 months. In comparison, the PEV40 phage powders generally had a ∼ 0.5 log10 pfu/ml loss upon storage regardless of temperature. When aerosolized, the total in vitro lung doses of PEV2 were of the order of 107 pfu, except the formulation containing 40% leucine stored at 20 °C which had a lower lung dose. The PEV40 powders also had lung doses of 106-107 pfu. The results demonstrate that spray dried Myoviridae and Podoviridae phage in a simple formulation of leucine and trehalose can be successfully stored for one year at 4 °C and 20 °C with vacuum packaging.
Cystic fibrosis (CF) is an inherited lung disease characterised by the accumulation of thick layers of dried mucus in the lungs which serve as a nidus for chronic infection. Pseudomonas aeruginosa is the predominant cause of chronic lung infection in cystic fibrosis. The dense mucus coupled with biofilm formation hinder antibiotic penetration and prevent them from reaching their target. Mucoactive agents are recommended in the treatment of CF in combination with antibiotics. In spite of the extensive research in developing novel drug combinations for the treatment of lung infection in CF, to our knowledge, there is no study that combines antibiotic, antibiofilm and mucoactive agent in a single inhaled dry powder formulation. In the present study, we investigate the possibility of adding a mucoactive agent to our previously developed ciprofloxacinquercetin (antibiotic-antibiofilm) dry powder for inhalation. Three mucoactive agents, namely mannitol (MAN), N-acetyl-L-cysteine (NAC) and ambroxol hydrochloride (AMB), were investigated for this purpose. The ternary combinations were prepared via spray drying without the addition of excipients. All ternary combinations conserved or improved the antibacterial and biofilm inhibition activities of ciprofloxacin against P. aeruginosa (ATCC 10145). The addition of AMB resulted in an amorphous ternary combination (SD-CQA) with superior physical stability as indicated by DSC and nonambient XRPD. Furthermore, SD-CQA displayed better in vitro aerosolization performance (ED ∼ 71 %; FPF ∼ 49 %) compared to formulations containing MAN and NAC (ED ∼ 64 % and 44 %; FPF ∼ 44 % and 29 %, respectively). In conclusion, a ternary drug combination powder with suitable aerosolization, physical stability and antibacterial/antibiofilm properties was prepared by a single spray drying step.