Production of highly stable spray dried phage formulations for treatment of Pseudomonas aeruginosa lung infection

Abstract

The potential of bacteriophage therapy for the treatment of pulmonary infections caused by antibiotic-resistant bacteria has been well recognised. The purpose of this study was to investigate the effect of excipients on stabilisation and aerosolisation of spray dried powders of morphologically different phages - PEV podovirus and PEV myovirus. Seven anti-pseudomonal phages were screened against 90 clinical strains of bacterial hosts and three of the phages were selected for formulation study based on the host range. Design of experiments was utilised to assess the effect of different excipients on the stabilisation and aerosolisation of spray dried phages. Both podovirus and myovirus phages were stable in spray dried formulations containing trehalose or lactose and leucine as excipients with less than 1-log₁₀ titre reduction during spray drying, with lactose providing superior phage protection over trehalose. Furthermore, the spray dried phage formulations dispersed in an Osmohaler at 85L/min produced a high fine particle fraction of over 50%. The results showed that the phages in this study can form respirable dry powder phage formulations using the same excipient composition. Spray dried various types of lytic phages hold significant potential for the treatment of pulmonary infections.

Keywords: Antibiotic-resistant bacteria; Bacteriophage therapy; Biotherapeutics; Powder aerosols; Pulmonary infections.

Figure 1

Schematic diagram of formulation development stages and screening criteria.

Figure 2

Host range of seven PEV phages against various clinical MDR P. aeruginosa strains. Host range was tested using standard spot test.

Figure 3

Main effects plots for means. PEV1, PEV20 and PEV61 were individually spray dried with either a) trehalose or b) lactose, together with leucine, pluronic F68 and PEG1000. Each excipient is ranked from 1 to 4 depending on the significance of their role in phage stabilisation (rank 1 being the most significant).

Figure 3

Main effects plots for means. PEV1, PEV20 and PEV61 were individually spray dried with either a) trehalose or b) lactose, together with leucine, pluronic F68 and PEG1000. Each excipient is ranked from 1 to 4 depending on the significance of their role in phage stabilisation (rank 1 being the most significant).

Figure 4

SEM images of phage (a) PEV1, (b) PEV20, and (c) PEV61 formulation spray dried with 12 mg/mL trehalose, 2.8 mg/mL leucine and 10 mg/mL PEG3000 (formulation 8), phage (d) PEV1, and (e) PEV61 formulation spray dried with 6 mg/mL lactose, 2.8 mg/mL leucine and 0.54% (v/v) pluronic F68 (formulation 14).

Figure 5

SEM images of powders spray dried from formulations containing high concentration of lactose or trehalose. Phage formulation spray dried with 12 mg/mL of sugar a) lactose or b) trehalose, 5.5 mg/mL of leucine and 0.27 mg/mL of pluronic F68.

Figure 6

SEM images of powders spray dried from formulations without leucine. Phage formulation spray dried with 12 mg/mL of sugar a) lactose or b) trehalose, 5 mg/mL of PEG1000 and 0.27 mg/mL of pluronic F68.

Figure 7

SEM images of formulations. The codes “23TH2LC” denotes 23 mg/mL trehalose with 2 mg/mL leucine; “23LT2LC” denotes 23 mg/mL lactose with 2 mg/mL leucine and so on.

Figure 8

Lung dose of different spray dried (a) PEV1, (b) PEV20, and (c) PEV61 formulations determined by in vitro aerosolisation followed by biological assay.

Figure 8

Lung dose of different spray dried (a) PEV1, (b) PEV20, and (c) PEV61 formulations determined by in vitro aerosolisation followed by biological assay.

Figure 9

X-ray powder diffraction patterns of spray dried phage formulations.

Figure 10

First sorption cycle data from dynamic vapour sorption (DVS) isotherm for representative phage spray dried formulations.