Pulmonary delivery of excipient-free tobramycin DPIs for the treatment of Pseudomonas aeruginosa lung infection with CF

Abstract

Pseudomonas aeruginosa infection has become a widespread problem in patients with cystic fibrosis (CF). A safe and effective manufacturing method is required to produce antibiotic dry powder inhalations (DPIs) which can be effectively delivered to treat lung infections. In this study, an excipient-free tobramycin inhalable powder was prepared using spray freeze-drying (SFD) method. The mass median aerodynamic diameters (MMAD) of optimized inhalable powder prepared by SFD was 1.30 µm, and the fine particle fractions (FPF) reached 83.31%. In both in vitro and in vivo safety and activity studies, the inhalable powder showed excellent safety performance at both animal and cellular levels, with a minimum inhibitory concentration (MIC) of 0.5 μg/mL. Compared with intravenous injection, inhalation of excipient-free tobramycin inhalable powder had a better effect in the infected mouse model because of its amorphous state. This study demonstrates that excipient-free tobramycin inhalable powder with good delivery and deposition performance can be successfully obtained using the SFD method. Inhalation of excipient-free tobramycin inhalable powder has the potential to be a promising strategy for treating pulmonary infections caused by P. aeruginosa in patients with CF.

Keywords: Pseudomonas aeruginosa infection; dry powder inhalations (DPIs); pulmonary drug delivery; spray freeze-drying (SFD); tobramycin.

FIGURE 1

The relationship between particle size with (a) tobramycin concentration C _tob ; (b) atomization pressure P and (c) volumetric feeding rate Q when applying SFD method.

FIGURE 2

Machine learning for screening key process parameters: (a) schematic diagram of the decision tree model by SFD; (b) proportion of importance of process parameters by SFD.

FIGURE 3

The SEM images and size distribution of (a) tobramycin API; (b) sample A4.

FIGURE 4

(a) ATR-FTIR spectrum; (b) XRPD spectrum; (c) DSC curves; (d) TGA curves of tobramycin API, and sample A4.

FIGURE 5

In-vitro aerosolisation performance of API and A4 by next-generation impactor (NGI).

FIGURE 6

EF (a), FPF (b), MMAD (c), and GSD (d) of A4 after stability testing after storage under different conditions for 28 days.

FIGURE 7

(a) MTT test results of optimum samples A4; (b) in vitro bacteriostatic test results of optimum samples A4.

FIGURE 8

In-vivo safety analysis. 4 x (a), 10 x (b), 40 x (c) Morphological images of the main organs of healthy and dosed mice.

FIGURE 9

In vivo anti-infection analysis: (a) Flow chart of the animal experiment; (b) Kaplan-Meier chart; (c) weight; and (d) body temperature during the animal experiment.

FIGURE 10

(a) Bioluminescence images in the mice at 6h and 72h (“×” means the experiment animals died because of infection.) (b) Total radiance count of bioluminescence images in mice of different groups; (c) OD₆₀₀ of bacterial solution from different groups; (d) Penh value in mice of each group after drug administration. Determination of inflammatory factors in mouse alveolar lavage fluid from different groups: (e) IL-4, (f) IL-6, and (g) TNF-α. (h) Photographs of bacterial colonies of lung homogenates from the P. aeruginosa-infected lung tissues of different groups. (Level of significance: * Indicating the significant difference with p < 0.05, ** Indicating the significant difference with p < 0.01, *** Indicating the significant difference with p < 0.001, **** Indicating the significant difference with p < 0.0001).

FIGURE 11

(a) Bioluminescence images of the main organs of mice; (b) morphological changes in the lungs of mice after dosing; (c) lung index of mice.