Leung et. al. Biotherapy Journal Review Spring 2016

Phage Therapy Reviews

Journal Citation:

Leung S, Parumasivam T, Gao F, Carrigy NB, Vehring R, Finlay WH, Morales S,  Britton WJ, Kutter E, Chan H. Production of Inhalation Phage Powders Using Spray Freeze Drying and Spray Drying Techniques for Treatment of Respiratory Infections. Pharm Res. 2016; Published on-line, February, 2016

Published Abstract

Purpose The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders. Method A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1=60:20:20 and F2=40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed. Results A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1=13.1±1.7×104 pfu and SD-F2=11.0±1.4×104 pfu) than from their counterpart SFD formulations (SFD-F1=8.3±1.8×104 pfu and SFDF2= 2.1±0.3×104 pfu). Conclusion Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.

Introduction & Background to Study

Pulmonary (lung) infections are deadly and communicable. These two facts alone make them an important target for antimicrobial therapy. Unfortunately, the frequent and repeated use of antibiotics for pulmonary infections such as tuberculosis, hospital-acquired pneumonias, or the pneumonias in patients with cystic fibrosis or lung cancers has created multi-resistant strains of those infections. Clearly, new treatments are needed for many of these resistant infections.

Bacteriophages (phages) are viruses that are bacterial parasites; they infect and replicate inside bacterial hosts, killing the bacteria in the process. There already exists a number of phages known to kill multi-resistant pulmonary pathogens; but getting those viruses to the site of infection remains a challenge.

Delivery of phages through inhalation could be an effective means of treating pneumonias. Most preparations for pulmonary delivery have been liquid aerosols delivered through intranasal instillation or nebulization. Formulating the phage as a dry powder has the advantage that it could be easily transported and stored until it could be inhaled.

In the past, the efficac of dry powder phage formulations have been dependent on phage type, the type and concentration of formulation excipients, and the production process. The aim of the current research was to compare and evaluate two methods of preparation – spray drying (SD) and spray freeze drying (SFD) – using different ratios of trehalose, mannitol and leucine as the bulking and stabilizing agents. The stability of phage after the powder production process and the in vitro aerosol performance of the formulation were both assessed.

Materials & Methods

A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine. The phage suspension was spray dried (SD) or sprayed, frozen in liquid nitrogen and then lyophilized (SFD).

The phage titer was calculated after each process, using the Miles-Misra surface droplet technique of spreading the phage preparation over a lawn of host bacteria (Pseudomonas aeruginosa) and counting plaques of bacterial killing after incubation overnight).

In vitro aerosol performance was assessed by dispersing 20 mg of powder into a Multi-stage    Liquid Impinger (MSLI) using an Osmohaler™ at 100 l/ min for 2.4 s. The fine particle fraction (FPF) was defined as the mass fraction of particles ≤5.0 μm with respect to the recovered dose.

Study Results

Each method had its advantages and disadvantages. The SFD method of production led to much greater loss of phage during droplet generation with the ultrasonic nozzle, but the phage were less vulnerable during the evaporative drying process. The SFD product also were associated with better phage recovery (80%) in the aerosol performance assay.

The two-fluid nozzle used in the SD method, on the other hand, was less destructive for the phage, but the phage were more vulnerable during the method’s evaporative drying process (0.75 log loss). The SD product was associated with lower phage recovery (20%) in the aerosol performance assay. Still, because of the lower powder loss during the powder production process, higher total lung doses were obtained for the SD formulations, compared with the SFD method overall.

Author’s Conclusions

“Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.”

In summary, the SD method produced more stable phage powder formulations and achieved better aerosol performance for the bacteriophage PEV2, and has the potential for providing viable phage for treating lung Pseudomonas infections.

Reviewer’s Conclusions

The data speaks for itself. For the PEV2 phage, SD is a better method for efficiently producing stable, aerosolizable powdered product for lung inhalation. The take home message should also be that preparation of phage must be individualized and carefully monitored throughout the process. Had the results of preparation been assessed only after spraying, or only after drying, a different conclusion may have been reached. Only by measuring the effect of each step in the preparation process was it possible to determine the efficacy of that process, and ultimately the optimal method for producing viable, inhalable, dry powdered phage for the treatment of pulmonary infections.