(T1330-06-40) Minimizing Mycobacteriophage Titer Loss During Inhaled Delivery by Developing Inhalable Phage Powders

Purpose: Pulmonary tuberculosis represents one of the greatest unmet needs in infectious disease; it is responsible for about 1.5 million deaths per year. Mycobacterium tuberculosis bacteria (Mtb) can hide and persist intracellularly inside immune cells like macrophages. These infections are notoriously difficult to treat, with the standard treatment course requiring at least 4 different antibiotics administered for at least 6 months. As the rise of antibiotic-resistance strains is further complicating tuberculosis treatment, innovative solutions are sorely needed. One potential alternative to antibiotics is using mycobacteriophages that are capable of infecting, lysing, and eradicating Mtb. However, delivering sufficient active phages to infection sites in the lungs is challenging as intravenously injected phages do not easily reach alveolar macrophages while pulmonary delivery of phages suspended in a solution by nebulization could damage the phages. The purpose of this study is to develop an inhalable dry powder of D29 mycobacteriophages to reduce phage titer loss during pulmonary delivery.

Methods: To develop an aerosolizable dry powder containing D29 phage, we leverage thin-film freeze-drying (TFFD) technology. Appropriate freezing temperature, reconstitution methods, and suitable stabilizing excipients were screened using a Plackett-Burman experimental design allowing accurate estimation of the main effects of each. The formulation was further optimized using a Box-Behnken design to determine the best levels of the selected excipients for phage titer recovery. Three optimal formulations were further investigated for titer recovery and powder aerosol performance. Finally, the effects of nebulization of D29 phages in liquid versus actuation of a D29 phage powder from a dry powder inhaler were evaluated and compared.

Results: The screening experiment suggested that utilizing a higher drum temperature during TFFD, reconstituting the powder using phage buffer instead of water, and high levels of lyoprotective excipients were beneficial for titer recovery. The formulations predicted to exhibit the best overall for titer recovery ranged between 0.3 and 0.4log-loss, and 65-70% fine particle fraction, with a single dose from a dry powder inhaler able to deliver over 10^8 pfu to the deep lung.  Nebulization of D29 in a solution caused over 2 log reduction in D29 activity. In comparison, phages in the TFFD powder only experienced a titer loss of 0.33 after the powder was loaded in capsules and actuated using a dry powder inhaler.

Conclusion: TFFD represents a viable method to prepare aerosolizable dry powders of phages for effectively delivery of viable phages into the lung for potential treatment of Mtb infections.

Acknowledgements: This work was supported in part by TFF Pharmaceuticals, Inc. and Via Therapeutics LLC.