(M1530-10-54) Development of an Aerosolizable Dry Powder of Mycobacteriophage D29 Using Thin-Film Freeze-Drying

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) are capable of hiding and persisting intracellularly inside of 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 desperately needed. One potential alternative to antibiotics is using mycobacteriophages such as D29, capable of infecting, lysing, and eradicating Mtb. However, delivering effective concentrations of active phage to infection sites in the lungs is a complex challenge.

Methods: In order 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.

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 leucine and trehalose were beneficial for titer recovery. The optimization experiment confirmed mild positive effects of increased leucine and trehalose content and relatively negligible effects of antioxidants, polymer content, and mannitol. The formulations predicted to exhibit the best overall for titer recovery ranged between 0.3 and 1.2 log-loss, and 42-72% fine particle fraction, with a single dose from a dry powder inhaler able to deliver between 10⁷ and 10⁸ pfu to the deep lung.

Conclusion: TFFD is a viable and scalable method to prepare aerosolizable dry powders for potential pulmonary phage therapy to treat antibiotic-resistant Mtb infections.

Acknowledgements: Z Cui reports a relationship with TFF Pharmaceuticals, Inc. that includes equity or stocks and research funding. RO Williams reports a relationship with TFF Pharmaceuticals, Inc. that includes consulting or advisory, equity or stocks, and research funding.