(M1430-07-39) Treatment of Non-Tuberculous Mycobacterial Infections via Spray Dried Tigecycline Dry Powder Aerosols

Purpose: Tigecycline, a broad-spectrum, glycylcycline antibiotic, has demonstrated potential in treating patients with non-tuberculous mycobacterial infections.^1,2 However, tigecycline’s instability when reconstituted in aqueous solution and severe adverse reactions upon intravenous delivery at high doses have led to both technical challenges and poor patient compliance.^1-3 Microparticle formulations of tigecycline for use in dry powder inhalers (DPIs) represent an alternative delivery method to localize delivery to the major site of infection, mitigating off-target effects.

Methods: Spray dried powder formulations were prepared using a Buchi-B290 spray dryer. Solutions of tigecycline and lactose were prepared with a total solids content of 15 mg mL^-1 in pH 6.5 phosphate buffer. The ratio of tigecycline:lactose:buffer salts (denoted as TGC:LAC:PB) utilized was either 90:0:10, 80:10:10, or 70:20:10 (w/w/w). Lactose monohydrate was included to prevent epimerization of tigecycline upon long term storage,⁴ and phosphate buffer salts are incorporated to maintain local physiological pH and increase patient tolerability. For all experiments, the B-290 aspirator, inlet temperature, peristaltic pump speed, and spray gas flow rate were maintained at 100%, 165 °C, 5 mL min^-1, and 473 L/h, respectively. The resulting outlet temperature ranged between 70-75 °C. The spray dried powders were collected and stored shielded from light at -20 °C under desiccant until analyzed. Particle morphology, crystallinity, and moisture content were analyzed using scanning electron microscopy (SEM), x-ray powder diffraction (XRPD), and thermogravimetric analysis (TGA), respectively. Aerodynamic properties were evaluated via release of 10 mg powder in a #3 HPMC capsule from an RS-01 inhaler into a Next Generation Impactor (NGI) for the determination of the aerodynamic particle size distribution (APSD). A preliminary in vivo efficacy study was performed in GM-CSF knockout mice exposed to Mycobacterium abscessus strain 21. Treatment with the 80:10:10 TGC:LAC:PB formulation in a custom-designed passive inhalation chamber was performed for 7 d beginning at 2 d post infection. On day 9, mice were sacrificed and bacterial content in the lungs was quantified.

Results: Spray dried particles were visualized using SEM (Figure 1). Corrugated particles, as shown in Figure 1, are desirable for use in DPIs as this morphology reduces contact points between particles and minimizes electrostatic cohesion, enhancing disaggregation and dispersibility. Upon spray drying, with or without lactose, completely amorphous behavior was observed. Amorphous particles are desired therapeutically as they are likely to be less stable and thus more bioavailable than crystalline materials. Moisture content was determined using TGA, where all formulations exhibited 5-6.5% water by mass. Aerodynamic properties of the three formulations were evaluated via cascade impaction (Figure 2). TGC:LAC:PB ratios of 90:0:10, 80:10:10, and 70:20:10 facilitated mass median aerodynamic diameters (MMADs) of 2.71 ± 0.04 µm, 3.01 ± 0.17 µm, 2.98 ± 0.06 µm, respectively. The addition of lactose significantly increased the MMAD, and as a result, decreased the fine particle fraction (percent of particles < 4.46 µm). While adding lactose is important to increase the stability of tigecycline upon long-term storage, lactose may decrease the amount of drug that can be deposited in the lungs, suggesting a balance of stability and lung deposition properties is necessary. For a preliminary in vivo evaluation, the 80:10:10 formulation was selected to maximize TGC dose but incorporate the potential stability imparted by lactose. Following 7-d treatment of M. abscessus-infected mice with the TGC formulation, there was a 0.91-log reduction in lung bacterial content compared to untreated mice (Figure 3). While not statistically significant, likely due to the short treatment course, this suggests the initial success of the spray dried formulation. A longer treatment course (2-4 weeks) is currently being evaluated.

Conclusion: Preliminary in vitro and in vivo analyses demonstrate the spray dried tigecycline formulations as promising candidates for the treatment of M. abscessus lung infections. The inclusion of leucine to aid dispersion is currently being investigated. Lactose/leucine concentration and spray drying parameters (solution concentration, feed rate) will be further optimized to maximize the efficiency of aerosolization with an ultimate goal of enhancing lung deposition in mice.

References: 1. Kwon, Y.-S.; Levin, A.; Kasperbauer, S. H.; Huitt, G. A.; Daley, C. L. Respir. Med. 2019, 158, 89-91.
2. Ferro, B. E.; Srivastava, S.; Deshpande, D.; Pasipanodya, J. G.; van Soolingen, D.; Mouton, J. W.; van Ingen, J.; Gumbo, T. Antimicrob. Agents Chemother. 2016, 60, 2895-2900.
3. Jitkova, Y.; Gronda, M.; Hurren, R.; Wang, X.; Goard, C. A.; Jhas, B.; Schimmer, A. D. PLoS One 2014, 9, e95281.
4. Fawzi, M. B.; Zhu, T.; Shah, M. US Patent 7,879,828, 2011.

Acknowledgements: This work was funded by the National Institutes of Health (R01AI155922). The in vivo studies were approved and carried out in compliance with the Institutional Animal Care and Use Committee standards.


Figure 1. Representative SEM images for spray dried powder with TIG:LAC:PB ratios of (A) 90:0:10, (B) 80:10:10, and (C) 70:20:10.


Figure 2. Mass distribution following actuation of 10 mg spray dried TGC:LAC:PB with ratios of 90:0:10 (purple), 80:10:10 (teal), and 70:20:10 (blue). The NGI was operated at an air flow of 60 L min-1 for 4 s.


Figure 3. Bacterial burden in the murine lung following infection with M. abscessus (Day 0), 2-d incubation period (Day 2), and 7-d treatment with spray dried TGC formulation (Day 9 untreated control vs. treatment).