(W1130-06-38) Formulation of a Teixobactin-Lipopeptide Hybrid as an Inhalable Dry Powder for the Treatment of Pulmonary Pan-Drug Resistant Bacterial Infections

Purpose: Antibiotic resistance remains an urgent threat to global health. The increasing prevalence of pan-drug resistant ‘superbugs’, or bacteria that are resistant to all classes of commercially available antibiotics, highlights the necessity of developing innovative antibacterial agents [1]. Teixobactin (TXB) is a depsipeptide-based antibiotic with narrow-spectrum activity against Gram-positive pathogens and limited potential for acquired resistance due to its novel mechanism of action [2]. Our research consortium has developed TXB-lipopeptide hybrids that expand activity to Gram-negative pathogens. Herein, we have formulated a lead TXB-lipopeptide candidate for localized delivery to the lungs via a dry powder inhaler (DPI) for the treatment of pan-drug resistant bacterial lung infections.

Methods: The TXB hybrid (APL-001) was spray dried using a Buchi-B290 spray dryer to prepare inhalable dry powders. The feed solution contained 25:15:60 w/w/w APL-001:L-leucine:excipient, where the excipient used was either lactose, mannitol, or trehalose. For all experiments, the B290 aspirator, inlet temperature, peristaltic pump speed, and nitrogen spray gas flow rate were set to 100%, 190 °C, 5 mL min^-1, and 1052 L/h (at standard temperature and pressure), respectively. The resulting outlet temperature was 75-85 °C. The dry powders were collected and stored at room temperature with desiccant until further analyzed. Physicochemical characteristics, including particle morphology, moisture content, and thermal properties, were evaluated using scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), respectively. Aerodynamic properties were assessed via cascade impaction using a Next Generation Impactor (NGI). Briefly, 10 mg of the spray dried powder was loaded into #3 hydroxypropylmethylcellulose (HPMC) capsules and released into the NGI from an RS-01 low resistance DPI for 4 s at a flow rate of 60 L/min. The aerodynamic particle size distribution (APSD) metrics of mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), emitted dose (ED), and fine particle fraction with respect to the emitted dose (FPF) were calculated.

Results: The TXB hybrid APL-001 was spray dried with leucine and a sugar excipient, either lactose, mannitol, or trehalose. L-leucine is frequently employed to improve dispersion of the aerosol powder. Lactose, mannitol, and trehalose have been reported previously as excipients that aid to preserve peptide activity despite the shear forces present during spray drying and subsequent storage. Spray dried APL-001 powders were visualized using SEM (Figure 1). Particles containing lactose appeared to have a smoother surface than those containing mannitol or trehalose. Moisture content was assessed using TGA. All three formulations contained ≤ 5 wt% moisture, ideal for long-term storage. Thermal properties as measured by DSC are shown in Figure 2. APL-001:leucine:lactose showed an endothermic event at 145 °C due to the lactose monohydrate excipient, likely a result of the release of bound water around 150 °C. An endothermic event (melting) occurred at 160 °C for APL-001:leucine:mannitol due to the mannitol component. However, no major events occurred on the DSC spectrum for APL-001:leucine:trehalose powder, where trehalose itself had endothermic events occur at 95 °C (melting of trehalose dihydrate) and 200 °C (melting of anhydrous trehalose). Leucine alone showed an absence of endothermic or exothermic events over the heating profile (0 to 200 °C). Aerodynamic properties of the three formulations were assessed via cascade impaction. The MMADs of spray dried APL-001 containing 60 wt% lactose, mannitol, and trehalose were 1.52 ± 0.55 µm (GSD = 1.97 ± 0.03), 2.25 ± 0.10 µm (GSD = 1.93 ± 0.07), and 2.11 ± 0.03 µm (2.03 ± 0.02), respectively (Figure 3). All three formulations exhibited an FPF of 80-85%, indicating that the powder that is released from the inhaler has great potential for lung deposition. However, the emitted dose varied between the three formulations, with lactose, mannitol, and trehalose excipients leading to EDs of 83 ± 8%, 50 ± 3%, and 62 ± 5%, respectively. With considerations to all APSD metrics, the use of lactose as the protective sugar excipient leads to the aerodynamically smallest particles with the greatest emitted dose.

Conclusion: The teixobactin-lipopeptide hybrid APL-001 was successfully formulated into a dry powder suitable for inhalation. The use of both lactose and leucine excipients generated the most aerodynamically favorable dry powder, with a high proportion of the powder in the respirable size range. Experiments to confirm APL-001 efficacy following spray drying are underway. Future efforts will include an in vivo pharmacokinetic study in order to compare lung exposure following inhaled or intravenous delivery of APL-001. Furthermore, pharmacokinetic/pharmacodynamic (PK/PD) modeling will be used to determine the excipient that results in the best flow dynamics and lung exposure [3].

References: [1] CDC, Antibiotic Resistant Threats in the United States, 2019, U.S. Department of Health and Human Services, CDC, Atlanta, GA, 2019.
[2] L.L. Ling, T. Schneider, A.J. Peoples, A.L. Spoering, I. Engels, B.P. Conlon, A. Mueller, T.F. Schäberle, D.E. Hughes, S. Epstein, M. Jones, L. Lazarides, V.A. Steadman, D.R. Cohen, C.R. Felix, K.A. Fetterman, W.P. Millett, A.G. Nitti, A.M. Zullo, C. Chen, K. Lewis, A new antibiotic kills pathogens without detectable resistance, Nature 517(7535) (2015) 455-459.
[3] S.B. Yeshwante, P. Hanafin, B.K. Miller, L. Rank, S. Murcia, C. Xander, A. Annis, V.K. Baxter, E.J. Anderson, B. Jermain, R. Konicki, A.A. Schmalstig, I. Stewart, M. Braunstein, A.J. Hickey, G.G. Rao, Pharmacokinetic Considerations for Optimizing Inhaled Spray-Dried Pyrazinoic Acid Formulations, Molecular Pharmaceutics 20(9) (2023) 4491-4504.

Acknowledgements: This work was funded by the National Institutes of Health (R01 AI170889). Microscopy was performed at the Chapel Hill Analytical and Nanofabrication Laboratory (CHANL), which is supported by the National Science Foundation, Grant ECCS-1542015.


Figure 1. Representative SEM images of (A) APL-001:leucine:lactose, (B) APL-001:leucine:mannitol, and (C) APL-001:leucine:trehalose at 10,000x magnification.


Figure 2. Representative DSC spectra for (A) APL-001:leucine:lactose, (B) APL-001:leucine:mannitol, (C) APL-001:leucine:trehalose, (D) lactose, (E) mannitol, and (F) trehalose.


Figure 3. Mass distribution of NGI experiments where 10 mg of spray dried APL-001 formulation was placed in a #3 HPMC capsule and released from an RS-01 low resistance inhaler at a flow of 60 L/min for 4 s.