Development, Characterization, and in vitro Efficacy Evaluation of an Inhalable H1B Antimicrobial Peptide Formulation...

Hymenochirin-1B (H1B) is a linear alpha-helical amphipathic anti-microbial peptide originally isolated from the skin secretions of the Congo-clawed frog Hymenochirus boettgeri (Pipidae). H1B has previously shown anticancer activity in non-small cell lung cancer (NSCLC; A549). Therefore, H1B was formulated into an inhalable dry powder (H1BSD) to stabilize the peptide for the treatment of NSCLC. H1B was spray-dried with the stabilizing excipients; mannitol, trehalose and L-leucine, keeping L-leucine constant at 20% w/w while varying trehalose and mannitol, resulting in H1BSD1-H1BSD5 powders. All H1BSD powders achieved a spray drying yield ranging between 43-63% w/w, drug content of ~ 100% and maintained a moisture content of less than 3% w/w. All dry powders were shaped with a distinct corrugated surface texture. The semi-crystalline nature of the powder was confirmed by the presence of an endothermic peak in DSC thermograms and reduced crystalline peaks in PXRD diffractograms. The mass median aerodynamic diameter fell within the optimal range of 1.7-2.5 µm, while the fine particle fraction exceeded 65% for all formulations, indicating efficient delivery to the respiratory airways. Based on physical characterization and aerosolization performance, H1BSD4 was selected as the optimal formulation. In vitro studies validated the therapeutic efficacy of H1BSD4. Stability studies confirmed that the H1BSD2-H1BSD5 powders remained stable at 25°C for 3 months, maintaining more than 90% of their drug content and preserving their semi-crystalline structure as validated by DSC and PXRD. In summary, H1BSD powder was successfully developed, maintaining anticancer activity while offering enhanced stability and optimized inhalation-based drug delivery.