(M0930-09-60) Drugs as Excipients: Evaluation of Colistin, a Cationic Antimicrobial Peptide, as a Multifunctional Stabilizer of Inhalable Niclosamide Microparticles

Purpose: Niclosamide and colistin exhibit potent synergistic activity against multidrug-resistant Gram-negative pathogens, including strains isolated from individuals with cystic fibrosis (CF). However, their clinical use is limited by pharmacokinetic challenges: niclosamide has extremely poor water solubility and undergoes extensive first-pass metabolism, while colistin suffers from low permeability and poor systemic bioavailability. To overcome these limitations and maximize local drug concentrations at the infection site, our study aimed to develop an inhalable dry powder formulation containing both drugs. A key goal of this work was to produce composite particles containing crystalline niclosamide, as in vitro studies suggest that its slow-release profile enhances therapeutic activity against CF-associated bacterial isolates. Traditional formulation strategies often rely on polymers, surfactants, or solvent mixtures to stabilize particles during spray drying. However, such excipients can reduce potency and raise safety concerns for pulmonary administration. As an alternative, we explored the use of colistin sulfate, a cationic antimicrobial peptide that synergizes with niclosamide, based on its amphiphilic and polycationic properties, which we hypothesized could support its role as a multifunctional excipient. Specifically, we investigated whether colistin could act as an electrostatic stabilizer during antisolvent precipitation of niclosamide, enabling the formation of stable crystalline suspensions without additional excipients.

Methods: Ultrasound-assisted antisolvent precipitation was used to prepare niclosamide particles. Particle size distributions were measured via dynamic light scattering (DLS) using quartz cuvettes and water as the dispersing medium. Spray drying was employed to obtain dry powders from optimized suspensions. Solid-state properties were analyzed by X-ray powder diffraction (XRPD), dry particle size distributions were measured by DLS at 3 bar, and aerodynamic behavior was assessed using next-generation impaction (NGI). Zeta potential was measured with a Zetasizer to evaluate surface charge modifications induced by colistin. To assess dissolution and diffusion, we conducted Transwell diffusion studies under niclosamide sink conditions using 0.12% w/v mucin in the apical compartment. Comparisons were made between niclosamide-colistin particles and the corresponding solution ratios.

Results: We systematically optimized formulation parameters to achieve stable niclosamide suspensions with controlled particle size by using ultrasound-assisted antisolvent precipitation (Fig. 1A and 1B). Particles prepared with colistin exhibited reduced particle size, as seen by a decreased median size (x₅₀) compared to particles in which no colistin was used, this effect became more pronounced after 1.5 hours (Fig. 1C). Increasing the colistin:niclosamide ratio further reduced particle size (x₅₀ decreases as colistin:niclosamide increases) (Fig. 1D). These suspensions were successfully spray dried achieving a 42% yield (Fig. 2A). The composite powders had favorable geometric properties (x₅₀ = 1.51 mm) and aerodynamic performance with a recovered niclosamide dose < 5mm = 76%, and < 3 mm = 70% for inhaled delivery (Fig. 2B). Solid-state characterization by XRPD confirmed the presence of crystalline niclosamide (Fig. 2B). Conceptually, drug behavior in the lung can be divided into an “absorption” phase, driven by diffusion through mucus, and a “clearance” phase, governed by epithelial permeation (Fig. 3A). Stabilization by colistin resulted in modulation of the surface charge of niclosamide crystals. Niclosamide alone exhibited negative surface charge, whereas niclosamide crystals coated with colistin exhibited positive surface charge (Fig. 3B). Transwell diffusion studies demonstrated that antisolvent-precipitated particles slowed the rate of niclosamide permeation compared to drugs in solution at the same ratio, ~10% for particles vs. ~30% for solution by 24h (Fig. 3C). These findings suggest that colistin content can be tuned to modulate niclosamide transport through mucus and its availability at the site of infection, impacting both the “absorption” and “clearance” phases.

Conclusion: Altogether, this study presents a dual-function formulation strategy in which colistin acts as both a therapeutic agent and an excipient, stabilizing crystalline particles. This approach enables a high-potency, excipient-free inhaled therapy. More broadly, our work demonstrates the potential of repurposing cationic peptides as functional excipients for mucosal drug delivery, offering a paradigm in which formulation and pharmacologic synergy are integrated at the molecular level to optimize local drug action.

Acknowledgements: This work was supported by the National Institutes of Health under Training Grant 5T32GM140223-02 (Pharmacological Sciences Training Program), the PhRMA Foundation Faculty Starter Grant (Brunaugh), a Cystic Fibrosis Foundation Pilot Grant (Brunaugh; BRUNAU23I0), and the Cystic Fibrosis Foundation Burkholderia cepacia Laboratory and Repository (Caverly; CAVERL25AB0).

Figure 1. Antisolvent precipitation utilizing colistin sulfate as an electrostatic stabilizer enables tunable niclosamide crystal size and dissolution kinetics

Figure 2. Composite niclosamide-colistin particles engineered for inhaled delivery.

Figure 3. Niclosamide-colistin particles modulate niclosamide exposure at the site of infection through dual-phase control