Background: Small molecule-based ion channels formed by the antifungal natural product amphotericin B (AmB) can act as functional replacements for CFTR in cultured cystic fibrosis (CF) airway epithelia, CFTR null pigs, and the noses of people with CF [1,2]. This “molecular prosthetics” (MP) approach is independent of the CFTR protein and thus might be effective for everyone with CF – including those who cannot benefit from CFTR modulators. Previous studies have shown that FDA-approved AmB is well-tolerated in the lungs. We found that combining AmB with Chol extends the range of effective concentrations over which AmB can restore airway host defenses in human CF airway epithelia . Building on this finding and additional structural , mechanistic, biophysical, and nanoparticle engineering studies, we have generated a new dry powder inhaler (Amphotericin B Cystetic for Inhalation – ABCI) for delivering AmB and Chol as a MP to the airways of people with CF.
Methods: We used established assays to study the structure of AmB aggregates and the capacity of ABCI to restore host defenses in CF airway epithelia:
- Solid State NMR (SSNMR), structure calculations , and microcrystal electron diffraction methods.
- Airway surface liquid (ASL) pH in cell line and primary cultures of airway epithelia .
- H14CO3− transport across NuLi and CuFi monolayers .
- ASL viscosity of AmB-treated primary cultures of airway epithelia .
- Antibacterial activity within the ASL of ABCI-treated primary cultures of airway epithelia .
- AmB aggregates consist of asymmetric homodimers arranged in a clathrate-like lattice encasing void volumes similar in size to Chol. These clathrates undergo a structural change to bind sterol guests. These data provide insight into how precomplexing Chol with AmB extends the concentration range over which AmB functions as an effective MP.
- Based on biophysical and nanoparticle engineering studies (see complementary abstract), we have generated a new dry powder inhaler, ABCI, for delivering AmB as a MP to the airways of people with CF.
- In immortalized cells and primary human airway epithelia, ABCI increases ASL pH at both low and high AmB concentrations. ASL pH increases within the first 6 hours post-treatment with ABCI and is sustained for at least 2 weeks.
- ABCI is well tolerated in rats and dogs at concentrations above those predicted to be efficacious in people with CF.
Conclusions: Key structural features of AmB aggregates reveal how this molecule binds Chol and suggests a dynamic equilibrium between cell surface clathrates and transmembrane ion channels. Insights from structural, mechanistic, biophysical, and nanoparticle engineering studies allowed us to rationally design a new AmB and Chol-containing dry powder inhaler, ABCI, that can increase host defenses in CF airway epithelia over a wide range of concentrations. These preclinical studies suggest that ABCI may serve as a novel treatment for people with CF, including those who cannot benefit from CFTR modulators.
Acknowledgements: Funded by cystetic Medicines
 Muraglia KA, et al. Small-molecule ion channels increase host defenses in cystic fibrosis airway epithelia. Nature 2019;567:405–8.
 Chorghade RS, et al. Amphotericin B induces epithelial voltage responses in people with cystic fibrosis. J Cyst Fibros 2021;20:540–50.
 Lewandowska A, et al. Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes. Nat Struct Mol Biol 2021;28:972–81.