The Cystic Fibrosis-Like Airway Surface Layer Is not a Significant Barrier for Delivery of Eluforsen to Airway Epithelial Cells

Abstract

Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo. To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo, no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.

Keywords: QR-010; airway surface layer; cystic fibrosis; delivery; deltaF508; eluforsen.

FIG. 1.

Representative XZ images of Cy5-labeled eluforsen (red; 100 μM) diffusion through a normal in vitro mucus layer. Green indicates time course of Cy5-labeled eluforsen (red) diffusion through calcein-stained cells. Images go from upper left to lower right. Time between pictures is 2 seconds each.

FIG. 2.

Diffusion velocity (A) and time to reach 60% Cy5-labeled eluforsen signal at the epithelial cells (B) for normal (2%–5% solids) and CF-like (5–11% solids) mucus; light gray bars indicate the 10 μM Cy5-labeled eluforsen dose, dark gray bars indicate the 25 μM Cy5-labeled eluforsen dose, and black bars indicate the 100 μM Cy5-labeled eluforsen dose. Bars indicate averages of 2–7 individual ALI cultures, error bars represent standard deviation. CF, cystic fibrosis; ALI, air-liquid interface.

FIG. 3.

Diffusion velocity (A) and time to reach 60% Cy5-labeled eluforsen signal at the epithelial cells (B) for CF-like mucus after different nebulization protocols; 1. PBS nebulization, followed by Cy5-labeled eluforsen nebulization 48 hours later (black bar), 2. eluforsen nebulization followed by Cy5-labeled eluforsen nebulization 48 hours later (gray checkered), 3. four times PBS nebulization with a 48-hour interval, followed by Cy5-labeled eluforsen nebulization (black horizontal stripes), and 4. four times eluforsen nebulization with a 48-hour interval followed by Cy5-labeled eluforsen nebulization (black vertical stripes). Bars indicate averages of nine individual ALI cultures, error bars indicate standard deviation. PBS, phosphate-buffered saline.

FIG. 4.

Stability of eluforsen in the presence of CF sputum (2%–5% solids) at 10 μg/mL concentration (gray bars) and 100 μg/mL concentration (black bars). Bars represent the average of seven sputum samples, error bars indicate standard deviation.

FIG. 5.

Relative eluforsen concentrations compared to t = 0 when incubated with dornase alfa (3 μg/mL) (A), salbutamol (2 μg/mL) (B), fluticasone (5 μg/mL) (C), N-acetylcysteine (100 mg/mL) (D), and aztreonam (6000 μg/mL) (E) for 24 hours. Striped bars show relative eluforsen concentrations in the presence of medication. Bars represent an average of four measurements, error bars indicate the standard deviation.

FIG. 6.

Eluforsen concentrations in the lung, trachea, liver, and kidneys of Scnn1b-Tg (gray bars) and WT mice (black bars) treated with eluforsen (n = 6), measured at 24 hours after the last dose. WT, wild type.

FIG. 7.

ISH; (A–D) 5 × objective and (E–H) 40 × objective of HPS (A, B, E, F) and ISH (C, D, G, H) of lung sections of Scnn1b-Tg and WT mice, which were treated with saline (n = 3). (A, B, E and F) show HPS staining, where nuclei are shown in dark purple (hematoxylin), cytoplasm is shown in pink (phloxine), and connective tissue is yellow (saffron). (C, D, G and H) show the same organ section subjected to ISH staining, wherein nuclei are stained in light purple (hematoxylin) and eluforsen is show in brown. Black arrows (A, Scnn1b-Tg) point out mucus plugs and macrophages in the lumen of bronchi. HPS, Hematoxylin/Phloxine/Saffron; ISH, in situ hybridization.

FIG. 8.

ISH; (A–D) 5 × objective and (E–H) 40 × objective of HPS (A, B, E, F) and ISH (C, D, G, H) of lung sections of Scnn1b-Tg and WT mice, which were treated with 10 mg/kg eluforsen (n = 3). (A, B, E and F) show HPS staining, where nuclei are shown in dark purple (hematoxylin), cytoplasm is shown in pink (phloxine), and connective tissue is yellow (saffron). (C, D, G and H) show the same organ section subjected to ISH staining, wherein nuclei are stained in light purple (hematoxylin) and eluforsen is shown in brown.

FIG. 9.

Eluforsen concentrations in the supernatant of control solution (gray bars) and in supernatants of bacterial suspensions of Pseudomonas aeruginosa (striped bars), Burkholderia multivorans (checkered bars), and Staphylococcus aureus (dotted bars). Bars are averages of two measurements, error bars indicate standard deviation.

FIG. 10.

Fluorescence signal at acceptor side after applying Cy5-labeled eluforsen to either a 1-day biofilm (A) or 5-day-old biofilm (B). Black lines indicate a biofilm composed of P. aeruginosa ATCC700888, dark gray lines indicate a biofilm composed of P. aeruginosa DSM 29305, and light gray lines correspond to control filters without biofilm.