Novel polymeric reducing agents as mucus modulators

Airway obstruction by mucus with abnormal viscoelastic properties is a key feature of chronic lung disease in patients with cystic fibrosis (CF, also known as mucoviszidosis) and a spectrum of other muco-obstructive lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma. Airway mucus is mainly composed of the two secreted mucins MUC5AC and MUC5B. These mucins organize into complex polymeric networks via the formation of covalent disulfide bonds, which in turn determine the viscoelastic properties of the mucus gel on airway surfaces. Emerging evidence suggests that increased oxidation of free thiol groups of mucin monomers by reactive oxygen species (ROS) released by leukocytes in chronic airway inflammation, leads to abnormal crosslinking and stiffening of mucus, and that this is a key mechanism underlying abnormal viscoelasticity of mucus in airway from patients with CF. These results suggest targeting of disulfide crosslinks with reducing agents as an effective mucolytic strategy in CF and potentially other muco-obstructive lung diseases. However, the only currently available reducing agent in the clinic, N-acetylcysteine (NAC), failed to show therapeutic benefit in clinical trials, likely because of its low potency and short duration of action as a small molecule.
In the first funding period, we developed a methodology for synthesis of dendritic polyglycerol sulfate (dPGS) with multiple free thiol groups (-SH) as mucolytic polymers. The new lead-candidate dPGS-SH was synthesized in gram-scale. Comprehensive in vitro testing of dPGS-SH demonstrated stability, low toxicity on highly differentiated human airway epithelial cultures and a significantly higher mucolytic potency compared to N-acetylcysteine (NAC, the only currently approved mucolytic drug) in airway mucus from patients with CF (Z02|Duerr/Siegmund/Weinhart), as determined by rheology measurements to assess effects on the storage and loss moduli (G’ and G’’) were performed. Furthermore, we established mucin Western blots to study effects on multimerization of MUC5AC and MUC5B polymers and measurements of mucus transport velocity (MTV) on highly differentiated primary human airway epithelial cultures (with A01|Mall/Gradzielski). Further, we established protocols for in vivo testing of mucolytic effects using an experimental small-molecule reducing agent (MUC-031) in βENaC-transgenic (βENaC-Tg) mice with muco-obstructive lung disease. Preliminary studies for a bifunctional polymer developed in the first funding period demonstrate effective mucolysis in vivo.