A novel thiol-saccharide mucolytic for the treatment of muco-obstructive lung diseases

Abstract

Background: Mucin disulfide cross-links mediate pathologic mucus formation in muco-obstructive lung diseases. MUC-031, a novel thiol-modified carbohydrate compound, cleaves disulfides to cause mucolysis. The aim of this study was to determine the mucolytic and therapeutic effects of MUC-031 in sputum from patients with cystic fibrosis (CF) and mice with muco-obstructive lung disease (βENaC-Tg mice).

Methods: We compared the mucolytic efficacy of MUC-031 and existing mucolytics (N-acetylcysteine (NAC) and recombinant human deoxyribonuclease I (rhDNase)) using rheology to measure the elastic modulus (G') of CF sputum, and we tested effects of MUC-031 on airway mucus plugging, inflammation and survival in βENaC-Tg mice to determine its mucolytic efficacy in vivo.

Results: In CF sputum, compared to the effects of rhDNase and NAC, MUC-031 caused a larger decrease in sputum G', was faster in decreasing sputum G' by 50% and caused mucolysis of a larger proportion of sputum samples within 15 min of drug addition. Compared to vehicle control, three treatments with MUC-031 in 1 day in adult βENaC-Tg mice decreased airway mucus content (16.8±3.2 versus 7.5±1.2 nL·mm^-2, p<0.01) and bronchoalveolar lavage cells (73 833±6930 versus 47 679±7736 cells·mL^-1, p<0.05). Twice-daily treatment with MUC-031 for 2 weeks also caused decreases in these outcomes in adult and neonatal βENaC-Tg mice and reduced mortality from 37% in vehicle-treated βENaC-Tg neonates to 21% in those treated with MUC-031 (p<0.05).

Conclusion: MUC-031 is a potent and fast-acting mucolytic that decreases airway mucus plugging, lessens airway inflammation and improves survival in βENaC-Tg mice. These data provide rationale for human trials of MUC-031 in muco-obstructive lung diseases.

FIGURE 1

Effect of recombinant human deoxyribonuclease (rhDNase), N-acetylcysteine (NAC) and MUC-031 on the elasticity of sputum from patients with cystic fibrosis (CF). a) Schematic of a cone-and-plate rheometer. Elastic (G′) and viscous (G′′) moduli are calculated from the measured response of the samples to the oscillating angular displacement. b) Schematic of the protocol for testing the mucolytic efficacy of MUC-031, rhDNase and NAC. The G′ and G′′ moduli of CF sputum samples are measured at baseline in a frequency sweep from 0.1 to 50 Hz at 5% strain, followed by addition and manual mixing of the test agents at 10% v/w (PBS control, rhDNase, NAC and MUC-031). c) Comparison of mucolytic effect of PBS control (n=15), rhDNase (n=15, 20 µg·mL⁻¹) and MUC-031 (n=15, 5 mM), in sputum not treated with protease inhibitors, as measured by change in G′ over 30 min and analysed using a generalised additive modelling (GAM) framework. d) Percentage of samples in each group (PBS, rhDNase and MUC-031) as function of time for which GAM-normalised G′ is decreased by 50%. e) Comparison of mucolytic effect of PBS control (n=8), NAC (n=16, 2.5 mM) and MUC-031 (n=16, 2.5 mM), in sputum treated with protease inhibitors Halt and EDTA, as measured by change in G′ over 30 min and analysed by GAM. f) Percentage of samples in each group (PBS, NAC and MUC-031) as function of time for which GAM-normalised G′ is decreased by 50%. In c) and e) the solid lines represent the model estimates for βj (referred to as normalised G′); the surrounding lighter shades indicate 95% pointwise confidence intervals over the time course. Nonoverlapping lines and surrounding colours are indicative of statistically significant differences between conditions. *: p<0.05, **: p<0.01, ****: p<0.0001 for comparison of cumulative curves for MUC-031 versus rhDNase, NAC or PBS.

FIGURE 2

Effect of recombinant human deoxyribonuclease (rhDNase), dithiothreitol (DTT) and MUC-031 on mucin size in sputum from patients with cystic fibrosis (CF). Freshly collected sputum samples from CF patients were treated with rhDNase (20 µg·mL⁻¹) or increasing concentration (0.1–10 mM) of DTT or MUC-031 at 37°C for 30 min; mucin was separated by gel electrophoresis; and Western blots were stained with antibodies against MUC5B and MUC5AC. a–d) Representative a, c) Western blots and b, d) summary of effect of rhDNase, and increasing concentrations of DTT and MUC-031 on high-molecular-weight intensity of a, b) MUC5B and c, d) MUC5AC expressed as percentage of untreated sputum aliquots (n=8–10 per group). NT: no treatment; D: rhDNase. ****: p<0.0001 compared with NT samples; ^#: p<0.05, ^###: p<0.001 compared with 0.1 mM concentration of same drug; ^¶: p<0.05, ^¶¶¶: p<0.001 compared with 1 mM concentration of same drug.

FIGURE 3

Acute treatment with MUC-031 reduces airway mucus plugging and inflammation in adult βENaC-Tg mice with chronic muco-obstructive lung disease. Adult βENaC-Tg mice and wild-type (WT) littermate controls were treated with MUC-031 or vehicle alone by intratracheal instillation three times in 1 day. a) Representative airway histology of βENaC-Tg and WT mice after acute treatment. Sections were stained with Alcian blue–periodic acid–Schiff (AB-PAS) to determine the presence of intraluminal and intraepithelial mucus. Scale bars=100 µm. b–d) Quantification of b) total, c) intraluminal and d) intraepithelial mucus content determined by measuring the volume density of AB-PAS positive material in proximal main axial airways (n=8–17 per group). e) Effects of acute treatment with MUC-031 on inflammatory cell counts in bronchoalveolar lavage (n=8–16 per group). *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001 compared with vehicle-treated WT mice; ^#: p<0.05, ^##: p<0.01 compared with vehicle-treated βENaC-Tg mice.

FIGURE 4

Chronic treatment with MUC-031 reduces airway mucus plugging and inflammation in adult βENaC-Tg mice with chronic muco-obstructive lung disease. Adult βENaC-Tg mice and wild-type (WT) littermates were treated with MUC-031 or vehicle alone by intratracheal instillation twice daily for 2 weeks. a) Representative airway histology of βENaC-Tg and WT mice after chronic treatment. Sections were stained with Alcian blue–periodic acid–Schiff (AB-PAS) to determine the presence of intraluminal and intraepithelial mucus. Scale bars=100 µm. b–d) Quantification of b) total, c) intraluminal and d) intraepithelial mucus content determined by measuring the volume density of AB-PAS positive material in proximal main axial airways (n=15–22 per group). e, f) Effects of e) chronic treatment with MUC-031 on inflammatory cell counts and f) concentrations of keratinocyte chemoattractant (KC), tumour necrosis factor (TNF)-α and interleukin (IL)-13 in bronchoalveolar lavage (n=10 per group). *: p<0.05, ***: p<0.001, ****: p<0.0001 compared with vehicle-treated WT mice; ^#: p<0.05, ^##: p<0.01 compared with vehicle-treated βENaC-Tg mice.

FIGURE 5

Preventive treatment with MUC-031 reduces mortality and airway mucus plugging in neonatal βENaC-Tg mice. Neonatal βENaC-Tg mice and wild-type (WT) littermates were treated with MUC-031 or vehicle alone by intranasal instillation twice daily from the first day of life for a period of 2 weeks. a) Effect of preventive treatment with MUC-031 on survival (n=49–95 per group). b) Representative airway histology of βENaC-Tg and WT mice after preventive treatment with MUC-031. Sections were stained with Alcian blue–periodic acid–Schiff (AB-PAS) to determine the presence of intraluminal and intraepithelial mucus. Scale bars=100 µm. c–e) Quantification of c) total, d) intraluminal and e) intraepithelial mucus content determined by measuring the volume density of AB-PAS-positive material in proximal main axial airways (n=14–36 per group). f) Representative agarose gel Western blots and corresponding densitometry of bronchoalveolar lavage samples stained with a murine anti-MUC5B antibody (n=23–32 per group). *: p<0.05, ****: p<0.0001 compared with vehicle-treated WT mice; ^#: p<0.05, ^##: p<0.01 compared with vehicle-treated βENaC-Tg mice.

FIGURE 6

Effects of preventive treatment with MUC-031 on airway inflammation in neonatal βENaC-Tg mice. Neonatal βENaC-Tg mice and wild-type (WT) littermates were treated with MUC-031 or vehicle alone by intranasal instillation twice daily from the first day of life for a period of 2 weeks. a) Inflammatory cell counts (n=26–53 per group) and b) concentrations of keratinocyte chemoattractant (KC), tumour necrosis factor (TNF)-α and interleukin (IL)-13 (n=8–12 per group) in bronchoalveolar lavage of βENaC-Tg and WT mice after preventive treatment. *: p<0.05, **: p<0.01, ****: p<0.0001 compared with vehicle-treated WT mice; ^#: p<0.05, ^##: p<0.01 compared with vehicle-treated βENaC-Tg mice.