An Improved Inhaled Mucolytic to Treat Airway Muco-obstructive Diseases

Abstract

Rationale: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them.

Objectives: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings.

Methods: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa.

Measurements and main results: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in βENaC-overexpressing mice, whereas NAC did not (n = 6-24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo.

Conclusions: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.

Keywords: mucins; mucociliary clearance; mucus; obstructive pulmonary diseases; reducing agents.

Figure 1.

The reduction potential of P3001 exceeds that of dithiothreitol and N-acetylcysteine. Spectrophotometric studies monitored formation of reduced 5′,5′-dithio-bis(2-nitrobenzoic acid) over time. Reaction rates, generated using second-order kinetics, for each compound were compared as a (A) function of reducing agent concentration at pH 6.5 or (B) over a relevant pH range. The x-axis reflects the pseudo–second-order kinetics of the reducing reaction rates per molar per second. DTT = dithiothreitol; NAC = N-acetylcysteine.

Figure 2.

Effect of N-acetylcysteine (NAC), dithiothreitol (DTT), and a novel reducing agent, P3001, on the biochemical and biophysical properties of chronic obstructive pulmonary disease sputum. Freshly harvested sputa were aliquoted and subjected to increasing concentrations (0.01–10 mM) of NAC, DTT, or P3001 at 37°C for 30 minutes and then quenched. (A) Electrophoretic separation reveals the biochemical effect of each compound on mucin multimerization. MUC5AC and MUC5B signals are shown individually or as an overlay (red indicates MUC5AC; green indicates MUC5B). (B) Graph indicates the degree of mucin reduction as percentage of monomeric signal intensity (e.g., full or 100% MUC5B reduction was achieved at 10 mM DTT). (C) Graph reveals changes in sputum biophysical properties as measured by spinnability, also referred to as capillary break. n = 6 for NAC and P3001 and n = 4 for DTT. **P < 0.01, ***P < 0.0001, ****P < 0.00001, two-way ANOVA Dunnett test. LMW = low molecular weight.

Figure 3.

Alterations of the mucin network following treatment of chronic obstructive pulmonary disease sputum. The same patient sputum was treated with N-acetylcysteine (NAC) or P3001 for 30 minutes at 37°C and analyzed to compare the structural changes of the mucin network. (A) Breakdown of multimeric MUC5B was assessed by agarose gel electrophoresis. Increasing concentration of NAC (0.2–200 mM) and P3001 (0.2–20 mM) were tested. (B) Effect of 20 mM NAC and P3001 on sputum elastic modulus (G′) as measured by a cone-and-plate rheometer and compared with PBS. **P < 0.01. n = 3 repeats per condition. (C) Effect of 20 mM NAC and P3001 on the mucin network topology as examined by immunocytochemistry and compared with PBS. Confocal images captured the disruption of MUC5AC (red) and MUC5B (green) threads as a result of sputum reduction. Nuclei are shown in blue (DAPI). Scale bar, 100 μm. PBS = phosphate-buffered saline.

Figure 4.

Effect of N-acetylcysteine and P3001 on cystic fibrosis (CF) human bronchial epithelial mucus transport. Human bronchial epithelial cells grown on air–liquid interface typically coordinate cilia beating and transport secreted material in a rotational manner (e.g., mucus hurricane). Mucus transport or velocity was tracked using fluorescently labeled microbeads (1 μm) while cells were maintained under thin-film conditions. (A) Long-exposure images of non-CF and CF cells nebulized with PBS (3 μl/cm²). Additionally, the same CF cells were nebulized with P3001 (5 mM). After P3001 treatment, beads cleared away from the initial anchor point (e.g., disruption of the mucus aggregate) but maintained the same overall directionality (e.g., clockwise rotational movement). Mucus transport was monitored for 2 hours. (B) Graphs show mucus velocity of non-CF and CF cells treated with PBS (i) or CF cells treated with 5 mM N-acetylcysteine or P3001 (ii). n = 3. *P < 0.05, **P < 0.01. NAC = N-acetylcysteine; PBS = phosphate-buffered saline.

Figure 5.

In vitro testing of N-acetylcysteine (NAC) and P3001 potency on mouse BAL fluid. BAL fluid from wild-type (WT) and βENaC-transgenic mice were subjected to increasing concentration of NAC and P3001 (0.02–20 mM). Samples were incubated at 37°C for 15 minutes and then quenched. (A) Western blotting was used to assess NAC and P3001 potency at different concentrations and at different stoichiometric ratios because mucin concentration was increased fourfold in βENaC mice as compared with WT animals. (B) Reduction of high-molecular-weight multimer intensity that reflects partial of full Muc5b reduction is graphed for NAC (gray circles) and P3001 (black squares) in the two models. NT = no treatment.

Figure 6.

Comparing N-acetylcysteine (NAC) and P3001 efficacy and toxicity in wild-type mice. Wild-type mice were treated via oropharyngeal aspiration (15 μl) with NAC at 50 mg/kg or P3001 at 1.5 mg/kg. BAL fluid was collected at various time points (15, 30, 45, and 60 min and 6 h). (A) NAC and P3001 efficacy to reduce Muc5b in vivo was assessed by agarose gel electrophoresis during a 60-minute period. (B) NAC pharmacokinetic properties in the murine lungs were established by measuring NAC concentrations in BAL fluid via mass spectrometry. n = 3–5 animals per time point. (C) Inflammatory responses were measured 6 hours after treatment and are displayed as total cell count (i) and percentage neutrophils (ii). n = 4–6 mice per group. *P < 0.05, **P < 0.01. (D) Representative images of hematoxylin and eosin–stained sections of mouse lungs harvested 30 minutes after treatment with NAC and P3001. Veh = vehicle.

Figure 7.

Comparing N-acetylcysteine (NAC) and P3001 efficacy to remove persistent mucus plugs from βENaC-overexpressing mice by aerosolization. βENaC-overexpressing mice were slightly anesthetized with isoflurane and subjected to repeated 15-minute nebulizations (nose only, three times, 2-h intervals) with normal saline, NAC, or P3001 at 200 mM. Lung sections were stained with alcian blue–periodic acid/Schiff (AB-PAS) and morphometric analysis was performed. (A) Representative images of AB-PAS sections of animals exposed to sham (n = 9) or treated with saline (n = 24), NAC (n = 6), and P3001 (n = 6). Images display the proximal region of the left main stem bronchus, a site routinely presenting significant mucus obstruction in βENaC-transgenic animals. Scale bar, 100 μm. (B) Morphometric measurement of AB-PAS–positive signal in whole-lung sections. **P < 0.01, ***P < 0.001, Mann-Whitney U test. NS = normal saline; Veh = vehicle.

Figure 8.

N-acetylcysteine (NAC) pharmacokinetics and mucolytic effects on cystic fibrosis (CF) sputum measured by microrheology. Five individuals with CF were treated with 20% (1.27 M) NAC via inhalation. Spontaneously expectorated sputa were collected 0, 15, and 90 minutes after treatment. (A) Sputum drug concentrations measured using mass spectrometry at 0, 15, and 90 minutes. (B) Sputum microrheology or mean square displacement (MSD) measured at baseline and 90 minutes after NAC treatment (in vivo) by tracking 1-μm fluorescence beads mixed in sputum specimens. In parallel, aliquots of sputa collected at baseline were incubated at 37°C with 100 mM NAC for 90 minutes (ex vivo). (C) MSD of baseline sputa incubated ex vivo at 37°C with 0.1 mg/ml rhDNase or 10 mM P3001 for 15 minutes. Passive bead diffusion (or MSD) is shown as scatter plot of MSD_τ=0.83s for tracer particles in sputum for each treatment condition. *P < 0.05. BSL = baseline; DiNAC = N,N′-diacetyl-l-cystine.