Targeting Trypsin-Like Proteases: A Mechanism to Restore Mucociliary Function in Cystic Fibrosis Airways

Dehydration of the airway surface liquid (ASL) and the resultant decline in function of the mucociliary escalator in cystic fibrosis airways is largely underpinned by the excessive flux of Na+ and water though ENaC. Proteolysis of the endogenous  and  subunits of epithelial sodium channels (ENaC) by channel activating proteases (CAPS) is the key regulatory mechanism for channel activation. Recent reports highlight that (1) CFTR (cystic fibrosis transmembrane conductance regulator) normally protects ENaC from the action of proteases and (2) a stark imbalance in proteases/protease inhibitor levels in CF airway cultures favour activation of normally inactive ENaC. The current study examines the potential therapeutic benefit of CAPS/ENaC inhibition in CF airways.
Our group has developed a panel of active-site directed affinity-based probes which target and inhibit trypsin-like proteases (potential CAPS); including the broad-spectrum inhibitor QUB-TL1. We have utilised this compound to interrogate the impact of trypsin-like protease inhibition on ENaC activity in differentiated primary airway epithelial cell cultures.
Electrophysiological data demonstrate QUB-TL1 selectively and irreversibly binds to extracellularly located trypsin-like proteases resulting in impaired ENaC-mediated Na+ transport. Visualisation of ENaC at the apical surface compartment of primary airway epithelial cells shows a large reduction in a low molecular weight (processed and active) form of ENaC, which was found to be abundant in untreated CF cultures. Consistent with the reduction in ENaC activity observed, QUB-TL1 treatment was subsequently shown to increase ASL height (performed in collaboration with Royal College of Surgeons in Ireland).
Our results are consistent with the hypothesis that targeting the CAPS-ENaC signalling axis may restore the depleted ASL seen in CF airways.

One-Year Safety of Olodaterol Once Daily via Respimat® in Patients with GOLD 2-4 Chronic Obstructive Pulmonary Disease: Results of a Pre-Specified Pooled Analysis

The novel long-acting β2-agonist olodaterol demonstrated an acceptable safety profile in short-term phase II clinical studies. This analysis of four randomized, double-blind, placebo-controlled, parallel-group, phase III studies (1222.11, NCT00782210; 1222.12, NCT00782509; 1222.13, NCT00793624; 1222.14, NCT00796653) evaluated the long-term safety of olodaterol once daily (QD) in a large cohort of patients with moderate to very severe (Global initiative for chronic Obstructive Lung Disease 2-4) chronic obstructive pulmonary disease (COPD). The studies compared olodaterol (5 or 10 μg) QD via Respimat®, formoterol 12 μg twice daily (BID) via Aerolizer® (1222.13 and 1222.14), and placebo for 48 weeks. Patients continued receiving background maintenance therapy, with ∼60% receiving concomitant cardiovascular therapy and 25% having a history of concomitant cardiac disease. Pre-specified analyses of pooled data assessed the adverse events (AEs) and serious AEs in the whole population, and in subgroups with cardiac disease, along with in-depth electrocardiogram and Holter monitoring. In total, 3104 patients were included in the safety analysis: 876 received olodaterol 5 μg, 883 received olodaterol 10 μg, 885 received placebos, and 460 received formoterol 12 μg BID. Overall incidence of on-treatment AEs (71.2%), serious AEs (16.1%), and deaths (1.7%) were balanced across treatment groups. Respiratory and cardiovascular AEs, including major adverse cardiac events, were reported at similar frequencies in placebo and active treatment groups. The safety profiles of both olodaterol 5 μg (marketed and registered dose) and 10 μg QD delivered via Respimat® are comparable to placebo and formoterol BID in this population, with no safety signals identified.

Efficacy response in CF patients treated with ivacaftor: Post-hoc analysis

Clinical studies in patients with cystic fibrosis and G551D-CFTR showed that the group treated with ivacaftor had improved clinical outcomes. To better understand the effect of ivacaftor therapy across the distribution of individual FEV1 responses, data from Phase 3 studies (STRIVE/ENVISION) were re-examined. In this post-hoc analysis of patients (n=209) who received 48 weeks of ivacaftor or placebo, patients were assigned to tertiles according to FEV1 response. These groups were then used to evaluate response (FEV1, sweat chloride, weight, CFQ-R, and pulmonary exacerbation). The number needed to treat (NNT) was calculated for specific thresholds for each outcome. Across all tertiles, numerical improvements in FEV1, sweat chloride, CFQ-R and the frequency of pulmonary exacerbations were observed in ivacaftor-treated patients: the treatment difference versus placebo was statistically significant for all outcomes in the upper tertile and for some outcomes in the lower and middle tertiles. The NNT for a≥5% improvement in %predicted FEV1 was 1.90, for a≥5% body weight increase was 5.74, and to prevent a pulmonary exacerbation was 3.85. This analysis suggests that the majority of patients with clinical characteristics similar to STRIVE/ENVISION patients have the potential to benefit from ivacaftor therapy.

Long-term macrolide maintenance therapy in non-CF bronchiectasis: Evidence and questions

An official American thoracic society research statement: Comparative effectiveness research in pulmonary, critical care, and sleep medicine

Background: Comparative effectiveness research (CER) is intended to inform decision making in clinical practice, and is central to patientcentered outcomes research (PCOR). Purpose: To summarize key aspects of CER definitions and provide examples highlighting the complementary nature of efficacy and CER studies in pulmonary, critical care, and sleep medicine. Methods: An ad hoc working group of the American Thoracic Society with experience in clinical trials, health services research, quality improvement, and behavioral sciences in pulmonary, critical care, and sleepmedicinewas convened. The group used an iterative consensus process, including a reviewbyAmerican Thoracic Society committees and assemblies. Results: The traditional efficacy paradigm relies on clinical trials with high internal validity to evaluate interventions in narrowly defined populations and in research settings. Efficacy studies address the question, "Can it work in optimal conditions?" The CER paradigm employs a wide range of study designs to understand the effects of interventions in clinical settings. CER studies address the question, "Does it work in practice?" The results of efficacy and CER studies may or may not agree. CER incorporates many attributes of outcomes research and health services research, while placing greater emphasis on meeting the expressed needs of nonresearcher stakeholders (e.g., patients, clinicians, and others). Conclusions: CER complements traditional efficacy research by placing greater emphasis on the effects of interventions in practice, and developing evidence to address the needs of the many stakeholders involved in health care decisions. Stakeholder engagement is an important component of CER. Copyright © 2013 by the American Thoracic Society.

Antibiotic and anti-inflammatory therapies for cystic fibrosis

Cystic fibrosis (CF) lung disease is characterized by chronic bacterial infection and an unremitting inflammatory response, which are responsible for most of CF morbidity and mortality. The median expected survival has increased from <6 mo in 1940 to >38 yr now. This dramatic improvement, although not great enough, is due to the development of therapies directed at secondary disease pathologies, especially antibiotics. The importance of developing treatments directed against the vigorous inflammatory response was realized in the 1990s. New therapies directed toward the basic defect are now visible on the horizon. However, the impact of these drugs on downstream pathological consequences is unknown. It is likely that antibiotics and anti-inflammatory drugs will remain an important part of the maintenance regimen for CF in the foreseeable future. Current and future antibiotic and anti-inflammatory therapies for CF are reviewed. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

BMP signalling: agony and antagony in the family

Bone morphogenetic proteins (BMPs) are secreted extracellular matrix (ECM)-associated proteins that regulate a wide range of developmental processes, including limb and kidney formation. A critical element of BMP regulation is the presence of secreted antagonists that bind and inhibit BMP binding to their cognate Ser/Thr kinase receptors at the plasma membrane. Antagonists such as Noggin, Chordin, Gremlin (Grem1), and twisted gastrulation-1 (Twsg1) have been shown to inhibit BMP action in a range of different cell types and developmental stage-specific contexts. Here we review new developments in the field of BMP and BMP antagonist biology during mammalian development and suggest strategies for targeting these proteins in human disease.

Reduced Bacterial Colony Count of Anaerobic Bacteria Is Associated with a Worsening in Lung Clearance Index and Inflammation in Cystic Fibrosis

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Identification of synergists that potentiate the action of polymyxin B against Burkholderia cenocepacia

Burkholderia cenocepacia and other members of the Burkholderia cepacia complex (Bcc) are highly multidrug-resistant bacteria that cause severe pulmonary infections in patients with cystic fibrosis. A screen of 2686 compounds derived from marine organisms identified molecules that could synergize with polymyxin B to inhibit growth of B. cenocepacia. At 1 μg/ml, five compounds synergized with polymyxin B and inhibited the growth of B. cenocepacia by more than 70% compared to growth in polymyxin B alone. Follow-up testing revealed that one compound from the screen, the aminocoumarin antibiotic novobiocin, synergized with polymyxin B and colistin against tobramycin-resistant clinical isolates of B. cenocepacia and Burkholderia multivorans. In parallel, we show that novobiocin sensitivity is common among Bcc species and these bacteria are even more susceptible to an alternative aminocoumarin, clorobiocin, which also had an additive effect with polymyxin B against B. cenocepacia. These studies support using aminocoumarin antibiotics to treat Bcc infections and show that synergizers can be found to increase the efficacy of antimicrobial peptides and polymyxins against Bcc bacteria.

The Role of Serine Proteases and Antiproteases in the Cystic Fibrosis Lung

Cystic fibrosis (CF) lung disease is an inherited condition with an incidence rate of approximately 1 in 2500 new born babies. CF is characterized as chronic infection of the lung which leads to inflammation of the airway. Sputum from CF patients contains elevated levels of neutrophils and subsequently elevated levels of neutrophil serine proteases. In a healthy individual these proteases aid in the phagocytic process by degrading microbial peptides and are kept in homeostatic balance by cognate antiproteases. Due to the heavy neutrophil burden associated with CF the high concentration of neutrophil derived proteases overwhelms cognate antiproteases. The general effects of this protease/antiprotease imbalance are impaired mucus clearance, increased and self-perpetuating inflammation, and impaired immune responses and tissue. To restore this balance antiproteases have been suggested as potential therapeutics or therapeutic targets. As such a number of both endogenous and synthetic antiproteases have been trialed with mixed success as therapeutics for CF lung disease.

A Secretory Leukocyte Protease Inhibitor Variant with Improved Activity against Lung Infection

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI’s proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o-; HBE) and CF cell lines (CFBE14o-; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40 mV and outwardly-rectifying I-V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers--NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterised the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, lipopolysaccharide (LPS) pattern, biofilm formation, urease activity, antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (p=0.037). Transmissible CF strains generally lacked O antigen, produced less pyocyanin, and had low virulence in G. mellonella. Further, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.