BackgroundTransforming growth factor-β1 (TGF-β1) contributes to more severe pulmonary disease in people with cystic fibrosis (pwCF). Our preclinical in vitro and in vivo studies demonstrated that losartan could reverse TGF-β1-mediated mucociliary dysfunction, albeit at doses difficult to achieve by oral administration. This study therefore examined airway losartan concentrations after oral and inhaled dosing to evaluate if oral losartan could be even expected to improve mucociliary clearance.MethodsWe conducted comparative pharmacokinetics studies in sheep assessing airway surface liquid (ASL) concentrations of losartan and its metabolites, EXP3179 and EXP3174, after oral and inhaled administration. Another pharmacokinetic study used oral losartan to compare serum levels in pwCF on and off CFTR modulators and healthy volunteers. Finally, an open-label clinical trial evaluated effects of oral losartan (50 mg twice daily for 14 weeks) on mucociliary and cough clearance (MCC/CC) in pwCF off CFTR modulators as they have a need for novel therapies (NCT03435939).ResultsIn sheep, oral losartan (50 mg twice daily for 4 days) did not achieve ASL levels expected to improve CF-associated mucociliary dysfunction based on our previous data. In humans, single dose pharmacokinetics of oral losartan showed differential results in pwCF and healthy volunteers, with pwCF off CFTR modulators achieving the lowest serum levels of losartan and EXP3174 with a complete absence of EXP3179, the anti-inflammatory metabolite of losartan. The open label study included seven participants. Serum concentrations of losartan and EXP3174 at week 14 matched levels observed in pwCF not on modulators in the pharmacokinetic study. As expected, no improvements in whole-lung or peripheral MCC/CC were found in participants with complete datasets. PpFEV1 remained unchanged, but nasal TGF-β1 levels significantly decreased with treatment, although systemic inflammatory markers were unaffected (C-reactive protein, calprotectin, and serum amyloid A).ConclusionReduced drug exposure compared to healthy volunteers reflects altered pharmacokinetics in pwCF. Nevertheless, oral losartan at clinically approved doses does not achieve airway concentrations needed to restore mucociliary function in any individual, and especially not in pwCF off modulators. Future studies should consider inhaled drug delivery to achieve therapeutically meaningful airway exposure to losartan and its metabolites.

Background/Objectives: The pulmonary administration of antibiotics can be advantageous in treating pulmonary infections by promoting high intrapulmonary drug concentrations with reduced systemic exposure. However, limited benefits have been observed for pulmonary administration versus other administration routes due to its rapid clearance from the lung. Here, the effects of structural modifications on the epithelial permeability and antibacterial potency of a third-generation cephalosporin were investigated to improve the understanding of drug properties that promote intrapulmonary retention and how they may impact efficacy. Methods: Ceftazidime was modified by attaching 18 hydrophobic, hydrophilic, and mucus-binding motifs to the carboxylic acid distant from the beta-lactam by amidation. Epithelial permeability was investigated by drug transport assays using human bronchial epithelial air–liquid interface cultures. Antibacterial potency was determined by microtiter MIC assays with B. pseudomallei, P. aeruginosa, E. coli, and S. aureus. Results: A 40–50% reduction in the transepithelial transport rate was exhibited by two PEGylated ceftazidime analogs (mPEG8- and PEG5-pyrimidin-2-amine-ceftazidime) and n-butyl-ceftazidime. An increase in the transport rate was exhibited by four analogs bearing small and hydrophobic or negatively charged motifs (n-heptane-, phenyl ethyl-, glutamic acid-, and 4-propylthiophenyl boronic acid-ceftazidime). The antibacterial potency was reduced by ≥10-fold for most ceftazidime analogs against B. pseudomallei, P. aeruginosa, and E. coli but was retained by seven ceftazidime analogs primarily bearing hydrophobic motifs against S. aureus. Conclusions: The covalent conjugation of PEGs with MW > 300 Da reduced the epithelial permeability of ceftazidime, but these modifications severely reduced antibacterial activity. To improve the pulmonary retention of antibiotics with low membrane permeability, this work suggests future molecular engineering studies to explore high-molecular-weight prodrug strategies.

Exposure to airborne particulate matter (PM) has been attributed to millions of deaths annually. However, the PM components responsible for observed health effects remain unclear. Oxidative potential (OP) has gained increasing attention as a key property that may explain PM toxicity. Using online measurement methods that impinge particles for OP quantification within seconds, we reveal that 60 to 99% of reactive oxygen species (ROS) and OP in secondary organic aerosol and combustion-generated PM have a lifetime of minutes to hours and that the ROS activity of ambient PM decays substantially before offline analysis. This implies that current offline measurement methods substantially underestimate the true OP of PM. We demonstrate that short-lived OP components activate different toxicity pathways upon direct deposition onto reconstituted human bronchial epithelia. Therefore, we suggest that future air pollution and health studies should include online OP quantification, allowing more accurate assessments of links between OP and health effects.

Introduction:In cystic fibrosis (CF), most CFTR mutations cause partial (Class II) or complete (Class I) loss of function. Modulators (VX) can improve CFTR function in Class II mutations but are ineffective for Class I mutations and may cause side effects, resulting in tolerability issues with concerns about long-term safety. Apical anion secretion, essential for maintaining airway surface liquid (ASL) homeostasis, is regulated by CFTR. Alternative anion channels, like ANO1 and SLC26A9, also contribute to ASL homeostasis. Our recent work indicates that specific amino acids can modulate ion channel expression, activity, and trafficking in epithelial cells. We developed a select amino acid formulation (SAA) to enhance anion secretion in primary human bronchial epithelial cells (HBEC) with CF, regardless of mutation. Methods:Transepithelial short-circuit current was measured in wildtype (WT)- and CF-HBEC with various Class I and Class II mutations. Cells were pretreated with DMSO or VX for 24 h before apical exposure to SAA in Ussing chambers. Benzamil-insensitive current was sequentially inhibited to determine the contributions of SLC26A9, CFTR, ANO1, and NKCC1. 36Cl unidirectional and net fluxes (J netCl) validated chloride secretion. Whole-cell patch-clamp studies determined the current density with SAA in WT- and CF-HBEC. CFTR, SLC26A9, and ANO1 mRNA and protein expression levels were assessed via qPCR and immunofluorescence. ASL volume, ciliary beat frequency (CBF), and mucociliary transport were also assessed. Results:SAA increased benzamil-insensitive current to 70%-85% of WT cells, and enhanced J netCl in both Class I and II mutations. J netCl contributed to 72%, 50%, and 39.5% of S9A13-inhibitable current in WT-, F508del+/+-, and G542X/R785X-HBEC, respectively. VX treatment increased current in Class II but did not affect Class I mutations. Increased chloride secretion with SAA was attributed to enhanced activity of SLC26A9 and partial CFTR restoration through elevated mRNA and membrane protein expression. SAA also increased ASL volume and CBF, confirming its effectiveness in Class I mutations. Discussion:SAA enhances chloride secretion through SLC26A9 and partial CFTR rescue in Class I and II mutations. These findings suggest SAA functions as a mutation-agnostic therapy to improve anion secretion and clinical symptoms, particularly in Class I mutations.

Cigarette smoke (CS) is a leading cause of chronic obstructive pulmonary disease (COPD). Here, we investigated whether the ion channel amplifier nesolicaftor rescues CS-induced mucociliary and ion channel dysfunction. As CS increases the expression of transforming growth factor-beta1 (TGF-β1), human bronchial epithelial cells (HBECs) from healthy donors were used for TGF-β1 and COPD donors (COPD-HBEC) for CS exposure experiments. CS and TGF-β1 induce mucociliary dysfunction by increasing MUC5AC and decreasing ion channel conductance important for mucus hydration. These include cystic fibrosis transmembrane conductance regulator (CFTR) and apical large-conductance, Ca2+-activated K+ (BK) channels. Nesolicaftor rescued CFTR and BK channel dysfunction, restored ciliary beat frequency (CBF), and decreased mucus viscosity and MUC5AC expression in CS-exposed COPD-HBEC. Nesolicaftor further reversed reductions in airway surface liquid (ASL) volumes, CBF, and CFTR and BK conductance, and blocked the increase in extracellular signal-regulated kinase (ERK) signaling in TGF-β1-exposed normal HBECs. Mechanistically, nesolicaftor increased, as expected, not only binding of PCBP1 to CFTR mRNA but also surprisingly to LRRC26 mRNA, which encodes the gamma subunit required for BK function. Similar to nesolicaftor, the angiotensin receptor blocker (ARB) losartan rescued TGF-β1-mediated decreases in PCBP1 binding to LRRC26 mRNA. In addition, the ARB telmisartan restored PCBP1 binding to CFTR and LRRC26 mRNAs to rescue CFTR and BK function in CS-exposed COPD-HBEC. Thus, nesolicaftor and ARBs act on the same target and were therefore neither additive nor synergistic in their actions. These data demonstrate that nesolicaftor and ARBs may provide benefits in COPD by improving ion channel function important for mucus hydration.NEW & NOTEWORTHY Cigarette smoke (CS) increases transforming growth factor-beta1 (TGF-β1) expression that causes mucociliary dysfunction by decreasing ion channel function. In our study, a CFTR amplifier (nesolicaftor) and angiotensin II receptor blockers (losartan and telmisartan) improve CS-induced ion channel dysfunction, by increasing binding of PCBP1 to CFTR and LRRC26 mRNAs. Therefore, nesolicaftor and ARBs, acting on the same target, may provide therapeutic benefits for treating smoking-related diseases.

To the Editor: Cystic fibrosis transmembrane conductance regulator (CFTR) modulators have significantly improved outcomes for people with cystic fibrosis (pwCF), yet those with unresponsive mutations remain without effective treatments. One promising strategy involves enhancing ion channels alternative to CFTR (reviewed in ref. 1), including apically expressed large conductance, calcium-activated potassium (BK) channels. BK channels, composed of the pore-forming subunit KCNMA1 (Slo1) and the regulatory subunit LRRC26 (γ1), are critical for airway surface hydration and mucociliary transport (MCT). Inhibition or knockdown of these components leads to reduced airway surface liquid, increased mucus concentration, and impaired MCT in CF models in vitro and in vivo. BK plays a crucial role in enhancing chloride exit, a phenomenon called apical loop current. Our simulations indicated that small apical increases in potassium conductance (from 0 to 0.002 S/cm2) enhance Cl– efflux approximately 3-fold. Reanalyzing airway single cell RNA-seq data from ref. 4, we show KCNMA1, LRRC26, and ANO1 expression in secretory cells. Among BK β-subunits, only KCNMB2/4 are detected, with KCNMB2 in ciliated cells and KCNMB4 in large airways, supporting the hypothesis that, in peripheral airways where CF disease starts, apical loop currents between ANO1 and BK, made of KCMNA1/LRRC26, can be enhanced therapeutically.

BackgroundInhibiting ENaC in the airways of people with cystic fibrosis (pwCF) is hypothesized to enhance mucociliary clearance (MCC) and provide clinical benefit. Historically, inhaled ENaC blockers have failed to show benefit in pwCF challenging this hypothesis. It is however unknown whether the clinical doses were sufficient to provide the required long duration of action in the lungs and questions whether a novel candidate could offer advantages where others have failed?MethodsDose-responses with the failed ENaC blockers (VX-371, BI 1265162, AZD5634, QBW276) together with ETD001 (a novel long acting inhaled ENaC blocker) were established in a sheep model of MCC and were used to predict clinically relevant doses that would provide a long-lasting enhancement of MCC in pwCF. In each case, dose predictions were compared with the selected clinical dose.ResultsEach of the failed candidates enhanced MCC in the sheep model. Translating these dose-response data to human equivalent doses, predicted that substantially larger doses of each candidate, than were evaluated in clinical studies, would likely have been required to achieve a prolonged enhancement of MCC in pwCF. In contrast, ETD001 displayed a long duration of action (≥16 h) at a dose level that was well tolerated in Phase 1 clinical studies.ConclusionsThese data support that the ENaC blocker hypothesis is yet to be appropriately tested in pwCF. ETD001 has a profile that enables dosing at a level sufficient to provide a long duration of action in a Phase 2 clinical study in pwCF scheduled for 2024.

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air–liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca 2+ -activated, and voltage-dependent K + (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 ( IL6 ), IL8 , and MMP9 , as well as MUC5AC . The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 ( FOXJ1 ), keratin 5 ( KRT5 ), and secretoglobin family 1A member 1 ( SCGB1A1 ) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

Propylene glycol (PG) is a common delivery vehicle for nicotine and flavorings in e-cigarette (e-cig) liquids and is largely considered safe for ingestion. However, little is known about its effects as an e-cig aerosol on the airway. Here, we investigated whether pure PG e-cig aerosols in realistic daily amounts impact parameters of mucociliary function and airway inflammation in a large animal model (sheep) in vivo and primary human bronchial epithelial cells (HBECs) in vitro. Five-day exposure of sheep to e-cig aerosols of 100% PG increased mucus concentrations (% mucus solids) of tracheal secretions. PG e-cig aerosols further increased the activity of matrix metalloproteinase-9 (MMP-9) in tracheal secretions. In vitro exposure of HBECs to e-cig aerosols of 100% PG decreased ciliary beating and increased mucus concentrations. PG e-cig aerosols further reduced the activity of large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. We show here for the first time that PG can be metabolized to methylglyoxal (MGO) in airway epithelia. PG e-cig aerosols increased levels of MGO and MGO alone reduced BK activity. Patch clamp experiments suggest that MGO can disrupt the interaction between the major pore-forming BK subunit human Slo1 (hSlo1) and the gamma regulatory subunit LRRC26. PG exposures also caused a significant increase in mRNA expression levels of MMP9 and interleukin 1 beta (IL1B). Taken together, these data show that PG e-cig aerosols cause mucus hyperconcentration in sheep in vivo and HBECs in vitro, likely by disrupting the function of BK channels important for airway hydration.

Secondary organic matter (SOM) formed from gaseous precursors constitutes a major mass fraction of fine particulate matter. However, there is only limited evidence on its toxicological impact. In this study, air-liquid interface cultures of human bronchial epithelia were exposed to different series of fresh and aged soot particles generated by a miniCAST burner combined with a micro smog chamber (MSC). Soot cores with geometric mean mobility diameters of 30 and 90 nm were coated with increasing amounts of SOM, generated from the photo-oxidation of mesitylene and ozonolysis of α-pinene. At 24 h after exposure, the release of lactate dehydrogenase (LDH), indicating cell membrane damage, was measured and proteome analysis, i.e. the release of 102 cytokines and chemokines to assess the inflammatory response, was performed. The data indicate that the presence of the SOM coating and its bioavailability play an important role in cytotoxicity. In particular, LDH release increased with increasing SOM mass/total particle mass ratio, but only when SOM had condensed on the outer surface of the soot cores. Proteome analysis provided further evidence for substantial interference of coated particles with essential properties of the respiratory epithelium as a barrier as well as affecting cell remodeling and inflammatory activity.