Ivacaftor pharmacokinetics and lymphatic transport after enteral administration in rats.

Background: Ivacaftor is a modern drug used in the treatment of cystic fibrosis. It is highly lipophilic and exhibits a strong positive food effect. These characteristics can be potentially connected to a pronounced lymphatic transport after oral administration. Methods: A series of studies was conducted to describe the basic pharmacokinetic parameters of ivacaftor in jugular vein cannulated rats when dosed in two distinct formulations: an aqueous suspension and an oil solution. Additionally, an anesthetized mesenteric lymph duct cannulated rat model was studied to precisely assess the extent of lymphatic transport. Results: Mean ± SD ivacaftor oral bioavailability was 18.4 ± 3.2% and 16.2 ± 7.8%, respectively, when administered as an aqueous suspension and an oil solution. The relative contribution of the lymphatic transport to the overall bioavailability was 5.91 ± 1.61% and 4.35 ± 1.84%, respectively. Conclusion: Lymphatic transport plays only a minor role in the process of ivacaftor intestinal absorption, and other factors are, therefore, responsible for its pronounced positive food effect.

Pulmonary tularaemia in a female adolescent with inflammatory bowel disease receiving infliximab: Do not miss the diagnosis.

We report the first described case of pulmonary tularaemia in the pediatric patient receiving infliximab for ulcerative colitis. We highlight the importance of considering Francisella tularensis in diagnostically challenging cases of persistent respiratory symptoms to facilitate early diagnosis and adequate therapy. The TCR-γδ + DN T cells are gaining important role in clinical practice. Polymerase chain reaction assays and serology guarantee early recognition.

Response to elexacaftor/tezacaftor/ivacaftor in intestinal organoids derived from people with cystic fibrosis.

Superior efficacy of elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) over tezacaftor/ivacaftor (TEZ/IVA) in people with cystic fibrosis (CF) and Phe508del/Phe508del genotype was shown in clinical trials. We utilized intestinal organoid approach to compare in vitro responses to these 2 CFTR modulator drug combinations and to check potential inter-individual variability in therapeutic response to the triple combination. Organoids from 17 subjects with Phe508del/Phe508del were screened with forskolin induced swelling assay. Significantly larger swelling, when exposed to ELX/TEZ/IVA as compared to TEZ/IVA, was observed in 16 of them. However, 1 sample showed no additional effect of ELX. The finding of unique CFTR variants in this sample indicates that genetic traits other than CF-causing CFTR mutation are worth exploring as they may have an impact on the definitive modulator drug response.

Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease.

Activation of the Ca2+ activated Cl- channel TMEM16A is proposed as a treatment in inflammatory airway disease. It is assumed that activation of TMEM16A will induce electrolyte secretion, and thus reduce airway mucus plugging and improve mucociliary clearance. A benefit of activation of TMEM16A was shown in vitro and in studies in sheep, but others reported an increase in mucus production and airway contraction by activation of TMEM16A. We analyzed expression of TMEM16A in healthy and inflamed human and mouse airways and examined the consequences of activation or inhibition of TMEM16A in asthmatic mice. TMEM16A was found to be upregulated in the lungs of patients with asthma or cystic fibrosis, as well as in the airways of asthmatic mice. Activation or potentiation of TMEM16A by the compounds Eact or brevenal, respectively, induced acute mucus release from airway goblet cells and induced bronchoconstriction in mice in vivo. In contrast, niclosamide, an inhibitor of TMEM16A, blocked mucus production and mucus secretion in vivo and in vitro. Treatment of airway epithelial cells with niclosamide strongly inhibited expression of the essential transcription factor of Th2-dependent inflammation and goblet cell differentiation, SAM pointed domain-containing ETS-like factor (SPDEF). Activation of TMEM16A in people with inflammatory airway diseases is likely to induce mucus secretion along with airway constriction. In contrast, inhibitors of TMEM16A may suppress pulmonary Th2 inflammation, goblet cell metaplasia, mucus production, and bronchoconstriction, partially by inhibiting expression of SPDEF.

Mutant CFTR Drives TWIST1 mediated epithelial-mesenchymal transition.

Cystic fibrosis (CF) is a monogenetic disease resulting from mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene encoding an anion channel. Recent evidence indicates that CFTR plays a role in other cellular processes, namely in development, cellular differentiation and wound healing. Accordingly, CFTR has been proposed to function as a tumour suppressor in a wide range of cancers. Along these lines, CF was recently suggested to be associated with epithelial-mesenchymal transition (EMT), a latent developmental process, which can be re-activated in fibrosis and cancer. However, it is unknown whether EMT is indeed active in CF and if EMT is triggered by dysfunctional CFTR itself or a consequence of secondary complications of CF. In this study, we investigated the occurrence of EMT in airways native tissue, primary cells and cell lines expressing mutant CFTR through the expression of epithelial and mesenchymal markers as well as EMT-associated transcription factors. Transepithelial electrical resistance, proliferation and regeneration rates, and cell resistance to TGF-ß1induced EMT were also measured. CF tissues/cells expressing mutant CFTR displayed several signs of active EMT, namely: destructured epithelial proteins, defective cell junctions, increased levels of mesenchymal markers and EMT-associated transcription factors, hyper-proliferation and impaired wound healing. Importantly, we found evidence that the mutant CFTR triggered EMT was mediated by EMT-associated transcription factor TWIST1. Further, our data show that CF cells are over-sensitive to EMT but the CF EMT phenotype can be reversed by CFTR modulator drugs. Altogether, these results identify for the first time that EMT is intrinsically triggered by the absence of functional CFTR through a TWIST1 dependent mechanism and indicate that CFTR plays a direct role in EMT protection. This mechanistic link is a plausible explanation for the high incidence of fibrosis and cancer in CF, as well as for the role of CFTR as tumour suppressor protein.

Organoids as a personalized medicine tool for ultra-rare mutations in cystic fibrosis: The case of S955P and 1717-2A>G.

BACKGROUND: For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717-2A > G leads to alternative splicing generating <1% normal CFTR mRNA and that S955P affects CFTR gating. Finally, studies in organoids predict that both patients are responders to VX-770 alone and even more to VX-770 combined with VX-809 or VX-661, although to different levels. CONCLUSION: This study demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs to patients with rare mutations.

Assessment of Distinct Electrophysiological Parameters in Rectal Biopsies for the Choice of the Best Diagnosis/Prognosis Biomarkers for Cystic Fibrosis.

Most cases of Cystic Fibrosis (CF) are diagnosed early in life. However, people with atypical CF forms pose diagnosis dilemmas, requiring laboratory support for diagnosis confirmation/exclusion. Ex vivo analysis of fresh rectal biopsies by Ussing chamber has been the best discriminant biomarker for CF diagnosis/prognosis so far. Here we aimed to evaluate different electrophysiological parameters from Ussing chamber analysis of rectal biopsies from people with CF (PwCF) to establish the one with highest correlations with clinical features as the best CF diagnosis/prognosis biomarker. We analyzed measurements of CFTR-mediated Cl- secretion in rectal biopsies from 143 individuals (∼592 biopsies), the largest cohort so far analyzed by this approach. New parameters were analyzed and compared with the previous biomarker, i.e., the IBMX (I)/Forskolin (F)/Carbachol (C)-stimulated short-circuit current (I'sc-I/F/C). Correlations with clinical features showed that the best parameter corresponded to voltage measurements of the I/F + (I/F/CCH) response (VI/F+I/F/C), with higher correlations vs. I'sc-I/F/C for: sweat chloride (59 vs. 52%), fecal elastase (69 vs. 55%) and lung function, measured by FEV1 (27 vs. 20%). Altogether data show that VI/F+I/F/C is the most sensitive, reproducible, and robust predictive biomarker for CF diagnosis/prognosis effectively discriminating classical, atypical CF and non-CF groups.

Ventilation Inhomogeneity and Bronchial Basement Membrane Changes in Chronic Neutrophilic Airway Inflammation.

BACKGROUND: Bronchial epithelial reticular basement membrane (RBM) thickening occurs in diseases with both eosinophilic (allergic bronchial asthma [BA]) and neutrophilic (cystic fibrosis [CF] and primary ciliary dyskinesia [PCD]) chronic airway inflammation; however, the lung function and airway remodeling relation remains unclear. The aim of this study was to test whether ventilation inhomogeneity is related to RBM thickening. METHODS: Multiple breath washout test, endobronchial biopsy, and BAL were performed in 24 children with CF, 11 with PCD, 15 with BA, and in 19 control subjects. Lung clearance index at 2.5% (1/40th) of starting nitrogen concentration (LCI2.5), RBM thickness, and lavage fluid cytology were quantified; their mutual associations were studied by using Spearman rank correlations (r). RESULTS: In asthma, ventilation inhomogeneity (mean ± SD) was mild (LCI2.5, 9.3 ± 1.4 vs 7.9 ± 0.9 in control subjects; P = .0391), and the RBM thickened (5.26 ± 0.98 µm vs 3.12 ± 0.62 µm in control subjects; P < .0001). No relation between RBM thickness and ventilation inhomogeneity or lavage cytology was found. In CF and PCD, RBM thickness was similar to that in asthma (4.54 ± 0.66 µm and 5.27 ± 1.11 µm, respectively), but ventilation inhomogeneity was significantly higher (LCI2.5, 12.5 ± 2.4 and 11.8 ± 2.5). Both in CF and PCD, RBM thickness correlated with LCI2.5 (r = 0.594, P = .015; r = 0.821, P = .023). In PCD only, RBM thickness was also related to the number of neutrophils in lavage fluid (r = 0.821; P = .023). CONCLUSIONS: Lung function impairment in relation to RBM thickness was milder in BA than in CF and PCD. In asthma, ventilation inhomogeneity did not correlate with RBM thickness, whereas it did in CF and PCD. This outcome suggests a different structure-function relation in these diseases.

TMEM16A in Cystic Fibrosis: Activating or Inhibiting?

The inflammatory airway disease cystic fibrosis (CF) is characterized by airway obstruction due to mucus hypersecretion, airway plugging, and bronchoconstriction. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is dysfunctional in CF, leading to defects in epithelial transport. Although CF pathogenesis is still disputed, activation of alternative Cl- channels is assumed to improve lung function in CF. Two suitable non-CFTR Cl- channels are present in the airway epithelium, the Ca2+ activated channel TMEM16A and SLC26A9. Activation of these channels is thought to be feasible to improve hydration of the airway mucus and to increase mucociliary clearance. Interestingly, both channels are upregulated during inflammatory lung disease. They are assumed to support fluid secretion, necessary to hydrate excess mucus and to maintain mucus clearance. During inflammation, however, TMEM16A is upregulated particularly in mucus producing cells, with only little expression in ciliated cells. Recently it was shown that knockout of TMEM16A in ciliated cells strongly compromises Cl- conductance and attenuated mucus secretion, but does not lead to a CF-like lung disease and airway plugging. Along this line, activation of TMEM16A by denufosol, a stable purinergic ligand, failed to demonstrate any benefit to CF patients in earlier studies. It rather induced adverse effects such as cough. A number of studies suggest that TMEM16A is essential for mucus secretion and possibly also for mucus production. Evidence is now provided for a crucial role of TMEM16A in fusion of mucus-filled granules with the apical plasma membrane and cellular exocytosis. This is probably due to local Ca2+ signals facilitated by TMEM16A. Taken together, TMEM16A supports fluid secretion by ciliated airway epithelial cells, but also maintains excessive mucus secretion during inflammatory airway disease. Because TMEM16A also supports airway smooth muscle contraction, inhibition rather than activation of TMEM16A might be the appropriate treatment for CF lung disease, asthma and COPD. As a number of FDA-approved and well-tolerated drugs have been shown to inhibit TMEM16A, evaluation in clinical trials appears timely.

Do linear logistic model analyses of volatile biomarkers in exhaled breath of cystic fibrosis patients reliably indicate Pseudomonas aeruginosa infection?

Non-invasive breath analysis has been used to search for volatile biomarkers of lungs and airways infection by Pseudomonas aeruginosa, PA, in cystic fibrosis patients. The exhaled breath of 20 PA-infected patients and 38 PA-negative patients was analysed using selected ion flow tube mass spectrometry, SIFT-MS. Special attention was given to the positive identification and accurate quantification of 16 volatile compounds (VOCs) as assured by the detailed consideration of their analytical ion chemistry occurring in the SIFT-MS reactor. However, the diagnostic sensitivity and specificity of the concentrations of any of the 16 compounds taken individually were found to be low. But when a linear combination of the concentrations of all 16 VOCs was used to construct an optimised receiver operating characteristics (ROC) curve using a linear logistic model, the diagnostic separation of PA-infected patients relative to the PA-negative patients was apparently good in terms of the derived sensitivity (89%), specificity (86%), and the area under the ROC curve is 0.91. Four compounds were revealed by the linear logistic model as significant, viz. malondialdehyde, isoprene, phenol and acetoin. The implications of these results to PA detection in the airways are assessed. Whilst such a metabolomics approach to optimise the ROC curve is widely used in breath analysis, it can lead to misleading indications. Therefore, we conclude that the results of the linear logistic model analyses are of limited immediate clinical value. The identified compounds should rather be considered as a stimulus for further independent studies involving larger patient cohorts.

Breath concentration of acetic acid vapour is elevated in patients with cystic fibrosis.

A study has been carried out of the volatile organic compounds present in the exhaled breath of 58 cystic fibrosis (CF) patients. An important observation is that the acetic acid vapour concentration measured by selected ion flow tube mass spectrometry (SIFT-MS) is significantly elevated in the exhaled breath of CF patients, independent of the Pseudomonas aeruginosa (PA) infection status (PA-infected median 170 ppbv; PA-negative median 182 ppbv), compared to that of healthy controls (median 48 ppbv). The cause for this may be decreased pH of the mucus lining the CF airways. Thus, we speculate that non-invasive measurement of breath acetic acid concentration could serve as an indicator of the acidity of the CF airways mucosa.