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Advances in Pharmacological Treatment of Cystic Fibrosis
Örebro University, School of Health and Medical Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cystic fibrosis (CF) is an inborn, hereditary disease, due to mutations in the gene for a cAMP-activated chloride (Cl-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). As a result of impaired ion and water transport,the airway mucus is abnormally viscous, which leads to bacterial colonization.Recurrent infections and inflammation result in obstructive pulmonary disease.Similar changes in the pancreas lead to pancreatic insufficiency.Several compounds have been tested to improve transepithelial ion transport in CF patients, either via activation of the mutant CFTR, or via stimulation of alternative chloride channels. The main purpose of this thesis was to find substances that might correct the defective ion transport in epithelial cells in CFand could be useful for the pharmacological treatment of CF patients. Long-term treatment with the macrolide antibiotic azithromycin (AZM)improved clinical parameters and lung function in CF patients and increased Cl- transport in CF bronchial epithelial cells (CFBE) (Paper I); although mRNA expression of the CFTR gene remained unchanged.In contrast, pre-exposure to the mucolytic antioxidant N-acetylcysteine (NAC) increased CFTR protein expression and was associated with increased Cl- efflux from CFBE cells (Paper II). Clinical trials of this substance might be warranted. Duramycin has been the subject of clinical trials that finished in June2009. Up till now, no results from this study are available. The effect of this substance on Cl- efflux from three CF and three non-CF cell lines (Paper III) was disappointing. An effect was found only in CFBE cells, the effect was minimal, occurred in a narrow concentration range, and was not associated with an increase in the intracellular calcium concentration [Ca2+]i. The fact that NO-donors stimulated Cl- efflux from CFBE cells (but did notchange [Ca2+]i) after several hours of preincubation suggests that these substances may be a potentially interesting group of compounds for the treatment of CF (Paper IV). A model for the effect of NO-donors on Cl- efflux is presented.

Abstract [sv]

Cystisk fibros (CF) är en medfödd, ärftlig, sjukdom, som förorsakas av en mutation i en gen som innehåller koden för en kloridkanal som aktiveras av cykliskt AMP (cystic fibrosis transmembrane conductance regulator, CFTR). Som en följd av otillräcklig transport av joner och vatten är slemmet i luftvägarna onormalt segt, vilket leder till att det koloniseras av bakterier. Upprepade infektioneroch inflammation av luftvägarna leder slutligen till obstruktiv lungsjukdom.Liknande förändringar i bukspottkörteln leder till att också detta organ inte fungerar. Flera kemiska ämnen har testats för sin förmåga att förbättra jontransporten över epitelet hos CF-patienter. Detta skulle kunna göras antingen genom aktivering av det muterade CFTR-proteinet, eller genom stimulering av alternativa kloridkanaler. Huvudsyftet med den forskning som beskrivs i denna avhandling var att hitta kemiska substanser som skulle kunna korrigera den defekta jontransporten i epitelceller hos CF-patienter, och därför vara nyttiga för behandlingen av patienterna. Behandling under längre tid med azithromycin (AZM), ett makrolidantibiotikum,förbättrade CF-patienternas kliniska status och lungfunktion,samt ökade kloridutflödet från CF bronkialepitelceller (CFBE-celler) (Arbete I).Däremot ändrades inte uttrycket av mRNA för CFTR-genen. I kontrast till detta ökade uttrycket av CFTR-proteinet om CFBE-cellerna utsattes för den slemlösande anti-oxidanten N-acetylcystein (NAC), vilket ledde till ökat kloridutflöde från denna cellinje (Arbete II). Det vore rimligt att utföra kliniska prövningar av detta ämne. Duramycin har testats i kliniska prov som slutade i juni 2009, men några resultatfrån dessa prov har inte offentliggjorts än. Effekten av detta ämne på kloridutflödet från tre CF-cellinjer och tre icke-CF cellinjer (Arbete III) var en besvikelse. Duramycin hade endast effekt på CFBE-celler, effekten var mycket liten, förekom endast i ett litet koncentrationsområde av duramycin, och var inte kopplad till en ökning av den intracellulära kalciumkoncentrationen [Ca2+]i. Att ämnen som avger kväveoxid (NO) stimulerade kloridutflödet från CFceller (men inte påverkade [Ca2+]i) efter några timmar, visar att denna grupp av ämnen kan vara potentiellt intressant för behandlingen av CF (arbete IV). En modell för effekten av NO på kloridtransporten i CF-celler presenteras.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2010. , 88 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 50
Keyword [en]
Cystic fibrosis, CFTR, chloride transport, N-acetylcysteine, NO-donors, duramycin, intracellular calcium, azithromycin
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
URN: urn:nbn:se:oru:diva-12424ISBN: 978-91-7668-773-4 (print)OAI: oai:DiVA.org:oru-12424DiVA: diva2:361214
Public defence
2010-12-17, Konferensrum Hjälmaren, USÖ, M-huset, S. Grev Rosengatan, Örebro, 09:15
Opponent
Supervisors
Available from: 2010-11-12 Created: 2010-11-08 Last updated: 2011-04-21Bibliographically approved
List of papers
1. Azithromycin increases chloride efflux from cystic fibrosis airway epithelial cells
Open this publication in new window or tab >>Azithromycin increases chloride efflux from cystic fibrosis airway epithelial cells
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2009 (English)In: Experimental Lung Research, ISSN 0190-2148, E-ISSN 1521-0499, Vol. 35, no 3, 210-221 p.Article in journal (Refereed) Published
Abstract [en]

It was investigated whether azithromycin (AZM) stimulates chloride (Cl−) efflux from cystic fibrosis (CF) and non-CF airway epithelial cells, possibly secondary to up-regulation of the multidrug resistance protein (MDR). CF and non-CF human airway epithelial cell lines (CFBE and 16HBE) were treated with 0.4, 4, and 40 μ g/mL AZM for 4 days. Cl− efflux was explored in the presence or absence of specific inhibitors of CFTR and alternative Cl−  channels. Six CF patients received AZM (500 mg daily) for 6 months. The percentage of predicted forced vital capacity (FVC%), forced expiratory volume (FEV1%), and the number of acute exacerbations were compared before and after treatment. Nasal biopsies were taken before and after treatment, and mRNA expression of MDR and CFTR was determined by in situ hybridization. A significant dose-dependent increase of Cl− efflux from CFBE cells (but not from 16HBE cells) was observed after AZM treatment. A CFTR inhibitor significantly reduced AZM-stimulated Cl−  efflux from CFBE cells. A significant improvement in FEV1%, and fewer exacerbations were observed. AZM treatment did not affect mRNA expression of MDR and CFTR. The stimulation of Cl− efflux could be part of the explanation for the clinical improvement seen among the patients.

Place, publisher, year, edition, pages
Taylor & Francis, 2009
Keyword
azithromycin, chloride transport, cystic fibrosis, lung function, Morphology, Medical cell biology, pathology
National Category
Medical and Health Sciences Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12440 (URN)10.1080/01902140802534967 (DOI)000264750200004 ()19337904 (PubMedID)2-s2.0-67149144260 (Scopus ID)
Available from: 2010-11-11 Created: 2010-11-11 Last updated: 2017-03-15Bibliographically approved
2. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells
Open this publication in new window or tab >>The effect of N-acetylcysteine on chloride efflux from airway epithelial cells
2010 (English)In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 34, no 3, 245-252 p.Article in journal (Refereed) Published
Abstract [en]

Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37°C. The effect of NAC on Cl transport was measured by Cl efflux measurements and by X-ray microanalysis. Cl efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl efflux with nearly 80% in CFBE cells. The intracellular Cl concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.

Place, publisher, year, edition, pages
London: Portland Press Ltd, 2010
Keyword
airway epithelium, chloride transport, cystic fibrosis, N-acetylcysteine, Medical cell biology, Morphology, cell biology, pathology
National Category
Cell and Molecular Biology Medical and Health Sciences Cell and Molecular Biology
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12451 (URN)10.1042/CBI20090007 (DOI)
Note
Georgia Varelogianni, Igor Oliynyk, Godfried M Roomans are also affiliated w. Department of Medical Cell Biology, Uppsala University, Box 571, SE-75123 Uppsala, SwedenAvailable from: 2010-11-12 Created: 2010-11-12 Last updated: 2012-02-28Bibliographically approved
3. Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non-cystic fibrosis epithelia
Open this publication in new window or tab >>Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non-cystic fibrosis epithelia
2010 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 118, no 12, 982-990 p.Article in journal (Refereed) Published
Abstract [en]

The lantibiotic duramycin (Moli1901, Lancovutide) has been suggested as a drug of choice in the treatment for cystic fibrosis (CF). It has been proposed that duramycin may stimulate chloride secretion through Ca2+-activated Cl channels (CaCC). We investigated whether duramycin exhibited any effect on Cl efflux and intracellular Ca2+ concentration ([Ca2+]i) in CF and non-CF epithelial cells. Duramycin did stimulate Cl efflux from CF bronchial epithelial cells (CFBE) in a narrow concentration range (around 1 μM). However, 100 and 250 μM of duramycin inhibited Cl efflux from CFBE cells. An inhibitor of the CF transmembrane conductance regulator (CFTRinh-172) and a blocker of the capacitative Ca2+ entry, gadolinium chloride, inhibited the duramycin-induced Cl efflux. No effect on Cl efflux was observed in non-CF human bronchial epithelial cells (16HBE), human airway submucosal gland cell line, human pancreatic epithelial cells, CF airway submucosal gland epithelial cells, and CF pancreatic cells. The [Ca2+]i was increased by 3 μM duramycin in 16HBE cells, but decreased after 1, and 3 μM of duramycin in CFBE cells. The results suggest that the mechanism responsible for the stimulation of Cl efflux by duramycin is mainly related to unspecific changes of the cell membrane or its components rather than to effects on CaCC.

Place, publisher, year, edition, pages
New York, USA: John Wiley & Sons, 2010
Keyword
Duramycin, cystic fibrosis, airway epithelium, chloride efflux
National Category
Medical and Health Sciences Immunology
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12442 (URN)10.1111/j.1600-0463.2010.02680.x (DOI)000284317500010 ()21091780 (PubMedID)2-s2.0-78649506897 (Scopus ID)
Available from: 2010-11-11 Created: 2010-11-11 Last updated: 2017-02-10Bibliographically approved
4. Effect of NO-donors on chloride efflux and intracellular Ca2+ concentration in cystic fibrosis airway epithelial cells
Open this publication in new window or tab >>Effect of NO-donors on chloride efflux and intracellular Ca2+ concentration in cystic fibrosis airway epithelial cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Previous studies have shown that the endogenous bronchodilator, S-nitrosglutathione (GSNO), causes a 4–fold increase in CFTR-mediated chloride (Cl) efflux and improves the trafficking of CFTR to the plasma membrane. The NO-donor GEA3162 had a similar, but smaller, effect on Cl efflux. In order to investigate whether the NO-donor properties of GSNO were relevant for its effect on Cl efflux from airway epithelial cells, the effect of a number of other NO-donors (SNP, SNAP, DETA-NO, and DEA-NONOate) on Cl efflux from CFBE (DF508/DF508) airway epithelial cells was tested. Cl efflux was determined using the fluorescent MQAE-technique. Possible changes in the intracellular Ca2+ concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like GSNO, SNP had no immediate effect on Cl efflux, but after 4h incubation with the NO-donor, an increased Cl efflux was found (in the order SNAP > DETA-NO > DEA- NONOate > SNP). The effect of DEA-NONOate on Cl efflux was not significant, and the compound may have (unspecific) deleterious effects on the CFBE cells. None of the compounds that caused significant Cl efflux caused significant changes in the intracellular Ca2+ concentration. It is concluded that the effect of GSNO is, at least in part, due to its properties as an NO-donor, and that the effect is likely to be mediated by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), not on Ca2+-activated Cl channels.

Keyword
Nitric oxide donors, cystic fibrosis, airway epithelium, chloride channels, medical cell biology
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12446 (URN)
Available from: 2010-11-11 Created: 2010-11-11 Last updated: 2016-12-08Bibliographically approved

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