Drugs new and old

Drugs new and old2023-11-21T12:35:58+00:00



Alpha-1 antitrypsin, acetylcysteine (see glutathione lower down), amiloride, aminopyrrolidine amide, amitriptyline, Antisense oligonucleotide splicing modulation, (PTC 124), curcumin, DENUFOSOL, Esc peptides, ENaC inhibitor AZD5634,  garlic, genistein, glutathione/ N-acetylcysteine, N-acetylcystiene for GI complications, icenticaftor (QBW251),  IgY egg yolk antibodies, Pseudomonas vaccines, marijuana, MOLI 1901 (duramycin leancovutide), miglustat (N-butyldeoxynozyremicin), phosphdiesterase inhibitors, probiotics, sodium-dependent glucose cotransporter 1/2 (SGLT1/2) inhibitor drugs phlorizin and sotagliflozin, SLC26A9 (membrane proteins involved in ion transport),      Thymosin A1,     CP-628006,                  inhibition of amiloride-sensitive sodium channels (with amiloride), C407 (bis-phosphinic acid derivative),


2011 Cuthbert AW. New horizons in the treatment of cystic fibrosis. [Review] Brit J Pharmacol 2011; 163:173-183. [PubMed]

Alan Cuthbert

Alan Cuthbert observes that at long last there appears to be progress with the pharmaco-therapeutic approach. Ongoing clinical trials have produced fascinating results in which clinical benefit appears to have been achieved. To arrive at this point ingenious ways have been devised to screen very large chemical libraries for one of two properties: (i) agents promoting trafficking of mutant CFTR to, and insertion into the membrane, and known as correctors or (ii) agents which activate appropriately located mutant CFTR, known as potentiators. The best compounds emerging from these programmes are then used as chemical scaffolds to synthesize other compounds with appropriate pharmaceutical properties, hopefully with their pharmacological activity maintained or even enhanced. In summary, this approach attempts to make the mutant CFTR function in place of the real CFTR. A major function of CFTR in healthy airways is to maintain an adequate airway surface liquid (ASL) layer. In CF the position is further confounded since epithelial sodium channels (ENaC) are no longer regulated and transport salt and water out of the airways to exacerbate the lack of ASL. Thus an additional possibility for treatment of CF is to use agents that inhibit ENaC either alone or as adjuncts to CFTR correctors and/or potentiators. Yet a further way in which a pharmacological approach to CF can be considered is to recruit alternative chloride channels, such as calcium-activated chloride channel (CaCC), to act as surrogates for CFTR. A number of P2Y(2) receptor agonists have been investigated that operate by increasing Ca(2+)(i) which in turn activates CaCC. Some of these compounds are currently in clinical trials. The knowledge base surrounding the structure and function of CFTR that has accumulated in the last 20 years is impressive. Translational research feeding from this is now yielding compounds that provide real prospects for a pharmacotherapy for this disease.

Professor Alan Cuthbert (1932-2016) was of the UK’s leading researchers in the field of CF. He was Professor of Pharmacology in the University of Cambridge (1991-1990) and Master of Fitzwilliam College from 1991-1999. After he retired he joined the Department of Medicine and continued his research until a year before he died. Alan was a very pleasant man and for some time served on the CF Trust’s Medical and Scientific Advisory Committee when I chaired that committee; his wry sense of humour, wise advice and support for myself as chair were greatly appreciated.

2007 Flume PA, O’Sullivan BP, Robinson KA, Goss CH, Mogayzel PJ Jr, Willey-Courand DB, Bujan J, Finder J, Lester M, Quittell L, Rosenblatt R, Vender RL, Hazle L, Sabadosa K, Marshall B. Cystic Fibrosis Foundation, Pulmonary Therapies Committee. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health. Amer J Respir Crit Care Med 2007; 176: 957-969.

Patrick Flume

In 2005 the Cystic Fibrosis Foundation established a committee to examine the clinical evidence for each therapy used for CF and to provide guidance for the prescription of these therapies. The committee members developed and refined a series of questions related to drug therapies used in the maintenance of pulmonary function. Questions were addressed in one of three ways, based on available evidence: (1) commissioned systematic review, (2) modified systematic review, or (3) summary of existing Cochrane reviews. The strength of the evidence and the estimate of benefit were recorded and the recommendations graded. Grade A-good evidence and substantial benefit. Grade B- fair evidence, benefit outweighs harm. Grade C – no recommendation, benefits and harm balance too close. Grade D – fair evidence not effective or harm outweighs benefit. Grade I – insufficient evidence”.

The following treatments received the following grades-
Nebulised tobramycin – grade A for severe, grade B for mild persistent Pseudomonas infection; evidence for other inhaled antibiotics insufficient; (surprisingly even in 2005 the important area of early Pseudomonas treatment was not dealt with); rhDNase grade A recommendation; hypertonic saline grade B; inhaled and oral steroids both grade D;
non-steroidal anti-inflammatory drugs grade B; leukotriene modifiers insufficient evidence ; macrolides grade B;
prophylactic anti-staphylococcal antibiotics grade D; ß-agonist bronchodilators grade B; anti-cholinergic bronchodilators Grade I; n-acetylcysteine grade I.

The treatments that have been the subject of a CF Foundation Phase III trial are awarded either A or B recommendation. It is quite surprising that even in 2007 the early eradication treatment of Pseudomonas is not considered in a document on inhaled therapy. Not all would agree with the grading of prophylactic anti-Staphylococcal antibiotics for which there is a European trial (Weaver et al, 1995 above) and a wealth of European experience.

Dr Patrick Flume (figure) works at the Medical University of South Carolina where he is Professor of Medicine and Pediatrics. He is also Director of the CF Centre there. He is co-chair of the Pulmonary Practice Guidelines Committee of the CF Foundation and very active in CF care and research.

Alpha-1 antitrypsin

1991 McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM, Crystal RG. Aerosol alpha-1-antitrypsin treatment for cystic fibrosis. Lancet 1991; 337:392-394. [PubMed]

Gerry McElvaney

A1-antitrypsin, the main inhibitor of neutrophil elastase, was given in aerosol form to 12 CF patients and found to suppress neutrophil elastase in respiratory lining fluid and restore its anti-neutrophil elastase capacity. Also, the treatment reduced the reversed inhibitory effect of CF epithelial lining fluid on Pseudomonas killing.

Apparently the material used in this trial (purified human plasma a1-antitrypsin – Prolastin, Cutter Biological) was very difficult to obtain in sufficient quantities. A subsequent trial, with a genetically engineered product which eventually became available, disappointingly failed to show significant benefit to patients and was not further developed as a treatment for cystic fibrosis (Martin SL, et al, 2006 below). However, some interest continues in Germany and there may be further developments.

2006 Martin SL, Downey D, Bilton D, Keogan MT, Edgar J, Elborn JS. Recombinant AAT Study Team. Safety and efficacy of recombinant alpha (1)-antitrypsin therapy in cystic fibrosis. Pediatr Pulmonol 2006; 41:177-183. [PubMed]
A previous study had been performed with plasma derived inhibitor (McElvaney et al, 1991 above). In this present study recombinant human ATT was used in a Phase II trial of rAAT at various dose levels. This drug was safe but, disappointingly, had little effect on neutrophil elastase activity and other markers of inflammation. As a result, there was no further work in CF from this group.

However, later a similar trial from Germany (Griese, M. et al, Eur Respir J 2007; 29:240-50. [PubMed] below) did show some reduction of inflammatory markers but no change of respiratory function. Griese et al suggested that the clear reduction of airway inflammation after ATT treatment may precede pulmonary structural changes; also the ATT deposition region, either bronchial or peripheral, may play a minor role for ATT inhalation in patients with cystic fibrosis. So there may be further developments in relation to CF.

2007 Griese M, Latzin P, Kappler M, Weckerle K, Heinzlmaier T, Bernhardt T, Hartl D. alpha1-Antitrypsin inhalation reduces airway inflammation in cystic fibrosis patients. Eur Resp J 2007; 29:240-250. [PubMed]
Although no effect on lung function was observed, the clear reduction of airway inflammation after alpha(1)-antitrypsin treatment may precede pulmonary structural changes. The alpha(1)-antitrypsin deposition region may play a minor role for alpha1-antitrypsin inhalation in cystic fibrosis patients.

2008 Griese M, Kappler M, Gaggar A, Hartl D. Inhibition of airway proteases in cystic fibrosis lung disease. [Review] Eur Resp J 2008; 32:783-795. [PubMed] Free article available.
This is a very detailed review of protease inhibition with detailed account of the previous studies up to this time with liberal references. A number of previous studies have shown that alpha1-AT genotype is not a major contributor to the variability of pulmonary disease severity in CF (Mahadeva R et al. Alpha1-antitrypsin deficiency alleles and the Taq-I G–>A allele in cystic fibrosis lung disease. Eur Respir J 1998; 11:873-879. [PubMed]; Henry MT et al. An alpha1-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis. Eur J Hum Genet 2001; 9:273-278. [PubMed];Frangolias DD et al. Alpha 1-antitrypsin deficiency alleles in cystic fibrosis lung disease. Am J Resp Cell Mol 2003; 29:390-396. [PubMed]).

2009 Brand P, Schulte M, Wencker M, Herpich CH, Klein G, Hanna K, Meyer T. Lung deposition of inhaled alpha1-proteinase inhibitor in cystic fibrosis and alpha1-antitrypsin deficiency. Eur Respir J 2009; 34:354-360. [PubMed]
This study aimed to determine the efficiency of delivering AAT using a novel inhalation device in subjects with AAT deficiency and CF compared with healthy subjects. In total, 20 subjects – six healthy, seven with AAT deficiency and seven with CF. Inhalation with controlled breathing patterns using the AKITA(2) device (lung function adapted) leads to high total lung deposition regardless of the degree of lung function impairment. Delivery of large amounts of AAT was achieved in a short period of time. This device may be an ideal option for aerosol therapy.

2010 Geller DE, Kesser KC.The I-neb Adaptive Aerosol Delivery System enhances delivery of alpha1-Antitrypsin with controlled inhalation. J Aeroso Med Pulm D 2010; Suppl 1:S55-9. [PubMed] Free article available.

David Geller

The I-neb AAD System enhanced alpha1-antitrypsin (AAT) delivery by inhalation-only aerosol generation and a low-residual dose. Predicted lung dose was high for both tidal breathing mode and target inhalation mode (TIM), but longer inspiratory times with TIM reduced the administration time to one-third that of tidal breathing. The authors concluded that slow, deep, controlled inspirations using the I-neb AAD System is an efficient method to deliver AAT.

– Alpha1-antitrypsin is not used in routine CF treatment. Apparently exogenous AAT is susceptible to both cleavage and oxidative inactivation. Following oxidation, its anti-NE capacity becomes negligible while both cathepsin1 and Pseudomonas elastase are known to cleave AAT (Chotirmall SJ et al. in Hodson and Geddes Cystic Fibrosis 4th edition, 2016).

2016 Griese M; Scheuch G. Delivery of Alpha-1 Antitrypsin to Airways. Ann Am Thorac Soc 2016; 13 (Suppl 4): S346-51, 2016 Aug. [PubMed]
Treatment with exogenous alpha-1 antitrypsin (AAT), a potent serine protease inhibitor, was developed originally for chronic obstructive pulmonary disease associated with AAT deficiency; however, other lung conditions involving neutrophilic inflammation and proteolytic tissue injury related to neutrophil elastase and other serine proteases may also be considered for AAT therapy. These conditions include bronchiectasis caused by primary ciliary dyskinesia, cystic fibrosis, and other diseases associated with an increased free elastase activity in the airways. Inhaled AAT may be a viable option to counteract proteolytic tissue damage. This form of treatment requires efficient drug delivery to the targeted pulmonary compartment. Aerosol technology meeting this requirement is currently available and offers an alternative therapeutic approach to systemic AAT administration. To date, early studies in humans have shown biochemical efficacy and have established the safety of inhaled AAT. However, to bring aerosol AAT therapy to patients, large phase 3 protocols in carefully selected patient populations (i.e., subgroups of patients with AAT deficiency, cystic fibrosis, or other lung diseases with bronchiectasis) will be needed with clinical end points in addition to the measurement of proteolytic activity in the airway. The outcomes likely will have to include lung function, lung structure assessed by computed tomography imaging, disease exacerbations, health status, and mortality.

– First used in 1991 when obtained from purified human plasma (McElvaney NG et al 1991). A subsequent trial, with a genetically engineered product, disappointingly failed to show significant benefit to patients and was not further developed as a treatment for cystic fibrosis (Martin SL, et al, 2006). However, some interest continued in Germany by the author of the present paper (Griese M et al. alpha1-antitrypsin inhalation reduces airway inflammation in cystic fibrosis patients. Eur Respir J 2007; 29:240-250.[PubMed] Free article). Apparently exogenous A1AT is susceptible to both cleavage and oxidation in the airways when its anti-neutrophil elastase capacity becomes negligible.[See Topics->Various old and new drugs ->Alpha-1-antitrypsin].

2016 McElvaney NG. Alpha-1 antitrypsin therapy in Cystic Fibrosis and the Lung Disease Associated with Alpha-1 antitrypsin Deficiency. Ann Am Thor Soc 2016; 13 Suppl 2:S191-6.[PubMed]
Cystic fibrosis and alpha-1 antitrypsin (AAT) deficiency are two of the commonest lethal hereditary lung diseases affecting white individuals. Although having quite different phenotypic extrapulmonary presentations, the lung disease associated with these conditions is exemplified by a neutrophil-dominated inflammation in which neutrophil elastase plays a major role. In AAT deficiency the diminution of the anti-neutrophil elastase protection, due to diminished AAT levels in the lung, predisposes the lung to an unopposed neutrophil elastase attack, whereas, in cystic fibrosis, the levels of AAT and other antiproteases are normal, but the neutrophil elastase burden is so large that it overwhelms the normal anti-neutrophil elastase protection. With this as background, it seems logical to augment the anti-neutrophil elastase defences of the lung in both conditions using exogenous AAT. The type of AAT, the route of administration, and the physiologic, radiologic, and clinical readouts for this type of therapy are discussed, along with the similarities and differences between the two conditions and their responses to AAT therapy.

Kim M, Cai Q, Oh Y.Therapeutic potential of alpha-1 antitrypsin in human disease.Ann Pediatr Endocrinol Metab.2018 Sep;23(3):131-135. doi: 10.6065/apem.2018.23.3.131. Epub 2018 Sep 28. [Pubmed] 30286568 Free PMC Article

Youngman Oh

Alpha-1 antitrypsin (AAT), an alpha globulin glycoprotein, is a member of the serine protease inhibitor (serpin) superfamily. The clinical significance of AAT is highlighted by AAT deficiency. Genetic deficiency of AAT can present as several neutrophilic diseases associated with emphysema, liver cirrhosis, panniculitis, and systemic vasculitis. Recently, animal and human studies have shown that AAT can control inflammatory, immunological, and tissue-protective responses. In addition, AAT treatment can prevent overt hyperglycemia, increase insulin secretion, and reduce cytokine-mediated apoptosis of pancreatic β-cells in diabetes. These multifunctional roles of AAT draw attention to the glycoprotein’s therapeutic potential for many inflammatory and autoimmune diseases beyond AAT deficiency. As underlying mechanisms, recent studies have suggested the importance of serine protease inhibitory activity of AAT in obesity-associated insulin resistance, chronic obstructive pulmonary disease, and cystic fibrosis. In this review, we explore the multiple functions of AAT, in particular, the anti-inflammatory and serine protease inhibitory functions, and AAT’s therapeutic potential in a variety of human diseases through published literature.

– With regard to alpha-1 antitrypsin for cystic fibrosis, the authors of this article comment as follows-Thus, in CF, the treatment focus is on decreasing neutrophil hyper activation and counteracting the effects of NE on the lung. To inhibit NE in the lung during CF progression, early studies have focused on the augmentation of systemic AAT levels by intravenous injection (McElvaney NG[PubMed]).Recently, a randomized, double-blind, placebo-controlled phase 2a study in CF patients has been further performed to evaluate the safety of 100 or 200 mg of inhaled AAT once daily for 3 weeks in 30 adult subjects and reported that inhalation is safe and well tolerated (Gaggar A et al, 2016 [PubMed]).Further multiple studies have demonstrated that AAT administered by inhalation can control neutrophil function and NE levels in a dose-dependent fashion as well as inflammation in the lung . However, many obstacles still remain in applying AAT in CF since mixed results have been observed depending on the devices used as well as lung condition and NE concentration of patients”

Dr. Min Sun Kim is Associate Professor in the Division of Endocrinology, Department of Pediatrics, Chonbuk National University Medical School, Jeonju, Korea.

Corresponding author is Professor Youngman OH, Department of Pathology, School of Medicine Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA


1990 Knowles MR, Church NL, Waltner WE, Yankaskas JR, Gilligan P, King M, Helms RW, Boucher RC. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Eng J Med 1990; 322:1189-1194. [PubMed]

Michael Knowles

Michael Knowles first discovered the increased bioelectrical potential difference across respiratory epithelium in CF (Knowles M et al. N Eng J Med 1981; 305:1489-1495 above). In this present study Knowles and Boucher investigate whether the inhibition of excessive absorption of sodium by inhaled amiloride might favourably affect the course of CF lung disease. Fourteen of 18 patients completed a one year double-blind, crossover trial comparing aerosolized amiloride (5 mmol per litre; 3.5 ml four times daily) with control solution. The mean (+/- SEM) loss of forced vital capacity (FVC) was reduced from 3.39 (+/- 1.13 ml) per day during treatment with vehicle alone to 1.44 (+/- 0.67) ml per day with amiloride (P <0.04). Sputum viscosity and elasticity, mucociliary and cough clearance improved during treatment with amiloride suggesting a beneficial effect.

– This trial created considerable interest but the effect of amiloride was modest and short-lived; also the loss of FVC in the control group seemed excessive. Apparently the action of inhaled amiloride is very transient and subsequently longer acting analogues were explored. However, this report did encourage us in Leeds to look at the effect of giving nebulised amiloride during the intravenous antibiotic treatment of exacerbations of respiratory infection in CF. We observed a definite but insignificant improvement in the early response to intravenous antibiotics in the amiloride group (Bowler et al, 1995 below). No benefit was seen from amiloride by Graham A et al (No added benefit from nebulised amiloride in patients with cystic fibrosis. Eur Respir J 1993; 6:1243-1248. [PubMed] ) nor in a French multi-centre randomized double blind placebo controlled trial in patients more than 5 years old (Pons G, et al, Pediatr Pulmonol 2000; 30:25-31. [PubMed]).

However, Kohler et al showed inhaled amiloride improved mucociliary clearance in patients with CF (Kohler et al, Eur J Respir Dis 1986; 69 (Suppl 146:319-326); also the same group confirmed this with a larger study (App AM et al. Am Rev Respir Dis 1990; 141:605-612. [PubMed]). Also Lindemann et al from Giessen had reported 50.4% more sputum was produced by autogenic drainage after inhalation of amiloride than after isotonic saline also visible liquefaction of secretions was noted by the physiotherapist and patients (Lindemann H et al. Elimination of secretions in CF patients under amiloride inhalation. Pneumologie 1990; 44:1148-1150[PubMed][German]).
Later more active and longer acting drugs (P- 680 & P- 522-O2-Parion Sciences/Gilead) that Inhibit excess Na absorption showed more promise and their development continues. Sadly, as is described later, the most promising of these was abandoned after a negative phase III trial.

1995 Bowler IM, Kelman B, Worthington D, Littlewood JM, Watson A, Conway SP, Smye SW, James SL, Sheldon TA. Nebulised amiloride in respiratory exacerbations of cystic fibrosis: a randomised controlled trial. Arch Dis Child 1995; 73:427-430. [PubMed]

Ian Bowler

As a result of Michael Knowles’s amiloride trial (Knowles et al, 1990 above), we assessed the benefit of nebulised amiloride added to the standard treatment of a respiratory exacerbation in people with cystic fibrosis. We performed a prospective, randomised, double blind, placebo controlled trial with 27 patients (mean age 12.8 years) in two hospitals in Leeds, UK. Both forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) showed improvements over the course of treatment as would be expected but there was no difference in respiratory function between the two groups at any of three time periods during the study. However, the time to reach peak FVC was significantly reduced in the amiloride group (4.2 v 7.6 days; 95% CI 0.4 to 6.4 days), but not in the time to reach peak FEV1 (5.7 v 7.9 days; 95% CI -1.2 to 5.6 days). Amiloride did not result in a greater overall improvement in respiratory function.

On the modest results of this trial we did not introduce amiloride into the treatment regimen for exacerbations. Apparently the duration of action of amiloride is so very short to the extent that it would be unlikely to have a significant beneficial effect. (Also negative trials by Graham et al Eur Respir J 1993; 6:1243-1248.[PubMed] ; Pons G et al, Pediatr Pulmonol 2000; 30:25-31. [PubMed]).

1996 Robinson M, Regnis JA, Bailey DL, King M, Bautovich GJ, Bye PTP. Effect of hypertonic saline, amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. Am J Respir Crit Care Med 1996; 153:1503-1509.[PubMed]
After inhalation of hypertonic (7%) saline alone, and with amiloride, the amount of radio aerosol cleared from the right lung at 60 and 90 minutes was significantly increased. The authors suggested that inhaled hypertonic saline was a potentially useful treatment for CF.
Peter Bye’s group at St Vincent’s Hospital in Sydney continued their work on hypertonic saline and eventually carried out a successful clinical trial of hypertonic saline in adults with CF and confirmed the value of hypertonic saline treatment (Elkins et al, 2006 below).

– Hypertonic (1.21 M) saline inhalations were originally reported to double the rate of removal of bronchial secretions in chronic bronchitis in an interesting study using radio aerosols (Pavia D et al, Enhanced clearance of secretions from the human lung after the administration of hypertonic saline aerosol. Am Rev Respir Dis 1978; 117:199-203.[PubMed]).

2000 Pons G, Marchand MC, d’Athis P, Sauvage E, Foucard C, Chaumet-Riffaud P. The Amiloride-AFLM Collaborative Study Group. Pediatr Pulmonol 2000; 30:25-31. [PubMed]
In this large French study 64 patients with CF, chronically infected with Pseudomonas aeruginosa, received either nebulised amiloride or placebo three times daily for 6 months in addition to their usual treatments. The study failed to demonstrate any significant benefit from the adding the amiloride to their treatment.

– This multicentre French study confirmed the lack of significant clinical effect of inhaled amiloride the use of which had been of interest as a treatment since Michael Knowles’s amiloride study in the early Nineties – using amiloride to reduce the excessive sodium absorption from the CF airways (Knowles MR et al. N Eng J Med 1990; 322:1189-1194. [PubMed] above). Apparently the usual preparation of amiloride is rapidly destroyed in vivo. Excessive Na absorption in now considered important in the pathophysiology of the respiratory tract, other ways of inhibiting excessive sodium absorption have replaced amiloride as treatment possibilities.

Longer acting analogues of amiloride may be more effective and subsequently became available. Subsequent development of one compound by Gilead (Gilead GS9411) which entered clinical trials in 2009 unfortunately failed a Phase III trial in 2010.

Pradeep Kota  Sustained inhibition of ENaC in CF: Potential RNA-based therapies for mutation-agnostic treatmentCurr Opin Pharmacol 2022 Apr 25;64:102209.doi: 10.1016/j.coph.2022.102209.Online ahead of print.[Pubmed]

Pradeep Kota

Disruption of the equilibrium between ion secretion and absorption processes by the airway epithelium is central to many muco-obstructive lung diseases including cystic fibrosis (CF). Besides correction of defective folding and function of CFTR, inhibition of amiloride-sensitive epithelia sodium channels (ENaC) has emerged as a bona fide therapeutic strategy to improve mucociliary clearance in patients with CF. The short half-life of amiloride-based ENaC blockers and hyperosmotic therapies have led to the development of novel RNA-based interventions for targeted and sustained reduction of ENaC expression and function in preclinical models of CF. This review summarizes the recent advances in RNA therapeutics targeting ENaC for mutation-agnostic treatment of CF.

Pradeep Kota is a Research Associate at the Cystic Fibrosis Research and Treatment Center, University of North Carolina at Chapel Hill, NC 27599, USA.


There is considerable interest in ceramide in recent years and the ill effects of its accumulation and inflammation within cystic fibrosis cells. (Teichgraber V et al, 2008.[PubMed]).

2009 Riethmüller J, Anthonysamy J, Serra E, Schwab M, Döring G, Gulbins E. Therapeutic efficacy and safety of amitriptyline in patients with cystic fibrosis. Cell Physiol Biochem 2009; 24:65-72.[PubMed]

Joachim Riethmueller

Amitriptyline, a blocker of acid sphingomyelinase and acid ceramidase, significantly reduces Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) mice with concurrent increase of survival ([PubMed]). The aim of the present study was to establish whether amitriptyline is safe and effective in the treatment of CF patients.
In a randomised, double-blinded, placebo-controlled, cross-over pilot study, 4 adult CF patients received 37.5 mg of amitriptyline or placebo twice daily for 14 days. Subsequently in a phase II study 19 adult CF patients were randomly allocated to three treatment groups receiving amitriptyline once daily for 28 days at doses of 25 mg (n=7), 50 mg (n=8), or 75 mg (n=8) or placebo (n=13). The primary outcome was the difference of forced expiratory volume in 1 sec (FEV(1)) at day 14 between amitriptyline and placebo.
The primary endpoint measures improved significantly in three of four patients in the pilot study after amitriptyline treatment vs placebo (relative FEV(1): 14.7+/-5%; p = 0.006) and in the 25 mg treatment group of the phase II study (relative FEV(1): 4.0+/-7%; p = 0.048). Amitriptyline was well tolerated in both studies and 96% of the patients completed the studies. Amitriptyline as a novel therapeutic option in patients with CF is safe and seems to be efficacious.

An example of “low hanging fruit” – a drug already in use which has a favourable effect on CF. More was published on this treatment.

2010 Becker KA, Tummler B, Gulbins E, Grassme H. Accumulation of ceramide in the trachea and intestine of cystic fibrosis mice causes inflammation and cell death. Biochem Bioph Res Co 2010; 403:368-374.[PubMed] Recent studies have demonstrated an accumulation of ceramide in the lungs of cystic fibrosis patients and in several mouse models. These findings showed that pulmonary ceramide concentrations play an important role in pulmonary inflammation and infection. In this study the authors investigated whether ceramide concentrations are also altered in the trachea and the intestine of cystic fibrosis mice and whether an accumulation of ceramide in these organs has functional consequences that are typical of cystic fibrosis. They found a marked accumulation of ceramide in tracheal and intestinal epithelial cells of cystic fibrosis mice.

– When acid sphingomyelinase activity is inhibited by treating the cystic fibrosis mice with amitriptyline or by genetic heterozygosity of acid sphingomyelinase in cystic fibrosis mice, ceramide concentrations in the trachea and the intestine are normalized. Moreover, increased rates of cell death and increased cytokine concentrations in the trachea, the intestine, or both were normalized by the inhibition of acid sphingomyelinase activity and the concomitant normalization of ceramide concentrations. These findings suggest that ceramide plays a crucial role in inflammation and increased rates of cell death in several organs of cystic fibrosis mice.

2010 Becker KA, Riethmuller J, Luth A, Doring G, Kleuser B, Gulbins E. Acid sphingomyelinase inhibitors normalize pulmonary ceramide and inflammation in cystic fibrosis. Am J Resp Cell Mol 2010;. 42:716-724. [PubMed]
Employing genetic mouse models the authors have recently shown that ceramide accumulation is critically involved in the pathogenesis of cystic fibrosis (CF) lung disease. Genetic or systemic inhibition of the acid sphingomyelinase (Asm) is not feasible for treatment of patients or might cause adverse effects. Thus, a manipulation of ceramide specifically in lungs of CF mice must be developed. They tested whether inhalation of different acid sphingomyelinase inhibitors does reduce Asm activity and ceramide accumulation in lungs of CF mice. The efficacy and specificity of the drugs was determined. Ceramide was determined by mass spectrometry, DAG-kinase assays, and fluorescence microscopy. They determined pulmonary and systemic Asm activity, neutral sphingomyelinase (Nsm), ceramide, cytokines, and infection susceptibility. Mass spectroscopy, DAG-kinase assays, and semiquantitative immune fluorescence microscopy revealed that a standard diet did not influence ceramide in bronchial respiratory epithelial cells, while a diet with Peptamen severely affected the concentration of sphingolipids in CF lungs.

Inhalation of the Asm inhibitors amitriptyline, trimipramine, desipramine, chlorprothixene, fluoxetine, amlodipine, or sertraline restored normal ceramide concentrations in murine bronchial epithelial cells, reduced inflammation in the lung of CF mice and prevented infection with Pseudomonas aeruginosa. All drugs showed very similar efficacy. Inhalation of the drugs was without systemic effects and did not inhibit Nsm.

The authors conclude that the present findings, employing several structurally different Asm inhibitors, identify Asm as primary target in the lung to reduce ceramide concentrations. They suggest that inhaling an Asm inhibitor may be a beneficial treatment for CF, with minimal adverse systemic effects.

2010 Becker KA, Grassme H, Zhang Y, Gulbins E. Ceramide in Pseudomonas aeruginosa infections and cystic fibrosis. [Review] Cell Physiol Biochem 2010; 26:57-66. [PubMed]
The authors discuss recent findings related to the role of sphingolipids, in particular ceramide, in cystic fibrosis and the bacterial infections associated with that disease. Ceramide accumulates in the lungs of cystic fibrosis mice and causes pulmonary inflammation, infection, and cell death, events that are corrected by the genetic deletion or pharmacological inhibition of acid sphingomyelinase; this inhibition normalizes ceramide concentrations in murine models of cystic fibrosis. Also initial clinical studies suggest that pharmacological inhibition of acid sphingomyelinase may be a novel strategy for treating patients with cystic fibrosis.

2011 Dechecchi MC, Nicolis E, Mazzi P, Cioffi F, Bezzerri V, Lampronti I, Huang S, Wiszniewski L, Gambari R, Scupoli MT, Berton G, Cabrini G. Modulators of sphingolipid metabolism reduce lung inflammation. Am J Resp Cell Mol 2011; 45:825-833. [PubMed]
Both miglustat and amitriptyline reduced the immune response, an effect that paralleled a decrease in the P. aeruginosa-induced accumulation of ceramide. Miglustat (100 mg/kg), given to C57BL/6 mice once daily for a period of 3 consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the number of neutrophils recruited in the airways and the expression of the keratinocyte-derived chemokine in lung extracts.

Collectively, these results indicate that targeting the metabolism of sphingolipids can down-modulate the recruitment of neutrophils into the lung.

2011 Wojewodka G, DE Sanctis JB, Radzioch D. Ceramide in cystic fibrosis: a potential new target for therapeutic intervention. J Lipids 2011; 2011:674968. Epub 2010 Dec 28. [PubMed] [A full review article the text of which is available to download via PubMed].
This group has recently found reduced levels of ceramides in CF patients and mice. Ceramides are sphingolipids involved in the structure of cell membranes but also participate in the inflammatory response, in cell signalling through membrane microdomains (lipid rafts), and in apoptosis. These characteristics of ceramides make them strong candidates for therapeutic intervention in CF. As more studies have come to evaluate the role of ceramide in CF, conflicting results have been described.

This paper discusses various views regarding the potential role of ceramide in CF, summarizes methods of ceramide detection and their role in the regulation of cellular and molecular processes.

2011 Yang Y, Uhlig S. The role of sphingolipids in respiratory disease. Ther Adv Respir Dis 2011;5:325-344.[PubMed]
Sphingolipids form a broad class of lipids with diverse functions ranging from membrane constituents to intracellular second messengers and extracellular mediators. They can be rapidly generated or converted into each other and they play pivotal roles in various cellular processes, many of which are broadly associated with inflammation and apoptosis. Among the numerous sphingolipids, ceramide and sphingosine-1-phosphate (S1P) have received the greatest attention. Ceramide is a hydrophobic molecule that is increased in the lungs of patients with cystic fibrosis and chronic obstructive pulmonary disease (COPD). Ceramide is the eponym for ceramide-rich membrane platforms. that need to form as a prerequisite to the uptake of several microorganisms including Pseudomonas aeruginosa, and as a prerequisite to many signaling processes including apoptosis and increased vascular permeability. Accordingly, abnormal amounts of enzymes involved in the synthesis of ceramide, such as neutral or acid sphingomyelinase, are found in emphysematic smokers and in patients with severe sepsis, and are considered as novel pharmacological targets. S1P acts as an extracellular mediator that opposes several actions of ceramide and acts by binding to G-protein coupled S1P receptors (S1P(1)-S1P(5)). Of particular interest are S1P(1) receptors that enhance vascular barrier functions and are antiapoptotic. Therefore, S1P(1)-receptor ligands are suggested as novel drugs for COPD and acute lung injury. S1P is a potent chemotaxin for many leukocytes, it organizes lymphocyte trafficking and is involved in several key symptoms of asthma such as airway hyperresponsiveness and pulmonary eosinophil sequestration. S1P is formed by sphingosine kinases that have been identified as possible drug targets for the treatment of asthma.

Based on these findings, several new drugs have recently been developed to specifically target sphingomyelinases, sphingosine kinases and S1P receptors for the treatment of COPD, cystic fibrosis, asthma and acute lung injury.

2012 Becker KA, Henry B, Ziobro R, Tummler, Gulbins E, Grassme H. Role of CD95 in pulmonary inflammation and infection in cystic fibrosis. J Mol Med (Berl). 2012 [PubMed]
This study found that Cftr deficiency in mice results in the upregulation and activation of CD95. CD95 activation is caused by increased ceramide concentrations in cystic fibrosis lungs, as revealed by genetic modifications that normalize pulmonary ceramide concentrations. The activation of CD95 in cystic fibrosis lungs further increases pulmonary ceramide levels and results in a vicious feedback cycle of CD95 activation and ceramide accumulation. Genetic studies reveal that CD95 is crucially involved in the induction of aseptic inflammation, an increase in the bronchial cell death rate, and an increased susceptibility to infection of Cftr-deficient mice. All of these pathologies are partially corrected by heterozygosity of CD95 in Cftr-deficient mice.

These findings identify CD95 as an important regulator of lung functions in cystic fibrosis and suggest that CD95 may be a novel target for treating cystic fibrosis.

2013 Nahrlich L. Mainz JG. Adams C. Engel C. Herrmann G. Icheva V. Lauer J. Deppisch C. Wirth A. Unger K. Graepler-Mainka U. Hector A. Heyder S. Stern M. Doring G. Gulbins E. Riethmuller J. Therapy of CF-patients with amitriptyline and placebo–a randomised, double-blind, placebo-controlled phase IIb multicenter, cohort-study. Cell Physiol Biochem 2013; 31:505-12. [PubMed]
Several recent studies revealed an accumulation of ceramide in bronchial, tracheal and intestinal epithelial cells of mice and patients with cystic fibrosis (CF). Normalization of ceramide concentrations in lungs of CF mice employing the functional acid sphingomyelinase inhibitor amitriptyline also normalized mucociliary clearance, chronic inflammation and infection susceptibility to pulmonary P. aeruginosa in these mice.

To test for a beneficial effect of amitriptyline in vivo, the authors performed a phase IIb randomised, double-blind, placebo-controlled study. Twenty-one CF patients were treated with 25 mg/d amitriptyline twice daily for 28 days. The placebo consisted of 19 patients and was also treated twice per day. The primary endpoint was the change in lung function in the intention-to-treat (ITT) population. Secondary endpoints were ceramide levels in epithelial cells and safety.

After treatment, forced expiratory volume in 1 sec predicted (FEV1) increased 6.3 +/- 11.5% (p=0.08) in the ITT population (36 of 40 CF patients) and 8.5 +/- 10% (p=0.013) in the per protocol (PP) population (29 of 40 patients). Ceramide levels decreased in nasal epithelial cells after amitriptyline treatment. Amitriptyline had no severe and only mild and mostly transient adverse effects, i.e. xerostomia and tiredness.

The authors concluded amitriptyline was is safe in CF-patients, increases FEV1 and reduces ceramide in lung cells of CF patients. There are no subsequent trials supporting the use of the drug in people with cystic fibrosis

Antisense oligonucleotide splicing modulatiom

Yifat S OrenOfra Avizur-BarchadEfrat Ozeri-GalaiRenana ElgrabliMeital R SchirelmanTehilla BlinderChava D StampferMerav OrdanOnofrio LaselvaMalena Cohen-CymberknohEitan KeremChristine E BearBatsheva Kerem  Antisense oligonucleotide splicing modulation as a novel Cystic Fibrosis therapeutic approach for the W1282X nonsense mutationJ Cyst Fibros 2022 Jul;21(4):630-636.doi: 10.1016/j.jcf.2021.12.012. Epub 2021 Dec 28.    34972649     Free article
Background: Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23.
Methods: ∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient.
Results: ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient.

Conclusion: The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.

Yifat S Oren is in with Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, Israel; SpliSense Therapeutics, Jerusalem, Givat Ram, Israel.

Ataluran (PTC124)

2000 Wilschanski M, Famani C, Blau H, Rivlin J, Augarten A, Vital A, Kerem B, Kerem E. A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations. Am J Resp Crit Care 2000; 161:860-865. [PubMed]

Michael Wilschanski

This study, by Michael Wilschanski and colleagues from Haddash University Hospital, Israel, was the first to determine if gentamicin in vivo can activate mutant CFTR in CF patients carrying stop mutations as had been suggested by Howard et al, (1996) and Bedwell et al, (1997) (both abstracts below).

Nine people with CF carrying stop mutations received gentamicin nasal drops for 14 days. The abnormal nasal potential difference improved after the gentamicin treatment suggesting that chloride transport had increased. The authors concluded that gentamicin may influence the underlying chloride transport abnormality in patients with CF carrying stop mutations (i.e. those mutations containing an X).

Aminoglycoside antibiotics can apparently increase the frequency of erroneous insertion of nonsense codons hence permitting the translation of CFTR alleles carrying missense mutations to continue reading to the end of the gene. It is appropriate that this study came from Israel as 64% of people with CF that country have at least one stop mutation.

– Previously Howard and co-workers had demonstrated in cells carrying CFTR nonsense mutations, that gentamicin induced a dose-dependent increase in expression of full-length CFTR. (Howard M, Frizzell RA, Bedwell DM. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nature Med 1996; 2:467-469. [PubMed]). Subsequently, Bedwell and co-workers showed in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation, that gentamicin was capable of restoring CFTR expression on the apical membrane (Bedwell DM, Keanjak A, Bebok Z, Bubien JK, Tousson A, Clancy JP, Sorscher EJ. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nature Med 1997; 3:1280-1284. [PubMed]). Intravenous gentamicin was also capable of producing small increases in CFTR conductance as judged by nasal PD measurements.
(Clancy JP, Bebok Z, Ruiz F, King C Jones J, Walker L, Hong J, Wing L, Macaluso M, Lyrene R, Sorscher EJ, Bedwell DM. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis. Am J Resp Crit Care 2001; 163: 1683-1692.[PubMed]).

Further work from Israel on gentamicin and stop mutations.

2003 Wilschanski M, Yahav Y, Yaacov Y, Blau H, Bentur L, Rivlin J. Aviram M, Bdolah-Abram T, Bebok Z, Shushi L, Kerem B, Kerem E. Gentamicin induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations. N Eng J Med 2003; 349:1433-41. [PubMed].
In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the DeltaF508 mutation received nasal gentamicin drops or placebo for two consecutive periods of 14 days. The gentamicin treatment caused a significant reduction in basal nasal potential difference in the 19 patients carrying one or two stop mutations (from -45 (+/-8) to -34 (+/-11) mV, P=0.005) and a significant response to chloride-free isoproterenol solution (from 0 (+/-3.6) to -5 (+/-2.7) mV, P<0.001). Also after gentamicin treatment, there was a significant increase in peripheral and surface staining for CFTR in the nasal epithelial cells of the patients carrying stop mutations.

– So in patients with CF, who have premature stop codons, gentamicin was confirmed as causing translational “read through,” resulting in the expression of full-length CFTR protein at the apical cell membrane, and corrected towards normal the typical electrophysiological abnormalities caused by CFTR dysfunction. Subsequently another compound, PTC 124, seemed to do the job more efficiently and went into clinical trials. Identified as PTC 124 (later Ataluren), the new chemical entity selectively induces ribosomal read through of premature but not normal termination codons.

2007 Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, Sweeney HL. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447 (7140):87-91. [PubMed]

Michael Welsh

PTC124 selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. The drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options including muscular dystrophy and cystic fibrosis.

2007 Linde L, Boelz S, Nissim-Rafinia M, Oren YS, Wilschanski M, Yaacov Y, Virgilis D, Neu-Yilik G, Kulozik AE, Kerem E, Kerem B. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest 2007; 117:683-692. [PubMed].
Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously the authors have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here the authors demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. They further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Their results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS.

Importantly, their results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin.

The authors suggest that their results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

2007 Clancy JP, Rowe SM, Bebok Z, Aitken ML, Gibson R, Zeitlin P, Berclaz P, Moss R, Knowles MR, Oster RA, Mayer-Hamblett N, Ramsey B. No detectable improvements in cystic fibrosis transmembrane conductance regulator by nasal aminoglycosides in patients with cystic fibrosis with stop mutations. Am J Resp Cell Mol 2007; 37:57-66. [PubMed].
A US multicenter study was conducted in two cohorts of patients with CF, those heterozygous for stop mutations in the CFTR gene and those without nonsense mutations, to investigate the effects of both gentamicin and tobramycin administered over a 28-day period on sequential nasal potential difference and airway cell immunofluorescence endpoints. Eleven patients with CF who had stop mutations were enrolled in a randomized, double-blinded, crossover fashion to receive each drug, while 18 subjects with CF without stop mutations were randomized 1:1 in a parallel fashion to receive one drug. After demonstration of drug delivery, neither aminoglycoside produced detectable changes in nasal ion transport or CFTR localization in brushed cells from either study group. These results with first-generation suppressive agents suggest the need for improved drug delivery methods and/or more potent suppressors of nonsense mutations to confer CFTR correction in subjects with CF heterozygous for nonsense mutations. The study provides valuable information on parameters of the nasal potential difference measurements for use in future multicenter clinical trials.

– The results appear to conflict with earlier work of Wilschanski et al (2000 and 2003 above) but variability of response has been attributed to the efficiency of nonsense-mediated mRNA decay which may vary and hence have an important role in governing the response to treatments aiming to suppress nonsense mutations. (2007 Linde L, et al. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest 2007; 117:683-692.[PubMed]).

2007 Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP. Restoration of W1282X CFTR activity by enhanced expression. Am J Resp Cell Mol Biol 2007; 37:347-356. [PubMed]
Various aminoglycosides induce “translational readthrough” of premature stop codons and have been shown to restore full-length functional protein in a number of preclinical and clinical settings. The authors studied two well-described premature termination codons found in the distal open reading frame of CFTR, W1282X and R1162X, expressed in polarizing and non-polarizing cells.

Their findings indicate that W1282X CFTR-expressing cells demonstrate significantly greater CFTR activity when over-expressed compared with R1162X CFTR cells, even when truncated protein is the predominant form. In addition, their results show that the combination of stimulated expression and stop codon suppression produces additive effects on CFTR-mediated ion transport.

– These findings are considered to provide evidence that W1282X CFTR exhibits membrane localization and retained chloride channel function after enhanced expression, and suggest that patients harbouring this mutation may be more susceptible to CFTR rescue.

2007 Sermet-Gaudelus I, Renouil M, Fajac A, Bidou L, Parbaille B, Pierrot S, Davy N, Bismuth E, Reinert P, Lenoir G. Les BMC Medicine 2007; 5:5. In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study. [PubMed]
A pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits. A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. The authors investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment.
After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations.

The authors concluded suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.

2008 Kerem E, Hirawat S, Armoni S, Yaakov Y, Shoseyov D, Cohen M, Nissim-Rafinia M, Blau H, Rivlin J, Aviram M, Elfring GL, Northcutt VJ, Miller LL, Kerem B, Wilschanski M. Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial. Lancet 2008; 372:719-727. [PubMed]

EItan Kerem

PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment.
The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle.

Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle’s treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle’s treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded.

The authors concluded that in patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

2010 Sermet-Gaudelius I, Boeck KD, Casimir GJ, Vermeulen F, leal T, Mogegnet A, Roussel D, Fritch J, hanssens L, Hirawat S, Miller NL, Constantine S, Reha A, Ajayi T, Elfring GL, Miller LL. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care 2010; 182:1262-1272.[PubMed]
A study to evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF. Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles. The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport.

Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults.

The authors concluded that in children with nonsense mutations CF, Ataluren can induce functional CFTR production and is well tolerated.

2011 Wilschanski M, Miller LL, Shoseyov D, Blau H, Rivlin J, Aviram M, Cohen M, Armoni S, Yaakov Y, Pugatch T, Cohen-Cymberknoh M, Miller NL, Reha A, Northcutt VJ, Hirawat S, Donnelly K, Elfring GL, Ajayi T, Kerem E. Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis. Eur Respir J 2011; 38:59-69. [PubMed]
Ataluren (PTC124) allows ribosomal read through of premature stop codons in mRNA. The authors evaluated drug activity and safety in patients with nonsense CF mutations who took ataluren three times daily for 12 weeks at either a lower dose (4, 4 and 8 mg.kg(-1)) or higher dose (10, 10 and 20 mg.kg(-1)). The study enrolled 19 patients (10 males and nine females aged 19-57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.

– Apparently the results in Duchenne muscular dystrophy have been disappointing and the benefits in CF were less evident in one study. However, further results in cystic fibrosis are awaited.

In June 2011 PTC therapeutics announced the results from a large multicentre Phase 3 study of ataluren (This section taken directly from http://ptct.client.share).(full reference below)

The data was presented at the ECFS Dublin Conference in 2012 by Michael Konstan.
This Phase 3 study, which was conducted across 11 countries, was a double-blind, placebo-controlled study comparing ataluren (n=116) to placebo (n=116) in CF patients. The primary endpoint, the relative change from baseline in %-predicted FEV1 at 48 weeks, showed a positive trend favoring ataluren versus placebo, and a larger effect in the patients not receiving chronic inhaled antibiotics. The effect of inhaled antibiotics was largely attributable to the use of inhaled aminoglycosides. In the intent-to-treat population, there was a 3% difference in the relative change from baseline in %-predicted FEV1 between the ataluren and placebo groups at Week 48 (-2.5% change on ataluren vs. -5.5% change on placebo; p=0.124). An analysis of the relative change from baseline in %-predicted FEV1 across all post-baseline study visits demonstrated an average difference between ataluren and placebo of 2.5% (-1.8% average change on ataluren vs. -4.3% average change on placebo; p= 0.0478)

The study was stratified by age, baseline FEV1, and the use of chronic inhaled antibiotics. A statistically significant effect (p=0.0072) was seen between treatment and use of inhaled antibiotics at baseline, indicating that inhaled antibiotics was a significant confounder of the overall results. A substantial treatment effect was seen in the patients not receiving chronic inhaled antibiotics at baseline; the Week 48 difference between the ataluren and placebo arms in FEV1 was 6.7% (-0.2% change on ataluren vs. -6.9% change on placebo). The secondary endpoint, the rate of pulmonary exacerbations (ie, the number of pulmonary exacerbations in 48 weeks) also showed a positive trend in favor of ataluren, with the rate in the ataluren group being 23% lower than the placebo group (p=0.0992). In the patients not receiving chronic inhaled antibiotics, the pulmonary exacerbation rate in the ataluren group was 43% lower than the rate in the placebo group. These results show a consistent treatment effect of ataluren on both pulmonary function and exacerbation rates. In patients not also on inhaled antibiotics there was a 43% reduction in pulmonary exacerbations.

In 2012 the FDA and the European Commission granted Ataluren Orphan Drug status for the treatment of nonsense mutation cystic fibrosis and nonsense mutation Duchenne and Becker muscular dystrophy.

2014 Kerem E, Konstan MW, De Boeck K, Accurso FJ, Sermet-Gaudelus I, Wilschanski M, Elborn JS, Melotti P, Bronsveld I, Fajac I, Malfroot A, Rosenbluth DB, Walker PA, McColley SA, Knoop C, Quattrucci S, Rietschel E, Zeitlin P, Barth J, Elfring GL, Welch EM, Branstrom A, Spiegel RJ, Peltz SW, Ajayi T, Rowe SM; for the Cystic Fibrosis Ataluren Study Group.Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Respir Med. 2014 May 15. pii: S2213-2600(14)70100-6. doi: 10.1016/S2213-2600(14)70100-6. [Epub ahead of print][PubMed]

This is the published summary of the Phase III study described above and the conclusions are the same

– Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin – an effect noted in a post-hoc analysis.

There has been some dispute as to how the drug was tested questioning whether PTC 124 did actually promote read through of stop mutations (Auld DS et al. Proc Natl Acad Sci USA 2010;107:4878-4883. [PubMed] fhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841876/pdf/pnas.0909141107.pdf; Thorne N et al. Chem Biol 2010; 17:646-657. [PubMed]http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925662/pdf/nihms217027.pdf). These suggestions were disputed (Peltz et al. Proc Nat Acad Sci USA 2009;106:E64http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700894/pdf/zpqe64.pdf) although a second study testing PTC 124 across a range of assays found no evidence of read through McElroy et al. PLos Biol 2013;11:e1001593http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692445/pdf/pbio.1001593.pdf) and problems relating to firefly luciferase used in the assay were discussed (Roberts RG. PLos Biol 2013;11:e1001458http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692443/pdf/pbio.1001458.pdf

(This account based in part on “Ataluran: reading through the controveries”. Courtesy CFNN (Cystic Fibrosis News Network), July 8,2014 – with permission)

2014 Kerem E. Konstan MW. De Boeck K. Accurso FJ. Sermet-Gaudelus I. Wilschanski M. Elborn JS. Melotti P. Bronsveld I. Fajac I. Malfroot A. Rosenbluth DB. Walker PA. McColley SA. Knoop C. Quattrucci S. Rietschel E. Zeitlin PL. Barth J. Elfring GL. Welch EM. Branstrom A. Spiegel RJ. Peltz SW. Ajayi T. Rowe SM. Cystic Fibrosis Ataluren Study Group. Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Respir Med 2014; 2(7):539-47.[PubMed]
A randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged > 6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV1] > 40% and < 90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV1. The primary endpoint was relative change in percent-predicted FEV1 from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205.
Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV1 did not differ significantly between ataluren and placebo at week 48 (-2.5% vs -5.5%; difference 3.0% [95% CI -0.8 to 6.3]; p=0.12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0.77 [95% CI 0.57-1.05]; p=0.0992).
However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5.7% difference (95% CI 1.5-10.1) in relative change from baseline in percent-predicted FEV1 between the ataluren and placebo groups at week 48 (-0.7% [-4.0 to 2.1] vs -6.4% [-9.8 to -3.7]; nominal p=0.0082), and fewer pulmonary exacerbations in the ataluren group (1.42 events [0.9-1.9] vs 2.18 events [1.6-2.7]; rate ratio 0.60 [0.42-0.86]; nominal p=0.0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group.
The authors concluded that although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, but it might be beneficial for patients not taking chronic inhaled tobramycin.

– As mentioned above there has been some difference of opinion as to the action of ataluren and as to whether the drug actually does promote read through of nonsense mutations. It has been suggested that the assay using luciferase as the reporter gene when positive, rather than indicating transcription, merely indicates direct stimulation of the reporter gene.

2014 Lentini L. Melfi R. Di Leonardo A. Spinello A. Barone G. Pace A. Palumbo Piccionello A. Pibiri I. Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol Pharm 2014; 11(3):653-64. [PubMed]
The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis, where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC read through even though its target has yet to be identified.

In the lack of a general consensus about its mechanism of action, the authors experimentally tested the ability of PTC124 to promote the read through of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harbouring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon.

– Apparently the conclusion is that, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific read through of internal TGA premature stop codons.

2016 Shoseyov D, Cohen-Cymberknoh M, Wilschanski M. Ataluren for the treatment of cystic fibrosis. Expert Rev Respir Med. 2016 Feb 24:1-5. [Epub ahead of print] [PubMed]

David Shoseyov

Ataluren is orally bioavailable and was shown to be effective in Cystic Fibrosis. Phase I and II studies established the safety and dosing regimens for Ataluren. The results of a short study showed modest improvements in pulmonary function and a reduction in quantitative cough assessment. There was improvement in nasal potential difference and nasal epithelial CFTR protein. In a phase III trial this effect was not observed in patients that were concomitantly treated with tobramycin inhalation. Following these positive findings, a multinational Phase III placebo-controlled efficacy trial is currently underway.

– This paper sums up the present situation regarding the use of ataluren for people with CF and stop mutations.

Dr David Shoseyov (figure) is Senior Physician and Advisor on Pediatric Lung Disease, Hadassah Medical Center, Jerusalem and Chair of Israeli Cystic Fibrosis Council’s Medical Advisory Committee

2017 Aslam AA, Higgins C, Sinha IP, Southern KW. Ataluren and similar compounds (specific therapies for premature termination codon class I mutations) for cystic fibrosis. Cochrane Database Syst Rev. 2017 Jan 19;1:CD012040. doi: 10.1002/14651858.CD012040.pub2. [Pubmed]
Objectives were to evaluate the benefits and harms of ataluren and similar compounds on clinically important outcomes in people with cystic fibrosis with class I mutations (premature termination codons). Searches identified 28 references to eight trials; five trials were excluded.

The authors concluded there is currently insufficient evidence to determine the effect of ataluren as a therapy for people with cystic fibrosis with class I mutations. Future trials should carefully assess for adverse events, notably renal impairment and consider the possibility of drug interactions. Cross-over trials should be avoided given the potential for the treatment to change the natural history of cystic fibrosis.

~A useful review on the present state of this treatment of these class 1 mutations.

2018 Pranke I, Bidou L, Martin N, Blanchet S, Hatton A, Karri S, Cornu D, Costes B, Chevalier B, Tondelier D, Girodon E, Coupet M, Edelman A, Fanen P, Namy O, Sermet-Gaudelus I, Hinzpeter A. Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons. ERJ Open Res.2018 Feb 23;4(1). pii: 00080-2017. doi: 10.1183/23120541.00080-2017. eCollection 2018 Jan.Full text available. [PubMed]
Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein.

The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Non-functional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient’s genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.

– A helpful review of the present situation regarding PTCs from a leading French group

Dr. Iwona Pranke is Post Doctoral Researcher at the Institut Necker, Paris

Konstan MW, VanDevanter DR, Rowe SM, Wilschanski M, Kerem E, Sermet-Gaudelus I, DiMango E, Melotti P, McIntosh J, De Boeck K; ACT CF Study Group. Efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis not receiving chronic inhaled aminoglycosides: The international, randomized, double-blind, placebo-controlled Ataluren Confirmatory Trial in Cystic Fibrosis (ACT CF). J Cyst Fibros. 2020 Jan 23. pii: S1569-1993(20)30030-8. doi: 10.1016/j.jcf.2020.01.007. [Epub ahead of print] [Pubmed]

Michael Konstan

Ataluren was developed for potential treatment of nonsense-mutation cystic fibrosis (CF). A previous phase 3 ataluren study failed to meet its primary efficacy endpoint, but post-hoc analyses suggested that aminoglycosides may have interfered with ataluren’s action. Thus, this subsequent trial (NCT02139306) was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation CF not receiving aminoglycosides.
Eligible subjects with nonsense-mutation CF (aged ≥6 years; percent predicted (pp) FEV1 ≥40 and ≤90) from 75 sites in 16 countries were randomly assigned in double-blinded fashion to receive oral ataluren or matching placebo thrice daily for 48 weeks. The primary endpoint was absolute change in average ppFEV1 from baseline to the average of Weeks 40 and 48.

279 subjects were enrolled; 138 subjects in the ataluren arm and 136 in the placebo arm were evaluable for efficacy. Absolute ppFEV1 change from baseline did not differ significantly between the ataluren and placebo groups at Week 40 (-0.8 vs -1.8) or Week 48 (-1.7 vs -2.4). Average ppFEV1 treatment difference from baseline to Weeks 40 and 48 was 0.6 (95% CI -1.3, 2.5; p = 0.54). Pulmonary exacerbation rate per 48 weeks was not significantly different (ataluren 0.95 vs placebo 1.13; rate ratio p = 0.40). Safety was similar between groups. No life-threatening adverse events or deaths were reported.

Neither ppFEV1 change nor pulmonary exacerbation rate over 48 weeks were statistically different between ataluren and placebo groups. Development of a nonsense-mutation CF therapy remains elusive.

– Disappointing result when earlier work seemed so promising.

Dr Michael Konstan is the Gertrude Lee Chandler Tucker Professor of Pediatrics, Department of Pediatrics, School of Medicine, Case Western Reserve University

Martin Y NgHong LiMikel D GhelfiYale E GoldmanBarry S CoopermanAtaluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.   Proc Natl Acad Sci U S A  2021 Jan 12;118(2):e2020599118.doi: 10.1073/pnas.2020599118.Free article   [Pubmed]

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers.
Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously, we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell’s protein synthesis machinery.

Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near cognate tRNA ± TRIDs.

Martin Y Ng is at the Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104.

Sylwia MichorowskaAtaluren-Promising Therapeutic Premature Termination Codon Readthrough FrontrunnerPharmaceuticals (Basel). 2021 Aug 9;14(8):785.doi: 10.3390/ph14080785. [Pubmed]Free PMC article

Sywia Michorowska

Around 12% of hereditary disease-causing mutations are in-frame nonsense mutations. The expression of genes containing nonsense mutations potentially leads to the production of truncated proteins with residual or virtually no function. However, the translation of transcripts containing premature stop codons resulting in full-length protein expression can be achieved using readthrough agents. Among them, only ataluren was approved in several countries to treat nonsense mutation Duchenne muscular dystrophy (DMD) patients.

This review summarizes ataluren’s journey from its identification, via first in vitro activity experiments, to clinical trials in DMD, cystic fibrosis, and aniridia. Additionally, data on its pharmacokinetics and mechanism of action are presented. The range of diseases with underlying nonsense mutations is described for which ataluren therapy seems to be promising. What is more, experiments in which ataluren did not show its readthrough activity are also included, and reasons for their failures are discussed.

Dr Sylwia Michorowska is in the Department of Bioanalysis and Drug Analysis, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland.

– The full article is an interesting very detailed review of the development and clinical trials of ataluren, initially known as PTC124, in muscular dystrophy, cystic fibrosis  and various metabolic disorders.

Curcumin2004 Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, Glöckner-Pagel J, Canny S, Du K, Lukacs GL, Caplan MJ. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science. 2004 Apr 23;304(5670):600-2. Free full text [PubMed]

       Marion Egan

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, DeltaF508, results in the production of a misfolded CFTR protein that is retained in the endoplasmic reticulum and targeted for degradation. Curcumin is a nontoxic Ca-adenosine triphosphatase pump inhibitor that can be administered to humans safely. Oral administration of curcumin to homozygous DeltaF508 CFTR mice in doses comparable, on a weight-per-weight basis, to those well tolerated by humans corrected these animals’ characteristic nasal potential difference defect. These effects were not observed in mice homozygous for a complete knockout of the CFTR gene. Curcumin also induced the functional appearance of DeltaF508 CFTR protein in the plasma membranes of transfected baby hamster kidney cells. The authors suggested that curcumin treatment may be able to correct defects associated with the homozygous expression of DeltaF508 CFTR.- This first report caused great excitement but the results were not confirmed. It was suggested that this may have been due to slight genetic differences in the CF miceMarion Egan is Professor of Pediatrics (Respiratory) and of Cellular And Molecular Physiology; Director, Cystic Fibrosis Center; Vice Chair for Research, Department of Pediatrics Yale School of Medcine– It was no surprise that this publication led to a great deal of interest and was widely reported in the media – for example “Daily curcumin slashed the death rate of CF-stricken mice”! Unfortunately these results could not be repeated (Song Y et al. J Biol Chem 2004; 279:40629-40633; Dragomir A et al. 2004. [PubMed]; Loo TW et al. 2004.[PubMed]).The subject of curcumin has been reviewed in detail in many publications elsewhere. It is a natural polyphenol used in ancient Asian medicine. Since the first article referring to the use of curcumin to treat human disease was published in The Lancet in 1937, more than 2,600 research studies using curcumin or turmeric have been published in English language journals (Strimpakos AS. Sharma RA. Curcumin: preventive and therapeutic properties in laboratory studies and clinical trials. Antioxid Redox Sign 2008; 10:511-545. [PubMed]). Although others could not reproduce these results of Egan et al in CF patients and a Phase 1 trial in CF patients did not show correction of CFTR there is continuing interest in the role of curcumin in the treatment of cystic fibrosis (Colas J et al. Disruption of cyto-keratin-8 interaction with F508-CFTR corrects its functional defect. Hum Mol Genet 2012; 21:623-634. [PubMed]).2004 Song Y, Sonawane ND, Salinas D, Qian L, Pedemonte N, Galietta LJ, Verkman AS.Evidence against the rescue of defective DeltaF508-CFTR cellular processing by curcumin in cell culture and mouse models.J Biol Chem.2004 Sep 24;279(39):40629-33. Epub 2004 Jul 26. [PubMed]
Curcumin, the yellow colored component of the spice turmeric, has been reported to rescue defective DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) cellular processing in homozygous mutant mice, restoring nasal potential differences and improving survival (Egan, M. E., Pearson, M., Weiner, S. A., Rajendran, V., Rubin, D., Glockner-Pagel, J., Canny, S., Du, K., Lukacs, G. L., and Caplan, M. J. (2004) Science 304, 600-602). Because of the implied potential use of curcumin or similar compounds in the therapy of cystic fibrosis caused by the DeltaF508 mutation, we tried to reproduce and extend the pre-clinical data of Egan et al. Fluorometric measurements of iodide influx in Fischer rat thyroid cells expressing DeltaF508-CFTR showed no effect of curcumin (1-40 microm) when added for up to 24 h prior to assay in cells grown at 37 degrees C. Controls, including 27 degrees C rescue and 4 mm phenylbutyrate at 37 degrees C, were strongly positive. Also, curcumin did not increase short circuit current in primary cultures of a human airway epithelium homozygous for DeltaF508-CFTR with a 27 degrees C rescue-positive control. Nasal potential differences in mice were measured in response to topical perfusion with serial solutions containing amiloride, low Cl-, and forskolin. Robust low Cl- and forskolin-induced hyperpolarization of 22 +/- 3 mV was found in wild type mice, with 2.1 +/- 0.4 mV hyperpolarization in DeltaF508 homozygous mutant mice. No significant increase in Cl-/forskolin hyperpolarization was seen in any of the 22 DeltaF508 mice studied using different curcumin preparations and administration regimens, including that used by Egan et al. Assay of serum curcumin by ethyl acetate extraction followed by liquid chromatography/mass spectrometry indicated a maximum serum concentration of 60 nm, well below that of 5-15 microm, where cellular effects by sarcoplasmic/endoplasmic reticulum calcium pump inhibition are proposed to occur. Our results do not support further evaluation of curcumin for cystic fibrosis therapy.- So these results failed to confirm the effect of circumin in rescuing defective dF508 CFTR. The authors conclude “Our results do not support further evaluation of curcumin as a therapy of CF caused by the ΔF508 mutation”.From the Department of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA.

2005 Berger AL. Randak CO. Ostedgaard LS. Karp PH. Vermeer DW. Welsh MJ. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. J Biol Chem 2005; 280:5221-6. [PubMed]
Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis. Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, the authors tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. They found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels.

– There is continuing interest in the role of curcumin in the treatment of cystic fibrosis (Colas J et al. Disruption of cyto-keratin-8 interaction with F508-CFTR corrects its functional defect. Hum Mol Genet 2012; 21:623-634.[PubMed]).

2006 Lipecka J, Norez C, Bensalem N, Baudouin-Legros M, Planelles G, Becq F, Edelman A, Davezac N. Rescue of DeltaF508-CFTR (cystic fibrosis transmembrane conductance regulator) by curcumin: involvement of the keratin 18 network. J Pharmacol Exp Ther 2006; 317:500-505. [PubMed]
Recently, curcumin was shown to rescue DeltaF508-CFTR localization and function in mice (Egan et al. Science 2004; 304:600-602 above). In previous work from the present authors, the keratin 18 (K18) network was implicated in DeltaF508-CFTR trafficking. Here, the authors hypothesize that curcumin could restore a functional DeltaF508-CFTR to the plasma membrane acting via the K18 network. They proposed K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.

– This study provides an explanation for the effect of curcumin but unfortunately the curcumin effect reported by Egan et al (2004 above) could not be reproduced by others although interest continued in the potential role of curcumin (Colas J et al. Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect. Hum Med Genet 2012; 21:623-634. [PubMed]).

2007 Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB.Bioavailability of curcumin: problems and promises. Mol Pharm. 2007 Nov-Dec;4(6):807-18. Epub 2007 Nov 14. [PubMed]
Curcumin, a polyphenolic compound derived from dietary spice turmeric, possesses diverse pharmacologic effects including anti-inflammatory, antioxidant, antiproliferative and antiangiogenic activities. Phase I clinical trials have shown that curcumin is safe even at high doses (12 g/day) in humans but exhibit poor bioavailability. Major reasons contributing to the low plasma and tissue levels of curcumin appear to be due to poor absorption, rapid metabolism, and rapid systemic elimination. To improve the bioavailability of curcumin, numerous approaches have been undertaken. These approaches involve, first, the use of adjuvant like piperine that interferes with glucuronidation; second, the use of liposomal curcumin; third, curcumin nanoparticles; fourth, the use of curcumin phospholipid complex; and fifth, the use of structural analogues of curcumin (e.g., EF-24). The latter has been reported to have a rapid absorption with a peak plasma half-life. Despite the lower bioavailability, therapeutic efficacy of curcumin against various human diseases, including cancer, cardiovascular diseases, diabetes, arthritis, neurological diseases and Crohn’s disease, has been documented. Enhanced bioavailability of curcumin in the near future is likely to bring this promising natural product to the forefront of therapeutic agents for treatment of human disease.

Dr Preetha Anand is from the Cytokine Research Laboratory and Pharmaceutical Development Center, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

2016 Day I, Shah K, Bradbury NA.Natural Compounds as Therapeutic Agents in the Treatment Cystic Fibrosis.
J Genet Syndr Gene Ther.
2016 Feb;7(1). pii: 284. Epub 2016 Jan 30.
[PubMed] Free PMC Article

               Neil Bradbury

The recent FDA approval of two drugs to treat the basic defect in cystic fibrosis has given hope to patients and their families battling this devastating disease. Over many years, with heavy financial investment from Vertex Pharmaceuticals and the Cystic Fibrosis Foundation, pre-clinical evaluation of thousands of synthetic drugs resulted in the production of Kalydeco and Orkambi. In view of the “egregious” cost of lumacaftor and ivacaftor (to quote Prof. Paul Quinton) the authors review alternative treatments which have been suggested to have a favourable effect on abnormal CFTR.

Yet, despite the success of this endeavor, many other compounds have been proposed as therapeutic agents in the treatment of CF. Of note, several of these compounds are naturally occurring, and are present in spices from the grocery store and over the counter preparations in health food stores. In this short review, the authors look at three such compounds, genistein, curcumin, and resveratrol,and evaluate the scientific support for their use as therapeutic agents in the treatment of patients with CF.

From the Dept. of Physiology and Biophysics, Chicago Medical School, North Chicago.

Professor Neil A Bradbury is corresponding author. His research interests lie in the area of regulation of membrane protein trafficking in polarized epithelia.

2017 Gonçalves C, Gomez JP, Même W, Rasolonjatovo B, Gosset D, Nedellec S, Hulin P, Huin C, Le Gall T, Montier T, Lehn P, Pichon C, Guégan P, Cheradame H, Midoux P. Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines.Eur J Pharm Biopharm. 2017 Aug;117:168-181. doi: 10.1016/j.ejpb.2017.04.015. Epub 2017 Apr 17.[PubMed]
Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. The authors reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx6-THF19-MeOx6) (TBCP2) can solubilize curcumin(Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, they provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and ΔF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on ΔF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl selective current at the membrane surface of F508del-CFTR cells.

The results show the great potential of the neutral amphiphilic triblock copolymer MeOx6-THF19-MeOx6 as carrier for curcumin in a Cystic Fibrosis context. They anticipate that other MeOxn-THFm-MeOxn copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients.

Dr Cristine Goncalves is a scientist at Centre de Biophysique Moléculaire, CNRS UPR4301 and Université d’Orléans, France.

2019 Chaudhary N, Ueno-Shuto K, Ono T, Ohira Y, Watanabe K, Nasu A, Fujikawa H, Nakashima R, Takahashi N, Suico MA, Kai H, Shuto T.Curcumin down-regulates toll-like receptor-2 gene expression and function in human cystic fibrosis bronchial epithelial cells.Biol Pharm Bull. 2019 Jan 10. doi: 10.1248/bpb.b18-00928. [Epub ahead of print] Free full text [PubMed]

     Niraj Chaudhary

In CF patients, up-regulation of toll-like receptor-2 (TLR2), a pattern recognition receptor that senses CF-pathogenic bacteria Staphylococcus aureus peptidoglycan (PGN), in airway epithelial cells is observed, and enhanced pro-inflammatory responses towards PGN may result in detrimental effects in CF patients. Here, the authors showed that curcumin, a well known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in CF bronchial epithelial cell line, CFBE41o- cells. Strong suppression of TLR2 gene and protein expression was observed at more than 40 μM of curcumin treatment in CFBE41o- cells. Consistent with decreased expression of TLR2, PGN-dependent IL-8 gene up-regulation was markedly reduced by 40 μM of curcumin treatment. Strong reductions of TLR2 gene expression and function were also observed in primary human CF bronchial epithelial cells, but not in human non-CF primary cells. Interestingly, curcumin treatment decreased nuclear expression of transcription factor specificity protein 1 (SP1), a factor that is critical for increased basal TLR2 expression in CF cell line and primary cells. Finally, curcumin-dependent SP1 reduction was diminished by anti-oxidant N-acetylcystein (NAC) and proteasomal inhibitor MG-132, suggesting the crucial roles of oxidative and proteasomal degradation pathways. Taken together, our study shows that curcumin down-regulates TLR2 gene expression and function in CF bronchial epithelial cells possibly by accelerating SP1 degradation via an oxidative process.

Dr Niraj Chaudhary in a scientist PhD student at Dept of Molecular Medicine, Kumanato University, Japan

– This paper describes clearly many of the biochemical mechanisms involved at a cellular level but does not deal with the earlier clinical work on CF by Egan and others.

There is currently little interest in the use of curcumin as such approaches have been overshadowed by the new modulators.


A selective P2Y2 agonist that stimulates ciliary beat frequency and Cl secretion in normal and CF airway epithelia enhanced by the effect of P2Y2 activation to also inhibit epithelial sodium transport. The early clinical trials were encouraging but the effect failed to achieve significance in the second Phase III study.

In January 2011 the CF Foundation announced that – “This Inspire Pharmaceuticals alternative chloride channel activator has been removed from the pipeline due to lack of efficacy”

2005 Deterding R, Retsch-Bogart G, Milgram L, Gibson R, Daines C, Zeitlin PL, Milla C, Marshall B, Lavange L, Engels J, Mathews D, Gorden J, Schaberg A, Williams J, Ramsey B. Safety and tolerability of denufosol tetrasodium inhalation solution, a novel P2Y2 receptor agonist: results of a phase 1/phase 2 multicenter study in mild to moderate cystic fibrosis. Pediatr Pulmonol 2005; 39:339-348. [PubMed]

Robin Deterding

Denufosol tetrasodium is a selective P2Y2 agonist that stimulates ciliary beat frequency and Cl secretion in normal and CF airway epithelia enhanced by the effect of P2Y2 activation to also inhibit epithelial sodium transport. Based on the results of this phase 1/phase 2 study, the Therapeutics Development Network (TDN) of the Cystic Fibrosis Foundation (CFF) and Inspire Pharmaceuticals, Inc., recently completed a multicenter, 28-day, phase 2 safety and efficacy clinical trial of denufosol inhalation solution in CF subjects with mild lung disease (Deterding RR et al. Am J Respir Crit Care Med 2007; 176:662-669 below; Kellerman D et al. Pulm Pharmacol Ther 2008; 21:600-607. [PubMed]).

2007 Deterding RR, Lavange LM, Engels JM, Mathews DW, Coquillette SJ, Brody AS, Millard SP, Ramsey BW, for the Cystic Fibrosis Therapeutics Development Network and the Inspire 08-103 Working Group. Phase 2 randomized safety and efficacy trial of nebulized denufosol tetrasodium in cystic fibrosis. Am J Resp Crit Care 2007; 176:362-369. [PubMed]
Denufosol tetrasodium is a selective P2Y(2) agonist that enhances mucosal hydration and mucus clearance by activating Cl(-) secretion and inhibiting epithelial Na(+) transport through a non-cystic fibrosis transmembrane conductance regulator mechanism in the lung. The study was a randomized, double-blind, multi-center, 28-day, phase 2 clinical trial of denufosol tetrasodium inhalation solution (20, 40, or 60 mg) versus placebo (normal saline). Denufosol patients (pooling active doses) had significantly higher changes from baseline in respiratory function than placebo patients at the end of the study.

– The authors concluded that Denufosol administered three times daily for 28 days appeared to be safe and well tolerated in this population with mild cystic fibrosis and provided preliminary evidence of potential benefit in lung function. This is one of a number of drugs showing promise to correct or reduce the effects of the basic defect on the airway surface liquid (also Deterding et al, 2005 above).

In June 2008, Inspire announced the results of its first Phase 3 trial with Denufosol (Tiger 1). The trial demonstrated statistical significance for its primary endpoint of change in FEV1 from baseline compared to placebo. By late 2010 Inspire had completed enrolment to a further Phase 111 trial (Tiger 2).

2008 Kellerman D, Rossi Mospan A, Engels J, Schaberg A, Gorden J, Smiley L. Denufosol: a review of studies with inhaled P2Y(2) agonists that led to Phase 3. [Review] Pulmon Pharmacol Therap 2008; 21:600-607.[PubMed]
The pharmacology of P2Y(2) agonists has been confirmed in several preclinical and clinical studies. Denufosol tetrasodium is a novel second-generation, metabolically stable, selective P2Y(2) receptor agonist currently in Phase 3 clinical development. In radio labelled deposition studies of P2Y(2) agonists in healthy non-smokers and smokers, approximately 7mg of a 40-mg nebulizer (PARI LC Star) load was deposited in the lungs. In a pharmacokinetic study in healthy volunteers, very limited systemic exposure was observed when doses of 200mg of denufosol were nebulized. Thus, it appears that high concentrations of denufosol can be achieved in the airways with very low systemic absorption. Denufosol has been generally well-tolerated in healthy volunteers and patients with CF. The most common adverse events were in the respiratory system, with cough having the highest frequency. Doses of 20-60mg have been evaluated in Phase 2 trials of up to 28 days duration, and superiority relative to placebo on FEV1 has been observed in patients with relatively normal lung function (FEV1 greater than or equal to 75% of predicted). The first Phase 3 trial is a comparison of denufosol 60mg and placebo in 350 patients with CF with FEV1 at study entry greater than or equal to 75% of predicted.

– Denufosol was one of the more hopeful new therapies with the particular aim of increasing hydration of the respiratory mucosa. However, the drug failed at the final Phase III trial for 48 weeks when the drug “did not improve pulmonary function or reduce the incidence of pulmonary exacerbations” so development was stopped (Ratjen F et al. J Cyst Fibros 2012. [PubMed]).

2010 Moss RB, Schaberg A, Tian W, Xue X, Ramsey B, Accurso FJ. Denufosol improved lung function and was well tolerated in adolescents with cystic fibrosis. NACFC Baltimore 2010. Pediatr Pulmonol 2010; Suppl 33: Abstract # 256
Analysis of an adolescent subgroup of the Tiger -1 trial 24 week trial followed by open label 24 weeks. Change FEV1 for denufosol group +112ml (+4%) compared with placebo group -10ml (-0.6%). At week 48 the denufosol group had increase in FEV1 +226ml (+8.8%) and the change from baseline FEF25-75 was +192ml (+10.5%). The treated and placebo groups were on a considerable amount of other therapy e.g. pulmozyme 81% vs 76%, macrolides 42/47, inhaled antibiotics 39/47. Approximately half the patients were chronically infected with P. aeruginosa.

2010 Accurso FJ. Denufosol efficacy in patients with minimally impaired baseline lung function and on minimal background therapy demonstrates its potential as an early intervention for cystic fibrosis lung disease. NACFC Baltimore 2010. Pediatr Pulmonol 2010; Suppl 33: Abstract # 226
Patients aged 5 years or older with FEV1> 75%. Subgroup from phase 3 Tiger-1 trial on only 0-2 background therapies. Denufosol demonstrated “clinically meaningful improvement relative to placebo in patients who had minimal background therapy in FEV1, FEV1%, FEF 25-75%, QOL respiratory domain. Highlight potential as an early intervention in CF.

Statement about the Denufosol Tetrasodium Clinical Program (from Inspire website November 2010)

TIGER-1, the first Phase 3 trial with denufosol for the treatment of CF, included a 24-week placebo-controlled portion, followed by a 24-week open-label safety extension. The placebo-controlled portion was a double-blind, randomized trial comparing 60 mg of denufosol to placebo, administered three times daily by jet nebulizer, in 352 patients with mild cystic fibrosis lung disease (baseline FEV1 = 75% of predicted normal).

Inspire is currently conducting TIGER-2, its second Phase 3 clinical trial with denufosol, and expects top-line results from this trial in the first quarter of 2011. Inspire is targeting a potential U.S. commercial launch for denufosol in the 2012 time frame assuming the results of TIGER-2 are positive, that Inspire subsequently files a New Drug Application (NDA) for denufosol with the FDA in the second half of 2011 and that the FDA approves such NDA under a priority review time line.
Inspire is conducting additional clinical trials in connection with its evolving denufosol franchise development plans and activities. Inspire is conducting DEFY (Denufosol Efficacy Over Four Years), a denufosol open-label clinical trial open to eligible patients that complete the year-long TIGER-2 trial. This three year trial will evaluate the potential disease-modifying impact of denufosol on rate of lung function decline. In August 2010, Inspire also initiated REACH (Researching an Early Approach to Cystic Fibrosis Health), a small safety and tolerability clinical trial of denufosol in CF patients ages 2 – 4 years old.

2011 Accurso FJ, Moss RB, Wilmott RW, Anbar RD, Schaberg AE, Durham TA, Ramsey BW. TIGER-1 Investigator Study Group. Denufosol tetrasodium in patients with cystic fibrosis and normal to mildly impaired lung function. Am J Resp Crit Care 2011; 183:627-634.[PubMed]

Frank Accurso

To evaluate the efficacy and safety of denufosol in patients with cystic fibrosis who had normal to mildly impaired lung function characteristic of early cystic fibrosis. A total of 352 patients greater than or equal to 5 years old with cystic fibrosis who had FEV(1) greater than or equal to 75% of predicted normal were randomized to receive inhaled denufosol, 60 mg, or placebo three times daily in a Phase 3, randomized, double-blind, placebo-controlled, 24-week trial. Main outcome measures included change in FEV(1) from baseline to Week 24 endpoint and adverse events.

Mean change from baseline to Week 24 endpoint in FEV(1) (primary efficacy endpoint) was 0.048 L for denufosol (n = 178) and 0.003 L for placebo (n = 174; P = 0.047). No significant differences between groups were observed for secondary endpoints including exacerbation rate and other measures of lung function. Denufosol was well tolerated with adverse event and growth profiles similar to placebo.

So Denufosol appeared to improved lung function relative to placebo in cystic fibrosis patients with normal to mildly impaired lung function.

A second Phase III trial (TIGER 2) 48 week trial did not confirm these findings and the primary endpoint of this TIGER 2 trial (change in FEV% at 48 weeks) did not achieve significance.

Charlie Johnson

Dr Charlie Johnson, CMO at Inspire said “The analysis of the primary endpoint, key secondary endpoints, and selected subgroup populations in TIGER-2 indicates an absence of meaningful treatment benefit in this patient population …we did not see the treatment effect at 24 weeks that was observed in the TIGER-1”.

As a result of these findings the open label study that followed was abandoned. This was very sad as Denufosol was one of the main drugs under development during the past decade from the first reports of the next generation P2Y(2) receptor agonists in CF in 2002 (Yerxa BR et al J Pharmacol Exp Ther 2002; 302: 871-880. [PubMed]).

January 3rd 2011 – Press release from CF Foundation

Inspire Announces Disappointing Results for CF Therapy Denufosol.
Inspire Pharmaceuticals, Inc., announced today that the top-line results from a second Phase 3 clinical trial of denufosol did not demonstrate a statistically significant improvement in lung function for those on the drug as compared to the placebo after 48 weeks. Improvement in lung function was the primary outcome measure for the trial. The Phase 3 trial included 466 patients in the United States, Australia, New Zealand and Canada. If the results had been positive, the data would have served as a basis for a New Drug Application (NDA) to the U.S. Food & Drug Association. (Rajen F et al. J Cyst Fibros 2012 Jun 8 [PubMed]).

“This is a serious disappointment to the entire CF community,” said Robert J. Beall, Ph.D., President and CEO of the Foundation. “We have long understood that drug development is not predictable, but we are always hopeful that promising therapies will prove effective for those with CF. Inspire’s latest data underscores the importance of our pursuing multiple new therapies that can control and cure CF.” The Foundation currently has more than 30 drugs in its drug development pipeline, including several in late stage clinical trials that aim to treat the basic defect in CF. Beall noted that the Foundation is “deeply indebted to the patients who participated in the denufosol trials, the caregivers who carried out the trials and the team at Inspire Pharmaceuticals for their commitment to bringing new CF therapies to patients.”

Esc peptides

Loretta FerreraFloriana CappielloMaria Rosa LoffredoElena PuglisiBruno CasciaroBruno BottaLuis J V GaliettaMattia MoriMaria Luisa Mangoni.Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.  Cell Mol Life Sci 2021 Dec 31;79(1):67.doi: 10.1007/s00018-021-04030-2.Free PMC article   [Pubmed]

Loretta Ferrera

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease.

Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects.

Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.

Lorreta Ferrera is at U.O.C. Genetica Medica, Istituto Di Ricovero E Cura a Carattere Scientifico (IRCCS) Istituto Giannina Gaslini, 16147, Genoa, Italy

ENaC inhibitor AZD5634

Cecilia KristenssonAnnika ÅstrandScott DonaldsonRon GoldwaterRaolat AbdulaiNaimish PatelPhilip GardinerUlrika TehlerAnne-Kristina MercierMarita OlssonEva ErsdalJukka MäenpääTobias BramerAnna MalmgrenWilliam BennettChristina KeenAZD5634, an inhaled ENaC inhibitor, in healthy subjects and patients with cystic fibrosisJ Cyst Fibros  2022 Feb 26;S1569-1993(22)00042-X. doi: 10.1016/j.jcf.2022.02.010.Online ahead of print.   [Pubmed]


Background: Epithelial sodium channel (ENaC) inhibitors may offer clinical benefit in cystic fibrosis (CF); however, data are limited. We report the outcomes of a Phase I (NCT02679729) and a Phase Ib (NCT02950805) study of AZD5634, a novel inhaled ENaC inhibitor.
Methods: A Phase I, first-in-human, single-blind, placebo-controlled, single ascending dose, sequential dose group study assessed the safety, tolerability, and pharmacokinetics of AZD5634 in healthy subjects (n=53) in part A following inhaled doses up to 1700 µg, and, in part B, following administration of single inhaled (1700 µg) and intravenous (65 µg) doses. A Phase Ib, randomized, double-blind, placebo-controlled, single-dose, 2-way cross-over study assessed the effects of a single dose (600 µg) of inhaled AZD5634 on mucociliary clearance (MCC), pharmacokinetics and safety and tolerability in patients with CF (n=11). Nasal potential difference (NPD) was assessed as an in situ target engagement exploratory biomarker.

Results: Absolute bioavailability of AZD5634 after inhalation was approximately 3%, indicating minimal distribution into the systemic circulation. Urinary excretion was a minor elimination pathway. Administration of inhaled AZD5634 did not improve MCC in CF patients, but AZD5634 inhibited ENaC in the nasal epithelium, as measured by NPD. AZD5634 was safe and well tolerated in both studies.
Conclusions: AZD5634 showed favourable pharmacokinetics and safety in healthy subjects and patients with CF. However, despite achieving target engagement, proof of mechanism was not achieved after a single dose in patients with CF. Further evaluation into multiple dose studies is warranted to explore its therapeutic potential.

Dr Cecilia Kristensson Clinical Programme Director with Early Respiratory & Immunology Early Clinical Development, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.


2005 Bjarnsholt T, Jensen PØ, Rasmussen TB, Christophersen L, Calum H, Hentzer M, Hougen HP, Rygaard J, Moser C, Eberl L, Høiby N, Givskov M. Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology 2005; 151(Pt 12):3873-3880.[PubMed]
Mice were treated with garlic extract or placebo for 7 days, with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology, mortality, histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation, and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin, respiratory burst and phagocytosis by PMNs, as well as leading to an improved outcome of pulmonary infections.
Subsequently a negative clinical trial of the use of garlic in CF was published in 2010 by Alan Smyth of Nottingham (Smyth AR et al. Pediatr Pulmonol 2010; 45:356-362.[PubMed]).(see below)

2010 Smyth AR, Cifelli PM, Ortori CA, Righetti K, Erskine P, Holland Ed, Giviskov M, Williams P, Camara M, Barrett DA, Knox A. Garlic as an inhibitor of Pseudomonas aeruginosa quorum sensing in cystic fibrosis pilot randomized controlled trial. Pediatr Pulmonol 2010; 45:356-362. [PubMed

                 Alan Smyth

The first clinical trial in man of a QS inhibitor. The authors randomized 34 patients to garlic or olive oil capsules (both 656 mg daily). Clinical outcomes and safety bloods were measured at baseline and after 8 weeks treatment. In this exploratory study, analysis was per protocol. Eight patients withdrew, leaving 26 for analysis (13 garlic). Garlic capsules were well tolerated. Although there was no significant effect of garlic compared to placebo in this pilot study, there was a suggestion of improvement with garlic which should be investigated in a larger trial.

– This is a disappointing result. Although there is a vast previous literature on the medicinal properties of garlic it is likely there would be few clinicians who would recommend their patients be included in a larger trial particularly as subjects for clinical trials are in short supply. However, there is continuing interest in garlic and its ability to block communication beteween bacteria – particularly between P. aeruginosa. Tim Holm Jakobsen and Michael Givskov of the University of Copenhagen, and their many collaborators have pinpointed a constituent of garlic (ajoene) that attacks a key step in the development of biofilms, in an effort they hope may offer help in particular for patients with cystic fibrosis (Jakobsen TH et al. Antimicrob Agents Chemother 2012; 56:2314-2325.[PubMed] Free PMC article). In 2012 theses researchers took out a patent on the use of ajoene to fight bacterial infections and Neem Biotech have bought the licence to the patent. They produce a medical product NXAS-401 intend for treating patients with cystic fibrosis.

2017 Jakobsen TH, Warming AN, Vejborg RM, Moscoso JA, Stegger M, Lorenzen F, Rybtke M, Andersen JB, Petersen R, Andersen PS, Nielsen TE, Tolker-Nielsen T, Filloux A, Ingmer H2, Givskov M. A broad range quorum sensing inhibitor working through sRNA inhibition. Sci Rep. 2017 Aug 29;7(1):9857. doi: 10.1038/s41598-017-09886-8. [PubMed]
For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here the authors show that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, they found that ajoene lowered expression of the sRNAs RsmY and RsmZ in P. aeruginosa and the small dual-function regulatory RNA, RNAIII in S. aureus, that controls expression of key virulence factors. We confirmed the modulation of RNAIII by RNA sequencing and found that the expression of many QS regulated genes encoding virulence factors such as haemolysins and proteases were lowered in the presence of ajoene in S. aureus. Importantly, their findings show that sRNAs across bacterial species potentially may qualify as targets of anti-virulence therapy and that ajoene could be a lead structure in search of broad-spectrum compounds transcending the Gram negative-positive borderline.

– So there is considerable continuing interest in this area, important in view of the increasing concerns regarding antibiotic resistance.

Michael Givskov

Prof. Michael Givskov of the Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore and the Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.

Dr. Tim H Jakobsen of the Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.


1997 Hwang TC, Wang F, Yang IC, Reenstra WW.Genistein potentiates wild-type and delta F508-CFTR channel activity. Am J Physiol. 1997 Sep;273(3 Pt 1):C988-98. [PubMed]

    Yzyh-Chang Hwang

Effects of genistein on wild-type (wt) and delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) were studied in NIH/3T3 cells stably transfected with wt or mutant CFTR cDNA. As measured by I- efflux, half-maximal concentration of agonist (K1/2) for forskolin-dependent activation was greater for delta F508-CFTR than wt-CFTR. Genistein decreased the K1/2 for both forms of the channel and increased the maximal activity of delta F508-CFTR by 3.7-fold. In cell-attached patches, 10 microM forskolin induced minimal delta F508-CFTR activity with characteristic prolonged closed times (estimated time constant, > 30 s). Genistein increased the forskolin-induced macroscopic currents of wt-CFTR and delta F508-CFTR by 3- and 19-fold, respectively. Variance analysis suggested that in the presence of forskolin and genistein the open probabilities (Po) of wt- and delta F508-CFTR were identical. In single-channel studies, at maximal adenosine 3′,5′-cyclic monophosphate (cAMP) stimulation, genistein increased the Po of wt-CFTR by prolonging the open time, but, at submaximal cAMP stimulation, the Po was increased by prolonging the open time and shortening the closed time. In excised patches with CFTR channels preactivated in the cell-attached mode, genistein increased ATP-dependent wt- and delta F508-CFTR current about twofold by prolonging the open time.

The results thus suggest that phosphorylation-dependent activation of delta F508-CFTR is defective and that genistein corrects this defect at least in part by binding to the CFTR protein.

Dr Yzyh-Chang Hwang is from the Department of Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia 65211, USA.

2008 Clunes MT, Boucher RC.Front-runners for pharmacotherapeutic correction of the airway ion transport defect in cystic fibrosis. Curr Opin Pharmacol. 2008 Jun;8(3):292-9. doi: 10.1016/j.coph.2008.04.006. Epub 2008 May 28. [PubMed] free article

            Mark Clunes

Although cystic fibrosis (CF) patients display multiorgan dysfunction (e.g. pancreas, gut, and lung) it is lung disease that is the leading cause of premature death in these patients. CF lung disease is characterized by persistent pulmonary infection and mucus plugging of the airways initiated by the failure of solute transport across the airway epithelium. Many drug therapies aim to alleviate the secondary characteristics of CF lung disease; however, new therapies in development are targeted at correcting the ion transport deficiency of CF. The goal is to hydrate airway surfaces by stimulating secretion (through activation of the CF transmembrane conductance regulator and calcium-activated chloride channels), and/or inhibiting absorption (through the epithelial sodium channel) thereby stimulating healthy mucociliary clearance. If mucociliary clearance can be stimulated sufficiently from an early age, then there is the possibility that secondary lung infection may be eradicated from the syndrome of CF disease.

From the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States.

Dr Clunes is now Associate Professor Physiology /Neuroscience St George’s University, Grenada. previously working at the University of North Carolina with Professor Richard Bouche

2011 Ying-Chun Yu, Haruna Miki, Yumi Nakamura, Akiko Hanyuda, Yohei Matsuzaki, Yoichiro Abe,Masato Yasui, Kazuhiko Tanaka, Tzyh-Chang Hwang, Silvia G. Bompadre, Yoshiro Sohma. Curcumin and genistein additively potentiate G551D-CFTRJ Cyst Fibros. 2011 Jul; 10(4): 243–252. [PubMed]
The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. The authors investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique.

Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR.

The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy.

2016 Day I, Shah K, Bradbury NA.Natural Compounds as Therapeutic Agents in the Treatment Cystic Fibrosis.
J Genet Syndr Gene Ther.
2016 Feb;7(1). pii: 284. Epub 2016 Jan 30.
[PubMed]Free PMC Article

               Isha Dey

(From Isha Dey et al 2016 summarising the information relating to genistein)

“Given its low toxicity, genistein appears to be a good candidate for the treatment of patients with CF. Importantly, the effective concentration for channel modulation (~2–3 μM), is within the range of achievable plasma levels of genistein (~1–2 μM) [21]. With regards to CFTR protein production, the observation that 100 μM genistein is inhibitory is somewhat irrelevant given achievable plasma concentrations. However, it also means that stimulatory concentrations of ~30 μM are also unlikely to be achievable. Although a clinical trial using a combination therapy of 4-phenylbutyrate and genistein has been planned, it was cancelled before the trial was initiated”.

Dr Isha Dey is a scientist working with Professor Neil Bradbury

Glutathione and N-acetylcysteine (a glutathione prodrug)

There are numerous publications on glutathione and cystic fibrosis since 1974. The main facts are that glutathione is transported via CFTR and also much of the published work concerns the antioxidant properties.

In 2000 Professor David Sheppard summarised the situation in reply to a question put to the CF Trust.

    David Sheppard

The main points of his reply were as follows – “In 1993 Dr Ron Crystal reported reduced levels of glutathione in the airway surface liquid of people with CF. In 1998 Drs Paul Linsell and John Hanrahan showed CFTR transports glutathione from inside to outside the cell but several less fold that chloride; Christine Bear suggests that CFTR may transport chloride and glutathione by different mechanisms. So glutathione transport is a normal function of CFTR suggesting that loss of CFTR contributes to the development and progression of CF. Dr Valerie Hudson of Brigham Young University has argued forcefully this point and in collaboration with Dr Clark Bishop has tested the effect on a small number of patients (this present trial).

1962 Webb WR. Clinical evaluation of a new mucolytic agent acetyl-cysteine. J Thorac Cardiovasc Surg 1962; 44:330-343. [PubMed] Acetyl cysteine, a derivative of the amino acid cysteine, is a drug that subsequently was widely used for CF in Europe but never popular in the UK. Here experience is reported of 285 patients with a variety of suppurative pulmonary conditions and “revealed it to be extremely effective with almost no associated complications. Seven patients with CF improved over 10 months with either inhalations or sleeping in a tent with the drug nebulised with saline and propylene glycol”. (figure in main text).

24 hour sputum collections from an adult with chronic bronchitis after starting N Acetylcysteine on day 2

Apparently N-acetylcysteine and other sulphydryl compounds act by depolymerising mucus in vitro by breaking disulphide bonds of the glycoproteins thereby lowering the viscosity (Sheffner AL et al. Ann N Y Acad Sci 1963; 106:298-310).

See impressive illustration of 24 hour sputum collections from a man with purulent bronchitis during the day prior to and the 4 days subsequent to use of acetyl-cysteine).

This paper contains a useful summary of all the many side effects of animal-derived trypsin and enzymatic treatments used to liquefy sputum – contrasted with the minimal side effects experienced with N-acetylcysteine.

Interestingly there has been a renewal of interest in acetyl cysteine in the Millennium and the drug has been used both for respiratory (6.5% of USA patients with CF take inhaled acetyl cysteine) and gastrointestinal problems in CF (Lillibridge CB et al. Oral administration of n-acetyl cysteine in the prophylaxis of “meconium ileus equivalent” J Pediatr 1967; 71:887-9; Gracey M et al. Treatment of abdominal pain in cystic fibrosis by oral administration of n-acetyl cysteine. Arch Dis Child 1969; 44:404-405).

More recently the relation to glutathione, in particular, has featured in recent references as N-acetylcysteine is an effective precursor of cysteine for tissue glutathione synthesis. Apparently CFTR is responsible for glutathione transport and there may be intracellular accumulation of glutathione in cystic fibrosis (Childers M et al. Medical Hypotheses 2007; 68:101-102. [PubMed]).

1963 Reas HW. The effect of N-acetylcysteine on the viscosity of tracheobronchial secretions in cystic fibrosis of the pancreas. J Pediatr 1963; 62:31-5. [PubMed] (Also Reas HW. South Med J. 1963; 56:1271-1278. [PubMed]). Viscosity measurements were determined on secretions obtained via tracheotomies from 2 patients with CF with the use of N-acetylcysteine aerosol which produced a greater fall in the viscosity of the secretions than did a control aerosol (also Webb 1962 above). The authors suggest that “The combination of this safe method of mucolysis with energetic postural drainage and physiotherapy may be very rewarding in the easier removal of retained pulmonary secretions”. (Also Suddarth SB. Acetylcysteine a new and effective mucolytic agent. Bulletin – Grainger Medical Center 1963; 15:65-69 above; Meeker IA Jr, Kincannon WN. Acetyl cysteine used to liquefy inspissated meconium causing intestinal obstruction in the newborn. Surgery, St Louis 1964; 56:419-425).

Obviously in the early Sixties there was considerable interest in this new mucolytic agent whose free sulphydryl group reduced the disulphide linkages of mucoproteins. Subsequently it was the subject of a number of publications in liquefying sputum, improving abdominal pain and treating meconium ileus but never became popular in the UK as part of the pulmonary treatment. Although a subsequent review found no evidence of benefit (Duijvestijn YC & Brand PL. Acta Paediatr 1999; 88:38-41.[PubMed), Ratjen et al (Eur J Pediatr 1985; 144:374-378.[PubMed]) found in a 12 week oral trial of NAC, ambroxal and placebo that “although no clinical differences could be observed between the three groups, significant impairment in the placebo group was found for trapped air and FEV1 when compared to the active groups, suggesting a therapeutic effect of ambroxal and NAC in CF”.

More recently the relation to glutathione has caused renewed interest in the drug which, when given orally in high doses, provides a source of glutathione and apparently reduces airway inflammation (Tirouvanziam R, et al. Proc Nat Acad Sc 2006; 103:4628-4633. below).

2005 Bishop C, Hudson VM, Hilton SC, Wilde C. A pilot study of the effect of inhaled buffered reduced glutathione on the clinical status of patients with cystic fibrosis. Chest 2005; 127:308-317. [PubMed]
To assess the impact of inhaled, buffered reduced glutathione (GSH) on clinical indicators of CF pathophysiology, a randomized, double-blind, placebo-controlled pilot study was conducted over an 8-week period with 19 subjects, age 6 to 19 years. The mean change for peak flow was – 6.5 L/min for the placebo group and +33.7 L/min for the GSH group (p = 0.04). 11 of 13 outcomes favoured the GSH group.

The authors concluded that this pilot study indicates the promise of nebulized buffered GSH to ameliorate CF disease, and suggest that longer, larger, and improved studies of inhaled GSH are warranted.

– Dr Clark Bishop is a Utah paediatrician who has studied nebulised glutathione. Its potential use as a treatment for CF is viewed with enthusiasm by some writers on the web. Certainly there seems to be increasing scientific backing for its use. There is a long article on Valerie Hudson (a political science professor) and glutathione (www.womansday.com 3/4/03) describing how she convinced Clark Bishop of the potential value of glutathione to the extent that although he had some resistance from other researchers he raised $50K to fund the present trial.

Interest in glutathione has gradually increased and the latest publication from the Bishop/Hudson collaboration was in 2008 (Visca A, Bishop CT, Hilton SC, Hudson VM. Improvement in clinical markers in CF patients using a reduced glutathione regimen: an uncontrolled, observational study. J Cyst Fibros 2008; 7:433-436. [PubMed] below). In this study the authors followed 13 patients (age range 1-27 years) with cystic fibrosis who were using a regimen of reduced glutathione (GSH), including oral glutathione and inhaled buffered glutathione in an uncontrolled, observational study. Dosage ranged from 66-148 mg/kg/day in divided doses, and the term examined was the initial 5.5 months of GSH use (45 days of incrementally adjusted dose, plus 4 months of use at full dosage). Baseline and post-measurements of FEV1 percent predicted, BMI percentile, and weight percentile were noted, in addition to bacterial status and pulmonary exacerbations.

Significant improvement in the following clinical parameters was observed: average improvement in FEV1 percent predicted (N=10) was 5.8 percentage points (p<0.0001), average weight percentile (N=13) increased 8.6 points (p<0.001), BMI percentile (N=11) improved on average 1.22 points (p<0.001). All patients improved in FEV1 and BMI, if measured in their case; 12 of 13 patients improved in weight percentile. Positive sputum cultures of bacteria in 11 patients declined from 13 to 5 (p<0.03) with sputum cultures of Pseudomonas aeruginosa becoming negative in 4 of 5 patients previously culturing PA, including two of three patients chronically infected with PA as determined by antibody status.

The authors concluded the use of a daily GSH regimen appears to be associated in CF patients with significant improvement in lung function and weight, and a significant decline in bacteria cultured in this uncontrolled study. These findings bear further clinical investigation in larger, randomized, controlled studies.

Subsequently a 12 weeks study from Germany of high dose n-acetylcysteine showed no effect on clinical parameters or inflammatory markers but it did increase the extracellular glutathione in the CF airways (Dauletbaev N et al. Eur J Med Res 2009; 14:352-358.[PubMed] below).

2005 Hartl D, Starosta V, Maier K, Beck-Speier I, Rebhan C, Becker BF, Latzin P, Fischer R, Ratjen F, Huber RM, Rietschel E, Krauss-Etschmann S, Griese M. Inhaled glutathione decreases PGE2 and increases lymphocytes in cystic fibrosis lungs. Free Radical Bio Med 2005; 39:463-472. [PubMed].
Reduced glutathione (GSH), a major antioxidant and modulator of cell proliferation, is decreased in the bronchoalveolar lavage fluid (BALF) of cystic fibrosis (CF) patients. GSH inhalation in CF patients significantly increased GSH levels in BALF and improved lung function (M. Griese et al., 2004, Am. J. Respir. Crit. Care Med.169, 822-828. [PubMed]). GSH depletion in vitro enhances susceptibility to oxidative stress, increases inflammatory cytokine release, and impairs T cell responses. BALF from 17 CF patients (median FEV1 67% (43-105%) of predicted) was assessed before and after GSH inhalation for total protein, markers of oxidative stress (8-isoprostane, myeloperoxidase, and ascorbic and uric acid), pattern of protein oxidation, prostaglandin E2 (PGE2), and proinflammatory cytokines. BALF cells were differentiated using cytospin slides, and lymphocytes were further analyzed by flow cytometry. Inhalation of GSH decreased BALF levels of PGE2 and increased CD4+ and CD8+ lymphocytes in BALF significantly but had no effect on markers of oxidative stress. BALF lymphocytes correlated positively with lung function, whereas levels of PGE2 showed an inverse correlation. The patients with the greatest improvement in lung function after GSH treatment also had the largest decline in PGE2 levels.

– So reduced glutathione inhalation in CF patients increases lymphocytes and suppresses PGE2 in the bronchoalveolar space. Thus, GSH primarily affected the pulmonary immune response rather than the oxidative status in CF patients. The authors suggest the effect of GSH inhalation on PGE2 levels and lymphocytes in CF warrants further investigation.

2006 Tirouvanziam R, Conrad CK, Bottiglieri T, Herzenberg LA, Moss RB, Herzenberg LA. High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis. Proc Nat Acad Sci USA 2006; 103:4628-33. [PubMed] Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high oxidant producers, airway neutrophils contribute largely to the systemic redox imbalance seen in CF. In turn, this chronic and profound imbalance can impact circulating neutrophils before their migration into airways. Indeed, in 18 CF patients with stable disease, blood neutrophils were readily deficient in the pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls). In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently, neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as was the number of airway neutrophils actively releasing elastase-rich granules (P = 0.005), as measured by flow cytometry. Pulmonary function measures were not improved, as expected with short-term treatment.

After excluding data from subjects without baseline airway inflammation, positive treatment effects were more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

– Conflicting views on the effect of N-acetylcysteine appear despite the Cochrane Review conclusion that it is of no benefit in CF.

2007 Childers M, Eckel G, Himmel A, Caldwell J. A new model of cystic fibrosis pathology: lack of transport of glutathione and its thiocyanate conjugates. Medical Hypotheses 2007; 68:101-112. [PubMed]
Understanding that the cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for glutathione (GSH) transport, the authors hypothesize that mutations of the CFTR, which create abnormal GSH transport, will lead to aberrations of GSH levels in both the intracellular as well as the extracellular milieu. These alterations in normal cellular GSH levels affect the redox state of the cell, thereby affecting the intracellular stress protein, metallothionein. The authors describe how this disruption of the redox state caused by excess cellular GSH, will naturally prevent the delivery of zinc as a cofactor for various enzymatic processes, and how these disruptions in normal redox may cause alterations in both humoral and cell-mediated immunity. Moreover, the symptom of thick sticky mucus in these patients might be explained through the understanding that oversulfation of mucus is a direct result of elevated cellular GSH and cysteine. The issues of hyperinflammation, altered pH and the imbalance of fatty acids that are typical in cystic fibrosis are addressed-all of which may also be linked to disruptions in GSH homeostasis. Additionally, this new model of cystic fibrosis pathology, clarifies the relationship between the CFTR and the multi-drug resistance proteins, and the lack of cell-mediated immunity by predicting that the substrate of these proteins is a glutathione adduct of thiocyanate. Finally, a new therapeutic strategy by using isothiocyanates to rectify the GSH imbalance and restore the immune system is suggested for the treatment of cystic fibrosis patients.
– Further work proposing a role for glutathione

2007 Kariya C, Leitner H, Min E, van Heeckeren C, van Heeckeren A, Day BJ. A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration. Am J Physiol Lung C 2007; 292:L1590-7. [PubMed]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase GSH levels in lung epithelial lining fluid (ELF) and whether CFTR plays a role in this process. There was a rapid elevation in the GSH levels after oral GSH dosing and a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice.

– Glutathione appears to be receiving increasing attention in the context of CF and these studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung (see also Bishop et al, 2005; Tirouvanziam et al, 2006; Childers M. 2007 – all above). There is evidence that CFTR is the main transporter of glutathione involved in maintaining basal epithelial lining fluid glutathione levels in the lung (Gould NS et al. Free Radic Biol Med 2012; 52:1201-1206. [PubMed]).

2008 Visca A, Bishop CT, Hilton SC, Hudson VM. Improvement in clinical markers in CF patients using a reduced glutathione regimen: an uncontrolled, observational study. J Cyst Fibros 2008; 7:433-436). [PubMed]
The CFTR mutation, which causes cystic fibrosis (CF), has also recently been identified as causing glutathione system dysfunction and systemic deficiency of reduced glutathione (GSH). Such dysfunction and deficiency regarding GSH may contribute to the pathophysiology of CF.
Thirteen patients (age range 1-27 years) with CF who were using a regimen of reduced glutathione (GSH), including oral glutathione and inhaled buffered glutathione in an uncontrolled study, were followed in an observational study. Dosage ranged from 66-148 mg/kg/day in divided doses, and the term examined was the initial 5.5 months of GSH use (45 days of incrementally adjusted dose, plus 4 months of use at full dosage). Significant improvement in the following clinical parameters was observed: average improvement in FEV1 percent predicted (N=10) was 5.8 percentage points (p<0.0001), average weight percentile (N=13) increased 8.6 points (p<0.001), BMI percentile (N=11) improved on average 1.22 points (p<0.001. Positive sputum cultures of bacteria in 11 patients declined from 13 to 5 (p<0.03) with sputum cultures of Pseudomonas aeruginosa becoming negative in 4 of 5 patients previously culturing PA, including two of three patients chronically infected with PA as determined by antibody status.

– The use of a daily GSH regimen appears to be associated in CF patients with improvement in lung function and weight, and a decline in bacteria cultured in this uncontrolled study. The authors of the study are convinced of the value of glutathione and considered these findings warrant further clinical investigation in larger, randomised, controlled studies as has been suggested by protagonists of the use of glutathione – Clark Bishop and Valerie Hudson. (see Bishop et al, 2005 above for more detail).

2009 Dauletbaev N, Fischer P, Aulbach B, Gross J, Kusche W, Thyroff-friesubger U, Wagner TO, Bargon J. A phase II study on safety and efficacy of high-dose N-acetylcysteine in patients with cystic fibrosis. Eur J med Res 2009; 14:352-358. [PubMed]
High-dose NAC was a well-tolerated and safe medication. High-dose NAC did not alter clinical or inflammatory parameters of the patients. However, extracellular glutathione in induced sputum tended to increase on high-dose NAC. A “prodrug approach” has been suggested as a means of increasing cellular glutathione levels as reduced glutathione has unfavourable biochemical and pharmacological properties (Cacciatore I et al. Molecules 2010; 15:1242-1264. [PubMed]).

– This study confirms the lack of clinical effect of N-acetylcysteine on the respiratory function and inflammatory parameters in CF but the increase in extracellular glutathione may be of some benefit. It is difficult to reconcile the lack of clinical effect with some of the early reports showing marked increase in the volume of sputum; also the clinical experience suggesting some benefit. The effects are complicated – even recently it has been reported that that NAC causes a significant efflux of Cl from CF bronchial epithelial cells (Varelogianni G et al. 2010 [PubMed] below).

2009 Nash EF, Stephenson A, Ratjen F, Tullis E. Nebulized and oral thiol derivatives for pulmonary disease in cystic fibrosis. Cochrane Database Syt Rev 2009 Jan 21;(1):CD007168 [PubMed] Assessed as up to date 27.10.2010
Searches identified 18 trials; eight (seven older than 10 years) (234 participants) are included. Three trials of nebulized thiol derivatives were identified (one compared 20% n-acetylcysteine to 2% n-acetylcysteine; another compared sodium-2-mercaptoethane sulphonate to 7% hypertonic saline; and another compared glutathione to 4% hypertonic saline). Although generally well-tolerated with no significant adverse effects, there was no evidence of significant clinical benefit in our primary outcomes in participants receiving these treatments. Five studies of oral thiol derivatives were identified. Three studies compared n-acetylcysteine to placebo; one compared n-acetylcysteine, ambroxol and placebo; and one compared carbocysteine to ambroxol. Oral thiol derivatives were generally well-tolerated with no significant adverse effects, however there was no evidence of significant clinical benefit in primary outcomes in participants receiving these treatments.

The reviewers found no evidence to recommend the use of either nebulized or oral thiol derivatives in people with cystic fibrosis. They considered there were very few good quality trials investigating the effect of these medications in cystic fibrosis, and further research is required to investigate the potential role of these medications in improving the outcomes of people with cystic fibrosis. So informed opinion sees no place for these drugs in CF management at the present time.

2010 Varelogianni G, Oliynyk I, Johannesson M. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells. Cell Biology International 2010; 34:245-252.[PubMed]
N-acetylcysteine (NAC) 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. CF bronchial epithelial cells (CFBE) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees 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.

– This is yet another interesting paper on NAC which although used as a mucolytic in Europe since the first reports (1962 Webb WR. Clinical evaluation of a new mucolytic agent acetyl-cysteine. J Thorac Cardiovasc Surg 1962; 44:330-343. [PubMed] above) has not found favour in the UK nor has performed well in CF patients in recent trials. The more recent relationship as a precursor to glutathione adds further interest to the NAC story. Apparently N-acetylcysteine and other sulphydryl compounds act by depolymerising mucus in vitro by breaking disulphide bonds of the glycoproteins thereby lowering the viscosity (Sheffner AL. Ann N Y Acad Sci 1963; 106:298-310. [PubMed]).

2011 Suk JS, Lai SK, Boylan NJ, Dawson MR, Boyle MP, Hanes J. Rapid transport of muco-inert nanoparticles in cystic fibrosis sputum treated with N-acetyl cysteine. Nanomedicine UK. 2011; 6:365-675. [PubMed] Sputum poses a critical diffusional barrier that strongly limits the efficacy of drug and gene carriers in the airways of individuals with cystic fibrosis. Previous attempts to enhance particle penetration of CF sputum have focused on either reducing its barrier properties via mucolytics, or decreasing particle adhesion to sputum constituents by coating the particle surface with non-mucoadhesive polymers, including polyethylene glycol (PEG). Neither approach has enabled particles to penetrate expectorated sputum at rates previously observed for non-mucoadhesive nanoparticles in human cervicovaginal mucus.

The authors sought to investigate whether a common mucolytic, N-acetyl cysteine (NAC), in combination with dense PEG coatings on particles, can synergistically enhance particle penetration across fresh undiluted CF sputum. They used high-resolution multiple particle tracking to measure the diffusion of uncoated and PEG-coated nanoparticles in native and NAC-treated CF sputum. They discovered that 200 nm particles, if densely coated with PEG, were able to penetrate CF sputum pretreated with NAC with average speeds approaching their theoretical speeds in water. Based on the rapid penetration of PEG-coated particles in NAC-treated sputum, they determined that the average spacing between sputum mesh elements was increased from 145 +/- 50 nm to 230 +/- 50 nm upon NAC treatment. Mathematical models based on particle transport rates suggest as much as 75 and 30% of 200 and 500 nm PEG-coated particles, respectively, may penetrate a physiologically thick NAC-treated CF sputum layer within 20 min. Uncoated particles were trapped in CF sputum pretreated with NAC nearly to the same extent as in native sputum, suggesting that NAC treatment alone offered little improvement to particle penetration.

The authors concluded that NAC facilitated rapid diffusion of PEG-coated, muco-inert nanoparticles in CF sputum and that their results provided a promising strategy to improve drug and gene carrier penetration in CF sputum, offering hope for improved therapies for CF.

The N-acetylcysteine story in relation to CF is quite remarkable. Although declared to be well tolerated with no significant adverse effects but without evidence of significant clinical benefit in CF, reports of its use keep appearing!

The CF Foundation Drug Pipeline states in 2012 – “A placebo controlled 12-week Phase 2 trial at Stanford University demonstrated a decrease in inflammatory cells in the lung and improvement in pulmonary function. Results from a Phase 2b multicentre trial did not reproduce these findings but an improvement in lung function was noted”.

A Cochrane Database Systematic Review in 2009 [PubMed] on nebulized and oral thiol derivatives for pulmonary disease in cystic fibrosis “found no evidence to recommend the use of either nebulized or oral thiol derivatives in people with cystic fibrosis. There are very few good quality trials investigating the effect of these medications in cystic fibrosis, and further research is required to investigate the potential role of these medications in improving the outcomes of people with cystic fibrosis”.

Two more recent studies failed to show significiant effects on inflammation, oxidative stress or clinical outcomes –

*2011 Tiruvanziam R et al. A Multi-center, phase IIB randomized placebo-controlled, double blind study of the effects of n-acetyl cysteine on redox changes and lung inflammation in cystic fibrosis patients. Pediatr Pulmonol Suppl 2011;280-281.

*2012 Griese M et al. Inhaled glutathione in cystic fibrosis. J Cyst Fibros 2012; 11:S11.
A controlled trial involving 153 subjects over 24 weeks. There was no significant change in FEV1 or other measures of clinical condition. Although free and total glutathione levels in sputum were increased in the treated group cellular, inflammatory, redox state and proteolytic markers remained unchanged.

– So this trial from a number of centres in Germany was essentiually negative.

2013 de Lima Marson FA, Bertuzzo CS, Secolin R, Ribeiro AF, Ribeiro JD. Genetic interaction of GSH metabolic pathway genes in cystic fibrosis. BMC Med Genet 2013; 14:60.[PubMed]
Cystic fibrosis (CF) is a monogenic disease caused by CFTR gene mutations, with clinical expression similar to complex disease, influenced by genetic and environmental factors. Among the possible modifier genes, those associated to metabolic pathways of glutathione (GSH) have been considered as potential modulators of CF clinical severity. In this way it is of pivotal importance investigate gene polymorphisms at Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC), Glutathione S-transferase Mu 1 (GSTM1), Glutathione S-transferase Theta 1 (GSTT1), and Glutathione S-transferase P1 (GSTP1), which have been associated to the GSH metabolic pathway and CF clinical severity.

In this study a total of 180 CF patients were included, which investigated polymorphisms in GCLC and GST genes (GCLC -129C>T and -3506A>G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G) by PCR and PCR-RFLP associating to clinical variables of CF severity, including variables of sex, clinical scores [Shwachman-Kulczycki, Kanga e Bhalla (BS)], body mass index, patient age, age for diagnosis, first clinical symptoms, first colonization by Pseudomonas aeruginosa, sputum’s microorganisms, hemoglobin oxygen saturation in the blood, spirometry and comorbidities. The CFTR genotype was investigated in all patients, and the genetic interaction was performed using MDR2.0 and MDRPT0.4.7 software.

The analysis of multiple genes in metabolic pathways in diseases with variable clinical expression, as CF disease, enables understanding of phenotypic diversity. This data show evidence of interaction between the GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G polymorphism with CFTR gene mutation classes, and BS (Balance testing accuracy=0.6824, p=0.008), which measures the commitment of bronchopulmonary segments by tomography.

The authors conclude polymorphisms in genes associated with metabolism of GSH act on the CF’s severity

2013 Griese M. Kappler M. Eismann C. Ballmann M. Junge S. Rietschel E. van Koningsbruggen-Rietschel S. Staab D. Rolinck-Werninghaus C. Mellies U. Kohnlein T. Wagner T. Konig S. Teschler H. Heuer HE. Kopp M. Heyder S. Hammermann J. Kuster P. Honer M. Mansmann U. Beck-Speier I. Hartl D. Fuchs C. Glutathione Study Group. Hector A. Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial. Am J Resp Crit Care 2013; 188:83-89. [PubMed]

Matthias Griese

Glutathione is the major antioxidant in the extracellular lining fluid of the lungs and depleted in patients with cystic fibrosis (CF). This study aimed to assess glutathione delivered by inhalation as a potential treatment for CF lung disease. This randomized, double-blind, placebo-controlled trial evaluated inhaled glutathione in subjects with CF 8 years of age and older and FEV1 of 40-90% of predicted. Subjects were randomized to receive 646 mg glutathione in 4 ml (n = 73) or placebo (n = 80) via an investigational eFlow nebulizer every 12 hours for 6 months. FEV1 (absolute values), both as pre-post differences (P = 0.180) and as area under the curves (P = 0.205), were the primary efficacy endpoints, and were not different between the glutathione group and the placebo group over the 6-month treatment period. Exploratory analysis showed an increase of FEV1 from baseline over placebo of 100 ml or 2.2% predicted; this was significant at 3 months, but not later. Subjects receiving glutathione had neither fewer pulmonary exacerbations, nor better scores for quality of life. Whereas increased glutathione and metabolites in sputum demonstrated significant delivery to the lungs, there was no indication of diminished oxidative stress to proteins or lipids, and no evidence for anti-inflammatory or antiproteolytic actions of glutathione supplemented to the airways. The adverse event incidence was similar between glutathione and placebo.

The authors concluded that inhaled glutathione in the dose administered did not demonstrate clinically relevant improvements in lung function, pulmonary exacerbation frequency, or patient-reported outcomes. Glutathione delivery to the airways was not associated with changes in markers of oxidation, proteolysis, or inflammation.

– Since 1974 there has been considerable interest in the use for the use of inhaled glutathione – and of a prodrug acetyl cysteine.The main facts are that glutathione is transported via CFTR and also much of the published work concerns the antioxidant properties.

Dr David Sheppard of Bristol summarised the situation in reply to a question put to the CF Trust. The main points of his reply were as follows – “In 1993 Dr Ron Crystal reported reduced levels of glutathione in the airway surface liquid of people with CF. In 1998 Drs Paul Linsell and John Hanrahan showed CFTR transports glutathione from inside to outside the cell but several less fold that chloride; Christine Bear suggests that CFTR may transport chloride and glutathione by different mechanisms. So glutathione transport is a normal function of CFTR suggesting that loss of CFTR contributes to the development and progression of CF. Dr Valerie Hudson of Brigham Young University has argued forcefully this point and in collaboration with Dr Clark Bishop has tested the effect on a small number of patients”.

2015 Calabrese C; Tosco A; Abete P; Carnovale V; Basile C; Magliocca A; Quattrucci S; De Sanctis S; Alatri F; Mazzarella G; De Pietro L; Turino C; Melillo E; Buonpensiero P; Di Pasqua A; Raia V. Randomized, single blind, controlled trial of inhaled glutathione vs. placebo in patients with cystic fibrosis. J Cyst Fibros 2015; 14(2):203-10. [PubMed]
54 adult and 51 pediatric patients were randomised to receive inhaled GSH or placebo twice daily for 12 months. Twelve months treatment with inhaled GSH did not achieve the predetermined primary outcome measure of 15% improvement in FEV1%. Only in patients with moderate lung disease, 3, 6 and 9 months therapy with GSH resulted in a statistically significant increase of FEV1 values from the baseline. Moreover GSH therapy improved 6-minute walking test in pediatric population. GSH was well tolerated by all patients.
The authors concluded that based on the results of this clinical trial, the treatment with inhaled GSH is assumed to lead an almost immediate improvement in the FEV1 in patients with moderate lung disease, a stabilization of BMI in adult population and an improvement of 6 minute walking test in children. In a prospective observational study that is currently ongoing in their center they have selected Lung Clearance Index as a more sensitive alternative to spirometry for detecting efficacy of GSH therapy also in patients with CF and mild lung disease. inhaled GSH has slight positive effects in CF patients with moderate lung disease warranting further study.

– The literature on the use of glutathione in CF is mounting but its apparent modest effects are not of a degree that would encouraged clinicians to introduce it into their patients’ already complicated treatment regimens.

2015 Conrad C; Lymp J; Thompson V; Dunn C; Davies Z; Chatfield B; Nichols D; Clancy J; Vender R; Egan ME; Quittell L; Michelson P; Antony V; Spahr J; Rubenstein RC; Moss RB; Herzenberg LA; Goss CH; Tirouvanziam R. Long-term treatment with oral N-acetylcysteine: affects lung function but not sputum inflammation in cystic fibrosis subjects. A phase II randomized placebo-controlled trial. J Cyst Fibros 2015; 14(2):219-27. [PubMed]
To evaluate the effects of oral N-acetylcysteine (NAC), which replenishes systemic glutathione, on decreasing inflammation and improving lung function in CF airways, a multicenter, randomized, double-blind proof of concept study was performed in which 70 CF subjects received NAC or placebo orally thrice daily for 24 weeks. Primary endpoints were change in sputum human neutrophil elastase (HNE) activity; secondary, FEV(1) and other clinical lung function measures; and safety, the safety and tolerability of NAC and the potential of NAC to promote pulmonary hypertension in subjects with CF.
Lung function (FEV(1) and FEF(25-75%)) remained stable or increased slightly in the NAC group but decreased in the placebo group (p=0.02 and 0.02). Log(10). Human neutrophil elastase activity remained equal between cohorts (difference 0.21, 95% CI -0.07 to 0.48, p=0.14).
The authors concluded NAC recipients maintained their lung function while placebo recipients declined (24 week FEV1 treatment effect =150 mL, p<0.02). However, no effect on HNE activity and other selected biomarkers of neutrophilic inflammation were detected. They suggested further studies on mechanism and clinical outcome are warranted.

– Most studies with glutathione or acetylcysteine (usually oral) in CF seem to show very modest improvement in either the antioxidant status or respiratory function. However, the treatment is still not recommended in the UK by the Cochrane Reviewers for treatment of people with CF (Cochrane Database Syst Rev2013;7:CD007168). More positive evidence is required for a drug to take it’s place amongst the vast number of treatments patients are expected to take.

2015 Visca A; Bishop CT; Hilton S; Hudson VM. Oral reduced L-glutathione improves growth in pediatric cystic fibrosis patients. J Pediatr Gastroenterol Nutr 2015; 60(6):802-10. [PubMed] The aim of the study was to determine whether oral GSH could improve growth in CF. Subjects were treated with oral GSH or placebo (calcium citrate), each 65 mg kg day divided into 3 doses per day at mealtimes, and administered daily for 6 months. The GSH treatment group gained an average of 0.67 standard deviation (SD) in weight-for-age-and sex z score (wfaszs), (19.1 weight percentile points) during the course of 6 months, with no adverse effects (vs placebo with an increase of 0.1 SD in wfaszs [2.1 weight percentile points], P < 0.0001). Fecal calprotectin improved, GSH -52.0 vs placebo 0.5), also BMI for GSH improved 0.69 SD BMI-adjusted-for-age-and-sex z score versus placebo 0.22 SD (BMI percentile 21.7 GSH vs 5.2 placebo), and height 0.2 SD in height-for-age-and-sex z score (hfaszs) GSH versus -0.06 SD hfaszs placebo [height percentile 7.0 GSH vs -2.6 placebo], all P < 0.0001). Secondary outcomes improved significantly, as well. The authors concluded that oral reduced L-GSH significantly improves measures of growth status and gut inflammation in CF.

– Glutathione just won’t go away! Despite the lack of interest in acetyl cysteine and glutathione in the UK, some published work seems to demonstrate some beneficial effects in people with CF.

2015 Muller M, Merrett ND. Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin. Chem Biol Interact 2015; 232:30-7. [PubMed]
From their studies (see abstract) the authors conclude the data suggest that intracellular glutathione (GSH) forms a cell-impermeant conjugate with pyocyanin and that availability of the thiol may be critical to minimising the toxicity of this important bacterial virulence factor at infection sites. Their data indicate that for GSH to have a clinically effective role in neutralising pyocyanin, the thiol needs to be available at millimolar concentrations.

– Further evidence relevant to the beneficial role of glutathione and indirectly the possible effect of acetyl cysteine?

2015 Kettle AJ, Turner R, Gangell CL, Harwood DT, Khalilova IS, Chapman AL, Winterbourn CC, Sly PD; AREST CF. Oxidation contributes to low glutathione in the airways of children with cystic fibrosis. Eur Respir J. 2014 ; 44(1):122-9. doi: 10.1183/09031936.00170213. Epub 2014 Mar 23.[PubMed] Glutathione is an important antioxidant in the lungs but its concentration is low in the airways of patients with cystic fibrosis. Whether this deficit occurs from an early age or how oxidative stress contributes to lowering glutathione is unknown. The authors measured glutathione, its oxidation products, myeloperoxidase, and biomarkers of hypochlorous acid in bronchoalveolar lavage from children with cystic fibrosis and disease controls using mass spectrometry and immunological techniques. The concentration of glutathione was lower in bronchoalveolar lavage from children with cystic fibrosis, whereas glutathione sulfonamide, a specific oxidation product of hypochlorous acid, was higher. Oxidised glutathione and glutathione sulfonamide correlated with myeloperoxidase and a biomarker of hypochlorous acid. The percentage of glutathione attached to proteins was higher in children with cystic fibrosis than controls. Pulmonary infections in cystic fibrosis resulted in lower levels of glutathione but higher levels of oxidised glutathione and glutathione sulfonamide in bronchoalveolar lavage.
The concentration of glutathione is low in the airways of patients with cystic fibrosis from an early age. Increased oxidation of glutathione by hypochlorous acid and its attachment to proteins contribute to this deficiency. The authors suggest therapies targeted against myeloperoxidase may boost antioxidant defence and slow the onset and progression of lung disease in cystic fibrosis.

2018 Das T, Simone M, Ibugo AI, Witting PK, Manefield M2, Manos J. Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and ViabilityFront Microbiol.2017 Dec 14;8:2429. doi: 10.3389/fmicb.2017.02429. eCollection 2017. [PubMed]Free PMC Article
Pyocyanin secreted by Pseudomonas aeruginosa is a virulence factor that damages epithelial cells during infection through the action of reactive oxygen species, however, little is known about its direct effect on biofilms. The authors demonstrated that pyocyanin-producing P. aeruginosa strains (PA14WT, DKN370, AES-1R, and AES-2) formed robust biofilms in contrast to the poorly formed biofilms of the pyocyanin mutant PA14ΔphzA-G and the low pyocyanin producer AES-1M. The addition of DNase I and reduced glutathione (GSH) significantly reduced biofilm biomass of pyocyanin-producing strains (P < 0.05) compared to non-pyocyanin producers. Subsequently they showed that a combined treatment comprising: GSH + DNase I + antibiotic, disrupted and reduced biofilm biomass up to 90% in cystic fibrosis isolates AES-1R, AES-2, LESB58, and LES431 and promoted lung epithelial cell (A549) recovery and growth. They also showed that exogenously added GSH restored A549 epithelial cell glutathione reductase activity in the presence of pyocyanin through recycling of GSSG to GSH and consequently increased total intracellular GSH levels, inhibiting oxidative stress, and facilitating cell growth and confluence.

They suggest these outcomes indicate that GSH has multiple roles in facilitating a return to normal epithelial cell growth after insult by pyocyanin. With increased antibiotic resistance in many bacterial species, there is an urgency to establish novel antimicrobial agents. GSH is able to rapidly and comprehensively destroy P. aeruginosa associated biofilms while at a same time assisting in the recovery of host cells and re-growth of damaged tissue.

2018 Ehre C, Rushton ZL, Wang B, Hothem LN, Morrison CB, Fontana NC, Markovetz MR, Delion MF, Kato T, Villalon D, Thelin WR, Esther CR Jr, Hill DB, Grubb BR, Livraghi-Butrico A, Donaldson SH, Boucher RC.An Improved Inhaled Mucolytic to Treat Airway Muco-Obstructive Diseases.Am J Respir Crit Care Med. 2018 Sep 13. doi: 10.1164/rccm.201802-0245OC. [Epub ahead of print] [Pubmed]

          Camille Ehre

Dithiothreitol (DTT) and a novel mucolytic agent, P3001, were directly compared to N-acetylcysteine (NAC) in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in βENaC over-expressing mice whereas NAC did not. In NAC-treated CF patients, deposited NAC was rapidly cleared from the lungs and was ineffective on sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than NAC, and was more effective than DNase, in CF sputum ex vivo.

The authors consider these results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P-3001 class mucolytic agents.

Dr. Camille Ehre is Assistant Professor at the Marsico Lung Institute, University of North Carolina School of Medicine

2018 Perra L, Balloy V, Foussignière T, Moissenet D, Petat H, Mungrue IN, Touqui L, Corvol H, Chignard M, Guillot L. CHAC1 Is Differentially Expressed in Normal and Cystic Fibrosis Bronchial Epithelial Cells and Regulates the Inflammatory Response Induced by Pseudomonas aeruginosa. Front Immunol. 2018 Nov 29;9:2823. doi: 10.3389/fimmu.2018.02823. eCollection 2018.[Pubmed]Free PMC Article
In cystic fibrosis (CF), Pseudomonas aeruginosa (Pa) colonizes the lungs, leading to chronic inflammation of the bronchial epithelium. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) mRNA is differentially expressed in primary human airway epithelial cells from bronchi (hAECBs) from patients with CF and healthy patients at baseline and upon infection with Pa. CHAC1 degrades glutathione and is associated with ER stress and apoptosis pathways. In this study, they examined the roles of CHAC1 in the inflammatory response and apoptosis in lung epithelial cells. First, they confirmed by reverse transcription quantitative polymerase chain reaction that CHAC1 mRNA was over expressed in hAECBs from patients without CF compared with the expression in hAECBs from patients with CF upon Pa (PAK strain) infection. Moreover, the Pa virulence factors LPS and flagellin were shown to induce CHAC1expression in cells from patients without CF. Using NCI-H292 lung epithelial cells, they found that LPS-induced CHAC1 mRNA expression was PERK-independent and involved ATF4. Additionally, using CHAC1 small interfering RNA, we showed that reduced CHAC1expression in the context of LPS and flagellin stimulation was associated with modulation of inflammatory markers and alteration of NF-κB signaling. Finally, they showed that Pa was not able to induce apoptosis in NCI-H292 cells.

Their results suggest that CHAC1 is involved in the regulation of inflammation in bronchial cells during Pa infection and may explain the excessive inflammation present in the respiratory tracts of patients with CF.

– In conclusion N-acetylcysteine receives only brief mention in the 2016 edition of the Hodson and Geddes Textbook. “N-acetylcysteine hydrolyses disulphide bonds connecting mucin oligomers and has been shown to decrease mucus viscosity in vitro. Clinical studies of aerosolised NAC however have not demonstrated benefits in chronic bronchitis (1992) or COPD (2005) and Cochrane Systemic Reviews in 1999 and 2009 found no evidence to recommend its use in CF. In addition longterm use (theoretically) may be damaging to the airway (Rubin BK. Respir Care 2011;56(9):1411-23).reducing mucin and inducing a cough.

The latest publications seem to be concentrating more on the effect on pseudomonal infection of the airways.

Molly Bozic , Christopher H Goss, Rabindra M Tirouvanziam, Arthur Baines, Margaret Kloster, Liebe Antoine, Drucy Borowitz, Sarah Jane Schwarzenberg, GROW study group. Oral Glutathione and Growth in Cystic Fibrosis: A Multi-Center, Randomized, Placebo-Controlled, Double-Blind Trial J Pediatr Gastroenterol Nut 2020 Sep 21.doi: 10.1097/MPG.0000000000002948.Online ahead of print. [Pubmed]

Molly Bozic

The nutritional status of children with cystic fibrosis (CF) is associated with mortality and morbidity. Intestinal inflammation may contribute to impaired digestion, absorption and nutrient utilization in patients with CF and oral glutathione may reduce inflammation, promoting improved nutritional status in patients with CF.

The GROW study was a prospective, multi-center, randomized, placebo-controlled, double-blind, Phase II clinical trial in pancreatic insufficient patients with CF between the ages of 2-10 years. Patients received reduced glutathione or placebo orally daily for 24 weeks. The primary endpoint was the difference in change in weight-for-age z-scores from baseline through week 24 between treatment groups. Secondary endpoints included other anthropometrics, serum and fecal inflammatory markers in addition to other clinical outcomes.Results: 58 participants completed the study. No significant differences were seen between glutathione (n = 30) and placebo (n = 28) groups in the 6 month change in weight-for-age z-score (-0.08; 95% CI: -0.22, 0.06; p = 0.25); absolute change in weight (kg) (-0.18; 95% CI: -0.55, 0.20; p = 0.35); or absolute change in BMI kg/m (-0.06; 95% CI: -0.37, 0.25; p = 0.69). There were no significant differences in other secondary endpoints. Overall, glutathione was safe and well tolerated.

Conclusion was that oral glutathione supplementation did not impact growth or change serum or fecal inflammatory markers in pancreatic insufficient children with CF when compared to placebo.

Dr Molly Bozie is a paediatric gastroenterologist and Associate Professor of Pediatrics at the Indiana University School of Medicine, Indianapolis, Indiana.

Marta GueriniGiorgia CondròValeria FriuliLauretta MaggiPaola Perugini . N-acetylcysteine (NAC) and Its Role in Clinical Practice Management of Cystic Fibrosis (CF): A ReviewPharmaceuticals (Basel 2022 Feb 11;15(2):217. doi: 10.3390/ph15020217.  Free PMC article [Pubmed]

       Marta Guerini

N-acetylcysteine is the acetylated form of the amino acid L-cysteine and a precursor to glutathione (GSH). It has been known for a long time as a powerful antioxidant and as an antidote for paracetamol overdose. However, other activities related to this molecule have been discovered over the years, making it a promising drug for diseases such as cystic fibrosis (CF). Its antioxidant activity plays a key role in CF airway inflammation and redox imbalance. Furthermore, this molecule appears to play an important role in the prevention and eradication of biofilms resulting from CF airway infections, in particular that of Pseudomonas aeruginosa.
The aim of this review is to provide an overview of CF and the role that NAC could play in preventing and eliminating biofilms, as a modulator of inflammation and as an antioxidant, restoring the redox balance within the airways in CF patients. To do this, NAC can act alone, but it can also be used as an adjuvant molecule to known drugs (antibiotics/anti-inflammatories) to increase their activity.

Dr Marta Guerini is in the Department of Drug Sciences, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy


1964 Meeker IA Jr. Acetyl cysteine used to liquefy inspissated meconium causing intestinal obstruction in the newborn. Surgery 1964; 56:419-425 [PubMed]
100ml 0f 20% acetyl cysteine and equal parts of 1% hydrogen peroxide were introduced into the lumen of the bowel where it remains for 30 minutes. Bowel resection was then not necessary.

1967 Lillibridge CB, Docter JM, Eidelman S. Oral administration of n-acetyl cysteine in the prophylaxis of “meconium ileus equivalent”. J Pediatr 1967; 71:887-889.[PubMed]
An adult with CF was chronically disabled by frequent abdominal pain and episodes of faecal impaction. Treatment with oral acetyl cysteine 30ml three time daily led to almost complete cessation of the attacks. The patients gained weight and became fully employed. This is the first report of this treatment in an older patient with CF.

1969 Gracey M, Burke V, Anderson CM. Treatment of abdominal pain in cystic fibrosis by oral administration of n-acetyl cysteine. Arch Dis Child 1969; 44:404-405.[PubMed]

Michael Gracy

Gracey later confirmed in 1975 that “this troublesome complaint may be relieved by giving n-acetyl cysteine by mouth and further personal experience confirms the value of this form of therapy. Sometimes up to 15ml of a 20% solution 3 or 4 times a day may be necessary at first”

1976 Hodson ME, Mearns MB, Batten JC. Meconium ileus equivalent in adults with cystic fibrosis of pancreas: a report of six cases. Br Med J 1976; 2(6039):790-791.[PubMed]
Eleven episodes of “meconium ileus equivalent” have been seen in six adults with cystic fibrosis of the pancreas. Three patients were initially treated surgically; one died and the other two developed serious postoperative chest infections. Six episodes were successfully treated medically with acetylcysteine orally and by enema, nasogastric suction, and intravenous fluids. Operation should be avoided if possible, and maintenance treatment with acetylcysteine may be necessary to prevent relapse.

– So oral acetylcysteine appears to be more effective as a treatment foe meconium ileus and it’s equivalent than for the chest

Icenticaftor (QBW251)

Kazani S, Rowlands DJ, Bottoli I, Milojevic J, Alcantara J, Jones I, Kulmatycki K, Machineni S, Mostovy L, Nicholls I, Nick JA, Rowe SM, Simmonds NJ, Vegesna R, Verheijen J, Danahay H, Gosling M, Ayalavajjala PS, Salman M, Strieter R.  Safety and efficacy of the cystic fibrosis transmembrane conductance regulator potentiator icenticaftor (QBW251).  J Cyst Fibros. 20(2):250-256, 2021 03.  Free article [Pubmed]
Background:This is the first-in-human study of icenticaftor, an oral potentiator of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel. Restoration of CFTR activity has shown significant clinical benefits, but more studies are needed to address all CFTR mutations.
Methods:Safety, pharmacodynamics/pharmacokinetics of icenticaftor were evaluated in a randomized, double-blind, placebo-controlled study in healthy volunteers. Efficacy was assessed in adult CF patients with ≥1 pre-specified CFTR Class III or IV mutation (150 and 450 mg bid), or homozygous for F508del mutation (450 mg bid). Primary efficacy endpoint was change from baseline in lung clearance index (LCI2.5). Secondary endpoints included %predicted FEV1 and sweat chloride level.
Results:Class IV mutations were present in 22 patients, Class III in 2 (both S549N), and 25 were homozygous for F508del. Icenticaftor was well-tolerated in healthy and CF subjects with no unexpected events or discontinuations in the CF groups. The most frequent study-drug related adverse events in CF patients were nausea (12.2%), headache (10.2%), and fatigue (6.1%). Icenticaftor 450 mg bid for 14 days showed significant improvements in all endpoints versus placebo in patients with Class III and IV mutations; mean %predicted FEV1 increased by 6.46%, LCI2.5 decreased by 1.13 points and sweat chloride decreased by 8.36 mmol/L. No significant efficacy was observed in patients homozygous for a single F508del.

Conclusions:Icenticaftor was safe and well-tolerated in healthy volunteers and CF patients, and demonstrated clinically meaningful changes in lung function and sweat chloride level in CF patients with Class III and IV CFTR mutations. (ClinicalTrials.gov: NCT02190604).

Dr Shamsah Kazani is at Novartis Institutes for BioMedical Research, Cambridge, MA, United States; Novartis Institutes for BioMedical Research, Basel, Switzerland.

IgY Egg Yolk Antibodies

2003 Kollberg H, Carlander D, Olesen H, Wejaker PE, Johannesson M, Larsson A. Oral administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections in patients with cystic fibrosis. Pediatr Pulmonol 2003; 35:433-440. [PubMed]

Hans Kollberg

Immunotherapy with specific egg-yolk antibodies (IgY) may be an alternative to antibiotics for the prevention of PA infections. CF patients gargled daily with an IgY-antibody preparation, purified from eggs of hens immunized with Pseudomonas bacteria. These patients were compared to a group who did not gargle with the preparation. Both groups had their first colonization with PA eradicated by antibiotics. The basic treatment for CF was essentially the same in both groups.
In the initial study, the period between the first and second colonization with PA was significantly prolonged for the treated vs. the control group. In the prolonged study, the treated group had only 2.5 sputum cultures positive for PA per 100 months of observation, and none of these patients became chronically colonized with PA. No adverse events were reported. In the control group, 13.7 cultures per 100 months of observation were positive for PA, and 5 (24%) patients became chronically infected with PA. This feasibility study shows that antipseudomonal IgY has the potential to effectively prevent PA colonization without any severe adverse effects

2007 Nilsson E, Kollberg H, Johannesson M, Wejaker PE, Carlander D, Larsson A. More than 10 years’ continuous oral treatment with specific immunoglobulin Y for the prevention of Pseudomonas aeruginosa infections: a case report. J Med Food 2007; 10:375-378. [PubMed]
Hans Kollberg’s group in Sweden have used orally administered immunoglobulin Y (IgY) preparations for 10 years (Kollberg et al, 2003 above) and the patients treated with IgY had only 2.5 P. aeruginosa-positive sputum cultures per 100 months, and none became chronically infected. In the control group, 13.7 of the cultures per 100 months were positive for PA and 24% of patients became chronically infected.

2008 Nilsson E, Larsson A, Olesen HV, Wejaker PE, Kollberg H. Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis. Pediatr Pulmonol 2008; 43:892-899.[PubMed]
This is an extended open study of oral prophylactic treatment with egg yolk antibodies against Pseudomonas aeruginosa, Anti-Pseudomonas IgY, of 17 Swedish patients with cystic fibrosis. They have been on prophylactic IgY treatment for up to 12 years and altogether for 114 patient years. A group of 23 Danish CF patients served as control. There has been a total absence of adverse events. Only 29 cultures have been positive for P. aeruginosa (cultures after chronic colonization not included), that is, 2.3/100 treatment months compared to 7.0/100 months in the control group (P = 0.028). In the IgY treated group only one pair of siblings (2/17) has been chronically colonized with P. aeruginosa compared to seven patients (7/23) in the control group. Atypical mycobacteria, S. maltophilia, A. xylosoxidans, and A. fumigatus have appeared only sporadically. There have been no cultures positive for B. cepacia. There was no decrease in pulmonary functions (P = 0.730) within the IgY group. Body mass index values were normal or close to normal for all IgY treated patients. In conclusion, Anti-Pseudomonas IgY has great potential to prevent P. aeruginosa infections.

Up to and including 2012 there are many studies published on the effect of IgY antibodies from egg yolks of immunised hens against a variety of organisms since the first publication in relation to plant viral IgY antibodies from the yolks of immunised hens (Polson A et al. Immunol Commun 1980; 9:475-493.[PubMed]). This particular product was recognised with Orphan Medicinal Product Designation by the EMeA in 2008.

A multicentre European clinical trial was in progress in 2012 (http://www.clinical trials.gov/ct2/show/NCT01455675).

2015 Thomsen K; Christophersen L; Bjarnsholt T; Jensen PO; Moser C; Hoiby N. Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils. Infect Immun 2015; 83(7):2686-93. [PubMed]
The authors report that Anti-P. aeruginosa IgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is attributed to IgY-facilitated formation of immobilised bacteria in aggregates, as visualised by fluorescence microscopy and the induction of increased bacterial hydrophobicity. The present study demonstrates that avian egg yolk immunoglobulins (IgY) targeting P. aeruginosa modify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis.

– Further supportive evidence for the beneficial effect of anti-P. aeruginosa IgY antibodies in reducing susceptibility to colonisation by P. aeruginosa infection and reducing the likelihood of chronic infection. Unfortunately the results of Hans Kohlberg’s multicentre study on IgY are overdue as the trial is still underway in mid-2015. (See also Topics section “Drug modulation and New Drugs” for further information).

2015 Müller S, Schubert A, Zajac J, Dyck T, Oelkrug C. IgY antibodies in human nutrition for disease prevention. Nutri J. 2015; 14(1):109. doi: 10.1186/s12937-015-0067-3. Free PMC article[PubMed]
Oral administration of preformed specific antibodies is an attractive approach against infections of the digestive system in humans and animals in times of increasing antibiotic resistance. Immunisation of chickens with specific antigens offers the possibility to create various forms of antibodies.
– The use of IgY egg yolk antibodies has been studied with regard to prevention of Pseudomonas aeruginosa infection in people with cystic fibrosis (see Kohlberg H. in Topics > New drugs > IgY egg yolk antibodies). The wider use of such treatments is reviewed in the present article.

– Unable to obtain information as to the results of the main trial of the antibodies in CF. There is no information on the website although the trial started in 2012.

2016 Thomsen K, Christophersen L, Bjarnsholt T, Jensen PØ, Moser C, Høiby N. Anti-Pseudomonas aeruginosa IgY antibodies augment bacterial clearanc`e in a murine pneumonia model. J Cyst Fibros. 2016 Mar;15(2):171-8. doi: 10.1016/j.jcf.2015.08.002. Epub 2015 Aug 21. [PubMed]

Oral prophylactic therapy by gargling with pathogen-specific egg yolk immunoglobulins (IgY) may reduce the initial airway colonization with Pseudomonas aeruginosa in cystic fibrosis (CF) patients. IgY antibodies impart passive immunisation and the authors investigated the effects of anti-P. aeruginosa IgY antibodies on bacterial eradication in a murine pneumonia model. P. aeruginosa pneumonia was established in Balb/c mice and the effects of prophylactic IgY administration on lung bacteriology, clinical parameters and subsequent inflammation were compared to controls.

Prophylactic administration of IgY antibodies targeting P. aeruginosa significantly reduced the bacterial burden by 2-log 24h post-infection compared to controls and was accompanied by significantly reduced clinical symptom scores and successive inflammatory cytokine profile indicative of diminished lung inflammation.Passive immunization by anti-P. aeruginosa IgY therapy facilitates promptly bacterial clearance and moderates inflammation in P. aeruginosa lung infection and may serve as an adjunct to antibiotics in reducing early colonization

– The results of a European trial of IgY therapy in people with CF which started in 2012 are awaited in 2019.

I had been unable to find any further information on the trial until late 2023 when  I contacted Dr Audrey Niemann Jönsson, (Co0ordinator, IMPACTT Project Coordination Office) who told me “There was no significant difference between placebo and the IgY, in the trial, I believe because the placebo (based on egg yellow extract) showed some effect as the IgY did. It was an interesting study which luckily developed good research in parallel. But of course we are disappointed.

Final Report Summary – IMPACTT (Immunoglobulin IgY pseudomonas A clinical trial for cystic fibrosis treatment) [more a description of the trial] Executive Summary: IMPACTT was supported by the European Union Seventh Framework Programme. IMPACTT started on January 1st 2011 as a collaboration between ten partners from seven European countries: Sweden Germany Denmark Belgium France Italy and Lithuania. The project has since expanded to include other countries which were financed by a Swedish National Grant and the ECRIN I-A grant in 2013 and 2014. The project’s primary goal was to provide an intervention therapy for prophylaxis and treatment of P. aeruginosa (PA) infections in CF patients. Anti-Pseudomonas IgY the drug candidate has an Orphan Drug Designation (European Medicines Agency (EMA) September 2008) and the project would provide technical data for an Orphan Drug registration for the avian antibody-based Drug Candidate Anti-Pseudomonas IgY. This technical data is a requirement for marketing authorization and for making the treatment available for CF patients within EU. The IMPACTT Project Coordinator is Professor Anders Larsson at the Uppsala Academic Hospital in Sweden (phone: +46 18 611-4271; Email: anders.larsson@akademiska.se). The Clinical Study Sponsor is Mukoviszidose Institut (MI) whose Scientific Manager WP2 leader and Global Project Manager for Anti-Pseudomonas IgY (also referred to as PsAer-IgY) (GPM) is Dr. Jutta Bend (email jbend@muko.infoor phone: +49 228 98780-47.

The clinical phase III study investigated the critical preventive and therapeutic effects of anti-Pseudomonas IgY in CF patients, by assessing the time to reinfection with PA after successfully treated acute or intermittent infection. The primary endpoint is the time to re-infection with PA. The samples are analyzed in the Central Lab in Copenhagen (Prof. Niels Høiby’s group) throughout the clinical study.
Anti-Pseudomonas IgY is the Drug candidate under investigation in all the IMPACTT work packages (WPs). IMPACTT has eight WPs, which supported either pre-clinical or clinical research.

The chore part of IMPACTT the pan-European clinical trial phase III is headed by the Sponsor MI WP2 leader. WP2 provided the infrastructure necessary for the clinical trial and ensured that the clinical study (ClinicalTrials.govIdentifier:NCT01455675) is performed efficiently with highest quality and according to GCP and the national and international regulations. Its goal was to investigate the recurrence of PA in the sputum of the patients who gargle with Anti-Pseudomonas IgY or a placebo formulae for two years. The clinical trial recruited 164 patients from 9 European countries: Germany Sweden Italy Belgium Ireland Poland Hungary Spain and Austria. The five newest countries were added with an Amendment to the EC GA in 2013. The project was prolonged by an amendment II to the EC Grant Agreement on2014-12-09 by two years. The recruitment was closed June 2015.The clinical study has been registered in the international clinical trial registry clinicaltrials.gov.

Pseudomonas Vaccines

2004 Lang AB, Rudeberg A, Schoni MH, Que JU, Furer E, Schaad UB. Vaccination of cystic fibrosis patients against Pseudomonas aeruginosa reduces the proportion of patients infected and delays time to infection. Pediatr Infect Dis J 2004; 23:504-510. [PubMed]
In 1989-1990, 30 young children with CF, mean age 7 years, with no prior history of infection with P. aeruginosa, were vaccinated against P. aeruginosa with a polyvalent conjugate vaccine. This is a report the follow-up of 26 of these patients from 1989 to 2001. The time to infection with P. aeruginosa was longer in the vaccination group than in the control group, and fewer vaccinated patients than controls became chronically infected (32% versus 72%; P < 0.001). The proportion of mucoid infections was higher in the control group (44%) than in the vaccinated group (25%). Patients >/=18 years of age at the end of the study had a lower mean forced expiratory volume at 1 s (FEV1) than did those 13-17 years of age, but this difference was small in the vaccinated group (73.6% versus 83.7%) compared with the controls (48.0% versus 78.7%). In the >/=18 year age category the mean FEV1% at 10 years was 73.6% (vaccinated) and 48.0% (controls) (P < 0.05). In the vaccinated group only 11 (44%) of 25 patients were underweight at the 10-year follow-up compared with 18 (72%) of 25 at the beginning of the study. In the control group 17 (68%) of 25 patients were underweight at 10-year follow-up compared with 16 (64%) of 25 at the beginning of the study.

Despite these encouraging results a subsequent large multicentre international European Phase III study failed to show any difference between the vaccinated and control children. This was attributed to the general fall in the prevalence of P. aeruginosa infections with increasing early eradication treatment. The results were a major disappointment to all concerned.

2007 Döring G, Meisner C, Stern M. for the Flagella Vaccine Trial Study Group. A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proceedings of the National Academy of Sciences of the United States of America 2007; 104:11020-5. [PubMed]
In a double-blind, placebo-controlled, multicenter trial, 483 European patients, 2-18 years of age without P. aeruginosa colonization were randomly assigned to receive four intramuscular injections of a bivalent P. aeruginosa flagella vaccine or placebo over a 14-month period. Patients were evaluated quarterly for P. aeruginosa-positive throat cultures and antipseudomonal serum antibody titers during the study period of 2 years. The vaccine was well tolerated, and the patients developed high and long-lasting serum antiflagella IgG titers.

In the intention-to-treat group (all patients enrolled), 82 of 239 vaccinated patients had P. aeruginosa infection and/or antipseudomonal serum titers compared with 105 of 244 patients in the placebo group (P = 0.05; relative risk: 0.80; 95% CI: 0.64-1.00). Analysis of the 381 patients in the per-protocol group, who received all four vaccinations or placebo treatments, revealed 37 of 189 patients with infection episodes in the vaccine group compared with 59 of 192 patients with such episodes in the placebo group (P = 0.02; relative risk: 0.66; 95% CI: 0.46-0.93). P. aeruginosa strains, exhibiting flagella subtypes included in the vaccine, were significantly less frequently isolated from vaccinates than from placebo controls (P = 0.016, relative risk: 0.319; 95% CI: 0.12-0.86). Chronic P. aeruginosa infection was rare because of recent institution of early antibiotic eradication regimes. Active immunization of patients with cystic fibrosis lowers the risk for infection with P. aeruginosa and therefore may contribute to a longer survival of these patients.

As in a similar trial of vaccine to protect against Pseudomonas (Laang et al. above) the chance of showing a convincing result was reduced by the widespread use of early eradication regimens resulting in a falling prevalence of chronic Pseudomonas infection. There is no regular vaccination against P. aeruginosa even in 2018.

2011 Sharma A, Krause A, Worgall S. Recent developments for Pseudomonas vaccines. Hum Vaccin 2011; 7:999-1011.[PubMed] Full article available. Infections with Pseudomonas aeruginosa are a major health problem for immune-compromised patients and individuals with cystic fibrosis. A vaccine against: P. aeruginosa has long been sought after, but is so far not available. Several vaccine candidates have been assessed in experimental animals and humans, which include sub-cellular fractions, capsule components, purified and recombinant proteins. Unique characteristics of the host and the pathogen have complicated the vaccine development. This review summarizes the current state of vaccine development for this ubiquitous pathogen, in particular to provide mucosal immunity against infections of the respiratory tract in susceptible individuals with cystic fibrosis.

This is a relatively recent comprehensive review of Pseudomonas vaccines. The full free text is available via the PubMed reference.

Meynet E, Laurin D, Lenormand JL, Camara B, Toussaint B, Le Gouëllec A. Killed but metabolically active Pseudomonas aeruginosa-based vaccine induces protective humoral- and cell-mediated immunity against Pseudomonas aeruginosa pulmonary infections. Vaccine. 2018 Mar 27;36(14):1893-1900. doi: 10.1016/j.vaccine.2018.02.040. Epub 2018 Mar 2. [Pubmed]
In this study, the authors developed an innovative and safer live-attenuated Pa vaccine based on a Killed But Metabolically Active (KBMA) attenuation method. KBMA Pa has been further rationally designed to overexpress beneficial effectors like the type 3 secretion system apparatus. They demonstrated that KBMA Pa elicits a high and broad humoral response in mice against several antigens of particular interest such as OprF and PcrV proteins. Moreover, they assessed cytokines in the serum of immunized mice and showed that KBMA Pa elicits Th1, Th2 and especially Th17 pathways of cell-mediated immune responses. Th17 pathway involvement was also confirmed after specific stimulation of helper T cells in immunized mice. Finally, they showed that this vaccine is safe and has a protective effect in a murine acute pulmonary infectious challenge. In conclusion, they consider that KBMA Pa is a new platform with high potential for the development of a vaccine against Pa.

– All previous vaccines against P. aeruginosa have been ineffective including one of the most promising, that of Dr. A B Lang and colleagues (Lang AB et al, Pediatr Infect Dis 2004; 23:504-510) where the apparently impressive results of their preliminary study prompted an international multicentre trial of the vaccine. Unfortunately the results of this major trial were entirely negative – there being no difference between the treated and control groups either in the prevalence of chronic P. aeruginosa infection or the number of new positive Pseudomonas respiratory cultures. It was possible that the increasing use of anti-Pseudomonas eradication therapy and the fall in the prevalence of chronic Pseudomonas infection in clinics (and the opportunity to become infected) contributed to the negative results (unpublished data).


Michael J Stephen, Jared Chowdhury, Luis Arzeno Tejada, Robert Zanni , Denis Hadjiliadis. Use of medical marijuana in cystic fibrosis patients. BMC Complement Med Ther 2020 Oct 27;20(1):323 doi: 10.1186/s12906-020-03116-x. Free PMC article [Pubmed]

Michael J Stephen

Background: The usage and attitudes towards medical marijuana in Cystic Fibrosis (CF) patients is unknown. Through the use of a survey we aim to clarify rates and reasons for use.
Methods: An anonymous survey was sent out to six centers in the Mid-Atlantic region of the United States. Use of and reason for medical marijuana was assessed, along with attitudes of the perceived utility of medical marijuana.
Results: A total of 637 surveys were sent out, and 193 surveys were returned (30.3% return rate). Three did not give consent, and one was empty, for a total of 189 completed surveys. 31 subjects (16.5%) reported having used marijuana for medical purposes in their lifetime, with 29 (15.4%) of these in the past year. The most used forms were edible and vaporized. The most common indications for usage were pain and stress. 28 out of 31 found marijuana to be a great deal effective for their symptoms. 21 of the 31 rated marijuana very important or important to their health. There were two reported side effects, both mild. Of 156 subjects who responded to the question if they would be interested in medical marijuana if available, 72 (46.2%) replied yes.
Conclusion: The use of marijuana for medical reasons was 15.4% in the past year in this sample CF population, although more expressed interest if it was available through prescription. Side effects were rare. CF physicians are going to have to familiarize themselves with advantages and disadvantages of medical marijuana as there is a great deal of interest within the community, and legalization becomes more common.

Dr Michael J. Stephen is an associate professor at Thomas Jefferson University in Philadelphia and director of the Adult Cystic Fibrosis Center.


Ballmann M, Junge S, von der Hardt H. Low-dose methotrexate for advanced pulmonary disease in patients with cystic fibrosis. Respir Med 2003; 97:498-500. [PubMed]
In the year before starting with low dose methotrexate (MTX), FEV1% decreased (median: 10% FEV1; range 9-15% P<0.005) after starting with MTX, FEV1% increased (median: 9% FEV1; range: 2-15% P<0.05). The IgG changed (median: -2 g/l; range: 0.2 to -7.3 g/l) in the first year with MTX.

The authors suggest that these preliminary data suggest a beneficial effect of MTX even in advanced pulmonary disease in CF patients.

Moli 1901- Duramycin Lancovutide

2004 Zeitlin PL, Boyle MP, Guggino WB, Molina L. A phase I trial of intranasal Moli1901 for cystic fibrosis. Chest 2004; 125:143-149. [PubMed]
The peptide drug Moli1901 activates an alternative chloride channel that is present in cystic fibrosis (CF) nasal and airway epithelia. Doing so bypasses the dysfunctional CF transmembrane regulator. Changes in the nasal potential difference (NPD) induced by chloride-free, amiloride-containing Ringers solution and by subsequent superfusion with the same solution plus 10 micro mol/L isoproterenol were consistent with both an acute and a sustained change in chloride transport in response to Moli1901. A similar analysis of nasal potential difference in the four CF participants demonstrated an acute response that resolved more quickly. A dose-response relationship to Moli1901 was observed in non-CF participants, but a greater range of variability within the CF participants contributed to the lack of a clear dose-response relationship in this group.

The authors concluded that Moli1901 stimulates chloride transport in normal and CF nasal epithelia in vivo, but may have a shorter duration of action in CF participants

2007 Grasemann H, Stehling F, Brunar H, Widmann R, Laliberte TW, Molina L, Doring G, Ratjen F. Inhalation of Moli 1901 in patients with cystic fibrosis. Chest 2007; 131:1461-1466. [PubMed]
Moli1901 stimulates an alternative chloride channel and may thus compensate for the CFTR deficiency in the airway epithelium of CF patients. A phase II, placebo-controlled, double-blinded, single-center, multiple (5 consecutive days), rising-dose (daily dose, 0.5, 1.5, or 2.5 mg of Moli1901) study in Germany was conducted to investigate the safety and tolerability of multiple doses of aerosolized inhaled Moli1901 in 24 patients with CF and stable lung disease. Moli1901 was well tolerated in all but one CF patient, in whom a transient significant decrease in FEV1 developed following inhalation, which resolved spontaneously, and in a second patient in whom transient throat numbness developed during drug inhalation.
A significant improvement of FEV1 was observed in the group receiving treatment with 2.5 mg/d Moli1901 compared to the group receiving placebo. Moli1901 was not detected in the plasma of the highest dose group.
The authors concluded that the inhalation of Moli1901 up to a total cumulative dose of 12.5 mg appears to be safe in adult patients with CF. In addition, Moli1901 had a sustained beneficial effect on pulmonary function, which supports further studies of its efficacy in CF patients.

Further studies with the drug, now called duramycin, followed and suggested that duramycin was suitable for intrapulmonary administration in cystic fibrosis. (Steiner I et al. Naunyn Schmiedebergs Arch Pharmacol 2008; 378:323-333.[PubMed]). Further in vitro work suggested that duramycin stimulated Cl efflux by inducing non-specific changes in the cell membrane rather than specific effects on the calcium activated chloride channels (Oliynyk I et al. 2010; APMIS: 118:982-990.[PubMed]).

Apparently although these early studies were held some promise for Duramycin (Moli1901) as a chloride channel activator, further development was not been persued due to “formulation issues”. However, a more recent review to access duramycin and it’s analogs by production in E. coli opens opportunities to improve duramycin as an antibiotic, phosphatidethanolamine probe, antiviral, or cystic fibrosis therapeutic (Huo L et al. Insights into the biosynthesis of Duramycin. Appl. Environ Microbiol2017; 83 (3). 27864176 [PubMed]. Free Full text).

Miglustat (N-butyldeoxynojyrimicin. Zavesca)

2008 Dechecchi MC, Nicolis E, Norez C, Bezzerri V, Borgatti M, Mancini I, Rizzotti P, Ribeiro CM, Gambari R, Becq F, Cabrini G. Anti-inflammatory effect of miglustat in bronchial epithelial cells. J Cyst Fibros 2008; 7:555-565.[PubMed]
The role of CFTR deficiency in promoting inflammation remains unclear. Recently Perez et al. (Perez A et al. 2007. [PubMed]) demonstrated that the inhibition of function of w/t CFTR produces an inflammatory profile that resembles that observed in CF patients, whereas the present authors found that correction of F508del-CFTR function with MPB-07 down-modulates the inflammatory response to P. aeruginosa in CF bronchial cells (Dechecchi MC et al. 2007.[PubMed]).

Since both evidence support a link between CFTR function and inflammation, they extended their investigation to other F508del-CFTR correctors, such as miglustat (Norez et al, 2006.[PubMed]), an approved drug for Gaucher disease, in comparison with the galactose analogue NB-DGJ.

They report here that miglustat but not NB-DGJ restores F508del-CFTR function in CF bronchial epithelial IB3-1 and CuFi-1 cells. Miglustat and NB-DGJ reduce the inflammatory response to P. aeruginosa in both CF and non-CF bronchial cells, indicating that the anti-inflammatory effect is independent of the correction of F508del-CFTR function. Miglustat also inhibits the inflammatory response induced by the supernatant of mucopurulent material obtained from the lower airway tract of cystic fibrosis patients with chronic bacterial colonization (Ribeiro, 2005). Both compounds do not interfere with the adherence of P. aeruginosa to the cells and reduce the expression of IL-8 not only after challenge with P. aeruginosa but also after exposure to TNF alpha or IL-1 beta, suggesting an effect on transduction proteins downstream and in common with different receptors for pathogens. Finally, miglustat has no major effects on overall binding activity of transcription factors NF-kappaBNF-kB and AP-1.

Since miglustat is an approved drug, it could be investigated as a novel anti-inflammatory molecule to ameliorate lung inflammation in CF patients. Increasing interest in miglustat since the first report of its effect on CF cells by Norez et al, 2006 [PubMed]. It is of particular interest as a “low hanging fruit” drug i.e. one that is already licensed for the treatment of Gaucher’s disease. This is a helpful review.

2009 Norez C, Antigny F, Noel S, Vandebrouck C, Becq F. A cystic fibrosis respiratory epithelial cell chronically treated by miglustat acquires a non-cystic fibrosis-like phenotype. Am J Respir Cell Mol Biol 2009; 41(2):217-225. [PubMed]
Miglustat (N-butyldeoxynojirimycin, Zavesca), an inhibitor of the alpha-1,2 glucosidase, has been proposed for clinical use in CF because of its effect as a corrector of the defective trafficking of F508del-CFTR. In the present study daily treatment for 2 months with low concentrations of miglustat on the human CF nasal epithelial cell line, JME/CF15 (F508del/F508del-CFTR), results in progressive, stable, reversible, and sustained correction of F508del-CFTR trafficking, down-regulation of sodium hyperabsorption, and regulation of the calcium homeostasis.

– The authors provide the first evidence that a respiratory CF cell can acquire a non-CF-like phenotype when chronically treated with low concentrations of a pharmacological drug.

*2012 Leonard A, Lebecque P, Dingemanse J, Leal T. A randomized placebo-controlled trial of miglustat in cystic fibrosis based on nasal potential difference. J Cyst Fibros 2012; 11:231-236.[PubMed]

Single-center, randomized, double-blind, placebo-controlled, crossover Phase II study in 11 patients (mean±SD age, 26.3±7.7 years) homozygous for the F508del mutation received oral miglustat 200 mg t.i.d. or placebo for two 8-day cycles separated by a 14-day washout period. The primary endpoint was the change in total chloride secretion (TCS) assessed by nasal potential difference. No statistically significant changes in TCS, sweat chloride values or FEV(1) were detected. Pharmacokinetic and safety were similar to those observed in patients with other diseases exposed to miglustat.

There was no evidence of a treatment effect on any nasal potential difference variable. The authors suggested that further studies with miglustat need to adequately address criteria for assessment of nasal potential difference. A miglustat study of longer duration and possible combination therapy with a potentiator compound could be considered.

Phosphodiesterase Inhibitors

2011 Lubamba B, Lebacq J, Reychler G, Marbaix E, Wallemacq P, Lebecque P, Leal T. Inhaled phosphodiesterase type 5 inhibitors restore chloride transport in cystic fibrosis mice. Eur Respir J 2011; 37:72-78.[PubMed]
Sildenafil and vardenafil, two selective inhibitors of phosphodiesterase type 5 (PDE5) are able, when applied by intraperitoneal injection, to activate chloride transport in cystic fibrosis (CF) mice homozygous for the F508del mutation. Oral treatment with the drugs may be associated with adverse haemodynamic effects. the authors hypothesised that inhaled PDE5 inhibitors are able to restore ion transport in F508del CF airway epithelium. They developed a restraint-free mouse chamber for inhalation studies. PDE5 inhibitors were nebulised for 15 min at concentrations adjusted from recommended therapeutic oral doses for male erectile dysfunction. They measured in vivo nasal transepithelial potential difference 1 h after a single inhalation of sildenafil, vardenafil or tadalafil in F508del CF and normal homozygous mice.

After nebulisation with the drugs in F508del mice, chloride transport, evaluated by perfusing the nasal mucosa with chloride-free buffer containing amiloride followed by forskolin, was normalised; the forskolin response was increased, with the largest values being observed with tadalafil and intermediate values with vardenafil. No detectable effect was observed on sodium conductance.

The authors’ results confirm the role of PDE5 inhibitors in restoring chloride transport function of F508del CF transmembrane conductance regulator protein and highlight the potential of inhaled sildenafil, vardenafil and tadalafil as a therapy for CF.

Publications on the effect of phosphodiesterase inhibitors such as sildenafil in CF have appeared since 2003 (Cobb BR et al. Am J Resp Cell Mol 2003; 29:410. [PubMed]) including a study from Wales of the effect of sildenafil on nasal cells from people with CF (Dormer RL et al. Thorax 2005; 60:55-59.[PubMed]). Most subsequent work seems to have been carried out on cells or CF mice.

2012 Lubamba B, Huaux F, Lebacq J, Marbaix E, Dhooghe B, Panin N, Wallemacq P, Leal T. Immunomodulatory activity of vardenafil on induced lung inflammation in cystic fibrosis mice. J Cyst Fibros 2012; 11:266-273. [PubMed]
The authors tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pre-treatment with vardenafil (0.14 mg/kg) was evaluated. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1β was observed in the CF group only. Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.

– This is a very detailed paper. The authors suggest that inflammation may occur in the absence of infection; that their data confirm the inflammatory responses are exaggerated in CF. Vardenafil in vivo in a clinical dose, blocks inflammation induced by P. aeruginosa endotoxin suggesting it may have a therapeutic potential. It is almost 10 years since the effects of various phosphodiesterase inhibitors in CF was described but it seems there is still some way to go before any of this group of drugs comes into clinical trials for CF.

*2012 Noel S, Dhooghe B, Leal T. PDE5 Inhibitors as Potential Tools in the Treatment of Cystic Fibrosis. Front Pharmacol 2012;3:167. Epub 2012 Sep 18.[PubMed]Free article available
Using phosphodiesterase type 5 (PDE5) inhibitors, these authors and others have provided evidence of rescued F508del-CFTR trafficking and corrected deficient chloride transport activity. Studies using PDE5 inhibitors in mice homozygous for the clinically relevant F508del mutation have been conducted with the aim of restoring F508del-CFTR protein function. They demonstrated, by measuring transepithelial nasal potential difference in F508del mice following intraperitoneal injection of sildenafil, vardenafil, or taladafil at clinical doses are able to restore the decreased CFTR-dependent chloride transport across the nasal mucosa. Moreover, vardenafil, but not sildenafil, stimulates chloride transport through the normal CFTR protein. They developed a specific nebulizer setup for mice, with which we demonstrated, through a single inhalation of PDE5 inhibitors, local activation of CFTR protein in CF. Significant potential advantages of inhalation drug therapy over oral or intravenous routes include rapid onset of pharmacological action, reduced systemic secondary effects, and reduced effective drug doses compared to the drug delivered orally; this underlines the relevance and impact of our work for translational science. More recently, they analyzed the bronchoalveolar lavage of CF and wild-type mice for cell infiltrates and expression of pro-inflammatory cytokines and chemokines; they found that the CFTR activating effect of vardenafil, selected as a representative long-lasting PDE5 inhibitor, breaks the vicious circle of lung inflammation which plays a major role in morbi-mortality in CF. Their data highlight the potential use of PDE5 inhibitors in CF. Therapeutic approaches using clinically approved PDE5 inhibitors to address F508del-CFTR defects could speed up the development of new therapies for CF.

*2012 Leier G, Bangel-Ruland N, Sobczak K, Knieper Y, Weber WM. Sildenafil acts as potentiator and corrector of CFTR but might be not suitable for the treatment of CF lung disease. Cell Physiol Biochem 2012; 29:775-790.[PubMed]
The phosphodiesterase-5 inhibitor sildenafil is an established and approved drug to treat symptoms of a variety of human diseases. In the context of cystic fibrosis (CF), a genetic disease caused by a defective CFTR gene (e.g. ΔF508-CFTR), it was assumed that sildenafil could be a promising substance to correct impaired protein expression. This study focuses on the molecular mechanisms of sildenafil on CFTR recovery. The authors used ΔF508-CFTR/wt-CFTR expressing Xenopus laevis oocytes and human bronchial epithelial cell lines (CFBE41o(-)/16HBE14o(-)) to investigate the pathways of sildenafil action. Cells were treated with sildenafil and cAMP-mediated current (I(m)), conductance (G(m)), and capacitance (C(m)) were determined. Sildenafil increased I(m), G(m), and C(m) of wt-CFTR and functionally restored ΔF508-CFTR in oocytes. These effects were also seen in CFBE41o(-) and 16HBE14o(-) cells. Transepithelial measurements revealed that sildenafil mediated increase (wt-CFTR) and restoration (ΔF508-CFTR) of channel activity. cGMP pathway blocker inhibited the activity increase but not CFTR/ΔF508-CFTR exocytosis.

From these data they conclude that sildenafil mediates potentiation of CFTR activity by a cGMP-dependent and initiates cGMP-independent functional insertion of CFTR/ΔF508-CFTR molecules into the apical membranes. Thus, sildenafil is a corrector and potentiator of CFTR/ΔF508-CFTR. Yet, the necessary high doses of the drug for CFTR recovery demonstrate that sildenafil might not be suited as a therapeutic drug for CF lung disease.

2015 Taylor-Cousar JL; Wiley C; Felton LA; St Clair C; Jones M; Curran-Everett D; Poch K; Nichols DP; Solomon GM; Saavedra MT; Accurso FJ; Nick JA. Pharmacokinetics and tolerability of oral sildenafil in adults with cystic fibrosis lung disease. J Cyst Fibros 2015; 14(2):228-36. [PubMed]
Pre-clinical models have shown that phosphodiesterase inhibitors (PDEi) like sildenafil have anti-inflammatory activity. An open-label pilot study of oral sildenafil administration was conducted in adults with mild to moderate CF lung disease. Subjects received oral sildenafil 20 or 40 mg p.o. t.i.d. for 6 weeks. Twenty subjects completed the study. Subjects with CF may eliminate sildenafil at a faster rate than non-CF subjects. The drug was safe and decreased sputum elastase activity. The authors suggested further study of sildenafil as an anti-inflammatory in cystic fibrosis.

– See also Topics -> Drug modulation, New drugs -> phosphodiesterase inhibitors. Also sildenafil is mentioned in various contexts in this History (pulmonary hypertension, activation of chloride transport) and these can be found via ‘Site Search’ in the Main Menu.

2018 Rodriguez Miguelez P, Seigler N, Tucker MA, Csanyi G, McKie KT, Forseen C, Harris RA. Sildenafil improves vascular endothelial function in patients with cystic fibrosis. Am J Physiol Heart Circ Physiol.2018 Aug 31. doi: 10.1152/ajpheart.00301.2018. [Epub ahead of print] [Pubmed]

Paula Rodriguez-Miguelez

Cystic fibrosis (CF), characterized by defective CFTR function, is associated with multiple systemic complications including vascular dysfunction. Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, not only enhances nitric oxide (NO) metabolism, it has been shown to improve CFTR functionality as well. Thus, sildenafil has been proposed as a potential therapy to improve vascular health in CF; however, its potential therapeutic role has yet to be determined.

To investigate the effect of sildenafil treatment on endothelial function in patients with CF.

Patients with CF completed a randomized, double blind, placebo controlled, crossover study with an acute dose of sildenafil (50 mg) and placebo followed by a four-week, open label extension with sildenafil (20 mg/day). Flow-mediated dilation (FMD) was used to evaluate endothelial function before and after treatments. In addition, phosphorylated (p) and total endothelial NO synthase (NOS3) protein expression was determined from endothelial cells that were exposed to plasma from the patients before and after four weeks of sildenafil.

No changes (p≥0.110) in endothelial function were observed after the acute dose of sildenafil. However, FMD significantly (p=0.029) increased after four weeks of treatment (∆FMD= 1.5 ± 2.2%). Moreover, p-NOS3 protein expression significantly (p=0.013) increased following 4 weeks of treatment (∆p-NOS3= 0.31 ± 0.39 AU) and was associated (r=0.593; p=0.033) with the observed change in FMD.

These data suggest that four weeks of sildenafil treatment can improve vascular endothelial function in patients with CF, likely through an increase in NOS3 phosphorylation.

Paula Rodriguez-Miguelez is Assistant Professor of Health Science, Department of Kinesiology and Health Sciences at Virginia Commonwealth University, Richmond


2007 Bruzzese E, Raia V, Spagnuolo MI, Volpicelli M, De Marco G. Maiuri L, Guarino A. Effect of Lactobacillus GG supplementation on pulmonary exacerbations in patients with cystic fibrosis. Clinical Nutrition 2007; 26:322-328. [PubMed] Nineteen children with CF received a probiotic Lactobacillus GG (LGG) for 6 months and then changed to a placebo of oral rehydration solution (ORS) for 6 months; in parallel nineteen received ORS and then changed to LGG. Patients treated with LGG showed a reduction of pulmonary exacerbations (Median 1 vs. 2)and of hospital admissions (Median 0 vs. 1, range 3 vs. 2,). LGG resulted in a greater increase in FEV1 (3.6% +/- 5.2 vs. 0.9% +/- 5; p=0.02) and body weight (1.5 kg +/- 1.8 vs. 0.7 kg +/- 1.8; p=0.02). The authors concluded that Lactobacillus GG reduces pulmonary exacerbations and hospital admissions in patients with CF, that probiotics may delay respiratory impairment and that a relationship exists between intestinal and pulmonary inflammation.

Bruzzese E et al had already shown that children with CF had evidence of intestinal inflamation as judged by faecal calprotectin levels and that these were reduced when they were given lactobacillus GG (Bruzzesse E et al. Aliment Pharmacol Ther 2004; 20: 813-819.[PubMed]).

There is an increasing interest on the part of patients, parents and doctors in the role probiotics in treating people with CF (Borowitz D et al, J Pediatr Gastroenterol Nutr 2005; 41:273-285. [PubMed]); however, as yet, few CF clinics advise their routine use, perhaps because the evidence of their value is still sparse but also there are so many other components to treatment that there is reluctance to add yet another medicine to gain a marginal benefit.

2010 Weiss B, Bujanover Y, Yahav Y, Vilozni D, Fireman E, Efrati O. Probiotic supplementation affects pulmonary exacerbations in patients with cystic fibrosis: a pilot study. Pediatr Pulmonol 2010; 45:536-540. [PubMed]
Probiotics reduce intestinal inflammation in, and Lactobacillus GG (LGG) reduces pulmonary exacerbation rate cystic fibrosis (CF) patients. the authors intended to determine the effect of a mixed probiotic preparation on pulmonary exacerbations and inflammatory characteristics of the sputum in CF patients. A prospective pilot study of 10 CF patients with mild-moderate lung disease and Pseudomonas aeruginosa colonization, were treated with probiotics for 6 months. Pulmonary function tests (PFT’s), sputum cultures with semi-quantitative bacterial analysis, and sputum neutrophil count and interleukin-8 (IL-8) levels were compared to pre-treatment and post-treatment values. The rate of pulmonary exacerbations was compared to 2 years prior to the study. The exacerbation rate was significantly reduced in comparison to the previous 2 years and to 6 months post-treatment (P = 0. 002). PFT’s have not changed at the end of treatment and during 6 months post-treatment. No change in sputum bacteria, neutrophil count, and IL-8 levels was observed.

The authors concluded that probiotics reduce pulmonary exacerbations rate in patients with CF and may have a preventive potential for pulmonary deterioration in CF patients.

The study involved few patients and there were historical controls, however there is increasing evidence that probiotics may have a favorable effect in people with CF and also in other conditions such as rosacea.

2017 Van Biervliet S, Declercq D, Somerset S.Clinical effects of probiotics in cystic fibrosis patients: A systematic review. Clin Nutr ESPEN.2017 Apr;18:37-43. doi: 10.1016/j.clnesp.2017.01.007. Epub 2017 Feb 22.[PubMed]
This study investigated the current evidence for probiotics as an adjunct to usual therapy for CF. Electronic clinical databases were interrogated for human randomised, controlled, intervention trials (1985-2015) testing the effects of probiotics on clinical endpoints in CF were reviewed. From 191 articles identified in initial searches, six studies met the critical inclusion criteria, and were reviewed in detail. These studies varied in size (n = 22 to 61) but were generally small and showed substantial diversity in protocol, specific probiotic species used and range of clinical outcomes measured. Probiotic administration showed beneficial effects on fecal calprotectin levels, pulmonary exacerbation risk, and quality of life indicators. In one study, such changes were associated with variations in gut microbiota composition. Despite encouraging preliminary results, the limited number of small and highly varied studies to date do not justify the addition of probiotics as an adjunct to current CF treatment protocols. Importantly, very minimal adverse effects of probiotics have been reported

2018 Van Biervliet S, Hauser B, Verhulst S, Stepman H, DelangheJ, Warzee JP, Pot B, Vandewiele T, Wilschanski M.Probiotics in cystic fibrosis patients: A double blind crossover placebo controlled study: Pilot study from the ESPGHAN Working Group on Pancreas/CF. Clin Nutr ESPEN. 2018 Oct;27:59-65. doi: 10.1016/j.clnesp.2018.06.008. Epub 2018 Jul 20 [Pubmed]

Stephanie Van Biervliet

A potential positive effect of probiotics in cystic fibrosis (CF) on fecal calprotectin (FCP), pulmonary exacerbations and weight has been described in small controlled trials. A double-blind multicenter cross-over study (2 × 4 m) was performed looking at abdominal pain, nutritional status, pulmonary function, pulmonary exacerbation, FCP and lactulose/mannitol gut permeability test. Patients kept a diary with daily scoring of abdominal pain, stool frequency and consistency as well as treatment changes.

31 CF patients entered the study of which 25 finished it. At start patients aged 9.3yrs (6.9-12.2), had a median BMI z-score of -0.5 (-1.5-0.08), height z-score of -0.4 (-1.1-0.05) and FEV1% of 100% (87.2-106.6). Median FCP at start was 61 μg/g (17-108) and gut permeability 0.079 (0.051-0.122). No significant changes were observed in the clinical parameters (BMI, FEV1%, abdominal pain, exacerbations). Despite being frequently abnormal (17/28 (61%) >50 mg/kg), FCP did not change significantly with probiotics. The proportion of patients with normal permeability was 8% during placebo and 32% during probiotic treatment (p = 0.031). FCP correlated to BMI z-score (p = 0.043) and gut permeability to abdominal pain (p = 0.015). The microbiome revealed a high predominance of Actinobacteria and Proteobacteriae. Probiotic supplementation did not result in a shift at the phylum nor at phylogenetic level.

The authors concluded that normalization of gut permeability was observed in 13% of patients during probiotic treatment. However, none of the previously described effects on fecal calprotectin (FCP), pulmonary exacerbations and weight, which have been described in previous small controlled trials, could be confirmed.

Prof.dr.Stephanie Van Biervliet works in the Department of Paediatrics and Medical Genetics Ghent University Hospital, and is coordinator of the CF centre.

Sodium/glucose cotransporters

Hiroyuki HiraiXiubin LiangYifei SunYihan ZhangJifeng ZhangY Eugene ChenHongmei MouYouyang ZhaoJie XuThe sodium/glucose cotransporters as potential therapeutic targets for CF lung diseases revealed by human lung organoid swelling assay.  Mol Ther Methods Clin Dev2021 Nov 24;24:11-19.doi: 10.1016/j.omtm.2021.11.008.eCollection 2022 Mar 10.   Free PMC article [Pubmed]

Hiroyuki Hiral

Cystic fibrosis (CF) is a lethal autosomal-recessive inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the present work, we derived human proximal lung organoids (HLOs) from patient-derived pluripotent stem cells (PSCs) carrying disease-causing CFTR mutations. We evaluated the forskolin (Fsk)-stimulated swellings of these HLOs in the presence of CFTR modulators (VX-770 and/or VX-809) and demonstrated that HLOs respond to CFTR modulators in a mutation-dependent manner. Using this assay, we examined the effects of the sodium-dependent glucose cotransporter 1/2 (SGLT1/2) inhibitor drugs phlorizin and sotagliflozin on the basis of our findings that SGLT1 expression is upregulated in CF HLOs and airway epithelial cells compared with their wild-type counterparts. Unexpectedly, both drugs promoted dF/dF HLO swelling. These results reveal SGLTs, especially SGLT1, as potential therapeutic targets for treating CF lung diseases and demonstrate the use of PSC-derived HLOs as a preclinical tool in CF drug development.

Dr Hiroyuki Hirai is Research Assistant Professor of Pediaitrics at the Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI 48109, USA.
Program for Lung and Vascular Biology, Stanley Manne Children’s Research Institute, Ann & Robert H. Lurie Children’s Hospital of Chicago, Division of Critical Care, Department of Pediatrics, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Chicago, IL 60611, USA.


Giulia GorrieriFederico ZaraPaolo ScudieriSLC26A9 as a Potential Modifier and Therapeutic Target in Cystic Fibrosis Lung DiseaseBiomolecules  2022 Jan 25;12(2):202.doi: 10.3390/biom12020202. Free article   [Pubmed]

      Giulia Gorrieri

SLC26A9 belongs to the solute carrier family 26 (SLC26), which comprises membrane proteins involved in ion transport mechanisms. On the basis of different preliminary findings, including the phenotype of SlC26A9-deficient mice and its possible role as a gene modifier of the human phenotype and treatment response, SLC26A9 has emerged as one of the most interesting alternative targets for the treatment of cystic fibrosis (CF). However, despite relevant clues, some open issues and controversies remain. The lack of specific pharmacological modulators, the elusive expression reported in the airways, and its complex relationships with CFTR and the CF phenotype prevent us from conclusively understanding the contribution of SLC26A9 in human lung physiology and its real potential as a therapeutic target in CF.

In this review, we summarized the various studies dealing with SLC26A9 expression, molecular structure, and function as an anion channel or transporter; its interaction and functional relationships with CFTR; and its role as a gene modifier and tried to reconcile them in order to highlight the current understanding and the gap in knowledge regarding the contribution of SLC26A9 to human lung physiology and CF disease and treatment.

Giulia Gorrieri  is a PhD student in the Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DiNOGMI), University of Genoa, 16132 Genoa, Italy. Medical Genetics Unit, IRCSS Giannina Gaslini Institute, 16147 Genoa, Italy

Jiafen GongGengming HeCheng WangClaire BartlettNaim PanjwaniScott Mastromatteo and 34 co-authors from Canada.Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung diseaseNPJ Genom Med 2022 Apr 8;7(1):28.doi: 10.1038/s41525-022-00299-9.   Free article [Pubmed]

     Jiafen Gong

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.

Dr Jiafen Gong is a biostatistician with the Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.


2017 Romani L, Oikonomou V, Moretti S, Iannitti RG, D’Adamo MC, Villella VR, Pariano M, Sforna L, Borghi M, Bellet MM, Fallarino F, Pallotta MT, Servillo G, Ferrari E, Puccetti P1, Kroemer G, Pessia M, Maiuri L, Goldstein AL, Garaci E. Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis. Nat Med. 2017 May;23(5):590-600. doi: 10.1038/nm.4305. Epub 2017 Apr 10. [Pubmed]

Luigina Romani

To date, no individual drug with pleiotropic beneficial effects is available for CF. Here the authors report on the ability of thymosin alpha 1 (Tα1), (a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent), to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the most common p.Phe508del mutation. Tα1 displayed two combined properties that favourably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for

– Apparently Tα1 is not currently available in the U.S. but it has been approved for clinical use for over 15 years in 35 countries in the treatment of other medical conditions (not CF) such as viral infections, immunodeficiency diseases, malignancies, and HIV/AIDS and has a good safety profile. Understandably there is considerable interest in this potential treatment for CF.

Professor Luigina Romani (figure) is Chair of the Department of Pathology, University of Perugia, and is an internationally recognised authority in the area of anti-fungal immunity.Professor Allan L Goldstein is Professor Emeritus in Residence of Biochemistry and Molecular Medicine at the George Washington School of Medicine and Health Sciences. He is a world authority on the thymus gland and the immune system and co-discoverer of thymosins. Professor Enrico Garaci is a distinguished scientist and Rector and Professor at the University of Rome. His research has mainly focused on the effects of specific and non-specific immune factors especially in viral diseases. He has held a number of distinguished national appointments.

– Although this the prospect of this drug having a role in CF treatment sounds encouraging there have been subsequent publications questioning the findings.

2018 Tomati V, Caci E, Ferrera L, Pesce E, Sondo ECholon DM, Quinney NL, Boyles SE, Armirotti ARavazzolo R, Galietta LJ, Gentzsch M, Pedemonte N. Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia JCI Insight. 2018 Feb 8;3(3). pii: 98699. doi: 10.1172/jci.insight.98699. [Epub ahead of print [Pubmed]
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated.

Marina M BelletMonica BorghiMarilena ParianoGiorgia RengaClaudia StincardiniFiorella D’OnofrioStefano BrancorsiniEnrico GaraciClaudio CostantiniLuigina Romani Thymosin alpha 1 exerts beneficial extrapulmonary effects in cystic fibrosis Eur J Med Chem 2021 Jan 1;209:112921.doi: 10.1016/j.ejmech.2020.112921. Epub 2020 Oct 9. [Pubmed]

Marina M Bellet

We have previously demonstrated that thymosin alpha1 (Tα1), a naturally occurring immunomodulatory peptide, displays multi-sided beneficial effects in CF that concur in ameliorating the lung inflammatory pathology. In the present study, by resorting to murine models of gut inflammation with clinical relevance for CF patients, we demonstrate that Tα1 can also have beneficial effects in extrapulmonary pathology. Specifically, Tα1 restored barrier integrity and immune homeostasis in the inflamed gut of CF mice as well as in mice with the metabolic syndrome, a disorder that may arise in CF patients with high caloric intake despite pancreatic sufficiency. The protective effects of Tα1 also extended to pancreas and liver, further emphasizing the beneficial effects of Tα1 in extra-pulmonary complications of CF. By performing wide-ranging multi-organ anti-inflammatory effects, Tα1 could potentially integrate current therapeutic approaches to tackle the complex symptomatology of CF disease

Marina M Bellet is in the Department of Experimental Medicine, University of Perugia, Perugia, 06132, Italy


Jia LiuAllison P BergYiting WangWalailak JantarajitKaty J SutcliffeEdward B StevensLishuang CaoMarko J PregelDavid N Sheppard.A small molecule CFTR potentiator restores ATP-dependent channel gating to the cystic fibrosis mutant G551D-CFTR.  J Pharmacol2022 Apr;179(7):1319-1337. doi: 10.1111 Br /bph.15709. Epub 2022 Jan 21.[Pubmed]
Background and purpose: 
Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP-628006 with a distinct chemical structure.
Experimental approach: Using electrophysiological assays with CFTR-expressing heterologous cells and CF patient-derived human bronchial epithelial (hBE) cells, we compared the effects of CP-628006 with the marketed CFTR potentiator ivacaftor.
Key results: CP-628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del-CFTR were larger than those with the gating variant G551D-CFTR. In excised inside-out membrane patches, CP-628006 potentiated wild-type, F508del-CFTR, and G551D-CFTR by increasing the frequency and duration of channel openings. CP-628006 increased the affinity and efficacy of F508del-CFTR gating by ATP. In these respects, CP-628006 behaved like ivacaftor. CP-628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP-628006 conferred ATP-dependent gating on G551D-CFTR, whereas the action of ivacaftor was ATP-independent. For G551D-CFTR, but not F508del-CFTR, the action of CP-628006 plus ivacaftor was greater than ivacaftor alone. CP-628006 delayed, but did not prevent, the deactivation of F508del-CFTR at the plasma membrane, whereas ivacaftor accentuated F508del-CFTR deactivation.

Conclusions and implications: CP-628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.

Jia Liu is at the Neuroscience and Pain Research Unit, Pfizer Inc., Cambridge, UK and Senior Research Assistant at the School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK.

c407  bis-phosphinic derivative

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