Various Old and New Drugs and Treatments
Alpha-1 antitrypsin, acetylcysteine, amiloride, amitriptyline, ATALUREN (PTC 124), curcumin, DENUFOSOL, garlic, genistein, glutathione/ N-acetylcysteine, N-acetylcystiene for GI complications, IgY egg yolk antibodies, Pseudomonas vaccines, marijuana, MOLI 1901 (duramycin leancovutide), miglustat (N-butyldeoxynozyremicin), phosphdiesterase inhibitors, probiotics, Thymosin a1
2011 Cuthbert AW. New horizons in the treatment of cystic fibrosis. [Review] Brit J Pharmacol 2011; 163:173-183. [PubMed]
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.
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.
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]
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.
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
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 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]
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.
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]
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
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]
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]
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]
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]
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]
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 Ng, Hong Li, Mikel D Ghelfi, Yale E Goldman, Barry S Cooperman. Ataluren 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 Michorowska. Ataluren-Promising Therapeutic Premature Termination Codon Readthrough Frontrunner. Pharmaceuticals (Basel). 2021 Aug 9;14(8):785.doi: 10.3390/ph14080785. [Pubmed]Free PMC article
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.