Progress in therapies for cystic fibrosis

Rapid Review

Progress in therapies for cystic fibrosis Kris De Boeck, Margarida D Amaral

Standard follow-up and symptomatic treatment have allowed most patients with cystic fibrosis to live to young adulthood. However, many patients still die prematurely from respiratory insufficiency. Hence, further investigations to improve these therapies are important and might have relevance for other diseases—eg, exploring how to increase airway hydration, how to safely downscale the increased inflammatory response in the lung, and how to better combat lung infections associated with cystic fibrosis. In parallel, development of modulators that target the underlying dysfunction in the cystic fibrosis transmembrane conductance regulator (CFTR) is fast moving forward. Existing treatments are specific to certain mutations, or mutation class, in CFTR. An effective, although not yet entirely corrective, treatment is available for patients with class III mutations, and a treatment with modest effectiveness is available for patients who are homozygous for Phe508del, albeit at a very high cost. Corrective treatments that are non-specific to mutation class and thus applicable to all patients—eg, gene therapy, cell-based therapies, and activation of alternative ion channels that bypass CFTR—are being explored, but they are still in early stages of development. In view of the large number of patients with very rare mutations, a plan to advance personalised biomarkers to predict treatment effect is also being investigated and validated.

Introduction Cystic fibrosis is the most common life-shortening rare disease, affecting around 32 000 individuals in Europe and about 85 000 individuals worldwide. In most European Union (EU) countries, adult patients now outnumber paediatric patients, but the median age at death remains low at roughly 28 years.1 However, for patients born in the past 15 years, median predicted survival in the UK is now greater than 50 years.2 The number of patients reported to disease registries worldwide rises steadily because of the widespread implementation of newborn screening, increased diagnosis in low-income and middle-income countries, and improved survival.3 Cystic fibrosis is caused by mutations in one gene— cystic fibrosis transmembrane conductance regulator (CFTR)—which encodes an epithelial chloride and bicarbonate ion channel.4,5 Most patients ultimately develop progressive lung disease with airway mucus obstruction, bacterial infection, and inflammation (figure 1) despite intense symptomatic (ie, standard) treatments, which do not treat the molecular cause of the disease (ie, defective CFTR protein). These symptomatic treatments include mucolytics to dissolve thick mucus, antibiotics to treat or prevent infections, and antiinflammatory agents to dampen chronic inflammation. However, treatments that act at the level of the CFTR molecular defect are necessary to block the series of events that lead to progressive lung disease. Patients have a large range of clinical phenotypes, which can be partly explained by the roughly 2000 CFTR gene mutations so far identified (see Cystic Fibrosis Mutation Database), of which only around 200 have been characterised in terms of disease liability (see CFTR2).7 Other genetic, cellular, and environmental factors, which remain largely unknown, also modify the clinical course of the disease and each individual’s response to therapy.8–11 Cystic fibrosis is often regarded as a model disease, since many pioneering studies in genetics, molecular

and cellular pathogenesis, and drug discovery that have been done in cystic fibrosis paved the way for other rare genetics disorders. Furthermore, evidence shows that the CFTR protein has a key role in several major respiratory conditions of high public health relevance that are rapidly becoming more prevalent in the EU, such as asthma and chronic obstructive pulmonary disease (COPD; estimated as the fourth leading cause of death worldwide), in which lack of functional CFTR at the cell surface has been demonstrated.12–14 The CFTR protein is even thought to have a role in smoking-related respiratory disease, since loss of CFTR at the plasma membrane is a major and early event in cells exposed to cigarette smoke and pollutants.15–18 These findings led some researchers to consider COPD and heavy smoking as so-called acquired CFTR deficiency, by contrast with genetic cystic fibrosis.

Lancet Respir Med 2016 Published Online April 1, 2016 http://dx.doi.org/10.1016/ S2213-2600(16)00023-0 Pediatric Pulmonology, Department of Pediatrics, University of Leuven, Leuven, Belgium (Prof K De Boeck PhD); and Faculty of Sciences, Biosystems and Integrative Sciences Institute (BioISI), University of Lisbon, Lisbon, Portugal (M D Amaral PhD) Correspondence to: Prof Kris De Boeck, Pediatric Pulmonology, Department of Pediatrics, University of Leuven, Leuven 3000, Belgium christiane.deboeck@uzleuven. be

For the Cystic Fibrosis Mutation Database see http://www.genet. sickkids.on.ca For more on CFTR2 see http://www.cftr2.org/

Key messages • 80 000 people worldwide have cystic fibrosis and will die prematurely from respiratory insufficiency • Standard follow-up and symptomatic treatments have allowed most patients to live to young adulthood • Improvement of conventional drugs continues to reduce disease complications by increasing airway hydration and ameliorate chronic lung inflammation and infection, thereby raising the expected age of survival • To further increase the lifespan and quality of life of patients, more effective treatments are needed and therapies correcting the underlying defect hold promise of achieving this goal • Modulators that target the underlying dysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) in a mutation-specific or mutation-class-specific way are being developed at a steady pace • An effective treatment is available for patients with class III mutations (around 5% of all patients) and is becoming available for patients who are homozygous for Phe508del (roughly 40–45% of all patients) • So-called mutation agnostic corrective treatments—such as gene therapy, cell-based therapies, and activation of alternative ion channels to bypass CFTR—are in early stages of development

www.thelancet.com/respiratory Published online April 1, 2016 http://dx.doi.org/10.1016/S2213-2600(16)00023-0

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Rapid Review

Symptomatic treatments

Molecular-defect treatments

6

Destructive cycle

7

Progressive loss of lung function

4 Decreased water content in airway surface liquid; thick mucus

3 Abnormal CI– permeability altered ionic transport

Inflammation and scarring

Bacterial infection

CI– 2 Defective CFTR protein

CFTR

5 Mucus obstruction and bronchiectasis

Na+

Na+

ENaC Na+

Na+

1 Two defective CFTR genes

Figure 1: Pathogenetic cascade that causes cystic fibrosis lung disease Cystic fibrosis is caused by mutations in the CFTR gene, which trigger a series of events that ultimately lead to severe lung deficiency. Most standard therapies treat the symptoms (eg, mucolytics to dissolve the thick mucus). However, treatments that target molecular defects acting earlier in this cascade of events (eg, CFTR modulators) can prevent progression to end-stage lung disease.6 CFTR=cystic fibrosis transmembrane conductance regulator. ENaC=epithelial sodium channel. Modified from Amaral.6

In this Review, we highlight progress in both standard symptomatic treatments and new therapies targeting the molecular defect in CFTR. In the fast-moving field of cystic fibrosis research, our discussion of these new therapies can be seen as an update to the 2013 Review in The Lancet Respiratory Medicine.19 Our focus here is to highlight recent and ongoing trials rather than past trials or preclinical studies.

Symptomatic therapies Understandably, most attention and resources are now focused on correction of the molecular defect with CFTR modulators—ie, correctors and potentiators. However, we should stress that none of these therapies are sufficiently effective to be used as stand-alone treatments at present. Even the most successful of these therapies (ie, ivacaftor in patients with a class III mutation) is used in addition to existing standard therapies.20 During treatment with ivacaftor, sweat chloride value, a marker of CFTR function, comes close to, but does not reach, the normal range.20,21 Additionally, although the clinical benefit of the drug is impressive (a 10% predicted improvement in forced expiratory volume in 1 s [FEV1]), disease progression is not stopped: the treatment is estimated to only halve the FEV1 rate of decline.22 Furthermore, during ivacaftor treatment, patients still have pulmonary exacerbations and other lung complications. Thus, until disease manifestations can be 2

prevented by eradicating the root cause and entirely blocking the pathophysiological cascade,23 the conventional symptomatic therapies, which enable most patients to live to adulthood, remain important.24 Further research to optimise these treatment modalities continues to be highly relevant for patients with cystic fibrosis and might also benefit patients with other chronic lung diseases, such as non-cystic fibrosis bronchiectasis and COPD. In this section, we discuss developments in the strategies to restore the airway surface liquid layer and improve mucociliary clearance, to dampen the excessive inflammatory response in the lung, and to control chronic lung infection.

Restoration of airway surface liquid and mucociliary clearance Central in the pathophysiology of cystic fibrosis lung disease are abnormally viscid secretions and deficient mucociliary clearance, leading to airway obstruction. The absence of chloride and bicarbonate secretion via the CFTR channel, coupled to excess sodium ion absorption via the epithelial sodium channel (ENaC, which is not downregulated by CFTR), leads to insufficient water secretion to the airway surface liquid layer. Whether this insufficiency results directly in decreased height of the periciliary layer, or whether the increased oncotic pressure in the overlying mucus layer pushes the

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Rapid Review

periciliary layer down (the new so-called gel-on-brush model),25 is under investigation. At present, inhalation of hypertonic saline and mannitol are used to improve airway hydration by altering osmolarity of the airway surface liquid.26,27 Dornase alfa (ie, recombinant human DNase-1) increases mucociliary clearance by decreasing sputum viscosity and is part of standard care for cystic fibrosis.24 As shown in the Epidemiologic Study of Cystic Fibrosis,28 the use of dornase alfa is associated with a reduction in the rate of FEV1 decline. However, around 30% of patients might be non-responders,29,30 probably because of insufficient sputum concentration of magnesium needed for DNase-1 activity or excessive sputum actin content, the naturally occurring inhibitor of DNase-1.30 When tested in vitro, PRX-110, a plant-cell-expressed recombinant form of human DNase-1, was more resistant than dornase alfa to the inhibitory effect of actin.31 Another strategy is to use actin depolymerising agents such as gelsolin or polyanions (eg, polyaspartate),32 which await proof-of-concept trials. To further restore mucociliary clearance, ENaC inhibitors are being considered as stand-alone therapy, in combination with existing hydrators such as hypertonic saline, or in combination with CFTR modulators. Indeed, ENaC inhibitors sit in between symptomatic therapy to improve mucociliary clearance and therapies aiming to improve the molecular defect by targeting non-CFTR channels (see below).

Safe reduction of excessive lung inflammation The complex association between inflammation and cystic fibrosis lung disease has been reviewed elsewhere.33,34 The hallmark of cystic fibrosis lung disease is infection by bacteria and other pathogens. However, compared with lung infection in other disorders, in cystic fibrosis the associated inflammation is increased but is still ineffective to clear pathogens from the lung. Lung inflammation in cystic fibrosis is driven by neutrophils, which contribute to structural lung damage, for example via release of neutrophil elastase. Although the mechanism is not fully understood, another hallmark is reduced invasiveness of pathogens despite a high local bacterial burden.35,36 Therefore, the balance between excessive inflammation (which contributes to lung damage) and insufficient inflammation (which possibly allows progression to more invasive disease) needs to be maintained, and potent anti-inflammatory therapies have been shown to worsen cystic fibrosis lung disease.37 Therapeutic strategies that only modestly decrease the inflammatory response, promote resolution of inflammation, and increase local antiprotease or antioxidant activity might prove safer and are now being explored. In a 4 year clinical trial in patients aged 6–14 years,38 the administration of high-dose systemic steroids (2 mg prednisone per kg bodyweight) on alternating days was

discontinued prematurely because of severe safety concerns. The low-dose treatment (1 mg prednisone per kg bodyweight) on alternating days did show a small benefit on repeated measurements of FEV1 (% predicted) but was again associated with serious side-effects, such as growth retardation, cataract, and more frequent pseudomonas infection.38 Inhaled steroids have no proven benefit.39 Leukotriene B4 (LTB4) is a potent activator of inflammatory responses mediated by neutrophils, macrophages, and monocytes, and its concentration is increased in the lung of patients with cystic fibrosis. In a clinical trial with the LTB4 receptor antagonist BIIL 284,37 active treatment was associated with increased pulmonary exacerbations in adult patients. In an animal model, BIIL 284 increased the incidence of bacterial infections in the mouse lung.40 Therefore, existing anti-inflammatory strategies are mainly restricted to non-steroidal drugs. Oral high-dose ibuprofen decreases the rate of decline of lung function, but fear of serious side-effects and the need for frequent drug level monitoring severely limit its use.41,42 In 2015, ibuprofen was identified to have some CFTR corrector activity in a human bronchial epithelial cell assay and in a mouse model.43 With treatments aiming to decrease inflammation, a more realistic expectation is to decrease pulmonary exacerbations and stabilise lung function (ie, less decline) rather than acute improvement in lung function.44 Obviously, a longer treatment period is needed to measure these outcomes.44 Several new compounds are being studied in clinical trials (table 1). Acebilustat is a small molecule that blocks the enzyme leukotriene A4 hydrolase, thereby decreasing the production of LTB4. The aim of acebilustat is to reduce airway obstruction by blocking excessive neutrophil influx and activation. In a phase 1 clinical trial with oral acebilustat (50 mg and 100 mg once daily) for 15 days in 17 adult patients with cystic fibrosis and mild to moderate lung disease, positive trends were seen in blood and sputum biomarkers, including LTB4, without changes in sputum microbiology.45 The planned phase 2 trial, EMPIRE-CF (ClinicalTrials.gov identifier NCT02443688), will aim to show a reduction in pulmonary exacerbations and in FEV1 decline. The oral anti-inflammatory compound ajulemic acid (Resunab) is designed to enhance the resolution of chronic inflammation by binding and activating cannabinoid receptor type 2, which is present on immune cells such as monocytes, T cells, and B cells. Ajulemic acid induces apoptosis of T cells, inhibits leucocyte migration, reduces cytokine release (eg, interleukin-6 and interleukin-8), decreases production of LTB4, and increases production of the anti-inflammatory lipoxin A4. The drug had a favourable safety profile in a phase 1 trial46 and will progress to phase 2 trials in cystic fibrosis (NCT02465450) and dermatomyositis. Research into strategies that increase local antiprotease and antioxidant activity to combat damage by neutrophils

www.thelancet.com/respiratory Published online April 1, 2016 http://dx.doi.org/10.1016/S2213-2600(16)00023-0

For more on Resunab see http:// www.corbuspharma.com/ product-pipeline/resunab

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Trial description

Intervention

Sponsor

Improving airway hydration NCT01619657

Phase 2 pilot study of safety and efficacy in newborns and infants

Preventive inhalation of 6% hypertonic saline (4 mL) versus 0·9% isotonic saline (4 mL), twice daily for 52 weeks

Heidelberg University, Heidelberg, Germany

NCT02378467

Phase 3 study of safety and efficacy (improvement in lung clearance index) in children aged 3–5 years

Inhalation of 7% hypertonic saline versus 0·9% isotonic saline, twice daily for 48 weeks

University of Washington, Seattle, WA, USA

NCT02343445

Phase 2 placebo-controlled trial of safety and efficacy in patients aged 12 years or older

Inhalation of ENaC blocker (P-1037) with saline or Parion Sciences hypertonic saline versus saline or hypertonic saline

Safely combatting inflammation NCT01270074

Phase 3 placebo-controlled trial of safety and efficacy in infants to prevent development of bronchiectasis at age 3 years

Azithromycin (10 mg/kg bodyweight) three times Queensland Children’s Medical per week, from age 3 months until 3 years Research Institute, Brisbane, QLD, Australia

NCT02443688

Phase 2 placebo-controlled trial of safety, tolerability, and efficacy in adult patients

Oral acebilustat, once daily for 48 weeks

NCT02465450

Phase 2 placebo-controlled trial of safety, tolerability, Ajulemic acid (JBT-101) pharmacokinetics, and efficacy in adult patients

Celtaxsys Corbus Pharmaceuticals

Improving control of lung infection NCT01455675

Phase 3 trial of safety and efficacy for prevention of re-infection with Pseudomonas aeruginosa

Avian polyclonal anti-P aeruginosa antibodies versus placebo

Mukoviszidose Institut, Bonn, Germany

NCT02526004

Phase 3 trial of microbiome-determined antibiotic therapy in cystic fibrosis exacerbations

Microbiome-guided therapy versus standard therapy

University College Cork, Cork, Ireland

ENaC=epithelial sodium channel.

Table 1: Key ongoing clinical trials of standard therapies

has been active for more than a decade, but progress is slow. The continued interest is explained by the safety of these approaches; unfortunately, efficacy has been more difficult to prove. In an open-label multicentre study in Germany in 52 patients with cystic fibrosis, pseudomonas lung infection, and raised free elastase levels in induced sputum,47 daily inhalation of α1 antitrypsin (25 mg) for 4 weeks resulted in raised sputum α1 antitrypsin levels and reductions in free elastase levels and concentrations of interleukin-8, TNF-α, interleukin-1β, and LTB4; decreased proportion of neutrophils; and a reduction in Pseudomonas aeruginosa colony count. However, these changes were not associated with any change in lung function.46 In a doubleblind phase 2 study (NCT01684410),48 higher doses (ie, 100 mg and 200 mg) of aerosolised α1 antitrypsin (a liquid preparation of purified α1 proteinase inhibitor from pooled human plasma) inhaled daily via a nebuliser for 3 weeks were safe and well tolerated. Increasing the amount of the antioxidant glutathione in the lungs has been attempted, either by inhalation of glutathione (which is deficiently transported by CFTR) or by oral administration of the prodrug N-acetylcysteine, which is then converted to glutathione.49–54 Although some changes in inflammatory parameters were noted with inhaled glutathione,49–54 no sustained change in lung function or exacerbations was seen in a subsequent 6-month trial.52 Conversely, in a 24-week double-blind trial with oral N-acetylcysteine (n=70),54 no change in inflammatory markers (eg, sputum neutrophil elastase or plasma interleukin-8) was seen, but lung function was stabilised without a significant change in pulmonary 4

exacerbations. However, in view of the small sample size, the possibility of this being a chance finding cannot be excluded. The authors hypothesised other potential mechanisms of action, such as a positive effect of N-acetylcysteine on other pathways of inflammation or on CFTR trafficking.

Efficient control of chronic lung infection: treatments beyond antibiotics Intensive use of oral, inhaled, and systemic antibiotics has undoubtedly improved survival of patients with cystic fibrosis and is still one of the mainstay therapies.24 Ultimately, however, Pseudomonas species will develop widespread antibiotic resistance, and pathogens that are increasingly more difficult to treat—such as Burkholderia cepacia complex, Achromobacter species, atypical mycobacteria, and fungi—might also emerge. The development of new inhaled antibiotics and better or faster inhalation devices has been the topic of several recent reviews.55–57 Therefore, we focus on strategies other than antibiotics to combat chronic lung infection in cystic fibrosis. Biofilm production is a common adaptation mechanism during chronic infection, and is typical for chronic pseudomonas infection in the cystic fibrosis lung. Disruption of the biofilm could thus help to eradicate Pseudomonas species, and several strategies that are being explored include saccharides such as liposomal β glycan58 and OligoG,59,60 an oligosaccharide derived from brown algae. Safety and tolerability of nebulised OligoG have been proven in healthy volunteers and in patients,61 and patterns

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of lung deposition after inhalation are now being studied. Another compound that has been reported to disrupt the biofilm (by a yet-unknown mechanism), oral cysteamine, is being tested in a phase 1 trial for tolerability and pharmacokinetics.62 Interestingly, this compound, in combination with a tea flavonoid, has been reported to rescue traffic of Phe508del-CFTR by restoring autophagy.63,64 Nitric oxide concentrations are decreased in exhaled air from patients with cystic fibrosis compared with healthy individuals.65 The importance of this finding is not entirely clear but has been linked to the increased susceptibility to infection.66 Attempts to increase nitric oxide production by inhalation of interferon-γ to stimulate nitric oxide synthase proved unsafe, since more exacerbations were seen in patients given the high dose; neither high-dose treatment nor low-dose treatment demonstrated benefit on lung function, sputum imflammatory markers, or bacterial density.67 Strategies to increase nitric oxide concentration without interfering with complex inflammatory cell signalling seem safer, but the effectiveness of these approaches is still to be proven. Direct inhalation of nitric oxide effectively decreased bacterial load in a rat model of P aeruginosa pneumonia.68 The safety and tolerability of nitric oxide inhalation (160 ppm for 30 min three times per day) have also been assessed in patients with cystic fibrosis, and at least transient reductions in bacterial load were noted.69 Nitric oxide concentrations in the airways can also be increased by repeated inhalations of L-arginine, the substrate for nitric oxide synthase. In a pilot trial,70 inhalation of 500 mg L-arginine twice daily for 2 weeks was well tolerated, and FEV1 increased by 56 mL on average, although this increase was not significant; no change in inflammatory markers in sputum was reported. Interest in treatment with bacteriophages—ie, viruses that infect and replicate in specific strains of bacteria— is mounting.71 Especially for patients colonised with multiresistant organisms, controlling the infection with bacteriophage treatment could be a last resort. At present, no clinical trial has started, but preparatory work specific for cystic fibrosis is advancing. AntiPseudomonas bacteriophages have been shown to retain their activity when nebulised.72 Strict safety regulations imposed by health authorities are one of the major hurdles to assess the efficacy of this therapy in the clinic. Finally, a new and rapidly expanding research area is the lung microbiota, but a full discussion of this topic is beyond the scope of this Review. Knowledge of lung microbiota has considerably changed the way lung infection in cystic fibrosis is viewed. The healthy airways are colonised by a wide spectrum of bacteria. By contrast, in the airways of patients with cystic fibrosis, bacterial diversity and species richness decrease as the disease progresses and lung function decreases.73 However, this improved knowledge of the microbial community has not yet translated into more judicious or efficacious use

of antibiotics during exacerbations. An EU-funded project and clinical trial have started to address these issues and fill this knowledge gap (see CF Matters).

For more on CF Matters see http://www.cfmatters.eu/

Therapies to correct molecular defects CFTR modification therapies: classes of CFTR mutations The roughly 2000 CFTR gene alterations that have so far been described consist of missense (39·6%), frameshift (15·6%), splicing (11·4%), and nonsense (8·3%) mutations; large (2·6%) and in-frame (2·0%) deletions or insertions; promoter mutations (0·7%); and presumed non-pathological variants (15·0%).6,74 However, the 3 base-pair deletion of phenylalanine 508 (Phe508del) is present in around 85% of patients worldwide, with a higher frequency reported in northern Europeans than in southern Europeans.75 All mutations that cause cystic fibrosis ultimately lead to a defect in cAMP-regulated chloride and bicarbonate secretion by epithelial cells, but many reasons exist for the different causative mechanisms.76 Not only is elucidation of the molecular and cellular effects brought about by CFTR mutations informative for structure–function studies, but it can also provide the scientific basis for mutation-specific corrective therapies.23 Therefore, these mutations can be grouped according to their functional defect into seven classes (figure 2).6,77,78 Class I mutations affect protein production and include mostly nonsense mutations (ie, those with premature stop codons), thus often causing degradation of mRNA by nonsense-mediated decay. Class II mutations include Phe508del and affect CFTR protein traffic as a result of protein misfolding and retention at the endoplasmic reticulum (ER) by the ER quality control mechanism. Such retention is followed by premature degradation, which prevents the protein from trafficking to the cell surface, thus severely reducing CFTR function.79,80 Class III mutations impair gating of the CFTR channel. Class IV mutations cause a substantial decrease in CFTR channel conductance (ie, flow) of chloride and bicarbonate ions. Class V mutations lead to a major reduction in the levels of normal CFTR protein, often because of alternative splicing that generates both aberrant and normal mRNA species, the proportion between which might vary among patients and in different organs of each patient. Class VI mutations destabilise CFTR at the cell surface, either by increasing CFTR endocytosis or by decreasing its recycling back to the cell surface. Finally, class VII mutations are so-called unrescuable mutations, because they cannot be pharmacologically rescued per se—eg, large deletions such as the dele2,3(21kb) mutation.81 However, promising so-called one-size-fits-all therapeutic strategies (also known as mutation agnostic; ie, independent of the mutation class) will also be suitable for this class of mutations (see below). Novel emerging drugs targeting the underlying defect—ie, mutant CFTR—open up new opportunities

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CI– CI– CI– – – CI CI– CI

CI–

CI–

CI–

CFTR

Wild-type CFTR Class I

Class II

Class III

Class IV

Class V

Class VI

Class VII

CFTR defect

No protein

No traffic

Impaired gating

Decreased conductance

Less protein

Less stable

No mRNA

Mutation examples

GLy542X, Trp1282X

Phe508del, Asn1303Lys, Ala561Glu

Gly551Asp, Ser549Arg, Gly1349Asp

Arg117His, Arg334Trp, Ala455Glu

Ala455Glu, 3272-26A→G, 3849+10 kg C→T

c. 120del23, rPhe508del

dele2,3(21 kb), 1717-1G→A

Corrective therapy

Rescue synthesis

Rescue traffic

Restore channel activity

Restore channel activity

Correct splicing

Promote stability

Unrescuable

Drug (approved)

Read-through compounds (no)

Correctors (yes)

Potentiators (yes)

Potentiators (no)

Antisense oligonucleotides, correctors, potentiators? (no)

Stabilisers (no)

Bypass therapies (no)

Figure 2: Classes of gene mutations and respective therapeutic strategies CFTR mutations are grouped into seven functional classes, with the expectation that the same modulators will be applicable to all the defects in one class. However, class VII mutations are not expected to be rescuable by any modulator. A therapeutic strategy for class VII mutations could involve stimulation of alternative chloride channels (ie, bypass therapies). CFTR=cystic fibrosis transmembrane conductance regulator. rPhe508del=rescued Phe508del. Modified from Amaral.6

for so-called curative treatment. At present, two of these drugs have been approved for clinical use: ivacaftor benefits only patients with gating mutations (around 5% of all patients) and, to some extent, patients with the Arg117His mutation; lumacaftor is beneficial for patients with the homozygous Phe508del genotype (ie, roughly 40–45% of all patients with cystic fibrosis; table 2).20,21,82–84 Ivacaftor was shown to decrease sweat chloride concentration by a mean of 50 mmol/L, augment predicted FEV1 by a mean of 10%, reduce the number of pulmonary exacerbations, and improve body-mass index (BMI), Z scores, and quality of life, not only in patients with the Gly551Asp mutation but also in patients with other class III mutations.20,21 Ivacaftor has now been used in patients with the Gly551Asp mutation for more than 5 years. Across age and disease severity groups, a robust benefit was seen after marketing authorisation by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA).85–87 Long-term benefits are starting to become apparent87—ie, less frequent infections with pseudomonas,88 reduced rate of lung function decline,21 improved glucose tolerance,89 sustained weight gain, and better growth in children.90 In vitro, ivacaftor potentiates not only class III CFTR 6

mutants but also the normal CFTR channel and some mutant proteins of classes IV and V.91 In a phase 3 trial,82 a 5% improvement in lung function was reported in adult patients with the class IV mutation Arg117His; however, this improvement was not seen in children with the same mutation. In the entire cohort with this mutation, improvements in sweat chloride (−24 mmol/L) and quality-of-life score were seen.82 However, clinical benefit in patients with other class IV or V mutations with residual function still needs to be firmly proven. New strategies are emerging for class V mutations that affect splicing, and a particular area of interest is the correction of alternative splicing with antisense oligonucleotides.92 The introduction of ivacaftor led to great optimism, even though this drug is indicated in only up to 10% of patients with cystic fibrosis. Ivacaftor provides proof of concept that small-molecule therapy that interferes with the molecular defect is possible, which encouraged patients to participate in clinical trials with CFTR modulators. Yet, many challenges remain to ensure that patients have access to new treatments. Ivacaftor is very expensive (around US$311 000 per patient per year), and some health authorities are slow to approve reimbursement, since the need for lifelong treatment poses a substantial burden on health-care budgets.93,94

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Rapid Review

Several companies are now developing other drugs that potentiate CFTR channel function, and clinical trials are ongoing (table 3). Results from phase 3 trials in patients homozygous for Phe508del showed that combination therapy of lumacaftor (a corrector that partly rescues Phe508del-CFTR traffic) and ivacaftor (which potentiates channel function) induced a significant but modest (2·6–4·0%) improvement in lung function and a Frequency

30–40% reduction in pulmonary exacerbations— defined as treatments with additional antibiotics because of an increase in pulmonary symptoms—over the 24 week treatment period.83 Nonetheless, this combination therapy has already been approved by the FDA and EMA for patients homozygous for Phe508del aged 12 years or older (table 2). Again, cost (around US$250 000 per patient per year) is a major issue, since 40–50% of patients qualify for this treatment.

Corrective therapy Limitations

Status

Gly551Asp and eight other gating mutations (class III)

4–5%

Ivacaftor

Very high cost (around US$311 000 per patient per year)

Approved in the EU and USA for patients aged 2 years or above

Arg117His (class IV)

1–2%

Ivacaftor

Very high cost; variable response

Approved in the EU for patients aged 18 years or above, and in the USA for patients aged 6 years or above

Lumacaftor plus ivacaftor

Very high cost (around US$250 000 per patient per year); low efficacy; variable response

Approved in the EU and USA for patients aged 12 years or above

Homozygous Phe508del

40–45%

Heterozygous Phe508del

40%

None

¨

¨

Nonsense mutations

10%

Ataluren

Benefit uncertain

Phase 3 trial (NCT02139306) ongoing in patients who are not using inhaled aminoglycosides

Other mutations (including class VII mutations)

10–15%

None

¨

¨

EU=European Union.

Table 2: Corrective therapies targeting the molecular defect in cystic fibrosis, by mutation type

Mechanism of action

Stage of development

Sponsor

Ataluren

Rescue CFTR protein synthesis by read-through of nonsense mutations

Phase 3 confirmatory study (NCT02139306) of efficacy and safety is ongoing in patients with nonsense CFTR mutations who are not taking inhaled aminoglycosides

PTC Therapeutics

VX-661 (corrector) plus ivacaftor (potentiator)

Rescue Phe508del-CFTR protein to the cell surface with a corrector and stimulate channel function with a potentiator

Phase 3 studies (NCT02347657, NCT02392234, and NCT02412111) of efficacy and safety are ongoing in patients aged 12 years or above with homozygous and heterozygous Phe508del-CFTR mutation

Vertex Pharmaceuticals

N91115 (proteostasis regulator)

Stabilise Phe508del-CFTR protein after being rescued to the cell surface

Phase 2 study (NCT02589236) is ongoing in adults who are homozygous for Phe508del and receiving lumacaftor–ivacaftor combination treatment

Nivalis Therapeutics

Riociguat

Improve CFTR expression

Phase 2 trial (NCT02170025) of safety, tolerability, and efficacy is underway in adults homozygous for Phe508del

Bayer

QR-010 (antisense oligonucleotide)

In-vivo correction of the defect in Phe508del-CFTR mRNA

Phase 1b trial (NCT02532764) is ongoing to assess the safety of single and multiple ascending doses of inhaled QR-010 in adult patients with homozygous Phe508del; phase 1 open-label exploratory study (NCT02564354) is underway to assess the efficacy of intranasal administration on nasal potential difference (a measure of CFTR function) in patients with homozygous and heterozygous Phe508del

ProQR Therapeutics

GLPG1837 (potentiator) Rescue CFTR function by stimulating channel function

Phase 2a trial of safety and tolerability is ongoing in adults with at least one class III mutation

Galapagos NV

QBW251 (potentiator)

Rescue CFTR function by stimulating channel function

Phase 1 (NCT02190604) and phase 2a (NCT02190604) trials are underway to assess safety, tolerability, and pharmacokinetics in healthy volunteers and adults with at least one class III–VI mutations

Novartis Pharmaceuticals

FDL169 (potentiator)

Rescue CFTR function by stimulating channel function

Phase 1 trial (NCT02359357) of safety, tolerability, and pharmacokinetics of single and repeat oral doses in healthy male volunteers is underway

Flatley Discovery Laboratory LLC

C-10355 and C-10358 (potentiators)

Rescue CFTR function by stimulating channel function

Phase 1 trial (NCT02392702) of safety, tolerability, and pharmacokinetics in healthy volunteers, with a pharmacokinetic comparison with ivacaftor, completed.

Concert Pharmaceuticals

Other correctors and potentiators

In the pipeline of several companies, pending results from preclinical and phase 1 studies of Vertex Pharmaceuticals, Rescue CFTR function at the cell surface by correctors with complementary action Galapagos NV, and Flatley two correctors and stimulate channel Discovery Laboratory LLC function with a potentiator in patients homozygous or heterozygous for Phe508del

CFTR=cystic fibrosis transmembrane conductance regulator.

Table 3: Treatments targeting molecular defects in CFTR and relevant clinical trials

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For more on the Cystic Fibrosis Trust see http://www. cysticfibrosis.org.uk/

At present, combination therapy has not shown efficacy in patients heterozygous for Phe508del. Few treatment options are available for patients with the predominant Phe508del mutation (present in roughly 85% of all patients),19,78,95 since the rescue of Phe508del by one corrector is indeed inefficient. An improved understanding of how the complex three-dimensional folding of the Phe508del-CFTR protein is disturbed points to the need for a combination of correctors with different mechanisms of action, together with a potentiator to allow more efficient rescue of the mutant protein.96,97 Ivacaftor potentiates normal CFTR, class III mutants, and several mutated CFTR proteins associated with residual function.91 However, different corrector–potentiator combinations are probably needed to rescue different misfolded class II mutant proteins.98 As has been shown for class V splicing defects,92 antisense oligonucleotides can also be used to correct the Phe508del mutation. Single-stranded antisense RNA-based oligonucleotides can act as guide sequences to repair the targeted abnormal mRNA. The repaired mRNA is then translated into a wild-type CFTR protein.99 For one such therapy (QR-010), clinical trials are underway to assess the safety, tolerability, and pharmacokinetics of single and multiple increasing doses of the inhaled drug in patients homozygous for Phe508del (NCT02532764 and NCT02564354). These studies will also investigate changes in sweat chloride, weight, Cystic Fibrosis Questionnaire Revised Respiratory Symptom Score, and FEV1. The so-called read-through strategy for patients with premature termination codons (ie, class I mutations) remains an important concept because it has potential in all diseases caused by a nonsense mutation. The EMA has granted conditional approval for ataluren in patients with Duchenne’s muscular dystrophy caused by a premature stop codon. For patients with cystic fibrosis, the 48 week double-blind phase 3 trial of ataluren100 did not lead to a firm conclusion; indeed, the primary endpoint of improvement in FEV1 was not met. However, a prespecified subgroup analysis showed that patients who were not using inhaled aminoglycosides had less decline in lung function than had those given placebo. In view of this finding, a confirmatory phase 3 trial in patients who are not using inhaled aminoglycosides (NCT02139306) is ongoing. Moreover, the number of new read-through drugs in preclinical development is growing.

Targeting non-CFTR channels: the bypass approach For patients with so-called unrescuable mutations (class VII), the most straightforward approach is to target alternative non-CFTR anion channels, such that the loss of CFTR-mediated chloride transport in airway epithelia can be bypassed to restore ion transport.21 These therapies, which are sometimes called mutation agnostic, might compensate not only for the loss of CFTR-mediated anion 8

conductance but also for other cellular defects, including ENaC hyperactivity and the reduced height and pH of airway surface liquid, which in turn impair bacterial clearance and killing,101 enhance sodium absorption,102 and reduce mucus fluidity.103–105 Many alternative ion channels and transporters are promising drug targets. Anoctamin 1 (ANO1; also known as TMEM16A) is a calcium-activated chloride channel present in many epithelial cells,106–110 and ANO6 (also known as TMEM16F) is an essential component of the outwardly rectifying chloride channel.111 Genetic variants of SLC9A3, which encodes a sodium–proton exchanger, have been associated with pseudomonas infections and decline in pulmonary function.110,112 The chloride–bicarbonate transporter SLC26A9 is associated with susceptibility to meconium ileus113 and diabetes,114 which are frequent complications in cystic fibrosis. However, the logical strategy of stimulating alternative chloride channels has been unsuccessful so far. Inhalation of denufosol, which stimulates calciumactivated chloride channels through the P2Y2 subtype of purinergic receptors, was promising in phase 2 investigation, but robust clinical benefit was lacking.115 Nevertheless, innovative approaches to find new pathways to stimulate these channels are underway (eg, INOVCF project116 and Cystic Fibrosis Trust in the UK). Another approach is to target ENaC, a major regulator of salt and water re-absorption. Since ENaC is downregulated by CFTR, the absence of functional CFTR leads to ENaC hyperactivity in cystic fibrosis,117,118 which in turn results in water depletion from the airway surface liquid and reduction in its height and fluidity.119 Mice that overexpressed β ENaC mimicked various aspects of human respiratory disease in cystic fibrosis,120 whereas mice with CFTR knockout or mutations did not develop lung disease.121 In view of the major role of ENaC in the airway, ENaC inhibitors have been developed to reduce ENaCmediated sodium hyperabsorption and increase hydration of airway surface liquid. These include specific inhibitors such as amiloride (the short-lived prototype ENaC blocker) and its derivatives benzamil or PS552, and activators of purinergic receptors (ATP, UTP, or denufosol) that inhibit ENaC through stimulation of calcium-activated chloride channels.23 However, repeated inhalations of amiloride did not show benefits in clinical trials.122 Development of longacting blockers was halted because of hyperkalaemia or other side-effects. Newer ENaC blockers, such as P-1037 and GS-9411 (NCT00999531), are being developed. The safety and tolerability of P-1037 (NCT02343445) are being studied as monotherapy and in combination with hypertonic saline. The inhaled combination had a transitory effect on mucus hydration and mucociliary clearance in healthy individuals and could lead to a greater or more prolonged benefit than that with inhaled hypertonic saline alone.123 Along these lines, a systems biology screen of more than 6000 genes identified a key ENaC regulator, DGK1,

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which normalises (but does not totally block) ENaC function and restores fluid absorption to normal levels in primary lung cells from patients with cystic fibrosis.124 However, excessive blocking of ENaC might cause severe harm due to hyperkalaemia or undesirable accumulation of fluid in the lungs (ie, pulmonary oedema).125

Gene, cell-based, and other mutation agnostic therapies After cloning the CFTR gene, gene therapy was originally the priority for therapy, but gene transfer into lung cells in vivo soon proved more challenging than anticipated. Nevertheless, research into gene therapy led to a phase 2b clinical trial (NCT01621867)126 of DNA plasmid containing the CFTR cDNA (ie, a copy of the CFTR mRNA) complexed with cationic liposomes (ie, a non-viral vector), which was delivered once a month to the lungs of patients with cystic fibrosis. Results from this double-blind randomised trial showed a significant, albeit modest, effect in the treatment group versus placebo at 12-month follow-up. The 3·7% difference in FEV1 resulted from stabilisation of lung function in treated patients and a decline in lung function in the placebo group.126 Despite the fact that the placebo was only 0·9% saline (ie, no empty vector, CFTR DNA, or liposomes), efficacy data are at least encouraging, and improved versions of this therapy might become a treatment option in the future. Cell-based therapy is another mutation agnostic therapeutic approach with high potential. Indeed, cellular reprogramming and genome editing technologies (based on targeting of nucleases) have made possible the correction of patient-specific pluripotent stem cells and induction of their differentiation into a specific cell type. This approach has been shown to be feasible for cystic fibrosis in intestinal organoids and induced pluripotent stem cells.127,128 Therefore, the disease-target tissues could potentially be repopulated with these corrected cells. However, safety issues still need to be resolved regarding the clinical use of stem cells before cell-based approaches can become a therapeutic option. Another class of emerging therapies focus on systems approaches to target both mechanisms of disease and their therapeutic correction.11 One such strategy targets proteostasis (ie, maintenance of the cellular proteome in a folded, functionally competent state) and can be applied not only for cystic fibrosis but also for a group of so-called protein misfolding diseases.129 A successful example is suberolyanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor and regulator of the chaperone protein Hsp90, which has been shown to restore the cell surface expression and channel activity of Phe508del-CFTR to 28% of wild-type levels in human primary airway epithelial cells.130

Repurposing existing drugs for so-called orphan mutations Despite the great breakthrough in drug development, the first CFTR modulator, ivacaftor, targets only 5–10% of patients at best, and its combination with lumacaftor

benefits another 40–45% of patients (ie, those who are homozygous for Phe508del). Thus, effective treatment for the remaining 50% of patients remains an unmet need. Indeed, the wide range of CFTR mutations consists of many uncommon variants (so-called orphan mutations), for which prediction of disease outcome is difficult, since the respective functional defect has not been defined. Although phenotypes vary widely, many of these mutations in classes IV–VI result in partial CFTR function and milder, so-called atypical, forms of disease.131,132 Since each of these mutations affect very few patients worldwide, they pose considerable challenges not only in establishing the diagnosis but also in assessing the potential of the new mutation-based therapies (so-called theranostics—ie, the use of molecular or functional diagnostic tests and targeted therapeutics in an interdependent, collaborative manner, which aims to individualise treatment by targeting therapy to an individual’s disease subtype and genetic or functional profile).131–133 Some of these patients with orphan mutations are likely to respond to existing CFTR modulators. Therefore, there is a need to functionally characterise these orphan mutations and to establish validated biomarkers for theranostics. Without any preselection of suitable candidates, the N-of-1 clinical trial that considers an individual patient as the sole unit of observation is an inefficient, costly, and time-consuming approach.

Using ex-vivo response to predict in-vivo response For many CFTR mutations, how they disturb the CFTR pathway is unknown, and most are very rare. For these rare mutations, use of ex-vivo models is a promising approach to predict in-vivo response. Bioelectric measurements of CFTR dysfunction in native or cultured tissues from patients that are already in use for cystic fibrosis diagnosis and prognosis134,135 have also been proposed for personalised therapies.6,136 The most promising models for treatment response are intestinal organoids and primary cultures of epithelial cells derived from bronchi or nasal scrapings.98,136,137 Primary intestinal organoids are grown from rectum crypts obtained via suction biopsy and contain stem cells; therefore, they can be expanded indefinitely to obtain a continuous source of patient-specific material. Forskolin induces swelling of organoids from healthy individuals via stimulation of the CFTR channel, but not in organoids from patients with cystic fibrosis, and the magnitude of swelling correlates with CFTR activity.137 Organoids from patients can be incubated with drug candidates to assess their effect on CFTR function. If CFTR function and organoid swelling are restored by a specific modulator in a patient’s tissue ex vivo, then testing the clinical benefit in vivo is the next logical step. There is therefore an unmet need to test compounds that improve organoid function ex vivo in patients via N-of-1 trials, so as to prove that a positive ex-vivo organoid response indeed correlates with a good in-vivo clinical response. Therefore, the research community

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Search strategy and selection criteria We searched PubMed for articles published in English from Jan 1, 2014, to Sept 30, 2015, with a focus on new areas of research for symptomatic therapies and therapies aiming to correct the molecular defect, and on recent treatments that are in or close to clinical development. We used the search terms “cystic fibrosis” and “novel therapies”, “novel drugs”, “treatments”, “correctors”, “potentiators”, “basic defect therapies”, or “innovative therapeutic strategies”. To complement our search on new drugs that are being developed, we also reviewed abstracts from the 38th European Cystic Fibrosis Conference and the 28th Annual North American Cystic Fibrosis Conference, which were both held in 2015.

has to move towards more standardised and accessible formats for the generalised use of these innovative approaches as surrogate markers in clinical trials and in personalised medicine. These major technological advances offer new possibilities for targeted therapies by precisely predicting which patients will or will not respond to a medical therapy.138

Conclusion On multiple fronts, treatments that improve outcome in patients with cystic fibrosis are emerging. Studies of many symptomatic treatments aiming to improve the effects of CFTR dysfunction (eg, abnormal mucociliary clearance and chronic lung infection; table 1) and treatments that target the underlying defect (table 3) are underway. Therapies targeting the molecular defect can be mutation agnostic (eg, gene therapy) or can be specific to mutation class or even particular mutations. With more than 2000 different CFTR mutations causing the disease, a precision medicine approach could offer great benefit. Contributors KDB reviewed articles on standard therapies, and MDA reviewed articles on treating the basic defect. Both authors wrote the manuscript. Declaration of interests KDB is a member of Steering Committee, an Advisory Board Member, and a principal investigator in studies funded by Vertex Pharmaceuticals; a member of the data monitoring committee in studies funded by Aptalis; a consultant and principal investigator in studies funded by Galapagos, PTC Therapeutics, and Boehringer; an Advisory Board Member and principal investigator in studies funded by Pharmaxis; and a consultant for Ablynx, Protalix, and Raptor during the conduct of the study. MDA reports personal fees from Vertex Pharmaceuticals, PTC Therapeutics, Gilead Sciences, and Galapagos; and grants from Gilead Génese outside of the submitted work. Acknowledgments KDB is supported by Instituut voor Wetenschap en Technologie (IWT) grant ZL356704 and Interne Fondsen KULeuven (IF) C32/15/027. Work in KDB and MDA’s laboratory is supported by UID/MULTI/04046/2013 centre grant (to BioISI) and research grants from FCT/MCTES Portugal (PTDC/BIM-MEC/2131/2014), Cystic Fibrosis Foundation USA (AMARAL15XX0 and AMARAL15XX1), Gilead GÉNESE-Portugal Programme (PGG/008/2015), and Cystic Fibrosis Trust UK (SRC 003).We thank Luka Clarke for revising this manuscript and to Els Aertgeerts for secretarial assistance.

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