CFTR Modulator Therapies for Cystic Fibrosis

Abstract

The cloning of cystic fibrosis transmembrane conductance regulator (CFTR) set into motion a cascade of discoveries that have helped to reveal the underlying pathophysiologic basis of cystic fibrosis (CF). This discovery and the knowledge that followed have also provided the opportunity to target this basic defect, with the hope of reversing or preventing the serious clinical consequences that result from absent CFTR function. With the recent approval of 2 therapies that directly modulate CFTR function in more than half of the CF population, we are now at the beginning of a pathway to providing increasingly effective therapies that have the potential to provide a fundamental change in the outcome of most patients with CF.

Introduction

Despite remarkable improvement in clinical outcomes, cystic fibrosis (CF) remains the leading inherited cause of death among Caucasians. The disease manifestations of CF are diverse, yet all result from reduced expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR). Patients typically experience symptoms that relate to respiratory, pancreatic, intestinal, biliary, and reproductive tract pathology, mirroring the tissue distribution of CFTR. Recently, the emergence of small-molecule CFTR modulating therapies has heralded a new era of CF clinical care, as the pathophysiologic basis of the disease is now beginning to be targeted.

CFTR is an ATP-dependent, voltage-gated anion channel belonging to the ATP binding cassette (ABC) class of transporters. Complex intracellular regulatory events, including ATP binding and phosphorylation, lead to conformational changes in the channel that permit transport of chloride and bicarbonate down their electrochemical gradients. In the lung, CFTR is believed to control the volume and/or composition of the airway surface liquid (ASL) that is produced by surface and glandular epithelia.^1–3 This ASL layer is critical to lung defenses, and CFTR dysfunction impairs these lung defenses through disruption of ASL homeostasis, setting the stage for the development of chronic airway infection and inflammation.^4,5

The precise nature of the CF host defense defect continues to be a matter of intense study and debate, however, and may in fact be multifactorial. Impaired chloride and water secretion into the ASL compartment, combined with mucus hypersecretion, leads to the accumulation of dehydrated airway mucus and, ultimately, compaction of the periciliary layer.⁶ These phenomena reduce cilia and cough-driven mucus clearance, thus impairing a key innate airway defense. In addition, absence of bicarbonate secretion through CFTR may alter ASL pH and reduce bacterial killing, providing another potentially important host defense defect.^7,8 Finally, reduced bicarbonate secretion and/or altered ASL pH may interfere with the release of mucin macromolecules from submucosal glands and goblet cells, leading to a nidus of mucus that may fail to detach from a gland opening, thus interfering with mucus transport through a distinct physiologic mechanism.^9–12 Separately or in combination, these pathogenic mechanisms lead to chronic airway infection and inflammation. While chronic neutrophilic inflammation could simply be the result of mucus stasis and infection, CFTR dysfunction has also been reported to contribute to a hyperinflammatory state in CF airways and neutrophil dysfunction,^13–15 providing an additional potential host defense abnormality. The development of novel therapies designed to directly modulate CFTR function is now available with more expected soon.¹⁶ This new class of therapies holds the potential to reverse the underlying basis of CF and change the face of this disease.

CFTR Structure and Function

An understanding of CFTR structure and function is guiding efforts to therapeutically target CFTR. Simplistically, CFTR has 5 domains: 2 membrane spanning domains (MSD1 and MSD2), 2 nucleotide binding domains (NBD1 and NBD2), and a regulatory domain (R domain). Together, the 2 membrane spanning domains form the pore through which anions traverse the channel, whereas the 2 intracellular NBDs form a binding site for ATP (Fig. 1A). The R domain has multiple sites that can be phosphorylated by protein kinase A (PKA), which is required for channel activation. The binding of ATP to the NBDs causes conformational changes in the MSDs, opening the channel pore and allowing the passage of anions through CFTR.

FIG. 1.

(A) Schematic of normal cystic fibrosis transmembrane conductance regulator (CFTR) function. CFTR is a transmembrane protein made up of 5 subunits: 2 membrane spanning domains forming a pore, 2 nucleotide binding domains, and a regulatory domain. CFTR allows for conductance of anions such as chloride when the regulatory domain is phosphorylated by protein kinase A, and the nucleotide binding domains bind and then hydrolyze ATP. (B) Mutations in position 551 interfere with the binding of ATP, and thus, the gating function of CFTR. Mutations in position 508 interfere with the interaction of NBD1 with the membrane spanning domains. Ivacaftor stabilizes the open confirmation of CFTR in an ATP-independent manner, but in a manner that is still subject to the activation of the R-domain by protein kinase A.

More than 1,900 different CFTR mutations have been identified to date. Modulation strategies that might be applied to any individual mutation are intimately linked to its effect on CFTR structure, biosynthesis, and function (Table 1). The most common mutation is a deletion that leads to the loss of phenylalanine at position 508 (F508del) in NBD1 and accounts for about 75% of CFTR mutant alleles worldwide.¹⁷ The absence of phenylalanine at this site causes misfolding of the protein during posttranslational processing in the rough endoplasmic reticulum, leading to degradation through the proteasome pathway and little or no protein reaching the cell surface. Any F508del CFTR that does escape endoplasmic reticulum (ER)-associated degradation and reaches the cell surface displays abnormal gating (ie, channel opening and closing) and a shortened life span on the cell surface due to protein instability.^18,19

Table 1.

CFTR Mutation Classification and Treatments

Mutation class	CFTR defect	Example mutations	Class-specific CFTR modulating therapies: available or hypothesized
Class I	Disruption of gene transcription or translation resulting in absent or incomplete CFTR protein	Nonsense- (“stop”) mutations	Read through promoters, including Ataluren
Class II	Defects in posttranslational processing of the protein and/or trafficking errors resulting in absent CFTR on cell membrane	F508del	Correctors such as lumacaftor and VX-661 + Potentiator
Class III	Defects in nucleotide binding leads to functional impaired ion permeability through the channel	G551D	Potentiators, such as ivacaftor
Class IV	Alteration in channel structure leading to restricted ion permeability (conductance defect)	R117H	Potentiators, such as ivacaftor, may influence CFTR function
Class V	Decreased quantity of normal CFTR	2789+5G>A, A455E, 3849+10KB C>T	(Ivacaftor is being studied)
Class VI	Functional CFTR with decreased life span at plasma membrane	N287Y, Q1412X, “corrected” F508del

CFTR, cystic fibrosis transmembrane conductance regulator.

Overview of CFTR Modulation—The Current Clinical Landscape

Small molecules designed to improve the biosynthesis, trafficking, or function of CFTR are called CFTR modulators and are further classified as either correctors or potentiators. Correctors increase the amount of CFTR expressed at the cell surface, whereas potentiators increase the time that the CFTR already localized to the cell surface will spend in the open state. A third class of compounds that encourage skipping of abnormal stop mutations during protein translation is called read through promoters. Presently, there are 2 CFTR modulators—1 potentiator and 1 corrector—that are available for the treatment of CF. Ivacaftor (VX-770; Kalydeco™; Vertex Pharmaceuticals, Cambridge, MA) is a CFTR potentiator for patients 2 years and older with specific class III/gating mutations, as well as for patients with R117H, a class IV mutation that has both gating and conductance abnormalities. This collection of mutations accounts for ∼5% of patients with CF. Lumacaftor (VX-809) is a CFTR corrector that has been developed as part of a combination product with ivacaftor (ORKAMBI™; Vertex Pharmaceuticals) for patients 12 years and older who are homozygous for the F508del mutation.

Ivacaftor

Despite a great deal of progress in understanding CFTR structure and function, the development of CFTR modulators has largely rested upon a strategy of high-throughput screening (HTS) using small-molecule libraries and cell-based functional assays. The identification of compounds that promote CFTR synthesis (eg, ataluren), trafficking (eg, lumacaftor, VX-661), or function (eg, ivacaftor)^20,21 from large chemical libraries is performed in the context of specific mutations. Following initial identification, compounds are further refined to improve potency, safety, and other pharmacologic properties.^22,23

The most successful CFTR modulating therapy thus far is ivacaftor, a CFTR potentiator initially developed for patients carrying at least 1 copy of the G511D mutation. Ivacaftor was developed using HTS methods to screen over 225,000 candidate compounds in cells containing the G551D CFTR mutation.²² Although the precise mechanism of action was not a consideration during the drug identification process, evidence now suggests that ivacaftor causes channel opening independent of ATP binding, even though CFTR activity remains dependent on phosphorylation by PKA (Fig. 1B).^24–28 It has been proposed that ivacaftor binding to CFTR shifts the energetic favorability of an open-channel confirmation of CFTR domains toward a coupling of the MSDs and NBDs. Ivacaftor binding allows for PKA-regulated anion conductance even in the setting of an abnormal nucleotide binding domain, as is seen in both the G551D and F508del mutations of CFTR.

An early Phase II clinical trial of ivacaftor in CF subjects with at least 1 G551D allele demonstrated significant improvements in CFTR activity, as evidenced by CFTR-dependent chloride transport measured through the nasal potential difference (NPD) assay, and a marked reduction in sweat chloride concentration (59.5 mM reduction from baseline). Lung function (FEV₁) also significantly improved.²⁹ These data led to the performance of 2 phase III clinical trials in patients >12 years of age (STRIVE) and between 6 and 11 years of age (ENVISION). In both studies, subjects experienced similar large improvements in lung function (absolute increase in FEV₁ of 10.6% and 12.5%, respectively), weight gain (2.7 and 2.8 kg), and significantly improved respiratory symptoms.^30,31 Individuals treated with ivacaftor in the STRIVE trial had a 55% risk reduction of pulmonary exacerbation. A reduction in exacerbations was not seen in the ENVISION study, likely because of a low event rate in this younger age group.^30,31

These impressive data led to the approval of ivacaftor by the FDA and the European Commission in 2012 for individuals over the age of 6 with at least 1 copy of G551D. Long-term data have demonstrated lasting improvement in clinical outcomes through 34 months of treatment.³² In 2015, an open-label study of ivacaftor in children ages 2 through 5 with CF and a gating mutation demonstrated improvements in sweat chloride, nutrition, and pancreatic function,³³ prompting the FDA to extend its approval of ivacaftor for use in children over the age of 2. Recent studies have shown improvements in extrapulmonary manifestations of CF in individuals with an appropriate genotype treated with ivacaftor, including improved exocrine pancreatic function,³⁴ insulin production,³⁵ sinus disease,³⁶ intestinal pH, and microbial profile.^37,38 There has also been a case report of improved hepatic steatosis following treatment with ivacaftor.³⁹

The ivacaftor indication for treatment was expanded in 2014 by both the FDA and the European Commission to include 8 other class III mutations (G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P, and G1349D) after similar improvements were demonstrated in these patients.⁴⁰

Finally, approval for use in patients (>6 years) with the R117H mutation, a class IV mutation with mixed conductance and gating defects, was granted following a trial in this patient group. Although R117H patients demonstrated lung function improvements that were considerably more modest than seen in the context of classic gating mutations, they as a group had much milder disease with potentially less room to improve.^40,41 Interestingly, older and more severely affected subjects did experience greater improvement with ivacaftor, whereas children aged 6–11 years, who on average had normal lung function, showed no improvement in lung function after 24 weeks of treatment.⁴¹ The impact of the intron 8 polythymidine sequence on disease manifestations of patients with this mutation is well described,⁴² but it is not known whether this genetic factor could also influence or predict treatment response to ivacaftor.

A clinical study of ivacaftor monotherapy in patients homozygous for F508del showed no significant improvement in lung function. Because F508del is a class II mutation, with abnormal protein folding and cellular trafficking as its primary abnormality, this result was not unexpected, although a minimal decrease in sweat chloride concentration was observed.⁴³ This study confirmed that potentiator monotherapy is insufficient to address F508del-CFTR defects.

Lumacaftor

The identification of correctors through high-throughput screening provided the opportunity to test molecules designed to modify F508del-CFTR's folding and trafficking defects. Lumacaftor, previously known as VX-809, was the first corrector to enter clinical trials after in vitro studies demonstrated its ability to increase the amount of mature CFTR at the cell surface and restore chloride transport in cultured CF airway epithelia to about 14% of normal levels.²³ Its mechanism of action is not well understood, but is believed to involve an interaction between NBD1 and MSD1 during protein folding, increasing the conformational stability of the molecule.^44–49

While an early investigation of lumacaftor monotherapy in patients homozygous for F508del demonstrated small but significant improvements in sweat chloride measurements, no difference in NPD measurements, the amount of mature CFTR measured by Western blot of rectal biopsy samples, or lung function was observed.⁵⁰ Given that appropriately trafficked F508del-CFTR still has significant gating and membrane stability defects that must be solved to restore full function, the lack of clinical efficacy was not unexpected.

Because in vitro studies demonstrated that combination therapy with lumacaftor and ivacaftor led to greater restoration of CFTR activity (∼25% of wild type²³), a phase II study was designed to test the hypothesis that lumacaftor with ivacaftor would yield greater clinical efficacy. In this study, patients were initially treated with lumacaftor alone (or placebo), followed by combination therapy to discern the impact of adding ivacaftor in the same patient groups.⁵¹ Interestingly, a modest reduction in sweat chloride was again demonstrated during lumacaftor monotherapy, but no additional effect on sweat chloride was observed after adding ivacaftor. Despite this fact, lung function tended to decline during lumacaftor monotherapy, but improved significantly during combination therapy.⁵¹ This apparent discordance between the sweat gland and lung responses could not be resolved, in part, because of the lack of pulmonary biomarkers that reflect CFTR function in the lung.

With these supportive phase II data, 2 nearly identical phase III studies in patients homozygous for F508del (TRAFFIC and TRANSPORT), together involving more than 1100 patients, were conducted concurrently. Each study included a low- and a high-dose lumacaftor group, each also receiving ivacaftor, in addition to a placebo group. Following 24 weeks of treatment, the absolute changes in FEV₁ in active treatment versus placebo groups ranged from 2.6% to 4.0%, corresponding to 4.3%–6.7% relative improvements.⁵² More impressive, however, was the observed reduction in pulmonary exacerbations in lumacaftor/ivacaftor treatment patients (30%–39% reduction), IV antibiotic use (45%–56% reduction), and hospitalizations (39%–61% reduction).⁵² Given the important role that exacerbations play in the progression of CF lung disease,^53,54 these data ultimately led to the approval in the United States by the FDA of lumacaftor/ivacaftor combination therapy (ORKAMBI; Vertex Pharmaceuticals) for F508del homozygous patients over the age of 12. Importantly, the safety profile of lumacaftor/ivacaftor also appeared to be favorable in the phase III program, although dyspnea and chest tightness did appear as a common adverse event.^50–52 These symptoms frequently occurred within the first few doses of medication, but resolved within 2–3 weeks for most patients.

The use of lumacaftor is complicated by its strong induction of the cytochrome P450 enzyme CYP3A, which increases the metabolism of many medications that are substrates of the enzyme, including azole antifungals and oral contraceptives.⁵⁵ Importantly, ivacaftor is also a CYP3A substrate. This fact complicates the pharmacology of the lumacaftor/ivacaftor combination product (ORKAMBI) and necessitates a higher ivacaftor dose when given in the presence of lumacaftor. This interaction also has the potential to reduce treatment efficacy if optimal ivacaftor levels are not achieved in an individual patient.

The apparent discrepancy between minimally changed sweat chloride concentration and improved pulmonary function has raised questions and complicated the assessment of new CFTR modulator therapies. The possibility that combination therapy with lumacaftor/ivacaftor yielded improvements in lung function by acting on targets other than CFTR has been raised, but this seems unlikely, given that neither agent used as monotherapy yielded positive effects in the F508del population. Alternatively, the F508del-CFTR response to correctors and potentiators may be tissue specific. A better understanding of the complete role that CFTR plays in the various tissues, as well as the highly complex downstream effects of these drugs, is needed to allow us to adequately interpret the drug responses observed in clinical trials. The lack of correlation between changes in sweat chloride concentration and FEV₁ in individual subjects with G551D-CFTR treated with ivacaftor also highlights this issue.⁵⁶ These observations clearly suggest that our ability to predict clinical benefit from sweat chloride responses is quite limited, at least in the case of F508del-CFTR, and better pulmonary-focused outcome measures are needed.

VX-661

VX-661 is the second corrector that has been identified and developed and is entering clinical trials. Although VX-661 is chemically similar to lumacaftor and shares a similar mechanism of action, it does not induce CYP3A and thereby does not impact ivacaftor levels. This pharmacologic benefit is highly desirable when considering the prospect of combining additional CFTR modulators in the future.

In a phase II study of VX-661 in F508del homozygous patients, monotherapy led to no significant improvement in lung function, as was observed with lumacaftor, whereas combination therapy with ivacaftor at the 2 highest doses yielded a 4.5%–4.8% absolute improvement from baseline in percent-predicted FEV₁ (7.5%–9% relative improvement) after 4 weeks of treatment.⁵⁷ Like lumacaftor/ivacaftor, sweat chloride reductions were modest during VX-661 monotherapy (4–5 mM), with little or no additional effect of adding ivacaftor.^57,58

Long-term randomized controlled trials of VX-661/ivacaftor combination therapy are currently underway in 4 patient groups, including those who are (1) homozygous for F508del, (2) heterozygous for F508del with a second mutation coding for CFTR with residual function, (3) heterozygous for F508del with a classic gating mutation (including G551D), and (4) heterozygous for F508del with a second mutation that is predicted to be nonresponsive to either agent (ClinicalTrials.gov identifiers NCT02516410, NCT02392234, NCT02412111, and NCT02347657). These trials should not only reveal whether VX-661/ivacaftor is safe and effective in F508del homozygous patients, as was shown for lumacaftor/ivacaftor, but also whether this treatment approach can be expanded to additional patient populations.

Preliminary data do suggest that combination therapy is likely to benefit heterozygous patients with a class III or partial functioning second mutation, as additional FEV₁ improvement was observed in G551D patients following the addition of a corrector to ongoing ivacaftor treatment.^58–60 Furthermore, the trial in F508del heterozygotes with nonresponsive second mutation tests the hypothesis that the more favorable pharmacologic properties of VX-661, compared to lumacaftor, will further increase CFTR function such that a clinical benefit may be achieved in these patients as well. The cumulative impact of these trials is large, given the possibility of increasing the number of patients with CF for whom a CFTR modulation therapy exists.

Ataluren

Although the development of CFTR correctors and potentiators has been a highly active area of drug development, the development of read-through promoters is also an important focus of research. Class I mutations, composed primarily of nonsense and splice site mutations, can be modulated by molecular mechanisms that suppress the recognition of abnormal termination signals during protein translation. Although F508del is responsible for about 75% of CF-causing mutations worldwide, 6 of the 8 next most common CF-causing mutations are nonsense mutations, and worldwide ∼10% of patients carry these mutations.¹⁷ It has long been recognized that aminoglycosides increase the translation of CFTR in the setting of premature stop mutations in vitro,^61–63 but clinical studies have yielded mixed results,^64,65 and concerns over toxicity during chronic use would be limiting.

Ataluren (PTC-124, Translarna™; PTC Therapeutics, South Plainfield, NJ) is an investigational small molecule drug identified using HTS being developed to promote nonsense suppression of CFTR transcription. However, there have been conflicting data, both in vitro⁶⁶ and from clinical trials,⁶⁷ complicating the evaluation of ataluren. Earlier unblinded crossover studies provided some evidence of increased CFTR function through the NPD assay, but sweat chloride values did not change.^67,68 A subsequent larger, multicenter blinded study was unable to reveal mechanistic evidence of improved CFTR function through NPD and sweat chloride changes and overall did not improve lung function.⁶⁹ However, because a subgroup of patients who were not using inhaled aminoglycosides did show improved lung function when compared to placebo-treated patients, an additional phase III clinical trial of ataluren in patients with nonsense mutations who are not taking chronic inhaled aminoglycosides is currently underway (ClinicalTrials.gov identifier NCT02139306).

Limitations of Current Therapies and Future Directions

Recent and ongoing studies demonstrate the rapid and significant progress that is occurring in the field of CFTR modulation. However, despite these successes, it is clear that CFTR modulating strategies that target F508del fail to achieve the same degree of CFTR functional restoration and clinical benefit that was achieved by ivacaftor in class III mutations. It is likely that the more complicated biological basis of CFTR dysfunction in the setting of F508del, compared to class III or IV mutations, explains the reduced therapeutic efficacy observed with current therapies.

Another important limitation of current corrector–potentiator therapy is a persistent problem of CFTR instability once F508del CFTR has arrived at the plasma membrane. In fact, several groups have demonstrated in vitro that prolonged exposure to ivacaftor may reduce the ability of correctors to rescue CFTR function, as the result of accelerated retrieval from the plasma membrane.^59,70 Ivacaftor, while increasing gating of F508del-CFTR, appears to also destabilize F508del-CFTR in the plasma membrane, thereby decreasing the potential effectiveness of corrector–potentiator combination therapy. This effect of ivacaftor may also be seen with wild-type CFTR as well.^70–72 When we try to reconcile these clinical and in vitro data, it is clear that they are not consistent. However, these in vitro data may be revealing an additional defect and treatment shortcoming that must be addressed to achieve optimal results with modulator therapies, and the development of drugs that improve the thermostability of F508del in the plasma membrane may become an important target of future “add-on” therapies.⁶⁶

The rapid evolution in this field, marked by the successful development of CFTR modulating therapies, inspires hope for longer healthier lives for individuals with CF. However, we are faced with multiple challenges. Although positive results from modulator therapy have been achieved in F508del homozygous patients, preliminary data suggest that lumacaftor/ivacaftor treatment of F508del heterozygotes (with nonresponsive mutations on the opposite chromosome) does not achieve significant clinical improvements, leaving roughly 50% of patients without an effective modulator treatment option.

Novel correctors and potentiators are currently being developed by a number of pharmaceutical companies, and there is great interest in identifying correctors with mechanisms of action different from lumacaftor and VX-661. In vitro studies suggest that these may have more than additive or synergistic effects on CFTR function when used in combination with currently available modulators (eg, lumacaftor and ivacaftor).⁴⁸ This prospect brings a great deal of excitement and optimism to this field with the hope of not only improving efficacy in F508del homozygotes but also reaching a threshold needed to achieve clinical benefit for F508del heterozygotes who currently are without an effective modulator option.

Another daunting challenge is the development of CFTR modulators for those individuals with rare CFTR mutations. For this to be feasible, a better understanding of CFTR biology is needed, in addition to the development of reliable biomarkers that reflect and predict clinical responses so that a personalized approach to therapy can be pursued.

Finally, the high costs of these medications have been a pointed area of concern, and we must grapple with their economic effect on the broader healthcare system while we balance cost–benefit concerns when deciding when and whether to use therapies that may be extremely costly while providing modest or uncertain long-term benefits.^73–75

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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