Download Modulating the cystic fibrosis transmembrane regulator – breaking

RCSIsmjstaff review
Modulating the cystic fibrosis
transmembrane regulator –
breaking the basic defects
Abstract
The defective allele responsible for cystic fibrosis (CF) is found in one in 19 people in Ireland.
Historically, treatment of CF comprised ameliorating patient symptoms, but recently there has
been much progress in CF therapy, not least of which are the CFTR gene modulators. These
target specific basic defects in the disease: that of deficient or abnormal synthesis; and,
processing of the CFTR channel, thus treating the disease, for the first time, systemically.
These compounds have implications for targeted CF therapy and mark a large step forward in
the field. So far, three targets have been overcome, with varying degrees of success. There
are six mutation classes in CF; Class I is most severe, but patient presentation depends on the
specific combination of alleles. Kalydeco was developed for the class III G551D mutation, and
demonstrated efficacy in homozygous patients, but the high cost of the drug remains an
issue. Lumacaftor and VX-661 were developed to address the ΔF508 Class II mutation;
lumacaftor was effective when used in conjunction with Kalydeco, and VX-661 is as yet
undergoing trials. Lastly, investigations are ongoing to assess Ataluren as a modulator of the
effects of the premature termination codons caused by Class I CF mutations; two of three
phase II studies showed varied improvement in patient symptoms following treatment with
Ataluren and a phase III trial is currently underway. This article discusses the progress in
CF-modulating therapy hitherto.
Royal College of Surgeons in Ireland Student Medical Journal 2013; 6(1): 79-83.
Introduction
Ramia Jameel
1
2
Dr Emer Reeves
2
Dr David Bergin
2
Prof. Gerry McElvaney
1
RCSI medical student
2
Department of Medicine, RCSI,
Beaumont Hospital, Dublin 9,
Ireland
Cystic fibrosis (CF) is a disorder that results from
mutations in the cystic fibrosis transmembrane
regulator (CFTR) gene. It causes a build-up of
mucus with resulting cycles of inflammation and
infection, and primarily affects the lungs and
digestive system.2 The disease was so named due
to the cyst formation in the pancreas after
pancreatic ducts became blocked in the course of
the disease’s pathology. The disorder is also termed
mucoviscidosis as a result of the thick mucus that
plugs the airways in its pulmonary pathology. It
has even been dubbed ‘65 roses’ by those patients
too young to pronounce its name or understand
its significance (Figure 1).
CF is a multisystemic hereditary disease with
protean manifestations and an autosomal recessive
pattern of inheritance. Since the discovery of the
CFTR gene in 1989,3 hopes of clinicians and
researchers have been focused towards the time
when knowledge of its effects could be built upon
to cure CF, but until just three years ago there had
been no significant progress.
CF has particular importance in Ireland, where
2.98 in 10,000 individuals have CF and one in 19
of the population is a carrier of a defective CFTR
allele.4 The incidence of CF in Ireland is the
highest in the world – nearly four times the rate
in other European countries and in the USA.4
Reflecting this, much work has been done in
Ireland directed towards individuals with the
disease, ranging from the RCSI Department of
Medicine’s significant participation in the CF
therapy trial of VX-7705 to the building of
dedicated CF centres in hospitals nationwide.6 To
date, the majority of treatment for CF has been
symptomatic, not curative, involving the use of
hypertonic saline, dornase alfa, pancreatic
enzymes, albuterol and inhaled antibiotics.7 These
are given to improve airway surface liquid
volume, break down mucus build-up, aid nutrient
absorption in the gastrointestinal tract, relax
bronchial muscle, and prevent and combat
infection. Life expectancy for CF patients has
increased and is now approaching 40 years,8 and
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FIGURE 1: ‘65 roses’ is how some young patients first learn to say
‘cystic fibrosis’.1
FIGURE 2: CFTR mutation classes and common mutations. Mutation
class correlates more closely to pancreatic function than to pulmonary
pathology.9,15
exciting new developments are underway as more discoveries are
being made with regard to the gene and its effects.
Mutation-specific CFTR-modulating therapy is among these advances
along with gene therapy and newer, more effective antimicrobials.
This article discusses the upcoming therapeutic CFTR modulators.
The presentations of CFTR mutation classes I-VI (Figure 2) range from
most severe to sub-clinical, respectively:
n Class I mutations result in a premature termination codon (PTC).
This causes the production of a truncated form of CFTR that is
degraded before it reaches the plasma membrane – thus it results
in a complete absence of CFTR on the membrane.9
n Class II mutations include the most common ΔF508 mutation. This
class causes abnormal post-translational modifications to the
complete protein – in this class, too, the mutated protein is
degraded before it can reach the cell membrane.9
n Class III mutations result in defective regulation of CFTR channel
function. This occurs due to impaired ATP binding and
hydrolysis, which stops energy-dependent channels from
opening. In Class III mutations there is a normal amount of CFTR
on the cell membrane but it is non functional.11 A prominent
example of this class is G551D (incidence of 4% worldwide, 10%
in Ireland).12 It is the second most prevalent mutation in
Ireland.13 The G551D mutation has recently received much
media attention as its respective modulator treatment, Kalydeco,
was, as of January 2012, approved by the FDA for use after an
accelerated Phase III trial.14
n Class IV mutations lead to a defective CFTR channel pore. These
mutations result in decreased protein function, and include the
relatively common R117H mutation.9
n Class V mutations cause a reduced amount of functional CFTR
protein to be present at the membrane.9
n Class VI mutations cause altered regulation of channels other than
the chloride channel. There is overlap of this class with other
classes, such as for the ΔF508 mutation, which is both a Class II
and Class VI mutation (Figure 2).9
CFTR channel and function
The CFTR gene encodes for a chloride channel found in the apical
membrane of secretory epithelia in respiratory, gastrointestinal,
reproductive and other exocrine glands.9 Its structure consists of two
transmembrane domains (of six α-helices each), two cytoplasmic
nucleotide binding domains (NBDs) and a regulatory domain (R
domain) containing protein kinase A and P phosphorylation sites.9 The
channel through which the chloride molecules pass is formed by the
two transmembrane domains.9 The CFTR protein is a member of the
ATP-binding cassette (ABC) family, a group of proteins that bind ATP in
order to transport molecules across cell membranes.9
The importance of CFTR – ion transport regulation
One of CFTR’s main functions is to allow the exit of Cl- ions from
epithelial cells into the lumen.10 However, it is a multifunctional protein
and many of its additional functions have only recently been described.
In addition to facilitating chloride transport, CFTR also regulates
transport of ions such as Na+ and CO3-, predominantly through
interactions involving its NBD.9 Thus, normal CFTR function is essential
on several levels to prevent duct blockage, mucous build-up, infection
and inflammation.
Mutation classes
Many of CF’s clinical presentations result from the varied effects on ion
conductance. The severity of the disease, in turn, depends upon the
extent to which CFTR function is impaired.9 There are six classes of
CFTR mutation based on their effects on the CFTR protein.9 The CFTR
modulators are an attempt at treatment specific to mutation class, and
thus understanding these mutations is key to understanding many of
the ongoing therapeutic advances in the disease.
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The phenotypic presentation of these mutations depends upon the
combination of an individual’s alleles. If both the alleles are of the
more severe genotypes (classes I-III), the individual is likely to present
with the classic CF phenotype, i.e., pancreatic insufficiency,
gastrointestinal symptoms and sinopulmonary infections.9
RCSIsmj staff review
Correcting and potentiating CFTR
Drugs that correct the problem of early degradation (as in Class I) are
called CFTR ‘correctors’.11 Those that correct aberrant trafficking or
gating of the channels once at the membrane (as in Classes II and III)
are known as CFTR ‘potentiators’ (Figure 3).11
Three different mutation targets are currently being addressed by two
pharmaceutical companies:
1. PTC124 (Ataluren) by PTC Therapeutics,7,8 to overcome Class I
mutations that result in PTCs.
2. VX-809 (Lumacaftor) by Vertex Pharmaceuticals,7,8 to correct CFTR
trafficking defects in Class II mutations.
3. VX-770 (Kalydeco/ivacaftor), also being developed by Vertex
Pharmaceuticals,7,8 which potentiates CFTR at the membrane in
Class III mutations.
PTC suppression and correction of aberrant CFTR trafficking are both
more difficult objectives to reach as they involve modulation issues on
several levels:
n PTCs can result from several mutations, which need to be targeted
individually;
n restoration of translation is also required; and,
n the functional protein must then be trafficked to the cell
membrane.
The three upcoming treatments are discussed here in order of
discovery, which also (understandably) reflected the targeting of the
least to most severe class.
Kalydeco – potentiating at the plasma membrane
The story of the first CFTR-modulating drug to be approved in the
therapy of CF, Kalydeco (also known as VX-770 and ivacaftor), is an
interesting one. This compound was a result of a commitment on the
part of the Cystic Fibrosis Foundation to, based on the knowledge of
CFTR accumulated to date, treat CF as a multisystem disorder and to
invest in the search for new therapies targeting basic CFTR defects.16 A
collaboration with Vertex Pharmaceuticals led to high-throughput
screening with CF lines to evaluate 228,000 molecules, from which
VX-770 was discovered.16 It demonstrated ability to potentiate CFTR in
homozygous G551D (Class III) and heterozygous G551D/ΔF508
(ΔF508 being of Class II) mutations.
The exact mechanism by which Kalydeco exerts its effects is as yet
unknown, but it has demonstrated positive results in all its pivotal
studies to date: one Phase II and two Phase III trials.17,18 It resulted in
notable and sustained improvements in forced expiratory volume in
one second (FEV1), which, it has been noted, is “the most well studied
and validated surrogate end point for patients with cystic fibrosis and
reflects airways obstruction”.16 A 10.6% increase in FEV1 from baseline
was demonstrated as compared with placebo, as well as improved
sweat chloride levels and nasal potential difference (NPD); the latter
reflects chloride transport.18 However, it would be valuable to measure
quantitative chest imaging and lung clearance index in future
investigations as they have been noted to be more sensitive as
measures of pulmonary disease in CF.16
Accurso et al. conducted a two-part Phase II trial evaluating Kalydeco’s
FIGURE 3: Targets of upcoming drugs.2
safety.17 The adverse events profile of Kalydeco was similar to placebo,
with serious adverse events being fewer in the treatment group (24%
vs. 42%).17 Ramsey et al.’s Phase III STRIVE study of 2011 was
conducted in patients over 12 years old. It demonstrated a significant
increase in FEV1, a decrease in sweat chloride levels (some out of the
threshold of CF diagnostic), a decrease in pulmonary exacerbation rate
and an increase in weight from baseline.18 This study was followed by
the two-year-long PERSIST open-label study, in which the long-term
safety and efficacy of Kalydeco were examined; the benefits of the
drug remained significant.19 The ENVISION study in six- to
11-year-olds demonstrated that Kalydeco was also safe and effective
for this age group.20 The weight gain in these trials’ results remains
unexplained and warrants further investigation – postulations include
that improvement in CFTR function in the digestive tract may augment
nutritional absorption.7
Although Kalydeco is a potentiator, it has its use in patients with the
ΔF508 mutation. On its own it has no efficacy in ΔF508 homozygous
patients21 but trials are ongoing to investigate its efficacy when
combined with other CFTR modulators. Further research gaps include
the appropriateness of its use for other Class III (gating) mutations, for
patients two to five years old, and for patients with one or more copies
of the Class IV R117H mutation.7
Kalydeco, approved by the FDA in January 2012, costs $294,000 per
year for its twice-daily regimen, though Vertex Pharmaceuticals has
programmes that allow patients with no insurance and a household
income of less than $150,000 per year to obtain the drug for free.
Those with insurance can receive up to 30% of the drug’s list
price.22 After being approved by the European Medicines Agency in
July of this year for individuals over the age of six years with at least
one copy of the G551D mutation,21 Kalydeco is currently being
reviewed for use in Ireland by the drug regulatory authorities.23
Kalydeco was approved by the Irish Minister for Health, Dr James
Reilly, in February 2013.24
Correcting ΔF508 – Lumacaftor and VX-661
Class II mutations result in abnormal CFTR folding and degradation of
the protein before it reaches the cell membrane. The ΔF508 mutation,
the most common mutation in Ireland (found in approximately 66%
of CF patients)25,26 belongs to this class. There is no CFTR present on
the cell membrane as a consequence of homozygous ΔF508
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mutations. Class II mutations therefore require more regulation – they
require a correctly formed protein to be produced as well as
translocation of the CFTR channel to the cell surface.
Due to the hypothesis that there may be residual defective ΔF508
CFTR at the membrane, Kalydeco was trialled in isolation for patients
homozygous for the ΔF508 mutation but, as discussed, did not prove
clinically effective. A Phase II randomised controlled trial (RCT) in a
similar group was conducted for VX-809, also known as Lumacaftor,
and it demonstrated safety and modest efficacy in reducing sweat
chloride, and concluded that Lumacaftor is a viable candidate for
further investigation.25
A Phase II RCT investigating the combined effect of Lumacaftor and
Kalydeco was conducted in 2011. In combination, the two drugs work
together to remedy the defect: Lumacaftor corrects the folding and
processing of CFTR and thus aids in transporting the protein to the
membrane, and Kalydeco then supports its function.2 The
62-participant study concluded that the combination improved CFTR
function significantly in patients homozygous for the ΔF508 mutation
and recommended further clinical investigation.27 In June 2012, the
results of a second part of that combination trial were announced,
enrolling 109 people over 18 years of age. Improvement in pulmonary
function was dose dependent, with those who received the greatest
dose of VX-809 demonstrating most improvement, and homozygous
individuals benefiting more than heterozygotes. A pivotal trial of the
Kalydeco–VX-809 combination is planned for homozygous and
heterozygous ΔF508 CF patients in 2013.28
Another drug is being investigated called VX-661, which is postulated
to increase CFTR trafficking to the membrane in the ΔF508 mutation.8
An exploratory Phase II clinical trial is in progress to evaluate the safety
and effects of VX-661 alone and in combination with Kalydeco. The
study is due to be completed in August 2013.29
Terminating PTCs – Ataluren
Class I comprises the most severe forms of mutation and represents
10% of CF genotypes.7 It consists of nonsense mutations that cause
the production of a truncated or unstable form of CFTR, which is
detected by chaperone proteins at the ER and degraded before it
reaches the cell surface. This class is associated with complete lack of
CFTR protein at the apical membrane of epithelial cells. G542X is the
most common of these mutations.2 Gene therapy and
aminoglycosides such as gentamicin30,31,32 are currently being
investigated for a therapeutic approach to Class I mutations.33
Aminoglycosides allow the attachment of a near-cognate tRNA to the
defective template, allowing a full-length protein to be produced. This
has been shown to be efficacious in vitro, in animal studies of CF and
muscular dystrophy, and in small numbers of CF patients.8
Of more relevance to this discussion of CFTR modulators, an orally
bioavailable non-glycoside compound specifically developed to
promote ribosomal read-through of nonsense mutations is under
review, designated PTC124 and also known as Ataluren.34 Ataluren
binds to eukaryotic ribosomes and suppresses PTCs in cell- and
animal-based disease models. There have been three randomised
open-label dose-ascending Phase II trials in CF. The results of all
showed tolerability to Ataluren in the short term. Improved function of
CFTR (measured by NPD) was seen in two.8 One study showed that
CFTR localisation to the nasal cell membrane was improved, and
another’s results demonstrated a reduction in cough over three
months.35,36 However, the third study did not demonstrate an
improvement in NPD and limitations of all studies included the small
sample size and lack of placebo controls.15 Concerns about Ataluren
include its potential to permit oncotic change, as its read-through
capabilities could potentially allow native mutations to go undetected.7
Adherence may also be an issue, as the medication needs to be taken
thrice daily.7 A Phase III RCT is in progress to determine the drug’s
safety, efficacy and tolerability for the duration of 48 weeks, to be
completed in October 2012.37
Conclusion
The advances made in CF therapeutics are many and encouraging, but
several questions remain to be answered. The long-term efficacy of
these medications is yet to be elucidated and more than 1,600
disease-associated mutations3 have to be tackled before CF can be
known as a curable disease. These drugs also provide the hope that the
damage CF can cause can be stopped in its early stages. Use of
Kalydeco has improved quality of life, for those who can afford it, with
decreased cough and sputum production and a reduced burden from
conventional treatment. Progressive lung disease and premature death
do not seem as imminent with the advent of such potentially curative
therapies, though longer-term studies remain to be conducted. The
issue also arises as to whether investment should focus solely on the
G551D patients by subsidising the drug or whether interventions
catering for all CF patients should be allocated the limited healthcare
budget – the ever-present ‘distribution problem’.38 It remains to be
seen whether PTC- and ΔF508-targeting modulators are potent
enough to have clinical effectiveness. Despite, or even because of,
these uncertainties, CFTR modulators, coupled with the introduction of
newborn screening in Ireland in July 2011,39 highlight the area of CF
therapy as one to watch in the near future.
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