Articles

Effectiveness of PTC124 treatment of cystic fibrosis caused

by nonsense mutations: a prospective phase II trial
Eitan Kerem, Samit Hirawat, Shoshana Armoni, Yasmin Yaakov, David Shoseyov, Michael Cohen, Malka Nissim-Rafinia, Hannah Blau,
Joseph Rivlin, Micha Aviram, Gary L Elfring, Valerie J Northcutt, Langdon L Miller, Batsheva Kerem, Michael Wilschanski

Summary
Background In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from Lancet 2008; 372: 719–27
nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane Published Online
conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes August 21, 2008
DOI:10.1016/S0140-
to selectively read through premature stop codons during mRNA translation, to produce functional CFTR.
6736(08)61168-X
See Comment page 691
Methods This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation
Hadassah Hebrew University
in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at Hospital, Jerusalem, Israel
16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, (E Kerem MD, S Armoni BSN,
patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The Y Yaakov MSc, D Shoseyov MD,
primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients M Cohen MD,
M Wilschanski, MBBS); PTC
who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential Therapeutics, South Plainfield,
difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial NJ, USA (S Hirawat MD,
was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380. GL Elfring MS, VJ Northcutt MS,
LL Miller MD); Department of
Genetics, The Life Sciences
Findings Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second Institute, The Hebrew
cycle. Mean total chloride transport increased in the first treatment phase, with a change of −7·1 (SD 7·0) mV University, Jerusalem, Israel
(p<0·0001), and in the second, with a change of −3·7 (SD 7·3) mV (p=0·032). We recorded a response in total (B Kerem PhD,
M Nissim-Rafinia PhD);
chloride transport (defined as a change in nasal PD of −5 mV or more) in 16 of the 23 patients in the first cycle’s
Schneider Children’s Hospital,
treatment phase (p<0·0001) and in eight of the 21 patients in the second cycle (p<0·0001). Total chloride transport Petach Tikvah, Israel
entered the normal range for 13 of 23 patients in the first cycle’s treatment phase (p=0·0003) and for nine of 21 in (H Blau MD); Carmel Hospital,
the second cycle (p=0·02). Two patients given PTC124 had constipation without intestinal obstruction, and four had Haifa, Israel (J Rivlin MD); and
Soroka Medical Center, Be’er
mild dysuria. No drug-related serious adverse events were recorded.
Sheva, Israel (M Aviram MD)
Correspondence to:
Interpretation In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration Dr Eitan Kerem, Hadassah
of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR Hebrew University Hospital,
dysfunction. Mount Scopus, Jerusalem,
Israel 91240
[email protected]
Funding PTC Therapeutics, Cystic Fibrosis Foundation Therapeutics.

Introduction UAG, or UGA stop codon in the protein-coding region of

Cystic fibrosis results from mutations in the gene that the corresponding messenger RNA (mRNA) transcript.
encodes the cystic fibrosis transmembrane conductance Such a stop codon causes premature cessation of
regulator (CFTR), an apical cell-surface epithelial chloride translation, with protein truncation leading to loss of
channel that promotes chloride efflux and secondarily function and consequent disease. Nonsense mutations
inhibits constitutive sodium influx via the epithelial sodium are responsible for about 10% of cystic fibrosis cases
channel (ENaC).1 Dysfunction of this regulator leads to worldwide.2 However, in Israel, nonsense mutations are
epithelial mucous dehydration and viscous secretions, the cause of cystic fibrosis in most patients.3 Because
which often cause chronic neutrophilic inflammation and people with such mutations produce little functional
occlusion of respiratory airways. Obstruction of pancreatic CFTR, these patients usually have a phenotype of severe
ducts, the biliary tract, and the vas deferens can occur. cystic fibrosis.4
Patients typically develop progressive respiratory dys- Certain aminoglycoside antibiotics (eg, gentamicin)
function and persistent pulmonary infections and often can induce ribosomes to read through a premature stop
have pancreatic insufficiency, diminished bodyweight, codon in mRNA, resulting in incorporation of an amino
chronic hepatobiliary inflammation, and male infertility. acid and continuation of translation to produce a
Respiratory failure is the most common cause of death. complete protein.5 We have previously shown that topical
No means for correcting the genetic defect has been application of gentamicin drops to the nasal mucosa can
available; current medical treatments are palliative. cause a local increase in CFTR-mediated chloride
A nonsense mutation is a single point alteration in transport as assessed by nasal transepithelial potential
DNA that results in the inappropriate presence of a UAA, difference (PD) in patients who have sufficient CFTR

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transcripts that contain a nonsense mutation.6,7 However, codons, but not normal stop codons.8 When tested in a
the inconvenience of parenteral administration and the mouse model of stop-mutation-mediated cystic fibrosis,
potential for serious toxic effects preclude long-term PTC124 generated production of full-length, functional
systemic use of gentamicin for supression of nonsense CFTR.9 Phase I studies in healthy volunteers established
mutations. the initial safety profile for PTC124,10 and defined dosing
PTC124 (3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]- regimens to achieve target trough plasma concentrations
benzoic acid) is a 284-Dalton, orally bioavailable, non- (of 2 to 10 μg/mL) that are known to be active in preclinical
aminoglycoside compound that was specifically developed models.8,9
to induce ribosomes to read through premature stop We aimed to use nasal PD to assess whether PTC124
could overcome the effects of a nonsense mutation by
N=23
restoring the functional activity of CFTR and increasing
total chloride transport. We also aimed to assess other
Age (years) 25 (18 to 56)
nasal PD measures of ion-channel activity, the cellular
Sex (male) 11 (48%)
levels of CFTR mRNA with a nonsense mutation,
Bodyweight (kg) 58 (36 to 83) disease-related clinical parameters, safety of this
Nasal transepithelial potential difference treatment, compliance with treatment, and PTC124
Basal potential difference (mV)* −40 (−67 to −29) pharmacokinetics.
Total chloride transport (mV)† 1·25 (−4·9 to 8·0)
Sweat chloride (mEq/L)‡ 88 (47 to 109) Methods
Pulmonary function§ Participants
FEV1 (predicted) 65% (41 to 117) Patients were referred by four participating cystic fibrosis
FVC (predicted) 81% (52 to 117) clinics in Israel for treatment at a single centre. All
Pathological bacterial or fungal colonisation 22 (96%) patients were aged 18 years or older, and had cystic
Pseudomonas aeruginosa 21 (91%) fibrosis as established by a typical clinical presentation,
Pseudomonas and methicillin-resistant 1 (4%) an abnormal sweat test (sweat chloride >40 mEq/L by
Staphylococcus pilocarpine iontophoresis), abnormal chloride transport
Mycobacterium abscessus 2 (9%) (nasal transepithelial PD more electrically positive than
None known 1 (4%) −5 mV during nasal perfusion with chloride-free
Exocrine pancreatic insufficiency 21 (91%) amiloride and isoproterenol),11–14 and the presence of two
Liver enzyme abnormalities disease-causing CFTR mutations, with at least one being
Alanine aminotransferase 0 a nonsense mutation as determined by gene sequencing.
Aspartate aminotransferase 1 (4%) Other criteria for eligibility were forced expiratory volume
Alkaline phosphatase 4 (17%) in 1 second (FEV1) of at least 40% of that predicted for a
Gamma-glutamyl transferase 1 (4%) patient’s age, sex, and height,15 and an oxygen saturation
Bilirubin 2 (9%) of 92% or greater in room air. Patients were excluded if
Lactate dehydrogenase 2 (9%) they had clinically unstable lung disease; a positive test
for infectious hepatitis; serum bilirubin greater than
Data are number (%) or median (range). FEV1=forced expiratory volume in
1 second. FVC=forced vital capacity. *Lower limit of normal=−28 mV.14 †Upper normal limits; serum transaminase values of twice
limit of normal=−5 mV.11,13,14 ‡Upper limit of normal=40 mEq/L. §Based on normal limits or more; or had recently used systemic or
normative data for age, sex, and height.15 inhaled aminoglycosides (within 14 days of start of
Table 1: Baseline characteristics treatment). Patients were permitted to continue stable
regimens of other inhaled drugs and oral pancreatic
enzymes. Female patients were excluded if they were
Allele 1 Allele 2 Stop codon Total Treatment response* Change to pregnant or breastfeeding. The institutional ethics
normal range†
committee and the Israeli Ministry of Health approved
G542X ΔF508 UGA 3 3 (100%) 3 (100%) the study protocol. All participants provided signed
G542X W1282X UGA/UGA 1 0 1 (100%) informed consent before initiation of study procedures.
G542X N1303K UGA 1 1 (100%) 1 (100%)
W1282X ΔF508 UGA 13 10 (77%) 9 (69%) Procedures
W1282X W1282X UGA/UGA 3 1 (33%) 1 (33%) We gave PTC124 orally, as a powder suspended in water, to
W1282X 3849+10kB C→T‡ UGA/UAA 1 1 (100%) 1 (100%) all eligible patients three times per day during each
3849+10kB C→T‡ ΔF508 UAA 1 1 (100%) 1 (100%) treatment phase. Doses in the treatment phase of the first
cycle comprised 4 mg PTC124 per kg of the patient’s
Data are n or n (%). *Change in nasal PD of at least –5 mV in either cycle, indicating increase in total chloride transport.
†Equal to or more electrically negative than −5 mV. ‡Insertional mutation resulting in an elongated messenger RNA, bodyweight with breakfast, 4 mg/kg with lunch, and
containing an in-frame premature UAA stop codon.35 8 mg/kg with dinner for 14 days. This treatment phase
was followed by 14 days without treatment. In the treat-
Table 2: Participants who had a treatment response or change to within normal range, by genotype
ment phase of the second cycle, patients received 10, 10,

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and 20 mg/kg PTC124 at breakfast, lunch, and dinner,

respectively, followed by 14 days without treatment. Treatment phase 1 Treatment phase 2
The primary outcome had three components: change 10
in CFTR-mediated total chloride transport; proportion of
patients who responded to treatment; and normalisation
of chloride transport. We assessed total chloride transport 5
Typical range for patients
by measurement of nasal PD at baseline, at the end of with cystic fibrosis†
each 14-day treatment course, and after 14 days without

Nasal potential difference (mV)

0
treatment. We assessed all outcome measures on day 0
and day 14 of each 14-day treatment phase. We obtained
pharmacokinetic blood samples on day 1 and day 14 of –5
each 14-day treatment phase. Nasal PD and CFTR mRNA
were also assessed at the end of the final 14-day follow-up
period. –10
We measured nasal transepithelial PD with standard
Normal range†
methods,16 by sequentially recording voltage tracings
from the left and right nostrils while warmed solutions –15
were perfused through a nasal catheter. Solutions
comprised Ringer’s lactate to assess basal PD, amiloride
to inhibit the epithelial sodium channel, chloride-free –20
0 14 28 42
calcium gluconate to induce an electrogenic chloride Time (days)
gradient, and isoproterenol to stimulate CFTR secretion
of chloride ions from the epithelium. We calculated the Figure 1: Total chloride transport* in individual participants during the two treatment cycles
primary outcome (total chloride transport) as the sum of *Total chloride transport was calculated as the sum of nasal potential difference during nasal perfusion with
chloride transport during the perfusions of calcium calcium gluconate (intrinsic chloride transport) and isoproterenol (stimulated chloride transport). †Data are from
reference 14. ‡During the treatment phase of the first cycle, patients received PTC124 at 16 mg/kg per day in
gluconate (intrinsic chloride transport) and isoproterenol three doses every day for 14 days, followed by 14 days without treatment. During the second treatment phase,
(stimulated chloride transport). We also measured basal patients were given 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without
nasal PD, sodium transport, intrinsic chloride transport, treatment. Data are shown for 23 patients in the first cycle, and for 21 in the second.
stimulated chloride transport, and the total change (∆) in
PD (change in voltage across the entire tracing from Cycle 1 p value Cycle 2 p value
(n=23) (n=21)
beginning to end).17 The nasal PD tracings were
independently reviewed by an external expert (Dr M Primary outcome measures
Sinaasappel, Rotterdam, Netherlands), who was unaware Patients with nasal PD within normal range* 0 2 (10%)
of the study timepoint at which each tracing was at baseline
recorded. The reported values are the averages from each Patients with nasal PD within normal range* 13 (57%) 0·0003† 9 (43%) 0·02†
at end of treatment
nostril as recorded by this independent reviewer.
Change in total chloride transport (mV) −7·1 (7·0) <0·0001‡ −3·7 (7·3) 0·032‡
We extracted total mRNA from nasal epithelium and
Total chloride transport response§ 16 (70%) <0·0001¶ 8 (38%) <0·0001¶
did real-time polymerase chain reaction (PCR) by
(90% CI 50–85) (90% CI 21–58)
published methods.7 All 19 patients who could be assessed
Secondary outcome measures
for mRNA analysis had either two nonsense mutations
Change in basal nasal PD (mV) +3·3 (7·3) 0·04‡ +3·1 (9·0) 0·13‡
or one nonsense mutation and one ΔF508 mutation.
Change in sodium transport (mV) −1·9 (8·8) 0·32‡ −2·0 (9·7) 0·36‡
Thus, CFTR transcripts with a nonsense mutation could
Change in intrinsic chloride transport (mV) −3·3 (2·8) 0·002‡ −1·1 (6·1) 0·43‡
be quantified by designing primers that recognised
Change in stimulated chloride transport (mV) −3·8 (3·8) <0·0001‡ −2·6 (3·3) 0·002‡
only non-ΔF508 transcripts (forward primer 5’-GCACCA
Change (Δ) in total PD (mV) −8·9 (7·3) <0·0001‡ −5·7 (8·6) 0·007‡
TTAAAGAAAATATCATCTT-3’; reverse primer
5’ -TTGTCTTTCTCTGCAAACTTGG-3’). We determined Data are number (%) or mean (SD) unless otherwise specified. *Equal to or more electrically negative than −5 mV.
the average of normalisation to two genes: keratin18 †McNemar’s test to compare end-of-treatment value with corresponding baseline value. ‡Paired t test to compare
end-of-treatment value with corresponding baseline value. §A change of at least –5 mV in nasal transepithelial
(forward primer 5’-TGATGACACCAATATCACACGA-3’, potential difference (PD). ¶χ2 test to compare observed response rate with null-hypothesis response rate of 10%.
reverse primer 5’-GGGGCCATCTACCTCCAC-3’) and
polymerase RNA II polypeptide A (forward primer 5’ Table 3: Changes in primary and secondary measures of nasal potential difference (PD)
GTCCAGTTCGGAGTCTGAG-3’, reverse primer
5’-GCCAGTCCGCTCAACA-3’). We derived the propor-
tion relative to that of a healthy person by comparing the Other outcome measures included FEV1, forced vital
normalised level of CFTR transcripts with a nonsense capacity (FVC), bodyweight, neutrophil counts,
mutation in each patient with the normalised level of sweat chloride concentrations, adverse events, and labora-
CFTR transcripts obtained from a volunteer who had no tory abnormalities, assessed with conventional clinical
known disease-causing CFTR mutation. techniques. Patients completed treatment logs and we

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counted bottles to calculate compliance (the proportion chloride transport as nasal PD of at least as electrically
of actual doses of PTC124 relative to planned doses). negative as −5 mV.11–14
PTC124 plasma concentrations were derived from a All patients were included in analyses of safety. Efficacy
validated bioanalytical method.10 analyses included all patients for whom we had
measurements at both baseline and end of treatment in
Statistical analysis each cycle. We used paired t tests to assess changes in
The planned sample size of 18 evaluable patients was measures of nasal PD, mRNA, pulmonary function,
designed to give 95% power (paired t test, two-sided bodyweights, neutrophil counts, and sweat chloride
α=0·05) to detect changes in total chloride transport of concentrations. We analysed the proportions of patients
similar magnitude and variability (−5 mV [SD 5]) at each who had responses in total chloride transport relative to
dose of PTC124, as we reported in our previous study the null hypothesis response rate (10%) with χ² tests, and
with topical gentamicin.6 This sample size allowed the proportion for whom total chloride transport entered
90% power (χ² test, one-sided α=0·05) to reject a a normal range relative to the corresponding baseline
conservatively estimated spontaneous total chloride with McNemar’s test. Pharmacokinetic parameters were
transport response or normalisation proportion of less calculated with non-compartmental methods. The trial
than 10% (null hypothesis) in favour of a pharmacologically was registered with who.int/ictrp and with clinicaltrials.
induced response or normalisation proportion of 40% or gov, number NCT00237380.
greater (alternative hypothesis) at each dose of PTC124.
On the basis of previous results,11–14 we predefined a total Role of the funding source
chloride transport response to treatment as a change of The primary funding source for the study was PTC
–5 mV or more. We also defined normalisation of total Therapeutics. Authors employed by PTC Therapeutics
(SH, GLE, VJN, and LLM) participated in the design of
the study; in the collection, analysis, and interpretation of
A Basal nasal PD B Sodium transport data; in the writing of the report; in the decision to submit
Treatment Treatment Treatment Treatment
the paper for publication; and in presentation of the
Change in nasal PD during perfusion

phase 1† phase 2† phase 1† phase 2†

–30 40 study results to regulatory authorities.
Basal nasal PD (mV)

with amiloride (mV)

–35 35
Results
–40 30 23 patients who had never been given PTC124 were
enrolled, and completed the treatment phase of the first
–45 25
cycle. One patient had an exacerbation of a pre-existing
–50 20 Mycobacterium abscessus infection in the first cycle, and
thus did not participate in the second cycle. Another pa-
C Intrinsic chloride transport D Stimulated chloride transport tient had rhinitis, which precluded baseline testing of nasal
Change in nasal PD during perfusion
Change in nasal PD during perfusion

4 4 PD before the second cycle; therefore we had paired

with calcium gluconate (mV)

with isoproterenol (mV)

3 3
2 assessments of nasal PD in cycle 2 for 21 patients. For
2 1
1 0 FEV₁ and FVC, 22 patients had paired assessments in each
0 –1
–2
cycle. Bodyweight, neutrophil counts, safety, compliance,
–1 –3 and pharmacokinetics could be assessed in 23 patients in
–2 –4
–5 cycle 1 and in 22 patients in cycle 2. Mean compliance with
–3 –6
–4 –7
assigned doses was 99% (range 98–100) in the first
treatment phase and 99% (93–100) in the second.
E Total chloride transport F Change (Δ) in total nasal PD Patients’ characteristics were generally typical of a
Change in nasal PD during perfusion

Change in nasal PD during perfusion

phenotype of severe cystic fibrosis (table 1). Most patients

with amiloride, calcium gluconate

6 40
with calcium gluconate and

4 had pulmonary dysfunction, infection of sputum with

and isoproterenol (mV)
isoproterenol (mV)

2 35
Pseudomonas aeruginosa or other pathogenic organisms,
0
–2 30
and pancreatic insufficiency. The predominant premature
–4 stop mutations in these 23 patients were W1282X and
–6 25 G542X (table 2). 18 patients had a premature stop
–8
mutation on a single CFTR allele and 5 patients had stop
–10 20
0 14 28 42 56 0 14 28 42 56 mutations on both alleles.
Time (days) Time (days) Before treatment began, all study participants had
values for total chloride transport within the typical range
Figure 2: Mean parameters of nasal potential difference (PD) for patients with cystic fibrosis—ie, more electrically
Error bars show standard error. *During the treatment phase of the first cycle, PTC124 was given at 16 mg/kg per
day in three doses for 14 days, followed by 14 days without treatment. During the treatment phase of the second
positive than −5 mV (figure 1 and tables 1 and 3).14 In
cycle, patients were given 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without both cycles of treatment with PTC124, we noted increases
treatment. Data are shown for 23 patients in the first cycle, and for 21 in the second. (p<0·0001 in cycle 1 and p=0·032 in cycle 2) in total

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chloride transport (as indicated by more electrically

negative voltages). The proportion of patients who Treatment phase 1* Treatment phase 2*
responded to treatment (predefined as an increase in 100
total chloride transport, indicated by a change of −5 mV 95
or more) was greater than the null-hypothesis response

Proportion of CFTR mRNA with a nonsense mutation relative to wild-type mRNA (%)
90
rate of 10% (p<0·0001 for both cycles). Of the eight
85
patients in the second treatment phase who had a total
80
chloride transport response, seven had shown a previous
response in the first cycle, and one had not shown a 75

previous response. Table 3 also shows that the proportion 70

of patients who had normal chloride transport (predefined 65
as nasal PD at least as electrically negative as −5 mV) 60
increased during PTC124 treatment (first cycle p=0·0003, 55
second cycle p=0·020).14 50
Results showed that participants who had all three 45
genotypes for premature stop mutations (G542X,
40
W1282X, and 3849+10 kB C→T), including patients who
35
had a nonsense mutation in a single CFTR allele or in
both CFTR alleles, had a total chloride transport response 30

or normalisation during at least one cycle of PTC124 25

treatment (table 2). 20

We assessed each of the parameters of nasal PD (table 3 15
and figure 2). PTC124 treatment was associated with 10
increases in intrinsic, stimulated, and total chloride 5
transport in at least one of the cycles (table 3). PTC124 0
was also associated with a more electrically positive basal 0 14 28 42 56
n=8 n=15 n=13 n=14 n=10
nasal PD (p=0·04), and with more electrically negative
Time (days)
changes in total (Δ) nasal PD (first cycle p<0·0001, second
cycle p=0·032). Comparison of changes in the first
treatment phase with those in the second cycle did not Figure 3: Mean proportion of CFTR mRNA that contained nonsense mutation
Error bars show standard error. CFTR=cystic fibrosis transmembrane conductance regulator. mRNA=messenger RNA.
show any dose response (p=0·14). *During the treatment phase of the first cycle, PTC124 was given at 16 mg/kg per day in three doses for 14 days,
19 patients had quantifiable amounts of CFTR mRNA followed by 14 days without treatment. During the treatment phase of the second cycle, patients were given
that contained a nonsense mutation. Figure 3 shows that 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. Data are shown for
the mean proportion of this mRNA, relative to wild-type all 19 patients for whom we could quantify mRNA and for whom data were available.

CFTR mRNA, did not change during either treatment

phase (first cycle p=0·18, second cycle p=0·32). We used was also assessed, since patients with cystic fibrosis can
regression analysis to average the proportion of CFTR have difficulty maintaining nutrition.1 Increases in
mRNA that contained a nonsense mutation, relative to bodyweight were seen in 19 of 23 patients in the first
wild-type CFTR mRNA, across all timepoints, and treatment phase, with a mean change of 0·6 kg (SD 0·6,
compared this with the most electrically negative value for p<0·0001), which persisted during the second cycle.
total chloride transport (after either treatment phase) Because chronic neutrophilic inflammation is a
(figure 4). Among all patients, proportions of CFTR prominent component of the pathophysiology of cystic
mRNA that contained a nonsense mutation were less fibrosis,1 we analysed circulating neutrophil counts. Mean
than 50% of wild-type mRNA. In the 13 patients who had neutrophil counts were lower after the treatment phase of
a W1282X mutation as the only type of nonsense mutation the second cycle, relative to a high baseline value for this
(homozygous W1282X or W1282X/ΔF508), the amount of cycle (p=0·015). Lymphocyte counts were not altered by
W1282X transcript was associated with the most PTC124 (data not shown). Sweat-chloride concentrations
electrically negative value for total chloride transport after did not change during the study (data not shown).
either treatment phase (r=0·57, R²=0·32, p=0·046). In Although we did not use a formal symptom survey, several
the two patients whose only nonsense mutation was patients reported reduced coughing and easier clearing of
G542X, total chloride transport became more electrically sputum during treatment with PTC124.
negative than –5 mV (ie, within the normal range) Adverse events were mild or moderate, and showed no
although in both patients the proportion of CFTR mRNA dose-dependent increase in frequency or severity (table 4).
that contained a nonsense mutation was less than 10% of Most were consistent with complications related to cystic
wild-type CFTR mRNA. fibrosis. In the treatment phase of the first cycle, two
Figure 5 shows that FEV1 and FVC increased by a small patients had symptoms consistent with a cystic-fibrosis-
amount in the first PTC124 treatment phase. Bodyweight related pulmonary exacerbation; one patient’s symptoms

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began 2 days before the start of PTC124 administration absence of sex-related differences in pharmacokinetics,
and were resolved with intravenous amikacin, and the and a lack of drug accumulation or metabolic autoinduction
other patient, who had a chronic infection of Mycobacterium between Day 1 and Day 14 (data not shown). The observed
abscessus, was removed from the study. In the treatment parameters were similar to those predicted from our
phase of the second cycle, two other patients had cystic- phase I study in healthy volunteers.10
fibrosis-related pulmonary exacerbations; one patient was
not treated and the other took oral ciprofloxacin. Transient Discussion
gastrointestinal events were sometimes noted but always The trial exemplifies the concept of personalised
abated within 1–2 days, despite continued use of PTC124. medicine:18 integrating selection of patients with a specific
Two patients had constipation without intestinal genetic defect, use of a treatment designed to overcome
obstruction and received enemas; this could have been that defect in gene expression, and direct assessment of
related to PTC124 treatment, although constipation is a protein function within disease-affected tissues. We used
recurrent complication of cystic fibrosis. Mild intermittent genotyping to identify patients in whom cystic fibrosis
dysuria with the first morning void was described by four was caused by a CFTR nonsense mutation. We
patients during the first treatment phase and two of the administered PTC124 to induce ribosomes to selectively
same patients during the second high-dose treatment bypass premature stop codon mutations in mRNA. We
phase. Urinalyses in these patients revealed no used nasal PD as a sensitive and established method to
proteinuria, pyuria, haematuria, bacteria, crystals, or assess the activity of full-length, functional CFTR protein
other abnormalities. Concentrations of blood-urea on epithelial cell surfaces, and CFTR-mediated chloride
nitrogen and creatinine in serum remained normal ion transport in nasal mucosa as an established surrogate
throughout the study (data not shown). Dysuria was for lower airway epithelium.12,13,16,20,21
usually resolved by increased hydration. One patient used Our results show that patients responded to treatment
phenazopyridine, with reduction of dysuric symptoms. with PTC124, as assessed by an increase in total chloride
Serum liver enzymes remained stable or decreased within transport, indicated by a change of −5 mV or more
the normal range during the course of the study (data not electrically negative. This was true for patients who had
shown). No patient discontinued PTC124 because of a all three mutant genotypes tested. In many patients,
drug-related adverse event. PTC124 shifted total chloride transport into the normal
We were able to analyse PTC124 pharmacokinetics in range. Treatment was also associated with reductions in
23 patients in the first cycle and 22 patients in the second intrinsic chloride transport, stimulated chloride
cycle. These data showed rapid oral absorption, dose- transport, and a more electrically negative total change
proportional increases in pharmacokinetic parameters, an (Δ) in nasal PD.
We noted a small electrically positive change in basal
nasal PD, suggesting that partial restoration of CFTR
0·0
function might have secondarily improved sodium
Typical range
for patients transport through ENaC epithelial sodium channels.22
–2·5
with cystic The magnitudes of changes in nasal PD is consistent
fibrosis*
with those recorded with topical application of gentamicin
–5·0
to the nasal mucosa6 and seems to exceed changes
Normal associated with topical gene transfer23,24 or other systemic
–7·5 r=0·57
range* drug therapy approaches.25
Nasal PD (mV)

R2=0·32
p=0·046 We used patients as their own controls on the basis that
–10·0
all study patients had abnormal nasal PD at baseline, and
that fewer than 5% of patients who have cystic fibrosis
–12·5
caused by nonsense mutations have either a spontaneous
total chloride transport response or a spontaneous return
–15·0
to within the normal range.6,11 Thus, our results can be
Nonsense mutation
attributed to the pharmacological activity of PTC124. The
–17·5
G542X only W1282X only 3849+10KB finding that improvements during PTC124 dosing in
3849+10KB+W1282X G542x+W1282X each of the two treatment phases reverted toward baseline
–20·0
0 10 20 30 40 50
during 14 days of no treatment supports the hypothesis
Proportion of CFTR mRNA with a nonsense mutation relative to wild-type mRNA (%) that the effect was mediated by PTC124. Changes within
secondary nasal PD parameters and review of nasal PD
Figure 4: Correlation of most normal nasal potential difference (PD) during treatment in either cycle with tracings by an independent expert also strengthen this
proportion of CFTR mRNA that contained a nonsense mutation relative to wild-type MRNA hypothesis. We did not include cystic fibrosis patients
CFTR=cystic fibrosis transmembrane conductance regulator. mRNA=messenger RNA. *Data are from reference 14.
Data are shown for all 19 patients for whom we had on-treatment values for total chloride transport and could
who did not have a CFTR nonsense mutation (eg, ΔF508
quantify mRNA. Correlation line is shown for the 13 patients in this analysis who had the W1282X mutation as the homozygous) on the basis that safety data for PTC124 in
only CFTR nonsense-mutation type. these patients were not sufficient at the beginning of the

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 Articles

study and that studies of nonsense suppression with

gentamicin showed that such patients have no prospect FEV1 FVC
100 100
for pharmacological benefit.6,11 95
p=0·037 p=0·350
95
p=0·027 p=0·212

To derive the doses for this trial, we had modelled 90 90

pharmacokinetic data from phase I studies in healthy 85 85
volunteers;10 we aimed to exceed the trough plasma 80 80

Proportion of predicted normal value (%)

Proportion of predicted normal value (%)

75 75
concentration range of 2–10 μg/mL that had been
70 70
associated with a PTC124 dose response in mouse models 65 65
of disease mediated by nonsense mutations.8,9 60 60
Pharmacokinetic assessments in this study confirmed 55 55
sustained, dose-proportional plasma exposures that were 50 50
45 45
largely consistent with the phase I data. Thus, we expected
40 40
total chloride transport to increase with increasing PTC124 35 35
dose; it did not. Given the lesser metabolism of PTC124 in 30 30
humans than in mice and preclinical evidence for 25 25
substantial retention of drugs and metabolites in 20 20
15 15
respiratory tissues (Miller L, unpublished data), we might
10 10
have overestimated the required plasma concentrations, 5 5
such that both study doses were at the upper end of a 0 0
sigmoidal dose-response curve, and thus we did not
record a dose response in this study. Bodyweight Absolute neutrophil count
65 p<0·0001 p=0·625 7000 p=0·448 p=0·015
The changes in nasal PD were less significant in the
60
second treatment phase than in the first cycle. A
55 6000
tachyphylaxis to drug effect in cycle 2 would seems
unlikely since no loss of activity was seen during 8 weeks 50

Absolute neutrophil count per mL

5000
of treatment with PTC124 in animal models,8 and effects 45
on total chloride transport seem to be sustained with 40
4000
Weight (kg)

longer term exposure in a separate study (Wilschanski M, 35

Kerem E; unpublished data). These findings could reflect 30
3000
decreased sensitivity for detection of changes in nasal PD 25
in the second cycle; such an effect could arise from 20
2000
β adrenergic desensitisation after repeated isoproterenol 15
stimulation during sequential assessments of nasal
10 1000
PD.6,26 In some individuals, excessive nasal inflammation,12
5
drugs,27,28 and technical factors29,30 can also decrease the
0 0
sensitivity of tests for nasal PD. 0 14 28 42 0 14 28 42
Repeated assessments of CFTR mRNA showed that Treatment Treatment Treatment Treatment
treatment with PTC124 did not affect the proportion of phase 1* phase 2* phase 1* phase 2*
transcripts that contained a nonsense mutation, relative Time (days) Time (days)
to wild-type mRNA. This finding suggests that PTC124
did not increase CFTR transcription or enhance stability Figure 5: Mean clinical measurements at baseline and end of each treatment phase
FEV1=forced expiratory volume in 1 second. FVC=forced vital capacity. Bars show standard error. Significance tests
of CFTR mRNA. These results accord with in vitro data show paired t test p values to compare end-of-treatment values with corresponding baseline values. FEV₁ and FVC
that show that PTC124 does not modify mRNA data are shown for 22 patients in each cycle. Bodyweight and absolute neutrophil count data are shown for 23
transcription or stability.8 Preclinical work has previously patients in cycle 1 and 22 patients in cycle 2. *During the treatment phase of the first cycle, PTC124 was given at
shown that it is possible to alter translational fidelity at 16 mg/kg per day in three doses for 14 days, followed by 14 days without treatment. During the treatment phase
of the second cycle, patients were given 40 mg/kg of PTC124 per day in three doses for 14 days, followed by 14
the site of a premature stop codon without modifying the days without treatment.
surveillance mechanism that is responsible for degrading
mutated mRNA through the process of nonsense-mediated mutation (relative to wild-type mRNA). The results
decay.31,32 suggested that the extent of change in chloride transport
Our pharmacological strategy was based on the depends on the cellular concentration of nasal epithelial
assumption that sufficient mRNA containing a nonsense CFTR transcripts, and that mRNA values of at least
mutation must be present to provide a template for 20% of wild-type predict whether total chloride transport
protein production during drug-induced ribosomal will be within a normal range during treatment. In
read-through.7,33 We tested this presumption in the largest two patients who had only the G542X mutation, total
subset of patients in our study with a single CFTR chloride transport changed to within the normal range
genotype—W1282X. We compared total chloride transport although the proportion of their CFTR mRNA that
with proportions of mRNA that contained a nonsense contained a nonsense mutation was less than 10% of

www.thelancet.com Vol 372 August 30, 2008 725

 Articles

of PTC124 over longer periods is warranted. The lowering

Cycle 1 (n=23) Cycle 2 (n=22)
of hepatically derived transaminase in serum suggests
Mild* Moderate* Mild* Moderate* that patients with cystic fibrosis-related liver disease
Exacerbation of cystic
1 (4·3%) 1 (4·3%) 2 (9·1%) ··
could be enrolled in future studies.
fibrosis The further development of PTC124 could offer a
Abdominal pain 3 (13·0%) 1 (4·3%) ·· 2 (9·1%) practical means to address the underlying cause of disease
Constipation ·· ·· ·· 2 (9·1%) in patients with nonsense mutations as the basis for cystic
Diarrhoea 2 (8·7%) ·· 3 (13·6%) ·· fibrosis. Because nonsense mutations are causative in
Dry mouth 1 (4·3%) ·· ·· ·· some patients with most inherited conditions, such an
Nausea 1 (4·3%) 1 (4·3%) ·· 1 (4·5%) approach could also be applied to other genetic disorders.
Vomiting ·· 1 (4·3%) ·· 1 (4·5%) Based on the foundation established by this study,
Asthenia ·· ·· 1 (4·5%) ·· additional trials of PTC124 have been initiated to assess its
Nasopharyngitis ·· ·· 1 (4·5%) ·· longer-term efficacy and safety, its use in paediatric
Increase in blood uric acid 1 (4·3%) ·· ·· ·· patients, and its activity in patients with Duchenne
Positive sputum culture 1 (4·3%) ·· ·· ·· muscular dystrophy. If this type of therapeutic approach
Arthralgia ·· ·· ·· 1 (4·5%) proves successful, clinicians might increasingly need to
Myalgia ·· 1 (4·3%) 2 (9·1%) ·· consider gene sequencing as an adjunct to other tests, not
Dizziness 1 (4·3%) ·· ·· ·· only to document the presence of genetic disease, but also
Headache 1 (4·3%) ·· 1 (4·5%) ·· to select genotypically focused treatments for individual
Dysuria 4 (17·4%) ·· 2 (9·1%) ·· patients.
Irregular menstruation 1 (4·3%) ·· ·· ·· Contributors
Cough 1 (4·3%) ·· 2 (9·1%) ·· EK, MW, SH, GLE, VJN, and LLM participated in the design of the study;
in the collection, analysis, and interpretation of nasal PD and clinical data;
Dyspnoea 1 (4·3%) ·· ·· ··
in the writing of the report; and in the decision to submit the paper for
publication. MN-R and BK did analysis and interpretation of the mRNA
Data are number (%) and show all reported events, irrespective of whether related to study drug. *Severity was graded
according to reference 19.
data. SA, YY, DS, MC, HB, JR, and MA participated in various aspects of
trial conduct and patient referral. SH, GLE, VJN, and LLM were involved
Table 4: Adverse events in regulatory interactions with the Israeli Ministry of Health, and
presented study results to the European Medicines Evaluation Agency and
to the US Food and Drug Administration. All authors had full access to all
wild-type CFTR mRNA. Various biological and testing of the data in this study and take complete responsibility for the integrity
factors could affect total chloride transport as a quantitative of the data and the accuracy of the data analysis.
functional surrogate for CFTR protein production; Conflict of interest statement
however, differences in read-through efficiency could also EK, MW, MN-R, BK, SA, YY, DS, and MC have received funding support
potentially result from differences in sequence of the stop from PTC Therapeutics for this study and have also received
compensation for time, effort, and travel expenses related to data
codon and the nucleotide following it. In this regard, discussions and presentations. SH, GLE, VJN, and LLM are employees
preclinical work with PTC124 shows that ribosomal of PTC Therapeutics, and hold financial interests in the company.
read-through of UGA-G (the sequence of G542X) is more Acknowledgments
efficient than that for UGA-A (the sequence of W1282X).8 This study was sponsored by PTC Therapeutics, and funded in part by a
Treatment with PTC124 was associated with small grant from the Cystic Fibrosis Foundation Therapeutics. We thank the
patients who committed their time and effort despite the uncertainties
increases in FEV₁, FVC, and bodyweight in most patients,
of testing a new medication; Martin Sinaasappel for his expert review of
and with a reduction in neutrophil counts. Some patients the nasal potential difference tracings; Greg Elfring and James Dancy,
also reported a decrease in pulmonary symptoms, such as Innovative Analytics, for their expertise in data management; Erella
cough. Although such changes suggest clinical effects Kenoshi for capable monitoring of the conduct of this study;
Elizabeth Colacino and Allen Reha for data review; and Peter Riebling
resulting from PTC124 treatment, alternative explanations
for support with the submission process.
include fluid retention, greater patient effort during
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