Yasmin Yaakov

Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial

BACKGROUND In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR. METHODS This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380. FINDINGS Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycles treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycles treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded. INTERPRETATION In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis

In a subset of patients with cystic fibrosis (CF), nonsense mutations (premature stop codons) disrupt production of full-length, functional CF transmembrane conductance regulator (CFTR). Ataluren (PTC124) allows ribosomal readthrough of premature stop codons in mRNA. We evaluated drug activity and safety in patients with nonsense mutation CF who took ataluren three times daily (morning, midday and evening) for 12 weeks at either a lower dose (4, 4 and 8 mg·kg−1) or higher dose (10, 10 and 20 mg·kg−1). The study enrolled 19 patients (10 males and nine females aged 19–57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.

Evaluation of the intestinal current measurement method as a diagnostic test for cystic fibrosis

The sweat test and nasal potential difference measurement are now established tools in the diagnostic work up of cystic fibrosis (CF). Intestinal current measurement (ICM) is under consideration as an aid in the diagnosis of CF especially in young children. The aim of this study is to evaluate the diagnostic reliability of ICM.

Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel Function Testing in Recurrent Acute Pancreatitis

Goals To understand the relationship between acute recurrent pancreatitis and cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Background An emerging number of patients present with a nonclassic phenotype of cystic fibrosis (CF) with partial features or single-organ disease only. The association between the phenotype of recurrent pancreatitis CFTR dysfunction is unclear. Methods Patients with idiopathic recurrent pancreatitis were referred for electrophysiologic investigation. Results Thirty-three patients (18 males) aged 20±12 years with recurrent pancreatitis were studied. Three patients had mild asthma and 1 patient had mild ulcerative colitis. There was no family history of CF. All patients had normal imaging of the pancreatic duct by endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography. No patient was pancreatic insufficient. Mean sweat chloride values were 41±14 meq/L (range: 18 to 64). Nasal potential difference (NPD) measurement was pathologic in 7 patients. Mean basal potential difference in these 7 patients was −33±13 mV and there was an abnormal response to chloride-free and isoproterenol solutions. There was no difference in sweat chloride concentration between the 2 groups. Mutation analysis revealed W1282X/5T, D1152H/5T, and W1282X/− in 3 patients with abnormal NPD and 1 W1282X allele was found in 1 patient with normal NPD. Conclusions In this series, 21% of patients with recurrent pancreatitis have abnormalities of CFTR function. Patients presenting with recurrent, “idiopathic” pancreatitis require CFTR function testing.

Nasal potential difference in non-classic cystic fibrosis—long term follow up†

Nasal potential difference (NPD) measurement is an electrophysiological test that assesses cystic fibrosis transmembrane conductance regulator (CFTR) activity and is a recognized diagnostic tool in CF. The aim of this study is to assess in the long term the role of NPD in patients whose diagnosis is questionable.

Genetic and electrophysiological characteristics of recurrent acute pancreatitis.

Objectives: The aim was to present the workup of patients with acute recurrent pancreatitis (ARP) for genetic analysis and electrophysiological testing. Methods: Patients with ARP with unknown etiology were referred for genetic testing and evaluation of cystic fibrosis transmembrane conductor regulator (CFTR) function by nasal potential difference (NPD) testing. Results: A total of 67 patients were evaluated. The mean age was 23 ± 17 years (median 17.0 years, range 1.5–72 years); 90% were Jewish and 10% Arab. Ten (15%) patients carried PRSS1 gene mutation (K23R(7), R122H(2), and D21A(1)). One patient had K172E/− (chymotrypsin C [CTRC]) mutation, 1 had I42M (serine protease inhibitor Kazal type 1 [SPINK1])/V235I (CTRC) together with &Dgr;F508/5T, 1 patient had R67H (SPINK1)/V235I (CTRC), and 1 patient had V235I (CTRC)/−. Ten of 67 (15%) patients submitted for CFTR gene testing carried mutations (&Dgr;F508/L997F, &Dgr;F508/5T(11TG), W1282/5T(12TG), W1282X/Y1014C, &Dgr;F508/R31C, R117H/−, R117H/Y1014C, D1152H/−, 5T(11TG)/−, and L997F/−). Fifty-four (80%) patients underwent sweat testing. Of these, 5 had sweat chloride ≥60 mEq/L, and 22 patients had sweat chloride from 40 to 60 mEq/L. Of the 56 (83%) patients had nasal potential difference testing, 4 (6%) with abnormal results. Conclusions: One-third (34%) of patients with ARP carry mutations for hereditary pancreatitis including rare mutations (K23R), and 12.5% have evidence of cftr mutations and 10% had CFTR dysfunction underscoring the importance of genetic and functional workup of these patients.

Comparison of Nasal Potential Difference and Intestinal Current Measurements as Surrogate Markers for CFTR Function.

Objectives: Nasal potential difference (NPD) measurement is part of the diagnostic criteria for cystic fibrosis (CF) and now used routinely as an endpoint in clinical trials of correcting the basic defect in CF. Intestinal current measurement (ICM), measured ex vivo on a rectal biopsy, has been used to study cystic fibrosis transmembrane conductance regulator (CFTR) function but has not been compared to NPD in the same subject in adults and children. The aim of the study is to evaluate the potential usefulness of ICM as a marker of CFTR function for treatment studies compared NPD in patients with CF and in healthy control subjects. Methods: ICM and NPD were performed on healthy controls and patients with CF. The healthy adults were individuals undergoing routine screening colonoscopy at the Beth Israel Deaconess Medical Center. The healthy children were undergoing colonoscopy for suspicion of inflammation in Hadassah Hebrew University Medical Center. The CF adults were recruited from Boston Childrens Hospital CF Center and CF Center Worcester Mass, the children with CF from Hadassah CF Center. Results: ICM measurements in healthy control subjects (n = 16) demonstrated a mean (±SE) carbachol response of 16.0 (2.2) &mgr;A/cm2, histamine response of 13.2 (2.1) &mgr;A/cm2 and a forskolin response of 6.3 (2.0) &mgr;A/cm2. Basal NPD of −15.9 (1.9) and response to Cl− free + isoproterenol of −13.8 (2.0). These responses were inverted in CF subjects (n = 12) for ICM parameters with carbachol response of −3.0 (0.5) &mgr;A/cm2, histamine −1.0 (0.8) &mgr;A/cm2 and a forskolin response of 0.5 (0.3) and also for NPD parameters; basal NPD of −42.2 (4.3) and response to Cl− free + isoproterenol of 4.3 (0.7). Pearson correlation test showed the comparability of ICM and NPD in assessing CFTR function. Conclusions: ICM is equivalent to NPD in the ability to distinguish patients with CF from controls and could be used as surrogate markers of CFTR activity in treatment protocols.

Influence of perfusate temperature on nasal potential difference

Nasal potential difference (NPD) quantifies abnormal ion transport in cystic fibrosis. It has gained acceptance as an outcome measure for the investigation of new therapies. To quantify the effect of solution temperature on NPD, we first examined the effect of switching from room temperature (20–25°C) to warmed (32–37°C) solutions and vice versa during each perfusion step. Secondly, standard protocols were repeated at both temperatures in the same subjects. Changing solution temperature did not alter NPD during perfusion with Ringer’s solution (<1 mV) (p>0.1). During perfusion with zero chloride solution, changing from room temperature to warmed solutions tended to decrease absolute NPD (i.e. it became less negative) by 0.9 mV (p>0.1); changing from warmed to room temperature increased NPD by 2.1 mV (p<0.05). During isoprenaline perfusion, changing from room temperature to warmed solutions increased NPD by 1.5 mV (p<0.01) and from warmed to room temperature decreased NPD by 1.4 mV (p<0.05). For full protocols at room temperature or warmed in the same subjects, mean values were similar (n = 24). During warmed perfusion, group results for total chloride response had a larger standard deviation. As this increased variability will probably decrease the power of trials, this study suggests that solutions at room temperature should be recommended for the measurement of NPD.

WS16.6 Is primary sclerosing cholangitis (PSC) a cystic fibrosis-related disorder? Electrophysiological testing and full sequencing of the CFTR gene

Objectives PSC and Cystic Fibrosis related liver disease (CFLD) have features in common: chronic inflammation and bile duct damage with similar cholangiographic findings. There is controversy if PSC is related to CFTR dysfunction. The aim of this study is to determine whether PSC is associated with mutations in the cftr gene and abnormalities in CFTR function assays. Methods Patients with PSC were referred for Nasal Potential Difference (NPD) measurement, sweat chloride concentration and complete cftr sequencing of the full coding sequence by NGS (new generation sequencing-PGM life technology). Results 32 patients aged 46±13 yrs (IBD in 17) were examined. Cftr sequencing showed 6 patients with cftr mutations on one allele and 19 with polymorphisms in the cftr gene. 28 patients had normal NPD results (Basal NPD −14.6±4.9, response to amiloride 9.2±4.7, response to Cl − free + isoproterenol −11.1±5.7 mV), sweat chloride 43±19 mmol/L and 4 patients had abnormal NPD results (Basal NPD −21.1±8.6, response to amiloride 15.7±7.4, response to Cl − free + isoproterenol −1.1±4.4 mV); sweat chloride was 69, 54, 65 and 44 mmol/L. Of note, one of these patients is infertile; one patient has chronic pancreatitis. Two patients with abnormal NPD carried W1282X/5T mutation and the 2620–26A Conclusion 4 patients out of 32 (12.5%) with PSC have been found to have some features of CFTR related disorder and 18.75% carry cftr mutations and 50% have polymorphisms of the cftr gene. This study will improve our understanding of the etiology of PSC and its relationship to cftr dysfunction.

WS20.3 Acquired CFTR dysfunction in patients with primary ciliary dyskinesia (PCD)

It has been shown that chronic bronchitis, cigarette smoke, hypoxia, and chronic airways inflammation are associated with reduced CFTR function. Primary ciliary dyskinesia (PCD) is characterized by chronic sino-pulmonary disease and chronic bronchiectasis. We have noticed that some patients with PCD have borderline or high sweat chloride test. This may suggest acquired CFTR dysfunction in these patients. Objectives To analyze CFTR function in a large cohort of PCD patients by measuring sweat chloride and nasal potential difference (NPD), and to compare the results with control individuals and patients with CF. Methods 30 PCD patients >6 years treated at our clinic performed sweat test and NPD. For all patients with above normal values in the sweat test and NPD tests, analysis for CFTR mutations or exome analysis was performed. Results The mean (±SD) value of sweat chloride of the 30 PCD patients was 51.5±18.6 mmol/L (normal 60 mmol/L) and 9/30 had normal values. 2/17 patients had abnormal NPD and genetically confirmed PCD. Conclusions PCD patients may have abnormal sweat chloride tests; therefore, it is important to perform NPD in patients with abnormal/borderline sweat test in order to differentiate between PCD and CF and to confirm the diagnosis with genetic analysis. It is suggested that PCD should be added to the list of diseases causing false-positive sweat tests. The significance of this reduced CFTR function needs further investigation since new therapies that augment CFTR function may be beneficial in these patients.