Ronald C. Rubenstein

Efficacy and safety of ivacaftor in patients with cystic fibrosis who have an Arg117His-CFTR mutation: a double-blind, randomised controlled trial.

BACKGROUND Ivacaftor has been previously assessed in patients with cystic fibrosis with Gly551Asp-CFTR or other gating mutations. We assessed ivacaftor in patients with Arg117His-CFTR, a residual function mutation. METHODS We did a 24-week, placebo-controlled, double-blind, randomised clinical trial, which enrolled 69 patients with cystic fibrosis aged 6 years and older with Arg117His-CFTR and percentage of predicted forced expiratory volume in 1 s (% predicted FEV1) of at least 40. We randomly assigned eligible patients (1:1) to receive placebo or ivacaftor 150 mg every 12 h for 24 weeks. Randomisation was stratified by age (6-11, 12-17, and ≥18 years) and % predicted FEV1 (<70, ≥70 to ≤90, and >90). The primary outcome was the absolute change from baseline in % predicted FEV1 through week 24. Secondary outcomes included safety and changes in sweat chloride concentrations and Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain scores. An open-label extension enrolled 65 of the patients after washout; after 12 weeks, we did an interim analysis. FINDINGS After 24 weeks, the treatment difference in mean absolute change in % predicted FEV1 between ivacaftor (n=34) and placebo (n=35) was 2·1 percentage points (95% CI -1·13 to 5·35; p=0·20). Ivacaftor treatment resulted in significant treatment differences in sweat chloride (-24·0 mmol/L, 95% CI -28·01 to -19·93; p<0·0001) and CFQ-R respiratory domain (8·4, 2·17 to 14·61; p=0·009). In prespecified subgroup analyses, % predicted FEV1 significantly improved with ivacaftor in patients aged 18 years or older (treatment difference vs placebo: 5·0 percentage points, 95% CI 1·15 to 8·78; p=0·01), but not in patients aged 6-11 years (-6·3 percentage points, -11·96 to -0·71; p=0·03). In the extension study, both placebo-to-ivacaftor and ivacaftor-to-ivacaftor groups showed % predicted FEV1 improvement (absolute change from post-washout baseline at week 12: placebo-to-ivacaftor, 5·0 percentage points [p=0·0005]; ivacaftor-to-ivacaftor, 6·0 percentage points [p=0·006]). We did not identify any new safety concerns. The studies are registered with ClinicalTrials.gov (the randomised, placebo-controlled study, number NCT01614457; the open-label extension study, number NCT01707290). INTERPRETATION Although this study did not show a significant improvement in % predicted FEV1, ivacaftor did significantly improve sweat chloride and CFQ-R respiratory domain scores and lung function in adult patients with Arg117His-CFTR, indicating that ivacaftor might benefit patients with Arg117His-CFTR who have established disease. FUNDING Vertex Pharmaceuticals Incorporated.

CFTR is functionally active in GnRH-expressing GT1-7 hypothalamic neurons.

We have demonstrated the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mRNA, and protein within the rat and human brains, in areas regulating sexual differentiation and function. We have found that GT1-7, a gonadotropin-releasing hormone (GnRH)-secreting hypothalamic neuronal cell line, expresses the CFTR gene, mRNA, and protein and cAMP-dependent 36Cl efflux. A linear 7-pS Cl- conductance, which is stimulated by ATP and cAMP analogs and inhibited by glibenclamide, consistent with CFTR activity, has been identified in GT1-7 cells. Antisense oligo(dN) generated against exon 10 of the CFTR gene transcript (mRNA) inhibit GnRH secretion into media [312 ± 73, 850 ± 150, 963 ± 304, and 912 ± 74 pg GnRH/4 × 106 cells for antisense, sense, missense, and no oligo(dN), respectively; P < 0.029 for antisense oligo(dN)-treated vs. normal cells]. No changes in intracellular synthesis of GnRH were noted [1,400 ± 371 and 1,395 ± 384 pg GnRH/4 × 106 cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent36Cl efflux. The expression of CFTR protein, detected by Western blotting, was also inhibited 68% by preincubation of cells with antisense oligo(dN). GT1-7 hypothalamic neurons express the CFTR gene, mRNA, and protein, which modulate neurosecretion. Abnormal neuropeptide vesicle trafficking by mutant CFTR may help to explain some of the diverse manifestations of cystic fibrosis.We have demonstrated the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mRNA, and protein within the rat and human brains, in areas regulating sexual differentiation and function. We have found that GT1-7, a gonadotropin-releasing hormone (GnRH)-secreting hypothalamic neuronal cell line, expresses the CFTR gene, mRNA, and protein and cAMP-dependent (36)Cl efflux. A linear 7-pS Cl- conductance, which is stimulated by ATP and cAMP analogs and inhibited by glibenclamide, consistent with CFTR activity, has been identified in GT1-7 cells. Antisense oligo(dN) generated against exon 10 of the CFTR gene transcript (mRNA) inhibit GnRH secretion into media [312 +/- 73, 850 +/- 150, 963 +/- 304, and 912 +/- 74 pg GnRH/4 x 10(6) cells for antisense, sense, missense, and no oligo(dN), respectively; P < 0. 029 for antisense oligo(dN)-treated vs. normal cells]. No changes in intracellular synthesis of GnRH were noted [1,400 +/- 371 and 1,395 +/- 384 pg GnRH/4 x 10(6) cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent 36Cl efflux. The expression of CFTR protein, detected by Western blotting, was also inhibited 68% by preincubation of cells with antisense oligo(dN). GT1-7 hypothalamic neurons express the CFTR gene, mRNA, and protein, which modulate neurosecretion. Abnormal neuropeptide vesicle trafficking by mutant CFTR may help to explain some of the diverse manifestations of cystic fibrosis.

Long-term treatment with oral N-acetylcysteine: Affects lung function but not sputum inflammation in cystic fibrosis subjects. A phase II randomized placebo-controlled trial

PURPOSE To evaluate the effects of oral N-acetylcysteine (NAC), which replenishes systemic glutathione, on decreasing inflammation and improving lung function in CF airways. METHODS A multicenter, randomized, double-blind proof of concept study in which 70 CF subjects received NAC or placebo orally thrice daily for 24 weeks. ENDPOINTS primary, change in sputum human neutrophil elastase (HNE) activity; secondary, FEV(1) and other clinical lung function measures; and safety, the safety and tolerability of NAC and the potential of NAC to promote pulmonary hypertension in subjects with CF. RESULTS Lung function (FEV(1) and FEF(25-75%)) remained stable or increased slightly in the NAC group but decreased in the placebo group (p=0.02 and 0.02). Log(10) HNE activity remained equal between cohorts (difference 0.21, 95% CI -0.07 to 0.48, p=0.14). CONCLUSIONS NAC recipients maintained their lung function while placebo recipients declined (24 week FEV1 treatment effect=150 mL, p<0.02). However no effect on HNE activity and other selected biomarkers of neutrophilic inflammation were detected. Further studies on mechanism and clinical outcomes are warranted.

Efficacy and safety of lumacaftor and ivacaftor in patients aged 6–11 years with cystic fibrosis homozygous for F508del-CFTR: a randomised, placebo-controlled phase 3 trial

BACKGROUND Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6-11 years homozygous for F508del-CFTR. METHODS In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV1) of 70 or more, and lung clearance index2·5 (LCI2·5) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (<25 kg vs ≥25 kg) and ppFEV1 severity (<90 vs ≥90) determined at the screening visit, and randomly assigned 1:1 to treatment using an interactive web response system to receive 200 mg lumacaftor and 250 mg ivacaftor every 12 hours or placebo for 24 weeks. Patients, all site personnel including the investigator and the site monitor, and the study team were blinded, with the exception of site personnel needing this information in the event of medical emergency or pregnancy and patient safety and regulatory affairs personnel to meet serious adverse event reporting requirements. The primary endpoint was the mean absolute change in LCI2·5 from all on-treatment study visits up to and including week 24. All randomly assigned patients who were exposed to any amount of study drug, with treatment assignment as assigned were included in primary and other efficacy analyses. All patients who were exposed to any amount of study drug, with treatment assignment as treated, were included in the safety analysis. This study was registered with ClinicalTrials.gov, number NCT02514473. FINDINGS Between July 23, 2015, and Sept 20, 2016, a total of 206 patients were enrolled and randomly assigned to receive lumacaftor and ivacaftor (n=104) or placebo (n=102). Two randomly assigned patients were never dosed with study drug (one in the placebo arm due to ineligibility arising from a streptococcal throat infection and one in the lumacaftor and ivacaftor arm due to withdrawal based on refusal to provide blood tests) and were not included in the analyses. 103 patients received at least one dose of lumacaftor and ivacaftor and 101 patients received at least one dose of placebo. For the primary endpoint, the average absolute change in LCI2·5 from baseline over all study visits up to and including the week 24 visit, least squares mean difference was -1·09 units (95% CI -1·43 to -0·75, p<0·0001) for lumacaftor and ivacaftor versus placebo. For the key secondary endpoint of sweat chloride concentration, the least squares mean difference versus placebo was -20·8 mmol/L (95% CI -23·4 to -18·2, average absolute change at day 15/week 4; p<0·0001). The least squares mean difference compared with placebo in absolute change in ppFEV1 from all on-treatment study visits until week 24 was 2·4 (95% CI 0·4-4·4, p=0·0182). 196 (96%) of 204 patients reported adverse events, most of which were mild (87 [43%]) or moderate (98 [48%]). Treatment was discontinued due to adverse events in three (3%) of 103 patients in the lumacaftor and ivacaftor group and two (2%) of 101 patients in the placebo group. Serious adverse events were reported in 13 (13%) of 103 patients in the lumacaftor and ivacaftor group and 11 (11%) of 101 patients in the placebo group. INTERPRETATION Treatment with lumacaftor and ivacaftor was associated with statistically significant improvements in lung function, as measured by LCI2·5 and ppFEV1, versus placebo in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR. The overall safety profile was consistent with previous phase 3 studies of lumacaftor and ivacaftor. FUNDING Vertex Pharmaceuticals.

Body Composition and Pulmonary Function in Cystic Fibrosis

Background: Lower body mass index (BMI) is associated with worse pulmonary function in cystic fibrosis (CF). Hypothesis: lean body mass (LBM) is more strongly associated with pulmonary function than BMI is. Methods: Anthropometrics, body composition by dual x-ray absorptiometry, and pulmonary function were determined in pancreatic insufficient CF (PI-CF) youth. Sex and age-adjusted Z-scores (BMI-Z, LBMI-Z, FMI-Z) were generated for CF and controls. (1) Associations of BMI-Z with LBMI-Z and FMI-Z and (2) age-adjusted associations of BMI-Z, LBMI-Z, and FMI-Z with FEV1%-predicted were tested. Results: Two hundred eight PI-CF subjects had lower BMI-Z, LBMI-Z, and FMI-Z compared to 390 controls. BMI-Z was associated with lower LBMI-Z (p < 0.0001) in PI-CF. In females, LBMI-Z and BMI-Z were positively associated with FEV1%-predicted; this relationship did not persist for FMI-Z after adjustment for LBMI-Z. In males, only LBMI-Z and BMI-Z were associated with FEV1%-predicted. Conclusion: In PI-CF youth, deficits in LBM were apparent. At lower BMI percentiles, BMI may not accurately depict LBM in PI-CF. In under-nourished PI-CF youth, this preservation of FM in preference to LBM is relevant since LBMI-Z, but not FMI-Z, is positively associated with FEV1%-predicted. Lean body mass index is more strongly associated with lung function compared to BMI, especially in the under-nourished child and adolescent with PI-CF.

IL-17A induces Pendrin expression and chloride-bicarbonate exchange in human bronchial epithelial cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.

Reduced β-Cell Secretory Capacity in Pancreatic Insufficient, But Not Pancreatic Sufficient, Cystic Fibrosis Despite Normal Glucose Tolerance.

Patients with pancreatic-insufficient cystic fibrosis (PI-CF) are at increased risk for developing diabetes. We determined β-cell secretory capacity and insulin secretory rates from glucose-potentiated arginine and mixed-meal tolerance tests (MMTTs), respectively, in pancreatic-sufficient cystic fibrosis (PS-CF), PI-CF, and normal control subjects, all with normal glucose tolerance, in order to identify early pathophysiologic defects. Acute islet cell secretory responses were determined under fasting, 230 mg/dL, and 340 mg/dL hyperglycemia clamp conditions. PI-CF subjects had lower acute insulin, C-peptide, and glucagon responses compared with PS-CF and normal control subjects, indicating reduced β-cell secretory capacity and α-cell function. Fasting proinsulin-to-C-peptide and proinsulin secretory ratios during glucose potentiation were higher in PI-CF, suggesting impaired proinsulin processing. In the first 30 min of the MMTT, insulin secretion was lower in PI-CF compared with PS-CF and normal control subjects, and glucagon-like peptide 1 and gastric inhibitory polypeptide were lower compared with PS-CF, and after 180 min, glucose was higher in PI-CF compared with normal control subjects. These findings indicate that despite “normal” glucose tolerance, adolescents and adults with PI-CF have impairments in functional islet mass and associated early-phase insulin secretion, which with decreased incretin responses likely leads to the early development of postprandial hyperglycemia in CF.

ERp29 regulates epithelial sodium channel functional expression by promoting channel cleavage.

The epithelial Na(+) channel (ENaC) plays a key role in the regulation of blood pressure and airway surface liquid volume. ERp29 is a 29-kDa thioredoxin-homologous endoplasmic reticulum (ER) protein that has only a single cysteine instead of the usual thioredoxin CXXC motif. Our group previously demonstrated that ERp29 promotes biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR). On the basis of similarities of CFTR and ENaC trafficking, we hypothesized that ERp29 would also regulate ENaC biogenesis and functional expression. In epithelial cells, overexpression of wild-type (wt) ERp29 increased ENaC functional expression [amiloride-sensitive short-circuit current (Isc)] in Ussing chamber experiments, as well as the abundance of the cleaved form of γ-ENaC in whole cell lysates. In contrast, siRNA-mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single cysteine (C157S ERp29) decreased ENaC functional expression. Cells in which wt ERp29 was overexpressed had a smaller fractional increase in amiloride-sensitive Isc when trypsin was applied to the apical surface to activate uncleaved ENaC, while cells in which C157S ERp29 was overexpressed or ERp29 was depleted had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin. Interestingly, these observations were not associated with altered expression of β-ENaC at the apical surface. Instead, ERp29 appeared to promote the interaction of β-ENaC with the Sec24D cargo recognition component of the coat complex II ER exit machinery. Together, these data support the hypothesis that ERp29 directs ENaC toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane.

Sexual and reproductive health care utilization and preferences reported by young women with cystic fibrosis

BACKGROUND This study aimed to describe and compare the sexual and reproductive health (SRH) care utilization among young women with cystic fibrosis (CF) with the general United States (U.S.) population. METHODS Women with CF ages 15-24years from five geographically diverse U.S. CF centers participated in a survey investigating SRH. Results were summarized and compared to a nationally representative sample of young women aged 15-24 who participated in the U.S. National Survey of Family Growth (NSFG) using logistic regression to adjust for confounders. RESULTS A total of 188 women with CF (19.7±2.7years) completed the survey; data were compared to 1997 NSFG respondents (19.6±0.10years). Women with CF had lower lifetime rates of ever obtaining a Pap smear or pelvic exam (26% vs. 57%; p<0.001) and similar rates of HPV vaccination (44% vs. 43%; p=0.64) compared to NSFG respondents. Thirty-seven percent of women with CF reported seeking contraception and <10% reported contraceptive counseling, STI testing/counseling, or pregnancy testing in their lifetime. In the prior 12months, 41% of NSFG respondents reported seeking contraception, 24% received contraceptive counseling, 22% STI testing/counseling, and 23% pregnancy testing. A minority of women with CF received or discussed SRH care in the CF setting, although 66% wanted to discuss SRH with their CF team. CONCLUSIONS Young women with CF report low rates of SRH care utilization and desire SRH discussions in the CF setting. Interventions should target improved SRH care delivery and encourage patient-provider communication around SRH in the CF care setting.

Patient and parent perceptions of the diagnosis and management of cystic fibrosis-related diabetes

Background Cystic Fibrosis Related Diabetes (CFRD) is an increasingly common complication in CF. CFRD introduces an additional complex chronic disease to individuals already attending to demanding treatment regimens. An improved understanding of the reaction to and coping mechanisms surrounding CFRD may facilitate management of CFRD. Methods Semi-structured interviews completed by 10 children with CFRD, 10 adults with CFRD and 10 parents of children with CFRD in a single large CF Care Center were analyzed utilizing phenomenological analysis. Results Patients and families reported having limited knowledge of CFRD prior to diagnosis. CFRD was considered an extension of their underlying CF and successful management depended upon integration into existing routines. Conclusions Health care professionals caring for patients with CFRD should be informed of the specific challenges that arise in CFRD. Education in advance of CFRD diagnosis may facilitate management.