Elizabeth Joseloff

Clinical Mechanism of the Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor in G551D-mediated Cystic Fibrosis

RATIONALE Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator recently approved for patients with CF age 6 and older with the G551D mutation. OBJECTIVES To evaluate ivacaftor in a postapproval setting and determine mechanism of action and response of clinically relevant markers. METHODS We conducted a longitudinal cohort study in 2012-2013 in G551D CF patients age 6 and older with no prior exposure to ivacaftor. Study assessments were performed at baseline, 1, 3, and 6 months after ivacaftor initiation. Substudies evaluated mucociliary clearance, β-adrenergic sweat secretion rate, gastrointestinal pH, and sputum inflammation and microbiology Measurements and Main Results: A total of 151 of 153 subjects were prescribed ivacaftor and 88% completed the study through 6 months. FEV1 % predicted improved from baseline to 6 months (mean absolute change, 6.7%; P < 0.001). Similarly, body mass index improved from baseline to 6 months (mean change, 0.8 kg/m(2); P < 0.001). Sweat chloride decreased from baseline to 6 months (mean change, -53.8 mmol/L; 95% confidence interval, -57.7 to -49.9; P < 0.001), reflecting augmented CFTR function. There was significant improvement in hospitalization rate (P < 0.001) and Pseudomonas aeruginosa burden (P < 0.01). Significant improvements in mucociliary clearance (P < 0.001), gastrointestinal pH (P = 0.001), and microbiome were also observed, providing clinical mechanisms underlying the therapeutic benefit of ivacaftor. CONCLUSIONS Significant clinical and physiologic improvements were observed on initiation of ivacaftor in a broad patient population, including reduced infection with P. aeruginosa. Biomarker studies substantially improve the understanding of the mechanistic consequences of CFTR modulation on pulmonary and gastrointestinal physiology.

Metabolomic Profiling Reveals Biochemical Pathways and Biomarkers Associated with Pathogenesis in Cystic Fibrosis Cells

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.

Considerations for the Conduct of Clinical Trials with Antiinflammatory Agents in Cystic Fibrosis. A Cystic Fibrosis Foundation Workshop Report

Inflammation leads to lung destruction and loss of pulmonary function in patients with cystic fibrosis (CF). Drugs that modulate the cystic fibrosis transmembrane conductance regulator (CFTR) have recently been approved. Although the impact of CFTR modulators on sweat chloride and lung function are exciting, they have not yet demonstrated an effect on inflammation. Therefore, CF antiinflammatory drug development must continue. Unfortunately, the lack of clarity with this process has left investigators and industry sponsors frustrated. The Cystic Fibrosis Foundation established a working group in early 2014 to address this issue. There are many inflammatory processes disrupted in CF, and, therefore, there are many potential targets amenable to antiinflammatory therapy. Regardless of a drugs specific mechanism of action, it must ultimately affect the neutrophil or its products to impact CF. The working group concluded that before bringing new antiinflammatory drugs to clinical trial, preclinical safety studies must be conducted in disease-relevant models to assuage safety concerns. Furthermore, although studies of antiinflammatory therapies must first establish safety in adults, subsequent studies must involve children, as they are most likely to reap the most benefit. The working group also recommended that pharmacokinetic-pharmacodynamic studies and early-phase safety studies be performed before proceeding to larger studies of longer duration. In addition, innovative study designs may improve the likelihood of adequately assessing treatment response and mitigating risk before conducting multiyear studies. Learning from past experiences and incorporating this knowledge into new drug development programs will be instrumental in bringing new antiinflammatory therapies to patients.

Serum metabolomics indicate altered cellular energy metabolism in children with cystic fibrosis

Cystic fibrosis (CF) is a multi‐system disease affecting multiple organs and cells besides the respiratory system. Metabolomic profiling allows simultaneous detection of biochemicals originating from cells, organs, or exogenous origin that may be valuable for monitoring of disease severity or in diagnosis.

Impact of CFTR Modulation on Intestinal pH, Motility, and Clinical Outcomes in Patients With Cystic Fibrosis and the G551D Mutation

Objectives:A defect in bicarbonate secretion contributes to the pathophysiology of gastrointestinal complications in patients with cystic fibrosis (CF). We measured gastrointestinal pH, clinical outcomes, and intestinal transit profiles in patients with the G551D mutation before and after treatment with ivacaftor, a CF transmembrane regulator channel (CFTR) potentiator.Methods:Observational studies of ivacaftor effectiveness were conducted in the United States and Canada. A subset of subjects ingested a wireless motility capsule (n=10) that measures in vivo pH, both before therapy with ivacaftor and 1 month after treatment; values obtained were compared for mean pH and area under the pH curve, and regional intestinal motility. We also queried subjects about abdominal pain and recorded body weight before and after treatment.Results:One month after administering ivacaftor, a significant increase in mean pH was observed after gastric emptying (P<0.05). Area under the pH curve analyses indicate increased bicarbonate mass (P<0.05 for select 5 min intervals and all segments >30 min); mean weight gain was 1.1 kg (P=0.08). No difference in abdominal pain or regional transit times was seen.Conclusions:CFTR modulation improves the proximal small intestinal pH profile in patients with the G551D CFTR mutation and we observed clinically relevant, contemporaneous weight gain, although it did not reach statistical significance. These data provide in vivo evidence that CFTR is an important regulator of bicarbonate secretion, which may be a translational link between CFTR function and clinical improvement.

Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel’s function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Human epididymis protein 4 (HE4) levels inversely correlate with lung function improvement (delta FEV1) in cystic fibrosis patients receiving ivacaftor treatment

BACKGROUND We have recently shown that human epididymis protein 4 (HE4) levels correlate with the severity of cystic fibrosis (CF) lung disease. However, there are no data on how HE4 levels alter in patients receiving CFTR modulating therapy. METHODS In this retrospective clinical study, 3 independent CF patient cohorts (US-American: 29, Australian: 12 and Irish: 19 cases) were enrolled carrying at least one Class III CFTR CF-causing mutation (p.Gly551Asp) and being treated with CFTR potentiator ivacaftor. Plasma HE4 was measured by immunoassay before treatment (baseline) and 1-6 months after commencement of ivacaftor, and were correlated with FEV1 (% predicted), sweat chloride, C-reactive protein (CRP) and body mass index (BMI). RESULTS After 1 month of therapy, HE4 levels were significantly lower than at baseline and remained decreased up to 6 months. A significant inverse correlation between absolute and delta values of HE4 and FEV1 (r = -0.5376; P < .001 and r = -0.3285; P < .001), was retrospectively observed in pooled groups, including an independent association of HE4 with FEV1 by multiple regression analysis (β = -0.57, P = .019). Substantial area under the receiver operating characteristic curve (ROC-AUC) value was determined for HE4 when 7% mean change of FEV1 (0.722 [95% CI 0.581-0.863]; P = .029) were used as classifier, especially in the first 2 months of treatment (0.806 [95% CI 0.665-0.947]; P < .001). CONCLUSIONS This study shows that plasma HE4 levels inversely correlate with lung function improvement in CF patients receiving ivacaftor. Overall, this potential biomarker may be of value for routine clinical and laboratory follow-up of CFTR modulating therapy.

Effectiveness of ivacaftor in cystic fibrosis patients with non-G551D gating mutations

BACKGROUND The cystic fibrosis transmembrane conductance regulator (CFTR) potentiator ivacaftor is approved for patients with CF with gating and residual function CFTR mutations. We report the results of an observational study investigating its effects in CF patients with non-G551D gating mutations. METHODS Patients with non-G551D gating mutations were recruited to an open-label study evaluating ivacaftor. Primary outcomes included: lung function, sweat chloride, weight gain, and quality of life scores. RESULTS Twenty-one subjects were enrolled and completed 6 months follow-up on ivacaftor; mean age was 25.6 years with 52% <18. Baseline ppFEV1 was 68% and mean sweat chloride 89.6 mEq/L. Participants experienced significant improvements in ppFEV1 (mean absolute increase of 10.9% 95% CI = [2.6,19.3], p = 0.0134), sweat chloride (-48.6 95% CI = [-67.4,-29.9], p < 0.0001), and weight (5.1 kg, 95% CI = [2.8, 7.3], p = 0.0002). CONCLUSIONS Patients with non-G551D gating mutations experienced improved lung function, nutritional status, and quality of life. This study supports ongoing use of ivacaftor for patients with these mutations.

CFTR modulator theratyping: Current status, gaps and future directions

BACKGROUND New drugs that improve the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with discreet disease-causing variants have been successfully developed for cystic fibrosis (CF) patients. Preclinical model systems have played a critical role in this process, and have the potential to inform researchers and CF healthcare providers regarding the nature of defects in rare CFTR variants, and to potentially support use of modulator therapies in new populations. METHODS The Cystic Fibrosis Foundation (CFF) assembled a workshop of international experts to discuss the use of preclinical model systems to examine the nature of CF-causing variants in CFTR and the role of in vitro CFTR modulator testing to inform in vivo modulator use. The theme of the workshop was centered on CFTR theratyping, a term that encompasses the use of CFTR modulators to define defects in CFTR in vitro, with application to both common and rare CFTR variants. RESULTS Several preclinical model systems were identified in various stages of maturity, ranging from the expression of CFTR variant cDNA in stable cell lines to examination of cells derived from CF patients, including the gastrointestinal tract, the respiratory tree, and the blood. Common themes included the ongoing need for standardization, validation, and defining the predictive capacity of data derived from model systems to estimate clinical outcomes from modulator-treated CF patients. CONCLUSIONS CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.

Take it to the Bank

In this issue of the Journal of Cystic Fibrosis, Beekman and colleagues report on discussions surrounding the establishment of European CF specimen biobanks for research use, which is complimented by a short communication by Garratt and the AREST CF research team [1,2]. These articles highlight the challenges and considerations of developing a multi-country CF biobank (the former), but also highlight the experience of managing a large biobank and the research advancements that a robust and well-conceived bank can enable (the latter). Research funding is often predicated on providing clear data sharing plans and greater access to research data and specimens [3,4]. With this push, biobanking should move from single investigator collections that are hoarded in personal freezers to more centralized collections available to the larger research community [5]. There are a number of key variables that need to be considered when developing and managing a specimen biobank, including the types of specimens obtained, standard operating procedures (SOPs) surrounding all steps (for the specimen) from the patient to the bank and ultimately the researcher (e.g.: sample acquisition, processing, handling, storage, transport, labeling, tracking, logging), ownership of specimens, processes for access to samples, systems to ensure quality of samples and accompanying information, location and management of the biorepository (e.g.: academic center, non-profit or for-profit storage facility), regulatory auditing, monitoring and ultimately the protection of patient health information. These considerations are certainly compounded in the context of a CF biobank with multiple contributing countries (and each with their accompanying regulations). However, establishing this type of multi-cultural and multi-ethnic biobank should be highly encouraged, particularly if the CF research community seeks to develop therapies and treatment approaches that allow all CF patients to live long, productive and fulfilled lives. The multi-cultural nature of a European CF biobank has great potential to shed light on cultural, environmental and societal impacts on patient outcomes, particularly since ~50% of disease heterogeneity is due to genetic influences and 50% is not [6]. Furthermore, as new therapies continue to enter care, banking of biospecimens is critical to maintain a ‘therapeutically relevant’ biobank that includes specimens from CF subjects with a variety of phenotypes and receiving a variety of therapies [7].