David A. Waltz

Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin.

The urokinase receptor (uPAR) coordinates plasmin-mediated cell-surface proteolysis and promotes cellular adhesion via a binding site for vitronectin on uPAR. Because vitronectin also binds plasminogen activator inhibitor type 1 (PAI-1), and plasmin cleavage of vitronectin reduces PAI-1 binding, we explored the effects of plasmin and PAI-1 on the interaction between uPAR and vitronectin. PAI-1 blocked cellular binding of and adhesion to vitronectin by over 80% (IC50 approximately 5 nM), promoted detachment of uPAR-bearing cells from vitronectin, and increased cellular migration on vitronectin. Limited cleavage of vitronectin by plasmin also abolished cellular binding and adhesion and induced cellular detachment. A series of peptides surrounding a plasmin cleavage site (arginine 361) near the carboxy-terminal end of vitronectin were synthesized. Two peptides spanning res 364-380 blocked binding of uPAR to vitronectin (IC50 approximately 8-25 microM) identifying this region as an important site of uPAR-vitronectin interaction. These data illuminate a complex regulatory scheme for uPAR-dependent cellular adhesion to vitronectin: Active urokinase promotes adhesion and also subsequent detachment through activation of plasmin or complex formation with PAI-1. Excess PAI-1 may also promote migration by blocking cellular adhesion and/or promoting detachment, possibly accounting in part for the strong correlation between PAI-1 expression and tumor cell metastasis.

A Phase I Study of Aerosolized Administration of tgAAVCF to Cystic Fibrosis Subjects with Mild Lung Disease

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.

A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial

BACKGROUND The phe508del CFTR mutation causes cystic fibrosis by limiting the amount of CFTR protein that reaches the epithelial cell surface. We tested combination treatment with lumacaftor, an investigational CFTR corrector that increases trafficking of phe508del CFTR to the cell surface, and ivacaftor, a CFTR potentiator that enhances chloride transport of CFTR on the cell surface. METHODS In this phase 2 clinical trial, we assessed three successive cohorts, with the results of each cohort informing dose selection for the subsequent cohort. We recruited patients from 24 cystic fibrosis centres in Australia, Belgium, Germany, New Zealand, and the USA. Eligibility criteria were: confirmed diagnosis of cystic fibrosis, age at least 18 years, and a forced expiratory volume in 1 s (FEV1) of 40% or more than predicted. Cohort 1 included phe508del CFTR homozygous patients randomly assigned to either lumacaftor 200 mg once per day for 14 days followed by addition of ivacaftor 150 mg or 250 mg every 12 h for 7 days, or 21 days of placebo. Together, cohorts 2 and 3 included phe508del CFTR homozygous and heterozygous patients, randomly assigned to either 56 days of lumacaftor (cohort 2: 200 mg, 400 mg, or 600 mg once per day, cohort 3: 400 mg every 12 h) with ivacaftor 250 mg every 12 h added after 28 days, or 56 days of placebo. The primary outcomes for all cohorts were change in sweat chloride concentration during the combination treatment period in the intention-to-treat population and safety (laboratory measurements and adverse events). The study is registered with ClinicalTrials.gov, number NCT01225211, and EudraCT, number 2010-020413-90. FINDINGS Cohort 1 included 64 participants. Cohort 2 and 3 combined contained 96 phe508del CFTR homozygous patients and 28 compound heterozygotes. Treatment with lumacaftor 200 mg once daily and ivacaftor 250 mg every 12 h decreased mean sweat chloride concentration by 9.1 mmol/L (p<0.001) during the combination treatment period in cohort 1. In cohorts 2 and 3, mean sweat chloride concentration did not decrease significantly during combination treatment in any group. Frequency and nature of adverse events were much the same in the treatment and placebo groups during the combination treatment period; the most commonly reported events were respiratory. 12 of 97 participants had chest tightness or dyspnoea during treatment with lumacaftor alone. In pre-planned secondary analyses, a significant decrease in sweat chloride concentration occurred in the treatment groups between day 1 and day 56 (lumacaftor 400 mg once per day group -9.1 mmol/L, p<0.001; lumacaftor 600 mg once per day group -8.9 mmol/L, p<0.001; lumacaftor 400 mg every 12 h group -10.3 mmol/L, p=0.002). These changes were significantly greater than the change in the placebo group. In cohort 2, the lumacaftor 600 mg once per day significantly improved FEV1 from day 1 to 56 (difference compared with placebo group: +5.6 percentage points, p=0.013), primarily during the combination period. In cohort 3, FEV1 did not change significantly across the entire study period compared with placebo (difference +4.2 percentage points, p=0.132), but did during the combination period (difference +7.7 percentage points, p=0·003). Phe508del CFTR heterozygous patients did not have a significant improvement in FEV1. INTERPRETATION We provide evidence that combination lumacaftor and ivacaftor improves FEV1 for patients with cystic fibrosis who are homozygous for phe508del CFTR, with a modest effect on sweat chloride concentration. These results support the further exploration of combination lumacaftor and ivacaftor as a treatment in this setting. FUNDING Vertex Pharmaceuticals, Cystic Fibrosis Foundation Therapeutics Development Network.

The impact of incident methicillin resistant Staphylococcus aureus detection on pulmonary function in cystic fibrosis

The incidence of methicillin resistant Staphylococcus aureus (MRSA) infection is increasing in cystic fibrosis (CF), but the impact of MRSA detection on clinical outcomes is unclear. Our objective was to determine whether incident detection of MRSA is associated with a change in pulmonary function over time in CF patients. We analyzed data from the Epidemiologic Study of Cystic Fibrosis (ESCF), a prospective observational study of CF patients in North America. Multivariable piecewise linear regression was used to model the impact of incident detection of MRSA on pulmonary function over time, measured by percent predicted forced expiratory volume in one second (FEV1% predicted), adjusting for potential confounders. There were 5,090 patients ≥6 years old who were MRSA negative for at least 2 calendar years. Five hundred ninety‐three (12%) of these patients acquired MRSA during the years 2001–2003, with detection rates of MRSA during those years rising from 4.4% to 6.9%. MRSA positive patients had a lower FEV1% predicted and received more antibiotic and other therapies than patients who remained MRSA negative. After adjusting for antibiotic therapy and other potential confounders, MRSA positive patients also had a higher rate of decline in FEV1% predicted both before and after the incident culture, although the rate of FEV1% predicted decline did not change significantly after MRSA detection. In conclusion, although MRSA in CF was a marker for more aggressive therapy and may reflect increased disease severity, incident MRSA detection was not associated with a changing rate of FEV1% predicted decline. Pediatr. Pulmonol. 2008; 43:1117–1123.

Assessment of safety and efficacy of long-term treatment with combination lumacaftor and ivacaftor therapy in patients with cystic fibrosis homozygous for the F508del-CFTR mutation (PROGRESS): a phase 3, extension study

BACKGROUND The 24-week safety and efficacy of lumacaftor/ivacaftor combination therapy was shown in two randomised controlled trials (RCTs)-TRAFFIC and TRANSPORT-in patients with cystic fibrosis who were aged 12 years or older and homozygous for the F508del-CFTR mutation. We aimed to assess the long-term safety and efficacy of extended lumacaftor/ivacaftor therapy in this group of patients in PROGRESS, the long-term extension of TRAFFIC and TRANSPORT. METHODS PROGRESS was a phase 3, parallel-group, multicentre, 96-week study of patients who completed TRAFFIC or TRANSPORT in 191 sites in 15 countries. Patients were eligible if they were at least 12 years old with cystic fibrosis and homozygous for the F508del-CFTR mutation. Exclusion criteria included any comorbidity or laboratory abnormality that, in the opinion of the investigator, might confound the results of the study or pose an additional risk in administering the study drug to the participant, history of drug intolerance, and history of poor compliance with the study drug. Patients who previously received active treatment in TRANSPORT or TRAFFIC remained on the same dose in PROGRESS. Patients who had received placebo in TRANSPORT or TRAFFIC were randomly assigned (1:1) to receive lumacaftor (400 mg every 12 h)/ivacaftor (250 mg every 12 h) or lumacaftor (600 mg once daily)/ivacaftor (250 mg every 12 h). The primary outcome was to assess the long-term safety of combined therapy. The estimated annual rate of decline in percent predicted FEV1 (ppFEV1) in treated patients was compared with that of a matched registry cohort. Efficacy analyses were based on modified intention-to-treat, such that data were included for all patients who were randomly assigned and received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01931839. FINDINGS Between Oct 24, 2013, and April 7, 2016, 1030 patients from the TRANSPORT and TRAFFIC studies enrolled in PROGRESS, and 1029 received at least one dose of study drug. 340 patients continued treatment with lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h; 176 patients who had received placebo in the TRANSPORT or TRAFFIC studies initiated treatment with lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h, the commercially available dose, for which data are presented. The most common adverse events were infective pulmonary exacerbations, cough, increased sputum, and haemoptysis. Modest blood pressure increases seen in TRAFFIC and TRANSPORT were also observed in PROGRESS. For patients continuing treatment, the mean change from baseline in ppFEV1 was 0·5 (95% CI -0·4 to 1·5) at extension week 72 and 0·5 (-0·7 to 1·6) at extension week 96; change in BMI was 0·69 (0·56 to 0·81) at extension week 72 and 0·96 (0·81 to 1·11) at extension week 96. The annualised pulmonary exacerbation rate in patients continuing treatment through extension week 96 (0·65, 0·56 to 0·75) remained lower than the placebo rate in TRAFFIC and TRANSPORT. The annualised rate of ppFEV1 decline was reduced in lumacaftor/ivacaftor-treated patients compared with matched controls (-1·33, -1·80 to -0·85 vs -2·29, -2·56 to -2·03). The efficacy and safety profile of the lumacaftor 600 mg once daily/ivacaftor 250 mg every 12 h groups was generally similar to that of the lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h groups. INTERPRETATION The long-term safety profile of lumacaftor/ivacaftor combination therapy was consistent with previous RCTs. Benefits continued to be observed with longer-term treatment, and lumacaftor/ivacaftor was associated with a 42% slower rate of ppFEV1 decline than in matched registry controls. FUNDING Vertex Pharmaceuticals Incorporated.

Role of bacteriocins in mediating interactions of bacterial isolates taken from cystic fibrosis patients

Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) lung infections are responsible for much of the mortality in cystic fibrosis (CF). However, little is known about the ecological interactions between these two, often co-infecting, species. This study provides what is believed to be the first report of the intra- and interspecies bacteriocin-like inhibition potential of Pa and Bcc strains recovered from CF patients. A total of 66 strains were screened, and shown to possess bacteriocin-like inhibitory activity (97 % of Pa strains and 68 % of Bcc strains showed inhibitory activity), much of which acted across species boundaries. Further phenotypic and molecular-based assays revealed that the source of this inhibition differs for the two species. In Pa, much of the inhibitory activity is due to the well-known S and RF pyocins. In contrast, Bcc inhibition is due to unknown mechanisms, although RF-like toxins were implicated in some strains. These data suggest that bacteriocin-based inhibition may play a role in governing Pa and Bcc interactions in the CF lung and may, therefore, offer a novel approach to mediating these often fatal infections.

Cutaneovisceral Angiomatosis With Thrombocytopenia

We describe 10 children with multiple vascular lesions of the skin and gastrointestinal tract associated with sustained, minor thrombocytopenia. In some children, there was involvement of the lung (n = 5), bone (n = 2), liver (n = 1), spleen (n = 1), and muscle (n = 1). The cutaneous lesions were congenital, multifocal, discrete, red-brown and variably blue macules and papules; in 3 children, a large dominant plaque was also present. All children developed hematemesis and/or melena and endoscopic evaluation revealed several to numerous small mucosal lesions that involved all levels of the gastrointestinal tract. Three of 5 children with pulmonary nodules had cough and 1 also had hemoptysis. Biopsies of cutaneous, gastrointestinal, and pulmonary lesions showed thin-walled, blood-filled vascular channels and variable endothelial hyperplasia. The endothelial nuclei were elongated, round, crescentic, or hobnailed. Cytoplasmic and extracellular periodic acid-Schiff positive deposits were often present in the zones of endothelial hyperplasia. The platelets were small in some children, suggesting a primary defect, possibly accounting for the thrombocytopenia. Gastrointestinal hemorrhage and hemoptysis required antiangiogenic therapy. The constellation of findings defines a congenital proliferative disorder of blood vessels with a distinctive microscopic appearance. We have termed this relatively indolent or slowly progressive disorder cutaneovisceral angiomatosis with thrombocytopenia because this designation incorporates its major clinical and histopathologic features.

Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis

BACKGROUND Lumacaftor/ivacaftor combination therapy has shown clinical benefits in patients with cystic fibrosis homozygous for the Phe508del CFTR mutation; however, pretreatment lung function is a confounding factor that potentially affects the efficacy and safety of this therapy. We aimed to assess the efficacy and safety of lumacaftor/ivacaftor therapy in these patients, defined by specific categories of lung function. METHODS Both trials (TRAFFIC and TRANSPORT) included in this pooled analysis were multinational, randomised, double-blind, placebo-controlled, parallel-group, phase 3 studies. Eligible patients from 187 participating centres in North America, Australia, and the European Union (both trials) were aged 12 years or older with a confirmed diagnosis of cystic fibrosis, homozygous for the Phe508del CFTR mutation, and with a percent predicted FEV1 (ppFEV1) of 40-90 at the time of screening. Patients were randomly assigned with an interactive web response system (1:1:1) to receive placebo, lumacaftor (600 mg once daily) plus ivacaftor (250 mg every 12 h), or lumacaftor (400 mg every 12 h) plus ivacaftor (250 mg every 12 h) for 24 weeks. Prespecified subgroup analyses of pooled efficacy and safety data by lung function, as measured by ppFEV1, were done for patients with baseline ppFEV1 (<40 and ≥40) and screening ppFEV1 (<70 and ≥70). The primary endpoint was the absolute change from baseline in ppFEV1 at week 24 analysed in all randomised patients who received at least one dose of study drug. Both trials are registered with ClinicalTrials.gov (TRAFFIC: NCT01807923; TRANSPORT: NCT01807949). FINDINGS Both trials were done between April, 2013, and April, 2014. Of the 1108 patients included in the efficacy analysis, 81 patients had a ppFEV1 that decreased to lower than 40 between screening and baseline and 1016 had a ppFEV1 of 40 or higher at baseline. At screening, 730 had a ppFEV1 of less than 70, and 342 had a ppFEV1 of 70 or higher. Improvements in the absolute change from baseline at week 24 in ppFEV1 were observed with both lumacaftor/ivacaftor doses in the subgroup with baseline ppFEV1 levels lower than 40 (least-squares mean difference vs placebo was 3·7 percentage points [95% CI 0·5-6·9; p=0·024] in the lumacaftor [600 mg/day]-ivacaftor group and 3·3 percentage points [0·2-6·4; p=0·036] in the lumacaftor [400 mg/12 h]-ivacaftor group). Improvements in ppFEV1 compared with placebo were also reported in the subgroup with baseline ppFEV1 levels of 40 or higher (3·3 percentage points [2·3-4·4; p<0·0001] in the lumacaftor [600 mg per day]-ivacaftor group and 2·8 percentage points [1·7-3·8; p<0·0001] in the lumacaftor [400 mg/12 h]-ivacaftor group). Similar absolute improvements in ppFEV1 compared with placebo were observed in subgroups with screening ppFEV1 levels lower than 70 and ppFEV1 levels of 70 or higher. Increases in body-mass index and reduction in number of pulmonary exacerbation events were observed in both lumacaftor/ivacaftor dose groups compared with placebo across all lung function subgroups. Treatment was generally well tolerated, although the incidence of some respiratory adverse events was higher with lumacaftor/ivacaftor than with placebo in all subgroups. In patients with baseline ppFEV1 levels lower than 40, these adverse events included cough, dyspnoea, and abnormal respiration. INTERPRETATION These analyses confirm that lumacaftor/ivacaftor combination therapy benefits patients with cystic fibrosis homozygous for Phe508del CFTR who have varying degrees of lung function impairment. FUNDING Vertex Pharmaceuticals.

Lumacaftor/Ivacaftor in Patients Aged 6–11 Years with Cystic Fibrosis and Homozygous for F508del-CFTR

Rationale: Combination lumacaftor/ivacaftor has been shown to improve lung function and other endpoints in patients aged 12 years and older with cystic fibrosis and homozygous for F508del‐CFTR, but it has not been assessed in younger patients. Objectives: In this open‐label phase III trial, we evaluated the safety, tolerability, pharmacodynamics, and efficacy of lumacaftor/ivacaftor combination therapy in patients aged 6‐11 years with cystic fibrosis who were homozygous for F508del‐CFTR. Methods: Patients (N = 58) received 200 mg lumacaftor/250 mg ivacaftor orally every 12 hours for 24 weeks in addition to their existing cystic fibrosis medications. Measurements and Main Results: Lumacaftor/ivacaftor was well tolerated; the safety profile was generally similar to that observed in larger lumacaftor/ivacaftor trials with older patients. Four patients discontinued (two because of drug‐related adverse events: elevated liver transaminases, n = 1; rash, n = 1). No safety concerns were associated with spirometry. No significant changes in percent predicted FEV1 were observed (change from baseline at Week 24, +2.5 percentage points; 95% confidence interval [CI], −0.2 to 5.2; P = 0.0671). At Week 24, significant improvements from baseline were observed in sweat chloride (−24.8 mmol/L; 95% CI, −29.1 to −20.5; P < 0.0001), body mass index z score (+0.15; 95% CI, 0.08 to 0.22; P < 0.0001), Cystic Fibrosis Questionnaire‐Revised respiratory domain score (+5.4; 95% CI, 1.4 to 9.4; P = 0.0085), and lung clearance index based on lung volume turnover required to reach 2.5% of starting N2 concentration (−0.88; 95% CI, −1.40 to −0.37; P = 0.0018). Conclusions: Lumacaftor/ivacaftor was well tolerated in this young population; no new safety concerns were identified. Improvements in lung clearance index, sweat chloride, nutritional status, and health‐related quality of life were observed after 24 weeks of treatment. Clinical trial registered with www.clinicaltrials.gov (NCT01897233).

MDCT evaluation of the prevalence of tracheomalacia in children with mediastinal aortic vascular anomalies.

Objective The purpose of this study is to determine the prevalence of tracheomalacia (TM) associated with different types of mediastinal aortic vascular anomalies in symptomatic children using paired inspiratory-expiratory multidetector computed tomography (MDCT). Materials and Methods The study group consisted of 15 consecutive symptomatic pediatric patients (12 males/3 females; mean age of 4.4 y; age range of 2 wk to 16 y) with mediastinal aortic vascular anomalies, who were referred for paired inspiratory-expiratory MDCT during a 35-month time period. Computed tomography (CT) angiography was also concurrently performed during the end-inspiration phase of the study. Two radiologists in consensus reviewed all CT images in a randomized and blinded fashion. End-inspiration and end-expiration CT images were reviewed for the presence and severity of tracheal narrowing and the type of mediastinal aortic vascular anomaly involved. TM was defined as ≥50% reduction in tracheal cross-sectional luminal area between end-inspiration and end-expiration. The presence of TM was correlated with the type of mediastinal aortic vascular anomaly and compared with the bronchoscopy results when available (n=9). Results Mediastinal aortic vascular anomalies included innominate artery compression (IAC) (n=6), a right aortic arch with an aberrant left subclavian artery (n=5), double aortic arch (n=3), and a left aortic arch with an aberrant right subclavian artery (n=1). Eight of 15 (53.3%) patients demonstrated TM. TM was seen in all 6 patients (100%) with IAC, 1 of 3 (33.3%) patients with double aortic arch, and 1 of 5 (20%) patients with a right aortic arch with an aberrant left subclavian artery. CT results were concordant with the results of bronchoscopy in all patients who underwent this procedure (n=9). Conclusions Symptomatic pediatric patients with mediastinal aortic vascular anomalies have a relatively high prevalence of TM, especially those with IAC. Paired inspiratory-expiratory MDCT should be considered part of the routine preoperative evaluation of TM in symptomatic children with IAC and also has the potential to play a role in evaluating patients with other mediastinal aortic vascular anomalies.