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Aztreonam for inhalation solution (AZLI) in patients with cystic fibrosis, mild lung impairment, and P. aeruginosa

  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    C.E. Wainwright
    Correspondence
    Corresponding author. Tel.: +61 7 36368111; fax: +61 7 36361958.
    Footnotes
    1 For the AIR-CF4 Study Group.
    Affiliations
    Queensland Children's Respiratory Centre, Royal Children's Hospital, Herston Rd. Herston, Queensland 4029, Australia
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    A.L. Quittner
    Footnotes
    1 For the AIR-CF4 Study Group.
    Affiliations
    University of Miami, 5665 Ponce de Leon Blvd., Coral Gables, FL, 33146, USA
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    D.E. Geller
    Footnotes
    1 For the AIR-CF4 Study Group.
    Affiliations
    Nemours Children's Clinic, 496 S. Delaney Ave., Suite 406A, Orlando, FL, 32803, USA
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    C. Nakamura
    Footnotes
    1 For the AIR-CF4 Study Group.
    Affiliations
    Children's Lung Specialists, 3820 Meadows Lane, Las Vegas, NV, 89107, USA
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    J.L. Wooldridge
    Footnotes
    1 For the AIR-CF4 Study Group.
    Affiliations
    Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 2021, Cincinnati, OH, 45229, USA
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    R.L. Gibson
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    1 For the AIR-CF4 Study Group.
    Affiliations
    Children's Hospital and Regional Medical Center, 4800 Sand Point Way NE, G0059, Seattle, WA, 98105, USA
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    S. Lewis
    Footnotes
    1 For the AIR-CF4 Study Group.
    Affiliations
    Gilead Sciences, Inc., 199 E. Blaine St., Seattle, WA, 98102, USA
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  • Author Footnotes
    1 For the AIR-CF4 Study Group.
    A.B. Montgomery
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    1 For the AIR-CF4 Study Group.
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    Gilead Sciences, Inc., 199 E. Blaine St., Seattle, WA, 98102, USA
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    1 For the AIR-CF4 Study Group.

      Abstract

      Background

      Previous aztreonam for inhalation solution (AZLI) studies included patients with cystic fibrosis, Pseudomonas aeruginosa (PA) airway infection, and forced expiratory volume in 1 s (FEV1) 25% to 75% predicted. This double-blind, multicenter, randomized, placebo-controlled trial enrolled patients (≥6 years) with FEV1>75% predicted.

      Methods

      AZLI 75 mg (n=76) or placebo (n=81) was administered 3-times daily for 28 days with a 14-day follow-up.

      Results

      Day 28 treatment effects were 1.8 points for CFQ-R-Respiratory Symptoms Scale (95%CI: −2.8, 6.4; p=0.443; primary endpoint); −1.2 for log10 sputum PA colony-forming units (p=0.016; favoring AZLI), and 2.7% for relative FEV1% predicted (p=0.021; favoring AZLI). Treatment effects favoring AZLI were larger for patients with baseline FEV1 <90% predicted compared to ≥90% predicted. AZLI was well-tolerated.

      Conclusions

      Effects on respiratory symptoms were modest; however, FEV1 improvements and bacterial density reductions support a possible role for AZLI in these relatively healthy patients. ClinicalTrials.gov identifier: NCT00712166.

      Keywords

      1. Introduction

      Cystic fibrosis (CF) is an autosomal-recessive disease characterized by thick pulmonary secretions and chronic airway infection with difficult to treat pathogens, the most significant of which is Pseudomonas aeruginosa (PA) [
      • Gibson R.L.
      • Burns J.L.
      • Ramsey B.W.
      State of the art: pathophysiology and management of pulmonary infections in cystic fibrosis.
      ]. The prevalence of infection increases with age; in 2008, 39.2% of US patients <18 years and 73.6% of patients ≥18 years of age were PA-positive [
      • Cystic Fibrosis Foundation Patient Registry
      2008 annual data report to the center directors.
      ]. Chronic airway infection is associated with progressive loss of lung function, which is the primary cause of death for CF patients [
      • Cystic Fibrosis Foundation Patient Registry
      2008 annual data report to the center directors.
      ,
      • Corey M.
      • Edwards L.
      • Levison H.
      • Knowles M.
      Longitudinal analysis of pulmonary function decline in patients with cystic fibrosis.
      ].
      Alternate-month, suppressive therapy with inhaled antibiotics is standard of care for CF patients infected with PA [
      • Flume P.A.
      • O'Sullivan B.P.
      • Robinson K.A.
      • et al.
      Cystic fibrosis pulmonary guidelines. Chronic medications for maintenance of lung health.
      ,
      • Heijermann H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Consensus working group. Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ]. Evidence for this practice came from studies of patients with moderately to severely impaired lung function. Currently available inhaled antibiotics in the US are approved for patients with CF and FEV1 25%–75% predicted, as these were the entry criteria for registration trials. Since the goal of such therapy is to preserve lung function and patients with high baseline lung function have a greater risk of steeper decline in lung function, CF patients with PA airway infection and normal to mildly compromised lung function should also benefit [
      • Konstan M.W.
      • Morgan W.J.
      • Butler S.M.
      • et al.
      Risk factors for rate of decline in forced expiratory volume in one second in children and adolescents with cystic fibrosis.
      ]. The prevalence of patients with mildly compromised lung function is increasing; in 2008, 56.6% of CF patients <18 years of age had FEV1>90% predicted [
      • Cystic Fibrosis Foundation Patient Registry
      2008 annual data report to the center directors.
      ].
      This study evaluated an inhaled antipseudomonal antibiotic, aztreonam for inhalation solution (AZLI; Cayston®, Gilead Sciences, Inc., Foster City CA). AZLI is indicated to improve respiratory symptoms in CF patients with PA [
      Prescribing information: Cayston® (aztreonam for inhalation solution).
      ]. Previous AZLI studies included patients with moderately to severely impaired lung function (FEV1 25%–75% predicted) and showed significant improvements relative to placebo on the Cystic Fibrosis Questionnaire—Revised (CFQ-R) Respiratory Symptoms Scale (RSS), time-to-need for intravenous (IV) or additional inhaled antipseudomonal antibiotics, forced expiratory volume in 1 s (FEV1), and log10 PA colony forming units (CFUs) in sputum [
      • McCoy K.S.
      • Quittner A.L.
      • Oermann C.M.
      • et al.
      Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • Gibson R.L.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway Pseudomonas in cystic fibrosis.
      ,
      • Oermann C.M.
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • et al.
      An 18-month study, AIR-CF3, of the safety and improvement in pulmonary function and respiratory symptoms with repeated courses of aztreonam for inhalation solution in patients with cystic fibrosis and airway Pseudomonas aeruginosa.
      ,
      • Quittner A.L.
      • Buu A.
      • Messer M.A.
      • Modi A.C.
      • Watrous M.
      Development and validation of the cystic fibrosis questionnaire in the United States: a health related quality of life measure for cystic fibrosis.
      ]. The current study repeats a previous study design (AIR-CF1), extending the efficacy and safety evaluation of AZLI to include patients with CF, PA airway infection, and milder impairment of lung function (FEV1>75% predicted) [
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • Gibson R.L.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway Pseudomonas in cystic fibrosis.
      ,
      • Wainwright C.
      • Nakamura C.
      • Geller D.
      • Montgomery A.B.
      A double-blind, multinational, randomized, placebo-controlled trial evaluating aztreonam for inhalation solution (AZLI) in patients with cystic fibrosis (CF), mild lung disease and P. aeruginosa (PA) [Abstract 81].
      ].

      2. Materials and methods

      2.1 Study design

      This double-blind, multicenter, multinational, randomized, placebo-controlled trial was conducted from June 2008 to June 2009 at 40 CF centers (US: 34; Canada: 1; Australia: 5). Eligibility was assessed at screening, ≤14 days before baseline (Day 0). Patients were randomly assigned (1:1) to a 28-day course of AZLI or placebo, administered three times daily (TID), with doses separated by ≥4 h. Randomization was stratified by age (6–13, 14–17, ≥18 years) and geographic region (North America, Australia; randomization code generated by Gilead Sciences). Additional study visits were at Day 14 (mid-treatment), Day 28 (end-of-treatment), and Day 42 (follow-up).
      A physical examination was performed at screening. Post-bronchodilator spirometry was performed according to American Thoracic Society guidelines at each study visit [
      • American Thoracic Society Committee on Diagnostic Standards for Non-Tuberculous Respiratory Diseases
      Standardization of spirometry 1994 update.
      ]. FEV1% predicted was calculated using the Knudson equation [
      • Knudson R.J.
      • Lebowitz M.D.
      • Holberg C.J.
      • Burrows B.
      Changes in the normal maximal expiratory flow-volume curve with growth and aging.
      ].
      AZLI (75 mg aztreonam, 52.5 mg lysine monohydrate) and placebo (5 mg lactose, 7.3 mg NaCl) were diluted in 0.17% saline (1 mL) and self-administered with the investigational eFlow® electronic nebulizer (PARI GmbH, Starnberg, Germany). Patients self-administered a short acting bronchodilator before each study drug dose.
      This study was conducted in compliance with the protocol, the Declaration of Helsinki, the International Conference on Harmonisation guideline for Good Clinical Practices, all applicable US Food and Drug Administration regulations, Canadian Health Products and Food Branch regulations, Australian Therapeutic Goods Administration regulations, and all other applicable local laws and regulations. Institutional review boards or ethics committees approved the study for each site. Patients and/or their legal representatives/guardians provided informed consent, or assent as applicable, before any study-related procedures, including screening procedures, were performed.

      2.2 Study population

      Eligible patients were ≥6 years of age, with documented CF and FEV1>75% predicted at screening. PA was present in expectorated sputum or throat swab culture samples at screening or documented in 2 samples within previous 12 months (1 positive sample obtained ≤3 months before screening). Eligible patients had experienced ≥2 of the following chronic and/or intermittent CF symptoms for ≥28 days before baseline with no worsening of symptoms within 7 days before baseline: chest congestion, daily cough, productive cough, wheezing, trouble breathing, or nocturnal wakening due to coughing. Eligible patients did not require immediate antipseudomonal antibiotic treatment of an impending exacerbation; investigators considered them to be in a stable condition. Patients were able to perform reproducible pulmonary function tests.
      Exclusion criteria included known hypersensitivity to monobactam antibiotics, inability to tolerate short-acting bronchodilators, lung transplantation history, or previous enrollment in an AZLI trial. Changes in baseline medications (azithromycin, hypertonic saline, or dornase alpha) or administration of any antipseudomonal antibiotic within 28 days before screening were excluded, as were changes in antimicrobial, bronchodilator, or corticosteroid medications or physiotherapy technique or schedule within 7 days before screening. Patients had a chest radiograph within 180 days before screening, with no significant acute findings (e.g., lobar infiltrate and atelectasis, pneumothorax, or pleural effusion). Females of child-bearing potential had a negative urine pregnancy test at screening, were practicing an acceptable method of birth control, and were not lactating. Patients who had received any investigational drug or device within 28 days (or 6-half lives) before screening were excluded, as were patients with any serious or active medical or psychiatric illness, which in the opinion of the investigator, would interfere with patient treatment, assessment, or compliance with the protocol.

      2.3 Study endpoints

      The primary efficacy endpoint was change from baseline at Day 28 on the CFQ-R RSS [
      • Quittner A.L.
      • Buu A.
      • Messer M.A.
      • Modi A.C.
      • Watrous M.
      Development and validation of the cystic fibrosis questionnaire in the United States: a health related quality of life measure for cystic fibrosis.
      ]. CFQ-R domains and scales are scored from 0 to 100, with higher scores indicating fewer symptoms, better functioning, or higher health-related quality of life. Secondary endpoints were: change from baseline at Days 14 and 42 on the CFQ-R RSS, change from baseline at Day 28 on the CFQ-R Physical Functioning Scale, use of additional antipseudomonal antibiotics, proportion of patients hospitalized, and change from baseline at Day 28 for log10 PA CFUs in sputum and FEV1% predicted. Additional efficacy endpoints included changes in other pulmonary function measures, weight, body mass index (BMI), other CFQ-R scores, and number of hospitalization days or missed school/work days.
      Safety endpoints included adverse events, acute airway reactivity, vital signs, serum chemistry, and hematology.
      Microbiological endpoints included the disappearance or appearance of respiratory pathogens and the change in minimum inhibitory concentration (MIC) of aztreonam for PA. The aztreonam concentrations required to inhibit the growth of 50% or 90% of PA isolates were reported as MIC50 or MIC90 values, respectively.

      2.4 Statistical methods

      Analyses included all randomly-assigned patients receiving ≥1 dose of study drug. Statistical tests were 2-tailed superiority analyses (α=0.05). Spirometry changes were calculated as relative values, calculated as a percentage of the baseline measurement. Microbiology assessments did not include data from all patients; some patients were not able to expectorate sputum samples, and some were PA-positive but CFUs were not reported.
      Based on the results from a previous AZLI study, a sample size of 140 patients provided ≥90% power to detect a 10-point difference between groups in mean change from baseline at Day 28 on the CFQ-R RSS, using a 2-sided 0.05-level test and assuming a standard deviation of 17.5 [
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • Gibson R.L.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway Pseudomonas in cystic fibrosis.
      ].
      For the primary efficacy analysis, treatment effect was assessed by a parametric analysis of covariance (ANCOVA); treatment and age group were fixed effects and baseline CFQ-R RSS score was a covariate. Analyses of other continuous variables used similar ANCOVA models, with respective baseline values as covariates. Exploratory analyses were performed for patients in mild (FEV1>75 to <90% predicted) and normal (FEV190% predicted) lung function categories as established by the Cystic Fibrosis Foundation (CFF), except the CFF “mild” category extends to 70% predicted [
      • Cystic Fibrosis Foundation Patient Registry
      2008 annual data report to the center directors.
      ].

      3. Results

      A total of 192 patients were screened and 157 patients received study-drug treatment (Fig. 1). Most patients used80% of study-drug doses (AZLI: 85.5% patients; placebo: 92.6%). One patient (AZLI) discontinued from the study and 6 patients discontinued treatment but remained in the study, discontinuing treatment was associated with pulmonary exacerbation for 3 patients (AZLI: 2; placebo: 1) and with the adverse event of FEV1 decreased for 1 patient (AZLI).
      Demographic characteristics were well balanced between treatment arms (Table 1). The majority of patients were 6–17 years of age (56.7%). Most patients were receiving dornase alfa (81.5%) and pancreatic enzymes (88.5%) at baseline. Patients had received a mean of 2.9 courses of TIS in the previous year; 65.0% of patients had received1 course.
      Table 1Baseline demographic and clinical characteristics.
      Placebo

      N=81
      AZLI

      N=76
      Total

      N=157
      Region; n (%)
       North America75 (92.6)72 (94.7)147 (93.6)
      Canada2 (2.5)02 (1.3)
      United States73 (90.1)72 (94.7)145 (92.4)
       Australia6 (7.4)4 (5.3)10 (6.4)
      Age, years; mean (SD)18.9 (9.1)19.5 (9.1)19.2 (9.1)
      Age group; n (%)
       6 to 13 years25 (30.9)22 (28.9)47 (29.9)
       14 to 17 years22 (27.2)20 (26.3)42 (26.8)
       ≥18 years34 (42.0)34 (44.7)68 (43.3)
       6 to 17 years47 (58.0)42 (55.3)89 (56.7)
      Male gender; n (%)44 (54.3)46 (60.5)90 (57.3)
      White race; n (%)77 (95.1)74 (97.4)151 (96.2)
      Weight, kg; mean (SD)53.5 (14.6)57.9 (17.8)55.6 (16.3)
      Body mass index, kg/m2; mean (SD)20.7 (3.0)21.7 (4.2)21.2 (3.7)
      CFTR genotype
      No. of patients with data=70 (placebo), 72 (AZLI), and 142 (total).
      ; n (%)
       Homozygous for ∆50840 (57.1)39 (54.2)79 (55.6)
       Heterozygous for ∆50826 (37.1)23 (31.9)49 (34.5)
       Unidentified2 (2.9)9 (12.5)11 (7.7)
       Other2 (2.9)1 (1.4)3 (2.1)
      TIS courses in previous year; mean (SD)3.0 (2.7)2.9 (2.5)2.9 (2.6)
      Use of medications at baseline; n (%)
       Azithromycin48 (59.3)41 (53.9)89 (56.7)
       Dornase alfa65 (80.2)63 (82.9)128 (81.5)
       Pancrelipase69 (85.2)70 (92.1)139 (88.5)
       Salbutamol54 (66.7)48 (63.2)102 (65.0)
      Baseline values; mean (SD)
       FEV1% predicted94.7 (12.9)95.5 (12.7)95.1 (12.8)
       FEV1 (L)2.9 (0.8)3.1 (0.9)3.0 (0.9)
       Log10 PA CFUs in sputum
      No. of patients with data=43 (placebo), 47 (AZLI), and 90 (total).
      4.4 (3.1)4.8 (2.6)4.6 (2.9)
       CFQ-R Respiratory Symptoms Score73.6 (17.9)70.3 (18.5)72.0 (18.2)
      MIC of aztreonam for all PA isolates at baseline
       MIC50; μg/mL111
       MIC90; μg/mL1688
       Minimum MIC; μg/mL111
       Maximum MIC; μg/mL128128128
       No. isolates tested
      No. of patients with data=63 (placebo), 64 (AZLI), and 127 (total) CFTR=cystic fibrosis transmembrane conductance regulator; CFQ-R=Cystic Fibrosis Questionnaire—Revised; CFUs=colony forming units; FEV1=forced expiratory volume in 1s; MIC=minimum inhibitory concentration; PA=P. aeruginosa.
      104104208
       Highest MIC
      No. of patients with data=63 (placebo), 64 (AZLI), and 127 (total) CFTR=cystic fibrosis transmembrane conductance regulator; CFQ-R=Cystic Fibrosis Questionnaire—Revised; CFUs=colony forming units; FEV1=forced expiratory volume in 1s; MIC=minimum inhibitory concentration; PA=P. aeruginosa.
      ; n (%)
      8 μg/mL53 (84.1)58 (90.6)111 (87.4)
      >8 μg/mL10 (15.9)6 (9.4)16 (12.6)
      a No. of patients with data=70 (placebo), 72 (AZLI), and 142 (total).
      b No. of patients with data=43 (placebo), 47 (AZLI), and 90 (total).
      c No. of patients with data=63 (placebo), 64 (AZLI), and 127 (total) CFTR=cystic fibrosis transmembrane conductance regulator; CFQ-R=Cystic Fibrosis Questionnaire—Revised; CFUs=colony forming units; FEV1=forced expiratory volume in 1 s; MIC=minimum inhibitory concentration; PA=P. aeruginosa.

      3.1 Efficacy

      Adjusted mean change at Day 28 from baseline CFQ-R RSS scores was 3.22 for AZLI-treated and 1.41 for placebo-treated patients (Table 2A; Fig. 2). The treatment effect (1.80; 95% confidence interval [CI]: −2.83, 6.44) was not statistically significant (p=0.443). Thus this study did not meet the primary efficacy endpoint. Subgroup analyses were performed for a pre-selected group of variables. Larger treatment effects were observed in favor of AZLI in female and adult patients, although these treatment effects were not statistically significant (Table 2A).
      Table 2AEfficacy results: primary and secondary endpoints.
      Placebo (N=81)AZLI (N=76)Treatment effect
      AZLI — placebo.
      P-value
      ANCOVA analyses. ANCOVA models included treatment, age group, and baseline values. Models for age subgroups included treatment and baseline values.
      Adjusted mean change from baseline to Day 28 (SE)
      No.No.
      Primary efficacy endpoint
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
       CFQ-R Respiratory Symptoms Score811.4 (1.6)753.2 (1.7)1.80.443
      Subgroup analyses
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
       Region
      North America752.0 (1.7)713.5 (1.8)1.40.562
      Australia6−4.0 (5.4)4−0.88 (6.7)3.20.725
       Age group
      6–13 years250.85 (3.4)221.3 (3.6)0.450.927
      14–17 years213.4 (2.7)191.3 (2.8)−2.10.593
      18 years340.44 (2.4)345.1 (2.4)4.70.183
      6–17 years461.8 (2.2)411.5 (2.4)−0.230.942
       Gender
      Male443.7 (2.3)453.7 (2.3)−0.040.990
      Female37−1.2 (2.4)302.7 (2.6)3.90.273
       CFQ-R version
      Child26−3.0 (8.7)22−2.6 (9.4)0.450.927
      Adolescent/adult551.8 (1.8)534.0 (1.9)2.30.385
       Highest aztreonam MIC for PA
      The parenteral breakpoint for aztreonam is 8μg/mL.
      8 μg/mL530.83 (2.3)573.1 (2.1)2.20.463
      >8 μg/mL10−2.1 (4.1)610.5 (5.3)12.60.103
       FEV1 at baseline
      Exploratory analysis.
      <90% predicted332.7 (2.7)299.5 (2.7)6.70.072
      90% predicted480.55 (2.1)46−0.81 (2.2)−1.40.658
      Secondary efficacy endpoints
       CFQ-R Respiratory Symptoms Score (baseline to Day 14)
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      810.28 (1.6)753.7 (1.6)3.40.133
       CFQ-R Respiratory Symptoms Score (baseline to Day 42)
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      812.9 (1.7)753.0 (1.7)0.100.965
       CFQ-R Physical Functioning Score (baseline to Day 28)
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      80−0.69 (1.5)761.8 (1.6)2.50.256
       Log10 PA CFUs in sputum (baseline to Day 28)31−0.14 (0.36)37−1.4 (0.36)−1.20.016
       Relative change in FEV1% predicted (baseline to Day 28)
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      81−2.5 (0.82)760.29 (0.85)2.70.021
      n (%); Baseline to End of StudyP-value
      Fisher's Exact Test.
       Use of Oral, IV, or Additional Inhaled Antipseudomonal Antibiotics21 (25.9)19 (25.0)>0.999
       Number of Patients Hospitalized
      Two additional patients (AZLI: 1, placebo: 1) were hospitalized off-study, each on Day 55, for pulmonary exacerbation.
      3 (3.7)8 (10.5)0.122
      CFQ-R=Cystic Fibrosis Questionnaire—Revised; CFUs=colony forming units; FEV1=forced expiratory volume in 1 s; IV=intravenous; MIC=minimum inhibitory concentration; PA=P. aeruginosa.
      a Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      b The parenteral breakpoint for aztreonam is 8 μg/mL.
      c Exploratory analysis.
      d Two additional patients (AZLI: 1, placebo: 1) were hospitalized off-study, each on Day 55, for pulmonary exacerbation.
      e AZLI — placebo.
      f ANCOVA analyses. ANCOVA models included treatment, age group, and baseline values. Models for age subgroups included treatment and baseline values.
      g Fisher's Exact Test.
      Figure thumbnail gr2
      Fig. 2Key efficacy endpoints. Mean changes in CFQ-R Respiratory Symptoms Scores (in points, on a scale of 0–100), mean relative changes in FEV1% predicted (percentage change from baseline values), and changes in log10 PA CFU/g sputum are plotted across the study visits.
      Statistically significant treatment effects favoring AZLI were observed for several secondary efficacy endpoints: change from baseline at Day 28 for adjusted mean log10 PA CFUs in sputum (AZLI: −1.4; placebo: −0.14; p=0.016) and adjusted mean relative change in FEV1% predicted (AZLI: 0.29%; placebo: −2.5%; p=0.021; Table 2A; Fig. 2). Amongst other efficacy endpoints, significant treatment effects favoring AZLI were observed for relative mean change from baseline FEV1 (L) at Day 28 and CFQ-R Social Functioning scores (Table 2B). Use of oral, IV, or additional inhaled antibiotics was similar for the AZLI (n=19) and placebo (n=21) groups during the entire study, with most use occurring during the follow-up period for both treatment groups (AZLI: 15; placebo: 17).
      Table 2BEfficacy results: additional endpoints.
      Additional efficacy endpointsPlacebo

      (N=81)
      AZLI

      (N=76)
      Treatment effect
      AZLI — placebo.
      P-value
      ANCOVA analyses. ANCOVA models included treatment, age group, and baseline values.
      Adjusted mean change (SE) from baseline to Day 28
      No.No.
      Relative change in FEV1 (L)
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      81−2.5 (0.83)760.36 (0.85)2.70.021
      Relative change in FVC (L)
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      81−1.4 (0.71)76−0.04 (0.73)1.40.171
      Relative change in FEF25–75
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      81−2.9 (2.2)762.6 (2.3)5.50.079
      Relative change in weight
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      81−1.9 (1.3)761.9 (1.3)3.00.111
      Change in BMI
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      (kg/m2)
      81−0.22 (0.24)760.33 (0.24)0.560.097
      Change on CFQ-R Scales
      Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      CFQ-R Health Perceptions, Role/School, Vitality, and Weight scales have only an adolescent/adult version.
       Body image801.1 (1.6)763.7 (1.7)2.60.259
       Digestion815.5 (1.9)753.6 (1.9)−1.90.470
       Eating disturbances81−1.2 (1.5)760.5 (1.5)−1.70.409
       Emotional functioning803.7 (1.2)763.3 (1.2)−0.370.828
       Health perceptions54−1.8 (2.0)541.8 (2.0)3.60.194
       Role/school551.2 (1.4)530.29 (1.5)−0.900.650
       Social functioning80−2.6 (1.3)751.9 (1.4)4.50.020
       Treatment burden815.1 (1.6)761.2 (1.6)−3.90.075
       Vitality54−2.2 (2.2)540.38 (2.2)2.60.402
       Weight552.6 (2.9)534.3 (2.9)1.70.667
      Mean (SD)
      Baseline to End of StudyP-value
      Wilcoxon Rank Sum Test.
      Number Hospitalization Days810.4 (2.5)760.9 (3.1)0.100
      Number Missed School/Work Days811.2 (3.0)761.6 (4.9)0.573
      BMI=body mass index; CFQ-R=Cystic Fibrosis Questionnaire—Revised; FEF25–75=forced expiratory volume from 25% to 75% of FVC; FEV1=forced expiratory volume in 1 s; FVC=forced vital capacity; PA=P. aeruginosa.
      a Missing postbaseline data were imputed with the last observation carried forward (LOCF) method.
      b CFQ-R Health Perceptions, Role/School, Vitality, and Weight scales have only an adolescent/adult version.
      c AZLI — placebo.
      d ANCOVA analyses. ANCOVA models included treatment, age group, and baseline values.
      e Wilcoxon Rank Sum Test.
      This study enrolled patients with mild disease severity (FEV1>75% predicted). Using the CFF's established lung function categories, the disease severity category was subdivided into “mild” (FEV1>75 to <90% predicted at baseline, 39.5% patients, n=62/157), and “normal” (FEV190% predicted at baseline, 60.5% patients, n=95/157), and patient characteristics and efficacy responses were characterized in an exploratory analysis. Patients with mild lung function impairment (FEV1>75 to <90% predicted) at baseline were older (mean [SD] age=20.7 [8.8] vs. 18.2 [9.1] years), with lower baseline scores on the CFQ-R RSS (67.4 [19.7] vs. 75.2 [16.5]) and higher baseline log10 PA sputum CFUs (5.30 [2.50] vs. 4.00 [3.01]), compared to patients with normal lung function (baseline FEV190% predicted). AZLI vs. placebo treatment effects for scores on the CFQ-R RSS were larger at Day 28 for patients with baseline FEV1<90% predicted than for patients with baseline FEV190% predicted (Table 2A). Likewise, AZLI vs. placebo treatment effects for mean relative change in FEV1% predicted were larger at Day 28 for patients with baseline FEV1<90% predicted (4.8% treatment effect; p=0.032) than for patients with baseline FEV190% predicted (1.4% treatment effect, p=0.302).

      3.2 Safety

      The majority of patients experienced one or more adverse events during the study (AZLI: 77.6%, n=59/76; placebo: 76.5%, n=62/81). Adverse events were mild to moderate in severity for most of these patients (AZLI: 91.5%, n=54/59; placebo: 95.2%, n=59/62). Cough and productive cough were the most commonly reported adverse events (Table 3). The incidence of the most commonly reported adverse events was similar for both treatment groups, except abdominal pain was reported for significantly fewer AZLI-treated patients. The most commonly reported adverse events considered by investigators to be treatment-related were cough (AZLI: 9.2%; placebo: 4.9%), productive cough (3.9%; 3.7%), respiratory tract congestion (3.9%; 0%), and pulmonary function test decreased (0%; 3.7%). Serious adverse events were reported for 11.8% (n=9/76) of AZLI-treated and 3.7% (n=3/81) of placebo-treated patients (p=0.073). These 12 patients experienced 20 serious adverse events; all were associated with hospitalizations and none were considered by investigators as treatment-related. The 12 on-study hospitalizations of 11 patients were associated with pulmonary exacerbation (AZLI: 2; placebo: 2); bronchitis, bronchiectasis, or bronchopneumonia (AZLI: 3), gastrointestinal conditions (AZLI: 2; placebo: 1), fever (placebo: 1), and influenza (AZLI: 1). No patients experienced an acute decrease in FEV1 of ≥15% after dosing at clinic visits and there were no reports of anaphylaxis. There were no significant differences between AZLI and placebo groups in clinical chemistry or hematology results or in vital signs except shifts of white blood cell, neutrophil, and platelet counts above the normal reference ranges were observed for fewer AZLI-treated patients. There were no deaths in this study.
      Table 3Adverse events reported for ≥10% of patients in either treatment group.
      Preferred termPlacebo

      (N=81)

      n (%)
      AZLI

      (N=76)

      n (%)
      P-value
      P-values are based on Fisher's Exact Test.
      Cough31 (38.3)35 (46.1)0.337
      Productive cough13 (16.0)18 (23.7)0.316
      Nasal congestion15 (18.5)13 (17.1)0.838
      Headache10 (12.3)14 (18.4)0.376
      Oropharyngeal pain11 (13.6)12 (15.8)0.822
      Rhinorrhoea12 (14.8)8 (10.5)0.479
      Respiratory tract congestion6 (7.4)11 (14.5)0.201
      Fatigue10 (12.3)6 (7.9)0.434
      Pulmonary function test decreased9 (11.1)7 (9.2)0.795
      Diarrhea9 (11.1)3 (3.9)0.133
      Abdominal pain10 (12.3)1 (1.3)0.010
      a P-values are based on Fisher's Exact Test.

      3.3 Microbiology

      Sputum samples were positive for PA for 86.5% of AZLI-treated patients at baseline (n=64/74); this decreased to 66.2% of patients at Day 28 (n=49/74) and increased to near baseline values at Day 42 (80.0%, n=60/75). The prevalence of PA-positive placebo-treated patients showed little change (baseline: 84.0%, n=63/75; Day 28: 83.5%, n=66/79; Day 42: 82.7%, n=62/75).
      For PA isolates from AZLI-treated patients, the MIC50 of aztreonam increased from baseline to Day 14 (≤1 μg/mL to 4 μg/mL) as did the MIC90 (8 μg/mL to 32 μg/mL); these increases were maintained at Days 28 and 42. For PA isolates from placebo-treated patients, the MIC50 (≤1 μg/mL) and MIC90 (16 μg/mL) of aztreonam remained unchanged during the study.
      The proportion of AZLI-treated patients who had PA isolates with aztreonam MIC values >8 μg/mL (parenteral breakpoint) increased from 9.4% at baseline (n=6/64) to 29.4% at Day 14 (n=15/51), and then remained fairly constant (Day 28: 26.5%, n=13/49; Day 42: 25.0%, n=15/60). The proportion of placebo-treated patients who had PA isolates with aztreonam MIC values >8 μg/mL remained fairly constant (baseline: 15.9%, n=10/63; Day 14: 20.3%, n=14/69, Day 28: 13.6%, n=9/66; Day 42: 16.1%, n=10/62). The PA isolate that was least susceptible to aztreonam was determined at baseline and at Day 28 for each patient. A ≥4-fold increase in aztreonam MIC for the least susceptible PA isolate was observed in 21 patients (AZLI: 13; placebo: 8; Fig. 3). Of these 21 patients, 4 patients in each treatment group had ≥4-fold increases in MIC that crossed or persisted above the aztreonam parenteral breakpoint of 8 μg/mL. No relationship was observed between the magnitude of the highest MIC of aztreonam for PA isolates for each patient at baseline and mean changes in CFQ-R RSS scores, FEV1% predicted, or log10 PA CFUs in sputum.
      Figure thumbnail gr3
      Fig. 3Four-fold or greater changes in minimum inhibitory concentrations (MICs) of aztreonam for P. aeruginosa (PA). Each horizontal bar represents data from a single patient. The left and right ends of a bar denote the aztreonam MIC of the least susceptible PA isolate at baseline and Day 28, respectively. Data are shown for all patients with ≥4-fold increases in aztreonam MIC values from baseline to Day 28. The vertical dotted line represents the parenteral susceptibility breakpoint of aztreonam for PA (8 μg/mL). Patients with MIC data: AZLI (N=44), placebo (N=54).
      There was no evidence for persistent increases in the isolation of Burkholderia spp., Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Aspergillus spp., or Staphyloccous aureus.

      4. Discussion

      Patients treated with AZLI showed a modest improvement in respiratory symptoms, as measured on the CFQ-R RSS. When compared with placebo, the treatment effect was not clinically or statistically significant, thus the study did not meet the primary efficacy endpoint. A significant treatment effect was observed at Day 28 for FEV1% predicted, a secondary endpoint. This primarily resulted from a decrease from baseline at Day 28 for placebo-treated patients; a small improvement was observed for AZLI-treated patients.
      On the CFQ-R RSS, larger AZLI vs. placebo treatment effects were observed for females compared with males, for adults compared with children, for patients with highest aztreonam MIC for PA >8 μg/mL compared with ≤8 μg/mL, and for patients with FEV1<90% predicted at baseline compared with ≥90% predicted, but none of these differences were statistically significant. The larger treatment effects in females and adults are in agreement with more compromised lung function typically seen in these subgroups [
      • Corey M.
      • Edwards L.
      • Levison H.
      • Knowles M.
      Longitudinal analysis of pulmonary function decline in patients with cystic fibrosis.
      ].
      The FEV1% predicted treatment effect observed in this study differed from the previously reported lack of significant lung function treatment effect observed for a subset of patients with baseline FEV175% predicted in the placebo-controlled AZLI Phase 2 study [
      • Retsch-Bogart G.Z.
      • Burns J.L.
      • Otto K.L.
      • et al.
      A phase 2 study of aztreonam lysine for inhalation to treat patients with cystic fibrosis and Pseudomonas aeruginosa infection.
      ]. Lack of significant effects on lung function were also reported in the literature for a placebo-controlled study of tobramycin inhalation solution (TIS) in patients with mild lung function impairment [
      • Murphy T.D.
      • Anbar R.D.
      • Lester L.A.
      • et al.
      Treatment with tobramycin solution for inhalation reduces hospitalizations in young CF subjects with mild lung disease.
      ]. Further, significant treatment effects were not observed for FEV1% predicted or on the CFQ-R RSS in a recent hypertonic saline trial in patients with FEV1>80% predicted [
      • Amin R.
      • Subbarao P.
      • Jabar A.
      • et al.
      Hypertonic saline improves the LCI in paediatric patients with CF with normal lung function.
      ]. The response of the placebo group in the current study also differed from responses observed in previous AZLI studies; in the current study both FEV1% predicted and scores on the CFQ-R RSS improved in the placebo group during the 14-day follow-up period and in previous studies both remained unchanged or decreased [
      • McCoy K.S.
      • Quittner A.L.
      • Oermann C.M.
      • et al.
      Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • Gibson R.L.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway Pseudomonas in cystic fibrosis.
      ]. The reason for this improvement in the placebo group is not known; it was not explained by differential use of oral, IV, or additional inhaled antipseudomonal antibiotics during the follow-up period as use of such antibiotics was similar for the placebo and AZLI treatment groups.
      AZLI was well tolerated, most adverse events were mild to moderate in severity and the most commonly reported adverse events were associated with respiratory symptoms.
      In contrast to previously reported AZLI trials, including one of 18-month duration, there were slight increases in this study for MIC50 and MIC90 of aztreonam for PA after AZLI treatment; these increases suggest an element of inducible resistance [
      • McCoy K.S.
      • Quittner A.L.
      • Oermann C.M.
      • et al.
      Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • Gibson R.L.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway Pseudomonas in cystic fibrosis.
      ,
      • Oermann C.M.
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • et al.
      An 18-month study, AIR-CF3, of the safety and improvement in pulmonary function and respiratory symptoms with repeated courses of aztreonam for inhalation solution in patients with cystic fibrosis and airway Pseudomonas aeruginosa.
      ]. This interpretation is supported by the observation that the baseline susceptibility of PA isolates in this study was generally greater than in the previous studies and the observation that, for most patients in this study, MIC values for PA isolates at Day 28 remained below the aztreonam parenteral breakpoint. Patients in previous studies had poorer lung function and were, on average, older than the patients in the current study, and thus likely had a history of more extensive treatment of PA airway infections. High aztreonam MICs have not been predictive of lack of efficacy, presumably due to the high aztreonam concentration achieved in sputum.
      Since the CF medical community recognizes the importance of preserving lung function in patients with chronic PA infection, it is important to explore why a drug like AZLI did not have a substantial treatment benefit on the CFQ-R RSS in this study. It seems unlikely that AZLI does not benefit this population; AZLI was effective in previous studies and there is no pathophysiologic reason to believe that an effective antibiotic would not benefit any CF population with chronic PA infection. It is possible that the sensitivity of the CFQ-RSS is not sufficient for patients with more modest symptoms at baseline (ceiling effect) or that the study may not have been adequately powered to detect a change. Sample size assumptions were based on observing a 10-point treatment difference on the CFQ-R RSS; however, the baseline CFQ-R RSS score was approximately 10 points higher than in previous AZLI studies [
      • McCoy K.S.
      • Quittner A.L.
      • Oermann C.M.
      • et al.
      Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • Gibson R.L.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway Pseudomonas in cystic fibrosis.
      ,
      • Oermann C.M.
      • Retsch-Bogart G.Z.
      • Quittner A.L.
      • et al.
      An 18-month study, AIR-CF3, of the safety and improvement in pulmonary function and respiratory symptoms with repeated courses of aztreonam for inhalation solution in patients with cystic fibrosis and airway Pseudomonas aeruginosa.
      ]. This indicates that patients in this study had fewer symptoms at baseline, and coupled with their high baseline FEV1% predicted values suggests that patients without measurable compromise of lung function will not have measurable improvement, in spite of chronic PA infection. This hypothesis is supported by analyses of the patient subset with FEV1>75 to <90% predicted; they had both measurable compromise and measured benefit.
      This study demonstrated a significant reduction in sputum bacterial density and maintenance of FEV1% predicted in AZLI-treated patients, compared with placebo. FEV1 has been shown to decline in the placebo arm of other studies as well; further supporting that maintenance of FEV1 should be a meaningful outcome even in a short term study. In spite of the general lack of demonstrated efficacy in patients with FEV1>75% predicted, prescribing guidelines do not specify a target level of lung compromise for azithromycin, hypertonic saline, or dornase alfa, but are limited in the US to patients with FEV1 25% to 75% predicted for TIS and AZLI.
      While this study of AZLI in CF patients with mild lung function impairment and airway PA did not meet its primary endpoint, much has been learned about this population. AZLI was well tolerated, FEV1% predicted declined in placebo-treated patients even over the short course of this study, and PA bacterial density decreased with AZLI treatment. In exploratory analyses, patients with FEV1>75 to <90% predicted had measurable improvement in respiratory symptoms and statistically significant improvements in FEV1% predicted, compared to patients with FEV190% predicted. Further studies of antipseudomonal therapies such as AZLI are warranted in patients with mild loss of lung function. Studies in patients with FEV1>75 to <90% predicted may be able to use short-term clinical outcomes, but measuring responses in patients with FEV190% predicted likely will require trial designs that use outcome measures that focus on the pathophysiology of the CF airway disease, or may need long-term studies measuring outcomes such as longitudinal decline in lung function.

      Conflict of interest statement

      CE Wainwright: The institution (Queensland Children's Medical Research Institute) received a research grant from Gilead to conduct this study. The institution also received compensation from Gilead for the author's participation in an advisory board meeting.
      AL Quittner: Receives consulting income from Gilead Sciences.
      DE Geller: The employer (Nemours Children's Clinic) receives research grants from Gilead, Novartis, Bayer and Mpex Pharmaceuticals.
      C Nakamura: None.
      JL Wooldridge: None.
      RL Gibson: No competing financial interests to declare. A portion of the pre-determined academic salary is from the implementation and conduct of clinical research studies. Recent funding sources have included the Cystic Fibrosis Foundation, National Institutes of Health, Vertex Pharmaceuticals, and Bayer Health Care.
      S Lewis and AB Montgomery: employees and stock holders of Gilead Sciences.

      Acknowledgments

      We thank the patients and their families as well as the additional investigators (F Accurso, The Children's Hospital Association, Aurora, CO, USA; R Anbar, State University of New York (SUNY) Upstate Medical University, Syracuse, NY, USA; P Anderson, University of Arkansas for Medical Sciences, Little Rock, AR, USA; D Bell, Prince Charles Hospital, Chermside, QLD, Australia; A Berlinski, Arkansas Children's Hospital, Little Rock, AR, USA; Y Berthiaume, Centre de Recherche du CHUM, Montreal, QC, Canada; J Billings, University of Minnesota Medical Center, Minneapolis, MN, USA; B Chatfield, University of Utah, Salt Lake City, UT, USA; B Clements, Princess Margaret Hospital for Children, Perth, WA, Australia; R Cohen, Long Island Jewish Medical Center, New Hyde Park, NY, USA; P Cooper, Childrens Hospital, Westmead, Westmead, NSW, Australia; C Daines, University of Arizona, Tucson, AZ, USA; G Graff, Pennsylvania State University, Hershey Medical Center, Hershey, PA, USA; A Hadeh, Drexel University School of Medicine, Philadelphia, PA, USA; D Hadjiliadis, University of Pennsylvania, Philadelphia, PA, USA; M Howenstine, Riley Hospital for Children, Indianapolis, IN, USA; C Lapin, Connecticut Children's Medical Center, Hartford, CT, USA; S McColley, Children's Memorial Hospital, Chicago, IL, USA; K McCoy, Nationwide Children's Hospital, Columbus, OH, USA; P Middleton, Westmead Hospital, Westmead, NSW, Australia; S Nasr, University of Michigan, Ann Arbor, MI, USA; B Nickerson, Children's Hospital of Orange County, Orange, CA, USA; C Oermann, Baylor College of Medicine, Houston, TX, USA; P Radford, Phoenix Children's Hospital, Phoenix, AZ, USA; S Reyes, Oklahoma City, OK, USA; J Rosen, Albany Medical College, Albany, NY, USA; A Sannuti, Indiana University Medical Center, Indianapolis, IN, USA; D Schaeffer, Nemours Children's Clinic, Jacksonville, FL, USA; J Sharp, The Women and Children's Hospital of Buffalo, Buffalo, NY, USA; G Shay, Kaiser Permanente Oakland Medical Center, Oakland, CA, USA; T Spencer, Children's Hospital Boston, Boston, MA, USA; D Toder, Children's Hospital of Michigan/Wayne State University, Detroit, MI, USA; L Varlotta, St. Christopher's Hospital for Children, Philadelphia, PA, USA; P Vauthy, Toledo Children's Hospital Cystic Fibrosis Center, Toledo, OH, USA; W Yee, Tufts — New England Medical Center, Boston, MA, USA). Medical writing assistance was provided by Kate Loughney, PhD, under the sponsorship of Gilead Sciences, Inc. This study was sponsored by Gilead Sciences, Inc and by NIH General Clinical Research Center grants M01 RR00400 , M01 RR10733 , and M01 RR00188 .

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