JCF-01114; No of Pages 8
Journal of Cystic Fibrosis xx (2014) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis☆,☆☆ C. Calabrese a,1 , A. Tosco b,1 , P. Abete b , V. Carnovale b , C. Basile b , A. Magliocca b , S. Quattrucci c , S. De Sanctis c , F. Alatri c , G. Mazzarella a , L. De Pietro a , C. Turino a , E. Melillo d , P. Buonpensiero b , A. Di Pasqua b , V. Raia b,⁎ b
a Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Italy Department of Translational Medical Sciences, Cystic Fibrosis Center, University "Federico II" of Naples, Italy c Department of Pediatrics, Cystic Fibrosis Center, Sapienza University of Rome, Italy d Pediatric Pneumology, Santobono-Pausilipon-Annunziata Children's Hospital, Naples, Italy
Received 25 May 2014; revised 28 September 2014; accepted 30 September 2014 Available online xxxx
Abstract Background: In cystic fibrosis (CF) the defective CF transmembrane conductance regulator protein may be responsible for the impaired transport of glutathione (GSH), the first line defense of the lung against oxidative stress. The aim of this single-blind, randomized, placebo-controlled trial was to evaluate the effect of inhaled GSH in patients with CF. Methods: 54 adult and 51 pediatric patients were randomized to receive inhaled GSH or placebo twice daily for 12 months. Results: Twelve month treatment with inhaled GSH did not achieve our predetermined primary outcome measure of 15% improvement in FEV1%. Only in patients with moderate lung disease, 3, 6 and 9 months therapy with GSH resulted in a statistically significant increase of FEV1 values from the baseline. Moreover GSH therapy improved 6-minute walking test in pediatric population. GSH was well tolerated by all patients. Conclusions: Inhaled GSH has slight positive effects in CF patients with moderate lung disease warranting further study. Trial registry: ClinicalTrials.gov; No.: NCT01450267; URL: www.clinicaltrialsgov. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Glutathione; Therapy
Abbreviations: CF, cystic fibrosis; GSH, glutathione; CFTR, cystic fibrosis transmembrane regulator; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; FEF25–75, flow at the mid-portion of forced vital capacity; 6MWT, six minute walking test; BMI, body-mass index; CCIQ, Chronic Cough Impact Questionnaire; CFQoL, Cystic Fibrosis Quality of Life Questionnaire; CRP, C-reactive protein; H2O2, hydrogen peroxide; EBC, exhaled breath condensate. ☆ Financial disclosure: Authors declare no conflicts of interest. ☆☆ Funding: The study was financed by AIFA (Italian Agency of Drugs) (FARM7K7XZB). ⁎ Corresponding author at: Department of Translational Medical Sciences University “Federico II” of Naples, Via S Pansini 5, 80131 Naples, Italy. Tel.: +39 0817463273; fax: + 39 0815463393. E-mail address: [email protected] (V. Raia). 1 Co-first author.
1. Introduction Glutathione (GSH) represents the first-line defense of the lung against oxidative stress-induced damage [1] enhancing the functional activity of different immune cells [2]. GSH is normally found in the epithelial lining fluid of lower airways at very high concentration. However, a depletion of its levels has been observed in the airways of patients with Cystic Fibrosis (CF) [3]. In vitro studies showed that CFTR protein plays a pivotal role in transmembrane glutathione transport [4]. In animal models, as CFTR KO mice, a strong evidence supports the role of CFTR on airway fluid GSH levels [5]. In patients with CF, the reduced
http://dx.doi.org/10.1016/j.jcf.2014.09.014 1569-1993© 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
transport of GSH induced by the defective CFTR protein prevents the counterbalance of excessive oxidative stress molecules released by inflammatory cells and bacteria into the airways [6]. The persistent GSH deficiency, reported also in the neutrophils of patients with CF [7], could explain the paradox of an overactive immune system that otherwise is ineffective in eradicating bacteria [8,9]. Based on these evidences, a role of GSH has been suggested as a new therapeutic option in CF. Several studies have investigated the potential therapeutic role of inhaled GSH in patients with CF. Three short-term clinical trials, of which only one was placebo-controlled, have shown the tolerability and efficacy of inhaled GSH on pulmonary function in these subjects [10–13]. A more recent [14] six-month randomized double-blind placebo-controlled study failed to demonstrate the efficacy of inhaled GSH therapy on lung function, pulmonary exacerbations, as well as on surrogate inflammation and oxidative biomarker. Thus, given the high expectations placed on this molecule in CF community, we investigated whether a longer term treatment with inhaled GSH could be as effective for controlling lung function and other clinical outcomes in both pediatric and adult CF patients. 2. Materials and methods 2.1. Trial design This was a prospective, single-blind, randomized, placebocontrolled trial comparing 12 months of treatment with inhaled GSH versus placebo in pediatric and adult patients affected by CF. All eligible patients were divided into two groups: 1) Pediatric Group: age ≥ 6 to b 18 years; 2) Adult Group: age ≥ 18 years. Patients were consecutively enrolled at the Pediatric and Adult Units of Campania Cystic Fibrosis Regional Centre, University
Federico II of Naples, and at Lazio Cystic Fibrosis Regional Centre, University La Sapienza Policlinico Umberto I. The study timeline has been shown in Fig. 1. At the enrollment (visit 0), patients of both groups were randomized to GSH or placebo arm. Clinical assessments were scheduled one, three, six, nine, and twelve months after the enrollment. At each visit, all patients underwent a complete medical examination, which included lung function, height and bodyweight; moreover sputum samples or deep pharyngeal suction (in 9 children) were collected for microbiological analysis. Spirometry was carried out in the morning (8.00 a.m. to 10.00 a.m.). All measures were made by a single operator using a single calibrated, dry wedge bellows spirometer. FEV1 and FVC were measured, using the best of 3 forced maneuvers fulfilling the American Thoracic Society standard recommendations [15]. Forced expiratory volume 1 s (FEV1) values and the % of the predicted values (Zapletal) were recorded, as well as forced expiratory flow at the mid-portion of forced vital capacity (FEF25–75) % of the predicted values. At the enrollment as well as at the end of the study the following parameters were recorded: exercise capacity by six-minute walking test (6MWT) [16], body mass index (BMI), BODE index [17], cough measured by Chronic Cough Impact Questionnaire (CCIQ) [18], and quality of life assessed by Cystic Fibrosis Quality of Life Questionnaire (CFQoL) [19]. The number of acute pulmonary exacerbations, defined following the criteria used by Fuchs [20,21], was evaluated during the study and in the year before the enrollment. As a surrogate biomarker of inflammation, serological C-reactive protein (CRP) was measured; in a subgroup of patients, hydrogen peroxide (H2O2) was determined by the Fenton's reaction [22,23] in both exhaled breath condensate (EBC) and serum as a marker of oxidative stress. Concomitant therapy was regularly recorded at each visit, as well as any change in antibiotic therapy.
Fig. 1. Study timeline. Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
2.2. Participants The inclusion criteria were: 1) age ≥ 6 years; 2) confirmed diagnosis of CF by sweat chloride test over 60 mmol/L and/or genetic analysis, and 3) stable clinical condition defined as the absence of substantial, acute clinical problems potentially affecting the outcome measures included in the protocol. Exclusion criteria were: 1) pregnancy and fertile women taking oral contraceptives; 2) cigarette smoking; 3) positive culture for Burkholderia cepacia; 4) history of hemoptysis and pneumothorax; 5) FEV1 ≤ 40% of the predicted value; and 6) hyperresponsiveness to GSH inhalation test defined as a decrease in FEV1 greater than 15% compared to basal value at a dynamic spirometry performed 10 and 60 min after the inhalation of GSH (dosage of 10 mg/kg body weight—max dose 600 mg). During the study, concomitant therapeutic approaches were continued by enrolled patients according to standardized guidelines. Thiolic mucolytic drugs, anti-oxidant drugs and Mesna (2-mercaptoethane sulfonate) were disallowed. Vitamins supplementation was not modified during the study. Chronic concomitant medications are listed in Supplemental Table 1 A–B. The study was conducted in accordance with the amended Declaration of Helsinki. Italian Agency of Drugs and Independent Local Ethics Committees of the involved centers approved the protocol. All subjects provided written informed consent before treatment. 2.3. Interventions The GSH arm received inhaled GSH formulated as vials containing lyophilized powder of reduced glutathione to be reconstituted with 8 ml of water, in order to achieve a tonicity of 435 milliosmol/kg, at the dose of 10 mg/kg of body weight (max dose 600 mg) twice daily for 12 months. The placebo group received inhaled 0.9% sodium chloride sterile solution (physiological solution) twice daily for 12 months. The administrations of GSH solution and placebo were performed through a compressed-air nebulizer (PARI turbo boy) or an equivalent device delivering aerodynamic particles of 3.5–5.5 micrometer diameter. The treatment and the placebo were kindly supplied by the company ®, Biomedica Foscama, Ferentino, Italy. The compliance with GSH or placebo was checked by the Investigator and recorded in CRF during the routine clinical controls on the basis of returned recipients. Percent level of compliance was calculated as follows: % compliance = number of vials or ampoules taken by the patient / number of vials or ampoules that should have been taken by the patient × 100. The minimum level of allowed compliance was 80%. 2.4. Outcomes Our primary outcome was a 15% change from baseline in percent predicted in FEV1, after 12 months of therapy with inhaled GSH compared to placebo. Secondary outcomes reported in the study included: change from baseline in GSH and placebo groups for FVC, FEF25–75,
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6MWT, BMI (kg/m2), BODE index, CCIQ, CFQoL, number of pulmonary exacerbations, antibiotic courses and number of days requiring i.v. antibiotics administration compared to the previous year. At the enrollment and at the end of the trial CRP was measured; in a subgroup of patients, H2O2 was evaluated in both EBC and serum [21,22]. 2.5. Sample size The sample size (N = 150) required for the statistical analysis was subsequently reduced to 100 patients, considering pediatric and adult patients as separate groups, on the basis of a target improvement of 15% in FEV1, from 50% to 65% of the predicted value, with a standard deviation of 19%, an alpha (type I) error of 5% and a beta (type II) error of 20% (power 80%). 2.6. Randomization and blinding All the participants were assigned to GSH or placebo groups according to a blind, computer-generated randomization list performed by a person not otherwise involved in the study. Each patient was assigned to a unique number related to his/her corresponding treatment. The treatment was single-blinded because the GSH has a distinct taste and smell that is difficult to reproduce as placebo. Patients of the two different groups were examined in different days. 2.7. Statistic methods Data were analyzed using the SPSS 13.0 statistic package. Baseline characteristics of the sample were expressed as mean ± standard deviation (SD). To evaluate differences between GSH and placebo arms during the study, a two way analysis of variance (ANOVA) for repeated measures was applied for the following variables: FEV1, FVC, FEF25–75 as absolute values and % of predicted values. Moreover, for all variables we compared the pre-post differences at each visit for spirometric variables and at last visit for non-spirometric variables from the baseline between the two study groups by an independent-T test. As ancillary analysis, in order to evaluate the differences between the baseline (Visit 0) and the end of the study (Visit 5), in each arm a one way ANOVA was used for the lung function parameters and a paired-T-test was performed for all the other variables. P values less than 0.05 were considered statistically significant. 3. Results During the study 105 patients were enrolled, 51 pediatric (24 F, mean age ± SD 12.8 ± 3.1, FEV1 %, mean ± SD 98.13 ± 20.20) and 54 adult patients (26 F, mean age ± SD 27.66 ± 8.25, FEV1 %, mean ± SD 64.52 ± 18.65). The participant flow is shown in Fig. 2. Patients were enrolled from June 2010 to September 2011. The last follow-up was completed in September 2012.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
Fig. 2. Flow diagram of study population.
3.1. Baseline data The baseline demographic, clinical and functional characteristics of the two groups were separately analyzed. No significant differences were found between the GSH and the placebo arms of both groups (Table 1). 3.2. Pediatric group 3.2.1. Outcomes In the pediatric patients with CF, twelve month treatment with inhaled GSH did not achieve our predetermined primary outcome measure of 15% improvement in FEV1 when compared to placebo. Moreover, GSH therapy did not lead to any significant statistical difference in the FEV1 (Supplemental Table 2A), even when the pre-post differences at each visit from baseline values were compared between the two groups (Fig. 3A, B, Supplemental Table 2B). No statistically significant difference between GSH and placebo groups was found for the other spirometric and non-spirometric variables after 12 months of therapy. 3.2.2. Ancillary analysis The analysis of all the variables at baseline and at the end of the study in each single arm (Supplemental Table 3A, 3B)
showed the following significant changes: the distance walked in the 6MWT increased in patients treated with inhaled GSH (GSH p = 0.01; placebo p = 0.096); the BMI increased in both the GSH and the placebo groups (p = 0.008 and p = 0.005, respectively); the number of pulmonary exacerbations increased in the placebo (p = 0.01) but not in the GSH arm (p = 0.44) during the study period compared to the previous year. The number of exacerbations of each arm and the time to first exacerbation (mean ± SD GSH vs placebo 92.9 ± 85.8 vs 104.0 ± 93.7 p = 0.730) were not significantly different during the study. 3.3. Adult group 3.3.1. Outcomes In adult CF patients, the lung function, assessed by an increase from baseline of at least 15% of FEV1%, did not improve after 12 months of treatment with inhaled GSH when compared to placebo. Moreover, no significant difference in the FEV1 was registered between GSH and placebo through the follow-up visits (Supplemental Table 4A). However, the changes in percentage predicted FEV1 measured at each visit from baseline were slightly higher in the GSH group than in the placebo group, reaching the statistical significance after 3, 6 and 9 months of therapy
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
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Table 1 Characteristics of the study population at baseline (Intention to treat N = 105). Variables
Age Median[range] Mean ± SD Males % Mutation DELTAF508/DELTAF508% DELTAF508/Other% Other Sputum colonization Pseudomonas aeruginosa % Staphylococcus aureus % Streptococcus pneumoniae% Acinetobacter xiloisodans % Hemophilus influenza % Stenotrophomonas maltophilia % Candida species % Aspergillus fumigatus % Spirometry FEV1 L/s, mean ± SD FEV1 %, mean ± SD FVC L/s, mean ± SD FVC%, mean ± SD FEV1/FVC%, mean ± SD FEF25–75 L/s, mean ± SD FEF25–75 %, mean ± SD BMI Mean ± SD BODE index Mean ± SD CCIQ Mean ± SD Pulmonary exacerbations Number, mean ± SD Duration, mean ± SD Cycles antibiotics Number, mean ± SD Days requiring i.v. antibiotics Number mean ± SD CRP mg/dl mean ± SD H2O2 Serum (CARR U) a EBC (μM)
Pediatric group GSH arm (n = 27)
Pediatric group Placebo arm (n = 24)
13.25 [6.0–17.9] 13.37 ± 3.17
12.55 [7.4–16.2] 12.29 ± 3.0
14 (51.2%)
P
Adult group GSH arm (n = 31)
Adult group Placebo arm (n = 23)
P
0.225
26 [19–52] 28.90 ± 9.40
24 [17–41] 26 ± 6.21
0.097
13 (54.2%)
0.869
18 (58. 1%)
10 (43.5%)
0.289
7 (25.9%) 13 (48.1%) 7 (25.9%)
6 (25%) 6 (25%) 12 (50%)
0.149
7 (22.5%) 15 (48.4%) 9 (29.1%)
7 (30. 4%) 6 (26.1%) 10 (43. 5%)
0.388
8 (29.6%) 18 (66.7%) 1 (3.7%) 1 (3.7%) 7 (25.9%) 4 (14.8%) 13 (48.1%) 0 (0%)
4 (16.7%) 13 (54.2%) 1 (4.2%) 1 (4.2%) 10 (41.7%) 2 (8.3%) 9 (37.5%) 1 (4.2%)
0.276 0.361 0.932 0.932 0.234 0.473 0.361 0.284
19 (61.3%) 18 (58.1%) 0 (0%) 0 (0%) 6 (19.4%) 4 (12.9%) 2 (6.5%) 1 (3.2%)
12 (52.2%) 13 (56.5%) 1 (4.3%) 3 (13%) 6 (26.1%) 0 (0%) 3 (13%) 0 (0%)
0.503 0.910 0.241 0.039 0.556 0.073 0.409 0.385
2.38 ± 0.95 95.38 ± 22.12 3.01 ± 1.12 102.2 ± 16.86 79.00 ± 10.85 2.28 ± 1.26 73.29 ± 32.73
2.47 ± 0.94 101.22 ± 17.74 3.17 ± 1.16 110.27 ± 12.25 78.14 ± 10.45 2.28 ± 1.18 75.81 ± 30.62
0.730 0.307 0.608 0.059 0.775 0.987 0.779
2.3 ± 0.76 64.94 ± 17.10 3.37 ± 0.90 76.04 ± 5.00 70.50 ± 9.89 1.70 ± 1.12 38.58 ± 25.59
2.17 ± 0.91 63.96 ± 18.87 3.10 ± 0.96 76.88 ± 16.71 71.16 ± 12.53 1.66 ± 1.40 41.04 ± 30.21
0.472 0.853 0.302 0.846 0.835 0.918 0.755
19.19 ± 3.12
19.82 ± 2.62
0.437
22.41 ± 2.61
22.48 ± 3.28
0.937
0.85 ± 0.60
0.75 ± 0.44
0.499
1.25 ± 1.06
1.34 ± 1.02
0.756
31.05 ± 8.21
32.13 ± 10.43
0.701
35.66 ± 10.29
39.47 ± 15.69
0.313
1.37 ± 1.54 7.89 ± 13.65
0.96 ± 1.16 5.00 ± 10.22
0.292 0.402
1.29 ± 0.82 1.03 ± 3.35
1.47 ± 1.27 2.91 ± 7.83
0.535 0.236
3.19 ± 2.22
2.83 ± 2.08
0.563
1.67 ± 1.42
2.13 ± 2.05
0.629
6.44 ± 5.64
4.13 ± 3.48
0.094
2.06 ± 2.14
2.65 ± 2.88
0.416
0.43 ± 0.18
0.45 ± 0.37
0.884
1.25 ± 1.03
1.23 ± 1.28
0.964
304.27 ± 69.96 3.66 ± 0.84
299.14 ± 77.18 3.62 ± 0.99
0.986 0.941
356.36 ± 75.47 3.48 ± 0.91
408.00 ± 103.52 3.57 ± 1.05
0.102 0.797
0.388
FEV1 forced expiratory volume in one second, FVC forced vital capacity,FEF 25–75 forced expiratory flows at 25–75% vital capacity; BMI Body mass index; CCIQ cough measured by the Chronic Cough Impact Questionnaire; CRP serological C-reactive protein; H2O2 hydrogen peroxide; EBC exhaled breath condensate; SD standard deviation. a 1 CARR U = 0.08 mg/100 ml H202 (n.v. 250–300).
(Fig. 3C, D, Supplemental Table 4B). There were no significant differences in the other spirometric and non-spirometric variables between the GSH and the placebo groups. 3.3.2. Ancillary analysis The analysis of all the variables at baseline and at the end of the study in each single arm showed the following significant changes: a decrease in the percentage predicted FEV1 values (p = 0.002),even when expressed as liter absolute values (p = 0.046), in the placebo arm that did not occur in the GSH
arm (FEV1% p = 0.132, and FEV1L p = 0.121, respectively) (Supplemental Table 5A); a decrease of FEF25–75 in both arms when measured as absolute value in l/s (p = 0.018 and p = 0.014, respectively), and only in the placebo group when expressed as percentage predicted (Supplemental Table 5A); a decrease of BMI in the placebo but not in the GSH group (p = 0.040 and p = 0.802 respectively) (Supplemental Table 5B). The time to first exacerbation (mean ± SD GSH vs placebo 195.8 ± 174.3 vs 191.36 ± 123.12 p = 0.942) was not significantly different during the study between the two arms.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
Fig. 3. Changes from baseline of FEV1 at each study visit measured in children as liter with standard error (A) and percentage predicted with standard error (B); in adults as liter with standard error (C) and percentage predicted with standard error (D); in patients with FEV1% b 81% as liter with standard error (E) and percentage predicted with standard error (F); in patients with FEV1% N81% as liter with standard error (G) and percentage predicted with standard error (H). Dark gray: glutathione; light gray: placebo.
3.4. Adults and children with FEV1 below 81% We mixed the data for adults and children determining the median baseline FEV1% for the group (Median 81.2%). We analyzed separately those with FEV1% above and below this median value. Only in patients with a FEV1% below the median we found a significant change of FEV1 (expressed as either percentage and liter) measured at visits 2, 3 and 4 from baseline in the GSH group compared to placebo (Fig. 3E, F, G, H). The exploratory analysis of the non-spirometric variables for the group of patients with FEV1% below 81% was provided in Supplemental Table 6. The time to first exacerbation was not
significantly different during the study between the two arms (mean ± SD GSH vs placebo 190.9 ± 122.24 vs 162.6 ± 177.07 p = 0.659). 3.5. Safety None of the enrolled patients showed a decrease in FEV1% values compared to baseline more than 15% after the GSH inhalation at the dosage of 10 mg/kg (Supplemental Table 7). Adverse events were reported as single events in some patients and none of them led to study drug discontinuation (Table 2a, b). No death occurred.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx Table 2a Summary of adverse events. Pediatric group (Intention to treat N = 51).
Abdominal pain Distal Intestinal Obstruction Hemoptysis Epistaxis Headache Chest Pain Rhinitis Pancreatitis Constipation Pityriasis Impaired glucose tolerance
Glutathione N = 27
Placebo N = 24
4 (14.8%) 1 (3.7%) 3 (11.1%) 0 (0%) 1 (3.7%) 1 (3.7%) 0 (0%) 0 (0%) 1 (3.7%) 1 (3.7%) 1 (3.7%)
2 (8.3 %) 0 (0%) 2 (8.3%) 2 (8.3%) 0 (0%) 1 (4.2%) 1 (4.2%) 1 (4.2%) 0 (0%) 0 (0%) 0 (0%)
4. Discussion In the pediatric group, a 12 months treatment with inhaled GSH did not lead to any significant increase from the baseline of the FEV1, expressed both in percentage and as absolute values, when compared with placebo. In adult patients, three months therapy with inhaled GSH resulted in a statistically significant improvement in percentage predicted FEV1, measured as a pre-post difference from baseline values, when compared to placebo, that persisted at 6 and 9 but not at 12 months. A reduced compliance to therapy of adult patients could explain the decrease in FEV1 values registered in the last visit. These results are in accordance with those reported by Griese et al. [14], who showed a significant increase of FEV1 absolute values from baseline after three months of GSH therapy during a double-blind, placebo-controlled, six-month study. The demographic and clinical characteristics of the CF patients enrolled by Griese were similar to our adult patients (mean age and FEV1 values). Furthermore, the regimen of GSH therapy was the same (600 mg twice daily). FEV1 still represents the most important single predictive factor of survival in CF [24,25]. In patients with CF and bacterial chronic infection an annual decline of lung function has been reported [26]. In a recent study, an age-dependent annual decline in percentage predicted FEV1 was assumed to be between 1% and 3% [27]. We confirmed these data, showing a significant decrease in FEV1 in the adult placebo group during the study period that did not occur in the GSH group. A decline of functional parameters in the placebo group, although not significant, was also observed by Griese [14]. GSH therapy did not improve the lung function in the pediatric population, differently from the results reported by Griese, who registered the best improvements in functional parameters in the pediatric subgroup. Several limitations of our trial could explain Table 2b Summary of adverse events. Adult group (Intention to treat N = 54).
Hemoptysis Distal intestinal obstruction Dehydration
Glutathione N = 31
Placebo N = 23
2 (6%) 2 (6%) 0 (0%)
2 (8%) 0 (0%) 1 (4%)
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these results. Differently from Griese, we administered the inhaled GSH according to body weight (10 mg/kg), probably thus reducing its therapeutic effects in children. Moreover, as inclusion criteria no upper limit was defined for FEV1 value, so that most enrolled children had a normal spirometry with no room of improvement. As a proof of this, when we mixed the results of adult and children sharing a FEV1% below 81%, we confirmed a significant improvement of the FEV1 expressed both as liter and percentage in the GSH arm compared to the placebo. Nevertheless, pediatric patients that assumed GSH showed a significant improvement of the distance walked in 6 min that is considered a marker of disease-severity according to previous data [28]. As expected, BMI increased in both pediatric groups; otherwise, in adults it decreased in the placebo but not in the GSH arm. Our results confirmed the presence of a marked oxidative stress by serum and exhaled H202 in all enrolled patients at baseline; however, no change in H2O2 and CRP was detected in the GSH group. We speculate that recommended treatment dosage during the study was not adequate to significantly modify the reduced endogenous GSH levels. Based on the results of this clinical trial, the treatment with inhaled GSH is assumed to lead an almost immediate improvement in the FEV1 in patients with moderate lung disease, a stabilization of BMI in adult population and an improvement of 6MWT in children. In a prospective observational study that is currently ongoing in our center we have selected Lung Clearance Index [29] as a more sensitive alternative to spirometry for detecting efficacy of GSH therapy also in patients with CF and mild lung disease. Registration number is NCT01450267. The full trial protocol can be accessed at www.clinicaltrials. gov. The study has been supported by AIFA. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jcf.2014.09.014. Financial/nonfinancial disclosures Author declares no conflicts of interest. Acknowledgments We are grateful to Rita MV Nobili from IRCSS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena-Milano for the permission to use Italian translation of CFQ-R from 6 to 13 years (Copyright 2009). References [1] Kelly FJ. Glutathione: in defence of the lung. Food Chem Toxicol 1999; 37(9–10):963–6. [2] Morris D, Khurasany M, Nguyen T, Kim J, Guilford F, Mehta R, et al. Glutathione and infection. Biochim Biophys Acta 2013;1830(5):3329–49. [3] Roum JH, Buhl R, McElvaney NG, Borok Z, Crystal RG. Systemic deficiency of glutathione in cystic fibrosis. J Appl Physiol 1993;75(6): 2419–24. [4] Linsdell P, Hanrahan JW. Glutathione permeability of CFTR. Am J Physiol 1998;275(1):C323–6.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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[5] Velsor LW, Kariya C, Kachadourian R, Day BJ. Mitochondrial oxidative stress in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice. Am J Respir Cell Mol Biol 2006;35(5):579–86. [6] McGrath LT, Mallon P, Dowey L, Silke B, McClean E, McDonnell M, et al. Oxidative stress during acute respiratory exacerbations in cystic fibrosis. Thorax 1999;54(6):518–23. [7] Franco R, Panayiotidis MI, Cidlowski JA. Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation. J Biol Chem 2007;282(42):30452–65. [8] Hudson VM. New insights into the pathogenesis of cystic fibrosis: pivotal role of glutathione system dysfunction and implications for therapy. Treat Respir Med 2004;3(6):353–63. [9] Hudson VM. Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione transport caused by CFTR mutation. Free Radic Biol Med 2001;30(12):1440–61. [10] Roum JH, Borok Z, McElvaney NG, Grimes GJ, Bokser AD, Buhl R, et al. Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis. J Appl Physiol 1999;87(1): 438–43. [11] Griese M, Ramakers J, Krasselt A, Starosta V, Van Koningsbruggen S, Fischer R, et al. Improvement of alveolar glutathione and lung function but not oxidative state in cystic fibrosis. Am J Respir Crit Care Med 2004; 169(7):822–8. [12] Bishop C, Hudson VM, Hilton SC, Wilde C. A pilot study of the effect of inhaled buffered reduced glutathione on the clinical status of patients with cystic fibrosis. Chest 2005;127(1):308–17. [13] Visca A, Bishop CT, Hilton SC, Hudson VM. Improvement in clinical markers in CF patients using a reduced glutathione regimen: an uncontrolled, observational study. J Cyst Fibros 2008;7(5):433–6. [14] Griese M, Kappler M, Eismann C, Ballmann M, Junge S, Rietschel E, et al. Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial. Am J Respir Crit Care Med 2013;188(1):83–9. [15] Standardization of Spirometry. Update. American Thoracic Society. Am J Respir Crit Care Med 1995;152(3):1107–36. [16] A.T.S. Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories. ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med 2002;166(1):111–7. [17] Mahler D, Wells C. Evaluation of clinical methods for rating dyspnoea. Chest 1988;93:580–6.
[18] Baiardini I, Braido F, Fassio O, Tarantini F, Pasquali M, Tarchino F, et al. A new tool to assess and monitor the burden of chronic cough on quality of life: Chronic Cough Impact Questionnaire. Allergy 2005;60(4):482–8. [19] Monti F, Lupi F, Gobbi F, Agostini F, Miano A, Gee L, et al. Validation of the Italian version of the Cystic Fibrosis Quality of Life Questionnaire (CFQoL), a disease specific measure for adults and adolescent with Cystic Fibrosis. J Cyst Fibros 2008;7(2):116–22. [20] Flume PA, Mogayzel Jr PJ, Robinson KA, Goss CH, Rosenblatt RL, Kuhn RJ, et al. Clinical Practice Guidelines for Pulmonary Therapies Committee. Cystic fibrosis pulmonary guidelines: treatment of pulmonary exacerbations. Am J Respir Crit Care Med 2009;180(9):802–8. [21] Fuchs HJ, Borowitz DS, Christiansen DH, Morris EM, Nash ML, Ramsey BW, et al. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis: the Pulmozyme Study Group. N Engl J Med 1994;331:637–42. [22] Horváth I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, et al. ATS/ ERS Task Force on Exhaled Breath Condensate. Exhaled breath condensate: methodological recommendations and unresolved questions. Eur Respir J 2005;26(3):523–48. [23] Cesarone MR, Belcaro G, Carratelli M, Cornelli U, De Sanctis MT, Incandela L, et al. A simple test to monitor oxidative stress. Int Angiol 1999;18(2):127–30. [24] Kerem E, Reisman J, Corey M, Canny GJ, Levison H. Prediction of mortality in patients with cystic fibrosis. N Engl J Med 1992;326:1187–91. [25] Liou TG, Adler FR, Fitzsimmons SC, Cahill BC, Hibbs JR, Marshall BC. Predictive 5-year survivorship model of cystic fibrosis. Am J Epidemiol 2001;153:345–52. [26] Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry 2006 Annual Data Report. Bethesda, Maryland: Cystic Fibrosis Foundation; 2007. [27] Liou TG, Elkin EP, Pasta DJ, Jacobs JR, Konstan MW, Morgan WJ, et al. Year-to-year changes in lung function in individuals with cystic fibrosis. J Cyst Fibros 2010;9:250–6. [28] Stollar F, Rodrigues JC, Cunha MT, Leone C, Adde FV. Six minute walk test Z score: correlations with cystic fibrosis severity markers. J Cyst Fibros 2012;11(3):253–6. [29] Kent L, Reix P, Innes JA, Zielen S, Le Bourgeois M, Braggion C, et al. Lung clearance index: evidence for use in clinical trials in cystic fibrosis. J Cyst Fibros 2014;13(2):123–38.
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Journal of Cystic Fibrosis xx (2014) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis☆,☆☆ C. Calabrese a,1 , A. Tosco b,1 , P. Abete b , V. Carnovale b , C. Basile b , A. Magliocca b , S. Quattrucci c , S. De Sanctis c , F. Alatri c , G. Mazzarella a , L. De Pietro a , C. Turino a , E. Melillo d , P. Buonpensiero b , A. Di Pasqua b , V. Raia b,⁎ b
a Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Italy Department of Translational Medical Sciences, Cystic Fibrosis Center, University "Federico II" of Naples, Italy c Department of Pediatrics, Cystic Fibrosis Center, Sapienza University of Rome, Italy d Pediatric Pneumology, Santobono-Pausilipon-Annunziata Children's Hospital, Naples, Italy
Received 25 May 2014; revised 28 September 2014; accepted 30 September 2014 Available online xxxx
Abstract Background: In cystic fibrosis (CF) the defective CF transmembrane conductance regulator protein may be responsible for the impaired transport of glutathione (GSH), the first line defense of the lung against oxidative stress. The aim of this single-blind, randomized, placebo-controlled trial was to evaluate the effect of inhaled GSH in patients with CF. Methods: 54 adult and 51 pediatric patients were randomized to receive inhaled GSH or placebo twice daily for 12 months. Results: Twelve month treatment with inhaled GSH did not achieve our predetermined primary outcome measure of 15% improvement in FEV1%. Only in patients with moderate lung disease, 3, 6 and 9 months therapy with GSH resulted in a statistically significant increase of FEV1 values from the baseline. Moreover GSH therapy improved 6-minute walking test in pediatric population. GSH was well tolerated by all patients. Conclusions: Inhaled GSH has slight positive effects in CF patients with moderate lung disease warranting further study. Trial registry: ClinicalTrials.gov; No.: NCT01450267; URL: www.clinicaltrialsgov. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Glutathione; Therapy
Abbreviations: CF, cystic fibrosis; GSH, glutathione; CFTR, cystic fibrosis transmembrane regulator; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; FEF25–75, flow at the mid-portion of forced vital capacity; 6MWT, six minute walking test; BMI, body-mass index; CCIQ, Chronic Cough Impact Questionnaire; CFQoL, Cystic Fibrosis Quality of Life Questionnaire; CRP, C-reactive protein; H2O2, hydrogen peroxide; EBC, exhaled breath condensate. ☆ Financial disclosure: Authors declare no conflicts of interest. ☆☆ Funding: The study was financed by AIFA (Italian Agency of Drugs) (FARM7K7XZB). ⁎ Corresponding author at: Department of Translational Medical Sciences University “Federico II” of Naples, Via S Pansini 5, 80131 Naples, Italy. Tel.: +39 0817463273; fax: + 39 0815463393. E-mail address: [email protected] (V. Raia). 1 Co-first author.
1. Introduction Glutathione (GSH) represents the first-line defense of the lung against oxidative stress-induced damage [1] enhancing the functional activity of different immune cells [2]. GSH is normally found in the epithelial lining fluid of lower airways at very high concentration. However, a depletion of its levels has been observed in the airways of patients with Cystic Fibrosis (CF) [3]. In vitro studies showed that CFTR protein plays a pivotal role in transmembrane glutathione transport [4]. In animal models, as CFTR KO mice, a strong evidence supports the role of CFTR on airway fluid GSH levels [5]. In patients with CF, the reduced
http://dx.doi.org/10.1016/j.jcf.2014.09.014 1569-1993© 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
transport of GSH induced by the defective CFTR protein prevents the counterbalance of excessive oxidative stress molecules released by inflammatory cells and bacteria into the airways [6]. The persistent GSH deficiency, reported also in the neutrophils of patients with CF [7], could explain the paradox of an overactive immune system that otherwise is ineffective in eradicating bacteria [8,9]. Based on these evidences, a role of GSH has been suggested as a new therapeutic option in CF. Several studies have investigated the potential therapeutic role of inhaled GSH in patients with CF. Three short-term clinical trials, of which only one was placebo-controlled, have shown the tolerability and efficacy of inhaled GSH on pulmonary function in these subjects [10–13]. A more recent [14] six-month randomized double-blind placebo-controlled study failed to demonstrate the efficacy of inhaled GSH therapy on lung function, pulmonary exacerbations, as well as on surrogate inflammation and oxidative biomarker. Thus, given the high expectations placed on this molecule in CF community, we investigated whether a longer term treatment with inhaled GSH could be as effective for controlling lung function and other clinical outcomes in both pediatric and adult CF patients. 2. Materials and methods 2.1. Trial design This was a prospective, single-blind, randomized, placebocontrolled trial comparing 12 months of treatment with inhaled GSH versus placebo in pediatric and adult patients affected by CF. All eligible patients were divided into two groups: 1) Pediatric Group: age ≥ 6 to b 18 years; 2) Adult Group: age ≥ 18 years. Patients were consecutively enrolled at the Pediatric and Adult Units of Campania Cystic Fibrosis Regional Centre, University
Federico II of Naples, and at Lazio Cystic Fibrosis Regional Centre, University La Sapienza Policlinico Umberto I. The study timeline has been shown in Fig. 1. At the enrollment (visit 0), patients of both groups were randomized to GSH or placebo arm. Clinical assessments were scheduled one, three, six, nine, and twelve months after the enrollment. At each visit, all patients underwent a complete medical examination, which included lung function, height and bodyweight; moreover sputum samples or deep pharyngeal suction (in 9 children) were collected for microbiological analysis. Spirometry was carried out in the morning (8.00 a.m. to 10.00 a.m.). All measures were made by a single operator using a single calibrated, dry wedge bellows spirometer. FEV1 and FVC were measured, using the best of 3 forced maneuvers fulfilling the American Thoracic Society standard recommendations [15]. Forced expiratory volume 1 s (FEV1) values and the % of the predicted values (Zapletal) were recorded, as well as forced expiratory flow at the mid-portion of forced vital capacity (FEF25–75) % of the predicted values. At the enrollment as well as at the end of the study the following parameters were recorded: exercise capacity by six-minute walking test (6MWT) [16], body mass index (BMI), BODE index [17], cough measured by Chronic Cough Impact Questionnaire (CCIQ) [18], and quality of life assessed by Cystic Fibrosis Quality of Life Questionnaire (CFQoL) [19]. The number of acute pulmonary exacerbations, defined following the criteria used by Fuchs [20,21], was evaluated during the study and in the year before the enrollment. As a surrogate biomarker of inflammation, serological C-reactive protein (CRP) was measured; in a subgroup of patients, hydrogen peroxide (H2O2) was determined by the Fenton's reaction [22,23] in both exhaled breath condensate (EBC) and serum as a marker of oxidative stress. Concomitant therapy was regularly recorded at each visit, as well as any change in antibiotic therapy.
Fig. 1. Study timeline. Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
2.2. Participants The inclusion criteria were: 1) age ≥ 6 years; 2) confirmed diagnosis of CF by sweat chloride test over 60 mmol/L and/or genetic analysis, and 3) stable clinical condition defined as the absence of substantial, acute clinical problems potentially affecting the outcome measures included in the protocol. Exclusion criteria were: 1) pregnancy and fertile women taking oral contraceptives; 2) cigarette smoking; 3) positive culture for Burkholderia cepacia; 4) history of hemoptysis and pneumothorax; 5) FEV1 ≤ 40% of the predicted value; and 6) hyperresponsiveness to GSH inhalation test defined as a decrease in FEV1 greater than 15% compared to basal value at a dynamic spirometry performed 10 and 60 min after the inhalation of GSH (dosage of 10 mg/kg body weight—max dose 600 mg). During the study, concomitant therapeutic approaches were continued by enrolled patients according to standardized guidelines. Thiolic mucolytic drugs, anti-oxidant drugs and Mesna (2-mercaptoethane sulfonate) were disallowed. Vitamins supplementation was not modified during the study. Chronic concomitant medications are listed in Supplemental Table 1 A–B. The study was conducted in accordance with the amended Declaration of Helsinki. Italian Agency of Drugs and Independent Local Ethics Committees of the involved centers approved the protocol. All subjects provided written informed consent before treatment. 2.3. Interventions The GSH arm received inhaled GSH formulated as vials containing lyophilized powder of reduced glutathione to be reconstituted with 8 ml of water, in order to achieve a tonicity of 435 milliosmol/kg, at the dose of 10 mg/kg of body weight (max dose 600 mg) twice daily for 12 months. The placebo group received inhaled 0.9% sodium chloride sterile solution (physiological solution) twice daily for 12 months. The administrations of GSH solution and placebo were performed through a compressed-air nebulizer (PARI turbo boy) or an equivalent device delivering aerodynamic particles of 3.5–5.5 micrometer diameter. The treatment and the placebo were kindly supplied by the company ®, Biomedica Foscama, Ferentino, Italy. The compliance with GSH or placebo was checked by the Investigator and recorded in CRF during the routine clinical controls on the basis of returned recipients. Percent level of compliance was calculated as follows: % compliance = number of vials or ampoules taken by the patient / number of vials or ampoules that should have been taken by the patient × 100. The minimum level of allowed compliance was 80%. 2.4. Outcomes Our primary outcome was a 15% change from baseline in percent predicted in FEV1, after 12 months of therapy with inhaled GSH compared to placebo. Secondary outcomes reported in the study included: change from baseline in GSH and placebo groups for FVC, FEF25–75,
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6MWT, BMI (kg/m2), BODE index, CCIQ, CFQoL, number of pulmonary exacerbations, antibiotic courses and number of days requiring i.v. antibiotics administration compared to the previous year. At the enrollment and at the end of the trial CRP was measured; in a subgroup of patients, H2O2 was evaluated in both EBC and serum [21,22]. 2.5. Sample size The sample size (N = 150) required for the statistical analysis was subsequently reduced to 100 patients, considering pediatric and adult patients as separate groups, on the basis of a target improvement of 15% in FEV1, from 50% to 65% of the predicted value, with a standard deviation of 19%, an alpha (type I) error of 5% and a beta (type II) error of 20% (power 80%). 2.6. Randomization and blinding All the participants were assigned to GSH or placebo groups according to a blind, computer-generated randomization list performed by a person not otherwise involved in the study. Each patient was assigned to a unique number related to his/her corresponding treatment. The treatment was single-blinded because the GSH has a distinct taste and smell that is difficult to reproduce as placebo. Patients of the two different groups were examined in different days. 2.7. Statistic methods Data were analyzed using the SPSS 13.0 statistic package. Baseline characteristics of the sample were expressed as mean ± standard deviation (SD). To evaluate differences between GSH and placebo arms during the study, a two way analysis of variance (ANOVA) for repeated measures was applied for the following variables: FEV1, FVC, FEF25–75 as absolute values and % of predicted values. Moreover, for all variables we compared the pre-post differences at each visit for spirometric variables and at last visit for non-spirometric variables from the baseline between the two study groups by an independent-T test. As ancillary analysis, in order to evaluate the differences between the baseline (Visit 0) and the end of the study (Visit 5), in each arm a one way ANOVA was used for the lung function parameters and a paired-T-test was performed for all the other variables. P values less than 0.05 were considered statistically significant. 3. Results During the study 105 patients were enrolled, 51 pediatric (24 F, mean age ± SD 12.8 ± 3.1, FEV1 %, mean ± SD 98.13 ± 20.20) and 54 adult patients (26 F, mean age ± SD 27.66 ± 8.25, FEV1 %, mean ± SD 64.52 ± 18.65). The participant flow is shown in Fig. 2. Patients were enrolled from June 2010 to September 2011. The last follow-up was completed in September 2012.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
Fig. 2. Flow diagram of study population.
3.1. Baseline data The baseline demographic, clinical and functional characteristics of the two groups were separately analyzed. No significant differences were found between the GSH and the placebo arms of both groups (Table 1). 3.2. Pediatric group 3.2.1. Outcomes In the pediatric patients with CF, twelve month treatment with inhaled GSH did not achieve our predetermined primary outcome measure of 15% improvement in FEV1 when compared to placebo. Moreover, GSH therapy did not lead to any significant statistical difference in the FEV1 (Supplemental Table 2A), even when the pre-post differences at each visit from baseline values were compared between the two groups (Fig. 3A, B, Supplemental Table 2B). No statistically significant difference between GSH and placebo groups was found for the other spirometric and non-spirometric variables after 12 months of therapy. 3.2.2. Ancillary analysis The analysis of all the variables at baseline and at the end of the study in each single arm (Supplemental Table 3A, 3B)
showed the following significant changes: the distance walked in the 6MWT increased in patients treated with inhaled GSH (GSH p = 0.01; placebo p = 0.096); the BMI increased in both the GSH and the placebo groups (p = 0.008 and p = 0.005, respectively); the number of pulmonary exacerbations increased in the placebo (p = 0.01) but not in the GSH arm (p = 0.44) during the study period compared to the previous year. The number of exacerbations of each arm and the time to first exacerbation (mean ± SD GSH vs placebo 92.9 ± 85.8 vs 104.0 ± 93.7 p = 0.730) were not significantly different during the study. 3.3. Adult group 3.3.1. Outcomes In adult CF patients, the lung function, assessed by an increase from baseline of at least 15% of FEV1%, did not improve after 12 months of treatment with inhaled GSH when compared to placebo. Moreover, no significant difference in the FEV1 was registered between GSH and placebo through the follow-up visits (Supplemental Table 4A). However, the changes in percentage predicted FEV1 measured at each visit from baseline were slightly higher in the GSH group than in the placebo group, reaching the statistical significance after 3, 6 and 9 months of therapy
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
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Table 1 Characteristics of the study population at baseline (Intention to treat N = 105). Variables
Age Median[range] Mean ± SD Males % Mutation DELTAF508/DELTAF508% DELTAF508/Other% Other Sputum colonization Pseudomonas aeruginosa % Staphylococcus aureus % Streptococcus pneumoniae% Acinetobacter xiloisodans % Hemophilus influenza % Stenotrophomonas maltophilia % Candida species % Aspergillus fumigatus % Spirometry FEV1 L/s, mean ± SD FEV1 %, mean ± SD FVC L/s, mean ± SD FVC%, mean ± SD FEV1/FVC%, mean ± SD FEF25–75 L/s, mean ± SD FEF25–75 %, mean ± SD BMI Mean ± SD BODE index Mean ± SD CCIQ Mean ± SD Pulmonary exacerbations Number, mean ± SD Duration, mean ± SD Cycles antibiotics Number, mean ± SD Days requiring i.v. antibiotics Number mean ± SD CRP mg/dl mean ± SD H2O2 Serum (CARR U) a EBC (μM)
Pediatric group GSH arm (n = 27)
Pediatric group Placebo arm (n = 24)
13.25 [6.0–17.9] 13.37 ± 3.17
12.55 [7.4–16.2] 12.29 ± 3.0
14 (51.2%)
P
Adult group GSH arm (n = 31)
Adult group Placebo arm (n = 23)
P
0.225
26 [19–52] 28.90 ± 9.40
24 [17–41] 26 ± 6.21
0.097
13 (54.2%)
0.869
18 (58. 1%)
10 (43.5%)
0.289
7 (25.9%) 13 (48.1%) 7 (25.9%)
6 (25%) 6 (25%) 12 (50%)
0.149
7 (22.5%) 15 (48.4%) 9 (29.1%)
7 (30. 4%) 6 (26.1%) 10 (43. 5%)
0.388
8 (29.6%) 18 (66.7%) 1 (3.7%) 1 (3.7%) 7 (25.9%) 4 (14.8%) 13 (48.1%) 0 (0%)
4 (16.7%) 13 (54.2%) 1 (4.2%) 1 (4.2%) 10 (41.7%) 2 (8.3%) 9 (37.5%) 1 (4.2%)
0.276 0.361 0.932 0.932 0.234 0.473 0.361 0.284
19 (61.3%) 18 (58.1%) 0 (0%) 0 (0%) 6 (19.4%) 4 (12.9%) 2 (6.5%) 1 (3.2%)
12 (52.2%) 13 (56.5%) 1 (4.3%) 3 (13%) 6 (26.1%) 0 (0%) 3 (13%) 0 (0%)
0.503 0.910 0.241 0.039 0.556 0.073 0.409 0.385
2.38 ± 0.95 95.38 ± 22.12 3.01 ± 1.12 102.2 ± 16.86 79.00 ± 10.85 2.28 ± 1.26 73.29 ± 32.73
2.47 ± 0.94 101.22 ± 17.74 3.17 ± 1.16 110.27 ± 12.25 78.14 ± 10.45 2.28 ± 1.18 75.81 ± 30.62
0.730 0.307 0.608 0.059 0.775 0.987 0.779
2.3 ± 0.76 64.94 ± 17.10 3.37 ± 0.90 76.04 ± 5.00 70.50 ± 9.89 1.70 ± 1.12 38.58 ± 25.59
2.17 ± 0.91 63.96 ± 18.87 3.10 ± 0.96 76.88 ± 16.71 71.16 ± 12.53 1.66 ± 1.40 41.04 ± 30.21
0.472 0.853 0.302 0.846 0.835 0.918 0.755
19.19 ± 3.12
19.82 ± 2.62
0.437
22.41 ± 2.61
22.48 ± 3.28
0.937
0.85 ± 0.60
0.75 ± 0.44
0.499
1.25 ± 1.06
1.34 ± 1.02
0.756
31.05 ± 8.21
32.13 ± 10.43
0.701
35.66 ± 10.29
39.47 ± 15.69
0.313
1.37 ± 1.54 7.89 ± 13.65
0.96 ± 1.16 5.00 ± 10.22
0.292 0.402
1.29 ± 0.82 1.03 ± 3.35
1.47 ± 1.27 2.91 ± 7.83
0.535 0.236
3.19 ± 2.22
2.83 ± 2.08
0.563
1.67 ± 1.42
2.13 ± 2.05
0.629
6.44 ± 5.64
4.13 ± 3.48
0.094
2.06 ± 2.14
2.65 ± 2.88
0.416
0.43 ± 0.18
0.45 ± 0.37
0.884
1.25 ± 1.03
1.23 ± 1.28
0.964
304.27 ± 69.96 3.66 ± 0.84
299.14 ± 77.18 3.62 ± 0.99
0.986 0.941
356.36 ± 75.47 3.48 ± 0.91
408.00 ± 103.52 3.57 ± 1.05
0.102 0.797
0.388
FEV1 forced expiratory volume in one second, FVC forced vital capacity,FEF 25–75 forced expiratory flows at 25–75% vital capacity; BMI Body mass index; CCIQ cough measured by the Chronic Cough Impact Questionnaire; CRP serological C-reactive protein; H2O2 hydrogen peroxide; EBC exhaled breath condensate; SD standard deviation. a 1 CARR U = 0.08 mg/100 ml H202 (n.v. 250–300).
(Fig. 3C, D, Supplemental Table 4B). There were no significant differences in the other spirometric and non-spirometric variables between the GSH and the placebo groups. 3.3.2. Ancillary analysis The analysis of all the variables at baseline and at the end of the study in each single arm showed the following significant changes: a decrease in the percentage predicted FEV1 values (p = 0.002),even when expressed as liter absolute values (p = 0.046), in the placebo arm that did not occur in the GSH
arm (FEV1% p = 0.132, and FEV1L p = 0.121, respectively) (Supplemental Table 5A); a decrease of FEF25–75 in both arms when measured as absolute value in l/s (p = 0.018 and p = 0.014, respectively), and only in the placebo group when expressed as percentage predicted (Supplemental Table 5A); a decrease of BMI in the placebo but not in the GSH group (p = 0.040 and p = 0.802 respectively) (Supplemental Table 5B). The time to first exacerbation (mean ± SD GSH vs placebo 195.8 ± 174.3 vs 191.36 ± 123.12 p = 0.942) was not significantly different during the study between the two arms.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
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C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx
Fig. 3. Changes from baseline of FEV1 at each study visit measured in children as liter with standard error (A) and percentage predicted with standard error (B); in adults as liter with standard error (C) and percentage predicted with standard error (D); in patients with FEV1% b 81% as liter with standard error (E) and percentage predicted with standard error (F); in patients with FEV1% N81% as liter with standard error (G) and percentage predicted with standard error (H). Dark gray: glutathione; light gray: placebo.
3.4. Adults and children with FEV1 below 81% We mixed the data for adults and children determining the median baseline FEV1% for the group (Median 81.2%). We analyzed separately those with FEV1% above and below this median value. Only in patients with a FEV1% below the median we found a significant change of FEV1 (expressed as either percentage and liter) measured at visits 2, 3 and 4 from baseline in the GSH group compared to placebo (Fig. 3E, F, G, H). The exploratory analysis of the non-spirometric variables for the group of patients with FEV1% below 81% was provided in Supplemental Table 6. The time to first exacerbation was not
significantly different during the study between the two arms (mean ± SD GSH vs placebo 190.9 ± 122.24 vs 162.6 ± 177.07 p = 0.659). 3.5. Safety None of the enrolled patients showed a decrease in FEV1% values compared to baseline more than 15% after the GSH inhalation at the dosage of 10 mg/kg (Supplemental Table 7). Adverse events were reported as single events in some patients and none of them led to study drug discontinuation (Table 2a, b). No death occurred.
Please cite this article as: Calabrese C, et al, Randomized, single blind, controlled trial of inhaled glutathione vs placebo in patients with cystic fibrosis, J Cyst Fibros (2014), http://dx.doi.org/10.1016/j.jcf.2014.09.014
C. Calabrese et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx Table 2a Summary of adverse events. Pediatric group (Intention to treat N = 51).
Abdominal pain Distal Intestinal Obstruction Hemoptysis Epistaxis Headache Chest Pain Rhinitis Pancreatitis Constipation Pityriasis Impaired glucose tolerance
Glutathione N = 27
Placebo N = 24
4 (14.8%) 1 (3.7%) 3 (11.1%) 0 (0%) 1 (3.7%) 1 (3.7%) 0 (0%) 0 (0%) 1 (3.7%) 1 (3.7%) 1 (3.7%)
2 (8.3 %) 0 (0%) 2 (8.3%) 2 (8.3%) 0 (0%) 1 (4.2%) 1 (4.2%) 1 (4.2%) 0 (0%) 0 (0%) 0 (0%)
4. Discussion In the pediatric group, a 12 months treatment with inhaled GSH did not lead to any significant increase from the baseline of the FEV1, expressed both in percentage and as absolute values, when compared with placebo. In adult patients, three months therapy with inhaled GSH resulted in a statistically significant improvement in percentage predicted FEV1, measured as a pre-post difference from baseline values, when compared to placebo, that persisted at 6 and 9 but not at 12 months. A reduced compliance to therapy of adult patients could explain the decrease in FEV1 values registered in the last visit. These results are in accordance with those reported by Griese et al. [14], who showed a significant increase of FEV1 absolute values from baseline after three months of GSH therapy during a double-blind, placebo-controlled, six-month study. The demographic and clinical characteristics of the CF patients enrolled by Griese were similar to our adult patients (mean age and FEV1 values). Furthermore, the regimen of GSH therapy was the same (600 mg twice daily). FEV1 still represents the most important single predictive factor of survival in CF [24,25]. In patients with CF and bacterial chronic infection an annual decline of lung function has been reported [26]. In a recent study, an age-dependent annual decline in percentage predicted FEV1 was assumed to be between 1% and 3% [27]. We confirmed these data, showing a significant decrease in FEV1 in the adult placebo group during the study period that did not occur in the GSH group. A decline of functional parameters in the placebo group, although not significant, was also observed by Griese [14]. GSH therapy did not improve the lung function in the pediatric population, differently from the results reported by Griese, who registered the best improvements in functional parameters in the pediatric subgroup. Several limitations of our trial could explain Table 2b Summary of adverse events. Adult group (Intention to treat N = 54).
Hemoptysis Distal intestinal obstruction Dehydration
Glutathione N = 31
Placebo N = 23
2 (6%) 2 (6%) 0 (0%)
2 (8%) 0 (0%) 1 (4%)
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these results. Differently from Griese, we administered the inhaled GSH according to body weight (10 mg/kg), probably thus reducing its therapeutic effects in children. Moreover, as inclusion criteria no upper limit was defined for FEV1 value, so that most enrolled children had a normal spirometry with no room of improvement. As a proof of this, when we mixed the results of adult and children sharing a FEV1% below 81%, we confirmed a significant improvement of the FEV1 expressed both as liter and percentage in the GSH arm compared to the placebo. Nevertheless, pediatric patients that assumed GSH showed a significant improvement of the distance walked in 6 min that is considered a marker of disease-severity according to previous data [28]. As expected, BMI increased in both pediatric groups; otherwise, in adults it decreased in the placebo but not in the GSH arm. Our results confirmed the presence of a marked oxidative stress by serum and exhaled H202 in all enrolled patients at baseline; however, no change in H2O2 and CRP was detected in the GSH group. We speculate that recommended treatment dosage during the study was not adequate to significantly modify the reduced endogenous GSH levels. Based on the results of this clinical trial, the treatment with inhaled GSH is assumed to lead an almost immediate improvement in the FEV1 in patients with moderate lung disease, a stabilization of BMI in adult population and an improvement of 6MWT in children. In a prospective observational study that is currently ongoing in our center we have selected Lung Clearance Index [29] as a more sensitive alternative to spirometry for detecting efficacy of GSH therapy also in patients with CF and mild lung disease. Registration number is NCT01450267. The full trial protocol can be accessed at www.clinicaltrials. gov. The study has been supported by AIFA. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jcf.2014.09.014. Financial/nonfinancial disclosures Author declares no conflicts of interest. Acknowledgments We are grateful to Rita MV Nobili from IRCSS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena-Milano for the permission to use Italian translation of CFQ-R from 6 to 13 years (Copyright 2009). References [1] Kelly FJ. Glutathione: in defence of the lung. Food Chem Toxicol 1999; 37(9–10):963–6. [2] Morris D, Khurasany M, Nguyen T, Kim J, Guilford F, Mehta R, et al. Glutathione and infection. Biochim Biophys Acta 2013;1830(5):3329–49. [3] Roum JH, Buhl R, McElvaney NG, Borok Z, Crystal RG. Systemic deficiency of glutathione in cystic fibrosis. J Appl Physiol 1993;75(6): 2419–24. [4] Linsdell P, Hanrahan JW. Glutathione permeability of CFTR. Am J Physiol 1998;275(1):C323–6.
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