Abstract
Chronic neutrophilic inflammation leads to oxidative damage, which may play an important role in the pathogenesis of cystic fibrosis lung disease. Bronchoalveolar lavage levels of the antioxidant glutathione are diminished in patients with cystic fibrosis. Here we evaluated the effects of glutathione aerosol on lower airway glutathione levels, lung function, and oxidative status. Pulmonary deposition of a radiolabeled monodisperse aerosol generated with a Pari LC Star nebulizer (Allergy Asthma Technology, Morton Grove, IL) connected to an AKITA inhalation device (Inamed, Gauting, Germany) was determined in six patients. In 17 additional patients bronchoalveolar lavage fluid was assessed before and after 14 days of inhalation with thrice-daily doses of 300 or 450 mg of glutathione. Intrathoracic deposition was 86.3 ± 1.4% of the emitted dose. Glutathione concentration in lavage 1 hour postinhalation was increased three- to fourfold and was still almost doubled 12 hours postinhalation. FEV1 transiently dropped after inhalation but increased compared with pretreatment values after 14 days (p < 0.001). This improvement was not related to the lavage content of oxidized proteins and lipids, which did not change with treatment. These results show that, using a new inhalation device with high efficacy, glutathione treatment of the lower airways is feasible. Reversion of markers of oxidative injury may need longer treatment, higher doses, or different types of antioxidants.
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is the most common lethal hereditary disorder of white individuals. The chronic airway inflammation in CF is characterized by an accumulation of large amounts of activated neutrophils and a persisting bacterial infection of the airways (1). Linked to this inflammation is an abnormal increase in oxidative stress that has been demonstrated by multiple markers, including elevated levels of protein oxidation (2), long-lived oxidants (3), prooxidant cytokines (4), increased oxidative damage to DNA (5), and lipid peroxidation (6). At the same time, the antioxidant capacity in patients with CF is severely reduced compared with control subjects (6). Glutathione in its reduced form is a tripeptide, l-γ-glutamyl-l-cysteinyl-glycine, containing a thiol group. Glutathione represents the normal first-line defense against oxidants released on the respiratory epithelial surface and has a pivotal role as a protective antioxidant against free radicals and other oxidants. In addition, glutathione has been implicated in modulation of redox-regulated signal transduction, regulation of cell proliferation, remodeling of extracellular matrix, apoptosis, and protective conservation of the antiprotease capacity of the lungs (7).
Glutathione is normally present in lower airway fluid at high concentrations (8). The glutathione level of the epithelial lining fluid is decreased in severe inflammatory lung diseases including CF (9, 10). In CF this likely results from the excessive oxidant burden and may also be linked to an abnormal glutathione transport associated with the defective CFTR (11–13). A number of in vitro models have demonstrated that exogenous glutathione or other cysteine-donating compounds are able to protect against inflammatory cell–mediated oxidant damage (14–19).
On the basis of these data augmentation of the extracellular glutathione level in the lungs may improve antioxidant capacity and counterbalance exaggerated oxidant stress. This objective was tested in pilot studies in patients with idiopathic pulmonary fibrosis (20), mild asthma (21), and CF (22). The latter study assessed the effect of six inhalations of 600 mg of reduced glutathione, each 12 hours apart, in seven adult patients with CF. Lung epithelial surface inflammatory cell–derived oxidants were suppressed and the level of glutathione increased 1.9-fold 1 hour after the last dose. Although large amounts of glutathione were nebulized in this study, only a relatively small increase in glutathione concentrations in the lung lining fluid was noted. This may be related to the relatively short half-life of glutathione in the respiratory tract (23) or to rapid oxidation of glutathione. In addition, the amount delivered to the lungs may also be of critical importance.
The goal of this study was to evaluate an optimized inhalation system to improve the intrapulmonary deposition of glutathione to assess its tolerance and effects on intrapulmonary glutathione concentrations as well as the state of oxidation of bronchoalveolar lavage (BAL) constituents.
Twenty-one adolescents or adults with CF participated in the study (for detailed clinical data, see Table E1 in the online supplement) (Figure 1)
The second part of the study assessed deposition of glutathione in 17 patients with mild to moderate cystic fibrosis defined as FEV1 greater than 45% of predicted, by means of BAL (Table 1
Subject | Age (yr) | Sex | CFTR Mutation | FEV1 (% predicted) | FEV1 Change during Study (% pre- dicted) | Inhaled Broncho- dilators | Glucocorticoids | Recombinant Human DNase | Inhaled Antibiotics | Microorganisms in Sputum | Clinical Signs of Asthma | Reversible Bronchoconstriction |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 18.0 | F | ΔF508/ΔF508 | 60.6 | 2.4 | Salbutamol, twice daily | No | 2.5 mg/d | Tobramycin, 150 mg/d | Pseudomonas aeruginosa, Candida albicans | No | Not tested |
| 2 | 16.4 | M | ΔF508/unknown | 43.0 | 6 | Salbutamol, twice daily | No | No | Tobramycin, 300 mg/d | P. aeruginosa | No | Not tested |
| 3 | 20.0 | M | ΔF508/ΔF508 | 57.0 | 15 | Formoterol, twice daily; ipratropium, three times daily | Budenoside, 400 μg twice daily | No | Tobramycin, 150 mg twice daily | P. aeruginosa, C. albicans, Aspergillus fumigatus | No | Not tested |
| 4 | 20.2 | M | ΔF508/ΔF5080 | 43.0 | 13 | Salbutamol, three times daily; ipratropium, three times daily | Budenoside, 400 μg twice daily; prednisone, 20 mg/d | No | No | P. aeruginosa | No | No |
| 5 | 19.3 | F | ΔF508/ΔF508 | 87.0 | 2.1 | Salbutamol, twice daily | No | 2.5 mg/d | Tobramycin, 300 mg/d | P. aeruginosa | No | No |
| 6 | 37.1 | F | ΔF508/ΔF508 | 56.0 | 9 | Salbutamol, twice daily | No | 2.5 mg/d | Tobramycin, 300 mg/d | P. aeruginosa, Staphylococcus aureus, A. fumigatus | No | No |
| 7 | 24.8 | M | ΔF508/ΔF508 | 79.0 | 16.1 | Ipratropium, twice daily; fenoterol, twice daily; salmeterol, twice daily | Fluticasone, 500 μg twice daily | No | Tobramycin, 300 mg twice daily | P. aeruginosa Geotrichum species, C. albicans, A. fumigatus | No | Not tested |
| 8 | 37.4 | M | R347P/Q493x | 99.0 | −4.2 | Formoterol, twice daily | Budenoside, 400 μg twice daily | No | Colistin, 79 mg twice daily | P. aeruginosa, S. aureus, C. albicans, A. fumigatus | No | Not tested |
| 9 | 18.8 | F | ΔF508/ΔF508 | 80.0 | −13.5 | Salbutamol, twice daily | Fluticasone, 500 μg twice daily | 2.5 mg/d | Tobramycin, 80 mg twice daily | A. fumigatus | Yes | No |
| 10 | 28.3 | F | ΔF508/ΔF508 | 72.3 | 12.4 | No | No | No | No | P. aeruginosa, S. aureus, C. albicans | No | No |
| 11 | 17.2 | M | ΔF508/unknown | 104.5 | −6.5 | No | No | No | No | Haemophilus influenzae | No | No |
| 12 | 22.4 | F | ΔF508/unknown | 88.1 | 4.6 | No | No | No | No | S. aureus | No | No |
| 13 | 29.9 | M | ΔF508/ΔF508 | 50.1 | −0.1 | Salmeterol, twice daily | Fluticasone, 500 μg/d | No | Tobramycin, 80 mg twice daily | P. aeruginosa, A. fumigatus, C. albicans | No | No |
| 14 | 37.0 | M | ΔF508/unknown | 61.0 | 11.5 | Ipratropium, twice daily | No | No | No | S. aureus | No | Not tested |
| 15 | 22.4 | M | ΔF508/unknown | 74.5 | 2.9 | Salbutamol, twice daily | Fluticasone, 500 μg twice daily | 2.5 mg/d | No | C. albicans, A. fumigatus, Stenotrophomonas maltophilia | No | No |
| 16 | 20.4 | F | ΔF508/unknown | 60.6 | 3.3 | Salbutamol, twice daily | Fluticasone, 500 μg twice daily | 2.5 mg/d | Tobramycin, 300 mg twice daily | P. aeruginosa, C. albicans, Candida glabrata | Yes | No |
| 17 | 21.9 | F | R553/unknown | 54.0 | 14 | No | No | 2.5 mg/d | No | C. albicans,
S. aureus,
H. influenzae | No | No |
Reduced glutathione sodium salt (Biomedica Foscana, Ferentino, Italy) was reconstituted at a concentration of 200 or 300 mg/ml, which resulted in a tonicity of 1,301 mOsm/L, or 1,952 mOsm/L, and a pH of about 7.0. The total emitted volume was set to 1.5 ml, which was delivered by an individualized number of breaths according to the patient's vital capacity by the AKITA inhalation device (see online supplement). This volume was selected to limit the inhalation time to 20 minutes or less.
BAL was performed in the middle lobe. The return from the first fraction was kept separate from the other three fractions, which were pooled. All manipulations of the samples were done immediately and on ice. After removal of the cells ice-cold 10% trichloroacetic acid was added to equal volumes, mixed, and centrifuged and the clear supernatant was stored at −70°C for analysis. Reduced and total glutathione were measured by reversed-phase high-performance liquid chromatography (26). The fraction of carbonyls was assessed by a sensitive slot-blot assay (27), thiols were determined with a thiol and sulfide quantitation kit (t-6060; Molecular Probes, Eugene, OR), lipid peroxides were assessed with a PeroXOquant quantitative peroxide assay kit (Pierce Biotechnology, Rockford, IL), and total protein and urea were determined as described (28).
Individual data and means ± standard error of the mean for n independent determinations are given. Comparisons were made by one-sided t-test, that is, we tested for an increase in glutathione concentrations. Multiple regression analysis, Pearson correlation analysis, and linear regression were performed. p < 0.05 was set as the level of significance and exact p values are reported (29).
Intrathoracic deposition in six patients was 85.5 ± 0.9% of the emitted aerosols. Extrathoracic deposition was only 5.5 ± 0.7%, and 9.0 ± 1.2% of the administered aerosol was found on the exhalation filter. Peripheral lung deposition (relative to the emitted dose), determined from the fraction of intrathoracic deposition found in the lungs 24 hours after the end of the inhalation, was 76.9 ± 1.9%. Regional deposition as assessed from the homogeneity of the scans in various areas, showed only minor interindividual variations between the different patients (see Table E2 in the online supplement).
The mean pretreatment level of reduced glutathione in BAL fluid was 2.9 ± 0.7 μmol/L and varied between 0.6 and 13.6 μmol/L in individual patients (Table 2
Reduced Glutathione (μmol/L) | Oxidized Glutathione (μmol/L) | Total Glutathione (μmol/L) | Percentage of Reduced Glutathione | |
|---|---|---|---|---|
| Inhaled dose 300 mg, lavage 1 h after end of inhalation (n = 4) | ||||
| Baseline | 4.01 ± 2.14 | 3.66 ± 2.50 | 7.68 ± 4.64 | 61 ± 6 |
| After 14 d of inhalation | 12.68 ± 3.59 | 12.08 ± 3.08 | 24.77 ± 7.05 | 50 ± 5 |
| p Value | 0.027 | 0.056 | 0.039 | 0.184 |
| Inhaled dose 450 mg, lavage 1 h after end of inhalation (n = 9) | ||||
| Baseline | 3.83 ± 1.33 | 1.24 ± 0.33 | 5.06 ± 1.63 | 70 ± 5 |
| After 14 d of inhalation | 15.59 ± 7.18 | 16.32 ± 4.27 | 31.91 ± 10.50 | 43 ± 6 |
| p Value | 0.017 | < 0.0001 | 0.001 | 0.004 |
| Inhaled dose 450 mg, lavage 12 h after end of inhalation (n = 4) | ||||
| Baseline | 1.37 ± 0.22 | 1.70 ± 0.13 | 2.44 ± 0.24 | 64 ± 6 |
| After 14 d of inhalation | 2.31 ± 0.46 | 2.45 ± 0.91 | 4.77 ± 1.19 | 54 ± 9 |
| p Value | 0.026 | 0.194 | 0.0651 | 0.138 |
Twelve hours after the inhalation of 450 mg of glutathione, the concentration of reduced lavage was increased 1.7-fold compared with the pretreatment level and the percentage of reduced glutathione was not different from baseline (Table 2; and see Table E3 in the online supplement).
The glutathione levels before or after inhalation of glutathione did not correlate with lung function or the number of polymorphonuclear leukocytes in BAL fluid (data not shown). Total protein (before, 470 ± 175 μg/ml; after 2 weeks of inhalation, 474 ± 175 μg/ml), the fraction of carbonyls (before, 17.0 ± 1.5 pmol/μg protein; after 2 weeks of inhalation, 16.0 ± 1.3 pmol/μg protein), reduced thiols (before, 2.0 ± 0.4 pmol –SH/μg protein; after 2 weeks of inhalation, 3.6 ± 1.1 pmol –SH/μg protein), and lipid peroxide content (before, 3.1 ± 0.4 μmol/L; after 2 weeks of inhalation, 2.8 ± 0.4 μmol/L) did not change significantly with treatment (Figure 2)
Figure 2. Lung function in 17 patients with mild to moderate cystic fibrosis before, 5 minutes after, and 40 minutes after the inhalation of a glutathione aerosol, as well as after 2 weeks of daily glutathione inhalation. A significant drop in FEV1 (top) 5 minutes after the inhalations was observed. This was completely reversible with time and without treatment after 40 minutes. After 2 weeks of inhalation, FEV1 was significantly higher compared with pretreatment levels and the levels after 5 and 40 minutes. The same pattern was observed for FVC (bottom). Data represent means ± SEM, and comparisons were made by repeated measures analysis of variance followed by Newman–Keuls post hoc test.
[More] [Minimize]Inhalation of glutathione with the AKITA device was well tolerated. A cough and an unpleasant odor of the glutathione solution were reported by some of the subjects as mild and were judged by all participants not to hinder them from continuing the inhalations (see Table E4 in the online supplement). Electronically assessed compliance (see online supplement) was 86.5 ± 3.3% of the number of inhalation breaths that had to be taken during the 2-week period to inhale the targeted dose of glutathione (see Table E1 in the online supplement). Noncompliance occurred among almost all patients, but only sporadically throughout the study period. The time needed to complete nebulization of the targeted dose with 300 or 450 mg of glutathione was 18 ± 1 minutes. Inhalation of glutathione induced an immediate reduction in lung function after 5 minutes that was reversible 40 minutes after the end of the inhalation (Figure 3)
Figure 3. Bronchoalveolar lavage carbonyls (top), thiols (middle), and lipid peroxides (bottom) were not different before and after treatment with inhaled glutathione in 15 patients with mild to moderate cystic fibrosis. Data points represent means of duplicate determinations andindividual values are given. Comparisons were made by paired t-test.
[More] [Minimize]In this study we showed that an improved method of aerosol delivery yielded an intrathoracic deposition of more than 80% of the emitted dose. Glutathione concentrations in BAL fluid were increased three- to fourfold 1 hour after inhalation and at 12 hours were still almost doubled. Unexpectedly, BAL fluid content of oxidized proteins and lipids did not change with treatment. These results show that supplementation of the lower airways with large amounts of glutathione is feasible. However, changes in the overall degree of oxidation of lavage constituents may need prolonged treatment periods or even higher doses than those used in the present study.
In patients with CF the CFTR defect results in chronic airway infection because of the inability to clear microorganisms from the respiratory tract. The predominating continuous and extensive neutrophilic cellular infiltrate overwhelms the physiological antioxidant capacity of the epithelial lining fluid and protection of the integrity of the pulmonary epithelia surface against oxidative damage is lost. The chronic excess oxidative stress results in alteration of structural proteins, DNA, membranes, and lipids (6), eventually leading to pulmonary damage and destruction. Decreased levels of glutathione have been found in plasma and lavage fluid of adult patients with CF with pronounced airway inflammation (9). Glutathione can scavenge a broad range of oxidant molecules generated during chronic airway inflammation, and thus improving the glutathione content of the epithelial lining fluid may be a valid and promising therapeutic approach to protect the lungs from oxidative damage. A previous study in seven subjects with CF has demonstrated the feasibility and tolerance of glutathione aerosol in patients with moderate to severe CF (22).
In the present study, further steps toward improved and efficient administration were undertaken. Optimized aerosol delivery, which resulted in high and homogeneous intrapulmonary delivery, was used to increase pulmonary glutathione level. The AKITA device used in this study individually controls a preset breathing pattern so that a slow and deep inhalation allows inhaled particles with an aerodynamic diameter between 2 and 5 μm to penetrate via the oropharynx and larynx into the lungs without being deposited by impacting in the upper respiratory tract. Within the lungs the particles are deposited mainly by sedimentation. Our patients showed pulmonary deposition of 85% of the emitted dose, which is much more than the values obtained without standardization in prior studies, which usually report deposition rates between 10 and 30% (30–36). The high pulmonary deposition and low extrapulmonary deposition were achieved without end-inspiratory breath hold times. This suggests that end-inspiratory breath holding is not necessary because of the slow and deep breathing, allowing the residence time of particles in the lungs to be long enough for sufficient deposition. In addition, the interindividual variability in lung deposition was low (see Figure E1 and Table E2 in the online supplement). In some patients ventilation in the upper lobe was poorer than in the other lobes and thus less aerosol reaches these upper lobes (see Figure E1 in the online supplement). This is a typical finding in patients with CF, who often have lung disease, predominantly affecting the upper lobes.
The inhalation of the two concentrations of glutathione was well tolerated. The average rate of adherence was good, sporadic inhalations being skipped, mainly at noon. The efficient intrapulmonary deposition delivered a fairly high amount of a hypertonic solution to the airways. Although this was well tolerated as judged from the oral and written comments of patients, lung function measurements indicated an acute drop in FEV1 that was spontaneously reversible in less than 1 hour. This effect was not observed in the study by Roum and coworkers (22), probably because the first lung function measurement was done 4 hours after the end of the inhalation. Our results are in agreement with those obtained in adult patients with mild asthma. Here, nebulized glutathione has been shown to cause major airway narrowing, induced cough, and breathlessness (21). The latter symptoms were not noted in our study (see Table E4 in the online supplement). In the patients with asthma, neither the high osmolarity nor the acidity of the glutathione accounted for the bronchoconstrictive effect, but it was suggested that sulfite formation from nebulized glutathione might be responsible for this effect. The bronchoconstriction promptly responded to inhaled β-agonists (37). In our study the bronchoconstrictive effect was spontaneously reversible, that is, without treatment with a β-agonist (Figure 1). Daily peak flow measurements in the morning and evening before the inhalation procedure (see Figure E2 in the online supplement) did not show any drop in baseline lung function. In contrast, with time peak expiratory flow increased significantly. During the study no β-agonists, in addition to those regularly taken by the patients before the study (Table 2), were administered. Previously, it has been shown that even the inhalation of physiological saline leads to a transient drop in FEV1 in patients with CF (38).
Interestingly, the overall lung function, that is, FEV1 and FVC, improved by 6–7% over the 14-day inhalation period. As we did not include a control group in this dose-finding trial, the reason(s) for this improvement cannot be determined. They include, among others, increased mucus clearing from the inhalation procedure itself and associated coughing (see Table E4 in the online supplement) or from the hypertonicity of the solution (39). Of interest in this context is the finding that both hyperosmolality and low millimolar concentrations of S-nitrosoglutathione increase the expression, maturation, and function of ΔF508 CFTR (40). The inhaled glutathione may serve as a precursor for the formation of nitrosoglutathione (41). In addition, glutathione may act directly as a mucolytic agent (42), and may modify cellular signaling events by transcriptional regulation of redox-sensitive transcription factors such nuclear factor κB and activator protein-1. Additional processes such as remodeling of the extracellular matrix, apoptosis of cells, and cytokine expression by local immune cells in the lungs have been shown to be influenced by reduced glutathione (7, 14). Large, direct antioxidant effects on BAL proteins and lipids were not observed in this study and are thus unlikely to be responsible for the observed changes in lung function.
The level of glutathione obtained with an optimized delivery system, that is, a three- to fourfold increase in reduced glutathione 1 hour after the end of inhalation, is substantially higher than the 1.9-fold increase reported by Roum and coworkers (22). Those authors nebulized a higher dose of glutathione (600 mg), which has an estimated intrapulmonary deposition rate of about 20% under normal nebulizing conditions in humans (23). Therefore, even considering the lower doses used in this study, the deposition rate of about 80% achieved high glutathione levels in the lower respiratory tract. As feasibility of the inhalations under everyday conditions was an important goal of this study, we limited the dose to have inhalation times of less than 20 minutes. If needed, an even higher amount of glutathione, that is, 600 mg, might be nebulized with the AKITA device to further increase pulmonary deposition. Alternatively, different approaches to increase alveolar glutathione, including oral or inhaled delivery of precursors of glutathione, such as N-acetylcysteine (43) or an increase in apical glutathione secretion by the pulmonary epithelium (19) may be used.
We reported all results obtained in BAL as concentrations in the recovered lavage fluid, as recommended by the European Task Force on BAL (44). This circumvents the problems associated with erroneous calculation of dilutional factors. For comparison, the urea correction was calculated and the level of reduced glutathione in the epithelial lining fluid before treatment (87 ± 16 nmol/ml) was diminished compared with previously reported values for control subjects (257 ± 21 nmol/ml) and was similar compared with adult patients with CF (78 ± 13 nmol/ml) (9). With glutathione treatment the levels achieved in this study (518 ± 201 nmol/ml) were higher than in these control subjects. Additional values available from normal subjects are 400 ± 21 nmol/ml (45) and 568 ± 53 nmol/ml (46). The level of oxidized glutathione in normal subjects is reported as 31.2 ± 6.5 (46) and 17.2 ± 0.3 nmol/ml (45). In patients with CF the corresponding value is 25 ± 11 nmol/ml, that is, 46% of total glutathione (22). This compares well with the value of 42 ± 7 nmol/ml of epithelial lining fluid, what we found in this study.
The ratio of reduced to oxidized glutathione 1 hour after the end of inhalation was clearly shifted toward an increased fraction of the oxidized form. This suggested a rapid usage of the nebulized glutathione within the lung as an antioxidant. Interestingly, at 12 hours after the last inhalation, the level of reduced glutathione was still almost doubled, suggesting a persistent increase in intraalveolar glutathione concentrations. At this time the ratio of reduced to oxidized glutathione was unchanged, suggesting that the glutathione-related redox state was not altered. These data are in agreement with the assessments of overall oxidation of BAL proteins, that is, the fraction of thiols and carbonyls, which were unaltered by the treatment. Similarly, concentrations of lipid peroxides, as indicators of lipid oxidation, were unchanged. We did not observe any effects of glutathione inhalation on the total number of cells, their viability, or the differential cell counts obtained. This is in agreement with the previous short-term study of glutathione inhalation in patients with CF (22). It still remains to be investigated whether a longer course of glutathione aerosol administration might change the number or pattern of inflammatory cells and of the oxidative state in the CF lungs. In addition to its mucolytic and antioxidant properties, glutathione may act on pulmonary epithelial cells and the local immune cells in the lungs (7, 14) through redox-sensitive transcription factors or indirectly via S-nitrosoglutathione, through modulation of CFTR function. All these effects, and also those on surrogate markers of the redox potential in BAL fluid, may be addressed in future studies.
The authors acknowledge the support of Prof. Peter Fürst (Hohenheim, Germany).
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