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Mitochondrial OXPHOS function is unaffected by chronic azithromycin treatment
Journal of Cystic Fibrosis 12 (2013) 682 – 687 www.elsevier.com/locate/jcf
Original Article
Mitochondrial OXPHOS function is unaffected by chronic azithromycin treatment Shelly Ben-Harush Negari a, c, e , Tzemach Aouizerat a, b , Ariel Tenenbaum c , Malena Cohen-Cymberknoh c, d , David Shoseyov c, d , Eitan Kerem c, d , Ann Saada a
a, b,⁎
Monique and Jacques Roboh Department of Genetic Research Hadassah-Hebrew University Medical Center, Ein Kerem, Jerusalem 91120, Israel b Department of Genetic and Metabolic Diseases Hadassah-Hebrew University Medical Center, Ein Kerem, Jerusalem 91120, Israel c Department of Pediatrics Hadassah-Hebrew University Medical Center, Jerusalem Israel d CF Center, Hadassah-Hebrew University Medical Center, Mt. Scopus Jerusalem 91240, Israel e Division of Adolescent Medicine Cincinnati Children's Hospital Medical Center, OH, USA Received 28 January 2013; received in revised form 11 March 2013; accepted 17 April 2013 Available online 13 May 2013
Abstract Background: Certain antibiotics may cause unwanted side effects due to the similarity of the mitochondrial translation system to the prokaryotic one. Children with cystic fibrosis (CF) are vulnerable to recurrent respiratory tract infections and azithromycin, a translation targeted antibiotic, is often used chronically to treat CF patients. No major clinical side effects were found with chronic treatment. However, mitochondrial function was not previously assessed. We evaluated oxidative phosphorylation (OXPHOS) in lymphocytes from children with CF receiving chronic azithromycin treatment using an improved ATP production assay. Method: Enzymatic activities of respiratory chain complexes II–IV and ATP production were measured in lymphocytes. Results: Relative to controls and to CF patients without azithromycin treatment, no significant difference in mitochondrial respiratory chain complexes II–IV was detected, and ATP production with pyruvate, glutamate and succinate, did not disclose any differences between the groups. Conclusion: We suggest that chronic treatment with azithromycin does not significantly affect OXPHOS function. © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Azithromycin; Chronic treatment; OXPHOS; Mitochondrial respiratory chain
1. Introduction Cystic fibrosis (CF) is the most common life shortening inherited disease in the population of European descent [1]. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in alteration in epithelial cell ion transport leading to increased sputum viscosity, stasis of secretions and impaired mucociliary clearance [2]. Dysregulation of respiratory epithelial cells, and innate immune dysfunction in ⁎ Corresponding author at: Department of Genetic and Metabolic Diseases, Hadassah-Hebrew University Medical Center, POB 12000, Jerusalem 91120, Israel. Tel.: + 972 2 6776844; fax: + 972 2 6 779018. E-mail addresses: [email protected], [email protected] (A. Saada).
the lung, leads to an exaggerated and ineffective airway inflammation which fails to eradicate pulmonary pathogens [3, 4]. The increased viscosity of the sputum impairs the effective clearance of inhaled microorganisms leading to recurrent endobronchial infections and bacterial colonization, mainly with Staphylococcus aureus and Pseudomonas aeruginosa, and resulting in an exaggerated inflammatory response that leads to the development of bronchiectasis and progressive obstructive airway disease [5]. Though abnormal CFTR function results in multi organ damage, the lung involvement has the most dramatic impact on quality of life and survival [6]. In the late '80s, it was shown that long term macrolide treatment substantially reduced morbidity and mortality in patients with diffuse panbronchiolitis [7, 8]. Since then, the potential benefit of macrolide antibiotics
1569-1993/$ -see front matter © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2013.04.006
S.B.-H. Negari et al. / Journal of Cystic Fibrosis 12 (2013) 682–687
has been evaluated in a variety of chronic respiratory diseases including CF [9]. Macrolides belong to the group of translation targeted antibiotic, interfering with protein synthesis, by reversibly binding to the 50s subunit of prokaryotic ribosomes and inhibiting the peptidyl transferase. The macrolide antibiotics achieve higher tissue than plasma concentrations as they penetrate the respiratory tissue and accumulate in the polymorphonuclear leukocyte extremely well [10]. Given their favorable bioavailability via oral route, their excellent tissue penetration and broad efficacy against many organisms affecting the lung, macrolides are used as first-line agents in the therapy of respiratory infections. Moreover, macrolides have documented to have wide spectrum of immunomodulatory effects on mammalian cells both in vivo and in vitro. These effects limit tissue damage by neutrophils, decrease mucus viscosity, and suppress angiogenesis, and support the rationale for using macrolide therapy for the treatment of chronic inflammatory airway diseases [11, 12]. Cochrane systematic review and meta-analysis of data from 10 clinical trials provide evidence that long term macrolide therapy in CF patients improves lung function, decreases frequency of pulmonary exacerbations, significantly reduce the need for oral antibiotics and improves nutritional status [13]. These studies have lead many CF centers, including ours, to treat patients with oral azithromycin. Macrolides are considered to be well-tolerated antibiotics; however, they have been associated with reversible ototoxicity [14], transaminitis and prolongation of the QT interval [11]. The most common side effect observed with Macrolide use is related to the GI tract and is thought to be a result of their ability to mimic the effects of motilin (a gastrointestinal tract polypeptide hormone) [10]. In the previously mentioned trials with CF patients, adverse events were uncommon and not obviously associated with azithromycin [9, 15–19]. Azithromycin along with many other antibiotics is targeted against the bacterial ribosome while sparing the eukaryotic translation system, as the cytoplasmic ribosome has a somewhat different structure. Contrarily, the mitochondrial translation system is separate from the cytosolic, resembling the prokaryotic, and the mitochondrial ribosome is more similar to the bacterial one [20]. Therefore it is not surprising that they can be affected by translation targeted antibiotics. In fact, side-effects such as ototoxicity, liver dysfunction and QTc prolongation can be a result of mitochondrial dysfunction [21]. Mitochondria are intracellular organelles, providing the vast majority of cellular energy in the form of ATP by oxidative phosphorylation (OXPHOS). The OXPHOS system, consist of the four respiratory chain complexes, consisting the electron transport chain (ETC) and the fifth complex ATP synthase. All complexes, with the exception of complex II, contain subunits encoded in the mitochondrial DNA (mtDNA) and are therefore dependent on intra-mitochondrial translation and the mitochondrial ribosome [22]. Hereto forth the effect of translation targeted antibiotics on mitochondrial function has been the subject of investigation in vitro but to our knowledge, it has not been systematically evaluated in children, and it is the objective of this study. As most children with CF in our CF center are chronically treated
683
with azithromycin as immunomodulator, we decided to focus our investigation on this group of patients. Since long term azithromycin treatment in CF patients infected with pseudomonas is becoming common, physician would benefit from an evaluation of such possible side effect. The mitochondrial function was evaluated in blood lymphocytes by measuring the enzymatic activity of the ETC complexes and by measuring ATP production by an improved method allowing minimal sample size. 2. Subjects, materials and methods 2.1. Subjects The study was done in the Hadassah medical center and approved by the IRB ethics committee. Exclusion criteria were refusal of the patient or his guardian, known mitochondrial disease, infection or malignancy. Blood samples were obtained by venipuncture with informed consent. Notably, For the CF patients, blood samples were drawn in conjunction with other periodic lab testing and no separate venipuncture was needed. Usual concomitant therapy, including physiotherapy, inhaled medications and pancreatic and vitamin supplements, continued throughout the trial and was monitored by the clinic physicians. 2.2. Materials Lymphoprep (Axis-Shield, Oslo, Norway) DMEM and fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), ATPlite (Perkin-Elmer Inc., USA), digitonin (Calbiochemicals, San-Diego USA) all other chemicals were of highest possible grade (Sigma-Aldrich USA). 2.3. Lymphocyte preparation Lymphocytes were prepared from 3–5 ml blood (drawn in EDTA) by differential centrifugation using Lymphprep (AxisShield Norway) according to the manufacturers' instructions. The lymphocyte suspension was divided into 2 aliquots in Eppendorf tubes and pelleted by centrifugation 1 min 10.000 rpm in a microfuge. The supernatant was removed and one batch was stored at − 70 °C for enzymatic assays. The other batch, for ATP production, was suspended in 0.5 ml freezing medium (glucose free DMEM supplemented with 40% fetal calf serum and 10% DMSO) placed in a freezing container (Mr. Frosty, Nalgene, Thermo Fischer Scientific Inc.), frozen at − 70 °C overnight and subsequently transferred for extended storage, into liquid nitrogen. 2.4. Enzymatic assays Enzymatic activities of complexes II–IV in lymphocyte homogenates were determined by spectrophotometry essentially as described [23–25]. Briefly, Complex II was measured as succinate dehydrogenase (SDH) based on the succinate-mediated phenazine methosulfate reduction of dichloroindophenol at 600 nm. Complex II + III was measured as succinate cytochrome
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c reductase, measuring the reduction of oxidized cytochrome c at 550 nm. Complex IV (cytochrome c oxidase) was measured by following the oxidation of reduced cytochrome c at 550 nm. Citrate synthase (CS), an ubiquitous mitochondrial matrix enzyme, serving as a control, was measured in the presence of acetyl CoA and oxaloacetate by monitoring the liberation of CoASH coupled to dithiobis (2-nitrobenzoic) acid at 412 nm. Protein concentration was determined by the Lowry method and calculated according to bovine serum albumin (BSA) standard curve. Measurements were performed on a dual beam UVKON spectrophotometer (Secomam, France). 2.5. ATP production ATP production was determined as described by Berger et al. [25] with the following modifications: lymphocytes were rapidly thawed by adding 1 ml phosphate buffered saline (PBS) and the suspension was microfuged for 1 min at 7000 rpm; the pellet was washed once with 1.5 ml permeabilization-substratemix buffer (potassium phosphate buffer 100 mM pH 7.4, KCl 150 mM, Tris25 mM pH 7.4 EDTA 2 mM, BSA fatty acid free 0.025%, ADP 1 mM, digitonin 40 μg/ml). The pellet was resuspended in 0.35 ml permeabilization-substrate-mix buffer and the reaction was started by adding 25 μl suspension to 25 μl permeabilization-substrate-mix buffer containing the substrates reaching the final concentrations of pyruvate 5mM + malate 1 mM (PM) or glutamate 5 mM + malate 1 mM (GM) or succinate 10 mM in the presence of 5 μg/ml rotenone (S). After an incubation of 30 min at 37 °C, the reaction was stopped by adding 25 μl ATPlite cell lysis solution (for background readings the cell lysis solution was added prior to the substrate). After 5 min shaking 50 μl was transferred to a white microtiter well plate and 20 μl substrate solution was added, after 10 min dark adaptation luminescence was read on a Synergy HT instrument (BioTek, USA). ATP production was calculated by comparing to a standard curve which was constructed on each separate occasion.
3.2. Evaluation of ATP production assay Initially we evaluated an improved version of the ATP production assay that we have previously utilized for studying the effect of anti epileptic drugs (Berger et al.). The major drawback with the initial method was the instability of the samples at − 70 °C. The gradual decrease of temperature and storage in liquid nitrogen, in the present study allowed the frozen lymphocytes to retain the ATP producing capacity for at least 6 months (results not shown). The ATP producing capability in lymphocytes was also stable in lymphocytes isolated from blood stored at room for up to 8 hours until lymphocyte preparation allowing the sample to be transported to the laboratory from the clinic. Immediate processing yielded a somewhat higher capacity while an overnight 15h delay between the blood drawing and lymphocyte preparation resulted in a severe drop in ATP production. Therefore, all samples in this study were processed between 4 and 8 hours (Fig. 1). The use of ATPlite in combination with microtiter well reader facilitated and shortened the assay process to less than 1 hour and allowed triplicate repeats with all substrates from a single batch of lymphocytes originating from 1.5 to 2 ml blood. 3.3. Mitochondrial function in patient and controls Lymhocyte ATP production capability with all substrates was not significantly different in patients chronically treated with azithromycin compared to the normal adult and pediatric control groups (Fig. 2). Rather ATP production was somewhat increased with pyruvate + malate and glutamate + malate relative to the pediatric control and the untreated CF groups. From the above data, we concluded that mitochondrial ATP production capability in lymphocytes is clearly not, negatively affected by chronic azithromycin treatment. On the other hand, the untreated CF group was slightly but not statistically significant decreased
400
2.6. Statistics
P+M
Data are expressed as mean ± SEM, and the results were evaluated by one-way ANOVA to compare means using the IBM SPSS statistics program version 20. 3. Results 3.1. Characteristics of the subjects
ATP [nmol/30min/mg]
350
G+M
300
S
250 200 150 100 50
The study group consisted of 16 pediatric patients (12 years ± 4.4 years) diagnosed with CF treated with azithromycin 250 mg (below 36 kg body weight) and 500 mg (above 36 kg of weight) once a day for at least 3 years. The three control groups consisted of 29 normal adult samples, 14 pediatric samples (obtained from children visiting the outpatient clinics for various reasons but without any signs of infections, malignancy, CF or mitochondrial disorders), and from 5 children with CF but not treated with azithromycin.
0 0h
4h
8h
15 h
24 h
Fig 1. ATP production in lymphocytes isolated from stored blood. Lymphocytes from blood from a healthy adult control was isolated and frozen in freezing medium, immediately or after different time periods of storage at room temperature. Subsequently to storage in liquid nitrogen, the lymphocytes were assayed for mitochondrial ATP production capability with the different substrates; pyruvate + malate (PM), glutamate + malate (GM) or succinate (S). The results are presented as mean ± SEM of at least triplicate measurements.
S.B.-H. Negari et al. / Journal of Cystic Fibrosis 12 (2013) 682–687 250
control adult
A
*
150
100
control adult
100
activity [mU/mg]
ATP [nmol/30min/mg]
CF az
* *
*
CF
200
*
120
control ped.
685
50
control ped. 80
CF CF az
60 40
* *
*
20 0 G+M
S
Fig. 2. ATP production in lymphocytes from patients and controls. Lymphocytes from blood from healthy adult controls (control adult), control pediatric patients (control ped) pediatric CF patients (CF) and pediatric CF patients chronically treated with azenil (CF az) and control were assayed for mitochondrial ATP production capability with different substrates; pyruvate + malate (PM), glutamate + malate (GM) or succinate (S). The results are presented as mean ± SEM of at least triplicate measurements in each subject. *p b 0.05.
compared with the other groups, however no specific conclusions could be drawn due to the small sample size and large SEM. Notably the adult control group was not significantly different from pediatric control group; therefore normal adult control samples (which are more readily available than normal pediatric samples) will be suitable as controls in future studies (Fig. 2). The ATP production assays were complemented with enzymatic measurements of individual ETC complexes. CS served as an internal control and estimation of mitochondrial content. Mitochondrial content was significantly increased in the pediatric controls relatively to the adult controls and both CF groups were decreased relatively the pediatric controls. However there was no significant difference between CF patient treated or not treated with Azenil. This was also reflected, but to a lesser extent in COX activity but not in II + III or SDH activities (Fig. 3A). The decrease in COX was diminished when normalized to citrate synthase activity. The II + III and SDH activities were slightly increased in the CF groups (Fig. 3B). Taken together no adverse effect of chronic azithromycin treatment on the measured parameters was observed.
4. Discussion Several studies have shown the beneficial effect of long term azithromycin treatment on the clinical status of cystic fibrosis patients. Chronic treatment with azithromycin for CF patients especially those infected with P. aeruginosa was shown to improve lung function, to improve quality of life, to decrease pulmonary exacerbation rate and to reduce the need for hospital admissions and for antibiotic treatment. These trials did not report major adverse events related to long term therapy. These trials evaluated clinical status only; neither of them evaluated the effect on mitochondrial function.
0 CS
B
COX
II+III
SDH
1.6
control adult 1.4
Activity [mU/mU CS]
P+M
control ped.
1.2
CF CF az
1 0.8 0.6
*
0.4
* *
* 0.2 0 COX/CS
II+III/CS
SDH/CS
Fig. 3. Enzymatic assay of ETC complexes. Lymphocytes from blood from healthy adult controls (control adult), control pediatric patients (control ped) pediatric CF patients (CF) and pediatric CF patients chronically treated with Azenil (CF az) and control were assayed for enzymatic activities of ETC complexes II–IV. Succinate dehydrogenase (SDH), succinate-cytochrome c reductase (II + III), cytochrome c oxidase (COX) and the matrix control enzyme citrate synthase. Fig. 3A depicts the actual activities while Fig. 3B depicts the relative activities compared to citrate synthase. The results are presented as mean ± SEM. *p b 0.05.
The concern from chronic treatment with azithromycin for CF patients has been the possible inhibitory influence of macrolides on the DNAse activity as seen in vitro, since most of the CF patients are treated with DNAse [11]. Another concern which still needs to be proven by designated research is the emergence of macrolide resistance strains of S. aureus but there is lack of long term data [13]. Drug induced mitochondrial toxicity is gaining recognition within the pharmaceutical industry; there are some in vitro methods for assessment of mitochondrial damage but to date there is lack of validated biomarkers for identifying drug induced mitochondrial dysfunction in patients [26]. Notably, some of the biochemical parameters of mitochondrial damage can mimic CF exacerbation or an acute infection in a CF patient (lactic acidosis, raised urea and creatinine, raised liver enzymes and hypo/hyperglycemia). Our research is a pilot assessment of the mitochondrial function in patients chronically treated with azithromycin. The mitochondrial OXPHOS was measured by
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two parameters in the peripheral lymphocytes; enzymatic activity of the respiratory chain complexes II–IV and ATP production. The improved freezing technique and storage in liquid nitrogen allowed reproducible ATP production after up to eight hour delay before lymphocyte preparation and prolonged storage in liquid nitrogen. The use of ATPlite kit with microtiter plates was much more convenient than the previous assay [25]. The improved method facilitates future studies allowing comparative measurement of ATP production in repeated samples from the same individual taken at different time intervals and transported from distant clinics. The ATP production assays were complemented by enzymatic measurements of the individual ETC complexes. We were not able to accurately measure rotenone sensitive complex I activity (despite numerous efforts), due to interfering NADH dehydrogenases in the homogenate. Notably mitochondrial isolation from small blood samples is not feasible. Nevertheless ATP production from both pyruvate and glutamate is complex I dependent and sufficient to detect complex I defects [23]. Moreover ATP production from pyruvate is proportional to pyruvate dehydrogenase activity [27]. Lymphocytes isolated from patients and control groups were analyzed with the improved ATP production method as well as with the enzymatic assays. Taken together, the results disclosed no significant effect of chronic azithromycin treatment on mitochondrial function in children with CF was detected. Rather some parameters were improved compared to untreated patients. Regretfully as the majority of CF patients in our center are treated with azithromycin, we were not able to include more non-treated CF patients in this study to confirm the significance of this observation with respect to ATP production. CS measured in lymphocyte homogenate showed a moderate but significant decrease in mitochondrial content observed in all patients relative to pediatric controls. This seems to be related to CF per se and not azithromycin as both treated and non-treated patients showed the same decrease. Nevertheless this decrease did not affect the overall OXPHOS as measured by ATP production. These results are in accord with our pervious results data, showing that azithromycin did not significantly affect OXPHOS dependent control fibroblast cell growth while exerting only a modest effect on fibroblasts derived from patients with mitochondrial translation disorders. On the contrary, chloramphenicol, impaired growth of both control and patients cells, even though both antibiotics bind to the exit tunnel of the prokaryotic large ribosomal subunit. Moreover, azithromycin, a macrolide, affects in-vitro mitochondrial translation; it does so to a much lesser extent than other classes of translation targeted antibiotics such as the aminoglycoside gentamycin and the tetracycline derivative doxycycline [28]. The variations between the antibiotics are most probably due to minor differences between the mitochondrial and the prokaryotic ribosome as well as their ability to penetrate cell membranes. Taken together, the data suggest that azithromycin is relatively harmless to mitochondrial function, and we therefore conclude that chronic treatment with azithromycin does not significantly impair OXPHOS function in lymphocytes. Further investigation is warranted in a larger cohort and ,in other tissues to confirm our data [29].
Conflict of interest There are no conflicts of interest to declare. Acknowledgements Corinne Belaiche is acknowledged for expert technical assistance. This work was financed by the Hadassah-Hebrew University Joint Research fund (to A.T. and A.S.). The sponsor had no involvement in the study design, collection, analysis and interpretation of the data. References [1] Spagnolo P, Fabbri LM, Bush A. Long-term macrolide treatment for chronic respiratory disease.Eur Respir J 2012 [Electronic publication ahead of print] [PMID: 23180583]. [2] Donaldson SH, Boucher RC. Update on pathogenesis of cystic fibrosis lung disease. Curr Opin Pulm Med 2003;9:486–91. [3] Watt AP, Courtney J, Moore J, Ennis M, Elborn JS. Neutrophil cell death, activation and bacterial infection in cystic fibrosis. Thorax 2005;60: 659–64. [4] Pillarisetti N, Williamson E, Linnane B, Skoric B, Robertson CF, Robinson P, et al. Infection, inflammation, and lung function decline in infants with cystic fibrosis. Am J Respir Crit Care Med 2011;184:75–81. [5] Chmiel JF, Berger M, Konstan MW. The role of inflammation in the pathophysiology of CF lung disease. Clin Rev Allergy Immunol 2002;23: 5–27. [6] Cohen-Cymberknoh M, Shoseyov D, Kerem E. Managing cystic fibrosis strategies that increase life expectancy and improve quality of life. Am J Respir Crit Care Med 2011;183:1463–71. [7] Koyama H, Geddes DM. Erythromycin and diffuse panbronchiolitis. Thorax 1997;52:915–8. [8] Kudoh S, Azuma A, Yamamoto M, Izumi T, Ando M. Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. Am J Respir Crit Care Med 1998;157:1829–32. [9] Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, et al. Macrolide Study Group. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. Jama 2003;290:1749–56. [10] Jain R, Danziger LH. The Macrolide Antibiotics: a pharmacokinetic and pharmacodynamic overview. Curr Pharm Des 2004;10:3045–53. [11] Adam L, Friedlander RK. Albert Chronic Macrolide Therapy in Inflammatory airway disease. Chest 2010;138:1202–12. [12] Martinez FJ, Curtis JL, Albert R. Role of macrolide therapy in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2008;3: 331–50. [13] Southern KW, Barker PM, Solis-Moya A, Patel L. Macrolide antibiotics for cystic fibrosis.Cochrane Database Syst Rev 2011;12 [CD002203]. [14] Wallace MR, Lk Miller, Nguyen MT, Shields AR. Ototoxicity with azithromycin. Lancet 1994;22(343):241. [15] Wolter J, Seeney S, Bell S, Bowler S, Masel P, McCormack J. Effect of long term treatment with azithromycin on disease parameters in cystic fibrosis: a randomised trial. Thorax 2002;57:212–6. [16] Clement A, Tamalet A, Leroux E, Ravilly S, Fauroux B, Jais JP. Long term effects of azithromycin in patients with cystic fibrosis: a double blind, placebo controlled trial. Thorax 2006;61:895–902. [17] Saiman L, Anstead M, Mayer-Hamblett N, Lands LC, Kloster M, Hocevar-Trnka J, et al. Effect of azithromycin on pulmonary function in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2010;303:1707–15. [18] Equi A, Balfour-Lynn IM, Bush A, Rosenthal M. Long term azithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial. Lancet 2002;360:978–84.
S.B.-H. Negari et al. / Journal of Cystic Fibrosis 12 (2013) 682–687 [19] McCormack J, Bell S, Senini S, Walmsley K, Patel K, Wainwright C, et al. Daily versus weekly azithromycin in cystic fibrosis patients. Eur Respir J 2007;30:487–95. [20] Christian BE, Spremulli LL. Mechanism of protein biosynthesis in mammalian mitochondria. Biochim Biophys Acta 1819;2012:1035–54. [21] Cohen BH, Saneto RP. Mitochondrial translational inhibitors in the pharmacopeia. Biochim Biophys Acta 2012;1819:1067–74. [22] Papa S, Martino PL, Capitanio G, Gaballo A, De Rasmo D, Signorile A, et al. The oxidative phosphorylation system in mammalian mitochondria. Adv Exp Med Biol 2012;942:3–37. [23] Saada A, Bar-Meir M, Belaiche C, Miller C, Elpeleg O. Evaluation of enzymatic assays and compounds affecting ATP production in mitochondrial respiratory chain complex I deficiency. Anal Biochem 2004;335:66–72. [24] Reisch AS, Elpeleg O. Biochemical assays for mitochondrial activity: assays of TCA cycle enzymes and PDHc. Methods Cell Biol 2007;80: 199–222.
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[25] Berger I, Segal I, Shmueli D, Saada A. The effect of antiepileptic drugs on mitochondrial activity: a pilot study. J Child Neurol 2010;25:541–5. [26] Nadanaciva S, Will Y. New insights in drug-induced mitochondrial toxicity. Curr Pharm Des 2011;17:2100–12. [27] Saada A, Aptowitzer I, Link G, Elpeleg ON. ATP synthesis in lipoamide dehydrogenase deficiency. Biochem Biophys Res Commun 2000;269: 382–6. [28] Jones CN, Miller C, Tenenbaum A, Spremulli LL, Saada A. Antibiotic effects on mitochondrial translation and in patients with mitochondrial translational defects. Mitochondrion 2009;9:429–37. [29] Ray WA, Murray KT, Hall K, Arbogast PG, Stein CM. Azithromycin and the risk of cardiovascular death. N Engl J Med 2012;366:1881–90.
Original Article
Mitochondrial OXPHOS function is unaffected by chronic azithromycin treatment Shelly Ben-Harush Negari a, c, e , Tzemach Aouizerat a, b , Ariel Tenenbaum c , Malena Cohen-Cymberknoh c, d , David Shoseyov c, d , Eitan Kerem c, d , Ann Saada a
a, b,⁎
Monique and Jacques Roboh Department of Genetic Research Hadassah-Hebrew University Medical Center, Ein Kerem, Jerusalem 91120, Israel b Department of Genetic and Metabolic Diseases Hadassah-Hebrew University Medical Center, Ein Kerem, Jerusalem 91120, Israel c Department of Pediatrics Hadassah-Hebrew University Medical Center, Jerusalem Israel d CF Center, Hadassah-Hebrew University Medical Center, Mt. Scopus Jerusalem 91240, Israel e Division of Adolescent Medicine Cincinnati Children's Hospital Medical Center, OH, USA Received 28 January 2013; received in revised form 11 March 2013; accepted 17 April 2013 Available online 13 May 2013
Abstract Background: Certain antibiotics may cause unwanted side effects due to the similarity of the mitochondrial translation system to the prokaryotic one. Children with cystic fibrosis (CF) are vulnerable to recurrent respiratory tract infections and azithromycin, a translation targeted antibiotic, is often used chronically to treat CF patients. No major clinical side effects were found with chronic treatment. However, mitochondrial function was not previously assessed. We evaluated oxidative phosphorylation (OXPHOS) in lymphocytes from children with CF receiving chronic azithromycin treatment using an improved ATP production assay. Method: Enzymatic activities of respiratory chain complexes II–IV and ATP production were measured in lymphocytes. Results: Relative to controls and to CF patients without azithromycin treatment, no significant difference in mitochondrial respiratory chain complexes II–IV was detected, and ATP production with pyruvate, glutamate and succinate, did not disclose any differences between the groups. Conclusion: We suggest that chronic treatment with azithromycin does not significantly affect OXPHOS function. © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Azithromycin; Chronic treatment; OXPHOS; Mitochondrial respiratory chain
1. Introduction Cystic fibrosis (CF) is the most common life shortening inherited disease in the population of European descent [1]. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in alteration in epithelial cell ion transport leading to increased sputum viscosity, stasis of secretions and impaired mucociliary clearance [2]. Dysregulation of respiratory epithelial cells, and innate immune dysfunction in ⁎ Corresponding author at: Department of Genetic and Metabolic Diseases, Hadassah-Hebrew University Medical Center, POB 12000, Jerusalem 91120, Israel. Tel.: + 972 2 6776844; fax: + 972 2 6 779018. E-mail addresses: [email protected], [email protected] (A. Saada).
the lung, leads to an exaggerated and ineffective airway inflammation which fails to eradicate pulmonary pathogens [3, 4]. The increased viscosity of the sputum impairs the effective clearance of inhaled microorganisms leading to recurrent endobronchial infections and bacterial colonization, mainly with Staphylococcus aureus and Pseudomonas aeruginosa, and resulting in an exaggerated inflammatory response that leads to the development of bronchiectasis and progressive obstructive airway disease [5]. Though abnormal CFTR function results in multi organ damage, the lung involvement has the most dramatic impact on quality of life and survival [6]. In the late '80s, it was shown that long term macrolide treatment substantially reduced morbidity and mortality in patients with diffuse panbronchiolitis [7, 8]. Since then, the potential benefit of macrolide antibiotics
1569-1993/$ -see front matter © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2013.04.006
S.B.-H. Negari et al. / Journal of Cystic Fibrosis 12 (2013) 682–687
has been evaluated in a variety of chronic respiratory diseases including CF [9]. Macrolides belong to the group of translation targeted antibiotic, interfering with protein synthesis, by reversibly binding to the 50s subunit of prokaryotic ribosomes and inhibiting the peptidyl transferase. The macrolide antibiotics achieve higher tissue than plasma concentrations as they penetrate the respiratory tissue and accumulate in the polymorphonuclear leukocyte extremely well [10]. Given their favorable bioavailability via oral route, their excellent tissue penetration and broad efficacy against many organisms affecting the lung, macrolides are used as first-line agents in the therapy of respiratory infections. Moreover, macrolides have documented to have wide spectrum of immunomodulatory effects on mammalian cells both in vivo and in vitro. These effects limit tissue damage by neutrophils, decrease mucus viscosity, and suppress angiogenesis, and support the rationale for using macrolide therapy for the treatment of chronic inflammatory airway diseases [11, 12]. Cochrane systematic review and meta-analysis of data from 10 clinical trials provide evidence that long term macrolide therapy in CF patients improves lung function, decreases frequency of pulmonary exacerbations, significantly reduce the need for oral antibiotics and improves nutritional status [13]. These studies have lead many CF centers, including ours, to treat patients with oral azithromycin. Macrolides are considered to be well-tolerated antibiotics; however, they have been associated with reversible ototoxicity [14], transaminitis and prolongation of the QT interval [11]. The most common side effect observed with Macrolide use is related to the GI tract and is thought to be a result of their ability to mimic the effects of motilin (a gastrointestinal tract polypeptide hormone) [10]. In the previously mentioned trials with CF patients, adverse events were uncommon and not obviously associated with azithromycin [9, 15–19]. Azithromycin along with many other antibiotics is targeted against the bacterial ribosome while sparing the eukaryotic translation system, as the cytoplasmic ribosome has a somewhat different structure. Contrarily, the mitochondrial translation system is separate from the cytosolic, resembling the prokaryotic, and the mitochondrial ribosome is more similar to the bacterial one [20]. Therefore it is not surprising that they can be affected by translation targeted antibiotics. In fact, side-effects such as ototoxicity, liver dysfunction and QTc prolongation can be a result of mitochondrial dysfunction [21]. Mitochondria are intracellular organelles, providing the vast majority of cellular energy in the form of ATP by oxidative phosphorylation (OXPHOS). The OXPHOS system, consist of the four respiratory chain complexes, consisting the electron transport chain (ETC) and the fifth complex ATP synthase. All complexes, with the exception of complex II, contain subunits encoded in the mitochondrial DNA (mtDNA) and are therefore dependent on intra-mitochondrial translation and the mitochondrial ribosome [22]. Hereto forth the effect of translation targeted antibiotics on mitochondrial function has been the subject of investigation in vitro but to our knowledge, it has not been systematically evaluated in children, and it is the objective of this study. As most children with CF in our CF center are chronically treated
683
with azithromycin as immunomodulator, we decided to focus our investigation on this group of patients. Since long term azithromycin treatment in CF patients infected with pseudomonas is becoming common, physician would benefit from an evaluation of such possible side effect. The mitochondrial function was evaluated in blood lymphocytes by measuring the enzymatic activity of the ETC complexes and by measuring ATP production by an improved method allowing minimal sample size. 2. Subjects, materials and methods 2.1. Subjects The study was done in the Hadassah medical center and approved by the IRB ethics committee. Exclusion criteria were refusal of the patient or his guardian, known mitochondrial disease, infection or malignancy. Blood samples were obtained by venipuncture with informed consent. Notably, For the CF patients, blood samples were drawn in conjunction with other periodic lab testing and no separate venipuncture was needed. Usual concomitant therapy, including physiotherapy, inhaled medications and pancreatic and vitamin supplements, continued throughout the trial and was monitored by the clinic physicians. 2.2. Materials Lymphoprep (Axis-Shield, Oslo, Norway) DMEM and fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), ATPlite (Perkin-Elmer Inc., USA), digitonin (Calbiochemicals, San-Diego USA) all other chemicals were of highest possible grade (Sigma-Aldrich USA). 2.3. Lymphocyte preparation Lymphocytes were prepared from 3–5 ml blood (drawn in EDTA) by differential centrifugation using Lymphprep (AxisShield Norway) according to the manufacturers' instructions. The lymphocyte suspension was divided into 2 aliquots in Eppendorf tubes and pelleted by centrifugation 1 min 10.000 rpm in a microfuge. The supernatant was removed and one batch was stored at − 70 °C for enzymatic assays. The other batch, for ATP production, was suspended in 0.5 ml freezing medium (glucose free DMEM supplemented with 40% fetal calf serum and 10% DMSO) placed in a freezing container (Mr. Frosty, Nalgene, Thermo Fischer Scientific Inc.), frozen at − 70 °C overnight and subsequently transferred for extended storage, into liquid nitrogen. 2.4. Enzymatic assays Enzymatic activities of complexes II–IV in lymphocyte homogenates were determined by spectrophotometry essentially as described [23–25]. Briefly, Complex II was measured as succinate dehydrogenase (SDH) based on the succinate-mediated phenazine methosulfate reduction of dichloroindophenol at 600 nm. Complex II + III was measured as succinate cytochrome
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c reductase, measuring the reduction of oxidized cytochrome c at 550 nm. Complex IV (cytochrome c oxidase) was measured by following the oxidation of reduced cytochrome c at 550 nm. Citrate synthase (CS), an ubiquitous mitochondrial matrix enzyme, serving as a control, was measured in the presence of acetyl CoA and oxaloacetate by monitoring the liberation of CoASH coupled to dithiobis (2-nitrobenzoic) acid at 412 nm. Protein concentration was determined by the Lowry method and calculated according to bovine serum albumin (BSA) standard curve. Measurements were performed on a dual beam UVKON spectrophotometer (Secomam, France). 2.5. ATP production ATP production was determined as described by Berger et al. [25] with the following modifications: lymphocytes were rapidly thawed by adding 1 ml phosphate buffered saline (PBS) and the suspension was microfuged for 1 min at 7000 rpm; the pellet was washed once with 1.5 ml permeabilization-substratemix buffer (potassium phosphate buffer 100 mM pH 7.4, KCl 150 mM, Tris25 mM pH 7.4 EDTA 2 mM, BSA fatty acid free 0.025%, ADP 1 mM, digitonin 40 μg/ml). The pellet was resuspended in 0.35 ml permeabilization-substrate-mix buffer and the reaction was started by adding 25 μl suspension to 25 μl permeabilization-substrate-mix buffer containing the substrates reaching the final concentrations of pyruvate 5mM + malate 1 mM (PM) or glutamate 5 mM + malate 1 mM (GM) or succinate 10 mM in the presence of 5 μg/ml rotenone (S). After an incubation of 30 min at 37 °C, the reaction was stopped by adding 25 μl ATPlite cell lysis solution (for background readings the cell lysis solution was added prior to the substrate). After 5 min shaking 50 μl was transferred to a white microtiter well plate and 20 μl substrate solution was added, after 10 min dark adaptation luminescence was read on a Synergy HT instrument (BioTek, USA). ATP production was calculated by comparing to a standard curve which was constructed on each separate occasion.
3.2. Evaluation of ATP production assay Initially we evaluated an improved version of the ATP production assay that we have previously utilized for studying the effect of anti epileptic drugs (Berger et al.). The major drawback with the initial method was the instability of the samples at − 70 °C. The gradual decrease of temperature and storage in liquid nitrogen, in the present study allowed the frozen lymphocytes to retain the ATP producing capacity for at least 6 months (results not shown). The ATP producing capability in lymphocytes was also stable in lymphocytes isolated from blood stored at room for up to 8 hours until lymphocyte preparation allowing the sample to be transported to the laboratory from the clinic. Immediate processing yielded a somewhat higher capacity while an overnight 15h delay between the blood drawing and lymphocyte preparation resulted in a severe drop in ATP production. Therefore, all samples in this study were processed between 4 and 8 hours (Fig. 1). The use of ATPlite in combination with microtiter well reader facilitated and shortened the assay process to less than 1 hour and allowed triplicate repeats with all substrates from a single batch of lymphocytes originating from 1.5 to 2 ml blood. 3.3. Mitochondrial function in patient and controls Lymhocyte ATP production capability with all substrates was not significantly different in patients chronically treated with azithromycin compared to the normal adult and pediatric control groups (Fig. 2). Rather ATP production was somewhat increased with pyruvate + malate and glutamate + malate relative to the pediatric control and the untreated CF groups. From the above data, we concluded that mitochondrial ATP production capability in lymphocytes is clearly not, negatively affected by chronic azithromycin treatment. On the other hand, the untreated CF group was slightly but not statistically significant decreased
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2.6. Statistics
P+M
Data are expressed as mean ± SEM, and the results were evaluated by one-way ANOVA to compare means using the IBM SPSS statistics program version 20. 3. Results 3.1. Characteristics of the subjects
ATP [nmol/30min/mg]
350
G+M
300
S
250 200 150 100 50
The study group consisted of 16 pediatric patients (12 years ± 4.4 years) diagnosed with CF treated with azithromycin 250 mg (below 36 kg body weight) and 500 mg (above 36 kg of weight) once a day for at least 3 years. The three control groups consisted of 29 normal adult samples, 14 pediatric samples (obtained from children visiting the outpatient clinics for various reasons but without any signs of infections, malignancy, CF or mitochondrial disorders), and from 5 children with CF but not treated with azithromycin.
0 0h
4h
8h
15 h
24 h
Fig 1. ATP production in lymphocytes isolated from stored blood. Lymphocytes from blood from a healthy adult control was isolated and frozen in freezing medium, immediately or after different time periods of storage at room temperature. Subsequently to storage in liquid nitrogen, the lymphocytes were assayed for mitochondrial ATP production capability with the different substrates; pyruvate + malate (PM), glutamate + malate (GM) or succinate (S). The results are presented as mean ± SEM of at least triplicate measurements.
S.B.-H. Negari et al. / Journal of Cystic Fibrosis 12 (2013) 682–687 250
control adult
A
*
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100
control adult
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* *
*
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* *
*
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Fig. 2. ATP production in lymphocytes from patients and controls. Lymphocytes from blood from healthy adult controls (control adult), control pediatric patients (control ped) pediatric CF patients (CF) and pediatric CF patients chronically treated with azenil (CF az) and control were assayed for mitochondrial ATP production capability with different substrates; pyruvate + malate (PM), glutamate + malate (GM) or succinate (S). The results are presented as mean ± SEM of at least triplicate measurements in each subject. *p b 0.05.
compared with the other groups, however no specific conclusions could be drawn due to the small sample size and large SEM. Notably the adult control group was not significantly different from pediatric control group; therefore normal adult control samples (which are more readily available than normal pediatric samples) will be suitable as controls in future studies (Fig. 2). The ATP production assays were complemented with enzymatic measurements of individual ETC complexes. CS served as an internal control and estimation of mitochondrial content. Mitochondrial content was significantly increased in the pediatric controls relatively to the adult controls and both CF groups were decreased relatively the pediatric controls. However there was no significant difference between CF patient treated or not treated with Azenil. This was also reflected, but to a lesser extent in COX activity but not in II + III or SDH activities (Fig. 3A). The decrease in COX was diminished when normalized to citrate synthase activity. The II + III and SDH activities were slightly increased in the CF groups (Fig. 3B). Taken together no adverse effect of chronic azithromycin treatment on the measured parameters was observed.
4. Discussion Several studies have shown the beneficial effect of long term azithromycin treatment on the clinical status of cystic fibrosis patients. Chronic treatment with azithromycin for CF patients especially those infected with P. aeruginosa was shown to improve lung function, to improve quality of life, to decrease pulmonary exacerbation rate and to reduce the need for hospital admissions and for antibiotic treatment. These trials did not report major adverse events related to long term therapy. These trials evaluated clinical status only; neither of them evaluated the effect on mitochondrial function.
0 CS
B
COX
II+III
SDH
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control adult 1.4
Activity [mU/mU CS]
P+M
control ped.
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CF CF az
1 0.8 0.6
*
0.4
* *
* 0.2 0 COX/CS
II+III/CS
SDH/CS
Fig. 3. Enzymatic assay of ETC complexes. Lymphocytes from blood from healthy adult controls (control adult), control pediatric patients (control ped) pediatric CF patients (CF) and pediatric CF patients chronically treated with Azenil (CF az) and control were assayed for enzymatic activities of ETC complexes II–IV. Succinate dehydrogenase (SDH), succinate-cytochrome c reductase (II + III), cytochrome c oxidase (COX) and the matrix control enzyme citrate synthase. Fig. 3A depicts the actual activities while Fig. 3B depicts the relative activities compared to citrate synthase. The results are presented as mean ± SEM. *p b 0.05.
The concern from chronic treatment with azithromycin for CF patients has been the possible inhibitory influence of macrolides on the DNAse activity as seen in vitro, since most of the CF patients are treated with DNAse [11]. Another concern which still needs to be proven by designated research is the emergence of macrolide resistance strains of S. aureus but there is lack of long term data [13]. Drug induced mitochondrial toxicity is gaining recognition within the pharmaceutical industry; there are some in vitro methods for assessment of mitochondrial damage but to date there is lack of validated biomarkers for identifying drug induced mitochondrial dysfunction in patients [26]. Notably, some of the biochemical parameters of mitochondrial damage can mimic CF exacerbation or an acute infection in a CF patient (lactic acidosis, raised urea and creatinine, raised liver enzymes and hypo/hyperglycemia). Our research is a pilot assessment of the mitochondrial function in patients chronically treated with azithromycin. The mitochondrial OXPHOS was measured by
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two parameters in the peripheral lymphocytes; enzymatic activity of the respiratory chain complexes II–IV and ATP production. The improved freezing technique and storage in liquid nitrogen allowed reproducible ATP production after up to eight hour delay before lymphocyte preparation and prolonged storage in liquid nitrogen. The use of ATPlite kit with microtiter plates was much more convenient than the previous assay [25]. The improved method facilitates future studies allowing comparative measurement of ATP production in repeated samples from the same individual taken at different time intervals and transported from distant clinics. The ATP production assays were complemented by enzymatic measurements of the individual ETC complexes. We were not able to accurately measure rotenone sensitive complex I activity (despite numerous efforts), due to interfering NADH dehydrogenases in the homogenate. Notably mitochondrial isolation from small blood samples is not feasible. Nevertheless ATP production from both pyruvate and glutamate is complex I dependent and sufficient to detect complex I defects [23]. Moreover ATP production from pyruvate is proportional to pyruvate dehydrogenase activity [27]. Lymphocytes isolated from patients and control groups were analyzed with the improved ATP production method as well as with the enzymatic assays. Taken together, the results disclosed no significant effect of chronic azithromycin treatment on mitochondrial function in children with CF was detected. Rather some parameters were improved compared to untreated patients. Regretfully as the majority of CF patients in our center are treated with azithromycin, we were not able to include more non-treated CF patients in this study to confirm the significance of this observation with respect to ATP production. CS measured in lymphocyte homogenate showed a moderate but significant decrease in mitochondrial content observed in all patients relative to pediatric controls. This seems to be related to CF per se and not azithromycin as both treated and non-treated patients showed the same decrease. Nevertheless this decrease did not affect the overall OXPHOS as measured by ATP production. These results are in accord with our pervious results data, showing that azithromycin did not significantly affect OXPHOS dependent control fibroblast cell growth while exerting only a modest effect on fibroblasts derived from patients with mitochondrial translation disorders. On the contrary, chloramphenicol, impaired growth of both control and patients cells, even though both antibiotics bind to the exit tunnel of the prokaryotic large ribosomal subunit. Moreover, azithromycin, a macrolide, affects in-vitro mitochondrial translation; it does so to a much lesser extent than other classes of translation targeted antibiotics such as the aminoglycoside gentamycin and the tetracycline derivative doxycycline [28]. The variations between the antibiotics are most probably due to minor differences between the mitochondrial and the prokaryotic ribosome as well as their ability to penetrate cell membranes. Taken together, the data suggest that azithromycin is relatively harmless to mitochondrial function, and we therefore conclude that chronic treatment with azithromycin does not significantly impair OXPHOS function in lymphocytes. Further investigation is warranted in a larger cohort and ,in other tissues to confirm our data [29].
Conflict of interest There are no conflicts of interest to declare. Acknowledgements Corinne Belaiche is acknowledged for expert technical assistance. This work was financed by the Hadassah-Hebrew University Joint Research fund (to A.T. and A.S.). The sponsor had no involvement in the study design, collection, analysis and interpretation of the data. References [1] Spagnolo P, Fabbri LM, Bush A. Long-term macrolide treatment for chronic respiratory disease.Eur Respir J 2012 [Electronic publication ahead of print] [PMID: 23180583]. [2] Donaldson SH, Boucher RC. Update on pathogenesis of cystic fibrosis lung disease. Curr Opin Pulm Med 2003;9:486–91. [3] Watt AP, Courtney J, Moore J, Ennis M, Elborn JS. Neutrophil cell death, activation and bacterial infection in cystic fibrosis. Thorax 2005;60: 659–64. [4] Pillarisetti N, Williamson E, Linnane B, Skoric B, Robertson CF, Robinson P, et al. Infection, inflammation, and lung function decline in infants with cystic fibrosis. Am J Respir Crit Care Med 2011;184:75–81. [5] Chmiel JF, Berger M, Konstan MW. The role of inflammation in the pathophysiology of CF lung disease. Clin Rev Allergy Immunol 2002;23: 5–27. [6] Cohen-Cymberknoh M, Shoseyov D, Kerem E. Managing cystic fibrosis strategies that increase life expectancy and improve quality of life. Am J Respir Crit Care Med 2011;183:1463–71. [7] Koyama H, Geddes DM. Erythromycin and diffuse panbronchiolitis. Thorax 1997;52:915–8. [8] Kudoh S, Azuma A, Yamamoto M, Izumi T, Ando M. Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. Am J Respir Crit Care Med 1998;157:1829–32. [9] Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, et al. Macrolide Study Group. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. Jama 2003;290:1749–56. [10] Jain R, Danziger LH. The Macrolide Antibiotics: a pharmacokinetic and pharmacodynamic overview. Curr Pharm Des 2004;10:3045–53. [11] Adam L, Friedlander RK. Albert Chronic Macrolide Therapy in Inflammatory airway disease. Chest 2010;138:1202–12. [12] Martinez FJ, Curtis JL, Albert R. Role of macrolide therapy in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2008;3: 331–50. [13] Southern KW, Barker PM, Solis-Moya A, Patel L. Macrolide antibiotics for cystic fibrosis.Cochrane Database Syst Rev 2011;12 [CD002203]. [14] Wallace MR, Lk Miller, Nguyen MT, Shields AR. Ototoxicity with azithromycin. Lancet 1994;22(343):241. [15] Wolter J, Seeney S, Bell S, Bowler S, Masel P, McCormack J. Effect of long term treatment with azithromycin on disease parameters in cystic fibrosis: a randomised trial. Thorax 2002;57:212–6. [16] Clement A, Tamalet A, Leroux E, Ravilly S, Fauroux B, Jais JP. Long term effects of azithromycin in patients with cystic fibrosis: a double blind, placebo controlled trial. Thorax 2006;61:895–902. [17] Saiman L, Anstead M, Mayer-Hamblett N, Lands LC, Kloster M, Hocevar-Trnka J, et al. Effect of azithromycin on pulmonary function in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2010;303:1707–15. [18] Equi A, Balfour-Lynn IM, Bush A, Rosenthal M. Long term azithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial. Lancet 2002;360:978–84.
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