A randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation

Study design

A randomised, double-blind, placebo-controlled trial of oral azithromycin, given in addition to conventional immunosuppression, was conducted at the Leuven University Hospital (Leuven, Belgium) with the approval of the institutional review board. Single-lung, bilateral lung or heart–lung transplant recipients who were ≥18 yrs of age were eligible. Patients were excluded if they had an intensive care unit stay of >30 days or had died <30 days post-transplantation, had an important bronchial stenosis, underwent retransplantation or multiorgan transplantation (lung plus a solid organ) or had undergone solid-organ or bone marrow transplantation in the past. In September 2005–December 2007, patients were offered enrolment if they met the study criteria at discharge after transplantation. Immediately after the provision of written informed consent, patients were randomly assigned to groups through permuted block randomisation in a 1:1 ratio by the Hospital Experimental Pharmacy (Leuven University Hospital) to receive either oral azithromycin or placebo for 2 yrs. Study treatment was initiated as soon as possible thereafter, but no more than 30 days later. Participants, nurses administering the study medication and principal investigators (L.J. Dupont and G.M. Verleden; both Katholieke Universiteit Leuven and University Hospital Gasthuisberg, Leuven, Belgium) were blinded to group assignment throughout the entire study period. All evaluations of outcomes were performed in a blinded manner by University investigators from the Laboratory of Pneumology (R. Vos and B.M. Vanaudenaerde; both Katholieke Universiteit Leuven and University Hospital Gasthuisberg). University investigators were solely responsible for the trial design, data collection, study management and data analyses presented in this article. The data and safety monitoring board (Clinical Trial Center, UZ Leuven, Leuven, Belgium) took no action regarding early trial cessation.

Administration of investigational medicinal product

Azithromycin (Zithromax™, azithromycin dihydrate, 250 mg hard gelatine capsule formulation) was purchased from Pfizer (Sandwich, UK) and overencapsulated by the University's Hospital Experimental Pharmacy. For placebo, lactose monohydricum Ph. Eur. was purchased from Fagron (Rotterdam, the Netherlands) and compounded into 300 mg hard gelatine capsules by the University's Hospital Experimental Pharmacy. Study medication was provided in numbered containers to the patients during routine follow-up visits at the outpatient clinic or during hospital admissions by a study nurse who also verified compliance and possible adverse events at each contact. The study drug was initiated at one dose a day for five consecutive days, followed by one dose three times a week on Mondays, Wednesdays and Fridays. Patients were instructed to continue treatment for 2 yrs. If a patient developed BOS, the study drug was stopped (without deblinding of the investigational drug) and the patient was initiated on open-label “rescue” treatment of azithromycin for ethical reasons, initiated at 250 mg daily for 5 days, followed by 250 mg three times a week.

Transplant monitoring and clinical management

Patients followed a typical, standardised outpatient care regimen after lung transplantation, with complete history and physical examination, blood work, urine, sputum and pharyngeal swab cultures, spirometry and chest radiography at twice-weekly intervals for the first two post-operative months, at weekly to biweekly intervals thereafter until 6 months post-transplantation, then every 2–4 weeks until the first post-operative year and, afterwards, life-long at intervals of 2–3 months. Surveillance bronchoscopy and bronchoalveolar lavage was routinely performed at specific time-points after transplantation (at discharge, and 3, 6, 12, 18 and 24 months post-transplantation, and later at intervals of 1 yr) in case of clinically suspected acute allograft rejection, infection or BOS. Transbronchial biopsies were routinely performed at discharge and 3 months post-transplantation in case of suspected acute rejection, infection or BOS. Bronchoscopic procedures and processing of specimens are comprehensively described in the supplementary material. Histologic rejection was defined according to established criteria 7. The presence of obliterative bronchiolitis was assessed at autopsy or on explant biopsies in case of retransplantation. Spirometry (Masterscreen; Jaeger, Hoechberg, Germany) was performed according to American Thoracic Society standards 27 and FEV₁ % predicted 28. BOS was defined as a sustained decrease in FEV₁ of ≥20% from the patient's maximum post-operative values in the absence of other causes 9. For patients initiated on open-label “rescue” treatment, response to azithromycin was defined as an increase in FEV₁ of ≥10% after 3–6 months of treatment and nonresponders were defined as having an increase of <10% or a further decrease 21. Survival data were obtained using mortality information in the Leuven University Hospital transplant database, in which all our lung transplant recipients since 1991 (n = 510 on January 1, 2010) are registered.

Therapeutic management

After discharge, both groups received conventional triple-drug immunosuppression with methylprednisolone, a calcineurin inhibitor (cyclosporine A or tacrolimus) and a cytostatic agent (azathioprine or mycophenolate mofetil), as comprehensively described in the supplementary material. Drug choice and dosing adjustments were made according to a standardised protocol at the discretion of the treating clinician on the basis of renal function, immunosuppressive trough levels, spirometry and biopsy results. Patients developing BOS were treated with azithromycin according to the open-label trial protocol and in the case of BOS progression with conversion of immunosuppressives or, less frequently, total lymph node irradiation or retransplantation.

All patients received conventional infectious prophylaxis for cytomegalovirus (CMV), Aspergillus spp. and Pneumocystis spp., as described in the supplementary material. CMV-related disease or pneumonitis was treated with Ganciclovir. Antibiotic treatment for bacterial infection was guided using bacterial cultures.

All patients received conventional prophylaxis for gastro-oesophageal reflux 29 and confirmed reflux was treated by a high-dose proton pump inhibitor, as described in the supplementary material. When the current study was initiated, no standardised protocol for the evaluation of reflux had been instituted at our centre; therefore, reflux was assessed either by pH impedance measurement or by gastroscopy at variable time-points during the first 2 yrs after transplantation.

End-points

Primary end-points were the prevalence of BOS and overall survival at 2 yrs after transplantation. Secondary end-points included the rate of histological acute rejection, lymphocytic bronchiolitis and pulmonary infection, and the prevalence of pseudomonal airway colonisation and gastro-oesophageal reflux, as well as change in FEV₁, and airway and systemic inflammation over time during the study.

Adverse events were defined as serious allergic reactions including skin reactions (rash, urticaria or Stevens–Johnson syndrome), angioneurotic oedema and anaphylaxis, cardiac arrhythmias (ventricular tachycardia or Torsades de Pointes), neurologic disorders (convulsions), gastrointestinal disorders including abdominal discomfort (nausea, dyspepsia, pain or cramps), diarrhoea or pseudomembranous colitis.

Statistical analysis

Power analysis stipulating a 30% difference in the prevalence of BOS after 2 yrs suggested enrolment of ≥82 patients based on a two-sided test (α = 0.05, β = 0.20), a BOS prevalence of 30% 2 yrs after transplantation and a dropout rate of 10%. The anticipated enrolment period was 3 yrs, based on a mean annual number of 39 transplantations in the preceding 3.5 yrs and an exclusion rate of 30%. Actual enrolment of 83 eligible patients could already be discontinued after 2.4 yrs due to a higher number of transplantations performed during this data collection period (mean annual number of 49 transplantations). Analyses were performed 2 yrs after the last subject had been enrolled, without unblinding of the randomisation to either the treating physicians or the enrolled patients and the study protocol specified a second analysis 3 yrs after enrolment of the last patient. All patients were in life-long follow-up and outcome variables were followed until the patient's death, independent of the continuation or discontinuation of study medication or the conclusion of the scheduled 2-yr study treatment period. Included patients were analysed according to the intention-to-treat principle and no patient was lost to follow-up.

Group means were compared using unpaired, two-tailed t-tests or Mann–Whitney tests for non-normally and normally distributed variables, respectively (Graphpad Prism 4.0 software, San Diego, CA, USA). The Chi-squared test was used to compare proportions. All reported p-values are two-sided and have not been adjusted for multiple testing. Kaplan–Meier estimates with log-rank tests were used for time-to-event analyses. For the end-point of BOS, survival times were censored at death, retransplantation or at study discontinuation if these preceded BOS, or else at 2 yrs after transplantation. For the end-point of death, survival times were not censored at retransplantation or at study discontinuation if these preceded death, or else at 2 yrs after transplantation. Additional Cox proportional-hazards analyses (SAS 9.1 Institute Inc. Software, Cary, NC, USA) were used to compare BOS-free survival and overall survival between groups with or without adjustment for type of transplantation (single or bilateral lung), recipient and donor age, recipient sex, recipient CMV mismatch status (positive donor to negative recipient), the rate of acute rejection (histological grade A), lymphocytic bronchiolitis (histological grade B), and CMV and non-CMV pneumonitis. The frequency or rate of specific events (i.e. acute rejection, lymphocytic bronchiolitis, CMV and non-CMV pneumonitis) was calculated by determining the number of events per yr of study time for each subject. Mixed-effect models were used to test longitudinal effects on repeated measurements of FEV₁, bronchoalveolar lavage neutrophilia and plasma C-reactive protein. Because within-individual repeated measures of outcomes are correlated, random effects were estimated at the subject level, nested within therapy group. To achieve normal distributions of neutrophilia and C-reactive protein, we applied Box–Cox transformations. Q-Q plots of the residuals, the residuals versus the fitted values and the residuals versus leverage were used to test the assumptions of all linear models.