Montelukast for bronchiolitis obliterans syndrome after lung transplantation: A randomized controlled trial

Bronchiolitis obliterans syndrome (BOS) remains the major problem which precludes long-term survival after lung transplantation. Previously, an open label pilot study from our group demonstrated a possible beneficial effect of montelukast in progressive BOS patients with low airway neutrophilia (<15%), and already on azithromycin treatment, in whom the further decline in pulmonary function was attenuated. This was, however, a non-randomized and non-placebo controlled trial. The study design is a single center, prospective, interventional, randomized, double blind, placebo-controlled trial, with a two arm parallel group design and an allocation ratio of 1:1. Randomization to additional montelukast (10 mg/day, n = 15) or placebo (n = 15) was performed from 2010 to 2014 at the University Hospitals Leuven (Leuven, Belgium) in all consecutive patients with late-onset (>2years posttransplant) BOS ≥1. Primary end-point was freedom from graft loss 1 year after randomization; secondary end-points were acute rejection, lymphocytic bronchiolitis, respiratory infection rate; and change in FEV1, airway and systemic inflammation during the study period. Graft loss at 1 y and 2y was similar in both groups (respectively p = 0. 981 and p = 0.230). Montelukast had no effect on lung function decline in the overall cohort. However, in a post-hoc subanalysis of BOS stage 1 patients, montelukast attenuated further decline of FEV1 during the study period, both in absolute (L) (p = 0.008) and % predicted value (p = 0.0180). A linear mixed model confirmed this association. Acute rejection, lymphocytic bronchiolitis, respiratory infections, systemic and airway inflammation were comparable between groups over the study period. This randomized controlled trial showed no additional survival benefit with montelukast compared to placebo, although the study was underpowered. The administration of montelukast was associated with an attenuation of the rate of FEV1 decline, however, only in recipients with late-onset BOS stage 1.


PROJECT SUMMARY
Lung transplantation is an accepted therapeutic option for patients with an end-stage lung disease. In Belgium a yearly average of 90 lung transplantations are performed (60% in Flanders). The long-term survival remains far below other solid organ transplantation with a median survival of only 5 years (1). This is attributed to chronic rejection or Bronchiolitis Obliterans Syndrome (BOS), which has an incidence of 10% per years and a prevalence of 50% after 5 years. Chronic rejection is the most important cause of late mortality after lung transplantation and leads to enormous morbidity and use of health care resources (2;3).
Few therapeutic options are available. Recently, we described a dichotomy within chronic rejection with a Neutrophilic Reversible Allograft Dysfunction (NRAD) phenotype and a Fibroproliferative BOS (fBOS) phenotype (4;5). Together with other groups, we demonstrated that NRAD accounts for 40% of chronic rejection and can be treated with the neo-macrolide azithromycin On the other hand, the remaining 60% of the patients not responding to azithromycin, "the fBOS phenotype", have no neutrophilic inflammation.
The current idea is that fibroproliferation alone drives this rapid deteriorating fBOS (6).
With this IWT-TBM project we aim to decrease chronic rejection (with 25%) and related mortality (with 15%) by tackling both phenotypes of chronic rejection: firstly, preventing neutrophilic inflammation with high dose Vitamin D, and secondly treating fibroproliferation (fBOS) with montelukast. Our proof of concept indicates the potential of this therapeutic strategy. Therefore, we will conduct two prospective, double-blind, randomized, placebo-controlled clinical trials to have the ultimate proof allowing to move forward to implementation in standard care for lung transplant patients.
In the first study, 100 patients will be randomised to Vitamin D or placebo after 1 month of transplantation and followed up for 2 to 3 years for the development of chronic rejection. In the second study, 30 patients with chronic rejection (fBOS) will be randomized to montelukast or placebo and followed for 1 to 2 years to evaluate survival and chronic rejection progression. 4 The project output will improved prevention and interventional therapy for chronic rejection after lung transplantation, which will: 1) decrease the incidence of chronic rejection, the progression of chronic rejection; 2) improve the long-term survival; 3) improve the quality of life and 4) decreased the direct and indirect use of health care resources. 5

PART II. PROJECT DESCRIPTION A. POSITIONING TOWARD THE TBM PROGRAMME
Few therapeutic options are available to treat chronic rejection and only azithromycin has proven to be really effective in 40% of affected patients (7). Moreover existing treatments are very expensive and not available in every centre. As a consequence, more intensive research is definitely needed to make further progress in the battle against chronic rejection after lung transplantation. Based on our current knowledge on chronic rejection, with its specific phenotypes and mechanisms, we want to target the most important mechanistic features of the two major phenotypes. It is our intention to prevent and to treat chronic rejection with adequate and tailored treatment strategies. Adding basic research will allow us to elucidate new mechanistical insights and perhaps explore new diagnostical tools again further tailoring the treatment strategy.
In this study we will focus on montelukast and vitamin D, as we recently observed a beneficial effect in a pilot study (a delay in the disease progression of chronic rejection) of montelukast in 11 patients with chronic rejection non-responsive to azithromycin.
Regarding vitamin therapy we observed that 60% of our lung tranplant patients have a 25-OH vitamin D deficiency. This project includes 2 parallel double blind placebo controlled randomized trials which may deliver absolute proof of its potential, allowing to progress to implementation in daily routine patient care and treatment.
In Belgium, around 800 lung transplantations have been performed since the first lung transplantation in 1969 by Prof F Derom. Nowadays about 90 lung transplantations are performed per year, of which 50 in UZleuven with a centre total of over 550. So the UZLeuven lung transplant centre, one of the top 10 most active lung transplant centres, represents a well positioned group and even the only one in Flanders/Belgium able to conduct this study and also able to later on implement this new strategy if succesfull.
Despite the fact that lung transplantation and hence, the development of chronic rejection, is rather infrequent compared with for instance COPD or asthma, the yearly costs are high.
In Belgium (Flanders=UZLeuven) the yearly cost for lung transplantation is about 29.6 6 million (17.7 million) euro of which 5.0 million (3.0 million) euro accounts for chronic rejection. These costs are enormous for an orphan disease but can be attributed to the frequent hospitalisations, extensive immunosuppressive treatment regimens and use of different diagnostic procedures (8;9). The impact of our previous research has already changed the concepts, the diagnostic procedures and the treatment strategies of chronic rejection not only in Belgium but also in the rest of the world. Our results with azithromycin have also changed concepts beyond transplantation in the respiratory field.
Currently a large clinical trail with azithromycin (AZISAST) in severe asthma is recruiting patients and basic research towards the potential of azithromycin to treat idiopatic pulmonary fibrosis is being conducted.
Despite all our efforts and encuraging results, it remains frustrating to obtain enough funding since lung transplantation is an "orphan" disease. Also the new study strategy of using "old, off label" medication already present for many years in other fields of (pulmonary) medicine (with generic drugs present on the market) prohibits interest from pharmaceutical companies. Our intention is only to improve patient care and to lower the health care costs. As a consequence, industrial interest is low or even non existing.
Because of all these arguments, we are confident that our project does fullfill all the criteria of the TBM programme.

B1. RATIONALE OF THE PROJECT
With this IWT-TBM project we will use a totally new treatment strategy to tackle the prevalence of chronic rejection and to treat it by interfering with both mechanisms involved namely neutrophilic inflammation and fibroproliferation.
With a median survival of 5 years, long-term survival for lung transplant patients remains far below other solid organ transplantation (10). This can mainly be attributed to the development of chronic rejection or Bronchiolitis Obliterans Syndrome (BOS). The incidence of chronic rejection is about 10% per year with a prevalence of 30% and 50% at 3 and 5 years post transplantation (Table I). Chronic rejection is the single most important cause of death accounting for 25-35% of the late mortality (Table I). Moreover, it leads to increased morbidity, loss of quality of life and increased use of health care resources (11). Few therapeutic options are available to treat chronic rejection and only azithromycin has proven to be really effective in some 40% of affected patients (13). Moreover existing treatments (such as photopheresis and total lymploid irradiation) are very expensive and not available in every centre. As a consequence, more intensive research is definitely needed to make further progress in the battle against chronic rejection after lung transplantation. The basis of this new therapeutic strategy is our recent findings of a dichotomy within chronic rejection. Up to recently chronic rejection was regarded as one homogeneous entity including two major elements: first, an abnormal inflammatory response of the airways and second, a fibroproliferative obliteration of these airways. Chronic rejection was clinically recognized by a persistent cough, sputum production and dyspnea and functionally assessed by a progressive loss of forced expiratory volume in one second (FEV 1 ) (14).  To further progress in the battle against chronic rejection, we need to address as well neutrophilic airway inflammation as well as fibropoliferation. The inflammation needs to be picked up and treated before the diagnosis of chronic rejection is made, at least when NRAD is concerned. With respect to the fBOS phenotype, we do not know yet what triggers the onset, but we do know that it may progress very fast with a steep decline in FEV 1 . As a consequence, the best we can do is trying to slow-down the progression.
Besides the known risk factors for the development of chronic rejection (HLA-mismatch, acute rejection episodes, receptor age, CMV and other viral infections), we and other 9 groups recently described new risk factors all acting via induction of a neutrophilic airway inflammation. These new risk factors are Pseudomonal colonisation, lymphocytic bronchitis/bronchiolitis, gastro-oesophageal reflux, air pollution, smoking and ischemiareperfusion injury (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). We also identified new markers of chronic rejection (the pH of exhaled breath condensate, exhaled carbon monoxide, airway neutrophilia and systemic Creactive protein) (28)(29)(30), which are all linked with innate airway inflammation with a prominent role for neutrophils (=NRAD phenotype). Patients who experience more neutrophilic inflammation after transplantation, become more prone to develop the NRAD phenotype of chronic rejection,. Therefore, new therapies that potentially prevent or treat this neutrophilic airway inflammation are mandatory, not only to reduce the severity but also the number of these events, which eventually may lower the prevalence of chronic rejection.
Current treatment strategies of chronic rejection have little effect and could at best arrest the deterioration of the FEV 1 and certainly do not discriminate for this dichotomy (31;32).
Only azithromycin has recently been demonstrated to have a beneficial effect on the FEV 1 evolution (33). Most of the current therapeutic agents focus on the adaptive immunity (lymphocytes) and on the fibroproliferation respons. Yet, most of these agents are toxic and associated with a lot of side-effect. Rapamycin, for instance, is a new anti-fibrotic agent which was not able to arrest the progression of chronic rejection, however, it was associated with an increase in infectious episodes (34). We rather believe that drugs with little or no side-effects such as azithromycin that specifically treat the innate inflammation and perhaps also affect fibroproliferation, are new targets we should focus on. Such drugs should not necessarily be newly designed agents, it is possible that renewed interest in some agents can be more useful (with azithromycin as the proof of the pudding). Therefore, we will focus on the potential role of Vitamin D and montelukast in prevention and treatment of chronic rejection.
The rational for the present study is based on this concept of the dichotomy, using a treatment and prevention option that better targets the 2 major mechanistic elements: neutrophilic inflammation and fibroproliferation. On top of this we want to use therapeutic agents which are less toxic, have few side-effects and are less expensive.

B2. STATE OF THE ART/ NOVELTIES
Vitamin D is generally accepted to be important in bone metabolism and calcium housekeeping. The choice to investigate Vitamin D as a potential chronic rejection-preventive drug is based on two important findings: firstly, it is know that mild vitamin D deficiency (revealed by low serum 25-OHD levels) is present in more than 50% of patients with advanced pulmonary disease and correlates with FEV 1 decline (COPD patients) and increased susceptibility for microbial infections (35). Secondly, vitamin D exerts pleotropic effects fully

11
The second aim of the study is to slow-down the progression of chronic rejection, especially the fBOS phenotype. As the present understanding of this phenotype is rather poor, there is a need for intensive research on this most lethal and frequent phenotype of chronic rejection, with a median survival of about 50% at 2 years after diagnosis ( Figure   II). ). LTA4 and LTB4 are involved in inflammation but cysteinyl-LTs (cysLTs) are more related to fibrosis mechanisms. CysLTs directly affect migration, proliferation, and extracellular matrix protein synthesis by fibroblasts (38). To target these elements we are interested in the use of montelukast, which is a cysteinyl-leukotriene1 receptor antagonist.
Montelukast is already widely used in the treatment of asthma where it reverses airway remodelling, including subepithelial collagen deposition (39). It has some antiinflammatory effects, especially on eosinophilic inflammation (40), but is also known as an antiproliferative agent (41;42). Moreover, in a recent pilot study montelukast improved pulmonary function in 60% of the patients with graft versus host disease after bone marrow transplantation, which is pathologically characterized as obliterative bronchiolitis, the hallmark of fBOS (43). As a consequence, a large NCI driven study with montelukast for OB associated with graft versus host disease after bone marrow or stem cell transplantation is ongoing in the States (clinicaltrials.gov NCT00656058).
To maximise the effect of montelukast, it is of crucial importance to start the therapy as early as possible after chronic rejection has been diagnosed. Therefore, randomization will occur as soon as fBOS is diagnosed (BOS grade 1 and a BAL neutrophilia <15%). We hypothesize that administration of montelukast may arrest the fibroproliferation and stabilize chronic rejection (and hence arrest the decline in FEV 1 ). As a consequence, we aim to reduce the BOS-related mortality. Mechanistical studies will accompany this clinical trial to give further insight into the underlying mechanisms allowing us to move forward in the treatment of this most devastating phenotype of chronic rejection.
As proof of concept, we investigated 22 lung transplant patients diagnosed with the fBOS phenotype of chronic rejection of which 11 patients received additional montelukast therapy and compared them to the 11 patients on standard therapy (figure III). In the montelukast group an arrest in the FEV 1 decline from 112±26 mL/month before BOS diagnosis to 13±13 mL/month (p=0.001) 6 months after diagnosis was observed. In the control group, we found no change in the rate of FEV 1 decline: 103±20 mL/month before BOS diagnosis versus 114±17 mL/month (p=0.74) after diagnosis was observed. The decline in FEV 1 during 6 months of montelukast treatment was different compared to the FEV 1 decline of the control group in the same period (p=0.001), whereas the 6 month decline before chronic rejection was not different between the control and montelukast group (p=0.74). This study is submitted for publication. After the start of the study drug, the decline in FEV 1 was arrested (p=0.001) in the MLK group while the control group showed no difference (p=0.74). The decline after diagnosis of fBOS was significant lower in the montelukast group (p=0.0025). 13 This project does not concern patents at all. Also this project has no connection with any other ongoing IWT project.
In summary: The vitamin D study starting just after lung transplantation is based on the presence of vitamin D deficiency within severe COPD patients (candidates to be listed for transplantation and representing 60% of the transplant population) and within a cohort of our own lung transplant patients. We hypothesize that vitamin D can downregulate important triggers like reflux, autoimmune reaction, dendritic cells, IL17 producing T cells, finally leading to a decreased airway neutrophilia and consequently a reduction of the prevalence of chronic rejection (especially the NRAD phenotype). Second, we will treat lung transplant patients diagnosed with chronic rejection (BOS grade 1 and 2, but not 3) with montelukast to arrest the fibroproliferation and deterioration of the disorder (specifically the fBOS phenotype).
It is our goal to find an optimal tailored treatment strategy for both phenotypes.

B.3 INNOVATION/AIMS
The primary goal of this project is to decrease the prevalence of chronic rejection and hence to increase the long-term survival after lung transplantation.
First aim: Vitamin D substitution to prevent chronic rejection Reduce chronic rejection with 25% Second aim:

Montelukast as rescue treatment for chronic rejection (fBOS) Reduce the chronic rejection related mortality with 15%
Within these treatment strategies we will investigate clinical outcome measures, BAL biopsies and blood to understand the underlying mechanisms and to find risk factors and early markers allowing even earlier and better diagnosis.
To study the therapeutic effect of Vitamin D and montelukast in the prevention and treatment of chronic rejection after lung transplantation we will conduct two parallel prospective placebo-controlled double blind randomised studies in lung transplanted patients. Furthermore, we want to explore the mechanisms of action of both Vitamin D and montelukast and the mechanism of chronic rejection. The combination of the clinical trial and the mechanistical research will allow us to explore whether Vitamin D and/or montelukast tackle one specific phenotype of chronic rejection allowing us to introduce a more tailored treatment strategy. 14 The duration of both studies is calculated to be 4 years with a reanalysis at 5 years.
Considering that the start of the study will be in October/November 2010 this project is conforming the duration range of the TBM period. The proof of concept study with the vitamin D deficiency in lung transplant population and the response to montelukast treatment for the fBOS phenotype indicates that this novel therapy strategy will likely be efficient for lung transplant patients.
Unique to our research centre, the study and the treatment strategy is that if this study would be successful, it will be instantaneously without any delay implemented in the standard patient care and treatment in our centre. One could consider government refunding as an issue but one needs to consider that these treatments are available as generics and moreover these therapies are mostly less expensive compared to other commonly used and experimental immunosuppressive therapies.

C.1.IMPACT OF THE STUDY
Our aim is to validate the therapy for preventing chronic rejection with vitamin D and treating chronic rejection (the fBOS phenotype) with montelukast as new gold standard of care in lung transplantation.
Our findings will have an immediate socio-economic impact.
As mentioned before, despite being an orphan disease, lung transplantation and chronic rejection cause a lot of morbidity/mortality and use of health care resources (see above). Yearly One could consider government refunding as an issue but one needs to consider that these treatments are available as generics and moreover these therapies are less expensive compared to other commonly used and experimental immunosuppressive therapies.

C.2. THE UTILISATION OF THIS PROJECT:
All lung transplant patients could benefit from the implementation of vitamin D to the standard care to prevent chronic rejection, and even if they do develop chronic rejection the patients can benefit from the effect of montelukast to stabilize the progression of chronic rejection. In this way we aim to improve the overall survival and quality of life of our lung transplantation cohort.
Our project will have an immediate medical impact for the individual patient. 16 If this study would be successful, the results would be instantaneously implemented, without any delay or hurdles to be taken, in the standard therapy in our centre. This is the biggest advantage of our lung transplant research centre where research findings can be without any delay translated to patient care and treatment, and that is why this project should be well considered for funding.
In It would be our goal to reach the same level with this study as we were able to reach with our findings on azithromycin which is now becoming standard therapy not only at UZLeuven and in Belgium but also world-wide. 17 Besides these direct utilisation potential also indirect potential needs to be considered as chronic rejection after lung transplantation serves as a model for other chronic lung disorder.
Our results with azithromycin have also been translated to other disorders like COPD, idiopatic pulmonary fibrosis, severe asthma (the AZISAST study) and bone marrow transplantation making the potential impact of our research on health care enormous.

C.3. INTELLECTUAL PROPERTY RIGHTS OF THE PROJECT
This project is not associated with own patent or intellectual rights.
There is freedom to operate for the utilization of the project results.

D1: A PREVENTION STUDY WITH HIGH DOSE VITAMIN D SUPPLEMENTS
For the prevention study with Vitamin D, after informed consent, patients will be To have a statistical power of 80% (α = 0.05; β = 0.20) to detect 25% reduction in chronic rejection (BOS1) after 2 years we estimate that 72 patients are needed. Considering the exclusion criteria (30%) and the drop-out (10%), a total population of 100 patients need to be enrolled in the study. The start of recruitment is October/November 2010. Important to mention is that, from previous experience, almost 100% of our patients are willing to participate in a study with a centre average of about 50 lung transplantations per year, the inclusion will take about 2 years. With a 2 and 3-year follow-up, the end of this part of the study will be reached after 4 to 5 years.

WITH AZITHROMYCIN
After informed consent, patients will be randomised at the time of diagnosis of chronic rejection (fBOS phenotype) to the montelukast (10 mg/day) or the placebo arm of the study.
This is a single centre prospective, interventional, randomized, double-blind, placebocontrolled trial in a clinical setting (tertiary University Hospital), which is investigatordriven and without a pharmaceutical sponsor. Lung transplant recipients will receive addon of study-drug (over-encapsulated placebo or montelukast) with a study-drug regime of 10 mg/day of montelukast until the end of the study-period to "standard of care" including azithromycin. The placebo: montelukast inclusion ratio is 1:1. The start of recruitment is November 2010 and with a centre average of 10 diagnosis of chronic rejection (specifically fBOS) per year, the inclusion will take about 3 years. Again, important to mention is that, from previous experience, almost 100% of our patients are willing to participate in a study. With a 1 and 2 -year follow-up, the end of this part of the study will be reached after 4 to 5 years.

D3. SPECIFIC DETAILS AND BASIC RESEARCH MODALITIES
As soon as the project will start patients will be included and randomised post transplant to the Vitamin D or placebo group or at diagnosis of chronic rejection to the montelukast group or to the placebo group. The lung transplant team will supply the study-drug to the patients via the routine follow-up at the outpatient clinic and at hospital admissions. There will be no additional costs for the patients or the hospital, since all examinations (except one) that will be performed (blood samples, lung function, X-rays, CT scans, fibre-optic  The Leuven Lung Transplant team also gathered substantial research funding over the last 5 years allowing some stability in the research projects. This has also led to multiple collaborations nationally and internationally. Lung transplantation at UZLeuven 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 These figures demonstrate that we are perfectly able to recruit the aforementioned numbers of patients for our study.

Official approval by the ethical commission:
Both arms of the study have been prepared separately and approved by the ethical comity of the

Other financial support:
This clinical study with an important research component is based on the extended expertise in a specific field of the project. This project is supported by the other projects within the lab certainly at the start as the studies are planned to start at 09/2010. But the requested funding is essential to accomplish the study.

Track record:
(i) Relevant publications: