Acute and chronic graft-versus-host disease (GvHD) is the primary causes of morbidity and mortality among patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT). Although the pathophysiology of GvHD has not been fully elucidated, it is well known that acute GvHD involves an immunocompetent donor T-lymphocyte attack on recipient cells, causing inflammation (1). The gastrointestinal (GI) tract, skin, and liver are the most common targets of GvHD, and thus skin rash, diarrhea, jaundice, and wasting are its most common manifestations. Chronic GvHD has features resembling autoimmune and other immunological disorders such as scleroderma, Sjögren syndrome, bronchiolitis obliterans, and chronic immunodeficiency, whose symptoms are often preceded by acute GvHD with possible multiorgan involvement. Although controversial, recent studies suggest that noninfectious lung injury occurring early after allo-HSCT, which is associated with significant morbidity and a poor response to therapy, may also be a manifestation of acute GvHD (2). In general, the clinical manifestations of bronchiolitis obliterans, compatible with GvHD, are part of the extensive chronic stage of the disease.
Inflammation plays a pivotal role in the underlying mechanism of the complex pathogenesis of GvHD. Eosinophil activation contributes to the onset and maintenance of tissue inflammation in several disease processes (3). Indeed, recent studies have shed some light on a potential role for eosinophils in the activation of GvHD (4).
The cysteinyl leukotrienes (cysLTs) LTC4, LTD4, LTE4, and LTB4 have important bronchoconstrictive and proinflammatory effects through their interactions with the cysLT1 and cysLT2 receptors (5). In particular, they appear to stimulate chemotaxis of eosinophils (6), increase small vessel permeability (7), and promote plasma leakage and leukocyte adhesion in postcapillary venules (8). They also regulate cytokine pathways during allergic inflammation (9). Multiple studies have shown that cysLTs influence the proliferation and differentiation of CD34+ progenitor cells through cysLT1 (10, 11). In addition, it was recently shown that cysLTs are potentiators of dendritic cell (DC) function, including antigen (Ag)-presenting capacity and cytokine production. Therefore, cysLTs and LTB4 seem to be major players in the initial stages of the immune response (12).
The leukotriene antagonists (LTRAs) are a class of drugs that block leukotriene receptors. Clinically, LTRAs have been used successfully to decrease the severity of chronic asthma, allergic rhinitis (13, 14), atopic dermatitis (15) and eosinophilic gastroenteritis (16). Sodium montelukast (Mk; Singulair, Merck) is an orally active LTRA that binds with high affinity to the cysLT1 receptor. A significant reduction in eosinophils in children with mild cystic fibrosis was observed after 21 days treatment with Mk (17).
Okunishi et al. (18) recently found that administration of the LTRA pranlukast in a murine model of asthma suppressed the activation of DC functions by cysLTs, and thereby indirectly inhibited subsequent Th1 and Th2 responses, as well as Ag inhalation-induced eosinophilic airway inflammation. These studies indicate that LTRAs may be useful in the suppression of the initial immune response in many immune- mediated disorders.
Thus, although the involvement of cysLTs in GvHD has not been fully investigated, the similarities between chronic GvHD and autoimmune disease—as well as the central role of initial host Ag-presentation, subsequent Th1 or Th2 activation, and eosinophilic infiltration in the pathophysiology of GvHD—provide the rationale for the use of LTRAs in patients with refractory chronic GvHD in order to suppress its development and progression.
On this basis, we added Mk to standard immunosuppressive therapy in 19 patients with steroid depended chronic GvHD.
PATIENTS AND METHODS
Study Population
Our prospective cohort consisted of 19 patients meeting the criteria for chronic GvHD after allogeneic HSCT from major histocompatibility complex–matched siblings in the Bone Marrow Transplantation Department at the Hadassah-Hebrew University Medical Center in Jerusalem, Israel between November 1998 and January 2004. The patients' clinical characteristics and GvHD involvement are described in Table 1. The patients were eligible and consecutively recruited for Mk supplementation based on standard chronic GVHD (cGVHD) scoring. For the retrospectively analysis of the study, we adapted the inclusion criteria of patients meeting the National Institute of Health Consensus Development Project for chronic GvHD (19). In addition, all patients' primary therapy included steroids with or without calcineurin inhibitor, azathioprine, thalidomide, and mycophenolate mofetil, whereas steroids could not be tapered off after a 3-month minimum period of therapy. Median period of persistent cGVHD prior to initiation of Mk therapy was 15 months (ranged 3–60 months). Each transplanted patient signed an approved informed consent.
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TABLE 1: Clinical characteristics of the patients
Diagnosis and Grading of GvHD
Global assessment of acute and chronic GvHD was based on standard clinical criteria and biopsy results according to the Severity Indices of the International Bone Marrow Transplantation Registry (19, 20). The acute and chronic GvHD grade was determined by the highest score in each complication. The diagnosis of cGVHD for each subject, was based on the scoring of each organ's involvement with typical manifestation, from day 100 posttransplant, and retrospectively analyzed according to the National Institutes of Health Consensus Development Project (19).
Mk was continued if the patient remained stable or showed improvement in the manifestations of chronic GvHD. In those patients whose GvHD grading was improved, other immunosuppressive medications, especially steroids, were gradually tapered off. The overall clinical response to Mk and outcome of chronic GvHD was assessed by clinical and laboratory measurements (21).
The primary and secondary endpoints were determined for each organ. Skin GvHD improvement and moderate improvement were considered >50% and 10–20% reduction in body surface area involvement, respectively. Liver function test improvement and moderate improvement was determined as >75% and <50% reduction in bilirubin and liver enzymes, respectively. Gastrointestinal tract improvement and moderate improvement was determined by elimination of symptoms and a gain in weight. Lung assessment was based on improvement of lung function tests (an increase of 30% in forced expiratory volume in 1 second).
Montelukast Protocol
Oral Mk, 10 mg once daily, was administered to patients with chronic GvHD without improvement after standard immunosuppressive therapy. All patients remained on their previous immunosuppressive regimen, including steroids, cyclosporine in combination with or without thalidomide, cyclophosphamide, azathioprine (Imuran), mycophenolate mofetil (Cellcept), and/or tacrolimus (Prograf) (Table 2).
TABLE 2: Treatment details and GVHD outcomes
In 15 patients, chronic GvHD developed spontaneously after acute GvHD, one patient (no. 3) suffered from de novo chronic disease, whereas in three patients (no. 4, 13, and 18) GvHD was induced primary in its chronic form after donor lymphocyte infusion (DLI).
RESULTS
Our patient population study group consisted of 19 patients, 12 males and 7 females, average age 38.5 (range 15–64) years, who underwent allogeneic HSCT from human leukocyte antigen–matched sibling, for a variety of hematological malignancies, including acute myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, chronic myelogenous leukemia, malignant melanoma, myelodysplastic syndrome, and thalassemia major (Table 1). Of the total 19 patients, 15 suffered from acute GvHD, including 10 who were diagnosed with grade I or II acute GvHD, three with grade III, and two who suffered from grade IV acute disease. All 19 patients suffered from extensive chronic GvHD. A total of 17, 19, and 16 patients showed skin, liver, and GI involvement, respectively, and five patients also suffered from lung involvement (Table 1).
Scoring of organ-specific chronic GvHD prior to Mk treatment is presented in Table 1. The median treatment period with Mk was 12 months (ranging from 2 to 30 months). Patients continued taking their immunosuppressive agents when Mk treatment was initiated; all patients were on steroids, 14 were taking cyclosporine, eight azathioprine, six thalidomide, two mycophenolate mofetil, one cyclophosphamide, and one tacrolimus.
During the combined treatment with Mk, the dosage of the immunosuppressive agents was reduced in responsive subjects. In patients with significant and resolved chronic GvHD, steroids were tapered off completely. In patients with moderate improvement, the steroid dosage was reduced by more than 50%.
The improvement under the combined treatment with Mk was not organ-specific and various systems showed significant amelioration.
In 3 of the 19 patients, there was resolving of liver involvement prior to initiation of Mk therapy. Of the 16 patients with active liver involvement at the time of Mk initiation, eight (50%) showed significant improvement in hepatic GvHD (>50% reduction of serum liver enzyme levels) while taking Mk, five (31%) showed a moderate improvement, in two (12%) hepatic GvHD was resolved, and one (6%) showed no change in the hepatic GvHD course.
With respect to GI involvement of the GvHD, in 3 out of the 16 patients, this resolved clinically prior to initiation of Mk. Of the other 13 patients there was complete resolution in three (23%), three (23%) improved significantly, four (30%) showed moderate improvement, and three (23%) patients did not show response to the supplementation of drug.
Of the 19 patients, 17 had GvHD of the skin. Of these, two (11%) with active skin involvement at the time of Mk initiation showed complete resolvement while taking Mk. Of the remaining patients, eight (47%) improved significantly while on Mk, five (29%) moderately, and two (11%) showed no response. Overall, 15 (88%) out of the 17 patients with active skin involvement at the time of Mk initiation showed clinical response.
Three of the five patients with lung involvement showed an objective improvement in pulmonary function test (PFT) values after initiation of Mk treatment; of which one improved mildly and two (patients 5 and 16) improved dramatically. Patients 10 and 17, both of whom had end-stage bronchiolitis obliterans at the time of Mk initiation, continued to deteriorate while on Mk and eventually died. It should be noted that in the remaining 14 patients without lung involvement while starting Mk, progression of the GvHD to the respiratory tract was not observed.
The overall clinical response of refractory chronic GvHD to Mk supplementation (Table 2) was based on clinical and laboratory evaluation. In 15 of 19 (79%) patients, there was an improvement in GvHD after Mk was added to their basic immunosuppressive regimens; in four (21%), the signs of chronic GvHD were resolved, two (10%) showed significant improvement, and nine (47%) showed moderate improvement of chronic GvHD. The four patients unresponsive to Mk treatment had resistant extensive GvHD. In two out of these four patients, the lung manifestation continued to progress. Of the total 19 patients, 16 are alive, whereas three died. Patients 10 and 17 died of end-stage bronchiolitis obliterans, whereas patient 8 died from a second malignancy (Table 2).
DISCUSSION
The results of our pilot study with 19 patients indicate that Mk is an effective supplementary agent in the treatment of chronic extensive drug-dependent GvHD and its influence on skin and liver is greater than on the GI symptoms of the disease. Mk treatment was more effective when administered in early stages rather than after longstanding resistant chronic GvHD. Furthermore, this preliminary investigation supports the potential role of Mk as a safe and toxicity-sparing supplement to standard therapy for patients with limited and extensive but drug-responsive disease. The known benefits of Mk treatment in respiratory conditions appear to be effective also for other organs involved in chronic GvHD. It bears mention that as described before (22, 23), no patients reported side effects from Mk supplementation.
The rationale behind the use of Mk addition in chronic GvHD is based on its mechanism of action and the pathogenesis of the disease. In a recent review, Iwasaki (24) described possible mechanisms for acute and chronic GvHD. It is considered that acute GvHD may be triggered before HSCT, when damaged tissues release inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1, and IL-6. These cytokines then increase the expression of host antigens which, in turn, are recognized by donor T cells, leading to T-cell activation and differentiation into the Th1 phenotype. Th1 cells then secrete interferon-gamma and IL-2, which expand the cytotoxic T cell and natural killer populations, while macrophages release inflammatory cytokines in response to endotoxins released from the damaged GI tract, resulting in damage to various host tissues. Chronic GvHD presents after day 100 postHSCT with symptoms similar to those seen in autoimmune diseases, and is thought to be mediated by alloreactive donor T cells that escape negative selection in the thymus because of damage caused by preconditioning or acute GvHD (24).
These CD4+ T cells may then differentiate into the Th2 phenotype and help B cells to synthesize alloantibodies against host tissue antigens. During acute GvHD, there is an increase in the number of eosinophils in the peripheral blood, the severity of its clinical presentation correlating with the histological eosinophilia in the biopsy from the GI tract (4). Furthermore, the hypereosinophilia in early bone marrow smears postHSCT may be predictive for the evolution of acute GvHD (25).
Stelmach et al. (26) showed that a 6-week treatment with Mk caused a clinically significant decrease in the serum concentration of the inflammatory markers IL-4, sICAM-1, eosinophilic cationic protein, and of eosinophil blood counts in children with asthma. Furthermore, Braccioni et al. (27) found that LTD4 can stimulate proliferation of peripheral blood and bone marrow eosinophil-basophil colony forming units (Eo-B-CFU) in atopic subjects, both of these processes were successfully suppressed by Mk. A subsequent study by Mao et al. (28) indeed concluded that the major effect of Mk might be its action on the eosinophils. Recently, Gelfand and Dakhama (29) described a novel interaction between CD8+ T lymphocytes and LTB4, in the persistent and progressive stages of asthma. They found that T cells express a high affinity receptor for LTB4 and that the expression of this receptor is essential to their accumulation in the lung and development of airway inflammation. In addition, it was suggested that Mk. inhibits eosinophil protease activity in vitro through a mechanism that might be independent of its antagonist effect on the CysLT1 receptor (30). These studies could partially explain the complexity of Mk anti-inflammatory effects in asthma and eventually extend its application to other immunological disorders such as GvHD.
Based on the known mechanisms of Mk, our preliminary results support the possible role of Mk therapy for GvHD of the lungs as well as of other affected organs. It is of interesting the positive impact of the drug on the other organs except of the respiratory system. Regarding the lungs, the use of the drug may serve as a major prevention approach for the development of bronchiolitis obliterans, which is associated with significant morbidity and mortality. This by itself should be considered as a randomized control study.
Although the results of our study are promising, there are several limitations that needed to be mention. This prospective cohort included only 19 heterogenic patients, with different localization of GVHD and a large variety of the immunosuppressive regimens used prior to Mk supplementation. This heterogeneity restricted our ability to compare the patients to an appropriate control group, but each patient constituted as internal control.
Our study proved the feasibility and safety of the drug and satisfactory efficacy that should be a base for future studies. Dosages of Mk higher than for asthma (10 mg twice daily instead of once a day) might be considered to further enhance the efficacy. The general therapeutic approach for chronic GvHD tends to shift the balance between the innate immune system (with its inflammatory components such as eosinophils, neutrophils, monocytes) and the adaptive arm (B and T cells) of the immune system towards the latter, whereas the inflammatory components are neglected as therapeutic targets for chronic GvHD. Therefore, Mk seems to be an attractive candidate for the therapeutic armentarium in the treatment and prevention of GvHD, especially sparing the toxic effects of prolonged and high dose steroid treatment. Future clinical studies are necessary to establish the role of Mk in the symptomatic treatment and prevention of GvHD after HSCT. Furthermore, additional studies should investigate the role and efficacy of other leukotriene receptor antagonists.
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