Published ahead of print on October 24, 2002, doi:10.1164/rccm.200205-455OC
© 2003 American Thoracic Society
In Vivo and In Vitro Effects of SAR 943, a Rapamycin Analogue, on Airway Inflammation and RemodelingNovartis Respiratory Research Centre, Horsham, United Kingdom Correspondence and requests for reprints should be addressed to Alexandre Trifilieff, Novartis Respiratory Research Centre, Wimblehurst Road, Horsham RH12 5AB, UK. E-mail: alexandre.trifilieff{at}pharma.novartis.com
No current therapy is considered to be satisfactory for severe asthma, and alternative approaches are still required for what is a major unmet medical need. In this study, we compared the effect of a rapamycin derivative, SAR 943, with budesonide, using a murine model of lung inflammation and remodeling. Allergen challenge of ovalbumin-sensitized BALB/c mice induced an increase in the levels of interleukin-5 and interleukin-4; numbers of eosinophil, neutrophil, and lymphocyte; cellular fibronectin; lung epithelial cell proliferation and mucus hypersecretory phenotype; as well as hyperreactivity to methacholine. Both SAR 943 and budesonide, when given intranasally 1 hour before and 24 hours after the aerosol challenge, inhibited all of these parameters with a similar potency (effective dose 50% of 1 mg/kg). In primary cultured smooth muscle cells from human airways, SAR 943 dose dependently inhibited epidermal growth factor–induced proliferation but did not affect the basal cell proliferation. Neither the basal nor stimulated proliferation of a human bronchial epithelial cell line (16HBE14o-) was affected by SAR 943. In conclusion, SAR 943 is as effective as budesonide in inhibiting both lung inflammation and remodeling in a murine model of asthma. Hence, this class of compound could offer beneficial effects in patients with severe asthma.
Key Words: mice • inflammation • remodeling • immunosuppressant
An unbalanced immune response and ensuing inflammation are acknowledged to be major contributors to the initiation and chronicity of the asthmatic process, as they are the basis of a cascade of cellular and mediator interactions resulting in a pulmonary inflammatory cell infiltrate (1). In addition to the inflammatory component, structural changes in the architecture of the airways have been reported. These changes include basement membrane thickening caused by collagen and fibronectin deposition, fibroblast proliferation, airway smooth muscle thickening as a result of both smooth muscle cell hyperplasia and hypertrophy, and excessive production of mucus glycoproteins (2). These modifications, collectively termed lung remodeling, lead to the thickening of the airway wall, which in turn could explain the chronic irreversible hyperresponsiveness that has been observed in this disease (3). Although inhaled corticosteroids and ß2 adrenergic agonists remain the drugs of choice for the majority in the treatment of chronic asthma, there remains a small group of patients who are characterized by ongoing symptoms and who experience frequent exacerbation despite the use of existing therapies. These patients, referred to as severe or difficult-to-treat subjects with asthma, are often treated with high doses of inhaled corticosteroids and/or oral corticosteroids and are at risk of developing unwanted side effects (4). Therefore, the identification of a novel therapy for these patients is urgently needed. One approach to treat these patients has been to use T cell immunomodulators such as cyclosporin A, a powerful immunosuppressant agent that is used mainly for the prevention of rejection during organ transplantation. In human studies, cyclosporin A has been shown to be a corticosteroid-sparing agent in severe asthma. However, this was associated with side effects such as an increase in diastolic blood pressure and a decrease in renal function (5, 6). Thus, the current therapy for severe and very severe asthma presently available is less than satisfactory, and an alternative approach is regarded as a major medical need. Rapamycin, an immunosuppressive macrolide, has attracted interest in recent years because of its potential in the treatment of various immunologic disorders, including asthma (7). In addition to its immunosuppressant properties, rapamycin also inhibits smooth muscle cell proliferation (8, 9), an important feature of remodeling. Unfortunately, this natural product exhibits unfavorable physicochemical properties. As a consequence, its formulation and administration in an appropriate therapeutic form have been proven to be rather difficult. SAR 943 (32-Deoxorapamycin), a novel rapamycin derivative with immunosuppressive properties, has been identified out of a series of rapamycin derivatives with chemical modifications designed to increase the chemical stability in galenical formulation (10). In view of its biologic properties, it is speculated that SAR 943 has a dual target in asthma, as it has the potential to work as an antiinflammatory and antiremodeling drug. To test this hypothesis, we have studied the effect of SAR 943 in a murine model of allergen-driven lung inflammation that presents some aspects of the remodeling seen in human asthma (11) and compared its potency with budesonide. Data illustrating the antiproliferative effect of SAR 943 on human epithelial and smooth muscle cells were also studied in vitro and are presented.
Animals and Experimental Design The studies reported herein conformed to the UK Animals (scientific procedures) Act of 1986. All of the procedures used in this article have been previously described by this laboratory (11, 12). In brief, female BALB/c mice were immunized on Days 0 and 14 and were exposed to a single aerosol of ovalbumin on Day 21. On Day 20, ALZET minipumps filled with 5-bromo-2'-deoxyuridine (10 mg/ml) were implanted subcutaneously in the scapular region of some of the mice. SAR 943 and budesonide were dissolved in dimethyl sulfoxide and then diluted with phosphate-buffered saline (PBS) (final concentration of dimethyl sulfoxide of 2%). Animals were dosed intranasally under halothane/oxygen/nitrous oxide anesthesia, with either test compound or vehicle (50 µl) 1 hour before and 24 hours after the aerosol challenge. On Day 22, airway hyperreactivity to aerosolized methacholine (0.1 M) was measured using barometric plesthysmography. Cytokine levels were determined (interleukin [IL]-4, IL-5, and interferon-  ), and differential cell counts were performed in bronchoalveolar lavage fluids. On Day 23, bronchoalveolar lavage differential cell counts and cellular fibronectin levels were measured. On Day 28, lungs were fixed with formalin, and 5-bromo-2'-deoxyuridine immunostaining and alcian blue/periodic acid Schiff staining were performed. Epithelial 5-bromo-2'-deoxyuridine nuclear labeling and mucin staining indexes were expressed as a percentage of positive cells versus total cells in at least 10 randomly chosen airways.
Cell Culture Smooth muscle cells were allowed to grow to 60% confluency in culture medium, followed by 24 hours in medium without fetal calf serum. All media were removed, and SAR 943 (in 100 µL minimum essential medium) was incubated for 20 minutes and washed out before the addition of epidermal growth factor (0.3 nM in 100 µL minimum essential medium). Cells were incubated for 20 hours before being pulsed with [3H]-thymidine (0.5 µCi/well) for 4 hours, trypsinized, and harvested onto glass filters. Scintillation fluid was added (50 µl/well), and counts per minute were measured using a Packard Topcount. Confluent epithelial cells were serum starved for 24 hours and then incubated for 24 hours with increasing concentrations of SAR 943 in serum-free minimum essential medium or minimum essential medium 10% fetal calf serum. Cell number was determined using a cell proliferation/viability kit (MTT kit; Boehringer, Rotkreuz, Switzerland) following the manufacturer instructions.
Data Analysis
Effect of SAR 943 and Budesonide on Ovalbumin-induced Lung Inflammation and Airway Hyperreactivity At 2 days after ovalbumin-aerosol challenge, a significant increase in the numbers of eosinophils, neutrophils, and lymphocytes in the bronchoalveolar lavage was observed when compared with sensitized animals challenged with PBS. The macrophage numbers were not affected by the ovalbumin challenge, and both compounds had no effect on this cell type (Figure 1) . Both SAR 943 and budesonide dose dependently inhibited the ovalbumin-induced infiltration of eosinophils and lymphocytes. At a dose of 1 mg/kg, the two compounds were equipotent (p = 0.83 and 0.54 for eosinophils and lymphocytes, respectively). SAR 943 had no significant effect on the allergen-induced increase in neutrophil numbers, whereas budesonide was effective at a dose of 3 mg/kg (Figure 1). As a control, the effect of SAR 943 (1 mg/kg) and budesonide (3 mg/kg) in PBS-challenged mice was studied. None of the compounds had an intrinsic effect on the airway resident cells (see Table E1 in the online supplement).
Because IL-4 and IL-5 were not detectable in the bronchoalveolar lavage 2 days after the challenge, a group of animals were challenged and killed 1 day after the challenge. At this time point and when compared with PBS-challenged animals, ovalbumin challenge induced a significant increase in both Th2 cytokines (Figure 2) . Both SAR 943 and budesonide inhibited the production of these cytokines in a dose-dependent manner. At a dose of 1 mg/kg, the two compounds were equipotent (p = 0.91 and 0.94 for IL-4 and IL-5, respectively) (Figure 2). Interferon- levels were not affected by the ovalbumin challenge, and neither compound had an effect on basal interferon- levels (data not shown). Both compounds also inhibited the ovalbumin-induced inflammatory cell influx observed on Day 1 postchallenge (Table 1)
.
When compared with animals challenged with PBS, a significant increase in airway hyperreactivity to aerosolized methacholine was observed after ovalbumin challenge. Both SAR 943 and budesonide dose dependently inhibited this hyperreactivity (Figure 3) . At a dose of 1 mg/kg, the two compounds were equipotent in their ability to abrogate the airway hyperreactivity (p = 0.78).
Effect of SAR 943 and Budesonide on Ovalbumin-induced Lung Remodeling The effects of SAR 943 and budesonide on the structural changes in the lung including epithelial cell proliferation, the appearance of a mucus hypersecretory phenotype, and bronchoalveolar lavage cellular fibronectin production were assessed after allergen challenge. When compared with PBS-challenged animals, the ovalbumin-challenged animals showed a threefold increase in epithelial cell proliferation (Figure 4) . Furthermore, the ratio of alcian blue–periodic acid-Schiff–positive epithelial cells was increased by 20-fold upon ovalbumin challenge (Figure 4 and representative picture in Figure 5) . We also observed 10-fold increase of cellular fibronectin levels in bronchoalveolar lavage fluid after ovalbumin challenge. SAR 943 and budesonide dose dependently inhibited the epithelial cell proliferation, mucus secretory phenotype (Figures 4 and 5), and cellular fibronectin production in bronchoalveolar lavage fluid (Figure 6) observed after the ovalbumin challenge. At a dose of 1 mg/kg, the two compounds were equipotent on ovalbumin-induced epithelial cell proliferation (p = 0.83), the mucus hypersecretion phenotype (p = 0.26), and cellular fibronectin production (p = 0.19) (Figures 4 and 6).
When applied to PBS-challenged animals, both SAR 943 (1 mg/kg) and budesonide (3 mg/kg) had a small but significant effect on epithelial cell proliferation, indicating that both compounds, at the dose used, affect basal epithelial turnover in vivo (see Table E2 in the online supplement). For both compounds, no significant effect was observed on the epithelial cell mucus secretory phenotype or on cellular fibronectin levels (see Table E2 in the online supplement).
Effect of SAR 943 on Human Cell Proliferation In Vitro
In this report, we show that SAR 943, when given locally to the airways potently inhibits airway inflammation, airway hyperreactivity, and some histologic markers of the remodeling process in a murine model of asthma. More importantly, we have shown that SAR 943 is as potent as budesonide, a well-recognized and widely used therapy to treat human asthma. SAR 943, like rapamycin, acts as an immunosuppressant by arresting T cells during the cell cycle in mid to late G1 phase. Therefore, it can inhibit lymphocyte activation/proliferation even when added after stimulation (13). Based on this property, it is not surprising that rapamycin is able to inhibit the proliferation and production of IL-5 from allergen-stimulated peripheral blood mononuclear cells isolated from atopic subjects with asthma (14). Apart from inhibiting T cell activation, rapamycin is also able to inhibit the differentiation of human B cells into antibody-producing cells in vitro, thereby decreasing antibody production (15). This has been confirmed in vivo, where rapamycin treatment inhibited the ovalbumin-induced immunoglobulin E and immunoglobulin G production (16). In our model, no evidence of any inhibitory activity of SAR 943 on serum immunoglobulin E levels was observed (data not shown). However, at the time of treatment, the serum immunoglobulin E levels are already well established, and no further increase is observed after the aerosol challenge (11). Moreover, in the same model, we have previously shown that budesonide was also unable to inhibit previously established immunoglobulin E levels (11). In contrast, SAR 943 potently inhibits the ovalbumin-induced production of bronchoalveolar lavage IL-4 and IL-5. It has been shown, in a similar murine model of lung inflammation, that IL-5–producing CD4+ cells play a pivotal role in lung inflammation and airway hyperreactivity (17). Therefore, the inhibition of inflammatory cell infiltration and airway hyperreactivity by SAR 943 is probably a reflection of its ability to inhibit T cell rather than B cell activation in our model. The inhibitory effect of SAR 943 on the Th2 cytokines production in our model is in contrast with previously reported effects of rapamycin in other disease models. In a graft-versus-host murine model, rapamycin inhibited the production of Th1 but not Th2 cytokines (18). On the other hand, combination therapy with IL-2 and rapamycin has been reported to prevent diabetes development in NOD mice, and this was associated with a decrease in number of Th1 cells and an increase in Th2 cells (19). This differential regulation of the Th1/Th2 balance by rapamycin probably reflects different effects on different disease mechanisms. In the context of asthma, rapamycin has been shown to inhibit proliferation of asthmatics' peripheral blood T lymphocytes (20) and also to inhibit allergen-induced proliferation and IL-5 production by peripheral blood mononuclear cells from patients with asthma (14), suggesting that in patients with asthma, rapamycin might prove efficacious in downregulating the Th2 response.
During the completion of this article, the effect of SAR 943 on the acute allergic inflammation and bronchial hyperresponsiveness in sensitized rats has been published (21). After allergen challenge, SAR 943 inhibited the bronchoalveolar lavage lymphocyte influx and the upregulation of mRNA expression for IL-2, IL-4, and interferon- A second facet studied in our model is the antiremodeling potential of SAR 943. Lung remodeling in asthma is characterized by thickening of the airway basement membrane caused by extracellular matrix deposition, smooth muscle mass increase, and mucus hypersecretion (2). We accept that our model does not mimic all of these aspects. However, the lung structural changes observed in human asthma are probably related to the chronicity of the disease, and it is difficult to model adequately chronic lung disease in laboratory animals. Nevertheless, it is our belief that our model might be a helpful aid to study the initial stages of airway remodeling. In support, cellular proliferation within the epithelium, the mucus hypersecretory phenotype and increases in the levels of cellular fibronectin were observed in the model. All of these parameters were potently inhibited by SAR 943, suggesting that in addition to its antiinflammatory activity, this compound has added potential as an antiremodeling therapy. Because epithelial cell destruction is a consistent observation in asthma (2), the antiproliferative activity of SAR 943 on the allergen-driven epithelial cell proliferation might be seen as an undesirable effect. However, in this model, we believe that epithelial proliferation more accurately reflects the activation state of the epithelium, rather than a repair process. Of more concern to us was the antiproliferative effect of SAR 943 on the basal epithelial turnover. However, the same observation was made with budesonide, which is considered to be a relatively safe therapy for the treatment of human asthma. Moreover, we have shown in vitro that SAR 943 was devoid of any inhibitory activity on either basal or stimulated proliferation of a human epithelial cell line. Airway smooth muscle thickening as a result of both smooth muscle cell hyperplasia and hypertrophy is one of the main characteristics of lung remodeling in human asthma (2). Because smooth muscle proliferation cannot be detected in our murine model, the antiproliferative effect of SAR 943 was studied on primary cultured human airway smooth muscle cells. Because corticosteroids are known to have antiproliferative effect on human smooth muscle cells in vitro (26, 27), the effect of budesonide was not studied in this system. SAR 943 potently inhibited smooth muscle cell proliferation induced by epidermal growth factor, a growth factor that has been shown to be elevated in asthmatic airways and has been associated with lung remodeling (28). These results suggest that SAR 943 could be an effective therapy against lung remodeling in human asthma without interfering with the epithelium repair process. Other immunosuppressants have been proposed as a treatment for severe asthma (7). Among them, cyclosporin has been tested in human studies. When given by inhalation, cyclosporin has been proven to be effective in mild and corticosteroid-dependent subjects with asthma (5, 6, 29). Like cyclosporin, rapamycin has been shown to inhibit, to an equivalent extent, the activation of T lymphocytes from patients with glucocorticoid-resistant and glucocorticoid-sensitive asthma, whereas, as expected, dexamethasone showed significantly lower activity on cells from the steroid-resistant patient (30). In contrast to dexamethasone and cyclosporin, rapamycin retains its inhibitory activity on activated lymphocytes and is effective in reducing the ability of IL-5 to maintain the survival of eosinophils in culture (31). Based on these in vitro observations, SAR 943, an immunosuppressive rapamycin derivative, might be even more effective than cyclosporin in treating human asthma. In summary, we have shown that SAR 943, a stable derivative of rapamycin, was as effective as budesonide in inhibiting both lung inflammation and remodeling in an acute murine model of asthma, and therefore, we propose that this class of compound could have beneficial effects in patients with severe asthma.
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form May 22, 2002; accepted in final form October 23, 2002
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ABSTRACT
INTRODUCTION
-smooth muscle actin. The human bronchial epithelial cell line (16HBE14o-) was obtained from Dr. D.C. Gruenert (University of California, San Francisco, CA). Cells were cultured at 37°C in a CO2 incubator (5% CO2/95% air) in minimum essential medium supplemented with 10% fetal calf serum, 2 mM of L-glutamine and penicillin-streptomycin (100 U/ml-100 mg/ml). SAR 943 (10 µM) was dissolved in dimethyl sulfoxide and further diluted with culture medium. 
