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Asthma and chronic obstructive pulmonary disease (COPD) are pulmonary disorders characterized by various degrees of inflammation and tissue remodeling. Adenosine is a signaling molecule that is elevated in the lungs of patients with asthma and COPD. Adenosine elicits its actions by engaging cell surface adenosine receptors, and substantial preclinical evidence suggests that targeting these receptors will provide novel approaches for the treatment of asthma and COPD. Studies in animal models of airway disease suggest that there may be clinical benefit to the use of A1, A3 and A2B adenosine receptor antagonists in the treatment of features of asthma and/or COPD, while A2A agonists may also prove effective. Several adenosine receptor based pharmacologic agents have entered clinical development for the treatment of asthma and COPD; however, the studies have been limited and the efficacy of such approaches is not yet clear.
Chornic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are prevalent inflammatory disorders of the lung that are poorly managed, and for which few new drugs have been developed (see Text Box). Pharmaceutical companies and research institutions are addressing the substantial potential for the development of novel therapeutic agents that modulate adenosine signaling with significant clinical advantage for chronic inflammatory airway diseases .
Asthma is a chronic inflammatory disease of the airways characterized by some degree of mast cell and eosinophilic airway infiltration/activation driven by specific cytokines and chemokines produced by CD4+ Th2 lymphocytes . Clinically, asthma is characterized by the sudden onset or deterioration of wheezing, cough, dyspnea, mucous production and concomitant airway obstruction in response to exposure to various stimuli. Asthma in most patients can be controlled by regular use of anti-inflammatory (i.e. inhaled corticosteroids) and bronchodilator maintenance agents (i.e. inhaled β2-agonists). Nonetheless, for some patients asthma continues to be poorly controlled in terms of ongoing symptoms, frequent exacerbations, persistent and variable airway obstruction, and frequent requirement for β2-agonists despite aggressive treatment.
In chronic obstructive pulmonary disease (COPD), the persistent inflammatory response of the airways is typically associated with smoking . This response cannot be reversed and generally leads to progressive airflow limitation, impairment of gas exchange, respiratory failure, cor pulmonale and death. The inflammatory infiltrate of COPD is distinct from that of asthma, with neutrophils, macrophages and CD8+ T-lymphocytes in small and large airways, as well as in lung parenchyma and pulmonary vasculature. For individuals whose asthma or COPD is not adequately controlled with conventional treatments, a few additional therapies, including methlyxanthines, anticholinergics, cromones and leukotriene modifiers, are available but have variable efficacy. Consequently, there is a compelling need for improved therapeutic strategies for these individuals, particularly oral therapies that enhance patient compliance.
Extracellular adenosine levels are elevated in response to cellular stress and injury through pathways that involve the release of adenine nucleotides and their subsequent dephosphorylation to adenosine . The production of extracellular adenine nucleotides and adenosine can regulate cell function by engaging purinergic type II or type I receptors respectively. This review will focus on the type I or adenosine receptors. These receptors are expressed on various inflammatory and stromal cells and have well characterized anti-inflammatory and wound healing activities that demonstrate the importance of these signalling pathways in tissue protection  (Figure 1). In some situations; however, adenosine generation and receptor engagement have pro-inflammatory and tissue destructive activities  that serve to amplify tissue injury (Figure 2). This appears to be the case with inflammatory lung diseases such as asthma and COPD. Much evidence suggests that adenosine is playing a detrimental role in these and perhaps other chronic disorders of the airways (Table 1). The observations summarized in Table 1 suggest that adenosine signaling has a potentially pathophysiological role in chronic inflammatory disorders of the airways. These important observations have driven research efforts to develop selective agonists or antagonists for adenosine receptor subtypes for use as novel therapies for asthma and COPD.
Here we review proposed mechanisms of action of adenosine receptors in preclinical studies and describe molecules with high affinity and selectivity for the human adenosine receptor subtypes that are under investigation at preclinical and clinical levels as treatments for respiratory diseases. Findings in preclinical studies suggest there may be efficacy in the use of A1R, A3R and A2BR antagonists in the treatment of key features of asthma and COPD, while A2AR agonist may prove beneficial as well. However, the results from clinical trials testing such hypotheses are limited and without firm conclusion.
Adenosine levels are elevated in the lungs of individuals with asthma and COPD, and adenosine receptors are known to be expressed on most if not all inflammatory and stromal cell types involved in the pathogenesis of these diseases. Extracellular adenosine elicits its effects by interacting with four receptors: A1R, A2AR, A2BR and A3R (Figure 1) . These receptors are differentially expressed in the human lung . The ability of adenosine, whose levels are raised in many pathophysiological conditions, to activate these four receptors varies depending on binding affinity, receptor density and the type of G protein involved . In particular, A1R and A2AR can be activated by physiological levels of adenosine, whereas A2BR and A3R require higher concentrations of adenosine for their activation. Studies in animal model systems provide evidence that complex yet specific receptor responses exist and are driven by cell type and tissue specific expression of these receptors.
As with all of the adenosine receptors, there is evidence that A1R exhibits both pro- and anti-inflammatory activities in cellular and animal models of inflammation . In the context of models directed at human asthma, however, the preponderance of studies suggests that this receptor has a pro-inflammatory role. Attention was drawn to this receptor’s role in asthma in studies demonstrating that treatment with A1R antagonists  and antisense oligodeoxynecleotides  directed against A1R could attenuate bronchoconstrictor responses in an allergic rabbit model. Whether these effects were mediated by blocking A1R on airway smooth muscle, inflammatory cells or airway nerves is not clear; however, recent studies in A1R knockout mice support the idea that a neuronal component is involved in adenosine-mediated bronchoconstriction . More recent studies in the allergic rabbit model have demonstrated that treatment with an A1R antagonist can attenuate pulmonary inflammation as well as bronchoconstriction . Additional evidence supporting a role for A1R in asthma is the ability of this receptor to influence mucin production and secretion. A1R signaling can enhance MUC2 mucin gene expression in human bronchial epithelial cells in vitro . Lastly, A1R levels are elevated in several preclinical models of chronic lung disease [11-14], and a recent study has demonstrated that A1R levels are elevated in bronchial epithelial and airway smooth muscle cells of asthmatics . Up-regulation of this receptor in asthma is supportive of a role for this signaling pathway in the pathophysiology of this disease.
Collectively, these preclinical studies build a compelling case that treatment with A1R antagonist might provide an avenue for attenuating features of asthma including airway inflammation, mucus production and bronchoconstriction. There is evidence; however, that A1R plays anti-inflammatory roles in the lungs of adenosine deaminase (ADA)-deficient mice (Table 2), . ADA is the major enzyme that controls endogenous adenosine levels and consequently ADA-deficient mice develop progressive increases in lung adenosine in association with pulmonary inflammation and tissue remodeling [17, 18]. Although this model is complex and its relevance to human asthma debatable, it does reveal the consequences of prolonged increases in adenosine on pulmonary inflammation, and it suggests that A1R signaling might serve different functions at different disease stages. In general, there is still much that is not understood about the specific cellular mechanisms of this receptor in regulating the features of inflammation and airway pathophysiology seen in asthma. Continued research efforts in models of airway inflammation are warranted in this regard, but it is likely that the results of clinical trials utilizing A1R antagonists will provide the most definitive pathway forward for understanding the role of this receptor in human airway disease.
Elevated expression of A1R has been reported in bronchial biopsy specimens obtained from asthmatic subjects , where immunoreactivity appears to be predominantly located in the bronchial epithelium and bronchial smooth muscle. Moreover, the methylxanthine bamiphylline might produce its anti-asthma effects in humans by blocking A1R . Given these findings, and given that activation of A1R on human airway epithelial, bronchial smooth muscle cells, neutrophils, macrophages and fibroblasts (reviewed in ) induces the release of mediators and cytokines that lead to airway hyperreactivity, inflammation and airway remodeling, it is not surprising that several companies are pursuing this target in human asthma and COPD.
EPI-2010, a 21-mer antisense oligodeoxynucleotide targeting human A1R, has been tested as an anti-asthma drug. In a small clinical trial conducted in patients with asthma, a single dose of EPI-2010 reduced the need for bronchodilator drugs to control asthma symptoms, concomitant with a reduction in asthma symptom scores . This effect was statistically and clinically significant and lasted for one week following a single dose. Because of disappointing results in a Phase II clinical trial, EPI-2010 was discontinued from clinical testing ; however, because of safety concerns with the use of antisense oligonucleotides as therapeutics in humans, it is possible that the doses of EPI-2010 used were sub-therapeutic in this study.
Another potential agent, currently in preclinical development as a once daily oral treatment for human asthma, is L-97-1, a small-molecule A1R antagonist . As compared with bamiphylline, L-97-1 has a considerably higher affinity and selectivity for human A1R . In a rabbit model of allergic asthma, L-97-1 blocks allergic airway responses, airway hyperresponsiveness to histamine, and airway inflammation [9, 19].
Increasing evidence suggest that A2AR mediates potent anti-inflammatory activities on specific cells and in various models of inflammation, which has promoted the development of A2AR agonists for the attenuation of inflammation in many disorders (reviewed in ). Given the central role of inflammation in asthma and COPD, there has been substantial preclinical research activity targeted at understanding the function of A2AR in models of airway inflammation.
Among the earliest, was a study by Fozard and colleagues demonstrating that intra-tracheal treatment with A2AR agonist CGS21680 inhibits airway inflammation in allergen sensitized and challenged brown Norway rats . Similar findings were reported in a mouse model of ovalbumin sensitization and challenge where CGS21680 treatment inhibited inflammatory cell recruitment in the lavage fluid . Interestingly, differences in mucus secretion and airway hyperresponsiveness were not seen. The same study demonstrated mixed effects of A2AR agonist treatment on the airway inflammation seen after exposure to LPS or cigarette smoke. Consistent with these findings, A2AR knockout mice that have been sensitized and challenged with ragweed exhibit enhanced airway inflammation and airway hyperresponsiveness as compared with wild-type mice . In addition, a recent study in ADA-deficient model demonstrated that genetic removal of A2AR leads to enhanced pulmonary inflammation, mucus production and alveolar airway destruction , (Table 2) further implicating A2AR signaling pathways as important anti-inflammatory networks in the lung. However, recent studies have described the ability of A2AR activation to promote fibrosis , suggesting there may be side effects associated with persistent A2AR activation.
Detailed characterization of the immunohistochemistry and binding of A2AR in human lung parenchyma of patients with COPD has revealed the presence of this receptor subtype in bronchiolar and alveolar epithelial cells, bronchiolar smooth muscle cells and endothelial cells with a high affinity and density . Because activation of A2AR increases intracellular cAMP similar to other agents that increase intracellular cAMP, e.g. PDE-IV inhibitors, beta-2 agonists or theophylline, A2AR agonists may represent a new class of anti-asthma drugs that produce bronchodilation and anti-inflammatory effects. Thus, selective A2AR agonists may also provide an alternative to corticosteroids for the treatment of airway inflammatory diseases.
Unfortunately, administration of the selective A2AR agonist GW328267X by inhalation at a dose of 25 μg twice daily, in 15 nonsmoking atopic asthmatics in a double-blind, placebo and fluticasone propionate (250 μg) controlled study, failed to protect against allergen-induced early or late asthmatic reactions, or the associated inflammatory response in the induced sputum . An obvious explanation is that the dose of the drug used was sub-therapeutic as suggested by the lack of adverse effects at a cardiovascular level. By increasing intracellular cAMP, activation of A2AR may also produce the cardiovascular side effects of hypotension and reflex tachycardia ; a factor that is likely to substantially reduce the therapeutic index of A2AR agonists as drug candidates in humans. GlaxoSmithKline has discontinued the clinical development of GW328267X. Conversely, the adenosine A2AR agonist UK371,104 is beneficial in the lung of anaesthetized guinea-pigs without any obvious cardiovascular side effects and may be valuable to explore the anti-inflammatory potential of inhaled adenosine A2AR agonists in clinical trials . UK432,097 is an analogue of UK371,104 with a similar lung focus of pharmacological activity in the anaesthetized guinea pig model discussed above. UK432,097 is currently in phase II trials for COPD .
The A2BR has the lowest affinity for adenosine, yet this receptor might be important in pathological environments where adenosine levels are elevated such as asthma . In support of this, multiple studies demonstrate the ability of A2BR engagement to promote the expression of pro-inflammatory mediators from various cell types critical to processes seen in chronic lung diseases. This includes the release of IL-8 from HMC-1 cells , IL-4 and IL-13 [32, 33] from HMC-1 cells and mouse bone-marrow-derived mast cells, IL-19 from airway epithelial cells  and MCP-1 from bronchial smooth muscle cells . In addition, A2BR activation promotes the production and release of IL-6 from airway epithelial cells , macrophages , pulmonary fibroblasts  and bronchial smooth muscle cells .
Adenosine potentiates the release of preformed mediators from sensitized mast cells, which is thought to be the underlying mechanism by which adenosine provocation elicits bronchoconstriction in asthmatics [30, 39]. The A2BR has been suggested to orchestrate key responses to adenosine in several respiratory conditions in humans, but direct evidence demonstrating the expression of this receptor on mast cells in the lung, or the existence of specific A2BR responses, is scarce in the literature. Studies utilizing antagonists and knockout mice suggest that the degranulation of mast cells in rodents is mediated through A3R [40, 41]; moreover, examination of isolated mast cells from A2BR knockout mice suggest this receptor is not involved in the degranulation process , but is important in promoting the production of IL-4 and IL-13 from these cells , which may affect late-stage or chronic features of airway diseases such as asthma. Thus, adenosine probably elicits important responses on mast cells that can influence acute and chronic features of asthma; however, additional studies are needed to more precisely define the specific adenosine receptors responsible for such activities on human mast cells.
A recent study revealed that treatment with A2BR antagonist CVT-6883 attenuated airway inflammation and airway hyperreactivity in a mouse model of ragweed sensitization and challenge . In addition, work in the ADA-deficient model suggests that A2BR signaling engages pathways that promote chronic airway inflammation and remodeling (Table 2) . Noted features include the accumulation of activated alveolar macrophages, airspace destruction, mucus cell metaplasia and pulmonary fibrosis. A2BR expression is elevated in the lungs of ADA-deficient mice and treatment of these mice with the selective A2BR antagonist CVT-6883 results in decreased production of pro-inflammatory mediators in association with diminished airspace enlargement and pulmonary fibrosis . Consistent with this is the observation that adenosine and A2BR levels are elevated in the lungs of IL-13  and IL-4  over expressing mice that develop pathological features of asthma, COPD and pulmonary fibrosis. Moreover, increases in A2BR have recently been described as a distinguishing feature of individuals with accelerated pulmonary fibrosis . Additional studies supporting a pro-fibrotic role for A2BR signaling include the demonstration that activation of A2BR can promote the differentiation of human pulmonary fibroblast into collagen-producing myofibroblasts , and engagement of A2BR can directly up-regulate the production of the pro-fibrotic molecule fibronectin in alveolar epithelial cells . The demonstration that adenosine can directly promote these processes, further strengthens the likelihood that this nucleoside is in itself pro-fibrotic and strongly implicates A2BR as a pro-fibrotic receptor. These studies suggest that up-regulation and engagement of A2BR might serve to enhance chronic remodeling in lung diseases.
These preclinical data suggests that A2BR antagonists may have utility in the treatment of chronic lung diseases where fibrosis is a major component, including the airway remodeling seen in asthma and in patients with interstitial lung diseases where fibrosis is detrimental. It must be realized, however, that A2BR signaling also serves important anti-inflammatory functions in acute lung injury that might make the timing of A2BR antagonist treatment important . In addition, A2BR expressed on airway epithelial cells has been implicated in the regulation of airway surface liquid volume mucous clearance . Disruption of these features should therefore be monitored closely as a side-effect of A2BR antagonist usage in the lungs.
Characterization of human lung parenchyma in patients with chronic inflammation of the airways suggests that A2BR might be present on mast cells and macrophages . As compared with control smokers, the affinity and density of these receptors in COPD patients appears to be substantially modified, suggesting adenosine signalling may be important in COPD. Such a role for A2BR signalling in the pathophysiology of asthma receives support from pharmacological studies using the adenosine receptor antagonists theophylline and enprophylline . Though these agents can inhibit phophodiesterase activity at high concentrations, the affective therapeutic concentrations used to improve lung function and symptoms in subjects with asthma were relatively low and in the range of their A2BR affinities .
The potent and highly selective A2BR antagonist CVT 6883 has completed Phase 1 clinical trials with no adverse events reported and has entered Phase 2 trials for human asthma . This compound is stated to have an A2BR Ki = 8nM with at least 1000-fold selectivity against the other adenosine receptors . Finally, Novartis has recently reported that their aminothiazole dual A2BR/A3R antagonist, QAF 805, failed to attenuate bronchial hyperresponisveness to inhaled AMP in a placebo controlled, double-blind, randomized, two-way crossover Phase 1b clinical trial of 24 AMP-sensitive asthmatics .
The A3R plays complex roles in inflammation, with both pro- and anti-inflammatory functions being described in multiple cellular and animal models with varying roles being dictated largely by species differences (for review see ). This receptor has also received attention in chronic inflammatory disorders of the airways. Transcript levels of A3R are elevated in lung biopsies of patients with asthma or COPD , where it is thought to localize to eosinophils. Functions attributed to A3R activation on human eosinophils include the inhibition of chemokine-induced migration  and the inhibition of degranulation and superoxide anion release , suggesting A3R agonists might have utility in the treatment of asthma.
In contrast to these findings in humans, pre-clinical studies in animal models suggest that A3R has a pro-inflammatory role in chronic inflammatory diseases of the airways. A3R is abundantly expressed on mouse eosinophils , and in vitro studies with mouse eosinophils confirm a role for this receptor in chemokine-induced eosinophil migration . However, genetic removal or pharmacological blockade of A3R in ADA-deficient mice results in decreased airway eosinophilia, but does not affect the degree of circulating eosinophilia, suggesting that A3R signaling is important in eosinophil trafficking in this model (Table 2). Moreover, a recent study demonstrated the absence of eosinophil peroxidase in the airways of A3R-deficient mice exposed to fibrosis inducing agent, bleomycin , suggesting that this receptor is needed for eosinophil degranulation in vivo. A3R is also abundant on rodent mast cells and is responsible for adenosine-mediated mast cell degranulation in vitro and in vivo in multiple models [40, 41]. In addition, A3R is markedly elevated in mucin-producing bronchial airway epithelial cells in several models of Th2-mediated airway inflammation and airway remodeling [12, 56, 58], and evidence from A3R over expressing and knockout mice suggests that this receptor serves to enhance mucus secretion in these environments . These findings suggest that treatment with A3R antagonists might have benefit in models of allergic airway inflammation. The discrepancies between findings in human cellular systems and those in animal models might be due to species differences or the inability to adequately assess the function of A3R in vivo in humans. It is likely that the usefulness of A3R agonists and antagonists in the treatment of asthma and COPD will only be revealed following appropriate clinical trials with such compounds.
As mentioned above, transcript levels of A3R are elevated in lung biopsies of patients with asthma or COPD where it appears to be involved in the inhibition of eosinophil chemotaxis. Because asthmatic inflammation is characterized by extensive infiltration of the airways by activated eosinophils, elevated adenosine concentrations associated with asthma may contribute to eosinophilic trafficking through modulation of A3R. King Pharmaceuticals published a patent application claiming a particularly potent and selective A3R antagonist for asthma characterized by a tricyclic xanthine derivative structure . As mentioned above, a dual A2BR/A3R antagonist, QAF 805 from Novartis failed to attenuate bronchial hyperresponsiveness to inhaled AMP in a placebo controlled, double-blind, randomized, two-way crossover Phase Ib study of AMP-sensitive asthmatics .
The pharmacological arsenal for allergy and asthma is fast growing, and significant endeavors in medicinal chemistry and pharmacology have yielded a number of safer and highly selective compounds directed against the four known adenosine receptor subtypes. Some of these compounds have entered clinical development and may be of assistance in the process of finding better therapies for patients with asthma and COPD (Table 3).
Although adenosine generation and the subsequent engagement of adenosine receptors on inflammatory and pulmonary cells appears to play an important pathogenetic role in regulating chronic lung disorders such as asthma and COPD (Figure 2), this knowledge has not yet been translated into effective treatments . It is likely that this failure reflects our limited comprehension of the complex interplay driven by the different pattern of receptor distribution and/or affinity of the four known adenosine receptor subtypes in specific cell types at different stages of the disease. Consequently, a complete and full characterization of adenosine receptor subtype distribution in the airways and their specific role in the response to adenosine in health and disease is mandatory. With this information, attempts at elucidating the role of specific adenosine receptors as valuable therapeutic targets in asthma and COPD may be conclusive.
Riccardo Polosa is full Professor of Internal Medicine and he is supported by the University of Catania, Italy. Research in M. R. Blackburn’s laboratory is supported by NIH Grants AI43572 and HL70952.
Disclosure Statement Riccardo Polosa has received lecture fees from CV Therapeutics, Merck, GSK, Astra-Zeneca, and Novartis; he has also served as a consultant to CV Therapeutics, Sanofi-Aventis, Merck, and Duska Therapeutics. Michael R. Blackburn has served as a consultant for CV Therapeutics.
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