Investigational A₃ adenosine receptor targeting agents

Abstract

Introduction

Adenosine is an endogenous nucleoside that accumulates in the extracellular space in response to metabolic stress and cell damage. Extracellular adenosine is a signaling molecule and it signals by activating four G-protein coupled receptors: the A₁, A_2A, A_2B and A₃ receptors. Since the discovery of A₃ adenosine receptors accumulating evidence has identified these receptors as potential targets for therapeutic intervention.

Areas covered

A₃ adenosine receptors are expressed on the surface of most immune cell types, including neutrophils, macrophages, dendritic cells, lymphocytes and mast cells. A₃ adenosine receptor activation on immune cells governs a broad array of immune cell functions, which include cytokine production, degranulation, chemotaxis, cytotoxicity, apoptosis, and proliferation. In accordance with their multitudinous immunoregulatory actions, targeting A₃ adenosine receptors has been shown to impact the course of a wide spectrum of immune-related diseases, such as asthma, rheumatoid arthritis, cancer, ischaemia, and inflammatory disorders.

Expert opinion

Given the existence of both pre-clinical and early clinical data supporting the utility of A₃ adenosine receptor ligands in treating immune-related diseases, further development of A₃ adenosine receptor ligands is anticipated.

1. Introduction

1.1. Adenosine and its receptors

The endogenous purine nucleoside, adenosine is a signaling molecule, which is present in the extracellular space at low concentrations in undisturbed tissue. The levels of adenosine are increased in stressful conditions, such as inflammation, ischaemia, hypoxia or trauma [1, 2]. Adenosine exerts organ protective actions through binding to one or more of the 4 transmembrane adenosine receptors, A₁, A_2A, A_2B and A₃ [3-8] Adenosine receptors are members of the family of G protein coupled receptors (GPCR) and their expression levels vary in different organs and cell types [9]. In this review, we focus on the A₃ adenosine receptor (A₃AR) and discuss its regulatory roles in the different cell types of the innate and adaptive immune systems, its role in regulating the course of various immune-related diseases, and the therapeutic potential of its activation or inactivation.

1.2. A₃ARs

The human A₃AR consists of 318 amino acids. The A₃AR shows large interspecies differences in its sequence; the homology between the rat and human A₃AR is only 74%. Interestingly, two splice variants of the A₃AR receptor can be found in rats but the significance of this phenomenon is unknown [10]. The A₃AR is expressed at the mRNA level in most organs of both rodents and human, including testis, lung, kidneys, placenta, heart, brain, spleen, and liver [11]. In addition, the A₃AR protein has been detected using radioligand binding or functional tests in all these organs in different species. Finally, most of the cell types of the immune system express functional A₃ARs on their surface [2].

1.3. A₃AR agonists and antagonists

The prototypical and most widely used A₃AR agonists are IB-MECA (figure 1a) and Cl-IB-MECA (figure 1b). Both IB-MECA and Cl-IB-MECA are adenosine derivatives bearing a lipophilic substituent (3-iodobenzyl) at the 6-amino group and ribose modification in the 5′ position [12]. There is an additional 2-chloro substituent in Cl-IB-MECA, which makes it more selective than IB-MECA [13]. Another highly selective agonist is CP532903 (figure 1c). While the methylxanthines caffeine and theophylline are classical antagonists for the A₁AR, A_2AAR, and A_2BAR, their affinity for the A₃AR is low. Therefore, antagonists for this subtype have been developed by modifying different molecules with heterocyclic structures. One family of selective A₃AR antagonists comprises the derivatives of 1.4-dihydropiridines, which are known as inhibitors of L-type Ca²⁺ channels. After various modifications, including the introduction of a 6-phenyl group, these molecules bind with high affinity and selectivity for the human A₃AR [14]. Since there are significant differences between the sequences of the human and rat A₃ARs, most of the antagonists developed for the human receptor bind with much lower affinity to rat and other rodent A₃AR. Prominent members of this family are MRS1191, MRS1334 and MRS1523 (figure 2a). The pyridylquinazoline derivative VUF5574 (figure 2b) and the triazoloquinazoline MRS1220 are also used as selective A₃AR antagonists [13], both with selectivity only in humans. There are also highly selective antagonists of the human A₃AR among the flavonoids, which are naturally occurring phenolic derivatives. MRS1067 is the most important member of this family [15].

Figure 1.

Chemical structures of A₃AR agonists: a. IB-MECA, b. Cl-IB-MECA, c. CP532903.

Figure 2.

Chemical structures of A₃AR antagonists: a. MRS1523, b. VUF5574.

1.4. A₃AR signaling

A₃AR receptor stimulation inhibits adenylyl cyclase activation via G_i protein, which in turn results in a decrease of cAMP levels [1, 2]. A₃AR activation can also stimulate the phospholipase C (PLC) pathway, which results in the elevation of intracellular inositol 1,4,5-trisphosphate and calcium (Ca²⁺) levels [11]. The A₃AR also can stimulate mitogen-activated protein kinases (MAPK), such as extracellular-signal regulated kinase 1/2 (ERK1/2) and p38 through the upstream activation of phosphatidylinositol-3-kinase (PI3K) [16]. The A3AR associated intracellular signaling pathways are summarized on Figure 3.

Figure 3.

Signaling pathways activated by A₃AR.

2. A₃ARs on immune cells

2.1. Neutrophils

Neutrophils are part of the first line of defense of the immune system; their function is to kill pathogens, remove debris of injured tissue, and secrete factors that regulate inflammation [17]. It was shown recently that the A₃AR, along with the P2Y₂ receptor, has a major role in the regulation of neutrophil migration to sites of inflammation. Chen and coworkers [18] showed that human neutrophils release ATP at the leading edge of the cell surface, and the released ATP is rapidly hydrolyzed to adenosine by the ecto-enzymes nucleoside triposphate dephosphorylase (CD39) and 5′-ectonuleotidase (CD73). Adenosine potentiates neutrophil migration by activating the A₃AR, which is recruited to the leading edge of the migrating neutrophils. This autocrine positive feedback mechanism provides signal amplification and regulates the chemotactic gradient sensing of the cells. Subsequently, the same group of investigators confirmed the potentiating role of A₃ARs in neutrophil migration using a mouse sepsis model [19]. In contrast, data published by Van der Hoeven and coworkers suggests that A₃AR activation inhibits neutrophil migration and superoxide production by suppressing the monomeric GTP-ase Rac, which small G protein is a central regulator of neutrophil functions [20, 21]. These conflicting results may be explained by the differences between the species and chemoattractants used. Chen et al. used both fMLP-stimulated human neutrophils and W peptide-stimulated neutrophils from mouse bone marrow. In contrast, Van der Hoeven and coworkers used neutrophils from mouse bone marrow that were stimulated with fMLP, C5a, IL-8, and PAF. The A₃AR agonists used were also different by the two groups, IB-MECA and CP-532,903 respectively. Although these differences provide a possible explanation for the opposite effects of A₃AR activation described by the two groups, further investigation is necessary to reveal more completely the regulatory role of A₃AR stimulation on neutrophil functions.

2.2. Macrophages

Macrophages are mononuclear phagocytes that produce high amounts of inflammatory mediators early during inflammation, and later participate in the resolution of inflammation by secreting anti-inflammatory cytokines, and by removal of pathogens, cell debris and apoptotic cells [4]. Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine produced by macrophages in response to different stimuli including toll-like receptor (TLR) activation. A₃AR agonists have been shown to inhibit TNF-α production in various macrophage populations [22-24]. Lee and coworkers studied the effect of A₃AR activation on LPS induced TNF-α production by microglia, the resident macrophage of the central nervous system, and found that A₃AR stimulation decreased TNF-α production by inhibiting the PI3K/Akt and NF-κB pathways [25]. Martin and coworkers confirmed the inhibitory effect of A₃AR activation on TNF-α production by the RAW264.7 mouse macrophage cell line [26]. They also found that the A₃AR-mediated inhibition of TNF-α production coincided with reductions in the calcium dependent activation of NF-κB and ERK 1/2. It is noteworthy that the conclusions of all these studies regarding a primary role of A₃ARs in decreasing TNF-α production are based on pharmacological A₃AR ligands. In contrast, the results of other studies employing adenosine receptor knockout mice suggest that the A_2AAR and A_2BAR rather than the A₃AR mediate the inhibitory effect of adenosine on macrophage TNF-α production [27-29].

In addition to decreasing TNF-α release, A₃AR ligands have been shown to inhibit other macrophage functions, as well. We showed using a pharmacological approach that IB-MECA reduced production of the chemokine macrophage inflammatory protein (MIP) 1α in mouse macrophages [28, 30]. Barnholt and coworkers found that A₃AR receptor stimulation inhibited IFN-γ–induced gene expression (including IRF1, iNOS and CD36 expression) in RAW264.7 murine and THP-1 human macrophages by decreasing STAT-1 phosphorylation [31]. In addition, IB-MECA inhibited the respiratory burst of human monocytes by blocking NADPH oxidase activity [32, 33]. Besides the regulation of the release of inflammatory mediators, A₃AR activation on human macrophages promotes tissue remodeling by enhancing the secretion of matrix metalloproteinases (MMP) [34].

In conclusion, A₃AR activation has mainly anti-inflammatory and tissue remodeling effects on macrophages.

2.3. Dendritic cells

Dendritic cells (DC) are specialized antigen-presenting cells that activate naive T cells thereby initiating adaptive immune responses. The expression level of adenosine receptors varies based on the maturation state of dendritic cells. In immature human dendritic cells, A₁ARs and A₃AR are expressed predominantly and stimulating these receptors induces Ca²⁺ mobilization from intracellular stores, actin polymerization, and chemotaxis [35]. Mature dendritic cells, however, down-regulate their A₃ARs and express mainly A_2AARs. In contrast, Dickenson et al found that A_2AARs and A₃ARs are expressed on the mouse dendritic cell line XS-106, and that stimulating these receptors inhibits TNF-α release [36]. Moreover, recent studies have uncovered an important anti-inflammatory role for the A_2BAR in dendritic cells [6, 37-39].

In conclusion, stimulating the A₃AR enhances the chemotaxis of human immature dendritic cells; however, the role of A₃ARs in regulating mature dendritic cell function is less well established.

2.4. Mast cells

Mast cells are important regulators of not only allergic responses but also many tissue functions like blood flow and coagulation, smooth muscle contraction, wound healing, and various innate and adaptive immune responses [40]. Ramkumar and coworkers showed for the first time that A₃AR activation augmented degranulation of the mast cell line RBL-2H3 [41]. Reeves and coworkers confirmed this result by reporting that IB-MECA increased the degranulation of rat mast cells in vitro [42]. Subsequently, Zhong and coworkers demonstrated that the A₃AR is highly expressed in murine primary lung mast cells, and that A₃AR stimulation in these cells induces degranulation and histamine release, which is mediated through the G_i and PI3K pathways and through an increase of intracellular Ca²⁺ levels [43].

In vivo studies also addressed the effect of A₃AR activation on mast cell degranulation. For example, IB-MECA injection into mice was found to augment plasma protein extravasation and since this effect was abolished in animals treated with the histamine-depleting compound 48/80, these results corroborated the stimulatory effect of IB-MECA on mast cell degranulation [42]. A similar study by Smith and coworkers [44] documented that Cl-IB-MECA treatment of mice augmented serum histamine levels and that this effect was absent in animals pre-treated with compound 48/80. Genetic studies confirmed the role of A₃ARs in activating mast cells, as Cl-IB-MECA potentiated antigen-dependent degranulation of murine bone marrow-derived mast cells, and this potentiating effect was not detectable in cells obtained from A₃AR deficient mice [45]. However, there is no evidence that the A₃AR plays a similar role in human mast cells and the A_2BAR appears to assume the role of A₃AR in increasing the release of inflammatory mediators in human mast cells [46, 47].

Jin and coworkers reported that inosine stimulated degranulation in RBL-2H3 cells, and caused mast cell-dependent constriction of arterioles in hamsters. These effects were attenuated by A₃AR blockade indicating a role for the A₃AR in mediating the mast cell-activating effect of inosine [48]. These observations were confirmed by Tilley and coworkers [49], who found that adenosine and inosine increased cutaneous vasopermeability in mice, effects that were abolished in A₃AR or mast cell deficient animals.

In addition to degranulation, there is evidence that A₃ARs can also control other mast cell functions. For example, Gao and coworkers demonstrated that IB-MECA protected rat RBL-2H3 cells from apoptosis caused by exposure to UV light [50]. Furthermore, Feoktistov and coworkers showed that A₃AR activation stimulated angiopoietin-2 expression in HMC-1 human mast cells [51]. In conclusion, A₃AR activation influences numerous mast cell functions in rodents and humans including degranulation, apoptosis and regulation of vasopermeability.

2.5. Lymphocytes

Studies using pharmacological approaches or knockout animals have shown that A₃ARs do not directly affect CD4⁺ lymphocyte function [2]. Nevertheless, it appears that A₃AR expression is up-regulated in human CD4⁺ lymphocytes after activation with phytohemagglutinin [52] and in murine macrophages stimulated with anti-CD3 antibody and antigen-presenting cells [53]. In contrast, there is evidence that A₃ARs can dictate CD8⁺ lymphocyte activation. For example, MacKenzie and coworkers reported that adenosine inhibited the adhesion of anti-CD3 antibody-activated cytotoxic T cells to adenocarcinoma cells through A₃ARs [54]. Hoskin et al. found that the A₃AR inhibited the tumoricidal activity of anti-CD3-activated cytotoxic T cells, and that this inhibition was achieved by reducing the mRNA levels of granzyme, perforin and FAS ligand, and by inhibiting interferon (IFN)-γ and interleukin (IL)-2 release [55]. In addition, Harish and coworkers showed that Cl-IB-MECA treatment potentiated natural killer (NK) cell activity in melanoma bearing mice [56] and Jeffe and coworkers found that IB-MECA increased IFN-α release by IFN-γ stimulated human NK cells [57].

Taken together, the A₃AR exerts inhibitory effects on cytotoxic T cell functions and stimulatory effects on NK cell functions.

3. A₃AR in disease states

3.1 Rheumatoid arthritis (RA)

RA is a chronic inflammatory disease, which affects the joints. During the course of RA, immune cells infiltrate the synovium, a delicate membrane on the joint surface, and release inflammatory mediators including TNF-α. This chronic inflammation ultimately leads to cartilage destruction, bone erosions, and joint deformities [58, 59]. Methotrexate is an anti-inflammatory drug that is widely used in the therapy of RA. An increasing body of evidence is available that methotrexate promotes adenosine release and that the anti-inflammatory effects of methotrexate are mediated by the released adenosine [60]. This observation coupled with the notion that adenosine receptor activation on innate immune cells inhibits TNF-α release made adenosine receptors promising targets in RA treatment. This idea was further underlined by data showing that the A₃AR was up-regulated in peripheral blood mononuclear cells of RA patients and cells of synovial tissue of rats with adjuvant induced arthritis [61-63].

We tested for the first time the potential of IB-MECA, the prototypical A₃AR agonist at the time, in alleviating the course of collagen-induced arthritis in mice. We found that IB-MECA reduced the severity of joint inflammation by inhibiting the formation of MIP-1α, the cytokine IL-12, and the infiltration of neutrophils [30]. These results were later confirmed by Baharav and coworkers, who found that both IB-MECA and Cl-IB-MECA ameliorated clinical and histological features of arthritis in various mouse and rat arthritis models [64]. Further experiments revealed that IB-MECA decreased the expression of PI3K, IKK, and NF-κB, implicating these signaling pathways as mediating the alleviating effect of IB-MECA on the course of arthritis. In addition to IB-MECA, another A₃AR agonist, CF502 also inhibited the clinical and pathological manifestations of adjuvant induced arthritis in rats [65, 66].

Ochaion et al. found that combined methotrexate and IB-MECA treatment was more effective than the two drugs alone. In addition, methotrexate augmented the expression of A₃AR on immune cells rendering them more sensitive to the effect of IB-MECA [67]. IB-MECA was found to be protective also in a rat osteoarthritis model, and the protective effect was associated with a down-regulation of the NF-κB pathway [68].

Finally, in agreement with animal model data, clinical trials using IB-MECA as a therapeutic agent for RA patients showed that it was safe and well-tolerated and that IB-MECA treatment resulted in improvements of symptoms of the disease [69].

3.2. Asthma

There is substantial evidence that adenosine can induce bronchoconstriction in patients with asthma or chronic obstructive pulmonary disorder (COPD) but not in healthy individuals, and that adenosine levels are elevated in the bronchoalveolar lavage fluid and exhaled breath condensate of patients with asthma [70-72]. In addition, A₃AR expression was found to be elevated in the airways of asthma patients, and in vitro studies suggest that A₃ARs on human eosinophils mediates inhibition of chemotaxis, degranulation and superoxide anion release, which are important pathophysiological events during airway inflammation [73-75]. Tilley and coworkers exposed mice to aerosolized adenosine and found that it caused bronchoconstriction, degranulation of airway mast cells and neutrophil infiltration in wild-type mice, but not in A₃AR deficient mice [76]. Also, 5′ N-ethylcarboxamide (NECA), a non-selective adenosine receptor agonist, caused airway hyperresponsiveness in wild-type mice, and this effect was abolished in A₃AR or mast cell deficient mice [77].

Mucin hypersecretion, besides airway hyperresponsiveness, is also an important feature of asthma and other chronic lung diseases. Young and coworkers investigated the role of A₃AR in regulating mucin hypersecretion in mice in which chronic inflammation in the lung is induced by deficiency in adenosine deaminase (ADA), which converts adenosine to inosine. Genetic or pharmacological inactivation of A₃AR in ADA deficient mice prevented mucin production and also decreased the migration of eosinophils into the airways [78]. In another model in which pulmonary inflammation was induced by ovalbumin, IB-MECA treatment increased mucin secretion in wild-type mice and this effect was absent in A₃AR deficient mice [79]. In conclusion, A₃ARs contribute to mucin hypersecretion in the setting of pulmonary inflammation.

From a translational point of view, it is noteworthy that there are marked differences in the role of A₃ARs in regulating certain aspects of airway inflammatory diseases between different species. In murine models, A₃AR activation causes eosinophil infiltration into the airways, which suggests the therapeutic potential of A₃AR antagonists in the treatment of asthma. On the other hand, A₃AR activation appears to inhibit the functions of human eosinophils, indicating that A₃AR agonists might be used in asthma therapy in humans. Further investigation is necessary to evaluate which approach is more promising in the treatment of human asthma.

3.3 Cancer

There are several lines of evidence indicating that the expression of A₃AR is higher in tumors, such as breast and colorectal carcinoma, as compared to relevant adjacent normal tissue [80, 81]. A₃AR expression is also elevated in the peripheral blood cells of patients with colorectal cancer compared to healthy inviduals [81]. These observations raise the possibility of using the A₃AR as a diagnostic marker and therapeutic target for the treatment of cancer [80, 81]. This idea is also supported by extensive data showing that A₃AR ligands can induce apoptosis in tumor cells. For example, Fishman and coworkers [82] injected mice with colon carcinoma cells, and then studied the effect of oral treatment with IB-MECA on tumor growth. They found that tumor growth was suppressed in the group treated with IB-MECA in comparison with the group treated with vehicle [82]. The beneficial effect of IB-MECA was associated with up-regulation of GSK-3β and down-regulation of NF-κB and the oncogenes cyclin D1 and c-Myc in extract prepared from tumor lesions. Similar to these results, Cl-IB-MECA induced apoptosis and inhibited tumor growth in rats with hepatocellular carcinoma [83]. Cl-IB-MECA treatment also inhibited tumor growth in mice injected with Hep-3B hepatoma cells and induced apoptosis in tumor cells in vitro [84]. Furthermore, A₃AR agonists inhibited cancer cell proliferation also in lung and thyroid cancer, and leukaemia [85-87]. In addition to the tumor growth inhibitory effect, the A₃AR is also able to suppress the formation of metastases in a model of rat prostate cancer and in the liver of mice inoculated with colon carcinoma cells [88, 89]. Finally, it is noteworthy that in some of these studies the agonists exerted effects only at high concentrations [85] or independently of the A₃AR [86].

In contrast, there is also evidence to suggest a pro-tumoral role for A₃AR activation. For example, Gessi et al. reported that A₃AR activation augmented the invasion of U87MG glioblastoma cells by increasing MMP-9 levels [90]. Also, Gessi and coworkers found that A₃AR activation stimulated the proliferation of colon cancer cell lines via a mechanism involving ERK1/2 activation [91]. It was recently shown that A₃ receptor stimulation elevates hypoxia inducible factor 1 (HIF-1) expression in A375 human melanoma and in human glioblastoma cells under hypoxic conditions [92-94]. In addition, A₃AR activation increases the production of vascular endothelial growth factor (VEGF) and angiopoietin-2 [92-94]. Since HIF-1, VEGF, and angiopoietin-2 are critical factors that support tumor angiogenesis, A₃AR activation may have undesired pro-tumor effects that warrant further investigation.

In summary, data about the role of the A₃AR in regulating tumor growth are somewhat controversial as there is evidence for both pro-tumoral and also anti-tumoral activity.

3.4. Ischaemia

Although A₃AR expression in myocardial tissue is low, A₃AR agonists have cardioprotective effects in myocardial ischaemia-reperfusion models [11]. Wan et al. found that the A₃AR agonist CP-532,903 reduced infarct size in an in vivo mouse model and was also protective in an isolated heart model of global ischaemia/reperfusion injury, and the protective mechanism involved activating sarcolemmal K_ATP channels [95]. Ge and coworkers investigated the effects of Cl-IB-MECA in an in vivo mouse model of infarction induced by 30 minutes of coronary occlusion and 24 hours of reperfusion. Treatment of wild type mice with Cl-IB-MECA during reperfusion reduced myocardial infarct size, but failed to reduce the infarct size in A₃AR knockout mice and in chimeric mice lacking the expression of A₃AR on bone marrow derived cells [96]. These results suggest a role of neutrophil A₃AR in the protective effect of Cl-IB-MECA and correlate with the earlier results of Jordan and coworkers who reported that IB-MECA inhibits neutrophil associated reperfusion injury [97].

Doxorubicin (DOX) is a drug used in the treatment of leukaemias and solid tumors but its clinical use is limited by its cardiotoxicity [98-103]. A₃AR activation has been shown to protect against DOX-induced cardiotoxicity by restoring Ca²⁺ homeostasis [104], which suggests that the combination of DOX with A₃AR agonists may prevent the side effects of DOX and allow wider use of this drug in anti-cancer therapy.

A₃AR activation protects not only against cardiac ischaemia but also against stroke. Chen et al. reported that Cl-IB-MECA pre-treatment was protective against hypoxia in primary cortical cultures and decreased the infarct size in an in vivo model of cerebral ischeamia induced by transient middle cerebral artery (MCA) ligation in wild-type mice but not in A₃AR knockout mice. In addition the infarct size induced by MCA ligation was increased in A₃AR knockout mice compared to wild-type controls [105]. Shen et al. found that inosine pre-treatment reduced MCA occlusion-induced cerebral infarction in rats and that this effect was inhibited by administering the A₃AR antagonist, MRS1191, which suggests that the effect of inosine is A₃AR dependent [106]. Finally, A₃AR activation was also found to be protective in a model of subarrachnoid hemorrhage [107].

In contrast, there are also data that support the neurotoxic effects of A₃AR activation. Sei and coworkers treated cultured rat cerebellar neurons with AR agonists and found that Cl-IB-MECA, but not A₁AR, A_2AAR or A_2BAR agonists, induced cell death in cultured neurons [108]. Cl-IB-MECA induced apoptosis also in primary cultures of rat astrocytes and in C6 glial cells by reducing the expression of Bcl-2 and enhancing caspase-3 activation [109]. Von Lubitz and coworkers studied the effect of IB-MECA on forebrain ischaemia in gerbils and found that although chronic IB-MECA treatment improved postischemic cerebral blood circulation, survival, and neuronal preservation, acute preischemic administration of IB-MECA impaired blood flow, enhanced mortality and neuronal damage [110]. The opposite effects of chronic and acute treatment may be related to the desensitization of A₃AR by chronic activation, which is the consequence of the down-regulation of Gi protein [111]. Taken together, both neuroprotective and neurotoxic effects of A₃AR activation have been described.

Unlike in the case of cardiac and cerebral ischeamia, A₃AR activation seems to be consistently harmful in renal ischaemic injury. Lee and coworkers studied the role of A₃ARs in ischaemia-induced renal failure in rats by inducing ischaemia with micro aneurysm clips after pre-treatment with IB-MECA or the A₃AR antagonist, MRS1191. The data showed that MRS1191 pre-treatment decreased blood urea nitrogen and creatinin concentrations and morphological damage in the kidney, and IB-MECA was harmful [112]. In further experiments, they confirmed these results in murine models of renal ischaemia-reperfusion and myoglobinuric injury: renal failure was attenuated in A₃AR deficient mice and WT mice pre-treated with an A₃AR antagonist [113].

3.5. Other inflammatory diseases

A₃AR activation has been found to be protective in a variety of inflammatory diseases including intestinal, pulmonary and systemic inflammation. IB-MECA attenuated colitis-induced inflammatory cell infiltration and colon inflammatory cytokine and chemokine levels in dextran sodium sulphate-induced colitis and also in the spontaneous colitis of IL-10 deficient mice [114]. A₃AR stimulation with IB-MECA has been found to be beneficial also in 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats [115]. In addition, Antonioli and coworkers found that the A₃AR inhibits colonic motility in both normal and inflamed colon [116]

A₃AR activation has been found to be anti-inflammatory in the lung. For example, Cl-IB-MECA inhibited the migration of neutrophils and attenuated microvascular permeability in the lung following LPS inhalation of mice [117]. The results of Morschl and coworkers suggest that along with neutrophil migration, A₃ARs also inhibit the invasion of eosinophils into the lung, as there was an increase in the number of eosinophils and the levels of related cytokines and chemokines in the lung of A₃AR deficient vs. wild-type mice exposed to bleomycin [118].

Dry eye syndrome is a disease caused by decreased tear production or increased tear film evaporation and it is associated with inflammation in the eye [119]. Data from a Phase 2 clinical trial show that orally administered IB-MECA improved various parameters of the disease, including corneal staining, tear break-up time and tear meniscus height and decreased intraocular pressure, in comparison to the group treated with placebo [120].

We studied the effects of A₃AR in a sepsis model induced by intraperitoneal administration of LPS and found that pre-treatment with IB-MECA decreased LPS induced plasma levels of IL-12, IFN-γ and nitrite, and enhanced the level of IL-10. In addition IB-MECA pre-treatment protected the mice from LPS-induced lethality [24]. Lee et al also investigated the effect of A₃AR on sepsis using a different model, cecal ligation and puncture. They found that the mortality, as well as the expression of inflammatory factors in plasma and renal cortices of A₃AR deficient mice was higher than that of wild-type littermates. IB-MECA treatment of WT animals subjected to cecal ligation and puncture improved survival and renal and hepatic function, and these protective effects were absent in A₃AR knockout animals [121].

4. Conclusion

In conclusion, a considerable amount of data from in vitro and in vivo experiments suggests that the A₃AR is expressed in various cell types of the innate and adaptive immune systems and that the A₃AR regulates the functions of both systems (Table 1). In addition the A₃AR regulates the course of numerous diseases, which include RA, cancer and asthma (Table 2).

Table 1.

In vitro effects of A₃AR activation

Cell type	Effect
neutrophil	chemotaxis ↑ or ↓, superoxide ↓
macrophage	TNF-α ↓, MIP-1α ↓, IRF1 ↓, iNOS ↓, CD36 ↓, NADPH ↓, MMP-9 ↑
dendritic cells	chemotaxis ↑, TNF-α ↓
mas cells	degranulation ↑, histamine ↑, apoptosis ↓, Ang-2 ↑
citotoxic T cells	tumoricidal activity ↓, granzyme ↓, perforin ↓, FAS ligand ↓, IFN-γ ↓, IL-2 ↓
NK cells	IFN-α↑
eosinophils	chemotaxis ↓, degranulation ↓, superoxide release ↓
Hep-3B hepatoma cells	apoptosis ↑
U87MG glioblastoma cells	MMP-9 ↑
A375 melanoma cells	HIF-1 ↑, VEGF ↑, Ang-2 ↑
neurons	hypoxia induced cell death ↓, apoptosis ↑

Table 2.

In vivo effects of A₃AR activation

Disease	Effect
Rheumatoid arthritis	Joint: MIP-1α ↓, IL-12 ↓, neutrophil infiltration ↓, deformation ↓
asthma	bronchoconstriction ↑, degranulation of airway mast cells ↑, neutrophil infiltration ↑, hyperresponsiveness ↑, mucin ↑
cancer	Tumor growth ↓, metastasis ↓
cardiac ischaemia	infarct size ↓
cerebral ischaemia	infarct size ↓
renal ischaemia	kidney damage ↑, blood urea ↑
colitis	neutrophil infiltration ↓, IL-1 ↓, IL-6 ↓, IL-12 ↓, MIP-1α ↓, MIP-2 ↓
sepsis	plasma IL-12 ↓, IFN-γ ↓, NO ↓, IL-10 ↑; lethality ↓

5. Expert opinion

Since the recent discovery of the A₃AR, it has become clear that this receptor represents a viable target for the treatment of various immune-related diseases. There are substantial preclinical and clinical data supporting the efficacy of A₃AR agonists in ameliorating the symptoms of RA. A₃AR ligands are also promising therapeutic agents in the treatment of COPD, inflammatory bowel diseases, and ischemia. In addition, a considerable amount of evidence has accumulated that A₃AR agonists are effective in inhibiting tumor growth and the formation of metastases in animal models of cancer.

However, there are numerous uncertainties with regard to the potential use of A₃AR ligands. The A₃AR displays the largest inter-species structural and pharmacological variation of all the adenosine receptors. The variation in the affinity of A₃AR agonists and antagonists, as well as the differential expression and function of the A₃AR among different species makes extrapolation from the preclinical to clinical scenario strenuous. This problem is further compounded by the broad expression of A₃ARs on the various immune cell types and the myriad roles of A₃ARs in regulating physiological functions. Side effects, such as a systemic suppression of the immune system causing augmented vulnerability to infections may limit the usefulness of A₃AR agonists in treating systemic diseases. Developing tissue or cell specific A₃AR ligands or targeted delivery of these ligands may assist in circumventing systemic side effects. Also problematic are the observations that in some of the preclinical studies, A₃AR agonists were effective only at high concentrations raising the possibility of effects mediated via other adenosine receptors. Overcoming these issues will require the development of more selective A₃AR-targeting drugs. These topics may be areas of significant progress in the coming years.

Optimism is, however, fueled by clinical trials, in which IB-MECA was tested in various conditions, which included RA, dry eye syndrome, and psoriasis. Oral administration of IB-MECA was safe and well-tolerated and resulted in improvements in some of the symptoms of diseases in the various trials. Further clinical trials are in progress with IB-MECA in RA and with Cl-IB-MECA in hepatocellular carcinoma and chronic hepatitis C.

Therefore we can be optimistic that new A₃AR ligands with greater specificity and affinity will offer a promising approach to treating inflammatory and immune-related diseases.

Article highlights box.

• A₃AR activation has mainly anti-inflammatory effects in various cell types of the immune system

• A₃AR agonist treatment is beneficial in multiple in vivo models of rheumatoid arthritis.

• In vitro data support the anti-tumoral and also the pro-tumoral effects of A₃AR activation, but in vivo experiments suggest the anti-tumoral role of A₃AR signaling.

• A₃AR activation can be both protective and harmful in different animal models of ischaemia.

• There are various completed and ongoing clinical trials for the use of A₃AR agonists in the treatment of numerous diseases like rheumatoid arthritis, cancer and hepatitis.

Abbreviations

A₁AR

A₁ adenosine receptor

A_2AAR

A_2A adenosine receptor

A_2BAR

A_2B adenosine receptor

A₃AR

A₃ adenosine receptor

ADA

adenosine deaminase

cAMP

cyclic adenosinemonophosphate

CD39

ectonucleoside triphosphate diphosphohydrolase 1

CD73

ecto-5′-nucleotidase

Cl-IB-MECA

2-chloro-N6-(3-iodobenzyl)-adenosine-5′-methylcarboxamide

COPD

chronic obstructive pulmonary disorder

DC

dendritic cell

DOX

doxorubicin

ERK

extracellular signal-regulated kinase

GPCR

G protein coupled receptors

HIF-1

hypoxia-inducible factor-1

IB-MECA

N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide

IKK

IκB kinase

IL-10

interleukin-10

IL-12

interleukin-12

IP₃

Inositol 1,4,5-trisphosphate

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

MIP-1α

macrophage inflammatory protein-1α

MMP

matrix metalloproteinase

MRS1067

3,6-dichloro-2′-isopropyloxy-4′-methyl-flavone

MRS1191

3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate

MRS1334

1,4-Dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl] ester

MRS1523

3-Propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate

MRS 3558

(1′S,2′R,3′S,4′R,5′S)-4-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide

NF-κB

nuclear factor κB

PI3K

Phosphatidylinositol 3-kinases

PLC

phospholipase C

RA

rheumatoid arthritis

TLR

toll-like receptor

TNF-α

tumor necrosis factor-α

VEGF

vascular endothelial growth factor

VUF5574

N-(2-Methoxyphenyl)-N’-[2-(3-pyrindinyl)-4-quinazolinyl]-urea