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Purinergic Ligands as Potential Therapeutic Tools for the Treatment of Inflammation-Related Intestinal Diseases
2018
Abstract
Inflammation-related intestinal diseases are a set of various conditions presenting an overactive enteric immune system. A continuous overproduction of pro-inflammatory cytokines and a decreased production of anti-inflammatory modulators are generally observed, while morpho-functional alterations of the enteric nervous system lead to intestinal secretory and motor dysfunctions. The factors at the basis of these conditions are still to be totally identified and current therapeutic strategies are aimed only at achieving and maintaining remission states, by using therapeutic tools like aminosalicylates, corticosteroids, immunomodulators, biological drugs (i.e., monoclonal antibodies), and eventually surgery.
Recent reports described a key role of purinergic mediators (i.e., adenosine and its nucleotides ATP and ADP) in the regulation of the activity of immune cells and enteric nervous system, showing also that alterations of the purinergic signaling are linked to pathological conditions of the intestinal tract.
These data prompted to a series of investigations to test the therapeutic potential for inflammation-related intestinal conditions of compounds able to restore or modulate an altered purinergic signaling within the gut.
This review provides an overview on these investigations, describing the results of preclinical and/or clinical evaluation of compounds able to stimulate or inhibit specific P2 (i.e., P2X7) or P1 (i.e., A2A or A3) receptor signaling and to modify the adenosine levels through the modulation of enzymes activity (i.e., Adenosine Deaminase) or nucleoside transporters.
Recent developments in the field are also reported and the most promising purine-based therapeutic strategies for the treatment of inflammation-related gastrointestinal disorders are schematically summarized.
Introduction
….
It is well established that during inflammation ATP is extracellularly released, a process involving pannexins or connexins and promoted by various stimuli (Eltzschig et al., 2012; Idzko et al., 2014).
Extracellular ATP (eATP) is then degraded to adenosine by the ectonucleotidases CD39 and CD73 (Allard et al., 2017).
While eATP generally plays a pro-inflammatory role through the activation of P2 (P2X and P2Y) purinergic receptors, the ATP degradation to adenosine usually represents a stop-signal for the inflammation process, with adenosine playing as anti-inflammatory agent through the activation of its P1 receptor targets.
Adenosine is then removed from the extracellular environment by nucleoside transporters and/or metabolic enzymes.
Over the years increasing evidences pointed out a critical involvement of the purinergic system in the pathophysiology of IBDs, thus spurring the research toward the evaluation of the potential therapeutic benefits in terms of anti-inflammatory activity, arising by pharmacological targeting of purinergic pathways (Hasko and Cronstein, 2004; Hasko and Pacher, 2008; Hasko et al., 2008; Burnstock, 2011, 2014; Burnstock et al., 2017). Furthermore, the involvement of ATP in the enteric motor dysfunctions associated with bowel inflammation is a hot topic deserving further investigations.
P2XR
…...
The potent, selective, and orally bioavailable P2X7R antagonist AZD9056 was studied in phase-two clinical trials for the treatment of rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD) showing to be well tolerated (2005-004110-32_Clinical_Trial_Results, 2005; Keystone et al., 2012). The efficacy and safety of AZD9056 was also clinically assessed in the management of patients affected by moderate/severe Crohn’s disease. Although the lack in change of inflammatory parameters, this study demonstrated that AZD9056 has the potential to improve symptoms, in particular abdominal pain, in patients with IBDs (Eser et al., 2015). Other P2X7R antagonists (CE-224,535 and GSK1482160) were studied in clinical trials for RA and inflammatory pain conditions or showed (JNJ47965567) ability to enter the CNS.
…
Antagonists of the P2X3R showed promising activity in alleviating inflammatory and neuropathic pain in preclinical studies. A-317491 reduced visceral hypersensitivity in an experimental model of colitis, suggesting P2X3R as target for the treatment of inflammation-related abdominal pain syndromes (Deiteren et al., 2015).
….
P1 Adenosine Receptors
Adenosine Receptors ... four subtypes (A1AR, A2AAR, A2BAR, and A3AR). Like the other Purinergic Receptors, ARs are widely expressed in the body and regulate many physiological functions. The endogenous ligand adenosine has a short half-life as it is internalized by nucleoside transporters and/or quickly modified to inosine by Adenosine Deaminase (ADA) or to AMP by Adenosine Kinase (ADK).
Reference A3AR agonists are IB-MECA and Cl-IB-MECA, in clinical trials for inflammation-related conditions (RA and psoriasis) (Borea et al., 2015; Jacobson et al., 2017).
….
About the molecules able to block the AR function, AR antagonists are generally divided in non-xanthine- and xanthine-based derivatives. Non-xanthine AR antagonists are based on a large variety of scaffolds (generally heterocycles). Reference compounds for pharmacological studies at ARs may be found within this heterogeneous group, some of which were developed also as water-soluble molecules, pro-drugs, and radiolabeled compounds. Xanthine-based AR antagonists contain the A2AAR inhibitor Istradefylline that was approved to market in Japan as antiparkinsonian tool (Jacobson and Muller, 2016).
At present, most of available studies investigating the role of AR signaling in several experimental models of colitis showed remarkable beneficial effects upon pharmacological modulation of A2AAR (Odashima et al., 2005; Cavalcante et al., 2006; Naganuma et al., 2006; Rahimian et al., 2010; Antonioli et al., 2011; Pallio et al., 2016). The A2AAR agonists ATL-146e or ATL-313 significantly reduced mucosal inflammation of colon, with a marked decrease in pro-inflammatory cytokine levels and in leukocyte infiltration and an increase of levels of anti-inflammatory cytokines (Naganuma et al., 2006; Odashima et al., 2006).
Recently Pallio et al. (2016) demonstrated the beneficial effects arising from A2AAR stimulation with polydeoxyribonucleotide in two experimental models of colitis. In the DSS model polydeoxyribonucleotide could counteract the hemorrhagic diarrhea, improve the weight loss, and restore the anatomic integrity of damaged epithelial and mucosal layers. In the DNBS model, polydeoxyribonucleotide markedly reduced the inflammatory response as well as the granulocytic infiltration into the mucosal and submucosal layers and, therefore, decreased the pro-inflammatory cytokines TNF and IL-1β, MPO activity and lipid peroxidation in colon samples. Noteworthy, polydeoxyribonucleotide treatment also affected Bax and Bcl-2 expression, reducing apoptotic and necrotic cells in all tissue layers. By contrast, no beneficial effects have been reported upon administration of CGS21680 in mouse model of DSS-induced colitis (Selmeczy et al., 2007). Further investigations are needed to better characterize the therapeutic potential of A2AAR agonists in IBDs.
A number of evidences reported that aging is often associated with a chronic, low-grade systemic inflammatory condition (Laflamme et al., 2017), that could predispose to the gastrointestinal alterations typical of the elderly subject (Remond et al., 2015). Recently, it has been demonstrated that the reduction of A2AAR in the digestive tract of aged mice, contributes to an increased inflammation and lower ability to counteract gut infection with deleterious effects in the elderly (Rodrigues et al., 2016).
Another promising option aimed at counteracting the bowel inflammation is the pharmacological stimulation of A3AR (Gessi et al., 2008; Antonioli et al., 2014a). The administration of IB-MECA revealed to afford a protective role in murine models of intestinal inflammation (Mabley et al., 2003; Guzman et al., 2006). In detail, the pharmacological engagement of A3AR determined the inhibition of several cytokine/chemokine/inflammatory genes, thus promoting a marked down-regulation of several pro-inflammatory mediators (MIP-1α and MIP-2, IL-1, IL-6, IL-12) and the production of reactive species of oxygen, determining an improvement of the intestinal damage (Guzman et al., 2006). A recent preclinical study by Ren et al. (2015) showed that the pharmacological stimulation of A3AR with Cl-IB-MECA inhibited the NF-κB pathway in the colonic epithelia of DSS colitis mice.
…
Regulation of Extracellular Adenosine Levels
Several authors investigated the efficacy of pharmacological treatments aimed at increasing the levels of endogenous adenosine, through the blockade of pivotal catabolic enzymes, as an alternative way to counteract intestinal inflammation. eATP is rapidly degraded to adenosine by ectonucleotidases CD73 and CD39.
Genetic deletion of these enzymes prompts a higher susceptibility to inflammatory states or more severe progression of inflammation in IBD experimental models (Idzko et al., 2014; Longhi et al., 2017).
Polymorphisms taking to lower ectonucleotidases expression takes to analog scenarios (Idzko et al., 2014).
By contrast, blockade of nucleoside transporters leads to an increase of extracellular adenosine levels, with a consequent improvement of the inflammation course in IBD models (Ye and Rajendran, 2009). The extracellular levels of adenosine are also regulated by the activity of metabolic enzymes like ADK and ADA (Cristalli et al., 2001).
The blockade of these enzymes is associated to anti-inflammatory effects and was preclinically tested as strategy to ameliorate intestinal inflammation-related conditions.
Siegmund et al. (2001) reported, for the first time, an anti-inflammatory effect of the ADK inhibitor GP515 in a mouse model of colitis.
Encouraging ameliorative effects were observed also following the inhibition of ADA in murine models of intestinal inflammation (Antonioli et al., 2007, 2010, 2012; La Motta et al., 2009), with reduction of the colonic inflammatory damage and decrease in tissue levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ, and chemokine C-X-C motif ligand 10), as well as a reduction of neutrophil infiltration and ROS production (Antonioli et al., 2007, 2010; Brown et al., 2008; La Motta et al., 2009).
Noteworthy, it was observed that the attenuation of colonic injury, following ADA blockade, was mediated mainly by the engagement of A2AAR and, to a lesser extent, A3AR (Antonioli et al., 2010).
…..
2018
Abstract
Inflammation-related intestinal diseases are a set of various conditions presenting an overactive enteric immune system. A continuous overproduction of pro-inflammatory cytokines and a decreased production of anti-inflammatory modulators are generally observed, while morpho-functional alterations of the enteric nervous system lead to intestinal secretory and motor dysfunctions. The factors at the basis of these conditions are still to be totally identified and current therapeutic strategies are aimed only at achieving and maintaining remission states, by using therapeutic tools like aminosalicylates, corticosteroids, immunomodulators, biological drugs (i.e., monoclonal antibodies), and eventually surgery.
Recent reports described a key role of purinergic mediators (i.e., adenosine and its nucleotides ATP and ADP) in the regulation of the activity of immune cells and enteric nervous system, showing also that alterations of the purinergic signaling are linked to pathological conditions of the intestinal tract.
These data prompted to a series of investigations to test the therapeutic potential for inflammation-related intestinal conditions of compounds able to restore or modulate an altered purinergic signaling within the gut.
This review provides an overview on these investigations, describing the results of preclinical and/or clinical evaluation of compounds able to stimulate or inhibit specific P2 (i.e., P2X7) or P1 (i.e., A2A or A3) receptor signaling and to modify the adenosine levels through the modulation of enzymes activity (i.e., Adenosine Deaminase) or nucleoside transporters.
Recent developments in the field are also reported and the most promising purine-based therapeutic strategies for the treatment of inflammation-related gastrointestinal disorders are schematically summarized.
Introduction
….
It is well established that during inflammation ATP is extracellularly released, a process involving pannexins or connexins and promoted by various stimuli (Eltzschig et al., 2012; Idzko et al., 2014).
Extracellular ATP (eATP) is then degraded to adenosine by the ectonucleotidases CD39 and CD73 (Allard et al., 2017).
While eATP generally plays a pro-inflammatory role through the activation of P2 (P2X and P2Y) purinergic receptors, the ATP degradation to adenosine usually represents a stop-signal for the inflammation process, with adenosine playing as anti-inflammatory agent through the activation of its P1 receptor targets.
Adenosine is then removed from the extracellular environment by nucleoside transporters and/or metabolic enzymes.
Over the years increasing evidences pointed out a critical involvement of the purinergic system in the pathophysiology of IBDs, thus spurring the research toward the evaluation of the potential therapeutic benefits in terms of anti-inflammatory activity, arising by pharmacological targeting of purinergic pathways (Hasko and Cronstein, 2004; Hasko and Pacher, 2008; Hasko et al., 2008; Burnstock, 2011, 2014; Burnstock et al., 2017). Furthermore, the involvement of ATP in the enteric motor dysfunctions associated with bowel inflammation is a hot topic deserving further investigations.
P2XR
…...
The potent, selective, and orally bioavailable P2X7R antagonist AZD9056 was studied in phase-two clinical trials for the treatment of rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD) showing to be well tolerated (2005-004110-32_Clinical_Trial_Results, 2005; Keystone et al., 2012). The efficacy and safety of AZD9056 was also clinically assessed in the management of patients affected by moderate/severe Crohn’s disease. Although the lack in change of inflammatory parameters, this study demonstrated that AZD9056 has the potential to improve symptoms, in particular abdominal pain, in patients with IBDs (Eser et al., 2015). Other P2X7R antagonists (CE-224,535 and GSK1482160) were studied in clinical trials for RA and inflammatory pain conditions or showed (JNJ47965567) ability to enter the CNS.
…
Antagonists of the P2X3R showed promising activity in alleviating inflammatory and neuropathic pain in preclinical studies. A-317491 reduced visceral hypersensitivity in an experimental model of colitis, suggesting P2X3R as target for the treatment of inflammation-related abdominal pain syndromes (Deiteren et al., 2015).
….
P1 Adenosine Receptors
Adenosine Receptors ... four subtypes (A1AR, A2AAR, A2BAR, and A3AR). Like the other Purinergic Receptors, ARs are widely expressed in the body and regulate many physiological functions. The endogenous ligand adenosine has a short half-life as it is internalized by nucleoside transporters and/or quickly modified to inosine by Adenosine Deaminase (ADA) or to AMP by Adenosine Kinase (ADK).
Reference A3AR agonists are IB-MECA and Cl-IB-MECA, in clinical trials for inflammation-related conditions (RA and psoriasis) (Borea et al., 2015; Jacobson et al., 2017).
….
About the molecules able to block the AR function, AR antagonists are generally divided in non-xanthine- and xanthine-based derivatives. Non-xanthine AR antagonists are based on a large variety of scaffolds (generally heterocycles). Reference compounds for pharmacological studies at ARs may be found within this heterogeneous group, some of which were developed also as water-soluble molecules, pro-drugs, and radiolabeled compounds. Xanthine-based AR antagonists contain the A2AAR inhibitor Istradefylline that was approved to market in Japan as antiparkinsonian tool (Jacobson and Muller, 2016).
At present, most of available studies investigating the role of AR signaling in several experimental models of colitis showed remarkable beneficial effects upon pharmacological modulation of A2AAR (Odashima et al., 2005; Cavalcante et al., 2006; Naganuma et al., 2006; Rahimian et al., 2010; Antonioli et al., 2011; Pallio et al., 2016). The A2AAR agonists ATL-146e or ATL-313 significantly reduced mucosal inflammation of colon, with a marked decrease in pro-inflammatory cytokine levels and in leukocyte infiltration and an increase of levels of anti-inflammatory cytokines (Naganuma et al., 2006; Odashima et al., 2006).
Recently Pallio et al. (2016) demonstrated the beneficial effects arising from A2AAR stimulation with polydeoxyribonucleotide in two experimental models of colitis. In the DSS model polydeoxyribonucleotide could counteract the hemorrhagic diarrhea, improve the weight loss, and restore the anatomic integrity of damaged epithelial and mucosal layers. In the DNBS model, polydeoxyribonucleotide markedly reduced the inflammatory response as well as the granulocytic infiltration into the mucosal and submucosal layers and, therefore, decreased the pro-inflammatory cytokines TNF and IL-1β, MPO activity and lipid peroxidation in colon samples. Noteworthy, polydeoxyribonucleotide treatment also affected Bax and Bcl-2 expression, reducing apoptotic and necrotic cells in all tissue layers. By contrast, no beneficial effects have been reported upon administration of CGS21680 in mouse model of DSS-induced colitis (Selmeczy et al., 2007). Further investigations are needed to better characterize the therapeutic potential of A2AAR agonists in IBDs.
A number of evidences reported that aging is often associated with a chronic, low-grade systemic inflammatory condition (Laflamme et al., 2017), that could predispose to the gastrointestinal alterations typical of the elderly subject (Remond et al., 2015). Recently, it has been demonstrated that the reduction of A2AAR in the digestive tract of aged mice, contributes to an increased inflammation and lower ability to counteract gut infection with deleterious effects in the elderly (Rodrigues et al., 2016).
Another promising option aimed at counteracting the bowel inflammation is the pharmacological stimulation of A3AR (Gessi et al., 2008; Antonioli et al., 2014a). The administration of IB-MECA revealed to afford a protective role in murine models of intestinal inflammation (Mabley et al., 2003; Guzman et al., 2006). In detail, the pharmacological engagement of A3AR determined the inhibition of several cytokine/chemokine/inflammatory genes, thus promoting a marked down-regulation of several pro-inflammatory mediators (MIP-1α and MIP-2, IL-1, IL-6, IL-12) and the production of reactive species of oxygen, determining an improvement of the intestinal damage (Guzman et al., 2006). A recent preclinical study by Ren et al. (2015) showed that the pharmacological stimulation of A3AR with Cl-IB-MECA inhibited the NF-κB pathway in the colonic epithelia of DSS colitis mice.
…
Regulation of Extracellular Adenosine Levels
Several authors investigated the efficacy of pharmacological treatments aimed at increasing the levels of endogenous adenosine, through the blockade of pivotal catabolic enzymes, as an alternative way to counteract intestinal inflammation. eATP is rapidly degraded to adenosine by ectonucleotidases CD73 and CD39.
Genetic deletion of these enzymes prompts a higher susceptibility to inflammatory states or more severe progression of inflammation in IBD experimental models (Idzko et al., 2014; Longhi et al., 2017).
Polymorphisms taking to lower ectonucleotidases expression takes to analog scenarios (Idzko et al., 2014).
By contrast, blockade of nucleoside transporters leads to an increase of extracellular adenosine levels, with a consequent improvement of the inflammation course in IBD models (Ye and Rajendran, 2009). The extracellular levels of adenosine are also regulated by the activity of metabolic enzymes like ADK and ADA (Cristalli et al., 2001).
The blockade of these enzymes is associated to anti-inflammatory effects and was preclinically tested as strategy to ameliorate intestinal inflammation-related conditions.
Siegmund et al. (2001) reported, for the first time, an anti-inflammatory effect of the ADK inhibitor GP515 in a mouse model of colitis.
Encouraging ameliorative effects were observed also following the inhibition of ADA in murine models of intestinal inflammation (Antonioli et al., 2007, 2010, 2012; La Motta et al., 2009), with reduction of the colonic inflammatory damage and decrease in tissue levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ, and chemokine C-X-C motif ligand 10), as well as a reduction of neutrophil infiltration and ROS production (Antonioli et al., 2007, 2010; Brown et al., 2008; La Motta et al., 2009).
Noteworthy, it was observed that the attenuation of colonic injury, following ADA blockade, was mediated mainly by the engagement of A2AAR and, to a lesser extent, A3AR (Antonioli et al., 2010).
…..