A2A Agonist Research Articles

Adenosine receptors differentially mediate enteric glial cell death induced by Clostridioides difficile Toxins A and B.

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.

The role of dorsomedial striatum adenosine 2A receptors in the loss of goal-directed behaviour.

Adenosine A2A receptors (A2AR) in the dorsal striatum have been implicated in goal-directed behaviour. While activation of these receptors with several methods has resulted in an insensitivity to outcome devaluation, particular explanations for how they disrupt behaviour have not been explored. We both confirm a role for A2A receptors in goal-directed responding and evaluate additional behavioural aspects of goal-directed control to more fully understand the role of A2A receptors in instrumental behaviour. To examine the effects of the adenosine A2A agonist CGS-21680 in the DMS on response-outcome encoding, updating representations of outcome value and on the ability to inhibit behaviour when reward is not available. Male rats were trained to lever press for food reward. The A2AR agonist CGS-21680 was infused into the dorsomedial striatum either before an outcome devaluation test, prior to training with two distinct response-outcome associations or prior to a test of discriminative stimulus control over instrumental performance. Intra-DMS administration of CGS-21680 impaired sensitivity to outcome devaluation. CGS-21680 treatment did not impair acquisition of specific response-outcome associations, selective control of responding based on the presence of stimuli that signaled when reward was or was not available, discrimination between stimuli or lever choices nor did it influence the effect of devaluation on the amounts of food eaten in a consumption test. CGS-21680 impairs the ability to modulate responding based on recent changes to outcome value, an effect that is not accounted for by impairments in behavioural inhibition, discrimination, encoding the specific outcome of a response or the effectiveness of specific satiety.

Impact of an Adenosine A2A Receptor Agonist and Antagonist on Binding of the Dopamine D2 Receptor Ligand [11C]raclopride in the Rodent Striatum.

Adenosine A2A and dopamine D2 receptors in the basal ganglia form heterotetrameric structures that are involved in the regulation of motor activity and neuropsychiatric functions. The present study examines the A2A receptor-mediated modulation of D2 receptor binding in vivo using positron emission tomography (PET) with the D2 antagonist tracer [11C]raclopride. Healthy male Wistar rats (n = 8) were scanned (60 min dynamic scan) with [11C]raclopride at baseline and 7 days later following an acute administration of the A2A agonist CGS21680 (1 mg/kg), using a MicroPET Focus-220 camera. Nondisplaceable binding potential (BPND) values were calculated using a simplified reference tissue model (SRTM), with cerebellum as the reference tissue. SRTM analysis did not show any significant changes in [11C]raclopride BPND (p = 0.102) in striatum after CGS21680 administration compared to the baseline. As CGS21680 strongly affects hemodynamics, we also used arterial blood sampling and a metabolite-corrected plasma input function for compartment modeling using the reversible two-tissue compartment model (2TCM) to obtain the BPND from the k3/k4 ratio and from the striatum/cerebellum volume of distribution ratio (DVR) in a second group of animals. These rats underwent dynamic [11C]raclopride scans after pretreatment with a vehicle (n = 5), a single dose of CGS21680 (1 mg/kg, n = 5), or a single dose of the A2A antagonist KW6002 (1 mg/kg, n = 5). The parent fraction in plasma was significantly higher in the CGS21680-treated group (p = 0.0001) compared to the vehicle-treated group. GCS21680 administration significantly reduced the striatal k3/k4 ratio (p < 0.01), but k3 and k4 estimates may be less reliable. The BPND (DVR-1) decreased from 1.963 ± 0.27 in the vehicle-treated group to 1.53 ± 0.55 (p = 0.080) or 1.961 ± 0.11 (p = 0.993) after the administration of CGS21680 or KW6002, respectively. Our study suggests that the A2A agonist CGS21680, but not the antagonist KW6002, may reduce the D2 receptor availability in the striatum.

Limonene restores A2AAR mediated‐vascular relaxation in asthmatic mice via nitric oxide

Limonene, a mono‐terpene flavonoid, has anti‐inflammatory effects in asthma by activating A2A adenosine receptors (A2AAR). Our objective was to identify the mechanism by which limonene restored A2A mediated aortic relaxation in asthmatic mice. Ex‐vivo tissue bath was used to study the effects of inhaled d‐limonene on isolated aortic rings from asthmatic mice. We used mice in which the A2A receptor was genetically knocked out (A2AKO) and C57BLJ/6 (wild‐type; WT) for the study. The mice were divided into control (CON) and allergen‐induced sensitized (SEN), control mice treated with limonene through aerosol route (CON+LIM) and allergen‐induced sensitized mice treated with limonene (SEN+LIM). Mice were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Control groups were given an i.p injection of vehicle (1,6 days) and the aerosol challenge of normal saline (11–13 days). SEN +LIM, CON +LIM groups were given 0.01% v/v aerosolized limonene via inhalation 90 min prior to challenges. Aorta were isolated on day 14 for further experiments. Tissue responses were studied in the form of contraction and relaxations in all groups. Concentration‐response curves (CRC) for selective A2Aagonist CGS‐21680 (10‐11‐10‐6M) were obtained for all groups. eNOS inhibitor L‐NAME (10‐4M) or prostaglandin inhibitor indomethacin (10‐5M) was added 30 min prior to starting CRC to study effect of nitric oxide and prostaglandins on vascular response. CGS‐21680 induced aortic relaxation was absent in WT SEN (5.62±3.72% WT SEN Vs 66.91 ± 6.8% CON, *p&lt;0.05). Limonene restored CGS 21680‐induced relaxation in asthmatic mice (63.8 ± 6.32% WT SEN+LIM Vs 5.62± 3.72% WT SEN). L‐NAME blocked CGS‐21680 response in CON, CON+ LIM and SEN+LIM, while indomethacin had minimal effect. In conclusion, it appears that limonene restores A2AAR‐mediated aortic relaxation induced through nitric oxide in asthmatic mice.

Regionally selective cardiovascular responses to adenosine A2A and A2B receptor activation.

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein‐coupled receptors (A1, A2A, A2B, A3). Here, we have investigated the effect of A2A and A2B‐selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague‐Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A2A‐selective agonist CGS 21680 (0.1, 0.3, 1 µg kg−1 min−1) or the A2B‐selective agonist BAY 60‐6583 (4,13.3, 40 µg kg−1 min−1) following predosing with the A2A‐selective antagonist SCH 58261 (0.1 or 1 mg kg−1 min−1), the A2B/A2A antagonist PSB 1115 (10 mg kg−1 min−1) or vehicle. The A2A‐selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B‐selective agonist BAY 60‐6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury.

Upregulation of Src Family Tyrosine Kinases in the Rat Striatum by Adenosine A2A Receptors.

Adenosine A2A receptors are Golf-coupled receptors and are predominantly expressed in the striatum of mammalian brains. As a mostly postsynaptic receptor, A2A receptors are implicated in the regulation of a variety of intracellular signaling pathways in striatopallidal output neurons and are linked to the pathogenesis of various neuropsychiatric and neurological disorders. This study investigated the possible role of A2A receptors in the modulation of the Src family kinase (SFK) in the adult rat striatum. In acutely prepared striatal slices, adding the A2A receptor agonist PSB-0777 induced a significant increase in phosphorylation of SFKs at a conserved autophosphorylation site (Y416) in the caudate putamen (CPu). This increase was also seen in the nucleus accumbens (NAc). Another A2A agonist CGS-21680 showed the similar ability to elevate SFK Y416 phosphorylation in the striatum. Treatment with the A2A receptor antagonist KW-6002 blocked the effect of PSB-0777 on SFK Y416 phosphorylation. In addition, PSB-0777 enhanced kinase activity of two key SFK members (Src and Fyn) immunoprecipitated from the striatum. These data demonstrate a positive linkage from A2A receptors to the SFK signaling pathway in striatal neurons. Activation of A2A receptors leads to the upregulation of phosphorylation of SFKs (Src and Fyn) at an activation-associated autophosphorylation site and kinase activity of these SFK members.

Selective Adenosine A2A Agonists May Change Myocardial Perfusion Imaging.

In recent years, the requirement for pharmacological stress myocardial perfusion imaging (SPECT) has increased, and adenosine stress testing is now the mainstream. Selective adenosine A2A receptor agonists will be applied clinically in the future. By selectively activating only A2A receptors, it can reduce complications such as bronchospasm, hypotension, and bradycardia, which have been problems with adenosine stress tests. In addition, since this drug can be administered in bolus injection, it has the advantage of being able to perform the test at one root.

Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing of Fibrinogen Density in Thrombus.

Blood platelets’ adenosine receptors (AR) are considered to be a new target for the anti-platelet therapy. This idea is based on in vitro studies which show that signaling mediated by these receptors leads to a decreased platelet response to activating stimuli. In vivo evidence for the antithrombotic activity of AR agonists published to date were limited, however, to the usage of relatively high doses given in bolus. The present study was aimed at verifying if these substances used in lower doses in combination with inhibitors of P2Y12 could serve as components of dual anti-platelet therapy. We have found that a selective A2A agonist 2-hexynyl-5’-N-ethylcarboxamidoadenosine (HE-NECA) improved the anti-thrombotic properties of either cangrelor or prasugrel in the model of ferric chloride-induced experimental thrombosis in mice. Importantly, HE-NECA was effective not only when applied in bolus as other AR agonists in the up-to-date published studies, but also when given chronically. In vitro thrombus formation under flow conditions revealed that HE-NECA enhanced the ability of P2Y12 inhibitors to decrease fibrinogen content in thrombi, possibly resulting in their lower stability. Adenosine receptor agonists possess a certain hypotensive effect and an ability to increase the blood–brain barrier permeability. Therefore, the effects of anti-thrombotic doses of HE-NECA on blood pressure and the blood–brain barrier permeability in mice were tested. HE-NECA applied in bolus caused a significant hypotension in mice, but the effect was much lower when the substance was given in doses corresponding to that obtained by chronic administration. At the same time, no significant effect of HE-NECA was observed on the blood–brain barrier. We conclude that chronic administration of the A2A agonist can be considered a potential component of a dual antithrombotic therapy. However, due to the hypotensive effect of the substances, dosage and administration must be elaborated to minimize the side-effects. The total number of animals used in the experiments was 146.

Ex‐vivo Effects of Limonene in Airways and Vasculature in Allergic Adenosine A2A receptor Knock‐out and Wild‐type mice

Limonene, a mono‐terpene flavonoid, has anti‐inflammatory effects in asthma by activating A2A adenosine receptors. We studied the effects of inhaled d‐limonene on isolated aortic and tracheal rings from asthmatic mice using tissue/organ bath system. We used mice in which the A2A receptor was genetically knocked out (A2AKO) and C57BLJ/6 (wild‐type; WT) for the study. The mice were divided into control (CON) and allergen‐induced sensitized (SEN), control mice treated with limonene through aerosol route (CON+LIM) and allergen‐induced sensitized mice treated with limonene (SEN+LIM). Mice were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Control groups were given an i.p injection of vehicle (1,6 days) and the aerosol challenge of normal saline (11–13 days). SEN +LIM and CON +LIM groups were given 0.01% limonene in the form of aerosol 90 min prior to the OVA and saline challenge on days 11,12,13. Trachea and aorta were isolated on day 14 for further experiments. Tissue responses were studied in the form of contraction and relaxations in all groups. Treatment with limonene significantly reduced the tracheal reactivity (80.68±20% in SEN+LIM vs SEN; p&lt;0.05) to methacholine (MCh) in WT mice that were allergic. MCh induced tracheal contractions were not altered in A2AKO limonene treated sensitized group compared to non‐treated A2AKO sensitized group (209.87 ± 41.33 A2AKO SEN+LIMSEN+LIM Vs 212.51 ± 43.18 in A2AKO SEN) In presence of A2A‐antagonist (SCH58651), a significant increase in the MCh induced contractility in WT SEN was observed(341.65 ± 2.664 SEN+SCH Vs 225.99 ± 67.34 SEN, pP&lt;0.05). A2A agonist CGS 21680 induced relaxation in WT CON was abolished in WT SEN but restored in WT SEN+LIM (37.8± 16.4% Vs 1.2± 11.1 in SEN, p&lt;0.05). The CGS‐induced tracheal relaxation in WT SEN+LIM and SEN groups was increased in the presence of the NADPH inhibitor apocynin (60.58 ± 17.20 SEN; 21.28 ± 16.02 SEN). In the aorta, acetylcholine‐induced aortic relaxation (endothelium‐dependent response) in WT SEN group was significantly lower than controls (29.82 ± 5.62 WT SEN Vs 51.51 ± 30.13 WT CON, p&lt;0.05) with limonene restoring the response (61.56 ± 11.34 WT‐SEN+LIM). CGS 21680 induced aortic relaxation in WT SEN was completely blunted compared to WT CON (5.62±3.72 WT SEN Vs 66.91 ± 6.8 WT‐CON, *p&lt;0.05). Treatment with limonene restored CGS 21680‐induced relaxation in asthmatic mice (63.8 ± 6.32 WT SEN+LIM Vs 5.62± 3.72 SEN) In conclusion, it appears that limonene restores the tracheal and aortic relaxation induced by adenosine‐mediated pathways by activating the A2A receptor in asthmatic mice.Support or Funding InformationSupported by LIU start‐up funds (DSP), HL027339 (SJM)

Regulation of Platelet Function By Adenosine Receptors

Introduction: We have recently shown that severe trauma and hemorrhage lead to inhibition of platelet aggregation and an elevation in cyclic adenosine monophosphate (cAMP). Adenosine is one of the few humoral agents known to stimulate cAMP in platelets. Because adenosine is released from damaged tissue, it may contribute to the platelet dysfunction seen after severe trauma. Platelets have four adenosine receptors (A1, A2a, A2b and A3). These receptors are G-Protein Coupled Receptors and have been proposed to stimulate adenylyl cyclase and increase intracellular cAMP. Although studies have shown that stimulate A2a can inhibit platelet aggregation and elevate cAMP, there is little data elucidating the function of the other receptors. Objective: Define which adenosine receptors affects platelet aggregation and cAMP production. Methods: Platelet-rich plasma (PRP) was isolated from whole blood of human volunteers, and centrifuged at 200g for 10min. Light transmission aggregometry was performed using a plate reader (Synergy Neo2 Multimode Reader, BioTek) with constant agitation. PRP was stimulated with adenosine diphosphate (ADP) with or without various adenosine agonists or antagonists, including the non-metabolizable adenosine agonist 5-(N-ethyl-carboxamido) adenosine (NECA), antagonists to receptors A1 (DPCPX), A2a (Sch 58261), A2b (GS 6201) and A3 (MRS 1220), or agonists for A2a (CGS 21680) A2b (BAY 60-6583) or agonist A1 (CCPA), A2a (CGS 21680), A2b (Bay 60-6583), A3 (2-Cl-IB-Meca). Cyclic AMP was extracted from 100ul of PRP after adding 1ml of EtOH, 10mM ammonium formate, with 10ug/ml cGMP-Br as an internal control. Samples were centrifuged at 20K g for 10min, and supernatant dried. Samples were brought up in 200ul of 0.1% formic acid for analysis by Reverse Phase liquid chromatography/ Tandem Mass Spectroscopy (Quantiva, ThrermoFisher). N-8/group. Results: Adenosine diphosphate (100uM) leads to platelet aggregation (change in mAbsorbance units, Table 1). The adenosine agonist NECA inhibited aggregation to ADP and elevated cAMP in a dose dependent manner (pg/ml per 1000 plt, Table 1). Platelet aggregation was inhibited and cAMP was elevated after stimulation with agonists for adenosine receptor A2a agonist, but not A1, A2b, or A3 (Table 2). Antagonists for A2a, but not A1, A2b, A3, blocked NECA inhibition of ADP aggregation (Table 3). Agonist for adenosine receptor A2a inhibited the ADP-induced aggregation and elevated cAMP in a dose response manner (Table 4). Discussion: Adenosine inhibits platelet aggregation to ADP. The mechanism appears to be due to elevation in intracellular cAMP, and works through the A2a receptor. These data suggest that the A2a receptor could be potential target for a resuscitation strategy that could attenuate or prevent platelet dysfunction after trauma by preventing stimulation of adenylate cyclase and synthesis of cAMP. This study was funded by the US Army medical Research and Development Command. Disclosures No relevant conflicts of interest to declare.

Effects of an adenosine A2A agonist on the rewarding associative properties of nicotine and neural plasticity in a rodent model of schizophrenia.

Adenosine A2a receptors form a mutually inhibitory heteromeric complex with dopamine D2 receptors such that each receptor exhibits lower sensitivity to its agonist after the opposing receptor agonist is bound. This study analyzed the effects of CGS 21680, an adenosine A2A agonist, on nicotine conditioned place preference (CPP) in adolescence using a rodent model of schizophrenia (SZ). Rats were treated from postnatal day (P) 1 to P21 with saline or the dopamine D2/D3 agonist quinpirole (NQ treatment) and raised to P41. After an initial preference test, rats were conditioned with saline or nicotine (0.6 mg/kg base) from P43 to P51. CGS 21680 (0.03 or 0.09 mg/kg) was given 15 minutes before nicotine was administered. The post-conditioning test was administered on P52. On P53, the nucleus accumbens (NAcc) was analyzed for brain-derived neurotrophic factor (BDNF) and glial cell-lined neurotrophic factor (GDNF). Results revealed that NQ treatment enhanced nicotine CPP, and both doses of CGS 21680 alleviated this enhancement. Nicotine also resulted in a CPP in controls, which was alleviated by both doses of CGS 21680. BDNF closely followed the behavioral results: CGS 21680 alleviated the enhancement in NAcc BDNF in NQ-treated animals, and eliminated the increase in NAcc BDNF produced by nicotine in controls. NQ-treated animals conditioned to nicotine resulted in an increase of NAcc GDNF, but this was eliminated by CGS 21680. Both BDNF and GDNF correlated with CPP performance. Results revealed that an adenosine A2A agonist decreased the rewarding aspects of nicotine and its accompanying neural plasticity changes in a model of SZ.

Deficiency of adenosine deaminase 2 triggers adenosine-mediated NETosis and TNF production in patients with DADA2

AbstractReduction of adenosine deaminase 2 (ADA2) activity due to autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1) results in a systemic vasculitis known as deficiency of ADA2 (DADA2). Neutrophils and a subset of neutrophils known as low-density granulocytes (LDGs) have been implicated in the pathogenesis of vasculitis, at least in part, through the formation of neutrophil extracellular traps (NETs). The study objective was to determine whether neutrophils and NETs play a pathogenic role in DADA2. In vivo evidence demonstrated NETs and macrophages in affected gastrointestinal tissue from patients with DADA2. An abundance of circulating LDGs prone to spontaneous NET formation was observed during active disease in DADA2 and were significantly reduced after remission induction by anti–tumor necrosis factor (TNF) therapy. Increased circulating LDGs were identified in unaffected family members with monoallelic ADA2 mutations. Adenosine triggered NET formation, particularly in neutrophils from female patients, by engaging A1 and A3 adenosine receptors (ARs) and through reactive oxygen species– and peptidylarginine deiminase–dependent pathways. Adenosine-induced NET formation was inhibited by recombinant ADA2, A1/A3 AR antagonists, or by an A2A agonist. M1 macrophages incubated with NETs derived from patients with DADA2 released significantly greater amounts of TNF-α. Treatment with an A2AAR agonist decreased nuclear translocation of NF-κB and subsequent production of inflammatory cytokines in DADA2 monocyte-derived macrophages. These results suggest that neutrophils may play a pathogenic role in DADA2. Modulation of adenosine-mediated NET formation may contribute a novel and directed therapeutic approach in the treatment of DADA2 and potentially other inflammatory diseases.

Contrast-enhanced ultrasound detects changes in microvascular blood flow in adults with sickle cell disease

In patients with sickle cell disease (SCD), poor outcome measures compromise the potential success of clinical trials. Contrast-enhanced ultrasound (CEUS) is a technique that can non-invasively quantify deep tissue microvascular blood flow. We tested the hypothesis that CEUS of forearm skeletal muscle could be used to: 1) assess microvascular abnormalities that occur during vaso-occlusive crisis; and 2) test new therapies for SCD that are targeted to improving the status of the microcirculation. We performed a prospective study, CEUS perfusion imaging of resting forearm muscle was performed in adults with SCD: 1) during and after a pain episode, and 2) before, during, and after a 24-hour infusion of the investigative agent, regadenoson, an adenosine A2A agonist. CEUS destruction-replenishment time-intensity data were analyzed to measure microvascular blood flow, as well as its components, microvascular blood volume and flux rate. Serial CEUS measurements were obtained in 32 adults with SCD. For the studies during crisis, there was a 30% reduction in microvascular blood flow compared to steady-state (p = 0.031), a reduction that was largely due to microvascular flux rate. For the regadenoson group, a non-significant 25% increase in flux rate and 9% increase in microvascular blood flow compared to baseline were detected during infusion. In a study of adults with SCD, CEUS detected changes in microvascular blood flow associated with vaso-occlusive crises. No changes were found during an infusion of the adenosine A2A agonist, regadenoson. This study provides preliminary evidence that CEUS could detect blood flow changes consistent with SCD physiology.

Abstract 5014: Profile of ARX001822, a highly potent, selective and orally bioavailable A2a antagonist effective in preventing adenosinergic suppression of T cell activation

Abstract Adenosine is elevated in the tumour microenvironment and plays a critical role in suppressing T cell function through high affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumour burden in syngeneic models. These effects are mimicked by small molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumours, particularly in combination with checkpoint inhibitors. However, the high levels of adenosine in the tumour microenvironment can dramatically reduce the effectiveness of competitive A2a antagonists. The challenge therefore is to identify highly potent and selective A2a antagonists which retain potency in the presence of high concentrations of adenosine and therefore have the potential to nullify the adenosinergic pathway within the tumour microenvironment ARX001822 binds A2a with high affinity (Ki=0.9 nM) and with greater than 660-fold selectivity over A1, A2b and A3. In a functional assay utilising CHO cells expressing recombinant A2a ARX001822 inhibited cAMP production in response to the selective A2a agonist CGS21680 in a competitive manner (KB= 0.3nM). Activation of A2a leads to the suppression of T cell-derived cytokine production and ARX001822 prevented this suppression even in the presence of high concentration of the adenosine receptor ligand NECA (IC50=38 nM). ARX001822 was also active in human whole blood, preventing NECA-mediated elevation of pCREB in CD8+ T cells and restoring production of interferon-γ with a potency 5-20 times higher than that of competitor molecules undergoing clinical evaluation in cancer. ARX001822 was orally bioavailable in rats and mice and was effective in inhibiting elevation of pCREB in mouse CD8+ T cells in an ex vivo pharmacodynamic assay. ARX001822 is a highly potent and selective A2a antagonist which is effective in preventing adenosinergic mediated suppression of cytokine production in the presence of high concentrations of adenosine receptor ligands and a full complement of plasma proteins. Knowledge of whole blood potency on both pCREB and interferon-γ modulation combined with the exposure required for activity in the pharmacodynamic model is helpful in estimating the clinical exposure required for A2a receptor blockade and downstream events related to modulating T cell function. Citation Format: Peter M. Finan, Roy Pettipher, Jonathan White, Viral Patel, Ben Moulton, Soraya Porres, Karolina Gherbi, Elizabeth M. Rosethorne, Steven J. Charlton, Clive McCarthy. Profile of ARX001822, a highly potent, selective and orally bioavailable A2a antagonist effective in preventing adenosinergic suppression of T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5014.

Reduction of repetitive behavior by co-administration of adenosine receptor agonists in C58 mice

Repetitive behaviors are diagnostic for autism spectrum disorder (ASD) and commonly observed in other neurodevelopmental disorders. Currently, there are no effective pharmacological treatments for repetitive behavior in these clinical conditions. This is due to the lack of information about the specific neural circuitry that mediates the development and expression of repetitive behavior. Our previous work in mouse models has linked repetitive behavior to decreased activation of the subthalamic nucleus, a brain region in the indirect and hyperdirect pathways in the basal ganglia circuitry. The present experiments were designed to further test our hypothesis that pharmacological activation of the indirect pathway would reduce repetitive behavior. We used a combination of adenosine A1 and A2A receptor agonists that have been shown to alter the firing frequency of dorsal striatal neurons within the indirect pathway of the basal ganglia. This drug combination markedly and selectively reduced repetitive behavior in both male and female C58 mice over a six-hour period, an effect that required both A1 and A2A agonists as neither alone reduced repetitive behavior. The adenosine A1 and A2A receptor agonist combination also significantly increased the number of Fos transcripts and Fos positive cells in dorsal striatum. Fos induction was found in both direct and indirect pathway neurons suggesting that the drug combination restored the balance of activation across these complementary basal ganglia pathways. The adenosine A1 and A2A receptor agonist combination also maintained its effectiveness in reducing repetitive behavior over a 7-day period. These findings point to novel potential therapeutic targets for development of drug therapies for repetitive behavior in clinical disorders.

The A2A receptor agonist CGS 21680 has beneficial and adverse effects on disease development in a humanised mouse model of graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for blood cancers and other blood disorders, but is limited by the development of graft-versus-host disease (GVHD). GVHD results in inflammatory damage to the host liver, gastrointestinal tract and skin, resulting in high rates of morbidity and mortality in HSCT recipients. Activation of the A2A receptor has been previously demonstrated to reduce disease in allogeneic mouse models of GVHD. This study aimed to investigate the effect of A2A activation on disease development in a humanised mouse model of GVHD. Immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (IL)-2 receptor γnull (NSG) mice injected with human (h) peripheral blood mononuclear cells (hPBMCs), were treated with either the A2A agonist CGS 21680 or control vehicle. Contrary to the beneficial effect of A2A activation in allogeneic mouse models, CGS 21680 increased weight loss, and failed to reduce the clinical score or increase survival in this humanised mouse model of GVHD. Moreover, CGS 21680 reduced T regulatory cells and increased serum human IL-6 concentrations. Conversely, CGS 21680 reduced serum human tumour necrosis factor (TNF)-α concentrations and leukocyte infiltration into the liver, indicating that A2A activation can, in part, reduce molecular and histological GVHD in this model. Notably, CGS 21680 also prevented healthy weight gain in NSG mice not engrafted with hPBMCs suggesting that this compound may be suppressing appetite or metabolism. Therefore, the potential benefits of A2A activation in reducing GVHD in HSCT recipients may be limited and confounded by adverse impacts on weight, decreased T regulatory cell frequency and increased IL-6 production.

Role of dopamine D2-like receptors and their modulation by adenosine receptor stimulation in the reinstatement of methamphetamine seeking.

Previous work has demonstrated that dopamine and adenosine receptors are involved in drug-seeking behaviors, yet the pharmacological interactions between these receptors in methamphetamine (MA) seeking are not well characterized. The present studies examined the role of the dopamine D2-like receptors in MA seeking and identified the interactive effects of adenosine receptor stimulation. Adult male Sprague-Dawley rats were trained to lever press for MA in daily 2-h self-administration sessions on a fixed-ratio 1 schedule for 10 consecutive days. After 1day of abstinence, lever pressing was extinguished in six daily extinction sessions. Treatments were administered systemically prior to a 2-h reinstatement test session. An increase in MA seeking was observed following the administration of the dopamine D2-like agonist, quinpirole, or the D3 receptor agonist, 7-OH-DPAT. Stimulation of D2 or D4 receptors was ineffective at inducing MA seeking. Quinpirole-induced MA seeking was inhibited by D3 receptor antagonism (SB-77011A or PG01037), an adenosine A1 agonist, CPA, and an adenosine A2A agonist, CGS 21680. MA seeking induced by a MA priming injection or D3 receptor stimulation was inhibited by a pretreatment with the adenosine A1 agonist, CPA, but not the adenosine A2A agonist, CGS 21680. These results demonstrate the sufficiency of dopamine D3 receptors to reinstate MA seeking that is inhibited when combined with adenosine A1 receptor stimulation.

Design, Synthesis, Experimental and Theoretical Characterization of a New Multitarget 2-Thienyl-N-Acylhydrazone Derivative.

Pulmonary arterial hypertension (PAH) is a chronic cardiovascular disease that displays inflammatory components, which contributes to the difficulty of adequate treatment with the available therapeutic arsenal. In this context, the N-acylhydrazone derivative LASSBio-1359 was previously described as a multitarget drug candidate able to revert the events associated with the progression of PAH in animal models. However, in spite of having a dual profile as PDE4 inhibitor and adenosine A2A receptor agonist, LASSBio-1359 does not present balanced potencies in the modulation of these two targets, which difficult its therapeutic use. In this paper, we describe the design concept of LASSBio-1835, a novel structural analogue of LASSBio-1359, planned by exploiting ring bioisosterism. Using X-ray powder diffraction, calorimetric techniques, and molecular modeling, we clearly indicate the presence of a preferred synperiplanar conformation at the amide function, which is fixed by an intramolecular 1,5-N∙∙∙S σ-hole intramolecular interaction. Moreover, the evaluation of LASSBio-1835 (4) as a PDE4 inhibitor and as an A2A agonist confirms it presents a more balanced dual profile, being considered a promising prototype for the treatment of PAH.

Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells

Anaphylatoxin C3a and adenosine receptors (AR) are implicated in the inflammatory process associated with allergic rhinitis and asthma by modifying mast cell (MC) responses. Possible interactions between these G-protein coupled receptor (GPCR) pathways in MCs have not yet been demonstrated. LAD2 human MC were stimulated with C3a in the presence or absence of AR agonists and antagonists and their adhesion, chemotaxis and mediator release were measured. The pan-specific AR agonist, 5′-N-Ethylcarboxamidoadenosine (NECA) inhibited C3a-induced LAD2 cell migration, adhesion, degranulation, production of CCL2, and ERK1/2 phosphorylation. The selective A2A receptor agonist CGS 21680 inhibited C3a-mediated degranulation, while the A2B and A3 receptor agonists BAY 60-6583 and IB-MECA, respectively, had no effect. Moreover, an A2A receptor antagonist SCH 58261 blocked the inhibitory effect of NECA on C3a-induced degranulation, suggesting that inhibition of degranulation was mediated through the A2A receptor. NECA increased intracellular cAMP in C3a-activated mast cells, suggesting that Gαs protein signals are required for adenosine-induced inhibition of C3a-mediated human mast cell activation. The adenylyl cyclase inhibitor SQ 22536 attenuated the inhibitory effect of NECA on C3a-activated degranulation, and the A2A agonist CSG 21680 potentiated the inhibition of mast cell activation mediated by the A2A receptor. Our results suggest that adenosine inhibits C3a-mediated activation of human mast cells, possibly through a Gαs protein-dependent pathway.

Adenosine Receptor-Mediated Cardioprotection-Current Limitations and Future Directions.

Since the seminal reports of adenosine receptor-mediated cardioprotection in the early 1990s, there have been a multitude of such reports in various species and preparations. Original observations of the beneficial effects of A1 receptor agonists have been followed up with numerous reports also implicating A2A, A3, and most recently A2B, receptor agonists as cardioprotective agents. Although adenosine has been approved for clinical use in the United States for the treatment of supraventricular tachycardia and coronary artery imaging, and the selective A2A agonist, regadenoson, for the latter, clinical use of adenosine receptor agonists for protecting the ischemic heart has not advanced beyond early trials. An examination of the literature indicates that existing experimental studies have several limitations in terms of clinical relevance, as well as lacking incorporation of recent new insights into adenosine receptor signaling. Such deficiencies include the lack of experimental studies in models that most closely mimic human cardiovascular disease. In addition, there have been very few studies in chronic models of myocardial ischemia, where limiting myocardial remodeling and heart failure, not reduction of infarct size, are the primary endpoints. Despite an increasing number of reports of the beneficial effects of adenosine receptor antagonists, not agonists, in chronic diseases, this idea has not been well-studied in experimental myocardial ischemia. There have also been few studies examining adenosine receptor subtype interactions as well as receptor heterodimerization. The purpose of this Perspective article is to discuss these deficiencies to highlight future directions of research in the field of adenosine receptor-mediated protection of ischemic myocardium.