A2A Agonist CGS Research Articles

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.

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.

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)

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.

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.

Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603

Endogenous adenosine A2B receptors (A2BAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A2BAR antagonist PSB 603 in HEK 293 cells. The A2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A2AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA EMAX with no significant effect on EC50 values. Kinetics analysis of the effect of PSB 603 on the A2BAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A2BAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.

Retinal A2A and A3 adenosine receptors modulate the components of the rat electroretinogram.

Adenosine is a neuromodulator present in various areas of the central nervous system, including the retina. Adenosine may serve a neuroprotective role in the retina, based on electroretinogram (ERG) recordings from the rat retina. Our purpose was to assess the role of A2A and A3 adenosine receptors in the generation and modulation of the rat ERG. The flash ERG was recorded with corneal electrodes from Sprague Dawley rats. Agonists and antagonists for A2A and A3 receptors, and adenosine were injected (5 µl) into the vitreous. The effects on the components of the single flash scotopic and photopic ERGs were examined, and ERG flicker. Adenosine (0.5 mM) increased the mean amplitudes of the scotopic ERG a-waves (68 ± 8 to 97 ± 14 µV, P = 0.042), and b-waves (236 ± 38 µV to 305 ± 42 µV). A2A agonist CGS21680 (2 mM) reduced the mean amplitude of the ERG b-wave, from 298 ± 21 µV in response to the brightest stimulus to 212 ± 19 µV (P = 0.005), and mean scotopic oscillatory potentials (OPs) from 100 ± 9 µV to 47 ± 11 µV (P = 0.023). ZM241385 [4 mM], an A2A antagonist, decreased the scotopic b-wave of the ERG. A3 agonist 2-CI-IB-MECA (0.5 mM) increased the a-wave, while decreasing the scotopic and photopic ERG b-waves, and the scotopic OPs. A3 antagonist VUF5574 (1 mM) increased the mean amplitude of the scotopic a-wave (66 ± 8 to 140 ± 29 µV, P = 0.046) and b-wave (224 ± 20 to 312 ± 39 µV, P = 0.0037). No significant effects on ERG flicker were found. We conclude that retinal neurons containing A2A and/or A3 adenosine receptors contribute to the generation of the ERG a- and b-waves and OPs.

Role of adenosine A2A receptor in ischemia reperfusion injury: signaling to extracellular signal‐regulated protein kinase (663.6)

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischemia. Out of the four known adenosine receptor subtypes (A1, A2A, A2B and A3), the A2A is of critical importance in stroke. Although, both A2A agonists and antagonists are proven protective during ischemia reperfusion (IR) injury, however, ,the mechanisms by which A2A receptors are noxious during IR remain elusive. The objective of the present study was to investigate the role of central A2A receptor in modulating cellular as well as behavioral responses to IR injury and the possible involvement of ERK signalling pathway. Hence, we hereby report that bilateral carotid occlusion for 45 min followed by 24h reperfusion period resulted in a significant memory impairment, motor incoordination as well as increased locomotor activity that were not affected by unilateral intrahippocampal injection of a selective A2A agonist CGS21680, but conversely mitigated by the selective A2A antagonist SCH58261. On the cellular level, IR increased the pERK, NFkB, cytochrome c, BDNF, Nrf2 and TNF‐α content in the hippocampus. With the exception of Nrf2 level, treatment with SCH58261 significantly reversed these effects, however, CGS21680 treatment resulted in a non‐significant change from IR insult. Accordingly these results suggest a possible protective role of the central A2A receptor blockade in IR injury with a prospective involvement of p42/44 pathway.

Chapter 5 - Dopamine D2 heteroreceptor complexes and their receptor–receptor interactions in ventral striatum: novel targets for antipsychotic drugs

This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-and-clasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptor-receptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing β-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex in the ventral striatum are proposed as one molecular mechanism for the potential antipsychotic effects of NT. Indications were obtained that the psychotic actions of the 5-HT2AR hallucinogens LSD and DOI can involve enhancement of D2R protomer signaling via a biased agonist action at the 5-HT2A protomer in the D2-5-HT2A heteroreceptor complex in the ventral striatum. Facilitatory allosteric D2likeR-OTR interactions in heteroreceptor complexes in nucleus accumbens may have a role in social and emotional behaviors. By blocking the D2 protomers of these heteroreceptor complexes, antipsychotics can fail to reduce the negative symptoms of schizophrenia. The discovery of different types of D2 heteroreceptor complexes gives an increased understanding of molecular mechanisms involved in causing schizophrenia and new strategies for its treatment and understanding the side effects of antipsychotics.

Trk and cAMP-dependent survival activity of adenosine A2A agonist CGS21680 on rat motoneurons in culture

The survival activity of adenosine A2A agonist CGS21680 on motoneurons in culture through the transactivation of neurotrophin receptor TrkB has been reported previously; however, since adenosine A2A receptor belongs to a Gs-protein-coupled receptor, we investigated the involvement of the cAMP pathway in the survival activity of CGS21680 using purified motoneurons in culture. CGS21680 alone showed only small survival activity, but the activity was significantly enhanced by the addition of a phosphodiesterase inhibitor, IBMX. This survival activity was partially inhibited by a protein kinase A inhibitor H89 or a neurotrophin receptor tyrosine kinase inhibitor K252a, and was completely inhibited by their combination. These results indicate that the survival activity of CGS21680 on motoneurons is exerted by the mixed effect of the adenylate cyclase–cAMP–PKA pathway and transactivation of Trk neurotrophin receptor. Under conditions in which the maximum survival of motoneurons was supported by sufficient concentrations of brain-derived neurotrophic factor (BDNF), a TrkB ligand, the addition of 100μM AMPA for 3 days led to significant cell death. Treatment with CGS21680 and IBMX protected motoneurons from the toxicity of AMPA, further supporting the presence of a TrkB-independent pathway of CGS21680 activity and suggesting a novel therapeutic approach to motoneuron diseases such as amyotrophic lateral sclerosis.

Cyp‐epoxygenases mediate adenosine A2A receptor induced vascular relaxation via KATP channels

A2A adenosine receptor (AR) causes aortic relaxation through generation of epoxyeicosatrienoic acids (EETs) formed via Cyp‐epoxygenases (Nayeem et al, AJP 2008). We hypothesize that A2AAR‐mediated aortic relaxation occurs via EETs through opening of KATP channels in smooth muscle cells. Organ bath experiments were done with isolated aortas from A2AKO and wild type (WT) mice. Pinacidil (KATP opener; 10−6M) caused higher relaxation in WT aorta compared to A2AKO (48.09±5.23% vs. 25.41±2.37%; p&lt;0.05). Cyp‐epoxygenase inhibitor MSPPOH (10−5M) caused significant decrease (p&lt;0.05) in pinacidil response in WT (48.09±5.23% vs. 24.93±5.34%) and A2AKO (25.41±2.37% vs. 14.66±3.88%) aorta. Adenosine analog NECA (10−5M) caused significant relaxation in WT aorta (19.65±3.25%) compared to contraction (13.94±6.57%) in A2AKO, that was lowered by KATP blocker glibenclamide in WT (0.17±6.43%) while higher contraction was observed in KO (28.47±5.1%). Relaxation to A2A agonist CGS 21680 (10−6M) in WT (19.02±2.75%) changed to contraction (23.66±6.31%; p&lt;0.05) with glibenclamide. Mitochondrial KATP opener diazoxide had no effect on precontracted tissues, while 5‐HD (mitochondrial KATP blocker, 100uM) had no effect on CGS 21680 response in WT and A2AKO aorta. These data suggest adenosine mediated aortic relaxation occurs via generation of EETs through sarcolemmal KATP channels. (HL027339, HL09444, HL07180)

Differential effects of the adenosine A2A agonist CGS-21680 and haloperidol on food-reinforced fixed ratio responding in the rat

Previous studies have shown that adenosine A(2A) receptors are colocalized with dopamine D(2) receptors on striatal neurons. Activation of these two receptors has antagonistic effects under a number of conditions suggesting that stimulation of adenosine A(2A) receptors may have behavioral effects resembling those produced by blockade of dopamine D(2) receptors, but this possibility has been investigated in a limited number of situations. We compared the effects of the adenosine A(2A) agonist CGS-21680 and the preferential D(2) dopamine antagonist haloperidol in a situation in which dopamine blockade produces a distinctive pattern of behavioral effects. Six rats were trained to lever press for food reward on a fixed ratio 15 schedule of reinforcement and then tested after being injected with various doses of CGS-21680 (0.064, 0.128, and 0.25 mg/kg) and haloperidol (0.25 and 0.1 mg/kg). Haloperidol produced a dose-dependent suppression of lever pressing with mean response rates declining across the duration of the test session. CGS-21680 also produced a dose-dependent suppression of responding, but this effect was not temporally graded, and responding was equivalently suppressed across the duration of the session. Additionally, CGS-21680 increased post-reinforcement pause duration to a much greater extent than did haloperidol. On this task, the behavioral effects of CGS-21680 do not resemble those produced by haloperidol. Several explanations of this discrepancy are possible, the most likely being that the observed behavioral effects of CGS-21680 result from an action at a site other than D(2) receptor-expressing striatal neurons.

Cholesterol 27-Hydroxylase but Not Apolipoprotein apoE Contributes to A2A Adenosine Receptor Stimulated Reverse Cholesterol Transport

Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNγ) CGS vs. IFNγ alone, n=4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 ± 2%; p<0.5113, inhibition vs. IFNγ alone, n=4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1.

Adenosine Can Mediate its Actions through Generation of Reactive Oxygen Species

Adenosine is an important cerebral vasodilator, but mediating mechanisms are not understood. We investigated the expression of adenosine receptor subtypes in isolated cerebral arterial muscle cells (CAMCs), and their role in adenosine-induced superoxide (O(2)(-)) generation and reduction in cerebral arterial tone. Reverse transcriptase-PCR, western blotting, and immunofluorescence studies have shown that CAMCs express transcript and protein for A1, A(2A), A(2B), and A(3) adenosine receptors. Stimulation of CAMCs with adenosine or the A(2A) agonist CGS-21680 increased the generation of O(2)(-) that was attenuated by the inhibition of A(2A) and A(2B) adenosine receptor subtypes, or by the peptide inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase gp91ds-tat, or by the mitochondria uncoupler 2,4-dinitrophenol. Application of adenosine or CGS-21680 dilated pressure-constricted cerebral arterial segments that were prevented by the antioxidants superoxide dismutase (SOD) conjugated to polyethylene glycol (PEG) and PEG-catalase or by the A(2B) adenosine receptor antagonist MRS-1754, or by the mixed A(2A) and A(2B) antagonist ZM-241385. Antagonism of the A(2A) and A(2B) adenosine receptors had no effect on cerebral vasodilatation induced by nifedipine. These findings indicate that adenosine reduces pressure-induced cerebral arterial tone through stimulation of A(2A) and A(2B) adenosine receptors and generation of O(2)(-) from NADPH oxidase and mitochondrial sources. This signaling pathway could be one of the mediators of the cerebral vasodilatory actions of adenosine.

Adenosine A2A and Beta-2 Adrenergic Receptor Agonist Synergy in B-Cell Malignancies: Selectivity, Breadth of Activity and Effects of Chronic Exposure.

Abstract 3762Poster Board III-698By using a high throughput combination screening strategy, we made the surprising discovery that adenosine A2A and beta-2 adrenergic receptor (b2AR) agonists have synergistic anti-proliferative activity in combination with dexamethasone, melphalan, lenalidomide, bortezomib and doxorubicin in preclinical multiple myeloma (MM) models. Both A2A and b2AR agonists are highly selective and demonstrate no single agent activity or synergy in normal cell types including human PBMCs, AoSMCs, HUVECs or HCAECs at concentrations 2-3 orders of magnitude greater than the IC50 in MM cell lines. To further examine the selectivity and breadth we have evaluated A2A and b2AR agonist combinations in a panel of 83 cell lines including solid tumor types and hematological malignancies. Single agents and combinations with dexamethasone and melphalan were systematically studied at multiple ratios of clinically relevant concentrations. Using a quantitative synergy score based on the Loewe model (Lehar et al. Nat Biotech 2009), we observe that combination activity for A2A or b2AR agonists is highly selective for hematologic malignancies with synergy observed most frequently in multiple myeloma and DLBCL cell lines. Synergy is also observed with the B-cell lines JM-1 (pre B-ALL) and GA-10 (Burkitt's lymphoma). Using a relative synergy cut-off (synergy score >1), we find that 13 of the 18 MM cell lines tested demonstrate a synergistic interaction between the A2A agonist CGS-21680 and dexamethasone and 11 demonstrate a synergistic interaction between CGS-21680 and melphalan. Using this same measure, 9 of 18 MM cell lines demonstrate synergy with combinations of the b2AR agonist salmeterol with either dexamethasone or melphalan. Nine and ten of the cell lines in this MM panel are insensitive or respond weakly to dexamethasone and melphalan as single agents respectively. All cell lines were treated with the same concentrations of dexamethasone or melphalan, pointing to A2A agonists having a higher breadth of activity across the MM cell line panel. An interesting observation is the strong synergy observed for A2A or b2AR agonists with dexamethasone in the glucocorticoid-insensitive cell lines EJM and ANBL-6, which suggests that these agents may help restore steroid sensitivity in refractory patients. In general, drug resistance is a recurrent problem for cancer drugs and development of resistance after chronic exposure can reduce drug efficacy and promote refractory disease. We therefore examined the effects of chronic exposure to either A2A or b2AR agonists in the MM.1S cell line. Exposure of cells to CGS-21680 or salmeterol for one month reduced single agent sensitivity >80%. Surprisingly, combinations of either agent with dexamethasone maintained similar amounts of synergy and cell killing as found with naïve untreated cells. As determined by Western blot analysis, the reduction in single agent activity after chronic exposure is not accompanied by a reduction in receptor levels. These data demonstrate that synergistic combinations of A2A and b2AR agonists are highly selective for B-cell malignancies and support the notion that synergistic drug combinations improve therapeutically relevant selectivity and circumvent drug resistance. This work further supports the rationalefor investigation of A2A and b2AR agonists in the treatment of B-cell malignancies and in particular, patients who have MM. Disclosures:Rickles:CombinatoRx, Inc.: Employment. Tam:CombinatoRx, Inc.: Employment. Necheva:CombinatoRx, Inc.: Employment. Giordano:CombinatoRx, Inc.: Employment. Borisy:CombinatoRx, Inc.: Employment. Lee:CombinatoRx, Inc.: Employment.

Adenosine A2A and A2B receptors work in concert to induce a strong protection against reperfusion injury in rat hearts

We aimed to test if stimulation of both adenosine A2A and A2B receptors is required to produce an effective cardioprotection against reperfusion injury. Isolated rat hearts were subjected to 30-min regional ischemia followed by 2 h of reperfusion. The adenosine A1/A2 receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size, an effect that was reversed by both the adenosine A2A antagonist SCH58261 and the A2B antagonist MRS1706. The A2B agonist BAY 60-6583 but not the selective A2A agonist CGS21680 reduced infarct size. Interestingly, a combination of BAY 60-6583 and CGS21680 further reduced infarct size. These results suggest that both A2A and A2B receptors are involved in NECA's anti-infarct effect at reperfusion. NECA attenuated mitochondrial swelling upon reperfusion and this was blocked by both SCH58261 and MRS1706, indicating that activation of A2 receptors with NECA can modulate reperfusion-induced mitochondrial permeability transition pore (mPTP) opening. In support, NECA also prevented oxidant-induced loss of mitochondrial membrane potential (ΔΨm) and matrix Ca2+ overload in cardiomyocytes via both the A2 receptors. In addition, NECA increased mitochondrial glycogen synthase kinase-3β (GSK-3β) phosphorylation upon reperfusion and this was again blocked by SCH58261 and MRS1706. In conclusion, A2A and A2B receptors work in concert to prevent reperfusion injury in rat hearts treated with NECA. NECA may protect the heart by modulating the mPTP opening through inactivating mitochondrial GSK-3β. A simultaneous stimulation of A2A and A2B receptors at reperfusion is required to produce a strong cardioprotection against reperfusion injury.

Regulation of hind-limb tone by adenosine A2A receptor in rats

Adenosine A2A receptor agonists produce a hypokinetic state (catalepsy) that is believed to reflect antagonistic interaction of A2A and dopamine D2 receptors in the basal ganglia. In addition to catalepsy, pharmacological blockade of D2 receptors produces rigidity. However there are conflicting data about the effect of A2A agonists on muscle tone, with some reports indicating an increase, while other data suggest that A2A catalepsy is dominated by muscle hypotonia. We investigated the effect on resistance to imposed movements of systemic cataleptic doses of the selective A2A agonist CGS21680 (5 mg/kg), and compared it with the effect of the D2 antagonist raclopride (5 mg/kg), in rats. Total resistance is made up of elastic and viscous components. The elastic component is velocity independent, and is referred to as “stiffness,” whereas viscosity, which dampens responses to imposed movements, is velocity dependent. Using a method for quantifying total joint resistance that enabled separate identification of stiffness and viscosity, we found that during catalepsy evoked by either drug there was a clear increase in joint rigidity. Both CGS21680 and raclopride significantly increased joint stiffness, the velocity independent component of rigidity that is most affected in Parkinsonism. In contrast, the effect of CGS21680 on the velocity-dependent viscosity component was less robust than for raclopride, and did not reach significance, possibly reflecting an interaction with sedative effects via extrastriatal receptors. The effect of CGS21680 and raclopride on joint stiffness is thus consistent with previous findings suggesting functional antagonism of A2A and D2 receptors in the basal ganglia.

Intra-accumbens injections of the adenosine A2A agonist CGS 21680 affect effort-related choice behavior in rats

Nucleus accumbens dopamine (DA) participates in the modulation of instrumental behavior, including aspects of behavioral activation and effort-related choice behavior. Rats with impaired accumbens DA transmission reallocate their behavior away from food-reinforced activities that have high response requirements and instead select less-effortful types of food-seeking behavior. Although accumbens DA is considered a critical component of the brain circuitry regulating effort-related processes, emerging evidence also implicates adenosine A(2A) receptors. The present work was undertaken to test the hypothesis that accumbens A(2A) receptor stimulation would produce effects similar to those produced by DA depletion or antagonism. Three experiments assessed the effects of the adenosine A(2A) agonist CGS 21680 on performance of a concurrent choice task (lever pressing for preferred food vs. intake of less preferred chow) that is known to be sensitive to DA antagonists and accumbens DA depletions. Systemic injections of CGS 21680 reduced lever pressing but did not increase feeding. In contrast, bilateral infusions of the adenosine A(2A) receptor agonist CGS 21680 (6.0-24.0 ng) into the nucleus accumbens decreased lever pressing for the preferred food but substantially increased consumption of the less preferred chow. Injections of CGS 21680 into a control site dorsal to the accumbens were ineffective. Taken together, these results are consistent with the hypothesis that local stimulation of adenosine A(2A) receptors in nucleus accumbens produces behavioral effects similar to those induced by accumbens DA depletions. Accumbens adenosine A(2A) receptors appear to be a component of the brain circuitry regulating effort-related choice behavior.