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This study was undertaken to determine whether the myocardial infarct-sparing effect of ATL-146e, a selective adenosine A(2A) receptor agonist, persists without a rebound effect for at least 48 h and to determine the optimal duration of ATL-146e treatment in anesthetized dogs. Reperfusion injury after myocardial infarction (MI) is associated with inflammation lasting 24-48 h that contributes to ongoing myocyte injury. We previously showed that an ATL-146e infusion, starting just before reperfusion, decreased inflammation and infarct size in dogs examined 2 h after MI without increasing coronary blood flow. In the present study, adult dogs underwent 90 min of left anterior descending coronary artery occlusion. Thirty minutes before reperfusion, ATL-146e (0.01 microg x kg(-1) x min(-1); n = 21) or vehicle (n = 12) was intravenously infused and continued for 2.5 h (protocol 1) or 24 h (protocol 2). At 48 h after reperfusion hearts were excised and assessed for histological risk area and infarct size. Infarct size based on triphenyltetrazolium chloride (TTC) staining as a percentage of risk area was significantly smaller in ATL-146e-treated vs. control dogs (16.7 +/- 3.7% vs. 33.3 +/- 6.2%, P < 0.05; protocol 1). ATL-146e reduced neutrophil accumulation into infarcted myocardium of ATL-146e-treated vs. control dogs (30 +/- 7 vs. 88 +/- 16 cells/high-power field, P < 0.002). ATL-146e infusion for 24 h (protocol 2) conferred no significant additional infarct size reduction compared with 2.5 h of infusion. A 2.5-h ATL-146e infusion initiated 30 min before reperfusion results in marked, persistent (48 h) reduction in infarct size as a percentage of risk area in dogs with a reduction in infarct zone neutrophil infiltration. No significant further benefit was seen with a 24-h infusion.

The activation of adenosine 2A receptors before reperfusion following coronary artery occlusion reduces infarct size and improves ejection fraction (EF). In this study, we examined the effects of delaying treatment with the adenosine 2A receptor agonist ATL146e (ATL) until 1 h postreperfusion. The infarct size and EF were serially assessed by gadolinium-diethylenetriaminepentaacetic acid-enhanced MRI in C57BL/6 mice at 1 and 24 h postreperfusion. The infarct size was also assessed by 2,3,5-triphenyltetrazolium chloride staining at 24 h. Mice were treated with ATL (10 microg/kg ip) either 2 min before reperfusion (early ATL) or 1 h postreperfusion (late ATL) following the 45-min coronary occlusion. The two methods used to assess infarct size at 24 h postreperfusion (MRI and 2,3,5-triphenyltetrazolium chloride) showed an excellent correlation (R=0.96). The risk region, determined at 24 h postreperfusion, was comparable between the control and ATL-treated groups. The infarct size by MRI at 1 versus 24 h postreperfusion was 25+/-1 vs. 26+/-1% of left ventricular mass (means+/-SE) in control mice, 16+/-2 versus 17+/-2% in early-ATL mice, and 24+/-2 versus 25+/-2% in late-ATL mice (intragroup, P=not significant; and intergroup, early ATL vs. control or late ATL, P<0.05). EF was reduced in control mice but was largely preserved between 1 and 24 h in both early-ATL and late-ATL mice (P<0.05). In conclusion, after coronary occlusion in mice, the extent of myocellular death due to ischemia-reperfusion injury is 95% complete within 1 h of reperfusion. The infarct size was significantly reduced by ATL when given just before reperfusion, but not 1 h postreperfusion. Either treatment window helped preserve the EF between 1 and 24 h postreperfusion.

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.

Background: Lung reperfusion injury is a significant problem in cardiothoracic surgery. Previous studies have demonstrated that an adenosine A(2A) agonist can attenuate lung reperfusion injury in a lung transplantation model. There has been little work, however, examining its effects in the setting of nontransplant ischemia reperfusion. Our hypothesis was that an A(2A) agonist would attenuate lung reperfusion injury in a warm ischemia hilar clamping model., Study Design: Sprague Dawley rats underwent 90 min of left hilar clamping followed by 4 h of reperfusion. Group 1 (n = 13) received an intravenous infusion of 0.06 ug/kg/min of ATL-146e, which was started 10 min before reperfusion. Group 2 (n = 16) received an equivalent saline infusion. A third sham group (n = 14) received the same protocol as Group 2 but no lung ischemia., Results: Animals receiving ATL-146e showed significant improvements in oxygenation (Group 1: 447 +/- 26.02 mmHg versus Group 2: 223 +/- 24.46 mmHg (P < 0.001) as well as ventilation (pCO2 Group 1: 48.78 +/- 3.88 versus Group 2: 63.56 +/- 4.80 (P = 0.009)). Total protein in the bronchoalveolar lavage was significantly higher in the saline group compared with the adenosine as well as a higher proportion of neutrophils. Histological analysis demonstrated a significantly higher number of neutrophils in the IR group compared with the adenosine group., Conclusions: ATL-146e, an adenosine analogue that is a specific agonist for the A(2A) receptor, attenuates reperfusion injury in an in vivo rat lung model. Arterial blood gas measurements demonstrate a statistically significant increase in oxygenation and improved ventilation.

Objectives: Adenosine receptor activation at reperfusion has been shown to ameliorate ischemia-reperfusion injury of the spinal cord, but the effects of therapy given in response to ischemic injury are unknown. We hypothesized that adenosine receptor activation with ATL-146e would produce similar protection from ischemic spinal cord injury, whether given at reperfusion or in a delayed fashion., Methods: Twenty-two New Zealand white rabbits were divided into three groups. All three groups, including the ischemia-reperfusion group (IR, n = 8), underwent 45 min of infrarenal aortic occlusion. The early treatment group (early, n = 8) received 0.06 mug/kg/min of ATL-146e for 3 h beginning 10 min prior to reperfusion. The delayed treatment group (delayed, n = 6) received ATL-146e starting 1 h after reperfusion. After 48 h, hind limb function was graded using the Tarlov score. Finally, lumbar spinal cord neuronal cytoarchitecture was evaluated., Results: Hemodynamic parameters were similar among the groups. Hind limb function at 48 h was significantly better in the early group (3.5 +/- 1.0) compared to the IR group (0.625 +/- 0.5, P < or = 0.01). There was a trend towards better hind limb function in the early group compared to the delayed group (2.4 +/- 1.1, P = 0.08). Hind limb function was similar between delayed and IR groups. Hematoxylin-eosin spinal cord sections demonstrated preservation of viable motor neurons in the early group compared to the delayed and IR groups., Conclusions: Early therapy with ATL-146e provided better protection in this study; therefore, therapy should not be delayed until there is evidence of ischemic neurological deficit. This study suggests that adenosine receptor activation is most effective as a preventive strategy at reperfusion for optimal protection in spinal cord ischemia-reperfusion injury.

We sought to determine whether administration of a very low, nonvasodilating dose of a highly selective adenosine A(2A) receptor agonist (ATL-193 or ATL-146e) would be cardioprotective in a canine model of myocardial stunning produced by multiple episodes of transient ischemia. Twenty-four anesthetized open-chest dogs underwent either 4 (n=12) or 10 cycles (n=12) of 5-min left anterior descending coronary artery (LAD) occlusions interspersed by 5 or 10 min of reperfusion. Left ventricular thickening was measured from baseline through 180 min after the last occlusion-reperfusion cycle. Regional flow was measured with microspheres. In 12 of 24 dogs, A(2A) receptor agonist was infused intravenously beginning 2 min prior to the first occlusion and continuing throughout reperfusion at a dose below that which produces vasodilatation (0.01 microg x kg(-1) x min(-1)). Myocardial flow was similar between control and A(2A) receptor agonist-treated animals, confirming the absence of A(2) receptor agonist-induced vasodilatation. During occlusion, there was severe dyskinesis with marked LAD zone thinning in all animals. After 180 min of reperfusion following the last cycle, significantly greater recovery of LAD zone thickening was observed in A(2A) receptor agonist-treated vs. control animals in both the 4-cycle (91 +/- 7 vs. 56 +/- 12%, respectively; P<0.05) and the 10-cycle (65 +/- 9 vs. 8 +/- 16%, respectively; P<0.05) occlusion groups. The striking amount of functional recovery observed with administration of low, nonvasodilating doses of adenosine A(2A) agonist ATL-193 or ATL-146e supports their further evaluation for the attenuation of postischemic stunning in the clinical setting.

Background and Aims: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production., Methods: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell., Results: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production., Conclusion: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.

Background: ATL-146e protects the spinal cord from ischemia/reperfusion injury, presumably via adenosine A(2A) receptor activation, but this relationship remains unproven. We hypothesized that spinal cord functional and cytoarchitectural preservation from ATL-146e would be lost with simultaneous administration of the specific adenosine A(2A) antagonist ZM241385 (ZM), thus proving that adenosine A(2A) receptor activation is responsible for the protective effects of this compound., Methods: New Zealand White rabbits underwent 45 minutes of infrarenal aortic cross-clamping. Groups (n = 10) included sham, ischemia, ischemia plus ATL-146e (ATL-146E), ischemia plus ZM, or ischemia with both compounds (agonist-antagonist). Tarlov scores were recorded every 12 hours. After 48 hours, the spinal cord was fixed for histology and microtubule-associated protein 2 immunohistochemistry., Results: Tarlov scores at 48 hours were significantly better in the sham and ATL-146E groups (5.0 and 3.9, respectively) compared with the other three groups (all < or =1.3; P < .001). On hematoxylin and eosin, neuronal viability was higher in the sham, ATL-146E, and agonist-antagonist groups compared with the control and ZM groups (P < .05). Microtubule-associated protein 2 expression was preserved in the sham and ATL-146E groups but was lost in the ATL + ZM, ZM241385, and control groups., Conclusions: ATL-146e preserves the spinal cord in terms of both cytoarchitecture and function after reperfusion of the ischemic spinal cord, but this preservation is not completely blocked by competitive adenosine A(2A) receptor antagonism. Although ATL-146e does seem to partially function through activation of the adenosine A(2A) receptor, the neuroprotective mechanism may not be limited to this particular receptor.

Background: Paraplegia remains a devastating complication of thoracic aortic surgery, which has been attenuated by retrograde adenosine and systemic adenosine A2A receptor activation. We hypothesized that despite retrograde spinal perfusion of an adenosine A2A agonist (ATL-146e), systemic therapy produces superior spinal cord protection with reduced inflammation., Methods: Forty pigs underwent 30-minute thoracic aortic cross-clamping. Pigs received: no therapy (control); retrograde saline (retrograde control); retrograde ATL-146e; systemic ATL-146e; systemic ATL-146e with retrograde saline; or systemic and retrograde ATL-146e. Retrograde therapies were given during ischemia. Systemic ATL-146e (0.06 microg.kg(-1).min(-1)) was given intravenously for 3 hours at reperfusion. At 24 hours, motor function was assessed using the Tarlov scale. Tissue was analyzed for neuronal viability, microtubule-associated protein-2 expression, and neutrophil sequestration (myeloperoxidase activity)., Results: Four pigs received retrograde barium showing both radiographic and histologic spinal cord perfusion. Tarlov scores at 24 hours were significantly improved versus both control groups in all ATL groups except the combined ATL-146e group (all p < 0.05). Neuronal viability by hematoxylin and eosin stain was significantly preserved in systemic ATL groups compared with both control groups (all p < 0.05). Microtubule-associated protein-2 expression was significantly preserved compared with both control groups in all systemic ATL groups. Systemic ATL significantly lowered myeloperoxidase activity versus both control groups (p < 0.01)., Conclusions: Both retrograde and systemic ATL-146e therapies attenuate ischemic spinal cord injury, but combining the two routes was less effective. Given comparable results between the two routes and the simplicity of systemic delivery, peripheral venous ATL-146e at reperfusion should be preferred for spinal cord protection in thoracic aortic surgery.

Object: Steroid agents remain the lone pharmacological treatment in widespread use for acute spinal cord injury (SCI), although their utility remains in dispute in the neurotrauma literature. Adenosine A2A receptor activation with ATL-146e, a selective A2A agonist, has shown potential benefit in treating SCI; however, it has not been compared with the gold standard, methylprednisolone. The authors of this study evaluated ATL-146e and methylprednisolone for their ability to preserve neuronal viability and motor function in experimental SCI., Methods: New Zealand White rabbits sustained SCI or sham injury via the Allen weight-drop technique. Ten minutes postinjury, animals received ATL-146e (ATL group, 0.06 microg/kg/min intravenously for 3 hours), methylprednisolone (steroid group, 30 mg/kg intravenously), or saline (trauma control group). Hindlimb motor function was recorded every 12 hours using the Tarlov motor grading scale (0, paralysis-5, normal hop). At 48 hours, fixed spinal cord tissue was evaluated for neuronal viability. Hindlimb motor function in animals treated with ATL-146e was equivalent to that of sham-injured animals and was significantly better than that of trauma control animals at all time points and that of steroid-treated animals at 12 hours (p = 0.05). Motor function in steroid-treated animals was worse than in those given ATL-146e and better than that of trauma control animals at later time points, but was not statistically significant (both p > 0.05). Neuronal viability (measured in neurons/hpf) was significantly higher in both treatment groups compared with the trauma control group (12.1 +/- 1.4 neurons/hpf for the ATL and 13.3 +/- 1.4 neurons/hpf for the steroid group compared with 7.5 +/- 1.5 neurons/hpf for the trauma control group; both p < 0.04). Neuronal viability did not differ among ATL-146e-treated, steroid-treated, and sham-injured groups., Conclusions: The use of ATL-146e is at least as effective as methylprednisolone in preserving function and is equivalent to methylprednisolone in preserving the structure of spinal cord tissue after blunt SCI. Adenosine A2A receptor activation may be an effective treatment for acute SCI while avoiding the adverse effects of steroid agents.

Background & Aims: Adenosine has been implicated as an important regulator of the inflammatory response. Four subtypes of adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) have been described, of which A 2A potentially inhibits inflammation. The aim of this study was to investigate the role of A 2A in mucosal inflammation by administering a selective A 2A agonist (ATL-146e) to experimental models of inflammatory bowel disease., Methods: The anti-inflammatory effects of ATL-146e were studied in the acute and chronic rabbit formalin-immune complex models of colitis and the SAMP1/YitFc mouse model of spontaneous ileitis., Results: ATL-146e significantly reduced the acute inflammatory index and tissue necrosis compared with vehicle ( P < .01) in the acute model of rabbit immune colitis. In the chronic rabbit immune colitis model, ATL-146e significantly suppressed inflammatory cell infiltration into the colonic mucosa ( P < .05) and prevented mortality. The administration of ATL-146e significantly decreased the chronic inflammatory index ( P < .01) and villus distortion index ( P < .01) in the ileum of SAMP1/YitFc mice, and ameliorated adoptively transferred ileitis in severe combined immunodeficient mice injected with CD4 + T cells from SAMP1/Yit mice ( P < .05). Tumor necrosis factor, interferon gamma, and interleukin 4 concentrations were significantly suppressed by ATL-146e treatment in supernatants from cultures of mesenteric lymph node cells of SAMP1/YitFc mice ( P < .05 vs vehicle-treated mice)., Conclusions: A 2A adenosine receptor activation by ATL-146e significantly reduced inflammation in the intestinal mucosa. This effect was associated with decreased leukocyte infiltration and inhibition of proinflammatory cytokines. Activation of A 2A by selective agonism may therefore serve as a novel therapy for the treatment of inflammatory bowel disease.

Background: Reperfusion injury continues to significantly affect patients undergoing lung transplantation. Isolated lung models have demonstrated that adenosine A 2A receptor activation preserves function while decreasing inflammation. We hypothesized that adenosine A 2A receptor activation by ATL-146e during the initial reperfusion period preserves pulmonary function and attenuates inflammation in a porcine model of lung transplantation., Methods: Mature pig lungs preserved with Viaspan (Barr Laboratories, Pomona, NY) underwent 6 hours of cold ischemia before transplantation and 4 hours of reperfusion. Animals were treated with (ATL group, n = 7) and without (IR group, n = 7) ATL-146e (0.05 microg kg -1 . min -1 ATL-146e administered intravenously for 3 hours). With occlusion of the opposite pulmonary artery, the animal was maintained for the final 30 minutes on the allograft alone. Recipient lung physiology was monitored before tissue evaluation of pulmonary edema (wet-to-dry weight ratio), myeloperoxidase assay, and tissue tumor necrosis factor alpha by means of enzyme-linked immunosorbent assay., Results: When the ATL group was compared with the IR group, the ATL group had better partial pressure of carbon dioxide (43.8 +/- 4.1 vs 68.9 +/- 6.3 mm Hg, P < .01) and partial pressure of oxygen (272.3 +/- 132.7 vs 100.1 +/- 21.4 mm Hg, P < .01). ATL-146e-treated animals exhibited lower pulmonary artery pressures (33.6 +/- 2.1 vs 47.9 +/- 3.5 mm Hg, P < .01) and mean airway pressures (16.25 +/- 0.08 vs 16.64 +/- 0.15 mm Hg, P = .04). ATL-146e-treated lungs had lower wet-to-dry ratios (5.9 +/- 0.39 vs 7.3 +/- 0.38, P < .02), lower myeloperoxidase levels (2.9 x 10 -5 +/- 1.2 x 10 -5 vs 1.3 x 10 -4 +/- 4.0 x 10 -5 DeltaOD mg -1 . min -1 , P = .03), and a trend toward decreased lung tumor necrosis factor alpha levels (57 +/- 12 vs 96 +/- 15 pg/mL, P = .06). The ATL group demonstrated significantly less inflammation on histology., Conclusion: Adenosine A 2A activation during early reperfusion attenuated lung inflammation and preserved pulmonary function in this model of lung transplantation. ATL-146e and similar compounds could play a significant role in improving outcomes of pulmonary transplantation.

Background: D-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure in which tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role. We examined the effects of a highly selective adenosine A2A receptor agonist (ATL-146e) on GalN/LPS-induced fulminant hepatic failure., Methods: Mice were given an intraperitoneal dose of GalN (800 mg/g body weight)/LPS (100 ng/g body weight) with and without ATL-146e (0.01 microg/kg) treatment. Liver injury was assessed biochemically and histologically. Also, TNF-alpha levels in the serum were determined., Results: The serum liver enzyme (ALT) level in vehicle-treated mice was 20 960 +/- 2800 IU/ml and was reduced by 63% to 7800 +/- 1670 IU/ml by treatment with 0.01 microg/kg per minute ATL146e, P < 0.05. Treatment with ATL-146e significantly reduced serum TNF-alpha and greatly reduced inflammation assessed by histopathologic examination compared with control mice treated with GalN/LPS. ATL-146e also reduced lethality at 12 h from 65% to 13%., Conclusion: The present findings suggest that the highly selective adenosine A2A receptor agonist (ATL-146e) prevents endotoxin-induced lethal liver injury by suppression of TNF-alpha secretion.

Adenosine and adenosine A(2A) receptor agonists have been shown to limit myocardial infarct size when given at vasodilatory doses during reperfusion. This beneficial effect is thought to be due, in part, to stimulation of adenosine A(2A) receptors on inflammatory cells. The specific aims of this study were to determine whether the anti-inflammatory and cardioprotective properties of a novel adenosine A(2A) receptor agonist, ATL-146e (ATL), alone or in combination with the phosphodiesterase IV inhibitor rolipram would occur using very low, nonvasodilating doses. In a canine model of reperfused myocardial infarction, low-dose ATL given alone reduced infarct size by 45% (P < 0.05 vs. control). When ATL was combined with a very low dose of rolipram (0.001 microg.kg(-1).min(-1)), a marked reduction in P-selectin expression and neutrophil infiltration (51% lower; P < 0.001 vs. control) was seen and the infarct size reduction (58% lower; P < 0.01 vs. control) was greater than observed with ATL (45% lower; P < 0.05) or rolipram (33% lower; P < 0.05) alone. In conclusion, a low, nonvasodilating dose of ATL, a highly selective adenosine A(2A) receptor agonist, reduced infarct size after reperfusion. Furthermore, combining ATL and the phosphodiesterase IV inhibitor rolipram reduced infarct size even more than either agent alone. Such combination therapy may be beneficial clinically by potentiating cardioprotection after coronary reperfusion at doses far below those producing vasodilatation or side effects.

Background: Ischemia-reperfusion (IR) injury negatively impacts patient outcome in lung transplantation. Clinically, we observed that lung transplant patients with ischemia-reperfusion injury tend to have cardiac dysfunction. Previous studies have shown that ATL-146e (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester), a selective adenosine A2A receptor agonist, reduces lung inflammation after ischemia-reperfusion. We hypothesized that pulmonary ischemia-reperfusion causes secondary heart dysfunction and ATL-146e will improve this dysfunction., Methods: We utilized an in vivo rabbit lung ischemia-reperfusion model. The Sham group underwent 120 minutes single lung ventilation. The IR and ATL groups underwent 90 minutes right lung ischemia with 30 minutes right lung reperfusion. The ATL-146e was given intravenously to the ATL group during reperfusion. Cardiac output and arterial blood gases were monitored, and neutrophil sequestration was measured by myeloperoxidase activity., Results: Upon reperfusion, cardiac output (mL/min) significantly dropped in the IR and ATL groups. By 15 minutes reperfusion, cardiac output in the ATL group improved significantly over the IR group and remained significant thereafter. Lung myeloperoxidase activity was significantly reduced by ATL-146e. Although never hypoxemic, arterial oxygenation was lower in the IR and ATL groups while central venous pressures and mean arterial pressures were similar among groups. A separate experiment demonstrated that reperfusion with the antioxidant N-(2-mercaptopropionyl)glycine prevented cardiac dysfunction., Conclusions: Pulmonary ischemia-reperfusion causes cardiac dysfunction independent of preload, afterload, and oxygenation. The ATL-146e improves this dysfunction presumably by the antiinflammatory effects of adenosine A2A receptor activation on neutrophils. One likely mechanism involves the release of oxidants from the ischemic lung upon reperfusion, which has immediate negative effects on the heart.

Background: Activation of adenosine A(2A) receptors reduces the production of various pro-inflammatory cytokines and suppresses neutrophil activation. Water-immersion restraint is well known to cause gastric mucosal lesions due to stress. The pathogenesis of stress-induced gastric mucosal lesions is characterized by activation of inflammatory cells and production of inflammatory cytokines. Agonists of adenosine A(2A) receptors are known to be anti-inflammatory, but the effects of these compounds on the development of gastric mucosal lesions has not been reported. In the present study, the effect of a potent and selective adenosine A(2A) receptor agonist, ATL-146e, on water-immersion stress-induced gastric mucosal lesions was studied., Methods: Rats were subjected to water-immersion stress with or without pretreatment with a single intraperitoneal injection of a potent and selective agonist of the adenosine A(2A) receptor. The gastric concentrations of myeloperoxidase (MPO), as an index of neutrophil accumulation, and the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), were measured., Results: The total length of gastric erosions (ulcer index) in control rats was 21.6 +/- 3.23 mm and was reduced by 86% to 3.1 +/- 0.83 mm by pretreatment with 5.0 microg/kg ATL146e (P < 0.001). The gastric content of MPO, TNF-alpha and IL-1beta were all increased after water-immersion stress and reduced to near normal levels by ATL-146e., Conclusion: A specific adenosine A(2A) agonist inhibits stress-induced gastric inflammation and damage. A(2A) agonist compounds may be useful for preventing ulcers and appear to act by blocking gastric inflammation., ((c) 2004 Blackwell Publishing Asia Pty Ltd.)

Background: Renal interstitial inflammation is a consequence of unilateral ureteral obstruction (UUO). Following ischemia/reperfusion, adenosine reduces renal inflammation and injury, effects which are potentiated by type 4 phosphodiesterase inhibitors. We therefore studied the effects of A2A adenosine receptor agonist (ATL146e), and PDE4 inhibitor (rolipram) in mice subjected to UUO., Methods: Mice were subjected to UUO or sham operation, and received either vehicle or ATL146e + rolipram by osmotic minipump for 1 or 7 days. At 1, 3, 7, or 14 days after operation, renal macrophage infiltration, apoptosis, proliferation, tubular atrophy, and interstitial fibrosis were quantitated, and expressions of IL-6 and TGF-beta mRNA were determined., Results: ATL146e + rolipram reduced macrophage infiltration by 40% after 3 days UUO (p < 0.05). Tubular apoptosis, tubular atrophy, and interstitial fibrosis were increased by 7 or 14 days UUO, but were unaffected by ATL146e + rolipram. However, cellular proliferation was increased by ATL146e + rolipram in the obstructed kidney. ATL146e + rolipram had no effect on the renal expression of IL-6 and TGF-beta mRNA., Conclusions: A2A receptor activation and PDE4 inhibition transiently reduce renal macrophage infiltration, but do not ameliorate the renal response to UUO. We speculate that the persistent stimulus for inflammation triggered by UUO cannot be reversed by agents that suppress inflammatory cell activation alone.

Unlabelled: Hextend (HEX) is a colloid solution that is FDA-approved for volume expansion during surgery. ATL-146e is a novel adenosine A2A receptor agonist that has anti-inflammatory, neuroprotective, and coronary vasodilator properties. Three series of experiments were designed to evaluate the therapeutic potential of HEX+/-ATL-146e for emergency resuscitation from traumatic brain injury (TBI) + hemorrhagic hypotension., Methods: In the first two studies in vivo, anesthetized, ventilated pigs (30-45 kg) received a fluid percussion TBI, 45% arterial hemorrhage, and 30 minutes shock period. In Series 1, resuscitation consisted of unlimited crystalloid (n = 8) or HEX (n = 8) to correct systolic arterial pressure >100 mm Hg and heart rate <100 bpm for the first 60 minutes ("emergency phase"), and then maintain cerebral perfusion pressure (CPP) > 70 mm Hg for 60-240 minutes. In Series 2 (n = 31), resuscitation consisted of a 1 L bolus of HEX + ATL-146e (10 ng/kg/min, n = 10) or HEX +placebo (n = 10) followed by crystalloid to the same endpoints. In Series 3 in vivo, the hemodynamic response evoked by 0, 10, 50, or 100 ng/kg/min ATL-146e was measured before or 60 minutes after HEX resuscitation from 45% hemorrhage., Results: Following TBI+hemorrhage, there were 4/22 deaths in series 1 and 11/31 deaths in series 2. In those alive at 30 minutes, mean arterial pressure, cardiac index, mixed venous O2 saturation, and cerebral venous O2 saturation were all reduced by 40-60%, while heart rate and lactate were increased 2-5 fold. With no resuscitation (n = 2), there was minimal hemodynamic compensation and progressive acidosis. Upon resuscitation, these values corrected but intracranial pressure progressively rose from <5 mm Hg to 15-20 mm Hg. Series 1: With HEX (n = 8) versus crystalloid (n = 8), CPP was less labile, acid/base was maintained, and the fluid requirement was reduced by 60% (all p < 0.05) Series 2: With ATL-146e (n = 10) versus placebo (n = 10), stroke volume and cardiac output were improved by 40-60%, and the fluid requirement was reduced by 30% (all p < 0.05). Series 3: ATL-146e caused a dose-related increase (p < 0.05) in stroke volume after, but not before, hemorrhage. The effects on pre-load, afterload, and heart rate were similar before and after hemorrhage., Conclusions: HEX alone is a safe and efficacious low volume alternative to initial crystalloid resuscitation after TBI. An adenosine A2A agonist combined with 1 L of HEX safely and effectively counteracted a decrease in cardiac performance noted after TBI+hemorrhage without causing hypotension or bradycardia.

Background: ATL-146e is an adenosine A(2A) agonist that has recently been demonstrated to improve neurological outcome in spinal cord injury in animals. In the current study, we extended the treatment paradigm and tested neurobehavioral functioning out to 1 week after injury to assess if early neurological improvement is sustained long term by an adenosine analogue., Materials and Methods: New Zealand White rabbits (3.0-3.5 kg) sustained mid-thoracic blunt spinal cord injury using a weight-drop model (10 g weight dropped from 6 cm directly onto dura). Animals received either (1) 3 h iv infusion of saline carrier (Trauma, N = 21); (2) 3 h iv infusion of 0.06 microg/kg/min ATL-146e followed by intraperitoneal bolus of 10.8 microg/kg ATL-146e at 3 h postinjury (ATL, N = 14); or (3) 3 h iv infusion of 0.06 microg/kg/min ATL-146e followed by intraperitoneal bolus injection of 10.8 microg/kg ATL-146e at 3, 12, and 24 h postinjury (ATL-PLUS, N = 11). Fourteen animals underwent sham injury. Hemodynamic parameters were monitored and hind limb motor functioning was assessed by Tarlov scores (0 = paralyzed to 5 = normal hop) for 7 days after injury., Results: ATL-146e significantly improved Tarlov scores of ATL-146e groups compared with saline-treated controls (P < 0.01 12, 24, 36, and 48 h). Control animals, severely neurologically impaired at 48 h (Tarlov 1.61 +/- 0.35), were euthanized early due to ethical concerns, thus not permitting later statistical comparisons. Early neurological improvements in both ATL-146e-treated groups were sustained longer term (7 day mean Tarlov, SHAM 4.9 +/- 0.30, ATL 5.0 +/- 0, ATL-PLUS 4.25 +/- 0.31)., Conclusions: ATL-146e given immediately after blunt spinal cord trauma significantly improves neurological outcome, which is sustained through 7 days. Early adenosine A2A receptor agonism may be critical since additional IP administration afforded no further neurological improvement. The current data further support the potential clinical utility of adenosine A(2A) agonists in the treatment of spinal cord injury.

Background: Sepsis is currently treated with antibiotics and various adjunctive therapies that are not very effective., Methods: Mouse survival (4-5 days) and peritoneal and blood bacteria counts were determined after challenge with intraperitoneal lipopolysaccharide (LPS) or live Escherichia coli., Results: The A(2A) adenosine receptor (AR) agonist 4-[3-[6-amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid methyl ester (ATL146e; 0.05-50 mu g/kg) protected mice from challenge with LPS, and protection occurred when treatment was delayed up to 24 h after challenge. Deletion of the A (2A) AR gene, Adora2a, inhibited protection by ATL146e. A putative A (3)AR agonist, N(6)-3-iodobenzyladenosine-5'-N-methyluronamide (IB-MECA; 500 mu g/kg but not 5 or 50 mu g/kg) protected mice from challenge with LPS. The protective effects of both ATL146e and IB-MECA were counteracted by the A(2A) AR selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)-phenol. In the live E. coli model, treatment with ATL146e (50 mu g/kg initiated 8 h after infection) increased survival in mice treated with ceftriaxone (5 days) from 40% to 100%. Treatment with ATL146e did not affect peritoneal numbers of live E. coli at the time of death or 120 h after infection but did increase numbers of peritoneal neutrophils and decreased the number of live E. coli in blood., Conclusions: AR agonists increase mouse survival in endotoxemia and sepsis via A(2A) AR-mediated mechanisms and reduce the number of live bacteria in blood.

Introduction: High pulmonary artery flow rates can result in severe reperfusion injury after lung transplantation. Our hypothesis was that selective activation of the adenosine A(2A) receptor with a highly specific analog (ATL-146e) would inhibit leukocyte activation and decrease reperfusion injury after high-flow reperfusion., Methods: Using our isolated, ventilated, blood-perfused rabbit lung model, all groups (n = 8 per group) underwent lung harvest, 4 hours of cold storage, and blood reperfusion for 30 minutes. Measurements of pulmonary artery pressure (in millimeters of mercury), arterial oxygenation (in millimeters of mercury), myeloperoxidase, peak inspiratory pressure, and wet/dry weight ratio were obtained. Groups 1 (high flow) and 2 (high flow ATL-146e) underwent reperfusion at 120 mL/min for 30 minutes. Groups 3 (controlled high flow) and 4 (controlled high flow ATL-146e) underwent controlled reperfusion with an initial reperfusion of 60 mL/min for the first 5 minutes, followed by a rate of 120 mL/min for 25 minutes. During reperfusion, groups 2 and 4 received ATL-146e at 4 microg. kg(-1). min(-1)., Results: ATL-146e significantly improved lung physiologic measurements under both high-flow (group 1 vs group 2) and controlled high-flow (group 3 vs group 4) conditions after 30 minutes., Conclusions: The adenosine A(2A) receptor analogue ATL-146e significantly decreases the severity of reperfusion injury in the setting of both high-flow and controlled high-flow reperfusion.

Background: The adenosine A2A agonist ATL-146e ameliorates reperfusion inflammation, reducing subsequent paralysis and neuronal apoptosis after spinal cord ischemia. We hypothesized that neuroprotection with ATL-146e involves inducible neuronal adenosine A2A receptors (A2A-R) that are upregulated after ischemia., Methods: Eighteen rabbits underwent laparotomy, and 14 sustained spinal cord ischemia from cross-clamping the infrarenal aorta for 45 minutes. One group (ischemia-reperfusion [I/R] + ATL) received ATL-146e intravenously for 3 hours during spinal cord reperfusion. A second group (I/R) received equivolume intravenous saline solution for 3 hours and served as an ischemic control, and a third group (Sham) underwent sham laparotomy. At 48 hours, all subjects were assessed for motor impairment using the Tarlov scoring system (0 to 5). Lumbar spinal cord sections were immunolabeled for A2A-R and graded in a blinded fashion using light microscopy., Results: There was a significant improvement in Tarlov scores in I/R + ATL animals compared with the I/R group. Sham-operated animals demonstrated no A2A-R immunoreactivity. There was a dramatic increase in A2A-R immunoreactivity in neurons of lumbar spinal cord sections from I/R compared with I/R + ATL and sham-operated animals., Conclusions: Reduction in paralysis in animals receiving ATL-146e correlates with the new finding of A2A-R expression on lumbar spinal cord motor neurons after ischemia. Adenosine A2A agonists may exert neuroprotective effects by binding to inducible neuronal A2A-R that are upregulated during spinal cord reperfusion, and reduced in response to administration of an A2A-R-specific agonist.

Background: ATL-146e, an adenosine A2A agonist, reduces paralysis after spinal cord ischemia-reperfusion. We hypothesized that systemic ATL-146e could improve neurologic outcome after blunt spinal cord trauma., Methods: Twenty rabbits survived a thoracic spinal cord impact of 30 g-cm. One group received 0.06 microg/kg/min ATL-146e for the first 3 hours after impact (A2A group), whereas a second group received saline carrier (T/C group). Neurologic outcome was measured using the Tarlov scale (0-5). Histologic sections from the A2A and T/C groups were compared for neuronal viability., Results: There was significant improvement in Tarlov scores of A2A animals compared with T/C animals at 12 hours (p = 0.007), with a trend toward improvement at 36 (p = 0.08) and 48 (p = 0.09) hours after injury. There was decreased neuronal attrition in A2A animals (p = 0.06)., Conclusion: Systemic ATL-146e given after spinal cord trauma results in improved neurologic outcome. Adenosine A2A agonists may hold promise as a rapidly acting alternative to steroids in the early treatment of the spinal cord injured patient.

Objective: Inflammation is likely a major contributor to spinal cord reperfusion injury after aortic reconstruction. Systemic 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL-146e), a selective adenosine A(2A) agonist, has been shown to reduce paralysis after spinal cord ischemia. We hypothesized that ATL-146e reduces cytokine production during spinal cord reperfusion, curtailing inflammation and decreasing spinal cord capillary platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression., Study Design: New Zealand White rabbits sustained spinal cord ischemia with 45-minute cross-clamping of the infrarenal aorta. One group of animals received intravenous ATL-146e at 0.06 microg/kg/min for 3 hours during reperfusion, beginning after 30 minutes of ischemia. A second group received saline solution vehicle alone for 3 hours, serving as an ischemic control. A third group served as sham-operated animals, undergoing laparotomy with anesthesia. Serum was assayed with enzyme-linked immunosorbent assay for tumor necrosing factor-alpha (TNF-alpha). Animals were allowed to recover for 48 hours and were evaluated for hind-limb motor function with the Tarlov (0 to 5) scoring system. At necropsy, animals from each group yielded spinal cords for immunohistochemical staining for PECAM-1. Data are expressed as mean +/- standard error of the mean, with statistical analysis with Student t test and Kruskal-Wallis nonparametric test., Results: Markedly improved Tarlov scores were seen in rabbits with ATL-146e (P <.001) during spinal cord reperfusion as compared with ischemic control animals. A significant reduction was found in TNF-alpha in the sera of rabbits with ATL-146e infusion (P <.01) as compared with ischemic control animals. Significantly reduced endothelial PECAM-1 staining intensity (P <.05) was seen in microscopic spinal cord sections from rabbits with ATL-146e., Conclusion: ATL-146e, an adenosine A(2A) agonist, reduces spinal cord reperfusion injury. The mechanism of the protection may involve a reduction in circulating TNF-alpha during a critical 3-hour reperfusion interval and reduction in spinal cord endothelial PECAM-1 upregulation.