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Articles in PresS. Am J Physiol Heart Circ Physiol (June 5, 2009). doi:10.1152/ajpheart.00705.2008 Reduction in Myocardial Infarct Size at 48 Hours after a Brief Intravenous Infusion of ATL-146e, a Highly Selective Adenosine A2A Receptor Agonist Rajan A.G. Patel, MD1, David K. Glover, PhD1,Alexis Broisat, PhD1, Hasan K. Kabul, MD1, Mirta Ruiz, MD1, N. Craig Goodman, MS1, Christopher M. Kramer, MD1,2, Denis J. Meerdink, PhD3, Joel Linden, PhD1, George A. Beller, MD1 1 Department of Medicine, Cardiovascular Division. University of Virginia, Charlottesville, VA USA 2 Department of Radiology. University of Virginia, Charlottesville, VA USA 3 Department of Physiology & Pharmacology, Thomas J. Long School of Pharmacy & Health Sciences, University of the Pacific, Stockton, CA, USA Running Head: ATL146e Mediated Infarct Size Reduction at 48 hours Post-MI Copyright © 2009 by the American Physiological Society. Abstract Objectives: To determine if the myocardial infarct sparing effect of ATL-146e, a selective adenosine A2A receptor agonist, persists without a rebound effect for at least 48hours. To determine the optimal duration of ATL-146e treatment in anesthetized dogs. Background: Reperfusion injury after MI is associated with inflammation lasting 24-48hours that contributes to ongoing myocyte injury. We previously showed that an ATL-146e infusion, starting just prior to reperfusion, decreased inflammation and infarct size in dogs examined 2hours post-MI without increasing coronary blood flow. Methods: Adult dogs underwent 90minutes of left anterior descending coronary artery occlusion. Thirty-minutes before reperfusion, ATL-146e (0.01 µg/kg/min) (n=21) or vehicle (n=12) was intravenously infused and continued for 2.5hours(protocol 1) or 24hours (protocol 2). At 48 hours post-reperfusion hearts were excised and assessed for histological risk area and infarct size. Results: Infarct size based on triphenyltetrazolium(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 ATL146e treated vs. control dogs (30±7 vs. 88±16 cells per high power field, p<0.002). ATL-146e infusion for 24hours (protocol 2) conferred no significant additional infarct size reduction compared to 2.5hrs of infusion. 2 Conclusions: A 2.5hour ATL-146e infusion initiated 30minutes before reperfusion results in marked, persistent(48hour) reduction in infarct size as a percent of risk area in dogs with a reduction in infarct zone neutrophil infiltration. No significant further benefit was seen with a 24hour infusion. Keywords: ATL146e, Adenosine, Reperfusion Injury, Myocardial Infarction 3 Introduction Reperfusion injury after coronary ischemia is an important pathophysiologic mechanism in myocardial infarction(MI)(19). It is a phenomenon of multi-factorial etiology including intense inflammation, damage from reactive oxygen species, shifts in metabolic substrate consumption, and perturbations of mitochondrial and cytostolic calcium ion homeostasis (29). During reperfusion after prolonged ischemia, myocytes and endothelial cells release metabolic products and cytokines that are pro-inflammatory(24). Neutrophils accumulate early in tissue undergoing reperfusion injury(27), followed by macrophages and other leukocytes (5). Adenosine infusion during reperfusion has been shown to reduce infarct size compared to placebo in clinical trials among patients suffering from MI with large areas of ischemic anterior myocardium at risk (16, 21). Perhaps due to limited sample size, this reduction in ischemic injury was not associated with a significant mortality reduction. Adenosine administration may lead to unwanted effects such as heart block or hypotension. These effects result from nonselective activation of four adenosine receptor subtypes (A1, A2A, A2B and A3). Selective activation of the adenosine A2A receptor may provide cardioprotection against reperfusion injury via an anti-inflammatory mechanism without nonselective activation of the other receptor subtypes caused by adenosine. In fact, a 2.5hour infusion of a non-vasodilating, yet anti-inflammatory dose of the A2A receptor agonist ATL-146e begining 30minutes prior to reperfusion in an open chest, anesthetized, canine model of myocardial infarction has been shown to 4 reduce infarct size as a percent of risk area by 45% when assessed at 2hours post-reperfusion (6). This low dose of ATL-146e did not cause increased regional myocardial blood flow as is observed with adenosine. Since the inflammatory response after myocardial infarction and reperfusion may last at least 24hours (3), the primary objective of this study was to determine whether the infarct reduction conferred by a 2.5hour infusion of a non-vasodilating, antiinflammatory dose of ATL-146e persists at 48hours without a rebound increase in infarct size. A secondary objective was to determine whether a 24hour infusion reduced infarct size to a greater extent than a 2.5hour infusion when assessed at 48hours post-MI. The data presented is critical in the justification and design of a randomized clinical trial to test if ATL146e therapy can attenuate reperfusion injury in patients suffering from acute myocardial infarction. Materials and Methods All animal experiments were performed with the approval of the University of Virginia Animal Care and Use Committee and were in compliance with the American Heart Association Position on Research Animal Use. Experimental protocol. Protocol 1 randomized thirty-two adult mongrel dogs of both sexes (mean weight 22.4 ± 2.6kg, range 19.5-29.5kg) to vehicle(n=16) or ATL-146e(n=16). Protocol 2 utilized 10 adult mongrel dogs of both sexes, all of which received ATL-146e for 24hours. Induction of general anesthesia was achieved with 5 sodium pentobarbital(30mg/kg, IV). After endotracheal intubation, dogs were mechanically ventilated on room air (Harvard Apparatus, Holliston, MA ). Anesthesia was maintained with a sodium pentobarbital intravenous infusion. One gram of cefazolin and 80mg of gentamicin were administered prior to surgery. A 5-French catheter was placed in the right femoral artery using the Seldinger technique and flushed with heparinized saline in order to monitor central arterial pressure and to serially sample arterial blood. The dogs were then placed in the right lateral decubitus position on a heating pad set to regulate and maintain a constant normal body temperature. Heart rate, electrocardiogram, blood pressure, oxygen saturation, expired pCO2 and body temperature were continuously monitored throughout the surgical procedure. In addition, arterial blood was periodically sampled and analyzed for pH, pO2 and pCO2 using a blood gas analyzer (Radiometer, Denmark). After sterile skin preparation and draping, a thoracotomy was performed at the 5th intercostal space. The pericardium was divided and the heart was suspended in a pericardial sling. All visible collaterals to the anterior wall of the left ventricle were ligated followed by complete occlusion of the left anterior descending artery (LAD). We have previously demonstrated that this technique produces a reproducible decrease in endocardial and transmural perfusion (i.e. risk area) during occlusion (6). After 60minutes of occlusion (i.e. 30 minutes prior to reperfusion) an intravenous infusion of ATL-146e (0.01µg/kg min) or vehicle (0.07% DMSO in saline) was randomly started in both the 2.5hr and 24hr infusion groups The infusion was started 30minutes before reflow in order to achieve a 6 steady state blood concentration before the ligatures were removed. The investigators performing the experiments were blinded to the identity of the solution being infused. Both groups of animals also received a metoprolol 5mg IV bolus 30minutes before reperfusion. If an animal was defibrillated, or if an animal experienced greater than 5minutes of frequent non-sustained ventricular tachycardia, an additional 5mg of IV metoprolol was administered at the time of the event. After 90minutes of occlusion all coronary ligatures were removed to allow reperfusion. Arterial blood was sampled at the times depicted in Figure 1. After 1hour of reperfusion, the chest was closed in layers. In the 24hr treatment group, the continuous infusion was maintained by subcutaneous implantation of an Alzet osmotic minipump (Model 2001D; Durect Co., Cupertino, CA) that had been preloaded with the ATL-146e solution (4.4 µg/uL) After the pentobarbital sodium infusion was discontinued, animals were given morphine analgesia (buprenorphine 0.15mg IV and 0.15mg subcutaneously). Animals were transferred to a heated, oxygenated recovery chamber and extubated once spontaneous respiration commenced. All investigators involved in the surgical procedure and data analysis were blinded from the solution being infused (ATL-146e or vehicle) via IV and mini-pump until after the post-mortem analysis was complete. Serum laboratory values Troponin I concentration from arterial blood collected at the time points shown in Figure 1 was analyzed on an automated blood chemistry machine 7 (Architect CI8200, Abbott Diagnostics, Abbott Park, Illinois). Complete blood cell counts with differentials were performed on samples collected at the time points shown in Figure 1. Analysis was performed on an automated hematology analysis machine (Cell Line 4000, Abbott Diagnostics, Abbott Park, Illinois). Histology Forty-eight hours post-MI, animals were placed under pentobarbital sodium anesthesia as described above. Risk area and infarct size were delineated using monastral blue dye and TTC, respectively, as previously described from our laboratory (6). The hearts were then harvested and the LV was sectioned from base to apex into four short axis slices, each approximately 1cm thick. These slices were stained with TTC (4). Sections were then digitally scanned. The infarct area, risk area, and normal myocardial area of each section were quantified using Sigmascan software (Systat Inc., San Jose, CA) by a blinded operator. The average of the infarct area from each of the four sections was divided by the average of the risk area to arrive at the infarct area as a percent of risk area as previously described from our laboratory (6). A priori, animals with a risk area less than 15% of the total myocardial area were excluded. Myocardial neutrophil quantification Transmural heart fragments from the grossly infarcted zone were randomly sampled, excised, and fixed in 3.7% paraformaldehyde solution. After 8 dehydration and clearing with xylene, tissue samples were embedded in molten paraffin at 60oC. The paraffin-embedded specimens were then sectioned at 5µm and stained for neutrophils with a commercially available mouse anti-human antibody (MCA8T4GT, Serotec, Raleigh, NC). Sixteen photomicrographs (20X) were taken serially every 2mm across each section. The number of neutrophils per field was quantified using ImagePro software (MediaCybernetics Inc., Bethesda, MD). The investigator quantifying neutrophil counts was blinded to whether the specimens were from dogs given ATL-146e or vehicle. Statistics One-way ANOVA with Tukey post-hoc correction was used to compare infarct sizes between the groups. A two-tailed Student’s t-test was used for comparisons of neutrophil counts between control and ATL-146e treated animals. Two-way repeated measures ANOVA was used to analyze the mean arterial pressure and heart rate responses between the groups over time. Fisher’s exact test was used when comparing the number of dogs in each group that died from VF and the number of dogs in each group that survived VF arrest. Statistical analysis was performed with SigmaStat v2.03 (SPSS Inc, Chicago, IL). RESULTS Sample Size Thirty-two dogs were originally randomized to protocol 1, control (n=16) or ATL-146e (n=16). The final study group included twenty-five dogs (control n=12; 9 ATL-146e n=13). Four dogs in the control group and 2 dogs in the ATL-146e group died secondary to lethal arrhythmias. One dog in the ATL-146e group was excluded from the study due to small risk area in accordance with predetermined criteria. Protocol 2 (24 hour infusion) included 10 adult mongrel dogs with a final study group of 8. One dog in protocol 2 died from lethal arrhythmia. Another dog was excluded due to small risk area. Hemodynamics There was no difference in heart rate or mean arterial blood pressure between ATL-146e treated or control animals at any monitored time point (Figure 2). Importantly no fall in systemic blood pressure was observed consequent to ATL-146e infusion. Ventricular fibrillation and metoprolol use In protocol 1, there was a trend toward a higher death rate from refractory ventricular fibrillation (VF) in the control dogs (ATL146e: 2/16 vs control: 4/16), however this trend did not reach statistical significance. Among the surviving animals there was no difference between groups in the number of dogs which experienced VF (ATL-146e: 7 vs. control: 6; p=1.000). There was no difference in the mean metoprolol dose administered in order to suppress ventricular tachycardia/ fibrillation between the two groups (ATL-146e: 6.9±1.2 vs. control: 8.3±1.4 mg, p=0.530). In protocol 2, one of the ten dogs died from refractory ventricular fibrillation. 10 Histologic Infarct Size Risk areas were similar between treatment groups (Figure 3). However infarct size by triphenyltetrazolium (TTC) staining as a percentage of risk area at 48 hours post-reperfusion was reduced by 49% in dogs receiving the 2.5 hour ATL-146e infusion compared to control animals (ATL-146e: 16.7±3.7% vs. control: 33.3±6.2%, p<0.05). Infusion of ATL-146e for 24hours did not show a greater reduction in infarct size than that achieved by just a 2.5 hour infusion (24hour: 17.1±4.3 vs. 2.5hour 16.7±3.7%, p=0.942). Myocardial Neutrophil Infiltration Infarct zone neutrophil infiltration (Figures 4 and 5) differed markedly between the two groups at 48hours post-MI (ATL-146e 30.5±6.8 vs. control 87.6±15.7 cells per field, p=0.002). As shown, the ATL-146e treated dogs had significantly fewer neutrophils in the reperfused infarct zone. Serum laboratory values Troponin I concentration trended lower in the ATL-146e group versus control at the 2.5 hour post-MI time point (ATL-146e 4.9±4.7 vs. control 12.7±13.4, p=0.135) and the 48 hour post-MI time point (ATL-146e 26.3±22.7 vs. control 40.0±39.2, p=0.334). There was no difference in serum white blood cell count, neutrophil count, and lymphocyte count between the treated and control dogs at any time point. 11 Discussion The present study demonstrates that a brief, 2.5hour infusion of a nonvasodilating, anti-inflammatory dose of the adenosine A2A receptor agonist ATL146e started 30minutes prior to reperfusion confers a sustained reduction in infarct size as a percent of risk area at 48hours after reflow. Histologic infarct size in treated dogs was significantly smaller than observed after reperfusion with vehicle alone (16.7% vs. 37.3%) as assessed by blinded observers. This was associated with a substantial reduction in inflammation as measured by infarct zone neutrophil infiltration. The current findings are in accordance with previously reported data at 2hours post-MI in which the radiolabeled leukotriene B4 antagonist RP517, an imaging agent that targets circulating neutrophils, showed reduced infarct zone neutrophil uptake in dogs treated with ATL-146e during reperfusion (6).There was no additional benefit from an extended 24 hour infusion of ATL-146e over the short 2.5hour infusion with respect to infarct size reduction. Other potential benefits of a more prolonged infusion were not explored in this study. Our findings also support the hypothesized mechanism of benefit of ATL-146e, which is a reduction in the acute inflammatory response consequent to reperfusion leading to a reduction in infarct size (25, 28, 29). The low ATL-146e dose used does not increase myocardial blood flow (7). Because dogs have variable coronary anatomy, each animal may have a slightly different coronary anatomic distribution and collateralization. It is speculated that infarct size estimated by serial Troponin I concentrations did not achieve statistical significance as these indices were not controlled for variations 12 in risk area as opposed to histologic infarct size measurements which were normalized to risk area. The importance of controlling for risk area is critical in human studies assessing reperfusion injury (17). Nevertheless the serial Troponin assessments were concordant with the histologic data and the myocardial neutrophil assessments. The current experimental model involved producing myocardial infarction at the time of thoracotomy. As expected surgical trauma to the chest wall caused a significant leukocytosis. Nonetheless, neutrophil counts from the infarct zone of ATL-146e treated animals were significantly lower than control animals. Mechanism of action of ATL-146e The adenosine receptors are G-protein coupled seven transmembrane receptors. The A1 receptor produces the bradycardia and heart block associated with adenosine. Additionally, the A1 receptor has a pro-inflammatory effect on neutrophil function. The A2B receptor contributes to the vasodilatory and hypotensive response of adenosine. A2B (dogs and primates) and A3 (rodents) receptor activation can be pro-inflammatory in bronchial smooth muscle tissue and may facilitate allergic reactions in susceptible subjects. On the other hand, there have also been reports showing both anti-inflammatory (8, 26) and antiinfarct (1) effects of A3 adenosine receptor activation. Nevertheless, the cardioprotective effects of adenosine during reperfusion injury appear to be mediated at least in part by A2A receptor activation (2, 13, 15, 18). Activation of A2A receptors results in increased intracellular cyclic adenosine monophospate 13 (cAMP) concentration resulting in activation of protein kinase A and inhibition of multiple steps in the inflammatory cascade. A2A receptors are present on a variety of cells including various types of leukocytes, cardiac myocytes, and endothelial cells. ATL-146e is a highly selective A2A receptor agonist that has been shown to have more potent binding activity to recombinant human and canine A2A receptors than the commercially available A2A receptor agonist CGS21680 (23). Data from Yang et al using A2A receptor knockout mice demonstrated that the cardio-protective mechanism of ATL-146e is via interaction with the A2A receptor (28). The leading hypothesis regarding the potent anti-inflammatory action of A2A receptor activation via ATL164e in reducing reperfusion injury involves A2A receptor mediated reduction of CD4+ lymphocyte activation. In the liver and kidney, NKT cells are the principle targets of A2A receptor activation. A2A receptor agonism on NKT cells has been shown to reduce neutrophil mediated reperfusion injury in these two organs (11, 14). It is not yet known if this is also the case in the heart, however the data presented here demonstrates that ATL164e treatment reduced inflammation in the reperfused zone, as evidenced by a reduction in neutrophil infiltration, compared to vehicle. In a recent study using an isolated, buffer-perfused heart model, Rork et al showed that, even in the absence of neutrophils, ATL146e reduced tissue resident mast cell degranulation and protected against post-ischemic myocardial necrosis (20). Thus, current evidence demonstrates that the anti-inflammatory and infarct sparing effects of A2A adenosine receptor activation result from inhibition of 14 multiple inflammatory cell types including NKT cells, mast cells, and neutrophils. An alternative or complimentary, yet non-exclusive, mechanism may be the concept of post-conditioning described by Kin, et al (10) as well as others that involves delayed washout of endogenous adenosine upon reperfusion which exerts a cardioprotective effect. Other investigators have also studied the role of the A2A receptor in reducing infarct size in acute models of reperfused myocardial infarction in large animals. CGS-21680, a commercially available A2A receptor agonist, is approximately 50 times less potent and less selective than ATL-146e. Schlack et al reported a 60minute LAD occlusion/ reperfusion study in which IC CGS-21680 was started 5minutes prior to reperfusion. After 6hours of reperfusion, infarct size as a percent of risk area was significantly smaller with CGS-21680 (22). Jordan, et al subsequently reported similar results in a canine model of LAD occlusion/reperfusion after 3 hours of reperfusion (9). More recently Lasley, et al reported data from a porcine model of infarction and reperfusion with intracoronary CGS-21680. Infarct size as a percent of risk area assessed after 3 hours of reperfusion was reduced with IC CGS-21680 (12). The porcine model is interesting in that pigs are generally regarded as having less collateral myocardial perfusion than dogs. Unlike adenosine, the dose of ATL-146e utilized in the current study conferred a beneficial effect on infarct size as a percent of risk area without requiring any increase in myocardial blood flow (7). Furthermore, unlike the current study, none of the aforementioned studies evaluated the impact of 15 adenosine receptor agonism in the presence of contemporary pharmacologic treatment with metoprolol at the time of reperfusion. Study Limitations Coronary flow was not assessed during ATL164e infusion in each of the experiments reported here. However, coronary flow during various infusions of ATL146e including the dose used here in the same experimental animal preparation have been previously published by our group (7). In addition, our conclusions are based on experiments in which collateral flow was not measured. Therefore, we cannot completely discount the possibility that differences in infarct size between control and ATL-146e-treated groups can be attributed to differences in collateral flow and not the effect of the drug. Clinical Implications The present experimental study demonstrated a sustained reduction in infarct size after administration of a 2.5hour intravenous ATL-146e infusion beginning 30minutes prior to reperfusion. It is hypothesized that ATL-146e administration to acute STEMI patients will be superior to the effect of adenosine since ATL-146e does not activate the A2B receptor causing hypotension and ATL-146e does not bind to the A1 receptor which can be associated with bradycardia, atrio-ventricular block and with pro-inflammatory effects. In recent clinical trials (AMISTAD I & II) conducted to assess whether adenosine would 16 limit myocardial reperfusion injury, patients in both studies assigned to adenosine treatment had an increased incidence of hypotension and heart block (16, 21). Additionally patients assigned to adenosine in AMISTAD I had an increased incidence of bradycardia (16). Adverse hemodynamic effects such as hypotension consequent to the A2a agonist infusion were not observed in this canine experimental study (see figures 2 and 3). Furthermore the benefit of ATL146e occurred in the presence of beta-blocker administration. Thus, the infarct reducing effect of ATL-146e is complementary to any infarct sparing effect of early intravenous metoprolol administration. This is important as early beta blocker therapy is part of the standard of care in treating patients with myocardial infarction. If ongoing chronic studies demonstrate no adverse effect of ATL-146e on infarct healing, as was seen with steroid administration post-MI, then a randomized clinical trial assessing the efficacy of ATL-146e in reducing myocardial infarction appears warranted. In summary, the highly selective adenosine A2A receptor agonist ATL-146e limits reperfusion injury when given as a brief 2.5hour infusion starting 30minutes prior to reperfusion. Despite the short time course of this infusion there is no rebound reperfusion injury when infarct size is assessed at 48hours post-MI. No added benefit with regard to infarct size was observed with the more prolonged 24hour infusion of the drug. The beneficial effect of ATL-146e appears to be via its anti-inflammatory properties and persists in the presence of intravenous metoprolol given at the time of reperfusion. This data will be useful for the design 17 of a randomized clinical trial assessing this novel treatment for attenuating reperfusion injury. Acknowledgement We are grateful to Ms. Susan Ramos who performed the histological preparation used for myocardial neutrophil counting and to the Adenosine Therapeutics Group, PGxHealth, LLC for supplying ATL146e. Grants: Supported by NIH RO1 HL075320 and T32EB003841 Disclosures: Rajan A.G. Patel – No relationships to disclose Alexis Broisat – No relationships to disclose Hasan K. Kabul – No relationships to disclose Mirta Ruiz – No relationships to disclose Craig Goodman – No relationships to disclose Christopher M. Kramer – No relationships to disclose David K. Glover - Equity Interest/Stock options in Adenosine Therapeutics, significant level relationship; Research grants, significant level relationship Denis J. Meerdink – No relationships to disclose Joel Linden - Equity Interest/Stock options in Adenosine Therapeutics, significant level relationship George A. Beller – Founders Stock in Adenosine Therapeutics and member of advisory board of Adenosine Therapeutics, significant level relationship 18 Reference List: 1.Auchampach JA, Ge ZD, Wan TC, Moore J, and Gross GJ. A3 adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs. Am J Physiol Heart Circ Physiol 285: H607-613, 2003. 2.Cargnoni A, Ceconi C, Boraso A, Bernocchi P, Monopoli A, Curello S, and Ferrari R. Role of A2A receptor in the modulation of myocardial reperfusion damage. J Cardiovasc Pharmacol 33: 883-893, 1999. 3.Dewald O, Ren G, Duerr GD, Zoerlein M, Klemm C, Gersch C, Tincey S, Michael LH, Entman ML, and Frangogiannis NG. 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Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury. Cardiovasc Res 61: 481-497, 2004. 28.Yang Z, Day YJ, Toufektsian MC, Ramos SI, Marshall M, Wang XQ, French BA, and Linden J. Infarct-sparing effect of A2A-adenosine receptor 23 activation is due primarily to its action on lymphocytes. Circulation 111: 21902197, 2005. 29.Yellon DM and Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med 357: 1121-1135, 2007. 24 Figure Legends: Figure 1. Timeline of the experimental protocol. Sixty minutes after LAD occlusion (30 minutes prior to reperfusion) a 2.5hour IV infusion of ATL-146e was started. Forty-eight hours after reperfusion, histologic assessment of risk area, infarct area, and neutrophil infiltration were performed as described in the methods section. Circles indicate time points at which arterial blood was sampled for analysis. Figure 2. Mean arterial pressure (MAP) and heart rate at baseline (prior to ATL146e or vehicle infusion) and during the infusion at 120 min of reperfusion (REP). There was no change in MAP or HR during the experimental protocol and no differences were observed in these parameters between the animals receiving ATL-146e or vehicle. BPM=beats per min; mmHg=millimeters of mercury. Figure 3. Infarct Size as a Percent of Risk Area. Myocardial risk area (left) and infarct size (right) in the experimental model of myocardial infarction. There is no difference in risk area between the 3 groups as measured by monastral blue dye. However the control animals had a larger infarct size compared to ATL-146e treated animals. There was no difference in infarct size between the animals that received a 2.5 hour infusion and a 24 hour infusion of ATL-146e. 25 Figure 4. Infarct Zone Neutrophil Count per 20X Field. The mean neutrophil count per 20X field from infarct zone tissue was significantly lower in ATL-146e treated dogs versus control. Figure 5. 20X photomicrographs from the infarct zone of a control animal (left) and an ATL-146e-treated animal (right). Neutrophils have been stained dark violet. Neutrophil infiltration of the infarct zone was visibly reduced in ATL-146e treated dogs. 26 Control (n=12) ATL146e-2.5hr (n=13) * p=0.027 vs Control # p=0.942 vs ATL146e-2.5hr ATL146e-24hr (n=8) 50 Percent 40 p = NS 30 * 20 # 10 0 Risk Area (%LV) Infarct Area (%RA) Neutrophil Count (cells/20X field) Control 100 ATL146e * p = 0.002 vs Control 75 50 25 0 *