Download Reduction in Myocardial Infarct Size at 48 Hours after a Brief

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. Of mice and dogs: speciesspecific differences in the inflammatory response following myocardial infarction.
Am J Pathol 164: 665-677, 2004.
4.Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC,
Corday E, and Ganz W. Early phase acute myocardial infarct size quantification:
validation of the triphenyl tetrazolium chloride tissue enzyme staining technique.
Am Heart J 101: 593-600, 1981.
5.Frangogiannis NG. Targeting the inflammatory response in healing
myocardial infarcts. Curr Med Chem 13: 1877-1893, 2006.
6.Glover DK, Riou LM, Ruiz M, Sullivan GW, Linden J, Rieger JM,
Macdonald TL, Watson DD, and Beller GA. Reduction of infarct size and
postischemic inflammation from ATL-146e, a highly selective adenosine A2A
19
receptor agonist, in reperfused canine myocardium. Am J Physiol Heart Circ
Physiol 288: H1851-1858, 2005.
7.Glover DK, Ruiz M, Takehana K, Petruzella FD, Riou LM, Rieger JM,
Macdonald TL, Watson DD, Linden J, and Beller GA. Pharmacological stress
myocardial perfusion imaging with the potent and selective A(2A) adenosine
receptor agonists ATL193 and ATL146e administered by either intravenous
infusion or bolus injection. Circulation 104: 1181-1187, 2001.
8.Jordan JE, Thourani VH, Auchampach JA, Robinson JA, Wang NP, and
Vinten-Johansen J. A(3) adenosine receptor activation attenuates neutrophil
function and neutrophil-mediated reperfusion injury. Am J Physiol 277: H18951905, 1999.
9.Jordan JE, Zhao ZQ, Sato H, Taft S, and Vinten-Johansen J. Adenosine A2
receptor activation attenuates reperfusion injury by inhibiting neutrophil
accumulation, superoxide generation and coronary endothelial adherence. J
Pharmacol Exp Ther 280: 301-309, 1997.
10.Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F, Zhao ZQ,
Guyton RA, Headrick JP, and Vinten-Johansen J. Postconditioning reduces
infarct size via adenosine receptor activation by endogenous adenosine.
Cardiovasc Res 67: 124-133, 2005.
11.Lappas CM, Day YJ, Marshall MA, Engelhard VH, and Linden J.
Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury
20
by inhibiting CD1d-dependent NKT cell activation. J Exp Med 203: 2639-2648,
2006.
12.Lasley RD, Jahania MS, and Mentzer RM, Jr. Beneficial effects of
adenosine A(2a) agonist CGS-21680 in infarcted and stunned porcine
myocardium. Am J Physiol Heart Circ Physiol 280: H1660-1666, 2001.
13.Lasley RD, Kristo G, Keith BJ, and Mentzer RM, Jr. The A2a/A2b receptor
antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist
pretreatment in in vivo rat myocardium. Am J Physiol Heart Circ Physiol 292:
H426-431, 2007.
14.Li L, Huang L, Sung SS, Lobo PI, Brown MG, Gregg RK, Engelhard VH,
and Okusa MD. NKT cell activation mediates neutrophil IFN-gamma production
and renal ischemia-reperfusion injury. J Immunol 178: 5899-5911, 2007.
15.Linden J. Molecular approach to adenosine receptors: receptor-mediated
mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775-787,
2001.
16.Mahaffey KW, Puma JA, Barbagelata NA, DiCarli MF, Leesar MA, Browne
KF, Eisenberg PR, Bolli R, Casas AC, Molina-Viamonte V, Orlandi C,
Blevins R, Gibbons RJ, Califf RM, and Granger CB. Adenosine as an adjunct
to thrombolytic therapy for acute myocardial infarction: results of a multicenter,
randomized, placebo-controlled trial: the Acute Myocardial Infarction STudy of
ADenosine (AMISTAD) trial. J Am Coll Cardiol 34: 1711-1720, 1999.
21
17.Micari A, Belcik TA, Balcells EA, Powers E, Wei K, Kaul S, and Lindner
JR. Improvement in microvascular reflow and reduction of infarct size with
adenosine in patients undergoing primary coronary stenting. Am J Cardiol 96:
1410-1415, 2005.
18.Norton ED, Jackson EK, Turner MB, Virmani R, and Forman MB. The
effects of intravenous infusions of selective adenosine A1-receptor and A2receptor agonists on myocardial reperfusion injury. Am Heart J 123: 332-338,
1992.
19.Rezkalla SH and Kloner RA. No-reflow phenomenon. Circulation 105: 656662, 2002.
20.Rork TH, Wallace KL, Kennedy DP, Marshall MA, Lankford AR, and
Linden J. Adenosine A2A receptor activation reduces infarct size in the isolated,
perfused mouse heart by inhibiting resident cardiac mast cell degranulation. Am
J Physiol Heart Circ Physiol 295: H1825-1833, 2008.
21.Ross AM, Gibbons RJ, Stone GW, Kloner RA, and Alexander RW. A
randomized, double-blinded, placebo-controlled multicenter trial of adenosine as
an adjunct to reperfusion in the treatment of acute myocardial infarction
(AMISTAD-II). J Am Coll Cardiol 45: 1775-1780, 2005.
22.Schlack W, Schafer M, Uebing A, Schafer S, Borchard U, and Thamer V.
Adenosine A2-receptor activation at reperfusion reduces infarct size and
22
improves myocardial wall function in dog heart. J Cardiovasc Pharmacol 22: 8996, 1993.
23.Sullivan GW, Rieger JM, Scheld WM, Macdonald TL, and Linden J. Cyclic
AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2propynylcyclohexyl adenosine A(2A) receptor agonists. Br J Pharmacol 132:
1017-1026, 2001.
24.Taqueti VR, Mitchell RN, and Lichtman AH. Protecting the pump:
controlling myocardial inflammatory responses. Annu Rev Physiol 68: 67-95,
2006.
25.Toufektsian MC, Yang Z, Prasad KM, Overbergh L, Ramos SI, Mathieu C,
Linden J, and French BA. Stimulation of A2A-adenosine receptors after
myocardial infarction suppresses inflammatory activation and attenuates
contractile dysfunction in the remote left ventricle. Am J Physiol Heart Circ
Physiol 290: H1410-1418, 2006.
26.van der Hoeven D, Wan TC, and Auchampach JA. Activation of the A(3)
adenosine receptor suppresses superoxide production and chemotaxis of mouse
bone marrow neutrophils. Mol Pharmacol 74: 685-696, 2008.
27.Vinten-Johansen J. 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
*