(Circulation. 1995;92:935-943.)
© 1995 American Heart Association, Inc.
Articles |
Infarct Salvage With Liposomal Prostaglandin E1 Administered by Intravenous Bolus Immediately Before Reperfusion in a Canine Infarction-Reperfusion Model
From the Division of Cardiology, University of Texas Health Science Center, Houston; the Texas Heart Institute, Houston (W.K.V.); and the Liposome Co, Inc, Princeton, NJ (C.S., A.J.).
Correspondence to Richard W. Smalling, MD, PhD, Professor of Medicine, University of Texas Medical School, MSB 1.246, 6431 Fannin, Houston, TX 77030.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background Prostaglandin E1 (PGE1) inhibits leukocyte and platelet function and reduces infarct size during left atrial infusion. Intravenous liposomal PGE1 (TLC C-53) accelerates thrombolysis and prevents reocclusion in canine coronary thrombosis. We tested the hypothesis that intravenous TLC C-53 would attenuate reperfusion injury in a canine infarction-reperfusion model.
Methods and Results Twenty-one open-chest dogs were randomized to receive a 10-minute intravenous infusion of either liposome diluent (placebo), free PGE1 (2 µg/kg), or TLC C-53 (2 µg/kg PGE1) after 2 hours of left anterior descending (LAD) occlusion just before reperfusion. Hemodynamic assessment, regional myocardial blood flow determination with radioactive microspheres, myocardial leukocyte infiltration by myeloperoxidase assay, and estimation of infarct size using triphenyl tetrazolium chloride staining were performed. Regional fractional shortening was measured with sonomicrometer crystals implanted in the midmyocardium. Infarct size as a percentage of the risk region was significantly reduced (P<.05) with TLC C-53 (37.9±17.4%) compared with PGE1 (56.7±13.9%) or placebo (58.0±9.9%) infusion. Infarct salvage with TLC C-53 was independent of collateral blood flow by ANCOVA. There was a dramatic reduction in myeloperoxidase activity in the infarct, risk, and border regions of dogs treated with TLC C-53 compared with placebo. Enzyme activity was also significantly reduced (P<.05) in the infarct zone with TLC C-53 (0.11±0.1 U/100 mg) treatment compared with PGE1 (0.38±0.3 U/100 mg). No significant differences in regional myocardial blood flow or myocardial function among treatment groups were identified, although there was a trend toward improved function in the TLC C-53 dogs.
Conclusions Bolus intravenous administration of TLC C-53 immediately before reperfusion results in reduced leukocyte infiltration and substantial infarct salvage. TLC C-53 may be useful in limiting reperfusion injury during treatment of acute myocardial infarction.
Key Words: liposomes • prostaglandins • reperfusion
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Clinical trials have documented enhanced survival with successful reperfusion in the setting of acute myocardial infarction, but improvement in left ventricular function (and implied functional infarct salvage) is less common.1 2 Recently, the MITI investigators3 found that very early administration of a thrombolytic agent resulted in marked improvement in both mortality and left ventricular function. To achieve this result, the lytic agent had to be administered within the first 70 minutes of chest pain, which would correspond to reperfusion within the first 2 hours of ischemia. Progressively less survival benefit is derived as the time from chest pain to initiation of thrombolytic therapy increases.4 5 6 Furthermore, results from a collaborative review of seven large randomized thrombolytic trials clearly demonstrate that administration of thrombolytic agents may be associated with an increased risk of mortality within the first 24 hours of hospital admission.7 This phenomenon has been called "early hazard" and probably represents an adverse outcome with reperfusion in patients presenting more than 2 hours after onset of symptoms.8 Moreover, the risk of early hazard increases with progressive delays from symptom onset to the administration of thrombolytic agents, limiting net mortality benefit.7 Although the mechanism of early hazard is unknown, reperfusion injury may play a role in its pathogenesis.9 In experimental animals, after 90 minutes of ischemic injury, reperfusion at the tissue level has been associated with extensive capillary damage and myocardial cell swelling.10 11 Despite restoration of epicardial coronary blood flow, microvascular blood flow was impaired, potentially further impairing infarct salvage. Thrombolytic agents such as tissue-type plasminogen activator are partially effective in restoring the blood flow in the epicardial vessels but do not appear to affect the "no-reflow" phenomenon.12 It is possible that reperfusion injury is more common in patients presenting >2 hours after onset of pain and limits the benefit of delayed reperfusion. A reduction in reperfusion injury could potentially improve the outcome of patients treated
3 to 6 hours after onset of pain. Prostaglandins E1 and I2 have shown promise in reducing infarct size in experimental animal models. Their infusion directly into the left atrium during continued ischemia results in infarct salvage in the absence of reperfusion.13 Prostaglandins E1 and I2 reduce free radical production in stimulated human neutrophils and may attenuate reperfusion injury.14 They also inhibit platelets15 and are vasodilators. Liposomal delivery of prostaglandin E1 (PGE1) may effectively target the PGE1 to white blood cells, platelets, and endothelial cells16 and possibly limit the hemodynamic impact of PGE1 until the liposomal preparation interacts with the target cellular elements.16 We have previously shown that repetitive administration of bolus doses of liposomal PGE1 reduces white blood cell activation and accumulation in ischemic tissue as well as infarct size in a 2-hour canine occlusion-reperfusion model. These effects were not associated with significant adverse hemodynamic effects, in contrast to continuous infusion of PGE1.17 In a more clinically relevant canine model with coil-induced coronary artery thrombi, we observed that liposomal PGE1 (TLC C-53), given intravenously 1 hour after clot maturation and just before intravenous streptokinase and heparin, accelerated thrombolysis, prevented reocclusion, and led to a significant reduction in infarct size.18 Furthermore, a dose of 2 µg/kg PGE1 of the TLC C-53 preparation administered as a bolus infusion did not result in significant differences in hemodynamic parameters compared with placebo.18
The purpose of this study was to test the hypothesis that intravenous bolus administration of liposomal PGE1 (TLC C-53) immediately before reperfusion would attenuate reperfusion injury, promote infarct salvage, and preserve regional myocardial function in a 2-hour canine infarction-reperfusion model. TLC C-53 containing 2 µg/kg PGE1 was compared with free PGE1 at a similar dose and with placebo.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
This study was reviewed and approved by the Animal Welfare Committee of the University of Texas Medical School at Houston. Thirty conditioned mongrel dogs weighing 25 to 35 kg were anesthetized with intravenous pentobarbital (30 mg/kg) and maintained on a volume ventilator with supplemental intramuscular and intravenous doses of pentobarbital administered for maintenance anesthesia. Before the procedure was begun, the dogs were randomized to receive an intravenous bolus over 10 minutes of TLC C-53, PGE1, or placebo at the time of reperfusion. A left lateral thoracotomy was performed using aseptic technique, and the heart was suspended in a pericardial cradle. Catheters were placed in the left atrium, ascending aorta, and left ventricle. The left anterior descending coronary artery (LAD) was gently dissected free of surrounding tissue just distal to the first diagonal branch, and a Doppler flow probe and coronary snare were placed around the exposed arterial segment. Sonomicrometer crystals were inserted at midwall in the circumferential plane in the ischemic region (supplied by the LAD) and a control region (supplied by the left circumflex artery). Visible epicardial collateral vessels from the distal circumflex artery were tied off outside the anticipated ischemic region. Baseline hemodynamic measurements, including aortic, left atrial, and left ventricular pressures and heart rate, were recorded. Radioactive microspheres 15 µm in diameter (New England Nuclear) were injected into the left atrium (3x106 to 5x106 microspheres per injection), and aortic blood was sampled continuously for 2 minutes to determine regional myocardial blood flow.19 After baseline measurements were obtained, the LAD was occluded with the snare, and flow cessation was documented by Doppler signal. After 10 minutes of LAD occlusion, hemodynamic and microsphere blood flow measurements were repeated. Hemodynamic measurements were also obtained 1 hour and 1 hour and 40 minutes after coronary occlusion. One hour and forty-five minutes after occlusion, either TLC C-53 (2 µg/kg PGE1), free PGE1 (2 µg/kg), or placebo diluent (0.01 mol/L buffered acetate solution) was administered over 10 minutes by continuous infusion. The PGE1 preparation used was Prostin VR Pediatric brand of alprostadil sterile solution (the Upjohn Co). TLC C-53 was supplied by the Liposome Co, Inc and consisted of 10 µg/mL prostaglandin E1 and 4 mg/mL egg phosphatidylcholine in 0.01 mol/L acetate buffer. When reconstituted, the liposomes have a mean particle size of approximately 150 nm (range, 50 to 500 nm) and possess a PGE1-to-lipid ratio of 1:400 by weight. Thus, for a 25-kg dog, a 5-mL solution containing 50 µg PGE1 and 20 mg egg phosphatidylcholine in buffered acetate would be administered. A dose-ranging study was performed with bolus infusions of TLC C-53. Doses of TLC C-53 containing 0.5 to 2.0 µg/kg PGE1 induced acceptable levels of tachycardia and reduction of mean arterial pressure and resulted in infarct salvage in previous studies.17 18
At the completion of infusion (just before reperfusion),
hemodynamic measurements were repeated. Precisely 2
hours after coronary occlusion, the LAD snare was released, and
reflow was established and documented by Doppler.
Hemodynamics were assessed 10 and 30 minutes after
reperfusion. Microsphere blood flow measurements were obtained
30 minutes after reperfusion. One hour after reperfusion,
hemodynamics were repeated, and at 2 hours after
reperfusion, hemodynamic and microsphere blood
flow measurements were obtained again. The protocol is outlined in Fig
1
.
|
After the 2-hour reperfusion measurements were completed, additional intravenous sedation with sodium pentobarbital was administered, and the dog was killed by intra-atrial injection of supersaturated potassium chloride. The heart was quickly excised and washed with tap water. A perfusion cannula was inserted into the LAD at the level of the snare, and the ascending aorta was attached to a perfusion stand. Simultaneously, the LAD was perfused with a 1% solution of triphenyl tetrazolium chloride (TTC) buffered to pH 8.5, and the aortic root was perfused with Evans blue at equal pressure (100 mm Hg) for 5 minutes. After perfusion, the atria and right ventricle were excised, the left ventricle was sliced from base to apex in a bread-loaf fashion into sections 1 cm thick, and the slices were weighed. The TTC stained the risk region containing residual viable tissue brick red, while the infarcted tissue remained unstained and appeared tan.20 The control region was stained by the Evans blue. After staining, 20- to 40-mg tissue samples were obtained from the infarct, border, and risk regions as well as the nonischemic (or control) region and flash-frozen at -70°C with liquid nitrogen. These samples were later assessed for myeloperoxidase activity to quantify neutrophil infiltration.21 After samples were obtained for the myeloperoxidase assay, the tissue slices were incubated in TTC at 37°C for an additional 30 minutes to ensure proper staining of ischemic but viable tissue. After TTC staining, both surfaces of each ventricular slice were traced on acetate film to show the histochemical demarcation of the infarct, risk, and control regions. Their respective areas were planimetered and multiplied by the slice weight to determine infarct size as a percentage of myocardium at risk and total left ventricular mass.20 The ventricular slices were then subdivided into approximately 1-g endocardial, midmyocardial, and epicardial pieces. Each piece was weighed and counted for radioactivity together with the reference arterial blood samples in an automated gamma scintillation counter (1282 Compugamma, LKB-Wallac). The energy windows were adjusted for the peak emission of the isotopes used for calculation of regional myocardial blood flow according to the method of Heymann et al.19 Percent segmental shortening was calculated by subtracting the end-systolic segment length determined at end ejection from the end-diastolic length, dividing by the end-diastolic length, and multiplying by 100.
The experimental procedure described conforms to the "Position of the American Heart Association on Research Animal Use" adopted on November 11, 1984.
Statistical Analysis
Results are expressed as mean±SD.
For multiple comparison
procedures including hemodynamic and coronary
flow data, ANOVA and the Newman-Keuls multiple-range test were
performed for repeated measures. ANCOVA was performed to compare
infarct size in the TLC C-53, PGE1, and placebo
groups, with collateral blood flow used as a covariate.
P<.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Of the 30 dogs studied, 9 were excluded from analysis. Seven dogs developed refractory ventricular arrhythmias and died during coronary occlusion before either study drug or placebo infusion. One dog had significant trauma to the LAD during dissection, and the experiment was aborted. The ninth animal was eliminated from analysis because of high transmural collateral blood flow in the ischemic region, which was in excess of 0.4 mL · min-1 · g-1 tissue (>35% of control region flow during LAD occlusion). (The mean transmural ischemic zone blood flow during occlusion for the 21 dogs analyzed was 15.6±8.1% of control region flow.) This left 7 animals randomly assigned to each of the three treatment groups acceptable for analysis.
Hemodynamics
Hemodynamic parameters, including
heart rate, mean arterial (aortic) pressure, left atrial
pressure, and left ventricular systolic and
end-diastolic pressures for placebo-, PGE1-,
and TLC C-53–treated dogs are shown in the Table. There
were no significant differences in heart rate or in the rate-pressure
product in the three treatment groups during coronary
occlusion or reperfusion. The mean arterial pressure and
systolic left ventricular pressure were similar for the
three groups at baseline and initially during coronary
occlusion before treatment. There was a modest but significant decline
in both variables during bolus intravenous
administration of TLC C-53 and PGE1 compared with placebo.
Significant differences among treatment groups in mean
arterial pressure and systolic left ventricular
pressure were no longer present 10 minutes after completion of
infusion, and there were no significant
physiological events during this transient period
of relative hypotension.
|
Regional Left Ventricular Function and
Dimensions
The myocardial segment end-diastolic lengths were
approximately 1.5 cm in the LAD and circumflex regions. Percent
fractional shortening in the circumflex region was equivalent in the
three study groups. The percent shortening in the LAD region was
approximately 13% for all three groups at baseline (Fig 2). The
LAD segment crystals were placed closer to the
apex than the circumflex crystals to maintain the crystals within the
ischemic region and hence had slightly increased segmental
shortening at baseline. Although the increase in left
ventricular end-diastolic length in the
ischemic region during occlusion was similar among
treatment groups, there was a significantly greater degree of
paradoxical systolic expansion (more negative values for percent
fractional shortening) for the PGE1 group compared with the
TLC C-53 group 10 minutes into coronary occlusion. With
reperfusion, the left ventricular end-diastolic
length tended to return toward normal and was not different in the
three study groups. After 2 hours of reperfusion, there was a trend
toward better segmental shortening in the TLC C-53 group, but
differences in segmental shortening among treatment groups during
reperfusion did not achieve statistical significance.
|
Effect of TLC C-53 on Myocardial Blood Flow
There were no
significant differences in transmural blood flow
among treatment groups during ischemia or reperfusion, as shown
in Fig 3. Mean transmural blood flow in the
ischemic region in TLC C-53 dogs declined from 0.82±0.27 to
0.09±0.06
mL · min-1 · g-1 tissue
during occlusion. The transmural blood flow ratio of the
ischemic to nonischemic regions for the TLC
C-53–treated dogs was 1.04±0.09 at baseline, declining to
0.15±0.09
during ischemia. Similar values were observed in the
other two groups. After an initial hyperemic phase upon release of the
LAD snare, the transmural blood flow to the ischemic region
tended to fall below baseline levels after 2 hours of reperfusion in
all three treatment groups.
|
Effect of TLC C-53 on White Blood Cell Infiltration
Measuring
the activity of the neutrophil-specific myeloperoxidase
enzyme is an indirect but accurate method of quantifying leukocyte
infiltration of tissue.21 As shown in Fig 4, there
was a dramatic reduction in myeloperoxidase
activity in the infarct, risk, and border regions of dogs treated with
TLC C-53 compared with placebo. Enzyme activity was significantly
reduced (P<.05) in the infarct zone with TLC C-53
(0.11±0.1 U/100 mg) treatment compared with PGE1
(0.38±0.3 U/100 mg). There seemed to be an intermediate response with
bolus administration of free PGE1 in the border zone and
risk region, which did not achieve statistical significance. The
nonischemic regions did not differ in enzyme activity.
|
Effect of TLC C-53 on Infarct Size
Infarct size expressed as
a percentage of the area at risk was
significantly lower (P<.05) with TLC C-53 (37.9±17.4%)
compared with PGE1 (56.7±13.9%) or placebo
(58.0±9.9%),
as shown in Fig 5. The area at risk as a percentage of
the left ventricle was similar (22%) for all three groups.
|
Infarct size
as a percentage of the area at risk was inversely related
to collateral blood flow into the ischemic zone during
coronary occlusion for the TLC C-53 group (Fig 6). The
correlation was relatively weak for the placebo
group. There was no correlation between infarct size and collateral
flow in the PGE1 group. When transmural collateral blood
flow to the ischemic region was used as a covariate, infarct
size (as a percentage of area at risk) was significantly smaller in the
TLC C-53 group compared with either PGE1 or placebo
(P<.05). The beneficial treatment effect of TLC C-53 in
reducing infarct size was especially evident in dogs with relatively
higher levels of collateral blood flow.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
In experimental models of temporary coronary occlusion, the severity and duration of ischemia are the principal determinants of the degree of reversible myocardial dysfunction that follows reperfusion (myocardial stunning)22 and the extent of irreversible myocardial damage that ensues.23 Other factors that significantly influence infarct size after reperfusion are the area of myocardium in jeopardy, presence of collateral blood flow into the ischemic region, and myocardial demand during occlusion.24 These findings reported in an NHLBI-sponsored cooperative study using a chronic canine model have recently been confirmed in patients with acute myocardial infarction who were injected with 99mTc sestamibi before reperfusion and tomographic imaging.25
Liposomal PGE1 and Reperfusion Injury
In the
present study, we found a significant reduction in
infarct size and leukocyte infiltration into the infarct, risk, and
border regions when TLC C-53 was administered as an
intravenous bolus after 2 hours of ischemia
immediately before reperfusion. Infarct size expressed as a percentage
of the area at risk and white blood cell infiltration of the infarct
zone were both significantly reduced with TLC C-53 compared with either
an identically administered dose of PGE1 or placebo
infusion. Furthermore, when transmural collateral blood flow was used
as a covariate according to the method of Reimer et al,24
infarct size (as a percentage of the area at risk) was significantly
smaller in the TLC C-53 group compared with either PGE1 or
placebo. The beneficial treatment effect of TLC C-53 on infarct salvage
was especially evident in dogs with relatively higher collateral blood
flow. In contrast to our previous experiments in which bolus infusion
of liposomal PGE1 beginning shortly after the onset of
ischemia seemed to improve myocardial blood flow in the
ischemic region,17 the present study failed to
demonstrate significant differences in ischemic zone transmural
blood flow among the three treatment groups. Furthermore, there was a
similar decline among treatment groups in transmural blood flow after 2
hours of reperfusion to below their respective baseline values,
suggesting that the no-reflow phenomenon was not significantly affected
by a single bolus administration of TLC C-53 before reperfusion.
Measurements of collateral blood flow were obtained after 10 minutes of ischemia. Although it is possible that further recruitment of collateral vessels might occur later into the ischemic period, in the canine model used, most collateral vessels will open shortly after the onset of ischemia. Measurement of collateral blood flow 10 minutes after occlusion was chosen for the ANCOVA in the Animal Models for Protecting Ischemic Myocardium (AMPIM) Cooperative Study,24 since these values were for the most part not significantly different from values obtained after 105 minutes of coronary occlusion for inner, middle, and outer thirds of the myocardium in either the conscious or unconscious models.
All dogs subject to analysis in this study had transmural collateral blood flow <0.25 mL · min-1 · g-1 tissue, which placed them well within the range for the development of myocardial necrosis. One dog with collateral flow in excess of 0.4 mL · min-1 · g-1 tissue was excluded from analysis. Mean transmural blood flow <0.4 mL · min-1 · g-1 myocardium has been shown to correlate with severe functional impairment (akinesia or dyskinesia) of the involved myocardium26 and corresponds to the level of flow at which myocardial necrosis is first evident.27 28 We used this value as a cutoff for exclusion in other recent studies.18 29
An area at risk of 35% to 45% of the left ventricle is typical when the occlusive snare is placed around the proximal circumflex artery.24 Our protocol involved occlusion of the LAD after the first diagonal branch, which subtends considerably less myocardium than the large, dominant circumflex artery of the dog. The area at risk in this study (22%) is consistent with the findings of other investigators whose experimental protocol involved LAD occlusion distal to the first diagonal branch in a canine model.30 31 Although postmortem myocardial staining occurred at a relatively short interval (2 hours) after coronary occlusion, gross examination of myocardial slices revealed a clear demarcation between the ischemic, risk, and control regions, which was substantiated by regional myocardial blood flow measurements. The ability of TTC staining to differentiate reversibly from irreversibly injured myocardium has been validated histologically after a 2-hour period of reperfusion.32 Moreover, if TTC staining of irreversible myocardium had occurred, as has been suggested for short periods of reperfusion,33 we would have expected a lower infarct size as a percentage of the risk region in the present study compared with other studies using longer reperfusion periods, which was not the case.
Regional myocardial segmental shortening can be reliably measured with sonomicrometer crystals and provides an accurate assessment of ventricular function during coronary ischemia.34 35 We found a trend toward improved recovery of ischemic myocardial dysfunction with free PGE1 and TLC C-53 treatment when measured 1 and 2 hours after reperfusion. Absolute differences in segmental shortening during early reperfusion between the PGE1 and TLC C-53 groups probably reflect pretreatment differences in wall motion rather than a beneficial effect of the liposomal formulation of PGE1. A chronic animal experimental design permitting repeated measurements over 7 to 10 days would be necessary to detect significant differences in recovery from myocardial stunning. Hemodynamic parameters were similar for the three groups except for a transient but significant drop in mean arterial pressure and left ventricular end-diastolic pressure that occurred during the 10-minute infusion of TLC C-53 or free PGE1. This finding was surprising, in light of previous studies that showed more hypotension and tachycardia associated with free PGE1 compared with TLC C-5317 36 and similar hemodynamic parameters during bolus infusion of TLC C-53 or placebo.17 18 No adverse clinical events during the infusion period or excessive bleeding after infusion were noted with the administration of either TLC C-53 or PGE1 in this study.
Since the major determinants of infarct size, including the duration and severity of ischemia, collateral blood flow to the ischemic region, and the rate-pressure product (a measure of myocardial oxygen demand), were similar in the three treatment groups, it is likely that the mechanism of action of TLC C-53 involves attenuation of reperfusion injury, possibly through inhibition of white blood cell activation or infiltration into the ischemic myocardial region. Measuring the activity of the neutrophil-specific azurophilic granule, which contains myeloperoxidase enzyme, is a sensitive method for accurately quantifying leukocyte infiltration during ischemic myocardial injury.21 The significant reduction in myeloperoxidase activity found in the ischemic, risk, and border regions in the TLC C-53–treated dogs would indicate a reduction in the number of infiltrating neutrophils in the ischemic territory after reperfusion.
Although experimental models and clinical trials have established that prompt, effective, and sustained restoration of blood flow after coronary occlusion can limit infarct size, improve ventricular function, and enhance survival,37 38 39 40 41 42 43 44 45 reperfusion triggers an active inflammatory response associated with an intense neutrophilic infiltration of ischemic myocardium that may produce further tissue injury.9 46 47 Because of the difficulty in differentiating ischemic injury from additional tissue damage incurred upon reperfusion of jeopardized myocardium, the concept of reperfusion injury is not universally accepted.47 Recent evidence for programmed cell death determined by the presence of internucleosomal DNA fragmentation in ischemic/reperfused rabbit myocardial tissue, but not in those animals subjected to ischemia alone, has strengthened the concept that reperfusion may also have injurious effects on ischemic, potentially viable myocardium.48 Many potential mechanisms for reperfusion injury have been explored in animal models. Suggested mechanisms include free radical liberation by the endothelium and white blood cells, leukocyte plugging of the microvasculature, the oxygen and calcium paradox, and many others.23 32 49 50 51 52 53 Most recent evidence has emphasized the importance of activation of white blood cells and attachment and transmigration through the endothelium, followed by irreversible myocyte damage.32 54 55 56 Entman and colleagues56 showed that the leukocyte respiratory burst, which irreversibly injures the myocyte, is dependent on adhesion involving CD11b/CD18 and intercellular adhesion molecule-1 (CD54) and is a local phenomenon. Lefer and colleagues showed that blocking adhesion molecule expression or activity effectively reduces reperfusion injury.57 58 59 Recent preliminary work in our laboratory suggests that TLC C-53 treatment may result in suppression of ICAM-1 and P-selectin expression on ischemic endothelium in a similar 2-hour canine infarct-reperfusion model.60
Since the effects of PGE1 are myriad, other avenues for explanation of the cardioprotective effects of TLC C-53 are open to investigation. TLC C-53 inhibits cyclic flow variations after endothelial injury or coil insertion in canine coronary arteries, in association with significant inhibition of ex vivo platelet aggregation.18 36 In the present study, we were not able to demonstrate differences in microvascular blood flow to the infarct zone among treatment groups. Attenuation of the no-reflow phenomenon would thus be an unlikely mechanism for infarct salvage with TLC C-53 in this experiment.
Pharmacokinetics and Mechanism of Action of TLC C-53
PGE1 is a naturally occurring eicosanoid synthesized
in virtually all mammalian tissues. During intravenous
infusion, free PGE1 is extensively extracted after a single
passage through the lungs (60% to 90% inactivation in patients with
normal lung function) and is rapidly metabolized to
15-keto-13,14-dihydro-PGE1, resulting in a half-life
of the parent compound of 
30 seconds.61 Further
metabolism to 13,14-dihydro-PGE1, which
is as effective as PGE1 in lowering blood pressure in
rabbits,62 also occurs in humans.63 The
presence of active, circulating metabolites of PGE1 could
contribute to antiplatelet, antineutrophil, and vasodilating
properties that cannot be attributed to the parent compound because of
pharmacokinetic considerations.61
TLC C-53 is a stable preparation of PGE1 associated with phospholipid microspheres, which may alter drug transport. Intravenously administered liposomes remain intravascular and are removed predominantly by the reticuloendothelial system. Disruption of the endothelium may permit escape of the liposomes and their contents from the intravascular space and thus enhance delivery to sites of inflammation. The distribution and pharmacokinetics of liposomal compounds are complex and depend on dose, size, and chemical composition, especially lipid content.64 These and other factors will influence the release of the encapsulated pharmacological agents as well as the ability of liposomes to escape the vascular space during various pathological conditions. Unpublished pilot studies performed in male Sprague-Dawley rats suggest that although the mean concentration in plasma of PGE1 was higher for rats given identical doses of the liposomal formulation, TLC C-53, than those given free PGE1, the difference was not large, and the rate and extent of excretion of PGE1 metabolites appeared comparable for the two dosage forms. The elimination half-life in plasma for PGE1 administered as TLC C-53 was approximately 1 minute. In the present study, the free PGE1 and TLC C-53 infusions ended 2 to 5 minutes before the onset of reperfusion. As a result of its short half-life, free PGE1 may have disappeared from the systemic circulation by the time of reperfusion. In addition, the pharmacological effects of PGE1 may be enhanced by selective targeting of white blood cells and neutrophils by the liposomal preparation. Recent evidence suggests that liposomes preferentially target activated leukocytes and potentiate the anti-inflammatory effects of PGE1 on neutrophil function.16 Combined therapy with liposomes and PGE1 was found to prevent lethal endotoxemic shock in rats. The administration of liposomes alone was partially protective, whereas PGE1 given alone adversely affected mortality. Anti-inflammatory effects of prostaglandins mediated by a rise in intracellular cAMP may be potentiated by phagocytosis.65 66 The synergistic anti-inflammatory effect of liposomes and PGE1 on neutrophil function may account for the reduction in infarct size and neutrophil infiltration in the infarct zone with TLC C-53 compared with free PGE1 observed in our study.
Study Limitations
A limitation to the present study involves
the choice of an
acute open-chest experimental design for evaluation of
ventricular function. A chronic animal model may provide
more accurate and reproducible measurements of ventricular
function during an awake and lightly sedated state as well as permit
detection of possible differences in recovery from myocardial stunning
among treatment groups. Moreover, the validity of TTC staining to
determine the ultimate extent of myocardial injury after a 2-hour
period of reperfusion is controversial.32 33 It is
possible that TLC C-53 delayed the onset of reperfusion injury without
influencing the extent of myocardial damage.
In this study, the free PGE1 and TLC C-53 infusions ended 2 to 5 minutes before the onset of reperfusion. Because of its short half-life, free PGE1 may have disappeared from the systemic circulation by the time of reperfusion, whereas some of the administered dose of liposomal PGE1 may remain bound in the liposomes. Although the effects of PGE1 and those of its metabolites on cellular elements responsible for reperfusion injury may have persisted, an experimental protocol involving the continuous infusion of the pharmacological agents tested well into the reperfusion period might have been a better choice for demonstrating enhanced delivery of one preparation of PGE1 over the other. We chose a more clinically relevant bolus dosing regimen to evaluate the potential usefulness of TLC C-53 as an adjunctive agent in limiting reperfusion injury after thrombolysis in humans.
Conclusions
Bolus intravenous administration of TLC C-53
immediately before reperfusion results in significant infarct salvage
and reduced leukocyte infiltration compared with reperfusion alone or
identical doses of PGE1. Thus, TLC C-53 may be a useful
adjunctive therapy for the treatment of acute myocardial infarction
with potential for reducing infarct damage due to reperfusion injury
independent of its ability to accelerate thrombolysis and
prevent reocclusion, possibly extending the time window for
intervention.
|
Acknowledgments |
|---|
This research was funded in part by the Liposome Co, Princeton, NJ. We wish to thank Carnell Parks for his technical assistance.
|
Footnotes |
|---|
Guest editor for this article was Joseph Loscalzo, MD, Boston University Medical Center Hospital, Boston, Mass.
Received January 3, 1995; accepted February 8, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Smalling RW, Fuentes F, Matthews MW, Freund GC, Hicks CH, Reduto LA, Walker WE, Sterling RP, Gould KL. Sustained improvement in left ventricular function and mortality by intracoronary streptokinase administration during evolving myocardial infarction. Circulation. 1983;68:131-138.
2.
Sheehan FH, Braunwald E, Canner P, Dodge HT, Gore J,
Van Natta P, Passamani ER, Williams DO, Zaret B, and Co-investigators.
The effect of intravenous thrombolytic
therapy on left ventricular function: a report on
tissue-type plasminogen activator and
streptokinase from the Thrombolysis in Myocardial
Infarction (TIMI Phase I) trial.
Circulation. 1987;75:817-829.
3.
Weaver WD, Cerqueira M, Hallstrom AP, Litwin PE,
Martin JS, Kudenchuk PJ, Eisenberg M, for the Myocardial Infarction
Triage and Intervention Project Group. Prehospital-initiated
vs hospital-initiated thrombolytic therapy: the Myocardial
Infarction Triage and Intervention trial. JAMA. 1993;270:1211-1216.
4. Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico (GISSI). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet. 1986;1:397-401. [Medline] [Order article via Infotrieve]
5. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet. 1988;2:349-360. [Medline] [Order article via Infotrieve]
6. Wilcox RG, von der Lippe G, Olsson CG, Jensen G, Skene AM, Hampton JR for the ASSET Study Group. Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction: Anglo-Scandinavian Study of Early Thrombolysis (ASSET). Lancet. 1988;2:525-530. [Medline] [Order article via Infotrieve]
7. ISIS, GISSI, EMERAS, USIM, ISAM, and AIMS Collaborative Groups. Indications for fibrinolytic therapy in suspected acute myocardial infarction: collaborative overview of results on mortality and major morbidity from the major randomised trials. Lancet. 1994;343:311-322. [Medline] [Order article via Infotrieve]
8. Lincoff AM, Topol EJ. Illusion of reperfusion: does anyone achieve optimal reperfusion during acute myocardial infarction? Circulation. 1993;87:1792-1805.
9. Braunwald E, Kloner RA. Myocardial reperfusion: a double-edged sword? J Clin Invest. 1985;76:1713-1719.
10. Kloner RA, Ganote CE, Jennings RB. The `no-reflow' phenomenon after temporary coronary occlusion in the dog. J Clin Invest. 1974;54:1496-1508.
11.
Willerson JT, Watson JT, Hutton I, Templeton GH, Fixler
DE. Reduced myocardial reflow and increased coronary
vascular resistance following prolonged myocardial ischemia in
the dog. Circ Res. 1975;36:771-781.
12.
Kloner RA, Alker K, Campbell C, Figures G, Eisenhauer
A, Hale S. Does tissue-type plasminogen
activator have direct beneficial effects on the
myocardium independent of its ability to lyse
intracoronary thrombi?
Circulation. 1989;79:1125-1136.
13.
Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC.
Dissimilar effects of prostacyclin, prostaglandin
E1 and prostaglandin E2 on
myocardial infarct size after coronary occlusion in conscious
dogs. Circ Res. 1981;49:685-700.
14. Fantone JC, Kinnes DA. Prostaglandin E-1 and prostaglandin I2 modulation of superoxide production by human neutrophils. Biochem Biophys Res Commun. 1983;113:506-512. [Medline] [Order article via Infotrieve]
15.
Vaughan DE, Plavin SR, Schafer AI, Loscalzo J.
PGE1 accelerates thrombolysis by tissue
plasminogen activator.
Blood. 1989;73:1213-1217.
16.
Eierman DF, Yagami M, Erme SM, Minchey SR, Harmon PA,
Pratt KJ, Janoff AS. Endogenously opsonized
particles divert prostenoid action from lethal to protective in models
of experimental endotoxemia. Proc Natl Acad Sci
U S A. 1995;92:2815-2819.
17. Li G, Amirian J, Felli P, Echols B, Smalling RW. Attenuation of the no-reflow phenomenon and improved infarct salvage by liposome bound prostaglandin-E1 prior to reperfusion. Clin Res. 1992;40:155A. Abstract.
18. Feld S, Li G, Amirian J, Felli P, Vaughn WK, Accad M, Tolleson TR, Swenson C, Ostro M, Smalling RW. Enhanced thrombolysis, reduced coronary reocclusion and limitation in infarct size with liposomal prostaglandin E1 in a canine thrombolysis model. J Am Coll Cardiol. 1994;24:1382-1390. [Abstract]
19. Heymann MA, Payne BD, Hoffman JIE, Rudolph AM. Blood flow measurements with radionuclide-labeled particles. Prog Cardiovasc Dis. 1977;20:55-79. [Medline] [Order article via Infotrieve]
20. Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC, Corday E, Ganz W. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J. 1981;101:593-600. [Medline] [Order article via Infotrieve]
21. Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Methods. 1985;14:157-167. [Medline] [Order article via Infotrieve]
22.
Bolli R. Mechanism of myocardial `stunning.'
Circulation. 1990;82:723-738.
23.
Opie LH. Reperfusion injury and its
pharmacologic modification. Circulation. 1989;80:1049-1062.
24.
Reimer KA, Jennings RB, Cobb FR, Murdock RH, Greenfield
JC Jr, Becker LC, Bulkley BH, Hutchins GM, Schwartz RP Jr, Bailey KR,
Passamani ER. Animal models for protecting ischemic
myocardium: results of the NHLBI Cooperative Study:
comparison of unconscious and conscious dog models.
Circ Res. 1985;56:651-665.
25.
Christian TF, Schwartz RS, Gibbons RJ.
Determinants of infarct size in reperfusion therapy for acute
myocardial infarction. Circulation. 1992;86:81-90.
26.
Gallagher KP, Kumada T, Koziol JA, McKown MD, Kemper
WS, Ross J Jr. Significance of regional wall thickening
abnormalities relative to transmural myocardial perfusion in
anesthetized dogs. Circulation. 1980;62:1266-1274.
27. Vokonas PS, Malsky PM, Paul SJ, Robbins SL, Hood WB. Radioautographic studies in experimental myocardial infarction: profiles of ischemic blood flow and quantification of infarct size in relation to magnitude of ischemic zone. Am J Cardiol. 1978;42:67-75. [Medline] [Order article via Infotrieve]
28.
Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC.
Myocardial infarction in the conscious dog: three-dimensional
mapping of infarct, collateral flow and region at risk.
Circulation. 1979;60:1141-1150.
29. Feld S, Ekas RD, Felli P, Amirian J, Smalling RW. Differential effects of synchronized coronary sinus retroperfusion on regional myocardial function during brief occlusion of the left anterior descending and circumflex coronary arteries. Cathet Cardiovasc Diagn. 1994;32:70-78. [Medline] [Order article via Infotrieve]
30. Matsuda M, Catena TG, Vander-Heide RS, Jennings RB, Reimer KA. Cardiac protection by ischaemic preconditioning is not mediated by myocardial stunning. Cardiovasc Res. 1993;27:585-592. [Medline] [Order article via Infotrieve]
31.
Murry CE, Richard VJ, Jennings RB, Reimer KA.
Myocardial protection is lost before contractile function
recovers from ischemic preconditioning. Am J
Physiol. 1991;260:H796-H804.
32.
Litt MR, Jeremy RW, Weisman HF, Winkelstein JA, Becker
LC. Neutrophil depletion limited to reperfusion reduces
myocardial infarct size after 90 minutes of ischemia: evidence
for neutrophil-mediated reperfusion injury.
Circulation. 1989;80:1816-1827.
33.
Richard VJ, Murry CE, Jennings RB, Reimer KA.
Therapy to reduce free radicals during early reperfusion does
not limit the size of myocardial infarcts caused by 90 minutes of
ischemia in dogs. Circulation. 1988;78:473-480.
34. Smalling RW, Kelley KO, Kirkeeide RL, Gould KL. Comparison of early systolic and early diastolic regional function during regional ischemia in a chronically instrumented canine model. J Am Coll Cardiol. 1983;2:263-269. [Abstract]
35. Smalling RW, Ekas RD, Felli PR, Binion L, Desmond J. Reciprocal functional interaction of adjacent myocardial segments during regional ischemia: an intraventricular loading phenomenon affecting apparent regional contractile function in the intact heart. J Am Coll Cardiol. 1986;7:1335-1346. [Abstract]
36.
Willerson JT, Yao S-K, McNatt J, Cui K, Anderson HV,
Swensen C, Ostro M, Buja LM. Liposome-bound
prostaglandin E1 often prevents cyclic flow
variations in stenosed and endothelium-injured canine
coronary arteries. Circulation. 1994;89:1786-1791.
37.
Reimer KA, Lowe JE, Rasmussen MM, Jennings RB.
The wavefront phenomenon of ischemic cell death, I:
myocardial infarct size vs. duration of coronary
occlusion in dogs. Circulation. 1977;56:786-794.
38. Theroux P, Ross J Jr, Franklin D, Kemper WS, Sasayama S. Coronary arterial reperfusion, III: early and late effects on regional myocardial function and dimensions in conscious dogs. Am J Cardiol. 1976;38:599-606. [Medline] [Order article via Infotrieve]
39.
Baughman KL, Maroko PR, Vatner SF. Effects of
coronary artery reperfusion on myocardial infarct size and
survival in conscious dogs. Circulation. 1981;63:317-323.
40.
The GUSTO Angiographic Investigators. The
effects of tissue plasminogen activator,
streptokinase, or both on coronary artery patency,
ventricular function, and survival after acute myocardial
infarction. N Engl J Med. 1993;329:1615-1622.
41.
Stadius ML, Davis K, Maynard C, Ritchie JL, Kennedy JW.
Risk stratification for 1 year survival based on characteristics
identified in the early hours of acute myocardial infarction.
Circulation. 1986;74:703-711.
42. Sheehan FH, Braunwald E, Canner P, Dodge HT, Gore J, Van Natta P, Passamani ER, Williams DO, Zaret B, and Co-investigators. The effect of intravenous thrombolytic therapy on left ventricular function: a report on tissue-type plasminogen activator and streptokinase from the Thrombolysis in Myocardial Infarction (TIMI Phase I) trial. Circulation. 1987;75:817-829.
43.
Anderson JL, Karagounis LA, Becker LC, Sorensen SG,
Menlove RL, for the TEAM-3 Investigators. TIMI perfusion grade 3
but not grade 2 results in improved outcome after
thrombolysis for myocardial infarction: ventriculographic,
enzymatic and electrocardiographic evidence from the TEAM-3
study. Circulation. 1993;87:1829-1839.
44. Serruys PW, Simoons ML, Suryapranata H, Vermeer F, Wijns W, van den Brand M, Bar F, Zwaan C, Krauss XH, Remme WJ, Res J, Verheugt FWA, van Domburg R, Lubsen J, Hugenholtz PG, for the Working Group on Thrombolytic Therapy in Acute Myocardial Infarction of the Netherlands Interuniversity Cardiology Institute. Preservation of global and regional left ventricular function after early thrombolysis in acute myocardial infarction. J Am Coll Cardiol. 1986;7:729-742. [Abstract]
45. Belenkie I, Thompson CR, Manyari DE, Knudtson ML, Duff HJ, Poon M-C, Smith ER. Importance of effective, early and sustained reperfusion during acute myocardial infarction. Am J Cardiol. 1989;63:912-916. [Medline] [Order article via Infotrieve]
46.
Go LO, Murry CE, Richard VJ, Weischedel GR, Jennings
RB, Reimer KA. Myocardial neutrophil accumulation during
reperfusion after reversible or irreversible ischemic
injury. Am J Physiol. 1988;255:H1188-H1198.
47. Forman MB, Virmani R, Puett DW. Mechanisms and therapy of myocardial reperfusion injury. Circulation. 1990;81(suppl IV):IV-69-IV-78.
48. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994;94:1621-1628.
49.
Chi L, Tamura Y, Hoff PT, Macha M, Gallagher KP, Schork
MA, Lucchesi BR. Effect of superoxide dismutase on myocardial
infarct size in the canine heart after 6 hours of regional
ischemia and reperfusion: a demonstration of myocardial
salvage. Circ Res. 1989;64:665-675.
50.
Przyklenk K, Kloner RA. `Reperfusion injury'
by oxygen-derived free radicals? Effect of superoxide dismutase plus
catalase, given at the time of reperfusion, on myocardial infarct size,
contractile function, coronary microvasculature, and regional
myocardial blood flow. Circ Res. 1989;64:86-96.
51.
Bolli R, Jeroudi MO, Patel BS, Aruoma OI, Halliwell B,
Lai EK, McCay PB. Marked reduction of free radical generation
and contractile dysfunction by antioxidant therapy begun at the time of
reperfusion: evidence that myocardial `stunning' is a
manifestation
of reperfusion injury. Circ Res. 1989;65:607-622.
52.
Ambrosio G, Weisfeldt ML, Jacobus WE, Flaherty JT.
Evidence for a reversible oxygen radical-mediated component of
reperfusion injury: reduction by recombinant human superoxide dismutase
administered at the time of reflow.
Circulation. 1987;75:282-291.
53.
Tamura Y, Chi L, Driscoll EM Jr, Hoff PT, Freeman BA,
Gallagher KP, Lucchesi BR. Superoxide dismutase conjugated to
polyethylene glycol provides sustained protection against myocardial
ischemia/reperfusion injury in canine heart.
Circ Res. 1988;63:944-959.
54. Engler RL, Schmid-Schonbein GW, Pavelec RS. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol. 1983;111:98-111. [Abstract]
55. Simpson PJ, Todd RF III, Fantone JC, Mickelson JK, Griffin JD, Lucchesi BR. Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-Mol, anti-CD11b) that inhibits leukocyte adhesion. J Clin Invest. 1988;81:624-629.
56. Entman ML, Youker K, Shoji T, Kukielka G, Shappell SB, Taylor AA, Smith CW. Neutrophil induced oxidative injury of cardiac myocytes: a compartmented system requiring CD11b/CD18-ICAM-1 adherence. J Clin Invest. 1992;90:1335-1345.
57.
Ma X-l, Weyrich AS, Lefer DJ, Buerke M, Albertine KH,
Kishimoto TK, Lefer AM. Monoclonal antibody to L-selectin
attenuates neutrophil accumulation and protects ischemic
reperfused cat myocardium.
Circulation. 1993;88:649-658.
58. Weyrich AS, Ma X-l, Lefer DJ, Albertine KH, Lefer AM. In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest. 1993;91:2620-2629.
59. Ma X-l, Tsao PS, Lefer AM. Antibody to CD-18 exerts endothelial and cardiac protective effects in myocardial ischemia and reperfusion. J Clin Invest. 1991;88:1237-1243.
60. Smalling R, Uthman M, Ramanna N, Yeh E, Amirian J, Felli P, Feld S, Accad M, Concoff A, Smith CW, Entman M, Janoff A. Suppression of ICAM-1 and P-selectin adhesion molecule expression by bolus iv liposomal PGE1 (TLC C-53) immediately prior to reperfusion in a two hour canine infarct/reperfusion model. J Am Coll Cardiol. 1995;249A. Abstract.
61. Simmet TH, Peskar BA. Prostaglandin E1 and arterial occlusive disease: pharmacological considerations. Eur J Clin Invest. 1988;18:549-554. [Medline] [Order article via Infotrieve]
62. Anggard E, Larsson C. The sequence of the early steps in the metabolism of prostaglandin E1. Eur J Pharmacol. 1971;14:66-70. [Medline] [Order article via Infotrieve]
63. Peskar BA, Hesse WH, Rogatti W, Diehm C, Rudofsky G, Schweer H, Seyberth HW. Formation of 13,14-dihydro-prostaglandin E1 during intravenous infusions of prostaglandin E1 in patients with peripheral arterial occlusive disease. Prostaglandins. 1991;41:225-228. [Medline] [Order article via Infotrieve]
64. Hwang KJ. Liposome pharmacokinetics. In: Ostro MJ, ed. Liposomes: From Biophysics to Therapeutics. New York, NY: Marcel Dekker; 1987:109-156.
65. Weissmann G, Zurier RB, Spieler PJ, Goldstein IM. Mechanisms of lysosomal enzyme release from leukocytes exposed to immune complexes and other particles. J Exp Med. 1971;134:149s-165s.
66. Zurier RB, Weissmann G, Hoffstein S, Kammerman S, Tai HS. Mechanisms of lysosomal enzyme release from human leukocytes. J Clin Invest. 1974;53:297-309.
This article has been cited by other articles:
 |
|
 |
|
  X Liu, Z Zhou, X Feng, Z Jia, Y Jin, and J Xu Cyclooxygenase-2 plays an essential part in cardioprotection of delayed phase of recombinant human erythropoietin preconditioning in rats. Postgrad. Med. J., September 1, 2006; 82(971): 588 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Calabresi, G. Rossoni, M. Gomaraschi, F. Sisto, F. Berti, and G. Franceschini High-Density Lipoproteins Protect Isolated Rat Hearts From Ischemia-Reperfusion Injury by Reducing Cardiac Tumor Necrosis Factor-{alpha} Content and Enhancing Prostaglandin Release Circ. Res., February 21, 2003; 92(3): 330 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bolli, K. Shinmura, X.-L. Tang, E. Kodani, Y.-T. Xuan, Y. Guo, and B. Dawn Discovery of a new function of cyclooxygenase (COX)-2: COX-2 is a cardioprotective protein that alleviates ischemia/reperfusion injury and mediates the late phase of preconditioning Cardiovasc Res, August 15, 2002; 55(3): 506 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. McCrory and S. G. E. Lindahl Cyclooxygenase Inhibition for Postoperative Analgesia Anesth. Analg., July 1, 2002; 95(1): 169 - 176. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Shinmura, X.-L. Tang, Y. Wang, Y.-T. Xuan, S.-Q. Liu, H. Takano, A. Bhatnagar, and R. Bolli Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits PNAS, August 29, 2000; 97(18): 10197 - 10202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-J. Rupprecht, J. v. Dahl, W. Terres, K. M. Seyfarth, G. Richardt, H.-P. Schulthei{beta}, M. Buerke, F. H. Sheehan, and H. Drexler Cardioprotective Effects of the Na+/H+ Exchange Inhibitor Cariporide in Patients With Acute Anterior Myocardial Infarction Undergoing Direct PTCA Circulation, June 27, 2000; 101(25): 2902 - 2908. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||