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Influence of sulfinpyrazone and naproxen on infarct size in the dog
Influence of Sulfinpyrazone and Naproxen on Infarct Size in the Dog ROBERTO BOLLI, MD‘ ROBERT E. GOLDSTEIN, MD. FACC NANCY DAVENPORT, RN, PhD+ STEPHEN E. EPSTEIN, MD, FACC Bethesda. Maryland
From ths cardiology Branch, National Heart, Lung, and Blood Instiie, National Institutes of Health, Bethesda, Marylend. Manuscript received June 30, 1980; revised manuscript received November 3, 1980. accepted November 7, 1980. Resberch Felbw, National institutesof Health. Bethesda, Maryland, at the time of this work. + Reclplent of Public Hea!th Service Research Fellowship !X32-NUO5170-01 from the U. S. Public Healtf~ Service, Bethesda, Maryland. Address for reprlnts: Roberto Bolli, MD, Cardiobgy Branch, Bullding 10. Room 7515. National lnstltutes of Heatth. Bethesda, Maryland 20205. l
To assess the potential role of platelet inhibitory agents in the treatment of myocardial infarction, the effect on infarct size of two platelet lnhibftors, suifinpyrazone and naproxen, was evaluated. In addition to platelet inhibition, sulfinpyrarone increases epicardial collateral flow and naproxen has lysosomal-stabilizing activity. Thirty-eight open chest dogs were given intravenously sulfinpyrazone (30 mg/kg, n = ll), naproxen (30 mg/kg, n = 14) or saline solution (n = 13) 10 minutes before and 3 and 6 hours after ligation of the mid iefl anterior descending coronary artery. Drug doses were sufficient to inhibit adenosine diphosphate-Induced platelet aggregation. The dogs were killed 72 hours after occlusion. Myocardium at risk of infarction-that is, the area supplied by the occluded artery (anatomic risk area)-was identlfied by simuftaneous perfusion of the aortic root with Evans blue and of the coronary artery distal to the occlusion with clear saline solution. Hearts were sliced horizontally and stained with triphenyi-tetrazolium-chloride. The infarcted area and anatomic risk area were measured with videoplanimetry. The percent of left ventricle infarcted was not significantly different among the control, sulfinpyrazone and naproxen groups (28 f 2, 30 f 1, 28 f 2 percent, respectively) nor was percent of anatomlc risk area infarcted significantly different (75 f 3, 79 f 3, 75 f 3 percent, respectively). Thus, neither suifinpyratone nor naproxen in platelet inhibitory doses altered infarct size. These results indicate that neither inhibitory effects on platelet function and prostaglandin synthesis nor associated lysosomai-stabflizlng properties identify agents with consistent infarct-sparing action.
Recently much attention has been focused on the role of platelets in coronary artery disease and the potential therapeutic efficacy of platelet of atherosclerotic inhibitory drugs. l-s Platelets may hasten development deposits or augment effects of preexisting coronary arterial narrowing. In addition, platelets may exacerbate the consequences of coronary occlusion either by aggregation within microvessels carrying collateral flow or by release of thromboxanes or other vasoactive materials. Hence, the hypothesis has been advanced that agents interfering with platelet function might reduce the extent of myocardial infarction after coronary occlusion.4J Recent reports of reduction in infarct size after coronary ligation in rats6v7 and dogssv” treated with ibuprofen, a potent platelet inhibitor, favor this hypothesis. In order to explore the hypothesis more completely, we evaluated the capability of two other platelet inhibitors, sulfinpyrazone and naproxen, to alter infarct size in a canine model similar to that used in studies of ibuprofen. Sulfinpyrazone has certain features of particular interest. In addition to interfering with platelet aggregationi possibly by inhibition of prostaglandin synthesis,” sulfinpyrazone also appears to interfere with platelet-endothelial interaction. l2 In the context of acute myocardial ischemia pretreatment with sulfinpyrazone has been found to improve epicardial collateral blood flow’s and to reduce the electrocardiographic evidence of ischemic injury14 as well as the incidence of ventricular fi-
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brillation.14 Reports of clinical trials of this drug have indicated beneficial effects in patients with coronary artery disease.*J Thus, assessment of the influence of platelet-inhibitory doses of sulfinpyrazone on infarct size after coronary occlusion seemed particularly useful. Naproxen, like ibuprofen, is a nonsteroidal anti-inflammatory agent derived from propionic acid. Naproxen shares many pharmacologic properties with ibuprofen: inhibition of platelet aggregation, interference with prostaglandin synthesis and lysosomal stabilizing activity.lsi2* However, it appears to have greater lysosomal stabilizing efficacy than ibuprofen.18Jg Thus, assessment of infarct-sparing potential of naproxen would be of value in estimating the role of these shared properties in minimizing myocardial necrosis. Methods Platelet Aggregation The effect of intravenous naproxen on in vitro platelet aggregation was studied in a separate series of five dogs; analogous data for sulfinpyrazone were available from previous studies.‘OJ” To assess the platelet-inhibitory properties of differing doses, increasing amounts (3,10,30 and 100 mg/kg body weight) of naproxen were given intravenously; blood samples for aggregation studies were drawn 10 minutes after each dose. In two dogs given 30 mg/kg aggregation studies were repeated 24 hours after administration of naproxen. The aggregation techniques used have previously been presented.*“,1”p2” In brief, aggregation was quantitated as change in optical transmission induced by addition of adenosine diphosphate to platelet-rich plasma under carefully controlled circumstances. Optical transmission was defined as 0 percent for platelet-rich plasma and 100 percent for platelet-poor plasma.
samples for oxygen saturation and hematocrit determination were obtained by left atria1 puncture. The left anterior descending coronary artery was isolated from the surrounding tissues and a snare placed around it. The site of occlusion of the left anterior descending coronary artery was chosen to produce ischemia in a zone approximating the apical half of the distribution of this vessel. Four electrodes mounted 5 mm apart on Dacron@ fabric were placed on the epicardial surface of the potentially ischemit area (Fig. l), and unipolar electrograms were recorded by a multichannel direct-writing recorder. The height of S-T segment was measured 100 ms after the onset of the QRS deflection. Sites showing intraventricular conduction abnormalities (that is, delay of the intrinsic deflection exceeding 40 ms or prolongation of the QRS complex beyond 65 ms) were excluded from the analysis. Two successive occlusions were carried out. The purpose of the first occlusion was selection of hearts with unequivocal myocardial ischemia. Electrograms were recorded 2 minutes and, if ischemia was not evident, 5 minutes after coronary occlusion; only dogs having S-T segment elevation (a sum total of at least 10 mV in the four leads) were admitted to study. The occlusion was then released. Two minutes later dogs were randomly assigned to three groups: naproxen ( 14 dogs, 30 mg/kg intravenously), sulfinpyrazone (1 I dogs, 30 mg/kg intravenously) or normal saline solution (13 dogs, 30 ml intravenously). Treatment was given 10 minutes before the second occlusion and repeated 3 and 6 hours later. The thorax was closed shortly after occlusion and the dogs were allowed to awaken. No attempt was made to suppress arrhythmic activity occurring after ligation. Blood samples for naproxen determinations were obtained 15 minutes after the first dose and immediately before the second and third doses. Plasma naproxen levels were determined by a gas-liquid chromatographic technique.*
Infarct Size Experimental preparation: Forty-one dogs of either sex, weighing 20 to 32 kg, were anesthetized with sodium thiamylal (25 mg/kg intravenously) and ventilated with room air. The heart was exposed under sterile conditions thoracotomy and suspended in a pericardial
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a left Blood
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FIGURE 1. Left lateralview of the heart showing the site of occlusion of the left anterior descending (LAD) coronary artery and the method used to obtain eplcardial electrograms from the ischemlc area. ECG = ektrocardiogram; LA = left atrium: LV = left ventricle: RV = right ventricle.
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Identification and measurement of region at risk of infarction: Approximately 72 hours after occlusion, dogs received heparin (15,000 U intravenously) to prevent clotting in the coronary vasculature; immediately afterward, they were killed with lethal doses of sodium pentobarbital and the hearts removed. The region at risk of infarction (that is, the portion of left ventricle normally supplied by the occluded artery) was identified as previously describ+dz4 In summary, one cannula was inserted into the left anterior descending coronary artery just below the site of ligation and a second cannula was inserted into the aortic root. The arterial bed distal to the occlusion was then perfused with colorless fluid (dextran 6 percent in saline solution) while the aortic root was simultaneously perfused with 1 percent Evans blue in the same fluid. Equal physiologic perfusion pressures were applied to the aortic and distal left anterior descending coronary arterial cannulas to prevent flow across any collateral vessels, either present prior to occlusion or developed after occlusion. As a result of this procedure, the myocardium normally perfused by the occluded portion of the coronary artery (subsequently termed anatomic region at risk of infarction) remained unstained, while the rest of the heart was stained blue. The risk region, as defined in this study, is an anatomic designation of the collateral-dependent portion of the heart. Our technique does not specify the competence of collateral vessels or the metabolic demands of the myocardium, factors that determine the actual amount of necrosis within the risk region. The heart was then sliced from apex to base into 1 cm thick slices in
’ These determinations were performed by Dr. S. l-l. Wan, University of California. Los Angeles.
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FlGURE 2. Anterior surface of the heart after perfusion of aortic root with Evans blue solution and of distal left anterior descending coronary artery with fluorescein solution. A, ordinary light. B, ultraviolet light. Note the reciprocal relation between Evans blue-stained (black) and fluorescein-stained (pale gray) myocardium.
planes parallel to the atrioventricular groove and the border between stained and unstained tissue-assumed to be the limit of the region at risk- was marked by a small incision on each cut surface. To verify that this border coincided with that of the myocardium actually perfused by uncolored dextran in saline solution, in a series of six hearts the staining technique described was modified in that the distal left anterior descending coronary artery was perfused with 0.1 percent sodium fluorescein in dextran in saline solution; perfusion of myocardium by this solution results in a distinct fluorescence under ultraviolet light without appreciable discoloration under ordinary light. Comparative examination of hearts with ordinary and ultraviolet light revealed a coincidence between the border of Evans blue-stained myocardium (region not at risk) and that of fluorescent-stained myocardium (region at risk) (Fig. 2 and 3), thus confirming the assumption on which our staining technique was based. Estimationof infarct size: The right ventricular free wall was then removed. To identify the infarct within the anatomic region at risk, slices were then weighed and incubated for 60 minutes in triphenyl-tetraxolium-chloride solution; this agent stains only viable, dehydrogenase-containing myocardium dark red, as described by Lie et als5 In the series of fluorescent-stained hearts, the nonfluorescent (and thus non-
perfused) portion of the anatomic region at risk coincidedwith the tetrasolium-negative (infarcted) tissue (Fig. 3, B and C). These findings indicate that, in the myocardium at risk, the presence of fluorescence detected by this technique is a reliable indicator of tissue survival. Lack of fluorescenceindicates nonperfusiondespite infusionof fluorescein into the coronary artery distal to the site of occlusion;this probably reflects high vascular resistance due to microvascular damage within infarcted myocardium. The weight of the anatomic region at risk and of the infarct was determined by photographing slices, projecting transparencies onto paper, and tracing portions at risk and necrotic portions. Each area was measured with videoplanimetry and the mass of the region at risk and the infarct mass were expressed as fractions of the weight of each slice. Total infarct weight was calculated as percent both of
the left ventricle and of the myocardium at risk. The latter estimate is of greater relevance, because the amount of necrotic tissue produced by coronary occlusion correlates directly with the magnitude of the arterial bed rendered ischemic.24*26v27Estimation of infarct weight as a percent of myocardium at risk also enables determination of whether the effect of a given intervention on infarct size varies with the size of the occluded coronary bed. Statisticalanalysis:One-way analysis of variance was used for statistical evaluation of the results. Values are reported as mean f standard error of the mean.
Results Platelet aggregation: In vitro adenosine diphosphate (ADP)-induced platelet aggregation showed a dose-related inhibition after intravenous administration of naproxen (Fig. 4). Platelet response, expressed as maximal percent light transmission in the presence of 2.34 X 10e4 M ADP, averaged 86 f 5 percent in control samples, was slightly decreased after 3 mg/kg (64 f 6 percent), but markedly inhibited after 10 mg/kg (19 f 4 percent) and 30 mg/kg (19 f 8 percent); the effect of the latter dose was still evident after 24 hours (30 f 12 percent). Each naproxen result was significantly different (p <0.05) from the control value. Larger doses (100 mg/kg) were associated with serious adverse effects (marked hemolysis, vomiting, neurologic symptoms). Similar degrees of inhibition were observed in the response to 2.34 X 10e5 A4 ADP. General observations: Arterial oxygen saturation and hematocrit, determined before the temporary coronary occlusion, were within normal limits in every dog. (A mild hemolysis was often observed in blood samples obtained for naproxen determination before the second and third doses of the drug; however, this did not cause any decrease of hematocrit below basal val-
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ues.) The mean heart rate at the time of the permanent occlusion was similar among control, naproxen-treated and sulfinpyrazone-treated dogs (152 f 6,137 f 9 and 148 f 9 beats/min, respectively). Left ventricular weight was 113 f 7 g in the control, 107 f 5 g in the naproxentreated and 106 f 4 g in the sulfinpyrazone-treated group. These values were not significantly different. Because the site of occlusion was based on the distribution of the left anterior descending coronary artery rather than on a fixed anatomic criterion, very similar fractions of the left ventricle were jeopardized by the occlusion (Fig. 5, left); the amount of myocardium at
risk was 37 f 2 percent (42 f 4 g) of the left ventricle in the control, 37 f 2 percent (40 f 3 g) in the naproxentreated and 39 f 2 percent (41 f 3 g) in the sulfinpyrazone-treated dogs. Three dogs were excluded from study before randomization because of insufficient S-T segment elevation (EST less than 10 mV) after the temporary coronary occlusion. Two (15 percent) of the 13 control dogs, 3 (21 percent) of the 14 naproxen-treated, and 1 (9 percent) of the 11 sulfinpyrazone-treated dogs manifested ventricular fibrillation within the initial 20 minutes of ischemia. These values did not differ sig-
FIGURE 3. Myocardial slice obtained from the heart shown in Figure 2. A, ordinary light. B. ultraviolet light. As in Figure 2, a reciprocal relation is observed between Evans blue-stained (black) and fluorescein-stained (pale gray) myocardium; the latter forms the outer boundary of the region at risk of infarction. Arrows indicate the border of fluorescent tissue. C after staining with triphenyltetraroliurn chloride. The shallow incisions (indicated by the pins) mark the borders of the region at risk. Comparison of B with C shows that the nonfluorescent portion of the region at risk is tetrazoliumnegative (white or pale gray) and thus necrotic, whereas the fluorescent part is tetrazoliumpositive (dark gray) and thus viable. 0, schematic digam illustratingB and C. Hetchsd area = normal myocardium; doMd erea = Infarct; white area sufroundln9 doned area = survivingmyocardium within the region at risk.
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nificantly. One control and one naproxen-treated dog died between 6 and 72 hours of occlusion, presumably of arrhythmias. Postmortem examination revealed no other apparent cause of death. This left 10 dogs for sacrifice in each treatment group. The plasma naproxen level was 153 f 10 I*g/ml (n = 9) 15 minutes after administration of the first dose; immediately before the second and third doses, the levels were 92 f 5 pg/ml (n = 8) and 108 f 7 pg/ml (n = 8), respectively. Infarct size: The control, naproxen-treated and sulfinpyrazone-treated groups did not show significant differences in infarct size expressed either as a percent of the left ventricle (28 f 2,28 f 2 and 30 f 1 percent, respectively; Fig. 5, middle), or as a percent of the myocardium at risk (75 f 3,75 f 3 and 79 f 3 percent, respectively, Fig. 5, right). For each of the three treatment groups the magnitude of the region of necrosis maintained a similar relation to the size of the occluded coronary beds (Fig. 6). Thus, in our dogs with documented ischemia, treatment before occlusion with either naproxen or sulfinpyrazone failed to reduce infarct size, irrespective of the size of the anatomic region at risk. Our data confirm previous studies24y26y27showing a linear correlation between infarct size and mass of myocardium at risk (r = 0.72, Fig. 6).
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FIGURE 4. Platelet aggregation 10 minutes after different intravenous doses of naproxen (NAP). Aggregation is quantitated as change in percent optical transmission induced by addition of adenosine diphosphate (final concentration 2.34 X low4 M) to platelet-rich plasma (0 percent corresponds to transmission through platelet-rich plasma and 100 percent to transmission through platelet-poor plasma). Results are expressed as mean f standard error of the mean. n = number of dogs studied. Asierlsks denote significant differences (p <0.05) from control (C).
Discussion
The results of our study demonstrate that neither naproxen nor sulfinpyrazone, administered in effective platelet-inhibitory doses, alters infarct size in the dog. Similar portions of left ventricle and, more importantly, similar portions of anatomic region at risk of infarction underwent necrosis in untreated and treated animals. “Platelet hypothesis”: The role of platelets in the evolution of ischemic injury after acute coronary occlusion is unclear. Accumulation of platelets in the ischemic area, particularly at the lateral margins, has been reported after coronary ligation in primates5 and induced coronary thrombotic obstruction in dogs.4 In the latter model, diffuse obstruction of the ischemic microvasculature by platelet aggregates was prevented
by aspirin. On the basis of these findings it has been hypothesized that platelet-mediated microcirculatory thrombosis reduces collateral flow to the ischemic zone, and therefore augments the extent of myocardial damage.4*5 However, subsequent experiments failed to show increased platelet deposition within infarcts in dogs undergoing nonthrombotic coronary occlusion.14*2s
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FIGURE 5. Weight of myocardium at risk as peroent of left ventricular mass (RISK/LV, left), weight of infarct as percent of left ventricular mass (MI/LV. mfddfe) and weight of infarct as percent of myocardium at risk (MI/RISK. right). C = control: NAP = naproxen-treated; SPZ = sulfinpyrazone-treated. Results are expressed as mean f standard error of the mean. Differences among the three groups are not significant.
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FIGURE 6. Myocardial infarct (MI) size as a percent of the left ventricle (LV) plotted as a function of the region at risk of infarction. C = control; NAP = naproxen-treated; SPZ = sulfinpyrazone-treated. The common linear relation observed for all groups is indicated by the regression line, which is calculated from individual data points from all groups.
Moreover, if the extent of ischemic necrosis is significantly enhanced by platelet aggregates, one would expect such necrosis to be limited by platelet-inhibitory drugs. However, aspirin, in platelet-inhibitory doses,24 and meclofenamic acid2g failed to reduce ischemic damage, and indomethacin enlarged the region of damage.27 Of the platelet-inhibitory drugs evaluated to date, only ibuprofen and its halogenated derivative fluribuprofen have been reported to diminish the extent of ischemic necrosis.6-g,30*31 Our protocol was designed to ensure effective platelet inhibition in dogs during the first hours of ischemia. Sulfinpyrazone, 30 mg/kg intravenously, was shown in previous experiments to abolish in vitro ADP-induced aggregation of dog platelets,13 and the current study indicated that naproxen, 30 mg/kg intravenously, also produced marked inhibition of platelet aggregation. With either drug, inhibition of platelet function was demonstrable 10 minutes after administration. Because we administered either drug 10 minutes before, and 3 and 6 hours after coronary occlusion, effective platelet inhibition was achieved at the onset of ischemia and maintained for at least several hours. Despite this, no change in infarct size was observed. Therefore, our data suggest that in this model (1) platelet inhibition in itself does not result in preservation of ischemic myocardium, and (2) platelet accumulation in the ischemic zone does not appear to play a significant role early in the course of myocardial infarction. Sulfinpyrazone: Another study from this laboratory13 showed that pretreatment with sulfinpyrazone (30 mg/kg intravenously) augments mean epicardial collateral flow both at 5 minutes and at 4 hours after coronary ligation in open chest dogs. However, the present study demonstrates that this potentially beneficial drug fails to reduce infarct size in a similar model, possibly
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because larger changes in collateral circulation are necessary to produce significant salvage of ischemic myocardium in open chest dogs. A reduction in the electrocardiographic evidence of ischemic injury that occurs acutely after coronary occlusion has been reported by Moschos et a1.14 in open chest dogs pretreated with sulfinpyrazone for 7 days. Although differences due to the prolonged pretreatment cannot be excluded, our data indicate that treatment with sulfinpyrazone, despite any acute electrophysiologic effects, does not reliably lead to a reduction in actual ischemic necrosis. Nevertheless, the increase in epicardial collateral blood flow13 and reduction in electrocardiographic evidence of injury14 may be related and possibly responsible for the antiarrhythmic effect of the drug that was also observed during acute myocardial ischemia.14 In our model the incidence of early ventricular fibrillation was too small to afford any inference about differences between treated and control dogs. Naproxen: Naproxen has a long plasma half-life in dogs (36 hours after intravenous injection32). Because we used a dose 10 times greater than that sufficient to produce significant platelet inhibition (Fig. 4), it seems reasonable to assume that pharmacologically effective levels of the drug were present in the blood throughout the 72 hours of coronary occlusion. This assumption is also supported by the marked platelet inhibition observed 24 hours after administration of naproxen. Naproxen resembles ibuprofen more closely than other nonsteroidal antiinflammatory agents in that (1) it stabilizes lysosomal membranes in vitro,18plg and (2) it inhibits inflammation, platelet aggregation and prostaglandin synthesis with a potency similar to that of ibuprofen.15-17*20-22 Th ese derivatives of propionic acid differ in that naproxen shows a more pronounced in vitro lysosomal stabilizing activity than does ibuprofen.lssls Lysosomal-stabilizing effect may be beneficial in acute myocardial ischemia during which activation of lysosomal hydrolases has been thought to play a significant role in cell death,33 although recent studies question the validity of this concept.34 Despite their close similarity the two drugs have different effects on myocardial ischemia. Naproxen failed to salvage ischemit myocardium in our study, whereas other investigations have shown that ibuprofen reduced ischemic injury in cats30 as well as infarct size in rats6,7 and dogs.8pg Differences in experimental models might account for such discrepancies. Infarct size was determined by Ribeiro et a1.8 after 24 hours of ischemia compared with 72 hours in the present study. It is therefore possible that ibuprofen (and possibly naproxen) might simply delay necrosis rather than prevent it. However, ibuprofen’s infarct-sparing action was demonstrable even 21 days after coronary arterial occlusion in rats7 In contrast, Jugdutt et a1.gstudied small infarcts (less than 15 percent of the left ventricle) in a closed chest model. Thus, the discrepancies cited could be explained if both ibuprofen and naproxen spared only small infarcts, in which the potentially salvageable border zone may be larger than the nonsalvageable center zone.
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Although any of these considerations is possible, it appears likely that naproxen lacks the infarct-sparing action of ibuprofen. Our data, therefore, suggest that properties other than those common to both ibuprofen and naproxen account for ibuprofen’s beneficial effect on myocardial ischemia. This inference is also supported by the failure to reduce ischemic damage seen with other nonsteroidal anti-inflammatory drugs (aspirin,24,2g meclofenamic acid2g and indomethacin27) that share an ability to block prostaglandin synthesis and platelet function.
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Implications: Our data provide evidence that antiinflammatory action, platelet inhibition, inhibition of prostaglandin synthesis and lysosomal-stabilizing activity are not in themselves sufficient to confer infarct-sparing action. Further investigation is needed to identify drug mechanisms that reliably lead to reduction in infarct size. Acknowledgment We thank John Bather, DVM, who kindly provided logistic support for sterile surgery. The technical assistance of Filippina Giacometti and Kirsti Kile is gratefully acknowledged.
References 1. Anturane Reinfarction Trial Research Group. Sulfinpyrazone in the prevention of cardiac death after myocardial infarction. N Engl J Med 1978;198:289-95. 2. Anturane Reinfarction Trial Research Group. Sulfinpyrazone in the prevention of sudden death after myocardial infarction. N Engl J Med 1980;302:250-8. 3. Braunwaid E. Treatment of the patient after myocardial infarction. The past decade and the next. N Engl J Med 1980;302:290-3. 4. Moschos CB, Lahlrl K, Lyons M, W&se AB, Oldewurtel HA, Regan RJ. Relation of microcirculatory thrombosis to thrombus in the proximal coronary artery: effect of aspirin, dipyridamole. and thrombolysis. Am Heart J 1973;88:61-8. 5. Rut W, McNamara JJ, Suehlro A, Suehlro G, Wkkllne SA. Platelet trapping in myocardial infarct in baboons: therapeutic effect of aspirin. Am J Cardiol 1980;46:405-12. 6. MacLean D, Flshbeln MC, Maroko PR, Braunwald E. Long-term salvage of ischemic myocardium by depleting catecholamin& and inhibiting inflammation (abstr). Clin Res 1977:25:455A. 7. MacLeai D, Ffahbeln mC, Bkan RI, Braunwald E, Maroko PR. Lor@erm preservation of ischemic myocardium by ibuprofen after experimental coronary artery occlusion (abstr). Am J Cardiol 1978;44:394 8. Rlbefro LOT, Yaeuda 1, Lowensfeln E, Braunwald E, Yaroko PR. Comparative effects on anatomic infarct size of verapamil, ibuprofen, and morphine-promethazine-chlorpromazine combination (abstr). Am J Cardiol 1979;43:396. 9. Jugdutt BI, Hutchlns GM, Bulkley BH, Becker LC. Salvage of ischemic myocardium by ibuprofen during infarction in the conscious dog. Am J Cardiol 1979;46:74-82. 10. Goldsteln RE, Davenport N, Llpson LC, et al. Relative effects of sulfinpyrazone and ibuprofen on canine platelet function and prostaglandin-mediated vasodilation. J Cardiovasc Pharm 1980; 2:399-409. 11. All M, McDonald JWD. Effects of sulfinpyrazone on platelet prostaglandin synthesis and platelet release of serontonin. J Lab Clin Med 1975;89:868-75. 12. Packham MA, Warrler ES, Glynn MF, Senyl AS, Mustard JF. Alteration of the response of platelets to surface stimuli by pyrazole compounds. J Exp bled 1967;126:171-88. 13. Davenport N, Gotdaieln RE, Capurro NL, et al. Sulfinpyrazone and aspirin increase epicardial coronary collateral flow in dogs. Am J Cardiol 198 1;47:848-54. 14. Moe&es C, Escoblnas A, Jorgensen 0 Jr, Regan T. Effect of sulfinpyrazone on survival following experimental non-thrombotic coronary occlusion (abstr). Am J dardiol 1979;43:372. 15. Crook D. Collfns AJ. Bacon PA. Chan R. Prostaalandin svnlhetase activity f&m human.rheumatoid synovial micros&es. Ann Rheum Dis 1976;35:327-32. 16. Brogden RN, Plnder RM, Sawyer PR, SpeQht TM, Avery CS. Naproxen: a review of its pharmacological properties and therapeutic efficacy and use. Drugs 1975;9:326-63. 17. McIntyre BA, Phlllp RB. Effect of three nonsteroidal anti-inflammatory agents on platelet function and prostaglandin synthesis in vitro. Thromb Res 1977;12:67-77.
18. Smith RJ. Modulation of phagocytosis by and lysosomal enzyme secretion from guinea-pig neutrophils: effect of nonsteroid antiinflammatory agents and prostaglandins. J Pharmacol Exp Ther 1977;200:647-57. 19. Smfih RJ, Sabln C, Gllchresl H, Wllllams S. Effect of anti-inflammatory drugs on lysosomes and lysosomal enzymes from rat liver. Biochem Pharmacol 1976;25:2171-7. 20. Meacock SCR, Kftchen EA. Some effects of non-steroidal antiinflammatory drugs on leucocyte migration. Agents Actions 1976;6:320-5. 21. Rfvkln I. The effect of bovine serum albumin on the in vitro inhibition of chemotaxis by anti-inflammatory agents. Agents Actions 1977;7:465-8. 22. Ollw E, Lunden I, Anggiird E. In vivo inhibition of prostaglandin synthesis in rabbit kidney by non-steroidal anti-inflammatory drugs. Acta Pharmacol Toxicol (Copenh) 1978;42:179-184. 23. Capurro NL, Marr KE, Aarnodt R, Goldsteln RE, Epstein SE. Aspirin-Induced increase in collateral flow following acute coronary occlusion in dogs. Circulation 1979;59:744-9. 24. Bonow RO, Upson LC, Sheehan FH, el al. Lack of effect of aspirin on myocardial infarct size in the dog. Am J Cardiol 1981;47: 258-64. 25. Lie JT, Palrolero PC, Holley KE, Thus JL. Macroscopic enzymemapping verification of large, homogenous experimental myocardial infarcts of predictable size and location in dogs. J Thorac Cardiovasc Surg 1975;69:599-605. 26. Lowe JE. Relmer KA, Jennlngs RB. Experimental infarct size as a function of the amount of myocardium at risk. Am J Pathol 1978;90:363-77. 27. Jugdutt BI, Hutchlns GM, Bulkley BH, Pltl B, Becker LC. Effect of indomethacin on collateral blood flow and infarct size in the conscious dog. Circulation 1979;59:734-43. 28. Moschos CB, Halder B, De La Cruz C, Lyons MM, Regan TJ. Antiarrhythmic effects of aspirin during nonthrombotic coronary occlusion. Circulation 1978;57:681-4. 29. Ogletree ML, Lefer AM. Influence of nonsteroidal anti-inflammatory agents on myocardial ischemia in the cat. J Pharmacol Exp Ther 1976;197:582-93. 30. Lefer AM, Crossley K. Optimal dose of ibuprofen in acute myocardial ischemia in the cat (abstr). Circulatory Shock 1979;6: 182. 31. Darsee JR, Kloner RA, Braunwald E. Demonstration of lateral and epicardial border zone salvage by fluribuprofen using an in vivo method for assessing myocardium at risk. Circulation 1981; 63: 29-35. 32. Runkel R, Chaplin M, Bousl G, Segre E, Forchlelll E. Absorption, distribution, metabolism and excretion of naproxen in various laboratory animals and human subjects. J Pharm Sci 1972;61: 703-8. 33. Wlldenthal K. Lyososomal alterations in ischemic myocardium: result or cause of myocellular damage? J Mol Cell Cardiol 1978; 10:593-603. 34. Boll1R, Davenport N, Gobleln RE, Epotefn SE. Effect of ischemia on myocardial proteolysis (abstr). Am J Cardiol 1980;45:414.
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From ths cardiology Branch, National Heart, Lung, and Blood Instiie, National Institutes of Health, Bethesda, Marylend. Manuscript received June 30, 1980; revised manuscript received November 3, 1980. accepted November 7, 1980. Resberch Felbw, National institutesof Health. Bethesda, Maryland, at the time of this work. + Reclplent of Public Hea!th Service Research Fellowship !X32-NUO5170-01 from the U. S. Public Healtf~ Service, Bethesda, Maryland. Address for reprlnts: Roberto Bolli, MD, Cardiobgy Branch, Bullding 10. Room 7515. National lnstltutes of Heatth. Bethesda, Maryland 20205. l
To assess the potential role of platelet inhibitory agents in the treatment of myocardial infarction, the effect on infarct size of two platelet lnhibftors, suifinpyrazone and naproxen, was evaluated. In addition to platelet inhibition, sulfinpyrarone increases epicardial collateral flow and naproxen has lysosomal-stabilizing activity. Thirty-eight open chest dogs were given intravenously sulfinpyrazone (30 mg/kg, n = ll), naproxen (30 mg/kg, n = 14) or saline solution (n = 13) 10 minutes before and 3 and 6 hours after ligation of the mid iefl anterior descending coronary artery. Drug doses were sufficient to inhibit adenosine diphosphate-Induced platelet aggregation. The dogs were killed 72 hours after occlusion. Myocardium at risk of infarction-that is, the area supplied by the occluded artery (anatomic risk area)-was identlfied by simuftaneous perfusion of the aortic root with Evans blue and of the coronary artery distal to the occlusion with clear saline solution. Hearts were sliced horizontally and stained with triphenyi-tetrazolium-chloride. The infarcted area and anatomic risk area were measured with videoplanimetry. The percent of left ventricle infarcted was not significantly different among the control, sulfinpyrazone and naproxen groups (28 f 2, 30 f 1, 28 f 2 percent, respectively) nor was percent of anatomlc risk area infarcted significantly different (75 f 3, 79 f 3, 75 f 3 percent, respectively). Thus, neither suifinpyratone nor naproxen in platelet inhibitory doses altered infarct size. These results indicate that neither inhibitory effects on platelet function and prostaglandin synthesis nor associated lysosomai-stabflizlng properties identify agents with consistent infarct-sparing action.
Recently much attention has been focused on the role of platelets in coronary artery disease and the potential therapeutic efficacy of platelet of atherosclerotic inhibitory drugs. l-s Platelets may hasten development deposits or augment effects of preexisting coronary arterial narrowing. In addition, platelets may exacerbate the consequences of coronary occlusion either by aggregation within microvessels carrying collateral flow or by release of thromboxanes or other vasoactive materials. Hence, the hypothesis has been advanced that agents interfering with platelet function might reduce the extent of myocardial infarction after coronary occlusion.4J Recent reports of reduction in infarct size after coronary ligation in rats6v7 and dogssv” treated with ibuprofen, a potent platelet inhibitor, favor this hypothesis. In order to explore the hypothesis more completely, we evaluated the capability of two other platelet inhibitors, sulfinpyrazone and naproxen, to alter infarct size in a canine model similar to that used in studies of ibuprofen. Sulfinpyrazone has certain features of particular interest. In addition to interfering with platelet aggregationi possibly by inhibition of prostaglandin synthesis,” sulfinpyrazone also appears to interfere with platelet-endothelial interaction. l2 In the context of acute myocardial ischemia pretreatment with sulfinpyrazone has been found to improve epicardial collateral blood flow’s and to reduce the electrocardiographic evidence of ischemic injury14 as well as the incidence of ventricular fi-
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brillation.14 Reports of clinical trials of this drug have indicated beneficial effects in patients with coronary artery disease.*J Thus, assessment of the influence of platelet-inhibitory doses of sulfinpyrazone on infarct size after coronary occlusion seemed particularly useful. Naproxen, like ibuprofen, is a nonsteroidal anti-inflammatory agent derived from propionic acid. Naproxen shares many pharmacologic properties with ibuprofen: inhibition of platelet aggregation, interference with prostaglandin synthesis and lysosomal stabilizing activity.lsi2* However, it appears to have greater lysosomal stabilizing efficacy than ibuprofen.18Jg Thus, assessment of infarct-sparing potential of naproxen would be of value in estimating the role of these shared properties in minimizing myocardial necrosis. Methods Platelet Aggregation The effect of intravenous naproxen on in vitro platelet aggregation was studied in a separate series of five dogs; analogous data for sulfinpyrazone were available from previous studies.‘OJ” To assess the platelet-inhibitory properties of differing doses, increasing amounts (3,10,30 and 100 mg/kg body weight) of naproxen were given intravenously; blood samples for aggregation studies were drawn 10 minutes after each dose. In two dogs given 30 mg/kg aggregation studies were repeated 24 hours after administration of naproxen. The aggregation techniques used have previously been presented.*“,1”p2” In brief, aggregation was quantitated as change in optical transmission induced by addition of adenosine diphosphate to platelet-rich plasma under carefully controlled circumstances. Optical transmission was defined as 0 percent for platelet-rich plasma and 100 percent for platelet-poor plasma.
samples for oxygen saturation and hematocrit determination were obtained by left atria1 puncture. The left anterior descending coronary artery was isolated from the surrounding tissues and a snare placed around it. The site of occlusion of the left anterior descending coronary artery was chosen to produce ischemia in a zone approximating the apical half of the distribution of this vessel. Four electrodes mounted 5 mm apart on Dacron@ fabric were placed on the epicardial surface of the potentially ischemit area (Fig. l), and unipolar electrograms were recorded by a multichannel direct-writing recorder. The height of S-T segment was measured 100 ms after the onset of the QRS deflection. Sites showing intraventricular conduction abnormalities (that is, delay of the intrinsic deflection exceeding 40 ms or prolongation of the QRS complex beyond 65 ms) were excluded from the analysis. Two successive occlusions were carried out. The purpose of the first occlusion was selection of hearts with unequivocal myocardial ischemia. Electrograms were recorded 2 minutes and, if ischemia was not evident, 5 minutes after coronary occlusion; only dogs having S-T segment elevation (a sum total of at least 10 mV in the four leads) were admitted to study. The occlusion was then released. Two minutes later dogs were randomly assigned to three groups: naproxen ( 14 dogs, 30 mg/kg intravenously), sulfinpyrazone (1 I dogs, 30 mg/kg intravenously) or normal saline solution (13 dogs, 30 ml intravenously). Treatment was given 10 minutes before the second occlusion and repeated 3 and 6 hours later. The thorax was closed shortly after occlusion and the dogs were allowed to awaken. No attempt was made to suppress arrhythmic activity occurring after ligation. Blood samples for naproxen determinations were obtained 15 minutes after the first dose and immediately before the second and third doses. Plasma naproxen levels were determined by a gas-liquid chromatographic technique.*
Infarct Size Experimental preparation: Forty-one dogs of either sex, weighing 20 to 32 kg, were anesthetized with sodium thiamylal (25 mg/kg intravenously) and ventilated with room air. The heart was exposed under sterile conditions thoracotomy and suspended in a pericardial
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a left Blood
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FIGURE 1. Left lateralview of the heart showing the site of occlusion of the left anterior descending (LAD) coronary artery and the method used to obtain eplcardial electrograms from the ischemlc area. ECG = ektrocardiogram; LA = left atrium: LV = left ventricle: RV = right ventricle.
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Identification and measurement of region at risk of infarction: Approximately 72 hours after occlusion, dogs received heparin (15,000 U intravenously) to prevent clotting in the coronary vasculature; immediately afterward, they were killed with lethal doses of sodium pentobarbital and the hearts removed. The region at risk of infarction (that is, the portion of left ventricle normally supplied by the occluded artery) was identified as previously describ+dz4 In summary, one cannula was inserted into the left anterior descending coronary artery just below the site of ligation and a second cannula was inserted into the aortic root. The arterial bed distal to the occlusion was then perfused with colorless fluid (dextran 6 percent in saline solution) while the aortic root was simultaneously perfused with 1 percent Evans blue in the same fluid. Equal physiologic perfusion pressures were applied to the aortic and distal left anterior descending coronary arterial cannulas to prevent flow across any collateral vessels, either present prior to occlusion or developed after occlusion. As a result of this procedure, the myocardium normally perfused by the occluded portion of the coronary artery (subsequently termed anatomic region at risk of infarction) remained unstained, while the rest of the heart was stained blue. The risk region, as defined in this study, is an anatomic designation of the collateral-dependent portion of the heart. Our technique does not specify the competence of collateral vessels or the metabolic demands of the myocardium, factors that determine the actual amount of necrosis within the risk region. The heart was then sliced from apex to base into 1 cm thick slices in
’ These determinations were performed by Dr. S. l-l. Wan, University of California. Los Angeles.
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FlGURE 2. Anterior surface of the heart after perfusion of aortic root with Evans blue solution and of distal left anterior descending coronary artery with fluorescein solution. A, ordinary light. B, ultraviolet light. Note the reciprocal relation between Evans blue-stained (black) and fluorescein-stained (pale gray) myocardium.
planes parallel to the atrioventricular groove and the border between stained and unstained tissue-assumed to be the limit of the region at risk- was marked by a small incision on each cut surface. To verify that this border coincided with that of the myocardium actually perfused by uncolored dextran in saline solution, in a series of six hearts the staining technique described was modified in that the distal left anterior descending coronary artery was perfused with 0.1 percent sodium fluorescein in dextran in saline solution; perfusion of myocardium by this solution results in a distinct fluorescence under ultraviolet light without appreciable discoloration under ordinary light. Comparative examination of hearts with ordinary and ultraviolet light revealed a coincidence between the border of Evans blue-stained myocardium (region not at risk) and that of fluorescent-stained myocardium (region at risk) (Fig. 2 and 3), thus confirming the assumption on which our staining technique was based. Estimationof infarct size: The right ventricular free wall was then removed. To identify the infarct within the anatomic region at risk, slices were then weighed and incubated for 60 minutes in triphenyl-tetraxolium-chloride solution; this agent stains only viable, dehydrogenase-containing myocardium dark red, as described by Lie et als5 In the series of fluorescent-stained hearts, the nonfluorescent (and thus non-
perfused) portion of the anatomic region at risk coincidedwith the tetrasolium-negative (infarcted) tissue (Fig. 3, B and C). These findings indicate that, in the myocardium at risk, the presence of fluorescence detected by this technique is a reliable indicator of tissue survival. Lack of fluorescenceindicates nonperfusiondespite infusionof fluorescein into the coronary artery distal to the site of occlusion;this probably reflects high vascular resistance due to microvascular damage within infarcted myocardium. The weight of the anatomic region at risk and of the infarct was determined by photographing slices, projecting transparencies onto paper, and tracing portions at risk and necrotic portions. Each area was measured with videoplanimetry and the mass of the region at risk and the infarct mass were expressed as fractions of the weight of each slice. Total infarct weight was calculated as percent both of
the left ventricle and of the myocardium at risk. The latter estimate is of greater relevance, because the amount of necrotic tissue produced by coronary occlusion correlates directly with the magnitude of the arterial bed rendered ischemic.24*26v27Estimation of infarct weight as a percent of myocardium at risk also enables determination of whether the effect of a given intervention on infarct size varies with the size of the occluded coronary bed. Statisticalanalysis:One-way analysis of variance was used for statistical evaluation of the results. Values are reported as mean f standard error of the mean.
Results Platelet aggregation: In vitro adenosine diphosphate (ADP)-induced platelet aggregation showed a dose-related inhibition after intravenous administration of naproxen (Fig. 4). Platelet response, expressed as maximal percent light transmission in the presence of 2.34 X 10e4 M ADP, averaged 86 f 5 percent in control samples, was slightly decreased after 3 mg/kg (64 f 6 percent), but markedly inhibited after 10 mg/kg (19 f 4 percent) and 30 mg/kg (19 f 8 percent); the effect of the latter dose was still evident after 24 hours (30 f 12 percent). Each naproxen result was significantly different (p <0.05) from the control value. Larger doses (100 mg/kg) were associated with serious adverse effects (marked hemolysis, vomiting, neurologic symptoms). Similar degrees of inhibition were observed in the response to 2.34 X 10e5 A4 ADP. General observations: Arterial oxygen saturation and hematocrit, determined before the temporary coronary occlusion, were within normal limits in every dog. (A mild hemolysis was often observed in blood samples obtained for naproxen determination before the second and third doses of the drug; however, this did not cause any decrease of hematocrit below basal val-
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ues.) The mean heart rate at the time of the permanent occlusion was similar among control, naproxen-treated and sulfinpyrazone-treated dogs (152 f 6,137 f 9 and 148 f 9 beats/min, respectively). Left ventricular weight was 113 f 7 g in the control, 107 f 5 g in the naproxentreated and 106 f 4 g in the sulfinpyrazone-treated group. These values were not significantly different. Because the site of occlusion was based on the distribution of the left anterior descending coronary artery rather than on a fixed anatomic criterion, very similar fractions of the left ventricle were jeopardized by the occlusion (Fig. 5, left); the amount of myocardium at
risk was 37 f 2 percent (42 f 4 g) of the left ventricle in the control, 37 f 2 percent (40 f 3 g) in the naproxentreated and 39 f 2 percent (41 f 3 g) in the sulfinpyrazone-treated dogs. Three dogs were excluded from study before randomization because of insufficient S-T segment elevation (EST less than 10 mV) after the temporary coronary occlusion. Two (15 percent) of the 13 control dogs, 3 (21 percent) of the 14 naproxen-treated, and 1 (9 percent) of the 11 sulfinpyrazone-treated dogs manifested ventricular fibrillation within the initial 20 minutes of ischemia. These values did not differ sig-
FIGURE 3. Myocardial slice obtained from the heart shown in Figure 2. A, ordinary light. B. ultraviolet light. As in Figure 2, a reciprocal relation is observed between Evans blue-stained (black) and fluorescein-stained (pale gray) myocardium; the latter forms the outer boundary of the region at risk of infarction. Arrows indicate the border of fluorescent tissue. C after staining with triphenyltetraroliurn chloride. The shallow incisions (indicated by the pins) mark the borders of the region at risk. Comparison of B with C shows that the nonfluorescent portion of the region at risk is tetrazoliumnegative (white or pale gray) and thus necrotic, whereas the fluorescent part is tetrazoliumpositive (dark gray) and thus viable. 0, schematic digam illustratingB and C. Hetchsd area = normal myocardium; doMd erea = Infarct; white area sufroundln9 doned area = survivingmyocardium within the region at risk.
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nificantly. One control and one naproxen-treated dog died between 6 and 72 hours of occlusion, presumably of arrhythmias. Postmortem examination revealed no other apparent cause of death. This left 10 dogs for sacrifice in each treatment group. The plasma naproxen level was 153 f 10 I*g/ml (n = 9) 15 minutes after administration of the first dose; immediately before the second and third doses, the levels were 92 f 5 pg/ml (n = 8) and 108 f 7 pg/ml (n = 8), respectively. Infarct size: The control, naproxen-treated and sulfinpyrazone-treated groups did not show significant differences in infarct size expressed either as a percent of the left ventricle (28 f 2,28 f 2 and 30 f 1 percent, respectively; Fig. 5, middle), or as a percent of the myocardium at risk (75 f 3,75 f 3 and 79 f 3 percent, respectively, Fig. 5, right). For each of the three treatment groups the magnitude of the region of necrosis maintained a similar relation to the size of the occluded coronary beds (Fig. 6). Thus, in our dogs with documented ischemia, treatment before occlusion with either naproxen or sulfinpyrazone failed to reduce infarct size, irrespective of the size of the anatomic region at risk. Our data confirm previous studies24y26y27showing a linear correlation between infarct size and mass of myocardium at risk (r = 0.72, Fig. 6).
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FIGURE 4. Platelet aggregation 10 minutes after different intravenous doses of naproxen (NAP). Aggregation is quantitated as change in percent optical transmission induced by addition of adenosine diphosphate (final concentration 2.34 X low4 M) to platelet-rich plasma (0 percent corresponds to transmission through platelet-rich plasma and 100 percent to transmission through platelet-poor plasma). Results are expressed as mean f standard error of the mean. n = number of dogs studied. Asierlsks denote significant differences (p <0.05) from control (C).
Discussion
The results of our study demonstrate that neither naproxen nor sulfinpyrazone, administered in effective platelet-inhibitory doses, alters infarct size in the dog. Similar portions of left ventricle and, more importantly, similar portions of anatomic region at risk of infarction underwent necrosis in untreated and treated animals. “Platelet hypothesis”: The role of platelets in the evolution of ischemic injury after acute coronary occlusion is unclear. Accumulation of platelets in the ischemic area, particularly at the lateral margins, has been reported after coronary ligation in primates5 and induced coronary thrombotic obstruction in dogs.4 In the latter model, diffuse obstruction of the ischemic microvasculature by platelet aggregates was prevented
by aspirin. On the basis of these findings it has been hypothesized that platelet-mediated microcirculatory thrombosis reduces collateral flow to the ischemic zone, and therefore augments the extent of myocardial damage.4*5 However, subsequent experiments failed to show increased platelet deposition within infarcts in dogs undergoing nonthrombotic coronary occlusion.14*2s
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FIGURE 5. Weight of myocardium at risk as peroent of left ventricular mass (RISK/LV, left), weight of infarct as percent of left ventricular mass (MI/LV. mfddfe) and weight of infarct as percent of myocardium at risk (MI/RISK. right). C = control: NAP = naproxen-treated; SPZ = sulfinpyrazone-treated. Results are expressed as mean f standard error of the mean. Differences among the three groups are not significant.
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FIGURE 6. Myocardial infarct (MI) size as a percent of the left ventricle (LV) plotted as a function of the region at risk of infarction. C = control; NAP = naproxen-treated; SPZ = sulfinpyrazone-treated. The common linear relation observed for all groups is indicated by the regression line, which is calculated from individual data points from all groups.
Moreover, if the extent of ischemic necrosis is significantly enhanced by platelet aggregates, one would expect such necrosis to be limited by platelet-inhibitory drugs. However, aspirin, in platelet-inhibitory doses,24 and meclofenamic acid2g failed to reduce ischemic damage, and indomethacin enlarged the region of damage.27 Of the platelet-inhibitory drugs evaluated to date, only ibuprofen and its halogenated derivative fluribuprofen have been reported to diminish the extent of ischemic necrosis.6-g,30*31 Our protocol was designed to ensure effective platelet inhibition in dogs during the first hours of ischemia. Sulfinpyrazone, 30 mg/kg intravenously, was shown in previous experiments to abolish in vitro ADP-induced aggregation of dog platelets,13 and the current study indicated that naproxen, 30 mg/kg intravenously, also produced marked inhibition of platelet aggregation. With either drug, inhibition of platelet function was demonstrable 10 minutes after administration. Because we administered either drug 10 minutes before, and 3 and 6 hours after coronary occlusion, effective platelet inhibition was achieved at the onset of ischemia and maintained for at least several hours. Despite this, no change in infarct size was observed. Therefore, our data suggest that in this model (1) platelet inhibition in itself does not result in preservation of ischemic myocardium, and (2) platelet accumulation in the ischemic zone does not appear to play a significant role early in the course of myocardial infarction. Sulfinpyrazone: Another study from this laboratory13 showed that pretreatment with sulfinpyrazone (30 mg/kg intravenously) augments mean epicardial collateral flow both at 5 minutes and at 4 hours after coronary ligation in open chest dogs. However, the present study demonstrates that this potentially beneficial drug fails to reduce infarct size in a similar model, possibly
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because larger changes in collateral circulation are necessary to produce significant salvage of ischemic myocardium in open chest dogs. A reduction in the electrocardiographic evidence of ischemic injury that occurs acutely after coronary occlusion has been reported by Moschos et a1.14 in open chest dogs pretreated with sulfinpyrazone for 7 days. Although differences due to the prolonged pretreatment cannot be excluded, our data indicate that treatment with sulfinpyrazone, despite any acute electrophysiologic effects, does not reliably lead to a reduction in actual ischemic necrosis. Nevertheless, the increase in epicardial collateral blood flow13 and reduction in electrocardiographic evidence of injury14 may be related and possibly responsible for the antiarrhythmic effect of the drug that was also observed during acute myocardial ischemia.14 In our model the incidence of early ventricular fibrillation was too small to afford any inference about differences between treated and control dogs. Naproxen: Naproxen has a long plasma half-life in dogs (36 hours after intravenous injection32). Because we used a dose 10 times greater than that sufficient to produce significant platelet inhibition (Fig. 4), it seems reasonable to assume that pharmacologically effective levels of the drug were present in the blood throughout the 72 hours of coronary occlusion. This assumption is also supported by the marked platelet inhibition observed 24 hours after administration of naproxen. Naproxen resembles ibuprofen more closely than other nonsteroidal antiinflammatory agents in that (1) it stabilizes lysosomal membranes in vitro,18plg and (2) it inhibits inflammation, platelet aggregation and prostaglandin synthesis with a potency similar to that of ibuprofen.15-17*20-22 Th ese derivatives of propionic acid differ in that naproxen shows a more pronounced in vitro lysosomal stabilizing activity than does ibuprofen.lssls Lysosomal-stabilizing effect may be beneficial in acute myocardial ischemia during which activation of lysosomal hydrolases has been thought to play a significant role in cell death,33 although recent studies question the validity of this concept.34 Despite their close similarity the two drugs have different effects on myocardial ischemia. Naproxen failed to salvage ischemit myocardium in our study, whereas other investigations have shown that ibuprofen reduced ischemic injury in cats30 as well as infarct size in rats6,7 and dogs.8pg Differences in experimental models might account for such discrepancies. Infarct size was determined by Ribeiro et a1.8 after 24 hours of ischemia compared with 72 hours in the present study. It is therefore possible that ibuprofen (and possibly naproxen) might simply delay necrosis rather than prevent it. However, ibuprofen’s infarct-sparing action was demonstrable even 21 days after coronary arterial occlusion in rats7 In contrast, Jugdutt et a1.gstudied small infarcts (less than 15 percent of the left ventricle) in a closed chest model. Thus, the discrepancies cited could be explained if both ibuprofen and naproxen spared only small infarcts, in which the potentially salvageable border zone may be larger than the nonsalvageable center zone.
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Although any of these considerations is possible, it appears likely that naproxen lacks the infarct-sparing action of ibuprofen. Our data, therefore, suggest that properties other than those common to both ibuprofen and naproxen account for ibuprofen’s beneficial effect on myocardial ischemia. This inference is also supported by the failure to reduce ischemic damage seen with other nonsteroidal anti-inflammatory drugs (aspirin,24,2g meclofenamic acid2g and indomethacin27) that share an ability to block prostaglandin synthesis and platelet function.
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Implications: Our data provide evidence that antiinflammatory action, platelet inhibition, inhibition of prostaglandin synthesis and lysosomal-stabilizing activity are not in themselves sufficient to confer infarct-sparing action. Further investigation is needed to identify drug mechanisms that reliably lead to reduction in infarct size. Acknowledgment We thank John Bather, DVM, who kindly provided logistic support for sterile surgery. The technical assistance of Filippina Giacometti and Kirsti Kile is gratefully acknowledged.
References 1. Anturane Reinfarction Trial Research Group. Sulfinpyrazone in the prevention of cardiac death after myocardial infarction. N Engl J Med 1978;198:289-95. 2. Anturane Reinfarction Trial Research Group. Sulfinpyrazone in the prevention of sudden death after myocardial infarction. N Engl J Med 1980;302:250-8. 3. Braunwaid E. Treatment of the patient after myocardial infarction. The past decade and the next. N Engl J Med 1980;302:290-3. 4. Moschos CB, Lahlrl K, Lyons M, W&se AB, Oldewurtel HA, Regan RJ. Relation of microcirculatory thrombosis to thrombus in the proximal coronary artery: effect of aspirin, dipyridamole. and thrombolysis. Am Heart J 1973;88:61-8. 5. Rut W, McNamara JJ, Suehlro A, Suehlro G, Wkkllne SA. Platelet trapping in myocardial infarct in baboons: therapeutic effect of aspirin. Am J Cardiol 1980;46:405-12. 6. MacLean D, Flshbeln MC, Maroko PR, Braunwald E. Long-term salvage of ischemic myocardium by depleting catecholamin& and inhibiting inflammation (abstr). Clin Res 1977:25:455A. 7. MacLeai D, Ffahbeln mC, Bkan RI, Braunwald E, Maroko PR. Lor@erm preservation of ischemic myocardium by ibuprofen after experimental coronary artery occlusion (abstr). Am J Cardiol 1978;44:394 8. Rlbefro LOT, Yaeuda 1, Lowensfeln E, Braunwald E, Yaroko PR. Comparative effects on anatomic infarct size of verapamil, ibuprofen, and morphine-promethazine-chlorpromazine combination (abstr). Am J Cardiol 1979;43:396. 9. Jugdutt BI, Hutchlns GM, Bulkley BH, Becker LC. Salvage of ischemic myocardium by ibuprofen during infarction in the conscious dog. Am J Cardiol 1979;46:74-82. 10. Goldsteln RE, Davenport N, Llpson LC, et al. Relative effects of sulfinpyrazone and ibuprofen on canine platelet function and prostaglandin-mediated vasodilation. J Cardiovasc Pharm 1980; 2:399-409. 11. All M, McDonald JWD. Effects of sulfinpyrazone on platelet prostaglandin synthesis and platelet release of serontonin. J Lab Clin Med 1975;89:868-75. 12. Packham MA, Warrler ES, Glynn MF, Senyl AS, Mustard JF. Alteration of the response of platelets to surface stimuli by pyrazole compounds. J Exp bled 1967;126:171-88. 13. Davenport N, Gotdaieln RE, Capurro NL, et al. Sulfinpyrazone and aspirin increase epicardial coronary collateral flow in dogs. Am J Cardiol 198 1;47:848-54. 14. Moe&es C, Escoblnas A, Jorgensen 0 Jr, Regan T. Effect of sulfinpyrazone on survival following experimental non-thrombotic coronary occlusion (abstr). Am J dardiol 1979;43:372. 15. Crook D. Collfns AJ. Bacon PA. Chan R. Prostaalandin svnlhetase activity f&m human.rheumatoid synovial micros&es. Ann Rheum Dis 1976;35:327-32. 16. Brogden RN, Plnder RM, Sawyer PR, SpeQht TM, Avery CS. Naproxen: a review of its pharmacological properties and therapeutic efficacy and use. Drugs 1975;9:326-63. 17. McIntyre BA, Phlllp RB. Effect of three nonsteroidal anti-inflammatory agents on platelet function and prostaglandin synthesis in vitro. Thromb Res 1977;12:67-77.
18. Smith RJ. Modulation of phagocytosis by and lysosomal enzyme secretion from guinea-pig neutrophils: effect of nonsteroid antiinflammatory agents and prostaglandins. J Pharmacol Exp Ther 1977;200:647-57. 19. Smfih RJ, Sabln C, Gllchresl H, Wllllams S. Effect of anti-inflammatory drugs on lysosomes and lysosomal enzymes from rat liver. Biochem Pharmacol 1976;25:2171-7. 20. Meacock SCR, Kftchen EA. Some effects of non-steroidal antiinflammatory drugs on leucocyte migration. Agents Actions 1976;6:320-5. 21. Rfvkln I. The effect of bovine serum albumin on the in vitro inhibition of chemotaxis by anti-inflammatory agents. Agents Actions 1977;7:465-8. 22. Ollw E, Lunden I, Anggiird E. In vivo inhibition of prostaglandin synthesis in rabbit kidney by non-steroidal anti-inflammatory drugs. Acta Pharmacol Toxicol (Copenh) 1978;42:179-184. 23. Capurro NL, Marr KE, Aarnodt R, Goldsteln RE, Epstein SE. Aspirin-Induced increase in collateral flow following acute coronary occlusion in dogs. Circulation 1979;59:744-9. 24. Bonow RO, Upson LC, Sheehan FH, el al. Lack of effect of aspirin on myocardial infarct size in the dog. Am J Cardiol 1981;47: 258-64. 25. Lie JT, Palrolero PC, Holley KE, Thus JL. Macroscopic enzymemapping verification of large, homogenous experimental myocardial infarcts of predictable size and location in dogs. J Thorac Cardiovasc Surg 1975;69:599-605. 26. Lowe JE. Relmer KA, Jennlngs RB. Experimental infarct size as a function of the amount of myocardium at risk. Am J Pathol 1978;90:363-77. 27. Jugdutt BI, Hutchlns GM, Bulkley BH, Pltl B, Becker LC. Effect of indomethacin on collateral blood flow and infarct size in the conscious dog. Circulation 1979;59:734-43. 28. Moschos CB, Halder B, De La Cruz C, Lyons MM, Regan TJ. Antiarrhythmic effects of aspirin during nonthrombotic coronary occlusion. Circulation 1978;57:681-4. 29. Ogletree ML, Lefer AM. Influence of nonsteroidal anti-inflammatory agents on myocardial ischemia in the cat. J Pharmacol Exp Ther 1976;197:582-93. 30. Lefer AM, Crossley K. Optimal dose of ibuprofen in acute myocardial ischemia in the cat (abstr). Circulatory Shock 1979;6: 182. 31. Darsee JR, Kloner RA, Braunwald E. Demonstration of lateral and epicardial border zone salvage by fluribuprofen using an in vivo method for assessing myocardium at risk. Circulation 1981; 63: 29-35. 32. Runkel R, Chaplin M, Bousl G, Segre E, Forchlelll E. Absorption, distribution, metabolism and excretion of naproxen in various laboratory animals and human subjects. J Pharm Sci 1972;61: 703-8. 33. Wlldenthal K. Lyososomal alterations in ischemic myocardium: result or cause of myocellular damage? J Mol Cell Cardiol 1978; 10:593-603. 34. Boll1R, Davenport N, Gobleln RE, Epotefn SE. Effect of ischemia on myocardial proteolysis (abstr). Am J Cardiol 1980;45:414.
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