Original Paper Pharmacology 1992;45:71-82
Department of Cardiovascular-Metabolic Disorders Pharmacology, Wyeth-Ayerst Research Laboratories, Princeton, N.J., USA
Keywords Myocardial ischemia Myocardial infarction Potassium channel activators Calcium channel blockers Hemodynamics
Potassium Channel Activators Cromakalim and Celikalim (W A Y-120,491 ) Fail to Decrease Myocardial Infarct Size in the Anesthetized Canine
Abstract The cardioprotective effects of the K channel activator drugs celikalim (WAY-120,491) and cromakalim were studied in a canine model of myocardial infarction consisting of 90 min of ischemia and 5 h of reperfusion. Intracoronary infusion of cromakalim and celikalim at 0.2 pg/kg/min beginning 10 min before occlusion of the left circumflex coronary artery and continuing throughout the duration of the reperfusion period appeared to exacerbate ischemic injury. Infarct size (percent of risk area) was 27.7 ± 5.6% in vehicle control animals (n = 5), 40.3 ± 6.2% for cromakalim (n = 5) and 55.7 ± 6.4% (p < 0.05 vs. vehicle) for celikalim-treated animals (n = 5). When these compounds were administered intravenously, using doses shown to increase total coronary flow in nonoccluded control animals, no exacerbation of ischemic injury was ob served. Anatomic infarct size was 32.8 ± 7.1 % for vehicle ani mals (n = 5) and 32.6 ± 13.3 and 30.9 ± 9.8% for cromaka lim- (n = 6) and celikalim-treated (n = 5) animals, respectively. Intravenous diltiazem decreased myocardial infarct size to 16.3 ± 7.3% (n = 5) of area at risk (p = NS vs. vehicle). The anatomic area at risk was similar in all three treatment groups, and no significant differences in rate-pressure product were observed. Results of this study suggest that K-channel-activating drugs such as cromakalim and celikalim may not be effec tive agents in the acute therapeutic management of myocar dial ischemic injury.
Received: August 29, 1991 Accepted: November 4, 1991
Jan M. Kitzen, PhD Departmen: of Cardiovascular Biology Rhonc-Poulenc Rorer Central Research 640 Allendale Road King of Prussia. PA 19406 (USA)
© 1992 S. Kargcr AG, Basel 0031 -7012/92/ 0452-0071 $2.75/0
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Jan M. Kitzen John D. McCallum Charlotte Harvey Mary Ellen Morin George T. Oshiro Thomas J. Colatsky
Methods
The potassium channel activators repre sent a new class of pharmacologic agents that may find clinical usefulness in a variety of therapeutic areas including hypertension, asthma and urinary incontinence [1], These agents arc thought to cause relaxation of vas cular and nonvascular smooth muscle by en hancing the efflux of potassium ions from smooth muscle, leading to a hyperpolariza tion that opposes the entry of calcium [2], The resulting vasodilation leads to a decrease in blood pressure and increases in local blood flow. Previous studies from our laboratories have identified WAY-120.491 (cclikalim) to be a potent K channel activator [3], Other studies have shown that celikalim and cromakalim were both potent coronary vasodilators in the anesthetized dog [4], The ability of these agents to increase coronary blood flow suggests that they may be useful in the man agement of myocardial ischemia. Recent studies have presented evidence demonstrat ing that several potassium channel activators including RP-52.891 and EMD-56,431 were able to reduce myocardial infarct size and attenuate myocardial stunning in the canine [5. 6], Other investigators have shown that cromakalim and pinacidil were able to im prove postischemic dysfunction in the glob ally ischemic rat heart [7] as well as reduce infarct size in the canine [7, 8]. These observa tions led us to examine the ability of celikalim [(-)-(3S-trans)-2-(3,4-dihydro-3-hydroxy-2,2dimethyl - 6 - [trifluoromethoxy] - 2H -1 - ben zopyran-4-yl)-2,3 dihydro-1H-isoindol-1-one] to reduce infarct size in a model of infarction similar to that reported on by Grover et al. [ 8],
Surgery Dogs were fasted overnight but allowed free access to water. On the day of the experiment, the animals were anesthetized with sodium pentobarbital 35 mg/kg, i.v. An endotracheal tube was inserted and connected to a Harvard respirator set at approximately 17 strokes/'min and 15 cmVkg/strokc to provide posi tive pressure ventilation with room air. The left femo ral artery and vein were cannulated with polyethylene tubing for measurement of arterial blood pressure and for intravenous administration of electrolyte solution to support blood pressure after defibrillation. An intra venous line was placed in the saphenous vein for con tinuous infusion of anesthetic. In order to maintain a constant depth of anesthesia during the procedure, the bolus dose of pentobarbital was supplemented with an intravenous infusion of pentobarbital solution (0.08 mg/kg/min, i.v.. infused at a rate of 0.039 ml/min) via a Harvard syringe pump. The right carotid artery was cannulated with a Millar Mikro-Tip pressure transducer and inserted into the left ventricle for direct measurement of left ventricular pressure. An intercostal incision was made between the fourth and fifth ribs on the left side to expose the heart, and the heart was suspended in a pericardial cradle. A monopolar plunge electrode was implanted in the circumflex distribution to monitor regional S-T segment changes from the lead V electrocardiogram (ECG). Lead II electrodes were also inserted, and waveforms for each lead were displayed throughout the experiment. The left circumflex coronary artery (LCCA) was dissected free from surrounding fat and connective tissue. A 1inch segment of the vessel was prepared, and the artery was instrumented proximally to distally with an elec tromagnetic flow probe, an occlusive snare and an intracoronary needle for drug infusion. The needle is prepared from a 25-gauge stainless-steel syringe needle that is curved to permit insertion into the coronary lumen parallel to the vessel. The needle is anchored to the epicardial surface of the heart, and the lumen is maintained patent by a continuous infusion of saline solution until the beginning of test substance infusion. The flow probe was connected to a Carolina Electro magnetic Flowmeter (Model 501), and left circumflex coronary flow was displayed throughout the experi ment. Experimental Protocol Protocol I: Intracoronary Drug Administration. Af ter the animal had stabilized, drug infusion was started 10 min before occlusion of the LCCA. Drugs were
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Introduction
Perfusion o f tlw Heart for Area at Risk and Staining the Heart for Infarct Measurements At the completion of reperfusion, the LCCA was ligated and cannulated with polyethylene tubing. The heart was perfused with 0.9% saline solution at a (low rate adjusted to yield a perfusion pressure equal to the animal's MAP at the end of reperfusion. Five minutes later, patent blue dye, I mg/kg (of 10 mg/ml solution) was administered directly into the left atrial appendage via a polyethylene cannula. After 2 min, if the heart had not arrested spontaneously, it was fibrillated elec
trically with a brief high-frequency pulse from a Grass S44 stimulator and immediately excised. The heart was rinsed under tap water, blotted dry and weighed. The atria and right ventricle were re moved, and the left ventricle was then weighed. The ventricle was sliced into four transverse 1-cm sections from base to apex. The slices were incubated for 15 min in a solution of 1.0% triphenyltetrazolium warmed to approximately 37 °C. Each slice was blot ted dry and weighed. The normal zone, area at risk and infarct zone were traced onto acetate sheets for planimetric measurements of infarct size. Exclusion Criteria In order for infarct data to be included in the data analysis, animals must have met the following criteria: (1) S-T elevation of at least 10 mV in lead V ECG dur ing LCCA occlusion - this did not have to be sustained throughout the occlusion period; (2) presence of ar rhythmias during reperfusion; (3) cyanotic appearance of epicardial surface following occlusion: (4) not more than four defibrillations (10-20 J) to restore cardiac rhythm in any animal developing ventricular fibrilla tion during occlusion or reperfusion.
Results Seventy-three animals were entered into this study. Thirty animals were used in pilot studies for determining drug dosages for in tracoronary and intravenous routes of admin istration. For the intracoronary drug adminis tration protocol in infarctcd animals, 16 ani mals were utilized. One animal was excluded due to failure to develop adequate ischemia. For the intravenous protocol. 15 animals were used in pilot studies, and 27 animals were entered into the infarct protocol. Seven ani mals were excluded for the following reasons (treatment groups included in parentheses): 2 failed to develop ischemia (celikalim), 2 died of severe hypotension before the completion of reperfusion ( 1 celikalim, 1 diltiazem) and 3 animals required an excessive number of defi brillations to resuscitate (celikalim). Results of the pilot studies showed that neither cromakalim nor celikalim at 0.1 pg/ 73
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infused at a flow rate adjusted to 0.055 ml/min. Base line hemodynamic measurements were obtained [blood pressure, heart rate (HR), lead II, lead V, left ventricular pressure, dP/dt and LCCA How], drug infusion was started and a postdrug hemodynamic measurement obtained. Ten minutes later the LCCA was occluded for 90 min. At the end of the occlusion period the occlusive snare was released while moni toring coronary How such that baseline How was restored gradually over a period of about 5-10 min. Reperfusion was carried out for 5 h. All hemody namic data were channeled to a Modular Instruments computer for on-line data acquisition, data display and statistical analysis. Doses for the study were selected from pilot studies where dmgs were infused into the coronary circulation of nonischemic animals. The major criterion for dose selection was to identify doses of celikalim and cromakalim that produced comparable effects on coronary blood flow. Since dmgs were infused directly into the coronary circula tion, systemic hemodynamic actions were minimized. Three treatment groups (n = 5) were included in this protocol: vehicle: celikalim 0.2 pg/kg/min. and cro makalim 0.2 pg/kg/min. Protocol II: Intravenous Drug Administration. The same protocol as described above was utilized except that drugs were administered into a femoral vein. Pilot studies were conducted in nonischemic animals in order to identify doses that produced the least amount of change in the rate-pressure product (RPP), i.c. mean arterial pressure (MAP) X HR. Four treatment groups were included in this portion of the study: vehicle: celi kalim 0.7 pg/kg/min; cromakalim 0.4 pg/kg/min. and diltiazem 0.18mg/kg (bolus) +7.5 pg/kg/min (infu sion). The vehicle used to solubilize all drugs was dimethylsulfoxide: polyethylene glycol 200: HiO (1.68:2.33:1.0). Appropriate volumes of this vehicle were then diluted into an infusion vehicle of 15 % poly ethylene glycol 200/saline to yield the exact doses required for each animal.
Table 1. H em odynam ic effects o f intracoronary adm inistration o f crom akalim and celikalim
Baseline Vehicle (n = 5) MAP. mm Hg HR. beats/min RPP, mm Hg X beats/min
116 ±4 138 ±6 16.027 ±538
After drug
Occlusion
Reperfusion
0 min
Oh
60 min 90 min
116 100 98 106 ±4 ±9 ±4 ±5 138 141 146 152 ±6 ±4 ±7 ±8 15.912 14.088 14.413 16.246 ±479 ±1,375 ±978 ±1.442
1h
2h
4h
5h
97 104 103 83 81 ±10 ± 6a ± 5a ±5 ±8a 160 133 156 179 165 ±10 ±11 ±9 ±19 ± 13a 15.410 13.668 16.443 14,913 13.348 ±1.156 ±1.307 ±2,388 ±1.680 ± 1,762
Cromakalim (n = 5) 0.2 yg/kg/min MAP, mm Hg 108 97 97 110 98 ±8 ±9 ±9 ±4 ±6 HR, beats/min 144 147 157 145 161 ±9 ±6 ±8 ±3 ±8 RPP. mm Hg 15,504 15.826 14.344 15.752 15,500 X beats/min ±1.293 ±1,478 ±1.903 ±1,168 ±1,315
86 85 81 65 63 ±7a ±3a ± 3a ±4a ±6a 143 158 161 196 193 ±11 ±7 ±4 ± 12a ±21a-b 12,357 13.396 13,137 12.654 12,562 ±1,158 ±800 ±1,311 ±1.346 ±2,300
Celikalim (n = 5) 0.2 yg/kg/min MAP. mm Hg 113 109 113 103 105 ±6 ±8 ±13 ±11 ±10 HR. beats/min 151 152 164 156 170 ±14 ±10 ±9 ±9 ±6 RPP. mm Hg 17,341 17,435 16.893 17.662 18.719 X beats/min ±2,001 ±2.199 ±3,316 ±2.572 ±2.242
99 90 88 78 76 ± 4 3 ±9 ±7a ± 5a 177 176 176 193 198 + 9a.b ±7 ± 8a-b ±8 ± 13a 17.574 16.070 15.625 15.220 15.052 ± 1.982 ±1.371 ±2,066 ±1.971 ±1.334 ± 9 3
kg/min, i.c., or 0.2 pg/kg/min, i.c., caused any significant changes in coronary blood flow. Since 0.2 pg/kg/min was approaching the up per limit of solubility for celikalim, no at tempt was made to increase the dose any fur ther. Other hemodynamic parameters (MAP. HR. dP/dt) were not significantly altered with these doses of the two K channel activators. Hemodynamics with Intracoronary Administration Mean Arterial Pressure, Heart Rale and Rate-Pressure Product. The effects of intracor onary administration of cromakalim and celi
74
kalim on these parameters are summarized in table 1. Baseline MAP ranged between 108 and 116 mm Hg for the three treatment groups. Vehicle-treated animals showed a steady de cline in MAP throughout the occlusion-reper fusion period. By the end of the experiment MAP had decreased by 35 mm Hg. Peak de creases in MAP for cromakalim and celikalim were 45 and 37 mm Hg (p = NS vs. vehicle) over the same time period. Baseline HR ranged from 138 to 151 beats/min. and no significant baseline differ ences were observed between the three treat-
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a p < 0.05 versus baseline;b p < 0.05 versus vehicle. All data presented as means ± SEM.
B
Tim e
(m in)
Tim e
(m in)
Fig. 1. Effects of intracoronary administration of cromakalim and cclikalim on LCCA blood fow (A) and regional ECG S-T segment (B). Regional S-T record ings were obtained from a unipolar plunge electrode placed in the posterolateral surface distal to the occlu-
sion site. Arrows denote time of drug administration. ■ = Vehicle; A = cclikalim. 0.2 gg/kg/min: • = cromaka lim, 0.2 gg/kg/min. Each point is the mean ± SEM of 5 observations. * p < 0.05 versus baseline.
inent groups. In general, HR tended to in crease over time in each treatment group; this was partially due to the fact that animals developed arrhythmias during the reperfu sion period. In vehicle-treated animals, HR reached a peak increase of +41 beats/min (p < 0.05 vs. baseline), while higher peak increases were attained in the cromakalim (+52 beats/min; p < 0.05 vs. baseline) and celikalim (+47 beats/min; p < 0.05 vs. base line) groups. RPP was calculated from the product of MAP and HR and was used to provide an index of myocardial oxygen consumption [9], No significant differences in RPP between groups were observed. Coronary Blood Flow and Regional S -T Segment. Baseline coronary blood flow ranged from 36 to 38 ml/min for all treatment groups (fig. IA). After complete obstruction
of coronary blood flow for 90 min. blood flow was slowly restored by carefully releasing ten sion on the occluder. In the first few minutes of reperfusion, significant hyperemia was ob served in all treatment groups (range: 62-64 ml/min). Coronary blood flow then declined toward baseline throughout the remainder of reperfusion. By the end of the experiment cor onary blood flow was 23-28 ml/min for the three treatment groups. In order to provide some index as to the severity of regional ischemia that developed in each animal, a unipolar electrode was im planted into the posterolateral wall in the region perfused by the LCCA to record local S-T elevation directly from the ischemic zone. Animals that did not display an S-T increase were assumed to be nonischemic and were excluded from the study. Previous stud ies [10] have shown this to be a reliable (al-
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A
from 160 ± 9 beats/min at baseline to 127 ± 7 beats/min at +90 min (p < 0.05 vs. base line). During reperfusion, HR was initially increased in the vehicle group and returned toward baseline by the end of the study. All animals displayed severe ectopy during reper fusion, and sinus HR was usually not ob served. Cromakalim- and celikalim-treated animals both displayed increases in HR dur ing reperfusion, while in diltiazem-treated an imals HR was maintained below baseline sinus rate throughout the reperfusion period. This effect on HR is consistent with results of Hemodynamics with Grover et al. [ 12] and Bush et al. [ 11 ]. Intravenous Administration There were no significant baseline differ Pilot studies were conducted in order to select doses of test drugs that produced the ences in RPP between the treatment groups least amount of systemic hemodynamic (baseline range = 16.001-19.485). RPP de changes. Diltiazem was included in this pro creased only by 2,753 units (17%) over the tocol to serve as a standard reference agent, time course of the experiment in vehiclesince several studies have shown diltiazem to treated animals. Cromakalim-treatcd animals reduce myocardial infarct size [11, 12]. The showed the smallest RPP decrease of all the diltiazem dose selected for the present investi drug treatment groups (peak decrease = 25 %). gation was the same as used by Grover et al. Peak decreases for celikalim and diltiazem [12]: 0.18 mg/kg i.v. (bolus) +0.0075 mg/kg/ were 37 and 34%, respectively. Although RPP min i.v. (infusion). This dose was shown to was significanlty decreased at several time exert less effect on the RPP than the dose used points in celikalim- and diltiazem-treated ani mals, these decreases were not significant in in the study by Bush et al. [11], Mean Arterial Blood Pressure, Heart Rate comparison with the vehicle group. Coronary Blood Flow and Regional S - T and Rate-Pressure Product. As shown in ta ble 2, no significant differences in blood pres Segment. Baseline coronary blood flow sure were observed between the three treat ranged from 40.0 ± 5.9 in vehicle-treated ani ment groups and the vehicle. Significant de mals to 57.0 ± 9.6 ml/min for celikalim. creases in diastolic blood pressure and MAP There were no significant differences in base were observed within each group, and the line flow between any of the treatment groups. peak decrease noted for the vehicle group was During reperfusion, coronary blood flow 23 mm Hg (MAP). Peak decreases in MAP (fig. 2A) never attained baseline level in the observed for the other treatment groups were vehicle-treated animals. Both K channel acti 36 mm Hg for cromakalim. 47 mm Hg for vators led to significant increases in coronary celikalim (due to slightly higher baseline blood flow that were sustained throughout most of the reperfusion period. This was MAP) and 27 mm Hg for diltiazem. HR was very stable throughout the occlu probably due to the coronary vasodilation sion period for the vehicle, cromakalim and action of these drugs which in the pilot studies celikalim treatment groups. In diltiazem- was observed to increase coronary blood flow treated animals, HR gradually decreased by 125 and 121 % above baseline for celikalim
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though imperfect) method for assessing re gional ischemia in animals where collateral flow cannot be measured. As shown in fig ure IB, animals in each treatment group de veloped comparable S-T segment elevation (range: 21-27 mV. p = NS between groups). In these studies, regional S-T segment changes were the only information available to evalu ate the extent of ischemia developed in all ani mals (see exclusion criteria in Methods) in response to occlusion of the LCCA.
Table 2. H em odynam ic effects o f intravenous adm inistration of crom akalint and celikalim
Base-
Vehicle in = 5) MAP, mm Hg HR. beats/min RPP. mm Hg X beats/min
After
Occlusion
Reperfusion
t*rU®
0 min
Oh
60 min 90 min
109 104 84 92 97 +6 ±7 ±11 ±8 ±8 146 151 146 156 151 ±10 ±8 ±16 ±7 ±6 16.001 15,828 12,956 14,580 14,824 ±1,878 ±1.813 ±2.769 ±1,813 ±1.565
Cromakalim (t,i -6 ) 0.4 fig/kg/min MAP. mm Hg 112 113 95 ±4 ±4 ±4 HR. beats/min 150 150 157 ±5 ±6 ±9 RPP. mm Hg 16.698 16.841 14,803 X beats/min ±558 ±505 ±611
Ih
2h
4h
5h
86 91 90 96 86 ±9 ±8 ±11 ±10 ± 8a 177 162 172 145 148 ±16" ±11 ±7 ±15 ± 19 15.642 14.981 16,682 13,460 13.248 ±2,713 ±2,251 ±2,463 ±2,735 ±2.880
83 88 80 87 89 77 76 ±4“ ±7“ ±9a ±6“ ±8a ± 6a ±6a 151 154 160 174 165 179 190 ±5 ±6 ±11 ±8 ±9 ±16a-b + 15a,b 12.523 13.591 12.860 14,176 15,603 13.514 14.308 ±522° ±1.229 ± l,774a ±937 ±1.732 ±1,356 ±1,417
Celikalim (n = 4) 0.7 fig /kg /min MAP. mm Hg 107 122 121 97 97 84 87 80 73 75 ±4 ±5 ±8 ±7a ± l l a ± I0a ± 7a ± 6a ± 6 a ± 6a HR. beats/min 160 164 170 164 168 156 201 172 165 173 ±5 ±7 ±5 ±6 ±7 ± I0a ±11 ±5 ±6 ±3 RPP. mm Hg 19.485 19,913 18,248 16.035 16.480 17.064 12,729 14.994 11.960 12,935 X beats/min ±927 ±1,370 ±1,751 ±1.720 ±2,433 ±2,227 ± 1,910 • 1.120 ±917 ± 818a Dihiazem (n =5) 0. IS mg/kg bolus + 0.0075 mg/kg/min MAP. mm Hg 97 112 107 92 88 94 85 102 95 88 ±5 ±6 ±6 ±5 ±5“ ±6a ±5 ±5 ±6 ±5a HR. beats/min 160 151 149 135 127 143 122 133 131 134 ±9 ±15 ±8 ± 7a ±8 ±19 ± 6a-b ±5b ±11 ±9 RPP. mm Hg 17.985 16.178 14,522 12.550 11.254 12.074 11,498 13.569 12,532 11.797 X beats/min ±1,461 ±2.033 ±1,425 ± l,340a ± l,059a ± 1.417a ±877a ± 1,047a ± l,627a ± 1,159a
and cromakalim, respectively. In diltiazemtreated animals coronary flow returned to baseline for the duration of the reperfusion period. S-T segments ranged from 11.8 to 15.5 mV during the 90-min occlusion period in vehicle-treated animals. S-T segments were similar for diltiazem-treated animals, ranging from 9.7 to 13.9 mV. In both groups of ani
mals treated with the K channel activators, ST segment increases were more pronounced than those in the vehicle and diltiazcm groups. Cromakalim-treated animals dis played the most pronounced increases in S-T segment (range: 14.3-26.6 mV) with several of these increases being significantly different from S-T changes observed in the vehicle group. The range of S-T segments observed
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a p < 0.05 versus baseline:b p < 0.05 versus vehicle. All data presented as means ± SEM.
B
A
■
1
50
F—
1
1
1
I
1
1
1 00 1 50 2 0 0 2 5 0 3 00 3 5 0 4 00 Tim e
Tim e
(m in)
(m in)
Fig. 2. Effects of intravenous administration of cromakalim and celikalim on LCCA blood flow (A) and regional ECG S-T segment (B). ECG recordings obtained as in figure 1. Arrows denote time of drug administration. □ = Vehicle (n = 5); ▲ = celikalim.
0.7 gg/kg/min (n = 4); • = cromakalim. 0.4 gg/kg/min (n = 6):*= diltiazem, 0.18 mg/kg(bolus) +0.0075 mg/ kg/min (n = 5). Each point is the mean ± SEM of 5 observations. * p < 0.05 versus baseline, @ p < 0.05 versus vehicle.
for celikalim-treated animals was 13.7-20.2 mV. Whether or not the exacerbated increases in S-T segments in animals treated with a K channel activator are due to severer ischemia or the K-channel-opening effects of these drugs cannot be determined from these data. Previous work in our laboratory with K. chan nel activator drugs in thrombosis studies has shown them to increase S-T changes induced by thrombotic occlusion [13]; however S-T segments were normal under conditions of physiological coronary perfusion. It should be pointed out that in this study, all 3 of the excluded animals treated with celikalim that developed ventricular fibrillation did so dur ing the reperfusion period. It is interesting to note that no differences in S-T segment be tween drug and vehicle were observed in the intracoronary administration protocol.
Effect o f Intracoronary Administration on Myocardial Infarct Size As shown in figure 3A. the anatomic size of the ischemic zone (area at risk) was similar in each of the three treatment groups (range for all groups: 35.7-48.5% of left ventricle). In farct size (expressed as percentage of area at risk) was 27.7±5.6% for vehicle-treated ani mals. Intracoronary administration of the K channel activators resulted in larger infarcts: 40.3±6.2% for cromakalim and 55.7±6.4% for celikalim (p < 0.05 for celikalim vs. vehi cle). A scatter plot (not shown) of the individ ual infarcts for each treatment group showed a trend for the K channel activator drugs to cause a wider distribution of infarct size in contrast to a more clustered distribution ob served in the vehicle group. The regional S-T segment changes, shown in figure 1B indicate
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I
0
B
AAR
l/AAR
l/LV
■
AAR
Vehicto
□
Celikalim
Cromakalim
||j
Diliiazem
l/AAR
l/LV
Fig. 3. Effects of K. channel activators and diltiazem on myocardial infarct size. AAR = Area at risk; I/AAR = infarct size/area at risk; I/LV = infarct size/ left ventricle, n = 5 for all treatments unless indicated otherwise. A Intracoronary administration. Test sub stances were infused directly into the coronary circula-
tion beginning 10 min before occlusion of the vessel and were infused for the duration of the experiment. * p < 0.05 versus vehicle, n = 5 for all treatments. B In travenous administration. Test substances were in fused beginning 10 min before occlusion of the vessel and were infused for the duration of the experiment.
that all animals developed comparable in creases in the S-T segment. Attempts to relate infarct size to S-T changes were unsuccess ful.
farct size was 32.8% of area at risk. This value is somewhat smaller than infarct size data reported by Grover et al. [7, 8,12] and Bush et al. [11] where size of vehicle infarcts ranged from 40 to 45% of risk area. In our studies, both K channel activators failed to affect myocardial infarct size: infarct sizes for these two groups were 32.6 ± 13.3 and 30.9 ± 9.8% for cromakalim and celikalim, respec tively (p = NS vs. vehicle). These infarct sizes correspond to decreases of 0.6% for cromakalim and 5.7% for celi kalim in comparison with the infarcts in vehi-
Effects o f Intravenous Administration on Myocardial In farct Size Area at risk ranged from 35.1 to 40.4% in the four treatment groups (fig. 3B). Risk area was slightly smaller in the diltiazem group (p = NS vs. vehicle) due to 1 animal in which the area at risk was only 30.4% of the left ven tricular mass. In the vehicle group mean in
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A
Discussion Results from this study demonstrate that the K channel activator drugs cromakalim and celikalim were unable to reduce myocar dial infarct size in this canine model of isch emic injury. Data further suggest that croma kalim and celikalim have the potential to ex acerbate ischemic injury. This conclusion is supported by the observation that both K channel activators produced larger infarcts (in comparison with vehicle) when these drugs were administered directly into the coronary circulation. In contrast, intravenous adminis tration of these compounds did not increase infarct size; however, the infarcts were similar to those in vehicle-treated animals. Exacerba tion of ischemic injury with K channel activa tors has been observed by other investigators, although under somewhat different experi mental conditions. Thus. Sakamoto et al. [ 14] observed that pinacidil decreased blood flow to the infarct zone and increased infarct size (4-hour occlusion of the left anterior descend
80
ing artery) in animals with a moderate steno sis placed on a non-infarct-related (LCCA) artery, while Imai et al. [15] showed that pin acidil was unable to decrease infarct size in animals subjected to a 4-hour occlusion of the left anterior descending artery. In these latter studies, pinacidil increased blood flow to the normal zone and had no effect on blood (low to the infarct zone during occlusion yet it increased flow to the epicardium during re perfusion. Nitroprusside was found to in crease blood flow within the infarct zone and was also shown to decrease myocardial infarct size by 31 %. Our results do not support the findings of Grover et al. [7, 8], who showed that intracor onary administration of cromakalim, 0.1 mg/ kg/min, decreased myocardial infarct size. Our studies used the same dosing regimen and experimental protocol as used by these investigators [7. 8], Our results are also in dis agreement with those reported by Auchampach et al. [5], who showed that the K channel activator RP-52891 significantly decreased myocardial infarct size in an experimental infarct model similar to the one reported on here. In our studies we were able to control or measure several of the factors known to affect myocardial infarct size. Thus, our data showed that the following factors were not responsible for the differences in myocardial infarct size between K channel activators and vehicle-treated animals: size of area at risk, blood oxygen content (as pO?, data not shown), and myocardial oxygen consumption (indirectly assessed as RPP). The decreases in RPP observed for the test drugs would, if any thing, be expected to lead to a decrease in infarct size since this parameter suggests that myocardial utilization of oxygen was de creased in these animals. Collateral blood flow to the infarct zone is a major determi nant of infarct size [16], and this variable was
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cle-treated animals. Diltiazem-treated ani mals showed the smallest infarcts of all four treatment groups: 16.3 ± 7.3%, correspond ing to a 50% decrease in infarct size compared to vehicle. These results are in good agree ment with those reported by Bush et al. [11] and Grover et al. [ 12], who found diltiazem to decrease infarct size by 35 and 45%. respec tively. The total number of animals that de veloped ventricular fibrillation among the treatment groups (including exclusions) was: vehicle, 1; cromakalim, 3; celikalim, 5, and diltiazem, 1. The relationship between drug and incidence of fibrillation cannot be estab lished from this study: however, there ap peared to be a tendency for the K channel activators to exacerbate arrhythmias during the occlusion period.
An important aspect of our results that lends support to our conclusion is the obser vation that our reference standard diltiazem was able to decrease myocardial infarct size. These results confirm earlier work by two sep arate investigators [11, 12] and therefore sug gest that our findings for the K channel acti vator drugs were probably not due to intrinsic differences (drug-independent effects) in col lateral How between the treatment groups. Although we did not examine the effects of intracoronary diltiazem to modify experi mental infarct size, a recent study by Higginson et al. [19] demonstrated that intracoro nary administration of diltiazem at the onset of ischemia was able to decrease infarct size significantly (68% decrease from control). In our study the effect of intravenous diltiazem on infarct size was not statistically significant; however, the decrease observed was pro nounced (50% decrease) and probably failed to attain significance due to the small sample size. In conclusion, our results for cromakalim and celikalim in this study suggest that K channel activators may not be effective agents for therapy of ischemic myocardial injury. It should be pointed out that both of these agents were benzopyran derivatives and it is possible that other chemical classes of K chan nel activators may not share these effects on the ischemic heart. It is interesting to note, however, that other investigators [20] have shown that various types of K channel activa tors can exert similar effects on myocardial refractoriness. Thus, Spinelli et al. [20] found that under normal physiological conditions cromakalim, pinacidil and nicorandil short ened only atrial refractoriness and were able to produce atrial arrhythmias but not ventric ular arrhythmias. However, Chi et al. [21] showed that in the postinfarction heart pin acidil was able to decrease ventricular refrac toriness and cause an increased incidence of
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not measured in these studies. Because of the importance of this factor in affecting ultimate infarct size, it is reasonable to suggest that dif ferences in collateral flow between experi mental subjects may have contributed to ob served differences in infarct size. Thus, it is possible that animals treated with intracoro nary cromakalim and celikalim may have had less tissue perfusion due to poor collateral cir culation than vehicle-treated animals during the occlusion period. However, since experi mental treatments were allocated in a ran domized fashion, it is unlikely that only those animals in these two treatment groups devel oped ischemia. The failure to demonstrate a reduction in infarct size with intracoronary administration of the K channel activators led us to repeat the study using systemically administered test drugs. For this portion of the study we also included the calcium-channel-blocking drug diltiazem, which has been demonstrated by several investigators to exert significant car dioprotective effects [11, 12, 17-19], Under similar experimental conditions as used in protocol I. we found that intravenous admin istration of the K channel activators resulted in no effect on infarct size. Pilot studies for each of these protocols showed that drug effects on coronary blood flow were affected differently by the two routes of administra tion. Thus, intracoronary administration of cromakalim and celikalim did not cause coro nary blood flow to increase during reperfu sion yet intravenous administration led to marked increases in coronary blood flow (fig. 1A, 2A). It seems possible that intracoro nary administration of these drugs, while not increasing total coronary flow, may have caused endocardial steal, an effect not ob served by Grover et al. [12] yet one that has been reported by others [13, 14], although under different experimental conditions (see above).
ventricular fibrillation when a second vascu lar bed was made ischemic. These observa tions suggest that drugs of the K channel acti vator type appear to exert complex pharmaco logic actions affecting myocardial conduction
and coronary blood flow. The usefulness of these agents in the ischemic heart will require additional characterization in order for their role in the therapy of cardiovascular disease to be better understood.
1 Edwards G. Weston A: Structureactivity relationships of K ' channel openers. Trends Pharmacol Sci 1990;11:417-422. 2 Cook N. Weir S, Danzeisen MC: Anti-vasoconstrictor effects of the K* channel opener cromakalim on the rabbit aorta - Comparison with the calcium antagonist isradipine. BrJ Pharmacol 1988;95:741-752. 3 Lodge N. Cohen R. Havens C. Colatsky TJ: The effects of the putative potassium channel activator WAY120,491 on s6Rb efflux from the rab bit aorta. J Pharmacol Exp Ther 1991;256:639-644. 4 Kitzen J. Stupienski R. DeSialo M. Rovnvak L, Pirozzi C. Oshiro G. Colatsky TJ: Hemodynamic effects of WAY-120,491 (WAY). [(-)-(3Strans)-2-(3.4-dihydro-3-hydroxy-2,2dimethyl-6-[trifiuoromcthoxy]-2H-lbenzopyran-4-yl)-2,3-dihydro-111isoindol-l-one). pinacidil (P). and cromakalim (C) in anesthetized dogs. FASEBJ 1990:4:2786. 5 Auchampach J, Maruvama M, Cavero 1, Gross G: The potassium channel agonist RP-52891 reduces infarct size in the anesthetized dog. Pharmacologist 1990:32:164. 6 Maruyama M, Gross G: Evidence for a role for potassium channels in stunned myocardium. FASEB J 1990;4:315. 7 Grover G, Sleph P. Dwonczyk S: Pharmacologic profile of croma kalim in the treatment of myocar dial ischemia in isolated rate hearts and anesthetized dogs. J Cardiovasc Pharmacol 1990:16:853-864.
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8 Grover G, Dwonczyk S, Parham C, Sleph P: The protective effects of cromakalim and pinacidil on reper fusion function and infarct size in isolated perfused rat hearts and anesthetized dogs. Cardiovasc Drug Ther 1990:4:465-474. 9 Gobcl F, Nordstrom L. Nelson R. Jorgensen C. Wang Y: The ratepressure product as an index of myocardial oxygen consumption during exercise in patients with an gina pectoris. Circulation 1978:57: 549-556. 10 Kitzen J. l.ucchesi B: Retrograde bleeding enhances incidence and se verity o f ischemia in the canine heart. FASEBJ 1989:3:2992. 11 Bush L, Romson J, Ash J, Lucchesi B: Effects of diltiazem on extent of ultimate myocardial injury resulting from temporary coronary artery oc clusion in dogs. J Cardiovasc Phar macol 1982:4:285-296. 12 Grover G, Sleph P. Parham C: Ef fect of diltiazem on infarct size, re perfusion flow and flow reserve. The effect of timing of treatment. J Phar macol Exp Ther 1988:246:263-269. 13 Kitzen J. McCallum J. Harvey C, Morin M: Antithrombotic activity of the phosphodiesterase III inhibi tor pclrinone in a canine model of coronary artery thrombosis: En hancement of efficacy with concur rent alpha-2 adrenergic antagonism. J Cardiovasc Pharmacol 1991:18: 777-790. 14 Sakamoto S, Liang C. Stone C, Hood W: Effects of pinacidil on myocardial blood flow and infarct size after acute left anterior descend ing coronary artery occlusion and reperfusion in awake dogs with and without a coexisting left circumflex coronary artery stenosis. J Cardio vasc Pharmacol 1989:14:747-755.
15 1mai N, Liang C, Stone C. Sakamoto S. Hood W: Comparative effects of nitroprusside and pinacidil on myo cardial blood flow and infarct size in awake dogs with acute myocardial infarction. Circulation 1988:77: 705-711. 16 Reimcr K, Jennings R. Cobb F, Murdock R. Greenfield J. Becker L, Healy-Bulklcv B. Hutchins G, Schwartz R. Bailey K. Passamani F.: Animal models for protecting isch emic myocardium. Results of the NHI.BI cooperative study. Compar ison of unconscious and conscious dog models. Circ Res 1985:56:651 — 665. 17 Wcishaar R. Ashikawa K. Bing R: Effect of diltiazem, a calcium antag onist. on myocardial ischemia. Am J Cardiol 1979;43:1137-1143. 18 Bush L, Li Y, Shlafer M. Jolly S, Lucchesi B: Protective effects of dilliazem during myocardial ischemia in isolated cat hearts. J Pharmacol Exp Ther 1981;218:653-661. 19 Higginson L., Tang A. Knoll G. Cal vin J: Effect of intracoronarv diltia zem on infarct size and regional myocardial function in the ischemic reperfused canine heart. J Am Coll Cardiol 1991:18:868-875. 20 Spinelli W, Follmer C, Parsons R. Colatsky T: Effects of cromakalim. pinacidil and nicorandil on cardiac refractoriness and arterial pressure in open-chest dogs. Eur J Pharmacol 1990:179:243-252. 21 Chi L, Uprichard A. Lucchesi B: Profibrillatory actions of pinacidil in a conscious canine model of sud den coronary death. J Cardiovasc Pharmacol 1990:15:452-464.
Kitzen/McCallum/Harvey/Morin/ Oshiro/Colatsky
K Channel Activators and Myocardial Infarct Size
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References
Department of Cardiovascular-Metabolic Disorders Pharmacology, Wyeth-Ayerst Research Laboratories, Princeton, N.J., USA
Keywords Myocardial ischemia Myocardial infarction Potassium channel activators Calcium channel blockers Hemodynamics
Potassium Channel Activators Cromakalim and Celikalim (W A Y-120,491 ) Fail to Decrease Myocardial Infarct Size in the Anesthetized Canine
Abstract The cardioprotective effects of the K channel activator drugs celikalim (WAY-120,491) and cromakalim were studied in a canine model of myocardial infarction consisting of 90 min of ischemia and 5 h of reperfusion. Intracoronary infusion of cromakalim and celikalim at 0.2 pg/kg/min beginning 10 min before occlusion of the left circumflex coronary artery and continuing throughout the duration of the reperfusion period appeared to exacerbate ischemic injury. Infarct size (percent of risk area) was 27.7 ± 5.6% in vehicle control animals (n = 5), 40.3 ± 6.2% for cromakalim (n = 5) and 55.7 ± 6.4% (p < 0.05 vs. vehicle) for celikalim-treated animals (n = 5). When these compounds were administered intravenously, using doses shown to increase total coronary flow in nonoccluded control animals, no exacerbation of ischemic injury was ob served. Anatomic infarct size was 32.8 ± 7.1 % for vehicle ani mals (n = 5) and 32.6 ± 13.3 and 30.9 ± 9.8% for cromaka lim- (n = 6) and celikalim-treated (n = 5) animals, respectively. Intravenous diltiazem decreased myocardial infarct size to 16.3 ± 7.3% (n = 5) of area at risk (p = NS vs. vehicle). The anatomic area at risk was similar in all three treatment groups, and no significant differences in rate-pressure product were observed. Results of this study suggest that K-channel-activating drugs such as cromakalim and celikalim may not be effec tive agents in the acute therapeutic management of myocar dial ischemic injury.
Received: August 29, 1991 Accepted: November 4, 1991
Jan M. Kitzen, PhD Departmen: of Cardiovascular Biology Rhonc-Poulenc Rorer Central Research 640 Allendale Road King of Prussia. PA 19406 (USA)
© 1992 S. Kargcr AG, Basel 0031 -7012/92/ 0452-0071 $2.75/0
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Jan M. Kitzen John D. McCallum Charlotte Harvey Mary Ellen Morin George T. Oshiro Thomas J. Colatsky
Methods
The potassium channel activators repre sent a new class of pharmacologic agents that may find clinical usefulness in a variety of therapeutic areas including hypertension, asthma and urinary incontinence [1], These agents arc thought to cause relaxation of vas cular and nonvascular smooth muscle by en hancing the efflux of potassium ions from smooth muscle, leading to a hyperpolariza tion that opposes the entry of calcium [2], The resulting vasodilation leads to a decrease in blood pressure and increases in local blood flow. Previous studies from our laboratories have identified WAY-120.491 (cclikalim) to be a potent K channel activator [3], Other studies have shown that celikalim and cromakalim were both potent coronary vasodilators in the anesthetized dog [4], The ability of these agents to increase coronary blood flow suggests that they may be useful in the man agement of myocardial ischemia. Recent studies have presented evidence demonstrat ing that several potassium channel activators including RP-52.891 and EMD-56,431 were able to reduce myocardial infarct size and attenuate myocardial stunning in the canine [5. 6], Other investigators have shown that cromakalim and pinacidil were able to im prove postischemic dysfunction in the glob ally ischemic rat heart [7] as well as reduce infarct size in the canine [7, 8]. These observa tions led us to examine the ability of celikalim [(-)-(3S-trans)-2-(3,4-dihydro-3-hydroxy-2,2dimethyl - 6 - [trifluoromethoxy] - 2H -1 - ben zopyran-4-yl)-2,3 dihydro-1H-isoindol-1-one] to reduce infarct size in a model of infarction similar to that reported on by Grover et al. [ 8],
Surgery Dogs were fasted overnight but allowed free access to water. On the day of the experiment, the animals were anesthetized with sodium pentobarbital 35 mg/kg, i.v. An endotracheal tube was inserted and connected to a Harvard respirator set at approximately 17 strokes/'min and 15 cmVkg/strokc to provide posi tive pressure ventilation with room air. The left femo ral artery and vein were cannulated with polyethylene tubing for measurement of arterial blood pressure and for intravenous administration of electrolyte solution to support blood pressure after defibrillation. An intra venous line was placed in the saphenous vein for con tinuous infusion of anesthetic. In order to maintain a constant depth of anesthesia during the procedure, the bolus dose of pentobarbital was supplemented with an intravenous infusion of pentobarbital solution (0.08 mg/kg/min, i.v.. infused at a rate of 0.039 ml/min) via a Harvard syringe pump. The right carotid artery was cannulated with a Millar Mikro-Tip pressure transducer and inserted into the left ventricle for direct measurement of left ventricular pressure. An intercostal incision was made between the fourth and fifth ribs on the left side to expose the heart, and the heart was suspended in a pericardial cradle. A monopolar plunge electrode was implanted in the circumflex distribution to monitor regional S-T segment changes from the lead V electrocardiogram (ECG). Lead II electrodes were also inserted, and waveforms for each lead were displayed throughout the experiment. The left circumflex coronary artery (LCCA) was dissected free from surrounding fat and connective tissue. A 1inch segment of the vessel was prepared, and the artery was instrumented proximally to distally with an elec tromagnetic flow probe, an occlusive snare and an intracoronary needle for drug infusion. The needle is prepared from a 25-gauge stainless-steel syringe needle that is curved to permit insertion into the coronary lumen parallel to the vessel. The needle is anchored to the epicardial surface of the heart, and the lumen is maintained patent by a continuous infusion of saline solution until the beginning of test substance infusion. The flow probe was connected to a Carolina Electro magnetic Flowmeter (Model 501), and left circumflex coronary flow was displayed throughout the experi ment. Experimental Protocol Protocol I: Intracoronary Drug Administration. Af ter the animal had stabilized, drug infusion was started 10 min before occlusion of the LCCA. Drugs were
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Introduction
Perfusion o f tlw Heart for Area at Risk and Staining the Heart for Infarct Measurements At the completion of reperfusion, the LCCA was ligated and cannulated with polyethylene tubing. The heart was perfused with 0.9% saline solution at a (low rate adjusted to yield a perfusion pressure equal to the animal's MAP at the end of reperfusion. Five minutes later, patent blue dye, I mg/kg (of 10 mg/ml solution) was administered directly into the left atrial appendage via a polyethylene cannula. After 2 min, if the heart had not arrested spontaneously, it was fibrillated elec
trically with a brief high-frequency pulse from a Grass S44 stimulator and immediately excised. The heart was rinsed under tap water, blotted dry and weighed. The atria and right ventricle were re moved, and the left ventricle was then weighed. The ventricle was sliced into four transverse 1-cm sections from base to apex. The slices were incubated for 15 min in a solution of 1.0% triphenyltetrazolium warmed to approximately 37 °C. Each slice was blot ted dry and weighed. The normal zone, area at risk and infarct zone were traced onto acetate sheets for planimetric measurements of infarct size. Exclusion Criteria In order for infarct data to be included in the data analysis, animals must have met the following criteria: (1) S-T elevation of at least 10 mV in lead V ECG dur ing LCCA occlusion - this did not have to be sustained throughout the occlusion period; (2) presence of ar rhythmias during reperfusion; (3) cyanotic appearance of epicardial surface following occlusion: (4) not more than four defibrillations (10-20 J) to restore cardiac rhythm in any animal developing ventricular fibrilla tion during occlusion or reperfusion.
Results Seventy-three animals were entered into this study. Thirty animals were used in pilot studies for determining drug dosages for in tracoronary and intravenous routes of admin istration. For the intracoronary drug adminis tration protocol in infarctcd animals, 16 ani mals were utilized. One animal was excluded due to failure to develop adequate ischemia. For the intravenous protocol. 15 animals were used in pilot studies, and 27 animals were entered into the infarct protocol. Seven ani mals were excluded for the following reasons (treatment groups included in parentheses): 2 failed to develop ischemia (celikalim), 2 died of severe hypotension before the completion of reperfusion ( 1 celikalim, 1 diltiazem) and 3 animals required an excessive number of defi brillations to resuscitate (celikalim). Results of the pilot studies showed that neither cromakalim nor celikalim at 0.1 pg/ 73
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infused at a flow rate adjusted to 0.055 ml/min. Base line hemodynamic measurements were obtained [blood pressure, heart rate (HR), lead II, lead V, left ventricular pressure, dP/dt and LCCA How], drug infusion was started and a postdrug hemodynamic measurement obtained. Ten minutes later the LCCA was occluded for 90 min. At the end of the occlusion period the occlusive snare was released while moni toring coronary How such that baseline How was restored gradually over a period of about 5-10 min. Reperfusion was carried out for 5 h. All hemody namic data were channeled to a Modular Instruments computer for on-line data acquisition, data display and statistical analysis. Doses for the study were selected from pilot studies where dmgs were infused into the coronary circulation of nonischemic animals. The major criterion for dose selection was to identify doses of celikalim and cromakalim that produced comparable effects on coronary blood flow. Since dmgs were infused directly into the coronary circula tion, systemic hemodynamic actions were minimized. Three treatment groups (n = 5) were included in this protocol: vehicle: celikalim 0.2 pg/kg/min. and cro makalim 0.2 pg/kg/min. Protocol II: Intravenous Drug Administration. The same protocol as described above was utilized except that drugs were administered into a femoral vein. Pilot studies were conducted in nonischemic animals in order to identify doses that produced the least amount of change in the rate-pressure product (RPP), i.c. mean arterial pressure (MAP) X HR. Four treatment groups were included in this portion of the study: vehicle: celi kalim 0.7 pg/kg/min; cromakalim 0.4 pg/kg/min. and diltiazem 0.18mg/kg (bolus) +7.5 pg/kg/min (infu sion). The vehicle used to solubilize all drugs was dimethylsulfoxide: polyethylene glycol 200: HiO (1.68:2.33:1.0). Appropriate volumes of this vehicle were then diluted into an infusion vehicle of 15 % poly ethylene glycol 200/saline to yield the exact doses required for each animal.
Table 1. H em odynam ic effects o f intracoronary adm inistration o f crom akalim and celikalim
Baseline Vehicle (n = 5) MAP. mm Hg HR. beats/min RPP, mm Hg X beats/min
116 ±4 138 ±6 16.027 ±538
After drug
Occlusion
Reperfusion
0 min
Oh
60 min 90 min
116 100 98 106 ±4 ±9 ±4 ±5 138 141 146 152 ±6 ±4 ±7 ±8 15.912 14.088 14.413 16.246 ±479 ±1,375 ±978 ±1.442
1h
2h
4h
5h
97 104 103 83 81 ±10 ± 6a ± 5a ±5 ±8a 160 133 156 179 165 ±10 ±11 ±9 ±19 ± 13a 15.410 13.668 16.443 14,913 13.348 ±1.156 ±1.307 ±2,388 ±1.680 ± 1,762
Cromakalim (n = 5) 0.2 yg/kg/min MAP, mm Hg 108 97 97 110 98 ±8 ±9 ±9 ±4 ±6 HR, beats/min 144 147 157 145 161 ±9 ±6 ±8 ±3 ±8 RPP. mm Hg 15,504 15.826 14.344 15.752 15,500 X beats/min ±1.293 ±1,478 ±1.903 ±1,168 ±1,315
86 85 81 65 63 ±7a ±3a ± 3a ±4a ±6a 143 158 161 196 193 ±11 ±7 ±4 ± 12a ±21a-b 12,357 13.396 13,137 12.654 12,562 ±1,158 ±800 ±1,311 ±1.346 ±2,300
Celikalim (n = 5) 0.2 yg/kg/min MAP. mm Hg 113 109 113 103 105 ±6 ±8 ±13 ±11 ±10 HR. beats/min 151 152 164 156 170 ±14 ±10 ±9 ±9 ±6 RPP. mm Hg 17,341 17,435 16.893 17.662 18.719 X beats/min ±2,001 ±2.199 ±3,316 ±2.572 ±2.242
99 90 88 78 76 ± 4 3 ±9 ±7a ± 5a 177 176 176 193 198 + 9a.b ±7 ± 8a-b ±8 ± 13a 17.574 16.070 15.625 15.220 15.052 ± 1.982 ±1.371 ±2,066 ±1.971 ±1.334 ± 9 3
kg/min, i.c., or 0.2 pg/kg/min, i.c., caused any significant changes in coronary blood flow. Since 0.2 pg/kg/min was approaching the up per limit of solubility for celikalim, no at tempt was made to increase the dose any fur ther. Other hemodynamic parameters (MAP. HR. dP/dt) were not significantly altered with these doses of the two K channel activators. Hemodynamics with Intracoronary Administration Mean Arterial Pressure, Heart Rale and Rate-Pressure Product. The effects of intracor onary administration of cromakalim and celi
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kalim on these parameters are summarized in table 1. Baseline MAP ranged between 108 and 116 mm Hg for the three treatment groups. Vehicle-treated animals showed a steady de cline in MAP throughout the occlusion-reper fusion period. By the end of the experiment MAP had decreased by 35 mm Hg. Peak de creases in MAP for cromakalim and celikalim were 45 and 37 mm Hg (p = NS vs. vehicle) over the same time period. Baseline HR ranged from 138 to 151 beats/min. and no significant baseline differ ences were observed between the three treat-
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K Channel Activators and Myocardial Infarct Size
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a p < 0.05 versus baseline;b p < 0.05 versus vehicle. All data presented as means ± SEM.
B
Tim e
(m in)
Tim e
(m in)
Fig. 1. Effects of intracoronary administration of cromakalim and cclikalim on LCCA blood fow (A) and regional ECG S-T segment (B). Regional S-T record ings were obtained from a unipolar plunge electrode placed in the posterolateral surface distal to the occlu-
sion site. Arrows denote time of drug administration. ■ = Vehicle; A = cclikalim. 0.2 gg/kg/min: • = cromaka lim, 0.2 gg/kg/min. Each point is the mean ± SEM of 5 observations. * p < 0.05 versus baseline.
inent groups. In general, HR tended to in crease over time in each treatment group; this was partially due to the fact that animals developed arrhythmias during the reperfu sion period. In vehicle-treated animals, HR reached a peak increase of +41 beats/min (p < 0.05 vs. baseline), while higher peak increases were attained in the cromakalim (+52 beats/min; p < 0.05 vs. baseline) and celikalim (+47 beats/min; p < 0.05 vs. base line) groups. RPP was calculated from the product of MAP and HR and was used to provide an index of myocardial oxygen consumption [9], No significant differences in RPP between groups were observed. Coronary Blood Flow and Regional S -T Segment. Baseline coronary blood flow ranged from 36 to 38 ml/min for all treatment groups (fig. IA). After complete obstruction
of coronary blood flow for 90 min. blood flow was slowly restored by carefully releasing ten sion on the occluder. In the first few minutes of reperfusion, significant hyperemia was ob served in all treatment groups (range: 62-64 ml/min). Coronary blood flow then declined toward baseline throughout the remainder of reperfusion. By the end of the experiment cor onary blood flow was 23-28 ml/min for the three treatment groups. In order to provide some index as to the severity of regional ischemia that developed in each animal, a unipolar electrode was im planted into the posterolateral wall in the region perfused by the LCCA to record local S-T elevation directly from the ischemic zone. Animals that did not display an S-T increase were assumed to be nonischemic and were excluded from the study. Previous stud ies [10] have shown this to be a reliable (al-
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A
from 160 ± 9 beats/min at baseline to 127 ± 7 beats/min at +90 min (p < 0.05 vs. base line). During reperfusion, HR was initially increased in the vehicle group and returned toward baseline by the end of the study. All animals displayed severe ectopy during reper fusion, and sinus HR was usually not ob served. Cromakalim- and celikalim-treated animals both displayed increases in HR dur ing reperfusion, while in diltiazem-treated an imals HR was maintained below baseline sinus rate throughout the reperfusion period. This effect on HR is consistent with results of Hemodynamics with Grover et al. [ 12] and Bush et al. [ 11 ]. Intravenous Administration There were no significant baseline differ Pilot studies were conducted in order to select doses of test drugs that produced the ences in RPP between the treatment groups least amount of systemic hemodynamic (baseline range = 16.001-19.485). RPP de changes. Diltiazem was included in this pro creased only by 2,753 units (17%) over the tocol to serve as a standard reference agent, time course of the experiment in vehiclesince several studies have shown diltiazem to treated animals. Cromakalim-treatcd animals reduce myocardial infarct size [11, 12]. The showed the smallest RPP decrease of all the diltiazem dose selected for the present investi drug treatment groups (peak decrease = 25 %). gation was the same as used by Grover et al. Peak decreases for celikalim and diltiazem [12]: 0.18 mg/kg i.v. (bolus) +0.0075 mg/kg/ were 37 and 34%, respectively. Although RPP min i.v. (infusion). This dose was shown to was significanlty decreased at several time exert less effect on the RPP than the dose used points in celikalim- and diltiazem-treated ani mals, these decreases were not significant in in the study by Bush et al. [11], Mean Arterial Blood Pressure, Heart Rate comparison with the vehicle group. Coronary Blood Flow and Regional S - T and Rate-Pressure Product. As shown in ta ble 2, no significant differences in blood pres Segment. Baseline coronary blood flow sure were observed between the three treat ranged from 40.0 ± 5.9 in vehicle-treated ani ment groups and the vehicle. Significant de mals to 57.0 ± 9.6 ml/min for celikalim. creases in diastolic blood pressure and MAP There were no significant differences in base were observed within each group, and the line flow between any of the treatment groups. peak decrease noted for the vehicle group was During reperfusion, coronary blood flow 23 mm Hg (MAP). Peak decreases in MAP (fig. 2A) never attained baseline level in the observed for the other treatment groups were vehicle-treated animals. Both K channel acti 36 mm Hg for cromakalim. 47 mm Hg for vators led to significant increases in coronary celikalim (due to slightly higher baseline blood flow that were sustained throughout most of the reperfusion period. This was MAP) and 27 mm Hg for diltiazem. HR was very stable throughout the occlu probably due to the coronary vasodilation sion period for the vehicle, cromakalim and action of these drugs which in the pilot studies celikalim treatment groups. In diltiazem- was observed to increase coronary blood flow treated animals, HR gradually decreased by 125 and 121 % above baseline for celikalim
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though imperfect) method for assessing re gional ischemia in animals where collateral flow cannot be measured. As shown in fig ure IB, animals in each treatment group de veloped comparable S-T segment elevation (range: 21-27 mV. p = NS between groups). In these studies, regional S-T segment changes were the only information available to evalu ate the extent of ischemia developed in all ani mals (see exclusion criteria in Methods) in response to occlusion of the LCCA.
Table 2. H em odynam ic effects o f intravenous adm inistration of crom akalint and celikalim
Base-
Vehicle in = 5) MAP, mm Hg HR. beats/min RPP. mm Hg X beats/min
After
Occlusion
Reperfusion
t*rU®
0 min
Oh
60 min 90 min
109 104 84 92 97 +6 ±7 ±11 ±8 ±8 146 151 146 156 151 ±10 ±8 ±16 ±7 ±6 16.001 15,828 12,956 14,580 14,824 ±1,878 ±1.813 ±2.769 ±1,813 ±1.565
Cromakalim (t,i -6 ) 0.4 fig/kg/min MAP. mm Hg 112 113 95 ±4 ±4 ±4 HR. beats/min 150 150 157 ±5 ±6 ±9 RPP. mm Hg 16.698 16.841 14,803 X beats/min ±558 ±505 ±611
Ih
2h
4h
5h
86 91 90 96 86 ±9 ±8 ±11 ±10 ± 8a 177 162 172 145 148 ±16" ±11 ±7 ±15 ± 19 15.642 14.981 16,682 13,460 13.248 ±2,713 ±2,251 ±2,463 ±2,735 ±2.880
83 88 80 87 89 77 76 ±4“ ±7“ ±9a ±6“ ±8a ± 6a ±6a 151 154 160 174 165 179 190 ±5 ±6 ±11 ±8 ±9 ±16a-b + 15a,b 12.523 13.591 12.860 14,176 15,603 13.514 14.308 ±522° ±1.229 ± l,774a ±937 ±1.732 ±1,356 ±1,417
Celikalim (n = 4) 0.7 fig /kg /min MAP. mm Hg 107 122 121 97 97 84 87 80 73 75 ±4 ±5 ±8 ±7a ± l l a ± I0a ± 7a ± 6a ± 6 a ± 6a HR. beats/min 160 164 170 164 168 156 201 172 165 173 ±5 ±7 ±5 ±6 ±7 ± I0a ±11 ±5 ±6 ±3 RPP. mm Hg 19.485 19,913 18,248 16.035 16.480 17.064 12,729 14.994 11.960 12,935 X beats/min ±927 ±1,370 ±1,751 ±1.720 ±2,433 ±2,227 ± 1,910 • 1.120 ±917 ± 818a Dihiazem (n =5) 0. IS mg/kg bolus + 0.0075 mg/kg/min MAP. mm Hg 97 112 107 92 88 94 85 102 95 88 ±5 ±6 ±6 ±5 ±5“ ±6a ±5 ±5 ±6 ±5a HR. beats/min 160 151 149 135 127 143 122 133 131 134 ±9 ±15 ±8 ± 7a ±8 ±19 ± 6a-b ±5b ±11 ±9 RPP. mm Hg 17.985 16.178 14,522 12.550 11.254 12.074 11,498 13.569 12,532 11.797 X beats/min ±1,461 ±2.033 ±1,425 ± l,340a ± l,059a ± 1.417a ±877a ± 1,047a ± l,627a ± 1,159a
and cromakalim, respectively. In diltiazemtreated animals coronary flow returned to baseline for the duration of the reperfusion period. S-T segments ranged from 11.8 to 15.5 mV during the 90-min occlusion period in vehicle-treated animals. S-T segments were similar for diltiazem-treated animals, ranging from 9.7 to 13.9 mV. In both groups of ani
mals treated with the K channel activators, ST segment increases were more pronounced than those in the vehicle and diltiazcm groups. Cromakalim-treated animals dis played the most pronounced increases in S-T segment (range: 14.3-26.6 mV) with several of these increases being significantly different from S-T changes observed in the vehicle group. The range of S-T segments observed
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a p < 0.05 versus baseline:b p < 0.05 versus vehicle. All data presented as means ± SEM.
B
A
■
1
50
F—
1
1
1
I
1
1
1 00 1 50 2 0 0 2 5 0 3 00 3 5 0 4 00 Tim e
Tim e
(m in)
(m in)
Fig. 2. Effects of intravenous administration of cromakalim and celikalim on LCCA blood flow (A) and regional ECG S-T segment (B). ECG recordings obtained as in figure 1. Arrows denote time of drug administration. □ = Vehicle (n = 5); ▲ = celikalim.
0.7 gg/kg/min (n = 4); • = cromakalim. 0.4 gg/kg/min (n = 6):*= diltiazem, 0.18 mg/kg(bolus) +0.0075 mg/ kg/min (n = 5). Each point is the mean ± SEM of 5 observations. * p < 0.05 versus baseline, @ p < 0.05 versus vehicle.
for celikalim-treated animals was 13.7-20.2 mV. Whether or not the exacerbated increases in S-T segments in animals treated with a K channel activator are due to severer ischemia or the K-channel-opening effects of these drugs cannot be determined from these data. Previous work in our laboratory with K. chan nel activator drugs in thrombosis studies has shown them to increase S-T changes induced by thrombotic occlusion [13]; however S-T segments were normal under conditions of physiological coronary perfusion. It should be pointed out that in this study, all 3 of the excluded animals treated with celikalim that developed ventricular fibrillation did so dur ing the reperfusion period. It is interesting to note that no differences in S-T segment be tween drug and vehicle were observed in the intracoronary administration protocol.
Effect o f Intracoronary Administration on Myocardial Infarct Size As shown in figure 3A. the anatomic size of the ischemic zone (area at risk) was similar in each of the three treatment groups (range for all groups: 35.7-48.5% of left ventricle). In farct size (expressed as percentage of area at risk) was 27.7±5.6% for vehicle-treated ani mals. Intracoronary administration of the K channel activators resulted in larger infarcts: 40.3±6.2% for cromakalim and 55.7±6.4% for celikalim (p < 0.05 for celikalim vs. vehi cle). A scatter plot (not shown) of the individ ual infarcts for each treatment group showed a trend for the K channel activator drugs to cause a wider distribution of infarct size in contrast to a more clustered distribution ob served in the vehicle group. The regional S-T segment changes, shown in figure 1B indicate
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I
0
B
AAR
l/AAR
l/LV
■
AAR
Vehicto
□
Celikalim
Cromakalim
||j
Diliiazem
l/AAR
l/LV
Fig. 3. Effects of K. channel activators and diltiazem on myocardial infarct size. AAR = Area at risk; I/AAR = infarct size/area at risk; I/LV = infarct size/ left ventricle, n = 5 for all treatments unless indicated otherwise. A Intracoronary administration. Test sub stances were infused directly into the coronary circula-
tion beginning 10 min before occlusion of the vessel and were infused for the duration of the experiment. * p < 0.05 versus vehicle, n = 5 for all treatments. B In travenous administration. Test substances were in fused beginning 10 min before occlusion of the vessel and were infused for the duration of the experiment.
that all animals developed comparable in creases in the S-T segment. Attempts to relate infarct size to S-T changes were unsuccess ful.
farct size was 32.8% of area at risk. This value is somewhat smaller than infarct size data reported by Grover et al. [7, 8,12] and Bush et al. [11] where size of vehicle infarcts ranged from 40 to 45% of risk area. In our studies, both K channel activators failed to affect myocardial infarct size: infarct sizes for these two groups were 32.6 ± 13.3 and 30.9 ± 9.8% for cromakalim and celikalim, respec tively (p = NS vs. vehicle). These infarct sizes correspond to decreases of 0.6% for cromakalim and 5.7% for celi kalim in comparison with the infarcts in vehi-
Effects o f Intravenous Administration on Myocardial In farct Size Area at risk ranged from 35.1 to 40.4% in the four treatment groups (fig. 3B). Risk area was slightly smaller in the diltiazem group (p = NS vs. vehicle) due to 1 animal in which the area at risk was only 30.4% of the left ven tricular mass. In the vehicle group mean in
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A
Discussion Results from this study demonstrate that the K channel activator drugs cromakalim and celikalim were unable to reduce myocar dial infarct size in this canine model of isch emic injury. Data further suggest that croma kalim and celikalim have the potential to ex acerbate ischemic injury. This conclusion is supported by the observation that both K channel activators produced larger infarcts (in comparison with vehicle) when these drugs were administered directly into the coronary circulation. In contrast, intravenous adminis tration of these compounds did not increase infarct size; however, the infarcts were similar to those in vehicle-treated animals. Exacerba tion of ischemic injury with K channel activa tors has been observed by other investigators, although under somewhat different experi mental conditions. Thus. Sakamoto et al. [ 14] observed that pinacidil decreased blood flow to the infarct zone and increased infarct size (4-hour occlusion of the left anterior descend
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ing artery) in animals with a moderate steno sis placed on a non-infarct-related (LCCA) artery, while Imai et al. [15] showed that pin acidil was unable to decrease infarct size in animals subjected to a 4-hour occlusion of the left anterior descending artery. In these latter studies, pinacidil increased blood flow to the normal zone and had no effect on blood (low to the infarct zone during occlusion yet it increased flow to the epicardium during re perfusion. Nitroprusside was found to in crease blood flow within the infarct zone and was also shown to decrease myocardial infarct size by 31 %. Our results do not support the findings of Grover et al. [7, 8], who showed that intracor onary administration of cromakalim, 0.1 mg/ kg/min, decreased myocardial infarct size. Our studies used the same dosing regimen and experimental protocol as used by these investigators [7. 8], Our results are also in dis agreement with those reported by Auchampach et al. [5], who showed that the K channel activator RP-52891 significantly decreased myocardial infarct size in an experimental infarct model similar to the one reported on here. In our studies we were able to control or measure several of the factors known to affect myocardial infarct size. Thus, our data showed that the following factors were not responsible for the differences in myocardial infarct size between K channel activators and vehicle-treated animals: size of area at risk, blood oxygen content (as pO?, data not shown), and myocardial oxygen consumption (indirectly assessed as RPP). The decreases in RPP observed for the test drugs would, if any thing, be expected to lead to a decrease in infarct size since this parameter suggests that myocardial utilization of oxygen was de creased in these animals. Collateral blood flow to the infarct zone is a major determi nant of infarct size [16], and this variable was
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cle-treated animals. Diltiazem-treated ani mals showed the smallest infarcts of all four treatment groups: 16.3 ± 7.3%, correspond ing to a 50% decrease in infarct size compared to vehicle. These results are in good agree ment with those reported by Bush et al. [11] and Grover et al. [ 12], who found diltiazem to decrease infarct size by 35 and 45%. respec tively. The total number of animals that de veloped ventricular fibrillation among the treatment groups (including exclusions) was: vehicle, 1; cromakalim, 3; celikalim, 5, and diltiazem, 1. The relationship between drug and incidence of fibrillation cannot be estab lished from this study: however, there ap peared to be a tendency for the K channel activators to exacerbate arrhythmias during the occlusion period.
An important aspect of our results that lends support to our conclusion is the obser vation that our reference standard diltiazem was able to decrease myocardial infarct size. These results confirm earlier work by two sep arate investigators [11, 12] and therefore sug gest that our findings for the K channel acti vator drugs were probably not due to intrinsic differences (drug-independent effects) in col lateral How between the treatment groups. Although we did not examine the effects of intracoronary diltiazem to modify experi mental infarct size, a recent study by Higginson et al. [19] demonstrated that intracoro nary administration of diltiazem at the onset of ischemia was able to decrease infarct size significantly (68% decrease from control). In our study the effect of intravenous diltiazem on infarct size was not statistically significant; however, the decrease observed was pro nounced (50% decrease) and probably failed to attain significance due to the small sample size. In conclusion, our results for cromakalim and celikalim in this study suggest that K channel activators may not be effective agents for therapy of ischemic myocardial injury. It should be pointed out that both of these agents were benzopyran derivatives and it is possible that other chemical classes of K chan nel activators may not share these effects on the ischemic heart. It is interesting to note, however, that other investigators [20] have shown that various types of K channel activa tors can exert similar effects on myocardial refractoriness. Thus, Spinelli et al. [20] found that under normal physiological conditions cromakalim, pinacidil and nicorandil short ened only atrial refractoriness and were able to produce atrial arrhythmias but not ventric ular arrhythmias. However, Chi et al. [21] showed that in the postinfarction heart pin acidil was able to decrease ventricular refrac toriness and cause an increased incidence of
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not measured in these studies. Because of the importance of this factor in affecting ultimate infarct size, it is reasonable to suggest that dif ferences in collateral flow between experi mental subjects may have contributed to ob served differences in infarct size. Thus, it is possible that animals treated with intracoro nary cromakalim and celikalim may have had less tissue perfusion due to poor collateral cir culation than vehicle-treated animals during the occlusion period. However, since experi mental treatments were allocated in a ran domized fashion, it is unlikely that only those animals in these two treatment groups devel oped ischemia. The failure to demonstrate a reduction in infarct size with intracoronary administration of the K channel activators led us to repeat the study using systemically administered test drugs. For this portion of the study we also included the calcium-channel-blocking drug diltiazem, which has been demonstrated by several investigators to exert significant car dioprotective effects [11, 12, 17-19], Under similar experimental conditions as used in protocol I. we found that intravenous admin istration of the K channel activators resulted in no effect on infarct size. Pilot studies for each of these protocols showed that drug effects on coronary blood flow were affected differently by the two routes of administra tion. Thus, intracoronary administration of cromakalim and celikalim did not cause coro nary blood flow to increase during reperfu sion yet intravenous administration led to marked increases in coronary blood flow (fig. 1A, 2A). It seems possible that intracoro nary administration of these drugs, while not increasing total coronary flow, may have caused endocardial steal, an effect not ob served by Grover et al. [12] yet one that has been reported by others [13, 14], although under different experimental conditions (see above).
ventricular fibrillation when a second vascu lar bed was made ischemic. These observa tions suggest that drugs of the K channel acti vator type appear to exert complex pharmaco logic actions affecting myocardial conduction
and coronary blood flow. The usefulness of these agents in the ischemic heart will require additional characterization in order for their role in the therapy of cardiovascular disease to be better understood.
1 Edwards G. Weston A: Structureactivity relationships of K ' channel openers. Trends Pharmacol Sci 1990;11:417-422. 2 Cook N. Weir S, Danzeisen MC: Anti-vasoconstrictor effects of the K* channel opener cromakalim on the rabbit aorta - Comparison with the calcium antagonist isradipine. BrJ Pharmacol 1988;95:741-752. 3 Lodge N. Cohen R. Havens C. Colatsky TJ: The effects of the putative potassium channel activator WAY120,491 on s6Rb efflux from the rab bit aorta. J Pharmacol Exp Ther 1991;256:639-644. 4 Kitzen J. Stupienski R. DeSialo M. Rovnvak L, Pirozzi C. Oshiro G. Colatsky TJ: Hemodynamic effects of WAY-120,491 (WAY). [(-)-(3Strans)-2-(3.4-dihydro-3-hydroxy-2,2dimethyl-6-[trifiuoromcthoxy]-2H-lbenzopyran-4-yl)-2,3-dihydro-111isoindol-l-one). pinacidil (P). and cromakalim (C) in anesthetized dogs. FASEBJ 1990:4:2786. 5 Auchampach J, Maruvama M, Cavero 1, Gross G: The potassium channel agonist RP-52891 reduces infarct size in the anesthetized dog. Pharmacologist 1990:32:164. 6 Maruyama M, Gross G: Evidence for a role for potassium channels in stunned myocardium. FASEB J 1990;4:315. 7 Grover G, Sleph P. Dwonczyk S: Pharmacologic profile of croma kalim in the treatment of myocar dial ischemia in isolated rate hearts and anesthetized dogs. J Cardiovasc Pharmacol 1990:16:853-864.
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8 Grover G, Dwonczyk S, Parham C, Sleph P: The protective effects of cromakalim and pinacidil on reper fusion function and infarct size in isolated perfused rat hearts and anesthetized dogs. Cardiovasc Drug Ther 1990:4:465-474. 9 Gobcl F, Nordstrom L. Nelson R. Jorgensen C. Wang Y: The ratepressure product as an index of myocardial oxygen consumption during exercise in patients with an gina pectoris. Circulation 1978:57: 549-556. 10 Kitzen J. l.ucchesi B: Retrograde bleeding enhances incidence and se verity o f ischemia in the canine heart. FASEBJ 1989:3:2992. 11 Bush L, Romson J, Ash J, Lucchesi B: Effects of diltiazem on extent of ultimate myocardial injury resulting from temporary coronary artery oc clusion in dogs. J Cardiovasc Phar macol 1982:4:285-296. 12 Grover G, Sleph P. Parham C: Ef fect of diltiazem on infarct size, re perfusion flow and flow reserve. The effect of timing of treatment. J Phar macol Exp Ther 1988:246:263-269. 13 Kitzen J. McCallum J. Harvey C, Morin M: Antithrombotic activity of the phosphodiesterase III inhibi tor pclrinone in a canine model of coronary artery thrombosis: En hancement of efficacy with concur rent alpha-2 adrenergic antagonism. J Cardiovasc Pharmacol 1991:18: 777-790. 14 Sakamoto S, Liang C. Stone C, Hood W: Effects of pinacidil on myocardial blood flow and infarct size after acute left anterior descend ing coronary artery occlusion and reperfusion in awake dogs with and without a coexisting left circumflex coronary artery stenosis. J Cardio vasc Pharmacol 1989:14:747-755.
15 1mai N, Liang C, Stone C. Sakamoto S. Hood W: Comparative effects of nitroprusside and pinacidil on myo cardial blood flow and infarct size in awake dogs with acute myocardial infarction. Circulation 1988:77: 705-711. 16 Reimcr K, Jennings R. Cobb F, Murdock R. Greenfield J. Becker L, Healy-Bulklcv B. Hutchins G, Schwartz R. Bailey K. Passamani F.: Animal models for protecting isch emic myocardium. Results of the NHI.BI cooperative study. Compar ison of unconscious and conscious dog models. Circ Res 1985:56:651 — 665. 17 Wcishaar R. Ashikawa K. Bing R: Effect of diltiazem, a calcium antag onist. on myocardial ischemia. Am J Cardiol 1979;43:1137-1143. 18 Bush L, Li Y, Shlafer M. Jolly S, Lucchesi B: Protective effects of dilliazem during myocardial ischemia in isolated cat hearts. J Pharmacol Exp Ther 1981;218:653-661. 19 Higginson L., Tang A. Knoll G. Cal vin J: Effect of intracoronarv diltia zem on infarct size and regional myocardial function in the ischemic reperfused canine heart. J Am Coll Cardiol 1991:18:868-875. 20 Spinelli W, Follmer C, Parsons R. Colatsky T: Effects of cromakalim. pinacidil and nicorandil on cardiac refractoriness and arterial pressure in open-chest dogs. Eur J Pharmacol 1990:179:243-252. 21 Chi L, Uprichard A. Lucchesi B: Profibrillatory actions of pinacidil in a conscious canine model of sud den coronary death. J Cardiovasc Pharmacol 1990:15:452-464.
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References
