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Selective antagonism of peptidoleukotriene response does not reduce myocardial damage opr neutrophil accumulation following coronary artery occlusion with reperfusion
PROSTAGLANDINS
SELECTIVE ANTAGONISM OF PEPTIDOLEUKOTRIENE RESPONSES DOES NOT REDUCE MYOCARDIAL DAMAGE OR NEUTROPHIL ACCUMULATION FOLLOWING CORONARY ARTERY OCCLUSION WITH REPERFUS~ON. J.W. Egan~, D.E. G r i s w o l ~ ~, L.M. H i l l e g ~ s s J.F. N e w t o n s , R.D. Eckardt ~, M.~. S l i v j a k ~, and E.F. Smith III
,
S m i t h ~ i n e & French Labs., l~pts, of Pharmacology I , In-nunology', and Drug Metabolism u King of Prussia, PA 19406
ABSTRACT This study was designed to assess the effect of a peptidoleukotriene receptor antagonist, SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MIIR). In conscious rats, SK&F 104353 (25 mglkg, i . v . ) antagonized LTD4-induced vasopressor responses by 90% and 60~ at l and 4 hr, respectively, indicating effective blockade of peptidoleukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil i n f i l t r a t i o n were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) a c t i v i t y , respectively, in the l e f t ventricular free wall (LVFN). Myocardial CPK levels were 8.1 ± 0.2 Ulmg protein in ShamMIIR vehicle-treated animals, and were significantly decreased to 6.4 ± 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 ± 0.5 Ulg LVFN in Sham-MIIR vehicle-treated animals, and significantly increased to 4.3 ± 0.6 Ulg LVFN in MI/R-vehicle animals. Administration of SK&F I04353 (25 mg/kg, i . v . ) l min prior to coronary occlusion and 3.5 hr post reperfusion had no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p > 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD4-1nduced vasopressor responses, SK&F I04353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/ reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury. INTRODUCTION The role of leukotrienes (LT) in myocardial ischemic/reperfusion injury and inflammatory cell accumulation is an area of intensive investigation. LTB4 increases vascular permeability and acts as a chemotaxin for polymorphonuclear leukocytes (PMN; I). Although less potent as chemotactic and chemokinetic substances, the peptidoleukotrienes LTC4, LTD4, and LTE4 are potent coronary vasoconstrictors
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PROSTAGLANDINS (2-4), bronchoconstrictors (5), and increase vascular permeability (6). In the reperfused myocardlum, the formation of peptidoleukotrienes and LTB4 are increased (7-9), although the time course of this increase has not been precisely characterized. Nonetheless, the nonselective 5-lipoxygenase inhlbitors BN 755C ( l O , l l ) , REV 590l (12) and AA-861 (g) have been reported to reduce myocardial reperfusion injury. Thus, there is evidence to support the suggestion that products of the 5-1ipoxygenase pathway may be involved in the sequelae of myocardial reperfusion injury and inflammatory cell accumulation. However, the specific role of peptidoleukotrienes as mediators of myocardial reperfusion injury remains undefined. The purposes of this study were f i r s t to characterize the pharmacokinetics and pharmacodynamics of a potent and selective peptidoleukotriene receptor antagonist, SK&F I04353 (2(S)-methoxy-3(R)-(2carboxyethylthio)-3-(2-(8-phenyloctyl)phenyl) propanoic acid; 5,13). Subsequently, we sought to determine the role of peptidoleukotrienes in myocardial Ischemic/reperfusion injury by examining the cardioprotective effects of SK&F I04353 in a well characterized rat model of ischemiclreperfusion injury (14-16). Myocardial injury was produced by coronary artery occlusion for 30 min followed by reperfusion for 24 hr. Additionally, myeloperoxidase (MPO) activity in the ischemic/ reperfused myocardlum was measured in order to assess the extent of polymorphonuclear leukocyte (PMN) accumulation. METHODS Pharmacoklnetic Evaluation Male Sprague-Dawley rats, weighing 375-450 g, were anesthetized with pentobarbital sodium (30 mglkg, i . p . ) and prepared, with modifications, as described previously (17). Briefly, catheters (0.60 mm i . d . , l.Ol mm o.d.) were placed into the vena cava and abdominal aorta through the l e f t femoral vein and artery, respectively. Both catheters were led subcutaneously along the tail and exteriorized through an incision 5 cm from the base of the t a i l . A lO ml syringe barrel, with the closed end removed, was placed over the tail and secured with tape. The syringe barrel served to protect the incision site and catheters. The animals were housed in individual cages and the syringe barrel used to tether the animals. Both catheters were kept patent with an infusion of 0.9% NaCl containing 2.4 Ulml heparin at a rate of 0.6 ml/hr. Blood pressure was continuously monitored (Statham P23 DC pressure transducer) on a Grass Model 7D multichannel recorder. Heart rate was determined by counting the deflections in arterial blood pressure from fast paper tracings. The animals were allowed to stabilize for I-2 days following the surgical procedures. The animals were permitted access to food and water ad libitum. The pharmacokinetics of SK&F I04353 were determined following the intravenous administration of a bolus of 25 mg/kg. Blood samples (200- 250 ~l; less than 2 ml total volume) were withdrawn via a venous catheter into heparinized syringes, and transfered to
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PROSTAGLANDINS Eppendorf mlcrotubes. Plasmawas separated by centrifugation at 4oc, and lO0 gl was transferred for extraction and determination of SK&F I04353 plasma concentrations as described below. Plasma concentrations versus time profiles for SK&F I04353 were analyzed using PHARM (18). The area under the plasma concentration curve (AUC) was determined using the log trapezoidal rule. The AUC from the last time point to i n f i n i t y was extrapolated by dividing the last data point by the terminal rate constant determined from the f i t t e d data. The AUC from the time of drug administration to the f i r s t sample point was determined using f i t t e d data. The data were f i t to the appropriate sum of exponentials (three compartmental model) by peeling, and the h a l f - l i f e for each disposition phase determined. The percentage of the dose irreversibly removed in the beta (% ~2 phase) and gamma phase (% ~3 phase) was calculated by the method of separate exponentials (19). The volume of d i s t r i bution at steady state was calculated by non-compartmental methods (20). Since multiple samples were taken from individual animals, the pharmacokinetic parameters were f i r s t estimated for individual animals and then averaged to obtain mean estimates. Pharmacodvnamic Evaluation The pharmacodynamics of SK&F I04353 were determined in a separate group of animals. Animals were prepared as described above, and received either LTD4 intravenously, at 45 min intervals, in increasing doses ranging from 0.17 - 51 nmoles/kg ( i . e . , 0.085 - 25 ~g/kg). LTD4 was administered in a volume of O.l ml/lOOg body weight and followed with a 0.5 ml saline flush. These i n i t i a l experiments indicated that the EDso dose of LTD4 ( i . e . , dose of LTD4 required to increase arterial blood pressure to BO% of the maximal LTD4 response) was 5.1 nmole/kg ( i . e . , 2.5 ~g/kg). On a subsequent day, animals were repeatedly injected with 5.1 nmole/kg LTD4, at 40 min intervals, to determine the reproducibility of hemodynamic responses to challenges of LTD4. On a separate day, animals were administered a dose of LTD4 (5.1 nmole/kg i . v . ) that caused a marked pressor response. Approximately l hr following this LTD4 challenge, animals were randomized into two groups and received either SK&F 104353 (25 mg/kg, i . v . ) or vehicle (0.9% NaCI). A second dose of either SK&F 104353 (25 mg/kg, i . v . ) or vehicle was administerd 4 hr later. Following the admini s t r a t i o n of either SK&F 104353 or vehicle, LTD4 (5.1 nmole/kg, i . v . ) was repeatedly administered and the acute increase in mean arterial blood pressure following each injection was monitored. Determination of Plasma SK&F 104353 Concentrations Plasma SK&F I04353 concentrations were determined according to methods previously described (17). Briefly, the internal standard SK&F I04652 (2(S)-methoxy-3(S)-(2-carboxyethylthio)- 3-(2-(8-phenyloctyl)phenyl) propanoic acid), was added to plasma samples, and extracted twice with methyl formate containing 0.5% trifluoroacetic acid. Samples were vortexed and centrifuged at lOO0 x g for 5 min.
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PROSTAGLANDINS The supernatants were combined, evaporated to dryness under nitrogen, and resuspended in HPLC mobile phase. Aliquots (25 ~l) were quantitated on an HPLC system using a 5 um NOVAPAK C18 cartridge (Waters Assoc., Inc., Milford, MA). HPLCeffluent was monitored at 215 nm with a Kratos Model 783 uv detector. SK&FI04353 and the internal standard were separated i s o c r a t i c a l l y using a solvent system consisting of 52~ acetonitrile in O.l~ trifluoroacetic acid at a flow rate of 2.0 mllmin. Plasmaconcentrations of SK&F I04353 were determined from peak area ratios (compared to internal standard) that were compared to extracted authentic standard curves. The detection l i m i t for the quantitation of SK&F I04353 was 0.25 ~glml, and the standard curve of the assay was linear to lO00 wglml. Coronary Artery Occlusion Coronary artery occlusion was produced in rats similar to procedures described previously (16). Briefly, male Sprague-Dawley rats, weighing 300 - 370 g, were anesthetized with ethyl ether. An i n c i sion was made on the l e f t side of the thorax over the pectoral muscles and the tissues retracted to expose the fourth intercostal space. Sutures were placed through the overlapping skin and muscles to permit rapid closure of the chest wall following the surgical procedure. The chest was then opened through the fourth intercostal space and the ribs were gently spread. The heart was quickly expressed out of the thoracic cavity, inverted, and a 6-0 s i l k ligature placed under the visualized l e f t main coronary artery. The ligature was then either ligated with a "shoe-string" knot (MI/R animals) or removed (Sham MI animals). In animals prepared for reperfusion of the ischemic myocardium (MIIR group), the ends of the suture used to produce the coronary ligation were exteriorized through the chest wall. The heart was returned quickly to the thoracic cavity, the a i r removed by s l i g h t l y compressing the chest wall, and the incision was closed by tying the previously placed sutures. The animals were returned to their cages and were observed to have recovered from the anesthetic within l to 2 min. The coronary artery was reperfused after 30 min of occlusion in conscious animals by releasing the knot and removing the suture. The animals were allowed food and water ad libitum. Animals were administered SK&F 104353 (25 mglkg) or vehicle (0.9% NaCl) intravenously via the dorsal penal vein l min prior to occlusion and again at 3.5 hr post reperfusion. The dose of SK&F I04353 refers to the free acid. All substances were prepared fresh daily and administered in a volume of approximately l m]. CPK Measurement Twenty four hr later, the rats were k i l l e d with CO2, weighed, and their hearts removed and placed into ice cold saline. Attached connective tissue was carefully removed and the hearts were dissected into right ventricular wall, septum and l e f t ventricular wall (LVFN) segments. All myocardial segments were then weighed. The complete LVFW was homogenized for 2 x 5 sec in cold 0.25 M sucrose containing I mM EDTA and lO mMmercaptoethanol with a Tekmar tissue homogenizer
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PROSTAGLANDINS (Cincinnati, OH). The homogenates were centrifuged at 30,000 x g for 30 min at 4oc. The supernatants were decanted and stored at 4°C until analyzed for creatine phosphokinase (CPK) activity and protein concentration, according to previously published techniques (21,22). CPK specific a c t i v i t y was expressed as units/mg protein. The remaining pellet was frozen on dry ice and stored at -20oc until assayed for MPO activity. The depletion of CPK specific activity has been shown in a number of previous studies to correlate with the histologic determination of infarct size in experimental animals ( i . e . , the rat; 23,24) and humans (25). Myeloperoxidase Activity Myeloperoxidase content of rat heart tissue was determined by the method of Bradley et at., (26), and modified as described previously (16,24). Briefly, MPO activity was determined from the pellet of the LVFH homogenate. The pellet was f i r s t resuspended in 20 ml 5 mM potassium phosphate buffer (pH 6) and then centrifuged for 30 min at 30,000 x g at 4°C. The resulting pellet was prepared as a lO~ homogenate with extraction buffer consisting of 0.5% hexacyltrimethylammonium bromide (Sigma Chemical Co., St. Louis, MO) dissolved in 50 mM potassium phosphate buffer (pH 6) at room temperature and immediately frozen on dry ice. Three freeze and thaw cycles were performed with sonication for lO sec between cycles. After the last sonication, the samples were allowed to chill on ice for approximately 30 min, and then centrifuged for 15 min at 12,500 x g at 4oc. An aliquot of the supernatant was then reacted with 0.167 mg/ml O-dianisidine dlhydrochloride (Sigma Chemical Co., St. Louis, MO) and 0.0005% HzO2, and the rate of change in absorbance was measured at 460 nm in an UV spectrophotometer. One unit of MPO a c t i v i t y is defined as that amount of MPO capable of degrading one micromole of peroxide/min at 25oc, and was expressed in units per g wet weight (U/g) of tissue. The usefulness of measuring MPO activity to assess PMN i n f i l t r a t i o n has been previously reported (26,28), and recently validated in the rat (16,27). Materials SK&F 104343 (2(S)-methoxy-3(R)-(2-carboxyethylthio)-3-(2-(8phenyloctyl)phenyl) propanoic acid) was dissolved in 0.9~ Natl. LTD4 (synthesized at Smith Kline and French Labs., King of Prussia, PA) was dissolved in d i s t i l l e d water and the stock stored at 4oc. On the day of use, a sample was removed, diluted in d i s t i l l e d water and scanned in a spectrophotometer (Beckman DU-50, Somerset, N.J.) from 200 - 350 nm. The absorbance at 282 nm was then measured, and the concentration of the sample determined using an extinction coefficient of 40,O00/M/cm. An aliquot of the stock was then diluted in o.g~ NaCI and stored on ice until used. All solutions were made fresh on the day of use. Statistics All data in the text and figures are expressed as the mean ± SEM of n observations. Statistical significance of the data was
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analyzed with the Kruskal-Hallis test. The differences between individual groups was determined by Student's t test. A p value of less than or equal to 0.05 was considered to represent statistical significance. RESULTS Pharmacokinetlcs Following intravenous administration, SK&F I04353 disappeared from plasma in a triphasic fashion (figure l ) . The f i r s t and second phases (tz/=~ z and tz/=~z), which accounted for 80% of the total area under the curve (AUC), had half-lives of approximately 2 and 16 min, respectively (Table I). The apparent terminal phase (tz/z~ 3) had a h a l f - l i f e of 2.2 ± 0.6 hr. At l and 4 hr following the administration of SK&F I04353, plasma concentrations had decreased to I000
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Table I. Pharmacoklnetic Parameters for SK&F I04353 Following Intravenous Administration to Male Sprague-Dawley Rats.
Parameterb
SK&F I04353a
tl12~ 1
_d
tI12~2
0.26 ± 0.06
tll2X3
2.24 ± 0.64
Vss
0.20 ± 0.03
AUC
89.53 ± If.51
AUC estrap, c
9.05 ± 1.30
X2 phase
62.78 ± 4.75
~3 phase
20.92 ± 2.07
CLp
4.85 ± 0.52
a Values represent the mean ± SEM (n=4) of pharmacokinetic parameters estimated for individual animals, administered a dose of 25 mg/kg. b Abbreviations of pharmacokinetic parameters are: AUC (~g-hr/ml), area under the plasma concentration time curve extrapolated to i n f i n i t y ; CLD (ml/min/kg), total systemic clearance from plasma; Vss (L/kg), volume of distribution at steady state; tl/2~ 2 (hr), h a l f - l i f e for the second phase; tl/2X3 (hr), h a l f - l i f e for the third phase; X2 phase, percentage of administered dose eliminated in the ~2 phase; ~ X3 phase, percentage of administered dose eliminated in the X3 phase; ~ AUC extrap., percentage of AUC that was extrapolated. c Includes area extrapolated from the time of drug administration until
the f i r s t sample point (approximately 60-I00 seconds) and from the last sample point to i n f i n i t y . The percentage of area extrapolated from the last time point to i n f i n i t y was 0.76 ± 0.24 %. d The h a l f - l i f e for the f i r s t phase is estimated to be approximately 2 min, and is not reported.
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PROSTAGLANDINS approximately 10 and 2 Ng/ml, respectively. The AUC for SK&F 104353 was 89.5 ± I I . 5 ~g-hr/ml. At the plasma concentrations attained after the intravenous (bolus) administration, the volume of d i s t r i b u t i o n of SK&F I04353 was 0.25 ± 0.05 I/kg. Pharmacodynamic$ The pharmacodynamics of a 25 mg/kg dose of SK&F 104353 against LTD4 pressor responses is shown in Figure 2. The i n i t i a l increase in blood pressure to a 5.1 nmolelkg dose of LTD4, prior to vehicle or drug administration, was approximately 40 mm Hg, and t y p i c a l l y had recovered to the baseline blood pressure within 5 min after injection of LTD4. There was no difference in the i n i t i a l LTD4 vasopressor response between the two groups. In the group of animals receiving the vehicle, the subsequent changes in blood pressure to a LTD4 challenge were similar to the i n i t i a l response over a 24 hr period. In comparison, 5 min and 1 hr after the administration of SK&F 104353 the LTD4 pressor response was i n h i b i t ed by approximately 90%. At 4 hr after the administration of SK&F I04353, the LTD4 hemodynamic response was s t i l l inhibited by 60%. At 4 hr a second injection of SK&F I04353 was given, and the subsequent i n h i b i t i o n of the LTD4 pressor responses (5-8 hr) were similar to those observed from l to 4 hr. Administration of SK&F I04353 had no effect on blood pressure in conscious rats. These
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TIME [hr] Figure 2. Pharmacodynamics of SK&F I04353 fo]lowing a bolus injection at time 0 and 4 hr (upward arrows). In animals receiving the vehicle, the LTD4 vasopressor response was 80-I00% of the i n i t i a l value. Administration of SK&F I04353 blocked the LTD4 challenge by 85-90% for I hr, and at 4 hr there was s t i | l 60% i n h i b i t i o n of the LTD4 response.
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data indicate that following the administration of a 25 mg/kg dose of SK&F I04353 at 0 and 4 hr, the LTD4 vasopressor response was inhibited by 50 - 90% for a period of at least 8 hr. Cardioprotection In the Sham MI-vehicle and Sham MI-SK&F I04353 groups the LVFH CPK specific activities were 8.I ± 0.2 Ulmg protein and 8.0 ± O.l Ulmg protein, respectively (p > 0.05, figure 3). Following coronary artery occlusion and reperfusion, myocardial CPK specific a c t i v i t y in the LVFH was significantly decreased to 6.4 ± 0.6 U/mg protein (p < 0.05). This represents approximately a 25% loss in the total depletable myocardial CPK level. In MIIR animals administered SK&F I04353, the LVFH CPK specific activity was 6.5 ± 0.6 Ulmg protein (p > 0.05, compared to the MIIR-vehicle group). These data indicate that administration of the peptidoleukotriene receptor antagonist did not protect the myocardium against the loss of CPK specific activity.
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Figure 3. Effect of SK&F I04353 on myocardial injury. Height of the bars represent the mean, brackets are SEM and number of animals are indicated at the bottom of the columns. CPK specific a c t i v i t y was reduced in both groups of animals subjected to coronary artery occlusion and reperfusion (MIIR).
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PROSTAGLANDINS
MyeloDeroxidase Activity To quantitate the PMN response to reperfusion injury, the accumulation of PMN was assessed by measuring MPO activity in the LVFH. In Sham MI-vehicle and Sham MI-SK&F ]04353 groups, MPO activities were l - 2 Ulg tissue (figure 4). After 0.5 hr of coronary artery occlusion and reperfusion for 24 hr, myocardial MPO activities were s i g n i f i cantly increased to 4.3 ± 0.6 U/g tissue and 4.4 ± 0.9 U/g tissue for the MIIR-vehicle and MI/R-SK&F I04353 groups, respectively. The administration of SK&F 104353 had no effect on the increase in myocardia| MPO activity (p > 0.05, compared to the MIIR-vehicle group). DISCUSSION There is evidence to suggest that products of the 5-lipoxygenase pathway may be involved in the progression of myocardial reperfusion
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Figure 4. Effect of SK&F 104353 on myocardial myeloperoxidase acti v i t y . For details, see legend to Figure 3. MPO activity of MIIR vehicle-treated animals was significantly increased compared to Sham MIIR vehicle animals: administration of SK&F I04353 had no effect on the increase in MPO activity In MI/R animals.
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injury and inflammatory cell accumulation. Mullane and Moncada (lO) f i r s t reported that BH 755C, an inhibitor of both the cyclooxygenase and 5-1ipoxygenase pathways, reduced myocardial infarct size and leukocyte accumulation in dogs subjected to reperfusion injury. This observation was subsequently confirmed by Jolly and Lucchesi ( l l ) . Moreover, we have reported that in rats subjected to coronary artery occlusion and reperfusion, administration of BH 755C (lO mg/kg, i . v . ) reduces myocardial damage (29). Although all 5-lipoxygenase inhibitors presently available concomitantly inhibit cyclooxygenase, the protective effects do not appear related to inhibition of cyclooxygenase since there are reports which have shown that aspirin, meclofenamate or indomethacin do not reduce ischemic myocardial damage (30,31). These findings suggest that a product of the 5lipoxygenase pathway is an important mediator of reperfusion injury and PMN accumulation, and that inhibition of cyclooxygenase activity per se does not explain the beneficial effects observed with these agents. The role of potential mediators in pathophysiologic disorders can be examined pharmacologically with the use of enzyme inhibitors or receptor antagonists. A disadvantage of enzyme inhibitors is that metabolism of substrate may be redirected to other biologically active products. This has been demonstrated with inhibitors of thromboxane synthase which may redirect substrate to prostacyclin, a substance with biologic activities which oppose that of thromboxane (32). Similarly, indomethacin, a cyclooxygenase inhibitor, may actually increase the amounts of lipoxygenase products formed (33). SK&F 104353 is a potent and highly selective antagonist of peptidoleukotriene responses. In guinea pig tracheal preparations, the pA2 of SK&F I04353 was B.6 (5). SK&F I04353 does not demonstrate i n t r i n s i c agonist activity, and does not inhibit guinea pig tracheal contractions produced by histamine, a thromboxanelprostaglandin endoperoxide, potassium chloride, PGD2 or platelet activating factor (5). Moreover, SK&F I04353 does not inhibit cyclooxygenase, 5-1ipoxygenase, phospholipase, or cyclic nucleotide phosphodiesterases, and does not interact with ~- or B-adrenergic receptors, angiotensin II receptors, dopaminergic receptors or LTB4 receptors (5,13). Therefore, SK&F I04353 is a highly potent and selective peptidoleukotriene receptor antagonist, and an appropriate agent with which to evaluate the specific role of peptidoleukotrienes in various pathophysiologic disorders. Following intravenous administration, SK&F I04353 disappeared from plasma In a triphasic fashion with a terminal h a l f - l i f e of 2 hr. At l and 4 hr, the plasma concentrations had decreased to lO and 2 wg/ml, respectively. In conscious rats administered an infusion of SK&F I04353 at 3 mg/kg/hr, steady state plasma concentrations of I-2 wglml are achieved (17), levels which are comparable to those measured at 4 hr after a bolus dose of 25 mg/kg. In conscious rats, these plasma concentrations of SK&F I04353 produce a lO fold rightward and parallel shift in the LTC4 or LTD4 vasopressor dose response curve and a 3 fold shift in the LTE4 dose response curve (data not shown). Therefore, SK&F I04353 is equieffective against LTC4 and LTD4
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PROSTAGLANDINS vasopressor responses, and only slight less effective against LTE4 responses. Indeed, in the present study the LTD4 pressor response was inhibited by approximately 90% for l hr, and was s t i l l inhibited by approximately 60% at 4 hr after the administration of SK&F I04353. Following a second injection, the pharmacodynamics of SK&F I04353 were unaltered from the i n i t i a l response. Therefore, these data indicate that the dose of SK&F I04353 was sufficient to antagonize peptidoleukotriene vasopressor responses, and that significant inhibition of vasopressin responses extended for a period of at least 8 hr. The primary purpose of this study was to examine the protective effects of a specific peptidoleukotriene receptor antagonist on the extent of myocardial injury produced by coronary artery occlusion followed by reperfusion. Peptidoleukotrienes may be suspected as mediators of myocardial reperfusion injury since the invading PMN have a capacity for synthesizing LTC4, LTD4 and LTE4 (34, 35). Moreover, both endothelial cells (35,36) and platelets (37), which lack the a b i l i t y to form LTA4, are capable of metabolizing LTA4 to LTC4. Potentially, peptidoleukotrienes could contribute to myocardial injury by coronary vasoconstriction (2,3), myocardial dysfunction (38) or by increasing neutrophil adherence within the myocardium (39). To optimize any potential benefit, SK&F 104353 was administered l min prior to coronary artery occlusion, and again at 3.5 hr post reperfusion. The extent of myocardial injury was assessed by measuring the depletion of CPK from the l e f t ventricle, a technique reported to correlate.with the histological and histochemical determination of infarct size (23-25). Figure 4 clearly indicates that SK&F 104353 had no effect on the loss of CPK from the l e f t ventricle. Consistent with the finding of a lack of protection against the extent of myocardial ischemiclreperfusion injury, myeloperoxidase activity, used as a marker for neutrophil accumulation, was similarly increased in both groups of animals subjected to coronary artery occlusion and reperfusion. There was no difference in l e f t ventrlcular CPK specific activity between the two Sham MI/R groups, and SK&F I04353 had no direct effects on the CPK assay. Therefore, these data indicate that antagonism of peptidoleukotriene responses was not associated with a diminution of myocardial damage or inflammatory cell accumulation. Furthermore, these results are further support for the supposition that other products of the 5-1ipoxygenase pathway, such as LTB4, are involved as a mediator of myocardial reperfusion damage. In the present study, measurement of MPO activity was used to assess PMN i n f i l t r a t i o n (26). This procedure is commonly used to assess inflammatory cell accumulation (28), and was recently v a l i dated for rat cardiac tissue (]6,27). In animals subjected to sham coronary artery occlusion, administration of SK&F I04353 had no effect on basal MPO levels, and in animals subjected to coronary artery occlusion and reperfuslon, administration of SK&F 104353 did not attenuate the increase in MPO activity. He have previously reported that in the rat, there exists a significant correlation between the degree of myocardial injury and the extent of PMN
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i n f i l t r a t i o n (16). In view of the fact that SK&F I04353 did not reduce the extent of myocardial injury, i t is not surprising that the increase in MPO activity was not altered. Therefore, the increase in MPO levels in the group of MIIR animals could be interpreted as confirming evidence that antagonism of peptidoleukotriene responses does not provide an effective means of protecting the ischemiclreperfused myocardium. In conclusion, administration of a potent and selective peptidoleukotriene receptor antagonist at a dose sufficient to provide significant inhibition of LTD4 vasopressor responses, did not provide protection of the myocardium following coronary artery occlusion and reperfusion. Since nonselective inhibitors of 5-lipoxygenase such as BW 755C and REV 5901 protect against myocardial reperfusion injury, these observations suggest that peptidoleukotrienes are not involved in the progression of myocardial injury and inflammatory cell response. Certainly, these results should be cautiously interpreted since only one, albeit extremely selective receptor antagonist was utilized, and the agent possibly may not gain access to the site(s) of peptidoleukotriene action. I t has been reported that REV 5901 is both a 5-1ipoxygenase inhibitor and an antagonist of peptidoleukotriene responses. Based upon the findings of the present study, i t does not seem likely that blockade of peptidoleukotriene receptors explains the mechanism of the cardioprotective effects of REV 5901. Nonetheless, the possibility remains that other products of the 5-1ipoxygenase pathway, such as LTB4 (9), are involved as mediators of myocardial damage and inflammatory cell accumulation. LTB4 is a potent chemotactic agent, and formation of this substance by the ischemic and reperfused myocardium could mediate the accumulation of injury producing inflammatory cells, such as neutrophils. The development of LTB4 receptor antagonists with high a f f i n i t y and specificity will permit the characterization of the role of this chemotaxin in the progression of myocardial reperfusion injury. ACKNOWLEDGMENTS All animals were housed in accordance with the "Guide for the care and use of laboratory animals", NIH Publication No. 85-23. Procedures involving the use of laboratory animals were approved by the Institutional Animal Care and Use Committee of Smith Kline and French Laboratories, and are in accordance with NIH guidelines for the use of experimental animals. REFERENCES I. Ford-Hutchinson, A.W., M.A. Bray, M.V. Doig, M.E. Shipley and M.J.H. Smith. Leukotriene B4, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264-265, ]980.
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PROSTAGLANDINS
2. Michelassl, F., L. Landa, R.D. H i l l , E. Lowenstein, W.D. Watkins, A.3. Petkau and W.M. Zapol. Leukotriene D4: a potent coronary artery vasoconstrictor associated with impaired ventricular contraction. Science 217:84]-843, 1982. 3. Woodman, O.L. and G.J. Dusting. Coronary vasoconstriction induced by leukotrienes in the anesthetized dog. Europ J Pharmaco186:125-128, 1983. 4. Roth, D.M. and A.M. Lefer. Studies on the mechanism of leukotriene Induced coronary artery constriction. Prostaglandins 26:573-581, 1983. 5. Hay, D.W.P., R.M. Muccitel|i, S.S. Tucker, L.M. Vickery-Clark, K.A. Wilson, O.G. Gleason, R.F. Hall, M.A. Wasserman and T.J. Torphy. Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guineapig and human airways. O Pharmacol Exper Therap 243:474-481, 1987. 6. Dahlen, S.E., J. Bjork, P. Hedqvist, K.E. Arfors, S. Hammarstrom, O. Liingren and B. Sameulsson. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inf|ammatory response. Proc Natl Acad Sci (USA) 78:3887-3891, 1981. 7. Barst, S. and K. Mullane. The release of a leukotriene D4 like substance following myocardial infarction in rabbits. Europ J Pharmacol I14:383-387. 1985. 8. Evers, A.S., S. Murphree, O.E. Saffltz, B.A. Jakschik and P. Neddleman. Effects of endogenously produced leukotrienes, thromboxane, and prostglandins on coronary vascular resistance in rabbit myocardial infarction. J Clin Invest 7__55:992-999, 1985. 9. Sasaki, K., A. Ueno, M. Katori and R. Kikawada. Detection of leukotriene B4 in cardiac tissue and its role in infarct extension through leukocyte migration. Cardiovasc Res 22:142-148, 1988. lO. Mullane, K. and S. Moncada. The salvage of ischemic myocardium by BW 755C in anesthetized dogs. Prostaglandins 24:255-266, 1982. I I . Jolly, S. and B.R. Lucchesi. Effect of BH 755C in an occlusionreperfusion model of ischemic myocardial injury. Amer Heart J I06:8-13, 1983. 12. Mullane, K., M.A. Hatala, R. Kraemer, W. Sessa and W. Westlin. Myocardial salvage induced by REV-5901: an inhibitor and antagonist of the leukotrienes. J Cardlovasc Pharmacol I0:398-406,
610
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PROSTAGLANDINS 1987. 13. Mong, S., H.-L. Hu, J. Miller, R.F. Hall, J.G. Gleason and S.T.
Crooke. SKF I04353, a high a f f i n i t y antagonist for human and guinea pig lung leukotriene D4 receptor, blocked phosphatidylinositol metabolism and thromboxane synthesis induced by leukotriene D4. Molec Pharmaco132:223-229, 1987. 14. Hale, S.L. and R.A. Kloner.
Effect of early artery reperfusion on infarct developement in a model of low collateral flow. Cardiovas Res 2_!:668-673, 1987.
15. Hochman, J.S. and H. Choo. Limitation of myocardial infarct
expansion by reperfusion independent of myocardial salvage. Circulation 75:299-306, 1987. 16. Smith, E.F., I I I , O.W. Egan, P.3. Bugelski, L.M. Hillegass, D.E.
Hill and D.E. Griswold. Temporal relation between neutrophil accumulation and myocardial reperfusion injury in the rat. Amer 0 Physio1255:HlO60-H1068, 1988. 17. Smith, E.F., I I I ,
L.B. Kinter, M. Jugus, M.A. Wasserman, R.D. Eckardt and O.F. Newton. Beneficial effects of the peptidoleukotriene receptor antagonist, SK&F I04353, on experimental endotoxemia in the conscious rat. Circ Shock 25:21-31, 1988.
18. Gomenl, R.
PHARM,an interactive graphics program for individual and population pharmacokinetic parameter estimation. Comput Biol Med L4:25-34, 1984.
19. Shand, D.G., R.E. Desjardins, T.D. Bjornsson, S.C. Hamill and
E.L.C. Procjett. The method of separate exponentials: a simple aid to devising drug-loading regimens. Clin. Pharm. Therap. 29:542-54?, 1981. 20. Benet, L.Z. and R.L. Galeazzi. Non-compartmental determination
of steady state volume of distribution. I077, 1979.
3 Pharm Sci 6_88:1071-
21. Rosalki, S.B.
An improved procedure for serum creatine phosphokinase determination. O Lab Clin Med 6__99:696-705, 1966.
22. Bradford, M.M. A rapid and sensitive method for the quantita-
tion of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 7_22:24B-256, 1976. 23. Maclean, D., M.C. Fishbein, E. Braunwald and P.R. Maroko. Long-
term preservation of ischemic myocardium after experimental coronary artery occlusion. 0 Clln Invest 61:541-553, 1978. 24. Chiariel]o, M., G. Brevetti, G. Derosa, R. Acunzo, F. Petillo,
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PROSTAGLANDINS F. Rengo and M. Condorelli. Protective effects of simultaneous alpha and beta adrenergic receptor blockade on myocardial cell necrosis after coronary arterial occlusion in rats. Amer Cardio146:249-254, 1980. 25. Roberts, R. and Y. Ishikawa. Enzymatic estimation of infarct size during reperfusion. Circulation 6__88(Suppl l ) : 183-I89, 1983. 26. Bradley, P.P., D.A. Priebat, R.D. Christensen and G. Rothstein. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. Anal Biochem 72:248-256, 1976. 27. Griswold, D.E., L.M. Hillegass, D.E. H i l l , J.H. Egan and E.F. Smith I I I . Method for quantification of myocardial infarction and inflammatory cell i n f i l t r a t i o n in rat cardiac tissue. J Pharmacol Methods 20:225-235, 1988. 28. Mullane, K., R. Kraemer and B. Smith. Myeloperoxidase activity as a quantitative assessment of neutrophil i n f i l t r a t i o n into ischemic myocardium. 3 Pharmacol Methods L4:157-167, 1985. 29. Smith, E.F., I I I , O.W. Egan, D.E. Griswold, L.M. Hillegass and M.O. Sliv~ak. Inhibition of peptidoleukotriene responses does not moderate ischemic damage and polymorphonuclear leukocyte i n f i l t r a t i o n in myocardium following coronary artery occlusion with reperfusion. In Proc Fourth Internat Prostoglandin Symposium, ed. by K. Schror, A.R. Liss, New York, New York, (accepted) 1989. 30. Ogletree, M.L. and A.M. Lefer. Influence of non-steroidal antiinflammatory agents on myocardial ischemia in the cat. J Pharmacol Exper Therap 197:585-593, 1976. 31. Jugdutt, B.I., G.M. Hutchins, B.H. Bulkley and L.C. Becker. Effect of indomethacin on collateral blood flow and infarct size in the conscious dog. Circulation 59:734-743, 1979. 32. Olson, R.H., D.S. Cohen, E.C. Ku, E.F. Kimble, H.B. Renfroe and E.F. Smith I I I . CGS 15435A, a thromboxane synthetase inhibitor with an extended duration of action: a comparison with dazoxiben. Europ 3 Pharmaco1133:265-273, 1987. 33. Docherty, J.C. and T.H. Wilson. Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils. Biochem Biophys Res Commun 148:534538, 1987. 34. Mita, H., Y. Yui and T. Shida. Effect of AA-861, a 5-1ipoxygenase i n h i b i t o r , on leukotriene synthesis in human polymorphonuclear ]eukocytes and on cyclooxygenase and ]2-1ipoxygenase a c t i v i t i e s in human p l a t e l e t s . Allergy 41:493-498, 1986.
612
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PROSTAGLANDINS 35. Feinmark, S.3. Cooperative synthesis of leukotrienes by leukocytes and vascular cells. Ln Biology of the Leukotrienes, ed. by R. Levi and R.D. Krell, pp 123-132, Ann. NY Academy Science, New York, NY, 1988. 36. Claesson, H.-E. and J. Haeggstrom. Metabolism of leukotriene A4 by human endothelial cells: evidence for leukotriene C4 and D4 formation by leukocyte-endothelial cell interaction. In Advances Prostaglandin, Thromboxane and Leukotriene Research, ed. by B. Samuelsson, R. Paoletti and P.W. Ramwell, pp 115-119, Raven Press, New York, NY. 1987. 37. Pace-Asciak, C.R., J. Klein and S.P. Spielberg. Metabolism of leukotriene A4 by human platelets. Ln Advances Prostaglandin, Thromboxane and Leukotriene Research, ed. by B. Samuelsson, R. Paoletti and P.H. Ramwell, pp 127-133, Raven Press, New York, NY, 1987.
38. Laurindo, F.R.M., C.K. Finton, D. Ezra, J.F. Czaja, G.Z. Feuerstein and R.F. Goldstein. Inhibition of eicosanoid-mediated coronary constriction during myocardial ischemia. FASEB 3 2:2479-2486, 1988. 39. Goetzl, E.J., L.L. Brindley and D.W. Goldman. Enhancement of human neutrophil adherence by synthetic leukotriene constituents of the slow-reacting substance of anaphylaxis. Immunology 50:35-41, 1983. Edltor: G. Kaiey
MAY 1989 VOL. 37 NO. 5
Received: 12-9-88
Accepted: 3-27-89
613
SELECTIVE ANTAGONISM OF PEPTIDOLEUKOTRIENE RESPONSES DOES NOT REDUCE MYOCARDIAL DAMAGE OR NEUTROPHIL ACCUMULATION FOLLOWING CORONARY ARTERY OCCLUSION WITH REPERFUS~ON. J.W. Egan~, D.E. G r i s w o l ~ ~, L.M. H i l l e g ~ s s J.F. N e w t o n s , R.D. Eckardt ~, M.~. S l i v j a k ~, and E.F. Smith III
,
S m i t h ~ i n e & French Labs., l~pts, of Pharmacology I , In-nunology', and Drug Metabolism u King of Prussia, PA 19406
ABSTRACT This study was designed to assess the effect of a peptidoleukotriene receptor antagonist, SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MIIR). In conscious rats, SK&F 104353 (25 mglkg, i . v . ) antagonized LTD4-induced vasopressor responses by 90% and 60~ at l and 4 hr, respectively, indicating effective blockade of peptidoleukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil i n f i l t r a t i o n were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) a c t i v i t y , respectively, in the l e f t ventricular free wall (LVFN). Myocardial CPK levels were 8.1 ± 0.2 Ulmg protein in ShamMIIR vehicle-treated animals, and were significantly decreased to 6.4 ± 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 ± 0.5 Ulg LVFN in Sham-MIIR vehicle-treated animals, and significantly increased to 4.3 ± 0.6 Ulg LVFN in MI/R-vehicle animals. Administration of SK&F I04353 (25 mg/kg, i . v . ) l min prior to coronary occlusion and 3.5 hr post reperfusion had no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p > 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD4-1nduced vasopressor responses, SK&F I04353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/ reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury. INTRODUCTION The role of leukotrienes (LT) in myocardial ischemic/reperfusion injury and inflammatory cell accumulation is an area of intensive investigation. LTB4 increases vascular permeability and acts as a chemotaxin for polymorphonuclear leukocytes (PMN; I). Although less potent as chemotactic and chemokinetic substances, the peptidoleukotrienes LTC4, LTD4, and LTE4 are potent coronary vasoconstrictors
MAY 1989 VOL. 37 NO. 5
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PROSTAGLANDINS (2-4), bronchoconstrictors (5), and increase vascular permeability (6). In the reperfused myocardlum, the formation of peptidoleukotrienes and LTB4 are increased (7-9), although the time course of this increase has not been precisely characterized. Nonetheless, the nonselective 5-lipoxygenase inhlbitors BN 755C ( l O , l l ) , REV 590l (12) and AA-861 (g) have been reported to reduce myocardial reperfusion injury. Thus, there is evidence to support the suggestion that products of the 5-1ipoxygenase pathway may be involved in the sequelae of myocardial reperfusion injury and inflammatory cell accumulation. However, the specific role of peptidoleukotrienes as mediators of myocardial reperfusion injury remains undefined. The purposes of this study were f i r s t to characterize the pharmacokinetics and pharmacodynamics of a potent and selective peptidoleukotriene receptor antagonist, SK&F I04353 (2(S)-methoxy-3(R)-(2carboxyethylthio)-3-(2-(8-phenyloctyl)phenyl) propanoic acid; 5,13). Subsequently, we sought to determine the role of peptidoleukotrienes in myocardial Ischemic/reperfusion injury by examining the cardioprotective effects of SK&F I04353 in a well characterized rat model of ischemiclreperfusion injury (14-16). Myocardial injury was produced by coronary artery occlusion for 30 min followed by reperfusion for 24 hr. Additionally, myeloperoxidase (MPO) activity in the ischemic/ reperfused myocardlum was measured in order to assess the extent of polymorphonuclear leukocyte (PMN) accumulation. METHODS Pharmacoklnetic Evaluation Male Sprague-Dawley rats, weighing 375-450 g, were anesthetized with pentobarbital sodium (30 mglkg, i . p . ) and prepared, with modifications, as described previously (17). Briefly, catheters (0.60 mm i . d . , l.Ol mm o.d.) were placed into the vena cava and abdominal aorta through the l e f t femoral vein and artery, respectively. Both catheters were led subcutaneously along the tail and exteriorized through an incision 5 cm from the base of the t a i l . A lO ml syringe barrel, with the closed end removed, was placed over the tail and secured with tape. The syringe barrel served to protect the incision site and catheters. The animals were housed in individual cages and the syringe barrel used to tether the animals. Both catheters were kept patent with an infusion of 0.9% NaCl containing 2.4 Ulml heparin at a rate of 0.6 ml/hr. Blood pressure was continuously monitored (Statham P23 DC pressure transducer) on a Grass Model 7D multichannel recorder. Heart rate was determined by counting the deflections in arterial blood pressure from fast paper tracings. The animals were allowed to stabilize for I-2 days following the surgical procedures. The animals were permitted access to food and water ad libitum. The pharmacokinetics of SK&F I04353 were determined following the intravenous administration of a bolus of 25 mg/kg. Blood samples (200- 250 ~l; less than 2 ml total volume) were withdrawn via a venous catheter into heparinized syringes, and transfered to
598
MAY 1989 VOL. 37 NO. 5
PROSTAGLANDINS Eppendorf mlcrotubes. Plasmawas separated by centrifugation at 4oc, and lO0 gl was transferred for extraction and determination of SK&F I04353 plasma concentrations as described below. Plasma concentrations versus time profiles for SK&F I04353 were analyzed using PHARM (18). The area under the plasma concentration curve (AUC) was determined using the log trapezoidal rule. The AUC from the last time point to i n f i n i t y was extrapolated by dividing the last data point by the terminal rate constant determined from the f i t t e d data. The AUC from the time of drug administration to the f i r s t sample point was determined using f i t t e d data. The data were f i t to the appropriate sum of exponentials (three compartmental model) by peeling, and the h a l f - l i f e for each disposition phase determined. The percentage of the dose irreversibly removed in the beta (% ~2 phase) and gamma phase (% ~3 phase) was calculated by the method of separate exponentials (19). The volume of d i s t r i bution at steady state was calculated by non-compartmental methods (20). Since multiple samples were taken from individual animals, the pharmacokinetic parameters were f i r s t estimated for individual animals and then averaged to obtain mean estimates. Pharmacodvnamic Evaluation The pharmacodynamics of SK&F I04353 were determined in a separate group of animals. Animals were prepared as described above, and received either LTD4 intravenously, at 45 min intervals, in increasing doses ranging from 0.17 - 51 nmoles/kg ( i . e . , 0.085 - 25 ~g/kg). LTD4 was administered in a volume of O.l ml/lOOg body weight and followed with a 0.5 ml saline flush. These i n i t i a l experiments indicated that the EDso dose of LTD4 ( i . e . , dose of LTD4 required to increase arterial blood pressure to BO% of the maximal LTD4 response) was 5.1 nmole/kg ( i . e . , 2.5 ~g/kg). On a subsequent day, animals were repeatedly injected with 5.1 nmole/kg LTD4, at 40 min intervals, to determine the reproducibility of hemodynamic responses to challenges of LTD4. On a separate day, animals were administered a dose of LTD4 (5.1 nmole/kg i . v . ) that caused a marked pressor response. Approximately l hr following this LTD4 challenge, animals were randomized into two groups and received either SK&F 104353 (25 mg/kg, i . v . ) or vehicle (0.9% NaCI). A second dose of either SK&F 104353 (25 mg/kg, i . v . ) or vehicle was administerd 4 hr later. Following the admini s t r a t i o n of either SK&F 104353 or vehicle, LTD4 (5.1 nmole/kg, i . v . ) was repeatedly administered and the acute increase in mean arterial blood pressure following each injection was monitored. Determination of Plasma SK&F 104353 Concentrations Plasma SK&F I04353 concentrations were determined according to methods previously described (17). Briefly, the internal standard SK&F I04652 (2(S)-methoxy-3(S)-(2-carboxyethylthio)- 3-(2-(8-phenyloctyl)phenyl) propanoic acid), was added to plasma samples, and extracted twice with methyl formate containing 0.5% trifluoroacetic acid. Samples were vortexed and centrifuged at lOO0 x g for 5 min.
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PROSTAGLANDINS The supernatants were combined, evaporated to dryness under nitrogen, and resuspended in HPLC mobile phase. Aliquots (25 ~l) were quantitated on an HPLC system using a 5 um NOVAPAK C18 cartridge (Waters Assoc., Inc., Milford, MA). HPLCeffluent was monitored at 215 nm with a Kratos Model 783 uv detector. SK&FI04353 and the internal standard were separated i s o c r a t i c a l l y using a solvent system consisting of 52~ acetonitrile in O.l~ trifluoroacetic acid at a flow rate of 2.0 mllmin. Plasmaconcentrations of SK&F I04353 were determined from peak area ratios (compared to internal standard) that were compared to extracted authentic standard curves. The detection l i m i t for the quantitation of SK&F I04353 was 0.25 ~glml, and the standard curve of the assay was linear to lO00 wglml. Coronary Artery Occlusion Coronary artery occlusion was produced in rats similar to procedures described previously (16). Briefly, male Sprague-Dawley rats, weighing 300 - 370 g, were anesthetized with ethyl ether. An i n c i sion was made on the l e f t side of the thorax over the pectoral muscles and the tissues retracted to expose the fourth intercostal space. Sutures were placed through the overlapping skin and muscles to permit rapid closure of the chest wall following the surgical procedure. The chest was then opened through the fourth intercostal space and the ribs were gently spread. The heart was quickly expressed out of the thoracic cavity, inverted, and a 6-0 s i l k ligature placed under the visualized l e f t main coronary artery. The ligature was then either ligated with a "shoe-string" knot (MI/R animals) or removed (Sham MI animals). In animals prepared for reperfusion of the ischemic myocardium (MIIR group), the ends of the suture used to produce the coronary ligation were exteriorized through the chest wall. The heart was returned quickly to the thoracic cavity, the a i r removed by s l i g h t l y compressing the chest wall, and the incision was closed by tying the previously placed sutures. The animals were returned to their cages and were observed to have recovered from the anesthetic within l to 2 min. The coronary artery was reperfused after 30 min of occlusion in conscious animals by releasing the knot and removing the suture. The animals were allowed food and water ad libitum. Animals were administered SK&F 104353 (25 mglkg) or vehicle (0.9% NaCl) intravenously via the dorsal penal vein l min prior to occlusion and again at 3.5 hr post reperfusion. The dose of SK&F I04353 refers to the free acid. All substances were prepared fresh daily and administered in a volume of approximately l m]. CPK Measurement Twenty four hr later, the rats were k i l l e d with CO2, weighed, and their hearts removed and placed into ice cold saline. Attached connective tissue was carefully removed and the hearts were dissected into right ventricular wall, septum and l e f t ventricular wall (LVFN) segments. All myocardial segments were then weighed. The complete LVFW was homogenized for 2 x 5 sec in cold 0.25 M sucrose containing I mM EDTA and lO mMmercaptoethanol with a Tekmar tissue homogenizer
600
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PROSTAGLANDINS (Cincinnati, OH). The homogenates were centrifuged at 30,000 x g for 30 min at 4oc. The supernatants were decanted and stored at 4°C until analyzed for creatine phosphokinase (CPK) activity and protein concentration, according to previously published techniques (21,22). CPK specific a c t i v i t y was expressed as units/mg protein. The remaining pellet was frozen on dry ice and stored at -20oc until assayed for MPO activity. The depletion of CPK specific activity has been shown in a number of previous studies to correlate with the histologic determination of infarct size in experimental animals ( i . e . , the rat; 23,24) and humans (25). Myeloperoxidase Activity Myeloperoxidase content of rat heart tissue was determined by the method of Bradley et at., (26), and modified as described previously (16,24). Briefly, MPO activity was determined from the pellet of the LVFH homogenate. The pellet was f i r s t resuspended in 20 ml 5 mM potassium phosphate buffer (pH 6) and then centrifuged for 30 min at 30,000 x g at 4°C. The resulting pellet was prepared as a lO~ homogenate with extraction buffer consisting of 0.5% hexacyltrimethylammonium bromide (Sigma Chemical Co., St. Louis, MO) dissolved in 50 mM potassium phosphate buffer (pH 6) at room temperature and immediately frozen on dry ice. Three freeze and thaw cycles were performed with sonication for lO sec between cycles. After the last sonication, the samples were allowed to chill on ice for approximately 30 min, and then centrifuged for 15 min at 12,500 x g at 4oc. An aliquot of the supernatant was then reacted with 0.167 mg/ml O-dianisidine dlhydrochloride (Sigma Chemical Co., St. Louis, MO) and 0.0005% HzO2, and the rate of change in absorbance was measured at 460 nm in an UV spectrophotometer. One unit of MPO a c t i v i t y is defined as that amount of MPO capable of degrading one micromole of peroxide/min at 25oc, and was expressed in units per g wet weight (U/g) of tissue. The usefulness of measuring MPO activity to assess PMN i n f i l t r a t i o n has been previously reported (26,28), and recently validated in the rat (16,27). Materials SK&F 104343 (2(S)-methoxy-3(R)-(2-carboxyethylthio)-3-(2-(8phenyloctyl)phenyl) propanoic acid) was dissolved in 0.9~ Natl. LTD4 (synthesized at Smith Kline and French Labs., King of Prussia, PA) was dissolved in d i s t i l l e d water and the stock stored at 4oc. On the day of use, a sample was removed, diluted in d i s t i l l e d water and scanned in a spectrophotometer (Beckman DU-50, Somerset, N.J.) from 200 - 350 nm. The absorbance at 282 nm was then measured, and the concentration of the sample determined using an extinction coefficient of 40,O00/M/cm. An aliquot of the stock was then diluted in o.g~ NaCI and stored on ice until used. All solutions were made fresh on the day of use. Statistics All data in the text and figures are expressed as the mean ± SEM of n observations. Statistical significance of the data was
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PROSTAGLANDINS
analyzed with the Kruskal-Hallis test. The differences between individual groups was determined by Student's t test. A p value of less than or equal to 0.05 was considered to represent statistical significance. RESULTS Pharmacokinetlcs Following intravenous administration, SK&F I04353 disappeared from plasma in a triphasic fashion (figure l ) . The f i r s t and second phases (tz/=~ z and tz/=~z), which accounted for 80% of the total area under the curve (AUC), had half-lives of approximately 2 and 16 min, respectively (Table I). The apparent terminal phase (tz/z~ 3) had a h a l f - l i f e of 2.2 ± 0.6 hr. At l and 4 hr following the administration of SK&F I04353, plasma concentrations had decreased to I000
I
I00
t
E
\
E
L
~L rr) ~t 0 U_
Q5 ~C Oo
0.1
O
I
I
I
I
I
I
2
3
4.
5
Time
6
I
I
7
8
[hr]
Figure I. Plasma concentrations of SK&F I04353 following the intravenous administration of a single bolus of 25 mg/kg.
602
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PROSTAGLANDINS
Table I. Pharmacoklnetic Parameters for SK&F I04353 Following Intravenous Administration to Male Sprague-Dawley Rats.
Parameterb
SK&F I04353a
tl12~ 1
_d
tI12~2
0.26 ± 0.06
tll2X3
2.24 ± 0.64
Vss
0.20 ± 0.03
AUC
89.53 ± If.51
AUC estrap, c
9.05 ± 1.30
X2 phase
62.78 ± 4.75
~3 phase
20.92 ± 2.07
CLp
4.85 ± 0.52
a Values represent the mean ± SEM (n=4) of pharmacokinetic parameters estimated for individual animals, administered a dose of 25 mg/kg. b Abbreviations of pharmacokinetic parameters are: AUC (~g-hr/ml), area under the plasma concentration time curve extrapolated to i n f i n i t y ; CLD (ml/min/kg), total systemic clearance from plasma; Vss (L/kg), volume of distribution at steady state; tl/2~ 2 (hr), h a l f - l i f e for the second phase; tl/2X3 (hr), h a l f - l i f e for the third phase; X2 phase, percentage of administered dose eliminated in the ~2 phase; ~ X3 phase, percentage of administered dose eliminated in the X3 phase; ~ AUC extrap., percentage of AUC that was extrapolated. c Includes area extrapolated from the time of drug administration until
the f i r s t sample point (approximately 60-I00 seconds) and from the last sample point to i n f i n i t y . The percentage of area extrapolated from the last time point to i n f i n i t y was 0.76 ± 0.24 %. d The h a l f - l i f e for the f i r s t phase is estimated to be approximately 2 min, and is not reported.
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PROSTAGLANDINS approximately 10 and 2 Ng/ml, respectively. The AUC for SK&F 104353 was 89.5 ± I I . 5 ~g-hr/ml. At the plasma concentrations attained after the intravenous (bolus) administration, the volume of d i s t r i b u t i o n of SK&F I04353 was 0.25 ± 0.05 I/kg. Pharmacodynamic$ The pharmacodynamics of a 25 mg/kg dose of SK&F 104353 against LTD4 pressor responses is shown in Figure 2. The i n i t i a l increase in blood pressure to a 5.1 nmolelkg dose of LTD4, prior to vehicle or drug administration, was approximately 40 mm Hg, and t y p i c a l l y had recovered to the baseline blood pressure within 5 min after injection of LTD4. There was no difference in the i n i t i a l LTD4 vasopressor response between the two groups. In the group of animals receiving the vehicle, the subsequent changes in blood pressure to a LTD4 challenge were similar to the i n i t i a l response over a 24 hr period. In comparison, 5 min and 1 hr after the administration of SK&F 104353 the LTD4 pressor response was i n h i b i t ed by approximately 90%. At 4 hr after the administration of SK&F I04353, the LTD4 hemodynamic response was s t i l l inhibited by 60%. At 4 hr a second injection of SK&F I04353 was given, and the subsequent i n h i b i t i o n of the LTD4 pressor responses (5-8 hr) were similar to those observed from l to 4 hr. Administration of SK&F I04353 had no effect on blood pressure in conscious rats. These
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TIME [hr] Figure 2. Pharmacodynamics of SK&F I04353 fo]lowing a bolus injection at time 0 and 4 hr (upward arrows). In animals receiving the vehicle, the LTD4 vasopressor response was 80-I00% of the i n i t i a l value. Administration of SK&F I04353 blocked the LTD4 challenge by 85-90% for I hr, and at 4 hr there was s t i | l 60% i n h i b i t i o n of the LTD4 response.
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data indicate that following the administration of a 25 mg/kg dose of SK&F I04353 at 0 and 4 hr, the LTD4 vasopressor response was inhibited by 50 - 90% for a period of at least 8 hr. Cardioprotection In the Sham MI-vehicle and Sham MI-SK&F I04353 groups the LVFH CPK specific activities were 8.I ± 0.2 Ulmg protein and 8.0 ± O.l Ulmg protein, respectively (p > 0.05, figure 3). Following coronary artery occlusion and reperfusion, myocardial CPK specific a c t i v i t y in the LVFH was significantly decreased to 6.4 ± 0.6 U/mg protein (p < 0.05). This represents approximately a 25% loss in the total depletable myocardial CPK level. In MIIR animals administered SK&F I04353, the LVFH CPK specific activity was 6.5 ± 0.6 Ulmg protein (p > 0.05, compared to the MIIR-vehicle group). These data indicate that administration of the peptidoleukotriene receptor antagonist did not protect the myocardium against the loss of CPK specific activity.
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Figure 3. Effect of SK&F I04353 on myocardial injury. Height of the bars represent the mean, brackets are SEM and number of animals are indicated at the bottom of the columns. CPK specific a c t i v i t y was reduced in both groups of animals subjected to coronary artery occlusion and reperfusion (MIIR).
MAY 1989 VOL. 37 NO. 5
605
PROSTAGLANDINS
MyeloDeroxidase Activity To quantitate the PMN response to reperfusion injury, the accumulation of PMN was assessed by measuring MPO activity in the LVFH. In Sham MI-vehicle and Sham MI-SK&F ]04353 groups, MPO activities were l - 2 Ulg tissue (figure 4). After 0.5 hr of coronary artery occlusion and reperfusion for 24 hr, myocardial MPO activities were s i g n i f i cantly increased to 4.3 ± 0.6 U/g tissue and 4.4 ± 0.9 U/g tissue for the MIIR-vehicle and MI/R-SK&F I04353 groups, respectively. The administration of SK&F 104353 had no effect on the increase in myocardia| MPO activity (p > 0.05, compared to the MIIR-vehicle group). DISCUSSION There is evidence to suggest that products of the 5-lipoxygenase pathway may be involved in the progression of myocardial reperfusion
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Figure 4. Effect of SK&F 104353 on myocardial myeloperoxidase acti v i t y . For details, see legend to Figure 3. MPO activity of MIIR vehicle-treated animals was significantly increased compared to Sham MIIR vehicle animals: administration of SK&F I04353 had no effect on the increase in MPO activity In MI/R animals.
606
M A Y 1989 VOL. 37 NO. 5
PROSTAGLANDINS
injury and inflammatory cell accumulation. Mullane and Moncada (lO) f i r s t reported that BH 755C, an inhibitor of both the cyclooxygenase and 5-1ipoxygenase pathways, reduced myocardial infarct size and leukocyte accumulation in dogs subjected to reperfusion injury. This observation was subsequently confirmed by Jolly and Lucchesi ( l l ) . Moreover, we have reported that in rats subjected to coronary artery occlusion and reperfusion, administration of BH 755C (lO mg/kg, i . v . ) reduces myocardial damage (29). Although all 5-lipoxygenase inhibitors presently available concomitantly inhibit cyclooxygenase, the protective effects do not appear related to inhibition of cyclooxygenase since there are reports which have shown that aspirin, meclofenamate or indomethacin do not reduce ischemic myocardial damage (30,31). These findings suggest that a product of the 5lipoxygenase pathway is an important mediator of reperfusion injury and PMN accumulation, and that inhibition of cyclooxygenase activity per se does not explain the beneficial effects observed with these agents. The role of potential mediators in pathophysiologic disorders can be examined pharmacologically with the use of enzyme inhibitors or receptor antagonists. A disadvantage of enzyme inhibitors is that metabolism of substrate may be redirected to other biologically active products. This has been demonstrated with inhibitors of thromboxane synthase which may redirect substrate to prostacyclin, a substance with biologic activities which oppose that of thromboxane (32). Similarly, indomethacin, a cyclooxygenase inhibitor, may actually increase the amounts of lipoxygenase products formed (33). SK&F 104353 is a potent and highly selective antagonist of peptidoleukotriene responses. In guinea pig tracheal preparations, the pA2 of SK&F I04353 was B.6 (5). SK&F I04353 does not demonstrate i n t r i n s i c agonist activity, and does not inhibit guinea pig tracheal contractions produced by histamine, a thromboxanelprostaglandin endoperoxide, potassium chloride, PGD2 or platelet activating factor (5). Moreover, SK&F I04353 does not inhibit cyclooxygenase, 5-1ipoxygenase, phospholipase, or cyclic nucleotide phosphodiesterases, and does not interact with ~- or B-adrenergic receptors, angiotensin II receptors, dopaminergic receptors or LTB4 receptors (5,13). Therefore, SK&F I04353 is a highly potent and selective peptidoleukotriene receptor antagonist, and an appropriate agent with which to evaluate the specific role of peptidoleukotrienes in various pathophysiologic disorders. Following intravenous administration, SK&F I04353 disappeared from plasma In a triphasic fashion with a terminal h a l f - l i f e of 2 hr. At l and 4 hr, the plasma concentrations had decreased to lO and 2 wg/ml, respectively. In conscious rats administered an infusion of SK&F I04353 at 3 mg/kg/hr, steady state plasma concentrations of I-2 wglml are achieved (17), levels which are comparable to those measured at 4 hr after a bolus dose of 25 mg/kg. In conscious rats, these plasma concentrations of SK&F I04353 produce a lO fold rightward and parallel shift in the LTC4 or LTD4 vasopressor dose response curve and a 3 fold shift in the LTE4 dose response curve (data not shown). Therefore, SK&F I04353 is equieffective against LTC4 and LTD4
MAY 1989 VOL. 37 NO. 5
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PROSTAGLANDINS vasopressor responses, and only slight less effective against LTE4 responses. Indeed, in the present study the LTD4 pressor response was inhibited by approximately 90% for l hr, and was s t i l l inhibited by approximately 60% at 4 hr after the administration of SK&F I04353. Following a second injection, the pharmacodynamics of SK&F I04353 were unaltered from the i n i t i a l response. Therefore, these data indicate that the dose of SK&F I04353 was sufficient to antagonize peptidoleukotriene vasopressor responses, and that significant inhibition of vasopressin responses extended for a period of at least 8 hr. The primary purpose of this study was to examine the protective effects of a specific peptidoleukotriene receptor antagonist on the extent of myocardial injury produced by coronary artery occlusion followed by reperfusion. Peptidoleukotrienes may be suspected as mediators of myocardial reperfusion injury since the invading PMN have a capacity for synthesizing LTC4, LTD4 and LTE4 (34, 35). Moreover, both endothelial cells (35,36) and platelets (37), which lack the a b i l i t y to form LTA4, are capable of metabolizing LTA4 to LTC4. Potentially, peptidoleukotrienes could contribute to myocardial injury by coronary vasoconstriction (2,3), myocardial dysfunction (38) or by increasing neutrophil adherence within the myocardium (39). To optimize any potential benefit, SK&F 104353 was administered l min prior to coronary artery occlusion, and again at 3.5 hr post reperfusion. The extent of myocardial injury was assessed by measuring the depletion of CPK from the l e f t ventricle, a technique reported to correlate.with the histological and histochemical determination of infarct size (23-25). Figure 4 clearly indicates that SK&F 104353 had no effect on the loss of CPK from the l e f t ventricle. Consistent with the finding of a lack of protection against the extent of myocardial ischemiclreperfusion injury, myeloperoxidase activity, used as a marker for neutrophil accumulation, was similarly increased in both groups of animals subjected to coronary artery occlusion and reperfusion. There was no difference in l e f t ventrlcular CPK specific activity between the two Sham MI/R groups, and SK&F I04353 had no direct effects on the CPK assay. Therefore, these data indicate that antagonism of peptidoleukotriene responses was not associated with a diminution of myocardial damage or inflammatory cell accumulation. Furthermore, these results are further support for the supposition that other products of the 5-1ipoxygenase pathway, such as LTB4, are involved as a mediator of myocardial reperfusion damage. In the present study, measurement of MPO activity was used to assess PMN i n f i l t r a t i o n (26). This procedure is commonly used to assess inflammatory cell accumulation (28), and was recently v a l i dated for rat cardiac tissue (]6,27). In animals subjected to sham coronary artery occlusion, administration of SK&F I04353 had no effect on basal MPO levels, and in animals subjected to coronary artery occlusion and reperfuslon, administration of SK&F 104353 did not attenuate the increase in MPO activity. He have previously reported that in the rat, there exists a significant correlation between the degree of myocardial injury and the extent of PMN
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i n f i l t r a t i o n (16). In view of the fact that SK&F I04353 did not reduce the extent of myocardial injury, i t is not surprising that the increase in MPO activity was not altered. Therefore, the increase in MPO levels in the group of MIIR animals could be interpreted as confirming evidence that antagonism of peptidoleukotriene responses does not provide an effective means of protecting the ischemiclreperfused myocardium. In conclusion, administration of a potent and selective peptidoleukotriene receptor antagonist at a dose sufficient to provide significant inhibition of LTD4 vasopressor responses, did not provide protection of the myocardium following coronary artery occlusion and reperfusion. Since nonselective inhibitors of 5-lipoxygenase such as BW 755C and REV 5901 protect against myocardial reperfusion injury, these observations suggest that peptidoleukotrienes are not involved in the progression of myocardial injury and inflammatory cell response. Certainly, these results should be cautiously interpreted since only one, albeit extremely selective receptor antagonist was utilized, and the agent possibly may not gain access to the site(s) of peptidoleukotriene action. I t has been reported that REV 5901 is both a 5-1ipoxygenase inhibitor and an antagonist of peptidoleukotriene responses. Based upon the findings of the present study, i t does not seem likely that blockade of peptidoleukotriene receptors explains the mechanism of the cardioprotective effects of REV 5901. Nonetheless, the possibility remains that other products of the 5-1ipoxygenase pathway, such as LTB4 (9), are involved as mediators of myocardial damage and inflammatory cell accumulation. LTB4 is a potent chemotactic agent, and formation of this substance by the ischemic and reperfused myocardium could mediate the accumulation of injury producing inflammatory cells, such as neutrophils. The development of LTB4 receptor antagonists with high a f f i n i t y and specificity will permit the characterization of the role of this chemotaxin in the progression of myocardial reperfusion injury. ACKNOWLEDGMENTS All animals were housed in accordance with the "Guide for the care and use of laboratory animals", NIH Publication No. 85-23. Procedures involving the use of laboratory animals were approved by the Institutional Animal Care and Use Committee of Smith Kline and French Laboratories, and are in accordance with NIH guidelines for the use of experimental animals. REFERENCES I. Ford-Hutchinson, A.W., M.A. Bray, M.V. Doig, M.E. Shipley and M.J.H. Smith. Leukotriene B4, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264-265, ]980.
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2. Michelassl, F., L. Landa, R.D. H i l l , E. Lowenstein, W.D. Watkins, A.3. Petkau and W.M. Zapol. Leukotriene D4: a potent coronary artery vasoconstrictor associated with impaired ventricular contraction. Science 217:84]-843, 1982. 3. Woodman, O.L. and G.J. Dusting. Coronary vasoconstriction induced by leukotrienes in the anesthetized dog. Europ J Pharmaco186:125-128, 1983. 4. Roth, D.M. and A.M. Lefer. Studies on the mechanism of leukotriene Induced coronary artery constriction. Prostaglandins 26:573-581, 1983. 5. Hay, D.W.P., R.M. Muccitel|i, S.S. Tucker, L.M. Vickery-Clark, K.A. Wilson, O.G. Gleason, R.F. Hall, M.A. Wasserman and T.J. Torphy. Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guineapig and human airways. O Pharmacol Exper Therap 243:474-481, 1987. 6. Dahlen, S.E., J. Bjork, P. Hedqvist, K.E. Arfors, S. Hammarstrom, O. Liingren and B. Sameulsson. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inf|ammatory response. Proc Natl Acad Sci (USA) 78:3887-3891, 1981. 7. Barst, S. and K. Mullane. The release of a leukotriene D4 like substance following myocardial infarction in rabbits. Europ J Pharmacol I14:383-387. 1985. 8. Evers, A.S., S. Murphree, O.E. Saffltz, B.A. Jakschik and P. Neddleman. Effects of endogenously produced leukotrienes, thromboxane, and prostglandins on coronary vascular resistance in rabbit myocardial infarction. J Clin Invest 7__55:992-999, 1985. 9. Sasaki, K., A. Ueno, M. Katori and R. Kikawada. Detection of leukotriene B4 in cardiac tissue and its role in infarct extension through leukocyte migration. Cardiovasc Res 22:142-148, 1988. lO. Mullane, K. and S. Moncada. The salvage of ischemic myocardium by BW 755C in anesthetized dogs. Prostaglandins 24:255-266, 1982. I I . Jolly, S. and B.R. Lucchesi. Effect of BH 755C in an occlusionreperfusion model of ischemic myocardial injury. Amer Heart J I06:8-13, 1983. 12. Mullane, K., M.A. Hatala, R. Kraemer, W. Sessa and W. Westlin. Myocardial salvage induced by REV-5901: an inhibitor and antagonist of the leukotrienes. J Cardlovasc Pharmacol I0:398-406,
610
MAY 1989 VOL. 37 NO. 5
PROSTAGLANDINS 1987. 13. Mong, S., H.-L. Hu, J. Miller, R.F. Hall, J.G. Gleason and S.T.
Crooke. SKF I04353, a high a f f i n i t y antagonist for human and guinea pig lung leukotriene D4 receptor, blocked phosphatidylinositol metabolism and thromboxane synthesis induced by leukotriene D4. Molec Pharmaco132:223-229, 1987. 14. Hale, S.L. and R.A. Kloner.
Effect of early artery reperfusion on infarct developement in a model of low collateral flow. Cardiovas Res 2_!:668-673, 1987.
15. Hochman, J.S. and H. Choo. Limitation of myocardial infarct
expansion by reperfusion independent of myocardial salvage. Circulation 75:299-306, 1987. 16. Smith, E.F., I I I , O.W. Egan, P.3. Bugelski, L.M. Hillegass, D.E.
Hill and D.E. Griswold. Temporal relation between neutrophil accumulation and myocardial reperfusion injury in the rat. Amer 0 Physio1255:HlO60-H1068, 1988. 17. Smith, E.F., I I I ,
L.B. Kinter, M. Jugus, M.A. Wasserman, R.D. Eckardt and O.F. Newton. Beneficial effects of the peptidoleukotriene receptor antagonist, SK&F I04353, on experimental endotoxemia in the conscious rat. Circ Shock 25:21-31, 1988.
18. Gomenl, R.
PHARM,an interactive graphics program for individual and population pharmacokinetic parameter estimation. Comput Biol Med L4:25-34, 1984.
19. Shand, D.G., R.E. Desjardins, T.D. Bjornsson, S.C. Hamill and
E.L.C. Procjett. The method of separate exponentials: a simple aid to devising drug-loading regimens. Clin. Pharm. Therap. 29:542-54?, 1981. 20. Benet, L.Z. and R.L. Galeazzi. Non-compartmental determination
of steady state volume of distribution. I077, 1979.
3 Pharm Sci 6_88:1071-
21. Rosalki, S.B.
An improved procedure for serum creatine phosphokinase determination. O Lab Clin Med 6__99:696-705, 1966.
22. Bradford, M.M. A rapid and sensitive method for the quantita-
tion of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 7_22:24B-256, 1976. 23. Maclean, D., M.C. Fishbein, E. Braunwald and P.R. Maroko. Long-
term preservation of ischemic myocardium after experimental coronary artery occlusion. 0 Clln Invest 61:541-553, 1978. 24. Chiariel]o, M., G. Brevetti, G. Derosa, R. Acunzo, F. Petillo,
M A Y 1989 VOL. 37 NO. 5
611
PROSTAGLANDINS F. Rengo and M. Condorelli. Protective effects of simultaneous alpha and beta adrenergic receptor blockade on myocardial cell necrosis after coronary arterial occlusion in rats. Amer Cardio146:249-254, 1980. 25. Roberts, R. and Y. Ishikawa. Enzymatic estimation of infarct size during reperfusion. Circulation 6__88(Suppl l ) : 183-I89, 1983. 26. Bradley, P.P., D.A. Priebat, R.D. Christensen and G. Rothstein. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. Anal Biochem 72:248-256, 1976. 27. Griswold, D.E., L.M. Hillegass, D.E. H i l l , J.H. Egan and E.F. Smith I I I . Method for quantification of myocardial infarction and inflammatory cell i n f i l t r a t i o n in rat cardiac tissue. J Pharmacol Methods 20:225-235, 1988. 28. Mullane, K., R. Kraemer and B. Smith. Myeloperoxidase activity as a quantitative assessment of neutrophil i n f i l t r a t i o n into ischemic myocardium. 3 Pharmacol Methods L4:157-167, 1985. 29. Smith, E.F., I I I , O.W. Egan, D.E. Griswold, L.M. Hillegass and M.O. Sliv~ak. Inhibition of peptidoleukotriene responses does not moderate ischemic damage and polymorphonuclear leukocyte i n f i l t r a t i o n in myocardium following coronary artery occlusion with reperfusion. In Proc Fourth Internat Prostoglandin Symposium, ed. by K. Schror, A.R. Liss, New York, New York, (accepted) 1989. 30. Ogletree, M.L. and A.M. Lefer. Influence of non-steroidal antiinflammatory agents on myocardial ischemia in the cat. J Pharmacol Exper Therap 197:585-593, 1976. 31. Jugdutt, B.I., G.M. Hutchins, B.H. Bulkley and L.C. Becker. Effect of indomethacin on collateral blood flow and infarct size in the conscious dog. Circulation 59:734-743, 1979. 32. Olson, R.H., D.S. Cohen, E.C. Ku, E.F. Kimble, H.B. Renfroe and E.F. Smith I I I . CGS 15435A, a thromboxane synthetase inhibitor with an extended duration of action: a comparison with dazoxiben. Europ 3 Pharmaco1133:265-273, 1987. 33. Docherty, J.C. and T.H. Wilson. Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils. Biochem Biophys Res Commun 148:534538, 1987. 34. Mita, H., Y. Yui and T. Shida. Effect of AA-861, a 5-1ipoxygenase i n h i b i t o r , on leukotriene synthesis in human polymorphonuclear ]eukocytes and on cyclooxygenase and ]2-1ipoxygenase a c t i v i t i e s in human p l a t e l e t s . Allergy 41:493-498, 1986.
612
MAY 1989 VOL. 37 NO. 5
PROSTAGLANDINS 35. Feinmark, S.3. Cooperative synthesis of leukotrienes by leukocytes and vascular cells. Ln Biology of the Leukotrienes, ed. by R. Levi and R.D. Krell, pp 123-132, Ann. NY Academy Science, New York, NY, 1988. 36. Claesson, H.-E. and J. Haeggstrom. Metabolism of leukotriene A4 by human endothelial cells: evidence for leukotriene C4 and D4 formation by leukocyte-endothelial cell interaction. In Advances Prostaglandin, Thromboxane and Leukotriene Research, ed. by B. Samuelsson, R. Paoletti and P.W. Ramwell, pp 115-119, Raven Press, New York, NY. 1987. 37. Pace-Asciak, C.R., J. Klein and S.P. Spielberg. Metabolism of leukotriene A4 by human platelets. Ln Advances Prostaglandin, Thromboxane and Leukotriene Research, ed. by B. Samuelsson, R. Paoletti and P.H. Ramwell, pp 127-133, Raven Press, New York, NY, 1987.
38. Laurindo, F.R.M., C.K. Finton, D. Ezra, J.F. Czaja, G.Z. Feuerstein and R.F. Goldstein. Inhibition of eicosanoid-mediated coronary constriction during myocardial ischemia. FASEB 3 2:2479-2486, 1988. 39. Goetzl, E.J., L.L. Brindley and D.W. Goldman. Enhancement of human neutrophil adherence by synthetic leukotriene constituents of the slow-reacting substance of anaphylaxis. Immunology 50:35-41, 1983. Edltor: G. Kaiey
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Received: 12-9-88
Accepted: 3-27-89
613