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Effects of cyclooxygenase and lipoxygenase inhibitors on digoxin-induced arrhythmias and haemodynamics in guinea-pigs
Pharmacological Research, Vol . 26, No . 3, 1992
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EFFECTS OF CYCLOOXYGENASE AND LIPOXYGENASE INHIBITORS ON DIGOXIN-INDUCED ARRHYTHMIAS AND HAEMODYNAMICS IN GUINEA-PIGS ILKER KANZIK, ICLAL ~AKICI, SIBEL ERSOY, MUSTAFA ARK, NURETTIN ABACIOGLU and HAKAN ZENGIL Departments of Pharmacology, Faculties of Pharmacy and Medicine, Gazi University, 06330 Etiler, Ankara, Turkey Received in final form 19 May 1992
SUMMARY Effects of cyclooxygenase and lipoxygenase inhibitors of digoxin-induced arrhythmias and haemodynamics were studied in guinea-pigs . ECG, mean arterial blood pressure heart rate, pressure rate index and arrhythmias were recorded, starting 15 min before digoxin administration and continuing for 30 min afterwards . The cyclooxygenase inhibitor aspirin (50 mg kg - ') and the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (0 .25-10 .0 mg kg - ') were found to produce a significant protection against the arrhythmias, whereas aspirin (100 mg kg') and CGS 8515 were found to be i neffective . S K &F 104 353, a potent and selective peptidoleukotriene receptor antagonist significantly attenuated the arrhythmias and mortality in a dose-dependent manner . It is concluded that production of both cyclooxygenase and lipoxygenase metabolites could favour the occurrence and/or the maintenance of digoxin-induced cardiac toxicity . KEY
woaos : arrhythmias, aspirin, BW 755C, SK&F 104 353, guinea-pig .
INTRODUCTION Possible involvement of eicosanoids in the pathogenesis of digitalis-induced cardiac arrhythmias has attracted considerable attention [1-3] . Release of PGs has been reported from canine hearts exposed to ouabain [4] . Similarities also exist betweeen the actions of glycosides and PGs . It has been suggested that both groups of agents inhibited Na+, K+-ATPase [5, 61 . In addition, some PGs have been demonstrated to induce arrhythmias [7] . Thromboxane A2 (TxA2 ), the major cyclooxygenase product of arachidonic acid (AA) in platelets, has been implicated in the genesis of arrhythmias due to myocardial ischaemia and subsequent reperfusion [8], as well as in digoxin-induced arrhythmias [2, 3] . Therefore we have investigated the effects of aspirin pretreatment . Correspondence to : Prof . Dr Ilker Kanzik . 1043-6618/92/070305-12/$08 .00/0
©1992 The Italian Pharmacological Society
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Furthermore, following the inhibition of cyclooxygenase, AA metabolism may be diverted towards the lipoxygenase pathway [9, 10] and/or leukocyte migration may be stimulated [11] . These effects may result in formation and release of cardiotoxic leukotrienes (LTs) [11-14] . Thus, at least theoretically, use of cyclooxygenase and lipoxygenase inhibitors may have an advantage over an inhibitor of either pathway alone, in prevention of digitalis-induced arrhythmias . In this connection, dose-dependent effects of a dual inhibitor of both cyclooxygenase and lipoxygenase [15] BW 755C was compared with the effects of cyclooxygenase inhibitor, aspirin in digoxin-induced arrhythmias, including haemodynamic changes . As 5-lipoxygenase products are among the mediators (possibly) released during cardiac arrhythmias [16], we have also assessed the effects of CGS 8515 which was recently reported to be a highly selective 5-lipoxygenase inhibitor, with negligible effects on cyclooxygenase, 12-lipoxygenase or 15-lipoxygenase [17] and a potent and selective peptidoleukotriene receptor antagonist, SK&F 104 353 [18] .
MATERIALS AND METHODS Surgical procedures
Local bred guinea-pigs (300-400 g) of either sex were anaesthetized with urethane (1 .5 g kg - ', i .p .) and kept warm with an overhead lamp to maintain a rectal temperature of 37 .3±0 .5 ° C . After induction of anaesthesia, tracheal cannulation was performed . Left common carotid arterial blood pressure was measured with a Nihon Kohden MPU 0 .5 A transducer via a catheter, filled with heparinized saline . A polyethylene catheter was placed in the right jugular vein for drug administration . The ECG was recorded from subcutaneous steel needle electrodes . ECG (lead II) and arterial blood pressure were continuously monitored on an oscilloscope and recorded on a Nihon Kohden 6000 polygraph . After completing surgical procedures, the animals were allowed to stabilize for at least 10 min prior to drug or vehicle administration . All guinea-pigs received heparin (20 IU kg- ') just after the cannulation of the vein . Measured variables
Cardiac rhythm and mean arterial blood pressure (MABP) were obtained every 5 min or more frequently when appropriate, using 4 or 6 s recording cycles beginning 10 min before drug administration and continuing for another 60 min after digoxin . Heart rate (HR) was measured from high speed recordings of pulsatile arterial blood pressure . The pressure rate index (PRI) calculated as the product of MABP and HR divided by 100 was used as an index of changes in myocardial oxygen consumption during the course of the experiment period [19] . The severity of arrhythmias detected from ECG and blood pressure tracings during this period was assessed by noting the onset of arrhythmias, the total number and onset of premature ventricular contractions (PVCs), ventricular tachycardia (VT, defined as a sequence of four or more consecutive ventricular extrasystoles), the incidence of ventricular fibrillation (VF, which in this species can spontaneously
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revert to sinus rhythm) and mortality . An arrhythmia score was calculated using the scale of Macleod et al. [20] with some modifications as described in detail previously [21] . The following values were given : 0 for 0-50 PVCs with no VT or VF over the observation period, I for 50-500 PVCs only ; 2 for 500 PVCs, or one episode of spontaneously reversible VT or VF : 3 for spontaneously reversible VT and/or VF between 2-30 episodes ; 4 for spontaneously reversible VT and/or VF more than 30 episodes ; 5 for irreversible VF causing death within 30-60 min ; 6 for fatal VF causing death within 15-30 min ; 7 for fatal VF causing death within 15 min . If normal sinus rhythm was re-established for at least 10 min, the experiment was discontinued . Drug treatment Digoxin was prepared according to the British Pharmacopeia (B .P . 1980) . In brief, digoxin was dissolved in 80% ethanol at a concentration of 5 mg/2 .5 ml and added to 8 ml of propylenglycol, then diluted to 20 ml with distilled water . The final concentration of digoxin stock solution was 250,ug/m1 - ' . Digoxin was given as an i .v . bolus injection at a dose of 0 .6 mg kg- ' . BW 755C was dissolved in 0 .9% NaCl just before use due to its rapid decomposition in aqueous solutions and given as an i .v . bolus injection 30 min prior to digoxin injection . Aspirin was dissolved in sodium bicarbonate solution (5% w/v) and injected intravenously I h before digoxin administration . CGS 8515 was dissolved in dimethylsulphoxide and administered 30 min before digoxin i njection . S K &F 104 353 was dissolved in saline and infused at a rate of 1, 3 or 10 mg kg- ' per hour following an initial i .v . bolus injection of I mg kg - ' . Urethane administration did not affect any cardiac haemodynamic parameter . The responses to the vehicles (n=3) alone were also not significantly different from the control values . Drugs The following drugs were used : digoxin (Sandoz, Istanbul), aspirin (Sigma), 3-amino-l-(3-trifluoromethylphenyl)-2-pyrozoline hydrochloride (BW 775C, Wellcome Research Laboratories, Beckenham, Kent), 4-N-(2-aminomethybenzoate)-l,2-o-naphthoquinone (CGS 8515, Ciba Geigy Corporation, Pharmaceuticals Division, New Jersey), 2(S)-hydroxy-3(R)-(2-carboxyethylthio)-3-[2-(8-phenyloctyl)phenyl] propanoic acid (SK&F 104 353, SmithKline and Welwyn, Hertfordshire) and Beecham Laboratories Research Ltd, dimethylsulphoxide (Sigma) . Statistical analysis The values in the text are presented as mean±sEM or percentage incidence . Fisher's exact test was used to detect significant differences in the incidence of VF and mortality between control and drug-treated groups . The Mann-Whitney U-test was used to estimate statistical significances between control and the arrhythmia
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scores and the time of onset of arrhythmias . All other data were analysed by using a two-tailed Student t-test . In all tests P<0 .05 was considered to be statistically significant .
RESULTS Drug effects on arrhythmias
Digoxin-induced arrhythmias appeared at 2 .9±0 .2 min as increased PR interval and PVCs which were followed by lethal VT and/or VFs . In the control group, 35 of 40 guinea-pigs died, representing a mortality rate of 87 .5% . The effect of drug treatment on these arrhythmias as measured by the time of onset of the first arrhythmia, incidence of VF, mortality rate are summarized in Table I . Aspirin (50 mg kg- ') attenuated significantly the incidence of VF (from 72 .5 to 16 .7%) . This effect was lost when the dose was increased to 100 mg kg - ' (Table I) and an increase in mortality occurred as a consequence of an increase in incidence of irreversible VF . The i .v . injection of 0 .25 mg kg- ' BW 755C, 30 min prior to digoxin administration statistically significantly increased the time required for the first
Table I Effects of drugs on the time of onset of first arrhythmias, incidence of VF and mortality obtained after the i .v . bolus injection of digoxin (0 .6 mg kg- ')
Saline Aspirin 50 mg kg- ' 100 mg kg - ' BW 755C 0 .25 mg kg- ' 2 .50 mg kg - ' 10 .00 mg kg- '
n
Time of onset of arrhythmias (min)
Incidence of VF (%)
Mortality (%)
40
2 .9±0 .2
72 .5
87 .5
6 6
3 .2±0 .4 2 .3±0 .4
16 .7* 50 .0
4 .5±0 .3* 5 .2±0 .5** 5 .0±0 .5**
17 .0*** 8 .0*** 0 .0***
58 .0 0.0*** 0 .0***
12 12 8
16 .7*** 100 .0
CGS 8515 0 .1 mg kg' 0 .3 mg kg - ' 3 .0 mg kg - '
3 5 6
1 .6±0 .8 3 .4±1 .1 2 .8±0 .5
66 .7 60 .0 50 .0
33 .3 60 .0 66 .6
SKF 1 1 1
7 7 7
4 .1±0 .3* 5 .3±0 .8** 6 .1±0 .8**
57 .1 14 .3** 28 .6*
71 .4 57 .1 28 .6**
104 353 mg kg- '+ 1 mg kg - ' h' mg kg '+3 mg kg - ' h - ' mg kg - '+ 10 mg kg- ' h - '
Data are expressed as mean±sEM ; n indicates the number of experiments . *P<0.05; **P<0.01 ; ***P<0 .001, compared with control values .
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arrhythmia to occur, reduced markedly the incidence of VF (Table I) and arrhythmia score (Fig . 1) . However, at this dose level, 58% of the animals died . In the presence of higher doses (2 .5 and 10 mg kg- ') of BW 775C, arrhythmogenic toxicity was almost completely prevented and none of the animals died at these dose levels . In paticular, no arrhythmia, except some single ectopic beats, was observed at the dose of 10 mg kg - ' . At 2 .5 mg kg- ', on the other hand, only one fibrillated out of 12 animals . A 5-lipoxygenase inhibitor CGS 8515, failed to produce any beneficial effect against the arrhythmia parameters, despite a tendency to reduce the occurrence of VF (Table I) . SK&F 104 353, a potent and selective peptidoleukotriene receptor antagonist, on the other hand, attenuated dose-dependently and significantly the arrhythmia parameters and the mortality rate (Table I, Fig . 1) . The appearance of the first ectopic beat and the consecutive arrhythmias was also delayed significantly . However, the beneficial effect of this treatment seemed to be limited, since the occurrence of VF and mortality could not be totally prevented .
Y
Fig . 1 . Effect of drugs on the arrthymia scores obtained after the i .v . bolus injection of digoxin (0.6 mg kg- ') . Control (n=40), aspirin (50 mg kg - ', n=6 ; 100 mg kg - ', n=6), BW 755C (0 .25 mg kg- ', n=12 ; 2 .5 mg kg -', n=12 ; 10 .0 mg kg - ', n=8), CGS 8515 (0 .1 mg kg- ', n=3 ; 0 .3 mg kg ', n=5 ; 3 .0 mg kg ', n=6), SKF 104 353 (1 .0 mg kg'+1 .0 mg kg - ' h - ', n=7 ; 1 .0 mg kg - '+ 3 .0 mg kg- ' h"', n=7 ; 1 .0 mg kg '+10 .0 mg kg' h- ', n=7) . Each bar represents the mean±sEM . *P<0 .05 compared with control values .
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Haemodynamic effects of the drugs The basal blood pressure of guinea-pigs anaesthetized with urethane was 46± 1 mmHg and the heart rate was 313±6 beats min - ' . These values are slightly lower than those reported by others [3] . The MABP values of BW 755C were shown in Fig . 2 . The MABP values obtained with the other agents used in this study were not different from each other . Only aspirin pretreatments were associated with
Table II Effects of drugs on heart rate (HR) before and after digoxin (0 .6 mg kg - ', i.v .) administration, measured at specific time points throughout the experiments Time (min)
Control
Aspirin 50 mg kg- ' 100 mg kg - '
BW 755C 0 .25 mg kg - ' 2 .5 mg kg- ' 10 .0 mg kg - '
SKF 104 353 1 mg kg'+ 1 mg kg h - '
-15
0
2
10
20
30
60
313±6 n=40
315±5 n=40
249±9 n=40
253±18 n=40
245±14 n=26
241±18 n=18
240±21 n=5
320±12 n=6 310±9 n=6
310±7 n=6 320±12 n=6
240±14 n=6
320±31 n=6
270±19 n=6
288±11 n=5
220±34 n=6
288±31 n=6
260±18 n=6 225±25
320±13 n=12 285±8** n=12 338±16
310±10 n=12 295±9 n=12 345±15*
295±14* n=12 280±11* n=12 285±10*
320±15** n=12 285±22 n=12 293±14
n=8
n=8
n=8
n=8
339±6** n=7
334±10 n=7
287±11* n=7
287±11 n=7
1 mg kg - '+ 3 mg kg h - '
296±20 n=7
287±19 n=7
244±18 n=7
257±14 n=7
1 mg kg - '+ 10 mg kg - ' h - '
304±14 n=7
304±15 n=7
257±16
236±25
300±0 n=3 318±12
300±0 n=3 312±15 n=5 280±16 n=6
260±10 n=3 282±18 n=5 264±18 n=6
n=7
n=7
n=4
315±18* n=12 290±7* n=12 308±8*** n=8
325±12*** n=6
240±n=2
273±15 n=9 295±5** n=12 315±10** n=8
252±35 n=5 289±10* n=12 308±14* n=8
330±17** n=6
300±n=2
287±22 n=7
249±16 n=6
240±n=3
189±45
260±17
252±24
n=7
n=6
n=5
285±10 n=3 270±n=5 290±27 n=4
285±12 n=2 270±n=2 240±n=2
CGS 8515 0 .1 mg kg- ' 0.3 mg kg -' 3 .0 mg kg- '
n=5 300±16 n=6
200±44 n=3 294±20 n=5 225±36 n=6
280±10 n=3 278±23 n=5 255±9 n=4
Data are expressed as mean±seen; n : indicates the number of experiments . *P<0 .05 ; **P<0.01 ; ***P<0 .001 ; compared with control values .
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higher pressures, although not statistically significantly different from the control group (data not shown) . Initially, HR (Table II) and computed PRI values (Table III) of all groups, also were not different from each other . Intravenous administration of digoxin (0 .6 mg kg- ') resulted in a dramatic elevation of MABP which reached a peak in 2 min . This was accompanied by a drop in arterial blood pressure at about 10 min and a second phase of rise which peaked at 20 min and persisted high throughout the remainder of the experiment (Fig . 2) . The initial acute rise in MABP could not be prevented by any drug intervention . In contrast, almost in all treatment groups the digoxin-induced second phase of rise of blood pressure was prevented and maintained between the limits of pre-digoxin values . In animals treated with higher dose of aspirin, however, MABP tended to decrease during the same period (data not shown) . Heart rate and computed PRI values generally did not vary significantly between all group of animals (Tables II and III) . However, in animals pretreated with BW 755C heart rate was significantly increased during the post-digoxin period . This increase in heart rate did not significantly affect the computed PRI values, suggesting no concomitant increase in oxygen demand (Table I11) . A similar finding was also observed in animals pretreated with the small dose of SK&F 104 353 .
E E a CO a
-15
0
15 Time (min)
30
60
Fig. 2 . Effects of BW 755C on mean arterial blood pressure (MABP) before and after digoxin (0 .6 mg kg - ', i .v .) administration in anaesthetized guinea pigs . (0) control (n=40), ( •) BW 755C (0 .25 mg kg', n=12) ; (Y) BW 755C (2 .5 mg kg', n=12 ; ( •) BW 755C (10 .0 mg kg', n=8) . All points represent mean and data points for all three points .
SEM .
*P<0 .05 compared with control values and refers to
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Table III Effects of drugs on pressure rate index (PRI=[(MABPxHR)/100]) before and after digoxin (0.6 mg kg- ', i .v .) administration, measured at specific time points throughout the experiments Time (min)
Control
Aspirin 50 mg kg- ' 100 mg kg- '
BW 755C 0 .25 mg kg - ' 2 .5 mg kg - ' 10 .0 mg kg - '
SKF 104 353 1 mg kg - '+ I mg kg h - '
-15
0
2
10
20
30
60
147±6 n=40
149±5 n=40
175±7 n=40
123±9 n=40
182±17 n=26
179±19 n=18
186±26 n=5
162±11 n=6 162±10 n=6
153±11 n=6 158±8 n=6
167±13 n=6 139±29 n=6
190±20** n=6 168±22 n=6
147±15 n=6 155±21 n=4
142±15 n=6 117±n=2
148±9 n=5 -
140±16 n=12 126±10 n=12 162±19 n=8
136±10 n=12 131±9 n=12 169±15 n=8
186±16 n=12 167±9 n=12 198±17 n=8
138±16 n=12 146±13 n=12 148±11 n=8
135±14* n=12 142±9* n=12 151±9 n=8
127±16* n=9 139±7 n=12 162±12 n=8
117±24 n=5 134±11 n=12 160±14 n=8
157±13 n=7
155±11 n=7
207±7** n=7
155±12* n=7
169±20 n=6
157±10 n=6
140±n=2
142±33 n=7
142±10 n=7
176±15 n=7
144±9 n=7
157±14 n=7
145±13 n=6
113±n=3
146±10 n=7
145±10 n=7
171±18 n=7
129±18 n=7
136±20 n=7
119±14 n=6
133±22 n=5
138±1 n=3 164±10 n=5 163±12 n=6
137±14 n=3 164±12 n=5 136±9 n=6
182±29 n=3 195±11 n=5 190±12 n=6
97±34 n=3 175±8** n=5 98±32 n=6
145±28 n=3 197±21 n=5 138±15 n=4
147±42 n=3 189±1 n=5 141±11 n=4
136±33 n=2 189±n=2 149±n=2
I mg kg - '+
3 mg kg h- ' I mg kg'+ 10 mg kg 'h"'
CGS 8515 0 .1 mg kg - '
0 .3 mg kg - ' 3 .0 mg kg'
Data are expressed as mean±sem ; n indicates the number of experiments . *P<0.05 ; **P<0 .01 ; compared with control values .
DISCUSSION At present, digitalis toxicity is thought to be related mainly to Na +/K+ -ATPase inhibition and subsequent development of oscillatory after potentials (OAPs) [22] . Under appropriate conditions, these OAPs reach threshold levels and initiate ectopic activity, thereby causing arrhythmias . Considerable evidence also
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indicates that eicosanoids are involved in arrhythmias induced by cardiac glycosides [1-3,23] . In this connection, it has been shown that glycosides stimulate cardiac prostaglandin biosynthesis [24] and that some nonsteroidal antiinflammatory drugs (aspirin, indomethacin) protect animals against arrhythmias due to glycoside intoxication [3] . In the present study, the potent cyclooxygenase inhibitor, aspirin (50 mg kg ') in good agreement with the above findings significantly attenuated but could not abolish the digitalis-induced arrhythmias and mortality . On the other hand, the protective effect of aspirin was lost at a higher dose (100 mg kg') . The mechanism by which aspirin and other nonsteroidal anti-inflammatory agents produce their anti-arrhythmic effect is not well established. In addition, since various PGs have been shown to be antiarrhythmic whereas drugs such as flurbiprofen potentiate the arrhythmias resulting from coronary artery ligation [25] or ketoprofen is ineffective against ouabain-induced arrhythmias in guinea-pigs [3], it appears that aspirin differs from other cyclooxygenase inhibitors . In a recent study we have shown that both Tx synthase inhibitor and Tx receptor antagonist significantly protected the guinea-pigs against digoxin-induced arrhythmias, although these effects were limited and dose-related with adverse effects [2, 26] . It is therefore possible to suggest that antiarrhythmic effect of aspirin in guinea-pigs against digoxin-induced arrhythmia may be due to inhibition of synthesis and release of TxA2 and subsequent increase of the production of the PGs with antiarrhythmic potential . In addition, our results on the pharmacological parameters indicate that aspirin has no significant direct effect on HR and ECG pattern of myocardium . Our findings also suggest that the dose of aspirin employed is crucial in obtaining a beneficial effect . This may be due to decreased biosynthesis of PGs with antiarrhythmic properties (such as PGE 2 and PGI2) in the presence of higher doses of aspirin . Additionally, cardiac glycosides have been shown to constrict the coronary arteries and thus may have potential to induce a condition similar to that of ischaemia in ventricles [27, 28] . There is also evidence that besides TxA 2 , a variety of potentially deleterious humoral mediators, including platelet activating factor [29], LTs [13, 16] and free oxygen radicals [30] are produced and released during ischaemia . Therefore, it is possible to suggest that products of lipoxygenase pathway of AA metabolism may also contribute to the pathogenesis of arrhythmias due to digitalis . This suggestion is strengthened by our observation that BW 755C, the dual inhibitor of cyclooxygenase and lipoxygenase, markedly and dosedependently protected animals against digoxin-induced arrhythmias (Table I, Fig . 1) . BW 755C in the dose range of 0 .25-10 .0 mg kg - ' had little or no measurable haemodynamic effect on its own, but significantly prevented animals from gradual decrease in MABP which was subsequent to rapid elevation due to digoxin (Fig . 2) . PRI was also higher in BW 775C treated groups compared to controls during this period . It is difficult, however, to attribute the protective action of the drug merely to improvements in haemodynamics, since these effects of BW 775C may in fact be the result of inhibition of the formation of noxious metabolites of AA . In the present study the lipoxygenase inhibitor CGS 8515 provided no significant protection in the guinea-pigs (Table I) . The lack of effect of the lipoxygenase inhibitor in this setting may be explained by the possibility that
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inhibition of lipoxygenase probably diverts endogenous AA to cyclooxygenase pathway and thus formed metabolites (especially TxA 2 ) exert a positive feedback on the toxicity of digoxin . This suggestion needs further investigation . However, the finding that peptidoleukotriene receptor antagonist provided significant protection against digoxin-induced arrhythmias indicated that products of lipoxygenase metabolism do contribute to the progression of these arrhythmias . When all these findings are taken together, it is interesting to note that, although aspirin at the dose of 50 mg kg -1 and peptidoleukotriene receptor antagonist, SK&F 104 353 tended to be antiarrhythmic by most measures, the inhibition of both cyclooxygenase and lipoxygenase by a dual inhibitor, BW 755C, provided a better protection than the other treatments . Furthermore, since none of the drug treatments could alter the computed PRI index significantly, it appeared that beneficial effects of these drugs are mainly due to the effects mediated via alterations in arachidonic acid metabolites rather than to improvement in myocardial oxygen supply . However, the beneficial action of drug therapies, at least in part, may also be due to improved blood pressure and reduced afterload in these animals . In conclusion, the initiation of arrhythmias by Na'/K'-ATPase inhibition and subsequent development of OAPs, combined with the production of noxious cyclooxygenase and to a lesser degree lipoxygenase metabolites, could favour the occurrence and/or the maintenance of cardiac toxicity under the influence of digoxin .
ACKNOWLEDGEMENTS The authors would like to thank Dr Lovell of Ciba-Geigy Corporation for the generous supply of CGS 8515 and Dr Skidmore of SmithKline and French Research Ltd for SKF 104 353, and Dr Moncada of Wellcome Research Laboratories for BW 755C .
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7 . Karmazyn M . Dhalla NS . A selective concentration-dependent dysrhythmogenic and antidysrhythmic action of prostaglandin E 2 , F,a and I, (prostacyclin) on isolated rat hearts . Experientia 1980; 36 : 996-8 . 8 . Coker SJ, Parratt JR, Ledingman IMcA, Zeitlin IJ . Thromboxane and prostacyclin release from ischaemic myocardium in relation to arrhythmias . Nature 1981 ; 291 : 323-4 . 9 . Lewis RA, Austen KF . The biologically active leukotrienes . J Clin Invest 1984 : 73 : 889-97 . 10 . Brune K, Peskar BA . Modulation by drugs of leukotriene and prostaglandin production from mouse peritoneal macrophages . Int J Tiss Res 1985 ; VII(2) : 97-103 . 11 . Mullane KM, Read N, Salmon JA, Moncada S . Role of leukocytes in acute myocardial infarction in anesthetized dogs : Relationship to myocardial salvage by anti-inflammatory drugs . . Pharmaco! Exp Ther 1984 ; 228(2) : 510-22 . 12 . Thiemermann C, Ney P, Schror K . The thromboxane receptor antagonists, daltroban, protects the myocardium from ischaemic injury resulting in suppression of leukocytosis . E,ur J Pharmacol 1988 ; 155 : 57-67 . 13 . Ikeda U, Toyo-Oka T, Arisaka H, Hosada S . Stimulated synthesis of prostaglandin E, or leukotriene C 4 from myocardial cells is not a cause but a result of their injury under hypoxia . J Mol Cel! Cardiol 1987 ; 19 : 523-6 . 14 . Pallandi RT, Perry MA, Campbell TJ . Proarrhythmic effects of an oxygen-derived tree radical generating system on action potentials recorded from guinea-pig ventricular myocardium : A possible cause of reperfusion-induced arrhythmias . Circ Res 1987 : 61 : 50-4 . 15 . Higgs GA, Flower RJ, Vane JR . A new approach to anti inflammatory drugs . Bioche m Pharmacol 1979 ; 28 : 1959-61 . 16 . Feuerstein G . Hallenbeck JM . Leukotrienes in health and disease . FASEB .l 1987 : 1 : 186-92 . 17 . Ku EC, Raychaudhuri A, Ghai G, Kimble EF, Lee WH, Colombo C, Dotson R, White D, Oglessby TD, Wasley JWF . Characterization of CGS 8515 as a selective 5-lipoxygenase (5-LO) inhibitor . Adr Prost Thromb Leu Res 1989 ; 19 : 87-9 . 18 . Hay DWP, Muccitelli RM, Tucker SS, Vickery-Clarck LM, Wilson KA, Gleason JG, Hall RF . Wasserman MA, Torphy TJ . Pharmacologic profile of SKF 104353 : A novel, potent and selective peptidoleukotriene receptor antagonist in guinea-pig and human airways . J Pharmacol Exp Ther 1987 ; 243(2) : 474-81 . 19 . Schror K . Thiemermann C . Treatment of acute mycoardial ischaemia with a selective antagonist of thromboxane receptors (BM 13177) . Br J Pharmacol 1986 ; 87 : 631-7 . 20 . Macleod BA, Augereau P, Walker MJA . Effects of halothane anaesthesia compared with fentanyl anaesthesia and no anaesthesia during coronary ligation in rats . Anesthesiolo,gi 1983 :58 :42-4 . 21 . Kanzik I, Cakici I, Demiryiirek AT, Abacioglu N, Dortlemez H . Digoxin-induced cardiac toxicity in the anaesthetized guinea-pigs and effect of heparin infusion . Arch Inr Pharmacod_vn Ther 1987 ; 290 : 165-72 . 22 . Ferrier GR . Digitalis arrhythmias : role of oscillatory after potentials . Progr Cardiorasc Dis 1977 ; 19 : 459-74 . 23 . Forster W . Prostaglandins and prostaglandin precursors as endogenous antiarrhythmic principles of the heart . Acta Biol Med Germ 1976 ; 35 : 1101-12 . 24 . Moffat P, Ferrier GR, Karmazyn M . A possible role for endogenous prostaglandins in the electrophysiological effects of acetylstrophanthidin on isolated canine ventricular tissues . Circ Res 1986 ; 58(4) : 486-94 . 25 . Coker SJ, Parratt JR . The effects of prostaglandins E 2 , F,a, prostacyclin, flurbiprofen and aspirin on arrhythmias resulting from coronary artery ligation in anaesthetized rats . Br J Pharmacol 1981 a ; 74 : 155-9 . 26 . Kanzik I, Zengil H, Ipek N, Cakici I, Abacioglu N, Dortlemez H . Synergism between UK 38 485 and ICI 185 282 against digoxin-induced arrhythmias in guinea-pigs . Arch In[ Pharmacodyn Ther 1991 ; 312(1/2) : 55-65 . 27 . Fleckenstein A, Byon KY . Prevention by Ca"-antagonistic compounds (verapamil, D
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600) of coronary smooth muscle contractures due to treatment with cardiac glycosides . Naunyn Schm Arch Exp Pathol Pharmacol 1974 ; 282 (Suppl) : R20 . 28 . Fleckenstein A, Fleckenstein-Grun G, Byon YK, Haastern HP, Spah F . Vergleichende Untersuchungen uber die Ca"-antagonistischen Grundwirkungen von Niludipin (Bay a 7168) and Nifedipin (Bay a 1040) auf Myokard, Myometrium and glatte Gefussmuskulatur. Arzneim-Forsch/Drug Res 1979 ; 29 : 230-46 . 29 . Braquet P, Touqui L, Shen TY, Vargaftig G . Perspectives in platelet-activating factor research . Pharmacol Rev 1987 ; 39 : 97-144 . 30 . Granger DN, Mollwarth MR, Parks DA . Ischaemia-reperfusion injury : role of oxygenderived free radicals . Acta Physiol Scand 1986 ; 548 : 47-63 .
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EFFECTS OF CYCLOOXYGENASE AND LIPOXYGENASE INHIBITORS ON DIGOXIN-INDUCED ARRHYTHMIAS AND HAEMODYNAMICS IN GUINEA-PIGS ILKER KANZIK, ICLAL ~AKICI, SIBEL ERSOY, MUSTAFA ARK, NURETTIN ABACIOGLU and HAKAN ZENGIL Departments of Pharmacology, Faculties of Pharmacy and Medicine, Gazi University, 06330 Etiler, Ankara, Turkey Received in final form 19 May 1992
SUMMARY Effects of cyclooxygenase and lipoxygenase inhibitors of digoxin-induced arrhythmias and haemodynamics were studied in guinea-pigs . ECG, mean arterial blood pressure heart rate, pressure rate index and arrhythmias were recorded, starting 15 min before digoxin administration and continuing for 30 min afterwards . The cyclooxygenase inhibitor aspirin (50 mg kg - ') and the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (0 .25-10 .0 mg kg - ') were found to produce a significant protection against the arrhythmias, whereas aspirin (100 mg kg') and CGS 8515 were found to be i neffective . S K &F 104 353, a potent and selective peptidoleukotriene receptor antagonist significantly attenuated the arrhythmias and mortality in a dose-dependent manner . It is concluded that production of both cyclooxygenase and lipoxygenase metabolites could favour the occurrence and/or the maintenance of digoxin-induced cardiac toxicity . KEY
woaos : arrhythmias, aspirin, BW 755C, SK&F 104 353, guinea-pig .
INTRODUCTION Possible involvement of eicosanoids in the pathogenesis of digitalis-induced cardiac arrhythmias has attracted considerable attention [1-3] . Release of PGs has been reported from canine hearts exposed to ouabain [4] . Similarities also exist betweeen the actions of glycosides and PGs . It has been suggested that both groups of agents inhibited Na+, K+-ATPase [5, 61 . In addition, some PGs have been demonstrated to induce arrhythmias [7] . Thromboxane A2 (TxA2 ), the major cyclooxygenase product of arachidonic acid (AA) in platelets, has been implicated in the genesis of arrhythmias due to myocardial ischaemia and subsequent reperfusion [8], as well as in digoxin-induced arrhythmias [2, 3] . Therefore we have investigated the effects of aspirin pretreatment . Correspondence to : Prof . Dr Ilker Kanzik . 1043-6618/92/070305-12/$08 .00/0
©1992 The Italian Pharmacological Society
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Furthermore, following the inhibition of cyclooxygenase, AA metabolism may be diverted towards the lipoxygenase pathway [9, 10] and/or leukocyte migration may be stimulated [11] . These effects may result in formation and release of cardiotoxic leukotrienes (LTs) [11-14] . Thus, at least theoretically, use of cyclooxygenase and lipoxygenase inhibitors may have an advantage over an inhibitor of either pathway alone, in prevention of digitalis-induced arrhythmias . In this connection, dose-dependent effects of a dual inhibitor of both cyclooxygenase and lipoxygenase [15] BW 755C was compared with the effects of cyclooxygenase inhibitor, aspirin in digoxin-induced arrhythmias, including haemodynamic changes . As 5-lipoxygenase products are among the mediators (possibly) released during cardiac arrhythmias [16], we have also assessed the effects of CGS 8515 which was recently reported to be a highly selective 5-lipoxygenase inhibitor, with negligible effects on cyclooxygenase, 12-lipoxygenase or 15-lipoxygenase [17] and a potent and selective peptidoleukotriene receptor antagonist, SK&F 104 353 [18] .
MATERIALS AND METHODS Surgical procedures
Local bred guinea-pigs (300-400 g) of either sex were anaesthetized with urethane (1 .5 g kg - ', i .p .) and kept warm with an overhead lamp to maintain a rectal temperature of 37 .3±0 .5 ° C . After induction of anaesthesia, tracheal cannulation was performed . Left common carotid arterial blood pressure was measured with a Nihon Kohden MPU 0 .5 A transducer via a catheter, filled with heparinized saline . A polyethylene catheter was placed in the right jugular vein for drug administration . The ECG was recorded from subcutaneous steel needle electrodes . ECG (lead II) and arterial blood pressure were continuously monitored on an oscilloscope and recorded on a Nihon Kohden 6000 polygraph . After completing surgical procedures, the animals were allowed to stabilize for at least 10 min prior to drug or vehicle administration . All guinea-pigs received heparin (20 IU kg- ') just after the cannulation of the vein . Measured variables
Cardiac rhythm and mean arterial blood pressure (MABP) were obtained every 5 min or more frequently when appropriate, using 4 or 6 s recording cycles beginning 10 min before drug administration and continuing for another 60 min after digoxin . Heart rate (HR) was measured from high speed recordings of pulsatile arterial blood pressure . The pressure rate index (PRI) calculated as the product of MABP and HR divided by 100 was used as an index of changes in myocardial oxygen consumption during the course of the experiment period [19] . The severity of arrhythmias detected from ECG and blood pressure tracings during this period was assessed by noting the onset of arrhythmias, the total number and onset of premature ventricular contractions (PVCs), ventricular tachycardia (VT, defined as a sequence of four or more consecutive ventricular extrasystoles), the incidence of ventricular fibrillation (VF, which in this species can spontaneously
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revert to sinus rhythm) and mortality . An arrhythmia score was calculated using the scale of Macleod et al. [20] with some modifications as described in detail previously [21] . The following values were given : 0 for 0-50 PVCs with no VT or VF over the observation period, I for 50-500 PVCs only ; 2 for 500 PVCs, or one episode of spontaneously reversible VT or VF : 3 for spontaneously reversible VT and/or VF between 2-30 episodes ; 4 for spontaneously reversible VT and/or VF more than 30 episodes ; 5 for irreversible VF causing death within 30-60 min ; 6 for fatal VF causing death within 15-30 min ; 7 for fatal VF causing death within 15 min . If normal sinus rhythm was re-established for at least 10 min, the experiment was discontinued . Drug treatment Digoxin was prepared according to the British Pharmacopeia (B .P . 1980) . In brief, digoxin was dissolved in 80% ethanol at a concentration of 5 mg/2 .5 ml and added to 8 ml of propylenglycol, then diluted to 20 ml with distilled water . The final concentration of digoxin stock solution was 250,ug/m1 - ' . Digoxin was given as an i .v . bolus injection at a dose of 0 .6 mg kg- ' . BW 755C was dissolved in 0 .9% NaCl just before use due to its rapid decomposition in aqueous solutions and given as an i .v . bolus injection 30 min prior to digoxin injection . Aspirin was dissolved in sodium bicarbonate solution (5% w/v) and injected intravenously I h before digoxin administration . CGS 8515 was dissolved in dimethylsulphoxide and administered 30 min before digoxin i njection . S K &F 104 353 was dissolved in saline and infused at a rate of 1, 3 or 10 mg kg- ' per hour following an initial i .v . bolus injection of I mg kg - ' . Urethane administration did not affect any cardiac haemodynamic parameter . The responses to the vehicles (n=3) alone were also not significantly different from the control values . Drugs The following drugs were used : digoxin (Sandoz, Istanbul), aspirin (Sigma), 3-amino-l-(3-trifluoromethylphenyl)-2-pyrozoline hydrochloride (BW 775C, Wellcome Research Laboratories, Beckenham, Kent), 4-N-(2-aminomethybenzoate)-l,2-o-naphthoquinone (CGS 8515, Ciba Geigy Corporation, Pharmaceuticals Division, New Jersey), 2(S)-hydroxy-3(R)-(2-carboxyethylthio)-3-[2-(8-phenyloctyl)phenyl] propanoic acid (SK&F 104 353, SmithKline and Welwyn, Hertfordshire) and Beecham Laboratories Research Ltd, dimethylsulphoxide (Sigma) . Statistical analysis The values in the text are presented as mean±sEM or percentage incidence . Fisher's exact test was used to detect significant differences in the incidence of VF and mortality between control and drug-treated groups . The Mann-Whitney U-test was used to estimate statistical significances between control and the arrhythmia
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scores and the time of onset of arrhythmias . All other data were analysed by using a two-tailed Student t-test . In all tests P<0 .05 was considered to be statistically significant .
RESULTS Drug effects on arrhythmias
Digoxin-induced arrhythmias appeared at 2 .9±0 .2 min as increased PR interval and PVCs which were followed by lethal VT and/or VFs . In the control group, 35 of 40 guinea-pigs died, representing a mortality rate of 87 .5% . The effect of drug treatment on these arrhythmias as measured by the time of onset of the first arrhythmia, incidence of VF, mortality rate are summarized in Table I . Aspirin (50 mg kg- ') attenuated significantly the incidence of VF (from 72 .5 to 16 .7%) . This effect was lost when the dose was increased to 100 mg kg - ' (Table I) and an increase in mortality occurred as a consequence of an increase in incidence of irreversible VF . The i .v . injection of 0 .25 mg kg- ' BW 755C, 30 min prior to digoxin administration statistically significantly increased the time required for the first
Table I Effects of drugs on the time of onset of first arrhythmias, incidence of VF and mortality obtained after the i .v . bolus injection of digoxin (0 .6 mg kg- ')
Saline Aspirin 50 mg kg- ' 100 mg kg - ' BW 755C 0 .25 mg kg- ' 2 .50 mg kg - ' 10 .00 mg kg- '
n
Time of onset of arrhythmias (min)
Incidence of VF (%)
Mortality (%)
40
2 .9±0 .2
72 .5
87 .5
6 6
3 .2±0 .4 2 .3±0 .4
16 .7* 50 .0
4 .5±0 .3* 5 .2±0 .5** 5 .0±0 .5**
17 .0*** 8 .0*** 0 .0***
58 .0 0.0*** 0 .0***
12 12 8
16 .7*** 100 .0
CGS 8515 0 .1 mg kg' 0 .3 mg kg - ' 3 .0 mg kg - '
3 5 6
1 .6±0 .8 3 .4±1 .1 2 .8±0 .5
66 .7 60 .0 50 .0
33 .3 60 .0 66 .6
SKF 1 1 1
7 7 7
4 .1±0 .3* 5 .3±0 .8** 6 .1±0 .8**
57 .1 14 .3** 28 .6*
71 .4 57 .1 28 .6**
104 353 mg kg- '+ 1 mg kg - ' h' mg kg '+3 mg kg - ' h - ' mg kg - '+ 10 mg kg- ' h - '
Data are expressed as mean±sEM ; n indicates the number of experiments . *P<0.05; **P<0.01 ; ***P<0 .001, compared with control values .
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arrhythmia to occur, reduced markedly the incidence of VF (Table I) and arrhythmia score (Fig . 1) . However, at this dose level, 58% of the animals died . In the presence of higher doses (2 .5 and 10 mg kg- ') of BW 775C, arrhythmogenic toxicity was almost completely prevented and none of the animals died at these dose levels . In paticular, no arrhythmia, except some single ectopic beats, was observed at the dose of 10 mg kg - ' . At 2 .5 mg kg- ', on the other hand, only one fibrillated out of 12 animals . A 5-lipoxygenase inhibitor CGS 8515, failed to produce any beneficial effect against the arrhythmia parameters, despite a tendency to reduce the occurrence of VF (Table I) . SK&F 104 353, a potent and selective peptidoleukotriene receptor antagonist, on the other hand, attenuated dose-dependently and significantly the arrhythmia parameters and the mortality rate (Table I, Fig . 1) . The appearance of the first ectopic beat and the consecutive arrhythmias was also delayed significantly . However, the beneficial effect of this treatment seemed to be limited, since the occurrence of VF and mortality could not be totally prevented .
Y
Fig . 1 . Effect of drugs on the arrthymia scores obtained after the i .v . bolus injection of digoxin (0.6 mg kg- ') . Control (n=40), aspirin (50 mg kg - ', n=6 ; 100 mg kg - ', n=6), BW 755C (0 .25 mg kg- ', n=12 ; 2 .5 mg kg -', n=12 ; 10 .0 mg kg - ', n=8), CGS 8515 (0 .1 mg kg- ', n=3 ; 0 .3 mg kg ', n=5 ; 3 .0 mg kg ', n=6), SKF 104 353 (1 .0 mg kg'+1 .0 mg kg - ' h - ', n=7 ; 1 .0 mg kg - '+ 3 .0 mg kg- ' h"', n=7 ; 1 .0 mg kg '+10 .0 mg kg' h- ', n=7) . Each bar represents the mean±sEM . *P<0 .05 compared with control values .
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Haemodynamic effects of the drugs The basal blood pressure of guinea-pigs anaesthetized with urethane was 46± 1 mmHg and the heart rate was 313±6 beats min - ' . These values are slightly lower than those reported by others [3] . The MABP values of BW 755C were shown in Fig . 2 . The MABP values obtained with the other agents used in this study were not different from each other . Only aspirin pretreatments were associated with
Table II Effects of drugs on heart rate (HR) before and after digoxin (0 .6 mg kg - ', i.v .) administration, measured at specific time points throughout the experiments Time (min)
Control
Aspirin 50 mg kg- ' 100 mg kg - '
BW 755C 0 .25 mg kg - ' 2 .5 mg kg- ' 10 .0 mg kg - '
SKF 104 353 1 mg kg'+ 1 mg kg h - '
-15
0
2
10
20
30
60
313±6 n=40
315±5 n=40
249±9 n=40
253±18 n=40
245±14 n=26
241±18 n=18
240±21 n=5
320±12 n=6 310±9 n=6
310±7 n=6 320±12 n=6
240±14 n=6
320±31 n=6
270±19 n=6
288±11 n=5
220±34 n=6
288±31 n=6
260±18 n=6 225±25
320±13 n=12 285±8** n=12 338±16
310±10 n=12 295±9 n=12 345±15*
295±14* n=12 280±11* n=12 285±10*
320±15** n=12 285±22 n=12 293±14
n=8
n=8
n=8
n=8
339±6** n=7
334±10 n=7
287±11* n=7
287±11 n=7
1 mg kg - '+ 3 mg kg h - '
296±20 n=7
287±19 n=7
244±18 n=7
257±14 n=7
1 mg kg - '+ 10 mg kg - ' h - '
304±14 n=7
304±15 n=7
257±16
236±25
300±0 n=3 318±12
300±0 n=3 312±15 n=5 280±16 n=6
260±10 n=3 282±18 n=5 264±18 n=6
n=7
n=7
n=4
315±18* n=12 290±7* n=12 308±8*** n=8
325±12*** n=6
240±n=2
273±15 n=9 295±5** n=12 315±10** n=8
252±35 n=5 289±10* n=12 308±14* n=8
330±17** n=6
300±n=2
287±22 n=7
249±16 n=6
240±n=3
189±45
260±17
252±24
n=7
n=6
n=5
285±10 n=3 270±n=5 290±27 n=4
285±12 n=2 270±n=2 240±n=2
CGS 8515 0 .1 mg kg- ' 0.3 mg kg -' 3 .0 mg kg- '
n=5 300±16 n=6
200±44 n=3 294±20 n=5 225±36 n=6
280±10 n=3 278±23 n=5 255±9 n=4
Data are expressed as mean±seen; n : indicates the number of experiments . *P<0 .05 ; **P<0.01 ; ***P<0 .001 ; compared with control values .
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higher pressures, although not statistically significantly different from the control group (data not shown) . Initially, HR (Table II) and computed PRI values (Table III) of all groups, also were not different from each other . Intravenous administration of digoxin (0 .6 mg kg- ') resulted in a dramatic elevation of MABP which reached a peak in 2 min . This was accompanied by a drop in arterial blood pressure at about 10 min and a second phase of rise which peaked at 20 min and persisted high throughout the remainder of the experiment (Fig . 2) . The initial acute rise in MABP could not be prevented by any drug intervention . In contrast, almost in all treatment groups the digoxin-induced second phase of rise of blood pressure was prevented and maintained between the limits of pre-digoxin values . In animals treated with higher dose of aspirin, however, MABP tended to decrease during the same period (data not shown) . Heart rate and computed PRI values generally did not vary significantly between all group of animals (Tables II and III) . However, in animals pretreated with BW 755C heart rate was significantly increased during the post-digoxin period . This increase in heart rate did not significantly affect the computed PRI values, suggesting no concomitant increase in oxygen demand (Table I11) . A similar finding was also observed in animals pretreated with the small dose of SK&F 104 353 .
E E a CO a
-15
0
15 Time (min)
30
60
Fig. 2 . Effects of BW 755C on mean arterial blood pressure (MABP) before and after digoxin (0 .6 mg kg - ', i .v .) administration in anaesthetized guinea pigs . (0) control (n=40), ( •) BW 755C (0 .25 mg kg', n=12) ; (Y) BW 755C (2 .5 mg kg', n=12 ; ( •) BW 755C (10 .0 mg kg', n=8) . All points represent mean and data points for all three points .
SEM .
*P<0 .05 compared with control values and refers to
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Table III Effects of drugs on pressure rate index (PRI=[(MABPxHR)/100]) before and after digoxin (0.6 mg kg- ', i .v .) administration, measured at specific time points throughout the experiments Time (min)
Control
Aspirin 50 mg kg- ' 100 mg kg- '
BW 755C 0 .25 mg kg - ' 2 .5 mg kg - ' 10 .0 mg kg - '
SKF 104 353 1 mg kg - '+ I mg kg h - '
-15
0
2
10
20
30
60
147±6 n=40
149±5 n=40
175±7 n=40
123±9 n=40
182±17 n=26
179±19 n=18
186±26 n=5
162±11 n=6 162±10 n=6
153±11 n=6 158±8 n=6
167±13 n=6 139±29 n=6
190±20** n=6 168±22 n=6
147±15 n=6 155±21 n=4
142±15 n=6 117±n=2
148±9 n=5 -
140±16 n=12 126±10 n=12 162±19 n=8
136±10 n=12 131±9 n=12 169±15 n=8
186±16 n=12 167±9 n=12 198±17 n=8
138±16 n=12 146±13 n=12 148±11 n=8
135±14* n=12 142±9* n=12 151±9 n=8
127±16* n=9 139±7 n=12 162±12 n=8
117±24 n=5 134±11 n=12 160±14 n=8
157±13 n=7
155±11 n=7
207±7** n=7
155±12* n=7
169±20 n=6
157±10 n=6
140±n=2
142±33 n=7
142±10 n=7
176±15 n=7
144±9 n=7
157±14 n=7
145±13 n=6
113±n=3
146±10 n=7
145±10 n=7
171±18 n=7
129±18 n=7
136±20 n=7
119±14 n=6
133±22 n=5
138±1 n=3 164±10 n=5 163±12 n=6
137±14 n=3 164±12 n=5 136±9 n=6
182±29 n=3 195±11 n=5 190±12 n=6
97±34 n=3 175±8** n=5 98±32 n=6
145±28 n=3 197±21 n=5 138±15 n=4
147±42 n=3 189±1 n=5 141±11 n=4
136±33 n=2 189±n=2 149±n=2
I mg kg - '+
3 mg kg h- ' I mg kg'+ 10 mg kg 'h"'
CGS 8515 0 .1 mg kg - '
0 .3 mg kg - ' 3 .0 mg kg'
Data are expressed as mean±sem ; n indicates the number of experiments . *P<0.05 ; **P<0 .01 ; compared with control values .
DISCUSSION At present, digitalis toxicity is thought to be related mainly to Na +/K+ -ATPase inhibition and subsequent development of oscillatory after potentials (OAPs) [22] . Under appropriate conditions, these OAPs reach threshold levels and initiate ectopic activity, thereby causing arrhythmias . Considerable evidence also
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indicates that eicosanoids are involved in arrhythmias induced by cardiac glycosides [1-3,23] . In this connection, it has been shown that glycosides stimulate cardiac prostaglandin biosynthesis [24] and that some nonsteroidal antiinflammatory drugs (aspirin, indomethacin) protect animals against arrhythmias due to glycoside intoxication [3] . In the present study, the potent cyclooxygenase inhibitor, aspirin (50 mg kg ') in good agreement with the above findings significantly attenuated but could not abolish the digitalis-induced arrhythmias and mortality . On the other hand, the protective effect of aspirin was lost at a higher dose (100 mg kg') . The mechanism by which aspirin and other nonsteroidal anti-inflammatory agents produce their anti-arrhythmic effect is not well established. In addition, since various PGs have been shown to be antiarrhythmic whereas drugs such as flurbiprofen potentiate the arrhythmias resulting from coronary artery ligation [25] or ketoprofen is ineffective against ouabain-induced arrhythmias in guinea-pigs [3], it appears that aspirin differs from other cyclooxygenase inhibitors . In a recent study we have shown that both Tx synthase inhibitor and Tx receptor antagonist significantly protected the guinea-pigs against digoxin-induced arrhythmias, although these effects were limited and dose-related with adverse effects [2, 26] . It is therefore possible to suggest that antiarrhythmic effect of aspirin in guinea-pigs against digoxin-induced arrhythmia may be due to inhibition of synthesis and release of TxA2 and subsequent increase of the production of the PGs with antiarrhythmic potential . In addition, our results on the pharmacological parameters indicate that aspirin has no significant direct effect on HR and ECG pattern of myocardium . Our findings also suggest that the dose of aspirin employed is crucial in obtaining a beneficial effect . This may be due to decreased biosynthesis of PGs with antiarrhythmic properties (such as PGE 2 and PGI2) in the presence of higher doses of aspirin . Additionally, cardiac glycosides have been shown to constrict the coronary arteries and thus may have potential to induce a condition similar to that of ischaemia in ventricles [27, 28] . There is also evidence that besides TxA 2 , a variety of potentially deleterious humoral mediators, including platelet activating factor [29], LTs [13, 16] and free oxygen radicals [30] are produced and released during ischaemia . Therefore, it is possible to suggest that products of lipoxygenase pathway of AA metabolism may also contribute to the pathogenesis of arrhythmias due to digitalis . This suggestion is strengthened by our observation that BW 755C, the dual inhibitor of cyclooxygenase and lipoxygenase, markedly and dosedependently protected animals against digoxin-induced arrhythmias (Table I, Fig . 1) . BW 755C in the dose range of 0 .25-10 .0 mg kg - ' had little or no measurable haemodynamic effect on its own, but significantly prevented animals from gradual decrease in MABP which was subsequent to rapid elevation due to digoxin (Fig . 2) . PRI was also higher in BW 775C treated groups compared to controls during this period . It is difficult, however, to attribute the protective action of the drug merely to improvements in haemodynamics, since these effects of BW 775C may in fact be the result of inhibition of the formation of noxious metabolites of AA . In the present study the lipoxygenase inhibitor CGS 8515 provided no significant protection in the guinea-pigs (Table I) . The lack of effect of the lipoxygenase inhibitor in this setting may be explained by the possibility that
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inhibition of lipoxygenase probably diverts endogenous AA to cyclooxygenase pathway and thus formed metabolites (especially TxA 2 ) exert a positive feedback on the toxicity of digoxin . This suggestion needs further investigation . However, the finding that peptidoleukotriene receptor antagonist provided significant protection against digoxin-induced arrhythmias indicated that products of lipoxygenase metabolism do contribute to the progression of these arrhythmias . When all these findings are taken together, it is interesting to note that, although aspirin at the dose of 50 mg kg -1 and peptidoleukotriene receptor antagonist, SK&F 104 353 tended to be antiarrhythmic by most measures, the inhibition of both cyclooxygenase and lipoxygenase by a dual inhibitor, BW 755C, provided a better protection than the other treatments . Furthermore, since none of the drug treatments could alter the computed PRI index significantly, it appeared that beneficial effects of these drugs are mainly due to the effects mediated via alterations in arachidonic acid metabolites rather than to improvement in myocardial oxygen supply . However, the beneficial action of drug therapies, at least in part, may also be due to improved blood pressure and reduced afterload in these animals . In conclusion, the initiation of arrhythmias by Na'/K'-ATPase inhibition and subsequent development of OAPs, combined with the production of noxious cyclooxygenase and to a lesser degree lipoxygenase metabolites, could favour the occurrence and/or the maintenance of cardiac toxicity under the influence of digoxin .
ACKNOWLEDGEMENTS The authors would like to thank Dr Lovell of Ciba-Geigy Corporation for the generous supply of CGS 8515 and Dr Skidmore of SmithKline and French Research Ltd for SKF 104 353, and Dr Moncada of Wellcome Research Laboratories for BW 755C .
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