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Effects of dantrolene sodium in rodent models of cardiac arrhythmia
European Journal of Pharmacology, 164 (1989) 521-530
521
Elsevier EJP 50723
Effects of dantrolene sodium in rodent models of cardiac arrhythmia R o b e r t R. B r o o k s *, J o h n F. C a r p e n t e r , S a n d r a M. J o n e s a n d C i n d a M. G r e g o r y Norwich Eaton Pharmaceuticals, Inc., Norwich, New York 13815, U.S.A.
Received 23 December 1988, accepted 3 January 1989
Dantrolene sodium has been compared with reference antiarrhythmic agents in rodent models of cardiac arrhythmia. In a coronary-artery-ligation model in rats, dantrolene sodium (3, 10 and 20 mg/kg i.v.) significantly decreased extrasystoles, episodes of ventricular tachyarrhythmia, and frequency, duration, and total episodes of ventricular fibrillation in a dose-dependent manner. In an electrically induced fibrillation model in rats, dantrolene sodium (10 and 20 mg/kg i.v.) significantly raised ventricular fibrillation threshold in a dose- and time-dependent manner. In contrast to its activity in these models, dantrolene sodium was not active in two chemically induced models involving automaticity. Aconitine-induced arrhythmias in rats and mice and ouabain-induced arrhythmias in guinea pigs were not suppressed by i.v. (10 or 20 mg/kg) or i.p. (100-3000 mg/kg) doses of the drug. These results show that the antiarrhythmic potential of dantrolene sodium, predicted by in vitro Class III and Class IV electrophysiological effects, is expressed in whole animal models. Dantrolene sodium; Antiarrhythmia agents; Cardiac arrhythmia models; Coronary artery ligation; Ventricular fibrillation threshold; Aconitine; Ouabain
1. Introduction
Heart disease remains a major health problem in the United States with significant mortality attributed to cardiac arrhythmias (Keefe et al., 1981). Despite mechanistic diversity, current antiarrhythmic drugs are inadequate (Bigger and Hoffman, 1980). Their use is hampered by undesirable side effects and lack of efficacy. One study showed that 40-60% of patients, whose deaths were attributable to abnormal cardiac electrical activity, were being treated with antiarrhythmic agents at the time of death (Schaffer and Cobb, 1975).
* To whom all correspondence should be addressed: Product Development, Norwich Eaton Pharmaceuticals, Inc., P.O. Box 191, Norwich, NY 13815-0191, U.S.A.
Dantrolene sodium; 1-[[[5-(4-nitrophenyl)-2furanyl]methylene]amino]-2,4-imidazolidinedione sodium salt hydrate, molecular weight 314.26) is a skeletal muscle relaxant approved for use in the treatment of spasticity and malignant hyperthermia (Pinder et al., 1977; Britt, 1984). In whole animals this drug produced only minimal effects on normal cardiac muscle and cardiovascular function (Ellis et al., 1976a; Ellis et al., 1976b). Yet, when administered intraveneously (10 m g / k g ) to anesthetized dogs, dantrolene sodium significantly increased atrial and ventricular effective refractory periods (Roewer et al., 1984). In in vitro experiments, using cardiac tissue from several species at high drug concentrations (300 /~M), dantrolene sodium caused both positive and negative inotropic effects (Meszaros et al., 1982; Butterfield et al., 1985). Electrophysiological changes induced by the drug (8-35/~M) have been studied
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
522
in dog (Salata and Jalife, 1982; Salata et al., 1983) and sheep (Lascault et al., 1982) Purkinje fibers. Dantrolene sodium lowered action potential plateau and prolonged action potential duration in a concentration- and frequency-dependent manner, which was reversed by elevation of calcium concentration in the perfusing medium. These findings suggested that dantrolene sodium might have a direct antiarrhythmic action, which has been realized in two animal models of cardiac arrhythmia. In a mouse model of ventricular fibrillation induced by chloroform vapor, dantrolene sodium administered i.p. protected in a dose-dependent manner (Butterfield et al., 1983). The median effective dose of dantrolene sodium was 52.6 mg/kg, compared with 62.9 mg/kg for lidocaine hydrochloride, 87.7 mg/kg for quinidine sulfate, and 113.6 mg/kg for procainamide hydrochloride. At the median effective dose, protection by dantrolene sodium began 15-30 rain after dosing and lasted beyond 90 rain. In a dog model of ventricular arrhythmia induced by 60 min ligation of the left anterior descending coronary artery followed by reperfusion, dantrolene sodium at 10 mg/kg i.v. and at 32 mg/kg p.o. suppressed ectopic ventricular extrasystoles without significantly affecting overall ventricular rate or blood pressure (Butterfield et al., 1983). Electrophysiological effects of dantrolene sodium have not been studied in rats, but prolongation of action potential duration has been shown in guinea pig ventricular muscle (Hiroaka et al., 1985). To further characterize its antiarrhythmic spectrum we have evaluated dantrolene sodium in standard rodent models of cardiac arrhythmia. A preliminary report of these results has appeared (Brooks et al., 1984).
2. Materials and methods
2.1. Animals
Male Sprague-Dawley rats weighing 250-450 g and male SW(BR) mice weighing 20-28 g were obtained from Taconic Farms, Inc. (Germantown, NY). Male Hartley strain guinea pigs were
purchased from Charles River Breeding Laboratories (Wilmington, MA). 2.2. Chemicals
Dantrolene sodium and nifedipine were synthesized by Norwich Eaton Pharmaceuticals, Inc. Ouabain octahydrate, aconitine, quinidine sulfate, and propranolol hydrochloride were obtained from Sigma Chemical Co. Saline and pentobarbital sodium (Nembutal ~) were purchased from Abbott Laboratories, methylcellulose (3000 cps) from Dow Chemical Co., urethane from Pfaltz-Baum, Inc., and heparin sodium (Hepalean®) from Harris Laboratories. The following reference antiarrhythmic agents were obtained as gifts from their manufacturers: lidocaine hydrochloride (Astra Pharaceutical Products); amefalone (ORG 6001) (Organon); metoprolol tartrate (CIBA Pharmaceutical Co.); bepridil (Wallace Laboratories); bretylium tosylate (DuPont Critical Care); melperone hydrochloride (Ferrosan); and mexiletine hydrochloride (Boehringer Ingelheim, Ltd.). 2.3. Coronary-artery ligation in rats
The model was run as described by Kane et al. (1979). In brief, respired pentobarbital-anesthetized rats were thoracotomized and the left descending coronary artery was ligated for 30 min. Arrhythmias indicated by a lead II electrocardiogram were enumerated. After the experiment, ligation was verified by visual inspection of the myocardium or by dye infusion. Dantrolene sodium as a 0.05% (w/v) solution in 4.44% (w/v) mannitol-0.008% (w/v) NaOH was administered i.v. over 5-8 min at an infusion rate of 5-8 ml/min per kg to give doses of 3, 10 and 20 mg/kg. Lidocaine hydrochloride as a 2% solution in 0.6% saline was infused i.v. over 30 s to give a 10 mg/kg slow bolus dose (1 ml/min per kg), followed immediately by a continuous 0.1 mg/min per kg infusion during the ligation. Corresponding vehicle doses were given to separate rats. Ligation was done after completing the drug or vehicle infusions.
523 2.4. Electrically induced fibrillation in rats A method similar to Marshall et al. (1983) was used. In brief, respired pentobarbital-anesthetized rats were thoracotomized and fitted with two barbed platinum electrodes positioned 3-6 mm apart on the left ventricular anterior wall such that the anode was 3 mm below the atrioventricular ring and the cathode was on or near the apex. Following a 15-min equilibration period, trains of rectangular pulses (lasting 5 s at 50 Hz with a pulse duration of 5 ms) were delivered every 10 s. Current intensity was then increased at a rate of 20-40 t~a/train until fibrillation occurred (defined as the ventricular fibrillation threshold, VFT) or until a maximum current of about 400 /zA was reached. Each rat received two stimulating sequences ( - 1 5 , 0 rain) prior to and four sequences ( + 15, + 30, + 45, + 60 min) following drug or vehicle administration at time 0. Rats were accepted only if BP was 50 nun Hg at the end of the equilibration period and fibrillation could be induced prior to treatment. Dantrolene sodium was prepared as an 0.8% ( w / v ) solution in a 50% organic vehicle containing 15.6% glycerin, 5.7% propylene glycol, 11.5% 2-pyrrolidone, 17.2% N-(2-hydroxyethyl)-2-pyrrolidone, 5.0% urea, and 10% glucose (all w / v ) in water. Nifedipine was prepared as a 0.1 m g / m l solution in this vehicle and protected from light. Metoprolol tartrate, amefalone, and melperone hydrochloride were used as 0.5% (w/v) solutions in 0.9% saline. Compounds or corresponding vehicles were administered i.v. in less than 3 min at 1 m l / m i n per kg. Doses used were 3, 5.4, 10 and 20 m g / k g for dantrolene sodium, 0.02 m g / k g for nifedipine, 5 m g / k g for metoprolol tartrate, and 10 m g / k g for amefalone and melperone hydrochloride. 2.5. Aconitine-induced arrhythmias in rats and mice Methods for aconitine arrhythmias were adapted from the literature (Miiller and Wilsmann, 1982; Nwangwu et al., 1977). For rats, the test substance or its vehicle was administered at a rate of 1-4 m l / m i n per kg followed within 5 min,
by an infusion of aconitine (20/~g/ml) at the rate of 0.1 m l / m i n (0.25-0.4 m l / m i n per kg). Conscious mice were pretreated i.p. (10 ml/kg) with test substance or vehicle at the times indicated before being anesthetized (60 m g / k g i.p. Nembutal) and infused with aconitine. Times chosen were the times of maximum effect of these substances in the mouse chloroform arrhythmia model (Butterfield et al., 1983). Aconitine (3 or 5 /~g/ml) was infused (tail vein) at the rate of 10 m l / m i n per kg. For rats, dantrolene sodium as a 0.05% (w/v) solution in mannitol-NaOH was administered i.v. at an infusion rate of 1 m l / m i n per kg over 20 min to give a dose of 10 mg/kg. Quinidine sulfate (1% w / v in saline) and propranolol hydrochloride (0.1% w / v in saline) were infused over 0.5 rain (2-4 m l / m i n per kg) to give doses of 10 and 20 m g / k g for the former and 2 m g / k g for the latter. For mice, compounds were suspended in 0.5% carboxymethylcellulose at concentrations to permit delivery of the indicated dose in 10 m l / k g body weight. Dantrolene sodium (100, 300, 1000 and 3000 m g / k g ) was given 60 rain before, quinidine sulfate (88 m g / k g ) or propranolol hydrochioride (43 m g / k g ) were given 30 min before, and lidocaine hydrochloride (53 mg/kg) was given 15 min before the start of aconitine. Aconitine was prepared fresh daily in 0.2% (v/v) dimethylformamide in water. 2.6. Ouabain-induced arrhythmias in guinea pigs A procedure modified from Sekiya and Vaughan Williams (1963) was used. Guinea pigs weighing 450-550 g were anesthetized with urethane (1.25 g / k g i.p.), intubated, and respired mechanically. After the preparation had stabilized as judged by blood pressure and electrocardiogram tracings, test compound or vehicle was administered i.v. in 7 min or less before starting the ouabain infusion (7.5/~g/min at 0.1 ml/min). Dantrolene sodium was also studied with administration at 30 and 60 min before ouabain. In separate experiments it was administered i.p. 60 min before ouabain. The onset of arrhythmia was the time of occurrence of the first extrasystole. The onset of fibrillation was identified as an asyn-
524
chronous irregular electrocardiogram pattern accompanied by a decrease in blood pressure to less than 15 mm Hg. Dantrolene sodium was administered i.v. at 10 and 20 m g / k g in both the 50% organic and alkaline mannitol vehicles described above. The 50% organic vehicle was also used for nifedipine. Bepridil, bretylium tosylate, and mexiletine hydrochloride were given in water. Propranolol was delivered in saline. All agents except dantrolene sodium were delivered i.v. in 0.25-2 mi. Doses are shown in table 5. For i.p. administration of 300 mg/kg, dantrolene sodium was suspended in 0.5% (w/v) methylceUulose at 10 mg/ml. Ouabain octahydrate was used as a 75 /~g/ml solution in saline.
1 s and with fewer than 3 peaks of at least 10 mm Hg per 5 s. Height was determined in reference to adjacent baselines of the peaks. Significance (at the 0.05 level) of differences between effects of drugs and vehicles on VFT change (posttreatment value minus pretreatment mean value for the same rat) were assessed by a t-test. Since there was no difference in threshold values for rats given different volumes of organic vehicle (corresponding to the dantrolene sodium and nifedipine doses), resuits from these vehicle-treated control rats were combined. 2. 7.3. Aconitine- and ouabain-induced arrhythmia models Treatment effects were assessed by a t-test.
2. 7. Data analysis 3. Results
2. 7.1. Rat coronary-artery-ligation model Only rats which survived the 30-min postligation period were included in the data sets (see Results). The number of ventricular extrasystoles (VES, irregular or unscheduled beats), number and duration of ventricular tachyarrhythmia (VT, runs of 7 or more VES as defined by Kane et al., 1982), and number and duration of ventricular fibrillations (VF, loss of sinus rhythm and decrease in BP to < 15 mm Hg sustained for more than 3 s) were extracted from the electrocardiogram. To stabilize variances, the following data were log-transformed prior to analysis: total VES/30 min, duration of VT + 1 and VF + 1, and number of episodes of V T + I and V F + I . Regression analysis was used to test for dose-response relationships, using log dose as the independent variable. Two-way analysis of variance was used to identify significant treatment and dose effects. Chi-square analysis was employed to test the significance of treatment effects on the incidence of VF (rats affected/total number of rats). 2. 7.2. Rat electrically induced fibrillation model The ventricular fibrillation threshold (VFT) was taken as the stimulus current which, during its delivery period, resulted in a BP tracing with pulsatiles not greater than 5 mm Hg for more than
3.1. Coronary-artery ligation in rats Based on mortality and timing and type of arrhythmia observed, our results from the rat model of coronary-artery-ligation-induced arrhythmia were comparable with those reported by others. Of 27 rats treated with vehicle (which include the control rats of table 1), 6% died from VF and 1% died from asystole. This is similar to the 10-25% mortality noted by others (Clark et al., 1980; Fagbemi and Parratt, 1981; Kane et al., 1980; Fagbemi et al., 1982), but less than the 89% found by Mertz and Kaplan (1982). All rats included in table 1 survived the 30-min ligation period. Dysrhythmic activity, including VT and VF, occurred in control rats between 4-14 min after ligation, and was maximal at approximately 9 min, similar to previous findings (Clark et al., 1980; Kane et al., 1980; Kane and Winslow, 1980). VES count varied considerably between rats. For 15 rats treated with mannitol-NaOH (table 1), a range of 46-3176 V E S / 3 0 min (mean 1004 _ 244 S.E.) was observed; for 5 saline-treated rats, the range was 248-1796 V E S / 3 0 min ( 7 4 1 _ 289). Mean VES values of 776 to 1483 for this same 30-min interval have been reported (Clark et al., 1980; Fagbemi and Parratt, 1981; Kane et al., 1980; Fagbemi et al., 1982; Mertz and Kaplan,
525 TABLE 1 Effect of i.v. dantrolene sodium and lidocaine hydrochloride in a rat coronary-artery-ligation model. Values are means__ S.E. (5 rats/group) and percentage of corresponding vehicle-treated control group. Infusion for 5-8 min of 0.05% (w/v) dantrolene sodium in 4.44% mannitol-0.008% NaOH at 5-8 ml/min per kg was completed just prior to ligation. Lidocaine hydrochloride, 2% in saline, was infused at 20 mg/min per kg to give 10 mg/kg, followed by continuous infusion of 0.1 mg/min per kg. Ligation was 5 min after the start of lidocaine, a Significant (P < 0.05) dantrolene sodium treatment effect (analysis of variance), b Significant (P < 0.025) dantrolene sodium treatment effect (chi-square). Treatment
Dose (mg) or volume (ml) per kg
Ventricular extrasystoles per 30 min
Ventricular tachyarrhythmia DuraNo. of tion episodes a (s)
Ventricular fibrillation DuraNo. of tion episodes a (s) a
Incidence rats b
Mannitol-NaOH Dantrolene sodium
6 ml 3 mg
9475:564 505+193 53%
625:47 255:18 39%
9.05:3.8 5.6+2.1 62%
30+30 65:6 20%
0.45:0.4 0.25:0.2 50%
1/5 1/5
Mannitol-NaOH Dantrolene sodium
20 ml 10 mg
999+386 4205:149 42%
795:38 14+ 8 18%
14.45:5.3 1.65:0.9 11%
75:4 05:0 0%
1.4+0.7 0 +0 0%
3/5 0/5
Mannitol-NaOH Dantrolene sodium
40 ml 20 mg
10665:395 1565:49 15%
365:18 105:5 26%
9.85:4.2 1.85:0.7 18%
75: 4 05:0 0%
0.6+0.2 0 +0 0%
3/5 0/5
Saline Lidocaine hydrochloride
0.5 ml 10 mg
741+289 9625:387 130%
48+20 225:13 47%
7.65:1.5 11.8+8.8 155%
1+ 1 0+ 0 0%
0.45:0.4 0 5:0 0%
1/5 0/5
1982; K a n e et al., 1979; M a r s h a l l et al., 1981). VT was o b s e r v e d in 95% ( 1 9 / 2 0 ) of c o n t r o l rats with a d u r a t i o n of 59.1 _+ 20.0 s for the m a n n i t o l N a O H - t r e a t e d rats a n d 4 7 . 8 _ 19.8 s for the saline-treated rats. T h e l i t e r a t u r e r e p o r t s 57-100% incidence of V T w i t h a d u r a t i o n of 67-99 s ( C l a r k et al., 1980; F a g b e m i a n d Parratt, 1981; F a g b e m i et al., 1982; K a n e et al., 1979; M a r s h a l l et al., 1981). V F was o b s e r v e d in 40% ( 8 / 2 0 ) of c o n t r o l rats with a d u r a t i o n of 14.5 +_ 9.8 s ( m a n n i t o l ) a n d 1.4 _+ 1.4 (saline). T h e literature r e p o r t s 40-89% incidence of V F with a d u r a t i o n of 24-62 s ( C l a r k et al., 1980; F a g b e m i a n d Parratt, 1981; F a g b e m i et al., 1982; M e r t z a n d K a p l a n , 1982; K a n e et al., 1979). L i d o c a i n e h y d r o c h l o r i d e (10 m g / k g i.v.) d i d n o t significantly reduce a n y m e a s u r e of a r r h y t h m i a i n d u c e d in rats b y ligation of the left d e s c e n d i n g c o r o n a r y artery (table 1). N o f i b r i l l a t i o n was seen in the l i d o c a i n e - t r e a t e d rats, b u t this d i d n o t differ significantly f r o m the vehicle-treated results, w h e r e two episodes of V F o c c u r r e d in 1 of 5 rats. L i d o -
caine's activity has b e e n r e p o r t e d in the c o r o n a r y a r t e r y - l i g a t i o n m o d e l in rats ( C l a r k et al., 1980; K a n e et al., 1979). Nevertheless, there is controversy over the efficacy of l i d o c a i n e in this exp e r i m e n t a l s i t u a t i o n a n d in the c o r r e s p o n d i n g clinical s i t u a t i o n of v e n t r i c u l a r e c t o p i c activity in the first few h o u r s after m y o c a r d i a l i n f a r c t i o n (Winslow, 1984). D a n t r o l e n e s o d i u m at doses of 3, 10 a n d 20 m g / k g i.v. s u p p r e s s e d VES in a d o s e - d e p e n d e n t m a n n e r to 53, 42 a n d 15%, respectively, of the m a n n i t o l - N a O H vehicle c o n t r o l counts (table 1). Because of the large v a r i a n c e in VES count, these r e d u c t i o n s were n o t quite significant at the P < 0.05 level (P < 0.1). V T d u r a t i o n was decreased. A t 3, 10 a n d 20 m g / k g i.v. doses the n u m b e r of e p i s o d e s of V T were significantly (P < 0.05) decreased to 62, 11, a n d 18% of the respective corres p o n d i n g vehicle values. T h e a n t i a r r h y t h m i c action of d a n t r o l e n e s o d i u m in this m o d e l was m a n i f e s t e d also as significant (P < 0.05) decreases in the n u m b e r of e p i s o d e s a n d average d u r a t i o n of
526 TABLE 2 Effect of i.v. dantrolene sodium and reference antiarrhythmic agents in the rat ventricular fibrillation threshold (VFT) model. For each rat two determinations ( - 15 and 0 min) were done to determine the pretreatment fibrillation threshold. A single determination was done at the times indicated posttreatment. Values are means+ S.E. for the number of rats shown. Dantrolene sodium and nifedipine were administered in a 50% organic vehicle (see Methods). Other agents were administered in saline, a Significant (P < 0.05) change in threshold (t-test). Treatment
Organic vehicle Dantrolene sodium
Nifedipine Saline Metoprolol tartrate Amefalone Melperone hydrochloride
Dose
No.
Pre-
Change in VFT posttreatment (/~A)
(mg/kg)
of rats
treatment threshold #A
+ 15 min
+ 30 min
+ 45 min
+ 60 min
0.4-2.5 (ml/kg) 3 5.4 10 20
21
170 ± 10
- 8 ± 15
3 + 15
19 ± 17
26 + 20
2 7 5 6
140 -/-50 191 + 21 150+24 173±30
0 ± 10 23 _ 12 74+43 a 133+33 a
0 + 10 80 + 52 98±53 a 163±54 a
0 + 30 91 ± 51 126±51 a 180±45 a
30 ± 40 54 ± 42 138±54 a 177±46 a
3
160 ± 23
- 33 ± 35
- 20 ± 12
- 7 ± 18
- 7 ± 24
1-2 (ml/kg) 5
3
110+ 6
-10+15
-23+
3
3
133± 5
-60±31
-53±
7a
10
3
93± 9
33± 9
40± 6
10
3
140+41
7+12
0+17
0.02
V F , as well as b y a s i g n i f i c a n t ( P < 0.025) d e c r e a s e
3±26
-17±
-67±18
a
9
-73±24
33± 5
13± 9
20±47
100±95
3.2. Electrically induced fibrillation in rats
i n t h e i n c i d e n c e o f V F . O n l y 1 o f 15 r a t s t r e a t e d with d a n t r o l e n e s o d i u m exhibited V F , a n d this
No significant changes of fibrillation threshold from baseline were observed during the hour after
o c c u r r e d i n a n a n i m a l g i v e n t h e l o w e s t (3 m g / k g ) d o s e . I n c o n t r a s t , 7 o f 15 r a t s t r e a t e d w i t h m a n n i -
g i v i n g v e h i c l e a l o n e ( t a b l e 2). O f t h e f o u r r e f e r -
tol-NaOH alone exhibited VF.
ence
antiarrhythmic
agents
tested,
two
signifi-
TABLE 3 Effect of i.v. dantrolene sodium, quinidine sulfate and propranol hydrochloride in the rat aconitine-induced arrhythmia model. Anesthetized rats were infused with compound or vehicle just before the start of the aconitine infusion (2 /tg/min). Dantrolene sodium was in mannitol-NaOH; quinidine sulfate and propranolol hydrochloride were in saline, a Significant (P < 0.05) treatment effect (t-test). Treatment
Dose (mg/kg)
No. of rats
Aconitine arrhythmia threshold dose Mean ± S.E. g/kg
% of control
Mannitol-NaOH Dantrolene sodium
20 (ml/kg) 10
10 11
27.2 + 4.0 35.3 ± 4.3
100 130
Saline Quinidine sulfate
1-2 (ml/kg) 10 20 2
10 5 5 5
20.4 + 1.6 25.3 + 2.2 32.4-t-5.7 24.5 ± 2.4
100 124 159 a 120
Propranolol hydrochloride
527
cantly changed VFT in the rat at the 30 min posttreatment test period (table 2). Amefalone (10 m g / k g i.v.) raised fibrillation threshold. In contrast, metoprolol tartrate (5 m g / k g i.v.) lowered VFT threshold - 5 3 + 7 /~A. Nifedipine and melperone did not significantly affect VFT. Dantrolene sodium (3 to 20 m g / k g i.v.) provided a dose-dependent increase in VFT (table 2). Increases were also time-dependent, the maximum effect occurring at 45-60 min after administration. The increases ( > 130 /zA) at all four posttreatment times after 10 and 20 m g / k g i.v. dosing were statistically significant (P < 0.05). 3.3. Aconitine-induced arrhythmias in rats and mice
In rats treated i.v. with dantrolene sodium (10 mg/kg), quinidine sulfate (10 mg/kg), and propranolol hydrochloride (2 mg/kg), the aconitine dose required to induce arrhythmias was 130, 124 and 120% of respective vehicle control values (table 3). These changes were not significant. A higher (20 mg/kg) dose of quinidine sulfate significantly (P < 0.05) raised the aconitine threshold dose to 139% of its control. However, in two of
seven rats, this dose caused AV block and death before the onset of VES. While a higher dose of dantrolene sodium may have given greater effects, we did not give more than 10 m g / k g i.v., since this is a clearly muscle-relaxant dose in rats, and this was also an antiarrhythmic dose in the above two rat models. In mice pretreated i.p. with lidocaine hydrochloride at 15 rain, or with propranolol hydrochloride at 30 min, before aconitine, the aconitine doses causing either single extrasystoles or tachyarrhythmia were not significantly raised (table 4). Quinidine sulfate (88 mg/kg) administered 30 min before the aconitine infusion, significantly (P < 0.05) raised both thresholds. Dantrolene sodium (100-3000 m g / k g i.p.) administered 60 min before aconitine, did not significantly increase the arrhythmogenic dose of aconitine (table 4). The significantly decreased aconitine threshold doses seen at 100 and 300 m g / k g i.p. dantrolene sodium may indicate a proarrhythmic action at high doses in this model. There also was no effect of dantrolene sodium (300 m g / k g i.p.) on thresholds determined with a slower rate of aconitine infusion (table 4).
TABLE 4 Effect of i.p. dantrolene sodium and reference agents in the mouse aconitine-induced arrhythmia model. Mice were pretreated with vehicle or compound at 60 min (dantrolene sodium), 30 rain (quinidine sulfate and propranolol hydrochloride), or 15 rain (lidocaine hydrochloride) before the start of the aconitine infusion, a Significant (P < 0.05) treatment effect (t-test). Treatment
Dose mg/kg
No. of mice
Aconitine arrhythmia threshold dose ( ~ g / k g ) Extrasystoles Mean _+S.E.
Tachyarrhythmia % of control
Mean -+ S.E.
% of control
Fast aconitine infusion (1.25 tt g / min) Carboxymethylcellulose Dantrolene sodium
Saline Lidocaine hydrochloride Propranolol hydrochloride Quinidine sulfate
10 ( m l / k g ) 100 300 1000 3000
9 5 5 5 4
83_+ 2 75+ 3 70_+ 2 a 97-+ 5 96_+11
100 90 84 117 116
108_+ 5 91_+ 5 a 91_+ 2 a 110+ 5 112_+11
100 85 84 102 104
10 ( m l / k g ) 53 43 88
5 5 4 5
63_+ 5 72_+ 4 78 _+14 109-+ 6 a
100 115 125 174
79_+ 4 103_+ 8 101 + 20 147-+ 8
100 130 128 187
10 10
60+ 1 73± 6
100 122
81+ 3 88+ 7
a
Slow aconitine infusion (0. 75 It g / min) Carboxymethylcellulose Dantrolene
10 m l / k g 300
100 109
528 TABLE 5 Effect of i.v. dantrolene sodium and reference agents in the guinea pig ouabain-induced arrhythmia model. Anesthetized guinea pigs were dosed with compounds in organic vehicle, mannitol-NaOH, saline or water (see Methods) over the 0.5-7 min period before the start of the ouabain octahydrate infusion (7.5 /~g/min, 0.1 ml/min). Mean ( + S.E.) arrhythmia and fibrillation threshold doses Otg/kg i.v.) of ouabain for the various vehicles were, respectively, as follows: 128+7 and 193+ 8 (n =17) for saline, 127+5 and 185 + 7 (n = 22) for water, 112 + 6 and 169 _+12 (n = 5) for mannitol-NaOH equivalent to the 10 m g / k g dose of dantrolene sodium, 128 + 11 and 199 + 14 (n = 5) for mannitol-NaOH equivalent to the 20 mg/kg dose, 125 + 6 and 187 + 7 (n = 16) for organic vehicle at -2.5 min, 111 + 8 and 171 + 12 (n = 7) at - 3 0 min, and 118 + 11 and 164+ 10 (n = 5) at - 6 0 min. a Significant (P < 0.05) change from the corresponding vehicle value (t-test). Treatment
Dose (mg/kg)
No. of guinea pigs
Percentage of control ouabain threshold dose (mean + S.E.) Arrhythmia
Fibrillation
5 5 3
102+ 6 105+11 82 + 4
114+ 7 100+ 9 107 + 10
5 6
110+ 7 147+23
92+ 5 124+22
1 5.6 10
3 4 4
92+ 8 115+ 3 152+ 9
a
106+13 122+14a 132+ 3 a
22
3
137+21
a
144+ 6
Bretylium tosylate
5
3
105+11
Bepridil
1.5
3
158+ 8 a
Dantrolene sodium in organic vehicle at - 60 min 20 at - 30 min 20 at - 2.5 min 20 Dantrolene sodium in mannitol-NaOH at - 2 min 10 at - 2 min 20 Mexiletine HC1
Propranolol HC1
3.4. Ouabain-induced arrhythmias in guinea pigs Because agents were tested in this model in s e v e r a l v e h i c l e s , r e s u l t s a r e e x p r e s s e d as p e r centage changes in arrhymogenic ouabain dose. Actual arrhythmia and fibrillation threshold doses of ouabain for guinea pigs dosed with saline, water, mannitol-NaOH, and an organic vehicle are g i v e n i n t h e n o t e s o f t a b l e 5. N o s i g n i f i c a n t d i f ferences in thresholds were attributable to different vehicles. Of the twelve reference antiarrhythmic agents e v a l u a t e d , n i n e s i g n i f i c a n t l y ( P < 0.05) r a i s e d either the arrhythmia or fibrillation threshold at o n e o r m o r e d o s e s ( t a b l e 5). T h e c l a s s I a g e n t m e x i l e t i n e w a s a c t i v e , as w a s t h e c l a s s II d r u g propranolol, and the class IV agent bepridil. The class III drug bretylium tosylate did not increase t h r e s h o l d s . I n fact, b r e t y l i u m s l i g h t l y l o w e r e d fibrillation threshold. Dantrolene sodium was evaluated under several conditions differing in vehicle, dose, route, and
a
86+ 4 a 140+ 8 a
time of dosing relative to the start of ouabain i n f u s i o n . I n 50% o r g a n i c v e h i c l e , d a n t r o l e n e s o d i u m ( 2 0 m g / k g i.v.) a d m i n i s t e r e d a t 60, 30, o r 2.5 m i n b e f o r e o u a b a i n h a d n o e f f e c t o n t h r e s h o l d s ( t a b l e 5). D a n t r o l e n e s o d i u m ( 1 0 o r 20 m g / k g i.v.) in mannitol-NaOH did not significantly change arrhythmia or fibrillation thresholds, although the h i g h e r d o s e r a i s e d t h e f o r m e r t o 147% o f c o n t r o l . To eliminate any effects of intravenous vehicles or
TABLE 6 Effect of i.p. dantrolene sodium in the guinea pig ouabain-induced arrhythmia model. Dantrolene sodium (10 m g / m l in 0.5% methylcellulose) or vehicle were administered to groups of five animals 60 rain before the start of the ouabain octahydrate infusion (7.5/~g/min). Treatment
Dose
Ouabain threshold dose (~ g/kg i.v.) Arrhythmia Fibrillation
0.5% Methylcellulose 10 (ml/kg) 135+ 13 Dantrolene sodium 300 (mg/kg) 138+44
199_+20 199+27
529 volumes, dantrolene sodium was administered i.p. in methylcellulose 60 min before the start of ouabain infusion. Arrhythmia and fibrillation thresholds were unchanged by this treatment in comparison with the corresponding vehicle-treated group (table 6).
4. Discussion The purpose of these experiments was to further characterize the antiarrhythmic activity of dantrolene sodium in several standard rodent models of cardiac arrhythmia. The compound was administered i.v. or i.p. in a dose range which included doses known to elicit skeletal muscle relaxation, i.e., 3-10 m g / k g i.v. Arrhythmias induced by mechanical (coronary artery ligation), electrical (ventricular fibrillation threshold), and chemical (aconitine and ouabain) stimuli were studied in mice, rats or guinea pigs. In the coronary artery-ligated rat, the antiarrhythmic activity of dantrolene sodium was manifested as a marked, dose-related suppression of VES, as reduction in VT (both number of episodes and duration), and, most strikingly, as complete elimination of VF at the 10 and 20 m g / k g i.v. dose levels. In the ventricular fibrillation threshold model in rats, dantrolene sodium also displayed dose-related antifibrillatory activity. The magnitude of the dantrolene sodium-induced increase in V F T was related to the time posttreatment. The maximum effect was seen 45 or 60 min after compound administration, but in a few rats given 10 and 20 m g / k g i.v., increases in V F T were still observed 120 rain after dosing. The arrhythmias occurring early after ligation of the coronary artery arise in epicardial layers where ischemic zone cells display shortened action potentials and depressed m a x i m u m rates of depolarization (Elharrar and Zipes, 1977). Although prolongation of action potential duration (APD) by dantrolene sodium has not been shown in rat cardiac tissue, this could be the basis of the drug's ability to suppress these early reentrant arrhythmias. The coronary artery ligation model may have clinical relevance. Early arrhythmias following
coronary artery ligation may mimic the period immediately following myocardial infarction in man, where estimates have been made of 22% mortality with an average time to death of 20 min (Paul, 1974; Gyfirffis, 1976). The lack of activity of dantrolene sodium in the aconitine- and ouabain-induced models is consistent with its lack of Class I effects. Both aconitine- and ouabain-induced arrhythmias are due to increased automaticity, rather than reentry, and involve elevation of intracellular sodium and the induction of afterdepolarizations (Winslow, 1984). These results demonstrate the antiarrhythmic action of dantrolene sodium in a variety of rodent arrhythmia models at doses which provide relaxation of skeletal muscle. While no direct relationship between prolongation of action potential duration in rodent cardiac tissue and the antiarrhythmic effect in whole animals has been shown, it is likely that the two properties of dantrolene sodium are related. Evidence supporting this relationship may be the lack of activity of both dantrolene sodium and a class III antiarrhythmic drug in the guinea pig ouabain model.
References Bigger, J.T., Jr. and B.F. Hoffman, 1980, Antiarrhythmic drugs, in: Goodman and Gilman's The Pharmacological Basis of Therapeutics, 6th ed., eds. A.G. Gilman, L.S. Goodman and A. Gilman (MacMillan Publishing Co., Inc., New York) p. 761. Britt, B.A., 1984, Dantrolene, Can. Anaesth. Soc. J. 31, 61. Brooks, R.R., J.F. Carpenter, S.M. Jones, C.M. Gregory and A.F. Moore, 1984, Evaluation of dantrolene sodium in rodent models of cardiac arrhythmia, Proceedings of the Ninth International Congress of Pharmacology, International Union of Pharmacology, London, England, p. 455P. Butterfield, J.L., R.R. Brooks and A.F. Moore, 1985, Contractile effects of dantrolene sodium (DS) on guinea pig atrial muscle in vitro, Fed. Proc. 44, 900 (No. 2861). Butterfield, J.L, J.F. Carpenter, S.M. Jones, R.R. Brooks, K.O. Ellis and A.F. Moore, 1983, Antiarrhythmic activity of dantrolene sodium (Dantrium), The Pharmacologist25, 246 (No. 751). Clark, C., M.I. Foreman, K.A. Kane, F.M. McDonald and J.R. Parratt, 1980, Coronary artery ligation in anesthetized rats as a method for the production of experimental dys-
530 rhythmias and for the determination of infarct size, J. Pharmacol. Meth. 3, 357. Elharrar, V. and D.P. Zipes, 1977, Cardiac electrophysiologic alterations during myocardial ischaemia, Am. J. Physiol. 233, H329. Ellis, K.O., J.L. Butterfield, F.L Wessels and J.F. Carpenter, 1976a, A comparison of skeletal, cardiac, and smooth muscle actions of dantrolene sodium - a skeletal muscle relaxant, Arch. Int. Pharmacodyn. Ther. 224, 118. Ellis, K.O., F.L. Wessels and J.F. Carpenter, 1976, Effects of intravenous dantrolene sodium on respiratory and cardiovascular functions, J. Pharm. Sci. 65, 1359. Fagbemi, O., K.A. Kane and J.R. Parratt, 1982, Creatine phosphate suppresses ventricular arrhythmias resulting from coronary artery ligation, J. Cardiovasc. Pharmacol. 4, 53. Fagbemi, O. and J.R. Parratt, 1981, Calcium antagonists prevent early post-infarction ventricular fibrillation, European J. Pharmacol. 75, 179. Gyhrf~s, I., 1976, Sudden death in AMI: a population study in Budapest, Seventh European Congress of Cardiology, Amsterdam, Netherlands, p. 780. Hiraoka, M., H. Kinoshita and S. Kawano, 1985, Action of dantrolene sodium on electrical and mechanical activity of guinea-pig ventricular muscles, Jap. J. Physiol. 35, 129. Kane, K.A., I. Lepr~m, F.M. McDonald, J.R. Parratt and L. Szekeres, 1980, The effects of prolonged oral administration of a new anti-dysrhythmic drug (ORG 6001) on coronary artery ligation dysrhythmias in conscious and anesthetized rats, J. Cardiovasc. Pharmacol. 2, 411. Kane, K., F. McDonald and J.R. Parratt, 1979, Coronary artery ligation in anesthetized rats as a model for the assessment of antidysrhythmic activity; the effects of lignocaine, propranolol and ORG 6001, Br. J. Pharmacol. 66, 463P. Kane, K., F. McDonald, J. Parratt, C. Timmer and J. Vink, 1982, Antiarrhythmic effects of ORG 6001 in rats: correlation with plasma and tissue drug concentrations, Br. J. Pharmacol. 75, 319. Kane, K.A. and E. Winslow, 1980, Antidysrhythmic and electrophysiological effects of a new antianginal agent, bepridil, J. Cardiovasc. Pharmacol. 2, 193. Keefe, D.ED., R.E. Kates and D.C. Harrison, 1981, New antiarrhythmic drugs: their place in therapy, Drugs 22, 363. Lascault, G., J. Asta, D. Cameron and M.B. Waxman, 1982, Electrophysiologic effect of sodium dantrolene on cardiac action potential, Circulation 66, I1-294 (No. 1175). Marshall, R.J., A.W. Muir and E. Winslow, 1981, Development of a severe model of early coronary artery ligation-induced dysrhythmias in the anesthetized rat, Br. J. Pharmacol. 73, 951.
Marshall, R.J., A.W. Muir and E. Winslow, 1983, Effects of antiarrhythmic drugs on ventricular fibrillation thresholds of normal and ischemic myocardium in the anesthetized rat, Br. J. Pharmacol. 78, 165. Mertz, T.E. and H.R. Kaplan, 1982, Pirmenol hydrochloride (CI-845) and reference antiarrhythmic agents: effects on early ventricular arrhythmias after acute coronary artery ligation in anesthetized rats, J. Pharmacol. Exp. Ther. 223, 580. Meszaros, J., V. Kecskemeti and J. Szegi, 1982, The mechanism underlying the dantrolene-induced positive inotropic effect in guinea pig atrial myocardium, Acta Physiol. Acad. Sci. Hung. 59, 373. M~iller, B. and K. Wilsmann, 1982, Effects of the optical isomers of D 600 on cardiovascular parameters and on arrhythmias caused by aconitine and coronary artery ligation in anesthetized rats, J. Cardiovasc. Pharmacol. 4, 615. Nwangwu, P.U., T.L. Holcslaw and S.J. Stohs, 1977, A rapid in vivo technique for preliminary screening of antiarrhythmic agents in mice, Arch. Intern. Pharmacodyn. 229, 219. Paul, O., 1974, Myocardial infarction and sudden death, in: The Myocardium: Failure and Infarction, ed. E. Braunwald (H.P. Publishing Co., New York) p. 273. Pinder, R.M., R.N. Brogden, T.M. Speight and G.S. Avery, 1977, Dantrolene sodium: a review of its pharmacological properties and therapeutic efficacy in spasticity, Drugs 13, 3. Roewer, N., K.H. Kuck, C. Nienaber, E. Schroeder and J. Schulte am Esch, 1984, Electrophysiologic effects of intravenous dantrolene on dog hearts, Anesthesiology 61 (3A, Suppl.), A254. Salata, J.J. and J. Jalife, 1982, Effects of dantrolene sodium on the electrophysiology properties of canine cardiac Purkinje fibers, J. Pharmacol. Exp. Ther. 220, 157. Salata, J.J., J.A. Wasserstrom and J. Jalife, 1983, Dantrolene sodium: effects on isolated cardiac tissues, J. Mol. Cell Cardiol. 15, 233. Schaffer, W.A. and L.A. Cobb, 1975, Recurrent ventricular fibrillation and modes of death in survivors of out-ofhospital ventricular fibrillation, New Engl. J. Med. 293, 260. Sekiya, A. and E.M. Vaughan Williams, 1963, The effects of pronethalol, dichloroisoprenaline and disopyramide on the toxicity to the heart of ouabain and anaesthetics, Br. J. Pharmacol. 21, 462. Winslow, E., 1984, Methods for the detection and assessment of antiarrhythmic activity, Pharmacol. Ther. 24, 401.
521
Elsevier EJP 50723
Effects of dantrolene sodium in rodent models of cardiac arrhythmia R o b e r t R. B r o o k s *, J o h n F. C a r p e n t e r , S a n d r a M. J o n e s a n d C i n d a M. G r e g o r y Norwich Eaton Pharmaceuticals, Inc., Norwich, New York 13815, U.S.A.
Received 23 December 1988, accepted 3 January 1989
Dantrolene sodium has been compared with reference antiarrhythmic agents in rodent models of cardiac arrhythmia. In a coronary-artery-ligation model in rats, dantrolene sodium (3, 10 and 20 mg/kg i.v.) significantly decreased extrasystoles, episodes of ventricular tachyarrhythmia, and frequency, duration, and total episodes of ventricular fibrillation in a dose-dependent manner. In an electrically induced fibrillation model in rats, dantrolene sodium (10 and 20 mg/kg i.v.) significantly raised ventricular fibrillation threshold in a dose- and time-dependent manner. In contrast to its activity in these models, dantrolene sodium was not active in two chemically induced models involving automaticity. Aconitine-induced arrhythmias in rats and mice and ouabain-induced arrhythmias in guinea pigs were not suppressed by i.v. (10 or 20 mg/kg) or i.p. (100-3000 mg/kg) doses of the drug. These results show that the antiarrhythmic potential of dantrolene sodium, predicted by in vitro Class III and Class IV electrophysiological effects, is expressed in whole animal models. Dantrolene sodium; Antiarrhythmia agents; Cardiac arrhythmia models; Coronary artery ligation; Ventricular fibrillation threshold; Aconitine; Ouabain
1. Introduction
Heart disease remains a major health problem in the United States with significant mortality attributed to cardiac arrhythmias (Keefe et al., 1981). Despite mechanistic diversity, current antiarrhythmic drugs are inadequate (Bigger and Hoffman, 1980). Their use is hampered by undesirable side effects and lack of efficacy. One study showed that 40-60% of patients, whose deaths were attributable to abnormal cardiac electrical activity, were being treated with antiarrhythmic agents at the time of death (Schaffer and Cobb, 1975).
* To whom all correspondence should be addressed: Product Development, Norwich Eaton Pharmaceuticals, Inc., P.O. Box 191, Norwich, NY 13815-0191, U.S.A.
Dantrolene sodium; 1-[[[5-(4-nitrophenyl)-2furanyl]methylene]amino]-2,4-imidazolidinedione sodium salt hydrate, molecular weight 314.26) is a skeletal muscle relaxant approved for use in the treatment of spasticity and malignant hyperthermia (Pinder et al., 1977; Britt, 1984). In whole animals this drug produced only minimal effects on normal cardiac muscle and cardiovascular function (Ellis et al., 1976a; Ellis et al., 1976b). Yet, when administered intraveneously (10 m g / k g ) to anesthetized dogs, dantrolene sodium significantly increased atrial and ventricular effective refractory periods (Roewer et al., 1984). In in vitro experiments, using cardiac tissue from several species at high drug concentrations (300 /~M), dantrolene sodium caused both positive and negative inotropic effects (Meszaros et al., 1982; Butterfield et al., 1985). Electrophysiological changes induced by the drug (8-35/~M) have been studied
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
522
in dog (Salata and Jalife, 1982; Salata et al., 1983) and sheep (Lascault et al., 1982) Purkinje fibers. Dantrolene sodium lowered action potential plateau and prolonged action potential duration in a concentration- and frequency-dependent manner, which was reversed by elevation of calcium concentration in the perfusing medium. These findings suggested that dantrolene sodium might have a direct antiarrhythmic action, which has been realized in two animal models of cardiac arrhythmia. In a mouse model of ventricular fibrillation induced by chloroform vapor, dantrolene sodium administered i.p. protected in a dose-dependent manner (Butterfield et al., 1983). The median effective dose of dantrolene sodium was 52.6 mg/kg, compared with 62.9 mg/kg for lidocaine hydrochloride, 87.7 mg/kg for quinidine sulfate, and 113.6 mg/kg for procainamide hydrochloride. At the median effective dose, protection by dantrolene sodium began 15-30 rain after dosing and lasted beyond 90 rain. In a dog model of ventricular arrhythmia induced by 60 min ligation of the left anterior descending coronary artery followed by reperfusion, dantrolene sodium at 10 mg/kg i.v. and at 32 mg/kg p.o. suppressed ectopic ventricular extrasystoles without significantly affecting overall ventricular rate or blood pressure (Butterfield et al., 1983). Electrophysiological effects of dantrolene sodium have not been studied in rats, but prolongation of action potential duration has been shown in guinea pig ventricular muscle (Hiroaka et al., 1985). To further characterize its antiarrhythmic spectrum we have evaluated dantrolene sodium in standard rodent models of cardiac arrhythmia. A preliminary report of these results has appeared (Brooks et al., 1984).
2. Materials and methods
2.1. Animals
Male Sprague-Dawley rats weighing 250-450 g and male SW(BR) mice weighing 20-28 g were obtained from Taconic Farms, Inc. (Germantown, NY). Male Hartley strain guinea pigs were
purchased from Charles River Breeding Laboratories (Wilmington, MA). 2.2. Chemicals
Dantrolene sodium and nifedipine were synthesized by Norwich Eaton Pharmaceuticals, Inc. Ouabain octahydrate, aconitine, quinidine sulfate, and propranolol hydrochloride were obtained from Sigma Chemical Co. Saline and pentobarbital sodium (Nembutal ~) were purchased from Abbott Laboratories, methylcellulose (3000 cps) from Dow Chemical Co., urethane from Pfaltz-Baum, Inc., and heparin sodium (Hepalean®) from Harris Laboratories. The following reference antiarrhythmic agents were obtained as gifts from their manufacturers: lidocaine hydrochloride (Astra Pharaceutical Products); amefalone (ORG 6001) (Organon); metoprolol tartrate (CIBA Pharmaceutical Co.); bepridil (Wallace Laboratories); bretylium tosylate (DuPont Critical Care); melperone hydrochloride (Ferrosan); and mexiletine hydrochloride (Boehringer Ingelheim, Ltd.). 2.3. Coronary-artery ligation in rats
The model was run as described by Kane et al. (1979). In brief, respired pentobarbital-anesthetized rats were thoracotomized and the left descending coronary artery was ligated for 30 min. Arrhythmias indicated by a lead II electrocardiogram were enumerated. After the experiment, ligation was verified by visual inspection of the myocardium or by dye infusion. Dantrolene sodium as a 0.05% (w/v) solution in 4.44% (w/v) mannitol-0.008% (w/v) NaOH was administered i.v. over 5-8 min at an infusion rate of 5-8 ml/min per kg to give doses of 3, 10 and 20 mg/kg. Lidocaine hydrochloride as a 2% solution in 0.6% saline was infused i.v. over 30 s to give a 10 mg/kg slow bolus dose (1 ml/min per kg), followed immediately by a continuous 0.1 mg/min per kg infusion during the ligation. Corresponding vehicle doses were given to separate rats. Ligation was done after completing the drug or vehicle infusions.
523 2.4. Electrically induced fibrillation in rats A method similar to Marshall et al. (1983) was used. In brief, respired pentobarbital-anesthetized rats were thoracotomized and fitted with two barbed platinum electrodes positioned 3-6 mm apart on the left ventricular anterior wall such that the anode was 3 mm below the atrioventricular ring and the cathode was on or near the apex. Following a 15-min equilibration period, trains of rectangular pulses (lasting 5 s at 50 Hz with a pulse duration of 5 ms) were delivered every 10 s. Current intensity was then increased at a rate of 20-40 t~a/train until fibrillation occurred (defined as the ventricular fibrillation threshold, VFT) or until a maximum current of about 400 /zA was reached. Each rat received two stimulating sequences ( - 1 5 , 0 rain) prior to and four sequences ( + 15, + 30, + 45, + 60 min) following drug or vehicle administration at time 0. Rats were accepted only if BP was 50 nun Hg at the end of the equilibration period and fibrillation could be induced prior to treatment. Dantrolene sodium was prepared as an 0.8% ( w / v ) solution in a 50% organic vehicle containing 15.6% glycerin, 5.7% propylene glycol, 11.5% 2-pyrrolidone, 17.2% N-(2-hydroxyethyl)-2-pyrrolidone, 5.0% urea, and 10% glucose (all w / v ) in water. Nifedipine was prepared as a 0.1 m g / m l solution in this vehicle and protected from light. Metoprolol tartrate, amefalone, and melperone hydrochloride were used as 0.5% (w/v) solutions in 0.9% saline. Compounds or corresponding vehicles were administered i.v. in less than 3 min at 1 m l / m i n per kg. Doses used were 3, 5.4, 10 and 20 m g / k g for dantrolene sodium, 0.02 m g / k g for nifedipine, 5 m g / k g for metoprolol tartrate, and 10 m g / k g for amefalone and melperone hydrochloride. 2.5. Aconitine-induced arrhythmias in rats and mice Methods for aconitine arrhythmias were adapted from the literature (Miiller and Wilsmann, 1982; Nwangwu et al., 1977). For rats, the test substance or its vehicle was administered at a rate of 1-4 m l / m i n per kg followed within 5 min,
by an infusion of aconitine (20/~g/ml) at the rate of 0.1 m l / m i n (0.25-0.4 m l / m i n per kg). Conscious mice were pretreated i.p. (10 ml/kg) with test substance or vehicle at the times indicated before being anesthetized (60 m g / k g i.p. Nembutal) and infused with aconitine. Times chosen were the times of maximum effect of these substances in the mouse chloroform arrhythmia model (Butterfield et al., 1983). Aconitine (3 or 5 /~g/ml) was infused (tail vein) at the rate of 10 m l / m i n per kg. For rats, dantrolene sodium as a 0.05% (w/v) solution in mannitol-NaOH was administered i.v. at an infusion rate of 1 m l / m i n per kg over 20 min to give a dose of 10 mg/kg. Quinidine sulfate (1% w / v in saline) and propranolol hydrochloride (0.1% w / v in saline) were infused over 0.5 rain (2-4 m l / m i n per kg) to give doses of 10 and 20 m g / k g for the former and 2 m g / k g for the latter. For mice, compounds were suspended in 0.5% carboxymethylcellulose at concentrations to permit delivery of the indicated dose in 10 m l / k g body weight. Dantrolene sodium (100, 300, 1000 and 3000 m g / k g ) was given 60 rain before, quinidine sulfate (88 m g / k g ) or propranolol hydrochioride (43 m g / k g ) were given 30 min before, and lidocaine hydrochloride (53 mg/kg) was given 15 min before the start of aconitine. Aconitine was prepared fresh daily in 0.2% (v/v) dimethylformamide in water. 2.6. Ouabain-induced arrhythmias in guinea pigs A procedure modified from Sekiya and Vaughan Williams (1963) was used. Guinea pigs weighing 450-550 g were anesthetized with urethane (1.25 g / k g i.p.), intubated, and respired mechanically. After the preparation had stabilized as judged by blood pressure and electrocardiogram tracings, test compound or vehicle was administered i.v. in 7 min or less before starting the ouabain infusion (7.5/~g/min at 0.1 ml/min). Dantrolene sodium was also studied with administration at 30 and 60 min before ouabain. In separate experiments it was administered i.p. 60 min before ouabain. The onset of arrhythmia was the time of occurrence of the first extrasystole. The onset of fibrillation was identified as an asyn-
524
chronous irregular electrocardiogram pattern accompanied by a decrease in blood pressure to less than 15 mm Hg. Dantrolene sodium was administered i.v. at 10 and 20 m g / k g in both the 50% organic and alkaline mannitol vehicles described above. The 50% organic vehicle was also used for nifedipine. Bepridil, bretylium tosylate, and mexiletine hydrochloride were given in water. Propranolol was delivered in saline. All agents except dantrolene sodium were delivered i.v. in 0.25-2 mi. Doses are shown in table 5. For i.p. administration of 300 mg/kg, dantrolene sodium was suspended in 0.5% (w/v) methylceUulose at 10 mg/ml. Ouabain octahydrate was used as a 75 /~g/ml solution in saline.
1 s and with fewer than 3 peaks of at least 10 mm Hg per 5 s. Height was determined in reference to adjacent baselines of the peaks. Significance (at the 0.05 level) of differences between effects of drugs and vehicles on VFT change (posttreatment value minus pretreatment mean value for the same rat) were assessed by a t-test. Since there was no difference in threshold values for rats given different volumes of organic vehicle (corresponding to the dantrolene sodium and nifedipine doses), resuits from these vehicle-treated control rats were combined. 2. 7.3. Aconitine- and ouabain-induced arrhythmia models Treatment effects were assessed by a t-test.
2. 7. Data analysis 3. Results
2. 7.1. Rat coronary-artery-ligation model Only rats which survived the 30-min postligation period were included in the data sets (see Results). The number of ventricular extrasystoles (VES, irregular or unscheduled beats), number and duration of ventricular tachyarrhythmia (VT, runs of 7 or more VES as defined by Kane et al., 1982), and number and duration of ventricular fibrillations (VF, loss of sinus rhythm and decrease in BP to < 15 mm Hg sustained for more than 3 s) were extracted from the electrocardiogram. To stabilize variances, the following data were log-transformed prior to analysis: total VES/30 min, duration of VT + 1 and VF + 1, and number of episodes of V T + I and V F + I . Regression analysis was used to test for dose-response relationships, using log dose as the independent variable. Two-way analysis of variance was used to identify significant treatment and dose effects. Chi-square analysis was employed to test the significance of treatment effects on the incidence of VF (rats affected/total number of rats). 2. 7.2. Rat electrically induced fibrillation model The ventricular fibrillation threshold (VFT) was taken as the stimulus current which, during its delivery period, resulted in a BP tracing with pulsatiles not greater than 5 mm Hg for more than
3.1. Coronary-artery ligation in rats Based on mortality and timing and type of arrhythmia observed, our results from the rat model of coronary-artery-ligation-induced arrhythmia were comparable with those reported by others. Of 27 rats treated with vehicle (which include the control rats of table 1), 6% died from VF and 1% died from asystole. This is similar to the 10-25% mortality noted by others (Clark et al., 1980; Fagbemi and Parratt, 1981; Kane et al., 1980; Fagbemi et al., 1982), but less than the 89% found by Mertz and Kaplan (1982). All rats included in table 1 survived the 30-min ligation period. Dysrhythmic activity, including VT and VF, occurred in control rats between 4-14 min after ligation, and was maximal at approximately 9 min, similar to previous findings (Clark et al., 1980; Kane et al., 1980; Kane and Winslow, 1980). VES count varied considerably between rats. For 15 rats treated with mannitol-NaOH (table 1), a range of 46-3176 V E S / 3 0 min (mean 1004 _ 244 S.E.) was observed; for 5 saline-treated rats, the range was 248-1796 V E S / 3 0 min ( 7 4 1 _ 289). Mean VES values of 776 to 1483 for this same 30-min interval have been reported (Clark et al., 1980; Fagbemi and Parratt, 1981; Kane et al., 1980; Fagbemi et al., 1982; Mertz and Kaplan,
525 TABLE 1 Effect of i.v. dantrolene sodium and lidocaine hydrochloride in a rat coronary-artery-ligation model. Values are means__ S.E. (5 rats/group) and percentage of corresponding vehicle-treated control group. Infusion for 5-8 min of 0.05% (w/v) dantrolene sodium in 4.44% mannitol-0.008% NaOH at 5-8 ml/min per kg was completed just prior to ligation. Lidocaine hydrochloride, 2% in saline, was infused at 20 mg/min per kg to give 10 mg/kg, followed by continuous infusion of 0.1 mg/min per kg. Ligation was 5 min after the start of lidocaine, a Significant (P < 0.05) dantrolene sodium treatment effect (analysis of variance), b Significant (P < 0.025) dantrolene sodium treatment effect (chi-square). Treatment
Dose (mg) or volume (ml) per kg
Ventricular extrasystoles per 30 min
Ventricular tachyarrhythmia DuraNo. of tion episodes a (s)
Ventricular fibrillation DuraNo. of tion episodes a (s) a
Incidence rats b
Mannitol-NaOH Dantrolene sodium
6 ml 3 mg
9475:564 505+193 53%
625:47 255:18 39%
9.05:3.8 5.6+2.1 62%
30+30 65:6 20%
0.45:0.4 0.25:0.2 50%
1/5 1/5
Mannitol-NaOH Dantrolene sodium
20 ml 10 mg
999+386 4205:149 42%
795:38 14+ 8 18%
14.45:5.3 1.65:0.9 11%
75:4 05:0 0%
1.4+0.7 0 +0 0%
3/5 0/5
Mannitol-NaOH Dantrolene sodium
40 ml 20 mg
10665:395 1565:49 15%
365:18 105:5 26%
9.85:4.2 1.85:0.7 18%
75: 4 05:0 0%
0.6+0.2 0 +0 0%
3/5 0/5
Saline Lidocaine hydrochloride
0.5 ml 10 mg
741+289 9625:387 130%
48+20 225:13 47%
7.65:1.5 11.8+8.8 155%
1+ 1 0+ 0 0%
0.45:0.4 0 5:0 0%
1/5 0/5
1982; K a n e et al., 1979; M a r s h a l l et al., 1981). VT was o b s e r v e d in 95% ( 1 9 / 2 0 ) of c o n t r o l rats with a d u r a t i o n of 59.1 _+ 20.0 s for the m a n n i t o l N a O H - t r e a t e d rats a n d 4 7 . 8 _ 19.8 s for the saline-treated rats. T h e l i t e r a t u r e r e p o r t s 57-100% incidence of V T w i t h a d u r a t i o n of 67-99 s ( C l a r k et al., 1980; F a g b e m i a n d Parratt, 1981; F a g b e m i et al., 1982; K a n e et al., 1979; M a r s h a l l et al., 1981). V F was o b s e r v e d in 40% ( 8 / 2 0 ) of c o n t r o l rats with a d u r a t i o n of 14.5 +_ 9.8 s ( m a n n i t o l ) a n d 1.4 _+ 1.4 (saline). T h e literature r e p o r t s 40-89% incidence of V F with a d u r a t i o n of 24-62 s ( C l a r k et al., 1980; F a g b e m i a n d Parratt, 1981; F a g b e m i et al., 1982; M e r t z a n d K a p l a n , 1982; K a n e et al., 1979). L i d o c a i n e h y d r o c h l o r i d e (10 m g / k g i.v.) d i d n o t significantly reduce a n y m e a s u r e of a r r h y t h m i a i n d u c e d in rats b y ligation of the left d e s c e n d i n g c o r o n a r y artery (table 1). N o f i b r i l l a t i o n was seen in the l i d o c a i n e - t r e a t e d rats, b u t this d i d n o t differ significantly f r o m the vehicle-treated results, w h e r e two episodes of V F o c c u r r e d in 1 of 5 rats. L i d o -
caine's activity has b e e n r e p o r t e d in the c o r o n a r y a r t e r y - l i g a t i o n m o d e l in rats ( C l a r k et al., 1980; K a n e et al., 1979). Nevertheless, there is controversy over the efficacy of l i d o c a i n e in this exp e r i m e n t a l s i t u a t i o n a n d in the c o r r e s p o n d i n g clinical s i t u a t i o n of v e n t r i c u l a r e c t o p i c activity in the first few h o u r s after m y o c a r d i a l i n f a r c t i o n (Winslow, 1984). D a n t r o l e n e s o d i u m at doses of 3, 10 a n d 20 m g / k g i.v. s u p p r e s s e d VES in a d o s e - d e p e n d e n t m a n n e r to 53, 42 a n d 15%, respectively, of the m a n n i t o l - N a O H vehicle c o n t r o l counts (table 1). Because of the large v a r i a n c e in VES count, these r e d u c t i o n s were n o t quite significant at the P < 0.05 level (P < 0.1). V T d u r a t i o n was decreased. A t 3, 10 a n d 20 m g / k g i.v. doses the n u m b e r of e p i s o d e s of V T were significantly (P < 0.05) decreased to 62, 11, a n d 18% of the respective corres p o n d i n g vehicle values. T h e a n t i a r r h y t h m i c action of d a n t r o l e n e s o d i u m in this m o d e l was m a n i f e s t e d also as significant (P < 0.05) decreases in the n u m b e r of e p i s o d e s a n d average d u r a t i o n of
526 TABLE 2 Effect of i.v. dantrolene sodium and reference antiarrhythmic agents in the rat ventricular fibrillation threshold (VFT) model. For each rat two determinations ( - 15 and 0 min) were done to determine the pretreatment fibrillation threshold. A single determination was done at the times indicated posttreatment. Values are means+ S.E. for the number of rats shown. Dantrolene sodium and nifedipine were administered in a 50% organic vehicle (see Methods). Other agents were administered in saline, a Significant (P < 0.05) change in threshold (t-test). Treatment
Organic vehicle Dantrolene sodium
Nifedipine Saline Metoprolol tartrate Amefalone Melperone hydrochloride
Dose
No.
Pre-
Change in VFT posttreatment (/~A)
(mg/kg)
of rats
treatment threshold #A
+ 15 min
+ 30 min
+ 45 min
+ 60 min
0.4-2.5 (ml/kg) 3 5.4 10 20
21
170 ± 10
- 8 ± 15
3 + 15
19 ± 17
26 + 20
2 7 5 6
140 -/-50 191 + 21 150+24 173±30
0 ± 10 23 _ 12 74+43 a 133+33 a
0 + 10 80 + 52 98±53 a 163±54 a
0 + 30 91 ± 51 126±51 a 180±45 a
30 ± 40 54 ± 42 138±54 a 177±46 a
3
160 ± 23
- 33 ± 35
- 20 ± 12
- 7 ± 18
- 7 ± 24
1-2 (ml/kg) 5
3
110+ 6
-10+15
-23+
3
3
133± 5
-60±31
-53±
7a
10
3
93± 9
33± 9
40± 6
10
3
140+41
7+12
0+17
0.02
V F , as well as b y a s i g n i f i c a n t ( P < 0.025) d e c r e a s e
3±26
-17±
-67±18
a
9
-73±24
33± 5
13± 9
20±47
100±95
3.2. Electrically induced fibrillation in rats
i n t h e i n c i d e n c e o f V F . O n l y 1 o f 15 r a t s t r e a t e d with d a n t r o l e n e s o d i u m exhibited V F , a n d this
No significant changes of fibrillation threshold from baseline were observed during the hour after
o c c u r r e d i n a n a n i m a l g i v e n t h e l o w e s t (3 m g / k g ) d o s e . I n c o n t r a s t , 7 o f 15 r a t s t r e a t e d w i t h m a n n i -
g i v i n g v e h i c l e a l o n e ( t a b l e 2). O f t h e f o u r r e f e r -
tol-NaOH alone exhibited VF.
ence
antiarrhythmic
agents
tested,
two
signifi-
TABLE 3 Effect of i.v. dantrolene sodium, quinidine sulfate and propranol hydrochloride in the rat aconitine-induced arrhythmia model. Anesthetized rats were infused with compound or vehicle just before the start of the aconitine infusion (2 /tg/min). Dantrolene sodium was in mannitol-NaOH; quinidine sulfate and propranolol hydrochloride were in saline, a Significant (P < 0.05) treatment effect (t-test). Treatment
Dose (mg/kg)
No. of rats
Aconitine arrhythmia threshold dose Mean ± S.E. g/kg
% of control
Mannitol-NaOH Dantrolene sodium
20 (ml/kg) 10
10 11
27.2 + 4.0 35.3 ± 4.3
100 130
Saline Quinidine sulfate
1-2 (ml/kg) 10 20 2
10 5 5 5
20.4 + 1.6 25.3 + 2.2 32.4-t-5.7 24.5 ± 2.4
100 124 159 a 120
Propranolol hydrochloride
527
cantly changed VFT in the rat at the 30 min posttreatment test period (table 2). Amefalone (10 m g / k g i.v.) raised fibrillation threshold. In contrast, metoprolol tartrate (5 m g / k g i.v.) lowered VFT threshold - 5 3 + 7 /~A. Nifedipine and melperone did not significantly affect VFT. Dantrolene sodium (3 to 20 m g / k g i.v.) provided a dose-dependent increase in VFT (table 2). Increases were also time-dependent, the maximum effect occurring at 45-60 min after administration. The increases ( > 130 /zA) at all four posttreatment times after 10 and 20 m g / k g i.v. dosing were statistically significant (P < 0.05). 3.3. Aconitine-induced arrhythmias in rats and mice
In rats treated i.v. with dantrolene sodium (10 mg/kg), quinidine sulfate (10 mg/kg), and propranolol hydrochloride (2 mg/kg), the aconitine dose required to induce arrhythmias was 130, 124 and 120% of respective vehicle control values (table 3). These changes were not significant. A higher (20 mg/kg) dose of quinidine sulfate significantly (P < 0.05) raised the aconitine threshold dose to 139% of its control. However, in two of
seven rats, this dose caused AV block and death before the onset of VES. While a higher dose of dantrolene sodium may have given greater effects, we did not give more than 10 m g / k g i.v., since this is a clearly muscle-relaxant dose in rats, and this was also an antiarrhythmic dose in the above two rat models. In mice pretreated i.p. with lidocaine hydrochloride at 15 rain, or with propranolol hydrochloride at 30 min, before aconitine, the aconitine doses causing either single extrasystoles or tachyarrhythmia were not significantly raised (table 4). Quinidine sulfate (88 mg/kg) administered 30 min before the aconitine infusion, significantly (P < 0.05) raised both thresholds. Dantrolene sodium (100-3000 m g / k g i.p.) administered 60 min before aconitine, did not significantly increase the arrhythmogenic dose of aconitine (table 4). The significantly decreased aconitine threshold doses seen at 100 and 300 m g / k g i.p. dantrolene sodium may indicate a proarrhythmic action at high doses in this model. There also was no effect of dantrolene sodium (300 m g / k g i.p.) on thresholds determined with a slower rate of aconitine infusion (table 4).
TABLE 4 Effect of i.p. dantrolene sodium and reference agents in the mouse aconitine-induced arrhythmia model. Mice were pretreated with vehicle or compound at 60 min (dantrolene sodium), 30 rain (quinidine sulfate and propranolol hydrochloride), or 15 rain (lidocaine hydrochloride) before the start of the aconitine infusion, a Significant (P < 0.05) treatment effect (t-test). Treatment
Dose mg/kg
No. of mice
Aconitine arrhythmia threshold dose ( ~ g / k g ) Extrasystoles Mean _+S.E.
Tachyarrhythmia % of control
Mean -+ S.E.
% of control
Fast aconitine infusion (1.25 tt g / min) Carboxymethylcellulose Dantrolene sodium
Saline Lidocaine hydrochloride Propranolol hydrochloride Quinidine sulfate
10 ( m l / k g ) 100 300 1000 3000
9 5 5 5 4
83_+ 2 75+ 3 70_+ 2 a 97-+ 5 96_+11
100 90 84 117 116
108_+ 5 91_+ 5 a 91_+ 2 a 110+ 5 112_+11
100 85 84 102 104
10 ( m l / k g ) 53 43 88
5 5 4 5
63_+ 5 72_+ 4 78 _+14 109-+ 6 a
100 115 125 174
79_+ 4 103_+ 8 101 + 20 147-+ 8
100 130 128 187
10 10
60+ 1 73± 6
100 122
81+ 3 88+ 7
a
Slow aconitine infusion (0. 75 It g / min) Carboxymethylcellulose Dantrolene
10 m l / k g 300
100 109
528 TABLE 5 Effect of i.v. dantrolene sodium and reference agents in the guinea pig ouabain-induced arrhythmia model. Anesthetized guinea pigs were dosed with compounds in organic vehicle, mannitol-NaOH, saline or water (see Methods) over the 0.5-7 min period before the start of the ouabain octahydrate infusion (7.5 /~g/min, 0.1 ml/min). Mean ( + S.E.) arrhythmia and fibrillation threshold doses Otg/kg i.v.) of ouabain for the various vehicles were, respectively, as follows: 128+7 and 193+ 8 (n =17) for saline, 127+5 and 185 + 7 (n = 22) for water, 112 + 6 and 169 _+12 (n = 5) for mannitol-NaOH equivalent to the 10 m g / k g dose of dantrolene sodium, 128 + 11 and 199 + 14 (n = 5) for mannitol-NaOH equivalent to the 20 mg/kg dose, 125 + 6 and 187 + 7 (n = 16) for organic vehicle at -2.5 min, 111 + 8 and 171 + 12 (n = 7) at - 3 0 min, and 118 + 11 and 164+ 10 (n = 5) at - 6 0 min. a Significant (P < 0.05) change from the corresponding vehicle value (t-test). Treatment
Dose (mg/kg)
No. of guinea pigs
Percentage of control ouabain threshold dose (mean + S.E.) Arrhythmia
Fibrillation
5 5 3
102+ 6 105+11 82 + 4
114+ 7 100+ 9 107 + 10
5 6
110+ 7 147+23
92+ 5 124+22
1 5.6 10
3 4 4
92+ 8 115+ 3 152+ 9
a
106+13 122+14a 132+ 3 a
22
3
137+21
a
144+ 6
Bretylium tosylate
5
3
105+11
Bepridil
1.5
3
158+ 8 a
Dantrolene sodium in organic vehicle at - 60 min 20 at - 30 min 20 at - 2.5 min 20 Dantrolene sodium in mannitol-NaOH at - 2 min 10 at - 2 min 20 Mexiletine HC1
Propranolol HC1
3.4. Ouabain-induced arrhythmias in guinea pigs Because agents were tested in this model in s e v e r a l v e h i c l e s , r e s u l t s a r e e x p r e s s e d as p e r centage changes in arrhymogenic ouabain dose. Actual arrhythmia and fibrillation threshold doses of ouabain for guinea pigs dosed with saline, water, mannitol-NaOH, and an organic vehicle are g i v e n i n t h e n o t e s o f t a b l e 5. N o s i g n i f i c a n t d i f ferences in thresholds were attributable to different vehicles. Of the twelve reference antiarrhythmic agents e v a l u a t e d , n i n e s i g n i f i c a n t l y ( P < 0.05) r a i s e d either the arrhythmia or fibrillation threshold at o n e o r m o r e d o s e s ( t a b l e 5). T h e c l a s s I a g e n t m e x i l e t i n e w a s a c t i v e , as w a s t h e c l a s s II d r u g propranolol, and the class IV agent bepridil. The class III drug bretylium tosylate did not increase t h r e s h o l d s . I n fact, b r e t y l i u m s l i g h t l y l o w e r e d fibrillation threshold. Dantrolene sodium was evaluated under several conditions differing in vehicle, dose, route, and
a
86+ 4 a 140+ 8 a
time of dosing relative to the start of ouabain i n f u s i o n . I n 50% o r g a n i c v e h i c l e , d a n t r o l e n e s o d i u m ( 2 0 m g / k g i.v.) a d m i n i s t e r e d a t 60, 30, o r 2.5 m i n b e f o r e o u a b a i n h a d n o e f f e c t o n t h r e s h o l d s ( t a b l e 5). D a n t r o l e n e s o d i u m ( 1 0 o r 20 m g / k g i.v.) in mannitol-NaOH did not significantly change arrhythmia or fibrillation thresholds, although the h i g h e r d o s e r a i s e d t h e f o r m e r t o 147% o f c o n t r o l . To eliminate any effects of intravenous vehicles or
TABLE 6 Effect of i.p. dantrolene sodium in the guinea pig ouabain-induced arrhythmia model. Dantrolene sodium (10 m g / m l in 0.5% methylcellulose) or vehicle were administered to groups of five animals 60 rain before the start of the ouabain octahydrate infusion (7.5/~g/min). Treatment
Dose
Ouabain threshold dose (~ g/kg i.v.) Arrhythmia Fibrillation
0.5% Methylcellulose 10 (ml/kg) 135+ 13 Dantrolene sodium 300 (mg/kg) 138+44
199_+20 199+27
529 volumes, dantrolene sodium was administered i.p. in methylcellulose 60 min before the start of ouabain infusion. Arrhythmia and fibrillation thresholds were unchanged by this treatment in comparison with the corresponding vehicle-treated group (table 6).
4. Discussion The purpose of these experiments was to further characterize the antiarrhythmic activity of dantrolene sodium in several standard rodent models of cardiac arrhythmia. The compound was administered i.v. or i.p. in a dose range which included doses known to elicit skeletal muscle relaxation, i.e., 3-10 m g / k g i.v. Arrhythmias induced by mechanical (coronary artery ligation), electrical (ventricular fibrillation threshold), and chemical (aconitine and ouabain) stimuli were studied in mice, rats or guinea pigs. In the coronary artery-ligated rat, the antiarrhythmic activity of dantrolene sodium was manifested as a marked, dose-related suppression of VES, as reduction in VT (both number of episodes and duration), and, most strikingly, as complete elimination of VF at the 10 and 20 m g / k g i.v. dose levels. In the ventricular fibrillation threshold model in rats, dantrolene sodium also displayed dose-related antifibrillatory activity. The magnitude of the dantrolene sodium-induced increase in V F T was related to the time posttreatment. The maximum effect was seen 45 or 60 min after compound administration, but in a few rats given 10 and 20 m g / k g i.v., increases in V F T were still observed 120 rain after dosing. The arrhythmias occurring early after ligation of the coronary artery arise in epicardial layers where ischemic zone cells display shortened action potentials and depressed m a x i m u m rates of depolarization (Elharrar and Zipes, 1977). Although prolongation of action potential duration (APD) by dantrolene sodium has not been shown in rat cardiac tissue, this could be the basis of the drug's ability to suppress these early reentrant arrhythmias. The coronary artery ligation model may have clinical relevance. Early arrhythmias following
coronary artery ligation may mimic the period immediately following myocardial infarction in man, where estimates have been made of 22% mortality with an average time to death of 20 min (Paul, 1974; Gyfirffis, 1976). The lack of activity of dantrolene sodium in the aconitine- and ouabain-induced models is consistent with its lack of Class I effects. Both aconitine- and ouabain-induced arrhythmias are due to increased automaticity, rather than reentry, and involve elevation of intracellular sodium and the induction of afterdepolarizations (Winslow, 1984). These results demonstrate the antiarrhythmic action of dantrolene sodium in a variety of rodent arrhythmia models at doses which provide relaxation of skeletal muscle. While no direct relationship between prolongation of action potential duration in rodent cardiac tissue and the antiarrhythmic effect in whole animals has been shown, it is likely that the two properties of dantrolene sodium are related. Evidence supporting this relationship may be the lack of activity of both dantrolene sodium and a class III antiarrhythmic drug in the guinea pig ouabain model.
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