ANTIARRHYTHMIC ACTIVITY OF DEXTRO AND LEVO ISOMERS OF 5-METHYL-8-(2-HYDROXY-3-T-BUTYLAMINO PROPOXY) COUMARIN HYDROCHLORIDE (BUCUMOLOL), A 18-ADRENERGIC BLOCKING AGENT, ON ACONITINE INDUCED ATRIAL AND OUABAIN-INDUCED VENTRICULAR ARRHYTHMIAS IN DOGS

Koichi NAKAYAMA, Takeshi OSHIMA* and Hiroyuki KOIKE* Department of ' Pharmacology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 and *Central Research Laboratories, Sankyo Co. Ltd., Shinagawa, Tokyo 140, Japan

Accepted August 22, 1979

Abstract-The (3-blocking, antiarrhythmic, and local anesthetic effects of the racemic mixture and optical isomers of bucumolol, 5-methyl-8-(2-hydroxy-3-t-butylamino propoxy) coumarin hydrochloride, were studied in dogs, guinea-pigs and frogs. In blocking the positive chronotropic response to isoproterenol, the levo-isomer of bucumolol was about 40 times more potent in dogs, and 270 times in guinea-pigs than its dextro-isomer and twice as effective in both species as the racemic mixture. In frog sciatic nerves bucumolol was 1/10-1115 as potent in local anesthetic action as propranolol on a weight basis. Dextro and levo-isomers and racemic bucumolol neither elevated electrical threshold for propagated impulses nor prolonged the effective refractory period of the dog right atrium. The levo-isomer and racemic bucumolol were capable of suppressing aconitine-induced atrial arrhythmia, while the dextro-isomer was less effective Both isomers and racemic bucumolol were capable of reversing ventricular arrhythmia caused by ouabain, but the effective dose of the levo-isomer was significantly less than that of the dextro-isomer. The results suggest that both specific jS-blocking activity and non-specific membrane action of bucumolol suppressed experimental arrhythmias in dogs produced by aconitine and ouabain.

Hashimoto and associates compared the potencies of various / -blocking agent in antagonizing positive chronotropic (1, 2) and inotropic responses (3, 4) to isoproterenol and to noradrenaline in excised and blood-perfused sino-atrial node and papillary muscle preparations of dogs. We later compared the antiarrhythmic effects of these compounds on aconitine-induced atrial and ouabain-induced ventricular arrhythmias in dogs, and concluded that some ;3-blocking agents were effective in suppressing these experimental arrhythmias but their effectiveness did not parallel the l3-blocking activity (5-7). Concerning antiarrhythmic effect, !3-blocking agents such as sotalol (8), N-isopropyl p-nitrophenyl ethanolamine (INPEA) and practolol (9, 10), which have practically no local anesthetic action, are effective in suppressing aconitine-induced atrial arrhythmias but not ouabain-induced ventricular arrhythmias in cats and dogs. On the other hand, Papp and Vaughan Williams (11) observed the in vivo activity of practolol in protecting anesthetized guinea-pigs against ouabain-induced ventricular fibrillation. Comparing INPEA and pindolol, Singh and Vaughan Williams (12) found that dose-response curves for percentage FIG. 1. Chemical structure of bucumolol. reduction in ouabain-induced ventricular fibrillation by those drugs were straight and parallel, indicating that 1NPEA was also effective but the effectiveness of INPEA was only 0.02 that of pindolol. This discrepancy may be due to the different animal species used or to the procedures for testing antiarrhythmic effects. If optical isomers, though difficult to obtain, were available, this discrepancy might be overcome. 5-Methyl-8-(2-hydroxy-3-t-butylamino-propoxy)coumarin hydrochloride (bucumolol) used in this study is a ,3-blocking agent which is approximately 3 times more potent than propranolol in blocking isoproterenol-induced positive chronotropic (7, 13) and inotropic responses (4) in dogs and guinea-pigs. The structural formula of bucumolol is shown in Fig. 1. Bucumolol has an asymmetrical carbon atom, and thus, (1) and (d)-di-0, 0-p toluoyl-tartaric acids were used as resolving agents for isolation of optical isomers. By a tedious fractional crystallization from methanol, two salts, i.e., d-bucumolol, m.p. 231-232 C [a]n 18.5 (c; 0.535 in methanol) and 1-bucumolol, m.p. 230-232-C, [a] 18.5 (c; 0.5 in methanol) were obtained as optically pure substances. Physical data for bucumolol and its optical isomers have already been reported in detail (13). In the present experiments, antiarrhythmic activities of d-, I-, and dl-bucumolol were tested on aconitine-induced atrial and ouabain-induced ventricular arrhythmias and their ;3-blocking potenties, local anesthetic activities and effects on the refractory period were compared.

MATERIALS AND METHODS Determination of,3-blocking activity in the chronotropic response to isoprenaline: Mongrel dogs of either sex weighing, 8 to 12 kg were anesthetized with an i.v. administration of 30 mg/kg of sodium pentobarbital. The femoral vein was cannulated for the injections. The heart rate was measured continuously by means of a cardiotachograph (Nihon Kohden RT-2) triggered by R wave of ECG lead II. The ;3-blocking activity was assessed against the positive chronotropic response to 0.3 /ig/kg i.v. of isoproterenol, which was administered about 3 min after a given dose of a 13-blocking agent from 3 to 1000 ,,tg/kg. Then, every 20 min, isoproterenol in the same dose was given until the positive chronotropic response to isoproterenol recovered to the control level. The guinea-pig right atria were isolated and suspended in Tyrode Solution. Rate of spontaneous contraction was augmented by 10-1 g/ml of isoproterenol and the degree of blockade was calculated according to the method of Saameli (14). Measurement of effective refractory period: Effective refractory period of the right atrium was studied in mongrel dogs of either sex weighing between 8 and 15 kg. Animals were anesthetized with 30 mg/kg i.v. of sodium pentobarbital and the hearts exposed by midsternal thoracotomy. Right atrial and and right ventricular electrograms were recorded with bipolar silver electrodes (1 mm apart) embedded in small acryl plaques (7 x 7 mm) attached to the epicardial surface of the myocardium by bio-tissue adhesive Aron alpha A (Sankyo). These electrograms were monitored on the screen of an oscilloscope (Nihon Kohden VC-7). The atrium was driven by square wave pulses of two or three times the threshold voltage and 1 msec duration with a cycle length of 400 msec which were delivered through stimulating bipolar electrodes attached to the right atrium. To measure the effective refractory period, an extra stimulus was interpolated via the same stimulating electrodes after every seventh regular pulse, with decreasing intervals. The effective refractory period was defined as the shortest interval which allowed the interpolated stimulus with the same current intensity as regular stimuli to elicit an impulse which propagates to the recording electrodes placed on the atrium. Data were analyzed statistically by paired comparison in which each dog was used as its own control. Measurement of local anesthetic activity: The local anesthetic activity of several com pounds were compared using the isolated sciatic nerve of frogs. The nerve was placed in a bath containing Ringer's solution at room temperature. A square wave pulse of 1 msec duration and 5 V strength was applied to the end of a nerve at a frequency of 1 Hz. The conducted action potential at the other end was displayed on the screen of an oscillograph (Nihon Kohden VC-7), and the height of the fastest action potential was measured before and after exposure for 30 min to each concentration of the drug used. Studies of cardiac arrhythmias: Mongrel dogs of either sex weighing 7 to 15 kg were anesthetized with 30 mg/kg i.v. of sodium pentobarbital. For induction of atrial arrhyth mia by aconitine, the authors previously devised a cup method (5) instead of the cotton pledget (15) to restrict the aconitine solution on the atrial surface. With this method a sustained atrial arrhythmia of a relatively uniform severity for several hours was obtainable without any significant disorder in cardiac function. Ventricular arrhythmia was induced by the administration of ouabain. Forty ag/kg of ouabain was injected intravenously and was followed by a dose of 20 //g/kg given 30 min later. Thereafter, an additional dose of 10 tig/kg was given every 15 min until ventricular arrhythmia developed (16). A total of from 60 to 801ig/kg of ouabain was usually sufficient to induce ventricular arrhythmia. The right vagal nerve was sectioned at the cervical level and the distal end was stimulated with I msec square wave stimuli at a frequency of 20 Hz and intensity of 2.5 V to suppress the sino-atrial pacemaker and the A-V conduction and induced ventricular automaticity (17). Test compounds were administered by intravenous infusion at a constant rate to block arrhythmia within a period of 10 to 30 min. When an antiarrhythmic effect did not appear after intravenous infusion for 40 min, the infusion of the drug was interrupted and the result was interpreted as negative. The rate of appearance of effective cases in d and 1-isomers was statistically analyzed by Fisher's x2 test. Difference of effective doses between d and 1-isomers was analyzed by Student's t-test. The drugs used as test compounds were a racemic mixture of bucumolol and its d and 1-isomers of hydrochloride (Sankyo), dl-isoproterenol sulfate (Boehringer Sohn Ingelheim), propranolol hydrochloride (ICI), quinidine sulfate (Tokyo Kasei) and procaine hydrochloride (Fujisawa). Drugs were dissolved in 0.9 °o physiological saline and doses referred to their salts. The drugs used to induce arrhythmias were aconitine (Nakarai Kagaku) and ouabain (Tokyo Kasei). Aconitine was dissolved in 0.01 N hydrochloride. Values were given in terms of means S.E.

RESULTS The data represent findings in 5-7 dogs. The mean heart rate was 16012 (N==20). The mean increase in heart rate produced by 0.3 /cg/kg i.v. of isoproterenol was 70--6 beats/min. The j3-blocking effect of bucumolol and its optical isomers were assessed against this positive chronotropic response. Figure 2 (upper panel) illustrates dose-response curves for percentage blockade of the isoproterenol-induced positive chronotropic responses by bucumolol or its optical isomers. The positive chronotropic response to isoproterenol was reduced to approximately 50 % with about 13 ; cg/kg of bucumolol and 6 /eg/kg of 1-bucumolol, while approximately 280 pg/kg of d-bucumolol was required to produce the same degree of inhibition. The relative potency of ~-adrenergic blocking activity determined

FIG. 2. Upper panel: dose-response curves for inhibition of isoproterenol-induced increase in heart rate of anesthetized dogs by racemic mixture of bucumolol and its d and 1-isomers. Inhibition is expressed as percentages of the positive chrono tropic response to isoproterenol, 0.3 pg/ kg i.v. Each point is the mean of 5-7 dogs and each bar is its standard error. Lower panel: dose-response curves for inhibition of isoproterenol-induced increase in beating rate of guinea-pig right atria. Concentration of isoproterenol used as agonist is 10_1 g/ml. Results are the mean of 4 observations for each point. by four point parallel assay was 1:2.26:0.06 of dl-, 1-, and d-isomers. The increase in contraction rate of the guinea-pig right atrium preparation with 10'8 g/ ml of isoproterenol was compared before and after increasing concentrations of bucumolol or its optical isomers (Fig. 2, lower panel). Four experiments were performed with each compound. The mean concentration required to produce 50% attenuation of the response to isoproterenol was 2.7x 10-9 g/ml of the 1-isomer, while it was 5.5 x 10-' g/ml of the d isomer. The ratio of !3-blocking activity of racemic mixture, 1 and d-isomers of bucumolol was 1:2.37:0.01. Effectsof bucumolol and its optical isomers, propranolol and quinidine on the effective refractory period of the dog right atrium : Racemic mixture of bucumolol at two dose levels, 0.3 and 5 mg/kg i.v., neither altered the threshold nor prolonged the refractory periods significantly (Table 1). Dextro and levo-isomers of bucumolol also were without significant effects on the threshold or effective refractory period. There was no significant difference in these effects between both isomers at a dose of 0.3 or 1 mg/kg i.v. On the other hand, propranolol at a dose of 0.3 or 5 mg/kg i.v. significantly increased both the threshold and refractory period (Table 1). Effects of quinidine increased significantly the refractory period but did not change the threshold (Table 1).

TABLE 1. Effect of bucumolol and other compounds on effective refractory period and threshold of atria of the dog Assessment of local anesthetic activity of racemic mixture, d and 1-isomers of bucumolol in isolated frog sciatic nerves: The local anesthetic activity of the compounds was assessed on the amplitude of action potentials of frog sciatic nerves. As shown in Table 2, the local anesthetic activity of bucumolol was about 1/17 that of propranolol and about 1/2 that of procaine on a weight basis. There was no statistical difference in local anesthetic activity between racemic mixture of bucurnolol and its optical isomers. Effects of racemic mixture and d and 1-isomers of bucumolol on aconitine-induced atrial arrhythmia in the dog: Atrial arrhythmia was produced by the local application of I aconitine solution by means of the cup method. d-, 1-Isomer or racemic mixture of bucumolol was given intravenously at a constant rate of 0.5 mg/kg per min until an antiarrhythmic effect was obtained. The infusion of the drug was interrupted when an antiarrhythmic effect appeared. However, if the effect was not observed even after administration of drug in a total dose of 15 mg/kg i.v., the result was regarded as negative. The 1-isomer and racemic mixture of bucumolol were found to be more potent in blocking atrial arrhythmias than was the d-isomer as shown in Table 3. In 5/7 dogs there was a conversion of sinus rhythm after application of the racemic mixture and 1-isomer of bucumolol. The effective doses of racemic mixture and 1-bucumolol were 6.2-'-1.4 and 4.6+0.6 mg/kg i.v. respectively (mean±S.E.). d-Isomer of bucumolol was less effective on the arrhythmia than 1-isomer and racemic mixture regarding effective cases (p<0.01). Atria] arrhythmia converted into normal sinus rhythm in 1/7 dogs tested. 1-Isomer and racemic bucumolol had no significant effects on the heart rate and systemic blood pressure. Effects of racendc mixture and d and 1-isomers of bucumolol on ouabain-induced ventricular arrhythmia in dogs: Ventricular arrhythmia was produced in dogs by intravenous adminis

TABLE 2. Effect of bucumolol and other compounds on the spike amplitude in isolated frog sciatic nerve TABLE 3. Effect of bucumolol and its optical isomers on aconitine-induced atrial and ouabain-induced ventricular arrhythmias

Fic. 3. Suppression of ouabain-induced ventricular arrhythmia by racemic bucumolol given by a titration procedure. The upper diagram shows the process of induction of ventricular arrhythmia by ouabain and successful suppression of the arrhythmia by racemic bucumolol given at the rate of 0.5 mg/kg per min. The dotted and hatched columns show nodal rhythm and ventricular arrhythmia, respectively. EC'G records at a, b, c, d, e, and fin the upper diagram are illustrated. SBP: the phasic change of systemic blood pressure, r.v.s.: right vagal stimulation and F CG lead It. tration of ouabain in a dose ranging from 60 to 80 'g/kg. Thirty minutes after an initial dose of 40 ; ig/kg, 20 , tg/kg was given and additional doses of 10 ,'jg/kg were given at 15 min intervals until rapid ventricular ectopic rhythm was not modified by electrical stimulation of the right vagal nerve. dl-Bucumolol or d or 1-isomer was infused at a constant rate of 0.5 mg/kg per min. In all cases except one with 1-isomer, the ventricular arrhythmia returned to regular sinus rhythm (Table 3) and right vagal stimulation induced prominent bradycardia, as shown in Fig. 3. The infusion of the drug was continued until it reached a total of 15 mg/kg. The duration of antiarrhythmic action lasted over 90 min, except for one case of dl-bucumolol. As summarized in Table 3, both d and ]-isomers were capable of sup pressing the heterotopic ventricular arrhythmia induced by ouabain. However, the anti arrhythmic dose of 1-isomer of bucumolol was significantly different from that of either d-isomer or racemic mixture (p<0.05). The antiarrhythmic dose had little effect on the systemic blood pressure and heart rate when the values before ouabain and after reversal to sinus rhythm were compared (p<0.01).

DISCUSSION Previous studies showed that /3-blocking activity of dl-bucumolol was three times that of propranolol (2, 4, 7, 13) but its local anesthetic activity was about 1/10 that of the latter (7). The present study confirmed these previous results. In frog sciatic nerve d-, 1 and dl-bucumolol all had local anesthetic activity, and in this respect were equipotent. In the dog right atrium, both isomers and racemic bucumolol failed to elevate the electrical threshold for propagated impulses to be elicited or to prolong the effective refractory period. In contrast to these findings, the /3-blocking effect of the 1-isomer was about 40 times that of the d-isomer and twice that of the racemic compound. Thus, unspecific membrane effects of the compound, such as those on the refractory period or local anesthetic action, did not parallel (3-blocking potency. The comparison of the activity of isomers of bucumolol in reversing either aconitine induced atrial or ouabain-induced ventricular arrhythmias are of particular interest in throwing light on the mechanism of experimental arrhythmias. On aconitine-induced atrial arrhythmia, dl-bucumolol and the 1-isomer were quite effective, while the d-isomer hardly ever reversed the atrial arrhythmia to sinus rhythm. Concerning the membrane effect of aconitine, Peper and Trautwein (18) found in voltage clamp experiments on sheep Purkinje fibers a specific increase in sodium conductance and a simultaneous inhibition of inactivation of the sodium carrier system. This could make the membrane unstable and an extrasystole would occur before repolarization is complete. As catecholamines accelerate the reactivation process, they may play a role in aconitine-induced arrhythmia by making the cardiac membrane unstable in the repolarization phase. As to ouabain-induced ventricular arrhythmia, opinions on the effect of /3-adrenoceptors are controversial. Howe and Shanks (19) reported that racemic propranolol and its optical isomers were equally effective in abolishing ouabain-induced arrhythmia, while the ]-isomer was a little more effective. Lucchesi et al. (20), and Whitsitt and Lucchesi (21) found that both i and d-isomer of either propranolol or pronethalol were capable of suppressing ectopic ventricular automaticity induced by ouabain, and such suggests that there is no special relation between fl-blocking action and prevention of ouabain-induced arrhythmia. Somani and others demonstrated that local anesthetic action was essential for antiarrhythmic activity of j3-blocking agents (22, 23). On the other hand, Raper and Wale (24) reported that in the presence of a sub-arrhythmogenic dose of ouabain, stimulation of the cardiac sympathetic nerve facilitated the arrhythmia and that this effect of sympathetic stimulation was effectively abolished by a low dose of propranolol. Dohadwalla et al. (25) and Singh and Vaughan Williams (12) reached the same conclusion. Involvement of sympathetic mechanisms in the development of ouabain-induced arrhythmias has been well documented (25-27). Arrhythmogenic doses of cardiac glycosides such as ouabain not only activate central sympathetic centers (28) but also stimulate sympathetic ganglia (29). Moreover, the catecholamine release from the adrenal medulla by ouabain infusion was clearly demonstrated by Hashimoto et al. using excised sino-atrial and papillary muscle preparations cross-circulated with blood from a donor dog (30). By means of electro physiological techniques Ferrier and Moe suggested that transient depolarization in Purkinje tissue was induced by acetylstrophanthidin, and that such may be due to Ca2+ influx (31). Tse and Han also found that ouabain enhanced the action of catecholamine on the auto maticity of Purkinje fibers causing ventricular arrhythmia (32). The mechanism by which ,3-blocking drugs act as antiarrhythmic is apparently complex. Evans et al. (33) demonstrated that the ;3-blocking agent, nadolol, lacking in membrane depressant action was relatively ineffective in suppressing existing arrhythmia induced by digoxin. However, in the present experiments the 1-isomer and racemic bucumolol were capable of suppressing aconitine-induced atrial arrhythmia, while the d-isomer was less effective. Furthermore, both isomers and racemic bucumolol reversed ventricular arrhythmia induced by ouabain but the effective dose of the 1-isomer was significantly less than that of the d-isomer. The differences in incidence of effective cases and effective doses between optical isomers of bucumolol in suppressing either aconitine-induced atrial or ouabain induced ventricular arrhythmia in dogs might to some extent be related to f3-blockade. It is probable that in dogs, the activity of sympathetic mechanisms played some role in the initiation as well as the maintenance of experimental arrhythmias produced by aconitine and ouabain and that both specific ,3-blocking activity and non-specific membrane action of bucumolol suppressed these arrhythmias.

Acknowledgements: We thank Mr. Y. Kobayashi and Dr. Y. Sato of Central Research

Laboratories, Sankyo Co. Ltd. for kindly preparing the optical isomers.

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