SD-3212 ((−)-(s)-2-[5-methoxy-2-[3-methyl-2-[3,4 (methylenedioxy) phenoxy] ethyl] amino] phenyl]-4-methyl-2H-1,4-benzothiazine-3(4H)-one hydrogen fumarate) is a newly developed compound with a molecular weight of 652.72, which exhibits potent antiarrhythmic effects on experimental ventricular tachyarrhythmias induced by chloroform, ouabain, adrenaline, and coronary occlusion and reperfusion (1-3). This compound is a stereoisomer of SD-3211, which is a nondihydropyridine-type calcium antagonist (4,5). These antiarrhythmic effects could be attributable to its inhibitory action on sodium and calcium channels (1-3). However, the effects of this drug on atrial tachyarrhythmias and atrial actionpotential characteristics have not been explored thoroughly. In our study we investigated effects of SD-3212 on canine atrial flutter induced after placing an intercaval obstacle and also on atrial action-potential characteristics in isolated canine atrial tissue by using standard microelectrode techniques.
In vivo study
Adult mongrel dogs, weighing 8-15 kg, were anesthetized with sodium pentobarbital (30 mg/kg) and intubated with a tracheal tube. The heart was exposed through a right thoracotomy in the fourth intercostal space under artificial respiration with a volume-cycled respirator. An anatomic obstacle was produced according to the previous study (6). The tissue on a line extending from the superior to inferior venae cavae was clamped and crushed. Three bipolar hook electrodes were placed in the right atrium, and another two or three, in the left atrium to record atrial electrograms (Fig. 1). A unipolar electrode was placed in the right atrium for programmed cathodal stimulation. The anode was placed in the abdominal wall. A venous cannula in the femoral vein was used to infuse normal saline solution to replace the spontaneous fluid loss and to inject SD-3212. Atrial electrograms and surface electrocardiographic lead II were recorded simultaneously on the thermal recorder (RTA1200; Nihon Kohden, Tokyo, Japan) at a paper speed of 100 mm/s. Pacing stimuli were provided by a digital electronic stimulator (SEN7103; Nihon Kohden) and a constant-current source (SS202J; Nihon Kohden) with a pulse duration of 2 ms at twice the late diastolic threshold.
The right atrial effective refractory period (RAERP) was determined by the extrastimulus technique at basic pacing cycle lengths of 300, 200, and 150 ms. After every eight basic stimuli, an extrastimulus was delivered in late diastole. The coupling interval of extrastimulation was shortened in 2-ms steps until a propagated atrial response was no longer produced. Determinations were repeated to assure reproducibility of the data. The ERP was defined as the longest coupling interval that did not produce a propagated atrial response. The interatrial conduction time (IACT) from the high right atrium to the lower left atrium was determined during atrial pacing at pacing cycle lengths of 300, 200, and 150 ms. Atrial flutter was induced by the burst atrial pacing at cycle lengths of 90-120 ms. Six consecutive atrial cycle lengths were averaged to represent the atrial flutter cycle length in each experiment.
After atrial flutter was initiated, a 3-min period was allowed for each dog to ensure that flutter was sustained. SD-3212 was injected intravenously in seven dogs at 2 or 3 mg/kg over a 3-min period. Drug injection was discontinued if atrial flutter was terminated during injection. The electrophysiologic study was repeated, and then reinitiation of atrial flutter was attempted. Arterial blood samples were obtained to determine plasma drug concentrations just after the termination of atrial flutter and after repeated measurements of electrophysiologic variables. The two plasma drug levels were averaged in each dog.
In vitro study
Adult mongrel dogs, weighing 7-15 kg, were anesthetized with sodium pentobarbital (30 mg/kg). The heart was rapidly removed, and atrial tissues (7 × 15 mm) obtained from both atria were fixed in a small tissue chamber (5 ml) and superfused continuously with Tyrode's solution gassed with 95% oxygen and 5% carbon dioxide. The solution was composed of the following (in mM): NaCl, 125; KCl, 4; NaH2PO4, 1.8; MgCl2, 0.5; CaCl2, 2.7; NaHCO3, 25; and glucose, 5.5. The preparations were stimulated at a basic frequency of 1.0 Hz with a bipolar electrode. Stimulation pulses of 1-ms duration and twice the diastolic threshold in intensity were delivered by an electronic stimulator (S-7272B; Nihon Kohden). Transmembrane action potentials were recorded by using glass microelectrodes filled with 3 M KCl (10-30 MΩ). The maximal upstroke velocity of the action potential (Vmax) was measured by using an electronic differentiator. After an equilibration time of 120 min, the amplified signals were displayed on an oscilloscope (VC-10; Nihon Kohden). The differentiated signal was passed through a peak detector, and the action potential at −50 mV level (APD−50) was measured by using an electronic APD monitor. These signals were continuously recorded on the chart recorder (RF-85A; Fukuda Denshi, Tokyo, Japan). Action potentials were recorded before and 30 min after drug administration at concentrations of 0.3, 1, and 3 μM. For study of the frequency-dependent effects of the drug, preparations were stimulated by trains of 30 stimuli at various frequencies (0.2, 0.5, 1, 2, and 3.3 Hz) before and after drug administration. We could not increase the stimulation frequency further because higher stimulus frequencies might cause the dislodgement of the microelectrode and make the superfused tissue hypoxic.
SD-3212 was dissolved in dimethyl sulphoxide as a stock solution of 10 mM, and it was diluted further with normal saline or Tyrode's solution to obtain the final concentration required. The final concentration of dimethyl sulphoxide did not exceed 0.03%, and this had no influence on the action-potential variables. Atrial flutter was not terminated with an injection of this concentration of dimethyl sulphoxide and was sustained >5 min in our preliminary study.
Results are presented as mean ± SEM. The analysis of data for significance was performed by an analysis of variance, and multiple comparisons were tested by Bonferroni's test. Paired data were compared by Student's t test. Statistical significance was set at p < 0.05.
In vivo study
Before drug injection, atrial flutter was inducible in seven of nine dogs tested. SD-3212 (1.9 ± 0.3 mg/kg) terminated atrial flutter in all of these seven dogs, with conduction block occurring in the low right atrium (Fig. 1). SD-3212 significantly increased atrial flutter cycle length from 126 ± 5 to 166 ± 14 ms (increase, 31 ± 8%; p < 0.005) just before termination of atrial flutter. After drug administration, atrial flutter was reinitiated in all six dogs in which reinitiation was tested, and the reinduced flutter cycle length was significantly increased to 154 ± 5 ms (p < 0.001). The plasma concentration of SD-3212 was 187 ± 56 ng/ml in four dogs.
Changes in atrial electrophysiologic variables were determined in nine dogs (seven dogs with and two dogs without inducible atrial flutter). These results are summarized in Figs. 2 and 3. At a basic atrial pacing cycle length of 150 ms, electrophysiologic variables were determined in four of nine dogs, because the atria were not captured by basic atrial pacing after SD-3212 injection. At each basic cycle length, SD-3212 increased RAERP, and the percentage increase in RAERP did not differ among three basic cycle lengths (18 ± 4% at 300 ms, 17 ± 3% at 200 ms, and 19 ± 3% at 150 ms). This indicates that SD-3212 does not have an apparent reversed use-dependent effect on RAERP. SD-3212 lengthened IACT at each basic cycle length, and the percentage increase showed a trend of usedependent block on interatrial conduction (8 ± 3% at 300 ms, 9 ± 3% at 200 ms, and 31 ± 6% at 150 ms).
In vitro study
The baseline characteristics of action potentials recorded from eight atrial preparations stimulated at 1 Hz are summarized in Table 1. As shown in Fig. 4, Vmax was decreased by SD-3212 in a concentration-dependent manner (change, 86 ± 8% at 0.3 μM, 76 ± 8% at 1 μM, and 68 ± 8% at 3 μM). APD90 was prolonged in a concentration-dependent manner (Fig. 4). The influence of stimulation frequency on the effects of SD-3212 on the APD at −50 mV and Vmax was evaluated in another seven preparations by using stimulation trains at different frequencies (0.2, 0.5, 1, 2, and 3.3 Hz). APD at −50 mV was continuously monitored on the chart recorder as mentioned previously. The decrease of Vmax was greatest at the highest stimulation frequency of 3.3 Hz, although prolongation of APD at −50 mV by SD-3212 did not show apparent frequency dependency (Table 2).
The major findings of our study are as follows:
- SD-3212 interrupted canine atrial flutter with the conduction block occurring in the low right atrium. However, atrial flutter was reinducible after termination by SD-3212 in all dogs tested.
- SD-3212 prolonged both IACT and atrial ERP significantly. IACT was prolonged in a use-dependent manner, but atrial ERP was not.
- In the in vitro study, SD-3212 decreased Vmax and prolonged APD of canine atrial cells in a concentration-dependent manner without affecting resting membrane potential.
Effects of SD-3212 on canine atrial flutter model
Atrial flutter in the canine model used in our study is caused by a macroreentry with an excitable gap (7). Antiarrhythmic effects of class I and III drugs were investigated by using this canine model. Prolongation of ERP is essential for termination of atrial flutter with class III drugs, but suppression of conduction is essential with class Ia and Ic drugs (6). Electrophysiologic effects of SD-3212 at the dosage used in our study were similar to those of disopyramide at doses of 1.6 ± 0.2 mg/kg (6). Antiarrhythmic drugs exert their action preferentially in the low right atrium irrespective of the mechanism for terminating atrial reentry (i.e., prolongation of ERP versus suppression of conduction) (8). The preferential action of antiarrhythmic drugs in the canine atrial reentry could be explained by different distribution of ionic currents, especially that of Ito (9). SD-3212 interrupted atrial flutter preferentially in the low right atrium as other drugs did so in our canine model (8).
Electrophysiologic effects of SD-3212
With SD-3212, prolongation of IACT was increased as the basic pacing cycle length was shortened, but percentage increase in atrial ERP did not change as the basic pacing length was changed from 300 to 150 ms in our study. These results indicate that SD-3212 has use-dependent block for suppression of conduction but do not show apparent reverse use-dependency for prolongation of ERP. However, the results of our in vitro experiment did not show apparent use-dependent suppression of Vmax with SD-3212 at frequencies of 0.2-3.3 Hz. In the in vivo study, we examined use-dependency at basic cycle lengths of 150-300 ms (i.e., 3.3-6.7 Hz), whereas in the in vitro study, we examined it from 0.2 to 3.3 Hz. Kodama et al. (10) reported that the time constant of SD-3212 for the recovery of Vmax from a use-dependent block was 1.3 s in guinea pig papillary muscles, suggesting that the use-dependent block of Vmax would be overt only at higher stimulus frequencies. The decrease of Vmax at low frequency might be attributable to the tonic block of the sodium channel by SD-3212. This difference in frequency range could contribute to the discrepancy between in vivo and in vitro study. Use-dependent suppression of Vmax with SD-3212 has been shown not only in papillary muscles of guinea pig (10) but also in those of rabbit (11). The lack of apparent reverse use-dependency for prolongation of APD with SD-3212 was reported by Takahashi et al. (11) by using rabbit papillary muscles. This indicates that antiarrhythmic effects of SD-3212 might not be diminished at shorter basic cycle lengths. Kodama et al. (10) reported that in guinea pig papillary muscle, SD-3212 has biphasic effects on APD, that is, moderate prolongation of APD at lower concentrations (1 μM) is reversed as the concentration is increased (10 μM). The biphasic effect of SD-3212 on APD is probably caused by changes in balances of the action on the outward potassium currents and the inward sodium and calcium currents (10). It has been demonstrated that in guinea pig atrial cells, SD-3212 inhibits the delayed rectifier potassium current (Ik) as well as the inward sodium and calcium currents (12). In our study, we did not find the biphasic dose-dependent effect of SD-3212. The drug concentrations used in our study were those exerting moderate prolongation of canine atrial APD. By use of patch-clamp techniques, Hara and Nakaya (12) revealed that SD-3212 had an inhibitory effect on the muscarinic acetylcholine-receptor-operated potassium current.
Our study is limited for several reasons. First, the mechanism for interruption of atrial reentry was not determined by assessing the width of excitable gap as in the previous study (6). However, changes in atrial ERP and IACT determined at a basic cycle length of 150 ms suggested that atrial reentry was interrupted with SD-3212 through suppression of conduction, as with disopyramide (6). Second, basic atrial pacing at a cycle length of 150 ms failed to capture the atria in five of nine dogs, and changes in electrophysiologic variables were not determined in these dogs at this particular basic cycle length. Finally, characteristics of ionic currents responsible for electrophysiologic changes observed in our in vivo and in vitro experiments were not investigated. Although limited for these reasons, our study suggests clinical efficacy of SD-3212 for the treatment of atrial tachyarrhythmias.
Acknowledgment: We thank Dai-ichi Pharmaceutical Co. for providing SD-3212 and also for measuring plasma concentration of this compound.
1. Miyawaki N, Yamazaki F, Furuta T, Shigei T, Yamauchi H. Antiarrhythmic effects of a novel Na and Ca channel blocker, SD-3212
: a comparison with its enantiomer (SD-3211). Drug Dev Res
2. Fukuchi M, Uematsu T, Nagashima S, Nakashima M. Antiarrhythmic effects of a benzothiazine derivative (SD-3211) and its stereoisomer (SD-3212
) in anesthetized rats and isolated perfused rat hearts compared with bepridil. Naunyn Schmiedebergs Arch Pharmacol
3. Hirasawa A, Haruno A, Matsuzaki T, Hashimoto K. Effects of a new antiarrhythmic drug
, on canine ventricular arrhythmia models. Jpn Heart J
4. Miyawaki N, Furuta T, Shigei T, Yamauchi H, Iso T. Electrophysiological properties of SD-3212
, a novel putative Ca antagonist, in isolated guinea pig and rabbit hearts. J Cardiovasc Pharmacol
5. Nakayama K, Morimoto K, Nazawa Y, Tanaka Y. Calcium antagonistic and binding properties of semotiadil (SD-3211), a benzothiazine derivative assessed in cerebral and coronary arteries. J Cardiovasc Pharmacol
6. Inoue H, Yamashita T, Nozaki A, Sugimoto T. Effects of antiarrhythmic drugs on canine atrial flutter
due to reentry: role of prolongation of refractory period and depression of conduction to excitable gap. J Am Coll Cardiol
7. Inoue H, Matsuo H, Takayanagi K, Murao S. Clinical and experimental studies of the effects of atrial extrastimulation and rapid pacing on atrial flutter
cycle: evidence of macro-reentry with an excitable gap. Am J Cardiol
8. Yamashita T, Inoue H, Nozaki A, Kuo T, Usui M, Sugimoto T. Role of anisotropy in determining the selective action of antiarrhythmics in atrial flutter
in the dog. Cardiovasc Res
9. Yamashita T, Nakajima T, Hazama H, et al. Regional differences in transient outward current density and inhomogeneities of depolarization in rabbits right atrium. Circulation
10. Kodama I, Suzuki R, Maruyama K, Toyama J. Electrophysiological effects of SD-3212
, a new antiarrhythmic agent with vasodilator action, on guinea-pig ventricular cells. Br J Pharmacol
11. Takahashi N, Ito M, Ishida S, Fujino T, Maruyama T, Saikawa T. Electrophysiological effects of SD-3212
, a novel antiarrhythmic agent, on rabbit hearts in vivo and in vitro. J Cardiovasc Pharmacol
12. Hara Y, Nakaya H. SD-3212
, a new class I and IV antiarrhythmic drug
: a potent inhibitor of the muscarinic acetylcholine-receptor-operated potassium current in guinea-pig atrial cells. Br J Pharmacol