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Antiarrhythmic Effects of a Novel Class III Drug, KCB-328, on Canine Ventricular Arrhythmia Models

Xue, Yixue; Tanabe, Shigeru*; Nabuchi, Yoshiaki*; Hashimoto, Keitaro

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Journal of Cardiovascular Pharmacology: August 1998 - Volume 32 - Issue 2 - p 239-247
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Abstract

Since the recent CAST study emphasized that the Na+ channel blocking antiarrhythmics, especially those subclassified as Ic, suppressed ventricular premature contractions (VPCs) effectively but increased the risk of sudden arrhythmic cardiac death (1-3), class III antiarrhythmic drugs have been rapidly developed with the expected outcome that they have different antiarrhythmic properties. These class III antiarrhythmic drugs have a common electrophysiologic feature, which is inhibiting the K+ channel, resulting in the prolongation of repolarization and refractoriness. Class III drugs have been proven to be effective in suppressing some experimental ventricular arrhythmias; however, all available drugs in this class have potentials to cause torsades de pointes-type ventricular tachycardias (VTs). We have been studying antiarrhythmic and proarrhythmic effects of class III drugs by using the same canine ventricular arrhythmia models. They are d-sotalol, MS-551, E-4031, intravenous amiodarone, sematilide, and dofetilide (4-8), and unlike the former three drugs, sematilide and dofetilide were not effective on the ventricular arrhythmias produced by coronary ligation and reperfusion in dog hearts. Sematilide at higher doses aggravated digitalis- and epinephrine (EPI)-induced ventricular arrhythmias from VT to ventricular fibrillation (VF) (7,8), so there is still a need for a novel class III drug with low arrhythmogenic potencies.

KCB-328, 1-(2-amino-4-methanesulfonamidophenoxy)-2-[N-(3,4-dimethoxyphenethyl)-N-methylamino]ethane hydrochloride (Fig. 1) is a new synthetic class III antiarrhythmic drug. The electrophysiologic test of this compound indicated that it remarkably increased the action-potential duration (APD) and effective refractory period (ERP) in isolated guinea pig papillary muscles. In the whole-cell patch-clamp experiments in isolated guinea-pig ventricular myocytes, KCB-328 at 10−6M inhibited Ik without a significant inhibition of Isi, like other class III drugs, but its APD-prolonging effect did not show a reverse frequency dependence (9,10). These results indicate that KCB-328 has unique electrophysiologic profiles different from most of the class III antiarrhythmic drugs. To determine whether this drug has antiarrhythmic or proarrhythmic effects and to compare it with other class III drugs, we conducted this investigation with in vivo arrhythmia models that had been used for other drugs.

FIG. 1
FIG. 1:
Chemical structure of KCB-328.

METHODS

Production of coronary-ligation and reperfusion arrhythmia

Thirty-two beagle dogs of either sex, weighing 6.0-13.0 kg, were used and were divided into two groups. Dogs of group 1 were anesthetized with halothane, whereas dogs of group 2 were anesthetized with pentobarbital. Dogs of both groups 1 and 2 were treated with an infusion of 1 mg/kg/h of KCB-328 or saline. Dogs of group 1 were anesthetized initially with intravenous thiopental sodium, 30 mg/kg, and intubated. Anesthesia was maintained by 1.0% halothane, vaporized with 100% oxygen by using a volume-limited ventilator (20 ml/kg, 15 strokes/min). The average values of arterial pH, Po2 and Pco2 were 7.38, 412 mm Hg, and 43.8 mm Hg the 30 min after reperfusion (n = 4). Dogs of group 2 were anesthetized with intravenous 30 mg/kg pentobarbital sodium followed by an infusion of 5 mg/kg/h.

In both groups, the chest was opened and the left anterior descending coronary artery (LAD) was isolated just proximal to the first diagonal branch. Because the incidence of occurrence of coronary ligation-reperfusion arrhythmias is known to be quite variable, experiments were randomized by using a pair of beagles (by coin-flip); one received the drug infusion and the other received 0.9% NaCl infusion. The speed of infusion was 1 ml/min. After 30 min from the start of either KCB-328 or saline infusion when the change in QTc became stable, the LAD ligation was performed by using a silk thread, and 20 min later released to examine the reperfusion responses.

A pair of epicardial electrodes was sutured on the border zone of the ischemic area of the left ventricle for continuous recording of the ventricular electrograms. The QT interval was assessed from the lead II electrocardiogram (ECG) and the ventricular surface electrogram. The QTc interval was calculated by using Bazett's formula, QTc = QT/√RR. The heart rate measured from the lead II ECG and the blood pressure were continuously monitored through a double-lumen arterial cannula in the femoral artery. Arterial blood samples were obtained through another lumen of the cannula (a) just before the start of KCB-328 infusion, (b) at LAD occlusion, and (c) at LAD reperfusion.

Production of programmed electrical stimulation (PES)-induced arrhythmias

Twelve beagle dogs of either sex weighing 8.0-11.5 kg were anesthetized initially with intravenous thiopental sodium, 30 mg/kg, and intubated. The chest was opened, and a two-stage coronary ligation of the LAD was performed under halothane anesthesia, and then the chest was closed. Six to 9 days after surgery, when myocardial infarction was established, the dogs were anesthetized initially with intravenous pentobarbital sodium, 30 mg/kg, and intubated. The chest was again opened under continuous administration of pentobarbital sodium (5 mg/kg/h) through a cannula in the femoral vein. PES was performed through the stainless-steel bipolar plunge electrodes, which were sutured on the noninfarcted myocardium of the left ventricular wall close to the myocardial infarct zone. A silver-coated copper wire was connected to a programmable stimulator (SS-201J; Nihon Kohden, Tokyo, Japan). The lead II ECG, instantaneous, and mean blood pressure from a cannula in the femoral artery were continuously recorded.

The basic pacing interval (S1) was set at 250 ms, which was shorter than the cycle length of the spontaneous heart rate. The excitation-threshold measurement was preceded by each PES procedure. After a train of 15 pacing stimuli, a single extrastimulus (S2) was delivered by shortening the S1-S2 interval in a 5-ms step until arrhythmia or refractoriness occurred. If S2 failed to induce arrhythmias, double extrastimuli (S2 and S3) were delivered with the S1-S2 interval just exceeding the ERP. S3 stimulus was introduced initially at the same interval as S2, which was then decreased again in 5-ms steps. If S3 failed to induce arrhythmias, S4 was added initially at the same interval as S3.

Arrhythmias that occurred at least twice during the control stimulation protocol were used further for pharmacologic analysis. The dogs with sustained ventricular tachycardia (SVT) and VF were recovered by using a DC defibrillator. KCB-328, 0.5 mg/kg/30 min, was given, and 30 min after the start of infusion, the same pattern of stimulation protocol was examined as before. By following the Lambeth Conventions (11), consecutive VPCs of >3 is defined as VT. NSVT is defined as VT lasting <30 s; SVT is a VT lasting >30 s.

Production of two-stage coronary ligation-induced arrhythmia

Six beagle dogs of either sex, weighing 8.0-11.0 kg, were anesthetized initially with an intravenous bolus of thiopental sodium, 30 mg/kg, and intubated. As reported earlier (12), the chest was opened and a two-stage coronary ligation of the LAD was performed under halothane anesthesia.

Experiments were done without anesthesia 24 and 48 h after coronary ligation. The lead II ECG, atrial electrogram from implanted electrodes sutured on the left atrial appendage, and the instantaneous and mean blood pressures were recorded continuously by using a telemetry system (Nihon Kohden). A constant-rate infusion of KCB-328, 0.1 mg/kg/min, was performed for 10 min, a total of 1 mg/kg, by using a syringe pump (Terumo, Tokyo, Japan), and arterial blood samples were drawn from one lumen of the arterial double-lumen cannula at 0, 5, 10, 30, and 60 min after the start of the infusion.

Production of digitalis-induced arrhythmia

Six beagle dogs of either sex, weighing 9-11 kg, were anesthetized with intravenous pentobarbital sodium (30 mg/kg) and intubated but were allowed to breath spontaneously with room air. The right femoral artery and vein were cannulated for recording blood pressure and for drug administration, respectively. The standard lead II ECG and atrial electrogram from catheter-tip electrodes placed in the right atrium through the external jugular vein were recorded continuously. As reported previously (13), 40 μg/kg ouabain was initially administered intravenously, followed by 10 μg/kg every 20 min until stable VT was produced. This arrhythmia usually continued for ≥1 h (6). KCB-328, 1 mg/kg, was administered intravenously for 10 min. Arterial blood samples were drawn 0, 5, 10, 15, 30, and 60 min after the start of infusion.

Production of epinephrine-induced arrhythmia

Five beagle dogs of either sex, weighing 10.0-11.0 kg, were anesthetized initially with intravenous thiopental sodium. As reported earlier (14), after intubation, 1.0% halothane, vaporized with 100% oxygen, was administered with a volume-limited ventilator (20 ml/kg, 15 strokes/min). EPI was infused through the left femoral vein at a rate of 2.0-2.5 μg/kg/min by using a syringe pump. If multifocal VT was not induced, a higher infusion rate was used. At 3 min after the start of EPI infusion, a constant-rate infusion of KCB-328, 1 mg/kg, was performed for 10 min by using a syringe pump, and arterial blood samples were drawn from one lumen of the arterial double-lumen cannula at 0, 5, 10, 15 min after the start of infusion.

The lead II ECG, atrial electrogram from catheter-tip electrodes in the right atrium, and the instantaneous and mean blood pressures were continuously recorded. The experimental and animal-care protocol was approved by the Animal Use and Care Committee of the Yamanashi Medical University.

Plasma KCB-328 assay

The arterial blood samples were collected into heparinized syringes at predetermined times and centrifuged at 3,000 g for 5 min. The plasma was stored at about −80°C until the assay. Plasma (200 ml) was mixed with 100 μl of 25% ammonia solution and extracted with 5 μl of dichloromethane. The organic layer was separated and evaporated to dryness under a stream of nitrogen. The dry residue was resuspended in 200 μl of high-performance liquid chromatographic (HPLC) mobile phase and 50-μl aliquot was injected into HPLC. HPLC analysis was performed with a Shimadzu LC-6A HPLC system equipped with a SPD-10A UV detector (Shimadzu, Kyoto, Japan). The samples were eluted with 27% acetonitrile containing a PIC-B8 ion pair reagent (Waters, Tokyo, Japan) at a flow rate of 1.0 ml/min. Detection wavelength at 283 nm was used.

Drugs

Drugs used in this study were KCB-328 (C & C Research Laboratories, Kyunggi-do, Korea), thiopental sodium (Tanabe Seiyaku, Tokyo, Japan), halothane (Takeda Chemical Industries, Osaka, Japan), pentobarbital sodium (Tokyo Kasei Kogyo, Tokyo, Japan) and (−)-ouabain octahydrate (Aldrich, Tokyo, Japan).

Evaluation of antiarrhythmic effects

The severity of ventricular arrhythmias was expressed by the arrhythmic ratio: the number of ventricular ectopic beats divided by the total heart rate, which is the number of all beats counted from the 5-s strip of ECG (i.e., the number of ventricular ectopic beats plus the number of conducted beats), and the ventricular beats were judged by the different shape of the ventricular complex from the normal QRS complex. The arrhythmic ratio before drug injection was almost 1, as shown in the control values of the figures, and there were no spontaneous improvements in these ratios. If the values after drug administration decreased significantly from the 0 time value, the drug was considered to have a significant antiarrhythmic effect.

Statistics

For the analysis of hemodynamic and ECG parameters and arrhythmic ratio, analysis of variance (ANOVA) was performed; when a statistical difference was detected, a Dunnett's multiple-comparison test was used to determine the difference between the 0 time value and the other values. All data are expressed as mean ± SD, and the statistically significant difference was determined at the p < 0.05 level unless otherwise stated.

For the PES-induced arrhythmia model, ANOVA was used to compare the QTc, heart rate, and blood pressure values before and after the drug treatment. Incidence of arrhythmias was compared by Wilcoxon signed-rank test and, in the case of p < 0.05, the drug was judged to have significant effects. Values are expressed as mean ± SD.

RESULTS

Coronary ligation and reperfusion arrhythmias

Group 1: KCB-328, 1 mg/kg/h, in halothane-anesthetized beagles. The heart rate and mean blood pressure of all beagles anesthetized with halothane were 127 ± 23 beats/min and 100 ± 20 mm Hg (n = 16). As shown in Fig. 2, KCB-328 prolonged the QTc interval significantly from 0.41 to 0.55 s1/2 (just before LAD ligation), as compared with that of the control group from 0.38 to 0.37 s1/2. KCB-328 decreased the heart rate by 18% (from 126 to 103 beats/min) just before ligation and by 18% (to 103 beats/min) just before reperfusion. During the 20 min of coronary ligation, five of eight dogs showed VPCs and VT. At the same time, all the dogs showed VPCs and VT in the control group. There was no change in the heart rate of the control group (130-128 beats/min just before occlusion). There were also no significant differences in the mean number of total VPCs between the drug and control groups (178 and 340 beats/20 min during the ligation period, respectively). KCB-328 had antifibrillatory effects after reperfusion (two of eight dogs in the drugtreated group compared with six of eight dogs in the control group; p < 0.05). In addition, during the 30 min of infusion of KCB-328 and before applying coronary occlusion, four of eight dogs showed VPCs or a short period of VT, but no dogs showed torsades de pointes-type VT. The KCB-328 plasma concentrations at 0, 10, 29 min (1 min before ligation), and 49 min (1 min before reperfusion) were 0 ± 0, 217 ± 44, 389 ± 267, and 403 ± 144 ng/ml (n = 6), respectively.

FIG. 2
FIG. 2:
Summary of the effect of KCB-328, 1 mg/kg/h, on the coronary ligation-reperfusion experiments in halothane-anesthetized beagles. Each column indicates the responses of each dog.

Group 2: KCB-328, 1 mg/kg/h, in pentobarbital-anesthetized beagles. Changing the anesthesia to intravenous pentobarbital, we repeated the experiment with KCB-328 by using the same dose as in group 1. The heart rate and mean blood pressure under pentobarbital anesthesia were 187 ± 40 beats/min and 121 ± 15 mm Hg (n = 16). As shown in Table 1, the QTc interval of the drug-treated group also increased significantly from 0.35 to 0.39 s1/2 just before ligation, whereas in the control group, there were no changes (from 0.33 to 0.33 before ligation). KCB-328 decreased the heart rate only 7% just before coronary ligation. In this pentobarbital-anesthetized condition, KCB-328 had antifibrillatory effects during ischemia and reperfusion (two of eight dogs in the drugtreated group compared with six of eight dogs in the control group; p < 0.05). The mean number of total VPCs in the drug and control groups was 391 and 651 beats during the 20 min of coronary occlusion (Table 1). In addition, during the 30 min of infusion of KCB-328 and before applying coronary occlusion, no dogs showed VPCs. The KCB-328 plasma concentrations were 0 ± 0, 177 ± 42, 215 ± 45, and 264 ± 54 ng/ml (n = 6) at the same time points obtained in the group 1 experiment.

TABLE 1
TABLE 1:
Effects of KCB-328 on the coronary ligation and reperfusion arrhythmia

PES-Induced arrhythmias in dogs with old myocardial infarction

By applying a series of PES, 12 dogs showed different types of ventricular arrhythmias: VF, SVT, NSVT, and VPC occurred in 1, 4, 2, and 5 dogs, respectively. As shown in Fig. 3, KCB-328, 0.5 mg/kg/30 min prolonged the QTc interval only by 11% (from 0.36 to 0.40 s1/2), but significantly reduced the severity of these arrhythmias. All types of ventricular arrhythmias became noninducible (NI; p < 0.05).

FIG. 3
FIG. 3:
Effects of intravenous infusion of KCB-328, 0.5 mg/kg/30 min, on the programmed electrical stimulation-induced arrhythmia models (n = 12). VF, ventricular fibrillation; SVT, sustained ventricular tachycardia; NSVT, nonsustained ventricular tachycardia; VPC, ventricular premature contraction; NI, noninducible.

Two-stage coronary ligation-induced arrhythmia

Approximately 24 h after two-stage coronary ligation of the LAD, all dogs showed continuously occurring multifocal VT. The arrhythmic ratio before KCB-328 administration was 0.94 ± 0.08 (n = 6), as shown in Fig. 4. KCB-328 significantly decreased the total heart rate, atrial rate, and increased the number of conducted beats and decreased the arrhythmic ratio at 2- and 4-min points. KCB-328 had no effect on the blood pressure. The KCB-328 plasma concentrations at 0, 5, 10, 15, 30, and 60 min were 0 ± 0, 898 ± 214, 1,070 ± 401, 245 ± 64, 125 ± 26, and 69 ± 28 ng/ml (n = 6), respectively.

FIG. 4
FIG. 4:
Effects of intravenous infusion of KCB-328, 1 mg/kg/10 min, on 24-h coronary ligation-induced arrhythmia (n = 6). *p < 0.05 against 0 time value. (□ total heart rate; ▪, atrial rate; ∇ conducted beats; •, mean blood pressure; ○, arrhythmic ratio; ▵, plasma concentrations of KCB-328).

The 48-h arrhythmia was less severe, and the arrhythmic ratio before KCB-328 administration was 0.84 ± 0.09. KCB-328 did not significantly change all the parameters, including the total heart rate, atrial rate, blood pressure, conducted beats, and arrhythmic ratio. The KCB-328 plasma concentrations at 0, 5, 10, 15, 30, and 60 min were 0 ± 0, 913 ± 72, 979 ± 291, 382 ± 135, 219 ± 74, and 141 ± 53 ng/ml (n = 6), respectively.

Digitalis-induced arrhythmias

After injection of a total dose of 40-60 μg/kg ouabain, almost all the beats were of ventricular origin. The arrhythmic ratio before KCB-328 administration was 1.00 ± 0.00 (n = 6). As shown in Fig. 5, KCB-328 significantly decreased the total heart rate and atrial rate. The mean blood pressure after 14 min was significantly decreased, but KCB-328 did not decrease the arrhythmic ratio. The KCB-328 plasma concentrations at 0, 5, 10, 15, 30, and 60 min were 0 ± 0, 981 ± 316, 1,110 ± 429, 271 ± 140, 140 ± 79, and 101 ± 85 ng/ml (n = 6), respectively.

FIG. 5
FIG. 5:
Effects of intravenous infusion of KCB-328, 1 mg/kg/10 min, on the digitalis-induced arrhythmia (n = 6). *p < 0.05 against 0 time value. The symbols are the same as in FIG. 4.

EPI-induced arrhythmia

As reported previously (14), EPI infusion at a rate of 2.0-2.5 μg/kg/min for 3 min produced VT. The arrhythmic ratio before KCB-328 administration was 0.90 ± 0.20 (n = 5). KCB-328, in a dose of 1 mg/kg/10 min, significantly decreased the total heart rate, atrial rate, blood pressure, and arrhythmic ratio, but only at the 15-min point (Fig. 6). The KCB-328 plasma concentrations at 0, 5, 10, and 15 min were 0 ± 0, 717 ± 89, 945 ± 128, and 270 ± 46 ng/ml (n = 4), respectively.

FIG. 6
FIG. 6:
Effects of intravenous infusion of KCB-328, 1 mg/kg/10 min, on the epinephrine-induced arrhythmia (n = 5). *p < 0.05 against 0 time value. The symbols are the same as in FIG. 4.

DISCUSSION

In this study, we showed that KCB-328 significantly prolonged the QTc interval and suppressed the reentry-type arrhythmias induced by PES, fatal VF on coronary ligation, and reperfusion under halothane or pentobarbital anesthesia, and decreased, although weakly, the arrhythmic ratio of the arrhythmia induced by 24-h coronary ligation. KCB-328 showed minor proarrhythmic effects: four dogs showed VPCs and VT by the direct action of KCB-328 in these halothane-anesthetized dogs subjected to coronary ligation and reperfusion. In addition, KCB-328 was not effective on automaticity arrhythmias (i.e., 48-h coronary ligation-, digitalis-, and EPI-induced arrhythmias), nor did it aggravate these arrhythmias.

In the coronary ligation and reperfusion-induced arrhythmias, KCB-328 was not effective in significantly suppressing the number of VPCs during the 20 min of LAD ligation but was effective in suppressing the occurrence of the VF either in the low-heart-rate halothane-anesthetized dogs or in the high-heart-rate pentobarbital-anesthetized dogs. The mechanisms of the coronary ligation and reperfusion VF may be due to reentry (15,16), but certainly automaticity may be an initiating factor (17,18). So class III antiarrhythmic drugs, being theoretically effective on narrow-excitable-gap reentry arrhythmias, may not necessarily be effective on this arrhythmia. We showed that class III antiarrhythmic drugs, E-4031 (5) and MS-551 (4), suppressed this reperfusion VF, simultaneously prolonging the QTc interval under halothane anesthesia. However, another class III drug, d-sotalol, did not suppress this reperfusion VF under halothane anesthesia but suppressed the reperfusion VF under pentobarbital anesthesia (4). Other class III drugs, dofetilide and sematilide, were not effective on this arrhythmia regardless of the anesthetics (7,8). Therefore KCB-328 seems to be different from other class III antiarrhythmic drugs in the pattern of effectiveness on the coronary ligation and reperfusion VF. Only KCB-328, so far as we have examined, suppressed ischemia and reperfusion VF in both the low-heart-rate halothane-anesthetized dogs and the high-heart-rate pentobarbital-anesthetized dogs. KCB-328 showed antifibrillatory effects irrespective of the degree of the QTc interval prolongation. Figure 7 shows a summary of our results on class III drugs on the reperfusion VF versus their QTc-prolonging effects. Percentage suppression of VF, calculated as (1-VF occurrence in the treated group/VF occurrence in the saline group) × 100, was plotted against QTc prolongation. Minus means increase in the VF occurrence. As shown in Fig. 7A of the pentobarbital-anesthetized experiments, statistically effective antifibrillatory drugs were KCB-328 and d-sotalol. The average plasma concentrations of KCB-328 at 1 min before coronary ligation and reperfusion were 215-264 ng/ml. Despite the higher average plasma concentrations of MS-551, sematilide, and d-sotalol at the same 1-min point (1,310-1,650, 1,070-2,180, and 8,250-12,400 ng/ml, respectively), they had no antifibrillatory effect. In Fig. 7B of the halothane-anesthetized experiments, the statistically significant antifibrillatory drugs were E-4031, MS-551, and KCB-328.

FIG. 7
FIG. 7:
Summary of our results on class III drugs on the reperfusion ventricular fibrillation (VF) versus their QTc-prolonging effects. A: The anesthetic was pentobarbital. B: The anesthetic was halothane. d-S, d-Sotalol; MS, MS-551; E, E-4031; Se, sematilide; KCB, KCB-328; Do, Dofetilide. The number under the drug indicates dose of the drug in mg/kg. The doses of KCB-328, dofetilide, MS-551, and E-4031 were 1 mg/kg/h, 0.1 mg/kg/h, 3.6 mg/kg/h, and 30 mg/kg + 3 mg/kg/min, respectively. Percentage suppression of VF was plotted against QTc prolongation.

Because the reperfusion-induced arrhythmias may be induced not only by the reentry mechanism but also by the increased automaticity, we used an arrhythmia model that was induced solely by the narrow-excitable-gap reentry (i.e., arrhythmias induced by PES in dogs with old myocardial infarction). In this experiment, KCB-328 prolonged the QTc interval only by 11%, but completely suppressed all types of arrhythmias including VF, SVT, NSVT, and VPC, that were induced in the control period. These results are consistent with other class Ia and class III drugs (propafenone, NE-10064, MS-551, sematilide. and dofetilide) by using similar models of a reentrant mechanism regardless of anesthetized or conscious conditions (7,8,19-21). These studies indicate that the QTc prolongation by KCB-328 must have contributed to the suppression of these reentry-type arrhythmias.

We reported effects of various antiarrhythmic agents by using canine automaticity ventricular arrhythmia models and classified antiarrhythmic agents based on their pharmacologic effectiveness (22,23). We concluded that EPI-induced arrhythmia is suppressed by Ca2+ channel blockers or β-blockers, whereas digitalis-induced and two-stage coronary ligation-induced arrhythmias are suppressed by Na+ channel blockers (22,23). By using the same arrhythmia models, we reported that d-sotalol aggravated the 48-h coronary-ligation arrhythmia, that E-4031 aggravated the EPI-induced arrhythmia (5), and that sematilide aggravated the digitalis- and EPI-induced arrhythmias (7).

In the 24-h coronary-ligation arrhythmias, KCB-328 significantly decreased the total heart rate and atrial beats, and also weakly decreased the arrhythmia, because there were only two time points, 2 and 4 min, when the arrhythmic ratio was significantly decreased in a concentration-independent manner. We have been using the arrhythmic ratio to evaluate antiarrhythmic effects of various antiarrhythmic drugs, and even when using the 5-s ECG strip, the mean values of the time-point data responded well to drug treatments, and so combining the effective plasma concentration data of several time points, we were able to determine the effective plasma concentration data to compare the potency of various antiarrhythmic drugs and to predict human effective or toxic plasma concentration of the newly developed drug (23,24). In digitalis-induced arrhythmia, KCB-328 significantly decreased the total heart rate, atrial beats, and mean blood pressure, but it did not change the arrhythmic ratio. The decrease of the total heart rate and atrial beats was observed also in the 24-h coronary-ligation arrhythmia model. In EPI-induced arrhythmia, KCB-328 significantly decreased the total heart rate, atrial beats, and mean blood pressure, and decreased the arrhythmic ratio only at the 15-min point. Because the 15-min point was when the EPI infusion was stopped, this effectiveness at only one time point indicates that this possible antiarrhythmic effect is a minor one. Other class III drugs, E-4031 and sematilide, aggravated this arrhythmia, and it is known that drug-induced or congenital QTc prolongation is often accompanied by the occurrence of torsades de pointes when sympathetic activation occurs. Thus with the QTc-prolonging activity of KCB-328, having no arrhythmogenic effect with EPI might be quite a favorable effect for KCB-328. The decrease in the blood pressure may not be a favorable effect, but KCB-328 did not decrease the blood pressure in the coronary-ligation and reperfusion arrhythmia models and also in the two-stage coronary-ligation arrhythmia models under a conscious condition. KCB-328 significantly decreased the number of atrial beats in three models, 24-h coronary ligation-, digitalis-, and EPI-induced arrhythmia models. This shows that KCB-328 has a negative chronotropic effect like other class III drugs (7,8). These results suggest that a K+ channel blocker can decrease the sinus node automaticity.

In the control period of the coronary ligation and reperfusion experiments, we demonstrated that KCB-328 prolonged QTc in a reverse rate-dependent manner and has a proarrhythmic effect in the low-heart-rate halothane-anesthetized condition; VPC and VT (not a torsades de pointes-type VT) occurred before applying the coronary ligation (four of eight dogs). Under the same conditions, MS-551 induced torsades de pointes-type VT, and E-4031 and dofetilide induced SVT. The KCB-328's reverse rate-dependent QT-prolonging effect is different from the report of Lee et al. (9), which showed that KCB-328 prolonged the ERP in a frequency-independent manner in the isolated guinea-pig papillary muscle. The similar inconsistency in the results of in vivo and in vitro studies were observed also for MS-551, for which we reported that MS-551 prolonged QTc in a reverse rate-dependent manner (4,25), but Cheng et al. (26) reported that MS-551 prolonged APD in a frequency-independent manner in isolated rabbit ventricular myocytes. This in vitro and in vivo difference may be related to the animal species, the protocol used, and the definition of the phenomenon. The mechanism for the reverse rate-dependent APD-prolonging effect of class III drugs was explained by the incomplete deactivation of Iks, which partially offset the rate-independent block of Ikr(27-29). However, Gintant (30) reported that the incomplete deactivation of Iks is not necessary for the reverse rate-dependent effect of the class III agents in canine myocytes. So the mechanism of reverse rate dependence by class III agents has not been fully explained, but it is well recognized that the risk of a high incidence of proarrhythmias, such as torsade de pointes, increases as the QT prolongation becomes greater at lower heart rates (27). Further studies are necessary to test and elucidate the reverse rate dependence of KCB-328.

In conclusion, KCB-328 prolonged the QTc interval in a reverse rate-dependent manner and significantly suppressed the coronary-ligation and reperfusion VF in both the low-heart-rate halothane-anesthetized dogs and the high-heart-rate pentobarbital-anesthetized dogs, and PES-induced arrhythmias, and weakly suppressed 24-h coronary-ligation arrhythmias. KCB-328 was not effective on digitalis-induced and did not aggravate EPI-induced arrhythmias. Comparing these effects with those of other class III drugs, KCB-328 can be said to have powerful antiarrhythmic effects with fewer arrhythmogenic potencies.

Acknowledgment: We thank C & C Research Laboratories for supplying KCB-328, and Mrs. Akiko Ozawa for excellent technical assistance.

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Keywords:

KCB-328; Ventricular arrhythmia; Ischemia arrhythmia; Reperfusion arrhythmia; Programmed electrical stimulation; Heart

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