Amiodarone is one of the most effective antiarrhythmic agents currently available in clinical use for the suppression of atrial fibrillation and life-threatening ventricular arrhythmias.1,2 3-7 8 9
SSR149744C (SSR; 2-butyl-3-{4-[3-(dibutylamino)propyl]benzoyl}-1-benzofuran-5-carboxylate isopropyl fumarate) is a new antiarrhythmic agent10 11 Na ), calcium (ICa(L) ), and several potassium (IKr , IKs , IK(ACh) and IKv1.5 ) currents with inhibition of responses to α1 - and β1 -adrenergic as well as angiotensin II (AT1 ) receptor stimulation. In anesthetized dogs, intravenously administered SSR149744C reduces heart rate (HR), increases PQ interval, and prolongs effective refractory periods (ERP) in atrial and atrioventricular node (AVN), without any change in the QTc interval. All these in vitro and in vivo electrophysiological properties of SSR149744C resemble those of dronedarone and amiodarone but with differences in their inhibitory potencies toward ionic currents.10
The aim of the present study was to investigate the potential antiarrhythmic activities of SSR149744C after acute intravenous (IV) or oral (PO) treatment in several in vitro and in vivo animal models of atrial and ventricular arrhythmias, in which amiodarone and dronedarone have been shown to be effective. At the atrial level, we assessed the activity of SSR149744C to terminate sustained AF maintained during continuous vagal nerve stimulation in anesthetized dogs12 + medium-induced AF in guinea pig hearts.13 14 15 16
METHODS
Our animal facilities and animal care and use programs are in accordance with the principles laid down in the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and its appendix. The Animal Care and Use Committee of Sanofi-Synthélabo Recherche approved all protocols.
Models of Atrial Arrhythmia
Vagal AF in Anesthetized Closed-Chest Dogs
Fourteen mongrel dogs of either sex, weighing 15-20 kg, obtained from Butler Farms Inc (Clyde, NY), were used for the study. Animals, fasted overnight, were anesthetized with α-chloralose (96 mg/kg IV) and artificially ventilated with a mixture of room air and supplemental oxygen (80/20) using a veterinary respirator (Alpha 100®, Minerve, Esternay, France).
Four limb electrodes were positioned to obtain standard leads of an electrocardiogram (ECG). Two bipolar electrodes were positioned in the right atrium via the jugular veins, one to record the atrial electrogram (low atrium) and the other to stimulate the atrium (high atrium). A third bipolar electrode was inserted into the right ventricle via a femoral vein to pace the ventricle when the ventricular rate was less than 90 bpm. These catheters were connected to a programmed stimulator (Jansen, JS1-SU2®, Beerse, Belgium) and to a physiograph recorder (Recor®, Siemens-Elema, Solna, Sweden) used to monitor and to record the various electrograms. Finally, the cervical vagal nerves were isolated and prepared for electrical stimulation with a home-made electrode.
Atrial fibrillation was produced by a brief burst (1-3 seconds) of atrial pacing (10 Hz frequency, 4 times the threshold current) in the presence of bilateral vagal stimulation. Vagal stimulation was delivered with a pulse of 0.1 millisecond and an applied voltage of 5V. The vagal stimulation frequency was adjusted in each dog to two thirds of the threshold for an asystole under control conditions (0.7 ± 0.1 Hz). Atrial fibrillation was defined as a rapid and irregular atrial rhythm (400-600 bpm).
The effects of vagal nerve stimulation on the cardiac cycle length were measured at various frequencies of stimulation (1, 2, 4, 6, and 8 Hz). The vagal nerves were stimulated for 30 seconds at each frequency, and the maximal value of cardiac cycle length was recorded. A 1-minute period was observed between 2 consecutive stimulations to allow the cardiac cycle length to return to its baseline value.
The atrial effective refractory period (AERP) was measured with a train of 7 basic (S1 ) stimuli followed by a premature (S2 ) stimulus. AERP was defined as the longest (S1 S2 ) interval failing to produce a propagated response and was assessed after pacing at basic cycle lengths (BCL) of 200, 250, 300, and 400 milliseconds.
The experimental protocol was the following: after a 30-minute stabilization period, AF was induced as previously described. Vagal stimulation was first maintained for 30 minutes to verify the stability of AF and then stopped to allow a return to sinus rhythm. Reinduction test was immediately attempted under vagal stimulation. When a cardioversion by the drug occurred before the thirtieth minute of AF, the reinduction test was immediately performed. Sinus heart rate (HR), vagal stimulation frequency-response of cardiac cycle length, and AERP were determined after each AF period/reinduction test in baseline (predrug), vehicle, or drug conditions. AF was considered as converted if a return to sinus rhythm was observed before the thirtieth minute following the onset of AF. AF was considered as reinducible if an episode of at least 1 minute of AF could be obtained.
Vehicle (injection 1 and injection 2 of PEG400, 75% in distilled water) or SSR149744C (3 mg/kg and 10 mg/kg, free base) was administered intravenously (1 mL/min) during a 10-minute infusion (Pump 22, Biotronik, Berlin, Germany) after 10 minutes of sustained atrial fibrillation in 7 animals per group. If either AF termination or AF prevention (or both of them) were not observed following the administration of the dose of 3 mg/kg of SSR149744C, a cumulative dose of 10 mg/kg was infused (60 minutes after the first dose) and evaluated according to the same experimental protocol.
Low-K+ Medium-Induced AF in Guinea Pig Hearts
The experiments were carried out in hearts from Hartley male (Centre d'élevage R. Janvier, 53940 Le Genest St-Isle, France) guinea pigs (424 ± 9 g). Fifteen minutes after an intraperitoneal injection of heparin sodium (1 μL/g of body weight, 5000 UI/mL), the animal was killed by cervical dislocation, and the heart was quickly excised. Contractions were prevented by immersion in ice-cold modified Krebs solution (composition in mM: NaCl 118; KCl 5.4; CaCl2 1.8; MgCl2 1.05; NaH2 PO4 1.2, NaHCO3 25.5; glucose 11.5; pyruvate 2.0). A cannula was inserted into the aorta and positioned close to the coronary ostia, and the heart was transferred to a perfusion apparatus for perfusion at constant flow (12 mL/min) with warmed (37°C), oxygenated (95% O2 , 5% CO2 ), carefully filtered Krebs solution at pH 7.35 using a peristaltic pump. The temperature in the organ chamber around the heart was maintained at 36°C. The electrical activity of the right atrium (RA) was recorded by means of platinum electrodes. The biopotential signal was digitized and amplified, and the data stream was simultaneously stored on computer with data acquisition software IOX (EMKA Technologies, Paris, France).
Initially, the heart was perfused with standard Krebs solution (KCl = 5.4 mM), and the preparation was allowed to equilibrate for at least 30 minutes. Then the heart was perfused with low-K+ Krebs modified solution (KCl = 1.4 mM) for 45 minutes to induce spontaneous and sustained AF (defined as a rapid irregular rhythm lasting for 3 minutes) in the presence of the vehicle or drug.
Vehicle (100% DMSO; n = 17) or SSR149744C (dissolved in 100% DMSO) was administered 20 minutes before and during low-K+ perfusion by injection through a side arm of the apparatus immediately above the cannula by means of a syringe pump (15 μL/min) in 12-mL/min flow. SSR149744C was studied at final (aortic) concentrations of 0.1 μM (n = 6), 0.3 μM (n = 5), and 1 μM (n = 6) in 0.125% DMSO with each heart receiving only 1 concentration.
The activity of SSR149744C was expressed as the percentage of hearts undergoing spontaneous atrial fibrillation and compared with the vehicle group. The time of occurrence of AF was also noted. All results were expressed as the ratio of the number of hearts showing AF to the number of heart preparations tested for each group.
Models of Ventricular Arrhythmia
Reperfusion-Induced Arrhythmias in Anesthetized Rats
The experiment was carried out in male Sprague-Dawley (Centre d'élevage R. Janvier, 53940 Le Genest-Saint-Isle, France) rats (255-300 g). The rats were anesthetized with sodium pentobarbital (60 mg/kg, IP) and maintained at 37°C with a homeothermic blanket system. The animals were ventilated with room air, through a tracheotomy, using a tidal volume of 2 mL/100 g body weight, at a rate of 60 strokes/min.
Arterial blood pressure (AP) was monitored via a catheter inserted into the carotid artery and connected to a Statham P23XL pressure transducer. Standard limb leads (I and II) electrocardiogram (ECG) and pulsatile arterial pressure were continuously recorded through relevant modules (Hugo Sachs Elektronik, March-Hugstetten, Germany) on a computer for subsequent analysis (software: HEM, Notocord System, Croissy, France). The jugular vein was catheterized for intravenous drug administration. The technique used for the occlusion and reperfusion of the left coronary artery in rats was a modification of the methods reported by Manning et al14 17
The drug was administered intravenously in anesthetized rats and orally in conscious rats at 5 and 120 minutes, respectively, before ligature of the left coronary artery. The coronary artery was occluded for 5 minutes by pulling both ends of the ligature, then reperfused for 10 minutes by releasing the tension.
Mean arterial blood pressure (MAP) and HR were measured from the pulsatile arterial blood pressure before coronary occlusion. These parameters were recorded together with limb lead I and II ECGs using the HEM software. The ECG was analyzed during reperfusion. The animals exhibiting significant ventricular (V) arrhythmias during ischemia were excluded from the study. Arrhythmias were evaluated according to the Lambeth Conventions,18
By intravenous route, six groups of rats were studied. One group received the vehicle (PEG400, 75% in distilled water; n = 10), and the other groups (n = 6) received a single dose of SSR149744C (0.1, 0.3, 1, 3, or 10 mg/kg, expressed as free base) in a randomized manner. The drug was dissolved in warm PEG400, 75% and given by intravenous route at a constant volume of 0.1 mL/100 g body weight.
By oral route, 5 groups of rats were studied. One group received the vehicle (methylcellulose, n = 10), and the other groups (n = 6 to 7) received a single dose of SSR149744C (3, 10, 30, or 90 mg/kg, expressed as free base), again in a randomized manner. The drug was suspended in 0.6% methylcellulose solution and given by oral route before anesthesia and surgery in a volume of 1 mL/100 g body weight.
Acute Sudden Death in Conscious Post-Myocardial Infarction (PMI) Rats
The experiments were carried out in male Sprague-Dawley (Charles River, Saint Aubin-les Elbœuf, France) rats (270-300 g). The technique to produce coronary occlusion in rats was similar to that described previously by Fabiani et al.19 15
The drug studied was suspended in methylcellulose (0.6% solution) and given orally, 2 hours before surgery, at constant volume of 1 mL/100 g body weight. Control and sham groups received vehicle only (0.6% methylcellulose; 1 mL/100 g body weight). Rats were distributed into 6 groups: sham (n = 13), PMI-vehicle (n = 55), PMI-SSR 30 mg/kg (n = 21), PMI-SSR 2 × 30 mg/kg (n = 17), PMI-SSR 2 × 45 mg/kg (n = 17), and PMI-SSR 90 mg/kg (n = 28). When the drug was given twice daily, the second administration was carried out 12 hours after the first one. Doses were expressed as free base.
Statistical Analysis
In the in vivo vagal AF model in dogs, values are given as mean ± SEM. To compare the incidence of cardioversion or prevention of reinduction following an injection of SSR149744C or vehicle, we used the Fisher exact test. For heart rate, to study the effect of each drug treatment, a 2-way analysis of variance (ANOVA) with repeated measures on drug injection was used. For the effects on vagal stimulation frequency responses and on atrial effective refractory period, comparisons were made using a 3-way ANOVA with 2 repeated factors, followed by a 2-way ANOVA with repeated measures on drug injection to evaluate the effect of each drug treatment. The repeated-measures analysis was used to take into account the fact that 3 measures were done on the same animal. The Dunnett test was also used to compare each injection to the predrug value. If the interaction “injection-group” was found significant, a complementary analysis (Winer analysis) was used to detect a group effect for each injection.
In the in vitro AF model in guinea pig hearts, comparisons were made between each pair, vehicle, and the SSR149744C groups using the Fisher exact test. The time (min) necessary to obtain AF was also measured and analyzed with a Log-rank test; medians with associated confidence intervals (CI 95%) were calculated only if more than half of the hearts produced induction of AF.
In the reperfusion-induced arrhythmia model, MAP and HR are expressed as mean ± SEM. A 2-way ANOVA on repeated measures followed by Dunnett test when necessary was used to analyze these parameters. A 1-way ANOVA followed by Dunnett test was used for time of onset of arrhythmias and rhythm disturbance duration. A Fisher exact test was used to determine the significance of differences for all results expressed as percentage incidence. All treated groups were compared with vehicle group.
In the sudden-death model, the Fisher exact test was used to compare the mortality rate between groups of animals. The mortality rate was expressed in percentage of animals subjected to permanent coronary artery occlusion and alive at the beginning of the period studied. All treated groups were compared with vehicle group.
For all studies, a P value of less than 0.05 was considered as statistically significant (SAS 8.2 software, SAS Institute Inc, Cary, NC; RS/1 software, BBN Software Product, Cambridge, MA).
RESULTS
Experimental Models of Atrial Arrhythmia
Vagal AF in Anesthetized Closed-Chest Dogs
Because SSR149744C inhibits the muscarinic receptor-operated K+ current (IK(ACh) ) in guinea pig atrial cells10 K(ACh) block, we assessed its effects on responses (maximal cardiac cycle length) to vagal stimulation at frequencies between 1 and 8 Hz. In the vehicle group, the maximal value of the cardiac cycle length increased with the increase in vagal stimulation frequency, and frequency-response curves were identical in the course of experiment (Fig. 1A ). Compared with predrug values, IV injections of 3 and 10 mg/kg SSR149744C produced no statistically significant effect on the maximal value of the cardiac cycle length at the vagal stimulation frequencies of 1, 2, and 4 Hz and induced a decrease in the maximal value of sinus cycle length (SCL) at 6 and 8 Hz of vagal stimulation frequency from −21% to −34% (P < 0.05) (Fig. 1B ). Thus, SSR149744C did not modify the maximal value of the SCL at the stimulation frequency used within the AF episodes (0.7 ± 0.1 Hz; two thirds of the threshold for asystole).
FIGURE 1: Changes in maximal value of sinus cycle length at various vagal stimulation frequencies under control conditions (A; n = 5-7) and after 3 then 10 mg/kg IV of SSR149744C (B; n = 6-7). Maximal value of sinus cycle length increased with the increase of stimulation frequency: There was no significant difference between predrug and vehicle curves (A). At both doses SSR149744C did not modify sinus rate at 1, 2, and 4 Hz of vagal stimulation frequency and attenuated the effects of vagal stimulation of 6 and 8 Hz. Each data point represents the mean ± SEM. Significant differences: *P < 0.05 versus vehicle, and °P < 0.05 versus predrug (3-way ANOVA on repeated measures followed by Dunnett test).
In the vehicle group, neither return to the sinus rhythm nor prevention of AF reinduction was observed after IV infusions of the vehicle: injections 1 and 2 (Fig. 2 ).
FIGURE 2: Effects of SSR149744C on vagal AF in anesthetized dogs. Vehicle and 3 mg/kg SSR149744C neither restored sinus rhythm nor prevented reinduction of AF in all 7 dogs; addition of 10 mg/kg SSR149744C 60 minutes later terminated AF in all dogs and prevented reinduction in 4 out of 7 dogs. Significant differences: *P ≤ 0.05 versus vehicle (Fisher exact test).
The dose of 3 mg/kg SSR149744C neither terminated nor prevented reinduction of AF in any of the 7 dogs evaluated (Fig. 2 ). Conversely, when given at the IV dose of 10 mg/kg following the dose of 3 mg/kg, SSR149744C terminated AF in all dogs (P < 0.05) and prevented reinduction in 4 out of 7 dogs (P = 0.07) (Fig. 2 ); the mean duration between the SSR149744C administration and the occurrence of cardioversion into sinus rhythm averaged 7.5 ± 0.8 minutes (n = 7). Although the experimental protocol was not suitable to allow a quantitative evaluation of the atrial fibrillation cycle length (AFCL), we systematically observed a slowing of AF (lengthening of AFCL) following the IV administration of 3 mg/kg SSR149744C.
Measurements of AERP after each AF episode showed that AERP remained statistically unchanged in the vehicle group (Fig. 3A ). In the treated group, where the predrug curve was not statistically different from that of the vehicle group, 3 mg/kg SSR149744C induced a slight and statistically nonsignificant lengthening of AERP. Addition of 10 mg/kg SSR149744C, however, prolonged AERP (P < 0.05 versus predrug and corresponding vehicle) at 400, 300, 250, and 200 milliseconds of BCL with a parallel shift of the curve (Fig. 3B ).
FIGURE 3: Changes in atrial refractory period (AERP) in anesthetized dogs. AERPs, measured at several stimulation cycle lengths, were not modified by vehicle injections (A). In the SSR149744C-treated groups, 3 mg/kg did not significantly modify AERPs, whereas 10 mg/kg lengthened AERPs with a parallel shift of the curve (B). Each data point represents the mean ± SEM (n = 7). Significant differences: *P < 0.05 versus vehicle, and °P < 0.05 versus predrug (3-way ANOVA on repeated measures followed by Dunnett test).
The determination of the spontaneous heart rate after each restoration of sinus control showed a significant, yet slight decrease in HR over time in the vehicle group and in the SSR149744C-treated group (Table 1 , Fig. 1 ). However, there was a trend for a greater reduction in HR in SR149744C-treated animals, particularly at 10 mg/kg with a significant effect compared with injection 2 of vehicle group (P < 0.05).
TABLE 1: Effects of SSR149744C on Sinusal Heart Rate (HR; b min−1 ) in Canine Model of Vagally AF
Low-K+ Medium-Induced AF in Guinea Pig Hearts
As shown in Figure 4 , sustained AF was observed in 94% (16/17) of vehicle (DMSO)-treated hearts, occurring within 19.0 minutes (CI 16-23) of perfusion with low-K+ Krebs solution (KCl = 1.4 mM). During perfusions of SSR149744C at concentrations of 0.1 μM and 0.3 μM, the incidence of AF was 83% and 60%, respectively (nonsignificant versus vehicle) with a mean time of AF induction of 20.5 (CI 18-28; ns) and 38.0 minutes (CI 29 to >45; P < 0.05). After perfusion of SSR149744C at the concentration of 1 μM, all hearts were protected against AF within the 45-minute period of observation.
FIGURE 4: Effects of SSR149744C on the prevention of AF induced by low-K+ medium in isolated guinea pig hearts: nonsignificant reductions of the incidence of AF were obtained at the two lowest concentrations, but 1 μM totally protected hearts from AF. Bar graph representing mean percentage AF inductions with number of observations of AF over number of hearts indicated above each bar; *P < 0.05 versus vehicle (Fisher exact test).
Experimental Models of Ventricular Arrhythmia
Reperfusion-Induced Arrhythmia in Anesthetized Rats
In the intravenous route study, the basal values of MAP and HR in the 5 groups, before SSR149744C administration, were not significantly different from those of the vehicle group (data not shown). Five minutes after injection, just before coronary ligature, the vehicle induced a slight but not statistically significant increase in MAP (from 116 ± 3 to 125 ± 2 mm Hg); 0.1 to 1 mg/kg SSR149744C did not modify MAP, whereas 3 and 10 mg/kg reduced MAP from 100 ± 8 to 96 ± 8 (−4%) and from 106 ± 9 to 79 ± 8 mm Hg (−25%) (P < 0.05 versus vehicle), respectively. Injection of the vehicle induced a slight yet significant decrease in HR (from 461 ± 10 to 430 ± 9 bpm; −7%). In comparison with the vehicle group, SSR149744C, at doses of 0.1, 0.3, 1, and 3 mg/kg, did not modify HR, whereas administration of 10 mg/kg significantly reduced HR from 452 ± 21 to 362 ± 12 bpm (−20%) (P < 0.05 versus vehicle).
Vehicle-treated animals exhibited a 100% incidence of reperfusion-induced premature ventricular beats, ventricular tachycardia, and a 90% incidence of ventricular fibrillation and mortality (Fig. 5 ). SSR149744C (0.1 to 10 mg/kg i.v.) reduced the incidence of ventricular arrhythmias and mortality in a dose-dependent relationship. The minimal effective dose was 0.3 mg/kg with a reduction in mortality from 90% to 33% (P < 0.05), and 10 mg/kg totally suppressed ventricular premature beats, VT, VF, and mortality during the reperfusion phase.
FIGURE 5: Effects of SSR149744C on the incidence of reperfusion-induced arrhythmias when administered intravenously 5 minutes before left coronary artery ligature in the anesthetized rat. Bar graphs indicate percentage of incidence of ventricular premature beats (VPB), tachycardia (VT), nonfatal fibrillation (VF), and mortality. SSR149744C (0.1 to 10 mg/kg IV) reduced and suppressed ventricular arrhythmias with a dose-dependent relationship. n = 10 (vehicle) and 6 (SSR149744C); *P < 0.05 versus vehicle (Fisher exact test).
In comparison with the vehicle group, 0.1, 0.3, 1, and 3 mg/kg SSR149744C did not significantly delay the onset of arrhythmias, but the higher dose of 10 mg/kg produced a delay greater than 600 seconds because there was no arrhythmia during the reperfusion phase (Table 2 ). Rhythm disturbance durations were significantly decreased by 1 and 3 mg/kg SSR149744C, whereas at the 10 mg/kg dose, the rhythm disturbance duration was equal to zero because of the absence of arrhythmia during the reperfusion phase.
TABLE 2: Effects of SSR149744C on Time to Onset of First Rhythm Disturbance (Delay) and on Duration of the Arrhythmias When Administered Intravenously (5 min) or Orally (120 min) Before Left Coronary Artery Ligature in the Rat
In the oral route study, after 2 hours of treatment and before coronary ligature, SSR149744C, in comparison to vehicle group, tended to decrease MAP at 30 and 90 mg/kg (−7% and −10% from 102 ± 6 mm Hg, respectively). For HR, a maximum effect was also obtained with the dose of 90 mg/kg: 461 ± 13 bpm in the vehicle group and 422 ± 12 bpm (−8%) in SSR149744C group. None of these differences were statistically significant.
In comparison to vehicle-treated animals, SSR149744C reduced the incidence of ventricular arrhythmias and mortality with a dose-dependent relationship. The minimal effective dose was 10 mg/kg PO with a reduction in mortality from 80% to 14% (P < 0.05) and in VF from 90% to 29% (P < 0.05), and at the 90-mg/kg dose, SSR149744C totally suppressed all ventricular arrhythmias during the reperfusion phase (Fig. 6 ).
FIGURE 6: Effects of SSR149744C on the incidence of reperfusion-induced arrhythmias when administered orally 2 hours before anesthesia and left coronary artery ligature in the rat. Bar graphs indicate percentage incidence of ventricular premature beats (VPB), tachycardia (VT), nonfatal fibrillation (VF), and mortality. SSR149744C (3 to 90 mg/kg PO) dose-dependently reduced and suppressed ventricular arrhythmias. n = 10 (vehicle), 6 (SSR 3, 30, and 90 mg/kg) and 7 (SSR 10 mg/kg); *P < 0.05 versus vehicle (Fisher exact test).
In comparison with the vehicle group, 3, 10, and 30 mg/kg SSR149744C (PO) did not significantly delay the onset of arrhythmias, but the 90 mg/kg dose delayed it by 600 seconds as there was no arrhythmia during the reperfusion phase (Table 2 ). Doses of 3, 10, and 30 mg/kg SSR149744C significantly decreased rhythm disturbance durations during this reperfusion phase. At the 90 mg/kg dose, the rhythm disturbance duration was equal to zero because of the absence of arrhythmia during the reperfusion phase.
Acute Sudden Death in Conscious PMI Rats
Occlusion of the left coronary artery under light anesthesia induced time-dependent occurrence of sudden cardiac deaths during the following 24 hours in conscious rats.15 Figure 7 .
FIGURE 7: Effects of SSR149744C on mortality during the first 24 hours after coronary artery occlusion in conscious rats. Bar graphs illustrate percentage mortality during three periods of observation (A, 0-0.5 hours; B, 0.5-6 hours; C, 6-24 hours) and 24 hours (D) in sham, vehicle, and SSR149744C groups. In the sham group there was only 1 death out of 13 rats, which occurred in the 0- to 0.5-hour period (A). In comparison to the vehicle group, where the mortality rate was high, SSR149744C administered orally at 30, 2 × 30, 2 × 45, and 90 mg/kg significantly decreased total mortality (D) at the two highest doses, mainly by conferring protection during the first two periods of observation (A and B); 30 mg/kg SSR149744C also reduced mortality in the 0.5- to 6-hour period (B). Number of deaths out of number of animals are indicated on each bar; *P < 0.05 versus vehicle (Fisher exact test).
In the vehicle group, 12 rats out of 55 (22%) died during the first 30 minutes, 18 rats out of 43 (42%) died during the 0.5- to 6-hour period, and 5 rats out of 25 (20%) died between 6 and 24 hours following coronary occlusion. In the sham group only 1 animal out of 13 died (8%) in the 0- to 0.5-hour period (Fig. 7 ).
Two hours after oral administration, SSR149744C clearly reduced early mortality during the 0- to 0.5-hour period: 30 mg/kg decreased mortality percentage to 5% (30 mg/kg group) and 6% (2 × 30 mg/kg group) compared with 22% in the vehicle group. SSR149744C at 45 mg/kg (2 × 45 mg/kg group) and 90 mg/kg totally prevented mortality (Fig. 7A ).
During the 0.5- to 6-hour period, the drug significantly reduced mortality from 42% in the vehicle group to 15%, 19%, 6%, and 4% for 30, 2 × 30, 2 × 45, and 90 mg/kg groups, respectively (Fig. 7B ). The late mortality in the 6- to 24-hour period was still present irrespective of the dose of SSR149744C (Fig. 7C ).
Administration of a second dose of 30 mg/kg 12 hours after the first (ie, 10 hours after coronary artery ligature) did not significantly change total mortality compared with the same dose given only once (41% versus 48%, respectively) but tended to reduce sudden death in the 6- to 24-hour period (23% versus 35%, respectively) (Fig. 7C ). In comparison to the dose of 90 mg/kg, experiments carried out with 2 × 45 mg/kg SSR149744C showed that the first administration of 45 mg/kg was as effective as the dose of 90 mg/kg on sudden death in both early periods (Fig. 7A,B ) and that the second administration of 45 mg/kg induced a reduction in late mortality, albeit not statistically significant compared with that observed with the dose of 90 mg/kg (19% versus 26%; Fig. 7C ).
Thus, SSR149744C reduced the total mortality with a dose-dependent relationship: beneficial effects were statistically nonsignificant at 30 and 2 × 30 mg/kg but significant and equipotent at 2 × 45 and 90 mg/kg (Fig. 7D ).
DISCUSSION
The present study of the acute antiarrhythmic effects of SSR149744C demonstrates that this new compound was effective by the IV or PO routes against experimentally induced atrial and ventricular arrhythmias. At the atrial level, SSR149744C restored sinus rhythm in a canine model of vagally mediated AF and prevented AF induced by low-K+ medium perfusion in the isolated guinea pig heart model. At the ventricular level, SSR149744C reduced reperfusion-induced arrhythmias in anesthetized rats and reduced early (0-24 hours) mortality following permanent left coronary artery ligature in conscious rats.
Compared with the results obtained in previous studies under similar experimental conditions (acute administration),11,20-22 Table 3 ). In the in vivo vagal AF model, SSR149744C was as potent as dronedarone and more effective (3 times) than amiodarone.22 + medium-induced sustained AF in isolated hearts in which amiodarone and dronedarone were ineffective at 1 μM (but effective in ex vivo conditions 2 hours after 90 mg/kg PO),21 11 20 Table 3 shows the minimal effective doses or concentrations of SSR149744C; it also shows that 0.3 μM is the in vitro minimal effective concentration, but it is difficult to compare with effective plasma concentrations in the in vivo situation because we did not measure this parameter. However, previous studies gave some pharmacokinetic data. After a 3 mg/kg IV injection, plasma concentration decreased in the first 30 minutes from 13 to 1.3 μM and from 22 to 3 μM in dogs and rats, respectively. After 30 mg/kg PO administration of SSR149744C, plasma concentration decreased from 11 μM (2 hours after administration) to 0.5 μM (24 hours after administration) in rats (C. Briot, unpublished data). All these observations suggest that we are in the same range of effective concentrations between the in vitro and in vivo animal arrhythmia models, which are close to the range of the IC50 values of SSR149744C against the majority of the ionic currents.10
TABLE 3: Comparison of the Antiarrhythmic Activities of SSR149744C, Amiodarone
The vagally mediated AF is a standard model in which several antiarrhythmic agents including D-sotalol, ambasilide, dofetilide, and azimilide have been shown to terminate AF induced by electric bursts during vagal stimulation in dogs.12,23
Because the drug did not modify the maximal cardiac cycle length response to vagal stimulation at the frequencies used in our experimental protocol, an antivagal action was unlikely to have been involved in the effect of SSR149744C. However, the drug reduced significantly the maximal cardiac cycle length at 6 and 8 Hz of vagal stimulation frequency (Fig. 1 ), and it inhibited IK(ACh) (IC50 = 0.09 μM) in guinea pig atria.10 24 K(ACh) -blocking effects, the anticholinergic action of SSR149744C may have positive consequences for its therapeutic use.
When the sinus rhythm was restored and vagal stimulation stopped, ERPs of the right atrium were dose-dependently lengthened by SSR149774C infusion. AERP prolongations for each dose were constant with a stimulation cycle length between 200 and 400 milliseconds, demonstrating a frequency-independent action on atrial refractoriness. These AERP prolongations may be caused by inhibitions of IKr , IKs , and IKur (or IKv1.5 ): currents present in canine right atrium25,26 10 Ks -induced action potential shortening at rapid rates.27
SSR149744C has Na+ channel-blocking activity,10 28 29 C drug (pilsicainide). Thus, the class I property of SSR149744C may also explain the termination of AF.
The model of low-K+ medium (1.4 mM)-induced sustained AF in isolated guinea pig hearts was firstly described by Manoach et al.13 30 31 32 + medium perfusion in guinea pig hearts, which induced subcellular alterations and heterogeneously impaired intercellular coupling. In guinea pig right atrium, we have observed that a reduction of extracellular K+ concentration from 4.0 mM to 1.4 mM hyperpolarized the resting potential (with appearance of a slow diastolic depolarization) and increased action potential amplitude and duration as well as contraction and spontaneous sinus cycle length (unpublished data). This positive inotropic effect in isolated atrium suggests an increase in intracellular Ca2+ concentration and a Ca2+ overload phenomena as described by Manoach et al13 10 2+ overload and reduce the automaticity of atrial cells and thus may have a cardioprotective action in this hypokalemic condition in hearts. However, AERP prolongations by the K+ repolarizing current-blocking properties of SSR149744C (discussed above) should not be excluded as a mechanism of the antiarrhythmic action in guinea pig.
In the rat ventricle, SSR149744C administered IV and PO exhibited significant antiarrhythmic properties against arrhythmias caused by sudden reperfusion of the ischemic myocardium. Although the exact mechanisms of reperfusion-induced arrhythmias are not fully understood, intracellular Na+ and Ca2+ loading have been implicated as contributing to reperfusion arrhythmogenesis.33-35 + channel blocker (class IB antiarrhythmic agent property as do lidocaine and amiodarone) and an L-type Ca2+ channel blocker (class IV property),10 + and Ca2+ influx. Class III agents, particularly the pure class IKr blockers such as d -sotalol, sematilide, and dofetilide, have less activity than class I and IV agents against reperfusion-induced arrhythmias in rats. However dl -sotalol reduces arrhythmias, indicating that β-blocking effects or a class II property may be antiarrhythmic in this model.36,37 10 Kr -blocking activity of SSR149744C may also be involved in its mechanism of action. However, recently, Sarraf et al38 B (lidocaine) actions are more effective than either agent alone for suppression of ischemia-induced arrhythmias in rats. This result suggests that combinations of its several antiarrhythmic class properties should be taken into account in the antiarrhythmic mechanism of action of SSR149774C.
Irreversible coronary artery ligature is accompanied by a rapid increase in electrical instability, often leading to fatal arrhythmia. In conscious rats implanted with an ECG telemetry system, Opitz et al15 animal model , we divided the observation period into 3 periods: the 2 first, 0-0.5 and 0.5-6 hours, corresponding to fatal arrhythmogenic periods, and the third, 6-24 hours, also an important phase where animal death is much less common in the untreated group but may become more frequent with short-acting antiarrhythmic agents, which may shift the mortality curve. A previous study has shown that amiodarone (30 and 90 mg/kg PO) and dronedarone (90 mg/kg PO) reduced total mortality in this sudden death model mainly by prevention of sudden death in the 0.5- to 6-hour period.20 15,39,40
The decreases in MAP and HR by SSR149744C that reduce myocardial O2 consumption (cardiac work) may explain the beneficial effects during permanent ischemia or ischemia-reperfusion. We cannot totally exclude these protective effects in its mechanisms of action; however, we observed a clear reduction of arrhythmias by the drug in the reperfusion model at doses (0.3-1 mg/kg IV or 10-90 mg/kg PO), which did not significantly change MAP and HR. Moreover, a relatively severe hypotension resulting in a decrease of the coronary perfusion may worsen myocardial oxygen supply-demand imbalance and thus favor arrhythmias. During this experiment in the reperfusion-induced arrhythmia model, we did not observe an increase in arrhythmias induced by SSR149744C even at the hypotensive dose of 10 mg/kg IV, which, on the contrary, totally suppressed arrhythmias.
Study Limitations
Our study has certain limitations for several reasons. First, we used an experimental model in which AF was induced by vagal stimulation. Atrial properties in vagotonic dogs are different from those in the diseased, dilated, and electrical remodeling atria as seen in humans AF.41 24
Second, AERP is measured only at 1 atrial site and after the vagal stimulation period. It is well known that the refractoriness in the atrium is regionally nonuniform and that vagal activity significantly increases this heterogeneity.
Third, the use of a low-K+ medium of 1.4 mM to induce spontaneous and sustained AF would not be expected to exist clinically. Thus, a low-K+ concentration may affect the action of SSR149744C in a manner that it would not occur under clinical conditions.
Fourth, the ion channels involved in repolarizing phase in dog and guinea pig atria and particularly in rat ventricle are different from those of human atrium and ventricle, respectively. It should be noted that amiodarone, a potent antiarrhythmic in human atrial and ventricular arrhythmias, is effective in the animal models used in this experiment.
Fifth, in animal studies with amiodarone, for which the effective therapeutic range in man is 1-2.5 mg/L (1.5-3.8 μM),42 43,44
CONCLUSION
In summary, the present paper is the first description of the antiarrhythmic profile of a new noniodinated benzofuran derivative. SSR149744C is effective in preventing and terminating hypokalemic and vagotonic AF. At the ventricular level, it suppresses reperfusion-induced arrhythmias after intravenous or oral treatment and reduces early mortality of conscious PMI rats. These results demonstrate that SSR149744C has atrial and ventricular antiarrhythmic activities, likely to result from its class I, II, III and IV antiarrhythmic actions described previously10
Thus, SSR149744C possesses potent antiarrhythmic properties in experimental animal models, which should provide therapeutic advantages (safety/efficacy) in humans over its parent compound, amiodarone.
ACKNOWLEDGMENTS
The authors are grateful to M. Lemallam, R. Libon, P. P. Mattei, D. Oliva-Briand, J. Planchenault, and G. Rizzoli, for their technical assistance and to Drs. Isabel Ann Lefevre, Stephen-Eric O'Connor and John Alexander for their help in reviewing the manuscript .
REFERENCES
1. Gill J, Heel RC, Fitton A. Amiodarone. An overview of its pharmacological properties, and review of its therapeutic use in cardiac arrhythmias.
Drugs . 1992;43:69-110.
2. Singh BN. Antiarrhythmic drugs: re-orientation in light of recent developments in the control of disorders of rhythm.
Am J Cardiol . 1998;81:3D-11D.
3. Charlier R. Cardiac actions in the dog of a new antagonist of adrenergic excitation which does not produce competitive blockade of adrenoceptors.
Br J Pharmacol . 1970;39:668-674.
4. Polster P, Broekhuysen J. The adrenergic antagonism of amiodarone.
Biochem Pharmacol . 1976;25:131-134.
5. Mason JW, Hondeghem LM, Katzung BG. Amiodarone blocks inactivated cardiac sodium channels.
Pflugers Arch . 1983;396:79-81.
6. Nishimura M, Follmer CH, Singer DH. Amiodarone blocks calcium current in single guinea pig ventricular myocytes.
J Pharmacol Exp Ther . 1989;251:650-659.
7. Kodama I, Kamika K, Toyama J. Cellular electropharmacology of amiodarone.
Cardiovasc Res . 1997;35:13-29.
8. Vaughan Williams EM. A classification of antiarrhythmic actions reassessed after a decade of new drugs.
J Clin Pharmacol . 1984;24:129-147.
9. Podrid PJ. Amiodarone: reevaluation of an old drug.
Ann Intern Med . 1995;122:689-700.
10. Gautier P, Guillemare E, Djandjighian L, et al.
In vivo and
in vitro characterization of the novel antiarrhythmic agent SSR149744C: electrophysiological, anti-adrenergic and anti-angiotensin II effects.
J Cardiovasc Pharmacol . 2004;44:244-257.
11. Manning AS, Bruyninckx C, Ramboux J, et al. SR33589, a new amiodarone-like agent: effect on ischemia- and reperfusion-induced arrhythmias in anesthetized rats.
J Cardiovasc Pharmacol . 1995;26:453-461.
12. Nattel S, Liu L, St-Georges D. Effects of the novel antiarrhythmic agent azimilide on experimental atrial fibrillation and atrial electrophysiologic properties.
Cardiovasc Res . 1998;37:627-635.
13. Manoach M, Varon D, Tribulova N, et al. Hypokalemia as a simple reproducible model for sustained atrial fibrillation.
J Mol Cell Cardiol . 1998;30:A4.
14. Manning AS, Coltart DJ, Hearse DJ. Ischemia and reperfusion-induced arrhythmias in the rat. Effects of xanthine oxidase inhibition with allopurinol.
Circ Res . 1984;55:545-548.
15. Opitz CF, Mitchell GF, Pfeffer MA, et al. Arrhythmias and death after coronary artery occlusion in the rat. Continuous telemetric ECG monitoring in conscious, untethered rats.
Circulation . 1995;92:253-261.
16. Cosnier-Pucheu S, Roccon A, Rizzoli G, et al. SSR149744C, a new antiarrhythmic drug, prevents experimental induced atrial fibrillation.
Eur J Heart Fail . 2003;2:53.
17. Kane KA, Parratt JR, Williams FM. An investigation into the characteristics of reperfusion-induced arrhythmias in the anaesthetized rat and their susceptibility to antiarrhythmic agents.
Br J Pharmacol . 1984;82:349-357.
18. Walker MJA, Curtis MJ, Hearse DJ, et al. The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia, infarction, and reperfusion.
Cardiovasc Res . 1988;22:447-455.
19. Fabiani JN, Deloche A, Camilleri JP, et al. Protocole d'étude de l'ischémie aiguë du myocarde chez le rat.
Ann Chir Thorac Cardiovasc . 1977;16:167-171.
20. Cosnier S, Guiraudou P, Grosjean J, et al. Amiodarone and dronedarone reduce early mortality in post MI rats.
Fund Clin Pharmacol . 1999;13:73s.
21. Cosnier-Pucheu S, Guiraudou P, Rizzoli G, et al. Effects of amiodarone and dronedarone in the prevention of atrial arrhythmias in guinea pig isolated heart.
Arch Mal Coeur Vaiss . 2001;94:21.
22. Finance O, Planchenault J, Bethegnies S, et al. Electrophysiological and anti-arrhythmic actions of a new amiodarone-like agent, dronedarone, in experimental atrial fibrillation.
J Mol Cell Cardiol . 1998;30:A251.
23. Wang J, Feng J, Nattel S. Class III antiarhythmic drug action in experimental atrial fibrillation.
Circulation . 1994;90:2032-2040.
24. Coumel P, Leclercq JF, Attuel P, et al. Autonomic influence in the genesis of atrial arrhythmias: atrial flutter and fibrillation of vagal origin. In: Narula DS ed.
Cardiac arrhythmias: electrophysiology, diagnosis and management . Baltimore: Williams & Wilkins. 1979:243-255.
25. Li D, Zhang L, Kneller J, et al. Potential ionic mechanism for repolarization differences between canine right and left atrium.
Circ Res . 2001;88:1168-1175.
26. Fedida D, Eldstrom J, Hesketh JC, et al. K
v 1.5 is an important component of repolarizing K
+ current in canine atrial myocytes.
Circ Res . 2003;93:744-751.
27. Jurkiewicz NK, Sanguinetti MC. Rate-dependent prolongation of cardiac action potentials by a methanesulfonamide class III antiarrhythmic agent. Specific block of rapidly activating delayed rectifier K
+ current by dofetilide.
Circ Res . 1993;72:75-83.
28. Nattel S, Kneller J, Zou R, et al. Mechanisms of termination of atrial fibrillation by class I antiarrhythmic drugs: evidence from clinical, experimental and mathematical modeling studies.
J Cardiovasc Electrophysiol . 2003;14:S133-S139.
29. Hayashi H, Fujiki A, Tani M, et al. Different effects of class Ic and III antiarrhythmic drugs on vagotonic atrial fibrillation in the canine heart.
J Cardiovasc Pharmacol . 1998;31:101-107.
30. Weissenburger J, Davy JM, Chezalviel F. Experimental models of torsades de pointes.
Fund Clin Pharmacol . 1993;7:29-38.
31. Helfant RH. Hypokalemia and arrhythmias.
Am J Med . 1986;80:13-22.
32. Tribulova N, Manoach M, Varon D, et al. Hypokalemia-induced ultrastructural, histochemical and connexin-43 alterations resulting in atrial and ventricular fibrillations.
Gen Physiol Biophys . 1999;18(Suppl):15-18.
33. Tani M, Shinmura K, Hasegawa H, et al. Effects of methylisobutyl amiloride on [Na+]i, reperfusion arrhythmias, and function in ischemic rat hearts.
J Cardiovasc Pharmacol . 1996;27:794-801.
34. Sweies J, Omogbai EK, Smith GM. Occlusion and reperfusion-induced arrhythmias in rats: involvement of platelets and effects of calcium antagonists.
J Cardiovasc Pharmacol . 1990;15:816-825.
35. Lu HR, Yang P, Remeysen P, et al. Ischemia/reperfusion arrhythmias in anaesthetized rats: a role of Na
+ and Ca
2+ influx.
Eur J Pharmacol . 1999;365:233-239.
36. Brooks RR, Carpenter JF, Miller KE, et al. Efficacy of the class III antiarrhythmic agent azimilide in rodent models of ventricular arrhythmia.
Proc Soc Exp Biol Med . 1996;212:84-93.
37. Chen J, Komori S, Li B, et al. IK independent class III actions of MS-551 compared with sematilide and dofetilide during reperfusion in anaesthetized rats.
Br J Pharmacol . 1996;119:937-942.
38. Sarraf G, Barrett TD, Walker MJA. Tedisamil and lidocaine enhance each other's antiarrhythmic activity against ischemia-induced arrhythmias in rats.
Br J Pharmacol . 2003;139:1389-1390.
39. Curtis MJ, Macleod BA, Walker MJ. Models for the study of arrhythmias in myocardial ischemia and infarction: the use of the rat.
J Mol Cell Cardiol . 1987;19:399-419.
40. Clements-Jewery H, Hearse DJ, Curtis MJ. Independent contribution of catecholamines to arrythmogenesis during evolving infarction in the isolated rat heart.
Br J Pharmacol . 2002;135:807-815.
41. Nattel S, Bourne G, Talajic M. Insights into mechanisms of antiarrhythmic drug action from experimental models of atrial fibrillation.
J Cardiovasc Electrophysiol . 1997;8:469-480.
42. Gill J, Heel RC, Fitton A. Amiodarone. An overview of its pharmacological properties, and review of its therapeutic use in cardiac arrhythmias.
Drugs . 1992;43:69-110.
43. Merot J, Charpentier F, Poirier JM, et al. Effects of chronic treatment by amiodarone on transmural heterogeneity of canine ventricular repolarization in vivo: interaction with acute sotalol.
Cardiovasc Res . 1999;44:303-314.
44. Varro A, Takacs J, Nemeth M, et al. Electrophysiological effects of dronedarone, a noniodinated amiodarone derivative in the canine heart: comparison with amiodarone.
Br J Pharmacol . 2001;133:625-634.
