Circ J 2008; 72: 1003–1011

Comparison of Electropharmacological Effects of Bepridil and Sotalol in Halothane-Anesthetized Dogs

Tomomichi Ishizaka, DVM*,**; Akira Takahara, PhD*; Hiroshi Iwasaki, MD*; Yoshitaka Mitsumori, MD*; Hiroaki Kise, MD*; Yuji Nakamura, MS*; Atsushi Sugiyama, MD*,**

Background Bepridil is known to have a multiple ion channel-blocking property in the heart, which has been applied for the treatment of atrial fibrillation and drug-refractory ventricular tachyarrhythmias. In this study, the electro-pharmacological effects of bepridil were compared with those of dl-sotalol, a representative class III anti- arrhythmic drug, using the halothane-anesthetized canine model. Methods and Results Cardiovascular and electrophysiological variables were measured under the halothane anesthesia. Intravenous administration of bepridil (0.3mg/kg, n=4) delayed the intraventricular conduction and prolonged the ventricular effective refractory period, whereas dl-sotalol (0.3mg/kg, iv, n=4) inhibited atrioven- tricular conduction and prolonged the atrial and ventricular effective refractory period. The additional adminis- tration of 10 times the higher dose of bepridil or dl-sotalol (ie, 3mg/kg, iv, n=4 for each group) decreased blood pressure, suppressed ventricular contraction and sinus automaticity, and prolonged the atrial and ventricular effective refractory period and monophasic action potential duration, in addition to the effects of the low dose. Conclusions The electropharmacological effects of bepridil and dl-sotalol were similar, although their potency for each cardiovascular variable varied significantly. These findings can be useful when selecting these drugs according to the pathophysiological condition of a patient. (Circ J 2008; 72: 1003–1011) Key Words: Bepridil; Electrophysiological effects; Hemodynamics; dl-Sotalol

epridil was initially introduced as an anti-anginal (β-adrenoceptor and IKr blocker) in terms of negative drug possessing a Ca2+ channel-blocking proper- chronotropic, inotropic and dromotropic actions, as well as B ty.1,2 The drug has been shown to also inhibit the effects on atrial and ventricular refractoriness.17,18 To clarify cardiac Na+ and K+ channels, which can prolong the atrial any similarity and differences in the cardiovascular pharma- and ventricular effective refractory periods.3–6 Recent clini- cological profile between the 2 drugs, in the present study we cal studies in Japan demonstrated that bepridil is efficacious precisely assessed the in vivo effects of bepridil in compari- against atrial fibrillation or drug-refractory ventricular son with a representative class III antiarrhythmic, drug dl- tachyarrhythmias.7–11 In vitro electrophysiological studies sotalol, on the cardiac repolarization process, together with have indicated that bepridil can block various types of K+ multiple cardiohemodynamic and electrophysiological vari- channels, including a rapid component of delayed rectifier ables. We used the halothane-anesthetized canine model in K+ currents (IKr),6 ultra-rapid component of delayed rectifier this study, which is useful in simultaneously analyzing in K+ current (IKur),12 slow component of delayed rectifier K+ vivo cardiovascular and electrophysiological profiles of current (IKs),6 transient outward current (Ito),13 inward recti- various drugs.19–24 fier K+ current (IK1) and muscarinic acetylcholine receptor- operated K+ current (IK,ACh).14 The in vivo cardiovascular and electrophysiological Methods profile of bepridil has been analyzed in comparison with Animals were obtained through the Animal Laboratory classical Ca2+ channel blockers such as verapamil and for Research, University of Yamanashi. All experiments diltiazem,15,16 because bepridil belongs to class IV anti- were performed according to the Guidelines for Animal arrhythmics. However, the pharmacological profile is con- Experiments, University of Yamanashi. Eight beagle dogs of sidered to be fundamentally similar to that of dl-sotalol either sex, weighing approximately 10kg, were divided into 2 animal groups: bepridil group (n=4) or dl-sotalol group (n=4). Dogs were initially anesthetized with thiopental (Received September 5, 2007; revised manuscript received December 19, 2007; accepted January 7, 2008) sodium (30mg/kg, iv). After intubation with a cuffed endo- *Department of Pharmacology, Interdisciplinary Graduate School tracheal tube, 1% halothane vaporized with 100% oxygen of Medicine and Engineering, University of Yamanashi, Chuo, was inhaled with a volume-limited ventilator (SN-408-3; **Yamanashi Research Center of Clinical Pharmacology, Fuefuki, Shinano, Tokyo, Japan). Tidal volume and respiratory rate Japan were set at 20ml/kg and 15strokes/min, respectively. Mailing address: Atsushi Sugiyama, MD, Department of Pharmacol- ogy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo 409-3898, Japan. Measurement of Cardiohemodynamic Parameters E-mail: [email protected] A heparinized catheter was inserted through the right All rights are reserved to the Japanese Circulation Society. For per- femoral artery for continuous monitoring of the systemic missions, please e-mail: [email protected] blood pressure. A thermodilution catheter (TC-704; Nihon-

Circulation Journal Vol.72, June 2008 1004 ISHIZAKA T et al.

Kohden, Tokyo, Japan) was positioned at the right side of stimulus of various coupling intervals. The duration of the the heart via the right femoral vein. The cardiac output was terminal repolarization period of the ventricle was calcu- measured by the standard thermodilution method, using a lated by the difference between the MAP90(CL400) and the cardiac output computer (MFC-1100; Nihon-Kohden). effective refractory period at the same site, which reflects Total peripheral vascular resistance was calculated using the extent of the electrical vulnerability of the ventricular the basic equation: Mean blood pressure/Cardiac output. A muscle.27 pig-tail catheter was positioned at the left ventricle through the right femoral artery to measure the left ventricular pres- Experimental Protocol sure (LVP). The maximal upstroke velocity of the LVP (ie, The cardiohemodynamic and electrophysiological param- LVdP/dtmax) and left ventricular end-diastolic pressure eters were monitored continuously using a polygraph sys- (LVEDP) were recorded during the sinus rhythm to estimate tem (RM-6000; Nihon-Kohden) and recorded on a thermal the contractility and preload of the left ventricle, respec- array recorder (WS-682G; Nihon-Kohden). These parameters tively. were analyzed using a real-time full automatic data analysis system (MP/VAS 3 for Macintosh ver.1.0; Physio-Tech, Measurement of Electrophysiologic Parameters Tokyo, Japan). Each measurement of the electrocardiogram, The surface lead II electrocardiogram was obtained from monophasic action potential signals, atrio-His and His-ven- the limb electrodes. Corrected QT intervals (QTc) were cal- tricular intervals was the mean of 3 recordings of consecu- culated using Bazett’s formula25 [QTc(B)=QT/(60/heart tive recordings. The cardiovascular variables were assessed rate)1/2], and Van de Water’s formula26 [QTc(V)=QT–87 in the following order at each dose. The electrocardiogram, (60/heart rate–1)], whereby a unit is given in seconds. A His-bundle electrogram, systemic and LVP, and monopha- quad-polar electrodes catheter was positioned at the non- sic action potential signals were recorded under the sinus coronary cusp of the aortic valves through the left femoral rhythm. The cardiac output was measured twice. The mono- artery to obtain the His-bundle electrogram. Another quad- phasic action potential signals were recorded during the polar electrodes catheter was positioned at the high right ventricle pacing at a cycle length of 400 and 300ms. The atrium through the right femoral vein for electrically pacing sinus node recovery time was assessed twice. Finally, the the right atrium. A bi-directional steerable monophasic Wenckebach block pacing cycle length, functional refrac- action potential recording/pacing combination catheter tory period of the atrioventricular node, and effective (1675P; EP Technologies Inc, Sunnyvale, CA, USA) was refractory period of the atrium and ventricle were measured positioned at the endocardium of the interventricular sep- as described above. tum in the right ventricle through the left femoral vein to After the basal assessment, bepridil was intravenously obtain the monophasic action potential signals, in addition administered at a low dose of 0.3mg/kg over 10min, and to electrically pacing the right ventricle. The signals were each parameter was assessed at 10, 20 and 30min after the amplified with a DC preamplifier (300, EP Technologies start of the drug infusion. Then, additional bepridil was Inc). administered at a high dose of 3mg/kg over 10min, and the The right atrium or right ventricle was electrically driven cardiovascular variables were assessed at 10, 20, 30, 45 and using a cardiac stimulator (SEC-3102; Nihon-Kohden) 60min after the start of the drug infusion. The effects of through the pacing electrodes catheter. The stimulation dl-sotalol at doses of 0.3 and 3mg/kg were assessed in a pulses were rectangular in shape, 1–2V (ie, about twice the manner similar to that of another series of experiments. threshold voltage) and 1ms in duration. The sinus node recovery time was obtained as a pause (in ms) from the last Assessment of theβ-Blocking Action paced atrial depolarization to the first sinus return cycle In the dl-sotalol group, 50ng/kg of isoproterenol, a β- after the electrical pacing of the right atrium for 30s at a adrenoceptor agonist, was intravenously injected before cycle length of 300 ms. The Wenckebach block pacing and 30min after the dl-sotalol was given. The extent of its cycle length was obtained as a length (in ms) at which a β-blocking activity was estimated by the inhibition of the Wenckebach phenomenon occurred. The effective refrac- isoproterenol-induced tachycardia and hypotension.22 tory period of the atrium was assessed by a pacing protocol that consisted of 5beats of basal stimuli in a cycle length of Measurement of the Plasma Drug Concentration 400ms followed by an extra stimulus of various coupling A volume of 3ml of blood was drawn from the left femo- intervals. The functional refractory period of the atrioven- ral artery to measure the plasma drug concentration before tricular node was obtained as the minimal interval between and at 10, 20 and 30min after the start of the low-dose infu- 2 consecutively conducted impulses through the atrioven- sion, and at 10, 20, 30 and 60min after the start of the high- tricular node, which was calculated from the curves relating dose infusion. The plasma was prepared from the blood sam- A1–A2 and H1–H2 intervals, as described elsewhere.22 ples, which were centrifuged at 1,500g for 30min at 4°C. The duration of the monophasic action potential signals The plasma was then stored at –80°C until the drug concen- was measured as an interval, along a line horizontal to the tration was measured. The plasma concentration of bepridil diastolic baseline, from the monophasic action potential was determined by LC/MS/MS (HPLC: Agilent 1100® upstroke to the desired repolarization level, and the interval Quaternary system, Agilent Technologies Japan, Ltd, Tokyo, (in ms) at 90% repolarization was defined as MAP90. In the Japan; MS/MS: API 4000, Applied Biosystems/MDS present study, the MAP90 was measured during the sinus SCIEX, Foster City, CA, USA). The plasma concentration rhythm (MAP90(sinus)) and at a pacing cycle length of of dl-sotalol was also measured by LC/MS/MS (HPLC: 400 ms (MAP90(CL400)) and 300 ms (MAP90(CL300)). The Agilent 1100® Quaternary system; MS/MS: Quattro Ultima, effective refractory period of the ventricle was assessed by Nihon Waters K.K., Tokyo, Japan). These measurements a programmed electrical stimulation to the right ventricle, were conducted at the laboratory of Hitachi High-Tech of which the pacing protocol consisted of 5beats of basal Science Systems, Ltd (Ibaraki, Japan). stimuli in a cycle length of 400ms followed by an extra

Circulation Journal Vol.72, June 2008 Comparison of Bepridil and Sotalol in Dogs 1005

Fig1. Time-courses of the (A) plasma drug concen- trations, and (B) heart rate (HR) and mean blood pres- sure (MBP) in the bepridil group (Left, n=4) and dl- sotalol group (Right, n=4). The inset figure shows the effects of dl-sotalol on the isoproterenol (ISO)-induced increase in HR and decrease in MBP (n=4). Data are presented as the mean±SEM. Closed symbols represent significant differences (p<0.05) from the corresponding pre-drug control value (C) for each parameter.

Fig2. Time-courses of maximum upstroke velocity of left ventricular pressure (LVdP/dtmax), left ventricular end-diastolic pressure (LVEDP), cardiac output (CO) and total peripheral vascular resistance (TPR) in the bepridil group (Left, n=4) and dl-sotalol group (Right, n=4). Data are presented as the mean±SEM. Closed symbols represent significant differences (p<0.05) from the corresponding pre-drug control value (C) of each parameter.

Circulation Journal Vol.72, June 2008 1006 ISHIZAKA T et al.

Fig 3. Typical tracings of the His-bundle electrogram (His), surface lead II electrocardio- gram (ECG) and monophasic action potentials (MAP) recorded from the right ventricle during the sinus rhythm. (A) Pre-drug control (Con- trol; Top) and 30min after the start of 3mg/kg of bepridil infusion (Bottom). (B) Pre-drug con- trol (Control; Top) and 30min after the start of 3mg/kg of dl-sotalol infusion (Bottom). HR, heart rate.

Drugs Plasma Drug Concentration Bepridil (molecular weight=366.54) and dl-sotalol (mo- The time-courses of the plasma drug concentrations of lecular weight=272.08) were extracted from the commer- bepridil (n=4) and dl-sotalol (n=4) are summarized in cial source Bepricor® (Daiichi Sankyo, Tokyo, Japan) and Fig1A. The peak plasma concentrations of bepridil after ad- Sotacor® (Bristol-Myers Squibb, Tokyo, Japan), respec- ministration at the low and high dose were 572±178ng/ml tively. They were dissolved in 0.9% saline, giving concen- (=1.6μmol/L) and 1,613±117ng/ml (=4.4μmol/L), respec- trations of 0.3 and 3mg/ml. These drugs were administered tively, and those of dl-sotalol were 1,156±148 ng/ml intravenously at a volume of 1ml/kg. The following drugs (=4.2μmol/L) and 12,052±944ng/ml (=44.3μmol/L), re- were used: isoproterenol (Kowa Pharmaceutical, Tokyo, spectively. Japan), thiopental sodium (Tanabe Seiyaku Co, Ltd, Osaka, Japan), halothane (Takeda Chemical Industries, Osaka, Effects on Hemodynamic Parameters Japan), and heparin calcium (Mitsui Pharmaceuticals Co, The time-courses of changes in the heart rate, mean blood Ltd, Tokyo, Japan). pressure, LVdP/dtmax, LVEDP, cardiac output and total pe- ripheral vascular resistance are summarized in Figs1B and Statistical Analysis 2. Their pre-drug control values (C) were 102±9beats/min, Data are presented as the mean±SEM. The statistical sig- 114±3 mmHg, 2,386±167 mmHg/s, 13±2 mmHg, 1.47± nificances within a parameter were evaluated by one-way, 0.15 L/min and 80±8 mmHg·(L/min)–1 for bepridil and repeated-measures analysis of variance, followed by Con- 130±12beats/min, 124±9mmHg, 3,000±159mmHg/s, 9± tract for mean values comparison, whereas those between 2mmHg, 1.95±0.21L/min and 65±4mmHg·(L/min)–1 for the groups were evaluated by unpaired t-test. A p-value of dl-sotalol, respectively. <0.05 was considered statistically significant. No significant change was detected in the bepridil group after the low dose for any of the hemodynamic parameters. The high dose decreased the heart rate for 20–60min, mean Results blood pressure at 10min and total peripheral vascular resis- There was no statistically significant difference in the re- tance at 10min, and increased the cardiac output at 10min. spective control values between the bepridil-administered The LVdP/dtmax increased at 10min and decreased at 20min group and the dl-sotalol-administered group except for after the high dose. No significant change was detected in LVdP/dtmax, QT interval, QTc(V) or MAP90. the LVEDP at either dose. In the dl-sotalol group, the low dose decreased the LVdP/dtmax for 10–30 min and cardiac output for 10–

Circulation Journal Vol.72, June 2008 Comparison of Bepridil and Sotalol in Dogs 1007

Fig4. Time courses of the PR interval (circles), QRS width (squares), QT interval (triangles) and QTc (trian- gles: Bazett; circles: Van de Water), duration of mono- phasic action potential at a level of 90% repolarization during the sinus rhythm (MAP90(sinus), circles), atrio- His interval (AH, circles) and His-ventricular interval (HV, squares) in the bepridil group (Left, n=4) and dl- sotalol group (Right, n=4). Data are presented as the mean ± SEM. Closed symbols represent significant differences (p<0.05) from the corresponding pre-drug control value (C) of each parameter.

20min, and increased the LVEDP at 10 and 30min, whereas interval increased at 10min after the low dose. After the no significant change was detected in the heart rate, mean high dose, the PR interval further increased for 10–60min, blood pressure or total peripheral vascular resistance after and the QT interval, QTc(B) and QTc(V) were prolonged the low dose. The high dose decreased the heart rate, mean for 10–60min after the high dose. Nonetheless, no signifi- blood pressure, LVdP/dtmax and cardiac output for 10– cant change was detected in the QRS width throughout the 60min and increased the LVEDP, but total peripheral vas- experiment. cular resistance increased for 10–60min. Effect on the His-Bundle Electrogram and MAP90 During Beta-Blocking Action of dl-Sotalol the Sinus Rhythm The effects of dl-sotalol on the isoproterenol-induced Typical tracing of the His-bundle electrogram and MAP cardiovascular responses are shown in the inset of Fig1B signal are depicted in Fig3, and the time-courses of changes (Right panel). At pre-drug control (C), iv injection of in the atrio-His and His-ventricular intervals and MAP90(sinus) isoproterenol increased the heart rate by 55beats/min and during the sinus rhythm are summarized in Fig4. Their pre- decreased the mean blood pressure by 44mmHg. dl-Sotalol drug control values (C) were 75±6, 27±1 and 249±11ms suppressed the isoproterenol-induced cardiovascular re- for bepridil and 64±3, 27±2 and 208±12ms for dl-sotalol, sponses in a dose-dependent manner. respectively. In the bepridil group, no significant change was detected Effects on the ECG Parameters During the Sinus Rhythm in the His-ventricular interval or MAP90(sinus) after the low Typical tracing of the ECG during the sinus rhythm are dose. After the high dose, the His-ventricular interval and depicted in Fig3, and the time courses of changes in the ECG MAP90(sinus) were prolonged at 20, 45 and 60min and for parameters are summarized in Fig4. The pre-drug control 20–60min, respectively. Meanwhile, no significant change values (C) of the PR interval, QRS width, QT interval, was detected in the atrio-His interval. In the dl-sotalol QTc(B) and QTc(V) were 104±9, 57±4, 286±15, 369±11 group, no significant change was detected in the atrio-His and 320±12ms for bepridil, and 91±3, 54±3, 233±11, 341± interval or MAP90(sinus) after the low dose. After the high 17 and 279±10ms for dl-sotalol, respectively. dose, the atrio-His interval and MAP90(sinus) were prolonged In the bepridil group, the QRS width and QTc(B) in- for 10–60min. However, no significant change was detected creased for 20–30min and 10–30min, respectively, after the in the His-ventricular interval. low dose. After the high dose, the QRS width, QT interval, QTc(B) and QTc(V) increased for 10–60min. Nonetheless, Effect on the Sinus Nodal and Atrioventricular Nodal no significant change was detected in the PR interval Functions throughout the experiment. In the dl-sotalol group, the PR The time-course of change in the sinus node recovery

Circulation Journal Vol.72, June 2008 1008 ISHIZAKA T et al.

Fig5. Time-courses of the sinus node recovery time (SNRT), functional refractory period of the atrioven- tricular node (FRP-AV) and Wenckebach block pacing cycle (WBB-PCL) in the bepridil group (Left, n=4) and dl-sotalol group (Right, n=4). Data are presented as the mean ± SEM. Closed symbols represent significant differences (p<0.05) from the corresponding pre-drug control value (C) of each parameter.

Fig6. Time-courses of the effective refractory period of the right atrium (AERP), effective refractory period of the right ventricle (VERP), MAP90 during the ven- tricular pacing at a cycle length of 400ms (MAP90(CL400), circles) and 300ms (MAP90(CL300), squares) and termi- nal repolarization period (TRP) in the bepridil group (Left, n=4) and dl-sotalol group (Right, n=4). Data are presented as the mean±SEM. Closed symbols represent significant differences (p<0.05) from the corresponding pre-drug control value (C) of each parameter. PCL, pacing cycle length. time, the Wenckebach block pacing cycle length and func- tricular node after the low dose. The high dose increased the tional refractory period of the atrioventricular node are sum- sinus node recovery time for 10–45min, and Wenckebach marized in Fig5. Their pre-drug control values (C) were block pacing cycle length and functional refractory period 583±32, 243±21 and 276±9ms for bepridil, and 537±26, of the atrioventricular node for 10–60min. 208±10 and 243±11ms for dl-sotalol. In the dl-sotalol group, the low dose increased the In the bepridil group, no significant change was detected Wenckebach block pacing cycle length and functional re- in the sinus node recovery time, Wenckebach block pacing fractory period of the atrioventricular node for 10–30min, cycle length or functional refractory period of the atrioven- whereas no significant change was detected in the sinus

Circulation Journal Vol.72, June 2008 Comparison of Bepridil and Sotalol in Dogs 1009

Table 1 Summary of Hemodynamic and Electrophysiological Effects Bepridil dl-Sotalol Low dose High dose Low dose High dose ∆Blood pressure (mmHg) –3±4 –20±5* –4±3 –10±2 ∆Heart rate (beats/min) 0±5 –16±4 –13±6 –35±8 ∆LVdP/dtmax (mmHg/s) 44±137* PIE: 391±29*, –639±76 –856±160 NIE: –292±84* ∆LVEDP (mmHg) 1±3–3±1* 3±15±1 ∆Cardiac output (L/min) 0.12±0.07 0.40±0.13* –0.17±0.12 –0.44±0.14 ∆FRP-AV (ms) 6±4* 73±17 25±5 74±1 ∆Atrial ERP(PCL400) (ms) 13±3 53±6 11±1 60±7 ∆Ventricular ERP(PCL400) (ms) 10±4 55±4 15±3 54±4 ∆Ventricular MAP90(PCL400) (ms) 18±9 60±14 4±350±9 ∆Ventricular MAP90(PCL300) (ms) 17±6 61±7* 6±639±6 *p<0.05, significant difference between the bepridil and dl-sotalol groups. Data represent the maximum changes from their corre- sponding pre-drug control value after the administration of a low (0.3 mg/kg) or high dose (3 mg/kg). After the high dose, bepridil induced a positive inotropic effect (PIE) and a negative inotropic effect (NIE). LVdP/dtmax, maximal upstroke velocity of the left ventricular pressure; LVEDP, left ventricular end-diastolic pressure; FRP-AV, functional refractory period of the atrioventricular node; ERP, effective refractory period; MAP90, monophasic action potential duration (in ms) at 90% repolarization. node recovery time at this dose. The high dose increased 5ms for dl-sotalol, respectively. the sinus node recovery time, Wenckebach block pacing In the bepridil group, the effective refractory period of cycle length and functional refractory period of the atrio- the ventricle was prolonged at 10min after the low dose, ventricular node for 10–60min. and it was further prolonged for 10–60min after the high dose. However, no significant change was detected in the Effect on the Effective Refractory Period of the Atrium terminal repolarization period at either dose. In the dl- The time-course of change in the effective refractory sotalol group, the effective refractory period of the ventricle period of the atrium is summarized in Fig6. The pre-drug was prolonged for 10–30min after the low dose, and it was control value (C) was 146±12ms for bepridil and 146±6ms further prolonged for 10–60min after the high dose. How- for dl-sotalol. ever, no significant change was detected in the terminal In the bepridil group, no significant change was detected repolarization period at either dose. in the effective refractory period of the atrium after the low dose, whereas it was prolonged for 10–60min after the high dose. In the dl-sotalol group, the effective refractory Discussion period of the atrium was prolonged for 10–30min after the In the present study, we assessed the effects of bepridil low dose, and it was further prolonged by 10–60min after and dl-sotalol using the halothane-anesthetized in vivo the high dose. canine model19–24 to better understand the differences in the in vivo cardiohemodynamic and electrophysiological Effects on the MAP90 During Ventricular Pacing profiles between the 2 drugs. The time-courses of changes in the MAP90 during ven- tricular pacing are summarized in Fig6. The MAP90(CL400) Doses of Bepridil and dl-Sotalol and MAP90(CL300) at the pre-drug control (C) were 231±7 The therapeutic plasma concentrations of bepridil and and 205±5ms for bepridil, and 215±10 and 197±5ms for dl-sotalol during repeated oral administration in humans dl-sotalol, respectively. have been reported to be 1.0–3.3μmol/L (367–1,210ng/ml) In the bepridil group, the MAP90(CL400) and MAP90(CL300) and 1.7–13.4μmol/L (456–3,647ng/ml), respectively.28 Thus, were prolonged at 10 min and 10–20 min, respectively, the plasma concentrations in the present study may corre- after the low dose. After the high dose, they were further spond to the therapeutic to supra-therapeutic levels of each prolonged for 10–60min. The maximum changes in the drug. dl-Sotalol effectively suppressed isoproterenol-induced MAP90(CL400) and MAP90(CL300) were 60 and 61ms, respec- tachycardia and hypotensive action, suggesting that the drug tively. In the dl-sotalol group, no significant change was can inhibit bothβ1- andβ2-adrenoceptors22 at therapeutic detected in the MAP90(CL400) or MAP90(CL300) after the low concentrations. dose, whereas they were prolonged for 10–60min after the high dose. The maximum changes in the MAP90(CL400) and Cardiohemodynamic Effects MAP90(CL300) were 59 and 46ms, respectively. In the pres- Bepridil decreased the mean blood pressure and total ent study, early afterdepolarization potential (ie, a hump in peripheral vascular resistance at the end of the high-dose the last phase of repolarization in the MAP recording) was infusion. As bepridil has been reported to suppress vascular not detected in any experiment. L-type Ca2+ channels above concentrations of 0.1μmol/L,29 the vasodilator action would be explained largely by such a Effects on the Effective Refractory Period of the Ventricle mechanism. The positive inotropic action was detected at and Terminal Repolarization Period the same time point, which may be associated with sympa- The time-courses of changes in the effective refractory thetic reflex resulting from its hypotensive and vasodilator period of the ventricle and terminal repolarization period actions.30,31 The negative inotropic action of bepridil was are summarized in Fig6. Their pre-drug control values (C) observed also after the high dose, which may be induced by were 203±3 and 29±5ms for bepridil, and 189±8 and 26± its cardiac L-type Ca2+ channel-blocking action and Na+

Circulation Journal Vol.72, June 2008 1010 ISHIZAKA T et al. channel inhibition.5,32,33 The drug also decreased the heart bepridil and dl-sotalol are summarized in Table1. Although rate and increased the sinus node recovery time after the the extent of the MAP90(CL400) prolongation effect was high dose, which may be associated with its cardiac L-type, similar between the bepridil group and dl-sotalol group, as well as T-type Ca2+ channel-blocking actions34,35 and bepridil had a greater effect on the MAP90(CL300) than dl- inhibition of IKr.6 sotalol, suggesting that bepridil may be useful in suppress- In the dl-sotalol group, negative inotropic and chrono- ing ventricular tachyarrhythmias as a result of re-entry.46 tropic effects and hypotensive action were observed in addi- The usefulness of bepridil and dl-sotalol for rate control in tion to increased preload to the left ventricular, which were patients with atrial fibrillation may be similar based on essentially in accordance with our previous study using their effects on the functional refractory period of the atrio- β-adrenoceptor blockers in the same animal model.21,22 ventricular node. Bepridil, as well as dl-sotalol, prolonged Considering that therapeutic doses of the IKr blockers the atrial effective refractory period, which might at least dofetilide and sematilide has been reported to decrease the suggest their clinical usefulness for the rhythm control of heart rate in this animal model,23,36 the bradycardiac effect atrial fibrillation.7–10 The negative inotropic and chronotropic of dl-sotalol may be partly associated with its IKr-blocking effects of dl-sotalol were greater than those of bepridil, property. whereas the reverse was true for hypotensive action, sug- gesting a difference in the hemodynamic adverse effects Electrophysiological Effects between the 2 drugs. As shown in the results, bepridil prolonged the QRS width and His-ventricular interval. Considering that bepridil has been reported to show a lidocaine-like fast kinetic block of Conclusions Na+ current,37,38 this mechanism may be associated with the The electropharmacological effects of bepridil and dl- ventricular conduction delay.24,39 Bepridil caused a negative sotalol were similar and, generally, in accordance with pre- dromotropic effect after the high dose, which is in accor- vious reports, although their potency for each cardiovascu- dance with previous reports,15,16,40 suggesting that the drug lar variable varied significantly, as summarized in Table1. can inhibit Ca2+ channels in vivo.21,41 However, its potency These findings can be useful when selecting these drugs during the sinus rhythm was weaker than that of dl-sotalol, according to the pathophysiological condition of a patient. based on the current results. Conversely, bepridil and dl- sotalol prolonged the functional refractory period of the Acknowledgments atrioventricular node and Wenckebach block pacing cycle The authors thank Mr Orii, Miss Sasaki and Miss Hasegawa at Hitachi length in an alike manner. Similar results were observed High-Tech Science Systems Ltd for carrying out the plasma concentration using therapeutic doses of typical Ca2+ channel blocker measurements for bepridil and dl-sotalol. This study was supported, in verapamil and β-adrenoceptor blocker propranolol in the part, by a Grant-in-aid from the Ministry of Education, Culture, Sports, same animal model.21 Science, and Technology of Japan (No. 19590532). dl Bepridil and -sotalol delayed ventricular repolarization in References a dose-dependent manner, which can be explained by the blockade of cardiac K+ channels, such as IKr and IKs6,42. Impor- 1. Hill JA, O’Brien JT, Scott E, Conti CR, Pepine CJ. Effects of bepridil on exercise tolerance in chronic stable angina: A double-blind, ran- tantly, bepridil prolonged the MAP90(CL300) and MAP90(CL400) domized, placebo-controlled, crossover trial. Am J Cardiol 1984; 53: to the same extent, whereas dl-sotalol prolonged the 679–683. MAP90(CL400) more potently than the MAP90(CL300), similar to 2. Verdouw PD, Scheffer MG. Cardiovascular actions after intravenous the IKr blockers dofetilide, nifekalant and sematilide.23,36,43 and intracoronary administration of the slow channel blocker bepridil. The results of bepridil are, generally, in accordance with a Arzneimittelforschung 1984; 34: 21–25. 32 3. Anno T, Furuta T, Itoh M, Kodama I, Toyama J, Yamada K. Electro- previous in vitro examination. On the other hand, the phar- mechanical effects of bepridil on rabbit isolated hearts. Br J Pharma- macological IKs blockade has been shown to enhance the col 1984; 81: 41–47. reverse rate-dependent prolonging action of the IKr blocker,44 4. Sassine A, Masse C, Fajuri A, Hirsch JL, Labrid C, Puech P. Elec- yet the reason why bepridil lacks a rate-dependent profile trophysiologic effects of bepridil in the anesthetized dog studied by 6 endocardiac electrodes. Am J Cardiol 1984; 53: 1707–1711. cannot be fully explained by its IKr and IKs blocking effects. 5. Yatani A, Brown AM, Schwartz A. Bepridil block of cardiac calcium In our previous study, the class Ia antiarrhythmic drug diso- and sodium channels. J Pharmacol Exp Ther 1986; 237: 9–17. pyramide and the class III antiarrhythmic drug amiodarone 6. Wang JC, Kiyosue T, Kiriyama K, Arita M. Bepridil differentially + prolonged the MAP90 in a rate-independent fashion in inhibits two delayed rectifier K currents, IKr and IKs, in guinea-pig + ventricular myocytes. Br J Pharmacol 1999; 128: 1733–1738. doses that blocked Na channels in the halothane-anesthe- 7. Yoshida T, Niwano S, Inuo K, Saito J, Kojima J, Ikeda-Murakami K, 24,45 + tized canine model. Thus, bepridil’s Na channel-block- et al. Bepridil prevents paroxysmal atrial fibrillation by a class III ing profile would be involved in the prolonging effect of antiarrhythmic drug effect. Pacing Clin Electrophysiol 2003; 26: the MAP90 at a faster pacing rate. 314–317. dl 8. Yoshida T, Niwano S, Inuo K, Saito J, Kojima J, Ikeda-Murakami K, Bepridil and -sotalol prolonged the atrial and ventricu- et al. Evaluation of the effect of bepridil on paroxysmal atrial fibril- lar effective refractory period in the present study, which lation: Relationship between efficacy and the f-f interval in surface can be explained by the IKr and Na+ channel-blocking ECG recordings. Circ J 2003; 67: 11–15. effects of bepridil and the IKr andβ-adrenoceptor blocking 9. Nakazato Y, Yasuda M, Sasaki A, Iida Y, Kawano Y, Nakazato K, et action of dl-sotalol.21,23,24,36,43 In the halothane-anesthetized al. Conversion and maintenance of sinus rhythm by bepridil in pa- tients with persistent atrial fibrillation. Circ J 2005; 69: 44–48. animal model, the extent of ventricular electrical vulnera- 10. Fujiki A, Tsuneda T, Sugao M, Mizumaki K, Inoue H. Usefulness and bility has been estimated by the terminal repolarization safety of bepridil in converting persistent atrial fibrillation to sinus period.20,23,27,36 As shown in the results, bepridil and dl- rhythm. Am J Cardiol 2003; 92: 472–475. sotalol hardly affected the terminal repolarization period. 11. Izumi D, Chinushi M, Watanabe H, Washizuka T, Okamura K, Komura S, et al. Bepridil for drug-refractory ventricular tachyar- rhythmias. Intern Med 2007; 46: 119–124. Comparison Between Bepridil and dl-Sotalol 12. Kobayashi S, Reien Y, Ogura T, Saito T, Masuda Y, Nakaya H. In- The hemodynamic and electrophysiological effects of hibitory effect of bepridil on hKv1.5 channel current: Comparison

Circulation Journal Vol.72, June 2008 Comparison of Bepridil and Sotalol in Dogs 1011

with amiodarone and E-4031. Eur J Pharmacol 2001; 430: 149– 1985; 235: 749–756. 157. 30. Takahara A, Sugiyama A, Satoh Y, Yoneyama M, Hashimoto K. 13. Berger F, Borchard U, Hafner D. Effects of the calcium entry blocker Cardiovascular effects of Y-27632, a selective Rho-associated kinase bepridil on repolarizing and pacemaker currents in sheep cardiac inhibitor, assessed in the halothane-anesthetized canine model. Eur J Purkinje fibres. Naunyn Schmiedebergs Arch Pharmacol 1989; 339: Pharmacol 2003; 460: 51–57. 638–646. 31. Takahara A, Iwasaki H, Nakamura Y, Sugiyama A. Cardiac effects 14. Hara Y, Nakaya H. SD-3212, a new class I and IV antiarrhythmic of L/N-type Ca2+ channel blocker cilnidipine in anesthetized dogs. drug: A potent inhibitor of the muscarinic acetylcholine receptor- Eur J Pharmacol 2007; 565: 166–170. operated potassium current in guinea-pig atrial cells. Br J Pharmacol 32. Nobe S, Aomine M, Arita M. Bepridil prolongs the action potential 1995; 116: 2750–2756. duration of guinea pig ventricular muscle only at rapid rates of stimu- 15. Kantelip JP, Alatienne M, Talmant JM, Ghyselinck N, Duchene- lation. Gen Pharmacol 1993; 24: 1187–1196. Marullaz P. Comparative electrophysiologic effects of bepridil, vera- 33. Ozaki H, Zaizen H, Kiyosue T, Nasu M, Arita M. Effect of bepridil pamil and diltiazem in conscious and anesthetized dogs. Arch Int on intracellular calcium concentration and contraction in cultured rat Pharmacodyn Ther 1986; 284: 5–18. ventricular myocytes. J Cardiovasc Pharmacol 1999; 33: 492–499. 16. Leboeuf J, Lamar JC, Massingham R, Ponsonnaille J. Electrophysiol- 34. Cohen CJ, Spires S, Van Skiver D. Block of T-type Ca channels in ogical effects of bepridil and its quaternary derivative CERM 11888 guinea pig atrial cells by antiarrhythmic agents and Ca channel an- in closed chest anaesthetized dogs: A comparison with verapamil and tagonists. J Gen Physiol 1992; 100: 703–728. diltiazem. Br J Pharmacol 1989; 98: 1351–1359. 35. Uchino T, Lee TS, Kaku T, Yamashita N, Noguchi T, Ono K. Volt- 17. Antonaccio MJ, Gomoll A. Pharmacologic basis of the antiarrhythmic age-dependent and frequency-independent inhibition of recombinant and hemodynamic effects of sotalol. Am J Cardiol 1993; 72: 27A– Cav3.2 T-type Ca2+ channel by bepridil. Pharmacology 2005; 74: 37A. 174–181. 18. Anderson JL, Prystowsky EN. Sotalol: An important new antiar- 36. Satoh Y, Sugiyama A, Tamura K, Hashimoto K. Effects of a class III rhythmic. Am Heart J 1999; 137: 388–409. antiarrhythmic drug, dofetilide, on the in situ canine heart assessed 19. Ando K, Sugiyama A, Takahara A, Satoh Y, Ishizaka T, Nakamura Y, by the simultaneous monitoring of hemodynamic and electrophysio- et al. Analysis of proarrhythmic potential of antipsychotics risperidone logical parameters. Jpn J Pharmacol 1999; 81: 79–85. and olanzapine in anesthetized dogs. Eur J Pharmacol 2007; 558: 37. Nawada T, Tanaka Y, Hisatome I, Sasaki N, Ohtahara A, Kotake H, 151–158. et al. Mechanism of inhibition of the sodium current by bepridil in 20. Satoh Y, Sugiyama A, Takahara A, Ando K, Wang K, Honsho S, et guinea-pig isolated ventricular cells. Br J Pharmacol 1995; 116: al. The antipsychotic and antiemetic drug prochlorperazine delays the 1775–1780. ventricular repolarization of the in situ canine heart. J Pharmacol Sci 38. Sato N, Nishimura M, Kawamura Y, Ward CA, Kikuchi K. Block of 2005; 97: 101–106. Na+ channel by bepridil in isolated guinea-pig ventricular myocytes. 21. Shiina H, Sugiyama A, Takahara A, Satoh Y, Hashimoto K. Compari- Eur J Pharmacol 1996; 314: 373–379. son of the electropharmacological effects of verapamil and propranolol 39. Takahara A, Sugiyama A, Satoh Y, Hashimoto K. Effects of mexile- in the halothane-anesthetized in vivo canine model under monophasic tine on the canine model of sparfloxacin-induced long QT syndrome. action potential monitoring. Jpn Circ J 2000; 64: 777–782. Eur J Pharmacol 2003; 476: 115–122. 22. Sugiyama A, Takahara A, Hashimoto K. Electrophysiologic, cardio- 40. Kawada M, Satoh K, Taira N. Profile of coronary vasodilator and car- hemodynamic andβ-blocking actions of a new ultra-short-actingβ- diac actions of bepridil revealed by use of isolated, blood-perfused blocker, ONO-1101, assessed by the in vivo canine model in com- heart preparations of the dog. J Cardiovasc Pharmacol 1983; 5: parison with esmolol. J Cardiovasc Pharmacol 1999; 34: 70–77. 604–612. 23. Takahara A, Sugiyama A, Satoh Y, Wang K, Honsho S, Hashimoto 41. Komori S, Ishii M, Hashimoto K. Antiarrhythmic effects of coronary K. Halothane sensitizes the canine heart to pharmacological IKr vasodilators on canine ventricular arrhythmia models. Jpn J Pharma- blockade. Eur J Pharmacol 2005; 507: 169–177. col 1985; 38: 73–82. 24. Yoshida H, Sugiyama A, Satoh Y, Ishida Y, Kugiyama K, Hashimoto 42. Ducroq J, Printemps R, Guilbot S, Gardette J, Salvetat C, Le Grand K. Effects of disopyramide and mexiletine on the terminal repolari- M. Action potential experiments complete hERG assay and QT inter- zation process of the in situ heart assessed using the halothane-anes- val measurements in cardiac preclinical studies. J Pharmacol Toxicol thetized in vivo canine model. Circ J 2002; 66: 857–862. Methods 2007; 56: 159–170. 25. Bazett HC. An analysis of the time-relations of electrocardiogram. 43. Satoh Y, Sugiyama A, Takahara A, Chiba K, Hashimoto K. Electro- Heart 1920; 7: 353–370. pharmacological and proarrhythmic effects of a class III antiarrhyth- 26. Van de Water A, Verheyen J, Xhonneux R, Reneman RS. An im- mic drug nifekalant hydrochloride assessed using the in vivo canine proved method to correct the QT interval of the electrocardiogram models. J Cardiovasc Pharmacol 2004; 43: 715–723. for changes in heart rate. J Pharmacol Methods 1989; 22: 207–217. 44. So PP, Hu XD, Backx PH, Puglisi JL, Dorian P. Blockade of IKs by 27. Sugiyama A, Hashimoto K. Effects of a typical IKr channel blocker HMR 1556 increases the reverse rate-dependence of refractoriness sematilide on the relationship between ventricular repolarization, prolongation by dofetilide in isolated rabbit ventricles. Br J Pharma- refractoriness and onset of torsades de pointes. Jpn J Pharmacol col 2006; 148: 255–263. 2002; 88: 414–421. 45. Sugiyama A, Satoh Y, Hashimoto K. Acute electropharmacological 28. Omata T, Kasai C, Hashimoto M, Hombo T, Yamamoto K. QT effects of intravenously administered amiodarone assessed in the in PRODACT: Comparison of non-clinical studies for drug-induced vivo canine model. Jpn J Pharmacol 2001; 87: 74–82. delay in ventricular repolarization and their role in safety evaluation 46. Nair LA, Grant AO. Emerging class III antiarrhythmic agents: Mecha- in humans. J Pharmacol Sci 2005; 99: 531–541. nism of action and proarrhythmic potential. Cardiovasc Drugs Ther 29. Suzuki H, Itoh T, Kuriyama H. Mechanisms of the bepridil-induced 1997; 11: 149–167. vasodilation of the rabbit mesenteric artery. J Pharmacol Exp Ther

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