Classification of Antiarrhythmic Drugs Keitaro Hashimoto1,* 1Professor Emeritus, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan

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Arrhythmia Models for Drug Research: Classification of Antiarrhythmic Drugs Keitaro Hashimoto1,* 1Professor Emeritus, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan

Received December 31, 2006

Abstract. The aim of this study was to classify antiarrhythmic drugs based on their effectiveness on 6 in vivo arrhythmia models, mainly using dogs. The models were produced by two-stage coronary ligation, digitalis, halothane-adrenaline, programmed electrical stimulation in old myocardial infarction dogs, coronary artery occlusion/reperfusion, or chronic atrioventricular block. Na+-channel–blocking drugs suppressed two-stage coronary ligation and digitalis arrhythmias. Ca2+-channel blockers and β-blockers suppressed halothane-adrenaline arrhythmia. Positive inotropic drugs aggravated halothane-adrenaline arrhythmia, but did not aggravate digitalis arrhythmia. K+-channel blockers suppressed programmed electrical stimulation induced arrhythmia, but induced torsades de pointes type arrhythmia in chronic atrioventricular block dogs and aggravated halothane-adrenaline arrhythmia. Na+/H+-exchange blockers suppressed coronary artery occlusion/reperfusion arrhythmias. This classification may be useful for predict- ing the clinical effectiveness in the preclinical stage of drug development.

Keywords: arrhythmia model, antiarrhythmic drug, proarrhythmic potential of drugs, ion channel, classification

1. Introduction sary. Also for applying the ablation technique to elimi- nate substrates of arrhythmias, only the proper area, Currently, interest in arrhythmia treatment is turning automatic foci, or a part or the entire re-entry circuit towards new devices and ablation techniques; and it is, need to be known. But what about pharmacological of course, certain that a failed sinus or Tawara’s (AV) treatments? Is there no room for developing new anti- node should be replaced by appropriate electronic pace- arrhythmic drugs? As Christ and Ravens discussed in makers and that cardiac collapse by ventricular fibrilla- her review (1), the relative importance of antiarrhythmic tion (VF) or potentially lethal torsades de pointes (TdP) drugs might have decreased, but they still are relied on should be stopped by electrical external or implantable for prevention of atrial fibrillation and sudden cardiac defibrillators. To implant pacemakers, only evidence of death by cardiologists world wide. More importantly, for malfunction of pacemaking and atrioventricular conduc- developing antiarrhythmic drugs, it has been necessary tion is necessary. To install a defibrillator, the genetic to know the precise mechanisms of the generation of preponderance of the occurrence of these arrhythmias, or action potentials, the different forms of action potentials, evidence of existence of ventricular arrhythmia types and the abnormalities of normal action potentials that are likely to develop into fatal sudden death or other precip- responsible for development into arrhythmias, and so on. itating factors should be known. Knowledge of pace- Thus introduction of newer drugs or understanding of making or precise mechanisms of generation of arrhyth- drug actions developed in parallel with the advances in mias in cellular and molecular levels is no longer neces- the knowledge about cardiac electrophysiology. Electri- cal mechanisms of the heart are generated by complex, multi-factorial phenomena simultaneously occurring *Corresponding author. [email protected] with channel- or receptor-related events. Molecular Published online in J-STAGE: April 4, 2007 doi: 10.1254/jphs.CRJ06013X research aims to elucidate the single function of a single molecule and the action of drugs on ion channels, trans- Invited article porters, or extracellular or intracellular receptors, but

333 334 K Hashimoto how these drugs work on arrhythmias is only known 2. Drug effects on arrhythmia models from experimental or clinical arrhythmias. After the CAST study (2) revealed the possible proarrhythmic 2-1. Canine two-stage coronary ligation arrhythmia effects of Na+-channel–blocking antiarrhythmic drugs, 2-1-1. Characteristics pharmacologists and cardiologists tried to make a new Ischemia, being a serious disorder of the myocardium, classification or guide for choosing proper drugs to treat causes pump and electrical failure; thus it is used often to clinical arrhythmias, namely the Sicilian Gambit, by produce experimental arrhythmias. Ischemia is a time- defining drug actions on cardiac tissues based on newer dependent phenomenon and starts with reversible molecular aspects of cardiac excitation and classifying damage finally reaching the stage of irreversible damage clinical arrhythmias to correlate or match the drugs and or scar formation; thus it is no wonder that ischemia the arrhythmias (3). However since the molecular related arrhythmias also show a variety of electrical mechanisms of arrhythmias are still not well known, and abnormalities. The two-stage coronary ligation tech- also comparative effectiveness of drugs on clinical nique was originally reported by Harris (9) and is arrhythmias are not fully known, we have been examin- characterized by consistent and stable sub-acute to ing drugs on various experimental animal arrhythmias chronic ischemic arrhythmias, showing severe ventri- models. It is obvious that the present antiarrhythmic cular tachycardia (VT), but never deteriorating to VF. drugs are not ideal ones, and also for many different This arrhythmia is thought to be an automaticity arrhythmias, many different types of drugs are neces- arrhythmia because overdriving the atria or ventricle, by sary. Drugs are still used to maintain sinus rhythm after electrical stimulation at rates higher than the rate of VT, conversion from atrial defibrillation, to stop the transi- suppressed the VT to conducted sinus rhythm (5). tion to VF clinically, and to target pathophysiological alterations to prevent the occurrence of lethal arrhyth- 2-1-2. Methods mias. It may be worthwhile to review our studies and try “Two-stage” means that the first stage is the start of to classify drugs by their effectiveness on arrhythmias of 30 min of ischemia, followed by the second stage of a dogs, rats, guinea pigs, and so on. Our previous results permanent occlusion of the left anterior descending on classical and investigational antiarrhythmic drugs and coronary artery (LAD) of the dog. In the first stage, our recent results on cardioprotective drugs have been isolated LAD soon after bifurcation with the left circum- summarized here. flex coronary artery (LCX) was surgically isolated from Antiarrhythmic drugs have been classified by the neighboring coronary veins or connective tissues and Vaughan Williams mainly based on their effects on then tied completely with one of double threads together cardiac action potentials into classes I to IV and later with a needle of about 1 mm outer diameter, which was correlated to their effects on Na+ channel, β-receptors, immediately withdrawn to make the needle size patency and K+ and Ca2+ channels (4). Here our animal experi- to produce a state of ischemia, but not result in complete mental arrhythmia models, both the automaticity in- occlusion. This usually produces ischemic changes in duced and re-entry induced arrhythmias are presented, the electrocardiogram (ECG) and produces only prema- which we used to quantitatively compare drug effects on ture ventricular contractions (PVC) lasting for a few min the arrhythmias. There have been many studies of anti- followed by a quiescent 30 min, and then another suture arrhythmic drug efficacies using animal arrhythmias, but is used to completely occlude the LAD. After the final most of them showed the effectiveness of a single drug ligature, the thoracotomized chest is closed, and the or made comparisons of that drug with one or two animal is left to recover from the anesthesia for use 24 or standard drugs. To make a preclinical prediction of the 48 h after coronary ligation. Though cardiac surgery usefulness of the drug, comparisons among many drugs under anesthesia in a sterile environment is needed, and were almost impossible. We used standard and repro- the technique of LAD isolation is not always easy, very ducible models that can occur clinically and tried to stable arrhythmias, PVC and VT (defined by more than study the antiarrhythmic drugs’ effects by comparing 3 continuous PVC), can be obtained which usually them quantitatively and qualitatively to find common appear several hours later, and the VT becomes fulmi- pharmacological properties and predict antiarrhythmic nant 24 h later in a conscious state, lasting up to 48 h; mechanisms of action of drugs. We have already written thus this arrhythmia can be used to evaluate and compare several summary review papers (5 – 8), but in this drug effects. The arrhythmias are continuous, and at the review, we have added new data obtained in recent peak of about 24 h after ligation, the PVC consists of years to compare a wide range of drugs. almost all the beats. To judge whether the beats are sinus beat or PVC, atrial electrodes were sutured on the epicardial surface of the right atrial appendage during Arrhythmia Models and Drugs 335 the operation, and ECG and atrial electrogram were channel–blocking drugs suppress this two-stage coro- simultaneously obtained. Preferably a His electrogram nary ligation arrhythmias and the effective plasma must be obtained to judge whether a beat is of a concentrations are closely related to the intraarterial conducted sinus rhythm or not, but fixing intracardiac doses to suppress intraventricular conduction (12), His electrodes usable in a conscious state is quite suggesting that they suppress arrthythmia through a difficult and invasive, so we chose the alternate atrial mechanism involving Na+-channel block (5, 10, 12 – 40). electrogram recordings. We used the arrhythmic ratio for Figure 1 shows the correlation between the reported expressing the severity of ventricular arrhythmias by the Na+-channel–blocking concentration in in vitro experi- number of PVC over a certain time, in our case usually ments mainly obtained in the isolated canine cardiac 10 sec, divided by the total number of beats including ventricular muscle preparations and their effective the PVC and the normal sinus beats. This arrhythmic plasma concentrations (EC50) for suppressing the two- ratio cannot differentiate the most severe VF from stage coronary ligation arrhythmia models of the effec- severe VT, but as drugs are used usually before VF tive 29 drugs; earlier data (before 1991) of Na+-channel– occurs, it is a good measure to express the severity of blocking concentration were those listed in Hashimoto arrhythmia for quantitative comparison. We found that et al. (5), those after 1991 are from the respective paper 24 h after LAD ligation, the arrhythmic ratio of the VT is (12, 34, 35), and data on disopyramide isomers were almost 1.0 in the two-stage coronary ligation arrhythmia those of Vanhoutte et al. (36). These canine effective model, and we calculated the effective plasma concen- plasma concentrations thus determined were also shown tration of a drug to decrease the control arrhythmic ratio to be very close to their clinical effective concentrations to 50% from the arrhythmic ratio-drug plasma concen- (5, 12, 34 – 37), thus from this series of experiments, we tration curve (EC50). For the drug plasma concentration could predict clinically effective drug plasma concentra- determinations, intra-arterial or intravenous catheters tion levels before these drugs went to clinical trials. were introduced, and the blood samples were drawn Though Na+-channel blockers are now classified accord- without the conscious animal being aware of the proce- ing to their mode and kinetics of Na+-channel inhibition, dure. Another advantage of this arrhythmia model is that our conclusion was that any drug that blocks Na+ the drugs are administered in a conscious state, so that it channels can suppress this two-stage coronary ligation is easy to evaluate the antiarrhythmic efficacy of drugs arrhythmia, but by using this arrhythmia model, we while simultaneously evaluating their central nervous could demonstrate the interaction between two Na+- system (CNS) side effects. Thus intravenous lidocaine channel blockers with different binding affinities (41) was not effective without CNS stimulant effects, even used in combination in an in vitro preparation (42). In though there has been a widespread belief that lidocaine the study, the actions of mexiletine and disopyramide is the most effective drug in the clinical setting of acute were additive, but those of mexiletine and aprindine ischemic arrhythmias (5, 10). We gave drugs either by were not. As shown in Table 1, TJN-505 is a chemically an intravenous or oral route. We tested many Na+- aconitine-like drug acting on Na+ channels and was channel–blocking drugs and other drugs. When a drug effective on the two-stage coronary ligation arrhythmia, was effective, the time-dependent drug plasma concen- but there is no data on this drug as a Na+-channel–block- tration curve, either after a bolus intravenous injection or ing drug in the in vitro cardiac tissue (38). HNS-32 is the curve at the rising phase after a constant infusion, also such a drug with Na+-channel–blocking activity gave almost the same effective plasma concentration and was shown to be effective on this arrhythmia (39). values; namely, there was almost no hysteresis in the Unlike most of the Ca2+-channel blockers, AH-1058, antiarrhythmic effect–EC50 relationships (5). suppressed two-stage coronary ligation arrhythmia This two-stage coronary ligation arrhythmia was possibly by inhibiting Ca2+ overload (43). stable and the success rate to obtain arrhythmias for drug evaluation is high, nearly 90%, in dogs. Pigs are prone to 2-1-4. Ineffective and proarrhythmic drugs develop VF after coronary ischemia, and smaller rats Two-stage coronary ligation arrhythmias differ from and mice do not show such stable VT. Guinea pigs never acute coronary occlusion/reperfusion arrhythmias, develop ischemia by in vivo coronary ligation (11). It described later, in that they almost never turn into VF or may still be the one of the best models for a new Na+- result in animal deaths, and there were no drugs aggra- channel–blocking antiarrhythmic drug to be tested pre- vating the two-stage coronary ligation VT to VF. Among clinically for further development. classical antiarrhythmic drugs, the Ca2+-channel–blocking drugs verapamil (10) and gallopamil (44) did not 2-1-3. Antiarrhythmic drugs suppress these arrhythmias and also β-blockers were not Drug effects can be summarized as follows: Na+- effective unless high doses to produce Na+-channel– 336 K Hashimoto

Fig. 1. Correlation of drug plasma effective concentrations (EC50) determined using canine two-stage coronary ligation arrhythmia and its Na+-channel blocking in vitro concentrations of ventricular muscle. Although Na+-channel–blocking concentration determinations were done in various institutions and drug protein binding is neglected, there is roughly a good relationship.

blocking effects were given (10, 13, 14, 17, 19, 45). Pure 2-2. Digitalis-induced ventricular arrhythmias K+-channel–blocking drugs that we tested did not 2-2-1. Characteristics suppress these arrhythmias (6, 46 – 49), but amiodarone The cardiotonic steroid digitalis is known to be given intravenously suppressed the two-stage coronary arrhythmogenic and its effect can be explained by intra- ligation arrhythmia, but the plasma concentration was cellular Ca2+ overload due 1) to intracellular Na+ not determined in this experiment (50). Since there accumulation by Na+-K+-ATPase inhibition, followed by was no prolongation of QT interval by intravenous 2) intracellular Ca2+ accumulation by enhanced Na+/Ca2+ amiodarone injection, this antiarrhythmic effect was exchange (5, 59, 60). Ca2+ overload, thus produced, thought to be due to amiodarone’s Na+-channel–block- can induce abnormal automaticity or delayed after- ing action (51). Positive inotropic drugs, catechola- depolarization-induced arrhythmia or cause re-entry mines, phosphodiesterase (PDE) inhibitors (52 – 54), the arrhythmia and conduction block by uncoupling the cell- forskolin derivative, colforsin daropate (55) or the Ca2+- to-cell connections and/or stimulating vagal influence sensitizer, MCI-154 (56), neither showed antiarrhythmic on the AV nodal conduction (60). However the precise effects nor aggravated the arrhythmia, unless used in mechanism of Ca2+ overload by the Na+/Ca2+ exchanger extreme doses. Zatebrazine, an If-blocking specific and the resultant increase in the inward current, leading negative chronotropic agent, did not suppress this to the development of delayed afterdepolarization is arrhythmia (57). As stated above, this arrhythmia was still not known, and emergence of new chemicals to suppressed by overdriving, so isoproterenol given at selectively inhibit the Na+/Ca2+ exchanger may solve the 1–3µg/kg produced sinus tachycardia and transiently problem and may also be useful for clinical digitalis suppressed this VT (our unpublished observation). A toxicity arrhythmias. The automaticity mechanism can cardioselective M2-receptor blocker, AF-DX 116, was be demonstrated by the overdrive suppression of this also examined on this arrhythmia, but it had no effect arrhythmia (5). (58). Arrhythmia Models and Drugs 337

Table 1. Classification of drugs using in vivo arrhythmia models

Aggravating drugs Effective drugs Ineffective drugs or procedure

Two-stage coronary I: A-2545, AFD-19, AFD-21 (NS-2), I: lidocaine ligation arrhythmia AHR 10718, AN-132, aprindine, AP-792, II: betaxolol, bupranolol, Ko 1400, bidisomide, bisaramil, cibenzoline, OPC-1427, oxprenolol, propranolol disopyramide, d-disopyramide, IV: gallopamil, verapamil l-disopyramide, etacizin, E-0747, flecainide, III: dofetilide, E-4031, KCB-328, HNS-32, KT-362, mexiletine, nicainoprol, sematilide, d-sotalol OPC88117, penticainide, phenytoin, pirmenol, M: colforsin daropate, milrinone, propafenone, SD-3212, tocainide, NS-2, OPC-18790, vesnarinone, MCI-154, procainamide, pilsicainide, TJN-505, zatebradine, AF-DX 116 TYB-3823, YUTAC II: pindolol, N-696 III: amiodarone (i.v.) M: dilazep

Digitalis arrhythmia I: A-2545, AFD-19, AFD-21 (NS-2), II: atenolol, betaxolol, N-696 AHR 10718, AN-132, AP-792, aprindine, III: E-4031, dofetilide, sematilide, bidisomide, bisaramil, cibenzoline, KCB-328 disopyramide, E-0747, etacizin, flecainide, M: bepridil, nicorandil, nitroglycerin, HNS-32, KT-362, mexiletine, nicainoprol, trimetazidine, cariporide, NS-2, OPC88117, penticainide, phenytoin, KB-R7943, milrinone, vesnarinone, pirmenol, procainamide, pilsicainide, MCI-154 propafenone, SD-3212, TJN-505, tocainide, trapidil, TYB-3823, YUTAC II: atenolol, carvedilol, pindolol, propranolol III: amiodarone (i.v.) M: magnesium sulphate, zadebradine, SEA0400, amrinone, colforsin daropate, sulmazole

Halothane-adrenaline II: atenolol, betaxolol, Ko 1400, N-696, I: AHR 10718, AN-132, tocainide I: disopyramide, etacizin, arrhythmia oxprenolol, pindolol, propranolol M: nicorandil, MCI-154, zatebradine pilsicainide, procainamide IV: AH-1058, AP-792, diltiazem, gallopamil, III: azimilide, dofetilide, nifedipine, nisoldipine, verapamil E-4031, KCB-328, III: amiodarone (i.v.) nifekalant, sematilide I: AFD-19, aprindine, cibenzoline, E-0747, M: amrinone, milrinone, flecainide, KT-362, mexiletine, nicainoprol, OPC-18790, sulmazole, OPC88117, penticainide, phenytoin, pirmenol, vesnarinone propafenone, SD-3212, TJN-505, TYB-3823, YUTAC M: dilazep, NCO-700, nitroglycerin, trapidil, trimetazidine, l-carnitine

PES-induced III: azimilide, dofetilide, KCB-328, nifekalant, I: flecainide arrhythmia sematilide I: A-2545, aprindine, disopyramide

Occlusion/reperfusion Halothane-anesthetized dogs Halothane-anesthetized dogs M: pacing-induced arrhythmia III: E-4031, nifekalant III: amiodarone (i.v., p.o.), dofetilide, heart failure Pentobarbital-anesthetized dogs KCB-328, sematilide, d-sotalol III: KCB-328, nifekalant, d-sotalol Pentobarbital-anesthetized dogs M: cariporide, KB-R9032 III: dofetilide, sematilide Rats M: KB-R7943, SEA0400, pravastatin M: cariporide, FR 186666, KB-R9032, DIDS, Rats pravastatin M: SITS, fluvastatin

Chronic AV block III: nifekalant, sematilide dog (TdP induction) M: cisapride, sparfloxacin, terfenadine

I: Na+-channel–blocking drugs; NIK-244 = etacizin, ME3202 (CM7857) = penticainide, SUN 1165 = pilsicainide. II: β-blocking drugs. III: K+- channel–blocking drugs; MS-551 = nifekalant. IV: Ca2+-channel–blocking drugs. M: Miscellaneous drugs with different mechanisms of action; AR-L115 = sulmazole, OPC8212 = vesnarinone, HOE642 = cariporide, NKH477 = colforsin daropate.

2-2-2. Methods animals except in rats, which are known to need thou- Digitalis can induce toxicity characterized by VT, VF, sands of times higher doses of digitalis than man and and death both by intermittent intravenous bolus injec- other animals (61). Test drugs are usually administered tion or continuous intravenous infusion in various before digitalis administration by intravenous bolus 338 K Hashimoto injection, and a simple test to record the occurrence of closely related to their in vitro Na+-channel–blocking PVC, VT, and VF using small animals, especially the concentrations (5, 15, 16, 18 – 40). Zatebradine did guinea pig, is often used for screening possible anti- not suppress the two-stage coronary ligation arrhythmia, arrhythmic effects of drugs, but precise quantitative data but digitalis arrhythmia was suppressed by a high dose are difficult to obtain from these experiments. We used of intravenous 1.5 mg/kg bolus injection (57). The dogs, and they were anesthetized with intravenous mechanism of this effect might be either due to If block pentobarbital, and then intravenous catheter atrial or other channel effects, but there was no supporting electrodes were introduced from the femoral vein (15, data of plasma concentration determination and also 62). Cumulative administration of digitalis, initially there were no other If blockers available for our study. 40 µg/kg intravenous ouabain followed by a 10 µg/kg Other drugs effective on digitalis arrhythmias without every 20 min were administered to produce continuously Na+-channel–blocking effect, but for which the exact occurring VT, lasting more than 1 h, and then the test mechanisms of action are unknown, are intravenous drugs were administered and the blood to determine magnesium sulfate (63), a possible Ca2+-channel drug plasma concentrations were drawn. As the drug blocker, and the Na+/Ca2+-exchange inhibitor SEA0400, concentration decreased, VT reappeared after effective reported to be relatively selective when suppressing drug administration, thus we could determine the canine inward Ca2+ movement (reverse mode) (64). Also effective plasma concentrations of drugs (5, 15). positive inotropic drugs with prominent tachycardic action, amrinone, colforsin daropate, and sulmazole, 2-2-3. Antiarrhythmic drugs suppressed this arrhythmia probably by overdriving the Drugs effective on our digitalis arrhythmias are seen increased automaticity (53, 55). + in Figure 2 with plasma EC50 values and in vitro Na - channel–blocking concentrations. The pattern of drug 2-2-4. Ineffective and proarrhythmic drugs effects are similar to that obtained using the two-stage Ca2+-channel–blocking drugs did not suppress this coronary ligation arrhythmias, namely, that the Na+- arrhythmia (44, 65 – 67) even though the mechanism of channel–blocking drugs suppressed these digitalis generation is thought to be increased intracellular Ca2+ arrhythmias and the effective plasma concentrations are concentration. The exception was magnesium sulfate,

+ Fig. 2. Correlation of drug plasma effective concentrations (EC50) determined using canine digitalis arrhythmia and its Na - channel blocking in vitro concentrations of ventricular muscle. There is a roughly a good relationship. Arrhythmia Models and Drugs 339 which acts as a Ca2+-channel blocker in vivo, and which maker activity both of the normal and latent pacemakers, suppressed digitalis arrhythmia (63). As for the ineffec- thus catecholamines in higher doses induce atrial and tiveness of Ca2+-channel blockers, its hypotensive effect ventricular arrhythmias along with sinus tachycardia. might have made it impossible to raise the doses high However some anesthetics, such as cyclopropane, enough to decrease influx of Ca2+ to the myocardium. chloroform and halothane, are known to highly sensitize We therefore tried intracoronary administration of a the myocardium to catecholamines and under those high-dose verapamil in digitalis-induced VT, and as a anesthetics, catecholamines in very low doses induce result, rapid pacing-induced and probably DAD- severe VT (72). We used halothane and adrenaline in induced PVC was suppressed by verapamil (68). In this dogs and guinea pigs to produce halothane-adrenaline model, Na+-channel blockers were tested, including arrhythmia. It is an automaticity arrhythmia because bisaramil, disopyramide, lidocaine, and flecainide, electrical overdrive suppresses the VT (5, 72, 73). which also suppressed this DAD-induced PVC. β- Blockers were not effective unless high doses to 2-3-2. Methods suppress Na+ channels were given (17, 19, 45, 69). K+- Dogs are initially anesthetized with thiopental sodium channel–blocking drugs did not suppress this arrhythmia and after intubation, 1% halothane vaporized by 100% (6, 46 – 49), but intravenous amiodarone, probably by oxygen is used for anesthesia and intravenous catheter its Na+-channel–blocking effect, suppressed this digitalis and intravenous atrial electrodes were introduced from arrhythmia (50). Also non-Ca2+-channel–blocking coro- the femoral vein (74). In the case of the guinea pig, under nary vasodilators such as bepridil, nicorandil, nitro- intraperitoneal thiopental anesthesia, intubation was glycerin, trimetazidine (65 – 67), the Na+/H+-exchange performed to inhale halothane, but due to its small size inhibitor cariporide (70), and the Na+/Ca2+-exchange the atrial electrodes were not used (73). For determining inhibitor KB-R7943, which has multichannel effects canine effective drug plasma concentrations, conti- and is not selective concerning reverse mode Na+/Ca2+ nuously occurring adrenaline arrhythmia was produced exchange as compared to SEA0400 (71), were not by intravenous infusion of adrenaline, adjusting the effective on the digitalis arrhythmia. infusion speed enough to produce stable VT lasting As for proarrhythmic drugs, adding more digitalis about 20 min, but not producing VF (74). The infusion changed the VT to lethal VF, so catecholamines must rate usually employed was around 2 – 3 µg/kg/min. theoretically aggravate digitalis-induced arrhythmia Most of the drugs were injected by an intravenous bolus because they add intracellular Ca2+ by opening Ca2+ and the plasma concentration-dependent decrease of the channels via β-receptor stimulation; thus, we did not arrhythmic ratio was examined for effective drugs. For systematically examine catecholamines. However we guinea pigs, a bolus adrenaline injection was used at examined PDE-inhibiting inotropic drugs in order to higher halothane concentrations of 2% – 4%, and drugs obtain safety pharmacology data taking into consider- were injected before adrenaline challenges (73). ation the possible combined use of different types of positive inotropic drugs. Milrinone, and vesnarinone 2-3-3. Antiarrhythmic drugs increased intracellular cyclic AMP concentration and Since adrenaline stimulates cardiac β receptors, all the increased intracellular Ca2+ concentration, but actually β blockers were thought to be and actually were effective had no aggravating effects (53). As compared to in low β-blocking doses at low plasma concentrations amrinone and sulmazole, milrinone and vesnarinone (13, 17, 19, 45, 69). Other important drugs that were may induce less tachycardia and might not have reached effective were Ca2+-channel blockers. All Ca2+-channel a level of atrial rate sufficient to overdrive digitalis- blockers, verapamil, gallopamil, diltiazem, or even so- induced ventricular automaticity. Also MCI-154, a called vaso-selective dihydropyridine Ca2+-channel positive inotropic drug having Ca2+ sensitizing and PDE blockers, such as nifedipine, nisoldipine, AP-792, and inhibiting action, did not aggravate digitalis arrhythmia AH-1058, suppressed adrenaline arrhythmia at Ca2+- (56). channel–blocking concentrations (5, 40, 43, 44, 65 – 67, 69). Gallopamil, given into LAD, also suppressed intra- 2-3. Halothane-adrenaline arrhythmia coronary adrenaline induced triggered rhythm (44). 2-3-1. Characteristics Some of the so-called coronary vasodilators, such as Another automaticity arrhythmia is the classical cate- dilazep, NCO-700, nitroglycerine, trapidil and trimetazi- cholamine-induced arrhythmia. Normal pacemaker dine also suppressed adrenaline arrhythmias (5), but activity of the heart is under the control of sympathetic this may not be due to their hypotensive effect, since and vagal influences and sympathetic stimulation and vasodilators with other mechanisms of action, such as sympathomimetic drugs are known to increase pace- the K+-channel opener nicorandil, did not suppress the 340 K Hashimoto adrenaline arrhythmia (65). Many other drugs also 2-4. Programmed electrical stimulation induced re- suppressed adrenaline arrhythmia by undetermined entry arrhythmia in old myocardial infarction dogs mechanisms whether due to additional Ca2+-channel– or 2-4-1. Characteristics β-receptor–blocking action or due to other mechanisms. This arrhythmia is produced in dogs given surgery to These include the Na+-channel blockers (5), AFD-19, produce myocardial infarction by two-stage coronary aprindine, cibenzoline, E-0747, flecainide, KT-362, ligation more than a week before, when there was no mexiletine, nicainoprol, OPC88117, penticainide, spontaneous PVC (47). Since arrhythmias were induced phenytoin, pirmenol, propafenone, SD3212, TJN-505, only by applying premature extrastimuli, the mechanism TYB-3823, and YUTAC, and other miscellaneous of generation is thought to be re-entry around the scar drugs, such as l-carnitine (66) and colforsin daropate tissue of the old infarction. Drug effects can be demon- (55). strated by the change of the severity of induced arrhythmias before and after the drug administration. The rank 2-3-4. Ineffective and proarrhythmic drugs of arrhythmias was used to evaluate drug effects, and Some of the Na+-channel blockers (5), such as AHR the order by their severity was VF as the most severe, 10718, AN-132, and tocainide, required higher concen- followed by sustained VT, non-sustained VT, PVC, and trations than needed to suppress two-stage coronary no arrhythmia. ligation and digitalis arrhythmias; and since those concentrations are not thought to be attainable in clinical 2-4-2. Methods use, those drugs could be said to be ineffective. Dogs with old myocardial infarction were anesthe- Furthermore, some Na+-channel blockers even aggra- tized with pentobarbital and after reopening the thoracic vated the adrenaline-induced VT to VF, namely, incision, epicardial stimulating bipolar electrodes were worsened this arrhythmia. They include commonly used sutured on the non-infarcted left ventricle, and up to Na+-channel blockers (5), such as disopyramide, trains of 3 premature extrastimuli were applied to induce etacizin, pilsicainide, and procainamide (29). K+- non-sustained VT (lasting less than 30 s), sustained VT channel blockers, azimilide (75), dofetilide (48), E-4031 (lasting more than 30 s), or VF (47). If sustained VT or (46), KCB-328 (49), nifekalant (76), and sematilide VF occurred, epicardial defibrillation was performed to (47), did not suppress, but instead aggregated this stop them. After 2 control runs to reproduce re-entry arrhythmia (6, 76). To demonstrate K+-channel blockers’ arrhythmias, an intravenous bolus injection of a drug proarrhythmic effect, we added another adrenaline was given and then the same protocol of arrhythmia arrhythmia experiment (77, 78). We constructed a dose- induction was applied. response relationship by administering bolus intravenous adrenaline and plotted the maximum arrhythmic 2-4-3. Antiarrhythmic drugs ratio attained against the doses. Then after bolus injec- Consistent effectiveness was observed for K+-channel tion of K+-channel blockers, we again constructed blockers, azimilide (75), dofetilide (48), KCB-328 (49), adrenaline dose-response curves. The K+-channel block- nifekalant (80), and sematilide (47), with simultaneous ers we tested, nifekalant and KCB-328 (77) shifted the QT prolongation in ECG (6). Usually the control curve to the left, showing that the proarrhythmic effect arrhythmias were set up as severe ones by applying 3 of adrenaline was intensified. However Na+-channel premature extrastimuli. K+-channel blockers decreased blockers with an action potential prolonging effect, such the severity of arrhythmias to PVC or not inducible (6). as class 1A drugs like disopyramide, did not shift the For this arrhythmia, the procedure to induce arrhythmias curve, showing that the proarrhythmic effect of the K+- took some time, 2 – 3 min, so it was difficult to deter- channel block seems to be suppressed by Na+-channel mine the effective plasma concentration for each drug so block (79). Since Ca2+-channel activation must be the only quantitative analysis was made by comparing the mechanism of the generation of adrenaline arrhythmia, intravenous doses. Besides K+-channel blockers, several not only β-receptor–mediated activators, catecholamine- Na+-channel blockers, A-2545 (37), aprindine, and type positive inotropic drugs, but also phospho- disopyramide (81), have also been reported to be diesterase-inhibiting positive inotropic drugs also aggra- effective. vated the adrenaline arrhythmia (52 – 54). Among the positive inotropic drugs, MCI-154, a phosphodiesterase 2-4-4. Ineffective and proarrhythmic drugs inhibitor with a Ca2+-sensitizing property (56), showed After demonstration by CAST of proarrhythmic the least proarrhythmic effect on this arrhythmia. potentials of Na+-channel blockers (2), this arrhythmia Zatebradine had no suppressing effect on the adrenaline model was used to demonstrate the potentials of these arrhythmia (57). side effects. The worst reputed drug flecainide has been Arrhythmia Models and Drugs 341 shown to aggravate EPS induced arrhythmias when the the normal variability of the time of occurrence of the control arrhythmias were set up as mild arrhythmias, arrhythmias. In our rat model, LAD was not isolated, such as PVC (37, 82); however it is difficult to judge instead a silk thread with a curved needle was placed whether this arrhythmia model is an appropriate and under the myocardium together with the coronary artery sufficient one to differentiate or select dangerous Na+- and vein and both the artery and vein were tied for coro- channel blockers. nary occlusion under intraperitoneal pentobarbital anesthesia; in this method, the ischemia period ranged 2-5. Coronary artery occlusion/reperfusion arrhyth- from 5 – 120 min for occlusion arrhythmia studies and mia usually 5 min for reperfusion studies (85, 86). In the case 2-5-1. Characteristics of rat experiments, the number of VF occurrence was This arrhythmia model mimics myocardial ischemia counted along with the number of PVC. related arrhythmias that are frequently observed in the clinic. Coronary occlusion can be done by thread liga- 2-5-3. Antiarrhythmic drugs ture of the isolated coronary artery of the anesthetized Some Na+-channel and K+-channel blockers have animal under thoracotomy, by inflating a balloon been reported to be effective in suppressing coronary occluder or ligature of a prepositioned thread around the occlusion/reperfusion arrhythmias, but in the canine coronary artery in a conscious state, or by intracoronary model, the control occurrence of reperfusion VF was balloon inflation. For this model, various animals could variable and differed by anesthesia, about 60% in be used, such as dogs, cats, pigs, rabbits, rats, and so on, halothane anesthetized dogs (our unpublished data of with the exception of the guinea pig whose coronary 43/76 dogs) and about 70% in pentobarbital anesthe- artery collateral circulation is too well developed (11). It tized dogs (our data of 146/217 dogs); we always is known that there are predominant time zones for the compared drug effects with the vehicle-treated control occurrence of acute coronary occlusion arrhythmias group using the same number of dogs (6). We examined (83). In the case of dogs, the first 5 and 30 min after several K+-channel blockers (6, 46 – 50, 87) because occlusion are those time zones (46, 84). The generation ischemia must activate the ATP-dependent K+ channel mechanism of this arrhythmia is thought to be auto- and shorten action potential duration, so the K+-channel maticity induced ectopic beats and subsequent re-entry blocker with the opposite effect to prolong action movement of the excitation. The severity of arrhythmias potential duration should be effective. Actually E-4031 can be categorized by the type of arrhythmias, like PVC, (46) and nifekalant (84) suppressed coronary occlu- VT, or VF, or by the number of ectopic beats over time, sion/reperfusion arrhythmia in halothane-anesthetized if VF does not occur. Reperfusion, after complete dogs, and KCB-328 (49), nifekalant (84) and d-sotalol coronary occlusion, often induced VF immediately. This (84) suppressed those in pentobarbital anesthetized spontaneous arrhythmia occurs in almost 60% – 70% in dogs. Also ischemia stops the action of Na+/K+ ATPase dogs and nearly 100% in pigs and rats, when the main followed by increase in the intracellular Na+ concentra- trunk of the LAD is occluded (83 – 86). The VF results tion by Na+/H+ exchange, which is then followed by in death in dogs and pigs, but small animals like rats, VF Na+/Ca2+ exchange, inevitably resulting in intracellular very often reverts to sinus rhythm (83 – 86). Ca2+ overload (7). Thus the two important exchange system blockers also are expected to overcome this 2-5-2. Methods intracellular Ca2+ overload. Among these drugs, only Our canine coronary occlusion/reperfusion arrhyth- the Na+/H+-exchange inhibitors cariporide (70) and mias were produced in halothane- or pentobarbital- KB-R9032 (89) showed a consistent antifibrillatory anesthetized animals (46). After the left thoracotomy, effect in pentobarbital anesthetized dogs (7) and cari- the LAD was isolated and a string was placed for tying poride (70, 90 – 92), FR 186666 (93), and KB-R9032 the artery for usually 30 min, but we also used 20-min (94), in rats. These Na+/H+ exchange inhibitors have occlusion in some studies (48). After stabilization been reported to be protective in various animal models following the surgical procedures, a drug was given by of coronary occlusion/reperfusion injuries, and the an intravenous bolus or by an infusion and then coronary antiarrhythmic effects are one of the common effects (7). occlusion followed by reperfusion was performed. In Other effective drugs in rat models were DIDS (95) and some experiments, chronic oral administration of drugs long-term pravastatin administration (88), but whether for days or weeks before the experiment was performed the effects were due to Cl/HCO3 exchange inhibition or (87, 88). The occurrence of PVC or VF was monitored HMG-CoA reductase inhibition is unknown. Our recent by continuous ECG recordings. Normally, drug plasma unpublished data on halothane anesthetized dogs, short concentration determinations were not done because of or long term pravastatin administration did not demon- 342 K Hashimoto strate the favorable rat results of suppressing the with K+-channel blockers in normal animals anesthe- coronary occlusion/reperfusion arrhythmias. tized with halothane or in a conscious state and also in the old myocardial infarction dogs in a conscious state 2-5-4. Ineffective and proarrhythmic drugs (99). For finding the proarrhythmic effects of QT- As stated above, some K+-channel blockers were prolonging K+-channel blockers, many laboratories effective, but others, amiodarone (50, 87), dofetilide demonstrated AV block dogs are suitable (100 – 102) (48), KCB-328 (49), sematilide (47) and, d-sotalol and probably other animals such as monkeys seem to be (84), were not effective in halothane-anesthetized dogs; useful. Already it has been demonstrated that in chronic and dofetilide (48) and sematilide (47) were not effec- AV block dogs, bradycardia plus myocardial remodeling tive in pentobarbital-anesthetized dogs (6). Also in the increased the baseline QT prolongation and QT-prolong- halothane anesthetized low heart rate open chest dogs, ing cardiac and non-cardiac drugs induced TdP and VF some of the K+-channel blockers, amiodarone (50), in high incidences (100 – 107). Since the occurrence of dofetilide (48), E-4031 (46), KCB-328 (49), nifekalant K+-channel blockers’ induced TdP and death occur (84), and sematilide (47), showed a proarrhythmic effect rarely in man, may be one in 10,000 or more, whether by spontaneously inducing PVC and VT before applying this very high incidence of TdP in chronic AV block coronary occlusion, but these arrhythmias before dogs really mimics the human situation is still open for applying coronary occlusion were not observed in high discussion. heart rate pentobarbital-anesthetized dogs by dofetilide Other models already mentioned are halothane (not (48), KCB-328 (49), nifekalant (84), sematilide (47), pentobarbital) anesthetized open chest dogs, and sensiti- and d-sotalol (84). Not like Na+/H+-exchange inhibitors, zation in halothane-adrenaline arrhythmia models (6, the Na+/Ca2+-exchange inhibitors KB-R7943 (71) and 46 – 49, 75 – 77). Since QT prolongation per se is SEA0400 (64) had no antiarrhythmic effect on the beneficial in increasing contractile force and prevention coronary occlusion/reperfusion arrhythmia in pentobar- from re-entry arrhythmias, to identify high risk patients bital-anesthetized dogs. It may be that the Na+ overload among the QT-prolonging drug users, the human gene by inhibition of the Na+/H+ exchange may be more analysis for the long QT syndrome using inquiry of arrhythmogenic than Ca2+ overload by inhibition of the hereditary channelopathy may be an alternative Na+/Ca2+ exchange, but there is no proof for the in vivo approach to prevent cardiac accidents by drugs with heart to support this idea. Other ineffective drugs were properties to alter cardiac excitation and contraction. SITS, a Cl/HCO3 inhibitor (95), and long-term fluvasta- This problem is still a central issue for developing tin, a lipid soluble HMG-CoA reductase inhibitor (88), new drugs, and the International Conference on administration in rat models. Because of the well known Harmonization (ICH) has been working successfully to fact that this arrhythmia is severe and often fatal, it is establish guidelines for finding QT-prolonging pro- difficult to evaluate or reveal proarrhythmic effects of perties of drugs in preclinical and early clinical experi- drugs using this model, except in the pacing-induced ments (108). Future efforts are needed to differentiate heart-failure dogs, where all halothane-anesthetized good or non-lethal QT-prolonging drugs from possible dogs fibrillated just after reperfusion (96). dangerous ones and also identifying precipitating factors making safe QT prolongation to TdP induction, such as 2-6. Animal models to reveal proarrhythmic poten- sex hormones, electrolyte imbalance such as hypopotas- tials of drugs for safety pharmacological studies semia, hereditary abnormalities, and so on. Arrhythmia models are not only useful and essential to find a means to suppress arrhythmias, but recent 3. Limitations demonstration of proarrhythmic effects of drugs, such as a CAST study showing more deaths in patients receiving The present review mainly summarizes our own Na+-channel blockers (2) and in patients receiving QT- experimental results on drugs using various animal prolonging non-cardiovascular drugs, highlighted needs arrhythmia models. Already, there have been many to demonstrate proarrhythmic effects of drugs (97). Most excellent reviews on antiarrhythmic drugs that made of the lethal QT-prolonging drugs produce tachyarrhyth- pharmacologically and pharmacokinetically sound use mias, thus put simply, animals at slower basal heart rates of the presently available antiarrhythmic drugs, but there help in the detection of proarrhythmia. So anesthetized have been few experiments to compare drugs using with bradycardic agents, such as halothane (6, 98), several different animal models and applying the same unanesthetized animals (99), sinus node–crushed, or AV protocol for quantitative comparisons. Presently, the node–crushed animals (100 – 102) have been used. pharmacological and non-pharmacological treatments However, it was difficult to observe TdP arrhythmias for arrhythmias have reached a mature state, where Arrhythmia Models and Drugs 343 dramatically different new drugs may not appear and 801–810. effective electronic devices are being refined, down- 11 Maxwell MP, Hearse DJ, Tellon DM. Species variation in the sized, and becoming less expensive. Additionally the coronary collateral circulation during regional myocardial resistance to use whole animals in medical research is ischemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res. 1987;21:737– making arrhythmia model studies difficult and expen- 746. sive, so summarizing our previous results and others 12 Wu ZJ, Awaji T, Hirasawa A, Motomura S, Hashimoto K. may help decrease the useless repetition of experiments Effects of a new class I antiarrhythmic drug bidisomide on and sacrifice of precious animals and hopefully promote canine ventricular arrhythmia models. Mol Cell Biochem. the refining of antiarrhythmic drug research and as a 1993;119:159–169. result promote the emergence of some dramatically 13 Hashimoto K, Tsukada T, Matsuda H, Matsubara I, Imai S. different new drugs in the future. Antiarrhythmic effect of oxprenolol on halothane-epinephrine and coronary ligation induced ventricular arrhythmias in beagle dogs. Jpn J Pharmacol. 1978;28:535–544. Acknowledgments 14 Hashimoto K, Tsukada T, Matsuda H, Matsubara I, Imai S. Antiarrhythmic effects of bupranolol against canine ventricular Many thanks to my former colleagues, secretaries arrhythmias induced by halothane-adrenaline or two-stage who devoted their efforts to help fulfill my research coronary ligation. J Cardiovasc Pharmacol. 1979;1:205–217. interests, and Ms Yasuko Hashimoto, who always 15 Hashimoto K, Komori S, Ishii M, Kamiya J. Effective plasma helped to improve the English of my papers including concentrations of aprindine in canine ventricular arrhythmias. J Cardiovasc Pharmacol. 1984;6:12–19. the present one. 16 Hashimoto K, Ishii M, Komori S. Antiarrhythmic plasma concentrations of mexiletine on canine ventricular arrhythmias. References J Cardiovasc Pharmacol. 1984;6:213–219. 17 Ishii K, Komori S, Satoh H, Ohta T, Motomura S, Hashimoto K. 1 Christ T, Ravens U. Do we need new antiarrhythmic compounds [Effects of N-696, a new β-blocking agent, on canine experi- in the era of implantable cardiac devices and percutaneous mental arrhythmias.] Folia Pharmacol Jpn (Nihon Yakurigaku ablation? Cardiovasc Res. 2005;68:341–343. Zasshi). 1984;84:259–266. (text in Japanese with English 2 CAST Investigators. Preliminary report: effect of encainide and abstract) flecainide on mortality in a randomized trial of arrhythmia 18 Hashimoto K, Ishii M, Komori S, Mitsuhashi H. Canine digitalis suppression after myocardial infarction. N Engl J Med. 1989; arrhythmia as a model for detecting Na-channel blocking 321:406–412. antiarrhythmic drugs: a comparative study using other canine 3 Task Force of the Working Group on Arrhythmias of the arrhythmia models and the new antiarrhythmic drugs, pro- European Society of Cardiology. The Sicilian gambit: a new pafenone, tocainide, and SUN 1165. Heart Vessels. 1985;1:29– approach to the classification of antiarrhythmic drugs based on 35. their actions on arrhythmogenic mechanisms. Circulation. 19 Ishii M, Komori S, Hashimoto K. [Effects of pindolol on canine 1991;84:1831–1851. experimental arrhythmias.] Coronary. 1985;2:103–108. (in Japa- 4 Vaughan Williams EM. Classifying antiarrhythmic actions: by nese) facts or speculations. J Clin Pharmacol. 1992;32:964–977. 20 Mitsuhashi H, Akiyama K, Hashimoto K. [Effects of new 5 Hashimoto K, Haruno A, Matsuzaki T, Sugiyama A, Akiyama antiarrhythmic drugs, AFD-21 and AFD-19 on canine ventri- K. Effects of antiarrhythmic drugs on canine ventricular cular arrhythmias.] Folia Pharmacol Jpn (Nihon Yakurigaku arrhythmia models: which electrophysiological characteristics of Zasshi). 1986;88:167P. (in Japanese) drugs are related to their effectiveness? Cardiovasc Drugs Ther. 21 Hashimoto K, Akiyama K, Mitsuhashi H. Antiarrhythmic 1991;5:805–818. plasma concentrations of cibenzoline on canine ventricular 6 Hashimoto K, Xue YX, Chen JG, Akie Y, Eto K, Ni C, et al. arrhythmias. J Cardiovasc Pharmacol. 1987;9:148–153. Effects of class III antiarrhythmic drugs on ventricular 22 Mitsuhashi H, Akiyama K, Hashimoto K, Sawa Y, Hatori Y. arrhythmias in dogs. Exp Clin Cardiol. 1997;2:25–29. Antiarrhythmic effects of a new drug, E-0747, on canine 7 Hashimoto K, Sugiyama A, Xue YX, Aye NN, Yamada C, ventricular arrhythmia models. Jpn J Pharmacol. 1987;44:155– Chino D. Antiarrhythmic effects of Na+/H+ exchange inhibitors. 162. Eur Heart J Suppl. 1999; 1 Suppl K:K31–K37. 23 Mitsuhashi H, Hashimoto K. Antiarrhythmic profile of a new 8 Hashimoto K, Nagasawa E, Zhu BN. Na/H exchange and class 1 drug, AHR 10718, on canine atrial and ventricular arrhythmia. In: Dhalla NS, Takeda N, Singh M, Lukas A, arrhythmia models. Jpn J Pharmacol. 1988;46:349–358. editors. Myocardial ischemia and preconditioning. Boston: 24 Hashimoto K, Watanabe K, Sugiyama A. Antiarrhythmic plasma Kluwer Academic Publishers; 2003, p. 389–397. concentrations of pirmenol on canine ventricular arrhythmias. 9 Harris AS. Delayed development of ventricular ectopic rhythms Jpn J Pharmacol. 1988;48:273–282. following experimental coronary occlusion. Circulation. 1950;1: 25 Matsuzaki T, Haruno A, Sugiyama A, Motomura S, Hashimoto 1318–1328. K. [Effects of AFD-21 and AFD-19 on canine coronary ligation 10 Hashimoto K, Satoh H, Shibuya T, Imai S. Canine-effective arrhythmia.] Folia Pharmacol Jpn (Nihon Yakurigaku Zasshi). plasma concentrations of antiarrhythmic drugs on the two-stage 1988;92:36 P. (in Japanese) coronary ligation arrhythmia. J Pharmacol Exp Ther. 1982;223: 26 Hashimoto K, Akiyama K, Mitsuhashi H. Antiarrhythmic effect 344 K Hashimoto

of a new class 1 antiarrhythmic drug, nicainoprol, on canine Yamada K. Combined application of class I antiarrhythmic ventricular arrhythmias. Jpn J Pharmacol. 1989;49:245–254. drugs cause “additive” or “synergic” sodium channel block in 27 Hashimoto K, Ishii M, Watanabe K. Relationships between cardiac muscles. Cardiovasc Res. 1990;24:925–931. ventricular arrhythmias and plasma concentrations of ME3202 43 Takahara A, Sugiyama A, Dohmoto H, Yoshimoto R, (CM7857) in dogs. J Cardiovasc Pharmacol. 1989;14:121–126. Hashimoto K. Antiarrhythmic and cardiohemodynamic effects 28 Akiyama K, Hashimoto K. Antiarrhythmic effects of the class 1c of a novel Ca2+ channel blocker, AH-1058, assessed in canine antiarrhythmic drug, flecainide, on canine ventricular arrhythmia arrhythmia models. Eur J Pharmacol. 2000;398:107–112. models. Jpn Heart J. 1989;30:487–495. 44 Matsuzaki T, Haruno A, Hashimoto K. Effects of gallopamil, a 29 Hashimoto K, Matsuzaki T, Haruno A. Antiarrhythmic plasma Ca2+ channel blocker in models of ventricular arrhythmia in concentrations of NIK-244 on canine ventricular arrhythmias. dogs. Eur J Pharmacol. 1993;231:363–370. J Cardiovasc Pharmacol. 1989;14:892–898. 45 Mitsuhashi H, Akiyama K, Hashimoto K. Effects of betaxolol, a 30 Hashimoto K, Watanabe K, Mitsuhashi H. Antiarrhythmic effect new beta 1 selective blocker, on canine ventricular arrhythmias. of a new class 1 antiarrhythmic drug, AN-132, on ventricular Jpn J Pharmacol. 1987;43:179–185. arrhythmias in beagles. Cardiovasc Drugs Ther. 1989;3:683– 46 Hashimoto K, Haruno A, Matsuzaki T, Hirasawa A, Awaji T, 690. Uemura Y. Effects of a new class III antiarrhythmic drug (E- 31 Hashimoto K, Watanabe K, Mochizuki S, Tomiyama A. Effects 4031) on canine ventricular arrhythmia models. Asia Pacific J of KT-362, a new Na and Ca influx and Ca release inhibitor, on Pharmacol. 1991;6:127–137. canine ventricular arrhythmias. Jpn J Pharmacol. 1989;51:475– 47 XueYX, Eto K, Akie Y, Hashimoto K. Antiarrhythmic and 482. proarrhythmic effects of sematilide in canine ventricular 32 Hashimoto K, Sugiyama A, Haruno A, Matsuzaki T, Hirasawa arrhythmia models. Jpn J Pharmacol. 1996;70:129–138. A. Effects of a new antiarrhythmic drug TYB-3823 on canine 48 Chen JG, Xue YX, Eto K, Ni C, Hashimoto K. Effects of ventricular arrhythmia models. J Cardiovasc Pharmacol. 1991; dofetilide, a class III antiarrhythmic drug, on various ventricular 17:336–342. arrhythmias in dogs. J Cardiovasc Pharmacol. 1996;28:576–584. 33 Hirasawa A, Haruno A, Matsuzaki T, Hashimoto K. Effects of a 49 Xue YX, Tanabe S, Nabuchi Y, Hashimoto K. Antiarrhythmic new antiarrhythmic drug, SD-3212, on canine ventricular effects of a novel class III drug, KCB-328, on canine ventricular arrhythmia models. Jpn Heart J. 1992;33:851–861. arrhythmia models. J Cardiovasc Pharmacol. 1998;32:239–247. 34 Haruno A, Hashimoto K. Antiarrhythmic effects of bisaramil in 50 Awaji T, Wu ZJ, Hashimoto K. Acute antiarrhythmic effects of canine models of ventricular arrhythmia. Eur J Pharmacol. intravenously administered amiodarone on canine ventricular 1993;233:1–6. arrhythmia. J Cardiovasc Pharmacol. 1995;26:869–878. 35 Nakamura M, Xue YX, Eto K, Hashimoto K. Antiarrhythmic 51 Kodama I, Kamiya K, Toyama J. Amiodarone: ionic and cellular effects of optical isomers of disopyramide on canine ventricular mechanisms of action of the most promising class III agent. Am arrhythmias. J Cardiovasc Pharmacol. 1996;27:368–375. J Cardiol. 1999;84(9A):20R–28R. 36 Vanhoutte F, Vereecke J, Carmeliet E, Verbeke N. Effects of the 52 Hashimoto K, Haruno A, Hirasawa A. [Effects of milrinone on enantiomers of disopyramide and its major metabolite on the canine experimental arrhythmias.] Coronary. 1990;7:445–450. electrophysiological characteristics of the guinea-pig papillary (in Japanese) muscle. Naunyn Schmiedebergs Arch Pharmacol. 1991;344: 53 Hashimoto K, Mitsuhashi H. Effects of OPC-8212, a new 662–673. positive inotropic agent, on canine ventricular arrhythmias. Br J 37 Xue YX, Arita J, Aye NN, Hashimoto K. Effects of an anti- Pharmacol. 1986;88:915–921. arrhythmic drug A-2545 on canine ventricular arrhythmia 54 Wu Z, Awaji T, Abe H, Motomura S, Hashimoto K. Effects of models; comparison with mexiletine and flecainide. Naunyn OPC-18790, a new positive inotropic agent, on canine ventri- Schmidebergs Arch Pharmacol. 1998;358:649–656. cular arrhythmias. Jpn J Pharmacol. 1993;63:399–404. 38 Arita J, Xue YX, Aye NN, Fukuyama K, Wakui Y, Niitsu K, 55 Hirasawa A, Awaji T, Hosono M, Haruno A, Hashimoto K. et al. Antiarrhythmic effects of an aconitine-like compound, Effects of a new forskolin derivatives, NKH477, on canine TJN-505, on canine arrhythmia models. Eur J Pharmacol. ventricular arrhythmia models. J Cardiovasc Pharmacol. 1993; 1996;318:333–340. 22:847–851. 39 Saitoh M, Sugiyama A, Hagihara A, Nakazawa T, Hashimoto K. 56 Eto K, Xue YX, Hashimoto K. Effects of MCI-154, a new Cardiovascular and antiarrhythmic effects of the azulene-1- cardiotonic Ca2+ sensitizer, on ventricular arrhythmias and carboxamidine derivative N1,N1-dimethyl-N2-(2-pyridylmethyl)- membrane ionic currents. Eur J Pharmacol. 1996;98:247–256. 5-isopropyl-3,8-dimethylazulene-1-carboxamidine. Arzneimittel- 57 Furukawa Y, Xue YX, Chiba S, Hashimoto K. Effects of forschung. 1997;47:810–815. zatebradine on ouabain-, two-stage coronary ligation- and 40 Takahara A, Hirasawa A, Dohmoto H, Shoji M, Yoshimoto R, epinephrine-induced ventricular tachyarrhythmias. Eur J Sugiyama A, et al. In vivo antiarrhythmic profile of AP-792 Pharmacol. 1996;300:203–210. assessed in different canine arrhythmia models. Jpn J Pharmacol. 58 Naito H, Furukawa Y, Hashimoto K. Effects of a cardioselective 2001;87:21–26. M2 receptor antagonist, AF-DX 116, on ventricular arrhythmias 41 Awaji T, Hashimoto K. Antiarrhythmic effects of combined in dogs. Heart Vessels. 1997;12:229–233. application of class I antiarrhythmic drugs; Addition of low-dose 59 Hashimoto K. Electrophysiological mechanisms for digitalis mexiletine-enhanced antiarrhythmic effects of disopyramide arrhythmias. Jpn Circ J. 1976;40:1451–1455. and aprindine in various-rate canine ventricular tachycardias. J 60 Roden DM. Antiarrhythmic drugs. In: Brunton LL, Lazo JS, Cardiovasc Pharmacol. 1993;21:960–966. Parker KL, editors. Goodman & Gilman’s The pharmacological 42 Kawamura K, Kodama I, Toyama J, Hayashi H, Saito H, basis of therapeutics. 11th ed. McGraw-Hill; 2006. p. 899–932. Arrhythmia Models and Drugs 345

61 Glitsch HG. Electrophysiology of the sodium-potassium- 78 Miyamoto S, Zhu B-M, Aye NN, Hashimoto K. Slowing Na+ ATPase in cardiac cells. Physiol Rev. 2001;81:1791–1826. channel inactivation prolongs QT interval and aggravates 62 Hashimoto K, Shibuya T, Satoh H, Imai S. Quantitative analysis adrenaline-induced arrhythmias. Jpn J Pharmacol. 2001;86:114– of the antiarrhythmic effect of drugs on canine ventricular 119. arrhythmias by the determination of minimum effective plasma 79 Miyamoto S, Zhu B-M, Teramatsu T, Aye NN, Hashimoto K. concentrations. Jpn Circ J. 1983;47:92–97. QT-prolonging class I drug, disopyramide, does not aggravate 63 Sugiyama A, Xue YX, Hagihara A, Saitoh M, Hashimoto K. but suppresses adrenaline-induced arrhythmias. Comparison Characterization of magnesium sulfate as an antiarrhythmic with cibenzoline and pilsicainide. Eur J Pharmacol. 2000;400: agent. J Cardiovasc Pharmacol Ther. 1996;1:243–254. 263–269. 64 Nagasawa Y, Zhu B-M, Chen J, Kamiya K, Miyamoto S, 80 Kamiya J, Ishii M, Katakami T. Antiarrhythmic effect of MS- Hashimoto K. Effects of SEA0400, a Na+/Ca2+ exchange inhibi- 551, a new class III antiarrhythmic agent, on canine models of tor, on ventricular arrhythmias in the in vivo dogs. Eur J Pharma- ventricular arrhythmias. Jpn J Pharmacol. 1992;58:107–115. col. 2005;506:249–255. 81 Ogawa S, Mitamura H, Katoh H. Effect of E-4031, a new class 65 Komori S, Ishii M, Hashimoto K. Antiarrhythmic effects of III antiarrhythmic drug, on reentrant ventricular arrhythmias in coronary vasodilators on canine ventricular arrhythmia models. comparison with conventional class I drugs. Cardiovasc Drugs Jpn J Pharmacol. 1985;38:73–82. Ther. 1993;7 Suppl 3: 621–626. 66 Hashimoto K, Mitsuhashi H, Nakamura T, Akiyama K. Anti- 82 Kigwell GA, Gonzales MD. Effects of flecainide and D-sotalol arrhythmic effects of possible anti-ischemic drugs. Yamanashi on myocardial conduction and refractoriness: relation to Med J. 1986;1:1–10. antiarrhythmic and proarrhythmic drug effects. J Cardiovasc 67 Hashimoto K, Mitsuhashi H, Nakamura T, Akiyama K. Anti- Pharm. 1993;21:621–632. arrhythmic effects of possible anti-ischemic drugs. In: Szekeres 83 Karagueuzian HS, Mandel WJ. Electrophysiologic mechanisms L, Papp JGy, editors. Proc 4th Cong Hung Pharmacol Soc, of ischemic ventricular arrhythmias. In: Mandel JW, editor. Budapest 1985, Vol 1 Sec 1, Pharmacological protection of Cardiac arrhythmias. Their mechanisms, diagnosis, and manage- the myocardium; 1985. p. 25–31. ment. Philadelphia: Lippincott; 1987. p. 452–474. 68 Haruno A, Hashimoto K. Antiarrhythmic effects of bisaramil on 84 Hashimoto K, Haruno A, Hirasawa A, Awaji T, Xue YX, Wu triggered arrhythmias produced by intracoronary injection of ZJ. Effects of the new class III antiarrhythmic drug MS-551 and digitalis and adrenaline in the dog. Jpn J Pharmacol. 1995;68: d-sotalol on canine coronary ligation-reperfusion ventricular 95–102. arrhythmias. Jpn J Pharmacol. 1995;68:1–9. 69 Hashimoto K. Electrophysiological correlates of antiarrhythmic 85 Komori S, Sano S, Li BH, Matsumura K, Naitoh A, Mochizuki effects of drugs and pharmacokinetic principles examined using S, et al. Effects of bidisomide (SC-40230), a new class I anti- canine ventricular arrhythmias. In: Papp JGy, editor. Cardio- arrhythmic agent, on ventricular arrhythmias induced by vascular pharmacology ’87. Budapest: Academiai Kiado; 1987. coronary artery occlusion and reperfusion in anesthetized rats; p. 79–93. comparison with mexiletine and disopyramide. Heart Vessels. 70 Xue YX, Aye NN, Hashimoto K. Antiarrhythmic effects of 1995;10:7–11. HOE642, a novel Na+-H+ exchange inhibitor, on ventricular 86 Clark C, Foreman MI, Kane KA, McDonald FM, Parratt JR. arrhythmias in animal hearts. Eur J Pharmacol. 1996;317:309– Coronary artery ligation in anaesthetised rats as a method for the 316. production of experimental dysrhythmias and for the determina- 71 Miyamoto S, Zhu B-M, Kamiya K, Nagasawa Y, Hashimoto K. tion of infarct size. J Pharmacol Methods. 1980;3:357–368. KB-R7943, a Na+/Ca2+ exchange inhibitor, does not suppress 87 Nagasawa Y, Chen J, Hashimoto K. Antiarrhythmic properties ischemia/reperfusion arrhythmias nor digitalis arrhythmias in of a prior oral loading of amiodarone in in vivo canine coronary dogs. Jpn J Pharmacol. 2002;90:229–235. ligation/reperfusion-induced arrhythmia model: comparison 72 Hashimoto K, Hashimoto K. The mechanism of sensitization of with other class III antiarrhythmic drugs. J Pharmacol Sci. the ventricle to epinephrine by halothane. Am Heart J. 1972; 2005;97:393–399. 83:652–658. 88 Chen J, Nagasawa Y, Zhu B-M, Ohmori M, Harada K, Fujimura 73 Noda Y, Hashimoto K. Development of a halothane-adrenaline A, et al. Pravastatin prevents arrhythmias induced by coronary arrhythmia model using in vivo guinea pigs. J Pharmacol Sci. artery ischemia/reperfusion in anesthetized normocholestrol- 2004;95:234–239. emic rats. J Pharmacol Sci. 2003;93:87–94. 74 Shibuya T, Hashimoto K, Imai S. Effective plasma concentra- 89 Yamada C, Xue YX, Chino D, Hashimoto K. Effects of tions of antiarrhythmic drugs against sustained halothane- KB-R9032, a new Na+/H+ exchange inhibitor, on canine adrenaline arrhythmia in dogs. J Cardiovasc Pharmacol. 1983; coronary occlusion/reperfusion-induced ventricular arrhyth- 5:538–545. mias. J Pharmacol Sci. 2005;98:404–410. 75 Xue YX, Yamada C, Chino D, Hashimoto K. Effects of 90 Aye NN, Xue YX, Hashimoto, K. Antiarrhythmic effects of + + azimilide, a KV(r) and KV(s) blocker, on canine ventricular arrhyth- cariporide, a novel Na -H exchange inhibitor, on reperfusion mia models. Eur J Pharmacol. 1999;376:27–35. ventricular arrhythmias in rat hearts. Eur J Phramcol. 1997; 76 Hashimoto K, Xue YX, Aye NN, Chino D. Worsening of canine 339:121–127. adrenaline arrhythmias by class III K channel blockers. J Cardio- 91 Aye NN, Komori S, Hashimoto, K. Effects and interaction, of vasc Pharmacol. 1999;34 Suppl 4:S39–S42. cariporide and preconditioning on cardiac arrhythmias and 77 Xue YX, Yamada C, Aye NN, Hashimoto K. MS-551 and infarction in rat in vivo. Br J Pharmacol. 1999;127:1048–1055. KCB-328, two class III drugs aggravated adrenaline-induced 92 Sugiyama A, Aye NN, Sawada N, Hashimoto K. Cariporide, a arrhythmias. Br J Pharmacol. 1998;124:1712–1718. highly selective Na+/H+ exchange inhibitor, suppresses the 346 K Hashimoto

reperfusion-induced lethal arrhythmias and “overshoot” pheno- 102 Weissenburger J, Davy J-M, Chezalviel F, Ertzbischoff O, menon of creatine phosphate in situ rat heart. J Cardiovasc Poirer JM, Engel F, et al. Arrhythmogenic activities of anti- Pharmacol. 1999;33:116–121. arrhythmic drugs in conscious hypokalemic dogs with atrio- 93 Zhu BM, Miyamoto S, Kamiya K, Komori S, Hashimoto K. ventricular block: Comparison between quinidine, lidocaine, Inhibitory effects of pre-ischemic and post-ischemic treatment flecainide, propranolol and sotalol. J Pharmacol Exp Ther. with FR 168888, a new Na+/H+ exchange inhibitor, on reperfu- 1991;259:871–883. sion-induced ventricular arrhythmias in anesthetized rat. Jpn J 103 Sugiyama A, Ishida Y, Satoh Y, Aoki S, Hori M, Akie Y, et al. Pharmacol. 2002;88:93–99. Electrophysiological, anatomical and histological remodeling of 94 Harada K, Sugimoto H, Shimamoto A, Watano T, Nishimura N. the heart to AV block enhances susceptibility to arrhythmogenic Cardioprotective effects of a novel Na+/H+ exchange inhibitor, effects of QT-prolonging drugs. Jpn J Pharmacol. 2002;88:341– KBR-9032, on cardiac ischemia-reperfusion injury in rats. Jpn J 350. Pharmacol. 1997;73 Suppl 1:232P. 104 Sugiyama A, Satoh Y, Takahara A, Nakamura Y, Shimizu- 95 Zhu B-M, Miyamoto S, Nagasawa Y, Saitoh M, Komori S, Sasamata M, Sato S, et al. Femotidine does not induce long QT − − Hashimoto K. Does Cl /HCO3 exchange play an important role syndrome: experimental evidence from in vitro and in vivo test in reperfusion arrhythmias in rats? Eur J Pharmacol. 2003;460: systems. Eur J Pharmacol. 2003;466:137–146. 43–50. 105 Yoshida H, Sugiyama A, Satoh Y, Ishida Y, Yoneyama M, 96 Arita J, Katahira S, Xue YX, Aye NN, Hashimoto K. Rapid Kugiyama K, et al. Comparison of the in vivo electrophysio- pacing-induced heart failure aggravates reperfusion arrhythmias logical and proarrhythmic effects of amiodarone with those of a in dogs. Asia Pacific J Pharmacol. 1998;13:137–144. selective class III drug, sematilide, using a canine chronic 97 Tamargo J. Drug-induced torsade de pointes: from molecular atrioventricular block model. Circulation J. 2002;66:758–762. biology to bedside. Jpn J Pharmacol. 2000;83:1–19. 106 Satoh Y, Sugiyama A, Takahara A, Chiba K, Hashimoto K. 98 Takahara A, Sugiyama A, Satoh Y, Wang K, Honsho S, Electrophysiological and proarrhythmic effects of a class III Hashimoto K. Halothane sensitizes the canine heart to antiarrhythmic drug nifekalant hydrochloride assessed using the pharmacological IKr blockade. Eur J Pharmacol. 2005;507:169– in vivo canine models. J Cardiovasc Pharmacol. 2004;43:715– 177. 723. 99 Akie Y, Ni C, Aye NN, Xue YX, Hashimoto K. Proarrhythmic 107 Takahara A, Sugiyama A, Ishida Y, Satoh Y, Wang K, effects of four class III antiarrhythmic drugs, MS-551, Nakamura Y, et al. Long-term bradycardia caused by atrio- sematilide, dofetilide and KCB328, examined using ambulatory ventricular block can remodel the canine heart to detect the ECG. Asia Pacific J Pharmacol. 2000;14:101–109. histamine H1 blocker terfenadine-induced torsades de pointes ar- 100 Chiba K, Sugiyama A, Satoh Y, Shiina H, Hashimoto K. rhythmias. Br J Pharmacol. 2006;147:634–641. Proarrhythmic effects of fluoroquinolone antibacterial agents: in 108 U.S. Department of Health and Human Services, Food and Drug vivo effects as physiologic substrate for torsades. Toxicol Appl Administration, Center for Drug Evaluation and Research, Pharmacol. 2000;169:8–16. Center for Biologics Evaluation and Research. S7B Nonclinical 101 Vos MA, Verduyn SC, Gorgels APM, Lipcsei GC, Wellens HJJ. Evaluation of the Potential for Delayed Ventricular Repolariza- Reproducible induction of early afterdepolarizations and torsade tion (QT Interval Prolongation) by Human Pharmaceuticals. de pointes arrhythmias by d-sotalol and pacing in dogs with October 2005 ICH. chronic atrioventricular block. Circulation. 1995;91:864–872.