Antiarrhythmic Efficacy of Combined IKs and -Adrenergic Receptor Blockade

Suppression of malignant ventricular arrhythmias by selective blockade of the cardiac slowly activating delayed rectifier current (IKs) has been demonstrated with the benzodiazepine L-768673 [(R)-2-(2,4-trifluoromethyl-phenyl)-N-[2-oxo-5-phenyl-1-(2,2,2trifluoro-ethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl]acetamide] in canine models of recent and healed myocardial infarction. The present study extends the initial antiarrhythmic assessment of IKs blockade by demonstrating prevention of ischemic malignant arrhythmias in dogs with recent (8.0 0.4 days) anterior myocardial infarction with the coadministration of a subeffective dose of L-768673 and a subeffective, minimally -adrenergic blocking dose of timolol. Administered individually, neither 0.3 g/kg i.v. L-768673 nor 1.0 g/kg i.v. timolol prevented the induction of ventricular tachyarrhythmia (VT) by programmed ventricular stimulation (PVS) or the development of malignant ventricular arrhythmia in response to acute coronary artery thrombosis. In contrast, coadministration of 0.3 g/kg i.v. L-768673 1.0 g/kg i.v. timolol suppressed the induction of VT by PVS (8/10, 80% rendered noninducible versus 1/10, 10% noninducible in vehicle group; p 0.01) and prevented the development of acute ischemic lethal arrhythmias (3/10, 30% incidence versus 8/10, 80% incidence in vehicle group; p 0.05). Concomitant administration of low-dose L-768673 timolol produced modest increases in QTc and paced QT intervals (4.5 1.2 and 5.5 1.4%; both p 0.01), increases in noninfarct zone relative and effective refractory periods (7.0 1.7 and 12.3 3.0%; both p 0.01), and lesser increases in infarct zone relative and effective refractory periods (5.3 1.6 and 5.8 1.4%; both p 0.01). These findings suggest that concomitant low-dose IKs and -adrenergic blockade may constitute a potential pharmacologic strategy for prevention of malignant ischemic ventricular arrhythmias. Delay of myocardial repolarization (class III electrophysiologic activity) via blockade of repolarizing potassium currents has been advanced as a potential antiarrhythmic mechanism. This is based on the premise that sufficient prolongation of myocardial refractoriness results in a wavelength of excitation that exceeds the path length of reentrant circuits, thereby preventing the initiation and/or maintenance of reentrant rhythms (Wellens et al., 1984). Myocardial repolarization in the majority of mammalian species studied, including humans, is controlled mainly by the interplay of the rapidly (IKr) and slowly (IKs) activating, delayed rectifier potassium currents (Sanguinetti and Jurkiewicz, 1990; Wang et al., 1994; Liu and Antzelevitch, 1995; Li et al., 1996; Salata et al., 1996a; Virag et al., 2001). The clinical assessment of selective blockers of IKr for the treatment of malignant ventricular arrhythmia has yielded disappointing results. d-Sotalol increased mortality in patients with previous myocardial infarction and left ventricular dysfunction (Waldo et al., 1996), and dofetilide displayed a neutral effect on mortality in patients with reduced left ventricular function and congestive heart failure (Torp-Pedersen et al., 1999). Characteristics of IKr blockers, which have been proposed to limit clinical antiarrhythmic efficacy, include reverse frequency dependence, whereby class III activity is diminished at faster heart rates and exaggerated at slower rates (Nattel and Zeng, 1984; Hondeghem and Snyders, 1990), and reduction of class III activity in the setting of sympathetic stimulation (Sanguinetti et al., 1991; Schreieck et al., 1997). A number of structurally distinct selective IKs blockers recently have become available for preclinical assessment. Initial studies indicate that selective IKs block may provide a profile of class III action differing significantly from that of IKr block, particularly with regard to frequency dependence ABBREVIATIONS: PVS, programmed ventricular stimulation; RVOT, right ventricular outflow tract; NZ, noninfarct zone; IZ, infarct zone; VRRP, ventricular relative refractory period; VERP, ventricular effective refractory period; NI, noninducible; VT, ventricular tachycardia; VF, ventricular fibrillation; LCX, left circumflex; APD, action potential duration; chromanol 293b, trans-6-cyano-4(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2dimethyl-chromane; LQT, long QT; L-768673, (R)-2-(2,4-trifluoromethyl-phenyl)-N-[2-oxo-5-phenyl-1-(2,2,2-trifluoro-ethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl]acetamide; L-735821, (R)-3-(2,4-dichlorophenyl)-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)acrylamide; HMR, (3R,4S)-( )-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methylmethanesulfonamide. 0022-3565/02/3021-283–289$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4823/990546 JPET 302:283–289, 2002 Printed in U.S.A. 283 at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from and activity during sympathetic stimulation, which may impart improved antiarrhythmic efficacy (Gerlach, 2001). To this point, the benzodiazepine IKs blocker L-768673 (Fig. 1) has been shown to prevent the development of malignant ventricular arrhythmia in anesthetized dogs with acute thrombotic coronary ischemia superimposed upon a recent myocardial infarction, and in conscious dogs with acute coronary ischemia and exercise superimposed upon a healed myocardial infarction (Lynch et al., 1999). Antiarrhythmic efficacy in the latter model was noteworthy because this conscious preparation possesses high sympathetic tone, and the IKr blocker d-sotalol was ineffective in this model (Vanoli et al., 1995). -Adrenergic receptor blockade also has demonstrated antiarrhythmic efficacy in preclinical animal models (e.g., Gang et al., 1984; Euler and Scanlon, 1988). In vitro studies have reported that -adrenergic stimulation increases the magnitude of IKs current (Sanguinetti et al., 1991; Han et al., 2001) and increases the effect of IKs blockers (Schreieck et al., 1997; Han et al., 2001), and that concomitant IKs block and -adrenergic stimulation results in exaggerated, inhomogeneous, and potentially proarrhythmic effects (Schreieck et al., 1997; Burashnikov and Antzelevitch, 2000; Shimizu and Antzelevitch, 2000). We therefore hypothesized that concomitant lowdose IKs and -adrenergic receptor blockade might provide enhanced and potentially safer antiarrhythmic activity than either mechanism alone. We tested this hypothesis by extending our previous antiarrhythmic assessment of the IKs blocker L-768673 in dogs with recent myocardial infarction (Lynch et al., 1999). The present study assesses the cardiac electrophysiologic and antiarrhythmic actions of a subeffective dose of L-768673 and a subeffective, minimally -adrenergic blocking dose of timolol, administered individually or in combination. The results of this study demonstrate significantly greater suppression of malignant arrhythmias with combination low-dose IKs and -adrenergic blockade. Materials and Methods All procedures related to the use of animals in these studies were reviewed and approved by the Institutional Animal Care and Use Committee at Merck Research Laboratories at West Point and conform with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996). Determination of -Adrenergic Blocking Dose of Timolol in Dogs with Recent Anterior Myocardial Infarction. A separate group of dogs (7.5–11.0 kg, 9.6 0.2 kg; total n 20) was used to determine a minimally -adrenergic blocking dose of timolol for use in subsequent antiarrhythmic studies. These animals possessed anterior myocardial infarctions produced surgically as described below, 10.1 0.8 days prior to study. The postinfarction animals were studied during anesthesia with -chloralose (80.0–100.0 mg/kg i.v.) and minimal sodium pentobarbital as needed (5 mg/kg i.v.), and ventilation using a cuffed endotracheal tube and a volume-cycled respirator. -Adrenergic activity was assessed by monitoring heart rate increases in response to bolus intravenous doses of 0.01 to 3.0 g/kg isoproterenol at 15, 75, 135, and 195 min after the following treatments: no timolol (n 5), 1.0 g/kg i.v. timolol (n 4), 10.0 g/kg i.v. timolol (n 6), and 100.0 g/kg i.v. timolol (n 5). The 15to 195-min time frame for assessment of -adrenergic blocking activity of timolol encompassed the time frame for response of postinfarction dogs to the development of acute, thrombotically induced posterolateral myocardial ischemia in the subsequent antiarrhythmic studies described below. Canine Model of Recent Anterior Myocardial Infarction. The anesthetized canine model of recent anterior myocardial infarction in which ventricular tachyarrhythmias may be induced by programmed ventricular stimulation (PVS) and in which malignant ventricular arrhythmias develop in response to acute coronary artery thrombosis was used to assess the antiarrhythmic efficacy of low-dose IKs and -adrenergic receptor blockade. This model was used previously to characterize the dose-dependent cardiac electrophysiologic and antiarrhythmic actions of the IKs blocker L-768673 (Lynch et al., 1999). The present study extends this initial evaluation through a comparison of the following four treatment groups: 1) microemulsion vehicle (n 10), 2) a subeffective 0.3 g/kg i.v. dose of L-768673 (n 10), 3) a minimally -adrenergic blocking dose of 1.0 g/kg timolol (n 8) determined in studies described above, and 4) coadministration of the subeffective 0.3 g/kg i.v. L-768673 the 1.0 g/kg timolol doses (n 10). These four treatment groups were studied concurrently and in randomized fashion. Surgical Preparation. Male or female purpose-bred mongrel dogs (7.2–11.4 kg; 9.1 0.1 kg; total n 38) were preanesthetized with sodium thiamylal (5.0 mg/kg i.v.), and general anesthesia was induced with isoflurane. A left thoracotomy was performed in the fourth intercostal space, the pericardium was incised, and the heart was suspended in a pericardial cradle. Anterior myocardial infarction was produced by a 2-h occlusion of the left anterior descending coronary artery followed by reperfusion. Surgical incisions were closed, and the animals were allowed to recover. Electrophysiologic Testing, Programmed Ventricular Stimulation, and Acute Posterolateral Myocardial Ischemia. Animals were studied at 8.0 0.4 days after anterior myocardial infarction. Postinfarction dogs were anesthetized with -chloralose (80.0–100.0 mg/kg i.v.) and minimal sodium pentobarbital as needed Fig. 1. Chemical structures of L-768673 and L-735821. 284 Lynch et al. at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from (5 mg/kg i.v.) and were ventilated by means of a cuffed endotracheal tube and a volume-cycled respirator. Systemic arterial pressure was monitored via the cannulated left common carotid artery, and the right femoral vein was isolated and cannulated for test compound administration. The heart was re-exposed via a left thoracotomy and suspended in a pericardial cradle. A surface bipolar electrode was sutured to the left atrial appendage for atrial pacing, and a bipolar plunge electrode was inserted into the interventricular septum near the right ventricular outflow tract (RVOT) adjacent to the site of left anterior descending coronary artery occlusion for the introduction of ventricular extrastimuli during PVS. One bipolar plunge electrode per zone was sutured into the infarcted anterior region of the left ventricle distal to the site of coronary artery occlusion and within the area of myocardial scarring as ascertained visually and by palpation (infarct zone, IZ) and into the noninfarcted posterolateral region of the left ventricle (noninfarct zone, NZ) for the measurement of ventricular excitation thresholds and refractory periods. Lead II electrocardiogram was monitored continuously. After stabilization of the preparation, the following baseline parameters were measured or derived: sinus heart rate, mean arterial pressure (diastolic plus one-third pulse pressure), electrocardiographic intervals including a rate-corrected QTc interval [QTc (QT milliseconds)(R-R seconds) ] and a paced QT interval determined during 2.5-Hz atrial pacing, NZ and IZ ventricular excitation thresholds (2-ms pulse duration, 300-ms coupling interval) during 2.5-Hz atrial pacing, and NZ and IZ ventricular relative (VRRP) and effective (VERP) refractory periods (2-ms pulse duration at 2 and 10 times the ventricular excitation thresholds, respectively) during 2.5-Hz atrial pacing. After the measurement of cardiac electrophysiologic parameters, PVS consisting of the introduction of one to three ventricular extrastimuli (S2–S4) during sinus rhythm and atrial pacing was performed at the RVOT site. If S2 was ineffective at exciting the RVOT site, PVS was attempted at the IZ site. S2 to S4 were applied at 2 times the diastolic threshold voltage or, if ineffective at this level, at 4 times the diastolic threshold voltage. Responses to baseline PVS were categorized as noninducible (NI), sustained ventricular tachycardia (VT, unimorphic or polymorphic), or VT degenerating into ventricular fibrillation (VT/VF) as defined previously (Lynch et al., 1999). PVS was continued until the initiation of either sustained VT or VT/VF, or until reaching S4 with no VT induction (NI). Baseline PVS-inducible sustained VT or VT/VF was the entry criterion for this study, i.e., all animals entered into the study responded to pretreatment baseline PVS with sustained VT or