Introduction

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Diheterabicyclo[3.3.1]nonane-based compounds have shown potential as antiarrhythmic drugs, suppressing sustained, reentrant ventricular tachycardias in the dog during subacute myocardial infarction (Scherlag et al., 1988; Smith et al., 1990; Bailey et al., 1984; Fazekas et al., 1993a; Fazekas et al., 1993b). Most of the diheterabicyclo[3.3.1]nonane compounds studied to date have little effect on blood pressure in the dog during subacute myocardial infarction (Scherlag et al., 1988, Smith et al., 1990; Bailey et al., 1984; Fazekas et al., 1993a; Fazekas et al., 1993b) and reduce the force of contraction in rabbit papillary muscles, only at supratherapeutic concentrations producing inexcitability (Chen et al., 1992, Fazekas et al., 1993c).

The first diheterabicyclo[3.3.1]nonane to be evaluated using intracellular microelectrode recordings was BRB-I-28. BRB-I-28 differs from the present drugs in having a 3-thia substituent (3-azo in GLG-V-13 and KMC-IV-84) and a 7-benzyl substituent on the 7-azo position (amide-linked imidazole in GLG-V-13 and sulfonamide-linked imidazole in KMC-IV-84) (fig. 1). BRB-I-28 reduces V_max and/or conduction times in normal cardiac tissues only at paced cycle lengths less than 300 msec in vivo (Scherlag et al., 1988; Patterson et al., 1993) and in vitro (Patterson et al., 1993). The depression of V_max and conduction velocity only at rapid HRs is consistent with the drug's molecular weight (223.1 daltons), pK_a (11.8), and low lipid solubility (octanol:water partition coefficient 2.82). Ischemic injury or subacute myocardial infarction increases both tonic conduction block and use-dependent conduction block (Patterson et al., 1993). BRB-I-28 produces little change in action potential duration in Purkinje tissue, right ventricular endocardium and left ventricular epicardium (Patterson et al., 1991; Patterson et al., 1993).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structures of BRB-I-28, GLG-V-13 and KMC-IV-84. In GLG-V-13 and KMC-IV-84, nitrogen has been substituted for sulfur in the diheterabicyclo[3.3.1]nonane ring. GLG-V-13 contains an amide-imidazole linkage, and KMC-IV-84 contains a sulfonamide-imidazole linkage.

Modifications were included into the diheterabicyclo[3.3.1]nonane structure of BRB-I-28 and related drugs to incorporate action potential prolongation. To increase separation of the two positively charged nitrogens, the positive charge of the 7-benzyl amine substituent on the diheterabicyclo[3.3.1]nonane ring was changed to a noncharged sulfonamide or amide linked through a benzene ring to a charged imidazole. This change was proposed to increase hydrophilicity and possibly to add class III electrophysiologic activity (inhibition of I_K) (Morgan et al., 1990; Lis et al., 1990) to the existing class Ib activity of BRB-I-28. The following experiments were performed to determine the cellular electrophysiologic actions of the imidazole-substituted diheterabicyclo[3.3.1]nonane drugs GLG-V-13 (3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane dihydroperchlorate) and KMC-IV-84 (7-[4-(1H-imidazol-1-yl)benzenesulfonyl]-3-isopropyl-3,7-diazabicyclo[3.3.1]nonane dihydroperchlorate) in canine ventricular tissues.

	Methods

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Purkinje tissues. Male mongrel dogs were anesthetized with i.v. sodium pentobarbital (30 mg/kg). Tissue sections containing free-running Purkinje fiber strands were removed from right (N = 12) or left (N = 12) ventricular endocardium. The tissues were superfused with Tyrode's solution (115 mM sodium chloride, 4.0 mM potassium chloride, 1.0 mM sodium dihydrogen phosphate, 1.5 mM magnesium chloride, 24 mM sodium bicarbonate and 1.35 mM calcium chloride) bubbled with 95% oxygen: 5% carbon dioxide (pH = 7.4). A Grass S-88 stimulator was used to initiate electrical activity at the proximal insertion of a free-running Purkinje fiber (2-msec stimuli, twice diastolic threshold). A glass microelectrode (10-20-Mohm resistance) was used to record intracellular potentials from the free-running Purkinje fiber. A bipolar electrode was used to record activation of the same fiber at its distal insertion. A differentiating amplifier with peak-and-hold detector was used to record V_max. Differentiation was linear in the range of 50 to 1000 V/sec. Membrane responsiveness curves were obtained by the introduction of premature stimuli. Electrical recordings were performed during pacing at rates from 0.2 to 4.0 Hz and at a pacing rate of 1.0 Hz with the introduction of premature stimuli from 500 msec to local refractoriness in 5-msec intervals. All electrical recordings were digitized and stored, and permanent records were obtained using a Gould ES 2000 recording system (Gould Electronics).

Subendocardial right ventricular myocardium. Male mongrel dogs were anesthetized with i.v. sodium pentobarbital (30 mg/kg). Subendocardial tissue sections measuring approximately 8 × 15 mm were removed from the right ventricle, immediately beneath the tricuspid valve annulus. The tissue preparations were superfused with Tyrode's solution (composition described previously). A Grass S-88 stimulator was used to pace the tissue preparation (2-msec stimuli, twice diastolic threshold). A glass microelectrode (10-20-Mohm resistance) was used to record intracellular potentials, and a bipolar electrode was used to record local activation near the microelectrode recording site. A differentiating amplifier with peak-and-hold detector was used to record V_max as described previously. Electrical recordings were obtained during pacing at rates from 0.2 to 5.0 Hz and at a pacing rate of 1.0 Hz with the introduction of premature stimuli from 500 msec to local refractoriness in 5-msec intervals.

Subepicardial left ventricular myocardium. Male mongrel dogs were anesthetized with i.v. sodium pentobarbital (30 mg/kg). Left ventricular epicardial tissue sections approximately 10 mm × 15 mm × 1 mm thick were removed from the anterior left ventricle. The long axis of the preparation was removed parallel to the diagonal branches of the anterior descending coronary artery and, hence, parallel to epicardial fiber orientation. The tissue preparations were superfused with Tyrode's solution as previously described. A Grass S-88 stimulator was used to pace the tissue preparation (2-msec stimuli, twice diastolic threshold) from bipolar electrode sites located in two opposing corners of the preparation. A glass microelectrode (10-20-Mohm resistance) was used to record intracellular potentials, and a bipolar electrode was used to record local activation near the microelectrode recording site. The stimulation sites and recording sites were arranged so that excitation could be performed both longitudinal and transverse to epicardial fiber orientation. A differentiating amplifier with peak-and-hold detector was used to record V_max as described previously. Electrical recordings were performed during pacing at rates from 0.2 to 5.0 Hz and at a pacing rate of 1.0 Hz with the introduction of premature stimuli from 500 msec to local refractoriness in 5-msec intervals.

Drug administration. Following a 1 hr period of superfusion with normal Tyrode's solution, GLG-V-13 and KMC-IV-84 (as hydroperchlorate salts) were administered in half-log increments from 0.32 to 10 mg/l. Each drug concentration was administered for 20 min before electrophysiologic testing at each drug concentration. After drug administration ended superfusion with normal Tyrode's solution was performed for 30 min and electrophysiologic testing was repeated.

Statistical analysis. Differences between drug treatment groups were determined using one-way analysis of variance for repeated measures. Individual differences were determined using Scheffé's test. P  .05 was the criterion for significance.

	Results

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Canine cardiac Purkinje fibers. GLG-V-13 and KMC-IV-84 administration failed to alter maximal diastolic potential (91 ± 1, 90 ± 1 mV), action potential amplitude (119 ± 2, 121 ± 2 mV), overshoot potential (29 ± 2, 31 ± 2 mV), V_max (486 ± 44, 524 ± 42 V/sec) or conduction time (24 ± 6, 15 ± 3 msec) from predrug values (respectively) in canine cardiac Purkinje fibers (table 1). V_max and conduction time were unchanged from predrug values over the entire range of cycle lengths studied (250-5000 msec). There was no shift in the membrane responsiveness curve (constructed using premature ventricular stimuli) (fig. 2) (cycle length = 1000 msec). Both GLG-V-13 and KMC-IV-84 produced a concentration-dependent prolongation of APD₂₅, APD₅₀, APD₉₀ and APD₁₀₀ in free-running Purkinje strands (table 1). The prolongation of the effective refractory period was proportional to APD₉₀. GLG-V-13 and KMC-IV-84 (at concentrations of 0.32, 1.0, 3.2, and 10 mg/l) failed to alter spontaneous automaticity (16 ± 5, 16 ± 1/min, respectively) or take-off potentials (76 ± 4, 75 ± 3 mV, respectively) in Purkinje tissues.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Membrane responsiveness curves for GLG-V-13 and KMC-IV-84. Membrane responsiveness curves are shown at predrug (), 10 mg/l drug () and washout () for GLG-V-13 (10 mg/l) and KMC-IV-84 (10 mg/l). No effect of drug administration was observed in canine Purkinje tissue.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Reverse use-dependence of action potential prolongation in canine Purkinje fiberseffects of GLG-V-13 and KMC-IV-84. The relationship between paced cycle length and APD90 is shown for GLG-V-13 and KMC-IV-84. No significant prolongation of action potential duration is observed at cycle lengths of 333 msec or less (P < .01 vs. predrug and washout for paced cycle lengths of 400, 500, 1000, 2000 and 5000 msec for both drugs).

Canine right ventricular endocardium. Both GLG-V-13 and KMC-IV-84 produced a concentration-dependent prolongation of APD₂₅, APD₅₀, APD₉₀ and APD₁₀₀ in subendocardium from the right ventricle (table 2). Prolongation of the action potential by GLG-V-13 and KMC-IV-84 was observed without alteration of resting membrane potential (85 ± 1, 83 ± 1 mV), action potential amplitude (110 ± 2, 112 ± 1 mV), overshoot potential (25 ± 2, 29 ± 1 mV), V_max (273 ± 40, 213 ± 14 V/sec) or conduction time (35 ± 6, 39 ± 3 msec) from predrug values (respectively). The prolongation of the effective refractory period was proportional to APD₉₀ (table 2).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Reverse use-dependence of action potential prolongation in canine right ventricular endocardiumeffects of GLG-V-13 and KMC-IV-84. The relationship between paced cycle length and APD90 is shown for GLG-V-13 and KMC-IV-84. No significant prolongation of action potential duration is observed at cycle lengths of 333 msec or less (P < .01 vs. predrug and washout for paced cycle lengths of 400, 500, 1000, 2000 and 5000 msec for both drugs).

Canine left ventricular epicardium. Both GLG-V-13 and KMC-IV-84 produced a concentration-dependent prolongation of APD₂₅, APD₅₀, APD₉₀ and APD₁₀₀ in subepicardium from the left ventricle (table 3). Prolongation of the action potential by GLG-V-13 and KMC-IV-84 was observed without change in resting membrane potential (83 ± 1, 83 ± 1 mV), action potential amplitude (108 ± 1, 110 ± 2 mV), overshoot potential (25 ± 1, 28 ± 2 mV), V_max longitudinal to epicardial fiber axis (178 ± 17, 218 ± 14 V/sec), V_max transverse to epicardial fiber axis (214 ± 16, 249 ± 14 V/sec), conduction velocity longitudinal to epicardial fiber orientation (0.54 ± 0.05, 0.59 ± 0.08 M/sec) and conduction velocity transverse to epicardial fiber orientation (0.20 ± 0.02, 0.17 ± 0.01 M/sec) from predrug values (respectively). The prolongation of the effective refractory period was proportional to APD₉₀ (table 3).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Reverse use-dependence of action potential prolongation in canine left ventricular epicardiumeffects of GLG-V-13 and KMC-IV-84. The relationship between paced cycle length and APD90 is shown for GLG-V-13 and KMC-IV-84. No significant prolongation of action potential duration is observed at cycle lengths of 333 msec or less (P < .01 vs. predrug and washout for paced cycle lengths of 400, 500, 1000, 2000 and 5000 msec for GLG-V-13 and for paced cycle lengths of 500, 1000, 2000 and 5000 msec for KMC-IV-84).

Depolarized canine left ventricular epicardium. Increasing extracellular potassium from 4.0 to 12 mM decreased resting membrane potential, action potential amplitude, overshoot potential, V_max and conduction velocity (table 4). GLG-V-13 and KMC-IV-84 (0.32, 1.0, 3.2 and 10 mg/l) failed significantly to alter these parameters over a range of paced cycle lengths from 250 to 5000 msec.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Reverse use-dependence of action potential prolongation in depolarized (12 mM) K+o canine left ventricular epicardiumeffects of GLG-V-13 and KMC-IV-84. The relationship between paced cycle length and APD90 is shown for depolarized left ventricular epicardium exposed to GLG-V-13 and KMC-IV-84. No significant prolongation of action potential duration is observed at cycle lengths of 400 msec or less (P < .05 vs. predrug and washout for paced cycle lengths of 2000 and 5000 msec for GLG-V-13 and for paced cycle lengths of 500, 1000, 2000 and 5000 msec for KMC-IV-84).

	Discussion

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Unlike previous diheterabicyclo[3.3.1]nonane compounds having two secondary amine moieties in the ring system (Scherlag et al., 1988; Patterson et al., 1991; Patterson et al., 1993; Fazekas et al., 1993a; Smith et al., 1990), neither GLG-V-13 nor KMC-IV-84 altered conduction in canine myocardium. The electrophysiologic actions of GLG-V-13 and KMC-IV-84 were limited to a rate-dependent prolongation of APD₅₀ and APD₉₀ in canine Purkinje, right ventricular subendocardium and left ventricular epicardium. Prolongation of initial repolarization, as reflected in prolongation of APD₂₅, was observed at higher dosages than those producing prolongation of APD₅₀ or APD₉₀. The present results are consistent with a selective inhibition of I_Kr current in canine myocardium. Such an action has been reported for GLG-V-13 in preliminary studies using voltage clamping in isolated ventricular myocytes from rabbit hearts (Fazekas et al., 1995). No inhibition of the slow component of the delayed rectifier was observed (I_Ks).

Reverse use-dependence of action potential prolongation. The reverse use-dependence observed with GLG-V-13 and KMC-IV-84 in the present experiments has been previously reported for methanesulfonalide class III drugs such as dofetilide (Gwilt et al., 1991; Jurkiewicz and Sanguinetti, 1993), E-4031 (Wettwer et al., 1991), d,l-sotalol (Strauss et al., 1970; Hafner et al., 1988), MK-499 (Baskin and Lynch, 1994; Krafte and Volberg, 1994) and sematilide (Krafte and Volberg, 1994). The actual mechanism(s) for reverse use-dependence is/are controversial. The earliest mechanism for reverse use-dependence of action potential duration was advanced by Hondeghem and Snyders (1990). Experimental data from their laboratory demonstrated a time- and voltage-dependent modulation of I_k with quinidine. Quinidine primarily reduced time-dependent outward potassium currents at negative membrane potentials, with blockade of outward potassium currents becoming less pronounced with depolarization (Roden et al., 1988). Later data, however, have failed to demonstrate a similar voltage-dependent or use-dependent reversal of I_Kr blockade with either of the methanesulfonalide class III antiarrhythmic drugs dofetilide (Jurkiewicz and Sanguinetti, 1993) and WAY-123,398 (Spinelli et al., 1993). Despite these observations, both dofetilide and WAY-123,398 produce reverse use-dependent prolongation of the action potential (Jurkiewicz and Sanguinetti, 1993; Spinelli et al., 1993).

Antiarrhythmic efficacy. The importance of reverse use-dependence as a determinant of antiarrhythmic activity is unknown. The relative absence of action potential prolongation at rapid atrial rates is clearly deleterious to the suppression of atrial fibrillation (Wang et al., 1994). Atrial fibrillation would not represent an ideal arrhythmia substrate for suppression by GLG-V-13 or KMC-IV-84 or by any class III agent with marked reverse use-dependence. The role of action potential prolongation in ischemically injured ventricular tissues constituting a reentrant pathway as a basis for antiarrhythmic drug efficacy is less well understood. In ischemically injured epicardium during the subacute phase of myocardial infarction in the dog, class III antiarrhythmic drugs produce little action potential prolongation. With clofilium (Patterson et al., 1992), the failure to prolong action potential duration in vitro is accompanied by an increase in refractoriness within the reentrant pathway in vivo and by postrepolarization refractoriness in vitro. With d,l-sotalol, little prolongation of action potential duration is observed in markedly damaged epicardial tissue examined 4 days after coronary artery ligation in the dog (Patterson, 1995), despite the presence of marked postrepolarization refractoriness in injured canine epicardium in vivo after d,l-sotalol administration (Cobbe et al., 1983; Patterson and Scherlag, 1996). GLG-V-13 has demonstrated conduction block and postrepolarization refractoriness in ischemically injured canine epicardium studied 4 to 5 days after anterior coronary artery ligation. The same experiments have failed to demonstrate a significant effect of GLG-V-13 on action potential duration, cellular determinants of conduction velocity or actual conduction velocity in the same injured epicardial tissue (unpublished data from our laboratory). Despite its inability to alter action potential duration in ischemically injured canine epicardium, GLG-V-13 increases refractoriness within injured canine epicardium and suppresses the induction of sustained ventricular tachycardia (Fazekas et al., 1993b).

Relationship to other antiarrhythmic agents. The chemical structures and chemical synthesis of GLG-V-13 and KMC-IV-84 were derived from earlier diheterabicyclo[3.3.1]nonane antiarrhythmic agents (Scherlag et al., 1988; Smith et al., 1990; Bailey et al., 1984). Another diheterabicyclo[3.3.1]nonane class III antiarrhythmic drug, ambasilide, has been developed and evaluated independently. The chemical structure is shown by Takanaka et al. (1992) and Zhang et al. (1992). Although ambasilide produces reverse use-dependence in canine Purkinje tissue, the drug prolongs action potential duration over a wide range of paced cycle lengths in canine subendocardium (Takanaka et al., 1992). Ambasilide inhibits both components of the delayed rectifier (I_Kr and I_Ks) in isolated guinea pig myocytes (Zhang et al., 1992). The lack of significant rate-dependence in canine ventricular muscle clearly distinguishes ambasilide from its chemical relatives GLG-V-13, KMC-IV-84 and tedisamil.

Proarrhythmia. The potential of GLG-V-13 and KMC-IV-84 to induce the formation of early afterdepolarizations and bradycardia-dependent ventricular arrhythmia has not been defined. Afterdepolarizations were not observed in the present experiments. Hypokalemia, acidosis and stimulation of sodium-calcium exchange were not, however, used in an attempt to provoke potential early formation of afterdepolarization. In a rabbit model of torsades de pointes using alpha adrenergic receptor stimulation to facilitate arrhythmia formation, GLG-V-13 has been shown to facilitate polymorphic ventricular arrhythmia formation (Fazekas et al., 1993c; Fazekas et al., in press). It has been suggested that the proarrhythmic effects of GLG-V-13 are slightly less than those observed with other class III drugs (Fazekas et al., 1993c; Fazekas et al., in press, 1997). In light of their similar ability to prolong the action potential at slow HRs, it is unclear why any one selective inhibitor of I_Kr should demonstrate more or less proarrhythmia from early afterdepolarization formation.

Conclusion. The present experiments demonstrate the selective class III electrophysiologic actions of the imidazole-substituted diheterabicyclo[3.3.1]nonane drugs GLG-V-13 and KMC-IV-84 in canine ventricular myocardium. Action potential prolongation in Purkinje, right ventricular subendocardium and left ventricular epicardium is rate-dependent, and little prolongation of action potential duration is observed at paced rates of 3 Hz or greater. The drugs had no effect on V_max or action potential amplitude, despite the derivation of the compounds from a diheterabicyclononane structure that has class I antiarrhythmic properties. The observed pharmacologic effects of GLG-V-13 and KMC-IV-84 are consistent with selective blockade of the rapid phase of the delayed rectifier current in canine myocardium (I_Kr). Further experiments are needed to further define both the antiarrhythmic efficacy and the proarrhythmic potential of GLG-V-13 and KMC-IV-84.