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Antifibrillatory Efficacy of Long-Term Tedisamil Administration in a Postinfarcted Canine Model of Ischemic Ventricular Fibrillation

Friedrichs, Gregory S.; Abreu, J. Nelson; Driscoll, Edward M. Jr.; Borlak, Jürgen*; Lucchesi, Benedict R.

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Journal of Cardiovascular Pharmacology: January 1998 - Volume 31 - Issue 1 - p 56-66
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Abstract

The identification of specific ion currents in cardiac muscle has provided the incentive to target specific sites for pharmacologic suppression in an effort to develop new antiarrhythmic agents. Increasing attention is being focused on Class III antiarrhythmic drugs as potential pharmacologic interventions for reducing the incidence of sudden cardiac death (1,2). Members of Class III antiarrhythmic drugs block cardiac K+ channels, thereby resulting in prolongation of the cardiac action potential, with minimal effects on conduction velocity. The strategy is supported by experimental studies in which selected Class III antiarrhythmic drugs are effective in attenuating reentrant cardiac rhythms associated with sudden cardiac death (3-6).

The development of new antiarrhythmic drugs has focused on identifying agents that inhibit the outward currents through voltage-gated K+ channels such as the delayed rectifier current (Ikr or Iks), the transient outward current (Ito), or the inward rectifier K+ current (Ik1) (1,7,8). Each current contributes to the repolarization phase of the normal cardiac cycle. Many of the new agents, however, have the potential to produce excessive prolongation of the action potential, leading to proarrhythmic activity (9). On the other hand, repolarizing currents expressed under pathophysiologic conditions may serve as more appropriate targets for potential antifibrillatory agents. Under normal conditions, the K(ATP) channel in ventricular myocardium is inhibited by intracellular adenosine triphosphate (ATP; 10,11). During hypoxia or ischemia, the K(ATP) channel in cardiac myocytes opens as a result of a decrease in intracellular ATP (10,12), increasing K+ efflux, accelerated repolarization, and shortening of the action-potential duration. In the ischemic myocardium, which favors reentrant arrhythmias, conditions that decrease the cardiac action-potential duration may facilitate arrhythmogenesis (1,13).

Tedisamil (KC-8857: 3,7-di-(cyclopropylmethyl)-9,9-tetramethylene-3,7-diazabicyclo[3.3.1]-nonane dihydrochloride) is reported to inhibit ionic currents from several cardiac ion channels: the delayed rectifier channel (Ik), the Ca2+-dependent potassium channel IK(Ca), and the transient outward current (Ito; 14-16). Tedisamil also modifies the functional responses to pinacidil, an opener of the K(ATP) channel (17). At high concentrations, tedisamil is capable of modulating Na+ channels as well (14). Tsuchihashi and Curtis (18) examined the effects of tedisamil on ventricular fibrillation elicited by regional ischemia and reperfusion in rat isolated hearts. Whereas tedisamil did not prevent the initiation of reperfusion-induced ventricular fibrillation, it did reduce the duration of the arrhythmia. Adaikan et al. (19) used tedisamil to suppress ectopy after ischemia/reperfusion, a finding supported by Bril et al. (20). Furthermore, tedisamil is capable of inducing chemical defibrillation (17,18).

In our previous investigation (21), we were able to demonstrate that a single oral dose of tedisamil (10 mg/kg) prolonged the ventricular effective refractory period and subsequently reduced the incidence of ischemia-induced ventricular fibrillation in the conscious dog. We observed a correlation between plasma concentrations of tedisamil and increases in the effective refractory period. However, we also reported that a single oral dose (10 mg/kg) of tedisamil was not well tolerated, and some dogs exhibited vomiting and diarrhea.

The goal of this investigation was to explore the possibility that a lower dose of the drug, administered over an extended period, would reduce unwanted side effects and maintain the "antifibrillatory" action of the drug. This would aid in an understanding of whether multiple dosing for several days would affect the pharmacokinetics and pharmacodynamics of the drug. Our study was conducted in the conscious, unsedated dog and was designed to (a) determine the effect of chronic oral tedisamil treatment on induction of ventricular arrhythmias by programmed electrical stimulation (PES) in the postinfarcted heart, and (b) determine whether the long-term administration of tedisamil could alter the incidence of ischemia-induced ventricular fibrillation.

METHODS

Guidelines for research involving the use of animals

The procedures followed in this study were in accordance with the guidelines of the University of Michigan University Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for Laboratory Animal Medicine. The University of Michigan is accredited by the American Association of Accreditation of Laboratory Animal Care, and the animal care and use program conforms to the standards in "The Guide for the Care and Use of Laboratory Animals," DHEW Publ. No.(NIH) 86-23, Rev. 1985.

Surgical preparation/long-term instrumentation

Male mongrel dogs (n = 23) weighing 13 ± 0.5 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and ventilated with room air with the use of a cuffed endotracheal tube and a Harvard respirator (Harvard Apparatus Co., S. Natick, MA, U.S.A.) adjusted to deliver a tidal volume of 30 ml/kg. By using aseptic technique, the left external jugular vein and left common carotid artery were isolated and cannulated for blood sampling and blood-pressure monitoring, respectively. A left thoracotomy was performed between the fourth and fifth ribs, the pericardium opened, and the heart suspended in a pericardial cradle. The left anterior descending (LAD) coronary artery was isolated at the tip of the left atrial appendage and the left circumflex (LCX) coronary artery was isolated ∼1 cm from its origin. A Teflon-insulated, silver-coated, copper wire electrode (27 gauge) was inserted into the LCX coronary artery and remained in contact with the intimal surface of the vessel.

A critical stenosis was applied to the LAD coronary artery by placing an 18- to 20-gauge hypodermic needle parallel to the vessel and tying a silk suture around the artery and the needle. The needle was removed immediately, thereby resulting in a narrowing of the arterial lumen. The diameter of the hypodermic needle was ∼75% of the diameter of the isolated LAD segment. Anterior wall ischemic myocardial injury was produced by total occlusion of the LAD coronary artery for 2 h, by using a snare formed from a loop of silicone rubber tubing pulled through a polyethylene cylinder. Regional ischemia was maintained for 2 h, after which the snare was released. Restoration of coronary artery blood flow was confirmed by visual inspection of the heart and normalization of the ST segment on the electrocardiogram (ECG).

A bipolar pacing electrode was sutured to the surface of the left atrium and used to maintain heart rate (HR) constant during refractory-period determinations. A bipolar plunge electrode (25-gauge stainless steel posts, 5 mm in length, 2-mm electrode separation) was sutured into the interventricular septum immediately to the right of the anterior descending coronary artery and adjacent to the right ventricular outflow tract (RVOT). The bipolar electrode was used for the introduction of ventricular extrastimuli during programmed ventricular stimulation and for the determination of the ventricular refractory period in the region of the RVOT. Silver disc electrodes were implanted subcutaneously for ECG monitoring, the thoracotomy incision was closed, and the animals were allowed to recover from surgical anesthesia. Postoperative care was maintained under the supervision of the veterinary staff and personnel of the Unit for Laboratory Medicine of the University of Michigan in consultation with the principal investigator. Antibiotic therapy (ampicillin, 2.0 mg/kg, s.c.) was maintained for 5 days after completion of the surgical procedure if deemed necessary on the basis of the animals' postoperative course of recovery.

Electrophysiologic studies and programmed electrical stimulation (PES) in the conscious canine 3-5 days after myocardial infarction

Electrophysiologic studies. Electrophysiologic monitoring and PES were performed between days 3 and 5 after anterior myocardial infarction and after the animal had recovered completely from the effects of surgical anesthesia and was considered to be free of postoperative complications (infection, arrhythmias, etc.). Animals were conscious and unsedated during the studies and rested comfortably and unrestrained throughout the experimental procedure.

ECG intervals and electrophysiologic parameters were determined immediately before PES. The electrocardiographic PR, QRS, and QTc intervals were determined during sinus rhythm, whereas a paced QT interval was measured during 2.5-Hz atrial pacing. During atrial pacing, an extrastimulus (S2) was introduced in late diastole (300 ms after the R wave) at a minimal current to elicit a ventricular response (V2). At twice excitation threshold, the basic S1-S2 coupling interval was decreased incrementally until S2 failed to elicit a V2. The ventricular effective refractory period (RVOT refractory period) was defined as the longest R-S2 interval at which a 2× RVOT excitation threshold voltage stimulus of 4-ms duration failed to elicit a V2 response.

Programmed electrical stimulation. PES consisted of the introduction of single (S2), double (S2-S3) and triple (S2-S3-S4) premature ventricular stimuli (4-ms duration, 2′ RVOT excitation threshold voltage) into the interventricular septum near the RVOT, by using a Grass model S8800 stimulator and an SIU5 stimulus isolation unit (Astro-Med Grass, West Warwick, RI, U.S.A.). The S2 extrastimulus was triggered from the R wave of the ECG, which served as the input for the stimulator. Thereafter, double and triple ventricular extrastimuli were introduced during sinus rhythm at S2-S3 and S2-S3-S4 coupling intervals ranging from 182 to 125 ms. Previous work showed that this method does not lead to the induction of ventricular dysrhythmias in animals without previous myocardial ischemic injury.

Only dogs responding to PES with either nonsustained or sustained ventricular tachycardia were entered into the study designed to assess the antifibrillatory activity of tedisamil. Ventricular tachycardia was defined as "nonsustained" if, by using the protocol described, five or more repetitive ventricular complexes were initiated reproducibly, but terminated spontaneously. Ventricular tachycardia was defined as "sustained" if it persisted ≥30 s or, in the event of hemodynamic compromise, required overdrive pacing for termination. The reproducibility of nonsustained and sustained ventricular tachycardia was confirmed by repeated attempts at PES, except in those cases in which the induced ventricular tachycardia was associated with hemodynamic compromise.

Resuscitative efforts were not attempted on animals in which ventricular fibrillation developed as a result of PES to avoid the confounding influence of repeated and occasionally prolonged resuscitation on the outcome of subsequent investigations. Survival of each dog during PES was of paramount importance. Therefore if nonsustained ventricular tachycardia was initiated reproducibly, more aggressive attempts to induce sustained ventricular tachycardia in the latter phases of the defined PES protocol were not attempted. Initiation of either nonsustained or sustained ventricular tachycardia by PES identified those dogs susceptible to the development of ventricular fibrillation in response to subsequent ischemia at a site remote from the previous myocardial infarction (non-infarct related myocardial region; 22). During PES, a successful drug response was defined as the failure after the drug administration to provoke either nonsustained or sustained ventricular tachycardia, after the PES protocol was complete, in a previously responsive animal. After determination of the baseline electrophysiologic values and reproduction of sustained or nonsustained ventricular tachycardia, the test drug was administered orally, and the electrophysiologic testing was repeated at the previously stated times.

Time-course studies: oral administration of tedisamil in the conscious dog

A separate group of dogs (n = 3 per dose) was instrumented to evaluate the effect of oral administration of tedisamil during 4 days of treatment by using three doses (1.5, 3.0, and 6.0 mg/kg, b.i.d.) for each regimen. The purpose of this study was to determine the pharmacokinetic profile of tedisamil after 4 days of dosing in a population of dogs comparable to those used in the sudden-death portion of the investigation. Blood samples were obtained for determination of drug concentration throughout the dosing period. Electrophysiologic variables and plasma samples were obtained at more frequent intervals (0, 5, 15, 30, 60 min; and 2, 3, 4, 5, 6, 7, 8, 12, and 24 h) on the final day of dosing. At the completion of the dosing regimen, each animal was killed, and tissue samples were obtained from various sites (cardiac apex, atrial tissue, papillary muscle, ventricular septum, and diaphragm).

Sudden-cardiac-death experimental protocol and drug-dosing regimens

A schematic diagram of the sudden-cardiac-death protocol is shown in Fig. 1. Two groups of conscious dogs were used in the sudden-death portion of the study, each randomly assigned to either the tedisamil-treatment group (n = 8) or the control group (n = 8). Both groups underwent identical LAD occlusion/reperfusion procedures and evaluation by PES.

FIG. 1
FIG. 1:
Tedisamil dosing regimen and protocol used in the investigation.

Because our previous investigation indicated that gastrointestinal side effects (vomiting/diarrhea) were associated with the administration of 10 mg/kg p.o., we chose a dosing regimen that would potentially alleviate the side effects observed in our earlier study (21). After baseline values were recorded and before-drug PES was performed, inducible dogs were administered tedisamil in capsule form (3.0 mg/kg) twice per day for 3 days. On the fourth day of dosing, the dog was returned to the electrophysiology laboratory where a capsule containing tedisamil was administered. Sixty minutes was allowed to elapse before PES was repeated.

Ischemia at a site remote from previous myocardial infarction for the induction of ventricular fibrillation

Posterolateral ischemia was initiated in the region of distribution of the LCX coronary artery by applying an anodal current of 150 μA to the intimal surface of the vessel, delivered through the previously implanted intraluminal electrode. This method has been shown repeatedly by our laboratory (3-5,22) to induce intimal injury, promoting oscillatory disturbances in coronary artery blood flow and ultimately thrombus formation in the LCX coronary artery. In the absence of an appropriate antifibrillatory intervention, the procedure results in a high incidence of ECG disturbances, leading to ventricular fibrillation in animals possessing an anterior-wall myocardial infarction. Thus the superimposition of an acute ischemic event in a region remote from a previous myocardial infarction was accompanied by sudden cardiac death resulting from ventricular fibrillation. Sudden cardiac death was defined as ventricular fibrillation occurring within 1 h of the onset of regional ischemia in the distribution of the LCX coronary artery, as determined from changes in the ST segment (depression or elevation or both) recorded from the lead II ECG.

On the death of the animal, the heart was removed, and the LCX coronary artery thrombus extracted and weighed. The heart was sectioned, and the transverse sections were placed in 0.4% triphenyltetrazolium chloride (TTC) for 10-15 min, maintained at 37°C. Exposure of the cut surface of the ventricular myocardium allows enzymatic reduction of TTC, leading to the formation of a brick-red formazan precipitate in regions where myocardial tissue remains viable. Infarcted regions of myocardium are unable to reduce TTC enzymatically to form the brick-red formazan precipitate and appear pale yellow. The regions demarcated by the TTC reaction and the nonreactive regions were traced on acetate sheets. The traced diagrams were digitized by using a flatbed scanner Macintosh Computer (Apple, Cupertino, CA, U.S.A.) and appropriate software (Mac-Draft; Innovative Data Design, Concord, CA, U.S.A.) was used to calculate the area of tissue that had undergone irreversible injury in the LAD and LCX regions of the heart.

High-pressure liquid chromatographic determination of tedisamil in plasma from venous blood

Venous blood samples were collected in sodium citrate (3.8%) solution at preselected intervals. Plasma was obtained by whole blood centrifugation at 2,000 g for 10 min. Plasma was frozen in liquid nitrogen and stored at −70°C until subsequent analysis. Determination of plasma tedisamil concentrations was performed by using high-pressure liquid chromatography (HPLC), described previously (21) and briefly outlined subsequently. Plasma samples were allowed to thaw at room temperature. Aliquots (0.1 and 0.5 ml dog plasma) were transferred into 10-ml polypropylene extraction tubes and sodium hydroxide solution (1N, 50 μl) added. Internal standard solution (gallopamil, 500 ng/ml; 100 μl dog plasma) and terbutyl methyl ether (3 ml) were then added, and the tubes capped before rotary mixing (rotator drive STR4; speed 10, 20 min). The organic layers were discarded and the aqueous layers transferred into microvials for subsequent HPLC analysis. The detector used was a Coulochem II electrochemical detector (model 5200; Bischoff Analysentechnik, Leonberg, Germany) with a Nucleosil NC100-C18AB (125 × 4.0 mm) column preceded by a Nucleosil NC100-C18AB (8 × 4 mm) column. The integrator and interface used were Gina-Chromatographysystem (Raytest, Straubenhardt, Germany). Peak height ratios (tedisamil/gallopamil) were used to construct the calibration curve. The concentration of tedisamil in calibration standards and quality-control samples was determined by using linear regression with 1/× weighting to improve accuracy at low concentrations.

Statistical analysis

The data are expressed as mean ± SEM. A one-factor analysis of variance (ANOVA) was performed within groups at appropriate time points to assess the effects of time. The difference between treatment groups for the incidence of sudden cardiac death was analyzed by Fisher's Exact test. Student's t test for paired replicates was performed within groups for electrophysiologic and hemodynamic results before and after drug treatment. Differences were considered significant at p < 0.05.

Drugs

Tedisamil was provided by Solvay Pharma Deutschland GmbH (Hannover, Germany). Capsules containing tedisamil or placebo were prepared immediately before oral administration. The content of the capsules was determined on the basis of animal body weight and the free base of the drug. Other reagents used in this study were obtained from commercial sources.

RESULTS

Time-course studies

Oral administration of tedisamil in the conscious dog. A separate group of dogs (n = 3 for each dose) was instrumented to evaluate the effect of oral administration of tedisamil over 4 days of treatment at three specific doses (1.5, 3.0, and 6.0 mg/kg, b.i.d.). Oral administration of tedisamil every 12 h for 4 days was unable to alter measured electrophysiologic variables significantly from baseline values (Table 1). Heart rate and arterial blood pressure was monitored on the day of testing in each group of dogs. A dose of 1.5 mg/kg was not associated with heart-rate alterations. However, after long-term dosing (3 and 6 mg/kg, b.i.d.), treatment produced a modest negative chronotropic effect in this group of animals (Table 2). Without significant electrophysiologic changes in the conscious dog with which to guide our selection of a long-term dosing regimen, we eliminated the highest dose to avoid potential gastrointestinal side effects and chose 3 mg/kg p.o., b.i.d, over 4 days to be used in the model of sudden cardiac death.

TABLE 1
TABLE 1:
Electrophysiologic variables after 4-day (b.i.d.) tedisamil treatment in the conscious dog
TABLE 2
TABLE 2:
Heart rate and mean arterial pressure after 4-day (b.i.d) tedisamil treatment in the conscious dog

Plasma concentrations.Figure 2 depicts the plasma concentrations of tedisamil and its metabolites from dogs pretreated with either 1.5, 3.0, or 6.0 mg/kg, b.i.d., for 3 days. On the fourth day, each animal was returned to the laboratory and administered tedisamil by the oral route. Blood samples from each dog were obtained at predetermined intervals. Peak plasma concentrations for pretreatment regimens of 1.5, 3.0, and 6.0 mg/kg were 58 ± 29 ng/ml, 181 ± 166 ng/ml, and 394 ± 267 ng/ml, respectively. Repeated twice daily, oral administration of tedisamil produced a dose-related increase in the peak plasma concentration. Plasma concentrations of tedisamil on day 4 were undetectable 12 h after the last dose.

FIG. 2
FIG. 2:
Plasma concentrations of tedisamil (left) and its metabolites (right) after oral treatment for 4 days, b.i.d., at doses of 1.5 (top), 3.0 (middle), and 6.0 mg/kg (bottom). Dashed line, a blood sample was not obtained after t = 12 h, until the conclusion of the experimental protocol (t = 24 h).

Tedisamil multiple oral dose treatment (3.0 mg/kg, b.i.d., 4 days) PES

A total of 23 postinfarcted dogs met the initial criteria (inducible ventricular tachycardia by PES) for inclusion in the sudden-cardiac-death protocol. The animals were allocated to two separate study groups. Eight animals were randomized to the control group, which received placebo. The other group of animals (n = 8) received tedisamil as a multiple oral dose (3 mg/kg, b.i.d., 4 days total). Anterior-wall infarct size was determined in each animal at the conclusion of the experimental protocol and expressed as percentage of the left ventricle. Dogs belonging to the placebo-treated group had a mean anterior-wall infarct of 22 ± 1%, compared with 20 ± 1% in tedisamil-treated dogs (Table 3). The remaining seven dogs were not included in the sudden-death analysis because of LCX electrode failure.

TABLE 3
TABLE 3:
Mean myocardial infarct size (percentage of left ventricle)

Tedisamil did not produce any observable untoward hemodynamic effects when administered as a multiple oral dose of 3.0 mg/kg, b.i.d. (4 days) to animals with infarcts. The recorded cardiac electrophysiologic effects at baseline and 60 min after the administration of tedisamil are summarized in Table 4. The recorded parameters included HR, atrioventricular (AV) conduction as determined by the PR interval, ventricular conduction as assessed from the duration of the QRS complex, ventricular refractoriness as calculated from changes in the QTc interval, and the effective refractory period determined by the extrastimulus method. As indicated in Table 4, tedisamil produced a negative chronotropic effect (although not significant at p < 0.05) and failed to prolong the effective refractory period or the QTc interval in the infarcted animals treated with a multiple-dose regimen of 3 mg/kg, b.i.d. × 4 days. Dogs that were at risk for sudden coronary death (i.e., inducible as determined by PES testing before tedisamil treatment) were affected by multiple administration of 3 mg/kg tedisamil. All animals (100%) were inducible before the administration of tedisamil. After 3 days of oral administration of tedisamil, 9% of the animals remained inducible (p < 0.05, compared with predrug inducibility). In contrast, 100% of the animals were inducible before placebo treatment; 63% of this group remained inducible after placebo treatment.

TABLE 4
TABLE 4:
Hemodynamic and electrophysiological parameters after long-term placebo or tedisamil treatment

Sudden-death studies

Plasma concentrations. Plasma concentrations of tedisamil and its major metabolites (M1 and M2) were determined from blood samples obtained at preselected time points after the administration of multiple doses of tedisamil (3 mg/kg, b.i.d.) from seven animals in this group. The data are presented in Fig. 3. On the test day (day 4), the maximal plasma concentration (Cmax) was 365 ± 35 ng/ml, which was achieved at t = 120 min after oral administration. After the final dose of tedisamil was administered (dose 8), a blood sample was not obtained until the conclusion of the experimental protocol (t = 24 h).

FIG. 3
FIG. 3:
Plasma concentrations of tedisamil after long-term oral dosing (3 mg/kg, b.i.d., 4 days) in those dogs entered into the sudden-cardiac-death protocol. Dashed line, a blood sample was not obtained after t = 12 h, until the conclusion of the experimental protocol (t = 24 h).

Inducible dogs. Eleven dogs were treated with tedisamil and entered into the sudden-death portion of the investigation. Three dogs did not develop posterolateral ischemia. On postmortem examination, we found electrode failure that precluded induction of endothelial injury and the subsequent transient ischemic events. The three dogs that had LCX electrode failure were excluded from the sudden-death portion of the analysis. Eight tedisamiltreated dogs remained in the sudden-death protocol. The results are shown in Fig. 4 (24-h survival rate). The percentage of animals surviving the first hour after developing posterolateral ischemia in the vehicle-treated group was 25%, compared with the group treated with tedisamil, in which all animals survived (100%; p < 0.05, Fisher's Exact test), despite the presence of ischemia in a region remote from the infarct-related artery. The difference in survival between groups, 24 h after posterolateral ischemia, remained statistically significant. The occurrence of delayed death during the latter portion of the observation period (2-24 h) was the result of acute pump failure and not ventricular fibrillation. The incidence of delayed death was also reduced in the tedisamil-treated group compared with the vehicle-treated group. Thrombus mass was determined at the conclusion of the experiment. There was no difference in thrombus mass between groups: 11 ± 3 and 16 ± 2 mg for placebo- and tedisamil-treated groups, respectively.

FIG. 4
FIG. 4:
Survival curve for tedisamil (multiple oral dose, 3.0 mg/kg) and vehicle-treated animals after the onset of posterolateral ischemia. Data shown are 24-h survival after the onset of posterolateral ischemia. Statistical analysis was performed with absolute numbers of animals dying of ventricular fibrillation (Fisher's Exact test). *p < 0.05 vs. vehicle treatment. Deaths occurring after 5-6 h are primarily the result of pump failure and not ventricular fibrillation.

Noninducible dogs. A second series of dogs (n = 6) that were noninducible (not responsive to PES; i.e., ventricular tachycardia could not be induced by PES), also was treated with tedisamil at a dose of 3 mg/kg, b.i.d., for 4 days. They were returned to the laboratory on the fourth day and subjected to PES. All of the dogs that were classified as noninducible remained noninducible after being treated with tedisamil over the long term. This group of low-risk dogs, with an anterior-wall infarct size of 7 ± 3% of the left ventricle, was entered into the sudden-cardiac-death protocol. None of the dogs in this group died of ventricular fibrillation (six of six survivors). Two dogs in this group died of delayed death, 9 and 12 h after posterior-wall ischemia.

Adverse drug reactions

Tedisamil, administered in a multiple-dose regimen, did not cause any apparent discomfort in any of the animals entered into the study. Unlike our previous investigation, there was no vomiting or diarrhea in any of the dogs studied. All the conscious animals receiving tedisamil rested quietly throughout the procedure and did not display any outward signs referable to abnormal alterations in central nervous system activity.

DISCUSSION

Antiarrhythmic compounds that prolong myocardial repolarization without prolonging conduction time are of interest as potential therapeutic agents for reducing the incidence of lethal ventricular arrhythmias. Class III antiarrhythmic drugs possessing a 2-aminobenzimidazole group (WAY 123,398) significantly increase the fibrillation threshold and terminate ventricular fibrillation in anesthetized dogs, an action that may be associated with inhibition of the delayed rectifier potassium current (23). Ambasilide, another investigational Class III agent, exerts differential effects on repolarization in Purkinje fibers and ventricular muscle and can lengthen action-potential duration over a wide range of pacing frequencies, presumably through inhibition of one or more potassium channels (24). Black et al. (5) showed NE-10064 (Azimilide), was effective in a conscious canine model of sudden cardiac death, a compound that has been shown to block both IKr and IKs(25-28). Other agents alleged specifically to modulate IKr and found effective in various models of ventricular arrhythmias include UK-68,798 (dofetilide; 4,29), E-4031 (6,30), and MK-499 (31). Despite their demonstrated efficacy in favorably modulating cardiac-rhythm disturbances in experimental animal models, agents that inhibit one or more of the outward K+ currents have the potential to exhibit proarrhythmic activity (9,32,33). Basic and clinical studies suggest that action-potential prolongation may result in an effective antiarrhythmic response. The most significant obstacle for further development of such drugs is the occurrence of enhanced QT prolongation and polymorphic ventricular tachycardia. IKr blockers share the characteristic that they can markedly prolong the QT interval and produce polymorphic ventricular tachycardia. Furthermore, QT-interval prolongation alone may not serve as a reliable indicator of whether or not a drug will reduce the incidence of ventricular fibrillation (32). With these considerations in mind, a suggested approach would be to focus on pharmacologic interventions that spare the physiologic channels involved in ventricular muscle repolarization while interdicting those channels (e.g., IK(ATP)) that become functional during ischemic stress. Previous studies indicated that tedisamil was able to inhibit the IK(ATP) channel in canine myocardial tissue, as well as to prevent ventricular fibrillation induced by pharmacologic IK(ATP)-channel agonists (17). Extension of this concept in the canine model of sudden cardiac death provided evidence that tedisamil could reduce the incidence of ventricular fibrillation associated with a transient ischemic event (21).

By using a model similar to that described here, Wallace et al. (34) administered tedisamil intravenously (0.1 and 1.0 mg/kg) and demonstrated significant increases in the ventricular effective refractory period of both normal and infarcted myocardium of anesthetized dogs, as well as a dose-dependent increase in QTc. In addition, the incidence of electrically induced ventricular tachycardia was reduced in 80% of the animals treated with tedisamil. Ventricular tachycardia, initiated as a result of PES testing, is believed to be a predictor of spontaneous ventricular tachycardia and sudden cardiac death after myocardial infarction, both clinically (35) and in experimental animal studies (36). Drug-induced suppression of PES-induced tachycardia has been a therapeutic goal, but it may not represent drug efficacy when survival is the end point being evaluated (37). In our study, the susceptibility to PES-induced ventricular tachycardia was reduced significantly after several days of oral administration of tedisamil. This characteristic of tedisamil may have contributed to reducing the incidence of ventricular fibrillation within 60 min after the onset of ischemia in a myocardial region remote from the infarct-related artery. Although tedisamil reduced the incidence of arrhythmia induced by programmed stimulation, this effect may not be compatible with block of the K(ATP) channel if the action of the drug was solely the result of inhibition of IK(ATP). Thus whereas block of IK(ATP), might account for inhibition of ischemia-induced ventricular fibrillation, other actions associated with tedisamil may be operative in altering the prevention of arrhythmias by PES (15,38,39). A more detailed analysis is needed specifically to demonstrate block of IK(ATP) by tedisamil. Most notable, however, was the finding that a significant reduction in ventricular fibrillation was observed in conscious dogs after oral administration of tedisamil when compared with vehicle-treated animals: eight of eight survivors versus two of eight survivors, respectively. Moreover, the number of tedisamil-treated animals completing the sudden-death protocol (24 h after onset of posterolateral ischemia) was significantly greater than those in the control group (75 vs. 0%, respectively). It is important to note that the surviving animals in the drug-treated group evolved a second myocardial infarct in the LCX region of the myocardium in addition to the previous surgically induced anterior-wall infarct. Thus despite an extensive degree of left ventricular wall injury, the tedisamil-treated animals had an enhanced 24-h survival rate compared with that of the control group.

Previous studies demonstrated that the infarcted canine heart provides a predictive model for identifying pharmacologic interventions having the potential for drug-induced arrhythmic activity (40,41). The lack of ectopic activity after tedisamil treatment also was observed in our previous study in which the drug was administered as a single 10-mg/kg oral dose, accompanied by gastrointestinal side effects (21). Our study differed in that the dosing regimen was 3.0 mg/kg every 12 h for 4 days along with repeated determinations of tedisamil plasma concentrations and its major metabolite. Neither post-drug-induced arrhythmic activity nor gastrointestinal side effects were observed in the conscious animal throughout the 4-day dosing regimen. Although tedisamil produced a modest slowing of HR, it was not associated with any degree of post-drug-induced arrhythmic activity. This finding is in agreement with those of previous investigators (34) who examined tedisamil in the anesthetized dog previously subjected to anterior-wall myocardial infarction. Only one published report indicates the development of arrhythmia after the administration of very high doses of tedisamil (2.5 mg/kg/min) in the rat (42). The latter study described direct sodium channel blockade culminating in AV block as the mechanism responsible for the observed proarrhythmic events.

Profibrillatory potential also can be evaluated by administering the test drug to infarcted animals that are noninducible in response to PES and are classified as at low risk of developing ventricular fibrillation (13,36). A separate group of infarcted conscious dogs determined to be noninducible (n = 6) did not display evidence of drug-induced arrhythmia during the 4-day oral-dosing regimen. Furthermore, ventricular fibrillation did not develop in any of the noninducible animals subjected to ischemia in a region remote from the infarct-related artery.

Our study demonstrates the antifibrillatory action of tedisamil in the infarcted dog at high risk for the development of ischemia-induced ventricular fibrillation. In addition, drug-induced arrhythmic activity was not observed in any of the tedisamil-treated animals. Furthermore, the dosing regimen did not produce observable changes in intraventricular conduction (QRS) or repolarization (QTc). The lack of an observed change on ventricular repolarization, while providing protection against ischemia-induced ventricular fibrillation, would be consistent with the suggestion that tedisamil is acting by modulating the opening of the K(ATP) channel in response to ischemic stress and the accompanying decrease in intracellular ATP. Alternatively, tedisamil may be exerting a stabilization effect on the electrophysiologic state of the ischemic myocardium that was not detected, but nevertheless acted against the development of ventricular fibrillation.

Published studies indicate that tedisamil blocks Ito(14,15,18,43), IK(15,38), the calcium-dependent potassium channel K(Ca)(16,39), and sodium channels at concentrations >20 μM(15). Our laboratory has reported that tedisamil may affect the ATP-dependent K+ channel under pathophysiologic conditions in which the K(ATP) channel would be expected to become functional, resulting in shortening of the ventricular action potential (17). The ability of tedisamil to modulate more than one membrane ion channel may contribute to its effectiveness in attenuating arrhythmias in experimental in vitro models and reduce the incidence of ventricular fibrillation when studied in the in vivo rodent models. The precise cellular electrophysiologic mechanism(s) responsible for the observed antifibrillatory action of tedisamil remains unknown. Despite this lack of understanding, we have demonstrated that long-term oral administration of tedisamil decreases the potential for induction of ventricular tachycardia by PES and, more important, reduces the incidence of ventricular fibrillation in the infarcted heart.

The absence of any cardiac electrophysiologic changes during the normoxic state is consistent with the concept that tedisamil inhibits the ATP-dependent potassium channel, which is closed when intracellular ATP content is normal. The superimposition of ischemia in a region (circumflex or posterior myocardium) remote from the infarct-related artery increases the probability of the K(ATP) channel opening as a result of a regional decrease in intracellular ATP. Opening of the K(ATP) channel results in shortening of the action-potential duration and a decrease in the refractory period. Therefore delayed activation within the damaged anterior myocardium is more likely to engage adjacent myocardium and establish a reentrant pathway capable of supporting ventricular tachycardia and ventricular fibrillation. We propose that tedisamil minimizes the reduction in ventricular refractoriness that would otherwise occur during regional ischemia. After several days of dosing, tedisamil may achieve its antifibrillatory action "in the background"; thus, its actions on cardiac electrophysiology are not manifested during normoxic perfusion of the heart.

In conclusion, tedisamil possesses electrophysiologic properties that may differ from that of current Class III antiarrhythmic drugs in that it lacks significant electrophysiologic effects in the normoxic heart in the doses used in our study. When administered over several days, significant alterations in QTc or ventricular effective refractory period could not be detected.

Despite the inability to affect electrophysiologic parameters associated with ventricular repolarization, tedisamil is able to provide a significant antifibrillatory effect in the infarcted heart. Furthermore, post-drug-induced arrhythmic activity was not observed in dogs treated with a multiple oral dosing regimen of tedisamil (3 mg/kg, b.i.d., 4 days). The suggestion that tedisamil acts as an antifibrillatory agent is supported by our experimental observations in the infarcted canine heart subjected to myocardial ischemia in a region remote from the infarct-related artery, a finding consistent with those of previously published studies (21,34). Through selection of an appropriate dosing regimen and establishment of a proper plasma concentration of tedisamil, it was possible to avoid undesirable gastrointestinal effects. Proper dosing may also serve to limit the cardiac electrophysiologic actions of the drug so that tedisamil functions in the background, whereby it modulates opening of the K(ATP) channel, thus allowing physiologic repolarizing currents to remain functional. The latter may explain the lack of drug-induced arrhythmogenicity. Tedisamil may provide an opportunity to explore the therapeutic potential of K(ATP) channel inhibition as a target site for antifibrillatory drug action.

Acknowledgment: This work was supported by a grant from the National Institutes of Health, Heart, Lung and Blood Institute, HL-05806-37 Merit Award. J.N.A. was supported by a fellowship from the Universidad de los Andes, Venezuela. We thank Solvay Pharma Deutschland GmbH (Hannover) for the generous educational gift. In addition, we acknowledge the assistance of Drs. H. Fritsch, B. Jeremic, and A. Lichtenberger for their determination of tedisamil concentrations in canine plasma.

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Keywords:

Class III antiarrhythmic; Sudden death; K+ channel blocker

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