ToxSci Advance Access originally published online on June 1, 2006
Toxicological Sciences 2006 93(1):205-212; doi:10.1093/toxsci/kfl025
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Use of a Failing Rabbit Heart as a Model to Predict Torsadogenicity
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* Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210; QTest Labs, LLC., 6456 Fiesta Drive, Columbus, Ohio 43235; and Department of Clinical Sciences, North Carolina State University, Raleigh, North Carolina 27606
1 To whom correspondence should be addressed at Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210. Fax: (614) 292-3646. E-mail: hamlin.1{at}osu.edu.
Received April 4, 2006; accepted May 9, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Humans with underlying cardiovascular disease are at greater risk than humans with normal hearts for developing torsade de pointes (TdP) following exposure to some drugs that prolong ventricular repolarization. This study was designed to test the hypothesis that rabbits with ischemic myocardial failure are at similarly increased risk of developing QTc prolongation and TdP following exposure to escalating doses of drugs, which is known to have a capacity to induce TdP in humans. Coronary artery ligation was performed in 28 rabbits, causing significant (p < 0.05) reduction in left ventricular shortening fraction and systolic myocardial dysfunction 4 weeks after ligation in all operated animals compared to 38 normal, nonoperated controls. All studies were performed on rabbits anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg). Rabbits were exposed to escalating doses of amiodarone (3, 10, 30 mg/kg/10 min), cisapride (0.10, 0.25, 0.50 mg/kg/10 min), clofilium (0.1, 0.2, 0.4 mg/kg/10 min), dofetilide (0.005, 0.01, 0.02, 0.04 mg/kg/10 min), quinidine (3, 10, 30 mg/kg/10 min), and verapamil (0.25, 0.5, 1.0 mg/kg/10 min). A greater percentage of rabbits with failing hearts developed TdP following intravenous infusion of escalating doses of dofetilide (85%), clofilium (100%), or cisapride (50%) than did normal rabbits exposed to the same drug protocol (20, 33, and 0%, respectively). None of the rabbits in either group developed TdP when exposed to escalating doses of amiodarone, verapamil, or quinidine. Two out of four test articles lengthened QTc more in rabbits with myocardial failure than in normals, and TdP occurred in 13 out of 28 rabbits with myocardial failure as opposed to only four out of 38 rabbits with normal myocardial function.
Key Words: rabbits; torsade de pointes; myocardial failure; myocardial infarction; vulnerability; QT interval.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In humans, heart disease appears to be an independent risk factor for the development of torsade de pointes (TdP) (Buja et al., 1993
; Kannel and Sorlie, 1981) and other life-threatening arrhythmias. Recently, TdP was produced in dogs (Kozhevnikov et al., 2002; Verduyn et al., 1997; Vos et al., 1995) and rabbits (Tsuji et al., 2002) with long-standing complete AV block, whereas in normal animals it can be produced only with the setting of dramatic alterations in plasma electrolytes (Fabritz et al., 2003) or by administering multiple drugs (Batey and Coker, 2002; Carlsson et al., 1990). Currently, most preclinical trials performed to assess the torsadogenicity of potentially therapeutic compounds test only surrogates for TdP, most commonly QTc prolongation. Prolongation of QTc is a common effect of certain cardiovascular and noncardiovascular drugs (Belardinelli et al., 2003). Not all drugs that prolong the QT interval are associated with an increased risk for TdP (Redfern et al., 2003). The mechanism of TdP development may involve altered calcium kinetics, possibly resulting from calmodulin kinase II (CaMKII) phosphorylation of ryanodine channels (Verduyn et al., 1995). This finding is supported by the fact that a calcium-calmodulin inhibitor, W-7, prevents TdP development in the setting of QTc lengthening induced by a torsadogenic drug without actually shortening the QTc interval (Gbadebo et al., 2002; Mazur et al., 1999). A model capable of developing TdP might thus permit more accurate and direct assessment of a compound's torsadogenic potential, rather than relying on the results of a surrogate test for TdP, such as prolongation of QTc, to identify risk. The hypotheses of this study were (1) QTc lengthens more in failing hearts than in nonfailing hearts in response to test articles that lengthen QTc and (2) failing hearts develop TdP more often than do nonfailing hearts. Of course, in developing models, it is important that the model has near-perfect sensitivity and specificity. That is, it should permit prediction of TdP in humans but should not produce TdP for a drug that does not do so in humans. The model proposed in this paper fulfills these requirements. This paper describes the effect of a coronary ligation model of myocardial ischemia leading to heart failure in rabbits, documents the differential effects of amiodarone, cisapride, clofilium, dofetilide, quinidine, and verapamil on QTc and torsadogenicity, and also establishes that this rabbit model of left ventricular myocardial failure manifests a more torsadogenic substrate than normal rabbits without heart failure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This study was approved by the Institutional Laboratory Animal Care and Use Committee of the Ohio State University and QTest Labs, LLC (Columbus, OH). A total of 71 rabbits were used (31 in the coronary ligation/myocardial failure group, 38 in the nonoperated control group, and 2 in the sham-operated control group). All rabbits weighed between 2.5 and 3.5 kg. All animal procedures were conducted in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals.
Surgical procedure.
Thirty-one male New Zealand White rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly. Animals received 100% oxygen (at a rate of 400–600 ml/min) and isoflurane (at a rate of 0.5–1.0%) through a loose-fitting face mask designed for small animals. Surgery was performed using a modified version of the method of Fujita et al. (2004). Rabbits were placed in dorsal recumbency, and surgical anesthesia was confirmed by the absence of the pedal reflex. Standard limb lead electrocardiograms (ECGs) were obtained during the period of surgery. Enrofloxacin (12 mg/kg) was given intramuscularly prior to surgery. The skin over the sternum was denuded of hair and prepared aseptically. The incision line was infiltrated with 2% lidocaine. A midline incision (approximately 3–4 cm) was made in the skin, and the sternum was split with a #10 scalpel blade, being careful to remain precisely on the midline so that injury to the parietal pleura was avoided. The sternum was gently retracted so that the pericardial sac could be observed, and the sac was incised to reveal the surface of the right and left ventricles and their respective coronary arteries. Both the left anterior descending and a major apical branch of the left circumflex artery were ligated at a level midway between the origin of the major apical branch and the cardiac apex. Ligatures were performed using 4/0 monofilament polypropylene suture. Production of myocardial ischemia was confirmed by ST-segment elevation on the ECG and observation of grossly visible regional cyanosis of the myocardial surface. Of the 31 rabbits subjected to this procedure, three died immediately following coronary ligation from ventricular fibrillation. In the remaining 28 rabbits, the pericardial sac was left open, but the sternum was closed with three simple interrupted sutures of 3/0 monofilament polydioxanone suture. Muscle layers were closed with simple interrupted sutures of 4/0 nylon, and the skin was stapled. All 28 rabbits that survived the surgery were given 0.03 mg/kg buprenorphine im TID and 12 mg/kg enrofloxacin im BID for 3 days postoperatively.
Echocardiographic assessment of left ventricular function.
Echocardiographic examination was performed under light ketamine/xylazine sedation (15 and 3 mg/kg, respectively) on the day prior to coronary ligation and again 4 weeks after surgery. Each rabbit was placed in right lateral recumbency, with an area of the right hemithorax denuded to allow echocardiographic images to be obtained from the dependent right hemithorax. Imaging was performed using an Aloka SSD-1400 Echocardiographic System (Aloka America, Wallingford, CT) with a 5-MHz transducer. Echocardiographic recordings included a simultaneously recorded ECG, and all raw data were captured digitally for offline measurement. Left ventricular structure and function were assessed by an evaluation of standard two-dimensional and M-mode imaging planes (Schiller et al., 1989). Measurements of left ventricular wall thickness and internal ventricular dimensions during systole and diastole were made from M-mode images obtained from the standard right parasternal short-axis view at a level just beneath the mitral valve, with the M-mode cursor directed between the papillary muscles. These measurements were subsequently used to calculate shortening fraction (SF). A reduction in SF of approximately 15% from the baseline preoperative value was required for entry into the myocardial failure group for the study. The significant but relatively small (
15%) reduction in SF was sought so that rabbits would not die from heart failure. All echocardiographic images were acquired and analyzed by a single experienced operator. Mean ± SEM was calculated for SF, left ventricular internal diastolic diameter (LVIDd), left ventricular internal systolic diameter (LVIDs), left ventricular posterior wall diastolic dimension (LVPWd), and left ventricular posterior wall systolic dimension (LVPWs). Values obtained before and 4 weeks after surgery were compared utilizing a paired t-test.
Before commencing the main experiments, pilot studies with dofetilide were performed to establish that there was no difference in QTc interval prolongation and TdP induction between normal rabbits and rabbits at 4 weeks after sham operations. In both cases, dofetilide (0.04 mg/kg/10 min) lengthened the QTc interval (371 vs. 357 ms, respectively, no statistical difference by t-test, p = 0.33) and failed to cause TdP in either group of animals.
Experimental protocol.
Animals were anesthetized with a combination of ketamine and xylazine (35 and 5 mg/kg im). The right marginal ear vein was cannulated for intravenous administration of drug. Rabbits were placed in dorsal recumbency. The right and left thoracic limb electrodes were attached to the right and left hemithoraces, the electrocardiograph was switched to limb lead I, and a bipolar transthoracic ECG was obtained on a Biopac MP100 Data Acquisition Unit (Biopac Systems, Inc., Santa Barbara, CA). The high-pass filter was set at 0.01 Hz and the low-pass filter at 1 kHz, and signals were digitally sampled at a frequency of 2 kHz. Anesthesia was maintained as necessary by administration of further doses of ketamine and xylazine (20 and 3 mg/kg im, respectively). All rabbits were allowed at least 10 min stabilization before starting any drug infusion.
Drugs were selected because they are either known torsadogens or known not to produce TdP. Torsadogens were selected from groups possessing varied pharmacological properties (e.g., class IA, class III, prokinetic). All drugs were infused intravenously over a period of 10 min, with a 20-min interval between doses. The infusion was stopped as soon as TdP started. The drugs tested in this study were amiodarone (3, 10, and 30 mg/kg; control group n = 6, myocardial failure group n = 4), cisapride (0.1, 0.25, 0.50 mg/kg; control group n = 4, myocardial failure group n = 4), clofilium (0.1, 0.2, 0.4 mg/kg; control group n = 6, myocardial failure group n = 5), dofetilide (0.005, 0.01, 0.02, and 0.04 mg/kg; control group n = 10, myocardial failure group n = 7), quinidine (3, 10, and 30 mg/kg; control group n = 6, myocardial failure group n = 4), and verapamil (0.25, 0.50, and 1.0 mg/kg; control group n = 6, myocardial failure group n = 4). The doses of each drug were chosen according to those used in previously published rabbit studies by other research groups (Batey and Coker, 2002; Carlsson et al., 1997; Farkas et al., 2002; Lu et al., 2000). RR and QT intervals were measured 15 min after each drug dose had been infused. The QT interval was corrected for heart rate by dividing the QT interval by the cube root of the preceding RR interval (Fridericia, 1920). Measurements were made from at least 12 consecutive cardiac cycles, and the average was used. The ECG intervals were measured in beats that originated from the sinoatrial node; however, at some points ECG intervals could not be obtained from all animals because of marked arrhythmia. The QT interval was defined as the time between the first deviation from the isoelectric line during the PR interval until the end of the T wave. TdP was defined as a polymorphic ventricular tachycardia where clear twisting of the QRS complexes around the isoelectric axis was seen.
Drugs.
Amiodarone hydrochloride (Sicor Pharmaceuticals, Irvine, CA) was prepared by diluting commercial amiodarone for injection (450 mg amiodarone in 9 ml of diluent) with 9 ml of 0.9% NaCl to a final concentration of 25 mg/ml. Cisapride (Medisca Inc., Plattsburgh, NY) was dissolved in 10% acetic acid to form a stock concentration of 10 mg/ml. Clofilium tosylate (Sigma, St Louis, MO) was dissolved in 0.9% NaCl solution to form a stock concentration of 3 mg/ml. Dofetilide (Pfizer, Groton, CT) was dissolved in 0.9% NaCl with the help of 0.1M hydrochloric acid to form a stock concentration of 0.1 mg/ml. Quinidine hydrochloride (Sigma) (stock concentration of 30 mg/ml) and verapamil hydrochloride (Sigma) (stock concentration of 2 mg/ml) were also dissolved in 0.9% NaCl. Analyses of dosing solutions were not performed; however, each drug was injected within 60 min of preparation.
Statistics.
Values are expressed as mean ± SEM. Fisher's exact tests were used to compare the incidence of TdP between groups. A probability value of p < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Effects of Coronary Ligation on Echocardiogram
Coronary ligation, followed by a 4-week healing period, provided a reproducible model of left ventricular systolic dysfunction in this study. Effects of myocardial infarction on regional wall motion and regional and global geometry were clearly detected 4 weeks after coronary ligation. Hypokinesis was observed at the posterior and lateral walls of the left ventricle. Figure 1 shows typical M-mode echocardiograms of the left ventricle before and 4 weeks after ligation of the coronary arteries. In all 28 surviving rabbits in the infarction group, left ventricular internal diastolic diameter (LVIDd) increased significantly (mean increase 11.8%; p < 0.01), as did left ventricular internal systolic diameter (LVIDs, mean increase 19.2%; p < 0.01) and the difference between LVIDd and LVIDs (SF, see Table 1). Mean SF decreased 14.8% (p < 0.05), from 30.41% before infarction to 25.90% afterward.
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Effects of Coronary Ligation on ECGs
No significant differences were observed in heart rate, QT, and QTc between ECGs recorded in the 28 rabbits prior to, immediately following, and 4 weeks after coronary ligation (Table 2). Immediately following coronary ligation, ST-segment deviation (Fig. 2) and a variety of ventricular ectopic beats were observed (i.e., ventricular premature beats and ventricular tachycardia).
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Arrhythmia Incidence
Four weeks following coronary ligation, no spontaneous arrhythmias (without provocation from drugs) were observed in the surgical ligation/myocardial failure group and none in the control group at any time. TdP (Fig. 3) was induced by drug administration in four of the 38 rabbits in the control group (10.5%). Of these four rabbits, TdP was produced by dofetilide in two rabbits and clofilium in the other two rabbits. In contrast, TdP was produced in 13 (> 46%; p = 0.001) of the 28 rabbits in the myocardial failure group. TdP was induced by cisapride, clofilium, and dofetilide (Fig. 4). The incidence of this arrhythmia was significantly higher in the myocardial failure group compared to the normal control group in response to clofilium (p < 0.05; 100% [five of five rabbits] of the myocardial failure group affected at the first or second dose level vs. 33% [two of six] of controls at the second dose level) and dofetilide (p < 0.05). Cisapride induced TdP only in rabbits with myocardial failure (50%; two of four). The occurrence of TdP in response to dofetilide was not directly proportional to dose. TdP occurred in six rabbits in the myocardial failure group overall (85%; six of seven) and was induced at the third dose (0.02 mg/kg) in three of six rabbits in the myocardial failure group, as well as in three of six rabbits in that group at the fourth dose (0.04 mg/kg). TdP was induced only by the higher fourth dose of dofetilide (0.04 mg/kg) in two of the 10 rabbits (20%) in the normal (nonoperated) control group.
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Effects of Compounds on QTc
The plots of difference in QTc interval from baseline values versus each dose of each compound for both myocardial failure and nonfailing control rabbits (Figs. 5a–5f) reveal that QTc lengthened more in rabbits in the myocardial failure group than in those in the control group in response to escalating doses of dofetilide (p < 0.01) and quinidine (p < 0.05). No significant differences in the effect of the other compounds on QTc interval were identified between the myocardial failure group and controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This study was designed to test the hypotheses that (1) for equivalent doses of torsadogenic compounds, QTc lengthens more in rabbits with failing hearts than in normal rabbits, and (2) rabbits with failing hearts are more prone to develop TdP than rabbits with normal hearts. Based on the results of this study, both hypotheses should be accepted; however, QTc prolonged more in rabbits with myocardial failure than in normal controls in response to cisapride (did not achieve statistical significance, p = 0.36), dofetilide, and quinidine, but not in response to clofilium. The fact that in some instance TdP occurred in the absence of exaggerated lengthening of QTc confirms the supposition that lengthening of QTc is merely a surrogate for torsadogenicity and not the proximate cause.
The statement that only sham-operated rabbits did not develop TdP when given dofetilide is based on the findings from only two sham-operated rabbits. Two out of 10 normal rabbits (20%) developed TdP when given dofetilide. Six out of seven rabbits ( 85%) with heart failure developed TdP. Therefore, the two sham-operated rabbits in this study were investigated to provide some assurance that sham operation alone would not be permissive to TdP when exposed to dofetilide.
Potential explanations of the pathophysiology that underpins these results remain speculative but might include the effects of infarction and/or myocardial failure on calcium metabolism. These effects include dysfunction of channels/pumps specific for calcium cycling between the extracellular and intracellular space (e.g., calcium channel, ICaL; sodium calcium exchanger, NCX; Ca-ATPase pump) and between the cytosol and sarcoplasmic reticulum (e.g., Inositol 1,4,5-trisphosphate, IP3; ryanodine receptor Ca2+ channel; cardiac sarco/endoplasmic reticulum Ca2+-ATPase, SERCA2). These channels/pumps may be affected by the degree of phosphorylation, possibly by CaMKII activated by the augmented calcium-calmodulin complex (Anderson, 2006). Myocardial failure characterized by a structurally diseased heart, in which the repolarizing currents are typically reduced in concert with diminished calcium handling capacity (Anderson, 2006), combined with delayed repolarization, may increase the free cytosolic calcium concentration, a factor that has been shown to be important in the induction of early afterdepolarizations (EAD) and triggered activity (Qin et al., 1996). CaMKII is upregulated in many animal models of cardiac failure (Anderson, 2006). Animals with myocardial failure may have impaired repolarization reserves (abbreviation of the duration for repolarization), a known risk of TdP in humans with failing heart patients (Roden and Yang, 2005).
Increased heterogeneity of ion channel physiology (Qin et al., 1996; Rozanski et al., 1998) is known to occur in the setting of myocardial failure. The importance of increased cardiac ion current heterogeneity and subsequently increased dispersion of repolarization and refractoriness across the left ventricular wall (i.e., transmural dispersion) to the potential genesis of TdP has been suggested in both clinical and experimental studies (Antzelevitch and Shimizu, 2002; Belardinelli et al., 2003; Lubinski et al., 1998). However, the exact relationship between dispersion of repolarization and TdP is not fully understood. Recent data suggest that spatial dispersion of ventricular repolarization provides the substrate for TdP, and EAD and the ectopic beats that result from them may provide the trigger (Belardinelli et al., 2003).
The lack of quinidine-induced TdP in our experiments is in accord with the results of previous in vivo animal studies (Chezalviel-Guilbert et al., 1995; Farkas et al., 2002; Lu et al., 2000). One possible explanation is that the proarrhythmic activity of quinidine was blunted in this rabbit model compared to its effect in humans by the rabbit's high heart rate (Farkas et al., 2002). Another potential explanation is that quinidine has multiple channel-blocking properties (i.e., at lower concentrations quinidine blocks IKr while it suppresses IKs and late INa at higher concentrations) and reduces transmural dispersion of ventricular repolarization (i.e., at higher concentrations quinidine produces a further prolongation of the epicardial and endocardial action potentials whereas an abbreviation of the action potential duration of M cells), a factor that has been shown to be responsible for TdP (Di Diego et al., 2003).
Intravenous amiodarone did not induce TdP in this study, a finding consistent with the findings of previous studies (Farkas et al., 2002; van Opstal et al., 2001). Amiodarone has the ability to inhibit a constellation of cardiac ionic currents (i.e., IKr, IKs, INa, ICaL), resulting in little proarrhythmia (Farkas et al., 2002). Amiodarone also produces a greater prolongation of the action potential duration in the epicardium and endocardium but less of an increase or decrease in the M cells, thereby reducing transmural dispersion of repolarization.
It appears that dofetilide, cisapride, and clofilium are associated with the lowest 50% inhibitory concentration (IC50) for the human ether-a-go-go-related gene (hERG), and these compounds are more torsadogenic in failing hearts than in normals. The IC50 for amiodarone and quinidine are 0.7 and 0.4 µM, respectively, whereas the IC50 for clofilium, dofetilide, and cisapride are 0.001, 0.012, and 0.02 µM, respectively (Diaz et al., 2004; Kim et al., 2005).
In the current study, clofilium, dofetilide, and cisapride induced TdP. This agrees with the results of other investigators (Batey and Coker, 2002; Carlsson et al., 1990, 1997; Lu et al., 2000). These compounds block the hERG current at very low concentrations, causing lengthening of action potential duration and allowing a longer time for cellular membrane depolarization within a voltage window that allows for repetitive ICaL channel reopening. These effects initiate EAD, the factor that has been shown to be the initiating mechanism of TdP (Anderson, 2006). Other in vivo and in vitro studies have shown that cisapride, clofilium, and dofetilide increase transmural dispersion of repolarization, providing a substrate for TdP (Di Diego et al., 2003; Faivre et al., 1999; Wu et al., 2005).
The dosage of dofetilide that induced TdP in this study is in the range that evoked TdP in other in vivo rabbit studies, i.e., rabbits with chronic AV block (0.02 mg/kg) or an 
1-adrenoceptor–stimulated rabbit (0.04 mg/kg) (Lu et al., 2000; Tsuji et al., 2006). The dofetilide concentration in this study was equal to or lower than that used to evoke TdP in isolated, perfused rabbit hearts stimulated with methoxamine and acetylcholine (0.1–0.7 µM) (D'Alonzo et al., 1999), based on the concentration of unbound dofetilide (Smith et al., 1992). In contrast, an efficacious dose of dofetilide (0.75 µg/kg po) in humans produces a plasma concentration of 2.26 ng/ml (approximately 0.005 µM). The concentration of dofetilide in the present study is approximately 10 times higher than that of effective human concentration (Tham et al., 1993).
Verapamil, a compound known to affect hERG physiology but because of multichannel effects does not result in TdP, was used as negative control in this study and did not produce TdP in normal rabbits or in rabbits with failing hearts. This finding could be supported by the previous study of Milberg et al. (2005), who found that verapamil prevented TdP by shortening of endocardial monophasic action potential duration and by reduction of left ventricular transmural of repolarization.
More than 90% of rabbits operated upon to produce myocardial infarction survived the duration of the study. No rabbit with heart failure has ever developed TdP unprovoked by torsadogenic articles. In fact, 1 week after ligation, rabbits never demonstrated even ventricular premature depolarizations. In both humans with spontaneous coronary occlusion and presumably in rabbits undergoing coronary ligation, the extent of resulting infarction may be highly variable (Cobb and Chu, 1988). Although not quantified by morphometry in this study, the extent of infarction produced by this anatomically defined technique appeared reasonably consistent from rabbit to rabbit. Although termed ischemic myocardial failure because an acute ischemic event precedes development of heart failure, this model is not a model of ischemic myocardial failure since the rabbits manifested myocardial failure weeks after the ischemic event, when ischemic myocardium could be expected to have been replaced by fibrous tissue. This pathophysiology is supported by the finding of J-point deviation that persists for days after coronary ligation but has completely resolved by the time the rabbits were exposed to test articles 4 weeks following coronary ligation.
The surgical procedure used in the study requires reasonable—but not exceptional—surgical skill and takes less than 1 h. Within 12 h after surgery, operated rabbits were jumping and eating normally. Although dramatic reductions in left ventricular SF indicated significant myocardial dysfunction, none of the 28 rabbits studied died of heart failure or manifested signs that might have indicated discomfort; thus, we believe that this model is humane. No postoperative discomfort was observed, possibly because of the aggressive use of analgesics and possibly because of the surgical approach (a midline sternotomy) combined with the small size of the incision. Because of the rabbits' unique pleural anatomy, neither endotracheal intubation nor positive pressure ventilation was required (Fujita et al., 2004), and the rabbits breathed with what appeared to be a nearly normal tidal volume under anesthesia. It is important that the incision be made precisely on the midline so that no incursion is made in either the left or the right thoracic cavities.
The rabbits that served as the control group for the group of operated rabbits were not sham operated. In light of our pilot results (see "Materials and Methods" section), it is unlikely that a thoracotomy alone could account for the differences in susceptibility to TdP between the operated and nonoperated rabbits.
The operated rabbits were not considered to be under threat of heart failure because they did not show any clinical signs (e.g., dyspnea, exercise intolerance); however, based on the echocardiographic findings of left ventricular enlargement and reduction in left ventricular SF, it is clear that they had myocardial failure (Katz, 1993). This model appears to share at least some of the important properties of human heart diseases that predispose to the development of TdP.
Advantages and Disadvantages of a Model of Myocardial Failure in Rabbits to Predict Torsadogenicity in Persons
As mentioned previously, the "perfect" surrogate for man is that which possesses sensitivities and specificities of 1.0, i.e., neither false positives nor false negatives. To determine these predictive values, hundreds of test articles must be investigated to assure that there are neither false negatives nor false positives. No model has been subjected to such scrutiny. Therefore, we believe that it is reasonable to claim that a model which has no false negatives or false positives for all drugs tested and which possesses the anatomical and physiological substrates that are torsadogenic in man (not substrates that may be torsadogenic but do not occur in the vast instances of TdP in humans) should be the "best" on a theoretical basis. Existing models (e.g., methoxamine-sensitized rabbit, third-degree AV block) do develop not only lengthening of QTc but also TdP in response to torsadogens; however, most instances of TdP do not occur because of the presence of an 1-adrenergic agonist or third-degree AV block (present in < 140,000 persons); rather TdP occurs with a substrate of heart failure (present in > 5,000,000 persons), left ventricular hypertrophy (present in 43,000,000 persons), or diabetes (present in 18,000,000 persons). Thus, the model of heart failure should be a much more realistic predictor for propensity of a test article to produce TdP in persons.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Our findings show that rabbits with failing myocardium are more susceptible to developing drug-induced TdP than rabbits without myocardial failure and that lower doses of torsadogens are required to produce this effect in the setting of ischemic myocardial dysfunction. Cisapride, clofilium, and dofetilide induced a higher incidence of TdP in rabbits with myocardial dysfunction, whereas amiodarone, quinidine, and verapamil failed to evoke TdP. This rabbit model of ischemic myocardial failure may be more useful than current surrogates for predicting torsadogenic risk in man since it tests directly for production of TdP rather than merely for lengthening QTc interval in response to drug administration.
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