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CARDIOVASCULAR

Electrophysiological Safety of Sertindole in Dogs with Normal and Remodeled Hearts

Department of Cardiology, Cardiovascular Research Institute Maastricht, Academic Hospital Maastricht, Maastricht, The Netherlands (M.B.T., P.G.A.V., M.S., R.L.H.M.G.S., J.D.M.B., M.A.V.); GENION, Evotec QAI AG, Hamburg, Germany (U.B.); and H. Lundbeck A/S, Copenhagen, Denmark (M.A.K., K.F., J.M.)

Received April 7, 2003; accepted July 10, 2003.

	Abstract

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Inhibition of the potassium current I_Kr and QT prolongation are associated with drug-induced torsades de pointes arrhythmias (TdP) and sudden cardiac death. We investigated the cardiac electrophysiological effects of sertindole, an antipsychotic drug reported to prolong the QT interval in schizophrenic patients. In cell cultures, sertindole seemed to be a selective blocker of I_HERG over other ion currents. For I_HERG, the IC₅₀ value was 64 ± 7 nM, whereas _ISCN5A, I_Ca,L, I_Ca,T, I_KCNQ1/KCNE1, and I_Kv4.3 were blocked in the micromolar range. In canine ventricular myocytes, the IC₅₀ value for I_Kr inhibition by sertindole was 107 ± 21 nM. Action potentials in these cells prolonged in a reverse rate- and concentration-dependent manner at 10 to 300 nM sertindole. In vivo, sertindole was administered to anesthetized dogs at clinically relevant (0.05-0.20 mg/kg) and high doses (1.0-2.0 mg/kg) i.v. At 0.05 to 0.20 mg/kg sertindole (plasma concentrations 30-157 nM), QT_c was prolonged by 1 to 5% in normal dogs and by 9 to 20% in dogs with remodeled hearts due to chronic atrioventricular block (CAVB). TdP was not induced at these doses in normal dogs or in CAVB dogs with reproducible induction of TdP by dofetilide in previous experiments. At 1.0 to 2.0 mg/kg sertindole (plasma concentrations 0.5-3.1 µM), QT_c prolonged by 6 to 11% in normal dogs and by 22% in dofetilide-sensitive CAVB dogs. TdP occurred in three of five animals in the latter group. Thus, at high i.v. doses sertindole can pose a serious proarrhythmic risk when electrical remodeling of the ventricles is present. At clinically relevant doses, however, sertindole does not cause TdP in anesthetized dogs with normal or remodeled hearts.

5-Chloro-1-(4-fluorophenyl)-3-(1-(2-(2-imidazolidinon-1-yl)-ethyl)-4-piperidyl)-1H-indole (sertindole) is an antipsychotic compound synthesized in the mid-1980s and introduced on the European market in 1996. Clinical phase III trials showed therapeutic effectiveness against both positive and negative symptoms of schizophrenia (Hale et al., 2000), whereas extrapyramidal symptoms were absent (Zimbroff et al., 1997). Sertindole has a high affinity for several serotonin and dopamine receptor subtypes and _1A-adrenergic receptors (Ipsen et al., 1997; Arnt, 1998; Bigliani et al., 2000). Furthermore, a high inhibitory effect on the current mediated by the potassium channel encoded by HERG has been shown in vitro (Rampe et al., 1998). HERG blocking properties with IC₅₀ values ranging from low nanomolar to low micromolar are common for most antipsychotic drugs (Frederiksen and Adamantidis, 2000; Haverkamp et al., 2002; Kongsamut et al., 2002) and may explain the drug-induced QT prolongation caused by some of them (Haverkamp et al., 2000).

In 1998, sertindole was withdrawn from the market due to concern about the high ratio of proven or suspected ventricular arrhythmias and sudden deaths in patients (Moore, 2002). QT_c intervals were prolonged in 4 to 5% of patients receiving sertindole (Kasper et al., 1998), and prolongation of action potential duration was confirmed in isolated feline (Drici et al., 1998) and rabbit hearts (Eckardt et al., 2002). After reevaluation of the existing data, and based on new preclinical, clinical, and epidemiological information, the concern about cardiac risk was outweighed by the therapeutic benefits of sertindole. This led to the reintroduction of sertindole on the European market in 2002 along with a prospective surveillance study of all patients taking the drug (Toumi, 2002).

In the present study, we investigated the cardiac electrophysiological effects of sertindole in vitro and in vivo to provide an ionic basis for repolarization prolongation by the drug in relation to possible proarrhythmic actions in intact dogs.

	Materials and Methods

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Animal handling was in accordance with the European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609/EU). The Committee for Experiments on Animals of Maastricht University approved all experiments.

Measurements on Ion Currents in Cell Cultures. Chinese hamster ovary cells were stably transfected with human cloned HERG (I_HERG; GENION, Hamburg, Germany) and SCN5A (I_SCN5A; obtained from Dr. R. Kallen, University of Pennsylvania, Philadelphia, PA), representing the rapidly activating delayed rectifier potassium current and fast inward sodium current, respectively. Maximal outward I_HERG and peak I_SCN5A were measured. KCNQ1 and KCNE1 were stably cotransfected in Chinese hamster ovary cells (I_KCNQ1/KCNE1; GENION) representing the slowly activating delayed rectifier potassium current. Human embryonic kidney 293 cells transiently expressing Kv4.3_2-39 (I_Kv4.3) were used to assess the transient outward potassium current. Calcium-current experiments were performed on a NG-108-15 neuroblastoma-glioma hybrid cell line expressing endogenous L- and T-type calcium channels. Standard voltage-clamp protocols, electrodes, superfusion, and internal solutions were used. Ion currents were measured at control and after 5-min incubation of sertindole at various concentrations. Each data point consisted of measurements from three to eight cells. IC₅₀ values were obtained by fitting the data to a two-parameter sigmoidal curve (I = cⁿ/(cⁿ + IC₅₀ⁿ)).

Experiments in Isolated Canine Ventricular Myocytes. Twelve mongrel dogs (body weight 29 ± 5 kg, 8 males) were sacrificed for myocyte isolation. Thoracotomy was performed under anesthesia. Heparin (10,000 IU) was administered i.v. to avoid intracoronary clotting. After quick excision, the heart was placed in cold oxygenated cardioplegic solution, and the coronary circulation was cannulated via the aorta. The heart was mounted to a constant-pressure Langendorff-like setup and perfused for 5 min using a Tyrode's solution with nominal [Ca²⁺]. Collagenase A (Roche Diagnostics, Mannheim, Germany) in 0.5 µMCa²⁺ Tyrode with 0.5 mg/ml bovine serum albumin perfused the heart for 30 to 35 min, followed by 0.2 mM Ca²⁺ Tyrode for 5 min to washout the collagenase. Midmyocardial cells were harvested from the free wall of both ventricles, gently minced, filtered, washed, and stored in 0.2 mM Ca²⁺ at room temperature until use within 24 h after isolation.

Myocytes were selected for experiments if they had sharp striations, clear contours, and transparent cytoplasms without granulations or blebs. Further criteria for action potential experiments included a stable resting membrane potential below -70 mV and a "spike-and-dome" morphology of the action potential.

Whole-cell currents were recorded (AxoPatch 1D; Axon Instruments, Union City, CA) using borosilicate glass patch pipettes filled with internal solution (125 mM K-asp, 20 mM KCl, 1.0 mM MgCl₂, 5 mM MgATP, 5 mM HEPES, 10 mM EGTA; pH adjusted to 7.2 with KOH) having a resistance between 1.0 and 3.0 M. Cells were superfused with a standard buffer solution (145 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl₂, 11 mM glucose, 10 mM HEPES, 1.8 mM CaCl₂, 0.005 mM nifedipine; pH adjusted to 7.4 with NaOH, 37°C). Sertindole was dissolved in dimethyl sulfoxide. The rapidly activating delayed rectifier potassium current I_Kr was measured as the tail current fraction fully blocked by 2 µM almokalant (Carmeliet, 1993).

Transmembrane action potentials (TAP) were recorded (Axo-Clamp 2B; Axon Instruments) using sharp glass microelectrodes filled with 3 M KCl and with a resistance between 20 and 60 M. Cells were superfused with the same solution as in the whole-cell current experiments except that nifedipine was left out. Addition of 2 µM almokalant to the superfusate was used to fully block I_Kr. Action potentials were recorded at each cycle length (CL) of 300, 400, 500, 1000, and 2000 ms. Action potential duration at 95% of repolarization (APD₉₅) is presented as the average of five beats >100 beats after a change in pacing CL.

In Vivo Experiments. Twenty-four anesthetized dogs (body weight 29 ± 4 kg, 11 males) were used for these experiments. In 13 animals, complete AV block was induced (van Opstal et al., 2001a). After 4 ± 1 weeks of AV block (chronic AV block; CAVB), the dogs were subjected to a TdP-susceptibility test using the I_Kr blocker dofetilide. Only if a dog showed reproducible TdP upon 25 µg/kg/5 min dofetilide, it was selected for the sertindole experiments. Thus, dofetilide was used as the positive reference compound. The average time between two experiments in a dog was 2 ± 1 weeks.

Anesthesia, perioperative care, signal processing, data recording, and off-line analysis have been described previously (van Opstal et al., 2001a). Standard and precordial ECGs were recorded. In addition, biventricular endocardial monophasic action potential (MAP) recordings were made (EP Technologies, Sunnyvale, CA).

RR and QT intervals in lead II, left and right ventricular MAP duration (LV MAPD and RV MAPD, respectively) at 100% repolarization were measured off-line and averaged from five consecutive beats. The interventricular dispersion of repolarization (MAPD) was calculated as the difference between the LV and RV MAPD. QT intervals were corrected for heart rate (QT_c) according to Van de Water's formula (Van de Water et al., 1989). The number of ectopic beats, defined as short-coupled beats arising from a new ventricular focus before complete repolarization of the previous beat, was counted during 10 min after administration of the drug. Both single and multiple ectopic beats were counted. The latter are considered more proarrhythmic. TdP was defined as a polymorphic ventricular tachycardia consisting of five or more beats twisting around the isoelectric line of the ECG in the setting of a prolonged QT interval.

Sertindole (mol. wt. 441 g/mol) was dissolved in 0.1 M HCl and diluted in 10% hydroxypropyl cyclodextrin and 0.05 M phosphate buffer (1:1). pH was adjusted to 7.4. The solution was filtered through a 22-µm pore filter before use. Sertindole was administered over 5 min through a cephalic vein and blood samples were taken from the contralateral cephalic vein to measure plasma concentrations.

Plasma Analysis. Blood samples were obtained 5, 10, and 25 min after drug administration and plasma was stored at -20°C until analysis at H. Lundbeck A/S (Copenhagen, Denmark). Sertindole plasma samples were extracted by solid mixed phase extraction. The sample extracts were analyzed by a normal phase high-performance liquid chromatography method with a mobile phase consisting of heptane, 2% piperidine in 2-propanol, and water (100:20:0.45) and quantified by fluorescence detection with excitation/emission wavelengths at 260 and 340 nm, respectively. The method had a mean recovery of 90% with a quantification limit of 0.5 ng/ml. Total plasma concentrations (free + bound) are reported.

Statistics. Electrophysiological parameters were compared with control using (repeated-measures) ANOVA followed by Bonferroni's test. Comparisons between controls were performed with an un-paired Student's t test. Data are reported as mean ± s.e.m. A P < 0.05 was considered statistical significant.

	Results

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Sertindole Is Selective for I_HERG. In Fig. 1A the molecular structure of sertindole is shown. Figure 1B shows concentration-response curves for the various cardiac ion channels expressed endogenously or by transfection in cell cultures. Sertindole inhibited I_HERG in a concentration-dependent manner over the range of 10-1000 nM, with 50% block at 64 ± 7 nM. I_KCNQ1/KCNE1 was inhibited by 10 ± 5% at 300 nM sertindole (IC₅₀ value = 6.9 ± 2 µM), whereas other currents (I_SCN5A, I_CaL, I_CaT, and I_Kv4.3) were only inhibited at micromolar concentrations. Representative examples of the six currents are shown in Fig. 2.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Selective block of IHERG by sertindole. A, chemical structure of sertindole (mol. wt. 441 g/mol). B, composite data on blocking effects of sertindole on IHERG, ISCN5A, ICa,L, ICa,T, IKCNQ1/KCNE1, and IKv4.3 in heterologous and endogenous expression systems. Data points represent the means for three to eight cells. Shown are the concentration-response curves, where the remaining current is plotted relative to its control level. IC50 values for the different currents are given in the legend. Hill coefficients are 1.16 for IHERG, 1.27 for ISCN5A, 1.15 for ICa,L, 3.98 for ICa,T, 0.99 for IKCNQ1/KCNE1, and 2.05 for IKv4.3.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Representative tracings of IHERG, ISCN5A, ICa,L, ICa,T, IKCNQ1/KCNE1, and IKv4.3 during treatment with sertindole. Concentrations of sertindole were chosen to indicate selectivity for IHERG block. Insets, voltage-clamp protocols.

Sertindole Blocks I_Kr in Canine Ventricular Myocytes. Activation of I_Kr occurred at depolarizations to higher than -10 mV and showed saturation at conditioning voltages (V_cond) 20 mV (Fig. 3B). Maximal I_Kr density at control was 0.14 ± 0.07 pA/pF. Boltzmann fit to the data revealed a half-maximal activation at 11 ± 1 mV and a slope factor of 5.9 ± 0.9 pA/pF/mV. Half-time for I_Kr deactivation upon repolarization to -50 mV was 294 ± 23 ms.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of sertindole on IKr in normal canine ventricular myocytes. A, representative current recordings at control (arrows) and during 100 nM sertindole (Cm = 207 pF). Left horizontal bar is at 600 pA. Illustrated are second-order exponential fits of the tail currents at control (black arrow), full IKr block (white arrow), and 100 nM sertindole. Current axis is enlarged, but time axis is identical to current trace in A. B, IKr tail densities (difference currents at control and during 300 nM sertindole minus full block by almokalant; ncells = 6). *, P < 0.05 versus control. Inset, voltage-clamp protocol. C, tail-current amplitudes in a representative cell during increasing concentrations of sertindole (Cm = 191 pF). Almokalant (2 µM) provides full IKr block. Voltage-clamp protocol as in A. D, concentration-response curve of IKr inhibition by sertindole. IC50 value is 107 ± 21 nM (Hill coefficient = 0.76, ncells = 10).

An example of I_Kr recorded under control conditions and under the influence of 100 nM sertindole is shown in Fig. 3A. Sertindole inhibited I_Kr tails in a concentration-dependent and voltage-independent manner. At 300 nM, the maximal I_Kr tail density had decreased to 0.08 ± 0.004 pA/pF (57 ± 4%; P < 0.05; Fig. 3B). Boltzmann fit of the remaining I_Kr at 300 nM sertindole showed a half-maximal activation at 8 ± 2 mV (P = N.S. versus control) and a slope factor of 4.2 ± 1.4 pA/pF/mV (P = N.S. versus control). Half-time for deactivation was 273 ± 48 ms (P = N.S. versus control). Figure 3C shows an example of the effects of accumulating concentrations of sertindole to illustrate the concentration dependence of the drug on I_Kr. Using multiple voltage protocols to analyze the properties of I_Kr under the influence of sertindole, a concentration-response relationship was obtained (Fig. 3D). Sertindole inhibited I_Kr in a concentration-dependent manner over the full range of 10 to 1000 nM, with 50% block at 107 ± 21 nM (n_cells = 10).

Sertindole Prolongs the Transmembrane Action Potential. TAP in normal canine ventricular myocytes prolonged from 166 ± 5 to 278 ± 13 ms by increasing pacing CL from 300 to 2,000 ms (n_cells = 11). Concentration-dependent prolongation of APD₉₅ was observed for 10 to 300 nM sertindole, reaching statistical significance at 100 nM and higher and for CL 400 ms (Fig. 4). Under the influence of 300 nM sertindole, APD₉₅ was prolonged to 197 ± 15 ms (18%) and 345 ± 44 ms (24%) at 300 and 2000 ms CL, respectively (P < 0.05 for both CL), showing clear reverse rate dependence. Early afterdepolarizations or abnormal automaticity were not observed.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Transmembrane APD95 during sertindole treatment. Concentrations used were 10, 30, 100, and 300 nM. Pacing CL of 300, 400, 500, 1,000, and 2,000 ms were applied. Left and right vertical axes are identical. *, P < 0.05, 100, and 300 nM versus control, ncells = 9. Inset, representative action potentials at control and during 300 nM sertindole, CL = 2000 ms (0 mV level indicated; scale bar, 100 ms at -80 mV).

Sertindole Causes Moderate Prolongation of Repolarization in Normal Hearts in Vivo. Cumulative doses of 0.05, 0.10, and 0.20 mg/kg sertindole (30-min intervals) were administered to five dogs. Plasma concentrations ranged from 33 ± 1 nM after 0.05 mg/kg to 157 ± 18 nM after 0.20 mg/kg. Reported plasma concentrations after human therapeutic dosing are 22 ± 12 to 158 ± 63 nM (Wong and Granneman, 1998); hence, we considered these doses in the dogs to be clinically relevant. Representative examples of the electrophysiological effects are shown in Fig. 5. QT_c interval did not prolong at 0.05 or 0.10 mg/kg sertindole. At 0.20 mg/kg QT_c prolonged from 277 ± 11 to 292 ± 20 ms (5%; P < 0.05; Fig. 6). At this dose, the RR interval increased from 465 ± 35 to 545 ± 47 ms (17%; P < 0.05) and the QT interval from 231 ± 7 to 252 ± 12 ms (9%; P < 0.05). The LV MAPD prolonged from 191 ± 8 to 213 ± 9 ms (10%; P < 0.05), whereas the RV MAPD remained unchanged (181 ± 9 to 197 ± 7 ms; P = N.S.), leaving the interventricular dispersion of repolarization unaltered.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Electrophysiological effects of cumulative doses of sertindole in an anesthetized normal dog. Three ECG leads, LV, and RV MAP recordings are shown in each panel. V6 refers to a precordial lead placed in the 6th intercostal space on the left lateral side of the thorax. RR intervals are above and QT times below lead II. MAPDs are below each signal. ECG calibrated to 1 mV/cm. Scale bar (right), 20 mV on the MAP signals. Horizontal scale bar, 1 s. In this example, the plasma concentrations measured at the time of these tracings (10 min after start of infusion) are 31, 77, and 121 nM after 0.05, 0.10, and 0.20 mg/kg, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Relation between QTc intervals and plasma concentrations of sertindole in anesthetized normal dogs. Cumulative doses of sertindole were administered i.v. as indicated (nlow doses = 5 dogs, nhigh doses = 6 dogs). Plasma samples were obtained 10 min after start of drug infusion at each dose. *, P < 0.05 versus respective QTc at control.

Cumulative doses of 0.5, 1.0, and 2.0 mg/kg sertindole (30-min interval) were administered to six other dogs. Plasma concentrations ranged from 0.5 ± 0.2 µM after 0.5 mg/kg to 3.1 ± 0.3 µM after 2.0 mg/kg. Twenty-four hours after the high dose-range experiments, the mean plasma concentration was 269 ± 31 nM. All high doses produced significant QT_c increases (Fig. 6) with a maximal QT_c prolongation from 294 ± 8 to 326 ± 19 ms (11%; P < 0.05) after 2.0 mg/kg sertindole. This involved a QT prolongation from 251 ± 10 to 289 ± 24 ms (15%; P < 0.05), whereas RR interval remained unchanged. LV MAPD increased from 214 ± 11 to 264 ± 28 ms (23%; P < 0.05) and RV MAPD from 208 ± 11 to 242 ± 22 ms (16%; P < 0.05). The interventricular dispersion of repolarization was not changed (8 ± 2 to 20 ± 12 ms; P = N.S.).

Sertindole induced no changes in the PQ interval or QRS duration. Apart from the QT prolongation, no major changes were seen in the T-wave morphology at low or high doses of administration (Fig. 5).

Sertindole Carries a Proarrhythmic Risk in Electrically Remodeled Hearts. Ten dofetilide-susceptible CAVB dogs received sertindole. In five animals, 0.10 mg/kg was administered, followed by another 0.20 mg/kg after 30 min. The QT_c interval prolonged more than in normal dogs (e.g., 20% after 0.20 mg/kg in CAVB dogs versus 5% in normal dogs). Electrophysiological data from these experiments are summarized in Table 1. The five other dogs were tested with 1.0 mg/kg sertindole (Table 1). Sertindole prolonged repolarization in a dose-dependent manner, whereas the CL of the idioventricular rhythm only increased at the high dose (Table 1). The high dose of sertindole caused reproducible TdP in three of five dogs (Fig. 7). In these three animals, the first TdP was seen on average 7 ± 2 min after start of the 1.0 mg/kg sertindole infusion (range 6-9 min). The two dogs not responding with TdP received another 1.0 mg/kg, which caused TdP in one dog. During the 1-h observation period after 1.0 mg/kg sertindole, a total of 19 TdP (6 ± 2; n_dogs = 3) were seen of which four TdP had to be cardioverted electrically. Single ectopic beats (16 ± 9) occurred in all dogs at high dosing, whereas multiple ectopic beats (5 ± 2) were seen in four dogs. Interventricular dispersion of repolarization tended to increase, e.g., from 45 ± 6 to 79 ± 19 ms at 1.0 mg/kg (P = 0.09).

View larger version (58K): [in this window] [in a new window]   Fig. 7. Proarrhythmia by sertindole at high doses in an anesthetized CAVB dog. TdP (right-most panel) occurred at 6 min after infusion of 1.0 mg/kg sertindole. Proarrhythmia was not observed at the low dose of 0.2 mg/kg (second-left panel, 10 min) in this or all other CAVB dogs. Three ECG leads, LV, and RV MAP recordings are shown in each panel. RR intervals are above and QT time below lead II. MAPDs are below each signal. ECG calibrated to 1 mV/cm. Scale bar (right), 20 mV on the MAP signals. Horizontal scale bar, 1 s.

Electrophysiological Data on the Positive Reference Compound Dofetilide. Concentration-response studies of dofetilide on I_Kr in native ventricular myocytes revealed an IC₅₀ value of 46 ± 9 nM (Fig. 8A). Prolongation of TAP in the myocytes was reverse rate-dependent (Fig. 8B). In normal anesthetized dogs, i.v. doses of 12.5, 25, and 50 µg/kg dofetilide (van Opstal et al., 2001a) caused significant QT_c prolongation (19-25%; P < 0.05 versus control; Fig. 8C). RR also increased, e.g., by 13% after 12.5 µg/kg (P < 0.05 versus control). Plasma concentrations of dofetilide are given in Fig. 8C. Dofetilide (25 µg/kg) induced TdP in 10/13 anesthetized CAVB dogs (Fig. 8D for n_dogs = 10 used for sertindole testing in vivo).

View larger version (24K): [in this window] [in a new window]   Fig. 8. Electrophysiological data on the positive reference compound dofetilide. A, concentration-response curve of the inhibition of IKr tails by dofetilide in normal canine ventricular myocytes (IC50 value, 46 ± 9; Hill coefficient, 0.76; mean, Cm = 180 ± 11 pF; ncells = 9). Almokalant (Almo; 2 µM) was used for full block of IKr. B, transmembrane APD95 upon increasing concentrations of dofetilide. *, P < 0.05, 100, and 300 nM versus control, ncells = 5. Vertical axes are identical. Inset shows two representative action potentials at control and during 300 nM dofetilide, CL = 2,000 ms (scale bar, 100 ms at -80 mV, 0 mV level indicated). C, QTc prolongation by increasing doses of dofetilide i.v. to six anesthetized normal dogs and corresponding plasma concentrations. *, P < 0.05 versus control QTc. For comparison, plasma concentrations after oral administration to humans are 5 to 23 nM (plasma protein binding, 60-70%; volume of distribution, 3 l/kg; Pfizer, 1999). D, electrophysiological effects of 25 µg/kg dofetilide i.v. to the 10 anesthetized dogs with CAVB that were TdP-inducible and used for sertindole testing. Values in milliseconds and percentage increases in brackets. *, P < 0.05 versus control. Singles and multiples, numbers of single and multiple ventricular ectopic beats. TdP and shocks, number of TdP and electrical cardioversions. Times after start of dofetilide infusion.

	Discussion

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The present study investigates the electrophysiological properties of sertindole from cloned cardiac ion channels to anesthetized dogs with normal and remodeled hearts. The results can be summarized as follows: 1) Sertindole is a selective blocker of I_HERG over other ion currents expressed in cell cultures. 2) Sertindole causes concentration-dependent block of native I_Kr, and this translates into reverse rate-dependent lengthening of myocyte action potentials. 3) In anesthetized dogs, dose-dependent prolongation of in vivo repolarization by sertindole is observed. 4) Clinically relevant doses of sertindole do not cause TdP in anesthetized normal dogs or in CAVB animals with reproducible induction of TdP by dofetilide in previous experiments. 5) High doses of sertindole induce multiple ectopic beats and TdP in the majority of these CAVB dogs.

Normal and Remodeled Hearts. To elucidate whether a drug is devoid of proarrhythmic properties, a reproducible animal model is essential. Testing drugs in normal hearts is necessary but is not sufficient for the recognition of proarrhythmic effects in the diseased heart. We used the canine model with CAVB, known to have acquired QT prolongation. Creation of CAVB results in a bradycardia-induced volume overload. Hypertrophy is observed in ventricular myocytes (Volders et al., 1998) as well as in the whole heart (Vos et al., 1998; Verduyn et al., 2001a). Contractile remodeling in vivo restores initially depressed cardiac output (compensated function), which is associated with an increased cytosolic Ca²⁺ transient in vitro (de Groot et al., 2000; Sipido et al., 2000). Down-regulation of I_Ks and I_Kr (Volders et al., 1999; Ramakers et al., 2003) and up-regulation of the sodium-calcium exchanger (Sipido et al., 2000) contribute to the electrical alterations in remodeled CAVB hearts. This ventricular remodeling predisposes to TdP and sudden cardiac death (van Opstal et al., 2001c).

Whereas most class III antiarrhythmic drugs cause TdP in 2 to 5% of patients (Haverkamp et al., 2000), an incidence in the order of 56 to 67% is encountered in anesthetized CAVB dogs, making the model very sensitive (Verduyn et al., 1997; van Opstal et al., 2001a). In the present study, the noncardiovascular drug sertindole was tested in a number of different ways and using a broad dose regimen. Based on earlier clinical reports of low proarrhythmia of sertindole in patients (0.3% cardiac mortality rate or 10% of antiarrhythmic drugs; Kasper, 2002), we anticipated a low TdP incidence in the CAVB dog. Serial testing in this model has shown reproducible induction of TdP (Verduyn et al., 2001b). Therefore, we chose to increase the sensitivity of the model and to evaluate the proarrhythmia of sertindole only in dogs that showed reproducible TdP after administration of 25 µg/kg dofetilide.

Cardiac Safety of Sertindole. This is the first report on sertindole in which both in vitro and in vivo investigations are combined. Sertindole caused prolongation of repolarization in both normal and CAVB dogs, although at variable degree, e.g., at 0.20 mg/kg, QT_c interval increased by up to 5% in normal hearts and by up to 20% in CAVB dogs. The plasma concentrations measured in dogs in this study at the low doses were comparable with plasma concentrations from human volunteers (4-20 mg/day sertindole p.o., range from 22 ± 12 to 158 ± 63 nM; Wong and Granneman, 1998). These doses did not cause TdP in dofetilide-sensitive CAVB dogs. Administration of 25 µg/kg dofetilide led to a plasma concentration of 79 ± 11 nM. Reported plasma concentrations from human volunteers receiving dofetilide ranged from 5 to 23 nM (Pfizer, 1999).

Eckardt et al. (2002) reported a low torsadogenic potential of sertindole in isolated rabbit hearts. They showed a 15 to 17% prolongation of the QT interval at a perfusion concentration of 1.5 µM sertindole without induction of TdP. In the present investigation in anesthetized dogs with normal hearts, 9% prolongation of the QT_c interval was observed at 1.3 ± 0.1 µM. No TdP was observed, confirming the results from Eckardt et al. (2002). Plasma protein binding in vivo and unknown levels of accumulation in cardiac tissue complicate comparisons between these models.

Relating plasma concentrations to concentrations used in the in vitro setting can only be done with great caution. Among the factors to be taken into account are plasma protein binding, tissue accumulation and the distance between the plasma protein and the receptor on the cardiomyocyte in situ. Plasma protein binding of sertindole in humans is high (>99%; Ereshefsky, 1996), indicating a free plasma concentration of maximally 1 to 2 nM after therapeutic administration, based on the plasma concentrations in human volunteers (Wong and Granneman, 1998). The level of accumulation in cardiac tissue is unknown, but a rather large volume of distribution is reported (20-40 l/kg; Ereshefsky, 1996), indicating accumulation of sertindole in various tissues. In our dogs, maximal QT prolongation was already seen 5 to 10 min after the start of infusion of sertindole, suggesting a rapid inhibition of I_Kr once the drug is present in the circulation. The relative I_Kr block induced by sertindole in vivo or in the clinic could be underestimated when plasma concentrations are compared with in vitro concentrations.

Apart from an inhibition of I_Kr, sertindole has also been reported to block the human dopamine D₂ and the 5-hydroxytryptamine_2A receptors (Arnt, 1998). It does also show _1A-blocking properties in rat mesenteric arteries (Ipsen et al., 1997). New studies are required to test possible additional electrophysiological properties of sertindole in the heart under conditions when physiological levels of these agonists are present.

Pharmacological Implications. Previous studies using chronic amiodarone administration have shown that TdP can be absent in CAVB dogs despite prolongation of the QT_c interval by 21% (van Opstal et al., 2001b). The present study indicates again a poor association between the degree of QT_c prolongation and the incidence of TdP (Table 1): at a comparable QT_c after 0.2 and 1.0 mg/kg sertindole, TdP were only induced after the higher dose. This stresses not only the importance of testing several doses when assessing the proarrhythmic potential of a drug but also the relevance of addressing other proarrhythmic factors such as ectopic beats and dispersion.

If our in vitro data from cell cultures and isolated canine myocytes would have determined the future for sertindole, the drug would have likely been abandoned from further development (e.g., based on the recommendations of the Policy Conference of the European Society of Cardiology; Haverkamp et al., 2000). The expansion of our study to in vivo testing showed a discrepancy between the in vitro finding of I_Kr inhibition and prolonged cellular repolarization and the absence of arrhythmias in normal anesthetized dogs. Our data strongly advocate the use of pathological animal models when testing for proarrhythmic properties of cardiovascular and noncardiovascular drugs.

A recent risk-benefit analysis of the preclinical and clinical data on sertindole by the European Committee for Proprietary Medicinal Products led to the reintroduction of sertindole to the European market in 2002.

Limitations. Steady-state plasma concentrations were not obtained in this study, as opposed to previous clinical studies, and the pharmacokinetic difference between acute i.v. and repeated oral dosing should be considered when extrapolating our data to humans. Differences in accumulated tissue concentrations after acute i.v. versus chronic oral administration will likely exist. Furthermore, species differences between dogs and patients should be taken into account.

In conclusion, in vitro studies clearly show sertindole's selective inhibition of I_HERG over other ion currents. Block of native I_Kr forms the ionic basis for action potential prolongation in canine ventricular myocytes and QT prolongation in vivo. At high i.v. doses, sertindole can pose a serious proarrhythmic risk when electrical remodeling of the ventricles is present, as in dogs with CAVB. At clinically relevant doses, sertindole does not cause TdP in anesthetized dogs with normal or remodeled hearts.

	Acknowledgements

 
We thank Lone Durup, Pia Maribo Sørensen and Vivian Lind for technical assistance and skills. Also, the efforts of Anne Reschkewitz, Jens-Jakob Karlsson, and Tina Dige to develop a plasma analysis method and measure sertindole and dofetilide concentrations in dog plasma are greatly appreciated. Dr. Rainer Netzer is acknowledged for providing the HERG and the KCNQ1/KCNE1 cell lines from GENION Forschungsgesellschaft GmbH (Hamburg, Germany). Paul G. A. Volders was supported by The Netherlands Organization for Health Research and Development (ZonMw 906-02-068).

	Footnotes

 
This study was sponsored by H. Lundbeck A/S (Copenhagen, Denmark).

DOI: 10.1124/jpet.103.052753.

ABBREVIATIONS: HERG, human ether-a-go-go-related gene; TAP, transmembrane action potential; CL, cycle length; APD, action potential duration; AV, atrioventricular; CAVB, chronic atrioventricular block; TdP, torsades de pointes; MAP, monophasic action potential; LV, left ventricle; RV, right ventricle.

¹ Current address: Department of Medical Physiology, Division Biomedical Genetics, University Medical Center, Utrecht, The Netherlands.

Address correspondence to: Dr. Paul G. A. Volders, Department of Cardiology, Cardiovascular Research Institute Maastricht, Academic Hospital Maastricht, P.O. Box 5800, NL 6202 AZ, Maastricht, The Netherlands. E-mail: p.volders{at}cardio.azm.nl

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