Effect of Clebopride,Antidopaminergic Gastrointestinal Prokinetics,on Cardiac Repolarization

Abstract

The inhibition of the potassium current I _Kr and QT prolongation has been known to be associated with drug-induced torsades de pointes arrhythmias (TdP) and sudden cardiac death. In this study, the authors investigated the cardiac electrophysiological effects of clebopride, a class of antidopaminergic gastrointestinal prokinetic, that has been reported to prolong the QT interval by using the conventional microelectrode recording techniques in isolated rabbit Purkinje fiber and whole-cell patch clamp techniques in human ether-à-go-go-related gene (hERG)-stably transfected Chinese hamster ovarian (CHO) cells. Clebopride at 10 μM significantly decreased the V _max of phase 0 depolarization (p < .05) and significantly prolonged the action potential duration at 90% repolarization (APD₉₀) (p < .01), whereas the action potential duration at 50% repolarization (APD₅₀) was not prolonged. For hERG potassium channel currents, the IC₅₀ value was 0.62 ± 0.30 μM. Clebopride was found to have no effect on sodium channel currents. When these results were compared with C _max (1.02 nM) of clinical dosage (1 mg, [p.o.]), it can be suggested that clebopride is safe at the clinical dosage of 1 mg from the electrophysiological aspect. These findings indicate that clebopride, an antidopaminergic gastrointestinal prokinetic drug, may provide a sufficient “safety factor” in terms of the electrophysiological threshold concentration. But, in a supratherapeutic concentration that might possibly be encountered during overdose or impaired metabolism, clebopride may have torsadogenic potency.

Both antidopaminergic gastrointestinal prokinetics and alternative 5-HT4 receptor agonists have been exploited worldwidely for the management of motor disorders of the upper gastrointestinal track, such as functional dyspepsia and gastric stasis of various origins. However, there has been a renewed interest in antidopaminergic prokinetics because of the worldwide withdrawal of cisapride, one of the most widely used alternative 5-HT4 receptor agonists, due to the well-known cardiac safety issues (Richter 2000). Since the withdrawal of cisapride, the treatment of upper gut motility disorders has rested mainly on antidopaminergic prokinetics (Tonini et al. 1999). However, various toxic effects such as hyperprolactinemia (Perez-Lopez et al. 1980; Rocco et al. 1990) and extrapyramidal dystonic reactions (Sol, Pelet, and Guignard 1980; Lopez et al. 1987) have also been widely reported with the antidopaminergic gastrointestinal prokinetics. No report has been issued with respect to the effects of antidopaminergic gastrointestinal prokinetic drugs on cardiac electrical activity. Clebopride, a substituted benzamide compound, is one of the most well-known antidopaminergic gastrointestinal prokinetic drugs in the market. The present study was undertaken to examine the effects of clebopride, an anti-dopaminergic gastrointestinal prokinetic drug, on the cardiac action potential duration recorded in rabbit purkinje fibers, on the ether-à-go-go-related gene (hERG) potassium channel, and on the sodium channel.

METHODS

Drugs

Clebopride used in this study were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A). Molecular formula of clebopride is C₂₀H₂₄CIN₃O₂·C₄H₄O₄ (CAS number is 84370-95-6) (Martindale 1996).

Recording of Action Potentials

This study was conducted in facilities approved by the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International. All procedures were approved by our Institutional Animal Care and Use Committee (IACUC). Female New Zealand white rabbits (1.8 to 2 kg) were anesthetized with pentobarbital sodium (45 mg/kg intravenously). Their hearts were rapidly removed and placed in Normal Tyrode solution (mM): 143 NaCl; 5.4 KCl; 5.0 HEPES; 0.33 NaH₂PO₄; 0.5 MgCl₂; 16.6 glucose; 1.8 CaCl₂; pH 7.4 aerated with O₂ gas. Purkinje fibers were excised from the left ventricle and stored in a chamber superfused with Normal Tyrode solution at 37.0°C ± 0.5°C. Action potentials were recorded using the conventional intracellular recording technique. The perfusing Tyrode solution was oxygenated with a gas mixture of 3% CO₂ and 97% O₂ and kept at 37°C with a temperature-controlled circulator. The perfusion speed was set at 5 ml/min. The tissue preparation was driven by electrical pulses (duration = 2 ms, frequencies of 1 Hz). The signals of action potentials were amplified with Geneclamp-200B (Molecular Devices, Sunnyvale, CA, U.S.A) and recorded by a recorder (Notocord Systems, Croissy-sur-Seine, France) for off-line analysis. The resting membrane potential (RMP), V _max of the phase 0 maximum depolarization, the total amplitude (TA), and the action potential duration at 50% (APD₅₀) and 90% (APD₉₀) repolarization were measured when they were stable. To test the drug effect in relation to concentrations, test articles were subsequently applied, and each concentration was allowed to perfuse for 30 min. All chemicals were obtained from Sigma-Aldrich, with the exception of KOH (Junsei Chemical, Tokyo, Japan).

Recording of hERG Currents

hERG-stably transfected Chinese hamster ovary (CHO) cells were used for the investigation. hERG CHO cell line was purchased from bSys GmbH (Witterswil, Switzerland). Ionic currents were recorded in whole-cell configuration using an Ax-opatch 200B amplifier (Molecular Devices). After the data were digitized and analyzed using a digidata (Molecular Devices) at a sampling rate of 5 kHz, they were low-pass filtered at 1 kHz. The patch pipettes were made from borosilicate glass capillaries (Harvard Apparatus, Edenbridge, Kent, UK) using a pipette puller (PP-830; Narishige, Tokyo, Japan). Their resistances were 3 to 3.5 MΩ when filled with hERG pipette solutions. The simulation frequency was 0.1 Hz. hERG potassium currents (I _hERG) was induced by single 2-s voltage pulse to +20 mV from the holding potential of −80 mV once every 10 s. The activation curve of I _hERG was obtained by plotting the peak tail current amplitude in response to 2-s voltage pulses to potentials between −80 and +60 mV from the holding potential of −80 mV in 10-mV increments and repolarization to −40 mV for 3 s, and the inactivation curve of I _hERG was obtained by plotting the peak tail current amplitude in response to voltage pulses to potentials between −110 and +20 mV from +20 mV in 10-mV increments for 20 ms. The composition of Tyrode solution for the recording of action potential and hERG currents was (in mM) NaCl, 143; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 0.5; HEPES, 5; NaH₂PO₄, 0.33; glucose, 16.6 (adjusted to pH 7.4 with NaOH). The hERG internal solution (pipette) contained (in mM) KCl, 130; EGTA, 5; HEPES, 10; MgCl₂, 1; Mg-ATP, 5 (adjusted to pH 7.25 with KOH). All chemicals were obtained from Sigma-Aldrich, with the exception of KOH (Junsei Chemical).

Recording of Na Currents

Transiently transfected human embryonic kidney (HEK) cells expressing the Na channel were used for the investigation. SCN5A cDNA in pCMV6-XL4 expression vector (purchased from Origene Technologies, Rockville, MD, U.S.A) was co-transfected with the surface marker protein green fluorescence protein (GFP) to allow assessment of the transfection efficiency and identification. The plasmid was sequenced and subsequently introduced into cells using lipofectAmin2000 (Gibco BRL, Carlsbad, CA, USA) as a transfection reagent according to the manufacturer’s instructions. Ionic currents were recorded in whole-cell configuration using an Axopatch 200B amplifier (Molecular Devices). After the data were digitized and analyzed using a digidata (Molecular Devices) at a sampling rate of 5 kHz, they were low-pass filtered at 1 kHz. The patch pipettes were made from borosilicate glass capillaries (Harvard Apparatus) using a pipette puller (PP-830; Narishige). Their resistances were 3 to 3.5 MΩ when filled with pipette solutions. The simulation frequency was 0.1 Hz. Sodium channel currents (I _Na) was induced by single 20-ms voltage pulse to −40 mV from the holding potential of −100 mV once every 10 s. The current-voltage relationship of I _Na was obtained by plotting the peak current amplitude in response to voltage pulses to potentials between −110 and +30 mV from the holding potential in 10-mV increments at 0.1 Hz. The composition of Tyrode solution was (in mM) NaCl, 143; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 0.5; HEPES, 5; NaH₂PO₄, 0.33; glucose, 16.6 (adjusted to pH 7.4 with NaOH). The internal solution (pipette) for measurement of sodium channel currents contained (in mM) CsF, 105; NaCl, 35; EGTA, 10; HEPES, 10 (adjusted to pH 7.25 with NaOH). All chemicals were obtained from Sigma-Aldrich.

Statistics

Statistical analysis was performed by comparing the differences between the drug treated groups and the control group using GraphPad InStat (version 3.05; GraphPad Software, CA, USA). Dunnett’s multiple comparison test was conducted and data was considered to be significant when p < .05 or p < .01.

Current amplitudes were measured before and after application of the respective compound. Relative remaining currents were calculated according to the following equation: Initial current amplitude/current amplitude in the presence of compound = relative remaining current.

Effects were calculated from the results of four experiments per concentration of the compound. Concentration response relations were calculated by a nonlinear least squares fit of equation (Hill equation; f = x ^b/(c^b + x ^b); b = Hill coefficient, c = IC₅₀) to the individual data points. Hill coefficient (H) and the half-maximum inhibiting concentration (IC₅₀) were calculated. Normalized curves of activation and inactivation were fitted using the Boltzmann distribution equation:

$$Y=1/1\{1+\text{exp}[(V_{1/2}-V\hspace{0.17em}m)/k]\},$$[1]

where Y is the normalized conductance (G/G _max) or current (I/Io), V _1/2 is the membrane potential at half-maximal conductance or current, and k is the slope factor. Data analysis was performed using pClamp 9 and SigmaPlot 2000. Results are presented as means ± SEM and error bars represent standard errors.

RESULTS

Recording of Action Potentials

Figure 1a shows that clebopride at the concentrations of 0.1, 0.3, and 1 μM had no significant effect on the action potential duration (APD) in rabbit Purkinje fiber when stimulated by frequencies of 1 Hz. Furthermore, it had no significant effects on the resting membrane potential (RMP), total amplitude, and V _max of phase 0 depolarization at concentrations up to 1 μM. However, clebopride at 10 μM significantly (p < .01) prolonged the action potential at 90% repolarization (APD₉₀) and (p < .05) decreased the V _max of phase 0 depolarization (Table 1) but had no effect on 50% repolarization (APD₅₀). Figure 1b shows representative illustrations of the effect of clebopride on rabbit purkinje fiber. Drug levels taken up by tissue were not measured.

Recording of hERG Potassium Channel Currents

Clebopride blocked hERG potassium channels expressed in CHO cells in a concentration-dependent manner as displayed in Figure 2. In these experiments, cells were depolarized for 2 s to +20 mV from the holding potential of −80 mV followed by a 3-s repolarization back to −40 mV. Tail currents were recorded at 100 ms after repolarization to −40 mV. When the effects of clebopride at various concentrations were evaluated, clebopride at concentrations of 0.001, 0.1, 1, and 10 μM was found to inhibit the amplitude of hERG currents by 3.14% ± 4.4%, 25.3% ± 1.7%, 52.6% ± 10.9%, and 96.5% ± 1.4%, respectively (n = 3–4). Figure 2a shows a representative tracing of the blocking effect of clebopride on hERG potassium channel currents. Figure 2b was the time course of the blocking effect of Figure 2a . Clebopride reduced the peak tail current amplitude measured at −40 mV in a concentration-dependent manner (Figure 2b ). The dose-response curve was shown in Figure 2c . The half-maximum inhibition concentration (IC₅₀) was 0.61 ± 0.30 μ M.

Figure 3a displays peak tail currents as a function of the preceding test pulse potential, resulting in activation curves. Cells were clamped at a holding potential of −80 mV. Depolarizing pulses were applied for 2 s to voltages between −80 and +60 mV in 10-mV increments, and tail currents were recorded during a constant repolarizing step to −40 mV for 3 s. The peak tail current increased with voltage steps from −80 to 60 mV and then plateaued with test pulse potentials positive to −20 mV. hERG peak tail currents were reduced by 1 μM clebopride by 69% ± 5% (n = 4), respectively. The changing mean values for the half-maximal activation potential (V _{1 / 2)} and slope factor are as follows: V _{1 / 2} from −25.62 ± 0.87 to −39.23 ± 2.02 mV; slope factor from 18.49 ± 0.74 to 24.77 ± 1.46). Both mean values for the half-maximal activation potential and the mean slope factor of the activation curve displayed statistically significant differences.

Figure 3b displays peak tail currents as a function of the preceding test pulse potential, resulting in inactivation curves. The peak tail current increase with voltage steps from −110 to −20 mV and then plateaued with test pulse potentials positive. The changing mean values for the half-maximal inactivation potential (V _{1 / 2}) and slope factor are as follows: V _{1 / 2} from −33.53 ± 0.36 to −41.52 ± 1.14 mV; slope factor from 6.41 ± 0.31 to 3.51 ± 1.21). Both mean values for the half-maximal activation potential and the mean slope factor of the activation curve displayed statistically significant differences.

Recording of Sodium Channel Currents

Clebopride had no effect on sodium channels expressed in CHO cells as displayed in Figure 4, whereas clebopride increased sodium currents to some extent. In these experiments, cells were depolarized for 20 ms to −40 mV from holding potential of −100 mV. When the effects of clebopride at various concentrations were evaluated, clebopride at concentration of 0.1, 1, and 10 μM was found to increase the amplitude of sodium currents by 1.4% ± 5.7%, 14.4% ± 23.6%, and 4.2% ± 16.9%, respectively (n = 4). Figure 4a shows a representative tracing of the effect of clebopride on SCN5A sodium channel currents. Figure 4b was the time course of the blocking effect of Figure 4a . The dose-response curve was shown in Figure 4c .

DISCUSSION

In the present study, we found that clebopride prolongs the APD₉₀ but not the APD₅₀ in the rabbit Purkinje fiber, although the blockade of I _hERG is induced by clebopride. Clebopride decreased I _hERG by shifting it to the negative direction in both activation and inactivation curves and by making the slope of activation slower and that of inactivation faster. In the present study, we were not able to identify the reason why V _max of 0 phase was decreased by clebopride at 10 μM. Clebopride did not have any effects on amplitude, activation, inactivation and time constant of the sodium channel current (data not shown). Further studies in the area of other ion channels associated with the cardiac action potential such as the transient outward potassium channel and calcium channel and integrating hERG assay data will be necessary to evaluate the effect of clebopride on the action potential in particular.

Clebopride has a high affinity value at the D2 receptor (K _i approximately 2 nM) (Tonini et al. 2004). Robinson et al. (1991) reported a human plasma level of clebopride of 0.5 ng/ml when measured up to 24 h following oral administration of 1 mg, the clinical dose of clebopride, using the capillary gas chromatography–negative-ion chemical ionization mass spectrometry. This value is equivalent to the molecular concentration of 1.02 nM (MW 490.0). The IC₅₀ value of clebopride on I _hERG was 0.61 μM, and its effect on the APD in the rabbit Purkinje fiber was 10 μM. These numbers were 610 and 1000 fold greater than the expected maximum concentration (C _max) of clebopride in human plasma after oral administration of 1 mg, the clinical dose of clebopride. (Redfern et al. 2003) reported that a 30-fold margin between C _max and hERG IC₅₀ may suffice for drugs currently undergoing clinical evaluation.

Based on the results of the study, it can be suggested that clebopride is safe at the clinical dosage of 1 mg from the electro-physiological aspect. These findings indicate that clebopride, an antidopaminergic gastrointestinal prokinetic drug, may provide a sufficient “safety factor” in terms of the electrophysiological threshold concentration. But, in a supratherapeutic concentration that might possibly be encountered during overdose or impaired metabolism, clebopride may have torsadogenic potency.