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Journal of Cardiovascular Pharmacology:
Original Article

The Utility of hERG and Repolarization Assays in Evaluating Delayed Cardiac Repolarization: Influence of Multi-Channel Block

Martin, Ruth L. PhD; McDermott, Jeff S. MS; Salmen, Heinz J. BS; Palmatier, Jason BA; Cox, Bryan F. PhD; Gintant, Gary A. PhD

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From the Department of Integrative Pharmacology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL.

Received for publication March 6, 2003; accepted October 30, 2003.

Reprints: Gary Gintant, PhD, Department of Integrative Pharmacology (R46R, Bldg AP-9), Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6119. E-mail: gary.gintant@abbott.com

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Abstract

Drug-induced delayed cardiac repolarization is a recognized risk factor for proarrhythmia and is associated with block of IKr (the potassium current encoded by the human ether-a- go-go-related gene [hERG]). To evaluate the utility of 2 in vitro assays widely used to assess delayed repolarization, we compared the effects of haloperidol and 9 structurally diverse drugs in a hERG and repolarization (canine Purkinje fiber action potential duration [APD]) assay over wide concentrations. Despite potent hERG current block (IC50 = 0.174 μM), haloperidol elicited a bell-shaped concentration–response relationship for APD prolongation, with lesser prolongation (and reduced plateau height) observed with concentrations eliciting maximal hERG block, consistent with multi-channel block at higher concentrations. Consistent with this hypothesis, APD prolongation with the specific IKr blocker dofetilide was a) reduced by concomitant administration of nifedipine (calcium current block) and b) reversed by lidocaine (late sodium current block). Additional studies demonstrated prominent (>50%) hERG inhibition with most (9/10) drugs despite wide APD changes (158% prolongation − 16% shortening), consistent with multi-channel block. The poor correlation between hERG and repolarization assays suggests that the hERG assay oversimplifies drug effects on the complex repolarization process for drugs demonstrating multi-channel block and that neither assay alone adequately predicts proarrhythmic risk.

Recent warnings, relabeling, and withdrawals of some drugs (including antihistamines, antifungals, and antipsychotic agents) from the market for cardiovascular safety concerns highlight the growing awareness of the potential risk for proarrhythmia by noncardiovascular drugs. At issue is a rare, drug-induced ventricular arrhythmia known as torsade-de-pointes, which may lead to syncope or sudden cardiac death. 1 The low incidence and potentially lethal consequences of this drug-induced arrhythmia pose a challenge to physicians, the pharmaceutical industry, and regulators alike. Dose-dependent prolongation of the QT interval has been used as a surrogate marker for proarrhythmia. 2 However, the extent of QT prolongation may be small (mean values sometimes <10 milliseconds above baseline values near 400 milliseconds) and difficult to detect above rate-dependent and diurnal variations in the QT interval. As a consequence, preclinical assays to assess delayed repolarization have been employed to assess the potential risk for drug-induced proarrhythmia.

At present, two functional in vitro assays are routinely employed when evaluating a drug's potential to delay cardiac repolarization. 1,3–5 One assay (termed a repolarization assay) characterizes drug-induced changes in the action potential duration (APD) of cardiac tissues (such as syncytial Purkinje fibers, papillary muscles, or isolated cardiac myocytes). Another approach evaluates drug-induced block of the rapidly activating delayed rectifier current (typically either hERG current expressed in heterologous systems or native IKr). The utility of this ionic current assay is based on the fact that most drugs that delay repolarization are associated with inhibition of the delayed rectifier current IKr (see ref. 4); this current plays a prominent role in defining terminal cardiac repolarization and is encoded (at least in part) by the hERG gene. 6,7 Using either the repolarization or hERG ionic current assay, a concentration-dependent “signal” is generally considered as evidence of proarrhythmic risk. However, the action potential reflects the effects of multiple ion channels, pumps, and exchangers. Thus, multiple drug effects could mask or modulate the potentially detrimental effects of hERG current inhibition, especially at higher drug concentrations.

Haloperidol is a butyrophenone antipsychotic linked clinically to QT prolongation and torsade-de-pointes. 8 Haloperidol has also been shown to block the cardiac delayed rectifier hERG current in Xenopus oocytes, 9 sodium channels, 10 and is associated with block of calcium current. 11 As part of a routine preclinical evaluation of cardiac electrophysiologic effects of drug candidates, we studied the actions of haloperidol (at and above therapeutic concentrations) in the hERG ionic current and canine Purkinje fiber repolarization (APD) assay. Despite potent concentration-dependent hERG block, haloperidol demonstrated a bell-shaped concentration–response curve for APD prolongation over the same concentration range, consistent with inhibition of multiple (non-hERG) cardiac currents. To explore this effect more fully, we compared haloperidol's effect on repolarization to that of the specific IKr blocking agent dofetilide alone and in combination with either the calcium channel blocking drug nifedipine (to mimic combined hERG and calcium current) or the late sodium current blocking drug lidocaine. A subsequent comparison of effects of 9 additional drugs in the hERG and APD assays revealed a monotonic relationship between action potential prolongation and hERG current inhibition for only 4 of 10 drugs, consistent with multi-channel block affecting cardiac repolarization with increasing drug concentrations. Two of 10 drugs eliciting prominent hERG block (fluoxetine, verapamil) are not linked to either QT prolongation or proarrhythmia clinically. Together, these studies suggest that neither an APD repolarization assay nor a hERG assay alone adequately predict delayed repolarization and proarrhythmic risk (especially for drugs affecting multi-channel block) and that the hERG assay oversimplifies drug effects on cardiac repolarization.

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METHODS

hERG Ionic Current Studies

hERG channels stably expressed in human embryonic kidney cells (HEK-293) 12 were maintained in Minimal Essential Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 200 mg/mL of G418 in a humidified atmosphere (95% air–5% CO2) at 37°C. Media were changed every 48 hours and cells were passaged weekly. Cells were not allowed to become more than 80% confluent. For electrophysiology recordings, cells were briefly trypsinized to release the cells from the plates, pelleted by centrifugation (1000 g), resuspended in culture media and studied within 8 hours.

The bath solution contained (in mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 5, and HEPES 20, pH = 7.4. Patch pipettes were constructed with borosilicate glass capillary tubes (resistance 1.8–3.8 MΩ). The pipette solution contained (in mM): K+ aspartate 125, KCl 20, EGTA 10, MgCl2 1, HEPES 5, and MgATP 5 (pH = 7.3). Experiments were performed at 36.5–37°C. Drugs were either diluted directly in bath solution or were dissolved in dimethyl sulfoxide (DMSO) before dilution in bath solution. Lower drug concentrations were prepared by serial dilution; DMSO concentrations never exceeded 0.1% (by volume). Cells studied were exposed to a single concentration of drug, with current block measured from drug-free control values.

Currents were recorded using either an Axopatch 200A or Axopatch Multiclamp 700A along with pClamp data acquisition software (version 8, Axon Instruments Inc., Union City, CA, U.S.A.). Drug effects were assessed using a voltage clamp protocol that stepped to −25, 0, 25, or 50 mV for 3 seconds, followed by a step to −50 mV for 4 seconds from a holding potential of −80 mV; clamp pulses were applied once every 15 seconds. IC50 values were calculated from tail currents measured at −50 mV following conditioning pulses to 0 mV (chosen to mimic plateau potentials of canine Purkinje fibers). IC50 values were derived after adjusting for current run-down evaluated from time-matched controls (E-4031, fluoxetine), DMSO-vehicle (cisapride, haloperidol, indomethacin, moxifloxacin, terfenadine, telithromycin, verapamil), or lactobionate vehicle (erythromycin). Current run-down in the presence of DMSO (measured following approximately 6–8 minutes exposure, analogous to drug-containing experiments) was approximately 9% and independent of the DMSO concentrations tested [8.29 ± 5.94% (n = 18), 9.07 ± 4.60% (n = 18) and 8.36 ± 3.11% (n = 26) for 0.001%, 0.01% and 0.1% DMSO (by volume, respectively)]. Current run-down for time (vehicle-free) alone (4.02 ± 2.05% [n = 11]) was less but not statistically different from DMSO control groups (ANOVA).

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Action Potential Repolarization Studies

Protocols used to evaluate drug effects on the Purkinje fiber action potential duration have been described previously. 13 Briefly, free-running canine (approximately 1–3 years of age, either gender, Marshall Farms) cardiac Purkinje fibers were excised, placed in a warmed chamber, and superfused (8–10 mL/min) with a Tyrode's solution containing (in mM): NaCl, 131; NaHCO3, 18; NaH2PO4, 1.8; MgCl2, 0.5; dextrose, 5.5; KCl, 4; CaCl2, 2 (aerated with 95% O2/5% CO2 [pH = 7.2 at room temperature]). Fibers were stimulated (2x threshold, biphasic waveform, typically 1–2 milliseconds in duration) using platinum electrodes located in the chamber floor and impaled with 3M KCl-filled microelectrodes (resistance 10–30 MΩ); electrical activity was monitored using high-input impedance electrometers (IE-210, Warner Instruments), recorded digitally (Digidata 1200, Axon Instruments), and analyzed using pClamp software. Studies were initiated after a minimum 30-minute equilibration period with stimulation. Fibers were considered suitable for study if (during stimulation at 2 seconds basic cycle length) the following criteria are satisfied: a) the membrane potential just prior to action potential upstroke was more negative than −80 mV, b) the APD ranged between 300 and 500 milliseconds (spanning approximately 1.2 standard deviations from the mean value of 405 milliseconds), and c) the normal automatic rate did not exceed the 2-second stimulation cycle length.

Electrophysiological effects on repolarization were evaluated during slow stimulation (2 seconds BCL [30 beats per minute]) chosen because repolarization changes are typically exaggerated during slow stimulation (reverse use-dependence 14) and are also more likely to reflect changes during bradycardia (a recognized risk factor for proarrhythmia with drugs that delay repolarization). During experiments, fibers were sequentially exposed to 3 ascending drug concentrations (typically, 1-, 10-, and 100-fold clinically encountered total plasma concentrations). The protocol consisted of pacing in a control (drug-free) solution for 20–25 minutes at a basic cycle length of 2 seconds, then consecutively paced at 800 milliseconds BCL (75 bpm) during the transition to a 400 milliseconds BCL for 2–3 minutes at each stimulation rate. Action potentials recorded at the end of each 25-minute equilibration period were used to define drug effects and generate cumulative concentration-response curves. The present in vitro studies were conducted in the absence of plasma proteins. For those drugs demonstrating high plasma protein binding (for example, haloperidol, fluoxetine, indomethacin, terfenadine, and verapamil), it is likely that concentrations tested in vitro are greater than free concentrations achieved in vivo. Differences in the free drug concentration in vitro versus in vivo will depend upon the characteristics of plasma protein binding, which can be highly nonlinear (and hence unpredictable) at higher drug concentrations. For these drugs, multi-channel blocking effects may occur at multiples of total plasma concentrations greater than expected from the present study.

For experiments with single drug exposure, the action potential duration was measured as the time from the maximum upstroke velocity to a potential 10 mV more positive than full repolarization from the average of 3 consecutive action potentials. For later experiments with 2-drug exposure, the action potential was measured to 90% of repolarization (APD90). Plateau height was measured as the voltage 100 milliseconds following the action potential upstroke (after transition of the action potential notch but before the initiation of terminal repolarization). No drugs elicited significant depolarization of the maximum diastolic potential.

Drugs evaluated previously for effects on Purkinje fiber repolarization included cisapride, erythromycin, fluoxetine, indomethacin, moxifloxacin, and terfenadine 13; newly evaluated compounds included the antipsychotic drug haloperidol, the antibiotic telithromycin, and anti-anginal agent verapamil, and the prototypic antiarrhythmic IKr channel blocking agent E-4031 15 (Table 1). With the exception of indomethacin, fluoxetine, and verapamil, all drugs have been linked clinically to either QT prolongation and/or torsade-de-pointes) at or above therapeutic concentrations on the basis of product labeling 16 or critical reviews. 17,18

Table 1
Table 1
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To experimentally “simulate” the effects of multichannel block on cardiac repolarization, we evaluated the actions of the specific IKr channel blocking drug dofetilide 19 in the absence and presence of either the l-type calcium current blocking agent nifedipine or the late sodium current blocking agent lidocaine. 20,21 A 100-nM concentration of dofetilide was chosen to block IKr based upon IC50 values below 10 nM for block of either native IKr or hERG current 22,23; for nifedipine, a concentration of 5 μM was chosen to reduce l-type calcium current. Prior studies have demonstrated that nifedipine does not affect hERG current expressed in HEK-293 cells, 24 and the structurally related compound nisoldipine does not block native cardiac K+ currents. 25 Lidocaine (5 and 25 μM) was used to block late sodium current; prior studies have shown that these concentrations preferentially affect late sodium current and modify repolarization at concentrations lower than those required to inhibit the fast inward sodium current and action potential upstroke. 20

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Statistical Analysis

Values are reported as mean ± standard error of the mean (s.e.m.). Differences in APD parameters were evaluated using repeated measures ANOVA followed by Dunnett's post-hoc test. IC50 values for hERG current block were obtained from fits to the logistic equation of the form y = [(A1 −A2)/{1 + (X/ Xo)P }] +A2, where P represents power, Xo represents the IC50 value, and A1 and A2 represent the initial (0) and final (100%) values of block using Origin (version 6.0).

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RESULTS

Effects of Haloperidol on hERG Current and Purkinje Fiber Repolarization

Figure 1 characterizes the typical effects of haloperidol on hERG current stably transfected in HEK-293 cells. Figure 1A illustrates the effect of 0.26 μM haloperidol on hERG current during a 3-second depolarizing test pulse to 0 mV followed by a 4-second repolarization step to −50 mV to elicit tail currents. Haloperidol elicited time-dependent block of outward current upon depolarization (consistent with open channel block), reduced tail current amplitude, and slowed tail current kinetics. Block of hERG current by haloperidol was also voltage-dependent. Figure 1B summarizes tail current block with low and intermediate haloperidol concentrations (0.026 and 0.26 μM) following 3-second conditioning test pulses to −25, 0.25, and 50 mV. Greater block is evident with stronger depolarizing test pulses with both haloperidol concentrations. Figure 1C summarizes concentration-dependent block of hERG current evaluated from tail currents following conditioning test pulses to 0 mV. Block of hERG tail current by haloperidol was fit with an IC50 value 0.174 μM.

Figure 1
Figure 1
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To compare hERG current block by haloperidol with its effect on ventricular repolarization, we evaluated haloperidol's effects on canine Purkinje fiber repolarization. Changes in the action potential configuration were evaluated during slow stimulation (2 seconds BCL) to mimic the slow voltage clamp protocol and to maximize repolarization delays; rates slower than 2 seconds BCL were not possible due to normal automaticity eliciting substantial phase 4 depolarization and occasional premature beats. Figure 2 demonstrates the typical effects of haloperidol on the canine Purkinje fiber action potential. Figure 2A illustrates superimposed action potentials recorded from a fiber exposed sequentially to 0.026, 0.26, and 2.66 μM haloperidol (concentrations matching those evaluated in the hERG ionic current assay). Concentration-dependent prolongation (extension of the plateau with minimal alteration in the action potential configuration) was observed at the lower and intermediate haloperidol concentrations (0.026 and 0.26 μM, respectively). These effects are qualitatively comparable to those observed with specific IKr blocking drugs E-4031 and dofetilide (unpublished observations). At the highest concentration (2.66 μM), haloperidol reduced the height at the start of plateau and affected moderate “triangulation” of the action potential. This resulted in action potential shortening with the highest (compared with intermediate) haloperidol concentration. Figure 2B summarizes the effects of haloperidol on the action potential duration and plateau height from 6 fibers. Action potential prolongation was maximal at the intermediate concentration (0.26 μM), and reduced at the highest concentration (Fig. 2B, upper graph). In contrast, the plateau height (measured 100 milliseconds after the upstroke) was significantly depressed only at the highest haloperidol concentration (Fig. 2B, lower graph). Changes in the action potential plateau configuration observed with the highest haloperidol concentration suggest effects on additional ionic currents (other than hERG) during the action potential plateau.

Figure 2
Figure 2
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Multi-channel Block: Combined Effects of IKr and Calcium Current Block

Changes in the action potential configuration with high haloperidol concentration resemble those observed with l-type calcium channel blocking agents verapamil and nifedipine; namely, a reduction in plateau height and triangulation of the action potential (unpublished observations). To investigate the effects of simultaneous IKr and l-type calcium current inhibition on cardiac repolarization, we evaluated changes in Purkinje fiber repolarization during superfusion with the selective IKr blocker dofetilide alone (0.1 μM) followed by superfusion with dofetilide plus nifedipine (5 μM) to block calcium current. Figure 3A illustrates representative changes in the action potential configuration with dofetilide alone, and during superfusion with dofetilide and nifedipine during slow stimulation (2 seconds BCL). Superfusion with dofetilide alone delayed final repolarization of the action potential to affect substantial prolongation without affecting the plateau height. Concomitant superfusion with nifedipine reduced the plateau height, triangularized the action potential, and dramatically reduced prolongation elicited by dofetilide. Figure 3B summarizes the effects of dofetilide alone and in combination with nifedipine on the action potential duration and plateau height. While dofetilide alone prolonged APD by 77% (365 ± 25 [control] versus 646 ± 24 milliseconds [dofetilide]), the addition of nifedipine (in the continued presence of dofetilide) mitigated these effects (22% prolongation versus control, 445 ± 27 milliseconds). Dofetilide alone elicited no change in the plateau height (−3.6 ± 1.4 versus −2.6 ± 1.3 mV [control versus dofetilide, respectively]). However, the addition of nifedipine (in the continued presence of dofetilide) significantly depressed the plateau height (−11.4 ± 2.6 mV compared with control value of −3.6 ± 1.4 mV). The maximum diastolic potential and maximum upstroke velocity were unaffected in these experiments. These results demonstrate that l-type calcium current block can significantly attenuate action potential prolongation elicited by IKr block.

Figure 3
Figure 3
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The local anesthetic lidocaine acts to shorten the action potential duration and lower the plateau height of Purkinje fiber before affecting the action potential upstroke. 20 (unpublished observations). These effects have been attributed to block of late sodium current prevalent in Purkinje fibers 21,26 and ventricular midmyocardial myocytes. 20,27 To evaluate the effect of late sodium current inhibition on delayed repolarization elicited by IKr blockade, we superfused fibers with dofetilide (0.1 μM) followed by dofetilide plus escalating lidocaine concentrations (5 and 25 μM) during slow stimulation (2 seconds BCL). Figure 4A illustrates representative changes in the action potential configuration with dofetilide alone, and during superfusion with dofetilide and the higher lidocaine concentration (25 μM). As expected (and as shown above), dofetilide alone delayed final repolarization without affecting the plateau height. Concomitant superfusion with 25 μM lidocaine reduced the plateau height, triangularized the action potential, and shortened the action potential duration compared with control values. Figure 4B summarizes the effects of dofetilide alone and in combination (with escalating lidocaine concentrations) on the action potential duration and plateau height. The low lidocaine concentration reduced action potential prolongation elicited by dofetilide alone (100% prolongation by dofetilide alone versus 47% prolongation in the presence of 5 mM lidocaine). The higher lidocaine concentration (25 μM) abolished APD prolongation by dofetilide, affecting a modest (9.5%) shortening that was not statistically different from control APD values. Neither dofetilide alone nor dofetilide plus the lower lidocaine concentration affected a change in the plateau height (−5.52 ± 2.38 mV versus −5.88 ± 2.09 mV versus −8.04 ± 2.61 mV [control versus dofetilide versus dofetilide + 5 μM lidocaine, respectively]). However, the higher lidocaine concentration significantly reduced the plateau height (−18.06 ± 1.82 mV compared with control mean value of −5.52 ± 2.38 mV). The maximum upstroke velocity was unaffected by all lidocaine concentrations used in these experiments (745 ± 72 and 691 ± 36 V/s for drug-free control and dofetilide periods versus 727 ± 77, and 731 ± 71 V/s for 5 μM and 25 μM lidocaine, respectively). These results demonstrate that inhibition of late sodium current can reverse action potential prolongation elicited by IKr block in canine Purkinje fibers.

Figure 4
Figure 4
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A Comparison of Assays: Action Potential Prolongation versus hERG Current Block

To further evaluate the role of multi-channel block in modulating delayed repolarization in vitro, we compared the effects of haloperidol and 9 additional drugs in the hERG and APD assay. Drugs selected included those linked to QT prolongation and proarrhythmia clinically as well as those not associated with delayed cardiac repolarization (Table 1). For each drug, matched concentrations were evaluated in both the hERG and APD assays; IC50 values for hERG block with all drugs are shown in Table 1. Figure 5 plots mean values for change in APD (abscissa) versus mean values for block of hERG current (percent change, ordinate) for each of 10 drugs. For most drugs (the exception being indomethacin), prominent (>50%) concentration-dependent hERG block was observed with higher concentrations (typically 10 and 100-fold total plasma values encountered clinically). Of drugs shown to block hERG, only 4 of 9 compounds (E-4031, erythromycin, moxifloxacin, and telithromycin) elicited convincing concentration-dependent APD prolongation along with incremental hERG block. Four other drugs (haloperidol, fluoxetine, verapamil, and terfenadine) elicited substantial concentration-dependent hERG block despite minimal APD prolongation (or, in some cases, APD shortening). The 1 drug demonstrating minimal hERG block (indomethacin) elicited APD shortening (−2.3%) at 50-fold therapeutic plasma concentrations (279.5 μM).

Figure 5
Figure 5
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It is evident that comparable levels of hERG block do not correlate with the extent of action potential prolongation when comparing different drugs. For example, action potential prolongation with cisapride was greater at the intermediate (1 μM) versus higher concentration (57.6 ± 7.3 versus 32.4 ± 4.1% for 1 and 10 μM concentrations, respectively) despite prominent (>96% block) hERG block (IC50 value = 0.018 μM;Table 1). Of all 10 compounds, only the prototypic antiarrhythmic agent E-4031 elicited prominent APD prolongation (>25%) at therapeutic concentrations. While most (9/10) drugs demonstrated concentration-dependent hERG block at or above therapeutic concentrations, only 5 of 9 hERG blocking drugs (cisapride, E-4031, erythromycin, moxifloxacin, telithromycin) affected appreciable (>25%) prolongation of the action potential duration for matching concentrations.

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DISCUSSION

Recent experiences suggest that delayed cardiac repolarization by non-cardiovascular drugs predisposes to proarrhythmia, including the potentially life-threatening cardiac arrhythmia torsade-de-pointes. 1,4 As these drug-induced events are rare, emphasis is placed on in vitro preclinical assays to detect delayed repolarization (a surrogate marker of proarrhythmia) testing therapeutic and supratherapeutic drug concentrations (the latter a risk factor for acquired long QT-syndrome). In general, most drugs that delay repolarization reduce the delayed rectifier current, IKr, at or above therapeutic plasma concentrations. 4 As the pore of the delayed rectifier channel in humans is encoded by hERG, 6,7 the hERG ionic current assay (along with repolarization assays) has assumed prominence in the in vitro evaluation of proarrhythmic liability.

The present study demonstrates concentration-dependent block of hERG by haloperidol, characterized with an IC50 value of 0.174 μM (Fig. 1). In contrast, APD prolongation with haloperidol was greatest at 0.26 μM, with lesser prolongation observed at a 10-fold greater concentration (Fig. 2). Haloperidol's effect to delay repolarization at the 2 lower concentrations was mirrored by the specific IKr blocking agent dofetilide, while haloperidol's effects on repolarization at the high concentration (mitigated prolongation and plateau height reduction) was mimicked by l-type calcium channel blockade (by nifedipine) in the presence of continued IKr block by dofetilide (Fig. 3). The mimicking of haloperidol's effects by coadministration of dofetilide and nifedipine is consistent with multi-channel block by haloperidol mitigating the effects of hERG block at supratherapeutic concentrations. Somewhat unexpectedly, we also observed that lidocaine reduced and then abolished action potential prolongation elicited by dofetilide in a concentration-dependent manner (Fig. 4). Lidocaine's reversal of dofetilide's effects was coincident with a reduction in the height of the action potential plateau and mirrored effects observed with the combined administration of nifedipine and dofetilide. The effect of lidocaine occurred without changes in the maximum rate of rise of the action potential upstroke, and is consistent with inhibition of late sodium current that supports the plateau in Purkinje fibers and some ventricular cells. 20,21,26,27 Our results with lidocaine in canine Purkinje fibers agree with an earlier rabbit Purkinje fiber study by Abrahamsson and coworkers that showed APD prolongation elicited by the iKr blocking agent almokalant was reversed by lidocaine. 28 However, our results with nifedipine are in disagreement with analogous studies by Abrahamsson in which nisoldipine did not reverse APD prolongation elicited by almokalant. These later results may be due to a lower concentration of calcium channel blocker employed by Abrahamsson or to species differences.

The modulation of delayed repolarization elicited by IKr block by concomitant block of inward plateau current(s) (for example, l-type calcium current or late sodium current) is likely to be complex and involve at least 4 factors. First, IKr activation is voltage dependent and spans the range of −40 to 0 mV for full activation in most species. 6,7,29 Thus, a drug that reduces (makes more negative) the plateau height by inhibiting an inward plateau current will reduce IKr activation during the action potential, thereby minimizing its contribution to repolarization and any further influence of IKr block. More negative plateau potentials will also reduce the extent of IKr blockade for drugs demonstrating voltage-dependent block (such as haloperidol). Similarly, the contribution of IKs is likely to be reduced with more negative plateau potentials (although activation of IKs is slower than IKr and is influenced more by adrenergic tone). Finally, reduction of ICa in the setting of IKr block may also reduce the extent of calcium current reactivation later during the action potential plateau, thereby antagonizing the initiation of early afterdepolarizations implicated in the genesis of torsade-de-pointes. 28,30 Computer simulations may be useful in elucidating these complex interactions.

It has been demonstrated that pretreatment with low concentrations of either lidocaine or nisoldipine directly inhibit EADs while minimally affecting APD. 28 Indeed, it has been suggested that inhibition of inward plateau current(s) may be protective against proarrhythmia with the novel antiarrhythmic drug BRL-32872 31, the antiarrhythmic drug verapamil, 24 and the antidepressant drug fluoxetine. 32,33 However, multi-channel block does not always guard against proarrhythmia, as excessive haloperidol concentrations have been linked to proarrhythmia. 34,35 Furthermore, inhibiting l-type calcium current may reduce contractility, leading to other changes. 32 Lowering the plateau height may also affect triangulation of the ventricular action potential that, according to Hondeghem, may be proarrhythmic. 36 Lesser effects of IKr blockade on repolarization resulting from multi-channel block with non-hERG channels will depend upon the characteristics of drug block of each channel, the properties and densities of individual currents contributing to the tissue-specific action potential configurations, and patterns of electrical activity. Finally, it must be recognized that delayed repolarization is a surrogate marker for proarrhythmia and torsade-de-pointes that can only be observed directly in intact heart preparations.

To our knowledge, this is the first study to systemically compare matched concentrations of a diverse set of drugs from different therapeutic areas in both a hERG and APD repolarization assay. Our results demonstrate prominent hERG current inhibition for 9 of 10 drugs at excessive concentrations (Fig. 5, Table 1). One group of 4 drugs (E-4031, erythromycin, moxifloxacin, and telithromycin) elicited concentration-dependent prolongation of the action potential duration paralleling incremental hERG block. A second group of four drugs (fluoxetine, haloperidol, terfenadine, and verapamil) minimally prolonged (or in some cases, shortened) the canine Purkinje fiber APD despite affecting substantial (>70%) hERG block at supratherapeutic concentrations. Cisapride provides a more complicated example, eliciting 94% block of hERG current at a concentration of 0.1 μM, maximal APD prolongation (57.6 ± 7.3%) at 1 μM, and lesser prolongation (32.4 ± 4.1%) at 10 μM concentration. These findings suggest that drugs that inhibit hERG current may be further categorized into two groups, with one group predominantly inhibiting only hERG current (affecting prominent APD prolongation), and a second group inhibiting multiple cardiac channels and thereby modulating hERG current block (especially at high concentrations). This conclusion is not surprising since one would expect higher drug concentrations to foster block of multiple channels as well as to potentially indirectly modulate block of other channels (for example, reducing plateau height, thereby affecting other currents later during the action potential).

We compared hERG block with changes in canine Purkinje fiber repolarization, as it is not possible to routinely evaluate hERG block and APD changes in native human ventricular preparations. Indeed, the generally low hERG current density in native cardiac preparations and presence of multiple overlapping currents provide formidable obstacles for evaluating hERG current inhibition in the same preparations as those used to record action potentials. We would expect that block of stably transfected hERG in HEK-293 cells would be comparable to block of canine IKr in Purkinje myocytes because a) the cERG polypeptide is 97% homologous to its human counterpart (with 100% identity in the N-terminal PAS domain, transmembrane segments, and cyclic nucleotide binding domain 37), and b) cERG current shows similar sensitivity to block by terfenadine, astemizole, and the methanesulfonanilide compound MK-499. 38 It is unlikely that differences in experimental conditions account for the lack of correlation between hERG block and APD prolongation in canine Purkinje fibers since identical concentrations of drugs (from identical lots) were evaluated in both assays under comparable experimental conditions (temperature, slow activity, roughly comparable voltage excursions). Whether the presence of channel subunits significantly affects drug block of hERG current remains unresolved. 39,40 While it is well known that differences in electrical activity exist between Purkinje fibers and various regions of ventricular myocardium, it is uncertain how these differences would affect the correlation between hERG and APD assays with different tissue types. It has been shown that high lidocaine concentrations reverse APD prolongation elicited by almokalant in rabbit Purkinje fibers but not rabbit ventricular papillary muscle. 28 What is clearly demonstrated in this study is that only a subset of drugs that elicit prominent concentration-dependent hERG block elicit prominent concentration-dependent prolongation of the action potential duration in vitro. These findings highlight the utility of repolarization studies with native tissues as simultaneous, integrated assays of drug effects on multiple cardiac ion channels.

In summary, the present studies demonstrate the difficulty of assessing potential proarrhythmic risk on the basis of concentration-dependent drug effects in 2 common in vitro preclinical assays. Only 1 of 10 drugs tested (indomethacin) does not block hERG at supratherapeutic concentrations. The extent of hERG block is poorly correlated with action potential prolongation in vitro, consistent with additional drug effects on non-hERG channels (multi-channel block), especially at high drug concentrations. While hERG block detects many drugs linked to delayed repolarization and torsade-de-pointes, it is not fully predictive of proarrhythmic risk; for example, both fluoxetine and verapamil elicit prominent hERG block but are not generally associated with either QT interval prolongation or torsade-de-pointes. Together, these observations demonstrate that the hERG assay is overly simplistic in predicting delayed repolarization and torsade-de-pointes for some drugs (especially at supratherapeutic concentrations), and that a repolarization assay provides additional information to interpret hERG assay results during the initial steps in the preclinical evaluation of potential proarrhythmic risk.

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ACKNOWLEDGMENTS

The authors thank Katina Daniell, Sandra Leitza, and Gilbert Diaz for their unfailing provisions of HEK-293 cells; Brian Ebert and Dr. Letty Medina for veterinarian assistance; and Dr. James Sullivan for continued support.

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REFERENCES

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Keywords:

hERG; torsade-de-pointes; action potential duration; Purkinje fibers; cardiac repolarization; haloperidol; arrhythmias

© 2004 Lippincott Williams & Wilkins, Inc.

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