Introduction

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Recently, there has been considerable attention focused on drugs that prolong the QT interval of the electrocardiogram. Examples can be found in many therapeutic classes, the danger being that excessive QT prolongation, which reflects delayed myocardial repolarization, may lead to potentially lethal ventricular arrhythmias (Stratmann et al., 1987; Zipes, 1987; Zehender et al., 1991; Benedict, 1993). The likelihood of developing deleterious adverse cardiac effects when exposed to QT-prolonging drugs can be enhanced under certain conditions such as electrolyte abnormalities, metabolic disturbances, and preexisting medical conditions such as heart disease and congenital long QT syndrome (Jackman et al., 1988).

The H₁-receptor antagonist class of drugs has figured prominently in regulatory agency concerns regarding QT-prolonging drugs. Several cases of QT prolongation and ventricular arrhythmias, in particular torsade de pointes, have been reported with the antihistamines astemizole and terfenadine (Craft, 1985; Bishop and Gaudry, 1989; Davies et al., 1989; Monahan et al., 1990; Lindquist and Edwards, 1997). These reports prompted warning of potentially serious adverse cardiac events with these agents. The most likely mechanism for these drug-related arrhythmias is blockage of one or more ion channels involved in cardiac repolarization. Several studies have demonstrated blockade of multiple cardiac K⁺ channels by terfenadine, including I_to, I_sus, I_K1, and I_Kr or human ether-a-go-go-related gene (HERG) (Table 1). In contrast to terfenadine, studies with nonhuman systems and/or under nonphysiological conditions have shown the antihistamine loratadine to be virtually free of cardiac ion channel-blocking effects (Table 2). However, recent case reports have appeared describing both atrial and ventricular arrhythmias associated with loratadine usage (Good et al., 1994; Haria et al., 1994; Lindquist and Edwards, 1997; de Abajo et al., 1999). Furthermore, a search of the World Health Organization database reveals an adverse cardiac event profile for loratadine, which includes ventricular arrhythmias, similar to that of terfenadine (Lindquist and Edwards, 1997), although examples of torsade de pointes associated with loratadine use have not been reported. This disparity in the lack of any significant cardiac ion channel-blocking effect and the existence of numerous adverse cardiac event reports for loratadine prompted the comparison of loratadine and terfenadine under physiological conditions with human cardiac ion channels. The effects of these drugs on ion channels were examined in either isolated human cardiac myocytes or a human cell line expressing the human K⁺ channel, HERG. This is the first study to examine and compare the ion channel-blocking profile and the rate dependence of loratadine and terfenadine under physiological conditions and with human cardiac ion channels.

	Materials and Methods

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Human tissue was obtained, in accordance with Tulane University School of Medicine institutional guidelines. Myocytes were isolated from specimens of human right atrial appendage obtained during surgery from hearts of five patients (ages 43-69 years) undergoing cardiopulmonary bypass. All atrial specimens were described as grossly normal at the time of excision and had no evidence of dilation on ECG, and all patients had normal P waves on ECG. Despite these qualifications, the atrial specimens may not represent "normal" atrial tissue. Some patients had received cardioactive drugs including Ca²⁺ channel blockers and digitalis. The cell-isolation procedure has been described in detail (Crumb et al., 1995a).

Transfection and Cell Culture. HEK 293 cells stably expressing HERG mRNA were maintained in minimum essential medium with Earle's salts supplemented with nonessential amino acids, sodium pyruvate, penicillin, streptomycin, and fetal bovine serum.

Solutions. Isolated human atrial myocytes or HEK cells were superfused with an "external" solution that consisted of 137 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 11 mM glucose, 10 mM HEPES; adjusted to a pH of 7.4 with NaOH. Glass pipettes were filled with an "internal" solution that consisted of 120 mM K-aspartate, 20 mM KCl, 4 mM Na-ATP, 5 mM EGTA, 5 mM HEPES; adjusted to a pH of 7.2 with KOH. Loratadine and terfenadine were kindly provided by Almirall Prodesfarma (Barcelona, Spain) and dissolved in 100% dimethyl sulfoxide (DMSO) to make concentrated stock solutions (1 mM). Loratadine was added to the bath solution from this concentrated stock (final DMSO concentration less than 0.1%) or from another more diluted stock solution (100 µM) made in distilled deionized water.

Conditions. Experiments were performed in the presence of 200 µM Cd²⁺ to block the L-type Ca²⁺ current and at a holding potential of 75 mV^. All experiments were performed at 36 ± 1°C. The transient outward current was measured by subtracting the amplitude of the current measured at the end of a depolarizing voltage pulse (sustained current) from peak current amplitude. The sustained current was measured at the end of a depolarizing pulse to +60 mV. Outward HERG tail current was measured to assess drug effects.

	Results

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Figure 1 illustrates the ion channel-blocking effects of loratadine and terfenadine at various pacing rates on I_to, I_sus, and I_K1 recorded from isolated human atrial myocytes. These experiments were performed under physiological conditions of temperature (36 ± 1°C), holding potential (75 mV), and external [K⁺] (4 mM). As indicated, even at pacing rates as high as 3 Hz, neither 1 µM terfenadine nor 1 µM loratadine had any effect on these human K⁺ currents. In contrast, both agents markedly inhibited HERG current amplitude recorded from stably expressing HEK cells (Fig. 2). HERG current was measured as an outward tail current elicited on repolarization to 40 mV from an immediately preceding depolarizing voltage pulse to +10 mV. At a pacing rate of 0.1 Hz, there was a significant reduction in outward HERG tail current amplitude compared with control on addition of either 100 nM loratadine (48.9 ± 3.8%, n = 9) or 100 nM terfenadine (41.1 ± 5.1%, n = 6) (p < .01). Fits of mean dose-response curves (n = 5-10) with the equation
where B(%) is the percent current blockage at drug concentration D, IC₅₀ is the concentration of drug that causes 50% block, and n is the Hill coefficient, yielded IC₅₀ values of 173 and 204 nM for loratadine and terfenadine, respectively (Fig. 2C). On pacing at higher frequencies (1-3 Hz), there was a slightly greater, but nonsignificant, reduction in HERG current amplitude compared with pacing at 0.1 Hz, indicating little use-dependent blockage of this current (Fig. 2D).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effects of 1 µM loratadine or terfenadine on Ito and Isus (A and B) and IK1 (C and D) recorded from isolated human atrial myocytes. Ito and Isus were elicited in response to 200-ms depolarizing pulses to +60 mV from a holding potential of 75 mV. IK1 was elicited by a voltage pulse to 100 mV (320 ms) from a holding potential of 75 mV. Overlapping currents are before and after addition of 1 µM indicated drug (pacing rate was 0.1 Hz). Plots show the rate dependence of loratadine and terfenadine blockade of Ito, Isus, and IK1. Symbols are means ± S.E. (n = 6).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Loratadine and terfenadine blockage of HERG stably expressed in HEK cells. HERG current was elicited by a 400-ms pulse to +10 mV followed by a pulse to 40 mV (400 ms) from a holding potential of 75 mV. HERG current was measured on return to 40 mV. HERG current amplitude was measured before and after addition of 100 nM terfenadine (A) or loratadine (B). Pacing rate was 0.1 Hz. C, mean dose-response curve for loratadine and terfenadine blockage of HERG. Fit is to a Hill equation. Symbols are means ± S.E. (n = 6-9). D, rate dependence of 100 nM loratadine or terfenadine blockage of HERG. Symbols are means ± S.E. (n = 6).

The results reported in this study are very different from those of Taglialatela et al. (1998), who recently reported that loratadine at a concentration of 3 µM produced only an approximately 30% reduction in HERG current amplitude in a human neuroblastoma cell line expressing HERG. The conditions used in this study (37°C, 400-ms depolarizing pulses, 4 mM [K⁺]_o, outward tail currents) and those in the Taglialatela et al. study (22°C, 10-s depolarizing pulses, 100 mM [K⁺]_o, inward tail currents) were quite different. When these conditions were mimicked in this study, results similar to those in the Taglialatela et al. study were observed, with 3 µM loratadine producing a 30.2 ± 2.1% (n = 4) reduction in HERG current amplitude (Fig. 3). These results were dramatically different from those obtained under more physiological conditions in this study, where 100 nM loratadine produced a greater than 45% reduction in HERG current amplitude, and 3 µM loratadine is predicted to produce an approximately 80% reduction (Figs. 2C and 3).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Blockage of HERG by 3 µM loratadine under different experimental conditions. A, inward HERG tail current before and after addition of 3 µM loratadine. Currents were elicited by a 10-s hyperpolarizing pulse to 140 mV from a holding potential of 40 mV (temperature, 22°C). B, comparison of the HERG-blocking actions of 3 µM loratadine under the indicated conditions. Data plotted for 22°C are means ± S.E. Data plotted for 37°C represent the predicted percent blockage obtained from the dose-response curve in Fig. 2C.

	Discussion

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The results of this study indicate that, of the four human cardiac K⁺ currents tested under physiological conditions, only HERG was blocked by either terfenadine or loratadine. The lack of any observed block of I_to, I_sus, and I_K1 by 1 µM terfenadine (Fig. 1) is in contrast to previous reports that indicate a 10 to 40% reduction in these currents with the same concentration of terfenadine (Table 1). Although it is not clear, these differences may be the result of species differences in cardiac ion channels or in experimental conditions (Table 1). It is interesting that terfenadine had no blocking effect on I_sus in human atrial cells because it has been shown to block Kv1.5, a channel cloned from human heart and expressed in mammalian cell lines (Rampe et al., 1993). The lack of terfenadine blockade of I_sus may reflect the fact that Kv1.5 is not the only current in human atria and may not be the major current underlying I_sus (Crumb et al., 1995b). In human ventricle, a Kv1.5-like current cannot be recorded (Mays et al., 1995). These observations make it unlikely that I_sus, I_to, or I_K1 are targets for terfenadine and loratadine at concentrations associated with arrhythmias. The lack of I_to blockade by terfenadine reported in this study is in contrast to a recent publication by Crumb (1999) in which terfenadine was found to potently and rate-dependently block I_to in human atrium recorded at 22°C. The likely reason for the disparity discussed in Results is that this study examined the effects of terfenadine on I_to at 37°C, at which drug unbinding kinetics are faster than those recorded at 22°C.

Another striking result of this study is the similarity in the HERG-blocking potency and rate dependence exhibited by loratadine and terfenadine (Fig. 2). Whereas the results indicating an IC₅₀ of 204 nM for terfenadine blockade of HERG are consistent with other studies on terfenadine (Table 1), previous studies have failed to observe any HERG or native I_Kr blocking action associated with submicromolar concentrations of loratadine (Table 2). This disparity might be explained by differences in experimental conditions. Indeed, when the experimental conditions of a previous study suggesting little HERG-blocking activity by loratadine were mimicked, similar results were obtained (Fig. 3). In their study, Taglialatela et al. (1998) performed experiments at room temperature, currents were elicited by very long depolarizing pulses (10 s), a high external [K⁺] was used (100 mM), and inward tail currents elicited by hyperpolarizing pulses to 140 mV were used to measure drug effects on current amplitude. In contrast, in this study, experiments were performed at 37°C, currents were elicited by much shorter voltage pulses (400 ms), 4 mM external [K⁺] was used, and outward tail currents elicited by pulses to 40 mV were used to measure drug effects on current amplitude. It is not known which of the experimental differences may be involved in the observed differences in loratadine-blocking affinity of HERG, but it has been reported that elevating external [K+] decreases drug affinity for I_Kr (Yang et al., 1996). Alternatively, the difference discussed in Results may be due to species differences in I_Kr. It has been shown that subtle changes in the amino acid sequence of ERG (ether-a-go-go-related gene) can result in dramatic changes in ERG pharmacology, as evidenced by the human and bovine forms of this channel, where a single amino acid change can result in a 100-fold difference in the sensitivity to the I_Kr blocker dofetilide (Ficker et al., 1998).

Loratadine blockage of HERG described herein provides a mechanism for the arrhythmias reported in association with loratadine usage (Good et al., 1994; Haria et al., 1994; Lindquist and Edwards, 1997; de Abajo et al., 1999). Indeed, a study examining the World Health Organization database for reporting of adverse drug reactions suggests that the incidence of arrhythmias reported in association with loratadine use is similar to that of terfenadine (Lindquist and Edwards, 1997), although life-threatening examples of torsade de pointes and incidences of sudden death have not been described with loratadine usage, whereas they have been with terfenadine use. Furthermore, a recent study including nearly 200,000 patients indicates that the risk of ventricular arrhythmias associated with terfenadine usage was no different than that observed for loratadine (de Abajo et al., 1999). The demonstration that terfenadine and loratadine share similar HERG-blocking potencies and rate dependence, over a concentration range associated with arrhythmias (Hilbert et al., 1987, 1988; Davies et al., 1989), is consistent with these clinical observations. Further studies correlating HERG blockade with myocardial concentrations of terfenadine and loratadine associated with arrhythmias would be beneficial. Differences in the incidence of severe, life-threatening arrhythmias between loratadine and terfenadine may rely more on differences in attainable myocardial concentrations of drug than differences in HERG blockade.