Interactions at Human Ether-A`-Go-Go–Related Gene Channels

TOXICOLOGICAL SCIENCES 114(2), 346–355 (2010) doi:10.1093/toxsci/kfq011 Advance Access publication January 13, 2010

Interactions at Human ether-a`-go-go–Related Gene Channels

Anne Friemel and Bernd J. Zu¨nkler1 Federal Institute for Drugs and Medical Devices, D-53175 Bonn, Germany

1 To whom correspondence should be addressed at Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, D-53175 Bonn, Germany.

Fax: þ49-228-207-3558. E-mail: [email protected]. Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021

Received November 23, 2009; accepted January 8, 2010

in Recanatini et al., 2005). Most of these torsadogenic drugs Several noncardiovascular drugs have the potential to induce block hERG currents. Hence, hERG channel blockade is an Torsades de Pointes cardiac arrhythmias via blockade of the rapid important indicator of potential proarrhythmic activity (review component of the cardiac delayed rectifier K1 current (I ), which Kr in Redfern et al., 2003). Therefore, the International Confer- is encoded by human ether-a`-go-go–related gene (hERG). The aim of the present study was to characterize possible interactions ence on Harmonization S7B Guideline (CPMP/ICH/423/02) between terfenadine, binding to a site located inside the central (2005) makes hERG current blockade studies mandatory in cavity, and the following substances with various binding sites: drug development. The nonclinical tests required there for dofetilide, fluvoxamine, chlorobutanol, and a hERG-specific toxin assessing the potential of a test substance to delay ventricular isolated from scorpion venom (CnErg1). The whole-cell configu- repolarization are major hurdles in drug development. ration of the patch-clamp technique was employed on hERG hERG channels have a unique structure, which makes them channels stably expressed in human embryonic kidney 293 cells. the binding target for many structurally diverse compounds. Terfenadine does not interact with dofetilide or fluvoxamine at Sanguinetti’s group used an Ala-scanning mutagenesis ap- hERG channels. Slight subadditive inhibitory effects on hERG proach to identify the amino acid residues, which are crucial for peak tail currents were observed when terfenadine and CnErg1 the interactions of the nonsedating H -histamine receptor were administered in combination. Terfenadine and chlorobutanol 1 synergistically inhibit hERG peak tail currents and enhance each antagonist, terfenadine, the gastroprokinetic drug, cisapride, other’s inhibitory effect in a concentration-dependent way. In and the methanesulfonanilide agent, MK-499, at hERG conclusion, terfenadine interacts with CnErg1 and chlorobutanol, channels (Mitcheson et al., 2000). The authors employed but not with dofetilide or fluvoxamine, at hERG channels. It is a homology model using the bacterial KcsA channel as shown that interactions between chlorobutanol and a hERG a template demonstrating that the high-affinity–binding site of channel blocker binding inside the central cavity (terfenadine) terfenadine is located in the central cavity of the channel produce synergistic effects on hERG currents. between the selectivity filter and the activation gate, at Tyr652 Key Words: hERG channel; interactions; terfenadine; and Phe656 in the S6 domain. Recently, also Thr623 and chlorobutanol; CnErg1. Ser624, located on the base of the pore helix, were shown to interact with terfenadine (Kamiya et al., 2008). The terfenadine-binding site overlaps with that of dofetilide, a methanesulfonanilide class III antiarrhythmic drug, which Torsades de Pointes (TdP), a life-threatening form of binds to Tyr652 and Phe656 and also to other amino acids ventricular arrhythmia, occurs under conditions where cardiac Thr623, Ser624, and Val625 located on the base of the pore repolarization is delayed (as indicated by prolonged QT inter- helix and Gly648 and Val659 in S6 of the hERG channel vals from electrocardiographic recordings). A likely mechanism subunit (Lees-Miller et al., 2000; Kamiya et al., 2006; reviews of QT interval prolongation and TdP is blockade of the rapid in Stansfeld et al., 2007, and in Thai and Ecker, 2007). component of the cardiac delayed rectifier Kþ current However, not all hERG channel blockers bind inside the (IKr; review in Keating and Sanguinetti, 1996). The Kv11.1 central cavity (review in Zu¨nkler, 2006). For the first time, protein a-subunit that underlies the IKr in the heart is encoded by Milnes et al. (2003) identified a drug with hERG channel block hERG (human ether-a`-go-go–related gene) (Sanguinetti et al., properties that are relatively insensitive to mutations of Tyr652 1995; Trudeau et al., 1995; Warmke and Ganetzky, 1994). and Phe656, the selective serotonin reuptake inhibitor fluvox- Several noncardiovascular drugs have the potential to induce amine. The rapid onset of hERG current block by fluvoxamine QT interval prolongations or TdP arrhythmias. Approximately seemed to be consistent with binding to an extracellular site, 2–3% of all prescriptions are estimated to involve medications possibly at the outer mouth of the channel (commentary in that may unintentionally cause the long QT syndrome (review Mitcheson, 2003).

Binding sites at the outer mouth of the channel have been a volume of 1 ml and was perfused at a rate of 2 ml/min; about 0.5 min was identified for hERG-specific toxins found in scorpion venom needed for the exchange of the bath solution. (ergtoxin-like peptides, e.g., CnErg1, isolated from scorpions Determination of the inhibitory effects of test substances on hERG currents was based on the following voltage-clamp protocol: a holding potential of 80 of the genus Centruroides; Gurrola et al., 1999). hERG mV and a voltage step to þ 20 mV applied for 2 s to evoke hERG currents, channels possess an unusually long S5-P linker (43 amino followed by a repolarization step to 40 mV for 2 s to induce hERG tail acids in comparison with 12–23 residues in other Kv channels), currents (stimulation frequency of 0.1 Hz). hERG tail currents were leak which presumably forms an amphipathic a-helix, which, corrected. Deactivating tail currents were fitted with two exponential functions together with the P-S6 linker, is responsible for a hydrophobic and extrapolated to the beginning of the repolarization step in order to calculate the peak tail current amplitude. CnErg1–binding site (Gurrola et al., 1999; Pardo-Lopez et al., The voltage dependence of hERG channel activation was determined from 2002; reviews in Korolkova et al., 2004, and in Wanke and peak tail currents measured at 40 mV following 2-s depolarizations to membrane voltages ranging from 40 to þ 40 mV in 10 mV steps. Peak tail

Restano-Cassulini, 2007). Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021 Chlorobutanol, which is used as a preservative in parenteral current amplitudes were normalized to the maximum peak tail current formulations, inhibits hERG currents at millimolar concen- amplitudes. The voltage dependence of channel availability was determined by fitting the values of the normalized peak tail currents with a Boltzmann trations (Kornick et al., 2003). Its binding site on hERG equation of the form: channels is unknown. Since it has a much lower affinity to I 1 ¼ ; ð1Þ hERG channels than the blockers binding inside the central V V Imax 1 þ exp 1=2 m cavity, it might be suggested that chlorobutanol inhibits hERG k currents via interaction with another binding site. where I was the peak tail current amplitude following the test potential Vm, Imax The existence of different binding sites raises the question was the maximal peak tail current amplitude, V½ was the potential at which whether these binding sites interact. If so, how does this channels were half-maximally activated, and k was the slope factor describing influence the effects of simultaneously administered drugs channel activation. acting at different binding sites. This has been investigated for The time dependence of hERG current blockade by terfenadine, chlorobutanol, and the terfenadine and chlorobutanol combination was methadone and chlorobutanol, each inhibiting hERG currents evaluated by using three different voltage protocols. First, an envelope of tails in a concentration-dependent manner; moreover, chlorobutanol protocol: cells were held at 80 mV and stepped to þ 40 mV for 50–600 ms in enhances the ability of methadone to block hERG currents 50-ms increments; peak tail currents after repolarization to 40 mV at each (Kornick et al., 2003). Some opioid agonists (L-a-acetyl- time point were measured as described above in the absence (control) and methadol (levacetylmethadol, LAAM) and methadone) are presence of test substances. Second, a long step pulse protocol: HEK 293 cells were stepped from a holding potential of 80 mV to þ 20 mV for 10 s; inhibitors of hERG currents (Katchman et al., 2002), but their stimulation was terminated and test substances were applied for > 5 min; and binding sites are not yet known. then the voltage protocol was applied again. Third, development and recovery The aim of the present study was to characterize possible of block were determined by applying 2-s voltage steps to þ 20 mV from interactions at hERG channels between a blocker bindinginside the a holding potential of 80 mV, followed by 2-s repolarization steps to 40 mV central cavity (terfenadine) and substances with various binding at a stimulation frequency of 0.1 Hz; peak tail currents were determined during the application of test substances until steady-state block was achieved and sites (dofetilide, fluvoxamine, CnErg1, and chlorobutanol). during the 10-min washout of test substances. Data analysis and statistical procedures. The concentration-response relationships for the inhibition of peak tail current amplitudes by test substances MATERIALS AND METHODS were calculated according to the logistic form of the Hill equation: I a ¼ 1 ; ð2Þ nðpxpKÞ Culture of human embryonic kidney 293 cells stably transfected with Ic 1 þ 10 hERG channels. Human embryonic kidney (HEK) 293 cells stably expressing hERG channels were kindly provided by Prof C. January where Ic is the peak tail current amplitude during the control periods before (University of Wisconsin). HEK 293 cells were plated at a density of 11 3 application of the test substances and I is the peak tail current amplitude in the 5 presence of test substances, a is the fraction of the hERG peak tail current 10 cells per dish (35-mm diameter) and cultured (at 10% CO2)in Dulbecco’s modified Eagle’s medium with 1 g/l glucose supplemented sensitive to inhibition by the test substance (a measure of the intrinsic activity; with 10% fetal calf serum, 100 U/ml penicillin, 100 lg/ml streptomycin, and this value was set to 1 in the case of terfenadine, dofetilide, fluvoxamine, and 2mM glutamine. chlorobutanol since these substances inhibit hERG currents completely), n is the slope parameter (Hill coefficient), x is the concentration of test Electrophysiological recording. hERG currents were recorded using the substances, and K (¼ IC50) is the midpoint of the curve with px ¼logx whole-cell patch-clamp configuration (Hamill et al., 1981) and an EPC 10 and pK ¼logIC50. patch clamp amplifier (Heka Elektronik, Lambrecht, Germany). Patch pipettes For calculation of the concentration-response relationships for the inhibition were pulled from borosilicate glass capillaries (Hilgenberg, Malsfeld, of hERG peak tail current amplitudes, the test substances were applied Germany) and had resistances between 3 and 5 MX when filled with pipette cumulatively at increasing concentrations and studied at the following solution B (see section Drugs and solutions). Stimulation protocols and data concentrations: 3, 10, 30, 100, and 300nM terfenadine; 1, 3, 10, 30, and acquisition were carried out using Pulsefit software (Heka Elektronik). The 100nM dofetilide; 0.3, 1, 3, 10, and 30lM fluvoxamine; 0.3, 1, 3, 10, and current sample frequency was 1 kHz. At least 50% series resistance 30mM chlorobutanol; 0.3, 1, 3, 10, 30, and 100nM CnErg1. The number of compensation was achieved in all experiments. Outward currents flowing from observations ranged from 3 to 12 per test concentration. the pipette to the bath solution are indicated by upward deflections. The combined effects of two substances A and B in the case of additivity Experiments were performed at room temperature (20–22°C). The bath had were calculated according to van den Brink (1977): 348 FRIEMEL AND ZU¨ NKLER

TABLE 1

IC50 Values, Hill Coefﬁcients (n), and Intrinsic Activities (a) Calculated for the Concentration-Dependent Inhibition of hERG Peak Tail Currents by Terfenadine, Dofetilide, Fluvoxamine, Chlorobutanol, and CnErg1

Test substance IC50 value (nM) n a

Terfenadine 27.7 (24.4–31.5) 1.13 (0.97–1.29) 1a Dofetilide 12.9 (11.4–14.6) 1.55 (1.27–1.83) 1a Fluvoxamine 3.6 3 103 (2.4–5.5 3 103) 0.89 (0.75–1.04) 1a Chlorobutanol 7.4 3 106 (6.1–9.1 3 106) 1.07 (0.85–1.30) 1a CnErg1 6.4 (3.8–10.7) 1a 0.84 (0.69–0.99)

aThese values were ﬁxed to 1. Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021

dependent inhibition of hERG peak tail currents by terfenadine, IAB a b ¼ þ ; ð3Þ dofetilide, fluvoxamine, chlorobutanol, and CnErg1. Figure 1 Ic 1 þ 1 þ ½B IC50A 1 þ 1 þ ½A IC50B IC50B ½A IC50A ½B shows representative experiments demonstrating the effects of the combined administration of terfenadine with high-affinity where Ic is the hERG peak tail current amplitude during the control periods before application of the test substances and IAB is the hERG peak tail current amplitude in the presence of the test substances, A and B, applied in combination, a and b are the intrinsic activities, and IC50A and IC50B are the half-maximally inhibitory concentrations of A and B. According to the diagonal constant ratio combination design proposed by Chou (2006), the combination ratio of two test substances applied simultaneously was kept at an equipotency ratio (IC50 ratio) since the contribution of each test substance to the combination is approximately equal under these conditions. The combination index (CI) for the quantification of synergism or antagonism for two test substances administered simultaneously was calculated according to Chou (2006): A B CI ¼ 1 þ 1 ; ð4Þ AðxÞ1 BðxÞ1 where the denominators A(x)1 and B(x)1 stand for the concentrations of test substances A and B, with each inhibiting hERG peak tail currents by x%, and the numerators A1 and B1 in combination also inhibit hERG peak tail currents by x%. The A(x)1 and B(x)1 values were calculated from Equation 2. A CI equaling 1 indicates additivity, a CI smaller than 1 indicates synergism, and a CI greater than 1 indicates antagonism. The results are expressed as means with the 95% confidence intervals given in parentheses. Equations were fitted using the Sigma Plot Windows program (Jandel Scientific). Significances were calculated by the two-tailed nonpaired t- test for single comparisons and by ANOVA with Bonferroni correction for multiple comparisons. p < 0.05 was considered to be significant. Drugs and solutions. The bath (solution A) contained (in mM):

140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, and 10 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) titrated to pH ¼ 7.40 with NaOH. The pipette (solution B) contained (in mM): 140 KCl, 5 MgCl2, 10 Ethylene glycol bis(b-aminoethyl ether)-N,N,N#,N#-tetraacetic acid, 2 CaCl2, 5 ATP, and 5 HEPES titrated to pH ¼ 7.15 with KOH (free [Ca2þ] ¼ 50nM; free [Mg2þ] ¼ 0.7mM). Terfenadine and fluvoxamine were purchased from Sigma (St Louis, MO), dofetilide from Sequoia (Pangbourne, UK), and CnErg1 from Alomone Labs (Jerusalem, Israel). Stock solutions of 30mM terfenadine and 3mM dofetilide were prepared in dimethylsulfoxide and stock solutions of 30mM fluvoxamine, 30mM chlorobutanol, and 1lM CnErg1 in solution A. FIG. 1. Effects on hERG currents of terfenadine combined with dofetilide, fluvoxamine, chlorobutanol, or CnErg1, each tested at half of their respective IC values. The voltage protocol is shown above. Arrows indicate zero current RESULTS 50 levels. The block of hERG peak tail currents is 38.6% at 13.8nM terfenadine þ 6.5nM dofetilide, 41.2% at 13.8nM terfenadine þ 1.8lM fluvoxamine, 62.7% Table 1 shows the IC50 values, Hill coefficients (n), and at 13.8nM terfenadine þ 3.7mM chlorobutanol, and 30.7% at 13.8nM intrinsic activities (a) calculated for the concentration- terfenadine þ 3.2nM CnErg1. INTERACTIONS AT hERG CHANNELS 349

TABLE 2 Effects on hERG Peak Tail Currents by Terfenadine in Combination with Dofetilide, Fluvoxamine, Chlorobutanol, or CnErg1

IC50 % Block in case multiples of additivity Dofetilide Fluvoxamine Chlorobutanol CnErg1

0.125 20 19.7 (15.4–24.0) (n ¼ 7) 0.25 33 (30.6 CnErg1) 29.2 (23.2–35.2) (n ¼ 7) 29.9a (27.6–32.2) (n ¼ 7) 33.8 (30.4–37.1) (n ¼ 9) 23.4a (18.7–28.0) (n ¼ 11) 0.5 50 (46.0 CnErg1) 47.3 (42.2–52.4) (n ¼ 9) 48.7 (42.9–54.4) (n ¼ 6) 63.2a (55.4–71.1) (n ¼ 8) 38.7a (33.1–44.2) (n ¼ 12) 1 67 (61.3 CnErg1) 78.5a (73.6–83.5) (n ¼ 8) 66.7 (63.7–69.7) (n ¼ 7) 90.8a (87.4–94.3) (n ¼ 7) 63.9 (58.7–69.0) (n ¼ 12) 2 80 (73.6 CnErg1) 81.1 (76.5–88.0) (n ¼ 5) 77.4 (73.8–81.1) (n ¼ 7) 95.9a (91.9–99.9) (n ¼ 8) 73.7 (67.8–79.5) (n ¼ 11)

Note. Block of hERG peak tail currents after application of terfenadine combined with dofetilide, fluvoxamine, chlorobutanol, or CnErg1, at multiples of the Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021 respective IC50 values (first column). aSignificantly different (p < 0.05) from the expected value in the case of additivity. hERG channel blockers with IC50 values in the nanomolar antagonism, respectively; 0.85–0.9, 0.7–0.85, 0.3–0.7, 0.1–0.3, concentration range (dofetilide and CnErg1), a low-affinity and < 0.1 indicate slight synergism, moderate synergism, hERG channel blocker with an IC50 value in the micromolar synergism, strong synergism, and very strong synergism, concentration range (fluvoxamine), or a very low-affinity respectively. When hERG peak tail currents were half- hERG channel blocker with an IC50 value in the millimolar maximally preblocked by terfenadine and CnErg1 at the concentration range (chlorobutanol) on hERG peak tail increasing concentrations of 1, 3, and 10nM was administered currents. The results of several experiments are summarized simultaneously, the calculated CI values were 1.13 (n ¼ 6), in Table 2. The inhibitory effects of combinations of 1.11 (n ¼ 7), and 0.83 (n ¼ 5), respectively, indicating additive terfenadine with dofetilide or fluvoxamine on hERG peak tail effects on hERG channels; by contrast, when first CnErg1 was currents did not deviate markedly from the expected values in administered and then terfenadine at increasing concentrations cases of additivity (Fig. 1, Table 2). The combination of of 1, 3, and 10nM was administered simultaneously, the terfenadine and CnErg1 had subadditive inhibitory effects on calculated CI values were 1.23 (n ¼ 5), 1.46 (n ¼ 5), and 1.20 hERG peak tail currents at low concentrations (Table 2). The (n ¼ 7), respectively, indicating subadditive effects. The effects interaction between terfenadine and CnErg1 was studied in of the combination of terfenadine and chlorobutanol on hERG detail, and CI values were calculated according to Chou peak tail currents showed superadditivity at high concentra- (2006): CI values of 0.9–1.1 indicate nearly additive effects of tions (Fig. 1, Table 2). These interactions between terfenadine a combination of test substances; 1.10–1.20, 1.20–1.45, and > and chlorobutanol on hERG channels were examined in detail 1.45 indicate slight antagonism, moderate antagonism, and (Fig. 2). hERG currents were preblocked by chlorobutanol at

FIG. 2. CI-Fa plot for the effects of terfenadine-chlorobutanol combinations on hERG peak tail current amplitudes. The CI value on the y-axis is plotted as a function of the effect levels (Fa: hERG peak tail current amplitude affected, 1 I/Ic) on the x-axis. CI < 1, ¼ 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. (A) The CI-Fa plot for the inhibition of hERG peak tail currents by simultaneous administration of 2.5 and 7.4mM chlorobutanol and increasing concentrations (1, 3, 10, 30, and 100nM) of terfenadine. The slopes of the regression lines are 0.62 and 1.35 in the presence of 2.5 and 7.4mM chlorobutanol, respectively, and increasing concentrations of terfenadine. (B) The CI-Fa plot for the inhibition of hERG peak tail currents by simultaneous administration of 9.2 and 27.7nM terfenadine and increasing concentrations (0.1, 0.3, 1, 3, and 10mM) of chlorobutanol. The slopes of the regression lines are 0.86 and 1.54 in the presence of 9.2 and 27.7nM terfenadine, respectively, and increasing concentrations of chlorobutanol. The number of observations was 6–14 per concentration of the combination tested. 350 FRIEMEL AND ZU¨ NKLER concentrations inhibiting the peak tail currents by 25 or 50% combination. The terfenadine-induced hERG current block was (2.5 and 7.4mM); additionally, terfenadine was administered at not signiﬁcantly different (p > 0.05) at depolarizing pulses increasing concentrations. The slopes of the regression lines of between 200 and 600 ms duration. the CI-Fa plot increased with increasing chlorobutanol Second, a long step pulse protocol was used (Fig. 5). The concentrations (from 0.62 at 2.5mM to 1.35 at 7.4mM time dependences of the fractional block of hERG current chlorobutanol), indicating that chlorobutanol enhanced the amplitudes (during the long pulse protocol) by both the inhibitory effects of terfenadine concentration dependently. chlorobutanol and the terfenadine and chlorobutanol combina- Similarly, hERG currents were preblocked by terfenadine at tion were ﬁtted with mono-exponential functions, yielding the concentrations inhibiting the peak tail currents by 25 or 50% following values for t½: 1.1 s for chlorobutanol and 1.7 s for the (9.2 and 27.7nM); additionally, chlorobutanol was adminis- terfenadine and chlorobutanol combination. Terfenadine-in-

tered at increasing concentrations. The slopes of the regression duced block increased slightly but not significantly (p > 0.05) Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021 lines of the CI-Fa plot increased with increasing terfenadine between seconds 1 and 10. concentrations (from 0.86 at 9.2nM to 1.54 at 27.7nM Third, when cells were repetitively stimulated at a frequency terfenadine), indicating that terfenadine enhanced the inhibitory of 0.1 Hz (Fig. 6), development of hERG current blockade effects of chlorobutanol concentration dependently. occurred at t½ values of 5.4 ± 0.5 min for terfenadine (n ¼ 6), In order to further characterize the mechanism of the 1.8 ± 0.2 min for chlorobutanol (n ¼ 5), and 4.0 ± 0.8 min for synergistic effects of terfenadine and chlorobutanol, the the terfenadine and chlorobutanol combination (n ¼ 7). On voltage- and time-dependent inhibitory effects were studied washout, the effects of chlorobutanol recovered rapidly with for separate administration of terfenadine and chlorobutanol at a t½ value of 1.3 ± 0.3 min. By contrast, the effects of both the their half-maximally inhibitory concentrations (IC50 values) of terfenadine and the terfenadine and chlorobutanol combination 27.7nM and 7.4mM, respectively, and of the combination at showed poor reversibility after a 10-min washout and the block half their IC50 values (13.8nM and 3.7mM). Block of hERG was still 43.5 ± 4.7% and 29.3 ± 5.2%, respectively, which was currents was strongly voltage dependent for chlorobutanol not significantly different (p > 0.05). (stronger at more positive membrane potentials), whereas In order to study the effects of chlorobutanol applied terfenadine-induced block demonstrated only slight voltage intracellularly, chlorobutanol was added to the pipette solution dependence. For the combination of terfenadine and chlor- at a concentration of 7.4mM to diffuse into the cell. obutanol, voltage dependence was intermediate (Fig. 3). Intracellularly applied chlorobutanol had no effects on hERG Voltage dependence of steady-state hERG current activation peak tail current amplitudes, which were 100.8 ± 2.9% (n ¼ 7) had the following V½ and k values: 9.1 ± 0.3 mV and 7.7 ± 0.3 of the control 10 min after the whole-cell configuration was mV in the absence of test substances (control; n ¼ 28); 5.7 ± established. 1.1 mV and 6.8 ± 0.9 mV for terfenadine (n ¼ 8); 15.2 ± 0.6 mV and 8.4 ± 0.5 mV for chlorobutanol (n ¼ 11); and 4.9 ± 0.7 mV and 7.4 ± 0.6 mV for the terfenadine and chlorobutanol DISCUSSION combination (n ¼ 9). Chlorobutanol and the terfenadine and chlorobutanol combination induced statistically significant It is shown in the present study that terfenadine does not hyperpolarizing shifts in the voltage dependence of channel interact with dofetilide or fluvoxamine at hERG channels. activation (p < 0.05); the k values did not significantly vary Slight subadditive inhibitory effects on hERG peak tail currents between the different experimental conditions (p > 0.05). The were observed when terfenadine and CnErg1 were adminis- voltage dependence of the effects of chlorobutanol and the tered in combination. Terfenadine and chlorobutanol in terfenadine and chlorobutanol combination coincided with the combination inhibited hERG peak tail currents synergistically, voltage range over the rising phases of the steady-state and each substance enhanced the inhibitory effect of the other activation curves corresponding to the membrane potential substance in a concentration-dependent manner. ranges over which hERG channel opening occurs (Fig. 3B). The IC50 value for block of hERG peak tail currents by The time-dependent block by terfenadine, chlorobutanol, terfenadine (27.7nM, Table 1) obtained in the present study and the terfenadine and chlorobutanol combination was first was slightly lower than the values reported previously (56– determined by an envelope of tails protocol for the study of the 204nM; review in Stansfeld et al., 2007). The lower IC50 value effects of progressive hERG channel activation on current might be due to the long period of administration of terfenadine blockade (Fig. 4). The time dependences of the fractional block (about 5–7 min). The administration was prolonged because of of hERG current amplitudes (after depolarization to a mem- the recent observation (Stork et al., 2007) that at a stimulation brane voltage of þ 40 mV) by both the chlorobutanol and the frequency of 0.1 Hz, steady-state inhibition of hERG currents terfenadine and chlorobutanol combination were fitted with by terfenadine is reached after about 5–10 min of drug mono-exponential functions, yielding the following half-life application. The IC50 value for block of hERG peak tail (t½) values for the onset of hERG current blockade: 251 ms for currents by dofetilide (12.9nM, Table 1) is within the range chlorobutanol and 277 ms for the terfenadine and chlorobutanol of the previously reported values (9.5–15nM; Snyders and INTERACTIONS AT hERG CHANNELS 351 Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021

FIG. 3. Voltage-dependent modulation of hERG currents by terfenadine, chlorobutanol, and the combination of terfenadine and chlorobutanol. The voltage protocol is shown above. (A) Representative hERG current traces in the absence (control, left traces) and presence of 27.7nM terfenadine (upper traces), 7.4mM chlorobutanol (middle traces), or the combination of 13.8nM terfenadine and 3.7mM chlorobutanol (lower traces). Arrows indicate zero current levels. (B) The normalized Boltzmann functions describing the voltage-dependent activation of hERG currents in the presence of 27.7nM terfenadine, 7.4mM chlorobutanol, and the combination of 13.8nM terfenadine and 3.7mM chlorobutanol and the mean data for the voltage dependence of fractional block of hERG peak tail currents indicated by the bars. Symbols represent means and the vertical lines the SE. The number of experiments was 8 for terfenadine, 11 for chlorobutanol, and 9 for the combination.

Chaudhary, 1996; review in Stansfeld et al., 2007). hERG peak pletely. The value for a was fixed to unity to calculate the IC50 tail currents were blocked by fluvoxamine with an IC50 value value and the Hill coefficient given in Table 1. The scorpion of 3.6lM (Table 1), thus similar to the previously reported toxin CnErg1 binds to hERG channels with a 1:1 stoichiometry value (3.8lM; Milnes et al., 2003). Also, the IC50 value for the (Gurrola et al., 1999; Hill et al., 2007; Pardo-Lopez et al., block of hERG peak tail currents by chlorobutanol (7.4mM, 2002), so that the value for the Hill coefficient was fixed to 1 in Table 1) is similar to the previously reported value (4.4mM; order to calculate the concentration-response relationship for Kornick et al., 2003). Fitting the concentration-response the inhibition of hERG peak tail current amplitudes by CnErg1 relationship for the inhibition of hERG peak tail current according to Equation 2. The IC50 value (6.4nM, Table 1) is amplitudes by chlorobutanol according to Equation 2 yielded within the range of the previously reported values (6–16nM; a value for a of 1.1 (0.6–1.6), which was not significantly Gurrola et al., 1999; Hill et al., 2007; Pardo-Lopez et al., different from unity; therefore, it was assumed that high 2002). Peptide toxins usually occlude the pore of the channel; concentrations of chlorobutanol inhibit hERG currents com- however, CnErg1 did not completely inhibit hERG currents at 352 FRIEMEL AND ZU¨ NKLER Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021

FIG. 4. Envelope of tails protocol used to study the time dependence of hERG current block induced by terfenadine, chlorobutanol, and the combination. The voltage protocol is shown above. (A) Representative current traces evoked by an envelope of tails protocol under control conditions (control, left traces) and in the presence of 27.7nM terfenadine (upper traces), 7.4mM chlorobutanol (middle traces), and the combination of 13.8nM terfenadine and 3.7mM chlorobutanol (lower traces). Arrows indicate zero current levels. (B) Mean data (± SE) for fractional block of hERG currents by 27.7nM terfenadine, 7.4mM chlorobutanol, and the combination of 13.8nM terfenadine and 3.7mM chlorobutanol plotted as a function of the depolarizing pulse durations. Block developed mono-exponentially for both the chlorobutanol and the terfenadine and chlorobutanol combination. The number of experiments was 11 for terfenadine, 7 for chlorobutanol, and 10 for the combination. high concentrations (Table 1), in accordance with previous at the outer mouth of the channel (as suggested by Milnes observations that the maximal effect of CnErg1 is about et al., 2003, and Mitcheson, 2003) was expected to interfere 90% suppression of the hERG current (Hill et al., 2007; with the transition from open to inactive (i.e., inactivation), Pardo-Lopez et al., 2002). which involves closure of an extracellular inactivation gate. The combination of terfenadine and dofetilide had additive This in turn was expected to antagonize terfenadine’s in- inhibitory effects on hERG peak tail currents (Fig. 1, Table 2), hibitory effects on hERG currents. However, using a molecular which seems to be due to overlapping binding sites in the central modeling approach, fluvoxamine was recently docked into the cavity of the hERG channel for both terfenadine and dofetilide hERG channel, so that its protonated nitrogen binds below the (Mitcheson et al., 2000; review in Stansfeld et al., 2007). Phe656 residues and outside the central cavity, which permits The lack of interactions between terfenadine and fluvox- the trifluoromethyl group of the compound to interact with amine (Fig. 1, Table 2) was surprising. Binding of fluvoxamine the nonaromatic binding site formed by Thr623 and Ser624 INTERACTIONS AT hERG CHANNELS 353 Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021

FIG. 5. Long step pulse protocol used to study the time dependence of hERG current block induced by terfenadine, chlorobutanol, and the combination of terfenadine and chlorobutanol. The voltage protocol is shown above. (A) Representative recordings of current traces prior to and immediately after application of 27.7nM terfenadine (upper traces), 7.4mM chlorobutanol (middle traces), and the combination of 13.8nM terfenadine and 3.7mM chlorobutanol (lower traces). Arrows indicate zero current levels. (B) Mean data (± SE) for the development of hERG current block induced by 27.7nM terfenadine, 7.4mM chlorobutanol, and the combination of 13.8nM terfenadine and 3.7mM chlorobutanol. Block developed mono-exponentially for both the chlorobutanol and the terfenadine and chlorobutanol combination, whereas block development for terfenadine was linear with time. The number of experiments was six for terfenadine, ﬁve for chlorobutanol, and seven for the combination.

(Stansfeld et al., 2007). This model suggests overlapping substances according to the Chou (2006) criteria. However, binding sites for fluvoxamine and terfenadine. The observation when terfenadine was administered in the presence of CnErg1, that the simultaneous administration of terfenadine and the calculated CI values were between 1.20 and 1.46, fluvoxamine has additive inhibitory effects on hERG peak tail indicating a moderate antagonism of two test substances currents may be compatible with both models. Further studies applied in combination according to the Chou (2006) criteria. are necessary for the elucidation of the fluvoxamine-binding In summary, the interactions of terfenadine and CnErg1 at site on hERG channels. hERG channels seem to be complex and cannot be described Recently, Margulis and Sorota (2008) found that cisapride simply by additive effects of two test substances. The with a binding site located within the central cavity has additive observations may indicate that CnErg1 slightly antagonizes inhibitory effects on hERG currents when combined with (1) the inhibitory effects of terfenadine on hERG peak tail currents. quinidine with an overlapping binding site, (2) fluvoxamine, The simultaneous administration of terfenadine and chlor- and (3) the ergtoxin BeKm-1. Whereas the first two interactions obutanol induced superadditive inhibitory effects on hERG correspond to those obtained in the present study, the third does peak tail currents (Fig. 1, Table 2). The observation that not. At low concentrations, terfenadine and CnErg1 adminis- synergism increased at increasing test concentrations of the tered in combination had subadditive inhibitory effects on combination can be explained by the higher probability that hERG peak tail currents (Table 2). In order to examine the both molecules bind simultaneously to the same channel at interaction between terfenadine and CnErg1 in detail and to higher test concentrations. Chlorobutanol and terfenadine calculate a CI-Fa plot, hERG currents were preblocked by one enhanced the inhibitory effects of the other substance test substance and the other test substance was administered at concentration dependently (Fig. 2). increasing concentrations in the presence of the first test Experiments studying the voltage- and time-dependent substance. When CnErg1 was administered in the presence of inhibitory effects of terfenadine and chlorobutanol on hERG terfenadine, the calculated CI values were between 0.83 and currents demonstrated that they have different mechanisms of 1.13, indicating a nearly additive effect of a combination of test action and, consequently, different binding sites on hERG 354 FRIEMEL AND ZU¨ NKLER

The binding site of chlorobutanol on hERG channels is not known. It might be suggested that hERG current block by chlorobutanol is caused by a modiﬁcation of the composition of membrane lipids. Hydroxypropyl b-cyclodextrins, which are cyclic oligosaccharides used to enhance drug solubility, inhibit hERG currents at a concentration of 6%; cyclodextrins modify the lipid environment and cholesterol composition of the plasma membrane, and it has been suggested that channels that are located in the lipid raft domains tend to be sensitive to the interaction with cyclodextrins (Mikhail et al., 2007). However,

the concentration-dependent enhancement of chlorobutanol’s Downloaded from https://academic.oup.com/toxsci/article/114/2/346/1673141 by guest on 29 September 2021 inhibitory effects by terfenadine (Fig. 2) seems to argue against nonspecific effects of chlorobutanol on hERG channels mediated via a modification of the composition of membrane lipids and might point toward a specific binding site of chlorobutanol on hERG channels. Two observations seem to indicate a binding site for chlorobutanol on the extracellular side of the hERG channel: first, intracellular application of FIG. 6. Time course of the development and recovery of hERG peak tail chlorobutanol had no effects on hERG currents, which argues current block induced by terfenadine, chlorobutanol, and the combination against a binding site accessible via the lipid phase of the obtained in three representative experiments. Bars indicate the duration of plasma membrane; second, the effects of chlorobutanol on application of 27.7nM terfenadine, 7.4mM chlorobutanol, and the combination hERG currents were rapidly reversible on washout (Fig. 6). of 13.8nM terfenadine and 3.7mM chlorobutanol. Further studies using mutagenesis approaches are required to characterize the chlorobutanol-binding site on hERG channels. channels. hERG channel blockade by terfenadine showed weak Chlorobutanol is used in parenteral dosage forms as an voltage dependence (Fig. 3), did not increase with depolarizing antimicrobial preservative at concentrations of up to 0.5% (about pulses > 200-ms duration during the envelope of tails protocol 30mM). Plasma concentrations of about 0.5mM chlorobutanol (Fig. 4), and increased only slightly during the long step pulse were reported in a patient receiving iv morphine preserved with protocol (Fig. 5). The observation that the inhibitory effect of 0.5% chlorobutanol (DeChristoforo et al., 1983). The ratio of the terfenadine on hERG currents is very slowly reversed on drug IC50 value for the block of hERG currents (7.4mM) to the washout (Fig. 6), an observation also made recently by Stork plasma concentration of chlorobutanol is about 15 and, therefore, et al. (2007), might be explained by a tight binding of below the margin of 30, which seems to be the line of demar- terfenadine to the inactivated state (Perrin et al., 2008) and cation between substances associated with TdP and those which a slow dissociation from the open state of the hERG channel are not (Redfern et al.,2003).However,thetorsadogenic (Stork et al., 2007). In contrast to terfenadine, voltage potential induced by block of hERG currents can be counter- dependence of the chlorobutanol effects coincided with the balanced by effects on other types of cardiac ion channels (e.g., membrane potential range over which hERG channel opening Naþ and L-type Ca2þ channels; Redfern et al., 2003). Further in occurs (Fig. 3). The results of the envelope of tails protocol vitro and in vivo electrophysiological studies are required to test indicate that channel opening is necessary for hERG current the torsadogenic potential of chlorobutanol. Depending on both block by chlorobutanol (Fig. 4), and the block developed the site and the speed of injection, it is not unlikely that the mono-exponentially during the long step pulse protocol (Fig. 5). concentration of chlorobutanol in the heart may approach the These observations indicate that chlorobutanol blocks level (2.5mM) at which synergistic inhibitory effects on hERG hERG channels primarily in the open state. Chlorobutanol currents were observed after simultaneous administration of also increased the hERG current amplitude in a membrane increasing terfenadine concentrations (Fig. 2B). potential range close to the threshold of channel activation (Fig. In conclusion, it is shown in the present study that terfenadine, 3B), i.e., it had an agonistic effect at this membrane potential which binds to the central cavity of the hERG channel, does not range. This effect might be due to chlorobutanol-induced interact with a substance with an overlapping binding site modification of the activation-gating process of the hERG (dofetilide) or with a substance with an unknown binding site channel. The observation that coapplication of terfenadine (fluvoxamine). Its inhibitory effects are slightly antagonized by markedly slowed the reversibility of the effects of chlorobu- the presence of a substance binding at the outer mouth (CnErg1). tanol (Fig. 6) might indicate that the slow dissociation of Terfenadine and the preservative chlorobutanol synergistically chlorobutanol in the presence of terfenadine is responsible inhibit hERG peak tail currents. It is not unlikely that similar for the synergistic effects on hERG peak tail currents (Figs. 1 synergistic effects on hERG currents are generated by interaction and 2, Table 2). between chlorobutanol, which may be an open state blocker INTERACTIONS AT hERG CHANNELS 355 binding to the extracellular side of the hERG channel, and other Mikhail, A., Fischer, C., Patel, A., Long, M. A., Limberis, J. T., Martin, R. L., hERG channel blockers binding inside the central cavity. Cox, B. F., Gintant, G. A., and Su, Z. (2007). 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