Introduction

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Despite the increasing popularity of nonpharmacologic approaches, antiarrhythmic drugs continue to be widely used in the treatment of cardiac arrhythmias. A major focus of antiarrhythmic drug development in the past decade has been compounds that prolong the cardiac action potential and refractoriness, commonly designated class III drugs, as a primary mechanism of action (Roden, 1993; Singh, 1996). Unfortunately, these agents have demonstrated only modest antiarrhythmic efficacy in both preclinical and clinical studies. In addition, many class III drugs block the rapid component of the delayed rectifier K⁺ current, I_Kr, an effect that prolongs cardiac repolarization in a potent manner (Sanguinetti, 1992; Roden, 1993; Singh, 1996). Block of I_Kr typically causes maximal action potential or QT prolongation at slowest heart rates, so-called reverse rate dependence, rather than the desired effect of greatest efficacy or block during a tachycardia (or rapid rates) (Hondeghem and Snyders, 1990). As a result, a major liability of I_Kr block has been the unpredictable development of excessive QT prolongation at normal heart rates, leading to polymorphic ventricular tachycardia, or Torsade de Pointes, which can cause syncope and sudden cardiac death (Roden, 1993; Singh, 1996). Although uncommon, such proarrhythmia can be catastrophic. However, its occurrence is not totally unexpected in light of recent findings that mutations in the human ether-a-go-go-related gene product (hERG), which is responsible for I_Kr, can cause the congenital long QT syndrome (Curran et al., 1995; Sanguinetti et al., 1995, 1996a; Zhou et al., 1998). It is currently not known what molecular component or components should be targeted to optimize efficacy and reduce toxicity during pharmacologic prolongation of the cardiac action potential. Nevertheless, it is clear that the development of more effective, less toxic drugs would be a significant advance in the treatment of cardiac arrhythmias.

The slow component of the cardiac delayed rectifier, I_Ks, has been shown to increase with -adrenergic stimulation (Bennett and Begenisich, 1987; Sanguinetti et al., 1991), as well as with rapid stimulation rates due to the slow time course of current deactivation (Jurkiewicz and Sanguinetti, 1993). Therefore, it has been proposed that the selective block of I_Ks may lead to greater drug effect at faster rates and thus improved efficacy, along with a reduction in the toxicity that occurs at slower or normal rates. Recently, agents such as azimilide that block I_Ks as well as I_Kr have been developed (Karam et al., 1998). Preliminary studies indicate that the clinical profile of such compounds may be associated with a reduced incidence of proarrhythmia (Pritchett et al., 1998; Page et al., 1999). More selective I_Ks blockers offer the promise of further improvements, as data from several laboratories demonstrate rate-independent class III activity and increased potency under the conditions of enhanced adrenergic tone (Schreieck et al., 1997; Fadayel et al., 1998; Bosch et al., 1998). Moreover, such compounds should more fully clarify the physiologic role of this K⁺ current in cardiac repolarization.

Previous work has suggested that racemic chromanol 293B blocks I_Ks with little effect on other cardiac ion currents (Lohrmann et al., 1995; Busch et al., 1996; Suessbrich et al., 1996; Loussouarn et al., 1997; Bosch et al., 1998). The purpose of this investigation was to synthesize the enantiomers of chromanol 293B and to determine their relative potency to block distinct recombinant human K⁺ channels that underlie the principal repolarizing K⁺ currents in human heart. These currents included the slow (I_Ks), rapid (I_Kr), and ultrarapid (I_Kur) components of the delayed rectifier, as well as the voltage-dependent transient outward current (I_To), and they were studied by heterologous expression of the recombinant K⁺ channels KvLQT1+minK (I_Ks; Barhanin et al., 1996; Sanguinetti et al., 1996b), hERG (I_Kr; Sanguinetti et al., 1995), Kv1.5 (I_Kur; Wang et al., 1993; Feng et al., 1997), and Kv4.3 (I_To; Dixon et al., 1996), respectively, in mammalian cells. Unexpectedly, our results demonstrate that the original stereochemical assignment of the chromanol 293B enantiomers was incorrect and that block by ()-[3R,4S]293B is selective for recombinant I_Ks.

	Experimental Procedures

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Materials. The enantiomers of chromanol 293B were synthesized by a ring opening reaction of the appropriate 6-cyano-2,2-dimethylepoxychromane with N-methyl-N-trimethylsilylethane sulfonamide in the presence of tetrabutylammonium fluoride (Lohrmann et al., 1995). Thus, (+)-[3R,4R]-6-cyano-2,2-dimethyl-epoxychromane gave rise to ()-[3R,4S]293B, and ()-[3S,4S]-6-cyano-2,2-dimethyl-epoxychromane gave rise to (+)-[3S,4R]293B. The requisite epoxychromanes were synthesized by enantioselective catalytic epoxidation of commercially available 6-cyano-2,2-dimethyl-chromane (Lee et al., 1991). The absolute stereochemistry of the epoxychromanes rests firmly on the x-ray crystallographically determined absolute stereochemical configuration of levcromakalim (Faruk, 1984, 1992; Ashwood et al., 1986). Although the physicochemical properties of both precursor epoxides were found to be identical to the literature values, the properties of the enantiomers of 293B differed from those reported by Lohrmann et al. (1995): for (+)-[3S,4R]293B, m.p. was 180-181°C and optical rotation (_D²⁰) was +0.382° (EtOH, c = 0.79) [versus m.p. 190-191°C, _D²⁰ +27.3° (EtOH, c = 10)]; for ()-[3R,4S]293B, m.p. was 181-182°C and _D²⁰ was 0.103° (EtOH, c = 0.93) (m.p. and _D²⁰ not reported). The chemical and stereochemical purities of ()-293B and (+)-293B were determined to be more than 97% by HPLC (ChiralPak AD 4.6 mm × 250 mm, 9:1 heptane/EtOH, 1 ml/min, 25°C), with a retention time of 12.2 min for ()-293B and 5.8 min for (+)-293B. The mass spectrum and ¹H and ¹³C NMR of the two enantiomers were found to be identical to each another and to those of racemic 293B and were consistent with the assigned structure. The elemental analyses of (+)-293B and ()-293B were found to be within 0.4% of predicted values.

Experimental Preparations. Chinese hamster ovary (CHO) cells were cultured in Ham's F-12 medium and transiently transfected using LipofectAMINE (Life Technologies, Inc., Grand Island, NY). Cells were transfected for 8 h in 35-mm dishes containing 6 µl LipofectAMINE and 2 µg of hERG cDNA or Kv4.3 cDNA. For the cotransfection of KvLQT1 and minK, 2 µg of each cDNA was used. The currents were recorded 48 to 72 h after transfection. The K⁺ currents derived from the human Kv1.5 channel were recorded using stably transfected mouse L cells (Snyders et al., 1993). L cells were cultured in Dulbecco's modified Eagle's medium. Subconfluent cultures of L cells were incubated with 2 µM dexamethasone to induce ion channel expression approximately 24 h before their use.

Voltage-Clamp Methods. Potassium currents were recorded using the whole-cell patch-clamp technique (Hamill et al., 1981). Only the cells that demonstrated minimal K⁺ current run-down initially were selected for experimentation. In addition, the experimental setup was designed to permit rapid solution changes with total bath solution turnover in 1 to 2 min. The patch-clamp methods that were used have been described previously (Snyders et al., 1993). Electrode resistances ranged from 1 to 2 M. Voltage-clamp command pulses were generated using pCLAMP software (v4.03; Axon Instruments, Inc., Foster City, CA). Currents were filtered at 5 kHz (3 dB, 4-pole Bessel filter). An Axopatch-1B patch-clamp amplifier (Axon Instruments, Inc.) was used with series resistance compensation. The holding potential for all pulse protocols was 80 mV. Experiments were performed at room temperature (20-22°C). The bath solution for all experiments contained 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, pH 7.35. The pipette intracellular solution contained 110 mM KCl, 5 mM K₂ATP, 2 mM MgCl₂, 10 mM HEPES, and 5 mM K₄BAPTA, pH 7.2. Data are presented as mean ± S.E.

Voltage-Clamp Protocols and Data Analysis. The cells were voltage-clamped to a holding potential of 80 mV. Voltage-clamp protocols were used to record K⁺ currents over a range of membrane potentials. Specific protocols are indicated in each figure. The voltage dependence of channel activation was fit with the Boltzmann equation:

(1)

where I_max is the maximum observed current, V_t is test membrane potential, V_1/2 is the membrane potential for 0.5*I_max, and k represents a slope factor and is equal to RT/zF (z is the apparent equivalent electrical charge; F, R, and T have their usual thermodynamic meanings).

(2)

(3)

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Stereochemical Configuration of the Enantiomers of Chromanol 293B. Contrary to previous reports (Lohrmann et al., 1995; Suessbrich et al., 1996), we observed that the levorotatory or ()-[3R,4S] enantiomer of chromanol 293B was more potent in its pharmacologic effects to block K⁺ channels, as detailed later. In addition, the physicochemical properties cited in the original study by Lohrmann et al. for the two enantiomers did not match our measurements for these materials, except with respect to the sign of their optical rotation. The assignment of stereochemical configuration in the present study is based on 1) the crystallographically determined absolute configuration of the synthetic precursor to levcromakalim (Faruk, 1984, 1992; Ashwood et al., 1986) and 2) the fact that the ring-opening reaction of this precursor with the sulfonamide nucleophile occurs stereospecifically with inversion of configuration at C4 of the chromane: ()-[3S,4S]-6-cyano-2,2-dimethyl-epoxychromane gives rise to (+)-[3S,4R]293B. These data indicate that the original stereochemical designation for these compounds was reversed (Lohrmann et al., 1995; Suessbrich et al., 1996).

Potent, Stereoselective Block of the KvLQT1+minK Channel Complex by ()-293B. Figure 1, A and B, demonstrates the effects of ()-293B on K⁺ currents derived from coexpression of KvLQT1 and minK in CHO cells. In Fig. 1A, families of outward K⁺ currents elicited by increasing voltage-step depolarizations are shown at baseline (control) and after the exposure to increasing concentrations of ()-293B. This enantiomer inhibited KvLQT1+minK current in a potent manner, with block averaging 69 ± 6 and 90 ± 4% at 3 and 30 µM, respectively, as shown in Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of ()- and (+)-[3R,4S]293B on K+ currents derived from coexpression of KvLQT1 and minK in CHO cells. A, outward K+ currents are shown under baseline or control conditions (left) and after bath superfusion of 3 and 30 µM concentrations of ()-293B (middle and right; voltage is stepped from 80 mV to a maximal potential of +60 mV in 20-mV increments for 4 s, with repolarization to 50 mV). ()-293B blocked KvLQT1+minK current in a potent manner, with nearly 90% suppression at 30 µM. B, steady-state K+ current amplitude (measured at the end of a depolarization to +60 mV) was recorded every 20 s during exposure to increasing concentrations of ()-293B. Inhibition of K+ current was apparent even at a concentration of the enantiomer of 0.3 µM. The time bar indicates a K+ current amplitude of 0. C, similar data are shown for 3 and 30 µM concentrations of (+)-293B (voltage-clamp protocol is the same as in A). Block by this enantiomer was less potent than that for ()-293B. D, time course of K+ current suppression is demonstrated during bath superfusion with increasing concentrations of (+)-293B.

View this table: [in this window] [in a new window]   TABLE 1 Percentage block of human cardiac potassium channels by the ()-[3R,4S] and (+)-[3S,4R] enantiomers of chromanol 293B

View larger version (23K): [in this window] [in a new window]   Fig. 2.   KvLQT1+minK: properties of drug block by the 293B enantiomers. Using the voltage-clamp protocol described in Fig. 1A, deactivating tail current amplitudes were plotted as a function of voltage for 0.3 µM ()-293B (A) and 3 µM (+)-293B (B). Averaged data (normalized to the maximal control value) for the resulting activation curves are shown before () and after () drug superfusion in both panels. C, concentration-response curves are shown for block of KvLQT1+minK currents by the enantiomers of chromanol 293B. The solid lines represent the best fit for the Hill equation, whereas the dotted line delineates the relationship when the Hill coefficient is fixed to 1 [the equation would not fit under this condition for (+)-293B].

Minimal Effect of the 293B Enantiomers on hERG. In contrast to KvLQT1+minK, the chromanol 293B enantiomers had little effect on K⁺ current derived from expression of the hERG channel. Figure 3 illustrates families of activating and deactivating K⁺ currents before and after exposure to 30 µM concentrations of ()-293B and (+)-293B (Fig. 3, A and B, respectively). As displayed in Table 1, ()-293B was slightly more potent than (+)-293B with respect to drug block: 30 µM ()-293B reduced maximum tail current by 10.8 ± 4%, a significant decrease, whereas a similar concentration of (+)-293B caused a decline of 7.6 ± 2.2%. The difference in percentage inhibition between the two enantiomers was not statistically significant. K⁺ currents at the end of a 1-s depolarizing pulse (Fig. 3C) and deactivating tail currents (Fig. 3D) were plotted as a function of voltage, with averaged values shown normalized to maximal predrug values. In Fig. 3D, activation curves were fit with the Boltzmann relationship. As for KvLQT1+minK, there was no significant shift in the voltage dependence of channel opening with either chromanol 293B enantiomer (V_1/2 = 1.1 ± 2.9, 7.1 ± 3.7, and 3.3 ± 1.5 mV for control, ()-293B, and (+)-293B, respectively).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Minimal effect of the chromanol 293B enantiomers on the hERG expressed in CHO cells. Outward K+ currents are shown before (left) and after (right) exposure to 30 µM concentrations of ()-293B (A) and (+)-293B (B) (voltage was stepped from 80 mV to a maximal potential of +60 mV in 10-mV increments for 1 s, with repolarization to 60 mV). Both enantiomers demonstrated very weak potency to block hERG current. K+ current at the end of the depolarizing voltage step (C) and deactivating tail currents (D) were plotted as a function of voltage and normalized to the maximal predrug value. , control measurements before drug exposure.  and , data obtained after the application of 30 µM ()-293B and (+)-293B, respectively. D, normalized data were fit with a Boltzmann function (solid curves) and scaled to control values (dotted lines).

Effects of ()-293B and (+)-293B on Kv1.5 and Kv4.3 Channels. Figure 4A shows families of Kv1.5 currents recorded during baseline or control conditions and after exposure to a 30 µM concentration of each enantiomer, with subsequent drug washout. It is apparent that ()-293B was more potent, with block averaging 53 ± 6% versus 29 ± 3% for (+)-293B at this concentration (Table 1). Figure 4B demonstrates the time course of steady-state K⁺ current amplitude during an individual experiment, again confirming the relative potencies of the two enantiomers and the stability of K⁺ currents during experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of ()-293B and (+)-293B on K+ current derived from expression of the human Kv1.5 channel. A, families of Kv1.5 currents are shown under control conditions and after exposure to 30 µM concentrations of each enantiomer, separated by washout periods (voltage was stepped from 80 mV to a maximal voltage of +60 mV in 10-mV increments for 100 ms, with repolarization to 40 mV). Block of Kv1.5 by ()-293B was nearly 50%, whereas 30 µM (+)-293B decreased K+ current by ~25%. B, time course of steady-state Kv1.5 current amplitude (at +60 mV) during bath superfusion of each enantiomer sequentially in the same cell, demonstrating the superior potency of ()-293B (time bar indicates K+ current amplitude of 0).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Block and the voltage dependence of channel activation of Kv1.5 in the presence of the 293B enantiomers. A, Kv1.5 currents (at +60 mV) under control conditions and after 30 µM ()-293B (left) or (+)-293B (right) are superimposed. The time-dependent decay of current during depolarization in the presence of drug and crossover of tail currents suggest open channel block. B, with the voltage-clamp protocol used in Fig. 5A, activation curves were generated by plotting tail current amplitude as a function of voltage. Averaged data normalized by the maximal predrug value are for control values () and for obtained after the application of 30 µM ()-293B () and (+)-293B (), respectively. Curves demonstrate fits of the Boltzmann equation.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of the chromanol 293B enantiomers on Kv4.3 currents. A and B, outward K+ currents during successive depolarizations are shown at baseline and after exposure to 30 µM concentrations of ()-293B (A) and (+)-293B (B) (voltage was stepped from 80 mV to a maximal potential of +60 mV in 10-mV increments for 300 ms). The degree of block was estimated by comparing peak outward current (C) or integrating the area under the current-time curve (D) in control and drug. The mean ± S.E. percentage block is shown in the bar graphs.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Given the remarkable advances in ion channel molecular biology, a logical strategy for antiarrhythmic drug development is to selectively target a particular cardiac ion channel. Based on existing data, as well as theoretical considerations, I_Ks blockers offer promise as antiarrhythmic agents with an improved clinical profile compared with currently available drugs. For example, the selective inhibition of I_Ks should produce maximal pharmacologic effect during the rapid heart rates associated with tachyarrhythmias. At normal heart rates, I_Kr plays a dominant role in repolarization due to the slow rate of I_Ks activation. However, at rapid rates, I_Ks is predicted to increase because deactivation is also slow, causing an accumulation of channels in the open state (Jurkiewicz and Sanguinetti, 1993). In addition, I_Ks is significantly enhanced during sympathetic activation and can contribute to a greater extent to repolarization under such conditions (Bennett and Begenisich, 1987; Sanguinetti et al., 1991). For these reasons, selective targeting of I_Ks may result in improved efficacy in terminating and preventing cardiac tachyarrhythmias. Experimental data indicate that I_Ks block is not associated with the reverse use dependence seen with I_Kr blockers, while demonstrating enhanced effect during -adrenergic stimulation (Schreieck et al., 1997; Bosch et al., 1998; Fadayel et al., 1998). These results imply that the excessive QT prolongation during sinus rhythm that results in proarrhythmia with I_Kr blockers may not occur with a drug that selectively blocks I_Ks. Enthusiasm for I_Ks block must be tempered by the knowledge that mutations in either KvLQT1 or minK can also cause the congenital long QT syndrome (Wang et al., 1996; Chouabe et al., 1997; Splawski et al., 1997; Duggal et al., 1998). Nevertheless, preclinical data indicate that I_Ks block can produce an antiarrhythmic effect (Billman et al., 1999). It is possible that although extensive or complete suppression of I_Ks (e.g., with the congenital long QT syndrome or drugs that bind with very high affinity) is proarrhythmic, pharmacologic inhibition of I_Ks with appropriate kinetics of drug-channel interaction can be antiarrhythmic. This concept can be tested with the availability of I_Ks-specific probes. Thus, I_Ks blockers offer the potential to provide improved antiarrhythmic drug therapy, as well as tools to better define the role of I_Ks in cardiac repolarization.

Chromanol 293B has been reported to be a selective blocker of I_Ks (Lohrmann et al., 1995; Busch et al., 1996; Suessbrich et al., 1996; Loussouarn et al., 1997; Bosch et al., 1998). However, as shown in Table 2, most previous studies have investigated the effects of racemic 293B on K⁺ currents in either guinea pig myocytes or after the expression of minK, which coassembles with endogenous KvLQT1, in Xenopus oocytes. This study represents the first analysis of the stereoselective effects of the enantiomers of chromanol 293B on the recombinant human K⁺ channels thought to represent not only I_Ks but also other major repolarizing K⁺ currents of the cardiac action potential. The effects of 293B on Kv1.5 and Kv4.3 had not been previously examined. Because these channels form the basis of an atrium-selective K⁺ current, I_Kur, and I_To, respectively, they were included in our analysis.

Importantly, after the unambiguous synthesis of the 293B enantiomers, our results demonstrate that the original (+)-[3S,4R]/()-[3R,4S] assignments are reversed (Lohrmann et al., 1995; Suessbrich et al., 1996) and that ()-293B is the more potent isomer for blocking KvLQT1+minK. Block is selective for I_Ks over hERG, whereas the enantiomers demonstrate weak potency against Kv1.5 and Kv4.3, as illustrated in Tables 1 and 2.

In this study, we chose to use recombinant human channels, because species-specific differences in channel protein sequences could alter drug binding. In addition, drug effects were examined after the heterologous expression in mammalian cells. This expression system was chosen because of the well-documented tendency for oocyte-based measurements to underestimate the IC₅₀ of membrane-bound ion channel targets (Grissmer et al., 1994; Po et al., 1999). The effects of both enantiomers were frequently tested in the same cell for each channel complex studied to confirm differences in potency between the two compounds. These methodologic considerations further strengthen our experimental results regarding the distinct pharmacologic effects of the 293B enantiomers and the potential relevance of these findings for human K⁺ currents.

Our results confirm and further extend the selectivity data previously obtained using racemic 293B (Table 2). Thus, the observed IC₅₀ values for the two enantiomers for inhibition of KvLQT1+minK current (~1.4 and 10 µM) are in reasonable agreement with the observed potency of racemic 293B for blocking the native I_Ks current in guinea pig myocytes (it should be noted that our measurements were performed at 22°C, whereas the I_Ks data were obtained at temperatures up to 36°C; Busch et al., 1996; Bosch et al., 1998) although lower than those reported in Xenopus oocytes (Busch et al., 1996; Suessbrich et al., 1996). We observed less than 50% inhibition at the highest concentrations tested (60 µM) for the block of Kv4.3. Although these results are in reasonable agreement with the observed IC₅₀ of 24 µM for racemic 293B against I_To current in human ventricular myocytes (Bosch et al., 1998), they suggest that factors other than the Kv4.3 subunit may be involved in determining the pharmacologic response of the native current. For Kv1.5, our data suggest that ()-293B will have only weak effects to block I_Kur current at concentrations that significantly inhibit I_Ks. The stereoselectivity for block that we observed [i.e., ()-293B was more potent] was reasonably well preserved for both channels for which the enantiomers had greatest affinity (i.e., KvLQT1+minK and Kv1.5). Although the molecular basis of these findings is not known, it is conceivable that part of a drug receptor site may be conserved between these K⁺ channels to explain these results.

In summary, we have synthesized the enantiomers of chromanol 293B and demonstrated that ()-[3R,4S]293B selectively inhibits the KvLQT1+minK ion channel complex. Based on these findings, this compound should serve as an important new tool to investigate the role of the I_Ks current in cardiac electrophysiology and pathophysiology.