Dronedarone (SR33589), a new noniodinated benzofuran derivative structurally related to amiodarone, is currently undergoing clinical trials for the treatment of atrial fibrillation. Despite deletion of iodine from its molecular structure, in vitro and in vivo studies have shown similar electrophysiologic effects of dronedarone to those of amiodarone after short-term^(1,2) as well as long-term⁽³⁾ treatments. Like amiodarone, dronedarone exerts sympatholytic effects⁽⁴⁾ and inhibits several ionic currents, namely: I_Na, I_CaL, I_CaT, I_f, I_Kr, I_Ks, and I_sus^(2,5,6). Both amiodarone and dronedarone have thus been termed "multichannel blockers," but it is not clear which ionic currents might have a fundamental role in the acute effects of amiodarone and dronedarone.

Clinical and experimental studies have indicated that vagal activation plays an important role in the genesis of atrial fibrillation^(7,8). Vagal nerve stimulation or application of acetylcholine induces nonuniform shortening of the atrial refractory period and thus favors conditions for occurrence and maintenance of atrial fibrillation^(9,10). Amiodarone is effective in treating atrial fibrillation⁽¹¹⁾ and inhibits the muscarinic acetylcholine receptor-operated K⁺ current (I_K(ACh)) in atrial cells⁽¹²⁾. Therefore, the present study was conducted to examine the effects of dronedarone, compared with amiodarone, onI_K(ACh) in guinea pig atrial cells.

METHODS

Cell isolation

Myocytes were enzymatically dissociated from a preparation of guinea pig atrial cells. During this procedure, all solutions were oxygenated and thermostated at 37°C. Hearts were removed from male guinea pigs weighing 250-300 g (Hartley, Charles River, France) euthanized per the Helsinki convention by cervical dislocation and placed in Ca²⁺-free Tyrode's solution (mM: NaCl, 137; KCl, 5.4; MgCl₂, 2; HEPES-NaOH, 10; glucose, 11.5; pyruvic acid, 5; thiamine, 0.1; pH 7.35), to which to 0.9 mM CaCl₂ was added.

The heart was mounted in a warmed jacketed Langendorff perfusion system and successively perfused with Ca²⁺-free Tyrode's solution for 5 min, Ca²⁺-free Tyrode's solution supplemented with collagenase (type II; 0.167 mg/ml) and protease (type IV; 0.083 mg/ml) for 6 min, and finally KB solution (mM: KCl, 25; L-glutamic acid (K), 70; MgCl₂, 5; KH₂PO₄, 10; HEPES-KOH, 10; glucose, oxalic acid, 10; pyruvic acid, 2; taurine, 10; β-hydroxybutyric acid, 2; ATP (K), 2; phosphocreatine (Na₂), 2; pH 7.2) for 10 min. The atria were excised from the heart and cut in KB solution. The supernatants were kept at 4°C and used after at least 1 h.

Experimental method and solution

The effects of amiodarone and dronedarone were studied in guinea pig atrial cells in whole cell configuration using patch clamp technique in normal Tyrode's solution (mM: NaCl, 137; KCl, 4; CaCl₂, 1.8; MgCl₂, 1; HEPES-NaOH, 10; glucose, 11.5; pyruvic acid, 5; thiamine, 0.1; pH 7.35) at 35 ± 1°C. Patch pipettes were filled with an intracellular-like solution (mM: KCl, 150; MgCl2, 1; HEPES-KOH, 5; β-hydroxybutyric acid, 2; ATP (K), 4; GTP (Na₂) 0.1; phosphocreatine (Na₂), 5; EGTA (K), 5). Amiodarone and dronedarone were daily dissolved at a concentration of 1 × 10⁻²M in a solution of acidified ethanol (0.5 ethanol + 0.15 ml HCl (N) + 0.35 ml H₂O; pH = 2) then diluted to 1 × 10⁻³M in Tyrode's solution containing 5% porcine serum albumin at pH = 3. Final concentrations were obtained with Tyrode's solution and pH was adjusted to 7.4 with NaOH (1 N). Corresponding ethanol and PSA concentrations were added to control Tyrode's solution.

Stimulation protocol

The holding potential was −50 mV. Carbachol (10 μM) was superfused for 10 s, every 2 min, to activateI_K(ACh). A potential ramp from −100 mV to +60 mV was applied during carbachol perfusion at 9 s. In another series of experiments, 100 μM GTP-γ-S (10 mM stock solution in H₂O at −80°C) was included in the patch pipette solution. The activatedI_K(ACh) gradually decayed probably because of desensitization. Assuming that this decay was a linear function of time, inhibition ofI_K(ACh) was calculated from the difference between the current level that was extrapolated from the current decay before the application of amiodarone or dronedarone and that changed by the drug.

All data are expressed in percentages of inhibition of control values as mean ± SEM independent ANOVA and Dunnett's test (RS1 computer program BBN Software Products Corp., Cambridge, MA, U.S.A.) were used for statistical appraisal of drug effects.

RESULTS

The effects of amiodarone and dronedarone on the carbachol-inducedI_K(ACh) in guinea pig atrial cells were examined in whole-cell voltage clamp configuration. Application of 10 μM carbachol activated an outward K⁺ current at a holding potential of −50 mV. After the activation,I_K(ACh) gradually declined because of desensitization^(12,13).Figure 1A illustrates effects of 0.1 μM dronedarone on carbachol-inducedI_K(ACh) at −50 mV. In a range of 6-8 min, dronedarone, as well as amiodarone (not shown), decreasedI_K(ACh). The potential ramps(Fig. 1B) indicate that the current reversed near to −90 mV, in close agreement with the predicted reversal potential for K⁺ ions (−96 mV); furthermore, these ramps show that the drug effects were observed at all potentials. The inhibition ofI_K(ACh) by amiodarone was also found to be voltage-independent (not illustrated).Figure 1C shows the concentration-dependent inhibitions by dronedarone and amiodarone ofI_K(ACh) (percentages ofI_K(ACh) inhibition were measured at least 6 min after the beginning of drug perfusion): dronedarone decreased the current by 44.7 ± 3.7% (n = 7), 76.7 ± 1.9% (n = 8), and 100 ± 0% (n = 3) of control at 0.01, 0.1, and 1 μM, respectively; amiodarone decreased the current by 31.4 ± 1.6% (n = 7), 45.0 ± 2.3% (n = 6) and 83.6 ± 5.6% (n = 11) at 0.1, 1, and 10 μM, respectively. Dronedarone and amiodarone blockedI_K(ACh) with an IC₅₀ slightly higher than 10 nM and 1 μM, respectively. This latter result of amiodarone is very close to that obtained by Watanabe et al.⁽¹²⁾ in the same preparation (IC₅₀ = 2.4 μM).

Because the activation ofI_K(ACh) depends on the G-protein-mediated interaction between muscarinic receptors and channel proteins, dronedarone could act by uncoupling this cascade of events at different levels. Amiodarone is known to inhibit the K_ACh channel itself or G proteins⁽¹²⁾. In order to dissect the effects of dronedarone on the receptor-G-protein interaction from a direct channel blockade, we tested the drug after the activation of the G-protein by intracellular perfusion with GTP-γ-S, a nonhydrolyzable GTP analog. Because the pipette solution contained GTP-γ-S (100 μM),I_K(ACh) rose after establishing the whole-cell configuration. Using the same protocol as previously but without carbachol application,Figure 2A illustrates the amplitudes of GTP-induced current in control medium or in the presence of 0.1 μM, dronedarone. The current was fully activated within 3 min (n = 21). The potential ramps presented inFigure 2B were measured on steadystate currents for the control and after 6-8 min of 0.1 μM, dronedarone perfusion. As shown above with carbachol, dronedarone inhibited the GTP-γ-S-inducedI_K(ACh) at all potentials. The histogram(Fig. 2C) shows that dronedarone inhibited this current by 27.6 ± 5.1% (n = 6) and 57.6 ± 3.2% (n = 8) at 0.01 and 0.1 μM, respectively.

DISCUSSION

The present study shows that dronedarone, in a concentration-dependent manner, blocks the carbachol-inducedI_K(ACh) in guinea pig atrial cells. This inhibition is effective at all potentials and the IC₅₀ is ≈ 10 nM, which is approximately 100 times lower than that of amiodarone (IC₅₀ ≈ 1 μM). Activation ofI_K(ACh) is a G-protein-mediated process triggered by the interaction of ACh with the muscarinic M₂-receptor⁽¹⁴⁾. Watanabe et al.⁽¹²⁾ have shown that the IC₅₀ of amiodarone was similar on carbachol-, adenosine- and GTP-γ-S-inducedI_K(ACh), indicating that amiodarone does not interact with the M₂-receptor but acts on the K_(ACh) channel itself and/or GTP-binding proteins. Dronedarone, with a molecular structure close to that of amiodarone, also blocks, in the same range of concentration, both the GTP-γ-S-and the carbachol-inducedI_K(ACh). These observations support the fact that dronedarone acts directly on K_ACh channels and/or GTP-binding proteins and not on M₂ receptors.

Amiodarone is not a selective channel blocker because it inhibitsI_K(ACh) and several inward (I_Ca(L)) and outward (I_Kr, I_Ks, I_Kl) currents in a narrow range of concentrations: 1 to 10 μM^(12,15,16). Compared with amiodarone, dronedarone is approximately three times more potent to inhibit each of the latter Ca²⁺ and K⁺ currents^(15,16), strengthening the greater in vivo activities of dronedarone observed in ventricular arrhythmia models^(17,18). The present data demonstrate that dronedarone is approximately 100 times more potent than amiodarone and also than class III antiarrhythmic drugs such as sotalol, E-4031, and MS-551 in guinea pig atrial cells⁽¹⁹⁾. Moreover, in contrast to amiodarone, dronedarone is a selectiveI_K(ACh) blocker, because its IC₅₀ value is smaller by at least one order of magnitude than those observed for Ca²⁺ and K⁺ currents^(15,16). It has been shown that dronedarone is three times more potent than amiodarone in terminating and preventing re-induction of experimental atrial fibrillation maintained during vagal nerve stimulation in anesthetized dogs⁽²⁰⁾. Although the inhibition ofI_K(ACh) by dronedarone observed in the present study may underlie in part the antiarrhythmic action of dronedarone in this experimental vagotonic atrial fibrillation, we cannot exclude participation of other channel-blocking properties of dronedarone to explain the higher potency of dronedarone than amiodarone.

At present, amiodarone is certainly the most effective drug on the market for treating and preventing recurrent atrial fibrillation, but this advantage is offset by its adverse noncardiac side effects^(21,22). Its noniodinated benzofuran derivative, dronedarone, which does not lead to phospholipid accumulation in lungs and thyroid hormone metabolism modifications in rats (M. Clinet, unpublished data, 1992) is a potent inhibitor ofI_K(ACh) in atrial cells, and should be a potent antiarrhythmic agent in human atrial fibrillation when vagal tone contributes to its genesis.

Acknowledgment: The authors wish to thank Dr. Isabel A. Lefevre for her help in reviewing the manuscript and S. Rognon for secretarial assistance.

REFERENCES