Differences of promethazine and terfenadine on ion channels in guinea pig ventricular myocytes

Promethazine, a first generation antihistamine, has an antiarrhythmic effect on ischemia-reperfusion inducing arrhythmias1 and experimental arrhythmias.2 However, terfenadine as a second generation of antihistamine, has been reported to elicit hypotension, bradycardia, prolongation of the QTc interval and torsades de pointes (TdP) like ventricular arrhythmia.3 This may be due to the blockage on rectifier postassium current (Ik) of terfenadine, resulting in the prolongation of the action potential duration (APD) and dispersion of the repolarization duration, which might provoke a specific form of polymorphic ventricular tachydysrhythmia, i.e. TdP.4 In clinical practice, however, the class III antiarrhythmic agents, which target on the Ik and prolong the action potential duration and QTc interval, rarely lead to arrhythmias. Other actions must be considered to underlie the arrhythmogenic tendency of terfenadine besides its inhibition on Ik. Though both promethazine and terfenadine block the H1 receptor, there must be a different pharmacology profile between the two compounds on ion channels of cardiac myocytes. Whole-cell patch clamp technique was used to investigate the effects of these two antagonists of the H1 receptor on the main ion currents in cardiac electrical activities. METHODS Materials Male guinea pigs, weighing 300 to 400 g, for isolating myocardial cells were sourced from the Center for Experimental Animals of Shanxi Medical University. Promethazine, terfenadine, and other chemicals such as bovine serum albumin, nicardipine, EGTA, were all purchased from Sigma Chemical Co. USA. N-2-hydroxy ethylpiperazine -N'-2-ethane-sulfonic acid (HEPES), was purchased from Beijing Chemical Reagents, China, and collagenase P, from Roche Diagnostics GmbH, Mannheim, Germany. Isolation of single ventricular myocytes Single ventricular myocytes were obtained by an enzymatic dissociation procedure similar to that described elsewhere5,6 after the guinea pig was anesthetized by an intraperitoneal injection of phenobarbital (30 mg/kg), then kept in the high-K+ storage solution containing KOH 85, L-glutamic acid 50, KCl 30, Taurine 20, KH2PO4 20, MgCI2 1.0, glucose 10.0, HEPES 10.0, EGTA 0.5 mmol/L (pH was titrated to 7.35–7.40 with 1.0 mol/L KOH) at room temperature ( 22 °C) for at least 1 hour before use. Recording of L-type calcium current, sodium current, and the delayed rectifier potassium current Cells, which were placed into a small chamber (volume 1.5 ml) on the stage of an inverted microscope, were constantly perfused with normal Tyrode’s solution containing 1.8 mmol/L CaCI2 at a rate of 2 ml/min. The pipette solution for recording calcium current (ICa-L) was composed of KCl 150, MgCI2 1.0, EGTA 5.0, HEPES 5.0, ATP-K2 3.0, GTP-T 0.1 mmol/L. The pipette solution for recording (INa) was composed of KCl 150, MgCI2 1.0, EGTA 5.0, HEPES 5.0, ATP-K2 3.0, ATP-Na 5.0 mmol/L. The pipette solution for recording IK, was composed of KCl 150, MgCI2 1.0, EGTA 5.0, HEPES 5.0, ATP-K2 3.0, ATP Na 5.0. The pH values of all mentioned solutions were titrated to 7.20–7.30 by 1 mol/L KOH. Under a voltage clamp, ICa-L was evoked by a voltage step pulse which depolarized from the holding potential of —40 mV to +10 mV for 600 ms. INa was elicited by a voltage pulse which depolarized to 0 mV from the holding potential of —80 mV for 10 ms, and Ik was induced by a voltage step pulse depolarized from —60 mV of holding potential to +40 mV for 3 seconds. Data were collected by pClampex 8.0 and analyzed using a pClampfit 8.0 program (Axon Instruments, USA). Statistical analysis Results were expressed as mean ± standard deviation (SD). ANOVA was used to analyze the differences between groups using SPSS. P value less than 0.05 was considered as significant. The concentrations for 50% of maximum inhibition (IC50), and 95% confidence interval (CI95) were calculated using linear regression. Plots were created with Sigmaplot 2003. RESULTS Inhibition of promethazine on ICa-L, INa and Ik Promethazine at 0.3 — 100 μmol/L inhibited the ICa-L,INa and Ik concentrationdependently (Table 1). The IC50 of promethazine on ICa-L, INa, and Ik was 5.08 μmol/L (CI95 was 1.87–14.88 μmol/L), 5.88 μmol/L (CI95 was 3.64 - 9.50 μmol/L) and 3.28 μmol/L (CI95 was 0.43—25.13 μmol/L), respectively.Table 1: Effects of promethazine on ICa-L, INa, Ik in ventricular myocytes of guinea pig (mean ± SD)Time-dependence of the effects of promethazine on ICa-L, INa and Ik The blockage of promethazine on ICa-L, INa and Ik was reversible. Currents were greatly depressed to <3 % of ICa-L and INa and to about 8% for IK in comparison with their initial amplitude at 6 minutes after the administration of promethazine at 100 μmol/L and were quickly restored to over 85 % at 12 minutes after being washed out with Tyrode’s solution (Fig. 1).Fig. 1.: Time-effect curve of 100 μmol/L promethazine on ICa-L, INa, IK. Drug was washed out at 6 minutes after perfusion. The effect of 100 μmol/L promethazine reached peak potential at 6 minutes and was restored quickly to over 80% at 18 minutes when the drug was washed out from 6 minutes.(Δ P<0.05, ΔΔ P<0.01 vs 0 min of Ik; # P<0.05, ## P<0.01 vs 0 min of INa; * P<0.05, ** P<0.01 vs 0 min of ICa-L)Inhibition of terfenadine on ICa-L, INa and Ik Terfenadine at 0.03 — 10.00 μmol/L also showed a concentration dependency in inhibiting cardiac ICa-L, INa, and Ik. Data in Table 2 show the currents at 16— 18 minutes after the drug perfusion which was washed out at 6 minutes. At 10 μmol/L, terfenadine inhibited INa and ICa to <1 % of their initial current amplitude while Ik was depressed to <10 % of it. The IC50 of terfenadine on ICa-L, INa and Ik was 0.60 μmol/L (CI95 was 0.13–2.83 μmol/L), 0.61 μmol/L (CI95 was 0.19 -1.94 μmol/L) and 0.79 μmol/L (CI was 0.13–4.76 μmol/L), respectively.Table 2: Effects of terfenadine on ICa-L, INa, Ik in ventricular myocytes of guinea pig (mean ± SD)Time-dependence of the effects of terfenadine on ICa-L, INa and Ik Compared to the inhibition characteristics of promethazine on ICa-L, INa and Ik, the blockage of terfenadine on these ion channels was slow and lasted much longer. About 16 — 18 minutes were needed for the effects of terfenadine 10 μmol/L on ICa-L, INa and Ik to reach their peak. Only 30%—40% of the initial current amplitude was restored at 30 minutes when washed out with Tyrode’s solution (Fig.2).Fig. 2.: Time-effect curve of 10 μmol/L terfenadine on ICa-L, INa, IK. Drug was washed out at 6 minutes after perfusion. 10 μmol/L terfenadine did not attain the most potent effect at 6 minutes but reached its peak at 18 minutes when the drug had been washed out for 12 minutes. The restoration to about 30 % to 40% was also very much slower when compared with promethazine. (Δ P<0.05, ΔΔ P<0.01 vs 0 min of Ik; # P<0.05, ## P<0.01 vs 0 min of INa;* P<0.05, ** P<0.01 vs 0 min of ICa-L)DISCUSSION The use of first generation antihistamines, such as diphenhydramine, hydroxyzine, chlorpheniramine, is limited by their anti-cholinergic and sedative properties. The second generation of antihistamines, which are called non-sedating antihistamines such as terfenadine, astemizole, are free of these side-effects. However, the cardiac effects implicated by the two generation antihistamines are different.7 It has been reported that promethazine has anti-arrythmic effects.1,2 Terfenadine produces significant disruption of the ECG wave form, including large amplitude, morphologically aberrant T-waves and, in some cases, torsades de pointes-type arrhythmias.8 However some researchers have indicated that the potential to cause ventricular arrhythmias is not a class effect because loratadine, cetirizine, and fexofenadine are not associated with QT prolongation, TdP, or other ventricular arrhythmias.9 Therefore, promethazine and terfenadine should be chosen to investigate their different actions on cardiac myocytes. This study showed that promethazine concentration -dependent inhibited ICa-L, INa, Ik which might be used to explain the anti-arrhythmic effect of the drug. The concentration used to inhibit cardiac ion channels, however, was 3.28 μmol/L for Ik, 5.88 μmol/L for ICa-L and 5.08 μmol/L for INa, which was much higher than those used for anti-allergy (19.3 ng/ml, which was 0.06 μmol/L).10 This can partially explain the fact that promethazine rarely induces arrhythmias when used in allergic diseases. Moreover, the fact that the inhibition of promethazine on cardiac ion channels is reversible and restored quickly may explain its anti-arrhythmic effect on patients. This study on guinea pigs showed that promethazine cured experimental arrhythmias induced by BaCI2 with its multiple ion channel blocking effects. The effect of promethazine on BaCI2 inducing experimental arrhythmias was no less potent or more potent than lidocaine. Terfenadine is a representative of the second-generation anti-histamine drugs which tend to cause lethal arrhythmias, according to some reports.9,11 Therefore, the studies on its actions on cardiac ion channels have attracted general attention.12–14 Though the inhibition effect of terferfenadine on cardiac ICa-L, INa, Ik has been found to be consistent with our present results, the time-dependence property of this compound was neglected before. In present study, results showed that terfenadine inhibited ICa-L, INa, Ik in a slow and long-lasting manner where the currents were difficult to restore. The blockage of terfenadine on Ik results in the prolongation of action potential duration and the dispersion of the repolarization duration, which provoke a lethal tachydysrhythmia, i.e. torsade de pointes, attribute to its serious arrhythmogenic properties. Furthermore, the potent and long-lasting inhibition of terfenadine on ICa-L and INa inducing a transduction block and local re-entry in the ventricular may aggravate the lethal arrhythmic effect of this compound. It must be noted that the concentration needed for terfenadine to block the cardiac ion-channels (0.79 μmol/L for Ik, 0.61 μmol/L for INa and 0.6 μmol/L for ICa-L, ) was close to that of its anti-allergic action (0.1 μmol/L) which underlie its tendency to cause lethal arrhythmias. In conclusion, promethazine can inhibit INa, Ik and ICa-L in a concentration-dependent manner. Terfenadine can block INa, Ik and ICa-L in a dose-dependent manner which is much more potent and long-lasting than that of promethazine, especially on INa and ICa-L.