Congrong Lin

The Effects of Chiral Isolates of Methadone on the Cardiac Potassium Channel IKr

Objectives: Methadone is a synthetic opioid, an analgesic and an antiaddictive. QT prolongation as well as torsade de pointes ventricular tachycardia and death have been reported with methadone. Methadone’s proarrhythmic toxicity is related to the inhibition of cardiac IKr channel and prolongation of the action potential. We hypothesized that the 2 isomers of methadone may have different effects on the IKr channel. Methods: The effects of the isomers on IKr were evaluated by using an oocytes system with heterogeneously expressed human ether-a-go-go-related gene (HERG) using the 2 electrode voltage clamp technique. r- and s-methadone were obtained by employing chiral high-performance liquid chromatography, separating methadone into 2 isolates, with optical rotations of –141 and +143 degrees. Results: At concentrations of 0.01, 0.03, 0.1, 1 and 3 mM, r/s-methadone produced a dose-dependent inhibition of HERG by 17 ± 5, 23 ± 4, 40 ± 4, 57 ± 3, 69 ± 3 and 80 ± 1%, respectively. The IC50 of r/s-methadone was 0.21 ± 0.02 mM. At 0.1, 0.3 and 1 mM, s-methadone reduced HERG current by 50 ± 4, 76 ± 5 and 87 ± 5%, respectively, while r-methadone reduced HERG current by 26 ± 4, 53 ± 3 and 77 ± 3%, respectively. Conclusions: There was a significant difference (p < 0.01) in the percentage of current inhibition between r- and s-methadone, at 0.1 and 0.3 mM (52% reduction). Thus, r-methadone may be a safer agent due to less QT effect.

The Additive Effects of the Active Component of Grapefruit Juice (Naringenin) and Antiarrhythmic Drugs on HERG Inhibition

Background: Grapefruit juice causes significant QT prolongation in healthy volunteers and naringenin has been identified as the most potent human ether-a-go-go-related gene (HERG) channel blocker among several dietary flavonoids. The interaction between naringenin and IKr-blocking antiarrhythmic drugs has not been studied. We evaluated the effect of combining naringenin with IKr-inhibiting antiarrhythmic drugs on cardiac IKr. Methods and Results:IKr current was studied by using HERG expressed in Xenopus oocytes, and the two-electrode voltage clamp technique was employed. Antiarrhythmic drugs (azimilide, amiodarone, dofetilide and quinidine) were tested. Experiments were performed at room temperature. Naringenin blocked HERG current dose dependently with an IC50 of 173.3 ± 3.1 µM. Naringenin 100 µM alone inhibited HERG current by 31 ± 6%, and this inhibitory effect was increased with coadministration of 1 or 10 µM antiarrhythmic drugs. When 100 µM naringenin was added to antiarrhythmic drugs, greater HERG inhibition was demonstrated, compared to the current inhibition caused by antiarrhythmic drugs alone. Addition of naringenin significantly increased current inhibition (p < 0.05). Conclusions: There is an additive inhibitory effect on HERG current when naringenin is combined with IKr-blocking antiarrhythmic drugs. This additive HERG inhibition could pose an increased risk of arrhythmias by increasing repolarization delay and possible repolarization heterogeneity.

The Influence of Extracellular Acidosis on the Effect of IKr Blockers

Background: Myocardial infarction causes the acidification of the cellular environment and the resultant acidosis maybe arrhythmogenic. The effect of acidosis on the action of antiar-rhythmic drugs, an important issue in the antiarrhythmic drug therapy after myocardial infarction, remains to be studied. Methods: To evaluate the effect of acidosis on rectifier potassium current (Ikr) blockers, the human ether-a-go-go-related gene (HERG), which encodes IKr, was expressed in Xenopus laevis oocytes. The two electrodes voltage clamp technique was used and the experiments were performed at room temperature. Results: Quinidine (10 µM) inhibited HERG tail current by 37% ± 5% at pH7.4. The block decreased to 5% ± 2% with extracellular pH at 6.2. Dofetilide (0.3 µM) inhibited HERG tail current by 34% ± 3% and 1% ± 2% at extracellular pH 7.4 and 6.2, respectively. Azimilide (10 µM) inhibited HERG tail current by 59% ± 3% and 17% ± 3% at extracellular pH 7.4 and 6.2. There were significant differences in the HERG inhibition by quinidine, dofetilide, and azimilide between pH 7.4 and pH 6.2 (P < .01). The drug concentration blocking 50% of current (IC50) was 5.8 ± 0.3 µM for azimilide, 9.9 ±1.0 µM for quinidine, and 0.5 ± 0.02 µM for dofetilide at pH 7.4. When extracellular pH was decreased from 7.4 to 6.2, the IC50 increased to 95.5 ± 11.3 µM for azimilide, 203.2 ± 15.7 µM for quinidine, and 12.6 ± 1.2 µM for dofetilide. Unlike quinidine, dofetilide, and azimilide, there was no significant difference in the percentage of current block by amiodarone between pH 6.2 and 7.4. For amiodarone, the IC50 was 38.3 ± 8.5 µM at pH 7.4 and 27.3 ± 1.6 µM at pH 6.2. Conclusion: Our data show that the Ikr blocking effect of azimilide, dofetilide, and quinidine was attenuated at acid pH, whereas this was not the case for amiodarone. These observations may explain the efficacy of amiodarone in reducing arrhythmic death in patients after a myocardial infarction compared with other IKr blockers.

A mechanism for the potential proarrhythmic effect of acidosis, bradycardia, and hypokalemia on the blockade of human ether-a-go-go-related gene (HERG) channels

Many drugs are proarrhythmic by inhibiting the cardiac rapid delayed rectifier potassium channel (IKr). In this study, we use quinidine as an example of highly proarrhythmic agent to investigate the risk factors that may facilitate the proarrhythmic effects of drugs. We studied the influence of pacing, extracellular potassium, and pH on quinidines IKr blocking effect, all potential factors influencing quinidines cardiac toxicity. Since the HERG gene encodes IKr, we studied quinidines effect on HERG expressed in Xenopus oocytes by the 2-electrode voltage clamp technique. When extracellular K+ was 5 mmol/L, quinidine blocked the HERG current dose dependently, with an IC50 of 6.3 ± 0.2 μmol/L. The blockade was much more prominent at more positive membrane potentials. The inhibition of HERG by quinidine was not use dependent. There was no significant difference between block with or without pacing. When extracellular K+ was lowered to 2.5 mmol/L, the current inhibition by quinidine was enhanced, and IC50 decreased to 4.6 ± 0.5 μmol/L. At 10 mmol/L extracellular K+, there was less inhibition by quinidine and the IC50 was 11.2 ± 3.1 μmol/L. Extracellular acidification decreased both steady state and tail currents of HERG. We conclude that the inhibitory effect of quinidine on IKr was decreased with extracellular acidification, which may produce heterogeneity in the repolarization between normal and ischemic cardiac tissue. Thus, the use-independent blockade of IKr by QT-prolonging agents such as quinidine may contribute to cardiac toxicity with bradycardia, hypokalemia, and acidosis further exaggerating the proarrhythmic potential of these agents.

Extracellular Acidification and Hyperkalemia Induce Changes in HERG Inhibition by Ibutilide

Background: A high incidence of proarrhythmia has been reported with ibutilide, especially in patients with underlying heart diseases. Our previous studies have shown that extracellular acidosis and hyperkalemia attenuate the HERG-inhibitory effect of proarrhythmic drugs, e.g. quinidine, but have little impact on the less-proarrhythmic drug amiodarone. We hypothesized that ibutilide would behave like quinidine in the presence of extracellular acidosis and hyperkalemia. Methods and Results: HERG was expressed on Xenopus oocytes, and the two-electrode voltage clamp technique was employed. Our results showed that ibutilide was a potent HERG inhibitor. When extracellular solution contained 5 mM KCl and pH was 7.4, the IC50 of ibutilide was 0.9 ± 0.1 µM. The inhibitory effect of ibutilide was attenuated when extracellular pH decreased to 6.2. There was a significant difference in current inhibition by ibutilide at pH 7.4 versus pH 6.2 (p < 0.01). When the extracellular potassium concentration was increased from 5 to 10 mM, ibutilide produced less current inhibition, and the IC50 was increased to 2.0 ± 0.1 µM. Conclusion: Extracellular acidosis and hyperkalemia attenuate the HERG-inhibitory effect of ibutilide. The differences in HERG inhibition between acidic and hyperkalemic regions compared to normal regions in the myocardium may result in heterogeneity in repolarization, which may contribute to the proarrhythmic toxicity of ibutilide.

The Effect of High Extracellular Potassium on IKr Inhibition by Anti-Arrhythmic Agents

Background: Hyperkalemia is a potentially life-threatening disorder frequently occurring in hospitalized patients. The ischemic myocardium releases potassium into the extracellular space which can cause regional hyperkalemia. These changes may modify the effects of anti-arrhythmic drugs acting on the rapid component of the delayed rectifier potassium current (IKr). We evaluated the influence of increased extracellular potassium concentration [K+]e on IKr inhibition by amiodarone, azimilide, dofetilide, quinidine and sotalol. Methods and Results: Experiments were performed at room temperature. IKr current was studied by using HERG gene expressed in Xenopus oocytes as a model of cardiac IKr. Two-electrode voltage clamp technique was employed. The recording bath solutions contained either 5 or 10 mmol/l KCl. Amiodarone, azimilide, dofetilide, quinidine and sotalol all produced a dose-dependent inhibition of HERG current. At 5 mmol/l [K+]e, the IC50 was 37.0 ± 12.5 µM for amiodarone, 5.8 ± 0.4 µM for azimilide, 1.5 ± 0. 2 µM for dofetilide, 9.1 ± 1.5 µM for quinidine, and 5.1 ± 0.8 mM for sotalol. Raising the extracellular potassium to 10 mmol/l, HERG block by azimilide, dofetilide, quinidine and sotalol was significantly decreased, while the block by amiodarone was unchanged. The differences in the percentage current block produced by 3 µM drugs at 5 and 10 mmol/l [K+]e were: –0.9% for amiodarone, 13.8% for quinidine, 20.5% for azimilide, and 16.2% for dofetilide. The differences in percentage block between 5 and 10 mmol/l [K+]e by sotalol 10 and 30 mM were 7.1 and 5.6%. At 10 mmol/l [K+]e, the IC50 was increased for azimilide, dofetilide, quinidine and sotalol but not for amiodarone; the IC50 was 24.7 ± 7.4 µM for amiodarone, 29.3 ± 3.9 µM for azimilide, 2.7 ± 0.2 µM for dofetilide, 27.6 ± 4.0 µM for quinidine, and 7.2 ± 1.7 mM for sotalol. Conclusion: Inhibition of IKr by azimilide, quinidine, dofetilide and sotalol was diminished by increasing [K+]e, while the inhibition by amiodarone was unchanged at normal and high [K+]e. The differential effects of azimilide, dofetilide, quinidine and sotalol at normal and high [K+]e could be pro-arrhythmic by favoring re-entry arrhythmias. These results further support the unique electrophysiological effect of amiodarone.

Recent advances in prodrugs as drug delivery systems.

Prodrugs are a class of drug derivatives with little or no pharmacological activity that are converted in vivo to therapeutically active compounds. The primary utility of a prodrug approach is to improve pharmaceutical properties. Because it does not alter the primary structure of the parent drug, the synthesis of prodrugs is usually much less difficult than the synthesis of analogs. The derived physicochemical properties of the resulting derivatives can be carefully tailored by means of structural modification of the promoiety. However, sufficient levels of intrinsic activity of the parent drug need to be assured through in vivo cleavage of the prodrug. The prodrug approach has been successfully applied to a wide variety of drugs. This article briefly discusses advances in strategies for development of prodrugs and their mechanisms of drug release.

The differential antibacterial and gastrointestinal effects of erythromycin and its chiral isolates.

The use of erythromycin has been limited by the gastrointestinal side effect properties, which include abdominal distress and diarrhea. To evaluate the possibility of reducing the toxicity of erythromycin, studies were undertaken to separate erythromycin into chiral isolates and then to test the activity of these chiral isolates on gastrointestinal contractility and bacteriostatic actions. Gastrointestinal contractility was obtained by the use of isolated strips of a rat colon. Antibacterial activity was used by obtaining the MICs of erythromycin and isolated agents against Enterococcus faecalis ATCC 29212. ANOVA was performed using the SPSS v.10 to determine statistical differences in the MICs and the amplitude and frequency of spike bursts. Results were expressed as mean ± SE (N = 5). The MICs (μg/mL) of erythromycin (racemate), chiral isolate X, and chiral isolate Y were 0.45 ± 0.29, 0.53 ± 0.24 (n.s.), and 0.2 ± 0.07 (P ≤ 0.001), respectively. Erythromycin (racemate) at 10−8 mol/L, 10−7 mol/L, 5 × 10−7 mol/L, 10−6 mol/L, and 10−5 mol/L concentrations caused the amplitude of spike bursts to increase by 18 ± 7% (P = n.s.), 43 ± 10% (P ≤ 0.05), 55 ± 12% (P ≤ 0.001), 121 ± 23% (P ≤ 0.001), and 163 ± 16% (P ≤ 0.001), respectively. The chiral isolate Y increased the amplitude of spike bursts at the same concentrations as tested above: 32 ± 11% (P ≤ 0.05), 48 ± 14% (P ≤ 0.001), 84 ± 13% (P ≤ 0.001), 112 ± 18% (P ≤ 0.001), and 121 ± 13% (P ≤ 0.001), respectively. Chiral isolate X caused much reduced effect on the amplitude of spike bursts: 9 ± 6% (P = n.s.), 27 ± 12% (P = n.s.), 27 ± 12% (P = n.s.), 30 ± 11% (P = n.s.), and 30 ± 11.2% (P = n.s.), respectively. EC50 for erythromycin (mixture) was 0.4 × 10−6 mol/L, and for erythromycin Y, it was 0.8 × 10−6 mol/L. The addition of erythromycin at 10−8 mol/L caused the frequency of spike bursts to increase 11 ± 7% at 10−7 mol/L, 5 × 10−7 mol/L, 10−6 mol/L, and 10−5 mol/L; the changes were 13 ± 10% (P = n.s.), 13 ± 10% (P = n.s.), 22 ± 13% (P = ns), and 39 ± 30% (P ≤ 0.05), respectively. Chiral isolate Y of erythromycin, changed the frequency of spike bursts by 26 ± 21% (P = n.s.); 35 ± 20% (P = n.s.), 39 ± 30% (P = n.s.), 41 ± 37% (P = n.s.), and 44 ± 36% (P = n.s.) at the respective concentrations as discussed above. Chiral isolate X altered the frequency of spike bursts at the same concentrations as 40 ± 30% (P = n.s.), 45 ± 30% (P = n.s.), 62 ± 41% (P = n.s.), 62 ± 41% (P = n.s.), and 52 ± 35% (P = n.s.), respectively. Data indicate that erythromycin (racemate) and chiral isolates X and Y possess similar antibacterial activity. It was also shown that erythromycin and chiral isolate Y increase significantly the amplitude of spike bursts compared with baseline. Isolate X does not increase the amplitude of spike bursts in a dose-dependent manner. The frequency of spike bursts is not significantly changed in the presence of erythromycin or the 2 chiral isolates.

83 IS THE PH-INDUCED CHANGE IN IKR INHIBITION BY IBUTILIDE ARRHYTHMOGENIC?

Ibutilide (I), a class III antiarrhythmic agent, is employed in conversion of atrial fibrillation and atrial flutter. Ibutilide inhibits the cardiac IKr channel and prolongs the Q-T interval and can give rise to ventricular tachycardia. In previous studies, we have shown that extracellular acidosis significantly attenuates the IKr inhibitory effect of proarrhythmic drugs (quinidine) and that extracellular acidosis has little impact on the inhibitory effect of less proarrhythmic drugs such as amiodarone. We hypothesized that I would behave more like quinidine than amiodarone with extracellular acidosis. Cardiac IKr was studied using human-ether-a-go-go-related gene (HERG) expressed on Xenopus oocytes and two-electrode voltage clamp technique. The recording solution contained 96 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl2, and 5 mM HEPES. The pH of the solution was adjusted to 6.2 or 7.4 to represent the acidic or normal conditions. At pH 7.4, I 0.3, 1, 3, and 10 μM inhibited current by 22 ± 5, 54 ± 5, 80 ± 3, and 93 ± 1%, respectively. When I was applied at pH 6.2, I 0.3, 1, 3, and 10 μM, I decreased HERG current by 10 ± 4, 29 ± 4, 32 ± 8, and 36 ± 5%, respectively. I 30 μM produced only a 48 ± 5% current block at pH 6.2. There were significant differences in the percentage current inhibition by 1, 3, and 10 μM I at normal pH versus pH 6.2 (p values < .01). The IC50 of I was 0.9 ± 0.1 μM at pH 7.4 and the IC50 was increased to 31 ± 6 μM at pH 6.2. Our results indicated that I is a potent IKr inhibitor and that extracellular acidosis markedly attenuated IKr inhibition. Diminished IKr inhibition in the ischemic region with low extracellular pH and potent IKr inhibition in the normal pH regions could result in heterogeneity in action potential duration, which may trigger and sustain arrhythmias and contribute to the proarrhythmic toxicity of ibutilide.

82 INHIBITION OF THE HERG CHANNEL BY ASPIRIN: PH OR DIRECT EFFECT?

Aspirin (ASA) has been widely used for many years for relieving pain and fever and in preventing heart attack and stroke. ASA overdose can result in an anion gap, metabolic acidosis, tinnitus, and, in severe cases, encephalopathy and cardiovascular collapse. There are studies showing the inhibitory effect of ASA on heat-evoked currents in rat dorsal root ganglion neurons and the augmented effect of ASA on the NMDA type of glutamate responses in spiral ganglion neurons. The effect of ASA on cardiac ion channels has not been studied. We evaluated the effect of ASA on cardiac IKr channel using HERG expressed on Xenopus oocytes. A two-microelectrode voltage clamp technique was used for recording, and the recording solution contained 96 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, and 5 mM HEPES, and the pH of the solution was adjusted with NaOH to 7.4. ASA was dissolved in the recording solution. At a concentration less than 1 mM, ASA has little effect on HERG current. ASA 1 mM and 2 mM inhibited current by 12 ± 2 and 22 ± 4%, respectively. ASA 3 mM inhibited current by 80 ± 6%. Considering the acidifying influence of ASA, the pH values of 1, 2, and 3 mM ASA solutions were determined as 7.1, 6.5, and 4.9. The pH of the recording solutions was adjusted to the corresponding pH, and the results showed that pH 7.1, 6.5, and 4.9 reduced HERG current by 6 ± 2, 10 ± 2, and 49 ± 5%. ASA 1, 2, and 3 mM caused greater inhibition of current than the recording solutions with the corresponding pH adjustment. There were significant differences in current inhibition between 2 mM ASA and the pH 6.5 recording solution (p < .05) and between 3 mM ASA and pH 4.9 recording solution (p < .01). ASA inhibits HERG current not only through acidification but also by a direct effect. The potent inhibition at 3 mM (54 mg/dL) further suggests that ASA at therapeutic doses and at doses seen with acute and chronic salicylism (40-120 mg/dL) may be arrhythmogenic owing to potent inhibition of the cardiac IKr channel.