The anti-malarial drug halofantrine and its metabolite N-desbutylhalofantrine block HERG potassium channels

OBJECTIVE: The antimalarial drug halofantrine has been associated with QT interval prolongation and with fatal and nonfatal arrhythmias in patients without known underlying cardiac abnormalities. A common target for QT interval-prolonging drugs is the human ether-a-go-go gene (HERG) which encodes the pore forming subunit of the rapidly activating delayed rectifier K(+) current (I(Kr)). METHODS: We studied the effects of halofantrine (0.1-1000 nM) and its major metabolite N-desbutylhalofantrine (3-1000 nM) on wild type HERG K(+) channels stably expressed in HEK 293 cells, using the whole cell patch-clamp recording technique. RESULTS: Halofantrine and N-desbutylhalofantrine blocked HERG K(+) channels in a concentration-dependent manner with a half-maximal inhibitory concentration of 21.6 nM (n=31 cells) and 71.7 nM (n=18 cells), respectively. The development of drug block for both halofantrine and N-desbutylhalofantrine required channel activation indicative of open and/or inactivated state block. Drug washout or cell hyperpolarization resulted in minimal current recovery consistent with virtually irreversible binding. Using a ventricular action potential voltage clamp protocol, halofantrine and N-desbutylhalofantrine block of HERG current was greatest during phases 2 and 3 of the action potential waveform. CONCLUSION: We conclude that both halofantrine and N-desbutylhalofantrine cause high affinity block of HERG K(+) channels. Although N-desbutylhalofantrine has been suggested to be a safer antimalarial agent compared to halofantrine, our results suggest that the gain in the safety margin for QT interval prolongation-related cardiotoxicity is minimal.     

Inhibition of HERG potassium channels by cocaethylene: a metabolite of cocaine and ethanol

OBJECTIVE: To investigate the mechanism by which cocaethylene, a metabolite of cocaine and alcohol inhibits a cardiac delayed rectifier potassium channel. METHODS: The cDNA of the HERG potassium channel that underlies I(Kr) in humans was transiently expressed in tsA201 cells and currents recorded using the patch clamp technique. RESULTS: The cocaethylene inhibition of HERG is concentration-dependent with an IC(50) of 4.0 microM. The inhibition increases over the range of voltages where the channels activate suggesting that cocaethylene binding may be linked to the activation or opening of the channels. Cocaethylene slows the deactivation of the tail current indicating that drug-modified channels are stabilized in the open conformation. Cocaethylene also accelerates inactivation but has no effect of the recovery from inactivation. CONCLUSIONS: Cocaethylene inhibits HERG by binding to the activated or open channels and by modulating the kinetics of inactivation. The cocaethylene inhibition of the channels occurs within the range of concentrations detected in the plasma of humans following the ingestion of cocaine and alcohol and is likely to contribute to the potent cardiotoxicity of this drug combination.     

Concentration-effect relations of 5-hydroxypropafenone in normal subjects

To evaluate the pharmacologic activity of 5-hydroxypropafenone, electrocardiographic changes (PQ and QRS duration) and blood pressure levels were measured in 6 healthy extensive metabolizers of debrisoquine after a single oral dose of 300 mg of this metabolite as a solution in a placebo-controlled, double-blind crossover study. Well-absorbed, with a lag time of 4.4 to 9.8 minutes, 5-hydroxypropafenone reached peak concentrations of 153 to 337 ng/ml after 20 to 51 minutes. The terminal half-life was 506 to 963 minutes. To describe the temporal aspects of the concentration-effect relation, a pharmacokinetic-pharmacodynamic model with a hypothetical effect compartment was applied. The relation between electrocardiographic changes and drug concentration at the effect site could be described by a linear regression model. Significant prolongations of PQ and QRS duration were found in 5 of 6 subjects. There were no changes in QTc interval, blood pressure measurements and heart rate in the supine position. However, blood pressure measurements in the upright position revealed a greater percent decrease of systolic blood pressure than with placebo (mean +/- standard deviation -25.6 +/- 13.8% vs -3.4 +/- 13.1%, p less than 0.05). It is concluded that 5-hydroxypropafenone exerts significant pharmacologic activity in humans as well as animals. Because QRS prolongation in patients treated with class IC antiarrhythmic drugs correlates with the antiarrhythmic effect, our data suggest that 5-hydroxypropafenone may contribute to the therapeutic activity of propafenone in humans.     

Effect of sulphasalazine and its active metabolite, 5-amino-salicylic acid, on toxic oxygen metabolite production by neutrophils.

The possibility that the mode of action of sulphasalazine and its active metabolite 5-amino-salicylic acid (5ASA) involves modification of toxic oxygen metabolite production by neutrophils has been investigated by measuring the effect of these drugs on luminol-dependent chemiluminescence, superoxide release and oxygen consumption by stimulated neutrophils in vitro. 5ASA, and to a lesser extent sulphasalazine, had profound inhibitory effects on the luminol dependent chemiluminescent response of neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (1 microM) + cytochalasin B (5 micrograms/ml). A concentration of 50 microM 5ASA or sulphasalazine produced 93.8 (2.3)% and 65.7 (3.7)% inhibition of control responses respectively. The concentration of 5ASA and sulphasalazine producing 50% inhibition of chemiluminescence were 3.6 (1.8) microM and 16.5 (6) microM respectively. Both drugs had little effect on the chemiluminescent response of neutrophils stimulated with phorbol myristate acetate (1 microgram/ml), producing only 11.4 (3.9)% and 34 (7)% inhibition respectively, at a concentration of 50 microM. Superoxide release from fMLP + CB stimulated neutrophils was also inhibited slightly by 5ASA (50 microM) by 35.6% and by sulphasalazine (50 microM) by 7.9%. Similarly, there was little inhibition in the rate of oxygen consumption by fMLP + CB stimulated neutrophils by either 5ASA or sulphasalazine at concentrations which produced near total abolition of luminol dependent chemiluminescence. These results show that sulphasalazine and 5ASA inhibit the reaction of toxic metabolites produced by stimulated neutrophils with luminol, without inhibition of the oxidase system producing these metabolites. The site of action of these drugs on neutrophils in vitro is thus extracellular, by scavenging a released metabolite, probably hypochlorite. This has important implications for their mode of action in vivo in inflammatory bowel disease.     

Blockade of HERG channels by HIV protease inhibitors

The HIV protease inhibitor class of antiretroviral drug causes unpredicted adverse effects by changing elements of normal cellular metabolism. A case of QT prolongation in a patient receiving protease inhibitors made us question whether these drugs might be responsible. We identified 24 patients with QT prolongation or torsade de pointes, or both, associated with protease inhibitors, using the Food and Drug Administration's voluntary adverse event reporting system. Attending physicians thought that protease inhibitors were the most probable cause of these symptoms in 14 of the patients. Drug-induced QT prolongation is usually caused by block of human ether-a-go-go-related gene (HERG) potassium channels, and we showed that lopinavir, nelfinavir, ritonavir, and saquinavir caused dose-dependent block of HERG channels heterologously expressed in HEK293 cells in vitro. We also recorded block by lopinavir of repolarising potassium current (I(Kr)) channels in neonatal mouse cardiac myocytes. Our data show that four protease inhibitors block HERG channels, suggesting that protease inhibitors could predispose individuals to QT prolongation and torsade de pointes.     

Effects of mexiletine, propafenone and flecainide on signal-averaged electrocardiogram.

The effects of mexiletine, propafenone and flecainide on the parameters of signal-averaged electrocardiogram in 40 subjects with symptomatic and repetitive ventricular arrhythmias were studied. Mexiletine (n = 16) suppressed ventricular arrhythmias in 10 patients and did not produce any significant changes in filtered QRS duration (fQRS), root mean square voltage of the final 40 ms of filtered QRS (RMS40) or low amplitude terminal component duration (LAS40). Acute (n = 8, 450 mg) and chronic (n = 16, 600-1200 mg.day-1) administration of propafenone determined a significant increase in fQRS (from 123 +/- 2.2 to 139 +/- 3 ms) and a reduction in RMS40 (from 54 +/- 8.8 to 34 +/- 6.7 microV); as a consequence the incidence of ventricular late potentials rose from 43 to 62%. The observed effects were independent of anti-arrhythmic efficacy, which was 86% for this drug. Acute (n = 8, 200 mg) and chronic (n = 13, 200-300 mg.day-1) administration of flecainide was associated with a marked prolongation in fQRS (from 123 +/- 2.8 to 138 +/- 4.1 ms) and a reduction in RMS40 (from 69 +/- 11.5 to 47 +/- 11 microV); thus determining an increase in the incidence of ventricular late potentials from 29 to 48%. Changes in signal-averaged electrocardiogram were not related to drug efficacy, which was 81%. These data indicate that 1c anti-arrhythmic drugs consistently modified the parameters of signal-averaged electrocardiogram; the observed changes might reflect an inhomogeneous slowing of intramyocardial impulse propagation.     

普罗帕酮对HERG钾通道孔胞膜外侧氨基酸突变前后的作用

目的:探讨普罗帕酮对人类ether-a-go-go相关基因(HERG)钾通道孔道胞膜外侧突变后结合能力的影响。方法:将HERG野生型通道(WT)和HERG突变型通道(MT)的互补核糖核酸(cRNA)注射到非洲爪蟾卵母细胞,孵育24～72h后以不同刺激程序用双微电极法记录通道电流的表达。用Clampfit9.2版本软件对数据进行分析。部分电流用相应的方程拟合。结果:HERGWT外侧的第628位点的甘氨酸突变为半胱氨酸(G628C)和第631位点的丝氨酸突变为半胱氨酸(S631C)成HERGMT后普罗帕酮与HERGMT通道的结合能力减小,50%抑制浓度(IC50)对HERGWT,HERGMT分别为5.31μmol/L和7.81μmol/L。普罗帕酮对HERGWT和HERGMT的阻滞效应都呈电压和浓度依赖性。普罗帕酮减小HERGWT,但未减少HERGMT通道的半数激活电压。结论:普罗帕酮是HERG通道的开放通道阻滞剂,HERG通道孔道膜外侧突变(G628C和S631C)能改变普罗帕酮与通道之间的结合能力,从而影响通道的激活。     

Effects of propafenone on coronary and systemic hemodynamics

This study was undertaken to evaluate the effects of intravenous Propafenone (2 mg/kg over 5') on Left Ventricular (LV) function and coronary blood flow. Twelve patients with coronary artery disease and post-ischemic LV disfunction were examined during routine cardiac catheterization. Serial measurements of central hemodynamics, LV high-fidelity pressure and coronary blood flow were recorded at rest and every 10' after Propafenone administration. Heart rate was unchanged, suggesting that Propafenone did not affect sympathetic tone. Cardiac index slightly decreased (from 3.3 +/- 0.9 L/min/m2 to 3.1 +/- 0.6 L/min/m2 at 10', p = ns), LV end-diastolic pressure rose significantly (from 17.7 +/- 2.1 mmHg to 22.7 +/- 4.2 mmHg at 20', p less than 0.01) and dP/dt max fell from 1897 +/- 291 mmHg/sec to 1577 +/- 312 mmHg/sec (p less than 0.02). Systemic vascular resistances had only minimal changes. Concomitantly, coronary vascular resistances decreased (from 0.77 +/- 0.17 mmHg/ml/min to 0.61 +/- 0.12 mmHg/ml/min, p less than 0.02) and coronary blood flow increased (from 138 +/- 29 ml/min to 172 +/- 21 ml/min, p less than 0.01). No significant difference was noted in myocardial oxygen consumption. No symptoms related to LV failure were observed during the study. In conclusion hemodynamic effects of Propafenone are characterized by moderate LV depression and by coronary artery dilatation, probably due to a calcium blocker-like activity.     

普罗帕酮和Ⅲ类新药Ambasilide对犬心房颤动的作用和电生理机制比较研究

目的应用心外膜标测技术观察Ⅲ类新药Ambasilide对迷走性持续心房颤动(房颤)的影响,并与普罗帕酮比较,研究其电生理作用机制。方法建立迷走神经性持续房颤模型,13只犬给予普罗帕酮(2mg/kg),另13只犬给予Ambasilide(2mg/kg),观察两药对房颤的终止情况,同时在用药前后进行心房电活动实时心外膜标测及心房有效不应期的测量。结果普罗帕酮2mg/kg负荷量可转复房颤11/13(84.6％),Ambasilide2mg/kg负荷量可转复13/13(100％)。当S1S1为400ms时普罗帕酮可使心房有效不应期从(149±6)ms增至(181±5)ms,增加率为23％±4％,而S1S1为200ms时,心房有效不应期从(120±6)ms增至(192±4)ms,增加率达66％±8％,心房有效不应期增加为正向频率依赖性(P＜0.01)。当S1S1为400ms时Ambasilide可使心房有效不应期从(164±3)ms增至(214±7)ms,增加率为31％±4％,而S1S1为200ms时,心房有效不应期从(126±5)ms增至(156±5)ms,增加率25％±6％,心房有效不应期增加为非频率依赖性(P＞0.05)。普罗帕酮在S1S1为200ms时可减慢心房传导速度达34％±4％,而Ambasilide对心房传导速度无影响。两药均可延长折返波长。结论(1)普罗帕酮和Ambasilide通过增加心房不应期和折返波长而终止迷走性持续房颤;(2)普罗帕酮延长心房不应期呈正向频率依赖性而Ambasilide呈非频率依赖性延长;(3)普罗帕酮减慢心房传导速度而Ambasilide对心房传导速度无影响。     

阿司咪唑阻断HERG通道的生物学特性及分子机制

目的 观察阿司咪唑对野生型和Y652突变型HERG通道阻断的生物物理学特性,探讨HERG通道分子位点改变对阻断的影响.方法 将HERG通道表达于非洲爪蟾卵母细胞,利用双电极电压钳技术测量其电流,观察阿司咪唑不同浓度、不同电压、不同作用时间下,对野生型和Y652A、Y652R突变型HERG通道电流的阻断作用.结果 阿司咪唑以电压、浓度、时间依赖性阻断HERG通道电流;与野生型比较,Y652A和Y652R突变型可显著减弱阿司咪唑对HERG通道的阻断作用.结论 阿司咪唑优先阻断开放状态的HERG通道,Y652是阿司咪唑与通道结合的关键位点,其极性和侧链长度改变可影响阿司咪唑与通道结合.     

致LQT2的无义突变的HERG通道的表达和药物拯救研究

目的本研究旨在探讨氨基糖苷类抗生素庆大霉素在异源表达系统中对导致长QT综合征2型的无义突变的HERG通道的作用。方法采用聚合酶链反应法制备相关突变体——W927X、R863X和E698X,并克隆到真核细胞表达载体中。将突变体的cDNA瞬时转染至HEK293细胞,应用全细胞膜片钳技术记录通道电流。药物拯救采用将转染24h后的HEK293细胞在含庆大霉素(400μg/mL)的培养液中孵育24h。结果W927X能表达典型的HERG电流,尽管与野生型HERG相比,电流幅值明显下降;R863X和E698X仅表达与未转染细胞上类似的内生性电流,说明未能形成功能性的HERG通道。庆大霉素能增强W927X的功能性表达,使其最大尾电流密度由11.6±2.4pA/pF(n=8)增至19.5±2.7pA/pF(n=7,P<0.05)。通道激活动力学特征在野生型HERG、W927X和W927X加庆大霉素干预组均没有明显差异。然而,庆大霉素对R863X和E698X并无明显作用。结论氨基糖苷类抗生素能部分恢复无义突变的HERG通道的功能性表达,且对不同的突变体其作用效应不同。     

Effects of L-leucine, its 2-keto acid metabolite and its non-metabolized analogue on rat tumoral islet cell function

L-Leucine and 2-ketoisocaproate stimulated insulin release from perifused rat tumoral islet cells (RINm5F line). The secretory response coincided with an increase in the intracellular ATP/ADP ratio, a stimulation of 45Ca outflow from cells perifused in the presence of extracellular Ca2+, and an increase in 32P efflux from cells prelabelled with radioactive orthophosphate. In contrast to D-glucose, however, L-leucine or 2-ketoisocaproate failed to decrease 86Rb outflow, to inhibit 45Ca outflow from cells perifused in the absence of Ca2+ and to enhance the labelling of inositol-containing phospholipids in cells exposed to myo-[2-3H]inositol. These findings suggest that D-glucose, L-leucine and 2-ketoisocaproate exert dissimilar effects on the subcellular distribution of adenine nucleotides and/or 86Rb. The non-metabolized analogue of L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also caused an initial stimulation of insulin release and 32P efflux, but this was soon followed by a severe and irreversible inhibition of insulin output, associated with a permanent enhancement of 86Rb outflow. The dual ionic and secretory response to BCH is interpreted in the light of its dual effect on the catabolism of endogenous amino and fatty acids, and raises the view that BCH could be used to interfere with the function of insulinoma cells.     

乌头碱阻断表达在卵母细胞上的HERG通道的电药理特性

目的观察乌头碱对表达在卵母细胞上的HERG电流的影响。方法HERG通道表达在非洲爪蟾卵母细胞上,利用双电极电压钳技术测量其电流。结果①HERG通道可以稳定表达在卵母细胞上;②乌头碱以浓度和电压依赖性方式阻断表达在卵母细胞上的野生型HERG通道,阻断的半数抑制浓度值是1.801±0.332μmol/L;③乌头碱对HERG电流的阻断呈时间依赖性;④峰电流幅值被1μmol/L乌头碱显著降低,而稳态失活的半数失活电压(-39.10±1.04 mV vs-41.61±2.66 mV,P>0.05,n=6)没有被乌头碱的阻断显著改变,而斜率k(32.37±1.04 mV vs 41.05±4.19 mV,P<0.05,n=6)出现正向移动。结论乌头碱对HERG电流呈浓度、电压和时间依赖性阻断,而其对失活状态下的HERG通道无明显阻断作用,HERG通道可能是乌头碱致心律失常的关键离子靶点之一。     

胺碘酮、普罗帕酮对QTc离散度的影响比较及其临床意义

目的比较胺碘酮（２７例）和普罗帕酮（３４例）分别治疗室性心律失常前后ＱＴｃ离散度（ＱＴｃｄ）的变化及对复发的影响。方法采用随机、单盲的方法，测定两组病人用药前及用药２周后的ＱＴｃｄ。结果（１）胺碘酮组用药后ＱＴｃ明显延长，但ＱＴｃｄ反而降低（４４．３±１４．９ｍｓ与３３．９±１６．１ｍｓ，Ｐ＜０．０５）；普罗帕酮组用药前后ＱＴｃ及ＱＴｃｄ无显著变化（Ｐ均＞０．０５）。（２）胺碘酮组和普罗帕酮组的有效率分别为８５．２％和８２．４％（Ｐ＞０．０５）；停药后１月内的室性心律失常复发率分别为１８．５％和５０％（Ｐ＜０．０５），普罗帕酮组复发者较未复发者的ＱＴｃｄ明显延长（４８．７±１５．３ｍｓ与３９．８±１２．５ｍｓ，Ｐ＜０．０１）。结论ＱＴｃｄ的降低可能是胺碘酮治疗室性心律失常复发率较低的重要因素