Electrophysiologic characterization of a novel hERG channel activator.

Activators of the human ether-a-go-go-related gene (hERG) K(+) channel have been reported recently to enhance hERG current amplitude (five synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound A-935142 ({4-[4-(5-trifluoromethyl-1H-pyrazol-3-yl)-phenyl]-cyclohexyl}-acetic acid) on guinea-pig atrial and canine ventricular action potentials (microelectrode techniques) and hERG channels expressed in HEK-293 cells (whole-cell patch clamp techniques). A-935142 shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current-voltage relationship revealed that this compound (60 microM) increased both outward and inward K(+) current as well as the slope conductance of the linear portion of the fully activated I-V relation. A-935142 significantly reduced the time constants (tau) of hERG channel activation at two example voltages (-10 mV: tau=100+/-17 ms vs. 164+/-24 ms, n=6, P<0.01; +30 mV: tau=16.7+/-1.8 ms vs. 18.9+/-1.8 ms, n=5, P<0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9 mV. The time course of hERG channel deactivation was slowed at multiple potentials (-120 to -70 mV). A-935142 also reduced the rate of inactivation and shifted the voltage-dependence of inactivation in the depolarizing direction by 15 mV. Recovery of hERG channel from inactivation was not affected by A-935142. In conclusion, A-935142 enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.

Hydroxypropyl beta-cyclodextrins: a misleading vehicle for the in vitro hERG current assay.

Delayed cardiac repolarization and fatal proarrhythmia have been linked to block of the repolarizing current, Ikr or hERG (human ether-a-go-go related gene) current. Thus, determining the potency of hERG block is critical in evaluating cardiac safety during preclinical development. Hydroxypropyl beta-cyclodextrins (HbetaC) are cyclic oligosaccharides used to enhance drug solubility. To evaluate the utility of HbetaC to enhance drug solubility in hERG screening assays, we studied the effect of HbetaC on hERG current and the sensitivity of the hERG assay to 3 structurally different hERG blocking drugs using whole-cell voltage clamp technique and HEK-293 cells expressing the hERG channel. HbetaC inhibited hERG activation and tail current and accelerated current deactivation in a concentration-dependent manner. HbetaC (6%) reduced the apparent potency of block by terfenadine (IC50 12000 nM vs 45 nM), cisapride (IC50 281 nM vs 28 nM), and E-4031 (163 nM vs 26 nM). Reduced potency of block was consistent with loss of activity as a result of complexation with HbetaC by terfenadine and cisapride (demonstrated in solubility studies) and interactions with HbetaC by E-4031 (demonstrated in absorbance studies). These results demonstrate that HbetaC is an unsuitable agent for enhancing compound solubility in the in vitro hERG current assay and may mask drug effects, allowing potentially dangerous drugs to advance into clinical development.

Identification of diamino chromone-2-carboxamides as MCHr1 antagonists with minimal hERG channel activity.

A series of potent 2-carboxychromone-based melanin-concentrating hormone receptor 1 (MCHr1) antagonists were synthesized and evaluated for hERG (human Ether-a-go-go Related Gene) channel affinity and functional blockade. Basic dialkylamine-terminated analogs were found to weakly bind the hERG channel and provided marked improvement in a functional patch-clamp assay versus previously reported antagonists of the series.

The effects of plasma proteins on delayed repolarization in vitro with cisapride, risperidone, and D, L-sotalol.

INTRODUCTION: Drug-induced long QT syndrome (LQTS) has been linked to arrhythmias (including Torsades de Pointes and sudden cardiac death), and has led to an increased awareness of the potential risk of delayed repolarization in vitro and in vivo. However, in vitro assessments of delayed repolarization have not been fully predictive of in vivo effects. METHODS: To define the extent to which plasma protein binding (ppb) contributes to such disparities in repolarization studies, we compared drug-induced prolongation of the canine Purkinje fiber action potential duration (APD(90)) in vitro during superfusion with 100% Tyrode's solution (Tyrodes), canine plasma [50% plasma/50% Tyrodes] and a 5% solution of recombinant human serum albumin in Tyrodes (HSA). Drugs evaluated included cisapride (>98% ppb), risperidone (90% ppb), and d, l-sotalol (negligible ppb). Effects on APD were monitored using standard microelectrode techniques under physiologic conditions and temperature ([K(+)]=4 mM, 37 degrees C) during slow stimulation (2 s basic cycle length). RESULTS: The effects of cisapride and risperidone on Purkinje fiber APD(90) were significantly attenuated in the presence of plasma proteins. However, with cisapride, the extent of reduction with plasma proteins was significantly less than predicted based on calculated free drug levels. DISCUSSION: We conclude that while plasma protein binding does reduce APD prolongation seen with bound drugs, this effect is not well correlated with the calculated plasma protein binding or expected clinical free fraction. Because of the complex drug interactions that occur in plasma, the electrophysiological effects seen with bound drugs are not well correlated with the calculated free fraction and thus caution should be exercised when assigning a predictive safety window. Thus, the canine Purkinje fiber assay is useful for defining the modulation of delayed repolarization due to plasma protein binding of novel therapeutic agents.

Optimization of chromone-2-carboxamide melanin concentrating hormone receptor 1 antagonists: assessment of potency, efficacy, and cardiovascular safety.

Evaluation of multiple structurally distinct series of melanin concentrating hormone receptor 1 antagonists in an anesthetized rat cardiovascualar assay led to the identification of a chromone-2-carboxamide series as having excellent safety against the chosen cardiovascular endpoints at high drug concentrations in the plasma and brain. Optimization of this series led to considerable improvements in affinity, functional potency, and pharmacokinetic profile. This led to the identification of a 7-fluorochromone-2-carboxamide (22) that was orally efficacious in a diet-induced obese mouse model, retained a favorable cardiovascular profile in rat, and demonstrated dramatic improvement in effects on mean arterial pressure in our dog cardiovascular model compared to other series reported by our group. However, this analogue also led to prolongation of the QT interval in the dog that was linked to affinity for hERG channel and unexpectedly potent functional blockade of this ion channel.