Predicting QT prolongation in humans during early drug development using hERG inhibition and an anaesthetized guinea-pig model.

BACKGROUND AND PURPOSE: Drug-induced prolongation of the QT interval can lead to torsade de pointes, a life-threatening ventricular arrhythmia. Finding appropriate assays from among the plethora of options available to predict reliably this serious adverse effect in humans remains a challenging issue for the discovery and development of drugs. The purpose of the present study was to develop and verify a reliable and relatively simple approach for assessing, during preclinical development, the propensity of drugs to prolong the QT interval in humans. EXPERIMENTAL APPROACH: Sixteen marketed drugs from various pharmacological classes with a known incidence -- or lack thereof -- of QT prolongation in humans were examined in hERG (human ether a-go-go-related gene) patch-clamp assay and an anaesthetized guinea-pig assay for QT prolongation using specific protocols. Drug concentrations in perfusates from hERG assays and plasma samples from guinea-pigs were determined using liquid chromatography-mass spectrometry. KEY RESULTS: Various pharmacological agents that inhibit hERG currents prolong the QT interval in anaesthetized guinea-pigs in a manner similar to that seen in humans and at comparable drug exposures. Several compounds not associated with QT prolongation in humans failed to prolong the QT interval in this model. CONCLUSIONS AND IMPLICATIONS: Analysis of hERG inhibitory potency in conjunction with drug exposures and QT interval measurements in anaesthetized guinea-pigs can reliably predict, during preclinical drug development, the risk of human QT prolongation. A strategy is proposed for mitigating the risk of QT prolongation of new chemical entities during early lead optimization.

Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

Interaction between neurotransmitters and exogenous norepinephrine in isolated rat anococcygeus muscle.

Continuous electrical field stimulation (EFS) elicited a sustained contraction and significantly increased the EC50 value of norepinephrine (NE), shifting the concentration-response curve for NE to the right. Tetrodotoxin significantly reduced the continuous EFS-evoked increases in basal tone and produced further dextral shift in the concentration-response curve for NE. N-methylhydroxylamine and NG-monomethyl-L-arginine (L-NMMA) attenuated the "dual" effects of continuous EFS on NE-induced contractions. L-Arginine partially reversed the inhibitory effect of L-NMMA. The results of the present study suggest that continuous EFS causes simultaneous release of both excitatory and inhibitory neurotransmitters, and the interaction (or functional antagonism) between the inhibitory neurotransmitter (endogenous nitric oxide, NO) and the excitatory neurotransmitter (endogenous NE), as well as exogenous NE, may occur at postjunctional site(s).

Depletion of intracellular Ca2+ stores activates a maitotoxin-sensitive nonselective cationic current in beta-cells.

Glucose stimulation of beta-cell insulin secretion is initiated by membrane depolarization coupled with an elevation in intracellular Ca2+ concentration ([Ca2+]i). Both depolarization-dependent Ca2+ entry and intracellular Ca2+ store release contribute to the sugar-induced rise in [Ca2+]i. Here we show that maneuvers depleting intracellular Ca2+ stores induce membrane depolarization and a sustained nitrendipine-sensitive Ca2+ influx, whereas interventions promoting Ca2+ store refilling produce a hyperpolarization and inhibit Ca2+ influx. Both intracellular Ca2+ store depletion and maitotoxin activated a depolarizing nonselective cation current carried principally by Na+ in the physiological range of membrane potentials. The activation of such a current may form the paradigm by which excitable cells refill depleted intracellular Ca2+ stores by depolarization-driven opening of voltage-activated Ca2+ channels.

Endoplasmic reticulum calcium store regulates membrane potential in mouse islet beta-cells.

Glucose stimulation of islet beta-cell insulin secretion is initiated by membrane depolarization and an elevation in intracellular free calcium concentration ([Ca2+]i) from a combination of influx through depolarization-activated Ca2+ channels and intracellular Ca2+ store release. Prevention of Ca2+ store refilling with thapsigargin produced a sustained depolarization, leading to enhanced Ca2+ influx and an elevation in [Ca2+]i in 12 mM glucose. Depletion of intracellular Ca2+ stores by external EGTA reduced [Ca2+]i and also caused a long-lasting depolarization. In single beta-cells, external EGTA activated an inward current, the voltage range and kinetic properties of which differed from those of voltage-dependent Ca2+ channels. A novel pathway thus exists in beta-cells by which depletion of endoplasmic reticulum Ca2+ stores results in the activation of an inward current that, by inducing depolarization, facilitates Ca2+ influx through voltage-gated Ca2+ channels. The physiological relevance of this pathway in the control of beta-cell function is indicated by the stimulation of insulin secretion by thapsigargin.

Dependence on NADH produced during glycolysis for beta-cell glucose signaling.

An increase in cytosolic ATP following glucose metabolism by pancreatic beta-cells is the key signal initiating insulin secretion by causing blockade of ATP-dependent K+ channels (KATP). This induces membrane depolarization, leading to an elevation in cytosolic Ca2+ ([Ca2+]i) and insulin secretion. In this report we identify the critical metabolic step by which glucose initiates changes in beta-cell KATP channel activity, membrane potential, and [Ca2+]i. The signal stems from the glycolytic production of NADH during the oxidation of glyceraldehyde 3-phosphate, which is subsequently processed into ATP by mitochondria via the operation of discrete shuttle systems.