KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting.

Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.

High Throughput Measurement of Ca++ Dynamics in Human Stem Cell-Derived Cardiomyocytes by Kinetic Image Cytometery: A Cardiac Risk Assessment Characterization Using a Large Panel of Cardioactive and Inactive Compounds.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful in vitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.

Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology.

Cellular and ionic causes of variability in the electrophysiological activity of hearts from individuals of the same species are unknown. However, improved understanding of this variability is key to enable prediction of the response of specific hearts to disease and therapies. Limitations of current mathematical modeling and experimental techniques hamper our ability to provide insight into variability. Here, we describe a methodology to unravel the ionic determinants of intersubject variability exhibited in experimental recordings, based on the construction and calibration of populations of models. We illustrate the methodology through its application to rabbit Purkinje preparations, because of their importance in arrhythmias and safety pharmacology assessment. We consider a set of equations describing the biophysical processes underlying rabbit Purkinje electrophysiology, and we construct a population of over 10,000 models by randomly assigning specific parameter values corresponding to ionic current conductances and kinetics. We calibrate the model population by closely comparing simulation output and experimental recordings at three pacing frequencies. We show that 213 of the 10,000 candidate models are fully consistent with the experimental dataset. Ionic properties in the 213 models cover a wide range of values, including differences up to ±100% in several conductances. Partial correlation analysis shows that particular combinations of ionic properties determine the precise shape, amplitude, and rate dependence of specific action potentials. Finally, we demonstrate that the population of models calibrated using data obtained under physiological conditions quantitatively predicts the action potential duration prolongation caused by exposure to four concentrations of the potassium channel blocker dofetilide.

High throughput measurement of Ca²âº dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry.

Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca²âº indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca²âº](i) at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca²âº dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background.

Pharmacological dissection of K(v)7.1 channels in systemic and pulmonary arteries.

BACKGROUND AND PURPOSE: The aim of this study was to characterize the functional impact of KCNQ1-encoded voltage-dependent potassium channels (K(v)7.1) in the vasculature. EXPERIMENTAL APPROACH: Mesenteric arteries, intrapulmonary arteries and thoracic aortae were isolated from adult rats. K(v)7.1 channel expression was established by fluorescence immunocytochemistry. Wire myography determined functionality of these channels in response to selective blockers and activators. Xenopus oocytes expressing K(v)7.1 channels were used to assess the effectiveness of selective K(v)7.1 channel blockers. KEY RESULTS: K(v)7.1 channels were identified in arterial myocytes by immunocytochemistry. K(v)7.1 blockers HMR1556, L-768,673 (10 µM) and JNJ39490282 (JNJ282; 1 µM) had no contractile effects in arteries, whereas the pan-K(v)7 channel blocker linopirdine (10 µM) evoked robust contractions. Application of two compounds purported to activate K(v)7.1 channels, L-364 373 (R-L3) and mefenamic acid, relaxed mesenteric arteries preconstricted by methoxamine. These responses were reversed by HMR1556 or L-768,673 but not JNJ282. Similar effects were observed in the thoracic aorta and intrapulmonary arteries. CONCLUSIONS AND IMPLICATIONS: In contrast to previous assumptions, K(v)7.1 channels expressed in arterial myocytes are functional ion channels. Although these channels do not appear to contribute to resting vascular tone, K(v)7.1 activators were effective vasorelaxants.

ECG arrhythmias in non-implanted vs. telemetry-implanted dogs: need for screening before and sufficient recovery time after implantation.

INTRODUCTION: The purpose of this study was: (1) to characterize and assess the incidence of spontaneous arrhythmias in totally naive Beagle dogs (n=51; 10 males+41 females): (2) to study the effects of permanent ECG- and LVP-probe telemetry implants both acutely and up to 233days after surgery in a subset of 11 female Beagle dogs. METHODS: Naive ECG assessments were conducted by means of 6 external telemetry leads in jacketed dogs. Telemetry ECG recordings were captured by means of implanted telemetry devices suitable for ECG, LVP and aortic blood pressure recording. Experienced laboratory personnel visually evaluated all 22h ECG recordings at different time points after implantation and evaluated the incidence and type of arrhythmia. RESULTS: The 51 healthy and totally naive Beagle dogs showed a prevalence of: 49.0% 2°AVB; 58.8% single atrial premature complexes; 17.6% junctional tachycardia; 27.5% ventricular complexes; 13.7% ventricular escape complexes; 21.6% ventricular premature complexes; 3.9% runs of ventricular complexes; 3.9% runs of ventricular escape complexes. As such, a high percentage of clinically normal Beagle dogs showed different types of arrhythmias when ECG's obtained by external telemetry leads were fully evaluated for a 22h period. The chronic implantation of a ventricular probe through the apex of the heart in 11 dogs only, resulted in higher incidences and frequencies of ventricular episodes, which (in some dogs) extended up to 8weeks. Eight months after surgery none of the implanted dogs showed ventricular tachycardia and only 10% had single ventricular or ventricular premature complexes at low frequencies. DISCUSSION: A thorough evaluation of the ECG's of Beagle dogs selected for telemetry implantation can help to avoid inherent arrhythmia-burdened dogs being implanted and used in studies where these arrhythmias will confound drug assessment by increasing the number of potential false positives.

Beyond the safety assessment of drug-mediated changes in the QT interval... what's next?

Assessing drug-induced changes (particularly prolongation) in the QT interval has been the major preoccupation of safety pharmacology since its inception, under the assumption that QT widening represents a surrogate biomarker for torsades de pointes (TdeP) liability. While evidence of changes in QT remains a bane to the development of novel therapeutic agents, non-clinical and clinical methods have been developed (with a certain amount of validation) to limit this potential liability of a new chemical entity (NCE). Because of the associated withdrawal of numerous drugs from clinical use, determining whether or not a drug development candidate exhibits a TdeP liability has been the motivation in the implementation of discussions between 'pharmaceutical companies', academicians, clinicians and regulatory authorities worldwide that has led to the development of the ICHS7A and ICHS7B guidance documents (Anon, 2001, 2005). Simultaneously, it has resulted in the firm establishment of safety pharmacology as a standalone discipline within the drug development scheme (Pugsley et al., 2008). As far as TdeP liability is concerned, QT widening remains the most poignant issue, in that QT widening in humans is immediately regarded as a cause for concern, yet QT widening in preclinical models (and indeed in man) is not a quantitative predictor of TdeP liability (and indeed may not even be a qualitative predictor by itself (Pugsley et al., 2008). The present focused issue of the journal returns to safety pharmacology, and contains papers arising from the 8th annual SPS Meeting that was held in Madison, WI in 2008. Indeed, so many papers have arisen from the meeting that this issue of the Journal is only part 1. Part 2 will be published as the next issue of the Journal. Some topics which have been addressed include whether an assessment method for drugs that produce a shortened QT interval is needed, what the role of the slow component of the delayed rectifier K current (I(Ks)) should be in a safety assessment and whether safety pharmacology endpoints can or should be added to repeat dose Toxicology studies.

Blockade of the I(Ks) potassium channel: an overlooked cardiovascular liability in drug safety screening?

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I(Ks) channel, which along with the transient outward current (I(to)) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I(Ks) blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I(Ks) channel blockade and describe the discovery of a potent I(Ks) blocker whose activity was not detected by standard hERG or invitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) invivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I(Ks) blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I(Ks) blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I(Ks)) activity. Assay Drug Development Technology 5, 617-627]). Because of the presence of I(Ks) channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I(Ks) blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I(Ks) liability before testing in humans. The possibility of undetected I(Ks) blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427-1438] in the ICH S7B regulatory guidelines.

Methods in safety pharmacology in focus.

This focused issue of the Journal of Pharmacological and Toxicological Methods is the fifth to highlight Methods in Safety Pharmacology and includes a number of articles from the 7th Annual Safety Pharmacology Society (SPS) meeting that was held in Edinburgh, Scotland, September 19-21, 2007. However, unlike issues of the past, in which content predominantly focused on cardiovascular issues (specifically QT interval prolongation, QT-HR correction methods and validation of non-clinical cardiovascular models) this issue is composed of a number of non-cardiovascular methods papers and review articles. Of particular interest to readers will be articles related to CNS studies, in particular neurobehavioral assessments in non-human primates and the effects of drugs in juvenile and adult rats (an article that may be relevant in light of recent EU/US pediatric legislation). While cardiovascular function may not dominate there are several useful methodological papers including an assessment of cardiovascular sensitivity of drugs in conscious and anesthetized non-human primates, and a mathematical model (fractal analysis) applied to canine heartbeat dynamics. A first for the journal is a paper by Vargas et al., (2008-this issue) in which members of the SPS formed a working group in order to assess and review safety pharmacology testing of biological therapeutic agents (specifically monoclonal antibodies, mAbs). The group provides recommendations that will likely shape regulatory strategy and discussions in the yet to be fully discussed area of biological safety testing. In the tradition of obtaining a perspective on industry safety pharmacology program practices Lindgren et al., (2008-this issue) provide the results of a recent SPS survey that examines ICH S7A and S7B trends, aspects of early 'frontloading' safety studies, abuse and dependence liability and Contract Research Organization (CRO) tests/assays used in safety assessment of core battery and supplementary organ systems. In keeping with the translation track aspect of the 2007 meeting is an overview of the Distinguished Service Award lecture to Dr. T. Hammond that discusses many aspects of safety pharmacology including its evolution, impact, value and translation of non-clinical findings to humans. Finally, perspectives are presented on the use of the zebrafish as an early safety pharmacology-screening assay.

In vivo mechanisms precipitating torsades de pointes in a canine model of drug-induced long-QT1 syndrome.

OBJECTIVE: Congenital loss of function and drug-induced inhibition of the slowly-activating delayed-rectifier K(+) current (I(Ks)) cause impaired cardiac repolarization. beta-Adrenergic-receptor stimulation contributes to sympathetically-induced torsades de pointes (TdP). An in vivo model of long-QT1 (LQT1) syndrome and TdP in a species with I(Ks) characteristics relevant to man is lacking. We investigated the in vivo mechanisms of TdP in a novel canine model of drug-induced LQT1 syndrome. METHODS: Adult beagle dogs (n=30; F/M) were anesthetized with lofentanil (0.075 mg/kg i.v.) and etomidate (1.5 mg/kg/hour). ECGs, left- (LV) and right-ventricular (RV) monophasic action potentials (MAPs), and intracavitary pressures were recorded simultaneously. Infusion of the I(Ks) blocker HMR1556 (0.025-0.050 mg/kg/min) mimicked LQT1, and bolus injections of isoproterenol (1.25-5 microg/kg) reproducibly triggered TdP in 94% of dogs (defibrillated if necessary). RESULTS: Isoproterenol evoked paradoxical repolarization prolongation during heart rate accelerations. Beat-to-beat variability [QT, LV MAP duration (MAPD(90))] and spatial dispersion of repolarization (T(peak)-T(end) interval, endo-minus epicardial MAPD(90), LV-RVMAPD(90)) were significantly increased. Early afterdepolarizations occurred predominantly in the endocardium and not the epicardium. During isoproterenol, secondary systolic contractions (aftercontractions; peak 25+/-6 mm Hg) arose in the LV (not RV) when TdP ensued. Prevention of TdP by esmolol (1.25 mg/kg), verapamil (0.4 mg/kg) or mexiletine (5 mg/kg) was only successful when repolarization prolongation was contained and aftercontractions remained absent. CONCLUSIONS: beta-Adrenergic challenges trigger TdP in a reproducible manner in this model of drug-induced LQT1. Paradoxical prolongation and increased temporal and spatial dispersion of repolarization precipitate TdP. Incremental LV systolic aftercontractions precede TdP, suggesting abnormal cellular Ca(2+) handling contributes to the arrhythmogenic mechanism.

Signs in vector-electrocardiography (VECG) predicting the fibrillatory propensity of iodixanol and mannitol solutions after injection into the left coronary artery of pigs.

RATIONALE AND OBJECTIVES: To find signs in vector-electrocardiography (VECG) predicting the ventricular fibrillatory propensity (VF-PROP) of iodixanol and mannitol solutions after injection into the left coronary artery (LCA) of pigs. MATERIALS AND METHODS: Five plasma-isotonic solutions perfused LCA: Iod 320 + Na/Ca (iodixanol 320 mg I/mL, 19 mM NaCl, 0.3 mM CaCl(2)), Iod 320 + Mann (iodixanol 320 mg I/mL, 50 mM mannitol), Mann + Na/Ca (240 mM mannitol, 19 mM NaCl, 0.3 mM CaCl(2)), Mann (275 mM mannitol), and Ringer (representing "physiologic electrolytes"). The first two solutions have at 37 degrees C viscosity 13 mPas and the others <1 mPas. In eight pigs, 20 mL of each solution was injected twice for 10 seconds, and in 15 pigs, each solution was injected for 11-40 seconds (0.5 mL/second) through a wedged catheter in the LCA. If ventricular fibrillation (VF) occurred, injection was stopped and heart was defibrillated. If VF did not occur, perfusion period was 40 seconds. A higher frequency of VF and a shorter period from start of injection until start of VF gave a solution a higher ranking of VF-PROP. RESULTS: The 10-second injections caused no VF. Ringer and Iod 320 + Na/Ca caused no VF after 40-second injections, whereas the other solutions caused VF. Ranking the solutions from lowest to highest VF- PROP gave: Ringer = Iod 320 + Na/Ca < Iod 320 + Mann < Mann + Na/Ca < Mann. Prolongation of QRS time and QTc time were the only VECG signs that showed significant differences (P < .05) between all solutions and correctly ranked the VF-PROP of all solutions in both animal groups. CONCLUSION: The results fit with the concept that a more physiologic electrolyte composition and a higher viscosity of a test solution will, after start of injection of that solution into LCA, delay changes in the electrolyte composition in myocardial interstitial fluid and also delay start of VF. If a plasma isotonic contrast medium (CM) with lower viscosity than that of iodixanol at 320 mgI/mL were created, we conclude that such a CM should have electrolyte composition closer to that of Ringer than present composition (19 mM NaCl and 0.3 mM CaC1(2)) to counteract the effects of faster diffusion of nonphysiologic electrolyte composition from the low-viscosity CM to myocardial interstitial fluid.