Utility of Normalized TdP Score System in Drug Proarrhythmic Potential Assessment: A Blinded in vitro Study of CiPA Drugs.

Drugs that prolong QT may cause torsade de pointes (TdP). However, translation of nonclinical assessment of QT prolongation or hERG channel, targeted by QT-prolonging drugs, into clinical TdP risk has been insufficient to date. In this blinded study, we confirmed the utility of a Normalized TdP Score System in predicting drug-induced TdP risks among 34 drugs, including 28 with low, intermediate, and high TdP risks under the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative plus six compounds with names blinded to the investigators, using the rabbit ventricular wedge assay. Concentration-dependent TdP scores were determined by drug-induced changes in QT, Tp-e , and proarrhythmias. Disclosure of the names and testing concentrations was made after completion of the experiments and report to the sponsors. Drugs' normalized TdP scores were calculated thereafter based on their respective free clinical maximum concentration (Cmax ). Drugs' normalized TdP scores were calculated and ranked for 33 drugs, excluding 1 investigational drug, and the TdP risks of the 28 CiPA drugs were correctly distinguished according to their respective categories of low, intermediate, and high TdP risks under the CiPA initiative. Accordingly, we are able to propose the cutoff values of the normalized TdP scores at 1 × Cmax : ≤ 0, > 0 to < 0.65 and ≥ 0.65, respectively, for low, intermediate, and high risk. This blinded study supports utility of our Normalized TdP Score System in predicting drug-induced TdP risks in 33 drugs, including 28 used for characterization of other assays under the CiPA initiative. However, these results need to be replicated in other laboratories.

Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay.

INTRODUCTION: Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model. METHODS: We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/µCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform. RESULTS: We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline). DISCUSSION: In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND AND PURPOSE: In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. EXPERIMENTAL APPROACH: Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. KEY RESULTS: Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. CONCLUSION AND IMPLICATIONS: We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. LINKED ARTICLES: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies.

INTRODUCTION: With the recent development of more sensitive biomarkers to assess kidney injury preclinically, a survey was designed i) to investigate what strategies are used to investigate renal toxicity in both ICH S7A compliant Safety Pharmacology (SP) studies after a single dose of a compound and within repeat-dose toxicity studies by large pharmaceutical companies today; ii) to understand whether renal SP studies have impact or utility in drug development and/or if it may be more appropriate to assess renal effects after multiple doses of compounds; iii) to ascertain how much mechanistic work is performed by the top 15 largest pharmaceutical companies (as determined by R&D revenue size); iv) to gain an insight into the impact of the validation of DIKI biomarkers and their introduction in the safety evaluation paradigm; and v) to understand the impact of renal/urinary safety study data on progression of projects. METHODS: Two short anonymous surveys were submitted to SP leaders of the top 15 pharmaceutical companies, as defined by 2012 R&D portfolio size. Fourteen multiple choice questions were designed to explore the strategies used to investigate renal effects in both ICH S7A compliant SP studies and within toxicology studies. RESULTS: A 67% and 60% response rate was obtained in the first and second surveys, respectively. Nine out of ten respondent companies conduct renal excretory measurements (eg. urine analysis) in toxicology studies whereas only five out of ten conduct specific renal SP studies; and all of those 5 also conduct the renal excretory measurements in toxicology studies. These companies measure and/or calculate a variety of parameters as part of these studies, and also on a case by case basis include regulatory qualified and non-qualified DIKI biomarkers. Finally, only one company has used renal/urinary functional data alone to stop a project, whereas the majority of respondents combine renal data with other target organ assessments to form an integrated decision-making set. CONCLUSION: These short surveys highlighted areas of similarity: a) urinary measurements are most commonly taken on repeat-dose toxicity studies, and b) renal SP studies are less often utilised. The two major differences are a) lack of consistent use of DIKI biomarkers in urinary safety studies and b) the way large pharmaceutical companies assess renal function. Finally, suggestions were made to improve the safety assessment methods for determining the safety of compounds with potential renal liability.

The concordance between nonclinical and phase I clinical cardiovascular assessment from a cross-company data sharing initiative.

It is widely accepted that more needs to be done to bring new, safe, and efficacious drugs to the market. Cardiovascular toxicity detected both in early drug discovery as well as in the clinic, is a major contributor to the high failure rate of new molecules. The growth of translational safety offers a promising approach to improve the probability of success for new molecules. Here we describe a cross-company initiative to determine the concordance between the conscious telemetered dog and phase I outcome for 3 cardiovascular parameters. The data indicate that, in the context of the methods applied in this analysis, the ability to detect compounds that affect the corrected QT interval (QTc) was good within the 10-30x exposure range but the predictive or detective value for heart rate and diastolic blood pressure was poor. These findings may highlight opportunities to refine both the animal and the clinical study designs, as well as refocusing the assessment of value of dog cardiovascular assessments beyond phase 1. This investigation has also highlighted key considerations for cross-company data sharing and presents a unique learning opportunity to improve future translational projects.

How do the top 12 pharmaceutical companies operate safety pharmacology?

INTRODUCTION: How does safety pharmacology operate in large pharmaceutical companies today? By understanding our current position, can we prepare safety pharmacology to successfully navigate the complex process of drug discovery and development? METHODS: A short anonymous survey was conducted, by invitation, to safety pharmacology representatives of the top 12 pharmaceutical companies, as defined by 2009 revenue figures. A series of multiple choice questions was designed to explore group size, accountabilities, roles and responsibilities of group members, outsourcing policy and publication record. RESULTS: A 92% response rate was obtained. Six out of 11 companies have 10 to 30 full time equivalents in safety pharmacology, who hold similar roles and responsibilities; although the majority of members are not qualified at PhD level or equivalent. Accountabilities were similar across companies and all groups have accountability for core battery in vivo studies and problem solving activities but differences do exist for example with in vitro safety screening and pharmacodynamic/pharmokinetic modeling (PK/PD). The majority of companies outsource less than 25% of studies, with in vitro profiling being the most commonly outsourced activity. Finally, safety pharmacology groups are publishing 1 to 4 articles each year. CONCLUSION: This short survey has highlighted areas of similarity and differences in the way large pharmaceutical companies operate safety pharmacology.

Direct effects of arsenic trioxide on action potentials in isolated cardiac tissues: importance of the choice of species, type of cardiac tissue and perfusion time.

INTRODUCTION: The aim of the present study was to evaluate direct/acute effects of arsenic trioxide on action potentials (APs) in isolated cardiac tissues, and to investigate if the choice of species and tissue and the duration of the perfusion play a role in arsenic-induced acute/direct prolongation of AP/QT. METHODS AND RESULTS: Direct electrophysiological effects of arsenic trioxide were measured in cardiac tissues isolated from four different species using micro-electrode recording. Arsenic (after 30 to 95 min perfusion at 10 µM) significantly prolonged APD(90), increased triangulation of the AP and elicited early afterdepolarizations (EADs) only in isolated guinea-pig and dog Purkinje fibers but not in rabbit and porcine (minipig) Purkinje fibers. Arsenic induced a prolongation of the APD(90) and increases in triangulation and the occurrence of EADs was not observed in papillary muscles of guinea-pigs and rabbits. Arsenic at 4 increasing concentrations from 0.1 µM to 10 µM at the standard perfusion-time of 15 min per concentration, and after a continuous 90-min perfusion at 1 µM and 1 Hz did not induce these direct effects on APD(90), triangulation and EADs in isolated guinea-pig Purkinje fibers, but it at 1 µM elicited EADs in 2 out of 7 preparations after 90 min at 0.2 Hz. DISCUSSION: The present study demonstrates that the choice of species and cardiac tissue as well as perfusion-time play important roles in arsenic-induced direct/acute effects on APD(90) and induction of EADs in vitro.

The fentanyl/etomidate-anesthetized beagle (FEAB) model in safety pharmacology assessment.

This unit describes a procedure for performing safety studies in the anesthetized beagle dog. Detailed are the anesthetic regime, the surgical procedure, and all materials needed to perform cardiovascular, central nervous system, and respiratory safety studies in these animals. An overview of all parameters that can be measured and calculated is provided, as are experimental protocols. Endpoints discussed include hemodynamic, electrocardiological, respiratory, arterial blood, and electroencephalogical parameters. Also presented are a formula to correct QT interval for changes in core body temperature and an overview of changes in ECG, MAP, and EEG traces that may occur during safety studies. The information provided yields a multiparametric model for performing reliable safety studies in anesthetized dogs.

Predicting drug-induced slowing of conduction and pro-arrhythmia: identifying the 'bad' sodium current blockers.

BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) for the identification of only drug-induced long QT and pro-arrhythmias have certain limitations. EXPERIMENTAL APPROACH: Conduction time (CT) was measured in isolated Purkinje fibres, left ventricular perfused wedges and perfused hearts from rabbits, and sodium current was measured in Chinese hamster ovary cells, transfected with Na(v)1.5 channels. KEY RESULTS: A total of 355 compounds were screened for their effects on CT: 32% of these compounds slowed conduction, 65% had no effect and 3% accelerated conduction. Lidocaine and flecainide, which slow conduction, were tested in more detail as reference compounds. In isolated Purkinje fibres, flecainide largely slowed conduction and markedly increased triangulation, while lidocaine slightly slowed conduction and did not produce significant triangulation. Also in isolated left ventricular wedge preparations, flecainide largely slowed conduction in a rate-dependent manner, and elicited ventricular tachycardia (VT). Lidocaine slightly slowed conduction, reduced Tp-Te and did not induce VT. Similarly in isolated hearts, flecainide markedly slowed conduction, increased Tp-Te and elicited VT or ventricular fibrillation (VF). The slowing of conduction and induction of VT/VF with flecainide was much more evident in a condition of ischaemia/reperfusion. Lidocaine abolished ischaemia/reperfusion-induced VT/VF. Flecainide blocked sodium current (I(Na)) preferentially in the activated state (i.e. open channel) with slow binding and dissociation rates in a use-dependent manner, and lidocaine weakly blocked I(Na). CONCLUSION AND IMPLICATIONS: Slowing conduction by blocking I(Na) could be potentially pro-arrhythmic. It is possible to differentiate between compounds with 'good' (lidocaine-like) and 'bad' (flecainide-like) I(Na) blocking activities in these models.

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.

The fentanyl/etomidate-anaesthetised beagle (FEAB) dog: a versatile in vivo model in cardiovascular safety research.

The purpose of conducting cardiovascular safety pharmacology studies is to investigate the pharmacological profiles of new molecular entities (NMEs) and provide data that can be used for optimization of a possible new drug, and help make a selection of NMEs for clinical development. An anaesthetised dog preparation has been used for more than two decades by our department to measure multiple cardiovascular and respiratory parameters and to evaluate different scientific models, leading to more in-depth evaluation of drug-induced cardiovascular effects. An anaesthetic regime developed in house (induction with lofentanil, scopolamine and succinylcholine, and maintenance with fentanyl and etomidate) gives us a preparation free of pain and stress, with minimal effects on the cardiovascular system. This anaesthetic regime had minimal influences on circulating catecholamine levels, on the baroreflex sensitivity, and on all measured basal parameters compared to conscious dogs. All parameters were stable for at least 3 h, with acceptable tolerance intervals, evaluated over 99 safety studies with 3 vehicle treatments (saline, 10% and 20% hydroxypropyl-beta-cyclodextrin). This translates into a highly sensitive model for detecting possible drug-induced effects of NMEs with different mechanisms of action such as: Ca-, Na-, I(Kr)-, I(Ks)-channel blockers, K- and Ca-channel activators, alpha1- and beta-agonists, and muscarinic antagonists. Fentanyl in combination with etomidate is a successful anaesthetic regime in humans [Stockham, R.J., Stanley, T.H., Pace, N.L., King, K., Groen, F. & Gillmor, S.T. (1987). Induction of anaesthesia with fentanyl or fentanyl plus etomidate in high-risk patients. Journal of Cardiothoracic Anesthesia. 1(1), 19-23.]. In the anaesthetised dog, QT correction factors (Van de Water correction and body temperature correction) and risk factors (total, short-term and long-term instability) have been evaluated, using this regime [Van de Water, A., Verheyen, J., Xhonneux, R. & Reneman, R. (1989). An improved method to correct the QT interval of the electrocardiogram for changes in heart rate. Journal of Pharmacological Methods, 22, 207-217.; van der Linde, H.J., Van Deuren, B., Teisman, A., Towart, R. & Gallacher, D.J. (2008). The effect of changes in core body temperature on the QT interval in beagle dogs: A previously ignored phenomenon, with a method for correction. British Journal of Pharmacology, 154, 1474-1481.; van der Linde, H.J., Van de Water, A., Loots, W., Van Deuren, B., Lu, H.R., Van Ammel, K., et al. (2005) A new method to calculate the beat-to-beat instability of QT duration in drug-induced long QT in anaesthetised dogs. Journal of Pharmacological and Toxicological Methods, 52, 168-177.]. Furthermore, this anaesthetic protocol has been used to create different scientific models (long QT, short QT) with different specific end-points (ventricular fibrillation, adrenergic- or pause-dependent TdP) and also their specific precursors: e.g. aftercontractions, phase 2 EADs, phase 3 EADs, DADs, T-wave morphology changes, T-wave alternans, R-on-T, transmural and interventricular dispersion [Gallacher, D.J., Van de Water, A., van der Linde, H.J., Hermans, A.N., Lu, H.R., Towart, R., et al. (2007). In vivo mechanisms precipitating torsade de pointes in canine model of drug-induced long QT1 syndrome. Cardiovascular Research, 76-2, 247-256.]. This paper gives a brief overview of the stability, reproducibility, sensitivity and utility of a well-validated anaesthetised dog model.

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.

Choice of cardiac tissue in vitro plays an important role in assessing the risk of drug-induced cardiac arrhythmias in human: beyond QT prolongation.

INTRODUCTION: Recently we have demonstrated that to the choice of tissue type is important in identifying I(Kr) and I(Ks)-induced prolongation of the action potential. However, the differential sensitivity of cardiac tissues to other ionic current blockers or modulators is relatively unknown. The aim of the present study was therefore to evaluate tissue-specific effects of different ion channel blockers or activators on the action potential (AP), which can affect other parameters in addition to drug-induced APD/QT prolongation or shortening. METHODS AND RESULTS: Electrophysiological effects were measured in isolated rabbit Purkinje fibers, papillary muscles and ventricular trabeculae using a microelectrode technique under the following conditions: block of I(to) with 4-AP (1 x 10(-3) M), block of Ca(2+) channels with diltiazem (1 x 10(-5) M), block of Na(+) channels with flecainide (1 x 10(-5) M), activation of Ca(2+) current with Bay-K-8644 (1 x 10(-5) M), activation of K(ATP) channels with levcromakalim (1 x 10(-5) M) or block of I(K1) current with BaCl(2) (n=8 to 12 for each group). 4-AP prolonged APD significantly more in the Purkinje fiber than in the papillary muscle or the ventricular trabecula. 4-AP elicited 63% incidence of early afterdepolarizations but 0% in the papillary or trabeculae. Diltiazem and flecainide shortened APD(40) and APD(50) and increased triangulation more in the Purkinje fiber, whilst having little effect on these parameters in the papillary muscle or the ventricular trabecula. Bay-K-8644 significantly prolonged APD in the ventricular trabecula, but not in the Purkinje fiber or the papillary muscle. BaCl(2) prolonged APD(90) in all tissues, but significantly shortened APD(40) only in the Purkinje fiber. Levcromakalim shortened APD in all tissues, but significantly less in the Purkinje fibers. CONCLUSION: The present study demonstrates that certain cardiac tissues respond differently to the same ion channel blockers/activators, which are not involved in APD/QT prolongation. As such the appropriate selection of tissue needs to be taken into careful consideration in cardiac safety assessments when exploring different mechanisms of drug-induced changes in the action potential.

Calmodulin antagonist W-7 prevents sparfloxacin-induced early afterdepolarizations (EADs) in isolated rabbit purkinje fibers: importance of beat-to-beat instability of the repolarization.

INTRODUCTION: The occurrence of early afterdepolarizations (EADs) has been related to the incidence of torsades de pointes in drug-induced long QT (LQT). The generation of EADs may be facilitated by Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). METHODS AND RESULTS: In the present study, we investigated a possible involvement of Ca(2+)/Calmodulin dependent protein kinase in the generation of sparfloxacin-induced EADs in isolated rabbit Purkinje fibers by means of a calmodulin antagonist W-7. EADs were evident in 8 of the 10 preparations perfused with sparfloxacin at 1 x 10(-4) M and stimulated at 0.2 Hz. The induction of EADs by sparfloxacin was associated with a large prolongation of the duration of the action potential (APD), an increase in the triangulation, and the short-term instability of the repolarization. CaM kinase blockade with the calmodulin antagonist W-7 inhibited sparfloxacin-induced EADs in a concentration-dependent manner (EADs were induced in 3 of 10, 1 of 10, and 0 of 8 preparations in the presence of W-7 at 5 x 10(-7) M, 5 x 10(-6) M, and 5 x 10(-5) M, respectively; P < 0.01 at 5 x 10(-6) M and 5 x 10(-5) M). The inhibition of sparfloxacin-induced EADs by W-7 at 5 x 10(-7) M and 5 x 10(-6) M was associated with a significant decrease in the beat-to-beat instability but not associated with a significant shortening of the APD and reduction of V(max). CONCLUSION: The present findings support the hypothesis that CaM kinase may be a proarrhythmic signaling molecule and demonstrate that CaM kinase may be involved in the generation of EADs in drug-induced LQT and enhanced beat-to-beat instability of repolarization is essential for the genesis of EADs in rabbit in vitro.

Choice of cardiac tissue plays an important role in the evaluation of drug-induced prolongation of the QT interval in vitro in rabbit.

INTRODUCTION: Regulatory guidelines (CPMP/986/96, ICHS7B) recommend the use of isolated cardiac tissues, including Purkinje fibers, papillary muscles and ventricular trabeculae, for detecting potential drug-induced long QT. However, the differential sensitivity of these tissues in experimental drug-induced long QT is relatively unknown. We investigated the electrophysiological characteristics of these tissue types in vitro together with their different responses to drugs that are known to induce prolongation of the QT interval in man. METHODS: Electrophysiological parameters were measured in vitro using a micro-electrode technique. The isolated rabbit Purkinje fibers, papillary muscles or ventricular trabeculae were superperfused with Tyrode's solution and stimulated according to different stimulation protocols. The effects of dofetilide (1 x 10(-8) M), sertindole (1 x 10(-6) M), erythromycin (3 x 10(-4) M) and sparfloxacin (1 x 10(-4) M) were evaluated relative to solvent (n=8 to 12 for each group). RESULTS: In isolated Purkinje fibers, action potential duration at 90% repolarization (APD(90) at 1 Hz) was markedly prolonged by 55% (erythromycin), 103% (dofetilide), 118% (sertindole) and 88% (sparfloxacin) from baseline. The prolongation of APD(90) caused by these 4 compounds was associated with a 28% to 78% incidence of early afterdepolarizations (EADs) at 0.2 Hz only in the Purkinje fiber. In contrast, APD(90) was altered by erythromycin, dofetilide, sertindole and sparfloxacin only by +15%, +6%, -7% or +15%, respectively, in isolated papillary muscles, and by 33%, +28%, +4% or +16%, respectively, in ventricular trabeculae. EADs were not induced by these four compounds in papillary muscles or in trabeculae. Reducing the stimulation rate to 0.2 Hz resulted in a 33% prolongation of APD(90) in Purkinje fibers, while APD(90) was shortened by 10% in papillary muscles and by 20% in ventricular trabeculae. CONCLUSION: The present study demonstrates that the differential sensitivity of tissue types play an important role in the detection of drug-induced long APD and EADs. Indeed the Purkinje fiber was the only tissue type to display the well known phenomenon associated with I(kr) channel blockade (inverse-use dependence), when the stimulation rate was decreased below 1 Hz. Rabbit Purkinje fibers constitute the most sensitive tissue type for detecting drug-induced long action potential duration and EADs. As such the selection of tissue type needs to be taken into careful consideration in cardiac safety assessments when exploring drug induced long QT.