David J. Gallacher

Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICHS7B Guidelines

The regulatory guidelines (ICHS7B) recommending inhibition of the delayed rectifier K+ current (IKr), carried by human ether‐a‐go‐go‐related gene (hERG) channels in cardiac cells (the hERG test), as a ‘first line’ test for identifying compounds inducing QT prolongation, have limitations, some of which are outlined here.

Blockade of the IKs 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.

The effect of changes in core body temperature on the QT interval in beagle dogs: a previously ignored phenomenon, with a method for correction

Body core temperature (Tc) changes affect the QT interval, but correction for this has not been systematically investigated. It may be important to correct QT intervals for drug‐induced changes in Tc.

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.

The Electro-Mechanical window: a risk marker for Torsade de Pointes in a canine model of drug induced arrhythmias.

BACKGROUND AND PURPOSE In cardiovascular pharmacology, electrical and mechanical events can be distinguished, and the phrase ‘electro‐mechanical window’ (EMw) describes the temporal difference between these events. We studied whether changes in EMw have potential predictive value for the occurrence of arrhythmias in fentanyl/etomidate‐anaesthetized beagle (FEAB) dogs.

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 Ca2+/calmodulin‐dependent protein kinase II (CaM kinase).

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.

EEG in the FEAB model: measurement of electroencephalographical burst suppression and seizure liability in safety pharmacology.

INTRODUCTION The purpose of this study was to explore the integration of electroencephalographical (EEG) measurements into the fentanyl/etomidate-anaesthetised Beagle (FEAB) model in order to detect burst suppression and/or seizure development caused by compounds, prior to new molecular entity (NME) declaration. Detecting such unfavourable side effects prevents their being found in conscious animals at a later stage of safety evaluation. In addition, this has the advantage of performing safety studies on the three vital organ systems (cardiovascular system, respiratory system and central nervous system) within one and the same animal model. METHODS Dogs were anaesthetized and instrumented according to the FEAB model requirements, and in addition three needle electrodes were placed on the cranium and a one lead EEG signal was measured. The raw signal was analysed by the Narcotrend® (MonitorTechnik, Bad Bramstedt, Germany) for depth of anaesthesia registration, visually analysed for burst suppression ratio calculation after different anaesthetics (pentobarbital and etomidate), and spiking and seizure activity were quantified after intravenous administration of different proconvulsant agents: pentylenetetrazole (PTZ), bicuculline (BCC), bupropion (BUP) and pilocarpine (PIL). RESULTS High doses of pentobarbital (60 mg/kg over 10 min) and etomidate (6 mg/kg over 10 min) induced dose-dependent burst suppression of 98 ± 2% and 61 ± 16%, respectively. Infusions of PTZ (1.5mg/kg/min), BCC (0.0625 mg/kg/min), BUP (0.5mg/kg/min) and PIL (5mg/kg/min) induced dose-dependent spiking and seizures: the thresholds were 34 ± 2, 0.15 ± 0.03, 10.0 ± 1 and 144 ± 9 mg/kg, respectively. In PTZ-treated dogs, spiking and seizures could be abolished with diazepam (2mg/kg i.v.) or with propofol (4 mg/kg i.v.). DISCUSSION The present study showed that a one lead EEG can be used reliably in the FEAB model to estimate the depth of anaesthesia, and to detect burst suppression and seizure risk in safety pharmacology studies.

Innovation in safety pharmacology testing

This issue of the Journal of Pharmacological and Toxicological Methods (JPTM) is themed. It is the eighth in a series, arising from the Annual Safety Pharmacology Society (SPS) meeting. The SPS is now in its 10th year as an independent branch of biological sciences (distinct from pharmacology and toxicology) and is the primary forum for driving advances in safety pharmacology. The theme of the meeting and this journal issue is innovation, and the focus is non-clinical safety assessment of new chemical entity (NCEs). The content is informed by regulatory guidance documents (S7A and S7B) prior to first in human (FIH) studies. The manuscripts cover a broad spectrum of safety pharmacology topics from theory to practice, with interrogation of state-of-the-art techniques, and profiling of methods that are in development for safety assessment. Philosophical and strategic issues are addressed, with consideration of the use of novel methods for population pharmacokinetic (PK) analysis, abuse liability, electrocardiogram (ECG) analysis algorithms, in vitro cardiac slice preparations, human pluripotent stem cells, and a brief discussion regarding the assessment of changes in the QRS complex of the ECG indicative of drug-induced blockade of cardiac sodium channels. Safety pharmacology methods continue to evolve.

Non-clinical models: Validation, study design and statistical consideration in safety pharmacology

The current issue of the Journal of Pharmacological and Toxicological Methods (JPTM) focuses exclusively on safety pharmacology methods. This is the 7th year the Journal has published on this topic. Methods and models that specifically relate to methods relating to the assessment of the safety profile of a new chemical entity (NCE) prior to first in human (FIH) studies are described. Since the Journal started publishing on this topic there has been a major effort by safety pharmacologists, toxicologists and regulatory scientists within Industry (both large and small Pharma as well as Biotechnology companies) and also from Contract Research Organizations (CRO) to publish the surgical details of the non-clinical methods utilized but also provide important details related to standard and non-standard (or integrated) study models and designs. These details from core battery and secondary (or ancillary) drug safety assessment methods used in drug development programs have been the focus of these special issues and have been an attempt to provide validation of methods. Similarly, the safety pharmacology issues of the Journal provide the most relevant forum for scientists to present novel and modified methods with direct applicability to determination of drug safety-directly to the safety pharmacology scientific community. The content of the manuscripts in this issue includes the introduction of additional important surgical methods, novel data capture and data analysis methods, improved study design and effects of positive control compounds with known activity in the model.