Gender differences in the slow delayed (IKs) but not in inward (IK1) rectifier K+ currents of canine Purkinje fibre cardiac action potential: key roles for IKs, beta-adrenoceptor stimulation, pacing rate and gender.

As the beagle dog is a commonly used preclinical species to test the effects of new drugs on cardiac repolarisation and Purkinje fibres have become an established in vitro preparation to assess the effects of these new drugs on action potential duration (APD), the main aim of this study was therefore to evaluate the relative contribution of the inward (I(K1)) and slow delayed (I(Ks)) rectifier cardiac K(+) currents to action potential repolarisation in beagle Purkinje fibres under three different experimental conditions: (i) selective block of I(K1) with BaCl(2), (ii) selective block of I(Ks) with (-) chromanol 293B under basal conditions and (iii) selective block of I(Ks) during beta-adrenoceptor stimulation. Furthermore, the dependence of this contribution on gender and pacing rate was investigated. Microelectrode techniques were employed to measure APD in Purkinje fibres from adult female and male dogs. At stimulation rates of 3.33, 1.0 and 0.2 Hz, the degree of prolongation of APD evoked by BaCl(2) (10 microM) was comparable in fibres from female and male dogs. At the same stimulation rates, 10 microM (-) chromanol 293B did not change the APD in fibres from female and male dogs. During beta-adrenoceptor stimulation with 0.1 microM isoproterenol, an APD prolonging effect of (-) chromanol 293B was detected. In the presence of isoproterenol, action potentials in fibres from male dogs get shorter when changing the stimulation rate from 1.0 to 0.2 Hz, while the opposite is seen in fibres from female dogs. This alteration was completely reversed by (-) chromanol 293B. In conclusion, our findings confirm that beta-adrenoceptor stimulation is one condition where there may be an increased role of I(Ks) in action potential repolarisation. Gender differences in the autonomic modulation of I(Ks) could be a contributing factor to the reported increased susceptibility of female hearts to arrhythmias.

Nonclinical proarrhythmia models: predicting Torsades de Pointes.

Prolongation of the QT interval and the cardiac action potential have been linked to a potentially fatal but rare tachyarrhythmia known as Torsades de Pointes (TdP). Nonclinical assays, such as those investigating the effect on I(Kr) (the hERG channel current), prolongation of the action potential duration (APD) and the QT interval, in vivo, have been developed to predict the risk of QT interval prolongation and TdP in man. However, there seems to be a dissociation between the risk of QT interval prolongation and the torsadogenic risk. There is an increasing mass of evidence showing that an increase in the QT interval does not necessarily lead to TdP. Thus, it appears that while standard assays are very good, although perhaps not infallible, at predicting the risk of QT interval prolongation in man they do not predict the proarrhythmic risk. Recently there has been a plethora of publications suggesting that there are electrophysiological markers associated with drug-induced TdP other than hERG channel activity, APD and the QT interval, and these markers may be better predictors of TdP. In this review, three in vitro and, briefly, three in vivo models or methods are discussed. These proarrhythmia models use electrophysiological markers such as transmural dispersion of repolarization, action potential triangulation, instability, reverse use-dependence, and the incidence of early after-depolarizations to predict the risk of TdP. Most of the models presented have been published widely. The particular variable or set of variables used by each model to predict the torsadogenic propensity of a drug has been reported to correlate with clinical outcome. While each variable/model has been shown to discriminate between antiarrhythmic and nonarrhythmic drugs, these reports should be interpreted cautiously since none has been independently (externally) assessed. Each model is discussed along with its particular merits and shortcomings; none, as yet, having shown a predictive value that makes it clearly superior to the others. Proarrhythmia models, in particular in vitro models, challenge current perceptions of appropriate surrogates for TdP in man and question existing nonclinical strategies for assessing proarrhythmic risk. The rapid emergence of such models, compounded by the lack of a clear understanding of the key proarrhythmic mechanisms has resulted in a regulatory reluctance to embrace such models. The wider acceptance of proarrhythmia models is likely to occur when there is a clear understanding and agreement on the key proarrhythmia mechanisms. Regardless of regulatory acceptance, with further validation these models may still enhance pharmaceutical company decision-making to provide a rational basis for drug progression, particularly in areas of unmet medical need.

Spectrum of effects detected in the rat functional observational battery following oral administration of non-CNS targeted compounds.

INTRODUCTION: The Functional Observational Battery (FOB) is a systematic evaluation of nervous system function in the rat, comprising more than 30 parameters across autonomic, neuromuscular, sensorimotor and behavioural domains. We have collated FOB outcomes from 50 compounds that were not targeted at CNS disorders, and would therefore be anticipated to have relatively few CNS side-effects, for evaluation of the FOB as part of the safety pharmacology 'core battery'. METHODS: Male Han Wistar rats (200-300 g) were used, with n=6 per treatment group. Each compound was tested acutely at 3 dose levels (oral route), from the therapeutic dose up to either 100 times this dose or to the maximal tolerated dose (MTD). A vehicle control group was included in each study. RESULTS: Effects were detected in the FOB for 94% of compounds tested. The commonest effects were weight loss/decreased body weight gain overnight post-dose (46% of compounds), and changes in core temperature (36%). Dose-related effects were observed with 62% of compounds; the commonest was decreased body weight gain (32%), followed by effects on tail flick latency (14%), landing foot splay (12%), decreased rectal temperature (10%), time to exit the centre circle in the open field (10%), diarrhoea/loose faeces (8%), respiratory effects (4%), grasping reflex (4%) and supported rears in the open field (4%). Remaining parameters were affected by < or =2% of compounds. DISCUSSION: The value of doing the FOB as part of the safety pharmacology 'core battery' is emphasised by the fact that, even for non-CNS targeted compounds, the majority affected at least one of the parameters in the FOB. These data may also help to anticipate the most frequently required 'follow-up' studies.

Evidence for gender differences in electrophysiological properties of canine Purkinje fibres.

Women are more prone to develop torsades de pointes, a rare life-threatening polymorphic ventricular tachycardia, than are men during administration of medicines that have the potential to block I(Kr) (rapid delayed rectifier cardiac K(+) current) and to prolong the QT interval. Blockade of I(Kr), hypokalaemia and extreme bradycardia were used to evaluate whether there are gender differences in cardiac repolarisation in canine Purkinje fibres (PFs). Microelectrode techniques were employed to measure action potential (AP) parameters in PFs from adult female and male dogs. Under control conditions, fibres from female animals in normal or low K(+) conditions exhibited significantly longer AP durations at 50% (APD(50)) and 90% (APD(90)) of repolarisation as compared with APDs of fibres from male animals. Gender-related difference to rate adaptation was also present in APD(90) of fibres from female animals compared to males. At a stimulation rate of 0.2 Hz, but not at 1.0 Hz, dofetilide elicited a significantly higher increase in APD(90), incidence of early afterdepolarisations, triggered and sustained-triggered activities (TAs) in fibres from female animals compared to males in either normal or low K(+) conditions. The sustained TAs were reversed by raising the concentration of [K(+)](0) in Purkinje preparations from both male (one out of one) and female (12 out of 12) dogs. In conclusion, our data provide experimental evidence pointing to gender differences in canine AP repolarisation. PFs from female dogs can be used in safety pharmacology studies as a sensitive model for evaluating the potential proarrhythmic events in vitro of a new medicinal product.

Review of the predictive value of the Langendorff heart model (Screenit system) in assessing the proarrhythmic potential of drugs.

Prolongation of the QTc interval of the electrocardiogram (ECG) is used as a surrogate marker for a rare, but life threatening, ventricular arrhythmia known as torsades de pointes (TdP). However, the clear link between QTc prolongation and the arrhythmogenic risk has not been demonstrated unequivocally. In the present review article, we examine (a) the current understanding of electrophysiological and pharmacological mechanisms linking changes in action potential (AP) properties with proarrhythmia and (b) the value of the isolated, paced Langendorff-perfused female rabbit heart model (Screenit system) in predicting the torsadogenic potential of drugs in man. The Screenit system records monophasic action potentials (MAPs) from which the following parameters are evaluated: action potential duration (APD), conduction, instability (indicative of beat to beat APD variability), triangulation (indicative of changes of Phase 3 repolarization), and reverse-use dependency (indicating that the APD is more prolonged at slow heart rates). So far, over 16,000 experiments have been conducted, including approximately 300 dedicated tests to evaluate, in a blinded manner, approximately 70 clinically used drugs. The drugs tested covered a wide range of compounds from various pharmacological and chemical classes with clinical torsadogenic propensity, as well as drugs without the latter effect in clinical settings. Overall, the Screenit system and its associated analysis classified the drugs based on their effects on AP morphology and conduction and additionally identified, in a qualitative manner, drugs clinically associated with TdP. Such an identification is based on the triangulation, reverse-use dependency, and instability of the AP, as well as on the direct indexes of proarrhythmia such as early afterdepolarization (EADs), ventricular tachycardia (VT), and ventricular fibrillation (VF). Overall, drugs that readily induce arrhythmia and/or EADs and/or causes triangulation, reverse-use dependency, and/or instability and/or a chaotic Poincaré plot in a range of concentrations likely to be achieved in man is likely to cause TdP in man, eventually. Only if none of these elements is present, at concentrations well exceeding the free therapeutic plasma concentration, can one expect that the drug will probably be devoid of torsadonenicity. Therefore, this in vitro model provides detailed information on the overall profile of drug-induced electrophysiological effects. In combination with other in vitro and in vivo repolarization assays and with pharmacokinetic data in man, it is a valuable tool to establish an integrated cardiovascular risk assessment of pharmaceutical compounds.

Sex differences in ventricular repolarization: from cardiac electrophysiology to Torsades de Pointes.

A number of non-cardiovascular drugs have been withdrawn from clinical use due to unacceptable adverse cardiac side-effects involving drug-induced Torsades de Pointes (TdP)--a rare, life-threatening polymorphic ventricular tachycardia associated with prolongation of the action potential duration of ventricular myocytes and, hence, prolongation of the QT interval, of the electrocardiogram (ECG), which measures the total time for activation of the ventricles and their recovery to the resting state. Research has suggested that women are more prone to develop TdP than men during administration of medicines that share the potential to prolong the QT interval, with 65-75% of drug-induced TdP occurring in women. Clinical and experimental studies show that female sex demonstrate differences in the electrocardiographic pattern of ventricular repolarization in human and other animal species and is associated with a longer rate-corrected QT (QTc) interval at baseline than males. Reports of a similar propensity towards drug-induced TdP in both premenopausal and postmenopausal women support factors in addition to those of female sex hormones eliciting sex-based differences in ventricular repolarization. However, conflicting evidence suggests sex hormones may have a role in increasing the susceptibility of women or ultimately reducing the susceptibility of men to TdP. Cyclical variations in hormone levels during the menstrual cycle have been associated with an increased and reduced risk of TdP. In contradiction to this finding, the male sex hormone is thought to be beneficial. Modulation of the ventricular repolarization by testosterone may explain why the QTc interval shortens at puberty, and might account for the tendency towards an age-dependent reduction in the incidence of drug-induced TdP in men. Mechanisms underlying these differences are not fully understood but a case for the involvement of gonadal steroids is obviously strong. Therefore, further non-clinical/clinical investigations ought to be a necessary step to elucidate any sex differences in cardiac repolarization characteristics, QT interval prolongation and susceptibility to cardiac arrhythmias. This may have implications for the development of the safest medicinal products and for the clinical management of cardiac arrhythmias.