Eric S. Hayes

Physicochemical Determinants for Drug Induced Blockade of HERG Potassium Channels: Effect of Charge and Charge Shielding

The data on the activities of all previously described HERG blockers and of the most widely cited I(Kr) blockers were analyzed with respect to the effect of potential charged center(s) and its shielding by surrounding structural elements. The following model was considered: the less shielding of the charged form of the drug occurs, the easier its deprotonation will be and the less potency of the blockade of HERG/I(Kr) channels will be. Tertiary amines which form ammonium ions shielded by two structural fragments of the drug molecule were found to be potent HERG/I(Kr) blockers with IC50 < 1 microM (16 of 19 compounds, 84%). However, if the charged center was found at the molecular periphery as such groups as dimethylamino, N-methylpiperidino, N-methylpiperazino, N-methylpyrrolidino, pyrrolidino, imidazolo and partial periphery (diethylamino), then only moderate potency for HERG blockade with 1 microM < IC50 < 10 microM (8 of 11 compounds; 73%) was observed. Similarly, 27 of 32 weak HERG blockers ( IC50 > 10 microM) were found to be primary or secondary amines, or neutral or very weakly basic compounds. Ions of primary and secondary amines are susceptible to the fast deprotonation of the charged center and they, as well as non-charged compounds, have a low probability of induction of Torsades de Pointes (TdP). Conformational analysis and modeling of the interaction of the charged fragment of the drugs with acetone, a system that mimics a ketone fragment of HERG/I(Kr) channel, supports preference of the conformation with the shielded charged center for potent HERG/I(Kr) blockers. The absence of stereospecificity of HERG/I(Kr) blockade observed in most of the published studies reinforces the importance of charged center shielding as a key parameter. We suggest that the introduction of a hydroxy group at position 3 relative to a tertiary ammonium charged center, or the introduction of hydroxy, alkoxy or amino groups at position 2 relative to the nitrogen center of an aromatic system, should provide easy access of a water molecule to the proton, thereby facilitating deprotonation and thus leading to a moderate or weak HERG/I(Kr) blockade and a reduced risk of TdP.

Sodium channel-blocking properties of spiradoline, a kappa receptor agonist, are responsible for its antiarrhythmic action in the rat

Spiradoline (U-62,066E), a selective kappa (kappa) receptor agonist, was examined for actions on the cardiovascular system and on myocardial ionic currents in rats. We initially characterized cardiac, hemodynamic, and antiarrhythmic actions of spiradoline in isolated perfused rat hearts and pentobarbital-anesthetized rats. Electrophysiologic studies in isolated myocytes were used to elucidate the mechanism for changes observed in vivo in the ECG, as well as for antiarrhythmic actions against electrical and ischemia-induced arrhythmias. In isolated rat hearts, spiradoline reduced heart rate and cardiac contractility and increased the PR interval and QRS width of the ECG in a concentration-dependent manner. In anesthetized rats, spiradoline dose-dependently reduced blood pressure and heart rate and prolonged the PR interval and QRS width. At slightly higher doses, it increased the QaT interval of the ECG. RSh, an index of sodium channel blockade in the rat, also was dose-dependently increased. Electrical stimulation of the left ventricle suggested that spiradoline may exert its antiarrhythmic action by blockade of myocardial sodium currents. The electrophysiologic actions of spiradoline on sodium currents, the transient outward (i(to)) and sustained plateau potassium (ik(sus)) currents were studied in isolated cardiac rat myocytes by whole-cell patch-clamp techniques. Spiradoline (15-500 microM) reduced peak sodium current in a rapid, reversible, and concentration-dependent manner; it also increased the rate of decay of I(to) and reduced the amplitude of Ik(sus). At a concentration of 150 microM, spiradoline produced a 24 +/- 2 mV hyperpolarizing shift in sodium current inactivation kinetics but did not alter activation processes. Spiradoline showed both tonic and frequency-dependent components of sodium current block. Thus spiradoline produced its antiarrhythmic actions via sodium channel blockade in myocardial tissue, although higher doses also block potassium currents. This combined ion channel-blocking property may be of added clinical benefit in the setting of myocardial ischemia.

The cardiac electrophysiological effects of sparteine and its analogue BRB-I-28 in the rat

This study compares the cardiovascular and antiarrhythmic effects of sparteine and a 3,7-diheterobicyclo[3.3.1]nonane analogue of sparteine, BRB-I-28, in pentobarbitone-anaesthetized rats subjected to left-ventricle electrical stimulation and occlusion of the left anterior descending coronary artery. Sparteine and BRB-I-28 produced a dose-dependent reduction in heart rate and blood pressure over the dose range 1-64 mumol/kg/min. As well, the P-R and Q-aT intervals of the electrocardiogram (ECG) were prolonged. The thresholds for induction of premature beats and ventricular fibrillation were dose-dependently increased and both drugs increased refractoriness. While sparteine and BRB-I-28 (at 16 and 64 mumol/kg/min, respectively) did not change the incidence of premature beats or ventricular tachycardia with coronary occlusion, both drugs equally reduced the incidence of ventricular fibrillation. We characterized the actions of sparteine and BRB-I-28 on cardiac Na+, transient outward and sustained outward plateau K+ currents of rat myocytes using the whole-cell patch-clamp. Sparteine and BRB-I-28 produced a concentration-dependent reduction in Na+ current with EC50 values of 110 and 230 microM, respectively. Both drugs produced hyperpolarizing shifts of 8 and 11 mV, respectively, for Na+ channel inactivation while neither produced a change in channel activation. Both drugs produced a concentration-dependent block of the sustained plateau K+ current and increased the rate of decay of the transient outward K+ current. Thus, sparteine and BRB-I-28 possess Na+ and K+ channel blocking properties which may account for their antiarrhythmic actions against electrical and ischaemic arrhythmias.

EFFECTS OF HALOTHANE AND ISOFLURANE ON RAT VENTRICULAR ACTION POTENTIALS RECORDED IN SITU

The effects of halothane and isoflurane on ventricular intracellular action potentials recorded in situ in pentobarbital anesthetized rats were studied. Halothane (0.5, 1 and 2 vol.%) and isoflurane (0.75, 1.5 and 3 vol.%) did not have identical effects on rat epicardial action potentials recorded by floating microelectrodes. However, over the concentration range tested, both anesthetics reduced blood pressure and heart rate to a similar extent. Isoflurane did not effect the maximum rate of rise of the action potential amplitude. However, 3 vol.% isoflurane reduced the resting membrane potential from -72+/-2 to -65+/-3 mV (mean+/-SEM, p<0.05) while the highest concentration of halothane had no effect. Halothane (2 vol.%) reduced action potential amplitude from 74+/-4 to 65+/-3 mV (p<0.05) and reduced the maximum rise rate of action potential from 175+/-21 to 133+/-8 V/s (p<0.05). Both isoflurane and halothane prolonged action potential duration at 10, 25 and 50% repolarization while only halothane significantly shortened action potential duration at 75% repolarization, Thus the effects of halothane and isoflurane on ventricular transmembrane action potentials were similar, but not identical. The relevance of such observations to the antiarrhythmic actions of halothane, but not isoflurane in this species is not clear.

SODIUM CHANNEL BLOCKING AND ANTIARRHYTHMIC ACTIONS OF THE NOVEL ARYLPIPERAZINE RSD992

Bolus doses (4-128 micromolkg(-1)) and infusions (2-32 micromolkg(-1)min(-1)) of the novel arylpiperazine drug RSD992 produced bradycardia in rats and guinea pigs but had minimal effect on ECG variables. RSD992 (2-32 micromolkg(-1)min(-1)) increased threshold current (I(T)) for induction of extra-systoles and induction of sustained ventricular fibrillation (VF(T)) and also increased the effective refractory period (ERP) and decreased the maximum following frequency (MFF) in rat and guinea pig hearts. RSD992 (32-512 microM) significantly increased PR and QRS intervals in isolated rat hearts subjected to conditions that mimic ischaemia (pH 6.4, K(+) 11mM) but not in isolated hearts under normal perfusion conditions (pH 7.4, K(+) 3mM). RSD992 (0.1-3.0mM) reduced peak sodium current in rat cardiac (rNa(v)1.5) sodium channels more potently than neuronal (rNa(v)1.2a) sodium channels expressed in Xenopus oocytes. The voltage-dependence of sodium channel activation was unaffected whereas inactivation was shifted in a hyperpolarized direction thus suggesting RSD992 may preferentially interact with the inactive state of the sodium channel, a state usually associated with myocardial cell depolarization in ischaemic myocardium. RSD992 (2-24 micromolkg(-1)min(-1)) decreased the incidence of ventricular arrhythmias and mortality in rats subject to coronary artery ligation. RSD992 exhibits frequency- and ischaemia-selective actions on myocardial sodium currents and antiarrhythmic actions in ischaemic rat myocardium.

Recalibration of nonclinical safety pharmacology assessment to anticipate evolving regulatory expectations.

Safety pharmacology (SP) has evolved in terms of architecture and content since the inception of the SP Society (SPS). SP was initially focused on the issue of drug-induced QT prolongation, but has now become a broad spectrum discipline with expanding expectations for evaluation of drug adverse effect liability in all organ systems, not merely the narrow consideration of torsades de pointes (TdP) liability testing. An important part of the evolution of SP has been the elaboration of architecture for interrogation of non-clinical models in terms of model development, model validation and model implementation. While SP has been defined by mandatory cardiovascular, central nervous system (CNS) and respiratory system studies ever since the core battery was elaborated, it also involves evaluation of drug effects on other physiological systems. The current state of SP evolution is the incorporation of emerging new technologies in a wide range of non-clinical drug safety testing models. This will refine the SP process, while potentially expanding the core battery. The continued refinement of automated technologies (e.g., automated patch clamp systems) is enhancing the scope for detection of adverse effect liability (i.e., for more than just IKr blockade), while introducing a potential for speed and accuracy in cardiovascular and CNS SP by providing rapid, high throughput ion channel screening methods for implementation in early drug development. A variety of CNS liability assays, which exploit isolated brain tissue, and in vitro electrophysiological techniques, have provided an additional level of complimentary preclinical safety screens aimed at establishing the seizurogenic potential and risk for memory dysfunction of new chemical entities (NCEs). As with previous editorials that preface the annual themed issue on SP methods published in the Journal of Pharmacological and Toxicological Methods (JPTM), we highlight here the content derived from the most recent (2015) SPS meeting held in Prague, Czech Republic. This issue of JPTM continues the tradition of providing a publication summary of articles primarily presented at the SPS meeting with direct bearing on the discipline of SP. Novel method development and refinement in all areas of the discipline are reflected in the content.

An examination of the cardiac actions of PD117,302, a κ-opioid receptor agonist.

These studies examined the opioid and non-opioid in vivo and in vitro actions of PD117,302 (((±)-trans-N-methyl-N-[2-(l-pyrrolidinyl)-cyclohexyl]benzo[b]thiophene-4-acetamide), a kappa (κ)-opioid receptor agonist. PD117,302 selectively labeled the κ-opioid receptor in guinea pig cerebellar membranes and in mice the ED50 for analgesia was 2.3µmol/kg. A non opioid cardiovascular assessment of PD117,302 showed that it dose-dependently increased left-ventricular peak systolic pressure in rat isolated perfused hearts but reduced heart rate and blood pressure in anaesthetized rats. Over the concentration range 0.3-30µM in vitro, and dose-range 0.25-4µmol/kg in vivo, PD117,302 dose-dependently prolonged the P-R interval, QRS width and Q-T interval of the rat heart ECG. Naloxone (either 1µM or 8µmol/kg) did not antagonize the observed ECG effects of PD117,302. Cardiac electrical stimulation studies in anesthetized rats showed that threshold currents for capture and fibrillation were increased and effective refractory period (ERP) prolonged. In rats subject to coronary artery occlusion PD117,302 reduced arrhythmia incidence. Intracellular cardiac action potential studies qualified the ECG changes produced by PD117,302 such that there was a dose-dependent reduction in the maximum rate of depolarization of phase 0 (dV/dtmax) and prolongation of the action potential duration (APD). In isolated cardiac myocytes PD117,302 dose-dependently (1-100µM) reduced peak Na(+) current and produced a hyperpolarizing shift in the inactivation curve. Transient outward and sustained outward K(+) currents were blocked by PD117,302. Thus, the ECG changes and antiarrhythmic effects observed in vivo result from direct blockade of multiple cardiac ion channels.

Pharmacological and toxicological activity of RSD921, a novel sodium channel blocker

BACKGROUND RSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors. METHODS The pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity. RESULTS In rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized. CONCLUSIONS RSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

Ventricular arrhythmia incidence in the rat is reduced by naloxone

This study characterized the antiarrhythmic effects of the opioid receptor antagonist naloxone in rats subject to electrically induced and ischemic arrhythmias. Naloxone (2, 8 and 32 μmol/kg/min) was examined on heart rate, blood pressure, and the electrocardiogram (EKG) as well as for effectiveness against arrhythmias produced by occlusion of the left anterior descending coronary artery or electrical stimulation of the left ventricle. Naloxone reduced blood pressure at the highest dose tested while heart rate was dose-dependently reduced. Naloxone dose-dependently prolonged the P-R and QRS intervals and increased the RSh amplitude indicative of effects on cardiac sodium (Na) channels. Naloxone prolonged the Q-T interval suggesting a delay in repolarization. Naloxone effects were comparable to the comparator quinidine. Naloxone (32 μmol/kg/min) reduced ventricular fibrillation (VF) incidence to 38% (from 100% in controls). This same dose significantly increased the threshold for induction of ventricular fibrillation (VFt), prolonged the effective refractory period (ERP) and reduced the maximal following frequency (MFF). The patterns of ECG changes, reduction in ischemic arrhythmia (VF) incidence and changes in electrically induced arrhythmia parameters at high doses of naloxone suggest that it directly blocks cardiac Na and potassium (K) ion channels.