Reverse Use-dependence Research Articles

State-Dependent Inhibition of Nav1.8 Sodium Channels by VX-150 and VX-548.

Nav1.8 sodium channels (Nav1.8) are an attractive therapeutic target for pain because they are prominent in primary pain-sensing neurons with little expression in most other kinds of neurons. Recently, two Nav1.8-targeted compounds, VX-150 and VX-548, have shown efficacy in clinical trials for reducing pain. We examined the characteristics of Nav1.8 inhibition by these compounds. The active metabolite form of VX-150 (VX-150m) inhibited human Nav1.8 channels with an IC50 of 15 nM. VX-548 (suzetrigine) was even more potent (IC50 0.27 nM). Both VX-150m and VX-548 had the unusual property of "reverse use-dependence," whereby inhibition could be relieved by repetitive depolarizations, a property seen before with another Nav1.8 inhibitor, A-887826. The relief of VX-548 inhibition by large depolarizations occurred with a time constant of ∼40 milliseconds that was not concentration-dependent. Reinhibition at negative voltages occurred with a rate that was nearly proportional to drug concentration, consistent with the idea that relief of inhibition reflects dissociation of drug from the channel and reinhibition reflects rebinding. The relief of inhibition by depolarization suggests a remarkably strong and unusual state-dependence for both VX-150m and VX-548, with very weak binding to channels with fully activated voltage sensors despite very tight binding to channels with voltage sensors in the resting state. SIGNIFICANCE STATEMENT: The Nav1.8 sodium channel (Nav1.8) is a current target for new drugs for pain. This work describes the potency, selectivity, and state-dependent characteristics of inhibition of Nav1.8 channels by VX-150 and VX-548, compounds that have recently shown efficacy for relief of pain in clinical trials but whose mechanism of interaction with channels has not been described. The results show that the compounds share an unusual property whereby inhibition is relieved by depolarization, demonstrating a state-dependence different from most sodium channel inhibitors.

Application of Machine Learning Techniques for Testing Sensitivity of Cardiac Dynamics to Arrhythmia Vulnerability Parameters

In this study, we developed a new approach to address the importance of arrhythmia vulnerability parameters for cardiac dynamics prediction in response to autonomic stimulation. We generated various case studies using a series of complex electrophysiology protocols for control versus stimulated single cardiac myocytes by autonomic nervous system (ANS). We collected the “TRiAD” related parameters (triangulation (APD TRI), reverse use dependence (RUD), beat‐to‐beat (bTb) instability of action potential duration and spatial APD dispersion (T‐wave area)). We used collected data to train different machine learning algorithms for classifying control versus ANS case studies. We used artificial neural network (ANN), support vector machine (SVM) with Gaussian kernels and random forest (RF) algorithms and observed that RF outperformed the other two algorithms with higher model accuracy for output prediction. Our results illustrated that bTb instability, APD TRI, RUD and T‐wave area are respectively the most important parameters for our classification problem. We also studied the sensitivity of cardiac dynamics to the listed TRiAD parameters. We observed that increase in bTb instability values leads to higher probability of case study being classified into control group while increase in RUD and TRI values result into higher probability of case study being classified into ANS group. Whereas T‐wave area values illustrated different trend compared to three other parameters, where low values could be misleading in decision making about the group that the case study will belong to and higher T‐wave values mostly belong to ANS classified case studies. Therefore, our study suggests the advantages of data analytics techniques for discoveries that may not have been evident.Support or Funding InformationThis study was funded by National Institutes of Health (Grant OT2OD026580).

Analysis of therapeutic utility of a multi-ion channel inhibitor acehytisine for paroxysmal supraventricular tachycardia in anesthetized guinea pigs

Acehytisine is a novel diterpene alkaloid inhibiting IK, INa and ICa, which has been developed as a novel drug for the treatment of paroxysmal supraventricular tachycardia. To better understand its therapeutic utility, we assessed the pharmacological and proarrhythmic effects of acehytisine in vivo using isoflurane-anesthetized guinea pigs in comparison with those of bepridil. We administered acehytisine (4 and 10 mg/kg) and beperidil (1 and 3mg/kg) intravenously to the isoflurane-anesthetized guinea pig (n=6 for each drug) for 10 minutes and observed its effects on cardiac electrophysiology. Acehytisine at therapeutic dose (4 mg/kg) decreased the heart rate, prolonged P wave duration, QRS width, QT interval and QTc. At high dose (10 mg/kg), it prolonged the PR interval besides enhancing the changes induced by the therapeutic dose. The peak changes in P wave duration by acehytisine were 6 and 10 ms at 4 and 10 mg/kg, respectively, which were both 1.5 times longer than those of bepridil. Additionally, acehytisine at therapeutic dose increased the beat-to-beat variability, whereas a dose-related increase was not detected at high dose. In contrast, bepridil increased the beat-to-beat variability in a dose-related manner. A reverse use-dependence was observed in the repolarization delay in both drugs, but the peak changes in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 29, 20 and 19 ms, respectively, which is 1.9, 1.5 and 1.2 times shorter than those of bepridil. These results indicate that acehytisine in comparison with bepridil exhibits a favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia.

Electropharmacological effects of amantadine on cardiovascular system assessed with J-Tpeak and Tpeak-Tend analysis in the halothane-anesthetized beagle dogs.

Since amantadine-induced long QT syndrome has been clinically reported, we investigated its electropharmacological effects to estimate the extent of proarrhythmic risk by using the halothane-anesthetized beagle dogs (n = 4). Amantadine in doses of 0.1, 1 and 10 mg/kg was infused over 10 min with a pause of 20 min under the monitoring of multiple cardiovascular variables. J-Tpeak and Tpeak-Tend were separately measured on the lead II electrocardiogram to precisely analyze the net balance between inward and outward current modifications by amantadine. The low dose increased the ventricular contractile force, but suppressed the intraventricular conduction. The middle dose prolonged the QT interval besides enhancing the changes induced by the low dose. The high dose increased the mean blood pressure, left ventricular end-diastolic pressure and total peripheral resistance, and accelerated the atrioventricular nodal conduction, but decreased the cardiac output besides enhancing the changes induced by the middle dose. A reverse use-dependence was confirmed in the repolarization delay. Amantadine hardly affected the J-Tpeak, but prolonged the Tpeak-Tend. Amantadine can be considered to stimulate Ca(2+) channel but inhibit Na(+) and K(+) channels in the in situ heart. J-Tpeak and Tpeak-Tend analysis suggests that amantadine may possess modest risk for arrhythmia.

LOWER PROGRAMMED PACING RATES REDUCES ATRIAL FIBRILLATION BURDEN IN PATIENTS ON DOFETILIDE

Dofetilide is one of the most effective anti-arrhythmic agents for Atrial Fibrillation (AF). Reverse Use Dependence (RUD) property of Dofetilide improves antiarrhythmic efficacy at lower heart rate. Several patients with AF have pacemakers or defibrillators for various reasons. The effectiveness of

Wenxin Keli Suppresses Ventricular Triggered Arrhythmias via Selective Inhibition of Late Sodium Current

Wenxin Keli is a popular Chinese herb extract that approximately five million Asians are currently taking for the treatment of a variety of ventricular arrhythmias. However, its electrophysiological mechanisms remain poorly understood. The concentration-dependent electrophysiological effects of Wenxin Keli were evaluated in the isolated rabbit left ventricular myocytes and wedge preparation. Wenxin Keli selectively inhibited late sodium current (INa) with an IC50 of 3.8 ± 0.4 mg/mL, which was significantly lower than the IC50 of 10.6 ± 0.9 mg/mL (n = 6, P < 0.05) for the fast INa. Wenxin Keli produced a small but statistically significant QT prolongation at 0.3 mg/mL, but shortened the QT and Tp-e interval at concentrations ≥ 1 mg/mL. Wenxin Keli increased QRS duration by 10.1% from 34.8 ± 1.0 ms to 38.3 ± 1.1 ms (n = 6, P < 0.01) at 3 mg/mL at a basic cycle length of 2,000 ms. However, its effect on the QRS duration exhibited weak use-dependency, that is, QRS remained less changed at increased pacing rates than other classic sodium channel blockers, such as flecainide, quinidine, and lidocaine. On the other hand, Wenxin Keli at 1-3 mg/mL markedly reduced dofetilide-induced QT and Tp-e prolongation by attenuation of its reverse use-dependence and abolished dofetilide-induced early afterdepolarization (EAD) in four of four left ventricular wedge preparations. It also suppressed digoxin-induced delayed after depolarization (DAD) and ventricular tachycardias without changing the positive staircase pattern in contractility at 1-3 mg/mL in a separate experimental series (four of four). Wenxin Keli suppressed EADs, DADs, and triggered ventricular arrhythmias via selective inhibition of late INa.

Markov Models of Use-Dependence and Reverse Use-Dependence during the Mouse Cardiac Action Potential

The fast component of the cardiac transient outward current, IKtof, is blocked by a number of drugs. The major molecular bases of IKtof are Kv4.2/Kv4.3 voltage-gated potassium channels. Drugs with similar potencies but different blocking mechanisms have differing effects on action potential duration (APD). We used in silico analysis to determine the effect of IKtof-blocking drugs with different blocking mechanisms on mouse ventricular myocytes. We used our existing mouse model of the action potential, and developed 4 new Markov formulations for IKtof, IKtos, IKur, IKs. We compared effects of theoretical IKtof-specific channel blockers: (1) a closed state, and (2) an open channel blocker. At concentrations lower or close to IC50, the drug which bound to the open state always had a much greater effect on APD than the drug which bound to the closed state. At concentrations much higher than IC50, both mechanisms had similar effects at very low pacing rates. However, an open state binding drug had a greater effect on APD at faster pacing rates, particularly around 10 Hz. In summary, our data indicate that drug effects on APD are strongly dependent not only on IC50, but also on the drug binding state.

The effects of allitridi and amiodarone on the conduction system and reverse use-dependence in the isolated hearts of rats with myocardial infarction

Ethnopharmacological relevanceAllium sativum L. (DaSuan in Mandarin) is a traditional Chinese herb that has been used to prevent and heal cardiovascular diseases. ObjectiveTo study the effects of allitridi (an active constituent of Allium sativum L.) and amiodarone on the conduction system and on reverse use-dependence in the isolated hearts of normal rats and rats with myocardial infarction (MI). Materials and methodsMale Sprague Dawley rats, with a ligated left anterior descending coronary artery, were used as myocardial infarction models to investigate the biological effects of the traditional Chinese herb. A single-phase electrode assay and isolated heart perfusion administration methods were employed to study and compare the electrophysiological effects of allitridi and amiodarone on normal and MI rats. Monophasic action potential (MAP) in vitro, effective refractory period (ERP) and monophasic action potential duration (MAPD)/ERP were measured to investigate reverse use-dependence (RUD) with allitridi and amiodarone. Moreover, bundle maps and heart rates were analyzed to evaluate the electrophysiological effects of allitridi on the conduction system of the cardiac muscles. Coronary flow was used to study the beneficial effects of the two drugs on the bundle of His in myocardial infraction. Results(1) Allitridi and amiodarone can reduce the infarction model of the His bundle (A-H, H-V) conduction and cardiac sinus rhythm in normal rats and isolated rat hearts. After washing in physiological solution (AK-H) for 15min, the allitridi group partially recovered, but the amiodarone group did not recover. (2) Allitridi and amiodarone had no significant effects on the change of MAPD90 or ERP in normal and MI rat hearts at different pacing frequencies (200, 250 and 300 beats/min), which indicated no RUD. In addition, the effects of allitridi on prolonging MAPD90 and ERP were weaker than those of amiodarone (P<0.01). The effects of allitridi on myocardial repolarization and its variation rate were also weaker than those of amiodarone (P<0.01). However, the prolonged administration of allitridi still did not cause RUD. Allitridi and amiodarone can significantly increase the ERP/APD90 rate of the isolated heart ventricles of normal rats and rats with MI. ConclusionWe propose that allitridi and amiodarone have similar effects on the cardiac conduction system and on the electrophysiology without RUD, which may be the result of the use of multi-channel blockers, such as calcium channel blockers and IKr and IKs channel blockers. Allitridi may be a promising antiarrythmic drug.

Modulation of the late sodium current by ATX-II and ranolazine affects the reverse use-dependence and proarrhythmic liability of IKrblockade

Drug-induced torsades de pointes (TdP) often occurs during bradycardia due to reverse use-dependence. We tested the hypothesis that inhibition or enhancement of late sodium current (I(Na,L) ) could modulate the drug-induced reverse use-dependence in QT and T(p-e) (an index of dispersion of repolarization), and therefore the liability for TdP. Arterially perfused rabbit left ventricular wedge preparations were used. Action potentials from the endocardium were recorded simultaneously with a transmural ECG. The effects of Anemonia sulcata toxin (ATX-II) (an I(Na,L) enhancer), d,l-sotalol, clarithromycin and ranolazine (an I(Na,L) blocker) on rate-dependent changes in QT, T(p-e) and proarrhythmic events were tested, either alone or in combination. Rate-dependent QT and T(p-e) slopes and TdP score (a combined index of TdP liability) were calculated at control and during drug infusion. ATX-II (30 nM) and sotalol (300 µM) caused a marked increase in QT and T(p-e) intervals, steeper QT-basic cycle length (BCL) and T(p-e) -BCL slopes (i.e. reverse use-dependence), and TdP. Addition of ranolazine (15 µM) to ATX-II or sotalol significantly attenuated QT-BCL, T(p-e) -BCL slopes and the increased TdP scores. In contrast, clarithromycin (100 µM) moderately prolonged QT and T(p-e) without causing R-on-T extrasystole or TdP, but addition of ATX-II (1 nM) to clarithromycin markedly amplified the QT-BCL and T(p-e) -BCL slopes and further increased TdP score. Modulation of I(Na,L) altered drug-induced reverse use-dependence related to QT as well as T(p-e) , indicating that inhibition of I(Na,L) can markedly reduce the TdP liability of agents that prolong QT intervals.

Contribution of late sodium current (INa-L) to rate adaptation of ventricular repolarization and reverse use-dependence of QT-prolonging agents

Abnormal rate adaptation of ventricular repolarization is arrhythmogenic. There is controversy on the underlying ionic mechanisms for rate-dependent change in repolarization. The purpose of this study was to examine the role of the late sodium current (I(Na-L)) in normal rate-dependence of ventricular repolarization and reverse use-dependence of QT-prolonging agents. The effects of I(Na-L) blockade, I(Na-L) enhancement, I(Kr) blockade, and changes in extracellular potassium concentration ([K(+)](o)) on rate adaptation of the QT interval and action potential duration (APD) were examined in isolated rabbit ventricular wedges and single myocytes. Rate dependence of I(Na-L), delayed rectifier potassium current (I(K)), and L-type calcium current (I(Ca)) was determined using a whole-cell, voltage clamp technique. At control, APD exhibited rate-dependent changes in the multicellular preparations as well as in the isolated single ventricular myocytes when [K(+)](o) remained constant. The rate dependence of APD was significantly enhanced by reduction of [K(+)](o) from 4 to 1 mM or by I(Na-L) enhancement but was markedly blunted by the selective sodium channel blocker tetrodotoxin. The I(Kr) blocker dofetilide (3 nM) amplified the QT to basic cycle length slope (71.2 ± 13.1 ms/s vs 35.1 ± 8.8 ms/s in control, n = 4, P <.05). This reverse use-dependence was abolished by tetrodotoxin at 5 μM (11.4 ± 4.3 ms/s, n = 4, P <.01). There were no significant differences in I(Ca) or I(K) over the range of basic cycle lengths from 2,000 to 500 ms. However, I(Na-L) exhibited a significant rate-dependent reduction. I(Na-L) is sensitive to rate change due to its slow inactivation and recovery kinetics and plays a central role in the rate dependence of APD/QT and in the reverse use-dependence of select APD/QT-prolonging agents.

Effects of Bepridil Versus E-4031 on Transmural Ventricular Repolarization and Inducibility of Ventricular Tachyarrhythmias in the Dog

Bepridil (a multiple channel blocker) may markedly prolong the QT interval and induce polymorphic ventricular tachyarrhythmias (VTA). We compared the transmural ventricular repolarization characteristics and inducibility of polymorphic VTA after administration of bepridil versus the pure I(Kr) blocker, E-4031, each administered to five open-chest dogs. We used plunge needle electrode to record transmural left ventricular (LV) repolarization and activation-recovery interval (ARI) to estimate local repolarization. The correlation between paced cycle length and ARI was separately examined in the LV endocardium, mid-myocardium (Mid), and epicardium. Attempts to induce VTA were made during bradycardia and sympathetic stimulation. Bepridil and E-4031 prolonged QT interval and ARI in all LV layers, though the magnitude of prolongation was greatest in Mid, increasing the transmural ARI dispersion, particularly during bradycardia. Compared with E-4031, bepridil caused mild, reverse use-dependent changes in ventricular repolarization, and less ARI dispersion than E-4031 during slow ventricular pacing. Both drugs increased ARI(max) and cycle length at 50% of ARI(max), though the changes were smaller after bepridil than after E-4031 administration. Bradycardia after the administration of each drug induced no VTA; however, sympathetic stimulation induced sustained polymorphic VTA in two of five dogs treated with E-4031 versus no dog treated with bepridil. Unlike the pure I(kr) blocker, E-4031, bepridil exhibited weak properties of reverse use-dependency and protected against sympathetic stimulation-induced VTA. It may be an effective supplemental treatment for recipients of implantable cardioverter defibrillator.

Validation of a guinea pig Langendorff heart model for assessing potential cardiovascular liability of drug candidates

IntroductionCardiac liabilities represent a major cause of recent withdrawal of marketed drugs and also for the high attrition rate evidenced during late stage drug development. To identify molecules with potential cardiovascular risks early in drug development, a screening model of ex vivo Langendorff hearts has been validated with 26 reference compounds of various chemical and therapeutic classes. MethodsThe hearts of adult guinea pigs were maintained by retrograde perfusion in Langendorff mode, beating spontaneously at sinus rhythm or paced via the right atrium at 200 and 300 beats per minute. Multiple parameters consisting of hemodynamic function (coronary and left ventricle pressure), cardiac electrophysiology (electrocardiogram and monophasic action potential) and indices of arrhythmogenesis (triangulation, reverse-use dependence, repolarization dispersion and beat-to-beat instability), together with overt arrhythmia were evaluated simultaneously. Ascending concentrations up to either 100-fold of the determined hERG IC50 or nominally 100 µM were routinely tested utilizing 4–6 hearts per compound. ResultsEach compound exhibited a unique cardiovascular profile: (i) the majority displayed concentration and heart rate-dependent mixed-ion channel or multiple-target effects that frequently resulted in bradycardia, atrioventricular block, negative inotropy, coronary vasodilatation, and QRS widening. (ii) Compounds associated with high arrhythmogenic risk in the clinic exhibited more “positive signals” at concentrations within 30-fold of their maximal therapeutic free plasma concentration than those with less arrhythmogenic potential. (iii) For several potent torsadogens, proarrhythmic indices other than the prolongation of QT/QTc and MAP duration appeared more sensitive in representing proarrhythmic liability. (iv) A scoring system was developed to assist in the rank-ordering of potential cardiotoxicants. DiscussionThe cardiovascular action of reference compounds profiled by this isolated heart model was generally consistent with their known mechanisms and, except for the sinus heart rate, correlated well with that observed in the clinic. Further, the overall cardiac liability estimated by the scoring system matched the clinical documentation, suggesting this model could serve as a valuable tool in early cardiovascular drug safety assessment.

Ultrafast sodium channel block by dietary fish oil prevents dofetilide-induced ventricular arrhythmias in rabbit hearts

Several epidemiologic and clinical studies show that following myocardial infarction, dietary supplements of omega-3 polyunsaturated fatty acids (omega3FA) reduce sudden death. Animal data show that omega3FA have antiarrhythmic properties, but their mechanisms of action require further elucidation. The effects of omega3FA supplementation were studied in female rabbits to analyze whether their antiarrhythmic effects are due to a reduction of triangulation, reverse use-dependence, instability, and dispersion (TRIaD) of the cardiac action potential (TRIaD as a measure of proarrhythmic effects). In Langendorff-perfused hearts challenged by a selective rapidly activating delayed rectifier potassium current inhibitor that has been shown to exhibit proarrhythmic effects (dofetilide; 1 to 100 nM), omega3FA pretreatment (30 days; n=6) prolonged the plateau phase of the monophasic action potential; did not slow the terminal fast repolarization; reduced the dofetilide-induced prolongation of the action potential duration; reduced dofetilide-induced triangulation; and reduced dofetilide-induced reverse use-dependence, instability of repolarization, and dispersion. Dofetilide reduced excitability in omega3FA-pretreated hearts but not in control hearts. Whereas torsades de pointes (TdP) were observed in five out of six in control hearts, none were observed in omega3FA-pretreated hearts. Docosahexaenoic acid (DHA) inhibited the sodium current with ultrafast kinetics. Dietary omega3FA supplementation markedly reduced dofetilide-induced TRIaD and abolished dofetilide-induced TdP. Ultrafast sodium channel block by DHA may account for the antiarrhythmic protection of the dietary supplements of omega3FA against dofetilide-induced proarrhythmia observed in this animal model.

Selective block of IKs plays a significant role in MAP triangulation induced by IKr block in isolated rabbit heart

Introduction The role of IKr (rapidly-activating delayed rectifier K + current) block in triangulation of monophasic action potentials (MAP) and in development of torsade de pointes (TdP) arrhythmia is known. Combined IKr and IKs (slowly-activating delayed rectifier K + current) block has been demonstrated to promote TdP. The aim of this study was to describe a possible implication of IKs block in MAP triangulation. Methods: Four contact electrodes were placed on the epicardium of the left ventricle of Langendorff-perfused rabbit hearts to record monophasic action potentials (MAP), with an IKr blocker d, l-sotalol (3 to 100 µM, n = 6) or a non-selective IKr blocker, quinidine (1 to 30 µM, n = 6). Their effects were assessed with or without a specific IKs blocker chromanol 293B (20 µM, n = 6), on MAP duration at 30, 60 and 90% of repolarization (APD30, 60 and 90, respectively) and MAP triangulation (APD90-APD30) at 1 and 0.2 Hz. Results: d, l-sotalol increased significantly APD90 and triangulation with reverse use-dependency for concentrations ≥ 10 µM. Quinidine markedly prolonged APD90 and triangulation with reverse use-dependency at concentrations ≥ 3 µM. Chromanol 293B alone had no effects on APD, but when combined with d, l-sotalol or quinidine (i) increased APD prolonging effects, (ii) lowered values of pro-arrhythmic concentrations, (iii) increased incidence and length of d, l-sotalol- or quinidine-induced Early Afterdepolarizations (EADs) and TdP. All these events were primarily due to an important slowing of final repolarization, i.e. a marked increased triangulation. Conclusion: IKs, even of low amplitude in rabbits, plays a key role in ventricular repolarization. IKs is involved in prolonged MAP duration mainly by triangulation and subsequent increased drug arrhythmogenicity. Therefore drug affinity for IKs must be evaluated with IKr studies as part of preclinical drug cardiac safety assessment.

Cardiac repolarization and its relation to ventricular geometry and rate in reverse remodelling during antihypertensive therapy with irbesartan or atenolol: results from the SILVHIA study

Hypertensive left ventricular (LV) hypertrophy is associated with a substantial risk for malignant arrhythmias and sudden death. According to recent results, antihypertensive therapy with the angiotensin II type 1 receptor blocker irbesartan reverses both structural and electrical remodelling. However, the relation between the LV geometric pattern (concentric vs eccentric) and electrical reverse remodelling has not been characterized, neither has the relation between repolarization and rate (QT/RR and JT/RR relation), which presumably reflects the propensity for bradycardia-dependent ventricular arrhythmia. In this study, repeat echocardiographic and electrocardiographic measurements were performed in hypertensive patients with LV hypertrophy, randomized to double-blind therapy with irbesartan (n = 44) or the beta(1)-adrenoceptor blocker atenolol (n = 48) for 48 weeks; 53 patients had concentric and 39 eccentric LV hypertrophy. In addition, 37 matched hypertensive subjects without LV hypertrophy and no current therapy served as controls. Irbesartan induced structural and electrophysiological reverse remodelling, independent of LV geometry. In contrast, atenolol had similar beneficial effect only in patients with concentric LV hypertrophy, while the response in those with eccentric hypertrophy was unfavourable with both prolonged repolarization time and an increased QT/RR slope (suggesting reverse-use dependence). In conclusion, there is a significant geometry-related difference in the reverse remodelling processes induced by irbesartan and atenolol. Echocardiographic characterization of the geometry in hypertension-induced LV hypertrophy might become an important step in the selection of optimal antihypertensive therapy.

Impact of study design on proarrhythmia prediction in the SCREENIT rabbit isolated heart model

Introduction Prediction of the propensity of a compound to induce Torsades de Pointes continues to be a formidable challenge to the pharmaceutical industry. Development of an in vitro model for assessment of proarrhythmic potential offers the advantage of higher throughput and reduced compound quantity requirements when compared to in vivo studies. A rabbit isolated heart model (SCREENIT) has been reported to identify compounds with proarrhythmic potential based on the observance of compound-induced triangulation and instability of the monophasic action potential (MAP), ectopic beats, and reverse-use dependence of prolongation of the MAP duration. Previous reports have indicated that this model qualitatively identifies proarrhythmic compounds and suggest the use of this model to assign safety margins for human clinical use. The intent of this series of studies was to evaluate the impact of study design on the proarrhythmic concentration predicted by this model. Methods Nine compounds of varying proarrhythmic potential and a negative control were tested in a blinded fashion using a series of different experimental protocols: Compounds were tested at multiple concentration ranges and extended perfusion times were also evaluated. Results In general when the dataset is viewed as a whole, the model did identify proarrhythmic compounds, however the concentration at which action potential prolongation, triangulation, instability, reverse-use dependence and ectopic beats occurred often varied based on the concentration range selected. Further analysis using extended compound perfusion times demonstrated that variability may be due in part to lack of adequate equilibration of compound with the cardiac tissue. Discussion We report that the model correctly identified proarrhythmic agents in a qualitative manner, but that study design impacts the proarrhythmic concentration derived from the model.

Proarrhythmia as a Class Effect of Quinolones: Increased Dispersion of Repolarization and Triangulation of Action Potential Predict Torsades de Pointes

Numerous noncardiovascular drugs prolong repolarization and thereby increase the risk for patients to develop life-threatening tachyarrhythmias of the torsade de pointes (TdP) type. The development of TdP is an individual, patient-specific response to a repolarization-prolonging drug, depending on the repolarization reserve. The aim of the present study was to analyze the underlying mechanisms that discriminate hearts that will develop TdP from hearts that will not develop TdP. We therefore investigated the group of quinolone antibiotics that reduce repolarization reserve via I(Kr) blockade in an intact heart model of proarrhythmia. In 47 Langendorff-perfused, AV-blocked rabbit hearts, ciprofloxacin (n = 10), ofloxacin (n = 14), levofloxacin (n = 10), and moxifloxacin (n = 13) in concentrations from 100 microM to 1,000 microM were infused. Eight monophasic action potentials (MAPs) and an ECG were recorded simultaneously. After incremental pacing at cycle lengths from 900 ms to 300 ms to compare the action potential duration, potassium concentration was lowered to provoke TdP. All antibiotics led to a significant increase in QT interval and MAP duration, and exhibited reverse-use dependence. Eight simultaneously recorded MAPs demonstrated an increase in dispersion of repolarization in the presence of all antibiotics. MAP triangulation (ratio: MAP(90/50)) and fluctuation of consecutive action potentials were increased for all tested drugs at high concentrations. In the presence of low potassium concentration, all quinolones led to TdP: ciprofloxacin, 4 out of 10 (40%); ofloxacin, 3 out of 14 (21%); moxifloxacin, 9 out of 13 (69%); and levofloxacin, 2 out of 10 (20%). Hearts that developed TdP demonstrated a significant greater influence on dispersion of repolarization and on triangulation as compared with hearts without TdP. Quinolone antibiotics may be proarrhythmic due to a significant effect on myocardial repolarization. The individual response of a heart to develop TdP in this experimental model is characterized by a greater effect on dispersion of repolarization and on triangulation of action potential as compared with hearts that do not develop TdP.

In-vitro experimental models for the risk assessment of antibiotic-induced QT prolongation

The prolongation of the ventricular repolarization and proarrhythmic effects (Torsade de Pointes: TdP) of five reference antibiotics were compared in four in-vitro models. 1. Using the patch clamp technique on the human ether-a-gogo-related gene (HERG) current, the rank order for blockade of the HERG-current (IC 50) was: sparfloxacin (44 μM) > telithromycin = moxifloxacin = erythromycin (± 100 μM). 2. Assessing their effects on action potential duration (APD 90) and incidence of early afterdepolarizations in isolated rabbit Purkinje fibers, the rank order was: sparfloxacin > moxifloxacin > telithromycin > erythromycin (prolongation of APD 90 at 100 μM: 83%, 48%, 33% and 17% from baseline compared to + 5% with solvent, P < 0.05, respectively). 3. Assessing the drug effects on the APD 60, triangulation, reverse use-dependency, and instability in isolated Langendorff-perfused rabbit hearts, the rank order was: moxifloxacin > erythromycin > sparfloxacin > telithromycin. 4. Assessing their torsadogenic potentials (scores of effects on QT-interval, peak of the T wave to end of T wave: T p–e, T p–e/QT ratio, R wave on T wave (R on T) and TdP in isolated rabbit left ventricular wedge preparations, the rank order for their TdP risk score was: sparfloxacin > erythromycin > moxifloxacin > telithromycin. Additional experiments with grepafloxacin indicate that the rank order to detect grepafloxacin-induced long QT was the wedge preparation > the Purkinje fiber > HERG > the isolated heart, where the isolated heart was unable to detect grepafloxacin-induced APD prolongation. The present study demonstrates that the first three in-vitro models can be used to assess the ability of antibiotic compounds to delay ventricular repolarization. However, with respect to their known clinical effects on QT and TdP incidence, the wedge preparation appears to be more predictive and suitable for detecting torsadogenic action of antibiotics.

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.

Azimilide dihydrochloride

Azimilide dihydrochloride is an antiarrhythmic drug with Vaughn Williams class III properties, which blocks both fast (IKr) and slow (IKs) components of the delayed rectifier cardiac potassium channel. The drug slows the heart rate slightly and, like other class III antiarrhythmic drugs, prolongs ventricular repolarization and thus, the QT interval. Unlike sotalol, another class III antiarrhythmic drug, azimilide does not exhibit reverse-use dependence, that is, its binding characteristics and effectiveness are not related to the heart rate. Azimilide is 85% bioavailable, reaches peak blood concentrations in 6–8 h and has a long elimination half-life of 114 h. Clinical trials have utilized once-daily dosing. These trials have tested the use of the drug for patients with supraventricular and ventricular arrhythmias.