3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Cardiovascular safety pharmacology profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor.

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 44.0µg/ml in HEK 293 cells. At 0.3 and 3µg/ml, etamicastat had no effects on the action potential (AP) in male dog Purkinje fibers. At 30µg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (-4%), AP duration at 60% (-14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious male dog, etamicastat (up to 20mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20mg/kg, the QTc interval was slightly prolonged (8-9% max, P<0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post-administration) were 1.4 and 3.7µg/ml at 10 and 20mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3µg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~0.2µg/ml).

Limitations of animal electrical cardiac safety models.

INTRODUCTION: Human electrical safety standards are based almost exclusively on animal studies and there is an unjustified assumption that ventricular fibrillation (VF) thresholds in animals are the same as those in humans. METHODS AND RESULTS: We analyzed differences between animals and humans in cardiac stimulation. A broad literature survey revealed that swine are a fragile electrophysiologic research species and have a dense intramural Purkinje fiber network, which is not found in some other species, including humans. Anesthesia agents have to be chosen carefully as swine are prone to malignant hyperthermia. Cardiac stimulation thresholds depend on weight and capture rates. Thus, the animal weight has to be representative of the weight of human subjects. Studies have shown significant ECG differences between humans and other species, including swine and canine. At least one study suggested that rabbit hearts tend to develop VF in a manner more similar to that seen in humans. CONCLUSION: Animal studies can play a role in conservatively evaluating cardiac safety. However, while still abiding by the precautionary principle, animal study design has to take into account the significant anatomical and electrophysiological differences between humans and other mammals. Data from multiple animal models may offer broader perspectives. If attempts are made to extrapolate animal results to humans then appropriate numerical correction factors should be applied, such as some of those discussed in this article.

A less aggressive therapeutic option for electrical storm.

Electrical storm (ES) describes the rapidly clustering ventricular fibrillation (VF) that requires multiple cardioversions. Emerging evidence suggests that Purkinje arborization and sympathetic nerve regeneration play a major role in initiating malignant arrhythmias. We report the case of two patients who, after having survived an acute myocardial infarction (MI), developed repetitive episodes of polymorphic ventricular tachycardia and VF one week after percutaneous revascularization, triggered by monomorphic premature ventricular contractions (PVCs). Owing to repetitive and drug-refractory VF episodes, temporary atrial overdrive pacing was attempted with complete suppression of VF. In the following month, recurrence of ventricular arrhythmia was inversely related to the atrial pacing rate. Although antiarrhythmic drugs other than beta-blockers had been discontinued, neither PVCs nor ventricular arrhythmias recurred at one-month follow-up when the lower pacing rate was set at 60 bpm. In conclusion in these patients, ES was likely related to nerve sprouting after ischemic injury. This chaotic phenomenon occurs early after tissue damage and shows a peak seven days after acute MI with degeneration of superfluous axon branches. High pacing rates can reduce early after depolarizations and suppress PVCs, thus preventing ES. On these grounds, ES patients may be treated with temporary overdrive pacing rather than early radiofrequency ablation.

Remodeling of the peripheral cardiac conduction system in response to pressure overload.

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.

Effects of nortriptyline on QT prolongation: a safety pharmacology study.

Nortriptyline, a second-generation tricyclic antidepressant, is an active metabolite of amitriptyline. Amitriptyline induces QT prolongation and torsades de pointes (TdP), which causes sudden death. We studied the cardiovascular safety of nortriptyline, including QT prolongation risk. We examined the effects of nortriptyline on the cardiovascular system in vivo and in vitro in accordance with the ICH-S7B guideline. We tested its effect on QT interval in conscious telemetered dogs. We also performed in vitro electrophysiological studies on hERG tail currents using stably transfected human embryonic kidney 293 (HEK293) cells. Action potential parameters were studied in isolated rabbit purkinje fibers. Nortriptyline dose-dependently blocked hERG current, with a tail IC(50) value of 2.20 ± 0.09 µM (n = 4). In the APD assay, total amplitude, Vmax, and resting membrane potential were not significantly changed by 1 µM nortriptyline, but nortriptyline at 0.3 and 1 µM shortened APD(50) and APD(90). Nortriptyline did not affect QTcV at 2 or 6 mg/kg, but slightly increased QTcV at 20 mg/kg. In conclusion, it is unlikely that nortriptyline affects the ventricular repolarization process at therapeutic dosages.

QTc shortening with a new investigational cancer drug: a brief case study.

INTRODUCTION: BAY-79 is an inhibitor of receptor tyrosine kinases with high selectivity versus other kinases. Species scaling, complicated by nonlinear pharmacokinetics, predicted a C(max.u) of 36-178nmol/L at the human efficacious exposure. METHODS: Preclinical cardiovascular safety pharmacology studies assessed currents (hERG, I(Na)), action potential (AP, rabbit Purkinje fiber), hemodynamic/ECG parameters (anesthetized Beagle dogs, intravenous infusion), and proarrhythmic potential (rabbit Langendorff heart Screenit model). RESULTS: Both hERG K(+) current and hNav1.5 Na(+) current were inhibited with low potency (IC(20)>10micromol/L). Purkinje fiber APs remained unaffected at 10micromol/L, but at 100micromol/L displayed reverse use-dependent AP duration shortening (APD(90)-33% at 1Hz) and triangulation. Infusion of BAY-79 into anesthetized dogs was associated with moderate hemodynamic effects (increased heart rate and diastolic blood pressure, reduced stroke volume) and marked QTcV shortening (-25ms) starting at approximately 0.65micromol/L (unbound); QRS was not changed. Assessment of the proarrhythmic potential in the Screenit model showed effects (AP duration shortening, triangulation, instability, reduced coronary flow, slowed conduction) at > or =30micromol/L (0.5h/concentration) and at 3micromol/L with longer exposure (2.5h/concentration). DISCUSSION: BAY-79 at plasma concentrations slightly higher than those predicted to be therapeutically efficacious in humans is associated with QTc shortening in dogs but of unclear mechanistic basis. The QTc shortening associated proarrhythmic potential of BAY-79 together with other considerations finally resulted in an unfavorable risk-benefit assessment.

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.

Droperidol and ondansetron in vitro electrophysiological drug interaction study.

Droperidol and ondansetron are potent anti-emetic agents which are often administered together. Although both drugs prolong QT interval in man by inhibition of Human Ether-a-go-go Related Gene-coded potassium channels, only droperidol was tested using more integrated experimental models. Therefore, we studied the effects of both compounds and their combination on action potentials (AP) of rabbit Purkinje fibers using conventional intracellular glass microelectrode. Purkinje fibers, driven at 1 Hz, were exposed to increasing concentrations (from 0.001 to 10 microm) of droperidol (n = 7) or ondansetron (n = 8) at 30 min intervals at 36.5 degrees C. Other fibers were exposed to a constant droperidol concentration (0.1 microm) alone (n = 7) or together with the same increasing concentrations of ondansetron (n = 6). Droperidol increased AP duration measured at 90% repolarization (APD90) in a concentration-dependent manner from 4.4 +/- 0.8% (mean +/- SEM) after 1 nm to a maximum of 158 +/- 72% after 1 microm. Ondansetron significantly increased APD90 by 5.3 +/- 2.1% at 100 nm up to 76 +/- 14% after 10 microm. Early after-depolarization occurred in 6/7 fibers exposed to droperidol and 1/8 fibers exposed to ondansetron. When given together, pure additive effects were observed. The concentrations that increased APD90 by 50% were 0.25 +/- 0.25 microm droperidol, 3.8 +/- 2.4 microm ondansetron and 1.5 +/- 0.8 microm ondansetron when given together with droperidol. Both ondansetron and droperidol prolong AP duration in Purkinje fibers, droperidol being 10 times more potent than ondansetron. Combination of ondansetron and droperidol exhibits an additive effect on AP duration. However, within clinically relevant concentrations, ondansetron does not further increase the AP prolongation caused by droperidol alone.

Mathematical models of the electrical action potential of Purkinje fibre cells.

Early development of ionic models for cardiac myocytes, from the pioneering modification of the Hodgkin-Huxley giant squid axon model by Noble to the iconic DiFrancesco-Noble model integrating voltage-gated ionic currents, ion pumps and exchangers, Ca(2+) sequestration and Ca(2+)-induced Ca(2+) release, provided a general description for a mammalian Purkinje fibre (PF) and the framework for modern cardiac models. In the past two decades, development has focused on tissue-specific models with an emphasis on the sino-atrial (SA) node, atria and ventricles, while the PFs have largely been neglected. However, achieving the ultimate goal of creating a virtual human heart will require detailed models of all distinctive regions of the cardiac conduction system, including the PFs, which play an important role in conducting cardiac excitation and ensuring the synchronized timing and sequencing of ventricular contraction. In this paper, we present details of our newly developed model for the human PF cell including validation against experimental data. Ionic mechanisms underlying the heterogeneity between the PF and ventricular action potentials in humans and other species are analysed. The newly developed PF cell model adds a new member to the family of human cardiac cell models developed previously for the SA node, atrial and ventricular cells, which can be incorporated into an anatomical model of the human heart with details of its electrophysiological heterogeneity and anatomical complexity.

Epicardial and intramural excitation during ventricular pacing: effect of myocardial structure.

Published studies show that ventricular pacing in canine hearts produces three distinct patterns of epicardial excitation: elliptical isochrones near an epicardial pacing site, with asymmetric bulges; areas with high propagation velocity, up to 2 or 3 m/s and numerous breakthrough sites; and lower velocity areas (<1 m/s), where excitation moves across the epicardial projection of the septum. With increasing pacing depth, the magnitude of epicardial potential maxima becomes asymmetric. The electrophysiological mechanisms that generate the distinct patterns have not been fully elucidated. In this study, we investigated those mechanisms experimentally. Under pentobarbital anesthesia, epicardial and intramural excitation isochrone and potential maps have been recorded from 22 exposed or isolated dog hearts, by means of epicardial electrode arrays and transmural plunge electrodes. In five experiments, a ventricular cavity was perfused with diluted Lugol solution. The epicardial bulges result from electrotonic attraction from the helically shaped subepicardial portions of the wave front. The high-velocity patterns and the associated multiple breakthroughs are due to involvement of the Purkinje network. The low velocity at the septum crossing is due to the missing Purkinje involvement in that area. The asymmetric magnitude of the epicardial potential maxima and the shift of the breakthrough sites provoked by deep stimulation are a consequence of the epi-endocardial obliqueness of the intramural fibers. These results improve our understanding of intramural and epicardial propagation during premature ventricular contractions and paced beats. This can be useful for interpreting epicardial maps recorded at surgery or inversely computed from body surface ECGs.

Action potential experiments complete hERG assay and QT-interval measurements in cardiac preclinical studies.

INTRODUCTION: The ICHS7B guideline focused on hERG and QT assays, although other factors have also been linked with the induction of severe arrhythmias. Thus, the aim of the present study was to demonstrate that two in vitro action potential recordings constitute convincing models of predictive drug-induced Torsades de pointes (TdP) and re-entry arrhythmias. METHODS: The effects of D,L-sotalol, flecainide and quinidine were investigated on potassium (hERG) and sodium (Na(V)1.5) currents transfected in HEK-293 cells to determine the repercussion of the blockade of these currents on rabbit Purkinje fibre (PF) and atrial action potentials. Atrial conduction velocity was also investigated as a model of re-entry arrhythmias. RESULTS: hERG channels were blocked by D,L-sotalol, quinidine and flecainide (IC(50): 69, 0.33 and 0.74 micromol/L, respectively). D,L-sotalol (30 micromol/L) induced reverse-use dependent increases in action potential duration (APD(90): +31.7% and +81.2% at 1 and 0.2 Hz) and triangulation (APD(90-40): +34.7% and +73.6% at 1 and 0.2 Hz) in PF but not in atria. Quinidine (10 micromol/L) also increased APD(90) (+14.5% and +68.5% at 1 and 0.2 Hz) and APD(90-40) (+73.3% and +152.1% at 1 and 0.2 Hz) in PF. Flecainide (10 micromol/L) shortened APD(90) in PF (-26.0% and - 22.2% at 1 and 0.2 Hz). Quinidine and flecainide blocked Na(V)1.5 channels by 32.3% and 73.1%, respectively, and produced decreases in dV/dt(max) which were more marked in atria (-20.4% and -31.9%) compared to PF (-12.8% and 22.4%) at 1 Hz. Finally, quinidine and flecainide decreased atrial conduction speed by 14.6% and 30.8%, respectively. CONCLUSION: Results obtained with flecainide demonstrate that use of the hERG channel alone should not be considered as a useful single assay. Rabbit Purkinje fiber action potentials can be considered as a comparable model for detection of reverse-use dependent APD prolongation and triangulation whereas the rabbit atria can be considered as a useful model for detection of sodium channel blockade associated with decreases in dV/dt(max) and conduction velocity.

Suitability of commonly used excipients for electrophysiological in-vitro safety pharmacology assessment of effects on hERG potassium current and on rabbit Purkinje fiber action potential.

INTRODUCTION: Regulatory guidelines require investigation of the liability for delayed ventricular repolarization by new chemical entities within a broad concentration range in-vitro. However, investigation can be limited by poor drug aqueous solubility, and by solvent physicochemical attributes that disrupt cell membrane integrity. Although excipients or solubilizing agents may aid to achieve the necessary high concentrations, no comprehensive overview on the suitability of solvents for in-vitro electrophysiological safety studies exists. METHODS: Excipients were tested for potential interference with the hERG (human ether-a-go-go-related gene) K(+) current (whole-cell voltage-clamp, 23+/-2 degrees C), and the shape of rabbit Purkinje fiber action potentials (conventional glass microelectrode technique, 37+/-1 degrees C). RESULTS AND DISCUSSION: Water-soluble complexation builders/carriers had little effect on hERG K(+) current at up to 50 mg/ml (BSA, bovine serum albumin) and 11 mg/ml (HP-beta-CD, hydroxypropyl-beta-cyclodextrin; IC(20), concentration of 20% inhibition). Water-soluble organic (co)solvents inhibited hERG K(+) currents (IC(20), %/mM): 0.7/152, ethanol; 0.9/67, Transcutol; 1.2/154, DMSO (dimethylsulfoxide); 1.6/389, acetonitrile; 1.9/48, polyethylene glycol 400; 2.1/660, methanol. Part of their inhibitory effect is attributed to the osmolality of extracellular solutions, because hERG IC(20) and extrapolated osmolality at the hERG IC(20) strongly correlate. Water-soluble non-ionic solubilizers/surfactants are potent inhibitors of hERG K(+) current with IC(20) concentrations of 0.07% (Cremophor EL) or lower (Tween 20, Tween 80: approximately 0.001%). Part of this inhibitory effect is attributed to their interaction with lipid membranes, because hERG inhibition occurs close to critical micelle concentrations (Cremophor, approximately 0.009%; Tween 20, approximately 0.007%). Purkinje fiber action potentials are little affected by HP-beta-CD at up to 2 mg/ml, while DMSO tends to shorten the action potential duration at 1%. CONCLUSION: When conducting electrophysiological in-vitro assessments of drug effects, solubilizers/surfactants (Cremophor EL, Tween 20, Tween 80) should be avoided. Instead, water-soluble organic (co)solvents (methanol, acetonitrile, DMSO) or complexation builders/carriers (HP-beta-CD, BSA) appear to be more favorable.

Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

sigma(2)-receptor ligand-mediated inhibition of inwardly rectifying K(+) channels in the heart.

The sigma(2)-receptor agonist, ifenprodil, was suggested as an inhibitor of G protein-coupled inwardly rectifying potassium channels. Nevertheless, an analysis of the role of sigma(2) receptors in cardiac electrophysiology has never been done. This work aims i) to identify the roles of cardiac sigma(2) receptors in the regulation of cardiac K(+) channel conductances and ii) to check whether sigma(2)-receptor agonists exhibit class III antiarrhythmic properties. The sigma(2)-receptor agonists ifenprodil, threo-ifenprodil, LNP250A [threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decane-4-one] (a derivative of ifenprodil devoid of alpha(1)-adrenergic and N-methyl-d-aspartate glutamate receptor-blocking properties), and 1,3-di(2-tolyl)guanidine were used to discriminate the effects linked to sigma(2) receptors from those of the sigma(1) subtype, induced by (+/-)-N-allylnormetazocine (SKF-10,047). The sigma(2)-receptor antagonist 3-alpha-tropanyl-2(pCl-phenoxy)butyrate (SM-21) was employed to characterize sigma(2)-mediated effects in patch-clamp experiments. In rabbits, all sigma(2)-receptor agonists reduced phenylephrine-induced cardiac arrhythmias. They prolonged action potential duration in rabbit Purkinje fibers and reduced human ether-a-go-go-related gene (HERG) K(+) currents. (+)-SKF-10,047 was completely inactive in the last two tests. The effects of threo-ifenprodil were not antagonized by SM-21. In HERG-transfected COS-7 cells, SM-21 potentiated the ifenprodil-induced blockade of the HERG current. These data suggest that sigma(2)-receptor ligands block I(Kr) and that this effect could explain part of the antiarrhythmic properties of this ligands family. Nevertheless, an interaction with HERG channels not involving sigma(2) receptors seems to share this pharmacological property. This work shows for the first time that particular caution has to be taken toward ligands with affinity for sigma(2) receptors. The repolarization prolongation and the early-afterdepolarization can be responsible for "torsades de pointe" and sudden cardiac death.

Effect of clebopride, antidopaminergic gastrointestinal prokinetics, on cardiac repolarization.

The inhibition of the potassium current I(Kr) and QT prolongation has been known to be associated with drug-induced torsades de pointes arrhythmias (TdP) and sudden cardiac death. In this study, the authors investigated the cardiac electrophysiological effects of clebopride, a class of antidopaminergic gastrointestinal prokinetic, that has been reported to prolong the QT interval by using the conventional microelectrode recording techniques in isolated rabbit Purkinje fiber and whole-cell patch clamp techniques in human ether-à-go-go-related gene (hERG)-stably transfected Chinese hamster ovarian (CHO) cells. Clebopride at 10 microM significantly decreased the Vmax of phase 0 depolarization (p < .05) and significantly prolonged the action potential duration at 90% repolarization (APD90) (p < .01), whereas the action potential duration at 50% repolarization (APD50) was not prolonged. For hERG potassium channel currents, the IC50 value was 0.62 +/- 0.30 microM. Clebopride was found to have no effect on sodium channel currents. When these results were compared with Cmax (1.02 nM) of clinical dosage (1 mg, [p.o.]), it can be suggested that clebopride is safe at the clinical dosage of 1 mg from the electrophysiological aspect. These findings indicate that clebopride, an antidopaminergic gastrointestinal prokinetic drug, may provide a sufficient "safety factor" in terms of the electrophysiological threshold concentration. But, in a supratherapeutic concentration that might possibly be encountered during overdose or impaired metabolism, clebopride may have torsadogenic potency.

Discovery of 3-methyl-N-(1-oxy-3',4',5',6'-tetrahydro-2'H-[2,4'-bipyridine]-1'-ylmethyl)benzamide (ABT-670), an orally bioavailable dopamine D4 agonist for the treatment of erectile dysfunction.

The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability. Further optimization with the (N-oxy-2-pyridinyl)piperidine template led to the discovery of compound 6b (ABT-670), which exhibited excellent oral bioavailability in rat, dog, and monkey (68%, 85%, and 91%, respectively) with comparable efficacy, safety, and tolerability to 1a. The N-oxy-2-pyridinyl moiety not only provided the structural motif required for agonist function but also reduced metabolism rates. The SAR study leading to the discovery of 6b is described herein.

ILSI-HESI cardiovascular safety subcommittee initiative: evaluation of three non-clinical models of QT prolongation.

INTRODUCTION: Drugs that delay cardiac repolarization pose potential safety risks to patients and cause serious regulatory concern because of the link between QT interval prolongation and the potentially fatal arrhythmia torsades de pointes (TdP). Predicting which drugs will cause TdP is an inexact and difficult science. The utility of non-clinical assays was not well understood due in part to variability in methods, species, and consistency in the assays reported in the literature. The Health and Environmental Sciences Institute of the International Life Sciences Institute (ILSI/HESI) outlined a set of studies to determine how well selected commonly used non-clinical assays identified compounds known to cause TdP and prolong QT interval in humans. METHODS: Compounds known to prolong ventricular repolarization and compounds considered safe by years of clinical use were tested in three assays: HERG ionic current, Purkinje fiber repolarization, and in vivo QT studies in conscious telemeterized dogs. RESULTS: The data from each of these assays demonstrate that compounds that may pose a proarrhythmia risk for patients can be distinguished from those that are considered safe. DISCUSSION: Taken collectively, the in-vitro and in-vivo preclinical results can be integrated to develop an accurate preclinical risk assessment to support clinical safety.

The time course of engraftment of human mesenchymal stem cells in fetal heart demonstrates that Purkinje fiber aggregates derive from a single cell and not multi-cell homing.

OBJECTIVE: To study the early time course of engraftment of human mesenchymal stem cells in fetal sheep heart and determine the relative roles of proliferation and homing in formation of aggregates of human Purkinje fiber cells. METHODS: The human sheep xenograft model was utilized for these studies. Prior to injection in the preimmune fetus, human cells were labeled with fluorescent dyes to be able to track human cells at early times of engraftment. RESULTS: Human stem cells were detected in fetal hearts between 29 and 39 hours after intraperitoneal injection. Engraftment was primarily in the Purkinje fiber system. By 45 hours engrafted human cells had a cardiac phenotype. When two groups of human mesenchymal stem cells, each labeled with a different fluorescent dye, were combined prior to injection, aggregates of human Purkinje fiber cells contained cells labeled with either one dye or the other, no aggregate contained cells labeled with both dyes. CONCLUSIONS: Human mesenchymal stem cells introduced into fetal sheep rapidly enter the myocardium. The swift differentiation into a cardiac phenotype indicates that the cardiac milieu has a strong influence on the fate of engrafting human mesenchymal stem cells. The absence of any aggregates of human Purkinje fiber cells containing both fluorescent dyes demonstrates that each aggregate of human Purkinje fiber cells is derived from a single mesenchymal stem cell and not from homing of multiple cells to a hotspot.

The D3-dopaminergic agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT) increases cardiac action potential duration and blocks human ether-a-go-go-related gene K+ channel.

The D3-dopaminergic agonist (+/-) 7-hydroxy-dipropylaminotetralin (7-OH-DPAT) prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced the upstroke velocity of the action potential of rabbit sinoatrial node cells. These effects were not mediated by D3-dopaminergic receptors. In cat Purkinje fibers, the drug increased action potential duration. In voltage-clamped cat ventricular myocytes, 7-OH-DPAT blocked the rapid component of the delayed rectifier potassium current, IKr. This effect was corroborated in experiments studying the effect of the drug on human Ether-a-go-go-related Gene channels expressed in Xenopus oocytes and in HEK293 cells. We conclude that the direct electrophysiologic effects of 7-OH-DPAT on cardiac tissues are caused by the blockade of the rapid component of the delayed rectifier potassium current, IKr.