In vivo and cellular antiarrhythmic and cardiac electrophysiological effects of desethylamiodarone in dog cardiac preparations.

BACKGROUND AND PURPOSE: The aim of the present study was to study the antiarrhythmic effects and cellular mechanisms of desethylamiodarone (DEA), the main metabolite of amiodarone (AMIO), following acute and chronic 4-week oral treatments (25-50 mg·kg-1 ·day-1 ). EXPERIMENTAL APPROACH: The antiarrhythmic effects of acute iv. (10 mg·kg-1 ) and chronic oral (4 weeks, 25 mg·kg-1 ·day-1 ) administration of DEA were assessed in carbachol and tachypacing-induced dog atrial fibrillation models. Action potentials were recorded from atrial and right ventricular tissue following acute (10 µM) and chronic (p.o. 4 weeks, 50 mg·kg-1 ·day-1 ) DEA application using the conventional microelectrode technique. Ionic currents were measured by the whole cell configuration of the patch clamp technique in isolated left ventricular myocytes. Pharmacokinetic studies were performed following a single intravenous dose (25 mg·kg-1 ) of AMIO and DEA intravenously and orally. In chronic (91-day) toxicological investigations, DEA and AMIO were administered in the oral dose of 25 mg·kg-1 ·day-1 ). KEY RESULTS: DEA exerted marked antiarrhythmic effects in both canine atrial fibrillation models. Both acute and chronic DEA administration prolonged action potential duration in atrial and ventricular muscle without any changes detected in Purkinje fibres. DEA decreased the amplitude of several outward potassium currents such as IKr , IKs , IK1 , Ito , and IKACh , while the ICaL and late INa inward currents were also significantly depressed. Better drug bioavailability and higher volume of distribution for DEA were observed compared to AMIO. No neutropenia and less severe pulmonary fibrosis was found following DEA compared to that of AMIO administration. CONCLUSION AND IMPLICATIONS: Chronic DEA treatment in animal experiments has marked antiarrhythmic and electrophysiological effects with better pharmacokinetics and lower toxicity than its parent compound. These results suggest that the active metabolite, DEA, should be considered for clinical trials as a possible new, more favourable option for the treatment of cardiac arrhythmias including atrial fibrillation.

Antiarrhythmic and cardiac electrophysiological effects of SZV-270, a novel compound with combined Class I/B and Class III effects, in rabbits and dogs.

Cardiovascular diseases are the leading causes of mortality. Sudden cardiac death is most commonly caused by ventricular fibrillation (VF). Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and heart failure. Pharmacological management of VF and AF remains suboptimal due to limited efficacy of antiarrhythmic drugs and their ventricular proarrhythmic adverse effects. In this study, the antiarrhythmic and cardiac cellular electrophysiological effects of SZV-270, a novel compound, were investigated in rabbit and canine models. SZV-270 significantly reduced the incidence of VF in rabbits subjected to coronary artery occlusion/reperfusion and reduced the incidence of burst-induced AF in a tachypaced conscious canine model of AF. SZV-270 prolonged the frequency-corrected QT interval, lengthened action potential duration and effective refractory period in ventricular and atrial preparations, blocked I Kr in isolated cardiomyocytes (Class III effects), and reduced the maximum rate of depolarization (V max) at cycle lengths smaller than 1000 ms in ventricular preparations (Class I/B effect). Importantly, SZV-270 did not provoke Torsades de Pointes arrhythmia in an anesthetized rabbit proarrhythmia model characterized by impaired repolarization reserve. In conclusion, SZV-270 with its combined Class I/B and III effects can prevent reentry arrhythmias with reduced risk of provoking drug-induced Torsades de Pointes.

The regulatory role of vasoactive intestinal peptide in lacrimal gland ductal fluid secretion: A new piece of the puzzle in tear production.

Purpose: Vasoactive intestinal peptide (VIP) is an important regulator of lacrimal gland (LG) function although the effect of VIP on ductal fluid secretion is unknown. Therefore, the aim of the present study was to investigate the role of VIP in the regulation of fluid secretion of isolated LG ducts and to analyze the underlying intracellular mechanisms. Methods: LGs from wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) knockout (KO) mice were used. Immunofluorescence was applied to confirm the presence of VIP receptors termed VPAC1 and VPAC2 in LG duct cells. Ductal fluid secretion evoked by VIP (100 nM) was measured in isolated ducts using videomicroscopy. Intracellular Ca2+ signaling underlying VIP stimulation was investigated with microfluorometry. Results: VIP stimulation resulted in a robust and continuous fluid secretory response in isolated duct segments originated from WT mice. In contrast, CFTR KO ducts exhibited only a weak pulse-like secretion. A small but statistically significant increase was detected in the intracellular Ca2+ level [Ca2+]i during VIP stimulation in the WT and in CFTR KO ducts. VIP-evoked changes in [Ca2+]i did not differ considerably between the WT and CFTR KO ducts. Conclusions: These results suggest the importance of VIP in the regulation of ductal fluid secretion and the determining role of the adenylyl cyclase-cAMP-CFTR route in this process.

Alpha-Adrenergic Agonists Stimulate Fluid Secretion in Lacrimal Gland Ducts.

Purpose: The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms. Methods: Fluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry. Results: Both norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [\(Ca_i^{2 + }\)]. Conclusions: Our results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of ß-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.

GIRK channel activity of Hungarian mushrooms: From screening to biologically active metabolites.

In the current study effects of fungal extracts on the G-protein-activated inwardly rectifying potassium channel (GIRK1/4) were screened using the automated patch-clamp method. 40 organic (n-hexane, chloroform, and 50% methanol) and aqueous extracts were prepared from 10 mushroom species native to Hungary. Among the examined fungal fractions of different polarities some n-hexane and chloroform extracts exerted considerable ion channel activity. One of the most active fungal species, Hypholoma lateritium was selected for further detailed examination to determine the compounds responsible for the observed pharmacological property. Evaluation of the ion channel activity of mushroom metabolites 1-10 revealed that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) demonstrates remarkable blocking activity on GIRK current (IC50 395.1 ±â€¯31.8 nM). Investigation of the selectivity of the GIRK inhibitory effect proved that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) has only weak inhibitory activity on hERG channel (7.9 ±â€¯2.8% at 100 µM), exerting more than three orders of magnitude lower blocking activity on hERG channel than on GIRK channel.

Evaluation of Possible Proarrhythmic Potency: Comparison of the Effect of Dofetilide, Cisapride, Sotalol, Terfenadine, and Verapamil on hERG and Native IKr Currents and on Cardiac Action Potential.

The proarrhythmic potency of drugs is usually attributed to the IKr current block. During safety pharmacology testing analysis of IKr in cardiomyocytes was replaced by human ether-a-go-go-related gene (hERG) test using automated patch-clamp systems in stable transfected cell lines. Aim of this study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch-clamp methods on HEK293 cells. The native ion currents (IKr, INaL, ICaL) were recorded from rabbit ventricular myocytes by manual patch-clamp technique. Action potentials in rabbit ventricular muscle and undiseased human donor hearts were studied by conventional microelectrode technique. Dofetilide, cisapride, sotalol, terfenadine, and verapamil blocked hERG channels at 37°C with an IC50 of 7 nM, 18 nM, 343 µM, 165 nM, and 214 nM, respectively. Using manual patch-clamp, the IC50 values of sotalol and terfenadine were 78 µM and 31 nM, respectively. The IC50 values calculated from IKr measurements at 37°C were 13 nM, 26 nM, 52 µM, 54 nM, and 268 nM, respectively. Cisapride, dofetilide, and sotalol excessively lengthened, terfenadine, and verapamil did not influence the action potential duration. Terfenadine significantly inhibited INaL and moderately ICaL, verapamil blocked only ICaL. Automated hERG assays may over/underestimate proarrhythmic risk. Manual patch-clamp has substantially higher sensitivity to certain drugs. Action potential studies are also required to analyze complex multichannel effects. Therefore, manual patch-clamp and action potential experiments should be a part of preclinical safety tests.

Diterpenoids from Euphorbia dulcis with Potassium Ion Channel Inhibitory Activity with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effect.

Nine new (1-9) and two known (10, 11) jatrophane diterpenoids were isolated from the methanol extract of Euphorbia dulcis. The structure elucidation of the compounds was performed by means of extensive spectroscopic analysis, including HRESIMS, 1D (1H, JMOD), and 2D (HSQC, HMBC, 1H-1H-COSY, NOESY) NMR experiments. The absolute configuration of compound 1 was determined by single-crystal X-ray diffraction. The electrophysiological effects of compounds 1-11 and the five diterpenoids (12-16) previously isolated from Euphorbia taurinensis were investigated on stable transfected HEK-GIRK1/4 (Kir3.1/3.4) and HEK-hERG (Kv11.1) cell lines using automated patch-clamp equipment. The majority of the diterpenoids showed significant blocking activity on GIRK channels (60.8-88.7% at 10 µM), while compounds 1, 2, 9-11, 13, and 14 exerted notable inhibitory effects even at 1 µM concentration. None of the jatrophane diterpenoids interfered with the function of hERG proteins; however, compound 14 remarkably hampered K+ flow through hERG channels. These selective activities suggest that jatrophane diterpenoids may represent a group of potential lead compounds for the development of novel therapeutic agents against atrial fibrillation.

Diterpene Lipo-Alkaloids with Selective Activities on Cardiac K+ Channels.

Aconitum diterpene alkaloids are known for their remarkable toxicity, which is due to their effect on ion channels. Activation of voltage-gated Na+ channels is the major cause of their cardiotoxicity, however, influence on K+ channels may also play a role in the overall effect.Here we report the synthesis of a series of lipo-alkaloids, including four new compounds, based on the 14-benzoylaconine structure, which is the core of a vast number of diterpene alkaloids naturally occurring in Aconitum species. The activities of these compounds were measured in vitro on K+ ion channels using the whole-cell patch-clamp technique. Structure-activity analysis was carried out based on the data of 51 compounds (32 genuine diterpene alkaloids, 5 fatty acids, and 14 lipo-alkaloids). Depending on their substitution, these compounds exert different activities on GIRK (G protein-coupled inwardly-rectifying potassium channel) and hERG (human ether-à-go-go-related gene) channels. Fatty acids and diterpene alkaloids show lower activity on the GIRK channel than lipo-alkaloids. Lipo-alkaloids also have less pronounced hERG inhibitory activity compared to the cardiotoxic aconitine. Considering the GIRK/hERG selectivity as an indicator of perspective therapeutic applicability, lipo-alkaloids are significantly more selective than the genuine diterpene alkaloids. 14-Benzoyl-8-O-eicosa-8Z,11Z,14Z-trienoate and 14-benzoyl-8-O-eicosa-11Z,14Z,17Z-trienoate are strong and selective inhibitors of GIRK channels, thus, they are promising subjects for further studies to develop diterpene alkaloid-based antiarrhythmic pharmacons.

Analgesic effect of dimethyl trisulfide in mice is mediated by TRPA1 and sst4 receptors.

TRPA1 receptors are calcium-permeable ligand-gated channels expressed in primary sensory neurons and involved in inflammation and pain. Activation of these neurons might have analgesic effect. Suggested mechanism of analgesic effect mediated by TRPA1 activation is the release of somatostatin (SOM) and its action on sst4 receptors. In the present study analgesic effect of TRPA1 activation on primary sensory neurons by organic trisulfide compound dimethyl trisulfide (DMTS) presumably leading to SOM release was investigated. Opening of TRPA1 by DMTS in CHO cells was examined by patch-clamp and fluorescent Ca2+ detection. Ca2+ influx upon DMTS administration in trigeminal ganglion (TRG) neurons of TRPA1 receptor wild-type (WT) and knockout (KO) mice was detected by ratiometric Ca2+ imaging. SOM release from sensory nerves of murine skin was assessed by radioimmunoassay. Analgesic effect of DMTS in mild heat injury-induced mechanical hyperalgesia was examined by dynamic plantar aesthesiometry. Regulatory role of DMTS on deep body temperature (Tb) was measured by thermocouple thermometry with respirometry and by telemetric thermometry. DMTS produced TRPA1-mediated currents and elevated [Ca2+]i in CHO cells. Similar data were obtained in TRG neurons. DMTS released SOM from murine sensory neurons TRPA1-dependently. DMTS exerted analgesic effect mediated by TRPA1 and sst4 receptors. DMTS-evoked hypothermia and hypokinesis were attenuated in freely-moving TRPA1 KO animals. Our study has presented original evidence regarding analgesic action of DMTS which might be due to TRPA1-mediated SOM release from sensory neurons and activation of sst4 receptors. DMTS could be a novel analgesic drug candidate.

Myrsinane, Premyrsinane, and Cyclomyrsinane Diterpenes from Euphorbia falcata as Potassium Ion Channel Inhibitors with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effects.

GIRK channels are activated by a large number of G protein-coupled receptors and regulate the electrical activity of neurons, cardiac atrial myocytes, and ß-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the pathophysiology of neuropathic pain, drug addiction, and cardiac arrhythmias. In the heart, GIRK channels are selectively expressed in the atrium, and their activation inhibits pacemaker activity, thereby slowing the heart rate. In the present study, 19 new diterpenes, falcatins A-S (1-19), and the known euphorprolitherin D (20) were isolated from Euphorbia falcata. The compounds were assayed on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cells. Blocking activity on GIRK channels was exerted by 13 compounds (61-83% at 10 µM), and, among them, five possessed low potency on the hERG channel (4-20% at 10 µM). These selective activities suggest that myrsinane-related diterpenes are potential lead compounds for the treatment of atrial fibrillation.

Effects of Chelidonium majus extracts and major alkaloids on hERG potassium channels and on dog cardiac action potential - a safety approach.

Chelidonium majus or greater celandine is spread throughout the world, and it is a very common and frequent component of modern phytotherapy. Although C. majus contains alkaloids with remarkable physiological effect, moreover, safety pharmacology properties of this plant are not widely clarified, medications prepared from this plant are often used internally. In our study the inhibitory effects of C. majus herb extracts and alkaloids on hERG potassium current as well as on cardiac action potential were investigated. Our data show that hydroalcoholic extracts of greater celandine and its alkaloids, especially berberine, chelidonine and sanguinarine have a significant hERG potassium channel blocking effect. These extracts and alkaloids also prolong the cardiac action potential in dog ventricular muscle. Therefore these compounds may consequently delay cardiac repolarization, which may result in the prolongation of the QT interval and increase the risk of potentially fatal ventricular arrhythmias.

Inhibition of G protein-activated inwardly rectifying K+ channels by extracts of Polygonum persicaria and isolation of new flavonoids from the chloroform extract of the herb.

The G protein-activated inwardly rectifying K+ channel-modulatory activities of Polygonum persicaria extracts were investigated by using an automated patch-clamp method, with the aim of identifying natural sources of promising ion channel-blocking compounds. The chloroform extract of the whole plant at 0.1 mg/mL exhibited high G protein-activated inwardly rectifying K+ channel-inhibitory activity. Fractionation of this extract by vacuum liquid chromatography on RP-silica gel resulted in 6 fractions, which were evaluated for G protein-activated inwardly rectifying K+ channel-modulatory activity. RP-HPLC of the most active fractions afforded the main compounds 1-4 in pure form and a mixture containing the minor constituents. The structures were identified by means of UV, HRMS, and advanced NMR methods as 3-O-senecioyl-isorhamnetin (1), 3-O-angeloyl-isorhamnetin (2), 5,3',4',5'-tetramethoxy-6,7-methylenedioxyflavone (3), and 3,5,3',4',5'-pentamethoxy-6,7-methylenedioxyflavone (4). Compounds 1-4 are new natural products, though 4 was reported earlier as a synthetic compound. Neither the individual, nor the combined application of compounds 1-4 modified the G protein-activated inwardly rectifying K+ channel activity. However, a marked G protein-activated inwardly rectifying K+ current-inhibitory effect was detected on use of the HPLC eluates containing the minor compounds. These results indicate the presence of electrophysiologically active agents among the minor compounds.

Identification of diterpene alkaloids from Aconitum napellus subsp. firmum and GIRK channel activities of some Aconitum alkaloids.

Diterpene alkaloids neoline (1), napelline (2), isotalatizidine (3), karakoline (4), senbusine A (5), senbusine C (6), aconitine (7) and taurenine (8) were identified from Aconitum napellus L. subsp. firmum, four (2-4, 6) of which are reported for the first time from this plant. The structures were determined by means of LC-MS, 1D and 2D NMR spectroscopy, including (1)H-(1)H COSY, NOESY, HSQC and HMBC experiments. Electrophysiological effects of the isolated compounds, together with nine diterpene alkaloids previously obtained from Aconitum toxicum and Consolida orientalis were investigated on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cell lines using automated patch clamp equipment. Significant blocking activity on GIRK channel was exerted by aconitine (7) (45% at 10 µM), but no blocking activities of the other investigated compounds were detected. The tested compounds were inactive on hERG channel in the tested concentration. The comparison of the previously reported metabolites of A. napellus subsp. firmum and compounds identified in our experiment reveals substantial variability of the alkaloid profile of this taxon.

Electrophysiological effects of ivabradine in dog and human cardiac preparations: potential antiarrhythmic actions.

Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (V(max)), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1-10 µM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent V(max) block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400 ms cycle length, V(max) block developed with an onset kinetic rate constant of 13.9 ± 3.2 beat(-1) in dog ventricular muscle. In addition to a fast recovery of V(max) from inactivation (τ=41-46 ms) observed in control, a second slow component for recovery of V(max) was expressed (offset kinetics of V(max) block) having a time constant of 8.76 ± 1.34 s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10 µM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm.

Cellular electrophysiological effect of terikalant in the dog heart.

The cellular mechanism of action of terikalant, an investigational antiarrhythmic agent known to block the inward rectifier and other potassium currents, has not yet been fully clarified. The aim of the present study was therefore to analyse the in vitro electrophysiological effects of terikalant in canine isolated ventricular muscle and Purkinje fibers by applying the standard microelectrode technique. The effects of terikalant on the duration of action potential at a stimulation cycle length of 1000 ms and on the maximum upstroke velocity of the action potential in right ventricular papillary muscle were examined at 1, 2.5, 10, and 20 microM concentrations. Terikalant significantly prolonged the action potential duration measured both at 50% and 90% of repolarization in concentration-dependent manner. The maximum upstroke velocity of the action potential was unaffected at 1 and 2.5 microM concentrations. However, this parameter was significantly reduced at 10 and 20 microM concentrations of terikalant. In Purkinje fibers terikalant (2.5 microM) also produced a marked action potential lengthening effect. Frequency dependence (cycle length of 300-5000 ms) of the action potential lengthening effect of terikalant was studied at a concentration of 2.5 microM. Prolongation of the duration of action potential occurred in a reverse frequency-dependent manner both in papillary muscle and Purkinje fibers, with a more pronounced frequency-dependence observed in Purkinje fibers. The onset kinetics of the terikalant (10 microM) induced block of the maximum upstroke velocity of the action potential was rapid (0.6+/-0.1 beat(-1), n=6) like that of Class I/B antiarrhythmics, and the offset (recovery) kinetics of the drug (2956+/-696 ms, n=6) best resembled that of Class I/A antiarrhythmic drugs. It was concluded that terikalant, unlike pure Class III antiarrhythmic drugs, has combined mode of action by lengthening repolarization and blocking the inward sodium current in a use-dependent manner.

Effect of a neuroprotective drug, eliprodil on cardiac repolarisation: importance of the decreased repolarisation reserve in the development of proarrhythmic risk.

1. The aim of this study was to analyse the effects of eliprodil, a noncardiac drug with neuroprotective properties, on the cardiac repolarisation under in vitro circumstances, under normal conditions and after the attenuation of the 'repolarisation reserve' by blocking the inward rectifier potassium current (I(K1)) current with BaCl(2). 2. In canine right ventricular papillary muscle by applying the conventional microelectrode technique, under normal conditions, eliprodil (1 microm) produced a moderate reverse rate-dependent prolongation of the action potential duration (7.4+/-1.5, 8.9+/-2.1 and 9.9+/-1.8% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=9). 3. This effect was augmented in preparations where I(K1) was previously blocked by BaCl(2) (10 microm). BaCl(2) alone lengthened APD in a reverse frequency-dependent manner (7.0+/-1.3, 14.2+/-1.6 and 28.1+/-2.1% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). When eliprodil (1 microm) was administered to these preparations, the drug induced a marked further lengthening relative to the APD values measured after the administration of BaCl(2) (12.5+/-1.0, 17.6+/-1.5 and 20.5+/-0.9% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). 4. In the normal Langendorff-perfused rabbit heart, eliprodil (1 microm) produced a significant QT(c) prolongation at 1 Hz stimulation frequency (12.7+/-1.8%, n=9). After the attenuation of the 'repolarisation reserve' by the I(K1) blocker BaCl(2) (10 microm), the eliprodil-evoked QT(c) prolongation was greatly enhanced (28.5+/-7.9%, n=6). In two out of six Langendorff preparations, this QT(c) lengthening degenerated into torsade de pointes ventricular tachycardia. 5. Eliprodil significantly decreased the amplitude of rapid component of the delayed rectifier potassium current (I(Kr)), but slow component (I(Ks)), transient outward current (I(to)) and I(K1) were not considerably affected by the drug when measured in dog ventricular myocytes by applying the whole-cell configuration of the patch-clamp technique. 6. The results indicate that eliprodil, under normal conditions, moderately lengthens cardiac repolarisation by inhibition of I(Kr). However, after the attenuation of the normal 'repolarisation reserve', this drug can induce marked QT interval prolongation, which may result in proarrhythmic action.

Pharmacologically activated migration of aortic endothelial cells is mediated through p38 SAPK.

Impairment in endothelial cell (EC) function plays a central role in vascular diseases (e.g. atherosclerosis, restenosis, diabetic angiopathies, microvascular angina, peripheral arterial disease). BRX-235 (a novel small molecule synthesized by Biorex, Hungary) has a potent vasculoprotective activity in different in vivo and in vitro studies. Since the importance of the p38 pathway in EC homeostasis and migration in particular is well documented, we have carried out studies to address the role of the p38 stress activated protein kinase (p38 SAPK) in the mode of action of BRX-235. In this study, Bovine aortic endothelial cells were used in a wounding migration assay (WMA) and for Western-blot analysis to study the effect and molecular mechanism of BRX-235-induced EC migration. The bovine aortic endothelial (BAE) cells were shown to be good models for EC migration. Both endothelial cell growth factor (ECGF)- and BRX-235-induced BAE cell migration were shown to be inhibited by SB 203580, a specific inhibitor of p38 SAPK. It was also shown that, BRX-235 induces phosphorylation of p38 SAPK without affecting p38 SAPK protein levels. Thus, BRX-235 acts upstream of p38 SAPK. In summary, we have shown that p38 SAPK is a potential pharmacological mediator for candidate drugs that target the endothelium.

Effect of BRX-220 against peripheral neuropathy and insulin resistance in diabetic rat models.

Bimoclomol (BML), a symptomatic antidiabetic agent, has been developed by Biorex R & D Co. to treat diabetic neuropathy and retinopathy. BRX-220, an orally active member of the BRX family, has been developed to treat diabetic complications and insulin resistance (IR) as a follow-up compound. The effect of BRX-220 on peripheral neuropathy was examined in rats with diabetes (type 1) induced by administration of a beta-cell toxin, streptozotocin (STZ, 45 mg/kg iv). Nerve functions were evaluated by electrophysiological measurements of muscle motor and sensory nerve conduction velocities (MNCV and SNCV, respectively). MNCV and SNCV decreased in diabetic rats by 25% (p < 0.001). A 1-month preventive treatment with BRX-220 (2.5, 5, 10, and 20 mg/kg po) dose-dependently improved diabetes-related deficits in MNCV (51.3%, 71.3%, 86.1%, and 91.3%) and SNCV (48.9%, 68.5%, 86.1%, and 93.2%). Insulin sensitivity was measured using the insulin tolerance test (ITT), both in STZ diabetic and in Zucker diabetic fatty (ZDF) rats (model of type 2 diabetes). Severe IR was detected in STZ diabetic and ZDF rats. This resistance was significantly (p < 0.05) reduced by BRX-220 treatment.