Christian Studenik

Differences in action potential and early afterdepolarization properties in LQT2 and LQT3 models of long QT syndrome.

Long OT syndrome has many causes from both acquired and congenital disorders. For the congenital disorders, their presentation and disease course are not identical. We studied two pharmacological models of long QT syndrome (LQT) to identify differences in cellular electrophysiological properties that may account for this. LQT2 was simulated by suppression of the rapidly activating delayed rectifier potassium current (IKr) with the drug E‐4031, and LQT3 was simulated by slowing of the sodium current (INa) decay with the toxin ATX II. Single rabbit ventricular cell action potentials were studied using the amphotericin B perforated patch clamp technique. Action potential and early afterdepolarization (EAD) properties were rigorously defined by the frequency power spectra obtained with fast Fourier transforms. The E‐4031 (n=43 myocytes) and ATX II (n=50 myocytes) models produced different effects on action potential and EAD properties. The major differences are that ATX II, compared with E‐4031, caused greater action potential prolongation, more positive plateau voltages, lower amplitude EADs with less negative take‐off potentials, greater time to the EAD peak voltage, and longer duration EADs. Despite causing greater action potential prolongation, the incidence of EAD induction was much less with the ATX II model (28%) than with the E‐4031 model (84%). Thus these two pharmacological models have strikingly different cellular electrophysiological properties. Our findings provide cellular mechanisms that may account for some differences in the clinical presentation of LQT2 and LQT3.

Proarrhythmic effects of antidepressants and neuroleptic drugs on isolated, spontaneously beating guinea-pig Purkinje fibers

Antidepressants and neuroleptic drugs are sometimes the reason for the occurrence of the polymorphic ventricular arrhythmia torsades de pointes in patients. Therefore, it was of interest to study the actions of some of these drugs such as imipramine, amitriptyline, doxepin, chlorpromazine, trifluoperazine and thioridazine in isolated, spontaneously beating Purkinje fibers of guinea-pig hearts using the intracellular microelectrode technique because experimentally induced early afterdepolarizations (EADs) may be associated with this special type of arrhythmia. If the extracellular K+ concentration was 2.7mM none of these drugs could elicit EADs. For that reason the K+ concentration was lowered to 1. 35mM and EADs were evoked by imipramine (2 and 5 microM). Amitriptyline (2 and 5 microM) and doxepin (2 microM) did not induce EADs. Only a concentration of 5 microM doxepin elicited EADs. Among the neuroleptic drugs, chlorpromazine at a concentration of 2 and 5 microM was responsible for the occurrence of EADs as well as thioridazine in the same concentrations. When trifluoperazine (2 and 5 microM) was applied no EADs could be observed. Tetrodotoxin (0. 2 microMl-1) abolished thioridazine-induced EADs. Several membrane depolarizing currents may participate in the initiation of these EADs. Our results demonstrate that in guinea-pig Purkinje fibers some tricyclic antidepressants and some neuroleptic drugs are responsible for the rare occurrence of EADs under hypokalemic conditions.

Cardiotoxicity of emetine dihydrochloride by calcium channel blockade in isolated preparations and ventricular myocytes of guinea‐pig hearts

1 The cardiotoxic effects of emetine dihydrochloride on mechanical and electrical activity were studied in isolated preparations (papillary muscles, sinoatrial and atrioventricular nodes, ventricular myocytes) of the guinea‐pig heart. 2 Force of contraction was measured isometrically, action potentials and maximum rate of rise of the action potential were recorded by means of the intracellular microelectrode technique. Single channel L‐type calcium current (Ba2+ ions as charge carrier) was studied with the patch‐clamp technique in the cell‐attached mode. 3 Emetine dihydrochloride (8–256 μm) reduced force of contraction in papillary muscles and spontaneous activity of sinoatrial and atrioventricular nodes concentration‐dependently; the negative inotropic effect was abolished when the extracellular Ca2+ concentration was increased. 4 Maximum diastolic potential, action potential amplitude, maximum rate of rise of the action potential and the slope of the slow diastolic depolarization were decreased by emetine in sinoatrial as well as atrioventricular nodes, while action potential duration was prolonged in both preparations (1–64 μm). 5 The amplitude of the L‐type calcium single channel current was not altered by emetine dihydrochloride, while average open state probability was decreased concentration‐dependently (10, 30 and 60 μm). 6 The most prominent effect of emetine dihydrochloride on single channel current was an increase of sweeps without activity. 7 At 60 μm, emetine dihydrochloride caused a decrease of the mean open time and an increase of the mean closed time. The number of openings per record and number of bursts per record were reduced. 8 It is concluded that emetine dihydrochloride produces an L‐type calcium channel block which might contribute to its cardiac side effects.

Studies on the Chemistry of Thienoannelated O,N- and S,N-Containing Heterocycles. Part 19[1]: Thieno[2,3-b][1,4]thiazines with Calcium Antagonistic and Potassium Opening Activities

In this study novel substituted 6‐benzyl‐thieno[2,3‐b][1,4]thiazines with an urea moiety were synthesized. Structural modifications of the amino side chain were carried out with the aim of finding tissue specific compounds. The effects on papillary muscles, right atria, aortic strips, and terminal ilea were investigated. Compounds 10c and 10d showed the most potent negative inotropic effect. The calcium antagonism of all derivatives occurred in a non‐competitive manner, which may indicate that they also have potassium channel opening activities.

Benzanilides with spasmolytic activity: Chemistry, pharmacology, and SAR

The following study describes the synthesis of new benzanilide derivatives and their pharmacological investigation on smooth muscle preparations of guinea pigs. All compounds were synthesized in good yields and showed a spasmolytic activity without significant effect on vascular smooth muscles and heart muscle preparations. Moreover, further pharmacological investigations as well as in silico studies were performed to elucidate the mechanism of action. Compound 3 showed the most potent spasmolytic activity with an IC(50) of 3.25microM.

Synthesis, spasmolytic activity and structure–activity relationship study of a series of polypharmacological thiobenzanilides

Recently we presented a series of benzanilide derivatives with a selective spasmolytic effect on terminal ileum preparations of the guinea pig. In this report we demonstrate a further development of these compounds. The exchange of the amide oxygen against a sulfur atom resulted in an up to 325 fold increase of the antispasmodic activity of the thiobenzanilide (IC(50) of 0.1 μM) compared to its benzanilide derivative. Considering their mode of action the compounds interacted with several molecular targets, suggesting that we identified a chemical identity able to modulate multiple targets simultaneously. Furthermore, based on this data set, we present a structure-activity relationship study supporting the important role of the sulfur atom.

Mechanism of sodium channel blockade in the cardiotoxic action of emetine dihydrochloride in isolated cardiac preparations and ventricular myocytes of guinea pigs

Emetine is used in the therapy of special forms of amebiasis and is abused as syrup of ipecac by persons with bulimia. Severe cardiac side effects were reported. Thus the intracellular microelectrode technique and the patch-clamp technique in the cell-attached mode were used to study the effects of emetine on the action potential and upstroke velocity (Vmax) in papillary muscles and Purkinje fibers of guinea pigs as well as on macroscopic and (S)-DPI 201-106-modified and unmodified single-sodium-channel current (I(Na)) of guinea-pig ventricular myocytes. Emetine caused a tonic block of Vmax and reduced I(Na) independent of frequency. Hill plots were linear, with slopes ranging from 0.96 to 1.06, suggestive of a first-order reaction. The current-voltage relation was not influenced, indicating a voltage-independent blockade of the sodium channels. The most prominent effects were an increase of sweeps without activity, a decrease of the fast component of the open-time distribution, an increase of the slow component of the closed-time distribution, and a reduction in the number of bursts per record. The amplitude of the unitary current was not changed. From the results, we conclude that I(Na) blockade contributes to the cardiotoxicity of emetine.

Novel screening test to assess the potential environmental toxicity of waste water samples

Biological effects of waste water samples were investigated using the method for isometric contraction measurements that is widely employed in the medical field for studying the effects of various drugs on heart and smooth muscle preparations. Raw waste water samples from a municipal treatment plant were extracted with chloroform and diluted in dimethylsulfoxide. Some samples were also digested with NaOH or microwave. Our results show that the test is sensitive for the investigation of waste water samples. The liquid part of the samples exerted a more potent relaxing effect on terminal ilea than the solids. Alkaline (NaOH) or microwave treated samples showed similar effects as untreated samples. Therefore we conclude that the major part of the reactive agent might be located in the liquid phase. We also demonstrate with this study that the used method enables the screening of waste water samples and their effect on terminal ilea. This method might be a tool to assess the environmental relevance of complex environmental samples.

Cardiotoxic effects of fenfluramine hydrochloride on isolated cardiac preparations and ventricular myocytes of guinea‐pigs

The cardiotoxic effects of fenfluramine hydrochloride on mechanical and electrical activity were studied in papillary muscles, Purkinje fibres, left atria and ventricular myocytes of guinea‐pigs. Force of contraction (fc) was measured isometrically, action potentials and maximum rate of rise of the action potential (Vmax) were recorded by means of the intracellular microelectrode technique and the sodium current (INa) with patch‐clamp technique in the cell‐attached mode. For kinetic analysis (S)‐DPI‐201‐106‐modified Na+ channels from isolated guinea‐pig ventricular heart cells were used. Fenfluramine (1–300 μM) produced negative chronotropic and inotropic effects; additional extracellular Ca2+ competitively antagonized the negative inotropic effect. Fenfluramine concentration‐dependently reduced Vmax and showed tonic blockade of sodium channels, shortened the action potential duration in papillary muscles and Purkinje fibres. In cell‐attached patches, fenfluramine decreased INa concentration‐dependently (10–100 μM), frequency‐independently (0.1–3 Hz; 30 μM). The h∞ curve was shifted towards hyperpolarizing direction. At 30 μM, fenfluramine blocked the sodium channel at all test potentials to the same degree, and neither changed the threshold and reversal potentials nor the peak of the curve. No effect on single channel availability, but a significant decrease in mean open times and increase in mean closed times was observed. Mean duration of the bursts decreased and number of openings per record increased with increasing drug concentration. It is concluded that the effect on INa plays an important role in the cardiotoxicity of fenfluramine in addition to primary pulmonary hypertension and valvular disorders.

A journey from benzanilides to dithiobenzanilides: Synthesis of selective spasmolytic compounds

A series of dithiobenzanilide derivatives was synthesized and each compound was evaluated for its ability to reduce KCl-induced contractions of smooth muscle preparations of the guinea pig. Starting from a recent publication describing benzanilide derivatives as antispasmodic agents, structure-activity guided synthesis was performed to obtain compounds with improved spasmolytic activity. First, compounds with two amide bonds were designed and second, both amide oxygens were replaced by two sp² sulfur atoms resulting in dithiobenzanilide derivatives. The most potent antispasmodic dithiobenzanilide 19 showed improved activity with an IC₅₀ value of 0.4 μM. Moreover, the study also demonstrated that these active compounds were able to antagonize the effect of spasmogens like acetylcholine and phenylephrine and that the activity is not mediated by activation of ATP-dependent potassium channels (K(ATP)-channels) or inhibition of endothelial nitric oxide synthase (eNOS).