Immunochemical properties of conjugated antigens from N-substituted phenothiazines and dibenzazepines with antiarrhythmic activity

The use of metabolites of antiarrhythmic drugs (ethmozine, ethacizine, and bonnecor) as haptens in the synthesis of conjugated antigens allowed us to induce the formation of antibodies with different specificity for certain metabolites. A new enzyme immunoassay was developed for the detection of phenothiazine and dibenzazepine derivatives (ethmozine, ethacizine, and bonnecor). Nanogram and subnanogram quantities of these substances may be detected in biological fluids.     

Differences in the efficiency of their interaction with membrane receptors of the dialkylaminoalkyl and dialkylaminoacyl derivatives of phenothiazine

New dialkylaminoacyl phenothiazine derivatives (DAC) were compared with their dialkylaminoalkyl analogues (neuroleptics chlorpromazine, trifluoperazine and fluphenazine) as well as with anti-arrhythmia drugs ethmozine and ethacizine for their receptor-blocking potencies. It was established that DAC are significantly less potent with dopamine alpha 1-adrenergic and H1-histamine receptors of calf and rabbit brain, which can explain the absence of neuroleptic effect of DAC drugs. DAC affinities to muscarinic and alpha-adrenergic receptors of both types are very similar to those of ethmozine and ethacizine. New DAC substance G-512 (chlorpromazine analogue) demonstrated high affinity to M1-muscarinic receptors of rabbit brain cortex (Ki = 4.2 nM) and to M2-muscarinic receptors of the rabbit heart (Ki = 48 nM).     

Characteristics of the protective action of ethacizin on the ischemic myocardium

A study was made of the effect of ethacizine, a new antiarrhythmic phenothiazint derivative, on the size of experimental myocardial infarction in rabbits 7 days after ligation of the coronary artery. Ethmozine was used as reference. Ethacizine diminished the extent of necrosis by 22.8% (P less than 0.05) when injected intravenously in divided doses beginning from the 30th minute of 2-hour ligation, the total dose being 1.5 mg/kg. The six-day cycle of ethacizine treatment instituted 24 h after coronary artery ligation (daily dose 1.2 mg/kg) provoked a more considerable reduction of the myocardial infarction size (by 44.9%). The effect of ethmozine was less pronounced though statistically significant. Ethacizine increased ATP content in both the ischemic and "intact" myocardium and minimized the impairment of membrane permeability in the occlusion zone 3 h after ligation when injected according to the first above-described scheme. It is assumed that these effects may contribute to the drug protective action on the ischemic myocardium.     

Inhibiting action of anti-arrhythmia agents of the phenothiazine series--etmozin and its diethylamino analog--on platelet aggregation and metabolism of endogenous arachidonic acid

Effect of the cardiotropic drugs of the phenothiazine series ethmozine, and its diethylamine analogue (DAAE), on platelet aggregation and formation of arachidonic acid metabolites has been studied. Both drugs inhibit the ADP-induced aggregation in the platelet-rich plasma. Ethmozine inhibits only the second (irreversible) wave of aggregation, while DAAE inhibits both the first (reversible) and the second one. 50% inhibition (ID50) of the second wave of aggregation is observed at the following concentrations of the two agents: 300-500 micrograms/ml (ethmozine) and 20 micrograms/ml (DAAE). DAAE completely inhibits the irreversible aggregation of platelets washed off plasma, induced by arachidonic acid (ID50 approximately 30 micrograms/ml) and Ca2+-ionophore A23187 (ID approximately 55 micrograms/ml); the aggregation, induced by thrombin is inhibited by 80-90% (ID approximately 130 micrograms/ml). Formation of arachidonic acid metabolites in platelets effected by these inducers was measured by the accumulation of malondialdehyde (MDA). DAAE fails to inhibit MDA formation induced by exogenous arachidonic acid, but completely prevents the synthesis of MDA induced by A23187 and thrombin. These data suggest that DAAE inhibits the release of endogenous arachidonic acid from membrane phospholipids catalysed by phospholipase A2, but does not affect its subsequent metabolic transformations. In all probability, ethmozine and DAAE, just as other phenothiazines, affect platelets via the inhibition of Ca2+-calmodulin-dependent reactions and processes.     

Effect of ethmozine on kidney function

Injection of ethmozine to rats (50 mg/kg subcutaneously) and to dogs (0,5 mg/kg intravenously) produced a substantial rise in diuresis, natriuresis and kaliuresis at the expense of the increased glomerular filtration rate and renal circulation. Injection of ethmozine (0.25-1.0 mg/min for 20 min) to the renal artery of dogs had no effect on the ipsilateral side. It is assumed that alterations in renal function induced by ethmozine injection are extrarenal in nature.     

Stimulus-dependent blockade of sodium channels of the Ranvier node membrane by the phenothiazine derivative ethmozine

The action of the antiarrhythmic drug ethmozine on sodium channels of the membrane was studied in experiments on single from Ranvier nodes by the voltage clamp method. Application of ethmozine to both the outer and the inner side of the membrane reduced the amplitude of the sodium current INa; the kinetics of this current and steady-state inactivation of the sodium channels were unchanged. Tonic and phasic (transient, stimulus-dependent) components can be distinguished in the ethmozine block of the sodium current. Tonic blockage of the sodium current develops slowly and can be potentiated by high-frequency stimulation of the membrane. The possible nature of the tonic block is discussed. The stimulus-dependent blockade of the sodium current deepens with an increase in the frequency and amplitude of depolarizing stimuli. Prolonged membrane depolarization does not evoke any additional blocking of the sodium current. It is concluded that the stimulus-dependent blockade is due to interaction between ethomizine and open sodium channels. Modification of the channels by batrachotoxin (preventing inactivation of the sodium channels) makes them insensitive to ethmozine. Increasing the potassium ion concentration on the outer side of the membrane was found to reduce the tonic effect of ethmozine and to potentiate the stimulus-dependent blockade. The action of ethmozine was compared with the effects of tertiary and quaternary local anesthetics.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 380–389, July–August, 1981.     

Changes in the sodium current under the action of etmozin and lidocaine inside and outside the membrane of single rat myocardial cells

The voltage clamp experiments were carried out on single internally perfused rat myocardial cells. The effect of ethmozine (8 x 10(-5) g/ml) and lidocain (8 x 10(-6) g/ml) on the fast maximum inward sodium current (INa) was studied. The drugs were tested inside and outside the cell. INa was inhibited insignificantly when ethmozine was added inside the cell. After 5 min of ethmozine action outside the cell INa dropped on the average to 43 +/- 6% of its initial value. Under these conditions the reactivation constrant of INa did not change significantly. Lidocain depressed INa both when added outside and inside the cell. However, when lidocain was added outside the cell a longer period was needed to depress INa. Comparison of lidocain and ethmozine action outside and inside the myocardial cell has shown that the sites of action of these antiarrhythmic drugs on the cellular membrane are different.     

Effect of ethmozine on action potentials and myocardial contraction in guinea pigs

Ethmozine decreased the maximum rate of action potential rise (Vmax) in a dose-dependent manner. Using the Scatchard plot the apparent dissociation constant was calculated to be 1.52 X 10(-5) g/ml. Ethmozine also decreased the force of contraction in the concentration range between 1 X 10(-6) and 1 X 10(-4) g/ml with the apparent dissociation constant obtained from the Scatchard plot being equal to 1.48 X 10(-5) g/ml. The linear correlation coefficient between the decrease in Vmax and the decrease in the force of contraction was found to be equal to 0.998. Negative inotropic action of ethmozine was less pronounced when the stimulation frequency had been switched from 0.8 to 0.1 Hz. The decrease in Vmax under the action of ethmozine (3 X 10(-5) g/ml) was diminished from 56 +/- 7% (0.8 Hz) to only 3 +/- 8% (0.1 Hz). This was accompanied by the decrease in the negative inotropic effect: from 58 +/- 9% (0.8 Hz) to 16 +/- 15% (0.1 Hz). It was assumed that the negative inotropic action of ethmozine was mediated by the Na--Ca exchange, which was inhibited by the decrease of the intracellular Na+ concentration due to the blockade of sodium channels by ethmozine.     

Electrophysiological effects of ethmozine in perfused rabbit hearts

His-bundle electrocardiography was used to evaluate the effects of ethmozine on cardiac conduction in isolated perfused rabbit hearts electrically driven at cycle lengths of 320 and 250 ms. There was no significant change in conduction until high concentrations of ethmozine were reached. His-Purkinje and atrioventricular (AV) nodal conduction were slowed significantly at 0.1 microgram/mL and atrial conduction at 1.0 microgram/mL. Conduction block occurred at 10.0 micrograms/mL in all the hearts treated. Effects of the drug (0.1 and 0.01 microgram/mL) on conduction of extrasystoles were also studied in hearts driven at a basic cycle length of 270 ms. No significant change was observed in atrial conduction of extrasystoles throughout the coupling intervals tested at both concentrations. Ethmozine (0.01 and 0.1 microgram/mL) caused slowing of His-Purkinje conduction of extrasystoles but the effect of the drug did not change as a function of the coupling interval. An interval-dependent increase in AV-nodal conduction time was observed, with the maximum slowing of conduction occurring at coupling intervals close to the effective refractory period of the AV node. AV-nodal functional refractory period was increased significantly by ethmozine (0.01 and 0.1 microgram/mL). The effective refractory period was significantly increased only at the higher concentration.     

Effect of diethylamine analog of ethmozine on parameters of sodium current in isolated rat cardiomyocytes

Voltage clamp experiments were made on ezymically isolated and internally perfused rat cardiac cells. The effect of a diethylamine analog of ethmozine (DAAE) on sodium current (INa) was tested when the drug was applied inside or outside the cell. It was found that the effect of DAAE (8 X 10(-6) g/ml) on INa was asymmetrical: after DAAE addition outside the cell, the amplitude of INa was effectively suppressed. Thus, 5 minutes after DAAE action the maximal value of INa in a voltage-current relationship was 20% of the control value without significant changes in the kinetics of INa. When the DAAE was added inside the cell preferentially, the inactivation time constant was increased without significant changes in the amplitude of the maximal INa. The same results were obtained with pronase (1 mg/ml) added inside the cell. It was supposed that as compared to ethmozine, the DAAE possesses a supplementary binding site on the cardiac cell membrane possibly linked to the structures responsible for inactivation processes.     

Influence of light on membrane potential of Neurospora crassa cells

In experiments with left atrial and right ventricular tissues fast sodium current (INa) was decreased by substituting sucrose for sodium in Tyrode's solution and with antiarrhythmic drugs--INa blockers (lidocaine, diphenylhydantoine and ethmozine). It was shown that INa decrease results in the growth of refractoriness (R) of the cardiac tissues. The hypothesis is advanced that the R growth caused by INa decrease is one of the mechanisms of selective sensitiveness of ischemic heart tissues to antiarrhythmic drugs--INa blockers.     

Biochemical basis for the spasmolytic effect of isoquinoline and phenothiazine derivatives

Experiments with isolated ring-like strips of pig heart veins and arteries and those with rat small intestinal strips made in the presence of hyperpotassium contracture have demonstrated that spasmolytic activity of papaverine, nospa, nonachlazine, and ethmozine is a consequence of the reduced respiratory intensity and phosphorylation in smooth muscle mitochondria. No changes in the anaerobic glycolysis rate have been found after drug administrations.     

Influence of ethacizin (the diethyl analog of ethmozine) on phase-dependent parasympathetic effects on the heart

The influence of ethacizin (a diethylamine analog of ethmozine) (1.10(-7)-1.10(-6) g/ml) upon the phase-dependent chronotropic parasympathetic effects was studied on the perfused frog heart. The vagolytic influence of ethacizin (5.10(-7) and 1.10(-6) g/ml) was detected; the concentration of 1.10(-7) g/ml was found ineffective. The vagolytic effect consisted of a decreased maximum of phase-dependent effect, reduced latency and time required for the manifestation of the maximum increase. The period of inhibitory vagal stimulus effectiveness did not change significantly.     

Frequency-dependent blockade of sodium channels in isolated rat myocardial cells by the anti-arrhythmia agent ethmozine

A patch-clamp method was used to study the effects of the phenotiazine antiarrhythmic drug ethmozine (E) on the fast sodium inward current (INa) in freshly isolated heart muscle cells of adult rats. At a concentration of 10(-5) M E caused INa inhibition that could be enhanced by increasing the frequency of depolarization. This inhibition was reversible. After the termination of repetitive depolarization the amplitude of INa recovered with a time constant of about 10 sec. These findings may help to explain the therapeutic efficiency of E in high frequency cardiac rhythm disturbances.     

Antagonists of calmodulin delay injury development in the severely ischemic perfused working rat heart

The effects of calmodulin antagonists trifluoperazine (TFP) and calmidazolium (CMZ) and of ethmozine (a phenothiazine without anticalmodulin activity) on the postischemic recovery in the perfused working rat hearts were studied. In the hearts subjected to 25 min zero-flow ischemia coronary flow, cardiac output, MVO2 and external work recovered to about 50% of the preischemic values during 40 min of reperfusion. TFP (5 x 10(-7) M and 10(-6) M) or CMZ (10(-7) M) improved the functional recovery to 75-94% whereas 5 x 10(-7) M ethmozine was not effective. In all experimental groups a prolongation of the ischemic period caused a progressive deterioration of the functional recovery while the total postischemic LDH release showed an initial gradual rise followed by a later decay. TFP and CMZ prolonged the time-to-half decay of the hemodynamic functions (tHF50) by 4-7 min and the time-to-peak of total LDH release (tLDHmax) by 5-10 min. In the hearts subjected to 0.2 ml/min low-flow ischemia tHF50 and tLDHmax were increased to 40 min, CMZ prolonged these times by further 5-10 min. Thus, TFP and CMZ delayed the development of the myocardial ischemic injury. Although other interpretations are possible, our data are consistent with the hypothesis that calmodulin-sensitive process is involved in the ischemic damage of the myocardium.     

Effect of tetrodotoxin and ethmozine on arrhythmias of a dog heart isolated in the late stage of experimental myocardial infarct

Dog hearts with ventricular extrasystole that developed 24 hours after coronary artery occlusion were isolated and perfused with blood from support dogs. After heart isolation the rhythm disturbances persisted regardless the decreased frequency of the ventricular beats. Administration of tetrodotoxin (4 X 10-8--10-7 g/ml) and ethmozine (3--5X X10-5 g/ml) abolished ventricular arrhythmias and restored the sinus rhythm. Potential mechanisms of the increased susceptibility of ischemic myocardial fibers to tetrodotoxin and antiarrhythmic drugs are discussed.     

Effect of diethylamine analog of ethmozine on parameters of fast sodium current in normal and depolarized myocardial fibers

The effect of a diethylamine analog of ethmozine (DAAE) on fast sodium current of normal and depolarized frog atrial trabeculae was studied by means of the double sucrose gap technique. The depolarization of the fibers was produced both by increasing extracellular potassium concentration up to 8 +/- 9 mM and by current passing. The resting potential of normal fibers was within the range of 75-80 mV, and the depolarized one was within 65 +/- 60 mV. DAAE (8 X 10(-7) g/ml) reduced sodium conductivity, slowed inactivation and reactivation of fast sodium current and shifted the steady-state activation curve (h infinity) to a more negative potential, but the steady-state activation curve (m infinity) to a more positive potential. All these effects were expressed more considerably in the depolarized fibers. The depressing ability of DAAE is assumed to be due to high concentration of the drug in the membrane owing to its high solubility in lipids. The slowing of sodium reactivation and inactivation shows the existence of a receptor for DAAE linked with a h-gate. Prolonged antiarrhythmic action is accounted for by a very slow recovery of sodium current after the drug superfusion. A stronger effect of DAAE on sodium current in the depolarized fibers is likely to point to its selective action on ischemic tissue.     

Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography-mass spectrometry

This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC-MS(n) analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MS(n) fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.     

Measurement of eight urinary metabolites of di(2-ethylhexyl) phthalate as biomarkers for human exposure assessment.

Human metabolism of di(2-ethylhexyl) phthalate (DEHP) is complex and yields mono(2-ethylhexyl) phthalate (MEHP) and numerous oxidative metabolites. The oxidative metabolites, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono(2-carboxymethylhexyl) phthalate (MCMHP), have been considered to be better biomarkers for DEHP exposure assessment than MEHP because urinary levels of these metabolites are generally higher than MEHP, and their measurements are not subject to contamination. The urinary levels of the above metabolites, and of three other recently identified DEHP oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono-2-(1-oxoethylhexyl) phthalate (MOEHP), and mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), were measured in 129 adults. MECPP, MCMHP and MEHHP were present in all the samples analysed. MEHP and the other oxidative metabolites were detected less frequently: MEOHP (99%), MECBP (88%), MECPrP (84%), MEHP (83%) and MOEHP (77%). The levels of all DEHP metabolites were highly correlated (p<0.0001) with each other, confirming a common parent. The ? and ?-1 oxidative metabolites (MECPP, MCMHP, MEHHP and MEOHP) comprised 87.1% of all metabolites measured, and thus are most likely the best biomarkers for DEHP exposure assessment. The percentage of the unglucuronidated free form excreted in urine was higher for the ester linkage carboxylated DEHP metabolites compared with alcoholic and ketonic DEHP metabolites. The percentage of the unglucuronidated free form excreted in urine was higher for the DEHP metabolites with a carboxylated ester side-chain compared with alcoholic and ketonic metabolites. Further, differences were found between the DEHP metabolite profile between this adult population and that of six neonates exposed to high doses of DEHP through extensive medical treatment. In the neonates, MEHP represented 0.6% and MECPP 65.5% of the eight DEHP metabolites measured compared to 6.6% (MEHP) and 31.8% (MECPP) in the adults. Whether the observed differences reflect differences in route/duration of the exposure, age and/or health status of the individuals is presently unknown.     

High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine

Oracine (I), a potential cytostatic drug, is enzymically converted to a number of metabolites whose formation has been studied in vitro and in vivo. The metabolites were separated by reversed-phase HPLC and characterized by UV spectra. Preparative TLC served for the isolation of the individual metabolites to allow their identification. Two metabolites were identified by Fourier transform NMR as 11-dihydrooracine (II) and a phenolic product (III). Two further metabolites (IV,V) were characterized. Some minor, presumably 11-dihydro metabolites and an 11-oxo metabolite produced in vitro and in vivo were revealed.