ANTICHOLINERGIC ACTIVITY AND PHARMACOKINETIC PARAMETERS OF AGENT SS-68 WITH PROPERTIES OF CLASS III ANTIARRHYTHMIC DRUGS

It was shown that the indole derivate SS-68 (50 and 250 μg/kg intravenous) in acute experiments on cats with neurogenic atrial fibrillation (AF) has a dose-dependent antiarrhythmic action which is associated with the neurotropic influence of this substance, since the suppression of the AF coincides with its anticholinergic effect observed for more than 2 hours, but cardiotropic action at this time was not observed. High antiarrhythmic activity of SS-68 to neurogenic AF may be due to the IKAch inhibition and blockade of M2-cholinergic receptors. SS-68 (50 μg/kg intravenous) in experiments on rabbits is characterized by the following pharmacokinetic parameters: Cmax = 14.5 ng/ml of blood plasma (duration of plasma exposure of SS-68 is 4 hours), T1/2 = 1,1 ± 0.06 hours, MRT(0 – t) = 1.6 ± 0.08 hours, AUC(0 – 1440) = 1006.1 ± 147.7 ng/ml×min, Vss = 4.8 ± 0.8 l/kg and CL = 0.051 l/h. Based on pharmacodynamic and pharmacokinetic parameters it was suggested that anticholinergic action of SS-68 in neurogenic AF will occur in a much lower dose than 50 mg/kg.


Introduction
Atrial fibrillation is the most common heart beat disorder.For the 50-year period of observation in the Framingham study, the morbidity of AF corrected to age increased fourfold [1].From 1990 to 2010, the prevalence of AF in the world and associated with it morbidity and mortality, despite all efforts of medical science and health care increased twofold [2].According to forecasts over the next 3 decades the number of patients with AF in Europe and the United States will increase by more than twofold, which is reason enough to call this arrhythmia "global epidemic" [3,4,5].
In recent years in the treatment of AF there is the preference for class III antiarrhythmic drugs (classification by E. M. Vaughan-Williams [6]), acting mainly at the expense of slowing repolarization and increasing the duration of refractory periods [7].
Despite the fact that to the present time in our country and abroad there were created a number of different on the effectiveness and mechanism of

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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY action of class III antiarrhythmic drugs (amiodarone, sotalol, bretylium, dofetilide, ibutilide, azimilide, vernakalant, dronedarone, nibentan, niferidil), search and development of representatives of this group of drugs continues, that is due to its not enough effectiveness and presence of side effects, the main of which is the torsades de pointes [7].
Experimental study of effectiveness and mode of action of new class III antiarrhythmic drugs is carried out using various models of auricular fluttering [8,9].Of these, greater attention should be a model of neurogenic AF [8,10], simulating in healthy animals without prior myocardial damage by steam stimulation of the atria on the back of vagal asystolism, initiated by irritation of the vagus nerve.This model in comparison with the known analogues, in which different approaches are used to development and maintenance of AF [8,11,12,13,14,15], is not associated with artificial irregularities of the internal environment and is control that brings it the most to the pathogenesis of AF in clinical practice [16,17].Simulation of neurogenic AF made possible to study antifibrillatory action of many substances, including representatives of class III antiarrhythmic drugs [18,19,20,21,22,23].
In previous studies it was shown that SS-68 on the mode of cardiotropic action may be fits into class III antiarrhythmic drugs [24].It should be noted that in experiments on dogs SS-68 suppresses auricular fluttering [25], which is simulated by a mechanical destruction of the sinus node and subsequent electrical stimulation of the right atrium according to the method described by P. A. Galenko-Yaroshevskii et al. [8] A. Rosenblueth, G. Ramos [26].Anticholinergic activity of SS-68 in the neurogenic AF has not been studied.In addition, the main pharmacokinetic parameters of the minimum effective doses of SS-68 have not been studied.
The aim of this study was to study anticholinergic activity of SS-68 in the neurogenic AF and identify its main pharmacokinetic parameters in minimum anticholinergic dose.
Experimental Experiments to study anticholinergic activity of SS-68 in neurogenic AF were performed on 16 cats weighing 2.6 to 3.4 kg with anesthesia by intraperitoneal administration of 1% of a mixture of α-chloralose and aethaminalum-natrium (75 and 15 mg/kg, respectively) and artificial pulmonary ventilation according to the method developed by Y. R. Sheikh-Zade, P. A. Galenko-Yaroshevsky [10].
Neurogenic AF was simulated by application to the endocardium of the right atrium 2 electric pulses (5 ms, 4 threshold) with an interval of 40 ms on the back of rhythmic stimulation of the cervical portion of the right vagus nerve (2 ms, 40 Hz, 6 thresholds) using the electrostimulator ESU-2.ECG tracing (intra-atrial) was performed using beat-to-beat ratemeter on the recording meter H-338-2, while visual inspection of the events was conducted by using an 8-channel indicator IM 789.Agent SS-68 was administered intravenous in doses of 50 and 250 mg/kg.
In the beginning of the experiment, and then after the administration of the SS-68 at 5, 30, 60 and 120 minutes there were determined the duration of the intervals P-P and P-Q ECG, sinoatrial conduction time [27,28], excitation thresholds of the atria and vagus nerve, atrial effective refractory period, the duration of AF, synchronizing and tonic components of the chronotropic effect of vagus nerve [8].
The research results were statistically analyzed.by the method of direct and indirect differences [29,30] with the definition of arithmetic average (M), standard error of the mean (± m) and indices of statistical significance (p).The differences were considered significant at p < 0.05.
The pharmacokinetics of SS-68 was studied on 12 chinchilla rabbits, which were pre-catheterized in the right ear vein.12 hours before the start of the experiment the animals were deprived of feed, leaving free access to water.The test substance was administered on the third day after catheterization intravenous bolus to 6 rabbits in the ear vein in the form of a solution of 500 µg/ml in water for injection at a dose of 50 mg/kg.Blood was sampled through a catheter in a volume of 0.3 ml in polypropylene tubes containing 20 µl of 5 % EDTA before administration and 5, 15, 30, 60, 120, 240, 480 and 1440 minutes after administration.Blood plasma was separated by centrifugation at 5600 g for 10 min and stored until analysis at -70 C.
To determine the concentration there was used previously validated method for determining SS-68 in the blood plasma of rabbits.Main pharmacokinetic parameters were calculated by A. A. Firsov et al. [31] in Microsoft Office Excel.Outliers in each time point were identified using a statistical test of Grubbs [32].This test is well-proven in similar studies [33].
Results and evaluation SS-68 at a dose of 50 mg/kg at 5, 30, 60 and 120 min after intravenous administration caused a statistically significant reduction in the duration of AF 85, 73, 50 and 33%, respectively.The intervals P-P and P-Q of ECG, the excitation threshold of the atrial effective refractory period and sinoatrial conduction of excitation were not be significantly changed.Against this background, SS-68 had vagolytic effect, manifested a significant increase in excitation thresholds of vagus nerve 16% (to the 5th min of the study), the synchronizing and tonic components of the chronotropic effect of vagus nerve, respectively, 50 and 47, 41 and 41, 41 and 37, 37 and 31% (to the 5, 30, 60 and 120 th min of the study) (table 1).
With increasing dose of SS-68 to 250 mg/kg at 5, 30, 60 and 120 min was a statistically significant reduction in the duration of AF 92, 84, 62 and 54%, respectively.Intervals P-P and P-Q of ECG, as well as the effective refractory period was significantly increased, there were P-P 13, 10 and 8% (5, 30 and 60th min), P-Q 22 and 14% (for 5 and 30 min) and effective refractory period 18, 13 and 12% (5, 30 and 60th min of the study).Excitation thresholds of the atria and sinoatrial conduction of excitation remained virtually unchanged.Vagolytic activity of SS-68 was manifested in the significant increase of the excitation thresholds of vagus nerve 24% and 14% (for 5 and 30 min) and the inhibition of the tonic and synchronizing components of the chronotropic effect of this nerve 62 and 58, 60 and 52, 44 and 58, 56 and 42%, respectively (to the 5, 30, 60 and 120 th min of the study) (table 2).
It is known that increased tone of the parasympathetic nerves causes a reduction in the duration of AF of cardiomyocytes of the atria due to the acetylcholine-activated outgoing K + -current (I KAch ) [34,35].It is shown that many class III antiarrhythmic drugs (sotalol, E-4031, MS-551, amiodarone, ambasilide, nibentan, niterider) have a preventive and neutralize effect against cholinergic AF, which is due to the inhibition of I KAch by blockade of M 2 -cholinergic receptors associated with I KAch by regulatory β -and γ-subunits of the G protein [34,35], and/or directly I KAch channel [36,37,38].
In recent experiments on mammalian cardiac myocytes (whole cell mode using the method of patch-clamp [8,39,40]) there was found that the greatest sensitivity to SS-68 has I KAch , L-type Ca 2+ current I CaL and fast K + -current of the detained straightening I Kr (median effective concentration IC 50 of SS-68 = 1.28, 1.78 and 2.75 µm, respectively) and moderate sensitivity characteristic for ultra-rapid delayed-rectifier K + -current I Kur and ATP-dependent inwardly rectifying K + -current I KATP (IC 50 of SS-68 = 19.8 and 20.0 µm, respectively), low sensitivity to transient outward potassium current Ito (IC 50 of SS-68 = 165.5 µm).Weakly susceptible to the action of SS-68 there were the fast Na + current I Na , inward rectifying K + -current I K1 , and slow component a current of the detained straightening IKs [24].In addition, in experiments on preparations of the left atrium of the rat (with the registration of the action potential by the method of intracellular lead of bioelectrical activity [41]) there was shown that SS-68 at a concentration of 10 µm (definition of minimum effective concentration was not conducted) weakens the effects of general stimulation of the M 2 and M 3 -cholinergic receptors by pilocarpine (at concentration of 10 µm) [42].Since the decline in

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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY SS-68 effect of stimulation of M 3 -cholinergic receptors may not be related to its effect on I KAch , because it is activated only by stimulation of M 2 and M 4 cholinergic receptors [43], the last of which in rat cardiomyocytes play a minor role, and M 1cholinergic receptors are absent [44,45,46], we can assume that the antiarrhythmic effect of SS-68 on neurogenic AF is associated with its inhibitory effect on I KAch and blocking M 2 -cholinergic receptors.
By the definition of the main pharmacokinetic parameters of SS-68 at a dose of 50 µg/kg has been established that its maximum concentration in plasma (C max ) is 14.5 ng/ml; duration of content of SS-68 in the blood is 4 hours.The shape of the concentration time curve is biexponential, suggesting rapid first phase of distribution, alternating a slower phase of elimination.The presence of a plateau indicates a multi-phase distribution, which occur sequentially.Two hours of the study, the concentration of SS-68 reduced 7.2 times (the second hour there was determined 2.0 ng/ml in blood plasma).It suggests that SS-68 in such dose rapidly eliminates in rabbits.Main pharmacokinetic parameters did not show high values of elimination half-life (T 1/2 = 1.1 ± 0.06 hours) and the middle residence time (MRT (0-t) = 1.6 ± 0.08 hours).Fast reduction of concentration in plasma causes a small value of the area under the curve (AUC (0-1440) = 1006.1 ± 147.7 ng/ml × min.The value of the stationary distribution volume (V ss ) equal to 4.8+0.8l/kg, that exceeds the amount of extracellular fluid in the body of rabbits, indicating a high ability of the drug to distribut and accumulate in the tissues.It is connected with the low value of systemic clearance (CL = 0.051 l/h).
Thus, SS-68 in conditions of neurogenic AF, caused by the irritation of the vagus nerve in cats, has a dose-dependent antiarrhythmic effect that is associated with the pre-emptive neurotropic influence of this agent, since the neutralize of the AF coincides with its anticholinergic effect observed over 2 hours and cardiotropic action at this time is absent.High antiarrhythmic activity of SS-68 in neurogenic AF may be due to the inhibition of I KAch and blockade of M 2 -cholinergic receptors.It is not excluded that antiarrhythmic activity of SS-68 in neurogenic AF will manifest itself in lower doses.This is shown by the pharmacodynamic and pharmacokinetic parameters of SS-68.To confirm this assumption more researches of the influence of SS-68 in neurogenic AF in the lower dose range are needed.
The method of synthesis of agent SS-68 modified and the necessary quantity acquired as part of the state assignment of the Ministry of education and science of the Russian Federation No.
4.129.2014/Kat the Department of chemistry of natural and macromolecular compounds, faculty of chemistry of Southern Federal University.

Table 1
The influence of SS-68 (50 µg/kg intravenous) on the cardiac output and the duration of AF when irritation of the vagus nerve in cats (M ± m, n = 8) the Р-Р interval of ECG, msec