Pharmacology 2 3 2016__.Pdf

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY

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The journal has been registered at the Federal service for supervision of communications information technology and mass media (Roskomnadzor) Mass media registration certificate El. № FS 77-55674 of October 28, 2013

Volume 2, № 3. 2016

ONLINE SCHOLARLY PEER-REVIEWED JOURNAL First published online: 2014 ISSN 2500-235X

EDITORIAL TEAM:

EDITOR-IN-CHIEF: Mikhail V. Pokrovskii, Academician of the Russian Academy of Natural, Doctor of Medical Sciences, Professor, Head of the Department of Pharmacology, Institute of Medicine, Belgorod State National Research University. DEPUTY EDITOR-IN-CHIEF: Pavel A. Galenko-Yaroshevsky, Corresponding member of Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Kuban State Medical University, Russia. EXECUTIVE SECRETARY: Oleg S. Gudyrev, Ph.D. of Medical Sciences, Associate Professor, Department of Pharmacology, Institute of Medicine, Belgorod State National Research University. ENGLISH TEXT EDITOR: Igor Lyashenko, Ph.D. in philology, Associate Professor, Department of English Philology and Intercultural Communication, Institute of Intercultural Communication and International Relations, Belgorod State National Research University.

EDITORIAL BOARD:

Tatyana G. Pokrovskaya, Doctor of Medical Sciences, Professor of the Department of Pharmacology, Institute of Medicine, Belgorod State National Research University. Mikhail V. Korokin, Doctor of Medical Sciences, Professor of the Department of Pharmacology, Institute of Medicine, Belgorod State National Research University.

CONSULTING EDITORS:

Elena B. Artyushkova, Doctor of Biology Sciences, Professor, Director of the Research Institute of Environmental Medicine, Kursk State Medical University, Russia. Tatyana V. Avtina, Ph.D in Pharmaceutical Sciences, Associate Professor, Department of Pharmacology, Institute of Medicine, Belgorod State National Research University. Natalia D. Bunyatyan, Doctor of Pharmacy Sciences, Professor, Deputy Director General for Research Federal State Budgetary Institution «Scientific Center for Expertise of Medical Application Products» of the Ministry of Health of the Russian Federation, Russia. Dr. Klaus Witte, Managing Director Abbot Laboratories GmbH, Germany Evgeny A. Konorev, Doctor of Medical Sciences, Professor, University of Kansas City, USA. Konstantin M. Reznikov, Doctor of Medical Sciences, Professor of the Department of Pharmacology, Voronezh State Medical University Named after N.N. Burdenko, Russia. Tatyana A. Savitskaya, PhD in Chemistry, Professor of the Department of Physical Chemistry, Belarusian State University, Belorussia. Lev N. Sernov, Honored Scientist of the Russian Federation, Doctor of Medical Sciences, Professor, General Director of "Farmkonsalting", Russia. Dmitry E. Skopin, Doctor of Physico-Mathematical Sciences, Professor, University "Al Balqa Applied", Jordan. Sergei Y. Shtrygol, Doctor of Medical Sciences, Professor, National University of Pharmacy, Ukraine.

Founder: Federal state autonomous educational establishment of higher professional education «Belgorod State National Research University» Publisher: Belgorod State National Research University Address of publisher: 85 Pobeda St., Belgorod, 308015, Russia Publication frequency: 4 /year

CONTENTS

EXPERIMENTAL PHARMACOLOGY

Bogus S.K., Kulikov A.L., Vinakov D.V., Suzdalev K.P., Galenko-Yaroshevskii P.A. Anticholinergic activity and pharmacokinetic parameters of agent SS-68 with properties of class III antiarrhythmic drugs 3 Gureev V.V. New approaches of morfofunktional pharmacological correction of violations of cardiovascular system in experimental preeclampsia 11

Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor 28

CLINICAL PHARMACOLOGY

Chuhareva N.A., Bontsevich R.A., Shchurovskaya K.V., Denisova D.S. The choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis 46 Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic factors 51 Kukes V.G. , Gorbach T.V. , Romashchenko O.V. , Rumbesht V.V. АT P as the marker of power exchange condition at the experimental ischemia of the myocardium due to metabolic drugs introduction 58

VETERINARY PHARMACOLOGY

Bui Quang Cu , Nguen Hoai Chao, Vesentsev A.I., Buhanov V.D. , Sokolovsky P.V., Mihaylyukova M.O. The antibacterial properties of modified bentonite deposit tam bo 63 Zuev N.P., Bukhanov V.D., Vezentsev A.I., Sokolovskiy P.V., Khmirov A.V., Zueva E.N., Salashnaya E.А., Mihaylyukova M.O. The etiological structure of mass diseases with young gastro and respiratory syndrome 75

PHARMACOLOGYCAL REVIEWS

Avdeeva N.V., Nikitina V.A., Kochkarova I.S., Litvinova A.S. The possibility of administration of glutamate receptors antagonists in the treatment of parkinson's disease 86 Danilenko L.M., Klochkova G.N., Kizilova I.V., Korokin M.V. Pharmacological preconditioning by recombinant erythropoietin as the possibility of increasing the stability of tissue of the retina to reperfusion ischemia in experiment 95 Martynov M.A., Martynova O.V., Shkileva I.Y., Tokarev I.A., Dovgan A.N. Thymosin β4 as basis for creation of a reparation preparation of new generation 101 Reznikov K.M. Paradigm of modern pharmacology: development and current approaches 107

Bogus S.K., Kulikov A.L., Vinakov D.V., Suzdalev K.P., Galenko-Yaroshevskii P.A. Anticholinergic activity and pharmacokinetic parameters of agent SS-68 with properties of class III antiarrhythmic 3 drugs. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 3-10.

Рус. Eng.

EXPERIMENTAL PHARMACOLOGY

UDC: 616.12-008.313:615.222:615.015]-092.9 DOI: 10.18413/2500-235X-2016-2-3-3-10

Bogus S.K.1 Kulikov A.L.2 ANTICHOLINERGIC ACTIVITY AND PHARMACOKINETIC Vinakov D.V.3 PARAMETERS OF AGENT SS-68 WITH PROPERTIES OF CLASS III Suzdalev K.P.4 ANTIARRHYTHMIC DRUGS Galenko-Yaroshevskii P.A.5

1) PhD of Medical Sciences, Assistant of the Department of Pharmacology of Kuban State Medical University. 4, Sedina st., Krasnodar, 350063, Russia, e-mail: [email protected] 2) Postgraduate student of the Department of Pharmacology of Medical institute of Belgorod State University 85, Pobedy St., Belgorod, 308015, Russia, e-mail: [email protected] 3) Postgraduate student of the Department of Pharmacology of Medical institute of Belgorod State University 85, Pobedy St., Belgorod, 308015, Russia, e-mail:[email protected] 4) PhD of Chemical Sciences, assistant professor of the Department of chemistry of natural and macromolecular compounds, Southern Federal University, 7, Zorge st., Rostov-on-Don, 344090, Russia, e-mail:[email protected] 5) Corresponding member of Russian Academy of Sciences, Doctor of Medicine, Prof. of of Kuban State Medical University 4, Sedina st., Krasnodar, 350063, Russia, e-mail: [email protected]

Abstract 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. Key words: vagus nerve, neurogenic atrial fibrillation, indole derivate SS-68, antiarrhythmic action, pharmacokinetics.

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

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Bogus S.K., Kulikov A.L., Vinakov D.V., Suzdalev K.P., Galenko-Yaroshevskii P.A. Anticholinergic activity and pharmacokinetic parameters of agent SS-68 with properties of class III antiarrhythmic 4 drugs. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 3-10. action of class III antiarrhythmic drugs (amiodarone, ventilation according to the method developed by sotalol, bretylium, dofetilide, ibutilide, azimilide, Y. R. Sheikh-Zade, P. A. Galenko-Yaroshevsky [10]. vernakalant, dronedarone, nibentan, niferidil), search Neurogenic AF was simulated by application to and development of representatives of this group of the endocardium of the right atrium 2 electric pulses drugs continues, that is due to its not enough (5 ms, 4 threshold) with an interval of 40 ms on the effectiveness and presence of side effects, the main of back of rhythmic stimulation of the cervical portion which is the torsades de pointes [7]. of the right vagus nerve (2 ms, 40 Hz, 6 thresholds) Experimental study of effectiveness and mode of using the electrostimulator ESU-2. ECG tracing action of new class III antiarrhythmic drugs is carried (intra-atrial) was performed using beat-to-beat out using various models of auricular fluttering [8, 9]. ratemeter on the recording meter H-338-2, while Of these, greater attention should be a model of visual inspection of the events was conducted by neurogenic AF [8, 10], simulating in healthy animals using an 8-channel indicator IM 789. Agent SS-68 without prior myocardial damage by steam was administered intravenous in doses of 50 and stimulation of the atria on the back of vagal 250 mg/kg. asystolism, initiated by irritation of the vagus nerve. In the beginning of the experiment, and then This model in comparison with the known analogues, after the administration of the SS-68 at 5, 30, 60 and in which different approaches are used to 120 minutes there were determined the duration of development and maintenance of AF [8, 11, 12, 13, the intervals P-P and P-Q ECG, sinoatrial conduction 14, 15], is not associated with artificial irregularities time [27, 28], excitation thresholds of the atria and of the internal environment and is control that brings vagus nerve, atrial effective refractory period, the it the most to the pathogenesis of AF in clinical duration of AF, synchronizing and tonic components practice [16, 17]. Simulation of neurogenic AF made of the chronotropic effect of vagus nerve [8]. possible to study antifibrillatory action of many The research results were statistically analyzed. substances, including representatives of class III by the method of direct and indirect differences antiarrhythmic drugs [18, 19, 20, 21, 22, 23]. [29, 30] with the definition of arithmetic average In previous studies it was shown that SS-68 on (M), standard error of the mean (± m) and indices of the mode of cardiotropic action may be fits into class statistical significance (p). The differences were III antiarrhythmic drugs [24]. It should be noted that considered significant at p < 0.05. in experiments on dogs SS-68 suppresses auricular The pharmacokinetics of SS-68 was studied on fluttering [25], which is simulated by a mechanical 12 chinchilla rabbits, which were pre-catheterized in destruction of the sinus node and subsequent the right ear vein. 12 hours before the start of the electrical stimulation of the right atrium according to experiment the animals were deprived of feed, the method described by P. A. Galenko-Yaroshevskii leaving free access to water. The test substance was et al. [8] A. Rosenblueth, G. Ramos [26]. administered on the third day after catheterization Anticholinergic activity of SS-68 in the neurogenic intravenous bolus to 6 rabbits in the ear vein in the AF has not been studied. In addition, the main form of a solution of 500 µg/ml in water for injection pharmacokinetic parameters of the minimum at a dose of 50 mg/kg. Blood was sampled through a effective doses of SS-68 have not been studied. catheter in a volume of 0.3 ml in polypropylene tubes The aim of this study was to study containing 20 µl of 5 % EDTA before administration anticholinergic activity of SS-68 in the neurogenic and 5, 15, 30, 60, 120, 240, 480 and 1440 minutes AF and identify its main pharmacokinetic parameters after administration. Blood plasma was separated by in minimum anticholinergic dose. centrifugation at 5600 g for 10 min and stored until Experimental analysis at -70 C. Experiments to study anticholinergic activity of To determine the concentration there was used SS-68 in neurogenic AF were performed on 16 cats previously validated method for determining SS-68 weighing 2.6 to 3.4 kg with anesthesia by in the blood plasma of rabbits. Main pharmacokinetic intraperitoneal administration of 1% of a mixture of parameters were calculated by A. A. Firsov et al. [31] α-chloralose and aethaminalum-natrium (75 and in Microsoft Office Excel. Outliers in each time point 15 mg/kg, respectively) and artificial pulmonary were identified using a statistical test of Grubbs [32]. This test is well-proven in similar studies [33].

Quantification of SS-68 in the blood plasma of 60th min), P-Q 22 and 14% (for 5 and 30 min) and rabbits was carried out on a liquid chromatograph LC effective refractory period 18, 13 and 12% (5, 30 and UltiMate 3000 (Thermo Fisher Scientific, USA). 60th min of the study). Excitation thresholds of the Detection of analyte was performed on a mass atria and sinoatrial conduction of excitation remained spectrometer Velos Pro (Thermo Scientific, USA) virtually unchanged. Vagolytic activity of SS-68 was with hot electrospray ionization (H-ESI-II). manifested in the significant increase of the Chromatographic separation was performed on a excitation thresholds of vagus nerve 24% and 14% column size 150 × 3.0 mm, filled by reversed-phase (for 5 and 30 min) and the inhibition of the tonic and sorbent Zorbax Eclipce XDB C18 with particle size synchronizing components of the chronotropic effect 3.0 mm with protective column Zorbax Eclipce XDB of this nerve 62 and 58, 60 and 52, 44 and 58, 56 and C18 12.5×3.0 mm with a particle size of 5.0 µm at a 42%, respectively (to the 5, 30, 60 and 120 th min of temperature of 40◦C in the mode of linear gradient of the study) (table 2). eluant at a flow rate of 0.4 ml/min by the following It is known that increased tone of the program: parasympathetic nerves causes a reduction in the Separation stage: Eluant A (5 mM Ammonium duration of AF of cardiomyocytes of the atria due to acetate + 0.1% formic acid) 55% → 45%; Eluant b the acetylcholine-activated outgoing K+-current

(acetonitrile) 45% → 55% for 4 minutes; (IKAch) [34, 35]. It is shown that many class III Washing stage: Eluant A (5 mM Ammonium antiarrhythmic drugs (sotalol, E-4031, MS-551, acetate + 0.1% formic acid) 45%; Eluant b amiodarone, ambasilide, nibentan, niterider) have a (acetonitrile) – 55% – 0,5 minutes; preventive and neutralize effect against cholinergic

Trim stage: Eluant A (5 mM Ammonium acetate AF, which is due to the inhibition of IKAch by + 0.1% formic acid) – 55%; Eluant b (acetonitrile) blockade of M2-cholinergic receptors associated with 45% for 2 minutes. IKAch by regulatory β – and γ-subunits of the The volume of injected sample was 5 µl. G protein [34, 35], and/or directly IKAch channel Scanning was performed by selected-ion monitoring [36, 37, 38]. (SIM). The transition mass for SS-68 – In recent experiments on mammalian cardiac 305,26→208,0, internal standard (fabomotizole) – myocytes (whole cell mode using the method of 307,41→114,0. patch-clamp [8, 39, 40]) there was found that the 2+ Results and evaluation greatest sensitivity to SS-68 has IKAch, L-type Ca + SS-68 at a dose of 50 mg/kg at 5, 30, 60 and 120 current ICaL and fast K -current of the detained min after intravenous administration caused a straightening IKr (median effective concentration IC50 statistically significant reduction in the duration of of SS-68 = 1.28, 1.78 and 2.75 µm, respectively) and AF 85, 73, 50 and 33%, respectively. The intervals moderate sensitivity characteristic for ultra-rapid + P-P and P-Q of ECG, the excitation threshold of the delayed-rectifier K -current IKur and ATP-dependent + atrial effective refractory period and sinoatrial inwardly rectifying K -current IKATP (IC50 of SS-68 = conduction of excitation were not be significantly 19.8 and 20.0 µm, respectively), low sensitivity to changed. Against this background, SS-68 had transient outward potassium current Ito (IC50 of SS- vagolytic effect, manifested a significant increase in 68 = 165.5 µm). Weakly susceptible to the action of + excitation thresholds of vagus nerve 16% (to the 5th SS-68 there were the fast Na current INa, inward + min of the study), the synchronizing and tonic rectifying K -current IK1, and slow component a components of the chronotropic effect of vagus current of the detained straightening IKs [24]. In nerve, respectively, 50 and 47, 41 and 41, 41 and 37, addition, in experiments on preparations of the left 37 and 31% (to the 5, 30, 60 and 120 th min of the atrium of the rat (with the registration of the action study) (table 1). potential by the method of intracellular lead of With increasing dose of SS-68 to 250 mg/kg at bioelectrical activity [41]) there was shown that SS- 5, 30, 60 and 120 min was a statistically significant 68 at a concentration of 10 µm (definition of reduction in the duration of AF 92, 84, 62 and 54%, minimum effective concentration was not conducted) respectively. Intervals P-P and P-Q of ECG, as well weakens the effects of general stimulation of the M2 as the effective refractory period was significantly – and M3-cholinergic receptors by pilocarpine (at increased, there were P-P 13, 10 and 8% (5, 30 and concentration of 10 µm) [42]. Since the decline in

SS-68 effect of stimulation of M3-cholinergic value of the stationary distribution volume (Vss) equal receptors may not be related to its effect on IKAch, to 4.8+0.8 l/kg, that exceeds the amount of because it is activated only by stimulation of M2 and extracellular fluid in the body of rabbits, indicating a M4 cholinergic receptors [43], the last of which in rat high ability of the drug to distribut and accumulate in cardiomyocytes play a minor role, and M1- the tissues. It is connected with the low value of cholinergic receptors are absent [44, 45, 46], we can systemic clearance (CL = 0.051 l/h). assume that the antiarrhythmic effect of SS-68 on Thus, SS-68 in conditions of neurogenic AF, neurogenic AF is associated with its inhibitory effect caused by the irritation of the vagus nerve in cats, has on IKAch and blocking M2-cholinergic receptors. a dose-dependent antiarrhythmic effect that is By the definition of the main pharmacokinetic associated with the pre-emptive neurotropic influence parameters of SS-68 at a dose of 50 µg/kg has been of this agent, since the neutralize of the AF coincides established that its maximum concentration in plasma with its anticholinergic effect observed over 2 hours

(Cmax) is 14.5 ng/ml; duration of content of SS-68 in and cardiotropic action at this time is absent. High the blood is 4 hours. The shape of the concentration antiarrhythmic activity of SS-68 in neurogenic AF time curve is biexponential, suggesting rapid first may be due to the inhibition of IKAch and blockade of phase of distribution, alternating a slower phase of M2-cholinergic receptors. It is not excluded that elimination. The presence of a plateau indicates a antiarrhythmic activity of SS-68 in neurogenic AF multi-phase distribution, which occur sequentially. will manifest itself in lower doses. This is shown by Two hours of the study, the concentration of SS-68 the pharmacodynamic and pharmacokinetic reduced 7.2 times (the second hour there was parameters of SS-68. To confirm this assumption determined 2.0 ng/ml in blood plasma). It suggests more researches of the influence of SS-68 in that SS-68 in such dose rapidly eliminates in rabbits. neurogenic AF in the lower dose range are needed. Main pharmacokinetic parameters did not show high The method of synthesis of agent SS-68 values of elimination half-life (T1/2 = 1.1 ± 0.06 modified and the necessary quantity acquired as part hours) and the middle residence time (MRT(0-t) = 1.6 of the state assignment of the Ministry of education ± 0.08 hours). Fast reduction of concentration in and science of the Russian Federation No. plasma causes a small value of the area under the 4.129.2014/K at the Department of chemistry of curve (AUC(0-1440) = 1006.1 ± 147.7 ng/ml × min. The 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 indicators and their Source Duration of the action, min dimension values 5 30 60 120

Baseline duration of the Р-Р interval of 388.0 ± 14.0 376.0 ± 13.0 387.0 ± 14.0 384.0 ± 15.0 385.0 ± 15.0 ECG, msec [97.0] [100.0] [99.0] [99.0]

Excitation threshold of atria, mV 438.0 ± 60.0 476.0 ± 90.0 458.0 ± 80.0 402.0 ± 70.0 415.0 ± 60.0 [109.0] [105.0] [92.0] [95.0]

Effective refractory period of a 142.0 ± 8.0 138.0 ± 6.0 135.0 ± 8.0 140.0 ± 6.0 139.0 ± 5.0 myocard, msec [97.0] [95.0] [100.0] [90.0]

Sinoatrial conduction of excitation. 28.0 ± 3.0 26.0 ± 2.0 29.0 ± 3.0 27.0 ± 2.0 25.0 ± 2.0 msec [93.0] [104.0] [96.0] [89.0]

P-Q interval of ECG, msec 75.0 ± 4.0 73.0 ± 2.0 76.0 ± 3.0 74.0 ± 2.0 77.0 ± 4.0 [97.0] [101.0] [99.0] [103.0]

Excitation thresholds of vagus nerve, 380.0 ± 40.0 440.0 ± 20.0* 410.0 ± 50.0 400.0 ± 40.0 410.0 ± 50.0 mV [116.0] [108.0] [105.0] [108.0]

Synchronizing component of the 259.0 ± 28.0 130.0 ± 10.0* 153.0 ± 24.0* 154.0 ± 20.0* 162.0 ± 25.0* chronotropic effect of vagus nerve, [50.0] [59.0] [59.0] [63.0] msec

Tonic component of the chronotropic 98.0 ± 12.0 52.0 ± 5.0* 58.0 ± 12.0* 62.0 ± 14.0* 68.0 ± 10.0* effect of vagus nerve, msec [53.0] [59.0] [63.0] [69.0]

Duration of AF, sec 140.0 ± 7.0 21.0 ± 4.0* 38.0 ± 6.0* 70.0 ± 12.0* 94.0 ± 14.0* [15.0] [27.0] [50.0] [67.0]

Comment: Here and in table 2: in brackets there are the indicators expressed in %; *p < 0.05 in comparison with baseline data. RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Bogus S.K., Kulikov A.L., Vinakov D.V., Suzdalev K.P., Galenko-Yaroshevskii P.A. Anticholinergic activity and pharmacokinetic parameters of agent SS-68 with properties of class III antiarrhythmic 8 drugs. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 3-10.

Table 2 The influence of SS-68 (250 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 indicators and their Source Duration of the action, min dimension values 5 30 60 120

Baseline duration of the Р-Р interval of 352.0 ± 8.0 398.0 ± 9.0* 387.0 ± 6.0* 379.0 ± 6.0* 360.0 ± 13 ECG, msec [113] [110.0] [108.0] [102.0]

Excitation threshold of atria, mV 540.0 ± 60.0 560.0 ± 70.0* 560.0 ± 80.0 530.0 ± 50.0 520.0 ± 60.0 [117.0] [104.0] [98.0] [96.0]

Effective refractory period of a 136.0 ± 5.0 160.0 ± 4.0 150.0 ± 5.0 152.0 ± 4.0* 144.0 ± 9.0 myocard, msec [104.0] [102.0] [112.0] [106.0]

Sinoatrial conduction of excitation. 22.0 ± 2.0 22.0 ± 2.0 22.0 ± 2.0 21.0 ± 1.0 20.0 ± 2.0 msec [100.0] [100.0] [95.0] [91.0]

P-Q interval of ECG, msec 72.0 ± 2.0 88.0 ± 2.0* 82.0 ± 3.0* 73.0 ± 3.0 70.0 ± 3.0 [122.0] [114.0] [101.0] [97.0]

Excitation thresholds of vagus nerve, 370.0 ± 40.0 460.0 ± 30.0* 420.0 ± 30.0* 390.0 ± 40.0 380.0 ± 30.0 mV [124.0] [114.0] [105.0] [103.0]

Synchronizing component of the 265.0 ± 22.0 101.0 ± 28.0* 106.0 ± 23.0* 111.0 ± 22.0* 117.0 ± 23.0* chronotropic effect of vagus nerve, [38.0] [40.0] [42.0] [44.0] msec

Tonic component of the chronotropic 94.0 ± 12.0 39.0 ± 6.0* 45.0 ± 12.0* 53.0 ± 14.0* 55.0 ± 13.0* effect of vagus nerve, msec [42.0] [48.0] [56.0] [58.0]

Duration of AF, sec 15.0 ± 8.0 12.0 ± 4.0* 24.0 ± 5.0* 58.0 ± 6.0* 7.0. ± 6.0* [8.0] [16.0] [38.0] [46.0]

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UDC:618.3-06-08:577.112.385.2 DOI: 10.18413/2500-235X -2016-2-3-11-27 Gureev V.V. NEW APPROACHES OF MORFOFUNKTIONAL PHARMACOLOGICAL CORRECTION OF VIOLATIONS OF CARDIOVASCULAR SYSTEM IN EXPERIMENTAL PREECLAMPSIA

Ph.D. Associate Professor, Department of Pharmacology of the Federal State Autonomous Educational Institution of Higher Education "Belgorod State National Research University" of the Ministry of Education and Science of the Russian Federation 85, str. Pobedy, Belgorod, 308015, Russia, e-mail: [email protected] Abstract. Experimental Modeling ADMA-like preeclampsia administration to rats was performed by N- nitro-L-arginine methyl ester, from 14 to 20 days of pregnancy. The animals were observed increase in blood pressure, proteinuria, impaired microcirculation in the placenta, the violation of the regulation of vascular tone and destructive changes in the placenta of ischemic origin. Introduction of tetrahydrobiopterin, a selective inhibitor of arginase II of, recombinant erythropoietin, tadalafil, erythromycin, azithromycin, and playback systems polivitaminnomineralnyh distant ischemic preconditioning resulted in a pronounced correction of morphological and functional disorders arise when modeling the experimental preeclampsia. This was reflected in the reduction of blood pressure, reduction of proteinuria, increase of microcirculation in the placenta, the restoration of vasodilator function of blood vessels and prevent destructive phenomena in the placenta compared with a group of untreated animals, increasing the concentration of NO end-metabolites in plasma. These data suggest a pronounced correction of morphological and functional disorders arise when modeling ADMA-like preeclampsia study medication and the prospects for further research to find new drugs have endoteleoprotektivnymi properties for correcting the second half of pregnancy preeclampsia. Keywords: ADMA, preeclampsia, endothelial dysfunction, rats, endotelioprotektory. INTRODUCTION methylated analogs of L-arginine asymmetric Preeclampsia is the most common obstetric dimethylarginine (ADMA) and monometilarginina pathology. In certain regions of the country, its (L-NMMA). The end finale is a complex frequency can be more than 30% of all cases of pathophysiological mechanisms and a violation of the pregnancy [1, 2]. In preeclampsia maternal mortality rheological properties of blood coagulation and is one of the first places. It accounts for up to 25% of endothelial dysfunction, which cause multiple organ cases [3, 4]. Children born to mothers who have had failure [14, 15, 16]. preeclampsia, perinatal morbidity is 30%, and the Thus, at the present time it has accumulated quite a mortality rate is 3-4 times higher than the population- lot of information on the impact of various factors on based [5]. In addition, suffering mental and physical the course of preeclampsia and their role in the development of the child, while women are more development of endothelial dysfunction as the main likely development of chronic kidney disease and pathogenetic link of this terrible disease and the hypertension [4, 6]. possibility of correction of endothelial dysfunction The pathogenesis of preeclampsia is still far from a pharmacological agents of different groups [17, 18, 19]. complete understanding, but more and more researchers However, the available literature there is no information are paying attention disangiogenesis in the placenta and about the study endothelioprotektive action in terms of impaired regulation of the tone of small arteries [7, 8, ADMA-like preeclampsia tetrahydrobiopterin, tadalafil, 9]. A morphological study of the placenta, many vitamin B6 and folic acid, multivitamins and mineral authors describe a specific histology, which is the complexes, erythromycin, azithromycin, recombinant imbalances of its border area between the maternal and erythropoietin, as well as on the effectiveness of the fetal parts [10, 11, 12, 13]. Incomplete cytotrophoblast direction, which is based on distant ischemic invasion occurs in maternal spiral arteries, leading to preconditioning. ischemic events in the placenta and increase the MATERIALS AND METHODS permeability barrier foetoplacental [11, 14]. The experiment was performed on female white rats Release in the systemic circulation of the of Wistar line weighing 250-300 g ADMA-like Gesto ischemic placenta free radicals, hormones, growth modeoirovali by administration of non-selective NO- factors, pro-inflammatory cytokines, fetal antigens synthase blocker of N-nitro-L-arginine methyl ester and other humoral factors causes an increase in the (L-NAME) was administered intraperitoneally in a dose content of cell adhesion molecules and blood of 25 mg / kg / day for seven days (day 14-20 of accumulation of endogenous inhibitors of eNOS – pregnancy) [10, 20, 21, 22]. To simulate the reduced

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Gureev V.V. New approaches of morfofunktional pharmacological correction of violations of cardiovascular system in experimental preeclampsia. Research result: pharmacology and 12 clinical pharmacology. Vol. 2, №3 (2016): 11-27. blood flow to the uterus on day 14 of gestation in 10 to the normal performance of animals weighing 250- animals were silver overlay clips directly above the 300 g at proteinuria, which values correspond to 0.93 ± bifurcation of the abdominal aorta (0.2 mm) on both 0.06 g / l, ie to 1.0 g / l. The introduction of L-NAME ovarian artery (0.1 mm). In 10 animals on day 14 of had no significant effect on 12-hour urine output. In the gestation performed silver overlay clip on the right iliac analysis of urine revealed a moderately pronounced artery (0.1 mm) and the right ovarian artery (0.1 mm) increase in proteinuria with values of 2,04 ± 0,22 g / l (p [23, 24]. <0,05). However, it should be noted that in the The degree of endothelial dysfunction by measurement of the microcirculation in the renal tissue calculating the ratio of endothelial dysfunction is statistically significant difference between the group (CED) [10, 20]. of intact animals, and pregnant animals with simulated The level of NO metabolites (m. E. The total ADMA-like preeclampsia was not observed. concentration of nitrite and nitrate, NOx) was Examination of endothelial NO-producing measured colorimetrically using a color development function showed that the simulation L-NAME- reaction diazotization of sulfanilamide nitrite forming induced deficit of NO resulted in a sharp decrease in part of the reagent. eNOS levels were determined in expression of eNOS and content of nitrite ion (NOx), the cell lysate by the method of R.J. Hendrickson respectively, 1.8 and 1.7-fold (Table 1.1.). with minor modifications [10, 25]. Histology of the kidneys intact pregnant rats (21 Measurement of the microcirculation in the days gestation) were as follows: glomeruli moderately placenta was performed using equipment companies congested, the basement membrane is thickened, Biopacsystems: polygraph MP100 module with laser inflammatory changes in the interstitium and glomeruli Doppler flowmetry (LDF) LDF100C and invasive are no pyramids; spastic and hypertrophic changes in needle sensor TSD144 [10, 20, 21]. the small arteries and arterioles were detected (Fig. 1.1). Collection of urine was performed for 12 hours using a special metabolic cages. The method Brandberg-Roberts-Stolnikova ring is sample Geller. To investigate the liquid in the greater omentum was performed weighing it, followed by drying at 37 0C for 24 hours and weighed again [5, 26]. Morphological studies were conducted under the guidance of prof. AA Dolzhikov and prof. VS Barsukov The embrions was removed from the uterine cavity, weighed, measured growth (craniocaudal size) followed by calculation of height-for-weight [27].

1. COMPARATIVE ASSESSMENT OF Figure 1.1. Kidney pregnant rats normal: small artery in the MORPHOFUNCTIONAL DISTURBANCES AT cortex is intact with no signs of hypertrophy of the walls, its ADMA-MODELING OF PREECLAMPSIA AND endothelium; glomerular capillary basement membrane is REDUCED BLOOD FLOW TO THE UTERUS thickened, the cellular inflammatory response is absent. H & With the introduction of ADMA-like agents – L- E stain. X 280. NAME to pregnant females (25 mg / kg once daily Placenta rats on day 21 of pregnancy with no from 14 to 20 days of pregnancy) on day 21 we signs of damage (Fig. 1.2). observed the development of pathology in its In rats with ADMA-like preeclampsia found to manifestations corresponding criteria of clinical increase the fluid content in the tissue of the greater manifestations of preeclampsia. There was a omentum with 49,89 ± 0,82% to 58,09 ± 1,73% statistically significant increase in systolic and (p <0.05). The glomeruli showed signs of ischemic diastolic blood pressure to 134,5 ± 2,3 and 92,0 ± 2,1 damage in the form of anemia, and degenerative to 186,3 ± 7 and 145,0 ± 5,0 mm Hg. Art., changes in the basal membrane of the glomerular respectively, and an increase in CED with 1,1 ± 0,11 capillaries with thickened in its form of wire loops (Fig. to 3,12 ± 0,17 (p <0,05) (Table. 1.1). 1.3 (b)). A notable reaction mesangium were observed: The value of the level of the microcirculation in the average number of cells per glomerulus was 39.20 ± the placenta intact pregnant rats on day 21 amounted to 2.99 (40.70 ± 3.80 normally). These changes may 446,3 ± 27,5 ped (perfusion units). In the simulation, explain the glomerular apparatus available to the rats in ADMA-like preeclampsia, it found a significant this group hyperfiltration with severe proteinuria. In the decrease to 218,3 ± 13,7 ped. In intact pregnant rats 12- small arteries and arterioles spasm observed and hour urine output was 5.8 ± 0.3 ml, which corresponded marked hypertrophy of the walls (Fig. 1.3 (a)).

Б . А

Figure 1.2. The structure of an intact placenta. spongy structure (villous) layer (a) and the border area between naps and a layer of giant trophoblast (b) of the placenta intact animals: uniform blood circulation capillaries of the villi and mezhkapillyarnyh spaces, relatively monomorphic structure trophoblast, the continuity of the transition from villous trophoblastic layer in the layer; Ochre. hematoxylin and eosin. Fig-. X 200

А Б

Figure 1.3. Kidney of pregnant rats with preeclampsia (pregnancy day 21). and – a spasm and hypertrophy of arterioles; b – a ball of anemia, capillary basement membrane thickened dramatically and have the form of wire loops (membranous glomerulopathy); Ochre. hematoxylin and eosin. Fig-. X 280

Morphology of the placenta at ADMA-like In some cases, this resulted in the emergence of preeclampsia manifested as ischemia villi and necrosis of decidual cells of placenta tissue. On the intervilleznyh spaces and desquamation of the outer surface of the placenta was observed massive endothelium in the vessels of the placenta (Fig. 1.4). deposits of fibrin.

А Б

Figure 1.4. Pathological changes in the placenta in modeling ADMA-like preeclampsia. A – uneven blood filling spongy layer; B – vacuolar degeneration of giant trophoblast; foci of necrosis on the border of giant trophoblast and decidua tissue; degenerative changes, anemia decidual layer. Ochre. hematoxylin and eosin. Fig-. X 200.

Silver overlay clips above the aortic bifurcation In the simulation, the reduced blood flow in both and 2 ovarian artery led to a rise in blood pressure up uterine horns in the placenta were observed to 155,4 ± 3,6 and 109,3 ± 5,7 mm Hg. Art. Factor of pronounced changes in ischemic (Fig. 1.5). endothelial dysfunction increased to 2,02 ± 0,23 (p Overlay clip on the artery supplying the right <0,05). In addition, there was a decrease of uterine horn only led to a decrease in microcirculation in the placenta of both uterine horns microcirculation in the placenta right before the horn to 229,7 ± 9,9 446,3 ± 27,5 at ped in intact pregnant and 204,4 ± 14,3 to 309,0 ± 15,8 ped in the left horn females. Simulation of blood flow reduction in both of the uterus (p <0,05). No statistically significant uterine horns resulted in a statistically significant as changes in blood pressure, CED, diuresis, proteinuria compared to intact pregnant animals increase in and NO – synthesizing endothelial function have proteinuria to 1,34 ± 0,11 g / l (p <0,05). been identified in these animals. NO-producing endothelial function tests in In the simulation, the reduced blood flow in the animals with reduced blood flow in both uterine right horn in the right uterine horn placentas appeared horns showed no statistically significant difference in ischemic changes and morphological picture is not the expression of eNOS and endothelial final fundamentally different from the placentas condition concentration of NO metabolites in blood plasma in a group of animals with the simulation of a compared with intact pregnant animals. reduced blood flow in both uterine horns

. Figure 1.5. Pathological changes in placental ischemia. A – a general view with extensive necrosis and hemorrhage in the labyrinth zone (LZ), B – and hemorrhagic necrosis of the lake in the LZ, B – a major focus of purulent detsiduita, tissue separation at the boundary of the placenta and the uterine wall. H & E stain. Fig-. X50 (A), X200 (B, C)

Integral characteristics of scoring changes in preeclampsia and simulate the reduced blood flow to morphological parameters for modeling ADMA-like the uterus is presented in Table. 1.1. Table 1.1 Values of morphological and functional parameters in pregnant rats at modeling ADMA-like preeclampsia and reduced blood flow in the uterus , (M ± m; n = 10) Group Pregnant Pregnant (reduced blood Pregnant intact + Pregnant (reduced blood flow to flow in the right uterine L-NAME Index the uterine horns 2) horn) SBP mm Hg. 134,5±2,3y 186,3±7* 155,4±3,6* 138,8±4,4y DBP, mm Hg. 92,0±2,1y 145,0±5,0* 109,3±5,7* 89,8±5,8y CED, cond. u 1,10±0,11y 3,12±0,17* 2,02±0,23* 1,29±0,08y microcirculation, 446,3±27,5y 218,3±13,7* 229,7±9,9* 204,4±14,3*R per. u 309,0±15,8*yL NOx, umol/dL 2,28±0,11y 1,28±0,08* 2,12±0,11y 2,15±0,09y eNOS, % 113,2±5,1y 68,5±3,3* 104,5±6,6y 105,4±3,2 y Assessment of pathomo– 0-1 5-6* 5-6* 5-6*P rphological shanges, in points 0-1L The liquid content in 49,89±0,82y 58,09±1,73 52,86±2,66 48,78±2,00y tissues gland % Weight , g 1,73±0,06y 1,52±0,06* 1,16±0,04* 1,13±0,06*R 1,31±0,08*L The growth, mm 24,59±0,42y 22,91±0,34* 20,10±0,27* 20,89±0,45*R 22,90±0,45*L Height/weight 14,91±0,28y 15,93±0,31* 18,25±0,55* 19,36±0,76*R mm/g 18,63±0,68*L Postimplantation death,% 0 0 36,53±2,86* 34,16±3,37*R 18,66±5,66*L Note: * – p <0.05 comparison with intact pregnant rats; y- at p <0.05 in comparison with pregnant rats with L-NAME, R- microcirculation in the right uterine horn, L – microcirculation in the left uterine horn. RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Gureev V.V. New approaches of morfofunktional pharmacological correction of violations of cardiovascular system in experimental preeclampsia. Research result: pharmacology and 16 clinical pharmacology. Vol. 2, №3 (2016): 11-27.

Comparative assessment of the fruit revealed a drug included with standard therapy of preeclampsia decrease in fetal weight at all modeled pathological methyldopa and improving endothelial dysfunction conditions as compared to the intact group of (CED) under the influence of all studied pregnant animals. In groups of animals with reduced pharmacological agents affect the way L-arginine – NO uterine blood flow malnutrition fruit it was more – cGMP (Table 2.1.). pronounced compared to the group with ADMA – similar to preeclampsia. In addition, in animals with When microcirculation research in placenta reduced uterine blood flow was observed were detected its improvement under the influence postimplantation fetal death (Table 1.1.). of tetrahydrobiopterin, a selective inhibitor of Thus, a comprehensive analysis of arginase 2 and tadalafil (Table 2.1.). In addition, morphological and functional changes in the under the influence of pharmacological agents that simulation of various pathologies of pregnant women affect the way L-arginine – NO – cGMP leads to the conclusion that the ADMA-like model is methyldopa and a decrease in proteinuria (see closest to the second half of pregnancy gestosis. This Table 2.2.). is supported by: high blood pressure, disturbance of In the study of the effect of the relationship vasoconstrictor and vasodilating tetrahydrobiopterin, ZB49-0010 and tadalafil on mechanisms, reduction of microcirculation in the the NO-synthesizing endothelial function in the placenta, increasing proteinuria, violation of NO- correction of ADMA-like preeclampsia was found synthesizing endothelial function, a set of a statistically significant improvement (p <0,05) morphological changes in the kidney and placenta, under the influence of methyldopa, that was the reason for using it as a base model. tetrahydrobiopterin and ZB49-0010 (Fig. 2.1). This 2. RESEARCH EFFICIENCY OF applies to both end metabolites content NO, and PHARMACOLOGICAL AGENTS AFFECTING the level of expression of eNOS. THE WAY L-ARGININE – NO – CGMP, THE Morphological and immunohistochemical CORRECTION OF ADMA-LIKE study revealed kidney and placenta protective PREECLAMPSIA effect of the application of methyldopa and Investigation of the effectiveness of pharmacological agents that affect the metabolic pharmacological agents that affect the way L-arginine – pathway of L-arginine – NO – cGMP, which NO – cGMP: tetrahydrobiopterin (BH4), a selective resulted in an integrated approach to the inhibitor of arginase 2 (ZB49-0010) and a PDE-5 assessment of the group intact pregnant animals inhibitor tadalafil in the correction of ADMA-like (Table 2.3.). preeclampsia showed a statistically significant reduction in blood pressure under the influence BH4, tadalafil and Table 2.1 Effect of pharmacological agents that affect the way L-arginine – of NO – cGMP in the blood pressure, CED and microcirculation in the placenta in the correction of ADMA-like preeclampsia in rats (M ± m; n = 10) Group SBP, DBP, CED , Microcirculation Index mmHg. mmHg. cond. u intact 134,5±2,3y 92,0±2,1y 1,10±0,11y 446,3±27,5y L-NAME 186,3±7* 145,0±5,0* 3,12±0,17* 218,3±13,7* L-NAME + methyldopa (2 x 0,43) 135,8±2,9y 103,8±3,8y 3,16±0,33* 248,4±10,7* mg / kg / day L-NAME + tetrahydro-biopterin 157,4±7,9*y 116,7±8,8*y 1,73±0,24*y 402,0±26,2y (10 mg / kg) L-NAME + ZB49-0010 (5 mg / kg) 192,5±8,7* 150,2±6,7* 1,49±0,14y 435,4±27,4y L-NAME + tadalafil (0.9 mg / kg) 149,7±2,2y 97,6±3,2y 1,85±0,08*y 398,7±24,9y Note: * – p <0,05 – compared to intact; y – p <0.05 – in comparison with L-NAME.

Table 2.2 Effect of pharmacological agents that affect the way L-arginine – NO – cGMP values of parameters a 12-hour proteinuria and urine output correction ADMA-like preeclampsia (M ± m; n = 10) Group Number of 12-hour urine Proteinuria Index (mL) (G / l) intact pregnant 5,8±0,3 0,93±0,06у L-NAME 5,6±0,3 2,04±0,22* L-NAME + methyldopa (2 x 0,43) mg / kg / day 5,4±0,3 1,45±0,14* у L-NAME + tetrahydrobiopterin (10 mg / kg) 5,6±0,3 1,16±0,09у L-NAME + ZB49-0010 (5 mg / kg) 5,6±0,3 1,19±0,10 у L-NAME + tadalafil (0.9 mg / kg) 5,7±0,3 0,99±0,05у

Note: * – p <0,05 – compared with intact pregnant animals; y – p <0.05 – compared with pregnant females receiving L- NAME.

2,50 120

% 100 2,00

1,50

60 107,7±4y

113,2±5,1y μmol/dL

1,00 108,2±4,2y 2,28±0,11

1,95±0,06y 40

1,86±0,07y

68,5±3,3* 1,48±0,08* 0,50 65,5±3,4*

1,28±0,08* 20

76,5±6,7* 1,36±0,10*

0,00 0 Int L-NAME Methyldopa ВН4 ZB49-0010 Tadalafilum Int L-NAME Methyldopa ВН4 ZB49-0010 Tadalafilum Note: * – p <0.05 compared with intact pregnant rats; Y- p <0.05 compared with the group of pregnant animals treated with L-NAME.

Figure 2.1. Study of the influence on NOx concentration in plasma and the expression of eNOS pharmacological agents that affect the way L-arginine – of NO – cGMP in the correction of ADMA-like preeclampsia Table 2.3 Effect of pharmacological agents that affect the way L-arginine – NO – cGMP to integral evaluation of the complex pathological changes in the kidney and placenta eNOS expression and the correction ADMA-like preeclampsia (n = 10)

Comprehensive assessment The expression of eNOS A series of experiments in points placenta, % Intact pregnant females 0-1 5,4±0,21 L-NAME 5-6* 0,04±0,01* L-NAME + methyldopa (2 x 0,43) mg / kg / day 3* 2,07±0,16* L-NAME + tetrahydrobiopterin (10 mg / kg) 2-3* 4,01±0,26 L-NAME + ZB49-0010 (5 mg / kg) 2* 4,45±0,21 L-NAME + tadalafil (0.9 mg / kg) 3* 5,04±0,26 Note. * – P <0,05 compared with intact pregnant females.

Microscopic study of the kidneys of animals Morphology of the placenta at the L-NAME- treated, showed the same kind of picture that is induced preeclampsia, the treatment of methyldopa sharply different from the control group of pregnant and pharmacological agents affect the metabolic rats. Small artery kidney cortex with signs of mild pathway of L-arginine – NO – cGMP, I approached hypertrophy of the muscular layer. The capillaries the group of intact animals. It noted monomorphic glomeruli congested, thickened basement membranes layer structure of the placenta without focal is not a symptom of "wire loops" were absent. destructive changes, mild hyperemia spongy layer. Mesangial significant reaction was observed. Thus, On the outer surface of the placenta were observed the average number of cells per glomerulus in the traces of fibrin deposits. group using Tadalafil was 39.80 ± 2.31 (40.70 ± 3.80 normally).

Thus, the use of tetrahydrobiopterin, a selective gestation, and the introduction of recombinant inhibitor of arginase 2 and tadalafil has a marked erythropoietin resulted in normalization of relations protective effect on the model of ADMA-like between vasodilating and vasoconstrictor responses preeclampsia, which was reflected in a statistically in of ADMA-like preeclampsia, as evidenced by significant antihypertensive effect, reducing the rate CED reduction to 1,52 ± 0,09, 1,56 ± 0,13 and 1,67 ± of endothelial dysfunction (CED), the prevention of 0,15 mustache. u, respectively (Table. 3.1). reduction of microcirculation in the placenta and in It is noteworthy that a single playback of ischemia- full prevention NOx reduction of nitrite ions and reperfusion episodes decreased CED on a background eNOS expression under the influence of tadalafil. At of L-NAME-induced pathology, without affecting the same time, the results of the placenta urine blood pressure. This is indicative of the distinguishing studies showed a reduction in proteinuria. features of the mechanisms of action of single and Morphological studies of kidney and placenta in multiple play episodes of ischemia-reperfusion and their these groups showed maximal approach to histology different effects on the process involved in the group intact pregnant animals. pathogenic elements of humoral and neurogenic The results of correction of morphological and contours regulation of the circulatory system. functional disorders of the cardiovascular system in Playing a single, 10-fold episode ischemia- such of ADMA-eclampsia suggest a promising reperfusion and administration of recombinant approach for the treatment and prevention of erythropoietin caused a significant improvement in preeclampsia aimed at overcoming of ADMA- microcirculation to 327,3 ± 17,2, 339,6 ± 20,4 and mediated inhibition of eNOS and correction caused 296,6 ± 27,1 cond. u, respectively, which was by its pathophysiological changes. significantly higher than that of pregnant females 3. RESEARCH EFFICIENCY SHORT with preeclampsia (p <0,05). EPISODE ISCHEMIA-REPERFUSION AND Playing 10-multiple episodes of ischemia- RECOMBINANT ERYTHROPOIETIN OF reperfusion 10 to 20 hours and the introduction of a ADMA-IN LIKE PREECLAMPSIA AND ROLE recombinant erythropoietin in pregnant animals with AND INOS ATP-DEPENDENT K + CHANNELS simulation of ADMA-like preeclampsia indicators IN THE REALIZATION OF THEIR POSITIVE normalized protein in the urine, the values of which EFFECT do not go beyond the norm and amounted to 1,02 ± Investigation of the effectiveness of short 0,04 and 1 01 ± 0,06 g / l, respectively, is statistically episodes of ischemia-reperfusion, and recombinant distinguishable (p <0.05) compared to untreated erythropoietin for the correction of morphological group of animals pregnant (Table 3.2.). and functional disorders of ADMA-while similar Effect of short episodes of ischemia-reperfusion, preeclampsia and recombinant erythropoietin for NO-producing Playing a single ischemic episode, for 90 endothelial function studied on the basis of data minutes before removing the samples did not lead to eNOS expression and increase NOx content of nitrite any significant change in blood pressure. ions (Fig. 3.1). It is found that after a single playback 10x playback ischemic episode and recombinant of ischemia-reperfusion episodes no increase erythropoietin administration resulted in a statistically expression or eNOS, any level of nitrite ions in the significant reduction in blood pressure: Systolic to blood plasma. Increased expression of eNOS 141.6 ± 5.5 and 143.5 ± 4.0 mmHg, diastolic to 104.2 ± parameters occurred under the influence of 5.7 and 98,1 ± 5,9 mm Hg (P <0.05), respectively recombinant erythropoietin, and playing back 10-fold (Table. 3.1). episode of ischemia-reperfusion. Increased content of Playing as a single ischemic episode in 90 final NO metabolites in blood plasma occurred only minutes to remove the samples and 10 times an at 10-fold playback episodes of ischemia-reperfusion. episode of ischemia-reperfusion 10 to 20 days of

Table 3.1 Effect of short episodes of ischemia-reperfusion and recombinant erythropoietin on blood pressure, CED, and on placenta mikrocirculation (M ± m; n = 10) Group SBP, DBP, CED , Microcirculation Index mmHg. mmHg. cond. u y y y y intact 134,5±2,3 92,0±2,1 1,10±0,11 446,3±27,5 L-NAME 186,3±7* 145,0±5,0* 3,12±0,17* 218,3±13,7* L-NAME + ischemia-reperfusion (1) 177,1±9,8* 124,9±8,4* 1,52±0,09y 327,3±17,2y L-NAME + ischemia-reperfusion (10) 141,6±5,5y 104,2±5,7y 1,56±0,13y 339,6±20,4y L-NAME + recombinant erythropoietin (50 U / kg) 143,5±4,0y 98,1±5,9y 1,67±0,15y 296,6±27,1y Note: SBP, DBP – systolic and diastolic blood pressure; CED – rate of endothelial dysfunction; microcirculation in the placenta; Ped – perfusion unit; * – P <0.05 – in comparison with a group of pregnant intact animals; y- P <0.05 – in comparison with a group of pregnant rats treated L-NAME.. Table 3.2 Effect of short episodes of ischemia-reperfusion and recombinant erythropoietin values of parameters a 12-hour proteinuria and diuresis in modeling ADMA-like preeclampsia (M ± m; n = 10) Group Number of 12-hour Proteinuria Index urine (mL) (G / l) 5,8±0,3 0,93±0,06у Intact

L-NAME 5,6±0,3 2,04±0,22* у L-NAME + ischemia-reperfusion (10) 5,5±0,3 1,02±0,04 L-NAME + recombinant erythropoietin (50 U / kg) 5,3±0,2 1,01±0,06у Note: * – p <0,05 – compared with intact pregnant animals; y – p <0.05 – compared with pregnant females receiving L-NAME. %

2,50 120

100 2,00

80 1,50

60 μmol/dL

1,00 116,5±4,4y 2,28±0,11y

40 87,3±6,5* 113,2±5,1y 1,92±0,18y

0,50 1,38±0,12* 68,5±3,3*

1,28±0,08* 20 1,11±0,09* 53,4±2,5*

0,00 0 Int L-NAME I-R1 I-R10 Erythropoetin Int L-NAME I-R1 I-R10 Erythropoetin Note: * – p <0.05 comparison with intact pregnant rats; Y- p <0.05 compared with the group of pregnant rats treated with L-NAME. Figure 3.1. Effect of short episodes of ischemia-reperfusion, and recombinant erythropoietin for NOx concentration in the plasma and in eNOS expression in endothelial correction ADMA-like preeclampsia

Morphologic study of kidney and placenta also playback episode ischemia-reperfusion revealed the found a protective effect of 10-fold playback ischemia- elimination of ischemic damage in the placental reperfusion episodes and administration of recombinant tissue (Fig. 3.3). There was a relatively even layer of erythropoietin. In animals with 10x playback of short spongy blood supply, no damage layer of giant episodes of ischemia-reperfusion picture dramatically trophoblast and decidua. On the outer surface of the different from the control group of pregnant rats. Small placenta observed weak fibrin deposits. artery kidney cortex with mild signs of hypertrophy and Integral characteristics of scoring morphological spasm of the muscle layer. The capillaries glomeruli parameters and results of immunohistochemical congested, thickened basement membranes is not a study are presented in Table. 3.3, which implies that symptom of "wire loops" is missing. A notable reaction a 10-fold playback brief episode of ischemia- mesangium were observed: the average number of cells reperfusion injury and administration of recombinant per glomerulus was 36.80 ± 3.16 (normally erythropoietin significantly reduced cumulative 40.70 ± 3.80). evaluation score and reduced in eNOS expression in Morphological examination of the placenta in placenta compared to ADMA-like preeclampsia. the correction of L-NAME-induced preeclampsia 10x

Table 3.3 Effect of 10 min ischemia-reperfusion episodes of pathological changes of the complex and expression of eNOS in modeling ADMA-like preeclampsia (n = 10) Comprehensive assessment The expression of eNOS № п/п A series of experiments in points placenta, % 1 Intact pregnant females 0-1 5,4±0,21 2 ADMA-like preeclampsia 5-6* 0,04±0,01* 3 L-NAME + ischemia-reperfusion (10) 2-3* 5,18+0,38 4 L-NAME + recombinant erythropoietin (50 U / kg) 3* 5,18+0,27 Note. * – P <0.05 compared to intact pregnant females.

А В

Figure 3.2. The placenta in animals with 10x playback of transient ischemic episodes on the background of ADMA-like preeclampsia. A – decidual layer: monomorphic structure, the absence of degenerative changes; B – even krovenapolnenie spongy layer; In – No degenerative changes trophoblast; Ochre. hematoxylin and eosin. Fig-. X 200 Study the role of iNOS and ATP-sensitive K+ proteinuria in untreated animals. Only CED, despite a channels in the implementation of the positive statistically significant increase in its remained below effects of brief ischemia-reperfusion episodes in the the group of untreated animals (p <0,05). correction of ADMA-like preeclampsia When administered aminoguanidine or Introduction of aminoguanidine, which is a glibenclamide pregnant animals fundamentally selective inhibitor of iNOS, or glibenclamide, which significant reduction of nitrite ions in the plasma and is a blocker of ATP-sensitive K+ channels, healthy the activity of eNOS did not happen. When pregnant animals did not result in a statistically administered to animals with them of ADMA-like significant change in blood pressure, CED, preeclampsia, the treatment of short episodes of microcirculation in the placenta and proteinuria. ischemia-reperfusion, increased levels of nitrite ions Introduction aminoguanidine or glibenclamide in the blood plasma of the final metabolites of NO pregnant animals with experimental preeclampsia, and eNOS expression under the influence of the latter the treatment of short episodes of ischemia- did not occur (Fig. 3.4). reperfusion injury, resulted in the total elimination of Noteworthy is that the negative effect of their antihypertensive effect, worsened circulation in aminoguanidine and glibenclamide on functional the placenta (Fig. 3.3), and increased to the level of indicators only appears when you attempt to correct

200 SBP 3,5 180 DBP

mmHg 160 CED 3 186,3±7* 198,5±13,1*

140 181,3±4,6* 2,5 120 2 100 141,6±5,5y 134,5±2,3y

80 1,5 3,12±0,17* 2,24±0,17y 60

145,0±5,0*

139,2±8,3* 1 2,34±0,09y

133,5±3,3* 40

104,2±5,7y

92,0±2,1y 0,5 1,56±0,13y

20 1,1±0,11y

0 0 Int L-NAME I-R (10) I-R (10) I-R (10) Int L-NAME I-R (10) I-R (10) I-R (10) +Glibenclamide +Aminoguanidine +Glibenclamide +Aminoguanidine

450 2,5 PE g/l 400 2 350

300 1,5

250 1 200 446,3±27,5y 1,49±0,06* 2,04±0,22* 150 0,5 1,61±0,07* 339,6±20,4y

100 1,02±0,04у 256,8±24,6* 218,3±13,7* 0,93±0,06у 233,2±21,6* 0 50 Int L-NAME I-R (10) I-R (10) I-R (10) +Glibenclamide +Aminoguanidine 0 Int L-NAME I-R (10) I-R (10) I-R (10) +Glibenclamide +Aminoguanidine Note: * – p <0.05 comparison with intact pregnant rats; Y- p <0.05 compared with the group of animals treated with L-NAME. Figure 3.3 Effect of aminoguanidine (300 mg / kg) and glibenclamide (50 mg / kg) on blood pressure, CED, micro-circulation in the placenta and proteinuria in the correction of ADMA-like preeclampsia short episodes of ischemia-reperfusion

2,50 120

2,00 100

80 1,50

60 μmol/dL

1,00 116,5±4,4y 2,28±0,11 113,2±5,1y

40 1,92±0,18y

0,50 1,28±0,08* 68,5±3,3* 1,29±0,07* 1,31±0,06* 20 62,6±2,7* 59,3±3,0* 0,00 Int L-NAME I-R (10) I-R (10) I-R (10) 0 +Glibenclamide +Aminoguanidine Int L-NAME I-R (10) I-R (10) I-R (10) +Glibenclamide +Aminoguanidine Note: * – p <0.05 comparison with intact pregnant rats; Y- p <0.05 compared with the group of animals treated with L-NAME. Figure 3.4. Effect of aminoguanidine (300mg / kg) and glibenclamide (50 mg / kg) on the NOx concentration in plasma and the expression of eNOS in the correction ADMA-like preeclampsia short episodes of ischemia-reperfusion

When administered aminoguanidine or erythropoietin has shown marked endotelioprotektivnoe glibenclamide experimental animals with effect on the model of ADMA-like preeclampsia, which preeclampsia, the treatment of short episodes of was reflected in the strengthening of endothelium- ischemia-reperfusion, there was almost complete dependent relaxation of blood vessels and reducing the elimination of the positive impact of the latter in the rate of endothelial dysfunction CED to the level of placental tissue and kidney. The kidneys were intact animals, as well as in reducing violations observed spasm and hypertrophy of the muscle layer microcirculation in the placenta. In addition, a 10-fold of small arteries. Flattened tubular epithelium, on reproduction of short ischemic episodes and the significant areas of desquamated. A notable reactions introduction of recombinant erythropoietin resulted in a were observed mesangium. decrease in blood pressure, normalization of Morphological picture of the placenta villi and proteinuria, restore function NO-producing system. ischemia manifested intervilleznyh spaces and Morphological study revealed the prevention of desquamation of the endothelium in the vessels of the destructive phenomena ischemic, caused by the placenta. There have been foci of necrosis of introduction of L-NAME when playing 10-fold decidual cells in the placental tissue. On the outer ischemic episodes and the introduction of recombinant surface of the placenta was observed massive erythropoietin. However, complete normalization of deposits of fibrin. morphological picture in the kidney and placenta did Integral characteristics of scoring morphological not come. parameters and results of immunohistochemical study It should be noted that the effects of a single are presented in Table. 3.4, from which it follows that a playback and 10x playback ischemic episodes NO- 10-fold reproduction of short episodes of ischemia- synthesizing function multidirectional. This fact reperfusion on the background of aminoguanidine or shows the different mechanisms for the glibenclamide positive effect on the morphological and implementation of their action functional disorders of ADMA-while such did not have endotelioprotektivnogo. preeclampsia. In the presented experiments showed that the Table 3.4 administration of aminoguanidine, an inhibitor of Effect of aminoguanidine and glibenklamina on iNOS, or glibenclamide, which is a blocker of ATP- complex pathological changes and the expression sensitive K + channels, the experimental animals of of eNOS in the correction of short episodes of ischemia- ADMA-like preeclampsia, treatment playback reperfusion of ADMA-like preeclampsia (n = 10) ischemia-reperfusion episodes, almost completely № eNOS Comprehensive eliminates the positive effect of the latter. This is A series of expression assessment in manifested in the removal of gipotezivnogo effect, experiments in the points reducing the severity of a positive effect on placenta,% endothelial function, reduction of microcirculation in 1 Intact pregnant 0-1 5,4±0,21 the placental tissue and indicators of endothelial NO- females producing function to the level of untreated animals, 2 ADMA-like 5-6* 0,04±0,01* preeclampsia as well as in the restoration of destructive damage to 3 L-NAME + the placental tissue and ischemic kidneys. This ischemia- 2-3* 5,18±0,38 demonstrates the important role of iNOS and ATP- reperfusion (10) sensitive K + channels in the implementation of the 4 L-NAME + protective effects of ischemia-reperfusion episodes in Ischemia- the correction of ADMA-like preeclampsia in the reperfusion injury 5-6* 0,03±0,01* experiment. (10) + aminoguanidine 4. STUDY OF EFFICIENCY (300mg / kg) ERYTHROMYCIN AND AZITHROMYCIN IN 5 L-NAME + ADMA-LIKE PREECLAMPSIA Ischemia- Introduction of azithromycin (30 mg / kg) reperfusion injury 5-6* 0,04±0,01* resulted in a statistically significant reduction in both (10) + systolic and diastolic blood pressure in animals with Glibenclamide (50 experimental ADMA-like preeclampsia (Fig. 4.1). mg / kg) Introduction erythromycin (30 mg / kg) did not have Note. * – P <0.05 compared to intact pregnant females. any significant effect on these parameters. Both drugs approximately equally CED significantly reduced to Thus, a single and 10x playback of short ischemic 1.88 ± 0.16 and 1.82 ± 0.09, respectively (p <0.05), episodes and the introduction of recombinant whereas in the group of untreated pregnant rats given RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Gureev V.V. New approaches of morfofunktional pharmacological correction of violations of cardiovascular system in experimental preeclampsia. Research result: pharmacology and 23 clinical pharmacology. Vol. 2, №3 (2016): 11-27.

L-NAME, it was 3,12 ± 0,17. However, the target different from the control group of pregnant rats. The level of reduction of CED is not reached. most pronounced beneficial effects of the therapy Course administration of erythromycin and was observed in the group of animals with the use of azithromycin caused a significant improvement in azithromycin. Small artery kidney cortex with microcirculation to 381,9 ± 16,6 and 362,1 ± 16,7 moderate signs of hypertrophy of the muscular layer. perfusion units, respectively, which was significantly The capillaries glomeruli congested, slightly higher than that of pregnant females with preeclampsia thickened basement membranes, symptom "wire (p <0,05), and statistically It did not differ from the loops" is missing. A notable reaction mesangium values of the microcirculation in intact pregnant rats. In were observed: the average number of cells per addition, there was a decrease in urine protein to the glomerulus was 39.40 ± 2.74 and 39.30 ± 2.55 level of intact animals (Fig. 4.1). respectively (normal 40.70 ± 3.80). Introduction of erythromycin and azithromycin Morphological examination of the placenta in caused improvement in NO-synthesizing endothelial the correction of erythromycin and azithromycin L- function, which resulted in an increase in the level of NAME-induced preeclampsia showed the same type nitrite ions in the plasma by the action of erythromycin of histology in both groups, but more positive effects and statistically significant increase in eNOS expression were found in a group of animals receiving under the influence of both drugs (Fig. 4.2). azithromycin. Placenta without pathological changes, A morphological study of the kidney and the naps and intervilleznye congested space. There are placenta also found a protective effect of signs of ischemia of the functional nature of the erythromycin and azithromycin. The renal vessels in placenta. On the outer surface of the placenta fibrin animals treated with L-NAME during treatment with deposits were observed. erythromycin and azithromycin, the pattern was

CED 200 3,5 180 SBP DBP 3 160 186,3±7* 140 2,5 180,8±11,1*

120 150,5±2,3y 2 100 134,5±2,3y 1,5 80 3,12±0,17* 60 1

145,0±5,0*

132,6±6,7* 40 1,88±0,16y 1,82±0,09y

107,7±4,8y 0,5

92,0±2,1y 20 0 1,1±0,11y 0 Int L-NAME Erythromycin Azithromycin Int L-NAME Erythromycin Azithromycin

PEd 450 2,5

400 2 350

300 1,5 250 g/l

200 446,3±27,5y 1

150 381,9±16,6y

100 2,04±0,22* 362,1±16,7y 0,5

50 218,3±13,7* 0,79±0,11у 0,96±0,06у

0 0 0,93±0,06у Int L-NAME Erythromycin Azithromycin Int L-NAME Erythromycin Azithromycin

Notes: * – p <0.05 comparison with intact pregnant rats; Y- p <0.05 compared with the group of pregnant animals treated with L-NAME.

Figure 4.1. Effect of erythromycin (30 mg / kg) and azithromycin (30 mg / kg) on blood pressure values, CED, micro-circulation in the placenta and proteinuria in the correction of ADMA-like preeclampsia

2,50 120

100 2,00

80 1,50

eNOS, % eNOS, 60

1,00 117,0±3,6y NOx, μmol/dL NOx, 2,28±0,11y 40 113,2±5,1y 105,4±4,2y 68,5±3,3* 1,48±0,10*

0,50 1,84±0,17y 20 1,28±0,08*

0,00 0 Int L-NAME Erythromycin Azithromycin Int L-NAME Erythromycin Azithromycin Note: * – p <0.05 compared to intact rat; Y- p <0.05 compared with the group of animals treated with L-NAME Figure 4.2. Effect of erythromycin (30 mg / kg) and azithromycin (30 mg / kg) on the NOx concentration in the plasma and in eNOS expression in endothelial correction ADMA-like preeclampsia. Integral characteristics of scoring morphological Improvement of microcirculation in the placenta parameters and results of immunohistochemical occurred in all treatment groups (Fig. 5.1). study are presented in Table. 4.1, which implies that The use of vitamin and mineral complexes erythromycin and azithromycin significantly reduced "Complivit® trimestrum Trimester 2" and scores in the evaluation of the integral (3.4) "Complivit® trimestrum trimester 3" in pregnant compared to ADMA-like preeclampsia and increased animals with simulation L-NAME-induced eNOS expression in placental vascular endothelium. preeclampsia statistically significantly reduced the Thus, erythromycin and azithromycin, 30 mg amount of protein in the urine, bringing its value in / kg showed a clear endothelioprotektive action the last group of animals to the value of intact model L-NAME-induced preeclampsia, as animals . Using a combination of folic acid and folic manifested in the reduction rate of endothelial acid and vitamin B6 are not resulted in a statistically dysfunction up to the level of intact animals, significant reduction in proteinuria pathology in this improving microcirculation in the placenta, and to model (Fig. 5.1). reduce the decrease eNOS expression on background Investigation of the effect of vitamin B9, complete prevention of proteinuria. Additionally, combined use of vitamins B6 and B9, drugs azithromycin showed moderate hypotensive effect, "Complivit® trimestrum Trimester 2" and erythromycin reduced drop stable metabolites in "Complivit® trimestrum trimester 3" to the NO- plasma NOx. Morphological studies have found a synthesizing endothelial function in the correction of decrease under the influence of erythromycin and ADMA-like preeclampsia showed that the use of azithromycin in the indicated doses of disorders of multivitamin-mineral preparations "Complivit® kidney and ischemic changes in the placenta, not trimestrum trimester 2" and "Complivit® trimestrum reaching the damage to structures. trimexter 3" significantly increased the concentration of nitrite ions (Fig. 5.2). Introduction of folic acid 5. STUDY THE EFFECTIVENESS OF and its combination with Vitamin B6 not result in a VITAMIN B9, COMBINING THE USE OF statistically significant increase in the concentration VITAMIN B9 AND B6, VITAMIN-MINERAL of nitrite ions in the blood plasma of animals with COMPLEX "COMPLIVIT® TRIMESTRUM ADMA-like preeclampsia. TRIMESTER 2" AND "COMPLIVIT® Table 4.1 TRIMESTRUM TRIMESTER 3" WITH ADMA- Effect of erythromycin and azithromycin in the complex LIKE PREECLAMPSIA pathological changes and the expression of eNOS in Introduction to the study drugs: folic acid, a placenta in modeling ADMA-like preeclampsia (n = 10) combination of folic acid and vitamin B6, Comprehen eNOS ® sive expression multivitamin-mineral complexes "Complivit № A series of experiments trimestrum Trimester 2" and " Complivit® trimestrum assessment in the Trimester 3" did not have any significant effect on in points placenta,% blood pressure in the background of the simulated 1 Intact pregnant females 0-1 5,4±0,21 2 L-NAME-induced pilot of ADMA-like preeclampsia (Fig. 5.1). 5-6* 0,04±0,01* Introduction of folic acid alone does not preeclampsia 3 L-NAME + erythromycin 3-4* 3,77±0,12* result in correction of CED. The introduction of folic (30 mg / kg) acid in combination with vitamin B6, as well as both 4 L-NAME + azithromycin 3* 4,28±0,21* multivitamin-mineral complexes led to an (30 mg / kg) improvement in endothelial function, which resulted Note. * – P <0.05 as compared with intact in a statistically significant reduction in CED. pregnant females.

CED

200 3,5 SBP 180 DBP

mmHg 3 160 186,3±7* 188,2±7,4* 182,1±5,1* 140 180,9±5,0* 2,5 170,2±7,2*

120 2

100 134,5±2,3y

80 1,5 3,12±0,17* 2,69±0,22*

60 2,0±0,1y

145,0±5,0* 2,10±0,1y 2,1±0,08y 138,9±6,1* 137,1±7,6* 1 133,2±5,8*

121,3±5,0* 40

92,0±2,1y 0,5 20 1,1±0,11y

0 0 Int L-NAME B9 B6+B9 CTr2Tr CTr3Tr Int L-NAME B9 B6+B9 CTr2Tr CTr3Tr

g/l 450 2 PE 1,8 400 1,6 350 1,4 300 1,2

250 1 2,04±0,22* 446,3±27,5y 0,8 1,33±0,06у 200 1,69±0,15* 1,66±0,11* 1,41±0,07у 0,6 389,3±27,3y

150 357,7±37,3y 284,4±10,4y 0,4 316,4±17,4y

100 0,93±0,06у

218,3±13,7* 0,2

50 0 Int L-NAME B9 B6+B9 CTr2Tr CTr3Tr 0 Int L-NAME B9 B6+B9 CTr2Tr CTr3Tr Note: Hereafter – СTr2Tr – «Complivit® trimestrum Trimester 2"; СTr3Tr – «Complivit® trimestrum trimester 3"; * – P <0.05 comparison with intact pregnant rats; Y- p <0.05 compared with the group of pregnant animals treated with L-NAME. Figure 5.1. Impact of the B9 vitamin (0,2mg / kg) combined use of vitamins B9 (0,2 mg / kg) and B6 (2 mg / kg) of drugs "Complivit® trimestrum 2 Trimester" (0.084 tab/kg) "Complivit® trimestrum 3 trimester" (Table 0.084 tab/kg) on blood pressure, CED, micro-circulation in the placenta and proteinuria in the correction of ADMA-like preeclampsia Dynamic expression of eNOS in the application of indicators described in this chapter preparations wore the same character as the concentration of stable NO metabolites (Fig. 5.2). There was a statistically significant increase in eNOS expression indices only in groups of animals treated with multivitamin-mineral preparations "Complivit® trimestrum Trimester 2" and "Complivit® trimestrum trimester 3"

2,50 120

eNOS, % eNOS, 100 2,00

80 1,50

60 113,2±5,1y 2,28±0,11y

NOx, μmol/dL NOx, 1,00 84,8±3,5y

40 82,8±3,7y 1,69±0,1y 68,5±3,3* 1,61±0,06y 63,4±3,5* 1,38±0,07* 1,40±0,06* 60,6±3,3* 0,50 1,28±0,08* 20

0,00 0 Int L-NAME B9 B6+B9 CTr2Tr CTr3Tr Int L-NAME B9 B6+B9 CTr2Tr CTr3Tr Note: Hereafter – СTr2Tr – «Complivit® trimestrum Trimester 2"; СTr3Tr – «Complivit® trimestrum trimester 3"; * – P <0.05 comparison with intact pregnant rats; Y- p <0.05 compared with the group of pregnant animals treated with L-NAME. Figure 5.2. Effect of vitamin B9 (0,2 mg / kg), the combined use of vitamins B9 (0,2 mg / kg) and B6 (2 mg / kg) of drugs "Complivit® trimestrum Trimester 2" ( 0.084 tab/kg) and "Complivit® trimestrum 3 trimester" (0.084 tab/kg) in the NOx concentration in plasma and the expression of eNOS in the correction of ADMA-like preeclampsia RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Gureev V.V. New approaches of morfofunktional pharmacological correction of violations of cardiovascular system in experimental preeclampsia. Research result: pharmacology and 26 clinical pharmacology. Vol. 2, №3 (2016): 11-27.

A morphological study of the kidney and the be data obtained by correcting the experimental placenta was found that administration of folic acid preeclampsia short episodes of ischemia-reperfusion and its combination with vitamin B6 did not result in and recombinant erythropoietin. In clinical situations improvement of histological kidney and placenta. where significantly increases the role of ischemic events With the introduction of vitamin and mineral in the placenta of their use will be increasingly complexes "Complivit® trimestrum Trimester 2" and important. "Complivit® trimestrum Trimester 3" observed Less pronounced therapeutic effect of moderately positive dynamics in the kidney and in macrolides and multivitamin-mineral complexes due placenta (Table. 5.1). In addition, there was increase to their influence on the part of the mediated in eNOS expression in placental vascular pathogenesis. However, this is not how does not endothelium. reduce the significance of the study. Both of these Thus, the data presented studies indicate that the groups of drugs can not be regarded as a drug of minimum protective properties have been identified choice for targeted treatment of preeclampsia, but by using folic acid as monotherapy and when that they have of the pleiotropic effects of vitamin combined with its use of vitamin B6. Intensity of increases the relevance of a balanced diet during positive effects from the use of multivitamin-mineral pregnancy, and in case of need for antibiotic preparations "Complivit® trimestrum Trimester 2" macrolides become an important property for the and "Complivit® trimestrum trimester 3" bore benefit of their choice. significant character and manifested in the Table 6.1 improvement of endothelial function, increase Integrated assessment of complex pathological microcirculation indices in the placenta and increase changes in the kidney and placenta in the correction of NO-synthesizing endothelial function, reduction of of ADMA-like preeclampsia (n = 10) proteinuria and positive dynamics of the Comprehensive A series of experiments assessment in morphological picture in the kidney and placenta. points Table 5.1 Intact pregnant females 0-1 Effect of vitamin B9, combined use of vitamins B6 and L-NAME 5-6* B9, drugs "Complivit® trimestrum Trimester 2" and ® L-NAME + tetrahydrobiopterin (10 mg / 2-3* "Complivit trimestrum trimester 3" in the complex kg) pathological changes and the expression of eNOS in the correction of ADMA-like preeclampsia (n = 10) L-NAME + ZB49-0010 (5 mg / kg) 2* eNOS L-NAME + tadalafil (0.9 mg / kg) 3* Comprehensive A series of expression L-NAME + ischemia-reperfusion (10) 2-3* № assessment in experiments in the L-NAME + recombinant erythropoietin 3* points placenta,% (50 U / kg) 1 Intact pregnant L-NAME + erythromycin (30 mg / kg) 3-4* 0-1 5,4±0,21 females L-NAME + azithromycin (30 mg / kg) 3* 2 L-NAME 5-6* 0,04±0,01* L-NAME + СTr2Tr (0.084 tab/kg) 4* 3 L-NAME + B (0,2 L-NAME + СTr3Tr (0.084 tab/kg) 4* 9 5-6* 0,04±0,01* mg/kg) Note: * – p <0,05 compared with intact pregnant females; 4 L-NAME + B9 (0,2 mg/kg)+ 5-6* 0,03±0,01* In conclusion, it should be noted that the results B6 (2 mg/kg) of this work provide experimental basis for new 5 L-NAME + СTr2Tr approaches of prevention and treatment of 4* 2,02±0,12* (0.084 tab/kg) preeclampsia pharmacological agents designed to 6 L-NAME + СTr3Tr 4* 1,83,±0,21* overcome or compensate ADMA-mediated inhibition (0.084 tab/kg) of NO-synthesizing endothelial function, as well as Note. * – P <0.05 compared to intact pregnant the activation of biological processes underlying females. mechanism of the positive effects of ischemic CONCLUSION. preconditioning. Comparative aspect analyzing the obtained data using an integrated indicator criterion morphological BIBLIOGRAPHY changes, it should be noted that the use of 1. Bell M. J. A historical overview of preeclampsia- pharmacological agents that affect the path of L- eclampsia. J. Obstet. Gynecol. Neonatal. Nurs. 39, №5 arginine was the most effective – NO – cGMP (Table (2010): 510-518. [Free PMC] 6.1.). This can be explained by a direct effect on the 2. De Falco S. The discovery of placenta growth main pathogenesis. In addition, special attention should factor and its biological activity. Exp. Mol. Med. 44, № 1 (2012): 1-9. [Free PMC] RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Gureev V.V. New approaches of morfofunktional pharmacological correction of violations of cardiovascular system in experimental preeclampsia. Research result: pharmacology and 27 clinical pharmacology. Vol. 2, №3 (2016): 11-27.

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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 28 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

Рус. Eng.

UDC: 615.224 DOI: 10.18413/2500-235X -2016-2-3-28-45 Yakushev V.I.1 CARDIOVASCULAR EFFECTS OF AN ARGINASE II SELECTIVE Pokrovskii M.V.2 INHIBITOR

1) Doctor therapist receptionist of Central District Hospital in Stroitel, Belgorod region Lenin Str., Stroitel, Yakovlevsky district, Belgorod region, 309070, Russia, e-mail: [email protected] 2) Doctor of Medical Sciences, Professor, Department of pharmacology of Medical University of NRU BelSU, 85 Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]

ABSTRACT For the first time in vitro experiments there were studied the inhibitory activity and safety of potential molecules arginase II selective inhibitors from the group of norleucine derivatives. Also first the substance under the code ZB49-0010C from the group of norleucine derivatives showed the greatest selectivity and inhibitory activity against arginase II in experiments in vitro. However, this substance in vivo exerts dose-dependent hypotensive action and cardioprotective and endothelial protective effects on the L-NAME induced and homocysteine-induced endothelial dysfunction (ED), which are most pronounced at a dose of 10 mg/kg in intragastric administration. Endothelial protective effect consists of in preventing the increase of coefficient of endothelial dysfunction (CED) and the decrease in the concentration of stable metabolites of nitric oxide in the blood plasma. Cardioprotective effect consists of in preventing the increase of the level of left ventricular pressure during the test for adrenoreactivity and reducing of the cardiac reserve during the overload resistance test, and also in preventing the development of the left ventricular hypertrophy. As part of the study there was investigated the dose-dependent anti-ischemic effect of the arginase II selective inhibitor, substance ZB49-0010C on the chronic limb ischemia in rats, which most pronounced at a dose of 10 mg/kg and consists of in preventing the fall in microcirculatory level on the 29th day of the experiment in the ischemic limb and a protective effect based on the morphological examination of the muscle tissue. Key words: arginase II selective inhibitors, ZB49-0010C, L-NAME, endothelial dysfunction, nitric oxide, methionine, homocysteine, ischemia, cardioprotective effect.

To date, the morbidity and, most importantly, factors having vasodilatatory and antiplatelet action the disablement and mortality from cardiovascular (NO, endothelium-derived hyperpolarizing factor, diseases have no tendency to decrease, despite the prostaglandins), and vasospastic and haemostatic presence of a huge number of tools and methods like system activation factors (endothelin-1, thromboxane medication and non-drug therapy, which in itself A2, superoxide anion). Thus, a key element of the reflects the limited therapeutic possibilities of pathogenesis of ED is the deficit of endogenous NO. modern medicine, or about the faulty and deadlock Normally functioning endothelium continuously the paradigm of pharmacotherapy of cardiovascular produces NO via endothelial NO synthase (eNOS) diseases. As long as modern medicine will not find from L-arginine [5, 6, 7, 8, 9]. adequate and effective treatment modalities of However, along with deficit of NO production, a cardiovascular diseases, the search for new decrease in its bioaccessibility has a significant therapeutic targets and molecules will not lose its importance in the pathogenesis of ED, which may be relevance and priority for innovation due to increase level in plasma asymmetric cardiopharmacology. dimethylarginine (ADMA), an endogenous According to the most progressive of existing competitive еNOS inhibitor. Another mechanism of paradigms, a key element in the pathogenesis of the NO reduction is associated with increased production majority of such cardiovascular diseases as of oxygen free radicals [4, 9, 10]. atherosclerosis, arterial hypertension, atherosclerotic As you know, L-arginine as the only substrate cardiovascular disease (CAD), acute cerebrovascular for NO synthesis, is actively biotransformed under event, dyscirculatory encephalopathy (DEP), chronic the influence of arginase. The concentration of L- arterial insufficiency of the lower extremities is an arginine in the blood plasma varies depending on a ED [1, 2, 3, 4]. ED is the imbalance between humoral RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 29 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. great many corrected and non-corrected factors [5, 6, arginase I in mice there were observed symptoms of 7, 8, 11,12, 13]. hyperammoniemia. All animals died within 10 to 14 It is known that the metabolism of L-arginine in days of postnatal development [26]. the cells occurs in two ways (the first way L-arginine That is why it is important to search for new is hydrolyzed by arginase to ornithine and urea. The highly selective inhibitors of arginase II, the study of second L-arginine is metabolized to nitric oxide and their cardioprotective and endothelial protective citrulline that is catalyzed by NO synthase. Enzymes effects. To date, the global pharmaceutical market arginase and NO-synthase compete for the common has no drugs from the group of arginase II selective substrate L-arginine [4, 9, 14, 15]. inhibitors. That is why the aim of our study is Arginase has a high activity exceeding the increase the efficiency of pharmacological correction activity of NO-synthase. It is represented as two of ED using arginase II selective inhibitors, isoforms: arginase I is liver and arginase II is the norleucine derivatives. extrahepatic form, localized more frequently in the RESEARCH OBJECTIVES kidney, prostate, small intestine. According to several 1. To carry out the selection of potential contemporary researchers, the increased activity of molecules arginase II selective inhibitors according arginase II has been observed in much pathology to the criteria of selectivity and safety tests in vitro. such as diabetes, asthma, glomerulonephritis, 2. In acute pharmacological experiments to psoriasis. As you know, arginase II inhibits еNOS, select antihypertensive doses of the most active and preventing the production of nitric oxide. A number safe agent from the group of norleucine derivatives. of studies have established that inhibition of this 3. To study endothelial protective and enzyme increases the nitric oxide production and the cardioprotective effects of arginase II selective prevention of dysfunctional disorders in the inhibitor under the code ZB49-0010C in dose endothelium [12, 13, 14, 16, 17, 18, 19, 23]. flexibility on the L-NAME induced deficit of Natural arginase inhibitors include amino acids endogenous NO. such as ornithine, leucine, valine, lysine, isoleucine 4. To study endothelial protective and and norvaline. All inhibitors of arginase are divided cardioprotective effects of arginase II selective into selective and non-selective, and specific acting inhibitor under the code ZB49-0010C in dose directly on the enzyme and block its active site, and flexibility on homocysteine-induced ED. non-specific, acting on the enzyme indirectly. With 5. To evaluate the antiischemic activity of the the aim of inhibiting arginase there was used Nω- arginase II selective inhibitor under the code ZB49- hydroxy-l-arginine (NOHA), which is an 0010C on the chronic limb ischemia on the intermediate in the synthesis of NO. However, due to background of L-NAME induced deficit of NO. the NOHA is a coenzyme of cytochrome P450, its MATHERIALS AND METHODS administration was very difficult, and in return, he The experiments were performed in the was synthesized Nω-hydroxy-nor-L-arginine (nor- laboratory of preclinical studies of the Center for NOHA) [10, 24]. Also as an arginase inhibitor there preclinical and clinical studies of Belgorod State was investigated £- difluoromethylornithine (DFMO) University in 520 male Wistar rats (weight 200±10 g, in vitro and in vivo. DFMO inhibits ornithine age 4-5 months) and 40 white mice of both sexes decarboxylase (ODC) and increases the amount of L- (weight 20±2 g; age 5-6 weeks). All experiments arginine through the urea cycle. However, to reduce were approved by the Ethics Committee of Federal the activity of arginase large concentrations of Autonomous Educational Institution of Higher DFMO are required, and it contributes to the Education Belgorod State University. Vivisection independent vascular responses and accumulation of was carried out in accordance with the ethical ornithine. Such substances as S-(2-boronoethyl)-L- principles for the treatment of laboratory animals of cysteine (BEC) and 2(S)-amino-6-boronohexanoic "The European Convention for the Protection of acid (ABH) swowed a high activity in reducing the Vertebral Animals Used for Experimental and Other activity of arginase. Another inhibitor of arginase is Scientific Purposes. CETS No. 123". The L-norvaline [1, 14, 15, 16, 24, 25]. investigated substances were synthesized by High- However, the described inhibitors of arginase Technology Center “CHEMRAR”, under the are low-selective or non-selective and inhibit the leadership of the director general Dmitry activity of arginase II and arginase I. A decrease in Vladimirovich Kravchenko. Structures of the the activity of arginase I associated with a number of synthesized potential molecules are given in table 1. side effects. In experiments with gene knockout of

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 30 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

Table 1 Chemical structure of the investigated substances

Laboratory Molecular Full chemical name of agent Structure code weight

O O O Z991-0104 1,2,4,7-tetraze- 5-tertbutylamino -6-[4H- 319 2,6- dimethoxyphenyl] pyrazine N N N N N

S 2-{1-[3-(thio isoxazole -5-yl) propyl] OH OH O B ZB49-0009 piperidine -4-yl}-6-( dihydroxyboryl) OH 378 norleucine N

NH2

H Cl Cl N O H Cl 2-{1-[3-(3-chloro isoxazole -5-yl) propyl] OH ZB49-0010С piperidine -4-yl }- 6-( dihydroxyboryl) OH O B 471.5 OH norleucine dihydrochloride N

NH2

Research methods in vitro. Study the specific was carried out using the following biochemical activity of ZB49-0010С reactions:

Arginase II hydrolyses L-arginine to Inc., San Diego, CA). To graph concentration mercaptovalerate, which in the presence of a 5, 5'- dependence there was chosen the equation: dithio-bis-(2-nitrobenzoic acid) (DTNB) is Y=Bottom curve + (Top curve-Bottom metabolized to the 5-thio-2-nitrobenzoic acid the curve)/(1+10^((LogIC50-X)* Curve slope)). characteristic yellow color. The activity of the To verify the most promising molecules arginase II is in direct ratio to the amount of 5-thio-2- according to the criterion of safety it is necessary nitrobenzoic acid and measures primary to study of the binding potential molecules spectrophotometrically by the intensity of yellow arginase II selective inhibitors hERG channel of color at a wavelength of 405 nm. A known arginase cardiomyocytes, as blocking of this channel leads to II inhibitor ABH was used as a positive control and fatal consequences such as the disturbance of to determine a value of minimum signal of the repolarization, QT interval increase and the experiment. IC50 (concentration of substance in µm subsequent development of asystole. which causes a 50% maximal effect) for ZB49- Determination of the effect of the studied 0010С was determined from the graph of the substances on the hERG channel was performed concentration dependence of the inhibiting effect of using in vitro test systems Invitrogen® PredictorTM the substance. The experimental data were analyzed hERG. There was investigated the binding substance in the GraphPad Prizm program (GraphPad Software, the hERG channel in the composition of the RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 31 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. membrane fraction when the concentration of the registration of hemodynamic parameters. Systolic drug 10 and 40 µmol. The method is based on blood pressure, diastolic blood pressure and heart rate changing the detection polarization of the were measured continuously by a sensor, the fluorescence, which decreases as the displacement computer program AcqKnowledge 4.1 and hardware- high-affinity fluorescent ligand tracer by the test software complex MP 150 "Biopac-systems" (USA). substance. Experimental procedures were performed There were performed functional tests for in accordance with the manufacturer's instructions to endothelium-dependent and endothelium- test the system PredictorTM hERG. independent vasodilatation with the following Research methods in vivo. Experiments to calculation of the CED [8, 27]. study general toxical action in the acute toxicity of To study the contractile activity of the the arginase II selective inhibitor under the code myocardium and hemodynamic parameters on the ZB49-0010С were performed on white mice of both back of ED, in rats with anesthesia and automatic sexes weighing 22±2 g in accordance with the breathing there was performed catheterization of the applicable guidelines on preclinical study of new left ventricle and there were measured continuously pharmacological substances (Mironov A. N., 2012, by a sensor, the computer program AcqKnowledge R. U. Khabriev, 2005). 4.1 and hardware-software complex MP 150 Therapeutic dose calculation of ZB49-0010С. "Biopac-systems" (USA) hemodynamic parameters, According to the study in vitro activity, ZB49-0010С such as left ventricular pressure, maximum at a concentration of 30 µmol has a 100% high contraction rate (+dp/dt max), maximum relaxation selective inhibitory activity against the arginase II. rate (-dp/dt max) and heart rate. There were On the basis of molecular weight 471.5, when the performed functional stress tests in the following concentration of the solution equal to 30 µmol, dose sequence: of the agent in the volume of distribution of entire 2. The test for adrenoreactivity body is 14 mg/kg. As the circulating blood volume in 3. The overload resistance test (the clamping of rats is approximately 8-10% of body weight, the the ascending aorta for 30 sec) [8, 27]. actual dose may be reduced to 10 times; therefore, a 4. Quantification of stable metabolites of NO. minimal therapeutic dose is 1 mg/kg. As part of the study there was used a modification of To study the hypotensive effect and the selection the method for the quantification of stable of effective doses there was performed acute metabolites of NO (Metelskaia V. A., 2005). pharmacological experiment. Thus arginase II Simulation the chronic limb ischemia of the hind selective inhibitor ZB49-0010С was administrated limb in rats was performed with anesthesia (chloral intragastric in doses of 1 mg/kg, 5 mg/kg and 10 hydrate 150 mg/kg and zoletil 60 mg/kg) by ligation mg/kg. Measurement and record of the systolic blood with the intersection of the femoral artery beneath the pressure, diastolic blood pressure and heart rate were inguinal ligament and removal of the portion of the made for 30 min before the intragastric great vessel, including the femoral, popliteal artery administration of ZB49-0010С, as well as after 15 and primary departments of the crural arteries and 30 min, 1 and 2 hours after on the tail by a sensor (Kolesnik, I. M., 2010). Assessment of the and hardware-software complex MP 150 "Biopac- microcirculation level in the tibiotarsus muscles of systems" (USA), in animals without anesthesia in a the rats was performed with noninhalation anesthesia holder at a temperature of 28◦ C, which was (chloral hydrate 150 mg/kg and zoletil 60 mg/kg) maintained with the help of a special heating using a laser Doppler flowmeter "Biopac-systems" chamber ("Biopac-systems", USA). MP 150, needle sensor TSD-144 and program 1. To simulate ED male rats were administrated AcqKnowledge 4.1 [28, 29]. intraperitoneal eNOS inhibitor L-NAME at a dose of To confirm the development of simulated 25 mg/kg of body weight daily, 1 time per day for 7 pathological processes and integrated assessment of the days. To simulate homocysteine-induced ED rats effectiveness of their correction in all series of were administrated intragastric methionine experiment there was performed histomorphometric (Polisintez, LLC, Belgorod) daily in a dose of 3 g/kg studies of the heart, kidneys and muscles of the hind body weight, 1 time per day. On the 8th day from the limb. start of the experiment under noninhalation The results of the studies were statistically anesthesia (chloral hydrate 150 mg/kg + zoletil 60 analysed. They calculated arithmetic means and mg/kg) the left carotid artery was catheterized for standard errors. The significance of differences was RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 32 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. evaluated in Student t-test, Wilcoxon and Mann- of 12.5 mg/kg daily for 28 days + ZB49-0010С at a Whitney tests. IC50 was calculated by linear regression dose of 1 mg/kg analysis. For the calculations there was used program 6. The group with simulated limb ischemia + for statistical analysis Microsoft Excel 2007. intraperitoneal administration of L-NAME at a dose THE RESEARCH DESIGN of 12.5 mg/kg daily for 28 days + ZB49-0010С at a To study dose-dependent pharmacological dose of 5 mg/kg activity, ZB49-0010C was administered 7. The group with simulated limb ischemia + intragastrically at doses of 1 mg/kg, 5 mg/kg and 10 intraperitoneal administration of L-NAME at a dose mg/kg in acute pharmacological experiment and in of 12.5 mg/kg daily for 28 days + ZB49-0010С at a the study of pharmacodynamic actionon in the long- dose of 10 mg/kg term experiment on the L-NAME induced and 8. The group with simulated limb ischemia + homocysteine-induced ED, as well as on the chronic intraperitoneal administration of L-NAME at a dose limb ischemia. of 12.5 mg/kg daily for 28 days + L-norvaline at a The study protocol of cardioprotective and dose of 1 mg/kg endothelial protective effects of the arginase II The study protocol of the antiischemic activity selective inhibitor ZB49-0010C included the of the arginase II selective inhibitor under the code following sections: ZB49-0010C included the following sections: 1. Simulation L-NAME induced and 1. Mixed noninhalation anesthesia (chloral homocysteine-induced ED and its correction by the hydrate intraperitoneally 150 mg/kg + zoletil 60 mg/kg) arginase II selective inhibitor ZB49-0010C for 7 days. 2. Dissection of skin and fascia 2. Assessment of endothelium-dependent and 3. Measurement of microcirculation by LDF endothelium-independent reactions of arterial 4. Blood sampling from the right ventricle for pressure on simultaneous intravenous introduction of test concentration of stable metabolites of nitric oxide acetylcholine and sodium nitroprusside with chloral 5. Sample collection of the muscles for hydrate anesthesia. morphological study. 3. Automatic breathing of animals, RESEARCH RESULTS catheterization of the left ventricle, registration of the Study of specific activity and mechanism of left ventricular pressure, +dp/dt, -dp/dt, heart rate. action of bioactive molecule ZB49-0010С. The There were performed functional stress tests to assess activity of the arginase II is directly proportional to functional reserves of the contractile activity of the the amount of 5-thio-2-nitrobenzoic acid and it was myocardium. measured spectrophotometrically by the intensity of 4. Blood sampling from the right ventricle for yellow color at a wavelength of 405 nm. A known biochemical analysis and euthanasia of animal. inhibitor of the arginase II ABH was used as a 5. Sample collection of the heart and kidneys positive control to determine value of minimum for morphological studies. signal of the experiment. IC50 for ZB49-0010С was In the study of the antiischemic activity of the determined from the graph of the concentration arginase II selective inhibitor ZB49-0010C there was dependence of the inhibitory effect of the substance developed the following research design: (figure 1 and table 2). Concentration dependence of 1. The group of intact animals the inhibitory effect of the substance ZB49-0010С 2. The sham-operated group of animals and control substance ABH in relation to the arginase 3. The group with simulated limb ischemia II is presented in figure 1. Each concentration point 4. The group with simulated limb ischemia + represents the average value for the two repetitions. intraperitoneal administration of L-NAME at a dose Specific activity of ZB49-0010С in respect of the of 12.5 mg/kg daily for 28 days. arginase II obtained in 2 independent experiments is 5. The group with simulated limb ischemia + shown in table 3. intraperitoneal administration of L-NAME at a dose

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 33 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

Figure 1. Concentration dependence of the inhibitory effect of the substance ZB49-0010С and control substance ABH in relation to the arginase II Table 2 Specific activity of ZB49-0010С in relation to the arginase II, obtained in two independent experiments

Max testing concentration Substance activity in max Numerical order of Substance IC , (nmol) (µmol) concentration (%) 50 the experiment ZB49-0010С 30 99 22.0 1 ZB49-0010С 30 100 19.7 2

As can be seen from figure 1 and table 2, ZB49- concentrations of 10 and 40 µm in the composition of 0010С has a nanomolar inhibitory activity against the membrane fractions using a test system arginase II, as a complete inhibitor of the enzyme. Invitrogen® PredictorTM hERG are presented in table The results of the binding potential molecules 3. arginase II selective inhibitors hERG channel at Table 3 The results of the binding potential molecules arginase II selective inhibitors hERG channel in the composition of the membrane fractions using a test system Invitrogen® PredictorTM hERG hERG- dependence fluorescence polarization Substance Polarization, mP. Mean Non-bindind Concentration (µmol) SD CV, % value (n=20) hERG, % Z991-0104 40 275 5.1 1.9 86 ZB49-0009 40 245 5.0 2.1 87 ZB49-0010 40 265 6.9 2.6 100 Z991-0104 10 287 10.1 3.5 96 ZB49-0009 10 271 5.4 2.0 97 ZB49-0010С 10 268 3,0 1,0 100

The data indicate the absence of the binding substance ZB49-0010C can be attributed to low-toxic ZB49-0010С hERG channel at concentrations up to 40 substances. µmol (table 3). The results of the dynamic of the systolic blood Conducted study of general toxical action in the pressure, diastolic blood pressure and heart rate in acute toxicity showed that after a single intragastric acute pharmacological experiment in animals without administration to mice of the active pharmacological anesthesia treated ZB49-0010C are presented in substance ZB49-0010C at the dose of 1438 mg/kg figure 2. animal deaths were not observed within two weeks As shown by the results of acute pharmacological after administration of ZB49-0010C. According to the experiment (figure 2), ZB49-0010C has a dose- classification Sidorov, K. K., active pharmacological dependent hypotensive effect, most pronounced at a RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 34 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. dose of 10 mg/kg, reaching its maximum after 30 min changing significantly up to 2 hours measurements of after a single intragastric administration, and not blood pressure.

A B

Figure 2. The dynamic of dose-dependent effect of the ZB49-0010C on the systolic blood pressure (A), diastolic blood pressure (B) and heart rate (C) in rats without anesthesia in the acute pharmacological experiment. Comment: * – p<0.05 in comparison with group of intact animals

Study of dose-dependent cardioprotective and dependent endothelial protective effect, most endothelial protective effects of the arginase II pronounced at a dose of 10 mg/kg, which manifested selective inhibitor substances under the code ZB49- in prevention of the increase in CED and approached 0010C on the L-NAME induced deficit of of its values to the group of intact animals. Also there endogenous NO. was found a dose-dependent antihypertensive action Dose-dependent effects of the ZB49-0010C at of ZB49-0010C, which is maximal at a dose of 10 baseline blood pressure, CED, Total NO, mg/kg on the back of L-NAME-induced deficit of adrenoreactivity, cardiac reserve and the diameter of endogenous NO. There was established a dose- cardiomyocytes in comparison with L-Norvaline in dependent cardioprotective effect of this rats with anesthesia on the back of L-NAME-induced pharmacological agent, which is most pronounced at ED are presented in figure 3. a dose of 10 mg/kg and manifested in preventing the Found that arginase II selective inhibitor increase of the left ventricular pressure during the test substance under the code ZB49-0010C has a dose- for adrenoreactivity and the reduction of cardiac

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 35 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. reserve during the overload resistance test, and dependent endothelial protective effect, most preventing the development of the left ventricular pronounced at a dose of 10 mg/kg, which manifested hypertrophy, identified by histological examination. in prevention of the increase in CED and approached of its values to the group of intact animals, and The study of the dose-dependent prevented the increase of the level of homocysteine cardioprotective and endothelial protective effects of in the blood plasma of rats and reduced the the arginase II selective inhibitor substances under concentration of stable metabolites of nitric oxide. At the code ZB49-0010C on the homocysteine-induced the same time there was discovered a dose-dependent endothelial dysfunction. cardioprotective effect of this pharmacological agent, Dose-dependent effect of ZB49-0010C at which is most pronounced at a dose of 10 mg/kg and baseline CED, Total NO, level of homocysteinemia, manifested in preventing the increase of the left adrenoreactivity, cardiac reserve and the diameter of ventricular pressure during the test for cardiomyocytes in comparison with L-Norvaline in adrenoreactivity and the reduction of cardiac reserve rats with anesthesia on the back of homocysteine- during the overload resistance test, and preventing induced ED is presented in figure 4. the development of the left ventricular hypertrophy, Found that arginase II selective inhibitor identified by histological examination. substances under the code ZB49-0010C has a dose-

A) Arterial pressure

B) Coefficient of endothelial dysfunction Figure 3. Dose-dependent effects of the ZB49-0010C compared with L-Norvaline on baseline arterial pressure (A), CED (B), Total NO (C), adrenoreactivity (D), cardiac reserve (E), the diameter of cardiomyocytes (F) in rats with anesthesia on the back of L-NAME-induced ED

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 36 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

B) Total NO

D) Adrenoreactivity 89.7%%** 100% 85.4%**

83.6%

82.0%**

73.7%**

66.0%*

E) Cardiac reserve Figure 3 (continued). Dose-dependent effects of the ZB49-0010C compared with L-Norvaline on baseline arterial pressure (A), CED (B), Total NO (C), adrenoreactivity (D), cardiac reserve (E), the diameter of cardiomyocytes (F) in rats with anesthesia on the back of L-NAME-induced ED

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 37 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

F) Diameter of cardiomyocytes Figure 3 (continued). Dose-dependent effects of the ZB49-0010C compared with L-Norvaline on baseline arterial pressure (A), CED (B), Total NO (C), adrenoreactivity (D), cardiac reserve (E), the diameter of cardiomyocytes (F) in rats with anesthesia on the back of L-NAME-induced ED Comment: * – p<0.05 in comparison with group of intact animals; ** – p<0.05 in comparison with the group of animals with L-NAME-induced ED.

A) Coefficient of endothelial dysfunction

B) Total NO Figure 4. Dose-dependent effects of the ZB49-0010C compared with L-Norvaline on baseline CED (A), Total NO (B), level of homocysteine in the blood plasma (C), adrenoreactivity (D), cardiac reserve (E), the diameter of cardiomyocytes (F) in rats with anesthesia on the back of homocysteine-induced ED.

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 38 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

C) Level of homocysteine in the blood plasma

D) Adrenoreactivity

89.5%**

88.4% 100% 83.3%**

80.1%**

79.2%**

68.2%**

E) Cardiac reserve Figure 4 (continued). Dose-dependent effects of the ZB49-0010C compared with L-Norvaline on baseline CED (A), Total NO (B), level of homocysteine in the blood plasma (C), adrenoreactivity (D), cardiac reserve (E), the diameter of cardiomyocytes (F) in rats with anesthesia on the back of homocysteine-induced ED

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 39 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

F) Diameter of cardiomyocytes

Figure 4 (continued). Dose-dependent effects of the ZB49-0010C compared with L-Norvaline on baseline CED (A), Total NO (B), level of homocysteine in the blood plasma (C), adrenoreactivity (D), cardiac reserve (E), the diameter of cardiomyocytes (F) in rats with anesthesia on the back of homocysteine-induced ED Comment: * – p<0.05 in comparison with group of Tween; ** – p<0.05 in comparison with the group of animals with homocysteine-induced ED.

The effects of the arginase II selective inhibitor, ischemic limb on the back of L-NAME-induced deficit substances ZB49-0010C at the microcirculation level of endogenous NO and its correction by arginase II in the ischemic limbs on the back of L-NAME- selective inhibitor, substances ZB49-0010C at doses of induced deficit of endogenous NO in experiment. 1, 5 and 10 mg/kg are presented in table 4. The results of the evaluation of the microcirculation level in animals on day 29 in the Table 4 The results of the evaluation of the microcirculation level in the tibiotarsus muscles and the concentration of stable metabolites of NO (Total NO) in the blood plasma of rats on day 29. (M±m), PU (perfusion units)

Group Perfusion units Total NО, umol/l Intact animals (n=20) 542.1 ±18.5 117.7 ±3.1 Sham-operated animals (n=20) 540±3.7 120.3±4.0 Animals with simulated limb ischemia (n=20) 360.8 ±8.7* 119.2 ±3.8* Simulated limb ischemia + L-NAME 12.5 mg/kg (n=20) 281.4±25.6** 61.3±4.2*** Simulated limb ischemia +L-NAME 12.5 mg/kg + 386.9 ±30.7*** 73.8 ±4.2*** ZB49-0010C 1 mg/kg (n=20) Simulated limb ischemia +L-NAME 12.5 mg/kg + 583.6±36.7*** 91.6±6.4*** ZB49-0010C 5 mg/kg (n=20) Simulated limb ischemia +L-NAME 12.5 mg/kg + 616.9±24.0*** 118.7±4.3*** ZB49-0010C 10 mg/kg (n=20) Simulated limb ischemia +L-NAME 12.5 mg/kg + L- 496.8±29.6*** 96.6±8.4*** norvaline 10 mg/kg (n=20) Comment: * – p<0.05 in comparison with group of intact animals; ** – p<0.05 in comparison with the group of animals with simulated limb ischemia; *** – p<0.05 in comparison with the group of animals with simulated limb ischemia+L-NAME. As can be seen from table 4, the minimum microcirculation level was dose-dependent, microcirculation level in the tibiotarsus muscles was maximum effect was observed at a dose of 10 mg/kg. noted in the group of animals with simulated limb Found that the arginase II selective inhibitor, ischemia+ L-NAME. The influence of the arginase II substance ZB49-0010C has dose-dependent selective inhibitor, substances ZB49-0010C at the antiischaemic effect, which most pronounced at a dose of 10 mg/kg and consists of in preventing the RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 40 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. fall in microcirculatory level, and also a protective one of the mechanisms of the antiischemic effect of effect on histoarchitecture of the striated muscle ZB49-0010C is the activation of synthesis of tissue in the ischemic limb on the results of endogenous NO due to increasing bioaccessibility of morphological studies (figure 5). It was found that L-arginine.

А B C

D E F Figure 5. Photomicrography 200 х: Morphological examination of the tibiotarsus muscles of rats, 29 days of the experiment. A – group of intact animals; B – group with simulated limb ischemia; C – group with simulated limb ischemia + L-NAME; D – group with simulated limb ischemia + L-NAME + ZB49-0010C 1 mg/kg; E – group with simulated limb ischemia + L-NAME + ZB49-0010C 5 mg/kg; F – group with simulated limb ischemia + L-NAME + ZB49-0010C 10 mg/kg. Hematoxylin and eosin stain. Thus, in the study of the 3 most promising cardiovascular diseases, a key role in the potential molecules, according to results of testing in pathogenesis of which, as already mentioned, is vitro, a substance under laboratory code ZB49- played the deficit of endogenous NO. As you 0010С from the group of norleucine derivatives know, L-arginine as the only substrate for NO shows most pronounced inhibitory activity (at a synthesis is actively metabolized by arginase II. concentration of 30 µmol) against arginase II as its Increased activity of arginase II leads to a decrease full inhibitor. This agent in vitro doesn’t bind in NO and, consequently, to the development of hERG- channel and not inhibit it at concentrations ED. According to several contemporary authors, up to 40 µmol, thus not affecting the membrane the increased activity of arginase is observed in potential and the cardiac repolarization that bronchial asthma, arthritis, glomerulonephritis, indicates its potential safety. psoriasis, with the development of diabetic erectile During a comprehensive in vivo studies, dysfunction. Also there was confirmed the link to simulation L-NAME induced and homocysteine- the high activity of arginase with the development induced ED, as well as chronic limb ischemia on of ED in rats. Inhibition of this enzyme increases the back of deficit of endogenous NO, there was the NO synthesis and prevents the dysfunctional found that arginase II selective inhibitor substance disorders in the endothelium (figure 6) [4, 13, 14, under laboratory code ZB49-0010С from the group 15, 16, 17, 18, 19, 20, 2121]. Therefore, the most of norleucine derivatives has a dose-dependent promising and expedient pathogenetically in the antihypertensive action, endothelial protective, prevention and complex therapy of cardiovascular cardioprotective, antiischaemic effects, most diseases is the use of highly selective arginase II pronounced at a dose of 10 mg/kg. inhibitors. As you know, today the world EVALUATION pharmaceutical market has no one drug from the According to current data, ED is the main risk group of arginase II selective inhibitors. There is a prediction of the overwhelming majority of variety of different reasons, the main of which, in

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 41 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. our opinion, is the failure of the search of accumulation in the cell membranes and molecules possessing on the one hand, the high intercellular space of LDL and VLDLa and their selectivity in respect of arginase II and oxidation and reduction the synthesis of sulfur pharmacological activity and, on the other hand, a containing glucosaminoglycans that leads to vessel wide therapeutic range and toxicological safety. wall laxity. Simulation of homocysteine-induced That is why the search for active highly selective deficit of endogenous NO leads to increase of CED and safe potential molecules arginase II selective to 3.4±0.2 (0.9±0.1 in the group of intact animals), inhibitors requires not only a huge set of and at the same time to increase the concentration mathematical, physicochemical methodology, of homocysteine in the blood plasma up to aimed at the synthesis and selection of the most 51.0±2.0 µmol/l (8.3±4.3 µmol/l in the control innovative and promising agents, but its long term group animals). Also simulation of homocysteine- test both in vitro and in vivo in various models of induced ED leads to a sharp decrease in the disease. Therefore, we have consistently used concentration of stable metabolites of NO (Total complex methodological approaches as NO) in plasma of rats to the level of 68.9±4.3 methodologies of selection of the most selective (19.0±4.3 in the group of intact animals). With the and safe substances in tests in vitro and with the administration of methionine there was observed a use of validated models of pathology in vivo, sharp decline in the functional parameters of the which are directed to the study of the myocardium after stress tests that shows the pharmacological activity of the most effective and development of latent cardiac insufficiency. safe molecule. Simulation of ED has also been performed In vitro tests established that the substance intraperitoneal injection of the eNOS inhibitor ZB49-0010С from the group of norleucine L-NAME to male rats at a dose of 25 mg/kg for 7 derivatives showed most pronounced inhibitory days. L-NAME in the body is converted to the activity at a concentration of 30 mmol against active inhibitor L-NNA, which inhibits NO arginase II, as its full inhibitor. This agent doesn’t synthesis by inhibiting the eNOS. Deficit of NO bind hERG channel of cardiomyocytes and not causes a reduction of endothelium-dependent inhibit it at concentrations up to 40 µmol that vasodilators effects and increases the indicates its potential safety in respect of cardiac vasoconstrictor influences, disturbances of electrical stability. systemic and regional hemodynamic, increases In our study, endothelial protective and blood pressure, leads to heart dysfunction, cardioprotective effects of arginase II selective increases the expression of endothelial cell inhibitor ZB49-0010C were studied on L-NAME adhesion molecules and others. induced and homocysteine-induced ED. To As a result of experiments, a seven-day simulate homocysteine-induced ED experimental blockade of NO-synthase with L-NAME led to the animals were administered intragastrical the development of arterial hypertension (systolic methionine at dose of 3 g/kg once a day for 7 days. blood pressure 191.2±6.4; diastolic blood pressure A metabolite of methionine homocysteine, due to 145.7±3.8 mm Hg, compared to the group of intact its high peroxide activity has a damaging effect on animals, systolic blood pressure138.2±3.6, the endothelium. Free radicals react with diastolic blood pressure 103.9±2.7 mm Hg). In synthesized in the endothelium NO and reduce its addition, develop of NO deficit manifested by the biological activity. Also homocysteine can increase in CED fivefold in comparison with its intervene in synthesis of disulfides that, in addition values in the group of intact animals, the negative to functional disorders of endothelial cells, dynamics in the stress tests, the decrease in the stimulates proliferation of vascular smooth muscle concentration of stable metabolites of NO in the cells, causing its remodeling. Endothelium- blood, myocardial hypertrophy and hypertrophy of independent vasodilatation in homocysteine- VSMC. However, CED in animals with induced ED is not as pronounced as in L-NAME- homocysteine-induced deficit of endogenous NO induced deficit of NO. Perhaps this is due to was lower than in animals with L-NAME – reduced availability of exogenous NO and the induced ED that makes you think about more smaller values of the initial arterial pressure. severe violation of the ratio of endothelium- Apparently, this can explain the higher values of dependent and endothelium-independent CED in L-NAME-induced ED. In the body, vasodilatation associated with administration of homocysteine promotes the synthesis and RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 42 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

L-NAME at a dose of 25 mg/kg than with the our opinion, may be associated with increase administration of methionine in dose 3 g/kg. endogenous synthesis of L-arginine by inhibiting The main objectives of our research were to activity of argininosuccinate synthetase and study the specific activity of the substance most accumulation of L-arginine due to inhibition of selective and safe molecule arginase II inhibitor arginase. In turn, L-arginine has several properties ZB49-0010C from the group of norleucine that are beneficial for the activity of the derivatives on the ED and limb ischemia in cardiovascular system, it facilitates membrane experiment. Mechanism of action of arginase II depolarization of endothelial cells and regulates selective inhibitor is due to its structural similarity the pH in these cells, as well as the pH of the with ornithine, which is one of the products of blood, reduces blood viscosity, reduces the metabolism of the urea cycle. Indirect effects of production of free radicals and removes them from the arginase II selective inhibitor to arginase the endothelial cells [3, 4, 5, 6, 7, 9, 14, 30]. activity are associated with inhibition of ornithine A morphological study demonstrates carbamyl transferase, which catalyzes the prevention under the influence of arginase II metabolism of ornithine citrulline in the urea cycle. selective inhibitor ZB49-0010C increase in The result is an excessive accumulation of transverse diameter of the cardiomyocytes in ornithine, which inhibits arginase. Moreover, animals with L-NAME-induced and homocysteine- arginase selective inhibitor increases the induced ED. Also under the action of arginase II endogenous synthesis of L-arginine from citrulline selective inhibitor ZB49-0010C has been observed by inhibiting argininosuccinate synthetase. The a prevention of development of negative suppression of the activity of argininosuccinate pathological processes in the kidneys which was synthetase by the arginase II selective inhibitor most pronounced at a dose of 10 mg/kg. leads to increase endogenous synthesis of In the study of biochemical markers of ED in L-arginine and restoration of NO synthesis [4, 10, the blood plasma there was observed a prevention 14, 15, 16, 17, 18, 19, 24, 25]. In our study of the reduce of the concentration of stable arginase II selective inhibitor ZB49-0010C were metabolites of NO in L-NAME – induced studied to identify its hypotensive action, pathology. In simulation of homocysteine-induced endothelial protective and cardioprotective effects pathology there was also observed a prevention of and anti-ischemic effect. Hypotensive effect of the reduce of the concentration of stable arginase II selective inhibitor ZB49-0010C was metabolites of NO and an increase the level of dose-dependent and most pronounced at a dose of homocysteine in the blood plasma which was most 10 mg/kg and was shown to prevent the increase in pronounced at a dose of 10 mg/kg. arterial pressure due to the blockade of eNOS. In the study of the anti-ischemic effect of Endothelial protective effect of arginase II arginase II selective inhibitor ZB49-0010C there selective inhibitor ZB49-0010C was also dose- was established prevention of falling of the dependent and most pronounced at a dose of microcirculation on the 29th day in the chronic 10 mg/kg, which was manifested by decrease of limb ischemia which was the most significant at a CED to 2.3±0.3 in L-NAME – induced ED and to dose of 10 mg/kg. A morphological study also 1.6±0.2 in homocysteine-induced ED. Endothelial revealed the most pronounced at a dose of protective effect of ZB49-0010C is due to the 10 mg/kg prevention the development of necrotic suppression of the activity of arginase II, thereby and atrophic processes in skeletal muscle of the there is an increase the synthesis of the main ischemic limb and the protective effect on vasodilator of endothelium NO. histoarchitecture of striated muscle. The results of the research have been These effects are primarily associated with the identified cardioprotective effect of the arginase II increase of bioaccessibility of L-arginine as the selective inhibitor ZB49-0010C in both models of main substrate for NO synthesis, due to the block ED, which manifested positive dynamic in the of its biotransformation by arginase II. It leads to stress tests (test for adrenoreactivity, overload an increase of NO synthesis, which is an important resistance test). It was possible to identify the factor that activates neoangiogenesis by increasing prevention of increase of adrenoreactivity and production of endothelial growth factor that reduce cardiac reserve. Cardioprotective effect of stimulates the proliferation, migration of endothelial the arginase II selective inhibitor ZB49-0010C, in cells and activates local blood flow [5, 28, 29, 30].

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 43 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

Figure 6. The pathogenesis of endothelial dysfunction and the application points of the action potential endothelial protective agents (L. Santhanam, D. W. Christianson, et al., 2008)

The obtained data fully coincide with the results of LDF – laser doppler flowmetry other authors [13, 15, 17, 18, 21, 25, 31, 32, 33, 34, 35, LDL – low-density lipoprotein 36] and indicate the prospects for drugs suppressing the NO – nitric oxide activity of arginase II. The selectivity of the studied nor-NOHA – Nω-hydroxy-nor-L-arginine drug makes it more preferable because there is no NOHA – Nω-hydroxy-l-arginine impact on the urea exchange [4, 14, 16, 26]. ODC – ornithine decarboxylase PU – perfusion units ABBREVIATION LIST VLDLa – very low-density lipoproteins +dp/dt max – maximum contraction rate VSMC – vascular smooth muscle cells -dp/dt max – maximum relaxation rate ABH – 2(S)-amino-6-boronohexanoic acid REFERENCES ADMA – asymmetric dimethylarginine 1. Babko A.V., Pokrovsky M.V., Terekhova E.G., et BEC – S-(2-boronoethyl)-L-cysteine al. Effect of combined use of an arginase inhibitor L- CAD – atherosclerotic cardiovascular disease Norvaline and a fixed combination of losartan and hydrochlorothiazide in one tablet on the endothelial CED – coefficient of endothelial dysfunction dysfunction in L-NAME-induced deficit of nitric oxide. DEP – dyscirculatory encephalopathy Bulletin of Belgorod state University. Series: Medicine. DFMO – £- difluoromethylornithine Pharmacy. Vol. 16. №. 22-2 (2011): 22-27. [Full text] DTNB – 5, 5'-dithio-bis-(2-nitrobenzoic acid) 2. Konopleva L.F. Endothelial dysfunction in the ED – endothelial dysfunction pathogenesis of cardiovascular disease and methods of its eNOS – endothelial NO synthase correction. Therapy. Ukrainian medical Bulletin. № 3 (56) L-NAME – L-Nitro-Arginine Methyl Ester (2011): 26-30. [Abstract] L-NNA – Nω-nitro-l-arginine RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 44 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45.

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Kuban protective effects of enhanced antibodies to endothelial scientific medical Bulletin. №3 (2015): 139-142. [Full text] growth factor of blood vessels. Dissertation in support of 16. Yakushev V.I., Pokrovsky M.V., candidature for a biological degree. Belgorod state national Beskhmelnitsyna E.A., et al. Arginase II is a new target to research University. Belgorod, 2011. [Abstract] create endothelial protective drugs. Bulletin of scientific 29. Artyushkova E.B. Pokrovsky M.V., Pokrovskaya T.G., et al. Comparative evaluation of the angiogenic RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Yakushev V.I., Pokrovskii M.V. Cardiovascular effects of an arginase II selective inhibitor. 45 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 28-45. effect of L-arginine and vascular endothelial growth factor hyperhomocysteineinduced endothelial dysfunction. (VEGF) in experimental limb ischemia. Pavlov Russian Research result: pharmacology and clinical pharmacology. medical-biological Bulletin. №1 (2009): 46-52. [Full text] 2016. Vol. 2, №1 (2): 42-45. [eLIBRARY] [Full text] 30. Sonin D.L., Syrensky A.V., Galagudza M.M., et 34. Skachilova S.Y., Kesarev O.G., Danilenko L.M., al. The role of nitric oxide in the regulation of Bystrova N.A., Dolzhikov A.A., Nikolaev S.B. distensibility of arterial vessels in normo – and Pharmacological correction of L-NAME-induced oxide hypertensive rats. Arterial hypertension. №6 (2002): deficiency with derivatives of 3-(2,2,2- 57-64. [Full text] trimethylhydrazinium) propionate. Research result: 31. Khadieva T.A., Dovgan A.P., Pokroskaya T.G. pharmacology and clinical pharmacology. 2016. Vol. 2, Method of correction of endothelial dysfunction with №1 (2): 36-41. [eLIBRARY] [Full text] combination of ademetionine and taurine. Research result: 35. Molchanova O.V., Pokrovskaya T.G., Povetkin pharmacology and clinical pharmacology. 2016. 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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Chuhareva N.A., Bontsevich R.A., ,Shchurovskaya K.V., Denisova D.S. The choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis. 46 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 46-50.

Рус. Eng.

CLINICAL PHARMACOLOGY

UDC: [656.071.61:616.08](470.325) DOI: 10.18413/2500-235X -2016-2-3-46-50 Chuhareva N.A. 1 Bontsevich R.A. 2 THE CHOICE OF ANTIMICROBIAL THERAPY AMONG PHYSICIANS Shchurovskaya K.V.3 IN THE TREATMENT OF GESTATIONAL PYELONEPHRITIS Denisova D.S.4

1) FGBU Scientific Center for Obstetrics, Gynecology and Perinatology. Academician VI Kulakov Russian Ministry of Health, 4, Oparin street, Moscow, Russian Federation, 117997, e-mail: [email protected] 2) NRU BSU, dep. pharmacology; Multidisciplinary clinic "Harmony Health" – the center of the safe pharmacotherapy pregnant and nursing; Belgorod, 85 Pobedy St., Belgorod, 308015, Russian Federation, e-mail: [email protected] 3) NRU BSU, dep. pharmacology, Belgorod, 85 Pobedy St., Belgorod, 308015, Russian Federation, e-mail: [email protected] 4) NRU BSU, Belgorod, 85 Pobedy St., Belgorod, 308015, Russian Federation, e-mail: [email protected]

Annotation. From 1 to 10% of the physiological pregnancy complicated by acute pyelonephritis. The purpose work- analyze the choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis. Materials and methods-analysis of the anonymous survey in the framework of the second stage of the All-Russian pharmacoepidemiological study, "Epidemiology of the use of drugs in pregnant women", which was conducted from February to April 2015. On the basis of this was carried out to compare the results with the results of the survey of doctors of the Belgorod region. Results and discussion. Approximately half of the doctors choose the treatment of pyelonephritis in pregnant cephalosporins of generations II and III, as well as amoxicillin / clavulanate. Up to 30% of physicians prescribe ineffective in this case macrolides. A third of doctors sent patients for treatment to other specialists. Conclusion. An analysis of the responses of doctors tactics purpose of antimicrobial therapy in gestational pyelonephritis showed that only half of practitioners prescribed therapy based on the rational use of antimicrobials. Keywords: pregnancy, pyelonephritis, protected penicillins, cephalosporins II and III generation, rational pharmacotherapy.

Introduction: the ureter enlarged uterus (particularly common in Gestational pyelonephritis – an inflammatory nulliparous due to the elasticity of muscles anterior process that occurs in the interstitial kidney tissue abdominal wall) [6], the strong growth of pathogenic and renal pelvis system, which can occur during microflora under the influence of estrogens [2, 4, 7]. pregnancy, childbirth or the postpartum period. The Under the influence of these causes may occur prevalence of the disease is sufficiently large – noted stagnation of urine with infection and the that during gestation they suffer from 1 to 10% of development of gestational pyelonephritis. women [1, 2]. Acute pyelonephritis is the most The causative agents of pyelonephritis well common inflammatory process (10-12%) [3, 4, 5] understood – most often found in the urine of occurring in the kidney during pregnancy. pregnant microorganisms of the family It is believed that a woman's body becomes Enterobacteriaceae (88%) [3, 4], the most typical more susceptible to the disease during pregnancy for representatives of which is Escherichia coli (E. coli) many reasons – a violation of passage of urine by the (65%) [3, 8], seldom – Klebsiella spp. (10%), Proteus ureters iz-za hormonal changes (relaxation of smooth spp, even rarer – Enterococcus faecalis (4,6%) [4, 7]. muscles of the ureter, renal pelvis expansion of the Gestational pyelonephritis and worsens the system under the influence of progesterone, the prognosis of pregnancy and can cause premature occurrence of vesicoureteral reflux) , compression of delivery, hypertensive disorders, pre-eclampsia and RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Chuhareva N.A., Bontsevich R.A., ,Shchurovskaya K.V., Denisova D.S. The choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis. 47 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 46-50. eclampsia, placental insufficiency, hypoxia and in pregnant women only after the culture results of infection of the fetus in utero, and even abortion [2]. urine on chuvsvitelnost to antibiotics. Timely detection and appropriate treatment Contraindicated for use at any stage of initiated pyelonephritis can significantly improve the pregnancy, fluoroquinolones, trimethoprim- quality of life of the pregnant, prevent most sulfamethoxazole (contraindicated in I and III complications both during pregnancy and in the trimester) and antibiotics group sulfonamides, postpartum period. tetracyclines, chloramphenicol, aminoglycosides According to clinical guidelines, the main individual (only for health reasons) [14, 15, 16, 17]. treatment of pyelonephritis in pregnant women is In view of the prevalence and social significance antibiotic therapy, which would meet the basic of the disease, a large number of complications, the requirements of safety and efficiency for both the empirical selection of antibacterial therapy at the mother and the fetus [9, 10]. Due to the large number beginning of treatment due to the timing of gestation of complications and the risk of pyelonephritis and the severity of the patient's condition [18], it is treatment should begin immediately after diagnosis, expedient to hold a pharmacoepidemiological study without waiting for the results of urine culture, that in order to identify the preferences of physicians in is, conduct empirical antibacterial drugs treatment of the selection of Antimicrobial Chemotherapy (ACT) a wide spectrum of action, based on the most pyelonephritis pregnant . common causative agents of the disease [6, 8]. According to the Federal Clinical guidelines in Objective: To analyze the choice of Belgorod 2014 the drugs of choice in the treatment of region ACT doctors in the treatment of pyelonephritis in pregnant women are cephalosporins pyelonephritis in pregnancy. Compare the data with III generation – ceftibuten, cefixime, cefotaxime, the results obtained as a result of the survey, which ceftriaxone, which have a broad spectrum of was conducted from February to April 2015, four antibacterial activity and can be used at any stage of federal districts of the Russian Federation – the pregnancy. According to the USA classification of Central, Volga, Urals and Far East. drugs FDA (Food and Drug Administration – FDA) Materials and Methods: In this study based on cephalosporins are classified in safety of use during the method of anonymous questionnaire in the pregnancy [11, 12]. In addition to the cephalosporins, framework of the second stage of the All-Russian as a rational pharmacotherapy is possible to use the pharmacoepidemiological study, "Epidemiology of drug group monobactams – aztreonam (Category B the use of drugs in pregnant women", which was classification FDA) [8]. conducted from February to April 2015. All-Russian With the ineffectiveness of these drugs, chronic Pharmacoepidemiological 1066 questionnaires were and long-term course of the disease, in pregnant analyzed in the study, of which 734 obstetricians and women for indications of alternative treatment may gynecologists, and 332 physician [19]. be applied -gentamicin + ampicillin (appointed after In a survey in the Belgorod region (BR) 94 the II trimester) [9], category C by the FDA, [8, 13]. doctors took part (28.7% and 69.1% inpatient If you have restrictions on the use of outpatient Profile, p <0, 01), of which 77 (81.9%) of aminoglycosides can be assigned to the group Obstetricians and Gynecologists, 17 (18, 1%), preparations of carbapenems, including meropenem, physicians (p <0,0001), with a total work experience of ertapenem, imipenem / cilastatin (Category B less than 5 years – 21.3% of physicians, 5-10 years – classification FDA) [9]. Also as can be seen 26,6%, 10-20 years – 20.2% of doctors more than altarnativnoy therapy drugs penicillins in 20 years and 26.6%. The survey was conducted on combination with β-lactamase inhibitors – the basis of women's clinics, city policlinics and piperacillin / tazobactam, ticarcillin / clavulanate (B municipal race. Home Belgorod, as well as in the according to FDA) [9] and generation cephalosporin central district hospital. IV – cefepime. Information obtained from the survey were The most prominent common and useful drug entered and processed using MicrosoftExel program. for gestational pyelonephritis – amoxicillin / Main part: clavulanate, currently can not be recommended as To determine the preferences of physicians in empirical therapy, because of the large kolichesvo the treatment of acute pyelonephritis in the isolated strains of E. coli, resistant to the drug (up questionnaire presented a list of the most commonly 43.0%) [3]. Amoxicillin / clavulanic acid, as well as used drugs: cephalosporin II generation (cefuroxime) appointed • Penicillin – benzyl penicillin, ampicillin, amoxicillin, amoxicillin / clavulanate;

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Chuhareva N.A., Bontsevich R.A., ,Shchurovskaya K.V., Denisova D.S. The choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis. 48 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 46-50.

• Cephalosporins – cefazolin, cefuroxime, (51.9% and 70.6% of obstetricians Physicians, p = cefotaxime, ceftriaxone, cefixime ("Supraks"), 0,161), the All-Russian doctors in the study 32.9% of cefepime; the questionnaires (p <0,0001). Cefazolin • Macrolides – erythromycin, spiramycin, (cephalosporin II generation) 27.7% of physicians josamycin, clarithromycin, azithromycin; chose the Belgorod region (32.5% of obstetrician- • Fluoroquinolones – norfloxacin, ofloxacin, gynecologist and 5.9% of the therapist, p = 0.02) and pefloxacin, ciprofloxacin, "Avelox", "tavanic"; 15.6% of physicians All-Russia study, p = 0.002. • Aminoglycosides – steptomitsin, gentamicin, Macrolides having low activity against gram- amikacin; negative microflora was given to 31.9% of the • Nitroxoline; responses in the BR (37.7% of obstetricians and • Fosfomycin (Monural); gynecologists and internists 5.9%, p = 0.01) and • Nitrofurans (furadonin, Furamat, Furagin); 18.4% of responses at All-Russia study, p = 0.0015. • lincomycin, clindamycin, vancomycin, Fosfomycin, a drug that is used only for the meropenem; treatment of cystitis and bacteriuria, indicated in their As it was provided for your answer and the questionnaires, 30.9% of physicians BR (32.5% and option "not to appoint, send to another specialist." 23.5% of obstetricians Physicians, p = 0.470) and Based on clinical guidelines for the treatment of 19.6% of doctors of All-Russian Research, p = 0.009. pyelonephritis in pregnant women as a starting Alternative drugs in the treatment of gestational cephalosporin III generation empirical therapy pyelonephritis – aminoglycosides, vancomycin – not (cefotaxime, ceftriaxone and cefixime), 56.4% of chosen any one of the doctors surveyed BR and in physicians chose BR (63.6% of obstetricians and rare cases, doctors noticed All-Russia study (0.4% gynecologists and 23,5% of the therapist, p = 0,002) and 0.5%, respectively), p> 0.5. The cephalosporin and 46.1% of physicians All-Russia study (p = 0,054). IV generation, notes BR 4,2% of doctors (3.9% and Amoxicillin / clavulanate (without preliminary 5.9%, obstetricians physicians) and 5.2% of urine culture sensitivity to the drug) 50.0% of physicians All-Russia study, p = 0,702. physicians chose the Belgorod region (57.1% of Absolutely contraindicated in the treatment of obstetricians and gynecologists and 17.7% of gestational pyelonephritis fluoroquinolones are listed physicians, p = 0,003) and 49.1% of the doctors at in the individual questionnaires BR physicians the All-Russia study (p = 0,861). (2.1%) and the All-Russia survey (2.2%). Cephalosporin II generation (cefuroxime) (also Send their patients for treatment to another without preliminary urine culture sensitivity to the drug) specialist doctors decide 26.6% BR (28.7% of chose only obstetricians Belgorod region (7.8%) and obstetricians and gynecologists and 17.6% of physicians All-Russia study (8,3%), p = 0.886. physicians, p = 0.356) and 32.3% of All-Russian A large number of responses were given research physicians (p = 0.257). Compare tactics ineffective as a therapy of pyelonephritis is not ACT doctors BR and All-Russia study are shown in protected against beta-lactamase penicillins – CP Table 1 and Figure 1. doctors pointed them in 55.3% of the questionnaires Table 1 Comparison of tactics AMT doctors Belgorod Region and All-Russia Research Doctors of All-Russia Doctors of Belgorod Region The drug Research р N=94 Abs. % N=1066 Abs. % Amoxicillin / clavulanate 47 50,0 523 49,1 0,861 Cephalosporin I generation 26 27,7 166 15,6 0,002 Cephalosporin II generation 6 6,4 88 8,3 0,523 Cephalosporin III generation 53 56,3 491 46,1 0,054 Cephalosporin IV generation 4 4,2 55 5,2 0,702 Not protected penicillins 52 55,3 350 32,9 <0,0001 Macrolides 30 31,9 196 18,4 0,0015 Fosfomycin 29 30,9 209 19,6 0,009 Fluoroquinolones 2 2,1 23 2,2 0,938 Aminoglycosides 0 0 4 0,4 >0,5 Vancomycin 0 0 5 0,5 >0,5 Do not prescribe pharmacotherapy 25 26,6 344 32,3 0,257 (Note: p -Hi-square Pearson)

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Chuhareva N.A., Bontsevich R.A., ,Shchurovskaya K.V., Denisova D.S. The choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis. 49 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 46-50.

Amoxicillin/clavulanate 50 49,1

Cephalosporin I generation 27,7 15,6

Cephalosporin II generation 6,4 8,3

Cephalosporin III generation 56,3 46,1

Cephalosporin IV generation 4,2 5,2

Not protected penicillins 55,3 32,9

Macrolides 31,9 18,4

Fosfomycin 30,9 19,6

Fkuoroquinolones 2,1 2,2

Aminoglycosides 0 0,5

Vancomycin 0 0,4

Do not prescribe 26,6 pharmacotherapy 32,3

0 60 Doctors of Belgorod Region (%)

Doctors of All-Russia Research (%)

Figure 1. The purpose frequency pyelonefrite drugs in pregnant women among obstetrician-gynecologists and internists Belgorod region of the Russian Federation Conclusion: All-Russian Research continue to prescribe their According to the data of the survey, about half patients previously ineffective drugs in the treatment of the doctors of the Belgorod region and the All- of pyelonephritis – macrolides (p = 0.0015) and Russian Research prescribe rational pharmacotherapy fosfomycin (p = 0.009). in the treatment of gestational pyelonephritis (in Reserve antimicrobials – amnoglikozidy, particular, Cephalosporin III generation), p = 0.054. vancomycin, meropenem – chose only doctors All- From about the same, but without specifying Russian Research; cephalosporin IV generation was what perparaty data can be allocated only after urine given roughly equal number of votes (p = 0.702), culture sensitivity operedeleniya rate doctors opt to however, none of the doctors did not indicate that amoxicillin / clavulanate (p = 0.861) and they prescribe them for health reasons, in the case cephalosporin II generation (p = 0.523). where the appointment of benefit to the mother Regardless of the national recommendations, the outweighs the potential risk for the development of canons of rational antibiotic therapy, doctors BR and the fetus. RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Chuhareva N.A., Bontsevich R.A., ,Shchurovskaya K.V., Denisova D.S. The choice of antimicrobial therapy among physicians in the treatment of gestational pyelonephritis. 50 Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 46-50.

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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 51 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 51-57.

Рус. Eng. UDC: 616.153.915:616.127-005.4-08 DOI: 10.18413/2500-235X -2016-2-3-51-57

Kornilov A.A.1 COMPREHENSIVE EVALUATION OF COMBINED Luneva J.V.2 PHARMACOTHERAPY OF CARDIAC PATHOLOGY CONSIDERING Povetkin S.V.3 EXOGENOUS AND PHARMACOGENETIC FACTORS 1) Assistant of the Department of Clinical Pharmacology , PhD . Kursk State Medical University. Str. Karl Marx, 3, Kursk , 305014 , Russia, e-mail: [email protected] 2) Associate Professor, Department of Clinical Pharmacology , PhD, Kursk State Medical University . Str . Karl Marx , 3 , Kursk , 305014 , Russia, e-mail: [email protected] 3) head of the Department of clinical pharmacology, doctor of medical Sciences, Professor. Kursk State Medical University. K. Marx street, 3, Kursk, 305014, Russia, e-mail: [email protected] Abstract. The study assessed changes in hemodynamic and biochemical parameters, vascular age and 5-year cardiovascular risk on the background of complex pharmacotherapy in patients with stable angina I-III functional class (FC), hypertension of I-III degree, chronic heart failure (CHF) II-III FC taking into account the influence of exogenous and pharmacogenetic factors. The study included 200 patients aged 45 – 65 years with concomitant cardiovascular diseases: hypertension of I-III degree, stable angina (I-III FC), with CHF (II-III FC). Patients of the 1st group (100 patients) received prestans, carvedilol, hydrochlorothiazide, verospiron, preparations of acetylsalicylic acid, atorvastatin. Patients of the 2nd group (100 patients) received monopril (fosinopril sodium), carvedilol, amlodipine, hydrochlorothiazide, verospiron, preparations of acetylsalicylic acid, atorvastatin. In hypertensiology part of the study to the end of the observation period the positive dynamics of the main hemodynamic parameters found in both groups. The main indicators of lipid spectrum were improved. Assessment of the impact of pharmacogenetic factors on the effectiveness of the therapy showed no influence of ACE gene polymorphisms (rs4344) and CYP2D6 gene (rs1135840) for the basic hemodynamic parameters, whereas carriers of the genotype (G;G) of the gene CETP (rs4783961) in both groups was associated with a less significant increase of cholesterol of high density lipoproteins (HDL cholesterol) compared with carriers of the allele (A;A) and (A;G). The significant improvement noted in the analysis of the data of vascular age as an integral indicator of the state of the cardiovascular system, as well as a 5-year cardiovascular risk on the background of the combined pharmacotherapy. . However, there were not detected statistically significant differences between 1-St and 2-nd groups Keywords: coronary heart disease, hypertension, heart failure, pharmacogenetic, lipid-transport system, the vascular age. Introduction. Cardiovascular diseases are permanent treatment and conscious self-control in the currently the subject of intense study because, despite outpatient setting. This term allows the patient to significant advances in prevention and therapy, they present a risk of future disease and complications, remain one of the main causes of death and disability in expressed in years, not percentages that are more most socially developed countries in the world [1, 2]. clear and accessible for most patients who do not The main goal of treatment of patients with have the medical education [5]. A modified scale arterial hypertension (AH), coronary heart disease SCORE (score 10-year risk of cardiovascular death) (CHD) is to reduce the risk of cardiovascular was the basis for the calculation. The modified scale complications and death from them. To achieve this SCORE for determining “vascular age” (VA) allows goal requires not only reduction in blood pressure to focus on the individual exogenous risk factors of (BP) to the target level, but the correction of all cardiovascular diseases and their complications modifiable risk factors, and treatment of associated (smoking, blood pressure, blood glucose, total diseases – diabetes mellitus (DM), CHF, etc. In turn, cholesterol and high-density lipoproteins) [6, 7]. the persistent commitment of these patients for Strong and significant correlation of the percentages continued medical treatment and lifestyle changes is of the risk of cardiovascular complications with a a major factor in the success of treatment of chronic calculated value of "vascular age" has already been cardiovascular diseases [3, 4]. demonstrated in some studies [8]. The term "vascular age” was developed a few "Vascular age" could potentially be reduced, years ago to promote the patient adherence to a since among its components are modified, exogenous RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 52 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 51-57. indicators (blood pressure, smoking, levels of total risk scale ASCORE in the composition of a cholesterol, lipoproteins of blood serum). comprehensive assessment of the various schemes of Consequently, the motivation to lower your “vascular combined pharmacotherapy in patients with age” for a few years with smoking cessation, concomitant cardiac pathology, given the exogenous modification of diet and constant medication is likely and pharmacogenetic factors. to be the most effective for the Russian patient [9]. Research tasks: The question of the influence of genetic factors 1) evaluation of the effectiveness of a 6-month controlling the pharmacokinetics and combined therapy using a variety of angiotensin pharmacodynamics of drugs, on the efficacy of converting enzyme inhibitors (ACE-I) (perindopril pharmacotherapy is also being actively discussed in the arginine and fosinopril) in achieving the target BP world in recent years. Personalized medicine originated values in patients with uncontrolled hypertension and in the depths of clinical pharmacology has become one concomitant cardiac pathology; of the most promising approaches in optimizing 2) evaluation of the effect of 6-month combined pharmacotherapy of socially significant diseases. The therapy on lipid level: total cholesterol, high density successes of molecular biology, in particular, the lipoproteins (HDL) and low density lipoproteins (LDL), sequencing of the human genome, has allowed and will triglycerides (TG), creatinine and glucose serum; allow in the future to identify the genes governing the 3) assessment of the impact of pharmacogenetic functioning of body systems and determining the factors, carriage of allelic variants of genes involved in efficacy and safety of pharmacological interventions the pharmacokinetics and pharmacodynamics of [10]. Thus, the pharmacogenetic approach is the most antihypertensive and hypolipidemic drugs on key modern among the methods of personalized medicine indicators of cardiovascular and lipid-transport systems; and improves the efficiency and increase safety of 4) the calculation of the indicator "vascular age" treatment of cardiovascular disease, determining the definition of a 5-year risk of complications on a scale prognosis of treatment. Unfortunately, only a few ASCORE and assessment of impact of combined pharmacogenetic tests approved for use in the Russian therapy on these parameters in patients after 6 Federation and in the world today, that are rarely used months of treatment. in clinical practice [11, 12]. The latter makes it relevant Materials and methods. as a search for new alleles responsible for the The study included 200 patients aged 45 – 65 years pharmacokinetics and pharmacodynamics of drugs, and with concomitant cardiovascular diseases: hypertension testing of pharmacogenetic studies have already of I-III degree, stable angina (I-III FC), with CHF (II-III identified the “alleles –candidates” in real clinical FC). Patients with myocardial infarction suffering for practice [13, 14]. In particular, the testing of less than 6 months, acute coronary syndrome, unstable pharmacogenetic tests in routine clinic allows you to angina, arrhythmias, CHF terminal (IV FC), valvular answer the questions of whether genotyping for already heart defects, history of stroke, chronic obstructive proposed in world literature the alleles given the pulmonary disease, DM of the 1st type or the potential benefits from such testing, including the decompensated 2nd type DM, pregnancy, patients who economic viability [15, 16, 17]. had serious cardiovascular disease or condition that It should be noted that neither the methodology affects duration of life (need for dialysis, cancer, drugs, of "vascular age" or the methodology of etc.) were excluded from the study. pharmacogenetic personalization of comorbid All patients randomized into two groups. pathology in the conditions of unorganized The main clinical characteristics of the study population not tested and possible benefit from their groups are presented in table 1. use is still under discussion, both at the international Table1 and Russian level. On the one hand, the dynamics of Clinical characteristics of study groups indicators of "vascular age" under the influence of Parameter 1st group 2nd group the most commonly applied schemes of combination The number of pharmacotherapy has not been studied. On the other patients 100 100 hand, data on the use of pharmacogenetic testing in • Men 48 54 conditions typical practices only begin to accumulate • Women 52 46 in the scientific community. All of the above makes it relevant to the topic undertaken for the study. Average age, years 60,5 ± 4,3 56,04±6,7 The purpose of this study was to investigate the dynamics of the indicator of “vascular age” based on the modified SCORE scale and the 5-year vascular

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 53 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 51-57.

Patients of the 1st group (100 patients) received heart rate (HR) and blood pressure (BP) in result of a fixed combination of ACE inhibitors and calcium titration of drug doses in all areas of channel blocker (CCB) – prestans (perindopril pharmacotherapy. arginine in a dose of 5-10 mg/day and amlodipine 5- Table 2 10 mg/day), carvedilol at a dose of 12.5-50 mg/day, Change hemodynamic variables in groups of hydrochlorothiazide – 12,5-25 mg/day, verospiron 25 observations on the background of treatment mg/day, acetylsalicylic acid drugs– 75 – 100 mg/day, (M±SD, n = 200) atorvastatin at a dose of 20-40 mg/day. 1st group (n=100) 2nd group (n=100) Patients of the 2nd group (100 patients) received Parameter At After 6 At After 6 monopril (fosinopril sodium) at a dose of 10-40 baseline months baseline months mg/day, carvedilol in the dose 12,5-50 mg/day, Systolic amlodipine 5-10 mg/day, hydrochlorothiazide – 12,5- BP, mm 152,7±10,9 134,2±7,6* 154,3±9,5 135,2±8,9* 25 mg/day, verospiron 25 mg/day, acetylsalicylic Hg. acid drugs– 75 – 100 mg/day, atorvastatin 20-40 Diastolic mg/day. BP, mm 92,2±7,6 81,1±6,3* 91,8±7,1 81,4±4,8* Hg The criteria for titration of the doses of medicine HR, 74,3±6,5 65,9±4,0* 72,6±4,2 64,8±3,5* were the target levels of hemodynamic, clinical and beats/min laboratory parameters. Note: here and in table 4 the significance of The observation period was 6 months. differences of indicators in the process of treatment: * – p< "Vascular age" was calculated by the modified 0.05, * * – p< 0,01. SCORE table, taking into account gender, age, smoking Further analysis of the changes in hemodynamic status, blood pressure and total cholesterol of blood parameters did not reveal statistically significant serum [5]. 5-year risk of cardiovascular complications differences (p > 0.05) between the groups (table 3). It was calculated on a scale ASCORE [5]. says comparable antihypertensive efficacy of the Pharmacogenetic testing for allelic variants of the proposed regimens, but also emphasizes their genes was carried out in pharmacogenetic laboratory of sufficiency in absolute terms. the сenter for preclinical and clinical studies, national Table 3 research university "BelSU", Belgorod, Russia. DNA Comparative analysis of efficiency of used extraction was performed using the reagents set pharmacotherapeutic schemes for changing the basic ThermoFisherScientific 250 discharge, to assess the hemodynamic parameters (M±SD, n = 200) quality and quantity of obtained DNA was used, the system Qubit with the set of necessary reagents. Hemodynamic 1st group 2nd group parameters (n=100) (n=100) Pharmacogenetic typing for alleles was conducted in ∆Systolic BP (%) 10,9±5,8 12,2±4,3 real time with the use of high-performance systems ∆Diastolic BP (%) 10,9±7,1 10,2±7,5 FluidigmBioMark™ HD System using a standard set of boot reagents, customized set of synthesized primers ∆ HR (%) 8,9±4,5 10,7±4,4 and a universal chip format is 48x48. Processing of the Seemed interesting to assess in practice the obtained results was performed in the software package influence of allelic variants of genes involved in the BiomarkDataAnalisys licensed to work in the pharmacokinetics and pharmacodynamics of drugs laboratory [18]. on the efficacy of pharmacotherapy. The impact of Statistical data processing was carried out by the allelic variant of the gene is a target of ACE methods of parametric and non-parametric statistics inhibitors – ACE allele (A/G) (rs4344) on the using Statistica 8.0. Differences were considered effectiveness of combination therapy containing ACE significant at values of bilateral p<0.05. Presenting the inhibitors – perindopril – in the 1st group, and results of the parametric statistics used the format fosinopril – in the 2nd group respectively were M±SD. estimated . Choice of single nucleotide The results of the study and their discussion. polymorphism rs 4344 caused, on the one hand, a Combined therapy was well tolerated by all small study of this polymorphism among alleles of patients. The drugs cancellation and adverse drug the gene for the ACE, and on the other hand, reactions were not recorded. contradictory data about its influence on the In hypertensiological part of the study the antihypertensive efficacy and safety of drugs that positive dynamics of the main hemodynamic inhibit this enzyme [19, 20]. Comparative analysis of parameters were found in both groups by the end of efficiency of used drug regimens subject to the the observation period (table 2). Patients in the carriage of allelic variants of the studied intervention groups achieved the target levels for polymorphisms is presented in table 4.

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 54 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 51-57.

Table 4 Comparative analysis of effectiveness of pharmacotherapy depending on the carriage of allelic variants of the gene ACE (rs4344) (M±SD, n=200) Group 1st group (n=100) 2nd group (n=100) rs4344 rs4344 (А;A), rs4344 (А;G), rs4344 (G;G), rs4344 (А;A), rs4344 (А;G), Allele (G;G), (n=23) (n=55) (n=22) (n=26) (n=53) (n=21) ∆ Systolic BP, 18,2±6,5 16,8±7,8 18,8±7,5 19,8±+7,6 19,5±7,9 19,4±7,4 mm Hg. ∆ Diastolic BP, 11,3±6,2 9,4±5,2 11,8±6,5 9,8±5,6 9,4±5,4 9,5±4,0 mm Hg ∆ HR, beats/min 7,2±2,5 7,0±2,9 7,1±2,8 9,8±6,6 9,8±6,2 9,4±5,1 Note: Difference between groups on relevant allelic variants in all hemodynamic parameters was not revealed. In the analysis of the data, within group genotypes determining the pharmacokinetics, and as a (A;A), (A;G), (G;G), nor between groups on relevant consequence, the effectiveness of most beta-blockers allelic variants of statistically significant differences is the cytochrome CYP2D6. The literature describes was not obtained (in all cases, the values of the two-way the impact of a number of alleles of this cytochrome p > 0.05). This is consistent with some of the literature on the efficacy and safety of pharmacotherapy with data about the absence of a clinically significant effect beta blockers. One of the most promising for the of this polymorphism for the realization of the study is single nucleotide polymorphism rs 1135840 hypotensive effect of ACE inhibitors. gene cytochrome CYP2D6, since in several studies it Further seemed interesting to assess the has been shown as sufficient frequency of mutant influence of genetic factors on the implementation of alleles, and its impact on the effectiveness of beta- another drug included in the scheme of blockers [21, 22]. In our study, we have attempted to pharmacotherapy in both groups of observation – evaluate the impact in terms of the Russian carvedilol. It is well known that a major enzyme population (table 5). Table 5 Comparative analysis of effectiveness of regimens depending on the carriage of allelic variants of CYP2D6 gene (rs1135840), (M±SD, n=200) Group 1st group (n=100) 2nd group (n=100) rs1135840 rs1135840 rs1135840 rs1135840 rs1135840 rs1135840 Allele (C;C), (n=9) (G;G), (n=81) (G;C), (n=10) (G;G), (n=79) (G;C), (n=11) (C;C), (n=10) ∆ Systolic BP, 18,2±6,5 17,8±7,8 16,1±7,4 19,4±7,2 18,2±7,1 17,4±7,4 mm Hg. ∆ Diastolic 11,4±6,2 11,1±5,2 9,5±6,4 9,5±5,4 9,2±5,3 9,5±4,0 BP, mm Hg ∆ HR, 7,2±2,5 7,1±2,6 5,4±2,1 9,9±5,6 9,2±5,1 8,4±4,8 beats/min Note: Differences between groups on relevant allelic variants in all hemodynamic parameters As can be seen from the table, or inside the levels of glucose and blood creatinine was not group by genotype (G;G), (G;C), (C;C) nor between observed, on the contrary, tended to their the groups on the respective allelic variants of improvement. The main indicators of lipid spectrum statistically significant differences was not received were improved (table 6). (in all cases, the values of the two-way p > 0.05). In Table 6 comparison of obtained results with available in the Dynamics of the main biochemical parameters in the world literature it should be noted that in some cases intervention groups (M±SD, n=200) carriers of the negative allele (C;C), this Parameter 1st group (n=100) 2nd group (n=100) polymorphism was associated with increased activity Total of CYP2D6, resulting in decrease in the effect of cholesterol, 5,37±0,71 4,62±0,63* 5,32±0,82 4,58±0,62* metabolizing them drugs, while in others such laws mmol/l has not been established. HDL, 1,57±0,41 1,82±0,45 1,6±0,47 1,77±0,43 In lipidological and biochemical part of the mmol/l study evaluated the effect of the lipid-lowering and LDL, 3,18±0,66 2,33±0,76* 3,03±0,84 2,36±0,67* antihypertensive drug therapy on lipid and mmol/l Creatinine, carbohydrate metabolism, levels of blood creatinine. 85,5±9,8 87,0±7,7 89,3±9,9 88,4±9,3 As a result of 6-months therapy in both groups mmol/l Glucose, pharmacological intervention negative impact on the 5,04±0,5 4,88±0,5 5,13±0,5 5,04±0,4 mmol/l RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 55 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 51-57.

Comparative analysis of the effectiveness of lipid- To assess the impact of genetic factors on the lowering intervention in patients in both groups are efficacy of lipid-lowering correction seemed presented in table 7. Note worthy adequate lipid- interesting to assess the impact of protein gene lowering effect on indicators of atherogenic fractions of polymorphism, transporting cholesterol esters CETP lipoproteins of blood serum and the absence of rs4783961 on the effectiveness of lipid-lowering statistically significant differences between the groups. correction in the groups of intervention. Table 7 This polymorphism choise is based on two Comparative analysis of the effectiveness of lipid- premises: first, a sufficient frequency of "minor" lowering intervention in patients with concomitant allele, and secondly, available in the world literature cardiac pathology (M±SD, n =200) 1st group data, not only about its effect on the lipid spectrum, Parameters 2nd group (n=100) (n=100) but also on the risk of early development of non-fatal lowering of total myocardial infarction [23, 24]. The results of the 13,4±2,2 13,9±2,26 cholesterol, % analysis of the influence of this allele on the basic increase parameters of lipid-transport system in patients in the 15,9±4,6 12,9±4,7 HDL cholesterol, % intervention groups are presented in table 8. reduction 26,7±5,3 24,8±4,6 LDL cholesterol, % Table 8 Dynamics of the main biochemical parameters depending on the carriage of allelic variants of the gene CETP (rs4783961), (M±SD, n = 200) Group 1st group (n=100) 2nd group (n=100) rs4783961 rs4783961 rs4783961 rs4783961 rs4783961 (А;A), rs4783961 (А;G), Allele (А;A), (G;G), (G;G), (А;G), (n=56) (n=23) (n=53) (n=22) (n=22) (n=26) lowering of total 13,4±2,2 13,8±2,5 12,9±2,1 13,9±2,3 13,5±2,4 12,9±1,9 cholesterol, % increase HDL cholesterol, 15,9±4,6 15,1±4,3 10,1±4,0* 12,9±4,7 12,1±4,3 9,3±4,0* % reduction 26,7±5,3 26,2±5,1 25,4±5,2 24,8±4,6 24,1±4,3 25,2±4,1 LDL cholesterol, % (А;A) / (А;G): p>0,05 (н/д) (А;A) / (А;G): p>0,05 (н/д) Differences within groups for LDL (А;G) / (G;G): p>0,05 (н/д) (А;G) / (G;G): p>0,05 (н/д) cholesterol, p (А;A) / (G;G): p>0,05 (н/д) (А;A) / (G;G): p>0,05 (н/д)

(А;A) / (А;G): p>0,05 (н/д) (А;A) / (А;G): p>0,05 (н/д) Differences within groups for HDL (А;G) / (G;G): p>0,05 (н/д) (А;G) / (G;G): p>0,05 (н/д) cholesterol, p (А;A) / (G;G): p<0,05 (А;A) / (G;G): p<0,05 Note: Differences between groups on the relevant alleles in the compared indicators were not identified.

As can be seen from the table, statistically and 12,1±4.3% for the allele (A;A) and (A;G), significant differences in dynamics of indicators of respectively in the second group). These differences HDL cholesterol have been identified as in the 1st are a subject for further study, make genotyping for and in the 2nd group. The carriers of the genotype this polymorphism is clinically significant, and (G;G), there was statistically significantly smaller confirm hypotheses about the influence of this increase of HDL cholesterol (10,1±4.0% in the first polymorphism on the risk of development of group, and 9.3±4.0% in the second group, cardiovascular complications, since HDL cholesterol respectively) compared with carriers of other alleles is "anti-risk” of coronary pathology. (15,9±4,6% and 15.1±4.3% for the allele (A;A) and In the part devoted to the assessment of the (A;G), respectively in the first group, and 12.9±4.7% dynamics of vascular age, we obtained the following RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 56 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 52-58. results. As among men, and among women the differences were not found, so the following is estimated vascular age and 5-year risk of cardiovascular summary data for groups in general. Comparative complications significantly decreased at 6 months analysis of dynamics of parameters of vascular age and treatment, with statistically significant gender 5-year cardiovascular risk are presented in table 9.

Table 9 Dynamics of indices of vascular age and 5-year cardiovascular risk in the intervention groups (M±SD, n =200) p 1st group (n=100) 2nd group (n=100) Parameters 1-2 At baseline After 6 months At baseline After 6 months 61,0 54,0** 60,0 54,0** ns Vascular age [55,8;66,0] [50,0;62,0] [52,8;65,0] [49,5;61,0] 2,7 2,0** 3,0 2,4** ns 5-year risk (abs.%) [2,3;4,0] [1,6;2,4] [2,1;3,6] [1,5;3,0] Notes: 1) abs.% – absolute ( % ) risk reduction, 2) the statistical significance of differences of indicators in dynamics in the same group: * p<0.05, * * p<0,01, 3) p 1-2 – intergroup differences in the indices, ns – the difference was not statistically significant. In the analysis of the parameters of vascular age disease. National Recommendations / Rational as an integral indicator of the state of the pharmacotherapy in cardiology. Vol. 5.(2011): P. 5-72. cardiovascular system, as well as a 5-year (In Russian). [eLIBRARY] cardiovascular risk on the background of the 3. Urazalina S. G., Rogoza A.N., Balahonova T.V. conducted pharmacotherapy there is a strong positive The relationship of "vascular age” with indicators of subclinical atherosclerotic lesion of the arterial wall in trend. However, statistically significant differences women with low and moderate cardiovascular risk on a between 1-St and 2-nd groups were not detected scale of "SCORE". Heart. Vol. 5 (2010): P. 271-276. (In Conclusion. Thus, the results of these studies Russian).[eLIBRARY] 4. Chazova I. E, Oschepkova E. V., Zhernakova Y. point to the pronounced clinical efficacy of combined V. Diagnosis and treatment of arterial hypertension. therapy in both groups, patients with concomitant Clinical guidelines. Cardiology. (2015) : P 3–30. (In cardiac pathology in the form of improved Russian). [Full text] hemodynamic parameters (significant decrease in 5. Karpov Yu. A. , Sorokin E. V.. Risk assessment systolic and diastolic BP), parameters of the lipid of complications of arterial hypertension and vascular age: spectrum, glucose and blood creatinine and new tools to improve the quality of treatment and better significant decrease of the indicator "vascular age" understanding of patient and doctor. News of cardiology. and the 5-year cardiovascular risk. All this Vol. 2 (2015): P 18-24. (In Russian). [eLIBRARY] emphasizes the necessity for an integrated approach 6. Cuende, J.I., Calaveras-Lagartos J. How to in the treatment of comorbid cardiovascular calculate vascular age with the SCORE project scales: a new method of cardiovascular risk evaluation. Eur. Heart pathology, primarily due to correction of modifiable J. Vol. 19 (2010): P. 2351-2358.[PubMed][Full text] risk factors. 7. D’Agostino R.B., Vasan R.S., Pencina M.J. [et On the other hand, the data obtained on the al.]. General cardiovascular risk profile for use in primary contribution of pharmacogenetic factors in the care. The Framingham Heart Study. Circulation. Vol.117 variation of the effect of drugs, emphasize that in (2008): P.743–753.[PubMed][Full text] some cases in real life, this influence is not clinically 8. Groenewegen K., den Ruijter H., Pasterkamp G. significant (as it was shown for ACE-I and beta- [et al].Vascular age to determine cardiovascular disease blocker), others may have a clinical value as it was risk: A systematic review of its concepts, definitions, and shown for lipid-lowering correction). The above clinical applications. Eur. J. Prev. Cardiol. Vol. 23 (2015): confirms the necessity for further research in this P. 264-274.[PubMed] 9. Karpov Yu. A., Sorokin E. V.. The effect of direction. combined antihypertensive therapy on the risk of References cardiovascular complications and vascular age: results of a 1. Mareev V. Y., Ageev F. T., Arutyunov G. P. [et multicenter open study ADVANT'AGE. News of al.]. Diagnosis and treatment of chronic heart failure (IV cardiology. Vol. 3 (2015): P. 18-26 (In Russian). revision). National recommendations. Heart Failure. Vol. [eLIBRARY] 14 (2013): P. 379-472. (In Russian). [Full text] 10. Caudle K.E., Dunnenberger H.V., Freimuth R.R.. 2. Martsevich S. Y., Kutishenko N. P., Tolpygino S. [et al]. Standardizing terms for clinical pharmacogenetic N. [et al.]. The efficacy and safety of drug therapy in test results: consensus terms from the Clinical primary and secondary prevention of cardiovascular RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kornilov A. A., Luneva J. V., Povetkin S.V. Comprehensive evaluation of combined pharmacotherapy of cardiac pathology considering exogenous and pharmacogenetic 57 factors. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 52-58.

Pharmacogenetics Implementation Consortium (CPIC). 18. BiomarkHDBrochure. [Electronic resource] // Genet Med. Vol. 21 (2016): P. 1-9.[PubMed][Full text] Fluidigm: site. – Mode of access: 11. Kukes V. G., Sychev D. A., Ignatiev I. V. Clinical https://www.fluidigm.com/products/biomark-hd-system pharmacogenetics and clinical practice: integration (date of access 24.07.2016.). [Full text] perspectives. Biomedicine. Vol. 5 (2006): P. 2-15 (In 19. Grilo A., Saes-Rosas M.P., Santos-Morano J. [et Russian). [eLIBRARY] al]. Identification of genetic factors associated with 12. Filippenko N. G. Povetkin S. V., Kornilov A. A. susceptibility to angiotensin-converting enzyme inhibitors- [et al.]. The possibilities of personalization of induced cough. Pharmacogenet Genomics. Vol. 1 (2011): pharmacotherapy for patients of a cardiological profile. P. 7-10. [PubMed] Cardio somatics. Vol. 2 (2011): P. 58-62 (In Russian). 20. Bhatnagar V., O'Connor D.T.,. Schork N.J. [et al]. [eLIBRARY] Angiotensin-converting enzyme gene polymorphism predicts 13. Kornilov A. A, Filippenko N. G. Povetkin S. V. the time-course of blood pressure response to angiotensin [et al.]. Prospects for personalization of pharmacotherapy converting enzyme inhibition in the AASK trial. J Hypertens. of cardiovascular diseases. Materials of international Vol.10 (2007): P.1-20. [PubMed][Full text] scientific-practical conference dedicated to the 81st 21. Yuan H., Yu M., Yang Y. [et al]. Association of anniversary of the Kursk state medical University and the CYP2D6 single-nucleotide polymorphism with response to 50th anniversary of pharmaceutical faculty. Kursk Vol. 1 ophthalmic timolol in primary open-angle Glaucoma--a (2016): P. 372-377. (In Russian). [eLIBRARY] pilot study. Ocul. Pharmacol. Ther. Vol. 5 (2010): P. 497- 14. Filippenko N. G. Povetkin S. V., Kornilov A. A. 501. [PubMed] [et al.]. From applied pharmacokinetic research to the 22. Suarez-Kurtz G., Pena S.D., Struchiner C.J. [et implementation of personalized pharmacotherapy in real al]. Pharmacogenomic Diversity among Brazilians: clinical practice. Biomedicine. Vol. 3 (2010): P. 158-160. Influence of Ancestry, Self-Reported Color, and (In Russian). [eLIBRARY] Geographical Origin. Front Pharmacol. Vol. 3 (2012): 15. Haga, S.B. Challenges of development and P. 191-198. [PubMed][Full text] implementation of point of care pharmacogenetictesting. 23. Sharafetdinova L.M., Ismagilova R.K., Maycova Expert Rev MolDiagn. (2016): P.1-12. [PubMed] E.V. [et al]. The influence of the polymorphism in the 16. Hicks J.K., Stowe D., Willner M.A. [et al]. promoter region of the gene CETP activity it protein in Implementation of Clinical Pharmacogenomics within a ischemic heart disease. Cytology. Vol. 6 (2014): Large Health System: From Electronic Health Record P. 462-464 (In Russian). [PubMed] Decision Support to Consultation Services. 24. Schierer A., Been L.F., Ralhan S. [et al]. Genetic Pharmacotherapy (2016): P.1-9.[PubMed] variation in cholesterol ester transfer protein, serum CETP 17. Filippenko N. G. Povetkin S. V., Kornilov A. A. activity, and coronary artery disease risk in Asian Indian [et al.]. Experience in the implementation of diabetic cohort. Pharmacogenet Genomics. Vol. 2 (2012): pharmacogenetic studies at the University hospital. P. 95-104. [PubMed][Full text] Biomedicine. Vol. 3 (2010): P. 79-80. (In Russian). [eLIBRARY]

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kukes V.G., Gorbach T.V., Romashchenko O.V., Rumbesht V.V. АТP as the marker of power exchange condition at the experimental ischemia of the myocardium due to metabolic drugs 58 introduction. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 58-62.

Рус. Eng. UDC: 616-005: 616-08 DOI: 10.18413/2500-235X -2016-2-3-58-62 Kukes V.G.1 АТP AS THE MARKER OF POWER EXCHANGE CONDITION Gorbach T.V.2 AT THE EXPERIMENTAL ISCHEMIA OF THE MYOCARDIUM DUE Romashchenko O.V.3 TO METABOLIC DRUGS INTRODUCTION Rumbesht V.V.4

1) Head of the Department of Clinical Pharmacology and Internal Medicine Propaedeutics of the First Moscow State Medical University named by I.M. Sechenov, academician of Russian Academy of Sciences, Professor; e-mail: [email protected] 2) Associate Professor, Department of Biochemistry of Kharkiv National Medical University; e-mail: [email protected] 3) Associate Professor, Department of Pharmacology, Medical University of the Belgorod State National Research University; e-mail: [email protected] 4) Associate Professor of mathematical and software security of information systems of the Institute of Engineering Technology and Natural Science of the Belgorod State National Research University; e-mail: [email protected] Abstract. For the purpose of definition of an adequate marker of a power exchange condition at the experimental ischemia of the myocardium due introduction of metabolic drugs, the experimental research on 60 rats-males of Wistar line has been spent. At experimental myocardial ischemia it has been found out energy-saving effects of trimetazidine, cytoflavin, meldonium, phosphocreatine, which were reached by different ways and in different degree. The indicator of ATP concentration in blood or erythrocytes closely correlated with the level of ATP in myocardial homogenates and with activity of mitochondrial heart enzymes – succinate dehydrogenase, citrate synthase, pyruvate dehydrogenase. Thus, this indicator can be recommended to study to assess the effectiveness of cardiocytoprotectors as the most appropriate marker of their energy-saving effect. Keywords: ATP, myocardial metabolism, cardiocytoprotection, energy-saving effect, metabolic drugs, trimetazidine, meldonium, cytoflavin, phosphocreatine. Introduction contained in standard conditions of vivarium, has The high indicators of prevalence and death rate been executed. The following groups of animals were from coronary heart disease (CHD), despite the used: 1) intact rats (n=10); 2) rats with an accepted standards of pharmacotherapy, cause necessity experimental ischemia of a myocardium (n=10); of modernisation of the accepted schemes of treatment 3) rats with an experimental ischemia of a [1, 2]. One of the promising directions of improving the myocardium entered trimetazidine (n=10); 4) rats pharmacotherapy of CHD considered with an experimental ischemia of a myocardium cardiocytoprotection [3, 4, 5, 6]. Metabolic-line drugs entered meldonium (n=10); 5) rats with an are widely used by doctors of the countries in post- experimental ischemia of a myocardium entered Soviet space as ones that enhance the patients’ life cytoflavin (n=10); 6) rats with an experimental quality, whereas in Europe they are administered not ischemia of a myocardium entered phosphocreatine more than in 1% of cases due to the lack of evidence of (n=10). Age of animals was chosen from a position their impact on the patients’ life expectancy [7]. One of of conformity of 10-month's rats – to middle age of the obstacles of overcoming the doubts of doctors in the human being. The choice of metabolic drugs spent effectiveness of the destination cardiocytoprotectors is from positions of their influence on various links of absence of adequate methods of myocardium the power exchange of cardiomyocytes. metabolism estimation in view of inaccessibility and Modelling of an ischemia of a myocardium has ethical impossibility of performance of researches on been made on the method described by Gaman D.V. human heart tissue. (2011) [8]: daily within 7 days subcutaneously the rats The purpose of the present research was were entered 0,1 ml of 0,1 % adrenaline solution. A definition of an adequate marker of a myocardium dose of medical drugs, entered with the therapeutic power exchange condition at an experimental purpose, was counted under formula of Rybolovlev J.R. ischemia of a myocardium due the introduction of (1979), it has been made 1) for trimetazidine (a pure metabolic drugs. solid of firm «Sigma Aldrich») – 0,5mg/100g rats in 2 Materials and methods of the research ml of 0,9% NaCl solution intragastric 2 times a day, For achievement of the purpose of work the that is equivalent to a recommended dose of experimental research which object were 60 rats- trimetazidine for a human (35 mg 2 times a day per os); males of Wistar line aged by 10 months, which 2) for meldonium (Мildronate of "Grindex" firms,

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kukes V. G., Gorbach T. V., Romashchenko O. V., Rumbesht V. V. АТP as the marker of power exchange condition at the experimental ischemia of the myocardium due to metabolic drugs 59 introduction. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 58-62.

Latvia) – 0,03ml/100g rats in 1,5 ml of 0,9 % NaCl increased ATP concentration in cardiomyocytes, but solution intravenously 1 time a day, that is equivalent to without reaching the level of intact rats. Introduction the recommended dose for a human (5 ml intravenously of meldonium led to the recovery of the amount of 1 time a day; 3) for cytoflavin – 0,07ml/100g rats in 1,5 ATP in the heart homogenates to the level of the ml of 0,9 % NaCl solution intravenously 1 time a day, intact rats, without excessive accumulation of that is equivalent to the recommended dose for a human nucleotides, which in our opinion, indicates the (10 ml intravenously in 200 ml of 0,9 % NaCl solution absence of doping properties of the drug. 1 time a day); 4) for phosphocreatine – 13mg/100g rats ATP levels in serum and red blood cells due to in 2 ml of 0,9 % NaCl solution intravenously 1 time a introduction of metabolic correctors have similar day, that is equivalent to the recommended dose for a dynamics, as reflected in Table 1, and indirectly human (2g/days intravenously of a medicine confirmed by correlation analysis. Positive "Neotone"). correlations were found between the concentration of The animals were deduced from experiment in ATP in myocardial homogenates and blood serum (r 10 days after introduction of medicines by = 0,66, p <0,0001), in red blood cells (r = 0,66, p decapitation. The heart was perfused with a cooled <0,0001). ATP levels in myocardial homogenate is solution of 0.9% NaCl. Preparation of myocardial also positively correlated with the activity of homogenates and isolation of mitochondria was mitochondrial enzymes – succinate dehydrogenase (r produced by the method described of M.V. Egorova, = 0,67, p <0,0001), citrate synthase (r = 0,65, p S.A. Afanasyev [9]. The erythrocytes were isolated <0,0001) and pyruvate dehydrogenase (r = 0,75 , p from heparinized blood by centrifugation. The <0,0001). There is an inverse relationship between washed erythrocytes were used for determination of ATP concentration in the myocardium and 2,3DPG 2,3-diphosphoglycerat (2,3-DPG) and free level in red blood cells (r = -0,33, p <0,01). Thus, the nucleotides (ATP and ADP) (N.P. Meshkova, S.E. more active oxidation-reduction reactions take place Severin, 1979). The content of ATP and ADP was in the mitochondria, the higher levels of ATP detect measured in the serum of blood (I.S. Mranova, 1975). in myocardial homogenates , blood serum and The activity of succinate dehydrogenase (SDH), erythrocytes with the lower level of tissue hypoxia. citrate synthase (CS) and pyruvate dehydrogenase So that, in terms of ATP concentrations in serum and (PDH) was investigated in the myocardial / or erythrocytes it can be indirectly judged the mitochondria. The level of ATP was determined in content of ATP in the myocardium and the intensity myocardial homogenate (M.I. Prochorova, 1982). of the oxidation-reduction reactions taking place in The data were treated by variation statistics with the mitochondria of the cardiomyocytes. arithmetic mean values and their errors, correlation We studied the effect of metabolic correctors on analysis, assessment of significant differences in the the activity of mitochondrial enzymes in comparative student t-test using the software "Microsoft Excel aspect. Thus, the succinate dehydrogenase activity in 2000" and "SPSS for Windows 11.0". experimental myocardial ischemia was significantly Results and their discussion reduced. Introduction of cytoflavin and meldonium At modelling of the ischemia of a myocardium led to partial activation of SDH, but not to the level of 10 months rats a significant increase in 2,3-DPG of the intact rats, administration of trimetazidine and and a decrease in ATP concentration in erythrocytes phosphocreatine was accompanied by decreased was found, suggesting the development of tissue activity of this Krebs cycle enzyme (Table 1). hypoxia and energy deficiency (Table 1). At the Activity of citrate synthase in experimental mitochondria a significant decrease in the activity of myocardial ischemia was significantly reduced. studied enzymes – SDG, CS and PDG was found, Introduction of trimetazidine, meldonium and which indicates a decrease in the intensity of cytoflavin led to a slight increase in CS activity in oxidative phosphorylation and oxidative mitochondria, while the introduction of decarboxylation of pyruvate. The consequence of this phosphocreatine was accompanied by inactivation fact a significant decrease in the concentration of citrate synthase in the mitochondria of rats. ATP in myocardial homogenates was observed. Pyruvate dehydrogenase activity in At the animal experiments the effectiveness of all mitochondria of cardiomyocytes in experimental 4 metabolic drugs due to chronic myocardial ischemia myocardial ischemia was significantly reduced. have been studied. We found significant differences in Introduction of cytoflavin to the animals was the mechanisms of action of each of them. contributed to increase the activity of PDH to the We considered the main indicator of energy level of the intact rats, administration of saving effect of metabolic drugs the ATP levels in phosphocreatine contributed to a slight increase in myocardial homogenates, which reflects the amount the activity of PDH, while trimetazidine had no of ATP in cardiomyocytes. Introduction of significant effect on the activity of this enzyme. trimetazidine, cytoflavin and phosphocreatine

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kukes V. G., Gorbach T. V., Romashchenko O. V., Rumbesht V. V. АТP as the marker of power exchange condition at the experimental ischemia of the myocardium due to metabolic drugs 60 introduction. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 58-62.

Table 1 Indicators of myocardial metabolism in 10 months rats in normal, at experimental myocardial ischemia and on the background of metabolic correctors (M ± m) Rats with Rats with Rats with Rats with Rats with myocardial myocardial myocardial myocardial Intact rats myocardial Indicators ischemia + ischemia + ischemia + ischemia + of 10 months, n=10 ischemia, cytoflavin, trimetazidine, meldonium, phosphocreatine, n=10 n=10 n=10 n=10 n=10

ATP, mkmol/l 664,54±14,49 ΔΔ## 594,44±5,75** 582,47±5,24** 587,05±6,40** 638,88±14,96Δ 589,28±5,43**

ADP, mkmol/l 315,11±8,78 330,53±16,05 316,05±3,01 323,57±5,40 319,27±7,51 328,86±2,93

ΔΔ Δ ΔΔ Erythrocytes 2,3DPG, mkmol/l 4,82±0,29 ## 7,21±0,32** 6,84±0,15** 6,12±0,19 5,70±0,18* 6,69±0,21**

ATP, mkmol/l 200,08±3,47 ΔΔ## 162,81±4,57**# 177,70±2,15**Δ 171,81±1,61** 192,32±5,36 ΔΔ 192,02±4,76ΔΔ

of ΔΔ ΔΔ blood Serum Serum ADP, mkmol/l 75,92±1,58## 79,31±1,13## 91,21±0,79** 85,00±3,22* 77,33±2,29 90,91±1,56** SDH, 17,82±1,10 ΔΔ## 11,83±0,47**# 13,45±0,47**Δ 10,77±0,68**ΔΔ 13,68±0,65* 10,12±0,29**Δ nmol/min·mg ΔΔ Δ Δ CS, nmol/min·mg 3,94±0,23 # 2,38±0,21**# 3,21±0,20* 3,08±0,27** 3,12±0,10* 2,00±0,08** PDH, mkmolNAD/min· 31,04±0,89 ΔΔ 21,68±0,90**## 31,34±0,73 ΔΔ 22,32±0,57** 27,30±0,45** ΔΔ 24,45±0,34** Δ mg

Mitochondria ATP in myocardial 3,08±0,24 ΔΔ# 1,18±0,08**## 2,09±0,16* ΔΔ 2,00±0,05**ΔΔ 3,16±0,09 ΔΔ 1,96±0,08**ΔΔ homogenate, mkmol/l

Note. ATP – adenosine triphosphate, ADP – adenosine diphosphate, 2,3DPG – 2,3-diphosphoglycerat, SDH – succinate dehydrogenase, CS – citrate synthase, PDH – pyruvate dehydrogenase. The significance of differences: *p<0,05; ** p<0,01 as compared to the intact rat; Δ p<0,05; ΔΔ p<0,01 as compared to the group of "myocardial ischemia"; # p<0,05; ## p<0,01 as compared to the group of "myocardial ischemia + cytoflavin"; $p<0,05; $$p<0,01 as compared to the group of "myocardial ischemia + meldonium";! p<0,05; !! p<0,01 as compared to the group of "myocardial ischemia + phosphocreatine".

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kukes V. G., Gorbach T. V., Romashchenko O. V., Rumbesht V. V. АТP as the marker of power exchange condition at the experimental ischemia of the myocardium due to metabolic drugs 61 introduction. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 58-62.

We found a strong direct correlations between all meldonium, which were achieved in different ways studied mitochondrial enzymes: SDH and CS (r = 0,75, and to varying degrees. p <0,0001), SDH and PDH (r = 0,70, p <0,0001), the 3. Introduction of meldonium to the animals CS and SDH (r = 0,74, p <0,0001), and between them with experimental myocardial ischemia resulted in and the ATP levels in the myocardium, erythrocytes recovery amount of ATP in heart homogenate to the and blood serum, what has been said above. level of intact rats without excessive accumulation of Our findings, firstly, confirm the presence of this nucleotide, indicating a lack of doping properties energy-saving effect in all we have studied metabolic of the drug. correctors – trimetazidine, cytoflavin, 4. To assess the effectiveness of phosphocreatine and meldonium, secondly, they cardiocytoprotectors in complex treatment of patients show that the achievement of energy economization with cardiac pathology, the study of ATP within cardiomyocytes occurs in different ways and concentration in erythrocytes and serum can be to varying degrees because of different mechanisms recommended during the treatment as the most of action. The studied results are consistent with appropriate marker of their energy-saving effect. published data [10, 11, 12] References Really, trimetazidine contributes switch in 1. Ratmanova A. Cardiovascular morbidity and energy metabolism from fatty acid oxidation to mortality – statistics on European countries (2008). glucose oxidation by inhibiting acetyl-CoA Medicine Review. №1(6) (2009): 6-12 (In Russian). acyltransferase, a key enzyme in fatty acid oxidation [Full text] 2. 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Treatment of patients with chronic practice index of ATP concentration in blood serum ischemic heart disease and the actual state of the problem in practical public health. Clinical Medicine. №8 (2007): or red blood cells as the most adequate and accessible 19-25 (In Russian). [eLIBRARY] marker of energy metabolism state in 8. Gaman D.V., Konopenko M.I., Tyubka T.Y. cardiomyocytes, and in the case of the definition of Features of morphofunctional ultrastructure of the heart in this indicator during the treatment of patients with experimental myocardial ischemia. Ukrainian cardiocytoprotectors it can be judged the energy- biopharmaceutical Journal. Vol.10, №5 (2011): 16-20. saving effect of the latest . [Full text] Conclusions 9. Egorova M.V., Afanasiev S.A. Isolation of 1. ATP concentration in serum or red blood mitochondria from the cells and tissues of animals and cells is the most appropriate measure of the energy humans: modern instructional techniques. Siberian metabolism in the heart muscle due to the close Journal of Medicine. Vol.26, №1 (2011): 22-28 (In Russian). [Full text] correlation with the level of ATP in myocardial 10. Yakushev V.I., Filippenko N.G., Kizilova I.V., homogenates, and the activity of mitochondrial Korokin M.V., Beskhmelnitsyna E.A., Litvinova A.S. enzymes of the heart – succinate dehydrogenase, Studying dosedependent endothelio- and cardioprotective citrate synthase, pyruvate dehydrogenase. activity of selective arginase II inhibitor in 2. In the experimental myocardial ischemia it hyperhomocysteineinduced endothelial dysfunction. has been detected energy-saving effects of Research result: pharmacology and clinical pharmacology. trimetazidine, cytoflavin, phosphocreatine and 2016. Vol. 2, №1 (2): 42-45. [eLIBRARY] RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Kukes V. G., Gorbach T. V., Romashchenko O. V., Rumbesht V. V. АТP as the marker of power exchange condition at the experimental ischemia of the myocardium due to metabolic drugs 62 introduction. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 58-62.

11. Molchanova O.V., Pokrovskaya T.G., Povetkin 15. The Russian register of medicines [Electronic S.V., K.M. Reznikov Endothelioprotective property of the resource] // Encyclopedia of medicines and products of combination of the thioctic acid and rosuvastatin shown in pharmaceutical range: website. Access: : the endothelial dysfunction models. Research result: http://www.rlsnet.ru/ (reference date of 09.21.2016) (In pharmacology and clinical pharmacology. 2016. Vol. 2, Russian). №1 (2): 9-15. [eLIBRARY] 16. Nikolaev A.A. Hemodynamic changes due to 12. Denisyuk T.A., Lazareva G.A., Provotorov cytoflavin using during coronary bypass surgery on the V.Yu., Shaposhnikov A.A. Endothelium and beating heart: Synopsis of dissert. of master degree. – St. cardioprotective effects of HMG-Co-Areductase in Petersburg. – 2005 – 20p (In Russian). [eLIBRARY] [Full combination with L-arginine in endothelial dysfunction text] modeling. Research result: pharmacology and clinical 17. Nedoshivin S.A. Cytoprotectors in the treatment pharmacology. 2016. Vol. 2, №1 (2): 4-8. [eLIBRARY] of heart failure in patients with coronary heart disease: 13. Kantor P.F., Lucein A., Kozak R., Lopaschuk Synopsis of dissert. of Dr. med., St. Petersburg. – 2002. – G.D. The antianginal drug trimetezidin shifts cardiac 40p (In Russian). [eLIBRARY] [Full text]. energy metabolism from fatty acid oxidation to glucose 18. Artyushkova E.V., Pokrovsky M.V., Artyushkova oxidation by inhibiting mitochondrial long-chain 3- E.B. et al. Endotelio- and cardioprotective effects of ketoacyl coenzyme A thiolase. Circ. Res. V.86 (2000): meldonium and trimetazidine in L-NAME-induced 580-588. [PubMed] endothelial dysfunction in experiment. Kursk scientific- 14. Kay L., Finelly C., Aussedat J. et al. Improvement practical herald "Man and his health". № 3 (2010): 5-10 of long term preservation of the isolated arrested rat heart (In Russian). [Full text] by trimetazidine effects on energy state and mitochondrial function. Am. J. Cardiol. V.76 (1995): 45B-49B. [PubMed]

VETERINARY PHARMACOLOGY Рус. Eng.

UDC: 666.322.4: 615.281.9 DOI: 10.18413/2500-235X -2016-2-3-63-74

Bui Quang Cu 1, Nguen Hoai Chao2, Vesentsev A.I.3, THE ANTIBACTERIAL PROPERTIES OF MODIFIED BENTONITE Buhanov V.D. 4, DEPOSIT TAM BO Sokolovsky P.V.5, Mihaylyukova M.O.6 1) Doctor of technical sciences, professor, researcher, Institute of environmental technology-vietnam academy of science and technology. 18 Hoang quoc viet – Hanoi, Vietnam, e-mail: [email protected] 2) Doctor of physico-mathematical sciences, professor, Head of Eco-friendly department, Institute of environmental technology-vietnam academy of science and technology. 18 Hoang quoc viet – Hanoi, Vietnam, e-mail: [email protected]. 3) Doctor of technical sciences, professor, head of the department of general chemistry, National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]. 4) Сandidate of veterinary sciences, associate professor of medical and biological bases of physical education National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]. 5) Graduate student, department of general chemistry, National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] 6) Graduate student, department of general chemistry, National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] Abstract. In this article, the antibacterial properties enriched bentonite Tam Bo deposit, Lam Dong Province (Vietnam) modified in various ways: alkali – by processing enriched bentonite LiOH solution at a temperature of 60°C for 30 min, brine – by processing enriched bentonite, 3% solution of Na2CO3 at 55°C for 30 min, and nanoparticles of silver – silver nanoparticles by immobilizing on the surface of the bentonite by precipitation of silver nanoparticles AgNO3 solution. Initially, the bentonite clay deposits Tam Bo contains 40 – 60 wt% of active sorption- montmorillonite. Enrichment, followed by modifying the content of montmorillonite can increase up to 70 – 80 wt. %. The modification allows the bentonite increase in its physical-chemical and colloid-chemical characteristics to 2 – 3 times, such as degree of swelling, the specific surface area, cation exchange capacity. The experimental data showed that the bentonite modified with silver nanoparticles can effectively inhibit the growth of opportunistic pathogens like Enterococcus feacalis, Escherichia coli, Proteus mirabilis, Pseudomonas aerugenosa, Salmonella typhimurium, Staphylococcus aureus and Candida albicans. Modification of bentonite clay and montmorillonite crystal lattice is included in its composition with silver nanoparticles allows increasing the bactericidal activity against aflatoxin to 65 times. Keywords: bentonite, montmorillonite containing sorbent, modification, enterosorbent, aflatoxin, pollutants, nano particle silver, opportunistic pathogens.

Introduction. radionuclides, pesticides, heavy metals), prevention The main directions of modern biotechnology and treatment of diseases. Chelators are widely used include the development of sorption materials for the in animal production [1-4]. Complex pathologic purpose of their further use in the design of high- changes in the composition of the intestinal performance products for hemodialysis and microflora with relevant clinical manifestations enterosorption, preparations of immobilized associated with dysbiosis, develops due to the use of enzymes, test kits for enzyme immunoassay and antibiotics is leveled exclusively at the expense of immunofluorescence analysis. Chelators are used to application enterosorbents [5]. In this situation, a bind metabolites, toxins and other substances in the serious alternative to antibiotics are the methods of digestive tract are promising in addressing human efferent medicine, allowing to correct the condition food regulatory issues, to reduce the intake of of the patient and reduce the toxic load on the body. environmentally harmful substances (including Creating a comprehensive veterinary drugs, using RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Bui Quang Cu , Nguen Hoai Chao, Vesentsev A.I., Buhanov V.D. , Sokolovsky P.V., Mihaylyukova M.O. The antibacterial properties of modified bentonite deposit tam bo. Research result: 64 pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 63-74. different sorbents groups in combination with of blood, in particular, increases the content of antibiotics, it extends the capabilities of calcium, magnesium, inorganic phosphorus [13]. extracorporeal methods of complex treatment of In the digestive tract the bentonite absorbs water acute intestinal infections, as chelators are the means and digestive juices, this increases the surface to a multi-faceted performance is determined not exposure of food bacteria, which improves the only by their symptomatic (antidiarrheal) and digestibility of food. Moreover, thanks to the ability pathogenetic (detoxification, and others.) etiotropic of the bentonite to the selective adsorption of the and effect as against pathogenic bacteria and viruses. chemical elements are removed from the digestive They are used to increase productivity, and most system of toxic substances, bacteria, alkaloids protein importantly, reduce the cost of production due to the compounds) [14]. higher efficiency of the use of nutritional food Prevalence containing montmorillonite clay is substances. This transformation is achieved by quite high. There are a number of large deposits increasing the feed of nutrients in the products due to containing montmorillonite clay around the world the use of advanced technology for feeding feed [15-19]. An urgent task of research is to develop preparations and additives stimulating the digestibility methods for the modification of montmorillonite clay and utilization of nutrients. One of the promising to improve their sorption characteristics and mineral adsorbents, are used as feed additives are bactericidal properties. Finding the solution of this natural clays containing montmorillonite [6-8]. The problem is devoted to this article. As a material for main purpose of them – animals detoxification of the the study was taken bentonite clay deposits Tam Bo body is achieved by enterosorption pathogens organic province of Lam Dong (Vietnam). Currently, the and inorganic nature. This feed supplement contains no deposit Tam Bo bentonite clay is mined from the chemicals that negatively affect the quality of animals quarry area 5,9 km2. Crude bentonite imeeet and products derived from them. It is not toxic and has heterogeneous and different shades from light gray to no cumulative properties. Natural minerals containing yellowish. According to the Vietnamese geologists rich composition of micro- and macronutrients, help to bentonite reserves in the deposit, Tam Bo is 178 improve digestion and utilization processes of nutrients, million m3, including 59 million m3 montmorillonite. thereby increasing resistance and productivity of Material and methods of research. animals. The study shows that the animals are removed For the study in this paper are taken samples of from the body of the missing minerals chemical bentonite clays Tam Bo deposit, which is assigned to elements, giving through the ion exchange mechanism the following markings TN 1, TN 5/1, VT 6. All redundant, thereby normalizing the mineral balance. samples were fine powders grayish-yellow color. The For example, a feed additive «M-Feed» (France mineralogical and chemical composition of bentonite manufacturer, Za du Haut du Bois 56580 Brehan clay Tam Bo deposit was determined by X-ray and France) on the basis of montmorillonite clay is designed X-ray fluorescence analysis using X-ray workstation to improve the quality of feed and improve the work of ARL 9900 series x-ray workstation with Co anode. the gastrointestinal tract of the animal. This supplement Morphological characteristics of crystals included in is a combination high-tech and totally natural product, the study clays determined using analytical created using nanotechnology. Penetrating into the transmission electron microscope of high resolution animal together with fodder, it absorbs harmful JEOL-2100 (Japan), and a deposit emission substances in the gastrointestinal tract; adsorbs large transmission electron microscope ultrahigh resolution molecules secreted by pathogenic bacteria, mycotoxins Technai G2F20 S-Twin, FEI Company and heavy metals; It stimulates specific and non- (Netherlands). To investigate the montmorillonite specific local immunity of the animal, and normalizes clay used a special method of sample preparation for the viscosity of the intestinal contents, protects the non-self-supporting samples and suspensions [20]. intestinal walls and helps to normalize the intestinal The carbon used as the substrate film which has been microflora. Extracts of herbs and essential oils that obtained by spraying a spectrally pure carbon layer make up the «M-Feed», causes appetite, improves the on the surface of the single crystal halite. Directly palatability of feed, promote better its digestibility and before spraying halite crystal cleaved along cleavage protect the body against pathogenic bacteria and planes for obtaining clean smooth surface. Spraying parasites. was carried out with the device for the deposition of Numerous studies have confirmed that the carbon films in vacuum Quarum Q150RE. Pulse addition of montmorillonite clay in animal feed sputtering was performed in a vacuum (pressure not increases milk yield and milk fat content, quality and exceeding 10.04 mbar) did 2-4 pulse for 2-5 seconds taste of meat in cattle; It provides an increase in with 30 seconds intervals. Cooked lowered carbon weight – in pigs; egg production and egg quality – a film on the surface of distilled water, poured into a bird [9-12], improves some biochemical parameters short flat cylinder with a large free surface area.

Clutching a copper mesh with tweezers, gently In a control tube was added only to the MIA caught carbon film so that it covered the mesh. study the culture of microorganisms, and in the next Further, the mesh was placed in a box for storage – only the MIA with the test Hinge of clay. meshs. To the resulting mesh with a carbon film The contents were then carefully resuspended deposited drop of an aqueous suspension of the and tubes were placed in a special holder to obtain sample. Moreover, according to the guidelines of MIA beveled surface. After sealing agar [21] to obtain reliable results of the study, analysis of experimental and control tubes were placed in an the samples was performed for three reticula sample incubator, and cultured at 37°C for 16-18 hours. deposited from the same sample, after thorough After culturing with the slant surface, followed mixing, each sample prior to application to the mesh. by washings were prepared in determining According to the guidelines of the [22] The concentration of microorganisms through the optical laboratory sample was formed in the amount of device – «Densilametr». Performance of the device in allocation of the average sample sufficient to perform the control swabs tubes, which contained MIA and a single analysis. studied clay, used to adjust the true concentration of Enrichment of bentonite was carried out by microorganisms in the control tubes swabs, because precipitation from an aqueous slurry, pouring the Optical turbidity difference, expressed in units of slurry of the upper layer, followed by drying. Further McFarland in washouts experimental and control carried out the modification of bentonite clay. For tubes reflect the true concentration of convenience, the notation method of modifying clays microorganisms in 1 ml. Washes produce sterile samples: alkaline – Li, salt – Na, the modification of isotonic sodium chloride solution, the volume of silver nanoparticles – Ag. Enrichment clay identified which was 5 ml. – En (sample name), the native form of clay Study of the sorption capacity of bentonite clays identified – Nat (sample name). in relation to mycotoxins performed as follows: a The modification was carried out in the sample of 10 mg of study 0,2 bentonite clay was following ways: placed in a beaker containing 10 ml of a solution of - Alkali (Li): bentonite enriched by treatment aflatoxin B1 (AfB1) with a concentration of 59 mg/l, with LiOH solution at 60°C for 30 min. pH = 7.0. After stirring, shaking for 60 min, the - Salt (Na): by treating bentonite enriched with solution was left for 24 hours for precipitation. The 3% solution of Na2CO3 at 55°C for 30 min. clarified solution was separated and the upper part - Silver nanoparticles (Ag): by immobilizing the was collected to determine the content of it remaining silver nanoparticles of bentonite surface by AfB1 ELISA method using a test-kit AgraQuant deposition of silver nanoparticles AgNO3 solution. Aflatoxin B1 firm Romer Labs (USA). To carry out Application of Silver Nanoparticles to bentonite is as the experimental work bution effectiveness inhibition follows: bentonite clay in powder form is suspended of development of mycotoxins using bentonite clay in an aqueous solution of silver nitrate, and the Ag+ modified with silver nanoparticles following ions are uniformly distributed in the pores of experimental parameters were used as a producer of montmorillonite, including inter-packet space lattice the mycotoxin using mold biomass peanuts on a montmorillonite as laminate silicate structure type 2: culture medium Sabouraud (4% agar from glukoza) 1 crystal lattice swelling. Then, the silver ions were from Merck (Germany) . bentonite nanosilver reduced to nano-sized metallic state using NaBH4. concentration was 2 wt.%. The content of nanosilver Adsorption characteristics of bentonite clay in the bentonite clay in the trials was 0; 5; 10; 20; 30 samples are determined in relation to the heavy metal and 50 mg in 10 ml culture medium Sabouraud (4% ions (Pb2+ and Cd2+) by spectrophotometry. glucose agar). Fungal spores were transferred into Sensitivity Enterococcus feacalis, Escherichia Petri dishes with Sabouraud culture medium (4% coli, Proteus mirabilis, Pseudomonas aerugenosa, glucose agar) containing bentonite clay modified Salmonella typhimurium, Staphylococcus Aureus with silver nanoparticles. The plates were placed in a and Candida albicans to the experimental samples thermostated oven for 24 – 48 h at 35°C. Circle performed sorbents meat infusion agar twice dilution diameter determined spread of fungi in a petri dish to method (MIA) containing 2 wt. % Agar. Clays determine the efficacy of inhibition of fungi under investigated sample was placed into tubes and the influence of bentonite clay modified with silver sterilized in an oven at a temperature of 160-180°C. nanoparticles. Then these tubes were added to 5 ml of molten MIA. Results and discussion. In the molten MIA, the temperature of which was 42 The first stage of implementation of the work – 43°C, the slurry was poured in advance of one of was to study the material composition of the clay the strains of microorganisms, the rate of 1*107 CFU samples submitted. Oxide chemical composition (colony forming units) in 1 ml of MIA. shown in table 1. RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Bui Quang Cu , Nguen Hoai Chao, Vesentsev A.I., Buhanov V.D. , Sokolovsky P.V., Mihaylyukova M.O. The antibacterial properties of modified bentonite deposit tam bo. Research result: 66 pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 63-74.

Table 1

Chemical composition of the oxide samples of clay from Tam Bo deposit

The content of oxides by wt.% Sample SiO2 Al2O3 Fe2O3 MgO CaO TiO2 K2O MnO Na2O Rh2O3 V2O5 Cr2O3 ZrO2 ZnO п.п.п. Sum TN 1 51,72 21,49 9,46 2,65 1,83 1,43 0,70 0,075 0,063 0,047 0,028 0,023 0,021 0,01 10,45 100 St.error, % 0,18 0,14 0,12 0,12 0,024 0,07 0,0019 0,044 0,0038 0,0015 0,0008 - 0,018 - 0,02 - TN 5/1 55,30 22,31 6,48 2,76 0,74 0,67 1,36 0,018 0,049 0,028 0,016 - 0,016 - 10,25 100 St.error, % 0,18 0,14 0,12 0,12 0,024 0,07 0,0019 0,044 0,0038 0,0015 0,0008 - 0,018 - 0,02 - VT 6 56,62 20,90 6,71 2,26 0,62 0,75 1,38 0,040 0,067 0,016 0,019 - 0,017 0,011 10,58 100 St.error , % 0,24 0,21 0,13 0,08 0,034 0,042 0,06 0,002 0,005 0,002 0,021 - 0,001 0,001 0,05 -

Submitted samples contain in its structure distribution of crystal montmorillonite and related silicas, aluminum and calcium (SiO2, Al2O3 and minerals in clays uneven (Fig. 1 a). An alternation of CaO), are typical for bentonite clay-based minerals individual crystals montmorillonite (S) and its montmorillonite groups, namely, dioctahedral components (A). The size of the aggregates is 10-70 montmorillonite aluminum ions, alkaline earth metal microns (Figure 1 b). ions, in this case Ca2 +. Fairly high content of aluminum oxide is typical for montmorillonite, kaolinite clays. The test samples of bentonite clay content of iron oxide (6.71 – 9.46 wt.%), slightly more than the previously published data [23, 24]. The mineralogical composition of the investigated clays are presented in Table 2. Table 2 Mineralogical composition clay samples

Content, wt.% Minerals TN 1 TN 5/1 VТ 6 Montmorillonite 51 55 45 Kaolinite 11 12 12 a) Illite 9 10 10 Dolomite 4 1 4 Quartz 10 10 21 Muscovite 5 3 missing Feldspar trace trace trace Goethite 3 5 3 Chlorite 4 4 3 Sum 100 100 100 The mineralogical composition clay samples from Tam Bo deposits submitted to montmorillonite (45-55 wt.%), Kaolinite (11-12 wt.%), Illite (9-10 wt.%), Low temperature trigonal quartz (10-21% by weight.) goethite, feldspars, chlorite, and b) others. It is found that the content of montmorillonite Figure 1. The distribution of particles in a sample of – essential mineral determining the adsorption montmorillonite clay VT 6 from Tam Bo deposit (S – capacity of bentonite clay – varies depending on the montmorillonite individual crystals; A – montmorillonite sampling site. The content of montmorillonite clay aggregates of crystals) deposit samples Tam Bo, selected near the reservoir, up to 70 wt.%. However, the average content of It can be seen (Figure 2.), that the form of montmorillonite clays from Tam Bo deposit is within montmorillonite crystals are plate isometric shapes, 50 wt.%. Based on these data we can conclude that sizes 1-4 microns. bentonite from Tam Bo deposit can be used for On the surface of montmorillonite crystals sorption-active material with high sorption capacity, crack unstable fixed mold (F), a thickness of 5 nm suitable for the preparation of feed additives used in and a length of 40 to 50 nm (Fig. 2a). Fixed to the animal husbandry and particularly in poultry [9-12]. inner surface cracks (Fig. 2 a) may be introduced and The study of structural and morphological various sorbed pollutants having a size less than 5 characteristics of the deposits of bentonite clays nm. montmorillonite crystals are torn, jagged edges samples from Tam Bo deposit revealed that the (Fig. 2 b). The edges of the crystals twist in tubes (T).

a) a)

b) b) Figure 2. deposits crystals in a sample of montmorillonite Figure 3. Montmorillonite (M) of sample clay VT 6 from Tam Bo deposit, T- twisted into a tubes edge clay VT 6 from Tam Bo deposit (F – crack unstable forms, of montmorillonite crystal T – twisted into a tubes edge crystal montmorillonite) Morphological characteristics of individual The methods of transmission electron particles of fine clay and related minerals determined microscopy confirmed that the edge by transmission (transmission) electron microscopy nanoplenochnogo montmorillonite single crystal can (Figure 3-6). It is found that crystal particles are twist (Fig. 3), which is probably due to the montmorillonite isometric views of films of size energetically more favorable state, because of the 100-200 nm, which tend to assemble into aggregates. surface tension forces are balanced. montmorillonite The thickness of the crystals determined by crystals can twist in tubes, which is probably due to a montmorillonite from darkdeposit transmission thermodynamically stable state of a cylindrical shape electron microscopy studies and of 3-5 nm. compared with the film (Fig. 3). This process intensifies the action of the flow of electrons. Low temperature trigonal crystal is shown in Figure 4 as a massive dark crystals.

Figure 4. Electron micrograph of a low-temperature trigonal quartz (Q) from a sample of clay VT 6 from Tam Bo deposit

Also kaolinite crystals are present in the plates and pretty clear crystallographic facet (Figure 5). composition of clay, having the form of hexagonal

a) b) Figure 5. Kaolin (K) from the sample of clay VT 6 from Tam Bo deposit. Figure 6 shows the results of experimental 30 minutes. The adsorption capacity of Pb2 + and studies on the adsorption Pb2+ ions and Cd2 + in the Cd2 + in samples of bentonite clay depends on pH and original sample bentonite VT 6 (Fig. 6 a) and increases with increasing pH from 2 to 6. In the enriched (Fig. 6 b), depending on the exposure time. region of pH 6 these metals undergo hydrolysis and It is found that the adsorption begins from the first form precipitates, whereby the adsorption process is moment of interaction, and reaches a maximum after terminated.

l l / /

mg mg , , ncentration Concentration Co

Time, min Time, min Figure 6. Dependence of concentration changes of the ions Pb2+ and Cd2+ on the adsorption on the duration of the original bentonite clay samples VT6 (A) and rich bentonite clay samples VT6 (B) Table 3 presents the results of a study of Cd2 + depending on the method of modification of sorption activity with respect to the ions Pb2 + and bentonite clay. Table 3 Adsorption of ions Pb2 + and Cd2 + on experimental samples Adsorption capacity, mg/g) Samples Pb2+ Cd2+ Nat (VT 6) 24,6 11,2 En (VT 6) 63,9 26,6 Na (VT 6) 62,7 29,5 Ag (VT 6) 58,8 26,0 From Table 3 it is established that the Table 4 shows the results of determination of the modification of bentonite clays in various ways sorptive capacity of clays from Lam Dong Province enhances its adsorptive capacity of clay with respect towards pathogenic microorganisms. to ions Pb2 + and Cd2 + from 2 – 3 times.

Table 4 The sorption capacity Tam Bo clay deposits in relation to conditionally pathogenic microorganisms

Native clay samples Clay concentration in Microorganisms mg/ml in MIA ТN 1 ТN 5/1 VТ 6 Control Cfu / ml 400 9.108 1.108 1.108 Enterococcusfeacalis 200 7.108 2.108 2.108 2.108 100 9.108 2.108 3.108 400 12.108 2.108 0,0 Escherichia coli 200 13.108 2.108 0,0 3.108 100 21.108 3.108 2.108 400 2.108 1.108 0,0 Proteus mirabilis 200 3.108 1.108 2.108 5.108 100 5.108 2.108 10.108 400 3.108 1.108 2.108 Pseudomonas aerugenosa 200 3.108 1.108 3.108 5.108 100 5.108 3.108 3.108 400 6.108 1.108 3.108 Salmonella typhimurium 200 8.108 2.108 4.108 4.108 100 9.108 3.108 5.108 400 6.108 0,0 0,0 Staphylococcus аureus 200 9.108 1.108 0,0 3,5.108 100 9.108 3.108 1.108 400 1.108 1.108 0 Candida albicans 200 3.108 5.108 1.108 5.108 100 3.108 6.108 3.108

Following the information given in the table, it For example, the number of microorganisms in should be concluded that seven Vietnamese the test tubes with clay TN 1 greater than the number containing montmorillonite clay the most effective of Cfu of microorganisms grown in test tubes in 1.5 – bacteriostatic effect in respect of the studied bacteria 7 times. The stimulation of microbial growth is and microscopic fungi of the genus Candida, had clay probably due to the presence of clay minerals, samples TN 5/1 and VT 6. In this test TN5/1 contributing to their growth. suppressed the growth of Staphylococcus aureus As the object of study to take a sample of (clay concentration of 400 mg/ml MIA), and showed bentonite clay VT 6, which showed the highest clay VT 6 effective inhibitory effect on Escherichia antibacterial properties. To enhance the antibacterial coli (400 mg/ml MIA), Proteus mirabilis (400 mg/ml activity conducted most effectively modifying clay MIA), Staphylococcus aureus (200-400 mg/ml MIA) sample VT 6 different ways: saline (Sample Na and Candida albicans (200-400 mg/ml MIA). (VT 6)), alkaline (Sample Li (VT 6)) and the silver Less productive remaining samples were clays nanoparticles (Sample Ag (VT 6)). As a control, the which are not delayed growth on investigated activated carbon is taken pharmaceutical, microorganism strains MIA containing a high manufacturing Pharmstandard, Russia. antibacterial concentration (400 mg / ml) of test samples (TN 1) activity results are shown in Table 5. and stimulate the growth and reproduction of microorganisms.

Table 5 Relationship of microorganisms washings concentration (Cfu / ml) on the presence of the ingredients in the composition and quantitative content of sorbent

Clay concentration in mg/ml in Modificated clay samples Microorganisms Control MIA Na (VT 6) Li (VT 6) Ag (VT 6) 200 18.108 0 0 100 - 0 0 50 - 0 0 Escherichia coli 25 - * 0 39.108 12,5 - 24.108 * 6,25 - - 2.108 3,125 - - 18.108 200 15.108 0 0 100 - 0 0 50 - * 0 25 - 9.108 0 Salmonella dublin 12.108 12,5 - - 0 6,25 - - * 3,125 - - 2.108 1,56 - - 9.108 100 18.108 0 0 50 - 0 0 25 - * 0 Salmonella enteritidis 12,5 - 6.108 0 22.108 6,25 - - * 3,125 - - 2.108 1,56 - - 18.108 200 30.108 0 0 100 - 0 0 50 - 0 0 . 8 Staphylococcus hyicus 25 - 2 10 0 36.108 12,5 - 30.108 0 6,25 - - * 3,125 - - 6.108 200 18.108 0 0 100 36.108 0 0 50 - 3.108 0 Staphylococcus 25 - 21.108 0 30.108 intermedius 12,5 - - * 6,25 - - 2.108 3,125 - - 24.108 Staphylococcus aureus, 200 33.108 0 0 метициллин 100 - 0 0 резистентный 50 - 0 0 25 - 2.108 0 36.108 12,5 - 20.108 * 6,25 - 22.108 2.108 3,125 - - 21.108 Proteus vulgaris 12,5 - - - 6,25 - - * 36.108 3,125 - - 2.108 1,56 - - 18.108 Note: 0 – the presence of microbial growth; * – Bactericidal effect; – - The study was conducted.

It is found that the sample Li (VT 6) at a dose of The control sample at a concentration of 100 mg/ml 25 and 50 mg/ml bactericidal acted on Salmonella MIA did not suppress the growth and development of and Escherichia. In this form of staphylococcus microbial strains investigated (tabl. 4). Instead sorbent at concentrations of 50-100 mg/ml provided inhibition of their growth and development, he on the only bacteriostatic effect, as after the sowing of the contrary stimulate their proliferation. Guided by the resulting washings are always marked growth of results obtained, it should be noted the high efficiency staphylococci used. of the sorbent shaped silver Ag (VT 6). Designed It is necessary to note the fact that the sample of complex preparation can be widely used in agriculture Na (VT 6) at a content of 200 mg/ml MIA not inhibited and veterinary. growth of test organisms. In turn, it enhances the The study of the ability of modified clay to growth of Staphylococcus intermedius and Salmonella inhibit the growth of fungi and the effect of dublin. Compared to control the number of microbial mycotoxins. Figure 7 shows the results of cells per 1 ml wash it was more respectively 1.2 and 1.3 determining the efficiency of inhibition of fungi times. Also increased the number of Cfu sorbent under the influence of bentonite clay, modified Staphylococcus aureus in 1 ml wash 1.1 times. nanoparticulate silver (Ag (VT 6)). The weight ratio More effectively inhibit the growth of bacteria in of the modified silver / Bentonite clay nanoparticles the study and act on bacteria bactericidal effect sample (Ag / Bent) / culture medium was varied between Ag (VT 6) in concentrations 6.25 – 12.5 mg/ml MIA. 0.05-0.5 wt.%.

Diameter of the mm Diameter circle, Content Ag/Bent in environment, % a) b) Figure 7. Effect of fungal growth inhibition sorbent Ag (VT 6) depending on the weight ratio of Ag / Bent

It was found that immobilized nanosilver The adsorption of aflatoxin B1 (AfB1) samples of bentonite clay are well inhibits the growth of fungal bentonite clays modified with other methods conducted mycotoxins. In Figure 7 it can be seen that increasing in water (bentonite clay concentration of 0.1 wt.%). the ratio of Ag / Bent from zero to 02 wt. % Circle AfB1 residual concentration in the filtrate was analyzed diameter of fungal growth is significantly reduced, by ELISA. The results are shown in Table 6. indicating that the inhibitory effects of silver nanoparticles with respect to mycotoxins. Table 6 Efficacy AfB1 adsorption on a sample of bentonite clay in water at AfB1, initial concentration 59 mg/l Concentration AfB Samples 1 Adsorption efficiency (%) (mg/l) Nat (VT 6) 59,00 0 En (VT 6) 6,590 88,8 Na (VT 6) 0,268 99,5 Li (VT 6) 0,105 99,8 Ag (VT 6) 0 100 CONCLUSION It was established that the modification of Established material (chemical, mineralogical) bentonite clay VT 6mestorozhdeniya Tam Bo leads to a composition, as well as the structural and substantial increase in the ability to inhibit the growth of morphological characteristics of the deposits of clay fungi and aflatoxin absorption (up to 65 times). samples Tam Bo Lam Dong province. The mineralogical composition of the clay samples (TN 1, RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Bui Quang Cu , Nguen Hoai Chao, Vesentsev A.I., Buhanov V.D. , Sokolovsky P.V., Mihaylyukova M.O. The antibacterial properties of modified bentonite deposit tam bo. Research result: 73 pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 63-74.

TN 5/1, VT 6) Tam Bo deposits represented (Vietnam): En (VT 6), Na (VT 6), Li (VT 6), Ag (VT montmorillonite (45 – 55 wt.%), Kaolinite (11 – 12 6) can be used as enterosorbent for detoxification wt.%), Illite (9 – 10 wt.%), trigonal quartz low humans and animals from ions of heavy metals, temperature (10 – 21 wt.%), goethite, feldspars, pathogenic and opportunistic bacteria, mycotoxins, chlorite, and others. It is found that the content of and hence in the production of feed additives for montmorillonite – essential mineral determining the livestock and ptitsevodstvodcheskih. adsorption capacity of bentonite clay – varies Work is performed at the expense of means of depending on the sampling site. The content of a grant of the Russian Federal Property Fund montmorillonite clay deposit samples Tam Bo, № 14-43-08021 «Research of processes selected near the reservoir, up to 70 wt.%. However, phaseformation and structurizations proceeding at the average content of montmorillonite clays Tam Bo joint pyrolysis of vegetable waste of agro-industrial deposit is within 50 wt.%. A modification of the clay complex of the Belgorod region with local samples presented in different ways: alkali – montmorillonite the containing clays and studying of bentonite enriched by treatment with LiOH solution influence of physical and chemical parameters of at 60°C for 30 min, brine – bentonite enriched by process of synthesis of effective composite sorbents treatment with 3% solution of Na2CO3 at 55°C for on absorption of heavy metals, pathogenic and 30 minutes and the silver nanoparticles – by opportunistic bacteria from water solutions and immobilization of silver nanoparticles on the surface cleaning of fertile soils of pesticides», 2015 – 2016, of the bentonite by the deposition of silver and State contract № 14.N08.11.0109 «Preclinical nanoparticles of AgNO3 solution. It was found that at research of medical product – bactericide a dose of 25 and 50 mg/ml of the alkaline form of enterosorbent based on montmorillonite», 2016-2018, clay (sample Li (VT 6)) bactericidal effect on and region grant «Development of an complex Salmonella and Escherichia. In this form of medical veterinary product for treatment and staphylococcus sorbent at concentrations of 50-100 prevention gastrointestinal infectious disease of pigs, mg/ml provided only bacteriostatic effect, as after the produced from in expensive raw materials of sowing of the resulting washings are always marked Belgorod region», 2016-2017. growth of staphylococci used. Clay treated with saline method (sample Na ЛИТЕРАТУРА (VT 6)) at a content of 200 mg/ml MIA, not inhibit 1. Abbès S., Z. Ouanes J. B. Salah-Abbes M. A. the growth of bacteria in the study. In turn, it Abdel-Wahhab R. Oueslati H. Bacha. Preventive roles of enhances the growth of Staphylococcus intermedius aluminosilicate clay against induction of micronuclei and and Salmonella dublin. Compared to control the chromosome aberrations in bone-marrow cells of Balbc mice treated with Zearalenone. Mutat. Res. 631 (2007): number of microbial cells per 1 ml wash it was more 85-92 [PubMed] [Full text]. respectively 1.2 and 1.3 times. It also increases the 2. Abbès S., Z. Ouanes, J. B. Salah-Abbès, number of Cfu of Staphylococcus aureus in 1 ml Z. Houas R. Oueslati, H. Bacha, O. Othman. The wash 1.1 times. The most effective proved to be the protective effect of hydrated sodium calcium clay modified with silver nanoparticles (sample Ag aluminosilicate against haematological, biochemical and (VT 6)). Even at concentrations 6.25 – 12.5 mg/ml pathological changes induced by Zearalenone in mice. MIA, it inhibits the growth of opportunistic Toxicon. 47 (2006): 567-574 [PubMed] [Full text]. microorganisms. The control sample at a 3. 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RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Bui Quang Cu , Nguen Hoai Chao, Vesentsev A.I., Buhanov V.D. , Sokolovsky P.V., Mihaylyukova M.O. The antibacterial properties of modified bentonite deposit tam bo. Research result: 74 pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 63-74. animals: health advantages and risks. Vet.Med. Czech 49, 16. Cheng-Che Tsai, Tzong-Yuan Juang, 10 (2004): 389 – 399 [Books. Google][Abstract] Shenghong A. Dai, Tzong-Ming Wu, Wen-Chiung Su, 7. J. H. Quisenberry. The use of clay in poultry Ying-Ling Liu, Ru-Jong Jeng. Synthesis and feed. Clays and Clay Minerals. 16 (1968): 267-270. [Clays montmorillonite-intercalated behavior of dendritic and Clay Minerals][Full text] surfactants. Journal of Material chemistry. 16 (2006): 8. Robert, A. Smectite-type clay minerals as 2056-2063 [Rsc][Astract]. nanomaterials, Schoonheydt. Clays and Clay Minerals. 50, 17. T. Grygar, D. Hradil, P. Вezdiеka, B. Doušová, 4 (2007): 411-420 [Reasearchgate][Full text]. L. Еapek, O. Schneeweiss. Fe(III)-modified 9. V. D. Bukhanov, A. I. Vezentsev, O. N. montmorillonite and bentonite: synthesis, chemical and Pankova, L. I. Naumenko, P. V. Sokolovsky. Influence UV-Vis spectral characterization, arsenic sorption, and montmorillonite of the containing clay on an organism of catalysis of oxidative dehydrogenation of propane. Clays laying hens when receiving organic eggs. Russian and Clay Minerals. 55, 2 (2007): 165-176 veterinary magazine. Series farm animals. 4 (2015): 8-11 [Geoscienceworld][Abstract]. (In Russian) [Cyberlinka][Full text] . 18. J.A. Kittrick. Montmorillonite equilibria and the 10. V.D. Bukhanov, A.I. Vezentsev, weathering environment. Soil Science Society of America A. A.Antipov, M. Z.Fyodorova, N. A. Volovicheva, N. F. Proceedings 35 (1971): 815-820 [Dl][Abstract]. Ponomaryova, N. A. Safonova, L. A. Kozubova. Use of 19. I.A. Afonina. Effect of copper and zinc on the the activated montmorillonitovy clay in a sharp experiment productive and biological indicators of laying hens cross on the broilers infected kolibakteriozy and salmonellosis «Rhodonite»: Dis ... Candidate. biol. Science. Novosibirsk Topical issues of veterinary biology. 4, 12 (2011): 51-57 State Agrarian University Institute of Veterinary Genetics (In Russian) [Twirpx][Full text]. and Breeding. Novosibirsk (2006): 208 p. (In Russian) 11. G.G. Onishchenko. Chemical safety as an [dissercat][Full text]. interdepartmental problem. The role of the State Sanitary 20. P. Hirsch, A. Hovi, R. Nicholson, D. Peshli, M. and Epidemiological Russia in chemical safety population. Whelan. Electronic microscopy of thin crystals. World Poison Gazette, № 1 (2002): 2-7 (In Russian). (1968): 575 (In Russian) [Twirpx][Full text]. [eLIBRARY][Full text] 21. Methodical recommendations of MP 1.2.2641- 12. Sycheva, L.V.. Stern and feeding of an 10 «Determination of priority types of nanomaterials in agricultural bird: Monograph. Perm, Perm SAA (2010): environment objects, foodstuff and live organisms». 126 p. (In Russian) [RSL][Abstract]. (2010): 85 (In Russian) [RPN][Full text]. 13. Davydova S.L., Tagasov V.I. Heavy metals as 22. Methodical recommendations of MP 1.2.0022- supertoxicants XXI century: a training manual. People's 11 «Sampling order for control of nanomaterials». (2011): Friendship University (2002): 140 (In Russian) 39 (In Russian) [Consultant][Full text]. [twirpx][Full text]. 23. K. Q. Nam, N. H. Toan Phan. Use of bentonite 14. Rolfe B. N., Miller R. F., Mcqueen I. S. and diatomaceous earth for treatment of municMIAl and Dispersion characteristics of montmorillonite, kaolinite animal waste . J.of Earth Sciences. 24, 4, (2005): 351-355 and hike clays in waters of varying quality, and their [Sciencedirect][Full text]. control with phosphate dispersants. Geological Survey 24. Y. F. Cheng, Y. P. Chen, X. H. Li, W. L. Yang, Professional Paper 334-G (1960): 48 [Usgs][Full text]. C. Wen, Y. M. Zhou. Effects of Palygorskite Inclusion on 15. Wai K.N, Banker G.S. Some physicochemical the Growth Performance, Meat Quality, Antioxidant properties of the montmorillonites. J. Pharm. Sci. 55, 11 Ability, and Mineral Element Content of Broilers. Biol (1966): 1215-1220 [Wiley][Abstract] Trace Elem Res 173, 1 (2016): 194-201 [Springer][Full text].

Рус. Eng. UDC:619: 616.24-002 DOI: 10.18413/2500-235X -2016-2-3-75-85 Zuev N.P.1, Bukhanov V.D.2, Vezentsev A.I.3, THE ETIOLOGICAL STRUCTURE OF MASS DISEASES WITH YOUNG Sokolovskiy P.V.4, GASTRO AND RESPIRATORY SYNDROME Khmirov A.V.5, Zueva E.N.6, Salashnaya E.А.7, Mihaylyukova M.O.8 1) Professor, Department of Parasitology and epizootiology VSAU name of Emperor Peter I. 394087, г. Voronez, 1Michurina Str., Russia. e-mail: [email protected]. 2) Сandidate of veterinary sciences, associate professor of medical and biological bases of physical education National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]. 3) Doctor of technical sciences, professor, head of the department of general chemistry, National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]. 4) Graduate student, department of general chemistry, National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] 5) Candidate of Biological Sciences, Veterinary Medicine Head of the Belgorod region, Chief State Veterinary Inspector of the area. 308000, г. Belgorod, 24 Pobeda Str.,Russia. e-mail: [email protected] 6) Student Belgorod State Agricultural University named V.Y. Gorin. 308503, Belgorod region., Belgorod district, p. Mayskiy, 1 Vavilova Str. 1, Russia. e-mail: [email protected] 7) Senior lecturer of the department of physical education Belgorod State Agricultural University named V.Y. Gorin. 308503, Belgorod region., Belgorod district, p. Mayskiy, 1 Vavilova Str. 1, Russia, Russia. e-mail: [email protected]. 8) Graduate student, department of general chemistry, National Research Belgorod State University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] Abstract. The study of the etiology and pathogenesis of diseases gastrointestinal and respiratory tract, the development of effective methods of these treatment and prevention are of great economic importance in ensuring the country's food animal population. Widespread gastrointestinal and respiratory diseases (25-76%) in large livestock farms due to the impact on the body of animals and birds of many technological-ray stress factors, reducing the natural resistance of the organism, pathogenic and opportunistic pathogenic microorganisms, both individually and in various associations. Etiology gastroenteritis and pneumonia animals studied complex, on the basis of epizootic, clinical, pathological data, bacteriological results, serology, hematology, immunobiochemical research methods. For detection of antibodies to pathogens of viral and chlamydial Institute infections using standard biofabrichnye antigens. Antimicrobial activity tilozinsoderzhaschih drugs against reference strains of mycoplasma, aholeplazm and field cultures of E. coli, Pasteurella and Staphylococcus aureus was investigated in liquid and solid culture media using an indicator 2,3,5 – triphenyltetrazolium chloride. Found, in the etiology of gastro-intestinal and respiratory diseases young farm animals participating Gram positive (Staphylococcus) and Gram negative (Escherichia, Salmonella, etc.) microorganisms, which usually stand in various combinations. The isolated microorganisms studyed degree of habituation to widely used in veterinary medicines practice (furazonalu, biovitu, sulgin, ampicillin, neomycin, streptomycin). Found that during several passages of them through the culture medium containing drugs (streptomycin, neomycin and ampicillin sulfates, tylosin tartrate, furazonal, Biovit, sulgin), microorganisms become resistant to them. Keywords: calves, lambs, pigs, gastroenteritis, pneumonia, etiology, stress, micro-organisms.

Introduction. liver and other organs, it is a process taking Bacteriological examination of pathological generalized. material from patients with gastroenteritis and Bacteriological examination of biological and pneumonia of pigs, lambs and chickens found that pathological material from patients with isolated them from the microflora often had non- gastroenteritis and pneumonia and died from these specific. When it pneumonia mostly present not only diseases of animals isolated microorganisms in the affected tissues but also in the kidney, spleen, association consisting: of Escherichia and

Mycoplasma, Staphylococcus and Salmonella studies can be concluded about the etiological role of (piglets); of Streptococcus and Pasteurella (calves); streptococci, staphylococci, Pasteurella, Salmonella, of Bordetella and Pasteurella (lambs). It should be zsherihy, Bordetella, Klebsiella, mycoplasma, noted that from animals belonging to a large cattle- influenza, enteroviruses, and corona- for gastroenteritis breeding complexes, flora isolated more often than and pneumonia in young farm animals . In the blood by young animals from farms with a complete serum of infected animals simultaneously detect production cycle, for gastroenteritis in the 2-3 times antibodies to Salmonella, esherihioznomu, with pneumonia and 1.5-2 times. bordetelleznomu, pasteurellosis and staphylococcal It should be noted that the pathogens were antigens [5, 6, 7, 8, 9]. isolated from the gastrointestinal tract and lungs, as Given that the level of total non-specific resistance well as other tissues and organs, indicating that the largely depends on the emergence and development of generalized nature of the infection. Of isolates in the disease, we conducted studies on the study-of some cases were resistant or slabochuvstvitelny to stressors affecting the natural resistance of the body of the most commonly used antibiotics in veterinary pigs. Transportation, rearrange ment and related medicine, in particular: Streptococcus – change and feeding conditions lead to a decrease in the erythromycin, staphylococcus – penicillin, number of blood erythrocytes, monocytes, Pasteurella – erythromycin and neomycin, lymphocytes, segmented neutrophils, hemoglobin, and tetracycline, chloramphenicol, Escherichia – to the phagocytic index of complementary, lysozyme, and polymyxin. serum bactericidal activity. The results of bacteriological studies also show Note that when transporting the total resistance that the microflora taking part in the etiology and level was reduced to a greater extent than on the farm pathogenesis of these diseases is the same taxonomic rearrangements. With increasing transportation characteristics. However, specific and generic duration piglets resistance level reduction was more composition mikrorganizmov allocated for pronounced, particularly in respect of its parameters gastroenteritis, is more diverse than in pneumonia, such as lymphocyte counts, hemoglobin, and and their pathogenicity was less pronounced than the lysozyme activity complementary serum [3, 10]. pathogens isolated from animals with pulmonary Our data showing direct correlation incidence disease. The microflora isolated from parenchymal piglets gastroenteritis and pneumonia with the level organs for gastroenteritis and pneumonia was of total non-specific resistance and conditions of practically identical. The clinical studies revealed a keeping and feeding technology, largely in line with certain pattern in the occurrence of gastroenteritis the results of research. and pneumonia. Recent usually recorded after Thus, gastroenteritis and pneumonia caused by gastroenteritis and proceed in a more severe form. young livestock associations viruses, mycoplasmas and Pneumonia more difficult to prevention and bacteria represented adenovirus, enterovirus, treatment, that to some extent be explained by the coronavirus, Escherichia, Salmonella, Pasteurella, emergence, in causing them mikroorganiz-atoms, Proteus, and Pseudomonas aeruginosa, exert their more active enzymes pathogenicity severe hemolytic pathogenic action amid falling under the influence of capacity and R-plasmids of resistance, including the different stressors general non-specific resistance of the cross, to drugs and protection factors microorganism. organism of young growth. Contributing factors and This is confirmed by our studies of patients with pathogens in the body cause immunological, pneumonia of pigs, calves and lambs caused by biochemical and morphological changes in the number Escherichia with the elements of anti-lysozyme of cases of morbidity and requiring their normalization. activity, which makes the animals less protected The concentration of animals in small areas, the against bacteria. change in the evolution Zion and economically The results of serological studies indicate the prevailing nature of their contents and feeding role of and others not identified by bacteriological contribute significantly to the spread of and virological research methods, agents of these gastrointestinal and respiratory diseases, decrease in diseases, such as influenza virus, enterovirus and total non-specific and specific resistance, and coronavirus in the etiology of gastroenteritis and inappropriate therapies – the emergence and spread pneumonia young farm animals. It should be noted of drug-resistant populations of microorganisms – that the number of seropositive animals in the off- pathogens . All this eventually contributes to farm enterprises for rearing and fattening livestock significant morbidity and death primarily young farm precast was significantly greater than in an economy animals. Reducing the level of natural resistance and with a closed production cycle [1, 2, 3, 4]. immunobiological reactivity, against which exerts its Based on the results of virological, effect of conditionally pathogenic microflora mikoplazmalogiche-ray, bacteriological and serological hampers prevention zhelu-sedimentary-intestinal and RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Zuev N.P., Bukhanov V.D., Vezentsev A.I., Sokolovskiy P.V., Khmirov A.V., Zueva E.N., Salashnaya E.А., Mihaylyukova M.O. The etiological structure of mass diseases with young gastro and 77 respiratory syndromeo. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 75-85. respiratory diseases. Moreover, the majority of coccal microflora was performed according to the pathologies of the gastrointestinal, respiratory tract, «Recommendations on the indication and gastroenteritis, pneumonia, with systemic lesions identification of staphylococci and streptococci»; pnevmoenterity, proceed with the participation of not Pasteurella – according to the «Guidelines for the one, but at the same time several agents [1, 4]. laboratory diagnosis of infectious pneumonia of Therefore, the study of the etiology and swine caused by Mycoplasma, Pasteurella and pathogenesis of these diseases, the development of Bordetella»; allocation of mycoplasma and effective methods of treatment and prevention are of aholeplazm – in accordance with the «Guidelines on great economic importance in ensuring the country's the isolation, cultivation and identification of food animal population. mycoplasmas and ureaplasma aholeplazm»; The main objective of this work was to study the Enterobacteriaceae – taking into account the «Manual etiological structure of the mass of young and gastro on the bacteriological diagnosis of colibacillosis diseases with respiratory syndrome.. farm, game animals and birds»; serological typing of Material and methods of research. Salmonella and Escherichia PA with O and The studies were performed in 1985-2007 years in H-agglutinating Salmonella syvorot kami the GNU Krasnodar NIVI, All-Russian Scientific manufactured Krasnodar biofabrika and O-coli Research Institute of Veterinary Pathology, serum-mi, made Armavir biofabrika – according to Pharmacology and Therapeutics research according to the «Manual on the use of agglutination O-coli sera» the thematic plan of the State Scientific and Technical and «guidelines for the use of Salmonella Program 0.51.09 (№ state. Registration 01.860113283, monoretseptornyh O and H-aglyutiniruyuschih 01.860113285) and branch scientific and technical adsorbed sera for the identification of Salmonella». 0.SKH.67 program (number of state. registration For detection of antibodies to pathogens of viral 01860113274), and when the research plan FGOU and chlamydial Institute infections using standard Belgorod State Agricultural Academy on R & D plan. biofabrichnye antigens: in the RSK at Hla-midioz Etiology gastroenteritis and pneumonia animals and adenoviral infection – Odessa venture for the studied com-plex, on the basis of epizootic, clinical, production of bacterial preparations in HAI influenza pathological data, bacteriological results, serology, and parainfluenza – Leningrad Scientific Research hematology, immunobiochemical research methods. Institute of Vaccines and Serums and Sverdlovsk To characterize the clinical condition of the animals Institute of viral vaccines for enterovirus and was measured body temperature (rectal) was coronavirus in the RN-strains of viruses produced in determined by pulse rate and respiration character Ukrainian and NIVI VGNKI, and for Phragmites – nasal secretions and stool. Before each experiment erythrocyte diagnostic kit VIEV. Diagnosticums used for preliminary morphological and bacteriological according to the supplied instructions to them. examinations were carried out on the slaughter of two To study the physiological and biochemical patients with gastroenteritis and Pneumatic- changes in the body of farm animals for moniyami pig, chicken, calf and lamb. In addition, gastroenteritis and pneumonia young farm animals, before and after the use of drugs from 3-5 animals in as well as the impact of technological factors each group were taking nasal discharge for the (transportation and regrouping) the emergence and isolation and identification of microorganisms. spread of gastroenteritis and pneumonia before stress Tampons with a material placed in tubes with exposure, and after 1, 10, 15, 30, 35 and 60 days mycoplasma growth medium. Sowing was carried from the blood vessels taking blood, which examined out on the BCH IPA with 5% sheep blood, chocolate the content of red blood cells and white blood cells in agar agar kazeinougolny (AMC), Endo agar, the particle counter Coulter counters (France), Wednesday Kitt Tarotstsi modified Wednesday hemoglobin – gemometrom Sali and VIEV with inhibitors to isolate mycoplasmas and gemoglobintsianidnym method hematocrit – on spiral aholeplazm. Cultures were incubated in an oven at centrifuge MPV-310 (Poland), erythrocyte 370°C. After 18-24 hours of incubation of culture- sedimentation rate – for Panchenkova, leukogram – studied biochemical, morphological and tinctorial by counting 200 cells stained by Giemsa method, a properties of isolated cultures. The pathogenicity of calculation of the percentage of each type of the bacteria except mycoplasma and aholeplazm bactericidal activity of blood serum – determined on white mice. Specific accessory O.V. Smirnovoy method and T.A. Kuzminoy isolated bacteria was adjusted using the determinant complementary activity – by O.V. Buharinu and D. Bergey and determinant zoopatogennyh N.V.Vasilevu (1974), lysozyme activity – for microorganisms edited by MA Sidorova. Typing K.A. Kagramanovoy and Z.V. Ermolevoy, the

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Zuev N.P., Bukhanov V.D., Vezentsev A.I., Sokolovskiy P.V., Khmirov A.V., Zueva E.N., Salashnaya E.А., Mihaylyukova M.O. The etiological structure of mass diseases with young gastro and 78 respiratory syndromeo. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 75-85. phagocytic activity of leukocytes, the phagocytic - Piglets – streptococci isolated from the blood index and phagocytic number – on V.S. Gostevu, of the heart; liver – Salma Nelly and unidentified S.I. Plyaschenko, V.G.Sidorov, total protein content – microflora; mesenteric lymph nodes – pathogenic refractometric, protein fractions sy-reversible blood – salmonella; by Olla and McCord in Karpyuk modification, - Calves – from the heart – Pasteurella; liver – quantitative determination of immunoglobulin classes – salmonella; Kidney – Escherichia; spleen – radial precipitation reaction on Mancini, the content salmonella, staphylococcus, Proteus; total immunoglobulins – A.D. Mak by Yuan et al. - Lambs – from the heart pathogenic Pasteurella; Antimicrobial activity tilozinsoderzhaschih Kidney – Salmonella; spleen – staphylococci; drugs against reference strains of mycoplasma, - Chickens – out of blood – negative aholeplazm and field cultures of E. coli, Pasteurella staphylococci; liver – salmonella, Escherichia and Staphylococcus aureus was investigated in liquid untyped and flora; mesenteric lymph nodes – and solid culture media using an indicator 2,3,5 – salmonella. triphenyltetrazolium chloride determination of Bacteriological examination of nasal secretions bactericidal and bacteriostatic action of drugs. with pneumonia animal was isolated and identified MBtsK determined by plating of the last two or three the following microorganisms: tubes in which no growth was observed. - From pigs – E. coli and Salmonella, Pharmaceutical research conducted in Staphylococcus and Streptococcus, Pasteurella and accordance with the dosage form technology and the Klebsiella, Bacillus subtilis. Pathogenicity of them requirements of the State Pharmacopoeia 13. were the first five generations; Designed 2 tilozinsoderzhaschih powdered drug. - Calves – Escherichia, Salmonella, At the beginning, middle and end of the Staphylococcus, Streptococcus, paste-Rell having experiments in the blood and serum were determined pathogenic and non-pathogenic Bacillus subtilis. Of above morphological and immunobiohimicheskie the 80% of the samples were not differentiable indicators, and in calves, piglets, lambs and chickens, microflora isolated, which had 50% of pathogenic in addition, the main carbohydrate, lipid, mineral and properties; protein metabolism. - From lamb – in 20% of cases, Escherichia, The resulting material was digitally processed Salmonella, Pasteurella, Streptococcus, Bacillus using mathematical methods of mathematical subtilis, 60% of unidentified microflora. The first four statistics, taken in Biology and Medicine (Microsoft kinds of pathogenic microorganisms possess properties Excel 97 application). in 20% of cases and untyped microflora 30%. Results and discussion. Bacteriological examination of pathological To determine the role of the microflora in the material (affected area of lung, bronchial and etiology and pathogenesis of calves diseases mediastinal lymph nodes, parenchymal organs) of performed bacteriology feces, nasal secretions of those killed for diagnostic purposes pneumonia patients with gastroenteritis and pneumonia in calves, patients piglets, lambs, calves, chickens and various lambs, piglets and chicks (5 animals of each species) flora was highlighted. From the lungs of pigs with and parenchymal organs from dead diagnostic pneumonia isolated Escherichia, Salmonella, purposes sick animals (n = 5). Pasteurella, Staphylococcus, Streptococcus. From the stool of patients with gastroenteritis Salmonella, Pasteurella and streptococci possess animal was isolated and identified the following pathogenic properties in 100% of cases, and microorganisms: Escherichia and Staphylococci 50; calves – - From pigs – enteropathogenic Escherichia, Escherichia, Salmonella, Pasteurella, Streptococcus. Salmonella, Staphylococcus, Streptococcus, Their pathogenicity was identified in 100, 100, 60 Enterobacter; and 50%, respectively. - Calves – pathogenic Escherichia, Salmonella, From the lungs of patients with pneumonia Proteus, Staphylococcus, Streptococcus, lambs were isolated and iden Rowan Escherichia - Lambs – pathogenic Escherichia, Salmonella, (pathogenic), Salmonella (50% of the pathogens); Staphylococcus and paste-Rell; streptococci (pathogenic). From pneumonic lungs - Chickens – pathogenic Escherichia, modified chickens isolated Escherichia, Salmonella Salmonella, and Staphylococcus. and Streptococcus spp. From parenchymal organs of patients with Studies show that for gastroenteritis and gastroenteritis: pneumonia young farm animals microflora feces, nasal secretions, and parenchymal organs represented

Staphylococci Streptococci Enterobacteriaceae Escherichia Salmonella Pasteurella Name of antibiotic В С V В С V В С V В С V В С V В С V 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Pneumonia pigs: p-5 p-8 p-3 p-7 p-4 Erythromycin 0 0 100 0 0 100 0 15 85 0 15 85 0 0 100 Chloramphenicol 0 100 0 0 25 75 0 33 67 0 71 29 0 75 25 Tetracycline 20 20 60 0 25 75 0 33 67 0 43 57 0 15 85 Penicillin 0 100 0 25 25 50 0 15 85 0 43 57 0 15 85 Streptomycin 20 80 0 0 37 63 0 25 75 0 14 86 0 15 85 Neomycin 0 20 80 0 37 63 0 33 67 0 14 86 0 15 85 Monomitsin 0 0 100 0 63 37 0 33 67 0 15 85 0 50 50 Polymyxin 0 0 100 0 0 100 0 50 50 0 15 85 0 85 15 Gentamicin 20 80 0 12 88 0 0 50 50 0 100 0 0 100 0 Pneumonia calves: p-5 p-4 p-3 p-4 p-4 Erythromycin 0 0 100 0 0 100 0 0 100 0 25 75 0 0 100 Chloramphenicol 0 100 0 0 25 75 33 33 33 0 50 50 0 75 25 Tetracycline 20 20 60 0 25 75 33 33 33 0 50 50 0 50 50 Penicillin 0 100 0 25 25 50 0 0 100 0 50 50 0 25 75 Streptomycin 20 80 0 0 25 75 0 0 100 0 50 50 0 15 85 Neomycin 0 20 80 25 25 50 0 0 100 25 25 50 0 15 85 Monomitsin 0 0 100 0 60 40 0 0 100 0 25 75 0 50 50 Polymyxin 0 0 100 0 0 100 0 100 0 0 100 0 0 85 15 Gentamicin 20 80 0 25 75 0 0 50 50 0 50 50 0 100 0 Gastroenteritis pigs: p-5 p-8 p-3 p-7 p-4 Erythromycin 0 0 100 0 0 100 0 0 100 0 15 85 0 0 100 Chloramphenicol 0 100 0 0 25 75 33 33 33 0 71 29 0 75 25 Tetracycline 20 20 60 0 25 75 33 33 33 0 42 58 0 50 50 Penicillin 0 100 0 25 25 50 0 0 100 0 42 58 0 25 75 Streptomycin 20 80 0 0 37 63 0 0 100 0 14 86 0 15 85 Neomycin 0 20 80 0 37 63 0 0 100 0 14 86 0 15 85 Monomitsin 0 0 100 0 63 37 0 33 67 0 15 85 0 50 50 Polymyxin 0 0 100 0 0 100 0 100 0 0 15 85 0 85 15 Gentamicin 20 80 0 12 88 0 0 100 0 0 100 50 0 100 0

Table 1 (continued) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Gastroenteritis calves: p-5 p-4 p-3 p-4 p-4 Erythromycin 0 0 100 0 0 100 0 0 100 0 25 75 0 0 100 Chloramphenicol 0 100 0 0 25 75 33 33 33 0 50 50 0 75 25 Tetracycline 20 20 60 0 25 75 33 33 33 0 50 50 0 50 50 Penicillin 0 100 0 25 50 25 0 0 100 0 50 50 0 15 85 Streptomycin 20 80 0 0 0 50 0 0 100 0 15 85 25 25 50 Neomycin 0 20 80 0 50 50 0 0 100 0 15 85 25 25 50 Monomitsin 0 0 100 0 50 50 0 33 67 0 15 85 0 50 50 Polymyxin 0 0 100 0 0 100 0 0 100 0 15 85 0 85 15 Gentamicin 20 80 0 25 75 0 0 100 0 0 100 50 0 100 0 Gastroenteritis lambs: p-3 p-3 p-4 p-3 Erythromycin 0 0 100 0 33 67 0 0 100 0 0 100 Chloramphenicol 0 33 67 0 67 33 0 75 25 33 33 33 Tetracycline 0 33 67 0 33 67 0 50 50 33 33 33 Penicillin 33 67 0 0 33 67 0 25 75 0 0 100 Streptomycin 0 33 67 0 15 85 0 15 85 0 0 100 Neomycin 0 33 67 0 33 67 0 25 75 0 0 100 Monomitsin 0 33 67 33 33 33 0 50 50 0 33 67 Polymyxin 0 0 100 33 67 0 0 80 20 0 0 100 Gentamicin 33 33 33 0 100 0 0 80 20 0 100 0 Pneumonia lambs: p-5 p-3 p-4 p-3 Erythromycin 0 20 80 0 33 67 0 25 75 0 0 100 Chloramphenicol 0 0 100 0 67 33 0 75 75 33 33 33 Tetracycline 20 20 60 0 33 67 0 50 50 33 33 33 Penicillin 50 50 0 0 33 67 0 25 75 33 33 33 Streptomycin 20 80 0 0 15 85 0 25 75 0 0 100 Neomycin 0 20 80 0 33 67 0 15 85 0 0 100 Monomitsin 0 20 80 33 33 33 0 50 50 0 33 67 Polymyxin 0 0 100 33 67 0 0 85 15 0 0 100 Gentamicin 20 80 0 0 100 0 0 100 0 0 100 0 Gastroenteritis chickens: p-4 p-3 p-4 Erythromycin 0 0 100 0 33 67 0 0 100 0 0 100 Chloramphenicol 0 25 75 0 67 33 0 75 25 Tetracycline 0 50 50 33 33 33 0 50 50 Penicillin 25 50 25 0 0 100 0 15 85 Streptomycin 0 75 25 0 0 100 0 15 85 Neomycin 0 75 25 0 0 100 0 25 75 Monomitsin 0 75 25 0 33 67 0 75 25 Polymyxin 0 0 100 0 0 100 0 85 15 Gentamicin 25 75 0 0 100 0 0 100 0 Thus, the microorganisms isolated from patients with gastroenteritis, pneumonia and animals presented as Gram-positive (S.aureus), and gram-negative microorganisms (E.coli, S.ch.suis, S.dublin etc.). However, it should

Based on these data the frequency of isolation of The transport of animals carried by road microorganisms and detection of antibodies to them, transport from supplier farms to inter-farm enterprise, pathogenicity and the sustainability of their antibiotic, it distant from each other in the second experiment on can be concluded about the etiological significance and 45 and 3rd of 15 kilometers. ehpizootologicheskaja primarily Pasteurella, Animals to stressors and after 15 and 30 days Salmonella, streptococci and staphylococci, after it was performed morphological, biochemical Escherichia, Klebsiella, and Bordetella. and immunological blood tests. Under the effect of To determine the degree of influence of various rearrangement (I experience) a decrease in serum stress factors on the occurrence and distribution of bactericidal activity through 4 chasa Studies 3 and young animals with diarrheal diseases and respiratory 4% (day 15 and 30), the phagocytic index and syndrome was conducted three series of experiments. erythrocyte content of 14% (15 days). In the first experiment we studied the effect of After transporting the animals marked decrease intra-rearrangements, in the second and third – in hemoglobin of 17 (I experience Day 60), 18 and 5% transportation and associated changing and feeding (2 experience, 20 and 30 days), red blood cells 16 and conditions on the natural resistance of the body of pigs. 13% (2 and 3 experiments, 20 days), leukocytes 75 %

(3 experiment), with an increase of 38% in the second 68,4%, скорость движения воздуха 0,16 и 0,12 м/с, experiment (20 days) decrease phagocytic number by содержание углекислого газа 0,18 и 0,15%, 22% (2 experience day 10), complementary activity by аммиака 7,6 и 7,71 мг/м3, бактериальная 33% (2 experience day 35), lysozyme 62% загрязненность воздуха 90,9 и 111,3 тыс. (2 experience day 35), 48 and 59% (3 experience, 10 микробных тел/м3. Заболеваемость поросят в них and 20 days), the bactericidal activity after 2 chasa соответственно составила 21,1 и 29,8%. research 33% (2 experience day 20) after 4 hours – Thus, all of us zoohygenic defined indicators, 6 and 6 watches – 4 % bactericidal strength – 13% the incidence of piglet rearing groups gastroenteritis (2 experience day 20) reduction of young neutrophils at and pneumonia are most correlated with the level of 150, 48 and 100% (2 experience, 10, 20, 35 days) with an bacterial contamination of indoor air. increase of the latter to 60 day 4 times, and 3 -em a conclusion can be drawn from the data that the experiment 3 and 2 times (days 10 and 20), a reduction of regrouping, transportation, poor detention conditions monocytes by 64 (2 experience day 10), segmented reduce the natural resistance of pigs in farms with neutrophils by 70% (3 experience 20den), lymphocytes, industrial technology: there is a decrease in red blood 9% (2 experience day 20) with an increase of 29% on the cells; (In which hemoglobin); complementary; last (3 experience day 20) and band neutrophils and lysozyme; serum bactericidal activity of leukocytes eosinophils by 88 and 15 (2 experience, 10 and 20 days). and opsonofagotsitarnoy. Ill 15 (43%) In the second experiment gastroenteritis and pneumonia, of which fell 9 The clinical picture and hematology patients (60.0%), 3-em 12 (76.0%), fell 7 (50.0%) and 1-5 gastroenteritis and pneumonia ohm (25.0%), fell 2 (40.0%). The average daily Clinical and hematological parameters were weight gain were, respectively, 276.0, 230.0, and studied in calves, piglets, lambs and chickens with 243.0 g. При исследовании зоогигиенических diarrheal and respiratory syndrome. The results of the показателей помещений установлено, что в 1-ом blood tests and patients gastroenteritmi pneumonia of и 2-ом корпусах температура воздуха равнялась pigs and calves are presented in Tables 4 and Table 5. 20,3 и 21,7 С, относительная влажность 68,1 и Table 4 Morphological and immunobiohimicheskie gastroenteritis patients blood counts and pneumonia of pigs (n = 15) and calves (n = 10)

Pneumonia Gastroenteritis Index piglets calves piglets calves Hemoglobin (g / L) 73,0+3,63 118,4+4,65 10,2+0,40 122,4+8,81 Leukocytes (109 / L) 16,0+1,39 8,2+0,41 17,9+2,40 5,8+0,54 Erythrocytes (1012 / L) 5,3+0,23 7,7+0,25 4,9+0,30 5,4+0,29 Neutrophils (%) Young 0,2+0,09 0,2+0,30 0,1+0,55 0,2+0,50 Stab 3,3+1,02 1,8+0,35 19,7+1,40 2,0+0,99 Segmented 29,5+4,11 17,6+0,93 17,8+0,60 18,0+4,74 Eosinophils (%) 3,9+2,20 2,5+0,60 3,7+0,35 1,6+0,50 Monocytes (%) 1,7+0,38 1,4+0,74 2,0+0,50 1,5+0,30 Lymphocytes (%) 61,4+4,59 69,8+6,08 62,5+0,30 70,5+0,50 Fal (%) 96,0+1,53 95,0+0,50 96,1+0,60 96,2+0,38 The FF 11,0+1,86 12,0+1,85 11,4+0,92 11,5+1,75- Phi 11,46+0,77 11,57+0,78 11,87+0,87- 11,43+0,47 Complementary 21,7+0,92 20,3+0,82- 28,4+11,90 22,7+0,83- Activity (%) 14,3+2,33 12,1+1,43- 7,1+0,40 12,3+1,43- Lysozyme activity (pg / ml) 19,0+19,15 17,0+1,25 17,0+8,15 17,7+1,15 Bactericidal. Assets. (%) 90,5+ 3,81 91,5+1,11 89,5+1,71 88,5+2,81 After 2 h 95,9+ 0,85 94,9+1,88 94,9+1,87 93,9+3,78 After 4 hours 6 hours 39,44+6,13 58,00+4,39 46,5+4,29 47,4+3,38 Protein fraction 12,90+4,11 4,17+0,99 14,2+2,04 12,1+1,04 Albumin (%) 22,77+3,34 11,21+0,96 13,0+1,05 15,2+1,25 Alpha-globulins (%) 24,88+4,02 26,16+4,61 24,6+2,20 21,7+1,20 Beta-globulins (%) - 6,9+0,50 5,62+3,47 7,2+3,40 7,4+1,43

Table 5 Features of clinical manifestations of gastroenteritis and blood parameters of calves at different ages Age, days Index 10-15 20-25 30-35 Temperature, ° C 38,1±0,1 38,5± 0,2 38,9±0,1 Pulse beats / min 116,5±7,8 128,6±8,2 104,4±3,9 Breath dyh.dvizheny / min 38,1±1,2 45,8±2,8 41,3±2,5 Hematocrit% 39,2±2,3 50,1±3,5 48,4±2,2 Hemoglobin, g / l 103,3±3,7 112,4±4,7 117,4±3,2 Red blood cells, mln / mm3 7,96±0,2 8,10±0,48 7,92±0,34 White blood cells, thousand / mm3 16,15±0,65 18,88±1,78 17,15±1,09 Neutrophils: Young 0,40± 0,02 0,70± 0,06 0,27± 0,02 Stab 6,4± 0,1 7,64± 1,31 8,77 ±1,15 Segmented 32,5± 1,89 35,3± 7,65 32,7 ±7,11 Lymphocytes 57,27± 5,8 51,31± 4,5 54,23± 5,5 Monocytes 2,76± 1,03 2,57± 0,55 2,43± 0,46 Eosinophils 0,77± 0,15 1,45± 0,41 0,72± 0,09 Basophils 0 0 0 Total protein, g / l 57,8± 1,61 53,8± 0,64 59,43± 0,73 Albumin,% 53,8± 0,82 53,8± 0,46 57,4± 0,46 Alpha globulins 12,99 ±1,7 17,981± 2,31 17,45± 1,46 Beta-globulins 17,84± 1,3 16,35± 0,86 15,63 ±1,2 Gamma globulins 15,21± 0,6 14,51± 1,04 12,51± 0,9 Glucose mol / l 2,89± 0,24 2,74± 0,17 2,69 ±0,14 Alkaline phosphatase, mol / tsp 1,45± 0,21 1,21± 0,45 0,85± 0,13 The performed studies of peripheral blood in Escherichia, Klebsiella, and Bordetella. patients with pneumonia and gastroenteritis of pigs CONCLUSION and calves found reduction of red blood cells and 1. Widespread gastro-intestinal and respiratory hemoglobin by increasing the number of white blood diseases (25-76%) in large livestock farms due to the cells and gamma-globulins (Tables 4 and 5). impact on the body of animals and birds of many Results of serum studies in RA with Salmonella, technological-ray stress factors (transportation, esherihioznymi, pasteurellosis, staphylococcal and rearrangement, crowding content, increased bordetelleznymi in HI with influenza virus antigens, contamination of the premises by microorganisms, Phragmites and pH on entero- and coronaviruses piglets and others.) , reducing the natural resistance of the gastroenteritis patients are presented in Table 10. organism, pathogenic and opportunistic pathogenic From Table 6 shows that in the middle (day 15) microorganisms, both individually and in various and the end of the study (day 30) there was an associations. increase in antibody titers to the antigens of 2. In the etiological structure of gastrointestinal Salmonella and esherihioznomu that indicates an diseases often take part: piglets St. aureus, E. coli. increase in the circulation of pathogens in pigs. S.ch. suis, Streptococcus pyogenes and pathogenic Thus, clinical, pathologoanatomic, haematological, genus Enterobacter; calves: St.aureus, E. coli. bacteriological and serological studies have established S. dublin, Streptococcus pyogenes, and Proteus that in the etiology of gastroenteritis and pneumonia of vulgaris; lambs: St.aureus, E. coli, S. dublin, pigs, calves and lambs in industrial farms participate P. multocida; chickens: E. coli. S.gallinarum, bacteria associations, under the action of which St.aureus. increases leukocyte content and gamma-globulin, a shift The causes of respiratory disease are: piglets – leukogram left core It decreases albumin and alpha- St. aureus, E. coli. S.ch. suis, Streptococcus globulins. pyogenes, P. multocida; calves – St.aureus, E. coli. The data about the frequency of isolation of S. dublin, Streptococcus pyogenes, P. multocida, microorganisms and the detection of antibodies to B.bronchiseptica; lambs – St.aureus, E. coli. them, pathogenicity and resistance of bacteria to S. dublin, B.bronchiseptica. antibiotics indicate the etiological role of Pasteurella, 3. Isolated microflora in the gastro-intestinal and Salmonella, streptococci and staphylococci, respiratory diseases of animals slabochuvstvitelna

REFERENCES 6. Rban V.P., Rudakov V.V., Karpenko L.Yu. 1. Zuev N.P., Shvecov N.N., Naumov M.M. Effect Effective of timogen in the prevention of gastrointestinal of technological stress factors on the incidence of diseases of pigs. Veterinary science. №10 (1991): 59-60. pneumonia and gastroenteritis of pigs. Vestnik of Kursk (In Russian) [Dissertcat][Abstract] State Agricultural Academy. №6 (2014): 70-71. (In 7. Zuev S.N., Martynova A.V., Buhanov V.D. Russian) [eLIBRARY] [AGRIS] Efficacy of combined combined with preparations 2. Mlynek, J. Effect of external factors before containing montmorillonite sorbents forswinedysentery. slaughter on meat quality of pigs / J. Mlynek , I. Imrich, E. Proceedings of Voronezh State University. Series: Mlyneková. Research in Pig Breeding. Vol.6, №2 (2012): Chemistry. Biology. Pharmacy. №2 (2013): 91-95. 41-45 [AGRIS] [Full text] (In Russian) [eLIBRARY] 3. Anisimova E.I., Gosteva E.R., Batargaliev A.S. 8. Mihajlov M.V., Potekhin K.I. Ways to improve Evaluation of cows of different genotypes for disease and stress resistance of poultry during their transplant. stress factor resistance, and general resistance. Materials Collection of Scientific Papers All-Russian Research Intern. scientific-practical. Conf. "Ways of extending the Institute of sheep and goat breeding. Vol.6, №2 (2013): product. Life moloch.h cows on the basis of optimization of 80-84. (In Russian) [eLIBRARY] breeding and feeding technologies (28-29 May, 2015): 9. Terekhov V.I., Ivanov A.V. The species 9-11. (In Russian) [eLIBRARY] [AGRIS] composition of bacteria isolated from pigs with acute 4. Marois C., Cariolet R., Morvan H., Kobisch M. intestinal diseases. Kuban Veterinary. №3 (2011): 23-25. Transmission of pathogenic respiratory bacteria to (In Russian) [Full text] specific pathogen free pigs at slaughter. Vet 10. Skorikov A.V., Malysheva T.V., Terekhov V.I. Microbiol. 129(3-4) (2008): 325-332. [PubMed] [AGRIS] Influence gidrogemola on natural resistance of pigs and 5. Zuev N.P., Shvecov N.N., Naumov M.M., et al. their incidence ehsherihiozom. Kuban Veterinary. Immune Biochemical parameters of lambs at technological №1(2013): 7-9. (In Russian) [Full text] stresses. Vestnik of Kursk State Agricultural Academy. №4 11. Terekhov V.I., Kravchenko V.M., Kramar P.V. (2014): 70-71. (In Russian) [Cyberlinka][Full text] Pathochemical and pathological changes in an organism of cows at nekrobakterioze. Kuban Veterinary. №3 (2011): 11-13. (In Russian) [Full text]

Рус. Eng.

PHARMACOLOGYCAL REVIEWS

UDC: 615.213 DOI: 10.18413/2500-235X -2016-2-3-86-94

Avdeeva N.V.1 THE POSSIBILITY OF ADMINISTRATION OF GLUTAMATE Nikitina V.A. 2 RECEPTORS ANTAGONISTS IN THE TREATMENT OF PARKINSON'S Kochkarova I.S.3 DISEASE Litvinova A.S. 4

1) PhD of Medical Sciences, Assistant of the Department of pharmacology, Belgorod State National Research University 85 Pobedy St., Belgorod, 308015, Russia, e-mail: [email protected] 2) 4 years student of medical and pediatric faculty of Medical University of NRU BelSU, 85 Pobedy St., Belgorod, 308015, Russia. e-mail: 999601@ bsu.edu.ru 3) Researcher at the Center of preclinical and clinical studies of the Belgorod State National Research University 85 Pobedy St., Belgorod, 308015, Russian Federation, e-mail: [email protected] 4) 6 years student of medical and pediatric faculty, Federal Autonomous Educational Institution of Higher Education Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, e-mail: [email protected]

ABSTRACT Parkinson's disease is the slow-progressing chronic neurodegenerative disease. It caused by the progressive destruction and death of neurons that produce the neurotransmitter dopamine, primarily in the substantia nigra and also in other parts of the Central nervous system. Insufficient production of dopamine leads to the activating influence of the basal ganglia to the cerebral cortex. Guiding symptoms are muscle rigidity, hypokinesia, tremor, postural instability. Modern medicine has not yet found methods of curing disease, however, the existing methods of conservative and surgical treatment significantly improve the patient’s quality of life and slow the progression of the disease. There is an assumption of the key role of glutamate receptors excessive activation in the pathogenesis of Parkinson's disease. It can be expected that glutamate receptors may be a new therapeutic target in the treatment of this pathology. The study of glutamate receptors blockers is an important task in the search for new pharmacological agents in the treatment of Parkinson's disease. Experiments on animal models suggest the change in the activity of these receptors can facilitate the primary motor symptoms of Parkinson's disease and also side effects caused by the levodopa replacement therapy. AMPA- NMDA receptors antagonists have shown the ability to reverse motor symptoms and levodopa-induced dyskinesia in preclinical models of Parkinson's disease. Metabotropic glutamate receptors antagonists are even more promising in the treatment of Parkinson's disease due to more "accurate" work in the synapse. These drugs also reduce motor deficits, as well as protecting the patient from neurodegeneration. Thus glutamate receptors are a promising target for the development of new pharmacological treatments of Parkinson's disease. Key words: Parkinson's disease, glutamate receptors, basal ganglia, NMDA receptors, AMPA receptors, metabotropic glutamate receptors, levodopa-induced dyskinesia.

INTRODUCTION worldwide. Recently, in connection with the increase in First the Parkinson's disease (PD) was described life expectancy and improvement of diagnostic by London physician James Parkinson in 1817 in “An capabilities of medicine, there is PD some incidence Essay on the shaking palsy”, therefore French rate. Guiding symptoms of PD are muscular rigidity, neurologist Jean Charcot proposed to name the disease hypokinesia, tremor, postural instability, and sleep after Parkinson [1]. This disease is a slow-progressing disturbance and cognitive defects [2]. Movement chronic neurodegenerative disease, which affects about disorders, which are major clinical implications, are 3% of the population aged 55 years and older caused by a sharp fall in synthesis of dopamine in the RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Avdeeva N.V., Nikitina V.A., Kochkarova I.S., Litvinova A.S. The possibility of administration of glutamate receptors antagonists in the treatment of parkinson's 87 disease. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 86-94. substantia nigra and striatum. Normally the amount of and pathological neurotransmission that is observed in dopamine in these formations is hundreds times higher parkinsonian brain. than the concentration in other brain structures, PATHOPHYSIOLOGIC MECHANISMS suggesting an important role of dopamine transmission OF PARKINSON'S DISEASE in the activities extra pyramidal system. Loss of Clinical symptoms of PD (akinesia, rigidity, dopamine innervation of the neostriatum plays a crucial tremor) are caused by a sharp fall in synthesis of role in the control of motor activity [3]. Today dopamine in the substantia nigra and striatum. The dopamine replacement therapy is background at the synthesis of dopamine takes place in the nerve cell unwrapped phase of PD. Because dopamine does not bodies of the nigrostriatal system. Here the mediator is pass through the blood-brain barrier (BBB), its formed in the form of small vesicles, which are predecessor levodopa was synthesized. Levodopa transported by the axonal transport to the presynaptic passes through the BBB and metabolized in the brain membane. Under the influence of nervous impulse under the action of dopamine decarboxylase to dopamine release in the synaptic cleft and impacting on dopamine. Therapy of early disease begins with the receptors of the postsynaptic membrane with an administration of dopamine receptor agonists [4]. effect of its depolarization. It is assumed that about 80% Dopamine receptor agonists due to its chemical of dopamine is absorbed in the presynaptic terminals on properties directly stimulate dopamine receptors, reuptake mechanism or are inactivated by COMT or reproducing the effect of dopamine. Historically, they monoamine oxidase type B (MAO-B). Such disorders were first administrated to patients with PD as add-on of intrinsic cellular metabolism as a violation of therapy to levodopa, but later it was found that in the mitochondrial respiration and energy failure of the early disease of dopamine receptor agonists have neuron, the strengthening of free-radical oxidation with comparable effect with levodopa. In most patients these the formation of aggressive peroxides, excessive drugs provide of motor symptomatic relief for several accumulation of free ions of Ca2+, increased activity of years. However, chronic exposure of these drugs leads glutamate excitotoxin are a key player in the factors to the emergence of motor fluctuations and drug- contributing to degeneration and apoptosis of neurons. induced dyskinesia that militate against their long-term L-glutamate is the major excitatory effectiveness [4, 5]. Adjuvant treatment to neurotransmitter in the brain of animals. Glutamate is monoaminooxidase inhibitors, anticholinergic drug and found in all departments of a CNS, as it is not only a COMT-inhibitors limited improves the effectiveness neurotransmitter but also a precursor of other amino and acceptability of the treatment to levodopa. acids. Bodies of glutamatergic neurons located in the However, more effective therapy for unwrapped cerebral cortex, olfactory bulbs, hippocampus, phase of motor symptoms of PD has not yet been substantia nigra, cerebellum, retina. Glutamatergic found [4]. Besides, used at present pharmacological synapses located in the tonsil of cerebellum, the agents (mainly drugs containing levodopa) do not striatum, granule neurons of the cerebellum. slow down the continued degeneration of Glutamate is dispensable amino acid, it doesn’t pass dopaminergic neurons, which functional activity through the BBB, does not arrive to the brain through causes the conversion of levodopa into dopamine by the blood. The synthesis is carried out in the brain, action of dopamine decarboxylase and requires a mainly intraneural. In addition to the main role of the gradual increase of the dose level to achieve the excitatory neurotransmitter, glutamate can cause effect. This ultimately leads to acerbation of PD. damage and death of dopaminergic neurons. This Sameness of therapeutic agents for the treatment of action of glutamate on neurons is designated by the PD largely associated with a deficit in knowledge on term excitotoxicity [6]. the cellular processes underlying the degeneration of Glutamate-mediated activity detected in almost dopaminergic neurons of the substantia nigra. The all brain structures. The greatest number of binding limitation of the pharmacological treatment of PD sites is located in the cerebral cortex, hippocampus, insists on the importance of non-dopaminergic striatum, mesencephalon and hypothalamus. therapeutic strategies for the treatment of the disease Glutamate receptors are divided into ionotropic and symptoms. Progress in the understanding of the metabotropic. There are several subtypes of anatomy and function of basal ganglia has given the glutamate receptors. Modern classification of opportunity to develop new strategies for treatment and ionotropic receptors based on their different slowing the progression of PD [3]. So the glutamate sensitivity to the action of NMDA, AMPA, kainic receptors have been proposed as promising therapeutic acid and quisqualic acid. There are two groups of target for treatment of PD, since they transmit fast receptors: NMDA and non-NMDA (it is divided into excitatory signals in fiber connection. Pharmacological AMPA and kainate). manipulation of these receptors can change both normal Glutamate excitotoxicity is mediated by NMDA receptors. After glutamate occupancies these RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Avdeeva N.V., Nikitina V.A., Kochkarova I.S., Litvinova A.S. The possibility of administration of glutamate receptors antagonists in the treatment of parkinson's 88 disease. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 86-94. receptors transport channels of neural membranes which in turn leads to increasing lipid open and the access of glutamate to the neuron peroxygenation, apoptosis and neuron death. enables. Extensive binding of glutamate by NMDA BASAL GANGLIA AND PARKINSON'S receptors leads to increasing of current Ca2+ into the DISEASE neuron through the channels of NMDA receptors. All the movements performed by man, are Due to the fact that the increasing of current Ca2+ is controlled by the Central CNS, which includes the brain one of the leading mechanisms of the neuron death, it and spinal cord. It is very difficult organized system that can be assumed that the mechanism of glutamate meets virtually all that happens in the body. The role of excitotoxicity in PD is associated with massive higher nervous activity belongs to the cerebral cortex. entrance of Ca2+ in dopaminergic neurons of the Once man only to think about some intentional action, substantia nigra [7]. There is a hypothesis about the the cortex is already leading to readiness all the systems integral role of hyperactivation of the glutamatergic that are responsible for this action. One of such system structures of Luys body in the pathogenesis of PD. is the so-called basal ganglia. The basal ganglia are an Luys body performs the relateral tell of corticonigral auxiliary motor system. It does not work independently, glutamatergic influences [8]. The hypothesis but in the close association with the cerebral cortex. The postulates the importance of glutamate damage basal ganglia are involved in performing complex neurons of substantia nigra, starting from the pre- movements such as writing, drawing, walking, kicking existing disease. the ball into the goal, lace shoes etc. It is responsible for NMDA receptors consist of five subunits that driving speed and for the accuracy and quality of are complexes of glycoprotein and lipids. The movements. Such movements are self-produced, so NMDA receptor is a receptor-ionophore complex, initially arising in the cerebral cortex. Hence, which includes 1) specific binding site of the information on these movements comes into the basal mediator (L-glutamic acid); 2) regulatory or ganglia that determine which muscles will be coactivativation specific binding site of glycine; participate in them and how each of the muscles must 3) allosteric modulatory sites located on the be stressed that the movements were as precise and membrane (polyamine) and ion channel focused. (phencyclidine binding sites, divalent cation and The basal ganglia consist of the nucleus caudatus voltage-gated Mg-binding site). Mg2+ ions selectively and neostriatum, globus pallidus internus and externus, block the activity of receptors at high hyper pars reticulate and pars compacta of substantia nigra polarization or depolarization. Glycine enhances the and the Luys body. Neostriatum is the main part of the responses of NMDA receptor by increasing the basal ganglia, which receives information from the opening frequency of the channel. In the absence of cortex in accordance with somatotopic organization, the glycine receptor is not activated by L-glutamate and intralaminar nucleus of thalamus. The information [6, 9]. It is known that NMDA receptors play an comes from the basal ganglia, mainly through the important role in learning and memory. Activation of globus pallidus internus and pars reticulate of the glutamate receptors is considered a universal substantia nigra, passes through the thalamus (ventral pathochemical process, causing damage to nerve intermediate nucleus and nucleus ventralis anterior), cells in a variety of pathological states [4]. The which are projected to the premotor cortex, the neurotransmitter role of the glutamic acid lies in the supplementary motor area and prefrontal cortex. The ability to cause membrane depolarization of the basal ganglia form several neuronal connections. There 2 neuron and therefore increase the conductivity of Ca + is striato-nigro-striatal connection, the end body of ions. Mediator function is in close connection with which is dopaminergic. This path degenerates in PD the exchange of Ca2+ that gives the opportunity to with the formation of cell inclusions Lewy bodies. talk about the glutamate-calcium cascade. The There is also the connection between globus pallidus glutamate action is manifested through the operation externus and Luys body, which is then projected in the with specific neuroreceptors with protein structure globus pallidus internus and pars reticulate of the and related in nature to cellular glycoproteins. substantia nigra. This path is by nature excitatory and Adverse of biochemical processes, observed in PD, monitors the level of pulsation of the inhibitory neurons triggers the glutamate-calcium cascade, leading to of the basal ganglia of thalamic areas. Despite the fact different changes in cellular metabolism caused that the structure of the basal ganglia there is some primarily by activation of the membrane degree of divergence and convergence relations, their permeability and the m7ovement of Ca2+ inside the projections form a parallel path, which in its most cell [9]. This phenomenon contributes to the adverse simplified form are classified on the motor way through of fundamental cellular processes, uncoupling the putamen, and non-motor way through the caudate of oxidative phosphorylation, free radical formation, nucleus.

The basal ganglia transmit their impulses with anterior and ventral intermediate nucleus of thalamus the help of special chemical compounds, called leads to a decrease in the activation, patients find it neurotransmitters. Their number and mechanism of difficult to start movement, because they have violation action (exciting or inhibiting) determine how muscles of activation of the supplementary motor area, while will work. Dopamine is main inhibitory explaining the rigidity and tremor more difficult. In the neurotransmitter which is secreted in the caudate early disease the condition of patients may improve on nucleus and the putamen, therefore the destruction of the top of administered therapy by the antiparkinsonian dopaminergic neurons in the substantia nigra a drugs. patient with PD theoretically could lead to Despite extensive studies, the cellular mechanisms hyperactivity of the caudate nucleus and the putamen. underlying the degeneration of dopaminergic neurons It may be accompanied by the permanent presence of of the mesencephalon in PD are understudied. Studies excitatory signals to the corticospinal motor control on genetic and toxic animal models suggest the system. Excessive excitation of many or all muscles possibility that oxidative stress, mitochondrial of the body leads to development of their rigidity. dysfunction, aberrant processing of proteins by the After the loss of inhibition in the feedback path, the ubiquitin-proteosome proteolytic system, inflammation excitement in some of this path might easily oscillate and activation of apoptosis play a role in the death of leading to tremor characteristic of PD. dopaminergic cells [10, 11]. More than 50-60% of dopaminergic nigrostriatal Simplified model of the basal ganglia motor neurocytes die before clinical implications of PD system provides an understanding of how indirectly (bradykinesia, increased muscle tone, resting tremor) degeneration of dopaminergic neurons of the substantia begins. There is an assumption of the existence of nigra is compensated by modulating synaptic multiple mechanisms by which brain cells may die transmission at other synapses within the basal ganglia, [2]. One of the mechanisms is the pathological can provide opportunities for non-dopaminergic accumulation of the protein alpha-synuclein that is therapeutic strategies. The striatum is the primary input associated with ubiquitin in the damaged cells. This to the basal ganglia. It receives excitation from multiple insoluble protein accumulates intracellular in neurons areas of the cerebral cortex, including primary motor forming inclusions called Lewy bodies. Lewy bodies cortex and other motor areas. initial manifest in the olfactory bulb, medulla TEST MODEL OF THE PARKINSON'S oblongata and the tegmentum of pons, and at this SYNDROME stage the disease is asymptomatic. During A problem for experimental studies of the progression of the disease, Lewy bodies manifest in pathogenesis of PD is the inability of biological the substantia nigra, areas of the mesencephalon and sampling from patients with this disorder, so there is basal forebrain, and in the last stage in the neocortex. practically no possibility of observing processes in These areas of the brain represent the main sites of living human cells in PD. There are well-known the neurons degeneration in PD; however, Lewy model of Parkinson's disease in vivo (in living bodies may not cause cell death, they can also carry a organisms) and in vitro ("in vitro"). Closest to the protective function. Other mechanisms of cell death processes of human cells are models of pathology in include proteasome and lysosomal systems vivo on animals, but when they are used there are dysfunction and reduced mitochondrial activity. The difficulties with results reproducibility, difficulty of accumulation of iron in the substantia nigra is usually the content of pure lines of animals and justify the registered in combination with protein inclusion. It use of models in vivo before the Ethics Committee. may be associated with oxidative stress, protein At the same time, in recent years use the models in aggregation and neuron death, but these mechanisms vitro are increasing. Thus, cell culture is a universal are understudied. The majority of patients have method for the study "physiological" and pathological cognitive defects, affective disorders and autonomic processes elucidate of the signal transfer mechanism, disturbances, which can relate to the damage of the gene regulation, cell proliferation and the mechanisms structures other than the nigrostriatal pathway. of their death. These models do not rule out in vivo Neurophysiologically patients with PD have an models, but it is a good supplement to them, allowing increase in the activity of neurons of the globus pallidus you to study the physiological processes and internus, which send excitation pulse to the Luys body mechanisms of pathogenesis of diseases, to understand as a result of loss inhibitory dopaminergic effects on the signal transfer mechanism, gene regulation, cell neostriatum. The increase of the deceleration pulse from proliferation and death. the globus pallidus internus (and, possibly, the pars It is not revealed to PD occurred naturally in other reticulate of the substantia nigra) to nucleus ventralis animal species than humans, although animal models

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Avdeeva N.V., Nikitina V.A., Kochkarova I.S., Litvinova A.S. The possibility of administration of glutamate receptors antagonists in the treatment of parkinson's 90 disease. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 86-94. which show some manifestations of the disease are used disrupting of mitochondrial function. In primates, in research. There are genetic models that are based on MPTP administration closely replicates the behavioral unique gene mutations observed in nepotism PD. problems of PD, and it is considered the best model for However, they are not able to recapitulate aspects of studying the antiparkinsonian effectiveness of new human disease, such as degeneration of dopaminergic drugs for the treatment of humans. Other models on the neurons, and appeared to be more useful for the study basis of toxins include the insecticide rotenone, the of heredity of PD and not for the evaluation of new herbicide paraquat and the fungicide maneb. Models therapeutic strategies [12]. Animal models of PD are a based on toxins are most commonly used on primates. means of assessing the ability of new drugs to reduce or Transgenic rodent models that replicate various aspects stop the motor symptoms and to slow progression of of PD have also been studied [16]. The use of the neurodegeneration associated with this condition. neurotoxin 6-hydroxydopamine (6-OHDA) produces a Evaluation of new therapeutic target for the model of Parkinson's disease in rats by targeting and symptomatic treatment of PD often depends on the destroying dopaminergic neurons in the nigrostriatal pharmacological and toxic models of PD in mice, rats pathway with administration in the substantia nigra. 6- and primates that are best suited to recreate the motor OHDA causes unilateral lesion of motor behavior of symptoms of the disease [13]. Pharmacological models rats, and new therapeutic agents can be evaluated for are used to explore the therapeutic potential of the treatment of these effects. Despite the fact that is no compounds, oriented on glutamate receptors, as they are animal model is not able to perfectly replicate human convenient and economical. Pharmacological agents disease, these models can be tools to assess the that induce the symptoms of PD include neuroplegics therapeutic potential of new targets and agents. such as the mixed D1\D2 dopamine receptor antagonist PRINCIPLES OF THERAPY haloperidol, which causes muscular rigidity, akinesia OF PARKINSON'S DISEASE and catalepsy. One more thing is reserpine, which Despite the stage of the disease, the modern causes profound akinesia in rodents [13]. Since there is approach to the treatment of PD involves two main a lack of standardization of these models by different strategies such as the search for agents that can slow, researchers, it is possible that this pharmacological delay or stop its progression (called neuroprotection) model will produce different results due to different and the creation of new, more effective drugs for methodologies. Therefore it is very important to give symptomatic treatment. The latter at the present time attention to the methods for the interpretation and is recognized the primary. Principles of drug therapy comparison of the results of these models. In addition, it of Parkinson's disease largely base on the concept of is important to keep in mind that parkinsonism which is dopaminergic deficit, and also on modern concepts induced by pharmacological agents in rodents are about the functioning of the dopaminergic synapse reversible and do not reproduce the morphological and the pathogenesis of neurodegenerative diseases. markers of disease, and therefore cannot simulate all In accordance with these principles therapy of PD aspects of the disease caused by the degeneration of involves the use of drugs that increase the synthesis dopaminergic neurons of the substantia nigra. Toxic of dopamine in the brain; stimulate the release of models of PD are useful for studying therapeutic dopamine from presynaptic terminals and inhibit its strategies to treat motor symptoms and neuroprotection reuptake of presynaptic elements; inhibit the and to study the mechanisms of the disease. The degradation (catabolism) of dopamine; stimulate the appearance of Parkinson's disease symptoms in a group postsynaptic dopamine receptors; prevent of of drug addicts in the 1980-ies who consumed a progressive neuronal death and slow the progression contaminated consignment of the synthetic opiate of the disease. The complex treatment of Parkinson's MPTP (dopaminergic neurotoxin 1-methyl-4-phenyl- disease should include antioxidants (for example, tetrahydropyridine), led to the fact that MPTP was natural vitamin E, not synthetic), exercise therapy. perceived as the agent that causes a parkinson's Because the disease is understudied, scientists still syndrome in primates, mice, and humans. MPTP is carry on polemics about possibility to slow the used to study the cellular mechanisms underlying the inevitable progression of the disease upon the cell death of dopaminergic system. MPTP can be existence of symptoms of PD. administrated systematically, but it needs to be Levodopa (precursor of dopamine) is a converted in the brain into an active metabolite 1- stereoisomer of dihydroxyphenylalanine (DOPA), Methyl-4-phenylpyridinium ion (MPP+) by which, as opposed to the dopamine, penetrates well monoamine oxidase. MPP+ is selectively transported into CNS. Under the action of the enzyme DOPA- into dopaminergic nerve termination with help from the decarboxylase levodopa is transformed into dopamine transporter and causing neurodegeneration by dopamine, thereby increasing its level in neostriatum.

The symptoms of Parkinson's disease disappear symptoms (tremor, bradykinesia, rigidity), however, within a short period of time. Levodopa is effective the exact mechanism by which this effect is achieved only constant concentration control in the body. With remains unclear. Unfortunately, surgery cannot be disease progression, and a decrease in the number of called widely available due to their relatively high cells of the neurons of the substantia nigra, its cost and a large number of contraindications for their effectiveness is sharply reduced. 97-99% levodopa conduct. The fact that the lesion of the Luys body transforms into dopamine in peripheral tissues, and globus pallidus with deep brain stimulation causing many side effects of the drug. With the aim improves the symptoms of Parkinson's disease, of reducing its number, levodopa is used in suggests that pharmacological drugs which are able combination with inhibitors of DOPA decarboxylase to reduce the activity of the Luys body can provide (carbidopa, benserazide) that doesn’t pass through significant relief of motor symptoms. the CNS. This combination metabolism of levodopa OVERVIEW OF TESTS OF DIFFERENT can occur only in the brain. The use of combinations TYPES OF GLUTAMATE RECEPTORS of levodopa and carbidopa ("Nakom", "Sinemet") ANTAGONISTS increases the likelihood of side effects from the CNS, In the last 20 years there have been many studies such as dyskinesia (80% of cases), anxiety, showing that glutamate receptors antagonists can be depression, delusions, hallucinations. used as drugs for the treatment of Parkinson's disease, MAO inhibitors (Selegiline, Rasagiline) as in combinations with levodopa and independently. metabolize dopamine, increasing its level in The following is the main conclusions and assumptions neostriatum. The use of these drugs with levodopa obtained during the testing of these drugs. can reduce the dose of the latter. On the basis of numerous studies it can be Tolcapone, entacapone are COMT inhibitors, an assumed antiparkinsonian effect of NMDA receptors enzyme that is responsible for the distribution of antagonists, since NMDA receptors mediate dopamine in the neurons. The administration of the glutamatergic excitation of neurons in the striatum combined precursor of dopamine leads to and Luys body, which is shown in various animal compensatory activation of this enzyme. The models (rats treated with haloperidol, monkeys treatment efficiency is lowered. Tolcapone and treated with MPTP) [7]. Receptor blockade may be entacapone inhibit COMT, allowing reducing the competitive and noncompetitive antagonists. dose of levodopa. Competitive antagonists of NMDA receptors (SDZ Dopamine receptor agonists are bromocriptine 220-581) directly block glutamate recognition site of (partial agonist of the dopamine receptors (D2)), the NMDA-receptor. Noncompetitive antagonists of lisuride (a derivative of ergot alkaloids), pergolide NMDA receptors (MK-801, dextrorphan, MSZ (dopamine receptors agonist (D1 and D2)). 2\579, CP-101606) connect the phencyclidine Drug that inhibits excitation of neostriatum recognition site of the NMDA receptors. It is known neurons by acetylcholine is trihexyphenidyl that these antagonists and antagonists of the glycine (Cyclodol) which is a muscarinic antagonist. Its site (MRZ 2\570, L-701324, (R)-HA-966)) revert effectiveness in PD is weaker than levodopa. It well catalepsy and muscular rigidity which are induced by eliminates tremor and muscular rigidity, but does not blockade of dopamine receptors in rats. Numerous affect bradykinesia. It is used in composition of studies show that NMDA receptors antagonists such complex therapy. as MK-801 and MRZ 2\579, also revert akinesia and Glutamate receptors antagonists (NMDA) are a other motor disorders in rodents, whereas antagonists relatively new group of drugs. Glutamate is of the glycine site of the NMDA-receptors is not exitotoxic transmitter in relation to pathways. Its effective in this model. Assume that the action on NMDA receptors induces the movement of antiparkinsonian effect of NMDA receptors Ca2+ inside the cell, which leads to a sharp increase antagonists consist in blockade of the activity of of stimulation and subsequent neuronal death in PD. these receptors in the striatum and the output nuclei Drugs that block glutamate receptors (amantadine, of the basal ganglia. Another possible mechanism by symmetrel) reduce toxic effects caused by which this effect is achieved is the reduction in the stimulation of NMDA receptors. release of acetylcholine from cholinergic neurons of Operative treatment of PD, which is aimed at the striatum. It is also interesting that some NMDA reducing the activity of the Luys body, and deep receptors antagonists can potentiate the effect of brain stimulation, has been very effective [14, 15]. levodopa suppressing motor fluctuations and Deep brain stimulation (Luys body or the globus dyskinesia that was shown in models of rodents and pallidus internus) significantly reduces motor primates, and consequently it can be most effective in

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Avdeeva N.V., Nikitina V.A., Kochkarova I.S., Litvinova A.S. The possibility of administration of glutamate receptors antagonists in the treatment of parkinson's 92 disease. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 86-94. combination with levodopa therapy. There is a further clinical studies using more selective drugs are perception that global inhibition of NMDA receptors very promising [7]. may lead to the development of serious side effects, AMPA receptors antagonists were also proposed such as psychosis, learning disability and disordered as potential therapeutic agents; however, preclinical motor function. This assumption leaves in doubt the studies have shown that they do not possess development prospect of NMDA receptors as new antiparkinsonian activity in monotherapy. For therapeutic target. Therefore testing of combinations example, systemic administration of NBQX and of NMDA receptor specific subunits has renewed GYKI 52466 does not prevent catalepsy induced by interest in this issue. NR2B is a subunit of NMDA haloperidol in rats. NBQX monotherapy has also not receptor in a large number is found in the striatum improved motor symptoms in rats induced by 6- and in nuclei of the basal ganglia, so it is assumed OHDA and MPTP affected primates. In addition that agents that selectively affect NMDA receptors GYKI 52466 has no effect on akinesia in rats with containing the NR2B subunit may have more specific monamine deficite, and does not intensify activities impact on the function of the receptor in areas of the of levodopa in the same model. However, other brain relevant to the pathophysiology of PD. Such studies have shown antiparkinsonian effect of AMPA agents have been developed and studied their receptors antagonists, independently and in antiparkinsonian effects in animal models. For combination with dopaminergic therapy. For example, traksoprodil (CP-101606) changes example, NBQX has set up muscular rigidity (but not catalepsy caused by haloperidol in rats and reducing akinesia) induced by reserpine in rats, and motor motor symptoms of Parkinson's disease in affected by deficite in MPTP affected primates. Also noted that MPTP monkeys. Another NMDA NR2B selective NBQX improves the ability of levodopa to set up receptor antagonist ifenprodil also reduces motor motor deficite in lesions of the substantia nigra in rats symptoms of PD in affected by MPTP monkeys. In and primates. It is assumed that the different results addition, these drugs improve the effectiveness of of the studies were due to the fact different methods levodopa that speaks about the clinical benefits of of induction of parkinsonism in animals (models with combination treatment. the use of neurotoxins has been more promising). At present amantadine is only used as the Preclinical studies of AMPA receptors NMDA receptors antagonist, which blocks the antagonists in the treatment of levodopa-induced noncompetitive NMDA-receptors in therapeutically dyskinesias gave more encouraging results for the effective concentrations. Amantadine stimulates potential use of these drugs in the treatment of PD. release of dopamine from presynaptic terminals, Noncompetitive AMPA receptors antagonist reduces the reuptake dopamine in the synapse, LY300164 (Talampanel) improves motor symptoms inhibits glutamatergic influence the frontal cortex on and reduces levodopa-induced dyskinesia in MPTP the striatum and has a distinct holinoblokirutm affected monkeys. Competitive AMPA receptors anticholinergic activity. As antagonist of glutamate antagonist – LY293558 in combination with receptors amantadine can implement its action at the levodopa reduces levodopa-induced dyskinesia in 6- level of the excitotoxic cascade [16, 17]. OHDA rats. Interestingly, combined blockade by low Although clinical benefit of amantadine (ineffective) doses of the AMPA and NMDA monotherapy is questionable, double-blind, placebo- receptors antagonists reduces levodopa-induced controlled study showed that amantadine reduces dyskinesia in rats and primates, suggesting the dyskinesia and motor fluctuations in patients treated by possibility of using low doses of glutamate receptors levodopa. These data suggest that further study NMDA antagonists to reduce dyskinesia [7]. receptors antagonists may be of utility to complex Metabotropic glutamate receptors (mGluR), in therapy with levodopa. Besides amantadine inhibits the contrast to the "fast" ionotropic, provide a slow development of dementia in patients with PD, response to glutamatergic signals, and also represent suggesting another positive effect of the blockade of a therapeutic target for the treatment of PD. NMDA receptors. Another NMDA receptors antagonist Metabotropic glutamate receptors consist of seven remacemide was also studied in patients with PD, both transmembrane domains connected to to G-proteins, as monotherapy and in complex with levodopa. which mediate most of the effects of these receptors However, despite its antiparkinsonian effect in rats and activation. Itself receptors consist of two subunits one primates, it has not shown the desired effect on people, of which binds glutamate. Metabotropic glutamate causing some doubts in the use of NMDA receptors receptors (mGluR) are divided into three groups antagonists in the symptomatic treatment of PD. Thus (mGluRI , mGluRII and mGluRIII ), although discovered eight genes that encode it (mGluRI

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Avdeeva N.V., Nikitina V.A., Kochkarova I.S., Litvinova A.S. The possibility of administration of glutamate receptors antagonists in the treatment of parkinson's 93 disease. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 86-94. includes mGluR1 and -5, mGluRII is mGluR2 and -3, mGluRIII agonist (L-AP4) reduces synaptic mGluRIII is mGluR4, -6, -7). Metabotropic transmission striato-pallidal and the hypothalamic- glutamate receptors are activated by lower nigral synapses. Also microdialysis studies have concentrations of glutamate than the main ionotropic shown that L-AP4 and L-SOP reduce the release of AMPA receptors. A group of mGluRI are usually GABA in the globus pallidus in rats. These data located on the postsynaptic membrane around the suggest that activation of mGluRIII can facilitate synaptic cleft. The effect of activation of these motor symptoms of PD, or by reducing the activity of receptors opposes the effect of dopamine and directly the indirect pathway, or by reducing the activity of stimulates the hyperactive nuclei in the Parkinsonian the nuclei in the hypothalamus. L-AP4 may be very brain [18]. Furthermore, its activation potentiates effective in the treatment of PD symptoms, because it currents of NMDA receptors in the striatum and Luys acts on the asymmetry of the forelegs 6-OHDA body. Antagonists of these receptors could have affected rats to the same extent as levodopa. antiparkinsonian effect by reducing the excitation of Intrapallidal administration of L-SOP facilitates the hyperactive basal ganglia. In accordance with this reserpine-induced akinesia in rats, which confirms hypothesis, several negative allosteric modulators of the hypothesis that the decrease in the activity of mGluR5 (MPTP and МТЕР) have antiparkinsonian these synapses can have therapeutic efficacy. effect on animal models. Interestingly, combined Glutamate receptors may be promising targets treatment by МРЕР and A2A adenosine receptors for treatment of neurological and psychiatric antagonists causes a noticeable reduction of akinesia complications associated with PD, such as anxiety, in 6-OHDA or reserpine affected rats. This suggests depression and cognitive defects. The mGluRII and the effectiveness of symptomatic therapy of PD with mGluR5 antagonists have anxiolytic action in the combination of mGluR5 antagonists and A2A preclinical models of anxiety, suggesting the adenosine receptors antagonists. A similar effect is possibility of their use for the treatment of both observed with the introduction of МРЕР and MK- motor and psychiatric symptoms of PD. Also 801 in 6-OHDA affected rats, which also indicates a mGluRIII, mGluR5 and mGluRII antagonists are possible antiparkinsonian effectiveness of effective in preclinical models of depression, simultaneous administration of NMDA receptors suggesting that they may be targets for treatment of antagonists and mGluR5 antagonists. Systemic depression in patients with PD. administration of MTEP to 6-OHDA affected rats CONCLUSION reduces levodopa-induced dyskinesias in animals for The main drugs in the treatment of PD are a long time treated with levodopa. These data levodopa and its derivatives, as well as the indicate that the possibility of mGluR5 antagonists phenothiazine type antipsychotics and anticholinergic may be useful for symptomatic treatment of PD in drugs. Unfortunately, they have quite a large number patients receiving long-term treatment by levodopa. of side effects, and over time, the dosage should be Despite the positive result in the study of increased, otherwise its cease to be effective. This antiparkinsonian effect of mGluR5 antagonists, the poses a lot of problems. The physician should mGluR1 antagonists (EMQMCM) failed to reduce determine the time of starting therapy and the group levodopa-induced dyskinesia, in connection with of drugs with which is more expedient to start the which they cannot be a promising target for the treatment to postpone the development of side effects treatment of motor symptoms of PD. at a later date. Until now, scientists and clinical The data of electrophysiological studies show physicians have failed to find ways of slowing down that the activation of mGluRII in the hypothalamic- the pathological process, and the more it stops or nigral synapses reduces their excitation. In prevent. Although PD is tried to cure, the experts can accordance with this intranigral or only partially eliminate the symptoms but not the intracerebroventricular adminisrtation of the cause itself. Today, all the efforts of scientists aim at mGluRII antagonist (DCG-IV, LY379268) restores finding drugs which are not only soften the the reserpine-induced akinesia in rats. Another symptoms of the disease, but also stop the mGluRII antagonist (LY354740) reverses degenerative processes responsible for its haloperidol-induced catalepsy and muscular rigidity progression. in rats. However mGluRII do not affect the The study of glutamate receptors antagonists is locomotor deficit caused by the reserpine to rats, an important task in the search for new which limits the therapeutic potential of this group to pharmacological agents for the treatment of PD. relief the motor symptoms of PD. Physiological Selectiveness of new agents is a key feature. studies on brain slice of rats showed that the selective Although the development of selective agents will

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UDC: 615.225:617.735-007.23 DOI: 10.18413/2500-235X -2016-2-3-95-100 Danilenko L. M. 1 METABOLIC CARDIOPROTECTION: NEW CONCEPTS Klochkova G.N. 2 IN IMPLEMENTATION OF CARDIOPROTECTIVE EFFECTS Kizilova I. V.3 OF MELDONIUM Korokin M.V.4 1) PhD in Pharmaceutical Sciences, Associate Professor of the Department of Pharmacology of Medical University of NRU BelSU, 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] 2) PhD, head of the clinical diagnostic laboratory, OGBUZ "Belgorod Regional Hospital St. Joasaph" Nekrasov St. 8/9, Belgorod, 308007, Russia. e-mail: [email protected] 3) head doctor of Central District Hospital in Stroitel, Belgorod region, Lenin Str., Stroitel, Yakovlevsky district, Belgorod region, 309070, Russia, e-mail: [email protected] 4) Doctor of Medicine, Professor of the Department of Pharmacology of pharmacology of Medical University of NRU BelSU, 85 Pobedy St., Belgorod, 308015, Russia, e-mail: [email protected] Abstarct. Recent studies confirm the need to find means to correct ischemic / reperfusion injury due to the hemodynamic medicine, which are already known do not have the proper cardioprotective effects. Key issue is the possibility of drug effects on the mitochondria of cardiomyocytes that controls the aerobic metabolism and maintenance of ATP admission into cardiomyocytes. Moreover mitochondria are the target of ischemia / reperfusion injury and involved in the cardioprotective mechanism ischemia and pharmacologic pre- and postconditioning. Effect of nitric oxide during ischemic reperfusion directed to mitochondria, is considered as the ultimate goal of cardioprotection. Preconditioning effects is begun from sarcolemmal membrane and then is directed to the cytoplasm through a plurality of stages of enzymes, including nitric oxide synthase (NOS), soluble guanylatecyclase (sGC) and protein kinase G (PKG). Thus, the signal is transmitted to the mitochondria, where it occurs cardioprotection. It is proved that the mitochondria provides protection of the heart against ischemia-reperfusion injury by opening the mitochondrial ATP-sensitive K + channels and by converting the capacity of mitochondria. Metabolic modulation concept can be used in the development of strategies cardioprotection for treatment in ischemic / reperfusion myocardial injury. Keywords: meldonium, pharmacological preconditioning, ischemic / reperfusion injury, heart. A brief review on the mechanisms of meldonium blocked toxicity of long-chain fatty acids during action. hypoxic myocardial injury [12, 13]. 1. Influence on metabolic rate However, under normal physiological Over the last few decades in medicine concentration carnitine stimulates the transport of "metabolic" trend has been developing intensively, fatty acids. However, at high concentrations of which aims at the analysis of cell metabolism carnitine it is pumping oxidized fatty acids from disorders in cardiovascular disease. Changes in cells, which lead to the destruction of the metabolism in ischemic cardiomyocytes can be myocardium. The ability to reduce the size of infarct regarded as the point of application of medical is caused by a decrease of L-carnitine content in the effects, particularly with drugs that can affect the tissues of the heart, and the subsequent inhibition of processes occurring in mitochondria directly. By fatty acid transport and protection of the outer now, it has been created a number of drugs that mitochondrial membrane of myocardial mitochondria directly affect the metabolic processes in Meldonium is a drug, which can reduce the level cardiomyocytes, which is known as "myocardial of carnitine. It blocks the synthesis of carnitine from cytoprotectors" [1, 2, 3, 4, 5, 6]. γ-buterobetaine by reversible competitive inhibition It is accumulated activated long-chain fatty acids of the enzyme gamma-butyrobetaine hydroxylase, in myocardial cells in ischemic heart disease [7, 8]. which reduces the carnitine-dependent transport of These conjugates of fatty acids inhibit additional fatty acids into the mitochondria [14, 15, 16]. myocardial oxygenation and intracellular production Under normal conditions, when there is no of ATP. As it has been shown in some studies, the shortage of oxygen, energy of the myocardium is delay of synthesis of ATP as well as the intracellular mainly formed from fatty acids (FA) and glucose to accumulation of long-chain fatty acids can reduce L- adenosine triphosphate(ATP).According to the needs, carnitine [9, 10]. In his studies on isolated porcine it is occurred the mobilization of FA by the signal the heart A. J. Liedtkeet al. [11] showed that carnitine nervous system via carnitine, which activated RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Danilenko L.M., Klochkova G.N., Kizilova I.V., Korokin M.V. Metabolic cardioprotection: new concepts in implementation of cardioprotective effects of meldonium. Research result: pharmacology and 96 clinical pharmacology. Vol.2, №3 (2016): 95-100. transport of FA across the mitochondrial membrane, of metabolism. According to modern concepts, "ideal" where is supplied a sufficient amount of oxygen [17]. metabolism corrector should prevent the accumulation The result of mitochondrial β-oxidation of FA is of a large amount of unoxidized FA in the cells formed acetyl-coenzyme A, which enters into the (prevention of cell membrane damage), activate the Krebs cycle, where ATP is synthesized. Another capture and oxidation of glucose by cells, inhibit the source of energy is a way of aerobic or anaerobic formation of lactate and stimulate the oxidation of oxidation of glucose. However, myocardial pyruvate, as well as being able to prevent the formation metabolism varies in ischemic conditions. Short- of activated oxygen species (prevention oxidative chain and long-chain FA enter the mitochondria, but stress). To the greatest degree, the "ideal" metabolism there is not enough oxygen for the oxidation in the corrector may include well-known meldonium, because cell. Consequently, in the mitochondria of ischemic it affects all three components [24, 25, 26]. tissue it is accumulated metabolites unoxidized FA (acylcarnitine and acyl-coenzyme A (acyl-CoA)), Cytoprotection mechanism of myocardial which block transport earlier synthesi zed ATP from ischemia mitochondria into the cytosol. It causes a devastating Meldonium belongs to a group of so-called effect on the cell membranes, which can lead cells to cytoprotectors–antihypoxants, which provide protection ischemic death. In addition, the accumulation of FA and power of the body cells in conditions of ischemia blocks the oxidation of glucose (it is studied during and increased load. Mildronat is a structural analogue of reperfusion of ischemic myocardium), and long-chain the gamma-butyrobetaine (GBB), and therefore it is a acylcarnitine contributes to reductions in ischemic competitive inhibitor of the GBB-hydroxylase – the last myocardium, which leads to a vicious circle [6, 18]. enzyme in the chain of carnitine biosynthesis in the The consequence of the toxic effect of non-oxidized body of humans and animals. Consequently, the drug metabolites FA is the blockade of Ca 2+ -ATPase of decreases concentration of carnitine in serum, and in the cytosol, and mitochondrial matrixreversibly. By several the sarcoplasmic reticulum – calcium pump required researchers it has been proposed another possible way for normal myocardial contractility. Consequently, it of meldonium influence on the content of carnitine in is necessary to restrict the flow of long-FA through the body. It has been proved, that meldonium is also an the mitochondrial membrane for the correction of inhibitor of carnitine reabsorption in the kidney, disturbed metabolism in the ischemic cells, because it reduces renal transport of carnitine [27]. This simultaneously activate an alternative oxidation mechanism provides a rapid decrease concentration of mechanism of the glucose for energy production in carnitinein the blood, which subsequently affects the the cells [19, 20, 21]. In conditions of an oxygen gradual decrease of its concentration in the tissues. As a shortage, it is more advantageous to use an oxidation result, carnitine is not reabsorbed in the kidneys and is of glucose than FA, since this process requires less not metabolized again and excreted from the body amount oxygen by the cells. The formation of ATP immediately. Reduced carnitine levels has a dual effect through aerobic glycolysis requires 12% less oxygen on the human body. Limiting the availability of than ATP production by the oxidation of FA. carnitine in the cytosol reduces the rate of activation On the one hand, due to oxygen deficiency, the and transport of long-chain FA to the place of their oxidized products of FA (acylcarnitine and acyl-CoA) oxidation in mitochondria. In other words, in ischemia accumulate in the myocardial ischemia, transport of meldonium slows the rate of penetration and ATP from mitochondria is blocked, cell membranes are accumulation of long-chain fatty acids in mitochondria. destroyed, ionic composition is changed, and Thereby it prevents blockage transport ATP from contracture of ischemic myocardium is developed. mitochondria to the cytosol and mitochondrial On the other hand, in hypoxia, glucose oxidation membrane integrity disruption due to the destroying only occurs to lactate, acidosis and electrical instability properties of activated FA (acylcarnitine and acyl- of myocardium are developed, and arrhythmias arise. CoA).Due to the limited transport and oxidation of fatty Thus, under ischemic conditions, it is essential to acids in the mitochondria, their concentration in the change the process of ATP production from FA cytosol increases, which is a signal of inclusion of oxidation to glycolysis. Thereby the need of the cells in alternative ways of energy production by aerobic oxygen for ATP production is reduced and it is glycolysis. It established, that meldonium increases the prevented the accumulation of activated forms FA in sensitivity of the insulin receptor to insulin and the cells mitochondria [22, 23]. Thus, it is important to stimulate the insulin controlled glucose uptake that use the oxygen sparing cytoprotection for the treatment promotes the availability of glucose for inclusion in the of ischemic heart disease what are partial inhibitors of energy production processes [28, 29]. oxidation, activators of FA transport and glucose At the same time meldonium activates the two oxidation. All the preparations for metabolic therapy in most important enzyme of aerobic glycolysis – cardiology can be divided into two groups: metabolic hexokinase and pyruvate dehydrogenase, which therapy preparations and preparations for the correction RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Danilenko L.M., Klochkova G.N., Kizilova I.V., Korokin M.V. Metabolic cardioprotection: new concepts in implementation of cardioprotective effects of meldonium. Research result: pharmacology and 97 clinical pharmacology. Vol.2, №3 (2016): 95-100. involve pyruvate formed from sugars into the Krebs A special role in the regulation of vascular tone cycle, and it prevents the formation of lactate. plays nitric oxide. Nitric oxide is the most potent Moreover, it is not only increased the activity of endogenous vasodilator. Nitricoxide, acting via these enzymes under the influence of meldonium, as guanylate cyclase, increases the formation of cyclic well as it is induced their biosynthesis. guanidin monophosphate, accumulation of which is the It is found, that the development of atherosclerosis causes of vascular relaxation. Oxidative stress and the and associated incidents of myocardial infarction, stroke high concentration of free radicals leads to accelerated and mortality directly correlated with the concentration degradation of nitric oxide [45]. The development of of carnitine in the plasma of patients and increased endothelial dysfunction in hypertension accompanied levels trimethylamine oxide, which is a metabolite of by apoptosis of vascular endothelial cells caused by free carnitine because of its degradation by intestinal radicals and a violation of the processes of intracellular microorganisms [30, 31]. The explanation is that energy transfer, so the correction of free-radical trimethylamine oxide, which is generated from processes and intracellular metabolism in the vascular carnitine, blocks macrophages capture of excessive endothelium is one of the conditions for the effective amounts of cholesterol at its peak concentration in the treatment of hypertension and endothelial dysfunction. blood, and return transport of cholesterol from plasma In this regard, meldonium may be the drug for into the liver and the bile. As a result, the formation of correction dysfunction of the vascular endothelium and cholesterol lesions can occur even at low levels of inhibition of free radical oxidation processes, as plasma average values in plasma cholesterol concentration. concentration of carnitinsprecursor – gamma- This explains the mechanism by which meldonium butyrobetaineincreasesunder the influence of significantly reduces plaque formation in the aorta in meldonium, which is first and foremost – its esters – animals predisposed to atherosclerosis [32, 33]. Also, in promotes NO biosynthesis – nitric oxide, which is the patients with diabetes mellitus type 2 (DM2) of disease main factor regulating vascular tone, and also affects severity is directly correlated with the concentration of platelet aggregation and flexibility of red blood cells. It carnitine in the blood plasma [34]. This indicates the has shown that the γ-butyrobetaine esters have potent need to review the use of carnitine or carnitine- acetylcholine-like effect on blood vessel containing products, as the degradation of carnitine to tonusMeldoniumhas the same effect [46, 47, 48]. trimethylamine oxide significantly increased risk of Consequently, there are carnitine-independent effects of atherosclerotic cardiovascular disease [30]. meldonium that cause a positive effect on the The only remedy in the world, which reduces the microcirculation. It should be emphasized, that the level of carnitine in humans, is meldonium. A pilot characteristic feature of NO is the ability rapidly (in less study in healthy volunteers, who received dietary than 5 seconds) to diffuse through the membrane in its trimethylamine, or its predecessors, has shown, that cells synthesize extracellular space and easily (without meldonium not only reduces the level of carnitine, but the receptors) to penetrate into the target cells. Inside also reduces the level of formation of pro-atherogenic the cell, it activates one and inhibits other enzymes factor – trimethylamine oxide. Thus, the use of participating in the regulation of cellular functions, and meldoniumto reduce to safe levels of carnitine and in fact acting as a local signaling molecule.NO is a trimethylamine oxide is pathogenetically justified and potent vasodilator, which is synthesized in the purposeful way of preventing the development of endothelium of the vascular wall, it quickly penetrates atherosclerosis, and possibly the DM2.Consequently, into the subendothelial space, and affects vascular meldonium is not only has unique cytoprotective smooth muscle cells.NO molecule reduces intracellular properties in ischemic lesions of different etiology and calcium by adenylate cyclase mechanism. This leads to localization, but also acts on the molecular mechanisms the relaxation of vascular smooth muscle cells, improve of atherosclerosis. Possible mechanisms of actions of microcirculation and endothelial function [49, 50, 51]. meldonium is discussed in the publications of several New concepts in implementation of authors [35, 36]. cardioprotective effects meldonium The points of action meldonium: protection of Another promising direction of studying the endothelial cells and cardioprotective effect mechanisms of action meldonium is a Another important pathogenetic part of the pharmacological preconditioning. One version is the defeat of the cardiovascular system is the inclusion of compensatory mechanisms by inhibiting development of dysfunction of vascular endothelium the synthesis of carnitine. [37, 38, 39, 40], the consequent remodeling of the In addition, a number of authors attempted to vascular wall and formation of angiopathy in arterial explore a new approach to the possible mechanism of hypertension, which creates conditions for stable action meldonium [52, 53, 54, 55]. In a number of high blood pressure, reducing the effectiveness of experimental studies have been proven the ability antihypertensive drugs, the development of meldonium activate a cascade of reactions through atherosclerotic lesions of arteries [41, 42, 43, 44]. ATP-dependent potassium channel system which

cardioprotection Protein synthesis Opioids The nucleus G белок of the cell L-NAME

Proteinсinase С Adenosine Bradykinin еNOS O2 Са++ L-arginine NO

Figure 1. The hypothetical role meldonium in the mechanism of ischemic preconditioning. Note.К+ АТФканалы – ATP-dependent potassium channel; eNOS – endothelial NO-synthase; iNOS – inducible NO-synthase; L-NAME – N- nitro-L-arginine-methyl ester; NO – Nitric oxide; РФК – reactive oxygen species; ЦОГ-2 – cyclooxygenase-2 ATP-dependent potassium channels are the of NO biosynthesis, vasoactive effects by reducing effector mechanism in the implementation of anti- peripheral vascular resistance and endotelioprotective ischemic effect, acts as a distant ischemic effect via induction of NO biosynthesis. preconditioning and meldonium/ Nitric oxide acts as a trigger of ischemic preconditioning [56, 57, 58]. References 1. Kosarev V.V., Babanov S.A. Clinical In either case, its synthesis is carried out by pharmacology of myocardial cytoprotection and their activation of the inducible and endothelial NO- place in the treatment of ischemic heart disease. Russian synthase. Meldonium competing for the receptors, Medical Journal. Cardiology. Vol. 20, № 4(2012): gamma-butyrobetaine, it reduces the concentration 187-188. (In Russian) [Full text] of carnitine. Due to the geomitric similarity gamma- 2. Mihin V.P. Cardiocytoprotectors – new direction butyrobetaine to acetylcholine it activates of Clinical Cardiology, heart. Arhiv Internal Medicine. endothelial acetylcholine receptors which lead to the № 1(2011): 21-27. (In Russian) [Full text] induction of NO synthesis. It follows that the 3. Skachilova S.J., Caesar O.G., Danilenko L.M. [et. meldonium can be recommended as the drug for al.]. Pharmacological ischemic myocardium protection of pharmacological preconditioning. 3- (2,2,2-trimethylhydrasine) propionate and evaluation of their antioxidant activity. Scientific statements Belgorod Thus, the therapeutic mechanism and protective State University. Series: Medicine. Pharmacy. Vol. 1, action of meldonium is its effect on carnitine levels, № 4 (6) (2015): 25-31. (In Russian) [Full text] trimethylamine oxide and metabolic energy pathways , 4. Lango R., Smolenski R.T., Narkiewicz M. [et. which provides a more economical and efficient al.]. Inflence of L-carnitine and its derivatives functioning of cells under conditions of oxygen onmyocardial metabolism and function in ischemic heart shortage. Antiischemic effects of meldonium are disease and during cardiopulmonary bypass. Cardiovasc achieved by reducing the intensity of fatty acid oxidation Res. Vol. 51(2001): 21-29. [PubMed] in ischemia (oxygen savings) , activation mechanisms to 5. Dambrova M., Liepinsh Е., Kalvinsh I. capture and oxidation of glucose for energy production Mildronate: cardioprotective action through pharmacological preconditioning, which includes carnitinelowering effct. Trends Cardiovasc Med. Vol. 12(2002): 275-279. [PubMed] compensatory mechanisms – training through the 6. Vilskersts R., Liepinsh E., Mateuszuk L. [et. al.]. suppression of the synthesis of carnitine, and induction Mildronate, a regulator of energy metabolism, reduces

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Рус. Eng.

UDC: 615.2 DOI: 10.18413/2500-235X -2016-2-3-101-106

Martynov M.A.1 Martynova O.V. 2 THYMOSIN β4 AS BASIS FOR CREATION Shkileva I.Yu.3 OF A REPARATION PREPARATION OF NEW GENERATION Tokarev I.A.4 Dovgan A.N.5

1) Surgeon, Belgorod Regional Clinical Hospital Saint Joasaph str. Gagarina 2, Belgorod, 308007 Russia e-mail: [email protected] 2) Graduate student of Department of pharmacology, clinical pharmacology of Medical Institute Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russia, e-mail: [email protected] 3) Student of Medical Institute Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russia e-mail: [email protected] 4) Oncologist Belgorod regional oncological clinic, 1, Kuibyshev St., Belgorod, 308010, Russia, e-mail: [email protected] 5) Graduate student of Department of pharmacology, clinical pharmacology of Medical Institute Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russia, e-mail: [email protected]

Abstract. In article is narrated about Thymosin β4. Its structure, a number of strategically important properties and opportunities is described. The list of researches and achievements over the past few years of researches is provided. On the basis of its multipurpose activities during regeneration of fabrics in various experiments on animals, Thymosin β4 has the potential for new researches, in kidneys and a liver, and also recovery of a spinal cord, bones and injury of ligaments. Besides, it can be useful in case of treatment of a wide range of other diseases, including concerning consequences of old bacterial damages and viral infections. Keywords: Thymosin β4, reparation, traumatism.

Today injury rate level constantly grows. Thanks to access to extreme sports, fixed haste, to emergencies people often are traumatized, both household, and production: whether it is small defect of integuments or extensive combined injury. Violation of healing of wounds is a problem for the immobilized patients sick with diabetes, and also elderly people. According to RosStat for 2015-2016 the registered surplus, both a production, and household injury rate on 1000 people – 92.2 persons, i.e. nearly 10% is observed. The surgery, orthopedics, traumatology is engaged in their diagnostics and treatment. For treatment and rehabilitation of patients a number of medicines such as is used: analgetic preparations, antihistaminic preparations, antibiotics, hormonal preparations, calcium preparations, vascular preparations, spazmolitik, locally – ointments. In case of inefficiency of conservative treatment resort to surgical. Now creation of the reparation preparation possessing a high reparative capability is very actual task for modern medicine and pharmacology as there is a great demand which becomes covered only for 40%. Proceeding from statistical data of the authoritative medical Pubmed portal (Figure) it is visible that every year scientists show the increasing interest concerning a Thymosin β4 (Tβ4). In spite of the fact that interest in studying of properties and structure of a Tβ4 has arisen 30 years ago – only the small part of properties of this peptide is studied. It proves a number of several experiments which are already made diversely and systems (Table 1).

Figure. Chronology of the publication on the subject Tβ4

Table 1 Some researches of properties of peptide of Tβ4 Тβ4 AUTHORS SUBJECT RESULT Gabriel Sosne, George W Ousler Tβ4 ophthalmologic solution for a dry eye. This research confirms efficiency of (randomized, placebo – controlled, the phase II 0,1% of Tβ4 as topical treatment for of clinical trials, is carried out with use of simpli-fication of signs and symptoms controlled medium) (CAE ™). of a dry eye. The therapeutic window of safety is defined. Tian-JiaoXu , Qi Wang Tβ4 represents a peptide from 43 Dimeasures of Tβ4 render a Xiao-Wen Ma, Zhen ZhangWei amino acids. Has crucial importan-ce for superactivity on an adhesion of Zhang, Xiao-Chang Xue, Cun restoration and reconstruction of tissues of a skin, wounds, than native Tβ4. Process of Zhang, QiangHao, Wei-Na Li, eyes, heart and a nervous system after a trauma. cleaning is simple and very effective Ying-Qi Zhang Meng Li Fully to use the efficiency as an agent for with the smallest expenses. treatment of the disease caused by a trauma it is recommended to spread out it to dimeasures. Ildiko Bock-Marquette, Ankur Tβ4 activates the integrin-sewed kinases and Participation of Tβ4 in migration of cells Saxena, Michael D. White activates migration of stem cells in heart, and survival of cardiomyocytes at distur- thereby increases survival of cardiomyocy-tes bance of a circulation in heart, them and restoration of normal work of heart. redistribution is mini-mized by losses of cardio-myocytes later myocar- dialinfarction. Yue Zhou, Eliana Martinez, Li- Tβ4 stimulates an angiogenesis and enlarges a Now research proved that Tβ4 makes Ping Su, Kok-Onn Lee1, Lei Ye pool of cells precursors of muscles at ischemic direct impact on neovascularization and option of sceletal muscles in model of an neo-genesis of sceletal muscles in model ischemia of back extremities of a mouse of an ischemia of a back extremity of a mouse. Tβ4 considerably enlarged density of capillaries. It is bound to its biological function on endothelial cells: migrations of endothelial cells and their proliferation. Deborah Philp, Tβ4 – the synthetic peptide containingits actin It is proved on experimental model at MahnazBadamchian, Brooke – the binding domain promote a fast adhesion mice with a diabetes mellitus and old Scheremeta, Mychi Nguyen, Allan of dermal defect. mice. L. Goldstein, Hynda K. Kleinman

In this article we will consider Thymosin also activation of cells of precursors, induction of derivatives β4 which can be a basis for creation of their migration, differentiation and integration in modern and innovative reparation drug and a processes of an angenesis, in particular induction of a vasoprotective. neoangiogenesis. Tβ4 can be the signal starting Adhesion of wounds includes several stages: immune system, induction of a chemotaxis, inhibition 1) inflammation (hemostases / vasoconstrictions), of a proliferation of stem cells of marrow, inhibition 2) proliferation (angiogenesis or vasifaction) and of an inflammation, an important property of 3) migration of keratinotsit and remodeling (collagen induction of a neoangiogenesis with the subsequent adjournment). reparative neogenesis of organs and tissues. A series Tβ4 represent family of highly conservative of the active centers in Tβ4 amino acids define the polar peptides with a molecular weight of 5kd. One action on physiological processes [8, 9]. of representatives of this family Tβ4. It consists of 43 Fragments in amino acid 1-4 have amino acids and the 4964th dalton has molecular antiinflammatory properties, 1-15 antiapoptozny and weight [1, 2]. With the N-trailer acetylated rest of a cytoprotective effect and 17-23 an angiogenesis and serine. Тβ4 is a larger water-soluble peptide which growth of hair is active for migration of cells, binding was for the first time allocated in 1966 by scientists by an actin, a dermal adhesion of wounds. Tβ4 Goldstein and Whyte from a bull tissue of a thymus. participates in a series of cellular reactions, such as an Researches in the field of hormones of a thymus led angiogenesis, an adhesion of wounds, body height of to a conclusion that Thymosin beta contain hair, an apoptosis and an inflammation [3, 10, 11, 12, practically in all tissues and various cells of a human 13, 14]. It inhibits an inflammation, microbial body body except for erythrocytes. High concentration height, formation of the cicatrix (due to depression of were noted in a lien, a thymus, mild and peritoneal level of miofibroblast) and an apoptosis, protects cells macrophages, and also in thrombocytes, a blood from cytotoxic damage, including glutamatneyronalny plasma, a wound exsudate. After a skin trauma, the toxicity [1, 15, 16, 17, 18, 19]. high Tβ4 levels naturally are present (13 mkg/ml) at Тβ4 represents multipurpose protein which wound liquid [3, 4]. He also acts as the main stimulates migration and a differentiation of stem molecule of a complexing actin in all eukaryotic cells, synthesis of a protease, and also an expression cells and is the potent regulator of an of various regulatory genes, such as laminin-332, a aktinapolimerization at mammals [5]. fibronectin, matrix metalproteases, factors of body Thymosin β4 is bound to a G-actin, blocks an height of hepatocytes and antioxidatic enzymes [11, actin polymerization with a factor of XIIIa of 18, 20, 21, 22, 23, 24, 25]. Migration of cells thrombocytes, exerting a great influence on process represents difficult process. Tβ4 promotes activity of of an adhesion of wounds [6, 7]. Two proteins matrix proteinases which is necessary for migration supporting a G-actin pool (a monomeric actin) are of epithelial cells [9, 26, 27, 28]. Тβ4 and a pro-eagle owl. In turn, the G-actin, is the Inhibitors of these enzymes reduce migration of water-soluble globular protein (weight 42 000 dalton) various types of cells. Such enzymes also degrade and consisting of 376 amino-acid remains. One molecule let out matrix molecules which can be hemotaktilny ATP is bound to each molecule of a G-actin. Тβ4 as factors of migration, thereby Tβ4 promotes chemotactic well as pro-eagle owls – low-molecular proteins with migration of cells to the place of the damaged site a molecular mass of a 12-15kd. They accelerate ADF [6, 16, 26, 30, 31,32, 33]. Such type of epithelial exchange mechanism for ATP, as well as Тβ4. migration has crucial importance for adhesion process. Acting as the buffer, it binds a monomeric actin from Migrations of Tβ4 it is carried out in several ways. where follows that it prevents polymerization the Direct migration assumes ability of Tβ4 to bind an aktin of microfilaments for maintenance of the actin. Proteases promote and improve migration, way general pool the monomers of aktin for needs of a the chemotoxicity factors of a matrix and inhibition of cell. Changes in Thymosin expression β4 are bound receptors of adhesion. Tβ4 induces synthesis of to a differentiation of cells, that is it is possible to laminin-332 which is important factors of adhesion and assume what Тβ4 suppresses energy release, at the migration of cells. Other mechanism includes same time keeping a cytoskeleton, cellular structure stabilization of a factor of a transcription of HIF1 which and cellular mobility from destruction. Release of is bound to the laminin-332 pro-motor, its chains. Thymosin from a complex with an actin and Proteases also reduce production of laminin-332, emergence it in a blood plasma, possessing a generate smaller chemotoxicity activity. Laminin-332 hypotoxicity and presence at rather high also stabilizes a complex: a cell and a cell matrix, their concentration at many types of cells of mammals, interaction which are important for migration of cells to both at a cytoplasma, and at an extracellular the damaged site of tissues. Tβ4 possesses environment. Tβ4 plays an important role in antiapoptichesky activity by conservation integral differentiation of stem cells in developments, and structure an epithelium for migration of a leaf. RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Martynov M.A., Martynova O.V., Shkileva I.Y., Tokarev I.A., Dovgan A.N. Thymosin β4 as basis for creation of a reparation preparation of new generation. Research result: 104 pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 101-106.

Cosecreted thrombocyte Tβ4 in the field of the injured regulator which controls migration of cancer cells to derma, can accelerate an adhesion of dermal wounds, places of an angiogenesis and innidiation of a tumor promoting migration of cells, accelerating collagen [49]. These various cellular answers are regulated by adjournment, inhibiting both an inflammation and an Tβ4-опосредованной of an expression of several apoptosis [34, 35]. Tβ4 reduces an inflammation in genes, such as specific proteases, laminin-5, and several tissues after different types of injuries by depression of inflammatory cytokines and chemokines. Tβ4 the beta migration of inflammatory factors; now are defined at (TGF beta) in the cultivated cornea cells strengthens an the molecular level [1, 16, 30, 38, 39]. This decrease of expression of laminin-5 and the transforming body an inflammation in fabric defect promotes Tβ4- height factor that in turn increases the activity of mediated of a reparation and is important for this laminin-5 and TGF-β- induced of migration of cells and process, and also solves concrete actions for migration, synthesis of a collagen, respectively [14]. Tβ4-mediated of cells for exercise of restoration of Tβ4 influences alarm ways of Want by an tissues. For example, Tβ4 reduces inflammatory factors, activation regulation a glycogen – synthase – cytokines and chemokines in many tissues, also reduces kinases-3 (GSK-3) in migration of cells of a inflammatory infiltration, activates anti-oxidizing carcinoma of the stomach [50]. enzymes, and reduces formation of active forms of The factor of body height of hepatocytes (HGF) oxygen. Тβ4 inhibits TNF-α- inducibility of a nuclear promotes rising a regulation of Tβ4 which influences factor an activation kappa In and blocks the Rela / a wound repair in endothelial cells of an umbilical translocation of p65 and the sensitizing effects of the vein of the person [51]. intracellular binding partners pinch-1 and integrin- Тβ4 promotes a full normal adhesion of all sewed kinazy. surface of a dermal wound at steroid treatment at Тβ4 selektivno bridges tissue XIIIa factor animals with a diabetes mellitus [1, 41, 43]. Tβ4 also transglutaminases for various molecules, including an activly restores and regenerates tissues of an eye, actin, collagen, fibrin and a fibrinogen [39, 41]. Other heart, a brain, a peripheric nervous system and a molecules, such as a plasmin, alcohol dehydrogenase, spinal cord and stimulates an angiogenesis in some a hexokinase, a pyruvatekinase, and a lactate tissues at systemic use, but not at local use on a dehydrogenase, can't be sewed Тβ4. Тβ4 it was wounded surface of an eye. identified as an angiogenic factor in the screen of the Two independent randomized double blind people early genes induced during a differentiation of clinical tests proved that experimental gel in structure endothelial cells of invitro [42]. with Tβ4 accelerates a dermal adhesion. Uses of Tβ4 in Tβ4 the best conservation of cardiomyocytes is treatment of other diseases caused by a trauma such as a promoted after an ischemia, by effect which is mediated myocardial infarction or reperfusion damage trauma by rising of an expression of a factor of body height of corneas, and an ischemia of a brain induces migration an endothelium of vessels (VEGF) and activation of cells to the place of damage [1, 6, 15, 16, 18, 41,]. It integrina-sewed kinases (ILK) [29]. It is surprising that was revealed that Тβ4 in any phosphatic and buffer it is little-known about potential receptors. Considering saline solution or hydrogel activly accelerates dermal an purinoceptor alarm ways of the active centers it would adhesion of wounds at healthy rats. At the mice sick be possible to expect several receptors [8]. A lot of with Diabetum where the slowed-down adhesion, was things are also unknown of Tβ4 role in a core. After an revealed that adjournment of a collagen were much incubation with cells, it quickly (in 30 minutes), is lower, than at the mice receiving Tβ4 as well as in any transported in a core where functions as a factor of a phosphatic and saline buffered solution or hydrogel. No transcription [1, 43]. The arising data demonstrate to differences were observed in migration of keratinotsit, what recent researches showed, чтоTβ4 is superfluous at all animals with Diabetum, showing almost full expresses in malignant tumors, and was suggested that wound repair for the 8th day. These researches show it is bound to metastatic ability and an angiogenesis [10, that Tβ4 is active for an adhesion of wounds in models 31, 44, 45, 46]. The induced expression of Tβ4 of disturbance of an adhesion and can be effective the strengthens body height of a tumor and an innidiation in relation of chronic diseases at the person. cellular lines of a melanoma and fibrosarcoma of a Тβ4 participate in activation the multipotent of mouse [47]. Besides, Tβ4 hyper expression in cancer cells precursors, integration of new cells at the place of cells of a large intestine of the person cause the a lesion and formation of their microvascular niche, strengthened body height and an invasion at increases neogenesis. Neogenesis of any tissue it isn't transplantation these cancer cells of mice [46, 48]. It is possible without a full-fledged neoangiogenesis. For proved what an excess expression of Tβ4 promoted this reason development and creation of the drugs penetration of cells and their migration through activating a neoangiogenesis and possessing ILK/AKT/β katenina-of an alarm way, causing endotelioprotektivny properties is one of the main tasks transition epithelial to mesenchymal cancer of modern pharmacology and medicine in general. in colorectal a blast. Tβ4 also works as the hypoxia Several additional biological activities were identified, RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Martynov M.A., Martynova O.V., Shkileva I.Y., Tokarev I.A., Dovgan A.N. Thymosin β4 as basis for creation of a reparation preparation of new generation. Research result: 105 pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 101-106. but not localized in a molecule, including its 10. Chappell D.C., Varner S.E., Nerem R.M., antimicrobic activity, induction of different genes Medford R.M. & Alexander R.W. 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Рус. Eng. UDC 615.11(042.5) DOI: 10.18413/2500-235X -2016-2-3-107-114 Reznikov K.M. PARADIGM OF MODERN PHARMACOLOGY: DEVELOPMENT AND CURRENT APPROACHES Professor of the Pharmacology Department, Doctor of Medical Sciences, Professor, Voronezh N.N.Burdenko State Medical University, 10 Studencheskaya Street, Voronezh 394036, Russia, e-mail: [email protected] Abstract. The article focuses on the current approach to understanding pharmaceutical products’ effect. It describes a paradigm shift in views on pharmaceutical action in correlation to the processes occurring under pathology. The paper discusses the role of regulatory processes in the development of a disease and a recovery. It points out the necessity of disease registration in order to early start a therapy. The author has also analyzed problems of modern pharmacology paying attention at the inability of monitoring the drug products effect during the treatment process and limited awareness of primary pharmaceutical interrelations with biological substrate. It covers the issues of therapeutical efficiency depending on body oxygen saturation. Special attention is paid to the water medium of a body directly related to the development of pharmaceutical effects. The article emphasizes the challenges of pharmaceutical effect depending on a dosage, structural-functional heterogeneity of tissues, consecution of pathological development, choice of its diagnostics etc. It outlines the most important ways of increasing the pharmaceutical efficiency based on the understanding of quantum- wave processes of the primary pharmacological effect. Key words: pharmacodynamics, pathology, pharmaceutical effects, therapy, diagnostics, reducing-oxidizing potential, regulation. Pharmacology is known as a science inventing development in Russia. The appearance of Molecular innovative, efficient and safe pharmaceutical Pharmacology Chair in the 2nd Moscow State products and studying processes resulted from their Medical University (Professor P.V. Sergeev, an introduction into a body. It creates the pillar of academician of RAMS; Candidate of Medical pharmaceutical therapy. Needs in safe and highly Sciences N.L.Shimanovsky, Head of this Chair) efficient pharmaceuticals claim complete appeared to be that very mainspring in Russia, which understanding of the fact in what way a led on a lot of scientists, and the 1st school of young pharmacological product improves recovery. scientists-pharmacologists held in Pushchino (in Pharmaceutical study goes back to the which the author participated) raised a great interest anthropogenic period and, having overcome several in this problem among the youth. Synthesis of a large stages of development, has been based for almost 200 amount of biologically active substances demanded a years on the scientific fundamental principles, though search of body molecules being able to interact with currently there is an opinion that it is not a science, these compounds resulting therapeutical effects. since effects observed under pharmaceutical action Thus, an original scientific direction “preparation – cannot be described mathematically. The target-receptor” has been striking out, which development of novel knowledge provided changes currently appears to be the leading direction in search in pharmacological science and realization of its final of effective pharmaceuticals. goals: assistance in recovery, pharmaceutical effects, In the 1970s the problem of the medical use of targeted development of pharmacologically active oxygen at an ambient pressure higher than molecules, study of body interaction with atmospheric pressure – hyperbaric oxygen therapy pharmaceuticals at molecular, nano-sized, quantum- (HBOT) – was being developed intensively. Fair wave levels, i.e. it ensured scientific paradigm shift amount of knowledge was available about in pharmacology. pharmaceuticals interaction under lack of oxygen in a Paradigm (derived from the Greek paradeigma, body; however, no valid scientific generalizations “an example”) is considered to be a conceptual model were made about pharmaceuticals effect under of the problem statement and solution dominating HBOT. Action mechanism of oxygen in a body, during a certain historic period in the scientific especially under pathological conditions, was not community. fully specified as well. The problem of metabolic Changes in scientific beliefs on the interaction processes changing under oxygen excess in a body between pharmaceuticals and an organism can be was being solved successfully at the Pathophysiology observed on the example of pharmacological chairs Chair of the Voronezh State Medical University

RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Reznikov K.M. Paradigm of modern pharmacology: development and current approaches. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 108 107-114. under the direction of Professor A.N. Leonov. The under direction of the Academician of the Russian novel data led to the conclusion that oxygen Academy of Sciences (RAS) V.I. Petrov, which gives saturation of a body can influence the process of an opportunity to reveal pharmacological properties pharmaceutical effect formation. of new chemical compounds promptly and with the Our first systematic research related to the action significant degree of reliability. of oxygen under increased pressure on However, body informational-energetic pharmacodynamics of cardio-vascular preparations interrelation with the environment takes place allowed to determine previously unknown regularities constantly, thus, ensuring principle “each and all know of toxic changeability, therapeutic effectiveness, about everything” to be realized. From this perspective, pharmacological properties of cardiotonic, antianginal, the idea of regulation of the body vital functions anti-arrhythmic, anticoagulant, vascular and other phenomenon has taken a different shape especially for agents under hyperbaric oxygenation. Oxygen has been understanding the origin of pathological processes and found to have alpha-adrenomimetic and beta- opportunities to influence them therapeutically, since adrenolytic action, i.e. produce receptor action pathology, besides structural-functional changes, also accompanied by appropriate effects. includes longstanding or permanent violation of photon Currently oxygen properties can generally be energetical and informational balance. Nevertheless, at presented in the following way: the present time the principal postulate explaining 1. An acceptor of electrons in the respiratory pharmaceutical effect is considered to be drug products chain etc. interaction with certain body receptors (“targets of 2. A reagent in reactions of free-radical action”). Better understanding of the processes of the oxidation of membrane lipids that provides body vital activity regulation under the constantly conformational restructuring of membranes and their changing environmental impact is impossible without permeability. taking into consideration the role of water in the body, 3. Being paramagnetic in its basic state it can the role of tissue biofields, the role of electron-photon result in agitation of electro-magnetic field within a interrelations at the cellular level. radius of 100 angstrom magnitude and influence According to the receptor pharmacology, a processes of electron transition in biomembranes. molecule of a drug product correlating with receptors 4. Oxygen can influence dipole orientation of results in their conformational restructuring, which interacting molecules in biomembranes and possibly causes changes of membrane permeability, enzymes the structured water. activity and, due to this, changes of cellular 5. Oxygen changes conformational properties of metabolism and function. Pharmacological receptors protein-clinging enzymes molecules. appear to be acceptors of natural mediators 6. It changes sensibility of alpha-beta- (adrenaline, noradrenaline, acetylcholine, dopamine, adrenoreceptors, muscarinic receptors and, probably, serotonin and others). Drug products are known to other bio-macromolecules. react with the same receptors. However, there are 10 7. Binding of oxygen with an iron atom heme is known non-receptor mechanisms of drug interactions accompanied by the electron density shift of both and therapeutical affects. They are as follows: reduced iron and adjacent nitrogen-bearing groups, i.e. 1. Physical mechanism (osmotic and adsorbent electrical current generation with relevant magnetic properties). field generation. Therefore, oxygen is considered being 2. Chemical mechanism (acids, alkalies). the most important ambient informational factor mating 3. Denaturation of proteins (antiseptics). structural and field components of living systems that is 4. Chelate bonds with metals (tetracycline). important in understanding of a great number of 5. Inclusion of a drug product into biomolecules pharmaceutical effects. (fluorouracil). This research work has developed a new 6. Inclusion of a drug product into membrane concept in the pharmacodynamics of drug products (narcotic substances). under HBOT [1]; the attempt to evaluate processes in 7. Competitive antagonism with the substance a body at the submolecular level being exclusively essential for the body (sulfanilamides). scientific result. 8. Action of pharmaceuticals through structured Currently, based on the pharmacological water (LLLT, homeopathy). paradigm: “pharmaceutical – receptor”, experimental 9. Resonance mechanisms (bioresonance study of biologically active compounds starts with therapy). the computerized search of their affinity to biological 10. Alteration of the informational flows in the substrate. Determination of the assumed organ or body (acupuncture, psychotherapy) etc. tissue activity of the investigated compounds is used As G Ling [2], V.L.Voeykov [3] et al stated, for this purpose. A complex “Microkosm” has been understanding the possibility of receptor developed in the Volgograd State Medical University interrelations giving sufficiently adequate RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Reznikov K.M. Paradigm of modern pharmacology: development and current approaches. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 109 107-114. explanation to action mechanism of a great number altering informational body processes as well. Thus, of pharmaceuticals demands detailed analysis of these regularities should be taken into consideration compliance of current physico-chemical, biophysical, in the therapeutical process. informational achievements to current status Initial diagnostic criteria of a pathological “biologically active substance – target”. A living process appear to be patient’s complaints and organism is reported to be an open system symptoms of a disease. J. Vitulkas [12] states that exchanging substance, energy and information with signs and symptoms are not the disease itself but the the environment; exogenic reactions are consumed by unique form of the human protective system of an organism and are included into informational attempt to eliminate fight a disease. It is structural exchange [4]. The fact is deemed to be proved that a and functional violations which are diagnostic major carrier of information both – inside a living criteria. To all intends and purposes it will be more system and between living organisms, – appears to be sensible to register and evaluate changes of electromagnetic radiation [4, 5, 6, 7, 8 and so on]. regulatory processes and choose adequate response Self-production is regarded as a general property measures before morphological changes develop. For of a living thing, and a system of receptor- exactly this reason the authors have developed a informational interrelations – intercellular, technique of regulatory changes registration. intracellular etc., which is inseparably associated Therapy should be aimed not only at the with the impact of various environmental factors, is elimination of a symptom and correction of structural reported to be an essential peculiarity to control this and functional alterations, but at deeper mechanisms of process. Endogenic informational consistency is restriction of self-reproduction processes with due provided in a body by physical (electromagnetic and account for predisposition Current therapeutical acoustic fields) and biological (neuromediators, procedures are mainly aimed at the correction of peptides, ions etc.) factors, herewith the leading place structural-functional changes and due to their low as concerning precision, inclusiveness, transmission selectiveness they significantly influence healthy tissues rate of information goes to physical factors. These that is not obligatory realized as unwanted (registered) factors are not considered to be the major objectives side effect, but may result in the changes which support of academic medicine since their quantum-wave abnormal condition of other organs and systems for a characteristics have not become either foundational long period of time. Recurrent course of a disease can in understanding the development of pathological also be related to these reasons. process or objectives of therapeutical exposure yet. It is necessary to take into account that “… any However, there is an opinion [9] that the future of body molecule, but for structural function, to this or that medicine belongs to the quantum mechanics field. It extent has a regulatory role as well, at this hormones, is even suggested to solve the challenges of brain and enzymes, biogenic amines, biologically active body interrelations based on quantum physics substances – they all are specific regulators” [13]. principles [10] that emphasizes importance of study (translation is ours) It is proved in the works about the of quantum-wave interrelations in processes of body mediator role of stimulating aminoacids by the living activity in general and pathology development academician V.I. Petrov, that is why one may talk of a in particular. Transition from information exchange fact that function continuity is provided by regulatory in the form of substance to the field level is assumed processes continuity, that the entire metabolism is a sort to occur by electromagnetic field of a certain range of state regulation, and, generally speaking, – never- [11], the fact that sill raises hot debate. ending regulatory interaction. An organism consisting of 50-100 trillion cells Since structural-functional changes cannot occur is constantly renewing, moreover, each tissue is without preceding violation of regulatory processes, it is renewing with its specific rate keeping the evident that treatment should be aimed at the restoration information about life of all previous generations in of normal tissue regulation at all levels: cellular (self- DNA. Changes of body functioning degree under the reproduction of biologically active structures), environmental factors can lead to changes of genetic intercellular (informational flows of ions, mediators, apparatus that, in turn, causes tissue restructuring peptides, aminoacids and others) and interorgan – giving a new grounding to informational neurohumoral. At the same time, the question remains interrelations. If force and rate of environmental unclear – how the succession of the recovery process is factor impact is comparable in time with processes of regulated with account of stereo-architectonic of not tissue self-renewal, it is such structural changes that only a single cell, but tissues and organs as well. occur; if the impact is too powerful, changes too Naturally, one of the factors of this process can be their rapidly or has continuous effect, then adaptive, function, but violated function cannot provide normal accomodational reactions fail to be formed in due process of self-reproduction. Therefore, there exists time and this result in structural-functional violations another mechanism of regulation of restoration RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Reznikov K.M. Paradigm of modern pharmacology: development and current approaches. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 110 107-114. processes in a body under pathological condition. This to be the norm, 6-12 and more have been observed mechanism should control processes of self- under pathology. These changes are detected and reproduction and react clearly to the exposing justified as necrotic patches under myocardium environmental factors. The authors suppose that there is infarction at the tissue level, and as the enhancement of a certain algorithm (dynamic plan, a graph) of cells, A-discs anisotrophy in the divisions of healthy animal tissues and organs self-reproduction in a living body, myocardium after being polarized. Even more distinct which can change itself during the pathology and not confirmation of the above-mentioned one can obtain perform the function it provides in its usual (non- analyzing myocardium functional characteristics, for pathological) state. Structured body water possessing example, cardiorhythmograms: inequality of R-R memory property and able to instantly transfer intervals in healthy people changes considerably after information between all cells, including the information the onset of a pathological process and only thorough obtained under environmental impact, can be material mathematical processing of cardiograms gives an basis for such an algorithm. A phenomenon of opportunity to make adequate conclusions. At the bioresonance can be a mechanism of perception of such biochemical level it is also very difficult to give a information, and changes of spiroid chain formations of definite answer about the benefit of changes in a biogenic water clusters can appear to be its transference, biochemical process under the influence of some though it might also be solitons displacement [14]. medications. The fact is that inequality of enzyme Therapeutical manipulations should start with activity can be clearly detected through tissue and it is restoration of this algorithm. There is an uninterrupted kept under therapeutical intervention, therefore, total and inseparable connection between the algorithm of enzyme activity cannot be a criteria of the therapeutic restoration and structural-functional interrelations. The benefit of a medication, but only 3-D histochemical algorithm is considered to be an integrating part of these image, which is unfortunately impossible to obtain in interrelations in time and in space, and if being exposed real time regimen, can be of greater use. it influences the structure and functions of a cell, tissue, Radical pathology on the basis of functional- an organ and a functional system. structural violations of cellular membrane is Disease patterns have repeatedly changed during considered to be a priority [15]. Free radicals appear the development of medicine. Humoral and solid body to be atoms or chemical compounds having an pathology prevailed for almost 100 years, but beginning unpaired electron. Such electrons give free radicals with the last mid-century cellular pathology took properties of high reactive ability and paramagnetism priority. Progress in the molecular biology and [16, 17]. These radicals define bactericidal and immune-histochemistry created a basis for the cytotoxic effects of leucocytes, cell proliferation and molecular pathology model. At the same time there regulation of vascular tone [18]; result in inhibiting appeared a danger that detailed study of molecular action of DNA and RNA synthesis in endothelial processes under various diseases can result in more cells [19], they are supposed to take part in nor- incomplete understanding of general regularities of a adrenaline synthesis [20, 21]. However, hydroxyl disease at the entire body level, an endless number of radical (HO*), which is able to result in DNA medicinal factors, including pharmaceuticals, and, violation, regenerate formation of organic radicals eventually, will bring medicine to a dead-lock. inducing processes of lipids perioxidation to The authors’ multiple thorough research of body participate in bactericidal and cytostatic leucocytes changes under simulation of a pathological process has action [22], is known to be the most chemically revealed that there are no qualitative changes between reactive. Together with toxic action attributable to indications of pathology and initial norm at the free radicals of oxygen, they are used by the cells in metabolic level, structural level of cells and tissues, physiological concentrations for modification of functional level of organs and systems. Practically all membrane enzymes (cytochrome P-450, cytochrome phenomena registered during simulation of a disease b, glucose-6-phosphatase, adenylate cyclase, also occur in healthy animals. Quantitative differences monoaminooxidase) and also influence ionic pumps depending on spatially-temporal are the only to be of adenosine triphosphatase [23] and other processes revealed. For example, the important role of sarcomere [3]. Therefore, it can be affirmed that one and the contracture changes in myocytes under the pathogenesis same phenomena can be observed under both of myocardial infarction have been detected by the physiological and pathological processes at the level authors at the cellular level (electron microscopy), of individual molecules, even so chemically reactive however, the same changes have also been found out in as free radicals. If not taking into consideration their the myocardium of healthy animals. Only the expressiveness and spatial-temporal character of quantitative differences have been observed: 2-5 changes they cannot be used for diagnostics or as a contractures at the cellular cross-section are considered RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Reznikov K.M. Paradigm of modern pharmacology: development and current approaches. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 111 107-114.

pharmaceutical targeted receptor. At the same time a distribution and respiratory enzyme activity in heart separate group of preparations – antioxidatives – cells, which is kept during myocardium infarction have been distinguished. occurrence as well. He also emphasizes the fact that Research of M.B. Shtark and O.I. Epstein [24] heart divisions with various contractile functions devoted to “bipathic phenomenon”, i.e. modifying have different heterogeneity index of glycogen effect of biologically active substances, applied in amount in cardiomyocytes. Considerable inequality small doses, related to the same substances applied in of glycogen at the cellular and cell sheet level have standard amounts, give new opportunities in been demonstrated experimentally, and treatment of pathological processes of various ultrastructural investigations of cardiomyocytes have character and localization. A lot of researches on given an opportunity to reveal temporal and spatial biological activity of small doses of preparations inequality of sarcomeres and their constituents [1]. have been performed in recent years [25, 26]. O.I. Structural inequality of heart cells related to location Epstein in his work [27] provides a detailed analysis of bioplasts: mitochondria, elements of of therapeutical effects and mechanisms of action of sarcoplasmatic reticulum, nuclear cell apparatus, – potentiated antibodies. The question, what can has been registered during electron-microscopic determinate a similar approach to pharmaceutical study, though one can observe certain regularity (i.e. influence on a body, still exists. mitochondria: sub-sarcolemmal, interfibrillar, The main theoretical background in understanding perinuclear) in their structure. To sum up, it may be such phenomena appears to be a consideration of the concluded that a living organism consisting of necessity of solution structuring in accordance with the various organs and tissues determinate inequality condition of initial substance being kept even under (heterogeneity) of the structure and distribution of its dilution of the initial substance up to zero concentration. components at the cellular level, which fact is of The idea of structuring in biology was suggested by great biological significance in terms of nonlinearity Clegg [28]. It is known that structured water possesses of registered phenomena. This phenomenon is properties significantly differentiating it from ordinary regarded to be very essential, particularly, to support water. The most important of the abovementioned continuity of cell functioning under the process of properties is water memory about the initial substance. persistent self-reproduction of its structures, being Certain research studies prove the fact that this discrete in time. phenomenon may take place in the immunological Unequal contractility of sarcomeres due to system [29]. On the other hand, application of a inequality of calcium ion flows may be the basis of pharmaceutical in very-low-doses is accompanied by heart rhythm variability. As it is shown in research hyperergic reaction to it realized possibly through works of R.M. Bayevsky [33] and Ary L. Goldberger et immunological mechanisms of hypersensitivity [27]. It al [34] heart rhythm fluctuations are defined by the is also known that natural antibodies participate in influence of sympatic and parasympatic nerves creating regulation of natural functions [30]. In 1999 O.I. characteristics of geometrical fractal in time that Epstein et al advanced an opinion about presence of supports the idea of chaotic nature of heart rhythm. At modifying properties in potentiated antibodies that was the same time, study of dynamic cardiointerval range brilliantly proved in their succeeding works. In the view changes allows not only determining the body of modern ideas about the role of natural antibodies he capability to adaptive reactions, e.g. under physical considers that it is possible to “regulate a regulator” exertion, but also controlling the status of regulatory specifically modifying functional activity of one or processes of the cardio-vascular system under certain another auto-antibodies with very-low-doses of pathological conditions and action of pharmaceuticals. antibodies. He has also developed a conception of Thus, in the applied aspect a phenomenon of substance release-activity giving an opportunity to heterogeneity may be the basis for search of novel understand mechanism of action of pharmaceuticals on approaches in diagnostics of donozological stages of the different basis. Such a therapeutical approach is diseases and monitoring therapeutical procedures. reported to be a significant breakthrough in modern The above-mentioned approach in studying pathogenic pharmacotherapy. structural-functional heterogeneity may be applied The authors’ long-term researches on not only to heart activity analysis but to investigation nonlinearity of processes in a body (illustrated by the of any periodic processes in a body and natural study of the cardio-vascular system) have enabled us phenomenon. Determination of heterogeneity index to formulate a few statements about the significance of metabolism findings or cell functions gives an of this phenomenon for better understanding of life- opportunity to reveal changes as early as at the level sustaining activity under pathology and treatment. of regulatory and even informational (the most E.G. Bykov has demonstrated in his researches [32] important) processes that, in turns, will allow using that there exists initial inequality of glycogen not foreign for the body molecules having a lot of RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Reznikov K.M. Paradigm of modern pharmacology: development and current approaches. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 112 107-114. side effects to correct changes, but absolutely principle of quantum mechanics is that molecules are harmless informational factors. never isolated, but rather united in the uninterrupted It is known that the system of acupunctural structure of charge density extending to the whole diagnostic gives an opportunity to evaluate a body Universe. Such properties as dipole moment, ionizing state from the perspective of its inseparable entirety potential and others are typical for isolated molecules, with the environment, and the system of acupoints and properties perceived at the macroscopic level, such (biologically active points – BAPs) is taken as as temperature, colour, viscosity, melting point play a bioenergetic and informational [35, 36] performing critical part in affecting biological substrate. However, specific energetic connection with the ambient informational role of pharmaceuticals is not fully environment due to intensity and direction changes of understood yet in terms of current molecular electro- and thermal exchange. The authors have conception, which evidently requires further detailed changed generally accepted method of BAPs study. characteristic measurements, which represents All the above mentioned proves the fact that specification of difference of potentials or human knowledge about influence of various factors temperature between BAP and intact skin zone at the on a body is not sufficient to be properly interpreted. distance no more than 2 cm during a certain period of Research of body water, which is significantly time. This indication is being registered for 2 minutes different from the environmental water, can and fluctuations of its value are analyzed dramatically contribute to the understanding of mathematically, i.e. changeability (heterogeneity) of pharmaceutical performance. The authors have been BAP status is investigated. Various researches, e.g. studying this problem for the recent 15 years. F.G. Portnov [37], R. Voll [38], have confirmed Research works of physico-chemists show that association between meridians’ points and a water molecules provide constant electrical corresponding system of organs; this fact being polarization around molecules of the diluted supported by numerous researches allows developing substance resulting in the appearance of sufficiently a technique for assessment of pharmaceutical actions large electrical dipole in water [39]. All living tissues (patent №2119296 with priority of 1994) and manifest bioelectromagnetism [40] and due to this receiving novel data about the action of cardio- fact they can be modified by various electromagnetic vascular, broncholytic, hormone and other fields as a result of free electric dipole laser water preparations on the regulatory processes in the body. effect. It has been proved theoretically and supported Further investigations of these processes with experimentally that water being the basis of inner the help of the differential thermometer (developed human body fluids (intercellular and intracellular) with the Voronezh State Politechnical Institute – from a physics perspectives is considered to exist in Professor Y.S.Balashov) demonstrated the possibility the state of boundary formed by biological objects of the temperature differences assessment (ΔТ) contacted with it. This water differs in many physical between BAP and an intact skin zone every second parameters from the corresponding ones of water in with subsequent storage of these findings in the unit the volume and possesses high biological activity. memory. Then 16 findings on fluctuations of The authors of “Nanopharmacology” temperature registered for 2 minutes are calculated, (Corresponding Member of the RAS and the regularity of functioning changes of the N.L.Shimanovsky et al) [41] state that “Currently it meridian, where the differences of temperature ΔТ must be taken as a fact that pharmaceuticals can be have been registered at the BAP, is determined. The active in very-low doses, what is more, these effects fact that indicated changes occur under both – regarding their directionality may differ from those allopathic and homeopathic substance introduction – that usually take place under action of is very important, it means that authors have pharmaceuticals applied in therapeutical doses.” developed a technique of registration of (translation is ours) informational impact on a body. The technique The data were published as early as 2006 [42] allows monitoring pharmaceutical effects in real time that there exist free monomers in water – water spin- regimen ensuring timely changes of preparation isomers with their own quantum differences dosage or its substitution for another one. (spinning – spin selectiveness to hydrogen Elements of pharmacological Informatiology as bounding). However, L.N. Gall [14] argues that total options of information data transfer and “water spin-isomery as itself hardly plays any role in transformation from environmental factors to internal functioning of the living system matter.” (translation structures of an organism can be found out as early as in is ours) Nevertheless, S.M. Pershin considers that scientific works of the 18th century. Fundamental hydrogen-bounding structures and ortho-para spin isomers provide water with long-lived condition RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY Reznikov K.M. Paradigm of modern pharmacology: development and current approaches. Research result: pharmacology and clinical pharmacology. Vol. 2, №3 (2016): 113 107-114.

(comparing to proton switching) after various considered to be natural for a healthy body, and its impacts, which can be defined as specific maintenance ensures variety of productive life. manifestations of small-capacity memory. To help a patient cure himself is quite as much Oxidation-reduction potential value is reported to art as science…Success or failure of a physician serve as a measurement of oxidation reduction depend on the doctor’s ability to adequately processes intensity in the system and is defined as communicate with patients at the quantum level, concentration ratio between oxidative and reductive where frequencies, images, coherence and wave forms of ions composing the given system. Direction functions are of greater importance than pathology and rate of chemical reactions can be specified by the and disease formula [9]. changes of oxidation-reduction potential. Investigations To sum up, the basis of modern paradigm of of pharmacological properties of ionized water with pharmacology can be a concept of quantum-wave different oxidation-reduction potential are scarce a interaction of pharmaceuticals with bio-substrates of considerable part of such investigations have been a body. This interaction exactly appears to be the carried out at the Pharmacology Department of the starting point of pharmacological effect formation. Voronezh State Medical University (2005 – 2015). These researches have demonstrated influence of References anolyte (oxidation-reduction potential +700) and 1. Reznikov K.M. Giperbaricheskaya farmakologiya catholyte (oxidation-reduction potential – 500 mV) on nekotorykh serdechno-sosudistykh sredstv: dokt. diss. the behavioral reactions of rats, their analgesic Hyperbaric pharmacology of some cardio-vascular medications: doct. dissertation, Voronezh, 1981, 520 p. 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Kravkov’s experiments [43], or testing and electrodiagnostics: clinical application of quantum mechanics. Vestnik biofizicheskoy meditsiny. Reporter of the pharmaceutical and a patient’s compatibility with biological medicine. № 1 (1992): 14 – 27. (in Russian) the Voll method widely applied nowadays. All these 10. Bass L. A quantum mechanical mind-body facts prove the idea that there exist mechanisms of interaction. Found. Phys. 55 (1975): 159. [Abstract] pharmaceutical molecule interactions with body 11. Smith C.W. Electromagnetic and magnetic vector living tissues, apart from psychological and receptor potential bio-information and water. Ultra High Dilution. mechanisms. Therefore, not only metabolic and Physiology and Physics, P.C. Endler, J. Schulte eds., functional, but also informational homeostasis is Dordrecht: Kluwer Acad. Publ., 1994. pp. 187-201.

12. Vitulkas Dzhordzh. Novaya model' zdorov'ya i 29. Benveniste J. et al.: Human basophil bolezni. A new model of health and disease. M. : Izd. degranulation triggered by very dilute antiserum against gruppa “ARNA”, 1997, 306 p. (in Russian) IgE. Nature. 333 (1988): 816-818. 13. Epshteyn O.I., Vorob'eva G.M. Informatsionno- 30. Ashmarin I.P., Freydlin I.S. Zhurn. evolyuts. optologicheskaya model' adaptatsii. Informational-optological biokhim. i fiziol. Journal of biochemical and physiological model of adaptation. M., 1997, 166 p. (in Russian) evolutions. Vol. 25, №2 (1989): 176-182. (in Russian) 14. Gall' L.N. Fizicheskie printsipy 31. Epshteyn O.I., Beregovoy N.A., Sorokina N.S. et funktsionirovaniya materii zhivogo organizma. Physical al. Influence of various potentiated antibody dilutions to principle of a living body functioning. SPb.: Izd-vo brainspecific protein S-100 on the dynamic of posttetanic Politekhn. un-ta, 2014, 400 p. (in Russian) potentiation in survival hippocampus sections. Byul. 15. Zhironkin A.G. Kislorod. Fiziologicheskoe i eksper. biol. Bulletin of experimental biology. Vol. 127, toksicheskoe deystvie. Obzor problem. Physiological and №3 (1999): 317-320. (in Russian) toxical action: a problem review. L.: Nauka, 32. Bykov E.G. Gistokhimicheskiy analiz Leningradskoe otdelenie, 1972, 172 p. (in Russian) ishemicheskogo porazheniya miokarda pri 16. Zenkov N.K. Men'shchikova E.B. Activated ekstrakorporal'nom shuntirovanii levogo zheludochka oxygen metabolites in biological systems. Uspekhi serdtsa v eksperi-mente. Avtoreferat diss. dokt. med. nauk. sovremennoy biologii. Achievements of modern biology. Histochemical analysis of ischemic myocardium damage Vol.113, issue. 3 (1993): 286-289. (in Russian) under experimental extracorporal bypass surgery of the left 17. Metelitsa D.N. Aktivatsiya kisloroda fermentnymi heart ventricle: research abstract of the doct. Diss., sistemami. Activation of oxygen by the enzyme systems. Voronezh, 1969, 38 p. (in Russian) M., Nauka, 1983, 256 p. (in Russian) 33. Baevskiy R.M. et al. Otsenka funktsional'nogo 18. Bast A., Goris R.G. Oxidative stress. sostoyaniya organizma na osnove matematicheskogo Biochemistry and human disease. Pharm. Weekbl. Sci. analiza serdechnogo ritma. Metodicheskie rekomendatsii. V.11, №3 (1989): 199-206. Evaluation of the functional body status on the basis of 19. Ljones N., Scotland N. Oxygen and life. Second mathematical analysis of the heart rhythm. Vladivostok, BOC Priestley Cjnf. L.: Roy. Soc. Chem., 1981, pp. 163-169. DVO AN SSSR, 1987, 72 p. (in Russian) 20. Nohl H. Physiologocal and pathophysiological 34. Goldberger Ary L. et al. Chaos and fractals in signficance of superoxide-radicals and regulatory role of human physiology. V mire nauki. In the world of science. the enzyme superoxide dismutase. Klin. Wochenschr. № № 4 (1990): 25-32. (in Russian) 19 (1981): 1081-1091. 35. Nechushkin A.I. Physiological fundamentals of 21. Noda Y., McGreer P.L., McGreer E.G. Lipid functional correction by acupunctural methods. Voprosy peroxide distribution in brain and the effect of hyperbaric meditsinskoy elektroniki. Issues of medicinal electronics. oxygen. J. Meurochem. Vol.40. №5 (1983): 1329-1332. Taganrog, 1981, issue 3, pp.52-56. (in Russian) 22. Vladimirov Y.A. Archakov A.I. Perekisnoe 36. Durinyan R.A.. Methodological and physiological okislenie lipidov v biologicheskikh membranakh. Lipides analysis of the problem of meridians‘ points and energy in peroxidation in biological membranes. M. Nauka, 1972, 234 reflexotherapy. Teoriya i praktika refleksoterapii, mediko- p. (in Russian) biologicheskie i fiziko-tekhnicheskie aspekty. Theory and 23. Archipenko Y.V., Kagan V.E., Kozlov Y.P. practice of reflexotherapy, medico-biological and physico- Modification of transport system enzymes Sa2+ in technical aspects. Saratov, 1981, pp.3-11. (in Russian) sarcoplasmotic reticulum under lipids peroxidation. 37. Portnov F.G. Elektropunkturnaya Biokhimiya. Biochemistry. Vol. 48, №3 (1983), pp. 433- refleksoterapiya. Electropunctural reflexotherapy. Riga: 441. (in Russian) Zinatne, 1987, 325 p. (in Russian) 24. Shtark M.B., Epshteyn O.I. Preambule. Byulleten' 38. Foll' R. Topograficheskoe polozhenie eksperimental'noy biologii i meditsiny. Prilozhenie. biologicheski aktivnykh tochek elektropunktury. Bulletin of experimental biology and medicine. Topography of biologicaly active acupunctural points. M.: Supplement. M., Izd. RAMN, 2003, pp.8-9. (in Russian) Tekhart, 1993, Vol.2, 285 p. (in Russian) 25. Burlakova E.B., Terekhova S.F., Grechenko T.N. et 39. Guidict E.D. et. al., Water as a free electric dipole al. Influence of ingibitors of lipide oxidation reactions on the laser. Physical Review Letter, 1988, 61, hh. 1085-1088. isolated neuron electrical activity of a French snail. Biofizika. 40. Frohlich H. The biological effects of microwaves Biophysics. Vol. 31. №5 (1986): 921-923. (in Russian) and related questions. Adv. Electr.,1980, 53, hh.85-152. 26. Benveniste J., Davenas E., Ducot B. et al. 41. Shimanovskiy N.L., Epinetov M.A., Mel'nikov L∙agitation de solutions hautement dilues n∙induit pas M.Y. Molekulyarnaya i nanofarmakologiya. Molecular and d∙activite biologique specifique. C.R. Acad. Sci. II. nanopharmacology. M.: FIZMATLIT, 2010, 624 p. (in Vol. 312 (1991): 461-466. Russian) 27. Epshteyn O.I. Very-low-dose regulatory capacity. 42. Bunkin A.F., Pershin S.M., Khusainova R.S. et al. Byulleten' eksperimental'noy biologii i meditsiny. Bulletin Spinisomeric selectiveness of water molecules under NA of experimental biology and medicine. M.: Izd. RAMN, hydration. Biofizika. Biophysics. Vol.54, №3 (2009): 2003, pp .10-16. (in Russian) 396-401. (in Russian) [eLIBRARY] 28. Clegg G.S. Intracellular water, metabolism and 43. Kravkov N.P. The limit of protoplasm sensitivity. cell architecture: Part 2. In: Coherent excitations in Uspekhi eksperimental'noy biologii. Achievements of Biological Systems. Heidelberg, 1983, pp.123-130. experimental biology, 1924, pp.3-4. (in Russian)

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Богус С.К., Куликов А.Л., Винаков Д.В., Суздалев К.Ф., Галенко-Ярошевский П.А. Холиноблокирующая активность и фармакокинетические аспекты соединения SS-68, обладающего свойствами антиаритмика iii класса 3 Гуреев В.В. Новые подходы фармакологической коррекции морфофункциональных нарушений сердечно-сосудистой системы при экспериментальном гестозе 11 Якушев В.И., Покровский М.В. Кардиоваскулярная активность селективного ингибитора аргиназы II 28

КЛИНИЧЕСКАЯ ФАРМАКОЛОГИЯ Чухарева Н.А., Бонцевич Р.А., Щуровская К.В., Денисова Д.С. Оценка предпочтений врачей акушеров-гинекологов и терапевтов белгородской области в лечении пиелонефрита беременных в сравнении с врачами российской федерации 46 Корнилов А.А., Лунева Ю.В., Поветкин С.В. Комплексная оценка эффективности фармакотерапии сочетанной кардиальной патологии с учетом экзогенных и фармакогенетическихфакторов 51 Кукес В.Г., Горбач Т.В., Ромащенко О.В., Румбешт В.В. АТФ как маркер состояния энергетического обмена при экспериментальной ишемии миокарда на фоне введения метаболических корректоров 58

ВЕТЕРИНАРНАЯ ФАРМАКОЛОГИЯ Быу К.К., Нгуен Х.Т., Везенцев А.И., Буханов В.Д., Соколовский П.В., Михайлюкова М.О. Антибактериальные свойства модифицированного бентонита месторождения «ТАМ БО» 63 Зуев Н.П., Буханов В.Д., Везенцев А.И., Соколовский П.В., Хмыров А.В., Зуева Е.Н., Салашная Е.А., Михайлюкова М.О. Этиологическая структура массовых болезней молодняка с гастроэнтеральным и респираторным синдромами 75

ФАРМАКОЛОГИЧЕСКИЕ ОБЗОРЫ Авдеева Н.В., Никитина В.А., Кочкарова И.С., Литвинова А.С. Возможности применения антагонистов глутаматных рецепторов при лечении болезни паркинсона 86 Даниленко Л.М., Клочкова Г.Н., Кизилова И.В., Корокин М.В. Метаболическая кардиопротекция: новая концепция в реализации кардиопротекторных эффектов мельдония 95 Мартынов М.А., Мартынова О.В., Шкилева И.Ю., Токарев И.А., Довгань А.Н. Тимозин β4 как базис для создания репарационного препарата нового поколения 101 Резников К.М. На пути создания парадигмы современной фармакологии 107

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УДК: 616.12-008.313:615.222:615.015]-092.9 DOI: 10.18413/2500-235X-2016-2-3-3-10

1 Богус С.К. ХОЛИНОБЛОКИРУЮЩАЯ АКТИВНОСТЬ И Куликов А.Л.2 ФАРМАКОКИНЕТИЧЕСКИЕ АСПЕКТЫ СОЕДИНЕНИЯ Винаков Д.В.3 Суздалев К.Ф.4 SS-68, ОБЛАДАЮЩЕГО СВОЙСТВАМИ АНТИАРИТМИКА III Галенко-Ярошевский П.А.5 КЛАССА

1) Кандидат медицинских наук, ассистент кафедры фармакологии Кубанского государственного медицинского университета, ул. Седина, 4, г. Краснодар, 350063, РФ, e-mail: [email protected] 2) Аспирант кафедры фармакологии Медицинского института Белгородского государственного научно- исследовательского университета, ул. Победы, 85, г. Белгород, 308005, РФ, e-mail: [email protected] 3) Аспирант кафедры фармакологии Медицинского института Белгородского государственного научно- исследовательского университета, ул. Победы, 85, г. Белгород, 308005, РФ, e-mail:[email protected] 4) Кандидат химических наук, доцент кафедры химии природных и высокомолекулярных соединений Южного федерального университета, ул. Зорге, 7, г. Ростов-на-Дону, 344090, РФ, e-mail:[email protected] 5) Член-корреспондент РАН, доктор медицинских наук, профессор Кубанского государственного медицинского университета, , ул. Седина, 4, г. Краснодар, 350063, РФ, e-mail: [email protected]

Аннотация. Показано, что производное индола SS-68 (50 и 250 мкг/кг внутривенно) в острых опытах на кошках в условиях нейрогенной фибрилляции предсердий (ФП) обладает дозозависимым антиаритмическим действием, которое связано с преимущественным нейротропным влиянием этого вещества, т.к. подавление ФП совпадает по времени с его холиноблокирующим эффектом, наблюдаемым в течение более 2-х часов, а кардиотропное действие к этому времени уже не проявляется. Высокая антиаритмическая эффективность SS-68 при нейрогенной ФП может быть обусловлена ингибированием IKAch и блокадой М2-холинорецепторов. Соединение SS-68 (50 мкг/кг внутривенно) в опытах на кроликах характеризуется следующими фармакокинетическими показателями: Сmax = 14,5 нг/мл плазмы крови (продолжительность содержания SS-68 в крови 4 часа), Т1/2 = 1,1 ± 0,06 часа, MRT(0 – t)= 1,6 ± 0,08 часа, AUC(0 – 1440) = 1006,1 ± 147,7 нг/мл ´ мин, Vss = 4,8 ± 0,8 л/кг и Сl = 0,051 л/час. Основываясь на фармакодинамических и фармакокинетических показателях высказано предположение, что холиноблокирующее действие SS-68 в условиях нейрогенной ФП будет проявляться в значительно меньшей дозе, чем 50 мкг/кг. Ключевые слова: блуждающий нерв, нейрогенная фибрилляция предсердий, производное индола SS-68, антиаритмическое действие, фармакокинетика.

УДК:618.3-06-08:577.112.385.2 DOI: 10.18413/2500-235X -2016-2-3-11-27

Гуреев В.В. НОВЫЕ ПОДХОДЫ ФАРМАКОЛОГИЧЕСКОЙ КОРРЕКЦИИ МОРФОФУНКЦИОНАЛЬНЫХ НАРУШЕНИЙ СЕРДЕЧНО- СОСУДИСТОЙ СИСТЕМЫ ПРИ ЭКСПЕРИМЕНТАЛЬНОМ ГЕСТОЗЕ

Кандидат медицинских наук, доцент кафедры фармакологии федерального государственного автономного образовательного учреждения высшего образования «Белгородский государственный национальный исследовательский университет» Министерства образования и науки Российской Федерации 85, ул. Победы, Белгород, 308015, Россия, e-mail: [email protected]

Аннотация. Моделирование экспериментального ADMA-подобного гестоза осуществляли путем введения крысам N-нитро-L-аргинин-метиловый эфир с 14 по 20 сутки беременности. У RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY 117

животных наблюдалось повышение артериального давления, протеинурия, нарушение микроциркуляции в плаценте, нарушение регуляции сосудистого тонуса и деструктивные изменения в плаценте ишемического генеза. Введение тетрагидробиоптерина, селективного ингибитора аргиназы II, рекомбинантного эритропоэтина, тадалафила, эритромицина, азитромицина, поливитаминноминеральных комплексов и воспроизведение дистантного ишемического прекондиционирования приводило к выраженной коррекции морфофункциональных нарушений возникающих при моделировании экспериментального гестоза. Это выразилось в снижении артериального давления, уменьшении протеинурии, увеличении показателя микроциркуляции в плаценте, восстановлении вазодилатирующей функции сосудов и предотвращение деструктивных явлений в плаценте по сравнению с группой не леченных животных, повышении концентрации конечных метаболитов NO в плазме крови. Приведенные данные позволяют говорить о выраженной коррекции морфофункциональных нарушений возникающих при моделировании ADMA-подобного гестоза исследуемыми препаратами и перспективности проведения дальнейших исследований по поиску новых лекарственных препаратов обладающих эндотелеопротективными свойствами для коррекции гестоза второй половины беременности. Ключевые слова: ADMA, гестоз, эндотелиальная дисфункция, крысы, эндотелеопротекторы.

УДК: 615.224 DOI: 10.18413/2500-235X -2016-2-3-28-45

Якушев В.И.1 КАРДИОВАСКУЛЯРНАЯ АКТИВНОСТЬ СЕЛЕКТИВНОГО Покровский М.В.2 ИНГИБИТОРА АРГИНАЗЫ II

1) Врач-терапевт приемного отделения ОГБУЗ «Яковлевская ЦРБ», ул. Ленина, г. Строитель, Яковлевский район, Белгородская обл., РФ, 309070, e-mail: [email protected] 2) Доктор медицинских наук, профессор; Медицинского института НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail: [email protected]

Аннотация. В работе впервые в опытах in vitro проведено изучение специфической ингибирующей активности и безопасности молекул-кандидатов, селективных ингибиторов фермента аргиназа II из группы производных норлейцинамида. Также впервые показано в опытах in vitro, что соединение под шифром ZB49-0010C, из группы производных норлейцинамида, обладает наибольшей селективностью и ингибирующей активностью в отношении фермента аргиназа II. При этом данное вещество in vivo оказывает дозозависимое гипотензивное, эндотелиопротективное и кардиопротективное действие на фоне L-NAME индуцированной и гипергомоцистеин-индуцированной ЭД, которое наиболее выражено в дозе 10 мг/кг при внутрижелудочном введении. Эндотелиопротективное действие проявляется в предотвращении повышения коэффициента эндотелиальной дисфункции (КЭД) и снижении концентрации стабильных метаболитов оксида азота в плазме крови. Кардиопротективное действие проявляется в предотвращении повышения уровня ЛДЖ в пробе на адренореактивность и снижения миокардиального резерва при проведении пробы на нагрузку сопротивлением, а также в профилактике развития гипертрофии миокарда левого желудочка. В ходе исследования изучено дозозависимое противоишемическое действие селективного ингибитора аргиназы II, вещества ZB49-0010C на модели хронической ишемии конечности у крыс, которое наиболее выражено в дозе 10 мг/кг и проявляется в предотвращении падения уровня микроциркуляции на 29-е сутки эксперимента в ишемизированной конечности и в протективном действии при морфологоическом исследовании гистоархитектоники мышечной ткани. Ключевые слова: ингибиторы аргиназы, ZB49-0010C, L-NAME, эндотелиальная дисфункция, оксид азота, метионин, гомоцистеин, ишемия, кардиопротекция.

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КЛИНИЧЕСКАЯ ФАРМАКОЛОГИЯ

УДК: [656.071.61:616.08](470.325) DOI: 10.18413/2500-235X -2016-2-3-46-50

1 Чухарева Н.А. ОЦЕНКА ПРЕДПОЧТЕНИЙ ВРАЧЕЙ АКУШЕРОВ- 2 Бонцевич Р.А. ГИНЕКОЛОГОВ И ТЕРАПЕВТОВ БЕЛГОРОДСКОЙ ОБЛАСТИ В Щуровская К.В.3 ЛЕЧЕНИИ ПИЕЛОНЕФРИТА БЕРЕМЕННЫХ В СРАВНЕНИИ С Денисова Д.С.4 ВРАЧАМИ РОССИЙСКОЙ ФЕДЕРАЦИИ

1) ФГБУ Научный центр акушерства, гинекологии и перинатологии им. академика В.И. Кулакова Минздрава России, ул. Академика Опарина, 4, г. Москва, РФ, 117997, e-mail: [email protected] 2) НИУ БелГУ, каф. фармакологии; многопрофильная поликлиника «Гармония здоровья» – центр безопасной фармакотерапии беременных и кормящих; Белгород, e-mail: [email protected] 3) НИУ БелГУ, каф. фармакологии, ул. Победы, 85, г. Белгород, РФ, 308000, e-mail: [email protected] 4) НИУ БелГУ, Белгород, ул. Победы, 85, г. Белгород, РФ, 308000, e-mail: [email protected]

Аннотация. От 1 до 10% случаев физиологически протекающая беременность осложняется острым пиелонефритом. Цель работы – проанализировать выбор антимикробной терапии среди врачей при лечении гестационного пиелонефрита. Материалы и методы – анализ анонимного анкетирования в рамках проведения второго этапа Всероссийского фармакоэпидемиологического исследования “Эпидемиология использования лекарственных средств у беременных”, которое проводилось с февраля по апрель 2015 года. На основе полученных данный было выполнено сравнение полученных результатов с результатами анкетирования врачей Белгородской области. Результаты и обсуждение. Примерно половина врачей выбирает для лечения пиелонефрита у беременных цефалоспорины II и III поколений, а так же амоксициллин/клавуланат. До 30% врачей назначают неэффективные в данном случае макролиды. Треть врачей направляют пациенток на лечение к другим специалистам. Заключение. Анализ ответов врачей по тактике назначения антимикробной терапии при гестационном пиелонефрите показал, что только половина практикующих специалистов назначают терапию, основываясь на рациональном применении антибактериальных препаратов. Ключевые слова: беременность, пиелонефрит, защищенные пенициллины, цефалоспорины II и III поколения, рациональная фармакотерапия.

УДК: 616.153.915:616.127-005.4-08 DOI: 10.18413/2500-235X -2016-2-3-51-57

1 Корнилов А.А. КОМПЛЕКСНАЯ ОЦЕНКА ЭФФЕКТИВНОСТИ Лунева Ю.В.2 ФАРМАКОТЕРАПИИ СОЧЕТАННОЙ КАРДИАЛЬНОЙ Поветкин С.В.3 ПАТОЛОГИИ С УЧЕТОМ ЭКЗОГЕННЫХ И ФАРМАКОГЕНЕТИЧЕСКИХФАКТОРОВ

1) Ассистент кафедры клинической фармакологии, кандидат медицинских наук, Курский государственный медицинский университет, ул. К.Маркса, 3, г. Курск, 305014, Россия, e-mail: [email protected] 2) Доцент кафедры клинической фармакологии, кандидат медицинских наук, доцент, Курский государственный медицинский университет, ул. К.Маркса, 3, г. Курск, 305014, Россия, e-mail: [email protected] 3) Заведующий кафедрой клинической фармакологии, доктор медицинских наук, профессор, Курский государственный медицинский университет, ул. К.Маркса, 3, г. Курск, 305014, Россия, e-mail: [email protected]

Аннотация. В работе проводилась оценка изменений гемодинамических и биохимических показателей, сосудистого возраста и 5-летнего сосудистого риска на фоне комплексной фармакотерапии у пациентов со стабильной стенокардией напряжения I-III функционального класса (ФК),

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гипертонической болезнью I-III степени, хронической сердечной недостаточностью (ХСН) II-III ФК с учетом влияния экзогенных и фармакогенетических факторов. В исследование было включено 200 пациентов в возрасте 45 - 65 лет с сочетанной сердечно- сосудистой патологией: АГ I-III степени, стабильной стенокардией напряжения (I-III ФК), ХСН (II-III ФК). Больные 1-й группы (100 человек) получали престанс, карведилол, гипотиазид, верошпирон, препараты ацетилсалициловой кислоты, аторвастатин. Больные 2-й группы (100 человек) получали моноприл (фозиноприл натрия), карведилол, амлодипин, гипотиазид, верошпирон, препараты ацетилсалициловой кислоты, аторвастатин. В гипертензиологической части исследования к концу срока наблюдения положительную динамику основных гемодинамических показателей выявили в обеих группах. По основным показателям липидного спектра была достигнута выраженная положительная динамика. Оценка влияния фармакогенетических факторов на эффективность проводимой терапии выявила отсутствие влияния полиморфизмов гена ACE (rs4344) и гена CYP2D6 (rs1135840) на основные параметры гемодинамики, в то время как носительство генотипа (G;G) гена CETP (rs4783961) в обеих группах вмешательства ассоциировалось с менее значимым повышением холестерина липопротеидов высокой плотности (ХС ЛВП) по сравнению с носителями аллелей (А;A) и (А;G). При анализе динамики показателей сосудистого возраста как интегрального показателя состояния сердечно-сосудистой системы, а также 5-летнего сосудистого риска на фоне проведенной фармакотерапии отмечена выраженная положительная динамика. Вместе с тем, статистически значимых различий между 1-ой и 2-ой группами выявлено не было. Ключевые слова: ишемическая болезнь сердца, гипертоническая болезнь, сердечная недостаточность, фармакогенетика, липид-транспортная система, сосудистый возраст.

УДК: 616-005: 616-08 DOI: 10.18413/2500-235X -2016-2-3-58-62

1 Кукес В.Г. АТФ КАК МАРКЕР СОСТОЯНИЯ ЭНЕРГЕТИЧЕСКОГО ОБМЕНА Горбач Т.В.2 ПРИ ЭКСПЕРИМЕНТАЛЬНОЙ ИШЕМИИ МИОКАРДА НА ФОНЕ Ромащенко О.В.3 Румбешт В.В.4 ВВЕДЕНИЯ МЕТАБОЛИЧЕСКИХ КОРРЕКТОРОВ

1) Заведующий кафедрой клинической фармакологии и пропедевтики внутренних болезней Первого Московского государственного медицинского университета им. И.М.Сеченова, академик РАН, профессор; e-mail: [email protected] 2) Доцент кафедры биохимии Харьковского национального медицинского университета; e-mail: [email protected] 3) Доцент кафедры фармакологии Медицинского института НИУ «БелГУ»; e-mail: [email protected] 4) Доцент кафедры математического и программного обеспечения информационных систем Института инженерных технологий и естественных наук НИУ «БелГУ»; e-mail: [email protected]

Аннотация С целью определения адекватного маркера состояния энергетического обмена миокарда при экспериментальной ишемии миокарда на фоне введения метаболических корректоров, было проведено экспериментальное исследование на 50 крысах-самцах линии Вистар. При моделированной ишемии миокарда были обнаружены энергосберегающие эффекты триметазидина, цитофлавина и фосфокреатина, которые достигаются разными путями и в разной степени. Показатель концентрации АТФ в сыворотке крови или эритроцитах тесно коррелировал с уровнем АТФ в гомогенате миокарда и с активностью митохондриальных ферментов сердца – сукцинатдегидрогеназы, цитратсинтазы, пируватдегидрогеназы, в связи с чем данный показатель можно рекомендовать к исследованию для оценки эффективности кардиоцитопротекторов как наиболее адекватный маркер их энергосберегающего эффекта. Ключевые слова: кардиоцитопротекция, энергосберегающий эффект, метаболические корректоры, триметазидин, цитофлавин, фосфокреатин, АТФ, метаболизм миокарда.

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ВЕТЕРИНАРНАЯ ФАРМАКОЛОГИЯ

УДК: 666.322.4: 615.281.9 DOI: 10.18413/2500-235X -2016-2-3-63-74

Быу К.К.1, Нгуен Х.Т.2, Везенцев А.И.3, АНТИБАКТЕРИАЛЬНЫЕ СВОЙСТВА МОДИФИЦИРОВАННОГО 4 Буханов В.Д. , БЕНТОНИТА МЕСТОРОЖДЕНИЯ «ТАМ БО» Соколовский П.В.5, Михайлюкова М.О.6

1) Доктор технических наук, профессор, исследователь, Институт экологических технологий Вьетнамской Академии наук и технологий. 18 Hoang quoc viet - Ханой, Вьетнам, e-mail: [email protected] 2) Доктор технических наук, профессор, директор экологического центра Института экологических технологий Вьетнамской Академии наук и технологий. 18 Hoang quoc viet - Ханой, Вьетнам, e-mail: [email protected]. 3) Доктор технических наук, профессор, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected]. 4) Кандидат ветеринарных наук, доцент кафедры медико-биологических основ физической культуры, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected]. 5) Аспирант, кафедры общей химии, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected] 6) Аспирант, кафедры общей химии, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected]

Аннотация. В данной статье изучены антибактериальные свойства обогащенной бентонитовой глины месторождения Там Бо, провинции Лам Донг (Вьетнам), модифицированной различными способами: щелочным - путём обработки обогащённого бентонита раствором LiOH при температура 60°С в течение 30 мин, солевым - путём обработки обогащённого бентонита 3%-ным раствором Na2CO3 при температуре 55°С в течение 30 мин и наночастицами серебра - путем иммобилизации наночастиц серебра на поверхности бентонита методом осаждения наночастиц серебра из раствора AgNO3. В исходном состоянии бентонитовая глина месторождения Там Бо содержит 40 – 60 масс.% сорбционно-активного монтмориллонита. Обогащение с последующим модифицированием позволяет увеличить содержание монтмориллонита до 70 – 80 масс. %. Модифицирование бентонитовой глины позволяет в 2 раза увеличить ее физико-химические и коллоидно-химические характеристики, такие как степень набухания, удельная поверхность, катионообменная емкость. Экспериментальные данные показали, что бентонит, модифицированный наночастицами серебра способен эффективно подавлять рост таких условно-патогенных микроорганизмов как Enterococcus feacalis, Escherichia coli, Proteus mirabilis, Pseudomonas aerugenosa, Salmonella typhimurium, Staphylococcus Aureus и Candida albicans. Модифицирование бентонитовой глины, а именно кристаллической решетки монтмориллонита, входящего в ее состав наночастицами серебра позволяет увеличить бактерицидную активность по отношению к афлатоксину до 65 раз. Ключевые слова: бентонит, монтмориллонит содержащий сорбент, модифицирование, энтеросорбент, афлатоксин, поллютанты, наночастицы серебра, условно-патогенные микроорганизмы.

УДК:619: 616.24-002 DOI: 10.18413/2500-235X -2016-2-3-75-85

Зуев Н.П.1, Буханов В.Д.2, 3 Везенцев А.И. , ЭТИОЛОГИЧЕСКАЯ СТРУКТУРА МАССОВЫХ БОЛЕЗНЕЙ Соколовский П.В.4, Хмыров А.В.5, МОЛОДНЯКА С ГАСТРОЭНТЕРАЛЬНЫМ И РЕСПИРАТОРНЫМ Зуева Е.Н.6, СИНДРОМАМИ Салашная Е.А.7, Михайлюкова М.О.8

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1) Профессор кафедры паразитологии и эпизоотологии ВГАУ имени императора Петра I. 394087, г. Воронеж, ул. Мичурина, 1, Russia. e-mail: [email protected]. 2) Кандидат ветеринарных наук, доцент кафедры медико-биологических основ физической культуры, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected]. 3) Доктор технических наук, профессор, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected]. 4) Аспирант, кафедра общей химии, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected] 5) Кандидат биологических наук, начальник управления ветеринарии Белгородской области, главный государственный ветеринарный инспектор области. 308000, г. Белгород, ул. Попова, 24,Russia. e-mail: [email protected] 6) Студент Белгородского государственного аграрного университета имени В.Я. Горина. 308503, Белгородская обл., Белгородский р-н, п. Майский, ул. Вавилова, 1, Russia. e-mail: [email protected] 7) Старший преподаватель кафедры физической культуры Белгородского государственного аграрного университета имени В.Я. Горина. 308503, Белгородская обл., Белгородский р-н, п. Майский, ул. Вавилова, 1, Russia. e-mail: [email protected]. 8) Аспирант, кафедры общей химии, Белгородский государственный национальный исследовательский университет. ул. Победы, 85, Белгород, 308015, Россия. e-mail: [email protected]

Аннотация. Изучение этиологии и патогенеза заболеваний желудочно-кишечного и респираторного трактов, а также разработка эффективных способов терапии и профилактики имеют важное народнохозяйственное значение в обеспечении населения страны продуктами животноводства. Широкое распространение желудочно-кишечных и респираторных болезней (25-76%) в крупных животноводческих хозяйствах обусловлено воздействием на организм животных и птиц многочисленных технологических стресс-факторов, снижающих естественную резистентность организма, патогенных и условно-патогенных микроорганизмов, как в отдельности, так и в различных ассоциациях. Этиологию гастроэнтеритов и пневмоний животных изучали комплексно, на основании эпизоотологических, клинических, патологоанатомических данных, результатов бактериологических, серологических, гематологических, иммунобиохимических методов исследований. Для выявления антител к возбудителям вирусных и хламидийной ин- фекций использовали стандартные биофабричные антигены. Антимикробную активность тилозинсодержащих препаратов в отношении референтных штаммов микоплазм, ахолеплазм, а также полевых культур эшерихий, пастерелл и золотистого стафилококка изучали на жидких и плотных питательных средах с применением индикатора 2,3,5- трифенилтетразолий хлорида. Установлено, что в этиологии желудочно-кишечных и респираторных болезней молодняка сельскохозяйственных животных принимают участие грамположительные (стафилококки) и грамотрицательные (эшерихии, сальмонеллы и т.д.) микроорганизмы, которые, как правило, выделяются в различных сочетаниях. У выделенных микроорганизмов изучена степень привыкания к широко применяемым в ветеринарной практике препаратам (фуразоналу, биовиту, сульгину, ампициллину, неомицину, стрептомицину). Установлено, что при проведении нескольких пассажей их через питательные среды, содержащие препараты (стрептомицина, неомицина и ампицилина сульфаты, тилозина тартрат, фуразонал, биовит, сульгин), микроорганизмы становятся устойчивыми к ним. Ключевые слова: телята, ягнята, поросята, гастроэнтериты, пеневмонии, этиология, стресс, микроорганизмы.

ФАРМАКОЛОГИЧЕСКИЕ ОБЗОРЫ

УДК: 615.213 DOI: 10.18413/2500-235X -2016-2-3-86-94

1 Авдеева Н.В. ВОЗМОЖНОСТИ ПРИМЕНЕНИЯ АНТАГОНИСТОВ Никитина В.А.2 Кочкарова И.С.3 ГЛУТАМАТНЫХ РЕЦЕПТОРОВ ПРИ ЛЕЧЕНИИ БОЛЕЗНИ Литвинова А.С.4 ПАРКИНСОНА

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1) Кандидат. мед. н., ассистент кафедры фармакологии, ассистент кафедры фармакологии, НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail: 999601@ bsu.edu.ru 2) Студентка факультета лечебного дела и педиатрии 4- го курса Медицинского института НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail: 999601@ bsu.edu.ru 3) Научный сотрудник центра доклинических и клинических исследований НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail: [email protected] 4) Студентка факультета лечебного дела и педиатрии 6- го курса Медицинского института НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail: [email protected] Аннотация. Болезнь Паркинсона – медленно-прогрессирующее хроническое нейродегенеративное заболевание. Вызывается прогрессирующим разрушением и гибелью нейронов, вырабатывающих нейромедиатор дофамин, – прежде всего в чёрной субстанции, а также и в других отделах центральной нервной системы. Недостаточная выработка дофамина ведет к активирующему влиянию базальных ганглиев на кору головного мозга. Ведущими симптомами являются: мышечная ригидность, гипокинезия, тремор, постуральная неустойчивость. Современная медицина пока не нашла методов излечения заболевания, однако существующие методы консервативного и оперативного лечения позволяющие значительно улучшить качество жизни больных и замедлить прогрессирование болезни. Существует предположение о ключевой роли гиперактивации глутаматных рецепторов в патогенезе болнзни Паркинсона. Это позволяет предположить, что глутаматные рецепторы могут быть новыми терапевтическими мишенями при лечении данной патологии. Изучение блокаторов глутаматных рецепторов является важной задачей, при поиске новых фармакологических средств, для лечения болезни Паркинсона. Исследования, проведенные на животных моделях, позволяют предположить, что изменение активности этих рецепторов может облегчить первичные двигательные симптомы болезни Паркинсона, а также побочные эффекты, вызванные заместительной терапией леводопы. Антагонисты АМРА- NMDA-рецепторов показали возможность реверсировать двигательную симптоматику и леводопа-индуцированные дискинезии в доклинических моделях болезни Паркинсона. Антагонисты метаботропных рецепторов глутамата являются еще более перспективными для лечения болезни Паркинсона, благодаря более «точной» работе в синапсе. Эти препараты также реверсируют двигательный дефицит, а так же защищают пациентов от нейродегенерации. Таким образом рецепторы глутамата представляют собой перспективные цели для разработки новых фармакологических методов лечения болезни Паркинсона Ключевые слова: болезнь Паркинсона, глутоматные рецепторы, базальные ганглии, NMDA рецепторы, AMPA рецепторы, метаботропные глутоматные рецепторы, леводопаиндуцированные дискинезии.

УДК: 615.225:617.735-007.23 DOI: 10.18413/2500-235X -2016-2-3-95-100

1 Даниленко Л.М. МЕТАБОЛИЧЕСКАЯ КАРДИОПРОТЕКЦИЯ: НОВАЯ Клочкова Г.Н.2 Кизилова И.В.3 КОНЦЕПЦИЯ В РЕАЛИЗАЦИИ КАРДИОПРОТЕКТОРНЫХ Корокин М.В.4 ЭФФЕКТОВ МЕЛЬДОНИЯ

1) Кандидат фармацевтических наук, доцент кафедры фармакологии Медицинского института НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail:[email protected] 2) Кандидат биологических наук, заведующая клинико-диагностической лаборатории, ОГБУЗ «Белгородская областная клиническая больница Святителя Иоасафа», ул. Некрасова 8/9, г. Белгород, РФ, 308007, e- mail:[email protected] 3) Главный врач ОГБУЗ «Яковлевская ЦРБ» ул. Ленина, г. Строитель, Яковлевский район, Белгородская обл., РФ, 309070, e-mail: [email protected] 4) Доктор медицинских наук, профессор; Медицинского института НИУ «БелГУ», ул. Победы, 85, г. Белгород, РФ, 308005, e-mail: [email protected] Аннотация. Результаты последних исследований подтверждают необходимость поиска средств для коррекции ишемического/ реперфузионного повреждения вследствие того, что уже RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY 123

известные гемодинамические препараты не оказывают должной кардиопротекции. Ключевым вопросом является возможность медикаментозного влияния на митохондрии кардиомиоцитов, что контролирует аэробный обмен веществ и обеспечение поступления АТФ в кардиомиоциты. Кроме того митохондрии являются мишенью ишемии/реперфузии и задействованы в кардиопротекторном механизме ишемического и фармакологического пред- и посткондиционирования. Влияние оксида азота при ишемической реперфузии, направленное на митохондрию, считается конечной целью кардиопротекции. При прекондиционировании путь начинается в мембране сарколеммы, а затем направляется в цитоплазму посредством множества каскадов энзимов, включая синтазу оксида азота (NOS), растворимую гуанилатциклазу (sGC) и протеинкиназу G (PKG). Таким образом, сигнал передается в митохондрии, где и происходит кардиопротекция. Доказано, что митохондрия осуществляет защиту сердца от ишемически-реперфузионных повреждений с помощью открытия митохондриальных АТФ-зависимых К + каналов и с помощью преобразования пропускной способности митохондрии. Концепция метаболической модуляции может быть использована при разработке стратегии кардиопротекции в клинике при ишемически/реперфузионных повреждениях миокарда. Ключевые слова: мельдоний, фармакологическое прекондиционирование, ишемически/реперфузионные повреждения, сердце.

УДК: 615.2 DOI: 10.18413/2500-235X -2016-2-3-101-106

Мартынов М.А.1 Мартынова О.В.2 3 ТИМОЗИН β4 КАК БАЗИС ДЛЯ СОЗДАНИЯ Шкилева И.Ю. Токарев И.А.4 РЕПАРАЦИОННОГО ПРЕПАРАТА НОВОГО ПОКОЛЕНИЯ Довгань А.Н.5

1) Врач-хирург, БОКБ святителя Иоасафа, Ул. Гагарина 2, Белгород, 308007, Россия, e-mail: [email protected] 2) Аспирант кафедры фармакологии, клинической фармакологии медицинского института Белгородского Государственного Национального Исследовательского Университета, 85, Ул. Победы, Белгород, 308015, Россия, e-mail: [email protected] 3) Студентка медицинского института Белгородского Государственного Национального Исследовательского Университета, 85, Ул. Победы, Белгород, 308015, Россия, e-mail: [email protected] 4) Онколог Белгородского областного онкологического диспансера, 1, Ул. Куйбышева, Белгород, Россия, e-mail: [email protected] 5) Аспирант кафедры фармакологии, клинической фармакологии медицинского института Белгородского Государственного Национального Исследовательского Университета, 85, Ул. Победы, Белгород, 308015, Россия, e-mail: [email protected]

Аннотация. В статье повествуется о тимозине β4. Описывается его строение, ряд стратегически важных свойств и возможностей. Приводится перечень исследований и достижений за последние несколько лет исследований. На основе его многофункциональной деятельности во время регенерации тканей в различных экспериментах на животных, тимозин β4 имеет потенциал для новых исследований, в области почек и печени, а также восстановления спинного мозга, костей и повреждения связок. Кроме того, он может быть полезен при лечении широкого спектра других заболеваний, в том числе в отношении последствий застарелых бактериальных повреждений и вирусных инфекций. Ключевые слова: тимозин β4, репарация, травматизм.

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УДК: 615.11(042.5) DOI: 10.18413/2500-235X -2016-2-3-107-114

Резников К.М. НА ПУТИ СОЗДАНИЯ ПАРАДИГМЫ СОВРЕМЕННОЙ ФАРМАКОЛОГИИ

Профессор кафедры фармакологии, доктор медицинских наук, профессор, Воронежский государственный медицинский университет им. Н.Н. Бурденко, ул. Студенческая, д.10, Воронеж, 394036, Россия, e-mail: [email protected]. Аннотация. В статье представлен современный подход в понимании формирования эффектов лекарственных средств. Прослежен путь изменения воззрений на механизм действия лекарств, в соответствии с процессами, возникающими при патологии. Обсуждены вопросы роли регуляторных процессов в формировании патологии и выздоровлении. Подчёркнута необходимость их регистрации для более раннего начала лечения. Проанализированы недостатки современной фармакотерапии с указанием на невозможность мониторирования действия лекарств в процессе лечения и на неполное понимание первичных взаимодействий лекарственного вещества с биологическим субстратом. Рассмотрены вопросы эффективности лекарственной терапии в зависимости от насыщенности организма кислородом. Особое внимание уделено водной среде организма, имеющей прямое отношение к формированию фармакологических эффектов. Затронуты такие вопросы, как зависимость действия лекарств от дозы, структурно-функциональная гетерогенность тканей, последовательность формирования патологического процесса и выбора способа его диагностики и другие. В работе названы наиболее важные возможности повышения эффективности лекарственной терапии, на основе понимания квантово-волновых процессов начального формирования фармакологического эффекта. Ключевые слова: фармакодинамика, патология, эффекты лекарств, лечение, диагностика, окислительно-восстановительный потенциал, регуляция.

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