Volume 3 (4) 2017 RESEARCH RESULT: PHARMACOLOGY and CLINICAL PHARMACOLOGY

Volume 3 (4) 2017 RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY

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; El. № FS 77-69077 of March 14, 2017.

The journal is supported by the Russian Scientific Society of Pharmacologists (RNOF), the Association of Clinical Pharmacologists of the Russian Federation, and is published in cooperation with the Association of Scientific Editors and Publishers (ASEP).

Volume 3, № 4. 2017

ONLINE SCHOLARLY PEER-REVIEWED JOURNAL First published online: 2015 ISSN 2500-235X

EDITORIAL TEAM:

EDITOR-IN-CHIEF: Mikhail V. Pokrovskii, Academician of the Russian Academy of Natural Sciences, Doctor of Medical Sciences, Professor, Head of the Department of Pharmacology, Institute of Medicine, Belgorod State National Research University, Russia. Expert in experimental pharmacology and history of pharmacology, member of the Association of Scientific Editors and Publishers (ASEP). DEPUTY EDITOR-IN-CHIEF: Pavel A. Galenko-Yaroshevsky, Corresponding member of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Kuban State Medical University, Russia. Expert in experimental pharmacology. EXECUTIVE SECRETARY: Tatyana V. Avtina, Сandidate of Pharmaceutical Sciences, Associate Professor, Department of Pharmacology, Institute of Medicine, Belgorod State National Research University, Russia. Expert in drug pharmacokinetics research. ENGLISH TEXT EDITOR: Igor Lyashenko, Candidate of Philology Sciences, Associate Professor, Department of English Philology and Intercultural Communication, Institute of Intercultural Communication and International Relations, Belgorod State National Research University. Expert in proofreading of English articles and texts.

EDITORIAL BOARD:

Branislava Miljković, PhD, Professor, Department of Pharmacokinetics and Clinical Pharmacy, University of Belgrade, Belgrade, Serbia. Expert in clinical pharmacology. Dmitry E. Skopin, Doctor of Physical and Mathematical Sciences, Professor, Al Balqa Applied University, Jordan. Expert in biomedical statistics and digital processing of biological signals. Elena B. Artyushkova, Doctor of Biological Sciences, Professor, Director of the Research Institute of Environmental Medicine, Kursk State Medical University, Russia. Expert in the field of biological research methods in pharmacology. Evgeny A. Konorev, Doctor of Medical Sciences, Professor, University of Kansas City, USA. Expert in preclinical drug research. Konstantin M. Reznikov, Honored Scientist of the Russian Federation, Doctor of Medical Sciences, Professor, Department of Pharmacology, Voronezh State Medical University named after N.N. Burdenko, Russia. Expert in experimental pharmacology. Lev N. Sernov, Honored Scientist of the Russian Federation, Doctor of Medical Sciences, Professor, General Director of "Farmkonsalting", Russia. Expert in the field of molecular screening of innovative molecules. Mikhail V. Korokin, Doctor of Medical Sciences, Professor, Department of Pharmacology, Institute of Medicine, Belgorod State National Research University, Russia. Expert in the field of pharmacology of innovative medicines. Natalia D. Bunyatyan, Doctor of Pharmaceutical 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. Expert in the field of pharmacological methods in pharmaceutical research. Oleg S. Gudyrev, Сandidate of Medical Sciences, Associate Professor, Department of Pharmacology, Institute of Medicine, Belgorod State National Research University, Russia. Expert in preclinical drug research. Radica Stepanović-Petrović, PhD, Full Professor, Department of Pharmacology, University of Belgrade, Belgrade, Serbia. Expert in preclinical drug research. Sergei Y. Shtrygol, Doctor of Medical Sciences, Professor, National University of Pharmacy, Ukraine. Expert in Clinical Pharmacology. Svetlana Ibrić, PhD, Vice-dean for science and international relations of the Faculty of Pharmacy, Department of Pharmaceutical Technology and CosmetologyBelgrade, University of Belgrade, Serbia. Expert in pharmacology, pharmaceutical technology. Tatyana A. Savitskaya, Сandidate of Chemistry Sciences, Professor, Department of Physical Chemistry, Belarusian State University, the Republic of Belarus. Expert in the field of chemistry and structure of action of biologically active substances. Tatyana G. Pokrovskaya, Doctor of Medical Sciences, Professor, Department of Pharmacology, Institute of Medicine, Belgorod State National Research University, Russia. Expert in clinical pharmacology. Zorica Vujić, PhD, Dean of the Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Belgrade, Belgrade, Serbia. Expert in the field of pharmacological methods in pharmaceutical research.

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

EDITORIAL Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yu.V. Oswald Schmiedeberg – the “father” of experimental pharmacology 3

EXPERIMENTAL PHARMACOLOGY

Bibik E.Yu., Saphonova A.A., Yeryomin A.V. , Frolov K.A., Dotsenko V.V., Krivokolysko S.G. Study of analeptic activity of tetrahydropyrido [2,1-b] [1,3,5] tiadiazine derivatives 20 Bogus S.K., Suzdalev K.F., Uvarov A.V., Galenko-Yaroshevsky P.A., Vasil'ev P.M., Vinakov D.V., Kiseleva N.V., Kochetkov A.N., Kiselev A.V. Anti-inflammatory and analgesic propertiesof SS-68 indole derivative 26 Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response 35 Dovgan A.P. Ligand of peripheral imidazoline receptors based on amides of heterocyclic acids C7070: effect on ishemized tissues 78 Efremova M.P., Ivasheva A.V., Korokin M.V. Study of pharmacological activity of Nigella damascena fixed oil in experiment 89 Miroshnichenko A.G., Perfil'yev V.Yu., Lysenko I.V., Zhernakova N.I. Interaction between some antibiotics and antioxidants 100 Peresypkina А.А. Correction of retinal angiopathy of hypertensive type by dimethylaminoethanol derivative 19-16 in experiment 113 Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver damages in carbon tetrachloride intoxication 120 Tarasova A.P., Danilenko L.M., Sernov L.N. Effect of pharmacological preconditioning with incretinomimetics exenatide and vildagliptin on the survival of ischemic tissues 132 Voronkov A.V., Gamzeleva O.Y. The effect of new forms for external application on the vasodilating function of the endothelium and the concentration of endothelial nitric oxide synthases in rats with an experimental model of a pathological scar at early healing times 151

CLINICAL PHARMACOLOGY

Zemskova V.A., Zoloedov V.I., Popova O.A. Optimization immunepharmacotherapy of pyoinflammatory diseases 160

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result: Pharmacology and Clinical 3 Pharmacology. 2017;3(4):3-19.

EDITORIAL

Rus.

UDC: 615:378 DOI: 10.18413/2313-8971-2017-3-4-3-19

Mikhail V. Pokrovskii1 Tatyana V. Avtina T. OSWALD SCHMIEDEBERG –THE “FATHER” OF Elena V. Zakharova EXPERIMENTAL PHARMACOLOGY Yulia. V. Belousova

Belgorod State National Research University, 85 Pobedy St., Belgorod, 308015 Russia Corresponding author, 1e-mail: [email protected]

“Our tribute to the memory of the Teachers and those who were pioneers of pharmacology is an invaluable gift to our descendants”

Abstract Biography. Oswald Schmiedeberg (1838-1921) was a son of a bailiff and a maid of honour, the eldest of the six children in the family. He was born and educated in the Russian Empire. Scientific activity. All his life he was completely devoted to science, making experimental pharmacology an independent scientific discipline, and was able to bring it to the international level. O. Schmiedeberg studied the action of muscarine and nicotine, digitoxin, hypnotics and analeptics. He was the first to introduce the concept of ―pharmacodynamics‖ and ―pharmacokinetics‖ of a drug. With his participation, the world‘s first pharmacological journal was founded, which is still published today. Science school. Working for many years at the University of Strasbourg, Schmiedeberg managed to educate about 120 students – professors from 20 countries of the world, many of whom later founded experimental pharmacology in their countries, for example, Abel in the USA, and N.P. Kravkov in Russia. Scientific activity of Schmiedeberg influenced scholars of his time and for generations to come, creating the preconditions for new high-profile discoveries and even for receiving Nobel prizes. But Oswald Schmiedeberg failed to obtain this high award himself, though he had been nominated 14 times.

Biography Gertrude Borchet. He worked as a bailiff in Oswald Johann Ernst Schmiedeberg Leidzen, later took charge of the forestry in (Figure 1) was born on 29.09.1838 (on Permisküla, and Paggar (Estonia), died in 1878 11.10.1838 New Style) in Gut-Laizane, in in Dorpat. The mother of O. Schmiedeberg, Courland (Laidze parish, Talsi municipality, Anna Lucy Bernard, was born in Lausanne Latvia), which was at that time part of the (Switzerland) in 1813, a daughter of Johann Russian Empire. Bernard, a watchmaker in Lausanne. She His father, Wilhelm Ludwig Schmiedeberg worked as a maid of honor and died in 1871. was born in 1809 in Vindau (Latvia), a son of His brother, Johann Julius Rudolf, was born in Johann Ernst (a mechanic in Libau) and 1840, worked as a forester in Estonia and was

never married. Oswald Schmiedeberg was the eldest of the six siblings [1, 2].

Fig. 1. Oswald Schmiedeberg [3]

After finishing a primary school in of the University of Dorpat, which was Permisküla, Schmiedeberg studied at a district connected with the hey day of Enlightenment in school of Dorpat (today Tartu, Estonia) from Russia in the second half of the 19th century. In 1852 to 1854. In 1855 he studied at the the 1860s, when in all spheres of scientific gymnasium in Dorpat, which he successfully activity there was a definite change for the finished in 1859, after which he entered the better, a system of freelance university lecturers Medical Faculty of the University of Dorpat, (privat-docents) started to develop at the where he was a student till 1866. The Imperial Medical Faculty, where only after 1863, young University of Dorpat (The Imperial University scientists (privat-docents) began to work on a of Yuryev from 1893 to 1918) was one of the constant basis. All this, undoubtedly, was due oldest universities in the imperial Russia; to the Enlightenment process in Russia at that nowadays it is The University of Tartu in period. The very development of the system of Estonia (Figure 2) [4, 5, 6]. freelance university lecturers (privat-docents) Schmiedeberg studied at the university at at Dorpat Medical Faculty proves that at that one of the best periods of the Medical Faculty December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result: Pharmacology and Clinical 5 Pharmacology. 2017;3(4):3-19. time it was in a flourishing condition, there wanted to study [2]. were those who could teach and those who

Fig 2. The Imperial University of Dorpat, 1860 [7]

After graduating from the University of independent of therapy, putting it on the Dorpat in 1866, O. Schmiedberg defended his experimental research basis, made him so doctoral thesis On Quantitative Determination famous that foreign universities such as The of Chloroform in Blood and Its Behavior University of Breslau, The University of Bonn Towards the Former (“Ueber die quantitative and The University of Giessen tried to hire him. Bestimmung des Chloroforms im Blute u. Sein R. Buchheim was the first scientist who put Verhalten gegen dasselbe”) under the pharmacology on sound scientific grounds, and supervision of Professor Rudolf Buchheim [8, the manual that he compiled included for the 9, 10, 11]. The title page of the dissertation of first time the description of the physiological the scientist can be seen in Fig. 3. effect of agents, on which their therapeutic Rudolf Buchheim was elected by the application was based. The vision of Buchheim Council of Dorpat University to the served as the cornerstone of modern Department of Pharmacology on December 30, pharmacology. One of the important 1846, and from October 1849 to 1867, he was achievements in Buchheim‘s scientific life is an ordinary professor at that department. His considered to have been the education of his fruitful professorship and his constant desire to follower in science, one of the greatest recognize pharmacology as a science scientists – Oswald Schmiedeberg [2, 13].

Fig 3. Title page of the thesis by O. Schmiedeberg [12]

While working at Dorpat University, laureate in Chemistry, when the latter was Schmiedeberg actively cooperated in his seeking a Doctor of Philosophy degree [2, 14]. research with outstanding scientists of that Georg Friedrich Carl Heinrich Bidder was time: a biochemist Karl Schmidt (1822-1894), a Russian physiologist and anatomist of anatomist Friedrich Heinrich Bidder (1810- German-Baltic descent, Professor and Rector of 1894), physiologist Karl Wilhelm von Kupffer the Imperial University of Dorpat (1857-1864), (1829-1902), etc. acorresponding member (1857) and an Karl Ernst Heinrich Schmidt was a Russian honorary member (1884) of St. Petersburg chemist of German-Baltic descent, a professor Academy of Sciences. In 1869, F.H. Bidder at the University of Dorpat, and a retired as Professor Emeritus [15, 16]. corresponding member of the Petersburg Carl Schmidt and Friedrich Bidder were Academy of Sciences (1873). He supervised a the first scientists who managed to dispel the degree project of Wilhelm Ostwald, a Nobel doubts of Henry Bence Jones, who had December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result: Pharmacology and Clinical 7 Pharmacology. 2017;3(4):3-19. published an article in The Lancet magazine in Pharmacology, Dietics and History of 1850, in which he wrote: ―The gastric juice is a Medicine. After being appointed an strongly acidic liquid secreted by the stomach Extraordinary Professor, Schmiedeber was ... . Which acid still remains unknown. Salt, appointed Director of the Pharmacological phosphoric, acetic, lactic and butyric acids are Institute, which he had held since the departure said to be present in gastric juice‖ [17]. In of Buchheim. 1852, F. Bidder and C. Schmidt published the Together with the renowned scientists of book Die Verdauungssaefte und der the University of Dorpat, Schmiedeberg made a Stoffwechsel (Digestive Juices and number of discoveries. Before 1870, his studies Metabolism), in which they presented the on physiological chemistry had led to the results of a quantitative analysis of gastric juice discovery of sulfuric acid in the urine of cats collected from various species of live animals, and dogs. Together with Ernest Bergman, he confirming the fact that the stomach normally conducted research on the poison of rotting secretes hydrochloric acid [15]. substances and discovered sepsin in form of its Karl Wilhelm Kupfer, a German anatomist, sulfuric acid salt. Together with Dr. Richard histologist and embryologist, was a student of Koppe, Schmiedeberg in 1868 studied the Friedrich Bidder, a prosector and an composition of the fly agaric (Agaricus extraordinary professor at the University of muscarius), which led to the isolation of Dorpat (1856–1866). His studies, conducted muscarin, the properties of which were studied jointly with Bidder, were on the structure of the in detail by both authors. Over that time, ten spinal cord. In honor of Kupfer, specialized scientific dissertations were completed under liver macrophages the main function of which the supervision by O. Schmiedeberg [2, 5]. is capturing and processing old nonfunctional In 1870, Schmiedeberg continued his blood cells were called Kupffer cells [18]. education in Germany, in Leipzig. He spent a After defending his dissertation in 1866, O. whole year at Leipzig University, working Schmiedberg became Assistant Professor to R. together with the outstanding physiologist Karl Buchheim at the Pharmacological Institute, and Friedrich Wilhelm Ludwig (1816-1895). O. in 1867 he received the title of Privat-docent. Schmiedberg successfully used the kymograph When Buchheim left Dorpat after getting an (device for recording blood pressure) invented invitation from the University of Giessen, by K. Ludwig in his scientific experiments and Schmiedeberg was asked to give lectures on reasonably considered K. Ludwig one of his pharmacology and dietics. In 1868, he was scientific teachers [19]. Fig. 4 shows a appointed Full-time Associate Professor, and in kymogram obtained on smoked paper. 1869 - an Extraordinary Professor of

Fig. 4. Example of kymogram on smoked paper [20]

In 1871, Oswald Schmiedeberg became time. Along with Schmiedeberg, there worked Full Professor of Pharmacology, Dietics and a number of prominent scientists. Among them History of Medicine at the University of was anatomist Heinrich Wilhelm Waldeyer Dorpat. From 1871 to 1872, Schmiedeberg (1836-1921), who was engaged in anatomical, visited Bern and Kцnigsberg (Prussia), and in histological, comparative-anatomical and 1872, Schmiedeberg resigned from the embryological studies. In 1884, he published a University of Dorpat after accepting a position paper in which he gave a detailed description of at the just-founded University of Strasbourg, the embryogenesis, structure and functional where later he was to get engaged in research significance of the pharyngeal lymphoid tissue and teaching for the next 46 years, started his (Pirogov-Waldeyer’s ring). In 1888, for the own scientific school and was the Director of first time he used the term ―chromosome‖, was the Pharmacological Institute [2]. one of the first supporters of Cajal‘s neural After arriving at the University of theory and suggested the term ―neuron‖ [23]. Strasbourg, his scientific laboratory was a very Other famous scholars were Felix Hoppe- small room in a hospital at Place de l'Hфpital. Seyler (1825-1895), one of the founders of the Along with promoting research in experimental Department of Biochemistry and the founder of pharmacology, in 1887 the University the journal of physiological chemistry and administration assigned a new spacious pathologist Friedrich Daniel von building for the laboratory, the building having Recklinghausen (1833-1910), whose research been designed by Schmiedeberg himself, in papers were on neurofibromatosis, parathyroid cooperation with the architect Otto Warth osteodystrophy and fibrous ostitis (these (1845-1918). Schmiedeberg‘s office and diseases, as well as a number of other personal laboratory were on the second floor, pathological processes were later called by his the laboratory at the corner with the balcony, name), and who studied rickets and the office immediately to the left with the bow osteomalacies, which later became classical. window (Figure 5) [21, 22]. Schmiedeberg was the youngest among The University of Strasbourg was one of colleague scientists. the most prestigious and best schools at the

Fig. 5 The Institute of Pharmacology in Strassburg,1877 [21]

In the period from 1918 to 1919, O. physiological antidote to the poison of fly- Schmiedeberg moved to Baden-Baden, where agaric, which will help avoid the life- he lived till his death. His friend in Baden- threatening consequences of accidental Baden was B. Naunyn. They were neighbors poisoning with this widespread species of and often would take a walk along a forest road mushrooms. Thus, poisoning can probably be in Baden-Baden, which is now called Schriever completely avoided. Lane [24]. Soon after studying the pharmacological His view of pharmacology as an affects of muscarine, O. Schmiedeberg began independent exact science, O. Schmiedeberg to study another pharmacological agent – laid in the third edition of his work nicotine. The Professor was the first in Dorpat Fundamentals of Pharmacology, for which he who got interested in this issue, and he further tried to provide a rational basis in contrast to continued his studies in Karl Ludwig‘s purely subjective empiricism [15]. laboratory in Leipzig. Schmiedeberg proved Schmiedeberg‘s Alma mater, The that nicotine suppressed the inhibitory effect of University of Dorpat, after the fall of the the vagus nerve on the heart and conlcluded Russian Empire in 1918 was intervened by the that this happened due to ganglionic blockade. Germans. Due to an increase of Russophobia That theory was examined thoroughly and was and the First World War, the University faculty later confirmed in the teachings by John members were evacuated to Voronezh, where Newport Langley (1852-1925) when studying they made up the basis for Voronezh State the autonomic nervous system [27, 28]. University [21, 22, 25]. About 150 years have passed since the Oswald Schmiedeberg died on discovery of the pharmacological effects of 12.07.1921, at the age of 83. muscarin and nicotine, but modern textbooks Scientific activity on pharmacology still describe vegetotrophic The vast knowledge acquired when agents basing on Schmiedeberg‘s cooperating with many professors in the field understanding of M- and N-cholinergic of medicine and chemistry allowed O. receptors, their agonists and antagonists. Schmiedeberg to make a number of Digitalis. One of the most famous discoveries. works by O. Schmiedeberg and the main Muscarine, nicotine. In 1869, in the direction of his research is rightly considered to monograph by Oswald Schmiedeberg and have been the study of digitalis, namely paramedic Robert Koppe, there appeared for alkaloids isolated from this flower. Despite the the first time an article about muscarin as a fact that there was little information about the toxic alkaloid extracted from fly-agarics medicinal properties of digitalis at that time, (Agaricus muscarius L.) [26]. When working this topic caused a lot of arguments and together, the scientists isolated pure poison controversy. It was O. Schmiedeberg who from fly-agarics picked in the vicinity of managed to work out many questions. Dorpat and described its pharmacological In 1874, when Schmiedeberg returned effects, named them muscarinic and proved that from France, he for the first time isolated a the poison possessed an antagonistic action separate substance from the collected red towards atropine. Antagonism manifested by flowers of the digitalis and called it digitoxin. atropine against muscarinic receptors was a This substance was proved to have a serious prototype of competitive antagonism. The work effect on the activity of the cardiovascular in question was of fundamental importance for system. His colleague, Robert Koppe, agreed to pharmacology and medicine as a whole and conduct an experiment to study the resulted in the discovery of the chemical pharmacological effects of digitalis on his own transfer of impulse in synapses by Otto Loewi body. During the experiment, R. Koppe (1873-1961). As O.Shmideberg and R. Koppe recorded the pulse on his wrist (Figure 6). wrote: ―These effects not only are of high Ingestion of 3.5 mg of digitoxin led to serious scientific, but also practical interest, since their poisoning, as well as to the side effects in form study has led to the discovery of a of arrhythmia (Pulsus bigeminus). December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result: Pharmacology and Clinical 10 Pharmacology. 2017;3(4):3-19.

Fig. 6. Radialis pulse curves of R. Koppe before (upper curve) and after (lower curve; pulsus bigeminus) ingestion of3.5 mg digitoxin over five days [21]

Later, R. Koppe described all the experiment purpose, Schmiedeberg first pharmacological effects of digitoxin removed the kidneys from the dog‘s body, and administration in his work, and Arthur then, with the help of blood released from Robertson Cushny (1866-1926), Professor of fibrinogen, studied kidney perfusion. This Pharmacology at the University of Edinburgh, experiment was more important for the future who worked with Schmiedeberg in Strasbourg of science than simply determining the precise for three years, in 1925 in his monograph - The location of the formation of hippuric acid. ―The Action and Uses in Medicine of Digitalis and fact that a number of other important issues Its Allies called Koppe‘s publication ―the best related to the process of metabolism in the description of the severe poisoning by digitalis animal‘s body, in particular, to the place and in a healthy person‖ and also translated the mechanism of urea formation, were solved with monograph into English [21, 29]. the help of tests on the removed kidney ... Resulting from his research, Schmiedeberg allows us to conclude that the value of this discovered 19 more alkaloids, including study can not be overestimated‖ , wrote about ouabain from parts of the oleander plant, as this Wldemar von Schroeder, Schmeideberg‘s well as substances from the bear's-foot and lily- student (1850-1898) [31, 32]. of-the-valley, and identified them as a single Camphor. Schmiedeberg and Hans pharmacological group, which he called a Handful Meyer successfully conducted a study group of digitalis [30]. of camphor. They found that camphor was Metabolism of xenobiotics. The first excreted from the body in the form of biochemical synthesis in the history of glucuronide. This study was the first to confirm biochemistry, namely, synthesis of hippuric the chemical transformations of drugs within acid from benzoic acid and glycine, was the body [2]. conducted in the laboratory by Schmiedeberg Sleeping pills. O. Schmiedeberg's in 1876, together with the outstanding dissertation of 1866 was on the anesthetic biochemist of the 19th century Gustav von effect of chloroform. Twenty years later, the Bunge, who worked at the University of scientist continued to develop this idea. He Dorpat. This fact proves that even after leaving suggested that some alcohols had a narcotic for Strasbourg in 1872 Schmiedeberg effect and slowed down breathing, whereas maintained scientific networks with the Russian ammonia, on the contrary, stimulated university and collaborated with its scientists in breathing. He supposed that some groups of the sphere of his biochemical studies. In 1877, atoms within a chemical compound, especially Schmiedeberg clearly demonstrated how this carbamates, could have analgesic and sedative reaction occurred in a dog‘s kidneys. For the pharmacological effects. Now, the hypnotic and December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result: Pharmacology and Clinical 11 Pharmacology. 2017;3(4):3-19. anesthetic effects of carbamates along with Schmiedeberg Research School stimulating the breathing have been confirmed. Oswald Schmiedeberg began his research ―This effect can be associated with the under the supervision of Rudolf Richard carbamate NH2 group, so that the character of Buchheim in Dorpat in the world‘s first the effects of this compound is preserved,‖ pharmacological research institute. At the Schmiedeberg wrote [33]. This work by beginning of his scientific career, Buchheim Schmiedeberg is noteworthy for three reasons. turned his own apartment into a research lab. First, it shows that the pharmacological Besides Schmiedeberg, Buchheim supervised principles and the biological effects of drugs about 90 post-graduate students and stated his depend on their chemical structure. Second, thoughts in essays and books. However, none this work described a completely new of Buchheim‘s initiatives and ideas would have anesthetic which is still used on animals. Third, ever been further developed, if pharmacologist the discovery led to a number of significant Oswald Schmiedeberg had not become one of discoveries of sleeping pills and sedatives such his doctoral students. Thanks to as bromisoval, barbiturates, and Schmiedeberg‘s own research and the fact that benzodiazepines. he had approximately 120 disciples from 20 Synthesis of urea. Schmiedeberg is countries – pharmacology and the considered the founder of the synthetic theory pharmacological school of Strasbourg got in of the formation of urea from ammonium vanguard and was studied worldwide, and the carbonate. According to this theory, urea is majority of well-known pharmacologists in the formed by the dehydration of carbamide-acid first half of the 20th century were his students ammonium, which can be considered as an (Fig. 7, 8). Schmeideberg‘s research activity intermediate stage of dehydration from was largely aimed at finding the correlation ammonium carbonate. When working in between the chemical structure of substances Dorpat, Schmiedeberg suggested that and their effectiveness as drugs. During his life, ammonium was part of urea. His further O. Schmiedeberg wrote over 200 scientific experiments in Strasbourg confirmed this books and articles, and his research is suggestion, and his student Waldemar von sometimes considered a major factor Schroeder demonstrated that the synthesis of determining the success of the German urea from ammonium carbonate took place in pharmaceutical industry prior to World War II the liver [34]. This knowledge was important [5, 24, 35, 36]. for understanding the process of reducing the Some disciples of Oswald Schmiedeberg acid-base balance in the formation of urea in [5, 21]: the liver in favor of increasing the formation of Otto Loewi (Nobel prize winner) [37], ammonia in the kidneys in acidosis. The John Jacob Abel (father of American synthetic theory of urea formation in the form pharmacology) [38], Heinrich Hermann Robert in which it was developed and substantiated by Koch (Nobel prize winner) [39], Rudolf the works of scientists of the 19th century, Gottlieb, Hans Horst Meyer, Carl Jacobj, Oskar existed with no change until the 30s of the 20th Minkowski, Alexander Ellinger, Heinrich century. In 1932, there appeared a new theory Dreser, Max Jaffe, George H. Whipple, of Krebs and Henseleit (1900-1981), which Corneel Heymans, Carl Ferdinand Cori; Arthur revealed the participation of new compounds in Robertson Cushny, Waldemar von Schroeder, the synthesis of urea. This theory was followed Sigmund Fraenkel, Franz Hofmeister, Alfred by a further discovery of the Krebs cycle, for Jaquet, Arthur Heffter, Max Arnold Cloetta, which Krebs was awarded the Nobel Prize in Vladimir Lindeman, Dickinson W. Richards , Physiology and Medicine in 1953. Vincenzo Cervello, Rudolf Eduard Kobert, Schmiedeberg also gave the first chemical Hermann Georg Fühner, Wolfgang Heubner, definition of the protein structure free from Ferdinand Siegert, Alessandro Baldoni, Edwin other impurities, by examining cartilage tissue. Stanton Faust, Louis Lewin. He managed to identify the structure of chondroitin [21]. December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result: Pharmacology and Clinical 12 Pharmacology. 2017;3(4):3-19.

Fig 7. Schmiedeberg with his disciples at a meeting to commemorate his 70th anniversary, Strasbourg, 1908 [21] 1. von Recklinghausen; 2. Reeb; 3. Cloetta; 4. Wallace; 5. Siegert;6. Heubner; 7. Fetzer; 8. Herlant; 9. Lindemann; 10. Faust; 11. Kobert; 12. Meyer; 13. Fühner; 14. Cervello; 15. Straub; 16. Jacobj; 17. Schmiedeberg; 18. Spiro;19. Hofmeister; 20. Harnack; 21. Muffat; 22. Heffter; 23. Cushny; 24. Huldschinsky; 25. His; 26. Minkowski; 27. Gottlieb; 28. Bethe; 29. Zinck.

The disciples of Oswald Schmiedeberg Bruzinskyy [43], Stanisіaw I. Czyrwiсski [44], in Russia Vladimir V. Nikolaev [45], Valerian O. Konstantin F. Arkhangelskyy [40], Podvysotskiy [46], Dmitryy M. Shcherbachev Nikolay P. Kravkov (1865-1924), the founder [47]. of the Russian pharmacology [41, 42], P.V.

Fig 8. O. Schmiedeberg with his disciples, 1905 [5]

O. Schmiedeberg's contribution to of this specialized pharmacological journal was enlightenment and his printed works published, which became a symbol of The idea of mass enlightenment was very combining physiological chemistry, pathology important for O. Schmiedeberg, since he and clinical science when studying the effects considered this an indispensable condition for of drugs. For a long time, the journal was the the development of science and society as a only source with the collection of the best whole. Together with his students, he wrote pharmacological studies and remained one of textbooks and popular books. the most important scientific journals. Despite With his friend Bernhard Naunyn and its interdisciplinary approach, O. Schmiedeberg pathologist and bacteriologist Edwin Klebs pursued the goal of developing pharmacology (1834-1913), O. Schmiedeberg founded the as a science independent of clinical and first pharmacological journal Archive of practical medicine. In 1925, the journal got a Experimental Pathology and Pharmacology, new name renamed, the modern name of the which was of paramount importance for archive is: Naunyn-Schmiedeberg's Archives promoting the development of theoretical of Pharmacology and it is still published medicine in Germany. In 1873, the first volume nowadays (Figure 9) [15, 21, 48].

Fig. 9 Title page of Archive of Experimental Pathology and Pharmacology, 1908. Leipzig [48]

O. Schmiedeberg also was the author of after he had developed a new drug for treating the book Grundriss der Pharmakologie in anemia ―Ferratin‖. In 1894, another work in Bezug auf Arzneimittellehre und Toxikologie English by O. Schmiedeberg was published: (Fundamentals of Pharmacology Through the Ferratin: the Ferruginous Element of Food. Doctrine of Medicinal Substances and Oswald Schmiedeberg wrote the book Toxicology), Leipzig, 1883 (Figure 10). In it, Arzneimittel und Genußmittel. (Medications Schmiedeberg developed his basic idea of and Pleasure), a treatise on stimulants, which perceiving the human body ―as a chemical was published in Leipzig in 1912 [50]. laboratory‖. The book was translated into most Über die Pharmaka in der Ilias und languages of the world in accordance with the Odyssee (Iliad and Odysseus in Pharmacology), translation by Hans Horst Mayer [49]. an essay by O.Schmiedeberg, which was very Another work by O. Schmiedeberg was popular, was published in Strasbourg in 1918 The Dietetic and Therapeutic Uses of Ferratin, [51]. published in English in Strasbourg in 1893

Fig. 10. Title page of O. Schmiedeberg‘s book Grundriss der Pharmakologie in Bezug auf Arzneimittellehre und Toxikologie (Fundamentals of Pharmacology Through the Doctrine of Medicinal Substances and Toxicology) [49]

Memoirs of the contemporaries about is his whole being, this is his whole life. After O.Schmiedeberg having some rest at home, at the beginning of a Unfortunately, no letters written by O. working day, he was already at his institute, Schmiedeberg have survived, not to mention and left his office only for a short lunch break, his autobiography. Nevertheless, we can learn leaving work late at night, usually being the last something about him, as a person, from the to leave‖. memoirs by Bernhard Naunyn and Hans Horst H.H. Meyer described Schmiedeberg‘s Meyer. teaching style: ―Schmiedeberg‘s teaching was O. Schmiedeberg never married. His not easy, he had a very strict style, he disciples said that he had once intended to demanded clear answers and clear thoughts. In marry and even bought a wedding cylinder for his lectures to the students, Schmiedeberg was that purpose, but his rival overtook him, and very serious about his teaching. His lectures the cylinder remained ―sitting on the shelf‖ for were, as well as his style of communication, the rest of his long life. B. Naunyn wrote about sober, thorough, very rich in content, and in the Schmiedeberg‘s attitude towards work: ‗"This process there were very bold judgments and December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Pokrovskii M.V., Avtina T.V., Zakharova E.V., Belousova Yulia V. Oswald Schmiedeberg – the “father” of experimental pharmacology. Research Result- : Pharmacology and Clinical 16 Pharmacology. 2017;3(4):3-19. assumptions, so he always impressed the pharmacology of the 19th-20th centuries. Of students, despite the fact that he avoided great importance is a system of specialized skillful speech patterns. When discussing scientific publications on pharmacology which scientific or political problems in the was created with his active participation. conversation, Schmiedeberg could expose Unfortunately, World War I destroyed extensive disproof or he alone could be against Schmiedeberg's Strasbourg Institute of everybody, but this exactly was his opinion. Pharmacology; however, the experimental This strictness also accounted for his success‖ pharmacology that he had created continued to [24, 27, 52]. develop. Such outstanding Schmeideberg's In 1956, the German Society for deciples as Heinrich Hermann Robert Koch and Experimental and Clinical Pharmacology and Otto Loewi were awarded the Nobel Prize; Toxicology (DGPT) established the highest Hans Horst Meyer, Rudolf Gottlieb, Heinrich award, the prize and the medal (Fig. 11) Dreser and many others pioneered the awarded ―for outstanding scientific industrial revolution in the pharmaceutical achievements in pharmacology, clinical industry. Special emphasis should be placed on pharmacology and toxicology‖. The award is the fact that in different years at named after Oswald Schmiedeberg and has Schmiedeberg's laboratory in Strasbourg there been presented since 1956 [53]. studied and did the internship a number of Russian scientists, such as Konstantin F. Arkhangelskyy, Nikolay P. Kravkov, the founder of the Russian pharmacology, P. V. Bruzinskyy, Stanisіaw I. Czyrwiсski, Vladimir V. Nikolaev, Valerian O. Podvysotskiy, Dmitryy M. Shcherbachev who stood at the origins of the Russian pharmacology.

Conflicts of Interest The authors have no conflict of interest to declare.

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EXPERIMENTAL PHARMACOLOGY

Rus.

UDC: 615.015.4 : 547.876 DOI: 10.18413/2313-8971-2017-3-4-20-25

Elena Yu. Bibik1 Anna A. Saphonova1 STUDY OF ANALEPTIC ACTIVITY OF Anton V. Yeryomin TETRAHYDROPYRIDO [2,1-b] [1,3,5] TIADIAZINE Konstantin A. Frolov1,2 DERIVATIVES Victor V. Dotsenko2,3 Sergey G. Krivokolysko1,2

1SI "Lugansk State Medical University the name of St. Luke ", 1g, 50th anniversary of the Defense of Lugansk block, Lugansk, 91045, LPR 2Research Institute "Himeх" GOU VPO "Lugansk State University. Vladimir Dal, Lugansk; LPR 3 FGBOU HPE "Kuban State University", Krasnodar) Corresponding author, 1e-mail: [email protected] Abstract Introduction: Our goal is to conduct an investigation of the analeptic activity of the tetrahydropyrido [2,1-b] [1, 3, 5] thiadiazine derivative group. Materials and Methods: Biological studies were carried out on 78 white pedigreed mature sexually transmitted rats of both sexes weighing 230-270 g in the autumn-winter period. The test substances were administered intragastrically at a dose of 5 mg/kg 1 hour prior to the induction of anesthesia. Animals of the control group received thiopental sodium at a dose of 70 mg/kg. As the reference preparation, sodium caffeine- benzoate was used intraperitoneally at a dose of 10 mg/kg. Results and Discussion: In the group of experimental animals that received intragastric substance 1 1 hour before thiopental anesthesia, a six-fold prolongation of the period of injection into anesthesia was found, the duration of anesthesia was comparable to that of the control. However, after 16 hours, 33.3% of the rats died, the rest of the animals were sharply inhibited. The original substance with laboratory cipher 2 significantly increases the time of introduction into anesthesia, has a pronounced analeptic activity, superior to that of caffeine. Conclusion: Thus, the conducted studies on the presence of 10 new biologically active compounds based on tetrahydropyrido [2,1-b] [1, 3, 5] thiadiazine derivatives in the spectrum of pharmacological activity showed the presence of the most pronounced analeptic and antinarcotic activity in compounds with laboratory ciphers 3, 6, 7 and 10. Compound 2, which is 6-oxo-8- {4 – [(2-chlorobenzyl) oxy] phenyl} -3- (2- ethoxyphenyl) -3,4,7,8-tetrahydro -2H, 6H-pyrido [2,1-b] [1, 3, 5] thiadiazine-9- carbonitrile, the analeptic effect is significantly superior to that of caffeine-benzoate that of sodium. Keywords: analeptic activity, derivatives of tetrahydropyrido[2,1-b][1,3,5]thiadiazine, thiopental sodium, caffein-sodium benzoate.

Introduction tetrahydropyrido [2,1-b] [1, 3, 5] thiadiazine The steady interest of various groups of derivatives are of particular interest. On the one researchers in the 1,3,5-thiadiazine derivatives hand, analeptics are antagonists of narcotic is due primarily to their biological activity and substances, they are used for respiratory high demand in agriculture and medicine. The depression due to carbon monoxide poisoning spectrum of practically important properties of and have a pronounced awakening effect in the most studied representatives of this group narcosis [14, 15]. On the other hand, the of compounds is very wide and includes respiratory analeptic caffeine is a part of antifibrinolytic, antituberculous, fungicidal, numerous combined paracetamol-containing bactericidal, anthelmintic, antitumor, medicines with antipyretic and anti- anticancer, hyperglycemic and other types of inflammatory activity. This increases mental activity. General questions of the chemistry and and physical performance, stimulates mental application of 1,3,5-thiadiazines are considered activity, motor activity, shortens the reaction in a number of fundamental reviews [1, 2, 3, 4, time, temporarily reduces fatigue and 5, 6, 7, 8, 9, 10, 11]. It‘s necessary to note that drowsiness in patients. the practical aspects of the use of condensed Our goal is to conduct an investigation of 1,3,5-thiadiazine derivatives have been studied the analeptic activity of the tetrahydropyrido to a much lesser extent up to the present. [2,1-b] [1, 3, 5] thiadiazine derivative group. Recently, we have shown that Materials and Methods tetrahydropyrido [2,1-b] [1, 3, 5] thiadiazines For the studies, (8R / 8S) -3-R-8-aryl-6- can act as effective inhibitors of the replication oxo-3,4,7,8-tetrahydro-2H, 6H-pyrido [2,1-b] of tick-borne flaviviruses, while in vivo in mice [1, 3, 5] thiadiazine-9-carbonitriles (1-10) they exhibit low total toxicity [12]; studies with synthesized by us from the noncatalyzed pronounced analgesic activity significantly Mannich reaction of substituted exceeding that of analgin have been observed tetramhydropyridine-2-thiolates of N- [13], studies of their anti-inflammatory and methylmorpholinium with primary amines and antipyretic activity have been carried out. excess formaldehyde [16, 17]: In this connection, screening studies to identify the analeptic activity of Ar O RNH2, HCHO CN EtOH, reflux R N N

O N S BH+ S Ar H CN

1-10 where, B = N-methylmorpholine; 1 – Ar = 3-MeO-4-EtOC6H3, R = 4-FC6H4; 2 – 4- (2-ClC6H4CH2O) C6H4, 2 – EtOC6H4; 3 – 4-MeOC6H4, cyclohexyl; 4 – 2-MeOC6H4, 3-Cl-4-MeC6H3; 5 – 2,4,5- (MeO) 3C6H2, 2-furfuryl; 6 – 3,4- (MeO) 2C6H3, 2-Me-3-Cl-C6H3; 7 – 3,4,5- (MeO) 3C6H2, benzyl; 8 – 3-MeO-4- EtOC6H3, 4-ClC6H4; 9 – 3-MeO-4-EtOC6H3, 2-FC6H4; 10 – 2-MeOC6H4, 2-EtC6H4.

Biological studies were carried out on 78 Animals throughout the study period were white pedigreed mature sexually transmitted kept in vivarium conditions on a standard diet rats of both sexes weighing 230-270 g in the of no more than six individuals in a cage in autumn-winter period in the certified accordance with the rules for working with morphological laboratory of the State laboratory animals. The conditions of keeping Institution "Lugansk State Medical University" animals and manipulations conducted with (certificate No. РЬ105 / 2008 from them met the requirements contained in the 30.12.2011). guidelines for ethical review of biomedical

Table The indices of the determination of analeptic activity in tetrahydropyrido [2,1-b] [1, 3, 5] thiadiazine derivatives Time of Duration of Characteristics of Group introduction in anesthesia, min the clinical picture anesthesia, min Control (sodium 6 20 thiopental) Comparison group 20 15 (sodium caffeine- benzoate) 1 2 20 Sharp slowing 2 55 15 Active 3 0 0 Animals did not enter anesthesia 4 6 20 Active 5 2 2 Death 50% within 1 hour 6 0 0 Animals did not enter anesthesia 7 0 0 Animals did not enter anesthesia 8 2 15 Active 9 4 960 Death 50% within 1 hour 10 0 0 Animals did not enter anesthesia

Substance 5, administered to the comparable to those in the control group of experimental animal 1 hour before the animals were recorded. experimental modeling of non-anional The original substance with laboratory anesthesia at a dose of 5 mg / kg, promoted cipher 2 significantly increases the time of acceleration of 3-fold occurrence in anesthesia. introduction into anesthesia, has a pronounced At the same time during the first hour, 50% of analeptic activity, superior to that of caffeine. the rats of this group died, and the remaining Attention is drawn to the triple extension of the 50% died for another 1.5 hours. period of introduction into anesthesia of In the group of experimental animals that animals of this experimental group. At the received intragastric substance 1 1 hour before same time, animals are not inhibited, active, thiopental anesthesia, a six-fold prolongation of show interest in food. the period of injection into anesthesia was Conclusion found, the duration of anesthesia was Thus, the conducted studies on the comparable to that of the control. However, presence of 10 new biologically active after 16 hours, 33.3% of the rats died, the rest compounds based on tetrahydropyrido [2,1-b] of the animals were sharply inhibited. [1,3,5] thiadiazine derivatives in the spectrum Comparing the parameters of injection and of pharmacological activity showed the the duration of anesthesia in rats that previously presence of the most pronounced analeptic and received substance 8 with those in the rats of antinarcotic activity in compounds with the control group, anesthesia appears 3 times laboratory ciphers 3, 6, 7 and 10. Compound 2, faster, and in duration is slightly shorter. In the which is 6-oxo-8- {4 – [(2-chlorobenzyl) oxy] experimental group with substance 4, indices phenyl} -3-(2-ethoxyphenyl) -3,4,7,8-

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Bibik E.Yu., Saphonova A.A., Yeryomin A.V. , Frolov K.A., Dotsenko V.V., Krivokolysko S.G. Study of analeptic activity of tetrahydropyrido [2,1-b] [1,3,5] tiadiazine derivatives. Research 24 Result: Pharmacology and Clinical Pharmacology. 2017;3(4):20-25. tetrahydro -2H, 6H-pyrido [2,1-b] [1,3,5] d]pyrimidine Derivatives. J. Agric. Food Chem. thiadiazine-9-carbonitrile, the analeptic effect is 2002;50(17):4839-4845. [Full text] significantly superior to that of caffeine- 9. Xia J, Zhimei Z, Xiaolin C, Ronge X, benzoate that of sodium. Song L, Lin W, Pengcheng L Preparation of 1,3,5-thiadiazine-2-thione derivatives of Conflicts of Interest chitosan and their potential antioxidant activity The authors have no conflict of interest to in vitro. Bioorganic & Medicinal Chemistry declare. Letters. 2007;17(15):4275-4279 [Full text] 10. Rodríguez-Fernándeza E, Manzanoa References JL, Benitoa JJ, Hermosab R, Monteb E, 1. Bermello JC, Piñeiro RP, Fidalgo LM, Criadoa J. Thiourea, triazole and thiadiazine Cabrera HR, Navarro MS. Thiadiazine compounds and their metal complexes as Derivatives as Antiprotozoal New Drugs. The antifungal agents. Journal of Inorganic Open Medicinal Chemistry Journal. 2011;5:51- Biochemistry. 2005;99(8):1558-1572. [Full 60. [PMC] text] 2. Dotsenko VV, Frolov KA, 11. Vicentini CB, Romagnol C, Andreotti Krivokolysko SG. Synthesis of partially E, Mares D. Synthetic Pyrazole Derivatives as hydrogenated 1,3,5-thiadiazines by the Growth Inhibitors of Some Phytopathogenic Mannich reaction. Chemistry of heterocyclic Fungi. J. Agric. Food Chem. compounds. [Khimiya geterotsiklicheskikh 2007;55(25):10331-10338. [Full text] soyedineniy]. 2015;51(2):109-127. (In Russian) 12. Osolodkin DI, Kozlovskaya LI, Dueva [Springer] EV, Dotsenko VV, Rogova YV, Frolov KA, 3. Kanno H. 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[Full text] antinarcotic effect of stimulants of the nervous 6. Rodríguez H, Suárez M, Albericio F. system. Leningrad: Medgiz; 1960. 269 p. (In Thiadiazines N, N-Heterocycles of Biological Russian) [Full text] Relevance. Molecules. 2012;17:7612-7628. 15. Arbuzov SYa, Nikiforov MI. Systemic doi: 10.3390/molecules17077612. [Full text] nerve anesthesia. Leningrad: Medicine; 1967. 7. Moody CJ. Polyoxa, Polythia and 224 p. (In Russian) [Full text] polyaza Six-membered Ring Systems. In 16. Dotsenko VV, Frolov KA, Pekhtereva Comprehensive Heterocyclic Chemistry. TM, Papainina OS, Suykov SYu, Krivokolysko Pergamon Press: Oxford; 1984, vol. 3. pp. SG. Design and Synthesis of Pyrido [2,1-b] 1039-1086. [Full text] [1,3,5] thiadiazine Library via Uncatalyzed 8. Vicentini CB, Forlani G, Manfrini M, Mannich-Type Reaction. 2014. ACS Comb. Sci. Romagnoli C, Mares D. Development of New 2014;(16):543-550. [Full text] Fungicides against Magnaporthe grisea: 17. Dotsenko VV, Krivokolyasko SG, Synthesis and Biological Activity of Chernega AN, Litvinov VP. Synthesis and Pyrazolo[3,4-d][1,3]thiazine, Pyrazolo[1,5- structure of pyrido [2,1-b] [1,3,5] thiadiazine c][1,3,5]thiadiazine, and Pyrazolo[3,4- December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Bibik E.Yu., Saphonova A.A., Yeryomin A.V. , Frolov K.A., Dotsenko V.V., Krivokolysko S.G. Study of analeptic activity of tetrahydropyrido [2,1-b] [1,3,5] tiadiazine derivatives. Research 25 Result: Pharmacology and Clinical Pharmacology. 2017;3(4):20-25. derivatives. Reports AN. 2003;389(6):763-767. Konstantin A. Frolov, PhD in Chemistry, (In Russian) [Full text] Leading Engineer of NIL "Himeks" GOU VPO 18. Belous YuB. Ethical examination of "Lugansk State University named after. biomedical research. Method. Vladimir Dal », 91034, Lugansk, apt. Youth, recommendations. Moscow; 2005. 156 p. (In 20a; Assistant of the Department Russian) [Full text] of Pharmaceutical Chemistry and Pharma- 19. Khubriev GU. Manual on cognosy of the State Institution "Lugansk State experimental preclinical study of new Medical University", 91045, Lugansk, apt. 50th pharmacological substances. M.: Medicine; nniversary of the Defense of Lugansk, 1d, + 2005. 832 p. (In Russian) [Full text] 38-066-569-38-37, [email protected]. 20. Kobzar AI. Applied mathematical Individual contribution: Took part in the statistics. M: Fizmatlit; 2006. 816 p. (In experimental part of the work, performed Russian) [Full text] statistical processing of the data obtained, interpreted and described the results of the Author Contributions study. Elena Yu. Bibik, Doctor of Medical Victor V. Dotsenko, Doctor of Chemical Sciences, Head of the Department of Sciences, Leading Researcher of the LLP Fundamental and Clinical Pharmacology, State "Himeks" GOU VPO "Lugansk State Institution "Lugansk State Medical University. Vladimir Dal », 91034, Lugansk, University", 91045, Lugansk, apt. 50th apt. Youth, 20a; Associate Professor of the anniversary of the Defense of Lugansk, 1d, Department of Organic Chemistry and + 38-099-711-33-71, [email protected]. Technologies of the Kuban State University, Individual contribution: Took part in the 350040, Krasnodar, ul. Stavropol, 149, + 7- experimental part of the work, performed 989-237-27-76, [email protected]. statistical processing of the data obtained, Individual contribution: Took part in the interpreted and described the results of the experimental part of the work, performed study. statistical processing of the data obtained, Anna A. Saphonova, Assistant of the interpreted and described the results of the Department of Fundamental and Clinical study. Pharmacology, State Institution "Lugansk State Sergey G. Krivokolysko, Doctor of Medical University", 91045, Lugansk, apt. Chemical Sciences, Scientific Leader of the 50th anniversary of the Defense of Lugansk, LLP "Himex" GOU VPO "Lugansk State 1g, + 38-050-422-31-48, [email protected] University. Vladimir Dal », 91034, Lugansk, Individual contribution: Took part in the apt. Youth, 20a; Head of the Department of experimental part of the work, performed Pharmaceutical Chemistry and Pharmacognosy statistical processing of the data obtained, of the State Institution "Lugansk State Medical interpreted and described the results of the University", 91045, Lugansk, apt. 50th study. anniversary of the Defense of Lugansk, 1d + Anton V. Yeryomin, Candidate of 38-050-422-61-19, [email protected] Sciences, Associative professor of the Individual contribution: He synthesized the Department of Fundamental and Clinical substances under study, took part in the Pharmacology, State Institution "Lugansk State experimental part of the work, performed Medical University", 91045, Lugansk, apt. statistical processing of the data obtained, 50th anniversary of the Defense of Lugansk, interpreted and described the results of the 1g, + 38-95-835-53-20, [email protected] study. Individual contribution: Took part in the experimental part of the work, performed Received: October, 19, 2017 statistical processing of the data obtained, Accepted: November, 30, 2017 interpreted and described the results of the Available online: December, 30, 2017 study.

Rus.

UDC: 616.12-008.313:615.222:615.015]-092.9 DOI: 10.18413/2313-8971-2017-3-4-26-34

Saida K. Bogus1 Konstantin F. Suzdalev2 A.V. Uvarov1 Pavel A. Galenko-Yaroshevsky1 ANTI-INFLAMMATORY AND ANALGESIC P.M. Vasil'ev4 PROPERTIES OF SS-68 INDOLE DERIVATIVE Dmitry V. Vinakov3 Natalia V. Kiseleva1 A.N. Kochetkov3 A.V. Kiselev5

1Kuban State Medical University, 2Southern Federal University, 3Belgorod State National Research University, 4Volgograd State Medical University, 5Krasnodar branch of S.N. Fyodorov Eye Microsurgery Complex Corresponding author, 1e-mail: [email protected]

Abstract The experiments with rats show that when administered intravenously (5 mg/kg) and intragastrically (5, 10, 15 and 20 mg/kg), SS-68 compound exerts an anti- inflammatory effect (AIE) on models of acute inflammation, caused by carrageenin and serotonin, and exerts no AIE in case of edema induced by complete Freund‘s adjuvant (CFA) and histamine, except for intragastric administration at a dose of 20 mg/kg. By its antiphlogogenic effect in case of carrageenin and serotonin edemas, SS-68 is comparable to diclofenac (5, 10 and 15 mg/kg intragastrically) and indomethacin (10 mg kg intragastriccally), and exceeds piroxicam (20 mg/kg intragastrically). By reference to its therapeutic index (TI), in the first case SS-68 exceeds diclofenac, indomethacin and piroxicam 2.2, 15.6 and 5.6 times, and in the second case it exceeds them 1.8, 12.8 and 4.4 times, respectively. In the mouse acetic writhing test, which reflects the predominant effect on κ (kappa)- opioid receptors, SS-68 when administered intraperitoneally at doses of 0.03, 0.06 and 0.12 mg/kg causes a dose-dependent decrease in the number of writhes by 40.0, 69.0 and 80.3%, respectively. Butorphanol, used for comparison in doses of 0.03, 0.12, 0.24, 0.48 and 0.96 mg/kg, exerts a dose-dependent analgesic effect (AE), with the number of writhes being 41.5, 52.4, 65.3, 71.1 and 80.6%, respectively. By its analgesic activity and TI, SS-68 exceeds butorphanol 1.9 and 2.7 times, respectively. Based on the results of the docking of affinity indicators for κ1-opioid receptors of SS-68, butorphanol (agonist-antagonist), as well as U-50488 compounds (selective agonist), it was established that, in accordance with the calculated values of the binding constant, the affinity in relation to SS-68 was 4.53 times higher than for butorphanol, and when compared with U-50488 it was 2.84 times lower. The suggestion is that AE of SS-68, like that of butorphanol, can be linked with both affinity for κ1-receptors and for other types of opioid receptors (mu, delta) and κ- receptor subtypes (κ2 or κ3).

Keywords: SS-68indole derivative, diclofenac sodium, indomethacin, piroxicam, anti-inflammatory effect, carrageenin, serotonin, histamine, complete Freund's adjuvant, analgesic effect, kappa opioid receptors

Introduction ml) were injected subplantarly into the right Earlier, we have shown that SS-68 indole hind paw of the rats. The compound SS-68, derivative has a high antiarrhythmic (in case of water-based for injection, was injected heart rhythm disorders of peripheral and central intravenously (iv) into the vein of the tail and genesis) and anti-ischemic (antianginal) effect intragastrically (IG) once for histamine and [1]. Besides, it is known that indole derivatives, serotonin-induced edemas and twice in case of in particular indomethacin and etodolac [2], edemas caused by carrageenin and CFA. have pronounced anti-inflammatory and Diclofenac sodium, indomethacin and analgesic properties. piroxicam (tablets crushed to homogeneous According to [3], of all opioid receptor mass in Tween-80), used as reference subtypes, the κ(kappa)-opioid receptor system preparations, were injected ig once through the takes the most significant part in developing the probe 1 hour before phlogogens. aforementioned pharmacological effects of The volume of the paws (the right one and many substances, and κ-receptor agonists can the left intact one) was measured potentially become promising in terms of being oncometrically using a plethysmometer used as a base for creating new medications (―UgoBasile‖, Italy) 4 and 24 hours after with antiarrhythmic, antianginal, anti- carrageenin and CFA administration, 30 and 45 inflammatory, analgesic and other effects. minutes after histamine and serotonin In view of the foregoing, it was of interest administration, respectively. to study the anti-inflammatory and analgesic For assessing the inflammatory response of (realized through the impact on κ-opioid the paw, the following formula (1) was used: receptors) effect of SS-68. The Goal was to evaluate the anti- , (1) inflammatory and analgesic effect of SS-68. where I – is an increase in paw volume Materials and Methods (%); V – paw volume after injection of The SS-68 compound was synthesized at t phlogogen (ml); and V – paw volume before the Department of Natural and High Molecular o phlogogone administration (ml). Compounds at the Southern Federal University (Rostov-on-Don, Russia). The therapeutic effect of SS-68 and The experiments were carried out on 87 reference preparations was evaluated in white non-linear male mice and 302 male rats comparison with the control; the calculations weighing 22-24 g and 210-230 g, respectively. were made by using the formula (2): The animals were kept in the standard conditions in a vivarium in accordance with GOST R 50258-92. , (2) Studies of anti-inflammatory and analgesic effects were carried out in compliance with the where e – are experimental animals, c – are ―The Guidelines for Conducting Pre-clinical control animals. Studies of Medications‖ [4]. To study the effect of SS-68 on exudative In addition, the antiphlogogenic affect of inflammation, there were used the models of substances was assessed by average effective inflammation caused by carrageenin [5], doses (ED50) and a range of therapeutic effect – serotonin, histamine and complete Freund‘s the therapeutic index (TI = LD50 / ED50). adjuvant [4]. Phlogogenic substances (made by The analgesic effect was studied using ―Sigma‖) – carrageenin (1%), serotonin (0.1%), mouse acetic writhing test. The chosen test is histamine hydrochloride (2 mM) and CFA (0.1 intended to study the acute visceral and deep December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Bogus S.K., Suzdalev K.F., Uvarov A.V., Galenko-Yaroshevsky P.A., Vasil'ev P.M., Vinakov D.V., Kiseleva N.V., Kochetkov A.N., Kiselev A.V. Anti-inflammatory and analgesic propertiesof SS-68 28 indole derivative. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):26-34. somatic pain [6, 7]. J/mol; T – temperature, the standard value in The SS-68 compound and butorphanol docking is 300 oK. tartrate (JSC Moscow Pharmaceutical Factory, Statistical processing of the data obtained Russia), taken for comparison, both water- in graded and alternative forms was carried out based for injections, were administered to the by means of computer software developed at animals once intraperitoneally (ip) to the right the Department of Pharmacology of Kuban lower third of the abdomen in doses ranging State Medical University of the Ministry of from 0.03 to 0.12 mg/kg and from 0.03 to 0.96 Healthcare of Russia. mg kg, respectively, 5 minutes before ip (on the left side) injection of 0.6% acetic acid solution Results and Discussion (0.1 ml/10 g of body weight) [6]. The degree of SS-68 compound administered iv to rats at analgesic effect (A) of substances was a dose of 5 mg/kg caused a marked inhibition expressed as a percentage, using the following of exudative inflammation triggered by formula (3): phlogogens carrageenin and serotonin, while inhibition of edema compared with the control , (3) group was 71.4 and 77.8 %, respectively (Table 1). No inhibition of edema was observed when administering SS-68 in the indicated dose and where Nc – is the number of writhes in the via the above channel in cases of inflammatory control group of animals, Ne – in the process caused by CFA and histamine (Table experimental group. 1). In cases of administering ig SS-68 and In addition, the analgesic effect was diclofenac in doses of 5, 10 and 15 mg/kg, the evaluated by ED and TI. 50 inhibition of carrageenin and serotonin edema Besides, the κ -opioid receptor SS-68, 1 was 36.2 and 27.5, 59.0 and 52.8, 75.2 and 79.2 butorphanol, a partial agonist of the κ 1 for the former substance, and 24.5 and 28.4, receptors, and U-50488, a highly selective κ 1 52.4 and 54.3, 67.8 and 72.5% for the latter receptor agonist, were docked into a specific substance, respectively, i.e., in terms of the binding site. The construction of 10 antiexudative effects of SS-68 and diclofenac conformations of each compound was carried in presence of marked phlogogens, they were out in Marvin Sketch 17.1.23 program [8]. close. It was of interest to compare the These conformations were optimized in antiphlogogenic effect of SS-68 under the MOPAC2016 [9] and the best one with accepted experimental conditions with that of minimal energy was selected. The docking of a indomethacin and pyroxicam in doses of 10 and human κ -opioid receptor (PDB code 4DJH) 1 20 mg/kg, which, according to our data and into an X-ray diffraction dimer model [10] was literature data [13], corresponds to ED . It was performed using AutoDock Vina 1.1.1 [11] 50 found that with carrageenin and serotonin (each compound was docked 5 times into each inflammation, indomethacin and piroxicam of the two dimer sites), along with calculating caused inhibition of edema by 45.4 and 97.1, the minimum docking energy ΔE. The 54.8 and 40.6%, respectively (Table 1), that is, ensemble docking procedure is described in the antiphlogogenic effect of these drugs was detail in [12]. close to that SS-68. The exception was the The calculation for the docking energy ΔE antiexudative effect of indomethacin in of the binding constants K was carried out serotonin edema, which was more significant using the formula (4): than that of SS-68, as well as of diclofenac and ΔE/RT- = eK , (4) piroxicam.

where R – is gas constant equal to 8.314

Table 1 The impact of SS-68, diclofenac, indomethacin and piroxicam on the development of acute exudative edema caused by carrageenin, CFA, serotonin and histamine in rats (M ± m)

Edema Inflammatio Substance No of Dose, Edema increase, Route inhibition, n initiator under study animals mg/kg % % Carrageenin Control 8 59.7 ± 6.5 SS-68 8 5 iv 16.9 ± 3.6*** 71.7 10 5 ig 38.1 ± 3.1** 36.2 10 10 ig 24.5 ± 4.1*** 59.0 10 15 ig 14.8 ± 3.8*** 75.2 Diclofenac 9 5 ig 45.1 ± 2.1* 24.5 10 10 ig 28.4 ± 4.1*** 52.4 10 15 ig 19.2 ± 3.5*** 67.8 Indomethacin 8 10 ig 32.6 ± 5.2** 45.4 Piroxicam 8 20 ig 27.0 ± 8.6*** 54.8 Serotonin Control 8 78.8 ± 3.4 SS-68 8 5 iv 17.5 ± 6.0*** 77.8 10 5 ig 57.1 ± 4.7** 27.5 10 10 ig 37.2 ± 4.3*** 52.8 10 15 ig 16.4 ± 3.8*** 79.2 Diclofenac 10 5 ig 56.4 ± 4.8** 28.4 9 10 ig 36.0 ± 5.6*** 54.3 10 15 ig 21.7 ± 4.2*** 72.5 Indomethacin 8 10 ig 2.3 ± 0.9*** 97.1 Piroxicam 8 20 ig 46.8 ± 5.8** 40.6 CFA Control 8 147.0 ± 9.0 SS-68 8 5 iv 154.9 ± 8.0 -5.4 8 10 ig 138.7 ± 7.6 5.6 10 20 ig 105.0 ± 8.2** 28.6 Diclofenac 10 10 ig 98.6 ± 6.3*** 32.7 Indomethacin 8 10 ig 67.7 ± 5.0*** 53.9 Piroxicam 8 20 ig 112.6 ± 17.6 23.4 Histamine Control 8 70.0 ± 9.7 SS-68 8 5 iv 54.1 ± 9.2 22.7 10 10 ig 68.3 ± 7.9 2.4 10 20 ig 37.4 ± 4.5** 46.6 Diclofenac 8 10 ig 29.0 ± 3.8*** 58.6 Indomethacin 8 10 ig 40.3 ± 8.6* 42.4 Piroxicam 8 20 ig 60.5 ± 4.7 13.6 Note: * р< 0.05, ** p< 0.01,*** p< 0.001 compared with control

To assess the antiexudative effect of SS-68 almost comparable. As for piroxicam, SS-68 and reference preparations, we compared their was 2.4 times more significant by this indicator ED50 (calculated for SS-68 and diclofenac and in relation to it. By their TI in the case of borrowed from literature sources for piroxicam carrageenin and serotonin inflammation, SS-68 [13]) and TI. It turned out that by activity exceeded diclofenac, indomethacin and (ED50) SS-68, diclofenac and indomethacin are piroxicam 2.2, 15.6 and 5.1 times in the former

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Bogus S.K., Suzdalev K.F., Uvarov A.V., Galenko-Yaroshevsky P.A., Vasil'ev P.M., Vinakov D.V., Kiseleva N.V., Kochetkov A.N., Kiselev A.V. Anti-inflammatory and analgesic propertiesof SS-68 30 indole derivative. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):26-34. case, and 1.8, 12.8 and 4.4 times in the latter case, respectively (Table 2). Table 2 Acute toxicity, anti-inflammatory effect and TI of SS-68, on carrageenan and serotonin models of exudative inflammation in rats

Exudative inflammation models carrageenan serotonin Substance LD 1, mg/kg 50 ED , ED , mg/kg TI 50 TI 50 mg/kg SS-68 608.0 8.3 73.3 9.5 64.0 Diclofenac 350.02 10.4 33.7 9.6 36.5 Indomethacin 47.03 10.0 4.7 9.4 5.0 (26.0 ’ 85.0) Piroxicam 290.04 20.04 14.5 20 14.5 (193.0 ’ 435.0) (13.0 ’ 32.0) 1 For mice via ig administration 2Neugodnova, O.P. et al. [14]. 3 Tsarichenko, G.V., Gan ina, G.A. [15]. 4Sigidin, Ya.A. et al. [13]. Note: In brackets is confident limit when p=0.05.

With CFA-and histamine-induced edema, certain extent attest to the ―universal‖ SS-68 (iv 5 mg/kg and/or 10 mg/kg) and mechanism of anti-inflammatory effect of SS- piroxicam (ig 20 mg kg) showed no statistically 68. significant anti-inflammatory effect, whereas Thus, SS-68 with iv and ig administration SS-68 (ig 20 mg/kg), diclofenac (ig 10 mg/kg) can exert an anti-inflammatory (dose- and indomethacin (ig 10 mg/kg) inhibited the dependent in the latter administration route) edema induced by the former phlogogen by effect on models of acute carrageenin- and 28.6, 32.7 and 53.9%, by the latter – by 46.6, serotonin-induced inflammation, and exerts no 58.6 and 42.4%, respectively. anti-inflammatory effect with CFA- and Our data on the anti-inflammatory effect of histamine-induced edema, except cases of ig SS-68 in the selected models of edema administration at a dose of 20 mg/kg. reflecting various pathogenetic mechanisms of According to antiphlogogenic activity in acute exudative inflammation (carrageenan carrageenan and serotonin edema, SS-68 is edema simulates the prostaglandin phase of comparable with diclofenac and indomethacin inflammation completed by affecting and exceeds piroxicam, and by TI it exceeds all cyclooxygenase; serotonin and histamine the reference preparations. edemas simulate the release of the marked In the mouse acetic writhing test, which biogenic amines increasing vascular reflects the predominant effect on κ-receptors, permeability from mast cells; CFA-induced the number of writhes in the control group of edema coincides with the initial phase of animals was 29.4 on average. The SS-68 inflammation involving cytokines, in particular compound at doses of 0.03, 0.06 and 0.12 IL-1, IL-2, IL-6, IL-8, TNF-α, IFN-γ, as well as mg/kg caused a dose-dependent decrease in the leukotrienes accumulated at that stage of number of writhes by 40.0, 69.0 and 80.3%, arachidonic acid metabolism, which is realized respectively (Table 3). via the 5-lipoxygenase pathway [16, 17]) to a

Table 3 Analgesic effect of SS-68 and butorphanol with ip administration in mouse acetic writhing test (M ± m)

Analgesic effect No of Dose, Substance number of writhes in relation to animals mg/kg control, % Control 12 29.4 ± 2.8 - 10 0.03 17.6 ± 2.6* 40.0 SS-68 10 0.06 9.1 ± 1.2** 69.0 10 0.12 5.8 ± 1.1** 80.3 10 0.03 17.2 ± 2.4* 41.5 8 0.12 14.0 ± 1.3** 52.4 Butorphanol 9 0.24 10.2 ± 1.5** 65.3 8 0.48 8.5 ± 1.6** 71.1 10 0.96 5.7 ± 1.2** 80.6 Note: * р< 0,01, ** p< 0,001compared with control

Butorphanol, used for comparison under and TI of SS-68 and butorphanol showed that the experimental conditions, at doses of 0.03, for the former their values are 0.038 mg/kg and 0.12, 0.24, 0.48 and 0.96 mg/kg had a dose- 7276.3, and for the latter – 0.072 mg/kg and dependent analgesic effect, with the number of 2666.7, that is, by its analgesic effect and TI, writhes in comparison with that in the control SS-68 exceeds butorphanol 1.9 and 2.7 times, group being 41.5, 52.4, 65.3, 71.1 and 80.6%, respectively (Table 4). respectively (Table 3). The calculation of ED50 Table 4 Acute toxicity, analgesic effect and TI of SS-68 and butorphanol when administered ip in mouse acetic writhing test

LD , Analgesic effect Substance 50 mg/kg ED50, mg/kg relative TI relative SS-68 276.5 0.038 1.9 7276.3 2.7 Butorphanol 192.01 0.072 1 2666.7 1 1Аnisimova, V.A. et al. [18].

To confirm the agonistic effect of SS-68 on accordance with the target value of the binding κ1-receptors, we compared the affinity values constant, the affinity of SS-68 for the κ1-opioid of SS-68, butorphanol, and U-50488 compound receptors was 4.53 higher than that of for these receptors, calculated basing on the butorphanol, and 2.84 times lower than that of docking results. It was found that, in U-50488 (Table 5). Table 5 Results of docking SS-68, butorphanol and U-50488 substance into specific binding site of κ1-opioid receptor

Docking energy ΔE, Substance Binding constant K, nM kcal/mol SS-68 -8.60 538.1 Butorphanol -7.70 2437.3 U-50488 -9.20 196.5

It should be noted that U-50488 is more Dynorphin Peptides Differentially Regulate the toxic than SS-68: when administered ip to Human Kappa Opioid Receptor. Life Sci. mice, the LD50 of the former being 25 mg/kg 2007;80(15):1439-1448. [19], whereas for the latter – 276.5 mg/kg. doi.org/10.1016/j.lfs.2007.01.018 [Abstract] Thus, SS-68 has an analgesic effect, 4. Mironov AN. et al. (2012). realized through the peripheral level of Rukovodstvo po provedeniyu doklinicheskix establishing pain sensitivity through agonistic issledovanij lekar-stvennyx sredstv. [A Guide action on κ1-receptors. It is possible that the to Preclinical Research of Medications]. analgesic effect of SS-68, like that of Moscow. (in Russian) butorphanol, can be linked to affinity for κ1- 5. Winter CA, Risley EA, Nuss GW. receptors, other types of opioid receptors (mu, Carrageenin-induced Edema in Hind Paw of delta) and for κ-receptor subtypes (κ2 or κ3). To the Rat as an Assay for Anti-inflammatory identify the possible involvement of certain Drugs. ProcSocExpBiol Med. 1962;111:544- opioid receptors in the mechanism of analgesic 547. [PubMed] action of SS-68, additional in-depth studies are 6. Spasov AA., Grechko OYu., Shtaryova needed, which will make it possible to classify DM., Anisimova VA. Analgesic Properties of SS-68 as either selective or non-selective κ- Morpholinoethylimidazobenzimidazole receptor agonist. Derivative RU-1205. Russian Journal of Experimental and Clinical Pharmacology Acknowledgements [Eksperimentalnaya i Klinicheskaya The SS-68 compound was synthesized and Farmakologiya]. 2013;76(9):15-18. (in developed within the grant project from Russian) [eLIBRARY] The Southern Federal University ВнГр – 7. Krylova, S. G., Zorikov, P. S., Zueva, 07/2017-11. The individuality and structure of E. P., Razina, T. G., Amosova, E. N., the compound were monitored by thin-layer Rybalkina, O. Yu. (2014). Experimental study chromatography and by NMR and IR of the analgesic activity of aconitum spectroscopy. NMR spectra were recorded at Kuznezoffi reichenb. Pacific Medical Journal the Educational and Scientific Laboratory of Tikhookeanskiy Medicinskiy Zhurnal. Resonance Spectroscopy of the Department of 2014;2:38-40. (in Russian) [eLIBRARY] Chemistry of Natural and High-Molecular 8. MarvinSketch, ChemAxonKft. (2017, Compounds of The Southern Federal June 19). Retrieved from University. IR spectra were recorded at the http://www.chemaxon.com/products/marvin/m Molecular Spectroscopy Center for Collective arvinsketch/ Use of The Southern Federal University. 9. MOPAC, Stewart Computational Chemistry. (2017, June 19). Retrieved from Conflicts of Interest http://openmopac.net. The authors have no conflict of interest to 10. Wu H, Wacker D, Mileni M, Katritch declare. V, Won HG, Vardy E, Liu W, Thompson AA, Huang XP, Carroll FI, Mascarella SW, References Westkaemper RB, Mosier PD, Roth BL, 1. Bogus SK., Galenko-Yaroshevskiy PA, Cherezov V, Stevens RC. (2010). Structure of Dukhanin AS, Shimanovskiy NL. Effect of the Human k-opioid Receptor in Complex with indole derivatives SS-68 having antiarrhythmic JDTic. Nature. 2010;485:327-332. doi: and antianginal properties on α 1-, β 1-and β 2 10.1016/j.ejmech.2014.12.016. [PubMed] adrenergic receptors. New technologies. [Novye 11. Trott O, Olson AJ. AutoDock Vina: Tekhnologii]. 2012.4:232 – 235. (in Russian) Improving the Speed and Accuracy of Docking [Full text] with a New Scoring Function, Efficient 2. Mashkovskiy MD. (2016). Optimization and Multithreading. J. Comp. Lekarstvennye sredstva. [Medications]. Chem. 2010;31(2):455-461. doi: Moscow: Novaya Volna. Print. (in Russian) 10.1002/jcc.21334. [PubMed] 3. Chen Y, Chen C, Liu-Chen LY. 12. Vasilyev PM, Spasov AA, Kochetkov December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Bogus S.K., Suzdalev K.F., Uvarov A.V., Galenko-Yaroshevsky P.A., Vasil'ev P.M., Vinakov D.V., Kiseleva N.V., Kochetkov A.N., Kiselev A.V. Anti-inflammatory and analgesic propertiesof SS-68 33 indole derivative. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):26-34.

AN, Vorfolomeeva VV, Yanalieva LR. (2016). Author Contributors Konsensusnyy podkhod k poisku insilico Saida K. Bogus, PhD in Medicine, protivodiabeticheskikh soedineniy. [A Assistant Professor, Department of Consensus Approach to Searching for insilico Pharmacology, Kuban State Medical University Antidiabetic Remedies]. In Mishen- of the Russian Ministry of Healthcare, e-mail: orientirovannyy Poisk Antidiabeticheskikh [email protected] Сontributor to all parts of Sredstv (pp. 126-181). Volgograd: Publishing the article. House of Volgograd State Medical University. Konstantin F. Suzdalev, PhD in (in Russian) [eLIBRARY] Chemistry, Associate Professor, Department of 13. Sigidin YaA, Shvarts GYa, Chemistry of Natural and High-molecular Arzamastsev AP, Liberman SS. (1988). Compounds, Faculty of Chemistry, Southern Lekarstvennaya terapiya vospalitelnogo Federal University, e-mail: protsessa: eksperimentalnaya i klinicheskaya [email protected]. Synthesis of SS-68 farmakologiya protivovospalitelnykh compound. preparatov. [Drug Therapy of the Inflammatory Aleksandr V. Uvarov, PhD in Medicine, Process: Experimental and Clinical Associate Professor, Department of Pharmacology of Anti-inflammatory Drugs]. Pharmacology, Kuban State Medical University M.: Medicina. Print. (in Russian) of the Russian Ministry of Healthcare, e-mail: 14. Neugodnova OP, Tsarichenko GV, [email protected] Contributed to the study Ryndina AE, et al. Toxicological Evaluation of of reference anti-inflammatory drugs. Voltaren. Pharmacol. i Toxicol. 1986;1:123. (in Pavel A. Galenko-Yaroshevsky, Doctor Russian) of Medical Science, Professor, Corresponding 15. Tsarichenko GV, Ganina GA. Toxicity Member of the Russian Academy of Sciences, of Indopan. Pharmacol. i Toxicol. 1986;2:117. Head of the Department of Pharmacology, (in Russian) Kuban State Medical University of the Russian 16. Shvarts G. (2004). Sovremennye Ministry of Healthcare, e-mail: nesteroidnye protivovospalitelnye preparaty. [email protected] Research [Modern Non-steroidal Anti-inflammatory project supervising. Drugs]. M: Reapharm. Print. (in Russian) Pavel M. Vasil'ev, Doctor of Biology, [eLIBRARY] Professor, Department of Pharmacology and 17. Galenko-Yaroshevskiy VP, Galka VV, Bioinformatics, Volgograd State Medical Varlashkina IA, Kovaleva VL. Influence of University of the Russian Ministry of Enoxifol on Acute Inflammation. Byulleten Healthcare, Senior researcher, e-mail: eksperimentalnoy biologii i meditsiny. [email protected]. ORCID: 0000-0002-8188- 2007;3:151-152.(in Russian) 5052. Supervised and contributed to molecular 18. Anisimova, V.A. et al. (2011). Patent docking study. of RF No. 2 412 187 C1 of 29.07.2009. Salts Dmitryy V. Vinakov, post-graduate pf 9-(2-morpholinoethyl)-2-(4- student, Department of Pharmacology, Institute fluorophenyl)imidazo[1,2-a]benzimidazole and of Medicine, Belgorod State National Research Salts of 9-aminoethyl-substituted 2-(4- University. Contributed to the study of anti- fluorophenyl)imidazo[1,2- a]benzimidazole inflammatory effect. with Analgesic Effect. Bul. №5. (in Russian) Nina V. Kiseleva, 6-year student, Faculty 19. Kuzeff, R.M., Topashka-Ancheva, of General Medicine, Kuban State Medical M.N., Mecheva, R.P. (2004). Inhibition of (-)- University of the Russian Ministry of trans-(1S,2S)-U50488 Hydrochloride by its Healthcare, e-mail: [email protected] Enantiomer in White Mice -- a Placebo- Contributed to the study of anti-inflammatory controlled, Randomized study. Forsch. effect. Komplementarmed. Klass. Naturheilkd. Andrey N. Kochetkov, research 2004;11(3):144-149. doi: 10.1159/000079443 technician, Department of Pharmacology and [PubMed] Bioinformatics, Volgograd State Medical University of the Russian Ministry of December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Bogus S.K., Suzdalev K.F., Uvarov A.V., Galenko-Yaroshevsky P.A., Vasil'ev P.M., Vinakov D.V., Kiseleva N.V., Kochetkov A.N., Kiselev A.V. Anti-inflammatory and analgesic propertiesof SS-68 34 indole derivative. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):26-34.

Healthcare, e-mail: [email protected] Received: October, 27, 2017 Contributed to molecular docking study. Accepted: November, 30, 2017 Aleksandr V. Kiselev, PhD in Medicine, Available online: December, 30, 2017 Head of the Laboratory for Tissue Preservation ―Eye Bank‖, Krasnodar branch of S.N. Fyodorov Eye Microsurgery Complex of the Russian Ministry of Healthcare, e-mail: [email protected] Contributed to the study of anti-inflammatory effect.

Rus. UDC: 615.37:616-018.74-008.6 DOI: 10.18413/2313-8971-2017-3-4-35-77

Tatiana A. Denisiuk PHARMACOTHERAPEUTIC STRATEGIES FOR ENDOTHELIAL DYSFUNCTION CORRECTION WITH USE OF STATINES IN SYNDROME OF SYSTEMIC INFLAMMATORY RESPONSE

Kursk State Medical University, 3, K. Marx St., Kursk, 305040, Russia Corresponding author, e-mail: [email protected]

Abstract Objectives: The ways of pharmacological correction of cardiovascular complications in the syndrome of the systemic inflammatory response (SIRS) are not fully developed. Goal: Determination of statins‘ new pharmacological effects and its combination with endothelioprotectors in the SIRS correction. Materals and Methods: In experiments on mice, rats and rabbits, the anti- inflammatory, cardioprotective and endothelioprotective effects of statins and endothelioprotectors were explored. The modeling of endotoxin-induced endothelial dysfunction (EIED) was created by infecting rats with Staphylococcus aureus (strain 13407), subcutaneously (60 billion microbial bodies). To determine the activity of the inflammatory process, the indices of the C-reactive protein used. The involvement of the cytokine link of inflammation was assessed according to the plasma levels of TNF-α and IL-6. Modeling of L-NAME-induced pathology and further evaluation of endothelial and endothelium-independent vascular reactions were carried through according to a standard protocol. Results: Simvastatin (9, 19, 35 mg/kg), atorvastatin (5, 9, 19 mg/kg), rosuvastatin (9, 19, 35 mg/kg) and nanoparticulated rosuvastatin (3, 6.3 and 11.6 mg/kg) proved a dose- dependent antiexudative effect on the model of formalin paw edema in mice. Similarly, the anti-inflammatory effect is evident in the exudative model on rabbits. The greatest effectiveness was demonstrated by rosuvastatin and its nanoparticularized form. Simvastatin 8.5 mg/kg, atorvastatin 4.3 mg/kg, rosuvastatin 8.5 mg/kg, and nanoparticulated rosuvastatin 11.6 mg/kg demonstrate a cardioprotective effect in the coronary-occlusive modeling of infarction in rats. In the process of cardioprotective effects‘ implementation the mechanisms of pharmacological preconditioning has significant importance. It was proved by the removal of effects with K + -ATP-as channels blockade with glibenclamide (5 mg/kg) and iNOS blockade with aminoguanidine (40 mg/kg). The use of inhibitors HMG-CoA reductase of simvastatin (2.2, 4.3 and 8.5 mg/kg), atorvastatin (1.1, 2.2 and 4.3 mg/kg), rosuvastatin (2.2, 4 , 3 and 8.5 mg/kg) and nanoparticulated rosuvastatin (3, 6.3 and 11.6 mg/kg) on the background of endotoxin- induced pathology modeling leads to the development of a dose-dependent endothelioprotective effect, which is expressed in normalization of coefficient of endothelial dysfunction (CED), to prevention of adrenoreceptivity increase and to the exhaustion of the myocardial reserve, as well as to the normalization of biochemical markers of inflammation (C-reactive protein) and the level of pro-inflammatory cytokines. At the same time, positive dynamics of the final products of NO and eNOS expression was defined.

Applying of monotherapy with donator NO L-arginine (70 and 200 mg/kg), a nonselective inhibitor of arginase BEC (5 and 10 mg/kg), a selective inhibitor of arginase-2 arginasine (1 and 3 mg/kg) and recombinant darbepoetin (50 and 500 μg/kg) in the modeling of endothelial dysfunction, has revealed their high activity, expressed in preventing the increase in CED, adrenoreactivity, preservation of the myocardial reserve and normalization of the values of biochemical markers (Total NO, eNOS expression, C-reactive protein, IL-6, TNF). Herewith, the drugs had a dose-dependent effect and were approximately equally effective. A vector analysis of the additive effects of the combined use of inhibitors of HMG-CoA reductase, simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin with L-arginine, inhibitors of arginase – BEC and arginasine, as well as darbepoetin demostrated that, with endotoxin-induced pathology, the highest probabilistic percentage of addiction was found in combination of rosuvastatin with arginasine (3 mg/kg) and darbepoetin (500 μg/kg), relatively, 31.9 ± 2.8 and 30.2 ± 2.9%. Discussion: The inhibitors of HMG-CoA reductase have demonstrated cardioprotective (decrease of adrenoreactivity and depletion of myocardial reserve, normalization of blood pressure) and endothelioprotective (amplification of eNOS expression, increase of NO) the attributes, which manifested itself as well as in monotherapy and in combination with some endothelioprotectors (L-arginine, arginasine, BEС, darbepoetin) in varying degrees. Keywords: endothelial dysfunction, nitric oxide, statins, endothelioprotectors, systemic inflammatory response syndrome.

Introduction of inflammatory mediators (bradykinin, Inflammation is a distinctly coordinated prostaglandins, histamine, proinflammatory systemic reaction of the body, focused on cytokines) and the change of the expression eliminating of the damaging agent. However, molecules profile of intercellular interactions rather often an inflammatory reaction causes (integrins, selectins) followed by leukocyte more damage to the body than the etiologic adhesion and iNOS activation; 3) the growth of factor that had induced it [1]. More than 100 cytotoxic NO concentrations with the formation years ago, William Osler in his reflections of active forms of nitrogen, the breakdown of about sepsis had correctly suggested that, intercellular contacts and the oxidation of LDL, except some rare cases, the cause of death in a the change of the antithrombotic properties of septic process is not an infection, but the body's the endothelium to prothrombotic and the response to it. A contemporary migration of leukocytes through the wall of pathophysiology explains this phenomenon micro vessels with further infiltration of tissues through the concept of a systemic inflammatory and the absorption of oxidized LDL; reaction (SIRS), considered as a response of the 4) hyperproduction of ROS, vasodilation, organism to various flogogenic effects of microthrombosis and systemic manifestations infectious and non-infectious origin [2]. The of SIRS; 5) death due to the gradual scientific researches of the last decade led to decompensation of life-support processes at the the formation of an integral picture describing systemic level or recovery [3]. It is easy to see SIRS through a cascade of neurohumoral that one of the central participants of this reactions, formed by complex mediator and continuum is the endothelium, which does not cytokine nets. Describing the pathogenesis of just produce the widest spectrum of humoral systemic inflammation, several main stages can factors, but also realizes the processes of be identified: 1) presentation of exudation, thrombosis and hemodynamics lipopolysaccharide on the surface of the control. The more interesting fact is that the endothelium, massive stimulation of the majority of patients who underwent abdominal pattern-recognition cells receptors of immune catastrophes [4, 5, 6, 7], sepsis [8, 9, 10] and and reticuloendothelial systems; 2) the release other infectious nosology associated with SIRS December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 37 Clinical Pharmacology. 2017;3(4):35-77.

[8, 11, 12] after regression of the pathological dysfunction (atherosclerotic vascular lesions, process on various terms are developing myocardial infarction, cerebral stroke) [13, 14] complications associated with endothelial (Fig. 1).

Fig. 1. Pathogenesis of vascular wall damage in SIRS. Note: Pathogenesis of the formation of an inflammatory injury of the vascular wall with subsequent atherogenesis and thrombus formation. LPS binds to the Toll-receptors of the endothelial cell, enhancing the release of pro-inflammatory cytokines and the expression of adhesion receptors, to which leukocytes are attracted by β- integrins followed by iNOS activation and massive NO release, the latter forming the peroxynitrite (ONOO-) and nitrosyl chloride (NOOCl) radicals, damaging cellular structures and dissociating eNOS. As a result, intercellular contacts are destroying; oxidized by free radicals of LDL induce the cascade of reactions, leading to an even greater dissociation of eNOS. Disturbance of cells structures contributes to increased vascular permeability and migration of leukocytes, their absorption of oxidized LDL and formation of foamy cells – harbingers of atherosclerotic plaques. The destruction of the endothelium and exposure of the subendothelial layer creates conditions for the activation and aggregation of thrombocytes with thrombosis

At the same time, with the coherence of the activity and reduce the level of pathogenetic regimens and the inclusion of proinflammatory cytokines, chemokines and multiple factors (VEGF, sFlt-1, autoantibody of adhesion molecules. Antithrombotic effects of receptor angiotensin II (type 1) (AT1-AA), statins can improve the coagulopathy caused by cytokines (tumor necrosis factor (TNF) -α), sepsis. By influencing on the modification of endothelin, active forms of oxygen (ROS), proteins, they prevent the presentation of the thromboxane, 20-hydroxyicosatetraenoic acid antigen by the endothelial cell and recognition (20-HETE), increased sensitivity to angiotensin by the immune cells. The ability of statins to II, etc.), the inadequacy of pharmacotherapeutic increase eNOS expression and reduce iNOS strategies pointed to correction of endothelial concentration has been also demonstrated. It dysfunction in acute systemic inflammation is has a great importance both for limiting the obvious. Besides, we can suppose the extent of damage in SIRS and for limiting the mechanisms underlying the ischemic extent of endotoxin-induced endothelial preconditioning may play a role in pathogenetic dysfunction as well [45]. approaches to the correction of multiple organ On the other hand, endothelium is a classic failure in endotoxin-induced pathology, target of such pharmacological agents as L- including myocardium, brain, kidney, retina arginine, BEC and darbepoetin. Theirs mono- [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25]. and combination therapy has been proved as a From this regard the inhibitors of 3-hydroxy-3- successful in treating of a wide range of methylglutaryl coenzyme A (HMG-CoA) – cardiovascular pathologies [46]. reductase are very perspective. Being the gold From this aspect, the pharmacological standard of lipid-lowering therapy [26, 27, 28, target "ADMA-eNOS", which was the research 29, 30, 31, 32, 33, 34], statins continue to object of Kursk's and after of Belgorod‘s attract the attention of researchers because of pharmacologists [47, 48, 49, 50, 51, 52], and their inherent pleiotropic effects [35, 36, 37], also of their colleagues from Volgograd, has a including cardioprotective [38, 39] anti- definite interest [53, 54, 55, 56]. inflammatory [40, 41] and endothelioprotective Methylated analogs of L-arginine – [42]. These effects do not depend upon asymmetric dimethylarginine (ADMA) and cholesterol decreasing, but caused by the main monomethylarginine (L-NMMA) – are mechanism of action – inhibition of HMG-CoA endogenous inhibitors of endothelial nitric reductase. Tit is concerned with the fact that a oxide synthase (eNOS). In recent years, it has number of intermediate products in the chain of been found that ADMA concentrations in cholesterol synthesis, such as farnesyl endotoxin shock are increasing and elevated pyrophosphate and geranylgeranil concentrations of ADMA are one of the key pyrophosphate, have pro-inflammatory, risk factors of the cardiovascular nosological prooxidant and several other negative attributes continuum [57]. are realizing through the activation of Taking into consideration the fact that one secondary mediators Ras Rac1, Rho [43]. of the main pathogenetic aspects of SIRS It is shown that activation of Rho is development is the imbalance between the associated with inhibition of eNOS, and an inducible and endothelial isoforms of NOS increase of leukocytes migration through the [58], pharmacotherapeutic correction of vascular wall. The activation of Ras is endothelial dysfunction in systemic connected with the synthesis initiation of pro- inflammation should be aimed at overcoming inflammatory cytokines, while the activation of the internal inhibition of eNOS, including Rac1 is connected with an increase in ADMA. mitochondrial NADPH oxidase activity, what The researches on overcoming the contributes to the oxidative stress development inhibitory effect of ADMA by the introduction of [44]. of L-arginine [59], tetrahydrobiopterin [60], a Numerous researches with the use of nonselective inhibitor of arginase L-norvaline different models had convincingly [61], selective inhibitors of arginase-2 demonstrated that statins possess anti-apoptotic December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 39 Clinical Pharmacology. 2017;3(4):35-77. arginasine [62] and others has got a of the Russian Academy of Medical Sciences. considerable development nowadays. The spread in groups according to the initial Based on this point of view, HMG-CoA mass did not exceed -10%. All experiments reductase inhibitors and endothelioprotectors were carried out at the same time of the day, can potentially have complementary effects in from 8 a.m to 12 a.m, in accordance with the prevention and treatment of endothelial principles set forth in the Convention for the dysfunction and other pathological conditions Protection of Vertebrates used for experimental associated with systemic inflammation. and other purposes (Strasbourg, France, 1986) Accordingly, there are significant and in accordance with the rules of laboratory theoretical prerequisites for the use of drugs practice in the Russian Federation (Order of the aimed at correcting endothelial function (in Ministry of Health of the Russian Federation particular, statins and endothelioprotectors) in No. 267 of June 19, 2003). complex treatment and in prevention of SIRS. The investigation of the anti- Hereby one of the main elements of the inflammatory effect on the model of proposed pharmacotherapeutic strategies is formalin edema of mice: The experiments statins. This group of drugs is in demand all were made on laboratory mice weighing 18-22 over the world and some of them are so-called g. The anti-inflammatory effect was evaluated blockbusters with sales volumes of more than in conditions of acute aseptic inflammation of 5-7 billion dollars per year in financial the formalin-induced mice, according to the equivalent. From the chemical point of view, degree of inhibition of the edema of the foot statins are represented by 8 international non- increasing by the drugs in comparison with the proprietary names, different in lipo- and control group of untreated animals. Exudative hydrophilicity and also in a number of edema was induced by subcutaneous injection pharmacokinetic and toxicological indicators. into the right rear foot of mice with 0.02 ml of Simvastatin and atorvastatin are the most a 2% aqueous solution of formalin. The mass of prevalent. But rosuvastatin is up-to-date. the foot was measured in 4 hours (at the peak Objective: to study the effectiveness of of the edema) after injection of phlogistics on pharmacological correction of endotoxin- electronic scales with an accuracy of 1 mg; as a induced pathology by statins using control measure the left paw of the same (simvastatin, atorvastatin, rosuvastatin, animal was used, which was injected with an nanoparticulated rosuvastatin) and their equal volume of isotonic NaCl solution in combinations with L-arginine, BEC arginase parallel with the injection of the flogogenic inhibitors and selective inhibitor of arginase-2 agent. The inhibitory effect was calculated by arginasine, and also by recombinant the formula: darbepoetin. ( MeMc %100) Eing , Mc Materials and Methods The experiments were made on healthy where Eing – the inhibitory effect, Me and matured rats of the Wistar line, weighing 180- Mc – the average increase of the edematous 250 g; laboratory mice weighing 18-22 g; and foot mass in the control and experimental also rabbits weighing 2-2.5 kg. In the groups. experiments, we used animals that passed The anti-inflammatory effect through the quarantine regime of the Kursk investigation according to I.A. Oivin State Medical University vivarium and did not method. The experiments were made out on have any external signs of any diseases. All laboratory rabbits (3 rabbits in each group) animals were kept in the same conditions, on a weighing 2-2.5 kg. To investigate the anti- normal diet. For the statistically reliable results, inflammatory activity of the drugs using the the groups were formed from 18-20 animals. Oivin method, the rabbits were fixed; The animals that were included into a control previously hair has been cut on the abdominal and into an experimental group were of the skin (13 x 5 cm piece). Tripanum coeruleum same age and came from the Stolbovaya cattery (permeability indicator) was injected into the December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 40 Clinical Pharmacology. 2017;3(4):35-77. edge vein of the ear in the form of a 1% followed by generalization of the infectious solution on a 0.9% solution of sodium chloride agent by daily injection place massage. Further, at a rate of 2 ml per 1 kg of animal weight. a compression, pneumatic massage of the Then 6-12 drops of o-xylene were applied to injection place was performed daily during 10 the area of the abdominal skin. The indicator of minutes [68]. the permeability of capillaries was the time of To determine the activity of the appearance of blue-stained spots on the skin inflammatory process, the indices of the C- and their diameter. By the difference in the reactive protein were used. The involvement of time of appearance of the spots and their the cytokine link of inflammation was diameter before and after the injection of the estimated according to the plasma content of drug, it was judged on its effect on the TNF-α and IL-6, determined by immunoassay permeability of capillaries. methods using the Alfa-TNF-α-IFA-Best A simulating model of coronary- reagents kits; "IL-6-IFA-Best" [69, 70, 71]. occlusive myocardial infarction in rats and Modeling of L-NAME-induced gestosis evaluation of the magnitude of the necrosis and estimation of endothelium-dependent zone. and endothelium-independent vascular The experiments were made on male reactions (M.V. Pokrovsky and co- auths., Wistar rats of 200-250 g. The drug was injected 2006). intraperitoneally 1 time per day and 30 minutes N-nitro-L-arginine methyl ether (L- before modeling myocardial infarction (MI). NAME) was injected to males daily, once a MI was reproduced on anesthetized animals by day, intraperitoneally at a dose of 25 mg/kg. On bandaging the descending branch of the left the 8th day from the beginning of the coronary artery on the lower edge of the left experiment, an L-NAME-induced and atrial appendage level, then the wound was hyperhomocysteine-induced endothelial layer-by-layer sutured. dysfunction a catheter was put in (under On the 4th day, the animal was killed, the anesthesia bychloral hydrate 300 mg/kg) into heart was removed, and crossed sections were the left carotid artery to record hemodynamic made every 4-6 mm, and then incubated in a parameters. Hemodynamic parameters: systolic 1% solution of triphenyltetrazolium of blue in blood pressure (SBP), diastolic blood pressure phosphate buffer pH 7.4 for 15 minutes at (DBP) and heart rate (HR) were measured 37 ° C. This method makes it possible to continuously, using the Biopac hardware visualize clearly the necrosis zone (not colored) complex (USA) and the computer program for further examination. "Acqknowledge 3.8.1". Bolus injection of Quantitative analysis and calculation of the pharmacological agents was made into the right area of necrotic tissue was carried out by means femoral vein. The following functional tests of the graphic editor Adobe Photoshop CS5 were performed: intravenous acetylcholine (40 [63]. μg/kg) [72] and sodium nitroprusside (30 To analyze the participation of K⁺-ATPase μg/kg) [73]. channels and iNOS in the realization of the A study of myocardial contractility after protective effects of pharmacological pathology modeling was made in narcotized preparations, glibenclamide [64, 65] and rats on controlled respiration. The left ventricle aminoguanidine [66, 67] were used. cavity was probed with a needle through the A simulating model of endotoxin- apex of the heart and by means of the hardware induced endothelial dysfunction. complex "Biopac" (USA), the computer In the modeling of endotoxin-induced program "Acqknowledge 3.8.1", the parameters endothelial dysfunction, the rats were infected of cardiohemodynamics (left ventricular with Staphylococcus aureus (strain 13407), pressure, maximum reduction rate (+ dp / dt subcutaneously with 60 billion microbial max), maximum relaxation rate (-dp / dt max), bodies, with followed by (after 24 hours) heart rate (heart rate) were registered. sensitization of the laboratory animal (0.1 ml of staphylococcal anatoxin subcutaneously), December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 41 Clinical Pharmacology. 2017;3(4):35-77.

To estimate the functional capabilities of diazotization followed by the development of the myocardium in animals, burthen tests were color. performed in the following sequence: The level of expression of endothelial nitric 1. Adrenoreactivity test – intravenous one- oxide synthase (e-NOS) was determined in a cell stage injection of an adrenaline hydrochloride lysate by using the Hendrickson method [77]. solution 1*10-5 mol/l, calculated on the basis of Detection of the eNOS band was carried out by 0.1 ml per 100 g of body weight [74]. the method of enhanced chemiluminescence 2. Resistance test – clamping of the (ECL). ascending aorta by 30 sec. [75]. Justification of doses and the experiment Biochemical markers of endothelial design: dysfunction. In this research, the study of endothelial A modification of the method for the and cardioprotective activity of statins in doses determination of stable metabolites of NO was was made; anti-inflammatory effects were used; it allows one-step quantitative detected before [78, 79]. In chronic determination of total nitrates and nitrites after experiments in the modeling of EIED and L- deproteinization of blood serum [76]. The NAME-induced endothelial dysfunction, drugs principle of the method is in simultaneous were injected at appropriate doses once a day reduction of nitrates to nitrites in the presence intraperitoneally or intragastrically for 7 days of chloride bath tubing and the reaction of 30 minutes after L-NAME injection.

Table 1 The correlation of doses of inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin for humans and experimental animals (rabbits, rats and mice) [80]

Human doze, Rabbit Rat Mouse Drug mg mg/kg mg/kg mg/kg Simvastatin 5 2.2 4.3 9 10 4.3 8.6 19 20 8.5 17.2 35 Atorvastatin 2.5 1.1 2.2 5 5 2.2 4.3 9 10 4.3 8.6 19 Rosuvastatin 5 2.2 4.3 9 10 4.3 8.6 19 20 8.5 17.2 35 Nano-rosuvastatin - 0.73 3 6.2 - 1.43 6.3 13.9 - 2.83 11.6 23.6

Simvastatin "Zokor"® tablets, film-coated Rosuvastatin "Crestor" Crestor® tablets, 20 mg, 28 pcs. packing contour mesh 14, film-coated 10 mg, 7 pcs. blister 7, a pack of cardboard pack 2 code EAN: 4601969006711 cardboard 1 code EAN: 7321838721251 No. П No. P N013094 / 01, 2011-10-24 Merck N015644 / 01, 2009-03-24 AstraZeneca UK Sharp & Dohme B.V. (Netherlands). Ltd. (United Kingdom). Atorvastatin "Lipitor" Tablets of round Nanoparticulated-rosuvastatin, substance. form 10, 20, 40 mg in a shell in a blister in Manufactured in V.N. Orekhovich Research cardboard bundle No. 30. Manufacturer: Institute of Biomedical Chemistry (Russian Gedeke / Parke-Davis, Pfizer, Inc. (Germany). Federation).

Glibenclamide Maninil® 5 tablets 5 mg, The protocol consists of the following 120 pcs. a bottle (bottle) 120, a pack of sections: cardboard 1 code EAN: 4013054000922 No. П 1. Simulation of EIED and or L-NAME- N011519 / 01, 2012-03-07 Berlin-Chemie AG / induced deficiency of nitric oxide and its Menarini Group (Germany). correction with the help of inhibitors of HMG- Aminoguanidine Aminoguanidine CoA reductase and L-arginine, BEC, arginasine hydrochloride (Sigma-Aldrich) CAS Number and darbepoetin. 1937-19-5 Linear Formula NH2 NHC (= NH) 2. On the 8th day in the conditions of NH2 • HCl9. etaminal-sodium anesthesia, the appraisal of A study of L-arginine produced by endothelium-dependent and endothelium- EUROBIOPHARM GmgH (Hamburg) was independent reactions of arterial pressure on made by intraperitoneal injection at doses of 70 intravenous one-stage administration of and 200 mg/kg/day. This dose is selected in acetylcholine and sodium nitroprusside. accordance with the literature data, where the 3. Connection of the animal to the artificial dosage range for the L-arginine activity is in ventilation of the lungs left ventricular the range of 30 mg/kg to 200 mg/kg [81]. catheterization, registration of the LDL, + dp / A nonselective inhibitor of arginase – S- dt, -dp / dt, HR, IFS. Stress tests for assess the (2-boro-ethyl) -L-cysteine (BEC), (WIRUD functional reserves of myocardial contractility. GmgH, Hamburg) was injected at a dose of 4. Removal of the animal from the 10 mg/kg/day. According to modern experiment and taking blood from the thoracic researchers, BEC is able to inhibit arginase [81] aorta for biochemical studies. exactly from this dosage. 5. Taking samples of myocardial tissue for Recombinant darbepoetin ―Aranesp‖ morphological examination and determination injection solution 0.3 mg, 1 unit syringes 0.6 of the diameter of cardiomyocytes. ml Amgen Europe B.V. (Netherlands). It was Statistical processing of the research injected intraperitoneally at doses of 50 and results 500 μg/kg for 7 days. (K.M. Reznikov, co- For graded data, the normality of the authors, 2017) [83] distribution was determined by using the Selective inhibitor of arginase-2 arginasine Shapiro-Wilk criteria. In the case of normal (laboratory code ZB49-0010) synthesized in distribution, the significance of the differences TVC "ChemRar" was administered was assessed using a one-dimensional analysis intraperitoneally at a dose of 1-3 mg/kg/day. of variance or dispersion analysis for repeated Design of the experiment on the study of measurements, followed by the Dunnet method cardio and endothelioprotective activity of of multiple comparisons. In a case if there inhibitors of HMG-Co-A-reductase wasn‘t normal distribution, a nonparametric simvastatin, atorvastatin, rosuvastatin and analogue of the Friedman dispersion analysis nanoparticulated rosuvastatin in combination was used with subsequent processing by the with L-arginine, BEC. Arginasine and method of multiple comparisons according to darbepoietin consists of the following blocks: Newman-Keils or Dunnet. The non-parametric 1. Control group. Wilcoxon test for coupled samples and Mana- 2. Simulation of EIED or L-NAME- Whitney were also used [84, 85]. induced deficiency of nitric oxide. The alternative data was worked on with 3. Estimation of endothelial and the help of Fisher's exact probability method cardioprotective effects of inhibitors of HMG- and the χ2 criterion. To estimate the correlation Co-A-reductase of simvastatin and L-arginine, between anti-inflammatory activity and BEC, arginasine and darbepoetin. endothelioprotection, the Pearson correlation 4. Appraisal of the effectiveness of coefficient was used. endothelial and cardioprotective effects of the The veracity of the observed changes in the combined use of HMG-CoA reductase parameters, caused by experimental drugs, both inhibitors and L-arginine, BEC, arginasine and as absolute and in percentage of the initial level darbepoetin. as well, was find out with the help of the December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 43 Clinical Pharmacology. 2017;3(4):35-77. residual method of the variable statistics by and nanoparticulated rosuvastatin causes a finding the mean weight of the shifts (M); the dose-dependent antiexudative effect, appeared arithmetic mean (+ m) and the probable error of in decreasing of the gain of weight degree of the mean (P) according to Student tables. the foot comparing to intact animals (Figure 2). Differences were estimated as significant, The calculated coefficient of inhibitory starting from p <0.05. For calculations, we used effect (CIE) with an increase in the dose of the program for statistical analysis Microsoft simvastatin from 9 to 35 mg/kg had been Excel 7.0. increasing from 12.7 + 4.6 to 46.3 + 4.9 cu. (p <0.05). For atorvastatin, the initial effective Discussion and Results of the Research anti-inflammatory dose was the less and Investigation of the anti-inflammatory amounted to 5 mg/kg for CIE 23.3 + 4.5 cu. effect of inhibitors of HMG-CoA reductase With dose increasing up to 19 mg/kg, CIE simvastatin, atorvastatin, rosuvastatin and became 39.7 + 4.5 cu. Rosuvastatin on this nanoparticulated rosuvastatin on the model exudative inflammation model also of formalin edema of mice foot and demonstrated a dose-dependent effect which exudative model of inflammation in rabbits can be compared with The most evident according to Oivin. antiexudative effect was seen in using of The use of inhibitors of HMG-CoA nanoparticulated rosuvastatin in the dose range reductase simvastatin, atorvastatin, rosuvastatin of 4 times lower than rosuvastatin (Figure 2).

Сoefficient of inhibitory action (c. u.)

6 46.3* 49.8* 5 46.8* 39.7* 4 34.5* 28* 26.7* 3 29.8* 12.7* 2 18.7* 16.4* 23.3* 1

0 rosuvastatin - Rosuvastatin Atorvastatin Atorvastatin Nano Simvastatin

* - P<0.05 in comparison with intact; #- Р<0.05 in comparison with EIED Fig. 2. The anti-inflammatory effect of inhibitors of HMG-CoA-reductase of simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the model of formalin edema of the foot of mice

The study results of the anti-inflammatory Simvastatin in doses of 4.3 and 8.5 mg/kg drugs activity according to the E.A. Oivin had a dose-dependent anti-inflammatory effect, method are shown in Figure 3. so the result of this was a decreasing of spots The study results of the anti-inflammatory area to 3.5 ± 0.3 and 3.1 ± 0.3 cm2; and same drugs activity according to the E.A. Oivin time an increasing the time of its coming out to method are shown in Figure 3. 397.1 ± 12, 3 and 397.1 ± 12.3 seconds. In comparison with the control values, 5.4 ± 0.3

Square of infiltration (cm2)

6 5.5 4.1* 5 3.6* 3.6* 4 2.7* 3.5* 2.9* 3 3.1* 2.2*

2 1.5* 1.0* 1.7*

0 - Nano rosuvastatin Rosuvastatin Atorvastatin Simvastatin

* - P<0.05 compared with intact; #- compared with EIED Fig. 3. Anti-inflammatory effect of inhibitors of HMG-CoA-reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the model of anti-exudative action in rabbits

The size of the spots was also minimal and rats in the control group of animals led to amounted to a larger dose of 1.5 ± 0.2 cm2. myocardial necrosis, the size of which was Nanoparticulated rosuvastatin (0.73, 1.43 and 12.71 ± 0.59% of the total myocardium size 2.83 mg/kg) showed comparable efficacy with (Figure 4). a dosage of rosuvastatin base substance 4 times The use of inhibitors of HMG-CoA exceeding that of nanoparticulated form. reductase simvastatin, atorvastatin, rosuvastatin Thus, statins demonstrate a dose-dependent and nanoparticulated rosuvastatin had a dose- anti-inflammatory (on the model of formalin dependent cardioprotective effect. In this edema of the foot in mice) and anti-exudative instance, the statins we were studying effect (on the model of exudative inflammation appeared to be approximately equally effective in rabbits according to Oivin's method). The (Figure 4). highest activity was evident in nanoparticulated The blockade of K + -ATPase channels by rosuvastatin in the dose range (0.73, 1.43 and glibenclamide (5 mg/kg) completely stopted the 2.83 mg/kg), which is 4 times lower than in the cardioprotective effect of inhibitors of HMG- experiments with the base substance (Figure 3). CoA reductase simvastatin, atorvastatin, The investigation of the cardioprotective rosuvastatin and nanoparticulated rosuvastatin effect of inhibitors of HMG-CoA-reductase in the simulation model of coronary-occlusive of simvastatin, atorvastatin, rosuvastatin infarction in rats (Figure 4). The use of and nanoparticulated rosuvastatin in the aminoguanidine (40 mg/kg) for iNOS blockade simulation model of coronary-occlusive had also completely stopted the myocardial infarction in rats. cardioprotective effect of the statins which The application of the ligature to the were studied (Figure 4). This indicates the descending branch of the left coronary artery in participation of the effects of mechanisms for December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 45 Clinical Pharmacology. 2017;3(4):35-77. the realization of pharmacological glibenclamide (5 mg/kg) and the "second preconditioning as the "first window" window" (iNOS blockade with aminoguanidine (blockade of K + -ATPase channels with 40 mg/kg).

Infarct size (%)

Nano- rosuvastatin Rosuvastatin

Atorvastatin

Simvastatin EIED + Intact + Statins Statins glibenclamide Statins aminoguanidine

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 4. Cardioprotective effect of inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the model of coronary-occlusive infarction in rats

Thereby, the written above indicates that infected with Staphylococcus aureus (strain statins have a dose-dependent cardioprotective 13407), subcutaneously with 60 billion effect in the simulation model of coronary- microbial bodies, followed by (after 24 hours) occlusive infarction in rats. In cardioprotective sensitization of the laboratory animal (0.1 ml of effects actualization, the mechanisms of staphylococcal anatoxin subcutaneously), after pharmacological preconditioning are of that generalization of the infectious agent by considerable importance, as it became evident means of a daily massage at the injection place with remove of effects during blockade of K + was made. A way of a chronic septic process in -ATPase-channels with glibenclamide and laboratory animals RU 2143749 C1 6 G09B23 blockade of iNOS with aminoguanidine. / 28 [86] learning. This model allows having a The investigation of endothelioprotective long-term dynamic process of endotoxin effects of inhibitors of HMG-CoA reductase intoxication with 100% survival of of simvastatin, atorvastatin, rosuvastatin experimental animals learning. and nanoparticulated rosuvastatin in the The temperature of the rats body during the simulation model of endotoxin-induced learning period was higher than the intact endothelial dysfunction. group for 2-3 ° C, reaching the maximum The simulation model of endotoxin- increase by the sixth day of the experiment induced endothelial dysfunction in (40.1 ± 0.2 ° C) and decreasing to the initial experimental animals values by the 15th days of the experiment (38.2 When a model of endotoxin-induced ± 0.1 оС). This fact demonstrates the dynamics endothelial dysfunction was created, rats were

Coefficient of Systolic blood Diastolic blood Group endothelial pressure pressure dysfunction (CED) Intact 129.4±2.2 89.2± 1.1 1.1 ± 0.1 Endotoxin-induced endothelial 117.6±2.3* 85.0±2.1* 3.7±0.5* dysfunction Note: SBP – systolic blood pressure (mmHg), DBP – diastolic arterial pressure (mmHg), CED – coefficient of endothelial dysfunction (unit units), * – significant difference with a group of intact animals (p <0.05).

Adrenoreactivity in animals with EIED This fact may indicate a more significant was 20% higher, and the myocardial reserve contribution of the inducible isoform of the was 30% lower than in intact animals. enzyme activation (iNOS) to an enlargement of Analyzing the values of biochemical NO content. Integral marker of inflammatory markers, reflecting the metabolism of nitric processes in the vascular wall C-reactive oxide, C-reactive protein and pro-inflammatory protein (CRP) has been increasing its level cytokines the most significant changes has been more than 7 times, which indicates the found. Thus, the total content of final development of systemic vasculitis. A similar metabolites of NO (Total NO) in animals with dynamics can be observed in the case of the EIDD from 116.8 ± 10.3 to 182.3 ± 12.4 μmol/l inflammatory cytokines IL-6 and TNF, the were highly increased. In contrast, eNOS values of which increased 16 and 2 times, expression has been decreasing significantly. respectively (Table 3). December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 47 Clinical Pharmacology. 2017;3(4):35-77.

Table 3 Changes in the values of biochemical markers (Total NO, Expression of eNOS, C-reactive protein, IL-6, TNF-α) in animals with endotoxin-induced endothelial dysfunction (EIED) (M ± m, n = 10) eNOS C-reactive Groups of animals NOх IL-6 TNF-α expression protein Intact 116.8±10.3 5.4±0.21 0.05±0.01 0.43±0.17 8.4±2.5 Endotoxin-induced endothelial 182.3±12.4* 0.04±0.01* 0.38±0.01* 6.87±1.93* 17.8±3.7* dysfunction Note: NOx is the final metabolite of NO (μmol/L); eNOS expression (%); level of CRP-C-reactive protein (mg/l); IL- 6 – interleukin 6 (pg/ml) TNF-α-tumor necrosis factor α (pg/ml), * – significant difference with a group of intact animals (p <0.05).

Thereby, the modeling of endotoxin- dysfunction. induced pathology by the injection of strain The use of inhibitors of HMG-CoA 13407 of Staphylococcus aureus leads to the reductase of simvastatin, atorvastatin, development of endothelial dysfunction with an rosuvastatin, and nanoparticulated rosuvastatin increase of CED 3.7-fold, a decrease in in EIED modeling revealed an evident dose- myocardial reserve and an augmentation of dependent endothelioprotective effect, adrenoreactivity, as well as an enlargement of manifested in a significant decrease in CED, stable NO metabolites against an increase in against normalization of systolic and diastolic inflammatory markers of C-reactive protein and blood pressure values. Thus, in the simulation pro-inflammatory cytokines IL-6 and TNF-α. of EIED, CED was 3.7 ± 0.5, whereas in large Decreased expression of eNOS indicates a doses of simvastatin (8.5 mg/kg), atorvastatin preferential involvement of the enzyme form (4.3 mg/kg), rosuvastatin (8.5 mg/kg) and iNOS in this process. nanoparticulated rosuvastatin (11.6 mg/kg), The endothelioprotective effects of the respectively, 2.3±0.5, 2.1±0.3, 1.7±0.5 and 1.5 inhibitors of HMG-CoA-reductase of ± 0.2 c.u., which was close to the values in simvastatin, atorvastatin, rosuvastatin and intact animals (1.1±0.1). Wherein, the most nanoparticulated rosuvastatin investigation effective drugs rosuvastatin and its in modeling endotoxin-induced endothelial nanoparticulated form were (Figure 5).

CED, c. u.

3.7* 3.6* 3.5* 4 3.3* 3.2*

# 2.9* # 2.7* # 3 2.4*# 2.5* 2.3*# 2.1*# # 2 1.7* # 1.1 1.5*

EIED + EIED + EIED + EIED + Intact EIED Simvastatin Atorvastatin Rosuvastatin Nano-rosuvastatin 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg mg/kg mg/kg mg/kg

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 5. Indices of endothelial dysfunction coefficient (CED) with the use of inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of endotoxin-induced endothelial dysfunction modeling

Parallel to this, conducting stress tests in be the most effective. The most evident animals with EIED indices a positive dynamics endothelial and cardioprotective effect of of contractility was observed. As a result, the inhibitors of HMG-CoA reductase of prevention of an increase in adrenoreactivity simvastatin, atorvastatin, rosuvastatin and and a decrease in the myocardial reserve was nanoparticulated rosuvastatin was found out in revealed. At the same time, as in the case of the values of biochemical markers in animals CED, rosuvastatin (8.5 mg/kg) and its with EIED (Figure 6). nanoparticulated form (11.6 mg/kg) proved to

Adrenoreactivity %

232.9* 240.3* 20 239.9* 238.9* 245.1* 18 16 240.1* 232.4* 225.3* 14 230.3* 12 232.0* 221.0*# 8.42 222.1*# 219.1*# 10 8 6 4 2 0

Intact EIED EIED + EIED + EIED + EIED + Simvastatin Atorvastatin Rosuvastatin Nano-rosuvastatin 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg mg/kg mg/kg mg/kg

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 6. Adrenoreactivity indicators with the use of inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of modeling endotoxin-induced endothelial dysfunction

The level of final metabolites of NOx and change in the volume of distribution of eNOS expression, subject to a high increase rosuvastatin by the blood stream restriction (NOx) (Figure 7) and decrease (Expression of (Figure 9). eNOS) (Figure 8) in the simulating of Systemic inflammatory reaction marker the endotoxin-induced pathology at the effect of C-reactive protein has showed a 7.5-fold statins were normalized on the 8th day and increase in EIED, but being under the influence reached values not differing from intact of medium and large doses of inhibitors of animals at the maximum doses of HMG-CoA HMG-CoA reductase simvastatin, atorvastatin, reductase. It should be noticed that the greatest rosuvastatin and nanoparticulated rosuvastatin, effect the nanoparticulated form of rosuvastatin it was reduced to values statistically not had. This fact supports the hypothesis of a different from intact animals (Figure 9).

Nox (µmol/l)

179.2* 182.3* 20 189.3* 171.1* 180.1* 18

16 # 141.1*# 142.0* 161.7* 14 152.9* 12 122.9*# 122.1*# 8.42 130.0*# 132.1*# 10 8 6 4 2 0

Intact EIED EIED + EIED + EIED + EIED + Simvastatin Atorvastatin Rosuvastatin Nano-rosuvastatin 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg mg/kg mg/kg mg/kg

* - P<0,05 -compared with intact, # - P<0,05-compared with EIED

Fig. 7. Concentration of nitrogen oxide metabolites (NOx) using the inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of endotoxin-induced endothelial dysfunction modeling.

eNOS expression (%)

54.1* 5.4* 20 27.9* 41.04* 26.1* 18

10016 *# 1.76*# 30.24 41.7*# 14 16.6*# 12 1.2*# 5.27*# 75.3 1.62*# 8.64*# 8010 8 6 40 4 2 0 0

Intact EIED EIED + EIED + EIED + EIED + Simvastatin Atorvastatin Rosuvastatin Nano-rosuvastatin 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg mg/kg mg/kg mg/kg

* - P<0.05 -compared with intact, # - P<0.05-compared with EIED

Fig. 8. Expression of NO-synthase (eNOS) using inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin against the background of endotoxin- induced endothelial dysfunction modeling

С-reactive protein (mg/l)

0.31* 0.38* 20 0.32* 0.30* 0.33*

16 0.19*# *# 0.18*# 0.17 0.21*# *# 12 0.08*# # 0.11 8.42 0.09 0.18*#

Intact EIED EIED + EIED + EIED + EIED + Simvastatin Atorvastatin Rosuvastatin Nano-rosuvastatin 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg mg/kg mg/kg mg/kg

*- P<0,05 -compared with intact, # - P<0,05-compared with EIED

Fig. 9. C-reactive protein (CRP) indices with the use of inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of endotoxin-induced endothelial dysfunction modeling

The proinflammatory cytokines IL-6 and rosuvastatin and nanoparticulated rosuvastatin TNF-α, during the EIED simulating, had had been exerting an evident normalizing effect increased 15 and 2.1 times. The use of medium and their values had been approximating to the and large doses of inhibitors of HMG-CoA same values in intact animals (Figure 10). reductase, simvastatin, atorvastatin,

IL-6 (pg/ml) TNF-α (pg/ml) 6.1* 19.13* 6.87* 17.83* 4.12* 5.95* 20 16.12* 17.01* 20 5.13* 15.45*

16 3.17*# 16 12.81*# 12.76*# 2.34*# 3.82*# 2.43*# 12.14*# 12.36*# 12 1.03*# 1.17*# 12 10.76*# 10.80*# 0.43 1.27# 1.48*# 8.42 9.89# 9.56*#

8 8

4 4

0 0

Intact EIED EIED + EIED + EIED + EIED+ Intact EIED EIED + EIED + EIED + EIED + simvastatin atorvastatin rosuvastatin Nano-rosuvastatin simvastatin atorvavastatin rosuvastatin Nano-rosuvastatin 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg 2.2; 4.3; 8.5 1.1; 2.2; 4.3 2.2; 4.3; 8.5 3.0; 6.3; 11.6 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED * - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 10. The concentration of proinflammatory cytokines IL-6 and TNF-α with the use of inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of endotoxin-induced endothelial dysfunction modeling

The most pronounced protective effect was increase in SBP and DBP, an increase in provided by the nanoparticulated form of adrenoreactivity, a decrease in the myocardial rosuvastatin 11.6 mg/kg. reserve, biochemical analysis showed a The modeling of L-NAME-induced disturbance in the metabolism of nitric oxide, endothelial dysfunction was accompanied by an histological findings revealed that modeling L-

NAME-induced NO deficiency on the 8th day simvastatin, atorvastatin, rosuvastatin and resulted in bright pronounced hypertrophy of nanoparticulated rosuvastatin effect on the myocardiocytes, and spasm of arterioles. model of L-NAME-induced deficiency of nitric Inhibitors of HMG-CoA reductase of oxide is shown in Figure 11.

АДBPsyst сист 175

125 MKDДМК АддиастBPdiast 48.6 75 4.9 25 28.5 ИнтактныеIntact -25 L-NAMEL-NAME LL-NAME-NAME+Simvastatin + Симвастатин MRМР146 -75 32.5 КЭД CED LL --NAMENAME+Atorvavastatin + Аторвастатин LL-NAME-NAME+Rosuvavastatin + Розувастатин -43.7 LL-NAME+-NAME+Nano НП розувастатин-rosuvastatin -0.4

27.5 AdRАДР Nox

eNOS

Fig. 11. Endothelioprotective effects of HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in L-NAME-induced endothelial dysfunction. Note: BPSyst – systolic blood pressure; BPdiast – diastolic blood pressure; CED – coefficient of endothelial dysfunction; NOx is the concentration of the nitrite ion in the plasma; eNOS – expression of eNOS; AdR – adrenoreactivity; MR – myocardial reserve; MKD is the myocardiocytes diameter (% of the group of intact animals)

HMG-CoA reductase inhibitors rosuvastatin had been also demonstrating simvastatin, atorvastatin, rosuvastatin, and cardioprotective effects and were preventing nanoparticulated rosuvastatin had been causing increasing in adrenoreactivity. Thus, in the a dose-dependent endothelial and control with simulating a model of L-NAME- cardioprotective effect. The result of it came induced nitric oxide deficiency, the maximum out in decreasing of systolic blood pressure intraventricular pressure during the test for values had been reaching to statistically adrenoreactivity was 247.3 ± 4.8 mm Hg, and significant values and were 167.2 ± 10.1, 140.3 with rosuvastatin and nanoparticulated ± 9.6, 132.2 ± 7.7 and 120.1 ± 6.4 mm Hg., but rosuvastatin correction 187.9 ± 10.2 and as for the controlled group, it was 190.3 ± 6.7 186.4 ± 10.7 mm Hg. respectively, which is mm Hg. A similar dynamics was found in the significantly less than in the control and close evaluation of diastolic blood pressure. At the to the values in intact animals. same time, CED dose-dependent had been The cardioprotective effect of inhibitors of decreasing reliably and under nanoparticulated HMG-CoA reductase of simvastatin, rosuvastatin influence had been reaching 1.9 ± atorvastatin, rosuvastatin and nanoparticulated 0.6 cu., what was almost corresponded to intact rosuvastatin had been also seen in improving animals and was practically 3 times less than in the indicators contractility during the resistance control 5.4 ± 0.6 cu. stress test. Thus, the values of intraventricular In experiments with stress tests, inhibitors pressure in the control group with L-NAME- of HMG-CoA reductase simvastatin, induced pathology were 66.0 ± 4.6% on 25th atorvastatin, rosuvastatin and nanoparticulated second of the test, whereas in the experimental

groups with simvastatin, atorvastatin, (C-reactive protein) and the level rosuvastatin and nanoparticulated rosuvastatin, proinflammatory cytokines normalization. respectively, they were 88.1 ± 5.8; 91.7 ± 6.3; During the experiment positive dynamics of the 92.4 ± 6.7 and 93.5 ± 7.4%, which is final products of NO and eNOS expression was significantly higher than in the control and does found. At the same time, the values of AD not differ from the values in intact animals of significantly were decreasing, and in the 83.6 ± 4.3%. experiments with nanoparticulated rosuvastatin Inhibitors of HMG-CoA reductase they had reached the set values. simvastatin, atorvastatin, rosuvastatin, and Investigation of the effect of nanoparticulated rosuvastatin had been causing monotherapy with NO donor L-arginine, a a dose-dependent endothelioprotective effect, nonselective inhibitor of arginase-S- (2-boro- which was consisted in preventing the increase ethyl) -L-cysteine (BEC), a selective in the values of the final metabolites of NOx inhibitor of arginase-2 arginasine and and reducing eNOS expression. Wherein darbepoetin in endotoxin-induced rosuvastatin and its nanoparticulated form had endothelial dysfunction in experimental the most significant effect. animals. So, the use of inhibitors of HMG-CoA Monotherapy with donator NO L-arginine reductase in simvastatin, atorvastatin, in the dose range of 70, 200 mg/kg daily rosuvastatin and nanoparticulated rosuvastatin intragastric on the background of the EIDE during simulating a model of EIED modeling normalized CED and did not (introduction of Staphylococcus aureus strain significantly affect the blood pressure 13407), and in simulating a model of ADMA- indicators. Inhibitors of arginase BEC (5 and like L-NAME-induced deficiency of NO leads 10 mg/kg) and arginasine (1 and 3 mg/kg) also to a dose-dependent endothelioprotective normalized CED and did not affect BP. The effect development, which consists in CED greatest effect was provided by recombinant normalization, adrenoreactivity encreasing erythropoietin at a dose of 500 μg/kg. So CED prevention and the myocardial reserve was 1.9 ± 0.2 USD, while in the group with depletion as well, and at the same time it leads EIED 3.7 ± 0.5 c. u. (Table 4). to biochemical markers of inflammation Table 4 Effect of monotherapy with L-arginine, BEC, arginasine and darbepoetin on the dynamics of hemodynamic parameters in animals with endotoxin-induced endothelial dysfunction (EIED) (M ± m, n = 10) Group SBP DBP CED Intact 129.4±2.2 89.2± 1.1 1.1 ± 0.1 EIED 117.6±2.3* 85.0±2.1 3.7±0.5* EIED + L-arginine 70 mg/kg 121.9±3.2 80.7±2.0 3.1±0.4* EIED + 200 mg/kg 118.5±2.1 76.3±2.1 2.1±0.3*#

EIED + BEC 5 mg/kg 114.1±3.0* 83.5±2.0 2.9±0.4* EIED + BEC 10 mg/kg 120.5±2.7 85.7±2.3 2.2±0.3*#

EIED + Arginasine 1 mg/kg 117.8±3.2* 88.2±2.7 3.0±0.3* EIED + Arginasine 3 mg/kg 126.2±3.4 85.1±2.1 2.1±0.3*#

EIED + Darbepoetin 50 µg/kg 121.2±3.1 86.9±2.1 2.5±0.3* EIED + Darbepoetin 500 µg/kg 126.3±3.2 83.4±2.3 1.9±0.2*# Note: SBP – systolic blood pressure (mmHg), DBP – diastolic arterial pressure (mmHg), CED – coefficient of endothelial dysfunction (unit units), * – significant difference with a group of intact animals (p <0.05); # – significant difference with the endotoxin-induced endothelial dysfunction (EIED) group (p <0.05). December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 53 Clinical Pharmacology. 2017;3(4):35-77.

The positive dynamics of the indices of BEC, arginasine and darbepoetin, the level of contractility in carrying out stress tests in final metabolites of NOx in the modeling of animals with EIDD was found in parallel. Thus, sepsis increased significantly less than in prevention of an increase in adrenoreactivity control experiments, and in large doses did not and a decrease in the myocardial reserve was differ from intact animals. Similarly, revealed. At the same time, as in CED, arginine monotherapy with these drugs prevented the (3 mg/kg) and darbepoetin (500 μg/kg) were decrease of eNOS expression. The most the most effective. significantly protective effect of monotherapy The most pronounced endothelial and with L-arginine, BEC, arginasine and cardioprotective effect of monotherapy with L- darbepoetin was manifested in relation to the arginine, BEC, arginine and darbepoetin was level of C-reactive protein and the values of manifested in the values of biochemical proinflammatory cytokines IL-6 and TNF-α markers in animals with EIDD. Thus, under the (Table 5). influence of monotherapy with L-arginine, Table 5 Effect of monotherapy with L-arginine, BEC, arginasine and darbepoetin on the dynamics of the values of biochemical markers (total NO, eNOS expression, C-reactive protein, IL-6, TNF-α) in animals with endotoxin-induced endothelial dysfunction (EIED) (M ± m, n = 10) Group NOх еNOS CRP IL-6 TNF-α Intact 116.8±10.3 5.4±0.21 0.05±0.01 0.43±0.17 8.42±2.51 EIED 182.3±12.4* 0.04±0.01* 0.38±0.01* 6.87±1.93* 17.83±3.79* EIED + L- arginine 165.4±9.7* 1.17±0.27* 0.13±0.01* 3.12±1.12* 11.13±2.17* 70 mg/kg 132.7±11.3*# 2.14±0.22*# 0.17±0.02*# 2.23±1.67*# 10.23±2.08*# 200 mg/kg EIED + BEC 5 mg/kg 167.1±12.4* 2.15±0.32* 0.12±0.02* 3.98±1.23* 10.16±2.37* 10 mg/kg 133.7±10.1*# 3.21±0.75*# 0.14±0.01*# 2.37±1.17*# 9.97±1.36*# EIED + arginasine 146.0±13.1* 2.39±0.74* 0.16±0.03* 2.24±1.27* 12.89±2.06* 1 mg/kg 125.1±97.3*# 4.16±1.15*# 0.10±0.02*# 2.03±0.98*# 10.54±1.72*# 3 mg/kg EIED + darbepoetin 50 145.1±11.7* 2.75±0.64* 0.21±0.02* 2.11±1.12* 9.58±2.29* µg/kg 122.5±10.5*# 4.19±0.72*# 0.17±0.01*# 1.72±0.97*# 8.20±2.26*# 500 µg/kg Note: NOx is the final metabolite of NO (μmol/L); eNOS expression (%); level of CRP-C-reactive protein (mg/l); IL-6 – interleukin 6 (pg/ml) TNF-α – tumor necrosis factor α (pg/ml), * – significant difference with a group of intact animals (p <0.05); # – significant difference with endotoxin-induced endothelial dysfunction (EIED) group (p <0.05).

The obtained results allowed to determine darbepoetin EIED on background modeling by the choice of doses. L-arginine 200 mg/kg, introducing strain 13407 Staphylococcus BEC-10 mg/kg, arginine-3 mg/kg and aureus exhibits endotelio- and cardioprotective darbepoetin 500 μg/kg for studies on the action, expressed in preventing increase CED effectiveness of combined use with inhibitors adrenoreactivity, conservation and of HMG-CoA reductase by simvastatin, normalization of myocardial reserve atorvastatin, rosuvastatin and nano-pararticular biochemical marker values (Totals NO, rosuvastatin. expression of eNOS, CRP, IL-6, TNF-α). In Thus, the use of monotherapy with a donor this case, the drugs had a dose-dependent effect NO L-arginine, inhibitors of arginase and and were approximately equally effective. December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Denisiuk T.A. Pharmacotherapeutic strategies for endothelial dysfunction correction with use of statines in syndrome of systemic inflammatory response. Research Result: Pharmacology and 54 Clinical Pharmacology. 2017;3(4):35-77.

Investigation of the effect of combined background of the EIED modeling normalized therapy with the L-arginine and inhibitors CED and did not significantly affect blood of HMG-CoA reductase by simvastatin, pressure (table 4). Inhibitors of HMG-CoA atorvastatin, rosuvastatin and reductase, simvastatin (8.5 mg/kg), atorvastatin nanoparticulated rosuvastatin in endotoxin- (4.3 mg/kg), rosuvastatin (8.5 mg/kg) and induced endothelial dysfunction. nanoparticularized rosuvastatin (11.6 mg/kg) at Monotherapy with donator NO L-arginine the most effective doses significantly improved (200 mg/kg) daily intragastric on the CED and did not affect blood pressure (Figure 5).

The protective effect of the combined use of L-arginine with HMG-CoA reductase inhibitors on the model of L-NAME-induced endothelial dysfunction BPsystАД сист 175

125 ИнтактныеIntact BPdiast MKDДМК 75 Аддиаст L-NAME 25 L-NAME

-25 LL--NAMENAME+Arginine + Ар -75

-125 LL--NAMENAME+Arginine + Аргинин + МР -175 CEDКЭД MR Симвастатин+Simvastatin

LL --NAMENAME+Arginine + Аргинин ++ АторвастатинAtorvastatin

L--NAMENAME+Arginine + Аргинин ++ РозувастатинRosuvastatin АДР Nox AdR NOx LL--NAME+NAME+Arginine Аргинин + + НанарозувастатинNano-rosuvastatin

eNOSeNOS

Fig. 12. Endothelioprotective effects of combined use of L-arginine with inhibitors of HMG-CoA reductase simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in L-NAME-induced endothelial dysfunction Note: BPsyst – systolic blood pressure; BPdiast – diastolic blood pressure; CED – coefficient of endothelial dysfunction; NOx – concentration of nitrite ions in plasma; eNOS – expression of eNOS; ADR – adrenoreactivity; MR – myocardial reserve; DMK is the diameter of myocardiocytes (% of the group of intact animals)

The combined use of L-arginine with reflected in the indices of hemodynamic HMG-CoA reductase inhibitors simvastatin, parameters of systolic and diastolic blood atorvastatin, rosuvastatin and nanoparticulated pressure, as well as CED, which in the EIED rosuvastatin showed the most pronounced group did not differ statistically from intact endothelioprotective effect, which was animals (Figure 13).

CED (c.u.) BP (mmhg)

160 129# 3.7* 140 129 127#130#135# 137#136# 117*118* 142# 137# 1.5*# 1.5*# 120 4 *# 1.7*# *# 2.1 2.1*# 1.7 100 92# 91# 88 89 85 76* 85# 83 84 94# 3 2.3*# 1.5*# 87# 1.1 80 1.6*# 2 60

1 40 20 0 0 kg + / + + 200 + + Intact EIED tatins mg + S arginine arginine arginine rosuvastatin - EIED - statins - L + + EIED L 200 + arginine arginine arginine+ arginine - - - EIED - EIED atorvastatin kg L nano L L rosuvastatin simvastatin L Intact / + + + arginine arginine + + + + + - EIED L mg EIED EIED simvastatin EIED EIED rosuvastatin EIED nanorosuvastatin EIED EIED EIED atorvastatin

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 13. The indices of endothelial dysfunction coefficient (CED) and blood pressure (BP) when combined use of L-arginine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of endotoxin-induced endothelial dysfunction (EIED) modeling

At the same time, a positive dynamics of and pro-inflammatory cytokines IL-6 and contractility indices was found in carrying out TNF-α, the values of which did not differ from stress tests in animals with EIED. Thus, the such in intact animals (Figures 15-17). prevention of an increase in adrenoreactivity Thus, the use of combined use of L- and a decrease in the myocardial reserve both arginine with HMG-CoA reductase inhibitors with the use of L-arginine and with its simvastatin, atorvastatin, rosuvastatin and combined use of inhibitors of HMG-CoA- nanoparticulated rosuvastatin against the reductase by simvastatin, atorvastatin, background of modeling of EIED and rosuvastatin and nanoparticularated L-NAME-induced deficiency of NO exhibits rosuvastatin was detected. At the same time, as endothelial and cardioprotective effect, with СED, the combined use of drugs led to the manifested in preventing CED, fact that the values did not differ from those of adrenoreactivity, preservation of the intact animals (Figure 14). myocardial reserve and normalization of the The most significantly protective effect of values of biochemical markers (Total NO, the combined use of the NO L-arginine donor expression of eNOS, CRP, IL-6, TNF-α). with the HMG-CoA reductase inhibitors Combined therapy was so effective that the simvastatin, atorvastatin, rosuvastatin and values obtained with combined therapy did not nanoparticulated rosuvastatin was manifested differ from those obtained in intact animals with respect to the level of C-reactive protein (Figure 12).

Adrenoreactivity (mmhg)

219.1*# 196.7*# 221.0*# 240.3* *# 211.4*# 222.1*# 193.8 201.5 201.0*# 232.0*# 250 204.8*#

200

150

100

0 kg + / EIED + EIED + L-arginine mg Intact EIED statins

arginine arginine 200 mg/kg EIED - L 200 + statins

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED Fig. 14. Adrenoreactivity indicators for combined use of L-arginine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin against the background of endotoxin-induced endothelial dysfunction modeling

In the experiments with loading tests, the a positive additive effect on the reduction of combined use of L-arginine with HMG-CoA hypertrophy of myocardiocytes was found. reductase inhibitors simvastatin, atorvastatin, Thus, the use of combined use of L- rosuvastatin and nanoparticulated rosuvastatin arginine with HMG-CoA reductase inhibitors also shows an additive cardioprotective effect, simvastatin, atorvastatin, rosuvastatin and prevention of adrenoreactivity growth and nanoparticulated rosuvastatin against the depletion of the myocardial reserve. background of the EIED and L-NAME-induced A similar additive effect of combined use deficiency shows NO endothelial and of L-arginine and HMG-CoA reductase cardioprotective effect, which is manifested in inhibitors simvastatin, atorvastatin, rosuvastatin preventing СED increase, adrenoreactivity, and nanoparticulated rosuvastatin forms, preservation of the myocardial reserve and interaction with metabolites of nitrogen NOx normalization of biochemical markers (Total and eNOS expression. NO, expression of eNOS, CRP, IL-6, TNF-α). In parallel with the dynamics of the Combined therapy was so effective that the additive effect of combined application of L- values obtained with combined therapy did not arginine and inhibitors of HMG-CoA differ from those in intact animals. reductase, simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin,

eNOS expression NOx

66.4*# 54.1*# 182.3* 132.1*# 117.8*# 75.3 *# 60.3 122.1*# 132.7*# 119.5*# 57.1*# 200 116.8 130.0*# *# 660 41.1*# 121.7 *# 123.5*# 55.6 150 122.9*# 28.9*# 4 27.9*# 100 30 26.1*# 5.4* 2 50

0 0 0 kg + EIED + + / kg + + kg / L-arginine + EIED / Intact mg 200 mg/kg + statins mg EIED arginine mg EIED Intact statins - statins statins EIED EIED arginine EIED L 200 - arginine arginine EIED + + - L 200 L 200

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 15. Nitric oxide concentration (NOx) and NO synthase expression (eNOS) in combined use of L-arginine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of endotoxin-induced endothelial dysfunction modeling

С-reactive protein (mg/l)

0.38*

0.18*# 0,40

0.06*# 0,30 0.17*# 0.11*# 0.06*# 0.09*# 0,20 0.05 0.07*# *# 0.08*# 0.08 0,10

0,00 kg + / EIED + + L-arginine mg Intact EIED 200 mg/kg arginine EIED statins EIED -

L + statins 200

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 16. C-reactive protein (CRP) indices with combined use of L-arginine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of endotoxin-induced endothelial dysfunction modeling

IL-6 TNF-α

6.87* 0.63*# 17.83* 9.56*# 7.56*# 8 0.78*# 20 10.80*# *# *# 1.48 0.90*# 10.23 8.42*# 8.42 9.89*# *# 6 15 8.79 2.23*# 1.17*# 10.76*# *# 0.92*# 9.12 4 0.43 1.27*# 10

2 1.03*# 5

0 0 + 200 + + kg + + kg + / kg / + / + kg / mg EIED mg statins statins mg Intact EIED EIED EIED arginine EIED statins EIED statins - arginine EIED mg arginine EIED - L Intact - arginine 200 L 200 - L 200 L

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 17. Concentration of proinflammatory cytokines IL-6 and TNF-α in combined use of L-arginine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of modeling endotoxin-induced endothelial dysfunction (EIED)

Study of the effect of combined therapy Parallel to this, a positive dynamics of with a nonselective inhibitor of arginase S- contractility indices was observed when (2-boro-ethyl)-L-cysteine (BEC), with carrying out stress tests in animals with EIED simvastatin, atorvastatin, rosuvastatin and (Figure 19). BEC was inferior in activity to nanoparticulated rosuvastatin in endotoxin- statins both in terms of preventing an increase induced endothelial dysfunction model. in adrenoreactivity, and in terms of conserving Monotherapy with a nonselective inhibitor an expansion reserve. The combined use of of arginase S-(2-boro-ethyl)-L-cysteine (BEC) BEC with simvastatin, atorvastatin, daily intragastric moderately normalized CED rosuvastatin, and nano-pararticular rosuvastatin moderately normalized CED and did not did not lead to an additive enhancement of the significantly affect blood pressure (Table 4). effect, and the values of intraventricular The CED value was 2.5 ± 0.4 c. u. Inhibitors of pressure when loading tests were close to those HMG-CoA reductase, simvastatin (8.5 mg/kg), in the BEC series and inferior to the HMG- atorvastatin (4.3 mg/kg), rosuvastatin CoA reductase inhibitors. A similar trend has (8.5 mg/kg) and nanoparticulated rosuvastatin been observed with regard to the values of (11.6 mg/kg) at the most effective doses biochemical markers in animals with EIED significantly improved CED and did not affect (Figures 20-22). blood pressure. The CED values were in the The most significantly protective effect range 2.3 – 1.5 c. u. of the combined use of BEC with inhibitors of The combined use of BEC with statins did HMG-CoA reductase by simvastatin, not show an additive effect with respect to atorvastatin, rosuvastatin and nanoparticulated CED and BP. Values were even slightly higher rosuvastatin was manifested with respect to the than with statin monotherapy, however, were level of C-reactive protein whose level was statistically significantly different from intact comparable in all series of experiments. animals (Figure 18). (Figure 21).

CED (c.u.) BP (mmhg)

160

140 135#129# 129 127#130# 125#126#127# 117*115* 122# 3.7* 120 100 1.5*# 2.5* 89 85# 92#91# 88 4 85 76* 87# 83 84 94# * 1.7*# * 80 2.5 # 2.1*# 2.7 3 2.3* 2.5* - 1.1 60 2.6* 2 40

1 20

0 0 kg + + mg / / BEC + Intact EIED + + mg EIED BEC mg Intact simvastatin atorvastatin statins rosuvastatin EIED simvastatin atorvastatin EIED + + rosuvastatin 10 + + + statins BEC + EIED 10 10 rosuvastatin - EIED + nanorosuvastatin + + BEC BEC BEC + + + + EIED EIED EIED BEC EIED Nano EIED EIED EIED EIED * - P<0.05 compared with intact; #- Р<0.05 compared with EIED Fig. 18. The indices of the endothelial dysfunction coefficient (CED) and arterial pressure (BP) when combined with the use of S- (2-boro-ethyl) -L-cysteine (BEC) and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in endotoxin-induced endothelial dysfunction model

Adrenoreactivity (mmhg)

226.5*# 240.3* 219.1*# 223.9*# 260 221.0*# 231.9*# 220.7*# 222.1*# 232.6*# 240 232.0*# 201.5 220

200

180 kg + / + + kg+ kg+ / mg BEC Intact EIED EIED statins 10 BEC mg EIED statins EIED 10 * - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 19. Adrenoreactivity in the combined use of S- (2-boro-ethyl) -L-cysteine (BEC) and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin against endotoxin-induced endothelial dysfunction modeling

eNOS expression NOx

75.3 54,1*# 132.1*# 40.1*# 182.3* 117.8*# 139.1*# *# 122.1*# 606 41.0*# 141.4 143.5*# *# 200 116.8 130.0*# 34.0 145.7 *# *# 32.5 4 26.6 27.9*# 150 122.9*# 30 29.6*# 26.1*# 100 0.54* 2 50 0 0 0 + + + + + BEC kg kg kg + + / / / + BEC kg / + statins mg mg EIED mg BEC statins statins BEC EIED EIED EIED Intact EIED 10 10 mg 10 10 EIED EIED statins 10 Intact 10 EIED

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 20. Nitric oxide concentration (NOx) and NO synthase expression (eNOS) when combined with the use of S-(2-boro-ethyl)-L-cysteine (BEC) and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of modeling endotoxin- induced endothelial dysfunction

С-reactive protein (mg/l)

0.83*#

1,0 0.57*#

0,8 0.18*# 0.38* 0.42*#

0,6 *# 0.11*# 0.21 0.38*#

0,4 0.05 0.09*#

0,2 0.08*#

0,0 + + kg + / + BEC kg / mg + EIED BEC Intact statins EIED mg EIED statins 100 100 10 10 EIED * - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 21. C-reactive protein (CRP) indices in the combined use of S-(2-boro-ethyl)-L-cysteine (BEC) and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin against endotoxin- induced endothelial dysfunction

IL-6 TNF-α

6.87* 17.83* 1.87*# 9.56*# 7.56*# 8 *# 20 *# 1.89 *# 10.80*# 8.42 1.48*# 10.23 *# 8.42 9.89*# *# 6 1.94 15 8.79 10.76*# *# 2.78*# 1.17*# 9.12 2.54*# 4 10 0.43 1.27*# 2 1.03*# 5

0 0 + + + + kg kg + / + + / kg kg / + ВЕС / EIED mg statins mg Intact EIED statins ВЕС EIED Intact ВЕС ВЕС EIED statins mg EIED 10 EIED 0 statins mg EIED ВЕС ВЕС 1 EIED 10 10

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 22. Concentration of proinflammatory cytokines IL-6 and TNF-α when combined use of S- (2-boro-ethyl) -L-cysteine (BEC) and inhibitors of HMG-CoA reductase of simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of modeling endotoxin- induced endothelial dysfunction (EIED)

Fig. 23. Protective effect of the combined use of S- (2-boro-ethyl) -L-cysteine (BEC) with HMG-CoA reductase inhibitors on the model of L-NAME-induced endothelial dysfunction Note: BPSyst – systolic blood pressure; BPdiast – diastolic blood pressure; CED – coefficient of endothelial dysfunction; NOx – concentration of nitrite ions in plasma; eNOS – expression of eNOS; ADR – adrenoreactivity; MR – myocardial reserve; DMK is the diameter of myocardiocytes (% of the group of intact animals)

The effect of combined therapy with a non- background of endotoxin-induced pathology selective inhibitor of arginase BEC (10 mg/kg) modeling, and L-NAME-induced NO with inhibitors of HMG-CoA reductase by deficiency, exhibits endothelial and simvastatin (8.5 mg/kg), atorvastatin cardioprotective effects in preventing CED, (4.3 mg/kg), rosuvastatin (8.5 mg/kg) and adrenoreactivity, preservation of the nanoparticulated rosuvastatin (11.6 mg/kg) on myocardial reserve and normalization of the the model of L-NAME-induced deficiency of values of biochemical markers (Total NO, nitric oxide is shown in Figure 23. eNOS expression, C-reactive protein, IL-6, BEC moderately reduced systolic and TNF-α). At the same time, combined therapy diastolic blood pressure and 2-fold decreased showed no additive effect of drugs (Figure 23). CED, the value of which was 2.5 ± 0.5 c.u., Study of the effect of combined therapy whereas in control 5.4 ± 0.6 c.u. (table 4). with a selective inhibitor of arginase-2 HMG-CoA reductase inhibitors simvastatin, arginasine with simvastatin, atorvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin and nanoparticulated rosuvastatin caused more pronounced rosuvastatin in endotoxin-induced endothelial and cardioprotective effects in endothelial dysfunction. preventing the increase in blood pressure and Monotherapy with a selective inhibitor of CED (Figure 5). arginase-2 arginasine (3 mg/kg) daily The combined use of a nonselective intragastric on the background of modeling inhibitor of BEC arginase with simvastatin, EIED moderately normalized CED and did not atorvastatin, rosuvastatin, and nanoparticulated significantly affect blood pressure (Figure 19). rosuvastatin also had a protective effect, but did The average value of CED was 2.1 ± 0.3 cu. not show an additive effect. In the experiments Inhibitors of HMG-CoA reductase, simvastatin with loading tests, the combined use of BEC (8.5 mg/kg), atorvastatin (4.3 mg/kg), with HMG-CoA reductase inhibitors also rosuvastatin (8.5 mg/kg) and nanoparticularized revealed a cardioprotective effect, preventing rosuvastatin (11.6 mg/kg) at the most effective an increase in adrenoreactivity and depletion of doses significantly improved CED and did not the myocardial reserve. At the same time, the affect blood pressure. The CED values were in combined use of drugs did not increase the the range 2.3 – 1.5 cu. cardioprotective effect, and the adrenoreactivity The combined use of a selective inhibitor values even increased slightly compared to of arginase-2 arginasine (3 mg/kg) with statins statin monotherapy. A similar additive effect of showed the additivity of the effect against CED the combined use of BEC and inhibitors of and BP. Values were even slightly higher than HMG-CoA-reductase of simvastatin, with monotherapy with statins, however, were atorvastatin, rosuvastatin and nanoparticulated statistically significantly different from intact rosuvastatin was also observed with respect to animals (Figure 24). In parallel, a positive the values of the final NOx metabolites and dynamics of contractility indices was found in eNOS expression. In parallel with the dynamics carrying out stress tests in animals with of the cardioprotective effect of the combined endotoxin-induced endothelial dysfunction. use of BEC with HMG-CoA reductase The selective inhibitor of arginase-2 arginasine inhibitors, a positive effect has been found with (3 mg/kg) was superior to statins both in terms respect to the reduction of hypertrophy of of preventing an increase in adrenoreactivity myocardiocytes. and in terms of conserving an expansion Thus, the use of the combined use of a reserve. The combined use of arginasine with nonselective inhibitor of arginase BEC with simvastatin, atorvastatin, rosuvastatin, and simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin resulted in an nanoparticulated rosuvastatin against the additive enhancement (Figure 25).

CED (c.u.) BP (mmhg) 160

140 135#129# 129 127#130# 126#127#128# 117*126* 124# 3.7* 120

100 91# 1.5*# 1.5* 89 85 85# 88# 88 4 85* 87# 83 84 87# * 1.7*# * 80 2.1 # 2.1*# 7.7 3 2.3* 1.5* 1.1 60 1.6* 2 40

1 - 20

0 0 + + + + + + kg kg / / EIED statins EIED simvastatin Intact Intact mg mg atorvastatin EIED rosuvastatin statins EIED + + EIED 3 + 3 3 arginasina arginasine arginasina rosuvastatin simvastatin - atorvastatin rosuvastatin + + arginasine + + + nano EIED EIED + EIED EIED arginasine EIED nanorosuvastatin arginasine + + EIED EIED EIED+arginasine EIED

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED Fig. 24. Indices of endothelial dysfunction coefficient (CED) and arterial pressure (BP) when combined use of arginasine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of endotoxin-induced endothelial dysfunction (EIED) modeling

A similar trend has been observed with pressure and reduced CED by a factor of 1.7 ± respect to the values of biochemical markers in 0.6, while in control 5.4 ± 0,6 c.u. Inhibitors of animals with endotoxin-induced endothelial HMG-CoA reductase simvastatin, atorvastatin, dysfunction (Fig. 26). rosuvastatin and nanoparticulated rosuvastatin The most significantly protective effect of caused more pronounced endothelial and the combined use of a selective inhibitor of cardioprotective effects in preventing the arginase-2 arginasine (3 mg/kg) with inhibitors of increase in blood pressure and CED. HMG-CoA reductase by simvastatin, The combined use of a selective inhibitor atorvastatin, rosuvastatin and nanoparticulated of arginase-2 arginasine (3 mg/kg) with rosuvastatin was manifested in relation to the simvastatin, atorvastatin, rosuvastatin and level of C-reactive protein and the values of nanoparticulated rosuvastatin also had a proinflammatory cytokines IL-6 and TNF- α, the protective effect and showed an additive effect, levels of which were comparable in all series of manifested in a decrease in the endothelial experiments that received pharmacotherapy dysfunction coefficient, prevention of NOx (Figures 27, 28). We did not find an additive concentration decrease, and improvement of effect when combined in this model of pathology. indices myocardial contractility in conducting In the model of L-NAME-induced nitric functional tests for adrenoreactivity and oxide deficiency, arginine (3 mg/kg) resistance loading, reduction of hypertrophy of moderately reduced systolic and diastolic blood myocardiocytes (Figure 29).

Adrenoreactivity (mm hg)

219.1*# 196.9*# 240.3* 221.0*# * 201.7*# 212 # 222.1*# 201.5 197.4*# 232.0*# 250 196.9*#

200

150

100

0 + + kg + + / + kg / mg Intact EIED statins EIED EIED 3 mg arginasine statins EIED 3 * - P<0.05 compared with intact; #- Р<0.05 compared with EIED Arginasine

Fig. 25. Adrenoreactivity in the combined use of arginasine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of modeling endotoxin-induced endothelial dysfunction (EIED)

eNOS expression NOx

*# 54.1 132.1*# 75.6 182.3* 107.9*# 67.1*# 119.8*# *# 122.1*# 125.1 123.1*# 6 41.0*# 200 116.8 130.0*# *# 180 119.4 605 61.7 *# 160 *# 60.3 122.9*# 4 56.2 27.9*# 140 55.4*# 120 3 26.1*# 100 30 0.54* 80 2 60 1 40 20 0 0 0 3 3 + + + + + + + + + kg + kg kg / / / kg / EIED statins statins mg EIED EIED mg Intact Intact mg statins EIED EIED EIED 3 statins EIED 3 EIED mg arginasine arginasine arginasine

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 26. Nitric oxide concentration (NOx) and NO synthase expression (eNOS) combined with the use of arginasine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of modeling endotoxin-induced endothelial dysfunction (EIED)

С- reactive protein (mg/l)

0.38* 0.23*# 0.18*# 0,4 0.17*# 0.19*# 0,3 0.11*# 0.10*# 0.09*# 0,2 0.05 0.12*# 0.08*# 0,1

0,0 + + kg + + / + kg mg statins EIED / EIED EIED Intact statins EIED Arginasine 3 mg Arginasine 3 * - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 27. Concentration of C-reactive protein (CRP) with the combined use of arginasine and HMG- CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of endotoxin-induced endothelial dysfunction (EIED) modeling

IL-6 TNF-α

17.83* 6.87* 3.89*# 9.56*# 8.63*# 8 *# 20 10.80*# 3.88 *# *# *# 10.54 8.47 1.48 *# 3.82 9.89*# *# 6 16 8.42 8.79 2.03*# 1.17*# 10.76*# 9.12*# 3.06*# 12 4 0.43 1.27*# 8 2 1.03*# 4 0 3 0 + + + + + kg 3 / kg + + + / + + kg mg statins / EIED EIED EIED statins Intact EIED kg 3 mg / Arginasine mg statins Intact Arginasine EIED EIED EIED statins EIED 3 mg Arginasine Arginasine

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 28. Concentration of IL-6 TNF-α with the combined use of arginasine and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of modeling endotoxin-induced endothelial dysfunction (EIED)

BPsystАД сист 175

125 BPdiast ИнтактныеIntact MKDДМК АДдиаст 75 L-NAMEL-NAME 25

-25 LL-NAME-NAME+Arginasine + Аргиназин

-75

L-NAME + Аргиназин + -125 CED L-NAME+Arginasine MRМР КЭД Симвастатин +Simvastatin L -NAME + Аргиназин + АторвастатинL-NAME+Arginasine +Atorvavastatin L-NAME + Аргиназин + РозувастатинL-NAME+Arginasine +Rosuvastatin АДР Nox L-NAME+ Аргиназин + AdR NOx НанарозувастатинL-NAME+Arginasine +Nano-rosuvastatin eNOS

Fig. 29. Protective action of combined arginine with HMG-CoA reductase inhibitors on the model of L-NAME-induced endothelial dysfunction Note: BPsyst – systolic blood pressure; BPdiast – diastolic blood pressure; CED – coefficient of endothelial dysfunction; NOx – concentration of nitrite ions in plasma; eNOS – expression of eNOS; ADR – adrenoreactivity; MR – myocardial reserve; DMK is the diameter of myocardiocytes (% of the group of intact animals)

Thus, the use of the combined use of a TNF-α). At the same time, combined therapy selective inhibitor of arginase-2 arginasine (3 revealed the additive effect of drugs. mg/kg) with simvastatin, atorvastatin, Study of the effect of combined therapy rosuvastatin and nanoparticulated rosuvastatin with darbepoetin, simvastatin, atorvastatin, against endotoxin-induced pathology modeling, rosuvastatin and nanoparticulated rosuvastatin and L-NAME-induced NO deficiency, exhibits in endotoxin-induced endothelial dysfunction. ectothelial and cardioprotective effects, The combined use of darbepoetin expressed in preventing the increase in CED, (500 μg/kg) with statins showed additivity of adrenoreactivity, preservation of the the effect against СED and BP. Values were myocardial reserve and normalization of the even slightly higher than with statin values of biochemical markers (Total NO, monotherapy, however, were statistically eNOS Expression, C-reactive protein, IL-6, significantly different from intact animals (Figure 30).

CED (c.u.) BP (mmhg)

160

140 135#129# 129 127#130# 122#123#124# 3.7* 117*126* 120# 120 1.5*# 1.5* 4 100 91# 1.7*# 89 85 85# 88# 88 1.9*# 2.1*# 1.6* 85*87# 83 84 87# 80 3 2.3* 1.5* 1.1 * - 1.6 60 2 40 1 20 0 0 kg + kg + + / / g g EIED rosuvastatin Intact - Intact µ EIED torvastatin statins Darbepoetin Darbepoetin statins A Rosuvastatin Simvastatin EIED EIED Darbepoetin EIED + + rosuvastatin Darbepoetin + + + - + + Nano Darbepoetin + 500 + 500 µ 500 + + Darbepoetin Darbepoetin Rosuvastatin EIED + EIED EIED EIED EIED EIED +Simvastatin EIED +Atorvastatin EIED EIED +Nano

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED Fig. 30. The indices of endothelial dysfunction coefficient (CED) with the combined use of darbepoetin and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of modeling endotoxin-induced endothelial dysfunction (EIED)

The most significantly protective effect of atorvastatin, rosuvastatin and nanoparticulated the combined use of darbepoetin (500 μg/kg) rosuvastatin against the background of with inhibitors of HMG-CoA reductase by endotoxin-induced pathology modeling, the simvastatin, atorvastatin, rosuvastatin and introduction of strain 13407 of Staphylococcus nanoparticulated rosuvastatin was manifested aureus shows endothelial and cardioprotective in relation to the level of C-reactive protein and effects, which is manifested in preventing CED the values of proinflammatory cytokines IL-6 and adrenoreactivity, preservation of the and TNF- were comparable in all series of myocardial reserve and normalization of the experiments that received pharmacotherapy. values of biochemical markers (Total NO, We did not find the additive effect of combined eNOS expression, C-reactive protein, IL-6, use on this model of pathology (Figures 31-33). TNF-α). At the same time, combined therapy Thus, the use of the combined use of revealed the additive effect of drugs darbepoetin (500 μg/kg) with simvastatin, (Figures 31-33).

eNOS expression NOx

66.8*# 182.3* 132.1*# 54.1*# 109.5*# 75.3 *# 63.0 122.1*# *# *# 119.4 *# 200 116.8 122.5 130.0*# *# 6 41.0*# 61.9 120.1 60 *# *# 56.3 160 122.9*# 119.3 56.6*# 4 27.9*# 120 80 30 26.1*# 0.54* 2 40 0 00 + kg + kg + + kg kg g/ + g/ + µ Intact g/ g/ EIED statins EIED µ EIED EIED Intact EIED statins IED 00 00 µ EIED 0 0 µ + + statins 500 E EIED 0 + + statins Darbepoetin Darbepoetin5 500 5 Darbepoetin Darbepoetin5

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 31. Nitric oxide (NOx) concentration and NO-synthase (eNOS) expression in combined use of darbepoetin and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin against endotoxin-induced endothelial dysfunction (EIED) modeling

С-reactive protein mg/l

0.38* 0.20*# 0.18*# 0,4

0.15*# *# 0,3 0.17 0.11*# 0.16*#

*# 0.09*# 0.18 0,2 0.05

0.08*# 0,1

0,0 kg + kg + + g/ µ Intact EIED EIED statins EIED EIED 500 µ g/ + statins + Darbepoeti n 500 Darbepoetin * - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 32. Concentration of C-reactive protein (CRP) in the combined use of darbepoetin and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of modeling endotoxin-induced endothelial dysfunction (EIED)

IL-6 TNFα

17.83* 6.87* 9.56*# 7.56*# 8 20 10.80*# *# *# *# *# 1.87 10.23 8.42 1.48 16 8.42 9.89*# 8.79*# 6 *# 2.78 1.89*# 10.76*# *# 1.17*# 12 9.12 1.94*# 4 1.27*# 0.43 2.54*# 8 2 1.03*# 4 0 0 + + + + + kg + + / kg + / g + kg kg g + µ / / statins µ EIED g EIED g Intact 0 µ µ EIED statins 0 EIED EIED Intact statins EIED 0 50 statins 0 EIED EIED 50 Darbepoetin Darbepoetin 50 50 Darbepoetin Darbepoetin

* - P<0.05 compared with intact; #- Р<0.05 compared with EIED

Fig. 33. Concentration of IL-6 and TNF-α with the combined use of darbepoetin and HMG-CoA reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin in the context of modeling endotoxin-induced endothelial dysfunction (EIED)

BPsystАД сист 175 125 Intact DMK Интактные ДМК 75 АДдиастBPdiast 25 L-NAMEL-NAME -25 -75 L-NAME + ДарбопоэтинДарбэпоэтин + -125 L-NAME+Darbepoetin MRМР -175 КЭД L-NAME + ДарбопоэтинДарбэпоэтин ++ СимвастатинL-NAME+Darbepoetin +Simvastatin L -NAME + ДабропоэтинДарбэпоэтин + + АторвастатинL-NAME+Darbepoetin +Atorvastatin АДР Nox L-L-NAME+DarbepoetinNAME + ДабропоэтинДарбэпоэтин + + ADR Розувастатин +Rosuvastatin L-L-NAME+DarbepoetinNAME+ ДабропоэтинДарбэпоэтин + + eNOS Нанарозувастатин +Nano-rosuvastatin

Fig. 34. Protective effect of the combined use of Darbepoetin with HMG-CoA reductase inhibitors on the model of L-NAME-induced endothelial dysfunction Note: BPyst – systolic blood pressure; BPdiast – diastolic blood pressure; CED – coefficient of endothelial dysfunction; NOx – concentration of nitrite ions in plasma; eNOS – expression of eNOS; ADR – adrenoreactivity; MR – myocardial reserve; DMK is the diameter of myocardiocytes (% of the group of intact animals)

In the model of L-NAME-induced of integral vector analysis, the values of which deficiency of nitric oxide, darbepoetin (500 are presented in Table 6. Comparing the areas μg/kg) moderately reduced systolic and of vector diagrams, we obtained distinct diastolic blood pressure and reduced CED by a differences between the group of intact factor of 1.8, with a value of 1.8 ± 0.3, whereas animals, animals with endotoxin-induced in control 5.4 ± 0.6 c.u. Inhibitors of HMG- endothelial dysfunction modeling and groups of CoA reductase simvastatin, atorvastatin, animals with pharmacological correction of the rosuvastatin, and nanoparticulated rosuvastatin above preparations. caused a more pronounced endothelial and Further, a probabilistic percentage of the cardioprotective effect in preventing the additions (Padd) of the tested combinations increase in blood pressure and CED (Figure (Table 6) was determined using an integral 34). vector analysis of combinations of HMG-CoA The combined use of darbepoetin (500 reductase inhibitors and endothelioprotectors. μg/kg) with simvastatin, atorvastatin, The highest probability percentage of the rosuvastatin, and nanoparticulated rosuvastatin additive for simvastatin was in combination also had a protective effect and showed an with the selective inhibitor of arginase-2 with additive effect (Figure 28), expressed in a arginine – 21.0 ± 1.9%. Similarly, atorvastatin decrease in the endothelial dysfunction worked at 22.6 ± 2.1%. It is noteworthy that the coefficient, prevention of NOx concentration maximum values of the additive effect were increase, and improvement in myocardial found for combinations of rosuvastatin with contractility when conducting functional tests arginine (3 mg/kg) and darbepoetin (500 for adrenoreactivity and resistance load; μg/kg), respectively, 31.9 ± 2.8 and 30.2 ± reduction of hypertrophy of myocardiocytes. 2.9%. Nanoparticulated rosuvastatin also Further analysis of combinations of showed the greatest addiction with arginasine inhibitors of HMG-CoA reductase and and darbepoetin 30.2 ± 2.9 and 26.0 ± 2.7%, endothelioprotectors was carried out on the respectively (Table 6). basis of the resulting areas obtained as a result Table 6 Probabilistic percent of additions (Padd) of the studied combinations in the modeling of ED

Experimental group Padd (%) ED + L-arginine 200 mg/kg + Simvastatin 8,5 mg/kg 3.4±0.3 ED + BEC 10 mg/kg + Simvastatin 8,5 mg/kg -0.01±0.1 ED + Arginasine 3 mg/kg + Simvastatin 8,5 mg/kg 21±1.9# ED + Darbepoetin 500 µg/kg + Simvastatin 8,5 mg/kg 9.2±1.1# ED + L-arginine 200 mg/kg + Atorvastatin 4,3 mg/kg 14.2±1.7# ED + BEC 10 mg/kg + Atorvastatin 4,3 mg/kg 0.0±0.1 ED + Arginasine 3 mg/kg + Atorvastatin 4,3 mg/kg 22.6±2.1# ED + Darbepoetin 500 µg/kg + Atorvastatin 4,3 mg/kg 13.4±1.8# ED + L-arginine 200 mg/kg +Rosuvastatin 8,5 mg/kg 23.5±2.7# ED + BEC 10 mg/kg + Rosuvastatin 8,5 mg/kg 0.01±0.1 ED + Arginasine 3 mg/kg + Rosuvastatin 8,5 mg/kg 31.9±2.8# ED + Darbepoetin 500 µg/kg + Rosuvastatin 8,5 mg/kg 30.2±2.9# ED + L-arginine 200 mg/kg Nanorosuvastatin 11,6 mg/kg 21±2.4# ED + BEC 10 mg/kg + Nanorosuvastatin 11,6 mg/kg 0.0±0.1 ED + Arginasine 3 mg/kg +Nanorosuvastatin 11,6 mg/kg 30.2±2.9# ED + Darbepoetin 500 µg/kg + Nanorosuvastatin 11,6 mg/kg 26.0±2.7# Note: ED – endothelial dysfunction; Padd – probability percentage of additions (%); * – p <0.005 in comparison with ED; # – p <0.005 compared with the endothelioprotective agent.

Discussion inflammation and level of proinflammatory The results of this research allow us to cytokines. The experiment had demonstrated affirm that simulating a model of endotoxin- positive dynamics of the final products of NO induced pathology created by injecting strain and expression of eNOS. It was proved that 13407 Staphylococcus aureus, leads to the rosuvastatin and its nanoparticulated form had development of endothelial dysfunction with the most significant effect. Statins has both changes in the response to endothelium- anti-inflammatory and cardioprotective effects. dependent and endothelium-independent In the implementation of anti-inflammatory and vascular tests; to increasing of stable NO cardioprotective effects, the mechanisms of metabolites with inflammatory markers values pharmacological preconditioning are of great raise: C-reactive protein, IL -6 and TNF-α. So, importance. This fact was proved with effects these phenomena correspond to the clinical removal in the blockade of К+-АТF-ase picture of endotoxin-induced multi-organ channels and blockade of iNOS. pathology typical for after abdominal disasters The combined use of L-arginine, a with the development of systemic vasculitis nonselective inhibitor of arginase, a selective and a further cascade of atherosclerotic changes inhibitor of arginase-2, arginasine, and in the vascular bed. The use of inhibitors of recombinant darbepoetin with HMG-CoA HMG-CoA reductase of simvastatin, reductase inhibitors had showed that the drugs atorvastatin, rosuvastatin and nanoparticulated enhance the endothelioprotective effects of rosuvastatin during simulating a model of each other. The combination of rosuvastatin endotoxin-induced pathology leads to a dose- with arginasine and darbepoetin gives the most dependent endothelioprotective effect additive effect. A possible explanation of the development, which is expressed in CED research results can be expressed as the normalization, adrenoreactivity prevention and analysis of application points of the drugs and myocardial reserve depletion, as well as to the suggestion of their pharmacodynamic normalization of biochemical markers of interaction (Figure 35).

Fig. 35. Hypothetical ways of pharmacodynamic interaction of HMG-CoA reductase inhibitors, erythropoietin preparations and endothelioprotectors

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Rus.

UDC: 615.275.4 DOI: 10.18413/2313-8971-2017-3-4-78-88

Anton P. Dovgan LIGAND OF PERIPHERAL IMIDAZOLINE RECEPTORS BASED ON AMIDES OF HETEROCYCLIC ACIDS C7070: EFFECT ON ISHEMIZED TISSUES

MAKS-Med Clinic "Garmonia Zdorovia", 32, Beregovaya str., 125284, Moscow, Russia Corresponding author, e-mail: [email protected]

Abstract Introduction: In this regard, the study of pleiotropic hepatoprotective properties of the agonist of peripheral imidazoline receptors C7070 seems interesting from an applied point of view. Materials and Methods: Models of a skin flap on a feeding leg, ischemia-reperfusion of the liver and rat heart isolated from Langendorff (ischemia-reperfusion and doxorubicin cardiomyopathy) were used. Results and Discussion: The I2 agonist C7070 at a dose of 10 mg/kg 4.5-fold prevents the increase in ALT and AST (332.56 ± 22.05/825.49 ± 22.46 ALT/AST 526.90 ± 17.97/1045.16 ± 80.02 units/l in control) and 2.5 times reduces the areas of ischmeic damage and necrosis (0.058 ± 0.029/0.046 ± 0.013 mm2) in the modeling of 15-minute ischemia liver. Moxonidine and metformin had a hepatoprotective effect: 44.99 and 36.88 for moxonidine (ALT and AST) and 34.20 / 21.02 for metformin (ALT / AST). The coefficients of histological hepatoprotective activity: 72.33 and 38.96 (moxonidine and metformin). C7070 (10.0 mg/kg) has a pronounced dermatoprotective activity and prevents the formation of necrosis on days 3 and 7 of the pathology by 40%. The dermatoprotective activity of metformin (50 mg/kg) from 3 to 10 days decreases from 81% to 92%. The dermatoprotective activity of moxonidine (1 μg/kg) was maximal on the 7th day and was 76%. In the isolated rat heart, the C7070 showed a protective effect in ischemia-reperfusion and on the model of doxorubicin cardiomyopathy. The STTi index: 8.3, 1.5; 7.9 and 7.8 U. in control, C7070, moxonidine and metformin. Keywords: С7070, metformin, moxonidine, ischemia, reperfusion, liver, skin flap, isolated heart, according to Langendorf.

Introduction Imidozoline receptors are localized in CNS Ischemia is a trigger and a pathogenesis (in the reticular formation cores, rostral stage simulteniously [1]. ventrolateral area of oblong brain) – type I. Diabetes mellitus and a metabolic Another type of imidazoline receptors – type II – syndrome lead to fat hepatic dysthrophy able to be finded on the peripheral tissues transferring into the necrosis of hepar [2]. (hepar, kidneys, pancreas) [4]. The last one was A drug therapy of diabetes mellitus and also founded based in mitochondrial metabolic syndrome have neither membranes. One more type of imidazoline hepatoprotective effects [3]. receptors was described as receptors in In this case, a study of additional sympatic nerve endings. The activation of these receptors decreases the noradrenaline pharmacological correction during the production [5]. I1−receptors was also detected biguanide therapy looks very interesting. on the platelet‘s membranes.

Rilmenidine and moxonidine are high independent factor of heart ischemic disease selected agonists of I1-receptors. Their affinity evolution [19]. to the I1-receptors more than to the α2- Pharmacological activity of metfrormin adrenoreceptors. Both of them has pronounced has not limited by positive influence to the hypotesive effect. It should be considered as a carbohydrate metabolism. sympatolitic activity of the I1-agonists [6]. Around at 2/3 patients with Imidozoline receptors were detected in hyperlypoproteinaemia IIB and IV types without smooth muscles of airways in the experiments diabetes metformin decreases triglyceride level on the dogs [7]. over 30%; decreasing of hypercholesterinaemia is Imidazoline receptors agonists have same less pronounced [9, 12]. pharmacokinetic parameters. It was noticed in Modern hypoglycemic drugs are able to number of studies, that their effect to the exert positive pleotropic effects to pancreas. imidazoline receptors continuing the whole day, But their activity is not enough to prevent despite their short elimination period. Most diabetes‘s complications. numbers of scintiests consider this property in In the view of foregoing, the vantage of connection with accumulation of these drugs in peripheral imidazoline receptors agonists, as a brain and other fat-containing tissues [8, 9, 10, medicines affecting early diabetic patient‘s 11]. survival, becomes obvious. Intolerance to glucose, overweight, The purpose of this study was detection of dyslipiaemia and arterial hypertension are vascular complication‘s protection of diabetic syndicated to the ―metabolic syndrome‖ [12]. patients. Probably, hyperinsulinaemia is able to be Materials and Methods related to the arterial hypertencion White Wistar rats were chosen for this development. Insuline increases sympatic study as standard animals for specific activity nervous system activity. This leads to the studies according to National pre-clinic studies increasing of cardiac output and periferial manual (2012). There were used 180 rats both vascular resistence [13, 14]. Agonists of central sexes. imidazoline receptors (I1) have a sympatolitic effect [15, 16]. Agonists of peripheral (I2) Doses of medicines imidazoline receptors decreases the tissue‘s Every used dosage was derived with help insuline resistence, decreasing the level of of the interspecific recount of doses according insulin [16]. to human minimal therapeutic doses. The studies of central imidazoline The minimal therapeutic dose for C7070 receptors agonists are more common, than the was detected as 100 mg for adult human. Using peripheral one [17]. the formula, the dose 10 mg/kg for rats was For example, placebo-controlled study by obtained. Haenni А., (1999) [16] in 72 patients with AH Metformin has 500 mg as a minimal and overweight moxonidine 8-week therapy therapeutic dose for human. According to leaded to the reliable insulin sensitivity. This formula, 50 mg/kg was selected as rat dose. effect was detected in glucose intolerance For moxonidine were obtained the rat dose group only. The group of patients without 20 µg/kg (minimal therapeutic dose for human glucose intolerance the modification of insulin – 0.2 mg) sensitivity was not detected. Data for minimal therapeutic doses were The basic role in pathogenesis of diabetes detected in instructions for use of the mellitus (II type) owned to the two independent medication. pathophysiologycal processes: the resistance to During the using of langenorff models the the insulin and decreasing of its secretion by β- equivalent doses of medicines were added into cells of pancreas [18]. The insulin resistance the prfusate. plays important role in metabolic syndrome Pharmacological methods. development. Also, the insulin resistance is For the study of ischemised skin flap accompanied by hyperinsulinaemia – survival 40 both sex rats were used. The

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Dovgan A.P. Ligand of peripheral imidazoline receptors based on amides of heterocyclic acids C7070: effect on ishemized tissues. Research Result: Pharmacology and Clinical 80 Pharmacology. 2017;3(4):78-88. animals were divided into the 4 groups (n=10): heartbeat (HB), the power of heart systole and contoll group; metformin (50 mg/kg) group; diastole (+dp/dt and – dp/dt). All the indexes moxonidine (20 µg/kg) group; C7070 were registered by Biopac Inc. system (10 mg/kg) group. hardware using the ACQ Knowledge software. Method of E.V. Kizhaev (1986) in P.A. Parameters were detecting during the whole Galenco-Yaroshevski was used in this model [20]. experiment [27, 28, 29, 30, 31]. The first one was the formation of skin flap The model of doxorubicine on the anterior abdominal wall. Then, studying cardiomyopathy was designed and patented medicines were administrated into the animals by group of scientists in Center of Pre-Clinic per os. Calculation of necrosis area at 3rd, 7th and Clinic Trials of Belgorod State National and 10th days with help of computer modeling Research University (including the author of was the third stage of this experiment. this article). The study of hepatotropic activity of 50 both sex animals were divided into the these medicines was carried out using the 5 groups (n=10): intact groups; control group D.A. Lopatin [21] method. (doxorubixine 20 mg/kg); doxorubixine 60 both sex rats were operated in this model. (20 mg/kg) + metformin (50 mg/kg) group; The animals were divided into the 6 groups doxorubixine (20 mg/kg) + moxonidine (n=10): intact groups; pseudooperated animals (20 µg/kg) group; doxorubixine (20 mg/kg) + (without vascular ligation); control group, C7070 (10 mg/kg) group. metformin (50 mg/kg) group; moxonidine (20 In 3 days before the modeling all µg/kg) group; C7070 (10 mg/kg) group. experimental animals started administration of All animals survived this experiment. studying medicines per os. After an hour ALT and AST were chosen as biochemical doxorubicine (20 mg/kg) was administrated markers of hepatic function. The structural into the rats intraperitoneal. changes evaluated with the help of hepatic histology. Results and Discussion The study of anti-ischemic cardio 1. The influence of peripheral protective activity was made using isolated imidazoline receptors agonists at skin flap heart by Langendorff [22, 23, 24, 25, 26]. The survival. animals were divided into the 4 groups (n=10): Intragastric administration of studying contoll group; metformin (50 mg/kg) group; drugs leads to the increasing of skin flap moxonidine (20 µg/kg) group; C7070 survival. The agonist of peripheral imidazoline (10 mg/kg) group. receptors (C7070) had the biggest anti- The next functional indexes for heart work ischemic activity (tab. 1). were chosen: left ventricular pressure (LVP), Table 1 THE influence of C7070 (10 mg/kg), moxonidine (20 µg/kg) and metformin (50 mg/kg) at the degree of skin flap necrosis at rats. (%) (M±m)

Skin flap necrosis’s degree, % Drugs and doses 3rd day 7th day 10th day Control 30.9±2.8 65.7±2.6 84.5±3.2 С7070 (10 mg/kg) 21.5±3.1* 41.4±3.2* 68.4±2.7* Moxonidine (20 µg/kg) 24.8±2.9* 50.5±3.0* 77.0±2.7* Metformin (50 mg/kg) 25.4±2.4* 59.2±2.2* 78.3±2.9* Note: *–p>0.05 in comparison with control group

Agonist of peripheral imidazoline dermatoprotective activity in comparing with receptors C7070 has more powerful moxonidine and metformin.

This difference is able to be controlled by So, laporotomy has no significant impact influence of C7070 at the vascular tonus. From to the ALT and AST levels. 15 minutes of the other side, we can expect this difference hepatic ischemia and following reperfusion due to mitochondrial protection of C7070. It reliably increase the ALN and AST levels. leads to maintaining of work capacity of skin Using studying drugs decrease hepatic cells. ferments‘ level. But even peripheral But, during the time, the difference imidazoline receptors agonist C7070 was not between studing drugs‘ effects increasing from able to return ferment level back to the control 5% at 3rd day to 10% at 10th day. group numbers. 2. The influence of peripheral Despite this, C7070 had the highest anti- imidazoline receptors’ agonist at hepar ischemic activity among all studied medicines damage due to ischemia-reperfusion. in this experiment. All animals survived the experiment. Hepatic ferments‘ levels are not enough to The levels of AST and ALT were evaluate anti-ischemic activity of these drugs. measured in blood of experimental animals That‘s why the rat liver was excise 3 days after after their euthanasia (3 days after modeling). modeling to describe its histological image. The next numbers were detected in Ischemic damage area and ischemic biochemical blood analysis (tab. 2). necrosis area was chosen as histology markers of structural damage (tab. 3). Table 2 The influence of C7070 (10 mg/kg), moxonidine (20 µg/kg) and metformin (50 mg/kg) at the mean values of ALT and AST in blood of experimental animals (U/ml) (M±m)

Animal group ALT, U/ml AST, U/ml Intact 102.8±8.8 284.1±19.3 Pseudo-operated 110.2±21.9* 289.8±16.2* Control 526.9±17.9** 1045.1±80.0** С7070 (10 mg/kg) 143.2±16.91 395.8±33.31 Moxonidine (20 µg/kg) 289.8±15.21 687.7±28.31 Metformine (50 mg/kg) 332.5±22.01 825.4±22.41 Note: *–p>0,05 in comparison with intact group; **– p<0,05 in comparison with psedo-operared group; 1 – p<0,05 05 in comparison with control group. Table 3 The influence of C7070 (10 mg/kg), moxonidine (20 µg/kg) and metformine (50 mg/kg) to the hepatic damage area, mm2 (M±m)

Animal group Iscemic damage area, mm2 Iscemic necrosis area, mm2 Intact n/a n/a Pseudo-operated n/a n/a Control 0.387±0.014 0.207±0.021 С7070 (10 mg/kg) 0.058±0.029* 0.046±0.013* Moxonidine (20 µg/kg) 0.090±0.025* 0.075±0.015* Metformin (50 mg/kg) 0.238±0.052* 0.125±0.020* Note: *–p>0.05 in comparison with control group

Histological examination did not detected Thus, peripheral imidaazolinee receptors‘ significant sings of ischemic damage or agonist C7070 has the highest hepatoprotective necrosis in intact or pseudo-operated animal properties in frames of this study. group.

3. The influence of peripheral level. That‘s why this medicine is able to work imidazoline receptors’ agonist C7070 to the in isolated heart. Transmembrane ion current functional ischemic isolated heart damage. control and ATP‘ase channels normalization (by Langendorff) allows C7070 significant decrease the heart It was detected, that decreasing of ischemic damage. perfusion in 10 times (ischemic hypoperfusion) In this connection, the question about all selected parameters (HB, LVP, +dP/dtmax, - th decreasing of cardiotoxic drugs effects by dP/dtmax) were significant decreased by 5 C7070 is very interesting. minute. They didn‘t back to the normal level by The most socially significant of them are 20th minute. antineoblastome antracycline drugs [32, 33]. During the reperfusion these parameters Doxorubicine cardiomyopathy model were less than start level by 5th minute of th has been used in this experiment. experiment. The same image we had by 20 minute of reperfusion, when most of This model uses the comparison of control parameters had less than half of start level (tab. animal group (doxorubicine 20 mg/kg 2 days 4). Besides, the recovery of perfusion volume before modeling) with intact group. And (reperfusion) leaded to the reperfusion comparison of experiment groups with control. arrhythmias (30% fibrillations in cases). The coefficient of AUC in time of 15‖ stimulating count was derived and patented Thus, moxonidine and metformin haven‘t specially for this model. significant influence to the heart muscle The stimulation of dilated by doxorubicine working. The same time, peripheral heart with help of electric stimulator leads to imidazoline receptors‘ agonist C7070 allowed diastolic dysfunction. It express in increasing of cardiomyocytes to contract with the almost minimal left ventrical pressure in time of same power and frequency as heart without electric stimulation. So, it leads to the ischemia. increasing AUC level. The coefficient STTi Metformin and moxonidine have a central shows the degree of diastolic dysfunction. Its and common activity to the whole organism. meaning inversely to the cardioprotective Peripheral imidazoline receptors‘ agonist activity of the drug. C7070 has the action realized on the cells‘ The results of experiment are able in fig. 1.

9 8 7 6 5 4 3 2 1 0 STTi (U) Control С7070 (10 mg/kg) Moxonidine (20 µg/kg) Metformin (50 mg/kg)

Fig. 1. The coefficient STTi for C7070 (10 mg/kg), moxonidine (20 µg/kg) and metformine (50 mg/kg) in modeling of doxorubicine (20 mg/kg) cardiomyopathy

Table 4 The influence of C7070 (10 mg/kg), moxonidine (20 µg/kg) and metformine (50 mg/kg) to the heart function measures (M±m, n=10) Metformin, 50 Animal group Control Moxonidine, 20 µg/kg С7070, 10 mg/kg mg/kg LVP 129.0±2.4 127.4±3.7 130.1±1.1 125.1±1.1 +dp/dt 2728.0±38.4 2694.3±46.1 2697.7±39.4 2799.3±20.0 Start level -dp/dt 1346.4±41.2 1342.1±25.0 1331.1±36.1 1235±31.2 HB 168.1±22.4 179.2±11.8 165.4±18.9 160.1±15.3 LVP 50.75±7.6 48.47±4.1 50.81±5.2 65.84±3.4* +dp/dt 1114.0±12.4 1181.2±21.2 1102.5±15.8 1276.4±11.7* 5‘ -dp/dt 613.1±11.2 625±1.2 617±7.6 656±4.1* HB 76.6±2.4 78.1±4.3 74.1±3.7 85.6±1.1* Hypo perfusion time LVP 28.2±1.4 23.48±4.2 26.94±3.1 39.63±2.3* +dp/dt 448.8±12.4 441.6±22.1 442.4±15.8 629.8±14.4* 20‘ -dp/dt 306.7±9.8 307.8±6.7 309.5±7.1 378.4±11.2* HB 213.1±7.2 211.8±6.4 215.6±8.2 280.4±4.5* LVP 101.9±2.5 115.9±4.9 128.7±7.1* 126.8±5.8* +dp/dt 2079.4±84.5 1991.5±86.1 1987±74.9 2564±81.2* 5‘ -dp/dt 1137.1±25.8 1195.0±29.7 989±15.3* 1215±20.8* HB 191.7±9.8 195.8±8.3 171.5±7.4* 161.9±9.1* Reperfusion time LVP 101.3±8.6 115.4±7.5 125.8±5.1* 129.5±4.3* +dp/dt 2121.1±30.8 2095.4±35.6 2184.6±29.3 2859±38.5* 20‘ -dp/dt 1137.0±25.4 1184.4±21.3 1121.1±36.4 1279±30.1* HB 181.8±10.8 185.4±9.6 169.5±11.2 160.2±3.6* Note: LVP – left ventrical pressuare, ―+dp/dt‖ – heart systolic power; ―-dp/dt‖ – heart diastolic power; HB – heartbeat. *–p>0,05 in comparison with control group

Conclusion effect was blocked by efaroxan and decreased Thus, peripheral imidazoline receptors‘ by α-adrenoblocker himbine [11]. agonist C7070 had the highest anti-ischemic Central I1-receptors of hypothalamic zone properties among all medicines included in this include in glycemia level control. It was study. showed in experiment with selective I1 agonist All differences are able to be explaned by – agmantin – reduce of glucose blood level. their mechanisms of action. The same action has a moxonidine too. Metformin‘s mechanism of action based on Besides, imidazoline receptors are able to decreasing of gluconeogenesis. Also, metformine plays one of the main roles in locate in pancreas and control the insulin insulin peripheral effects increasing, anaerobe secretion [35]. glucose metabolic way increasing, lipless The using of moxonidine at Zucker rats decreasing. leaded to hypothalamic neuropeptid Y level In number in vivo and in vitro studies the decreasing. It is able to be one of the weight- metformin influence to the cell AMP-kinase reducing mechanisms of this drug [15]. was detected. This enzyme plays a role in Probably, some part of these actions realize glucose transport through the membranes with through the α2-adrenoreceptors. The peripheral help of GLUT4 enzyme. Besides, metformin actions of these drugs are able to lead these has showed the ability to cell membrane changes too. rigidity decreasing. It is one of the frequent Peripheral imidazoline receptors‘ agonist condition able to lead to the diabetes‘s C7070 realize its action through the complications [20]. mitochondrial protection. The influence to the Metformin actives AMP-activated ATP-channels of external and internal proteinkinase (AMPK) – hepatic enzyme of mitochondria membranes. Slowing and insulin signalization. AMPK also playing a role blocking of avalanche current of iron ions leads in common energy balance and glucose and to the oxidative stress decreasing [. lipid metabolism. AMPK activation is Imidazoline receptors activation leads to necessary stage for metformin inhibition effect the arachnid acid synthesis increasing and to hepatic gluconeogenesis [14]. Na+/H+ ion change inhibition [17]. Resuming the foregoing, anti-ischemic Scientists from Kharkov paid their metformin action based on energy reserves attention to the peripheral imidazoline cumulating and decreasing of current nutrial receptors‘ agonists ability to glycemia control. reserves spending. According to their studies, anti-diabetic effects Moxonidine is a central imidazoline of these drugs not less than metformin. But, receptors‘ agonist. It takes a part in C7070 has more favorable toxic profile in redistribution of hepatic bloodstream through comparison with biguanides [39]. Besides, the collateral vessels from a. gastrica sinistra. imidazoline receptors‘ agonists don‘t lead to They were free from flow reducing. Also, the hypoproteinaemia and hyperlactacidaemia moxonidine‘s anti-ischemic activity is able to progression [40]. base on vessels‘ opening in reperfusion Modern anti-diabetic drugs can lead some moment [35]. pleiotropic effects to the pancreas. But their There were enough number of pre-clinic activity is not enough to prevent the and clinic trials of imidazoline receptors complications. agonists. So, imidazoline receptors‘ agonists For example, Mukaddam-Daher in his trial advantage as drugs with vascular prevent detected the increasing of diuresis, Na and K properties becomes obvious. excretion at rats in moxonidine group. The

Resuming the foregoing, it is able to scheme (fig. 2). combine all mechanisms of action into one

С7070 Moxonidine

The influence to The influence to the peripheral the central imidazoline imidazoline receptors I2 receptors I1

Ion-change Stimulation of: processes lipolisis, glycolisis and inhibition Bloodflow other katobilc (including processes, going Fe2+/Fe3+ optimization in basicly without oxygen exchange) microcirculation participation vesseles

Fig. 2. The differences between C7070 and moxonidine mechanisms of action

Findings 0.090±0.025/0.075±0.015 mm2 for ischemic Peripheral imidazoline receptors‘ agonist damage/necrosis areas respectively. C7070 (10 mg/kg) has pronounced dermatoprotective activity and prevents the Acknowledgement skin flap necrosis progression by 3rd and 7th day A study produced in the framework of the to 40%. collaborative work on the state contract 10 Dec Peripheral imidazoline receptors‘ agonist 2014 №14411.2049999.19.109 "Preclinical C7070 (10 mg/kg) shows 4.5 times less studies of anti-diabetic drugs – agonist increasing of ALT and AST (332.56±22.05/ imidazoline receptors C7070" by 825.49±22.46 U/ml ALT/AST in C7070 group ―Pharma2020‖. versus 526.90±17.97/1045.16±80.02 U/ml in control group) and decreases in 2.5 times Conflicts of Interest ischemic damage and ischemic necrosis area The authors have no conflict of interest to (0.058±0.029 и 0.046±0.013 mm2 respectively) declare in modeling of 15-minute hepatic ischemia. Dermatoprotective activity of metformin References (50 mg/kg) decreases from 81% at 3rd day to 1. Abrashova TV, Gushchin YA, 92% at 10th day. Kovaleva MA, Rybakova AV, Selezneva AI, Dermatoprotective activity of moxonidine Sokolova AP, Khodko SV. Directory. (20 µg/kg) was maximal by 7th day – 76%. Physiological, biochemical and biometric Moxonidine and metformine had a indicators of the norm of experimental animals. hepatoprotective activities too. SPb.: Publishing house "LEMA", Metformin decreases the level of [SPRAVOCHNIK. Fiziologicheskie, ALT/AST to 332.5±22.0/825.4±22.4 U/ml and biohimicheskie i biometricheskie pokazateli ischemic damage/necrosis area to normy ehksperimental'nyh zhivotnyh.] 2013. 0.238±0.052/0.125±0.020 mm2. 116 p. (in Russian) [Fulltext] Moxonidin parameters were: 2. Zhernakova NI, Alechin SA, Lomykov 289.8±15.2/687.7±28.3 U/ml for AST/ALT and DI, Dolzhikov AA. Preconditioning for ischemic and reperfusion injury of the liver.

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December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Efremova M.P., Ivasheva A.V., Korokin M.V. Study of pharmacological activity of Nigella damascena fixed oil in experiment. Research Result: Pharmacology and Clinical 89 Pharmacology. 2017;3(4):89-99.

Rus.

UDC 528.675.1:547.915:615.322/.076.9:616.12-008.46 DOI: 10.18413/2313-8971-2017-3-4-89-99

Marina P. Efremova1 Aelita V. Ivasheva2 STUDY OF PHARMACOLOGICAL ACTIVITY Mikhail V. Korokin3 OF NIGELLA DAMASCENA FIXED OIL IN EXPERIMENT

1 Рyatigorsk Medical Pharmaceutical Institute of Volgograd Medical State University of the Ministry of Health Care of Russia. 11, Kalinin Av., Pyatigorsk, 357532, Russia 2Interregional center of vocational postgraduate training and professional development of experts "Development". 178, Mira St., Pyatigorsk, 357532, Russia 3Belgorod National Research University. 85, Pobedy St., Belgorod, 308015, Russia Corresponding author, 1e-mail: [email protected]

Abstract Introduction: Literature data, rich chemical macro-microelement composition set the stage for experimental pharmacological study of adaptogene and lipid protective activity of Nigella damascena essential oil. Objectives: study of pharmacological activity of Nigella damascena essential oil while simulating pathology on laboratory animals. Methods: irritant action – HET-CAM test and DRAIZE-test, acute toxicity, open field test, simulated hypoxia method, swimming with the load, No-way-out and Rotating-rod tests, acute lipid pathology under Tween 80 (tween lipid pathology), vitamin D2 hyperlipidemia, simulated chronic heart failure (CHF). Results and Discussion: Nigella damascena essential oil have low irritant effect along with almost non-toxicity. Nigella oil increase life duration with the presence of hypoxia and endurance while swimming-with-the-load test. Course administration of Nigella oil normalize hormone-mediatore metabolism on the background of acute stress. Course administration of Nigella damascena essential oil normalize lipids on the background of CHF, acute tween and subchronic vitamin D2 hyperlipidemias. Conclusion: stress protective, actoprotective and adaptogene properties of Nigella damascena essential oil was pharmacologically proved. It was substantiated experimentally that Nigella damascena essential oil has an impact on lipid metabolism on the background of chronic heart failure (CHF), acute tween, subchronic vitamin D2 lipid pathologies and blood serum lipoprotein lipase (LPL) activity. Keywords: Nigella damascena essential oil, actoprotector, adaptogene, hyperlipidemia.

Introduction (thymoquinone); lipase enzyme; macro- According to variety of literature data (potassium, calcium, magnesium, ferrum) and Nigella damascena seed essential oil contain: microelements (manganese, magnesium, zinc, essential oil (31-42%); essential oil with copper, molybdenum, strontium, selenium, nigellon and thymoquinone as main chrome, lead, boron, iodine) [1, 2, 3, 4, 5]. components (0.37 – 0.5%); sum of alkaloids Literature data, rich chemical, macro- and (damascene and damascenine 0.1-0.3% microelement composition set a base for respectively); flavonoids; sesquiterpene experimental pharmacological research of compounds; tocopherols; steroids; triterpenoid Nigella damascena essential oil adaptogene and saponins; coumarins; quinones lipid protective activity [6, 7].

Objectives: study of pharmacological essential oil was administered intragastrically at activity of Nigella damascena essential oil the doses of 1mg/kg and 10mg/kg one time 40 while simulating pathology on laboratory min before experiment or as a course during 2 animals. weeks. Control animals received distilled water [11, 12]. Methods For examination of actoprotective activity The research was conducted in accordance of Nigella damascena essential oil were used: with the policy of Declaration of Helsinki. forced swimming in 33-35oC water, with load – Experiments were carried out with 246 white 7.5% of rat bodyweight to refusal and rotating male Wistar rats 180-200 g weight, 12 white rod tests with mice (animals were placed on mice 30-33 g weight, 4 guinea pigs 600-700 g horizontal rod, rotating at a rate of 10 rpm) [13, weight and 12 Leggron chicken embryo 14]. 9-10 days. Stress has been evoked by fixation rats on Experiment started with oil irritant effect its back for 6 hours resulting in development of on chicken embryo – HET-CAM test. Fixed oil pain-emotional stress type of No-way-out was applied on chorioallantoic membrane at a situation. Stress resistance was determined by dose of 0.3 ml and monitored for 120 sec. following values: thymus and adrenal glands DRAIZE-test was conducted on guinea pigs weight, gastric mucosal ulceration and during 2 min. Sunflower oil GOST R 52465- ulceration rate. Biochemical values were 2005 was used as a control (solvent). studied by spectrofluorometric Mice were administered once by Nigella (spectrofluorometer ―Hitachi‖ MPF-4) and damascena essential oil at a dose of 8.5 mg/kg colorimetric (photoelectric colorimeter КФК-2 as the rats at a dose of 30 mg/kg intragastrically – УХЛ4,2) methods. Rat‘s blood, hearts, to determine acute toxicity. Animals were cerebral cortex were analyzed and monitored for 14 days. homogenized. Concentration of adrenalin Open field test was conducted 24 hours (AD), noradrenaline (NAD), dopamine (DA), since mice were administrated with 0.17 ml of 11-oxycorticosteroid, serotonin (S) and Nigella damascena essential oil to examine histamine (H) [15, 16, 17, 18, 19]. motion behavior features and emotional state. Pharmacological effect of Nigella The experiment lasted for 3 min. Amount of damascena essential oil was studied on the center and squares crossing, postures, grooming background of simulated hyperlipidemia by (washing), defecation, diuresis served as setting acute tween lipid pathology. Were indexes of behavior [8]. Experimental dose was studied two types of Nigella damascena defined during active rat swimming. Mature essential oil administration schedule at the male rats were divided into 7 groups of 6 background of ―tween‖ model of animals which were selected on the base of hyperlipidemia: one-time administration and a bodyweight and swimming ability. Oil had course administration. In a course oil was been administered intragastrically once a day administered oral using tube for 14 days. before the experiment for 14 days at the doses Untreated animals and animals who got of 1, 5, 10, 15, 20, 25 mg/kg in accordance with physiological solution at a dose of 1mg/100g groups. Control animals received distilled were used as a control. After 14 days Tween-80 water [9, 10]. was administered intraperitoneally and 12 The aim of the second stage was to hours after that decapitation was performed determine adaptogene activity of Nigella [20]. Subchronic vitamin model was simulated damascena essential oil. The aim was gained by by course administration of vitamin D2 along a series of experiments. Antihypoxic effect was with everyday administration of cholesterol modeling by normobaric normocapnic hypoxia. alimentary and mercazolilum for metabolism Rats were placed into hermetic chamber 1.5 l inhibition. Vitamin model of hyperlipidemia vol. Carbon dioxide was eliminated by soda was induced using Vsilenko‘s method [21]. lime. Animals life duration indicated the Experimental animals‘ blood serum and liver hypoxia impact resistance. Nigella damascena were examined for biochemical values to make December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Efremova M.P., Ivasheva A.V., Korokin M.V. Study of pharmacological activity of Nigella damascena fixed oil in experiment. Research Result: Pharmacology and Clinical 91 Pharmacology. 2017;3(4):89-99. a judgment about lipid metabolism. Those functioning properly. Next was local irritating values are: rat‘s blood serum cholesterol, blood action. DRAIZE-test showed hyperemia and serum triglyceride, blood serum beta- and pre- chemosis score <2. Results obtained allows for beta-lipoproteins, blood serum lipoprotein the conclusion that Nigella damascena essential lipase, liver cholesterol, liver triglyceride oil has low irritant effect. [22, 23]. Animals‘ survival ability after injection of Right ventricular chronic heart failure was Nigella damascena essential oil for acute simulated by intermittent admission of silicone toxicity determination was 100% in 2 weeks oil into each pleural cavity at a dose of monitoring. According with K. Sidorov‘s 1.5ml/100g rat weight according with the classification essential oil refers to 5 class as method of N. Pyatnitskiy and Y. Blinkov. Oil non-toxic compound. administration was performed under hexenalum Thereby while valuing using safety low anesthesia. After 30 days oil administrations irritating effect was determined along with were repeated at a dose of mg/100g of rat practically non-toxic features. weight. Course of Nigella damascena essential Open field test administration showed oil has been administered for 14 days 24 hours 42.9% mood increase after Nigella damascena after second administration [24, 25]. administration resulting in defecation, diuresis and posturing. Physical activity had a tendency Results and Discussion to increase up to 9.5% resulting in center, First step was to determine irritant effect. squares crossing and grooming amount Chicken embryo coat was intact, clear, thin, (Figure 1). with blood vessel and capillaries system

% 160 142.9 * 140 120 110 100 90.5 100 100 100 100100 100 100 18.2 80 59.3 * 2.5 1.0 60 42.3 3.3 40 3.0 20 * 0.3 0.3 0.7 38.3 10.8 20 0.5 0 square center posture grooming diuresis defecation

Untreated NDEO 8.5 mg/kg

Fig. 1. The influence of Nigella damascena essential oil on nervous system. Note: NDEO – Nigella damascena essential oil, * – p>0.05 reliable relatively to untreated animals

Experimental dose of Nigella damascena increased in comparison with swimming time essential oil was chosen by the results of the of control group. What is more, increase had research on dose addiction depended on linear character from 1 to 15 mg/kg. Following dynamic performance rising. As the result was dose escalation had no valued dynamic determined that in a 10mg/kg dose of Nigella performance rising (Figure 2) [8]. damascena essential oil swimming time

min. 70

0 Control NDEO 1 NDEO 5 NDEO 10 NDEO 15 NDEO 20 NDEO 25 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Swimming time day 1, min Swimming time day 14, min

Fig. 2. Ergotropic effect escalation after Nigella damascena essential oil administration at the doses from 1 to 25 mg/kg. Note: NDEO – Nigella damascena essential oil, min – minutes

Thereby according to results of 2 weeks‘ Experimental data gathered while swimming oil administration research there were chosen shows that one-time administration of Nigella experimental groups with 1 mg/kg and 10 damascena essential oil at a dose of 10 mg/kg 2 mg/kg one-time and course administration. times increases rat‘s performance in the For the second stage of the research the comparison to control group and 3 times aim was to determine adaptogene Nigella increases performance after course damascena essential oil activity. The administration. Rotating rod test showed adaptogene medications tend to combine at increase in mice‘ muscular load tolerance up to least three types of activity – actoprotective, 81.1% after one-time administration and up to antihypoxic and stress protective [26]. 209.1% after course administration (Figure 3). Antihypoxic effect showed in rat‘s life Stress tolerance experiment showed that duration increase up to 63.5 ± 3 min with both Nigella oil at a dose of 10 mg/kg prevents types of administration – one-time and course thymus involution, decreasing its presence up in comparison to control group. However to 35% after one-time administration and reliable data became available for Nigella normalizing this value after course damascena essential oil at a dose of 10 mg/kg, administration. Adrenal gland examination which increased rat‘s life duration on the showed prevention of gland hypertrophy background of hypoxia up to 35% in acute accumulation after one-time and course Nigella experiment and up to 26% after course oil administration up to 28.78% and 38.85% administration (Fig. 3). respectively. Gastric mucosa erosion reduced Actoprotective activity of Nigella 2.6 times after one-time administration and 7.8 damascena essential oil resulted in forced times after course administration in comparison swimming with the load and Rotating rod test. with control (Figure 4).

100 min., 86.8* sec. 82.2* 80 70.5*# 75.1*# 63.5 60 53.4 44.3 44.5# 39.6 40 38.2# 40 30.8# 30# 16.8 20 12.1 0 Control NDEO 10mg/kg NDEO 10mg/kg NDEO 1mg/kg NDEO 1mg/kg one-time course one-time course

Hypoxia/min. Swimming/min. Rotating rod/sec

Fig. 3. The influence of Nigella damascena essential oil 10 mg/kg on muscular load and hypoxia. Note: NDEO – Nigella damascena essential oil; min. – minutes; sec. – seconds; * – p>0.05 reliable relatively to control animals; # – p>0.05 reliable relatively to animals who got NDEO 10mg/kg one-time

180 % 167.47 157 160 144.6 148.88# 136.1# 140 119.28# 120 100 100.5# 100 80 72.58 57.61# 59.17 63.03# 59# 60 57.93# 50.61# 43.22# 39.02 45.08 40 39.76 20 12.41# 12.8# 0 Control NDEO 10mg/kg NDEO 10mg/kg NDEO 1mg/kg NDEO 1mg/kg Buckthorn oil 2.0 Buckthorn oil 2.0 one-time course one-time course mg/kg 1-time mg/kg cour. Thymus Adrenal gland GM damage level – Untreated animals

Fig. 4. Stress protective activity of Nigella damascena essential oil at a dose of 10 mg/kg and 1 mg/kg. Note: NDEO – Nigella damascena essential oil; cour – course administration; GM – gastric mucosa; # – p>0.05 reliable relatively to control animals

Stress resistance reflected in changings of Blood plasma showed 2.23 times reducing biochemical values: adrenalin (AD), concentration of DA mediator at the noradrenaline (NAD), dopamine (DA), serotonin background of increasing AD 1.74 times and (S), histamine (H) and protective effect index NAD 2.24 times. There was an increasing of (PEI) in blood, heart, cerebral cortex. concentration of NAD and DA in heart muscle December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Efremova M.P., Ivasheva A.V., Korokin M.V. Study of pharmacological activity of Nigella damascena fixed oil in experiment. Research Result: Pharmacology and Clinical 94 Pharmacology. 2017;3(4):89-99. of stressed animals 2.03 and 1.62 times For objective interpretation of gathered respectively. Adrenalin level in heart truly 1.77 data were used protective effect index (PEI), times reduced in comparison to untreated rats. which shows the ability of preparation to In cerebral cortex was seen truly increasing of increase organism resistance against various adrenalin concentration – 1.87 times, interferences and negative effectiveness noradrenaline – 6.1 times and dopamine – 1.56 indicator (NEI). Preparation can be counted as times compared with the normal range. adaptogene if its PEI>0.2 (Figure 5).

Blood Heart Cerebral cortex NAD DA 0.63 2,5 1.02 AD S 2 * H 1.02 * 1,5 0.4 * 0.4 1 * * * * 0,5 * 0 * UOCUOCUOCUOCUOC

PEI – protective effect index PEI>0.2; NEI = 0

Fig. 5. The influence of Nigella damascena essential oil on hormone-mediator metabolism under simulated acute stress. Note: U – untreated; O – one-time administration; C – course administration; AD – adrenaline; NAD – noradrenaline; DA – dopamine; H – histamine; S – serotonin; * – p>0.5 reliable relatively untreated animals

Stress protective research would be in comparison to untreated group. Course incomplete without research of adrenal gland administration of Nigella oil at a dose of 10 activity marker. During the experiment 1.9 mg/kg led to complete normalizing of hormone times increase of 11-oxycorticosteroids (11- level in blood. OXY) concentration in blood was determined Table 1 The influence of Nigella damascena essential oil 10 mg/kg on 11-OXY metabolism in rat’s blood under acute stress

Value Untreated Control (stress) NDEO 10 ml/kg NDEO 10 ml/kg one-time course. 11-OXY, mkg/ml 0.575 0.041 1.108±0.062* 0.851±0.114* 0.592±0.056# Note: NDFO – Nigella damascena essential oil, 11-OXY – 11-oxycorticosteroid; * – p>0.05 – reliable relatively untreated animals; # – p>0.05 – reliable relatively control animals.

Thereby Nigella oil 10 mg/kg one-time and administration at a dose of 10 mg/kg prevented course administration promoted normalization lipid metabolism failure and reflected on lipid of catecholamine in all examined tissues. What profile. In experimental rat group normalization is more, corrective oil effect became more of lipid profile was related to reliable reducing evident after course administration. of low-density lipoprotein concentration by Studying the influence of Nigella 68.38% herewith high-density lipoprotein damascena essential oil on rat‘s lipid profile concentration was increased by 22.3% under simulated chronic heart failure (CHF), relatively control. At the background of Nigella acute tween and subchronic vitamin D2 lipid damascena essential oil administration also pathologies was the next stage of the research. total cholesterol reducing by 13.2% was At the background of simulated CHF observed (Figure 6). pathology Nigella damascena essential oil

2,5 2.285 2.021*# 1.98*# 2 1.783 1.762

1,5 0.965 0.829 1.001*# 1.014*# 0.989 1 0.583 0.661 0.582 0.569 0.453*# 0.572*# 0,5 0.341* 0.394*# 0.234 0.146 0 Untreated Control NDEO 10mgг/kg NDEO 10mg/kg Lipanor® 1-time Cour. Ch HDLP LDLP VLDLP

Fig. 6. The influence of Nigella damascena essential oil on rat's lipid metabolism under chronic heart failure. Note: NDEO – Nigella damascena essential oil; Ch – cholesterol; HDLP – high-density lipoprotein; LDLP – low-density lipoprotein; VLDLP – very low-density lipoprotein; 1-time – one-time administration; Cour. – course administration; * – p>0.05 reliable relatively untreated animals; # – p>0.05 – reliable relatively control animals

The cholesterol level reducing was reduced by 10.43% and 24.63% respectively after detected in blood serum by 39.6% and by one-time administration and by 22.44% and 22% 50.6% after one-time and course respectively after course administration. administrations respectively as in liver – by Triglyceride level reduced in blood serum by 94.1% and 96.2% after one-time and course 25.73% and by 36.73% in liver after course administrations respectively under acute tween Nigella oil administration (Figure 8). hyperlipidemia. Triglyceride content tended to To improve gathered data lipoprotein reduce in blood serum by 36% and 78.5% after lipase (LPL) activity was examined by separate one-time and course administration set of experiments. LPL activity increased after respectively and in liver – by 56.9% (one-time) Nigella damascena fixed oil administration to and 71.5% (course) rats with acute tween and D2 subchronical (Figure 7). intoxication up to 1.321 0.20 lipase unit which Same lipid metabolism values changes in relative units would be 36% relatively happened under vitamin D2 hyperlipidemia. control (Figure 9). Cholesterol in rat‘s blood serum and liver

8 7.16 7 6 5.49* 5 4.21 4.05 4.12 4.12 4 3.28 2.95# 3.16* 3 2.28 2.28 1.82 2.12*# 2 1.34 1.35*# 1.04 0.97* 1.26# 0.95 1.18 1 0 Untreated Control NDEO 10mg/kg NDEO 10 mg/kg Lipanor® 13 1-time, path cour. path. mg/kg

Cholesterol BS Triglyceride BS Cholesterol L Triglyceride L

Fig. 7. Lipid metabolism values changing in blood serum and liver after Nigella damascena essential oil administration under acute tween lipid pathology. Note: NDEO – Nigella damascena essential oil; 1-time – one time administration; cour. – course administration; path. – pathology; BS – blood serum; L – liver; * – p>0.05 reliable relatively untreated animals; # – p>0.05 reliable relatively control animals.

8 7.57 6.78# 7 6.05 6 5.72*# 4.56*# 4.72* 5 4.12 4.23# 4.11 4 3.57 3.26 3.24 3.25 3.42 3.06* 2.85 3 2.18 1.78 2.05 2 1.04 1 0 Untreated Control NDEO 1time NDEO cour. path. Lipanor® 13 path. ml/kg Cholestrol BS Triglycerides BS Cholesterol L Triglycerides L

Fig. 8. Lipid metabolism values changing in blood serum (BS) and liver (L) after Nigella damascena essential oil administration at a dose of 10 mg/kg under subchronic vitamin D2 lipid pathology. Note: NDEO – Nigella damascena essential oil; 1time – one-time administration; cour. – course administration; path. – pathology; BS – blood serum; L – liver; * – p>0.001 difference reliability relatively untreated animals; # – p>0.001 difference reliability relatively control animals

2.833* 3 Lipoprotein lipase 2.45* 2,5 1,99 2 1.784# +135 1,5 1.312# 1.213 +102 1.09 1 0.829 +47% +10.1 +64% 0,5 100% +8.9% -32% 0 Untreated NDEO NDEO Control NDEO NDEO Lipanor® Lipanor® 10mg/kg 10mg/kg 10mg/kg 10mg/kg 13mg/kg 13mg/kg 1time cour 1time, course, 1time cour. path. path.

Fig. 9. The influence of Nigella damascena essential oil at a dose of 10 mg/kg on blood serum lipoprotein lipase activity. Note: NDEO – Nigella damascena essential oil; 1time – one-time administration; cour. – course administration; path. – pathology; * – p>0.05 reliable relatively untreated animals; # – p>0.05 reliable relatively control animals

Conclusion subchronical vitamin D2 lipid pathology The experimental research resulted in Nigella damascena essential oil‘ therapeutic conclusion that Nigella damascena essential oil effect at a dose of 10 mg/kg stays equal to has high activity while being safe for oral comparator drugs. administration by mammal. The active impact on blood serum During the research adaptogene activity of lipoprotein lipase was determined, after one- Nigella damascena essential oil was determined time administration and course administration by size of antihypoxic, actoprotective and stress alike. protective effects whose intensity increases after course oil administration. Conflicts of Interest High stress protective effect of Nigella The authors have no conflict of interest to damascena essential oil was achieved at an declare. experimental dose of 10 mg/kg. Course administration of Nigella References damascena essential oil normalized 1. Al-Kayssi AW, Shihab RM, Mustafa catecholamine level in blood, heart and brain SH. Impact of soil water stress on Nigellone oil under simulated stress. Changes of hormone- content of black cumin seeds grown in mediatore balance have positive compensatory calcareous-gypsifereous soils. Agricultural character. Nigella damascena essential oil Water Management. 2011;(100):46-57. practically non-toxic and has no irritant effect. [Fulltext] It was determined experimentally that Nigella 2. Kokoska J, Havlik I, Valterova et al damascena essential oil at a dose of 10 mg/kg Comparison of chemical composition and has positive impact on lipid profile by antibacterial activity of Nigella sativa seed normalizing it under simulated CHF. Also, it essential oils obtained by different extraction was determined that after lipid metabolism methods. Journal of Food Protection. values were changed under acute tween and 2008;71(12):2475-2480. [Fulltext]

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Rus. UDC 615.015.2 DOI: 10.18413/2313-8971-2017-3-4-100-112

Alexandr G. Miroshnichenko1 Vyacheslav Yu. Perfil'yev INTERACTION BETWEEN SOME ANTIBIOTICS Ilya V. Lysenko AND ANTIOXIDANTS Nina I. Zhernakova

Altai State University, 61, Prospect Lenina St., Barnaul, 656049, Russia Corresponding author,1e-mail: [email protected] Abstract Introduction: The bactericidal action of many antibacterial agents has a common mechanism associated with the generation of hydroxyl radicals and the activation of oxidative stress. This is confirmed by the ratio between the degree of bactericidal effect and the level of hydroxyl radicals. The study of the effect of antioxidants on the antibacterial activity of chemotherapeutic agents can open new prospects for improving the treatment of infectious diseases. Objectives: Study of the effect of antioxidants on the activity of chemotherapeutic agents in relation to opportunistic bacteria Methods: The study examined the interaction of antibiotics (gentamicin, ciprofloxacin, ceftazidime) and antioxidants (ascorbic acid, N-acetylcysteine, methylethylpyridinol) in vitro and in vivo. We conducted a dynamic study of the effect of antioxidants at concentrations of 0.5, 1, 2 and 4 mM on the activity of antibacterial agents in vitro for three strains of Klebsiella pneumoniae and three strains of Escherichia coli. The effect of antioxidants on the effectiveness of antibacterial therapy was studied in Wistar rats with experimental bacterial peritonitis. Results and Discussion: In vitro studies have shown that all antioxidants reduce the activity of gentamicin. Methyl ethylpyridinol increases the effect of ciprofloxacin, ceftazidime. Ascorbic acid enhances the action of ceftazidime. Ascorbic acid, methylethylpyridinol and N-acetylcysteine (80 mg / kg) reduce the antibacterial activity of gentamicin (30 mg / kg) and ciprofloxacin (50 mg / kg) and do not change the intensity of peroxidation processes in combination with these antibacterial agents under experimental infection caused by K. pneumoniae. These antioxidants reduce the prooxidant effect of ciprofloxacin (50 mg / kg), without affecting its antibacterial activity in escherichiosis peritonitis. Ascorbic acid, methylethylpyridinol and N- acetylcysteine (80 mg / kg) do not reduce the nephrotoxic effect of gentamicin (30 mg / kg). Conclusion: Antioxidants have anmultidirectional effect on the efficacy of antibacterial agents in vitro and under experimental bacterial peritonitis. Combination of antibiotics with antibacterial agents should be accompanied by preliminary in vitro studies. The rational combination of antibacterial agents and antioxidants increases the effectiveness of anti-infective chemotherapy and prevents the formation of resistant strains. Keywords: antibacterial agents, antioxidants, Klebsiella pneumoniae, Escherichia coli, drug interactions.

Introduction microorganisms, in some cases resistant to The use of antibacterial agents has caused several classes of antibacterial drugs [1, 2, 3]. the emergence and wide spread of resistant Many researchers note that if existing trends do

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Miroshnichenko A.G., Perfil'yev V.Yu., Lysenko I.V, Zhernakova N.I. Interaction between some antibiotics and antioxidants. Research result: Pharmacology and Clinical Pharmacology. 101 2017;3(4):100-112. not change in a positive way, then medicine processes [14, 15, 16]. Consequently, will face a problem half a century old, when antioxidants can reduce the antimicrobial antibiotics were not yet available [4, 5]. In this activity of chemotherapeutic agents and reduce regard, the improvement of antimicrobial the effectiveness of treatment [17]. Thus, the chemotherapy, aimed at increasing the advisability of using antioxidants in effectiveness of antibacterial drugs and combination with antibiotics is controversial. reducing the number of strains with multiple The study of the effect of antioxidants on the drug resistance, remains a very urgent task. antibacterial activity of chemotherapeutic Pharmacotherapy of a bacterial infection is agents is relevant and can open new prospects usually not limited to the use of an antibacterial for improving the treatment of infectious agent and includes other drugs whose effects patients. are directed to the macroorganism. Objectives Antioxidants are used in the pharmacotherapy Study of the influence of antioxidants on of bacterial infections because of the significant the activity of chemotherapeutic agents with role of enhancing the processes of free radical respect to opportunistic bacteria (Escherichia oxidation in pathogenesis [6, 7, 8]. The use of coli and Klebsiella pneumoniae). the latter is considered justified, since bacterial infection is accompanied by the production of Methods active forms of oxygen damaging biomolecules We studied the interaction of antioxidants and making a significant contribution to the and antibiotics (Table 1). The invitro studies development of disorders of cellular used periodic cultures of control and clinical metabolism [9, 10, 11]. According to modern strains of the Enterobacteriaceae family: data, the bactericidal action of many Escherichiacoli (control strain ATCC 25922 antibacterial agents has a common mechanism and two clinical strains) and Klebsiella associated with the generation of hydroxyl pneumoniae (control strain ATCC 13883 and radicals and the activation of oxidative stress in two clinical strains). Daily cultures of the bacterial cells. This is confirmed by the ratio strains were prepared by incubation on slanted between the degree of bactericidal effect and agar at 35 ° C and used to prepare inoculum – the level of hydroxyl radicals [12, 13]. In bacterial suspensions in a 0.9% sodium addition, radical particles in low concentrations chloride solution with an optical density of 1.0 act as intracellular signaling molecules, McFarland. supporting the normal course of biochemical Table 1 Antioxidants and antibiotics Antioxidants Antibiotics Ascorbic acid (Panreac, Spain) Gentamicin (AppliChem, Germany) N-acetylcysteine (Sigma-Aldrich, USA) Ciprofloxacin (Sigma-Aldrich, USA) Methylethylpyridinol (MEZ, Russia) Ceftazidime (Sigma-Aldrich, USA)

Incubation mixtures for studying the effect thermostat at 35 ° C. for 24 hours. To assess the of antioxidants on the activity of antibiotics growth of strains, a device was used to were prepared on the basis of the mineral determine the optical density of bacterial medium M9. The incubation mixture contained suspensions of Densi-la-meter (Erba Lachema an antioxidant, an antibiotic and a periodic s.r.o., Czech Republic). The measurements bacterial culture. Antioxidants were studied at were carried out every 2 hours for 12 hours, concentrations of 0.25, 0.5, 1, 2 and 4 mM. and also after 24 hours of incubation. The Antibiotics were kept at concentrations equal to obtained data were compared with the data of half the minimum inhibitory concentrations control incubation mixtures containing no established for each strain. After inoculating antioxidants. the bacterial suspension, the mixture containing In vivo studies were performed on 595 the antioxidants was incubated in an air male Wistar rats aged 2-3 months weighing December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Miroshnichenko A.G., Perfil'yev V.Yu., Lysenko I.V, Zhernakova N.I. Interaction between some antibiotics and antioxidants. Research result: Pharmacology and Clinical Pharmacology. 102 2017;3(4):100-112.

190-290 g grown in the nursery of Institute of glutathione, the activity of glutathione Cytology and Genetics of the Siberian Branch peroxidase and catalase. Ceruloplasmin was of the Russian Academy of Sciences also determined as a marker of the intensity of (Novosibirsk, Russia). The maintenance of the inflammatory process. animals met the requirements of the Helsinki To assess liver function in blood plasma, Declaration of the World Medical Association the activity of alanine aminotransferase and (2000), the European Convention on the alkaline phosphatase was determined. The renal Protection of Vertebrates used for experimental function was assessed by plasma concentrations or other scientific purposes (Strasbourg, 1986). of urea and creatinine. These parameters were All animals were kept in the same room in determined using diagnostic kits (Vital individual cages at an air temperature of 20-25 Diagnostics SPb, Russia). ° C without restriction of access to food and Computer programs Microsoft Office water. To carry out the research, healthy Excel 2003 (Microsoft Corporation, USA) and animals were selected who had undergone a SigmaStat 3.5 (Systat Software Inc., USA) for two-week quarantine in the vivarium. The Windows were used for calculations. The experimental groups were formed by random statistical processing of the results was carried sampling, taking into account the body weight out using the nonparametric criteria of Kraskel- as the determining index. Wallis and Mann-Whitney. The data were The in vivo experiments were divided into presented as a median and interquartile range – 2 parts according to the number of Me (25%, 75%). Differences were considered microorganisms studied (Escherichia coli, significant at P≤0.05 (comparison of two Klebsiella pneumoniae). Each part was divided independent groups) and at P≤0.017 (pairwise into 5 series in accordance with the comparison of three independent groups). antibacterial agents studied. For each series of 35 animals, groups (1-5) of 7 individuals were Results and Discussion formed. Interaction between antibacterial agents In each series, the animals of all groups and antioxidants in vitro were injected intraperitoneally with a bacterial The results of a study of the modulation of suspension (2.5 units by McFarland, 5 ml / kg) the activity of antibacterial agents by obtained from a daily pure culture of a clinical antioxidants in vitro are presented in Table 2. strain of Escherichia coli or Klebsiella There is a pronounced antagonism between pneumoniae. 3 hours after injection, a sterile gentamicin and antioxidants, which indicates solvent of antibacterial agent was administered an important role of enhancing the processes of to animals of Group 1, Group 2 – solution of peroxidation in the course of the bactericidal antibacterial agent, Group 3 – sequential action of the antibiotic. Note that solutions of antibacterial agent and ascorbic aminoglycosides – this is the only class of acid, Group 4 – solutions of antibacterial agent inhibitors of protein biosynthesis, which has a and methylethylpyridinol, Group 5 – solutions bactericidal effect. This is confirmed by of antibacterial agent and N-acetylcysteine. M.A. Kohansky et al. (2007), which proved the Antibiotics were administered in doses: leading role of free radical oxidation in the gentamicin 30 mg/kg, ciprofloxacin 50 mg/kg, realization of this effect [12]. ceftazidime 120 mg/kg. Antioxidants were Glutathione, as a natural metabolite of the administered in a dose 80 mg/kg. After 18 microorganisms under consideration, provides hours, the blood of experimental animals universal protection against active forms of underwent biochemical studies. oxygen [18]. In connection with this, the To assess the free radical oxidation in the expected decrease is the activity of blood plasma of rats, the concentrations of ciprofloxacin and ceftazidime, antibiotics with ceruloplasmin, thiobarbiturate-reactive a proven prooxidant component in the products of the plasma were measured, in mechanism of action [12], under the action of erythrocytes – the concentration of reduced N-acetylcysteine, a precursor of glutathione.

Table 2 Interaction between antibacterial agents and antioxidants in vitro

Escherichiacoli Klebsiellapneumoniae Antioxidant Strain 1 Strain 2 Strain 3 Strain 1 Strain 2 Strain 3 Gentamicin Ascorbic acid А++ А++ А++ А++ А++ А++ Methylethylpyridinol А++ А++ А++ А++ А++ А++ N-acetylcysteine А++ А++ А++ А++ А++ А++ Ciprofloxacin Ascorbic acid 0 0 0 0 А+ А++ Methylethylpyridinol S 0 S S++ S S N-acetylcysteine А++ А++ А++ А А++ А++ Ceftazidime Ascorbic acid S S S S S S Methylethylpyridinol S S S S S S N-acetylcysteine А++ А++ А++ А++ А++ А+ Note. Antagonism: A ++ – within 24 hours of incubation; A + – in most cases, the dependence on the concentration of the antioxidant may be absent; A – episodic development of culture is higher than control, dependence on antioxidant concentration may be absent; 0 – no influence, the development of culture does not change, or there are point changes with respect to control. Synergy: S – episodic development of culture below control, dependence on antioxidant concentration may be absent; S + – in most cases, the dependence on the concentration of the antioxidant may be absent; S ++ – within 24 hours of incubation.

The effect of ascorbic acid on the activity an antibacterial mechanism of action similar to of antibacterial agents has no obvious picture. aminoglycosides, although less pronounced. The reason for this is the chemical instability of Interaction between antibacterial agents the ascorbic acid molecule under aerobic and antioxidants in vivo conditions and the ability of the substance to Consider the results of a study modulating act as a pro-oxidant [19]. chemotherapy with the use of gentamicin The independent antibacterial activity of (Tables 3 and 4).It should be noted that this methyl ethylpyridinol was demonstrated by us particular antibiotic causes the greatest earlier [20]. It has been established that methyl alertness when administered with antioxidants, ethylpyridinol enhances the action of all since the latter resulted in the appearance of antibacterial agents studied, with the exception almost complete antibiotic resistance in vitro. of gentamicin, against most strains, with the The described effect eventually manifested strongest effect being manifested in high itself in animals with experimental peritonitis – concentrations of the antioxidant. This fact a significant decrease in the concentration of testifies, first, the lack of competition for a ceruloplasmin is observed only in the regime of pharmacological target, and secondly, the monotherapy with an antibiotic, the insignificant effect of the antioxidant properties introduction of antioxidants completely of methyl ethyl pyridinol on the action of pro- neutralizes this effect. The severity of leaking oxidants. At the same time, with the incubation peritonitis is enhanced by renal dysfunction with gentamicin, methyl ethyl pyridinol developing in animals receiving gentamicin due showed the most pronounced antagonistic to the implementation of the nephrotoxic activity compared to other antioxidants. In this properties of the antibiotic. regard, it appears that methylethylpyridinol has

Table 3 Biochemical indices of rats with bacterial peritonitis caused by Escherichia coli (with gentamicin administration) P Group 1 Group 2 Group 3 Group 4 Group 5 Erythrocyte catalase activity, %/mg Hb 0.030 0.049 0.041 0.036 0.046 (0.026;0.043) (0.045;0.054) (0.037;0.047) (0.029;0.060) (0.043;0.060) P x 0.038 0.097 0.383 0.038 Concentration of reduced glutathione in erythrocytes,umol/g Hb 0.56 0.78 0.74 0.70 0.70 (0.49;0.61) (0.68;0.79) (0.56;0.82) (0.63;0.77) (0.55;0.73) P x 0.004 0.097 0.026 0.128 Erythrocyte glutathione peroxidase activity,umol/(min× g Hb) 18.11 19.62 17.15 18.84 19.32 (17.37;20.80) (17.61;22.31) (13.48;21.54) (16.72;20.80) (17.28;22.36) P x 0.710 0.535 1.000 0.620 Concentration of thiobarbiturate-reactive products,umol/l 4.88 4.65 4.26 3.76 3.64 (3.26;5.11) (3.63;5.88) (3.73;4.61) (3.44;4.41) (3.22;4.24) P x 0.710 0.805 0.535 0.535 Concentration of ceruloplasmin,mg/l 512.8 455.0 511.0 518.0 522.4 (481.7;571.8) (418.0;489.1) (440.1;523.5) (499.2;598.5) (414.5;541.8) P x 0.017 0.456 0.535 0.902 Alanine aminotransferase activity in plasma,umol/(sec×l) 1.13 1.22 1.21 1.26 1.28 (0.85;1.48) (0.77;1.29) (1.02;1.36) (1.08;1.59) (0.96;1.40) P x 0.902 1.000 0.620 1.000 Activity of alkaline phosphatase in plasma,nmol/(sec×l) 1758 1517 1665 1625 1517 (1609;1920) (1287;1713) (1124;1744) (1238;1903) (1378;1638) P x 0.073 0.209 0.383 0.073 Urea concentration in plasma,mmol/l 2.48 5.08 4.26 5.13 4.89 (2.23;2.67) (4.14;7.03) (3.62;4.65) (4.06;6.47) (3.74;5.21) P x <0.001 <0.001 <0.001 0.011 Concentration of creatinine in plasma,umol/l 66.3 76.4 80.4 89.9 93.9 (60.7;70.3) (75.3;94.5) (72.9;88.3) (77.8;110.0) (81.9;106.2) P x 0.001 0.026 0.002 0.011

The administration of gentamicin, as well reactive products in plasma under experimental as its combination with methylethylpyridinol, peritonitis. This can be due, firstly, to the pro- prevents the formation of a deficit of reduced oxidant properties of gentamicin, which do not glutathione in animals with E. coli-induced allow to reduce the intensity of the processes of peritonitis. In animals with peritonitis caused free radical oxidation even when the pathogen by Klebsiella pneumoniae, this effect is absent. is destroyed, and secondly, the renal function is However, neither the antibiotic nor its impaired, and accordingly, the elimination of combination with antioxidants in both cases lipid peroxidation products. reduces the concentration of thiobarbiturate-

Table 4 Biochemical indices of rats with bacterial peritonitis caused by Klebsiella pneumoniae (with gentamicin administration)

P Group 1 Group 2 Group 3 Group 4 Group 5 Erythrocyte catalase activity, %/mg Hb 0.048 0.030 0.049 0.049 0.050 (0.044;0.052) (0.023;0.035) (0.047;0.052) (0.045;0.051) (0.044;0.059) P x 0.001 0.535 0.805 0.805 Concentration of reduced glutathione in erythrocytes, umol/g Hb 0.55 0.61 0.46 0.52 0.47 (0.47;0.67) (0.45;0.63) (0.42;0.57) (0.48;0.56) (0.45;0.54) P x 1.000 0.456 0.710 0.383 Erythrocyte glutathione peroxidase activity, umol/(min× g Hb) 18.28 16.27 16.92 17.40 17.43 (14.44;19.22) (14.01;17.64) (16.78;17.48) (16.21;18.35) (16.47;19.17) P x 0.259 1.000 0.902 0.710 Concentration of thiobarbiturate-reactive products, umol/l 3.57 3.49 3.52 3.60 3.45 (3.43;3.67) (2.70;3.59) (3.14;3.76) (2.96;4.15) (3.36;3.92) P x 0.318 1.000 0.805 0.620 Concentration of ceruloplasmin, mg/l 496.1 406.0 462.9 450.6 447.1 (459.6;592.2) (382.8;423.7) (408.4;528.7) (446.0;518.4) (438.1;500.5) P x 0.017 0.259 0.259 0.209 Alanine aminotransferase activity in plasma, umol/(sec×l) 2.32 1.73 1.96 2.27 1.97 (1.98;2.56) (1.71;1.96) (1.76;2.17) (1.90;2.31) (1.95;2.22) P x 0.017 0.128 0.383 0.318 Activity of alkaline phosphatase in plasma, nmol/(sec×l) 1493 1301 1286 1304 1431 (1281;1558) (1183;1486) (1210;1492) (1221;1482) (1222;1522) P x 0.318 0.383 0.383 0.456 Urea concentration in plasma, mmol/l 2.25 4.67 4.81 5.29 4.81 (1.94;2.56) (4.48;5.15) (4.60;5.39) (4.40;5.62) (4.41;5.40) P x <0.001 <0.001 <0.001 <0.001 Concentration of creatinine in plasma, umol/l 55.8 95.6 96.1 98.1 96.9 (49.4;60.0) (88.3;105.7) (89.5;102.4) (90.9;107.6) (86.1;105.0) P х <0.001 <0.001 <0.001 <0.001

The administration of ciprofloxacin by E. coli, is preserved both in the reduces the oxidase activity of ceruloplasmin, monochemotherapy regimen and in co- and this effect in rats with peritonitis induced administration with antioxidants (Table 5).

Table 5 Biochemical indices of rats with bacterial peritonitis caused by Escherichia coli (with ciprofloxacin administration)

P Group 1 Group 2 Group 3 Group 4 Group 5 Erythrocyte catalase activity, %/mg Hb 0.042 0.038 0.039 0.042 0.038 (0.033;0.051) (0.035;0.044) (0.028;0.048) (0.030;0.050) (0.029;0.055) P x 0.456 0.318 0.535 0.710 Concentration of reduced glutathione in erythrocytes, umol/g Hb 0.55 0.73 0.72 0.63 0.62 (0.52;0.65) (0.65;0.76) (0.63;0.82) (0.50;0.88) (0.54;0.67) P x 0.011 0.026 0.456 0.456 Erythrocyte glutathione peroxidase activity, umol/(min× g Hb) 13.31 14.69 18.90 19.39 19.23 (12.92;16.99) (12.26;17.07) (16.07;19.74) (17.55;20.41) (17.78;21.12) P х 0.902 0.026 0.038 0.026 Concentration of thiobarbiturate-reactive products, umol/l 4.69 3.95 4.65 3.64 4.11 (4.52;4.92) (3.59;4.49) (4.53;4.68) (3.39;4.42) (3.75;4.31) P x 0.017 0.535 0.004 0.004 Concentration of ceruloplasmin, mg/l 536.4 383.3 471.6 461.1 410.4 (499.8;601.3) (350.4;465.7) (393.1;491.8) (437.5;476.7) (394.4;488.9) P x 0.004 0.026 0.017 0.026 Alanine aminotransferase activity in plasma, umol/(sec×l) 1.93 1.87 1.77 1.80 1.77 (1.84;2.53) (1.83;1.90) (1.62;2.09) (1.74;1.99) (1.67;1.96) P x 0.383 0.209 0.165 0.128 Activity of alkaline phosphatase in plasma, nmol/(sec×l) 1458 1464 1304 1458 1319 (1438;1866) (1397;1530) (1280;1589) (1292;1560) (1250;1821) P x 0.456 0.259 0.456 0.165 Urea concentration in plasma, mmol/l 1.56 1.78 1.93 1.78 1.62 (1.41;2.08) (1.55;2.08) (1.72;2.03) (1.64;1.89) (1.57;1.80) P x 0.456 0.383 0.383 0.805 Concentration of creatinine in plasma, umol/l 46.8 51.8 50.8 51.3 50.3 (45.2;49.3) (48.9;54.5) (49.7;53.1) (48.6;53.0) (50.1;53.2) P x 0.165 0.209 0.259 0.165

The concentration of thiobarbiturate- action of ciprofloxacin, and the elimination of reactive products in animals with E. coli- products of peroxidation and endotoxins is not induced peritonitis decreases with hampered by the preserved functions of the ciprofloxacin, indicating a decrease in the liver and kidneys. Important is the fact that the intensity of peroxidation processes. This effect administration of antioxidants increases the is observed despite the known pro-oxidant activity of glutathione peroxidase. Untypical is properties of ciprofloxacin. In this regard, it can the profile of peroxidation markers in animals be argued that a decrease in the intensity of with escherichial peritonitis who received peroxidation is associated with the antibacterial ciprofloxacin with ascorbic acid. On the one December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Miroshnichenko A.G., Perfil'yev V.Yu., Lysenko I.V, Zhernakova N.I. Interaction between some antibiotics and antioxidants. Research result: Pharmacology and Clinical Pharmacology. 107 2017;3(4):100-112. hand, the concentration of thiobarbiturate- depending on the conditions, can be both anti- reactive products is maintained at the control and pro-oxidant. level, which indicates the lack of realization of The administration of antioxidants to its antioxidant properties by ascorbic acid. On animals with K. pneumoniae infection retains the other hand, the concentration of reduced the activity of ceruloplasmin at the control glutathione is at the same level as the level, which indirectly may indicate a decrease monotherapy group. This is probably due to the in the effectiveness of treatment (Table 6). dual properties of ascorbic acid, which, Table 6 Biochemical indices of rats with bacterial peritonitis caused by Klebsiella pneumonia (with ciprofloxacinadministration)

P Group 1 Group 2 Group 3 Group 4 Group 5 Erythrocyte catalase activity, %/mg Hb 0.053 0.046 0.046 0.040 0.048 (0.048;0.057) (0.036;0.049) (0.034;0.048) (0.038;0.050) (0.039;0.053) P x 0.038 0.026 0.038 0.209 Concentration of reduced glutathione in erythrocytes, umol/g Hb 0.64 0.63 0.68 0.84 0.66 (0.61;0.72) (0.59;0.69) (0.63;0.80) (0.59;0.87) (0.63;0.81) P x 0.620 0.318 0.383 0.535 Erythrocyte glutathione peroxidase activity, umol/(min× g Hb) 11.63 16.94 13.04 12.35 15.11 (10.37;15.27) (14.26;18.44) (11.89;14.10) (11.06;14.03) (13.16;15.25) P х 0.097 0.535 0.805 0.620 Concentration of thiobarbiturate-reactive products, umol/l 4.26 4.26 4.34 4.34 3.84 (3.71;4.93) (3.74;4.84) (3.91;4.47) (3.94;4.47) (3.71;4.36) P x 0.805 0.902 0.902 0.535 Concentration of ceruloplasmin, mg/l 468.1 427.0 406.0 415.6 445.4 (446.3;529.8) (399.9;446.3) (404.0;439.5) (397.0;491.3) (424.6;468.3) P x 0.038 0.053 0.165 0.209 Alanine aminotransferase activity in plasma, umol/(sec×l) 1.99 1.86 2.25 2.08 2.03 (1.94;2.40) (1.73;2.00) (2.00;2.56) (1.83;2.40) (1.74;2.25) P x 0.165 0.535 1.000 0.456 Activity of alkaline phosphatase in plasma, nmol/(sec×l) 1515 1328 1541.8 1325 1524 (1368;1942) (1287;1619) (1361;1718) (1270;1757) (1316;1641) P x 0.209 0.620 0.318 0.620 Urea concentration in plasma, mmol/l 1.98 1.96 1.99 2.23 2.01 (1.88;2.30) (1.89;2.29) (1.86;2.11) (1.76;2.35) (1.87;2.29) P x 0.902 0.805 0.902 0.902 Concentration of creatinine in plasma, umol/l 48.3 45.1 55.3 57.1 53.8 (46.6;55.5) (45.1;50.2) (45.6;56.2) (46.1;57.5) (47.7;59.1) P x 0.128 1.000 0.710 0.620

Table 7 Biochemical indices of rats with bacterial peritonitis caused by Escherichia coli (with ceftazidimeadministration)

P Group 1 Group 2 Group 3 Group 4 Group 5 Erythrocyte catalase activity, %/mg Hb 0.048 0.027 0.029 0.031 0.031 (0.035;0.051) (0.019;0.038) (0.026;0.032) (0.021;0.033) (0.029;0.035) P x 0.026 0.001 0.007 0.017 Concentration of reduced glutathione in erythrocytes, umol/g Hb 0.52 0.67 0.67 0.68 0.68 (0.38;0.58) (0.56;0.76) (0.62;0.74) (0.64;0.72) (0.67;0.71) P x 0.038 0.038 0.017 0.011 Erythrocyte glutathione peroxidase activity, umol/(min× g Hb) 16.77 19.07 20.09 21.57 21.48 (14.48;17.72) (18.11;22.71) (19.73;22.26) (19.71;22.11) (18.63;22.91) P х 0.007 0.002 0.002 0.026 Concentration of thiobarbiturate-reactive products, umol/l 4.38 2.79 2.87 3.29 2.91 (3.89;4.60) (2.34;3.71) (2.51;3.30) (3.23;3.58) (2.27;3.49) P x 0.038 0.011 0.038 0.011 Concentration of ceruloplasmin, mg/l 497.9 389.4 443.6 357.0 347.4 (462.9;509.5) (331.2;414.3) (328.3;529.6) (314.1;456.1) (291.8;400.5) P x 0.038 0.456 0.128 0.007 Alanine aminotransferase activity in plasma, umol/(sec×l) 1.32 1.23 1.10 1.11 1.11 (1.27;1.64) (1.17;1.29) (0.85;1.25) (0.96;1.22) (0.87;1.18) P x 0.026 0.007 0.004 <0.001 Activity of alkaline phosphatase in plasma, nmol/(sec×l) 1193 1301 1301 1295 1310 (1103;1722) (1179;1763) (1153;1532) (1216;1370) (1207;1383) P x 0.710 0.902 0.710 0.805 Urea concentration in plasma, mmol/l 1.84 1.77 1.73 1.66 1.72 (1.74;1.91) (1.76;1.86) (1.71;1.97) (1.65;1.71) (1.63;2.30) P x 1.000 0.535 0.128 0.620 Concentration of creatinine in plasma, umol/l 54.8 50.6 50.6 46.3 48.3 (44.2;54.8) (42.1;58.0) (40.1;54.8) (42.1;57.0) (42.9;51.8) P x 0.805 0.620 0.620 0.318 The administration of ciprofloxacin, as combination with ascorbic acid and well as its combination with antioxidants, does methylethylpyridinol) is a decrease in catalase not lead to a decrease in the concentration of activity. Thus, despite a decrease in the plasma thiobarbiturate-reactive products and an concentration of ceruloplasmin, ciprofloxacin increase in the content of reduced glutathione. does not cause a decrease in oxidative stress in The activity and glutathione peroxidase does animals with peritonitis caused by Klebsiella not change. This indicates a persistent oxidative pneumoniae. Probably, this is due to its more stress even against antibiotic chemotherapy. severe course compared to infection caused by The only effect of ciprofloxacin (including in E. coli. Antioxidants also had a weak effect.

Hence it should be concluded that the but its combination with antioxidants affect this effectiveness of the use of antioxidants in the parameter ambiguously (Tables 7 and 8). complex therapy of bacterial infection depends In particular, the effect of the antibiotic is not only on the mechanism of action of the preserved in combination with ascorbic acid antibacterial agent, but also on the specific and methylethylpyridinol, but is lost in pathogen. combination with N-acetylcysteine. In animals Just like ciprofloxacin, ceftazidime causes with an escherichiosis infection, on the a decrease in oxydase activity of ceruloplasmin, contrary, the effect of ceftazidime is retained only in combination with N-acetylcysteine.

Table 8 Biochemical indices of rats with bacterial peritonitis caused by Klebsiella pneumoniae (with ceftazidimeadministration)

P Group 1 Group 2 Group 3 Group 4 Group 5 Erythrocyte catalase activity, %/mg Hb 0.042 0.018 0.028 0.028 0.033 (0.035;0.057) (0.015;0.024) (0.021;0.030) (0.021;0.034) (0.028;0.036) P x 0.002 0.004 0.011 0.073 Concentration of reduced glutathione in erythrocytes, umol/g Hb 0.51 0.68 0.56 0.60 0.60 (0.45;0.60) (0.60;0.74) (0.53;0.67) (0.53;0.74) (0.50;0.66) P x 0.007 0.128 0.128 0.318 Erythrocyte glutathione peroxidase activity, umol/(min× g Hb) 18.52 20.95 20.19 20.98 19.02 (17.44;19.98) (19.07;22.38) (18.37;21.36) (19.12;21.57) (18.20;19.42) P х 0.053 0.209 0.053 0.710 Concentration of thiobarbiturate-reactive products, umol/l 3.80 3.10 3.33 3.53 3.33 (3.50;4.58) (2.84;3.53) (3.27;3.72) (3.22;3.78) (3.27;3.60) P x 0.017 0.097 0.165 0.073 Concentration of ceruloplasmin, mg/l 500.5 406.0 383.3 380.6 409.5 (425.9;532.0) (381.3;416.9) (356.6;409.7) (367.3;400.3) (388.3;432.3) P x 0.026 0.011 0.011 0.073 Alanine aminotransferase activity in plasma, umol/(sec×l) 1.89 1.52 1.69 1.73 1.76 (1.77;2.25) (1.25;1.76) (1.30;1.82) (1.27;1.75) (1.21;1.81) P x 0.026 0.073 0.053 0.038 Activity of alkaline phosphatase in plasma, nmol/(sec×l) 1557 1389 1404 1572 1464 (1315;1748) (1187;1442) (1302;1493) (1306;1751) (1292;1628) P x 0.209 0.318 0.902 0.456 Urea concentration in plasma, mmol/l 1.81 2.04 2.27 2.17 2.14 (1.60;2.33) (1.96;2.12) (1.93;2.54) (1.91;2.36) (2.08;2.39) P x 1.000 0.209 0.383 0.259 Concentration of creatinine in plasma, umol/l 45.3 44.81 47.6 47.8 51.3 (43.7;47.9) (44.2;46.0) (46.6;49.9) (46.4;51.1) (48.6;52.7) P x 0.902 0.097 0.097 0.073

A characteristic effect that is caused by ciprofloxacin, ceftazidime. Ascorbic acid ceftazidime is a decrease in plasma activity of enhances the action of ceftazidime. alanine aminotransferase, which indicates a Ascorbic acid, methylethylpyridinol and decrease in liver damage in conditions of N-acetylcysteine (80 mg/kg) reduce the peritonitis, and is probably also a consequence antibacterial activity of gentamicin (30 mg/kg) of the antioxidant effect of the antibiotic. and ciprofloxacin (50 mg/kg) and do not To confirm this, consider the peroxidation change the intensity of peroxidation processes markers. In animals under the influence of when combined with these antibacterial agents ceftazidime, the concentration of under experimental infection conditions, caused thiobarbiturate-reactive products actually by K. pneumoniae. These antioxidants reduce decreases. In this case, in combination with the pro-oxidant effect of ciprofloxacin (50 mg / antioxidants, the effect does not increase in kg) without affecting their antibacterial activity animals with escherichial peritonitis, and in under experimental escherichiosis infection. animals with peritonitis caused by Klebsiella Ascorbic acid, methylethylpyridinol and N- pneumoniae, on the contrary, it is lost. Similar acetylcysteine changes are observed when considering the (80 mg/kg) do not reduce the nephrotoxic concentration of reduced glutathione. effect of gentamicin (30 mg/kg), but decrease In animals with peritonitis caused by its antibacterial efficacy. Klebsiella pneumoniae, antibiotic and its The use of substances that alter the activity combination with antioxidants cause a decrease of antibacterial agents has not only an obvious in the activity of erythrocyte catalase and an clinical, but also epidemiological significance. increase in the activity of glutathione The use of any antibacterial agent leads to a peroxidase. With infection caused by Klebsiella gradual increase in the frequency of resistance pneumoniae, the activity of the latter does not of microorganisms. The time interval from the change, and the combination with N- beginning of application of the drug to the acetylcysteine retains catalase activity at the moment of occurrence of 100% resistance is control level. the time of effective use of the antibacterial Thus, antioxidants in complex therapy of agent. Inclusion in the chemotherapy of bacterial infection using ceftazidime have an modulators-synergists, increasing the ambiguous effect, which is due to the effectiveness of treatment, leads to an extension properties of the pathogen strain.If they did not of this time. Modulators-antagonists, which affect the activity of the antibiotic in the reduce the activity of antibacterial agent, therapy of escherichial peritonitis, then in the reduce the time of its effective use. case of infection caused by Klebsiella The activity of each antioxidant essentially pneumoniae, there is no definite regular depends on the environment and the conditions influence. In addition, because of its instability, of its functioning [21]. Another reason ceftazidime can have a very unstable complicating the evaluation of antioxidant pharmacokinetics. However, the decrease in defense mechanisms is the interdependence of antibiotic activity caused only N-acetylcysteine the content and activity of antioxidants and only under conditions of infection caused combined into antioxidant systems. The effect by Klebsiella pneumoniae. This confirms the of some antioxidants can be inverted when their data obtained by us in vitro and demonstrates a content changes. In this regard, the effects decrease in the bactericidal effect of exhibited by antioxidants are determined by the ceftazidime by N-acetylcysteine. applied dose, by administration, Conclusion biotransformation and elimination. In vitro studies found that all antioxidants The obtained data demonstrate that the significantly reduce the activity of gentamicin. existing traditional approach to determining the N-acetylcysteine also reduces the activity of expediency of prescribing an antibacterial agent ciprofloxacin and ceftazidime. by microbiological evaluation of the Methylethylpyridinol increases the effect of susceptibility to the pathogen is insufficient in the context of complex pharmacotherapy. This December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Miroshnichenko A.G., Perfil'yev V.Yu., Lysenko I.V, Zhernakova N.I. Interaction between some antibiotics and antioxidants. Research result: Pharmacology and Clinical Pharmacology. 111 2017;3(4):100-112. is due, above all, to the fact that other drugs can 5. Kapil A. The chalenge of antibiotic significantly change the sensitivity of bacteria resistance: need to contemplate.Indian J. Med. to the chemotherapeutic agent until the Res.2005;121(2):83-91. [PubMed] development of resistance. Therefore, in order 6. Nechayeva GI, Vershinina MV, to increase the effectiveness of Vysokogorskiy VE, Pritykina pharmacotherapy and reduce the occurrence of TV.Substantiation of the need for antioxidant antibiotic resistant strains, it is necessary to therapy in community-acquired pneumonia in conduct in vitro microbiological studies with patients with connective tissue all drugs planned for the treatment of the dysplasia.Proceedings of the Chelyabinsk patient, as well as the causative agent that Scientific Center of the URO RAS. [Izvestiya caused the disease, since the type of interaction Chelyabinskogo nauchnogo tsentra URO of antibacterial agents and modulator RAN]. 2004;1(22):220-224. (In Russian) substances is determined not only by [eLIBRARY] [Full Text] mechanisms the effects of these drugs, but also 7. Nevskaya NA.Antioxidants in intensive the characteristics of a particular strain of the therapy of peritonitis. Far Eastern Medical microorganism. Journal. [Dal'nevostochnyy meditsinskiy zhurnal]. 2009;4:126-130. (In Russian) Conflicts of Interest [eLIBRARY] [Full Text] The authors have no conflict of interest to 8. Lowes DA, Webster NR, Murphy MP, declare. Galley HF.Antioxidants that protect mitochondria reduce interleukin-6 and References oxidative stress, improve mitochondrial 1. Hoban DJ, Nicolle LE, Hawser S, function, and reduce biochemical markers of Bouchillon S, Badal R.Antimicrobial organ dysfunction in a rat model of acute susceptibility of global inpatient urinary tract sepsis. British Journal of Anaesthesia. isolates of Escherichia coli: results from the 2013;110(3):472-480. [PubMed] [Full Text] Study for Monitoring Antimicrobial Resistance 9. Exline MC, Crouser ED.Mitochondrial Trends (SMART) program: 2009-2010. Diagn mechanisms of sepsis induced organ Microbiol Infect Dis. 2011;70(4):507-511. failure.Frontiers Biosc. 2008;13:5031-5041. [PubMed] [Full Text] [PubMed] [Full Text] 2. Rice, L. B. The clinical consequences of 10. Ding S, Minohara Y, Fan XJ [et antimicrobial resistance.Curr. Opin. Microbiol. al.].Helicobacter pylori infection induces 2009;12:476-481. [PubMed] [Full Text] oxidative stress and programmed cell death in 3. Kozlov RS, Golub AV, Dekhnich AV, human gastric epithelial cells. Infection and Sukhorukova MV.Antimicrobial Resistance of Immunity. 2007. [PubMed] [Full Text] Gram-negative Microorganisms Causing 11. Ruggieri AJ, Levy RJ, Deutschman Complicated Intra-abdominal Infections in CS.Mitochondrial dysfunction and resuscitation Russia. Clinical microbiology and in sepsis. Crit. Care Clin. 2010;26:567-575. antimicrobial chemotherapy. [Klinicheskaya [PubMed] [Full Text] mikrobiologiya i antimikrobnaya 12. Kohanski MA, Dwyer DJ, Hayete B, khimioterapiya]. 2015;3:227-234. (in Russian) Lawrence CA, Collins JJ. A common [eLIBRARY] [Full Text] mechanism of cellular death induced by 4. Kosinets AN, Frolova AV, Bulavkin bactericidal antibiotics. Cell. 2007;130:797- VP, Okulich VK.Antibiotic resistance. New 810. [PubMed] [Full Text] opportunities for antibacterial action.Bulletin of 13. Dwyer DJ, Kohanski MA, Hayete B, Vitebsk State Medical University.[Vestnik Collins JJ. Gyrase inhibitors induce an Vitebskogo gosudarstvennogo meditsinskogo oxidative damage cellular death pathway in universiteta]. 2014;13(2):70-77. (in Russian) Escherichia coli. Molecular Systems Biology. [eLIBRARY] [Full Text] 2007;3:91.[PubMed] [Full Text] 14. D'Autreaux B, Toledano MB. ROS as signalling molecules: mechanisms that December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Miroshnichenko A.G., Perfil'yev V.Yu., Lysenko I.V, Zhernakova N.I. Interaction between some antibiotics and antioxidants. Research result: Pharmacology and Clinical Pharmacology. 112 2017;3(4):100-112. generate specificity in ROS homeostasis. 21. Men'shchikova EB,Lankin VZ, Nature Reviews Molecular Cell Biology. Zenkov NK, Bondar' IA, Krugovykh NF, 2007;8:813-824. [PubMed] [Full Text] Trufakin VA. Oxidative stress. Prooxidants and 15. Bledsoe G, Crickman S, Mao J, Xia antioxidants(Moscow: Slovo, 2006), 556. (In CF, Murakami H, Chao L, Chao Russian) [eLIBRARY] J.Kallikrein/kinin protects against gentamicin- induced nephrotoxicity by inhibition of Author contributors inflammation and apoptosis. Nephrology Alexandr G. Miroshnichenko, Candidate Dialysis Transplantation. 2006;21:624-633. of Medical Sciences, Leading Researcher, [PubMed] [Full Text] Institute of Biological Medicine, Altai State 16. Tonks NK. Redox redux: revisiting University, e-mail: [email protected]. The author PTPs and the control of cell signaling.Cell. was the primary role in planning and 2005;121:667-670. [PubMed] [Full Text] conducting experiments, analyzing and 17. Goswami M, Mangoli SH, Jawali N. compiling the study results. Effects of glutathione and ascorbic acid on Vyacheslav Yu. Perfiliev, researcher, streptomycin sensitivity of Escherichia coli. Laboratory of Industrial Pharmacy and Antimicrobial agents and chemotherapy. supercritical fluid technologies, Altai State 2007;51(3):1119-1122. [PubMed] [Full Text] University, e-mail: [email protected] author 18. Große C, Schleuder G, Schmole C, took part in analyzing and interpreting the Nies DH. Survival of Escherichia coli cells on research results. solid copper surfaces is increased by Ilya V. Lysenko, researcher, Institute of glutathione. Applied and Environmental Biological Medicine, Altai State University, e- Microbiology. 2014;80(22):7071-7078. mail: [email protected]. The author took [PubMed] [Full Text] part in analyzing and interpreting the research 19. Matsuoka Y, Yamato M, Yamada results. 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December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Peresypkina А.А. Correction of retinal angiopathy of hypertensive type by dimethylaminoethanol derivative 19-16 in experiment. Research Result: Pharmacology and 113 Clinical Pharmacology. 2017;3(4):113-119.

Rus.

UDC: 57.084.1 DOI: 10.18413/2313-8971-2017-3-4-113-119

Anna A. Peresypkina CORRECTION OF RETINAL ANGIOPATHY OF HYPERTENSIVE TYPE BY DIMETHYLAMINOETHANOL DERIVATIVE 19-16 IN EXPERIMENT

Federal State Autonomous Educational Institution of Higher Education «Belgorod State National Research University», 85, Pobedy str., Belgorod, 308015, Russia Corresponding author, e-mail: [email protected]

Abstract Introduction: 63% of hypertensive patients have evidence of hypertensive angiopathy. Increase of effectiveness of drug therapy of retinal angiopathy of hypertensive type is an important task of pharmacology. Objectives: To increase the effectiveness of pharmacological correction of retinal angiopathy of hypertensive type using a new dimethylaminoethanol (DMAE) derivative 19-16. Materials and Methods: We studied protective effects of DMAE derivative 19-16 in doses 25 mg/kg, 50 mg/kg on a model of retinal angiopathy of hypertensive type on Wistar male rats, which was carried out by N-nitro-L-arginine-methyl ester (L-NAME) introducing in a dose 12.5 mg/kg/day within 28 days. To study the fundus of experimental animals, direct ophthalmoscopy was used. Electroretinography (ERG) was recorded in response to a single stimulation. The induced biopotentials were amplified, averaged and presented graphically with the help of Biopac-systems MP-150 with the program AcqKnowledge 4.2 (USA). To assess the degree of development of functional damage to the retina, the ratio of the amplitudes of the b- and a-waves, the coefficient b/a, was estimated. Results and Discussion: DMAE derivative 19-16 in a dose 50 mg/kg prevented the development of ischemic damage and retinal vascular changes caused by introduction of L-NAME, to a greater extent than in a dose 25 mg/kg. The observed retinoprotective effects are confirmed by ophthalmoscopy and ERG. Conclusion: The search of new ways of correction of the retinal angiopathy as a complication of hypertension is an urgent task of pharmacology, which can be solved by using a new DMAE derivative 19-16. Keywords: retinal angiopathy of hypertensive type, dimethylaminoethanol derivatives, ophthalmoscopy, electroretinography.

Introduction Hypertension, existing for a long time, The picture of the fundus and indicators of leads to serious complications in retina. 63% of local hemodynamics significantly complement hypertensive patients have evidence of the representation of researchers about the hypertensive angiopathy [2]. Hypertensive features of the disease, can detect early signs of angiopathy is characterized by the first phase of organic changes in the retinal vessels and judge hypertension, in which there are only functional by their state with a certain degree of vascular disorders and the pressure is unstable. confidence about the changes of regional The above changes in the retina, of course, lead vascular bed and on the vascular system of the to disruption of blood supply and the body as a whole [1]. development of ischemic conditions.

The search for innovative molecules [3, 4] fourth (n = 10) – the correction of is an important task of pharmacology. pathology by DMAE derivative 19-16 in a dose Moreover, their study should be carried out in 50 mg/kg. vivo models [5, 6]. L-NAME was injected in a dose Derivatives of DMAE (precursors of 12.5 mg/kg/day within 28 days i/p daily [7]. acetylcholine) are classified as nootropic DMAE derivative 19-16 was administered agents. In ophthalmology, in the complex to rats in the solution form in doses 25 treatment of retinal vascular diseases, the mg/kg/day and 50 mg/kg/day intragastrically nootropic drugs are used, for example, (i/g) for 60 min prior to administration of nicotinoyl-GABA, ginkgo biloba, vinpocetine. L-NAME daily for 28 days. In the treatment of retinal angiopathy of To measure blood pressure in rats (tail) hypertensive type antihypertensive drugs, a system of non-invasive measurement of symptomatic treatment are used, which do not blood pressure for small animals NIBP200 always allow to achieve the desired result. was used in the complex Biopac-systems Therefore, increasing the effectiveness of MP-150. pharmacological correction of retinal To study the fundus in experimental angiopathy of hypertensive type is an important animals, direct ophthalmoscopy was used on task of pharmacology and ophthalmology. the 29th day of the experiment (Bx a Neitz In connection with the foregoing, it should ophthalmoscope, Japan). To expand the be noted the relevance of studying the pupil, eye drops Irifrin 2.5% were used. The protective properties of new DMAE derivative ophthalmoscope was approached to the rat`s 19-16 on the model of retinal angiopathy of eye and sent a beam of light at a distance of hypertensive type in experiment. 0.5-2 cm to obtain a clear picture of the Objectives fundus. To increase the image, the lens To increase the effectiveness of Osher MaxField 78D model OI-78M was pharmacological correction of retinal used [8]. angiopathy of hypertensive type using a new ERG was performed at once after DMAE derivative 19-16. ophthalmoscopy. For this purpose, the animals were kept in the dark within 30 min Materials and Methods [9], then anesthetized and fixed on a table. Experiments were carried out on Wistar The corneal silver electrode was placed on male rats weighing 225-275 g. The rats were the cornea, the reference needle electrode taken for the study with no external signs of EL452 was placed subcutaneously in the disease, passed quarantine regime. Ethical skull region, the ground needle electrode principles of handling laboratory animals are EL450 was placed in the base of the tail. A observed in accordance with the «European stroboscope with a flash of white light, Convention for the Protection of Vertebral connected to the stimulator STM200 by Animals Used for Experimental and Other Biopac System, Inc. (USA) were placed Scientific Purposes. CETS No. 123». behind the animal back; the ERG was Manipulations were performed on rats under recorded in response to a single stimulation. general anesthesia by intraperitoneal (i/p) The induced biopotentials were amplified, introducing an aqueous solution of chloral averaged and presented graphically on the hydrate in a dose 300 mg/kg. screen with help of Biopac-systems MP-150 The study includes the following groups: and program AcqKnowledge 4.2 (USA). To first (n = 10) – the group of intact animals, evaluate the degree of development of second (n = 10) – the group with the functional retinal damage, the ratio of the modeling of retinal angiopathy (control), amplitudes of the b- and a-waves of the third (n = 10) – the correction of pathology ERG, the coefficient b/a, was estimated [10]. by DMAE derivative 19-16 in a dose 25 mg/kg, From the 10 values obtained in each group, the output was the average value, which was recorded in the protocol. December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Peresypkina А.А. Correction of retinal angiopathy of hypertensive type by dimethylaminoethanol derivative 19-16 in experiment. Research Result: Pharmacology and 115 Clinical Pharmacology. 2017;3(4):113-119.

For all data, the descriptive statistics which pathogenesis is associated with the were applied: the data were checked for the development of hypertension in rats on the normality of the distribution. The type of background of daily i/p administration of distribution was determined by the Shapiro- L-NAME in a dose 12.5 mg/kg/day for 28 days Wilk criterion. In the case of a normal [7] (SBP 204.8 mmHg, DBP 164.2 mmHg in a distribution, the average value (M) and the group with pathology; SBP 139.2 mmHg, DBP standard error of the mean (m) were 104.2 mmHg in the intact group, p<0.05). The calculated. In cases of abnormal distribution, confirmation of the formation of vascular the median (Me) and the quarterly range changes of hypertensive type in the retina were (QR) were calculated. Intergroup differences results of ophthalmoscopy and ERG on the 29th were analyzed by parametric (Student's t- day of the experiment. test) or nonparametric (Mann-Whitney test) In accordance with the protocol the methods, depending on the distribution type. anesthesia of animals was carried out on 29th Differences were determined at 0.05 day of the experiment. significance level. Statistical analysis is Example of ophthalmoscopy on intact performed using the software Statistica 10.0. animal is shown in fig. 1. Results and discussion. We used a model of retinal angiopathy of hypertensive type,

Fig. 1. Example of ophthalmoscopy on intact Wistar rat. Optic disc is circular or oval shape and stands out from the fundus in pale – pink. The boundaries of the optic nerve disc are clear. It lies in the plane of the retina. From the middle of the optic nerve exit the central vessels of the retina. Blood vessels of the retina don`t have anastomoses. The veins and arteries are straightforward, caliber is uniform, not crimped. The general background is pink

Example of ophthalmoscopy on animal vasoconstriction, "the phenomenon of chiasm", with retinal angiopathy of hypertensive type is a symptom Salus-Hun I. Veins are crimped at shown in fig. 2. the periphery. The background is slightly paly. In the group with correction by DMAE Example of ophthalmoscopy on animal derivative 19-16 in a dose 25 mg/kg/day it was with correction by DMAE derivative 19-16 in a found during ophthalmoscopy: optic disc is dose 50 mg/kg/day is shown in fig. 3. circular or oval in shape and stands out from The results of the electrophysiological the fundus in pale – pink. The boundaries of the activity evaluation of rat retina on day 29 of the optic nerve disc are clear. There is a slight experiment are presented in tab. 1.

Fig. 2. Example of ophthalmoscopy on Wistar rat with retinal angiopathy of hypertensive type (control). Optic disc is circular or oval shape and stands out from the fundus in pink. The boundaries of the optic nerve disc are clear. Veins are congested, full-blooded, crimped at the periphery. Arteries are narrowed, slightly crimped. Retina is palely (ischemic). Symptom Salus-Hun I

Fig. 3. Example of ophthalmoscopy on animal with correction by DMAE derivative 19-16 in a dose 50 mg/kg/day. Optic disc is circular or oval shape and stands out from the fundus in pale – pink. The boundaries of the disc are clear. Blood vessels of the retina don`t have anastomoses. The veins and arteries are straightforward, no crimping. Veins are slightly dilated. The general background is pink, not ischemic Table 1 Results of electroretinography on day 29th of the experiment (M ± m; n = 10), r.u.

Experimental groups b/a Intact 2,6±0,07у Control 2,2±0,05* Correction by DMAE derivative 19-16, 25 mg/kg/day 2,4±0,06*у Correction by DMAE derivative 19-16, 50 mg/kg/day 2,5±0,08 у Note: * – p<0.05 compared with the group of intact animals, у – р<0.05 compared with the control group

Thus, the results of fundus studies and Angiopathy of the retina of hypertensive ERG in experimental groups revealed type is correlated with a high risk of cardio – pronounced protective properties of the DMAE vascular diseases in general, but not included in derivative 19-16 in a dose 50 mg/kg/day, the risk assessment tools. Currently abroad the exceeding its effect in a dose 25 mg/kg/day, research of hypertensive retinopathy as consisting in a reduction in the development of determinant of target organ damage in vascular changes in the retina on the hypertension are actively conducted [18, 19, background of arterial hypertension, which 20]. were noted in the control group; an increase in In connection with the foregoing, the study the coefficient b/a in the groups with the of the protective properties of the new DMAE correction of pathology, which is caused by the derivative 19-16 on the model of retinal restoration of the positive wave b on the ERG angiopathy of hypertensive type in the and indicates the preservation of the experiment was topical. electrophysiological function of the bipolar and Conclusion müllerian cells. Results of ocular fundus studies revealed The main factors in the development of the most pronounced protective effect of the retinal angiopathy of hypertensive type are new DMAE derivative 19-16 in a dose 50 disorders of common hemodynamics, local mg/kg/day on the model of retinal angiopathy changes in the vessel walls. From local changes of hypertensive type in Wistar male rats. are the most important violations of the Correction of retinal angiopathy of vascular endothelium [11]. hypertensive type by DMAE derivative 19-16 In this regard, there is a need to find new in a dose 50 mg/kg/day leads to higher values methods of retinoprotection for possible of the coefficient b/a of ERG on the 29th day of reduction of the damaging effect of ischemia, the experiment compared to the group with formed in the retinal angiopathy of correction by DMAE derivative 19-16 in a dose hypertensive type. Segment of drugs for the 25 mg/kg/day, which indicates the restoration treatment of vascular diseases of the eye as a of the electrophysiological state of the retina. complication of systemic diseases is expedient to expand due to the increasing of incidence Conflicts of interest and lack of funds for targeted correction of The authors have no conflict of interest to ischemic lesions of the eye vessels [12]. declare. Based on the fact that electrophysiological studies often have a decisive importance in the References early and differential diagnosis of retinal 1. Savina YN, Dolgikh VV, Pogodin disorders [13], to study the correction of AV, et al. The early manifestations of functional changes in the retina, researcher hypertensive angiochorioretinopathy in must conduct a comprehensive analysis, adolescents with essential hypertension. including ophthalmoscopic, Ophthalmosurgery. 2014;3:48-52. (in Russian) electroretinography, microcirculation research. [Full text] Current literature data indicate the 2. Stepushina OA. Study of the effective use of nootropics in ophthalmic diagnostic capabilities of combined use of practice: clinical and functional examinations adaptive optics and method of retinal vascular showed a faster and more stable improvement calibrometry [dissertation]. [Moscow]: in visual acuity in the group of patients with Research Institute of Eye Diseases; 2012. 96 p. chronic ocular ischemic syndrome with the use (in Russian) [eLIBRARY] [Full text] of choline alfoscerate [14]; the use of citicoline 3. Beskhmelnitsyna EA, Kravchenko has shown a positive effect in the treatment of DV, Pokrovsky MV, et al. Molecular screening amblyopia and improves the function of the for potential selective antagonists of TRPA1 retina [15, 16]; vinpocetine modulates ion channel. The Bulletin of the Scientific metabolic activity and function during retinal Centre for Expert Evaluation of Medicinal ischemia [17], etc. Products. 2016;7(3):186-189. [Full text] December. 2017. 3(4). Research Results in Pharmacology rrpharmacology.ru Peresypkina А.А. Correction of retinal angiopathy of hypertensive type by dimethylaminoethanol derivative 19-16 in experiment. Research Result: Pharmacology and 118 Clinical Pharmacology. 2017;3(4):113-119.

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Rus.

UDC: 616.36-005.4-089.843-092.9 DOI: 10.18413/2313-8971-2017-3-4-120-131

Marina S. Razumova1 STUDY OF PHARMACOLOGICAL ACTIVITY IN SUBLIMATED Ekaterina S. Litvinova CULTURE LIQUID OF ALLOGENIC, XENOGENEIC Vassili P. Gavriliouk HEPATOCYTES AND FIBROBLASTS FOR THE CORRECTION OF LIVER DAMAGES IN CARBON TETRACHLORIDE INTOXICATION

Kursk State Medical University, 3, K. Marx St., Kursk, 305041, Russia Corresponding author, 1e-mail: [email protected]

Abstract Introduction: At the present time many clinical and experimental studies are devoted to the correction of hepatocytes and liver functional abnormalities with the use of cellular technologies. Materials and Methods: Experimental studies were performed on 175 Wistar rats weighing from 120 to 140 g. Acute toxic liver damages (ATLD) in laboratory animals was simulated by intramuscular injection of carbon tetrachloride (CTC) at a dose of 3 ml/kg as 50% solution in olive oil five times with an interval of 24 h. Results and Discussion: The introduction of Essentiale H and hypoxen to the animals with ATLD enabled to normalize PTI, TAA and the activity of antioxidant defense enzymes (SOD and catalase) and corrected the concentrations of MDA and SMON. Simultaneous introduction of CLAH proteins, Essentiale H and hypoxen to animals with acute toxic liver damage, in comparison with the previous groups of experimental animals, additionally normalizes the concentration of MDA and SMON in blood plasma. Combined administration of Essentiale N, hypoxen and CLAH proteins, in comparison with the previous groups of experimental animals, normalizes the concentration of MDA, SOD activity, sorption capacity of erythrocytes. Conclusion: The administration of hepatocytes culture liquid of intact rats in combination with pharmacological preparations (Essentiale Forte H and Hypoxen) to allogeneic recipients intoxicated with CTC corrects systemic metabolic disorders arising from the action of hepatotrophic poison more efficiently, in comparison with their separate use. Keywords: allogenic hepatocytes, xenogeneic hepatocytes, fibroblasts, sublimated culture liquid, liver damages in carbon tetrachloride intoxication, Essentiale forte H, Hypoxen.

Introduction the diagnostics complexity and the difficulty The incidence of acute and chronic liver choosing the most effective therapies in this diseases in the overall structure of human category of patients as well. Frequent liver diseases and the mortality from this type of pathologies include acute toxic liver damages pathology keeps steadily growing even in (ATLD), complicated by the development of economically developed countries [1, 2, 3]. The hepatic insufficiency under intoxication with problems of diagnostics, pathogenesis and hepatotoxic poisons, taking large doses of treatment of chronic and acute liver diseases analgesics, anti-inflammatory, antibacterial, remain relevant these days. This is due not antimetabolic and other medications [4, 5, 6]. only to the continuing tendency for increase in Unsatisfactory results of ATLD treatment the number of patients with this nosology, but are currently due to the lack of effective December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 121 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. pathogenetically substantiated Experiments included rats which underwent the pharmacotherapy, that is why, a promising quarantine regimen of Kursk State Medical direction for such pathological conditions University vivarium, which had no signs of correction is in the use of cellular technologies, somatic and infectious diseases. In addition, 65 and one of such directions is the use of humoral hepatocytes donors were involved 5-6 days factors obtained during the cultivation of xeno- after birth, including 30 Wistar rats and 35 and allogeneic hepatocytes, fetal fibroblasts [7, white mice. All the investigations were carried 8, 9]. This is due to the prevailing opinion that out at the same time of day, from 8 am to 12, the effect of whole cells transplantation is subjected to all the principles set out in the associated not so much with their organ- Convention for the Protection of Vertebrate substituting function as with the humoral and Animals used for experimental and other molecular mechanisms responsible for purposes (Strasbourg, France, 1986) and increasing not only the activity of recipient‘s according to the rules of laboratory practice in hepatic cells (hepatocytes), but also their the Russian Federation (Directive of the regeneration due to the induction of regulatory Ministry of Health of the Russian Federation peptides production, such as growth factor [10, No. 267 of June 19, 2003). 11, 12]. ATLD in laboratory animals was simulated Research studies conducted during recent by intramuscular injection of carbon years confirm that the damage and regeneration tetrachloride (CTC) at a dose of 3 ml/kg as of hepatocytes by different physical, chemical 50% solution in olive oil five times with an and biological factors are predominantly caused interval of 24 h (Smakhtin M.Yu. et al., 2003). by immune, oxidant disorders and structural- Xenogeneic (murine) and allergenic functional changes in the properties of red hepatocytes (XH, AH) were isolated from blood cells, which, in their turn, are a kind of animals 5-6 days after birth by M.N. Berry, "cellular dosimeter" of pathogenic endo- and/or D.S. Friend method (1969). exogenous factors action [13, 14, 15]. At the To get culture liquid of xenogeneic and present time many clinical and experimental allogeneic hepatocytes (CLXH, CLAH), 5x107 studies are devoted to the correction of cells per 3 ml of medium were cultivated in hepatocytes and liver functional abnormalities medium 199 for 6 hours. Following the with the use of cellular technologies, there are a incubation period termination, the cells were few works on the study of oxidative, immune precipitated by centrifugation (15 minutes at and erythrocytic disorders and their 400 g). Protein concentration in culture liquid pharmacological and non-pharmacological was determined by the Kjeldahl method. The correction in various types of liver pathology, obtained CLXH and CLAH were administered but there are practically no researches studying intraperitoneally five times with the first humoral factors in culture liquid of xeno- and injection of hepatotrophic poison (with a allogenic hepatic cells (hepatocytes) [16, 17, 24-hour interval) to the rats with ATLD in the 18]. Analyzing the fractions of humoral factors amount of 5 m/ kg of protein. contained in the liquid after xeno- and FB were isolated from corpora and allografts cultivation in it seems to be a extremities fragments of 8-12-week-old human promising direction, since it is them that are the abortus tissues by their mechanical active principles in the repair of liver cells [19, disintegration to microfragments of 0.1-0.2 mm 20, 21, 22]. and further cultivation of cells. The resulting Aim of the research: to estimate the culture liquid of FB (CLFB) was introduced proteins efficacy in culture liquid of xeno-, intraperitoneally to rats with ATLD in the allogeneic hepatocytes, and fibroblasts in the amount of 5 mg/kg, simultaneously with the correction of metabolic and morphological first injection of CTC. disorders in acute toxic liver damage. To obtain proteins from 50-100 ml of Materials and Methods culture liquid, they were precipitated with an Experimental studies were performed on equal volume of 10% trichloracetic acid; the 175 Wistar rats weighing from 120 to 140 g. resulting precipitate was separated by 20 December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 122 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. minutes centrifugation at 1500 g. After times (with a 24-hour interval) to the rats with supernatant separation, the precipitate was ATLD in the amount of 5 mg/kg of protein. diluted in normal saline and dialyzed with two Poisoning with hepatotrophic toxin at the changes of phosphate-buffered saline pH 7.2- doses used and frequency of administration 7.4 for 18 hours. according to literature data and in our After the protein concentration had been experiments did not result in their death within determined and brought by 0.9% solution of the experiment, the animals were removed from sodium chloride to 5 mg/ml, the obtained the experiment in 24 hours after the last solution was filtered through sterilizing administration of CTC, culture liquid and their membranes of 0.2 μm, packed in sterile 2 ml proteins AH, FB, XH, essentiale forte H and vials and lyophilized in a freeze drying plant hypoxen. "VIRTIS". The study groups included 12-14 animals; The obtained proteins of CLXH, CLAH or the control group included 19 healthy rats of KLFB were intraperitoneally introduced with the same age, sex and body weight (Table 1). the first injection of hepatotrophic toxin five

Table 1 Groups of animals Groups of animals Amount Intact 19 CTC 14 CTC + culture fluid of allogeneic hepatocytes 12 CTC + culture fluid of xenogeneic hepatocytes 14 CTC + culture liquid of fibroblasts 13 CTC + proteins of culture fluid of allogeneic hepatocytes 14 CTC + proteins of culture fluid of xenogeneic hepatocytes 12 CTC + proteins of culture liquid of fibroblasts 14 CTC + proteins of culture fluid of allogeneic hepatocytes after lyophilization 13 CTC + proteins of culture fluid of xenogeneic hepatocytes after lyophilization 14 CTC + proteins of culture liquid of fibroblasts after lyophilization 14 CTC + essentiale + hypoxen 12 CTC + culture fluid of allogeneic hepatocytes + essentiale + hypoxen 14 Total sum: 175

Functional status evaluation of the liver Vitalab Flexor E (the Netherlands) with cells (hepatocytes) was carried out by the Analyticon® Biotechnologies AG reagents determination in the blood serum of alanine (Germany). Fibrinogen content was aminotransferase, gamma-glutamyl determined by means of a semi-automatic transpeptidase, alkaline phosphatase, analyzer of hemostatic profile STart4 (France), prothrombin index activities, thymol turbidity reagents Diagnostica Stago (France). test using standard reagent set. The intensity of lipid peroxidation Liver enzymes activity was evaluated by processes (LPO) was assessed by acyl means of automatic biochemical analyzer hydroperoxides (AHP) and malonic dialdehyde December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 123 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. (MDA) content in the blood plasma and slices were prepared in thermostatic cooler. erythrocytes. Antioxidant system state was Further on the microtome, paraffin slices of 5-7 determined by direct/ competitive solid-phase μm were prepared; they were subjected to enzyme immunoassay (EIA) with the detection paraffin removing, followed by staining with of reaction products in the wave-length hematoxylin and eosin. Cryostat sections were spectrum of 405-630 using ready-made stained with Sudan IV. commercial kits: superoxide dismutase activity Alongside with routine histological (SOD) "Bender Medsystems" (Austria) and methods of investigation, a morphometric catalase "Cayman Chemical" (the USA). Total research was used to morphologically evaluate antioxidative activity (TAA) was determined the liver tissue condition on histologic sections, by a method based on the degree of inhibition allowing to objectively estimate the degree and of ascorbate- and ferro-induced oxidation of peculiarities of ongoing processes in hepatic Tween-80 to MDA. The level of stable parenchyma and its potential regenerative metabolites of nitric oxide (SMNO) was capabilities. detected using two analytical procedures: Statistical processing of the results endogenous nitrite measurement and nitrate obtained in the thesis research was conducted conversion into nitrite using nitrite reductase using the criteria of variational and statistical followed by total nitrite measurement by azo analysis with the calculation of mean values dye absorption in Griess test at wave-length of (M), arithmetic means error (m). The 540 nm using a kit for solid-phase EIA of "R & significance of differences was evaluated D" firm (England). All the results of the according to Mann-Whitney U-test. enzyme-linked immunoassay were recorded by Differences with p <0.05 were considered means of automatic reader for the EIA ―Efos statistically significant. 9305‖ (Russia). The total number of peripheral blood Results and Discussion erythrocytes and their hemoglobin content were Intoxication with CTC for 5 days results in calculated according to generally accepted the development of the following biochemical syndromes of hepatic tissue damage: cytolysis standard methods. To this end, heparinized syndrome (an increased activity of AST, ALT, blood was centrifuged, plasma and erythrocytes which, in its turn, reduces De Ritis ratio), the were obtained. General sorption capacity of syndromes of extrahepatic and intrahepatic erythrocytes, due to the external architectonics cholestasis (increased GGT and APT activity) of cell membrane and the sorption capacity of and toxic hepatocytes damage (an increase in erythrocytes glycocalyx for alcian blue were bilirubin concentration, AST, ALT, GGT, APT determined. activity, GGT /AST rate, and De Ritis ratio Phagocytic activity of polymorphonuclear becomes less than 1), decline in synthetic leukocytes was assessed by the following processes (reduction of PTI and FB) and indices: phagocytic index (PI), phagocytic inflammatory syndrome (an increase in TTT) number (PN) and index of phagocytosis (Table 2). activity (IPA). The activity of oxygen- The introduction of the culture medium dependent systems of polymorphonuclear 199, on the basis of which the culture liquid of leukocytes was evaluated fibroblasts was obtained, to the animals spectrophotometrically by the reaction of nitro intoxicated with CTC does not affect the blue tetrazolium reduction (NBT-test) with the parameters of hepatocytes functional activity use of PD 303 S Apel (Japan) altered with the administration of hepatotrophic spectrophotometer. poison (Table 2). After animals had been removed from the The use of CLFB under ATLD conditions experiment, the liver was taken for normalizes the GGT/AST rate, partially morphological study. Pieces of the studied corrects the activity of APT, ALT, GGT, AST organs were fixed in 10% neutral formalin. We /ALT rate, GGT /AST rate, bilirubin and used tissues embedding in paraffin, partly in fibrinogen concentrations towards the gelatin; in some cases, non-fixed freshly frozen parameters of healthy animals (Table 2). December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical 124 Pharmacology. 2017;3(4):120-131. Table 2 Function of hepatocytes, the plasma oxidative status, and metabolic status in erythrocytes and FMA of peripheral blood neutrophils in acute toxic hepatopathy when transplanting the culture liquid of fibroblasts (M ± m)

1 2 3 4 Unit Indicators Introduction of CTC and: measure Control – Medium 199 CLFB AST U/l 28.9±2.3 53.9±4.2*1 50.1±4.2*1 51.2±4.0*1 ALT U/l 22.2±1.8 88.3±4.9*1 83.2±5.4*1 60.8±4.7*1-3 APT U/l 232.1±16.8 458.2±34.1*1 401.3±17.2*1 306.7±16.1*1-3 GGT U/l 5.8±0.2 20.1±2.1*1 19.5±1.4*1 8.9±0.6*1-3 Bili umol/l 5.3±0.3 19.1±1.1*1 17.5±0.4*1 10.9±0.9*1-3 PTI % 61.9±3.7 45.9±3.2*1 43.5±2.9*1 47.8±3.1*1 FBG h/l 4.2±0.1 2.2±0.03*1 2.3±0.05*1 3.4±0.02*1-3 TTT Unit S-Н 2.4±0.3 4.2±0.1*1 4.1±0.2*1 4.0±0.3*1 MDA umol/l 2.1±0.05 5.7±0.3*1 5.3±0.2*1 3.4±0.2*1-3 standard 0.8±0.03 2.0±0.1*1 1.9±0.05*1 0.94±0.03*1-3 AHP unit SOD stand.U/ml 10.9±0.7 8.3±0.6*1 8.1±0.4*1 9.2±0.5*1-3 TAA % 39.2±1.0 32.9±1.9*1 33.1±2.6*1 41.0±3.2*2.3 Kat μkat/l 11.9±0.5 9.1±0.7*1 8.9±0.4*1 11.8±1.2*2.3 *1 *1 *1-3 SMON umol/l 6.7±0.3 2.8±0.07 2.9±0.1 3.8±0.2 MDA umol/l 0.3±0.02 0.6±0.03*1 0.7±0.07*1 0.3±0.02*2.3 AHP stand.U 0.13±0.02 0.5±0.02*1 0.6±0.08*1 0.2±0.02*1-3 SOD stand.U/ml 24.4±1.6 8.9±0.1*1 8.5±0.4*1 16.7±0.8*1-3 SCE % 50.5±1.8 28.5±3.5*1 29.1±2.7*1 48.7±2.5*2.3 SCG 1012 g/eryth. 2.8±0.04 1.7±0.05*1 1.6±0.1*1 2.4±0.08*1-3 Erythrocytes 4.2±0.07 3.2±0.03*1 3.3±0.07*1 4.1±0.2*2.3 count 1012 /l Hemoglobin h/l 14.5±0.4 13.5±0.4*1 13.2±0.3*1 14.3±0.5*2.3 NBT – sp. mOD 0.7±0.05 1.1±0.03*1 1.2±0.06*1 0.8±0.05*2.3 NBT-ind. n/z mOD 0.9±0.05 1.6±0.05*1 1.7±0.05*1 1.3±0.06*1-3 NBT-ind. o/z mOD 1.1±0.06 1.5±0.05*1 1.6±0.06*1 1.6±0.05*1 PI absolute 47.5±1.0 74.8±2.6*1 72.7±1.9*1 52.8±1.7*1-3 PN absolute 2.0±0.1 2.7±0.05*1 2.6±0.1*1 2.7±0.1*1 Note. The asterisk marked significant differences of average arithmetical (p <0.05); figures close to the star are in relation to that of a group of these differences.

When evaluating oxidative parameters of corrects, but not to the control level, the the blood plasma in experimental animals with concentration of lipid peroxidation products ATLD, without and by the introduction of the and stable metabolites of nitrogen oxide and culture medium 199, the activation of LPO SOD activity (Table 2). processes (an increase in MDA and AHP Having analyzed the indices of metabolic levels), a decrease in antioxidant defense activity in red blood cells, we found out that the indices (TAA), activity of SOD and Kat) and introduction of hepatotoxic poison to SMON content (Table 2) have been revealed. experimental animals reduces their total count, The use of CLFB, in contrast to the indices hemoglobin content, increases endoglobular in the 2nd and 3d groups of animals, processes of lipid peroxidation (an increase in completely restores TAA, catalase activity and MDA and AHP concentrations), reduces SOD

December. 2017. 3(4). Research Results in Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 125 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. activity and sorption indexes (SCE and SCG) The CLAH is more effective, as in (Table 2). comparison with the CLXH, its use under the The introduction of the culture medium conditions of ATLD further normalizes the 199 does not affect the parameters of activity of the AST, GGT/AST and PTI rate, erythrocytes metabolic status, altered by the corrects ALT activity, De Ritis ratio and FBG intake of CTC. The use of CLFB normalizes content (Table 3). the total count of erythrocytes, the level of The study of oxidant status at the systemic hemoglobin in them, MDA concentration, the level showed that the use of CLXH normalizes total sorption capacity of erythrocytes (SCE), TAA and catalase activity and corrects, but not and corrects, but not to the parameters of up to the parameters of healthy animals, the level healthy rats, the activity of SOD, the level of of LPO products and the activity of SOD (Table AHP, the sorption capacity of glycocalyx in red 3). blood cells (SCG) (Table 2). Introduction of CLAH, in comparison with The study of innate immunity factors gave the previous group of animals, additionally the following results. In rats with ATLD, normalizes the concentration of AHP, SOD without or by the introduction of the culture activity, corrects the concentration of SMNO to medium 199, an increase in all the studied a greater extent (Table 3). indices of the functional-metabolic activity of The study of erythrocytic parameters peripheral blood neutrophils was revealed, i.e., revealed that the use of CLXH normalized the oxygen-dependent (an increase in NBT – sp., total number of erythrocytes, the level of NBT – ind. n/z, NBT – ind. o/z) and phagocytic hemoglobin in them and corrected the parameters of LPO and the sorption capacity of activity (an increase of PI, PN and EIA) red blood cells towards the control group, the (Table 2). activity of SOD as well (Table 3). The introduction of CLFB normalized the The use of CLAH in animals under the NBT-sp. and corrected NBT – ind. n/z, PI and conditions of CTC poisoning, in addition to the EIA towards the parameters of healthy animals effects of CLXH, normalizes SCG, MDA level (Table 2). and corrects to a greater degree SCE and SOD During histologic examination of liver activity of peripheral blood erythrocytes tissues of animals intoxicated with carbon (Table 3). tetrachloride, extensive areas of centrolobular, Further, it was found that compounds with large-drop adipose degeneration of hepatocytes, the corrective effect on the function of multiple focal necrosis with inflammatory peripheral blood polymorphonuclear leukocytes neutrophilic-lymphocyte infiltration are extracellular, since the introduction of predominantly, and disturbances in beam CLXH to intoxicated recipients normalized cell structure of lobules were detected. activity rate KAo and correlated, but not to the Microscopically, in animals with ATLD control values, the other studied indices of receiving CLFB, there is a disturbance in liver neutrophils FMA (Table 3). histoarchitecture, leukocyte-lymphocyte The administration of CLAH, in infiltration, and centrolobular large-drop comparison with the CLXH, additionally adipose degeneration. In the center of the normalizes NBT-sp., NBT-ind. o/z and lobule plethora is observed. normalizes and corrects to a greater degree the The use of culture liquid of xeno- and phagocytic activity of polymorphonuclear allogeneic hepatocytes for metabolic disorders leukocytes of peripheral blood (Table 3). correction in acute toxic hepatopathy. The use The nature of morphological liver changes of CLXH in animals with CTC poisoning, in in animals with ATLD, which received culture comparison with the group of rats with ATLD liquid of XH is basically similar to the only, normalizes the activity of GGT and APT, pathological morphology of the liver tissue that corrects the other hepatotrophic poison-induced occurs in rats with ATLD: large-drop adipose parameters, characterizing functional-metabolic degeneration of hepatocytes in the central parts activity of hepatocytes, towards the control of lobes, necrotic changes with inflammatory group of animals (Table 3). December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 126 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. cellular infiltration and discomplexation of lobules remain unchanged. Table 3 Function of hepatocytes, plasma oxidative status, and metabolic status of erythrocytes and FMA of peripheral blood neutrophils in acute toxic hepatopathy under the transplantation of culture liquid of xenogeneic and allogeneic hepatocytes (M ± m)

1 2 3 4 Unit Introduction of CTC and culture liquid of: Indicators measure Control Xenogeneic Allogeneic – hepatocytes hepatocytes AST U/l 28.9±2.3 53.9±4.2*1 40.1±2.1*1.2 30.3±2.6*2.3 ALT U/l 22.2±1.8 88.3±4.9*1 53.0±3.5*1.2 34.8±3.2*1-3 APT U/l 232.1±16.8 458.2±34.1*1 262.3±14.1*2 263.3±11.1*2 GGT U/l 5.8±0.2 20.1±2.1*1 0.76±0.04*1.2 0.87±0.06*1-3 Bili umol/l 5.3±0.3 19.1±1.1*1 6.7±0.3*1.2 6.3±0.4*1.2 PTI % 61.9±3.7 45.9±3.2*1 53.2±3.3*1.2 56.1±3.0*2.3 FBG h/l 4.2±0.1 2.2±0.03*1 3.3±0.04*1.2 3.8±0.1*1-3 TTT Unit S-Н 2.4±0.3 4.2±0.1*1 3.1±0.05*1.2 3.0±0.05*1.2 MDA umol/l 2.1±0.05 5.7±0.3*1 3.2±0.1*1.2 3.1±0.06*1.2 standard 0.8±0.03 2.0±0.1*1 0.9±0.05*1.2 0.74±0.04*2.3 AHP unit SOD stand.U/ml 10.9±0.7 8.3±0.6*1 9.9±0.6*1.2 12.0±0.5*2.3 TAA % 39.2±1.0 32.9±1.9*1 42.6±2.6*2 43.8±3.3*2 Kat μkat/l 11.9±0.5 9.1±0.7*1 12.6±0.7*2 12.4±1.1*2 *1 *1 *1-3 SMON umol/l 6.7±0.3 2.8±0.07 2.7±0.1 4.9±0.1 MDA umol/l 0.3±0.02 0.6±0.03*1 0.4±0.04*1.2 0.3±0.02*2 AHP stand.U 0.13±0.02 0.5±0.02*1 0.2±0.01*1.2 0.21±0.01*1.2 SOD stand.U/ml 24.4±1.6 8.9±0.1*1 14.3±0.7*1.2 19.8±1.3*1-3 SCE % 50.5±1.8 28.5±3.5*1 36.2±3.2*1.2 44.2±2.2*1-3 SCG 1012 g/eryth. 2.8±0.04 1.7±0.05*1 2.5±0.2*1.2 2.7±0.08*2 Erythrocytes 4.2±0.07 3.2±0.03*1 4.1±0.2*2 4.2±0.2*2 count 1012 /l Hemoglobin h/l 14.5±0.4 13.5±0.4*1 14.5±0.4*2 14.7±0.8*2 NBT – sp. mOD 0.7±0.05 1.1±0.03*1 0.9±0.06*1.2 0.8±0.06*2 NBT-ind. n/z mOD 0.9±0.05 1.6±0.05*1 1.3±0.05*1.2 1.2±0.05*1.2 NBT-ind. o/z mOD 1.1±0.06 1.5±0.05*1 1.4±0.04*1.2 1.2±0.04 *2.3 PI absolute 47.5±1.0 74.8±2.6*1 58.7±2.0*1.2 51.1±2.5*1-3 PN absolute 2.0±0.1 2.7±0.05*1 2.4±0.06*1.2 1.8±0.04 *1-3 Note. The asterisk marked significant differences of average arithmetical (p <0.05); figures close to the star are in relation to that of a group of these differences.

In animals with ATLD receiving CLAH, in The use of proteins of culture liquid of contrast to the group of animals with ATLD fibroblasts, xenogeneic and allogeneic without correction, histoarchitecture of the liver hepatocytes in correction of metabolic tissue is morphologically basically preserved, disorders in acute toxic hepatopathy. The but in the central sections of the lobules there is introduction of CLFB proteins to experimental a fine-focal predominantly small-drop adipose rats with ATLD prior or after their degeneration of hepatocytes, necrotic changes lyophilization, in comparison with the group were not detected. intoxicated with CTC, normalizes GGT/ AST rate, TAA and catalase activity, corrects, but

Fig. Influence of proteins of culture liquid of allogeneic hepatocytes, essentiale forte H and hypoxen on hepatocyte function, plasma oxidative status, metabolic status of erythrocytes and FMA of peripheral blood neutrophils in acute toxic hepatopathy

Combined introduction of CLAH proteins The simultaneous use of CLAH and and intoxication with CTC normalizes the pharmacological agents in animals with acute activity of AST, GGT, APT, GGT/ AST rate, toxic liver damage normalizes, with the PTI and corrects, but not up to the norm, other exception of De Ritis ratio, CTC-changed studied parameters reflecting the functional- biochemical measurement in the blood plasma metabolic activity of liver cells (Figure).

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 128 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. reflecting the functional activity of hepatocytes and metabolic activity in peripheral blood (Figure). neutrophils except for NBT-sp. and NBT-ind. The administration of Essentiale H and n/z (Figure). hypoxen in animals intoxicated with CTC In animals that received Essentiale H and normalized TAA and the activity of antioxidant hypoxen under ATLD conditions, histological defense enzymes (SOD and catalase) and examination shows focal changes in the liver corrected the concentrations of MDA and tissue in the form of large and small-drop SMON (Figure). adipose degeneration of hepatocytes, single The use of CLAH proteins in CTC- small areas of necrosis with a weak intoxicated animals normalized the level of inflammatory reaction. AHP, the activity of SOD and catalase, TAA, Morphologically, in animals with ATLD, corrected MDA and SMON concentrations which received CLAH proteins, Essentiale H (Figure). and hypoxen, the histoarchitecture of the liver Simultaneous introduction of CLAH tissue is preserved, the beam structure of the proteins, Essentiale H and hypoxen to animals lobules is clear, nuclei are moderately with acute toxic liver damage, in comparison basophilic in hepatocytes, the cytoplasm is with the previous groups of experimental oxyphilic, only small foci with granular animals, additionally normalizes the dystrophy of the hepatocytes occur by places. concentration of MDA and SMON in blood During morphological study of the liver plasma (Figure). tissue in groups of animals with acute toxic The administration of pharmacological liver damage and intoxicated animals that agents to the animals with ATLD normalizes received the proteins of CLFB, CLXH, CLAH, the hemoglobin content in erythrocytes and CLAH in combination with essentiale and brings the activity of SOD and the sorption hypoxen, in addition to generally accepted capacity of cells closer to the normal histological methods of research, to objectively parameters (Figure). verify and evaluate changes severity and The introduction of CLAH proteins also peculiarities occurring in the liver parenchyma, normalizes the level of hemoglobin and morphometric methods of investigation were corrects the other studied parameters of performed which were to determine the metabolic status in peripheral blood parameters characterizing the degree and erythrocytes (Figure). peculiarities of ongoing processes and enabling Combined administration of Essentiale N, to evaluate regenerative potential of the liver hypoxen and CLAH proteins, in comparison parenchyma. with the previous groups of experimental In animals with acute toxic liver damage animals, normalizes the concentration of MDA, with CTC, in comparison with intact rats, SOD activity, sorption capacity of erythrocytes changes in the values of morphometric indices and their count in the blood, corrects the level are represented by a significant decrease in PD of AHP to a greater degree towards the norm by 1.4 times, FCM by 2.1 times, NM by 2.2, (Figure). BCMI by 2 times and an increase in MMI by The introduction of Essentiale H and 2.5 times (Table 4). These changes in indices hypoxen to CTC intoxicated animals corrects are characterized by the development of the indices of phagocytic activity (PI, PN, congestive phenomena (plethora, cholestasis) in EIA), NBT-ind. o/z and NBT-test of peripheral the liver tissue, an increase in the current blood neutrophils and corrects the other studied (relatively short-term) intensity of hepatocyte parameters of oxygen-dependent metabolic functioning, their pronounced degenerative activity in peripheral blood neutrophils changes in the form of dystrophy and necrosis, (Figure). an increase in the ongoing intensity of The use of CLAH both separately and in reparative processes (due to "binuclear reserves combination with Essential H and hypoxen consumption") and a decrease in the depth of resulted in normalization or additional hepatic tissue reparative reserves. correction of the studied indices of functional December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical 129 Pharmacology. 2017;3(4):120-131. Table 4 Morphometric parameters of the hepatic tissue status in animals that received proteins of the CLFB, CLXH, CLAH and CLAH in combination with essentiale H and hypoxen under the conditions of acute CTC poisoning (M ± m)

Item Indices PD FCM NM BCMI MMI No groups х105 х105 х103 1. Intact animals 0.53±0.03 10.54±0.8 11.73±0.9 2.27±0.1 53.8±2.9 2. CTC 0.39±0.02*1 4.96±0.3*1 5.35±0.4*1 1.16±0.08*1 133.5±8.9*1 CTC + CLFB 3. 0.44±0.02*1.2 5.88±0.4*1.2 6.40±0.4*1.2 1.48±0.08*1.2 128.9±9.2*1 proteins CTC + CLXH 4. 0.45±0.02*1.2 6.41±0.5*1.2 6.98±0.5*1.2 1.51±0.08*1.2 110.0±9.3*1.2 proteins CTC + CLAH 5. 0.50±0.02*2-4 9.05±0.6*2-4 10.10±0.9*2-4 2.22±0.09*2-4 64.7±4.2*2-4 proteins CTC + CLAH proteins + 6. 0.51±0.03*2-4 9.52±0.6*2-4 10.70±0.8*2-4 2.4±0.1*2-4 59.0±3.9*2-4 essentiale + hypoxen Note. The asterisk marked significant differences of average arithmetical (p <0.05); figures close to the star are in relation to that of a group of these differences.

The introduction of CLFB and CLXH of the liver cells damage, an increase in the proteins into the animals under the conditions phagocytic and oxygen-dependent activity of of acute carbon tetrachloride intoxication peripheral blood neutrophils, oxidative stress resulted in a positive dynamics of development, and the disturbance in morphometric indices compared to animals erythrocytes metabolic activity have been without correction with proteins, however the revealed. In these conditions the use of culture absolute values of PD, FCM, NM, BCMI and liquid of fibroblasts in the presence of carbon MMI were significantly different from those in tetrachloride intoxication partially normalizes intact animals, that indicates the presence of and corrects metabolic disorders at the systemic congestive phenomena and degenerative and local levels. Administration of culture changes in the liver tissue, intensive "binuclear liquid of allogeneic hepatocytes in case of reserves consumption" and a decrease in the carbon tetrachloride poisoning normalizes and depth of reparative reserves. corrects metabolic disorders at the systemic and The administration of CLAH proteins and local levels more effectively, in comparison CLAH proteins in combination with essentiale with the administration of culture liquid of and hypoxen in acute toxic hepatopathy xenogeneic hepatocytes. manifested by insignificant changes in In integral estimation of disturbed morphometric parameters, whose values did parameters amount under various experimental not differ with assurance from the group of conditions with the determination of these intact animals. This bears evidence to high disorders pronouncement by their degree, it invariance of the histological structure, the was established that the use of pharmacological absence of morphologically significant agents (Essentiale H and hypoxen) and culture alternative changes and disturbances in liquid of allogeneic hepatocytes enabled to reparative capabilities of the liver tissue in the correct and normalize the immune and animals of these groups. metabolic status disorders at the systemic level, whereas their concomitant use normalizes the Conclusion studied indices practically entirely. In ATLD caused by carbon tetrachloride The development of biochemical intoxication, the main biochemical syndromes syndromes of the liver tissue damage, the December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 130 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. activation of metabolic and functional activity 6. Terekhova SV, Bystrova NA, of polymorphonuclear leukocytes, and the Litvinova ES, Gavrilyuk EV. Correction by activation of lipid peroxidation in the blood allogenny hepatocytes of immunometabolic plasma have been revealed in CTC violations at experimental ischemia of a liver. intoxication. The administration of hepatocytes The system analysis and management in culture liquid of intact rats in combination with biomedical systems [Sistemnyj analiz i pharmacological preparations (Essentiale Forte upravlenie v biomedicinskih sistemah]. H and Hypoxen) to allogeneic recipients 2012;11(2):414-417. (In Russian) intoxicated with CTC corrects systemic [eLIBRARY] metabolic disorders arising from the action of 7. Si-Tayeb K, Noto FK, Nagaoka M, Li hepatotrophic poison more efficiently, in J, Battle MA, Duris C, North PE, Dalton S, comparison with their separate use. Duncan SA. Highly efficient generation of human hepatocyte-like cells from induced Conflicts of Interest pluripotent stem cell. Hepatology. The authors have no conflict of interest to 2010;51(1):297-305. [PubMed] declare. 8. Paradhan SC, Girisn C. Hepatoprotective herbal drug, silymarin from References experimental pharmacology to clinical 1. Litvinova ES., Razumova MS, medicine. Indian. J. Med. Res. Kharchenko AV, Bystrova NA, Gavrilyuk VP. 2006;124(5):491-504. [PubMed] Immune-correcting effects of culture liquid of allogeneic hepatocytes and essentiale in acute 9. Razumova MS, Litvinova ES, toxic liver damage. Allergology and Gavrilyuk EV. Efficacy of culture liquid of Immunology [Allergologiya i immunologiya]. fibroblasts, xeno- and allogeneic hepatocytes 2016;17(4):293. (In Russian) and their proteins isolate in the correction of 2. Berkos AS, Nikolaev GV. The metabolic disorders in acute toxic hepatopathy. mechanism of development of a humoral Journal of Scientific Articles "Health and immune response to allogeneic organ Education in the 21st Century" [Zhurnal transplantation. Bulletin of transplantology and nauchnykh statej «Zdorov`e i obrazovanie v artificial organs [Vestnik transplantologii i XXI veke»]. 2017;19(10):313-316. (In Russian) iskusstvennyh organov]. 2017;19(2):139-151. [eLIBRARY] [Full text] (In Russian) [eLIBRARY] [Full text] 10. Terekhova SV, Bystrova NA, 3. Onishchenko ON. Cellular Litvinova ES, Gavrilyuk EV. Pharmacological technologies and modern medicine. correction of immunometabolic disturbances Pathological physiology and experimental geptraly and meksikory at animals against the therapy [Patologicheskaja fiziologija i background of an ischemic lesion of a liver. jeksperimental'naja terapija]. 2004;4:2-11. Scientific sheets of the Belgorod state 4. Razumova MS., Litvinova ES, university. Series: Medicine. Pharmaceutics Kharchenko AV, Dudka VT, Konoplya AI. [Nauchnye vedomosti Belgorodskogo Morphological liver changes under gosudarstvennogo universiteta. Serija: experimental acute toxic hepatopathy and their Medicina. Farmacija]. 2012;20(22-1):179-182. pharmacological correction. Morphology (In Russian) [eLIBRARY] [Full text] [Morfologiya]. 2017;151(3):99. (In Russian) 11. Burda YU, Konoplya AI, Ershov DV. [eLIBRARY] Effect of a complex of humoral factors 5. Yuyou D, Andreea C, Ying M, Naoki produced by fibroblasts on the adhesive ability Y, Songqing H, Sanjeev G, Sanjiv SG, Mark of inflammatory cells in vitro. Modern high A., Zern MD. Differentiation and enrichment of technologies [Sovremennye naukoemkie hepatocyte-like cells from human embryonic tehnologii]. 2005;4:86-88. (In Russian) stem cells in vitro and in vivo. Stem. Cells. [eLIBRARY] [Full text] 2007;25(12):3058-3068. [PubMed]

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Razumova M.S., Litvinova E.S., Gavriliouk V.P. Study of pharmacological activity in sublimated culture liquid of allogenic, xenogeneic hepatocytes and fibroblasts for the correction of liver 131 damages in carbon tetrachloride intoxication. Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):120-131. 12. Roskams T, Katoonizadeh A, Komuta 20. Valdes F. lvarez AM, Locascio A, M. Hepatic progenitor cells: an update. Clin Vega S, Herrera B, Fernández M, Benito M, Liver Dis. 2010;14(4):705-718. [PubMed] Nieto MA, Fabregat I. The epithelial 13. Ivanova EU, Stolyarevich EU, mesenchymal transition confers resistance to Gichkun OE, Bogdanova NB, Baranova FS, the apoptotic effects of transforming growth Artiukhina LY, Tomilina NA. Analysis of factor beta in fetal rat hepatocytes. Mol. screening methods for antibodies to HLA in the Cancer. Res. 2002;1(1):68-78. [PubMed] diagnosis of antibody mediated rejection of a 21. Baptista PM, Siddiqui MM, Lozier G, kidney transplant. Bulletin of Transplantology Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the and Artificial Organs [Vestnik transplantologii generation of a vascularized liver organoid. i iskusstvennyh organov]. 2016;18(3):39-49. Hepatology. 2011;53(2):12-18. [PubMed] (In Russian) [eLIBRARY] [Full text] 22. Razumova MS, Litvinova ES, 14. Paqan R, Martín I, Llobera M, Vilaró Kharchenko AV, Bystrova NA, Gavrilyuk VP. S. Epithelial-mesenchymal transition of Immune and metabolic effects of culture fluids cultured rat neonatal hepatocytes is of allogeneic hepatocytes, essentiale and differentially regulated in response to hypoxen in acute toxic liver injury. Journal of epidermal growth factor and dimethyl Scientific Articles "Health and Education in the sulfoxide. Hepatology. 1997;25(3):598-606. 21st Century" [Zhurnal nauchnykh statej [PubMed] «Zdorov`e i obrazovanie v XXI veke»]. 15. Jackson DA, Elsawa SF. Factors 2016;18(12):117-119. (In Russian) Regulating Immunoglobulin Production by [eLIBRARY] [Full text] Normal and Disease-Associated Plasma Cells. Biomolecules. 2015;5:20-40. [PubMed] Author Contributors 16. Anishchenko VV, Trubacheva AV, Marina S. Razumova, Postgraduate Razumakhina M.S. The basic calculated Student, Department of Pathological Anatomy, characteristics of the electric signal of pancreas Kursk State Medical University, е-mail: at modelling of acute pancreatitis by ethyl [email protected]. Collection, analysis and alcohol. Journal of Siberian Medical Sciences interpretation of data for publication. [Medicina i obrazovanie v Sibiri]. 2012;1:53- Ekaterina S. Litvinova, Candidate of Medical Sciences, Associate Professor, 60. (In Russian) [eLIBRARY] [Full text] Department of Pathological Anatomy, Kursk 17. D‖Amour KA, Agulnick AD, Eliazer State Medical University. E-mail: S, Kelly OG, Kroon E, Baetge EE. Efficient [email protected]. Contribution to the concept differentiation of human embryonic stem cells and design of the work. to definitive endoderm. Nat. Biotechnol. Vassili P. Gavriliouk, Doctor of Medical 2005;23:1534-1541. [PubMed] Sciences, Head of the Department of of 18. Youchev MI, Grozdanov PN, Zhou H, Children's surgery and pediatrics of Faculty of Racherla H, Guha C, Dabeva MD. Postdegree Education, Kursk State Medical Identification of adult hepatic progenitor cells University, e-mail: [email protected]. Critical capable of repopulating lnjured rat liver. analysis of intellectual content. Hepatology. 2008;47:636-647. [PubMed] 19. Mo Lee H, Kwon UH, Kim H, Kim HJ, Kim B, Park JO, Moon ES, Moon SH. Received: October, 05, 2017 Pulsed Electromagnetic Field Stimulates Accepted: November, 30, 2017 Cellular Proliferation in Human Intervertebral Available online: December, 30, 2017 Disc Cells. Yonsei Med. J. 2010;51(6):954- 959. [PubMed]

Rus.

UDC: 615.225: 617.735-007.23 DOI: 10.18413/2313-8971-2017-3-4-132 -150

Alla P.Tarasova1 EFFECT OF PHARMACOLOGICAL PRECONDITIONING Lyudmila M. Danilenko WITH INCRETINOMIMETICS EXENATIDE AND Lev N. Sernov VILDAGLIPTIN ON THE SURVIVAL OF ISCHEMIC TISSUES

Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russia Corresponding author, 1e-mail: [email protected]

Abstract Introduction: Currently, much attention is paid to the pleiotropic effects of entretenimiento. Purposes: Study of the protective effect exenatide and valdiation with pharmacological correction of ischemic myocardial damage, damage of liver and skin graft during the experiment. Methods: During the experimental study we used a comprehensive approach to the study of the antiischemic effects of entretenimento: doksorubitsinola model of cardiomyopathy, hypo/reperfusion of the isolated heart, ischemia/reperfusion of the liver and the modeling of the skin flap on the supply leg. Results and discussion: Exenatide (10 mcg/kg/day) and vildagliptin (0.2 mg/kg/day) demonstrate a cardioprotective effect on doxorubicinol model of pathology that is reflected in the decline in the rate of diastolic dysfunction (StТТI), respectively, to 5.3±0.1 standard units. and 6.5±0.2 standart units in comparison with the control group 8.3±0.1 standart units in the model hypo/reperfusion of the isolated hearts of rats, exenatide (10-6 mol/l) and vildagliptin (10-4 mol/l), prevent the decrease of left ventricular pressure (LG). Exenatide (10 µg/kg) and vildagliptin (0.2 mg/kg) prevent necrotization of the skin flap 1.5 and 1.3 times in comparison with the control group. In the model of ischemia/reperfusion of the liver exenatide possess dose related hepatoprotective effect. All protective effects of entretenimiento leveled combined with a blocker of ATP-sensitive potassium channels glibenclamide (0.4 mg/kg). Conclusion: During the study it was found that exenatide dose of 10 µg/kg/day and vildagliptin dose of 0.2 mg/kg, have a pronounced cardioprotective, hepatoprotective, and a pronounced cytoprotective effect on a model of isolated skin flap on the supply leg. ATP-dependent potassium channels are effector mechanism in the implementation of the protective effects of entretenimiento. Keywords: entretenimiento, exenatide, vildagliptin, doksorubitsinola cardiomyopathy, isolated heart of rats, ischemia.

Introduction of increase of the risk of cardiovascular disease New substances with cardiotropic effects (CVD) in patients with T2DM when using are being identified among various classes of drugs incretin series[9, 10, 11]. Furthermore, chemical and pharmacological groups [1, 2, 3, the results of experimental and clinical studies 4, 5, 6].Currently aspects of cardiovascular highlight the potential cardioprotective safety are a priority when choosing individual properties of these drugs [12, 13]. tactics of patients with type 2 diabetes mellitus Accumulated results in recent experimental (T2DM) [7, 8]. It is possible to assume the lack and clinical studies allow to speak about

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Tarasova A.P., Danilenko L.M., Sernov L.N. Effect of pharmacological preconditioning with incretinomimetics exenatide and vildagliptin on the survival of ischemic tissues. Research 133 Result: Pharmacology and Clinical Pharmacology. 2017;3(4):132-150. pleiotropic effects of entretenimiento [14]. The from the Laboratory animal nursery receptors of GLP-1 are detected in endothelia "Stolbovaya" of Governmental Institution The and cardiomyocytes, monocytes, macrophages, Scientific Center of Biomedical Technologies neurons, bone tissue, adipose tissue and other Russian Academy of Medical Sciences target organs [15, 16]. (Moscow region). The contents and their care The great interest represents the study of were carried out according to the cardioprotective and antiischemic effects of recommendations of State Standard – 53434- entretenimiento exenatide and vildagliptin. The 2009 "Principles of good laboratory practice" exact mechanisms underlying their effects on International guidelines "of the European different organs and tissues, are still not Convention for the protection of vertebrate established [13, 15, 17, 18, 19]. The discussed animals used for experimental and other effect is the one of ischemic preconditioning, scientific purposes" (The European where the implementation of effects can be Convention, 1986). All the experiments were done through opening of mitochondrial K-ATP approved by the local Ethics Committee channels, which can be a state trigger (Protocol No. 12-2014 dated 21st January preconditioning [20]. The evidence of the 2015). activation of antiapoptotic pathways as one of Modeling of doxorubicinol the possible mechanisms of the cardiomyopathy cardioprotective effect of early ischemic phase During the simulation of the doxorubicinol of preconditioning are obtained. The most cardiomyopathy, all the rats were divided into 4 likely is the protective effect of peptide growth experimental groups of 10 animals. The first factors including insulin, insulin-like growth group (n=10) the control one, which was factor 1, transforming growth factor beta-1, injected intraperitoneally with physiological cardiotrophin-1 and growth factors of solution. The second group (n=10) was injected fibroblasts, caused by the relatively reperfusion intraperitoneally with doxorubicin (Teva) in a injury of the myocardium associated with cumulative dose of 20 mg/kg, once. The third apoptosis through inhibition of P42/P44 MAR- one (n=10) was injected doxorubicin and kinazy and the P13-channals/Аkt-signaling [21, intraperitoneal vildagliptin ("Galvus", Novartis, 22, 23, 24]. The pharmacological Switzerland) at a dose of 0.2 mg/kg/day. The preconditioning with incrimination can also be fourth group (n=10) was injected doxorubicin realized via NO/cGMP-dependent mechanism and exenatide subcutaneously once per day which is also involved in cardioprotective ("Beta", Eli Lilly and Company, USA) at a effects of entretenimiento [25, 26]. dose of 10 mg/kg/day. The doses were It is anticipated that GLP-1 may also have calculated considering the coefficient of a positive impact on oxidative stress and interspecies transfer of doses of the human endogenous antioxidant defense mechanisms, body on the body of the rat. Animals were through the increased expression of heme- taken out from experiment after 48 hours. Their oxygenase-1(HO-1) [27, 28], while having a hearts were removed under zolotilova positive impact in myocardial cardioprotection anesthesia (30 mg/kg) and placed in the ice (2- [29, 30]. 4°C) solution of Krebs-Henseleit the following Purposes: The study of the protective composition (mmol): NaCl 118.5; KCl – 4.7; effect of pharmacological preconditioning with MgSO4/7Н2О – 1.2; KH2PO4 – 1.2; CaCl2 – incrimination exenatide and valdiation when 1.5; glucose, 11.1; NaHCO3 -25.0. The pH of pharmacological correction of ischemic the solution during the whole experiment was myocardial damage, the damage of liver and 7.4. The aorta of the heart was coulibalied and skin graft in the experiment. produced by retrograde perfusion of the heart method of Langendorff in the mode of flow Methods perfusion for 20 min with a solution of Krebs- The experiments were performed on male Henseleit, saturated with Carbogen (95 % O2 + and female rats of Wistar line weighing 200- 5% CO2) at 37°C and at a pressure of 100 mm 250 g . The experimental animals are obtained Hg and speed perfusate 10 ml/min. The December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Tarasova A.P., Danilenko L.M., Sernov L.N. Effect of pharmacological preconditioning with incretinomimetics exenatide and vildagliptin on the survival of ischemic tissues. Research 134 Result: Pharmacology and Clinical Pharmacology. 2017;3(4):132-150. contractile function of the heart was recorded effect by the dynamics of indices of with the help of the inserted into the cavity of contractility and reperfusion fibrilace. the left ventricle a latex balloon connected to a Hepatotropic antiischemic activity pressure sensor embedded in the device for The study of hepatotropic antiischemic physiological studies МР150 company activity entretenimiento was carried out in "BiopacSystems, Inc" (California, USA). The anesthetized animals, performing median balloon was filled with distilled water, the laparotomy on the white line of the abdomen. volume of which was sufficient to create end- Ischemia was simulated for 15 minutes. After diastolic pressure in the left ventricle at the that, the contents of the abdomen were laid level of 3-5 mm Hg. Using the original back, and the operative wound was sutured in software program, AcqKnowledge company layers. The animals for 3 days were "BiopacSystems, Inc" (California, USA), all administered exenatide (10 mcg/kg/day) and rats were checked for the indices of vildagliptin (0.2 mg/kg/day) control group with contractility: left ventricular pressure (LG, mm 0.9 % solution of sodium chloride Hg), heart rate (HR, beats/min), the maximum intraperitoneally. rate of contraction (+dp/dtmax, mm Hg/sec), At the end of 3 days after the experiment the maximum speed of myocardial relaxation (- the animals were euthanized with subsequent dp/dtmax, mm Hg.St./sec). To create a high blood sampling from the heart for biochemical frequency (480 beats/min.) a connector (ground analysis and selection of the whole liver to electrical stimulator) was attached to a to the conduct morphological analysis. To assess the metallic cannula, and a left atrial appendage liver function there were chosen biochemical was joined by a connector. After 20 minutes of markers of damage to hepatocytes of ALT and perfusion with a solution with a high content of AST. To assess structural changes there were Ca2+(5 mmol/l), the heart was subjected to made histological sections of specimens of electrical stimulation pulses using the STM liver of the experimental rats with subsequent device 200-1 of the company "BiopacSystems, morphological assessment [33]. Inc" (California, USA) for 15 seconds. Modeling of the skin flap on the supply To assess the functionality of the leg myocardium, the diastolic dysfunction ratio or The possibility of optimizing the survival "diastole defect" (StTTI) calculated from the of the tissues was investigated in a model of intraventricular pressure curve was used. The isolated skin flap on the supply leg. All animals area under the curve was calculated by folding were performed the modeling of the skin flap the trapezium areas, which is equal to the on the leg on the second day of the experiment. product of its height on the middle line. After anesthesia the animals were fixed in "diastole defect" (StTTI) expressed in the us supine position. The hair on the abdomen was [31]. carefully cut out, the skin was treated with 70% Integrated assessment of myocardial solution of ethyl alcohol. Margins of 1 cm from damage in doxorubicinol cardiomyopathy in the xiphoid process on the white line of the animals have identified the isoenzyme CPK abdomen, made the skin flap 1cm – base, 4 cm (CPK-MB) and lactate dehydrogenase (LDH). long, (keeping the supply vessel) insulated in a The activity of lipid peroxidation (LPO) was plastic bag, the edges of the skin sutured with a evaluated by the content of malondialdehyde continuous suture. The estimation of the area of (MDA), diene conjugates (DC). the surviving tissue was performed Hypo-reperfusion in isolated heart of planimetrically on the fifth day. Further we rats calculated the survival rate (ratio of the area of In еру experiments on perfused according the surviving fabric to the original square flap to Langendorf isolated rats‘ hearts we ×100%) [34]. simulated hypo – and reperfusion injury (hypoperfusion a 10 – fold decrease in Results and Discussion perfusion with normal content of Ca2+ (2.5 Dynamics of functional, biochemical and mmol/l) [32]. It was judged about the damaging morphological indicators in the simulation December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Tarasova A.P., Danilenko L.M., Sernov L.N. Effect of pharmacological preconditioning with incretinomimetics exenatide and vildagliptin on the survival of ischemic tissues. Research 135 Result: Pharmacology and Clinical Pharmacology. 2017;3(4):132-150.

doxorubicinol cardiomyopathy, minute for 15 seconds on the background of hypo/reperfusion of the isolated heart, increasing concentrations of Ca2+ and 5 mmol ischemia/reperfusion of the liver and the in perfusate on the isolated heart of rats as an modeling of the skin flap on the supply leg additional criterion of assessment of For the evaluation of cardioprotective cardioprotective actions of drugs used ratio actions of pharmacological agents, including StТТI reflecting"defect diastole" area under the the modeling of the pathology of curve of the rise of end-diastolic pressure. cardiomyopathy by administration of While intact rats have StTTI is 1.4±0.1 standard doxorubicin intraperitoneally at a dose of 20 units, and in rats with modeling of the mg/kg, 48 hours later, the assessment of pathology of 8.3±0.3 standard units parameters of left ventricular contractility in (Fig 1.) conditions of high rate reductions 480 beats per

140 120 100

80 60 mmHg 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 sec a 200 180 160

140 120 100 mmHg 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 sec b Fig. 1. Dynamics of pressure in the left ventricle (mm. Hg.) with the imposition of rapid heart rhythm contractions (480 BPM) for 15 seconds. The concentration of Ca2+ in perfusate 5 mmol/L. Intact group (a), doxorubicin (20 mg/kg) (b)

The fundamental difference in the area the necessity of introducing a factor StТТI, under the curve of the rise of end-diastolic which is quite revealing and informative. pressure in the intact group and the control on For a comprehensive evaluation of the background of doxorubicin, naturally led to myocardial damage it was determined by the

IU/L IU/L ** 800 ** 1600 700 1400 600 1200 500 1000 400 800 300 600 200 * 400 * 100 200 0 0 Intact Control Intact Control CPK-MB LDH

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Fig. 2. The content of creatine phosphokinase CPK-MB, LDH, MDA, DK, in the model gypo/reperfusion in isolated hearts of rats. Note: * p< 0.05 compared with control.** – p< 0.05 compared with intact animals

Investigation of the stability of the values. Restore the original volume of myocardium to ischemia/reperfusion damage perfusion (reperfusion) was accompanied by were studied on the model of gypo/reperfusion the development of reperfusion arrhythmias in isolated heart of rats under register pressure that in 3 cases out of 10 lead to fibrillation. in the left ventricle (LG). In studying the At the 5th min of reperfusion LG, cardioprotective activity in the model Hypo- +dP/dtmax, -dP/dtmax remained below the initial and reperfusion was discovered that a decrease level. A similar falling trend in the parameters in perfusion in 10 times (coronary of contractility was preserved and further to the hypoperfusion) occurs a marked drop in heart 20th min of reperfusion, where LG, +dP/dtmax, rate and contractility indices during the first -dP/dtmax were less than half the initial value 5min.By the 20th min of hypoperfusion HR, (tab. 1). LR,+dP/dtmax, -dP/dtmax were below the initial

In the simulation laparotomy, there were ischemia with subsequent reperfusion of the statistically significant indicators of liver liver statistically significantly raised levels of enzyme values. The imposition of a 15-minute transaminases in average 5 times (tab. 2). Table 1 Functional parameters of myocardium in gypo/reperfusion in isolated heart of rats (%of initial level) (M±m; n=10)

Hypoperfusion time Reperfusion time Indicators The outcome 5 minutes 20 minutes 5 minutes 20 minutes Heart rate, heartbeats 224.3±8.4 -64.6±3.2 -58.3±2.3 -16.6±10.3 -10.8±16.1 per min LG, mm Hg. 120.0±9.8 -65.8±7.5 -69.3±9.1 -19.8±8.1 -19.5±14.1 + dp/dtmax, mm Hg. 2498±4.7 -76.7±2.5 -78.8±1.5 -19.8±14.1 -15.0±19.0 - dp/dtmax, mm Hg. -1346±1.8 -71.6±2.0 -74.8±10.2 -36.6±10.4 -55.2±12.1 Note: LG – left ventricular pressure (mm. Hg.); +dp/dtmax maximal rate of reduction (mm Hg/sec); -dp/dtmax maximal rate of relaxation (mm Hg/h); HR – heart rate (beats/min).* – p<0.05 in comparison with the control group. Table 2 Indicators of the level of transaminases in the simulation of Ieremii/reperfusion of the liver (U/l) (M±m; n=10) Group of animals ALT AST Intact 68.1±13.8** 102.8±8.9** Lineameriloan 72.8±12.3** 110.2±10.3** Control 216.9±16.8*1 189.1±13.2*1 Note: *–p < 0.05 compared with the group of intact animals; **– p < 0.05 in comparison with the false-operated group; 1– p>0,05 in comparison with the control group.

According to the study design, the we calculated the survival rate (ratio of the area assessment of the degree of survival of the skin of the surviving fabric to the original square flap on the leg was performed at 3, 7, 10 a day, flap 100 %. The result revealed that the area of then planimetrically after Avtandilov, necrosis on the 10 day increases by 2.5 times measuring the area of surviving tissue. Further (table. 3). Table 3 Survival rates of the isolated skin flap on the supply leg at 3, 7, 10 days after its modeling. (M±m; n=10) Drugs and their The area of necrosis KL, % dosages 3 day 7 day 10 day Saline (Control) 30.98±2.84 65.76±2.64 84.55±3.23 Note:* – p<0,05 in comparison with the control group

Thus, doksorubitsinola model of in the model of isolated skin flap on the cardiomyopathy, Hypo/reperfusion in isolated supply leg from exenatide and vildagliptin hearts of rats, ischemia/reperfusion of the liver Entretenimiento exenatide at doses and the modeling of the skin flap on the supply (1 mg/kg/day and 10 mg/kg/day) and leg are easily repeatable and informative vildagliptin (0.02 and 0.2 mg/kg/day) did not models to assess of protective effects of drugs affect the degree of decrease of contractility in ischemic tissues. (table. 4). 2. The study of cardioprotective, However, dose-dependently we prevented hepatoprotective, and cytoprotective effect the decrease in contractility during the tests

Table 4 The influence of entretenimiento exenatide and vildagliptin on the performance of the contractile function of the hearts of rats under doxorubicinol cardiomyopathy (M±m; n=10) Groups of animals LG +dp/dtmax -dp/dtmax HR IntactAnimals 87.3±9.2* 1423±162.2* -1265.2±173.2* 248±32.1 Control Doxorubicin 64.5±11.2** 1025.7±154.3** -1031.1±159.4** 247±29.4 (20mg/kg) Doxorubicin (20mg/kg)+ 60.2±9.4** 1165,7±134.3** -1109.9±119.4** 232±29.4 Exenatide (1 µg/kg/day.) Doxorubicin(20mg/kg)+ 76.8±7.4* 1302±169.2* -1157.4±137.3* 231±26.9 Exenatide (10mcg/kg/day.) Doxorubicin (20mg/kg) + 59.1±10.7** 1107,7±15.,3** -984.9±129.1** 227±29.4 Vildagliptin(0.02 mg/kg/day.) Doxorubicin (20mg/kg)+ 73.2±51* 1219±145.4* -1108±169.3* 232±36.1 Vildagliptin (0.2 mg/kg/day.) Note: the LG – left ventricular pressure (mm. Hg); +dp/dtmax maximal rate of reduction (mm Hg/sec); -dp/dtmax maximal rate of relaxation (mm Hg/h); HR – heart rate (beats/min). Doxorubicin was administered intraperitoneally, 48 hours before the experiment. Entretenimiento and vildagliptin exenatide were administered twice with an interval of 24 hours, subcutaneously and intraperitoneally, respectively, * * p<0.05 compared with the group of intact animals;* – p<0.05 in comparison with control group.

9 StTTI 8.3** 8 7 6.5* 6 5.4** 5 4 3 1.4* 2 1 0 Intact Control Exenatade (10 Vildagliptin mcg/kg/day) (0.2 mg/kg/day) Fig. 3. The impact exenatide (10 mg/kg/day) and vildagliptin (0.2 mg/kg/day) for the coefficient of diastolic dysfunction (StТТI), with doxorubicinol cardiomyopathy. Note: ** p<0.05 compared with the group of intact animals; * – p<0.05 in comparison with control group

-6 -4 The values StТТI for exenatide at a dose of 1 10 mmol/l and vildagliptin 10 mmol/l, mcg/kg/day and vildagliptin 0.02 mg/kg/day were statistically znachimyh changes LG, +dP/dtmax, not significantly different from the control group. -dP/dtmax and heart rate in the period of During the study of cardioprotective hypoperfusion to 20 min in comparison with activity of incrimination on the model of the control was not detected. In the period of Hypo/reperfusion it discovered that the addition reperfusion, both entretenimiento prevented the of perfusiology solution exenatide at a dose of fall in contractility and on the 5th and 20th min December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Tarasova A.P., Danilenko L.M., Sernov L.N. Effect of pharmacological preconditioning with incretinomimetics exenatide and vildagliptin on the survival of ischemic tissues. Research 139 Result: Pharmacology and Clinical Pharmacology. 2017;3(4):132-150.

differed little from the original values, whereas In addition, unlike the control, the number of in control, they fell by more than 50% (tab. 5). reperfusion arrhythmias decreased markedly. Table 5 Cardioprotective action exenatide (10-6 mmol/l) and vildagliptin (10-4 mmol/l) with Hypo- reperfusion in isolated heart of rats (%of initial level)(M±m; n=10) Group of Hypoperfusion time Reperfusion time Indicators Outcome animals 5 minutes 20 minutes 5 minutes 20 minutes Heart rate, Control 224.3±8.4 -64.6±7.2 -58.3±8.3 -16.6±10.3 -10.8±14.1 heartbeats Exenatide 233.0±7.3 -3.2±6.4* -53.5±5.3 +1.4±9.1* +2.8±13.7 * per min Vildagliptin 221.3±7.8 -45.3±4.9 * -54.6±7.2 -8.9±6.2* -3.2±9.1 * LG, mm Hg Control 120.0±9.8 -65.8±7.5 -69.3±9.1 -19.8±8.1 -19.5±14.1 Exenatide 116.8±7.1 -78.7±9.3 -62.6±8.8 -8.1±10.2* -1.8±11.2* Vildagliptin 114.0±11.1 -67.0±8.7 -61.1±9.2 -6.4±9.4 * -1.4±10.0* + dp/dtmax, Control 2498±4.7 -76.7±2.5 -78.8±6.5 -19.8±11.1 -15.0±12.0 mm Hg Exenatide 2309±7,5 -77.6±7.9 -75.7±10.1 -12.9±9.4* +5.4±9.2* Vildagliptin 2325±6.9 -78.6±9.4 -76.8±5.5 -10.8±8.8* +3.5±8.7* - dp/dtmax, Control 1346±1.8 -71.6±5.0 -74.8±10.2 -36.6±10.4 -55.2±12.1 mm Hg Exenatide 1345±2.4 -69.1±9.7 -69.6±9.2* -12.9±7.9* +3.7±11.0* Vildagliptin 1265±5.7 -67.3±6.8 -70.5±7.4 -10.8±9.4* +4.5±7.9* Note: The LG – left ventricular pressure (mm. Hg.St); +dp/dtmax maximal rate of reduction (mm Hg./sec); -dp/dtmax maximal rate of relaxation (mm Hg./h); HR – heart rate (beats/min).* – p<0,05 in comparison with the control group.

The ability of exenatide 10 µg/kg/day and and vildagliptin contributed to the decline in vildagliptin 0.2 mg/kg/day to prevent damage levels of creatine kinase-MB by 27% and 19% to cell membranes was estimated by the change and LDH of 11.8% and 9.6% relative to the in the activity of ck-MB and LDH. Exenatide control group (figure 4).

900 800 ** 2000 700 ** 600 500 * 1500 * 400 * 1000 * 300 200 * 500 * 100 0 0 Intact Control Exenatade Vildagliptin Intact Control Exenatade Vildagliptin (10 (0.2 (10 (0.2 mcg/kg/day) mg/kg/day) mcg/kg/day) mg/kg/day) LDH CPK-MB Fig. 4. The content of creatine phosphokinase CPK-MB and LDH in groups with exenatide (10 mg/kg/day.) and vildagliptin (0.2 mg/kg/day). Note: ** p<0.05 compared with the group of intact animals; * – p<0.05 in comparison with control group Similar changes were detected in the The correction of the ischemic liver marker of the products of lipid peroxidation damage by exenatide (10 mg/kg/day) and (figure 5). vildagliptin (0.2 mg/kg) reduces the level of

4 0,6

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2,5 * * DC * * ( 2 0,3 /g of tissueof /g

1,5 tissue of

0,2 1 g 232/ conjugates nmole 0,1

0,5 ∆Е

Malondialdehyde 0 Diene 0 Intact Control Exenatade (10 Vildagliptin Intact Control Exenatade Vildagliptin mcg/kg/day) (0.2 (10 (0.2 mg/kg/day) mcg/kg/day) mg/kg/day) Fig. 5. The contents of MDA and DC in homogenate in groups with exenatide (10 mg/kg/day.) and vildagliptin (0.2 mg/kg/day) Note: ** p<0.05 compared with the group of intact animals; * – p<0.05 in comparison with control group

Table 6 Indicators of the level of transaminases in modeling of schemia/reperfusion of the liver (U/l) (M±m; n=10)

Group of animals ALT AST Intact 102.89±8.9** 68.1±13.8** Lineameriloan 110.2±10.3** 72.8±12.3** Control 189.1±13.2*1 216.9±16.8*1 Exenatide (10 µg/kg/day) 130.2±10.9** 107.1±11.7** Vildagliptin (0.2 mg/kg) 157.2±11.4** 145.8±13.4** Note: *– p < 0.05 compared with the group of intact animals; **– p<0,05 in comparison with the false-operated group; 1 – p<0,05 in comparison with the control group.

The same trend is confirmed by the group at 3, 7 and 10 day. In the control group, morphological picture of the liver (figure 6). the amount of necrosis amounted to When modeling the skin flap on the supply 84.55±3.23%, in the group with exenatide and leg, both studied entretenimiento contributed to vildagliptin 63.18±2.42 and 76.08±2.53, the reliable correction to increase the area of respectively. surviving tissue in comparison with the control

a b Fig. 6. Morphological picture of the liver in a series of false-operated animals (a) and control group (b) micro photos. X400

Thus, on the totality of functional, The prior blockade of the ATP-sensitive biochemical and morphological indicators, potassium channels using intraperitoneal entretenimiento exenatide (10 mg/kg/day) and injection of glibenclamide dose of 5 mg/kg on vildagliptin (0.2 mg/kg) has a pronounced the background modeling of the doxorubicinol cardioprotective, hepatoprotective, and cardiomyopathy, applications exenatide and cytoprotective effects on the studied models of vildagliptin led to the leveling effects of pathologies. cardioprotection. The ratio of diastolic 3. Defining of the role of ATP dependent dysfunction StTTI in the group of animals potassium channels in the implementation of treated with glibenclamide and exenatide and the protective effects of entretenimiento vildagliptin weren‘t significantly different from The most likely effector link cytoprotective the control group and amounted to 7.5±0.6 and in the phenomenon of ischemic preconditioning 7.9±0.7 standard unit (figure 7). are the ATP-dependent potassium channels.

StTTI 9 8.3** 8 7.5** 7.9** 7 6.5* 6 5.4* 5 4 3 1.4* 2 1 0 Intact Control Exenatade Exenatade Vildagliptin Vildagliptin (10 (10 (0.2 mg/kg/day) (0.2 mg/kg/day) mcg/kg/day) mcg/kg/day) + + Glibenclamide Glibenclamide (0.4 mg/kg) (0.4 mg/kg) Fig. 7. The impact of the exenatide and vildagliptin on a factor of diastolic dysfunction (StТТI), with doxorubicinol cardiomyopathy. Note: ** p<0.05 compared with the group of intact animals; * – p<0.05 in comparison with control group

Cardioprotective effects of incrimination Hypo/reperfusion in isolated hearts of rats. The were leveled with the introduction of the level of biochemical markers and products of perfusate glibenclamide (10-6ммоль) fashion lipid peroxidation didn‘t reduce (figure 8).

Vildagliptin (0.2 mg/kg/day) + Glibenclamide (0.4 mg/kg) **

Vildagliptin (0.2 mg/kg/day) *

Exenatade (10 mcg/kg/day) + Glibenclamide (0.4 mg/kg) **

Exenatade (10 mcg/kg/day) *

Control **

Intact *

0 200 400 600 800

CPK-MB IU/L

Vildagliptin (0.2 mg/kg/day) + Glibenclamide (0.4 mg/kg) **

Vildagliptin (0.2 mg/kg/day) *

Exenatade (10 mcg/kg/day) + Glibenclamide (0.4 mg/kg) **

Exenatade (10 mcg/kg/day) *

Control **

Intact *

0 500 1000 1500 2000 LDH IU/L

Vildagliptin (0.2 mg/kg/day) + ** Glibenclamide (0.4 mg/kg)

Vildagliptin (0.2 mg/kg/day) *

Exenatade (10 mcg/kg/day) + ** Glibenclamide (0.4 mg/kg)

Exenatade (10 mcg/kg/day) *

Control **

Intact * 0 1 2 3 4

Malondialdehyde (MDA) nmole/g of tissue

Vildagliptin (0.2 mg/kg/day) + ** Glibenclamide (0.4 mg/kg)

Vildagliptin (0.2 mg/kg/day) *

Exenatade (10 mcg/kg/day) + ** Glibenclamide (0.4 mg/kg)

Exenatade (10 mcg/kg/day) *

Control **

Intact * 0 0,1 0,2 0,3 0,4 0,5 0,6

Diene conjugates (DC) ∆Е232/g of tissue

Fig. 8. The content of CPK-MB, LDH, MDA, DK in the exenatide group (10 mg/kg/day.) and vildagliptin (0.2 mg/kg/day) in the model of Hypo/reperfusion in isolated hearts of rats. Note: ** p<0.05 compared with the group of intact animals; * – p<0.05 in comparison with control group

Entretenimento didn‘t show the the control group. That shows the lack of effect pronounced hepatoprotective actions in the pre- on the background of administration of blockade of ATP-sensitive potassium channels. glibenclamide (5 mg/kg) (table 7). The values of ALT and AST is comparable to

Table 7 Protective action of exenatide and vildagliptin on transminase on the background of glibenclamide during ischemia/reperfusion of the liver (U/l). (M±m; n=10) Group of animals ALT AST Intact 68.1±13.8** 102.89±8.9** Lineameriloan 72.8±12.3** 110.2±10.3** Control 216.9±16.8*1 189.1±13.2*1 130.2±10.9** 127.1±11.7** Exenatide 10 µg/kg/day Exenatide 10мкг/kg/day 211.3±15.1** 175.6±13.2** +Glibenclamide 0.4 mg/kg 157.2±11.4** 145.8±13.4** Vildagliptin 0.2 mg/kg Vildagliptin 0.2 mg/kg+ 202.90±16.9 169.4±8.1 Glibenclamide 0.4 mg/kg Note: *–p < 0.05 compared with the group of intact animals; **– p<0.05 in comparison with the false-operated group; 1 – p<0.05 in comparison with the control group.

The prior blockade of ATP-sensitive modeling of a skin graft on feeding pedicle potassium channels, completely negate the in rats. protective effect of entretenimiento when

Table 8 Protective action of exenatide and vildagliptin on the background of glibenclamide survival of skin graft (%). (M±m; n=10) The area of necrosis KL, % Drugs and their dosages 3 day 7 day 10 day Saline (control) 30.98±2.84 65.76±2.64 84.55±3.23 Exenatide (10 µg/kg/day) 24.37±3.12* 44.13±3.15* 63.18±2.42* Exenatide (10мкг/kg/day) + Glibenclamide (0.4 29.18±2.04 59.16±2.53 81.35±3.20 mg/kg) Vildagliptin (0.2 mg/kg) 26.10±2.83* 52.19±3.00* 76.08±2.53* Vildagliptin (0.2 mg/kg) + 33.22±2.12 60.71±2.59 83.51±3.13 Glibenclamide (0.4 mg/kg) Note: *- p<0.05 in comparison with the control group

Nowadays, there is a large amount of data became the basis for the study of its on the impact of GLP-1 and DPP on the cardiovascular effects [35, 36]. The receptors cardiovascular system and various organs and of GLP-1 (along with receptors for glucagon, tissues, the mechanisms of these effects differ secretin, calcitonin, somatoliberin, parathyroid from those mediated by the decrease of hormone, vasointestinal peptide) belong to the glycemia. The receptors of GLP-1 are widely class of the family of receptors associated with represented in the body and, in addition to G-proteins (GCPR) [37]. In terms of the gastrointestinal tract, nervous system, lungs, damage of ischemia—reperfusion kidneys, lymphocytes were found also in cytoprotection is mainly due to the anti- vascular smooth muscle cells, cardiomyocytes, apoptotic effect. GLP-1 binding to the receptor, endocardium and endothelial cells, which suppresses apoptosis of β-cells and

Fig. 9. Schematic representation of the proposed pathways by which GLP-1 may exert its cardiovascular actions. The combination of GLP-1 effects on the myocardium (i.e. apoptosis and necrosis prevention in cardiomyocytes through the activation of the RISK pathway, increased glucose metabolism, vasodilatory and anti-inflammatory actions) with GLP-1 metabolic and vascular effects at the systemic level contributes to cardiac survival and function improvement Note: cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; Cyt c, cytochrome c; DPP-4, dipeptidyl peptidase-4; ERK, extracellular signal-regulated kinase; GLUT, glucose transporter; GSK, glycogen synthase kinase; LDH, lactate dehydrogenase; MEK1/2, MAP kinase kinase; MPTP, mitochondrial permeability transition pore; NOS, nitric oxide synthase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; ROS, reactive oxygen species

However, the effects on the receptors /reperfusion of the liver (15 min) leads to an GLP-1 and inhibition of DPP-4 require the increase in transaminases for 5 times (ALT and further study. AST) and the characteristic dynamics of Conclusion morphological patterns. The survival rate of Thus, in the course of the study it was skin flap on the leg are, respectively, 50, 60 and revealed that doksorubitsinola cardiomyopathy 70%. The proposed methods allow to provide a (20 mg/kg, intraperitoneally for 48 hours) leads comprehensive evaluation of effects of to the development of «the defect of diastole» pharmacological agents on the survival of increasing the area under the curve of left ischemic tissues. Exenatide (10 µg/kg) and ventricular pressure 8 times. gypo/reperfusion vildagliptin (0.2 mg/kg) exert a (10:1, 20 min) in isolated hearts of rats leads to cardioprotective effect in the models of a decrease in LG by 2 times. Ischemia doxorubicinol cardiomyopathy, expressed in a

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Research results in Pharmacology. 2017;3(4):Х-Х!!!! Rus. UDC: 615.014.22:616.5-003.923 DOI: 10.18413/2313-8971-2017-3-4-151 -159

Andrey V. Voronkov, THE EFFECT OF NEW FORMS FOR EXTERNAL APPLICATION Olesya Yu. Gamzeleva1 ON THE VASODILATING FUNCTION OF THE ENDOTHELIUM AND THE CONCENTRATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASES IN RATS WITH AN EXPERIMENTAL MODEL OF A PATHOLOGICAL SCAR AT EARLY HEALING TIMES

Pyatigorsk Medical and Pharmaceutical Institute – branch of Volgograd State Medical University, 11, Kalinin av., Pyatigorsk, 357532, Russia. Corresponding author, 1e-mail: [email protected]

Abstract Introduction: According to statistical data, the tendency towards an increase in the number of patients with keloid and hypertrophic scars at the site of skin lesions remains in the world, which in most cases is due to the lack of effective methods for preventing this type of complications. Therefore, the development of new methods of pharmacological correction, which allow us to normalize the wound healing process at an early stage, seems very relevant. One of the promising areas for the search for substances suitable for the prevention of pathological scars is the study of natural compounds possessing endothelioprotective activity, since, according to modern ideas, it is the endothelial dysfunction that underlies the pathogenesis of this complication. Materials and Methods: All studies were performed on 60 male rats of the Wistar line, divided into 6 experimental groups. New forms for external use based on flavonoids PMFI-92 and PMFI-93 and comparison preparations "Kontraktubeks", "Egallohit" were used as research objects. The effect of new PMFI-92 and PMFI-93 formulations on the vasodilating function of the endothelium of the skin of animals with chemical burn was introduced with the introduction of nitric oxide synthesis modifiers: acetylcholine (ACS), L-arginine, nitro-L-arginine methyl ester (L-NAME). The effect of local pharmacological support in this pathology on the level of endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) nitric oxide syntheses was determined by the method of solid phase enzyme immunoassay (ELISA). Results and Discussion: Application of the proposed new forms based on natural compounds PMFI-92 and PMFI-93 allows to improve the vasodilating function of the endothelium already at the early stages of wound healing, with an increase in the concentration of eNOS and nNOS, as well as a decrease in the concentration of iNOS. Conclusion: The experimental data obtained by us testify to the pronounced positive effect of the local application of PMFI-93 and PMFI-92 compositions on the vasodilating function of the skin vessels, and also to restore the balance between the activities of various forms of nitric oxide synthases. Keywords: Endothelial dysfunction, keloid and hypertrophic scars, PMFI-92, PMFI-93, eNOS, nNOS, iNOS.

Introduction of 10-40% of patients [1, 2, 3]. In connection Skin lesions leading to the formation of with the widespread prevalence of this pathological scars are one of the main causes of problem, various scientific approaches have aesthetic and functional defects in an average been developed that explain the pathogenesis of

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Voronkov A.V., Gamzeleva O.Y. The effect of new forms for external application on the vasodilating function of the endothelium and the concentration of endothelial nitric oxide synthases in rats with an experimental model of a pathological scar at early healing times. 152 Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4):151-159. the formation of pathological scars [4, 5, 6, 7, groupResearch– pseudo-operated results in Pharmacology. animals2017;3(4):Х -Х!!!! (L/O), 2 8, 9]. So, their formation is associated with a group – negative control (NK), Group 3 – violation of microcirculation in the area of animals receiving the composition PMFI-92, 4 damage, with excessive intensity of group – PMFI-93, 5 group – comparison inflammation, with the accumulation of excess preparation "Kontraktubeks", 6 group – collagen, etc. [4, 5, 6, 7, 8, 9]. In recent years, preparation of comparison "Egallohit". For the greatest scientific interest is dysfunction of narcosis, chloral hydrate was used, which was the endothelium of damaged vessels as a target administered intraperitoneally at a dose of 350 of pharmacological action in the prevention of mg / kg, slaughter of animals was carried out keloid and hypertrophic scars, since it is this under ether anesthesia. New forms for external pathological process that leads to a disruption use based on flavonoids PMFI-92 and PMFI-93 in the metabolism of biologically active and comparison preparations "Kontraktubeks", substances and mediators in the damaged "Egallohit" were used as research objects. surface [4, 5, 10]. In the modern A chemical burn was modeled in rats on a pharmaceutical market, there are a number of pre-depilated area of the skin of the back by drugs that affect these pathological subcutaneous injection with 0.2 ml of glacial mechanisms, with the exception of drugs acetic acid. The investigated objects were capable of restoring the damaged endothelial applied to the damaged area of the skin 2 times function in the early stages of wound healing a day for 1.0 g for 14 days, the registration of [4, 5, 11, 12, 13, 14]. So, to reduce the already the indices was carried out on the 7th and 14th formed shallow defect and prevent the days of the experiment. The analysis of the formation of scars after uncomplicated damage, vasodilating function of the endothelium was the gels Kontraktubeks and Egallohit use performed by ultrasound dopplerography with natural compounds [15, 16, 17, 18, 19]. At the the intravenous administration of ASC (Sigma- same time, the search for ways of emergency Aldrich, USA) at a dose of 0.1 mg / kg; L- prevention of the formation of a pathological arginine (Sigma-Aldrich, USA) at a dose of scar is still relevant. In connection with the 150 mg/kg and nitro-L-arginine methyl ester above, the creation of new forms for external (L-NAME) (Sigma-Aldrich, USA) at a dose of application capable of restoring the endothelial 15 mg / kg [21]. The UZOP-010-01 sensor with function is very promising, and, in our opinion, an operating frequency of 25 MHz was located it is expedient to consider natural compounds on the inside of the area of the skin of the of the flavonoid series as objects for the rumen, the indices were recorded with the help creation of such preparations [4, 5, 20]. of the MM-D-K-Minimax Doppler v.1.9 The purpose of this study was to study the working computer program. (Saint-Petersburg, effect of new forms for external use on the Russia). ELISA was performed on a microtiter basis of the flavonoids PMFI-92 and PMFI-93 plate reader Tecan Infinite F50 (Austria) using on the vasodilating function of the endothelium species-specific nitric oxide synthetase assays as one of the components of the endothelial of Cloud Clone Corp (USA). All studies function, as well as the indirect determination corresponded to the rules of laboratory practice of the activities of various isoforms of nitric (GLP) in pre-clinical studies and the oxide synthases, since restoring the balance International Recommendations of the between eNOS, nNOS and iNOS can act as one European Convention for the Protection of of the possible mechanisms for restoring both Vertebrates (1997). The results were processed the vasodilating and endothelial functions in using the variational statistics methods using general. the parametric t-test of the Student (t-test) and Materials and methods the nonparametric U-test (Mann-Whitney test) In the experiment, three-month-old male using the Statistica 6.0 and StatPlus 2009 Wistar rats weighing 220-250 g were used, application packages, as well as Microsoft without external signs of diseases, divided into Office Excel 2007. 6 experimental groups of 10 animals each: 1

Results and Discussion significantlyResearch results exceeded in Pharmacology. by 1.17 2017;3(4): timesХ- Х!!!!the same The initial velocity of blood flow in the index in animals that received " Egallohit », in vessels of the skin of the back of the L/O which the blood flow velocity in the zone of animals was 2.366 ± 0.242 cm / sec, while in damage prior to the introduction of analyzers the NC animals on the 7th day of the was 0.52 ± 0.089 cm/s. At the same time, in the experiment this index was significantly lower animals of group 4, the initial blood flow (p <0.001) 5.54 times compared to the first velocity did not differ from that in the group group , which is an unfavorable prognostic receiving "Kontraktubeks" (0.6 ± 0.080 cm/s). indicator [21]. It should be noted that local In the rats receiving PMFI-92 during the week, pharmacological stimulation of wound healing the initial blood flow velocity was 0.57 ± 0.154 already at this stage of the experiment led to cm/sec, which is 1.33 times higher than in the some improvement in the analyzed index, with rats of group 1 (p<0.05), but was somewhat the highest initial blood flow rate recorded in lower, than in animals, which during the week rats receiving the composition PMMI-93. In were applied PMFI-93 and Kontraktubeks. The this group of rats, it was 0.606 ± 0.198 cm/sec, results of the analysis of this indicator are which exceeded the similar result in animals clearly reflected in Figure 1. with pathology (p <0.05) by 1.42 times, and

Fig. 1. Initial velocity of blood flow in the damaged area of the skin, cm/s Note: α – is statistically significant in relation to the group of pseudo-operated animals (p <0.001), single-factor analysis with the Bonferroni amendment

An important diagnostic criterion for the and indirectly suggests prevalence of state of the vasodilating function of the vasoconstriction over vasodilation. In contrast endothelium is the response of the vessels to to the control group of rats with a rumen intravenous administration of the ACX, which model, the response to the administration of the stimulates the production of nitric oxide and, ACX is preserved in animals receiving the accordingly, causes vasodilation [21]. Thus, in composition PMFI-93, as evidenced by an the animals of group 1, a classical response of increase in blood flow velocity by 17.39% of the skin vessels to a given analyzer was the baseline (p <0.05), which is 2, 14 times observed, expressed in an increase in the higher than in NK animals. In addition, the average linear blood flow velocity by 47.61% blood flow velocity of this group of rats was (p <0.01) from its baseline level. Significantly significantly different from the group of the less pronounced changes were recorded in comparison drug "Kontraktubeks", where the group 2 – an increase in the blood flow velocity blood flow velocity increased by 16.87% was only 8.14% of the baseline, which was compared to its baseline level, and was 1.18 5.84 times (p <0.05) lower than in rats and rats, times higher than in the group receiving

«Egallohit» (+ 14.76% of the initial level). In significantlyResearch results different in Pharmacology. from 2017;3(4): the NDХ-Х!!!! and the response to the introduction of the ACS, the groups of animals that received the comparator. blood flow velocity in the group of animals Changes in the rate of cutaneous blood flow receiving PMFI-92 increased by 17.62% (p with intravenous administration of the ACS in <0.05) from its baseline, the index was all experimental groups are shown in Figure 2.

Fig. 2. Increase in blood flow velocity with intravenous injection of acetylcholine Note: * – statistically significant with respect to the initial SC (p <0.05), ** – statistically significant with respect to the initial SC (p <0.01), γ – statistically significant in relation to the group of pseudo- operated animals (p < 0.05), Δ – statistically significant in relation to the group of negative control animals (p <0.05), single-factor analysis with the Bonferroni amendment

When L-arginine (substrate in the synthesis the comparative drugs "Kontraktubeks" and of nitric oxide synthesis) was administered to "Egallohit", the analyzed index had the animals of group No. 1, there were no significantly higher values (p <0.05) than in significant changes in blood flow velocity, groups No.3, No.4: an increase was observed in which agrees with available literature data [22]. comparison with the initial value against L- A different reaction of the vessels to this arginine by 17.69% and 25.12% respectively analyzer was recorded in the group of NK rats, (Fig. 3). where the blood flow velocity increased by Following the introduction of L-arginine 31.78% (p <0.05) relative to its baseline level, blockade of the nitric oxide synthesis system by which is described in the literature as the intravenous administration of L-NAME, the "L-arginine paradox" phenomenon and is one blood flow velocity in the damaged area of the of the important markers of disturbance of the skin in L / O animals decreased by 35.28% (p vasodilating function [21, 22]. Already at this <0.05). At the same time, in rats with a model period of the experiment, it is possible to note of a pathological scar, the blood flow rate the significant differences between the animals decreased by only 6.75% from its baseline that received PMFI-92 and PMFI-93 in this level. The lack of influence of L-NAME on the indicator. Thus, the administration of L- indicator under study may be due to the already arginine to group No. 4 caused an unreliable existing deficiency in eNOS activity as a result increase in blood flow by only 8.06%, which is of the simulated pathology and also supports 3.94 times lower (p <0.05) than in Group 2. In the theory of the "L-arginine paradox" [21, 22]. turn, when L-arginine was administered to The response of the vessels to L-NAME in all group 3, the rate of cutaneous blood flow animals receiving PMFI-93 was more increased (in comparison with the initial value) significant and was expressed in a decrease in by 12.35%, which was statistically significant the linear velocity of cutaneous blood flow: in (p <0.05) less than those in negative control rats with CEPR – by 16.29% (p <0.05), which groups in 2, 56 times. In animals that received was more than the same indicator in rats NK in

2.41 times. Against the backdrop of L-NAME, group.Research The results use in of Pharmacology. comparator 2017;3(4): drugsХ-Х!!!! led to a a significant decrease (p <0.05) in the rate of less pronounced vascular response (p <0.05): in cutaneous blood flow was observed in rats the animals treated with Kontraktubeks, the receiving the PMFI-92 composition at 16.12% blood flow rate decreased by 15.27%, and in of the baseline, which did not differ rats that received Egallohit 14.50% its initial significantly from animals of the PMFI-93 value (Fig. 4).

Fig. 3. Increase in blood flow velocity with intravenous administration of L-arginine Note: * – statistically significant with respect to the initial SC (p <0.05), ** – statistically significant with respect to the initial SC (p <0.01), γ – statistically significant in relation to the group of pseudo- operated animals (p < 0.05), Δ – statistically significant in relation to the group of negative control animals (p <0.05), single-factor analysis with the Bonferroni amendment

Fig. 4. The drop in blood flow velocity with intravenous injection of L-NAME Note: * – statistically significant with respect to the initial SC (p <0.05), ** – statistically significant with respect to the initial SC (p <0.01), γ – statistically significant in relation to the group of pseudo- operated animals (p < 0.05), Δ – statistically significant in relation to the group of negative control animals (p <0.05), single-factor analysis with the Bonferroni amendment

Thus, it can be concluded that the chemical the administration of ACX and L-NAME and burn provoked a negative change in the the enhancement of the response to L-arginine, vasodilating function of the vascular while the local pharmacological the support of endothelium on the 7th day of the experiment, experimental compounds allowed to correct the expressed in the weakening of the response to described violations. At the same time, at the

Changes in blood flow velocity with the introduction Group of of analyzers from the baseline, in % animals Increase Fall ATSX L-arginine L-NAME Group 1 47.61±0.711** 5.00±2.063 35.28±11.022* Group 2 8.55±1.631γ 22.73±5.673*γ 7.53±2.037γ Group 3 18.64±1.881*Δ 9.44±1.258Δ 17.67±2.005*Δ Group 4 19.49±2.746*Δ 7.89±2.375Δ 18.46±2.647*Δ Group 5 18.52±2.290 14.04±1.349Δ 17.30±2.088*Δ Group 6 15.53±1.660 17.78±10.278 15.77±1.653Δ Note: * – statistically significant with respect to the initial blood flow velocity (p <0.05), single- factor analysis with Bonferroni correction; ** – statistically significant with respect to the initial blood flow velocity (p <0.01), single-factor analysis with Bonferroni correction; *** – statistically significant with respect to the initial blood flow velocity (p <0.001), single-factor analysis with Bonferroni correction; γ – statistically significant in relation to the group of pseudo-operated animals (p <0.05), single-factor variance analysis with Bonferroni amendment; Δ – statistically significant in relation to the group of negative control animals (p <0.05), single-factor analysis with the Bonferroni amendment; p is the level of the reliable difference.

At this stage of the study, we can note the an increase in the concentration of constitutive preservation of pathological changes in the NOS isoforms with a simultaneous decrease in group of rats # 1, as well as the presence of iNOS content already on the 7th day of the recovery dynamics when using local experiment compared to animals not receiving pharmacological support. At the same time, pharmacological support. Thus, when PMFI-92 after analyzing all the indicators listed above on was used in the indicated time interval, the the 7th and 14th day of the experiment, the concentrations of eNOS and nNOS were objects of investigation can be arranged in the significantly higher than those in group No. 2, following order, reflecting the increase in the respectively, by 1.31 and 1.02 times, and when effect on the vasodilating function of the PMFI-93 was applied, 1.25 and 1 , 14 times. endothelium: "Egallohit" <"Kontraktubeks" The concentration of the inducible form of the <"PMFI-92" <"PMFI-93" . enzyme, in turn, decreased by 1.29 times in Based on the well-known data that the comparison with the NK animals. As for the process of improving the vasodilating function comparison drug "Egallohit", it caused only a of the endothelium is primarily due to the significant change in the concentration of restoration of the balance between the eNOS, which was less pronounced than in constitutive forms of eNOS, nNOS and the groups Nos. 3 and 4. In animals that received inducible form of iNOS, in the second stage of the reference preparation "Kontraktubeks", a the studies we determined their concentration significant increase in eNOS activity was in the tissue homogenate of the damage zone noted, and a decrease in iNOS activity in the on days 7 and 14 after the modeling of PMFI-92 and PMFI-93 groups was observed. It pathology [23, 24, 25, 26]. It was found that the should be emphasized that a similar trend is compositions PMFI-93 and PMFI-92 are observed on the 14th day of the experiment characterized by a unidirectional effect on (Table 2). vascular endothelial function, characterized by

Research results in Pharmacology. 2017;3(4):Х-Х!!!! Table 2 Determination of the concentrations of different isoforms of nitric oxide synthases at 7 and 14 days of the experiment

Concentration NOS Group 2 Group 3 Group 4 Group 5 Group 6 Group 1 7 days 14 days 7 days 14 days 7days 14 days 7 days 14 days 7 days 14 days eNOS, 97.33±2.5 76.80±0.61 73.94±3.07 100.45±1.0 264.69±9.96 96.34±0.6 126.58±5.5 93.74±0.94 98.98±1.11 84.56±1.4 84.99±2.7 ng/ml 77 5# 1# 04▲ 4▲ 95▲ 81▲ 7▲ 6▲ 61▲ 15▲ nNOS, 17.71± 31.77± 26.97± 32.27± 43.58± 36.28± 39.46± 27.03± 24.93± 31.61± 21.78± ng/ml 2.329 0.997# 0.389# 9.187▲ 8.616▲ 6.149▲ 3.192▲ 3.564 0.240 5.010 5.539 iNOSin 14.16± 31.89± 30.31± 24.79± 24.03± 24.78± 24.75± 21.88± 22.05± 35.64± 36.20± plasma, 0.448 4.587# 1.986# 1.672▲ 2.857▲ 4.299 3.566▲ 4.259▲ 2.347▲ 2.503 4.977 ng/ml

Note: # – statistically significant in relation to the group of pseudo-operated animals (p <0.05), U – Mann-Whitney test; ▲ – statistically significant in relation to the negative control group (p <0.05), U – Mann-Whitney test.

Conclusion the pathogenesis of cheloid scars formation. Thus, the selected model of the Journal of postgraduate medical education. pathological scar is accompanied by a [Vestnik poslediplomnogo medicinskogo persistent violation of the vasodilating function obrazovaniya]. 2015;3:50-55. (In Russian) of the endothelium, as evidenced by the change [eLIBRARY] [Fulltext] in the response of the vessels of the skin to the 5. Voronkov AV, Stepanova EF, Zhidkova administration of the ACS, L-NAME, L- YY, Gamzeleva OY. Modern approaches of arginine. In this group of animals there was a pharmacological correction of pathological decrease in the concentration of eNOS with a scars. Fundamental research. simultaneous increase in the content of iNOS. [Fundamental'nye issledovaniya]. Using PMFI-93 and PMFI-92 formulations on 2014;2(3):301-308. (In Russian) [eLIBRARY] the basis of natural compounds, early in wound [Fulltext] healing (7th day), it is possible to improve 6. Ong CT, Khoo YT, Tan EK. Epithelial- vasodilation and endothelial function, which is mesenchymal interactions in keloid important for the prevention of the formation of pathogenesis modulate vascular endothelial pathological scars. In this case, the growth factor expression and secretion. J. normalization of the vasodilatation process may Pathol. 2007;211(2):95-108. [PubMed] be related to the enhancement of eNOS 7. Hawkins HK. Pathophysiology of the activity, and also to the inhibition of iNOS burn scar. Total Burn Care. Saunders Elsevier: under the action of the listed formulations. The Philadelphia; 2007. 608-619 p. [Scienceopen] results of this study indicate the prospects of 8. Bran GM, Goessler UR, Hormann K. these compounds for further deeper study with Keloids: current concepts of pathogenesis. Int J the goal of creating on their basis an external Mol Med. 2009;24(3):283-293. [PubMed] [Full dosage form with endothelioprotective activity text] for preventing the formation of pathological 9. Sarrazy V, Billet F, Micallef L. scars. Mechanisms of pathological scarring: role of myofibroblasts and current developments. Conflicts of Шnterest Wound Repair Regen. 2011;19:10. [PubMed] The authors have no conflict of interest to 10. John E, Julian P, Ton J. Endothelial declare. function and dysfunction: testing and clinical relevance. Circulation. 2007;1286-1295. References [PubMed] [Full text] 1. Kobets MV, Vasiljeva LS, Mikhalevich 11. Gorokhov VG, Tikhonova LV. IM, Malyshev VV. Reactivity of an organism at Postoperative complications of skin and gross hypertrophic skin scars. Acta Biomedica scars. Acta Biomedica Scientifica. [Byulleten' Scientifica. [Byulleten' vostochno-sibirskogo vostochno-sibirskogo nauchnogo centra nauchnogo centra sibirskogo otdeleniya sibirskogo otdeleniya rossijskoj akademii rossijskoj akademii medicinskih nauk]. medicinskih nauk]. 2011;4:40-41. (In Russian) 2014;97(3):35-40. (In Russian) [eLIBRARY] [eLIBRARY] [Fulltext] [Fulltext] 12. Namazi MR, Fallahzadeh MK, 2. Bock O, Schmidd-Ott G, Malewski P. Schwartz RA. Strategies for prevention of Quality of life of patients with keloid and scars: what can we learn from fetal skin? Int. J. hypertrophic scarring. Arch. Dermatol. Res. Dermatol. 2011;50(1):85-93. [PubMed] [Full 2006;297(4):433-438. [PubMed] text] 3. Furtado F, Hochman B, Ferrara S. What 13. Naylor MC, Brissett AE. Current factors affect the quality of life of patients with concepts in the etiology and treatment of keloids? Rev. Assoc. Med. Bras. keloids. Facial Plastic Surgery. 2009;55(6):700-704. [PubMed] [Full text] 2012;28(5):504-512. [PubMed] 4. Majorova AV, Voronkov AV, 14. Ogawa R. The most current algorithms Voronkova MP, Gamzeleva OYu, Zhidkova for the treatment and prevention of YuYu. Position of endothelium dysfunction in hypertrophic scars and keloids. PlastReconstr December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Voronkov A.V., Gamzeleva O.Y. The effect of new forms for external application on the vasodilating function of the endothelium and the concentration of endothelial nitric oxide 159 synthases in rats with an experimental model of a pathological scar at early healing times. Research result: Pharmacology and Clinical Pharmacology. 2017;3(4):151-159.

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CLINICAL PHARMACOLOGY

Rus.

UDC: 616-002.3:615.03+615.37 DOI: 10.18413/2313-8971-2017-3-4-160 -176

Veronica A. Zemskova, OPTIMIZATION IMMUNEPHARMACOTHERAPY Vladimir I. Zoloedov1, OF PYOINFLAMMATORY DISEASES Olesya A. Popova

The N.N. Burdenko Voronezh state medical university of Ministry of Health of Russian Federation, 10, Studencheskay str., Voronezh, Russia, 394036 Corresponding author, 1e-mail: [email protected]

Abstract Introduction: The results of clinical investigation of several pyoinflammatory diseases are presented. Objectives: On clinical data of various models of pyoinflammatory diseases – deep pyodermia (UCP), chronic salpingooforitis (OHSO), chronic pyelonephritis (CPN), authors found a significant mechanism of influence the pathogenesis on the manifestation of immunopathology, efficacy and mechanisms of pharmacological immunocorrection action. Methods: The study included 300 patients with purulent-inflammatory diseases of various genesis – UCP, CPN, OHSO. All the patients before and after traditional treatment with mono-and combination of immunomodulators of different origin – Roncoleukin, Licopide, Superlimph, Derinate, Polyoxidonium, Timogen, Galavit were subjected to a standard hemato-immunological examination and additionally model for each nosologic forms of diseases, bacteriological and clinical research. Mathematical analysis of obtained data was determined the significant differences of parameters from a predetermined level, and optionally, key, signal tests, formalized in the form of formulas. Results and Discussion: A significant impact of three types of the pathogenesis of inflammatory diseases on the nature and severity of the hemato-immunological disorders in patients are established; the effectiveness and mechanisms of action of the modulator Galavit; a lock of a normalizing effect of the traditional treatment of patients; increase activity due to additional assignments of 7 options of monocorrectors and three of their combinations; the ability of modulating drugs affect not only immunological, but also on haematological, bacteriological and clinical characteristics of patients. In results, authors are documented a hole effects the action of immune correcting drugs. A formalized assessment of the variations in clinical and laboratory parameters of patients allowed us to determine diagnostically significant target of each immune factor integrally to compare the effect of individual therapy, to establish mechanisms for the modification of the function of the lymphoid system in mono- and combination immune therapy, due to the inversion of mathematical analysis to identify laboratory evidence for selection of specific variants of complex treatment of patients. Conclusion: The result of clinical investigation and mathematical analysis of the results achieved was the formulation of a 6-level algorithm to identify and addressing treatment

immunocompromising persons in the form of 7 approved programs for computer-based introduction to computer outcome of laboratory examination of patients. Keywords: pyoinflammatory diseases, immunotherapy, immunological disorders, formalized assessment.

Introduction wide spectrum antibiotics) and increased Under natural conditions, when infection allergization, it is necessary to free the develops, it forms a complicated pathogenic organism from infectious agents extremely complex that includes accumulation of a lien quickly and restore its destroyed homeostasis antigens, aggressive factors (endotoxins, acute- [8, 9, 10, 11] phase proteins, low-molecular nucleic acids By origin, immunotherapeutic drugs etc.), and ultra boundary suppression and subdivide into 4 groups: 1). obtained from stimulation of immune reactivity, and also blood of various organs of humans and animals competition between extra- and intra-cellular (plasma, immunoglobulins, thymus drugs, parasites for defense reaction regulators, myelopeptides, interferons, splenin, placenta cytokines, distortion of metabolic processes extract, antilymphocytic serum etc.), (peroxidation of lipids and proteins), dystrophic 2).obtained from plants (tinctures of and other processes. The main method of eleuterococcus, schizandra etc.), 3).stimulators infectious diseases treatment by antibacterial, of microbial origin (pyrogenal, prodigiosan, antiviral and other drugs that lyse or limit the zymosan, sodium nucleinate, bificol, reproduction of causative agents, haven‘t bacteriophages etc.), 4).synthetic drugs turned to account because these factors have a (levamisole, pentoxyl, methyluracil, hemodez, series of substantial weaknesses. Firstly, they polyoxidonium, licopid, diuciphonum etc.). have no physical ability completely eliminate Вy the nature of immunotherapeutic pathogens; secondly, they stimulate the action, immunotherapeutic drugs subdivide adaptation of the pathogens, for instance, to into drugs with specific (directed) action – antibacterial drugs, and realize corruption vaccines, anatoxins, immune serums, distortion of defense reactions towards immunoglobulins, and nonspecific organism deficiency, allergization and autoaggression. At resistance stimulators– blood and its the same time, in case of repeated infection of preparations, plasma, bificol etc.) [12, 13]. larger causative agents (bacteria) by smaller Indications for prescription of immune ones (viruses), when the causative agents are stimulators are flaccid course of infectious destroyed, the etiology of infection changes, process, its chronization and recurring, sharp etc. All this requires additional comprehensive long-term suppression of the indices of therapeutic influence on the mentioned and nonspecific antiinfection resistance and specific other mechanisms [1, 2, 3, 4, 5, 6, 7]. immunity in patients. It may also include Immunotherapy of infections. It implies changes in the nature and increased intensity of that in treatment of chronic and poorly pathological changes, threat of secondary responding to conventional therapy diseases, infection development and curative use of they use vaccines, anatoxins, immunoglobulins, drugs with immunosuppressing properties [12, i.e. etiotropic drugs and pathogenetic treatment 14, 15]. Immunotherapy prescribed in a with use of blood, blood substitutes, plasma, complex with other medical drugs (antibiotics, nonspecific immunity stimulators etc. A series sulfanilamides, corticosteroids). Its efficiency of drugs simultaneously have antimicrobial and depends on the correct assessment of the initial immune stimulating actions (immunoglobulins, condition of the patient's immune reactivity, the plant extracts etc.). Presently, because of nature and intensity of pathological changes, changes in the natures of infectious diseases the choice of optimal drug and dosing schedule. course, extensive clinical use of drugs that It is also necessary to have an idea about the suppress immune reactions (corticosteroids, December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Zemskova V.A., Zoloedov V.I., Popova O.A. Optimization immunepharmacotherapy of pyoinflammatory diseases. Research result: Pharmacology and Clinical Pharmacology. 162 2017;3(4):160-176. action mechanisms of prescribed drugs, their Clinical and immune criteria of effectiveness side effects, compatibility with other infection are determined by respective specialists with treatment methods, allergic properties etc. due account for preliminary approbation of the Sometimes, vaccine therapy prescribed in case recommended actions. Immunotropic drugs, of immune tolerance to a certain AB, may according to some classifications, divide into render no positive clinical effect and even groups that induce various effects. These aggravate the condition of immune depression; effects are quite numerous and are presented: simultaneously, there risk of anaphylactic 1. Stimulation of immune cells formation shock is possible, as well as induction of due to the influence on the hemopoiesis system autoimmune diseases and toxic shock. Blood (colony-stimulating factors). and plasma transfusion is a good means 2. Interaction with specific receptors of instrument for stimulation of the patient's immunocompetent cells. reactivity. However, this method of treatment is 3. Stimulation or suppression of cytokines restrained by stable indications and must be release. performed on the basis of controlling its effect 4. Formation of active (vaccine) or passive on the course of the disease, immunity indices (serum) antiinfection immunity. and the possibility of allergy. Drugs of 5. Normalization of microecological status etiotropic (immune serums, immunoglobulines, of organism (eubiotics). bacteriophages, interferon) and desintoxication 6. Provision of energy needs and plastic action (drugs of blood, plasma, blood components for immune reactions (macro-, substitutes) should be prescribed as early as microelement, vitamins, biological additives, possible after the onset of the disease. A series antihypoxic drugs). of nonspecific stimulators are used at the height 7. Activation of the processes of of disease and in the convalescence period detoxification of immune reaction products (pentoxyl, vitamins, methyluracil) or for (hepatoprotectors, enteral sorbents, afferent treatment of complications (ferrocalum, phytin, methods). levamisole). Curative vaccines are added to 8. Elimination of antigens (adsorbents) patients with lingering and chronic forms of from organism. disease. The use of polysaccharide preparations 9. Substitution therapy (thymus drugs, is contraindicated for fever conditions. immunoglobulins, white-cell-rich suspension). Eubiotics are not prescribed simultaneously 10. Direct action on antigens (antiviral with antibiotics and other similar drugs [14, 15, drugs) [21, 22]. 16, 17]. Objectives: On clinical data of various Nonspecific immunomodulation models of pyoinflammatory diseases – deep (immunotherapeutic support) of infections pyodermia, chronic salpingooforititis, chronic It implies not only correction of immune, pyelonephritis, authors found a significant but also normalization of hematological, mechanism of influence the pathogenesis on bacteriological, clinical and other indices in the manifestation of immunopathology, patients. Under today's conditions of collective efficacy and mechanisms of pharmacological immunity decrease of the humanity, substantial immunocorrection action. growth of infectious diseases rate, chronization, uncontrolled and irrational use of antibacterial Materials and Methods drugs, the problem of immune reactivity In investigation was included of 300 modulation acquires special relevance. patients:100 patients suffered from deep Diagnostic criteria of control over prescription pyodermia in the stage of exacerbation (EDP), of immunotropic drugs are implemented 100 – from chronic pyelonephritis in the stage through assessment of the complex of clinical of exacerbation (ECPN), 100 women – from and laboratory indices in patients [18, 19, 20]. chronic salpingooopharitis (ECSO) at the age

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Zemskova V.A., Zoloedov V.I., Popova O.A. Optimization immunepharmacotherapy of pyoinflammatory diseases. Research result: Pharmacology and Clinical Pharmacology. 163 2017;3(4):160-176. from 18 to 60 and 30 healthy patients - taking into account the criteria of including and momentary donors. The patients in each excluding, randomization, representation, and nosologic form were divided into two equal statistic processing of data with a proper groups and were undergone a standard distribution of parameters: frequency and treatment and a supplementary one which resulting frequency, graphical. Ranging included a modulators: galavit, roncoleukin, analysis, formalization of immunopathology lycopide, roncoleukin with lycopide, and modulation mechanisms by formulas of superlymph, derinate, superlymph with immune system disorders (FDIC) and targets of derinate, polyoxidonium, thymogen, thymogen correction (FTC) [22, 23, 23, 25, 26, 27]. with polyoxidonium in reglamented doses. Results and discussion Before and after the treatment the patients During our study we have determined a were undergone a standard examination to universal standard reaction of routine reveal routine hematological markers of hematological tests on the inflammatory inflammation, common immunological factors conditions in patients with EDP, ECPN and (population, subpopulation of lymphocytes, ECSO: leukocytosis, neutrophilia, eosinophilia, serum immunoglobulins, cytokines, monocytosis, lymphopenia, ESR acceleration. absorptional and metabolic ability of In the case of PID relatively to TCPN higher phagocytes) and also specific ones for concrete leukocytosis and the neutrophilia have been diseases – bacteriological and clinical registrated, relatively to ESCO – a sufficiently parameters. As a result, the diagnostic material, less monocytosis and ESR have been observed, taken from skin foci, urine, vaginal secrete, was the patients with ESCO had an advantage over treated by general methods and pathogenic and those with ECPN – according to the number of saprophyte staphylococcus, colibaccillus, neutrophiles and monocytes. proteus, klebsiella have been sowed and A typical character in all the cases of PID identified. The number of sterile tests was also has been expressed in the tendency to defined. Clinical parameters included: disbalance or suppression of T-section intoxicational syndrome, subphebrilitis, immunity, humoral activation, suppression of lymphadenitis, renal colic, leukocyturia, phagocytes defense, a definite risk of the leucorrhea discharge, hypertrophy, adnexa uteri development of autoagressive and toxic disorders, etc. conditions, an accumulation of The accuracy of the results was provided proinflammatory cytokines, see fig. 1-3. by the rational arrangement of patients groups,

А. APP

Б. ECPN

В. ESCO

Fig. 1. Differences in hematological parameters from the normal level in the acute period of PID of various genesis Note: Leuk. – leukocytes, Lymph – lymphocytes, Neut. – neutrophils, EOS – eosinophils, Mon. – monocytes, ESR – exacerbation sedimentation rate, * – reliability of differences from the norm level at P <0.05, on the vertical axis – % of patients with 2-3 DIF / HIS

Fig. 2. Graphic visualization of differences in immunologic indexes from the normal level in patients with EDP before the treatment Note: Tr-T-regulators, B – B-cells, IgA, IgM, IgG – immune globulins, CIC – circulating immune complexes, MMM – molecules of moderate mass, LIN – leucocytes with integreen receptors, PI, PN – phagocyte index and number, NBTsp, NBTac – spontaneous and activated tests with nitro blue tetrasolium, IL-4, IL- 6, I IL- 8 – interleukins, TNFα – tumor necrosis factor, Ma – apoptosis marker, T – T-cells, Th – T-helpers, Tc – T-cytotoxics, Tac – T-active, NKr – natural killers regulators, NKc – natural killers cytotoxics, NKt – natural killers thymus dependent, * – evidence of differences from norm in p<0.05, punctual line – normal meaning of parameters of healthy people, broken line – meanings of parameters of sick people

Fig. 3.Graphic visualization of differences in immunologic indices from norm in patients with ECPN before the treatment

Note: Tr-T-regulators, B – B-cells, IgA, IgM, IgG – immune globulins, CIC – circulating immune complexes, MMM – molecules of moderate mass, LIN – leucocytes with integreen receptors, PI, PN – phagocyte index and number, NBTsp, NBTac – spontaneous and activated tests with nitro blue tetrasolium, IL-4, IL- 6, I IL- 8 – interleukins, TNFα – tumor necrosis factor, Ma – apoptosis marker, T – T-cells, Th – T-helpers, Tc – T-cytotoxics, Tac – T-active, NKr – natural killers regulators, NKc – natural killers cytotoxics, NKt – natural killers thymus dependent, * – evidence of differences from norm in p<0.05, punctual line – normal meaning of parameters of healthy people, broken line – meanings of parameters of sick people.

While assessing the dynamics of moderate ECPN (50), ESCO (45), EDP-38 tests. The numbers of immunological indices from normal determination of the level of immunologic significance, and the outcomes of frequency indices changes has shown its moderate and resulting frequency analysis, the most expressiveness in IDP cases and sufficient – in variations have been revealed in patients with ECPN and ESCO cases.

Fig. 4. Graphic visualization of differences in immunologic indices from norm in patients with ESCO before the treatment Note: Tr-T-regulators, B – B-cells, IgA, IgM, IgG – immune globulins, CIC – circulating immune complexes, MMM – molecules of moderate mass, LIN – leucocytes with integreen receptors, PI, PN – phagocyte index and number, NBTsp, NBTac – spontaneous and activated tests with nitro blue tetrasolium, IL-4, IL- 6, I IL- 8 – interleukins, TNFα – tumor necrosis factor, Ma – apoptosis marker, T – T-cells, Th – T-helpers, Tc – T-cytotoxics, Tac – T-active, NKr – natural killers regulators, NKc – natural killers cytotoxics, NKt – natural killers thymus dependent, * – evidence of differences from norm in p<0.05, punctual line – normal meaning of parameters of healthy people, broken line – meanings of parameters of sick people

A detailed study of the immunologic of inflammation, disbalance of mechanisms in concrete nosologic forms of immunoglobulins of class G and T-cells. The pyoinflammatory diseases (PID) has revealed data obtained have the evidence that despite the their dependence from pathogenesis. Thus typical immunological body reaction on the FDIS decoding showed, that in EDP there was general inflammation, there are concrete observed the accumulation of markers of the pathologies, dependent upon pathogenesis of autoimmune processes (CIC), cytotoxic natural diseases having a diagnostic significance. killers and anti-inflammatory cytokine (IL4). In Since the assessment and direct ECPN – activation of humoral section of comparison of action effectiveness in the immunity in B-cells and MCM, the increase of differentiated complex immunotherapy PID of cytotoxic lymphocytes, in TSCO – the presence different genesis with galavite between various

Fig. 5. Graphic visualization of galavite‘s own effect in patients with EDP

Note: Tr-T-regulators, B – B-cells, IgA, IgM, IgG – immune globulins, CIC – circulating, MMM – molecules of moderate mass, LIN – leucocytes with integreen receptors, PI, PN – phagocyte index and number, NBTsp, NBTac – spontaneous and activated tests with nitro blue tetrasolium, IL-4, IL- 6, IIL- 8 – interleukins, TNFα – tumor necrosis factor, Ma – apoptosis marker, T – T-cells, Th – T-helpers, Tc – T-cytotoxics, Tac – T-active, NKr – natural killers regulators, NKc – natural killers cytotoxics, NKt – natural killers thymus dependent, * – evidence of differences from normin, p<0.05, punctual line – normal meaning of parameters of healthy people, broken line – meanings of parameters of sick people. Circumference – normal parameters of patients after traditional treatment

Fig. 6. Graphic visualization of galavite‘s own effect in patients with ECPN

Fig. 7. Graphic visualization of galavite‘s own effect in patients with ESCO

It was found out that under the galavite stimulating vector and NBTsp, IgG –with influence the patients with EDP have undergone suppressing. The main points in the immune some evident changes from the initial level 5 system of galavite modulator in ECPN were + + - laboratory indices in 24 studied. The tests (PI 3NBTsp 3 IgG 3). concentrations of IgM, phagocytes number have In women suffering from ESCO under the increased but NBTsp, the level of FNO and cells influence of galavite modulator has been with apoptosis marker have decreased. The key achieved a mathematically significant change - - targets of modulator in EDP were TNF 3Ma of 7 indices but their spectrum turned out to be + - 3IgM 2 . different. The number of Th, Tac, Tr, NKt, In patients with ECPN the same IgA, -M concentrations have increased, CIC – preparation resulted in a significant dynamics have decreased. The main effect of the + - from the initial level of 7 immunologic tests – a corrector has been produced on NKt 3CIC - phagocyte index NBTac, IL-4, -6, -8 with a 3IgM 3. December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Zemskova V.A., Zoloedov V.I., Popova O.A. Optimization immunepharmacotherapy of pyoinflammatory diseases. Research result: Pharmacology and Clinical Pharmacology. 171 2017;3(4):160-176.

These results show that the key targets in provide a complete correction of the changed the immune system of the galavite modulator in clinical laboratory indices as a supplementary patients with IDP, ECPN, ESCO have been method of treatment a galavite modulator has different. been used. Preliminary studies have shown that the traditional therapy PID, in general, did not Table 1 Rating assessment of differentiate treatment effectiveness in DIP in ranges

Indices Summa of Nosoform Treatment Hem. Bact. Clin. Imm. N.L. Effectiveness ranges of PID М N М N М N М N ТT 1 2 1 2 1 1 3 2 4 17 6 +Gal 1 1 1 2 1 1 3 1 3 14 4 EDP ТT 1 2 1 2 1 1 3 2 4 17 6 +Gal 1 1 1 2 1 1 3 1 3 14 4 ECPN ТT 2 3 3 2 1 3 3 2 3 22 5 +Gal 1 3 2 1 1 1 3 1 3 16 2 ESCO

Note: ТT – traditional treatment, Gal – galavite, Hem – hematologic, Bact – bacteriologic, Clin - clinical, Imm – Immunologic indices, МN – mobile and normalizing effects, N.L. – number of strong correlation links, 1 2 – maximal and moderate ranges of effectiveness.

Due to the table data, a supplementary role It was also found that the traditional of galavite at the background of the traditional uncorrected treatment of different nosoforms of treatment of all the PID nosoforms according to DGT as a whole proved to be insufficiently the outcome rating has provided an evident effective, and in particular – differentiated increase of its effectiveness in hematological, according to the effect on the grouped and bacteriological, immunological and clinical individual indicators of patients. At the same indices. Besides, the preparation activity was time, normalization of clinical, bacteriological, higher in ESCO patients. and less often hematological and While analyzing the obtained data, we immunological parameters was observed, have documented the phenomena of typical which puts additional immunotherapy of variations formation of hemato-immunological patients on the agenda. As its variants, inflammation markers, independent from PID modulators of various origins were chosen; pathogenesis. They were in the form of stimulants of local and systemic immunity; its leukocytes masses, granulocytes, monocytes, various links [28]. accelerated ESR, disbalance or suppression of As a result of the application of formalized some immunity sections and the induction of analytical approaches, the clinical and the diagnostically significant laboratory laboratory effectiveness of complex treatment changes on the subpopulation, interleukin, of patients was shown to increase and the immunoglobulin levels, given in the formulary declining overall rating of its variants was forms. determined. With UCP: 1 – roncoleukin with We have also determined qualitatively and lycopide, 2 – lycopide, 3 – galavite, quantatively differentiated clinic laboratory roncoleukin; with ECPN: 1 – superlymph with effects of combinations of traditional treatment derinatom, 2 – galavite, 3 – derinate, 4 – with galavite, dependant from the pathogenesis superlimph; with ESCO: 1 – polyoxidonium, 2 of disorders and analyzed parameters. – thymogen with polyoxidonium or galavite, 3 – thymogen. The effectiveness of one

Table 2 Signal targets in the lymphoid system of differentiated immunotherapy of PID of various genesis

Treatment FIT FISD EXTENSION OF DEEP PYODERMIA + + + + + + Тraditional treatment В 3Тс 3NBTsp 3 IL8 3NKc 3Ма 3 + ¯ + + + + +roncoleukin Таc 3IgG 3Т 3 IgM 3 CIC 2Таc 2 + + - + + - +lycopide FI 3NBTac 3IL6 3 Тr 3IgA 3Тc 2 + ¯ + + + + +galavit IL4 3TNF 3Ма 3 IL6 3NBTsp 3IL4 3 + + + + + + +roncoleukin +lycopide NBTsp 3Тc 3Тh 3 NKc 3IgG 3IL4 3 EXTENSION OF CHRONIC PYELONEPHRITIS + + ¯ + + + Тraditional treatment Т 3IgA 3 CIC 2 NKc 3IgM 3TNF 2 + + + + + + +superlimph Тr 3В 3Тc 3 Тr 3IgM 3NKt 3 + + + ¯ + + +derinate NBTsp 3FN 3NKr 3 Т 2FI 3IgG 3 + + ¯ + + + +galavit FI 3IL4 3Тr 2 FI 3IL4 3Тr 3 + + ¯ + + + +superlimph +derinate Тh 3Тr 3 CIC 3 В 3FI 3Тc 3 EXTENSION OF CHRONIC SALPINGFOORITIS ¯ + ¯ + + + Тraditional treatment Таc 2IgM 2Тh 2 IL8 3NKc 3Ма 3 + + + + + + +thymogen Тh 3Тc 3 NKr 3 IgM 3 CIC 2Таc 2 + + + + + - +polyoxidonium Тr 3NBTsp 3FN 3 Тr 3IgA 3Тc 2 + + + + + + +galavit FN 3IL4 3NKt 3 TNF 3В 3FI 3 + + - + + + +thymogen +polyoxidonium Тh 3NBTac 3МSМ 3 NKc 3IgG 3IL4 3 Note: ТT – traditional treatment, Gal – galavite, Hem – hematologic, Bact – bacteriologic, Clin - clinical, Imm – Immunologic indices, МN – mobile and normalizing effects, N.L. – number of strong correlation links, 1 2 – maximal and moderate ranges of effectiveness.

Detailing the targets of correctors and The third, unified on the basis of the creating programs for computers has allowed to formulas of the immune system disorders, the formulate a simplified method of selecting certificate for the computer program active drugs for individual nosoforms for key №2015614977. laboratory markers. A six-level algorithm for The fourth, summarized on the basis of the detection and treatment of grouped indicators, certificate for the computer immunocompromised contingents has been program No. 2015612811. singled out and protected. The fifth, detailed, based on a complete The first, prelaboratory, preliminary, on analysis of the immune status, certificate for the basis of the questionnaire, the certificate for the computer program No. 2014619846. the computer program No. 2014519643. The sixth, personalized, based on the The second, auxiliary on the basis of formulas of immunocorrection targets, the immunotropic treatment, the certificate for the certificate for the computer program No. computer program № 2015612811. 201466056 and 2016619036.

Table 3 Additional laboratory indications for the selection of differentiated immunotherapy PID Recommended Nosoform Critical values of benchmarks treatment APP Тraditional В<0,2*109l,Тc<0,14*109l, NBTsp<2,9 CU treatment +roncoleukin Таc<0,04*109l, Т<0,5*109, IgG>18,0 g/l +lycopide FI<24,3%, NBTac<7,5%,IL6<пкг/ml +galavit IL-4<5,5pkg/ml, TNF>0,32 pkg/ml, Ма>0,14*109л +roncoleukin+ В<0,2*109l,Тc<0,14*109l, NBTsp<2,9% lycopide ECPN Тtraditional Т<0,5*109l, IgА<0,5g/k, CIC>45,9 CU treatment +superlimph NKr<0,11*109l, NKt<0,18*109l, IL4<6,6 pkg/ml +derinate Т<0,03*109l, Тc<0,14*109l, В>0,6*109l +galavit FI<24,3%, IL4<6,6 pkg/ml Тr<0,19*109l +superlimph+derean Тh<0,38*109l, Тr<0,03*109l, CIC< 45,9 CU t ESCO Тtraditional Таc>0,2*109l, Тh>1,82*109l, IgМ<0,6 g/l treatment +thymogen Тh<0,38*109l, Тc<0,14*109l, NKr<0,11*109l +polyoxidonium Тr<0,03*109l, NBTsp<2,9%, FN<2,4 mt +galavit FN<2,4 mt, IL4<6,6 pkg/ml, NKt<0,18*109l +thymogen+ Тh<0,38*109l, NBTsp<2,9%, МSМ>9,1 CU polyoxidonium

Conclusion accumulation of the proinflammatory 1. Patients with exacerbation of deep cytokines. pyoderma, chronic pyelonephritis, and 3. It has been revealed a diagnostically salpingooophoritis were found to have standard significant influence of pathogenesis of each hematological changes (leukocytosis, disorder on the concrete character and neutrophilia, eosinophyllosis, monocytosis, expressiveness of the immunopathology which lymphopenia, accelerated ESR), immunological was shown in individual FDIS patients: in EDP + + + (imbalance of cell, humoral, phagocytic –CIC 3NKc 3Il6 3; in ECPN – + + + + + - defense, accumulation of CIC, MSM, pro- B 3MMM 2Tc 3; in ESCO – TNF 3IgG 3T 2. inflammatory cytokines), metabolic 4. Traditional nonimmunotropic treatment (suppression of antioxidant system factors, of PID causes uneven normalization of clinical stimulation – free radical oxidation of lipids and laboratory status: with APP (acute purulent and proteins). Key formulas of laboratory pyelonephritis) – significant (> 66%) disorders in patients have been determined, elimination of changes in bacteriological, with the help of the diagnostic value clinical and secondary (33-66%) – hemato- coefficient. immunological indicators; with ECPN – 2. Three clinical models with PID have significant – bacteriological, secondary – shown a qualitative typical character of the immunological, insignificant (<33%) – immunopathology – a tendency to disbalance hematological, biochemical, clinical tests; at and suppression of T-section immunity, the ECSO – average – bacteriological, activation of humoral, the suppression of immunological and insignificant – phagocyte, the increased risk of autoaggressive hematological and clinical parameters of and toxic conditions development, the patients. December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Zemskova V.A., Zoloedov V.I., Popova O.A. Optimization immunepharmacotherapy of pyoinflammatory diseases. Research result: Pharmacology and Clinical Pharmacology. 174 2017;3(4):160-176.

5. A differentiated distribution of galavite of individual correctors and their combinations action was established for laboratory for determining signal laboratory markers. parameters of patients with different PID: 11. A six-stage method for identifying hematologic – significant for all nosoforms; on immunocompromised individuals and choosing immunological – the average for ECPN, the optimal differential therapy for purulent- inessential – with APP and ECSO. The key inflammatory diseases (an electronic assistant effect of the modulator in the immune system to a doctor) was developed based on the with APP appeared to be oriented toward pro- introduction of digital results of a laboratory and anti-inflammatory cytokines, CD95 + examination of patients in a computer. lymphocytes; with ECSO – in PI, IL4, Tr; at ECSO – on PN, IL4, NKt. Conflict of Interest 6. Additional application of differentiated The authors have no conflict of interest to immunocorrection in complex treatment of declare. APP increases its final clinical and laboratory significance with the following decreasing References rating of individual drugs: 1-roncoleukin + 1. Vorobejv AA. Medical microbiology, lycopide, 2-lycopide, 3-roncoleukin, 4-galavit, virology and immunology. Moscow: Publishing 5-traditional therapy. house Moscow information agency; 2004. 720 7. Additional appointment of patients with p., (in Russian) [BooksMed] ECPN immunocorrectors causes stimulation of 2. Pokrovsky VI. Handbook of Clinical the overall activity of complex treatment with Immunology. Allergologie, Immunogenetics the following rating of individual effects: and Immunopharmacology for general 1-derinate + superlimph, 2-galavite, 3-derinate, practitioners. Part 1. Moscow: Triada; 2005. 4-superlimph, 5-traditional therapy. 512 p. (In Russian) [eLIBRARY] 8. Additional inclusion in the conventional 3. Pokrovsky V.I. Medical Microbiology. treatment of ECSO immunotropes potentiates Textbook for High School. Moscow: Publishing its resulting efficacy with the following rating House GEOTAR – Media; 2007. 765 p. (In of the declining efficacy of specific drugs: Russian) [Abstract] 1-polyoxidonium, 2-thymogen + polyoxidonium or galavite, 3-thymogen, 4. Pokrovsky VI. Immunology and 4-traditional therapy. epidemiology of infections: study guide for 9. With the combination of modulators in medical students. Moscow: Publishing house patients with PID, it is not mechanical Triada; 2015. 375 p. (In Russian) [eLIBRARY] summation of the properties of individual drugs 5. Novikov DK. Clinical that is realized, but an increase in the overall Immunopathology. Moscow: Publishing House efficiency and transformation of the reference Mеdical Literature; 2009. 449 p. (In Russian) targets of the complex with respect to mono- [Abstract] actions. Thus, in case of APP, roncoleukin + 6. Tsarev VN. Microbiology, virology and lycopid causes activation of neutrophil immunology: textbook for colleges. Moscow: metabolism, accumulation of regulatory Publishing house GEOTAR-Media; 2010. 5436 subpopulations of T cells; with ECPN, p. (In Russian) [eLIBRARY] superlymph + derinate – increase in the level of 7. Labinskaya AS, Volina EG. Medical Th, NBTac and decrease in MSM; at ECSO microbiology: manual. General and sanitary timogen + polyoxidonium – stimulation of microbiology. Book 1. Moscow: Publishing NBTsp, the number of Tc and Th. house BINOM; 2008. 1077 p. (In Russian) 10. As an auxiliary method of selecting the [eLIBRARY] optimal variants of immunotherapy for 8. Medunitsyn NV, Pokrovsky VI, Osno purulent-inflammatory diseases, it is proposed VV. Immunoprofilactic and immunotherapy of to detail, with the help of the diagnostic value infections diseases. Moscow: GEOTAR-Мedia; coefficient, formalized targets of action (FIT) 2005. 512 p. (In Russian) [Abstract]

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Author Contributors Olesya A. Popova, Assistant of the Veronica A. Zemskova, Associate Department of Microbiology, PhD, The N.N. professor of the Department of Microbiology, Burdenko Voronezh state medical university of PhD, The N.N. Burdenko Voronezh state Ministry of Health of Russian Federation, e- medical university of Ministry of Health of mail: [email protected]. Design Russian Federation, e-mail: of the work. [email protected]. Contribution to the concept, collection, analysis and Received: October, 17, 2017 interpretation of data for publication. Accepted: November, 30, 2017 Vladimir I. Zoloedov, Professor of the Available online: December, 30, 2017 Department of Hospital therapy and Endocrinology, MD, professor, The N.N. Burdenko Voronezh state medical university of Ministry of Health of Russian Federation, e- mail: [email protected]. ORCID: 0000-0002-6314-0962. Critical analysis of intellectual content.

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РЕДАКТОРСКАЯ СТАТЬЯ

Покровский М.В., Автина Т.В., Захарова Е.В., Белоусова Ю.В. Освальд Шмидеберг – «отец» экспериментальнои фармакологии 3

ЭКСПЕРИМЕНТАЛЬНАЯ ФАРМАКОЛОГИЯ

Бибик Е.Ю., Cафонова А.А., Еремин А.В. ,Фролов К.А., Доценко В.В., Кривоколыско С.Г. Изучение аналептической активности производных тетрагидропиридо [2,1-b][1,3,5] тиадиазина 20 Галенко-Ярошевский П.А., Васильев П.М, Винаков Д.В., Киселева Н.В., Кочетков А.Н., Киселев А.В. Противовоспалительные и анальгетические свойства производного индола SS-68 26 Денисюк Т.А. Фармакотерапевтические стратегии коррекции эндотелиальной дисфункции с использованием статинов при синдроме системного воспалительного ответа 35 Довгань А.П. Лиганд периферических имидазолиновых рецепторов на основе амидов гетероциклических кислот С7070: влияние на ишемизированные ткани 78 Ефремова М.П., Ивашева А.В., Корокин М.В. Изучение фармакологической активности жирного масла чернушки дамасской в эксперименте 89 Мирошниченко А.Г., Перфильев В.Ю., Лысенко И.В., Жернакова Н.И. Взаимодействие между некоторыми антибиотиками и антиоксидантами 100 Пересыпкина А.А. Коррекция ангиопатии сетчатки по гипертоническому типу производным диметиламиноэтанола 19-16 в эксперименте 113 Разумова М.С., Литвинова Е.С., Гаврилюк В.П. Изучение фармакологической активности сублимированной культуральной жидкости аллогенных, ксеногенных гепатоцитов и фибробластов для коррекции поражений печени при отравлении четыреххлористым углеродом 120 Тарасова А.П., Даниленко Л.М., Сернов Л.Н. Влияние фармакологического прекондиционирования инкретиномиметиками эксенатидом и вилдаглиптином на выживаемость ишемизированных тканей 132 Воронков А.В., Гамзелева О.Ю. Влияние новых форм для наружного применения на вазодилатирующую функцию эндотелия и концентрацию эндотелиальных синтаз оксида азота у крыс с экспериментальной моделью патологического рубца на ранних сроках заживления 151

КЛИНИЧЕСКАЯ ФАРМАКОЛОГИЯ

Земскова В.А., Золоедов В.И., Попова О.А. Оптимизация иммунофармакотерапии гнойно-воспалительных заболеваний 160

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 178

РЕДАКТОРСКАЯ СТАТЬЯ

УДК: 615:378 DOI: 10.18413/2313-8971-2017-3-4-3-19

1 Покровский М.В. ОСВАЛЬД ШМИДЕБЕРГ – «ОТЕЦ» ЭКСПЕРИМЕНТАЛЬНОЙ Автина Т.В. ФАРМАКОЛОГИИ Захарова Е.В. Белоусова Ю.В.

ФГАОУ ВО «Белгородский государственный национальный исследовательский университет», 85, ул. Победы, Белгород, 308015, Россия Автор для корреспонденции, 1e-mail: [email protected]

Аннотация Биография. Освальд Шмидеберг (1838-1921) был сыном судебного исполнителя и фрейлины, старшим из шести детей в семье. Родился и получил образование в Российской империи. Научная деятельность. Всю свою жизнь он полностью посвятил науке, сделав экспериментальную фармакологию самостоятельной научной дисциплиной, и смог вывести еѐ на мировой уровень. О. Шмидеберг изучал действие мускарина и никотина, дигитоксина, снотворных и аналептиков, первым ввел понятия «фармакодинамики» и «фармакокинетики» лекарственного средства. При его участии был создан первый в мире фармакологический журнал, который выпускается и сегодня. Научная школа. Шмидеберг долгие годы работал в университете Страсбурга, на базе которого им воспитано более 120 учеников – профессоров из 20 стран мира, многие из которых стали основателями экспериментальной фармакологии в других странах, например, Абель в США, Н.П. Кравков в России. Научная деятельность Шмидеберга повлияла на ученые умы своего времени на несколько поколений вперед, дав предпосылки для новых открытий и даже получения Нобелевских премий. А вот сам Освальд Шмидеберг этой высокой награды удостоен не был, хотя его номинировали на неѐ 14 раз.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 179

ЭКСПЕРИМЕНТАЛЬНАЯ ФАРМАКОЛОГИЯ

УДК: 615.015.4 : 547.876 DOI: 10.18413/2313-8971-2017-3-4-20-25

Бибик Е.Ю.1 Cафонова А.А.1 ИЗУЧЕНИЕ АНАЛЕПТИЧЕСКОЙ АКТИВНОСТИ Еремин А.В.1 ПРОИЗВОДНЫХ Фролов К.А.1,2 ТЕТРАГИДРОПИРИДО[2,1-b][1, 3, 5]ТИАДИАЗИНА Доценко В.В.2,3 Кривоколыско С.Г.1,2

1ГУ «Луганский государственный медицинский университет», Луганск; 2НИЛ «Химэкс» ГОУ ВПО «Луганский государственный университет им. Владимира Даля», Луганск; 3ФГБОУ ВПО «Кубанский государственный университет», Краснодар Автор для корреспонденции, 1e-mail: [email protected]

Аннотация Введение. Цель нашего исследования – провести изучение аналептической активности производных тетрагидропиридо [2,1-b] [1,3,5] тиадиазина. Методы. Биологические исследования проводились на 78 белых беспородных половозрелых крысах обоих полов весом 230-270 г в осенне-зимний период. Испытуемые вещества вводили внутрижелудочно в дозе 5 мг/кг за 1 час до введения анестезии. Животные контрольной группы получали тиопентал- натрий в дозе 70 мг/кг. В качестве эталонного препарата использовали кофеин-бензоат натрия внутрибрюшинно в дозе 10 мг/кг. Результаты и их обсуждение. В группе экспериментальных животных, получавших внутрижелудочное вещество за 1 час до анестезии тиопенталом натрия, было обнаружено шестикратное удлинение периода анестезии, инъекция в анестезию, продолжительность анестезии. Однако через 16 часов умерли 33,3% крыс, остальные животные были резко подавлены. Исходное вещество с лабораторным шифрованием 2 значительно увеличивает время введения в анестезию, обладает выраженной аналептической активностью, превосходящей эффективность кофеина. Выводы. Таким образом, проведенные исследования по 10 новым биологически активным соединениям на основе производных тетрагидропиридо [2,1-b] [1,3,5] тиадиазина показали наличие наиболее выраженной аналептической и антинаркотической активности в соединениях с лабораторными шифрами 3, 6, 7 и 10. Соединение 2, которое представляет собой 6-оксо-8- {4 – [(2-хлорбензил) окси] фенил} -3- (2-этоксифенил) -3,4, 7,8-тетрагидро-2H, 6H-пиридо [2,1-b] [1,3,5] тиадиазин-9-карбонитрила, по аналептическому эффекту значительно превосходит аналог коффеина-бензоата натрия. Ключевые слова: аналептическая активность, производные тетрагидропиридо [2,1-b] [1,3,5] тиадиазина, тиопентал натрия, бензоат натрия.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 180

УДК: 616.12-008.313:615.222:615.015]-092.9 DOI: 10.18413/2313-8971-2017-3-4-26-34

Богус С.К.1 Суздалев К.Ф.2 Уваров А.В.1 Галенко-Ярошевский П.А.1 ПРОТИВОВОСПАЛИТЕЛЬНЫЕ Васильев П.М.4 И АНАЛЬГЕТИЧЕСКИЕ СВОЙСТВА ПРОИЗВОДНОГО Винаков Д.В.3 ИНДОЛА SS-68 Киселева Н.В.1 Кочетков А.Н.3 Киселев А.В.5

1Кубанский государственный медицинский университет, 2Южный федеральный университет, 3Белгородский государственный национальный исследовательский университет, 4Волгоградский государственный медицинский университет, 5Краснодарский филиал МНТК "Микрохирургия глаза" им. акад. С.Н. Федорова

Аннотация. Показано, что в экспериментах на крысах соединение SS-68 при внутривенном (5 мг/кг) и внутрижелудочном (5, 10, 15 и 20 мг/кг) введениях оказывает противовоспалительное действие (ПВД) на моделях острого воспаления, вызванного каррагенином и серотонином, и не проявляет ПВД в условиях отѐков, индуцированных полным адъювантом Фрейнда и гистамином, за исключением внутрижелудочного введения в дозе 20 мг/кг. По антифлогогенной активности при каррагениновом и серотониновом отеках SS- 68 сопоставимо с диклофенаком (5, 10 и 15 мг/кг внутрижелудочно) и индометацином (10 мг/кг внутрижелудочно), более значимо, чем пироксикам (20 мг/кг внутрижелудочно). По терапевтическому индексу (ТИ) SS-68 в первом случае в 2,2, 15,6 и 5,6 раза, а во втором – в 1,8, 12,8 и 4,4 раза соответственно превосходит диклофенак, индометацин и пироксикам. В опытах на мышах в тесте "уксусные корчи", отражающем преимущественное воздействие на κ(каппа)-опиодные рецепторы, SS-68 при внутрибрюшинном введении в дозах 0,03, 0,06 и 0,12 мг/кг вызывает дозозависимое снижение числа корчей на 40,0, 69,0 и 80,3% соответственно. Буторфанол, взятый для сравнения, в дозах 0,03, 0,12, 0,24, 0,48 и 0,96 мг/кг обладет дозозависимым болеутоляющим действием (БУД), при этом число корчей составляло 41,5, 52,4, 65,3, 71,1 и 80,6% соответственно. По болеутоляющей активности и ТИ SS-68 в 1,9 и 2,7 раза соответственно превосходит буторфанол. По результатам докинга показателей аффинитета к κ1-опиоидным рецепторам SS-68, буторфанола (агонист-антагонист), а также соединения U-50488 (селективный агонист) установлено, что в соответствии с расчетными значениями константы связывания, аффинность в отношении SS-68 была в 4,53 выше, чем буторфанола, а по сравнению с U-50488 – в 2,84 раза ниже. Высказано предположение, что БУД SS-68, подобно буторфанолу, может быть связано как с аффинитетом к κ1-рецепторам, так и другим типам опиоидных рецепторов (мю, дельта) и подтипам κ-рецепторов (κ2 или κ3). Ключевые слова: производное индола SS-68, диклофенак натрия, индометацин, December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 181

пироксикам, противовоспалительное действие, каррагенин, серотонин, гистамин, полный адъювант Фрейнда, болеутоляющее действие, каппа-опиодные рецепторы.

УДК: 615.37:616-018.74-008.6 DOI: 10.18413/2313-8971-2017-3-4-35-77

Денисюк Т.А. ФАРМАКОТЕРАПЕВТИЧЕСКИЕ СТРАТЕГИИ КОРРЕКЦИИ ЭНДОТЕЛИАЛЬНОЙ ДИСФУНКЦИИ С ИСПОЛЬЗОВАНИЕМ СТАТИНОВ ПРИ СИНДРОМЕ СИСТЕМНОГО ВОСПАЛИТЕЛЬНОГО ОТВЕТА

ФГБОУ ВО «Курский государственный медицинский университет» Минздрава России Kursk State Medical University, 3, K Marksa St., Kursk, 305040, Russia Corresponding author, 1e-mail: [email protected]

Аннотация Введение. Пути фармакологической коррекции сердечно-сосудистых осложнений при синдроме системного воспалительного ответа (ССВО) разработаны не в полной мере. Цель. Установление новых фармакологических эффектов статинов и их комбинации с эндотелиопротекторами при коррекции ССВО. Материалы и методы. В экспериментах на мышах, крысах и кроликах исследовались противовоспалительные, кардиопротекторные и эндотелиопротекторные эффекты статинов и эндотелиопротекторов. Моделирование эндотоксин-индуцированной эндотелиальной дисфункции (ЭИЭД) производили путем заражения крыс золотистым стафилококком (штамм 13407), подкожно (60 миллиардов микробных тел). Для определения активности воспалительного процесса использовали показатели С-реактивного белка. Участие цитокинового звена воспаления оценивалось по данным содержания в плазме крови ФНО-α и ИЛ-6. Моделирование L-NAME- индуцированной патологии и оценку эндотелийзавиимых и эндотелийнезависимых сосудистых реакций проводили по стандартному протоколу. Результаты. Симвастатин (9, 19, 35 мг/кг), аторвастатин (5, 9, 19 мг/кг), розувастатин (9, 19, 35 мг/кг) и нанопартикулированный розувастатин (3, 6,3 и 11,6 мг/кг) проявляют дозозависимое антиэкссудативное действие на модели формалинового отека лапы у мышей. Аналогично противовоспалительное действие проявляется на экссудативной модели у кроликов. Наибольшую эффективность продемонстрировали розувастатин и его нанопартикулированная форма. Симвастатин 8,5 мг/кг, аторвастатин 4,3 мг/кг, розувастатин 8,5 мг/кг и нанопартикулированный розувастатин 11,6 мг/кг проявляют кардиопротективное действие при моделировании коронароокклюзионного инфаркта у крыс. В реализации кардиопротективных эффектов существенное значение имеют механизмы фармакологического прекондиционирования, о чем свидетельствует снятие эффектов при блокаде К+-АТФ-азных каналов глибенкламидом (5 мг/кг) и блокаде iNOS аминогуанидином (40 мг/кг). Использование ингибиторов ГМГ-КоА-редуктазы симвастатина (2,2, 4,3 и 8,5 мг/кг), аторвастатина (1,1, 2,2 и 4,3 мг/кг), розувастатина (2,2, 4,3 и 8,5 мг/кг) и нанопартикулированного розувастатина (3, 6,3 и 11,6 мг/кг) на фоне моделирования эндотоксин-индуцированной патологии, приводит к развитию дозозависимого эндотелиопротективного действия, выражающегося в December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 182

нормализации КЭД, предотвращении повышения адренореактивности и исчерпания миокардиального резерва, а также в нормализации биохимических маркеров воспаления (С-реактивный белок) и уровня провоспалительных цитокинов. При этом обнаружена положительная динамика конечных продуктов NO и экспрессии eNOS. Использование монотерапии донатором NO L-аргинином (70 и 200 мг/кг), неселективным ингибитором аргиназ BEC (5 и 10 мг/кг), селективным ингибитором аргиназы-2 аргиназином (1 и 3 мг/кг) и рекомбинантным дарбэпоэтином (50 и 500 мкг/кг) при моделировании эндотелиальной дисфункции, обнаружило их высокую активность, выражающуюся в предотвращении увеличения (коэффициента эндотелиальной дисфункции) КЭД, адренореактивности, сохранении миокардиального резерва и нормализации значений биохимических маркеров (Тотал NO, экспрессия eNOS, С-реактивный белок, ИЛ-6, ФНО-α). При этом, препараты показали дозозависимый эффект и были примерно равноэффективны. Векторный анализ аддитивных эффектов сочетанного использования ингибиторов ГМГ-КоА-редуктазы симвастатина 8,5 мг/кг, аторвастатина 4,3 мг/кг, розувастатина 8,5 мг/кг и нанопартикулированного розувастатина 11,6 мг/кг с L-аргинином, ингибиторами аргиназ – BEC и аргиназином, а также дарбэпоэтином показал, что при эндотоксин-индуцированной патологии наибольший вероятностный процент аддиции оказался при сочетании розувастатина с аргиназином (3 мг/кг) и дарбэпоэтином (500 мкг/кг), соответственно, 31,9±2,8 и 30,2±2,9%. Обсуждение. Ингибиторы ГМГ-КоА-редуктазы продемонстрировали кардиопротективный (снижение адренореактивности, исчерпания миокардиального резерва, нормализация АД) и эндотелиопротективный (усиление экспрессии eNOS, возрастание уровня NO) эффекты, как в монотерапии, так и в комбинации с некоторыми эндотелиопротекторами (L-аргинин, аргиназин, ВЕС, дарбэпоэтин) в различной степени. Ключевые слова: эндотелиальная дисфункция, оксид азота, статины, эндотелиопротекторы, синдром системного воспалительного ответа.

УДК 615.275.4 DOI: 10.18413/2313-8971-2017-3-4-78-88

Довгань А.П. ЛИГАНД ПЕРИФЕРИЧЕСКИХ ИМИДАЗОЛИНОВЫХ РЕЦЕПТОРОВ НА ОСНОВЕ АМИДОВ ГЕТЕРОЦИКЛИЧЕСКИХ КИСЛОТ С7070: ВЛИЯНИЕ НА ИШЕМИЗИРОВАННЫЕ ТКАНИ

Белгородский государственный национальный исследовательский университет. РФ, г. Белгород, ул. Победы, 85 e-mail: [email protected] Аннотация. Введение. Ишемия является как пусковым моментом, так и этапом патогенеза многих патологических состояний. В связи с этим изучение плейотропных гепатопротекторных свойств агониста периферических имидазолиновых рецепторов C7070 видится интересным с прикладной точки зрения. Материалы и методы. Использованы модели кожного лоскута на питающей ножке (по Кижаеву Е.В.), ишемии-реперфузии печени (по Лопатину Д.А.) и December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 183

изолированного по Лангендорфу сердца крыс (ишемия-реперфузия и доксорубициновая кардиомиопатия). Препараты вводились в дозах: 10 мг/кг С7070; 2 мкг/кг моксонидин и 50 мг/кг метформин. Полученные результаты и их обсуждение: Агонист имидазолиновых рецепторов II типа С7070 в дозе 10 мг/кг в 4,5 раза предотвращает повышение АЛТ и АСТ (332,56±22,05 и 825,49±22,46 АЛТ и АСТ против 526,90±17,97 и 1045,16±80,02 Ед/л в группе контроля) и в 2,5 раза уменьшает площади ишмеических повреждений и некроза (0,058±0,029 и 0,046±0,013 мм2 соответственно) при моделировании 15-ти минутной ишемии печени. Моксонидин и метформин так же обладали гепатопротекторным действием и их коэффициенты составили 44,99 и 36,88 для моксонидина (АЛТ и АСТ) и 34,20/21,02 для метформина (АЛТ/АСТ). Коэффициенты гистологической гепатопротекторной активности составили 72,33 и 38,96 для моксонидина и мтформина соответственно. С7070 (10,0 мг/кг) обладает выраженной дерматопротекторной активностью и предотвращает формирование некроза на 3 и 7 сутки патологии, соответственно на 40%. Дерматопротекторная активность метформина (50 мг/кг) от 3-х к 10-м суткам снижается с 81% до 92%. Дерматопротекторная активность моксонидина (1 мкг/кг) оказалась максимальна на 7-е сутки и составила соответственно 76%. На изолированном сердце крыс С7070 показал протективное действие при ишемии-реперфузии и на модели докирубициновой кардиомиопатии. При этом индекс STTi составил соответственно 8,3; 1,5; 7,9 и 7,8 у.е. в контроле, С7070, моксонидине и метформине. Ключевые слова: С7070, метформин, моксонидин, ишемия, реперфузия, печень, кожный лоскут, изолированное сердце, по Лангендорфу.

УДК: 528.675.1:547.915:615.322/.076.9:616.12-008.46 DOI: 10.18413/2313-8971-2017-3-4-89-99

Ефремова М.П.1 ИЗУЧЕНИЕ ФАРМАКОЛОГИЧЕСКОЙ АКТИВНОСТИ Ивашева А.В.2 ЖИРНОГО МАСЛА ЧЕРНУШКИ ДАМАССКОЙ Корокин М.В.3 В ЭКСПЕРИМЕНТЕ

1Пятигорский медико-фармацевтический институт филиал ФГБОУ ВО ВолгГМУ Минздрава России, г. Пятигорск, пр. Калинина 11, 357532, Россия 2 Межрегиональный центр профессиональной послевузовской подготовки и повышения квалификации специалистов "Развитие", г. Пятигорск, ул. Мира 178, 357532, Россия 3 Белгородский государственный национальный исследовательский университет, г. Белгород, ул. Победы 85, 308015, Россия Автор для корреспонденции, 1e-mail: [email protected]

Аннотация. Введение. Экспериментальному фармакологическому изучению адаптогенной и липидопротекторной активности жирного масла чернушки дамасской послужили литературные данные, богатый химический, макро – и микроэлементный состав. Цель. Изучение фармакологической активности жирного масла чернушки дамасской при моделированной патологии на лабораторных животных. Методы. Раздражающее действие – НЕТ – САМ test и DRAIZE-test, острая токсичность, тест «Открытое поле», метод моделированной гипоксии, плавание с отягощением, тесты «Безвыходная ситуация», «Вращающийся стержень», острая твиновой липидопатология, Д2 витаминная гиперлипидемия, моделированная хроническая сердечная недостаточность (ХСН). December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 184

Результаты и их обсуждение. Жирное масло чернушки дамасской обладает слабым раздражающим действием, при практически не токсичных свойствах. Масло чернушки повышает продолжительность жизни крыс в условиях гипоксии и выносливость в тесте принудительного плавания с отягощением. Курсовое введение масла чернушки нормализует гормонально-медиаторный обмен, при моделированном остром стрессе. Курсовое ведение жирного масла чернушки дамасской нормализует липидный спектр на фоне моделированной ХСН, острой твиновой и субхронической Д2-витаминной гиперлипидемиях . Выводы. Фармакологически доказаны стресспротекторные, актопротекторные и адаптогенные свойства жирного масла чернушки дамасской. Экспериментально обосновано влияние жирного масла чернушки дамасской на липидный обмен при хронической сердечной недостаточности (ХСН), острой твиновой, субхронической Д2 -витаминной липидопотологиях и активность липопротеидлипазы (LPL) в сыворотке крови. Ключевые слова: жирное масло чернушки дамасской, актопротектор, адаптоген, гиперлипидемия.

УДК 615.015.2 DOI: 10.18413/2313-8971-2017-3-4-100-112

Мирошниченко А.Г.1 ВЗАИМОДЕЙСТВИЕ МЕЖДУ НЕКОТОРЫМИ Перфильев В.Ю. АНТИБИОТИКАМИ И АНТИОКСИДАНТАМИ Лысенко И.В. Жернакова Н.И.

Алтайский государственный университет, Проспект Ленина, 61, Барнаул, 656049, Россия Автор для корреспонденции 1e-mail: [email protected]

Аннотация Введение. Бактерицидное действие многих антибактериальных агентов имеет общий механизм, связанный с генерацией гидроксильных радикалов и активацией окислительного стресса. Это подтверждается соотношением между степенью бактерицидного эффекта и уровнем гидроксильных радикалов. Изучение влияния антиоксидантов на антибактериальную активность химиотерапевтических агентов может открыть новые перспективы для улучшения лечения инфекционных заболеваний. Цель. Изучение влияния антиоксидантов на активность химиотерапевтических агентов в отношении условно-патогенных бактерий. Методы. В исследовании изучалось взаимодействие антибиотиков (гентамицин, ципрофлоксацин, цефтазидим) и антиоксидантов (аскорбиновая кислота, N-ацетилцистеин, метилэтилпиридинол) in vitro и in vivo. Мы провели динамическое исследование влияния антиоксидантов в концентрациях 0,5, 1, 2 и 4 мМ на активность антибактериальных агентов in vitro в отношении трех штаммов Klebsiella pneumoniae и трех штаммов Escherichia coli. Влияние антиоксидантов на эффективность антибактериальной терапии изучалось у крыс линии Wistar с экспериментальным бактериальным перитонитом. Результаты и их обсуждение. Исследования in vitro показали, что все антиоксиданты снижают активность гентамицина. Метилэтилпиридинол усиливает действие ципрофлоксацина, цефтазидима. Аскорбиновая кислота усиливает действие цефтазидима. Аскорбиновая кислота, метилэтилпиридинол и December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 185

N-ацетилцистеин (80 мг/кг) снижают антибактериальную активность гентамицина (30 мг/кг) и ципрофлоксацина (50 мг/кг) и не изменяют интенсивность процессов пероксидации в сочетании с этими антибактериальными агентами в условиях экспериментальной инфекции, вызванной K. pneumoniae. Эти антиоксиданты уменьшают прооксидантное действие ципрофлоксацина (50 мг/кг), не влияя на его антибактериальную активность при эшерихиозном перитоните. Аскорбиновая кислота, метилэтилпиридинол и N-ацетилцистеин (80 мг/кг) не снижают нефротоксическое действие гентамицина (30 мг/кг). Выводы. Антиоксиданты оказывают разнонаправленное воздействие на эффективность антибактериальных агентов как in vitro так и in vivo. Комбинирование антиоксидантов с антибиотиками должно сопровождаться предварительными исследованиями in vitro. Ключевые слова: антибактериальные агенты, антиоксиданты, Klebsiella pneumoniae, Escherichia coli, лекарственные взаимодействия.

УДК: 57.084.1 DOI: 10.18413/2313-8971-2017-3-4-113-119

Пересыпкина А.А. КОРРЕКЦИЯ АНГИОПАТИИ СЕТЧАТКИ ПО ГИПЕРТОНИЧЕСКОМУ ТИПУ ПРОИЗВОДНЫМ ДИМЕТИЛАМИНОЭТАНОЛА 19-16 В ЭКСПЕРИМЕНТЕ

ФГАОУ ВО «Белгородский государственный национальный исследовательский университет», 85, ул. Победы, Белгород, 308015, Россия e-mail: [email protected]

Аннотация Введение. У 63% пациентов с гипертонической болезнью есть признаки ангиопатии сетчатки по гипертоничскому типу. Повышение эффективности лекарственной терапии ангиопатии сетчатки является важной задачей фармакологии. Цель. Повысить эффективность фармакологической коррекции ангиопатии сетчатки по гипертоническому типу с использованием нового производного диметиламиноэтанола (ДМАЭ) 19-16. Материалы и методы. Изучены протективные эффекты производного 0ДМАЭ 19-16 в дозах 25 мг/кг, 50 мг/кг на модели ангиопатии сетчатки по гипертоническому типу у крыс-самцов Wistar на фоне введения N-нитро-L- аргинин-метилового эфира (L-NAME) в дозе 12,5 мг/кг/сут в течение 28 суток. Для исследования глазного дна экспериментальных животных применена прямая офтальмоскопия. Электроретинография (ЭРГ) регистрировалась в ответ на одиночную стимуляцию. Биопотенциалы были усилены, усреднены и представлены графически на экране с помощью Biopac-systems MP-150 с программой AcqKnowledge 4.2 (США). Для оценки степени развития функционального расстройства сетчатки использовали отношение амплитуд волн b и a на ЭРГ, коэффициент b/a. Результаты и их обсуждение. Производное ДМАЭ 19-16 в дозе 50 мг/кг предотвращает развитие ишемического повреждения и сосудистых изменений сетчатки по гипертоническому типу, вызванных введением L-NAME в течение 28 суток, в большей степени, чем в дозе 25 мг/кг. Наблюдаемые ретинозащитные эффекты подтверждаются результатами офтальмоскопии и ЭРГ.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 186

Выводы. Поиск новых способов коррекции ангиопатии сетчатки по гипертоническому типу как осложнения артериальной гипертензии является неотложной задачей фармакологии, которую можно решить с использованием нового производного ДМАЭ 19-16. Ключевые слова: ангиопатия сетчатки по гипертоническому типу, производные диметиламиноэтанола, офтальмоскопия, электроретинография.

УДК: 616.36-005.4-089.843-092.9 DOI: 10.18413/2313-8971-2017-3-4-120-131

Разумова М.С.1 ИЗУЧЕНИЕ ФАРМАКОЛОГИЧЕСКОЙ АКТИВНОСТИ Литвинова Е.С. СУБЛИМИРОВАННОЙ КУЛЬТУРАЛЬНОЙ ЖИДКОСТИ Гаврилюк В.П. АЛЛОГЕННЫХ, КСЕНОГЕННЫХ ГЕПАТОЦИТОВ И ФИБРОБЛАСТОВ ДЛЯ КОРРЕКЦИИ ПОРАЖЕНИЙ ПЕЧЕНИ ПРИ ОТРАВЛЕНИИ ЧЕТЫРЕХХЛОРИСТЫМ УГЛЕРОДОМ

Курский государственный медицинский университет, 3, Карла Маркса ул., Курск, 305041 Российская Федерация Автор для корреспонденции, 1e-mail: [email protected]

Аннотация Введение. В настоящее время множество клинико-экспериментальных работ посвящены коррекции нарушений функции гепатоцитов и печени в целом с применением клеточных технологий. Материалы и методы. Экспериментальные исследования выполнены на 175 крысах Вистар с массой от 120 до 140 г. Острое токсическое поражение печени (ОТПП) у лабораторных животных моделировали путем внутримышечного введения четыреххлористого углерода (ЧХУ) в дозе 3 мл/кг в виде 50% раствора в оливковом масле пятикратно с интервалом 24 ч. Результаты и их обсуждение: Применение эссенциале и гипоксена у животных, отравленных ЧХУ, нормализовало общую антиоксилительную активность (ОАА) и активность ферментов антиоксидантной защиты (СОД и каталазу) и корригировало концентрацию малонового диальдегида (МДА) и стабильных метаболитов оксида азота (СМON). Введение животным с острым токсическим поражение печени одновременно белков КЖАГ, эссенциале и гипоксена, по сравнению с предыдущими группами экспериментальных животных, дополнительно нормализует в плазме крови концентрацию МДА и СМON. Совместное применение эссенциале, гипоксена и белков культуральной жидкости аллогенных гепатоцитов, по сравнению с предыдущими группами экспериментальных животных, нормализует концентрацию МДА, активность СОД, сорбциенную емкость эритроцитов и их колическтво в крови, корригирует в большей степени в сторону нормы уровень ацилгидроперекисей. Выводы. Использование культуральной жидкости гепатоцитов интактных крыс аллогенным реципиентам при отравлении ЧХУ в сочетании с фармакологическими препаратами (эссенциале форте Н и гипоксеном) более эффективно, по сравнению с их раздельным использованием, корригирует системные метаболические нарушения, возникающие вследствие воздействия гепатотропного яда. Ключевые слова: аллогенные гапатоциты, ксеногенные гепатоциты, фибробласты, культуральная жидкость, поражение печени четыреххлористым углеродом, эссенциале форте Н, гипоксен. December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 187

УДК: 615.225:617.735-007.23 DOI: 10.18413/2313-8971-2017-3-4-132-150

Тарасова А.П.1 ВЛИЯНИЕ ФАРМАКОЛОГИЧЕСКОГО Даниленко Л.М. ПРЕКОНДИЦИОНИРОВАНИЯ ИНКРЕТИНОМИМЕТИКАМИ Сернов Л.Н. ЭКСЕНАТИДОМ И ВИЛДАГЛИПТИНОМ НА ВЫЖИВАЕМОСТЬ ИШЕМИЗИРОВАННЫХ ТКАНЕЙ

ФГАОУ ВО «Белгородский государственный национальный исследовательский университет», 85, ул. Победы, Белгород, 308015, Россия Автор для корреспонденции 1e-mail: [email protected]

Аннотация. Введение. В настоящее время большое внимание уделяется плейотропным эффектам инкретиномиметиков. В работе представленны имеющиеся данные о противоишемических эффектах инкретиномиметиков. Кроме того, обсуждается реализация защитных эффектов по типу ишемического прекондиционирования, где конечным эффекторным звеном можно считать митохондриальные АТФ – зависимые калиевые каналы. Цели. Изучение протективных эффектовэксенатида и вилдаглиптина дляфармакологической коррекции ишемических повреждений миокарда, печени и кожного лоскута в эксперименте. Методы. В экспериментальном исследовании использовался комплексный подход к изучениюпротивоишемических эффектов инкретиномиметиков: доксорубициновая модель кардиомиопатии, гипо/реперфузия изолированного сердца, ишемия/реперфузия печени и моделирование кожного лоскута на питающей ножке. Результаты и их обсуждение. Эксенатид (10 мкг/кг/сутки) и вилдаглиптин (0,2 мг/кг/сутки) проявляют кардиопротективный эффект на доксорубициновой модели патологии, что выражается в снижении коэффициента диастолической дисфункции (StТТI), соответственно до 5,3±0,1 ус.ед. и 6,5±0,2ус.ед. посравнению с группой контроля 8,3±0,1 ус.ед. и на модели гипо/реперфузии изолированного -6 -4 сердца крыс эксенатид (10 моль/л) и вилдаглиптин (10 моль/л), препятствуют снижению левожелудочкового давления (ЛЖД).Эксенатид (10 мкг/кг) и вилдаглиптин (0,2 мг/кг) предотвращают некротизацию кожного лоскута в 1,5 и 1,3 раз в сравнении с группой контроля.На модели ишемии/реперфузии печени эксенатид обладают гепатопротекторным эффектом. Все протективные эффекты инкретиномиметиков нивелировались при совместном применении с блокатором АТФ-зависимых калиевых каналов глибенкламидом (0,4 мг/кг). Выводы. В ходе проведенного исследования было выявлено, что эксенатид в дозе 10 мкг/кг/сут ивилдаглиптин в дозе 0.2 мг/кг обладают выраженным кардиопротекторным, гепатопротекторными выраженным цитопротекторным эффектом на модели изолированного кожного лоскута на питающей ножке. АТФ- зависимых калиевые каналы являются эффекторным механизмом в реализации протективных эффектов инкретиномиметиков. Ключевые слова: инкретиномиметики, эксенатид, вилдаглиптин, доксорубициноваякардиопатия, изолированное сердце крыс, ишемия.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 188

УДК: 615.014.22:616.5-003.923 DOI: 10.18413/2313 -8971-2017-3-4-151-159

Воронков А.В.1 ВЛИЯНИЕ НОВЫХ ФОРМ ДЛЯ НАРУЖНОГО ПРИМЕНЕНИЯ Гамзелева О.Ю. НА ВАЗОДИЛАТИРУЮЩУЮ ФУНКЦИЮ ЭНДОТЕЛИЯ И КОНЦЕНТРАЦИЮ ЭНДОТЕЛИАЛЬНЫХ СИНТАЗ ОКСИДА АЗОТА У КРЫС С ЭКСПЕРИМЕНТАЛЬНОЙ МОДЕЛЬЮ ПАТОЛОГИЧЕСКОГО РУБЦА НА РАННИХ СРОКАХ ЗАЖИВЛЕНИЯ

1Пятигорский медико-фармацевтический институт – филиал ФГБОУ ВО ВолгГМУ Минздрава России, 11, пр. Калинина, г. Пятигорск, 357532, Россия. Автор для корреспонденции, 1e-mail: [email protected]

Аннотация Введение. Согласно статистическим данным, во всем мире сохраняется тенденция к росту числа пациентов с келоидными и гипертрофическими рубцами на месте повреждения кожных покровов, что в большинстве случаев связано с отсутствием эффективных методов профилактики данного рода осложнений. Поэтому разработка новых способов фармакологической коррекции, позволяющих уже на ранней стадии нормализовать процесс заживления раны, представляется весьма актуальным. Одним из перспективных направлений поиска веществ, пригодных для профилактики патологических рубцов, является изучение природных соединений, обладающих эндотелиопротекторной активностью, поскольку, согласно современным представлениям, именно дисфункция эндотелия лежит в основе патогенеза данного осложнения. Цель. Все исследования были выполнены на 60 крысах-самцах линии Wistar, разделенных на 6 экспериментальных групп. В качестве объектов исследования выступили новые формы для наружного применения на основе флавоноидов ПМФИ-92 и ПМФИ-93 и препараты сравнения «Контрактубекс», «Эгаллохит». Материалы и методы. Изучено влияние новых составов ПМФИ-92 и ПМФИ-93 на вазодилатирующую функцию эндотелия сосудов кожи животных с химическим ожогом при введении модификаторов синтеза оксида азота: ацетилхолина (АЦХ), L-аргинина, нитро-L-аргинин метилового эфира (L-NAME). Методом твердофазного иммуноферментного анализа (ИФА) установлено влияние местной фармакологической поддержки при данной патологии на уровень эндотелиальной (eNOS), нейрональной (nNOS) и индуцибельной (iNOS) синтаз оксида азота. Результаты и их обсуждение. Применение предложенных новых форм на основе природных соединений ПМФИ-92 и ПМФИ-93 позволяет улучшить вазодилатирующую функцию эндотелия уже на ранних сроках заживления ран, при этом наблюдается увеличение концентрации eNOS и nNOS, а также снижение концентрации iNOS. Вывод. Полученные нами экспериментальные данные свидетельствуют о выраженном позитивном влиянии местной аппликации составов ПМФИ-93 и ПМФИ-92 на вазодилатирующую функцию сосудов кожи, а также позволяют восстановить баланс между активностями различных форм синтаз оксида азота. Ключевые слова: Эндотелиальная дисфункция, келоидные и гипертрофические рубцы, ПМФИ-92, ПМФИ-93, eNOS, nNOS, iNOS.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 189

КЛИНИЧЕСКАЯ ФАРМАКОЛОГИЯ

УДК 616-002.3:615.03+615.37 DOI: 10.18413/2313-8971-2017-3-4-160-176

Земскова В.А. ОПТИМИЗАЦИЯ ИММУНОФАРМАКОТЕРАПИИ Золоедов В.И.1 ГНОЙНО-ВОСПАЛИТЕЛЬНЫХ ЗАБОЛЕВАНИЙ Попова О.А.

Воронежский государственный медицинский университет им. Н.Н. Бурденко Министерства здравоохранения Российской Федерации, 10, ул. Студенческая, Воронеж, Россия, 394036 Автор для корреспонденции, 1e-mail: [email protected]

Аннотация Введение. В работе представлены результаты клинических исследований гнойно- воспалительных заболеваний. Цель. Изучить механизм влияния патогенеза на манифестацию иммунопатологии, эффективность и механизмы фармакологического иммунокорригирующего действия на клинических моделях гнойно-воспалительных заболеваний – глубокой пиодермии (ОГН), хронического сальпингоофорита (ОХСО), хронического пиелонефрита (ОХПН). Методы. Под наблюдением находилось 300 больных, страдающих гнойно- воспалительными заболеваниями различного генеза – ОГП, ОХПН, ОХСО. Все пациенты до и после традиционного лечения с моно- и комбинацией иммуномодуляторов различного происхождения – ронколейкином, ликопидом, суперлимфом, деринатом, полиоксидонием, тимогеном, галавитом подвергались стандартному гемато-иммунологическому обследованию и дополнительно типовым для каждой нозоформы заболеваний – бактериологическим и клиническим исследованиям. Математический анализ полученных данных определял достоверные отличия параметров от заданного уровня, а также дополнительно – ключевые, сигнальные тесты, формализованные в виде формул. Результаты и их обсуждение. Установлено значимое влияние трех видов патогенеза гнойно-воспалительных заболеваний на характер и выраженность гемато-иммунологических расстройств у больных, эффективность и механизмы действия модулятора галавита; недостаточное нормализующее действие традиционного лечения больных; повышение его активности за счет дополнительного назначения 7 вариантов монокорректоров и трех их сочетаний; способность модулирующих препаратов влиять не только на иммунологические, но и на гематологические, бактериологические и клинические характеристики больных, т. е реализовать общеорганизменное действие. Формализованная оценка вариаций клинико-лабораторных параметров пациентов позволила определить диагностически значимые мишени каждого иммунотропного фактора, интегрально сопоставить эффективность влияния отдельных методов терапии, установить механизмы модификации функции лимфоидной системы при моно- и комбинированной иммунокоррекции, за счет инверсионного математического анализа идентифицировать лабораторные показания для выбора конкретных вариантов комплексного лечения больных.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru Research Result: Pharmacology and Clinical Pharmacology. 2017;3(4) 190

Выводы. Результатом обобщения и математического анализа достигнутых результатов явилось формулирование 6 уровневого алгоритма выявления и адресного лечения иммунокомпрометированных лиц в виде 7 утвержденных программ для ЭВМ на основе введения в компьютер итогов лабораторного обследования больных. Ключевые слова: гнойно-воспалительные заболевания, иммунотерапия, иммунологические расстройства, формализованная оценка.

December. 2017. 3(4). Research Result: Pharmacology and Clinical Pharmacology rrpharmacology.ru