A Comparative Evaluation of the Efficacy of Dimethylaminoethanol

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Volume 6 (1) 2020

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Research Results in Pharmacology

CONTENTS

Spasov A.A., Kucheryavenko A.F., Gaidukova K.A., Kosolapov V.A., Zhukovskaya O.N. Antiplatelet activity of new derivatives of benzimidazole containing sterically hindered phenolic group in their structure 1 Bratchikov O.I., Tyuzikov I.A., Dubonos P.A. Clinical and experimental rationale for antioxidant therapy of chronic bacterial prostatitis 11 Bobrakova A.A. Effect of complex therapy with Cortexin on the state of the body’s regulatory systems in patients with rosacea 21 Lokteva T.I., Rozhkov I.S., Gureev V.V., Gureeva A.V., Zatolokina M.A., Avdeyeva E.V., Zhilinkova L.A., Prohoda E.E., Yarceva E.O. Correction of morphofunctional disorders of the cardiovascular system with asialized erythropoietin.and arginase II selective inhibitors KUD 974 and KUD 259 in experimental preeclampsia 29 Palikov V.A. Palikova Yu.A., Dyachenko I.A. Study of protective properties of butyrylcholinesterase in acute anticholinesterase poisoning on BChE-KO and BALB/c mice 41 Demchenko S.A., Koklin I.S., Koklina N.Yu. Role of Arginase 2 as a potential pharmacological target for the creation of new drugs to correct cardiovascular diseases 47 Pokrovskaya L.A., Zubareva E.V., Nadezhdin S.V., Lysenko A.S., Litovkina T.L. Biological activity of mesenchymal stem cells secretome as a basis for cell-free therapeutic approach 57 Severina H.I., Georgiyants V.A., Kovalenko S.M., Avdeeva N.V., Yarcev A.I., Prohoda S.N. Molecular docking studies of N-substituted 4-methoxy-6-oxo-1-aryl- pyridazine-3-carboxamide derivatives as potential modulators of glutamate receptors 69 Lebedev A.A., Bessolova Yu.N., Efiov N.S., Bychkov E.R., Droblenkov A.V., Shabanov P.D. Role of orexin peptide system in emotional overeating induced by brain reward stimulation in fed rats 81 Bontsevich R.A., Gavrilova A.A., Adonina A.V., Vovk Ya.R., Goncharova N.Y., Batisheva G.A., Cherenkova O.V., Myronenko O.V., Luchinina E.V., Barysheva V.O., Ketova G.G., Bochanova E.N., Tilekeeva U.M, Dauletbekov N.D. Pharmacotherapy and other aspects of senior medical students’ knowledge in community-acquired pneumonia: the final results of the KNOCAP II project 93

Research Results in Pharmacology 5(4) 2019

Research Results in Pharmacology 6(1): 1–9 UDC: 615:547.785.5:616.155.2 DOI 10.3897/rrpharmacology.6.50373

Research Article

Antiplatelet activity of new derivatives of benzimidazole containing sterically hindered phenolic group in their structure

Alexander A. Spasov1, Aida F. Kucheryavenko1, Ksenia A. Gaidukova1, Vadim A. Kosolapov1, Olga N. Zhukovskaya2

1 Volgograd State Medical University, 1 Pavshikh Bortsov Sq., Volgograd 400131, Russia 2 Research Institute of Physical and Organic Chemistry, Southern Federal University, 194/2 Stachki Av., Rostov-on-Don 344090, Russia

Corresponding author: Ksenia A. Gaidukova ([email protected])

Academic editor: Oleg Gudyrev ♦ Received 3 December 2019 ♦ Accepted 10 January 2020 ♦ Published 28 February 2020

Citation: Spasov AA, Kucheryavenko AF, Gaidukova KA, Kosolapov VA, Zhukovskaya ON (2020) Antiplatelet activity of new derivatives of benzimidazole containing sterically hindered phenolic group in their structure. Research Results in Pharmacology 6(1): 1–9. https://doi.org/10.3897/rrpharmacology.6.50373

Abstract Introduction: Cardiovascular diseases are currently the leading cause of global disability and mortality. According to the centers for disease control and prevention, the average life expectancy of a person would be 10 years longer but for a high prevalence of cardiovascular diseases, and if antiplatelet drugs and special therapy were used. Materials and methods: Antiplatelet activity of the novel benzimidazole derivatives containing a sterically hindered phenolic group in their structure has been investigated in vitro, using a model of ADP-induced platelet aggregation of rabbit’s plasma. The compounds exhibiting high antiplatelet activity and acetylsalicylic acid, as a reference drug, were examined for antioxidant properties in an ascorbate-dependent model of lipid peroxidation. Results: It was established that the compounds with high antiplatelet activity demonstrated the pronounced antioxidant action. The compound RU-1144 (1-(3,5-ditretbutyl-4-hydroxyphenyl) -1-hydroxypropyl)-phenyl-pyrimidobenzimidazole hydrochloride), in in vitro experiments, had a pronounced antiplatelet activity, surpassing the reference drug acetylsalicylic acid by 21.8 times; in the study of antioxidant activity, the leader compound was inferior to the reference drug dibunol by 1.7 times. By inhibiting intravascular platelet aggregation in vivo, this compound exceeded acetylsalicylic acid by 1.5 times and was slightly inferior to clopidogrel by 1.4 times. Discussion: Benzimidazole derivatives with a hindered phenolic substituent in their structure exhibited antiplatelet and antioxidant properties. It was established that the compounds with high antiplatelet activity demonstrated the pronounced antioxidant action. Conclusion: The chemical class of benzimidazole derivatives with a hindered phenolic substituent in their structure is promising for the search for new antiaggregant and antioxidant drugs.

Keywords benzimidazole, antiplatelet activity, acetylsalicylic acid, antioxidant activity.

Copyright Spasov AA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Spasov AA et al.: Study of antiplatelet and antioxidant activity of new benzimidazole derivatives

Introduction Considering that oxidative stress is caused by the interaction of platelets and blood vessels (Sies 2015, Fuentes One of the most relevant causes of deaths all over the et al. 2019), it is of importance to search for and develop world are cardiovascular diseases and their consequences. combination antiplatelet and antioxidant drugs (Reinisch Even with the constantly improving quality of life and et al. 2001). development of the pharmaceutical industry, the number It is known that in the treatment of pathological condi- of people suffering from cardiovascular diseases is gro- tions of the cardiovascular system, combination therapy is wing every day. The process of blood clotting plays one used, not only when using antiplatelet agents of various of the most important roles in the pathogenesis of ische- groups, but also in combination with antioxidant drugs to mic disorders in various organs and tissues of the human prevent hypoxic conditions associated with heart attacks, body and, thus, making the use of antiplatelet agents and strokes, etc. (Szabó et al. 2017, Gaba et al. 2018). That is other therapies for their treatment and prevention of vi- why the dual use of antiplatelet and antioxidant drugs is tal importance (Gaba et al. 2018). The basic pathologies highly recommended nowadays in the prevention of patho- accompanied by inflammatory and atherosclerotic com- genetic development of thrombosis (Anjum et al. 2018). plications are the processes of macro- and microthrombo- The chemical class of substituted heterocyclic ben- genesis, which can be the consequence of hyperreactivity zimidazoles is considered to be the base structure of new of platelets and contribute to an increased risk of ather- drugs based on it, which was shown by a wide range of othrombotic events (Szabó et al. 2017). Recently active biological activity shown before (Spasov et al. 1997, forms of oxygen and nitrogen have been identified as the Anisimova et al. 2002, 2006, Spasov et al. 2009, Kuch- main causal agents of this pathology. Reactive oxygen eryavenko et al. 2014, Kucheryavenko 2016). The pre- species, such as active forms of oxygen and nitrogen, play vious studies determined the ability of benzimidazole an important role in regulating platelet responses to col- derivatives containing spatially hindered phenol in their lagen and are collagen-dependent on thrombus formation. structure to inhibit oxidative stress. In this connection, it But at the same time, they play a vital role in the lipid appeared interesting to study antiplatelet activity in these peroxidation, which can lead to an abnormally increased compounds, since the creation of drugs combining antithrombogenic potential of blood. As a result, the rate of platelet, antioxidant activities may be promising for the damaging the vascular endothelium and of atherosclero- treatment of pathologies associated with increased blood tic complications is increasing. Currently, the search for thrombogenic potential. new antioxidant drugs to prevent the excessive formation Some other previous studies determined the ability of of free radicals that are involved in the pathogenesis of benzimidazole derivatives containing spatially hindered many pathological conditions, such as hypoxic, ischemic phenol in their structure to exhibit pronounced antioxi- and reperfusion injuries of organs, especially the brain, dant activity (Baldisserotto et al. 2020). That is why, in myocardium, and also ageing processes, is highly rele- addition to studying an antiplatelet activity, these com- vant (Spasov et al. 2013, Jang et al. 2015, Bisht et al. pounds were studied in the ascorbate-dependent lipid per- 2017, Kattoor et al. 2017). oxidation (LPO) test. The literature review (Baldisserotto et al. 2020), as well However, the well-known antiplatelet drugs very often as the results of the studies previously conducted at the De- do not have a required activity, and also have a lot of side partment of Pharmacology and Bioinformatics at Volgograd effects of varying severity (Guthrie 2011). That is why State Medical University showed that there were heterocy- the need for searching for new inhibitors of the platelet clic nitrogen-containing compounds capable of blocking aggregation process with a more pronounced activity and the aggregation of platelets and reducing lipid peroxidation fewer side effects, remains highly relevant. (Kucheryavenko et al. 2014, Kucheryavenko 2016). There have been also studies that proved that the main mechanism of antiplatelet action of the class of benzim- Materials and methods idazole derivatives was the inhibition of thromboxane synthesis; the similar results were also obtained by for- This paper reports on an experimental study of the effect eign researchers (Chang et al. 2017, Houston et al. 2017, of 24 new benzimidazole derivatives containing spatially Baldisserotto et al. 2020). hindered phenols (Research Institute of Physical and Or- Oxidative stress is proved to contribute to the develop- ganic Chemistry of Southern Federal University) on pla- ment of cardiovascular diseases (Fuentes et al. 2019) and telet aggregation and ascorbate-dependent lipid peroxi- may play an important role in platelet activation. This may dation in vitro. The experiments were performed on 10 be due to the direct effect of oxidative stress on platelets, rabbits, weighing 3–3.5 kg, and 40 white outbred male as well as to its indirect effect that causes the destruction rats kept in the vivarium (temperature 22–24 °C, relative of labile vessels, vascular agents originating from the en- humidity 40–50%) with natural illumination on a standard dothelium, such as nitric oxide. Oxidative stress caused by diet, following the rules of good laboratory practice when platelets through several intracellular sources, which also conducting preclinical studies in the Russian Federation, affect vascular tone, is important for blood flow and blood as well as the rules and international recommendations of clotting in blood vessels (Li et al. 2014, Kattoor et al. 2017). The European Convention for the Protection of Vertebrate Research Results in Pharmacology 6(1): 1–9 3

Animals Used in experimental Studies (1997). All the pro- The method for analyzing the relationship between cedures with the animals were carried out in accordance the antiplatelet and antioxidant activities of hindered with the standards set forth in the eighth edition of Guide phenols was carried out by the probabilistic histogram for the Care and Use of Laboratory Animals and ARRIVE method (Mandel 1988). For this, all the studied sub- (Animal Research: Reporting of In Vivo Experiments). stances were divided into classes with different levels The antiplatelet activity was studied on the model of of activity. To determine the boundaries of the class of ADP-induced platelet aggregation according to the meth- the compounds with a high antiplatelet activity, a cluster od described in (Вorn 1962) in modification of Gabbasov analysis of data was performed, according to the Δ% in- V.A. (Gabbasov 1989) on a laser platelet aggregation ana- dicator in the studied concentrations: highly active – Δ% lyzer Biola 220 LA (Russia). The studies were performed ≥ 50%; moderately active – Δ% ≥ 25% and low-active on platelet-rich rabbit plasma, which had been obtained – Δ% ≥ 20%. by the method of VA Lyusov. and Belousova Yu.B. (1971). The toxicity study of the most active compounds was Adenosine-5-diphosphoric acid (ADP) (Sigma, USA), at carried out following the requirements and instructions a final concentration of 5 μM, was used as an aggregation of the Federal Service for Supervision of Healthcare and inducer. The tested compounds and the reference prepa- Social Development (Makarov et al. 2012). Acute toxicity ration – acetylsalicylic acid – were studied at a concen- was determined on 75 white nonlinear male mice, weigh- tration of 100 μM. The level of aggregation was evaluating 20–22 grams, with intraperitoneal administration. The ed by a degree of aggregation, defined as the maximum deaths of animals were recorded within two weeks. The increment of light transmission after the addition of the toxicological indicator – LD50 was calculated according to inductor. To evaluate the activity of the compounds, Δ% the Litchfield-Wilcoxon’s method. inhibition of the functional activity of platelets was deter- In the last stage of the experiment, the dependence mined. For the most active substances, a dose-dependent of the antiplatelet activity of benzimidazole derivatives antiplatelet activity was studied to calculate IC50 (inhibito- having hindered phenolic substituent in their chemical ry concentration, inhibiting platelet aggregation by 50%), structure was determined. Statistical processing of the ex- using the regression analysis method in Microsoft Excell. perimental data was carried out using the Mann-Whitney The antioxidant activity of substances was studied in criterion by means of GraphPad 5.0 and Microsoft Excell experiments in vitro on the model of ascorbate-dependent 2007 statistical software package. lipid peroxidation (Lankin 1975). The compounds were studied in a concentration range of 1×10-7-1×10-5 M. As a substrate, 4% rat liver homogenate was used. The reaction Results was initiated with 50 mM ascorbic acid (Chemapol, Czech Republic). The oxidation rate was judged by the accumu- While searching for compounds with antiplatelet and an- lation of malondialdehyde in the reaction with thiobarbi- tioxidant activity, 13 highly active compounds were iden- turic acid (Fluka, Switzerland). The optical density of the tified among 26 new benzimidazole derivatives having coloured product was measured at a wavelength of 532 a shielded phenolic substituent in their structure, which nm on a PD-303 UV spectrophotometer (APEL, Japan) in statistically significantly exceed acetylsalicylic acid. The a cuvette with an optical path length of 10 mm. The activ- antiplatelet effect of 2 substances was comparable to that ity of the substances was evaluated in% in relation to the of the reference drugs, the other 12 compounds were infe- sample without the compound. Dibunol (Merck, Germa- rior to it by activity (Table 1). ny) was used as a reference drug. The calculation of IC50 In addition to the study of antiplatelet activity, these (inhibitory concentration, suppressing LPO by 50%) was compounds were studied in the ascorbate-dependent li- performed using regression analysis in Microsoft Excell. pid peroxidation test. Regarding the inhibition of lipid The effect of the substance on the functional activity of peroxidation, among the 26 tested compounds, 12 highly platelets in an in vivo test, with biological material being active substances were revealed that were comparable to examined ex vivo according to the Born (1962) method, dibunol (Table 1). modified by Gabbasov (1989) on a Biola LA-220 laser Among the most active 13 compounds, in relation to platelet aggregation analyzer. For the test, blood was sam- the inhibition of platelet aggregation in vitro, a dose-ef- pled from the abdominal aorta of 72 outbred adult male fect relation was studied for calculating IC50, presented in rats, weighing 250.0–300.0 g (anaesthetized with a solu- Table 2. As you can see, the first three compounds showed tion of chloral hydrate (400 mg/kg)). Two hours before the greatest activity. the study, the compounds and comparison drugs were ad- The next study was on the correlation dependence of ministered intragastrically, using a metal atraumatic intra- antiplatelet and antioxidant activities. In the group of gastric probe. All the investigated samples were dissolved the compounds with high antiplatelet activity, a posi- in distilled water. The control group of the animals was tive correlation was observed towards the second type injected with a solvent in an equivalent volume. Next, of activity (Table 3). The correlation coefficient for this platelet-rich plasma was obtained, and the studies were group was positive and amounted to 0.73. When com- carried out according to the method for studying the func- paring other groups, this indicator did not confirm the tional activity of platelets in vitro described above. correlation dependence. 4 Spasov AA et al.: Study of antiplatelet and antioxidant activity of new benzimidazole derivatives

Table 1. Effect of Benzimidazole Derivatives Having a Hindered Phenolic Substituent on ADP-induced (5 μM) rabbit platelet aggregation and on lipid peroxidation (LPO) in vitro (M ± m) (n = 6).

№ Tested compound Inhibition of platelet aggregation (Δ%) at a Antioxidant activity at a concentration of 100 μm concentration of 100 μm (Mean ± SEM) (Mean ± SEM) 1 RU-873 91.9 ± 4.31*# 61.8 ± 3.41* 2 RU-1144 91.9 ± 2.53*# 87.6 ± 6.52*@ 3 RU-1263 86.5 ± 3.72*# 80.7 ± 2.34* 4 RUP-4b 86.1 ± 2.85*# 36.8 ± 5.21*@ 5 RUP-7b 84.4 ± 6.36*# 35.4 ± 4.54*@ 6 RUS-193 84.3 ± 4.39*# 87.0 ± 6.65*@ 7 RU-871 82.0 ± 6.33*# 73.8 ± 2.23* 8 RU-1261 80.0 ± 8.11*# 67.9 ± 5.13* 9 RU-1249 77.7 ± 6.61*# 77.2 ± 7.85* 10 RU-903 69.9 ± 8.34* 76.1 ± 2.12* 11 RUP-6b 69.8 ± 7.91* 36.3 ± 4.65*@ 12 RU-1180 67.7 ± 5.83* 88.8 ± 2.51*@ 13 RUP-5b 65.9 ± 6.25* 43.9 ± 8.75*@ 14 RUP-3b 45.3 ± 1.73* 31.1 ± 4.89*@ 15 RUS-191 40.5 ± 4.52* 48.0 ± 3.98*@ 16 RU-1250 36.4 ± 5.53* 0 17 RUP-2b 35.0 ± 1.85*# 46.8 ± 7.98*@ 18 RUS-190 34.1 ± 4.89*# 19.5 ± 6.71*@ 19 RU-1265 27.9 ± 5.37*# 87.6 ± 7.12*@ 20 RUP-2 27.3 ± 4.33*# 61.7 ± 3.67* 21 RUCH-6 26.0 ± 1.91*# 20.4 ± 6.81*@ 22 RU-1260 19.4 ± 7.02*# 65.4 ± 5.45* 23 RU-1251 13.7 ± 4.80*# 0 24 RU-887 12.8 ± 3.43# 0 25 RUS-198 9.1 ± 3.53# 30.1 ± 5.15*@ 26 RUCH-2 3.7 ± 0.46*# 49.6 ± 6.49*@ 27 Acetylsalicylic acid 53.1 ± 4.40* – 28 Dibunol – 85.8 ± 2.78* Note: * – (p≤0.05) changes are statistically significant in relation to the control, Mann-Whitney test; # – (p < 0.05) changes are statistically significant in relation to the effect of the reference drug acetylsalicylic acid; @ – (p < 0.05) changes are statistically significant in relation to the effect of the reference drug dibunol; n – number of the animals tested.

Table 2. Inhibiting Activity (IC50) of New Benzimidazole Derivatives and Acetylsalicylic Acid (Mean ± SEM) (n = 6).

№ Tested compound Inhibition of platelet aggregation, Δ% (Mean ± SEM) IC50, μM Tested concentration, μM 100 10 1 1. RU-1263 86.5 ± 3.72† 45.4 ± 3.96† 38.3 ± 4.40† 5.3 2. RU-1144 91.0 ± 2.53† 52.8 ± 0.96† 29.9 ± 4.60† 5.5 3. RU-1261 80.0 ± 8.11† 53.5 ± 1.77† 32.8 ± 5.54† 5.9 4. RU-871 82.0 ± 6.33† 49.5 ± 2.72† 25.5 ± 1.33† 8.3 5. RUP-7b 84.4 ± 6.36† 49.9 ± 1.00† 15.1 ± 2.73† 10 6. RU-873 91.9 ± 4.31† 31.0 ± 2.56† 18.6 ± 1.05† 12 7. RUP-4b 86.1 ± 2.85† 38.0 ± 4.36† 19.2 ± 3.13† 12 8. RUP-6b 69.8 ± 7.91† 44.0 ± 2.22† 22.7 ± 7.39† 16 9. RU-903 69.9 ± 8.34† 36.0 ± 5.19† 29.5 ± 5.40† 17 10. RUS-193 84.3 ± 4.39† 28.9 ± 5.70† 5.9 ± 0.97 18 11. RU-1249 77.7 ± 6.61† 28.5 ± 2.44† 15.4 ± 2.16† 20 12. RUP-5b 65.9 ± 6.25† 40.7 ± 3.04† 18.7 ± 4.59† 22 13. RU-1180 67.7 ± 5.83† 34.7 ± 3.46† 26.4 ± 1.10† 23 14. Aspirin 53.1 ± 5.40† 26.8 ± 1.77† 5.6 ± 1.25 120 Note: † – (p≤0.05) changes are statistically significant in respect to the control, the Mann-Whitney test; n – number of the animals tested.

Table 3. Ranking of Tested Substances by Correlation Indicators The substances that showed the highest antiplatelet

Between Antiplatelet and Antioxidant Activities on the Models activity were selected to study the IC50 antioxidant activ- of ADP-induced Platelet Aggregation and Ascorbate-dependent ity, compared with that of dibunol (Table 4). The study Lipid Peroxidation in Vitro. showed that all the substances were inferior in this activity to the reference drug; however, the sample under code Activity types Correlation coefficient Highly active Moderately Low-active RU-1144 turned out to be the closest in this value to IC50 active To determine the leader compound. In vivo studies Antiplatelet activity 0.730582 -0.65583 0.61985 were performed to inhibit platelet aggregation of the Antioxidant activity three compounds under codes RU-1144, RU-1261, and Research Results in Pharmacology 6(1): 1–9 5

Table 4. Antioxidant Activity of Compounds RU-1144, RU-1261, RU-1263 on the Model of Ascorbate-dependent Lipid Peroxida- tion (in Vitro Experiments) (Mean ± SEM, n = 6).

Code Inhibition of antioxidant activity (∆%, Mean ± SEM) IC50 1×10-5 1×10-6 5×10-6 2.5×10-6 1×10-6 RU-1144 87.6 ± 6.52* 79.71 ± 0.7* 61.13 ± 1.1* 48.53 ± 1.6* 35.36 ± 2.8 2.12×10-6 RU-1261 67.9 ± 5.13* 59.71 ± 0.7* 44.71 ± 1.4* 24.20 ± 1.9* 6.25 ± 1.4 5.53×10-6 RU-1263 80.7 ± 2.34* 76.38 ± 0.8* 53.30 ± 2.6* 31.09 ± 1.8* 17.33 ± 2.7* 4.13×10-6 Dibunol 85.8 ± 2.78* 74.1 ± 3.12* – – 45.3 ± 1.8* 1.23×10-6 Note: * – (p < 0.05) statistical reliability of differences in comparison with baseline

RU-1263, which showed the highest antiplatelet activity in vitro, in order to calculate the ED50 index. As a result, it was shown that, by this indicator, compound RU-1144 was superior to the other two substances and the reference drug acetylsalicylic acid, and was also comparable to clopidogrel (Fig. 1). The presence of data from the in vitro studies and acute daily toxicity made it possible to further calculate the con- ditinoal range of the therapeutic effect (conditional therapeutic index (CTI)) (Table 5). Tested compound RU-1144 exceeded acetylsalicylic acid by CTI by 3.7 times. Next, the dependence of antiplatelet activity on the chemical structure of the compounds was studied. Figure 1. ED50 of antiplatelet activity of the compounds, All the tested compounds that were studied are conju- acetylsalicylic acid (ASA) and clopidogrel with a single gates of 2,6-di-tret-butylphenol and a fused heterocyclic intragastric administration to male rats on the model of ADP- nucleus. The structure of the latter makes it possible to iso- induced (5 μM) platelet aggregation. late 6 scaffolds: 1H-benzimidazoles and salts of 1H-benzimidazolium-3, N9-2,3-dihydroimidazobenzimidazoles, Table 5. Antiplatelet Activity (IC50), Acute Daily Toxicity 3,5-dihydrotriazinobenzimidazoles, 2,3,4,10-tetrahydro- (LD50), and the Conventional Therapeutic Index (CTI) of New pyrimido-benzimidazoles, 2,3- dihydroimidazobenzimi- Benzimidazole Derivatives and Acetylsalicylic Acid. dazoles and 4H-triazole-benzimidazoles (Fig. 2). № Code ED , mg/kg LD mg/kg CTI LD /ED The highest level of activity was more specific for 50 50 50 50 1 RU -1144 18.8 749.2 39.9 2,3,4,10-tetrahydropyrimidobenzimidazole derivatives. 2 Aspirin 28.5 310.0 10.9 All 5 compounds of this group at a concentration of 100 μM blocked platelet aggregation by more than 70% and exceeded the reference drug acetylsalicylic acid. The most to the level of antiplatelet activity, which seem to indicate active of all the tested compounds of this class were RU- the presence of non-additive interactions among radicals. 873 and RU-1144, which are hydrochlorides. RU-871 hy- The only derivative of 2,3-dihydrothiadiazinobenzimi- drobromide and RU-1249 succinate are 10% less active. dazole RUS-193, where the hindered phenolic substituent The introduction of methyl substituents in the 7,8-dime- is in the second position, showed a pronounced antiag- thyl-2,3,4,10-tetrahydropyrimido-benzimidazole hyd- gregant activity (84.3%). The least active (suppression of robromide molecule RU-903 led to a loss of 20% of the platelet aggregation by 40% or less at a concentration of activity. 2,3-dihydroimidazobenzimidazole hydrochloride 100 μM) are 1H-benzimidazole derivatives, containing a RU-1180, which is the closest homolog of the leader com- phenolic substituent in the N1 position, and 3,5-dihydrotri- pound RU-1144, also had rather a high activity (67.7%). azinobenzimidazole derivatives RUS-190 and RUS-191, Besides, a high activity (65.9–86.19% in relation to the 4H-triazole-benzimidazole RUS- 198. It can be concluded suppression of ADP-induced platelet aggregation) was that the increased electron density in the 3rd ring of the noted in the compounds of the 1H-benzimidazolium salt condensed heterocyclic system is an unfavorable factor. group, especially those that are dihydrobromides (RUP- Thus, the highest number of highly active compounds 4b, RUP-5b, RUP-6b, RUP-7b) and hydrochlorides con- belongs to the group of derivatives of 2,3,4,10-tetrahydro- taining propyl (RU-1261) or propenyl (RU-1262) radicals pyrimidobenzimidazole and 1H-non-imidazolium salts, in the N1 position. The other 1H-benzimidazolium hyd- whereas the representatives of the other scaffolds had no robromides showed a significantly less activity or were pronounced activity. An exception is active 2,3-dihydrothi- hardly active (RUCh-2, RU-1260). Other substituents adiazinobenzimidazole RUS-193, which, due to its planar (alkyl, benzyl, amino) were found in the cluster of both structure, is very different from the other tested derivatives. active and inactive compounds. The limited number of de- The group of substances derived from 1H-benzimida- rivatives available for the study did not make it possible to zoles is the largest in terms of the number of representa- make a conclusion about the contribution of each of them tives, most of which showed a pronounced antiplatelet ac- 6 Spasov AA et al.: Study of antiplatelet and antioxidant activity of new benzimidazole derivatives

Figure 2. General formula of derivatives of N-7-ditretbutyl-4-hydroxyphenyl pyrimidobenzimidazoles (A), N-9-ditretbutyl-4-hydroxyphenyl benzimidazoles (B), N-9-ditretbutyl-4-hydroxyphenyl triazinobenzimidazoles (C), 11- 2,6-ditretbutyl-1-hydroxyphenyl-2,3-thiadiazinobenzimidazoles (D); N-7-ditretbutyl-4-hydroxyphenyl-N9-2,3-dihydroimidazobenzimidazoles (E) and N-7-ditretbutyl-4-hydroxyphenyl-triazolobenzimidazoles (F). tivity. Most of the representatives of this group had a salt methyl or propanoic acid residue – in the second has no residue represented by hydrobromide in their structure. high antiplatelet properties. However, the compounds with dihydrobromides in the The last two groups of substances, where the main structures showed a higher activity. The tested samples structures were N9-2,3-dihydroimidazobenzimidazoles of this group, in which the hindered phenol substituent and triazolobenzimidazoles, and in the first position of was in the first position, did not show pronounced activity each derivative there was a hindered phenolic substituent and were inferior to the reference drug. Compound 1-me- represented by 1-(3,5-ditertbutyl-4-hydroxyphenyl)-pro- thyl-2- (3,5-di-tert-butyl-4-hydroxyphenyl) -propane-1- pane-1-on, showed a very low antiplatelet activity. one-3-amine, in which the hindered phenol substituent However, a compound derived from N9-2,3-dihydro- was in the second position, while in positions 1 and 3 imidazobenzimidazole, having 2,6-di-tretbutyl-4-(1-hy- were methyl and amino substituents, did not have a pro- droxypropyl)-phenyl in the first position and a hydrochlo- nounced effect on its antiplatelet activity either. The most ride salt as hydrochloride in the first position, showed a active were the compoundes that in the second position pronounced activity towards inhibiting ADP-induced had 1- 3,5-ditertbutyl-4-hydroxyphenyl)-propane-1-one. platelet aggregation. The activity of these compounds in the test of ADP-induced platelet aggregation was highest relative to the other compounds. The introduction of the methyl derivative Discussion into position 3 and N-ethyl piperidine or N, N-diethyl am- inoethyl into position 1 led to a sharp increase in this type Activation of platelet hemostasis and oxidative stress are of activity. Benzyl in the 3rd position also increased the among the main reasons for an increased blood thrombo- activity of the compounds. The other compounds of this genic potential. group showed a low antiplatelet activity. Thus, the inclu- The study of 26 new benzimidazole derivatives with sion of benzyl into position 3 and 4-ethyl morpholine into a sterically hindered phenol in their structures showed position 1 of the structure reduced the activity. The only that more than half of all compounds had a potential an- benzimidazole derivative, having the hindered phenolic tiplatelet effect in vitro, which significantly exceeded the substituent in the 3rd position and methyl and amine – in activity of the reference drug, acetylsalicylic acid. Earlier positions 1 and 2, showed no pronounced antiplatelet ac- studies revealed a pronounced antioxidant activity in ben- tivity either. Moreover, the inclusion of benzyl and prope- zimidazole derivatives with a sterically hindered phenol nyl-1 into the structure of the compounds in the first posi- in their structures (Venkatesan and Rao 2000, Wright et tion did not lead to an increase in an antiplatelet activity. al. 2001). That is why besides studyinh the antiplatelet The next group of compounds represented by the com- activity, these compounds were studied in the test of mon structure of triazinobenzimidazoles, where the hin- ascorbate-dependent lipid peroxidation (LPO). When dered phenolic substituent was in the first position, and searching for the most active compounds with antioxidant Research Results in Pharmacology 6(1): 1–9 7 activity, it was found that 12 benzimidazole derivatives Therefore, it was impossible to make any conclusions about were more active than dibunol, the other compounds were this radical influencing the studied types of activities. less active or inactive. Thus, scaffolds based on N-7-ditretbutyl-4-hydroxyphe- Moreover, in order to fully take into account the co- nyl pyrimidobenzimidazoles and N-9-ditretbutyl-4-hydroxy- operative effect of the above factors on the formation of phenyl benzimidazoles exhibited pronounced antiplatelet blood plate aggregates in the vascular bed and to evaluate and antioxidant activities, which were superior to those of more accurately the antiplatelet effect of the most active following scaffolds: N-9-ditretbutyl-6,6-4-ditretbutyl-1-hy- compounds, an in vivo study was performed. As a result, droxyphenyl-2,3-thiadiazinobenzimidazoles, N9-2,3-di- it was shown that substances under codes RU-1144, RU- hydroimidazobenzimidazoles, N-7-ditretbutyl-4-hy- 1261 and RU-1263 had a high antiplatelet activity, but the droxyphenyl-N9-2,3-dihydroimidazobenzimidazoles and most active compound in terms of ED50 was connection N-7-ditretbutyl-4-hydroxyphenyl-triazolobenzimidazoles. RU-1144. The tested compound RU-1144 in terms of CTI With dislocating the ditretbutyl radical from positions N-7; exceeds acetylsalicylic acid by 3.7 times. N-9 and C-11, there was a decrease in antiplatelet and anti- The 26 compounds selected in this study belonged to the oxidant activities in the above groups. following 6 scaffold groups: 1H-benzimidazoles and salts Thus, the ability of benzimidazole derivatives, having of 1H-benzimidazolium-3, N9-2,3-dihydroimidazobenzim- a ditretbutyl radical in their structures, to inhibit plate- idazoles, 3,5-dihydrotriazinobenzimidazoles, 2,3,4,10-tet- let aggregation processes and to prevent oxidative stress, rahydropyrimidobenzimidazoles, 2,3-dihydrothiadiazin- makes them promising for further study of their antiplate- obenzimidazoles and 4H-triazole-benzimidazoles. let activity. All pyrimidobenzimidazole derivatives having ditretbutyl-4-hydroxyphenyl radical in position R1 had a pronounced antiplatelet activity and, in terms of Δ% of plate- Conclusions let aggregation inhibition, at a concentration of 100 μM, exceeded the reference drug acetylsalicylic acid. Also, the Thus, as a result of studying 26 new benzimidazole deri- study propved the high antioxidant activity of these com- vatives with spatially hindered phenol in their structure, pounds, which was comparable to that of dibunol. it was shown that three compounds under the codes RU- Derivatives of 1H-benzimidazole showed an anti- 1144, RU-1261 and RU-1263 showed high antiaggregant platelet activity of various intensity. Most compounds and antioxidant activity. of this group were derivatives containing 1- (3,5-ditret- Scaffolds based on N-7 ditretbutil-4-hydroxyphenyl butyl-4-hydroxyphenyl) propane-1-on in the R2 posi- of pyrimidinemethanol and N-9 ditretbutil-4-hydroxy- tion, which to various extents effected both antiplatelet phenyl of benzimidazoles showed pronounced anti- and antioxidant activities. When this radical was moved platelet and antioxidant activity, surpassing scaffolds: to the R1 position, the compounds lost these two types N-9 ditretbutil-4-hydroxyphenyl triazenoimidazole of activities, and when the hindered phenolic substituent With-11-2,6-ditertbutyl-1-hydroxyphenyl-2,3-mediasen- was moved to the R3 position, the antioxidant activity in- timent, N9-2,3-dihydroimidazole, N-7 ditretbutil-4-hy- creased, whereas the antiplatelet activity decreased. Thus, droxyphenyl-N9-2,3-dihydroimidazole and N-7 ditret- only 6 compounds showed a pronounced antiplatelet ac- butil-4-hydroxyphenyl triazolopyrimidines. When the tivity superior to that of the reference drug acetylsalicylic ditretbutyl radical is shifted to the position in N-7; N-9 acid. What these compounds had in common was having and C-11 are represented in the groups there was a de- a hindered phenolic substituent represented by 1-(3,5-dit- crease in antiplatelet and antioxidant activity. retbutyl-4-hydroxyphenyl) propane-1-on in position R2. Compound RU-1144 exhibits a pronounced antiplate- Triazinobenzimidazole derivatives were represented let effect, combined with a high antioxidant activity, by 2 substances, having 1-(3,5-ditretbutyl-4-hydroxyphe- which makes it attractive for further in-depth study as a nyl) -propane-1-on in the R1 position, though no reliable drug with a multi-target mechanism of action for the treat- data were obtained about this radical influencing in any ment and prevention of thrombosis. was the activities under study, due to a small sampling of the substances. When studying the group of derivatives of 2,3-thiazin- Conflict of interest obenzimidazoles, which contain a hindered phenolic substituent in position R2, only one active compound was found. The authors declare no conflict of interest.

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Author contributions

Alexander A. Spasov, Doctor of Medical Sciences, Full Professor, Academician of the Russian Academy of Sci- ences, Head of the Department of Pharmacology and Bioinformatics, e-mail: [email protected], ORCID ID http:// orcid.org/0000-0002-7185-4826. The author defined the idea of research.

Aida F. Kucheryavenko, Doctor of Medical Sciences, Professor of the Department of Pharmacology and Bioin- formatics, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-1406-6919. The author consulted on the research idea, the concept and design of the study.

Ksenia A. Gaidukova, Assistant professor of the Department of Pharmacology and Bioinformatics, e-mail: kse- [email protected], ORCID ID http://orcid.org/0000-0003-4376-6332. The author was engaged in conducting experimental work, analysis of the material, results and conclusions, writing and editing the text of the article.

Vadim A. Kosolapov Doctor of Medical Sciences, Professor of the Department of Pharmacology and Bioinfor- matics, e-mail: [email protected], ORCID ID http://orcid.org/0000-0002-6702-1207. The author consulted on the research idea.

Olga N. Zhukovskaya, Candidate of Chemical Sciences, Researcher in the Laboratory of Organic Synthesis, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-0865-6656. The author took part in synthesis of the substances. Research Results in Pharmacology 6(1): 11–19 UDC: 616.65–002.–615.272: 615.035.1 DOI 10.3897/rrpharmacology.6.50940

Research Article

Clinical and experimental rationale for antioxidant therapy of chronic bacterial prostatitis

Oleg I. Bratchikov1, Igor A. Tyuzikov2, Pavel A. Dubonos1

1 Urology Department, Kursk State Medical University, 3 Karl Marx St., Kursk 305041, Russian Federation 2 Tandem-Plus Medical Center, 3V Pervomayskiy Lane, Yaroslavl 150000, Russian Federation

Corresponding author: Oleg I. Bratchikov ([email protected])

Academic editor: T. Pokrovskaya ♦ Received 8 December 2019 ♦ Accepted 19 January 2020 ♦ Published 5 March 2020

Citation: Bratchikov OI, Tyuzikov IA, Dubonos PA (2020) Clinical and experimental rationale for antioxidant therapy of chronic bacterial prostatitis. Research Results in Pharmacology 6(1): 11–19. https://doi.org/10.3897/rrpharmacology.6.50940

Abstract Introduction: Literature data prove the important role of oxidative stress in the pathogenesis of Chronic Bacterial Prostatitis (CBP) and its recurrence, which reduces the effectiveness of standard etiotropic therapy of the disease. Aim of study: To improve the results of the pharmacotherapy of CBP by a comprehensive assessment of oxidative disorders in the prostate gland in a clinical and experimental study to provide evidence for antioxidant support. Material and methods: The results of experimental simulation of CBP in 60 male rats and examination of 90 patients with CBP (average age 38.2 ± 1.4; main group) and 30 clinically healthy men (average age 35.5±1.5; control group), which included history-taking, collecting complaints, questioning, general and special examinations, biochemical, cytological, microbiological, sonographic studies. In some experimental animals and patients with CBP, different modes of pharmacotherapy were tested (antimicrobial monochemotherapy; antimicrobial chemotherapy+zinc picolinate; antimicrobial chemotherapy+L–carnitine tartrate in standard doses). The data were processed using descriptive and comparative statistics. Results and discussion: Clinical and experimental findings showed the compensatory nature of the prostatic oxidative disorders after a standard antimicrobial monochemotherapy of the first episode of CBP and their continued persistence with a high risk of decompensation and development of mitochondrial dysfunction after a course of standard antimicrobial monochemotherapy in CBP recurrence. Zinc deficiency in the patients with CBP was detected on average 2.7 times more often than in the healthy men, so zinc determination in the prostatic fluid and subsequent drug compensation should be considered as first–line diagnostic and treatment measures. In the patients with CBP withoutzinc deficiency, L-carnitine may be an effective alternative to pharmacological correction of the prostatic oxidative disorders. Conclusion: To increase the effectiveness of standard etiotropic therapy of CBP, simultaneous antioxidant support is necessary, using differentiated administration of antioxidants/antihypoxants zinc( or L-carnitine).

Keywords Chronic bacterial prostatitis, experimental simulation, antimicrobial chemotherapy, free radical oxidation, lipid peroxidation, superoxide dismutase, succinate dehydrogenase, antioxidant, antihypoxant, zinc, L-carnitine.

Copyright Bratchikov OI et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 12 Bratchikov OI et al.: Clinical and experimental rationale for antioxidant therapy of chronic...

Introduction to “kill” the excessive reactive oxygen species formed under oxidative stress on the background of inflammato- Chronic prostatitis (CP) is one of the most common uro- ry process, to optimize and improve the effectiveness of logical diseases in men of different ages, but the results traditional etiotropic antimicrobial chemotherapy, which of its complex pharmacotherapy, even applying modern remains a first-line therapy in CBP. This would reduce the pharmacological achievements, in many cases can not be negative effects of inflammation and associated oxidative considered as satisfactory, which results, first of all, in a stress for prostate homeostasis and would be laying the low quality of life of this category of patients (Nickel and groundwork for faster and more complete anatomical and Weidner 2000; Loran et al. 2002; Kogan et al. 2009; Be- functional recovery of the organ after eradication of the lousov et al. 2013; Dosta and Sevostianov 2013). pathogen, since this problem is not solved by any of the One of the key reasons for this situation is the complex antibiotics due to their lack of such a mode of action (Vi- etiology and multi-factor nature of the CP pathogenesis, cari et al. 2002; Balercia et al. 2017). including its infectious (bacterial) type – chronic bacterial In modern urological practice, several pharmacologi- prostatitis (CBP), which makes up about 5–7% of the to- cal agents are used as effective antioxidants, of whichzinc tal structure of the disease types (Litwin et al. 1999; Enge- and selenium are the most studied. However, despite their ler et al. 2018; Grabe et al. 2018). popularity among urologists, zinc and selenium medica- Currently, it is understood that, in the etiopathogenesis tions are often used in clinical practice without clear indi- and outcomes of CBP, an important role is played not only cations, without laboratory confirmation of the deficiency/ by direct negative effects of pathogenic microorganisms insufficiency of these essential micro-elements, - empiri that cause direct reactions of alterations and inflammato- cally and “blindly” (Cui et al. 2015; Sivkov et al. 2015; ry infection in the prostate tissue (inflammatory infection Mo et al. 2016). Such medicinal substance as L-carnitine, mechanisms), but also by a cascade of secondary non-in- which is widely used in other fields of medicine and is a fectious inflammatory biochemical reactions, which are proven universal intracellular antioxidant with a known induced by the infectious matter and inevitably involved mechanism of action at the level of mitochondria of cells, in pathogenetic interaction with it and have adaptive and which are the main cellular organelles that resist oxidative reparative nature (Kullisaar et al. 2012; Tyuzikov et al. stress, is less studied in terms of its effectiveness as an an- 2013; Aoun et al. 2015; Molochkov et al. 2015). The level tioxidant in the complex pharmacotherapy of CBP (Zhou of prostate cells protection from the free radical oxidation et al. 2007; Ramasamy et al. 2012). The results of modern arising as a result of inflammatory infection, as well as its foreign studies show that due to the presence of high lev- further development and especially the outcomes deter- els of oxidative stress in patients with CBP, L-carnitine mined by a degree of severity of structural damage and administration in this disease can be explained from a functional deficiencies in the prostate tissue after eradi- pathogenetic point of view (Vicari et al. 2002; Condorelli cation of the pathogen from it and clinical and laboratory et al. 2017; Shang et al. 2017). However, in the Russian relief of CBP exacerbation depends on the safety, adequa- scientific literature, the effectiveness of L-carnitine as an cy and direction of these secondary adaptive reactions antioxidant in the treatment of CBP has been hardly stud- (Tyuzikov et al. 2013; Grabe et al. 2018). ied, and L-carnitine medications available in Russia are One of the key natural mechanisms of cellular adaptive used for a single indication – male infertility. reactivity is the antioxidant support network (ASN), con- Currently, an active search for effective antioxidant sisting of various enzymatic and non-enzymatic natural an- drugs and rational modes of their administration in CBP is tioxidants and present in all cells of the human body without continuing (Balercia et al. 2017). However, according to the exception (Kostyuk and Potapovich 2004; Mentschikova available Russian literature, a number of important practical et al. 2006). An infectious agent in the prostate gland in issues of complex pharmacotherapy of CBP remain open, in CBP naturally leads to the primary activation of ASN in particular, the sequence of applying etiotropic antibacterial cells, which resists inflammatory infection and associated and pathogenetic antioxidant therapies, the possibility of free radical oxidation, so a “biochemical scenario” of their accurate laboratory diagnosis of deficiency/insufficiency of development and outcomes largely depends on a degree of the most important natural antioxidants (for example, zinc) ASN compensation. In case of ASN failure or deficient, and the correlation of their plasma and prostatic concentra- pathological processes in the prostate cells enhance and ox- tions, the definition of clear indications for the administra- idative stress develops, often inducing additional anatomi- tion of the antioxidant depending on the clinical and labo- cal and functional disorders in the organ (increased cellular ratory features of CBP and a number of other treatment and alterations, violation of apoptosis in the cell, mitochondrial diagnostic issues important for clinical practice. cell dysfunction, excessive peroxidation of cell membrane lipids, intracellular acidosis and hypoxia, etc.) (Kovalchuk et al. 2007; Kullisaar et al. 2012; Tyuzikov 2017). Aim of study In light of the above, it becomes evident that it is necessary to consider co-administration, along with antimi- Improving the results of the pharmacotherapy of Chro- crobial chemotherapy, of pathogenetic medicines that nic Bacterial Prostatitis by a comprehensive assessment support the activity of ASN in cell at the level sufficient of the local oxidative disorders in the prostate gland in a Research Results in Pharmacology 6(1): 11–19 13 clinical and experimental study and to provide rationale was ongoing, prospective and full-design. The results of the for a differentiated combined etiopathogenetic pharmaco- complex examination of men in the control group were tak- therapy with antioxidant support in this disease. en as normal reference values of the studied indicators.

Entry criteria: Material and methods • Presence of clinical CBP symptoms (the main The study, which consisted of two parts – experimental clinical sign is chronic pelvic/prostatic pain with and clinical, was organized and performed in accordance typical irradiation) in combination with appropri- with the regulatory acts and guidelines governing the con- ate laboratory validation (identification of signifi- duct of experimental and clinical research in the Russian cant pathogens in the prostatic fluid in a diagnosti- Federation. Laboratory animals were treated in accor- cally significant titer >103 CFU/ml) dance with the current Rules for the Use of Experimental • Absence of a history of surgery or trauma of the Animals, International Guiding Principles for Biomedical pelvis and perineum Research Involving Animals (1985) and Russian Guideli- • No symptoms of any neurological disease nes for Experimental (Pre-clinical) Studies of New Phar- • No diabetes mellitus type 1 or type 2 macological Agents (2000). All the patients who entered • Absence of anamnesis and clinical and laboratory the main group of the clinical part of the study and com- signs of infections (STIs) at the time of the study pleted it, as well as the men of the control group, had been • Age of men up to 50 previously informed of the aims and objectives of the study, and each of them filled in an informed consent for par- Exclusion criteria: ticipation in the study and for using his personal results of the study for further statistical analysis. • Presence of any clinical and sonographic signs of The experimental part of study was performed in 60 infravesical obstruction of any genesis outbred mature healthy male rats weighing 180–200 g, • Presence of lower urinary tract symptoms (LUTS) after a 14-day quarantine regime, without any signs typical for overactive bladder of acute and chronic diseases. To simulate a laboratory • Therapy of any LUTS or chronic pain earlier than model (CBP), a modified method by Nickel J. C.-Goto 3 months ago, without any positive results T. (1991) was used. The prostate gland and posterior ure- • Taking medications that can affect the bladder thra of male rats were infected by introducing a 0.05 ml and/or prostate less than 6 months before the start and 0.1 ml of suspension of E.coli NIHJ JC-2 culture at of the study the concentration of 108 CFU/ml into the prostatic part • Known or suspected prostate cancer (total blood of the urethra through a catheter. At the same time, 20 PSA > 4 ng/ml) animals were infected once (experimental model of “CBP • Individual drug intolerance or contraindications to episode”), 20 animals were re-infected 30 days later (ex- the medications used in this study. perimental model of “CBP recurrence”), and the remaining 20 intact animals made up the control group. After Statistical processing of the age index of the control euthanasia, preparation and necessary studies of prostate group (n = 30) showed an average age of 35.5 ± 1.5 years homogenates of the laboratory animals were performed. (confidence interval 0.95| 20–45), and for the patients of The clinical part was based on clinical observations, the the main group (n = 90) the average age was 38.2 ± 1.4 results of complex examination and pharmacotherapy of 90 years (confidence interval 0.95| 24–46). Thus, the control men (average age 38.2±1.4) with proven criteria for the diag- and main groups were homogeneous in age, since the nosis of chronic bacterial prostatitis, which were performed confidence intervals overlapped in each group. The mini- in the period of 2016–2018 in the outpatient departments of mum duration of CBP was 1 year, the maximum duration the Urology Clinic of Kursk State Medical University. The was 13 years (the average duration of CBP was 7.5 ± 1.4 control group consisted of 30 clinically healthy men (av- years; confidence interval 0.95| 1–13). The frequency and erage age 35.5±1.5) without prostate pathology. The total structure of the clinical symptoms of CBP in the main number of men included in the study was 120. The study group are presented in Table 1.

Table 1. Frequency and Structure of the Clinical Symptoms of CBP in the Main Group (n = 90).

Clinical symptoms Absolute number (people) Percentage (%) Pelvic/prostate pain syndrome with or without pain irradiation to adjacent anatomical regions 90 100.0 Increased anxiety and depression levels 38 42.2 Impaired productivity due to the chronic pain 36 40.0 Reduced sex drive 32 35.5 Reduced frequency and degree of morning and adequate erections 31 34.4 Orgasmic disorders 21 23.3 Inappropriate urination 15 16.7 14 Bratchikov OI et al.: Clinical and experimental rationale for antioxidant therapy of chronic...

Complaints and anamnestic data had been collected (levofloxacin); antimicrobial chemotherapy + zinc picol- from all the men before the start of examination and treat- inate; antimicrobial chemotherapy + L–carnitine tartrate ment, in accordance with the generally accepted medical in standard doses for 28 days). Statistical processing was methods. To objectify and evaluate the severity of CBP performed in Microsoft Excel-2007 and Statistica 6.0. and the quality of life of the patients after collecting an- (StatSoft, USA). Data processing was performed using amnesis and complaints, a questionnaire was conducted descriptive and comparative statistics. The results of the using a valid questionnaire – NIH–CPSI–QL (National study were entered into a personal computer based on Mi- Institute of Health Chronic Prostatitis Symptom Index crosoft Excel–2007 and Statistica 6.0. Spearman correla- – Quality of Life). All men underwent general physical tion coefficient (r) was determined to study the interaction and special urological examination using standard meth- among the quantitative features. The Student’s t–test was ods. The laboratory examination of the prostatic fluid was used to evaluate the intergroup differences in the values performed using standard cytological and microbiologi- of indicators that have a continuous distribution. To solve cal studies. To exclude concomitant sexually transmitted the problems of studying the influence of two or more infections (STIs), urethral smears of all men in the main conditions on a certain random variable, various statistical and control groups were examined using two methods: methods of multivariate analysis were used. The critical enzyme immunoassay (ELISA) and polymerase chain confidence level of the null statistical hypothesis (about reaction (PCR). To determine the level of reactive oxy- the absence of significant intergroup differences or factor gen species (ROS) in biological substances, the method influences) was assumed to be 0.05. The value of p<0.05 of luminal-dependent chemiluminescence (LDCL) was generally accepted in biomedical research was considered used, based on the use of a mixture of a chemiluminescent as statistically significant for all indicators. probe – luminol (3-aminophthalhydrazide) – and horse- radish peroxidase for accurate measurements of hydrogen peroxide formation. Determination of lipid peroxidation Results and discussion products (LPP) in biological substrates was performed using the following methods: diene conjugates were de- In the experiment with simulated CBP episode, in the pros- termined by the method of Stalnaya I.D. (1977); malond- tate tissue of the laboratory animals a significant increase ialdehyde was determined using spectrofluorometry after in the processes of free radical oxidation was revealed (a its reacting with thiobarbituric acid by the method of Stal- significant 4.6-time increase in the number of ROSs, a naya I.D. and Garishvili T.G. (1977). Superoxide dismu- 2-time increase in their activity, an increase in the level of tase (SOD) activity was determined by a spectrophotomet- intermediate lipid peroxidation products (diene conjugates ric method, using the method of Mistra H.P., Fridovich I. and malondialdehyde) by 34.6% and 42.0%, respectively, (1972) modified by Kostyuk V.A. et al. (1990). Succinate a 1.5-time increase in the activity of superoxide dismutase dehydrogenase activity was determined by the method (SOD) compared with those in the intact animals of the of Storozhuk P.G. and Storozhuk A.P. (2004), based on control group (p < 0.05), without significant dynamics of the ability of this enzyme to restore nitroblue tetrazolium mitochondrial succinate dehydrogenase (SDH)). After the (NBT) to formazan. In order to exclude prostate cancer, course of antimicrobial monochemotherapy, the number all the men of both groups at the initial stage of the study of ROSs in the prostate tissue was normalized, but their were determined the blood level of total PSA by a heter- activity remained 1.6 time higher, the level of malondial- ogeneous two-stage enzyme immunoassay, using stand- dehyde was 20.1% higher and the activity of SOD – 19.7% ard Enzymun-Test PSA assays by BoehringerMannheim higher, compared those of the control group (p < 0.05). Corp. (Germany). The zinc content in the blood serum In the prostate gland of the laboratory animals with was determined by colorimetric method (IFCC), based simulated CBP recurrence, significantly more pronounced on the formation of a colored complex compound of zinc cellular oxidative disorders were detected compared to with dithizone. The content of zinc in the prostatic fluid the CBP episode model (a 7.6-time increase in the number was determined by Х-ray fluorescence analysis, based on of ROS, a 3.5-time increase in their activity, a 33.3% in- the spectrum features of secondary fluorescence radiation crease in SDH activity (p < 0.05) against the background of the sample, which occurs under the influence of hard- of multidirectional changes in the level of lipid peroxida- er Х-rays. To test the reference parameters of the healthy tion products and a 32.0% lower SOD activity than in the men (prostate volume and its structure), as well as to as- control group (p < 0.05)). After a course of antimicrobial sess the initial morphometric parameters of the prostate monochemotherapy, in the prostate tissue there remained and their dynamics during observation and pharmacother- significantly higher levels of ROS (3.45 times) and their apy, all men of the main and control groups included in activity (2.1 times) against a significantly lower (32.0%) the study were subjected to transrectal US by a 5.5–7- level of SOD activity (p < 0.05) and unreliably lower MHz rectal biplane sensor (Ultramark-9, USA) and an ul- (30.1%) level of mitochondrial SDH compared with those trasound complex Logiq 500 Pro Series (USA). To assess of the control group (p < 0.1). the effectiveness of CBP treatment in the experimental The comparative clinical and laboratory characteristics and clinical parts of the study, several pharmacotherapy of the control and main groups of the clinical part of the modes were tested (antimicrobial monochemotherapy study are presented in Table 2. Research Results in Pharmacology 6(1): 11–19 15

Table 2. Clinical Characteristics and Indicators of the Oxidative Status in the Prostate Gland of the Men of the Control and Main Groups (n = 120).

Studying indicator (unit of measurement) Control group (n=30) (mean value M ± m Main group (n = 90) (mean value M ± m and confidential interval CI 0.95) and confidential interval CI 0.95) Symptom severity assessment (points) 0.86 ± 0.2 (0–1) 8.5 ± 2.1* (5–17) Overall symptoms score (points) 0.61 ± 0.1 (0–1) 12.2 ± 2.1* (9–27) Pain index (points) 0.67 ± 0.2 (0–1) 4.9 ± 1.6* (6–14) Life quality index (points) 0.62 ± 0.2 (0–1) 3.3 ± 0.8* (2–5) Leucocytosis in the prostatic fluid (units/LPF) 6.5 ± 1.5 (0–10) 38.8 ± 10.2* (15–60) Number of lecithin granules (units/LPF) Moderate quantity in 27/30 (90.0%) Reduced Moderate quantity in 35/90 (38.9%)* quantity in 3/30 (10.0%) Reduced quantity in 55/90 (61.1%)* Prostatic Fluid Crystallization Test Positive in 26/30 (86.7%) Positive in 31/90 (34.4%)* Light sum of the prostatic fluid (relative units) 1.35 ± 0.14 (1.11–1.78) 8.51 ± 0.35* (4.89–7.43) Fluorescence peak amplitude of the prostatic fluid 0.6 ± 0.1 (0.4–0.8) 2.1 ± 0.4* (0.9–2.8) (relative units) Diene conjugates in the prostatic fluid (nmol/ml) 8.2 ± 1.5 (4.5–10.1) 12.5 ± 1.2* (8.4–15.7) Malondialdehyde in the prostatic fluid (nmol/ml) 0.56 ± 0.04 (0.50–0.62) 0.75 ± 0.03* (0.56–0.87) SOD activity in the prostatic fluid (relative units/ml) 42.19 ± 2.14 (35.43–50.23) 78.34 ± 8.25* (63.56–92.55) Note: *– statistically significant difference in comparison with the control group

As follows from Table 2, the differences in the clin- the patients with CBP without it showed non-significantly ical and laboratory indicators between the control and worse clinical characteristics of pain and quality of life, main groups were statistically significant (p < 0.05). In non-significantly higher levels of leukocytosis, malondi- contrast to the healthy men of the control group, the pa- aldehyde and a lower content of lecithin granules in the tients with CBP had oxidative imbalance in the prostate prostatic fluid (p < 0.1), but significantly lower (by 20.2%) gland (increased number and activity of ROS, increased activity of SOD in the prostatic fluid (p < 0.05), between leukocytosis of the prostatic fluid, and more lipid perox- the activity of which and the concentration of zinc in the idation and SOD activity in the prostatic fluid) and its prostatic fluid a significant moderate positive connection secretory disorders (reduced number of lecithin granules was revealed (r = 0.389; n = 90; p = 0.001). in the prostatic fluid, the high frequency of the disorders The integrative results of comparative evaluation of the of crystallization of the prostatic fluid), which, from the effectiveness and tolerability of the CBP pharmacother- clinical point of view, corresponded to more pronounced apy modes tested in this study are presented in Table 3. symptoms of pain syndrome and more inferior quality of As follows from Table 3, the standard antimicrobi- life in the patients with CBP compared with the healthy al monochemotherapy mode in contrast to the modes of men in the control group (p < 0.05). In the patients with combined pharmacotherapy with additional prescription CBP, significant positive correlations were found between of zinc and L-carnitine showed significantly worse results the amount of ROS and SOD activity in the prostatic fluid of microbiological eradication of pathogens and treatment (n = 90; r = 0.413; p = 0.001) and between the amount of in relation to the clinical characteristics of the disease, the ROS in the prostatic fluid and the clinical pain index (n = quality of patients’ life, secretory function of the prostate 90; r = 0.304; p = 0.001). gland and especially its oxidative status (p < 0.05), which The frequency of the absolute serum zinc deficiency violations in the form of a hyperproduction of ROS and in the patients with CBP was 28.9%, which is 2.89 times accumulation of the lipid peroxidation products (malon- significantly higher than in the control group of healthy dialdehyde) – along with a decreased SOD activity in the men (10.0%, respectively; p < 0.05). The frequency of the prostatic fluid continued to persist after a course of anti- absolute zinc deficiency in the prostatic fluid in- thepa microbial monochemotherapy. tients with CBP was 41.1%, which is 2.5 times significant- Thus, the experimental findings showed the compensa- ly higher than in healthy men of the control group (16.7%, tory nature of oxidative changes in the prostate cells after respectively; p < 0.05). In general, the healthy men of the standard antimicrobial monochemotherapy of the first ep- control group had a statistically significant weak posi- isode of CBP (ASN compensation phase) and the contin- tive correlation between the zinc levels in the serum and uing persistence of the oxidative disorders in the prostate the prostatic fluid (r = 0.156; n = 30; p = 0.001), which cells after a course of standard antimicrobial monochemo- turned out to be more statistically strong (r=0.204; n=7; therapy with a high risk of mitochondrial dysfunction in p = 0.001) in the range of subnormal values (< 543 mcg/L) CBP recurrence (ASN decompensation phase). and the lower tercile of the reference normal values of se- The obtained results of the clinical part of the study rum zinc level (543–738 mcg/L). A statistically significant also confirmed the negative impact of the infectious agent moderate positive correlation (r = 0.345; n = 37; p = 0.001) on the oxidative status of the prostate gland and reflected was also found in the patients with CBP with absolute a significant role of free-radical prostatic aggression as an zinc deficiency and lower limit of normal serum levels. additional non-infectious component in the pathogenesis The patients with CBP with zinc deficiency compared to of pain syndrome in CBP. 16 Bratchikov OI et al.: Clinical and experimental rationale for antioxidant therapy of chronic...

Table 3. Integrative Comparative Evaluation of the Effectiveness and Tolerability of Various CBP Pharmacotherapy Modes (n = 75).

Studying indicator (unit of Control group (n = 30) Group 1 Etiotropic Group 2 Combination therapy Group 3 Combination therapy measurement) (average value M ± antimicrobial (antimicrobial chemotherapy + (antimicrobial chemotherapy m and confidential monochemotherapy (n = zinc picolinate) (n = 15) (mean + L–carnitine tartrate) (n = interval CI 0.95) 15) (mean value M ± m and value M ± m and confidential 15) (mean value M ± m and confidential interval CI 0.95) interval CI 0.95) confidential interval CI 0.95) Microbiological efficiency (%) – 86.7 93.3 100.0 Frequency of side effects (%) – 20.1 6.7 6.7 Symptoms severity assessment 0.86 ± 0.1 (0–1) 0.92 ± 0.3* (0–2) 0.76 ± 0.2 (0–1) 0.78 ± 0.3 (0–1) (points) Overall symptoms score 0.61 ± 0.1 (0–1) 0.63 ± 0.3 (0–3) 0.62 ± 0.2 (0–2) 0.61 ± 0.3 (0–1) (points) Pain index (points) 0.67 ± 0.2 (0–1) 1.13 ± 0.2* (0–2) 0.71 ± 0.5 (0–1) 0.68 ± 0.3 (0–1) Life quality index (points) 0.62 ± 0.2 (0–1) 0.98 ± 0.2* (1–3) 0.67 ± 0.4 (0–1) 0.63 ± 0.3 (0–1) Leucocytosis in the prostatic 6.5 ± 1.5 (0–10) 11.5 ± 4.5 (8–14) 8.5 ± 2.5 (4–12) 6.5 ± 2.5 (0–10) fluid (units/LPF) Number of lecithin granules Moderate quantity in Moderate quantity in 6/15 Moderate quantity in 10/15 Moderate quantity in 9/15 (units/LPF) 27/30 (90.0%) Reduced (40.0%) Reduced quantity in (66.7%) Reduced quantity in 5/15 (60.0%) Reduced quantity in 6/15 quantity in 3/30 (10.0%) 9/15 (60.0%) (33.3%) (40.0%) Light sum of the prostatic fluid 1.35 ± 0.14 (1.11–1.78) 2.06 ± 0.09*/** (1.61–2.83) 1.47 ± 0.05 (1.22–1.89) 1.37 ± 0.05 (1.15–1.81) (relative units) Fluorescence peak amplitude 0.6 ± 0.1 (0.4–0.8) 0.8 ± 0.2 (0.6–1.3) 0.7 ± 0.2 (0.5–1.1) 0.7 ± 0.1 (0.5–0.9) of the prostatic fluid (relative units) Diene conjugates in the 8.2 ± 1.5 (4.5–10.1) 8.1 ± 0.4 (6.4–10.5) 8.1 ± 0.2 (5.8–9.9) 7.8 ± 0.3 (5.2–9.1) prostatic fluid (nmol/ml) Malondialdehyde in the 0.56 ± 0.04 (0.50–0.62) 0.68 ± 0.03*/** (0.72–0.90) 0.59 ± 0.03 (0.53–0.69) 0.58 ± 0.04 (0.51–0.70) prostatic fluid (nmol/ml) SOD activity in the prostatic 42.19 ± 2.14 (35.43– 35.19 ± 2.87*/** (32.12–42.34) 40.76 ± 2.32 (37.12–48.54) 40.74 ± 2.17 (35.12–48.43) fluid (relative units/ml) 50.23) Note: * – differences are statistically significant when comparing the indicators of the group treated by antimicrobial monochemotherapy with the indicators in the control group and both combination therapy groups (p < 0.05); ** –differences are statistically significant when comparing the indicators of the group treated by antimicrobial monochemotherapy and those of both combination therapy groups (p < 0.05)

Compared with the healthy men, the patients with tive disorders in the prostate gland. The study showed a CBP were on average 2.7 times more likely to have a significant negative role of free-radical reactions in the deficiency of serum and/or prostatic zinc. Against the formation of anatomical and functional disorders in the background of this, they had worse clinical and labora- prostate gland in CBP, which are able to complete the tory parameters of the disease and persisting oxidative “vicious loop” of its pathogenesis and maintain the or- disorders in the prostatic fluid in contrast to the patients gan alterations after a course of standard antimicrobial with CBP without zinc deficiency. In routine clinical monochemotherapy (Fig. 1). practice, direct zinc determination in the prostatic fluid Based on the results of the study, the following prac- should be considered as the most objective and inform- tical algorithm for zinc deficiency diagnosing in patients ative method of zinc deficiency diagnosing in patients with CBP is proposed (Fig. 2.). with CBP. Standard etiotropic antimicrobial chemo- An optimized algorithm for the sequence and differen- therapy of CBP does not guarantee a complete pharma- tiated administration of combined etiopathogenetic phar- cological cure of the prostate and has no affect on the macotherapy in CBP, based on the results of the study, can course and outcomes of free-radical oxidation, which be presented as follows (Fig. 3). naturally develops against the background of the entry The proposed practical algorithms are designed to im- of an infectious agent into the prostate tissue. In this prove the traditional diagnostic and pharmacotherapy of regard, additional prescription of medicinal agents that CBP carried out in a routine urological use and to imple- can safely and effectively neutralize the negative impact ment an individual approach to the management of pa- of oxidative stress on the prostate gland (antioxidants tients with this disease. and/or antihypoxants) is pathogenetically substantiated to improve the results of modern CBP pharmacotherapy. Taking into account the crucial physiological role of Conclusion zinc in prostate metabolism and the high frequency of zinc deficiency in patients with CBP, the first-line thera- The obtained results of the clinical and experimental stu- py in zinc-deficient patients with CBP is a medical cor- dy convincingly confirmed the significant negative role of rection of the deficiency of this essential micro-element. free radical oxidation (oxidative stress) induced by an in- For patients with CBP without zinc deficiency, the ad- fectious agent in the multifactorial pathogenesis of CBP, ministration of L–carnitine tartrate may be an effective which has been reflected in the scientific literature of -re and safe front-line therapy to correct the existing oxida- cent years. In this connection, it is possible to substantia- Research Results in Pharmacology 6(1): 11–19 17

Figure 1. The role of oxidative local disorders in the prostate gland in the formation of the “vicious loop of pathogenesis” of CBP. Abbre- viations: LP – lipid peroxidation; SOD – superoxide dismutase; SDG – succinate dehydrogenase; ASN – antioxidant support network.

Figure 2. Diagnostic algorithm for zinc deficiency detecting in CBP patients in routine urological use. 18 Bratchikov OI et al.: Clinical and experimental rationale for antioxidant therapy of chronic...

Figure 3. Algorithm of the sequence and differentiated choice of personalized combined etiopathogenetic pharmacotherapy in patients with CBP. te with a high degree of confidence the need to optimize As an effective and safe antioxidant support for stand- the existing methods of diagnostic and pharmacotherapy ard antimicrobial chemotherapy of CBP, a targeted zinc of this disease by personalized additional administration replacement therapy (in zinc-deficient patients with CBP) with etiotropic antimicrobial chemotherapy of medici- and L-carnitine therapy (in all patients with CBP) can nal agents with antioxidant/antihypoxant activity. At the be recommended, which, according to the results of the same time, it should be noted that the practical side of clinical and experimental study described in the present the problem of pharmacological correction of oxidative paper, significantly increase the effectiveness of pharma- cell disorders in CBP, namely, selecting specific drugs cotherapy of this disease and are characterized by both and their prescribing modes, as well as their effectiveness good tolerance and compliance. It should also be noted and tolerability in this type of the disease still remains that in the conditions of unsatisfactory results of standard understudied. In our opinion this is due to the obvious antimicrobial chemotherapy of CBP, it quite promising underestimation of the role of free–radical oxidation in to further search for, to study and to test new or already this type of the disease by urologists (in clinical practice, known medicinal agents with the ability to freely pene- when treating CBP, the main emphasis is placed solely on trate into the prostate tissue during the development of antimicrobial monochemotherapy), as well as to the fact an inflammatory infection process in it and significantly that on the pharmaceutical market there is a large num- reduce the severity of oxidative stress in it, since there are ber of medicinal agents with claimed antioxidant/antihy- a few antioxidants/antihypoxants currently used in urolo- poxant activity, many of which do not yet have sufficient gy, and the effectiveness of the corresponding drugs is not evidence of a high degree of their efficiency in general always reliably proven. and in CBP, in particular. As follows from the available foreign and Russian literature, various medicinal agents with the above-stated mechanism of action have been te- Conflict of interest sted and continue to be tested as an antioxidant support for pharmacotherapy of chronic prostatitis. The authors declare no conflict of interest.

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Author contributions

Oleg I. Bratchikov, Doctor of Medical Sciences, Professor, Head of the Department of Urology, e-mail: bra- [email protected] The author was engaed in research design, analysis of the obtained data, and the article-writing

Igor A. Tyuzikov, Doctor of Medical Sciences, Professor, urologist, e-mail: [email protected] The author obtained the data for the analysis, analyzed the obtained data, and was engaged in the article writing.

Pavel A. Dubonos, Postgraduate Student, Department of Urology, e-mail: [email protected] The author reviewed the relevant literature and obtained the data for analysis. Research Results in Pharmacology 6(1): 21–27 UDC: 615.331 DOI 10.3897/rrpharmacology.6.50533

Research Article

Effect of complex therapy with Cortexin on the state of the body’s regulatory systems in patients with rosacea

Alexandra A. Bobrakova1

1 State Budgetary Institution of Healthcare “Oryol Regional Skin and Veneralogic Clinic” 68 Pushkina St., Oryol 302030, Russia

Corresponding author: Alexandra A. Bobrakova ([email protected])

Academic editor: T. Pokrovskaya ♦ Received 28 November 2019 ♦ Accepted 19 December 2019 ♦ Published 5 March 2020

Citation: Bobrakova AA (2020) Effect of complex therapy with Cortexin on the state of the body’s regulatory systems in patients with rosacea. Research Results in Pharmacology 6(1): 21–27. https://doi.org/10.3897/rrpharmacology.6.50533

Abstract Introduction: For treating patients with rosacea, it is important to study the effectiveness of drugs with both neuromodulating activity and immunomodulatory properties. The aim of the study is to examine the nature of biochemical parameters, the state of the blood antioxidant system, and the state of the immune system in patients with rosacea before and after a traditional treatment and the treatment using Cortexin. Materials and methods: 216 people participated in the study. All the patients received a traditional therapy. The patients in the study group additionally received Cortexin at a dose of 10 mg intramuscularly № 10. The clinical examination of the patients included a thorough collection of complaints, life histories, medical histories, and physical examination. The state of the skin, the hepatobiliary system, the blood antioxidant system, the immune and the endocrine systems were studied in detail. Results and discussion: The therapy using Cortexin made it possible to achieve a better clinical effect in terms of dermatological status. Thanks to the innovative treatment, 40% of the patients got rid of the clinical manifestations of the disease, and none of patients was reъъported to have no clinical effect. The patients with rosacea had disorders in the hepatobiliary system, an imbalance in the lipid peroxidation system and in the blood antioxidant system, as well as hyperactivation of the cellular and humoral parts of the immune system. The therapy with Cortexin did not have a significant advantage over the traditional treatment in the processes of normalization of the hepatobiliary system and the antioxidant system of the blood. However, Cortexin, being a complex of natural low-molecular-weight peptides, had a corrective effect on the cellular and humoral parts of the immune system. Unlike the traditional treatment, which maintained elevated levels of helper T-lymphocytes, lymphocytic index and interleukin 12, against the background of a slightly reduced level of T-lymphocyte suppressors. Conclusion: As a result of the Cortexin use in complex therapy, the patients with rosacea had a faster normalization of their dermatological and immune statuses.

Keywords rosacea, Cortexin.

Copyright Bobrakova AA This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Bobrakova AA: Effect of complex therapy with Cortexin...

Introduction and ratio of CD4+ and CD8+ cells in blood without any pronounced changes in the concentration of immunoglo- Rosacea is a common inflammatory dermatosis that af- bulins A, M, and G, as well as the content of circulating fects the skin of the face (Marson and Baldwin 2019). immune complexes) (Khorshev et al. 2008). In (Khors- According to Russian authors, rosacea has the highest hev et al. 2008), Cortexin is considered as a corrector proportion (36%) in the structure of acne-like dermato- of the neuroimmune component of the pathological ses (Yutskovskaya et al. 2010). In the Russian Federation, process. This observation is important for the treatment rosacea accounts for about 5% of all dermatological di- of rosacea, since the immunological aspects of rosacea agnoses. However, according to Russian cosmetologists, pathogenesis are much discussed in the literature. And the real figure reaches 20.6% (Tan et al. 2016). Despite the presence of the cytoprotective, membrane-stabilizing the large number of scientific studies on the problem of and antioxidant effects increases the relevance of consi- rosacea, there is still no clear concept of the etiology and dering neuropeptide drug Cortexin as an adjunct in the pathogenesis of this disease, and the mechanisms of its treatment of rosacea. formation have not been fully studied. The aim of the study is to study the nature of biochemi- Many authors associate the occurrence of rosacea with cal parameters, the state of the lipid peroxidation system, disorders in the dermal matrix (Steinhoff et al. 2013), with the blood antioxidant system, and the immune system in the action of microorganisms, the genetic predisposition the patients with rosacea before and after the traditional (Aldrich et al. 2015; Chang et al. 2015), with disorders of treatment and a treatment using Cortexin. the gastrointestinal tract and the hepatobiliary system (Pa- rish and Witkowski 1995; Gravina et al. 2015; Egeberg et al. 2017; Yang 2018), the endocrine (Spoendlin et al. Materials and methods 2013a) and nervous systems (Jafferany 2007; Spoendlin et al. 2013b; Egeberg et al. 2016a), with changes in the The study was conducted on the basis of the state bud- immune status (Schmidt et al. 1983; Yamasaki and Gallo getary healthcare institution “Oryol Regional Skin and 2011; Egeberg et al. 2016b), as well as with the impact of Veneralogic Clinic” in 2014–2017. The work was organi- the environmental factors. zed and conducted in accordance with the legal acts and One of the key links in the mechanisms of the rosacea guidelines governing clinical trials in the Russian Fede- development is the development of the local angioneuro- ration. Before the start of the study, all the patients were sis (Mimov 2013), associated with the disruption of the informed of the goals and objectives of the study, each peripheral nerve endings. Involvement of the nervous of them gave written informed consent to participate in system in the pathogenesis does not end with the damage it and to have the results of their diagnosis and treatment to these segments. A cosmetic defect in the form of dry published, while treating the information about the pa- and itchy skin is the most significant permanent irritation tients as confidential. The study involved 216 people,of trigger for the nerve endings, involving the brain in the both sexes, aged from 20 to 60 years, who received an chain of pathogenesis by forming an imbalance in brain outpatient treatment. All the patients were divided into 2 functioning and involving the entire body in the patholo- groups: a study group with an innovative treatment con- gical process, in particular the human adaptation system. sisted of 109 people, and the control group – 107 people. In this regard, it is important to study the effectiveness The general inclusion criteria for the study: of the drugs with both neuromodulating activity and immunomodulatory properties (Irwin 2008; Schwab et al. • informed consent of a patient to participate in the 2011), affecting the body’s regulatory systems in patients study and follow the doctor’s instructions; with rosacea. • having a clinically confirmed diagnosis of rosacea Cortexin is a neuropeptide drug, being a complex of in the acute stage; low-molecular-weight peptides isolated from the cerebral • age over 18 years. cortex of cattle and pigs. A set of Cortexin components provides neurotropic, cytoprotective, immunomodulato- The exclusion criteria for the study: ry, membrane-stabilizing, and antioxidant effects (Coulter et al. 1993; Horshev et al. 2002; Studenikin 2006). Cor- • not meeting the inclusion criteria for the study; texin is widely used in clinical practice as a cerebropro- • having physiological conditions (pregnancy, tective, nootropic, and neurometabolic agent. lactation); However, in the literature there are indications of the • neoplastic process; immunomodulatory effect of Cortexin in the treatment • individual intolerance to the drugs of the treatment of the neurological diseases, its corrective effect on the regimen. cellular and humoral links of the immune system (Khors- hev et al. 2008). The use of Cortexin in the treatment of A compulsory criterion for inclusion in the group of organic mental disorders was accompanied by normali- the innovative treatment was the prescription of Cortexin zation of immune parameters (an increase in the number by the neurologist for medical reasons (consequences of of T and B lymphocytes, the restoration of the number traumatic brain injury, encephalopathy of various origins, Research Results in Pharmacology 6(1): 21–27 23 cognitive disorders (memory and thought disorders), epi- Results and discussion lepsy, or asthenic conditions). All the respondents received a traditional therapy in accordance with the Federal Clinical Guidelines for the In order to evaluate the impact of the internal and exter- Management of Rosacea Patients by the Russian Society nal factors on the pathological process, the anamnestic of Dermatologists and Cosmetologists (2010). The pa- data of patients from both study groups were analyzed. tients of the innovative treatment group additionally re- The duration of the disease in the patients was on average ceived Cortexin at a dose of 10 mg intramuscularly № 10. from 3 to 18 years. The majority of the patients, 57.8%, The clinical examination of patients included a tho- reported the duration of the pathological process to have rough collection of complaints, life histories, medical lasted from 6 to 11 years. The duration of the disease from histories, and a physical examination. The states of the 1 to 3 years was reported by 9.3% of the patients. In this skin, the hepatobiliary system, the lipid peroxidation group of patients, the pathological process proceeded in system, the blood antioxidant system, the immune and a lighter form, without involving new skin areas. A third endocrine systems were studied in detail. For that, in –32.9%, had a history of the disease from 12 to 20 years. addition to the complaints about the skin condition, the They were mostly middle-aged and elderly patients. Du- following laboratory indicators were evaluated: bioche- ring these age periods, there was a significant decrease mical blood analysis (total cholesterol, total bilirubin, in the recovery functions of the body, which resulted in a alanine transaminase (ALT), aspartate aminotransferase longer process, a poor response to the therapy, and a de- (AST), gamma-glutamyltranspeptidase (GGT), thymol crease in the duration of remission periods. A large group sample the level of pro-oxidants and antioxidants in of the surveyed, 43%, reported exacerbations once a year, blood systems (catalase, superoxide dismutase (SOD), mainly in spring and summer periods, which can be ac- total antioxidant activity of blood plasma (TAA), counted for by an increased insolation (Jansen 2011). malone dialdehyde (MDA) and acetyl hydroperoxi- Another 26% of the patients recorded the exacerbations de (AHP), performance indicators of the cellular and twice a year, 19% – less than once a year, and 12% – 3 or humoral links of the immune system, of the monocy- more times a year. tic-macrophage link and of the inflammatory mediators. The analysis of the clinical picture revealed that before The assessment and determination of the severity of the treatment, all the patients reported dry skin. The majori- the dermatological status were based on the standard ty, 80%, complained of itching of various intensity. Telan- classification of rosacea (Wilkin et al. 2002) and the giectasias were visualized in 90% of the patients. Erythema Rosacea Diagnostic Evaluation Score (RDES) (Adas- was observed in 35% of the patients, papular exanthemums kevich 2004). Examinations were performed before and – in 16%. Facial swelling was detected in 8.8%. after the treatment according to standard regimens and During the study, the Rosacea Diagnostic Evaluation using Cortexin. Score was applied before and after the treatment for the The obtained data was processed using descriptive sta- purpose of statistical evaluation of the clinical manifesta- tistics of the Microsoft Excel 2007 data package. tions of rosacea (Fig. 1).

Figure 1. Dynamics of clinical manifestations of rosacea against the background of the traditional and investigational therapy with Cortexin. 24 Bobrakova AA: Effect of complex therapy with Cortexin...

Most cases of rosacea were of Stage II and Stage III.. ples, which indicated disorders of the hepatobiliary sys- The analysis of the indicators of the Rosacea Diagnostic tem in the rosacea patients. During the therapy in both Evaluation Score revealed a significant decrease of- ne groups, there was a tendency to normalize biochemical gative signs of rosacea in the patients who had received parameters, with the cholesterol level and the values of the complex therapy using Cortexin in contrast to the pa- the thymol sample in the patients of the study group of in- tients who had received the traditional treatment. Thus, novative treatment getting significantly lower than those the therapy using Cortexin made it possible to get rid of in the patients of the control group. This indicates the pro- the clinical manifestations of the disease in 40% of the cess of normalization of the structure and the dynamics of patients, and none of the patients in this group lacked a the inflammation processes. clinical effect. The lipid peroxidation system (LPS) and the blood The next stage of the study was an analysis of comorbi- antioxidant system (BAS) play an essential role in orga- dities in the rosacea patients (Fig. 2). Out of the examined nizing the proper functioning of the body. Thus, a distur- patients, 65.1% suffered from a concomitant pathology. bance of balance of the pro- and antioxidant systems of Among the most common pathologies, there were disea- blood is a provoking factor for activating the pathogenesis ses of the gastrointestinal tract and nervous system. of many chronic conditions, including rosacea. Rosacea, being a multifactorial disease, implies the Observing the initial parameters of the lipid peroxida- presence of the predisposing and provoking changes in tion products and the oxidative modification of plasma the functioning of various organs and systems of the body. proteins, superoxide dismutase, and total antioxidant acti- Assuming a significant role of the digestive system and, vity of plasma, an imbalance in the LPS-BAS system was in particular, the hepatobiliary system in the pathogenesis found in rosacea patients (Table 2). The LPS-BAS system of disease, the key indicators of the liver were examined reacted to the inflammatory process by increasing shifts (Table 1). in MDA and AHP indicators, which showed activation of Before the treatment, the indicators in both groups the neutrophilic link in the peripheral blood. The MDA did not differ significantly. There was a slight increase index in the rosacea patients was almost 2 times higher in cholesterol, bilirubin, transaminases, and thymol sam- than that of the healthy donors. The level of AHP was sig-

Figure 2. Concomitant somatic pathology in rosacea patients. Note: A – diseases of the gastrointestinal tract; B – neurological pathology; C – endocrine dysfunction; D – diseases of the cardiovascular system; E – combined pathology; F – no pathology.

Table 1. Dynamic Changes in Biochemical Parameters in Patients with Rosacea During the Treatment.

Indicants Study group Control group Before treatment After treatment Before treatment After treatment Cholesterol, mm /L 5.75+0.28 3.52+0.09* # 6.09+0.26 4.22+0.11* Bilirubin, mmol/L 20.66+0.96 11.06+1.1* 19.96+0.96 12.08+1.15* ALT, IU/L 38.34+1.64 20.9+1.95* 37.36+1.64 22.7+1.84* AST, IU/L 37.22+1.62 20.65+0.97* 37.12+1.62 22.04+0.88* GGT, IU 33.03+0.68 19.95+0.93* 34.91+0.68 19.37+0.96* Thymol turbidity test, IU 6.55+0.55 1.8+0.07 *# 5.95+0.55 2.0+0.10* Note: * – p<0.05 – compared to the pre-treatment group; # – p<0.05 – compared to the control group. Research Results in Pharmacology 6(1): 21–27 25

Table 2. Dynamic Changes in Indicators of the LPS-BAS System in Patients with Rosacea During Treatment.

Indicants Study group Control group Before treatment After treatment Before treatment After treatment Catalase, mcg/ml 8.9+1.07 18.86+1.3* 9.2+1.16 17.54+1.6* TAA, % 40.26+2.4 50.07+1.7* 44.29+2.4 52.01+1.5* SOD, IU/ml 9.98+0.61 10.3+1.3 10.08+0.61 11.1+1.21 MDA, uIU/ml 5.75+0.8 2.83+0.3* 5.95+0.89 2.96+0.6* AHP, IU 0.88+0.04 0.18+0.02* 0.89+0.05 0.21+0.04* Note: * – p<0.05 – compared to the group before treatment.

Table 3. Dynamic Changes in Immune System Parameters in Patients with Rosacea During Treatment.

Indicants Study group Control group Control values Before treatment After treatment Before treatment After treatment (norm) CD4+, % 51.0+0.5° 45.0+0.3* 49.0+0.1° 46.0+0.4*° 45.0+0.2 CD8+, % 17.0+0.3° 21.0+0.4*# 17.2+0.6° 19.0+0.3*° 21.0+0.2 CD4+/CD8+ 2.8+0.1° 2.2+0.3*# 2.8+0.2° 2.4+0.2*° 2.1+0.1 CD16+, % 11.9+0.9° 13.2+0.6*# 10.2+0.1° 12.7+0.2*° 14.3+0.7 CD22+, % 12.7+0.4° 14.1+0.9* 11.8+0.2° 14.4+0.6* 14.3+0.1 IgG, g/L 18.5+1.1° 14.0+0.1* 18.5+0.8° 13.7+0.9* 13.5+0.3 IgА, g/L 3.9+0.9° 2.2+0.3* 3.2+1.0° 2.0+0.2* 2.0+0.6 IgМ, g/L 1.7+0.4° 1.2+0.3* 1.5+0.5° 1.2+0.4* 1.1+0.3 НСТ test, % 27.6+2.0° 23.6+1.2*# 27.6+7.3° 27.8+1.1° 22.5+1.6 IL10, % 18.5+1.3° 14.5+2.1* 18.4+2.2° 14.9+3.2* 12.3+1.9 IL12, % 41.8+6.1° 36.6+5.0* 41.6+4.9° 38.0+5.0*° 32.3+2.3 Note: * – p<0.05 - compared to the pre-treatment group; # – p<0.05 - compared to the control group; ° – p<0.05 - compared to the control values. nificantly (3 times) higher in all forms of rosacea than in Cortexin. In the patients of the control group, the levels the group of the healthy individuals. of helper T-lymphocytes, lymphocytic index, and inter- During the treatment, there was a marked tendency leukin 12 remained elevated, against the background of to normalize the functioning of the LPS-BAS system in slightly reduced levels of suppressor T-lymphocytes. both groups. This was expressed in an increase in the ac- The neurotrophic drug Cortexin is a lyophilizer of the tivity of enzymes of the antioxidant system (catalase and cerebral cortex of calves. It consists of neuropeptides, plasma TAA), as well as a decrease in the content of li- amino acids, and trace elements. All these components pid peroxidation products (MDA, AHP). There were no of Cortexin determine the spectrum of its indications for significant differences in the indicators of the LPS-BAS specific correction of molecular and cellular processes system in the study and control groups. The comparison at various stages of the pathological process. The thera- of the data showed a decrease in the activity of the inflam- py using Cortexin significantly optimized the treatment matory process against the background of the therapy, by prognosis and made it possible to achieve the best the- normalizing the indicators of the LPS-BAS system. rapeutic result in the treatment of patients with rosacea. The state of the immune cell link was evaluated before treatment by the levels of helper T-lymphocytes, suppressor T-lymphocytes, lymphocytic index, killer T-lympho- Conclusion cytes, and B-lymphocytes (Table 3). Before treatment, the level of the immunologic profile 1. Therapy using Cortexin made it possible to achieve in the patients with rosacea showed a significant incre- a better clinical effect in terms of dermatological sta- ase in the immune-regulatory index – the ratio of CD4+/ tus, compared with the traditional therapy. The inno- CD8+ by increasing the level of T-helpers (CD4+) and vative treatment helped 40% of the patients get rid of reduced T-suppressor (CD8+), which reflects the intensity the clinical manifestations of the disease, and none of immune responses and suggests the hyperactivity of of the patients in this group lacked a clinical effect. the immune system. There was also a decrease in the level 2. The therapy with Cortexin does not have a signifi- of natural killer cells and B-lymphocytes, and an increase cant advantage over the traditional treatment in the in the phagocytic activity of neutrophils (NBT test). processes of normalization of the hepatobiliary sys- In the process of evaluating the humoral immunity indi- tem and the blood LPS-BAS. cators (Table 3), the majority of patients showed an increase 3. Cortexin, being a complex of natural low-molecu- in the production of the main classes of immunoglobulins lar-weight peptides, has a corrective effect on the – IgG, IgA, IgM in blood serum, as well as an increase in cellular and humoral parts of the immune system. the level of anti-inflammatory cytokines IL-10 and IL-12. The therapy with Cortexin resulted in normaliza- After the therapy, the normalization of immune system tion of immune system parameters in the patients parameters was recorded in the patients who had taken with rosacea. In contrast to the traditional treatment, 26 Bobrakova AA: Effect of complex therapy with Cortexin...

which maintained elevated levels of helper T-lym- Conflict of interests phocytes, lymphocytic index and interleukin 12, against the background of slightly reduced levels of T-lymphocyte suppressors. The author declares no conflict of interest.

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Author contributions

Alexandra A. Bobrakova, dermatovenerologist, Head of the State Budgetary Institution of Healthcare ”Oryol Regional Skin and Veneralogic Clinic”, e-mail: [email protected] The author defined the purpose and objectives of the study, developed its design, analyzed the current scientific literature on the topic of the study, collected the material, analyzed and interpreted the results of the study. Research Results in Pharmacology 6(1): 29–40 UDC: 618.3-008.6-08-039.71 DOI 10.3897/rrpharmacology.6.50851

Research Article

Correction of morphofunctional disorders of the cardiovascular system with asialized erythropoietin and arginase II selective inhibitors KUD 974 and KUD 259 in experimental preeclampsia

Tatyana I. Lokteva1, Il’ya S. Rozhkov1, Vladimir V. Gureev1, Anastasia V. Gureeva3, Marija A. Zatolokina3, Elena V. Avdeyeva3, Lyudmila A. Zhilinkova2, Evgenij E. Prohoda4, Elizoveta O. Yarceva3

1 Belgorod State University, 85 Pobedy St., Belgorod 308015, Russia 2 Kursk Academy of State and Municipal Service, 9 Stantsionnaya St., Kursk 305044, Russia 3 Kursk State Medical University, 3 K. Marx St., Kursk 305041, Russia 4 Kursk Regional Clinical Hospital, 45a Sumskaya St., Kursk, 305007, Russia

Corresponding author: Vladimir V. Gureev (produmen@yandex)

Academic editor: Oleg Gudyrev ♦ Received 5 January 2020 ♦ Accepted 3 March 2020 ♦ Published 20 March 2020

Citation: Lokteva TI, Rozhkov IS, Gureev VV, Gureeva AV , Zatolokina MA, Avdeyeva EV, Zhilinkova LA, Prohoda EE, Yarceva EO (2020) Correction of morphofunctional disorders of the cardiovascular system with asialized erythropoietin and arginase II selective inhibitors KUD 974 and KUD 259 in experimental preeclampsia. Research Results in Pharmacology 6(1): 29–40. https:// doi.org/10.3897/rrpharmacology.6.50851

Abstract Introduction: Preeclampsia remains one of the most common causes of maternal and perinatal mortality worldwide. A significant role in the pathogenesis of this pathology is assigned to placental ischemia and endothelial dysfunction. Therefore, the aim of the present study was to study the effectiveness of asialized erythropoietin and arginase II selective inhibitors: KUD-259 and KUD-974 in the correction of morphofunctional disorders of the cardiovascular system in experimental preeclampsia. Materials and methods: The study was performed in 260 female Wistar rats, each weighing 250–300 g. An AD- MA-like preeclampsia was reproduced in the experiment. To assess the emerging morphofunctional disorders, the following parameters were used: blood pressure, coefficient of endothelial dysfunction, microcirculation in the placenta, proteinuria, fluid content in the omentum, concentration of terminal metabolites in the blood plasma, and morphometric parameters of fetuses. Results and discussion: The administration of arginase II selective inhibitor KUD-974 in combination with asialized erythropoietin leads to a pronounced correction of emerging changes: a decrease in systolic and diastolic blood pressure in 1.5 and 1.7 times, a decrease in proteinuria in 3.6 times and a decrease in fluid content in the omentum. When arginase II selective inhibitor KUD 974 and asialized erythropoietin are used with methyldopa, the positive effects of the former are enhanced. Conclusion: Arginase II selective inhibitors KUD-259 and KUD-974 and asialized erythropoietin have a pronounced positive effect on the correction of morphofunctional disorders in animals withADMA-like preeclampsia.

Keywords arginase II selective inhibitor, asialized erythropoietin, preeclampsia, endothelial dysfunction.

Copyright Lokteva TI et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 30 Lokteva TI et al.: Correction of morphofunctional disorders of the cardiovascular system...

Introduction In view of the fact that placental ischemia is another component of the pathogenesis of preeclampsia (George The frequency of hypertensive pregnancy complicati- et al. 2017; Tomimatsu et al. 2019), erythropoietin deriv- ons is 5–30%, according to the Ministry of Health of the atives not having an erythropoietic effect are a promising Russian Federation, while they occupy the fourth place group of drugs in the treatment and prevention of preec- in the structure of maternal mortality rate. From 2 to 8% lampsia (Mofidi et al. 2011; Ishii et al. 2012; Kaneko et of pregnancies are complicated by preeclampsia in the al. 2013). These circumstances created the prerequisites world, according to rough estimates (Alkema et al. 2016; for the present study. Roberge et al. 2017; Conti-Ramsden et al. 2019). Hyper- Objective: To prove the effectiveness of asialized tensive disorders are the cause of the 16% of maternal erythropoietin and selective arginase II inhibitors: KUD- losses in countires with high levels of socio-economic de- 259 and KUD-974 in the correction of morphofunctional velopment (Pankiewicz et al. 2019). disorders of the cardiovascular system arising from ex- According to modern concepts, the development of perimental preeclampsia. preeclampsia is based on the disorder of the endothelium structural and functional state (Abalos et al. 2013; Kittur et al. 2013; Yanagawa et al. 2013). It arose as a result of Materials and methods placentation disorder on the background of incomplete remodeling of the uterine arteries, which ultimately leads The experimental study was conducted at the Research to a decrease in blood supply and placental ischemia Institute of Pharmacology of Living Systems of Belgorod (George et al. 2017; Tomimatsu et al. 2019). State National Research University. The study was per- In spite of a huge amount of the studies conducted, the formed in compliance with the General Requirements for only method of treating this condition recognized by all the Competence of Testing and Calibration Laboratories experts is still timely delivery, aimed primarily at min- 17025-2009, GOST R ISO 5725-2002 and the Rules of imizing the threat to pregnant woman’s life. All other Laboratory Practice, approved by Order of the Ministry of recommendations for the management of patients with Healthcare and Social Development of the Russian Federa- preeclampsia can be reduced to the following principles: tion dated August 23rd, 2010 № 708n. All the experiments correction of hypovolemia, normalization of blood pres- were approved by the Ethical Committee of Belgorod State sure, restoration of the blood rheological properties, and National Research University. Vivisection was performed prevention of the progression of endothelial dysfunction in compliance with the ethical principles of treating labora- (Pankiewicz et al. 2019; Tomimatsu et al. 2019). tory animals of the European Convention for the Protection Nowadays, the only proven effective method for the of Vertebrates Used for Experimental and Other Scientific prevention of preeclampsia is the administration of low Purposes. CETS No. 123 (European Treaty Series 1986). doses of aspirin. A number of international recommenda- ADMA-like preeclampsia was simulation. The study tions, including those by WHO, state that after 12 weeks was performed in 260 female Wistar rats, each weigh- of pregnancy, aspirin is required at a dose of 75–100 mg ing 250–300 g. ADMA-like preeclampsia was simulat- per day. More recent studies concluded that the dose of ed by administration of a nonselective eNOS blocker aspirin should be more than 100 mg per day, and its ad- N-nitro-L-arginine methyl ether (L-NAME) at a dose of ministration after 16 weeks of pregnancy had more ad- 25 mg/kg/day intraperitoneally from the 14th to 20th day vantages for the prevention of preeclampsia (Toppozada of pregnancy. One day after the last injection under an- et al. 1991; Montfort et al. 2020). esthesia (chloral hydrate 300 mg/kg), hemodynamic pa- Preclinical studies in L-NAME induced preeclampsia rameters were recorded, and the endothelial function was in rats demonstrated the positive effects of resveratrol (Zou studied using a sensor and a hardware complex for inva- et al. 2014), recombinant erythropoietin (Gureev 2016) sive measurement of hemodynamic parameters by Biopac and tadalafil (Yoshikawa et al. 2017). Another promising (USA) and the AcqKnowledge 3.8.1 computer software group of drugs for the treatment of hypertensive condi- (Stupakova et al. 2019). tions in pregnant women is arginase inhibitors (Nguyen et Assessment of an endothelial dysfunction degree al. 2016; Severinova et al. 2019). At the same time, argin- in the simulated pathology. Endothelial dysfunction ase II inhibitors as being more selective are of particular was evaluated by calculating a coefficient of endothelial interest (Pokrovskii et al. 2017; Gureev et al. 2015). dysfunction (CED), which is the ratio of endothelium-de- So, despite a wide range of drugs, objective reality dic- pendent vaso-relaxation (acetylcholine) and endotheli- tates the need to search for and study the effectiveness um-independent vaso-relaxation (sodium nitroprusside) of new and currently existing drugs aimed at the preven- (Korokin et al. 2019). tion and treatment of preeclampsia and its complications. Estimation of the terminal NO metabolites. The lev- There is evidence of endothelial protective properties of el of NO metabolites (that is the total concentration of selective arginase II inhibitors KUD-259 and KUD-974 nitrates and nitrites, NOx) was determined by the color- (Pokrovskii et al. 2017); however, no studies of their ef- imetric method, according to the staining pattern in the fectiveness in experimental preeclampsia have been con- diazotization reaction of sulfonamide nitrite, which is a ducted yet. part of the reagent (Stupakova et al. 2019). Research Results in Pharmacology 6(1): 29–40 31

Analysis of the placental microcirculation. Microcir- 3. Simulation of experimental preeclampsia + meth- culation in the placenta was measured using Biopac sys- yldopa (2 × 0.043 g/kg twice a day intragastrically) tems equipment: an MP100 polygraph with laser Doppler 4. Simulation of experimental preeclampsia + recom- flowmetry module (LDF) LDF100C and an invasive needle binant erythropoietin (50 IU/kg/day intraperitoneal- probe TSD144 (Korokin et al. 2015), which was mounted ly, on days 7, 10, 13, 16, and 19). directly in the projection of the placental disk. Registration 5. Simulation of experimental preeclampsia + asialized and processing of LDF results were carried out using Ac- erythropoietin (0.4 μg/kg/day intraperitoneally). qKnowledge 3.8.1 software; microcirculation values were 6. Simulation of experimental preeclampsia + asialized expressed in perfusion units (PU) (Gureev 2016). erythropoietin (2.4 μg/kg/day intraperitoneally). The study of swelling of the omentum. To study the 7. Simulation of experimental preeclampsia + L-Nor- liquid content in the omentum, it was weighed, followed valine (10 mg/kg/day intragastrically). by drying at 37 °C for 24 hours and another weighing. 8. Simulation of experimental preeclampsia + selec- Morphological methods for assessing changes in the tive arginase II inhibitor KUD-259 (1 mg/kg/day placenta and kidneys in ADMA-like preeclampsia. A intragastrically). histological examination (in all series of the experiment) 9. Simulation of experimental preeclampsia + selec- of the kidneys and placenta was performed for morpholog- tive arginase II inhibitor KUD-974 (1 mg/kg/day ical confirmation of the development of simulated patho- intragastrically). logical processes and in a comprehensive assessment of 10. Simulation of experimental preeclampsia + meth- the drugs effectiveness. The material was fixed in 10% yldopa (2 × 0.043 g/kg twice a day intragastrically) + formalin, followed by paraffin embedding. The kidneys asialized erythropoietin (2.4 μg/kg/day intraperitone- were sliced perpendicular to the main axis of the organ ally). through the pelvis. Histological sections of the placenta 11. Simulation of experimental preeclampsia + meth- were performed in a strictly vertical direction through the yldopa (2 × 0.043 g/kg twice a day intragastrically) middle of the placental disc, capturing all layers of the + selective arginase II inhibitor KUD-974 (1 mg/kg/ placenta and the walls of the uterine horn. The study of the day intragastrically). slides, photoprotocoling and morphometry were carried 12. Simulation of experimental preeclampsia + asial- out using a Leica DM4000B microscope with a video re- ized erythropoietin (2.4 μg/kg/day intraperitoneal- cording and image processing system. For all the morpho- ly) + selective arginase II inhibitor KUD-974 (1 mg/ logical studies, hematoxylin and eosin staining was used. kg/day intragastrically). The study of embryos. The embryos were removed from the uterine cavity; their weight and growth (cranio- caudal size) were measured, followed by calculating the Results and discussion growth-weight coefficient. Statistical processing of the research results. De- scriptive statistics were applied to all the data. The data Effect of asialized erythropoietin on morphofunctional were checked for normal distribution. The type of dis- disorders resulting from experimental preeclampsia tribution was determined by the Shapiro-Wilk criterion. In the case of a normal distribution, the mean value (M) Female white Wistar rats, each weighing 250–300 g, and the standard error of the mean (m) were calculated. were used in the experiment. To simulate experimental The intergroup differences were analyzed using Student’s preeclampsia, N-nitro-L-arginine methyl ether (L-NA- t-test or Mann-Whitney U-test, depending on the type of ME), which has the biological properties similar to tho- distribution. The statistical significance of the differences se of asymmetric dimethylargenin (ADMA) – an eNOS between morphological changes after their ranking was blockade – was administered at a dose of 25 mg/kg intra- evaluated using Mann-Whitney nonparametric method of peritoneally from the 14th to 20th day of pregnancy. This data analysis. All the calculations were performed using a resulted in a statistically significant (p < 0.05) increase in Microsoft Excel 7.0 statistical package. systolic and diastolic blood pressure from 123.2 ± 3.46 and 76.3 ± 5.71 to 194.8 ± 7.88 and 149.8 ± 4.73 mmHg, Experiment design respectively, in comparison with the group of the intact animals (Table 1). According to the objective, all animals were divided into Administration of asialized erythropoietin to the an- the following groups: imals with experimental preeclampsia at a dose of 0.4 μg/kg/day intraperitoneally did not lead to a statistical- 1. Control group (animals with oral administration of ly significant decrease in blood pressure. A statistically NaCl at equivalent doses from the 14th to 20th -day significant (p < 0.05) decrease in systolic and diastolic of pregnancy). blood pressure to 167.3 ± 3.43 and 129.4 ± 4.17 mmHg 2. Simulation of experimental preeclampsia (L-NAME was observed when administering asialized erythropoi- (25 mg/kg once daily intraperitoneally) from the etin intraperitoneally at a dose of 2.4 μg/kg/day to the 14th to the 20th day of pregnancy). animals with experimental preeclampsia, but it did not 32 Lokteva TI et al.: Correction of morphofunctional disorders of the cardiovascular system...

Table 1. Experimental results of the correction of experimental preeclampsia with asialized erythropoietin in rats (M ± m; N = 10).

Indicator Group SBP, mm hg DBP, mm hg CED, cond. un. Microcircula-tion, PU Intact 123.2 ± 3.46y 76.3 ± 5.71y 1.21 ± 0.08y 493 ± 21.1y L-NAME 194.8 ± 7.88* 149.8 ± 4.73* 3.17 ± 0.22* 212 ± 6.0* Methyldopa (2× 0.043 g/kg) 134.9 ± 1.89y* 102.0 ± 3.71y* 2.51 ± 0.18y* 272 ± 12.2y* EPo (50 IU/kg) 179.3 ± 3.84y* 133.4 ± 2.62y* 2.12 ± 0.21y* 301 ± 10.1y* AsEPo (0.4 μg/kg) 183.1 ± 6.71y* 139.7 ± 3.72y* 2.09 ± 0.14y* 357 ± 22.6y* AsEPo (2.4 μg/kg) 167.3 ± 3.43y* 129.4 ± 4.17y* 1.67 ± 0.12y* 413 ± 20.0y* Note: SBP, DBP – systolic and diastolic blood pressure (mmHg); CED – coefficient of endothelial dysfunction; EPo – recombinant erythropoietin; AsEPo – asialized erythropoietin; PU – perfusion units; * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group. reach the target level. It is worth noting that the intensity metabolites in plasma from 2.23 ± 0.04 μmol/dl (in intact of the hypotensive effect of asialized erythropoietin was animals) to 1.25 ± 0.01 μmol/dl (Fig. 1A). The admin- comparable with the hypotensive effect of recombinant istration of asialized erythropoietin to the animals with erythropoietin, but was inferior to the hypotensive effect experimental preeclampsia at a dose of 0.4 μg/kg/day and of methyldopa, a drug used in the treatment of hyperten- 2.4 μg/kg/day intraperitoneally led to a statistically sig- sive conditions in pregnant rats. nificant (p < 0.05) increase in this indicator to 1.49 ± 0.02 Simulation of experimental preeclampsia was accom- μmol/dl and 1.61 ± 0.02 μmol/dl, respectively, but it did panied by a change in the rate of reactions to vasodilat- not reach the target level. ing humoral factors, which indicated the disorder of the Simulated preeclampsia did not change the daily di- regulatory mechanisms of vascular tone and is explained uresis, but it led to a statistically significant (p < 0.05) by the resulting endothelial dysfunction. The coefficient increase in proteinuria compared with the intact pregnant of endothelial dysfunction (CED) increased from 1.21 ± animals from 0.18 ± 0.05 g/L to 2.02 ± 0.20 g/L (Fig. 1B). 0.08 (intact animals) to 3.17 ± 0.22 relative units. Admin- The administration of asialized erythropoietin at a dose of istration of asialized erythropoietin to the animals with 0.4 μg/kg/day and 2.4 μg/kg/day intraperitoneally led to a experimental preeclampsia at a dose of 0.4 μg/kg/day and statistically significant (p < 0.05) decrease in protein in the 2.4 μg/kg/day intraperitoneally led to a statistically signif- urine to 1.4 ± 0.06 g/L and 1.03 ± 0.08 g/L, respectively. icant (p < 0.05) decrease in CED to 2.09 ± 0.14 and 1.67 A study of the liquid content in the omentum of the ± 0.12 relative units, respectively, but CED did not reach animals with experimental preeclampsia revealed its in- the target level. This indicates a decrease in violations of crease (p < 0.05) compared with the intact pregnant ani- the mechanisms controlling blood pressure. mals from 44.20 ± 1.08% to 54.27 ± 0.64%. The admin- It is important to emphasize that a decrease in CED istration of asialized erythropoietin at the studied doses under the influence of asialized erythropoietin at a dose of led to a decrease in the level of swelling of the omentum 0.4 μg/kg/day was comparable with the effect of recombi- tissues (p < 0.05) to 49.89 ± 0.75% and 48.73 ± 0.81%, nant erythropoietin and exceeded the effect of methyldo- respectively, but it did not reach the target level. The liq- pa, a comparative drug for the treatment of hypertensive uid content in the omentum decreased in the animals with disorders in pregnant rats. The decrease in СED under the ADMA-like preeclampsia treated with methyldopa and influence of asialized erythropoietin at a dose of 2.4 μg/ recombinant erythropoietin (p < 0.05) to 50.51 ± 0.57% kg/day was more pronounced than the effect of both com- and 50.74 ± 0.51%, respectively. parison drugs. A histological study of placental microsections in In the animals with experimental preeclampsia, a sta- the group of intact animals on the 21st day of gestation tistically significant (p < 0.05) decrease in the placenta revealed two well-visualized parts of the placenta: fetal microcirculation was observed from 493 ± 21.1 perfusion and maternal. A microscopic examination of the histolog- units (PU) (intact animals) to 201 ± 6.0 PU. The admin- ical sections of the placenta in the animals with experi- istration of asialized erythropoietin to the animals with mental preeclampsia revealed distinct signs of destruc- experimental preeclampsia at a dose of 0.4 μg/kg/day and tive-dystrophic changes in the placenta. The administration 2.4 μg/kg/day intraperitoneally led to a statistically signif- of the studied pharmacological agents for the correction of icant (p < 0.05) increase in the placenta microcirculation experimental preeclampsia led to positive dynamics with to 357 ± 22.7 PU and 413 ± 20.0 PU, respectively, but the pronounced effect in the group of animals treated with did not reach the target level. However, it is of impor- asialized erythropoietin at a dose of 2.4 μg/kg/day. tance that under the influence of asialized erythropoietin The results of the study of the features of fetal de- at both doses, the improvement in microcirculation was velopment in the animals with ADMA-like preeclamp- more pronounced in comparison with the effects of both sia revealed fetal malnutrition (Table 2). The fetal mass comparison drugs (Table 1). changed to a greater extent. The most accurate indicator A biochemical study showed that the simulation of ex- reflecting this pattern is the fetal height-to-weight ratio, perimental preeclampsia led to a decrease in the final NO which statistically significantly increased for these- em Research Results in Pharmacology 6(1): 29–40 33

Table 2. Effect of asialized erythropoietin on statural-weight values of fetal development in ADMA-like preeclampsia (M ± m; N = 20).

Indicator Group Weight of embryos, g Stature of embryos, mm Statural- weight value, mm/g Intact 1.69 ± 0.03y 24.65 ± 0.37 14.57 ± 0.13y L-NAME 1.47 ± 0.03* 23.95 ± 0.40 16.33 ± 0.10* Methyldopa (2 × 0.043 g/kg) 1.52 ± 0.02* 23.80 ± 0.28 15.65 ± 0.08y* EPo (50 IU/kg) 1.54 ± 0.03* 24.25 ± 0.47 15.73 ± 0.09y* AsEPo (0.4 μg /kg) 1.54 ± 0.03* 24.20 ± 0.45 15.72 ± 0.09y* AsEPo (2.4 μg /kg) 1.63 ± 0.03y 24.80 ± 0.40 15.20 ± 0.09y* Note: * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group; EPo – recombinant erythropoietin; AsEPo – asialized erythropoietin.

Figure 1. Effect of asialized eryropoietin on the concentration of final nitric oxide metabolites in plasma during experimental preeclampsia (A), proteinuria (B). Note: * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group; EPo – recombinant erythropoietin; AsEPo - asialized erythropoietin. bryos. No post-implantation deaths were observed in any In the animals with experimental preeclampsia, the ad- groups. The administration of the studied pharmacologi- ministration of arginase II selective inhibitors: KUD-259 cal agents led to positive dynamics, namely an increase and KUD-974 – at a dose of 1 mg/kg/day intragastrical- in the mass of the fetus, though the level of the intact ani- ly led to a statistically significant (p < 0.05) increase in mals was not reached. the placenta microcirculation to 410 ± 18.7 PU and 394 ± 24.4 PU, respectively, but did not reach the target level. Effect of selective arginase II inhibitors: KUD-259 and However, it is worth noting that under the influence of KUD-974 on morphofunctional disorders in experi- both arginase II selective inhibitors: KUD-259 and KUD- mental preeclampsia 974, the improvement of microcirculation was comparable to the effect of the non-selective arginase II inhibitor The administration of arginase II selective inhibitors: – L-Norvaline – and was more pronounced in comparison KUD-259 and KUD-974 – to the animals with expe- with the drugs included in the treatment standards for hy- rimental preeclampsia led to a statistically significant pertensive conditions in pregnant women (Table 3). (p < 0.05) decrease in blood pressure compared with the A biochemical study found that the administration of group of the untreated animals only under the influence of arginase II selective inhibitors: KUD-259 and KUD-974 KUD-974 (Table 3). at a dose of 1 mg/kg/day intragastrically to the animals The use of arginase II selective inhibitors in the an- with experimental preeclampsia led to a statistically sig- imals with ADAM-like preeclampsia: KUD-259 and nificant (p < 0.05) increase in the content of terminal NO KUD-974 – at a dose of 1 mg/kg/day orally led to a sta- metabolites in blood plasma up to 1.60 ± 0.02 μmol/dl and tistically significant (p < 0.05) decrease in CED to 1.53 ± 1.80 ± 0.03 μmol/dl, respectively, but it did not reach the 0.10 and 1.70 ± 0.12 relative units, respectively, but did target level (Fig. 2A). not reach the target level. This indicates a decrease in vio- The administration of arginase II selective inhibitors: lations of the mechanisms controlling blood pressure. It is KUD-259 and KUD-974 – to the pregnant animals with of importance that a decrease in CED under the influence experimental preeclampsia did not lead to a change in di- of arginase II selective inhibitors: KUD-259 and KUD- uresis, but led to a decrease in proteinuria (p < 0.05) to 974 – at a dose of 1 mg/kg/day was comparable with the 0.76 ± 0.08 g/L and 0.89 ± 0.08 g/L, respectively. effect of the non-selective arginase II inhibitor – L-Nor- The use of arginase II selective inhibitors: KUD-259 valine – and exceeded the effect of the comparison drug and KUD-974 – led to a decrease in the level of swelling included in the treatment regimen of hypertensive condi- of the omentum tissues (p < 0.05) to 49.30 ± 0.26% and tions in pregnant – methyldopa. 48.92 ± 0.68%, respectively, but the target level was not 34 Lokteva TI et al.: Correction of morphofunctional disorders of the cardiovascular system...

Table 3. Correction results of experimental preeclampsia with arginase II selective inhibitors: KUD-259 and KUD-974 (M ± m; N = 10).

Indicator Group SBP, mm Hg DBP, mm Hg CED, relative units. Microcircula-tion, PU Intact 123.2 ± 3.46y 76.3 ± 5.71y 1.21 ± 0.08y 493 ± 21.1y L-NAME 194.8 ± 7.88* 149.8 ± 4.73* 3.17 ± 0.22* 212 ± 6.0* Methyldopa (2 × 0.043 g/kg) 134.9 ± 1.89y* 102.0 ± 3.71y* 2.51 ± 0.18y* 272 ± 12.2y* L-Norvaline (10 mg/kg) 201.3 ± 2.89* 153.4 ± 3.27* 1.81 ± 0.12y* 406 ± 19.8y* KUD-259 (1 mg/kg) 195.8 ± 5.52* 144.9 ± 6.88* 1.53 ± 0.10y* 410 ± 18.7y* KUD-974 (1 mg/kg) 148.1 ± 2.96y* 105.7 ± 4.85y* 1.70 ± 0.12y* 394 ± 24.4y* Note: SBP, DBP – systolic and diastolic blood pressure (mmHg); CED – coefficient of endothelial dysfunction; PU – perfusion units; * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group.

Figure 2. The effect of arginase II selective inhibitors: KUD-259 and KUD-974 – on the concentration of terminal nitric oxide metabolites (A) in plasma and proteinuria during experimental preeclampsia (B). Note: * – p < 0.05 in comparison with the group of the intact animals; y – p < 0.05 compared with the L-NAME group. reached. When the animals with ADMA-like preeclamp- combination with methyldopa decreased systolic and dia- sia were administered with L-Norvaline, the fluid content stolic blood pressure to the level not statistically different in the omentum tissues was 49.57 ± 0.70%. from that in the group of the intact animals. In the group After the pharmacological correction, there was a pos- of the animals treated with asialo-EPO and methyldopa, itive dynamics of the previously detected reactive mor- only systolic pressure decreased to the level statistically phological changes in the placenta structures on the 21st indistinguishable in comparison with the group of the in- day of gestation during experimental preeclampsia in all tact animals. the experimental groups . The most pronounced positive The combined use of asialized erythropoietin and ar- effects were observed in the group of the animals treated ginase II selective inhibitor KUD-974 led to a statistically with arginase II selective inhibitor KUD-794. significant (p < 0.05) decrease in systolic and diastolic The administration of the studied arginase II selective blood pressure compared with the group of the untreat- inhibitors: KUD-259 and KUD-974 – led to an increase in ed animals, while the level of diastolic pressure was not fetal weight in the animals with ADMA-like preeclamp- statistically indistinguishable from the group of intact an- sia, but the level of the intact animals was not reached imals. A decrease in blood pressure in the combined use (Table 4). of the pharmacological agents to the level statistically indistinguishable from the group of the intact animals can Effect of the combination of asialized erythropoietin be regarded as potentiation of their action, since this was and selective arginase II KUD-974 inhibitor and their not observed in the monotherapy. combined use with methyldopa on morphofunctional The use of a combination of asialized erythropoietin disorders in ADMA-like pre-eclampsia with arginase II selective inhibitor KUD-974 in the animals with experimental preeclampsia, and their combina- The administration of asialo-EPO and arginase II selec- tion with methyldopa, led to a more pronounced decrease tive inhibitor KUD-974 in combination with a standard in CED compared with groups of the animals where these therapy of hypertensive conditions methyldopa in the pharmacological agents were used as a monotherapy (Ta- pregnant animals with experimental preeclampsia led to ble 5). In all the groups with combined use of the studied a statistically significant (p < 0.05) decrease in systolic pharmacological agents, CED reached the level of that in and diastolic blood pressure compared with the group of the intact animals. the untreated animals (Table. 5). It is important to note The administration of asialized erythropoietin and that the use of arginase II selective inhibitor KUD-974 in arginase II selective inhibitor KUD-974 in combination Research Results in Pharmacology 6(1): 29–40 35

Table 4. Effect of arginase II selective inhibitors: KUD-259 and KUD-974 – on statural-weight value of fetal development with ADMA-like preeclampsia (M ± m; N = 20).

Group Indicator Weight of embryos, g Stature of embryos, mm Statural-weight value, mm / g Intact 1.69 ± 0.03y 24.65 ± 0.37 14.57 ± 0.13y L-NAME 1.47 ± 0.03* 23.95 ± 0.40 16.33 ± 0.10* L-NAME + L-Norvaline 1.58 ± 0.03y* 24.45 ± 0.53 15.51 ± 0.06y* L-NAME + Methyldopa 1.52 ± 0.02* 23.80 ± 0.28 15.65 ± 0.08y* L-NAME + KUD-259 1 mg/kg/day 1.60 ± 0.02y* 24.30 ± 0.31 15.16 ± 0.10y* L-NAME + KUD-974 1 mg/kg/day 1.61 ± 0.03y* 24.20 ± 0.39 15.07 ± 0.08y* Note: * – p < 0.05 in comparison with the group of the intact animals; y – p < 0.05 compared with the L-NAME group.

Table 5. Effect of asialized erythropoietin and arginase II selective inhibitor KUD-974 Combined with Methyldopa, as well as the combined use of asialized erythropoietin and arginase II selective inhibitor KUD-974 on functional parameters in experimental preeclampsia (M ± m; N = 10).

Indicator Group SBP, mm Hg DBP, mm Hg. CED, relative units Microcircu-lation, PU Incact 123.2 ± 3.46y 76.3 ± 5.71y 1.21 ± 0.08y 493 ± 21.1y L-NAME 194.8 ± 7.88* 149.8 ± 4.73* 3.17 ± 0.22* 212 ± 6.0* Methyldopa (2 × 0.043 g/kg) 134.9 ± 1.89y* 102.0 ± 3.71y* 2.51 ± 0.18y* 272 ± 12.2y* AsEPo (2.4 μg/kg) 167.3 ± 3.43y* 129.4 ± 4.17y* 1.67 ± 0.12y* 413 ± 20.0y* KUD-974 (1 mg/kg) 148.1 ± 2.96y* 105.7 ± 4.85y* 1.70 ± 0.12y* 394 ± 24.4y* KUD-974 + Methyldopa 130.9 ± 4.43y 91.4 ± 5.07y 1.36 ± 0.10y 492 ± 15.8y AsEPo + Methyldopa 130.1 ± 3.70y 97.4 ± 4.93y* 1.44 ± 0.12y 480 ± 10.9y AsEPo + KUD-974 133.0 ± 2.50y* 88.1 ± 6.45y 1.30 ± 0.12y 481 ± 16.8y Note: SBP, DBP – systolic and diastolic blood pressure (mmHg); CED – coefficient of endothelial dysfunction (relative units); PU – perfusion units; EPo – recombinant erythropoietin; AsEPo – asialized erythropoietin; * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group.

with methyldopa, as well as a combination of asialized arginase II selective inhibitor KUD-974 in combination erythropoietin and arginase II selective inhibitor KUD- with methyldopa to 44.25 ± 0.87% and 43.24 ± 1.31%, 974 to the animals with experimental preeclampsia, led to respectively, as well as with the combined use of asial- an improvement in placental microcirculation to the level ized erythropoietin and arginase II selective inhibitor of the intact animals (Table 5). KUD-974 (41.95 ± 0.88%). Moreover, it is of importance A biochemical study of the concentration of terminal that this indicator in the described experimental groups NO metabolites in plasma revealed a statistically signifi- reached the level of that in the intact animals. cant increase (Fig. 3A) with the administration of asialized A histological examination of the placenta showed that erythropoietin and arginase II selective inhibitor KUD-974 the administration of asialized erythropoietin and argin- in combination with methyldopa, as well as with the com- ase II selective inhibitor KUD-974 in combination with bined use of asialized erythropoietin and arginase II selec- methyldopa, as well as a combination of asialized eryth- tive inhibitor KUD-974. Moreover, it is important to note ropoietin and arginase II selective inhibitor KUD-974 in that this indicator in the described experimental groups the animals with experimental preeclampsia, led to pro- reached the level of that in the intact animals. nounced positive dynamics of the morphological pattern The use of asialized erythropoietin and arginase comparable with that in the group of the intact animals. II selective inhibitor KUD-974 in combination with The results of the study of the fetal development in the methyldopa, as well as a combination of asialized eryth- animals with ADMA-like preeclampsia treated with the ropoietin and arginase II selective inhibitor KUD-974 combination of asialized erythropoietin and arginase II se- in the animals with experimental preeclampsia, led to a lective inhibitor KUD-974 with methyldopa, as well as the statistically significant decrease in protein concentration combination of asialized erythropoietin and arginase II se- in the urine. At the same time, proteinuria in the groups lective inhibitor KUD-974 revealed an increase in embry- with combined use of the studied pharmacological agents os’ weight (Table 6). In both groups in which the combined decreased to a level comparable to that in the group of the therapy included arginase II selective inhibitor KUD-974, intact animals. No changes in diuresis in the described the fetal weight reached the level of the intact animals. experimental groups were observed. Thus, the results of the experiments indicate a pro- When assessing the fluid content in the omentum, a nounced protective activity of asialized erythropoietin and statistically significant (p < 0.05) decrease was revealed arginase II selective inhibitor KUD-974 in combination with the administration of asialized erythropoietin and with methyldopa, as well as the combined use of asialized 36 Lokteva TI et al.: Correction of morphofunctional disorders of the cardiovascular system...

Table 6. Effect of asialized erythropoietin and arginase II selective inhibitor KUD-974 in combination with Methyldopa, as well as the combined use of asialized erythropoietin and arginase II selective inhibitor KUD-974 on fetal statural-weight values in AD- MAc-like preeclampsia (M ± m; N = 20).

Group Indicator Weight of embryos, g Stature of embryos, mm Statural-weight value, mm/g Intact 1.69 ± 0.03y 24.65 ± 0.37 14.57 ± 0.13y L-NAME 1.47 ± 0.03* 23.95 ± 0.40 16.33 ± 0.10* Methyldopa (2 × 0,043 g/kg) 1.52 ± 0.02* 23.80 ± 0.28 15.66 ± 0.08y* AsEPo (2.4 μg /kg) 1.63 ± 0.03y 24.80 ± 0.40 15.20 ± 0.09y* KUD-974 (1 mg/kg) 1.61 ± 0.03y* 24.20 ± 0.39 15.06 ± 0.08y* KUD-974 + Methyldopa 1.62 ± 0.03y 23.95 ± 0.52 14.81 ± 0.10y AsEPo + Methyldopa 1.62 ± 0.02y 24.20 ± 0.40 14.94 ± 0.10y* AsEPo + KUD-974 1.65 ± 0.03y 24.30 ± 0.41 14.78 ± 0.09y Note: * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group; EPo – recombinant erythropoietin; AsEPo – asialized erythropoietin.

Figure 3. The effect of asialized erythropoietin and arginase II selective inhibitor KUD-974 in combination withmethyldopa , as well as the combined use of asialized erythropoietin and arginase II selective inhibitor KUD-974 on the concentration of terminal nitric oxide metabolites in plasma (A) and proteinuria (B) in experimental preeclampsia. Note: * – p < 0.05 in comparison with the group of intact animals; y – p < 0.05 compared with the L-NAME group; EPo – recombinant erythropoietin; AsEPo – asialized erythropoietin. erythropoietin and arginase II selective inhibitor KUD-974 Another important factor in endothelial dysfunction is on correction of morphofunctional disorders that occur in ischemia. Endothelial cells, in comparison with other cells animals with simulated preeclampsia. It is worth noting of the body, are quite resistant to ischemia. It is assumed that the used combinations provide a more pronounced that during ischemia, endotheliocytes switch to anaerobic correction of morphofunctional disorders compared to us- energy metabolism and also produce heat shock proteins, ing each drug from this combinations as a monotherapy. glyceraldehyde-3-phosphate dehydrogenase enzymes and non-neuronal enolase, which are involved in glycolysis, which in turn increase cell resistance to damage (Lutsen- Conclusion ko et al. 2015). However, hypoxia alters the expression of a number of genes involved in the regulation of vascular One of the foundations of the modern concept of the de- tone, which leads to a shift in the balance between vaso- velopment of preeclampsia is a violation of the structural dilation and vasoconstriction towards the latter. Besides, and functional states of the endothelium (Pankiewicz et under conditions of hypoxia, the activity of phospholipas- al. 2019; Tomimatsu et al. 2019) as a result of placenta- es A and C, diacylglycerol lipase, increases; the cascade tion disorder on the background of incomplete remode- of arachidonic acid is launched, and the concentration of ling of the uterine arteries, which ultimately leads to a its derivatives increases, which also affect vascular tone. decrease in blood supply and placental ischemia (George One of the promising groups of drugs for the treatment et al. 2017; Tomimatsu et al. 2019). The endothelium is a of preeclampsia are arginase inhibitors. At present, a great powerful endocrine organ involved in regulating vascular deal of data has been accumulated on their pronounced tone and maintaining their normal structure, monitoring endothelial protective properties, including those obtained the rheological properties of blood and local inflammati- using various experimental models of preeclampsia (Gu- on. One of the manifestations of the development of en- reev 2016; Nguyen et al. 2016). Arginase II inhibitors are dothelial dysfunction is a deficiency of nitric oxide (NO), of particular interest, as being more selective. The pre- which is a powerful vasodilator (Aouache et al. 2018; clinical studies of the selective arginase II inhibitor ZB49- Than et al. 2018). 0010 in various pathology models showed its pronounced Research Results in Pharmacology 6(1): 29–40 37 endothelial protective properties that are superior to those pharmacological agents, having various mechanisms of for the non-selective inhibitor of L-norvaline (Gureev et action, affect a greater number of pathogenetic points. al. 2015; Nguyen et al. 2016; Severinova et al. 2019). The Thus, the administration of the drugs with a different literature contains data on the endothelial protective prop- mechanism of action to animals with ADMA-like preec- erties of arginase II selective inhibitors KUD-259 and lampsia leads to more pronounced protective effects com- KUD-974 on the functional state of vascular endothelium pared to the groups in which these drugs were used in a (Pokrovskii et al. 2017); however, to date, studies of their monotherapy. It is logical that the use of new pharma- effectiveness have not been conducted in experimental cological agents in complex therapy for the treatment of preeclampsia yet. preeclampsia is most appropriate and has great prospects Thus, it becomes obvious that short-lived erythropoi- for further research. etin derivatives that do not have an erythropoietic effect and selective arginase II inhibitors can serve as another 1. The use of asialized erythropoietin at the doses of promising area for searching for new drugs to prevent and 0.4 μg/kg and 2.4 μg/kg is accompanied by a pos- correct preeclampsia. In this regard, the purpose of this itive dynamics of morphofunctional disorders in study was to prove the effectiveness of asialized eryth- experimental preeclampsia, which is expressed in a ropoietin and arginase II selective inhibitors: KUD-259 decrease in blood pressure, a decrease in CED by and KUD-974 – in the correction of morphofunctional about 1.5 and 2 times, respectively, an increase in disorders of the cardiovascular system arising from ex- microcirculatory indicators by 1.7 and 2 times, a perimental preeclampsia. decrease in proteinuria by 31% and 49%, and an in- The course administration of asialized erythropoietin crease in the concentration of terminal metabolites led to dose-dependent protective effects, which resulted in of nitric oxide in blood plasma by 20% and 30%. the correction of morphofunctional disorders arising from 2. The use of arginase II selective inhibitors: KUD- this experimental pathology. There was a decrease in blood 259 and KUD-974 – at a dose of 1 mg/kg in exper- pressure, an improvement in the placental microcircula- imental preeclampsia is accompanied by a positive tion, a noticeable restoration of the regulatory mechanisms dynamics of morphofunctional disorders, which is of vascular tone, a decrease in proteinuria, a decrease in manifested in a decrease in blood pressure under swelling of the omentum, and a decrease in morphological the influence of KUD-974, a decrease in CED by abnormalities of the ischemic genesis in the placenta. about 2 times, and a 1.9-time increase in indicators The presence of pronounced protective effects of asial- of microcirculation, a decrease in proteinuria by ized erythropoietin can be explained by the ability to bind 63% and 56%, and an increase in the concentration to the erythropoietin heterodimeric receptor in the absence of final metabolites of nitric oxide in blood plasma of an erythropoietic effect (Joshi et al. 2010; Mofidi et al. by 25% and 44%. 2011; Kaneko et al. 2013). This is confirmed in the ex- 3. The combined use of arginase II selective inhibitor periments on various organs and tissues and is explained KUD-974 and asialized erythropoietin with meth- by anti-apoptotic and antioxidant properties, the ability to yldopa (2 × 0.043 g/kg/day) is accompanied by a restore the endothelial function (Joshi et al. 2010; Ishii positive dynamics of morphofunctional disorders, et al. 2012; Kaneko et al. 2013; Kittur et al. 2013). The which is manifested in a decrease in blood pressure pronounced activity during ischemic injuries (Yanagawa which is more pronounced in the combination of et al. 2013) logically follows from the ability to activate, KUD-974 with methyldopa, and a decrease in CED like recombinant erythropoietin, the processes of natural by about 2.2 and 2.4 times, an increase in microcir- cytoprotection occurring in ischemic preconditioning. culation to the level of the intact animals, a decrease Based on this, it is fair to assume that the effects observed in proteinuria and an increase in the concentration upon administration of recombinant erythropoietin to rats of terminal metabolites of nitric oxide in blood plas- with ADMA-like preeclampsia could be expected from ma to the level of the intact animals. asialized erythropoietin (Gureev 2016). 4. The combined use of arginase II selective inhibitor Positive effects are associated with blockade of the en- KUD-974 with asialized erythropoietin in experi- zyme – arginase II. The potential effects of the blockade mental pre-eclampsia is accompanied by a positive of this enzyme have been confirmed in many experiments dynamics of morphofunctional disorders, which is (Xiong et al. 2014; Sobolev et al. 2018). It was fair to ex- manifested in a decrease in blood pressure, a decrease pect that the positive effects obtained in the correction of in CED by about 2.4 times, an increase in microcir- morphofunctional disorders of ADMA-like preeclampsia culation, a decrease in proteinuria, and an increase in by other arginase II inhibitors would be found in the stud- the concentration of terminal nitric oxide metabolites ied pharmacological agents (Gureev et al. 2015; Nguyen in blood plasma to the level of the intact animals. et al. 2016; Severinova et al. 2019). The administration of the studied pharmacological agents in various combinations led to a more pronounced Conflict of interests correction of morphofunctional disorders in ADMA-like preeclampsia. This is explained by the fact that the used The authors state no conflict of interest to declare. 38 Lokteva TI et al.: Correction of morphofunctional disorders of the cardiovascular system...

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Author contributions

Tatyana I. Lokteva, postgraduate student, Department of Pharmacology and Clinical Pharmacology, e-mail: tati- [email protected] ORCID ID http://orcid.org/0000-0003-4072-1980. The author had a leading role in planning and performing the experiment, analyzing the data and literature, and writing the article.

Il’ya S. Rozhkov, postgraduate student, Department of Pharmacology and Clinical Pharmacology, e-mail: [email protected] ORCID ID http://orcid.org/0000-0002-9092-229X. The author had a leading role in planning and performing the experiment, analyzing the data and literature, and writing the article.

Vladimir V. Gureev, Doctor of Medical Sciences, Associate Professor, Professor of the Department of Pharmacol- ogy and Clinical Pharmacology, e-mail: [email protected]. ORCID ID http://orcid.org/0000-0003-1433-1225. The author took part in planning the experiments, analyzed the literature and participated in interpreting the data.

Anastasia V. Gureeva, 3-year student, Faculty of Medicine, e-mail: [email protected]. ORCID ID http:// orcid.org/0000-00031719-7316. The author took part in planning the experiments, analyzed the literature and participated in interpreting the data.

Marija A. Zatolokina, Doctor of Medical Sciences, Assistant Professor, Professor of the Department of Histology, Embryology, Cytology; e-mail: [email protected], ORCID ID http://orcid.org/0000-0002-9553-1597. The author took part in planning the experiments, analyzed the literature and participated in interpreting the data.

Elena V. Avdeeva, Doctor of Biological Sciences, Professor of the Department of Normal Physiology, e-mail: [email protected], ORCID ID http://orcid.org/0000-0002-7152-5483. The author took part in planning the experiments, analyzed the literature and participated in interpreting the data. 40 Lokteva TI et al.: Correction of morphofunctional disorders of the cardiovascular system...

Lyudmila A. Zhilinkova, PhD in Technical Sciences, Associate Professor, Department of Philosophy, Social, Le- gal and Natural Sciences, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-0443-7130. The author took part in planning the experiments, analyzed the literature and participated in interpreting the data.

Evgenij E. Prohoda, medical doctor of the Kursk Regional Clinical Hospital, e-mail: prohoda_evgenij@yandex. ru. The author analyzed the literature and participated in interpreting the data.

Elizoveta O. Yarceva, 6-year student, Faculty of Medicine, e-mail: [email protected]. The author analyzed the literature and participated in interpreting the data. Research Results in Pharmacology 6(1): 41–45 UDC: 577.151.45, 57.084 DOI 10.3897/rrpharmacology.6.50941

Research Article

Study of protective properties of butyrylcholinesterase in acute anticholinesterase poisoning on BChE-KO and BALB/c mice

Viktor A. Palikov1, Yuliya A. Palikova1, Igor A. Dyachenko1,2

1 Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Prospekt Nauki,, Pushchino, Moscow Region 142290, Russia 2 Pushchino Scientific Center, Russian Academy of Sciences, 3 Prospekt Nauki, Pushchino, Moscow Region 142290, Russia

Corresponding author: Viktor A. Palikov ([email protected])

Academic editor: Oleg Gudyrev ♦ Received 15 August 2019 ♦ Accepted 25 November 2019 ♦ Published 20 March 2020

Citation: Palikov VA, Palikova YuA, Dyachenko IA (2020) Study of protective properties of butyrylcholinesterase in acute anticholinesterase poisoning on BChE-KO and BALB/c mice. Research Results in Pharmacology 6(1): 41–45. https://doi. org/10.3897/rrpharmacology.6.50941

Abstract Introduction: The article presents the results of studying the protective properties of recombinant human butyrylcholinesterase (rhBChE) in a model of acute anticholinesterase poisoning in mice knocked out for the BChE gene. Balb/c inbred mice were also used to demonstrate the important role of BChE. Materials and methods: In the study, BChE-ko and Balb/c mice were used. An organophosphorus compound (OPC) paraoxon was used as a toxic agent causing acute anticholinesterase poisoning. rhBChE was used as an antidote for OPC poisoning. To obtain rhBChE, an expression system based on CHO cell lines was chosen. In order to suppress BChE in Balb/c mice, a carboxyl esterase blocker cresylbenzodioxaphosphorin oxide (CBDP) was used. Two parameters were used to study the recovery after toxicity modeling: the end time of the animal tremor and the distance covered in open-field for 3 minutes. Results and discussion: The acute poisoning model using the CBDP blocker showed that the sensitivity of Balb/c mice increased significantly. The use of rhBChE against the background of CBDP allowed achieving 100% survival of animals with the minimum lethal dose of paraoxon. Knockout mice are expected to be more sensitive to the toxin, and the use of a biological trap in the form of rhBChE made it possible for 70% of the animals to survive with the minimum lethal dose of paraoxon. Besides, the use of rhBChE facilitated reducing the recovery time after OPC poisoning. Conclusion: The results of the study showed that the use of rhBChE as a protective agent in acute OPC poisoning significantly increased the survival of the animals and reduced the clinical manifestations of poisoning.

Keywords organophosphorus compound, butyrylcholinesterase, knockout mice, in vivo model.

Copyright Palikov VA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 42 Palikov VA et al.: Study of protective properties of butyrylcholinesterase in acute...

Introduction ing high-fat feed unlike wild-type animals (Lockridge et al. 1987; De Vriese et al. 2005). In (Iwasaki et al. 2007), OPC is widely used both in agriculture and everyday life it was shown that the activity of serum BChE correlates as a means of combating insects, rodents, and weeds. with an obesity degreeof patients, with the lipid profile of Poisoning can be seasonal and massive. The pathways blood serum and with adegree of insulin resistance. of OPC in the human body are absorption by the skin, eyes and respiratory tract. Absorption can also take place through the gastric tract (self-poisoning). OPC molecules Materials and methods are distributed throughout the body from blood elements to organs and tissues, including natural depots, physio- Paraoxon was used as OPC. Paraoxon inhibits cholines- logical targets, and excretory organs (liver and kidneys). terase enzymes, mainly acetylcholinesterase (AChE), ne- OPC inhibit cholinesterase enzymes, mainly acetylcho- cessary for the destruction of acetylcholine (Terekhov et linesterase, necessary for the destruction of acetylcholine. al. 2015b). As a result, acetylcholine accumulates, which As a result, acetylcholine accumulates, which leads to leads to excitation, and subsequently to exhaustion and excitation, and subsequently to exhaustion and persistent persistent paralysis of cholinergic structures. The functi- paralysis of cholinergic structures (Masson et al. 2008). on of AChE is to terminate the action of acetylcholine at The main function of acetylcholinesterase (AChE) is the the joints of various cholinergic nerve endings with their hydrolysis of acetylcholine and, as a result, the cessation effector organs or postsynaptic sites. Organophosphorus of neurotransmission. When AChE is inhibited, the work compounds and carbamates are the most important AChE of the diaphragm and other respiratory muscles, as well as inhibitors; they are often called anticholinesterase inhi- the central rhythm generator in the brain stem, is disrupt- bitors. In the presence of inhibitors, AChE is gradually ed, resulting in respiratory failure and death of the body. suppressed and can no longer hydrolyze acetylcholine Butyrylcholinesterase, also called serum cholinester- (Terekhov et al. 2015a, Mokrushina et al. 2017; Terek- ase or pseudocholinesterase, is abundant in human plas- hov et al. 2017). As a result, acetylcholine does not form ma (3 mg/L) and has a half-life of 12 days (Lockridge choline and acetic acid, which causes the accumulation of et al. 2005; Ostergaard et al. 2006). BChE is present in acetylcholine on cholinergic receptor sites. This leads to almost all body tissues – liver, intestines, pancreas, pla- excessive stimulation of cholinergic receptors throughout centa, heart, in the central and peripheral nervous system, the central and peripheral nervous systems. etc. (Silver 2005). Serum BChE is synthesized in the liver To determine the effectiveness of butyrylcholinester- and from there enters the bloodstream. The physiological ase as a prophylaxis of poisoning with organophosphorus role of BChE is still not fully understood. Unlike AChE, compounds, a specific biomodel was used, which takes BChE does not have a unique physiological function that into account the difference in the esterase status of mice could not be compensated by other enzymes. People with- and humans. In human blood, there is twice as much out BChE activity are healthy, fertile, and live to old age BChE than in mouse blood (5 and 2.6 mg/L, respectively), (Manoharan et al. 2007). Experiments on knockout mice whereas the content of AChE is 25 times smaller (0.008 for the BChE gene also showed that the complete absence and 0.2 mg/L, respectively). Unlike many animals, there of BChE activity does not affect the health and fecundity is no carboxyl esterase in human plasma, which can lead of animals (Li et al. 2008; Lockridge et al. 2008). In total, to a false interpretation of the data when assessing the in the human and mouse bodies, there is on average 10 toxicity of OPC. In human plasma, there are two main es- times more BChE than AChE (Ashani and Pistinner 2005; terases, butyrylcholinesterase (BChE, 5 mg/L) and PON1 Rudakova et al. 2011; Kurdyukov et al. 2012). The high- (50 mg/L). In addition, in plasma there is a small amount est concentrations of BChE were found in the liver, blood of acetylcholinesterase (AChE), which practically has no plasma, skin, lungs, and small intestine, which indicate contribution to the esterase activity of the blood. In or- the protective role of the enzyme and its participation in der to minimize the background activity of endogenous the detoxification of xenobiotics that enter the body with carboxyl esterase in mice, a cresylbenzodioxaphosphorin food and air. oxide inhibitor (CBDP) was used at a dose of 1.5 mg/kg, As said above, the physiological function of BChE is which completely suppressed the action of this enzyme. not fully understood. By hydrolyzing various compounds This inhibitor was administered subcutaneously. and binding to OPC, BChE performs primarily protective The study was conducted on Balb/c and BChE-ko male functions in the body and also participates in the metabo- mice in accordance with the requirements of the current lism of drugs (Saxena et al. 2006; Masson and Lockridge Guidelines for the Preclinical Study of New Pharmacolog- 2010). There is evidence that BChE also plays a role in ical Substances and the Rules of Laboratory Practice in the development of type 2 diabetes mellitus (Kamal et al. the Russian Federation (National Standard of the Russian 2009). BChE is one of the esterases that inactivates the Federation, GOST 33647-2015). The procedures with the appetite-stimulating hormone octanoyl-ghrelin, turning it animals were reviewed and approved of by the Bioethical into an inactive form (pure peptide – ghrelin). The role Commission of the Institute of Bioorganic Chemistry of of BChE in fat metabolism is confirmed in experiments the Russian Academy of Sciences (RAS). The number of on BChE (-/-) mice that developed obesity when receiv- animals in the group was at least 8. Research Results in Pharmacology 6(1): 41–45 43

The experiment on mice of the Balb/c line was divided into 3 stages:

• At the first stage, groups of mice were injected with paraoxon intravenously at doses of 0.5; 0.55; 0.6; and 0.7 mg/kg. • At the second stage, the CBDP blocker was injected subcutaneously, and 30 minutes later paraoxon was administered intravenously at the doses similar to those at the first stage. • According to the results of the first two stages the doses of paraoxon from which the animals had died were selected for further research. A CBDP blocker Figure 1. Animal survival in an experimental model of acute an- was administered subcutaneously, then 15 minutes ticholinesterase poisoning. Note: Balb/c, BChE-ko – male mice, later, the mice were intravenously administered OPC – organophosphorus compound, CBDP – cresylbenzodiox- with BChE at a dose of 60 mg/kg, after which para- aphosphorin oxide. oxon was administered intravenously.

Further, the study of BChE as a prophylaxis of OPC poisoning was carried out on the knockout mice using the BChE-ko butyrylcholinesterase gene. This part of the experiment was divided into 2 stages:

• At the first stage, a CBDP blocker was subcutaneously administered to the groups of mice, Paraoxon was administered intravenously at doses 0.4; 0.5 and 0.55 mg/kg 30 minutes later. • According to the results of the first stage, two doses of paraoxon from which 100% and 50% of the animals had died were selected. A CBDP blocker was administered subcutaneously, then 15 minutes Figure 2. Survival of animals using BChE in an experimen- later the mice were intravenously administeredwith tal model of acute anticholinesterase poisoning. Note: Balb/c, BChE at a dose of 60 mg/kg, after which paraoxon BChE-ko – male mice, OPC – organophosphorus compound, was administered intravenously. CBDP – cresylbenzodioxaphosphorin oxide, BChE – butyrylcholinesterase. Clinical observations the action of the blocker.It was decided to continue a fu- Tremor is the main clinical sign of anticholinesterase rther study using CBDP. poisoning. For all the test systems, the end time of tre- BChE gene knockouts makes it possible to see how mor was recorded. To study a locomotor activity, the open the absence of this esterase affects OPC poisoning. The field test was used on an OptoVarimex-ATM3 unit. The sensitivity of the mice is increased by 30% relative to the observation time was 3 minutes, during which the distan- Balb/c mice using an inhibitor. 1These results confirm the ce covered was automatically recorded. Testing was per- importance of this esterase. formed 24 hours after poisoning. After confirming the theory associated with the use of a carboxyl esterase inhibitor, it was time to use this experimental model to prove the protective activity of BChE Results and discussion in acute anticholinesterase poisoning. Work with BChE began with a paraoxon dose of 0.55 mg/kg at which 70% The first priority was to test the theory using a CBDP of the animals had died. As a result of using BChE, the en- carboxyl esterase inhibitor. Since this esterase also acts tire group survived (Fig. 2). The next group was a group as a natural OPC biotrap, it can affect the interpretati- of animals, where, with the introduction of 0.6 mg/kg of on of the results. Figure 1 shows that a prior subcuta- paraoxon, the entire group died without exception. Using neous administration of CBDP significantly reduces the BChE made it possible for all the animals from this group chances of survival in the animals treated with paraoxon. to survive. It was further decided to continue increasing When using CBDP together with paraoxon at a dose of the dose of paraoxon. When using BChE together with 0.5 mg/kg, 35% of the animals die, at a dose of 0.55 mg/ paraoxon at a dose of 0.65 mg/kg, 40% of the animals kg, 85% of mice die, and a dose of 0.6 mg/kg is a lethal died, whereas an increase in the dose of paraoxon to dose. An increase in sensitivity to OPC is associated with 0.7 mg/kg caused the death of the entire group. 44 Palikov VA et al.: Study of protective properties of butyrylcholinesterase in acute...

group. The use of BChE raised the survival rate to 50%. The use of BChE at a paraoxon dose of 0.6 mg/kg did not affect survival; with all the animals from this group dying. In the death of animals, clear dose/effect dependence was observed on the administration of paraoxon. The locomotor activity showed similar results. When simulating poisoning using a CBDP inhibitor, the difference in distance in the group of animals treated with paraoxon at a dose of 0.5 mg/kg was especially noticeable (Fig. 3). One day after poisoning, the animals without carboxyl esterase were 60% less mobile than the animals in the group with this esterase. The locomotor activity implies the severity Figure 3. Locomotor activity of animals in an experimental of poisoning and recovery time. As expected, the knockout model of acute anticholinesterase poisoning. Note: Balb/c, mice, even 24 hours later, felt worse than the Balb/c mice. BChE-ko – male mice, OPC – organophosphorus compound, In addition to the fact that preliminary administration of CBDP – cresylbenzodioxaphosphorin oxide. BChE at a dose of 60 mg/kg can significantly increase the chances of survival, the recovery time after poisoning is significantly reduced (Fig. 4). The Balb/c mice with BChE doubled the distance traveled. In the same way, the BChE- ko mice improved their rate by 2 times. It can be argued with confidence that the introduction of BChE will lead to a further decrease in delayed clinical signs OPC poisoning.

Conclusion

According to the results of the study, it can be argued that the intravenous use of BChE at a dose of 60 mg/kg can significantly increase the chances of survival of animals Figure 4. Locomotor activity of animals using BChE in an ex- that have received a minimal lethal dose of OPC. In ad- perimental model of acute anticholinesterase poisoning. Note: dition to the survival, the recovery time after poisoning Balb/c, BChE-ko – male mice, OPC – organophosphorus com- is reduced, which indicates a decrease in the delayed ad- pound, CBDP – cresylbenzodioxaphosphorin oxide, BChE – bu- verse effects of poisoning. From the mechanism of acti- tyrylcholinesterase. on of BChE it is clear that the larger the dose, the more pronounced the effect. The question naturally arises of the maximum tolerated dose. In the future, more research As for working with the knockout mice, 0.5 mg/kg was is needed to study the issue of BChE activity in chronic chosen to study the activity of BChE as the starting dose OPC poisoningand to check the safety of this esterase. of paraoxon, in accordance with which the death of 50% of the animals from the group was observed. The use of BChE at this dose of paraoxon can increase the survival of Conflict of interest animals up to 100%. In the experimental model, paraoxon at a dose of 0.55 mg/kg caused the death of the entire The authors declare no conflict of interest.

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Author contributions

Viktor A. Palikov, researcher, Laboratory of Biological Tests, e-mail: [email protected], ORCID ID https://orcid.org/0000-0003-2989–4477. The author participated in planning the experiments, analysis of the literature and interpreting the data.

Yuliya A. Palikova, researcher, Laboratory of Biological Tests, e-mail: [email protected], ORCID ID https://orcid.org/0000-0001-9547-0686. The author participated in planning the experiments, analysis of the literature and interpreting the data.

Igor A. Dyachenko, PhD in Biological Sciences, senior researcher, Laboratory of Biological Tests, e-mail: [email protected], ORCID ID https://orcid.org/0000-0002-3053-2804. The author participated in planning the experiments, analysis of the literature and interpreting the data. Research Results in Pharmacology 6(1): 47–55 UDC: 611.018.74:615.256.4 DOI 10.3897/rrpharmacology.6.50942

Review Article

Role of Arginase 2 as a potential pharmacological target for the creation of new drugs to correct cardiovascular diseases

Sergey A. Demchenko1, Ivan S. Koklin2, Natalia Yu. Koklina2

1 Belgorod Regional Clinical Psychoneurological Hospital, Privolnaya St., Belgorod 308010, Russia 1 Kursk State Medical University, 3 Karl Marx St., Kursk 305004, Russia

Corresponding author: Ivan S. Koklin ([email protected])

Academic editor: M. Korokin ♦ Received 8 January 2020 ♦ Accepted 22 February 2020 ♦ Published 23 March 2020

Citation: Demchenko SA, Koklin IS, Koklina NYu (2020) Role of Arginase 2 as a potential pharmacological target for the creation of new drugs to correct cardiovascular diseases. Research Results in Pharmacology 6(1): 47–55. https://doi.org/10.3897/ rrpharmacology.6.50942

Abstract Introduction: The review provides relevant information about arginase 2, the role of this enzyme in the formation of endothelial dysfunction and, as a consequence, the development of cardiovascular diseases. History of the discovery of arginase and its functions: The discovery of arginase took place long before its active study as a substance that affects the formation of endothelial dysfunction. Role of arginase 2 in the development of a number of cardiovascular diseases: The role of NO synthase and arginase 2 in the formation of oxidative stress is determined. The pathophysiological mechanisms of the development of a number of cardiovascular diseases, such as coronary heart disease, atherosclerosis, and aortic aneurysm, are described. The modern possibilities of treatment of endothelial dysfunction in the pathology of the cardiovascular system and the possibility of creation of new drugs are considered. An increase in the activity of arginase 2 was proven to occur in the case of the development of coronary heart disease (CHD), hypertension, type II diabetes mellitus, hypercholesterolemia, as well as in the process of aging. According to the WHO, coronary heart disease and apoplectic attack have topped the list of causes of death worldwide over the past 15 years. Arginase 2 as a potential pharmacological target: The purpose of this literature review is to determine the possibilities of use of arginase 2 as a new target for the pharmacological correction of cardiovascular diseases.

Keywords arginase 2, cardiovascular diseases, endothelial dysfunction.

Introduction which is manifested in a decrease in the bioavailability of nitric oxide (Durante et al. 2007). This causes research Cardiovascular and metabolic diseases are at the moment interest in the pathophysiological mechanisms of the oc- recognized by the WHO as the most important global currence and development of the endothelial component health problem. The leading role in the pathogenesis of of these diseases (Korokin et al. 2011; Pokrovskii et al. these pathologies is played by endothelial dysfunction, 2012; Danilenko et al. 2017).

Copyright Demchenko SA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 48 Demchenko SA et al.: Role of Arginase 2 as a potential pharmacological target for...

Speaking about the bioavailability of nitric oxide, one The development of an inflammatory reaction is- ac cannot disregard the role of the essential arginine amino acid, companied by macrophages producing lipopolysaccha- which is a substrate for NO synthase and arginase. These are rides, interleukins (IL-4, IL-6) and interferons-gamma, the metabolic enzymes that support the homeostasis of the which are inducers of arginase activity (Eelen et al. 2018). NO-synthase-arginine-arginase system and provide a sta- In addition, inflammation disrupts the function of carriers ble level of nitric oxide. Two fundamental mechanisms for of cationic amino acids that are involved in the transport reducing of the amount of NO have been described: a de- of arginine, which also leads to a decrease in NO synthe- crease in its synthesis and excessive deactivation of reactive sis. The discovery of such an endogenous vasodilator as oxygen species (ROS) (Durante et al. 2007). nitric oxide fundamentally changed the understanding of Mammal arginase, a manganese-metalloenzyme that endothelial functions. hydrolyzes L-arginine to L-ornithine and urea, participates Furchgott and Zawadzki (1980) demonstrated the in- in the regulation of nitric oxide synthesis, while compet- volvement of endothelial cells in support of hemovascular ing for the substrate with nitric oxide synthase. Three homeostasis. These authors showed that the endothelium isoforms of NO synthase are currently known: neuronal performs not only a barrier function, but also participates NOS (nNOS, or NOS-1), neuronal NOS (iNOS, or NOS- in the most complicated scheme of regulation of vascular 2) and endothelial NOS (eNOS, or NOS-3) (Berkowitz et tone, producing a number of vasodilating and vasocostric- al. 2003). An increased expression of arginase leads to an tor factors. The imbalance of these factors leads to the increase in the consumption of arginine and the synthesis development of endothelial dysfunction, which underlies of urea and ornithine; at the same time the availability of the formation of atherosclerotic vascular disease, as well arginine for NO synthase decreases and, as a result, the as the development of cardiovascular, renal and metabolic production of nitric oxide decreases. A number of studies diseases (Pokrovsky et al. 2008). have shown the critical contribution of arginase to the de- Taking into account the importance of NO metabolism velopment of cardiovascular and metabolic diseases: an in the formation of cardiovascular pathology and the fact increase in arginase activity has been revealed in the case that arginase causes a decrease in the bioavailability of of hypertension, type II diabetes mellitus, hypercholes- nitric oxide, the reason why the relationship between NO terolemia, atherosclerosis, and as the aging process pro- and arginase is currently recognized as a key regulatory gresses (Gerstein et al. 2008). pathway of the vascular system becomes clear. There is For now, two isoforms of arginase are known that cata- only one substrate for arginase – L-arginine, the active lyze the same reaction. Arginase 1 is a protein that con- use of which can provoke not only a deficiency of NO, sists of 322 amino acids and is conventionally considered but also a decrease in its protective effects on endotheli- to be a liver isoform. Although arginase 1 was originally um: prevention of abnormal vasoconstriction, inhibition detected only in the liver, it has now been proven that it of platelet aggregation and a decrease in the expression can be found in endothelial cells and vascular myocytes, of adhesion molecules on the surface of endothelial cells being a cytosolic enzyme. Arginase 2, in turn, is distrib- (Verdegem et al. 2014). uted in many organs and tissues and is a mitochondrial Recent studies have shown that an increased arginase enzyme, which is by 58% identical in structure to argin- activity accompanies the development of cardiometabolic ase 1 (Ash et al. 2000). Due to the active involvement of diseases, such as hypertension, ischemic reperfusion le- arginine in the synthesis of ornithine and urea, competi- sions, diabetes mellitus and the aging process (Mapanga tive to NO synthase, arginase causes excessive formation and Essop 2016). These data have become the prerequi- of reactive oxygen species, which, in turn, contributes to site for a more active study of the possibility of inhibiting oxidative stress. In addition, this enzyme is an inducer of arginase in order to correct endothelial dysfunction. the synthesis of polyamines and proline (Li et al. 2001), History of the Discovery of Arginase and Its Functions and also contributes to the processes of proliferation and In 1904, Kossel and Dakin, when administrating the remodelling of smooth muscle fibres of a vessel wall. studied enzyme which would later be known as “arginase” There are studies suggesting the anti-inflammatory into the liver of mammals, noted a decrease in arginine effect of superexpression of arginase 1 by its interaction levels and a hydrolysis process linked to it, resulting in with endothelial NO synthase in rabbits. the formation of ornithine and urea. After that event, other Pro-inflammatory mediators, reactive oxygen and ni- researchers began to report the presence of arginase in var- trogen species (RONS), glucose, and oxidized low-den- ious organs and tissues of animals. So Clementi discov- sity lipoprotein (ox-LDL) are endogenous stimulators of ered this enzyme in the liver of amphibians, fish, turtles arginase expression. Reducing the level of L-arginine, the and kidneys of birds. The obtained data were further sup- above factors are predictors of the formation of endothe- plemented by Edlbacher and Rottler, who in their turn re- lial dysfunction. After oxidation, low-density lipoproteins vealed arginase in the liver, kidneys, thymus, testicles and bind to the lectin-like receptors of LDL-1 and stimulate placenta of mammals, and also showed that the amount the activation of arginase. Oxidized LDL also block the of enzyme in males was higher (Kossel and Dakin 1904). production of NO through increased synthesis of caveolin In 1927, Chaudhuri carried out studies on 32 birds I, which interferes with the activity of NO synthase (John and showed the presence of arginase in their kidneys and and Schmeider 2003). testicles, and also confirmed the previously expressed as- Research Results in Pharmacology 6(1): 47–55 49 sumption about the gender difference in the distribution of a cause of the excessive activity of the transcription factor this enzyme: arginase was present in the genitals of only 1, which contributes to neovascularization in the area of males (Chaudhuri 1927). In the future, the sexual differ- ischemia. Reperfusion tissue injuries are also accompa- ence in the metabolism of arginase and the role of steroid nied by activation of oxidative stress and a decrease in the hormones in the realization of its effects were actively activity of NO synthase (Hein et al. 2003; Villalba et al. discussed; this enzyme was also assumed to participate in 2016). There is evidence of increased glycolysis process- some pathological processes (Tousoulis et al. 2002). es in endothelial cells under the conditions of hypoxia. Subsequent studies proved not only the presence of ar- As soon as the relationship between endothelial dys- ginase in organs and tissues in various animal species, but function and an early development of atherosclerosis was also an active participation of this enzyme in the forma- described (Drexler et al. 1991), the connection between tion of cardiovascular disorders. For example, Buga et al. impaired metabolism of endothelial cells and elementary (1996) for the first time described the process of arginase changes in a vessel wall became apparent. Dhawan et al. expression in rat aortic endothelial cells and noted that en- (2005) created an experimental model on monkeys fed a dogenous inhibition of this enzyme by NG-hydroxy-L-ar- high-cholesterol diet in order to demonstrate the interde- ginine (an intermediate compound in the synthesis of NO pendence of NO and endothelial dysfunction. In the ani- from L-arginine) could provide a sufficient amount of ar- mals that were at the same time injected with L-arginine, an ginine for NO synthesis. expressed decrease in formation of ateromas was observed, The relationship between the arginase activity and en- due to an adequate endothelial function and an increased dothelial dysfunction due to the aging process was shown NO level (Chicoine et al. 2004). In a similar vein, several by Berkowitz et al. (2016), who performed a compara- other experimental studies were carried out, and all of them tive analysis of old and young Wistar rats (Steppan et al. showed the relationship between a low level of arginase, 2016). The dependence of atherosclerotic vascular chang- accompanied by the formation of endothelial dysfunction, es on the arginase activity was actively studied by a team and the development of the atherosclerotic process, as well of researchers under the supervision of Ryoo. They were as a more serious cardiovascular pathology in the future. the first to describe a high potential of pharmacological Atherosclerosis is a chronic progressive condition, in inhibition of arginase in order to protect a vessel wall which atherosclerotic plaques are deposited on the en- from atherosclerotic changes (Aldemir et al. 2003). dothelial surface of the major arteries, which causes nar- rowing of the vessel lumens and damage to the subendothelial layers. This is a multifactorial lesion of a vessel Role of arginase 2 in the wall, but cholesterol and inflammatory mediators make development of a number of the largest contribution to the formation of atheromas. At the initial stage of an atherosclerotic plaque formation, cardiovascular diseases functional or structural damage to the vessel wall occurs. This contributes to the accumulation of adhesion mole- The direct connection between the development of endo- cules with subsequent fixation of monocytes on endothe- thelial dysfunction and the formation of cardiovascular lial cells, after which they migrate to the subendothelial pathology is the fact proved by many experimental and space and turn into macrophages. Along with this process, clinical studies (Demougeot et al. 2005; Morris et al. there is an active accumulation of LDL in the intima. Ac- 2005; Yakushev et al. 2015; Koklin and Danilenko 2019). tivation of oxidative stress and an increase in the number Ludmer et al. (1988) in their unique work described the of reactive oxygen species, which include superoxide ani- vasoconstrictive effect of acetylcholine in patients with on, leads to the oxidation of these lipoproteins to oxidized atherosclerosis. Using angiography of the coronary arte- LDL (Kovamees et al. 2015). ries, they found that intracoronary administration of this As mentioned above, a decrease in the bioavailability drug causes a vascular spasm, which provokes the forma- of nitric oxide is a key link in the pathogenesis of cardio- tion of endothelial dysfunction. vascular diseases. However, the development of endothe- Coronary heart disease (CHD), hypertensive disease, lial dysfunction against the background of deficiency of advanced atherosclerosis, vascular disorders in type II NO, is not always accompanied by atherosclerotic chang- diabetes mellitus, in pulmonary hypertension, and in hy- es in blood vessels (Lefer and Lefer 1996). A complex poestrogenic conditions, as well as a number of other pro- cascade of reactions and interactions between circulating cesses are accompanied by hypoxia and, consequently, the substances, cellular receptors and intracellular signaling transition of endothelium to hypoxic metabolism (Hein et mechanisms always leads to a disrupted hemovascular al. 2003; Demougeot et al. 2005). Under conditions of homeostasis. The main mechanism to support the bio- oxygen deficiency, mitochondrial respiration is impaired availability of nitric oxide is to maintain a constant bal- and the ability of cytochrome to bind oxygen is reduced, ance between the activity of endothelial NO synthase and due to which a large number of electrons are formed, arginase isoforms. The first signs of atherosclerosis are re- which cause the synthesis of ROS, rather than NO, from corded when an imbalance occurs, which involves exces- arginine. It is these metabolic changes that are fundamen- sive production of ROS, a decrease in the availability of tal in the formation of endothelial dysfunction. Hypoxia is NO, or both of these processes (Pokrovskaya 2008). Ar- 50 Demchenko SA et al.: Role of Arginase 2 as a potential pharmacological target for... ginase is involved in the process of atherogenesis mainly researchers agree that the formation of ROS is stimulat- due to the creation of deficiency of nitric oxide, which ed by pro-inflammatory factors and leads to an impaired causes vasoconstriction, inhibition of platelet aggregation endothelial function. and adhesion of leukocytes to a blood vessel wall and the Pro-inflammatory conditions in blood vessels have a formation of atheromas. direct relationship with impaired endothelial function and Another scenario for the development of atherosclero- the development of atherosclerosis. The production of sis is through the oxidation of low density lipoproteins, cytokines stimulates the activity of arginase and reduces which facilitate the conversion of phagocytes into foam the expression of the NO synthase gene, ultimately lead- cells. This process leads to the synthesis of reactive oxy- ing to an increase in the production of reactive oxygen gen species catalyzed by NADPH oxidase. In the experi- species. In this vein, Spillmann et al. (2014) studied the mental models with monkeys, the data were also obtained relationship between liver X-receptors (LXR, a hormone demonstrating that hypercholisterinemia stimulated the receptor involved in cholesterol reverse transport) and an synthesis of asymmetric dimethyl-L-arginine (ADMA), increased activity of tumor necrosis factor. The results which is an endogenous inhibitor of NO synthase. obtained by these researchers suggest that LXR agonists Ryoo et al. (2013), studying the role of arginase 2 in decrease mRNA expression and arginase 2 activity, and the development of atherosclerosis, found that oxidized therefore, restore the bioavailability of NO. LDL stimulated the release of this enzyme and reduced Cai et al. (2020) focused on the study of inflammatory the production of NO. In that study, mice with the de- factors responsible for the formation of endothelial dys- letion of the arginase 2 gene were used, which received function. They showed an increase in the arginase activity a diet rich in cholesterol. As a result, in such animals a in the aorta of rats when exposed to serum amyloid A-li- decreased arginase function improved the functional state poprotein produced by the liver. of endothelial cells, compared with the intact mice. Thus, Aneurysms are local balloon-like expansions in an ar- the study showed that genetic inhibition of arginase had a terial wall, which are mainly caused by an impaired en- protective effect on endothelium (Loscalzo 2001). dothelial function, a destroyed extracellular matrix and The hypothesis of oxidative modification as a critical a decreased number of smooth muscle cells. The forma- stage in the development of atherosclerosis is also worth tion of an aneurysm is based on the development of an mentioning. According to this theory, for atherosclerotic inflammatory process, accompanied by the activation of changes to happen, it is not enough just to increase the oxidative stress and, as a result, by an increase in ROS amount of LDL; these lipoproteins have to undergo oxi- originating from macrophages, smooth muscle cells, fi- dative changes: oxidized LDL are recognized by specific broblasts, and endothelial cells. A recent study provides macrophage receptors, absorbed by phagocytes and accu- the data confirming the crucial role of endothelial ROS mulate in them. in aortic dissection in transgenic mice with endothelium The special role of arginase 2 is confirmed by the study specific increased expression of NADPH oxidase 2. In the on mice with artificial inhibition of arginase gene expres- studied cases, an increase in the reactive oxygen species sion. The activity of arginase 2 in the endotheliocytes of in blood was connected with an increase in episodes of such animals was significantly reduced, which suggested aortic dissection in response to stimulation by angiotensin the primacy of this isoform of this enzyme (Topal et al. 2 (Vandekeere et al. 2015; Rafii et al. 2016) ROS induce 2006). In other studies, inhibition of this very arginase 2, endothelial cells to secrete cyclophillin A, which, in turn, including in the animals fed a high cholesterol diet, led to causes the activation of the inflammatory process of a the restoration of endothelial function, increased NO level vessel wall, the production of matrix metalloproteinases and vasodilation (Ignarro et al. 2001). and remodelling of a vessel wall, which ultimately leads One of the known mechanisms of activation of ather- to its dissection. Thus, in the prevention of aneurysm and osclerotic changes is apoptosis of endothelial cells. Con- aortic dissection, the most important step is the correction firming this, Sushek et al. (2003) demonstrated that the of metabolic disorders of endothelial cells. expression of interleukin-1, tumor necrosis factor α and Another mechanism contributing to the develop- interferon gamma in rats with the blocked arginase 2 gene ment of endothelial dysfunction is shear stress, which led to apoptosis of endothelial cells at the administration is a predisposing moment in the atheroma formation of hydrogen peroxide. At the same time, at high levels of process (Sonin et al. 2002). It is the stress acting along nitric oxide, no cell death was observed, which suggested the surface area (a vessel wall) and is part of the total a protective effect ofarginine on endothelium. stress. Teupser et al. (2006), when studying pig carotid An increase in the amount of ROS caused by a high endothelial cells, demonstrated an increased expression activity of arginase 2 is one of the reasons for the for- of arginase 2 after induction of shear stress by an oscilla- mation of endothelial cell dysfunction in animals with tory change compared to the level of this stress over the atherosclerosis. The same direct relationship is observed previous three days. To confirm the role of this enzyme in the development of coronary heart disease and vas- in the described changes, a control group of animals was cular disorders in patients with type 2 diabetes mellitus. introduced into the study, to which the arginase inhibitor – However, the effect of arginase on endothelial cells in hydroxy-nor-L-arginine (Nor-NOHA) was administered, vivo has not been fully studied yet. Nevertheless, many which led to a decrease in ROS production and an increase Research Results in Pharmacology 6(1): 47–55 51 in the proliferation of smooth muscle cells of the vessel this enzyme is realized through a competitive effect on wall. Regarding this effect, Xiong et al. (2017) studied NO synthase and a decrease in NO production (Iyer et al. the role of arginase 2 in the proliferation of smooth mus- 2002; Boger 2014). cle fibres of a vessel wall using human umbilical veins as In the early 1990s, arginase inhibitors were developed, an example and demonstrated that activation of this en- initially, to study the effects that occur when the activity zyme potentiated proliferative processes in vascular cells. of this enzyme decreases. However, the observed effects Xiong et al. (2017) also observed the progression of aging brought promising results in terms of correcting the en- and activation of apoptosis in the absence of arginine 2, dothelial function, decreasing a cholesterol level, stabiliz- emphasizing the progression of atherosclerosis due to the ing blood pressure and normalizing metabolic disorders resulting weakness of the vascular layers. The adminis- (Morris et al. 1997). tration of thrombin into cells of a human umbilical vein Arginase inhibitors are classified into selective and increased the expression of arginase 18 hours later, and non-selective, as well as into specific, directly blocking the a peak effect was reached 24 hours later. In that study, enzyme itself, and non-specific, acting indirectly. In order an HMG-CoA inhibitor fluvastatin, which distorts the to inhibit arginase, there was an attempt made to use an in- RhoA-ROCK pathway and leads to a decrease in argin- termediate compound of NO synthesis – N-hydroxy-L-ar- ase expression under the influence of thrombin, was also ginine (NOHA). But the attempt failed due to the fact administered. The similar effects were observed in that that this substance was also a coenzyme of the P450 cy- study with the administration of other ROCK inhibitors. tochrome, and Nω-hydroxy-nor-L-arginine (nor-NOHA) Studying the role of arginase 2 in inflammatory -re was synthesized to substitute it. Difluoromethylornithine, actions, Ming et al. (2012) found its protective effect in which blocks ornithine decarboxylase and, accordingly, the development of insulin resistance, type 2 diabetes increases the amount of arginine via uric acid metabolism, and atherosclerosis in mice with a deficiency of this iso- was studied in vitro and in vivo as an arginase inhibitor. form. The group of researchers supervised by Weissman, A side effect of this substance was the development of when comparing a vascular function between transgenic independent vascular reactions and the accumulation of C57Bl/6 mice with enhanced expression of the arginase 2 ornithine (Shi et al. 2001). Such substances as S-(2-bo- gene and control groups of animals, revealed an endothe- ronoethyl)-L-cysteine, 2(S)-amino-6-hexanoic acid and lium-mediated vasodilation disorder induced by ACh in L-norvaline are also interesting in terms of blocking the the transgenic group. Those authors also proved that an effects of arginase. The currently known arginase inhibi- increased activity of arginase 2, regardless of the level of tors are low-selective or non-selective and affect both ar- lipids in plasma, was a sufficient reason for the develop- ginase 2 and arginase 1. In the experiment, hyperammone- ment of inflammatory changes and the formation of ather- mia was observed in the mice with an arginase 1 knockout osclerosis (Zhang et al. 2015). gene and all the animals died on the 10–14th days of post- There is evidence in the literature that arginase has not natal development (Kasten et al. 2013). The identification only a harmful effect on the functional state of endotheli- of such side effects of a decreased activity of arginase 1 um, but this enzyme can also have a beneficial effect on indicates the relevance of searching for a highly selective the vessel wall. This ability, as the case may be, to play arginase 2 inhibitor with the expressed cardioprotective either a protective or damaging role puts arginase in an and endothelioprotective properties. Moreover, at the mo- even more interesting perspective. For example, Teupser et ment there are no drugs from the group of selective argin- al. (2006), studying genes for predisposition to atheroscle- ase 2 inhibitors at the global pharmaceutical market (Jung rosis, showed that an increased expression of the arginase et al. 2005; Salmito et al. 2015). 1 gene in macrophages promotes resistance to atheroscle- One of the recent clinical studies showed that arginase rotic changes, presumably by implementation of anti-in- inhibitors had a positive effect on endothelium in patients flammatory mechanisms. The atheroprotective role of this with familial hypercholesterolemia. In patients with ath- isoform of arginases is realized in several ways, such as erosclerosis, endothelial dysfunction correlates with the differential activation of macrophages, modulation of an plasma content of natural arginine analogues, competitive inflammatory response in smooth muscle cells of a vessel endogenous inhibitors of NO synthase. There are two such wall, and an impaired stability of an atherosclerotic plaque. substances: asymmetric dimethylarginine (ADMA) and monomethylarginine (L-NMMA). A number of previously mentioned studies suggested a possible productive use Arginase 2 as a potential of L-arginine, both in monotherapy and in combination pharmacological target with antihypertensive drugs in cases of endothelial dysfunction caused by ADMA and/or L-NMMA. In (Loya- ga-Rendon et al. 2005), it was shown that such a therapy As it follows from all the above data, an increase in the (latter) clearly increased the activity of endothelial NO arginase gene expression and/or a direct increase in its synthase and, as a result, increased NO synthesis, which activity undoubtedly leads to the formation of endothe- was expressed in correcting endothelial dysfunction. lial dysfunction and the development of atherosclerotic The data of the studies already completed as of todate vascular changes. The main pathway for the action of directly or indirectly confirm a high potential of even 52 Demchenko SA et al.: Role of Arginase 2 as a potential pharmacological target for... non-specific inhibition of arginase. For example, in rats The main obstacle to a more accurate understanding of with metabolic syndrome treated with citrulline, norva- the difference in the effects of the two isoforms of arginas- line or ornithine, normalization of blood pressure levels es seems to be the difficulty in creating a specific inhibitor was observed compared with the control group. Such an for each of them. This difficulty is due to the great simi- effect was achieved directly by the increase in - thebio larity of the chemical structure of these enzymes (58%), availability of nitric oxide and indirectly by reversing which have almost identical metal clusters and active site hypertriglyceridemia and insulin resistance (Durante et configurations in their compositions. Nonspecific- inhi al. 2007; Xu et al. 2014). Holowatz and Kenney (2007) bition of the activity of both arginases is a problem of proved the connection of essential arterial hypertension current studies, since it does not make it possible to deter- with the reduced reflex vasodilation of skin vessels and mine which isoform the observed effects belong to. found that acute nonspecific inhibition of arginase- re Clinical trials are strictly limited, so few researchers have stored this reflex. Atorvastatin has a similar effect, its -ad evaluated the role of arginase in humans. The study of reac- ministration for months resulted in the restoration of the tions catalyzed by isoforms of this enzyme is a vast area for function of skin microcirculation vessels in patients with the development of new methods of treatment and preven- hypercholesterolemia, and the observed effect was medi- tion of many clinical conditions. A large number of effective ated by a decrease arginase activity. The analysis of all the studies which have been carried out in recent decades in- studies whose results are available at the moment shows dicate the participation of arginase in the formation of en- that there are a number of metabolic processes catalyzed dothelial dysfunction, and, consequently, of cardiovascular by this enzyme, which can be potentially influenced in and metabolic diseases. The introduction of a pharmacologi- order to correct cardiometabolic disorders. cal agent selectively inhibiting the activity of arginase 2 into a therapy of such conditions will significantly expand the prospects for the correction of these pathological processes. Conclusion Thus, to identify the details of the mechanisms of transcription, transduction, as well as the action of arginase it- At the moment, there are a large number of studies confir- self and to develop pharmacological methods of affecting ming the participation of arginase in the regulation of the these processes in the treatment and prevention of cardio- bioavailability of nitric oxide, the formation of endotheli- vascular and metabolic diseases are a very promising area al dysfunction, the development of atherosclerosis, and a for further research. number of other cardiovascular and metabolic disorders. However, the pathophysiological mechanisms of the arginase in various conditions are not completely clear, which Conflict of interest makes it imperative to describe in detail the molecular pathways of the metabolism of this enzyme. The authors have no conflict of interest to declare.

References

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Author contributions

Sergey A. Demchenko, PhD student, Department of Pharmacology and Clinical Pharmacology, e-mail: demchen- [email protected] The author was engaged in collecting, analyzing and interpreting the data for the paper.

Ivan S. Koklin, surgeon of the Surgical department, e-mail: [email protected]. Under the supervision of the scientific consultant, the author conducted an analysis of Russian and foreign literature.

Natalya Y. Koklina, assistant of the Department of Traumatology and Orthopedics, e-mail: [email protected], OR- CID ID http://orcid.org/0000-0002-2109-8882. The author prepared final copy of the manuscript to be sent to the editorial office. Research Results in Pharmacology 6(1): 57–68 UDC: 576.5, 57.04, 615.324, 604.4 DOI 10.3897/rrpharmacology.6.49413

Review Article

Biological activity of mesenchymal stem cells secretome as a basis for cell-free therapeutic approach

Liubov A. Pokrovskaya1, Ekaterina V. Zubareva2, Sergey V. Nadezhdin2,3, Anna S. Lysenko2, Tatyana L. Litovkina2

1 National Research Tomsk State University, 36 Lenin Ave., Tomsk 634050, Russia 2 Belgorod State National Research University, 85 Pobedy St., Belgorod 308015, Russia 3 Research Laboratory of Cellular, Assisted Reproductive and DNA Technologies, Belgorod State National Research University, 85 Pobedy St., Belgorod 308015, Russia

Corresponding author: Ekaterina V. Zubareva ([email protected])

Academic editor: Mikhail Korokin ♦ Received 14 December 2019 ♦ Accepted 20 January 2020 ♦ Published 30 March 2020

Citation: Pokrovskaya LA, Zubareva EV, Nadezhdin SV, Lysenko AS, Litovkina TL (2020) Biological activity of mesenchymal stem cells secretome as a basis for cell-free therapeutic approach. Research Results in Pharmacology 6(1): 57–68. https://doi. org/10.3897/rrpharmacology.6.49413

Abstract Mesenchymal stem (stromal) cells (MSCs) are self-renewing, cultured adult stem cells which secrete a complex set of multiple soluble biologically active molecules such as chemokines, and cytokines, cell adhesion molecules, lipid mediators, interleukins (IL), growth factors (GFs), hormones, micro RNAs (miRNAs), long non-coding RNAs (lncRNAs), messenger RNAs (mRNAs), exosomes, as well as microvesicles, the secretome. MSCs of various origin, including adipose-derived stem cells (ASCs), bone marrow derived mesenchymal stem cells (BM-MSCs), human uterine cervical stem cells (hUCESCs), may be good candidates for obtaining secretome-derived products. Different population of MSCs can secret different factors which could have anti-inflammatory, anti-apoptotic, anti-fibrotic activities, a neuroprotective effect, could improve bone, muscle, liver regeneration and wound healing. Therefore, the paracrine activity of conditioned medium obtained when cultivating MSCs, due to a plethora of bioactive factors, was assumed to have the most prominent cell-free therapeutic impact and can serve as a better option in the field of regenerative medicine in future.

Keywords mesenchymal stem cells; pre-conditioning; secretome; regeneration; immunomodulation; therapeutic potential.

Introduction 2015; Leavitt et al. 2016; Del Papa et al. 2019; Kuchar- zewski et al. 2019; L et al. 2019; Lombardi et al. 2019). Mesenchymal stem (stromal) cells (MSCs) are self-rene- Besides, MSCs also have anti-tumorigenic, anti-fibrotic, wing, culture expandable adult stem cells that have been anti-apoptotic, anti-inflammatory, pro-angiogenic, neuro- shown to be a promising candidate for cell-based thera- protective, anti-bacterial and chemo-attractive effects py (Ferreira et al. 2018). Using mesenchymal stem cells (Maumus et al. 2013; Bartosh et al. 2016; L et al. 2019). for regenerative purposes is possible due to their trophic, MSCs harvested from numerous anatomical locations, paracrine, and immunomodulatory properties (Stagg including the bone marrow, adipose tissue, Wharton’s jel- and Galipeau 2013; Del Papa et al. 2015; Marfia et al. ly of the umbilical cord, display similar immunopheno-

Copyright Pokrovskaya LA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 58 Pokrovskaya LA et al.: Biological activity of mesenchymal stem cells secretome... typic profiles. However, there is a large body of evidence There is evidence that the molecules produced by showing that, despite the similarity in their immunophe- MSCs (secretomes), especially those packaged in extra- notypes, MSCs secrete a complex set of multiple soluble cellular vesicles (EVs), influence the tissue repair even biologically active molecules, the secretome, composi- better than the cells themselves (Lepperdinger et al. 2008; tion of which varies significantly, depending on the age Madrigal et al. 2014; Dubey et al. 2018; Ferreira and of the host and niches where the cells reside (Baksh et al. Gomes 2018; Pelizzo et al. 2018; Campanella et al. 2019; 2004; Traktuev et al. 2008; Amos et al. 2010; Daquinag Lombardi et al. 2019; Mitchell et al. 2019). et al. 2011; Cheng et al. 2012; Daquinag et al. 2013; Ka- Therefore due to the paracrine activity, MSCs condi- pur and Katz 2013; Kyurkchiev et al. 2014; Madrigal et tioned medium (CM) or purified MSC-derived extracel- al. 2014; Dubey et al. 2018; Ferreira et al. 2018; L et al. lular vesicles having a plethora of bioactive factors are 2019; Lombardi et al. 2019; Meiliana et al. 2019). The assumed to have the most prominent cell-free therapeutic MSCs secretome in general consists of such biologically impact and can serve as a better option in the field of reactive factors as chemokines and cytokines, cell adhesion generative medicine in the future (Maguire 2013; Zhou molecules, lipid mediators, interleukins (ILs), growth et al. 2013; Vizoso et al. 2017; Park et al. 2018; L et al. factors (GFs), hormones, micro RNAs (miRNAs), long 2019; Lombardi et al. 2019). non-coding RNAs (lncRNAs), messenger RNAs (mR- This new frontier of research provides several key ad- NAs), exosomes, as well as microvesicles (Kyurkchiev et vantages over cell based applications: (a) the administra- al. 2014; Madrigal et al. 2014; Dubey et al. 2018; Ferreira tion of proteins instead of whole cells as a new therapeu- et al. 2018; Lombardi et al. 2019; Meiliana et al. 2019). tic option in regenerative medicine; (b) CM can be stored It is revealed that MSC secretion include in particular without any toxic cryopreservatives, such as dimethyl vascular endothelial growth factor (VEGF), insulin-like sulfoxide (DMSO), for a relatively long period; (c) prepa- growth factor 1 (IGF-1), basic fibroblast growth factor ration of CM is more economical as it can be mass-pro- (bFGF), transforming growth factor beta 1 (TGF-b1), duced from the available MSC populations under current nerve growth factor (NGF), placental growth factor good manufacturing practice (cGMP) conditions; (d) (PGF), stromal-derived growth factor (SDF-1/CXCL12), evaluation of CM for safety and efficacy will be much monocyte chemo-attractant protein-1 (MCP-1/CCL2), simpler and analogous to conventional pharmaceutical IL-6, IL-8, IL-10 and IL-13 (Meiliana et al. 2019), bone agents (Bermudez et al. 2015; L et al. 2019). morphogenetic proteins (BMP), CC chemokine ligand 5/ Regulated on activation, normal T cell expressed and secreted (CCL5/RANTES), epidermal growth factor (EGF), Anti-inflammatory activity granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), It is well known that there are anti-inflammatory factors hepatocyte growth factor (HGF), inter-cellular adhesion in the MSCs secretome, including tumor necrosis factor molecules (ICAM), indoleamine-2,3-dioxygenase (IDO), β1 (TGFβ1) (Zagoura et al. 2012), interleukin 13 (IL13) leukemia inhibitory factor (LIF), matrix metalloproteas- (Bermudez et al. 2016), interleukin 18 binding protein es (MMP-1, MMP-2, MMP-3, MMP-7), platelet-de- (IL18BP), ciliary neurotrophic factor (CNTF), neurot- rived growth factor (PDGF), metalloproteinase inhibitors rophin 3 (NT-3) factor, interleukin 10 (IL10), interleukin (TIMP-1, TIMP-2) (Polacek et al. 2011; Osugi et al. 2012; 12 p70 (IL12p70), interleukin-25 (IL-25), which is also Inukai et al. 2013; Kyurkchiev et al. 2014; Pereira et al. known as interleukin-17E (IL17E), interleukin 27 (IL27), 2014; Ferreira et al. 2018; Linero and Chaparro 2014). or interleukin 1 receptor antagonist (IL1RA). On the other Herewith, mesenchymal stem cells of various origin, hand, pro-inflammatory cytokines are also present in including bone-marrow-derived mesenchymal stem cells MSCs conditioned medium, for example, IL1b, interleu- (BM-MSCs), adipose tissue-derived stem cells (ADSCs) kin 6 (IL6) (See et al. 2011; Cantinieaux et al. 2013), in- or human uterine cervical stromal stem cells (hUCESCs), terleukin 8 (IL8) (Mirabella et al. 2011), and interleukin 9 may be good candidates for obtaining products from se- (IL9) (Lee et al. 2011). Thus, the balance between the anti- cretome (Zhao et al. 2013; Vizoso et al. 2017). However and pro-inflammatory factors will determine the final -ef different populations of MSCs should be used for specific fect of conditioned medium on the inflammatory process. purposes because the composition of the secretome de- However, the numerous studies highlight MSCs an- pends on the stromal cells source. For instance, ADSCs ti-inflammatory effect. Yi and Song (2012) described that have higher expression of mRNA, VEGF-D, IGF-1 and MSCs inhibited proinflammatory cytokines, such as inter- IL-8, while dermal-sheath- and dermal-papilla-derived feron (IFN)-γ and tumour necrosis factor α (TNFα), while cells secrete higher concentrations of CCL2 and leptin increasing release of anti-inflammatory IL10. Legaki et al. (Hsiao et al. 2012). It is known that placenta-derived (2016) showed that hUCESC-CM treatment significantly MSCs are characterized with increased expression levels reduced mRNA expression of pro-inflammatory cytokines of HGF, bFGF, IL-6, IL-8, IL-1a and IL-1b, while in se- (IL6, IL8, TNFα and macrophage inflammatory protein-1 cretome obtained from bone marrow-derived MSCs the alpha (MIP-1α)), but increased mRNA expression of the levels of VEGF-A, NGF and angiogenin are higher (Du et anti-inflammatory cytokine (IL10). The similar results al. 2016; Meiliana et al. 2019). were obtained during experiments with MSC-CM from Research Results in Pharmacology 6(1): 57–68 59 amniotic fluid in a mice colitis model (Legaki et al. 2016). endothelial growth factor (VEGF), and stromal cell-de- It was also found that hUCESC-CM reduced the infiltration rived factor-1 (SDF-1), which is also called chemokine of leucocytes in ocular tissues (Vishnubhatla et al. 2014). (C-X-C motif) ligand 12 (CXCL12) (Tang et al. 2005). Moreover, anti-inflammatory cytokines in MSC-CM There are also studies revealing a pro-apoptotic effect of can contribute to the beneficial effects seen in animal mod- hUCESC-CM on malignant cells. In accordance with these els of diabetes, acute colitis, inflammatory arthritis, etc. data, the effects of MSCs on normal and cancer cells are dif- (Brini et al. 2017; Kay et al. 2017; Pouya et al. 2018). It ferent. Along with an antiapoptotic effect of hUCESC-CM was revealed that single intravenous injection of a condi- on normal cells (Bermudez et al. 2016), the apoptosis oc- tioned medium derived from adipose tissue (hAT-CM) into curred in cancer cells under the influence of conditioned streptozotocin-(STZ)-treated diabetic mice relieved the di- medium obtained from human uterine cervical stem cells abetic neuropathic pain by re-establishing the Th1/Th2 bal- in vitro and in vivo (Eiró et al. 2014; Vizoso et al. 2017). ance with a long-lasting relief of sensory hypersensitivity. In the experiments with the STZ-treated diabetic mice, it was demonstrated that the content of anti-inflammatory and Anti-fibrotic activity immunomodulatory cytokines (IL-1β, IL-6 and TNF-α) in dorsal root ganglia, sciatic nerves and spinal cord restored An anti-fibrotic effect of stem cells conditioned medium to basal levels after 1 week of hAT-CM injection. The el- is mediated by bioactive molecules in MSCs secretome evated level of IL-10 also confirmed realization of an an- which decrease accumulation of extracellular proteins ti-inflammatory mechanism (Brini et al. 2017). Pouya et al and, therefore, lead to reduced scar formation. An et al. (2018) showed that an intraperitoneal injection of MSC- (2017) studied the influence of umbilical cord-derived CM in C57BL/6 mice with colitis mediated a significant mesenchymal cells (UCMSC) secretome on formation of decrease in colon inflammation and an increase in colon fibrotic areas in mice with hepatic fibrosis. A decrease in weight and length, which led to the disease activity index the number of activated hepatic stellate cells (HSCs) ex- and mortality rate reducing. Furthermore, the mesenteric pressing α- smooth muscle actin (α-SMA) was shown af- lymph nodes and spleen of the mice infused with MSC-CM ter an injection of the UCMSC-CM in the diseased mice, demonstrated increased levels of the anti-inflammatory cy- which was accompanied by reducing fibrotic areas. The tokines IL-10 and TGF-β and reduced levels of the pro-in- researchers analysed the UCMSCs secretome using na- flammatory cytokine IL-17 confirming the anti-inflamma- no-chip-LC/QTOF-MS and discovered the presence of tory role of CM. Similarly, in the antigen-induced model of milk fat globule EGF factor 8 (MFGE8), an anti-fibrotic inflammatory arthritis, it was shown that an intra-articular protein known to down-regulate the expression of TGF- injection of murine MSC-CM was effective in reducing βR1 (transforming growth factor β type 1 receptor) at the disease severity and cartilage damage. The high levels of mRNA and protein level, thereby decreasing the activati- IL10 in CM were revealed, which correlated with an an- on of human hepatic stellate cells (An et al. 2017). ti-inflammatory response (Kay et al. 2017; L et al. 2019). In order to study whether the anti-inflammatory potential of ADSC secretome is higher than EV-enriched fraction Paracrine effect of MSCs in bone of ADSCs secretome, the effect of both fractions was inves- regeneration tigated on the TNF-α-induced nuclear translocation of the NF-κB subunit p65 in U251 cells. It is interesting to note that the level of nuclear NF-κB p65 was significantly in- It was revealed that secretome synthesized by different creased by TNF-α treatment compared to control cells. The stem cells, including rat bone marrow-derived MSCs effect of the total secretome fraction on TNF-α stimulated (rBM‑MSCs), human adult mesenchymal stem cells cells was accompanied by a non-significant reduction of (haMSCs) and human fetal mesenchymal stem cells nuclear p65, whereas the influence of EV fraction led to a (hfMSCs), promoted osteogenic differentiation of significantly reduced amount of p65 (Mitchell et al. 2019). rBM-MSCs. It is quite important that the human MSC conditioned medium effects in the same way as rat MSCs secretome, or even better. Xu et al. (2016) described that Anti-apoptotic activity the hfMSCs secretome is characterised as a conditioned medium with the strongest osteogenic induction ability There are studies which illustrate that MSCs produce in- compared to the conditioned medium obtained when hibitor proteins of apoptosis to restore local environment cultivating rBM-MSC or haMSC. It was also revealed and prevent therefore cell death (Li et al. 2015). Tang et that hfMSC conditioned medium does not induce any al. (2005) reported that MSCs decreased the pro-apoptotic significant immune response, which makes it different factors expression (Bax and cleaved caspase-3) and stimu- from the haMSC secretome. At the same time, conditioned lated at the same time the anti-apoptotic compounds levels media at different concentrations did not affect rBM- (Bcl-2). It is noteworthy that MSCs treatment of hearts MSC viability or cell proliferation. Furthermore, hfMSCs led to elevated level of pro-angiogenic factors expression, secretome at the concentration of 100 μg/μl could enhance including basic fibroblast growth factor (bFGF), vascular mineralization during rBM-MSC osteogenic induction 60 Pokrovskaya LA et al.: Biological activity of mesenchymal stem cells secretome... via increasing of alkaline phosphatase (ALP) activity and It was shown that the MSCs secretome mediated neu- formation of calcium nodules. roprotective and neurotrophic effects (Caseiro et al. 2016; The expression levels of osteogenic marker genes, Luarte et al. 2016; Ratajczak et al. 2016) due to a number including runt-related transcription factor 2 (Runx2), os- of neurotrophic factors (de Almeida et al. 2014; Mead et teocalcin (OCN), osteopontin (OPN), and osterix (Osx), al. 2014). There are studies which demonstrate on nerve were significantly upregulated on Days 3 and 10 after the injury models that the MSC-based approach generated hfMSCs secretome treatment. ALP is responsible for os- healing effects, including enhanced vascularization of teoblastic differentiation at an early stage; it hydrolyses the regenerating site, increased thickness of the myelin pyrophosphyte and generates inorganic phosphate which sheaths, modulation of the Wallerian degeneration stage, promotes the process of mineralization. Runx2 produces accelerated fibre regeneration, reduction of fibrotic scar- bone matrix proteins and is essential for osteoblast differ- ing, and improved fibre organization (Caseiro et al. 2016). entiation. Growth and differentiation factors regulate ex- It was discovered on experimental animal models that pression levels of OPN and mediate bone formation and stem cells obtained from bone marrow (BM-MSCs) and its remodeling. Osx acts downstream of Runx2, this fac- adipose tissue (ASCs) improved the healing process after tor takes part in the processes of osteoblast differentiation stroke (Wei et al. 2009; Honmou et al. 2012; Teixeira et and bone tissue formation. It was shown that conditioned al. 2014), demyelination (Constantin et al. 2009; Cristo- medium obtained from hfMSC stimulated differentiation fanilli et al. 2011; Teixeira et al. 2014), Parkinson’s dis- of rBM-MSCs in osteogenic direction in vitro. Further- ease (Cova et al. 2010; Erba et al. 2010), and spinal cord more, application of the hfMSCs secretome locally into injury (Arboleda et al. 2011; Park et al. 2012). To restore distraction osteogenesis gap in rats led to accelerated new the central nervous system (CNS), the stem/progenitor bone formation and consolidation (Xu et al. 2016). cells from different sources could be used. For example, It is known that during the distraction osteogenesis pro- stem cells present in the Warton jelly of the umbilical cedure proliferation of bone progenitor cells increases and cord, known as Wharton jelly stem cells (WJ-MSCs) and their recruitment to the target site occurs. The healing pro- human umbilical cord perivascular cells (HUCPVCs), cess is accompanied by the angiogenesis and bone tissue have a great potential in healing CNS injuries (Salgado formation/mineralization. At the same time, the mecha- et al. 2010; Datta et al. 2011; Taghizadeh et al. 2011). nisms of the hfMSCs conditioned media effect on bone tis- Populations of WJ-MSCs and HUCPVCs are also iden- sue regeneration, including vascular network formation and tified as mesenchymal stem cells (Sarugaser et al. 2005; remodelling, remain unknown. There is evidence that the Weiss and Troyer 2006; Baksh et al. 2007; Sarugaser et MSCs secretome mediates the release of vascular endothe- al. 2009). The major effects of MSCs are supposed to lial growth factor (VEGF) at the site of tissue repair, being be determined by their secretomes (Salgado et al. 2010; stimulated by hypoxia or normoxia. It is known that the ex- Carvalho et al. 2011; Ribeiro et al. 2012; Teixeira et al. pression of VEGF enhances bone tissue and blood vessels 2013; Teixeira et al. 2014). Both neural stem cells (NSCs) formation during distraction osteogenesis, and this biolog- and MSCs secrete a variety of growth factors (Salgado et ical active factor is required for osteogenic differentiation. al. 2015). It was shown that the molecular content of the Moreover, osteogenic lineage commitment of MSCs MSCs secretome depended on the culture duration and is accompanied by the Osx and OCN osteoprogenitors tissue sources of MSCs, and it influenced significantly the markers expression. Treatment of the damaged area with changes of primary cultures of hippocampal neurons and the hfMSCs secretome upregulates the number of Osx- glial cells viability (Kim et al. 2013). and OCN-positive osteoprogenitors in the distraction During the in vitro experiments, the ability of the zone in comparison with the control group. Different sig- HUCPVCs conditioned media to modulate the survival naling pathways may be involved in VEGF production and viability of both neuronal and glial cells populations following hfMSC conditioned medium influence, and was shown (Salgado et al. 2015). It was demonstrated that further experiments are required to make the molecular application of HUCPVCs-CM to human telencephalon mechanisms of these processes clear (Xu et al. 2016). neural precursor cells (htNPCs) in vitro led to an increase of neuronal cell differentiation, which was characterised by higher densities of immature (DCX+ cells) and ma- Neuroprotective effect and impact ture neurons (MAP-2+ cells). Moreover, an injection of on neural/glial proliferation HUCPVCs and their secretome into the dentate gyrus (DG) was accompanied by increasing the endogenous proliferation (BrdU+ cells) in a week. It was revealed that There have been several studies in which adult stem cells, application of HUCPVCs led to an increased number of including mesenchymal stem cells, were used as a pos- newborn neurons (DCX+ cells). And an injection of CM sible tool for central nervous system (CNS) regenerati- or HUCPVCs into the DG tissue promoted an elevated on (Lindvall and Kokaia 2010; Shihabuddin and Aubert level of fibroblast growth factor-2 (FGF-2) and, to a lesser 2010; Kan et al. 2011; Teixeira et al. 2014), which could extent, of nerve growth factor (NGF). Thus, either trans- be a promising therapeutic option (Pittenger et al. 1999; plantation of HUCPVCs or the application of their condi- Zuk et al. 2002; Wang et al. 2004; Teixeira et al. 2014). tioned media potentiated enhanced neuronal viability and Research Results in Pharmacology 6(1): 57–68 61 differentiation in vitro and in vivo (Teixeira et al. 2014; media obtained from ASCs stimulated angiogenesis in the Salgado et al. 2015). ischemic environment. The following bioactive molecules The effect of intranasal application of CM derived from were detected in the secretome after the analysis: G-CSF, stem cells of human exfoliated deciduous teeth (SHEDs) in TGF-β, VEGF, HGF, and bFGF (Rehman et al. 2004; Ka- an animal model of Alzheimer’s disease was studied. And pur and Katz 2013). The impact of HGF on the formation the cell-free treatment was accompanied by an improve- of blood vessels was proved through restricting the produc- ment of cognitive function and induced neuroregenerative tion of this factor by ASCs, which resulted in a decreasing effects, for example, an attenuated pro-inflammatory -re effect by ASCs on endothelial cell proliferation, migration sponse induced by amyloid plaques, and anti-inflammato- and survival in the ischemic environment (Cai et al. 2007). ry M2-like microglia (Mita et al. 2015; Vizoso et al. 2017). It was revealed that application of a secretome obtained from hypoxic-preconditioned MSCs promoted reduce of Cutaneous wound healing neuronal cell loss and apoptosis and production of VEGF which stimulated recovery processes in the organisms of A cutaneous wound healing is a fascinating process, traumatic brain injury-induced rats (Chang et al. 2013). which requires cell migration, proliferation, matrix protein synthesis, and tissue remodelling. In particular, keratinocytes are involved in the epithelialization and dermal Angiogenesis and revascularization repair, and endothelial cells promote angiogenesis (Kober et al. 2016). The migration and proliferation of fibroblasts Angiogenesis is a process of new vasculature sprouts are the key processes in a wound healing mechanism. In formation from pre-existing blood vessels. This process the early stage of wound repair, they move to the damaged normally occurs during wound healing. Numerous studies region and promote blood vessels regeneration and gra- illustrated a particular impact of the MSCs secretome at nulation tissue formation. In the advanced trauma stage, the different stages of angiogenesis (Burlacu et al. 2013). fibroblasts mature into myofibroblasts which are respon- The effect of MSCs on the process of angiogenesis is sible for wound closure process (Zhao et al. 2013). studied in different spectrum of diseases, including- im It was revealed that the adipose stem cell-conditioned paired vessel growth during atherosclerosis and wound medium (ASC-CM) had a marked stimulating effect on cu- healing. Several studies demonstrated that the MSCs ap- taneous wound healing, via affecting the mechanism for this plication led to stimulation of blood vessels formation in response by influencing effector cells (Lombardi et al. 2019). animal models of myocardial infarction, neurogenic blad- An increased proliferation and migration activity of der, peripheral artery disease, stress urinary incontinence, primary human dermal fibroblasts (HDFs) as well as type and cerebral ischemia/stroke (Hsieh et al. 2013; Liu et al. I collagen secretion was shown after ASCs secretome ap- 2013; Sharma et al. 2013). plication (Kim et al. 2007). Kober et al. (2016) revealed The MSCs secretome contains numerous biological- a stimulatory effect of ASC-CM on ASCs, and the- pro ly active molecules which act as angiogenic stimulators liferation of keratinocytes after application of ASCs se- and inhibitors (Kinnaird et al. 2004; Di Santo et al. 2009; cretome was significantly reduced. Furthermore, addition Boomsma et al. 2012; Ho et al. 2012). An extensive pro- of ASC-CM did not affect cell migration, which had been teomic analysis of the conditioned media of mesenchy- tested with in vitro scratch assays. Collawn et al. (2016) mal stem cells stimulated with inflammatory cytokines demonstrated on a 3D skin model that application of ASC- revealed the presence in a secretome of tissue inhibitor CM as well as ASCs promoted an acceleration of wound of metalloproteinase-1 (TIMP-1) which is responsible for repair. Seo et al. (2017) showed that ASC-CM, similarly to the MSCs antiangiogenic impacts (Zanotti et al. 2016). ASCs, stimulated the proliferation, migration, and invasion Moreover, some studies showed the dependence of the properties of HUVECs. An addition of ASC-CM led to in- pro- and anti-angiogenic factors secretion on chemokines creasing HaCaT cell proliferation and migration, as well and hypoxic conditions. In particular, to describe the ef- as vascular endothelial growth factor (VEGF) secretion. fect of bioactive molecules on MSCs secretion ability, it is Cooper et al. (2018) demonstrated that application of ASC- important to note that TGFα increases the level of growth CM stimulated HDF migration ability in vitro. Park et al. factors in the secretome (i.e., VEGF, hepatocyte growth (2018) collected CM samples from cultured ASCs isolated factor (HGF), platelet-derived growth factor (PDGF), IL6 from adipose tissues of breast cancer patients and added the and IL8). And a conditioned medium from MSCs treat- secretome to HDFs, normal adult human primary epider- ed with TFG-α induces blood vessel growth in an in vivo mal keratinocytes (HEKa), or HUVECs cultures. It result- assay (De Luca et al. 2011; Vizoso et al. 2017). The ob- ed in increased cell proliferation, migration, and invasion. tained data demonstrate the complicated set of bioactive Kim et al. (2018) showed that ASC-CM from 3D-cultures molecules in the MSCs secretome which can be balanced influenced more significantly on the proliferation- ofHa under the different interventions to promote angiogenesis. CaT cells than the one obtained from 2D-cultivated ASCs. The ASCs secretome also has a proangiogenic effect, Noverina et al. (2019) analyzed the growth factor profile in which was demonstrated in acute myocardial infarction ASC-CM via immunosorbent assay (ELISA) method and models. Rehman et al. (2004) showed that the conditioned revealed a higher concentration level of fibroblast growth 62 Pokrovskaya LA et al.: Biological activity of mesenchymal stem cells secretome... factor (FGF), which is involved in wound healing and re- ly formed muscle fibres during the regeneration process generation. Stojanovic and Najman (2019) demonstrated revealed that the application of EV fraction had a greater the immunomodulatory and wound healing potential of effect than the total ASCs secretome (Mitchell et al. 2019). the secretome collected from stem cells previously isolated from adipose tissue, or lipoma (Lombardi et al. 2019). Some studies described the presence of growth factors Liver regeneration in the MSCs secretome that promoted tissue regeneration with a special focus on proliferation (Lee et al. 2011; Lee et al. (2014) demonstrated that liver regeneration in Zagoura et al. 2012; Turner et al. 2013; Bhang et al. 2014; partially hepatectomised models occurred after systemic Bermudez et al. 2015). The experimental myocardial in- infusion of the ASCs secretome. It was shown that appli- farction models showed that anti-fibrotic and angiogenic cation of ASCs-CM increased mRNA expression of Lgr5 effects of the MSCs secretome inhibited scar formation (a Wnt target gene), which was an indicator of actively process (Cargonini et al. 2012; Preda et al. 2014) and dividing stem cells. Expression of Lgr5 occurred in small stimulated the synthesis and consequent secretion of bi- cells located near bile ducts as a result of a liver cell injury oactive molecules responsible for remodelling (Williams (Huch et al. 2013; Lee et al. 2014). During the repair pha- et al. 2013; Vizoso et al. 2017). se, those cells were able to generate significant numbers of It is known that such components of the ASCs se- hepatocytes and biliary duct cells and, thus, could be con- cretome as bFGF significantly stimulate migration and sidered a class of liver progenitor cells. Higher expression proliferation of functional cells in wound healing; however of Lgr5 in small cells located near bile ducts is an indicator PDGF-AA and VEGF influence only fibroblasts migration of liver regeneration (Huch et al. 2013; Lee et al. 2014). (Zhao et al. 2013). It was revealed that EGF could acceler- Lee et al. (2014) showed an increased expression of ate reepithelialisation process via stimulating the keratino- p-Akt, p-Erk1/2, which are the downstream effector com- cytes proliferation and migration in an acute wound. Zhao ponents of HGF signaling pathways, and p-STAT3, which et al. (2013) demonstrated an increased proliferation of is responsible for cell cycle progression from G1 to S fibroblasts under the EGF influence. However, the mech- phase, after application of the ASC-secretome (Lee et al. anism of the EGF effect on cells migration is still unclear. 2014), which indicated that the ASC-secretome promoted Hu et al. (2013) showed that wound healing was mediat- liver regeneration. ed via application of the ASCs secretome which stimulated migration of vascular endothelial cells 4 hours later, fibroblasts 12 hours later and then keratinocytes 24 hours later. Conclusion Park et al. (2018) reported that elevated levels of EGF, bFGF and HGF in conditioned media promoted The MSC-conditioned medium, or secretome, contains a wound healing. plethora of cytokines and a wide array of bioactive fac- And finally, it was revealed that increasing prolifera- tors, such as chemokines, cell adhesion molecules, lipid tive and migratory characteristics of different dermis cel- mediators, IL, growth factors, hormones, exosomes, mi- lular components, including dermal fibroblasts, keratino- crovesicles, etc., which are secreted by MSCs (Lee et al. cytes, and endothelial cells, in vitro occurred by activating 2019). These factors have been considered as protagonists PI3K/Akt and FAK-ERK1/2 signaling (Park et al. 2017). to participate in tissue repair and regeneration through their paracrine actions that mediate cell-to-cell signalling (Madrigal et al. 2014). Muscle regeneration It is critical for the success of such a cell-free therapy to identify, analyze and elucidate the mechanism of action Tissue regeneration and homeostasis are mediated by cell of each component of the secretome. proliferation, migration and stem cell differentiation processes. Mitchell et al. (2019) showed that application of the total secretome mouse myoblast cell line C2C12 for Conflict of interest statement 48 hours resulted in increased cell proliferation in comparison with the control group. Moreover, the total secre- The authors declare that the research was conducted in tome stimulated the differentiation of C2C12 cells into the absence of any commercial or financial relationships myofibers (Mitchell et al. 2019). that could be construed as a potential conflict of interest. Mitchell et al. (2019) studied the effect of the total ASCs secretome and its EV fraction on tissue repair in a mouse model of acute cardiotoxin-induced muscle injury. Acknowledgments It was revealed that the total ASCs secretome stimulated the process of tissue regeneration, which was confirmed The research was carried out with the financial support of by a significant decrease in the activity of lysosomes in the the Ministry of Science and Higher Education of the Rus- group of animals treated with the total ASC-CM (Mitch- sian Federation, Agreement № 14.575.21.0164, identifier ell et al. 2019). Studying the cross-sectional area of new- RFMEFI57517X0164. Research Results in Pharmacology 6(1): 57–68 63

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Author contributions

Liubov A. Pokrovskaya, Junior Researcher of Laboratory of Polymers and Composite Materials, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-4764-0665. The author made substantial contributions to the conceptualization of the article and later participated in drafting the article.

Ekaterina V. Zubareva, PhD in Biology, Associate Professor of the Department of Biology, e-mail: zubareva@ bsu.edu.ru, ORCID ID http://orcid.org/0000-0002-6480-7810. The author made substantial contributions to the conceptualization of the article and later participated in drafting the article, prepared the final version of the paper.

Sergey V. Nadezhdin, PhD in Biology, Associate Professor, Head of the Scientific Research Laboratory of Cellular, Assisted Reproductive and DNA Technologies, e-mail: [email protected], ORCID ID http://orcid.org/0000- 0001-8444-6330. The author made substantial contributions to the conceptualization of the article, participated in drafting the article, and gave the final approval of the version to be submitted.

Anna S. Lysenko, undergraduate student, e-mail: [email protected]. The author participated in drafting the article.

Tatyana L. Litovkina, undergraduate student, e-mail: [email protected]. The author participated in drafting the article. Research Results in Pharmacology 6(1): 69–82 UDC: 616.858 DOI 10.3897/rrpharmacology.6.52026

Research Article

Molecular docking studies of N-substituted 4-methoxy-6-oxo-1-aryl-pyridazine-3-carboxamide derivatives as potential modulators of glutamate receptors

Hanna I. Severina1, Victoriya A. Georgiyants1, Sergiy M. Kovalenko2, Natalia V. Avdeeva3, Artem I. Yarcev3, Svetlana N. Prohoda3

1 National University of Pharmacy, 53 Pushkinskaya St., Kharkiv 61002, Ukraine 2 V.N.Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61077, Ukraine 3 Kursk State Medical University, 3 K. Marksa St., Kursk 305041, Russia

Corresponding author: Natalia V. Avdeeva ([email protected])

Academic editor: Oleg Gudyrev ♦ Received 13 January 2020 ♦ Accepted 19 February 2020 ♦ Published 30 March 2020

Citation: Severina HI, Georgiyants VA, Kovalenko SM, Avdeeva NV, Yarcev AI, Prohoda SN (2020) Molecular docking studies of N-substituted 4-methoxy-6-oxo-1-aryl-pyridazine-3-carboxamide derivatives as potential modulators of glutamate receptors. Research Results in Pharmacology 6(1): 69–82. https://doi.org/10.3897/rrpharmacology.6.52026

Abstract Introduction: The virtual target-oriented screening is a necessary stage of modern drug-design. In the present study, the affinity of pyridazine derivatives for the most promising antiparkinsonian biotargets – I–III groups of metabotropic and ionotropic NMDA-glutamate receptors – was evaluated. Materials and methods: Docking of the studied ligands to the active sites of biotargets – mGluR5, mGluR3, mGluR8, NMDA GluN2B – was performed using AutoDockVina. Base of the preparation of ligands and proteins – AutoDock- Tools-1.5.6. A Discovery Studio Visualizer 2017/R2 was used to visualize the interpretation of the results. Results and discussion: A high degree of the affinity is predicted for group III of the metabotropic mGlu8 receptors – binding energy from -5.0 to -8.7 kcal/mol, compared to -6.1 kcal/mol of that of the reference drug (L-AP4), as well as for the ionotropic NMDA GluN2B receptors –binding energy from -8.7 to -11.6 kcal/mol, compared to -11.3 kcal/mol of that of ifenprodil. Conclusion: The prospects of the searching for glutamate receptor modulators in a number of n-substituted 4-methoxy-6-oxo- 1-aryl-pyridazine-3-carboxamide derivatives are proved. Some aspects of the structure-affinity relationship are discussed.

Keywords pyridazine, antiparkinson agents, docking, mGluR, NMDA.

Copyright Severina HI et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 70 Severina HI et al.: Molecular docking studies of...

Introduction studying the compounds affecting mGluR as potential antiparkinsonian agents was proved in 2003 (Marino et al. Scientific achievements of the last decades concerning 2003) and continues to be relevant nowadays (Avdeeva et the mechanisms of the antiparkinsonian action, the crystal al. 2017; Zhang et al. 2019). structure of target proteins and the amino acid compositi- Structurally, mGluR belong to the C-class of G-pro- on of active sites of receptors, the developed arsenal of in tein-coupled receptors. According to the functional activ- silico methods for analyzing and evaluating the affinity of ity, structure homology, and the list of selective ligands, the ligand for the receptor makes it possible to rationalize all mGlu receptors are divided into 3 groups, which in the search for new biologically active substances, in par- turn are subdivided into 8 more subtypes (Kunishima et ticular, antiparkinsonian ones. al. 2000; Kravchenko et al. 2016; Avdeeva et al. 2018). The feasibility and prospects of searching for antipar- All glutamate receptors have a modular architecture (Jin kinsonian substances among n-substituted 4-methoxy- and Ma 2017), wherein each of the subunits is made up of 6-oxo-1-aryl-pyridazine-3-carboxamide derivatives was 4 main parts: an amino-terminal domain (ATD) located in evaluated using the virtual screening tools, namely, mo- the extracellular space, a ligand-binding domain (LBD) lecular docking to the active sites of the known antipar- also located in the extracellular space, a transmembrane kinsonian biotargets. domain (TD) located inside the membrane, and a C-termi- In accordance with the research algorithm, the first nal domain (CTD) located in the intracellular space. stage of research is to assess the affinity for the metabo- Objective of the study: To conduct the docking and tropic glutamate receptors (mGluR). The prospect of to evaluate the affinity of the virtual ligand base among

Flowchart 1. The algorithm of the searching for the antiparkinsonian agents. Research Results in Pharmacology 6(1): 69–82 71 n-substituted 4-methoxy-6-oxo-1-aryl-pyridazine-3- Results and discussion carboxamide derivatives for the active sites of the most promising antiparkinsonian biotargets – the metabotrop- Stage I of the study: the assessment of the compounds ic receptors of groups I-III and ionotropic NMDA recep- affinity for metabotropic glutamate receptors of Group I tors of glutamate. The affinity of the virtual data base of compounds for Group I of the metabotropic glutamate receptors was Materials and methods evaluated by docking of the mGlu5 receptor into a active site. The crystal structure of the transmembrane domain The affinity for a biological target was studied using flexi- of mGlu5 receptor in a closed conformation with the ne- ble molecular docking. For this purpose, the AutoDock gative allosteric modulator mavoglurant in the active site Vina program was used, which gives a good correlation was established in 2014 (Doré et al. 2014). Despite the indicators between the calculated and experimentally ob- fact that mavoglurant passed the second phase of the cli- tained data (Wang et al. 2016). The ability of the used nical trials for the treatment of Levodopa-induced dys- docking algorithm to reproduce the experimental data kinesia, the affinity of potential antiparkinson agents for was evaluated by the reference docking of the native li- the active site of the transmembrane domain of mGlu5 gands. Macromolecules (proteins) from the Protein Data (PDB 6FFH) was evaluated in comparison with feno- Bank (PDB) were used as the biological targets, which bam – an mGluR5-selective negative allosteric modula- are freely available: metabotropic glutamate receptors: tor (Christopher et al. 2019). The choice of fenobam as a mGlu5 – PDB ID 6FFH, mGlu3 – PDBID 4XAR, mGlu8 reference drug for assessing the affinity for the mGluR5 – PDBID 6BT5, and ionotropic NMDAgluN2B receptors receptor is due to some structural similarity with the sub- of glutamate – PDBID 3QEL. stances to be synthesized (Fig. 1). The transmembrane domain of the mglu5 receptor Preparation of the ligands consists of seven transmembrane α-helices (Fig. 2A). The configuration of the helical bundle together with extracel- The substance structures were obtained using MarvinS- lular loops strongly restricts the entrance to the alloster- ketch 18.23 and saved in mol format. After that, they ic pocket, which results in its relatively small size – the were optimized by Chem3D, using the molecular mecha- entrance radius is about ~7 A°. In addition, the allosteric nics (MM2) algorithm and saved as pdb files. Using Au- site itself is located at a distance of about 8 A° from the toDockTools-1.5.6, pdb files were converted to PDBQT receptor surface and is a fairly narrow hydrophobic space. (Trott and Olson 2010). This size of the pocket entry and the location of the active site condition a small size of the ligand and can impair the Preparation of the proteins scoring functions during docking. According to the literature data (Doré et al. 2014, PDB files were downloaded from The Protein Data Christopher et al. 2019), the main hydrophobic pocket Bank. Discovery Studio Visualizer 2017/R2 was used of the active site is formed by residues of the follow- to remove water and ligand molecules from the crys- ing amino acids: valine (Val806), methionine (Met802), tal. Protein structures were saved as pdb files. In -Au phenylalanine (Phe788), tryptophan (Trp785), leucine toDockTools-1.5.6, polar hydrogens were added to the (Leu744), isoleucine (Ile651), proline (Pro655), asparag- protein structure and stored as PDBQT. Gridboxes were ine (Asn747), and glycine (Gly652). The main ”shrink- established relative to native ligands. AutoDock Vina age” of the ligand occurs between alanine (Ala810) and was used for docking (Trott and Olson 2010). Disco- proline (Pro 655), bordered by isoleucine (Ile625), gly- very Studio V17.2. 0.16349 was used to visualize the cine (Gly624), serine (Ser654 and 658) on one side, and docking results. by tyrosine (Tyr659) – on the other. Hydrophobic interac-

Figure 1. The structure of the mGluR5 negative allosteric modulators in comparison with the ligands under study. 72 Severina HI et al.: Molecular docking studies of...

A B

Figure 2. А. 3D structure of mGlu5 with a negative allosteric modulator fenobam in the active site B. 3D image of the fenobam conformation in the mGluR5 active site.

tion occurs with tyrosine (Tyr659), serial (Ser 809) valine Stage 2 of the study: the evaluation of the compounds (Val806), and proline (Prp 655). affinity for the metabotropic glutamate receptors of Despite the location of the allosteric site and its nar- Group II row size, when evaluating the reproducibility of the docking technique in mGluR5 of the native ligand, it At the next stage, the virtual database of compounds was was possible to achieve a suitable conformation and a docked into the active site of the metabotropic glutamate satisfactory evaluation function, which was -8.7 kcal/ receptor of Group II subtype 2 – mGluR3 (PDB ID 4XAR). mol for fenobam. As seen in Fig. 2B, fenobam is com- A strong allosteric agonist of the mglur2/3 receptor (1S, 2S, pletely immersed in a narrow hydrophobic cleft of the 5R, 6S)–2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic active site of the receptor, forming a stable conformation acid (LY354740)) was used as a reference ligand (Schoepp by entering a hydrophobic interaction; this conformation et al. 2003, Linden et al. 2005, Menezes et al. 2013). is further stabilized by the hydrogen bonds, in particular The recombinant protein of the mGlu3 amino-terminal with the hydroxyl groups of serine and tyrosine (Ser809, domain in the conformation with Ly354740, from the mac- Tyr659), which is consistent with the literature data roscopic point of view, is a closed topology (Monn et al. (Christopher et al. 2019). 2015), where the upper (LB1) and lower (LB 2) lobes of At the stage of the selecting compounds for synthe- the protein are closely related to the ligand in the hinge re- sis, no detailed analysis of conformational placement was gion (Fig. 3A). The binding of the agonist induces the pro- carried out, with only the binding energy (the scoring cess of joining the protein lobes, which in turn leads to the function) being taken into account in comparison with opening of the channel and the activation of the receptor. the native ligand. The results of the docking of the com- The visualization of the experimentally established pounds with the active site of the mGluR5 receptor are interaction and the image of the ligand-binding pocket presented in Table 1. are shown in Figure 3B. Describing the binding site, it As can be seen from the results of the docking, a sat- should be noted that there is a fairly spacious entrance to isfactory level of affinity for the active site of the mGlu5 the binding pocket, the predominant majority of hydrogen receptor is predicted for pyridazine derivatives: the bind- interactions, including the tetrahedral network of ligand ing energy was from -11.2 to -5.2 kcal/mol, versus -8.7 amino group bonds with the carboxyl groups of alanine kcal/mol for the native fenobam ligand. The highest(un- and asparagine (Ala172, Asp301), and the threonine hy- favorable) binding energy level was demonstrated by the droxyl (Thr 1744), participation in the interaction of both compound with the N-(4-diethylamino)phenyl substit- protein lobes (LB1 and LB2), and the possibility of the uent (0182), and the lowest – with the diphenylpropyl ligand conformational mobility. in the carboxamide fragment (0131), which may be ex- The amino acids of the binding site, according to the plained by the conformational mobility of the radical and literature data, are (Fig. 4A): by the additional centers of the hydrophobic interaction. The compounds with a benzyl amide fragment have a • the upper lobe (LB 1) – arginine (Arg68), lysine higher affinity than the substances with a phenylamide (Lys 389), alanine (Ala172), threonine (Thr174), radical. A decrease in the affinity is predicted for all serine (Ser 151), which participate in the forma- the compounds that have a substituted NH-group of the tion of hydrophilic bonds; tyrosine (Tyr222) – carboxamide residue (for example, 0129, 0265, 0249), 4-hydroxyphenyl radical enter into hydrophobic which is probably due to the ability of the NH-group to interaction (Fig. 5A); form hydrogen bonds to stabilize the ligand in the cavity • the lower lobe (LB2) – aspartic acid (Asp301) and of the active site. tyrosine (Tyr 150). Research Results in Pharmacology 6(1): 69–82 73

Table 1. Results of docking of N-substituted 4-methoxy-6-oxo-1-arylpyridazine-3-carboxamides with the glutamate receptors.

Ligand R/R1 Receptors mGluR5 (6FFH) mGlu3 (4XAR) mGlu8 NMDA Glu N2B (6BT5) (3QEL) Binding energy (kcal/mol) Native reference ligand -8.7 (fenobam) -8.2 (Ly235) -6.1 (L-AP4) -11.3(Ifenprodil)

O O

R H N N N

R1 O

0057 4-Me/H -7.5 -6.7 -7.5 -10.4 0077 4-F/H -9.2 -6.1 -7.9 -10.3 0098 4-Cl/H -8.9 -6.5 -7.4 -9.4 0102 2-OMe/H -8.9 -7.2 -7.9 -8.9 0128 H/Me -8.0 -6.5 -8.2 -10.0 0129 H/Et -6.1 -5.2 -6.7 -8.4

O O

H N N N R O

0058 2-Me,5-Cl -8.2 -5.5 -7.9 -10.1 0066 3,5-diMe -8.7 -6.6 -8.1 -10.1 0067 2,6-diMe -7.2 -6.6 -6.0 -10.3 0070 4,5-diMe -9.4 -5.1 -8.6 -10.7 -6.7 -6.7 -10.2 -5.8 -6.7 -10.5 -6.7 -6.9 -10.2 0095 3-Me,6-OMe -7.5 -5.3 -7.4 -9.9 0101 2-Me,3-Cl -8.6 -5.3 -6.6 -10.4 0105 2-Me, 4-Br -8.7 -5.5 -6.2 -10.6

0060 CH(Ph)2 -7.5 -5.7 -8.7 -9.7

0126 CH(CH2)2(Ph)2 -5.6 -6.7 -6.8 -10.2

0131 (CH2)2 CH(Ph)2 -11.2 -5.1 -8.8 -12.3

R O O

H N N N

R1 O

0001 4-Cl/H -6.7 -3.9 -8.3 -11.1 0175 H/H -8.7 -6.0 -8.0 -9.5 0176 4-OMe/H -8.6 -5.8 -7.2 -9.4 0197 4-Me/H -8.7 -6.1 -7.3 -9.9 0233 2-Cl/H -7.9 -7.3 -8.1 -9.5 0241 3-OMe/H -8.3 -7.1 -7.4 -9.7 0244 4-I/H -7.4 -3.9 -6.4 -9.4 0258 H/Me -6.7 -6.7 -7.2 -10.0 0265 H/H/ NMe -6.4 -6.0 -6.0 -9.6

O O

H R N N N

0181 H -8.3 -7.0 -6.2 -10.1

0182 4-N(Et)2 -5.2 -4.8 -5.8 -9.6 74 Severina HI et al.: Molecular docking studies of...

Ligand R/R1 Receptors mGluR5 (6FFH) mGlu3 (4XAR) mGlu8 NMDA Glu N2B (6BT5) (3QEL) Binding energy (kcal/mol) 0184 3-OMe -8.1 -5.4 -6.7 -9.6 0185 4-OMe -7.8 -5.1 -6.4 -9.6

0190 3-COCH3 -8.3 -6.0 -7.2 -9.7 0192 2-COOMe -5.7 -5.9 -6.4 -8.7 0198 2-Me, 4-Cl -6.2 -4.8 -6.6 -9.9 0199 2,5-diCOOMe -5.4 -4.4 -5.0 -9.3 0201 2-OH -8.0 -7.0 -6.6 -9.7 0205 4-OEt -6.7 -5.6 -6.5 -9.2 0207 2,6-diMe -6.4 -7.0 -5.4 -10.7 0208 2-OEt -6.1 -6.6 -5.7 -8.7 0209 3,4-diMe -6.1 -6.7 -6.5 -8.7 0210 3-Cl -7.8 -7.6 -7.4 -9.6 0213 4-Et -6.3 -5.8 -6.5 -9.4 0215 2-Et -6.3 -6.6 -5.9 -8.7 0216 2,4,6-triMe -5.6 -7.2 -4.8 -8.5 0218 4-Bu -6.2 -5.3 -6.5 -9.6 0219 2-naphtyl -8.6 -5.4 -7.6 -10.4 0220 3-Me -9.1 -5.0 -6.8 -10.4 0222 2,3-diMe -6.9 -7.5 -6.7 -11.1 0224 2,4-diMe -7.0 -7.6 -6.7 -10.9 0226 2-OMe, 5-Me -6.1 -7.2 -6.9 -9.3 0228 4-Br -7.1 -4.6 -6.2 -9.8 0230 3-Cl, 4-Me -7.6 -7.3 -7.3 -9.6 0231 2-Me, 3-Cl -6.3 -5.2 -6.3 -10.3

0234 3,5-diCF3 -8.0 -5.8 -8.1 -10.9 0235 2-Me,4-Br -6.5 -5.8 -6.9 -9.5 0239 3,5-diCl -6.9 -5.6 -6.6 -10.9 0245 4-Me -7.8 -5.8 -6.2 -9.6 0246 3-Br -8.9 -6.0 -6.5 -9.9 0248 2-OMe, 5-Cl -5.7 -5.2 -6.5 -9.7 0260 4-O-Ph -7.6 -6.3 -7.3 -11.0

0268 2-CONH2 -6.2 -6.7 -6.7 -9.3 O O

R N N N

R1 O

0163 N -6.5 -6.1 -6.6 -7.9

0168 N -6.2 -5.3 -7.5 -9.9

0200 CH(Ph)2 -7.4 -5.5 -7.9 -10.4 0243 N -6.4 -3.6 -5.6 -9.7

Ph Ph 0249 Ph/Bn -6.7 -4.4 -7.5 -9.1 0256 Bn/Bn -7.5 -3.2 -7.0 -9.8 0194 -8.1 -6.9 -6.5 -9.6

R O O

H N N N

R1 O

0305 H/H -8.8 -5.8 -7.9 -10.5 0325 4-Me/H -9.4 -5.0 -7.5 -9.8 0357 4-Cl/H -9.1 -5.0 -7.4 -9.7 0360 2-OMe -9.2 -5.8 -7.6 -9.8 0386 H/Me -8.2 -5.4 -7.9 -10.7 0387 H/H/NMe -7.2 -5.1 -6.2 -9.1 Research Results in Pharmacology 6(1): 69–82 75

Ligand R/R1 Receptors mGluR5 (6FFH) mGlu3 (4XAR) mGlu8 NMDA Glu N2B (6BT5) (3QEL) Binding energy (kcal/mol)

O O

H R N N N

0311 H -7.8 -6.2 -6.4 -10.9 0332 2-OH, 5-Cl -7.9 -4.5 -6.8 -10.6 0335 2,6-diMe -7.0 -5.6 -6.6 -10.5 0337 3,4-diMe -9.3 -5.5 -7.8 -11.3 0338 3-Cl -8.7 -7.4 -6.6 -10.8 0339 2-Cl -8.0 -6.5 -6.5 -11.3 0348 3-Me -8.1 -5.3 -7.1 -10.6 0349 2-Me -8.0 -5.5 -6.7 -11.6 0354 2-OMe, 5-Me -7.2 -5.6 -6.0 -9.6 0355 4-Me -8.0 -6.4 -6.7 -10.7 0358 3-Cl,4-Me -9.1 -5.5 -7.0 -11.2 0359 3-Cl,2-Me -8.4 -5.4 -6.7 -10.7 0373 4-Me -8.5 -5.5 -6.7 -10.7 0376 2-OMe, 5-Cl -6.1 -5.4 -6.8 -10.7

O O

R N N N

R1 O

0328 CH(Ph)2 -7.6 -4.4 -8.4 -10.3 0377 Ph/Bn -6.7 -4.9 -7.5 -9.3

O O

H N N N

R O F

0419 H -9.1 -7.0 -8.4 -10.3 0484 Me -8.4 -6.1 -8.0 -10.6

O O

H R N N N

O F

0435 2-Me,5-Cl -7.7 -6.3 -7.2 -10.3 0455 2-Me -7.9 -5.3 -6.9 -10.9 0458 4-Cl -8.6 -5.5 -6.9 -10.2

The ability of the docking algorithm used in the study to ic agonist of mGlu3. No clear patterns of dependence of the reproduce the experimental data in the case of the mGlu3 affinity level on structural features were identified. The -low receptor is demonstrated in Figure 4B. The location of the est affinity is predicted for the N,N-dibenzylcarboxamide ligand and all the interactions agree with the experimental derivative with a 2-methylphenyl radical in the 1st position data, except the missing hydrogen bond with threonine (0256), and the highest for the N-3-Chlorobenzyl-substitut- (Thr174), but its close and correct location indicates the ed with a 4-methylphenyl radical in the 1st position. correct location of the ligand in the active site. The success of the method is also confirmed by the low binding Stage 3 of the study: the evaluation of the compounds energy of the native ligand -8.2 kcal/mol. affinity for Group III of the metabotropic glutamate No compound exceeded the affinity index of the native receptors ligand, demonstrating higher binding energy values from -3.2 to -7.4 kcal/mol in the result of the virtual database of The first ligand that exhibits a strong selective agonism to compounds docking to the active binding site of the alloster- mGluIII group receptors, but at the same time not being 76 Severina HI et al.: Molecular docking studies of...

A B

Figure 3. А. 3D macroscopic structure of mGluR3 with allosteric agonist of mGlu2/3 receptors – LY354740 B. 3D molecular structure of the active mGluR3 site with the LY354740 ligand (green-colored molecule).

A B

Figure 4. А. Experimentally established interaction of LY354747 with the amino acids in the active mGluR3 site. B. Reference interaction of LY354747 with amino acids in the active site of the mGlu3 receptor.

selective of certain subtypes of Group III mGlu receptors, molecules of alanine (Ala 155 and 177), serine (Ser156), is L-2-amino-4-phosphonobutyric acid (L-AP4), descri- threonine (Thr179) from the upper lobe of the protomer bed back in 1997 (Thomsen 1997). The crystal structure of (LB1), tyrosine (Tyr227), and aspartic acid (Asp309) from the recombinant human amino-terminal domain mGlu8 in the lower lobe (LB2). The important interactions which may combination with the selective l-AP4 agonist was isolated be responsible for the selectivity of L-AP4 are postulated to and described only in 2018 (PDBID 6BT5) (Avdeeva et be ionic interactions of phosphate with the formation of salt al. 2019). Unfortunately, at the moment, the crystal struc- bridges with lysine 71 and 401, arginine 75 (LB1), and its ture of the closed conformations with agonists of other bidentant hydrogen interaction with lysine 314 (LB2). subtypes of Group III mGlu receptors – mGlu4, mGlu6 In this study, the binding energy was -6.1 kcal/mol, or mGlu7 – has not been established. However, there is when docking the reference ligand l-AP4 into the active evidence of a significant similarity of the amino acid se- site of the mGlu8 amino-terminal domain. The visuali- quence of active sites of Group III mGlu receptors, which, zation of the docking results (Fig. 6А, В) demonstrates accordingly, allows predicting to some extent the affinity almost a complete compliance of the obtained conforma- for all receptors in this group. tion of l-AP4/mGlu8 with the experimentally established The mGlu8 amino-terminal domain consists of two data: a characteristic tetrahedral network of hydrogen asymmetric protomers that form a homodimer (Fig. 5A) bonds between the amino group of the ligand and the thre- (Schkeryantz et al. 2018). The binding center of the se- onine hydroxyl (Thr179), the alanine carbonyl (Ala 155), lective LAP 4 agonist is the hinge region of the protein, and the carboxyl group of aspartic acid (Asp309), as well located between the complementary globules (LB1/LB2), as all salt bridges between the phosphate. The exception which are connected to each other by three short loops. was the hydrogen bond with a water molecule, since the The following amino acid residues are the experimentally automatic docking methodology involves removing water established binding site of the L-AP4 agonist (Fig. 5B): two molecules from the protein globule. Research Results in Pharmacology 6(1): 69–82 77

A B

Figure 5. Spatial structure of the mGlu8 receptor with an L-AP4 agonist at the binding site А. 3D MGlu8 structure with an L-AP4 agonist in the binding site B. Interaction of L-AP4 with amino acid residues of the binding site. Hydrophilic bonds are indicated by blue lines, hydrophobic bonds are indicated by gray lines.

A B

Figure 6. 3D (A) and 2D (B) interactions of L-AP4 with the amino acids of the mGlu8 binding site.

The binding energy of the native ligand was -6.1 kcal/ teric site of ionotropic NMDA (N-methyl-D-aspartate) mol. For derivatives of N-substituted 4-methoxy-6-oxo- glutamate receptors. The ionotropic NMDA glutamate 1-aryl-pyridazine-3-carboxamides, a higher degree of receptor is a heterotetramer of two subunits – N1R and affinity for the active site of the mGlu8 receptor is pre- N2R. Each of the subunits is made up of 4 parts: an ami- dicted: the binding energy of all the compounds that were no-terminal domain (ATD), a ligand-binding domain selected for synthesis is either at or above the reference (LBD), a transmembrane domain (TD), and a C-termi- drug value (from -5.0 to -8.7 kcal/mol). The lowest affini- nal domain (CTD). The activity of the NMDA receptors ty (binding energy -5.0 kcal/mol) is predicted for dimethyl in the ion channels is regulated by the allosteric binding 2 - [[4-methoxy-1-(2-methylphenyl)-6-oxo-pyridazine-3- of small molecules to the amino-terminal domain, or the carbonyl]amino]benzene-1,4-dicarboxylate (0199). It can ligand-binding domain, in a subtype-specific manner: in also be noted that benzylamine derivatives have some of particular, after the simultaneous binding of glycine and the best indicators of the affinity in comparison with the glutamate to the GluN1 and GluN2 subunits, respectively phenylsubstituted carboxamide derivatives. (Mothet et al. 2015). Ifenprodyl-4 - [(1R, 2S) - 2 - (4-ben- zylpiperazine-1-yl)-1-hydroxypropyl phenol is consi- Stage 4 of the study: the evaluation of the affinity of dered to be such a molecule, specifically inhibiting the the compounds for NMDA glutamate receptors NMDA receptor of the GluN1 and GluN2 subtypes (Wil- liams 1993; Gallagher et al. 1996). The negative alloste- The next stage of the research was to study the affinity ric modulation of the NMDA receptors occurs by binding of the virtual database of the compounds for the allos- ifenprodil to active sites of the amino-terminal domain. 78 Severina HI et al.: Molecular docking studies of...

A B

Figure 7. Macroscopic 3D (A) and schematic (B) images of the structure of the amino-terminal domain of the NMDA receptor with a negative allosteric negative modifier in the active site.

A B

Figure 8. Experimentally established (A) and reference interaction (B) of ifenprodil with amino acids in the active site of the NMDA glutamate receptor.

It is the complex of amino-terminal domains GluN1/ Comparing the experimental literature data and the re- GluN2B in a closed conformation with ifenprodil (PDB sults of the reference docking (Fig. 8A, B), the reproducibil- ID 3QEL) that were used to dock the virtual database of ity of the method becomes obvious: all hydrophobic and hy- the compounds (Fig. 7A, B). drophilic bonds are present, and the locations of ifenprodyl It was experimentally established that the hydrophobic fragments relative to amino acid residues are comparable. pocket where ifenprodil is immersed consists of the fol- The difference in the reference docking is - theab lowing amino acid residues of both subunits: sence of a single hydrogen bond between phenylalanine (Phe176) and ifenprodyl hydroxyl, whereas the hydro- • GluN1b subunit – threonine (Thr 110), tyrosine phobic interaction is preserved. The binding energy of (Tyr109), phenylalanine (Phe 113), serine (Ser132), and the native ifenprodil ligand was -11.3 kcal/mol, which leucine (Leu135); demonstrates a high affinity for the receptor. • GluN2B subunit – (Pro 177), isoleucine (Ile As for the studied derivatives of N-substituted 4-meth- 111), glutamine (Gln110), alanine (Ala107), glutamic oxy-6-oxo-1-aryl-pyridazine-3-carboxamides, they acid (Glu 236), and phenylalanine (Phe176). demonstrate a high affinity for the NMDA glutamate receptor: the binding energy ranges from -8.7 to-11.6 kcal/mol. Next to the binding pocket is an empty space that is When docked to this receptor, it is substituted phenyl car- surrounded by hydrophobic residues, including alanine boxamide derivatives that show more stable results, where- (Ala75) of the GluN1b subunit and isoleucine (Ile 82), as benzyl-substituted ligands show a bit worse results. phenylalanine (Phe114) of the GluN2B subunit. The accuracy of the docking methodology and the ability to reproduce the parameters of the experimental data Conclusion were confirmed when evaluating the affinity of the native ifenprodil ligand for the active site of the amino-terminal • According to the results of docking, a high degree of domain of the NMDA glutamate receptor. affinity of 4-methoxy-6-oxo-1-arylpyridazine-3-car- Research Results in Pharmacology 6(1): 69–82 79

boxamide derivatives is predicted for the following • SAR analysis of the docking results shows that glutamate receptors: the N-benzyl-substituted derivatives have better ◦ metabotropic mGluR8 (Group III) – binding ener- affinity, compared to that of the N-phenyl-sub- gy from -5.0 to -8.7 kcal/mol, versus -6.1 kcal/mol stituted carboxamide derivatives. Substitution of in the reference drug (L-AP4); hydrogen of the NH-group of the carboxamide ◦ ionotropic NMDA of the GluN2 subtype – the bind- residue with a methyl or ethyl radical leads to a ing energy from -8.7 to -11.6 kcal/mol, compared to decrease in the affinity, which is probably due -11.3 kcal/mol in the native ifenprodyl ligand. to the ability of the NH-group to form hydrogen • A satisfactory level of the affinity is predicted with bonds to stabilize the ligand conformation in the the active mGluR5 site (Group I): binding energy cavity of the active site. There is no clear relation- 11.2–5.2 kcal/mol versus -8.7 kcal/mol in the ref- ship between the substituent in the phenyl ring erence ligand fenobam. and the affinity level. • The results of the docking to the active mGluR3 • According to the binding energy values for me- site (Group II) were less satisfying. No compound tabotropic and ionotropic glutamate receptors, 96 exceeded the affinity of the native ligand: binding substances were selected for synthesis, with the energies from -3.2 to -7.4 kcal/mol compared to best affinity predicted for at least two of the four -8.2 kcal/mol, respectively. types of the glutamate receptors.

References

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Author contributions

Natalia V. Avdeeva, PhD in Medical Sciences, Associate Professor, e-mail: [email protected], ORCID ID https:// orcid.org/0000-0003-1405-4555. The author suggested the idea of the article, made substantial contributions to the design of the article and participated in drafting the article.

Hanna I. Severina, PhD in Pharmaceutical Sciences, Associate Professor of the Department of Pharmaceutical Chemistry, e-mail: [email protected], ORCID ID https://orcid.org/0000-0003-2894-9384. The author played a leading role in conducting the experiment, interpreting the data, and writing the article.

Victoriya A. Georgiyants, Doctor of Sciences (Pharmacy), Professor, Head of the Department of Pharmaceutical Chemistry, e-mail: [email protected], ORCID ID https://orcid.org/0000-0001-8794-8010. The author played a leading role in the design of the experiment and analysis of the data, and participated in the experiment.

Sergiy M. Kovalenko, Doctor of Sciences (Chemstry), Professor, e-mail: [email protected], ORCID ID https://orcid.org/0000-0003-2222-8180. The author analyzed and interpreted the data, wrote and edited the article.

Artem I. Yarcev, 6-year student, Faculty of Medicine, e-mail: [email protected]. The author analyzed the literature and participated in interpreting the data.

Svetlana N. Prohoda, medical doctor of the Kursk Regional Clinical Hospital, e-mail: prohoda_svetlana@yandex. ru. The author analyzed the literature and participated in interpreting the data. Research Results in Pharmacology 6(1): 81–91 UDC: 616-092.9 DOI 10.3897/rrpharmacology.6.52180

Research Article

Role of orexin peptide system in emotional overeating induced by brain reward stimulation in fed rats

Andrei A. Lebedev1, Yulia N. Bessolova1, Nikolai S. Efimov1, Eugeny R. Bychkov1, Andrei V. Droblenkov1, Petr D. Shabanov1,2

1 Institute of Experimental Medicine, 12 Academician Pavlov St., St.-Petersburg 197376, Russia 2 Kirov Military Medical Academy, 6-Zh Academician Lebedev St., St.-Petersburg 194044, Russia

Corresponding author: Andrei A. Lebedev ([email protected])

Academic editor: Mikhail Korokin ♦ Received 18 December 2019 ♦ Accepted 24 February 2020 ♦ Published 31 March 2020

Citation: Lebedev AA, Bessolova YuN , Efimov NS, Bychkov ER, Droblenkov AV, Shabanov PD (2020) Role of orexin peptide system in emotional overeating induced by brain reward stimulation in fed rats. Research Results in Pharmacology 6(1): 81–91. https://doi.org/10.3897/rrpharmacology.6.52180

Abstract Introduction: The purpose of this work was to prove that the reaction of food self-deprivation in “fed up” rats is a suitable model for studying the emotional overeating in the experiment. Methods: The self-deprivation reaction, i.e. self-isolation of an animal from food during electrical self-stimulation of the brain, was studied in animals with food deprivation. To reproduce the self-stimulation of the lateral hypothalamus, the male Wistar rats were trained to press a pedal in a Skinner box. After training, the rats received food deprivation, then a feeder was placed in the Skinner box, and a conditioned food reflex was developed in rats within 5 days. Results and discussion: The food self-deprivation reaction was observed in the ”satiated” rats with a current intensity of 10% and above the threshold for self-stimulation. Hungry animals pressed the pedal for hypothalamic self-stimulation and took no notice of the feeding trough. Sulpiride, a dopamine D2 antagonist (5 and 20 mg/kg i.p.), administered to the “satiated” rats decreased both the eating behavior and self-stimulation in food self-deprivation testing. SB- 408124, an orexin A receptor antagonist (0.5 mg/ml, 20 μl intranasally) reduced only the number of pellets eaten, but not the number of pedal presses. Conclusion: The orexin A receptors are preferably involved in emotional eating compared with orexin B (OX2R TCS- OX2-29) and D2 dopamine receptors. Because emotional eating is significantly related to clinical eating disorders, like bulimia and binge eating disorder, it seems promising to use drugs of the orexin system to treat and prevent the issue.

Keywords orexin, dopamine, self-stimulation, self-deprivation, overeating, SB-408124, TCS-OX2-29, sulpiride, rats.

Introduction to clinical eating disorders, like bulimia and binge eating disorder (BED), as well as obesity and malnutrition, there Emotional eating is a phenomenon in which a person is growing interest in learning how to treat and prevent consumes food for reasons other than being physically the issue. More specifically, emotional eating is linked to hungry. Because emotional eating is significantly related a tendency to overeat in response to negative emotions,

Copyright Lebedev AA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 82 Lebedev AA et al.: Role of orexin peptide system in emotional overeating induced by brain... but little is known about emotional eating and positive and lateral hypothalamus. Both receptors are present in emotions, like happiness or excitement (Sultson et al. the prefrontal cortex, amygdala, bed nucleus of stria ter- 2017). Negative emotional eaters also increase eating af- minalis, paraventricular thalamic nucleus, dorsal raphe, ter experiencing positive emotions, suggesting that these ventral tegmental area, and laterodorsal tegmental nucle- emotions may contribute to food consumption in emoti- us – peduncolo pontine nucleus (Trivedi et al. 1998; Lu onal eaters as much as negative emotions (Bongers et al. et al. 2000; Marcus et al. 2001). These findings suggest 2013, 2016). New assessment tools, which in addition to that orexins and their receptors are likely to play a broad negative emotional eating also address positive emotional regulatory role in the central nervous system. Injection of eating, could be of potential help in planning interven- OXA into the lateral ventricle of rats during early light tion. Further, the tendency to overeat in response to po- phase induced a dose-related increase in a food intake in sitive emotions could be integrated into current models rats (Sakurai et al. 1998), which was blocked by pre-treat- of eating disorders, especially when addressing relapse ment with the OX1R antagonist SB-334867 (Haynes et prevention (Pompili and Laghi 2017; Sultson et al. 2017). al. 2000; Rodgers et al. 2001). Hypothalamic OX neu- Episodes of binge eating (BE) in humans are charac- rons are activated by cues associated with consummatory terized by compulsive, non-homeostatic consumption rewards, such as food (Harris et al. 2005), suggesting a of an unusually large quantity of highly palatable food potential role of the orexin system in response to external in a short period of time. Even though they are not hun- environmental cues linked to cognitive aspects of feed- gry, subjects eat more rapidly than normally until feel- ing. Recent reports support a role for orexin signaling ing uncomfortably full. As described by the DMS-IV-TR in the neurobehavioral and motivational effects of drugs (American Psychiatric Association 2000), these episodes of abuse (Harris and Anthenelli 2005; Jupp and Dalley are accompanied by a subjective sense of loss of control 2014; Baimel and Borgland 2015; Matzeu et al. 2018; over eating, and are associated with the feelings of dis- Schmeichel et al. 2018). Neural systems that motivate and tress, disgust, depression, being guilty about overeating, reinforce drug abuse have also been proposed to underlie and eating alone because of embarrassment. Lisdexamfe- behaviors associated with compulsive food seeking and tamine (LDX), a pro-drug of d-amphetamine, is the only food intake (Volkow and Wise 2005; Johnson and Kenny pharmaceutical drug currently approved for treatment 2010; Corwin et al. 2011; Schulte et al 2019). of BED, and works through modulation of monoamine A number of binge eating testing methods are current- transmission. LDX has been shown to directly decrease ly being used in animal models (Corwin and Buda-Levin compulsive eating in rats as well as humans, as measured 2004; Corwin et al. 2011). These models are considered by the Yale–Brown obsessive compulsive scale modified within the context of their effects on brain reward- sys for binge eating (Griffiths et al. 2019). LDX administra- tems, including dopamine, opioids, cholinergic systems, tion produces sustained increases in dopamine in rats, serotonin, and GABA. All of the models demonstrate that which could recover low dopaminergic states character- binge-type consumption of palatable food can occur in- istic of compulsive overeating to relieve a negative emo- dependently of obesity (Boggiano and Chandler 2006). tional state (Griffiths et al. 2019). Current medications, There are three commonly used models. First, a model like topiramate (McElroy et al. 2009) or sibutramine of sugar bingeing in which animals with repeated, inter- (Appolinario et al 2003; Wilfley et al. 2008), have been mittent access to a sugar solution develop behaviors and reported to reduce BED in clinical studies. However, their brain changes that are similar to the effects of some drugs administration is associated with a variety of adverse side of abuse, serving as the first animal model of food addic- effects, which represent serious problems during chronic tion (Avena et al. 2008). Second, another model in which treatment (Yager 2007; McElroy et al. 2009). a history of dieting and stress can perpetuate further binge In 1998, two groups independently identified a new eating of palatable and non-palatable food (Boggiano and class of neuropeptides originating in hypothalamic nuclei Chandler 2006). Lastly, a limited access model in which (De Lecea et al. 1998; Sakurai et al. 1998). These pep- non-food deprived rats with sporadic limited access to a tides, called orexin-A (OXA) and orexin-B (OXB), also high-fat food develop binge-type behaviors (Corwin and denoted as hypocretin 1 and hypocretin 2, are produced Buda-Levin 2004). When using these models, periods of from proteolytic processing of the pre-pro-OX peptide elements of stressful, negative emotional effects are as- and bind to two GPCRs, namely OX-1 and OX-2 recep- sumed: food deprivation, foot-shock, intermittent access tors (OX1R and OX2R), also denoted as HcrtR1 and Hcr- to a sugar solution, or sporadic limited access to a high-fat tR2. OX1R is coupled to Gq/11, whereas studies using food. At the same time, models with positive emotion- neuronal cells suggest that OX2R is coupled to Gq, Gs, al effects, like happiness or excitement, often associated and Gi proteins. In the central nervous system, OX1R and with emotional eating, have not been used previously. OX2R show partially overlapping, but largely distinct This study used the reaction of self-stimulation of the and complementary, distribution patterns (Sakurai 2007). lateral hypothalamus with the threshold current inten- Brain areas, such the infralimbic cortex, hippocampus, sity as a positive emotional component of the environ- and locus coeruleus, exhibit high expression of OX1R, ment for eating behavior in fed rats. The self-deprivation whereas OX2R is the only receptor expressed in arcuate phenomenon shown earlier, assessed as a maladaptive nucleus, tuberomammillary nucleus, and dorsomedial preference in a hungry animal, was retrieved after pos- Research Results in Pharmacology 6(1): 81–91 83 itively reinforcing electrical stimulation of certain brain trodes were lowered into the left or right medial forebrain areas rather than food. The earliest account of the phe- bundle as it passes through the lateral hypothalamic area nomenon stemmed from the pioneering work of J. Olds (coordinates: 2,5 mm posterior to bregma, 2.0 mm lateral (1958), who demonstrated that rats receiving rewarding to the midline suture, 8.4 mm below the dorsal surface of hypothalamic electric stimulation would self-stimulate the skull). The electrodes were attached to the skull with to exhaustion, neglecting other bodily needs. According optical screws and dental acrylic, and one week was allo- to J. Olds, the highly rewarding properties of the hypo- wed for recovery from surgery. thalamic electric stimulation represented the activation of the neural systems that sub serve conventional reward Apparatus (Margules and Olds 1962). G. Spies (1965) demonstrated that rats having rewarding electrodes in the so-called Standard operant conditioning chambers (RITEC, St. Pe- “feeding center” of the lateral hypothalamus preferred tersburg, Russia) were housed in ventilated, sound-atte- hypothalamic electric stimulation to food in a discrete nuating cubicles, equipped with a 2.8-W house light and choice situation even during prolonged periods of food a lever. An operant chamber (29.2×30.5×24.1 cm) had a deprivation. The issues of modeling and studying the ef- response lever 4.5 cm wide, 2.0 cm inside, 3.0 cm abo- fects of hypothalamic activation from the point of view of ve the floor, with a stimulus light centered 7.6 cm above reproducing the non-chemical dependence of emotional the lever (Bespalov et al. 1999; Shabanov and Lebedev overeating, in particular, binge eating, have not been pre- 2013). The chamber was connected to an IBM clone mi- viously conducted. The aim of this work was to prove the crocomputer through an MED interface and controlled role of orexin brain system in mechanisms of BED in the by MED-PC software (MED Associates Inc., East Fair- model of food self-deprivation in well-fed rats. To date, field, Vermont, USA). Electrical pulses were produced the self-deprivation reaction has been studied exclusively by the constant current stimulators (PHB-150B; MED in animals with food deprivation (Frank et al. 1982; Frank Associates Inc.). The electrical stimuli were delivered to and Stutz 1984). the animal through an electrical swivel assembly (Plastic One, Roanoke, Virginia, USA), which extended into the test chamber. Depression of a lever resulted in a single Material and methods 500-msec of rectangular bipolar waves with a pulse frequency of 100 Hz and a pulse duration of 0.1 ms (FR1). Animals The animals were tested for self-stimulation for 10 min each day for 5 days. During these daily 10-min sessions, The experiments were performed on 29 Wistar male the current level was adjusted to produce maximum le- rats weighing 240±30 g (12–13 weeks of age) each, in ver pressing rates in each animal without disruptive mo- accordance with EU Directive 2010/63/EU for animal tor involvement or convulsions. The animals that did not experiments and were approved by the local biomedical lever-press for intracranial self-stimulation (ICSS) more ethics committee. The animals were obtained from the than 50 times in the 10-min sessions were dropped from Rappolovo laboratory animal nursery (Leningrad region, the experiment. The remaining animals were run for 30- Russia). At the beginning of the experiment, the rats were min sessions on 5 additional days in order to establish housed individually in standard rodent cages (43×28×15 stable base rates of responding. cm, length-width-height) with wood-chip bedding. Stan- dard rat lab chow (Rappolovo) and filtered tap water were ICSS/food competition freely available throughout the experiment. Artificial light was provided daily from 08.00 am to 08.00 pm; the room After stable self-stimulation rates had been established, temperature and humidity were maintained at 21±21°C the animals were placed on a food-deprivation schedule. and 40–70%, respectively. When food-deprived, the spe- They were trained to reach a feeder with a lot of pellets cial diet-fed animals daily had limiting access to food for (45-mg Noyes food pellet) within 30 min for 5 days. This 4 hours, with free access to water. Accordingly, prior to training was conducted in the box for self-stimulation tes- each test, food deprivation was maintained for 20 hours. ting. The ICSS lever was inactive during this phase of training. Further experiments were carried out using food/ Surgery ICSS competition: the feeder was placed and the ICSS lever was active with the same current intensity. All the The rats were anesthetized with Xyla (Xylazine hy- animals were required to maintain a stable weight level drochloride 20 mg/1 ml, Metaalweg 8, 5804 CG Venray, for 3 consecutive days. Netherlends) and Zoletil 100 (tiletamine hydrochloride/ zolazepam hydrochloride, 250 mg/5 ml, Valdepharm, Test for stimulus-bound behavior France). Bipolar stainless steel electrodes of 0.2 mm thickness (Plastic One), insulated except at the cross sec- After a series of ICSS/food competition, the animals were tion at the tip, were stereotaxically implanted using a mi- switched for access to a pelleted standard rat chow diet cromanipulator (IEM, St. Petersburg, Russia). The elec- and ad libitum water. The satiated experimental animals 84 Lebedev AA et al.: Role of orexin peptide system in emotional overeating induced by brain... were individually placed into the chamber which contain- the three trials) and current intensities that failed to sup- ed a dish of food pellets and some small wooden blocks port responding. The threshold for an ascending series was and allowed to explore the chamber for a 5-min period. defined as the midpoint between stimulation intensities At the end of this familiarization period, brain stimula- that did not support responding and current intensities that tion was turned on for 30 sec at the threshold intensities supported responding for two consecutive blocks of tri- for behavior changes used during ICSS/food competition. als. Four threshold estimates were recorded, and the mean The 30-sec train of stimulation was followed by 60 sec of these values was taken as the final threshold (Koob et of “no-stimulation”. In total, 10 stimulation periods were al. 1993). After the stimulation threshold were found, the presented. Records were made of any eating or gnawing feeder was placed in an experimental box, and the ICSS responses elicited by the stimulation. Although stimu- lever was active. The number of approaches to the fee- lus-bound behaviors are usually controlled by different ding trough, the number of pellets eaten, and the number stimulation parameters, this test was included to ensure of pedal pressings at a fixed threshold current intensity that the parameters actually used did not elicit consum- were determined, and the parameters of eating behavior matory behaviors. and self-stimulation reactions at various current intensities were determined for 10 min. On the following days, drugs Test for ICSS induced emotional overeating were injected to an animals in an individual cage 20 minutes before testing, and then the rat was placed into a cham- The current threshold for pedal pressings was used for ber to study overeating caused by self-stimulation of the ICSS-induced emotional overeating in well-fed rats. ICSS brain; the threshold current in the satiated rats was used. blocks of three trials are presented to the rat for evaluating a threshold at a given stimulation intensity (Koob et al. Histology 1993), and the intensity is altered systematically between blocks of trials by 5 μA steps. The initial stimulus intensity When the tests were completed, the animals were sacri- is set 40 μA above the baseline current threshold for each fied with an overdose of Nembutal and perfused with animal. Each test session typically lasts 30–40 min. The saline followed by Formalin. The brains were removed, current threshold for a descending series was defined as embedded in celloidin, sliced and stained with cresylfast the midpoint between stimulation intensities that suppor- violet (Fig. 1). The electrode placements were determined ted responding (i.e. positive responses to at least two of using the atlas (Konig and Klippel 1963).

Figure 1. The trace after the electrode in the lateral hypothalamus (arrow), located below the conditional border between fornix (f) and the forebrain inner capsule (ci). Note: A line drawn from the fornix down to the brain surface separates the lateral hypothalamus from the medial hypothalamus with an extended complex of the arcuate nucleus (Arc). Stained by cresyl violet. The scale bar is 500 μm. Research Results in Pharmacology 6(1): 81–91 85

Drugs and chemicals

A dopamine D2 antagonist sulpiride (Sigma, USA) at doses of 5 and 20 mg/kg i.p., a selective orexin A receptor (OX1R) antagonist SB-408124 (N-(6,8-Diflu- oro-2)-methyl-4-quinolinyl)-N′-[4-(dimethylamino) phenyl]urea, cat. No. S2694) and a selective antagonist of the orexin B receptors (OX2R) TCS-OX2-29 (2S)-1-(3,4-Dihydro-6,7-dimethoxy-2(1H)-isoquinoli- nyl)-3,3-dimethyl-2-[(4-pyridinylmethyl)amino]-1-bu- tanone hydrochloride (Sigma, USA), diluted in distilled Figure 2. The effect of current intensity on the number of pedal water, 0.5 mg/ml, were used as pharmacological analy- pressings for self-stimulation and pellets eaten in the self-depri- zers. Solutions of orexin antagonists were administered vation test in “well-fed” rats. intranasally at a dose of 20 μl (10 μl in each nostril). As a control, the intranasal administration of a 0.9% sodium chloride solution (isotonic solution) was used. animals ate out of the 20 stimulations at optimal current intensities was 13.2. Self-stimulation rates for the animals Statistical analysis ranged between 62 and 350 presses per 10 minutes and av- eraged 135.4 and 154.2 for the 6 positive and 12 negative The data were expressed as the mean ± standard error of stimulation-bound feeders, respectively. This difference the mean (SEM). Differences among groups were statis- was not statistically significant. The slight trend being in tically tested by oneway analysis of variance (ANOVA) the opposite direction to that seen in the rats supports the followed by the Tukey-Kramer post hoc multiple compa- conclusion that there is no relationship between behavior risons test. The behavior data were presented as the me- in the competition and stimulus-bound eating tests. dian (interquartile range) and analysed using a non-para- In the next series of experiments, 12 non food-de- metric Kruskal-Wallis test followed by Dunn’s post-test prived, negative stimulus-bound eaters were used in ICSS/ for multiple comparisons. All statistical analyses were food competition. Mean lever press rates for ICSS and the conducted using Graph Pad Prism version 6.0 (Graph Pad number of pellets eaten during competition are shown in Software, San Diego, California, USA). Statistical signi- Figure 2. It is apparent from this figure that the animals ficance was assumed at P<0.05. almost totally neglected food, even during the competition phases. At the same time, when assessing ICSS/food competition by threshold currents for the self-stimulation Results reaction, there were observed transitions from the pedal to the feeder, and the animals ate up to 60 pellets in 10 The five animals that did not lever-press for ICSS more than minutes of the experiment. 50 times in the 10-min sessions were dropped from the experiment. In food/ICSS competition test, the food-deprived Drug investigation animals were classed as self-deprivers when using current strength causing maximum lever-pressing rates without dis- After administration of a D2 dopamine antagonist sulpiri- ruptive motor involvement or convulsions. All 18 animals de at a dose of 5 mg/kg i.p., the number of approaches to deprived themselves of food during the food/ICSS compe- the feeder practically did not change, the number of pellets tition: the animals almost totally neglected food during the eaten and the number of pedal pressings did not decrease competition phases. Eating behavior was not observed after significantly. After administration of sulpiride at a dose of an increase in the duration of the experiment for 1.5 hours. 20 mg/kg, the number of approaches to the feeder and the A food reaction with self-stimulation reaction intervals was number of pellets eaten decreased by more than 2 times, and observed only when threshold currents were used. the number of pedal-pressings by more than 3 times (Fig. 3). The rats that had completed an extensive series of com- After intranasal administration of a selective antagonist petition tests over 3 days were later tested for stimula- of orexin A receptor (OX1R) SB-408124 (20 μl, 0.5 μg), tion-bound feeding using the procedure described above. there was a significant decrease in the number of pellets Twelve of the 18 tested animals showed evidence of any eaten – from 54.2 to 32.1, and the number of pedal-press- of the three responses measured during the tests for stimu- ings significantly increased from 97.6 to 138.2 (Figs 3, lus-bound behavior. Neither eating, nor gnawing could be 4). The number of approaches to the feeding trough did elicited by the administration of long durations (30 sec) of not change significantly. After intranasal administration stimulation adjusted to the current level used in the self-dep- of a selective antagonist of the orexin B receptor (OX2R) rivation tests. Subsequently, these 12 rats were taken for TCS OX2 29 (20 μl, 0.5 mg/ml), there was no decrease experiments with overeating induced by self-stimulation. in the number of pellets eaten, and the number of pedal The average threshold intensity capable of evoking eat- pressings did not change (Fig. 5). Neither significantly ing was 26 μA, and the average number of times when the changed the number of approaches to the feeder. 86 Lebedev AA et al.: Role of orexin peptide system in emotional overeating induced by brain...

strated when ICSS was placed in competition with food reward in food-deprived animals (Spies 1965; Routten- berg 1968; Frank et al. 1982; Wise 2005). No effect from the noncontingently administered electrical stimulation of the brain (ESB) on food intake in 12 experimental rats indicated that self-deprivation was not due to drive-reducing, transient satiety, or consummatory response suppression functions of the ESB. At the same time, in 6 out of 18 rats, noncontingently administered ESB caused food intake. The slight trend being in the opposite direction to that seen in the rats supports the conclusion that the- Figure 3. The effect ofsulpiride , SB-408124 and TCS OX2 29 on re is no relationship between behavior in the competition the number of pedal pressings for self-stimulation in the self-dep- and stimulus-bound eating tests, which is consistent with rivation test at a threshold current strength in “well-fed” rats. other research (Frank et al. 1982). Note: * p≤0.05 – compared with the control group of animals. The results of the competition test indicate that stimulation of the medial forebrain bundle in the lateral hypothalamic-ventral tegmental area has a greater reward value than primary food reward, thus producing a general self-deprivation phenomenon. In ICSS/food competition test, 18 of the animals were classed as self-deprivers. The animals deprived themselves of food during the ICSS/ food competition. The current work evaluated for the first time the self-derivation reaction in “well-fed” rats, using threshold current values. When current was reduced to a threshold value for the self-stimulation reaction, ICSS/food competition was transformed into ICSS/food cooperation. In this case, excessive food intake is modelled under conditions of an emotiogenic environment looking like production Figure 4. The effect of sulpiride, SB-408124 and TCS OX2 29 of binge eating (DMS-V-TR) in humans. This study used on the number of approaches to the feeder in the self-depriva- the self-stimulation reaction to the lateral hypothalamus tion test at a threshold current strength in “well-fed” rats. Note: of the threshold current intensity in the satiated rats as an * p≤0.05 – compared with the control group of animals. additional positive emotional component of the environment for food reinforcement. Research on positive emotional eating is largely based on data on its effects in humans. Тhе Positive-Negative Emotional Eating Scale (PNEES) was constructed and tested on 531 women, who also completed Eating Dis- orders Assessment Scale. The results showed that a two-factor model constituting Positive emotional eating (PNEES-P) and Negative emotional eating (PNEES-N) fit the data well (Sultson et al. 2017). The analysis showed that after controlling for the mediating effect of PNEES-N, PNEES-P continued to significantly predict binge eating. The tendency to overeat in response to positive emotions could be integrated into current models of eating disorders, especially when addressing relapse pre- Figure 5. The effect of sulpiride, SB-408124 and TCS OX2 29 vention (Pompili and Laghi 2017; Sultson et al. 2017). In on the number of pellets eaten in the self-deprivation test at a the present experiments, out of 18 animals, only 12 were threshold current strength in “satiated” rats. Note: * p≤0.05 – used in which direct stimulation of the hypothalamus did compared with the control group of animals. not cause a food reaction, but only an exploratory reaction. Thus, the feeding of the satiated rats in ICSS/food cooperation was not observed as a result of direct elec- Discussion trical stimulation of the hypothalamus. The feeder and pedal turn out to be pragmatically different environmental The current studies of ICSS/food competition are consis- signals causing a general activation background of the tent with the results of the studies conducted by a number emotional-motivational arousal. It can be assumed that of authors. The self-deprivation phenomenon was demon- this condition is similar to that which occurs during the Research Results in Pharmacology 6(1): 81–91 87 development of emotional overeating in humans. Multi- their receptors modulate food intake, arousal, stress and ple runs to the feeder during self-stimulation reaction with the use of addictive drugs (Shabanov et al. 2016; Ayra- threshold current in the satiated rats were seen (Fig. 4). petov et al. 2018; Thyssen et al. 2018). OX1R antagonist As currently known, BED refers to non-chemical de- GSK1059865 and OX1/OX2R antagonist SB-649868, pendence and, apparently, is formed like other types of but not the OX2R antagonist JNJ-10397049, have been addictive behavior (Iemolo et al. 2012; Pompili and Laghi shown to reduce binge eating in female rats at doses that 2017). It was shown that an activating effect of post-train- do not cause sleep. However, in this work, the highly ing lateral hypothalamic self-stimulation on aversive and preferred food was served in the regime (Boggiano and appetitive classical conditioning (Ettenberg and White Chandler 2006), with three cycles of food restriction and 1978), as well as post-training ICSS could also be an ef- a foot-shock, i.e. negative emotional influences were fective treatment for improving implicit visual discrim- summarized (Piccoli et al. 2012). ination learning and memory (Garcia-Brito et al. 2017). Appropriate responding to aversive and rewarding Stimulation of the medial forebrain bundle was linked to stimuli is essential for survival, and subcortical neural sys- activation of general arousal systems, due to activity of tems for regulating emotions are evolutionarily conserved dopaminergic, noradrenergic and serotoninergic ascend- (Coulombe and White 1980). The precise mechanisms ant fibers (Wise 2005). Only indirect evidence exists for underlying approach and avoidance behaviors are begin- addictive behavior on natural environmental stimuli. In ning to be uncovered, with efforts focused on the intricate particular, сonditioned taste preferences were observed in connectivity of the lateral hypothalamus (Giardino 2018). rats which drank flavored water, followed by a session of The lateral hypothalamus receives dense synaptic inputs self-stimulation. The control groups neither self-stimulat- from the amygdala, a brain region critical for emotional ed, nor exhibited сonditioned taste preferences (Ettenberg processing. Multiple distinct cell types are co-mingled in and White 1978). It was shown that self-stimulation of the lateral hypothalamus, and posterodorsal lateral hypo- brain zones with higher thresholds and lower pedal press- thalamic neurons expressing the neuropeptide hypocretin es than medial forebrain bundle, lateral hypothalamus (Hcrt) are particularly important for motivated behaviors. and ventral tegmental area activated feeding, contributing HcrtR signaling has been hypothesized to promote both to obesity. In particular, prefrontal cortex self-stimula- negative and positive emotional states, but a nuanced tion induced feeding and weight gain over several weeks theory for how Hcrt-lateral hypothalamic neurons pro- of exposure to stimulation. Overall, it appears that pre- cess oppositely-valenced stimuli remains under devel- frontal cortex self-stimulation modulates energy balance opment. Inputs to the lateral hypothalamus originating (McGregor and Atrens 1991). The use of moderate stimu- from the extended amygdala may govern complex behav- lation by threshold currents in the present work confirms ioral responses to emotional stimuli. Bed nuclei of stria the data obtained. terminalis (BNST) is sufficient to drive emotion-related In the present work, it was shown that after adminis- behaviors (Giardino 2018). Corticoliberin and cholecys- tration of D2 dopamine receptors antagonist sulpiride, tokinin neurons of BNST respectively provide abundant the number of approaches to the feeder, the number of and sparse inputs onto orexin lateral hypothalamic neu- pellets eaten and the number of pedal pressings during rons, display discrete physiological responses to salient self-stimulation decreased dose-dependently. In other stimuli, drive opposite emotionally valenced behaviors, words, both feeding and reinforcing properties of electri- and receive different proportions of inputs from upstream cal stimulation decreased during food self-deprivation. It networks. The data provide an advanced model for how was demonstrated that another D2 antagonist raclopride parallel BNST-lateral hypothalamus pathways promote reduced pedal-pressing responses reinforced with elec- divergent emotional states via connectivity patterns of trical stimulation of the ventral tegmental area and those genetically defined, circuit-specific neuronal subpopula- reinforced with food within 30 min after injection. An in- tions (Giardino 2018). crease in the frequency of brain-stimulation pulses and In recent years, excessive food consumption in an a change in the schedule of food reinforcement, which emotiogenic environment (contributing to the develop- respectively increased the baseline rate of responding, did ment of binge eating) has been associated with changes not alter the effectiveness of raclopride (Nakajima and in the brain, similar to those observed during the forma- Baker 1989). tion of dependence (Gearhardt et al. 2011). It has been At the same time, after administration of orexin A re- shown that in patients with bulimia nervosa and BED in ceptor (OX1R) antagonist SB-408124 (but not after ad- the striatum, the dopamine content can change, similar to ministration of orexin B receptor antagonist TCS OX2 that observed after taking the drug of abuse (Wang et al. 29) only a decrease in the number of eaten pellets was 2011). The release of dopamine and its receptor binding observed, whereas the number of pedal pressings was not in animals with binge eating also resembles a response significantly changed or mainly increased (see Figs 3, 5). to drug administration (Avena et al. 2008). Orexigenic Thus, the current studies have shown the selectivity neuropeptides, which are synthesized in the lateral hypo- of OX1R (SB-408124) in relation to food reactions dur- thalamus, can modulate the activity of neurons in the ven- ing self-deprivation in the well-fed rats. This is largely tral tegmental region and ventral striatum. In particular, consistent with the literature. It is known that orexins and orexin-containing neurons send projections from the lat- 88 Lebedev AA et al.: Role of orexin peptide system in emotional overeating induced by brain... eral hypothalamus to the ventral tegmental region, where in-A) was first shown to be delivered from the nose to the OX1R play a key role in regulating the release of dopa- brain and proposed as a new strategy to treat narcolepsy mine by the mesolimbic system of the brain and are thus (Scranton et al. 2011). A study in non-human primates involved in the effects of various drugs and mechanisms demonstrated that intranasal hypocretin-1 reduces cogni- of dependence (Cason et al. 2010). In addition, episodes tive performance deficits resulting from sleep deprivation of binge eating can be controlled through the influence on (Deadwyler et al. 2017). However, intranasal hypocretin-1 the processes of reward and reinforcement of highly pre- administration is just starting to be explored in humans. ferred food (goodies). In this regard, neurons of the lateral This line of research has focused on narcolepsy, a disorder hypothalamus containing orexin are a key link for food characterized by impaired or absent CNS hypocretin sign- reward (Koob 2008) and are activated by reward signals, aling and has shown promising results (Baier et al. 2008). such as food or drugs. Moreover, stimulation of neurons Animal studies have demonstrated that intranasal admin- of the lateral hypothalamus containing orexin leads to the istration of hypocretin-1 leads to significantly greater tis- reinstatement of drug use in rats (Stuber and Wise 2016). sue-to-blood concentration ratios in all brain regions over In terms of practical orientation, the intranasal introduc- 2 h as compared to intravenous administration. Intranasal tion that was used in the work has obvious prospects. One administration also increased drug targeting to the brain of the most challenging problems facing modern medicine and spinal cord 5- to 8-fold (Dhuria et al. 2009). is how to deliver a given drug to a specific target at the exclusion of other regions. For example, a variety of compounds have beneficial effects within the central nervous Conclusion system (CNS), but unwanted side effects in the periphery. For such compounds, traditional oral or intravenous drug Thus, it is necessary to conclude the selectivity of the or- delivery fails to provide benefit without cost. However, exin A antagonist SB-408124 with respect to binge ea- intranasal delivery is emerging as a noninvasive option ting compared with the action of orexin B antagonist TCS for delivering drugs to the CNS with minimal peripheral OX2 29 and dopamine D2 receptors antagonist sulpiride, exposure. Additionally, this method facilitates the delivery i.e. blockade of only one dopaminergic link in the regu- of large and/or charged therapeutics, which fail to effec- lation of brain supporting mechanisms (Lebedev et al. tively cross the blood-brain barrier. Thus, for a variety of 2013). Food self-deprivation caused by electrical self-sti- growth factors, hormones, neuropeptides and therapeutics mulation of the lateral hypothalamus in well-fed rats is a including insulin, oxytocin, orexin, and even stem cells, promising and adequate model for studying the mechanis- intranasal delivery is emerging as an efficient method of ms of binge eating in an experiment. administration, and represents a promising therapeutic strategy for the treatment of diseases with CNS involvement, such as obesity, Alzheimer’s disease, Parkinson’s Conflict of interests disease, Huntington’s disease, depression, anxiety, autism spectrum disorders, seizures, drug addiction, eating disor- The authors declare no competing financial interests and ders, and stroke. In 2004, intranasal hypocretin-1 (orex- no conflict of interests.

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Author contributors

Andrei A. Lebedev, Dr. Biol. Sci. (Pharmacology), Professor, Head, Laboratory of General Pharmacology, Dept of Neuropharmacology, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-0297-0425 (main idea, organization of the experimental study).

Yulia N. Bessolova, post-graduate student, Dept of Neuropharmacology, e-mail: [email protected], ORCID ID http://orcid.org/0000-0001-8605-6758 (experimental work, obtaining the main results, statistical analysis).

Nikolai S. Efimov, post-graduate student, Dept of Neuropharmacology, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-1068-4701 (experimental work, obtaining the main results, statistical analysis).

Eugeny R. Bychkov, PhD (Pathophysiology), Head, Laboratory of Pharmacology of the Central Nervous System, Dept of Neuropharmacology, e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-1068-4701 (organization of the experimental study).

Andrei V. Droblenkov, Dr. Med. Sci. (Anatomy and Histology), Professor, Leading Researcher, Dept of Neurop- harmacology, e-mail: [email protected], ORCID ID http://orcid.org/0000-0001-5155-1484 (morphological research).

Petr D. Shabanov, Dr. Med. Sci. (Pharmacology), Professor and Head, Dept of Neuropharmacology; Head, Dept of Pharmacology (Kirov Military Medical Academy), e-mail: [email protected], ORCID ID http://orcid. org/0000-0003-1464-1127 (general supervision of the experiment, writing the article, proofreading). Research Results in Pharmacology 6(1): 93–99 UDC: 615.035+616.24-002 DOI 10.3897/rrpharmacology.6.49977

Research Article

Pharmacotherapy and other aspects of senior medical students’ knowledge in community-acquired pneumonia: the final results of the KNOCAP II project

Roman A. Bontsevich1, Anna A. Gavrilova1, Anna V. Adonina1, Yana R. Vovk1, Natalya Y. Goncharova2, Galina A. Batisheva2, Olga V. Cherenkova2, Olena V. Myronenko3, Elena V. Luchinina4, Valeriya O. Barysheva5, Galina G. Ketova5, Elena N. Bochanova6, Ulankul M. Tilekeeva7, Nurbek D. Dauletbekov7

1 Outpatient Clinic “Garmoniya Zdorov’ya” LLC “MAKSBelmed”, 50 M. Ordynka St., Moscow 115184, Russia 2 Voronezh State Medical University named after N.N. Burdenko, 10 Studencheskaya St., Voronezh 394036, Russia 3 Dnipropetrovsk Medical Academy (State Establishment), 9 Vernandsky St., Dnipro 49044, Ukraine 4 Saratov State Medical University named after V.I. Razumovsky, 112 Bolshaya Kazachia St., Saratov 410012, Russia 5 South Ural State Medical University, 64 Vorovsy St., Chelyabinsk, 454092, Russia 6 Krasnoyarsk State Medical University named after Professor V.F. Voino-Yasenetskiy, 1 Partizana Zheleznyaka St., Krasnoyarsk 660022, Russia 7 Kyrgyz State Medical Academy, 92 Akhunbaev St., Bishkek 720020, Kyrgyz Republic

Corresponding author: Roman А. Bontsevich ([email protected])

Academic editor: Mikhail Korokin ♦ Received 8 January 2020 ♦ Accepted 24 February 2020 ♦ Published 31 March 2020

Citation: Bontsevich RA, Gavrilova AA, Adonina AV, Vovk YR, Goncharova NY, Batisheva GA, Cherenkova OV, Myronenko OV, Luchinina EV, Barysheva VO, Ketova GG, Bochanova EN, Tilekeeva UM, Dauletbekov ND (2020) Pharmacotherapy and other aspects of senior medical students’ knowledge in community-acquired pneumonia: the final results of the KNOCAP II project. Research Results in Pharmacology 6(1): 93–99. https://doi.org/10.3897/rrpharmacology.6.49977

Abstract Introduction: Community-acquired pneumonia (CAP) remains an extensive medical and social problem. It is the most common human disease and one of the leading causes of death from infectious diseases. Increasing the level of senior medical students’ knowledge of the diagnosis, treatment and prevention of CAP will improve the level of medical care to the population. The aim of the study: to determine the level of senior medical students’ basic knowledge of CAP prevention, diagnosis and treatment with the help of a pharmacoepidemiological study. Materials and methods: The multicenter study “KNOCAP” (the full name of the project “The Assessment of Phy- sicians’ and Students’ Knowledge of Community-acquired Pneumonia Basics”) presents the results of an anonymous prospective survey aimed at assessing the knowledge and preferences of senior medical students in terms of the CAP pharmacotherapy. In the second stage of the project (2017–2019). The results from 394 senior students from 8 centers of Russia, Ukraine and Kyrgyzstan were received and analyzed. An original questionnaire was developed for this study on the basis of the current clinical guidelines. Conclusion: The final results of a prospective survey revealed an insufficient level of students’ basic knowledge of diagnosis, treatment and prevention of CAP. The study revealed a statistically significant heterogeneity of knowledge levels in different centers, which indicates the need for the introduction of unified and in-depth training programs in this area.

Copyright Bontsevich RA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 94 Bontsevich RA et al.: Pharmacotherapy and other aspects of senior medical students’ knowledge...

Keywords survey, community-acquired pneumonia, clinical recommendations, knowledge level, pharmacotherapy, pharmacoep- idemiology.

Introduction each respondent, the average scores for individual questions, and the average scores for the entire questionnaire Community-acquired pneumonia (CAP) is an acute di- were evaluated. The patterns of answers to individual sease that occurs under community-acquired conditions questions were also analyzed; statistically non-systemic and is accompanied by the symptoms of a lower respi- question skippings were allowed. No answer to open-end ratory tract infection (fever, cough, sputum production, questions rendered 0 points. The main questions of the chest pain, labored breathing) and radiological signs of questionnaire are presented below (without answers): focal-infiltrative changes in the lungs in the absence of obvious diagnostic alternative (Chuchalin et al. 2010). 1. Indicate the main pathogen(s) of CAP. Currently, the mortality rate from the above pathology 2. Choose the most effective way to prevent CAP. remains high, despite a large number of research articles 3. Indicate the main diagnostic sign of CAP when ex- on the treatment of this disease and success in this area, amining a patient. which is associated with the developed pharmacotherapy 4. Determine the diagnostic minimum of a mild form schemes for CAP. Due to the prevalence of this disease of CAP. throughout the world and the social and economic im- 5. Choose criteria without which the diagnosis of CAP portance of CAP, the pharmacoepidemiological studies cannot be confirmed among senior students are especially relevant in order to 6. Indicate the rational time for a repeated X-ray ex- further optimize their knowledge and, as a result, improve amination with positive dynamics in the treatment the implementation of clinical guidelines. of CAP. 7. Indicate possible reasons for delaying an antimicrobial therapy (AMT). Materials and methods 8. Choose a key criterion for terminating AMT. 9. Indicate the correct definition of the “sequential The multicenter KNOCAP study (the full name of the therapy” in the management of patients with CAP. project is ”The Assessment of Physician‘ and Students’ 10. Indicate typical mistakes in the initial AMT for a Knowledge of Community-acquired Pneumonia Basics”) non-severe CAP. presents the results of an anonymous prospective survey 11. Write a drug/treatment regimen for a mild form of on the assessment of senior medical students’ knowledge CAP without risk factors and/or concomitant dis- in matters of the diagnosis, treatment, prevention of CAP eases, indicating the dosage, frequency and mode of and also their accordance with the modern clinical guide- administration. lines (Bontsevich et al. 2015, 2017; Russian Respiratory 12. Write a drug/treatment regimen for a mild form of Society 2018). According to the outcomes of the second CAP with risk factors and/or concomitant diseases, stage of this study (2017–2019), the results of a survey indicating the dosage, frequency and mode of ad- of 394 senior medical students from 8 centers of Russia, ministration. Ukraine and Kyrgyzstan were obtained and analyzed. An original questionnaire was developed for this All the information of the questionnaire was entered into study. It consisted of multiple choice questions and open- an electronic database and processed using the application end questions. This questionnaire is based on the cur- programs of Microsoft Excel and Statistica 10. Statistical rent clinical guidelines for managing patients with CAP processing showed that the analyzed distribution of data (Chuchalin et al. 2010). The following point system was from the sample of the senior medical students was expect- used to calculate the results of the survey: the respond- edly normal: Kolmogorov-Smirnov test d=0.04982, p>0.20; ent received 0 point for an incorrect answer; depending Lilliefors test p<0.05. The statistical significance of the dif- on the completeness of the answer, for an incomplete or ferences when comparing these samples were recorded at a partially correct answer – from 0.25 to 0.75 points; for the bilateral level of p<0.05 based on the analysis of arbitrary correct answer – 1 point. Therefore, with all the correct contingency tables, using the Pearson’s chi-square (×2) test. answers, the maximum average score was 1.0. The av- The centers from the Ukraine (Dnipro and Kiev) were erage completeness rate for the correct, partially correct combined to correctly calculate the statistical significance. and wrong answers was defined as the average response This method of knowledge evaluation was specially completeness (ARC) rate, which is an equivalent for the developed for the KNOCAP project and cannot fully re- average level of correct answers. The average scores of flect the general level of education quality at universities. Research Results in Pharmacology 6(1): 93–99 95

Results and discussion

The survey involved 394 senior medical students (23.6% from Belgorod, 7.6% from Bishkek, 29.0% from Voronezh, 10.5% from the Dnipro and Kiev (the united center of the Ukraine), 5.3% – from Krasnoyarsk, 6.8% – from Saratov and 1.2% – from Chelyabinsk). The average completeness rate for all the questionnaires was 43.7%, from 33.1% to 51.9% for different centers (p<0.05). The minimum level of correct answers was received to questions No.6 (time for a Figure 1. The minimum, maximum and average levels of re- repeated X-ray examination) – 24.4% (from 10.0 to 40.0% sponse completeness of knowledge in CAP among the centers. among centers, p<0.01), No.10 (typical mistakes in the ini- Note: ARC – average response completeness rate. tial AMT) – less than 1% (from 19.0 to 40.0%, p<0.01), No.12 (treatment regimen for patients with risk factors and/ or concomitant diseases) – 6% (from 0 to 25.0%, p<0.01). Today the most effective way to prevent CAP is pneu- The maximum average level of correct answers was recei- mococcal and influenza vaccines (Chuchalin et al. 2010; ved to questions No.2 (the most effective way to prevent McLaughlin et al. 2018; Russian Respiratory Society CAP) – 63.7% (from 31.0 to 90.0%, p<0.01), No.7 (the re- 2018). The second question of the questionnaire suggested asons for delaying an AMT) – 63.2% (from 52.0 to 79.0%, choosing the highly efficient methods of CAP prevention p> 0.05), and No.9 (“sequential therapy” of CAP) – 61.6% from the proposed options: cold water treatment, homeo- (from 48.0 to 90.0%, p<0.01). The major results are pre- pathic and immunomodulatory therapy, vaccination with sented in Fig. 1. A detailed analysis with comments on each pneumococcal and influenza vaccines. The majority of the question of the questionnaire is presented below. respondents (63.7%) chose the correct answer, indicating At the beginning of the questionnaire, the students had vaccinal prevention, 16.6% answered partially correctly, to choose the preferred regulatory documents when man- and 19.7% of the respondents did not complete the task. aging patients with CAP. The following answers were of- ARC was from 31.0 to 90.0% at different centers (p<0.01). fered: “order”, “standard”, “guidelines”, “treatment based In the third question, the students had to indicate the on own experience”, “neither agree nor disagree” and main diagnostic sign of CAP when examining a patient. “other”, where the respondents could provide their own Classic objective symptoms are: dullness of percus- answer option. The majority of the respondents preferred sion sound over the affected areas of the lung, bronchi- to use a standard as a regulatory document (25.0%), 19.5% al breathing, rhonchi crackles, or crepitation, increased choose 2 or more regulatory documents for the treatment bronchophony and vocal trembling. All of these clinical of CAP, the order was chosen by 10.5% of the respond- manifestations are the main components of the syndrome ents, 4.6% choose the category “other ”, 27.0% preferred of pulmonary consolidation. One-third (32.1%) of the guidelines, and 13.4% of senior students found it difficult students chose the correct answer, 14.8% gave a partial- to answer. The answer “treatment based on own experi- ly correct answer, and 53.2% answered incorrectly. ARC ence” was not chosen by any of the respondents, which among the centers was from 15.0 to 77.0%, p<0.01. may be due to the lack of experience in practical health- In the fourth question of the questionnaire, the students care among the respondents. It is important to note that were asked to determine the diagnostic minimum of a there is no correct answer to this question; therefore it was mild form of CAP. The following answer options were possible to assess the level of senior students’ awareness suggested: of the use of the regulatory framework only indirectly. In the first question, potential pathogens of CAP were • Single-plane radiography; presented – staphylococcus (including S. aureus and oth- • Biplane radiography; ers); streptococcus – S. рyogenes и S. haemoliticus; S. • Biochemical blood assay; pneumoniae; Enterobacteriaceae; Haemophilus influen- • Complete blood count; za; viruses, fungi; and atypical microorganisms. The stu- • Clinical analysis of sputum; dents had to choose one or more pathogens, which, in their • Microbiological analysis of sputum. opinion, are the most common pathogens of CAP or write their own version. According to the current clinical guide- The correct answer (biplane radiography, complete lines (Russian Respiratory Society 2018; Bontsevich et al. blood count) was given by 10.2% of the respondents, par- 2019b), the most common causative agent of CAP is S. tially correct answers – by 73.9%, and 15.9% of the stu- pneumoniae, this answer was the only true one. The cor- dents answered incorrectly. ARC among the centers was rect answer was given by 33.5% of the respondents, 47.7% from 28.0 to 64.0% (p<0.01). answered partially correctly, indicating several pathogens It must be remembered that the diagnosis of CAP is along with S. pneumonia, 18.8% answered incorrectly. determined if the patient has an X-ray confirmation of fo- ARC among the centers was from 48.0 to 68.0%, p<0.01. cal lung infiltration and at least two clinical signs from 96 Bontsevich RA et al.: Pharmacotherapy and other aspects of senior medical students’ knowledge... among the following: acute fever at the onset of the dis- cefazolin; the use of ampicillin per os; the use of respira- ease (tо > 38.0 °С), wet cough, physical symptoms (rhon- tory fluoroquinolones in patients without risk factors and chi crackles or crepitation, bronchial breathing, dullness a “not sure” option. According to the clinical guidelines of percussion sound), leukocytosis >10*109/L and/or shift (Chuchalin et al. 2010), all the options referred to an er- to the stab neutrophils (>10.0%) (Chuchalin et al. 2010; roneous strategy in managing non-severe CAP patients: Russian Respiratory Society 2018). the use of ampicillin per os is accompanied by low bi- In the next question, the respondents had to choose the oavailability (40.0%) in comparison with amoxicillin criteria without which the diagnosis of CAP could not be (75–93.0%); cefazolin has a low activity against pneu- confirmed. Fewer than half of the respondents (42.9%) mococci, as well as the absence of clinically significant chose the correct answer, indicating the absence of X-ray activity against H. influenzae; ciprofloxacin is low-active confirmation of focal lung infiltration as the main reason against S. pneumoniae and M. pneumonia; it is inappro- of problems with CAP diagnosing, 21.1% of the respond- priate to prescribe respiratory fluoroquinolones as drugs ents answered partially correct, 36.0% of the students to patients without risk factors; ampicillin/oxacillin (Am- gave the wrong answer. ARC was from 36.0 to 70.0% at piox) should not be used in medical practice because of the different centers, p<0.01. an irrational combination of antibiotics (Çilli et al. 2018; The sixth question of the questionnaire asked the re- Cillóniz et al. 2018). The majority of students gave an spondents to indicate the rational time for repeated X-ray incomplete correct answer – 90.5%, fewer than 1.0% examination with positive dynamics in the treatment of of the respondents were able to give the correct answer, CAP. Only 24.4% of the students answered the question and 9.3% of the respondents gave a wrong answer. ARC correctly, choosing “at least 14 days later”, 1.0% answered among the centers was from 19.0 to 40.0%, p<0.01. partially correctly, and 74.6% did not complete the task. The next two questions required a “written” answer ARC in the centers was from 10.0 to 40.0% (p<0.01). from each student. In the first question, it was necessary In the seventh question, the students were asked to to indicate the optimal starting therapy for a non-severe choose the possible reasons for delaying an antimicrobial CAP in patients without risk factors and/or concomitant therapy (AMT). The majority (62.3%) of the respondents diseases (diabetes, chronic renal insufficiency, congestive coped with this task, indicating that there were no rea- heart failure, COPD, chronic alcoholism, cachexy, drug sons for the delay of AMT with a confirmed diagnosis of addiction, liver cirrhosis) and/or in patients who had been CAP, 35.4% gave an incorrect answer, and 2.3% gave an taking systemic antimicrobial drugs (AMD) in the pre- incomplete answer. ARC in the different centers was from vious 3 months for more than 2 days. In this case, ac- 52.0 to 79.0%, p> 0.05. cording to the clinical guidelines, the drugs of choice are The leading indication for terminating AMT for a amoxicillin or macrolides. Despite the fact that in vitro non-severe CAP is a stable normalization of body tem- aminopenicillins do not cover the full range of potential perature over a period of 48–72 hours, combined with pathogens, clinical trials did not reveal differences in both a positive clinical picture and the absence of signs the effectiveness of these antibiotics in comparison with of clinical instability (Pertseva and Sanina 2013; Rachi- macrolides and respiratory fluoroquinolones McLaugh( - na et al. 2016; Sinopalnikov 2018,2019). This question lin et al. 2018; Wicha et al. 2019). Macrolides should be was answered correctly by 33.2% of students, 12.0% of preferred if it is impossible to use aminopenicillins (idi- the respondents answered partially correctly, and 54.7% osyncrasy, allergy), and also with a suspected ”atypical” answered incorrectly. ARC among the centers was from etiology of the disease (S. pneumoniae and M. рneumo- 11.0 to 57.0%, p<0.01. niae) (Breitling et al. 2018; Lee et al. 2018). It is worth The ninth question of the questionnaire concerned noting that parenteral forms of drugs during out-patient the ”sequential therapy” for CAP. This type of AMT in- treatment do not have proven advantages over oral ones, volves the consistent administration of two dosage forms at the same time, they pose a threat to the development of (for parenteral and oral administration) of the same anti- post-injection complications in the form of abscesses and bacterial drug. The best option for this type of AMT is a require additional costs for administration (Chuchalin et two-stage administration of antimicrobial drugs: first, the al. 2010; Bontsevich et al. 2019a). Only 9.1% of the sen- use of parenteral form and then the transition to the oral ior students answered this question correctly, 27.2% of administration right after the stabilization of the patient’s the students answered partially correctly, and 63.7% gave condition, normalization of the body temperature and the the wrong answer. ARC among the centers was from 4.0 improvement of the CAP clinical picture (Pertseva and to 32.0%, p<0.01. Avramenko 2017; Spichak 2019). The correct answer was The next question required to indicate the optimal given by 61.6% of the respondents, 3.3% answered par- starting therapy for a non-severe CAP in patients with risk tially correctly, and 35.1% did not cope with the question. factors and/or concomitant diseases (diabetes, chronic re- ARC among the centers was from 48.0 to 90.0%, p<0.01. nal insufficiency, congestive heart failure, COPD, chronic In the next question, the students had to indicate typi- alcoholism, cachexy, drug addiction, liver cirrhosis) and/ cal mistakes in the initial AMT for a non-severe CAP. The or in patients who had been taking systemic antimicrobial following answers were proposed: the use of ampicillin/ drugs (AMD) in the previous 3 months for more than 2 oxacillin (Ampiox); the use of ciprofloxacin; the use of days. It is recommended to prescribe tableted forms of Research Results in Pharmacology 6(1): 93–99 97

• acquiring basic scientific, theoretical and practical knowledge to solve theoretical and practical problems; • mastering the technology of pharmacoepidemiological research; • improving professional skills throughout professional career; • learning to work independently; • having skills associated with the use of modern information on clinical guidelines and treatment standards for community-acquired pneumonia; • using a computer; • having research skills.

Figure 2. Variability of ARC for the studied centers. Conclusions

AMD to such patients, but the therapy strategy in these A survey of the senior medical students within the frame- patients is different, because the probability of the etio- work of the KNOCAP multicenter study showed that the- logical role of gram-negative flora increases (Ewig et al. re are significant gaps in knowledge of the correct ma- 2009; Sligl et al. 2014). Combined AMD is recommended nagement of patients with CAP. The greatest difficulties as a starting therapy: amoxicillin + clavulanic acid. If there for students were caused by open-end questions, which is a risk factor of “atypical” microflora, a combination of involved determining the starting AMT in the treatment of β-lactam and macrolide may be prescribed. An alternative a non-severe CAP in patients with or without risk factors to the combined therapy is the administration of respira- and concomitant diseases, the question of determining tory fluoroquinolones (levofloxacin, moxifloxacin, gemi- the “typical mistakes” in the initial AMT for a non-severe floxacin) or oral cephalosporins (cefditoren) (Chuchalin CAP, as well as the question that suggested setting the ra- et al. 2010; Russian Respiratory Society 2018). tional time for a repeated X-ray examination with positive Fewer than half of the students answered partially cor- dynamics in the treatment of CAP. rectly – 21.6%, 72.4% gave the wrong answer and only Therefore there is a need for educational activities 6.0% of the respondents gave a correct answer. ARC among senior students: additional seminars, lectures, de- among the centers was from 0% to 25.0% (p<0.01). velopment of educational materials for self-training in clin- In conclusion of the study, the variability of the ARC ical pharmacology. And the therapeutic disciplines should range was analyzed among the centers which participated comply with current regulatory documents developed for in the study. The maximum value of ARC was 51.9%, and the diagnosis and treatment of various nosological forms, it was registered in center No. 6, whereas the minimum and an increasing number of academic hours will help to value of ARC – 33.1% was registered in center No. 8. solve the problem of insufficient awareness of future doc- These results are presented in Fig. 2. tors of basic issues of managing patients with CAP. Summarizing this study, in order to optimize pharmacotherapy and other aspects of senior students’ knowledge in community-acquired pneumonia, the following algo- Conflict of interest rithms for mastering the material to form the professional competence of future doctors should be considered: The authors have no conflict of interest to declare.

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Author contributions

Roman A. Bontsevich, MD, PhD, Associated Professor, pulmonologist, clinical pharmacologist and therapist; e-mail: [email protected], ORCID ID http://orcid.org/0000-0002-9328-3905. Being the author of the idea and the project coordinator, he analyzed the general results and provided the final conclusions, finalizing the article.

Anna A. Gavrilova, MD, therapist; e-mail: [email protected], ORCID ID http://orcid.org/0000-0002-4335- 5165. The author conducted an analysis of the results, suggested the conclusions, was engaged in the discussion and editing the article.

Anna V. Adonina, senior student, intern; e-mail: [email protected], ORCID ID http://orcid.org/0000-0002- 9735-7270. The author was engaged in the discussion and editing the article.

Yana R. Vovk, senior student, intern; e-mail: [email protected], ORCID ID http://orcid.org/0000-0002- 7741-9745. The author was responsible for translating the final version of the article into English.

Natalya Y. Goncharova, MD, PhD, Associated Professor; e-mail: [email protected], ORCID ID http://orcid. org/0000-0002-4113-5206. The author was responsible for conducting the regional study in Voronezh, Russia. Research Results in Pharmacology 6(1): 93–99 99

Galina A. Batisheva, MD, PhD, Professor; e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-4771- 7466. The author was responsible for conducting the regional study in Voronezh, Russia.

Olga V. Cherenkova, MD, PhD, Associated Professor; e-mail cherenkova [email protected], ORCID ID http://orcid.org/0000-0001-5320-2720. The author was responsible for conducting the regional study in Voronezh, Russia.

Olena V. Myronenko, MD, PhD, Associated Professor: e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-3514-3338. The author was responsible for conducting a regional study in Dnepr (Dni- pro), Ukraine.

Elena V. Luchinina, MD, PhD, Associated Professor; e-mail: [email protected], ORCID ID http://orcid. org/0000-0002-3120-8491. The author was responsible for conducting the regional study in Saratov, Russia.

Valeriya O. Barysheva, MD, PhD, Assistant Professor; e-mail: [email protected], ORCID ID http://orcid. org/0000-0001-7762-7854. The author was conducting the regional study in Chelyabinsk, Russia.

Galina G. Ketova, MD, PhD, Professor; e-mail: [email protected], ORCID ID http://orcid.org/0000-0002- 4678-6841. The author was responsible for conducting the regional study in Chelyabinsk, Russia.

Elena N. Bochanova, MD, PhD Doctor of Medical Sciences, Associated Professor; e-mail: [email protected], ORCID ID http://orcid.org/0000-0003-4371-2342. The author was responsible for conducting the regional study in Krasnoyarsk, Russia.

Ulankul M. Tilekeeva, MD, Professor Head of the Department of Basic and Clinical Pharmacology; e-mail: ul- [email protected], ORCID ID http://orcid.org/0000-0002-8407-8248. The author was responsible for conducting the regional study in Bishkek, Kyrgyz Republic.

Nurbek D. Dauletbekov, Senior Lecturer of the Department of Basic and Clinical Pharmacology; e-mail: nur- [email protected], ORCID ID http://orcid.org/0000-0002-3457-5537. The author was responsible for conducting the regional study in Bishkek, Kyrgyz Republic.

﻿A Comparative Evaluation of the Efficacy of Dimethylaminoethanol

A Comparative Evaluation of the Efficacy of Dimethylaminoethanol