RU2514632C1 - Antioxidant, stress- and neuroprotective pharmacological agent potassium comenate - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and medicine and concerns using comenic acid potassium salt as a preventive and therapeutic antioxidant, stress- and neuroprotective agent in the amount of 2 to 8 mg per 1 kg of body weight daily on the empty stomach for 3 days.

EFFECT: agent possess high efficacy.

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Description

The invention relates to experimental medicine and pharmacology, in particular to means for the prevention and treatment of neurodegenerative diseases caused by oxidative damage to the brain.

Many CNS pathologies in which the destructive processes of brain neurons are observed are associated with the activation of lipid peroxidation (LP). Oxidative stress leading to overproduction of free radicals (SR) and membrane destruction associated with activation of phospholipase hydrolysis plays a major role in the pathogenetic mechanisms of cerebral ischemia. Cerebral ischemia initiates a cascade of biochemical reactions that underlie tissue damage. The main mechanisms of neuronal damage include depletion of energy resources under conditions of acidosis of brain tissue, disruption of ionic homeostasis, depolarization of cell membranes, accumulation of exciting amino acids, and hyperproduction of reactive oxygen species (ROS). Chronic oxidative stress damages all classes of biomolecules, including lipids, proteins, and nucleic acids. An excess of free radicals that adversely affects the DNA of brain neurons can cause a change in the genetic code and cell death [Popova MS, Stepanichev M.Yu. Induction of the cell cycle, amyloid beta and free radicals in the mechanism of development of neurodegenerative processes in the brain // Neurochemistry. 2008.V.25. Number 3. S.170-178; Zozulya Yu.A., Baraboy V.A., Sutkova D.A. Free radical oxidation and antioxidant protection in brain pathology. M .: Knowledge. 2000. S.226-221; Smirnova I.N., Fedorova T.N., Tanashyan M.M., Suslina Z.A. Clinical efficacy and antioxidant activity of Mexidol in chronic cerebrovascular diseases // Nervous diseases. 2006. No1. S.33-36; Fedorova T.N. Oxidative stress and brain protection against ischemic damage: Diss. Dr. biol. Sciences: 03.00.04: Moscow. 2004.298 s. RSL OD. 71: 05-3 / 6; Calabrese V., Bates T.E., Stella A.M. NO synthase and NO-dependet signal pathways in brain aging and neuredegenerative disorders: the role of oxidant / antioxidant balance // Neurochem. 2000. V.25. P.1315-1341].

Another important factor causing the death of neurons is the prolonged stimulation of CNS neurons by the neurotransmitter glutamate. Being an exciting neurotransmitter, glutamate is found in most brain neurons. In conditions of ischemia, glutamate is released from the ends of ischemic neurons into the intercellular space. Under normal conditions, neurons and glia cells absorb excess glutamate from the intercellular space. However, ischemic cells lack the necessary energy for this. As a result of the accumulation of a large amount of glutamate, saturation of the receptors of neighboring neurons occurs and the damage zone expands. Overexcitation of glutamate receptors, accompanying hypoxia, ischemia, has a damaging effect on neurons, leads to impaired calcium homeostasis. Delayed calcium dysregulation is one of the key signals leading to increased generation of ROS and lipid peroxidation after the toxic effects of glutamate, a decrease in the level of endogenous antioxidants, damage and death of brain cells in ischemic conditions [Suslina ZA., Maksimova M.Yu. The concept of neuroprotection: new opportunities for urgent therapy of ischemic stroke. Symposium of the Research Institute of Neurology of the Russian Academy of Medical Sciences "Treatment of stroke: the state of the problem" // Nervous diseases. 2004. No3. C.4-7; Vergun O., Keelan J., Khodorov BI, Duchen MR Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurons // J. Physiol. 1999. Vol. 519. P.451-466; Pinelis V.G., Bykova L.P., Bogachev A.P., Isaev N.K., Viktorov I.V., Khodorov B.I. The toxic effect of glutamate on cultured cerebellar granular cells reduces the intracellular ATP content. The role of Ca ^{2+ ions} // Bull. an experiment. biol. and honey. 1997.V. 123. No. 2. S.162-164; Nishizawa Y. Glutamate release and neuronal damage in ischemia // Life Sciences. 2001. Vol. 69. P.369-381].

The antioxidant defense of the body during oxidative stress is not able to completely neutralize the resulting excess of ROS. Under these conditions, the feasibility of using antioxidant drugs that can protect brain tissue from damage [Nonaka Sh., Katsube N. and Chuang D.-M. Lithium Protects Rat Cerebellar Granule Cells against Apoptosis Induced by Anticonvulsants, Phenytoin and Carbamazepine // JPET. 1998. V.286. P.539-547].

Currently, for the prevention and treatment of degenerative diseases with ischemic damage, antioxidants are used, which by their origin are divided into two main groups: natural and synthetic.

Natural antioxidants: enzymes, proteins, low molecular weight compounds (vitamins E and C, carotenoids, ubiquinone, etc.).

The group of synthetic antioxidants is represented by synthetic analogues of vitamin E, aromatic phenols and polyphenols (ionol, probucol), emoxipine and mexidol, some derivatives of barbituric acid and phenothiazine, iron and zinc preparations, organic acids and their derivatives, some amino acids and amino steroid derivatives [Karneev A. N., Solovyova E.Yu. et al. Use of alpha-lipoic acid preparations as a neuroprotective therapy of chronic brain ischemia. Handbook of outpatient physician. http://old.consilium-medicum.com/media/refer/06_08/76.shtml]. In this case, intoxication with certain drugs, including barbiturates, can cause reversible cognitive impairment - dysmetabolic dementia [Chukhlovina M.L. Dementia Diagnosis and treatment. St. Petersburg: 2010. S.60].

The use of various potassium compounds as drugs is known. These are mainly organic potassium salts (potassium orotate, potassium asparaginate, potassium acetate) and inorganic potassium salts (potassium chloride) [Cru J. Biochemistry, Medical and biological aspects, trans. with french M., 1979. p. 46; Laboratory research methods in the clinic, ed. V.V. Menshikov. M., 1987. S. 261, 263; http://dic.academic.ru/]

Potassium - the main cation of intracellular fluid, is important for a variety of biological processes. It is important for nervous and muscle activity, since it forms an electrolytic environment, it is necessary to maintain osmotic pressure and acid-base balance, for the normal functioning of nerve endings and muscle cells in conducting nerve impulses in the nervous and muscle tissue. The main function of potassium is the formation of a transmembrane potential and the propagation of potential changes along the cell membrane by exchange with sodium ions along a concentration gradient. Potassium is involved in maintaining the electrical activity of the brain, the functioning of nerve tissue, the contraction of skeletal and cardiac muscles, slows down the rhythm of heart contractions and, acting similarly to the vagus nerve, is involved in the regulation of heart activity, and is involved in the normalization of blood pressure. It is known that lowering the sodium / potassium ratio by adding potassium to food reduces pressure in hypertensive patients. Potassium supplements can reduce the risk of diseases of the vessels of the brain, as well as the kidneys and heart [Skalny A.V., Rudakov I.A. Bioelements in medicine. M .: 2004.272 s. http://www.adlshop.ru/node/121].

The use of potassium chloride (Kalii chloridum) is known. Potassium is involved in the process of conducting nerve impulses and transferring them to innervated organs. The introduction of potassium into the body is accompanied by an increase in the content of acetylcholine and excitation of the sympathetic department of the nervous system. With intravenous administration, an increase in adrenaline excretion of adrenaline is noted.

Contraindications: with careless intravenous administration, the drug can cause intoxication (paresthesia). In rare cases, a paradoxical reaction can be observed - an increase in the number of extrasystoles. When taken orally, nausea, vomiting, and diarrhea may appear. The use of potassium chloride is contraindicated for violations of atrioventricular conduction, with complete blockade of the heart, renal excretory function. In these cases, potassium accumulates in the blood plasma, which can lead to intoxication. There is evidence of severe complications that occurred after taking poorly soluble dosage forms of potassium chloride. In order to avoid possible complications, it is not recommended to use potassium chloride in the form of conventional tablets

It is known to use a medicinal product aspartame containing potassium asparaginate, etc. This preparation has properties similar to those of potassium chloride. However, unlike it, it has a significantly less pronounced local irritant effect on the digestive tract and is better tolerated by patients. This is due to the fact that Asparkam tablets contain potassium in the form of an organic salt, which dissociates less than potassium chloride [http://www.mordovnik.ru/solkalia. Potassium salts; Small medical encyclopedia. M .: Medical Encyclopedia. 1991-1996; First aid. M .: Big Russian Encyclopedia. 1994; Encyclopedic dictionary of medical terms. M .: Soviet Encyclopedia. 1982-1984 http://dic.academic.ru].

Closest to the claimed antioxidant neuroprotective agent is comenic acid (5-hydroxy-γ-pyrone-2-carboxylic acid).

Comenic acid has antioxidant and neurotropic properties [Shurygin A.Ya., Kolendo SV, Yugay G.A. Antioxidant effect of comenic acid. University News. North Caucasus region. Natural Sciences. 2000. No1. S.100-101, Gusaruk L.R. The effect of transferred stress on morphological and functional features of cultured rat neurons. Abstract. diss. Cand. biol. sciences. M. 1998; Shurygin A.Ya. The drug is balis. 2002. 416 s].

Its pronounced growth-stimulating effect on the culture of neurons of the cerebral cortex of prenatally stressed animals was established (A. Shurygin, Drug baliz, 2002, 416 p.). Comenic acid has a mild sedative effect that is not addictive [RF patent for invention No. 2209062, IPC (7) A61K 31/351, A61P 25/20], is a highly effective non-narcotic analgesic that does not have negative side effects that are not addictive and addictive leading to long-term withdrawal of pain [RF patent No. 2322977 A61K 31/351 (2006.01), A61P 29/02 (2006.01)]. It has anti-withdrawal, anxiolytic and antidepressant properties [Panova TI Mechanisms of the influence of comenic acid on the integrative activity of the brain / Theoretical and experimental medicine. Medicine today and tomorrow. 2005. No1. S.28-33].

The technical result is the creation of a drug that helps to reduce the death of neurons under conditions of oxidative stress in neurogenital diseases of the brain.

To achieve a technical result, it is proposed to use, as a pharmacological agent for the prevention and treatment of neurodegenerative diseases caused by oxidative damage to the brain, the potassium salt of comenic acid (potassium coment), obtained by mixing a solution of comenic acid, heated to a temperature of 70-80 ° C, with a hydroxide solution potassium at volumes taken from the calculation made stoichiometrically, to a pH value of 4.6. Potassium comentate is used in an amount of 2 to 8 mg per 1 kg of body weight as an antioxidant, stress and neuroprotective agent once daily, on an empty stomach, for 3 days.

To obtain the potassium salt of comenic acid, we take 50 ml of a 3 mM hot solution of comenic acid (70-80 ° C) and add, stirring, 15 ml of a 3 mM potassium hydroxide solution. The reaction proceeds quickly, the solution turns yellow. Potassium salt of comenic acid is isolated from the solution by distillation of water under vacuum or by evaporation of water from a salt solution in a water bath. The yield of potassium comentate is about 90%.

The antioxidant stress and neuroprotective properties of the potassium salt of comenic acid were studied using a model system generating free radicals (CFL system), a culture of brain neurons, and a stress model in experimental animals.

Antioxidant properties of potassium salt of comenic acid

The effect of potassium comentate on the generation of reactive oxygen species (ROS) in the model system was studied in a CFL medium (citrate-phosphate buffer with the addition of luminol) of the following composition: 4 ml of citrate-phosphate buffer (105 mM KCl, 20 mM KH ₂ PO ₄ , 4 mM sodium citrate; pH 7.45) with the addition of luminol (10 mM). The formation of ROS was initiated by the introduction with constant stirring of 30 μl of a 35 mm solution of iron sulfate. In this model, the oxidation of iron ions in the presence of orthophosphate and citrate is accompanied by the formation of ROS and chemiluminescence (CL) occurs, selectively enhanced by luminol, which is suppressed in the presence of antioxidants. ChL was recorded with a SmartLum 5773 instrument for 5 minutes. The light sum of chemiluminescence was evaluated.

The antioxidant activity of the potassium salt of comenic acid was evaluated by the inhibition of CL of the model system with the addition of aqueous solutions of the drug in comparison with a solution of comenic acid. The final concentration of the substance in the cuvette was 0.1 mg / ml and 0.01 mg / ml. The chemiluminescence of free radicals in the CFL model system (control) is taken as 100%. The experimental results were determined by the intensity of chemiluminescence (in cu) and calculated as a percentage of the control [Farkhutdinov P.P., Likhovsky V.A. Chemiluminescent research methods of free radical oxidation in biology and medicine. 1995, Ufa, 90 pp.]. Processing of the data was carried out using PowerGraph software version 3.3. Significance of differences was evaluated using Student's criterion. The results of the study are presented in table 1.

Table 1 The level of reduction of free radicals in the model system of CFL in the presence of comenic acid and its potassium salt (% of control) No. p / p A drug Concentration, mg / ml 0.01 0.1 one coment potassium 34.24 ± 1.22 ^* 64.6 ± 0.95 ^* ** 2 comenic acid 33.29 ± 1.36 ^* 69.39 ± 1.16 ^* ** Note:
1 ^* p <0.001 in comparison with the control;
2 ^** p <0.001 compared with 0.01 mg / kg.

Analysis of the data presented in table 1 shows that the potassium salt of comenic acid significantly reduces the content of free radicals in comparison with the control in the model CFL system. Moreover, the level of reduction of free radicals by potassium coment is practically the same as the level of free radical quenching by comenic acid. The antioxidant properties of potassium comentate and comenic acid depend on the concentration of the substance. Thus, an increase in their concentration in the test solution from 0.01 mg / ml to 0.1 mg / ml contributes to a significant (more than 30%) increase in the level of quenching of free radicals.

Thus, the potassium salt of comenic acid has a pronounced antioxidant effect, while its antioxidant properties practically do not differ from those of comenic acid.

To assess the state of oxidative mechanisms in the brain, the most commonly used are the determination of lipid peroxidation products (LPO), the intermediate product of which is malondialdehyde (MDA), as well as the study of chemiluminescence of biological media.

The effect of potassium coment on the intensity of free radical oxidation and the content of MDA in the brain of stressed animals.

The level of free radical oxidation in the brain was determined by the chemiluminescent method [Farkhutdinov U.R., Farkhutdinov P.P. // Bull. an expert. biol. and honey. 2000, Vol. 129. No. 3, S.260-264] on the SmartLum 5773. The experimental results were evaluated by the intensity of chemiluminescence (the sum of chemiluminescence in cu) and was calculated as a percentage of the control. Statistical analysis of the results was carried out using Student's criterion [Lakin G.F. Biometrics. M. 1990. 352 p.]. The content of MDA was determined by the method of Gavrilov B.V. et al. [Gavrilov VB, Gavrilova A.R., Mazhul L.M. Determination of the content of lipid peroxidation products in the test with thiobarbituric acid // Questions of medical chemistry. 1987. No. 1. S.19-21]. Statistical analysis of the results was carried out using Student's criterion [Lakin G.F. Biometrics. M. 1990. 352 s] in MO Excel 2003.

Potassium comentate was administered to mice per os, for 3 days, once a day on an empty stomach, at concentrations of 1, 2, 4 and 8 mg per 1 kg of body weight before stressing the mice. Stress in mice was induced by hanging from the cervical fold for five hours. After 5 hours, the mice were decapitated, the brain was placed in liquid nitrogen, and then the level of free radical oxidation (CPO) and MDA were determined in the brain.

The calculation of the range of amounts of potassium comentate 1-8 mg / kg body weight was carried out taking into account its effectiveness and the absence of harmful effects on the body.

It is known that the toxic dose of potassium for humans is 6 g [Skalny A.V., Rudakov I.A. Bioelements in medicine. M. 2004.272 p.]. The maximum single dose of potassium contained in the potassium comenta per person in our experiments is 0.11 g. In addition, we compared the K ⁺ content in our preparation and in the KCl and potassium asparaginate drugs. So a single dose of potassium for a person in KCl is 0.21 g, and in a drug potassium asparaginate a single dose is 0.04 g, and a daily dose is 0.12 g. In this regard, the use of potassium coment in amounts of more than 8 mg / kg body weight consider inappropriate.

The following groups of males of outbred mice were formed:

1) intact;

2) intact + coment potassium 1 mg / kg;

3) intact + potassium coment 2 mg / kg body weight;

4) intact + potassium coment 4 mg / kg;

5) intact + potassium coment 8 mg / kg body weight;

6) stressed;

7) stressful + coment potassium 1 mg / kg;

8) stressful + potassium coment 2 mg / kg body weight;

9) stressful + coment potassium 4 mg / kg;

10) stressful + coment potassium 8 mg / kg body weight. The experiment was conducted on 54 white outbred male mice weighing 23-25 g, 9 mice in each group. The results are presented in table 2.

table 2 The effect of potassium coment on SRO and the content of MDA in the brain (trained mice (M ± m) No. p / p Groups of animals SRO (light sum, c.u.) MDA (nM / 1 g protein) one intact 208.23 ± 4.31 3.17 ± 0.14 2 intact + potassium coment 1 mg / kg 214.32 ± 2.17 3.37 ± 0.52 3 intact + potassium coment 2 mg / kg 215.89 ± 1.25 3.69 ± 0.48 four intact + potassium coment 4 mg / kg 221.12 ± 4.91 3.32 ± 2.17 5 intact + potassium coment 8 mg / kg 218.01 ± 4.26 3.49 ± 1.23 6 stressed 247.08 ± 7.71 ^** 4.46 ± 0.20 ^*** 7 stressful + coment potassium 1 mg / kg 234.03 ± 6.52 3.42 ± 1.07 8 stressful + coment potassium 2 mg / kg 228.13 ± 5.12.01 3.31 ± 0.31.01 9 stressful + potassium coment 4 mg / kg 218.00 ± 3.62 retention 3.28 ± 0.28▪ 10 stressful + coment potassium 8 mg / kg 204.99 ± 2.27▪▪ 3.21 ± 0.15 retribution Note: ^* p≤0.05; ^** p≤0.01; ^*** p≤0.001 - the reliability of statistically significant differences compared with intact control
▪ p≤0.05; ▪▪ p≤0.01; ▪▪▪ p≤0.001 - reliability compared to stressed animals (stress control).

An analysis of the data presented in Table 2 shows that when potassium comentate is used in amounts of 1-8 mg / kg body weight in intact animals (groups 2, 3, 4, 5), the level of free radical oxidation in the brain is practically the same as in intact control (1 Group). A sharp increase in the level of free radicals (p≤0.01) is observed after stress exposure (group 6) on the animal organism. When using potassium coment in amounts of 1-8 mg / kg body weight, the intensity of CPO in the brain of stressed animals decreases. At the same time, in group 7 (1 mg / kg body weight), a rather slight decrease in the level of SRO is observed. The potassium salt of comenic acid exhibits the most pronounced antioxidant activity in an amount of 8 mg / kg body weight (group 10).

The study of the effect of potassium comentate on the content of MDA in the brain of the above groups of animals showed that potassium comentate in amounts of 1-8 mg / kg of body weight has practically no effect on the content of MDA in the brain of intact animals (groups 2, 3, 4, 5). Significant and significant, in comparison with intact animals, an increase in the level of MDA is noted after stressful effects on the body (group 6). While in mice stressed and receiving potassium coment in amounts of 1, 2, 4, and 8 mg / kg of body weight, a decrease in the content of MDA is observed, in comparison with group 6 (stress control). In this case, the most pronounced and almost the same decrease in the level of MDA is observed in groups of mice 8, 9 and 10 (2, 4 and 8 mg / kg body weight).

Thus, it was found that potassium coment in amounts of 2, 4, and 8 mg / kg body weight exerts a pronounced antioxidant stress-protective effect, which manifests itself in an intensive decrease in the overproduction of free radicals and the content of lipid peroxidation products in the brain of stressed animals. In this case, the most pronounced antioxidant, stress-protective effect of potassium salt of comenic acid has an amount of 8 mg / kg of body weight. In this regard, for further studies, we used potassium salt of comenic acid in an amount of 8 mg / kg body weight.

The effect of potassium salt of comenic acid on the antioxidant glutathione system in the brain of stressed animals

It is known that the composition of the antioxidant system necessary to control the production of reactive oxygen species and prevent free radical reactions includes both enzymes and numerous low molecular weight antioxidants or compounds that prevent the formation of free radicals. Different antioxidants play a different role in tissues.

An important link in the protection of cells from the toxic effects of primary lipid peroxidation products is the antioxidant glutathione system [Baraboy V.A., Sutkova D.A. Oxidative-antioxidant homeostasis in norm and pathology / Under. ed. Acad. Academy of Medical Sciences of Ukraine Yu.A. Zozuli. K., 1997, 413 pp.]. The effect of the potassium salt of comenic acid on the antioxidant glutathione system was evaluated by the activity of the enzymes glutathione peroxidase, glutathione reductase and the content of reduced glutathione in the brain of stressed animals.

Glutathione acts as a hydrogen donor and cofactor in a number of antioxidant enzyme systems. The decrease in the intracellular content of reduced glutathione significantly reduces the resistance of cells and the body to intoxication. The determination of free sulfhydryl groups (reduced glutathione (GSH)) in an acid-soluble supernatant was determined by the method of [Sedlak J., Lindsay R.H. Estimation of total, proteinbound and nonprotein sulthydryl groups in tissue with Ellman′s reagent // Anal. Biochem. 1968. Vol. 25. P.192-205].

The enzyme glutathione peroxidase (GP) is one of the main enzymes of the antioxidant defense of the body against endogenously or exogenously induced formation of peroxides, including lipid peroxides. Using glutathione, GP restores the main product of lipid peroxidation — lipid hydroperoxide, neutralizes their toxic effect on membranes and prevents the initiation of secondary lipid oxidation reactions [Gavrilova AR, Khmara N.F. Determination of erythrocyte gutathione peroxidase activity at saturating substrate concentrations // Laboratory. 1986. No. 12. S.721-724].

Glutathione peroxidase was determined by the oxidation rate of glutathione reduced in the presence of tertiary butyl hydroperoxide [Moin V.M. A simple and specific method for determining the activity of glutathione peroxidase in red blood cells // Laboratory. 1986. No. 12. S.724-727]. The essence of the method lies in the fact that during the incubation of the sample, glutathione peroxidase from the supernatant oxidizes the saturating concentrations of reduced glutathione - the substrate. Glutathione peroxidase activity is judged by the amount of reduced oxidized glutathione restored.

The functional role of the highly specific cytoplasmic glutathione reductase is to generate reduced glutathione from its disulfide form.

The activity of glutathione reductase (GR) is determined by the method of [Yusupov LB On improving the accuracy of determining the activity of erythrocyte glutathione peroxidase // Laboratory work. 1989. No. 4. S.100-101].

The experiment was carried out on white outbred mice - males, weighing 18-20 g. The following groups of animals were formed:

1) intact;

2) stressed;

3) intact + potassium coment 8 mg / kg body weight;

4) stressful + coment potassium 8 mg / kg body weight.

Potassium comentate was administered to mice at a concentration of 8 mg / kg body weight for 3 days before stressing the animals, once a day on an empty stomach. Stress was caused by immobilization by hanging mice by the cervical fold for five hours. After 5 hours, the mice were decapitated, the brain was placed in liquid nitrogen, and then the content of reduced glutathione, the activity of the enzymes glutathione peroxidase and glutathione reductase were determined.

The results of the study are presented in table 3.

Table 3 The effect of potassium coment on the antioxidant glutathione system of the brain of stressed animals No. p / p Groups of animals GSH μmol / 1g ow. mk GP μmol / 1 min 1 g protein GR micromol / 1 sec 1 g protein one intact 2.00 ± 0.05 664.00 ± 12.00 1.28 ± 0.06 2 stressed 1.82 ± 0.04 ^* 745.00 ± 30.00 ^* 1.47 ± 0.05 ^* 3 intact + coment potassium 8 mg / kg body weight 2.07 ± 0.06▪▪ 612 ± 24 1.18 ± 0.16 four stressful + potassium coment 8 mg / kg body weight 2.04 ± 0.08 ret 619.00 ± 22.00▪▪ 1.24, ± 0.10▪ Note:
^* p≤0.05; ^** p≤0.01 - the reliability of statistically significant differences compared with intact control;
▪ p≤0.05; ▪▪ p≤0.01 - compared with stressed animals (stress control).

Analysis of the data presented in table 3 showed that after stress exposure (group 2) in the brain of animals, the activity of glutathione peroxidase significantly increases (p≤0.001), the activity of glutathione reductase increases (p≤0.05), a significant decrease in the concentration of reduced glutathione is noted ( p≤0.05).

The use of potassium coment in an amount of 8 mg / kg body weight 3 days before stress exposure contributes to the normalization of antioxidant processes in the brain of experimental animals (column 4). Glutathione metabolism did not change significantly after administration of potassium comentate to intact animals. That is, potassium coment in an amount of 8 mg / kg body weight does not adversely affect the glutathione metabolism of intact animals. Thus, it was found that the immobilization stressful effect on the body of mice leads to the intensification of lipid peroxidation (increased content of CPO and MDA) in the brain and mobilization of glutathione AOS (a statistically significant increase in the activity of glutathione peroxidase and glutathione reductase enzymes). The use of potassium salt of comenic acid in an amount of 8 mg / kg body weight for three days before stressing the animals helps to normalize the oxidative mechanisms of lipid peroxidation and, accordingly, to restore the antioxidant defense of the brain, which is manifested in a decrease in the activity of the enzymes glutathione peroxidase and glutathione reductase, and an increase in the concentration of reduced glutathione. At the same time, the level of antioxidant protection when using potassium comentate practically does not differ from that in the brain of intact animals (intact control group).

The effect of potassium salt of comenic acid on glutamate cytotoxicity in the culture of granular cells of the cerebellum of rat pups

The development of methods of prevention and treatment is based on the assumption that a significant number of brain cells can be saved by blocking the initiation or course of intra-neural pathochemical destructive processes that have certain temporal and spatial characteristics.

The effect of the potassium salt of comenic acid on glutamate cytotoxicity in the culture of neurons of the cerebellum of rat pups was studied in comparison with comenic acid.

Studies were performed on Wistar rat pups. Cerebellar granule cell cultures were obtained from the brain of 7–9-day-old rat pups by the method of enzyme-mechanical dissociation [Viktorov IV, Khaspekov LG, Shashkova N.A. // Guide to the cultivation of nerve tissue: Methods, techniques, problems. 1986. M. 266 p.]. Cell cultures after 7 days of cultivation were exposed to glutamate and / or potassium salt of comenic acid, comenic acid.

Potassium comentate and comenic acid were introduced into the neuron culture groups without exposure to glutamate and after 10 minutes of exposure (100 μM) of glutamate in balanced salt solution (CCP), after placing them in the original nutrient medium. After that, the cultures were incubated in the CO ₂ incubator for another 4.5-5 hours. The effect of potassium comentate at concentrations of 10 ^-2 M, 10 ^-3 M, 10 ^-4 M, 10 ^-5 M, 10 ^-6 M and comenic acid at concentrations of 10 ^-3 M, 10 ^-4 M was studied. The cultures served for 10 min in the Soviet Socialist Republic, as well as cultures treated with glutamate in the Soviet Socialist Republic. After fixing the cultures, the number of living and dead neurons was taken into account on an Invertoscopes ID 03 inverted microscope. Reliability of differences was evaluated using Student's criterion in Excel 2003 MO.

Cerebellar neuron cultures obtained from intact rat pups were divided into the following groups, respectively:

1.1 control of the SSR;

1.2 SSR + coment potassium 10 ^-2 M;

1.3 SSR + coment potassium 10 ^-3 M;

1.4 SSR + coment potassium 10 ^-4 M;

1.5 SSR + coment potassium 10 ^-5 M;

1.6 SSR + coment potassium 10 ^-6 M;

1.7 SSR + comenic acid 10 ^-3 M;

1.8 SSR + comenic acid 10 ^-4 M;

2.1 control of SSR with glutamate (Glu) 100 μM;

2.2 SSR with glutamate (Glu) 100 μM + potassium coment 10 ^-2 M;

2.3 SSR with glutamate (Glu) 100 μM + potassium comentate 10 ^-3 M;

2.4 SSR with glutamate (Glu) 100 μM + potassium comentate 10 ^-4 M;

2.5 SSR with glutamate (Glu) 100 μM + potassium coment 10 ^-5 M.

2.6 SSR with glutamate (Glu) 100 μM + coment potassium 10 ^-6 M;

2.7 SSR with glutamate (Glu) 100 μM + comenic acid 10 ^-3 M;

2.8 SSR with glutamate (Glu) 100 μM + comenic acid 10 ^-4 M. The results of the study are presented in table 4.

Table 4 - Effect of potassium comentate and comenic acid on the cytotoxic effect of glutamate in cerebellar neuron culture No. p / p Crop groups The number of living neurons (%) The control Coment potassium (M) Comenic acid (M) 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 10 ^-3 10 ^-4 one 2 3 four 5 6 7 8 one SSR 95.12 ± 0.62 94.82 ± 1.05 95.13 ± 0.97 96.14 ± 0.76 95.92 ± 0.71 95.60 ± 1.11 85.8 ± 3.3 85.2 ± 6.0 2 glutamate 29.04 ± 1.56 _# ^** 48.28 ± 2.14 _# ^** 46.88 ± 1.35 _# ^** 64.97 ± 3.27 _# ^** 54.65 ± 3.51 _# ^** 50.37 ± 2.37 _# ^** 46.7 ± 3.1 _# ^** 35.9 ± 2.9 _# ^* Note:
1 _# p <0.001 - in comparison with the control of the SSR;
2 ^* p <0.05 - in comparison with the glutamate control group;
3 ^** p <0.001 - in comparison with the glutamate control group.

Analysis of the research results showed (Table 4) that the number of surviving neurons after exposure to potassium comentate (groups 1.2-1.6) and comenic acid (groups 1.7, 1.8) on intact cerebellar granular cells, regardless of the concentration of the substance, practically does not differ from the control ( group 1.1). After exposure to glutamate, the survival of granular cells of the cerebellum decreases significantly (by 66.08%) (group 2.1), p <0.001. The introduction of cerebellum neurons into the cultures treated with glutamate potassium comentate (10 ^-2 M - 10 ^-6 M), contributed to a significant increase in the resistance of neurons to glutamate cytotoxicity in all groups of cultures (groups 2.2-2.6). Moreover, the maximum neuronal survival was observed in culture group 2.4 with the addition of potassium comentate at a concentration of 10 ^-4 M. With the use of comenic acid, neuronal survival in the culture groups treated with glutamate (2.7 and 2.8) was significantly higher than in the control-glutamate group. however, its maximum was significantly lower than when adding potassium salt of comenic acid to the culture.

Thus, it was found that the use of potassium salt of comenic acid against the background of glutamate cytotoxicity contributes to a significant increase in the survival of cerebellar neurons in culture. At the same time, the resistance of neurons to the toxic effects of glutamate in cultures with the addition of comenic acid salt exceeds that when using comenic acid, taken as a prototype for the greatest number of matches.

We evaluated the toxicity of the potassium salt of comenic acid by indicators of free radical oxidation and lipid peroxidation in the brain of experimental animals (Table 2), as well as control cultures of cerebellar neurons with the addition of the studied doses of the above salts. The results of the studies showed that the potassium salt of comenic acid in the doses we are studying is not toxic.

It is known that the basis for conducting clinical trials of antioxidants in the treatment of diseases with degenerative disorders are studies conducted on cultured cells and model animals [Andersen Julie K. Oxidative stress in neurodegene ration: cause or consequence? // Nature Reviews Neuroscience 2004. Vol. 5. S 18-S25].

We studied the effect of various doses of potassium salt of comenic acid on the level of free radicals in the CFL model system and in the brain of stressed animals, on the antioxidant glutathione system in the brain (trained animals (Tables 1, 2, 3), as well as on the stability of cultured neurons cerebellum to glutamate cytotoxicity (table 4).

Our data indicate that the stressful effect on the body of experimental animals is accompanied by an increase in oxidative processes in the brain, which manifests itself in an increase in the level of free radicals and an increase in the content of one of the products of lipid peroxidation, MDA, in the brain, and a decrease in the level of glutathione protection.

It was found that the use of potassium salt of comenic acid in amounts of 2-8 mg / kg body weight before stress on the body significantly reduces the level of free radicals and stimulates the antioxidant glutathione system in the brain of stressed animals. At these doses, the potassium salt of comenic acid when administered orally has a pronounced antioxidant, stress-protective effect.

The effect of potassium salt of comenic acid on the resistance of cultured cerebellum neurons to glutamate cytotoxicity was studied.

It was found that the use of potassium comentate at concentrations of 10 ^-2 M - 10 ^-6 M in a culture of cerebellar nerve cells against the background of glutamate cytotoxicity significantly increases the survival of neurons. The maximum survival of neurons is observed in the group of cultures after exposure to potassium coment at a concentration of 10 ^-4 M (group 2.9, p <0.001).

Obtaining potassium comenta and the study by the authors of the elements of a subtle mechanism of its effect on activated oxidative processes in the brain of stressed animals and the development of glutamate excitotoxicity allows us to conclude that it is promising for use as an antioxidant, stress and neuroprotective drug for ischemic brain damage, including with violation of ionic homeostasis and on the background of cardiovascular pathology. Analysis of the prior art did not allow to find a solution that completely coincides in the totality of essential features with the claimed, which confirms the novelty of the obtained compounds as antioxidant, stress and neuroprotective agents. Thus, the range of highly effective drugs with antioxidant, stress and neuroprotective properties for the prevention and treatment of neurodegenerative diseases caused by oxidative damage to the brain is expanding.

Therefore, the claimed technical solution also satisfies the criteria: novelty, inventive step and industrial applicability.

Claims (1)

The use of potassium salt of comenic acid as a prophylactic and therapeutic antioxidant, stress and neuroprotective agent in an amount of from 2 to 8 mg per 1 kg of body weight, once on an empty stomach, for 3 days.