EA027170B1 - Pharmaceutical composition for correcting symptoms of or treating asthenia and/or chronic fatigue syndrome - Google Patents

Abstract

The invention relates to the field of medicine, and more particularly to pharmacology, and even more particularly to pharmaceutical compositions for treating various conditions associated with a lack of energy potential resulting from a pathological process. A pharmaceutical composition for correcting symptoms of or treating asthenia and/or chronic fatigue syndrome, when isolated or combined with various diseases and/or premorbid and/or postmorbid conditions in mammals, is characterized in that it contains, as active ingredients, 2-ethylthiobenzimidazole hydrobromide and/or a pharmaceutically acceptable salt thereof, and levocarnitine in therapeutically effective amounts.

Description

The invention relates to medicine, in particular to pharmacology, in particular to pharmaceutical compositions for the treatment of various conditions associated with a lack of energy potential, resulting from a pathological process.

Bemitil was synthesized and studied at the Department of Pharmacology of the Military Medical Academy in the 1970s under the guidance of Professor V.M. Vinogradova. The experiment was established (Bobkov, Yu.G. et al., 1981, etc.), and then confirmed in clinical studies (Losev A.S. et al., 1990; Smirnov A.V. et al., 1990; Stroganov V.P. et al., 1990; Shakhnazarov A.S. et al., 1990; Sytnik S.I., Pastushenkov V.A., 1993), that even with a single application, bemitil significantly increases the physical performance of animals and accelerates it recovery after extreme loads, especially in complicated conditions (high-altitude hypoxia, overheating, marching throws, etc.).

With a course application, the effect of bemitil increases in the first 3-5 days, and then it is steadily maintained at the achieved level (Smirnov A.V., 1993; Bobkov Yu.G. et al., 1993), surpassing the effect of piracetam and pyriditol (Aleksandrovsky Yu.A. et al., 1988).

The experiment revealed the hepatoprotective effect of bemitil (Aleksandrova A.I. et al., 1988), obtained positive results in the treatment of acute cerebrovascular disorders in the model of cranial hemorrhage (Plotnikova T.M. et al., 1993), the reparative effect of relation to the mucous membrane of the small intestine with experimental peritonitis (Starokon P.M., Larin Yu.N., 1999). The following abilities of bemitil have also been described:

increase the activity of antioxidant enzymes, especially superoxide dismutase (Katkov V.F. et al., 1989, Shakhnazarov A.S. et al., 1990);

to inactivate ίη νίίτο hydroperoxide is more effective than glutathione (Diezhe A.A. et al., 2002); prevent the decrease in the content of reduced glutathione, δΗ groups, the activity of glutathione reductase, and liver glutathione peroxidase in acute hypoxia in the experiment and enhance the synthesis of antioxidant enzymes of the glutathione system (Zarubina I.V., Mironova O.P., 2002);

induce mixed-type cytochrome P-450 activity, increasing the total content of cytochrome P-450 in microsomes and monooxygenase activity (Sorokina EA et al., 2002);

to reduce the level of blood lactate in patients (Leosco V. A et al., 1996) and the LPO intensity determined by the indirect antiradical route (Plotnikov MB et al., 1988, 1990);

to promote liver regeneration after partial hepatectomy in the experiment (Gayvoronskaya V.V. et al., 2002);

have an antimutagenic effect (Durnev A.D. et al., 1988).

A hypothesis was put forward according to which the mechanism of action of bemityl (2 ethylthiobenzimidazole hydrobromide) and its analogues, derivatives of 2-thiobenzimidazole, is based on the activation of RNA synthesis in different cells, leading to increased protein synthesis. Such an action may be due to their interaction with the genome, probably due to the structural similarity of benzimidazole with purine bases - adenine and guanine (Smirnov A.V., 1993; Smirnov A.V. et al., 1994).

This effect is not organ-specific or tissue-specific, but it is always more pronounced in those organs and tissues in which the processes of RNA synthesis and, therefore, protein are actively proceeding.

It is curious that among the drugs derived from benzimidazole there are many drugs of various uses, with different medical orientations: with antipsychotic (pimozide), actoprotective (bemitil, etomerzole), antiarrhythmic (mibefradil, rhythmidazole), antiulcer (omeprazole, gastrozole, p antiallergic (astemizole), uricosuric (irtemazole), anthelmintic (mebendazole, thiabendazole). A group of benzimidazole derivatives (fenbendazole, oxafendazole, parbendazole, triclabendazole) used in veterinary medicine as anthelmintic agents is also represented. Does this mean that they all stimulate protein synthesis?

Interestingly, imidazole itself, as shown in an experiment on a model of myocardial ischemia (διηίΐΐι E.P.A., 1980), has a cardioprotective effect due to the specific inhibition of thromboxane-A2 synthetase. The latter, being a coronary constrictor and increasing in activity with ischemia, can cause further deterioration of coronary blood flow according to the vicious circle principle.

A.V. Smirnov and V.Yu. Gancho (1990) showed that enhanced protein synthesis occurs mainly in the brain, which explains the improvement in memory when using bemitil.

One way or another, but it has been established that in order to maintain a high level of metabolism, as well as physical performance, gluconeogenesis in the liver and kidneys is necessary, which ensures utilization of lactic acid and glucose resynthesis in various conditions with its high production, for example, with increased muscle activity. Gluconeogenesis enzymes are relatively short-lived proteins and their renewal is ongoing.

Strengthening the synthesis of precisely these enzymes is that component in the mechanism of action of boehmil, which is most important for increasing physical performance. In addition, bemitil and its analogues activate the synthesis of proteins that provide the economizing effect characteristic of these preparations: a decrease in oxygen consumption and heat production, a decrease in the expenditure of energy resources 1,027,170, including per unit of work performed.

Another hypothesis of the mechanism of action of bemitil was put forward by Yu.G. Bobkov et al. (1993) emphasizes the ability of a drug in an experiment to accelerate the transition of superoxide radical and other free radicals to molecular oxygen, followed by its direct use in the metabolic cycle due to the activation of antioxidant enzymes - SOD, catalase, glutathione peroxidase. Let us leave this mechanism to the conscience of the authors.

Although bemithil is an effective means of restorative and rehabilitative, rehabilitative action, and in the most diverse pathological conditions, it relieves asthenia, increases working capacity, and it can also be used in acute hypoxia.

It is curious that in the modern training method of interval hypoxic therapy, in which patients create artificial hypoxic hypoxia with a prophylactic (preconditioning) and therapeutic purpose, it is envisaged to use, say, proofreading methods. In model experiments with such a peculiar therapeutic hypoxia, bemitil proved to be a reliable protector, stimulating the development of adaptive metabolic changes in the brain, heart, liver, kidneys and skeletal muscles (2, IV, 2001).

Encouraging data were obtained in the clinic, indicating the antihypoxic effect of bemitil to prevent myocardial damage in acute heart attack, in connection with cardiac operations with cardiopulmonary bypass, with chronic fetal hypoxia in pregnant women with gestosis, with chronic respiratory failure and viral hepatitis (Lobzin Yu. V., Smirnov A.V., 1993; Shevchenko Yu.L. et al., 1995; Semigolovsky N.Yu. et al., 1995, 1996, 1998; Leosko V.A. et al., 1996; Ryabinin G .B. et al., 1999).

In the clinic, Bemitil was also used for neuromuscular diseases (progressive myodystrophy, atrophic myotonia, and secondary amyotrophy). An increase in muscle strength, creatine index, a decrease in lipid peroxidation products, and fermentemia were noted (Lobzin V.S., Pustozerov V.G., 1993). There is some reason to rely on the cerebroprotective properties of bemitil, since other benzimidazole derivatives in the experiment (models with carotid ligation, gravity) were, according to the data of L.M. Gaeva and T.N. Scherbakova (1994) in this aspect even more active than cavinton, piracetam, phenibut and GHB.

Bemitil is used to increase and restore working capacity, including in extreme conditions (heavy loads, hypoxia, overheating, etc.); to accelerate and strengthen the development of adaptation to the effects of various extreme factors; for the treatment of asthenic disorders of various nature (with neurasthenia, somatic diseases, after severe infections and intoxications, in the pre- and postoperative period during surgical interventions, etc.); in the complex treatment of the effects of traumatic brain injury, meningitis, encephalitis, cerebrovascular accident, as well as memory impairment.

Observations of the use of bemitil in patients with acute myocardial infarction date back to 1994-1995 (Semigolovsky N.Yu., 1995,1996, 1998). The drug was prescribed to patients twice a day - in the morning and in the morning, for fear of excessive psychostimulating action at night.

Patients receiving boehmil were somewhat more active, more alert. A hypothermic effect of the drug was observed - the body temperature in patients on the 5-6th day of the disease sometimes turned out to be lower than 36 ° C, which corresponds to the description of the properties of bemitil (Smirnov A.V., 1993, Bobkov Yu.G. et al., 1993, etc.) and generally characteristic of thiourea derivatives (Pastushenkov L.V., 1968; Uryupov O.Yu., 1969).

The drug did not show any noticeable antianginal and antiarrhythmic effects. The dynamics of enzyme activity during its use did not differ significantly from the control, excluding the statistically significant decrease in AlAT for 3-4 days of heart attack (p <0.05), compared with the control indicator. The latter corresponds to the observations of Yu.V. Lobzina and A.V. Smirnova (1993) on the hepatoprotective properties of bemitil.

A study of the protective activity of the drug revealed its hypocoagulation properties in patients with AMI, as indicated by a significant decrease in the main group of the average prothrombin index over the entire time of observation (p <0.01-0.05) and the average fibrinogen content on days 5-6 of AMI (p <0.05 ) Rapid reduction of stress hyperglycemia under the influence of bemitil was also noted, which coincides with a description of its effect in other conditions (M.D. Mashkovsky, 1993) and, possibly, implies the need for substrate support of treatment with this antihypoxant.

The accelerated decrease in the initially reliably increased average systolic ECG indicator (p <0.05) coincides with the data of L. A. Novikov (1994) on the inotropic activity of bemitil.

Side effects when using the drug were not observed.

The experiment showed the ability of bemitil, etomerzole, and yakton to accelerate liver regeneration after partial hepatectomy (an acceleration of the increase in the mass of the liver, an increase in the content of nucleic acids and glycogen in it, an improvement in the functional state, which is manifested by a decrease in the level of bilirubin in the blood and a decrease in the duration of hexenal sleep, were revealed). Reparative

- 2,027,170 the activity of these agents exceeded the effect of the combination of regeneration stimulants, derivatives of purine and pyrimidine bases, riboxin and potassium orotate (Gayvoronskaya V.V. et al., 2002).

Carnitine.

The international non-proprietary name of the drug is levocarnitine (Leuosatsepe). Carnitine chloride is available in a 10% solution for injection (1ml contains 100 mg of the substance).

For the first time V.S. Gulevich (1905), but only in 1959 Ρτίΐδ established the leading role of carnitine in the process of β-oxidation of fatty acids. In 1969, the physiological form of carnitine, represented by L-carnitine, was identified.

The human body contains 20-25 g of this substance, of which about 98% is found in skeletal muscle and myocardium. The need for b-carnitine in an adult is 200-500 g per day. The greatest amount of it is found in lamb (190 mg / 100 g) and in avocados. The biosynthesis of L-carnitine takes place mainly in the liver (from lysine and methionine with the participation of reduced iron and cofactors of the synthesis of vitamins C, B6, B3), and its main consumers are cardiac and skeletal muscles. It is curious that scurvy causes a deficiency of carnitine in muscle tissue at a constant concentration in the liver, kidneys and plasma.

The antihypoxic properties of carnitine have been proven by L. Katei and colleagues (1987) and A.L. Karaev et al. (1991). Carnitine refers to drugs that actively affect metabolic processes. In addition to antihypoxic, it has anabolic, antithyroid, stimulating fat metabolism regenerative effects.

The drug restores the alkaline reserve of blood, does not affect the blood coagulation system, reduces the formation of keto acids, increases the resistance of tissues to the influence of toxic decomposition products, activates aerobic processes and inhibits anaerobic glycolysis, stimulates and accelerates reparative processes.

It has a fat-mobilizing effect - it includes a fatty acid metabolic shunt, the activity of which is not limited by oxygen, so carnitine is effective in acute hypoxia of the brain, myocardium and other critical conditions.

ZA Suslina et al (2003) studied the antioxidant effect of L-carnitine in the treatment of discirculatory encephalopathy in patients with diabetes mellitus. In addition to basic therapy, L-carnitine (2 g daily) increased the resistance of patients' blood lipoproteins to peroxidation, which indicates its antioxidant properties. The authors noted the hypoglycemic effect of L-carnitine: its use allowed to reduce the dose of sugar-lowering drugs by 42%. During treatment with L-carnitine, the examined patients showed an improvement in abstract and practical thinking and memory. The results allowed the authors to recommend the inclusion of L-carnitine in the treatment complex of patients with vascular diseases of the brain.

The drug has a neurotrophic effect: it has been shown that carnitine inhibits the development of apoptosis, limits the affected area and restores the structure of the nervous tissue. There is other information about the effectiveness of the drug in discirculatory encephalopathy, various traumatic and toxic lesions of the brain, in acute cerebrovascular accident - ischemic stroke, transient ischemic attack. Prescribe the drug in the acute, subacute and recovery periods.

Many authors have found a decrease in the level of carnitine in the myocardium in various heart diseases - from ischemic disease, including myocardial infarction, to cardiomyopathies, hypertension, valvular defects and congestive heart failure (ZradpoB L.O.E.A., 1982; BoH1e§ H E.A., 1986; Thing! / V. E.A., 1990).

U. Makishiga et al. (1990) showed that myocardial carnitine deficiency is mainly related to free carnitine, while total carnitine levels often remain normal due to an increase in mainly long chain ester bound carnitine. V. Veshch1 / et al. (1990) noted a direct (although not linear) relationship between carnitine deficiency and a decrease in left ventricular ejection fraction — its level was especially low in patients with ejection fraction below 30%. And A. Ra! E1 et al. (1990) found the prognostic significance of decreasing myocardial carnitine content by observing 40 patients over 5 years, of which 18 carnitine-deficient mortality was 33.3%, in contrast to 22 patients with normal carnitine levels with mortality 4.6%.

One of the causes of carnitine deficiency in the myocardium is considered to be its release into the plasma during ischemia, followed by leaching. So, Κί.Κίζζοη et al. (1989) found an initial increase in the plasma concentration of carnitine in the first 48 hours after acute myocardial infarction, and O. VayeN and XU.Tvette (1990) recorded a decrease in plasma free carnitine after atrial pacing in the group of patients, experienced myocardial ischemia in contrast to patients without ischemia or lactic free ischemia. Deficiency of carnitine in the myocardium as a result of ischemic damage to cells impedes the transfer of fatty acids to mitochondria and leads to their accumulation in the cytoplasm (Poryadin G.V., 1997; LauraksNik O., 2000).

In 1979, L.N. Orge proposed the use of carnitine for myocardial ischemia. According to

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A.I. Toleikis et al. (1986), carnitine in the experiment with myocardial ischemia had a protective effect, filling up the deficiency (A.L. 8bid, 1978), which was also confirmed in the clinic for patients with angina pectoris and congestive heart failure (Savchuk V.I. et al., 1991 ; Belozerov Yu.M., 1992; 1 <appka \ ua ea, 1984; Sketsy ea, 1985; Retiaibe / S. ea, 1985; Og1appbo S., Kiksosh S, 1986; C Assyugue L.E.A., 1991; Vogskagb I.E.A., 1994 and others). The usual dosage was 2 g per day for 1-6 months. The authors noted a decrease in the patient's need for nitrates, an increase in the antianginal action of beta-blockers and calcium antagonists, an increase in tolerance to loads.

The positive effect of carnitine was also observed in patients with acute myocardial infarction, including its complicated course: cardiogenic shock and cardiac arrhythmias (KeBi / ζί A.S. e.a., 1984; SyabePo S. e.a., 1986; K ^ ζζоη R.A.A., 1989; Oauyi R.E.A., 1992; Sotisch S.S., Beitep V, 1992; Sotischs S.S., Loske R., 1993). The drug was administered intravenously up to 21 g in the first hours of the disease and orally (up to 6 g per day for 30 days). A decrease in the zone of necrosis and 12-month mortality, stabilization of hemodynamics, antiarrhythmic effect of the drug, an increase in the contractility of the myocardium and excretion of fatty acids in the urine were revealed. With cardiogenic shock, the use of the drug contributed to the elimination of acidosis and a reduction in mortality from 80 to 50% (Sotis S.S., Bosque R., 1993).

In the treatment of chronic heart failure, the efficacy of daily oral administration of 2 g of L-carnitine for 1.5-12 months was studied (Sybsh1 O.E.A., 1988; Re ^ ηaηbeζ C, 1991; Vogskagb I.E.a., 1994 ) The authors noted in patients a subjective improvement, an increase in myocardial contractility, a decrease in the consumed doses of cardiac glycosides, diuretics, vasodilators, antiarrhythmics and nitrates.

The purpose of the drug as a cardioprotector in the treatment of cytostatics, especially the anthracycline series, is also shown. According to G.A. Lazareva et al. (2002), carnitine reduces the severity of changes in immunological functions in cases of poisoning with hemolytic poisons (phenylhydrazine).

The drug is also used for coronary heart disease, various cardiomyopathies, obesity, diabetes mellitus, and sports medicine. There are modern recommendations for the use of zinc-carnitine (150 mg twice a day) for the prevention and treatment of peptic ulcer disease, to increase the effectiveness of N.ru1op erradication (Atakama T.E.A., 1990; MkgoaI N.E.a., 1999 )

Carnitine deficiency syndrome is described (Grinio L.P., 1988; Katrab S. e.a., 1975; \ Uage A.E. ea, 1978, etc.), manifested by muscle pain, renal and heart failure. It is known that cardiomyopathy in children refers to severe myocardial diseases with a continuously progressive course and a severe prognosis with high mortality (Leontyeva I.V., 2001). The systemic form of primary carnitine deficiency is clinically manifested by severe hypoglycemia, renal failure and encephalopathy according to the type of Reye's syndrome (Belozerov Yu.M., 1992). In this case, a substitution therapy with carnitine gives a certain effect.

In pediatrics, indications for using the drug are: the consequences of birth injury and asphyxiation; hypotrophy and hypotension of the newborn; respiratory distress syndrome of the newborn; nursing of premature infants who are on a full parenteral nutrition, and children who undergo hemodialysis; a syndrome complex similar to Reye's syndrome (hypoglycemia, hypoketonemia, coma), which develops in children with valproic acid; lack of body weight in children and adolescents under 16 years of age; propionic and other organic acididemia, primary (genetic) carnitine deficiency.

The drug is also used for anorexia, malnutrition, growth retardation in children. Carnitine increases the secretion and enzymatic activity of digestive juices (gastric and intestinal), improves the absorption of food. It reduces excess body weight and reduces muscle fat.

On the other hand, secondary acquired carnitine deficiency develops with its active elimination (hemodialysis, valproic acid intake), with excessive expenditure (ischemic lesions, trauma, recovery, pregnancy), with food intake disorders (parenteral nutrition, malabsorption, intestinal diseases), violation of reabsorption in the kidneys (chronic renal failure).

The drug increases the threshold of resistance to physical exertion, leads to the elimination of post-loading acidosis and, as a result, the restoration of efficiency after prolonged depletion of stress. It increases the reserves of glycogen in the liver and muscles, contributes to its more economical use.

The drug is prescribed independently or in combination therapy.

With intravenous administration after 3 hours, carnitine is no longer detected in the blood. It easily penetrates the liver, myocardium, and more slowly - into the muscles. It is excreted by the kidneys mainly in the form of acyl ethers.

Carnitine is administered intravenously slowly (no more than 60 drops per minute!). Before administration, every 100 mg of the carnitine chloride preparation (1 ml of a 10% solution) is dissolved in 50 ml of an isotonic 4,027,170 NaCl solution or 5% glucose solution.

In the acute period of the disease, in the first 3 days, carnitine is administered at 10-14 mg / kg of body weight of the patient, in the following days - at 7 mg / kg of body weight. The general course of treatment is 7-10 days. If necessary through

10-12 days spend a repeated course of 7 mg / kg of body weight for 3-5 days.

In acute myocardial infarction, the drug is used in a daily dose of 100-200 mg / kg in 4 divided doses or as a continuous intravenous infusion for 48 hours, followed by a 2-fold dose reduction. Patients on chronic hemodialysis are given 2 g once immediately after the completion of the next session.

When prescribing the drug in the subacute and recovery periods of dyscirculatory encephalopathy and various brain lesions, patients are administered 0.5-1.0 g of carnitine chloride once a day for 3-5 days. If necessary, after 12-14 days, a second course is prescribed.

The use of carnitine during hemodialysis allows you to level out deviations in the blood lipid spectrum (reduces the level of triglycerides and cholesterol, increases the content of high density lipoproteins), reduces the frequency of hypotension and cardiac arrhythmias, helps to increase the number of red blood cells and hemoglobin, and reduces the dose of erythropoietin.

As for the side effects, when prescribing carnitine, allergic reactions are possible. In the case of rapid intravenous administration, the appearance of pain along the veins, passing with a decrease in the rate of administration, is possible. In diabetes, people receiving insulin and oral hypoglycemic agents may develop hypoglycemia. They also describe pain in the epigastric region, dyspeptic symptoms, muscle weakness.

Glucocorticoids contribute to the accumulation of carnitine in tissues (except the liver), other anabolics enhance its effect.

Contraindications with the exception of increased individual sensitivity to the drug are not described.

As was said, substances related to ethylthiobenzimidazole are widely described, including in patents KI 1251374, the use of 2-ethyl-mercaptobenzimidazole bromide as a psychotropic agent with an actoprotective psycho-energizing and antihypoxic effect.

KI 2061686, the use in the pharmaceutical compositions of 2-ethylthiobenzimidazole derivatives of hydrobromide and hydrochloride, exhibiting weak tranquilizing activity.

KI 2157684, a drug belonging to the group of actoprotectors, providing a moderate psychostimulating effect, in which the active ingredient is 2-ethylmercaptobenzimidazole hydrobromide, used to treat systemic lupus erythematosus. As a compound with antihypoxic activity, it is used in borderline psychiatric disorders, when stimulation of mental and physical functions is indicated.

Ki 2188012, the use of 2-ethyl-mercaptobenzimidazole bromide in the treatment of patients with primary biliary cirrhosis as an immunomodulating agent that acts on the cellular component of the immune system and helps increase the effectiveness of other targeted drugs used in the treatment of cirrhosis.

KI 2173144, the use of actoprotectants containing 2-ethylthiobenzimidazole hydrobromide monohydrate derivative as an active ingredient for the treatment of Duchenne-Becker myodystrophy.

This derivative, due to structural similarities with purine bases, adenine and guanine, enhances the synthesis of mitochondrial enzymes, increases the energy potential of muscle tissue and stimulates redox processes. An increase in the energy potential of muscle tissue contributes to the activation of protein and enzyme synthesis, which leads to a decrease in the permeability of myocyte membranes, promotes muscle regeneration, and slows down the myodystrophic process.

Ki 2017483, the use of a medicinal product containing 2-ethylthiobenzimidazole hydrobromide monohydrate as a means of increasing the body's resistance to hypoxia and restoring performance when performing heavy physical exertion and when exposed to adverse factors, as well as to eliminate polypharmacy, to prevent occupational hearing loss; KI 2019161 for the prevention of acute glaucoma attacks in geomagnetic storms.

KI 2138263, the use of 2-ethylthiobenzimidazole hydrobromide for the treatment of hypothalamic-pituitary insufficiency in women using the effect of increasing the resistance of the brain to asphyxia and circulatory hypoxia, which is manifested by a marked increase in cerebral circulation, improved oxygen supply to the brain, as well as an inhibition of the degradation of the energy functions of neurogly mitochondrial cells.

The bemactor preparation may be indicated as the closest analogue (Terra Medica Nova, No. 4 '98 PHARMACY NEWS Bemactor is a new drug for general medical practice). The drug causes a pronounced anti-asthenic effect and accelerates the healing process in various diseases. Bemaktor was produced in the form of a tablet form (manufactured by TSY-October). However, its release has been discontinued since 1999. It is not available to doctors and patients. As written in recommendation 5,027,170 for use, it is taken orally in doses of 250-500 mg for 2–3 or 5–7 days (but not more than 10–12 days because of the possibility of excessive psychoactivating effect).

When creating the invention, the task was to find a drug capable of exerting a combined biological effect under conditions of energy deficiency in the body.

The problem is solved by a new combination of two agents that activate energy supply systems for the functioning of metabolic processes, and at the same time they do not intersect each other in terms of impact on activation mechanisms. Based on this combination, a pharmaceutical composition was developed for the correction of symptoms of treatment of asthenia and / or chronic fatigue syndrome both in isolation and in various diseases and / or pre and / or postmorbid conditions in mammals, characterized in that it contains 2 ethylthiobenzimidazole and / or its pharmaceutically acceptable salt and levocarnitine in therapeutically effective amounts.

Acceptable therapeutically effective amounts are 50-250 mg for 2-ethylthiobenzimidazole and / or its pharmaceutically acceptable salts, 150-750 mg of levocarnitine.

As pharmaceutically acceptable salts, it is possible to use, for example, salts such as ethylthiobenzimidazole hydrobromide (bemityl), 2-ethylthiobenzimidazole hydrochloride.

The pharmaceutical composition may contain targeted additives to formulate a convenient dosage form. In particular, it may contain excipients selected from the group of sugars, sugar substitutes, flavor substitutes, disintegrating agents, disintegrant, flavor, in the following ratios, wt.%:

Ethylthiobenzimidazole hydrobromide 5.46-15.15.

Levocarnitine 21.86-44.55.

A filler selected from the group of sugars 9.09-42.62.

A filler selected from the group of sugar substitutes 9.09-16.39.

A filler selected from the group masking the taste of 2.42-6.56.

A filler selected from the group of disintegrating components 1.21-4.37.

A filler selected from the group of substitutes for loosening agents 1,21-2,19.

A filler selected from the group of flavors 0.30-0.55.

As a filler, sugars can be used, for example, lactose, glucose, sucrose, fructose, cyclodextrin.

As a filler, sugar substitute groups can be used, for example, mannitol, xylitol, sorbitol, isomalt, fructooligosaccharides.

Lemon juice, honey, steviosides, maltol, etc. can be used as a filler in the taste masking group.

As a filler, groups of disintegrating components can be used, for example, croscarmellose sodium, starch, microcrystalline cellulose, agar, gelatin.

As a filler, a group of substitutes for disintegrating agents can be used, for example, sodium starch glycolate, silicate microcrystalline cellulose.

As a filler in a group of flavors, food flavors can be used.

The composition may be presented in solid dosage form. An example of such a form are lozenges.

The compositions of the invention are used to correct the symptoms of the treatment of asthenia and / or chronic fatigue syndrome both in isolation and in various diseases and / or pre and / or post-morbid conditions by administering the drug by administering from 5 to 20 days if necessary with repeated use of a break lasting 2 or more days.

Effect: agents can significantly enhance the effects of each other, including those related to the energy supply of the functioning of metabolic processes, and accordingly improve the condition of patients with a wide variety of diseases or extramorbid conditions. When combined, an increase in antiasthenic, antistress action is observed, physical and mental performance is increased, and the antihypoxic effect is significantly increased.

The use of a combination in the form of a quick-release absorbable tablet avoids such side effects of ethylthiobenzimidazole as a cumulative effect and an increased psycho-activating effect, which can be considered as an additional result.

The possibility of carrying out the invention can be demonstrated by the following examples.

Example 1

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Tablets, wt.%

Ethylthiobenzimidazole hydrobromide 10 Levocarnitine 30 lactose 34 xylitol 16 maltitol 4.7

Microcrystalline cellulose 2.9 Sodium starch glycolate 2.1

Menthol flavoring agent 0.3

Example 2

Capsules, mass. 5 Ethylthiobenzimidazole 15.15 Levocarnitine 21.86 Mannitol rest

Example 3

Tablets for resorption, wt.%

Ethylthiobenzimidazole hydrobromide 5.46 Levocarnitine 44.55

Maltitol 49

Sodium saccharin 0.09

Example 4. Pharmacological activity.

The aim of this study was to study the effect of repeated administration of a combination of bemitil with levocarnitine in two models of chronic fatigue syndrome in mice. As the first model, physical fatigue syndrome caused by repeated physical exertion was used. Fatigue syndrome caused by repeated stress in mice was used as the second model.

Riboxin was used as a substance for comparison.

Research Methods.

4.1. Experimental animals.

All experiments were carried out in accordance with the principles of working with laboratory animals (Rpps1p1e§ o £ Ashsha1 Saga, directive 86/609 / EEC) according to the Helsinki Declaration. The purchase of animals and the conduct of experiments were made on the basis of an appropriate license from the Ethics Committee (Ministry of Agriculture of the Republic of Estonia). All persons caring for animals and conducting experiments have personal licenses allowing them to conduct experiments on animals. During the experiments, every effort was made to minimize the number of animals and their suffering.

All experiments were performed on 120 male mice of the C57VB / 6 line. Animals were purchased from the company Nag1ap (England). The average age of the animals at the time of arrival at the vivarium of the Biomedical Center of the University of Tartu was 6 weeks (120 mice). Upon arrival, the animals were quarantined for 2 weeks in the vivarium of the Biomedical Center of the University of Tartu. After which the animals were transferred to the vivarium of the Institute of Pharmacology (room number 3028). The animals were kept in plastic cages (5 mice in a cage) measuring 25x45x12cm (wxdxh) without restriction in food and water, with a 12-hour light cycle (the light turns on automatically at 8.00). Cells with animals were kept in special climatic containers (Zapnig, Denmark). The containers are equipped with HEPA filters for air purification and have 24-hour monitoring of humidity and air temperature. Relative humidity in containers is maintained within 50 ± 2%, temperature 22 ± 1 ° С. Access to animals is allowed only to personnel who have the appropriate license. Cell cleaning and provision of food and water are carried out once a day. Animal feed: granulated K70 (Eac1ash1p, 31oscoyo1sh, Sweden). Animals were kept in climatic containers for 1 week before the start of the experiments. 2 series of experiments were carried out and 60 mice were used in each series. In the first series of experiments, the effect of substances on fatigue syndrome caused by repeated physical exertion (swimming in the pool) was studied. In the second series of experiments, the effect of substances on the development of behavioral syndrome caused by stress exposure (chronic variable stress) was studied. For experiments with repeated physical exertion, mice aged 8-8.5 weeks were taken; for experiments where variable stress was used, the age of the mice was 10-10.5 weeks.

In the first series of experiments, the body weight of mice at the beginning of the experiment was 22.7 ± 0.3 g (n = 60), in the second series of experiments, the mass of mice was 23.1 ± 0.2 g (n = 60).

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All test substances were administered as a suspension in a 1% starch paste using a gastric tube once a day during the day (12.00-13.00) for 7 days. Solutions of substances were prepared daily just before the start of administration. Doses of the investigated substances are given in table. one.

Doses of the investigated substances used in the studies, and their designations in the text of the report.

Table 1

Doses (mg / kg) Doses (mg / kg) of substances, where Designation substances levocarnitine recalculated combinations of substances in provided by by customer on levocarnitine l-tartrate report Riboxin 148.6 mg / kg Riboxin 148.6 mg / kg Riboxin Bemitil 74.2 mg / kg + Levocarnitine 185.8 mg / kg mg Bemitil 74.2 mg / kg Levocarnitine B-tartrate 272.7 mg / kg Low Dose (MD) Bemitil 148.4 mg / kg + Levocarnitine 371.6 mg / kg Bemitil 148.4 mg / kg Levocarnitine B-tartrate 545.4 mg / kg Average Dose (DM) Bemitil 222.6 mg / kg + Levocarnitine 557.4 Bemitil 222.6 mg / kg + Levocarnitine B-tartrate 818.0 mg / kg Large dose (DB)

mg / kg

mg / kg

Physical fatigue syndrome caused by repeated physical exertion.

Before starting the experiments, animals were encoded and divided into 6 groups of 10 animals each using a randomization program.

Groups of animals, studied substances and experimental design are given. The time after the last administration of the test substances is indicated.

table 2

No. column Zhi in the The introduction of substances 1 time per day in within 7 days Physical kaya load 2 times a day in within 7 days Test rotate the ongoing rod Test open go field Depression test different held not Test recognized Avania new facility one The control - 24 hours 48 hours 48 hours 48-50-72 hours 2 The control: physical load + 24 hours 48 hours 48 hours 48-50-72 hours 3 Riboxin + 24 hours 48 hours 48 hours 48-50-72 hours 4 Bemityl + levocarnitine (MD) + 24 hours 48 hours 48 hours 48-50-72 hours 5 Bemityl + levocarnitine (SD) + 24 hours 48 hours 48 hours 48-50-72 hours 6 Bemityl + levocarnitine (DB) + 24 hours 48 hours 48 hours 48-50-72 hours

The method of physical activity.

Physical activity in mice was carried out 2 times a day. To do this, we used a pool measuring 25x12x25 cm, filled with water (25 ° C). Animals were placed in the pool and were in the water for 6 minutes. The load was carried out in the morning from 8.00 to 11.00 and in the evening from 17.00 to 20.00 on all animals, with the exception of the first control group of mice. During the last swimming session in the pool, the time was measured during which the animals were stationary when swimming. At the same time, 4 identical pools were used in the experiment and swimming sessions were recorded on videotape. The behavior analysis was carried out on the basis of a video recording by an employee who was not familiar with the protocol of substance administration.

The test substances were administered daily in between swimming sessions (12.00-8,027,170

13.00). 24 hours after the last injection of the test substances, behavioral experiments were carried out according to the scheme shown in Table. 2.

Fatigue syndrome caused by repeated variable stress exposure in mice.

Before starting the experiments, animals were encoded and divided into 6 groups of 10 animals each using a randomization program. The stress effect was carried out according to the scheme presented in table. 3, twice a day for seven days. The test substances were administered once a day (12.0013.00) for 7 days.

Table 3

Characteristics of stress

Days experiences Stressful effect (30 min) 08.00-08.30 Stressful effect (30 min) 17.00-17.30 one Immobilization of animals in the re-trainer Swimming in cold water 3 sessions of 5 min 2 Swimming in cold water 3 sessions of 5 min Striking electric current (0.1 A, 30

sec, 10 strokes) 3 Electric shock (0.1 A, 30 sec, 10 strokes) Bright light 120 lux 4 Bright light 120 lux Immobilization of animals in the re-trainer 5 Room in a cold room (+ 4 ° C) Hanging by the tail 3 sessions of 5 minutes 6 Hanging by the tail 3 sessions of 5 minutes Wet litter 7 Wet litter Hanging by the tail 3 sessions of 5 minutes

24-72 hours after the last injection of the substances, the behavior of animals was evaluated: physical performance, the presence of symptoms of depressive behavior, fear, anxiety, and mnemonic functions.

Assessment of behavioral reactions.

Assessment of physical performance was carried out according to the duration of the stationary state of the animals during the last swimming session, as well as using the test of a rotating rod. Testing of animals using the rotating rod technique was carried out 24 hours after the last injection of substances. In our experiments, we used a rotating rod with a diameter of 30 mm manufactured by T8E 8u§1esz, Toure Νο. 337500-M / A; 8epa1 Νο. 120402-06 (Germany) with automatic registration of the retention time of mice on the rod. In preliminary experiments, it was found that for a given diameter of the rod, the optimal rotation speed should be 20 rpm. The ability to retain animals on the rod depended on the degree of their fitness. Therefore, during the experiment, on the 1st, 3rd and 5th days of administration, training sessions were conducted for all mice, including control animals without physical activity. In this case, the rotation speed during each session with the help of software gradually increased from 0 to 20 rpm. The device allowed simultaneous testing of up to 5 animals. The device was connected to a computer, allowing you to automatically record data on the retention time of animals on the rod.

Anxiety and fear were assessed using an open field test 48 hours after the last injection of substances. The open field is a box measuring 40x40x40 cm, divided into 16 squares. The number of crossed squares (motor activity) and the time spent by the mice in the central squares were estimated. Animals with low anxiety spend more time in the central squares.

Assessing the ability of animals to actively avoid stressful effects (test for depressive state) was carried out in the test of hanging by the tail. In this state, animals actively seek to climb the stand and get out of this state. Animals with depressive symptoms do not resist and remain motionless. The time during which the animals were stationary for 180 s of observation was determined. Testing was carried out 48 hours after the last injection of substances.

Evaluation of episodic memory on the model of recognition of a new object in mice was carried out using the test of recognition of a new object. This test is widely used to evaluate episodic memory in animals. The test is based on the fact that a healthy animal examines a new object much more time than an old one.

The experiment consisted of three phases: the addictive phase, the training phase and the retention phase (see table. 2).

1) The addictive phase.

During this phase, the mice were individually placed in a wooden box measuring 50x50x50 cm (length and width height), located in an experimental room dimly lit by incandescent lamps, with constant illumination of 60 lux. The box floor was divided into 16 identical squares with a side length of 12.5 cm. The animal was in the box for 5 minutes and the experimenter recorded the number of crossed squares. This indicator was subsequently used to assess the motor activity of mice. After 5 minutes, the animal was removed from the crate and the floor of the crate was wiped with a 5% ethanol solution to eliminate odor.

2) Training phase.

2 hours after the end of the addictive phase, the training phase was carried out. For this, the animal was again placed in the center of the box, on the floor of which two identical objects were installed. The objects were two wooden cubes located in opposite corners of the box (Fig. 1).

Scheme of experience in the study of episodic memory in the test of recognition of a new object in mice.

The animal was given the opportunity to examine objects for 5 minutes, and at the same time, the time during which the animal examined each of the objects was recorded. These data are necessary to assess the degree of motivation and research activity of animals. After each animal, the box floor was wiped with a 5% ethanol solution. At the end of the experiment, the animal was placed in a home cage.

3) Retention phase.

72 hours after the last physical activity, the animals were again placed in a study box in which one object was replaced with a new object of a different shape and color (Fig. 1).

The animal was again given the opportunity to explore the old and new objects for five minutes, and the time of investigation of each object was recorded by the experimenter.

The research preference coefficient of a new object was presented as the ratio of the time of research of a new object with respect to the total time of research of the old and new objects according to the formula (Tnovx100) / (Tst + Tnov), where Tst and Tnov are the time of investigation of the old and new objects.

Statistical analysis.

For each group of mice, the mean values (M) were calculated ± the standard error of the mean (t). Further, the data were analyzed using a one-way analysis of variance followed by a retrospective Bonferroni test (action of the test substances, dose effect). Differences between groups were considered significant at p <0.05. Statistical data processing was carried out using the statistical software SgarBab ΡΚΙ8Μ-5 (USA).

Research results and discussion of the results.

The rationale for the selection of doses of bemitil and levocarnitine for research

Doses of the test substances were provided by the customer of the study. After agreeing on the dose of levocarnitine, the bases were counted on levocarnitine tartrate (see research methods, Table 1).

The influence of the studied substances on the behavior of mice on the background of repeated physical exertion for 7 days.

The influence of the studied substances on the body weight of animals during repeated physical exertion.

During the experiments, one animal in the control group with physical activity died on the 3rd day of the substance administration. An autopsy revealed an esophageal perforation. Therefore, in this group the number of animals was 9 mice.

The effect of physical activity and the studied substances on the body weight of animals are given in table. 4 and FIG. 2.

Table 4

The influence of the studied substances on the dynamics of animal body mass during repeated physical exertion

Group No. Body weight (g) on the 1st day of administration Body weight (g) on the 7th day of administration The dynamics of the mass (g) of the body of animals The control 22.6 ± 0.6 23.1 + 0.6 0.5 + 0.1 Control (physical activity) 22.4 ± 0.4 21.7 + 0.7 - 0.7 ± 0.3 ** Riboxin 23.5 ± 0.6 21.9 + 0.6 -0.5 ± 0.2 * Bemitil + Levocarnitine (MD) 23.1 + 0.5 23.2 ± 0.5 0,1 + 0,11 Bemitil + Levocarnitine (SD) 21.8 + 0.9 21.4 + 0.8 -0.4 ± 0.2 Bemitil + Levocarnitine (DB) 22.3 ± 0.6 21.9 + 0.7 -0.5 ± 0.2

The mean values and standard errors of the mean (M ± m) for a group of 9-10 mice are presented. * p <0.05; ** p <0.01 compared with control (Bonferroni test).

Analysis of variance revealed a significant effect of physical activity and the action of substances on the dynamics of body weight: P5.53 = 4.763, p = 0.001. Subsequent analysis of intergroup differences using the Bonferroni test showed that repeated physical activity significantly (p <0.01) reduced body weight

- 10,027,170 mice. The studied combination of boehmyl + levocarnitine with repeated administration reduced the drop in body weight. The effect of substances is most clearly shown in FIG. 2. As can be seen from FIG. 2, the combination of bemitil + levocarnitine in small doses had the most noticeable effect on weight loss as a result of physical exertion. The effect of medium and large doses of a combination of bemitil + levocarnitine was manifested to a somewhat lesser extent. Riboxin did not significantly affect the dynamics of body weight during physical fatigue of animals (table. 4, Fig. 2).

The influence of the studied substances on the physical performance of mice after repeated physical exertion.

The physical fatigue of mice was evaluated by two tests: firstly, by the time of a stationary state during swimming in the pool in the last swimming session and, secondly, by the time of keeping animals on a rotating rod. The results of the first experiment are presented in table. 5.

Table 5

The influence of the studied substances on the duration (s) of the stationary state of mice when swimming in the pool during the last swimming session

Test substances The number of mice in the group Time (sec) motionless state when swimming in the pool 1. Control 10 84.2 + 12.3 2. Control: physical activity nine 154.1 + 12.2 ** 3. Riboxin 10 142.5 + 11.1 * 4. Bemitil + Levocarnitine (MD) 10 100.8 + 11.3 5. Bemitil + Levocarnitine (DM) 10 124.5 + 13.0 B. Bemitil + Levocarnitine (DB) 10 114.2 + 18.2

The mean values and standard errors of the mean (M ± m) for a group of 9-10 mice are presented. ** P <0.01 compared with the control without physical activity (Bonferroni test).

As experiments showed, after repeated physical exertion (swimming in the pool), the animals developed fatigue syndrome, which manifested itself in an increase in the immobility time during the last swimming session in the pool. Analysis of variance showed statistically significant differences: P5.53 = 3.746; p = 0.0056. A statistical analysis of intergroup differences using the Bonferroni test also revealed a statistically significant (p <0.01) effect of physical activity on the time of immobility of the animals (Table 5). The combination of boehmyl + carnitine in small doses (MD) reduced the immobility time of mice compared to the control group receiving exercise, but the data did not reach a statistically significant level. On the other hand, a statistically significant difference with control without physical exertion also disappeared, which indicates a partial effect of the combination of bemitil + levocarnitine on physical performance. The average effect (DM) and high dose (DB) of the combination of bemitil + levocarnitine had the same effect. Riboxin had no effect on physical performance in the swimming test (Table 5).

In the following experiment, the effect of substances on the physical performance of animals was evaluated in a rotating rod test. The results of the experiments are given in table. 6.

Table 6

The influence of the studied substances on the retention time of mice on a rotating rod after repeated physical exertion

Group No. and test substances The number of mice in the group The time (sec) retention of mice on rotating rod 1. Control 10 160.3 + 12.3 2. Control: physical activity nine 105.2 + 12.4 * 3. Riboxin 10 110.2 + 10.0 4.Bemitil + Levocarnitine (MD) 10 160.6 + 12.9 # 5. Bemitil + Levocarnitine (DM) 10 119.4 + 14.35 6 Bemitil + Levocarnitine (DB) 10 107.7 + 3.8

The mean values and standard errors of the mean (M ± m) for a group of 9-10 mice are presented. * P <0.05 compared with control without physical activity; # p <0.05 compared with control after

- 11 027170 physical activity (Bonferroni test).

As experiments showed, after repeated physical exertion (swimming in the pool) the animals developed fatigue syndrome, which manifested itself in a reduction in the retention time on a rotating rod. Analysis of variance showed statistically significant differences: B5.53 = 5.212; p = 0.0006. Subsequent intergroup statistical analysis using the Bonferroni test revealed a statistically significant (P <0.05) effect of physical activity on the retention time of animals on a rotating rod (Table 6). The combination of boehmyl + carnitine in small doses (MD) statistically significantly (p <0.05) prevented physical fatigue syndrome, which manifested itself in an increase in the retention time of mice on a rotating rod. Medium (DM) and higher doses (DB) of bemitil + levocarnitine combination did not affect the physical performance of mice after exercise. Also, the effects of riboxin were not detected.

The influence of the test substances on locomotor activity and the level of anxiety in mice in an open field test after repeated physical exertion.

Data on the influence of the studied substances on the behavior of animals in the open field test are presented in table. 7.

Table 7

The effect of the test substances on locomotor activity (number of sectors crossed) and anxiety level (time in seconds spent in central sectors) of mice after repeated physical exertion in an open field test

Group No. and test substances The number of mice in the group number nipped sectors Time (s) being in central sectors 1. Control 10 123.8 + 11.9 11.0 + 1.7 2. Control: physical activity nine 140.8 + 15.3 8.4 + 1.0 3.Riboxin 10 132.6 + 8.2 11.3 + 1.8 4.Bemitil + Levocarnitine (MD) 10 135.7 + 10.8 12.9 + 3.9 5. Bemitil + Levocarnitine (DM) 10 128.6 + 9.7 9.4 + 1.6 6. Bemitil + Levocarnitine (DB) 10 107.7 + 3.8 10.2 + 2.1

The mean values and standard errors of the mean (M ± m) for a group of 9-10 mice are presented. Statistical analysis showed that repeated physical activity does not affect motor activity (the number of sectors crossed) and the level of anxiety (time spent in the central sectors) in an open field test. There was also no statistically significant effect of substances in the open field test (Table 6).

The effect of the test substances on the depressive state of mice after repeated physical exertion in the tail suspension test.

The results of this test are given in table. 8.

Table 8

The effect of the test substances on the depressive state in mice after repeated physical exertion. Assessment of a depression-like state was determined by the time (s) of immobility when hanging by the tail

Test substances Qty mice Time (sec) of immobility in the tail suspension test 1. Control 10 157.0 + 11.7 2. Control: physical activity nine 142.0 + 5.0 3. Riboxin 10 152.1 + 12.3 4. Bemitil + levocarnitine (MD) 10 136.0 + 16, 3 5. Bemitil + levocarnitine (DM) 10 135.3 + 19.7 6. Bemitil + levocarnitine (DB) 10 118.6 + 19.2

The mean conceptions and standard errors of the mean per group of 9-10 mice are given.

Repeated physical exertion as well as the studied substances did not affect the immobility time of mice in the tail suspension test (Table 8).

The effect of repeated physical exertion and the test substances on episodic memory in mice after repeated physical exertion.

The results of studying the effect of repeated physical exertion (swimming in the pool) and 12,027,170 test substances on episodic memory in mice are presented in Table. nine.

Animals of the control group without physical activity showed a pronounced preference in the study of a new object: the preference index in the control group was 76.0 ± 2.3%. Repeated physical activity in the control group did not significantly affect the episodic memory of animals. Also, the action of the test substances on episodic memory was not detected (Table 9).

Table 9

The influence of the studied substances on the episodic memory of mice in the test of recognition of a new object after repeated physical exertion (swimming in the pool)

Group No. and Test Substances Number animals New Object Preference Index

1. Control 10 76.0 + 2.3 2. Control: physical activity nine 71.9 + 3.2 3. Riboxin 10 60.8 + 2.6 4. Bemitil + levocarnitine (MD) 10 70.8 + 3.4 5. Bemitil + levocarnitine (DM) 10 72.1 ± 4.6 6. Bemitil + levocarnitine (DB) 10 62.4 + 4.1

Presents the average values of the preference index of the new object ± standard errors of the mean (M ± m) for a group of 9-10 animals.

Conclusion

Repeated physical activity (swimming in the pool twice a day for 7 days) caused physical fatigue syndrome in mice, which manifested itself in an increase in the duration of a stationary state when swimming in the pool during the last session and in a decrease in the retention time on a rotating rod. The combination of the studied substances bemitil + levocarnitine when re-administered in small doses (74.2 + 185.8 mg / kg) eliminated physical fatigue in both tests. However, with repeated administration of medium (148.4 + 371.6 mg / kg) and large doses (222.6 + 557.4 mg / kg) combinations of bemitil + levocarnitine, this effect manifested itself to a much lesser extent or disappeared. Riboxin at a dose of 148.6 mg / kg, administered once a day in parallel with physical activity for 7 days, did not affect the performance of animals.

Repeated physical exertion did not affect the level of spontaneous motor activity, anxiety level, episodic memory, and did not cause depressive symptoms in mice. The studied combinations of boehmyl + levocarnitine and riboxin did not affect these behavior parameters of mice either.

The influence of the studied substances on the dynamics of the body mass of mice with repeated stress.

The influence of the studied substances on the dynamics of body weight during repeated variable stress is shown in table. 10.

Table 10

The influence of the studied substances on the dynamics of body weight of animals with repeated stress

Group No. Body mass (d) on the 1st day introductions Body mass (d) on the 7th day introductions Dynamics body weight (g) animals The control 23.2 ± 0.5 24.9 ± 0.7 1.7 + 0.4 Control (repeated stress) 23.0 ± 0.5 23.9 ± 0.5 0.9 ± 0.3 Riboxin 23.9 + 0.6 26.2 ± 0.6 2.3 ± 0.4 # Bemitil + Levocarnitine (MD) 23.4 ± 0.5 25.1 + 0.5 1.7 + 0.3 Bemitil + Levocarnitine (SD) 22.4 ± 0.8 23.8 + 0.8 1.5 + 0.3 Bemitil + Levocarnitine (DB) 22.8 ± 0.6 23.6 + 0.5 0.8 ± 0.3

- 13 027170

The mean values and standard errors of the mean (M ± m) for a group of 9-10 mice are presented.

# p <0.05 compared with a stressed group of mice.

Repeated stress tended to decrease the gain in body weight of mice. Under the influence of riboxin and a combination of boehmyl + levocarnitine in a small dose, the increase in body weight was somewhat accelerated (Fig. 3). It should be noted that, although the analysis of variance showed the presence of a statistically significant effect of the group (P5.54 = 3.421, p <0.01), further analysis using the Bonferroni test revealed a statistically significant difference in the dynamics of body weight of mice between stressed animals and animals treated Riboxinum (tab. 10, fig. 3).

The influence of the studied substances on the physical performance of mice after repeated stress loads.

The physical fatigue of mice was evaluated by the time of immobility when swimming in the pool and by the retention time on a rotating rod. The results of the experiments are presented in table. 11 and 12.

Table 11

The influence of the studied substances on the duration (c) of the stationary state of mice when swimming in the pool after repeated stress

Group No. and investigated substance The number of mice in the group Time (sec) motionless state when swimming in the pool 1. Control 10 97.4 + 12.3 2. Control: stress 10 190.0 + 5.3 *** 3. Riboxin 10 107.9 + 12, Z # 4. Bemitil + levocarnitine (MD) 10 100.9 + 8.2 ## 5. Bemitil + levocarnitine (DM) 10 111.4 ± 10.9 ## 6. Bemitil + levocarnitine (DB) 10 91.8 ± 7.9 ##

The mean conceptions and standard errors of the mean (M ± m) for a group of 10 mice are presented. *** P <0.001 compared with the control without stress; # p <0.05, ## p <0.01 compared with the stress control (Bonferroni test).

As experiments have shown, repeated stress caused fatigue syndrome in animals, which manifested itself in an increase in the immobility time during swimming in the pool. Analysis of variance showed statistically significant differences: P5.54 = 16.15; p = 0.0001. Subsequent statistical analysis using the Bonferroni test revealed a statistically significant (p <0.01) effect of stress on the time of immobility of animals (table. 11). The combination of boehmyl + levocarnitine in all doses (MD, DM and DB) statistically significantly (p <0.001) reduced the immobility time of mice when swimming in the pool compared to the control group, which was subjected to stress. Riboxin also significantly (p <0.05) reduced the immobility time of mice when swimming in the pool (table. 11).

Table 12

The influence of the studied substances on the retention time of mice on a rotating rod after repeated stress

Group No. and investigated substance The number of mice in the group Time (sec) mouse retention on rotating pivot 1. Control 10 172.7 + 27.7

- 14,027,170

2. Control: stress 10 101.4 ± 10.4 * 3. Riboxin 10 104.9 ± 14.6 4. Bemitil + levocarnitine (MD) 10 130.4 ± 15.9 5. Bemitil + levocarnitine (DM) 10 87.9 ± 12.6 * 6. Bemitil + levocarnitine (DB) 10 95.7 ± 11.0 *

The mean values and standard errors of the mean (M ± m) for a group of 10 mice are presented. * P <0.05 compared with the control without stress (Bonferroni test).

As experiments showed, after repeated stressful loads, animals developed fatigue syndrome, which manifested itself in a reduction in retention time on a rotating rod. Analysis of variance showed statistically significant differences: B5.54 = 3.665; p = 0.0063. Subsequent statistical analysis of differences between the individual groups using the Bonferroni test revealed a statistically significant (p <0.05) effect of stress on the retention time of animals on a rotating rod (Table 12). The combination of boehmyl + levocarnitine in small doses (MD) partially reduced physical fatigue, which was manifested in an increase in the retention time of mice on a rotating rod. At the same time, the statistically significant difference between the control group without stress and the group with stress load disappeared. However, the combination of bemitil + levocarnitine in medium and large doses (DM and DB) did not affect the retention time of stressed animals on a rotating rod. Riboxin did not affect retention time on a rotating rod in stressed animals.

The effect of the test substances on locomotor activity and anxiety level in mice in an open field test after repeated stress.

Data on the influence of the studied substances on the behavior of animals in the open field test are presented in table. 13. Repeated variable stress significantly (p <0.05) increased the locomotor activity of animals in an open field (Table 13). The studied substances partially suppressed motor activity, however, the data did not reach the level of reliability. Repeated stress exposure did not affect the time spent by mice in the central sectors of the open field (anxiety indicator) and there was also no significant effect of the test substances on this indicator. Analysis of variance was as follows: B5.54 = 2.19; p = 0.067.

Table 13

The effect of the test substances on locomotor activity (number of sectors crossed) and anxiety level (time spent in central sectors) after repeated stress exposure in mice in an open field test

Group No. and investigated substance number mice number crossed sectors Time (sec) finding in central sectors 1. Control 10 86.0 ± 8.2 8.5 ± 2.3 2. Control: physical load 10 141.8 ± 12.5 * 15.3 ± 2.3

3. Riboxin 10 111.6 ± 11.9 10.3 ± 2.4 4. Bemitil + levocarnitine (MD) 10 135.9 ± 11.2 15.9 ± 1.9 5. Bemitil + levocarnitine (SD) 10 116.8 ± 13.6 8.7 ± 1.7 6. Bemitil + levocarnitine (DB) 10 109.8 ± 6.2 12.7 ± 1.1

- 15 027170

The mean conceptions and standard errors of the mean (M ± m) for a group of 10 mice are presented.

* P <0.05 compared with the control without stress (Bonferroni test).

The effect of the test substances on the depressive state of mice after repeated stress exposure in the tail suspension test. The results of the experiments are given in table. 14.

Table 14

The effect of the test substances on the depressive state in mice after repeated stress. Assessment of a depression-like state was determined by the time (s) of immobility when hanging by the tail

Group No. and subjects number Time (sec) of immobility in substance mice tail hanging test 1. Control 10 91.5 ± 2.6 2. Control: repeated stress 10 169.9 ± 7.1 *** 3. Riboxin 10 128.5 ± 8.3 #

4. Bemitil + levocarnitine (MD) 10 126.9 ± 10, B ## 5. Bemitil + levocarnitine (SD) 10 121.6 ± 12.2 ## 6. Bemitil + levocarnitine (DB) 10 107.7 ± 4.3 ###

The mean conceptions and standard errors of the mean (M ± m) for a group of 10 mice are presented. *** P <0.001 compared with the control without stress; # p <0.05, ## p <0.01, ### p <0.001 compared with the stress control (Bonferroni test).

Analysis of variance showed a significant effect of the group: E5.54 = 10.48; p <0.0001. An analysis of intergroup differences revealed a significant (p <0.001) effect of stress on the development of a depression-like state, which manifested itself in an increase in the immobility time in the tail suspension test. The studied combination of substances in all doses significantly reduced the immobility time in the tail suspension test (Table 14). Riboxin also reduced immobility time in this test. Thus, we can conclude that the studied substances weaken the depressive state in mice, which develops as a result of repeated stress exposure.

The effect of repeated stress and test substances on episodic memory in mice.

The results of a study of the effect of repeated stress and test substances on episodic memory in mice are presented in table. fifteen.

In animals of the control group, preference was observed in the study of a new object: the index of preference in the control group was 67.7 ± 4.4%. Repeated stress exposure in the control group did not significantly affect the episodic memory of animals. Also, the action of the test substances on episodic memory was not detected (Table 15).

Table 15

The influence of the studied substances on the episodic memory of mice in the test of recognition of a new object after repeated stress exposure

Group No. and subjects substance number animals Preference index new facility 1. Control 10 67.7 ± 4.4 2. Control: stress 10 71.5 ± 2.7 3. Riboxin 10 60.8 ± 2.6 4. Bemitil + levocarnitine (MD) 10 70.8 ± 3.3 5. Bemitil + levocarnitine (SD) 10 72.1 ± 4.6 6. Bemitil + levocarnitine (DB) 10 62.4 ± 4.1

Presents the average values of the preference index of the new object ± standard errors of the mean (M ± t) for groups of 10 animals.

Conclusion

In mice, repeated stress exposure led to increased physical fatigue in mice, which was manifested in a slight decrease in body weight gain, an increase in the time of an inactive state when swimming in the pool, and a reduction in the retention time on a rotating rod. Repeated stress exposure also led to the development of a depression-like state, which manifested itself in an increase in the immobility time in the tail suspension test. The combination of the studied substances boehmyl + levocarnitine when re-administered in small doses (74.2 + 185.8 mg / kg) slightly increased the gain in body weight during stress and statistically significantly eliminated physical fatigue in both swimming and rotating rod tests. Repeated administration of medium (148.4 + 371.6 mg / kg) and large doses (222.6 + 557.4 mg / kg) combinations of boehmil + levocarnitine also reduced the immobility time when swimming in the pool, however, in the rotating rod test this effect manifested to a much lesser extent or disappeared. Riboxin at a dose of 148.6 mg / kg, administered once a day for 7 days, increased the physical performance of animals when swimming in the pool, but did not affect the ability of mice to stay on a rotating rod.

The combination of bemitil + levocarnitine over the entire dose range reduced the depressive state of animals caused by repeated stress. However, it should be noted that this effect may be associated with their improving effect on physical performance. Rather, their effect on physical performance predominates in the spectrum of action of these substances, and not their antidepressant effect.

Claims (4)

CLAIM 1. A pharmaceutical composition for treating asthenia and / or chronic fatigue syndrome, characterized in that the active components contain 2-ethylthiobenzimidazole hydrobromide and / or its pharmaceutically acceptable salt and levocarnitine in therapeutically effective amounts. 2. The pharmaceutical composition according to claim 1, characterized in that as the target additives contains substances selected from the group of sugars, sugar substitutes, flavor substitutes, disintegrating agents, disintegrant, flavoring agent in the following ratios, wt.%: Ethylthiobenzimidazole hydrobromide: 5.46-15.15. Levocarnitine: 21.86-44.55. Filler selected from the group of sugars: 9.09-42.62. Filler selected from the group of sugar substitutes: 9.09-16.39. Filler selected from the taste masking group: 2.42-6.56. A filler selected from the group of disintegrating components: 1.21-4.37. Filler selected from the group of substitutes for loosening agents: 1.21-2.19. Filler selected from the group of flavorings: 0.30-0.55. 3. The composition according to claim 1, characterized in that it is presented in solid dosage form. 4. The composition according to claim 3, characterized in that the solid dosage form is a tablet for resorption. ^one