RU2377237C1 - 2-ethyl-6-methyl-3-hydroxyliridiniumhydroxybutanedioat expressing anti-ishemic, cerebroprotective, neurotropic and lipid-regulating activity, pharmaceutical compositions and medical product - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention refers to medicine, specifically to a new chemical compound - 3-hydroxypyridine derivative of formula

expressing anti-ischemic, cerebroprotective, neurotropic and lipid-regulating activity, and to solid and liquid pharmaceutical compositions based thereon, and to application in manufacturing of medical products for prevention and treatment of cardiovascular, mental diseases, cerebral ischemia and obesity.

EFFECT: new compound which can be perspective in manufacturing of medical products for prevention and treatment of cardiovascular, mental diseases, cerebral ischemia and obesity.

12 cl, 11 ex, 16 tbl

Description

The invention relates to medicine, specifically to a new chemical compound - a derivative of 3-hydroxypyridine having anti-ischemic, cerebroprotective, neurotropic and lipid-regulated activities, pharmaceutical compositions based on this compound.

These properties suggest the possibility of using the proposed pharmaceutical compositions based on the new compound for the prevention and treatment of cardiovascular, mental diseases, cerebral ischemia and obesity.

The well-known anti-ischemic agent from the group of β-blockers - anaprilin (M.D. Mashkovsky. Medicines, M., Novaya Volna LLC, 2002, vol. I, pp. 254-256) realizes its positive properties mainly on background of oppression of the heart muscle (cardiodepressive effect), has a number of undesirable side effects (dizziness, bronchospasm, red blood cell aggregation, etc.).

A well-known nootropic agent that is widely used in medical practice is piracetam (MD Mashkovsky. Medicines, M., New Wave LLC, 2002, vol. I, pp. 111-113).

The purpose of the invention is a new compound with anti-ischemic, cerebroprotective, nootropic and lipid-regulated properties, which, unlike anaprilin, does not have cardio-depressant effects and undesirable side effects, pharmaceutical compositions based on the new compound, as well as the possibility of using the proposed pharmaceutical compositions based on the new compound for the prevention and treatment of cardiovascular, mental illness, cerebral ischemia and obesity.

This goal is achieved by a new compound, namely: 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula:

as well as pharmaceutical compositions containing a new compound with the working name Ethoxidol: tablets, injectable solutions.

The new compound is prepared in a known manner, namely by reacting 2-ethyl-6-methyl-3-hydroxypyridine with hydroxybutanedioic acid in the presence of a solvent by heating.

A solid dosage pharmaceutical composition in the form of a tablet containing the active substance and the desired additives is obtained by preliminary granulation followed by tableting. The ratio of active substance and target additives is, wt.%:

active substance 0.05-40.0 targeted supplements 60.0-99.95

As target additives, it optionally contains simultaneously starch, croscarmellose sodium, polyvinylpyrrolidones, sugars, cellulose derivatives, aerosil, glycine, N-acetyl-L-glutamic acid, 2- (dimethylamino) ethanol, stearic acid salts, corrective substances, in the following ratio components, wt.%:

starch 0,0-15,0 croscarmellose sodium 0,0-10,0 polyvinylpyrrolidones 0,0-10,0 Sahara 0,0-20,0 cellulose derivatives 0,0-50,0 aerosil 0,0-10,0 glycine 0,0-10,0 N-Acetyl-L-Glutamic Acid 0,0-15,0 2- (dimethylamino) ethanol 0,0-10,0 stearic acid salts 0,0-1,0 corrective substances 0,0-10,0

The presence in the solid dosage forms of starch, sugars, polyvinylpyrrolidones, croscarmellose sodium, cellulose derivatives provides an increase in the release rate and the absorption of the active substance, increases the disintegration and dissolution of the dosage form, and also creates conditions for the penetration of water and enzymes into the tablet. Starch is preferably corn or potato.

The presence of stearic acid salts provides the sliding effect required at the stage of tablet compression. It is advisable to use magnesium or calcium stearate as salts.

Glycine, N-acetyl-L-glutamic acid, 2- (dimethylamino) ethanol as targeted additives to the active substance contribute to the intensification of cellular metabolism and metabolism, redox processes, cell regeneration, lipid metabolism, which should positively affect the course treatment of coronary heart disease, cerebral ischemia and neuroprotection.

A pharmaceutical composition in the form of an injection solution containing the active substance and the desired additives is prepared by gradually dissolving the components in water for injection.

The ratio of active substance and target additives is, wt.%:

active substance 0.05-40.0 targeted supplements 60.0-99.95.

As target additives, it optionally contains simultaneously glycine, N-acetyl-L-glutamic acid, 2- (dimethylamino) ethanol, Trilon B in the following ratio, wt.%:

glycine 0,0-10,0, N-acetyl-L-glutamic acid, 0,0-50,0 2- (dimethylamino) ethanol 0,0-20,0 Trilon B 0,0-5,0

Trilon B as a target additive enhances the stability of the solution.

The claimed ratio of the components found experimentally, are optimal and allow you to get a technical result corresponding to the task:

pharmaceutical compositions meet the requirements of the State Pharmacopoeia of the 11th edition, have a shelf life of at least 2 years and provide a high pharmacotherapeutic effect of the active substance.

The synthesis of a new substance, the composition and methods of manufacturing the claimed pharmaceutical compositions are given in the following examples.

Example 1. 2-Ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate.

To 380 ml of 2-propanol, 31.28 g (0.288 mol) of 2-ethyl-6-methyl-3-hydroxypyridine, 31.49 g (0.235 mol) of hydroxybutanedioic acid are added. The reaction mass is heated and stirred under reflux at 65-70 ° C for 5-10 minutes, filtered, the filtrate is cooled to 5-10 ° C. The precipitate formed is separated, washed with 40 ml of chilled 2-propanol, and dried. Get 58.76 g of compound I (yield 95%), a white crystalline product with a melting point of 106-107 ° C.

C ₁₂ H ₁₇ NO ₆

Found,%: C 53.05; H, 6.14; N, 5.14.

Calculated,%: C 53.14; H, 6.27; N, 5.17.

IR spectrum, cm ^-1 : 1656, 1558, 1298, 1168, 1092, 854 (in liquid paraffin).

UV spectrum, nm: 223, 291 (in ethanol).

Example 2. A solid pharmaceutical composition in the form of tablets.

268.56 g of microcrystalline cellulose, 8.63 g of aerosil, 31.20 g of starch and 37.440 g of milk sugar are loaded into the apparatus, the mixture is moistened with an aqueous solution of 2- (dimethylamino) ethanol and N-acetyl-L-glutamic acid in an amount of 280, 97 g and mix thoroughly.

The resulting mass is granulated through a sieve with a hole diameter of 2 mm The granules are dried at 45-50 ° C to constant weight and wiped through a sieve with a hole diameter of 1 mm. Get 446.70 g of granulate No. 1. The output at the stage is 99.03%.

Similarly receive 125.98 g of granulate No. 2 from 105.05 g of Ethoxidol, 20.80 g of glycine and 13.45 g of a 10% aqueous solution of polyvinylpyrrolodone. The output at the stage is 99.03%.

To obtain a tablet mass, granules No. 1 and No. 2 are loaded into the apparatus, mixed thoroughly and dusted with a mixture of powders: aerosil - 1.24 g, croscarmellose sodium - 13.18 g, magnesium stearate - 6.18 g and flavoring - 6.18 g.

To obtain tablets, the resulting mixture is tabletted. Receive 600 g (1000 pieces) of Ethoxidol tablets on average 100 mg each. The yield is 95.2%, counting on the starting Ethoxidol.

Example 3. A solid pharmaceutical composition in the form of tablets.

Analogously to example 2, granulate No. 1 is obtained on the basis of 278.56 g of microcrystalline cellulose, 8.63 g of aerosil and 37.44 g of milk sugar, the mixture is moistened with an aqueous solution of 2- (dimethylamino) ethanol and N-acetyl-L-glutamic acid the amount of 280.97 g. Granulate 2 is obtained on the basis of 105.05 g of Ethoxidol and 33.12 g of a 10% aqueous solution of polyvinylpyrrolodone. To obtain tablets, a mixture of granules No. 1 and No. 2 was dusted with a mixture of croscarmellose powders - 14.21 g, calcium stearate - 6.07 g, flavoring - 6.31 g and tabletted.

Receive 600 g (1000 pieces) of Ethoxidol tablets on average 100 mg each.

The yield is 95.2%, counting on the starting Ethoxidol.

Example 4. A solid pharmaceutical composition in the form of tablets.

Analogously to example 2, granulate No. 1 is obtained on the basis of 306.00 g of cellulose, 8.63 g of aerosil and 37.44 g of sugar, the mixture is moistened with an aqueous solution of 2- (dimethylamino) ethanol and N-acetyl-L-glutamic acid in an amount of 280 , 97 g. Granulate No. 2 was prepared on the basis of 105.05 g of Ethoxidol, 10.40 g of glycine and 24.11 g of a 10% aqueous solution of polyvinylpyrrolodone. To obtain tablets, a mixture of granules No. 1 and No. 2 was dusted with a mixture of croscarmellose powders - 10.33 g, calcium stearate - 4.07 g, aerosil - 1.04 g and flavoring - 5.83 g and tabletted.

Receive 600 g (1000 pieces) of Ethoxidol tablets on average 100 mg each.

The yield is 95.5%, counting on the starting Ethoxidol.

Example 5. A solid pharmaceutical composition in the form of tablets.

Analogously to example 2, granulate No. 1 is obtained on the basis of 269.00 g of cellulose, 8.63 g of aerosil, 30.3 g of starch and 37.44 g of sugar, the mixture is moistened with an aqueous solution of 2- (dimethylamino) ethanol and N-acetyl- acid L-glutamine in an amount of 280.97 g. Granulate No. 2 was prepared on the basis of 105.05 g of Ethoxidol, 33.98 g of glycine and 13.45 g of a 10% aqueous solution of polyvinylpyrrolodone. To obtain tablets, a mixture of granules No. 1 and No. 2 was dusted with a mixture of croscarmellose powders - 10.33 g, magnesium stearate - 6.18 g, aerosil - 1.24 g and flavoring - 6.07 g and tabletted. Receive 600 g (1000 pieces) of Ethoxidol tablets on average 100 mg each. The yield is 97.2%, counting on the starting Ethoxidol.

Example 6. The pharmaceutical composition in the form of a solution for injection.

700.0 ml of water for injection is poured into a 2.0 L flask and 50.0 g of Ethoxidol, 16.0 g of 2- (dimethylamino) ethanol, 34.0 g of N-acetyl-L-glutamic acid are gradually added with stirring, 0.1 g of glycine and 0.5 g of Trilon B. After dissolving all the components, the solution was brought to a volume of 1000 ml, then the solution was filtered under aseptic conditions through a millipore 0.22 μm in diameter. 500 ampoules of 2 ml are obtained.

Example 7. A pharmaceutical composition in the form of a solution for injection.

Analogously to example 6 load of 50.0 g of ethoxidol, 20.0 g of 2- (dimethylamino) ethanol, 0.05 g of glycine. 500 ampoules of 2 ml are obtained.

Example 8. The pharmaceutical composition in the form of a solution for injection.

Analogously to Example 6, 50.0 g of Ethoxidol, 1.0 g of N-acetyl-L-glutamic acid and 0.05 g of Trilon B are loaded. 500 ampoules of 2 ml are obtained.

table 2 Pharmaceutical composition Ethoxidol in the form of a solution for injection Name of quality indicator Quality Standards Actual figures Example 6 Example 7 Example 8 Description Colorless or slightly colored liquid Colorless liquid Lightly colored liquid Colorless liquid Transparency The drug should be transparent (GFH1, issue 1, p.198) Transparent Transparent Transparent Color The color of the solution is not more intense than standard No. 7b (GF X1, issue 1, p.194) Not more intense than standard 7b Not more intense than standard 7b Not more intense than standard 7b Foreign matter Glutamic acid no more than 0.5% Less than 0.5% Less than 0.5% Less than 0.5% Stability 2 years at 20 ° C 2 years 2 years 2 years

Anti-ischemic, cerebroprotective, nootropic and lipid-regulated properties of the claimed substance are installed on generally accepted standard models.

Example 9. The study of anti-ischemic activity.

The experiments were carried out on white non-linear male rats, mice, rabbits and cats in comparison with Anaprilin.

The effect on the ratio of the necrosis zone to the ischemic zone during ischemic damage to rat myocardium was studied at a dose of 5% of LD ₅₀ . At the same dose, the effect on the development of reperfusion arrhythmias was studied. Ventricular fibrillation was caused by coronary artery occlusion. The effect of the drug on cardiohemodynamics and lipid peroxidation (LPO) induced by immobilization stress was determined.

It was shown that ethoxidol at a dose of 10 mg / kg eliminates ventricular fibrillation during occlusion of the coronary arteries and reduces the number of animals that experience ventricular fibrillation to 2 out of 8 experimental animals at a dose of 10 mg / kg and 2 out of 9 at a dose of 23 mg / kg.

Ethoxidol significantly increases the threshold for cat ventricular fibrillation at a dose of 23.0 mg / kg from 5 to 60 minutes after occlusion of the coronary arteries, while Anaprilin has this effect in an isotoxic dose from 30 to 60 minutes.

In conditions of acute myocardial ischemia at a dose of 15 mg / kg, it has a distinct cardioprotective effect on cardiodynamics, bringing the values of contractility (dP / dt), blood pressure and others to the level of intact control.

Ethoxidol at a dose of 1 mg / kg has the ability to reduce the intensity of manifestations of lipid peroxidation processes caused by rabbit immobilization for 30 days, as evidenced by a decrease in the level of malondialdehyde (MDA) in blood plasma by 46% and in kidney tissue. At the same time, the level of total cholesterol is reduced by 41.7%, and the atherogenic index is reduced by 82.9%.

Table 4 Changes in cardiodynamic parameters (%) in rats with acute myocardial ischemia in an open chest, n = 8 Time from the beginning of the experiment, min Recorded Parameters dp / dt max,% GARDEN, mmHg Heart rate, beats / min Exodus 100 ± 4.0 98 ± 5.2 432 ± 17.0 5 -15 ± 2.5 -15 ± 4.4 * -5 ± 1.4 10 -14 ± 5.0 -18 ± 4.1 * -1 ± 3.6 fifteen -19 ± 4.7 -18 ± 5.5 * -1 ± 4.0 twenty -17 ± 4.0 -22 ± 5.8 * -3 ± 2.7 thirty -18 ± 4.7 -22 ± 5.8 * -4 ± 2.2 40 -21 ± 4.7 -23 ± 6.5 * -5 ± 2.7 fifty -18 ± 4.7 -24 ± 6.2 * -5 ± 3.0 60 -19 ± 4.9 -20 ± 7.0 * -7 ± 3.0 Note: * - differences with the initial level are significant at p <0.05.

Thus, in an experiment, ethoxidol exhibits a pronounced cardioprotective effect, which is manifested in a decrease in the alteration of myocardial tissue under ischemic conditions, and restoration of cardiac hemodynamics parameters damaged by ischemia. In addition, it is characterized by an antioxidant effect and a lipid-regulating effect when modeling stress-conditioned prooxidant states.

Example 10. The study of neuroprotective activity.

The study of the nootropic effect of Ethoxidol was carried out using 2 methods:

- study of the effect of Ethoxidol on the learning ability of the conditioned reflex of passive avoidance of rats of under-education;

- study of the effect of ethoxidol on impaired memory - amnesia test of the conditioned passive avoidance reflex caused by scopolamine in rats.

The study of nootropic effects was carried out in a certified installation "Lafayette Instrument Co" (USA). The study was performed on white mongrel sexually mature rats males weighing 250-280 g

The test for reproducing learning was carried out 24 hours after training, the latent period of the animal's entry into the dark compartment was recorded, and then the number of animals that did not enter the dark dangerous compartment at all and remained on the illuminated hanging platform (rats that remember the situation well) for 180 s was recorded.

Ethoxidol and the reference drug Piracetam was administered once inside (intragastrically) using a special probe in a volume of 0.2 ml per 100 g of rat weight 40 minutes before training. The animals in the control group were injected with distilled water.

All the studied substances did not significantly change the latent time for completing the skill of entering the dark chamber (Table 3). The latent period of entry into the dark chamber during the performance of the mink reflex when the animals were first placed in the chamber did not change compared to the control (Table 5).

After this, passive avoidance reaction was trained, for which the rat received slight pain irritation in the dark chamber.

Control animals after training and with the subsequent reproduction of the trained 24 hours after training remembered the pain irritation received in the dark chamber and entered it with a long latent period. Only 1 rat out of 15 did not enter the dark chamber at all.

According to the results in Table 5, ethoxidol at a dose of 200 mg / kg and tablets at a dose of 100 mg / kg improve learning of the conditioned reflex of passive avoidance in rats, which is expressed in a significant increase in the latent time of entry into the dark chamber when the reflex is reproduced. Piracetam at a dose of 200 mg / kg also improved the learning of the conditioned reflex of passive avoidance in terms of the latent period of the reflex and in terms of increasing the percentage of animals that did not enter the dark chamber at all. Thus, ethoxidol at a dose of 200 mg / kg and tablets at a dose of 100 mg / kg improve the learning of the conditioned reflex of passive avoidance in rats of under-education and, in terms of the latency period of the reflex, has a similar effectiveness with Piracetam at a dose of 200 mg / kg.

The study of the antiamnestic effect of ethoxidol in the method of scopolamine amnesia.

The effect of substances on amnesia was studied in a certified Lafayette Instrument Co installation (USA).

The rat was seated on a brightly lit platform with its tail toward the open guillotine door leading into a dark chamber. Due to the mink reflex, after finding the entrance to the dark compartment of the chamber, the rat passed into the dark compartment. The latent time to complete this skill was recorded. Then the rat was left for 180 s in a dark chamber with the aim of assimilating it, and the rat spent almost all the time in a dark chamber.

To obtain amnesia of passive avoidance reaction, animals were injected with scopolamine (1.0 mg / kg ip) 30 minutes before training. The test for reproducing the trained and amnesia was performed 24 hours after the training, for which the rat was again placed on the platform with its tail to the hole and recorded:

- the latent period of entry of the animal into the dark compartment and

- then, for 180 s, the number of animals that did not enter the dark dangerous compartment at all and remained on the illuminated hanging platform (rats who remember the situation well).

Ethoxidol and Piracetam were administered once inside in a volume of 0.2 ml per 100 g of rat weight 40 minutes before training. The animals in the control group were injected with distilled water.

In control experiments, when a rat was placed on an illuminated platform, animals with a short latent period entered the dark chamber and received pain irritation (training) there. When the reflex is reproduced 24 hours after training, the rats remember the trained one, and when they are placed on the illuminated platform, the animals remain there and do not enter the dark dangerous chamber, where they received pain irritation (training) the day before (Table 6).

Scopolamine reliably shortens the duration of the latent period of entry into the dark chamber during the reproduction of the reflex and significantly reduces (up to 20%) the number of animals that did not enter the dark compartment of the chamber at all during reproduction, i.e. remembering the aversive stimulus applied there the day before (Table 6).

Ethoxidol at a dose of 100 mg / kg significantly (p≤0.05) increases (1.92 times) the duration of the latent period of rat entry into the dark compartment when reproducing the reflex compared with animals treated with one scopolamine. Along with this, under the influence of the drug, a significant increase (up to 50%) in the number of animals that did not enter the dark compartment of the chamber when reproducing the amnesized reflex, i.e. remembering the applied aversive stimulus (Table 6). The data obtained indicate the presence of a clear antiamnestic effect in Ethoxidol at a dose of 100 mg / kg.

Piracetam in a dose of 100 mg / kg significantly (p≤0.05) increases (1.8 times) the duration of the latent period of entry into the dark compartment when playing the reflex, but does not significantly increase the number of animals that did not go into the dark compartment of the chamber when playing the amnestied reflex.

Table 6 Anti-Amnestic Activity of Ethoxidol on the Model of Scopolamine Amnesia of the Passive Avoidance Conditioned Reflex Drug dose Latent period of entry into the dark chamber (s) Percentage of animals not entering the dark chamber Training Play The control
Dist water inside 16.2 ± 2.5 163.0 ± 17.0 90 Scopolamine 1 mg / kg, ip 10.1 ± 1.54 55.4 ± 21.25 * twenty* Ethoxidol 100 mg / kg + Scopolamine 14.6 ± 2.63 106.42 ± 22.4 # fifty# Ethoxidol 200 mg / kg + Scopolamine 11.3 ± 2.15 75.82 ± 23.0 27 Ethoxidol Solution 100 mg / kg + Scopolamine 13.9 ± 2.63 107.42 ± 22.4 # fifty# Piracetam 100 mg / kg + Scopolamine 12.3 ± 1.91 99.23 ± 14.7 # 40 Piracetam 200 mg / kg + Scopolamine 13.5 ± 1.52 105.73 ± 21.4 # fifty# * - p <0.05, the significance of differences by Student's t-test relative to the group of animals that received distilled water; * - p <0.05, the reliability of the values according to the student criterion with respect to control with amnesia.

Thus, a dose of 100 mg / kg of ethoxidol (solution) has an antiamnestic effect, which is characterized by a significant increase in the latent time of entry into the dark chamber and an increase in the number of animals that have not entered the hazardous chamber at all compared with the control animals with amnesia. Piracetam in doses of 100 and 200 mg / kg has a similar effect. Ethoxidol at a dose of 100 mg / kg is not inferior to Piracetam at a dose of 100 mg / kg in terms of anti-amnestic effect.

The study of the anti-alcohol effect of Ethoxidol was carried out in experiments on sexually mature outbred white male rats according to 2 tests: open field techniques and rotating rod techniques.

Ethoxidol and Piracetam were administered once orally (intragastrically) 40 minutes before the experiment.

We recorded horizontal locomotor activity by the number of crossed squares, vertical locomotor activity by the number of uprights and research activity by the number of openings (peeks).

Ethanol was administered at a dose of 1.5 mg / kg intraperitoneally and then after 10 minutes the test substance was administered at a dose of 100 mg / kg orally (intragastrically). Open field behavior was recorded 40 minutes after the administration of Ethoxidol or Piracetam. With the introduction of distilled water or ethanol alone (control studies), the behavior in the open field was recorded 40 minutes after their introduction.

As a result of the studies, it was found that ethanol at a dose of 1.5 g / kg does not change horizontal motor activity, but at the same time significantly reduces vertical motor activity. In addition, ethanol significantly and reliably reduces research activity, reducing the number of holes examined (Table 7).

Ethoxidol, administered at a dose of 100 mg / kg (inside) to intact animals, causes an unreliable decrease in horizontal and vertical locomotor activity and the number of holes examined.

Ethoxidol, when introduced after ethanol, restores disturbances in the behavior of rats in an open field, which is expressed in a significant increase in the uprights and the number of holes examined (Table 7). When studying the effect of Piracetam at a dose of 100 mg / kg on the effects of ethanol, it was found that Piracetam, like Ethoxidol, causes a significant increase in the number of uprights.

Thus, ethoxidol in a dose of 100 mg / kg orally has the ability to restore impaired motor activity and orientational research behavior of animals caused by ethanol in an open field, which is reflected in a significant increase in vertical struts and the number of holes examined.

Table 7 The Antialcoholic Effect of Ethoxidol on the Ability to Recover Ethanol-Disturbed Behaviors in an Open Field Test Substance, dose, route of administration The number of horizontal movements The number of vertical movements Number of holes examined The control is intact, dist. water 24.6 ± 2.02 9.5 ± 1.19 15.7 ± 1.54 Control - Ethanol (1.5 g / kg, w / b) 29.6 ± 2.8 5.0 ± 0.7 * 6.9 ± 1.23 * Ethoxidol (100 mg / kg, by mouth) 22.1 ± 7.4 6.8 ± 1.9 9.3 ± 4.7 Ethanol (1.5 g / kg, w / w) + Ethoxidol (100 mg / kg, inside) 32.3 ± 4.9 8.6 ± 1.2 # 9.7 ± 1.4 # Ethanol (1.5 g / kg, w / w) + Ethoxide (100 mg / kg, by mouth) tablets 34.0 ± 4.9 7.0 ± 1.9 9.8 ± 1.4 # Piracetam (100 mg / kg, by mouth) 26.4 ± 8.3 8.7 ± 2.1 12.4 ± 2.1 Ethanol (1.5 g / kg, ip) + Piracetam (100 mg / kg, by mouth) 21.9 ± 7.1 8.2 ± 1.3 # 9.5 ± 2.2 * - p <0.05, the significance of differences by student t-test relative to the group of intact animals; # - p <0.05, the significance of differences by student t-test relative to the group of animals treated with ethanol.

Piracetam at a dose of 100 mg / kg causes only an increase in the number of uprights. Ethoxidol is superior to Piracetam in anti-alcohol action in the open field test.

Violation of coordination of movement was assessed in the test of a rotating rod (rota rod test) with a revolution speed of 3 revolutions per minute. When a rat is placed on a rotating rod, the animal is normal, fingering with paws, held on it. Animals with side effects of a motor, sedative nature and other disorders are not kept on the rod and fall from it. The retention time on the rod and the number of animals that fell from the rotating rod within 2 minutes were recorded.

It was found that in the control group of rats that received distilled water, only one out of ten animals fell from the rod within 2 minutes of registration. Ethanol at a dose of 1.5 mg / kg dramatically disrupts the coordination of movements in rats: after the introduction of ethanol, 100% of the animals could not stay on the rotating rod (Table 8).

Ethoxidol at a dose of 100 mg / kg inward did not interfere with the coordination of movements in rats on a rotating rod. The introduction of ethoxidol against the background of ethanol led to the restoration of coordination of movements impaired by ethanol. Under the influence of Ethoxidol, an increase of up to 50% in the number of animals retained on the rod was observed (control after ethanol — 0%) and a significant increase (4.6 times compared with the control after ethanol) in the retention time on a rotating rod.

Table 8 The anti-alcohol effect of ethoxidol in its ability to restore impaired coordination of movements caused by ethanol in a rotating rod test Substance, dose, route of administration Retention time on the rod, s The percentage of animals held on the rod for 120 s Control intact, distilled water 103.5 ± 12.02 90 Control - Ethanol (1.5 g / kg, w / b) 18.5 ± 7.82 * 0 * Ethoxidol (100 mg / kg, by mouth) 110.7 ± 11.9 80 Ethanol (1.5 g / kg, w / w) + Ethoxide (100 mg / kg, solution) 85.8 ± 16.05 # 60 # Piracetam (100 mg / kg, by mouth) 108.5 ± 17.1 90 Ethanol (1.5 g / kg, ip) + Piracetam (100 mg / kg, by mouth) 58.3 ± 12.8 # fifty# * - p <0.05, the significance of differences by Student's t-test relative to the group of intact animals. # - p <0.05 significance of differences relative to the group of animals treated with ethanol.

Piracetam at a dose of 100 mg / kg orally did not disturb coordination in rats on a rotating rod. The introduction of Piracetam on the background of ethanol led to the restoration of coordination of movements impaired by ethanol. Under the influence of Piracetam, an increase of up to 50% in the number of animals not retained on the rod was observed (control after ethanol — 0%), and a significant increase (3.1 times compared with the control after ethanol) of the retention time on the rotating rod.

Thus, at a dose of 100 mg / kg, ethoxidol has a pronounced anti-alcohol effect in the test of a rotating rod, which is expressed in the restoration of coordination of movements impaired by ethanol under the influence of the drug. Under the influence of Ethoxidol, the retention time of rats on a rotating rod significantly increases (4.6 times) and the number of animals retained on a rod increases up to 60% (control with ethanol - 0%). Ethoxidol is superior to Piracetam in influencing ethanol-impaired motor coordination.

Example 11. An experimental study of the effectiveness of Ethoxidol for cerebral ischemia (cerebroprotor activity).

For the study, we used healthy sexually mature animals (rats) that were quarantined for 14 days, and animals of the same age group (6 months) were used in the experiment.

Studies were carried out on 3 groups of laboratory animals:

- control - animals with brain ischemia modeling;

- animals with cerebral ischemia, which were administered Ethoxidol at a dose of 5 mg / kg;

- animals with cerebral ischemia, which were administered Ethoxidol at a dose of 20 mg / kg.

Ethoxidol was administered intraperitoneally prophylactically (30 minutes before modeling the pathology) and therapeutically (immediately after ischemia).

1. To create cerebral ischemia, several standard models were used [Guide to the experimental (preclinical) study of new pharmacological substances. / Ed. R.U. Khabrieva. - 2nd ed., Revised. and add. - M .: OJSC "Publishing house" Medicine ", 2005. - 832 p .; Sernov L.N., Gatsura V.V. Elements of experimental pharmacology. - M., 2000. - 352 p.]:

gravitational overloads in cranio-caudal position;

clamping of both carotid arteries in white rats for 10-15 minutes against a background of systemic arterial hypotension up to 40 mm Hg;

Bilateral occlusion of both carotid arteries for 24 hours was used as a model of brain ischemia.

Total cerebral ischemia was modeled using gravitational overloads in a cranio-caudal position. Studies have shown that in the simulation of gravitational overloads [Gayevoy M.D., Adzhienko L.M., Makarova L.M. Cerebral ischemia caused by gravitational overload. // Expert. and wedge. pharmacol. - 2000. - T.63, No. 3, p.63-64] in the cranio-caudal direction, there is a decrease in blood pressure in the carotid artery to 0, ie animals have total brain ischemia. To simulate gravitational overloads, a centrifuge with a diameter of 2 m was used. Awake rats were placed in special container containers, fortified to the ends of the centrifuge levers, and a powerful engine created the necessary degree of gravity for 10 s. At the first stage of work, studies were carried out to identify the most effective doses of Ethoxidol, which increase the survival of animals with hypergravity. For this purpose, animals were injected with Ethoxidol intraperitoneally at doses of 5, 10 and 100 mg / kg prophylactically for 3 days before modeling the pathology. The value of gravitational overloads in this case was selected so that the lethality of animals in the control group was no more than 20%. The criterion of cerebroprotective action was an increase in the survival of animals (in%) in comparison with control animals.

The volumetric rate of cerebral blood flow (MK) in rats was recorded by the method of hydrogen clearance using a platinum electrode located on the surface of the sagittal sinus in the region of the sinus drain. The method is based on recording the rate of leaching of previously introduced hydrogen from the brain tissue and allows us to determine the volumetric velocity of MK [Gayevoy MD, Adzhienko LM, Makarova LM Cerebral ischemia caused by gravitational overload. // Expert. and wedge. pharmacol. - 2000. - t. 63, No. 3, p. 63-64].

MK was evaluated according to the curve of the change in the voltage of hydrogen at the electrode by the polarographic method and was calculated by the formula (1) to calculate the organ blood flow per 100 g of the brain.

where T _^1/2 - the time (in minutes) during which the value of the clearance curve decreases twice on a logarithmic scale;

λ is the ratio of the amount of hydrogen in 1 g of tissue to the amount of hydrogen in 1 ml of blood.

A boxed breathing apparatus was used to stabilize respiration in anesthetized rats.

Determination of autoregulatory responses of cerebral vessels

A necessary condition for studying the regulatory responses of cerebral vessels is the modeling of acute changes in systemic blood pressure (SBP). For this purpose, bloodletting was used, which allows to reduce the SBP to any level, and subsequent infusion of blood into the arterial bed allows you to raise the SBP to the initial level.

Given that deep anesthesia inhibits the regulatory responses of cerebral vessels, studies were conducted under mild anesthesia. Since the condition for studying the regulation of cerebral blood flow is a stable regime of blood oxygenation, experiments were carried out with artificial respiration under the control of pO ₂ and blood pH using a biological microanalyzer.

The nature of cerebrovascular reactions was judged by the testimony of MK and the resistance of brain vessels (CCM) at various levels of perfusion pressure. Assessment of the regulatory capabilities of the intracranial circulatory system was carried out on the basis of the regulation coefficient, which was calculated by the formula (2):

where K is the regulation coefficient;

R _o and P _o are the initial values of resistance (mm Hg / 100 g / min / ml) and pressure (mm Hg) in the vessels of the brain;

ΔR and ΔР are changes in resistance (mmHg / 100 g / min / ml) and pressure (mmHg) in the vessels of the brain when exposed to the system.

When analyzing the coefficient of regulation, it was taken into account that if K = 0, there is no regulation in the system, a value of K> 0 indicates the presence of autoregulation, and when K <1, incomplete autoregulation occurs, when K = 1 - full regulation. When K> 1 - this indicates hypercompensation, K <0 indicates perverse pathological reactions leading to a deepening of the disturbances caused by an external acting force.

STUDY OF THE INFLUENCE OF ETOXIDOL ON SURVIVAL OF EXPERIMENTAL ANIMALS WITH TOTAL BRAIN ISCHEMIA

The study of ethoxidol on the model of total cerebral ischemia was carried out on a model caused by gravitational overloads in the cranio-caudal position. The criterion for cerebroprotective action was% survival of animals. Ethoxidol was administered intraperitoneally prophylactically in doses of 1, 5, 20 and 100 mg / kg (substance, tablets and solution).

Table 9 The effect of ethoxidol on the survival of animals under gravitational overload in cranio-caudal position Condition of experience Survival rate,% The control 33.3 ± 6.7 Ethoxidol 1 mg / kg 50.0 ± 7.1 Ethoxidol 5 mg / kg 66.6 ± 6.7 * Ethoxidol 20 mg / kg 66.6 ± 6.7 * Ethoxidol 100 mg / kg 50.0 ± 7.1

In the control series of experiments, animal survival was on average 33.3% (Table 9). The introduction of ethoxidol at a dose of 5 and 20 mg / kg had a positive effect on the studied parameter, increasing the survival rate to 66.6% (Table 9), i.e. 33.4% relative to animals of the control group. The use of ethoxidol in doses of 1 and 100 mg / kg did not lead to a statistically significant change in the survival of rats under conditions of total cerebral ischemia (Table 9).

Thus, the data obtained indicate the feasibility of studying Ethoxidol as a potential cerebroprotective agent in doses of 5 and 20 mg / kg.

Example 12. STUDY OF THE INFLUENCE OF ETHOXIDOL ON THE BRAIN BLOOD AND ITS AUTO-REGULATION IN THE POST-CHEMICAL PERIOD

To assess the effect of ethoxidol on cerebral blood flow and its autoregulation in conditions of acute brain ischemia, we used a model of circulatory cerebral ischemia, which was created by occlusion of both carotid arteries in rats for 12 minutes with a simultaneous decrease in SBP to 40 mm Hg.

Control experiments on the study of changes in the dynamics of indicators of cerebral blood flow (MK), systemic blood pressure (SBP) and vascular resistance of the brain (CCM) were carried out on 2 groups of animals. In the first group there were animals in which ischemia was not modeled, in the second - animals with ischemia, in which indicators were studied in the postischemic period.

In the 1st group of rats during the entire experiment (60 min), no significant changes were revealed by MK, SAD, or SSM (Table 10). The opposite picture was observed in animals after ischemia (Table 10). So, already at the 5th minute of reperfusion, pronounced changes in all studied parameters are observed: a decrease in the volumetric velocity of MK by 47.6% and an increase in CCM by 71% compared to the outcome (Table 10). In addition, at 5-15 minutes of reperfusion in animals of the control group, a decrease in SBP by 19.9% was recorded (Table 10). Marked cerebral hemodynamic disturbances in animals with acute cerebral ischemia persisted until the end of the observation period: a decrease in MK (by 49.2%) and SBP (by 32.7%), as well as an increase in CCM (by 33.7%) (Table 10 ) Thus, in the postischemic period, a significant decrease in the total blood flow in the brain was observed, due to both hypotension and an increase in the tone of cerebral vessels.

Table 10 The dynamics of changes in the indicators of MK, CAD and SSM in control experiments in the pre- and postischemic period Observation Time (min) Research indicators Before ischemia (1 group) After ischemia (2 group) Baseline MK 89.2 ± 6.1 101.4 ± 8.4 GARDEN 119.4 ± 2.3 122.5 ± 3.9 SSM 1.34 ± 0.10 1.24 ± 0.10 Changes in indicators in% of the initial values 5 MK -4.8 ± 2.9 -47.6 ± 1.8 ^x * GARDEN -2.7 ± 2.1 -18.2 ± 2.6 ^x * SSM + 2.2 ± 4.3 + 71.0 ± 20.1 ^x * fifteen MK -8.7 ± 5.2 -37.7 ± 5.2 ^x * GARDEN -4.1 ± 3.1 -19.9 ± 3.9 ^x * SSM + 5.2 ± 4.6 + 40.3 ± 16.7 ^x * thirty MK -8.3 ± 2.8 -40.8 ± 4.5 ^x * GARDEN -6.20 ± 4.2 -27.1 ± 6.1 ^x * SSM + 2.2 ± 2.5 + 60.1 ± 17.8 ^x * 60 MK -8.5 ± 5.2 - 51.8 ± 5, 5 ^x * GARDEN -8.30 ± 3.8 -31.8 ± 6.1 ^x * Ccm 0,0 ± 4,1 + 49.5 ± 13.2 ^x * Note: statistically significant (P <0.05) parameter shifts are indicated x - relative to the original values, * - in the 2nd group in comparison with the 1st group.

In the study of autoregulatory responses of cerebral vessels, it was found that in animals without ischemia, a decrease in SBP to 60 mm Hg was accompanied by a decrease in cerebral blood flow by an average of 17.4 ± 6.4% and a significant decrease in vascular resistance of the brain (36.3 ± 4.8%) [B. Vilensky Stroke: prevention; diagnosis, treatment. / SPb .: LLC Publishing House FOLIANT, 2002. - 327 p.]. This indicates the ability of the cerebral vessels to maintain regulatory mechanisms when the SBP changes within the specified limits. Decrease in SBP to 40 mm Hg accompanied by a significant decrease in vascular resistance of the brain by 47.9 ± 0.6 (table. 11). Consequently, the disruption of autoregulatory reactions of cerebral vessels in anesthetized animals without ischemia occurs with a SBP below 60 mm Hg.

Table 11 Dynamics of changes in indicators of autoregulatory reactions of cerebral vessels in control experiments GARDEN mm Hg Research indicators Animals without ischemia Animals after ischemia Baseline 120 MK 127.0 ± 8.1 107.6 ± 4.3 SSM 0.98 ± 0.07 1.10 ± 0.04 Changes in indicators in% of the initial values one hundred MK -0.1 ± 2.0 -26.6 ± 2.3 ^x * SSM -16.7 ± 0.1 ^x + 18.8 ± 3.6 ^x * K _a 0.75 -0.80 80 MK -11.4 ± 6.7 -38.3 ± 2.1 ^x * SSM -21.7 ± 5.7 ^x * + 13.8 ± 4.0 ^x * K _a 0.85 -0.63 60 MK -17.4 ± 6.4 ^x -44.1 ± 1.2 ^x * SSM -36.3 ± 4.8 ^x -6.8 ± 4.3 * K _a 0.97 0.20 40 MK -15.6 ± 6.1 ^x * -53.3 ± 1.0 ^x * SSM -58.5 ± 4.8 ^x -25.5 ± 3.4 ^x K _a 0.70 0.51 Note: statistically significant (P <0.05) parameter shifts are indicated x - relative to the original values, * - in comparison with animals without brain ischemia.

In control experiments in the postischemic period in rats, a decrease in SBP to 100 mm Hg led to a significant decrease in cerebral blood flow (26.6 ± 2.3%) and an increase in the resistance (18.8 ± 3.6%) of cerebral vessels relative to the initial level, as well as to a significant decrease in the autoregulation coefficient (0.80) (Table. eleven). Thus, in this group of animals, even when the SBP is reduced to 100 mm Hg. there is a distortion of the autoregulatory reactions of the vessels of the brain. With a further decrease in SBP, cerebral blood flow continued to decline and at 40 mmHg, was less than 50% of the initial level (Table 11). The data obtained indicate a disorder in the postischemic period of autoregulation of cerebral circulation.

The introduction of ethoxidol in a dose of 5 mg / kg under experimental conditions at the 60th minute of the experiment reduces the tone of the cerebral vessels by 19.2 ± 2.5% relative to the initial values. At a dose of 5 mg / kg, ethoxidol had a statistically significant effect on cerebral blood flow only at 15 minutes, increasing it by 7.0 ± 3.6% above the initial values (Table 12).

When studying the effect of Ethoxidol at a dose of 5 mg / kg on the reactivity of cerebral vessels with a stepwise decrease in SBP, it was found that, under the experimental norm, the object of study significantly contributes to the maintenance of MK even at a SBP of 40 mm Hg, as evidenced by Ka, equal to 0, 84 (tab. 13).

The prophylactic administration of the test object at a dose of 5 mg / kg prevents the development of postischemic hypoperfusion (Table 12). In the analysis of the GARDEN indicator, it was found that Ethoxidol prevents the development of postischemic hypotension and pathological vasoconstriction during the entire period of reperfusion.

When evaluating autoregulatory reactions in the postischemic period against the background of preliminary administration of Ethoxidol at a dose of 5 mg / kg, it was found that a stepwise decrease in SBP to 60 mm Hg leads to hyperactoregulatory responses of cerebral vessels (Table 13).

The therapeutic administration of ethoxidol not only contributes to the preservation of autoregulatory responses of cerebral vessels, but even leads to hyperactoregulation with a decrease in SBP from 100 to 40 mm Hg, i.e. with therapeutic use is more effective than with prophylactic. Ethoxidol contributes to maintaining stable cerebral blood flow with dosed hypotension (Table 13).

Table 13 The effect of ethoxidol at a dose of 5 mg / kg on the indices of autoregulatory responses of brain vessels GARDEN mm. Hg Research indicators Experimental norm Preventative Therapeutic introduction Baseline 120 MK 101.0 ± 7.0 88.6 ± 5.7 74.8 ± 7.3 Ccm 1.20 ± 0.07 1.37 ± 0.14 1.66 ± 0.13 Changes in indicators in% of the initial values one hundred MK + 1.5 ± 1.0 -2.2 ± 1.2 * + 20.5 ± 9.0 ^x * Ccm -14.0 ± 1.5 ^x -6.8 ± 1.3 ^x * -18.5 ± 5.7 K _a 1.20 0.96 1.40 80 MK + 4.7 ± 4.5 + 2.4 ± 7.3 * + 42.0 ± 10.8 ^x * Ccm -19.3 ± 3.6 ^x -24.0 ± 5.8 ^x * -47.0 ± 4.0 ^x * K _a 1.26 0.91 1,64 60 MK -0.8 ± 11.9 + 8.8 ± 2.8 ^x * + 37.5 ± 6.9 ^x * Ccm -36.7 ± 8.8 ^x -49.6 ± 1.2 ^x * -60.3 ± 1.8 ^x * K _a 1,08 1.41 1,53 40 MK -15.5 ± 2.2 ^x -15.4 ± 3.1 ^x * + 6.3 ± 4.0 * Ccm -45.2 ± 3.4 ^x * -55.2 ± 2.9 ^x * -66.0 ± 1.5 ^x * K _a 0.84 0.72 1.37 Note: Significant shifts (P <0.05) are indicated relative to: x - initial values, * - animals of the control group.

Example 13. The study of lipid-regulating activity of Ethoxidol

MATERIALS AND METHODS

1. Evaluation of the lipid-regulating activity of Ethoxidol. Since a necessary condition for studying the lipid-lowering activity of new compounds is to evaluate their effect on lipid metabolism in animals with normal serum lipids (normolipidemic animals), the effect of ethoxidol on this indicator was studied. The lipid-regulating activity of the studied drug was evaluated in vivo with its 10-day course administration to normolipidemic rats at a dose of 10.0 mg / kg (intragastric in 2% starch gel).

2. The lipid-regulating activity of Ethoxidol in vitro was studied on a model of ethanol-induced liposynthesis in rat liver homogenate. The liver washed with saline was homogenized using Krebs-Ringer solution (128 mmol NaCl, 1.4 mmol CaCl ₂ , 1.4 mmol MgSO ₄ , 5.2 mmol KCl, 0.18 mmol NaH ₂ PO ₄ , pH 7.4) as the medium and in samples containing 100 ml of homogenate in 1 ml, ethoxidol alcohol solution was added at a concentration of 1 × 10 ^-5 M. Samples containing 20 μl of ethanol were used as a control. After incubation (1 hour, 37 ° C), lipids were extracted from the homogenate and analyzed by thin layer chromatography (TLC).

3. Evaluation of the lipid-lowering activity of Etoxidol in a model of experimental hyperlipidemia.

Hyperlipidemia was induced in outbred male rats weighing 220-250 g with an atherogenic diet containing 10% cholesterol and 1% cholic acid in an oil suspension (based on 1.0 ml of fat load per 100 g of body weight) [Parfentieva EP, Vasilenko Yu.K., Lisevitskaya L.I. et al. The effect of ursolic acid on some indicators of lipid metabolism in experimental atherosclerosis. - Q. honey. Chemistry. - 1980, No. 2, p. 174-179].

The experimental animals were injected with a fat suspension (tube, intragastrically) for 10 days and ethoxidol (in a 2% starch gel) was administered intragastrically at a dose of 10.0 mg / kg and the same dose of nicotinic acid used as a reference preparation.

For the study, blood, liver, brain and aorta samples were extracted after decapitation after 16-18 hours of fasting.

1.2. Biochemical research methods

The hypolipidemic activity of Ethoxidol was evaluated by the degree of inhibition of induced hyperlipidemia, analyzing the following biochemical parameters:

1) in blood serum

- total cholesterol (cholesterol-total),

- HDL cholesterol (cholesterol-HDL)

- LDL cholesterol (HS-LDL),

- cholesterol VLDLP (HS-VLDLP),

- triglycerides (TG)

2) in the liver

- HS-general,

- lipid composition: phospholipids (PL), free cholesterol (CXC), non-esterified fatty acids (NEFA), triglycerides (TG) and cholesterol esters (ECS).

The content of cholesterol in the blood serum was determined by the Ilka method [Rodionova L.P. Modification of the method for determining serum triglycerides. - Lab. A business. - 1980, No. 5, p.297-299], HDL-C was evaluated in the supernatant after heparin - manganese precipitation of VLDL + LDL [Laboratory research methods in the clinic. Ed. V.V. Menshikova, Moscow: Medicine, 1987]. Determination of LDL-C was carried out by calculation according to the formula [Cates M. Technique of lipidology. - M .: Mir, 1975, p. 74-76]:

HS-LDL = HS-total - (TG / 5 + HS-HDL),

where the ratio of TG / 5 corresponds to the content of cholesterol - VLDL.

The cholesterol coefficient of atherogenicity (KHS) was calculated according to [Velichko L.N., Timofeev V.P., Shefer I.A. Micrometer of densitometric determination of fractions of blood phospholilides by thin layer chromatography on silufol UV-254 plates. - Q. honey. chemistry. - 1980, No. 2, p.275-277].

The serum content of TG was determined by the conventional method [Gribanov GA., Sergeev S.A. Express microanalysis of total serum lipids and their fractions. - Q. honey. Chemistry, - 1975, v.21, No. 6, p. 652-655].

The content of cholesterol in the liver and aorta was evaluated by the modified Ilka method, conducting preliminary lipid extraction [Miroshkichenko VP, Gromashevskaya LA, Kasatkina MG et al. Determination of bile acids and cholesterol in the liver. - Lab. Case, 1978, No. 3, p.149-153].

Lipid extraction from liver homogenate was carried out according to the method of Folch in the modification of Cates. Analysis of the lipid composition of tissues and organs was carried out by TLC on silofol plates, using a hexanediethyl ether-acetic acid solvent system (80:20: 2) to evaluate total lipids, and a chloroform-methanol solvent system (65:25: four). Individual lipid fractions were evaluated spectrophotometrically (Spekol-221) after elution of the lipid zones detected with a 10% alcohol solution of phosphor-molybdenum acid.

A statistical assessment of the reliability of the results was carried out using the parametric student criterion.

RESEARCH RESULTS.

1. The study of the lipid-lowering activity of Ethoxidol.

Studies have shown that ethoxidol did not cause changes in the content of TG and total cholesterol in the blood serum and did not affect the ratio of atherogenic and antiatherogenic lipoprotein fractions in rats with normal lipid levels. With the course of Ethoxidol administration, there was also no increase in the cholesterol content in the liver of experimental animals (Table 14).

2. An initial in vitro evaluation of the lipid-regulating activity of Ethoxidol was carried out on a model of ethanol-induced liposynthesis in rat liver homogenate. The results of the influence of ethoxidol on the lipid composition of the liver homogenate are presented in table 15. As can be seen from the data presented, in the model system, as compared with intact material, there was a tendency to increase the content of all lipid components. Incubation of liver homogenate in the presence of 1 × 10 ^-5 M Ethoxidol was accompanied by a decrease in the content of lipid fractions. A positive effect of the drug was expressed in a decrease in the content of total cholesterol due to its free form, as well as a significant decrease in the content of TG - more than 2 times in comparison with the control group (table 15).

Table 14 The effect of ethoxidol at a dose of 10 mg / kg on cholesterol and triglycerides in the blood serum and liver of normolipidemic rats (M ± m, n = 10) Indicator Units Intact animals Ethoxidol Blood serum Total cholesterol mg / 100 ml 82.3 ± 5.1 85.9 ± 6.2 HS-HDL - "- 56.3 ± 5.8 55.5 ± 4.9 HS- (LDL + VLDL) - "- 24.2 ± 3.3 28.7 ± 3.8 Triglycerides - "- 66.1 ± 6.5 70.5 ± 6.2 Liver HS-General mg / g tissue 1.67 ± 0.14 1.88 ± 0.18

Table 15 The effect of ethoxidol at a concentration of 10 ^-5 M on the lipid composition of rat liver homogenate, mg / g of tissue (M ± m, n = 10) Indicator 1. Homogenate of the liver of intact animals 2. Liver homogenate + ethanol (control) 3. Liver homogenate + ethanol + ethoxide Total cholesterol 1.52 ± 0.11 1.70 ± 0.11 1.14 ± 0.012 ** Phospholipids 1.94 ± 0.16 2.25 ± 0.25 1.63 ± 0.26 Cholesterol free 1.24 ± 0.08 1.40 ± 0.08 0.91 ± 0.11 ** NEZHK 0.71 ± 0.06 0.85 ± 0.07 0.70 ± 0.05 Triglycerides 0.85 ± 0.08 1.15 ± 0.07 0.50 ± 0.11 *** Esters of cholesterol 0.60 ± 0.07 0.69 ± 0.08 0.52 ± 0.07 Note: ** - P <0.01 to group 2 control, *** - P <0.001 to group 2 control.

3. Comparative study of the lipid-lowering activity of ethoxidol and nicotinic acid

The study of the lipid-lowering effect of ethoxidol and the comparison drug: the well-known lipid-lowering drug nicotinic acid, was carried out on a model of experimental hyperlipidemia in rats.

As can be seen from the data given in Table 16, the effect of atherogenic load for 10 days was accompanied by an increase in the level of TG and total cholesterol in the blood serum of rats, and the observed hypercholesterolemia developed due to atherogenic fractions of lipoproteins with a decrease in the level of HDL-C. The atherogenicity index increased to 2.66 ± 0.39, exceeding this indicator of the group of intact animals by more than 3 times. Hypercholesterolemia was accompanied by an accumulation of cholesterol in the liver (7.12 ± 0.70 compared to 1.85 ± 0.17 in the control group).

The introduction of 10 mg / kg of ethoxidol in experimental animals for 10 days slowed the development of the pathological process: the level of TG (1.6 times) and cholesterol in the atherogenic fractions of lipoproteins (1.8 times) decreased. Due to a significant increase in the level of HDL-C, the value of the atherogenic coefficient decreased by 4.2 times.

Under similar conditions, a marked lipid-lowering effect was noted for the comparison drug, nicotinic acid: nicotinic acid reduced the level of TG and total cholesterol, contributing to the accumulation of anti-atherogenic lipoproteins.

It should be noted that with comparable lipid metabolism correction indicators found for Ethoxidol and nicotinic acid, Ethoxidol had the advantage of contributing to the normalization of cholesterol in the liver to normal, while with the introduction of nicotinic acid a 4-fold increase compared to the norm was observed.

Thus, the studies showed that, in contrast to Anaprilin, Ethoxidol does not exhibit an undesirable cardio-depressive effect, on the contrary, it exhibits a pronounced cardioprotective effect, in addition, it has an antioxidant effect and a lipid-regulating effect when modeling stress-conditioned prooxidant states.

Ethoxidol is not inferior in terms of its nootropic effect to the drug Piracetam widely used in medical practice, and surpasses the latter in terms of anti-alcohol effect.

In a laboratory experiment, the positive effect of ethoxidol in the prevention and treatment of cerebral ischemia as a cerebroprotector has been shown.

It was shown that ethoxidol has a pronounced lipid-lowering activity under experimental hyperlipidemia, contributing to a decrease in blood levels of triglycerides and cholesterol, atherogenic lipoproteins and does not cause lipid metabolism changes when normolipidemic rats are given on a course.

An important component of the anti-atherogenic effect of Etoxidol is its ability to increase the cholesterol content in the anti-atherogenic fraction of lipoproteins, which provides the drug an advantage over the well-known anti-oxidative lipid-lowering agent - probucol, for which a potentially unfavorable decrease in HDL levels is known.

It was established that the severity of the lipid-lowering effect of Ethoxidol is similar to the well-known drug - nicotinic acid, but it has the advantage of being able to significantly reduce the cholesterol in the liver, while nicotinic acid enhances the accumulation of cholesterol in this organ due to hyperlipidemia.

The registered effects of Ethoxidol determine its potential effectiveness when used in humans as a drug with lipid-regulating activity, reducing the content of atherogenic fractions of cholesterol and triglycerides and increasing the level of anti-atherogenic fractions of cholesterol in high-density lipoproteins. The results obtained allow us to consider Ethoxidol, including as a means of preventing and treating vascular lesions when they are damaged by atherosclerotic plaques - coronary, cerebral and others, which play a decisive role in heart attacks and strokes.

All of the above properties of Ethoxidol make it promising in the production of drugs for the prevention and treatment of cardiovascular, mental illness, cerebral ischemia and obesity.

Claims (12)

1. 2-Ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula

2. The use of compounds 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate, characterized in that it has anti-ischemic activity. 3. The use of compounds 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate, characterized in that it has cerebroprotective activity. 4. The use of compounds 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate, characterized in that it has neurotropic activity. 5. The use of compounds 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate, characterized in that it has lipid-regulating activity. 6. A solid dosage pharmaceutical composition, characterized in that it contains a compound 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate having anti-ischemic, cerebroprotective, neurotropic and lipid-regulating activity. 7. The solid dosage pharmaceutical composition according to claim 6, characterized in that it is made in the form of a tablet with a ratio of active substance and target additives, wt.%:
active substance 0.05-40.0 targeted supplements 60.0-99.95 8. A solid dosage pharmaceutical composition according to any one of claims 6 and 7, characterized in that as target additives it optionally contains simultaneously starch, croscarmellose sodium, polyvinylpyrrolidones, sugars, cellulose derivatives, aerosil, glycine, N-acetyl-L-glutamine acid, 2- (dimethylamino) ethanol, salts of stearic acid, corrective substances in the following ratio of components, wt.%:
starch 0,0-15,0 croscarmellose sodium 0,0-10,0 polyvinylpyrrolidones 0,0-10,0 Sahara 0,0-20,0 cellulose derivatives 0,0-50,0 aerosil 0,0-10,0 glycine 0,0-10,0 N-Acetyl-L-Glutamic Acid 0,0-15,0 2- (dimethylamino) ethanol 0,0-10,0 stearic acid salts 0,0-1,0 corrective substances 0,0-10,0 9. A liquid pharmaceutical composition, characterized in that it contains a 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate compound having anti-ischemic, cerebroprotective, neurotropic and lipid-regulating activity. 10. The liquid pharmaceutical composition according to claim 9, characterized in that it is made in the form of a solution for injection with a ratio of active substance and target additives, wt.%:
active substance 0.05-40.0 targeted supplements 60.0-99.95 11. A liquid pharmaceutical composition according to any one of claims 9 and 10, characterized in that, as target additives, it optionally contains simultaneously glycine, N-acetyl-L-glutamic acid, 2- (dimethylamino) ethanol, Trilon B in the following ratio of components , wt.%:
glycine 0,0-10,0, N-acetyl-L-glutamic acid, 0,0-50,0 2- (dimethylamino) ethanol 0,0-20,0 Trilon B 0,0-5,0 12. The use of the compound 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate according to any one of claims 1 to 11 in the manufacture of medicaments for the prevention and treatment of cardiovascular, mental illness, cerebral ischemia and obesity.