RU2326665C1 - Medicinal agent with antihypoxic, antistroke, memory-refining action - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: 2,4,6-trimethyl-3-oxypyridine succinate (trimexidine) is offered as specified agent. Agent is applied for treatment of acute and chronic strokes including ischemic and hemorrhagic stokes, abnormalities caused by other conditions and diseases accompanied with cognitive function reduction and neurodegeneration. Improved neurologic deficit, high muscular tonus, and improved coordination of movements after stroke, restored memory impairments and reduced number of animal death caused by stroke.

EFFECT: antihypoxic action of trimexidine surpasses one of mexidol.

4 ex, 11 tbl

Description

The invention relates to medicine, namely to the creation of an anti-stroke, antihypoxic and memory-improving agent intended for the treatment of acute and chronic cerebrovascular disorders, including ischemic and hemorrhagic strokes, aging disorders and other conditions and diseases, accompanied by a decrease in cognitive functions and neurodegeneration.

At present, calcium channel antagonists are used for strokes, but they have significant side effects and disadvantages, one of which is the presence of cardiovascular effects leading to "robbing" the brain (Russian Drug Register. Encyclopedia of drugs. Ed. G.L. Vyshkovsky, Vol. 14, M., Radar, 2006).

The closest analogue of the claimed drug both in chemical structure and in action is Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate), which is used to treat cerebrovascular disorders (Russian Drug Register. Encyclopedia of drugs. Ed. G .L. Vyshkovsky. Issue 14, M., Radar, 2006).

However, known drugs have the following disadvantages, namely calcium channel blockers: cinnarizine (stugeron), nimodipine, flunarizin causes drowsiness, headache, dry mouth, gastrointestinal upset, hypotension, and therefore they are not indicated in patients with hypotension; in addition, these drugs have cardiovascular effects, leading to "robbing" the brain. Mexidol has insufficient antihypoxic activity and effectiveness in stroke conditions.

The aim of the invention is the creation of funds with anti-stroke, antihypoxic and improves memory action.

This goal is achieved by the use of 2,4,6-trimethyl-3-hydroxypyridine succinate of the formula as an anti-stroke, antihypoxic and memory-improving action

2,4,6-trimethyl-3-hydroxypyridine succinate is described in RF patent No. 2276138, publ. 06/10/2006, however, as a substance with anti-stroke, antihypoxic and memory-improving effects, it has not been studied.

2,4,6-trimethyl-3-hydroxypyridine succinate (trimexidine) has an anti-stroke, antihypoxic, memory-improving effect and is superior to mexidol in these types of activity, having significant advantages over it, namely: trimexidine is superior to mexidol in antihypoxic and antiamnestic activity in all indicators, has an effect in a lower dose than Mexidol, and when comparing the same dose, it has a more pronounced and deep effect. Trimexidine significantly exceeds Mexidol in anti-stroke action, has a more pronounced effect on indicators of neurological deficit, impaired memory, muscle tone, motor coordination, significantly reduces mortality after stroke and has a greater therapeutic breadth.

The following examples illustrate the effectiveness of 2,4,6-trimethyl-3-hydroxypyridine succinate (trimexidine) as an anti-stroke, antihypoxic, learning and memory enhancing agent compared to the closest analogue of mexidol.

Example 1. Antihypoxic effect of trimexidine on a model of hypobaric hypoxia in a pressure chamber.

Acute hypobaric hypoxia was modeled in a flow-exhaust pressure chamber in experiments on outbred white male mice weighing 24-28 g, according to the well-known certified methodology ("Guidelines for the study of antihypoxic activity of pharmacological substances" (Manual on experimental (preclinical) study of new pharmacological substances ", Moscow, 2005). Pressure was recorded with an altimeter, ascent rate with a variometer. Animals were" raised "at a speed of 20 m / s to a platform" height "11000 m. To eliminate hyperk The apnya contained 30-35% alkali in the chamber. The exposure was 10 minutes. Then, the animals were "lowered" to the initial conditions for 5 minutes. During the entire exposure, the state and behavior of the mice were evaluated, the life expectancy of the animals and the percentage of surviving mice in relation to the control were recorded taken as 100%.

To create identical hypoxia conditions, the animals of the experimental (1 mouse) and control (1 mouse) groups were simultaneously placed in the chamber, and thus, for each dose of the drug, its own control group was used. In each group there were 10-16 animals.

Statistical processing of data was performed with the calculation of arithmetic mean and their confidence intervals at P <0.05. To assess the reliability of the results, we used the parametric criterion of statistical data processing by the Student method (t-test)) using the Biostat computer program.

Trimexidine was administered intraperitoneally at doses of 50, 100 and 200 mg / kg 40 minutes before the experiment. Mexidol was administered intraperitoneally at doses of 100 and 200 mg / kg 40 minutes before the experiment. The control groups of animals were injected intraperitoneally with distilled water in an equivalent volume.

It was found that under conditions of hypobaric hypoxia in 100% of the control animals, twitching of the body and severe tremor are already observed at an altitude of 7 thousand m. In mice treated with trimexidine at doses of 100 and 200 mg / kg, tremor was observed only at an altitude of 10 thousand m During the time spent at an altitude of 11 thousand m, control mice showed periodic tremor (11 episodes), whereas in animals treated with trimexidine, tremor was observed once (only when elevated).

After administration of mexidol at a dose of 200 mg / kg, tremor also decreased, and at an altitude of 11 thousand meters, against the background of mexidol, only 4 episodes of tremor were observed. Compared to Mexidol, Trimexidine at doses of 100 and 200 mg / kg completely eliminated tremor.

Table 1 shows the results showing that under hypobaric hypoxia, trimexidine in doses of 100 mg / kg and 200 mg / kg significantly increases the life expectancy of animals compared to the control.

At a dose of 100 mg / kg, he increased life expectancy by 1.5 times, and at a dose of 200 mg / kg, by 2.1 times compared with the control. At a dose of 50 mg / kg, trimexidine also increased (1.3 times) the life expectancy of mice under hypoxia, but this effect was statistically unreliable at P≤0.05.

Mexidol did not have a significant antihypoxic effect at a dose of 100 mg / kg and was inferior in activity to trimexidine at a dose of 200 mg / kg.

After a 10-minute exposure of mice in a pressure chamber at a critical height of 11,000 m, all animals died in the control (100%). Trimexidine at a dose of 50 mg / kg increased the number of surviving animals to 25%, however, this effect was statistically unreliable (Table 2). At a dose of 100 mg / kg, trimexidine significantly (up to 37.5%) increased the percentage of surviving mice compared to the control. With an increase in the dose to 200 mg / kg, the antihypoxic effect of trimexidine was enhanced, was reliable compared with the control, and the percentage of surviving animals was 50% (Table 2).

Mexidol in doses of 100 and 200 mg / kg significantly reduced the percentage of death of animals during hypoxia, but its effect was weaker than the effect of trimexidine.

Table 1 The effect of trimexidine on the life expectancy of mice on a model of hypobaric hypoxia Substance Dose mg / kg Life expectancy of animals in minutes The control Distil. water 1.97 ± 0.51 Trimexidine fifty 2.52 ± 1.17 The control Distil. water 2.15 ± 0.77 Trimexidine one hundred 3.32 ± 0.34 * The control Distil. water 1.83 ± 0.61 Trimexidine 200 3.87 ± 0.71 * The control Distil. water 1.89 ± 0.81 Mexidol one hundred 2.32 ± 1.12 The control Distil. water 1.63 ± 0.56 Mexidol 200 2.72 ± 0.37 * * - significance of differences from control at P <0.05

table 2 The effect of trimexidine on the survival of mice in a model of hypobaric hypoxia Substance Dose The number of surviving animals after 10 minutes Surviving / Total number of mice % The control Distil. water 0/16 0 Trimexidine fifty 4/16 25 The control Distil. water 0/16 0 Trimexidine one hundred 6/16 37.5 * The control Distil. water 0/16 0 Trimexidine 200 8/16 fifty* The control Distil. water 0/16 0 Mexidol one hundred 5/16 31.5 * The control Distil. water 0/16 0 Mexidol 200 7/16 43.7 * * - significance of differences from control at P≤0.05 (χ ² )

Thus, trimexidine in doses of 100 and 200 mg / kg has a pronounced significant antihypoxic effect in the hypobaric hypoxia model. Trimexidine improves the general condition of animals when climbing to a height, increases life expectancy and reduces the death of animals under conditions of hypobaric hypoxia.

Trimexidine is superior to mexidol in antihypoxic activity in the hypobaric hypoxia test both in its effect on the general condition of animals and in terms of life expectancy under hypoxia, having an effect in lower doses than mexidol, and when comparing the same dose of 200 mg / kg, it is more pronounced and deep effect.

Example 2. Antihypoxic effect of trimexidine on a hypoxia model with hypercapnia in a pressurized volume.

The studies were carried out according to the well-known certified methodology ("Guidelines for the Study of the Antihypoxic Activity of Pharmacological Substances" (Manual on the Experimental (Preclinical) Study of New Pharmacological Substances ", Moscow, 2005). The experimental procedure was that the mouse was placed in a sealed glass container with a volume 200 ml and then the life span of the animal was recorded, 16 animals were used in each group. techniques, are arithmetic means and their confidence intervals at P <0.05.

To assess the reliability of the results, we used the parametric criterion of statistical data processing by the Student method (t-test)) using the Biostat computer program.

Trimexidine was administered intraperitoneally at doses of 50, 100 and 200 mg / kg 40 minutes before the experiment. Mexidol was administered intraperitoneally at doses of 100 and 200 mg / kg 40 minutes before the experiment. The control groups of animals were injected with distilled water in an equivalent volume.

A study of the antihypoxic properties of trimexidine in a model of hypoxia with hypercapnia in a hermetic volume showed that the drug at doses of 100 and 200 mg / kg significantly (P <0.05) increases the life expectancy of animals compared to the control group of mice, respectively, by 1.5 and 1.8 times (table 3). With a decrease in the dose of trimexidine to 50 mg / kg, a significant antihypoxic effect is not observed.

Mexidol at a dose of 100 mg / kg does not have an antihypoxic effect, and at a dose of 200 mg / kg it has an antihypoxic effect, significantly increasing the life expectancy by 1.4 times. Mexidol is inferior in activity to trimexidine, which at this dose increases the life expectancy of mice by 1.8 times.

Thus, trimexidine has antihypoxic activity in the hypoxia test with hypercapnia in a pressurized volume of 100 and 200 mg / kg, which is expressed in the ability of the substance to increase the life span of animals under hypoxia. Mexidol has a lesser effect in the conditions of this test, since it is ineffective at a dose of 100 mg / kg and has a less pronounced effect than 200 mg / kg in trimexidine.

Table 3 Antihypoxic effect of trimexidine on a model of hypoxia with hypercapnia in a hermetic volume Substance Dose mg / kg Life expectancy of animals in minutes The control Distil. water 16.1 ± 0.41 Trimexidine fifty 18.7 ± 0.86 The control Distil. water 13.2 ± 0.7 Trimexidine one hundred 19.7 ± 1.04 * The control Distil. water 14.3 ± 0.6 Trimexidine 200 26.1 ± 0.71 * The control Distil. water 17.7 ± 0.8 Mexidol 200 24.7 ± 0.5 * * - significance of differences between control and experience at P <0.05 (t-test)

Example 3. The anti-stroke effect of trimexidine.

The anti-stroke effect of trimexidine was investigated in experiments on rats on a model of hemorrhagic stroke (intracerebral post-traumatic hematoma). Modeling of local hemorrhage in the brain - creating a hemorrhagic stroke with cerebral ischemia (intracerebral post-traumatic hematoma) was carried out according to the well-known method (Copyright certificate No. 1767518, 1990; Jackowski A., Crockard A., Burnstock G., Ross Russell R., Kristek F. The time course of intracranial pathophysiological changes following experimental subarachnoid haemorrage in the rat., Journal of cerebral blood flow and metabolism, 1990, v.10, p. 835-849).

The experiments were carried out on white outbred male rats weighing 260-280 g. Rats were kept under vivarium conditions with free access to food and water.

In order to create a stroke in rats anesthetized with chloral hydrate (400 mg / kg, w / m), craniotomy was performed and then using a special device (mandrin-knife) and stereotaxis, brain tissue was destroyed in the area of the internal capsule, followed by (2- 3 minutes) by introducing blood taken from under the tongue of the animal (0.02-0.03 ml) into the site of damage. In this way, a local autohemorrhagic bilateral stroke in the area of the inner capsule (diameter 2 mm, depth 3 mm) is achieved without significant damage to the superior formations of the brain and neocortex.

During the operation and immediately after it, 40% of rats with stroke died.

In surviving animals with hemorrhagic stroke, neurological, cognitive and other behavioral disorders were subsequently recorded.

The dynamics of the development of disorders caused by intracerebral post-traumatic hematoma, and the effect of trimexidine on the behavior of rats were evaluated within 14 days after the operation with recording the behavior and condition of the animals on the first, third, seventh and fourteenth days after the operation.

Animals were divided into several groups: 1 - intact rats; 2 - falsely operated rats, which were anesthetized and then performed craniotomy, but did not destroy the brain tissue; 3 - animals with hemorrhagic stroke; 4 - animals with hemorrhagic stroke, which was administered trimexidine; 5 - animals with hemorrhagic stroke, which was administered mexidol.

Trimexidine was administered to animals at a dose of 100 mg / kg, intraperitoneally 2 hours after surgery and then another 3 times every 4 hours (4 injections per day after surgery). Then, trimexidine was administered once daily for 7 days. Mexidol at a dose of 100 mg / kg was intraperitoneally administered in a similar manner.

Control animals were injected intraperitoneally with physiological saline in an equivalent volume.

A complex of methods traditionally used for these purposes was used to assess the violation of the behavior and condition of animals after a hemorrhagic stroke.

To assess neurological status, we used the method of recording neurological deficit indicators according to the McGrow stroke-index scale modified by I.V. Gannushkina (1996) and the methods for recording muscle tone and coordination of movements.

To evaluate cognitive functions, the method of conditioned reflex of passive avoidance in the dark / light chamber was used (installation by Laffayette Instrument Co, USA). Within 14 days after surgery, the ability of trimexidine to increase animal survival was assessed.

Statistical processing of data was carried out with the calculation of arithmetic mean and their confidence intervals at P <0.05. To assess the reliability of the results, we used the parametric criterion of statistical data processing by the Student method (t-test) and the χ-square (χ ² ) criterion of the Biostat computer program.

3.1. The effect of trimexidine on indicators of neurological deficit according to the Me Grow scale after hemorrhagic stroke.

Neurological deficit in animals after a stroke was determined a day after a stroke on the Stroke-index McGrow scale (stroke index) modified by I.V. Gannushkina (Gannushkina I.V. Functional angioarchitectonics of the brain, Medicine, Moscow, 1997, 224 pp.)

The severity of the condition was assessed by the sum of the corresponding points.

Registered the percentage of animals in which it was noted:

1. Mild symptoms up to 2.5 points on the Stroke-index scale (lethargy, weakness of the extremities, one-sided half-throat, tremor, manege movements);

2. Severe symptoms of neurological disorders - from 3 to 10 points (paresis and paralysis of the limbs, as well as a lateral position) (Table 4).

Table 4 Neurological dysfunctions on the Me Grow scale Neurological symptoms Stroke index (stroke index) in points Lethargy, slow motion 0.5 Tremor one One-sided semidose one Bilateral Semi Disease 1,5 Limb weakness 1,5 Unilateral ptosis 1,5 Bilateral ptosis 1,5 Maneuvering movements 2 Paresis of 1-4 limbs 2.0-5.0 Paralysis of 1-4 limbs 3.0-6.0 Coma 7 Death 10

It was found that in false-operated control rats without stroke on the first day after surgery, mild neurological disturbances in the form of lethargy, slow movement, weakness of the limbs are observed only in 10-20% of the animals. Severe neurological impairment, manifested in the form of ring maneuvers, paresis and paralysis of the extremities, was not observed in mock operated control rats.

In rats that survived an experimental hemorrhagic stroke, on the first day after the operation, all (100%) animals showed mild neurological disorders and distinct severe manifestations of neurological deficit were observed: ring movements - in 30%, paresis - in 40%, paralysis limbs - in 30%.

Trimexidine after 4-fold administration at a dose of 100 mg / kg significantly reduced the appearance of a neurological deficit after a stroke. So, mild disorders after the administration of trimexidine were recorded only in 40% of animals (100% in the control). Along with this, trimexidine significantly reduced severe neurological disorders - manege movements and paralysis of 1-4 limbs (Table 5).

Mexidol in a similar dose of 100 mg / kg was inferior in activity to trimexidine in its ability to reduce mild neurological disorders, in particular lethargy and slow movement, and also to a lesser extent reduced severe disturbances (maneuvering movements).

Table 5 The effect of trimexidine on neurological deficit indicators (according to the McGrow scale) in the first days after a stroke in rats surviving a hemorrhagic stroke Neurological symptoms The number of animals with neurological disorders,% The control
False Operations. The control
After a stroke Trimexidine
100 mg / kg Mexidol
100 mg / kg Lethargy, slow motion twenty 100 ^ 40 * 70 Limb weakness 10 100 ^ 40 * 60 * Maneuvering movements 0 30 ^ 0 * 0 * Paresis of 1-4 limbs 0 40 ^ twenty twenty Paralysis of 1-4 limbs 0 30 ^ 0 * 0 * ^ - significance of differences from false-operated rats at P≤0.05 (χ ² )
* - significance of differences from stroke rats at P≤0.05 (χ ² )

Thus, trimexidine causes a significant decrease in the manifestations of neurological deficiency in rats a day after a hemorrhagic stroke and is superior in activity to Mexidol.

3.2. The effect of trimexidine on impaired coordination of movements after a stroke.

Neurological deficit, expressed in impaired coordination of movements, was evaluated in a test of a rotating rod ("Guidelines for the study of the activity of pharmacological substances". Guidelines for the experimental (preclinical) study of new pharmacological substances ", Moscow, 2005).

Rats were placed on a horizontal rod 4 cm in diameter, rotating at a speed of 3 revolutions per minute. The inability of animals under the influence of the drug to maintain balance on the rod for 2 minutes was considered as a manifestation of impaired coordination of movements.

It was found that in control rats after a hemorrhagic stroke, a pronounced violation of coordination of movements is observed. The severity of these changes varies somewhat depending on the period of their registration after a stroke. On the 1st day after a stroke, impaired coordination of movements was observed in 50% of the animals, on the 3rd day also in 50%, on the 7th in 55% and on the 14th in 44% of rats (Table 6).

Trimexidine at a dose of 100 mg / kg weakened impaired coordination in rats. Pronounced and statistically significant results were obtained on the 3rd, 7th and 14th day after a stroke (Table 6).

Mexidol at a dose of 100 mg / kg also reduced the severity of coordination disorders on the seventh and 14th day after a stroke. However, on 1-3 days after a stroke, Mexidol did not have a significant effect on these disorders in comparison with Trimexidine, which improved these disorders.

Thus, trimexidine has the ability to restore impaired motor coordination after a stroke and is superior to mexidol in this effect.

Table 6 The effect of trimexidine on impaired coordination of movements after a stroke Groups of animals The number of animals that could not stay on a rotating rod (3 rpm) for 2 minutes, in abs. and% 1st day 3rd day 7th day 14th day a.e. % a.e. % a.e. % a.e. % Intact (no operation) 0/10 0 0/10 0 0/10 0 0/10 0 Falsely operated 2/10 twenty 1/10 10 1/10 10 1/9 10 Stroke 6/12 50 ^ 5/10 50 ^ 5/9 55 ^ 4/9 44 ^ Stroke + Trimexidine 100 mg / kg 4/10 40 2/10 twenty* 0/10 0 * 0/10 0 * Stroke + Mexidol 100 mg / kg 5/10 fifty 4/10 40 2/10 twenty* 0/10 0 * but. e. - absolute units.
^ - significance of differences from false-operated rats at P≤0.05 (χ ² )
* - significance of differences from rats with stroke at P≤0.05 (χ ² )

3.3. The effect of trimexidine on the weakening of muscle tone after a stroke

To assess muscle tone, we used the pull-up test on a horizontal bar ("Guidelines for the Study of the Activity of Pharmacological Substances". Guide to the Experimental (Preclinical) Study of New Pharmacological Substances ", Moscow, 2005).

The rat was hung with its front paws on a wire stretched at a height of 20-30 cm from the table surface. Intact rats with undisturbed muscle tone are quickly pulled up and held on the crossbar with four paws. Failure of this reflex by the animals of the experimental group indicates a violation of muscle tone and neurological deficit.

In rats after a hemorrhagic stroke, muscle tone is significantly weakened. On the 3rd day after a stroke, muscle tone was weakened on average in 55%, on the seventh in 44% and on the fourteenth in 44% of the animals.

After a course of administration of trimexidine at a dose of 100 mg / kg, an improvement in muscle tone was observed. Significant restoration of muscle tone after the administration of trimexidine was observed on the 7th and 14th day after a stroke (Table 7).

Mexidol at a dose of 100 mg / kg also had the ability to restore muscle tone of animals after a stroke, but was inferior in activity to trimexidine, since it had no effect on the 7th day after a stroke (Table 7).

Thus, trimexidine has the ability to restore muscle tone of animals after a stroke and is superior in activity to Mexidol.

Table 7 The effect of trimexidine on muscle tone of animals after a stroke Group of animals The number of animals that did not catch up on the horizontal bar, in abs. units and% 1st day 3rd day 7th day 14th day a.e. % a.e. % a.e. % a.e. % Intact (no operation) 0/10 0 0/10 0 0/10 0 0/10 0 Falsely operated 1/10 10 1/10 10 0/10 0 0/9 0 Stroke 5/10 50 ^ 5/9 55 ^ 4/9 44 ^ 4/9 44 ^ Stroke + Trimexidine 100 mg / kg 4/10 40 4/10 40 1/10 10* 1/10 10* Stroke + Mexidol 100 mg / kg 3/10 thirty 3/10 thirty 3/10 thirty 1/10 10* a.e. - absolute units.
^ - significance of differences from false-operated rats at P≤0.05 (χ ² )
* - significance of differences from stroke rats at P≤0.05 (χ ² )

3.4. The effect of trimexidine on cognitive impairment after a stroke.

The studies were carried out according to the method described in the "Guidelines for the study of the nootropic activity of pharmacological substances" (Guide to the experimental (preclinical) study of new pharmacological substances ", Moscow, 2005). The experiment was carried out on a standardized installation of the conditioned passive avoidance reflex (Passive Avoidance by Lafayette Instrument Co, USA) Structurally, the installation consists of a platform located at a distance of 1 m from the floor, illuminated by a special lamp and connected to it by a dark camera with electric native floor. The rat was placed on illuminated platform in front of the dark tail installation chamber to the inlet.

To assess the natural mink skill of running a rat into a dark place, the latent time of the first entry into the dark chamber was recorded.

It was established that control rats (intact and false-operated), when placed on an illuminated platform, quickly, with a short latent period, pass into a dark chamber. A stroke does not violate the mink reflex, but increases the latent time of its execution. Trimexidine and Mexidol also do not interfere with the implementation of the mink reflex and the latent time to complete the reflex.

In order to train the conditioned passive avoidance reflex (passive avoidance reaction), a rat in a dark chamber was exposed to a single pain irritation by electric current (0.45 mA) until the rat ran out of the dark, dangerous compartment. The rat remembered that it was dangerous to enter a dark chamber (training). The preservation of the memorial trail (passive avoidance reaction) was carried out 24 hours after training, as well as 3, 7 and 14 days after a stroke. For this purpose, the animal was again placed on the illuminated platform and the latent period of the first entry into the dark chamber and the time the rat was in the dark dangerous chamber were recorded for 3 minutes.

It was found that in the control groups of intact and pseudo-operated rats that received distilled water during the entire experiment, when reproducing passive avoidance reaction 24 hours after training (receiving pain irritation in the dark compartment of the chamber) 90% of the animals remembered the electric shock and did not enter the dark “dangerous” »The camera during the entire observation time.

In control rats, memory was impaired 3, 7, and 14 days after hemorrhagic stroke. In these animals, when assessing the safety of the memorial trail one day after training, the latent time of entry into the dark chamber was significantly reduced. So, on the 7th day after a stroke, only 25% of the animals did not enter the dark chamber at all, i.e. they remembered the electric shock, and in 75% of the rats the memory was impaired, and as a result of this they entered a dark dangerous chamber.

After the course of the introduction of trimexidine, a restoration of cognitive impairment by stroke was noted. The effect of trimexidine on memory depended on the duration of registration of the safety of the commemorative trace and the course of administration of the drug. So, on the 1st and 3rd day after a stroke, the improvement in cognitive functions after the administration of trimexidine at a dose of 100 mg / kg was unreliable, but on the 7th and 14th day, against the background of the use of trimexidine, a significant improvement in memory indices was observed (Tables 8 and 9) .

It can be seen from the presented data that, under the influence of trimexidine, there was a significant increase in both the latent time of entry into the dark chamber 7 and 14 days after the stroke (Table 8), and the increase in the number of animals that did not enter the dark dangerous chamber 14 days after the stroke ( table 9).

Mexidol at a dose of 100 mg / kg had no effect on the cognitive functions of animals 7 days after a stroke, but restored them only 14 days after a stroke (Tables 8 and 9).

Table 8 Improvement of memory impaired by a stroke (according to the latent time passive avoidance reaction index) after course use of trimexidine. Groups of animals Latent time (sec) of entering a dark chamber when playing passive avoidance reaction at different times after a stroke 1 day 3 days 7 days 14 days Intact 123.1 ± 18.7 134.5 ± 23.4 129.1 ± 28.5 139.1 ± 19.3 Falsely operated. 132.3 ± 12.0 118.7 ± 20.7 120.5 ± 19.2 115.7 ± 21.4 Stroke 24.1 ± 3.2 ^ 21.7 ± 6.1 ^ 18.7 ± 4.2 ^ 20.7 ± 7.1 ^ Stroke + Trimexidine 35.4 ± 14.1 42.2 ± 21.7 63.2 ± 16.7 * 71.2 ± 18.7 * Stroke + Mexidol 31.2 ± 11.7 36.1 ± 11.9 33.2 ± 21.1 78.9 ± 14.5 * ^ - significance of differences from false-operated rats at P≤0.05 (t-test student; χ ² )
* - significance of differences from rats with stroke at P≤0.05 (t-student test; χ ² )

Thus, trimexidine, administered to rats at a dose of 100 mg / kg for 7 days, restores memory impairment in the conditioned passive avoidance reflex model at 7 and 14 days after the creation of a hemorrhagic stroke. Under the influence of trimexidine, a statistically significant increase (up to 40%) in the number of animals remembering the negative situation (in control with a stroke - 10%) is observed and the latent time of entry into the dark dangerous chamber is increased by 3.4 times compared with stroke rats. Trimexidine is superior to Mexidol in the ability to restore cognitive function impaired after a stroke.

3.5. The effect of trimexidine on the survival of rats with hemorrhagic stroke

It was found that in the group of false-operated rats, only one out of ten rats died on the 7th day after the false operation.

During the operation of creating a stroke and directly (within 5 hours) after a stroke, 20 rats from 50 operated animals were killed, i.e. 40% of the animals died.

30 rats who survived after a stroke were observed for 14 days. Of these, 10 rats were injected with trimexidine at a dose of 100 mg / kg for 7 days, and distilled water was administered to 10 rats of mexidol at a dose of 100 mg / kg and 10 rats (control).

It was established that in the group of control animals another 30% of stroke animals died during the first days after a stroke, and by the 14th day the death rate reached 60% (Table 10).

Trimexidine at a dose of 100 mg / kg intraperitoneally daily for 7 days prevented the death of rats with stroke (Table 10).

Against the background of the drug, death was observed only in 20% of rats that had a stroke (60% in the control). Thus, trimexidine reduced death after a stroke by 3 times compared with untreated animals.

After administration of Mexidol at a dose of 100 mg / kg, the death of 30% of the animals was observed, i.e. the drug reduced death by 2 times compared with untreated control animals.

Thus, trimexidine significantly reduces (3 times) the death of animals after a stroke and exceeds Mexidol by this indicator.

Table 10 The effect of trimexidine on the survival of animals after a stroke Group of animals The number of dead animals within 14 days after hemorrhagic stroke 1st day 3rd day 7th day 14th day a.e. % a.e. % a.e. % a.e. % Falsely operated. 0/10 0 0/10 0 0/10 0 1/10 10 Stroke 3/10 30 ^ 4/10 40 ^ 5/10 50 ^ 6/10 60 ^ Stroke + Trimexidine 1/10 10 1/10 10* 2/10 twenty* 2/10 twenty* Stroke + Mexidol 1/10 10 2/10 twenty 2/10 twenty* 3/10 thirty* ae are absolute units.
^ - significance of differences from false-operated rats at P≤0.05 (χ ² )
* - significance of differences from rats with stroke at P≤0.05 (χ ² )

Thus, a dose of 100 mg / kg of trimexidine, administered to rats according to the scheme: 2 hours after a stroke, then 3 injections every 4 hours and then every day for 7 days, causes a significant improvement in post-stroke disorders. The drug improves the indicators of neurological deficit within a day after a stroke, and on the 7th and 14th day after a stroke it increases muscle tone, improves coordination of movements, restores memory impaired by a stroke, improves the reproduction of the conditioned reflex of passive avoidance on the 7th and 14th day after a stroke, and at 3, 4 times reduces the death of animals after a stroke.

Example 4. Antiamnestic activity of trimexidine.

The studies were carried out on white outbred male rats weighing 250-280 g according to the method described in the "Guidelines for the study of the nootropic activity of pharmacological substances" (Guide to the experimental (preclinical) study of new pharmacological substances ", Moscow, 2005).

The study was conducted on a standard automated installation of the conditioned reflex of passive avoidance (Passive Avoidance, Lafayette Instrument Co, USA). Structurally, the installation consists of a small platform located at a distance of 1 m from the floor, illuminated by a special lamp and a dark chamber connected to it with an electrode floor. The rat was placed on the illuminated platform before entering the dark chamber of the installation with its tail to the inlet, and the animal, preferring the dark chamber, quickly passes into it.

In order to teach the conditioned passive avoidance reflex (passive avoidance reaction), the rat in the dark chamber received a single pain irritation by electric current (0.45 mA) through the electrode floor, and the animal remembered that it was dangerous to enter the dark chamber. The duration of irritation was determined by running out of the animal from the dark compartment.

To obtain amnesia, the maximum electroshock (50 Hz, 0.2 sec) was used, which was carried out through corneal electrodes immediately after training. Electroshock erases a commemorative footprint.

A test for the reproduction of passive avoidance reaction (to preserve a memorable trace) was carried out 24 hours after training and electric shock. The animal was again placed on the illuminated platform, and the animal was kept for 2 minutes on the illuminated platform and in a dangerous dark compartment (no current is fed to the floor when playing passive avoidance reaction).

Trimexidine was administered at a dose of 100 mg / kg ip. Mexidol at a dose of 100 mg / kg intraperitoneally was used as a comparison drug. Substances were administered 40 minutes before training. The control groups of animals were injected with distilled water in an equivalent volume. Each dose of the substance was studied in 10 animals.

It was established that control trained animals, when reproducing passive avoidance reaction without the action of electric shock, prefer to be on a bright platform. Erasing the training using electroshock causes amnesia in animals, as a result of which animals with a short latent period enter the dark chamber and remain there much more than animals without amnesia (Table 11).

Trimexidine at a dose of 100 mg / kg had a pronounced antiamnestic activity, which was manifested in the fact that the time spent by animals in a dark dangerous chamber was reduced, and the time spent on a safe, illuminated platform was significantly increased compared with the control animals with amnesia (Table 11 )

In terms of antiamnestic activity, trimexidine is superior to mexidol used in a similar dose of 100 mg / kg (Table 11).

Table 11 Anti-amnestic action of trimexidine Groups of animals Dose mg / kg The time spent by rats in the reproduction of the reflex, in seconds on a lighted platform in a dark chamber Control without amnesia Distil. water 87.4 ± 11.2 32.6 ± 4.8 Amnesia Control Distil. water 28.2 ± 6.1 ^ 91.8 ± 14.5 ^ Trimexidine one hundred 74.2 ± 9.1 * 45.8 ± 8.7 * Mexidol one hundred 56.2 ± 13.4 * 63.8 ± 5.3 * ^ - significance of differences between control without amnesia and control with amnesia at P <0.05.
* - significance of differences between the control with amnesia and the effects of substances at P <0.05.

Thus, trimexidine has a pronounced antiamnestic activity and is superior to mexidol in its effectiveness.

As follows from the presented data, trimexidine in doses of 100 and 200 mg / kg (intraperitoneally) has a significant significant antihypoxic effect in an experiment in mice under conditions of hypobaric hypoxia and hypoxia with hypercapnia in a hermetic volume. With hypobaric hypoxia, trimexidine improves the general condition, increases the life expectancy and reduces the death of animals, and with hypoxia with hypercapnia in the hermetic volume increases the life expectancy of animals. Trimexidine is superior to mexidol in antihypoxic activity in all respects, has an effect in a lower dose (100 mg / kg) than mexidol (200 mg / kg), and when compared with the same dose, 200 mg / kg has a more pronounced and deep effect.

Trimexidine (100 mg / kg, intraperitoneally, 7 days course) on a hemorrhagic stroke model (intracerebral post-traumatic hematoma) causes an improvement in neurological status, restores muscle reflexes impaired by stroke, muscle tone, coordination of movements, behavior, cognitive functions, reduces animal death after a stroke and thus has a complex anti-insulin action. In terms of anti-stroke action, trimexidine is superior to mexidol in all respects.

Trimexidine at a dose of 100 mg / kg has a pronounced antiamnestic activity, which is expressed in the ability to eliminate the amnesia of the conditioned reflex of passive avoidance in rats and restore memory impaired by maximum electric shock. In terms of antiamnestic activity, trimexidine is superior to mexidol used in a similar dose.

Claims (1)

The use of 2,4,6-trimethyl-3-hydroxypyridine succinate as an anti-stroke, antihypoxic and memory enhancing agent.