EA011238B1 - A substance having anti-hypoxic, anti-apoplexy, improving memory activity - Google Patents

Abstract

The invention relates to the field of medicine, namely to a substanse having anti-apoplexy, anti-hypoxic and improving memory activity intended for treating acute and chronic derangements of cerebral circulation, including ischemic and haemorrhagic strokes, derangements while aging and other conditions and diseases attended by decreasing cognitive functions and by neurodegeneration. As the substanse having anti-apoplexy, anti-hypoxic and improving memory activity 2,4,6-trimethyl-3-oxypiridine succinate is used. 2,4,6-trimethyl-3-oxypiridine succinate (trimexidine) has an anti-hypoxic effect and anti-amnesic activity. Trimexidine exceeds mexidol at all points in anti-hypoxic, anti-apoplexy and improving memory activity.

Description

The invention relates to medicine, in particular to the creation of an agent with anti-stroke, anti-hypoxic and memory-improving effect, intended for the treatment of acute and chronic disorders of the cerebral circulation, including ischemic and hemorrhagic strokes, disorders during aging 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 brain stalling (Russian Medicines Register. Drug Encyclopedia. Ed. GL Vyshkovsky, Issue 14, M., RLS, 2006).

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

However, the known remedies have the following disadvantages, namely: calcium channel blockers: cinnarizine (stugerone), nimodipine, flunarizin causes drowsiness, headache, dry mouth, gastrointestinal disorders, hypotension, and therefore they are not indicated in patients with hypotension; In addition, these drugs have cardiovascular effects that lead to brain stalling. Mexidol has insufficient antihypoxic activity and efficacy in stroke conditions.

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

This goal is achieved by using as a tool with anti-stroke, antihypoxic and improves memory effect, 2,4,6-trimethyl-3-oxypyridine succinate formula

n

he

NOEP-CH2CH2-COOH

2,4,6-Trimethyl-3-hydroxypyridine succinate is described in the RF patent №2276138, published on 10.06.2006, however, it has not been studied as a substance with anti-insult, anti-hypoxic and memory-improving effect.

2,4,6-Trimethyl-3-hydroxypyridine succinate (Trimexidine) has an anti-insult, antihypoxic, memory-improving effect and surpasses Mexidol in these types of activity, having significant advantages over it, namely: Trimexidine exceeds Mexidol in anti-hypoxic and anti-amnestic activity in all indicators, has an effect in a lower dose than mexidol, and when comparing the same dose has a more pronounced and profound effect. Trimexidine significantly exceeds Mexidol in its anti-insult action, has a more pronounced effect on the indices of neurological deficit, impaired memory, muscle tone, coordination of movements, significantly reduces mortality after a stroke and has a greater therapeutic breadth.

The examples below illustrate the effectiveness of 2,4,6-trimethyl-3-hydroxypyridine succinate (trimexidine) as an anti-insult, antihypoxic, and memory improving remedy in comparison with the closest analogue of mexidol.

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

Acute hypobaric hypoxia was modeled in a flow-exhaust pressure chamber in experiments on outbred white mice-males weighing 24-28 g, according to well-known certified methods (Guidelines for the study of antihypoxic activity of pharmacological substances (Guidelines for experimental (preclinical) study of new pharmacological substances, Moscow , 2005) .The pressure was recorded with an altimeter, the ascent rate was measured with a variometer. The animals were raised at a speed of 20 m / s to a platform 11000 m high. To eliminate the hypercap and 30-35% alkali was in the chamber.The exposure was 10 minutes, then the animals were lowered to the initial conditions for 5 minutes.The whole condition and behavior of the mice was assessed, the lifespan of the animals and the percentage of surviving mice were recorded in relation to the control taken for 100%.

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

Statistical data processing was performed with the calculation of arithmetic averages and their confidence intervals at P <0.05. To assess the reliability of the results used parametric

- 1 011238 Student's statistical data processing criterion (1-1C81) using the Βίοδίπΐ computer program.

Trimexidine was administered intraperitoneally in doses of 50, 100 and 200 mg / kg 40 minutes before the experiment. Mexidol was administered intraperitoneally in 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 has been established that in conditions of hypobaric hypoxia in 100% of control animals already at an altitude of 7 thousand meters body twitches and strong tremor are observed. In mice treated with trimexidine in doses of 100 and 200 mg / kg, the tremor was observed only at an altitude of 10 thousand meters. During the time spent at an altitude of 11 thousand meters, the control mice had periodic tremor (11 episodes), whereas in animals received trimeksidin, tremor was observed only once (only when lifting).

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

In tab. 1 shows the results showing that under conditions of hypobaric hypoxia, Trimexidine in doses of 100 mg / kg and 200 mg / kg significantly increases the life expectancy of animals compared with the control.

At a dose of 100 mg / kg, it increased life expectancy by 1.5 times, and at a dose of 200 mg / kg, it increased 2.1 times compared with the control. At a dose of 50 mg / kg, trimexidine also increased (1.3 times) the lifespan of mice under hypoxic conditions, but this effect was not statistically significant 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 altitude of 11,000 m, all animals died in control (100%). Trimexidine at a dose of 50 mg / kg increased the number of surviving animals by up to 25%, but this effect was not statistically significant (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 dose of up to 200 mg / kg, the antihypoxic effect of trimexidine increased, 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 lifespan of mice on a model of hypobaric hypoxia

Substance Dose, mg / kg The lifespan of animals in minutes Control Distilled. Water 1.97 ± 0.51 T rimexidine 50 2.52 ± 1.17 Control Distilled. water 2.15 ± 0.77 Trimexidine 100 3.32 ± 0.34 * Control Distilled. Water 1.83 + 0.61 Trimexidine 200 3.87 ± 0.71 * Control Distilled. Water 1.89 ± 0.81 Mexidol 100 2.32 + 1.12 Kon grol Distilled. Water 1.63 ± 0.56 Mexidol 200 2.72 ± 0.37 *

* - reliability of differences from control at P <0.05

Table 2. The effect of trimexidine on the survival of mice on a model of hypobaric hypoxia

Substance Dose Number of survivors animals in 10 minutes Survivors / Total Mice % Control Distilled. Water 0/16 0 Trimexidine 50 4/16 25 Control Distilled. Water 0/16 0 Trimexidine 100 6/16 37.5 * Control Distilled. Water 0/16 0 Trimexidine 200 8/16 50* Control Distilled. Water 0/16 0 Mexidol 100 5/16 31.5 * Control Distilled. Water 0/16 0 Mexidol 200 7/16 43.7 *

* - reliability of differences from the control at P <0.05 (x ² )

- 2 011238

Thus, trimexidine in doses of 100 and 200 mg / kg has a pronounced reliable antihypoxic effect under the conditions of the hypobaric hypoxia model. Trimexidine improves the general condition of animals when rising to a height, increases life expectancy and reduces the death of animals in 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 life expectancy under hypoxic conditions, having an effect in smaller 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 the hermetic volume.

The studies were carried out according to a well-known certified method (Guidelines for the study of the antihypoxic activity of pharmacological substances (Guidelines for the experimental (preclinical) study of new pharmacological substances, Moscow, 2005). The experimental procedure consisted in placing the mouse in a 200 ml hermetically sealed glass container then the life span of the animal was recorded.In each group, 16 animals were used. Statistical data processing was performed with arithmetic means and their confidence intervals at Р <0.05.

To assess the reliability of the results, we used a parametric criterion for statistical data processing using the Student’s method (Nei) using the computer program VüyiG.

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

The study of the antihypoxic properties of trimexidine on a model of hypoxia with hypercapnia in the pressure capacity 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, 1.5 and 1.8 times (tab. 3). When reducing the dose of trimexidine to 50 mg / kg, a reliable 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 in this dose increases the life expectancy of mice by 1.8 times.

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

Table 3. Antihypoxic effect of trimexidine on the model of hypoxia with hypercapnia in the hermetic volume

Substance Dose, mg / kg The lifespan of animals in minutes Control Distilled. Water 16.1 ± 0.41 Trimexidine 50 18.7 ± 0.86 Control Distilled. water 13.2 ± 0.7 Trimexidine 100 19.7 ± 1.04 * Control Distilled. Water 14.3 ± 0.6 Trimexidine 200 26.1 ± 0.71 * Control Distilled. water 17.7 ± 0.8 Mexidol 200 24.7 ± 0.5 *

* - reliability of differences between control and experience at P <0.05 (1-1)

Example 3. 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 - the creation of a hemorrhagic stroke with cerebral ischemia (intracerebral post-traumatic hematoma) was performed according to a well-known technique (Author's certificate No. 1767518, 1990; 1asco \\ lk | A., Cgoscagb A., Vysyosk S., Bo88 V55ye11 V. , Kpysk R. T1ge Dshe soigay οί sh1gasgasha1 ra111or11uio1oschsa1 yayidek Gootheshd ekhreptep1a1ybaghokhoyenb yaeshottade 11η 11g tah!

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

In order to create a stroke in rats, anesthetized with chloral hydrate (400 mg / kg, in / m), conducted

- 3 011238 trephine and then, using a special device (mandrin - knife) and stereotaxis, carried out the destruction of brain tissue in the area of the inner capsule followed by (after 2-3 minutes) the introduction of blood from the tongue of the animal ( 0.02-0.03 ml). In this way, a local autohemorrhagic bilateral stroke is achieved in the area of the inner capsule (diameter - 2 mm, depth - 3 mm) without significant damage to the above-located brain and neocortex formations.

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

Survived animals with hemorrhagic stroke subsequently recorded neurological, cognitive, and other behavioral disorders.

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 surgery with recording the behavior and condition of animals on the first, third, seventh and fourteenth days after surgery.

Animals were divided into several groups: 1 - intact rats; 2 - sham-operated rats, which were anesthetized and then trepanned the skull, but did not destroy brain tissue; 3 - animals with hemorrhagic stroke; 4 - animals with hemorrhagic stroke, which was injected 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 a day after surgery). Then Trimexidine was administered daily once for 7 days. Mexidol at a dose of 100 mg / kg was intraperitoneally injected in a similar way.

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

To assess the behavioral and behavioral disorders of animals after a hemorrhagic stroke, a set of methods traditionally used for these purposes was used.

To assess the neurological status, we used the method of recording indicators of the neurological deficit on the scale of stroke assessment (ikeke-shbeh) by Msgott modified by I.V. Gannushkina (1996) and methods for recording muscle tone and coordination of movements.

To assess the cognitive functions, we used the method of the conditioned reflex of passive avoidance in a dark / light chamber (installation BAHGAYAE 1 Pigital Co., USA). Within 14 days after surgery, the ability of trimexidine to increase the survival rate of animals was evaluated.

Statistical data processing was performed with the calculation of arithmetic averages and their confidence intervals at P <0.05. To assess the reliability of the results, we used a parametric criterion for statistical data processing using the Student's method (1-1ec1) and the χ-square test (χ ² ) of the VuYa computer program.

3.1. The effect of trimexidine on neurological deficit scores on the MS Co / u scale after hemorrhagic stroke.

The neurological deficit in animals after a stroke was determined a day after a stroke on a 81toke-MsStote scale (stroke index) modified by I.V. Gannushkina (Gannushkina IV, Functional angioarchitecture of the brain, Medicine, Moscow, 1997, 224 p.).

The severity of the condition was estimated by the sum of the corresponding points. The percentage of animals registered was noted:

1. Mild symptomatology up to 2.5 points on a scale of hoki-shbeh (lethargy of movements, weakness of limbs, one-sided half-shoes, tremor, manege movements);

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

Table 4. Neurological dysfunctions according to the MS scale;

Neurological symptoms 81ke 1ps3eh (stroke index) in points Lethargy, slow movements 0.5 Tremor one One-sided semi one Two-way half 1.5 Limb weakness 1.5 Unilateral ptosis 1.5

Bilateral ptosis 1,5

Manezh movements 2

Paresis of 1-4 limbs 2,0-5,0

Paralysis of 1-4 limbs 3.0-6.0

Coma

Death

- 4 011238

It was established that in the sham-operated control rats without a stroke on the first day after the operation, light neurological disorders in the form of lethargy, slowness of movements, weakness of the limbs are observed only in 10-20% of animals. Severe neurological disorders, manifested in the form of manezhnye movements in a circle, paresis and paralysis of the limbs, in the sham-operated control rats were noted.

In the rats that survived the experimental hemorrhagic stroke, on the first day after surgery, all (100%) animals had mild neurological disorders and there were clear severe manifestations of neurological deficit: manege movements in a circle - in 30%, paresis - in 40%, paralysis limbs - in 30%.

Trimexidine after 4-fold administration at a dose of 100 mg / kg reliably reduced the manifestations of neurological deficit after a stroke. So, slight violations after 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 trimexidine activity in its ability to reduce mild neurological disorders, in particular lethargy and slowness of movements, and also to a lesser extent, reduce severe disorders (manege movements).

Table 5. The effect of trimexidine on the indices of neurological deficit (according to the MS scale) on the first day after a stroke in rats that survived a hemorrhagic stroke

Neurological symptoms The number of animals with neurological disorders,% Control False Opera. Control after stroke Trimexidine 100 mg / kg Mexidol 100 mg / kg Lethargy, slow movements 20 100 ^liters 40 * 70 Limb weakness ten _ 100 ^liters 40 * 60 *

Manege movements 0 30 ^L 0 * 0 * Paresis 1-4 limbs 0 40 ^L 20 20 Paralysis of 1-4 limbs 0 30 ^l 0 * 0 *

^Λ - reliability of differences from sham-operated rats with P <0.05 (x ² ) * - reliability of differences from stroke rats with P <0.05 (x ² )

Thus, Trimexidine causes a significant decrease in the manifestations of neurological deficit in rats a day after hemorrhagic stroke and surpasses Mexidol in activity.

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

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

Rats were placed on a horizontal rod with a diameter of 4 cm, 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 established that in control rats after a hemorrhagic stroke, a marked impaired coordination of movements was observed. The severity of these changes varies somewhat depending on the period of their registration after a stroke. On the 1st day after the stroke, a lack of coordination of movements was observed in 50% of animals, on the 3rd day also in 50%, on the 7th day - in 55% and on the 14th day - in 44% of rats (Table 6).

Trimexidine in a dose of 100 mg / kg weakened impaired coordination of movements in rats. Pronounced and statistically reliable results were obtained on days 3, 7, and 14 after a stroke (Table 6).

Mexidol at a dose of 100 mg / kg also reduced the severity of impaired coordination of movements on the seventh and 14th day after a stroke. However, for 1-3 days after the 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 movement coordination disturbed after a stroke and is superior to Mexidol in this effect.

- 5 011238

Table 6. The effect of trimexidine on impaired coordination of movements after a stroke

Group The number of animals that could not resist on animals I rotating rod (3 rpm) for 2 minutes, in abs. units and%

1st day 3rd day 7th day 14th day au % au % au % au % Intact (without surgery) 0/10 0 0/10 0 0/10 0 0/10 0 False operated 2/10 20 1/10 ten 1/10 ten 1/9 ten Stroke 6/12 50 ^A 5/10 50 ^l 5/9 55 ^l 4/9 44 ^l Stroke + Trimexidine 100 mg / kg 4/10 40 2/10 20* 0/10 0 * 0/10 0 * I Stroke + Mexidol 100 mg / kg 5/10 50 4/10 40 2/10 20* 0/10 0 *

but. e. - absolute units of ^L - reliability of differences from falsely operated rats with P <0.05 (x ² ) * - reliability of differences from rats with stroke with P <0.05 (x ² )

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

For the assessment of muscle tone, a tightening test on a horizontal crossbar was used (Methodological guidelines for the study of the activity of pharmacological substances. Guidelines for 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 surface of the table. Intact rats with undisturbed muscle tone are quickly tightened and held on the crossbar with four legs. Failure of this reflex by animals from the experimental group indicates a violation of muscle tone and neurological deficit.

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

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

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

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

Table 7. The effect of trimexidine on the 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 au % au % au % au % Intact (without surgery) 0/10 0 0/10 0 0/10 0 0/10 0 False operated 1/10 ten 1/10 ten 0/10 0 0/9 0 Stroke 5/10 50 ^l 5/9 55 ^l 4/9 ₄₄ l 4/9 44 ^ Stroke + Trimexidine 100 mg / kg 4/10 40 4/10 40 1/10 ten* 1/10 10 ^ Stroke + Mexidol 100 mg / kg 3/10 thirty 3/10 thirty 3/10 thirty 1/10 ten*

but. e. - absolute units of l - reliability of differences from falsely operated rats with P <0.05 (x ² ) * - reliability of differences from stroke rats with P <0.05 (x ² )

- 6 011238

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

Studies were performed according to the method described in the Guidelines for the study of the nootropic activity of pharmacological substances (Guidelines for experimental (preclinical) study of new pharmacological substances, Moscow, 2005). The experiment was carried out on a standardized setting of the passive avoidance conditioned reflex (Razzue LuoIaise company LaRauye 1n81Gnept1So, USA). Structurally, the installation consists of a 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 an illuminated platform before entering the dark chamber of the installation with its tail to the inlet.

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

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

In order to train the conditioned passive avoidance reflex (passive avoidance response), a single pain stimulus (0.45 mA) was applied to a rat in a dark chamber until the rat ran out of the dark, dangerous compartment. The rat remembered that it was dangerous to enter the dark cell (training). The preservation of the memorial trace (reproduction of passive avoidance reaction) was carried out 24 hours after the training, and also 3, 7 and 14 days after the stroke. To this end, 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 stayed in the dark dangerous chamber was recorded for 3 minutes.

It was established that in the control groups of intact and sham-operated rats who received distilled water during the entire experiment, when reproducing the passive avoidance reaction in 24 hours after training (getting pain stimulation 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 time of observation.

In control rats, 3, 7, and 14 days after hemorrhagic stroke, memory was impaired. In assessing the preservation of the memorial trace, the latent time of entry into the dark chamber was significantly reduced in these animals a day after the training. So, on the 7th day after the 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 disturbed and, as a result, they entered the dark dangerous chamber.

After the course of administration of trimexidine, recovery of cognitive functions impaired by the stroke was noted. The memory effect of trimexidine depended on the registration period for the preservation of the memory trace and the course of drug administration. So, on the 1st and 3rd day after the stroke, the improvement of cognitive functions after administration of Trimexidine in a dose of 100 mg / kg was not significant, but on the 7th and 14th day, against the background of using Trimexidine, there was a significant improvement in memory indicators (Tables 8 and 9) .

The data presented show that under the influence of Trimexidine, a significant increase was observed 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 stroke (in terms of the latent time of passive avoidance reaction) after the course application of trimexidine.

Groups Latency time (s) of entering a dark cell with animals playing passive avoidance reactions at various 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 ^L 21.7 + 6.1 ^liters 18.7 + 4.2 ^L 20.7 ± 7.1 ^liters 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 ’

^L - reliability of differences from sham-operated rats at P <0.05 (1-1e1 Student; χ ² ) * - reliability of differences from rats with stroke at P <0.05 (ΐ-Student's test; χ ² )

- 7 011238

Table 9. Improvement of memory impaired by stroke (in terms of% of rats remembering training and not entering a dangerous dark chamber) after the course of administration of Trimexidine

Animal groups % of rats remembering training and not entering a dangerous dark cell 1 day 3 days 7 days 14 days Intact 60 80 70 60 False operated 50 70 60 50 Stroke 20 ^l 10 ^l 20 ^l 10 ^l Stroke + trimexidine 40 thirty thirty 40 * Stroke + Mexidol 40 thirty 40 40 *

^L - reliability of differences from sham-operated rats at Р <0.05 (1-1С51 Student's; χ ² ) * - reliability of differences from rats with stroke at P <0.05 (ΐ-Student's test; χ ² )

Thus, Trimexidine, administered to rats at a dose of 100 mg / kg for 7 days, restores memory impairment on the model of the conditioned passive avoidance reflex 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 the control with stroke - 10%) is observed and increases by 3.4 times, compared with the stroke rats, the latent time of entry into the dark dangerous chamber. Trimexidine is superior to Mexidol in its ability to restore cognitive functions impaired after a stroke.

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

It was established that in the group of falsely 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 immediately (within 5 hours) after a stroke, 20 rats died from 50 operated animals, i.e. 40% of animals died.

rats who survived a stroke were observed for 14 days. Of these, 10 rats were given trimexidine at a dose of 100 mg / kg for 7 days, and the other 10 stroke rats, mexidol at a dose of 100 mg / kg, and distilled water (10) were administered to 10 rats.

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

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

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

After the introduction of Mexidol in a dose of 100 mg / kg, 30% of animals died, i.e. the drug reduced death by 2 times compared with untreated control animals.

Thus, Trimexidine reliably reduces (3 times) the death of animals after a stroke and surpasses Mexidol in 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 au % au % au % au % Falsely operated 0/10 0 0/10 0 0/10 0 1/10 ten Stroke 3/10 30 ^L 4/10 40 ^l 5/10 50 ^l 6/10 60 ^l Stroke + Trimexidine 1/10 ten 1/10 ten* 2/10 20* 2/10 20* Stroke + Mexidol 1/10 ten 2/10 20 2/10 20* 3/10 thirty*

but. e. - absolute units of l - reliability of differences from sham-operated rats at P <0.05 (χ ² ) * - reliability of differences from rats with stroke at P <0.05 (χ ² )

- 8 011238

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

Example 4. Antiamnesic activity of trimexidine.

Studies were conducted 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 (Guidelines for experimental (preclinical) study of new pharmacological substances, Moscow, 2005).

The study was carried out on a standard automated installation of the passive avoidance conditioned reflex (Rakuye Auo1bapee, La Gaouye 1n51Tell Co., USA). Structurally, the installation consists of a small platform located at a distance of 1 m from the floor, lit by a special lamp and a dark chamber connected to it with an electrode floor. The rat was placed on a lighted 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 conditional reflex of passive avoidance (passive avoidance reaction), a rat in a dark chamber received a single painful stimulation with 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 the stimulation was determined by running the animal out of the dark compartment.

To obtain amnesia, a maximum electric shock (50 Hz, 0.2 s) was used, which was conducted through the corneal electrodes directly after training. An electric shock causes erasure of a memorable trace.

The test for the reproduction of passive avoidance reaction (for the preservation of a memorial trail) was carried out 24 hours after training and electric shock. The animal was again placed on the illuminated platform and for 2 min the time spent on the illuminated platform and in the dangerous dark compartment was recorded (no current is applied to the floor when playing passive avoidance reaction).

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

It is established that the control trained animals during the reproduction of passive avoidance reaction without an electric shock prefer to be on a light platform. Erasing training with electroshock causes animals to amnesia, with the result that animals with a short latency period enter the dark chamber and remain there much more of the time than animals without amnesia (Table 11).

Trimexidine in a dose of 100 mg / kg had a pronounced anti-amnesic activity, which was expressed in that the time spent by animals in a dark dangerous cell decreased, and the time spent on a safe, illuminated platform increased significantly compared with the control animals with amnesia (Table 11). ).

In its antiamnesic activity, trimexidine is superior to mexidol, used at a similar dose of 100 mg / kg (Table 11).

Table 11. Antiamnesic effect of trimexidine

Animal groups Dose, mg / kg The time spent by the rats when playing back the reflex, in seconds on the illuminated platform in a dark cell Control without amnesia Distiller water 87.4 ± 11.2 32.6 ± 4.8 Control with amnesia Distiller water 28.2 ± 6.1 ^liters 91.8 ± 14.5 ^L Trimexidine 100 74.2 ± 9.1 * 45.8 + 8.7 * Mexidol 100 56.2 ± 13.4 * 63.8 ± 5.3 *

^L - significance of differences between the control without amnesia and control with amnesia at P <0.05 * - significance of the differences between control with amnesia and the effects of substances at P <0.05 Thus, Trimexidine has a pronounced anti-amnesic activity and exceeds Mexidol in its effectiveness.

As follows from the presented data, trimexidine in doses of 100 and 200 mg / kg (intraperitoneally)

- 9 011238 has a pronounced reliable antihypoxic effect in an experiment on mice under conditions of hypobaric hypoxia and hypoxia with hypercapnia in hermetic volume. With hypobaric hypoxia, trimexidine improves the general condition, increases life expectancy and reduces the death of animals, and during hypoxia with hypercapnia in the hermetic volume increases the life expectancy of animals. Trimexidine is superior to mexidol in antihypoxic activity in all indicators, has an effect in a lower dose (100 mg / kg) than mexidol (200 mg / kg), and when comparing the same dose, 200 mg / kg has a more pronounced and deep effect.

Trimexidine (100 mg / kg, intraperitoneally, course of 7 days) on a model of hemorrhagic stroke (intracerebral post-traumatic hematoma) causes an improvement in neurological status, restores muscle stroke reflexes damaged by stroke, muscle tone, coordination of movements, behavior, cognitive functions, reduces the death of animals after insult and thus has a complex anti-insulin action. In anti-stroke effects, trimexidine surpasses Mexidol in all indicators.

Trimexidine in a dose of 100 mg / kg has a pronounced anti-amnesic activity, which is reflected in the ability to eliminate the amnesia of the conditioned reflex of passive avoidance in rats and restore memory impaired by the conduct of maximum electroshock. In its antiamnesic activity, trimexidine is superior to mexidol, which is used in a similar dose.

Claims (1)

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