EA040876B1 - L-ENANTIOMER OF 2-ETHYL-6-METHYL-3-HYDROXYPYRIDINUM - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to the field of medicine, specifically to a new chemical compound - L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate, which has cerebroprotective, hepatoprotective, lipid-regulating, anti-ischemic and neurotropic activity.

Prior Art

One of the promising areas developed by domestic pharmacologists refers to innovative domestic drugs in the series of hydroxypyridine derivatives.

Among the derivatives of 2-ethyl-6-methyl-3-hydroxypyridine, a number of drugs are known that have a wide spectrum of biological activity.

In the early 1980s in the State Research Institute of Pharmacology of the Russian Academy of Medical Sciences Smirnov L.D. and Kuzmin V.I. synthesized mexidol - 2-ethyl-6-methyl-3-oxypyridine succinate. Mexidol is a drug with a multicomponent spectrum of pharmacological effects and a multifactorial mechanism of action. The most important components of the activity of mexidol are its antioxidant, membranotropic effects, the ability to modulate the functioning of receptors and membrane-bound enzymes and restore neurotransmitter balance. (Mexidol: main neuropsychotropic effects and mechanism of action. T.A. Voronina. State Research Institute of Pharmacology named after V.V. Zakusov. M., RAS).

Mexidol is widely used in medicine, as well as veterinary medicine as an antioxidant and antihypoxic agent, characterized by a wide spectrum of pharmacological action and high efficiency (nootropic and tranquilizing action - US Pat. RU 2065299, anti-ischemic and anti-atherosclerotic effect - US Pat. RU 2144822, antianginal - US Pat. RU 2168993, hepatoprotective - patent RU 2189817, antimicrobial - patent RU 2157686, etc.).

Compositions based on 2-ethyl-6-methyl-3-hydroxypyridine succinate are known in the form of various dosage forms: a solution for parenteral administration (Order of the Ministry of Health of the Russian Federation dated December 31, 1996 No. 432 Mexidol® 5% solution for injection, Pat. RU 2205640, 2380089 , 2398583), tablets (Order of the Ministry of Health of the Russian Federation dated January 26, 1998 No. 21 Mexidol® coated tablets, 0.125 g), capsules (US Pat. RU 2144822, 2145855), dressings (US Pat. RU 2149648), etc.

A pharmaceutical composition in the form of tablets for oral use based on 2-ethyl-6-methyl-3-hydroxypyridine succinate is proposed (RU 2444359).

It is also known the drug ethoxydol - 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate (racemate), which has anti-ischemic, cerebroprotective, neurotropic and lipid-regulating activities (RU 2377237), formula:

Pharmaceutical compositions containing ethoxidol are also known: tablets, injection solutions.

It has been shown that etoxidol at a dose of 10 mg/kg eliminates ventricular fibrillation in coronary artery occlusion and reduces the number of animals with ventricular fibrillation to 2 out of 8 experimental animals when administered at a dose of 10 mg/kg and to 2 out of 9 - when administered at a dose of 23 mg /kg.

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

In conditions of acute myocardial ischemia at a dose of 15 mg/kg, it has a pronounced cardioprotective effect on cardiac hemodynamics, bringing the values of contractility (dP/dt), arterial blood pressure (BP), etc. to the level of intact control.

Ethoxydol at a dose of 1 mg/kg has the ability to reduce the intensity of lipid peroxidation in laboratory rabbits caused by their 30-day immobilization, as evidenced by a decrease in the level of malonic dialdehyde (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%.

The successes of stereopharmacology achieved over the past decades have enriched science with an understanding of the general patterns: structure - activity and structure - toxicity, for substances that are racemic mixtures of left- and right-handed isomers. D- and L-enantiomers with different pharmacological properties were obtained. Studies have shown that levorotatory isomers tend to be more toxic, but also more active, while dextrorotatory and racemates are inferior to them in terms of pharmacological effects.

The classic example is L-adrenaline: its intracardiac concentration is 11 times higher than the dextrorotatory isomer. High stereospecificity is characteristic of receptor interactions, including processes of hepatic metabolism, which are also determined by the nature of the drug-receptor relationship.

Clinical studies of S-amlodipine. The antihypertensive and antianginal effects of Samlodipine are confirmed by the results of clinical studies. It has been shown that half the doses of the drug are required to achieve the optimal therapeutic effect of S-amlodipine.

Currently, only 15% of drugs are chirally pure substances, that is, they consist of enantiomers of the same type. The use of optical isomers of known cardiovascular drugs is a way to increase their effectiveness and tolerability (M.I. Lutai, A.F. Lysenko, O.I. Moiseenko. National Research Center Institute of Cardiology named after N.D. Strazhesko of the Academy of Medical Sciences of Ukraine, Kiev City).

Currently, such derivatives of 3-hydroxypyridine as mexidol, emoxipin are used in clinical practice (G.I. Klebanov et al. Antioxidant properties of 3-hydroxypyridine derivatives: mexidol, emoxipine and proxipin. Questions of Medical Chemistry No. 3 2001). These drugs have antioxidant, antihypoxic, nootropic, anticonvulsant, anxiolytic effects.

The essence of the invention

The aim of the invention is to develop a new chemical compound of the L-enantiomer of 2-ethyl-6methyl-3-hydroxypyridinium L-hydroxybutanedioate (compound 1), formula (I):

its use as a cerebroprotective agent, as well as the study of its antihypoxic activity, taking into account the requirements for preclinical studies of drugs. The compounds show cerebroprotective activity, which provides a prolongation of the bioelectrical activity of the myocardium, compared with the racemate.

Also, the aim of the invention is to create a new agent with hepatoprotective lipid-regulating, anti-ischemic and neurotropic activity, containing the compound Lenantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate (compound 1).

Compound 1 in the form of a composition with pharmaceutically acceptable carriers (target additives) can be administered in various dosage forms. The compound is used in therapeutically effective doses, which depend on the weight, sex and age of patients, the etiology and severity of their diseases. Preferred doses are 20 to 300 mg.

EFFECT: effectiveness of a new derivative of 3-hydroxypyridine, namely L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate for the treatment and prevention of a wide range of diseases as a means of exhibiting hepatoprotective lipid-regulating, anti-ischemic and neurotropic activity, providing a prolonged period bioelectrical activity of the myocardium, compared with the racemate.

Information confirming the possibility of carrying out the invention

Example 1

1. Obtaining the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate.

To 13.7 g (0.1 m) of 2-ethyl-6-methyl-3-hydroxypyridine was added 13.41 g (0.1 m) of L-hydroxybutanedioic acid, mixed thoroughly and heated to a melting point of 100-110°C . About 120 ml of water was gradually added to the resulting mixture, the resulting solution was filtered, and 6070% of water was removed by vacuum distillation. Then, 80 ml of acetone was added to the resulting residue and crystallized, the resulting precipitate was filtered and dried in a vacuum. Received 23.8 g of a white crystalline substance with high hygroscopicity, deliquescent in air.

C12H17N6

Found, %: С 53.07; H 6.38; N 5.11.

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

IR spectrum, ν cm-1: 3510, 3230, 1660, 1610, 1560, 1292, 1161.

UV spectrum, nm: 228.291 (water)

Angle of rotation [a]D20 -13.6 (4.5% acetonitrile).

2. Biological research.

General characteristics of laboratory animals.

The studies were carried out on 69 male and 69 female white mice of the SHK line weighing 20-22 g, obtained from the nursery of the Andreevka Branch of the Federal State Budgetary Institution of Science, the Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency of Russia.

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The number of animals.

Experimental animal groups of 6 mice of each sex per group for studying acute toxicity and antihypoxic activity are a representative sample that allows you to obtain statistically reliable data, as evidenced by many years of practice in toxicological studies. During the studies, the animals were kept under controlled conditions: ambient temperature 20-24°C, relative humidity 50-60%.

2.1. Determination of acute toxicity of compound 1.

In the present study, acute toxicity was evaluated in laboratory mice of both sexes after a single intravenous injection of compound 1 or the reference drug Ethoxydol.

White outbred mice (6 mice of each sex per group) were injected with compound 1 or the reference drug in increasing doses of 300, 400, 500, 600, 700 mg/kg as a solution in water for injection with a total volume of 0.5 ml, acceptable for this animal species.

Animals were observed for at least 14 days after administration. The toxicity of drugs was judged by the death of animals and the general picture of intoxication.

The general condition of the animals was recorded: the features of their behavior, the intensity and nature of motor activity, the presence and nature of seizures, coordination of movements, skeletal muscle tone, response to tactile or pain stimuli, the frequency and depth of respiratory movements, the condition of the hair and skin. The number of dead animals was counted during the experiment.

The data obtained were processed by the generally accepted method of variation statistics with the calculation of the mean value (M) and the standard error of the mean value (m). Paired Student's t-test with Bonferroni's correction was used as a criterion for assessing the significance of differences in mean values at a significance level of P<0.05; to determine the significance of differences in absolute values, a nonparametric chi-square test was used; with a sample size of less than 10 - Fisher's exact test; to determine the significance of differences before and after administration of the pharmacological agent, the Wilcoxon test was used.

Based on the results obtained, the characteristics of acute toxicity were determined using the Miller-Tainter probit analysis [10] (Tables 1-4).

The clinical picture of intoxication in mice with the introduction of both compared pharmacological agents was identical. With the introduction of toxic doses of compound 1 and the reference drug Ethoxydol, Russia, a decrease in the motor activity of animals was noted. When the dose was increased, a short-term (2-3 min) period of hyperkinesia with anxiety was observed. Movement coordination was impaired, skeletal muscle tone was increased, tonic and clonic convulsions were observed. During this period, involuntary acts of defecation and urination were observed. Some of the animals died on the background of convulsions. The surviving animals were in an inhibited state, which was expressed in a reduced reaction to light, sound, tactile and pain stimuli (stimulation of the tail root). At the same time, convulsive muscle twitches, decreased appetite, diarrhea or constipation were observed against the background of increasing water consumption. Breathing was rapid and superficial, hyperthermia was recorded. The animals were in the above state for the first two days, after which their physiological state gradually returned to the initial state.

Over the next 14 days, the phenomena of intoxication in animals manifested themselves in the form of a reduced reaction to stimuli. The hairline lost its luster, characteristic of healthy animals, the skin remained unchanged. In the future, the condition of the animals gradually returned to normal.

When observing the animals for 14 days, no deaths were recorded among the surviving animals.

The results of the study of mortality in mice after administration of compound 1 or the reference drug Ethoxydol are presented in tables 1-4.

Table 1. Dose-response relationship (lethal outcomes) with intragastric administration of compound 1 to male mice.

Dose mg/kg 300 400 500 ; | 600 700 Number of animals in group 6 6 6 6 6 Qty Survivors 6 3 2 0 0 Number of dead 0 3 4 6 6 % lethality 0 50.0 66.4 100.0 100.0

The calculation of the acute toxicity parameters gave the following results: DL ₅₀ =419.74 (364.2-474.3) mg/kg, DL ₁₆ =304.3 mg/kg, DL84 =523.8 mg/kg.

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Table 2. Dose-effect relationship (lethal outcomes) with intragastric administration of the reference drug Ethoxydol to male mice.

Dose mg/kg 300 400 500 600 700 Number of animals in group 6 6 6 6 6 Number of Survivors 6 5 3 1 0 Number of dead 0 1 3 5 6 % lethality 0 16.7 50.0 83.3 100.0

The calculation of the acute toxicity parameters gave the following results: DL50 =498.93 (414.4-643.9) mg/kg, DL ₁₆ =391.2 mg/kg, DL ₈₄ =622.3 mg/kg.

Table 3. Dose-effect relationship (lethal outcomes) with intragastric administration of compound 1 to female mice

Dose mg/kg 300 400 500 600 700 Number of animals in group 6 6 6 6 6 Number of Survivors 6 3 3 0 0 Qty ] dead .......... ..............] ------g---------- G........Ί ________—______J 50.0; ......................... □ , 6 3 I 00.0 ;

The calculation of the acute toxicity parameters gave the following results: DL50 =428.81 (291.8-554.4) mg/kg, DL ₁₆ =313.4 mg/kg, DL ₈₄ =574.7 mg/kg.

Table 4. Dose-effect relationship (lethal outcomes) with intragastric administration of the reference drug Ethoxydol to female mice

Dose mg/kg 300 400 500 600 700 Number of animals in group 6 6 6 6 6 Number of Survivors 6 4 3 3 0 Mortality 0 2 3 3 6 % lethality 0 33.3 50.0 50.0 100.0

The calculation of the acute toxicity parameters gave the following results: DL50 =499.36 (350.1-628.3) mg/kg, DL ₁₆ =367.3 mg/kg, DL ₈₄ =628.0 mg/kg.

The dynamics of body weight of surviving mice treated with toxic doses of compound 1 or the reference drug Ethoxydol did not differ significantly from those in the control groups. When analyzing the dynamics of water and food consumption after intravenous administration of toxic doses of compound 1 or the reference drug Ethoxydol, no significant difference was also found in comparison with the control groups of mice (Tables 5-10).

Table 5. Daily consumption of dry food (g) and water (ml) per mouse (male) after intragastric administration of toxic doses of compound 1

No. Group, dose mg/kg Underconsumption, ml Feed intake, g Background 7 days 14 day Background 7 days 14 day 1 300.0 6.0 6.9 6.8 9.1 8.9 9.2 2 400.0 6.1 6.7 7.0 9.0 8.7 9.1

3 j 500.0 6.1 7.3* 6.7 9.1 8.9 9.0 1 4 | Control 6.1 6.4 6.7 9.0 9.2 9.4 j i

* Differences when compared with control are significant at p<0.05 (chi-square test).

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Table 6. Daily consumption of dry food (g) and water (ml) per mouse (male) after intragastric administration of toxic doses of the reference drug Ethoxydol

Ks Group, dose mg/kg Water consumption, ml Feed intake, g Background 7 days 14 day Background 7 days 14 day 1 300.0 6.1 6.4 6.6 9.2 9.1 9L 2 400.0 6.1 6.8 6.5 9.2 8.9 8.9 3 500.0 6.0 6.4 7.0 9.1 8.8 9.0 4 600.0 6.2 7.3* 7.4* 9.0 8.3* 8.7* 5 Control 6.1 6.3 6.6 9.1 9.3 9.6

* Differences when compared with control are significant at p<0.05 (chi-square test).

Table 7. Daily consumption of dry food (g) and water (ml) per mouse (female) after intragastric administration of toxic doses of compound 1

No. Group, dose, mg/kg Water consumption, ml Feed intake, g Background 7 days 14 day Background 7 days 14 day 1 300.0 6.0 6.5 6.7 9.1 8.9 9.0 1 2 400.0 6.2 6.9 6.6 9.2 9.3 9.3 3 500.0 6.1 7.2* 6.7 9.2 9.0 9.1 4 Control 6.0 6.3 6.5 9.2 9.5 9.7

* Differences when compared with control are significant at p<0.05 (chi-square test).

Table 8. Daily consumption of dry food (g) and water (ml) per mouse (female) after intragastric administration of toxic doses of the reference drug Ethoxydol

No. Group, dose, mg/kg Water consumption, ml Feed intake, g Background 7 days 14 day Background 7 days 14 day 1 300.0 6.2 6.4 6.6 9.2 8.9 9.4 2 400.0 6.0 6.9 6.6 9.2 9.0 9.2 3 500.0 6D 6.4 6.8 9.0 8.3* 9.1 4 600.0 6.0 7.2* 6.9 9.0 7.5* 9.0 5 Control 6.3 6.6 6.7 9.1 9.3 9.7

* Differences when compared with control are significant at p<0.05 (chi-square test).

Table 9. Dynamics of the body weight of surviving mice (males, g), when determining the acute toxicity of compound 1 and the reference drug Ethoxydol

№№ The dose of the drug №№ STOMACH Compound 1 Comparator drug Exodus. Through Through Exodus Through Through ipynn mg/kg puff 7 days 14 days 7 days 14 days 1 21.2 20.2 20.8 20.5 18.8 20.2 2 20.9 18.3 20.6 21.7 18.6 20.8 1 300.0 3 21.3 19.9 21.0 21.7 19.8 20.2 4 21.4 18.6 20.6 21.9 18.2 20.4 5 20.7 18.4 20.4 20.6 18.6 21.0 6 21.6 193 21.2 20.2 17.8 19.7 M 21.18 19.12* 20.77 21.10 18.63* 20.38 0.14 ozz 0.12 ODO 038 0.19 1 20.3 18.6 19D 21.0 18.3 20.6 2 21.7 18.2 20.0 21.4 17.3 20.1 2 400.0 3 22.4 18.6 20.2 20.8 16.2 18.7 4 - - - 20.6 16.0 18.8 5 - - - 21.8 17.5 20.3 m 20.78 17.84* 19.40 21.12 17.06* 19.70 ±t 0.65 0.41 0.29 0.22 0.43 0.40 1 20.8 15.2 18.0 21.4 17.5 18.2 2 19.5 16.1 18.8 20.8 15.9 18.0 3 500.0 3 - 19.7 16.4 19.8 m 20.8 153 18.0 19.5 16.1 18.8 20.63 16.60* 18.67 ±t 0.50 0.47 0.57 600.0 1 - • • 20.4 16.6 193 m 20.4 16.6 193 ±sh - - - 1 21.6 21.8 22.0 20.0 21.5 22.7 2 21.8 21.9 22.4 20.4 21.3 23.0 5 Control 3 20.9 21.3 21.8 22.4 22.8 23.9 4 19D 20.7 22.4 21.5 22.1 24.1 5 20.4 21.9 23.2 20.7 21.7 23.9 6 21.5 21.9 23.3 22.0 22.9 24.3 M 20.92 21.58 22.52 21.17 22.05 23.65 ±t 039 ODO 0.25 039 0.28 036

* Differences when compared with baseline values (Wilcoxon test) and corresponding control values (Student's t-test) are significant at p<0.05.

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Table 10. Dynamics of body weight of surviving mice (females, g), when determining the acute toxicity of compound 1 and the reference drug Ethoxydol

№№ Dose of the drug No. belly Compound 1 Ethoxydol Exodus. Through Through Exodus. Through Through groups mg/kg nyh 7 days 14 days 7 days 14 days 1 21.5 20.8 20.9 21.3 20.4 21.4 2 20.7 18.7 20.6 19.8 18.6 20.8 1 300.0 3 22.2 20.0 20.6 22.3 20.8 21.1 4 21.8 19.4 20.6 18.9 18.3 19.7 5 20.9 18.6 19.3 21.5 20.1 19.6 6 21.2 20.2 19.5 22.0 20.2 20.4 M 2138 19.62* 2035 20.97 19.73* 20.50 0.23 036 037 034 0.42 ozo I 21.1 18.6 19.4 20.5 17.7 19.2 2 400.0 2 22.4 19.6 19.8 20.9 18.0 19.8 3 20.7 16.7 17.6 20.8 16.4 20.0 4 - - - 22.3 17.3 18.8 m 20.65 1730* 18.75 21.13 1735* 19.45 ±t 0.68 0.64 0.50 0.40 035 0.28 1 22.4 17.6 18.4 22.0 19.4 20.0 3 500.0 2 21.8 17.3 19.3 20.2 17.0 20.1 3 19.0 16.4 19.0 20.4 15.8 17.9 m 21.07 17.10* 18.90 20.87 17.40* 1933 ±t 1.05 036 036 037 1.06 0.72 1 - - 21.6 15.0 17.8 4 600.0 2 - - - 21.3 17.1 20.2 3 - - - 19.9 16.0 20.1 M - - • 20.93 16.03* 193? - - - 0.52 0.61 0.78 1 21.4 21.9 22.5 18.2 19.8 21.2 2 20.3 21.0 23.5 19.4 21.0 23.2 5 Control 3 22.0 23.6 24.1 21.3 22.7 24.0 4 21.7 22.9 23.7 22.1 22.9 23.8 5 19.4 20.6 23.0 22.0 22.7 24.2 6 19.1 21.5 22.6 19.1 21.0 22.6 M 20.65 21.92 2333 2035 21.68 23.17 ±sh 0.50 0.47 036 0.68 0.52 0.46

* Differences when compared with baseline values (Wilcoxon test) and corresponding control values (Student's t-test) are significant at p<0.05.

Thus, the analysis of the obtained results allows us to conclude that compound 1, in terms of acute toxicity, belongs to the 3rd toxicity class (moderately toxic) [11] according to GOST 12.1.007-76.

2.2. Determination of antihypoxic activity.

Experiments to determine the antihypoxic activity of substances were carried out on white mice weighing 20-22 g, anesthetized with sodium thiopental (50 mg/kg intraperitoneally).

Compound 1 or the reference drug was administered intravenously (in the tail vein) for 3 min at a dose corresponding to 5% of the LD ₅₀ value using an electronic dispenser (Kent Scientific, USA) 5 min before tracheal ligation. The results of the study are presented in table. eleven.

As the results of the experiments showed, the reference drug Ethoxydol, used in the highest therapeutic dose, led to an increase in the life time of experimental animals - the shortest time of maintaining bioelectric activity was 592 s, and the longest - 610 s, which significantly differed from similar indicators in mice of the control group. In addition, when taking the drug, there was no noticeable decrease in heart rate (Table 11).

Table 11. The effect of compound 1 and the reference drug Ethoxydol on the lifespan of mice (the time of myocardial bioelectrical activity) against the background of acutely developing asphyxia

Substance (drug) Dose, mg/kg (%ots ₀ ) Animal number Lifespan, s Heart rate, beats/min 1 2 3 4 5 0.9% NaCl solution (control) - 1 429 384 2 388 352 3 416 367

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4 394 393 5 410 348 6 422 366 M±t 41017 36817 Ethoxydol 25.0 (5) 7 585 376 8 592 412 9 617 399 10 574 405 eleven 631 386 12 609 404 M±t 60119' 397110 13 716 380 14 722 396 15 693 374 16 674 361 1 2 3 4 5 connection 1 21.2(5) 17 709 385 18 663 390 M±t 696110 ³⁶ 381±9

^a Significant difference from the control group (Gehan survival test with Yates correction);

^b The difference from the Ethoxidol group is statistically significant at p<0.05 (univariate analysis of variance, Newman-Keuls test);

* The difference from the control is statistically significant at p<0.05 (univariate analysis of variance, Newman-Keuls test).

Compound 1, when administered to mice at a dose of 5% of the LD ₅₀ value, extended the period of myocardial bioelectric activity to 696±10 s, which is statistically significantly more than the same indicator in the control group and in the comparator group. In addition, under the influence of the study drug, myocardial contractility was 381 ± 9 bpm.

A study of the acute toxicity of the enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate (compound 1) in comparison with the drug Ethoxydol showed that after a single intravenous administration to mice of the SHK line, the DL ₅₀ index of compound 1 for male mice was 419, 7 mg/kg, for female mice - 428.8 mg/kg; the DL ₅₀ index of the reference drug Ethoxidol for male mice was 498.9 mg/kg, for female mice - 499.4 mg/kg. Compound 1 belongs to the 3rd class of toxicity when administered parenterally according to GOST 12.1.007-76.

When studying the antihypoxic activity of compound 1 in comparison with the finished dosage form of the drug Ethoxydol, it was found that the levorotatory isomer with a single prophylactic injection into the tail vein of mice 5 minutes before hypoxia is reproduced at a dose of 5% of the DL ₅₀ value, has antihypoxic activity. At the same time, the indicated activity of compound 1 statistically significantly exceeds the corresponding activity of the reference drug, as evidenced by the duration of the bioelectrical activity of the heart. Neither the reference drug nor compound 1 has a bradycardic effect.

Example 2

Solution for injection (mass/volume):

L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate - 5.00%;

sodium metabisulphite - 1.0%;

M solution of hydrochloric acid to pH 4.5;

Water for injection - up to 100%.

Example 3

Capsules:

L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate - 30 wt.%;

starch - 25 wt.%;

polyvinylpyrrolidone - 3.5 wt.%;

microcrystalline cellulose - 12.5 wt.%;

lactose - 29.0 wt.%;

Example 4. Study of the lipid-regulating action of the L-enantiomer.

The studies were carried out on an experimental model of dyslipidemia in Wistar rats (males, weighing 250–280 g, 10 animals per group). The model of cholesterol dyslipidemia was induced by the introduction of an oil solution of cholesterol at a dose of 40 mg/kg and vitamin D3 at a dose of 25,000 IU/kg (injections were carried out daily for 20 days). Before the start of the experiment and in dynamics (after 20 and 30 days), the following indicators were determined in rats:

1. Total cholesterol (TC) in blood plasma.

2. β-lipoproteins (β-LP).

3. High density lipoprotein cholesterol (HDL cholesterol).

4. The concentration of triglycerides (TG).

5. Atherogenic index (AI).

AI was calculated using the formula: AI = (TC - HDL-C)/HDL-C.

The content of high density lipoprotein cholesterol was determined by the Abel method after heparin-manganese precipitation and precipitation of low density lipoproteins from serum. The content of triglycerides was determined using diagnostic test kits.

The research results are given in table. 12.

Table 12. The effect of L-enatinomer on the parameters of the lipid spectrum of the blood plasma of rats with dyslipidemia induced by cholesterol and vitamin D3

group of animals total cholesterol, mmol/l HDL cholesterol, mmol/l IA TG, mmol/l β-lp, c.u. Intact 1.02 ± 0.40 ± 1.50 ± 0.56± 6.25± nye 0.07 0.02 0.01 0.14 0.05 Control 1.77 ± 0.39± 3.53± 0.67± 16.7±

l 20 days 0.22 0.02 0.21 0.22 1.12 Control 30 days 1.49 ± 0.14 0.39± 0.04 3.04± 0.32 1.12 ± 0.21 14.8± 2.31 L- enantiomer, 25 mg/kg 1.09 ± 0.11 0.53± 0.12 1.05 ± 0.04 0.42± 0.01 6.83± 1.87

The significance of differences compared to intact and control animals is p<0.001 and p<0.01, respectively.

The obtained results indicate that the administration of cholesterol in combination with vitamin D3 causes negative changes in the blood plasma spectrum of animals, which are characteristic of dyslipidemia IIA (according to the WHO classification). After 20 days, these disorders were manifested in a significant increase in the level of β-LP (p<0.001) and an increase in AI (p<0.001). After 30 days in animals of the control group, in addition to these proatherogenic changes, an increase in TG (p<0.01) and total cholesterol (p<0.01) was also observed.

In addition, a pronounced imbalance of the redox system was observed. Plasma catalase levels were significantly reduced. Accumulation of products of lipid peroxidation (LPO) was found. The administration of the L-enantiomer at a dose of 25 mg/kg orally (from days 20 to 30) to animals that were previously injected with cholesterol with vitamin D3 had a pronounced lipid-correcting effect: the HDL-C fraction significantly increased, the content of total cholesterol, βLP decreased, and the AI value decreased to normal. (value in intact animals). Thus, the L-enantiomer is an effective lipid-regulating agent.

Example 5 Acute Toxicity and Antihypoxic Activity Studies.

The studies were carried out on 69 white mice-males and 69 white mice-females of the SHK line weighing 20-22 g. receive statistically reliable data, as evidenced by the long-term practice of toxicological studies. During the experiment, the animals were kept under controlled conditions: ambient temperature 20-24°C, relative humidity 50-60%.

In the present study, acute toxicity was evaluated in white outbred mice of both sexes after a single intravenous injection of compound 1 and the reference drug. White outbred mice (6 mice of each sex per group) were injected with compound 1 or the reference drug in increasing doses of 300, 400, 500, 600, 700 mg/kg of animal weight as a solution in water for injection with a total volume of 0.5 ml, which acceptable for this type of animal.

Animals were observed for at least 14 days after administration. About toxicity

- 8 040876 drugs were judged by the death of animals and the general picture of intoxication.

The general condition of the animals was recorded: the features of their behavior, the intensity and nature of motor activity, the presence and nature of seizures, coordination of movements, skeletal muscle tone, response to tactile or pain stimuli, the frequency and depth of respiratory movements, the condition of the hair and skin. The number of animals that died during the experiment was counted.

The data obtained were processed by the generally accepted method of variation statistics with the calculation of the mean value (M) and the standard error of the mean value (m). Paired Student's t-test with Bonferroni's correction was used as a criterion for assessing the significance of differences in mean values at a significance level of P<0.05; to determine the significance of differences in absolute values, a nonparametric chi-square test was used; with a sample size of less than 10 - Fisher's exact test; to determine the significance of differences before and after administration of the pharmacological agent, the Wilcoxon test was used.

Based on the results obtained, the acute toxicity characteristics were determined using the Miller-Tainter probit analysis.

The clinical picture of intoxication in mice with the introduction of both compared pharmacological agents was identical. With the introduction of toxic doses of compound 1 and the reference drug Ethoxydol (racemate) (Russia), a decrease in the motor activity of animals was noted. When the dose was increased, a short-term (2-3 min) period of hyperkinesia with anxiety was observed. Movement coordination was impaired, skeletal muscle tone was increased, tonic and clonic convulsions were registered. During this period, involuntary acts of defecation and urination were observed. Some of the animals died due to convulsions. The surviving animals were in an inhibited state, which was expressed in a reduced reaction to light, sound, tactile and pain stimuli (irritation of the root of the tail), while convulsive muscle twitches, decreased appetite, diarrhea or constipation were observed against the background of increasing water consumption. Breathing was rapid and shallow, hyperthermia was recorded. The animals were in the above state for the first two days, after which their physiological state gradually returned to the initial state.

Over the next 14 days, the phenomena of intoxication in animals manifested themselves in a reduced reaction to stimuli. The hairline lost its luster, characteristic of healthy animals, the skin remained unchanged. In the future, the condition of the animals gradually returned to normal.

When observing the animals for 14 days, no death was recorded among the surviving animals.

Compound 1, when administered to mice at a dose of 5% of the LD ₅₀ value, extended the period of myocardial bioelectric activity to 696±10 s, which is statistically significantly greater than similar values in the control group and in the comparator group. In addition, under the influence of the study drug, myocardial contractility was 381±9 bpm. While the racemate, used in the highest therapeutic dose, led to an increase in the life time of experimental animals, in which the shortest time of maintaining bioelectric activity was 592 s, and the longest - 610 s.

Example 6 Study of anti-ischemic activity.

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

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

Compound 1 at a dose of 5 mg/kg was shown to eliminate ventricular fibrillation in coronary artery occlusion and reduce the number of animals in which ventricular fibrillation occurs to 1 out of 10 experimental animals.

The drug at a dose of 10 mg/kg has a distinct cardioprotective effect on cardiohemodynamics, bringing contractility (dP/dt), blood pressure and other values closer to the level of intact control.

Example 7. Study of neuroprotective activity.

Neurotropic activity was confirmed in the passive avoidance conditioned reflex trainability model in untrained rats and the passive avoidance conditioned amnesia test induced by scopolamine.

The study was carried out on white outbred mature male rats weighing 250-280 g.

The test for reproducing learning ability was carried out 24 h after training, the latent period of the animal entering the dark chamber was recorded, and then the number of animals that did not enter the dark dangerous chamber at all and remained on the illuminated hanging platform (rats,

Claims (5)

sho remembering the situation). Compound 1 or reference drug Ethoxidol was administered once (intragastrically) using a special probe in a volume of 0.2 ml per 100 g of rat weight 40 minutes before training. Animals of the control group were injected with distilled water. All the studied substances did not significantly change the latent time of performing the skill of entering a dark chamber. The latent period of entry into the dark chamber during the performance of the mink reflex at the first placement of the animals in the chamber did not change compared to the control. After that, the conditioned passive avoidance reflex (CRPA) was trained, for which the rat received a slight pain stimulus in a dark chamber. After 24 h of training, during the subsequent reproduction of the trained one, the rats remembered the pain stimulus received in the dark chamber and entered there with a long latent period. Only 1 rat out of 15 did not enter the dark chamber at all. The experimental results show that compound 1 at a dose of 200 mg/kg improves the learning of the passive avoidance conditioned reflex in untrained rats, which is expressed in a significant increase in the latent time of entering the dark chamber during reflex reproduction. The comparator at a dose of 200 mg/kg also improved the learning of the passive avoidance conditioned reflex in terms of the latent period of the reflex reproduction and in terms of an increase in the percentage of animals that did not enter the dark chamber at all. Thus, the drugs have similar neuroprotective efficacy at a dose of 200 mg/kg. The enantiomer at a dose of 100 mg/kg showed greater activity than the racemate at the same dose. Example 8. Study of hepatoprotective properties. The study was conducted on 30 volunteer patients with chronic hepatitis who received standard therapy. Compound 1 was administered intravenously as a 5% solution in 0.9% sodium chloride solution, 2 ml once a day for 2 weeks. All patients before and after the prescribed therapy underwent biochemical studies regarding their levels of alanine aminotransferase (ALT), aspartate aminotransferase (ACT), cyclophosphamide, lactate dehydrogenase (LDH), malondialdehyde, bilirubin, total protein and protein fractions. By day 14, ALT activity returned to normal in 22 patients, which amounted to 1.30±0.2 mmol/l. In 89% of patients, the concentration of bilirubin returned to normal. In patients who were injected with compound 1, the hepatocyte damage index was significantly lower than in patients who were injected with the reference drug (Etoksidol, 5% solution). Thus, the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate showed hepatoprotective activity, which was more significant than that of the reference drug Ethoxydol. CLAIM 1. Use of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula Ci ₂ H ₁₇ NO ₆ as a cerebroprotective agent. 2. Use of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula Ci ₂ H ₁₇ NO ₆ as a hepatoprotective agent. 3. Use of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula CnH^NOg as a lipid-regulating agent. 4. Use of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula C^HnNOg as an anti-ischemic agent. 5. Use of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula ΟηΗρΝΟβ as a neurotropic agent with neuroprotective activity. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2