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Title:
L-ENANTIOMER 2-ETHYL-6-METHYL-3-HYDROXYPYRIDINIUM L-HYDROXYBUTANDIOATE WITH CEREBROPROTECTIVE, HEPATOPROTECTIVE, LIPID-REGULATING, ANTI-ISCHEMIC AND NEUROTROPIC ACTIVITIES
Document Type and Number:
WIPO Patent Application WO/2019/117820
Kind Code:
A1
Abstract:
The subject of this study is a new chemical compound, L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxyethane butanedionate and its antihypoxic activity. The estimation of its acute toxicity showed that enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L- hydroxypyridinium refers to toxicity class 3 for its toxicological properties according to the classification specified in GOST 12.1.007 – 76. Comparative studies of antihypoxic actions of enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate and of Ethoxidol drug after a single intravenous prophylactic injection in a dose, corresponding to 5% of DL50 value, determined for this mode of administration for mice, showed that the duration of the cardiac electrobiological activity in the animals that were administered the enantiomer was higher than that in the mice that were administered prophylactically Ethoxidol drug in a dose 25.0 mg/kg.

Inventors:
SKACHILOVA SOFIYA YAKOVLEVNA (RU)
MYASNIKOV DMITRIJ GEORGIEVICH (RU)
IVANOV ANDREJ NIKOLAEVICH (RU)
Application Number:
PCT/SK2018/050013
Publication Date:
June 20, 2019
Filing Date:
November 14, 2018
Export Citation:
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Assignee:
MADMIT TRADE A S (SK)
International Classes:
A61K31/44; A61P1/16; A61P3/06; A61P9/10; A61P25/28
Foreign References:
EA024773B12016-10-31
RU2663836C12018-08-10
RU2065299C11996-08-20
RU2144822C12000-01-27
RU2168993C12001-06-20
RU2189817C12002-09-27
RU2157686C12000-10-20
RU2205640C12003-06-10
RU2380089C22010-01-27
RU2398583C22010-09-10
RU2145855C12000-02-27
RU2149648C12000-05-27
RU2444359C12012-03-10
RU2377237C12009-12-27
Other References:
DATABASE WPI Week 201051, Derwent World Patents Index; AN 2010-C56259, XP002787431
VLASOV A P ET AL: "NEW METHODICAL APPROACHES TO PREVENTING SYSTEMIC TOXIC DAMAGES", FARMATSIYA, IZDATEL'SKII DOM RUSSKII VRACH, RU, no. 4, 1 January 2011 (2011-01-01), pages 42 - 46, XP009509940, ISSN: 0367-3014
MEXIDOL: "Research Pharmacological Institute named after V.V. Zakusov's", RUSSIAN ACADEMY OF MEDICAL SCIENCES, article "Main Neuropsychotropic Effects and Mode of Action, T.A. Voronina State Institution"
ORDER OF THE MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION AS OF, 31 December 1996 (1996-12-31)
ORDER OF THE MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION AS OF, 26 January 1998 (1998-01-26)
G.I. KLEBANOV ET AL.: "ANTIOXIDANT CHARACTERISTICS OF 3-OXYPYRIDINE DERIVATIVES: MEXIDOL, EMOXYPINE AND PROXYPINE", MEDICAL CHEMISTRY
Attorney, Agent or Firm:
MAJLINGOVA & PARTNERS, S.R.O. (SK)
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Claims:
Claims:

1. L-Enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate, of formula (I):

for use as a cerebroprotective medicament.

2. L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate of formula (I):

for use as a hepatoprotective medicament.

3. L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate of formula (I):

for use as a lipid-regulating medicament.

4. L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate of formula (I):

for use as an anti-ischem ic medicament.

5. L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate of formula (I):

for use as a neurotropic medicament.

Description:
L-Enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate with Cerebroprotective, Hepatoprotective, Lipid-Regulating, Anti-Ischemic and Neurotropic Activities

Techical Field

This invention relates to an area of medicine, namely, a new chemical compound - L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L- hydroxybutandioate, which has a cerebroprotective, hepatoprotective, lipid-regulating, anti-ischemic and neurotropic activities.

Background Art One of the promising areas, explored by domestic pharmacologists refers to innovation domestic medicinal drugs in the line of hydroxypyridine derivatives.

Among the derivatives of 2- ethyl-6-methyl-3- hydroxypyridine there is a number of drugs with a wide spectrum of biological activities.

At the beginning of the 1980-s, Smirnov L.D. and Kuzmin V.l. from the State Scientific Pharmacological Research Institute of the Russian Academy of Medical Sciences synthesized Mexidol - succinate 2-ethyl-6-methyl-3-oxypyridine. Mexidol is a drug with a poly-component spectrum of pharmacological effects and with a multi- factor mode of actions. The most important parts of its activity are its antioxidant and membrane-trophic effects, its ability to modulate functioning of receptors and membrane-bound enzymes and to restore the neurally mediated balance. (Mexidol: Main Neuropsychotropic Effects and Mode of Action, T.A. Voronina State Institution «Research Pharmacological Institute named after V.V. Zakusov's» of the Russian Academy of Medical Sciences, Moscow).

Mexidol is widely used in human medicine and in veterinary medicine as well as an antioxidant and as an antihypoxic drug with a wide range of pharmacological actions and with high efficacy (nootropic and tranquilizing actions - patent RU 2065299, anti-ischemic and antiatherosclerotic actions - patent RU 2144822, antianginal action - patent RU 2168993, hepatoprotective action - patent RU 2189817, antimicrobial action - patent RU 2157686 and etc). Chemical compound 2-ethyl-6-methyl-3-hydroxypyridine succinate has the following pharmaceutical forms: solutions for parenteral infusions (Order of the Ministry of Health of the Russian Federation as of December 31 , 1996 No. 432‘Mexidol® solution 5% for injections’, patents RU 2205640, 2380089, 2398583), tablets (Order of the Ministry of Health of the Russian Federation as of January 26, 1998 No. 21 ‘Mexidol® coated tablets, 0,125g’), capsules (patents RU 2144822, 2145855), bandaging material (patent RU 2149648) and etc.

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

Besides, a medicinal drug Ethoxidol - 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutandioate (racemate) with anti-ischemic, cerebroprotective, neurotropic and lipid-regulating actions is also known (RU 2 377 237). It has the following formula:

There are also pharmaceutical compositions, containing Ethoxidol: tablets, solutions for injections.

It was shown that Ethoxidol in a dose 10 mg/kg neutralizes ventricular fibrillation in a coronary occlusive disease and reduces the number of laboratory animals with ventricular fibrillation to 2 of 8 experimental ones, when administered in a dose 10mg/kg and to 2 of 9 ones, when administered in a dose 23mg/kg.

Ethoxidol statistically increases the ventricular fibrillation threshold of cats in a dose 23.0 mg/kg within the period from 5 to 60 minutes of the occlusion of coronary arteries, while Anaprilin has such action in an iso-toxic dose within the period from 30 up to 60 minutes.

In acute myocardial ischemia, a dose 15 mg/kg has an expressed cardio- protective action on cardiac hemodynamics, approximating the value of contractibility (dP/dt), of arterial blood pressure etc. to the level of the intact control. Ethoxidol in a dose 1 mg/kg has the ability to reduce intensity of lipid peroxidation in laboratory rabbits, caused by their 30-day immobilization, which is evidenced by the reduction of the level of malondialdehyde (MDA) by 46% in blood plasma and in kidney tissues. In this case, the general cholesterine is reduced by 41.7% and atherogenicity index is reduced by 82.9%.

The advances of stereo-pharmacology in recent decades enriched science with understanding of the general pattern of structure-activity and structure-toxicity for the substances that represent racemic mixtures of levorotary and dextrorotary isomers. D- and L-enantiomers were obtained with various pharmacological properties. Studies showed that levorotary isomers are generally more toxic, but more active too, while dextrorotary isomers and racemates are inferior as compared to the levorotary ones as for the intensity of pharmacological effects.

A classic example for it is L-adrenalin: its intracardial concentration is 11 times higher than that of a dextrorotary isomer. Interactions of receptors, including processes of hepatic metabolism, which are also determined by the character of a drug-receptor correlation, are characterized by a high stereo-specificity.

Clinical studies of S-amlodipine. The results of clinical studies substantiate the antihypertensive and antianginal effects of S-amlodipine. It was shown that for the optimum therapeutic action of S-amlodipine, half as less doses of the drug are required.

Now, only 15% of medicinal drugs are chirally pure substances, i.e. they contain enantiomers of the same type.

The use of optical isomers (enantiomers) of well-known cardiovascular agents increases their efficacy and acceptability.

M.l. Lutay, A.F. Lysenko, O.l. Moiseyenko.

National Research Center named after N.D. Strazhesko’s Institute of Cardiology of the Academy of Medical Sciences of Ukraine, Kiev.

Today such derivatives of 3-hydroxypyridine, as mexidol and emoxypin, are used in clinical practice (G.l. KLEBANOV et al. ANTIOXIDANT CHARACTERISTICS OF 3- OXYPYRIDINE DERIVATIVES: MEXIDOL, EMOXYPINE AND PROXYPINE, Medical Chemistry Issues No.3 2001 ). These drugs have antioxidant, antihypoxic, nootropic, antispastic and anxiolytic actions.

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

(I):

its use as a cerebroprotective medicament and to study its antihypoxic activity, while taking in consideration the requirements for pre-clinical studies of pharmaceuticals. The compound has a cerebroprotective activity that provides a more prolonged period of myocardial bioelectrical activity as compared to the activity of the racemate.

Further, the aim of the invention is the development of a new pharmaceutical product with hepatoprotective, lipid-regulating, anti-ischemic and neurotropic activites, coprising compound L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L- hydroxybutandioate (compound 1 ).

Compound 1 , as a composition with pharmaceutically acceptable carriers (target additives), can be administered in different pharmaceutical forms. The compound is used in therapeutically effective doses, which depend on patients’ body weight, gender, age, etiology and severity of their diseases. The preferable doses comprise from 20 up to 300 mg.

Technical result: Efficiency of the new derivative of 3-hydroxypyridine, namely, L- enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate, used for treatment and for preventive care of a wide range of diseases as a pharmaceutical product with hepatoprotective, lipid-regulating, anti-ischemic and neurotropic actions that provide a more prolonged period of myocardial bioelectrical activity as compared to the action of the racemate.

Detailed description

Example 1 1. Synthesis of L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L- hydroxybutandioate.

13.41 g (0.1 m) L-hydroxybutandioic acid is added to 13.7 g of (0.1 m) 2-ethyl-6- methyl-3-hydroxypyridine, mixed up thoroughly and heated up to the melting temperature of 100°C - 110°C. About 120 ml of water is added gradually to the obtained mixture, the solution is filtered, and 60-70% of water is removed by vacuum distillation. Then 80ml of acetone is added to the remainder, the bulk is crystallized, the obtained residual matter is filtered and dried in vacuum. 23.8 g of white crystalline substance with a high hygroscopicity is obtained, which deliquesces in air.

C12H 17NO6

Found, %: C 53.07; H 6.38; N 5.11.

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

IR-spectrum, v erm 1 : 3510, 3230, 1660, 1610, 1560, 1292, 1161.

UV-spectrum, nm: 228, 291 (water)

Optical rotation [O]D 20 -13.6 (4.5% acetonitrile)

2. Biological Studies

General Characteristics of Laboratory Animals

For these studies, 69 white male SHK mice and 69 white female mice of weight 20-22g were delivered from the breeding nursery of Andreyevka Branch of Federal State Budgetary Institution of Science «Scientific Center of Biomedical Technologies» of the Federal Medical and Biological Agency of Russia.

Number of animals Experimental groups of animals with six mice of each gender in a group to study acute toxicity and antihypoxic activity is a representative selection, which enables to obtain statistically reliable data, which is evidenced by a multi-year practice of toxicological studies. During the studies, the animals were kept under controlled conditions: air temperature 20°C - 24°C, relative humidity 50 - 60%. 2.1. Acute Toxicity Testing of Compound 1

In this study, acute toxicity was estimated with the help of the laboratory mice of both genders that were injected intravenously a single dose of compound 1 or of comparative drug Ethoxidol.

White outbreed mice (per 6 mice of each gender in a group) were injected compound 1 or comparative drug in ascending doses of 300, 400, 500, 600, 700 mg/kg in the form of a solution in injection water of the total volume of 0.5ml, which is acceptable for this species of animals.

Following the injection of the drugs, the animals were monitored for not less than 14 days. Basing on fatalities and on the general picture of intoxication, toxicity of the drugs was estimated.

The performance status of animals was estimated, for example, specifics of their behavior, intensity and character of motor activity, presence and character of spasms, motion coordination, skeletal muscles stretch, responses to tactile or pain stimuli, frequency and depth of respiratory movements, pelage and skin state. The number of animals that died during the studies was registered.

The obtained data was processed with the help of a general variation statistics method with the calculation of the mean value (M) and standard error of the mean (m). As a criterion of confidence in estimation of the mean value differences, a paired Student t-criterion was used with Bonferroni adjustment with significance level P<0.05; to determine reliability of absolute value differences, non-parametric criterion‘chi- square’ was used; with the selection number less than 10, Fisher's exact test was employed; to determine reliability of differences before and after injection of the pharmaceutical agents, Wilcoxon criterion was applied.

Then basing on the obtained results, the characteristics of‘acute’ toxicity were determined with the help of Miller-Tainter’s probit analysis [10] (Tables 1-4).

The clinical presentations of intoxication upon the administration of the compared pharmacological agents were identical. When toxic doses of compound 1 or of comparative drug Ethoxidol, Russia, were injected, a decrease of motor activity of animals was observed. With the escalation of a dose, there was a short period (2- 3 minutes) of hyperkinesia with unrest. The motion coordination was disturbed, the skeletal muscle tonus was increased, clonic and tonic spasms were registered. During this period, involuntary acts of defecation and urination were observed. Some of the animals died, affected by spasms. The survived animals were sluggish with impaired responses to light, sound, tactile and noxious stimuli (tail head stimuli). They also developed muscle jactation, loss of appetite, diarrhea or obstipation and they consumed water increasingly. They had hurried shallow breathing, hyperthermia was registered. The animals had this condition for the first two days, whereafter their physiological condition gradually returned to the initial one.

During the subsequent 14 days, intoxication of the animals manifested itself in the form of decreased responses to stimuli. The pelage of the animals lost its gloss, characteristic of healthy animals; their skin condition did not change. Later their condition gradually returned to normal.

The animals were followed up for 14 days, with no deaths of the survived animals registered.

The results of mice lethality studies after the administration of compound 1 or of comparative drug Ethoxidol are shown in Tables 1 -4.

Table 1

Dose-effect dependence (fatal cases) after intragastric administration of compound 1 to male mice

Calculation of acute toxicity parameters resulted in the following:

DL 5O =419.74 (364.2÷474.3) mg/kg, DL I6 = 304.3 mg/kg, DL 84 =523.8 mg/kg.

Table 2

Dose-effect dependence (fatal cases) after intragastric administration of comparative drug Ethoxidol to male mice

DI_5O=498.93 (414.4÷643.9) mg/kg. DL I6 = 391 .2 mg/kg. DL 8 =622.3 mg/kg.

Table 3

Dose-effect dependence (fatal cases) after intragastric administration of compound 1 to female mice

DI_5O=428.81 (291 8÷554.4) mg/kg. DL I6 = 313.4 mg/kg. DL 8 =574.7 mg/kg.

Table 4

Dose-effect dependence (fatal cases) after intragastric administration of comparative drug Ethoxidol to female mice

DI_5O=499.36 (350.1 ÷628.3) mg/kg. DLi 6 = 367.3 mg/kg. Dl_ 84 =628.0 mg/kg.

Body weight dynamics of the survived mice that were administered the toxic doses of compound 1 or of comparative drug Ethoxidol did not differ significantly from that of the control group. When analyzing dynamics of water and feed consumption after the intravenous injections of compound 1 or of comparative drug Ethoxidol, there were no significant differences as compared to the control groups of mice (Tables 5 - 10).

Table 5

Daily feed (g) and water (ml) consumption per mouse (male species) after intragastric administration of toxic doses of compound 1

Note: * - When compared to the control, d ifferences are significant with p<0.05 (chi- square criterion).

Table 6 Daily feed (g) and water (ml) consumption per mouse (male species) after intragastric administration of toxic doses of comparative drug Ethoxidol

Note: * - When compared to the control, d ifferences are significant with p<0.05 (chi- square criterion).

Table 7

Daily feed (g) and water (ml) consumption per mouse (female species) after intragastric administration of toxic doses of compound 1

Note: * - When compared to the control, differences are signr leant with p<0.05 (chi- square criterion).

Table 8

Daily feed (g) and water (ml) consumption per mouse (female species) after intragastric administration of toxic doses of comparative drug Ethoxidol

Note: * - When compared to the control, differences are significant with p<0.05 (chi- square criterion)

Table 9

Body weight dynamics of the survived mice (males, g) for determining acute toxicity of compound 1 and of comparative drug Ethoxidol

Note: * - Differences as compared to the initial values (Wilcoxon criterion) and the corresponding control values (Student t-criterion) are significant with p<0.05

Table 10

Body weight dynamics of the survived mice (females, g) for determining acute toxicity of compound 1 and of comparative drug Ethoxidol

Note: * - Differences as compared to the initial values (Wilcoxon criterion) and the corresponding control values (Student t-criterion) are significant with p<0.05 Thus, the performed analysis of the obtained results enables to refer compound

1 to toxicity class 3 (mildly toxic) as for its acute toxicity factor [1 1 ] GOST 12.1 .007 - 76.

2.2. Determination of antihypoxic action

Experiments on determining antihypoxic actions of the substances were performed on white mice of weight 20-22g, narcotized with thiopent sodium (50 mg/kg abdominally).

Compound 1 or the comparative drug were being injected for three minutes intravenously (the tail vein) in a dose, corresponding to 5 % of LD50 value, with the help of an electronic meter (‘Kent Scientific’, USA), five minutes prior to the trachea ligation. The results are shown in Table 1 1.

The results of the experiments show that the administration of comparative drug Ethoxidol in the highest therapeutic dose, resulted in an increase of a life span of experimental animals; the shortest period of preserving of electrobiological activity was 592s, and the longest period was 610s, which differed significantly from the analogous values of the mice of the control group. Besides, administration of the drug did not cause a significant decrease of the cardiac rate (Table 1 1 ).

Table 1 1

Effects of compound 1 and of comparative drug Ethoxidol on the life span of mice

(myocardium bioelectrical activity) following an abruptly developing asphyxia

Note: a - Variations from the control group are significant (Gehan’s survival criterion with Yates correction)

b - Variations from Ethoxidol group are statistically significant, if p<0.05 (one- dimensional dispersion analysis, Newman-Keuls Test)

* - Variations from the control group are statistically significant, if p<0.05 (one- dimensional dispersion analysis, Newman-Keuls Test).

Compound 1 , injected to the experimental mice in a dose that comprised 5% of LD50 value, prolonged the bioelectrical activity period of the myocardium to 696±10s, which is statistically significantly more than that of the control group or of the comparative drug group. Besides, the administration of the drug resulted in the myocardial contractility of 381 ±9 bpm.

The comparative studies of acute toxicity of enantiomer 2-ethyl-6-methyl-3- hydroxypyridinium L-hydroxybutandioate (compound 1 ) and of Ethoxidol drug showed that after a single intravenous injection of compound 1 to SHK mice, DL50 value for male mice was 419.7 mg/kg, for female mice - 428.8 mg/kg; and after a single intravenous injection of comparative drug Ethoxidol, DL50 value for male mice was 498.9 mg/kg, for female mice - 499.4 mg/kg. Compound 1 refers to toxicity class 3 for a parenteral mode of administration according to GOST 12.1.007 - 76.

Studies of antihypoxic actions of compound 1 and of comparative finished pharmaceutical forms of Ethoxidol showed that a single prophylactic injection of a levorotating isomer into the tail vein of mice in a dose of 5% of DL50, 5 minutes before stimulating hypoxia, results in its antihypoxic activity. This activity of compound 1 significantly exceeds statistically the corresponding activity of the comparative drug, which is evidenced by the duration of the cardiac bioelectrical activity. Neither comparative drug, nor compound 1 have bradycardia effects.

Example 2

Solution for injections (weight/volume)

L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate 5.00% Sodium metabisulfite 1.0%

1 M hydrochloric acid solution to pH 4.5

Water for injections to 100% Example 3

Capsules

L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutandioate - 30 % mass

Starch - 25 % mass

Polyvinyl pyrrolidone - 3.5 % mass

Microcrystalline cellulose - 12.5 % mass

Lactose - 29.0 % mass Example 4

Lipid-regulating activity of L-enantiomer.

Studies were carried out with the help of an experimental model of dyslipidemia in Wistar line rats (males, weight 250-280g, 10 animals in a group). The model of cholesterine dyslipidemia was induced by injecting of the oil solution of cholesterine in a dose 40 mg/kg and of vitamin D 3 in a dose 25000 lU/kg (the injections were made for 20 days daily).

The following values were determined in rats before the experiment and over time (after 20 and after 30 days):

•Total cholesterine (TCH) in blood plasma,

•b-lipoproteids (b-LP),

•Cholesterine of high-density lipoproteins (CH HDL),

•Concentration of triglycerides (TG),

• Atherogenic index (Al).

The value of the Atherogenic Index was calculated from the following formula: Al = (TCH— CH HDL) / CH HDL.

The concentration of the cholesterine of high-density lipoproteins was determined with the help of the Abelian method, following heparin-manganic precipitation and subsidence of low-density lipoproteins from plasma. The concentration of triglycerides was determined with the help of diagnostic test kits.

The results of the studies are shown in Table 12. Table 12

Effect of L-enantiomer on lipid specter values of the blood plasma of rats with dyslipidemia, induced by cholesterine and vitamins D 3

Significance of differences as compared to intact and control animals is p< 0.001 and p< 0.01 accordingly.

The obtained results show that the injection of cholesterine in combination with D 3 causes negative changes in the specter of blood plasma of animals, characteristic of dyslipidemia IIA (WHO classification). After 20 days, the disorders manifested themselves in a significant increase of the level of b-LP (p< 0.001 ) and in an increase of Al (p< 0.001 ). After 30 days, the animals of the control group had, in addition to the specified proatherogenic changes, an increase of TG (p< 0.01 ) and of TCH (p< 0.01 ).

Besides, a significant imbalance of the oxidant-antioxidant system was registered. Catalase concentration in blood plasma was reduced significantly. Lipid peroxidation products were accumulated. The introduction of L-enantiomer in a dose 25 mg/kg orally (from the 20 th to the 30 th days) to the animals that had been preliminarily introduced cholesterine and vitamin D 3 , resulted in an apparent lipid-correcting action: CH HDL fraction increased significantly, TCH, b-LP values were decreased, and Al value was reduced to the norm (intact animals’ values).

Thus, L-enantiomer is an effective lipid-regulating agent. Example 5

Studies of acute toxicity and of antihypoxic action

The experiments were carried out on 69 white male SHK mice and 69 female SHK mice of weight 20-22g. Experimental groups of animals with six mice of each gender in a group to study acute toxicity and antihypoxic activity is a representative selection, which enables to obtain statistically reliable data, which is evidenced by a multi-year practice of toxicological studies. During the studies, the animals were kept under controlled conditions: air temperature 20°C- 24°C, relative humidity 50 - 60%.

To assess acute toxicity in this study, white outbreed mice of both genders were administered a single intravenous injection of compound 1 and of the comparative drug. White outbreed mice (6 mice of each gender in a group) were injected compound 1 or comparative drug in ascending doses of 300, 400, 500, 600, 700 mg/kg in the form of a solution in injection water of the total volume of 0.5ml, which is acceptable for this species of animals.

Following the injection of the drugs, the animals were monitored for not less than 14 days. Basing on the fatalities and on the general picture of intoxication, toxicity of the drugs was estimated.

The performance status of animals was estimated, for example, specifics of their behavior, intensity and character of motor activity, presence and character of spasms, motion coordination, skeletal muscles stretch, responses to tactile or pain stimuli, frequency and depth of respiratory movements, pelage and skin state. The number of animals that died during the studies was registered.

The obtained data was processed with the help of a general variation statistics method with the calculation of the mean value (M) and standard error of the mean (m). As a criterion of confidence in estimation of the mean value differences, a paired Student t-criterion was used with Bonferroni adjustment with significance level P<0.05; to determine reliability of absolute value differences, non-parametric criterion‘chi- square’ was used; with the selection number less than 10, Fisher's exact test was employed; to determine reliability of differences before and after injection of the pharmaceutical agents, Wilcoxon criterion was applied.

Then basing on the obtained results, the characteristics of‘acute’ toxicity were determined with the help of Miller-Tainter’s probit analysis.

The clinical presentations of intoxication upon the administration of the compared pharmacological agents were identical. When toxic doses of compound 1 or of comparative drug Ethoxidol (racemate), Russia, were injected, a decrease of motor activity of animals was observed. With the escalation of a dose, there was a short period (2-3 minutes) of hyperkinesia with unrest. The motion coordination was disturbed, the skeletal muscle tonus was increased, clonic and tonic spasms were registered. During this period, involuntary acts of defecation and urination were observed. Some of the animals died, affected by spasms. The survived animals were sluggish with impaired responses to light, sound, tactile and noxious stimuli (tail head stimuli). They also developed muscle jactation, loss of appetite, diarrhea or obstipation, and they consumed water increasingly. They had hurried shallow breathing and hyperthermia. The animals had this condition for the first two days, whereafter their physiological condition gradually returned to the initial one.

During the subsequent 14 days, intoxication of the animals manifested itself in the form of decreased responses to stimuli. The pelage of the animals lost its gloss, characteristic of healthy animals; their skin condition did not change. Later their condition gradually returned to normal.

The animals were followed up for 14 days, with no deaths of the survived animals registered.

Compound 1 , injected to the experimental mice in a dose that comprised 5% of LD50 value, prolonged the bioelectrical activity period of the myocardium up to 696±10s, which is statistically significantly more than that of the control group or of the comparative drug group. Besides, the administration of the drug resulted in the myocardial contractility of 381 ±9 bpm. While the racemate, administered in the highest therapeutic dose caused a prolongation of the life span of experimental animals, the shortest bioelectrical activity period was 592s, and the longest period was 610 s.

Example 6:

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

The effect of a 5% dose of LD50 on the correlation of the necrosis zone and the ischemia zone in ischemic myocardial injuries of rats was studied. Besides, the effect of the same dose on the development of reperfusion arrhythmias was under study. Ventricular fibrillation was caused with the help of the occlusion of coronary arteries. Then the effect of the drug on cardiac hemodynamics and on lipid peroxidation, induced by immobilization stress, was determined.

It was shown that compound 1 in a dose 5mg/kg corrects ventricular fibrillation in case of the occlusion of coronary arteries and decreases the number of animals that develop ventricular fibrillation to one of ten experimental ones.

The drug in a dose 10 mg/kg has an apparent cardioprotective action on cardiac hemodynamics, approximating the contractility values (dP/dt), the values of the arterial blood pressure and other values to the level of the intact control.

Example 7:

Neuroprotective activity study.

The neuroprotective action was demonstrated with a help of a model‘trainability of untrained rats for a conditioned passive avoidance reflex’ and with the help of a scopolamine-caused amnesia test of a conditioned passive avoidance reflex.

The experiments were carried out on outbred mature male rats of weight 250- 280g.

A trainability demonstration testing was carried out 24 hours after the training. The latent period of animals that entered the dark section was registered, and then the number of animals that did not enter the dark dangerous section and that stayed on a hanging lit platform was registered (rats well remembering the situation).

0.2 ml per 100g of weight of compound 1 or of comparative drug Ethoxidol were injected to the experimental rats once (intragastrically) with the help of a special probe 40 minutes before the training. The animals of the control group were injected distilled water.

No injected substances changed significantly the latent time of performing the skills of entering the dark section. The latent time of entering the dark section while exhibiting‘hole exploratory behavior’ during the first placement of the animals into the chamber did not differ from the control.

After that, the rats were trained for a conditioned passive avoidance reflex, wherefore they got a mild pain stimulation, while in the dark section.

After 24 hours of the training during the subsequent representation of their skills, the rats remembered the pain simulation in the dark section, and they entered it with a longer latent period. Only one rat of 15 did not enter the dark section at all.

The results of the experiments show that compound 1 in a dose 200 mg/kg improves trainability of untrained rats for a conditioned passive avoidance reflex, which is expressed in a significant increase of the latent time of entering the dark section. The comparative drug in a dose 200 mg/kg also improves trainability for a conditioned passive avoidance reflex in terms of the latent period of reproducing the reflex and in terms of an increase of the number of animals that did not enter the dark section at all. Thus, the drugs have a similar neuroprotective efficiency in a dose 200 mg/kg. The enantiomer in a dose 100 mg/kg was more active than the racemate in the same dose.

Example 8

Hepatoprotective activity

Trials were carried out on 30 volunteers with chronic hepatitis, who were administered standard therapy. Two ml of compound 1 were injected intravenously in the form of 5% solution for 0.9% of sodium chloride solution once a day for two weeks. Before and after the therapy, biochemical studies were carried out for all the patients as for the levels of their alanine aminotransferase, aspartate aminotransferase, cyclophosphan, lactic dehydrogenase, malondialdehyde, bilirubin, total protein and protein fractions.

By the 14 th day, 22 patients had a normalized activity of alanine aminotransferase, which was 1 .30±0.2 mmole/l. 89% of the patients had a normalized bilirubin concentration The hepatocyte impairment index in patients, who administered compound 1 was reliably lower than that in the patients, who administered the comparative drug (Ethoxidol 5% solution).

Thus, L-enantiomer 2-ethyl-6-methyl-3-hydroxypyridinium L- hydroxybutandioate showed a hepatoprotective activity that was more significant than that of the comparative drug Ethoxidol.