RU2705575C1 - Agent exhibiting hepatoprotective, lipid-regulating, antiischemic and neurotropic activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and clinical pharmacology. Disclosed is use of L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate as an agent exhibiting hepatoprotective, lipid-regulating, antiischemic and neurotropic activity.

EFFECT: compound provides a prolonged period of bioelectric activity of the myocardium compared to the racemate.

1 cl, 1 tbl, 7 ex

Description

The invention relates to the field of chemistry, medicine and clinical pharmacology.

A promising direction in the chemistry of organic compounds is the search and creation of innovative domestic drugs in a series of hydroxypyridine derivatives exhibiting a wide range of pharmacological activity.

Currently, 3-hydroxypyridine derivatives such as mexidol, emoxipin (G.I. KLEBANOV et al. ANTIOXIDANT PROPERTIES OF 3-OXYPYRIDINE DERIVATIVES: MEXIDOL, EMOXIPINE AND PROXIPINE, Issues of medical chemistry No. 3 2001) are used in clinical practice. These drugs have antioxidant, antihypoxic, nootropic, anticonvulsant, anxiolytic effects.

Known drug Ethoxidol - 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate in the form of a racemate, which has anti-ischemic, cerebroprotective, neurotropic and lipid-regulated activities (RU 2377237), of the formula:

as well as pharmaceutical compositions based on it.

The specified patent can be selected as the closest analogue of the prototype.

The aim of the invention is the creation of a new tool that shows hepatoprotective lipid-regulating, anti-ischemic and neurotropic activity and is a compound-L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate (compound 1):

Compound 1 can be administered in various dosage forms as a composition with pharmaceutically acceptable carriers (target additive). The compound is used in therapeutically effective doses, which will depend on the weight, gender, age of the patients, etiology and severity of the disease. Preferably, the dosage is from 20 to 300 mg.

Effect: the effective use of a new derivative of 3-hydroxypyridine, namely the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate for the treatment and prevention of a wide range of diseases as an agent that exhibits hepatoprotective lipid-regulating, anti-ischemic and neurotropic activity, which provides a prolonged period of myocardial bioelectric activity, in comparison with the racemate.

The invention can be illustrated by the following examples of the invention.

Example 1 Synthesis of 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, 13.41 g (0.1 m) of L-hydroxybutanedioic acid are sprinkled, the mass is thoroughly mixed and heated until melting at 100-110 ° FROM. To the obtained melt, 120 ml of water are gradually added with stirring, the resulting solution is filtered, 60-70% of water is distilled off in vacuo, 80 ml of acetone is added to the obtained residue, the mass is crystallized, the resulting precipitate is filtered off and dried in vacuum. Obtain 23.8 g of a white crystalline substance with high hygroscopicity, spreads in air. C12H17N06

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

Calculated,%: C 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)

Rotation angle [α] D20 -13.6 (4.5% acetonitrile).

Example 2 Solution for injection (weight / vol.)

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

Sodium metabisulfite 1.0%

1 M hydrochloric acid solution to pH 4.5

Water for injection up to 100%

Example 3 Capsules

L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate - 30 mass. %

Starch - 25 mass. %

Polyvinylpyrrolidone - 3.5 mass. %

Microcrystalline cellulose - 12.5 mass. %

Lactose - 29.0 mass. %

Example 4 Study of the Lipid-Regulating Effect of the L-Enantiomer

The studies were conducted on an experimental model of dyslipidemia in Wistar rats (males, weight 250-280 g, 10 animals per group). The model of cholesterol dyslipidemia is induced by the administration of an oil solution of cholesterol at a dose of 40 mg / kg and vitamin D ₃ at a dose of 25,000 IU / kg (administration to animals daily for 20 days).

Prior to the experiment and in dynamics (after 20 and 30 days), the following indicators were determined in a rat:

1. The content of total cholesterol (OXS) in blood plasma,

2. β-lipoproteins (β-LP),

3. High density lipoprotein cholesterol (HDL cholesterol),

4. The content of triglycerides (TG),

5. Atherogenicity index (IA).

IA was calculated by the formula: IA = (OXC - HDL cholesterol) / HDL cholesterol.

The cholesterol content of high density lipoproteins 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 kits.

The research results are shown in table 1.

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

The results obtained indicate that the introduction of cholesterol in combination with vitamin D ₃ causes negative changes in the blood plasma spectrum of animals, 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 IA (p <0.001). After 30 days, in animals of the control group, in addition to these pro-atherogenic changes, an increase in TG (p <0.01) and GC (p <0.01) was also observed.

In addition, a pronounced imbalance of the oxidant-antioxidant system was observed. Plasma catalase levels decreased significantly. The accumulation of lipid peroxidation products (LPO) was detected. The administration of 25 mg / kg orally (from day 20 to day 30) to animals that had previously received cholesterol with vitamin D ₃ , the L-enantiomer had a pronounced lipid-correcting effect: the fraction of HDL cholesterol increased significantly, the content of total cholesterol, β-LP decreased, and IA decreased to norms (level of intact animals).

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

Example 5 Acute Toxicity and Antihypoxic Activity Studies

The studies were performed on 69 white SHK male mice, 69 white SHK female mice weighing 20-22 g. The formation of animal groups of 6 mice of each sex for the experiment on the study of acute toxicity and antihypoxic activity is a representative sample and allows for many years of experience in toxicological studies to obtain statistically reliable data. 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 mice of both sexes with a single intravenous administration of compound 1 and the reference drug. White outbred mice (6 animals of each sex in the group) were injected with compound 1 or a comparative preparation in increasing doses of 300, 400, 500, 600, 700 mg / kg of animal weight in the form of a solution in water for injection with a total volume of 0.5 ml, permissible for this type of animal.

Observation of animals was carried out for at least 14 days after administration. The toxicity of the 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, reaction to tactile, pain stimuli, the frequency and depth of respiratory movements, the condition of the hair and skin. Counted the number of dead animals 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 standard error of the mean value (m). The paired Student's t test with Bonferroni correction at a significance level of P <0.05 was used as a criterion for assessing the reliability of differences in average values at a significance level of P <0.05, the non-parametric chi-square test was used to determine the significance of differences in absolute values, with a sample size of less than 10, Fisher’s exact test Wilcoxon test was used to determine the significance of the differences before and after administration of the pharmacological agent.

Based on the results obtained, the characteristics of “acute” toxicity were determined using a probit analysis according to Miller and Taint. 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 Ethoxidol (racemate), Russia, a decrease in the motor activity of animals was noted. With an increase in dose, a short-term (2-3 min) period of hyperkinesia with anxiety symptoms was observed. The coordination of movements was disturbed, the skeletal muscle tone was increased, tonic and clonic convulsions were observed. During this period, involuntary acts of defecation and urination were observed. Some animals died on the background of seizures. Surviving animals were in a "inhibited" state, which was expressed in a reduced response to light, sound, tactile and pain stimuli (tail root irritation), with convulsive muscle twitching, decreased appetite, diarrhea, or stumping due to increased water consumption. Breathing was rapid and superficial; hyperthermia was recorded. In the above state, the animals were in the first two days, after which they gradually assumed their initial physiological position.

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

During dynamic observation of animals for 14 days, death among surviving animals was not noted.

Compound 1 prolonged the period of myocardial bioelectric activity, being administered to mice at a dose of 5% of the LD50 value to 696 ± 10 s, which is statistically significantly more than in the control and in the comparison drug group. In addition, under the influence of the analyte, myocardial contractility was maintained up to 381 ± 9 beats / min. While the racemate used in the highest therapeutic dose led to an increase in the life time of the experimental animals, the shortest bioelectric activity conservation time was 592 s and the largest - 610 s.

Example 6. The study of anti-ischemic activity.

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

The effect on the ratio of the zone of necrosis to the zone of ischemia during ischemic damage to rat myocardium was studied at a dose of 5% of LD50. At the same dose, the effect on the development of reperfusion arrhythmias was studied. Ventricular fibrillation was caused by occlusion of the coronary arteries. The effect of the drug on cardiohemodynamics and lipid peroxidation (LPO) induced by immobilization stress was determined.

It was shown that compound 1 at a dose of 5 mg / kg eliminates ventricular fibrillation during occlusion of the coronary arteries and reduces the number of animals that have ventricular fibrillation to 1 out of 10 experimental animals.

In conditions of acute myocardial ischemia at a dose of 10 mg / kg, it has a clear cardioprotective effect on cardiodynamics, bringing contractility (dP / dt), blood pressure and others to the level of intact control.

Example 7. The study of neuroprotective activity.

The activity was confirmed on the model "learning of the conditioned reflex of passive avoidance of rats of under-education" and with the help of the test of amnesia of the conditioned reflex of passive avoidance caused by scopolamine in rats.

The study was performed on white mongrel sexually mature rats males weighing 250-280 g.

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

Compound 1 and the reference drug Ethoxidol was administered once orally (intragastrically) using a special probe in a volume of 0.2 ml per 100 g of rat weight 40 minutes before training. The animals in the control group were injected with distilled water.

All the studied substances did not significantly change the latent time of completing the skill of entering the dark chamber. The latent period of entry into the dark chamber when performing the mink reflex when the animals were first placed in the chamber did not change compared to the control.

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

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

According to the results, compound 1 at a dose of 200 mg / kg improves learning of the conditioned reflex of passive avoidance in rats, which is expressed in a significant increase in the latent time of entry into the dark chamber when the reflex is reproduced. The comparison drug at a dose of 200 mg / kg also improved the learning of the conditioned reflex of passive avoidance in terms of the latent period of the reflex and in terms of increasing the percentage of animals that did not enter the dark chamber at all. Thus, the drugs have similar neuroprotective efficacy at a dose of 200 mg / kg. At a dose of 100 mg / kg, the enantiomer showed greater activity than racemate in the same dose.

Example 7 Study of Hepatoprotective Properties

The study was conducted in 30 volunteer patients with chronic hepatitis who received standard therapy. Compound 1 was administered as a 5% solution in a 0.9% sodium chloride solution, 2 ml iv once a day for 2 weeks. In all patients, before and after the prescribed therapy, biochemical studies were performed: ALT, ACT, CF, LDH, MDA, bilirubin, total protein and protein fractions.

By the 14th day in 22 patients normalization of ALT activity was observed, and amounted to 1.30 ± 0.2 mmol / l, the bilirubin content increased to normal in 89% of patients. Against the background of the introduction of compound 1, the hepatocyte damage index was significantly lower than the control series (Ethoxidol 5% solution).

Thus, the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate showed a hepatoprotective effect that is superior to that of the reference drug Ethoxidol.

Claims (3)

   The use of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate of the formula 1

as a means of showing hepatoprotective lipid-regulating, anti-ischemic and neurotropic activity.