RU2702003C1 - Antysteatosis agent - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to use of sodium 4,4'-(propanediamido)dibenzoate as an anti-steatosis agent, supporting a beam structure of liver parenchyma, preserving portal tracts without fibrosis, preventing periportal necroinflammatory activity.

EFFECT: wider range of low-toxicity anti-stethosics, having protective and regenerative properties, causing significantly more pronounced and early reparative and regenerative exposure on the liver.

1 cl, 1 tbl, 4 ex

Description

The invention relates to medicine and pharmacy and relates to a new antistatic agent 4.4 '- (propanediamido) sodium dibenzoate of the formula I, which can be used in medicine as a potential treatment for non-alcoholic fatty liver disease.

The compound itself and its anti-atherosclerotic, anti-alcohol, antioxidant and antihypertensive properties are known [A. from. 1773234 USSR, A61K 31/19; A. s. 1787030 USSR, A61K 31/19].

Currently, there is a steady increase in the frequency of diseases, which are based on associated disorders of carbohydrate and fat metabolism (metabolic syndrome, type 2 diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease). According to the Russian DIREG 2 study, non-alcoholic fatty liver disease (NZHLP) was found in 37.3% of patients who turned to the therapist for help; as a result, it ranked first among liver diseases - 71.6% [V.T. Ivashkin, O.M. Drapkina, I.V. Mayev et al. // Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2015, 25 (6), 31-38].

NLBF is a polyetiological disease, which is based on disorders of carbohydrate and fat metabolism, both in the body itself and in the body as a whole in the framework of the metabolic syndrome, obesity, and diabetes mellitus. NLBP is manifested in the form of steatosis or steatohepatitis with outcomes in cirrhosis or hepatocellular carcinoma.

Effective pharmacotherapy of fatty hepatosis has not yet been developed. Derivatives of malonic (methanedicarboxylic) acid are of interest as antistatosis drugs. 4,4 '- (Propanediamido) sodium dibenzoate, as a derivative of malonic acid, has a certain similarity with succinate and therefore can competitively inhibit the excess succinylation of regulatory proteins observed in a wide range of metabolic diseases (obesity, metabolic syndrome, diabetes mellitus, fatty liver disease )

The objective of the invention is the expansion of the arsenal of antistatic agents with low toxicity.

This task is achieved by using 4,4 '- (propanediamido) sodium dibenzoate as an antistatic agent.

The invention is illustrated by examples of synthesis and research of pharmacological properties, the description includes one table and is accompanied by two synthesis schemes.

Example 1. Obtaining 4,4 '- (propanediamido) sodium (I) dibenzoate.

Synthesis takes place in two stages.

Stage 1. Obtaining N, N'-malonyl-di-p-aminobenzoic acid (MPABA).

The stage is the key one and consists in N-acylation of PABA with malonic ester with the formation of the target MPABA. The process proceeded in the environment of o-xylene at a temperature of 145-150 ° for 8-10 hours.

Stage 2. Obtaining sodium salt MPABK

The chemistry of the process of obtaining from MPABA the corresponding sodium salt under the action of sodium hydroxide is described by the following scheme:

The method of obtaining 4,4 '- (propanediamido) sodium dibenzoate was studied and carried out in laboratory conditions using standard commercial raw materials.

The composition of the synthesized compound is confirmed by elemental analysis.

Gross formula: C ₁₇ H ₁₂ N ₂ O ₆ Na ₂ . Found,%: C - 52.62, H - 3.07, N -7.19. Calculated%: C - 52.86, H - 3.13, N - 7.25.

The structure of the synthesized substance was proved by spectral methods of analysis of ¹ H and ¹³ C NMR, IR spectroscopy.

Example 2. Determination of acute toxicity.

Determination of LD50 4,4 '- (propanediamido) sodium dibenzoate was carried out with a single intravenous administration in various experimental doses to males and females of laboratory mice and determination of the toxicity class of the claimed substance in a single intragastric administration to males and females of laboratory outbred rats [Prozorovsky VB Statistical processing of the results of pharmacological studies // Psychopharmacology and Biological Addiction. 2007. - T. 7. - No. 3-4. - S. 2090-2120].

It was found that when determining, according to the Prozorovsky method, 1LE50 of the claimed compound when administered to mice intravenously is 2060 ± 160 mg / kg for males and 1900 ± 170 mg / kg for females. When determining acute toxicity according to the OECD method, Test No. 423, “Acute oral toxicity - a method for determining the acute toxicity class”, a dose of 2000 mg / kg did not cause death if used in animals of both sexes, which allows the claimed agent to be classified as a toxicity class “toxicity class 5 or not classified. ”

Example 3. The effect of the claimed funds on the content of total lipids in the liver of mice with experimental steatosis.

All experiments were carried out in accordance with the Order of the Ministry of Health of the Russian Federation dated 01.04.16, No. 199n "On approval of the rules of good laboratory practice." The animals were kept under standard vivarium conditions on a normal diet with free access to water.

Euthanasia of animals was carried out by euthanasia with carbon dioxide in the CO ₂ -box THF3481-V01 (Bioscape, Germany).

Statistical processing of the obtained data was carried out using the Statistica 6.0 software package. The significance of differences in the normal distribution of quantitative traits was evaluated using Student's t-test (for independent samples). Numerical data are presented as: arithmetic mean (M) ± standard deviation (SD). The confidence level was set to 95%.

The study of lipid content in the liver was performed on 36 linear mice of C57bl / 6 males weighing 20-28 g, obtained from the Rappolovo nursery (Leningrad region). To model liver steatosis, a hypercaloric high-fat diet was used with an excess of easily digestible carbohydrates, which was created by adding melted pork fat (19%), sucrose (10%) and isolated soy protein (8%) to standard feed (Chi C. L. et al. Effects of caloric intake on learning and memory function in juvenile C57BL / 6J mice // BioMed research international. - 2015. - T. 2015]. Such a diet promotes the development of hepatic steatosis in animals and the formation of dysglycemia. Animals were divided into three groups of 12 mice each:

1. An intact group that received standard food and orally purified water;

2. The control group received a high-calorie high-fat diet and orally purified water;

3. The experimental group received a high-calorie, high-fat diet and orally 4.4 '- (propanediamido) sodium dibenzoate at a dose of 10 mg / kg.

After 24 weeks of modeling the pathology of the animals, they were taken out of the experiment and total lipids were quantified in the liver tissue by the method of Folch [Methods of biochemical studies (lipid and energy metabolism): Textbook / ed. M. Prokhorova. - L .: publishing house of Leningrad State University, 1982.- 272 p. - S. 54-57].

As a result, it was found that a hypercaloric high-fat diet leads to a significant increase in the lipid content in the liver tissue by 6.3%. Under the influence of 4,4 '- (propanediamido) sodium dibenzoate, a significant (p <0.05) decrease in total lipids in the liver was observed by 16% (table).

Designations: * - significant differences from the corresponding indicator of the intact group (p <0.05); ** - significant differences from the corresponding indicator of the control group (p <0.05).

Against the background of a hypercaloric high-fat diet, the influx of free fatty acids into the liver increases. This creates not only prerequisites for the development of steatosis, but also mitochondrial dysfunction, leading to the loss of cell control of lipid peroxidation and activation of lipid peroxidation, which ultimately leads to the death of hepatocytes by the mechanism of apoptosis or necrosis, the development of inflammatory infiltration and liver fibrosis [Cherkashina E.A., Petrenko L.V. Evstigneeva A.Yu. Non-alcoholic fatty liver disease: pathogenesis, diagnosis, treatment // Ulyanovsk Medical Biological Journal. - 2014. - No. 1. - S. 35-47]. Therefore, even a slight decrease in liver steatosis is a factor that improves the prognosis of the course of the disease.

Example 4. Histological examination of the liver of animals with experimental steatosis on the background of the proposed drug.

Histological examination of the liver is the modern “gold standard” for the diagnosis of steatosis, inflammation and the assessment of the stage of fibrosis in case of damage within the framework of fatty liver disease [V.T. Ivashkin, O.M. Drapkina, I.V. Mayev et al. // Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2015, 25 (6), 31-38]. The study of the effect of 4,4 '- (propanediamido) sodium dibenzoate on the histological picture of the liver was carried out similarly to the experiment described above. Additionally, metformin (Sigma, USA) at a dose of 300 mg / kg / day was used as a comparison drug.

Liver samples were fixed with a 10% normal (pH-7.4) formaldehyde solution for 24 hours. Fixed tissue samples were sequentially incubated in isopropanol followed by paraffin immersion on an ExelsiorES automatic histoprocessor (Thermo, UK). Dehydrated and paraffin-saturated tissue samples were poured with paraffin. Sections of 4 μm thickness were prepared from prepared paraffin blocks using a semi-automatic rotational microtome НМ340Е (Thermo, Germany) and stained with hematoxylin and eosin. Morphological changes were studied in transmitted light using an AxioImagerA2 research microscope (Thermo, Germany) equipped with a digital color camera and image acquisition software with a total magnification of × 100, × 200, × 400.

Histological examination of the liver of animals from the control group with experimental steatosis showed the preservation of the structure of the liver tissue, portal tracts without fibrosis, with varying degrees of severity of lymphocytic or lympho-macrophage stromal infiltration. In the periportal sections of the hepatic lobules, predominantly small-focal lymphohistiocytic infiltration was detected, located among groups of hepatocytes with necrobiotic changes. The cytoplasm of hepatocytes, mainly in the centrolobular departments of the hepatic lobules, with the phenomena of pronounced protein dystrophy in the form of uneven enlightenment, heterogeneity, lumpiness, some hepatocytes contain small or medium vacuoles in the cytoplasm.

In some samples, in addition, there are large foci of necrosis, abundantly infiltrated with lymphocytes, neutrophilic and eosinophilic granulocytes, located in the liver parenchyma, mainly in the immediate vicinity of the walls of the central (hepatic) veins. In addition to necrotic inflammatory changes in the cytoplasm of hepatocytes, small drops of fat were determined.

The inventive compound on the background of a hypercaloric diet allowed to preserve the structure of the liver tissue, portal tracts without fibrosis and mainly without pathological cell infiltration. The stroma of single portal tracts is focally infiltrated by lymphocytes. There is no periportal necroinflammatory activity. The beam structure of the liver parenchyma is preserved. The cytoplasm of hepatocytes with the phenomena of mild protein granular dystrophy. Inside the hepatic lobules, the proliferation of Kupffer cells and small focal lymphohistiocytic infiltration with an admixture of eosinophilic granulocytes are uneven, generally weakly expressed. In some samples, there are small foci of hepatocyte proliferation in areas of damage to the parenchyma. Scanty uneven polymorphic cell infiltration around the central (hepatic) veins.

In the group of animals that received metformin on the background of the diet, the structure of the liver tissue was preserved, portal tracts without fibrosis. The stroma of part of the portal tracts is infiltrated by lymphocytes. The beam structure of the liver parenchyma is preserved. Hepatocyte nuclei are moderately polymorphic, often with enlightened nuclei and uneven distribution of chromatin in them, with distinct hyperchromic nucleoli. The cytoplasm of hepatocytes with the phenomena of mild protein dystrophy. Inside the hepatic lobules, the proliferation of Kupffer cells and small focal lymphohistiocytic or mixed with eosinophilic granulocytes infiltration, located mainly in the immediate vicinity of the central veins, are uneven, generally weak.

Thus, in the group of animals treated with high-calorie food, the phenomena of damage to the liver parenchyma and a more pronounced pathological cellular reaction predominate, while in the group of mice that received, along with high-calorie food 4.4 '- (propanediamido) sodium dibenzoate, the manifestations of the current organization predominate ( with a macrophage productive reaction), as well as regenerative changes in hepatocytes with obviously less pronounced effects of damage to the liver parenchyma. In addition, it should be noted a decrease in fatty hepatocyte infiltration when using a pharmacological agent. Apparently (ceteris paribus), 4,4 '- (propanediamido) sodium dibenzoate has protective and regenerative properties, causing a significantly more pronounced and early reparative and regenerative effect on the liver. In contrast to the claimed compounds, in the group receiving metformin, regenerative changes were not detected.

Thus, the presence of antistatic activity in 4,4 '- (propanediamido) sodium dibenzoate was proved when modeling experimental liver steatosis in animals based on the study of the content of total lipids in the liver and histological examination of the organ. It was found that the claimed compound reduces the amount of total lipids in the liver tissue by 16% compared with the control. Comparison of existing morphological changes suggests the predominance of productive and proliferative (reflecting the processes of repair and regeneration) changes in the group that received the claimed compound over alterative-exudative changes (reflecting damage) in the group with experimental liver steatosis against the background of a high-fat load. According to the pharmacological activity of the claimed drug (4,4 '- (propanediamido) sodium dibenzoate) is superior to the comparison drug metformin. With the unidirectional results of the studies, 4.4 '- (propanediamido) sodium dibenzoate works in an effective dose of 10 mg / kg, while for metformin a dose of 300 mg / kg is required.

The scope of the claimed funds may be the treatment of non-alcoholic fatty liver disease.

Claims (1)

The use of 4,4 '- (propanediamido) sodium dibenzoate as an antistatosis agent supporting the beam structure of the liver parenchyma, preserving portal tracts without fibrosis, preventing periportal necro-inflammatory activity.