RU2614691C2 - Hepatoprotective substance - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: application of a taurine derivative, N-isopropylamide-2-benzylamine ethanesulfonic acid hydrochloride as a hepatoprotective agent for prevention of acute or chronic hepatitis, liver cirrhosis. Pre-clinical testing of drug showed lower intensity of infiltrative-sclerotic lesions and steatosis, hepatocytes necrosis frequency.

EFFECT: N-isopropylamide-2-benzylamine ethanesulfonic acid hydrochloride is not worse than Carsil in terms of hepatoprotective activity and can find application in medical practice.

4 dwg, 2 tbl

Description

The invention relates to biologically active compounds used in medicine and in pharmacology.

The invention relates to the expansion of the arsenal of drugs that have hepatoprotective effects in acute and chronic hepatitis and cirrhosis. For this purpose, it is proposed to use the compound - N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride. This structural analogue of taurine has been previously described as an inhibitor of platelet aggregation [Taurine derivatives exhibiting the ability to inhibit platelet aggregation AS No. 1540228]. The compound is non-toxic, the synthesis is simple and affordable. Conducted preclinical trials of the drug allow us to recommend it as a hepatoprotective agent for the prevention and treatment of acute and chronic hepatitis and cirrhosis.

The problem of prevention and treatment of diseases of the hepatobiliary system remains one of the urgent tasks of medical science. Recently, there has been a high incidence of toxic hepatitis among people of working age, which determines the importance of the search for new drugs for the treatment of this pathology [SD Podymova Liver Disease / A Guide for Physicians. - M.: Medicine. - 1999. - 704 p .; Rizzetto M. Therapy of chronic viral hepatitis. // Ital. J. Gastroenterol. Hepatol. - 1999. - Vol. 31. - P. 781-793; Okovity S.V. Experimental and clinical justification for the use of drugs with metabolic and immunotropic effects in various liver pathologies. Abstract. dis ... doctor honey. sciences. - SPb., 2003 .-- 41 p. ]. In the complex treatment of diseases of the liver and biliary tract, a variety of medications are used. Among them, an important place is occupied by drugs that have a hepatoprotective effect. Basically, they are of plant origin: phytopreparations of flavonoid nature from spotted milk thistle (legalon, silymar, karsil), from tansy - tanacechol, etc. [Mashkovsky MD Medicines 15th ed., 2005. - S. 526-527]. Due to their nature, these agents, as a rule, have a complex of pharmacological activity [SD Podymova Liver Disease / A Guide for Physicians. - M.: Medicine. - 1999. - 704 p. ]. As a hepatoprotective agent, the drug Karsil is often used [Mashkovsky M.D. Medicines 15th ed., 2014. - S. 526-527]. The hepatoprotective effect of carcil is associated with antioxidant properties, normalization of phospholipid metabolism, and stimulation of protein synthesis. The clinical effect of this compound begins to appear after its long-term use. However, prolonged use of carcil can cause dyspeptic symptoms (heartburn, retching, heaviness in the stomach, laxative effect) [Mashkovsky MD Medicines 13th ed., 1998. - S. 515-516]. Therefore, with the rapid course of the disease, this approach may be ineffective and insufficient [Podymova SD Liver Disease / A Guide for Physicians. - M.: Medicine. - 1999. - 704 p. ]. Therefore, the problem of finding new drugs to eliminate liver damage remains relevant. It seems promising to find such agents among compounds with a wide spectrum of pharmacological activity.

According to modern concepts, the pathogenesis of hepatitis is polyetiological and, ultimately, reduces to an imbalance of a number of aggressive and protective factors of the body. It is known that toxic damage to the liver is accompanied by a violation of the permeability of hepatocyte membranes and activation of lipid peroxidation processes with subsequent impaired liver function.

The chemical compound N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride (C ₁₂ H ₂₀ N ₂ O ₂ S HCl) obtained by us was tested on an experimental model of hepatitis.

The effect of its use exceeded expectations, after which the drug was subjected to a detailed study using a complex of modern methods (biochemical and histological) on a model of experimental liver pathology caused by the introduction of CCl ₄ (carbon tetrachloride). The hepatoprotective effect of the test drug was evaluated in two series of experiments:

series 1 - prophylactic use of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride in an acute experiment;

series 2 - course (within 21 days) use of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride under conditions of a chronic experiment.

Materials and methods

The comparative hepatoprotective activity of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride was studied in male rats weighing 180-200 g using the CCl ₄ model of toxic liver damage.

The comparison drug is carlsil. It is a total preparation containing silymarin, silibinin and extract of the fruit of milk thistle [Mashkovsky M.D. Medicines 15th ed., 2005. - S. 526-527].

Series 1. Prophylactic use of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride Modeling of toxic liver damage CCl4

The experiments were carried out on 40 white male rats weighing 180-200 g, which were divided into 4 groups. Group 1 was composed of intact animals. Group 2 rats were dosed daily with saline, group 3 — the claimed compound of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride (25 mg / kg), group 4 — carlsil comparison drug (20 mg / kg). Starting from day 8, animals of groups 2, 3, and 4 caused liver damage by administering CCl ₄ (50% solution in liquid paraffin) at a dose of 0.4 ml / kg. CCl ₄ was administered subcutaneously (1 time per day) for four days.

On the 7th day from the beginning of the introduction of carbon tetrachloride, when, according to the literature [Strechen SB, Stresyun V.N. The mechanisms of hepatoprotective action of new derivatives of nicotinic acid in experimental liver damage CCl ₄ // Bull. exp biol. and honey. - 1972. - No. 7. - S. 58-60], histological and functional changes corresponding to toxic hepatitis develop in the liver, rats were decapitated. After opening the abdominal cavity, the liver was quickly removed, a piece was cut off from it (weighing 1 g) and frozen in liquid nitrogen. Tissue sampling in each experiment was carried out from the same lobe of the liver. Glycogen content was determined in liver homogenates [Kemp A., Heijningen V. Van // Biochem. J. - 1954. - Vol. 56. - P. 646-648], in the blood serum of transaminase - alanine aminotransferase (AlAT), aspartate aminotransferase (AsAT) and total bilirubin using reagent kits according to the manufacturer's instructions [Komarov F.I., Korovin B.F., Menshikov V. AT. Biochemical studies in the clinic. Elista: APP "Dzhangar", 1998. - 250 s]. For histological examination, liver pieces were fixed in 10% neutral formalin solution and embedded in paraffin. Serial sections were stained with hematoxylin and eosin.

The effect of the preparation of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride on the change in biochemical parameters during prophylactic use.

The research results are presented in table 1. It can be seen that with toxic liver damage on the 7th day from the beginning of CCl ₄ administration in rats treated with saline, the glycogen content in the liver tissue significantly decreased (from 4.80 ± 0.25 to 1, 4 ± 0.3 g%). In this case, the concentration of AlAT increased in blood serum (from 65.5 ± 4.0 to 85.3 ± 3.2 U / L), AcAT (from 165.3 ± 4.0 to 199.4 ± 4, 7 U / L.) And the level of total bilirubin (from 27.8 ± 2.4 to 52.7 ± 4.5 μmol / l).

In rats that were administered the claimed compound - N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride (group 3) or carlsil comparison drug (group 4), the glycogen content in the liver tissue was significantly higher, the level of total bilirubin was lower than in animals not treated treatment (group 2), and the concentration of transaminases (AlAT and AsAT) did not differ from that in intact animals.

From the results obtained, it can be concluded: a preliminary, prior to toxic effect, administration of the claimed compound of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride, as well as the reference drug carcil, prevented depletion of glycogen stores in hepatocytes and an increase in the content of AlAT, AsAT, total bilirubin in blood serum.

The effect of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride on the histological picture of the liver with prophylactic use.

The control

The results of the studies showed that the histological picture of the liver tissue of animals that were injected with physiological saline before CCl ₄ injection (Fig. 1, photo B), in comparison with that observed in the liver of intact rats (Fig. 1, photo A), was characterized by small-drop fatty degeneration, irregular step necrosis, sclerotic changes and lymphomacrophage infiltration, localized in the portal tracts; adaptation changes were absent.

Reference carlsil

The prophylactic administration of carzil did not prevent the development of small-drop fatty degeneration, but reduced the intensity of lymphomacrophagous infiltration and promoted the appearance of adaptive changes in the form of nuclear polymorphism of hepatocytes (Fig. 1, photo D).

The inventive compound hydrochloride N-isopropylamide-2-benzylaminoethanesulfonic acid

The microstructure of rat liver tissue was consistent with moderate hepatitis activity (Fig. 1, photo B). Histological examination showed that stepwise necrosis in the liver parenchyma is small, fatty droplet dystrophy is unstable within one observation with a predominance of hydropic dystrophy, adaptive-compensatory changes in the form of nuclear polymorphism of hepatocytes were noted.

From the obtained data it can be concluded: preliminary administration of the claimed compound of the hydrochloride of N-isopropylamide-2-benzylaminoethanesulfonic acid has a positive effect on the histological picture of the liver with its toxic damage to CCl ₄ . This is evidenced by a decrease in the degree of activity of the pathological process, a decrease in the intensity of infiltrative sclerotic lesions and fatty degeneration, the frequency of necrosis, and an increase in adaptive changes in the form of nuclear polymorphism of hepatocytes.

Series 2. The study of the hepatoprotective effect of N-isopropylamide hydrochloride - 2-benzylaminoethanesulfonic acid with a course (within 21 days) administration in a chronic experiment

Simulation of toxic liver damage CCl ₄

The experiments were performed on 100 white male rats weighing 180-200 g. The inventive compound N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride and the comparison drug carlsil were started to be administered to animals with already formed toxic hepatitis, which was simulated by subcutaneous injection of CCl ₄ (50% solution in liquid paraffin ) at a dose of 0.4 ml / kg. On the 7th day from the start of CCl administration, ₄ animals were divided into 3 groups. Group 1 rats were injected with saline, group 2 — the claimed compound N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride (25 mg / kg). Group 3 animals - carlsil comparison drug (20 mg / kg). The inventive compound N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride and the reference preparation Karsil were administered daily orally (via tube). The therapeutic effect was evaluated in dynamics. For this, on the 7th, 14th and 21st day from the start of treatment, 10 rats were decapitated from each experimental group. Total bilirubin was determined in blood serum, and glycogen content in liver tissue homogenates. On histological examination, serial sections were stained with hematoxylin and eosin.

The effect of the preparation of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride on the change in biochemical parameters in a chronic experiment with a course (within 21 days) administration

The research results are presented in table 2. They showed that in animals with toxic damage caused by carbon tetrachloride, in comparison with intact rats, the glycogen level in the liver decreased by 71% and the concentration of total bilirubin in blood serum increased by 134%. Established facts indicate a violation of carbohydrate metabolism and liver pigment formation function. Further observations of the dynamics of the development of toxic damage showed that in untreated animals, the glycogen content in the liver increased from the 14th day of observation, and the total serum bilirubin level decreased from the 7th day (Table 2). At the same time, it should be noted that on the 21st day these indicators did not return to the values observed in intact rats.

In the group of rats with toxic liver damage that received the carlsil comparison drug, the total bilirubin content decreased to the values observed in intact rats on the 14th day, and glycogen stores were practically restored - by the 21st day of therapy.

In animals with simulated toxic hepatitis, against the background of the introduction of the claimed agent, N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride, the glycogen content in the liver increased by the 21st day, and the total bilirubin level decreased to the values observed in intact animals by the 7th day of treatment , indicating the restoration of glycogenesis and pigmentation function of hepatocytes.

Thus, the results of a comparative study of the therapeutic effect of the claimed compound of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride with liver damage caused by carbon tetrachloride indicate that the test compound (N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride) restores the total bilirubin content in the blood shorter time than carcil comparison drug.

The effect of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride on the histological picture of the liver in a chronic experiment with a course (within 21 days) administration

The control

In the liver of animals of the control group treated with CCl ₄ and physiological saline, on the 7th and 14th day of observation, coarse granularity, as well as step necrosis in the central and intermediate zones (Fig. 2, photo B and C) were revealed compared with liver of intact rats (Fig. 2, photo A). On the 21st day of observation, binuclear cells appeared, indicating a weakly expressed adaptation (Fig. 2, photo D). Toxic hepatitis at this observation period was characterized by an average degree of activity.

The inventive compound hydrochloride N-isopropylamide-2-benzylaminoethanesulfonic acid

The histological picture during therapy with N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride corresponded to chronic hepatitis of moderate activity on the 7th and 14th day of treatment (Fig. 3, photos A and B), and on the 21st day it was characterized by a low degree of activity (Fig. 3, photo B). Damage processes correlated with distinct adaptive changes in hepatocytes, in the form of nuclear polymorphism. In portal tracts moderate macrophage infiltration was noted.

Reference carlsil

According to the results of histological analysis, the comparison drug was slightly inferior to N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride. So, on the 7th and 14th day of carlsil therapy, structural abnormalities in the animal liver corresponded to moderate hepatitis of moderate activity (Fig. 4, photos A and B). Step serial necrosis was observed on serial sections of the liver, eosinophilic degeneration and hydropic degeneration of hepatocytes predominated in most lobules. On the 21st day of treatment with Karsil against the background of adaptive changes in the form of polymorphism of nuclei, step necrosis occurred (Fig. 4, photo B).

From the histological studies, it can be concluded that in case of CCl ₄ liver damage, a comparative assessment of the therapeutic effect of the claimed compound — N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride — and the reference carcil preparation revealed a positive trend. So, on the 14th day of therapy with the claimed compound, there was a clear tendency to decrease the intensity of damage processes, a low degree of cellular infiltration, a significant severity of compensatory changes (adaptive and regenerative processes), the tendency of connective tissue to mature. By the 21st day of treatment, these processes intensified. The results of a histological examination of the liver during poisoning with carbon tetrachloride indicate that under the conditions of a chronic experiment with a course (within 21 days) administration of the hepatoprotective effect of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride is comparable with the effect of the standard preparation of carcil.

Thus, biochemical and histological studies have shown that the claimed substance is superior in hepatoprotective activity to the comparison drug carlsil.

Claims (1)

The use of N-isopropylamide-2-benzylaminoethanesulfonic acid hydrochloride as a hepatoprotective agent for the prevention of acute or chronic hepatitis, liver cirrhosis.

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