RU2800017C1 - Way to treat liver cirrhosis - Google Patents

Abstract

FIELD: medicine; therapy; hepatology.

SUBSTANCE: invention can be used to treat liver cirrhosis in rats. The method includes administration of ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor. The medicinal products are administered at the following times and in the following sequence: ketanserin is administered from the 85th to the 95th day at a dose of 0.2 mg/kg from the first injection of carbon tetrachloride, reserpine is administered on the 96th day and on the 100th day at a dose of 1 mg/kg, vascular endothelial growth factor is administered on days 96–102 at a dose of 200 ng/animal, hepatocyte growth factor is administered on day 100 at a dose of 300 ng/animal.

EFFECT: use of the invention allows to treat liver cirrhosis due to the consistent correction of typical pathological processes: inflammation, fibrosis, as well as stimulation of angiogenesis and acceleration of neogenesis of hepatocytes.

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Description

SUBSTANCE: invention relates to medicine, namely to therapy and hepatology, and can be used to increase the efficiency of treatment of experimental liver cirrhosis and accelerate regeneration.

The liver is a vital organ and its diseases remain a major public health problem. More than 2 million deaths from liver diseases are recorded annually in the world, of which 1 million are associated with liver cirrhosis (LC) or its complications [Asrani S.K., 2019]. In Russia, liver pathology, including cirrhosis, occupies a leading place in the structure of gastroenterological mortality [Tsukanov V.V., 2021]. LC is a form of liver fibrosis caused by various liver diseases, including viral hepatitis, alcoholic liver disease, non-alcoholic steatohepatitis, and autoimmune liver diseases [Nishikawa K, 2018]. In 50% of cases, the development of cirrhosis is associated with the toxic effects of alcohol [Bataller R., 2015]. cirrhosis occurs as a result of various mechanisms of liver damage, but all of them lead to necroinflammatory process and fibrogenesis. Histologically, LC is characterized by diffuse nodular regeneration surrounded by dense fibrous septa, followed by parenchymal death and collapse of liver structures [Tsochatzis EA, 2014]. Therapy for cirrhosis is symptomatic, and in the terminal stage the only treatment is liver transplantation. cirrhosis leads to various complications (liver failure, portal hypertension and an increased risk of carcinogenesis), leading to a poor prognosis; therefore, the development of new treatments for cirrhosis is an important task [Nishikawa K, 2018].

A known method for correcting disorders in the liver tissue under the influence of a specific 5-HT 2 receptor antagonist - ketanserin [Vorobioff J, 1989]. Ketanserin is a serotonin inhibitor, reduces pressure in the portal vein of the liver and, by improving hemodynamics of the liver, increases the effectiveness of therapy [Vorobioff J, 1989].

This method is close to the claimed and is selected as a prototype.

The disadvantage of this method are the frequent side effects of ketanserin and its lack of effectiveness, which may limit its clinical use [Vorobioff J, 1989].

Promising for the treatment of liver cirrhosis may be a complex effect on the liver tissue - multicomponent therapy (ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor) has a pronounced anti-inflammatory and antifibrotic effect, stimulates liver regeneration processes and restores the function of hepatocytes.

The objective of this invention is to develop a method for the treatment of liver cirrhosis due to the consistent correction of typical pathological processes: inflammation, fibrosis, as well as stimulation of angiogenesis and acceleration of neogenesis of hepatocytes.

To achieve a new technical result in the method of treating liver cirrhosis by administering ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor are additionally administered, while the drugs are administered at the following times and in the following sequence: ketanserin is administered from the 85th to the 95th day at a dose of 0.2 mg/kg from the first injection of carbon tetrachloride, reserpine is administered on the 96th day and on the 100th day at a dose of 1 mg/kg, vascular endothelial growth factor is administered on days 96-102 at a dose of 200 ng/animal, hepatocyte growth factor is administered on day 100 at a dose of 300 ng/animal.

New in the present invention is the joint sequential use of ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor in the treatment of experimental liver cirrhosis.

Ketanserin (Ketanserinum, Sigma, USA) is a specific antagonist of 5-HT2 receptors; in addition, it has a moderate alpha-adrenergic blocking effect. Ketanserin blocks various effects of serotonin, affects platelet aggregation. When blocking 5-HT2A receptors with ketanserin, the content of collagen, procollagen 1 and 3 in the lungs of animals with pneumofibrosis decreases, thus. its antifibrotic effect is manifested [Fabre A., 2008].

Reserpine ("Reserpine", "Sigma", USA) - 3,4,5-trimethoxybenzoate of methyl reserpate. An indole alkaloid isolated from the plant Rauvolphia serpentina. The main pharmacological property of reserpine is its sympatholytic effect, due to the fact that under its influence catecholamines are rapidly released from the granular depot of presynaptic nerve endings. The released catecholamines are subjected to the inactivating action of monoamine oxidase (MAO), which leads to a decrease in catecholamines in the synaptic cleft and a weakening of adrenergic influences on the effector systems of peripheral organs [Mashkovsky, 2020]. The sympatholytic reserpine reduces the infiltration of lung tissue by inflammatory cells and prevents the deposition of connective tissue in the lung parenchyma in mice under conditions of bleomycin administration [Dygai A.M., 2018].

Vascular Endothelial Growth Factor (VEGF, Sigma, USA) - secreted by endothelial cells and pericytes in response to hypoxia, stimulates angiogenesis and causes increased microvascular permeability.

Hepatocyte growth factor (HGF, Sigma, USA) is a powerful mitogen for hepatocytes and various cell types, including endothelial and epithelial cells. It also indirectly affects the morphogenesis of the epithelium.

Thus, the objective of the invention is to develop a method for treating liver cirrhosis and accelerating regeneration by sequentially correcting typical pathological processes: fibrosis with ketanserin, inflammation with reserpine, and further stimulation of angiogenesis by vascular endothelial growth factor (VEGF) and acceleration of hepatocyte neogenesis by hepatocyte growth factor (HGF).

Distinctive features in the claimed combination showed new properties that do not explicitly follow from the prior art in this field and are not obvious to a specialist. An identical set of features was not found in the analyzed patent and medical scientific literature.

The method is based on data sources of information and analysis of the results of experimental studies. The effectiveness of the method has been proven in an experiment on male Wistar rats at the age of 12 weeks. The animals were obtained from the Department of Experimental Biological Models of the NIIFiRM. E.D. Goldberg of the Tomsk NIMC (certificate available). The study was approved by the Ethics Committee of E.D. Goldberg of the Tomsk Research Center of the Russian Academy of Sciences. The animals were divided into 4 groups: 1 - control group (8 rats), 2 - pathological control group (8 rats), 3 - group with liver cirrhosis treated with ketanserin (8 rats). 4 - group with liver cirrhosis treated with polytherapy (8 rats).

The method will be clear from the following description and figures 1 and 2 attached thereto.

The figure 1 shows the liver of male Wistar rats on the 102nd day of the experiment, when the tissue was stained with hematoxylin and eosin: a - intact, 6 - treated with alcohol and TCA, c - treated with ketanserin; d - treated with ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor against the background of modeling liver cirrhosis. Magnification × 100.

The figure 2 shows the liver of male Wistar rats on the 102nd day of the experiment, with tissue staining for connective tissue according to Van Gieson: a - intact, b - treated with alcohol and TCA, c - treated with ketanserin; d - treated with ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor against the background of modeling liver cirrhosis. Magnification × 100.

The method is carried out as follows.

Cirrhosis of the liver is modeled by oral administration of a 40% oily solution of carbon tetrachloride (THU, ECOS-1, Russia) for 12 weeks in a volume of 0.2 ml per 100 grams of body weight once a week. To potentiate the hepatotropic effect, the animals are given free access to a 5% solution of ethanol and glucose throughout the experiment. [Myadelets O.D., 2017; Evseenko D.A., Dundarov Z.A., 2018]. Tetrachlorocarbon is an organochlorine compound, SCC. Poisonous in both liquid and vapor form. When ingested, it is rapidly excreted and is not detected after 48 hours. Metabolized to chloroform and carbon dioxide. It has a toxic effect on the liver, the result may be yellow atrophy of the liver, as well as its cirrhosis.

The specific 5-HT2 receptor antagonist ketanserin (Ketanserinum, Sigma, USA) is administered intraperitoneally at a dose of 0.2 mg/kg, starting from the 85th to the 95th day from the beginning of the simulation of liver cirrhosis, daily.

The sympatholytic reserpine (Reserpine, Sigma, USA) is administered intraperitoneally on days 96 and 100 at a dose of 1 mg/kg.

Vascular endothelial growth factor (VEGF, Sigma, USA) is administered intravenously on days 96-102 at a dose of 200 ng/animal.

Hepatocyte growth factor (HGF, Sigma, USA) is injected intravenously once at a dose of 300 ng/animal on the 100th day from the start of modeling liver cirrhosis.

The claimed doses and mode of administration are selected empirically and are optimal for obtaining the claimed technical result. Increasing the dose and frequency of administration cancel the claimed technical result. Intact animals are used as a background (intact control). On the 102nd day after the start of the simulation of liver cirrhosis, rats are euthanized by an overdose of CO ₂ and the morphological picture of the liver and biochemical parameters of blood serum are studied. For histological examination of the liver, paraffin blocks are prepared according to the standard method. Sections are obtained from each block, which are stained with hematoxylin and eosin to study the content of lymphocytes, macrophages, neutrophils and according to Van Gieson for connective tissue [Merkulov G.A., 1969]. Determination of serum alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total and direct bilirubin, cholesterol is carried out according to generally accepted methods, using an automatic biochemical analyzer "Wesktap Coulter AU480" (USA) and standard kits from "Wesktap Coulter" (USA).

Mathematical processing of the results is carried out using standard methods of variation statistics. Significance of differences is assessed using the parametric Student's t test or the nonparametric Mann-Whitney U test.

Example

With the course administration of carbon tetrachloride (TCA) and alcohol, a pronounced inflammatory reaction develops in the liver tissue, including lymphohistiocytic infiltration of the portal tracts, fatty and hydropic degeneration of hepatocytes (Fig. 1b), fibrosis with the formation of false lobules (Fig. 2b), stepwise necrosis of hepatocytes along the periphery of the lobules, while signs of cholestasis are revealed. At the same time, modeling of liver cirrhosis causes a significant decrease in the number of sinusoidal capillaries in the liver (Table 1).

Monotherapy with ketanserin in animals with toxic alcoholic cirrhosis of the liver shows a mild therapeutic effect: a slight decrease in the intensity of fatty and protein degeneration, and a decrease in the deposition of fibrotic masses (Fig. 1c, 2c). In addition to mononuclear cells, neutrophils appeared in the composition of the inflammatory infiltrate. The use of ketanserin has a positive effect on the microvascular bed of the liver, as evidenced by a significantly greater number of sinusoidal capillaries in the liver (table 1).

Multicomponent therapy of experimental liver cirrhosis with ketanserin, reserpine, VEGF and HGF leads to a significant decrease in the severity of fatty and protein degeneration of hepatocytes (Fig. 1d). At the same time, a decrease in the intensity of inflammatory infiltration of the liver parenchyma is observed, and a smaller number of necrotic hepatocytes is additionally detected. The area of fibrous tissue decreases, large areas of hepatic tissue with a normal structure are revealed (Fig. 2d). There is an increase in the processes of regeneration of liver cells, which is confirmed by the appearance of large hepatocytes with nuclear hypertrophy and binuclear liver cells. In the study of the microvascular bed of the liver of rats treated with ketanserin, reserpine, VEGF and HGF, there is an increase in the number of sinusoidal capillaries in the liver, while the value of this indicator exceeds that in animals treated with ketanserin alone (Table 1).

Massive destruction of hepatocytes under the influence of alcohol and TCA leads to a significant increase in the levels of alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, and cholesterol in the blood plasma of rats (Table 2).

Under conditions of modeling liver cirrhosis, blockade of C2 serotonin receptors by ketanserin ambiguously affects the biochemical parameters of blood serum. Thus, monotherapy significantly increases the level of AST, ALT, total and direct bilirubin in rats with liver cirrhosis, while ketanserin significantly reduces the concentration of cholesterol (Table 2).

The result of polytherapy is a significant decrease in the levels of serum ALP, AST and ALT (Table 2).

Modeling of cirrhosis leads to a significant increase in the area of connective tissue in the liver (Table 3). With the introduction of ketanserin, a decrease in the area of connective tissue in the liver of animals with cirrhosis is observed. Polytherapy leads to a significant decrease in the area of connective tissue in the liver (Table 3).

Thus, on the model of toxic liver cirrhosis, it was shown that the introduction of ketanserin, reserpine, vascular endothelial growth factor and hepatocyte growth factor has a more pronounced anti-inflammatory, antifibrotic effect than in the case of using ketanserin in isolation, in addition, stimulation of angiogenesis in the liver and hepatocyte neogenesis is observed, restoration of liver function occurs.

Sources of information taken into account when compiling the description

1. Asrani S.K., Devarbhavi H., Eaton J., Kamath P.S. Burden of liver diseases in the world // Journal of hepatology. -2019. - Vol. 70, no. 1. - P. 151-171.

2. Tsukanov B.V., Vasyutin A.V., Tonkikh Yu.L. The burden of liver cirrhosis in the modern world // Doktor.Ru. - 2021. - Volume 20, No. 4. - S. 21-25.

3. Nishikawa K, Osawa Y, Kimura K. Wnt/p-Catenin Signaling as a Potential Target for the Treatment of Liver Cirrhosis Using Antifibrotic Drugs // Int J Mol Sci. - 2018. - Vol. 19(10). - P. 3103.

4. Bataller R., Gao B. Liver fibrosis in alcoholic liver disease // Semin. LiverDis. - 2015. Vol. 35, No. 2. - P. 146-156.

5. Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet. - 2014. - Vol.383(9930). - P. 1749-61.

6. Vorobioff J, Garcia - Tsao G, Gamen M, Picabea E. Long-term hemodynamic effects of ketanserin, a 5-hydrotrjptamine blocker, in portahypertenzie patients // Hepatology. - 1989. - Vol. 9, #1. - P. 88-91.

7. Fabre, A. Modulation of bleomycin-induced lung fibrosis by serotonin receptor antagonists in mice / A. Fabre, J. Marchal-Somme, S. Marchand-Adam, et al. // Eur Respir J. - 2008. - Vol. 32. - P. 426-436.

8. Mashkovsky M. D. Medicines. - 16th ed., Revised, corrected. and additional, - M .: New wave: Publisher Umerenkov, 2020, - 1216 p.

9. Dygai A.M., Skurikhin E.G., Krupin V.A. Pulmonary fibrosis and stem cells: new treatment approaches. Moscow: RAN, 2018. - 200 p.

10. Myadelets O.D., Lebedeva E.I. Degenerative and regenerative processes in the liver of white rats in the simulation of toxic cirrhosis. Changes in oval cells // Journal of GrGMU. - 2017. - No. 3. - S. 294-300.

11. Evseenko D.A., Dundarov Z.A. Experimental formation of liver cirrhosis in animals in laboratory conditions [Electronic resource] // Hepatol and Gastroenterol. - 2018. - V. 2, No. 2. - S. 122-128.

12. Merkulov G.A. Pathological technique course. - St. Petersburg: Medicine, 1969. - S. 14, 36-38, 58, 98-100, 163, 170.

Claims (1)

A method for treating liver cirrhosis in rats by administering ketanserin, characterized in that reserpine, vascular endothelial growth factor and hepatocyte growth factor are additionally administered, while the drugs are administered at the following times and in the following sequence: ketanserin is administered from the 85th to the 95th day at a dose of 0.2 mg/kg from the first injection of carbon tetrachloride, reserpine is administered on the 96th day and on the 100th day at a dose of 1 mg/kg, vascular endothelial growth factor is administered on days 96-102 at a dose of 200 ng/animal, hepatocyte growth factor is administered on day 100 at a dose of 300 ng/animal.