RU2244554C1 - Agent for treatment of viral hepatitis c - Google Patents

Abstract

FIELD: medicine, virology, pharmaceutical industry, pharmacy.

SUBSTANCE: invention proposes the preparation used for treatment of viral hepatitis C that comprises birch bark extract with the content of betulin above 70% and a pharmaceutically acceptable carrier. The preparation is administrated to patient by oral route. The preparation promotes to the effective treatment of viral hepatitis C. Invention can be used in treatment of viral hepatitis C.

EFFECT: valuable medicinal properties of agent.

4 cl, 2 tbl

Description

Technical field

The present invention relates to medicine, and more specifically relates to the search for new, effective treatments for hepatitis C.

State of the art

Viral hepatitis is one of the most dangerous and common infectious diseases. About 2 million people die every year from pathologies associated with this disease in the world.

At least 5 different viruses are the cause of viral hepatitis - enteric viruses A and E and parenteral viruses (B, C, D).

Hepatitis C virus is the youngest, it was discovered only in 1989. With its discovery, an intensive study of this type of hepatitis began, which is the main cause of the development of chronic diffuse diseases of the liver and hepatocytic carcinoma.

A distinctive feature of the hepatitis C virus is the ability to prolong persistence in the body, which leads to a high level of chronic infection (50-80% of cases). The stability of the virus is due to its ability to replicate with a high level of "mutation", as a result of which several immunologically different variants arise, due to which the virus avoids immune surveillance. The resulting neutralizing antibodies are highly specific and are not able to neutralize newly emerging variants of the virus.

The extremely high incidence of viral hepatitis and, in particular, hepatitis C causes the urgent task of finding and creating effective therapies for this infection.

Currently, two drugs officially used in the treatment of viral hepatitis C (interferon and ribavirin or virazole) are officially known in practical medicine, but this does not make the task of finding new treatments for this disease irrelevant, in particular, due to the side effects that accompany the use of known drugs, such as the development of anemia, neutropenia, leukopenia, dyspeptic symptoms.

SUMMARY OF THE INVENTION

The objective of the present invention is to find a new, highly effective agent for the treatment of hepatitis C.

Another objective of the present invention is to find a highly effective agent for the treatment of hepatitis C of natural origin, since this eliminates a number of problems associated with the use of chemotherapy.

These problems are solved by the present invention due to the establishment by the authors of high antiviral activity against hepatitis C virus in an extract of birch bark (birch bark) with a betulin content of more than 70% in the extract.

Betulin is one of the first natural substances that was first isolated from plant materials in 1778. In 1876, W. Hausmann published the results of elementary analyzes of betulin (Hausmann U., Annalen der chemie, 182, p. 365).

The main source of natural betulin is birch. The white part of the birch bark in individual species of birch (Betula verrucosa, Betula papyfera) contains up to 30% or more betulin (Hayek, E.W. et al. (1989) A Bicentenmal of Betulin, Phytochemistry, 28 (9) p. 229-2242).

Betulin is a pentacyclic triterpen with a carbon skeleton of a series of lupan, which is also called betulinol, trochotone, birch camphor and (cili-) rubber. A characteristic feature of the lupane series is a ring with five carbon atoms in the pentacyclic system, which at position C-19 has an α-isopentenyl group. Betulin is characterized by high thermal stability, its melting point is between 250 and 261 ° C, and even higher values are achieved after sublimation of the recrystallized product. Its molecular weight is 442.7, it is soluble in pyridine and tetrahydrofuran, but slightly soluble in dichloromethane, chloroform and cold organic solvents, and its solubility increases significantly with increasing temperature. Betulin is practically insoluble in water and cold petroleum ether. In addition, kinetic studies have shown a very low reactivity of betulin hydroxyl groups.

As early as 1899, J. Wheeler showed the antiseptic properties of betulin and was therefore used to sterilize dressings and plasters (Wheeler J., (1899), Pharm. J., Die Darstellung des Betulin durch Sublimation, 494, Ref. Chem. Centr 1900 I, p. 353). In naturopathy and traditional medicine, birch bark decoctions have been received and are still being used, which are used to treat malaria, dropsy, gout and skin diseases, as well as tinctures for compresses in the treatment of abscesses.

A large number of studies are devoted to the hepatoprotective activity of betulin. It was found that betulin reduces the activity of alanine aminotransferase, alkaline phosphatase, the content of maleic dialdehyde, prevents the inflammatory process in the liver (Yu.K. Vasilenko et al. "Pharmacological properties of birch bark triterpenoids" Experimental and Clinical Pharmacology, 1993, v. 54 No. 4 pp. . 53-55).

However, there is no reliable information about the antiviral activity of betulin, in contrast to its various synthetic derivatives. Thus, in vitro 3-substituted glutaryl derivatives of betulin have been shown to have cytotoxicity against HIV transformed lymphocytes (Synthesis and anti-HIV activity of betulin derivaties, Bioorg Med Chem Lett. 1998 May 19, 8 (10) p. 1267 -1272).

A large number of publications are devoted to the preparation of a number of betulinic acid derivatives as potential AIDS control agents (see, for example, “Synthesis and anti-HIV activity of betulinic acid and dihidrobetulinic acid derivaties”, Bioorg Med Chem lett, 1997, Dec 5 (12) p .2133-2143).

The feasibility of using betulin derivatives has also been investigated in relation to hepatitis in non-viral models of hepatitis caused by Cll ₄ , tetracycline and acetone. (O.B. Flekhter "Synthesis of lupane group triterpenoids and their hepatoprotective activity", Bioorganic Chemistry, 2000, Volume 26, No. 3, pp. 215-223).

Disclosure of invention

The authors of the present invention, it was unexpectedly discovered that the birch bark extract has antiviral properties. This effect was found in a model of an experimental infection caused by human hepatitis C virus in mice.

Despite the fact that the antiviral activity of betulin derivatives was shown by several authors on an in vitro model of HIV infection, the surprise of this result is not only due to the fact that previous studies of betulin did not reveal antiviral activity against hepatitis C in its derivatives, but and the fact that the authors of the invention themselves in the study of birch bark extract in cell cultures (in vitro) did not find this activity in him. It appeared only in vivo.

The implementation of the invention

The following describes the results of studies of the effect of birch bark extract on the reproduction of hepatitis C virus (hereinafter abbreviated as HCV) in laboratory animals.

Material and methods:

Virus: A cytopathogenic variant of HCV was isolated from a serum of a chronically infected HCV patient. According to genotyping data, an isolated HCV variant belongs to the most common HCV genotype 1b in Russia. In the studies, infectious doses of HCV equal to 10.0 TCD 50 were used.

Antiviral drug. An extract with a betulin content determined by HPLC — 72% was used in the work. In the experiment, various dilutions from 50 to 150 mg per 1 kg of weight were used, which were administered to the animals by the oral method daily for 4 days.

Animals. To study the antiviral activity of betulin, white mongrel sexually mature mice weighing 18-20 g were used.

Methods HCV-containing material was administered to animals intraperitoneally. Some animals were left as controls. On the 9th day after infection, the mice were “fed” (administered orally) with a solution of betulin (physiological solution, with 10% Tween-80) (hereinafter referred to as betulin, the drug described above) in a volume of 50 μl containing 50, 100 and 150 mg betulin. Betulin was given to mice once a day for 4 days. In the experiment, 10 animals were used for each experiment variant. After 2 days, as well as 10 days after the last injection, 50% of the animals were opened, the liver and brain were removed, and blood was taken. Blood serum samples, supernatant obtained after centrifugation at 3000 rpm for 15 min of homogenates of liver and brain tissue were used to determine HCV antigens and infectious activity of hepatitis C virus. To this end, virus-containing samples were titrated in cultures of transplanted kidney cells of a pig embryo SPEV. The HCV antigen in these samples was studied in a hemagglutination reaction using the classical method described by Clarke and Casals (1968).

Cell culture. The cultures of transplantable pig embryonic kidney cells (SPEV) highly sensitive to the cytopathogenic effect of HCV obtained from the company Narvak, Moscow. They were used as a one-day monolayer of cells grown in 24-well plastic panels. SPEV cell cultures were grown on medium 199 with 10% serum of calf embryo, supplemented with glutamine and antibiotics (100 U / ml). To titrate the residual infectious activity of the virus, the same pig embryonic kidney cell line (SPEV) was used.

Infectious activity of HCV contained in samples of organs and tissues of infected mice was taken into account by titration, usually 6-7 days after infection, when the maximum cytopathogenic effect of the virus developed, using the Reed and Mench formula to calculate hepatitis C virus titer.

Results.

The research data are shown in tables 1 and 2. As can be seen from the data in table. 1a, all blood serum samples obtained from HCV infected mice belonging to different groups contain both antigenic and infectious activities of HCV. Moreover, the maximum manifestations of these indicators were found in the control group of HCV infected animals that did not receive betulin. In animals treated with the drug at a dose of 50 mg per 1 kg of weight in 50 μl, there was a noticeable tendency to lower HCV hemagglutinin titers, and infectious serum HCV titers decreased by 2.6 lg (average data). A significant decrease in antigenic and infectious activities of HCV in the blood serum was noted in the group of animals treated with Betulin daily at a dose of 100 mg / kg. In this case, the titers of HCV infection activity fell by 3.4 lg and the antigenic activity of HCV in mouse serum samples decreased by more than 2 times. To a lesser extent, the drug was active when used at a dose of 150 mg / kg. The antigenic activity of the virus decreased, the titers of infectious activity decreased slightly under the influence of the drug (1.8 lg). Close results indicating the effectiveness of the drug “betulin” were obtained in the study of liver tissue from all groups of animals (table 1b). In particular, it was shown that the maximum decrease in HCV antigens and its infectious activity was observed in the case of using the drug in a single dose of 50 and 100 mg per 1 kg of weight with 4-fold administration of the drug. From the data of table 1b it can be seen that the antigenic activity of HCV in this case decreased by 2 or more times and up to 2 lg a decrease in the infection activity of HCV in the liver tissues was noted. Decreased and liver dimensions HCV infected mice under the influence of the therapeutic effect of the drug highest titers of HCV (7,0 lg to TCD _50/20 ul) was observed in brain tissue of HCV infected mice (Table 1). However, in this case, mice treated with betulin in doses of 50 and 100 mg per 1 kg showed a decrease in titers of HCV infection activity by 2.0-2.5 lg, respectively. Moreover, a decrease in HCV antigen was noted in all groups of animals treated with the drug. In the group of mice treated with betulin at a dose of 150 mg per 1 kg, there was a tendency to an increase in titers of HCV infection activity in the brain.

Thus, the data obtained in the study of the organs and tissues of mice on the 17th day after HCV infection and on the 3rd day after 4-fold use of the betulin preparation indicate a high antiviral activity of the drug against infection caused by HCV in animals.

In this regard, the duration of positive changes in the body of HCV infected mice after drug withdrawal was revealed. Therefore, the 2nd group of HCV infected animals was examined on day 28 after infection and on day 11 after cancellation of treatment with Betulin.

The results are shown in tables 2a, b, c. A study of the antigenic activity of HCV in the organs and tissues of mice during this period of time revealed that treatment with betulin in doses of 50 mg and 100 mg per 1 kg, as a rule, led to a significant decrease in the content of HCV hemagglutinin in serum, liver and the brain of HCV infected animals (at a dose of 100 mg per 1 kg up to 4 times in the liver of mice, 2 or more times in the blood serum and brain of animals). A dose of 150 mg per 1 kg, as a rule, led to a slight decrease in the content of HCV antigen in the organs and tissues of animals infected with HCV.

A study of the infection activity of HCV in the organs and tissues of HCV infected mice showed that in the long term after the treatment was completed, the antiviral effect of the administered drug persisted for more than 10 days. However, an analysis of the data obtained revealed other patterns in the parameters of HCV infection at these times. In particular, it was shown that the HCV titers contained in the blood serum and in the liver of the control group of animals significantly exceeded these values detected early after infection of mice (1.5 lg - in blood serum, 2.4 lg - in the liver). At the same time, on the 28th day after infection in the brain tissue, titers of the virus began to decrease markedly.

The antiviral effect of the “betulin” drug was somewhat reduced during these periods after drug withdrawal, however, the greatest effect was observed during the period when a dose of 50 mg per 1 kg of weight was used for treatment. In this case, in the blood serum of animals, virus titers decreased on average by 1.9 lg, in the liver - by 2.9 lg, in the brain - by 2.4 lg. A 4-fold administration of the “betulin” preparation in a dose of 100 mg per 1 kg led to less significant changes in the HCV titer in the direction of reduction (see tables 2a, b, c).

Thus, the positive effect of the drug “betulin” on the decrease in the rates of infection caused by HCV in mice was established, and 10 days after its cancellation.

The results obtained by the inventors suggest that the birch bark extract has high antiviral activity against infection caused by hepatitis C virus, which allows us to offer it as a means for treating this type of hepatitis.

However, there is no reason to argue that the content of betulin in the extract of at least 70% is critical and limit the scope of the invention to it. Practically significant results can be obtained with lower values of betulin content. The establishment of these boundary values can be carried out by specialists in carrying out routine routine work, in particular, based on the above in the description of the present invention.

The preferred form of application of birch bark extract are solid dosage forms: tablets, pills, capsules, powders. In this case, pharmaceutically acceptable carriers customary for the species may be used. A preparation may include one or more excipients, such as microcrystalline cellulose, lactose, sugars, starches, mannitol, sorbitol and other polyols, calcium carbonate, calcium phosphate, calcium sulfate, polyethylene glycols, etc.

The preparation may also include surfactants and other conventional components for solid pharmaceutical preparations, such as colorants, flavors, adsorbents.

In the manufacture of a coated tablet preparation, the latter can be obtained from film-forming substances such as hydroxypropyl cellulose, a plasticizer, such as polyethylene glycols, and other auxiliary additives such as, for example, pigments.

Claims (3)

1. The use of birch bark extract as a treatment for viral hepatitis C. 2. A drug for the treatment of viral hepatitis C, including birch bark extract with a betulin content of more than 70% and a pharmaceutically acceptable carrier. 3. A method of treating viral hepatitis C, characterized in that the patient is orally administered the drug according to claim 2.