Pharmaceutical composition and method for treating the cases of progressing hepatic fibrosis and cirrhosis

FIELD: medicine; pharmaceutical engineering.

SUBSTANCE: pharmaceutical composition COMPRISES 5-(2-pyrazinyl)-4-methyl-1,2-dithyol -3-thion (oltipraze) and dimethyl-4,4'-dimetoxi-5,6,5',6'-dimethylene-dioxybiphenyl-2,2' dicarboxilate (DDB) as the main components. Oltipraze: DDB proportion is preferentially equal to 50-1:1-50, the most preferential being 5:1.

EFFECT: enhanced effectiveness of treatment.

6 cl, 6 dwg, 9 tbl

The technical field to which the invention relates.

The present invention relates to the field of biotechnology and, in particular, relates to pharmaceutical compositions for the treatment and prevention of fibrosis and cirrhosis. In particular, the present invention relates to a pharmaceutical composition for the treatment and prevention of fibrosis and cirrhosis, including 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-tion (oltipraz) and dimethyl-4,4’-dimethoxy-5,6,5’,6’-dimethylene-dioxybiphenol-2,2’, in primary forms (DDB) as main active ingredients.

The level of technology

The liver plays a key role in the metabolism of xenobiotics and the metabolism of endogenous substances and is an important organ with the corresponding enzymatic reactions and energy metabolism. Among the many chronic diseases in Korea the most common and life-threatening diseases are hepatitis, cirrhosis and liver cancer after cardiovascular diseases. Because Korea has a relatively large population of drinkers compared with developed countries and is caused by drinking liver damage occur quite frequently, much attention is paid to the treatment of liver diseases. Chronic liver damage caused by viral infections or alcohol often lead to cirrhosis or liver cancer. Given the physiologically the ski characteristics and significance of liver tissue, and also because of the importance of treatment and prevention of liver diseases, there is a great need in the final development of therapeutic and prophylactic agents against liver damage.

A variety of substances, including some synthetic compounds and herbal medicines, have a protective effect on the liver in vitro and in vivo. It is known that silymarin (silymarin) and betaine have a protective effect on the liver, and their mechanism of action is the inhibition of cytokines and increased levels of glutathione, however, it is difficult to expect therapeutic effect due to low efficiency. These tools are often used in clinical trials, because now is not a good therapeutic agents against liver disease. Malotilate (malotilate) and its derivatives, which are indicated for the treatment of liver fibrosis, protect the liver from toxic chemicals and their possible mechanism of action involves the induction of conjugating enzymes phase II and inhibition of cytochromes P450. However, these connections indiscriminately inhibit certain cytochrome P450 and exhibit only a preventive effect.

It is known that certain substituted sulfur containing dithiolthione occurring naturally in cruciferous plants have a protective effect on the liver. Of them oltipraz (oltipraz) was used as a therapeutic means is about schistosomiasis; it has the following formula:

Oltipraz increases the cellular content of thiols and induces the expression of enzymes responsible for maintaining a pool of glutathione (GSH) and tissue detoxification from electrophilic molecules. Oltipraz increases the activity of the following enzymes: NAD(P)H-honoredarts, microsome of epexegetical, glutathione-S-transferase (GST) and UDP-GT. In particular, GST protects the liver from such toxic chemicals, such as carbon tetrachloride or acetaminophen (Ansher SS, Dolan P, and E. Bueding Chemoprotective effects of two dithiolthiones and of butylhydroxyanisole against carbon tetrachloride and acetaminophen toxicity. Hepatology 1983 3, 932-935).

In addition, oltipraz suppresses carcinogenesis induced by benzo[a]pyrene, NDEA and brazilbim with mustard gas, as well as that induced by aflatoxin B1 carcinogenesis in the liver and induced azoxymethane carcinogenesis in the colon (Bolton MG, Munoz A, Jacobson LP, Groopman JD, Maxuitenko YY, Roebuck BD, and Kensler TW. Transient intervention with oltipraz protects against aflatoxin on-induced hepatic tumorigenesis. 1993, Cancer Res.53, 3499-3504).

Known mechanisms of suppression of carcinogenesis by oltipraz. First, it increases the level of antioxidant restored GSH in the tissues. Secondly, it inhibits bioactivation carcinogens such phase I enzymes like cytochrome P450. Thirdly, oltipraz enhances the detoxification of carcinogens, inducyruya detoxifying phase II enzymes, including GST and UDP-GT. In-fourth the s, it inhibits the replication of human immunodeficiency virus (HIV) type I in vitro. Fifthly, it eliminates reactive intermediates in cells by increasing the level of thiols and enhances DNA repair. It was reported that oltipraz increases the level of GSH in most tissues and eliminates free radicals formed during irradiation or xenobiotics. It is also known that oltipraz acts as a protective agent against radiation, contributing to the maintenance of cellular homeostasis.

Given the above, the description hereinafter will be described more detailed information. Cancer is an uncontrolled growth and differentiation of cells, probably due to DNA damage in somatic cells (Cancer Biology, 3rd ed. Raymond W. Ruddon, pp. 61-95, 497-507, Oxford Press). Anticancer action of chemicals depends mainly on their antimutagenic properties or from their activity to suppress transformation in cancer cells or proliferation of cancer cells. Oltipraz studied as a means for cancer chemoprophylaxis (Ansher et al., 1983; Bolton et al., 1993). Anticancer chemoprophylactic action oltipraz is due not only to inhibition of cytochrome P450 3A, but also with the induction of detoxification enzymes phase II. Oltipraz increases the expression of mutation-S-transferase (GST) in cells and in animals (Clapper et al., 1994; Davidson et al., 1990), Thu is connected with suppression caused by toxic substances of tissue damage and carcinogenesis (Kensler et al., 1987; Maxuitenko et al., 1998). Oltipraz protects the liver from tissue damage caused by irradiation (Kim et al., 1997), as known from previous studies of the induction of GST means the adaptive response of the cells. Oltipraz also protects the liver from toxic substances (Ansher et al., 1983). Oltipraz suppresses induced by aflatoxin B1 carcinogenesis through catalyzed by cytochrome P450 3A metabolic activation of the carcinogen. According to recent clinical trials oltipraz reduces the level of aflatoxin B1 in the blood plasma of people with a higher risk of liver cancer. The use of oltipraz also led to the reduction induced by aflatoxin In 1 of carcinogenesis in animals.

It was reported that oltipraz inhibits replication of hepatitis b virus (HBV) in 2.2.15 cells infected with the plasmid containing HBV DNA. So, oltipraz inhibits transcription of the gene of hepatitis b virus increases the expression of p53 protein (Chi et al., 1998) and inhibits replication of human immunodeficiency virus (HIV) (Prochaska et al., 1995).

Was tested against chemoprophylactic actions oltipraz against carcinogenesis in the liver in China. The results showed that oltipraz has a weak protective effect against carcinogenesis in the liver. It is also known that oltipraz protects the liver from hepatitis toxic substances, at least a small degree. In addition, it was proved the security of oltipraz in toxicity studies conducted in rats and dogs (Fund. Appl. Toxicol. 1997 Jan; 35(1): 9-21).

Originating from Shizandrae connection DDB (dimethyl-4,4’-dimethoxy-5,6,5’,6’-dimethylene-dioxybiphenol-2,2’, in primary forms) is a drug for the treatment of hepatitis, which is used clinically in East Asia, including Korea. DDB protects the liver from damage caused by carbon tetrachloride, galactosamine, thioacetamide, and prednisolone, and also increases the production of antibodies. Since it is known that DDB was effectivnes in clinical trials on patients with hepatitis, it is widely used in the clinical setting. The authors of the present invention have reported that the pharmacological action of DDB is associated with inhibition of activation κNF and products α-TNF. In addition, it was found that DDB has no effect on the expression of enzymes of drug metabolism. Because κNF is known as a transcription factor mediating the inflammatory response, the inhibitor κNF must possess the ability to suppress systemic inflammatory processes.

Liver fibrosis is a pre-pathological state in which the affected tissue of the liver in such a chronic liver diseases like hepatitis, are not restored to normal tissue, and prevrashalis is in fibrous tissue type of collagen in the process of adaptive response in vivo. Although liver fibrosis is a consequence of the regenerative process in vivo in response to tissue damage, the liver tissue is replaced by fibrous tissue, which cannot perform normal functions (metabolism in vivo or production of bile). Because of the constant and repetitive fibrogenic liver leads to cirrhosis and eventually leads to death, is of great importance to develop new drugs for the treatment of liver fibrosis. However, because the mechanism of fibrogenesis liver does not exactly known, good drugs have not yet been developed.

Recent studies have shown that β-transforming growth factor (β-TGF) - cytokine secretory cells Kupffer and ito cells in the liver, is an important factor in liver fibrosis. In addition, it was reported that blocking the activity of p-TGF using antibodies or antisense JfTHLR, as well as the modification of the receptor P-TGF significantly reduces liver fibrosis. However, these studies were confirmed only at the level of experiments. There were no reports of drugs against fibrosis and cirrhosis, suitable for clinical application.

The invention

The purpose of the present invention provides for pharmaceutical compositions, the most effective in the treatment of fibrosis and cirrhosis the liver, which can be used as a preventive tool.

Another objective of the present invention provides the use of pharmaceutical composition used to produce drugs for the treatment and prevention of fibrosis and cirrhosis.

Another objective of the present invention provides a method of treatment or prevention of fibrosis and cirrhosis of the liver, involving the introduction of a pharmaceutical composition to a mammal.

The present invention provides a pharmaceutical composition for the treatment and prevention of fibrosis and cirrhosis, which includes 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-tion (oltipraz) and dimethyl-4,4’-dimethoxy-5,6,5’,6’-dimethylene-dioxybiphenol-2,2’, in primary forms (DDB) as main components. The ratio of oltipraz:DDB in this composition is preferably 50-1:1-50, most preferably 5:1. Composition oltipraz/DDB according to the present invention show extremely good effect in the treatment and prevention of fibrosis and cirrhosis of the liver and are safe drugs with low toxicity in humans.

List of figures

Figa is a photograph showing the inhibitory effect of oltipraz

the expression of mRNA β1-TGF in the liver, with the introduction of the rat DMN.

Figa is a photograph of liver tissue normal animal (staining H&E).

F. the .2b - a photograph of liver tissue normal animal (staining H&E).

Figa is a photograph of liver tissue in the group treated with DMN (staining H&E).

Fig.3b is a photograph of liver tissue in the group treated with DMN (three-color staining by Masson).

Figa is a photograph of liver tissue in the group receiving both DMN and oltipraz (25 mg/kg)/DDB (5 mg/kg) (staining H&E).

Fig.4b is a photograph of liver tissue in the group receiving both DMN and oltipraz (25 mg/kg)/DDB (5 mg/kg) (three-color staining by Masson).

Figa is a photograph of liver tissue in the group receiving both DMN and oltipraz (15 mg/kg)/DDB (15 mg/kg) (staining H&E).

Fig.5b is a photograph of liver tissue in the group receiving both DMN and oltipraz (15 mg/kg)/DDB (15 mg/kg) (three-color staining by Masson).

Figa is a photograph of liver tissue in the group receiving both DMN and oltipraz (5 mg/kg)/DDB (25 mg/kg) (staining H&E).

Fig.6b is a photograph of liver tissue in the group receiving both DMN and oltipraz (5 mg/kg)/DDB (25 mg/kg) (three-color staining by Masson).

Detailed description of the invention

The authors of the present invention made an unprecedented discovery that oltipraz has unexpected extraordinary effect in the treatment and prevention of fibrosis and cirrhosis. In addition, they discovered that the effectiveness of alteplase in the treatment of profilaktike increases significantly in combination with DDB.

Fibrosis is the preliminary stage of cirrhosis, it occurs in severe liver damage under the action of a number of factors. Cirrhosis has some relationship to carcinogenesis: it significantly increases the likelihood of liver cancer patients. However, the pathological mechanism of cirrhosis clearly differs from liver cancer. Thus, fibrosis of the liver occurs when chronic and severe damage to the liver tissue. The factors that cause tissue damage in the liver, including viruses, parasites, alcohol, chemicals and medicines. Liver fibrosis occurs due to excessive production of extracellular matrix (collagen I, III and type IV) due to the activation neprinimajemi cells in the tissue of the liver: Kupffer cells, stellate cells, etc. In particular, fibrosis occurs when the activation and the subsequent transformation of stellate cells in myofibroblast. Activated stellate cells then produce excessive amounts of extracellular matrix. Moreover, fibrosis and cirrhosis as pathological phenomena are clearly differentiated from viral hepatitis and liver cancer. Thus, methods of treatment and prophylaxis of them also differ. However, there is currently no suitable for clinical application of drugs from cirrhosis of the liver.

The present invention is based on the discovery that oltipraz known as an effective media is the primary objective in the prevention of liver cancer, also effective against fibrosis and cirrhosis, which are completely different pathological mechanisms of liver cancer. In addition, the present invention is applied oltipraz together with DDB, which was previously known only as a tool against hepatitis that makes them so effective in the treatment and prevention of fibrosis and cirrhosis, which surpasses all expectations. These facts are confirmed by the experiments described below.

Oltipraz or oltipraz/DDB reduce the number of points on a scale of fibrosis and scale Knodell, employees performance caused by dimethylnitrosamine (DMN) fibrosis. This coincides with the results of microscopic examination of tissue samples. In addition, with the introduction of oltipraz or oltipraz/DBB significantly reduced these levels of hepatotoxicity, as ALT, AST, bilirubin and γ-glutamyltranspeptidase (γ-GT). This indicates that oltipraz or oltipraz/DBB can facilitate fibrosis, slowing down the processes. The mechanism of suppression of fibrosis by oltipraz or oltipraz/ DBB revolves around inhibiting the expression β-TGF. According to the results of quantitative RT-PCR, oltipraz or oltipraz/DBB completely inhibit the increased expression of β-TGF caused by dimethylnitrosamine. This is an indication that oltipraz as a drug capable of suppressing arose ovenia and the development of fibrosis and cirrhosis. Oltipraz induces detoxification enzymes of the liver GST and men, increases GSH and shows the conjugation of the radicals, while DDB does not induce detoxification enzymes. With this in mind, we can assume that oltipraz and DDB have complementary pharmacological effects. In particular, since it is believed that inhibition of DDB metabolic activity of CYP3A in the human liver microsomes leads to the inhibition of metabolism of oltipraz, even low-dose alteplase can provide strong protection of the liver and to prolong the time of action of this drug.

In the present invention therapeutic effects of the combination oltipraz/DBB was observed at different ratios on rats, which were previously introduced DMN. In these experiments, the weight ratio oltipraz/DBB was 25:5, 15:15 and 5:25 (mg/kg). After the introduction of the DMN (the measurements were carried out 1 week after its introduction within 3 weeks) significantly increased ALT, AST, bilirubin, γ-glutamyltranspeptidase (γ-GT), the number of points on a scale of fibrosis and the Knodell scale compared with the control group. On the contrary, when the introduction of oltipraz/DBB these values decreased. Oltipraz (25 mg/kg) / DDB (5 mg/kg) showed the greatest suppression of fibrosis. This proves that oltipraz and DBB are synergies in the suppression of liver damage and fibrosis, and shows that the optimal ratio is avno 5:1. In addition, with the introduction of combination oltipraz/DBB blood biochemical parameters (number of points on a scale of fibrosis and the Knodell scale) decreased stronger than the introduction of one oltipraz (50 mg/kg). Accordingly, it can be assumed that the use of oltipraz together with DDB will reduce side effects such as disorders of the gastrointestinal tract, paresthesia of the hands and feet, etc. that occur when high doses of oltipraz. DDB suppresses inflammatory response, which causes the DMN and oltipraz prevents the expression β-TGF, increases the expression of antioxidant enzymes and causes an increase in GSH. This mechanism oltipraz/DBB increases to limit the effectiveness of treatment and prevention of liver fibrosis. So is implemented complementarity effects oltipraz/DBB to protect the liver. In addition, oltipraz/DBB can be applied in the clinic, as this is almost not discernible side effects.

The pharmaceutical composition of the present invention preferably is applied by combining compiled independently drugs or by obtaining a combined preparation containing a mixture of drugs. In practice, the pharmaceutical composition of the present invention is applied in the form of finished dosage forms suitable for oral administration in accordance with the rules of the relevant field f is rmaceutical. At this dosage form for oral administration (oral) include hard and soft capsules, tablets, powders, etc. Oral compositions, along with oltipraz/DBB as a pharmacologically active agent, can contain one or more pharmaceutically inactive, standard media. For example, the oral composition may contain additives such as starch, lactose, carboxymethyl cellulose, kaolin and other fillers, water, gelatin, alcohol, glucose, gum Arabic, tragacanth gum and other binders, starches, dextrin, sodium alginate and other dezintegriruetsja substances; talc, stearic acid, magnesium stearate, mineral oil and other lubricants. You can also add substances that promote dissolution.

The daily dosage of the present invention depends on various factors such as the degree of liver of the patient, duration of hepatitis, age, health status, complications, etc. However, usually an adult composition oltipraz/DBB appoint one or two times daily for a total dose of from 5 to 200 mg / day, more preferably from 25 to 50 mg. However, in patients with severe liver disease or when applying as a remedy against recurrence after removal of cancerous liver tumors present invention allows us to go beyond these before the crystals, pharmaceutical compositions and to appoint another large doses. It is most preferable to take orally twice a day one or two prepared containing 25 mg of alteplase and 5 mg DDB.

In the pharmaceutical compositions of the present invention optimally combines oltipraz, possesses remarkable qualities suppression of fibrosis and protect the liver, and DDB, having excellent effect of suppressing inflammatory reactions. This composition provides suppression of fibrosis and cirrhosis, has low toxicity and has almost no side effects. Thus, the present invention can be safely used for a long time for the treatment and prevention of fibrosis and cirrhosis.

Hereinafter the present invention will be disclosed in more detail in the working examples. However, the present invention is not limited to these examples.

Information confirming the possibility of carrying out the invention

Test cases

The control example 1. Antifibrosis action oltipraz (1)

In rats, which was continuously introduced dimethylnitrosamine (DMN) for 4 weeks, was observed 4-fold increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma. In the preliminary introduction of alteplase dose of 50 mg/kg increased activities of ALT and AST in the blood plasma is inhibited by 50% (table 1).

Activity γ-glutamyl is antipeptide (γ -GT) and the content of bilirubin in the blood plasma are the indicators of the functional state of the liver. Oltipraz inhibited the increase of activity γ-GT 70-80% in rats treated with DMN. On the other hand, with the introduction of DMN content of bilirubin were increased 8-fold compared with the control group. When DMN was administered oltipraz (50 mg/kg), increased bilirubin in the plasma was reduced by 65%.

Each value represents the average value of the ± the standard deviation. The number of animals used ranged from 8 to 16. Statistical significance between groups was determined using the criterion Newman-Kelisa for multivariate analysis. Indicators of significance: * = p<0.05 compared with control;^#= p&t; 0.05 compared with the group treated with DMN.

Control example 2. Antifibrosis action oltipraz (2)

Histopathological effect of oltipraz on DMN-induced liver fibrosis was investigated in laboratory animals. Explicit fibrosis was observed in rats treated with DMN for 4 weeks 3 times a week. When DMN was administered oltipraz (oral doses of 5-50 mg/kg 3 times per week for 4 weeks), fibrosis of the liver tissue was decreased in comparison with the introduction of one DMN. Necrosis and fibrosis of the liver tissue was determined histologically using such indicators histopathology of the liver, as a painting by van gieson (Van Gieson) and trichrome staining according to Masson (Masson) (table 2).

Oltipraz dose of 50 mg/kg effectively reduced DMN-induced fibrosis (table 2). The degree of fibrosis was determined by the scale of fibrosis and the Knodell scale, which show the degree of liver damage and fibrosis. Compared with the group treated only DMN, DMN group+oltipraz noted fewer points on a scale of fibrosis and the Knodell scale, which indicates the recovery of liver damage and fibrosis.

Each value represents the average value of the ± the standard deviation. The number of animals used ranged from 8 to 16. Statistical significance between groups was determined using the criterion Newman-Kelisa for multivariate analysis. Indicators of significance: * = p<0,05, ** = p<0,01. The degree of fibrosis: 0 = normal, 1 = weak presence of fibrous tissue, 2 = moderate presence of fibrous tissue, 3 = presence of fibrous tissue, 4 = strong signs of fibrosis. The Knodell scale is the sum of the scores in the periportal jumpers (maximum = 10), vnutridolkovom cell death (maximum = 4), portal inflammation (maximum = 4) and fibrosis (maximum = 4).

Control example 3. Pharmacological mechanism antifibrotic actions oltipraz

β-TGF - this is the main cytokine, the expression of which increases with fibrosis as tissue damage. The expression of mRNA β1-TGF animals was measured by the method of RT-PCR with the introduction of one DMN while the introduction is tipaza and DMN. In animals treated with DMN for 4 weeks, irreversible excessive fibrogenesis did not give the possibility to measure the expression of mRNA β1-TGF. The expression of mRNA β1-TGF was measured in animals treated with DMN one. After 18 hours after administration of DMN was administered oltipraz. After that, we measured the expression of mRNA β1-TGF 24 hours. In rats treated with DMN, mRNA β1-TGF was significantly increased in liver tissue. DMN-induced expression of mRNA β1-TGF fully ingibirovalos with the introduction of oltipraz at a dose of 100 mg/kg of the Expression of GAPDH mRNA was not changed nor with the introduction of one DMN, nor with the simultaneous introduction of oltipraz and DMN. Consequently, this means that the pharmacological mechanism of suppression of liver fibrosis by oltipraz is to reduce the expression β1-TGF (Fig 1).

Reference example 4. Assessment of inhibition by oltipraz products β-TGF

Conducted experiments on cells RAW264.7 macrophages to verify that oltipraz directly inhibits the production β-TGF, which is expressed at high levels in macrophages, and to assess the associated molecular pharmacological mechanisms. When oltipraz directly added to RAW264.7 cells with high level expression β-TGF, it turned out that oltipraz inhibits the expression β-TGF dose-dependent manner. These results show that oltipraz can action the SQL as antifibrotic agent in hepatic Kupffer cells by inhibiting the production β -TGF. Moreover, the increased expression of β-TGF inhibited by EGTA or genistein, which is an inhibitor of tyrosine kinases. This result indicates that the inhibition of oltipraz products β-TGF may be due to the regulation of intracellular calcium and changing the activity of protein kinases (table 3).

Reference example 5. Anti-inflammatory mechanism DDB (inhibition of activation κ-NF)

It is known that activation κNF is associated with inflammatory processes. To compare the activity κNF in liver tissue with the introduction of DDB and without the introduction of DDB, conducted the following experiments. Used male SD rats weighing about 150 g were included In each group 7 rats. In the control group (the introduction of the solvent) and the experimental group (administration of DDB in doses from 20 to 100 mg/kg/day) activity κNF was measured after injection of endotoxin (lipopolysaccharide, or LPS) method bias movably the tee during electrophoresis (EMSA).

The introduction of LPS (1 µg/kg) led to increased levels of the transcription complex of P50 and P65 molecules κNF in nuclei. However, the introduction of DDB (20-100 mg/kg) 2 hours prior to LPS injection blocked the translocation complex κNF in the nucleus and its activity. These results show that DDB has the ability to inhibition of LPS-indicated activation κNF in the liver of rats.

Stimulation of Raw264.7 cells by adding 1 µg/kg LPS caused an increase in binding κNF with DNA in 30 min or 1 hour. DDB at concentrations of 0.1, 0.3 and 1.0 mm inhibited the activity κNF concentration-dependent manner. These results coincide with the results obtained in the liver of rats treated with DDB (table 4).

Inhibition of degradation of subunit I-κα NF

Translocation κNF in the nucleus is preceded by the phosphorylation and proteolytic degradation of the sub is denizy I-κ Inα. This experiment was conducted in order to check, does the effect of DDB on activation κNF with inhibition of the degradation of I-κα. The level of protein I-κα decreased after 30 min of rats after administration of LPS (1 µg/kg). On the contrary, the introduction of DDB 2 hours before the injection of LPS reduced the decrease in protein I-καcaused by LPS. Also on the RAW264.7 cells DDB inhibited the degradation of protein I-kVA concentration-dependent manner in the concentration range from 0.3 to 1 mm. These results show that DDB inhibits the degradation of I-κα and thereby activation κNF (table 5).

Reference example 6. Anti-inflammatory mechanism DDB (inhibition of the production and mRNA expression α-TNF)

α-TNF is a major mediator of the response to LPS, which is involved in inflammatory processes. The ELISA method was defined, is reduced if the selection in the blood α-TNF during inhibition DDB activity κIn-NF. Once in the introduction LPS (1 μg/kg, intravenous) led to increased levels α-TNF from 35 to 5160 PG/ml plasma 2 hours after injection. Introduction DDB doses of 20, 50 or 100 mg/kg prevented LPS caused increased levels α-TNF, reducing it to 1350, 1230 and 690 PG/ml plasma, respectively. In RAW264.7 cells DDB reduced caused by LPS products α-TNF. These results show that DDB inhibits the expression α-TNF in macrophages and liver (table 6).

Reference example 7. Antifibrosis action combinations oltipraz/DD(1)

Antifibrosis action combination was studied by changing the ratio of components in this combination. In the study of the effect of this combination with different proportions of components was partially modified dosage schedule DMN. Whereas in the control examples 1 and 2 DMN was administered to rats at a dose of 10 μl/kg in total 12 times over a period of 4 weeks, 3 times a week, when evaluating the effect of combination with different proportions of the component is used in the model with less liver damage in animals in which DMN was administered to rats at a dose of 10 μl/kg in total 9 times over a period of 3 weeks, 3 times a week, then in this control example, the DMN was not introduced in the remaining 1 week.

After 1 week after administration of DMN for 3 weeks, the animals were marked with a 2.9 score of fibrosis and 11.7 points in the Knodell scale. Thus, in these animals, a decrease in liver fibrosis compared with animals treated with DMN for 4 weeks, which was 3.7 points on a scale of fibrosis and 16.1 points in the Knodell scale.

After 1 week after the introduction of the DMN during 3 weeks 3 times a week activity of ALT and AST in the blood plasma of animals was increased 4-fold compared with the control group.

Animals treated with oltipraz and DBB in the dose ratio of 25:5, 15:15 and 5:25 mg/kg for 3 weeks (3 times a week the day after each injection DMN), increased activities of ALT and AST in the blood plasma of animals were considerably reduced compared to animals receiving only DMN. When comparing the effectiveness of different proportions of components in combination turned out that the animals in the group treated with oltipraz and DBB in the ratio of 15 doses:15 mg/kg showed significant inhibition of the activities of ALT and AST in plasma, however, the degree of inhibition was less than that in animals treated with oltipraz and DBB in the dose ratio of 25:5 mg/kg (table 7).

Akti is ness γ -GT and the content of bilirubin in the blood plasma are representative indicators of the functional state of the liver. In rats treated with oltipraz and DBB in the dose ratio of 25:5, 15:15 and 5:25 mg/kg for 3 weeks, DMN-induced increase of bilirubin in the plasma was reduced to a statistically significant extent. Like the results for ALT and AST of those groups of animals, which were introduced oltipraz and DBB, increased bilirubin most strongly suppressed in animals treated with oltipraz and DBB in the dose ratio of 25:5 mg/kg

Total protein and albumin levels in the blood are indicators of protein synthesis in liver tissue. With the development of cirrhosis of the liver total protein content in the blood is usually reduced. After 1 week after administration of DMN for 3 weeks total protein content in animals decreased significantly, however, it was at the normal level of the control group in those groups of animals that received oltipraz and DBB in the dose ratio of 25:5 and 15:15 mg/kg (table 7).

These results show that the proportions of the components of oltipraz and DBB, which was used in this example, the greatest suppression of liver fibrosis caused by DMN showed the group of animals treated with oltipraz and DBB in the ratio of 25:5 mg/kg Should also be noted that the effect in the group of animals treated with oltipraz (25 mg/kg) / DDB (5 mg/kg), comparable or pre is oshodi the what was observed in the control example 1 in animals treated with one oltipraz dose of 50 mg/kg. Indeed, the suppression of DMN-induced increase in ALT was 40% in the group treated with one oltipraz dose of 50 mg/kg, whereas suppression of liver fibrosis was 43% with the introduction of oltipraz and DBB in a dose of 25:5 mg/kg, Similar results were obtained in the case of other indicators.

Each value represents the average value of the ± the standard deviation. The number of animals used ranged from 8 to 10. Statistical significance between groups was determined using the criterion Newman-Kelisa for multivariate analysis. Indicators of significance: * = p<0.05 compared with control;^#= p<0.05 compared with the group treated with DMN.

Reference example 8. Antifibrosis action oltipraz/DDB (2)

Histopathological effect of different combinations oltipraz/DBB on DMN-induced liver fibrosis was investigated in laboratory animals. Liver fibrosis was not observed in normal rats (figa and 2b). Explicit fibrosis of the liver tissue was observed in rats after 1 week after the introduction of the DMN during 3 weeks 3 times a day (figa and 3b). With the introduction of oltipraz and DDB in the dose ratio of 25:5 and 15:15 mg/kg for 3 weeks (3 times a week the next day after the introduction of the DMN), fibrosis of the liver tissue was significantly decreased in comparison with the introduction of one DMN. In particular, the group of animals treated with oltipraz and DBB in the dose ratio of 25:5 mg/kg (figa and 4b), was more effective than the drug group of animals treated oltipraz DBB in the ratio of 15 doses:15 mg/kg (figa and 5b) and 5:25 mg/kg (figa and 6b), and the development of liver fibrosis was strongly suppressed.

Necrosis and fibrosis of the liver tissue was determined histologically using this indicator accumulation of collagen as a three-color staining by Masson. The degree of liver fibrosis was determined by the scale of fibrosis and the Knodell scale, which show the degree of liver damage and fibrosis.

Each value represents the average value of the ± standard deviation, and the number of animals used ranged from 8 to 10. Statistical significance between groups was determined using the criterion Newman-Kelisa for multivariate analysis. Indicators of significance: ** = p<0,01 compared with the group treated with DMN. The degree of fibrosis: 0 = but the mA 1 = weak presence of fibrous tissue, 2 = moderate presence of fibrous tissue, 3 = presence of fibrous tissue, 4 = strong signs of fibrosis. The Knodell scale is the sum of the scores in the periportal jumpers (maximum = 10), vnutridolkovom cell death (maximum = 4), portal inflammation (maximum = 4) and fibrosis (maximum = 4).

Reference example 9. The effect of combination oltipraz/DDB on the expression β-TGF

As indicated above, β-TGF - a is a cytokine that causes fibrosis of the liver, whose connection with the development of liver fibrosis is well known. In table presents changes in the content of mRNA β-TGF obtained by the method of RT-PCR.

In the group treated with DMN, there was a significant increase in production β-TGF compared with the control group. In animals treated with a combination of oltipraz/DBB in different proportions, quantitative determination of the expression β-TGF method RT-PCR showed that the introduction of oltipraz at a dose of 30 mg/kg and joint introduction of oltipraz and DBB in a dose of 25:5 mg/kg completely inhibited the expression of β-TGF. However, the increasing ratio of DDB:oltipraz suppression of expression β-TGF consistently reduced. In the group of animals treated with one DDB, was not observed suppression of expression β-TGF. Therefore, we can assume that the suppression of expression β-TGF called by oltipraz. On this basis, the best with the compared between oltipraz and DDB is 5:1 (table).

Working examples

Working example 1

Oltipraz 25 mg

DDB 1 mg

Lactose 50 mg

Starch 10 mg

Magnesium stearate proper number

The above components were mixed and received the tablets of conventional tabletting methods.

Working example 2

Oltipraz 50 mg

DDB 1 mg

Lactose 50 mg

Starch 10 mg

Magnesium stearate proper number

The above components were mixed and received the tablets of conventional tabletting methods.

Working example 3

Oltipraz 5 mg

DDB 10 mg

Lactose 50 mg

Starch 10 mg

Magnesium stearate proper number

The above components were mixed and received the tablets of conventional tabletting methods.

Working example 4

Oltipraz 25 mg

DDB 5 mg

Lactose 30 mg

Cu is hmal 28 mg

Talc 2 mg

Magnesium stearate proper number

The above components were mixed and received capsules by filling this mixture of capsules of hard gelatin conventional methods of producing capsules.

Working example 5

Oltipraz 25 mg

DDB 5 mg

Lactose 30 mg

Starch 28 mg

Talc 2 mg

Magnesium stearate proper number

The above components were mixed and received capsules by filling this mixture of capsules of hard gelatin conventional methods of producing capsules.

Working example 6

Oltipraz 1 mg

DDB 50 mg

Lactose 30 mg

Starch 28 mg

Talc 2 mg

Magnesium stearate proper number

The above components were mixed and received capsules by filling this mixture of capsules of hard gelatin conventional methods of producing capsules.

Working example 7

Oltipraz 5 mg

DDB 25 mg

Lactose 30 mg

Starch 28 mg

Talc 2 mg

Magnesium stearate proper number

The above components were mixed and received capsules by filling this mixture of capsules of hard gelatin conventional methods of producing capsules,

Working example 8

Oltipraz 250 mg

DDB 50 mg

Sugar isomerized 10 g

Sugar 30 mg

CMC sodium 100 mg

Lemon fragrance proper quantity

(add purified water on the total volume 100 ml)

Of the above components was prepared suspension according to the conventional methods of producing suspensions. Filled with a slurry dark bottle 100 ml and sterilized.

Working example 9

Oltipraz 50 mg

DDB 250 mg

Sugar isomerized 20 g

Sugar 20 g

Sodium alginate 100 mg

Orange flavouring proper number

(add purified water to a total volume of 100 ml)

Of the above components was prepared suspension according to the conventional methods of producing suspensions. Filled with a slurry dark bottle 100 ml and sterilized.

Working example 10

Oltipraz 25 mg

DDB 5 mg

Lactose 30 mg

Starch 20 mg

Magnesium stearate proper number

The above components were mixed, made in polyethylene coated shell and tightly closed, receiving the drug in powder form.

Working example 11

1 soft capsule:

Oltipraz 25 mg

DDB 5 mg

The polyethylene glycol 400 400 mg

Glycerin is concentrated 55 mg

Purified water 35 mg

The polyethylene glycol was mixed with concentrated glycerin, and then added purified water. After adjusting the mixture to 60°it was added oltipraz. The mixture was stirred at about 1500 rpm After achieving a homogeneous mixture was cooled at room temperature with slow stirring. Removed Ozerki air using a vacuum pump, receiving content for soft capsules.

The shell is soft capsules produced according to conventional methods, using the well-known formula: 132 mg of gelatin, 52 mg of concentrated glycerin, 6 mg, 70%solution deorbital 1 capsule, the corresponding number of ethylaniline as a flavoring and Carnauba wax as a coating material.

Industrial application

Composition oltipraz/DBB according to the present invention show extremely good effect in the treatment and prevention of fibrosis and cirrhosis. Thus, they can be used in the clinic for the treatment and prevention of fibrosis and cirrhosis.

1. Pharmaceutical composition for the prevention and treatment of progressive fibrosis and cirrhosis, including 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-tion (oltipraz) and dimethyl-4,4’-dimethoxy-5,6,5’,6’-dimethylene-dioxybiphenol-2,2’, in primary forms (DD).

2. The pharmaceutical composition according to claim 1, manufactured in a dosage form selected from the group consisting of capsules, tablets, soft capsules, suspensions, syrups, injectable solutions and powders.

3. The pharmaceutical composition according to claim 1, intended for oral administration.

4. The pharmaceutical composition according to claim 1, in which the weight ratio of oltipraz and DDB is 50-1: 1-50.

5. The pharmaceutical composition is about any one of claims 1 to 4 for the manufacture of drugs for the prevention and treatment of progressive fibrosis and cirrhosis.

6. Method for the prevention and treatment of progressive fibrosis and cirrhosis of the liver, involving the introduction of a mammal the pharmaceutical composition according to any one of claims 1 to 4.