Pharmaceutical composition containing 1,2-dithiol-thione derivative for preventing or treating diseases mediated by high lxr-alpha expression. RU patent 2491065.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and medicine, and concerns a pharmaceutical composition for preventing and treating diseases mediated by high expression or high activity of liver X receptor (LXRa), high expression or high activity of a sterol regulatory element-binding protein-1c (SREBP-1).

EFFECT: preventing and treating renin hypertension, hyperaldosteronism, adrenoleukodystrophy, glomerular sclerosis, proteinuria, nephropathy, hepatic steatosis, hypertriglyceridemia or hyperreninemia, possessing high activity.

6 cl, 11 dwg, 9 ex

The technical field to which the invention relates

The present invention relates to a derived 1,2- for inhibition of expression or activity of the liver X receptor alpha (LXRα) and expression or activity of the protein-1, with Sterol-sensitive regulatory element (SREBP-1). The present invention also refers to the pharmaceutical compositions, including derivative 1,2-. Pharmaceutical composition is effective for prevention and treatment of diseases caused by increased expression LXRα or SREBP-1. Such diseases may include liver steatosis, medications known to exacerbate hypertriglyceridemia, , hypertension caused by , aldosteronism, , glomerulosclerosis, and nephropathy.

The present invention represents the culmination of a research project supported by the center for engineering research (Engineering Research Center, ERC) of the Ministry of education, science and technology (project no R11-2007-107-01001-0, research in the field of metabolism and inflammation).

The level of technology

Liver X receptor (LXR), the receptor is activated by proliferator-activated (PPAR) and X receptor (FXR) are nuclear hormone receptors belonging to the family of receptor type II. These receptors form d) an heterodimer with X receptor (RXR) and bind to DNA. In the absence of bound ligand d) an heterodimer binds to DNA and forms a complex together with co- protein; on the other hand, when the binding of a ligand structural changes, protein co-repressor separated and associated protein co-activator, resulting in the transcription of the gene target (Hermanson et al., Trends Endocrinol. Metab., 2002, 13: 55-60). Among nuclear hormones LXR plays an important role in the control of gene transcription associated with the metabolism and cholesterol homeostasis. Examples of this include gene genes apolipoprotein E (apoE), ABCA1, ABCG1, ABCG5, ABCG8, cholesterol-7α-hydrolases and EFSA-receptor class In type I (Schwartz et al., Biochem. Biophys. Res. Commun., 2000, 274: 794-802). Additionally, LXR directly affects the gene SREBP-1c and controls lipid metabolism (Yoshikawa et al., Mol. Cell. Biol., 2001, 21: 2991-3000).

The LXR there are two isomers, LXRα and LXRβ. In most cases LXRα is present in the liver and LXRβ is present in most organs. LXRα activated , being natural ligands, high glucose concentration and 0901017 and GW3965, are artificial ligands, and controls the expression of genes associated with the synthesis of lipids and cholesterol homeostasis. When lipids are produced by the liver, LXRα functions sensor of lipids and significantly increases the expression and activity of SREBP-1c, which is a key transcription factor controlling the expression of genes, and thus promotes the synthesis of fatty acids in the tissues of the liver and increased the number of triglycerides in the blood.

Induced LXRα nealkogolny liver steatosis may develop in two different ways: by SREBP-1c-dependent path and SREBP-1c-independent way. SREBP-1c-dependent hepatic steatosis developed thanks to the increase of expression of genes through mediated LXRα activation of transcription of SREBP-1c. SREBP-1c-independent steatosis of the liver develops, as the activity of the LXRα leads to increase of CD36 protein, which is a carrier of fatty acids, resulting in greater quantities of fatty acids moves in the liver. As described above, the activity LXRα contributes to the development of nonalcoholic steatosis. However, medications that inhibit the development of steatosis by an activity control LXRα, yet to be established.

SREBP is a protein that is associated with Sterol-dependent regulatory element (SRE), which is a plot of the control of gene transcription, controlled , and exists in three isoforms: SREBP-1A, SREBP-1c and SREBP-2. Transcription of SREBP-1A and SREBP-1c is carried out with the same gene, and SREBP-2 is synthesized by the expression of another gene. SREBP-1c is a transcription factor, controls the transcription of genes associated with the synthesis of fatty acids and SREBP-2 is a transcription factor, controlling transcription of genes associated with the synthesis of cholesterol, Nonoperating SREBP is located in the membrane of the endoplasmic and its size is 125 kDa. Before activation, caused by the disadvantage , SREBP - associated with the membrane in an inactive form. When you activate SREBP moves in the Golgi apparatus and then split with the formation of the active form of the protein size 65 kDa. After activation of SREBP moves into the nucleus where it binds with the Sterol-dependent regulatory elements of target genes and increases the gene expression of enzymes lipid synthesis. Genes, which are the targets of SREBP-1c, represent genes enzymes synthesis of fatty acids. Examples of these enzymes include fatty acid synthase (FAS), acetyl-COA (ACC) -COA (SCD). When the number of free fatty acids coming from the blood to the liver and synthesized de novo liver becomes more of fatty acids, secreted in the form of very low density lipoprotein (VLDL) and subjected β-oxidation, the balance of lipid metabolism in the liver broken and develops liver steatosis. Thus, SREBP-1C, which induces and monitors production of proteins FAS, ACC and SCD involved in the synthesis of fatty acids, is an important factor contributing to the alcoholic or not hepatic steatosis (Kohijma et al., Int. J. Mol. Med., 2008, 21(4): 507-511, Donohue, J. World Gastroenterol. 2007, 13(37): 4974-4978). Hepatic steatosis is a painful condition in which the content of fat in the liver is 5% or more of the total weight of the liver. Liver disease, including liver steatosis, are the most common cause of death, not counting cancer among adults aged 40 to 50 years. In industrial countries, about 30% of the population have symptoms of steatosis and about 20% of cases develop cirrhosis of the liver through the stage of liver fibrosis. 50% of cirrhotic patients die from liver disease within 10 years after the diagnosis of liver cirrhosis. Number of cases of nonalcoholic steatosis of the liver increased due to the «Pro-Western» diet with a high content of lipids and lack of physical activity. Currently the only method of treatment of steatosis is to fix the style of life, such as diet.

Practically no available medicines are effective in the treatment of steatosis, and sick recommend only diet and exercise. Although received therapeutic effects of too low. Thus, there is a need to create medicine for effective treatment of steatosis. At the same time as a maintenance drug therapy is used betaine, , methionine, choline, drugs, medical or pharmaceutical effects of these compounds are not proven. Hence there is a need to develop drugs for effective treatment of steatosis without side effects.

SREBP-1 and SREBP-2 was more expressed in kidney old people and due to the increased expression of SREBP-1 and SREBP-2 synthesis of lipids and accumulation of triglycerides and cholesterol in the kidneys increases, which may cause glomerular sclerosis, and nephropathy (Jiang et al., Kidney Int., 2005, 68(6): 2608-2620).

It is shown that LXRα plays an important role in renin secretion in the kidneys. Both LXRα and LXRβ sufficiently expressed in cells that produce renin. According to Morello et al., 0901017 and GW3965, are agonists LXRα, increase the mRNA expression of renin in the kidneys and renin activity of blood (Morello et al., J. Clin. Invest., 2005, 115: 1913-1922). When blood is present excess of renin, occurs and as a result, developing hypertension and aldosteronism.

LXR also controls the expression of a gene ABCD2 associated with (ALD), and thus inhibitor LXR effective for the treatment of ALD, where ALD is a very rare disease that occurs when fatty acids with very long chains (VLCFA) in vivo are not destroyed and enter the brain, where they damage the nerve cells (Weinhofer et A1., J. Biol. Chem., 2005, 280: 41243-41251).

The authors of the present invention have screened the effects of various drugs to address technical tasks described above. As a result, they found that the expression and activity of LXRα - and LXRβ-dependent SREBP-1 inhibited the introduction of drugs containing derivatives of 1,2-, such as . They also found that the inhibition of SREBP-1 derivatives of 1,2- significantly inhibited the expression of genes are genes-targets, and therefore inhibited the accumulation of triglycerides in the liver tissue induced by diet rich in lipids. Based on these findings, the authors of the present invention are the pharmaceutical composition containing the derivative 1,2-, for the prevention and treatment of diseases caused by increased expression LXRα or SREBP-1.

Advantages

Pharmaceutical composition according to the present invention, contains derivatives of 1,2- as an effective component. Pharmaceutical composition is effective for prevention and treatment of diseases caused by increased expression or increased activity LXRα, or diseases caused by increased expression or increased activity of SREBP-1. Examples of derivatives of 1,2- include (4-methyl-5-(2-)-1,2-dithiol-3-tion), 3-methyl-1,2-dithiol-3-tion and 5-(6-)-4-methyl-1,2-dithiol-3-tion. As a result of the introduction of pharmaceutical compositions inhibited the expression and activity of SREBP-1, where SREBP-1 is key transcription factor controlling gene expression by enzymes in regulation of activity of LXRα, and therefore inhibited the expression of genes, resulting inhibited the accumulation of triglycerides in the liver tissue, which is due steatosis, caused by the violation of metabolism. Accordingly, pharmaceutical compositions containing derivatives of 1,2- as an effective component according to the present invention, are effective for the prevention and treatment of steatosis. Also, pharmaceutical compositions are effective for the prevention and treatment of hypertriglyceridemia , high blood pressure due to , , , glomerulosclerosis, proteinuria and nephropathy.

The present invention provides for the additional method of inhibiting the expression or activity of the LXRα or SREBP-1 using compounds or compositions presented here. Inhibition may lead to the prevention or treatment of disease or condition associated with increased expression or increased activity LXRα or SREBP-1.

The present invention provides a method of treatment of a disease or condition is associated with increased expression or increased activity LXRα or SREBP-1, such as, for example, hepatic steatosis, hypertriglyceridemia, , hypertension caused by , aldosteronism, , glomerulosclerosis, proteinuria and nephropathy, using compounds and compositions presented here.

Short description of the shapes

Figure 1 shows the scheme of introduction of .

Figure 2 shows the effect of the treatment on LXRα, the expression of which improved as a result of a diet with a high content of lipids. The results are shown as a value relative to the level of mRNA LXRα in the group with normal diet. (ND: normal diet, HFD: a diet with a high content of lipid, Olt: , **: p<0.01 compared to ND group and ##: p<0.01 compared with the group receiving only HFD).

Figure 3 shows the effect of the treatment activity LXRα, increased as a result of enhanced expression LXRα (S.S: ).

Figure 4 shows the effect of the treatment on the activity of SREBP-1, the expression of which improved as a result of a diet with a high content of lipids. (ND:normal diet, HFD: a diet with a high content of lipid, Olt: , **: p<0.01 compared to ND group and ##: p<0.01 compared with the group receiving only HFD).

Figure 5 shows the effect of the treatment on the expression of protein SREBP-1 after processing H4IIE and HepG2, representing a line of liver cells and primary culture cells of rat liver, respectively, using 0901317 representing activator LXRα. Protein SREBP-1 identify by Western blotting. (S.E: short-term exposures films, L.E: long exposure films As: lysate intact cells and nuclear faction HepG2 cells).

The figures 6-8 shows the results of the Western blot in the case of the derivatives of 1,2- on the level of SREBP-1, the expression of which increased as a result of processing activator LXRα.

Figure 9 shows the comparative number of genes enzymes (FAS, ACC), expressed in the liver tissue in the diet with a high content of lipids in the introduction of animals with the simulated obesity liver caused by a diet rich in lipids.

Figure 10 shows the effect of the treatment on the amount of triglycerides in the liver tissue in animal models of obesity liver caused by a diet rich in lipids, with the introduction of animals with the simulated obesity liver caused by a diet rich in lipids.

The present invention, based on the fact that the derivatives of 1,2-, such as , inhibit the activity LXRα and the expression and activity of the protein 1, with Sterol-dependent regulatory element (SREBP-1), which is a cellular protein, the controlling expression genes. The authors of the present invention found that the expression of LXRα in mice with elevated LXRα as a result of application of a diet with a high content of lipids inhibited (figure 2), and that the binding capacity LXRα towards LXR-linking element DNA is also reduced (figure 3). Also, the authors of the present invention found that the expression of SREBP-1, which increases when added to cell line hepatocytes 0901317, known as the activator LXRα, inhibited or other derivatives of 1,2- (figure 4-8).

With the introduction of derivatives of 1,2-, such as , inhibited the expression and activity of SREBP-1, which is a key transcription factor controlling gene expression enzymes, due to the control of activity LXRα, additionally, as a result of inhibition of the expression of genes prevented accumulation of triglycerides, which is due to violation of metabolism induced steatosis of the liver tissue. Thus, since pharmaceutical composition according to the present invention, contains derivatives of 1,2- as an effective component, pharmaceutical composition can be effective for prevention and treatment of steatosis. The authors of the present invention found that with the introduction of the fabric of the liver, in which the amount of triglycerides increased as a result of application of a diet with a high content of lipids, the amount of triglycerides, reduces significantly (figure 10 and 11) and the expression of fatty acid synthase (FAS) and acetyl-COA carboxylase (ACC), which are enzymes synthesis of fatty acids that are promoted in mice that were fed a diet with a high content of lipids, also significantly inhibited (figure 9).

Based on these findings, the authors of the present invention to provide a pharmaceutical composition for prevention and treatment of diseases caused by increased expression or increased activity LXRα or SREBP-1, where pharmaceutical composition contains as an effective component of the derivatives of 1,2- formula 1 below, its pharmaceutically acceptable salt, or hydrate, or prodrug and pharmaceutically acceptable media.

Formula 1

where X is the carbon or nitrogen, Q is a sulfur, oxygen or-S=O and R 1 and R 2 each independently choose from the group consisting of a hydrogen atom, C 1-7-alkyl, 3-7 C-cycloalkyl, C 1-7-, C 1-7-alkoxy, 3-7 C-, C 1-7-, C 3-7-, C 1-7-, C 1-7-, C 1-7-alkylsulfonyl, C 1-7-, HO-C 1-7-alkyl, HS-C 1-7-alkyl, hydroxy, thiol, halogen, , nitro, cyano, C 1-7-, C 1-7-, C 1-7-, 1-4 C--1-4 C-alkyl, C 1-4 -alkoxy-1-4 C-alkyl, 1-4 C--1-4 C-alkyl amino, C 1-7-, C 1-7-, 1-4 C-alkoxy-1-4 C-, 1-4 C--1-4 C-, 1-4 C-, phenyl, , phenyl-1-4 C-alkyl, -1-4 C-alkyl, phenyl-1-4 C-alkoxy-1-4 C-alkyl, phenyl-1-4 C--1-4 C-alkyl, phenoxy-1-4 C-alkyl, -1-4 C-alkyl, , phenoxy-1-4 C-, phenyl-1-4 C-alkoxy-1-4 C-, -1-4 C-alkyl, -1-4 C-alkyl and -1-4 C--1-4 C-alkyl where refers to the 5 - or 6- cyclic connection, containing at least one heteroatom, selected from the group consisting of nitrogen, sulfur, and oxygen; phenyl and can be substituted or and affordable Deputy can be selected from the group consisting of halogen, C 1-7-alkyl, C 1-7-alkoxy, C 1-7-, C 1-7-, C 1-7-, 1-4 C-, 1-4 C-, nitro, amino, cyano, BUT-1-4 C-alkyl, HS-C 1-4-alkyl, HO-C 1-7-alkoxy, HO-C 1-7-, HS-C 1-7-, HS-C 1-7-alkoxy, thiol, hydroxy and , and if filled, and phenyl can have one or many deputies; phenyl and each independently can be with at least one benzene or described above; and condensed phenyl and can be substituted or and available Deputy can be selected from the group consisting of halogen, C 1-7-alkyl, C 1-7-alkoxy, C 1-7-, C 1-7-, C 1-7-, 1-4 C-, 1-4 C-, nitro, amino, cyano, HO-1-4 C-alkyl, HS-C 1-4-alkyl, HO-C 1-7-alkoxy, HO-C 1-7-, HS-C 1-7-, HS-C 1-7-alkoxy, thiol, hydroxy and , and if filled, phenyl and can have one or many of the deputies.

Diseases caused by increased expression or increased activity LXRα or SREBP-1, may include, but are not limited to, hypertension caused by , aldosteronism, , glomerulosclerosis, , nephropathy, hepatic steatosis, medications known to exacerbate hypertriglyceridemia or . So, the present invention provides a pharmaceutical composition for prevention and treatment of hypertension caused by , , , glomerulosclerosis, proteinuria, nephropathy, steatosis, hypertriglyceridemia or , where pharmaceutical composition contains derivatives of 1,2- formula 1 as an effective component m pharmaceutically acceptable carrier.

The present invention also provides a way of inhibiting the expression or activity of the LXRα or SREBP-1 in vitro or in vivo, comprising an introduction to pharmaceutical compositions, where pharmaceutical composition contains as an effective component of the derivative 1,2- formula 1 below, its pharmaceutically acceptable salt, its prodrug, it or hydrate and a pharmaceutically acceptable carrier.

Formula 1

where X is the carbon or nitrogen, Q is a sulfur, oxygen or-S=O and R 1 and R 2 each independently choose from the group consisting of a hydrogen atom, C 1-7-alkyl, 3-7 C-cycloalkyl, C 1-7-, C 1-7-alkoxy, 3-7 C-, C 1-7-, 3-7 C-, C 1-7 -, C 1-7-, C 1-7-alkylsulfonyl, C 1-7-, HO-C 1-7-alkyl, HS-C 1-7-alkyl, hydroxy, thiol, halogen, , nitro, cyano, C 1-7-, C 1-7-, C 1-7-, 1-4 C--1-4 C-alkyl, 1-4 C-alkoxy-1-4 C-alkyl, C 1-4 --1-4 C-alkyl amino, C 1-7-, C 1-7-, 1-4 C-alkoxy-1-4 C-, 1-4 C--1-4 C-, 1-4 C-, phenyl, , phenyl-1-4 C-alkyl, -1-4 C-alkyl, phenyl-1-4 C-alkoxy-1-4 C-alkyl, phenyl-1-4 C--C 1-4 -alkyl, phenoxy-1-4 C-alkyl, -1-4 C-alkyl, , phenoxy-1-4 C-, phenyl-1-4 C-alkoxy-1-4 C-, -1-4 C-alkyl, -1-4 C-alkyl and -1-4 C--1-4 C-alkyl where refers to the 5 - or 6- cyclic connection, containing at least one heteroatom, selected from the group consisting of nitrogen, sulfur, and oxygen; phenyl and can be substituted or and affordable Deputy can be selected from the group consisting of halogen, C 1-7-alkyl, C 1-7-alkoxy, C 1-7 -, C 1-7-, C 1-7-, 1-4 C-, 1-4 C-, nitro, amino, cyano, HO-1-4 C-alkyl, HS-C 1-4-alkyl, HO-C 1-7-alkoxy, HO-C 1-7-, HS-C 1-7-, HS-C 1-7-alkoxy, thiol, hydroxy and , and if filled, phenyl and may have one or many of the deputies; phenyl and each independently can be with at least one benzene or described above; and condensed phenyl and can be substituted or and available Deputy can be selected from the group consisting of halogen, C 1-7-alkyl, C 1-7-alkoxy, C 1-7-, C 1-7-, C 1-7-, 1-4 C-, 1-4 C-, nitro, amino, cyano, HO-1-4 C-alkyl, HS-C 1-4-alkyl, HO-C 1-7-alkoxy, HO-c 1-7-, HS-C 1-7-, HS-C 1-7-alkoxy, thiol, hydroxy and , and if replaced, phenyl and can have one or many of the deputies.

The present invention also provides a way to prevent or cure diseases or conditions associated with increased expression or increased activity LXRα or SREBP-1, including the introduction of pharmaceutical composition where the pharmaceutical composition contains as an effective component of the derivative 1,2- formula 1 below, its pharmaceutically acceptable salt, its prodrug, it or hydrate and a pharmaceutically acceptable carrier.

Formula 1

where X is the carbon or nitrogen, Q is a sulfur, oxygen or-S=O and R 1 and R 2 each independently choose from the group consisting of a hydrogen atom, C 1-7-alkyl, 3-7 C-cycloalkyl, C 1-7-, C 1-7-alkoxy, 3-7 C-, C 1-7-, 3-7 C -, C 1-7-, C 1-7-, C 1-7-alkylsulfonyl, C 1-7-, HO-C 1-7-alkyl, HS-C 1-7-alkyl, hydroxy, thiol, halogen, , nitro, cyano, C 1-7-, C 1-7-, C 1-7-, 1-4 C--1-4 C-alkyl, C 1-4 -alkoxy-1-4 C-alkyl, 1-4 C--1-4 C-alkyl amino, C 1-7-, C 1-7-, 1-4 C-alkoxy-1-4 C-, 1-4 C--1-4 C-, 1-4 C-, phenyl, , phenyl-1-4 C-alkyl, -1-4 C-alkyl, phenyl-1-4 C-alkoxy-1-4 C-alkyl, phenyl-1-4 C--1-4 C-alkyl, phenoxy-1-4 C-alkyl, -1-4 C-alkyl, , phenoxy-1-4 C-, phenyl-1-4 C-alkoxy-1-4 C-, -1-4 C-alkyl, -1-4 C-alkyl and -1-4 C--1-4 C-alkyl where refers to the 5 - or 6- cyclic connection, containing at least one heteroatom, selected from the group consisting of nitrogen, sulfur, and oxygen; phenyl and can be substituted or and affordable Deputy can be selected from the group consisting of halogen, C 1-7-alkyl, C 1-7-alkoxy, C 1-7-, C 1-7-, C 1-7-, 1-4 C-, 1-4 C-, nitro, amino, cyano, HO-1-4 C-alkyl, HS-C 1-4-alkyl, HO-C 1-7-alkoxy, HO-C 1-7-, HS-C 1-7-, HS-C 1-7-alkoxy, thiol, hydroxy and , and if filled, and phenyl can have one or many deputies; phenyl and each independently can be with at least one benzene or described above; and condensed phenyl and can be substituted or and available Deputy can be selected from the group consisting of halogen, C 1-7-alkyl, C 1-7-alkoxy, C 1-7-, C 1-7-, C 1-7-, 1-4 C-, 1-4 C-, nitro, amino, cyano, HO-1-4 C-alkyl, HS-C 1-4-alkyl, HO-C 1-7-alkoxy, HO-C 1-7-, HS-C 1-7-, HS-C 1-7-alkoxy, thiol, hydroxy and , and if filled, phenyl and can have one or many of the deputies.

In some realizations of ways to treat or prevent diseases or conditions, mediated LXRα or SREBP-1 disease or condition is, for example, liver steatosis, medications known to exacerbate hypertriglyceridemia, , hypertension caused by , aldosteronism, , glomerulosclerosis, or nephropathy. In some realizations connection disclosed here apply to facilitate and/or slow down the development of one or more of these diseases or conditions. Derivatives of 1,2- contained in the pharmaceutical composition according to the present invention may include organic compound, including 1,2-dithiol-3-tion and bicyclic molecule and derived organic compounds. Б can be , , , thiazole or (table 1).

Table 1

Examples of compounds containing 1,2-dithiol-3-tion, for inhibition of the expression of SREBP-1

R 1 :N, -alkyl(C 1-8 ) R 2 :N, -halogen, alkoxy R 3 :N, -O R4:-N-alkyl(C 1-8 ) one of the R 2 replaced

R 1 to : -H, -alkyl(C 1-8 ) One of X, Y and Z represent - N and others represent C

R 1 to :-H, -alkyl(C 1-8 ) R 2 :N, -alkyl(C 1-8 )

R 1 :N, -alkyl(C 1-8 ) R 2 :-H,-alkyl(C 1-8 ) - halogen

R 1 :N, -alkyl(C 1-8 ) R 2 :-H,-alkyl(C 1-8 ) R 3 :-N-alkyl(C 1-8 ) R 4 :-H, -alkyl(C 1-8 )

R 1 :N, -alkyl(C 1-8 ) R 2 :-H, -alkyl(C 1-8 ) R 3 :H - alkyl(C 1-8 ) R 4 :-H, - alkyl(C 1-8 )

A daily dose of pharmaceutical compositions according to the present invention may vary in accordance with the severity of disease, time of development of the disease, age, health and complexity of the condition of the patient. For example, the pharmaceutical composition according to the present invention can be administered in a dose of 1-500 mg, preferably 30-200 mg per adult per day. Dose can be administered as a bolus or more servings.

The present invention described in detail by the following experimental examples. Experimental examples are only illustrative purposes and do not limit the scope of the claims of the present invention.

EXAMPLES

A comparative example 1. Experimental animals and diet

Male mice of C57BL/6 (average weight of 25-30 g) as experimental animals receive from Charles River Orient (Seoul, Korea). For at least one week before testing mice will have acclimatised to the environment in the research center of testing animals pharmaceutical College, Seoul national University, which represent humidity 55±5%, the temperature of 22±2 degrees C and controlled ventilation. Mice again and alternately exhibited in the light, and then placed in the darkness with the intervals of 12 hours (from 7 am to 7 PM). During testing, the amount of food and water, absorbed mice, has not significantly changed. Weight and the condition of mice measure every week. Two groups of mice, respectively, are grown on a diet with a high content of lipids (Dyets Inc., Bethlehem) and on a normal diet for 10 weeks. During the last four weeks in each case of mice injected (10 or 30 mg/kg, 3 times a week) (figure 1). Each group will consist of 10 mice.

A comparative example 2. Receive samples

and derivatives of 1,2- receive from CJ Co., Ltd. Derivative 1,2- used in the present invention, it is possible to receive by way of, revealed in the KR №10-0604261. A diet with a high content of lipids for the induction of obesity liver receive from Dyet Co., USA. bred 40% 200 to obtain the required concentration.

A comparative example 3. Real-time PCR

To obtain cDNA use the General RNA (2 mg) and primer d(T) 16 , which is extracted from the liver of mice, and reverse transcriptase AMV. The relative number of genes evaluated quantitatively using real-time PCR using dye CyBr green. Real-time PCR is carried out with the help of installation of Light-cycler 2.0 company Roche (Mannheim, Germany). PCR performed according to the method manufacturer with application of the Light-cycler software 4.0 for analyzing the relative number of relevant genes.

A comparative example 4. Western blotting

Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate (SDS-PAGE) is conducted with the help of the apparatus Mighty Small II SE 250 by the method of (Laemmli UK (1970)). Cultivation of aliquots liver samples are diluted with sample buffer (63 mm Tris (pH 6,8), 10% of glycerin, 2% LTOs, 0,0013% bromophenol blue, 5% β ) and then spend electrophoresis using 7.5% and 9% gel prepared on a solution electrode buffer (15 g of Tris, 72 g glycine and 5 g LTOs 1 l of solution). After the completion of electrophoresis of proteins from the gel is transferred to nitrocellulose membranes in the buffer solution (25 mm Tris, 192 mm glycine, 20% v/v methanol (pH 8.3 a)) in the device for the samples at 190 mA for one hour. A reaction to anti-SREBP-1 as the primary antibodies and then with antibodies goats against rabbit IgG, with horseradish peroxidase as the secondary antibodies within 1 hour. Then apply system ECL (Amersham, Gaithesberg, MA) for the visualization of immunoreactive protein. Equal load on the number of protein in their respective samples identify with the use of antibodies against β-actin (Sigma, St. Louis, MO).

A comparative example 5. A method of analysis

The data contained in the following experimental examples, obtained with the help of the program for pharmaceutical calculations. So, the significance of the differences between different experimental groups is assessed through a one-way analysis of the quadratic deviation (mono-direction quadratic variance assay) (Fisher, R.A., Statistical Methods for Research Workers, Edinburgh: Oliver & Boyd, 1925) and the results are then processed by the method of Newman Coil (Newman Keuls) (Norman GR et al., Biostatistics: The Bare Essentials, 2000) (*p<0.05, **p<0,01).

Experimental example 1. Action the increased expression of LXRα

Mice raises on a diet with a high content of lipids and normal diet for 10 weeks. Evaluate expression LXRα in the liver tissue of a group of mice fed a diet with a high content of lipids and were (10-30 mg/kg, 3 times a week), which is a combination of 1,2-, within the last four weeks. Allocate mRNA of liver tissue, cDNA is synthesized using real-time PCR and then hold real-time PCR using the appropriate primer (mouse LXR, 5'-TGCCATCAGCATCTTCTCTG-3' (semantic) and 5'-GGCTCACCAGCTTCATTAGC-3' (antisense)). The level of mRNA expression of LXRα in the group with normal diet (ND) gives a value of 1 and determine the relative expression levels in the group fed a diet with a high content of lipids, or in the group fed a diet with a high content of lipids, and . The results are shown in figure 2. So, you can see that the expression of LXRα, which is the intracellular sensor lipids significantly increased in the group fed a diet with a high content of lipids (p<0.01) and the increased expression of LXRα inhibited by introducing (p<0.01).

Experimental example 2. Inhibitory effect of the introduction of activity LXRα

LXRα controls gene expression through education dimer with RXRα and bind it to the relevant district (LXRE) promoter of the target. Does the binding capacity LXRE with the introduction of identify by electrophoretic analysis of the shift. oligonucleotide LXRE gene SREBP-1 mark using radioisotope the 5′-end with [γ - 32 P]ATP and T4 Polina and then sample (1 ml >10 6 cpm) are incubated with nuclear fraction of the proteins with a buffer solution for linking. The reaction solution is subjected to electrophoresis in 4% polyacrylamide gel and analyzed by . sequence LXRE used in this test is a 5′-CAGTGACCGCCAGTAACCCCAGC-3′. Specificity of binding to DNA identify with titration («cold») breakdown and analysis . Titration breakdown in advance a reaction with 20- surplus (on a molar basis) oligonucleotides. For the analysis of a reaction of antibodies against LXRα or RXRα (2 mg) with the reaction mixture at room temperature for about 30 min and then the same type of sample, labelled with a radioisotope, and additionally incubated for 30 min, and then subjected to electrophoresis.

When the overexpression of LXRα and RXRα intensity slowly moving bands increases compared with the control (see first and second band on the figure 3A). When analysing the using antibodies against LXRα and RXRα protein binding with DNA reduced due to the presence of antibodies against LXRα and RXRα and formed a band with (see third and fifth band on the figure 3A). Such results confirm the specificity of binding LXRα/RXRα with DNA.

With the introduction of increase the intensity of the bands with delayed mobility (from the first to the third band in the figure 3) decreases (see the fourth and fifth stripes on figure 3). Such results show that the binding capacity LXRα in relation to DNA is significantly reduced with the introduction of .

Experimental example 3. The act of putting the increased SREBP-1

Assess the levels of SREBP-1 in the liver tissue of mice used in experimental example 1. mRNA extracted from the liver tissue, cDNA are using real-time PCR and then with her conduct real-time PCR using the appropriate primers (mouse SREBP-1, 5'-AACGTCACTTCCAGCTAGAC-3' (semantic) and 5'-CCACTAAGGTGCCTACAGAGC-3' (antisense)). The level of mRNA expression of SREBP-1 in the group with normal diet (ND) gives a value of 1 and determine the relative expression levels in the group fed a diet with a high content of lipids, or in the group fed a diet with a high content of lipids and . The results are shown in figure 4. So, you can see that the expression of SREBP-1 increased significantly in the group fed a diet with a high content of lipids (p<0.01) and the increased expression of SREBP-1 inhibited by introducing (p<0.01).

Experimental example 4. Inhibitory effect on the expression and activity of SREBP-1

H4IIE and HepG2, representing a line of liver cells and primary culture cells of rat liver, respectively, process 0901317 as activator LXRα and then identify protein SREBP-1 by Western blotting. Expression of SREBP-1 significantly increased for 12 hours after treatment 0901317 (see the fourth track on the respective images gels (shown in figure 5A). Increased expression of the protein SREBP-1 decreases dependent on the concentration of manner when processing (fifth and sixth track on the respective images gels on the figure 5A), which demonstrates the inhibition of the expression of SREBP-1 .

Additionally, the increase in nuclear localization of SREBP-1 in HepG2 cells treated 0901317, reduced dependent on the concentration of manner when processing (figure 5), which demonstrates the inhibition of the activity of SREBP-1 .

Cellular fractions receive the following way. Buffer solution with low osmotic pressure (10 mm HEPES (pH 7,9), 10 mm KCl, 0.1 mm EDTA, 0,5% Nonidet P-40, 1 mm DTT and 0.5 mm (PMSF)) add to the line of liver cells and put on ice for 10 minutes Then gained solution centrifuged at 7200 g for 5 min and used as cytoplasmic faction. Separately, buffer solution with high osmotic pressure (20 mm HEPES (pH 7,9), 400 mm NaCl, 1 mm EDTA, 10 mm DTT and 1 mm PMSF) add to the line of liver cells and put on ice for one hour. Then gained solution centrifuged at 15000 g for 10 min and used as a nuclear faction. Separately buffered solution (10 mm HEPES (pH 7,9), 100 mm NaCl, 1 mm EDTA, 10% of glycerin, a 0.5% Triton X-100, 0,5% Nonidet P-40, 1 mm DTT and 0.5 mm PMSF) are added to the cells, washed PBS, and then are lysed for 1 hour. Then gained solution centrifuged at 10,000 g for 10 min and applied as a whole cell extract. These cell faction kept at -70 OC before use. The concentration of the protein is determined by the method of Bradford, through a combination of (Bio-Rad protein assay kit, Hercules, CA, USA).

Experimental example 5. Inhibitory effect of derivatives of 1,2- on the expression of SREBP-1

The derivatives of 1,2- the increased expression of SREBP-1, induced activator LXRα (0901317), estimated with the use of cell lines H4IIE. Expression of SREBP-1, increased as a result of processing cell line H4IIE using 0901317, inhibited by processing derivatives of 1,2- (figures 6-8).

Experimental example 6. Inhibitory effect on the expression of FAS and ACC, representing enzymes synthesis of fatty acids

Assess the levels of expression of FAS and ACC, which are the products of target genes SREBP-1, in the liver of mice used in the experimental example 1. mRNA extracted from the liver tissue, cDNA are using real-time PCR and then with her conduct real-time PCR using appropriate primers (mouse ACC1, 5'-GTCAGCGGATGGGCGGAATG-3' (semantic) and 5'-CGCCGGATGCCATGCTCAAC-3' (antisense); mouse FAS, 5'-AGCGGCCATTTCCATTGCCC-3' (semantic) and 5'-CCATGCCCAGAGGGTGGTTG-3 (antisense). The level of mRNA expression of FAS and ACC1 in the group with normal diet (ND) gives a value of 1 and determine the relative levels of mRNA expression of FAS or ACC in the group fed a diet with a high content of lipids, or in the group fed a diet with a high content of lipids and . The results are shown in figure 9. So, you can see that the expression of FAS and ACC significantly increased in the group fed a diet with a high content of lipids (p<0.01) and the increased expression of FAS and ACC inhibited by introducing (p<0.01).

Experimental example 7. Inhibitory effect on the amount of triglycerides, accumulated in liver tissue by application of a diet with a high content of lipids

Evaluate action on the amount of triglycerides in the liver tissue of mice in the experimental example 1. The amount of triglycerides in the liver tissue is the index of the degeneration of the liver. After the introduction of (Bae et al., Hepatology, 2007, 46: 730-739) measure number liver tissue. In mice that were fed a diet with a high content of lipids in 10 weeks, the amount of triglycerides in the liver tissue is significantly increased compared with mice in the group receiving normal diet (p<0.01), but after the introduction the amount of triglycerides in the liver tissue is significantly reduced (p<0.01) (figure 10).

Experimental sample 8. The therapeutic effect of on fabric liver in an animal model of steatosis of the liver caused by a diet with a high content of lipids

Therapeutic effects the degeneration of the liver caused by a diet with a high content of lipids used in the experimental example 1 identify dyeing by Oil Red-O using specific mass to fat dye. The liver of animals with obesity, the liver is fixed with the help of a neutral solution 10% formalin and subjected to the process of fixing the known and the process of dehydrogenation/dehydration and then the liver tissue in paraffin. Placed in paraffin pieces of fabric cut into slices 4 mm thick, painted Oil Red-O and then identify with the help of an optical microscope. In the group receiving a diet with a high content of lipids (HFD + media), a considerable red staining; in the group receiving (HFD + ) painted in the red part is significantly reduced (figure 11). Thus, you can see that the therapeutic effect of is high.

Are received various formulations containing derivatives of 1,2- as an effective component.

An example of a 1

25 mg

Lactose

50 mg

Starch

10 mg

Magnesium stearate

By necessity

These components are mixed and then apply a well-known method for obtaining tablets, thus receiving a preparation in tablets.

An example of a 2

3-methyl-1,2-dithiol-3-tion

50 mg

Lactose

50 mg

Starch

10 mg

Magnesium stearate

By necessity

These components are mixed and then apply a well-known method for obtaining tablets, thus receiving a preparation in tablets

An example of a 3

These components are mixed and then fill up the soft gelatinous capsules according to the known procedure manufacture of capsules with the purpose of reception of the drug in the form of capsules.

An example of a 7

100 mg

Isomerized sugar

10 g

Sugar

30 mg

Sodium carboxymethylcellulose

100 mg

Lemon fragrance

By necessity

Purified water

to the desired volume

(The total volume of all components 100 ml)

The suspension obtained using these components are in a known method of reception of suspensions and then fill the bottle dark glass of 100 ml and sterilized, thus receiving the drug in the form of suspension.

An example of a 8

3-methyl-1,2-dithiol-3-tion

250 mg

Lactose

30 mg

Starch

20 mg

Magnesium stearate

By necessity

These components are mixed up homogeneity and fill them covered with a polyethylene bag and sealed it, thus receiving the drug in powder form.

An example of a 9

One soft capsule contains:

100 mg

Polyethylene glycol 400

400 mg

Concentrated glycerin

55 mg

Purified water

35 mg

Polyethylene glycol and concentrated glycerin mix and then add purified water. Temperature of the mixture is maintained at the level of about 60 C, it adds derivative 1,2- and evenly mixed by mixing at a speed of about 1500 rpm Then slowly stirring reduce the temperature to room and remove bubbles using a vacuum pump, receiving thus contents of soft capsules. Surface film of soft capsules are obtained by soft processing famous in the art gelatin or superplasticising admixture. One capsule obtained using 132 mg gelatin, 52 mg concentrated glycerin, 6 mg 70% solution , as an additional agent odorants and wax as covering the agent according to the generally accepted method of reception of capsules.

Industrial application

Pharmaceutical composition according to the present invention is effective for prevention and treatment of diseases caused by increased expression or increased activity LXRα or diseases caused by increased expression or increased activity of SREBP-1. With the introduction of pharmaceutical compositions of the expression and activity of SREBP-1 inhibited, where SREBP-1 is the key transcription factor controlling gene expression enzymes by changing the activity LXRα, and additionally inhibited the expression of genes, resulting in inhibited the accumulation of in the liver tissue, which is due steatosis of the liver caused by metabolic disorders. Accordingly, pharmaceutical compositions containing derivatives of 1,2- as the active components according to the present invention, are effective for the prevention and treatment of steatosis. Also, pharmaceutical compositions are effective for the prevention and treatment of hypertriglyceridemia , hypertension caused by , , , glomerulosclerosis, proteinuria and nephropathy.

1. Pharmaceutical composition for prevention and treatment diseases caused by increased expression or increased activity of liver X receptor (LXRα), containing as an effective component of a compound selected from a group of compounds listed below, its pharmaceutically acceptable salt, it hydrate or prodrug of:

2. Pharmaceutical composition for prevention and treatment of diseases caused by increased expression or increased activity of protein 1, with Sterol-dependent regulatory element (SREBP-1), which contains as an effective component of a compound selected from the group below its pharmaceutically acceptable salt, it hydrate or prodrug of:

3. Pharmaceutical composition for prevention and treatment of hypertension caused by , , , glomerulosclerosis, proteinuria, nephropathy, steatosis, hypertriglyceridemia or containing as an effective component of a compound selected from the groups below its pharmaceutically acceptable salt, it hydrate or prodrug of:

4. Pharmaceutical composition for prevention and treatment of steatosis, containing as an effective component of a compound selected from the groups below its pharmaceutically acceptable salt, it hydrate or prodrug of:

5. Pharmaceutical composition on any one of claims 1 to 4, wherein the connection formula 1 is a (4-methyl-5-(2-)-1,2-dithiol-3-tion).

6. Pharmaceutical composition for prevention and treatment of steatosis, containing as an effective component (4-methyl-5-(2-)-1,2-dithiol-3-tion), its pharmaceutically acceptable salt, it hydrate or prodrug.