Carboxylic acids and their derivatives containing pharmaceutical compositions

 

In the application described therapeutically effective compounds, representing amphipatic carboxylate of the formula R-COOH or a salt, ester or amide of such compounds, where R denotes a saturated or unsaturated alkyl chain containing 10 to 24 carbon atoms, one or more of which may be substituted by heteroatoms, where one or more members of the chain of carbon atoms or heteroatoms optionally form part of a ring, and where said chain is optionally substituted hydrocarbonyl radical, heterocyclyl radical, (ness.) alkoxygroup substituted by hydroxyl (ness.)by alkyl, hydroxyl, carboxyla, halogen, phenyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil by phenyl,3-C7cycloalkyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil3-C7cycloalkyl, and specified amphipatic carboxylate has the ability to endogenous conditions become appropriate thioether of coenzyme A. the Invention also relates to modulation of the activity of HNF-4and by the way necessiate. The invention explains the modulation of expression of genes involved in initiation and development of pathologies in humans. 3 N. and 24 C.p. f-crystals, 4 Il., 1 PL.

The technical field to which the invention relates.

The invention relates to therapeutically effective compounds and to methods for treating certain diseases/syndromes using these compounds.

Links

In the following description, the references given in parentheses or as a Superscript of the corresponding part of the description, refer to the following publications:

1. Sladek F. M., Zhong, W. M., Lai, E., Darnell J. E., Jr. Gene Dev. 4, 2353-2365 (1990).

2. F. M. Sladek in: Liver Gene Expression (Ed. Tronche F., and M. Yaniv), pp. 207-230, R. G. Landes Co., Austin, TX (1994)

3. The Metabolic and Inherited Bases of Inherited Disease (edited by Scriver C. R., Beaudet, A. L., Sly, W. S., Valle, D.), vol II, part 8, 1995 (McGraw-Hill, Inc.)

4. Yamagata, K., and others, Nature 384, 458-460 (1996).

5. DeFronzo R. A. and Eleaterio F., Diabetes Care, 14, 173-194 (1991).

6. Leff T., Reue K., Melian, A., Culver H. and Breslow J. L., J. Biol. Chem. 264, 16132-16137 (1989).

7. Cave W. T., FASEB J., 5, 2160-2166 (1991).

8. Chin J. P. F., Prost. Leuk. Essent. Fatty Acids 50, 211-222 (1994).

9. Grundy S. M. and Think M. A., J. Lipid Res. 31, 1149-1172 (1990).

10. Storlien L. H., and others, Science 237, 885-888 (1987).

11. Unger, R. H., Diabetes 44, 863-870 (1995).

12. Morris, M. C., Saks F. and Rosner Century, Circulation 88, 523-533 (1993).

13. Hultin, M. B. The Bar-Tana J., Diabetes 37, 1618-1624 (1988).

16. Tzur, R., Smith, E., and Bar-Tana J., Int. J. Obesity 13, 313-326 (1989).

17. Russel J. C., P. M. Arny, Graham S. E., Dolphin P. and J. Bar-Tana J., Arterioscler. Thromb. Biol. 15, 918-923 (1995).

The above publications describe to patents and patent applications are fully incorporated in the present description by reference, if it is not listed specifically for each individual publication, respectively, for each separate description to the patent and patent application.

Background of invention

Hepatocytes nuclear factor-41(HNF-4) (data summarized in reference 2) is orthonovum representative of the superfamily of nuclear receptors. HNF-4expressed in liver, kidney, intestine and pancreas adults and embryos and causes various abnormalities in mice. Homologous recombination HNF-4leads to death of the embryo. Like other members of the superfamily receptor HNF-4consists of a modular structure, including the highly conserved N-terminal DNA-binding domain, through which the hinge is connected with vibrophones C-end landscapebased domain. Previously held Cloner 1 and HNF-42, which is a variant splicing, with the insertion of 10 amino acids at the C-terminal domain.

HNF-4is an activator of gene expression. Activation of transcription through HNF-4mediated by its binding in the form of glycosilated with reactive sequences promoter DR-1 of target genes, leading to activation of the complex of transcription initiation. Activated HNF-4genes (data summarized in reference 2), encode different enzymes and proteins involved in the metabolism of lipoproteins, cholesterol and triglycerides (apolipoprotein AI, AII, AIV, B, CIII, microsomal protein transfer of triglycerides, cholesterol-7-hydroxylase), lipid metabolism (mitochondrial acyl-COA-dehydrogenase fatty acids with medium chain length, proximally acyl-COA-oxidase fatty acids, cytochrome P450 isoenzymes involved in-oxidation of fatty atilov and hydroxylation of steroids, protein, fatty acid binding, cell rathinasamy protein II, tractility), glucose metabolism (phosphoenolpyruvate-Karbala), in blood coagulation (factors VII, IX, X) in the metabolism of iron (transferrin, erythropoietin) and in the activation of macrophages (protein type hepatocytes growth factor/stimulating macrophage protein, proteins of the nucleus of the virus of hepatitis B and X are proteins, long terminal repeat of human HIV-1,-1-antitripsin).

Some genes that are activated by HNF-4play a major role in the emergence and development of atherogenesis, cancer, autoimmune and some other diseases [3]. So, for example, overexpression of apolipoprotein, AIV and III, and protein transfer microsomal triglyceride levels can lead to dyslipoproteinemia (combination of hypertriglyceridemia and hypercholesterolemia) due to increased production of lipoproteins of very low density (ONPL) and chylomicrons in combination with a decrease in their clearance from plasma. Similarly, the increase in speed of glycolysis in the pancreas, leading to induced HNF-4/HNF-1 overexpression/insulin secretion in the pancreas can lead to hyperinsulinemia, which cause resistance to insulin. To date, found that mstitelnosti to insulin in combination with induced HNF-4the overexpression of phosphoenolpyruvate-carboxykinase and increased production of glucose in the liver can lead to impaired glucose tolerance (IGT) and result in non-insulin-dependent diabetes mellitus (NIDDM). In addition, currently hyperinsulinemia is considered as the main etiological factor in the emergence and development of essential hypertension, and therefore, the controlled overexpression of HNF-4genes can additionally lead to hypertension. Induced by HNF-4overexpression of coagulation factors in combination with possible sverkhekspressiya inhibitors of fibrinolysis blood (e.g., inhibitor-1 plasminogen activator) can also lead to increased formation of blood clots and decrease fibrinolysis, which is accompanied by increased susceptibility to atherosclerotic processes.

It was also established that dyslipoproteinemia, obesity, IGT/NIDDM, hypertension and disorders of coagulation/fibrinolysis can be combined with one syndrome (X-syndrome, metabolic syndrome, syndrome of resistance to insulin) [5]. The high activity of HNF-4in relation Out to cause etiology, characteristic X-syndrome. According to current data category X syndrome and the syndrome as a whole are considered as major risk factors for atherosclerotic cardiovascular disease in Western countries, suggesting that HNF-4plays a role in the origin and development of atherogenesis. In addition, because people with X-syndrome, occurs and develops breast cancer, colon and prostate cancer, it can be assumed that the controlled overexpression of HNF-4genes may participate in the emergence and development of these malignant diseases.

In addition to the role that HNF-4plays in the expression of genes associated with X-syndrome, HNF-4activates the expression of genes that encode proteins involved in the modulation process of autoimmunity. So, for example, induced by HNF-4overexpression of stimulating macrophage protein may lead to sensitization of macrophages in relation to a self-antigen or antigens with cross-reactivity, Thu sclerosis and psoriasis. In addition, since the transcription of the protein core of the hepatitis B X protein, as well as the long terminal repeat of human HIV-1 are controlled by HNF-4, HNF-4may participate in the modulation process of the infection caused by these viral agents.

Because overexpression induced by HNF-4genes can lead to dyslipoproteinemia, IGT/NIDDM, hypertension, disorders of coagulation and fibrinolysis blood, atherogenesis, cancer, inflammation, immune deficiency and other diseases, it can be expected that inhibition of the activity of HNF-4in relation to the transcription will decrease due to HNF-4pathologies. However, it is still not detected ligands for HNP-4that could serve as the basis for the creation of inhibitors of the activity of HNF-4in relation to transcription. In the present invention proposed ligands for HNF-4low molecular weight, which have been specially created for the purpose to act as modulators induced by HNF-4genes, or involving controlled HNF-4the genes.

Summary of the invention

According to the present invention proposed a therapeutically effective compounds, including amphipatic carboxylate of the formula R-COOH or a salt, ester or amide of such compounds, where R denotes a saturated or unsaturated alkyl chain containing 10 to 24 carbon atoms, one or more of which may be substituted by heteroatoms, where one or more members of the chain of carbon atoms or heteroatoms optionally form part of a ring, and where said chain is optionally substituted hydrocarbonyl radical, heterocyclyl radical, (ness.)alkoxygroup substituted by hydroxyl (ness.)by alkyl, hydroxyl, carboxyla, halogen, phenyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil by phenyl,3-C7cycloalkyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil3-C7cycloalkyl sootvetstvujushij tiefer of coenzyme A.

In a preferred embodiment, amphipatic carboxylate is xenobiotics amphipatic carboxylate. In a more preferred embodiment, xenobiotics amphipatic carboxylate may be a dicarboxylic acid with a long chain,HE-carboxylic acid-In(OH)2-carboxylic acid, similar lovebrimbo acid or non-steroidal anti-inflammatory drugs. In the most preferred embodiment, amphipatic carboxylate selected from the group consisting of stearoyl(18:0)-COA, oleoyl(18:1)-COA, linoleoyl(18:2)-COA, linoleoyl(18:3)-COA, eicosapentaenoic(20:5)-COA, docosahexaenoyl(22:6)-COA, 1,16-hexadecanedioic acid, 1,18-octadecadienoic acid, 2,2,15,15-tetramethoxy-Dean-1,16-diNovo acid, 2,2,17,17-tetramethylhexadecane-1,18-diNovo acid, 3,3,14,14-tetramethylhexadecane-1,16-diNovo acid, 3,3,16,16-tetramethylhexadecane-1,18-diNovo acid, 4,4,13,13-tetramethylhexadecane-1,16-diNovo acid, 4,4,15,15-tetramethylhexadecane-1,18-diNovo acid, 16-(OH)2-hexadecanoyl acid, 18-IN(OH)2-octadecanoic acid, 16-(OH)2-2,2-dimethylhexylamine N)2-3,3-dimethyloctadecyl acid, 16-(OH)2-4,4-dimethylhexylamine acid, 18-IN(OH)2-4,4-dimethyloctadecyl acid, 16-hydroxyhexadecanoic acid, 18-hydroxyoctadecadienoic acid, 16-hydroxy-2,2-dimethylhexylamine acid, 18-hydroxy-2,2-dimethyloctadecyl acid, 16-hydroxy-3,3-dimethylhexylamine acid, 18-hydroxy-3,3-dimethyloctadecyl acid, 16-hydroxy-4,4-dimethylhexylamine acid and 18-hydroxy-4,4-dimethyloctadecyl acid.

Another object of the present invention is a method of treatment X syndrome, introducing a therapeutically effective amount of amphipatic carboxylate. In the preferred embodiment, each of the diseases related to the category of X-syndrome, can be treated separately.

Another object of the present invention are methods of modulating the activity of HNF-4.

And finally, another object of the invention are methods of treatment of a disease or syndrome, introducing a therapeutically effective amount of amphipatic carboxylate. By using the methods according to the invention it is possible to treat diseases such as, for example, breast cancer, rry atilov long chain and COA (atilov long-chain-COA) are ligands for HNF-4. Fused protein GST-HNF-4(LBD) (I) consists of HNF-4(LBD) fused with glutathione-S-transferase (GST). His-HNF-4(n) consists of full-HNF-4labeled with 6 histidine residues.

A. Saturation curve of binding to the Palmitoyl(16:0)-COA

The corresponding recombinant proteins incubated until reaching equilibrium with [3H]-Palmitoyl(16:0)-COA (of 0.05 µci) and with increasing concentrations of unlabeled, Palmitoyl(16:0)-COA, as shown in the drawing. The dissociation constant (Kd), is 2.6 μm, and the maximum binding of 1 mol of Palmitoyl(16:0)-COA to 1 mol HNF-4determine by the method of Scatchard.

B. Competitive binding with myristoyl (14:0)-COA

The corresponding recombinant proteins incubated with nm [3H]-Palmitoyl(16:0)-COA (60 CI/mmol) and increasing concentrations of the unlabeled myristoyl(14:0)-COA, as shown in the drawing. The percentage of binding corresponds to the number of radioactively labeled with [3H]-Palmitoyl(16:0)-COA in the associated faction. 100% binding occurs when the concentration of [3H]-Palmitoyl(16:0)-COA 0.3 nmole. Meant the of the EU50for other thioesters of fatty atilov and Koa, and xenobiotics atilov and COA are shown in the table.

In Fig.2 shows that the ligands HNF-4representing the thioesters of fatty atilov and COA, modulate the binding of HNF-4with-specific DNA enhancer.

A. Binding of His-HNF-4(14 ng) with SR in the absence (lane 1) or presence of either 10 μm myristoyl(14:0)-COA (lane 2) or 10 μm Palmitoyl(16:0)-COA (lane 3)

B. Binding of His-HNF-4(20 ng) with SR in the absence (lane 1) or presence of either 10 μm stearoyl(18:0) COA (lane 2) or 10 μm linoleoyl(18:3, w-3)-KoA (lane 3)

C. Activating binding His-HNF-4(14 ng) with HDN using increasing concentrations of myristoyl(14:0)-COA.

Shows the slice of gel containing radioactively labeled chemicals associated with the dimer His-HNF-4.

In Fig.3 shows the modulation of the activity of HNF-4in relation to in vitro transcription using thioesters of fatty atilov long chain and COA.

A. The data are representative of experiments, in oncentrate His-HNF-4and without His-HNF-4(lanes 1-3, 7-9) or adding 10 μm Palmitoyl(16:0)-COA (lanes 4-6) or 10 μm stearoyl(18:0) COA (lanes 10-11). Transcripts of the test and control matrices with the correct start of transcription is indicated by a simple arrow (and bold arrow (respectively.

B. Induced HNF-4a transcription in the absence (empty bars) or presence of 10 μm Palmitoyl(16:0)-COA (shaded bars) or 10 μm stearoyl(18:0) COA (hatched bars). The multiplicity of transcription refers to the ratio of specific transcripts obtained with the test matrix, and transcripts obtained using a control matrix, standardized relative to the value obtained without HNF-4. In the drawing summarized data on the 5 independent experiments for each acyl-COA. The symbol * is marked valid data obtained compared to the corresponding value in the experiments without added ligand.

In Fig.4 shows the modulation of the activity of HNF-4using fatty acids with long chain and using xenobiotics amphipatic 232D/chr/945.gif" border="0">using fatty acids with long-chain

The frequency of induction of CAT activity by transfection HNF-4determine, by comparing the CAT-activity adding pSG5-HNF-4and adding the plasmid pSG5, and depending on the applicable designated on the drawing of fatty acids, added to the culture medium. In the drawing, has summarized data on 3-4 independent experiments for each fatty acid. Data are given as mean values ± standard deviation (SCO).

B. Suppresse HNF-4using xenobiotics dicarboxylic acids

The multiplicity of induction is defined as CAT activity in cells incubated with such ligands as 3,3,12,12-tetramethylhexadecane-about acid (), 3,3,14,14-tetramethylhexadecane acid (g) and 3,3,16,16-tetramethylethylenediamine acid (5), standardized in relation to the activity of cells incubated without added ligands. Values EU50for the above and for other xenobiotics ligands to the following:

3,3,12,12-tetramethylethylenediamine acid >300 μm

3,3,14,14-tetramethylhexadecane acid 230 microns

2,2,15,15-tetramethylhexadecane acid 150 microns

2,2,17,17-tetramethylethylenediamine acid 150 microns

4,4,13,13-tetramethylhexadecane acid 150 microns

4,4,15,15-tetramethylethylenediamine acid 150 microns

bezafibrat 260 microns

nafenopin 160 microns

indometacin 130 microns

Detailed description of the invention

When creating the present invention, it was found that fatty acids with long chain directly modulate the activity of HNF-4in respect of transcription by binding the corresponding thioesters of fatty atilov and COA with landscapebased domain of HNF-4. Modulation of the activity of HNF-41 in respect of transcription when used as agonistic or antagonistic ligands of thioethers acyl-COA can lead to two completely independent induced by ligands reactions, namely, to shift the balance between oligomeric and dimeric forms of HNF-4or to effects inherent in dimeric form HNF-4the affinity for binding with-specific enhancers.

In the context of the present description, these terms have the following values.

The concept of "am is the concept of "xenobiotics" refers to compounds that alien with respect to the intermediate metabolism of mammals.

The concept of "X-syndrome" refers to a syndrome that includes some or all of the following diseases, such as 1) dyslipoproteinemia (combination of hypercholesterolemia and hypertriglyceridemia, low IDPs-cholesterol), 2) obesity (in particular expressed obesity), 3) impaired glucose tolerance (IGT), leading to non-insulin-dependent diabetes mellitus (NIDDM), 4) essential hypertension and 5) thrombogenic/fibrinolytic defects.

The term "modulation" refers to either increase or decrease the activity of HNF-4. Modulation of HNF-4can be direct, for example, when binding to HNF-4or indirect, for example, associated with another mechanism, such as kinase activity. Compounds of the present invention that are associated with HNF-4depending on chain length and/or degree of saturation can either activate or inhibit its binding with-specific enhancers.

Treatment X syndrome fall under the scope of the present invention. These the lady of inhibiting the activity of HNF-4in respect of transcription, also falling under the scope of the invention, represent inhibition using antisense transcript, using antibodies or by using any other method to reduce excessive activity of HNF-4.

Methods

Recombinant proteins HNF-4

cDNA rat HNF-4l (pLEN4S) [1] was subcloned into the coding glutation-S-transferase (GST) plasmid pGEX-2T (firm Pharmacia) and the resulting plasmid was digested with SmaI and Ass and re-ligated, receiving slit plasmid GST-HNF-4(LBD). Fused plasmid expressed in a strain of E. coll BL21(DE3) by induction with 0.2 mm IPTG for 60 min and the product was purified by affinity chromatography using glutathione-agarose pellet (firm Sigma), getting fused protein GST-HNF-4(LBD), consisting of 96-455 amino acid sequence HNF-4wild-type, fused with GST. Full-size cDNA HNF-41, cloned in the vector 6His-pET11d, expressed in E. coli strain BL21(DE3)plysS.

Analysis of the ligand binding

Recombinant GST-HNF-41(LBD) (100 pmole) or His-HNF-4[3H]-Palmitoyl(16:0)-COA were separated using charcoal coated with a Dowex and determined the amount of bound ligand using a liquid scintillation counter. Nonspecific binding of [3H]-Palmitoyl(16:0)-COA was determined by its binding to a fragment of the GST or the carbonate-dehydrogenase as especificados protein.

The analysis of changes in mobility in gel

His-HNF-4and the acyl-COA pre-incubated for 30 min at 22°C in PMM Hepes (pH of 7.9) containing 50 mm KCl, 1 mm of dithiothreitol, 2.5 mm MgCl2, 10% glycerol, 1 μg poly(dI-dC) in a final volume of 20 µl. Then added labeled with32P oligonucleotide (0.1 ng), including the element SR sequence of the promoter of apolipoprotein CIII (APO CIII) person (-87/-66)6and the incubation continued for another 15 minutes Complexes protein-DNA was dissolved in 5% sedentarism polyacrylamide gel 0.6-fold TBE and quantitatively assessed by imaging using devices Phosphor Imager.

Analysis of in vitro transcription

Reaction of 20 units RNasin, 0.5 μg treated with ultrasound DNA salmon sperm and His-HNF-4and the specified test ligand. The mixture is pre-incubated for 30 min at 22°C, then added 10 ng of the control matrix pAdML200, including last main promoter of adenovirus (-400/+10) associated with not containing G cassette length of 200 base pairs, and 200 ng of the test matrix, including three copies of the element SR sequence of the promoter of the APO CIII (-87/-66) located against the course of transcription relative to the synthetic promoter of Boxing Hogness of ovalbumin and before does not contain G cassette length 377 base pairs. The mixture is additionally pre-incubated for 10 min at 22°C, and then added to 40 μg of nuclear extract of HeLa and additionally pre-incubated for 30 min at 30°C. Then was added to 0.5 mm ATP, 0.5 mm TTF, 25 μm UTP and 10 µci [32-P]-UTP (i.e., 800 CI/mol, the firm Amersham) and the reaction mixture was incubated for 45 min at 30°C at a final concentration of 25 µl. The reaction was stopped by adding 175 μl of stop mix (0.1 M sodium acetate (pH 5.2), 10 mm etc, 0.1% of LTOs, 200 μg/ml tRNA), and then were extracted with phenol and precipitated with ethanol. RNA is laminam gel containing 7 M urea in TBE. By tomography using devices Phosphor Imager quantitatively evaluated correctly initiated transcripts. Test DNA template was designed, embedded in plasmid RS2AT amplificatory using PCR oligonucleotide obtained using as template a plasmid (SR)3-TK-CAT and three copies of an item SR sequence of the promoter of the gene, APO CIII (-87/-66), with sites EcoRI and SSTI 5'- 3'-ends, respectively. The resulting plasmid was digested with SphI and Sad and ligated with a synthetic oligonucleotide (5'-CGAGGTCCACTTCGCTATATATTCCCCGAGCT-3'), containing the promoter sequence timedancing herpes virus simple (HSV) (-41/-29) and the promoter of the chicken ovalbumin (-33/-21).

Analysis using transfection

Cell line COS-7, together transfection for 6 h reporter plasmid (SR)3-TK-CAT (5 μg) and either expressing the plasmid pSG5-HNF-4(0,025 g), or the plasmid pSG5 (0,025 g), which was added by precipitation using calcium phosphate, cultured in serum-free medium with the addition of the above fatty acids (in the form of complexes with albumin in a molar tp://img.russianpatents.com/chr/946.gif" border="0">-galactosidase vector pRSGAL (1 µg). Design (SR)3-TK-CAT received by embedding synthetic oligonucleotide including the sequence of the promoter of the gene Aro CIS(-87/-66) (5'-GCAGGTGACCTTTGCCCAGCGCC-3'), flanked by the restriction site HindIII, pBLCAT247progress against transcription relative to the promoter timedancing length 105 base pairs. Selected design, containing three copies of a synthetic oligonucleotide in direct orientation, and its structure was confirmed by sequencing.

The thioesters of fatty atilov and COA

The thioesters of fatty atilov and COA was obtained by interaction of free fatty acids dissolved in anhydrous acetonitrile, with 1,1’-carbonide-midazolam. The reaction mixture was evaporated to dryness and the corresponding conjugate acyl-imidazole was subjected to interaction with one equivalent restored COA dissolved in a mixture of THF with water in the ratio 1:1. Upon completion of the reactions were analyzed by TLC on plates of silica gel N (Merck) (butanol/acetic acid/water 5:2:3). Derived in the form of acyl-COA besieged by using 0.1 M Hcl and the precipitate was washed three times with 0.1 M Hcl, three times with a simple ether that does not contain peroxide, and three times with acetone. Spektrofotometrichesky when using soo and his realization of additional advantages are illustrated in the examples, which do not limit its scope.

Example 1

The thioethers atilov long chain and COA are ligands for HNF-4

It is established that the thioethers atilov with different chain length and degree of saturation and COA specific contact HNF-4. Binding was assessed either by the example of binding landscapebased domain of HNF-4fused with glutathione-S-transferase (GST-HNF-4(LBD), or for example, linking to the full-size protein HNF-4“, marked by six histidine residues (His-HNF-4).

Binding of Palmitoyl(16:0)-COA with landscapebased domain or full-length protein HNF-4reached saturation when Kdof 2.6 μm and had a tendency to saturation at a ratio of 1 mol of tiefer fatty acyl-COA/1 mol HNF-4(Fig.1A). The binding of acyl-COA appeared to be specific, while the free fatty acid and free COA were inactive. Linking thioesters atilov with different chain length and with varying degrees of saturation and Koa was assessed by competitive binding of the radioactively labeled Palmitoyl(16:0)-COA with Ryoko who was not identified when using tiefer saturated fatty acyl with a chain length shorter With12and COA. However, the affinity for binding to HNF-4tiefer fatty acyl long chain and COA is not significantly dependent on the chain length or degree of saturation of the respective ligands, remaining in the range of 0.5 to 0.3 microns. The specificity of binding to HNF-4tiefer fatty acyl long chain and Koa also assessed by analyzing the estimated binding of Palmitoyl(16:0)-COA with recombinant labeled with histidine, activated using proliferated peroxisomesreceptor (His-PPAR). In contrast to the results obtained when using the HNF-4the thioesters of fatty atilov long chain and COA was not associated with PPARorreceptor retinoic acid X (RXRa). These results indicate that the thioethers natural for the body fat atilov long chain and COA can contact landscapebased domain of HNF-4and to serve as a specific for this protein ligands.

Linking thioethers acyl-COA with HNF-4not limited preveden also detected when using thioesters xenobiotics atilov and COA (RCOSKoA), where R is a moiety comprising a saturated or unsaturated alkyl chain comprising from 10 to 24 carbon atoms, one or more of which may be substituted by a heteroatom, where one or more of these members of the chain of carbon atoms or heteroatoms optionally form a ring part and where this chain optionally may be substituted (Fig.1B).

Example 2

Modulation of the activity of HNF-4using thioesters atilov long chain COA

Effects on the activity of HNF-4binding thioesters atilov long chain and Koa was evaluated by studying the binding of HNF-4with specific to it element SR sequence of the promoter of the gene, APO CIII (-87/-66)6in the presence of thioethers atilov with different chain length, degree of saturation and the degree of substitution, and Koa, and without the thioethers. Binding was determined using analysis of changes in mobility in the gel. As can be seen in Fig.2, the binding SR with HNF-4ci increased with increasing concentrations of His-HNF-4and activated when using thioesters natural for the body of the saturated fatty acyl residues with a chain length12-C16 the e concentrations, necessary for its binding to HNF-4. In addition, I found that some of the thioesters of fatty atilov and Koa, and xenobiotics atilov and Koa are the true antagonists, HNF-4that is , they inhibit its inherent link with-specific enhancers. Thus, in particular, incubation HNF-4in the presence of either stearoyl(18:0)-COA, or-linoleoyl(18:3)-COA resulted in a pronounced inhibition of its binding to the oligonucleotide SR (Fig.2). Similarly, incubation of HNF-4in the presence of various thioethers xenobiotics atilov and COA resulted in inhibition of its binding with specific to it oligonucleotide SR. Thus, thioethers natural for the body or xenobiotics atilov and COA that are associated with HNF-4depending on chain length, degree of saturation or degree of substitution can serve as agonists, partial agonists or antagonists of the activity of HNF-4in respect of transcription.

Example 3

Modulation induced by HNF-4aktivnosta ligands, which are agonists or antagonists for HNF-4also was assessed by analysis catalyzed addition of nuclear extract of HeLa and induced recombinant HNF-4the speed of transcription in vitro test matrix, including not containing G cartridge length 377 base pairs under the control of promoters representing a sequence timedancing HSV and chicken ovalbumin, and enhanced three copies of the element SR promoter gene APO CIII. The ability of HNF-4to activate the transcription was assessed by adding representative tiefer acyl long chain COA and without it. To assess transcription as an internal control matrix used matrix that includes not containing G cassette length of 200 base pairs under the control of the last main promoter of adenovirus (AdML) and lacking the enhancer HNF-4. As can be seen in Fig.3, transcription in vitro test matrix was increased depending on the concentration of HNF-4, reaching saturation at a concentration of HNF-4200 ng. Induced by HNF-4transcriptases with data about the effect of agonists and antagonists, HNF-4from the experiments on the evaluation of changes in the mobility in the gel. Thus, thioethers atilov COA that are associated with HNF-4, can directly modulate its activity against transcription in a cell-free system.

The intracellular effects of ligands HNF-4on mediated HNF-4transcription was evaluated on the cell line COS-7, which was transfectional jointly by the vector expression of HNF-4and reporter plasmid CAT under the control of the promoter timedancing and enhanced one of the three copies SR promoter gene APO CIII. Transfection cells were incubated in the presence of free fatty acids and xenobiotics amphipatic carboxylates which represent ligands for HNF-4with agonistic or antagonistic activity. As can be seen in Fig.4A, when the culture medium was added fatty acids, expression enhanced SR reporter plasmids were activated with HNF-47 times. Activation of transcription in transfection HNF-4can �45.gif" border="0">or may be the result of the binding of HNF-4activated in endogenous conditions acyl-COA. Adding to the culture medium myristic(14:0) and palmitic(16:0) acid resulted in a dose-dependent activation of HNF-4-dependent transcription, whereas stearic (18:0),-linolenic(18:3) or eicosapentaenoate (20:5) acid inhibited the transcription, which is consistent with data on agonistic or antagonistic activity of the corresponding thioesters of fatty atilov and COA obtained as in the experiments for the evaluation of changes in the mobility in the gel (Fig.2), and in the analysis in a cell-free transcription system (Fig.3). Similarly, the inhibition of the activity of HNF-4in respect of the transcription can be detected in the experiments for transfection (Fig.4B) adding to the culture medium xenobiotics amphipatic carboxylates (RCOOH), where R denotes a radical, which represents a saturated or unsaturated alkyl chain containing 10 to 24 carbon atoms, one or more of which may be substituted by heteroatoms, where one or more members of the chain of atoms is hydrocarbonyl radical, heterocyclyl radical, (ness.)alkoxygroup, (ness.)the alkyl substituted by hydroxyl, hydroxyl, carboxyla, halogen, phenyl, substituted phenyl, C3-C7cycloalkyl or substituted C3-C7cycloalkyl. Thus, intracellular mediated HNF-4the expression can be modulated using a natural for the body of fatty acids, as well as using xenobiotics amphipatic carboxylates, in which endogenous conditions have the ability to transform into the corresponding COA thioesters (RCOSKoA). Highly effective inhibitors are compounds in which R is substituted by an-carboxyla: 2,2,15,15-tetramethylhexadecane-1,16-Dianova acid, 2,2,17,17 - tetramethylhexadecane-1,18-Dianova acid, 3,3,14,14-tetramethylhexadecane-1,16-Dianova acid, 3,3,16,16-tetramethylhexadecane-1,18-Dianova acid, 4,4,13,13-tetramethylhexadecane-1,16-Dianova acid, 4,4,15,15-tetramethylhexadecane-1,18-Dianova acid. To another group of effective compounds include the following compounds, in which R is substituted by anthe hydroxyl: 16-hydroxyhexadecanoic acid, 18-hydroxyoctadecadienoic acid, 16-hydroc Canova acid, 18-hydroxy-3,3-dimethyloctadecyl acid, 16-hydroxy-4,4-dimethylhexylamine acid, 18-hydroxy-4,4-dimethyloctadecyl acid. Another group of somewhat less effective compounds include analogues lovebrimbo acid (fibrates) or nonsteroidal anti-inflammatory drugs. General services action can be determined mainly by the composition of nuclear thioethers acyl-COA and agonistic/antagonistic activity of each of them in binding to HNF-4.

Example 4

Physiological mechanisms of action

Inhibition of the activity of HNF-4in respect of transcription using a natural for the body or xenobiotics amphipatic carboxylates, in which endogenous conditions have the ability to transform into the corresponding COA thioesters can serve as therapeutic targets in the treatment of diseases that are caused by and/or developed by overexpression controlled HNF-4the genes. The specific action of amphipatic carboxylate as an inhibitor of the activity of HNF-4in respect of transcription may depend primarily on 945.gif" border="0">. Currently it is impossible to predict which of the amphipatic carboxylates having the ability to turn into endogenous conditions in the corresponding COA thioesters can be considered as the true antagonists, HNF-4. For example, it is proved that myristoyl(14:0)-COA or Palmitoyl(16:0)-COA are activators ability HNF-4to activate transcription, while the next homologue in this series, i.e. stearoyl(18:0)-COA, is the true antagonist. However, it should be noted that partial agonists can cause a marked inhibition of the activity of HNF-4with the substitution of the endogenous conditions of strong agonists, HNF-4or in competition with the more active agonists for binding to HNF-4.

The total activity of amphipatic carboxylate as an inhibitor of the activity of HNF-4in relation to transcription in vivo may depend not only on inherent to its corresponding tiefere COA ability to act as an antagonist of HNF-4but also depend on the particular cell type and so what rofiles nutritional/pharmacological availability of appropriate acids, the availability of each of them for COA-diatrypaceae, as well as the availability of the corresponding acyl-COA for the hydrolysis of acyl-COA-hydrolases, the ability to esterification with lipids to oxidation in the products, elongation, desaturation, or to bind with other acyl-COA-binding proteins. In addition, endogenous acyl-COA, resulting COA-diatrypaceae amphipatic carboxylates, not related to fatty acids (such as retinoic acid, prostaglandins, leukotrienes, and others), you can contact HNF-4and to modulate its activity as agonists or antagonists. The resulting action may also depend on additional nuclear factors that can affect the balance between oligomeric and dimeric forms of HNF-4on the affinity of binding of HNF-4with-specific enhancers or on the interaction of HNF-4with a protein complex of transcription initiation. In particular, since HNF-4and activated peroxisomal activator receptor (PPAR) have similar consensus sequences DR-1 and acids with long-chain, than their corresponding COA-thioesters, activity-specific acyl-COA and indirectly affect HNF-4may be similar to the activity detected by activation of PPAR corresponding free acid, or can be found antagonism.

Despite the above hypotheticall, agonistic/antagonistic profile of ligands for HNF-4from the group acyl-COA, demonstrated in the example in the present description, the examples, may contribute to the development of molecular basis of impacts identified in vivo using dietary fatty acids, which affect some of the adjustable HNF-4the genes. Fatty acyl long chain consists of dietary fat, which accounts for 30-40% of calories absorbed from food in Western countries. In addition to their role as substrates because they are most easily oxidized with the release of energy or transformed as a result of esterification into triglycerides and phospholipids necessary for the formation of fatty tissue and cell membranes, respectively, certain dietary fatty acids have long been recognized as PI is inu [10,11], hypertension [12], defects in blood clotting and fibrinolysis [13], inflammation, immune deficiency and other diseases. These previously unexplained effects now can be decrypted based on the data about the influence of the corresponding acyl-COA on the activity of HNF-4in respect of transcription, which leads to modulation of expression of genes involved in initiation and development of such pathologies. Specific effects of dietary fatty acids with long chain on blood lipids and blood coagulation should be considered with the light exactly proven effects of HNF-4genes encoding proteins that are involved in the metabolism of lipoproteins (apolipoprotein AI, AII, B, CIII, microsomal protein transfer triglycerides) and blood coagulation (factors IV, IX, X). In fact just proved the increase of the content in the plasma OVPL-NPL - and IDP-cholesterol caused by dietary saturated fatty acids with chain length With12-C16in General, and myristic acid in particular, consistent with activation of HNF-4under the influence of the corresponding thioethers saturated atilov and COA and the absence of effects of tpri using saturated stearic (18:0) acids may also be due to an antagonistic effect of stearoyl(18:0)-COA on the activity of HNF-4. Similarly, the decrease in the level of lipids under the influence of mono - and polyunsaturated fatty acids [9], associated with the replacement of saturated dietary fatty acids, consistent with the activity of thioethers poly - or monetising atilov and COA compared with thioethers nasishenna fatty atilov and COA, which is also complemented by direct inhibition of HNF-4using linoleoyl(18:3)-COA, eicosapentaenoic (20:5)-COA or docosahexaenoyl(22:6)-COA. In addition, the increase in blood clotting induced saturated dietary fatty acids with chain length With12-C16and correlated with a corresponding increase in factor VII, decrease blood clotting induced polinenasyschennami dietary fatty acids, as well as unexpectedly installed the lowering of the content of factor VII and blood coagulability specific induced food stearic(18:0) acid, similarly may be due to the influence of the corresponding thioesters of fatty atilov and COA on the activity of HNF-4that leads to modulation of the expression of controlled HNF-4genes encoding dependent on vitamin K factors of SWE xenobiotics amphipatic carboxylates, which in endogenous conditions can be tarifitsirovana with the formation of the corresponding COA thioesters and which act as agonists or antagonists of HNF-4may be pharmacological therapeutic mechanism of diseases, emerging and developing as a result of overexpression of controlled HNF-4the genes. The possibility of using xenobiotics substituted amphipatic carboxylates should be considered in light of the generalized information obtained through simulation on animals [14-17], which relates to their pharmacological characteristics in relation to changes in the flow of dyslipoproteinemia, obesity, resistance to insulin and atherosclerosis, i.e., diseases associated with overexpression of some controlled HNF-4the genes. Therapeutic effectiveness of these medicines may be due to the inhibition of the activity of HNF-4in respect of transcription, as described in the examples given in this description.

Claims

1. Application xenobiotics the compounds of formula R-COOH or salts, f is the volume of carbon one or more of which may be substituted by heteroatoms, where one or more members of the chain of carbon atoms or heteroatoms optionally form part of a ring, and where said chain is optionally substituted hydrocarbonyl radical, heterocyclyl radical, (ness.)alkoxygroup substituted by hydroxyl (ness.)by alkyl, hydroxyl, carboxyla, phenyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil by phenyl,3-C7cycloalkyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil3-C7-cycloalkyl, with the specified connection has the ability to endogenous conditions to turn into a corresponding thioether of coenzyme A, i.e., in RCOSKoA, for the treatment of X-syndrome and/or any disease, including X-syndrome, regardless of activation Rliver.

2. Application under item 1, where R is chosen from the group including-carboxyl and-hydroxyl chain.

3. Application under item 1, where RCOOH denotes a fatty acid with a long chain.

4. Use the https://img.russianpatents.com/chr/969.gif" border="0">-carboxyl and RCOOH is chosen from the group comprising 1,16-hexadecanedioic acid, 1,18-octadecadienoic acid, 2,2,15,15-tetramethylhexadecane-1,16-diNovo acid, 2,2,17,17-tetramethylhexadecane-1,18-diNovo acid, 3,3,14,14-tetramethylhexadecane-1,16-diNovo acid, 3,3,16,16-tetramethylhexadecane-1,18-diNovo acid, 4,4,13,13-tetramethylhexadecane-1,16-diNovo acid and 4,4,15,15-tetramethylhexadecane-1,18-diNovo acid.

5. Application under item 1, where R denotesis hydroxy, and RCOOH is chosen from the group including a 16-hydroxyhexadecanoic acid, 18-hydroxyoctadecadienoic acid, 16-hydroxy-2,2-dimethylhexylamine acid, 18-hydroxy-2,2-dimethyloctadecyl acid, 16-hydroxy-3,3-dimethylhexylamine acid, 18-hydroxy-3,3-dimethyloctadecyl acid, 16-hydroxy-4,4-dimethylhexylamine acid and 18-hydroxy-4,4-dimethyloctadecyl acid.

6. Use one of the PP.1-5, intended for the treatment of dyslipoproteinemia (hypertriglyceridemia, hypercholesterolemia, low content of IDPs-cholesterol).

7. Use one of the PP.1-5, designed to ensure resistance to insulin, disorders of glucose tolerance and non-insulin dependent diabetes mellitus (NIDDM).

8 is the resultant of PP.1-5, designed to reduce blood clotting or enhance fibrinolysis blood.

10. Use one of the PP.1-5, intended for the treatment of coronary or peripheral atherosclerosis.

11. Use one of the PP.1-6, intended for the treatment of rheumatoid arthritis, multiple sclerosis, psoriasis or inflammatory bowel disease.

12. Use one of the PP.1-6, intended for the treatment of breast cancer, colon cancer or prostate cancer.

13. Pharmaceutical composition for oral administration, intended for the treatment of X-syndrome and/or any disease, including X-syndrome, regardless of activation Rliver, comprising a therapeutically effective amount xenobiotics the compounds of formula R-COOH or a salt, ester or amide of such compounds, where R denotes a saturated or unsaturated alkyl chain containing 10 to 24 carbon atoms, one or more of which may be substituted by heteroatoms, where one or more members of the chain of carbon atoms or heteroatoms optionally form part of a ring, and where said chain is optionally substituted hydrocarbonyl radika what baxrom, a phenyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil by phenyl,3-C7cycloalkyl or substituted by a hydroxy-group, (ness.)the alkyl, (ness.)alkoxygroup, (ness.)alkenyl or (ness.)the quinil3-C7cycloalkyl, with the specified connection has the ability to endogenous conditions to turn into a corresponding thioether of coenzyme A, i.e., in RCOSKoA and targeted supplements.

14. The composition according to p. 13, where R is chosen from the group including-carboxyl and-hydroxyl chain.

15. The composition according to p. 13, RCOOH where denotes a fatty acid with a long chain.

16. The composition according to p. 13, where the active substance is chosen from the group comprising the following compounds in which R stands for-carboxyl and RCOOH is chosen from the group comprising 1,16-hexadecanedioic acid, 1,18-octadecadienoic acid, 2,2,15,15-tetramethylhexadecane-1,16-diNovo acid, 2,2,17,17-tetramethylhexadecane-1,18-diNovo acid, 3,3,14,14-tetramethylhexadecane-1,16-diNovo acid, 3,3,16,16-tetramethylhexadecane-1,18-diNovo acid, 4,4,13,13-tetramethylhexadecane-1,16-diNovo acid and 4,4,15,15-those who>hydroxy, and RCOOH is chosen from the group including a 16-hydroxyhexadecanoic acid, 18-hydroxyoctadecadienoic acid, 16-hydroxy-2,2-dimethylhexylamine acid, 18-hydroxy-2,2-dimethyloctadecyl acid, 16-hydroxy-3,3-dimethylhexylamine acid, 18-hydroxy-3,3-dimethyloctadecyl acid, 16-hydroxy-4,4-dimethylhexylamine acid and 18-hydroxy-4,4-dimethyloctadecyl acid.

18. Composition according to one of paragraphs.13-17, intended for the treatment of dyslipoproteinemia (hypertriglyceridemia, hypercholesterolemia, low content of IDPs-cholesterol).

19. Composition according to one of paragraphs.13-17, designed to ensure resistance to insulin, disorders of glucose tolerance and non-insulin dependent diabetes mellitus (NIDDM).

20. Composition according to one of paragraphs.13-17, intended for the treatment of essential hypertension.

21. Composition according to one of paragraphs.13-17, designed to reduce blood clotting or increased fibrinolysis blood.

22. Composition according to one of paragraphs.13-17, intended for the treatment of coronary or peripheral atherosclerosis.

23. Composition according to one of paragraphs.13-17, intended for the treatment of rheumatoid arthritis, multiple sclerosis, psoriasis or inflammatory ), colon cancer or prostate cancer.

25. The method of treatment X syndrome, introducing a therapeutically effective amount of a compound that inhibits controlled HNF-4the transcription.

26. A method of modulating the activity of HNF-4that includes introducing an effective amount of amphipatic carboxylate.

27. The method according to p. 26, characterized in that the modulation is an inhibition of the activity of HNF-4.

28. The method according to p. 26, characterized in that the modulation represents the activation activity of HNF-4.



 

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