Peptides inhibiting transforming growth factor tgfБЕТА1

 

The invention relates to synthetic peptide antagonists of the binding of transforming growth factor TGF1 with its receptors in the body with a sequence containing from 2 to 15 amino acids that are identical or similar to amino acids natural transforming growth factor TGF1 and/or its receptors. The peptides can be used as active compounds for the preparation of compositions used for liver diseases, in particular for liver fibrosis (cirrhosis). 1 N. and 9 C.p. f-crystals, 7 tab., 28 Il.

The LEVEL of TECHNOLOGY

Cell growth is regulated by various proteins belonging to the group of growth factors (Schalch DS et al. (1979) Endocrinolody 104:1143-1151). The most important growth factors associated with the development of cells and could have implications for autocrine and paracrine mechanisms involving transforming growth factors (TGFs) (L. Braun et al. (1988) Cell Biol. 85:1539-1543; Lyons RM and Moses HL (1990) Eur. J. Biochem. 187:467-473).

The term "transforming growth factor TGF" was first used to describe the activity produced by the cell line, transformed under the action of sarcoma virus in mice or rats (deLarco JE Ayu to induce normal growth in soft agar of such cells, for growth which was necessary solid media. More detailed studies revealed two classes of transforming growth factors TGF called TGFand TGFthat, in turn, comprise a family of related proteins. A family of proteins TGFincludes 5 isoforms (Brand T. and Schneider MD (1995) J. Mol. Cell Cardiol. 27:5-18) dimeric structure (Schlunneger MP and Grutter MG (1992) Nature 358:430-434; Brand, T. and Schneider MD (1995) J. Mol. Cell Cardiol. 27:5-18). Studies of Mature purified proteins derived from the same species shows a high degree of identity of the sequences of these proteins (table 1).

TGF1, which is synthesized as a precursor of 390 amino acids, referred to as Pre-Pro-TGF1. When you first hydrolysis released hydrophobic fragment, consisting of 29 amino acids, which leads to the emergence of Pro-TGF1. Then the next time you break the chain at the site, which is located before the end of TGF1 and which consists of two arginine residues, released Mature TGF1 - there is a protein consisting of 112 amino acids with a molecular mass of 12 kDa. Dsow, while receiving dimer with a molecular mass of 25 kDa. Changes of this structure leads to loss of biological function (Barnard JA et al. (1990) Biochim. Biophys. Acta 1032:79-87).

As is known, within the framework of TGF1 there are different domains. One of these domains, as found, is located between amino acids 40 and 82 and is involved in the binding of TGF1 with its cellular receptors. (Quian SW et al. (1992) Proc. Natl. Acad. Sci. 89:6290-6294; Burmester JK et al. (1993). Proc. Natl. Acad. Sci. 90:8628-8632).

Receptors of the transforming growth factor TGF1 and contacted other proteins

Characterized by five types of specific receptors, binding with TGF1 (Cheifetz S et al. (1988) J. Biol. Chem. 263:17225-17228 Casillas and Lopez F. et al. (1991) Cell 67:785-795). These receptors have different affinities for different types of TGF1. To date the best studied receptor types I, II, and III (an overview is given Attisano L et. al. (1994) Biochim. Biophys. Acta 1222:71-80; Derynck R. (1994) Trends Biochem. Sci. 19:548-553; Yingling et al. (1995) Biochim. Biophys. Acta 1242:115-136). Also described receptors type IV (Maskow K. and Daniel-pour D. (1991) J. Biol. Chem. 266:9907-9911) and the receptor V type Ichijo H. et al. (1991) J. Biol. Chem. 266:22459-22464). It was also reported that the transmembrane and cytoplasmic domain of the al. (1996) J. Immunol. 156:564-573)) by about 70% similar to receptor type III, both human and rat.

RIII could be suitable for binding to TGF1 and delivery to the RII, which, in turn, could form a complex with RI (Yamashita et al. (1994) J. Biol. Chem. 269:29172-20178) or complexes in which different molecules RI combined with RII (Weiss G. and Massague J. (1996) EMBO J 15:276-289). RII-RI interactions could cause Feofilova RI and subsequent activation they serine/trionychinae that would Feofilova other molecules that carry genetic information, such as protein molecules MADR2 (Macias-Silva M, et al., (1996) Cell 87:1215-1224) (1).

The role of transforming growth factor TGF1 in the differentiation and regeneration of liver cells

The impact varies depending on the time of development and cell type.

The increase in extracellular matrix when exposed to the stellate cells of the liver (Ito cells), the main source of extracellular matrix proteins (Mustce TA et al. (1987) Science 237:1333-1336).

- Differentiation of epithelial cells and hepatocytes (Florini JR et al. (1986) J. Biol. Chem. 261:16509-16513).

- Inhibition of cell growth in the process of liver regeneration. This effect is of great importance for the conservation of cleto growth factor (EGF), as observed in the case of cultures of hepatocytes of rat embryo (Noda M. and Rodan GA (1987) J. Cell Physiol. 133:426-437).

The role of transforming growth factor TGF1 when fibrose liver

As it was found that TGF1 is associated with the process of liver fibrosis (Czaja MJ et al. (1989) J. Cell Biol. 108:2477-2482; G. Annoni et al. (1992) J. Hepatol. 14:259-264), causing an increase in the formation of extracellular matrix proteins with the participation of hepatic stellate cells (lipocytes or Ito cells) or their receptors and inhibition of the synthesis of proteolytic enzymes, which cause the destruction of the matrix (Ignotz RA and Massague J. (1986) J. Biol. Chem. 261:4337-4345). In the liver, TGF1 induces the synthesis of collagen and fibronectin in stellate cells of the liver (Weiner FR (1990) Hepatology 11:111-117). Is autoregulated by increasing its own synthesis by inducing him under the action of mRNA.

It was also found that TGF1 is associated with increased education2-macroglobulin, synthesized by hepatocytes and activated stellate cells of the liver. Upon binding with TGF1, causing its inactivation (Bachem MG (1994) Ann NY Acad. Sci. 737:421-424), A2-macroglobulin, as I believe, removes TGF1 and the expression of mRNA for procollagen type I and level in serum peptide of type III procollagen (Castilla A. et al. (1991) N. Engl. J. Med. 324:933-940).

The life expectancy of patients with liver cirrhosis is lower than normal, due to complications that arise during the course of the disease, such as portal hypertension and liver failure.

The effects of transforming growth factor TGF1 on the extracellular matrix

The interaction of TGF1 with the receptor cells produces:

- Activation of the synthesis of procollagen, fibronectin (Ignotz RA et al. (1987) J. Biol. Chem. 262:6443-6446) and related proteins, including membrane proteins that can interact with components of the intercellular matrix (Carter WG (1982) J. Biol. Chem. 257:13805-13815).

- Inhibition of the synthesis of proteolytic enzymes capable of causing the destruction of the matrix (Fukamizu H. and Grinnell F. (1990) Exp. Cell Res. 190:276-282).

- The stimulation of the synthesis of inhibitors of proteolytic enzymes (Fukamizu H. and Grinnell F. (1990) Exp. Cell Res. 190:276-282).

These effects lead to an increase in the interaction of cells with the extracellular matrix, which combined with more pronounced rearrangement of proteins from Kotor-4592). These data confirm that TGF1 participates in the processes of scarring (Fukamizu H. and Grinnell F. (1990) Exp. Cell Res. 190:276-282; Barnard JA et al. (1990) Biochim. Biophys. Acta 1032:79-87).

Peptides as inhibitors of the interaction of the ligand-receptor

There is the possibility of using small molecules, synthetic peptides, as analogues of molecules that are present in the body, in order to simulate their actions. Research conducted by Leste with TCS. (LeSateur), show the possibility of using cyclic analogs of nerve growth factor (NGF), imitatingring fragment, causing the binding of nerve growth factor with its receptor (LeSateur L. et al. (1996) Nature Biotechnology 14:1120-1122). It is also possible to use peptides as antagonists of these molecules, preventing the natural interaction of the protein with its receptor blocking participation peptide (La sarte JJ et al. (1994) J. Acquired Immune deficiency = MKD Sydromes 7:129-134; LeSateur et al. (1995) J. Biol. Chem. 270:6564-6569). Previous studies have shown the possibility of using synthetic peptides as inhibitors of the interaction of the ligand-receptor, even when recognizing the epitope is not continuous (Daniels AJ et al. (1995) Mol. Pharmacol. 48:425-432). Other research is and, belonging to the group of receptor type II, showed the possibility of using cyclic peptides as inhibitors of the interaction of TGF1 RII (Demetriou, M. et al. (1996) J. Biol. Chem. 271:12755-12761). In this cyclization is possible to obtain peptides having the structure similar to the structure that might exist in vivo.

DETAILED description of the INVENTION

In accordance with the above reasons, the authors of the present invention is believed that peptides derived from both TGF1 and its receptors, or proteins having the ability to bind to TGF1 could inhibit the action of TGF1. Thus, the authors of the present invention decided to consider this possibility.

Selection of peptides that need to synthesize

Peptides for synthesis were chosen in different ways, depending on, was whether they derived TGF1 or its derivatives receptors.

In the case of a sequence of TGF1, the peptides synthesized from 15 amino acids, which are included in the complete sequence of TGF1. Each peptide contained 10 amino acids podanie software developed in the laboratory of the authors of the present invention. One of the computer programs allowed to compare two amino acid sequences with the goal of predicting partially complementary regions. Other programs were also used, was it possible to predict the region of proteins that could be most affected on the basis of hydrophobicity and hydrophilicity of the amino acids constituting the amino acid sequence.

Synthesis of peptides

Peptides were synthesized by a solid phase method (Merrifield (1963) J. Am. Chem. Soc. 85:2149-54) using fluorenylmethoxycarbonyl (Fmoc) as a temporary protective group for the alpha-amino group (Atherton et al. (1989) Journal of the Chemical Society Perkins Transactions 1: 538-546). For the synthesis of small quantities of a large number of peptides used in the power synthesizer that allows you to simultaneously synthesize 96 peptides (Borras)-Cuesta et al. (1991) Biologi-cals 19: 187-190). To use the peptides were stored at a temperature of -80In the solid state.

Purification of peptides by high performance liquid chromatography (HPLC)

The synthesized peptides are analyzed and purified by high performance liquid chromatography (HPLC), using a systems-PakC18300, 15 μm, 8100 mm (Millipore Corp., Bedford, USA). The peptide was dissolved in 0.1% solution triperoxonane acid (TFA) in distilled water, up to a maximum concentration of 1 mg/ml Solution of peptide (100 μl) was injected into the column and elute with a gradient of water/acetonitrile (Fig.15) (Romil Ltd., Cambridge, USA), both in 0.1% solution of TFA at a flow rate of 1 ml/min Fractions containing the peptide, determined by their absorption at 220 nm and 280 nm (matrix photodiode detector. Waters 991, Millipore Corp., Bedford, USA).

For the purification of peptides using column Waters DeltaPakC18300, 15 μm, 25100 mm (Millipore Corp., Bedford, USA). The peptide is dissolved and injected (2 ml) in the same conditions as in the previous case, using the same gradient at a flow rate of 5 ml/min Fraction, which contains pure peptide is collected in the flask.

IN VITRO TESTS, the RESEARCH activity of the PEPTIDES

Cell line

Use line derived from lung epithelium mink, MV-1-Lu, (CCL-64, American Type Cell Culture, Virginia, USA). Cells are grown in flasks for crops area 162 cm2(Costar Corporation, Cambridge, USA) in an incubator at 37C and 5% CO2to merge. Use Rodi (FCS, Biological Industries, Kubbutz Beit Haemek, Israel), 10 mm N-2-hydroxyethylpiperazine-N'-2-econsultancy acid, HEPES (1 M HEPES Buffer, Bio-Whittaker, Verviers, Belgium) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).

Test for inhibition of cell growth MV-1-Lu

Cell line MV-1-Lu grown, as shown above, is removed from the bottom of the flask culture using 5 ml of a mixture of trypsin-EDTA (Biological Industries, Kubbitz Beit Haemek, Israel), resuspended in complete medium and centrifuged at 1500 rpm for 8 minutes. After centrifugation cells resuspended in complete medium to a concentration of 50,000 cells in 1 ml of the test take 10 ml of the cell suspension and distributed in 96-well flat-bottomed tablet for cell cultures by placing 100 μl in the wells (Costar Corporation, Cambridge, USA), and incubated overnight at 37C and 5% CO2that leads to the adhesion of cells to the bottom of the hole. By the end of this interval, peptides, want to explore make in RPMI to a final concentration of 200 μg/ml in the presence of TGF1 in RPMI at a concentration of 200 PG/ml (R&D Systems Europe Ltd., Abingdon, UK). The final concentration of fetal calf serum in the hole is 2.5%. After 24 hours incubation each well add 1 µci soderjaschegosya within 12 hours (Grubeck-Loebenstein Century et al. (1989) J. Clin. Invest. 83:764-770; Brennan FM et al. (1990) Clin. Exp. Immunol. 81:278-285).

After the end of the incubation period the cells are removed from the bottom of the hole, using a mixture of trypsin-EDTA and collected using a hand-held harvester cells (Titertek cell harvester, Skatron Instruments Inc., Sterling, USA), which destroys the cells, collecting DNA on nitrocellulose filters (Filter MAT 11731, Skatron Instruments Inc., Sterling, USA), where they perform fixation. The filters are placed individually in 5 ml polypropylene test tube, add 4 ml of scintillation fluid (Biogreen-11, Reactivos S. A. Scharlau, Barcelona, Spain). The activity of each tube count for 90 seconds usingLKB scintillation counter (Beta plate system, LKB, Uppsala, Sweden).

The study of inhibition of binding of transforming growth factor TGF1 with the receptor cells

Introduction selective labels in the receptors of the cells (introduction affine label)

Cell line MV-1-Lu is removed from the bottom flasks with culture, incubare them at 37C for 10 minutes with 10 ml of solution 1 (128 mm NaCl, 5 mm KCl, 25 mm 4-(2-hydroxyethyl)-1-piperazine-econsultant at pH 7.5, 5 mm glucose and 1 mm EDTA). Deleted thus cells resuspended in solution 2 (128 mm NaCl, 5 mm KCl, 50 mm 4-(2-hydroxyethyl)-1-pinjem when 1000g for 5 minutes. After centrifugation cells resuspended in solution 2 to a concentration of 106cells in 1 ml

From the resulting suspension of cells selected aliquots of 0.5 ml and placed in 24-well plates (Greider GmbH, Frickenhausen, Germany), add the peptides in 50 μl of a solution of a concentration of 0.8 mg/ml, and then the obtained product is incubated for 2 hours at 4With under stirring. Then add125I-TGF1 (2 µci) to a final concentration of PM 277.2 M. (recombinant125I-TGF1 person, 800-2200 CI/mmol, produced by Amersham Life Science, Buckinghamshire, UK) and then incubated for two hours at 4With under stirring.

After incubation, the cells are transferred into centrifuge tubes and centrifuged in the cold at HD within 1 minute. Then twice washed with a cold solution of 2 and resuspended in 0.5 ml of a cold solution of 2 with 5 ál of dimethyl-sulfoxide (DMSO 99.5% pure, produced by Sigma Chemical Co.", St. Louis, USA) and disuccinimidylsulfite (DSS, manufacturing firms "Pierce Chemical Co.", Rockford, USA), while setting a final concentration of 0.25 mm DSS. The reaction is stopped after 15 minutes by dilution, centrif the shape in 0.5 ml of Triton X-100 (produced by Bio-Rad Laboratories, Hercules, USA), 1% vol./about., containing 10 mm Tris at pH 7.0, 1 mm EDTA, 0.1 mm of phenylmethylsulfonyl, 1 μg/ml of pepstatin and 1 µg/ml leupeptin (produced by Sigma Chemical Co.", St. Louis, USA), and incubated for 40 minutes at 4C. the Fraction that is insoluble in the detergent, separated by centrifugation at 12000g for 15 minutes. The fraction that is soluble in the washing liquid (supernatant), and the fraction that is insoluble (residue), frozen at -20(Massague J. and Like C. (1985) J. Biol. Chem. 260:2636-2645).

Electrophoresis of proteins in polyacrylamide gel with dodecyl sulfate

Fractions, soluble and insoluble in the detergent used for analysis by electrophoresis in acrylamide gels/bisacrylamide with a concentration of 7.5% for 5-6 hours at a voltage of 220 volts.

Proteins visualize staining by the dye solution, Kumasi brilliant blue (comassie brilliant blue(produced by "Serva Feinbiochemica GmbH, Heidelberg, Germany) in methanol (50%), acetic acid (10%) and distilled water for 30 minutes. Subsequent washing carried out with a solution of methanol (50%), acetic acid (10%) and distilled water for at least 15 minutes for the first wash, and is to remove the background color.

Flow cytometry

Inhibition of binding of TGF1 with the receptor cells in the mediation of peptides measured by direct fluorescence. To do this, use fluorescent kit (Fluorokine rh TGF-biotin, R&D Systems Europe Ltd., Abingdon, UK). This technique is based on the ability of the biotinylated TGF1 to contact with the receptor cells in a special way and the subsequent interaction of Biotin with Avidya containing fluorescent label, so that the intensity of the signal will depend on the amount of TGF1, associated with the receptors of the cells.

Cell line MV-1-Lu grown in flasks with square 162 cm2remove, using raster 1 (described above) and resuspended in physiological solution for subsequent centrifugation at 500g for 5 minutes. After centrifugation the cells again resuspended in physiological solution at a cell concentration of 4106cells in 1 ml 25 ál of this cell suspension added to borosilicate tubes size 1275 mm, which are then added to the peptide, which should be investigated in 40 μl of RPMI medium 1640, 1. To control the specificity add 10 ál of the biotinylated reagent set, 10 ál of biotinylated TGF1 added as a positive control, and 20 μl of anti-TGF1 blocking antibody added as a negative control. Saline and added to all samples to obtain the total volume of 75 μl. All tubes are incubated in the dark for 1 hour at 4C.

At the end of the incubation period, add 10 ál avidin containing fluorescent label, incubated for 30 minutes in the dark at 4With, then add 2 ml of wash solution (RDF1), then centrifuged at 500g for 6 minutes. Sediment cells resuspended in 0.2 ml cold PBS for cytometry (FACScan, Becton Dickinson Immummocytometry Systems, California, USA). This technique allows to measure the fluorescence emitted by each cell in those cases, when the laser beam falls on it, using a computer program (LisysII, Becton Dickinson Immunocytometry Systems, California, USA). In Fig.16 shows a typical image obtained through analysis by flow cytometry.

For cmd src="https://img.russianpatents.com/chr/8243.gif">the positive controlused to delimit the areas corresponding to the labeled cells that are associated with TGF1-Biotin (M2), and areas that correspond to unlabeled cells (M1). As soon as the boundaries of areas is set, counting the percentage of cells contained in each of them. The same procedure is performed with the data obtained in those cases where the peptides are incubated with TGF1-Biotin or cells, depending on if the peptides of the receptor or TGF1 respectively. Using these data, calculate the magnitude of the inhibition percentage for each of the peptides using the following formula: 100-((M2 Peptide - M2 "Negative control")100/(M2 "Positive control" - M2 "Negative control")).

EXPERIMENTS IN VIVO. EXPERIMENTAL MODEL of FIBROSIS

Used white rats-males (albino Wistar rats) of the same age (5 weeks ± 1.5 weeks) in order to receive the group is homogeneous in age and initial weight. Throughout the experiment, animals were kept at constant temperature (22(C) ini (NA) caused by inhalation tetrachloride hydrogen for 11 weeks, twice a week (Novoa JM et al. (1976) Patologia IX:223-240; Camps, J. et al. (1987) Gastroenterology 93:498-505). The shutter speed under the action of CCL4carried out by passing compressed air at a speed of 3 l/min through a wash bottle for gas. Originally used a shutter speed equal to one minute, then increasing the shutter speed for one minute per week, reaching 4 minutes on the fourth week. During the fifth week CCL4did not enter, and at the sixth week started again with the shutter speed set to 5 minutes. This time was maintained until week 11. To water were added to 400 mg/ml phenobarbital (Luminal, Bayer, Leverkusen, Germany), starting one week before the start of the exposure under the action of CCL4and until the end of the experiment.

Before treatment was left for one week, during which the rat did not enter CCL4. During treatment, the rats were injected weekly dose of CCL4as indicated (Fig.2).

The distribution of animals in groups

Before induction of liver cirrhosis animals were divided into 4 groups.

A control group of healthy animals (From): animals that were not subjected to the process of fibrosis.

A control group of healthy animals, the cat is in the last 3 weeks (coinciding in time with the time of treatment, rats in group Tto2).

Control group 1 animals subjected to cirrhosis (Ci1): animals were subjected to induction of the process of fibrosis by inhalation CCL4two times a week. To achieve the fifth week of these animals were divided into 2 groups:

Control group 1 animals subjected to cirrhosis (Ci1): animals that continued to be subjected to the process of inducing fibrosis until week 11 and which have not entered the peptide R. These animals during the induction through the day put the serum in saline solution (weeks 5 to 11).

Group 1 animals subjected to cirrhosis and treatment (Tto1): animals, which were injected peptide R obtained from the sequence of the receptor type III, through the day, during the process of inducing fibrosis, week from 5 to 11. Control group 2 animals subjected to cirrhosis (CI2): animals that continued to be subjected to the process of inducing fibrosis, and not receiving any peptide R or serum in saline solution. This group reached week 11 was divided into two other groups.

Control group 2 animals subjected to cirrhosis (Ci2): animals subjected to cirrhosis who did not receive any treatment, remained as the 13 to 15).

Group 2 animals received treatment (Tto2): animals subjected to cirrhosis treated with a peptide derived from the sequence of the receptor type III (R) for 3 weeks (weeks 13 to 15).

Treatment animals

Group Tro1: these animals were subjected to treatment during the process of fibrosis. The treatment with peptide began at the fifth week (before curing under the action of CCL4within 5 minutes) and continued until the end of the eleventh week of the process of induction of cirrhosis.

Group Tto2: these animals were subjected to treatment after completion of the process of induction of cirrhosis (11 weeks). Treatment started one week after the last inhalation CCL4and continued for 21 days.

Before treatment and after its completion in all animals treated with the peptide, took the blood. The peptide was administered by subcutaneous injection in the abdominal region at a dose of 70 μg per animal in 500 μl of saline.

Sacrificare animals and excision of the liver

After injection of the peptide animals, as in the case of the model in rats, and in the case of the model in mice, animals were sacrificially by decapitation, after taking their blood from ZAPADNOGO plexus with the cap is the dominant solution formalin for subsequent histological research. Other fragments were placed in cryoprobes, which was placed in liquid nitrogen and then kept at -80C. Anatomopathological study of the liver

For histological studies used parts of the liver, pre-fixed in formalin for at least 24 hours, then placed in ethanol (70%).

After dehydration, the sections were closed up in paraffin blocks. From the obtained blocks were prepared serial sections with a thickness of 3 μm using the disk microtome of Leitza and steel blades. Before staining, the slices were deparaffinization in xylene (AnalaR, BDH, Poole, UK) for 15 minutes, after heating them at 60With in a heating Cabinet for 15 minutes and then was hydrational sequential treatments by decreasing alcohol concentrations: 100%, 96%, 80% and 70%, and finally with water. Used the following dyes:

The hematoxylin-eosin.

- Trichromacy Masson (Locquin M. and Langeron, (1985) in Manual de Microscopia Ed. Labor S. A. Barcelona): Uses a specific dye for collagen proteins (green light).

- Sirius red dye that is specific for collagen.

Confirmation of liver fibrosis: analysis images

For image analysis the floor of the Tokyo, Japan), which received photographs of various parts of the drug. Six plots of each of the drugs, selected randomly, were stained by the dye Sirius red. Different acquired images were analyzed using computer software (Visilog 4.1.5, Noesis, Orsay, France), which allowed to calculate the area of sites subjected to fibrosis, and the total area of the drug. From these data for each plot was calculated the rate of fibrosis (parcel size, subject to fibrosis/total area). In order to use this program, it was necessary to modify the system to obtain images through the use of polarizing filters (Olympus U-POT, Tokyo, Japan), and light green filters (Olympus IF550, Tokyo, Japan), which will automate the process of analyzing the samples.

Determination of collagen in tissue sections treated with paraffin, a thickness of 14 μm

Slices with a thickness of 14 μm were used for analysis by this method were obtained in the same manner as described above, the slice thickness of 3 μm. These sections were subjected to deparaffinizing within 12 hours in xylene. After the paraffin was removed, samples were hydrational serial transmission through has spirtually prior to staining in a solution of 160 mg of green dye Fast Green FCF (Fluka Chemica-BioChemica, Buchs, Switzerland) in 160 ml of a saturated solution of picric acid (Merck, Darmstadt, Germany) for 15 minutes in the dark. The samples were washed by immersing up until they will not cease to paint the washing water. After the excess dye was removed, the samples were stained for 30 minutes in the dark in a solution of red dye 160 mg Direct Red 80 (Fluka Chemica-BioChemica, Buchs, Switzerland) and 64 mg of green dye Fast Green, both dye in 160 ml of a saturated solution of picric acid. The samples were again washed, immersed in water up until they will not cease to paint wash water, and then the samples were removed from the slides, scraping a small spatula. Taken thus, the sections were placed in separate tubes containing 3 ml of 0.1 n NaOH solution (Quimon, Montplet&Esteban S. A., Barcelona, Spain) and methanol (1:1). Selected aliquots from different tubes for spectrophotometric studies (Lambda 2 UV/VIS spectrophotometer, Perkin-Elmer, Norwalk, USA) at a wavelength of 540 nm and 630 nm, using as the reference solution an aliquot of a mixture of 0.1 G. of NaOH and methanol (Lopez de Leon A. and Rojkind (1985) Histochem. Cytochem. 33:737-743; Gaudio E. et al. (1993) Int. J. Exp. Path. 74:463-469).

According to the English OU in house and researcher. (1993) Int. J. Exp. Path. 74:463-469), to determine the amount of collagen and total protein was used following the obtained in vivo, were subjected to statistical analysis. Compliance with the quantitative variables with normal distribution was checked using the test Shapiro-Wilks.

In cases where data did not conform to a normal distribution, applied nonparametric statistical analysis. Comparison between groups was performed using the criterion of Kruskal-Wallis (Well-Wallis) followed by a comparison with the U criterion by Mann-Whitney (Mann-Whitney). The obtained data are presented graphically as rectangles, with the image of the average value of the received data (bold line inside each rectangle) along with the interquartile breadth (height of the rectangle), where the "whiskers" of each of the rectangles represent the largest and smallest value that is related to the interquartile latitude.

The relationship between variables was examined using Fisher's exact test. To determine the independence of these variables used logistic regression.

The values of R equal to or less than 0.05 are considered as statistically significant.

All of these methods statistical analysis was performed using the program SPSS for Windows version 6.1.3.

INHIBITION of the activity of the TRANSFORMING FACTOR is almyroudis growth factor TGF1 is a cytokine, which can inhibit growth of the cell line MV-1-Lu in vitro (Grubeck-Loebenstein Century. et al. (1989) J. Clin. Invest. 83:764-770; Brennan FM et al. (1990) Clin. Exp. Immunol. 81:278-285), so this line was used to study the blocking action of peptides on TGF1. After combining different environment, cells and thymidine authors of the present invention investigated the effect of different concentrations of TGF1 on the binding of [methyl-3H] thymidine cell line MV-1-Lu in the culture, in order to determine the most suitable conditions for the test. These conditions are shown in Fig.3.

After it was determined the optimal concentration of the cell line MV-1-Lu (5000 cells per well) and the lowest concentration of TGF1, can cause inhibition equal to about 90% (200 PG/ml, Fig.18), the inhibitory effect of synthetic peptides was studied at a concentration of 200 μg/ml Inhibition of the activity of transforming growth factor TGF1 in vitro with synthetic peptides

Synthetic peptides that are potential inhibitors of the activity of TGF1, is chosen in such a way as shown above in the section "src="https://img.russianpatents.com/chr/946.gif">1, and peptides derived from the TGF1) were investigated using cell line MV-1-Lu. The peptides were dissolved in buffered RPMI medium containing no fetal calf serum, and used the following method:

peptides belonging to the sequence of the receptor or complementary high hydrophilicity TGF1, incubated for 30 minutes in the presence of the cytokine and connect with the culture of cells. Peptides derived from the sequence of TGF1, is added to the cell culture before the addition of TGF1, because they interact with receptors on the cell surface.

This incubation is carried out in 100 µl of the same medium, which was used when adding cells. Active peptides affect cell growth to a greater or lesser extent depending on their ability to inhibit TGF1.

The inhibition activity of TGF1 peptides derived from TGF1

In the first stage were synthesized peptides with overlapping sequences derived from TGF1. These peptides (tabldt thus linking natural TGF1 with these receptors.

In Fig.4 shows the inhibitory activity of the peptides shown in table 6, on the activity of TGF1. Because TGF1 inhibits growth of the cell line MV-1-Lu, inhibition of this cytokine peptides leads to changes in the growth of the cell line MV-1-Lu.

As you can see from the data shown in Fig.4, the peptide R obtained from a sequence of TGF1, is one of those peptides that have shown the greatest inhibitory activity against TGF1. For a more detailed study of the inhibitory effect of peptide R was conducted to investigate the effect of concentration of peptide for inhibition of activity of the cytokine, as described below.

The influence of the dose of the peptide to inhibit the activity of TGF1 under the action of peptide n

Investigated the effect of the concentration of peptide R on the inhibition activity of TGF1 under the action of R. Since this peptide is not very soluble in the test environment, the original solution or suspension was prepared at a nominal concentration of peptide (i.e. a concentration that would be achieved if the peptide dissolved in orestano in studies of inhibition.

In Fig.5 shows the evaluation of the inhibitory effect of the nominal concentration of the peptide before and after filtering. You can see that the peptide R after the filter and without filter has the same activity.

After data were obtained for peptide R, the authors of the present invention decided to extend the peptide, both from the N-Terminus, and the C-end, and to explore the impact of this on the activity of the peptide. In addition, changes were made in the amino acid sequence of the peptide in order to increase its solubility and to determine the value of two cysteine residues in its amino acid sequence for inhibiting the activity of TGF1. The structure of the synthesized peptides are shown in table 3.

In Fig.6 shows the results of inhibiting the activity of TGF1 under the action of the peptides listed in table 3.

From the data shown in Fig.6, it can be seen that the peptide R29 is active. This peptide includes the previously studied peptide R and contains 9 additional amino acids from N-Terminus (Fig.4). Research conducted by a number of authors (Quian SW et al. (1992) Proc. Natl. Acad. Sci. 89:6290-6294) and by Burmester JK et al. (1993) Proc. Natl. Acad. Sci. 90:8628-8632) with the which is necessary for the activity of this cytokine (amino acids 40 to 82 in the sequence of the Mature TGF full1). It was assumed that the peptide R29 (amino acids 34 to 56 in the sequence of the Mature TGF full1), more extensive than the peptide R (amino acids 43 to 56), can acquire a three-dimensional structure, more similar in action to the structure of the TGF1. For this reason, the peptide R29 used for studies of binding to receptors of the cells, based on the introduction of affinity tags.

The study of inhibition of binding of TGF1 with its receptors in the presence of peptide R29 (introduction affine label)

Peptide R29 derived from the amino acid sequence of TGF1, used in studies related to the introduction of affinity labels to confirm the ability of this peptide to inhibit the binding of TGF1 with its corresponding receptor cells (Materials and methods).

Due to the different activity used parties125I-TGF1, the concentration of peptide used in the study, in each case brought into line with that used by the party125I-TGF1. The results of these studies are showing is necessary to block binding125I-TGF1 with the receptor cells.

The inhibition activity of TGF1 peptides derived from the amino acid sequence of the receptor type III rats

With the aim of finding new peptides that are inhibitors of the activity of transforming growth factor TGF1, were synthesized peptides derived from the receptor type III rats. Based on the structure of regions of amino acid sequences were selected peptides, which, as expected, are complementary blocks of amino acids in the amino acid sequence of TGF1. The authors present invention hoped that these peptides are able to bind with the free TGF1 binding to it and preventing the binding of TGF1 with the receptor cells.

Were other peptides synthesized by overlapping 10 amino acids, and "covering" part of the extracellular region of the receptor type III (amino acids 45 to 410). It was reported that there is a soluble receptor type III, which corresponds to the extracellular region of the receptor, this area cut out from the membrane and it acts as the 5-795). In later works described two possible site responsible for binding to transforming growth factor TGF1, one of which is localized at the N-Terminus of the receptor (Lopez-Casillas et al. (1994) J. Cell Biol. 124:557-568), and the other is located in the area closest to the membrane in the direction From the end of the Fukushima D. et al. (1993) J. Biol. Chem. 268:22710-22715; Pepin MC et al. (1995) FEBS Lett 377:368-372). Based on this, we synthesized peptides of the extracellular region of the receptor, with the assumption that these peptides might be able to bind in circulation TGF1.

The peptides listed in table 4, were investigated for their ability to block the inhibition of growth of the cell line MV-1-Lu. Because TGF1 is able to inhibit the growth of cells that line, inhibition of transforming growth factor TGF1 peptides could return the cells ability to grow. These studies are shown in Fig. from 9 to 12.

As can be seen in Fig. 9 to 12, there are various peptides, which are able to inhibit growth of the cell line MV-1-Lu to a greater or lesser extent, but only peptide R capable of almost completely and the s study of the effect of different concentrations of the peptide on the activity of TGF1 at a fixed concentration of TGF1, equal to 200 PG/ml.

The study of the influence of dose on the inhibition activity of TGF1 under the action of peptide N

Investigated the effect of the concentration of peptide R on the inhibition activity of TGF1. Due to the low solubility of this peptide was prepared by the original solution of the nominal concentration of peptide in the same manner as in the case of peptide R, took aliquots of this solution and was filtered or even used directly in studies of inhibition.

In Fig.13 shows the inhibitory activity of the peptide used in the nominal concentrations before and after filtration. You can see that when using the filtrate peptide R not observed measurable inhibition activity.

Confirming the ability of the peptide R to inhibit the activity of TGF1 so that the inhibition depends on the dose of the peptide, the authors of the present invention moved to the synthesis of new peptides based on the amino acid sequence of the peptide R, with the aim of trying to improve the solubility and, therefore, increase the activity of the peptide when used in menow (R) was equivalent to the peptide R. Another peptide (R), similar to the peptide R receptor type III rats, which also showed activity. These new peptides are shown in table 5.

The results of the research activity of the peptides listed in table 5, shown in Fig.14.

The study of the influence of dose inhibition of TGF1 under the action of peptide n

Conducted a study of the influence of dose on the magnitude of the achieved effect of inhibition under the action of peptide R obtained from the sequence of the receptor type III person, in order to determine whether the activity of this peptide concentration (Fig.15). You can see that the activity of the peptide decreases with decreasing concentrations of peptide used in the tests.

The study of inhibition of binding of TGF1 with its receptors under the influence of peptide R (introduction affine label)

Peptide R obtained from the sequence of the receptor type III person, used in studies related to the introduction of affinity labels, to confirm the ability of this peptide to inhibit the binding of TGF1 with its cellular receptors (Materials and methods).

Due to the different activity used parties125I-TGF1. The results of these studies are shown in Fig.15.

After confirmation of inhibition of binding of transforming growth factor TGF1 with its corresponding receptors under the influence of peptide R, further studies were performed in order to attirbute" peptide R. It was found that the peptide loses its activity at a concentration equal to 2105molar concentrations125I-TGF1.

Inhibition of transforming growth factor TGF1 under the action of peptides derived from other proteins that bind transforming growth factor TGF1 and according to the assumptions, which are complementary transforming growth factor TGF1

In this series of synthesized peptides, the structure of which is shown in table 6, derived from proteins capable of binding to TGF1.

In Fig. 17 and 18 shows the inhibitory activity of the peptides listed in table 6.

As can be seen in figures 17 and 18, only the peptide P150 shows activity greater than 50%. However, it was found that the peptides R and activity in the conditions of this study.

Measurement by flow cytometry inhibitory effect of synthetic peptides on the binding of transforming growth factor TGF1 with its receptors

The peptides produced from the previous syntheses, as the peptides that were synthesized from the sequence of TGF1, and the peptides that were synthesized from receptor type III, used for measurement by flow cytometry for their ability of inhibiting the binding of TGF1 with the receptor cells. When conducting these studies, cells were incubated with peptide before addition of avidin-FITS (Materials and methods). Then measured the fluorescence intensity avidin-FITS: the intensity of fluorescence is directly proportional to the amount of TGF1, associated with the cells, and is inversely proportional to the activity of the peptide. The results obtained for the peptides of interest, shown in Fig.19 and in table 7.

INHIBITION of the activity of TRANSFORMING GROWTH FACTOR TGF1 IN VIVO

Peptide R obtained from the sequence of the receptor type III human activity for a specified peptide was confirmed bioinactive peptide for induction of experimental cirrhosis under the influence of CCL4on the model in rats. Experiment simulating cirrhosis in rats of Wistar line

In this model of liver cirrhosis induced by inhalation of carbon tetrachloride for 11 weeks, twice a week (Lopez Novoa JM et al. (1976) Patologia IX:223-240; Camps, J. et al. (1987) Gastroenterology 93:498-505), as described in "Materials and methods".

Peptide R was introduced in accordance with two methods:

1. Method 1: the peptide was injected intraperitoneally every other day during induction of cirrhosis (11 weeks). Fig. 20 and 21.

2. Method 2: the peptide was injected intraperitoneally every other day for 3 weeks, after cirrhosis has developed, namely, 12 weeks after the start of the process of induction of cirrhosis. Fig. 22 and 23.

Collagen formation using the first and second methods were determined using two methods:

In Fig. 36 and 38 shows the total number of newly formed collagen, as measured by staining of liver slices (two per animal) dyes green Fast Green and red Direct Red, with subsequent elution staining and spectrophotometric detection (materials and methods) (Lopez de Leon A. and Rojkind (1985) Histochem. Cytochem. 33:737-743; Gaudio E. et al. (1993) Int. J. Exp. Path. 74:463-469).

In Fig. 21 and 23 show the number of newly formed collagen, measured by the method of the Academy of Sciences of the OPA (materials and methods).

As can be seen in Fig. 20, when analyzing the ratio between collagen and total protein observed statistically significant differences (P<0,05) between the groups of rats treated with the peptide R (Tto1), and a control group of rats subjected to liver cirrhosis (Ci1). In Fig.37, in the case, when considering the area of sites subjected to fibrosis, showing statistically significant differences (P<0,001) between the groups of rats treated with the peptide R (Tto1), and a control group of rats subjected to liver cirrhosis (Cl1).

As can be seen in Fig. 22 and 23, which shows the results related to rats treated with the peptide after cirrhosis has developed, the differences between the group of rats treated with the peptide R (Tto2), and a group of rats not treated with the peptide (Ci2), are not statistically significant in the cases when for the assessment of fibrosis you are using any of these two methods.

A comparison was made between the two methods used to determine the amount of collagen, using regression analysis, to test the randomness of the selection fields for research in each of the drugs and, therefore, to check the accuracy of image analysis, Fig. 24 and 25.

CAA is statistically significant (F<0,001). This confirms that the choice of images for the study was carried out quite by accident and, thus, confirms the reliability of the data obtained by means of image analysis.

In Fig. 26 and 27 shows the images obtained by the optical microscope preparations of liver stained with the dye Sirius red when the magnification of X10, obtained from the liver of rats treated with the peptide during the induction process cirrhosis (Ci1and Tto1).

Image shown in Fig. 26, were obtained without using any filter.

In Fig. 27 shows the images after they have been modified for research using special computer programs. These modifications consist in the use of two filters, one polarizing filter, and a second green color filter to improve image quality and facilitate their assessment.

Fig. 26 and 27 can detect the differences between the images of the preparations obtained from rats subjected to cirrhosis (Ci1), and rats treated with the peptide R (Tto1).

Differences in the effectiveness of methods 1 and 2 can be a consequence of the fact that the production of TGF

When comparing groups of rats not treated with the peptide, at the end of the process of induction of cirrhosis (CI1), with a group of rats not treated with the peptide subjected to cirrhosis, in the fourth week after the completion of the process of induction of cirrhosis (Ci2), the authors present invention have found that there are statistically significant differences (P=0,016) between the two groups (Fig.28), which may mean that there is a partial regression of cirrhosis in those cases, when removing the effect of the agent that causes cirrhosis that has been emphasized by several authors (Szende-In et al. (1992) In Vivo 6:355-361; Columbano A (1996) Carcinogenesis 17:395-400).

These differences in the effectiveness of the two techniques may also be the result of methods, because according to method 2 animals subjected to treatment only for 3 weeks every other day, while according to the method 1 animals subjected to treatment for longer periods of time (7 weeks, every other day).

The results obtained indicate that it is possible to inhibit the activity transtv, derived from different proteins. In the future would be of great interest to try to improve the biological activity of these peptides. This could be done by systematically replacing each of the amino acids included in the sequence, other 19. Once the peptide with higher activity is obtained, it is necessary to obtain their mimotope (McConnell-SJ (1994) Gene 151:115-118; Steward-MW (1995) J. Virol. 69:7668-7673), in order to increase life expectancy inhibitory agent in the body.

DESCRIPTION of FIGURES

Fig.1. Inhibition of binding of TGF1 cell line MV-1-Lu under the action of peptide R measured by flow cytometry. The image As obtained in the study of cells, incubated with biotinylated TGF1 and grown with avidin-FITC. The image obtained in the study of cells, incubated with avidin-TCF1 without prior addition of TGF1. The image obtained in the study of cells incubated with TGF1 and pre-incubated with peptide R at a concentration of 0.42 μg/ml, and grown with avidin-FITC. On the x-axis indicated esmera the value of the fluorescence intensity. It also shows the areas corresponding to the cells labeled TGF1-Biotin and avidin-FITC (M2), and cells that do not contain a label (M1).

Fig.2. Schematic representation of the process of cirrhosis under the action of CCL4. Black arrows show when the rats were introduced two weekly dose of CCL4and black arrows with a dotted line to show when the rats were administered a single weekly dose. Gray arrows indicate the introduction of peptide R. A: control group of healthy rats; In: control group of healthy rats + R, B1: peptide-70 µg/day; With: a group of rats exposed to cirrhosis; C1with a saline solution; C2with the peptide in the amount of 70 mg/day; D: group of rats exposed to cirrhosis under the influence of CCL4+phenobarbital; D1plus saline; D2 plus peptide in the amount of 70 mg/day.

Fig.3. The effect of TGF1 on the growth of cell line MV-1-Lu. Cells were grown at a density of 5000 cells per well and at the indicated concentrations of TGF1, PG/ml On the x-axis: concentration of TGF1 (PG/ml); y-axis: number of cycles per minute (S. R. m.).

Fig.4. The percentage inhibition of TGF1 (200 PG/ml) under the ml Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.5. The percentage inhibition of TGF1 (200 PG/ml) in the presence of peptide R in various nominal concentrations, with filtration (and without filtering ().

Fig.6. The percentage inhibition of TGF1 (200 PG/ml) under the action of peptides derived from TGF1. All peptides were tested at a concentration of 200 μg/ml Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.7. The autoradiograph obtained in the study of receptor transforming growth factor TGF1 with the introduction of affinity tags. Track C1: effect of incubation of cells with125I-TGF1 at a concentration of 0.16 μm, which corresponds to the radioactivity of 0.3 µci (positive control). Track C2: effect of pre-incubation of cells with nonradioactive TGF1 at a concentration of 10 times Bo is Noah incubation was carried out with the peptide R29 at a concentration of 106times higher than the molar concentration of125I-TGF1. You can see that the observed inhibition of binding125I-TGF1 with the receptors of the cells I, II and III types as under the action of peptide R29, and under the action of non-radioactive TGF1.

Fig.8. The autoradiograph obtained in the study of receptors TGF1 with the introduction of affinity tags. Paths from C1 to C6: the influence of pre-incubation with cell line MV-1-Lu with various concentrations of peptide R29 (106, 8105, 6105, 4105, 2l05and 105from molar concentrations125I-TGF1, respectively), before adding the125I-TGF1. Track 7: the effect of pre-incubation of cell line MV-1-Lu with unlabeled TGF1 (102from molar concentrations125I-TGF1) before adding the125I-TGF1 (negative control). Track C8: effect of pre-incubation of cell line MV-1-Lu125I-TGF1 in the concentration of the).

Fig.9. The percentage inhibition of TGF1 (200 PG/ml) under the action of peptides derived from the receptor, presumably being complementary areas of TGF1. All peptides were tested at a concentration of 200 μg/ml Inhibition activity of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.10. The percentage inhibition of TGF1 (200 PG/ml) under the action of overlapping peptides derived from the extracellular region of the receptor type III. All peptides were tested at a concentration of 200 μg/ml Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.11. The percentage inhibition of TGF1 (200 PG/ml) under the action of overlapping peptides derived from the extracellular region of the receptor type III. All peptides were tested at a concentration of 200 μg/ml Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.13. The percentage inhibition of TGF1 (200 PG/ml) in the presence of peptide R in various nominal concentrations, with filtration (and without filtering ().

Fig.14. The percentage inhibition of TGF1 (200 PG/ml) under the action of the overlapping peptides, obtained by modification of the peptide R (R to R), and peptides derived from the receptor type III person (R and R). All peptides were tested at a concentration of 200 μg/ml Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.15. The percentage inhibition activity of TGF1 (200 PG/ml) in the presence of peptide R in various nominal concentrations, without filtering.

Fig.16. The autoradiograph obtained in the study of receptors tranformirovanie in the presence of peptide R at a concentration of 106times higher than the molar concentration of125I-TGF1. Track C2 and C3: effect of pre-incubation of cells with nonradioactive TGF1, present at a concentration 10 times higher than the concentration of125I-TGF1 (negative control). Track C4 and C5: effect of incubation of cells with125I-TGF1 in a concentration of 0.1 μm, which corresponds to the activity of 0.2 µci (positive control). You can see that the observed inhibition of binding125I-TGF1 with the receptors of the cells under the action of peptide R, and under the action of non-radioactive TGF1.

Fig.17. The percentage inhibition of TGF1 (200 PG/ml) under the action of peptides derived from the receptor type II person (S), fetuin (R to R) and endoglin (from P150 to R). All peptides were tested at a concentration of 200 μg/ml Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.18. The percentage inhibition of TGF1 (200 PG/ml) under the action of the peptide is Inhibition of TGF1, 100% corresponds to the growth of the cell line MV-1-Lu, which is observed in the absence of TGF1.

Fig.19. The percentage inhibition of binding of TGF1 cell line MV-1-Lu under the action of various synthetic peptides. Inhibition was studied by measuring the percentage of labeled cells (emit fluorescence) and unlabeled cells (not emit fluorescence) for each of the peptides (materials and methods).

Fig.20. The impact of the introduction of peptide R on collagen synthesis during experimental induction of cirrhosis under the influence of CCL4. The ratio of collagen and total protein are shown on the ordinate axis. The abscissa shows the different groups of rats: With=the group of healthy rats;+R=group of healthy rats treated with the peptide R; Tto1=the group of rats subjected to liver cirrhosis under the influence of CCL4and treated with the peptide R during this period in a day; Ci1=the group of rats subjected to induction of cirrhosis under the action of CCL4for 11 weeks and not treated with the peptide R.

Fig.21. The impact of the introduction of peptide R on collagen synthesis during experimental induction of cirrhosis under the influence of CCL4drug colored dye Sirius red. The abscissa shows the different groups of rats: With=the group of healthy rats;+R=group of healthy rats treated with the peptide; Tto1=the group of rats subjected to liver cirrhosis under the influence of CCL4, which was introduced peptide R every other day during this period and Ci1=the group of rats subjected to induction of cirrhosis under the influence of CCL4for 11 weeks and not treated with the peptide R.

Fig.22. The impact of the introduction of peptide R on collagen synthesis after being induced cirrhosis under the influence of CCL4. On the axis of ordinate shows the ratio of collagen and total protein. The abscissa shows the different groups of rats: From=healthy rats;+R=healthy rats treated with the peptide; Tto2=rats subjected to cirrhosis under the influence of CCL4, which was introduced peptide R through the day at the end of this period, and Ci2= rats subjected to induction of cirrhosis under the influence of CCL4for 11 weeks and not treated with the peptide R.

Fig.23. The impact of the introduction of peptide R on collagen synthesis after being induced cirrhosis under the influence of CCl4. On the axis of ordinate shows the ratio of areas prone to cirrhosis of the total area of tissue is Ted; Tto2=rats subjected to cirrhosis under the influence of CCL4, which was introduced peptide R through the day at the end of this period, and Ci2=rats subjected to induction of cirrhosis under the influence of CCL4for 11 weeks and not treated with the peptide R.

Fig.24. The comparison of data relating to the amount of collagen and square areas prone to fibrosis, received two methods used. The abscissa shows the ratio of the areas prone to fibrosis, to the total area obtained by the method of image analysis. The ordinate shows the ratio of the quantity of collagen, mg, to the amount of total protein, mg, obtained by the method of spectrophotometric analysis of sections of liver stained with the dye Direct Red and Fast Green. Given R2. (F0,001).

Fig.25. The comparison of data relating to the amount of collagen and square areas prone to fibrosis, received two methods used for samples obtained at the end of the implementation method 2. The abscissa shows the ratio of the areas prone to fibrosis, to the total area obtained by the method of image analysis. The ordinate shows the ratio of the number of mechani, colored dyes Direct Red and Fast Green. Given R2. (F0,001).

Fig.26. The image, which correspond to the 24 areas of drugs rat liver stained with the dye Sirius red, obtained by optical microscopy (10). Rats subjected to cirrhosis (Ci1), at the end of the induction of cirrhosis under the influence of CCL4and rats subjected to cirrhosis treated with the peptide R (Tto1in the process of induction of cirrhosis under the influence CC14. Of the products obtained for each animal taken different areas (R=rat, and s=the area).

Fig.27. The image, which correspond to the 24 areas of drugs rat liver stained with the dye Sirius red, obtained by optical microscopy (10). Rats subjected to cirrhosis (Ci1), at the end of the induction of cirrhosis under the influence of CCL4and rats subjected to cirrhosis treated with the peptide R (Tto1in the process of induction of cirrhosis under the influence of CCL4. Of the products obtained for each animal taken different areas (R=rat, and s=area). In order to detect collagen fibers used polarized light and the green is tositsa to rats, subjected to cirrhosis, at the end of 12 weeks of induction of cirrhosis under the influence of CCL4Ci2refers to rats subjected to cirrhosis, in week 4, after completion of the process of induction of cirrhosis. P=0,016. Y-axis: parcel size, subject to fibrosis/total area.

Claims

1. Peptides, which are antagonists of the binding of transforming growth factor TGF1 with its receptors in the body, characterized in that they are synthetic peptides with the sequence containing from 2 to 15 amino acids that are identical or similar to amino acids natural transforming growth factor TGF1 and/or its receptors.

2. Peptides under item 1, characterized in that they contain the amino acid sequence of SEQ ID NO: 3.

3. Peptides under item 1, characterized in that they contain the amino acid sequence of SEQ ID NO: 4.

4. Peptides under item 1, characterized in that they contain the amino acid sequence of SEQ ID NO: 5.

5. Peptides under item 1, characterized in that they contain the amino acid sequence of SEQ ID NO: 6.

6. Peptides under item 1, characterized in that they contain Amin is the sequence of SEQ ID NO: 8.

8. Peptides under item 1, characterized in that they contain the amino acid sequence of SEQ ID NO: 9.

9. Peptides on PP.1-8 for use as active compounds (active compounds) for the preparation of compositions used for liver diseases.

10. The peptides according to p. 9 for use as active compounds (active compounds) for the preparation of compositions used in the liver fibrosis (cirrhosis).

 

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