MXPA01005122A - TGF&bgr;1 INHIBITOR PEPTIDES - Google Patents

Abstract

Antagonists synthetic peptides which are obtained from TGF&bgr;1 or from its receptors in the organism. It is possible to use either the peptides themselves or the genic sequences which code them and the recombinant systems which express them for the fabrication of compositions used in the treatment of hepatic diseases and more particularly in cases of fibrosis. These compositions may optionally include mimotopes of said active peptides.

Description

"PEPTIDOS INHIBITORES DE TGFBETA1 ' DESCRIPTION OF THE STATE OF THE ART The control of cell growth is regulated by different proteins from the group of growth factors (Schalch DS et al (1979) Endocrinology 104: 1143-1151). Among the most important growth factors involved in cell development, capable of acting in an autocrine and paracrine manner, are the transforming growth factors (TGF) (Braun L. et al. (1988) Cell Bi'ol 85: 1539-1543; Lyons RM and Moses HL (1990) Eur. J. Biochem. 187: 467-473). The term TGF was used, for the first time, to describe the activity produced by a cell line transformed with the murine sarcoma virus (deLarco JE and To-daro GJ (1978) Proc. Nati. Acad. Sci. 75: 4001-4005; Mizel SB et al (1980) Proc. Nati, Acad. Sci. 77: 2205-2208). The supernatant of these cells was able to induce the normal growth, in soft agar, of cells that need a solid support to grow. More specific studies revealed two classes of TGF, which were called TGFa and TGFβ, which in turn encompass families of related proteins. The TGFß family consists of 5 iso- REF: 129561 forms (Brand T. and Schneider MD (1995) J. Mol.Cell Cardiol.27: 5-18) of 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 proteins, purified from the same species, have shown a high degree of identity between their sequences (Table 1). Table 1. Homology between the different types of TGFßs. TGFβ1, TGFβ2 and TGFβ3 from humans, TGFβ4 from chicken and TGFβ5 from frog. (Roberts AB and Sporn MB, 1990). % of TGFßl TGFß2 TGFβ3 TGFβ4 TGFβ5 ta.t? fl? 100 TGFß2 71 100 TGFß3 72 16 100 TGFß4 82 64 71 100 TGFß5 76 66 69 72 100 TGFßl is synthesized as a precursor of 390 amino acids called Pre-Pro-TGFßl In a first hydrolysis the release of a hydrophobic fragment of 29 amino acids occurs, which gives rise to Pro-TGFßl. Subsequently, the mature TGFßl is released by another cut in a region that precedes the amino end of TGFβ1 and consists of two arginines, giving rise to a protein of 112 amino acids with a molecular weight of 12 kDa. To give rise to the form biologically active, two of these monomers are joined together by means of disulfide bridges, obtaining a dimer of 25 kDa. Modifications of this structure cause the loss of biological function (Barnard JA et al (1990) Biochim Biophys, Acta 1032: 79-87). The existence of several domains within the structure of TGFβ1 is known, one of these domains is located between amino acids 40 and 82 and is involved in the binding of TGFβ1 to its cellular receptors (Quian SW et al (1992) Proc. Nati, Acad Sci 89: 6290-6294, Burmester JK et al (1993) Proc Nati Acad Sci 90: 8628-8632). Receptors of TGFßl and other binding proteins • Five types of specific receptors have been characterized - for TGFßl (Cheifetz S et al (1988) J. Biol. Chem. 263: 17225-17228 and López Casillas F. et al. 1991) Cell 67: 785-795). These receptors have different affinities for different types of TGFßl. Type I, II, and III receptors are the most well-known so far (reviewed in 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). Type IV receptors have also been described (MacKay K. and Danielpour D. (1991) J. Biol. Chem. 266: 9907- 9911) and type V (Ichijo H. et al (1991) J. Biol. Chem. 266: 22459-22464). It has also been described that the transmembrane and cytoplasmic domains of the endoglin (Cheifetz S et al (1993) J. Biol. Chem. 267: 19027-19030; Bellón T. et al. (1993) Eur. J. Immunol. : 2340-2345; Yamashita et al (1995) J. Biol. Chem. 269: 1995-2001; Zhang H. et al. (1996) J. Immunol. 156: 564-573)) has about 70% analogy with both human and rat type III receptors. The RUI would be responsible for binding the TGFßl and presenting it to RII which in turn would form a complex with Rl (Yamashita et al (1994) J. Biol. Chem. 269: 20172-20178) or complexes in which several molecules of Rl are associated with the RII (Weiss G. and Massagué J. (1996) EMBO J 15: 276-289). The RII-RI interaction would cause the phosphorylation of Rl and the subsequent activation of its serin / threonine kinase, which would phosphorylate second messengers such as MADR2 proteins.

(Macías-Silva M et al., (1996) Cell 87: 1215-1224). Role of TGFßl in liver differentiation and regeneration? The effects produced are different depending on the time of development and cell type. . Increase of the extracellular matrix, acting on the stellar hepatic cells (Ito cells), main source of matrix proteins (Mustoe TA et al (1987) Science 237: 1333-1336). . Differentiation of epithelial cells to hepatocytes (Florini JR et al (1986) J. Biol. Chem. 261: 16509-16513). . Inhibition of cell growth during the process of liver regeneration. This effect is of great importance in the maintenance of cellular rest in vivo (Kato Y et al (1988) Proc. Nati, Acad. Sci. 85: 9552-9556). . Inhibition of endocytosis of the epithelial growth factor receptor (EGF) as observed in cultures of rat fetal hepatocytes (Noda M. and Rodan GA (1987) J. Cell Physiol. 133: 426-437). Role of TGFßl in hepatic fibrosis TGFßl has been associated with hepatic fibro-sis processes (Czaja MJ et al (1989) J. Cell Biol. 108: 2477-2482; Annoni G. et al. (1992) J. Hepatol 14: 259-264) causing an increase in the production of extracellular matrix proteins, by hepatic stellate cells (lipocytes or Ito cells), their receptors and inhibiting the synthesis of proteolytic enzymes that degrade the matrix (Ignotz RA and Massagué J. (1986) J. Biol. Chem. 261: 4337-4345). In the liver TGFßl induces the synthesis of collagen and fibronectin in liver stellate cells (Weiner FR (1990) Hepatology 11: 111-117). As well there is a self-regulation increasing its own synthesis, through the induction of its mRNA. TGFßl is also implicated in the increased synthesis of a2-macroglobulin synthesized by hepatocytes and activated hepatic stellate cells. By binding to TGFßl and causing its inactivation (Bachem MG (1994) Ann NY Acad. Sci. 737: 421-424) a2-Ma-croglobulin would eliminate TGFβ1 from the extracellular compartments. The study of patients affected by chronic liver damage has shown that there is a correlation between the expression of TGFβ1 and mRNA expression for procollagen type I and serum levels of procollagen type III peptide (Castilla A. et al (1991). )., Engl. J. Med. 324: 933-940). Patients with liver cirrhosis have a shorter life expectancy than normal due to complications that appear in the course of the disease, such as portal hypertension or liver failure. Effect of TGFßl on the extracellular matrix The interaction of TGFßl with cellular receptors causes:. Activation of the synthesis of procollagen, fibronectin (Ignotz RA et al (1987) J. Biol. Chem. 262: 6443-6446) and Related proteins, among which we find membrane proteins capable of interacting with the components of the extracellular matrix (Cárter WG (1982) J. Biol. Chem. 257: 13805-13815). . Inhibition of the synthesis of proteolytic enzymes capable of degrading the matrix (Fukamizu H. and Grinnell F. (1990) Exp. Cell Res. 190: 276-282). . Stimulation of the synthesis of proteolytic enzyme inhibitors (Fukamizu H. and Grinnell F. (1990) Exp. Cell Res. 190: 276-282). All this induces an increase in the interactions of the cell with the extracellular matrix, which together with the greater reorganization of the proteins that compose it, results in an increase in the total amount of extracellular matrix (Roberts CJ et al (1988) J. Biol. Chem. 263: 4586-4592). These evidences confirm the implication of TGFßl in healing processes (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 receptor ligand interaction There is the possibility of using small molecules, synthetic peptides, as analogs of existing molecules in the organism, in order to emulate their function. Studies performed by LeSateur et al demonstrate the possibility of using cyclic analogues of nerve growth factor (NGF), emulating the ß-turn region, allowing its binding to the 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 native factor from interacting with its receptor by a blockade mediated by the peptide (Lasarte JJ et al (1994) J. Acquired Immune Deficiency Syndromes 7: 129-134; LeSateur and col (1995) J. Biol. Chem. 270: 6564-6569). Previous studies have demonstrated the utility of synthetic peptides as inhibitors of the ligand-receptor interaction even if the recognition epitope is not continuous (Daniels AJ et al (1995) Mol.Pharmacol. 48: 425-432) . Other studies carried out with the type II receptor of TGFßl and with fetuin, a glycoprotein from the group of type II receptors, have demonstrated the possibility of using cyclized peptides as inhibitors of the interaction of TGFßl with RII (Demetriou M. et al. 1996) J. Biol. Chem. 271: 12755-12761). With this cyclization it is possible to obtain peptides with a structure similar to that which could be given in vivo.

DETAILED DESCRIPTION OF THE INVENTION For the reasons indicated above, we believe that peptides from both TGFβ1 and their receptors, or from proteins capable of binding to TGFβ1, could be inhibitors of the action of TGFβ1. So we decided to explore this possibility.

Choice of the peptides to be synthesized The choice of peptides to be synthesized was made differently depending on whether they came from TGFßl or its receptors. In the case of the sequence of TGFßl, peptides of 15 amino acids were synthesized that encompassed the entire TGFßl sequence. Each peptide had 10 amino acids in common with its two immediate neighbors. In the case of the sequences of their receptors, the peptides were chosen based on computer programs designed in our laboratory. One of these programs allows to compare two amino acid sequences with each other, in order to predict partially complementary zones. Other programs capable of predicting the areas of the proteins that would be most exposed were also used, based on the hydrophobicity and hydrophilicity of the amino acids that make up its sequence. Synthesis of Peptides The peptides were synthesized by the solid phase method (Merrifield (1963) J. Am. Chem. Soc. 85: 2149-54), using fluorenylmethyloxycarbonyl (Fmoc) as a temporary protective group of the alpha-amino group (Atherton et al. al. (1989) Journal of Chemical Society Perkins Transactions 1: 538-546.). For the synthesis of small quantities of a large number of peptides, a multiple synthesizer was used that allows the simultaneous synthesis of 96 peptides (Borras-Cuesta et al (1991) Biologicals 19: 187-190). The peptides were stored at -80 ° C to the solid state until their use. Purification of the peptides by HPLC The peptides synthesized were analyzed and purified by high pressure liquid chromatography (HPLC), using a Waters 600E-900 system (Millipore Corp., Bedford, United States). For analysis of the peptides, by analytical HPLC, a Waters Radial-Pak ™ C18 300A 15 μm, 8x100mm column (Millipore Corp., Bedford, United States) was used. The peptide was dissolved in a solution of 0.1% TFA in distilled water, at a maximum concentration of 1 mg / ml. The solution of peptide was injected (100μl) into the column and eluted in a water / acetonitrile gradient (Figure 15) (Ro il Ltd., Cambridge, United States) both with 0.1% TFA at a flow of 1 ml / minute. The fractions containing the peptide were detected by their absorbance at 220 nm and 280 nm (photo diode array detector, waters 991, Millipore Corp., Bedford, United States). A Waters column was used for its purification Delta-Pak ™ C18 300 at 15 μm, 25xl00mm (Millipore Corp., Bedford, United States). The peptide was dissolved and injected (2 ml) under the same conditions as in the previous case, using the same gradient at a flow of 5 ml / min. The fraction containing the pure peptide was collected in a flask.

EXPERIMENTATION JN VITRO. STUDY OF THE ACTIVITY OF THE PEPTIDES Cell lines A line from the lung epithelium of vison, MV-l-Lu (CCL-64, American Type Cell Cul ture, Virginia, United States) was used. The cells were cultured in 162 cm2 culture flasks (Costar Corporation, Cambridge, United States) in an oven at 37 ° C and 5% C02, until subconfluence was achieved. A complete medium was used: RPMI 1640 with L-glutamine (GibcoBRL, Life Technologies Ltd., Paisley, Scotland) supplemented with 5% fetal calf serum (FCS, Biological Industries, Kibbutz Bei t Haemek, Israel), 10 mM HEPES (1M HEPES Buffer, Bio-Whi ttaker, Verviers, Belgium) and antibiotics (penicillin lOOU / ml and streptomycin 100 μg / ml). Growth inhibition assay of the MV-1 -Lu cell line The Mv-1-Lu cells grown as indicated above, were detached from the bottom of the culture flasks using 5 ml trypsin-EDTA (Biological Industries, Kibbutz Bei t Haemek, Israel), were resuspended in complete medium and centrifuged at 1500 rpm for 8 minutes. After centrifugation the cells were resuspended in complete medium at a concentration of 50000 cells / ml. To perform the assay, 10 ml of the cell suspension was taken and dispensed into 96-well flat bottom plates (Costar Corporation, Cambridge, United States) adding 100 μl / well, and incubated overnight at 37 ° C. ° C and 5% of C02, which allows the adhesion of the cells to the bottom of the wells. Once this time had elapsed, the peptides to be tested in RPMI were added at a final concentration of 200 μg / ml in the presence of a concentration of 200 pg / ml of TGFßl in RPMI (R &D Systems Europe Ltd., Abingdon, United Kingdom ). The final concentration of FCS in the well was 2.5%. After 24 hours of incubation, 1 μCi of tritiated thymidine was added per well ([methyl-3 H] -thymidyne 25 Ci / mmol, Amersham Life Science, Buckinghamshire, United Kingdom) and incubated for an additional 12 hours (Grubeck-Loebenstein B. Col. (1989) J. Clin. Invest. 83: 764-770; Brennan FM et al (1990) Clin. Exp. Immunol., 81: 278-285). Once the incubation periods were finished, the cells were detached from the bottom of the wells with trypsin-EDTA and collected using a manual harvester (Ti tertek cell harvester, Skatron Instruments Inc., Sterling, United States) that lyses the cells by collecting the DNA in nitrocellulose filters (Filter MAT 11731, Skatron Instruments Inc., Sterling, United States) where it is fixed. The filters were individually placed in 5 ml polypropylene tubes to which was added 4 ml of scintillation fluid (Biogreen-11, Reactants Scharlau S.A., Barcelona, Spain). The activity of each tube was quantified for 90 seconds in a scintillation counter ß LKB (Beta píate system, LKB, Upssala, Switzerland). Study of the inhibition of TGFßl binding to cell receptors Selective labeling of cell receptors (Affinity labeling) The MV-l-Lu cells were peeled off from the culture flasks by incubating them at 37 ° C for 10 minutes, with 10 ml of solution 1 (128 mM NaCl, 5 mM KCl, 4- (2-hydroxyethyl) -1-piperazineethanesulfonate 25 mM at pH 7.5, 5 mM glucose and 1 mM EDTA). The cells thus removed were resuspended in solution 2 (128 mM NaCl, 5 mM KCl, 50 mM 4- (2-hydroxyethyl) -1-piperazinelethanesulfonate at pH 7.5, 1.2 mM CaCl 2, 1.2 mM MgSO 4 and 5 mg / ml BSA) and collected by centrifugation at 1000 x g. for 5 minutes. After centrifugation the cells were resuspended in solution 2 at a concentration of 10 cells / ml. From this cell suspension aliquots of 0.5 ml were made in 24-well plates (Greiner GmbH, Frickenhausen, Germany) where the peptides were added, in 50 μl of a 0.8 mg / ml solution, were incubated for 2 hours. hours at 4 ° C in agitation. Subsequently 125I-TGFßl was added (2μCi) at a final concentration of 277.2 pM (12SI-TGFβ1 human recombinant 800-2200 Ci / mmol, Amersham Life Science, Buckinghamshire, United Kingdom) and incubated for another two hours at 4 ° C under agitation. After incubation, the cells were transferred to a centrifuge tube where they were centrifuged cold at 12000 x g. for 1 minute. Subsequently, they were washed 2 times in cold 2 solution and resuspended in 0.5 ml of solution 2 cold, 5 μl of dimethyl sulfoxide (DMSO 99, 5%, Sigma Chemical Co., St. Louis, United States) and disuccimidyl suberate (DSS, Pierce Chemical Co., Rockford, United States) giving a final concentration of 0.25 mM of DSS. The reaction was stopped at 15 minutes by dilution, centrifugation and washing with a solution containing 0.25M sucrose, 10 mM Tris and 1 mM EDTA at pH 7.4. The cell pellet was resuspended in 0.5 ml of Triton x-100 (Bio-Rad Laboratories, Hercules, United States) 1% v / v, 10 mM Tris pH 7.0, 1 mM EDTA, Phenylmethylsulfonyl fluoride 0, lmM, Pepsatin lμg / ml and Leupeptin lμg / ml (Sigma Chemical Co., St. Louis, United States) and incubated for 40 minutes at 4 ° C. The fraction insoluble in detergent is separated by centrifugation at 12000 x g. during 15 minutes. The soluble detergent (supernatant) and insoluble (precipitate) fractions were frozen at -20 ° C (Massagué J. and Like B. (1985) J. Biol. Chem. 260: 2636-2645). Electrophoresis of polyacrylamide-dodec ilsulf ato-sodium gel proteins The soluble and insoluble detergent fractions were used for electrophoretic analysis in 7.5% acrylamide / bisacrylamide gels for 5-6 hours at 220 volts. The protein staining was performed with a solution of comassie brillant blue "R250 (Serva Feinbiochemica GmbH, Heidelberg, Germany) in 50% methanol, 10% acetic acid and distilled water, for 30 minutes The subsequent washes were carried out with a 50% methanol solution, 10% acetic acid. and distilled water for 15 minutes, in a first wash and 2.5% methanol, 0.5% acetic acid and distilled water, in the following washes, until the background color is eliminated. of the TGFßl, mediated by the peptides, to the cell receptors was measured by the direct immunofluorescence method, using an immunofluorescence kit (Fluorokine rh TGFß-biotin, R &D Systems Europe Ltd., Abingdon UK). The assay is based on the binding caty of biotinylated TGFßl to cellular receptors, specifically and the subsequent interaction of biotin with fluoresceinated avidin, so that the intensity of the signal will depend on the the amount of TGFβ1 bound to cellular receptors. MV-l-Lu cells grown in 162 cm2 flasks were peeled off using solution 1 (described above) and resuspended in physiological saline for centrifugation at 500 x g. for 5 minutes. After centrifugation The cells were resuspended again in physiological saline at a concentration of 4 × 10 6 cells / ml. 25 μl of the cell suspension was added to 12x75 mm borosilicate tubes to which the peptide to be tested was added in 40 μl of RPMI 1640 medium, giving a final concentration of 0.42 μg / μl and 10 μl of TGFβl biotinylated As a control of the specificity, 10 μl of a biotinylated reagent supplied by the Kit was added, as a positive control, 10 μl of biotinylated TGFβ1 was added and 20 μl of an anti-TGFβ1 blocking antibody was added as a negative control. In all controls, physiological saline was added until reaching a total volume of 75 μl. All tubes were incubated for one hour at 4CC in the dark. After the incubation period, 10 μl of fluorescein avidin was added and incubated for 30 minutes at 4 ° C in the dark, after which 2 ml of a washing solution (RDF1) was added and centrifuged at 500 x g for 6 minutes. The cell pellet was resuspended in 0.2 ml of cold PBS for cytometric analysis (FACScan, Becton Dickinson Immunocy take try Systems, California, United States). This procedure allows the measurement of the fluorescence emitted by each cell when a laser beam is struck by a computer program (Lisys'll, Becton Dickinson Immunocy take try Systems, California, United States). two) . A typical image of the flow cytometric analysis is shown in Figure 16. In order to obtain the inhibition data of the binding of TGFßl to the receptors, the positive control of the assay was used to delimit the fields corresponding to the labeled cells, which have bound to the TGFßl-biotin, (M2) and to the cells not marked (Ml). Once the fields were delimited, the percentage of cells that were inside each one was calculated. The same was done with the data obtained when the peptide was incubated with TGFβ-biotin or with the cells, depending on whether they were from the receptors or TGFβ1 respectively. With these data, the percent inhibition of each peptide was calculated using the following formula: 100 - ((M2 Peptide-M2 Negative) xlOO / (M2 Positive-M2 Negative)).

IN VIVO EXPERIMENTATION. EXPERIMENTAL FIBROSIS MODEL Male white rats (Wistar albina breed), from simultaneous litters (5 weeks ± 1.5 weeks), were used in order to obtain a homogenous group in age, and initial weight. Throughout the experimental period, the animals were kept under constant temperature conditions (22 ° C) and with a light / dark cycle of 12 hours. They had free access to water and food.

Hepatic cirrhosis (CH) was induced by inhalation of carbon tetrachloride for 11 weeks, twice a week (López Novoa JM et al (1976) Pathology IX: 223-240, Camps J. et al (1987) Gastroenteroiogy 93: 498-505). Exposure to CC14 was done by blowing compressed air, at a rate of 3 liters / minute, through a gas scrubber flask. It began with a minute of exposure, increasing by one minute per week to reach 4 minutes in the fourth week. During the fifth week, CC14 was not administered, starting again at the sixth week with a 5-minute exposure. This exposure time was maintained until week 11. In the drinking water 400 mg / l of phenobarbital (Luminal®, Bayer, Leverkusen, Germany) was added, from one week before starting exposure to CC14 until the end of the period of experimentation. Before the treatment was started, one week was left in which they were not administered CC14. During the treatment they were given a weekly dose of CC14, as a recall (Figure 2). Distribution of the animals The animals were divided into 4 groups before the induction of liver cirrhosis began.

Healthy Controls (Co): Animals that were not subjected to the process of fibrosis. Healthy controls treated (Co + P144): Animals that were not submitted to the process of fibrosis and were administered the peptide P144 during the last 3 weeks (coinciding in time with the treatment of the Tto2 group of rats). Cirrhotic Controls 1 (Ci1): Animals subjected to the process of induction of cirrhosis by inhalation of CC14 twice a week. These animals were separated into 2 groups at the end of the fifth week: Cirrhotic controls 1 (Ci.,): Animals that continued to undergo the process of induction of fibrosis until week 11, without administering the P144 peptide. They were given saline on alternate days, throughout the induction process (weeks 5 to 11). Cirrhotic Treatments 1 (Tto: Animals that were administered peptide P144 from the sequence of the type III receptor, on alternate days, during the process of induction of fibrosis, from week 5 to week 11. Cirrhotic controls 2 (Ci2): Animals that remained subject to the process of induction of fibrosis without receiving the P144 peptide or saline, this group was subdivided into two others at week 11. Cirrhotic controls 2 (Ci?): Cirrhotic animals They were not subjected to any type of treatment, and were kept as controls. saline injections for 3 weeks (weeks 13 to 15). Cirrhotic Treatments 2 (Tto,): Cirrhotic animals that were treated with the peptide from the type III receptor sequence (P144), for 3 weeks (weeks 13 to 15). Treatment of animals. Tto Group: These animals were subjected to treatment during the process of fibrosis. Treatment with the peptide was started in the fifth week, (before exposure to CC14 for 5 minutes) and continued until the end of eleven weeks of the cirrhosis induction process. • Tto2 Group: These animals were subjected to treatment after the end of the cirrhosis induction process (11 weeks). Treatment was started one week after the last inhalation of CC14 and continued for 21 days. Before starting the treatment and at the end of the treatment, all the animals subjected to the treatment with the peptide were bled. The peptide was administered by subcutaneous injection, in the abdominal area at a dose of 70 μg / animal in 500 μl of physiological saline. Sacrifice of the animals and dissection of the liver After the treatment of the animals with the peptide, both in the model with rats and mice, they were sacrificed by decapitation, after having extracted blood from the retrorbital plexus with a capillary. Immediately afterwards, the liver was dissected and samples collected. The samples were cut and introduced into formaldehyde as fixative solution, for further histological analysis. Other fragments were introduced into cryotubes, which after immersion in liquid nitrogen were stored at -80 ° C. Pathological evaluation of the liver The histological study was performed on liver fragments previously fixed in formaldehyde for at least 24 hours, after which they were introduced into ethanol (70%). After dehydration, it was included in paraffin blocks. Serial cuts of 3 μm thickness were made from the obtained blocks, using a Leitz rotation microtome and steel blades. Prior to staining, the sections were dewaxed in xylol (AnalaR, BDH, Poole, United Kingdom) for 15 minutes, after heating at 60 ° C in an oven for 15 minutes, and hydrated by successive passages with concentrated alcohols. decreasing trend 100%, 96%, 80% and 70% ending in water. The following stains were made: Hematoxylin-Eosin. Masson's trichrome (Locquin M, and Langeron, (1985) in Manual of Microscopy Ed. Labor S.A Barcelona): Uses a specific dye for collagenic proteins (light green). Sirius red: Specific stain for collagen. Confirmation of liver fibrosis: image analysis For the image analysis of the samples obtained, a light microscope (Olympus BH-2, Tokyo, Japan) connected to a video camera (Sony DXP-950P, Sony Co., Tokyo, Japan), with which the different fields of each preparation were captured. 6 fields were randomly taken from each preparation stained with Syrian red. The different captured images were analyzed by means of a computer program (Visilog 4, 1, 5, Noesis, Orsay, France) able to calculate the area of fibrosis and the total area of the preparation. With these data, an index of fibrosis (area of fibrosis / total area) of each field was calculated. To be able to use this program it was necessary to modify the acquisition of the images by using polarized light filters (Olympus U-POT, Tokyo, Japan) and green light (Olympus IF550, Tokyo, Japan).

It allowed the automation of the process of analysis of the samples. Detection of collagen in 14 μm sections of tissue for deceased The 14 μm sections that were used for this technique were obtained in the same way as the 3 μm cuts mentioned above. These cuts were subjected to a deparaffinization process for 12 hours in xylol. Once the paraffin was eliminated, the samples were hydrated by passing them through different degrees of alcohol 96%, 80%, 50%, finishing the process in distilled water. Once hydrated they were subjected to a pre-staining process in a 160 mg solution of Fast Green FCF (Fluka chemika -BioChemika, Buchs, Switzerland) in 160 measured pyruvic acid (Merk, Darmstadt, Germany) saturated for 15 minutes in darkness. The samples were washed by immersion in water until they stopped coloring the washing water. Once the remaining dye was eliminated, the samples were stained for 30 minutes in the dark in a solution of 160 mg of Direct Red 80, (Fluka Chemika-BioChemika Buchs, Switzerland) and 64 mg of Fast Green, both dyes in 160 ml of acid saturated picric. They were washed again until the excess dye was removed and the samples were removed from the slides by scraping the sample with a small spatula. The cuts thus detached were introduced in different tubes containing 3 ml of 0.1 N NaOH solution (Quimón, Montplet &Esteban S. A., Barcelona, Spain) and Methanol (1: 1). Aliquots of the different tubes were taken for reading in the spectrophotometer (Lambda 2 UV / VIS spectrophotometer, Perkin-Elmer, Norwalk, United States) at wavelengths of 540 nm and 630 nm using an aliquot of the NaOH solution as target 0.1 N and Methanol (López de León A. and Rojkind (1985) Histochem Cytochem 33: 737-743; Gaudio E. et al. (1993) Int. J. Exp. Path. 74: 463-469). According to the works of Gaudio E. et al. (1993) Int. J. Exp. Path. 74: 463-469) the following formulas were used to obtain the amounts of collagen and total protein: mg Collagen = absorbance at 540 nm - absorbance at 630 nm 37 mg Collagen / mg total protein = mq Collagen mg Collagen + mg non-collagenic proteins Non-collagenic proteins = absorbance at 630 nm Statistical treatment of the results The data obtained in the in vivo experimentation were subjected to statistical analysis. The normality of the quantitative variables was checked by the Shapiro-Wilks test. Because the data did not fit a normal distribution, nonparametric statistics were performed. The comparison between groups was made by the Kruskal-Wallis H followed by the Mann-Whitney U comparison. The data were plotted by boxes representing the median of the data, thick line inside each box, along with the interquartile range, height of the box, while the whiskers of each box represent the highest and lowest observations within a given inter-quartile range. The association between variables was studied by Fisher's exact test. A logistic regression was performed to study the independence of the association of these variables. The value of P equal to or less than 0.05 was considered significant. All statistical analyzes were performed using the SPSS program for Windows V 6.1.3.

INHIBITION JN yjT.RO OF THE ACTIVITY OF TGFßl Cell Growth Inhibition Assay of the MV-l-Lu Line TGFßl is a cytokine capable of inhibiting the growth of the MV-1-Lu cell line (Grubeck-Loebenstein B. et al (1989) J. Clin. Invest. 83: 764-770; Brennan FM et al (1990) Clin. Exp. Immunol. 81: 278-285), whereby this line was used to test the blocking effect of the peptides on the TGFßl. After different combinations of media, cells and thymidine, the effect of different concentrations of TGFßl on the incorporation of [methyl-3H] thymidine, by the MV-l-Lu cells in culture, was studied until the most suitable conditions for the test. These conditions are shown in Figure 3. Once determined both the optimal concentration of MV-l-Lu cells (5000 cells / well) and the lowest concentration of TGFßl capable of producing an inhibition of around 90% (200 pg / ml , Figure 18) the inhibitory effect of the synthetic peptides at the concentration of 200 μg / ml was tested. In vitro inhibition of TGFßl activity by synthetic peptides The synthetic peptides potentially inhibiting the activity of TGFβ1, chosen as indicated above in the section: choice of the peptides to be synthesized (both those coming from proteins that bind to TGFβ1 and TGFβ1 itself) were tested using the cell line MV-l-Lu. The peptides were dissolved in buffered RPMI medium, free of fetal calf serum and the procedure was as follows: Peptides belonging to the receptor sequence, or complementary to the hydrophilicity peaks of TGFβ1, were incubated for 30 minutes in the presence of This cytokine was then added to the cell culture. Peptides from the TGFßl sequence were added to the cell culture before the addition of TGFβ1, to interact with the cell surface receptors. These incubations were carried out in 100 μl of the same medium as that used to add the cells. The active peptides allowed cell growth to a greater or lesser extent depending on their capacity to inhibit TGFßl. Inhibition of TGFβ1 by peptides from TGFβ1 In a first step, overlapping peptides from TGFβ1 were synthesized. These peptides (Table 2) were synthesized thinking that some of them could join the cellular receptors, thus preventing the binding of natural TGFßl to these receptors.

Table 2. Peptides from TGFßl. The number of the peptide is indicated next to its position in the complete sequence, as well as its amino acid sequence. For convenience of synthesis all the peptides were synthesized with an alanine added at the C-terminal end which is not indicated in the table.

Peptide Sequence Pl (280.293, AlaLeuAspThrAsnTyrCysPheSerSerThrGluLysAsn P2 (284-297) AsnTyrCysSerSerThrGluLysAsnCysCysValArg P3 (288_301) SerSerThrGluLysAsnCysCysValArgGlnLeuTyrlle P4 (294_307) CysCysValArgGlnLeuTyrlleAspPheArgLysAspLeu P5 (298,311) GlnLeuTyrlleAspPheArgLysAspLeuGlyTrpLysTrp p6 (302-3is) AspPheArgLysAspLeuGlyTrpLysTrpIleHisGluPro P7 (306_319) AspLeuGlyTrpLysTrpIleHisGluProLysGlyTyrHis P8 (308_321) GlyTrpLysTrpIleHisGluProLysGlyTyrHisAlaAsn P9 (312,325) IleHisGluProLysGlyTyrHisAlaAsnPheCysLeuGly P1 ° (316-329 ) LysGlyTyrHisAlaAsnPheCysLeuGlyProCysProTyr P11 (319-333) HisAlaAsnPheCysLeuGlyProCysProTyrlleTrpSerLeu P12 (322.335) PheCysLeuGlyProCysProTyrlleTrpSerLeuAspThr P13 (326_339) ProCysProTyrlleTrpSerLeuAspThrGlnTyrSerLys P14 (330_343) IleTrpSerLeuAspThrGlnTyrSerLysValLeuAlaLeu p15 (335-349) P16 ThrGlnTyrSerLysValLeuAlaLeuTyrAsnGlnHisAsnPro _349) GlnTyrSerLysValLeuAlaLeuTyrAsnGlnHisAsnPro P17 (340,353) ValLeuAlaLeuTyrAsnGlnHisAsnProGlyAlaSerAla p18 (343- 58) LeuTyrAsnGlnHisAsnProGlyAlaSerAlaAlaProCysCys P19 (344-358) TyrAsnGlnHisAsnProGlyAlaSerAlaAlaProCysCys P20 (348-360) P21 AsnProGlyAlaSerAlaAlaProCysCysValProGln (350,363) GlyAlaSerAlaAlaProCysCysValProGlnAlaLeuGlu P22 (3S4 367) AlaProCysCysValProGlnAlaLeuGluProLeuProIle P23 (358,371) ValProGlnAlaLeuGluProLeuProIleValTyrTyrVal P24 (364-377) P25 ProLeuProIleValTyrTyrValGlyArgLysProLysVal (368-38D ValTyrTyrValGlyArgLysProLysValGluGlnLeuSer P26 (372-385) p27 GlyArgLysProLysValGluGlnLeuSerAsnMetlleVal (378-39D GluGlnLeuSerAsn-MetlleValArgSerCysLysCysSer In Figure 4 the inhibitory effect of the peptides of Table 6 on the activity of TGFßl is shown. Since TGFβ1 inhibits the growth of MV-1-Lu cells, the inhibition of this cytokine by the peptides leads to the restoration of the growth of MV-1-Lu cells. As can be seen in Figure 4, peptide P12, from the sequence of TGFßl, is the one with the highest inhibitory activity of TGFßl. With the purpose of To study in more detail the inhibitory effect of the P12 peptide, a study was made of the effect of the concentration of the peptide on the inhibition of the cytokine, which is indicated below. Dose-response assay of the inhibition of TGFβ1 by the P12 peptide The effect of the concentration of the P12 peptide on the inhibition of TGFβ1 activity was studied. Because this peptide was not readily soluble in the assay medium, solutions or suspensions of nominal concentration of peptide were prepared (that which would have been achieved if the peptide had completely dissolved) and aliquots were taken from them which were filtered or they were used directly for the inhibition assays. In Figure 5 the inhibitory effect of nominal concentrations of peptide is studied, before and after filtering. It is observed that the filtered and unfiltered P12 peptide has practically the same activity. Once obtained the results with peptide P12 it was decided to lengthen the peptide both, in the N-terminal direction as C-terminal and study the effect on its activity. In addition, modifications were made in its sequence to improve its solubility and study the importance of the two Cysteines of its sequence on the inhibitory activity of TGFßl. The synthesized peptides are indicated in Table 3. < Table 3. Peptides from the modification of the P12 peptide.

Peptide Sequence p12 (322-335) PheCysLeuGlyProCysProTyrlleTrpSerLeuAspThr P28 (322-344) PheCysLeuGlyProCysProTyrlleTrpSerLeuAspThrGlnLysVal LeuAlaLeuTyr P29 (313-335) P30 IleTrpSerLeuAspThr HisGluProLysGlyTyrHisAlaAsnPheCysLeuGlyProCysProTyr PheSerLeuGlyProCysProTyrlleTrpSerLeuAspThr P31 P32 PheCysLeuGlyProSerProTyrlleTrpSerLeuAspThr PheSerLeuGlyProSerProTyrlleTrpSerLeuAspThr P33 P34 PheCysLeuGlyProCysProTyrlleTrpSerAspAspAsp AspAspAspGlyProCysProTyrlleTrpSerLeuAspThr P35 P36 AspAspAspGlyProCysProTyrlleTrpSerAspAspAsp GlyProCysProTyrlleTrpSerAspAspAsp P37 P38 AspAspAspGlyProCysProTyrlleTrpSer AspGlyProCysProTyrlleTrpSerAsp Figure 6 shows the results of the inhibition of TGFßl by the peptides of Table 3.

In Figure 6 it is observed that peptide P29 is active. This peptide encompasses the P12 peptide tested previously and has 9 amino acids more towards the N-terminus (Figure 4). Studies conducted by Quian SW et al. (1992) Proc. Nati Acad. Sci. 89: 6290-6294) and by Burmester JK et al. (1993) Proc. Nati Acad. Sci. 90: 8628-8632) by using recombinant chimeric proteins identified a region of TGFßl necessary for the activity of this cytokine (amino acids 40 to 82, in the sequence of mature TGFßl). It was speculated that peptide P29 (amino acids 34 to 56), in the sequence of the mature TGFßl) to cover a larger area than the peptide P12 (amino acids 43 to 56), could acquire a three-dimensional structure more similar to the structure of the TGFßl in circulation. For this reason peptide P29 was used for binding assays to cellular receptors, based on affinity labeling. Assays of inhibition of TGFßl binding to its receptors by peptide P29 (affinity tagging) P29 peptide from the TGFßl sequence was used in affinity tagging assays to test its ability to inhibit TGFßl binding to its cellular receptors (Material and Methods). Due to the different activity of the lots of 125I-TGFßl used, the peptide concentrations usedin the trials they were adjusted according to the concentration of the 15I-TGFßl lot used in each case. The results of these tests are shown in Figures 7 and 8. Subsequent tests were conducted to look for the minimum concentration necessary to block the binding of 125 I-TGFβ1 to the cellular receptors. Inhibition of TGFßl by peptides from the rat type III receptor sequence In order to find new peptides inhibiting the activity of TGFßl, peptides were synthesized from the rat type III receptor. Some peptides were chosen based on regions of their sequence that were predicted to be complementary to blocks of amino acids in the TGFßl sequence. It was hoped that these peptides would be able to bind free TGFßl, sequestering it and preventing its binding to cellular receptors. Other peptides were synthesized by overlapping 10 amino acids and covering part of the extracellular zone of the type III receptor (amino acids 45 to 410). It has been described that there is a soluble type III receptor that corresponds to the extracellular area of the receptor, this area is cut off from the membrane and acts as a sequestrator of the TGFßl in circulation (López Casillas F. et al (1991) Cell 67: 785-795). Subsequent studies have described two possible areas of binding to TGFßl, one of them is found at the N-terminal end of the receptor (López-Casillas et al (1994) J. Cell Biol. 124: 557-568) and the other is in the area closest to the membrane, towards the C-terminus (Fuku-shima D. et al (1993) J. Biol. Chem. 268: 22710-22715; Pepin MC et al (1995) FEBS Lett 377: 368-372). For these reasons peptides were synthesized from the extracellular zone of this receptor assuming that these peptides could be able to sequester the circulating TGFβl. Table 4 shows the peptides synthesized.

Table 4. Peptides from rat type III receptor. The number of the peptide and its sequence are indicated. P39 to P65 are predicted peptides as complementary to TGFβ1 and P66 to P138 are overlapping peptides that cover the extracellular region of the receptor. For convenience of synthesis all the peptides were synthesized with an alanine added at the C-terminal end which is not indicated in the table.

Peptide Sequence P39 (91-102) AsnProIleAlaSerValHisThrHisHisLysPro P40den-lis) ValPheLeuLeuAsnSerProGlnProLeuValTrp P41 (109.120) SerProGlnProLeuValTrpHisLeuLysThrGlu P42 (U0-121) ProGlnProLeuValTrpHisLeuLysThrGluArg P43 1 ((333-344) TrpAlaLeuAspAsnGlyTyrArgProValThrSer P44 'P45 ProIleValProSerValGlnLeuLeuProAspHis' (, 555-566) GlyAspGluGlyGluThrAlaProLeuSerArgAla P46' (, 563-574) LeuSerArgAlaGlyValValValPheAsnCysSer P47 ((603-614) p48 LeuPheLeuValProSerProGlyValPheSerVal (605,616) LeuValProSerProGlyValPheSerValAlaGlu P49 (707-71ß) GluLeuThrLeuCysSerArgLysLysGlySerLeu P50 (712-723) P51 SerArgLysLysGlySerLeuLysLeuProArgCys (717_728) SerLeuLysLeuProArgCysValThrProAspAsp P52 (722-733) ArgCysValThrProAspAspAlaCysThrSerLeu P53 (727-738) P54 AspAspAlaCysThrSerLeuAspAlaThrMetile (73l-742) ThrSerLeuAspAlaThrMetlleTrpThrMetMet P55 (732-743) P56 SerLeuAspAlaThrMetlleTrpThrMetMetGln (737 -748) MetlleTrpThrMetMetGlnAsnLysLysThrPhe P57 (742-752) MetGlnAsnLysLysThrPheThrLysProLeuAla P58 (747-758) ThrPheThrLysProLeuAlaValValLeuGlnVal P59 ', (761-775) LysGluAsnValProSerThrLysAspSerSerProIleProPro P60 '((766-780) SerThrLysAspSerSerProIleProProProProProGlnlle "" 1 (771-785) SerProIleProProProProProGlnllePheHisGlyLeuAsp P62 (776-790) ProProProGlnllePheHisGlyLeuAspThrLeuThrValMet P63 (781-795) PheHisGlyLeuAspThrLeuThrValMetGlylleAlaPheAla P64 (786-800) ThrLeuThrValMetGlylleAlaPheAlaAlaPheValIleGly P65 (797-809) p66 LeuLeuThrGlyAlaLeuTrpTyrlleTyrSerHis (45.59) LeuMetGluSerPheThrValLeuSerGlyCysAlaSerArgGly P67 (50_64) ThrValLeuSerGlyCysAlaSerArgGlyThrThrGlyLeuPro P68 (55.69 ) CysAlaSerArgGlyThrThrGlyLeuProArgGluValHisVal P69 (60-74) ThrThrGlyLeuProArgGluValHisValLeuAsnLeuArgSer P70 (65-79) ArgGluValHisValLeuAsnLeuArgSerThrAspGlnGlyPro P71 (70-84) LeuAsnLeuArgSerThrAspGlnGlyProGlyGlnArgGlnArg P72 (75-89) ThrAspGlnGlyProGlyGlnArgGlnArgGluValThrLeuHis P73 (80-94) GlyGlnArgGlnArgGluValThrLeuHisLeuAsnProIleAla P74 (85-99) GluValThrLeuHisLeuAsnProIleAlaSerValHisThrHis P75 (90-104) LeuAsnProIleAlaSerValHisThrHisHisLysProIleVal P76 (95.?09) SerValHisThrHisHisLysProIleValPheLeuLeuAsnSer P77 (100-114) HisLysProIleValPheLeuLeuAsnSerProGlnProLeuVal P78 (P79 PheLeuLeuAsnSerProGlnProLeuValTrpHisLeuLysThr 105-119 (110-124) ProGlnProLeuValTrpHisLeuLysThrGluArgLeuAlaAla P 0 (1i5_? 29) TrpHisLeuLysThrGluArgLeuAlaAlaGlyValProArgLeu P81 (120-134) P82 ArgLeuAlaAlaGlyValProArgLeuPheLeuValSerGluGly (125-139 GlyValProArgLeuPheLeuValSerGluGlySerValValGln "3 (130-144 PheLeuValSerGluGlySerValValGlnPheProSerGlyAsn P84, P85 GlySerValValGlnPheProSerGlyAsnPheSerLeuThrAla 135-149 (? the 4th-154 PheProSerGlyAsnPheSerLeuThrAlaGluThrGluGluArg P86 (145-159 PheSerLeuThrAlaGluThrGluGluArgAsnPheProGlnGlu P 7 (150.?6 GluThrGluGluArgAsnPheProGlnGluAsnGluHisLeuVal P 8 (155-169 AsnPheProGlnGluAsnGluHisLeuValArgTrpAlaGlnLys P89 (? 174 AsnGluHisLeuValArgTrpAlaGlnLysGluTyrGlyAlaVal the 6th-P90 (165-179 ArgTrpAlaGlnLysGluTyrGlyAlaValThrSerPheThrGlu P l (170-184 GluTyrGlyAlaValThrSerPheThrGluLeuLysIleAlaArg P # 2 ( 175-189 ThrSerPheThrGluLeuLysIleAlaArgAsnlleTyrlleLys P ° 3 (180-194 LeuLysIleAlaArgAsnlleTyrlleLysValGlyGluAspGln P94 (185-199 AsnlleTyrlleLysValGlyGluAspGlnVa lPheProProThr P95 (190-201) ValGlyGluAspGlnValPheProProThrCysAsnlleGlyLys ° 6 (195-209) ValPheProProThrCysAsnlleGlyLysAsnPheLeuSerLeu P97 (200-214) CysAsnlleGlyLysAsnPheLeuSerLeuAsnTyrLeuAlaGlu P98 1 (205-219) AsnPheLeuSerLeuAsnTyrLeuAlaGluTyrLeuGlnProLys °° P (210-224) AsnTyrLeuAlaGluTyrLeuGlnProLysAlaAlaGluGlyCys P100 (P101 TyrLeuGlnProLysAlaAlaGluGlyCysValLeuProSerGln 215-229 (220-234 AlaAlaGluGlyCysValLeuProSerGlnProHisGluLysGlu P102 (225_239 ValLeuProSerGlnProHisGluLysGluValHisIlelleGlu P103 (P104 ProHisGluLysGluValHisIlelleGluLeulleThrProSer 230-244 (235-249 ValHisIlelleGluLeulleThrProSerSerAsnProTyrSer P105 (240-254 LeuIleThrProSerSerAsnProTyrSerAlaPheGlnValAsp PllO (265-279 AspProGluValValLysAsnLeuValLeulleLeuLysCysLys plll (270-284 LysAsnLeuVa1Leul1eLeuLysCysLysLysSerVa1AsnTrp P112 (275-289 IleLeuLysCysLysLysSerValAsnTrpValIleLysSerPhe H3 (280-294 LysSerValAsnTrpValIleLysSerPheAspValLysGlyAsn PH4 (285-299 VallleLysSerPheAspValLysGlyAsnLeuLysValIleAla P115 (290-304 AspValLysGlyAsnLeuLysValIleAlaProAsnSerlleGly P106 '(245-259 SerAsnProTyrSerAlaPheGlnValAspIlelleValAspIle P107 (250-264 AlaPheGlnValAspIlelleValAspIleArgProAlaGlnGlu P108 (255-269 IlelleValAspIleArgProAlaGlnGluAspProGluValVal P109 (P116 ArgProAlaGlnGluAspProGluValValLysAsnLeuValLeu 260-274 (295-309 LeuLysValIleAlaProAsnSerlleGlyPheGlyLysGluSer PH7 (300-314 ProAsnSerlleGlyPheGlyLysGluSerGluArgSerMetThr P118 (305-319 PheGlyLysGluSerGluArgSerMetThrMetThrLysLeuVal H 9 (3i0-32 GluArgSerMetThr etThrLysLeuValArgAspAspIlePro P120 (P121 MetThrLysLeuValArgAspAspIleProSerThrGlnGluAsn 315-329 (320-334 ArgAspAspIleProSerThrGlnGluAsnLeuMetLysTrpAla P122 (325-339 SerThrGlnGluAsnLeuMetLysTrpAlaLeuAspAsnGlyTyr P123 (330-344) P124 LeuMetLysTrpAlaLeuAspAsnGlyTyrArgProValThrSer (335-3 LeuAspAsnGlyTyrArgProValThrSerTyrThrMetAlaPro 9 125 (340-354 ArgProValThrSerTyrThr etAlaProValAlaAsnArgPhe P126 '(345- 359 TyrThrMetAlaProValAlaAsnArgPheHisLeuArgLeuGlu P127 (350- 364 ValAlaAsnArgPheHisLeuArgLeuGluAsnAsnGluGluMet P128 (P129 HisLeuArgLeuGluAsnAsnGluGluMetArgAspGluGluVal 355,369 (360- 374 AsnAsnGluGluMetArgAspGluGluValHisThrlleProPro P130' (365-379 ArgAspGluGluValHisThrlleProProGluLeuArglleLeu P131 (P132 HisThrlleProProGluLeuArglleLeuLeuAspProAspHis 37o-384 (375-389 GluLeuArglleLeuLeuAspProAspHisProProAlaLeuAsp 1 P133 (380-394 LeuAspProAspHisProProAlaLeuAspAsnProLeuPhePro P134: (385 -399 ProProAlaLeuAspAsnProLeuPheProGlyGluGlySerPro P135 (390-404 AsnProLeuPheProGlyGluGlySerProAsnGlyGlyLeuPro P136 (395,409 GlyGluGlySerProAsnGlyGlyLeuProPheProPheProAsp Pli / (400-414 AsnGlyGlyLeuProPheProPheProAspIleProArgArgGly P13 8 (405-419 PheProPheProAspIleProArgArgGlyTrpLysGluGlyGlu The peptides of Table 4 were tested for their ability to block TGFβ1 in the inhibition model of the MV-l-Lu cell line. Since TGFßl is able to inhibit the growth of this line, the inhibition of TGFßl on the part of the peptides would be able to restore cell growth. These tests are shown in Figures 9 to 12. As can be seen in Figures 9 to 12, there are several peptides capable of inhibiting to a greater or lesser extent the cre- of the MV-l-Lu cell line, although only the P54 peptide is able to inhibit almost by. complete the activity of TGFßl. In order to carry out a more in-depth study of this peptide, tests were performed using different concentrations of peptide against a fixed concentration of TGFßl of 200 pg / ml. Dose-response assay of the inhibition of TGFβ1 by the peptide P54 The effect of the concentration of the P54 peptide on the inhibition of TGFβ1 activity was studied. Due to the low solubility of this peptide, stock solutions of nominal concentration of peptide were prepared, as was done in the case of peptide P12, from which aliquots were taken that were filtered or used directly for the assays. of inhibition. Figure 13 studies the inhibitory effect of nominal peptide concentrations, before and after filtering. It is observed that in the filtrate of the P54 peptide there is no measurable inhibitory activity. Once the ability of the P54 peptide to inhibit TGFßl activity in a dose-dependent manner was confirmed, new peptides were synthesized, based on the P54 sequence, in order to try to improve the solubility and thus its activity to lower doses. Two peptides from the human type III receptor were also synthesized. One of these peptides (P144) is equivalent to the P54 peptide. The other peptide (P145) is similar to the P106 peptide of the rat type III receptor that had also shown activity. These new peptides are indicated in Table 5.

Table 5. Peptides from the modification of peptide P54 (peptides P139 to P143) and the human type III receptor (peptides P144 and P145).

Peptide Sequence Provenance "4 (731,742) ThrSer euAspAlaThrMetlleTrpThrMetMet Receptor Type III Rat P139 P140 ThrSerLeuAspAlaThrMetlleTrpAspAspAsp AspAspAspAlaThrMetlleTrpThrMetMet P141 P142 AspAlaT rMetI1eTrpAsp ThrSer euMetlleTrpThrMetMet P143 P144 ThrSerLeuAspAlaThrThrMetMet (729-, 42, ThrSer euAspAlaSerllelleTrpAlaMetMet Receptor Type III Human GlnAsn P145, SerAsnProTyrSerAlaPheGlnValAspIleThr Receptor Type IleAsp III Human The activity assay of the peptides of Table 5 is indicated in Figure 14.

Dose-response assay of TGFßl inhibition by the Peptide P144 A dose-response assay was performed with peptide P144 from the human type III receptor sequence, in order to check whether its activity was concentration dependent (Figure 15). It can be seen how the activity of the peptide decays as the concentration of peptide used in the assays decreases. Assays of inhibition of TGFßl binding to its receptors by peptide P144 (affinity labeling) P144 peptide from the sequence of the human type III receptor was used in the affinity-mark je assays to check its ability to inhibit the binding of TGFßl to its cellular receptors (Material and Methods). Due to the different activity of the 125μ-TGFßl batches used, the peptide concentrations used in the assays were adjusted according to the concentration of the 15I-TGFβl pool used in each case. The results of these assays are shown in Figure 15. Once the inhibition of the binding of TGFßl to its cellular receptors was verified by peptide P144, a new assay was carried out in order to titrate the P144 peptide. It was observed that the peptide lost its activity to the concentration of 2xl05 times the molar concentration of 125I- TGFßl. Inhibition of TGFßl by peptides from other proteins capable of binding to TGFβ1 and predicted to be complementary to TGFβ1. In this series, the peptides of Table 6 were synthesized from proteins capable of binding to TGFβ1.

Table 6 Peptides from different caput proteins bind to TGFβ1 (type II receptor P146, fetuin P147 to P149, endoglin P150 to P154 and a2 -Macroglobulin P155 to P179). The number of the peptide is indicated next to its position in the complete sequence, its amino acid sequence, as well as its origin. For convenience of synthesis all the peptides were synthesized with an alanine added at the C-terminal end which is not indicated in the table.

Peptides Sequence Provenance P146 (84.101) CysValAlaValTrpArgLysAsnAspGluAsnlleThr Type II Receiver LeuGluThrValCys II P147 (H4-132) CysAspPheGlnLeuLeuLysLeuAspGlyLysPheSer Fetuin ValValTyrAla ysCys P148,? I4.132) CysAspPheHisIleLeuLysGlnAspGlyGlnPheArg Fetuin ValCysHisAlaGlnCys P149 (xl4.i32) CysAspIleHisValLeuLysGlnAspGlyPheSerVal Fetuin LeuPheThrLysCysAsp P150 (27-26D GluAlaVal eulle euGInGlyProProTyrValSer Endoglin TrpLeu P151 (289-303) ValAsn euProAspThrArgGlnGly euLeuGluGlu Endoglin AlaArg P152 (445-459) LeuAspSerLeuSerPheGln euGlyLeuTyrLeuSer Endoglin ProHis P153 481-495) ProSerlleProGluLeuMetThrGlnLeuAspSerCys Endoglin Gln eu P154,479,493) MetSerProSerlleProGluLeuMetThrGlnLeuAsp Endoglina SerCys P155 (13-24) LeuLeuLeuLeuValLeuLeuProThrAspAlaSer GC2 -Macroglobulin P156, ProThrAspAlaSerValSerGly and sProGlnTyr 012-Macroglobulin P157,44_55) ThrGluLysGlyCysValLeuLeuSerTyrLeuAsn 0-2 -Macroglobulin P158,? 66,177) TyrlleGlnAspPro ysGlyAsnArglleAlaGln (X2 -Macroglobulin P158,166,177, TyrlleGlnAspProLysGlyAsnArglleAlaGln a2 -Macroglobulin P159, i92.203, PheProLeuSerSerGluProPheGlnGlySerTyr a2 -Macroglobulin P160 2 7-258) AsnValSerValCysGlyLeuTyrThrTyrGlyLys a2 -Macroglobulin P16 (2 8-259) ValSerValCysGlyLeuTyrThrTyrGly and sPro 012 -Macroglobulin P162 (2S0-26D ValCysGly euTyrThrTyrGlyLysProValPro 012 -Macroglobulin P163 (267-278) SerlleCysArgLysTyrSerAspAlaSerAspCys 0C2 -Macroglobulin P164 ¡469-480) ProCysGlyHisThrGlnThrValGlnAlaHisTyr 012 -Macroglobulin P165 (55-565) AspSerAlaLysTyrAspValGluAsnCysLeuAla CX2 -Macroglobulin P167 (790.ß0i) GlnProPhePheValGluLeuThrMetProTyrSer a2 -Macroglobulin P168 (827-838) GlnLeuGluAlaSerProAlaPhe euAlaValPro 012 -Macroglobulin P169 (835_836) SerValGlnLeuGluAlaSerProAlaPheLeuAla CX2 -Macroglobulin P170 (876-887) AlaLeuGluSerGlnGluLeuCysGlyThrGluVal a2 -Macroglobulin P171, LysSerLysIleGlyTyrLeuAsnThrGlyTyr a2 -Macroglobulin P172, 1005-1016) HeGlyTyrLeuAsnThrGlyTyrGlnArgGlnLeu CC2 -Macroglobulin P173 (1062-1073) LysArgLysGluVal eu ysSerLeuAsnGluGlu a2 -Macroglobulin P174 (1193-1204) ValGlyHisPheTyrGluProGlnAlaProSerAla 0C2 -Macroglobulin P175 (1209-1220) T rSerTyrValLeu euAlaTyrLeuThrGlnAla CL2 -Macroglobulin P176 (12U-1222) TyrValLeuLeuAlaTyrLeuThrAlaGlnProAla 0C2 -Macroglobulin P177 (1256.1267) ValAlaLeuHisAlaLeuSerLysTyrGlyAlaAla 0 2 -Macroglobulin P178 (1232-1243) TyrGlyArgAsnGlnGlyAsnThrTrpLeuThrAla OL2 -Macroglobulin Pl79 (i234-i2 5) ArgAsnGlnGlyAsnThrTrpLeuThrAlaPheVal a2 -Macroglobulin The inhibitory activity of the peptides from Table 10 is indicated in Figures 17 and 18. As can be seen in Figures 17 and 18 only peptide P150 showed activity greater than 50%. However, peptides P146 and P149 that had been described as active by Demetriou M et al (1996) J Biol Chem 271: 12755-12761 were not active under the conditions used for this assay.

Measurement by flow cytometry of the inhibitory effect of synthetic peptides on the binding of TGFßl to its cell receptors Peptides from previous syntheses, both those synthesized from the TGFßl sequence and the type III receptor, were used to measure, by flow cytometry, their inhibitory capacity of the binding of TGFßl to cellular receptors. In these tests the. cells are incubated with the peptide before adding the TGFβ-biotin that will be revealed using avidin-FITC (Material and Methods). Subsequently, the fluorescence emitted by the avidin-FITC is measured, which will be directly proportional to the amount of TGFβ1 bound to the cells and inversely proportional to the activity of the peptide. The results obtained with the most relevant peptides are shown in Figure 19 and Table 7.

Table 7. Comparison of the inhibitory activity of TGFβ1, of some peptides, measured by the growth inhibition bioassay of MV-1-Lu1 cells (200 μg / ml peptide concentration) with the inhibition of TGFβl binding to their cell receptors measured by flow cytometry2 (420 μg / ml peptide concentration).

Peptides bioassay Cysitometry Sequence ^ inhibition) 1 (% inhibition) 2 P29 77.6 92.34 HisGluProLysGlyTyrHis AlaAsnPheCysLeuGlyPro CysProTyrlleTrpSerLeu AspThr Pll 40 86 HisAlaAsnPheCysLeuGly ProCysProTyrlleTrpSer Leu P12 96 77 PheCysLeuGlyProCysPro TyrIleTrpSerLeuAspThr P18 18.2 6.5 LeuTyrAsnGlnHisAsnPro GlyAlaSerAlaAlaProCys Cys P54 97 82.3 ThrSerLeuAspAlaThrMet IleTrpThrMetMet P140 -1.7 69.8 AspAspAspAlaThrMetIle TrpThrMetMet P142 70 72 ThrSerLeuMe111eTrpThr MetMet P106 40 91 SerAsnProTyrSerAlaPhe GlnValAspIlelleValAsp He P145 21 74.35 SerAsnProTyrSerAlaPhe GlnValAspIleThrlleAsp P144 88 80 ThrSerLeuAspAlaSerlle IleTrpAlaMetMetGlnAsn P150 64 73 GluAlaValLeuIleLeuGln GlyProProTyrValSerTrp Leu P152 45 68.4 LeuAspSerLeuSerPheGln LeuGlyLeuTyrLeuSerPro His IN VIVO INHIBITION OF THE TGFßl ACTIVITY The P144 peptide from the sequence of the human type III receptor, which had been active in the growth inhibition bioassays of the cell line MV-l-Lu, was used in the in vivo tests to study its inhibitory effect in the induction of experimental cirrhosis with CC14, in a rat model.

Experimental cirrhosis model in Wistar rats In this model liver cirrhosis is induced by inhalation of carbon tetrachloride, for 11 weeks, twice a week (López Novoa JM et al (1976) Pathology IX: 223-240; Camps J. et al (1987) Gastroenteroiogy 93: 498-505) as indicated in Material and Methods. Peptide P144 was administered according to two protocols: 1. Protocol 1: The peptide was administered every other day intraperitoneally during the process of induction of cirrhosis (11 weeks). Figures 20 and 21. 2. Protocol 2: The peptide was administered every other day intraperitoneally for 3 weeks, once the cirrhosis had been established, that is 12 weeks after the onset of cirrhosis induction. Figures 22 and 23. The production of collagen in both protocols was measured by two techniques: Figures 36 and 38 show the production of total collagen measured by staining liver slices (two per animal) stained with Fast Green and Direct Red , elution of color and reading in spectrophotometer (Material and Methods) (López de León A. and Roj ind, (1985) Histochem, Cytochem, 33: 737-743, Gaudio E. et al (1993) Int. Path. 74: 463-469). Figures 21 and 23 reflect the production of collagen measured by image analysis from liver sections stained with Syrian red, using light microscopy (Material and Methods). As can be seen in Figure 20, significant differences (P <0.05) are observed between the group of rats treated with the peptide P144 (Ttox) and the group of control cirrhotic rats (CÍ when studying the collagen ratio vs total tein In Figure 37 the differences between the group of rats treated with the peptide P144 (Tto and the group of control cirrhotic rats (Ci ^ are also significant (P <0.001) when studying the area of fibrosis, as can be seen in figures 22 and 23, in which show the results of the treated rats once cirrhosis is established, the differences between the groups of rats treated with the peptide P144 (Tto2) and the untreated cirrhotics (Ci2) are not significant when either of the two measurement techniques are used. fibrosis The two techniques used for the measurement of collagen were compared by means of a linear regression in order to verify the randomness in the choice of the fields under study in each preparation and with it the validity of the image analysis, Figures 24 and 25. As can be seen in graphs 24 and 25, there is a correlation between both techniques with an R >; 0.85 in both cases, being highly significant (F < 0.001). This confirms that the acquisition of the images under study was carried out in a completely random manner and with it the validity of the data obtained through the image analysis. Figures 26 and 27 show the images obtained by light microscopy from liver preparations dyed with Syrian red at an increase of 10X obtained from livers of rats treated during the process of induction of cirrhosis (Ci, and Tto,). The images of Figure 26 were obtained without applying any type of filter. Figure 27 shows the images once modified for study by a specific software. These modifications consist of the application of two filters, one of polarized light and the other of green light, in order to increase the quality of the images and facilitate their study in an automated way. In figures 26 and 27 it is observed that there are differences between the images coming from the cirrhotic rats (Cix) and those coming from the rats treated with the peptide P144 (Ttox). The differences in effectiveness between protocols 1 and 2 could be due to the fact that the production of TGFßl could be much lower once cirrhosis has been induced (protocol 2) than during the process of induction of cirrhosis with CC14 (protocol 1), and could even be be at normal levels, so that the effect of treatment with peptide P144 would be less noticeable in protocol 2 than in protocol 1. When comparing groups of untreated cirrhotic rats, at the end of the process of induction of cirrhosis ( Cix) with the untreated cirrhotic, at 4 weeks At the end of the induction (Ci2) it is observed that there are significant differences (P = 0.016) between both groups (Figure 28), which would indicate that there is a partial regression of cirrhosis when eliminating the cirrhotising agent, an observation that has been published by various authors (Szende-B et al (1992) In Vivo 6: 355-361; Columbano A (1996) Carcinogenesis 17: 395-400). These differences in effectiveness between the two protocols could also be due to the protocol itself since the animals of protocol 2 were treated only for 3 weeks on alternate days, while the animals of protocol 1 were treated for a longer period of time (7). weeks, also on alternate days). The results obtained show that it is possible to inhibit TGFßl both in vi tro and in vivo by synthetic peptides from different proteins. In the future it would be of great interest to try to increase the biological activity of these peptides. This could be done by systematically replacing each of the amino acids in their sequences with the remaining 19. Once the most active peptide is reached, it would be convenient to prepare mimotopes (McConnell-SJ (1994) Gene 151: 115-118; Steward-M (1995) J.

Virol. 69: 7668-7673) thereof in order to increase the half-life in the organism of the inhibitory agent.

DESCRIPTION OF THE FIGURES Figure 1. Inhibition of TGFßl binding to MV-1-Lu cells by peptide P144, as measured by flow cytometry. A, image obtained by analyzing the cells incubated with biotinylated TGFβ1 and revealed with avidin-FITC. B, image obtained by analyzing the cells incubated with avidin-FITC without prior addition of TGFβ1. C, image obtained by analyzing the cells incubated with TGFβ1 previously incubated with peptide P144 at a concentration of 0.42 μg / μl, the development was performed with avidin-FITC. In abscissas the emitted fluorescence is indicated and in ordinate the number of cells for each fluorescence value. Fields corresponding to the cells labeled with TGFßl-biotin and avidin-FITC (M2) and unlabeled cells (Ml) are also indicated. Figure 2. Representative diagram of the cirrhosis process by CC14. With black arrows it is indicated when the rats were administered two weekly doses of CC14 and with black discontinuous arrows when it was a weekly dose. The gray arrows indicate the administration of peptide P144. A: Healthy controls; B: Healthy controls + P144, B,: with peptide 70 μg / day; C: Cirrhotic; Cx with saline; C2 with peptide 70 μg / day; D: Cirrhotic with CC14 + Phenobarbital; O1 and saline; D2 and peptide 70 μg / day.

Figure 3. Effect of TGFβ1 on the growth of MV-1-Lu cells. Cells were cultured at a density of 5000 cells / well at the indicated TGFßl concentrations, in pg / ml. Abscissa: TGFßl concentration (pg / ml); Ordered: c.p.m. Figure 4. Percentage of inhibition of TGFβ1 (200 pg / ml) by TGFβ1 peptides. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 5. Percentage of inhibition of TGFßl activity (200 pg / ml) in the presence of different nominal concentrations of filtered and unfiltered P12 peptide (•). Figure 6. Percentage of inhibition of TGFβ1 (200 pg / ml) by TGFβ1 peptides. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 7. Autoradiography of an affinity label assay of TGFβ1 receptors. Cl street: effect of incubation of cells with a concentration of 0.16 μM of 125TGFßl that corresponds to an activity of 0.3μCi (positive control). Street C2: effect of pre-incubation of cells with a non-radioactive TGFßl concentration times higher than that of 125I-TGFßl (negative control). C3 lane: preincubation was performed with peptide P29 at a concentration 106 times higher than the molar concentration of 125I-TGFßl. The inhibition of the binding of 125 I-TGFβ1 to the cellular receptors type I, II and III can be observed both by the P29 peptide and by the non-radioactive TGFβ1. Figure 8. Autoradiography of an affinity label assay of TGFβ1 receptors. Streets Cl a C6: effect of preincubation of MV-1-Lu cells, with different concentrations of peptide P29 (106, 8x105, 6x105, 4x10s, 2x105, 105 times the molar concentration of 125 I-TGFßl respectively), prior to the addition of 125I-TGFßl. Lane C7: effect of preincubation of MV-1-Lu cells with unlabeled TGFβ1 (102 times the molar concentration of 125 I-TGFβ1) prior to the addition of 125 I-TGFβ1 (negative control). C8 street: effect of incubation of MV-l-Lu cells with a concentration of 0.42 μM of 12SI -TGFßl that corresponds to an activity of 0.4 μCi, without previous preincubations (positive control). Figure 9. Percentage of inhibition of TGFßl (200 pg / ml) by receptor peptides predicted as complementary to TGFßl areas. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 10. Percentage of inhibition of TGFβ1 (200 pg / ml) by overlapping peptides from the extracellular region of the type III receptor. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 11. Percentage of inhibition of TGFβ1 (200 pg / ml) by overlapping peptides from the extracellular region of the type III receptor. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 12. Percentage of inhibition of TGFβ1 (200 pg / ml) by overlapping peptides from the extracellular region of the type III receptor. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1.

Figure 13. Percentage inhibition of the activity of the TGFßl (200 pg / ml) in the presence of different nominal concentrations of the P54 peptide filtered (eo) and unfiltered (•). Figure 14. Percentage of inhibition of TGFβ1 (200 pg / ml) by receptor peptides from the modification of peptide P54 (P139 to P143) and peptides from the human type III receptor (P144 and P145). All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 15. Percentage of inhibition of TGFßl activity (200 pg / ml) in the presence of different nominal concentrations of unfiltered P144 peptide. Figure 16. Autoradiography of an affinity label assay of TGFβ1 receptors. Cl street: preincubation was performed with peptide P144 at a concentration 106 times higher than the molar concentration of 125I-TGFßl Streets C2 and C3: effect of preincubation of cells with a concentration of non-radioactive TGFβ1 10 times higher than that of 15I-TGFβl (negative control). C4 and C5 streets: effect of the incubation of the cells with a concentration of 0.1 μM of 125TGFßl that corresponds to an activity 0.2μCi (positive control) The inhibition of 125I-TGFβl binding to cellular receptors can be observed both by peptide P144 'and non-radioactive TGFβ1. Figure 17. Percentage of TGFßl inhibition (200 pg / ml) by peptides from the human type II receptor (P146), from fetuin (P147 to P149) and endoglin (P150 to P154). All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 18. Percentage of inhibition of TGFßl (200 pg / ml) by peptides from α2-macroglobulin. All peptides were tested at the concentration of 200 μg / ml. An inhibition of TGFβ1 of 100% corresponds to the growth of MV-1-Lu cells obtained in the absence of TGFβ1. Figure 19. Percentage of inhibition of TGFßl binding to MV-1-Lu cells by different synthetic peptides. Inhibition was studied by measuring the percentage of cells labeled (emit fluorescence) and unlabeled (no fluorescence) for each peptide (Material and Methods). Figure 20. Effect of the administration of peptide P144 on the synthesis of collagen during the induction of cirrhosis. experimental sis with CC14. In ordinate, the ratio of collagen to total protein is indicated. In the abscissas the different groups of rats are indicated: Co = healthy rats; Co + P144 = healthy rats treated with peptide P144; Tto rats subjected to induction of cirrhosis with CC14 and to which the peptide P144 is supplied on alternate days during this period and Cix = rats subjected to induction of cirrhosis with CC14 for 11 weeks and which are not treated with the P144 peptide. Figure 21. Effect of the administration of peptide P144 on collagen synthesis during the induction of experimental cirrhosis with CC14. In ordinates the quotient between the area of fibrosis and the total area in tissue preparations dyed with Syrian red is indicated. In the abscissas the different groups of rats are indicated: Co = healthy rats; Co + P144 = healthy rats treated with the peptide; Tto rats subjected to induction of cirrhosis with CC14 and to which the peptide P144 is supplied on alternate days during this period and Cix = rats subjected to induction of cirrhosis with CC14 for 11 weeks and which are not treated with the P144 peptide. Figure 22. Effect of P144 peptide administration on collagen synthesis once cirrhosis was induced with CC14. In ordinate, the ratio of collagen to total protein is indicated. In the abscissas the different groups of rats are indicated: Co = healthy rats; Co + P144 = healthy rats treated with the peptide; Tto2 = rats subjected to induction of cirrhosis with CC14 and to which peptide P144 is supplied on alternating days at the end of this period and Ci2 = rats subjected to induction of cirrhosis with CC14 for 11 weeks and that are not treated with peptide P144. Figure 23. Effect of P144 peptide administration on collagen synthesis once cirrhosis was induced with CC14. In ordinates the quotient between the area of fibrosis and the total area in tissue preparations is indicated. In the abscissas the different groups of rats are indicated: Co = healthy rats; Co + P144 = healthy rats treated with the peptide; Tto2 = rats subjected to induction of cirrhosis with CC14 and to which the peptide P144 is supplied on alternating days at the end of this period and Ci2 = rats subjected to induction of cirrhosis with CC14 for 11 weeks and which are not treated with the peptide P144 . Figure 24. Comparison between the data on amount of collagen and area of fibrosis, obtained by the two techniques used. On the abscissa axis, the quotient values between the area of fibrosis and the total area obtained by image analysis are indicated. The ordinate shows the quotient values between the μg of collagen and the mg of total protein, obtained by spectrophotometric analysis of liver sections stained with "Direct Red and Fast Green". The R2 is indicated. (F < 0.001).

Figure 25. Comparison between the data on amount of collagen and area of fibrosis, obtained by means of the two techniques used for the study of the samples at the end of protocol 2. On the abscissa axis, the quotient values between the area of fibrosis are indicated and the total area, obtained through image analysis. The ordinate shows the quotient values between the μg of collagen and the mg of total protein, obtained by spectrophotometric analysis of liver sections stained with "Direct Red and Fast Green". The R2 is indicated. (F < 0.001). Figure 26. Representative images of the 24 fields obtained by light microscopy (10X) from preparations of rat livers stained with Syrian red. Cirrhotic rats (CÍ at the end of the induction of cirrhosis with CC14 and cirrhotic treated (Tto with peptide P144 during the process of induction of cirrhosis with CC14.) Different fields were taken from the preparations from each animal (R = rat and C = field) Figure 27. Representative images of the 24 fields obtained by light microscopy (10X) from preparations of rat livers stained with Syrian red Cirrhotic rats (Ci-) at the end of the induction of cirrhosis with CC14 and cirrhotic treated (Tto with peptide P144 during the process of induction of cirrhosis with CC14. Different fields were taken from the preparations from each animal (R = rat and C = field). Polarized light and green filter have been used in order to highlight the collagen fibers. Figure 28. Comparison between the two groups of untreated cirrhotic rats. Ci1 are cirrhotic rats at the end of 12 weeks of induction of cirrhosis with CC14, Ci2 are cirrhotic rats at 4 weeks after the end of the process of induction of cirrhosis. P = 0.016. Ordered: Area fibrosis / Total area.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. - 1 - LIST OF SEQUENCES < 110 > Scientific and Technological Institute of Navarre (ICTN) < 120 > Peptides inhibitors of TGFßl < 160 > 10 < 210 > SEQ ID NO: 1 < 211 > 15 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Coming from TGFßl, position 319-333 < 400 > His Wing Asn Phe Cys Leu Gly Pro Cys Pro Tyr lie Trp 5 10 Ser Leu 15 < 210 > SEQ ID 'NO: 2 < 211 > 14 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > From TGFßl, position 322-335 < 400 > Phe Cys Leu Gly Pro Cys Pro Tyr lie Trp Ser Leu Asp 5 10 Thr < 210 > SEQ ID NO: 3 < 211 > 12 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Derived as a complement to TGFßl, position 731-742 < 400 > Thr Ser Leu Asp Ala Thr Met lie Trp Thr Met Met 5 10 < 210 > SEQ ID NO: 4 < 211 > fifteen < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Overlapped with the extracellular region of rat type III receptor position 245-259 < 400 > Ser Asn Pro Tyr Ser Ala Phe Gln Val Asp lie lie Val 5 10 Asp lie 15 < 210 > SEQ ID NO: 5 < 211 > 9 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Modification P54 deduced as complementary to TGFßl, position 731-742 < 400 > Thr Ser Leu Met lie Trp Thr Met Met 5 < 210 > SEQ ID NO: 6 < 211 > 14 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > From the modified human type III receptor, position 729-742 < 400 > Thr Ser Leu Asp Ala Ser lie lie Trp Ala Met Met Gln 5 10 Asn < 210 > SEQ ID NO: 7 < 211 > 14 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > From the human type III receptor modified position 241-254 - 3 - < 400 > Being Asn Pro Tyr Being Wing Phe Gln Val Asp lie Thr lie 5 10 Asp < 210 > SEQ ID NO: 8 < 211 > 15 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Position 247-261 of the Endoglina < 400 > Glu Ala Val Leu lie Leu Gln Gly Pro Pro Tyr Val Ser 5 10 Trp Leu 15 < 210 > SEQ ID NO: 9 < 211 > 15 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Position 445-459 of the Endoglina < 400 > Leu Asp Ser Leu Ser Phe Gln Leu Gly Leu Tyr Leu Ser 5 10 Pro His 15 < 210 > SEQ ID NO: 10 < 211 > 23 < 212 > Peptide < 213 > Artificial sequence < 220 > Domain < 223 > Modification P12, situation 322-335 of TGFßl < 400 > His Glu Pro Lys Gly Tyr His Wing Asn Phe Cys Leu Gly 5 10 Pro Cys Pro Tyr lie Trp Ser Leu Asp Thr 15 20

Claims (15)

1. - 63 - CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Peptides antagonists of the binding of TGFβ1 to its receptors in the organism, characterized as being synthetic peptides with sequences less than or equal to 15 amino acids, identical or similar to those of natural TGFB1 and / or its receptors.
2. 2. - active peptide according to claim 1, characterized by possessing the amino acid sequence SEQ ID NO: 3.
3. 3. Active peptide according to claim 1, characterized by having the amino acid sequence SEQ ID NO:
4. 4. 4 Active peptide according to claim 1, characterized by possessing the amino acid sequence SEQ ID NO: 5.
5. 5. Active peptide according to claim 1, characterized by possessing the amino acid sequence SEQ ID NO: 6.
6. 6. Peptide active according to claim 1, characterized in having the amino acid sequence SEQ ID NO: 7.
7. 7. Active peptide according to claim 1, characterized by possessing the amino acid sequence SEQ ID NO: 8. - 64 -
8. 8. Active peptide according to claim 1, characterized by possessing the amino acid sequence SEQ ID No:
9. 9. 9. Mimotopos of any of the active peptides according to claims 1 to 8, characterized by having an antagonistic effect similar to them. and a longer half-life in the organism than these.
10. 10. Method of using at least one of the active peptides according to claims 1 to 8 and / or at least one of its mimotopes to make an application composition for liver diseases.
11. 11. Method of using at least one DNA encoding at least one of the active peptides according to claims 1 to 8 for manufacturing an application composition in hepatic diseases that optionally includes at least one of the mimotopes of said active peptides .
12. 12. Method of using at least one recombinant expression system that encodes at least one of the active peptides according to claims 1 to 8 for manufacturing an application composition in liver diseases that includes - optionally at least one of the mimotopes of said active peptides.
13. 13. Method according to claim 12, characterized in that the recombinant system is a defective adenovirus.
14. 14. Method according to claim 12, characterized in that the recombinant system is a plasmid.
15. 15. Method according to claims 11 to 14 for application to hepatic fibrosis.