MXPA01006229A - &agr;v - Google Patents

Abstract

The invention relates to novel peptides which are biologically active as ligands of&agr;v&bgr;6 integrin. Said peptides have a common structural motif, i.e. Asp Leu Xaa Leu - or in a preferred form Arg Xaa Asp Leu Xaa Xaa Leu Arg-, wherein Xaa represents any amino acid radical. The peptides according to the invention can be used as efficient&agr;v&bgr;6 integrin receptor inhibitors and consequently in the treatment of different diseases and pathologies.

Description

INHIBITORS OF THE INEGRINE ALPHA-V-BETA-6 DESCRIPTION OF THE INVENTION The invention describes new peptides that from the biological point of view act as ligands of integrin v & 6. All these peptides have a structure in common, - that is, the structure - Asp Leu Xaa Xaa Leu -, being - Arg Xaa Asp Leu Xaa Xaa Leu Arg - the preferred form of this structure, in which Xaa represents a residue of any amino acid. The peptides of the invention act as effective inhibitors of the integrin receptor avßß and > therefore, they can be used for the treatment of different diseases and pathological conditions. Integrins belong to the family of class I heterodimeric transmembrane receptors, and have an important role in numerous cell-matrix or cell-cell adhesion processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47). In general, they can be classified into three classes: ßj. Integrins, which are receptors for the extracellular matrix; β2 integrins, which are activatable on leukocytes and are "fired" during inflammatory processes; and av mtegrins, which influence the cellular response in riratri zacióp and nf -'- x * pathological processes (Marshall and Hart, 1996, Semin. Cancer Eiol., 7, 191). All integrins as?, A ?? ß3, aßß_. avß ?, avß3 and v & 6 Ref: 128T66 bind to the peptide sequence Arg-Gli-Asp (RGD) found, for example, in the natural fibronectin ligand. Soluble peptides containing the RGD sequence can inhibit the interaction between each of these integrins with fibronectin. Av 6 is a relatively poor integrin (Busk et al., 1992, J. Biol. Chem. 267 (9), 5790) that increases in number in the presence of repair processes in the epithelial tissue, and that unites preferably fibronectin and tenascin which are natural matrix molecules (Wang et al., 1996, Am. J. Respir Cell Mol. Biol. 15 (5), 664). The physiological and pathological functions of avß6 are still not known with precision, but it is assumed that this integrin plays a fundamental role in physiological processes and diseases (for example, inflammation, scarring, tumors) in which epithelial cells intervene. Accordingly, av ß is expressed on keratinocytes in wounds (Haapasalmi et al., 1996, J. Invest. Dermatol. 106 (1), 42), from which it is deduced that, in addition to the healing processes and of inflammations, there are also other pathological skin processes such as, for example, psoriasis, which can be influenced by agonists or antagonists of the aforementioned integrin. Avßí also plays an important role in the epithelium of the respiratory tract (Weinacker et al., 1995, Am. J. Respir, Cell Mol. Biol. 12 (5), 547), so that in the case of diseases of the Respiratory pathways, such as bronchitis, asthma, pulmonary fibrosis and tumors in the respiratory tract, should be able to successfully employ the corresponding agonists / antagonists of this integrin. Finally it is known that av 6 also intervenes in the intestinal epithelium, so that the corresponding agonists / antagonists of this integrin should also be appropriate for the treatment of inflammations, tumors and wounds of the gastrointestinal tract. So far, no low molecular weight inhibitor has been found that binds selectively to integrin v & 6. Therefore, the purpose of the invention was to find other ligands for avß6, preferably peptides, other than the already known natural ligands or antibodies of high molecular weight which from a therapeutic and diagnostic point of view are difficult to manipulate and apply, which are potent , specific and selective and of low molecular weight, and that can be used both for the mentioned therapeutic areas as well as for diagnosis or as a reagent. It was discovered that the peptide compounds that respond to the formulas indicated below and their salts are soluble molecules that act on cells that carry the mentioned receptor, or that when they are united on surfaces, they constitute synthetic ligands for cellular adhesion promoted by av ß. First of all they act as inhibitors of the integrin av ß, in particular, by inhibiting the interactions of the receptor with other ligands such as, for example, the adhesion of fibronectin. This effect can be checked, for example, according to the method of J.W. Smith et al. which is described in J. Biol. Chem. 265, pgs. 12267 to 12271 (1990). P.C.Brooks, R.A.Clark and D.A.Cheresh describe in Science 264, pgs. 569 to 571 (1994), the dependence of the beginning of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins.

It was also discovered that the new substances have valuable pharmacological properties, are well tolerated and can be used as a medicine. This is described later in detail. The peptides of the invention can also be used as diagnostic agents for the detection and localization in vivo of disease states in the epithelial system, for which they must be provided with corresponding markers (for example, the biotinyl moiety) as described in the state of the art. The invention also encompasses conjugates formed with other active substances, such as cytotoxic active substances, and also conjugates with radioactive labels for X-ray therapy or PET diagnosis, and also fusion proteins with marker proteins such as GFP or antibodies, or therapeutic proteins such as IL-2. Accordingly, the object of the invention is constituted by the peptides of formula I W1 - X1n Arg X2 Asp Leu X3 X * Leu X5 X6m - W2 I, wherein X1, X2, X3, X4, X5, X6 independently represent an amino acid residue, in which the amino acids are independently chosen from the group formed by Ala , Asn, Asp, Arg, Cis, Gln, Glu, Gli, Fen, His, Lie, Leu, Lis, Met, Nle, Homo-Fen, Fgli, Pro, Ser, Tr, Tri, Tir or Val, and in the which amino acids mentioned can also be derivatized, W2 is selected from the group consisting of OH, OR, NHR, NR2, NH2, W1 represents H or an acyl residue, R represents alkyl of 1 to 6 carbon atoms and ynym are independently a number - from 0 to 15. In cases in which mon has a value greater than 1, the residues X1 and X6 can independently be the same or different from each other. The amino acids or amino acid residues that have been formed by derivatization of natural amino acids, or which are homologues or isomers of the latter, are also included according to the invention. The amino acid residues are usually linked together through their alpha-amino and alpha-carboxyl groups (peptide bond). In addition, the invention has as its preferred object those peptides in which X2 represents an amino acid residue selected from the group consisting of Tr, Ser, Asp or glycine, then also those peptides in which X3 represents an amino acid residue selected from the group consisting of Asp, Glu, Arg, Lis, His or Tir, and finally those peptides in which X4 represents an amino acid residue selected from the group consisting of Ser, Tir, Tr, Gli or Val. Among the preferred compounds (meanings and abbreviations are indicated above and below) are the peptides of general formula II x Xxn Arg Tr Asp Leu X3 X4 Leu Arg Xßm - W2 lia, W1 X ^ Arg Ser Asp Leu X3 X4 Leu Arg X6m - W "Hb, W1 X1n Arg Asp Asp Leu X3 X4 Leu Arg Xdra - W ^ líe, W1 Xta Arg Ser Asp Leu X3 X4 Leu Arg x - w¿ Hd, W1 X1n Arg Gli Asp Leu X3 X4 Leu Arg Xßm - W2 le, and the peptides of general formula III W1 - Xxn Arg X2 Asp Leu Asp X4 Leu Arg Xem - W2 Illa, W1 - X ^ Arg X2 Asp Leu Glu X4 Leu Arg Xßm - W2 Illb, W1 - Xxn Arg X2 Asp Leu Arg X4 Leu Arg Xsm - W2 lile, W1 - Xxn Arg X2 Asp Leu Lis X4 Leu Arg Xem - W2 I ld, W1 - X1n Arg X2 Asp Leu His X4 Leu Arg X 6 'm - w * lile, W1 - X1,! Arg X2 Asp Leu Tir X4 Leu Arg Xsm W Il f, and the peptides of general formula IV W1 - X1n Arg X2 Asp Leu X3 Ser Leu Arg Xsm - W2 IVa, W1 - x Arg X2 Asp Leu X3 Tir Leu Arg Xßm - W2 IVb, W1 - Arg X2 Asp Leu X3 Tr Leu Arg Xsm - W2 IVe, W1 - X1 !, Arg X2 Asp Leu X3 Gli Leu Arg Xfim - W2 Vd, W1 - X1,! Arg X2 Asp Leu X3 Val Leu Arg Xßm - W2 IVe. The peptides particularly preferred by the invention are those of formula V W1 Xxn Arg Tr Asp Leu Asp Ser Leu Arg X6"- W < v, and of the latter, those of formula VI are particularly preferred.

W1 Xxn Arg Tr Asp Leu Asp Ser Leu Arg Tr X m-l - Wa VI, Finally, the individual compounds indicated below are particularly preferred, among which are also those that are modified in the terminal N or C (a) H-Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-Tr-Tir-Tr-Leu-OH (b) H-Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-OH (O Ac-Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-Tr-OH (d) Ac- Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-Tr-NH2 (e) H-Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-Tr-OH (f) H-Arg- Tr-Asp-Leu-Asp-Ser-Leu-Arg-Tr-NH2 (g) H-Arg-Tr-Asp-Leu-Tir-Tir-Leu-Arg-Tr-T? R-OH (h) Ac- Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 The abbreviations used in this text correspond to the following amino acid residues: Ala A alanine Asn N asparagine Asp D aspartic acid Arg R arginine Cis C cysteine Gln Q glutamine Glu E glutamic acid Gli G glycine His H histidine He I isoleucine Leu L leucine Lis lysine Met M methionine Nle norleucine Om ornithine Fen F phenylalanine Fgli phenylglycine Pro P proline Ser S serine Tr T threonine Tri w triptophan Tir Y tyrosine Val V If the amino acids indicated above have several enantiomeric forms, then the present text also covers - for example, as a component of the compounds of formulas I to VI - to give these forms and their mixtures. In addition, the amino acids can be protected - for example, as components of the compounds of formulas I to VI - with protective groups known per se. The compounds of formulas 1 to VI can have one or more chiral centers and, therefore, present various stereoisomeric forms. The formulas mentioned cover all these forms, in particular the D and L forms, either in enantiomeric mixtures or in racemic mixtures. Finally, the formulas I and II indicated in this text also include the corresponding salts, in particular, the corresponding salts that are physiologically acceptable. The compounds of the invention also include so-called "pro-fatty acids", that is, the compounds of formula I have been derivatized with, for example, alkyl or acyl groups, sugars or oligopeptides, which are rapidly cleaved in the body thus releasing the active compounds of the invention. The compounds of the invention also include derivatives that are composed of the peptides of the invention and known marker compounds that allow peptides to be easily identified. As examples of these derivatives, • can name the biotinylated peptides or the fluorescein-labeled peptides. The peptides of the invention are generally linear, but they can also form a cycle. The invention does not only cover the mentioned peptides of formulas I to VI, but also the mixtures and compositions which, in addition to these compounds of the invention, also contain other pharmacological active substances or auxiliary substances which can conveniently influence the primary pharmacological effect of the peptides of the invention. invention. In general, both the compounds of the invention and the starting materials for their preparation are prepared according to known and customary methods such as those described in the literature (for example, in certain works such as that of Houben-Weyl, "Methoden der organischen Chemie "(Methods of Organic Chemistry), Georg-Thieme-Verlag, Stuttgart), and under reaction conditions that are known and suitable for the reactions mentioned. It is also possible to make use of known variants of these methods. The peptides of the invention can be prepared preferably by means of solid phase synthesis, followed by cleavage and purification, as described, for example, Jonczyk and Meienhofer (Peptides, Proc. 8ch Am. Pept. Symp., Eds. V. Hruby and DHRich, Pierce Comp.III, pp. 73-77, 1983, or Angew. Chem. 104, 1992, 375) or according to Merrifield (J. Am. Chem. Soc. 94, 1972, 3102). On the other hand, these compounds can also be prepared according to the usual methods of an amino acid and peptide synthesis, as disclosed, for example, in Novabiochem-1999 Catalog & Peptide Synthesis Handbook der Calbiochem - Novabiochem GmbH, D-65796 Bad Soden, in numerous standard works and in various published patent applications. Peptides "or biotinylated or labeled proteins in fluorescent form can also be prepared according to standard methods [e.g., EABayer and M.Wilchek in Methods of Biochemical Analysis Vol 26," The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology "(? l use of the avidin-biotin complex as a tool in molecular biology); and Handbook of Fluorescent Probes and Research Chemicals, ß 'edition, 1996, by R.P.Haugland, Molecular Probes, Inc .; or also in the world patent No. 97/14716). It goes without saying that the peptides of the invention, of formulas I to VI, can also be released from their functional derivatives by solvolysis, in particular hydrolysis, or by hydrogenolysis of the latter. As starting substances for solvolysis or hydrogenolysis, those substances are preferred which instead of one or more free amino groups and / or free hydroxyl groups contain correspondingly protected amino and / or hydroxyl groups, preferably those which instead of an H atom attached to an N atom carry an amino protecting group, or instead of the H atom of a hydroxyl group they carry a hydroxylc protecting group. The same applies to carboxylic acids, which by substitution of their hydroxyl function in -CO-OH can be protected with a protecting group by forming, for example, an aster. The term "amrno protecting group" is known and refers to groups that are suitable to protect (block) an amino group from chemical reactions, but which can easily be cleaved after having carried out the desired chemical reaction in other places of the molecule. The term "hydroxyl protective group" is also known and refers to groups that are 1 Suitable for protecting a hydroxyl group from chemical reactions, but which are easily cleaved once the desired chemical reaction has been carried out at other sites in the molecule. The release of the compounds from their functional derivatives is carried out according to the protective group used, for example, with strong acids, conveniently with TFA or perchloric acid, but also with other strong inorganic acids such as hydrochloric acid or sulfuric, with strong organic carboxylic acids such as trichloroacetic acid, or with sulfonic acids such as benzenesulfonic or p-toluenesulfonic acid. Protective groups which are separated by hydrogenolysis (for example, CBZ or benzyl) can be cleaved, for example, by treatment with hydrogen in the presence of a catalyst (for example, a noble metal catalyst such as palladium, conveniently on a support like coal). The procedures are carried out in a known manner, so it is considered unnecessary to describe them in this text in more detail. As mentioned above, the peptides of the invention include physiologically acceptable salts thereof, which can also be prepared according to standard methods. Thus a base of formula I can be converted into its salt by the addition of an acid, for example, by reaction of equivalent amounts of the base and the acid in an inert solvent such as ethanol and then by subsequent evaporation. Acids which form physiologically acceptable salts are particularly suitable for this reaction. Therefore, inorganic acids may be used, such as, for example, sulfuric acid, nitric acid, hydrocides such as hydrochloric or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, then also organic acids, in particular mono- or polybasic aliphatic, alicyclic, araliphatic, aromatic or heterocyclic carboxylic, sulphonic or sulphonic acids such as, for example, formic, acetic, propionic, pivalic, diethylacetic, malonic, succinic, pimelic, fumaric acids maleic, lactic, tartaric, malic, citric, gluconic, ascorbic, nicotinic, isonicotinic, methanesulfonic or ethanesulphonic, ethanedisulfonic, 2-hydroxyethanesulfonic, benzene sulfonic, p-toluenesulfonic, naphthalene monosulfonic, naphthalenedisulfonic and lauryl sulfuric. Salts of physiologically unacceptable acids, for example picrates, can be used to isolate and / or purify the compounds of the invention. On the other hand, an acid of formula I can also be converted into one of its metal or ammonium salts, physiologically acceptable, per treatment with a base. Among the salts, sodium, potassium, magnesium, calcium and ammonium salts are particularly preferred, and also substituted ammonium salts, for example, dimethylammonium, diethylammonium or diisopropylammonium salts, monoethanolammonium, diethanolammonium, citric acid salts, -cyclohexylammonium, dicyclohexylammonium, dibenzylethylenediammonium, and also, for example, the salts formed with arginine or lysine. As mentioned above, the peptides of the invention can be used in medicine and veterinary medicine as active substances of drugs, in particular, for the prophylaxis and / or therapy of diseases in which the epithelial cells intervene. These include, in particular, diseases or inflammations or the healing processes of the skin, the organs of the respiratory tract and the gastrointestinal tract, among which may be mentioned, for example, stroke, angina pectoris, Tumor diseases, osteolytic diseases such as osteoporosis, pathological angiogenic diseases such as, for example, inflammations, pulmonary fibrosis, diseases of dialysis, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatic arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Morbus Crohn, atherosclerosis, psoriasis, restenosis after an angioplasty, lds acute kidney failure or kidney infections. Accordingly, the object of the invention is constituted by the peptides of the formulas defined above, below and in the claims, including their physiologically acceptable salts, which act as medicaments, diagnostic agents or reagents. A particular object of the invention are those drugs that act as inhibitors and that are intended to combat diseases directly or indirectly based on the "expression of the avβ6 integrin receptor, in particular, in the cases of pathological angiogenic diseases, thrombosis, infarction. of myocardium, coronary heart disease, arteriosclerosis, tumors, osteoporosis, inflammations, infections, and to influence the healing processes of wound healing.The invention also relates to pharmaceutical compositions containing at least one medically from formulas I to VI and optionally excipients and / or auxiliary substances Another object of the invention is the use of the physiologically acceptable peptides and / or their salts, as defined in the claims and in the specification descriptive, to prepare a drug designed to combat diseases based directly or indirectly in an integrin v & receptor expression6, in particular, pathological angiogenic diseases, thrombosis, myocardial infarction, coronary heart disease, arteriosclerosis, tumors, osteoporosis, inflammations, infections, and also to influence the healing healing processes of wounds. These medicaments of the invention or the pharmaceutical compositions containing them can be used in medicine and veterinary medicine. Among the excipients there may be mentioned organic or inorganic substances which are suitable for enteral (for example, oral), parenteral or topical application or for application in the form of inhalant aerosol solution, and which do not react with the new compounds. Examples of these excipients are water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerin triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc and petrolatum. For oral administration, tablets, pills, capsules, powders, granules, syrups, juices or drops are used in particular for rectal administration, suppositories for parenteral administration, solutions, preferably oily solutions or aqueous, and also suspensions, emulsions or implants, and for topical application ointments, creams or powders. The new compounds can also be lyophilized and the resulting lyophilized products can be used, for example, for the preparation of injectable preparations. The aforementioned preparations can be sterilized and / or contain auxiliary substances such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifying agents, salts to influence the osmotic pressure, pH regulating substances, dyes, flavor correction substances and / or several additional active substances, such as, for example, one or several vitamins. For the application in the form of aerosol inhaling solution, aerosols containing the active substance either dissolved or suspended in a carrier gas or a mixture of gases (for example, C02 or chlorofluorocarbons) can be used. The active substance is usually conveniently used in micronized form, and one or more additional solvents which are physiologically acceptable, such as, for example, ethanol, can also be added. Inhalant solutions can be administered using conventional inhalers. The substances of formula I of the invention are generally administered analogously to the other known and commercial peptides (for example, the compounds described in US Pat. No. 4,472,305), preferably in doses of approximately between 0.05 and 500 mg, in particular between 0.5 and 100 mg per dosage unit. The daily dose sc is preferably between about 0.01 and 20 mg / kg of body weight. However, the particular dose for each patient depends on a wide variety of factors, for example, the effectiveness of the particular compound used, age, body weight, general state of health, sex, diet , the timing and method of administration, the rate of excretion, the combination of medications and the severity of the particular disease to which the therapy is applied. Parenteral administration is preferred. The invention also includes recombinant DNA sequences that contain fragments encoding peptide regions that have the structural peptide motifs of the invention of formulas I to VI. Such DNA can be transferred to the cells by means of particles, as described by Ch. Andree et al. in Proc.

Nati cad Sci. 91, 12188 to 12192 (1994), or the transference to the cells can be increased by means of other auxiliary substances such as liposomes (A.l.

Aronsohn and J.A. Hughes J. Drug Targeting, 5, 163-169 (1997)).

Therefore, the transfer of such DNA can be used in yeast, by virus-Bacculus or in mammalian cells to produce the peptide substances of this invention. When an animal or human organism is infected with a Recombinant DNA of this type, the peptides of the invention, which finally form spontaneously by means of the infected cells, can bind directly to the VH6 inte-grin receptor of, for example, tumor cells and block it. The corresponding recombinant DNA, which can be prepared using conventional and known techniques, can also be presented, for example, in the form of a viral DNA containing fragments coding for the protein of the viral capsule. By infection of the organism of a host with recombinant viruses, and preferably non-pathogenic viruses of this type, host cells expressing the av ß integrin can be preferentially attacked. Suitable viruses are, for example, the adenovirus species that have already been repeatedly used as vectors for foreign genes in mammalian cells. Thanks to their numerous properties, these viruses are considered good candidates for genetic therapy, as indicated by S.J.Watkins et al. in Gene Therapy 4, 1004-1012 (1997) (see also the text by J. Engelhardt et al in Hum. Gene Ther.4, 759-769 (1993)). As expressed by A. Fasbender et al. in J. Clin. Invest. 102, 184-193 (1998), a common problem in genetic therapy both through viral vectors and nonviral vectors is the limited efficiency of genetic transfer. With the additional sequence of the ligand for the integrin vñ6 in the adenovirus viral capsule protein, sequence described above, the transfer, for example, of the cDNA of the transmembrane conductance regulatory protein of cystic fibrosis can be improved (CTFR - Cystic Fibrosis Transmembrane Condunctance Regulator). Similar to the work of T. Tanaka et al. in Cancer Research 58, pgs. 3362-3369 (1998), instead of DNA for angiostatin DNA can also be used for the sequences of this invention for cellular transfections by retroviral or adenoviral vectors. The peptides of the invention can also be applied within a liposome complex formed by lipid / peptide / DNA for the transfection of cell cultures, together with a liposome complex composed of lipid / DNA (without peptide) for use in genetic therapeutics applied to the human being. The preparation of a liposome complex formed by lipid / DNA / peptide is described, for example, by Hart S.L. and col. 1998: Lipid-mediated Enhancement of Transfection by a Non-Viral Integrin-Targeting Vector; Human Gene Therapy 9, 575-585. A liposome complex formed by lipid / peptide / DNA can be prepared, for example, from the following solutions: 1 μg / μl lipofectin (equimolar mixture of DOTMA (= N- [1- (2, 3- dioleylexy) -propyl] -N, N, N-trimethylammonium) and DOPE (dioleyl-phosphatidylethanolamine), 10 μg / ml of plasmid DNA and 100 μg / ml of the peptide, both the DNA and the peptide are dissolved in the cell culture The liposome complex is obtained by mixing the three components in a given weight ratio (lipid: DNA: peptide, for example, 0.75: 1: 4) .The complexes with liposomal DNA that are destined for therapeutics Genetics applied to humans have already been described [Caplen NJ et al 1995: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis (Transfer of CFTR genes to the nasal epithelium of patients with cystic fibrosis by means of liposomes) Nature Medicine 1, 39-46). Accordingly, another object of the invention is the use of a correspondingly modified recombinant DNA of gene delivery systems, in particular of a virus DNA, to combat diseases based directly or in-directly on the expression of integrin v &; 6, in particular, pathological angiogenic diseases, thrombosis, myocardial infarction, coronary heart disease, arteriosclerosis, tumors, osteoporosis, inflammations, infections, and to influence the healing processes of wound healing. wounds The novel compounds of the invention can also be used as integrin ligands in the preparation of columns for affinity chromatography in order to obtain integrins at the pure state. The compound formed by avidin-a derivatized support material, for example Sepharose, and the new compounds of formula I is prepared according to known methods [for example, E.A. Bayer and M.Wilchek in Methods of Biochemical Analysis Vol 26"The Use of the Avidin-Biotin Complex as a Tool m Molecular Biology" (The use of the avidin-biotin complex as a tool in molecular biology)]. As polymeric support materials, the fixed polymer phases which have preferably hydrophilic properties and which are known and customary in peptide chemistry can be used. Examples of these fixed phases are crosslinked polyazugars such as cellulose, Sepharose or Sephadex®, acrylamides, polymers based on polyethylene glycol or Tentakel® polymers.

Example 1 Preparation and purification of the peptides of the invention Preparation and purification was carried out, in principle, by means of the Fmoc strategy to protect the acid-labile side chains, on acid-labile resins and by employing a commercial "continuous flow" peptide synthesizing apparatus that responds to the indications of Haubner et al. (J.Am. Chem. Soc.118, 1996, 17703). By way of example, the synthesis and purification of the peptide amide Ac-RTDLDSLR-NH2 is described below. For the synthesis of peptide acids, an o-chlorotryl chloride-resin (Novabiochem) with the corresponding amino acid was combined with an Fmoc group at its C-terminal and used inside the apparatus for synthesis according to the processing instructions (Milligen). The main steps are: washing - cleavage of the Fmoc protective group - washing - coupling with the next Fmoc-amino acid - acetylation - washing. If after the last coupling of the amino acid it is desired to carry out a N-terminal acetylation, this will be carried out once the last Fmoc protecting group is cleaved and using the corresponding activated acyl residue, for example, acetic anhydride. In a commercial apparatus for synthesis and applying a usual procedure (apparatus and manual of Milligen 9050, PepSynthesizer ™, 1987) 2 g of 9-Frr.oc-aminoxanteniloxy-resin (Novabiochem, 0.37 mmol / g) were subjected in each case. ) successively with 0.45 g of hydroxybenzotriazole hydrate (HOBt), 0.5 ml of ethyldiisopropylamine, 4-diisopropylcarbodiimide equivalents (DIC) and Fmoc-amino acid in dimethylformamide (DMF) at a coupling step of 60 minutes each time. The washing steps were carried out in DMF for 10 minutes, the cleavage steps were carried out in piperidine / DMF (1: 4 vol) for 5 minutes, and the N-terminal acetylation steps (capping) were carried out with anhydride acetic / pyridine / DMF (2: 3: 15 vol) for 15 minutes. Then the amino acids Fmoc-Arg (Pmc), then Fmoc-Leu, then Fmoc-Ser (But), then Fmoc-Asp (OBut), then Fmoc-Leu, then Fmoc-Asp (Obut), then Fmoc-Tr were incorporated. (But) and finally Fmoc-Arg (Pmc). After washing with DMF and isopropane and drying under vacuum, 3.48 g of the acetylated peptidyl-acetyl in the N-terminal Ac-Arg (Pmc) -Tr (But) -Asp (OBut) -Leu-Asp (OBut) were obtained. ) -Ser (But) -Leu-Arg (Pmc) -aminoxanthyloxy -resin. By treatment of this peptidyl-resin with tri-fluoroacetic acid / anisole / dichloromethane (74 ml / 3.7 ml / 74 ml) for 4 hours at room temperature, filtration, vacuum concentration and trituration with diethyl ether, a precipitate was obtained of 0.6 g of Ac-Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 peptide. The product was purified by reverse phase HPLC on Lichrosorb RP18 (250-25.7 um, Merck KGaA) in 0.3% TFA with a gradient of 4% to 24% 2-propanol in 2 hours at 8 ml. / min, was analyzed by means of a UV photometer at 215 nm. The fractions containing the product were lyophilized. The analysis of FAB-MS (Fast Atom Bombardment Mass Spectroscopy) of the obtained product responded to the expected results: C41 H73 NI5 015 M 1015, -5 g / mol; (M + H) + was 1016. The retention time of the pure product Ac-Arg-Tr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 in analytical HPLC chromatography on Superspher RP18e (250-4, Merck KGaA) and with a gradient of 0-99% A (0.08 M phosphate pH 3.5, 15% acetonitrile) to B (0.03 M phosphate pH 3.5, 70% acetonitrile) at 50 min at 1 ml / min and with detection at 215 nm, was 7.22 min.

Other HPLC analyzes were carried out in the two systems indicated below: System A: 0.3% trifluoroacetic acid with a gradient of 0-80% 2-propanol in 50 min. on LichroSpher 60RP-Select B® (250-4) (Merck KGaA, Darmstadt, Germany), at 1 ml / min. and with detection at 215 nm.

System B: 0.1% trifluoroacetic acid with a gradient of 30-70% acetonitrile in 50 min. on SuperSpher 100RP18ef (250-4) (Merck KGaA, Darmstadt, Germany), at 1 ml / min. and with detection at 215 nm.

Example 2 The peptides indicated in Table 1 were prepared and purified in a manner analogous to that described in Example 1. Table 1 Structure MW F? B- S Rt (HPLC) min Rt (HPLC) / mcn (S / mol) [M + H] (System A) (System A) eaconcx-ao RTDIDSLRTYTL 1453.6 1456 21.9 DSLRTYTL 968.1 969 18.6 j RTD DS 818.9 820 18.6 23.6 DLOSLRTY 982.1 983 16.6 RTDLDSLR 975.1 975 13.5 | RTD DSLRTY 1239.3 1239 16.6 Ac-RTDLDSLRT 1118.2 1119 16.2 15.6 RTDLDSLRT 1076.2 1076 13.9 | RTD PSLRTY 1221.4 1221 19.2 j RTDLDLRT-NH2 988.1 989 13.4, Ac- RTDLDLRT-NH2 1030.2 1031 15.3 RTDLYYLMDL 1302.5 1302 28.2 RTDLDSLRT-NH2 1075.2 1076 11.1 13.8 RTDUDPLRTY 1249.4 1250 16.3 RTDLYYLRT? 1363.5 1363 11.5 Ac-RTDLDSLRT-NH2 1117.2 1118 13.2 15.0 Ac-RTDLDSLR-NHj 1015.5 1016 see ex. 1 TD DSLRT 920.0 920 ¡14.8 PVDLYYLMDL 1241.5 1241 36.1 As the comparative compounds, known RGD p-peptides such as GRGDSPK, cyclo- (RGDfV) and the linear peptide DLYYLMDL were used. Example 3 Obtaining an avßß integrin preparation Av 6 was prepared and purified in "a shortened, transmembrane and soluble form (Weinacker et al., 1994, J. Biol. Chem. 269, 6940) from an expression system of a baculovirus, using for this purpose recombination techniques that are known for av & 2 (Metha et al., 1998, Biochem. J. 330, 861) and making use of affinity chromatography with a 14D9.F8 antibody (Mitjans and col., 1995, J Cell Sci. 108, 2825.) Human cDNA clones for vy & e are known and can be easily acquired.To express the transmembrane avs and shortened from recombinant Baculovirus cells, the transfer vector pAcUW31 (Clontech Lab. Inc., USA) that allows the simultaneous expression of two cDNAs for different targets, for which an av transfer vector was prepared, the (? TM) v transmembrane and shortened starting from plasmid a? TM (pBAc9) using to restriction enzyme EcoRI and Xbal (Metha et al., Lit. see above), and by ligation through the "blunt ends" was cloned into the BamHl separation site of PAcUW31 and down ("downstream") of the polyhedrin promoter. The transmembrane-shortened β6-cDNA was cut and separated from the plasmid pCDNAneoß6 (Weinacker et al., Lit. indicated above) using the restriction enzyme EcoRI and Xbal, and by ligation through the "blunt ends" cloned also within the Ba Hl separation site of PAcUW31 and downstream ("downstream") of the polyhedrin promoter. The tandem vectors containing shortened cDNA for v and ßs were used to obtain the recombinant Baculovirus (Metha et al., Lit. see above). The recombinant baculoviruses were used to infect insect "High Five" cells. The soluble receptor was isolated after 48 to 71 hours of care, for which the supernatant of the cell culture was passed through columns for affinity chromatography of the type mentioned above and eluted at pH 3.1. All steps of the procedure were carried out at room temperature and in the absence of any type of detergent. Peak fractions were neutralised, concentrated and dialyzed at 4 ° C, and finally stored at -80 ° C. The human receptor, soluble and recombinant, thus obtained, is biologically active and retains its specificity for the ligands. European Patent No. 0846 702 describes a similar method used to prepare soluble avß3.

Example 4: avß6 / fibronectin receptor binding assay The peptides prepared according to the invention were linked to the immobilized receptor for av ß in solution and together with competitively acting fibronectin, and the value was determined Q as a measure of the selectivity of the binding to av e of the peptide to be tested. The Q value is calculated from the quotient of the IC50 values of the test specimen and a standard. The linear peptide hepta-RGD GRGDSPK (Lit. / Patent) was used as a standard substance., see Pytela et al. Science 231, 1559 (1986)). In particular, the binding assay was carried out in the following manner: The immobilization of the soluble avß6 receptor on microtiter plates was carried out by dilution of the protein solution in TBS ++ and subsequent incubation overnight at 4 ° C ( 100 ul / well). Non-specific binding sites were blocked by incubation (2 h, 37 ° C) with 3% BSA (weight / volume) in TBS ++ (200 ul / well). Excess BSA was removed by washing three times with TBSA + *. The peptides were serially diluted (1:10) in TBSA- + and incubated together with the biotinylated fibronectin (2 μg / ml) and with the immobilized integrin (50 μl of peptide + 50 μl of ligand per well).; 2 h; 37 ° C). Fibronectin and unbound peptides were separated by washing three times with TBSA ++. The detection of bound fibronectin was carried out by incubation (1 h, 37 ° C) with an anti-biotin antibody coupled to an alkaline phosphatase (Biorad) (1: 20,000 in TBSA ++, 100 μl / well). After washing three times with TBSA ++, colorimetric detection was carried out by incubation (10-15 min, 25 ° C, in the dark) with substrate solution (5 mg nitrophenyl phosphate, 1 ml ethanolamine, 4 ml H20; 100 μl / well). The enzymatic reaction was stopped by the addition of 0.4M NaOH (100 μl / well). The intensity of the color was determined at 405 nm in a measuring device in ELISA and compared to the value zero. As a zero value, wells that were not coated with the receptor were used. The compound GRGDSPK was used as the standard. The ICSO values corresponding to the peptides tested were read from a graph and, when the latter were added to the ICS0 value of the standard peptide, the Q value of the peptide of the invention was determined. The results of the described trial are summarized in the following table: Table 2 Values of Q less than 1 indicate that the peptides exhibit a relatively better binding to the receptor than the comparison standard peptide, considering that said receptor, seen in absolute form, already exhibits in the competition a good binding with the natural fibronectin ligand. Example 5 For comparative purposes and analogously to that described in the previous example, binding assays of integrin ligands were carried out with different integrins (for example, avß3, av s) and their respective ligands (for example, vitronectin, fibrinogen).

EXAMPLE 6 General Preparation of a DNA-Liposome Complex and its Use for Genetic Therapy: The lipid and DNA are mixed in a weight ratio of 5: 1 (lipid: DNA) in HEP? S solution (140 mM NaCl, lmM MgCl 2, 2mM CaCl 2, 6mM KCl, 10mM H? P? S, 10mM D-glucose, pH 9.0). The individual dose is 30 ug DNA / 200 μl. 200 μl of this lipid-DNA complex is applied to the nasal epithelium by means of a nebulizer. This is repeated 10 times at 15 minute intervals. The total dose of DNA is 300 μg.

The following examples relate to pharmaceutical preparations.

Example A: vials for injections The pH of a solution of 100 g of an active substance of formula I and 5 g of disodium hydrogen phosphate in 3 1 of bidistilled water is adjusted to 6.5 with 2N hydrochloric acid, then filtered in sterile conditions, this solution is introduced into the bottles, lyophilized and finally the bottles are closed in sterile conditions. Each bottle for injection contains 5 mg of the active substance. Example B: suppositories A mixture composed of 20 g of an active substance of formula I, 100 g of socy lecithin is melted} a and 1400 g of cocoa butter, the molten mass is poured into the molds and allowed to cool. Each suppository contains 20 mg of active substance. Example C: solution A solution is prepared with 1 g of active substance of formula I, 9.38 g of NaH2P04 x 2 H20, 28.48 g of Na2HP04 x 12 H20, 0.1 g of benzalonium chloride and 940 ml of bidistilled water. The pH is adjusted to 6.8, brought to a volume of 1 1 and sterilized by irradiation. This solution can be used in the form of eye drops. Example D: ointment Under aseptic conditions 500 mg of an active substance of formula I are mixed with 99.5 g. of Vaseline Example E: Tablets A mixture composed of 1 kg of an active substance of formula I, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is compressed into tablet form, such that each tablet contains 10 mg of the active substance. Example F: Dragees The tablets are formed analogously to that described in Example E and then coated in the usual manner with a bath of sucrose, potato starch, talc, tragacanth and dye. Example G: capsules With 2 kg of an active substance of formula I, hard gelatine capsules are filled, so that each capsule contains 20 mg of the active substance. Example H: ampoules A solution of 1 kg of an active substance of formula I in 60 1 of bidistilled water is filtered under sterile conditions. The ampoules are filled with this solution and then lyophilized and closed under sterile conditions. Each ampoule contains 10 mg of the active substance. Example I: inhalant aerosol solution 14 g of an active substance of formula I are dissolved in 10 1 of isctonic NaCl solution. With this solution, commercial containers that have a vaporizing mechanism are filled. The solution can be inhaled through the mouth or nose. Each spray (approx 0.1 ml) corresponds to a dose of approx. 0.14 mg.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (16)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. Peptides of the formula W1 - X1 !. Arg X2 Asp Leu X3 X4 Leu X5 X6m-I, characterized in that X1, X2, X3, X4, X5, X6 independently represent an amino acid residue, in which the amino acids are independently chosen from the group consisting of Ala, Asn, Asp, Arg, Cis, Gln, Glu, Gli, Fen, His, Lie, Leu, Lis, Met, NIe, homo- Fen, Fgli, Pro, Ser, Tr, Tri, Tir or Val, and in which the mentioned amino acids also they can be derivatized, W1 represents H or Ac, Wt2 represents OH, OR, NHR, NR2, NH2, R represents alkyl of 1 to 6 C atoms, and n and m are independently a number from 0 to 15.

2. Peptides according to claim 1, characterized in that? 2 represents an amino acid residue selected from the group consisting of Tr, Ser, Asp or glycine.

3. Peptides according to claim 1, characterized in that? 3 represents an amino acid residue selected from the group consisting of Asp, Glu, Arg, Lis, His or Tir.

4. Peptides according to claim 1, characterized in that? 4 represents an amino acid residue selected from the group consisting of Ser, Tir, Tr, Gli or Val.

5. Peptides according to claim 1, which correspond to formula V 1 - . 1 - . 1 - Arg Tr Asp Leu Asp Ser Leu Arg X6ffi - W2 V, with the meanings indicated in claim 1.

6. Peptides according to claim 5, which correspond to formula VI 1 - X1 !, Arg Tr Asp Leu Asp Ser Leu Arg Tr X6m-? - 2 VI,

7. Peptides of formula I or II, according to claims 1 to 6, and their physiologically acceptable salts as medicaments.

8. A drug according to claim 7, as an inhibitor to combat diseases based on an expression and pathological function of the integrin ocvßß receptors.

9. - Medicine according to claim 8, to combat thrombosis, myocardial infarction, coronary heart disease, arteriosclerosis, tumors, osteoporosis, fibrosis, inflammations, infections, psoriasis, and to influence the healing processes of wounds.

10. - Pharmaceutical composition containing at least one medicament according to one of claims 7 to 9, and optionally excipients and / or auxiliary substances and optionally also other active substances.

11. Use of the peptides according to Claims 1 to 6, and / or of their physiologically acceptable salts, for preparing a medicament for the purpose of combating diseases based on the expression and pathological function of avβ6 integrin receptors.

12. - Use according to claim 11 for preparing a medicament intended to combat thrombosis, myocardial infarction, coronary heart disease, arteriosclerosis, tumors, osteoporosis, fibrosis, inflammations, infections, psoriasis, and to influence the processes of wound healing.

13. - Recombinant DNA, which contains a sequence coding for a peptide fragment corresponding to a peptide according to claims 1 to 6.

14. - Recombinant viral DNA according to the claim 13,

15. - Virus, characterized in that it contains a capsule protein having a sequence corresponding to a peptide according to claims 1 to 6.

16. - Use of a virus according to claim 15, for preparing a medicament for the purpose of combating diseases based on expression and pathological function of interim avß6 receptors. INHIBITORS OF INTEGRINA ALFA-V-BETA-6 SUMMARY OF THE INVENTION The invention describes new peptides that from the pur.tc of biological view act as ligands of the mtegr na av 6 • All these peptides have a structure in common, that is, the structure - Asp Leu Xaa Xaa Leu -, being -Arg Xaa Asp Leu Xaa Xaa Leu Arg - the preferred form of this structure, in which Xaa represents any ammácidc residue. The peptides of the invention act as effective inhibitors of the vβg integrin receptor and, therefore, can be used for the treatment of different diseases and pathological conditions.