JP2002533064A - Integrin αvβ6 inhibitor - Google Patents

Abstract

(57) Abstract The present invention relates to novel peptides that are biologically active as ligands for α _v β ₆ integrins. The peptides have a common structural motif, namely Asp Leu Xaa Leu- or a preferred form of Arg Xaa Asp Leu Xaa Xaa Leu Arg-(where Xaa represents any amino acid residue). The peptide of the present invention can be used as an effective α _v β ₆ integrin receptor inhibitor, and thus can be used in the treatment of various diseases and medical conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes novel peptides that are biologically active as ligands for integrin α _v β ₆ . All of these peptides have a common structural motif, -Asp Leu Xaa Xaa Leu- , or a preferred form of -Arg Xaa Asp Leu Xaa Xaa Leu Arg-(where Xaa represents any amino acid residue). The peptide of the present invention can be used as an effective α _v β ₆ integrin receptor inhibitor, and thus can be used in the treatment of various diseases and pathological findings.

[0002] Integrins belong to the class I family of heterodimeric-transmembrane receptors that play important roles in many cell matrices and cell-cell adhesion processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47). They can be roughly divided into three classes: integrin β ₁ , an extracellular matrix receptor, capable of activating leukocytes and “triggered” during inflammation processes, integrin β ₂ , and during wound healing and other pathological processes, an integrin alpha _V acting on cellular response (Marshall and Hart, 1
996, Semin. Cancer Biol. 7, 191).

[0003] Integrin α₅β₁, Α_IIbβ₃, Α₈β₁, Α_Vβ₁, Α_Vβ₃And α _V β₆Are all Arg-Gly-Asp (RGD) peptide sequences, i.
Binds to Bronectin. Soluble RGD-containing peptides are identified as
It can inhibit the interaction between tegulin and fibronectin. α_Vβ₆Is relatively rare
A large integrin (Busk et al., 1992 J. Biol. Chem. 267 (9), 5790)
During the repair process, an augmentation is formed, preferably a natural matrix molecule.
Binds to ibronectin and tenascin (Wang et al., 1996, Am. J. Respir.
Cell Mol. Biol. 15 (5), 664). α_Vβ₆Physiological and pathological functions are still positive
Not exactly known. However, this integrin is associated with epithelial cells.
Important in physiological processes and diseases (eg, inflammation, wound healing, tumors)
It is assumed that it plays a role. Therefore, α_Vβ₆Is the keratin rhino in the wound
(Haapasalmi et al., 1996, J. Invest. Dermatol. 106 (1), 42)
This suggests that, in addition to the wound healing process and inflammation,
Skin events can be affected by agonists or antagonists of the integrin
Is assumed. Furthermore α_Vβ₆Plays a role in the airway epithelium (
Weinacker et al., 1995, Am. J. Respir. Cell Mol. Biol. 12 (5), 547),
In addition, suitable agonists / antagonists of this integrin include bronchitis, asthma, pulmonary fibrosis
And in airway disorders such as airway tumors. After all, α_Vβ₆But
Also plays a role in the intestinal epithelium and is therefore a suitable integrin agonist / antagonist
It is known that can be used to treat gastric / intestinal inflammation, tumors and wounds.

[0004] Until now, no low molecular weight inhibitor that selectively binds to integrin α _V β ₆ has been found. Thus, apart from the therapeutically and diagnostically intractable natural high molecular weight ligands and antibodies, which are not only powerful, specific or selective, can be used in the therapeutic field, but also diagnostic or The task was to find a low molecular weight ligand, preferably a peptide, for α _v β ₆ that could also be used as a reagent.

[0005] Peptide compounds as soluble molecules of the formula shown below and their salts affect the action on cells bearing the above receptors or bind to the surface, and thus are directed against cell adhesion mediated α _v β ₆ It was found to be an artificial ligand. They are,
In particular, it acts as an integrin α _V β ₆ inhibitor, and in particular acts on the interaction of the receptor with other ligands, such as inhibiting the binding of fibronectin. This effect has been demonstrated by Smith et al. (JW Smith et al. In J. Biol. Chem. 265, 12267-12271 (
1990)). The dependence of the origin of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins has been described by Brooks et al. (PC Brooks, RA Clark and DA Cheresh in Science 264, 569-71 (199).
4)) indicated by

In addition, the new substances have very valuable pharmacological properties with good tolerance (tolerabili
ty) and found to be usable as a medicament. this is,
This is described in more detail below.

Further, the peptide compound of the present invention, if provided with a suitable marker (for example, a biotin residue), can be used as a diagnostic agent for detecting and localizing pathological conditions in vitro according to the prior art. , Can be used in epithelial systems. The invention also includes conjugation with other active compounds, for example cytotoxic active compounds, and conjugation with radioactive labels for radiotherapy or PET diagnostics, as well as G
It also includes a fusion protein with a marker protein such as FP or antibody, or a therapeutic protein such as IL-2.

Accordingly, the present invention provides a compound of formula I

Embedded image In the formula, X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ are each independently an amino acid residue, and the amino acids are each independently Ala, Asn, Asp, Arg
, Cys, Gln, Glu, Gly, Phe, His, Ile, Leu, Lys
, Met, Nle, Homo-Phe, Phg, Pro, Ser, Thr, Trp,
W ² is selected from the group consisting of Tyr or Val, and the above amino acids that may be derivatized, W ² is selected from OH, OR, NHR, NR ₂ , and NH ₂ groups; and W ¹ is H or an acyl group. Wherein R is alkyl having 1 to 6 C atoms, and n and m are each independently a number from 0 to 15, and When m or n is a value greater than 1, residues X ¹ and X ⁶ may each be, independently of one another, identical or different.

According to the present invention, those amino acids or amino acid residues are derived from natural amino acids, are derivatized, or their analogs or isomers are also encompassed. Amino acid residues are usually linked to each other via their α-amino group and α-carboxyl group (peptide bond).

[0010] Further, the present invention preferably comprises X²From Thr, Ser, Asp and Glycine
Those peptide compounds which are amino acid residues selected from the group consisting of:
X³Is selected from the group consisting of Asp, Glu, Arg, Lys, His and Tyr.
For those peptide compounds that are selected amino acid residues, and finally X ⁴ Is selected from the group consisting of Ser, Tyr, Thr, Gly and Val.
These peptide compounds are amino acid residues.

Accordingly, preferred compounds (symbols or abbreviations see above and below) include compounds of the general formula
II compounds

Embedded image And compounds of general formula III

Embedded image And compounds of general formula IV

Embedded image Is included.

Particularly preferred peptide compounds of the invention are compounds of formula V

Embedded image And in this connection especially compounds of the formula VI

Embedded image It is.

In conclusion, the following compounds are particularly preferred, which also include N-terminal and C-terminal modifications:

Embedded image

The above and following abbreviations are abbreviations for the following amino acid residues:

Embedded image

Where the amino acids can give rise to multiple enantiomers, all of these enantiomers and mixtures thereof are included above and below, for example, as constituents of the compounds of Formulas I-VI. In addition, amino acids are provided with suitable protecting groups known per se, for example, as constituents of the compounds of Formulas I-VI. Compounds of Formulas I-VI can have one or more chiral centers, thus giving rise to various stereoisomers. The formula shown includes all these stereoisomers, especially D
And the L-isomer, especially the enantiomers and racemic mixtures. Consequently, the above and below formulas I and II of the present invention also include the corresponding salts, especially the corresponding physiologically acceptable salts.

The so-called prodrug derivatives are included in the compounds of the invention, ie, for example, compounds of the formula I modified with alkyl or acyl groups, sugars or oligopeptides, which are rapidly cleaved in the body, Donate the active compound. Furthermore, derivatives are also included in the compounds of the present invention consisting of the actual peptides of the present invention and marker compounds that can easily detect the peptides. Examples of such derivatives include biotinylated or fluorescently labeled peptides.

In general, the peptides of the invention are linear, but can be cyclized. The present invention shows the formula
Not only the peptides I to VI, but also these compounds of the invention, as well as mixtures and preparations containing other pharmaceutically active compounds or adjuvants which can sensitize the initial pharmacological activity of the invention in the desired manner. .

The starting materials of the compounds according to the invention and their formulations can be obtained in a manner known per se otherwise, as described in the literature (eg, Houben-Weyl, Methoden de
r organischen Chemie (method of organic chemistry), Georg-Thieme-Verlag, Stuttgart, et al.), ie, are known and are often prepared and used under suitable reaction conditions for the reaction. The use of variants known per se is also made here.

Preferably, the peptides of the invention are, as already described, for example, Jo
nczyk and Meienhofer ^{(Peptides, Proc. 8 th Am. Pept} . Symp., Eds. V. Hruby
And DH Rich, Pierce Comp. III, p. 73-77, 1983, or Angew. Chem. 104 , 1
992, 375) or according to Merrifield (J. Am. Chem. Soc. 94 , 1972, 3102) by solid-phase synthesis followed by removal and purification. Alternatively, for example, Novabiochem-1999 Calbiochem Catalog & Peptide Synthesis Handbook-Novabiochem G
mbH, D-65796 from Bad Soden, which can be prepared by conventional methods of amino acid and peptide synthesis, as is known from many standard studies and published patent applications. Biotinylated or fluorescently labeled peptides / proteins can also be obtained using standard methods (eg, EA Bayer and M. Wilchek, Methods of Biochemica).
l Analysis, Vol. 26, The Use of the Avidin-Biotin Complex as a Tool in M
olecular Biology, and Handbook of Fluorescent Probes and Research Cemica
ls 6th edition, 1996, by RP Haugland, Molecular Probes, Inc. or other WO 97
/ 14716).

Of course, the peptides of formulas I-VI can also be released by solvolysis, preferably by hydrolysis, or by hydrogenolysis of their derivatives. Preferred starting materials for solvolysis or hydrogenolysis are those in which one or more free amino and / or hydroxyl groups are replaced by the corresponding protected amino and / or hydroxyl groups, preferably an H atom bonded to an N atom Is a compound having an amino-protecting group, or a compound having a hydroxyl-protecting group in place of the H atom of the hydroxyl group. The same applies to carboxylic acids which can be protected by a substituent of the hydroxyl group of -CO-OH, for example by a protecting group such as an ester.

The expression “amino protecting group” is commonly known and is suitable for protecting (blocking) an amino group from a chemical reaction, but where the desired chemical reaction occurs elsewhere in the molecule. It relates to groups that can be easily removed after they have been lost. The expression "hydroxyl protecting group" is also commonly known and is suitable for protecting a hydroxyl group from a chemical reaction,
It relates to groups that can be easily removed after the desired chemical reaction has occurred elsewhere in the molecule. Separation of the compound from the functional derivative can be performed, for example, using a strong acid, for convenience using TFA or perchloric acid, and further using other strong inorganic acids such as hydrochloric acid or sulfuric acid, strong organic acids such as trichloroacetic acid, Or using a sulfonic acid such as benzenesulfonic acid or p-toluenesulfonic acid—depending on the acid used. Hydrogenolytically removable protecting groups (eg, CBZ or benzyl) can be removed by hydrotreatment in the presence of a catalyst (eg, a noble metal catalyst such as palladium on a support such as carbon for convenience). it can. The method is generally known and is not described here in more detail.

As already mentioned, the peptides according to the invention include their physiologically acceptable salts, which can likewise be prepared by standard methods. Thus, a base of formula I can be converted with an acid to the relevant acid addition salt, for example, by reaction of an equal amount of the base and acid in an inert solvent such as ethanol and subsequent evaporation. Due to this reaction, suitable acids yield, in particular, physiologically acceptable salts. So, for example,
Hydrohalic acids such as sulfuric acid, nitric acid, hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, inorganic acids such as sulfamic acid, and further organic acids, especially aliphatic acids, alicyclic acids, or araliphatic (Araliphatic), aromatic or heterocyclic monobasic or polybasic carboxylic acids, or sulfonic acids or sulfuric acids, such as formic acid, acetic acid, propionic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, Maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethanesulfonic acid, ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene mono- and disulfonic acids, lauryl sulfuric acid can be used. Salts of physiologically unacceptable acids, such as, for example, picrates, can be used for the isolation and / or purification of the compounds according to the invention. On the other hand, an acid of formula I can be converted to one of its physiologically acceptable metal or ammonium salts by reaction with a base. Possible salts here are, in particular, sodium, potassium, magnesium, calcium and ammonium salts, furthermore, for example, dimethyl-, diethyl- or diisopropylammonium salts, monoethanol-, diethanol- or diisopropylammonium salts, cyclohexyl- or dicyclohexylammonium Salts, for example salts of arginine or lysine.

As already mentioned, the peptide compounds of the invention can be used as pharmaceutically active compounds in human and veterinary medicine, in particular in the prevention and / or treatment of disorders involving epithelial cells. In particular, the emphasis in this context is on skin diseases or inflammation or wound healing processes, respiratory and stomach and intestinal areas, and thus, for example, stroke, angina, oncosis, osteoporosis, etc. Osteolytic diseases such as inflammation, pulmonary fibrosis,
Ocular diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma (rubeot)
ic glaucoma), ulcerative colitis, Crohn's disease, atherosclerosis, psoriasis,
In restenosis after angioplasty, acute renal failure or nephritis.

Accordingly, the present invention relates to a peptide compound of the formula as defined above and below and as a medicament, a diagnostic or a reagent, including the physiologically acceptable salts.

In particular, the present invention relates to diseases based indirectly or directly on the expression of α _v β ₆ integrin receptor, in particular pathological angiogenic diseases, thrombosis, myocardial infarction, coronary heart disease, atherosclerotic artery The present invention relates to suitable medicaments as inhibitors for inhibiting sclerosis, tumors, osteoporosis, inflammation, infections and wound healing processes.

The invention also relates to a medicament of at least one formula I to VI and, where appropriate, to a suitable pharmaceutical formulation comprising a vehicle and / or excipient.

Furthermore, the present invention relates to diseases in which a peptide compound and / or a physiologically acceptable salt thereof as described in the claims and the specification are indirectly or directly based on the expression of α _V β ₆ integrin receptor. And therefore pathological angiogenic diseases, thrombosis, myocardial infarction, coronary heart disease, atherosclerosis, tumors, osteoporosis, inflammation, among others
It relates to the use for the manufacture of a medicament for the control of infectious diseases and diseases for promoting the wound healing process. The medicament of the present invention or a pharmaceutical preparation containing it can be used in human or veterinary medicine. Possible excipients are organic or inorganic substances which are suitable for enteric (eg oral) or parenteral administration or topical application or administration in inhalation form, and which do not react with the novel compounds, eg water Vegetable oil, benzyl alcohol, alkylene glycol, polyethylene glycol, glycerol triacetate, gelatin, carbohydrates such as lactose and starch, magnesium stearate, talc, petrolatum and the like. Especially tablets, pills, coated tablets, capsules, powders, granules, syrups,
Juices or drops are used for oral administration, suppositories are used for enteral administration, solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants are used for parenteral administration, and ointments , Cream or powder is used for topical application. The novel compounds can be lyophilized, and the lyophilizates obtained are used, for example, in the manufacture of injection preparations. The indicated formulations can be sterilized and / or lubricants, preservatives, stabilizers and / or surfactants, emulsifiers, salts for regulating osmotic pressure, buffers, flavorings and / or for example 1 or 1 or 2 such as 2 or more vitamins
Vehicles such as the additional active compounds described above may be included.

For administration as an inhalation spray, the spray may be used containing the active compound dissolved or suspended in a propellant or a propellant mixture such as CO ₂ or chlorofluorohydrocarbon. For convenience, the active substance is used here in finely divided form,
It can be present in one or more additional physiologically tolerable solvents, for example ethanol. Inhalation solutions can be administered using conventional inhalers.

The substances according to the invention can be administered, as specified, analogously to other findings, commercially available peptides (eg as described in US Pat. No. 4,472,305), preferably It can be administered between approximately 0.05 and 500 mg, in particular between 0.5 and 100 mg, per dosage unit. The daily dose is preferably between approximately 0.01 and 20 mg / kg of body weight. However, the particular dose for each patient will depend on all types of factors, such as the effect of the particular compound employed, age, weight, general health and gender, diet, time and route of administration, excretion rate, It depends on the severity of the particular disorder to which the pharmaceutical combination and treatment applies. Parenteral administration is preferred.

Finally, the present invention also includes a recombinant DNA sequence comprising a section encoding a peptide comprising the peptide structural motif of formulas I to VI of the present invention.

[0031] Such DNA can be obtained from Ch. Andree et al. Proc. Natl. Acad. Sci. 91, 12188-12.
192 (1994).
Other vehicles, such as Aronsohn and JA Hughes J. Drug Targeting, 5, 163-169 (1997)) can increase transport to cells.

This transfer of DNA is therefore used for the production of the peptide substances according to the invention in yeast, with baculoviruses or in mammalian cells.

[0033] When the body of an animal or human has undergone infection such recombinant DNA, peptide itself ultimately present invention formed by infected cells, for example tumor cells, the alpha _V beta ₆ integrin receptor Join directly and block it.

However, suitable recombinant DNAs that can be produced by known and conventional techniques include:
For example, it can be in the form of viral DNA containing a section encoding a viral coat protein. Infection of the host organism with a recombinant, preferably a non-pathogenic virus of this type, preferably attacks (targets) host cells expressing the integrin α _V β ₆ .

Suitable viruses are, for example, adenovirus species that have already been used many times as vectors for foreign genes in mammalian cells. Many properties make it a good candidate for gene therapy, which is described in SJ Watkins et al. Gene Therapy 4
, 1004-1012 (1997) (J. Engelhardt et al. Hum. Gene Ther. 4, 175-769 (1993
) Can also be inferred from). A. Fasbender et al. J. Clin. Invest. 102, 184-19
3 (1998), the ability of limited gene transfer is a common problem in gene therapy with viral and non-viral vectors. Using the additional ligand sequence of α _V β ₆ integrin in the adenovirus coat protein, for example, cystic fibrosis transmembrane regulation
ane conductance regulator (CFTR) can improve the transfer of cDNA.

As in the study of T. Tanaka et al. Cancer Research 58, 3362-3369 (1998),
Instead of angiostatin DNA, the DNA of the sequence of the present invention can also be used for cell transfection with a retroviral or adenoviral vector.

The peptides of the present invention may also be used in humans together with lipid / DNA (excluding peptide) liposome complexes to prepare lipid / peptide / DNA liposome complexes prepared for cell culture transfection. Used during gene therapy. The preparation of lipid / peptide / DNA liposome complexes is described, for example, in Hart S .;
L., et al 1998: Lipid-mediated Enhancement of Transfection by a Non-Viral
Integrin-Targeting Vector, Human Gene Therapy 9, 575-585.

The lipid / peptide / DNA liposome complex can be prepared, for example, from the following stock solution: 1 μg / μl lipofectin (DOTMA (= N- [1- (2,3-dioleyloxy) propyl] -N , N, N-trimethylammonium chloride) and DOPE (equimolar mixture of dioleylphosphatidylethanolamine)), 10 μg / ml plasmid DN
A and 100 μg / ml peptide. For this reason, both DNA and peptide are dissolved in the cell culture medium. The liposome complex is prepared by mixing the three components in a specific weight ratio (eg, 0.75: 1: 4 for lipid: DNA: peptide). Liposome DNA complexes for human gene therapy have been described previously (Caplen NJ, et a
l 1995: Liposome-mediated CFTR gene transfer to the nasal epithelium of
patients with cystic fibrosis, Nature Medicine 1, 39-46).

Thus, the present invention relates to diseases, indirectly or directly based on the expression of the α _V β ₆ integrin receptor, of recombinant DNA, in particular of viral DNA, of a suitably modified gene release system, and thus in particular of pathology. Vascular disease, thrombosis, myocardial infarction,
It relates to the use for controlling coronary heart disease, atherosclerosis, tumors, osteoporosis, inflammation, infections and promoting the wound healing process.

The novel compounds of the present invention can also be used as ligands for integrins for the preparation of affinity chromatography columns for producing integrin in pure form. Avidin-derivatized supports such as, for example, Sepharose and novel compounds of formula I can be formed in a manner known in the art (eg EA Ba).
yer and M. Wilchek, Methods of Biochemical Analysis, Vol. 26, The Use of
the Avidin-Biotin Complex as a Tool in Molecular Biology). Suitable polymeric supports in this case are polymer solid phases conventionally known in peptide chemistry, preferably having hydrophilic properties, for example, cross-linked polysaccharides such as cellulose,
Polymers based on Sepharose or Sephadex®, acrylamide, polyethylene glycol or tentakel polymers (Tentakel polym
ers (registered trademark)).

Example 1 Preparation and Purification of the Peptides of the Invention: In principle, the preparation and purification were carried out according to Haubner et al. (J. Am. Chem. S.
oc. 118, 1996, 17703) using an Fmoc method with acid-labile resins protected by acid-labile resins using a commercially available continuous-flow peptide synthesizer. Was.

In the following, the synthesis and purification are described by taking the peptide amide Ac-RTDLDSLR-NH ₂ as an example. For the synthesis of peptide acids, o-chlorotrityl chloride resin (Novabiochem) is covered with the appropriate C-terminal Fmoc amino acid according to the manufacturer's instructions and used on the synthesizer according to the manufacturer's instructions (Milligen). The principle steps are: washing-removal of Fmoc protecting group-washing-coupling with the next Fmoc amino acid-capping (acetylation)-washing. If N-terminal acylation is desired after the last amino acid coupling, this is done after removal of the last Fmoc protecting group using a suitable activated acyl group such as, for example, acetic anhydride.

2 g of 9-Fmoc-aminoxanthinyloxy resin (Novabiochem, 0.37 mmol / g) are subjected to a coupling step, in each case for 60 minutes, in a commercial synthesizer and in a typical manner (apparatus and Milligen 9050 PepSynthesizer ^™ Handbook, 1987), dimethylformamide (DMF), 0.45 g of hydroxybenzotriazole hydrate, 0.5 ml of ethyldiisopropylamine, 4 equivalents of diisopropylcarbodiimine (DIC) and 4 equivalents of Fmoc-amino acid each. It was offered continuously. The washing step was performed with DMF for 10 minutes, the removal step was with piperidine / DMF (1: 4 volume) for 5 minutes, and N-terminal acetylation (capping) was performed with acetic anhydride / piperidine / DMF (2: 3).
: 15 volumes) for 15 minutes. Amino acids Fmoc-Arg (Pmc) followed by Fmoc-Arg
For Leu, then Fmoc-Ser (But), then Fmoc-Asp (OBut), then Fmoc-Leu, then Fmoc-Asp (OBut), then Fmoc-Thr (But), and finally Fmoc-Arg (Pmc) Was. After washing with DMF and isopropanol and subsequent drying under reduced pressure, 3.4
8 g of N-terminal acetylated peptidyl resin, Ac-Arg (Pmc) -Thr (But) -Asp (OBut) -Leu-
Asp (OBut) -Ser (But) -Leu-Arg (Pmc) -aminoxanthinyloxy resin was obtained.

For 4 hours at room temperature, trifluoroacetic acid / anisole / dichloromethane (74 ml / 3.7
of the peptide, Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ 0.6 g by treatment of the peptidyl resin with 0.1 ml / 74 ml), filtration, concentration under reduced pressure and trituration with diethyl ether. Was obtained. Purification of the product was performed using a gradient of 4% to 24% 2-propanol for 2 hours at 8 ml / min in 0.3% TFA in Lichrosorb RP18 (250-25
RP-HPLC at 7 μm, Merck KGaA), 215 nm with UV flow photometer.
Was evaluated.

The product-containing fraction was lyophilized. FAB-MS (Fast Ato Mass Spectrometer)
According to m Bombardment Mass Spectroscopy)), the product obtained met the expected value: C ₄₁ H ₇₃ N ₁₅ O ₁₅ M 1015.5 g / mol, (M + H) ⁺ 1016. 0 to 99% A (0.08
M phosphate pH 3.5, 15% acetonitrile) to B (0.03 M phosphate pH 3.5
, 70% acetonitrile), SuperSpher RP18e (250-4, M
purified product Ac-Arg by analytical HPLC on erck KGaA) and detection at 215 nm.
-Thr-Asp-Leu-Asp- Ser-Leu-Arg-NH 2 has a retention time of 7.22 minutes.

Further HPLC analysis was performed on two systems: System A : Lichrosorb 60 RP-Select B® (250-4) (Merck KGaA,
Darmstadt, Germany) with a gradient of 0-80% 2-propanol for 50 minutes
0.3% trifluoroacetic acid, 1 ml / min, and detection at 215 nm.

System B : SuperSpher 100 RP18e® (Merck KGaA, Darmstadt, Germany) for 50 minutes, 0.1% trifluoroacetic acid with a gradient of 30-70% acetonitrile, 1 ml / min and detection at 215 nm.

Example 2 The following peptides shown in Table 1 were prepared and purified as in Example 1.

[Table 1] Table 1: The comparison compounds used were GRGDSPK, cyclo- (RGDfV), and the linear peptide DLYYL.
It was a known RGD peptide such as MDL.

[0049] Example 3 alpha _V beta ₆ integrin Formulation Preparation:. 14D9.F8 antibody affinity chromatography (Mitjans et al, 1998, Bio
chem. J, 330, 861) using a known recombinant techniques to the α _V β ₃ (Mehta e
α _v β ₆ was obtained and purified from the baculovirus expression system in a soluble truncated form according to T al., 1998, Biochem. J. 330, 861). Human alpha _V and beta ₆ cDNA clones, commonly known and used for conventional. 2
Delivery vector pAcUW31 (Clontech La) that allows the simultaneous expression of two different target cDNAs
b. Inc., the USA), was used to express the transmembrane tip lack alpha _V beta ₆ from recombinant baculovirus cells. For this, alpha and _V transfer vector was prepared, with the limit transmembrane absence (.DELTA.TM) alpha _V enzymes EcoRI and XbaI (see above as Mehta et al. Reference), was excised from the plasmid α _V ΔTM (pBAc9) . It was cloned into the BamHI cleavage site of pAcUW31 downstream of the polyhedrin promoter with blunt end ligation.
Using restriction enzymes EcoRI and XbaI transmembrane tip lack beta ₆ of the cDNA pCDNAneoβ _{6 (Weina}
cker et al., supra for reference). And similarly, it was cloned into the BamHI cleavage site of pAcUW31 downstream of the polyhedrin promoter by blunt end ligation.

[0050] The tandem vector comprising a tip lack alpha _V and beta ₆ was used to obtain the recombinant baculovirus (see above as Mehta et al. Reference). Recombinant baculovirus was used to infect High Five insect cells. 48
After ７１71 hours of culture, the soluble receptor was obtained by passing the supernatant from the cell culture through an affinity column of the type described above and eluting at pH 3.1. All process steps were performed at room temperature without surfactant. The peak fractions were neutralized, concentrated, dialyzed at 40 ° C and finally stored at -80 ° C. Thus, the resulting recombinant soluble human receptor is biologically active and retains its ligand properties. A similar preparation method used for soluble α _v β ₃ is described in EP 0846 702.

[0051] EXAMPLE 4 α _V β _{6 /} fibronectin receptor binding test: The prepared peptides of the present invention, together with fibronectin acting antagonism, was bound to immobilized alpha _V beta ₆ receptor in solution, and, alpha _V beta _The Q value was measured as a measure of the selectivity of the binding of the peptides tested for ₆ . Q value in this case is calculated from the quotient of an IC ₅₀ value of the test peptide and the standard. The standard used was the linear hepta-RGD peptide GRGDSPK (cf./patent cf. Pytela et al. Science 231, 1559, (
1986)).

[0052] Specifically, binding studies were performed as follows: The immobilization of soluble alpha _V beta ₆ receptor in microtiter plates, carried out by dilution of the protein solution in TBS ++, then overnight at 4 ° C. incubation (10
0 μl / well). Non-specific binding sites were blocked (200 μl / well) by incubation with 3% (w / v) BSA in TBS ++ (2 hours, 37 ° C.). Excess BS
A was removed by washing three times with TBSA ++. Peptides were serially diluted in TBSA ++ (1:10
) Incubated with biotinylated fibronectin (2 μg / ml) with immobilized integrin (50 μl peptide + 50 μl ligand per well, 2 h, 37 ° C.). Unbound fibronectin and peptide were removed by washing three times with TBSA ++.
Bound fibronectin was detected by incubation with alkaline phosphatase-conjugated anti-biotin antibody (Biorad) (1: 20,000, 100 μl / well in TBSA ++, 1 hour, 37 ° C.). After three washes with TBSA ++, colorimetric detection was performed using a substrate solution (nitrophenyl phosphate 5 mg, ethanolamine 1 ml, H ₂ O 4 ml; 100
μl / well) (10-15 minutes, 25 ° C., in the dark). The enzymatic reaction was stopped by the addition of 0.4 M NaOH (100 μl / well). Color intensities were measured at 405 nm in an ELISA instrument and equalized to zero values. Wells that were not coated with the receptor were assigned a zero value. GRGDSPK was used as the standard. The IC ₅₀ values for the tested peptides were read from the graph, and the Q values of the peptides of the invention were determined together with the IC ₅₀ values of the standard peptides. The results of the tests described are summarized in the following table:

[0053]

[Table 2] Table 2: A Q value of less than 1 indicates that they relatively antagonize the natural ligand fibronectin and have relatively better binding to the receptor than the standard peptide found in the constant term and already having good binding. Means to show.

[0054] Similar to the example of embodiment 5 destination, for comparative purposes, integrin different binding test integrin ligand _{(e.g., α V β 3, α V} β 5) with a ligand corresponding to them (e.g., vitronectin, Fibrinogen).

Example 6 General Preparation of DNA-Liposome Complexes and Use for Gene Therapy Lipids and DNA were converted to Krebs-HEPES solution (140 mM NaCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 6 mM
KCl, 10 mM HEPES, 10 mM D-glucose; pH 9.0), weight ratio 5: 1 (lipid: DNA)
) And mix. Each dose here is 30 μg / 200 μl of DNA. This lipid-DNA
200 μl of the conjugate is applied to the nasal epithelium using a pump nebulizer. Do this 10 times every 15 minutes
Repeat several times. The total dose of DNA is 300 μg.

The following examples relate to pharmaceutical formulations: Example A: Injection vial A solution of 100 g of the active compound of the formula I and 5 g of disodium hydrogenphosphate is brought to pH 6.5 in 3 l of double-distilled water with 2N hydrochloric acid. Conditioned, sterile filtered, filled into injection vials, lyophilized under sterile conditions, and sealed aseptically. Each injection vial contains the active compound
Contains 5 mg.

Example B: Suppository A mixture of 20 g of the active compound of the formula I is mixed with 100 g of soy lectin and 1400 cocoa butter.
Melted with g, poured into molds and cooled. Each suppository contains 20 mg of active compound.

Example C: Solution 1 g of a solution of the active compound of the formula I, 9.38 g of NaH ₂ PO ₄ .2H ₂ O in 940 ml of double-distilled water, Na ₂ HP
28.48 g of O ₄ .12H ₂ O and 0.1 g of benzalkonium chloride were prepared. Adjusted to pH 6.8, made up to 11 and sterilized by radiation. This solution can be used in the form of eye drops.

Example D: Ointment 500 mg of the active compound of the formula I are mixed with 99.5 g of petrolatum under aseptic conditions.

Example E Tablets A mixture of 1 kg of the active compound of the 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 in a conventional manner to give tablets, each of which is obtained. Tablets contain 10 mg of active compound.

Example F: Coated tablets As in Example E, the tablets were pressed and then coated in a conventional manner with a coating of sucrose, potato starch, tragacanth and a colorant.

Example G: Capsules 2 kg of the active compound of the formula I are filled in a customary manner into hard gelatin capsules, each capsule containing 20 mg of the active compound.

Example H Ampoules A solution of 1 kg of the active compound of the formula I in 60 l of double-distilled water is sterile filtered, filled into ampoules, lyophilized under sterile conditions and sealed aseptically. Each ampoule contains 10 mg of active compound.

Example I: Inhalation spray 14 g of the active compound of the formula I are dissolved in 10 l of isotonic NaCl solution and the solution is filled into a spray container having a conventional pump mechanism. The solution can be sprayed on the mouth or nose. One spray (approximately 0.1 ml) corresponds to a dose of approximately 0.14 mg.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 7/02 A61P 9/10 101 9/10 13/12 101 17/02 13/12 17/06 17 / 02 19/02 17/06 19/10 19/02 27/02 19/10 27/06 27/02 29/00 27/06 101 29/00 31/00 101 35/00 31/00 37/08 35 / 00 43/00 111 37/08 C07K 7/06 43/00 111 C12N 7/00 C07K 7/06 15/00 ZNAA C12N 7/00 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY) , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, S , SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG , BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Frankfighter Str. 250, D-64293 Darmstadt, Federal Republic of Germany (72) Inventor Jonzig, Alfred Germany Day-64295 Darmstadt, Shepp-Alley 57 (72) Inventor Kraft, Sabine Germany Day-65668 Rimbach Ha, Gieselherstrasse 8 (72) Inventor Mehta, Ray Mill, UK, England UK 7 A.I.D, Bastan Hall Lane 1-3, MRC Collaboration Center F-term (reference) 4B024 AA01 AA11 BA32 CA02 HA11 HA17 4B065 AA95Y AB01 CA44 CA46 4C084 AA01 AA02 AA03 AA07 BA01 BA08 BA16 BA17 BA18 BA19 BA20 BA21 BA23 CA56 DC50 NA14 ZA332 ZA362 ZA402 ZA452 ZA542 ZA812 ZA892 ZA962 ZA972 ZB112 ZB152 ZB262 ZB322 ZA352 ZA33 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 ZA352 AB ZA97 ZB11 ZB15 ZB26 ZB32 ZB35 ZC35 4H045 AA10 AA30 BA15 BA16 CA01 DA86 EA29 EA53 FA74

Claims (16)

[Claims] 1. A compound of the formula I In the formula, X ¹ , X ² , X ³ , X ⁴ , X ⁵ , and X ⁶ are each independently an amino acid residue, and the amino acids are each independently Ala, Asn, A
sp, Arg, Cys, Gln, Glu, Gly, Phe, His, Ile, L
eu, Lys, Met, Nle, Homo-Phe, Phg, Pro, Ser, Th
selected from the group consisting of r, Trp, Tyr or Val, and the above-mentioned amino acids which may be derivatized; W ¹ is H or Ac; W ² is OH, OR, NHR, NR ₂ , NH is _2, R is alkyl having 1-6 C atoms, n, m are each, independently of one another, a number from 0 to 15, in represented by peptide compounds. 2. The peptide compound according to claim 1, wherein X ² is an amino acid residue selected from the group consisting of Thr, Ser, Asp and glycine. Is wherein ^{X 3, Asp, Glu, Arg} , Lys, His or Ty
The peptide compound according to claim 1, which is an amino acid residue selected from the group consisting of r. 4. The peptide compound according to claim 1, wherein X ⁴ is an amino acid residue selected from the group consisting of Ser, Tyr, Thr, Gly and Val. 5. Formula V The peptide compound according to claim 1, wherein the symbols are as shown in claim 1. 6. Formula VI The peptide compound according to claim 5, which is represented by: 7. Formula I or I according to claim 1 as a medicament
The peptide compounds represented by I and their physiologically acceptable salts. 8. The medicament according to claim 7, which is an inhibitor for disease suppression based on the expression and pathological function of α _v β ₆ integrin receptor. 9. A method for controlling thrombosis, myocardial infarction, coronary heart disease, atherosclerosis, tumor, osteoporosis, fibrosis, inflammation, infection, psoriasis, and promoting the wound healing process. 9. The medicament according to 8. 10. Pharmaceutical formulation comprising at least one medicament according to any of claims 7 to 9, if appropriate a vehicle and / or excipient, and if appropriate other active compounds. 11. The peptide compound according to any one of claims 1 to 6 and / or a physiological agent thereof for the manufacture of a medicament for disease suppression based on the expression and pathological function of α _v β ₆ integrin receptor. Use of acceptable salts. 12. Production of a medicament for controlling thrombosis, myocardial infarction, coronary heart disease, atherosclerosis, tumor, osteoporosis, fibrosis, inflammation, infection, psoriasis, and promoting the wound healing process. The use according to claim 11, for the use. 13. A recombinant DNA comprising a sequence encoding a peptide portion corresponding to the peptide compound according to any one of claims 1 to 6. 14. The recombinant viral DNA according to claim 13. A virus comprising a coat protein having a sequence corresponding to the peptide compound according to any one of claims 1 to 6. 16. Use of the virus according to claim 15, for the manufacture of a medicament for controlling a disease based on expression of an α _v β ₆ integrin receptor and pathological function.