WO2001000660A1 - INHIBITORS OF THE INTEGRIN αvβ¿6? - Google Patents

INHIBITORS OF THE INTEGRIN αvβ¿6? Download PDF

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Publication number
WO2001000660A1
WO2001000660A1 PCT/EP2000/005404 EP0005404W WO0100660A1 WO 2001000660 A1 WO2001000660 A1 WO 2001000660A1 EP 0005404 W EP0005404 W EP 0005404W WO 0100660 A1 WO0100660 A1 WO 0100660A1
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WO
WIPO (PCT)
Prior art keywords
asp
leu
arg
ser
thr
Prior art date
Application number
PCT/EP2000/005404
Other languages
German (de)
French (fr)
Inventor
Alfred Jonczyk
Beate Diefenbach
Ulrich Groth
Gunther Zischinsky
Original Assignee
Merck Patent Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Patent Gmbh filed Critical Merck Patent Gmbh
Priority to CA002377224A priority Critical patent/CA2377224A1/en
Priority to MXPA01013247A priority patent/MXPA01013247A/en
Priority to EP00949177A priority patent/EP1189930A1/en
Priority to SK1872-2001A priority patent/SK18722001A3/en
Priority to BR0011954-7A priority patent/BR0011954A/en
Priority to KR1020017016580A priority patent/KR20020015704A/en
Priority to PL00352374A priority patent/PL352374A1/en
Priority to JP2001507066A priority patent/JP2003503422A/en
Priority to AU62630/00A priority patent/AU771099B2/en
Publication of WO2001000660A1 publication Critical patent/WO2001000660A1/en
Priority to NO20016341A priority patent/NO20016341L/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to novel peptides of the formula I which are biologically active as ligands of the integrin ⁇ v ⁇ ⁇ ,
  • X 6 denotes Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp,
  • amino acids mentioned can also be derivatized, the amino acid residues are peptide-like linked via the ⁇ -amino and ⁇ -carboxy groups, the D- and the L-forms of the optically active amino acid residues are included, as well as their physiologically acceptable salts, and wherein Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 is excluded.
  • the invention was based on the task of finding new compounds with valuable properties, in particular those which can be used for the production of medicaments.
  • the peptides according to the invention can be used as effective inhibitors of the ⁇ v ß integrin receptor and thus for the treatment of various diseases and pathological findings.
  • Other inhibitors of the integrin ⁇ v ß 6 are in DE 19858857 and by S.Kraft et al. in J. Biol. Chem. 274, 1979-85 (1999).
  • the compounds according to the invention are to be regarded as selection inventions in relation to the application mentioned.
  • Integrins belong to the family of heterodimeric class I transmembrane receptors, which play an important role in numerous cell matrix or cell-cell adhesion processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47) , They can be roughly divided into three classes: the ⁇ -integrins, which are receptors for the extracellular matrix, the ⁇ 2 integrins, which can be activated on leukocytes and are "triggered” during inflammatory processes, and the ⁇ v integrins which influence the cell response in wound healing and other pathological processes (Marshall and Hart, 1996, Semin. Cancer Biol. 7, 191).
  • the integrins ⁇ sß-i, ⁇ u ß3, antei, ⁇ v ß ⁇ , ⁇ v ß3, ⁇ v ßs, ⁇ v ßs and ⁇ v ß6 all bind to the Arg-Gly-Asp (RGD) peptide sequence in natural ligands such as fibronectin or vitronectin. Soluble peptides containing RGD are capable of the interaction of each of these integrins with the corresponding natural one
  • ⁇ v ß ⁇ is a relatively rare integrin (Busk et al., 1992 J. Biol. Chem. 267 (9), 5790), which is increasingly formed in epithelial tissue during repair processes and preferentially binds the natural matrix molecules fibronectin and tenascin (Wang et al ., 1996, Am. J. Respir. Cell Mol. Biol. 15 (5), 664).
  • the physiological and pathological functions of ⁇ v ß ⁇ are not yet exactly known, but it is believed that this integrin is important in physiological processes and diseases (e.g. inflammation, wound healing, tumors) in which epithelial cells are involved Role play.
  • ⁇ v ß ⁇ is expressed on keratinocytes in wounds (Haapasalmi et al., 1996, J. Invest.
  • ⁇ v ß ⁇ plays a role in the respiratory epithelium (Weinacker et al., 1995, Am. J. Respir. Cell Mol. Biol. 12 (5), 547), so that corresponding agonists / antagonists of this integrin in respiratory pathological diseases such as bronchitis, asthma, pulmonary fibrosis and respiratory tumors could be used successfully.
  • ⁇ v ⁇ 6 also plays a role in the intestinal epithelium, so that corresponding integrin agonists / antagonists could be used in the treatment of inflammation, tumors and wounds of the gastrointestinal tract.
  • the peptide compounds according to the invention and their salts act as soluble molecules on cells which carry the receptor mentioned or, if they are bound to surfaces, are artificial ligands for ⁇ v ⁇ 6 -mediated cell attachment. Above all, they act as ⁇ v ß 6 integrin inhibitors, in particular inhibiting the interactions of the receptor with other ligands, such as. B. the binding of fibronectin. This effect can be demonstrated, for example, by the method described by JW Smith et al. in J. Biol. Chem. 265, 12267-12271 (1990).
  • the peptide compounds according to the invention can also be used as diagnostics for the detection and localization of pathological conditions in the epithelial system in vivo if they are equipped with appropriate markers (for example the biotinyl residue) according to the prior art.
  • the invention also includes combinations with at least one other active ingredient and / or conjugates with other active ingredients, such as cytotoxic active ingredients and conjugates with radio markers for X-ray therapy or PET diagnosis, but also fusion proteins with marker proteins such as GFP or antibodies, or therapeutic proteins such as IL-2.
  • other active ingredients such as cytotoxic active ingredients and conjugates with radio markers for X-ray therapy or PET diagnosis, but also fusion proteins with marker proteins such as GFP or antibodies, or therapeutic proteins such as IL-2.
  • X 1 is Ser, Gly or Thr;
  • X 2 means Leu
  • X 3 represents Asp or D-Asp
  • X 4 represents Gly, Ala or Ser; in e) X 1 Ser, Gly or Thr,
  • X 5 represents Leu
  • X 6 represents Arg; as well as their salts.
  • the invention relates in particular to peptide compounds selected from the group
  • Trt trityl (T phenylmethyl).
  • amino acids can occur in several enantiomeric forms, all of these forms and also their mixtures (for example the DL forms) are included above and below. Furthermore, the amino acids can be used with corresponding known per se
  • prodrug derivatives are also included in the compounds according to the invention, i. H. with z. B. alkyl or acyl groups, sugars or oligopeptides modified compounds that are quickly cleaved in the organism to the active compounds of the invention.
  • This also includes biodegradable polymer derivatives of the compounds according to the invention, as described, for. B. in Int. J. Pharm. 115, 61-67 (1995).
  • amino acids and amino acid residues mentioned can also be derivatized, the N-methyl, N-ethyl, N-propyl, N-benzyl or C ⁇ - Methyl derivatives are preferred. Further preferred are derivatives of Asp and Glu, in particular the methyl, ethyl, propyl, butyl, tert-butyl,
  • the compounds according to the invention also include compounds in which Ac is replaced by another acyl function, such as propionyl, butyryl or also benzoyl.
  • the compounds according to the invention also include derivatives which consist of the actual peptides according to the invention and known marker compounds which make it possible to easily detect the peptides.
  • derivatives are radio-labeled, biotinylated or fluorescence-labeled peptides.
  • Fluorescent dye residue preferably means 7-acetoxycoumarin-3-yl, fluorescein-5- (and / or 6-) yl, 2 ', 7'-dichlorofluorescein-5- (and 6-) yl, dihydrotetramethylrosamin-4-yl, tetramethylrhodamine 5- (and / or 6-) yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-ethyl or 4,4-difluoro-5,7 diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl.
  • Suitable functionalized fluorescent dye residues that can serve as reagents for the preparation of the compounds of formula I according to the invention are, for. B. described in "Handbook of Fluorescent Probes and Research Chemicals, 5th Edition, 1992-1994, by R.P. Haughland, Molecular Probes, Inc.”.
  • the peptides according to the invention are linear, but they can also be cyclized.
  • the invention includes not only the peptides mentioned but also mixtures and preparations which, in addition to these compounds according to the invention, also contain other pharmacological active ingredients or adjuvants which can influence the primary pharmacological action of the peptides according to the invention in a desired manner.
  • the peptides according to the invention can preferably be prepared by means of solid phase synthesis and subsequent cleavage and purification, as is the case for by Jonczyk and Meienhofer (Peptides, Proc. 8th
  • the peptides according to the invention can be prepared on a solid phase (manually or in an automatic synthesizer) in an Fmoc strategy with acid-labile side-protecting groups and purified by RP-HPLC. Peak uniformity can be measured by RP-HPLC and substance identity using FAB-MS.
  • the peptides can be prepared by conventional methods of amino acid and peptide synthesis, such as. B. from Novabiochem - 1999 Catalog & Peptide Synthesis Handbook from Calbiochem-Novabio-chem GmbH, D-65796 Bad Soden, is known from numerous standard works and published patent applications.
  • Gradual couplings and fragment condensation can be used. Different N-terminal, C-terminal and side protection groups can be used, which are preferably selected to be orthogonally cleavable. Coupling steps can be carried out with different condensation reagents such as carbodiimides, carbodiimidazole, those of the uronium type such as TBTU, mixed anhydride methods, and acid halide or active ester methods. activated Esters are conveniently formed in situ, e.g. B. by adding HOBt or N-hydroxysuccinimide.
  • a cyclization of a linear precursor molecule with side protecting groups can also be carried out with such condensation reactions, such as. B. in DE 43 10 643 or in Houben-Weyl, I.e., Volume 15/11, pages 1 to 806 (1974).
  • Resins can e.g. on polystyrene or
  • Polyacrylamide based, anchor functions such as Wang, o-chlorotrityl are for the production of peptide acids, aminoxanthenoxy anchors e.g. usable for the production of paptidamides.
  • Biotinylated or fluorescence-labeled peptides / proteins can also be produced using standard methods (e.g. EA Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology; and Handbook of Fluorescent Probes and Research Chemicals , 6 th Edition, 1996, by Haugland RP, Molecular Probes, Inc .; or WO 97/14716).
  • the peptides according to the invention can also be released by solvolysis, in particular hydrolysis, or by hydrogenolysis of their functional derivatives.
  • Preferred starting materials for solvolysis or hydrogenolysis are those which contain corresponding protected amino and / or hydroxyl groups instead of one or more free amino and / or hydroxyl groups, preferably those which instead of an H atom which is connected to an N atom is an amino protective group or which carry a hydroxy protective group instead of the H atom of a hydroxy group.
  • carboxylic acids which can be protected by substitution of their -CO-OH hydroxy function by means of a protective group, for example as an ester.
  • amino protecting group is generally known and refers to groups which are suitable for protecting (blocking) an amino group from chemical reactions, but which are easily removable after the desired chemical reaction at other locations on the
  • hydroxyl protecting group is also generally known and refers to groups which are suitable for protecting a hydroxyl group against chemical reactions, but which are easily removable after the desired chemical reaction has been carried out elsewhere in the molecule.
  • the in freedom is also generally known and refers to groups which are suitable for protecting a hydroxyl group against chemical reactions, but which are easily removable after the desired chemical reaction has been carried out elsewhere in the molecule.
  • Carboxylic acids such as trichloroacetic acid or sulfonic acids such as benzene or p-toluenesulfonic acid.
  • Hydrogenolytically removable protective groups e.g. CBZ or benzyl
  • a catalyst z. B. a noble metal catalyst such as palladium, advantageously on a support such as coal.
  • Typical • protecting groups for N-termini and for pendant amino groups are Z, BOC, Fmoc, those for C-termini or the Asp or Glu side chains are O-prim.-alkyl (eg OMe or OEt), O-tert .-Alkyl (e.g. OBut) or OBenzyl.
  • Z, BOC, N0 2 , Mtr, Pmc or Pbf are suitable for the guanidino function of Arg.
  • Alcoholic functions can be protected by tert-alkyl radicals or trityl groups.
  • the groups BOC, OBut and Mtr can e.g. B. preferably with TFA in dichloromethane or with about 3 to 5N HCl in dioxane at 15-30 °, the FMOC group with an about 5 to 50% solution of dimethylamine, diethylamine or piperidine in DMF at 15 -30 °.
  • the trityl group is used, for example, to protect the amino acids histidine, asparagine, glutamine and cysteine.
  • the cleavage is carried out with TFA / 10% thiophenol, the trityl group being cleaved from all the amino acids mentioned; when using TFA / anisole or TFA / thioanisole, only the trityl group of His, Asn and Gin is cleaved, whereas they are remains on the Cys side chain.
  • Hydrogenolytically removable protective groups can e.g. B. by treatment with hydrogen in the presence of a catalyst (z. B. a noble metal catalyst such as palladium, advantageously on a support such as coal).
  • a catalyst z. B. a noble metal catalyst such as palladium, advantageously on a support such as coal.
  • Suitable solvents are the above, especially z. B. alcohols such as methanol or ethanol or amides such as DMF.
  • the hydrogenolysis is generally carried out at temperatures between about 0 and 100 ° and pressures between about 1 and 200 bar, preferably at 20-30 ° and 1-10 bar.
  • Hydrogenolysis of the CBZ group succeeds e.g. B. good on 5 to 10% Pd / C in methanol or with ammonium formate (instead of hydrogen) on Pd / C in methanol / DMF at 20-30 °.
  • the peptides according to the invention comprise their physiologically acceptable salts, which can also be prepared by standard methods.
  • a base of a compound according to the invention can be converted with an acid into the associated acid addition salt, for example by reacting equivalent amounts of the base and the acid in an inert solvent such as ethanol and subsequent evaporation.
  • acids that provide physiologically acceptable salts are suitable for this implementation.
  • inorganic acids can be used, for example sulfuric acid, nitric acid, hydrohalic acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, and also organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or poly-based carbon atoms.
  • sulfuric acid nitric acid
  • hydrohalic acids such as hydrochloric acid or hydrobromic acid
  • phosphoric acids such as orthophosphoric acid
  • sulfamic acid and also organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or poly-based carbon atoms.
  • Sulfonic or sulfuric acids e.g.
  • acetic Acid formic acid, acetic Acid, propionic acid, pivalic 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, 2-dodesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, 2-sulfonic acid, 2-diacid, , p-toluenesulfonic acid, naphthalene mono- and disulfonic acids, laurylsulfuric acid. Salts with physiologically unacceptable acids, such as picrates, can be used
  • Isolation and / or purification of the compounds according to the invention can be used.
  • an acid of the compounds according to the invention can be converted into one of their physiologically acceptable metal or ammonium salts by reaction with a base.
  • Suitable salts are in particular the sodium, potassium, magnesium, calcium and ammonium salts, and also substituted ammonium salts, e.g. B. the dimethyl, diethyl or diisopropylammonium salts, monoethanol, diethanol or diisopropylammonium salts, cyclohexyl, dicyclohexylammonium salts, dibenzylethylenediammonium salts, z. B. salts with arginine or lysine.
  • the peptide compounds according to the invention can be used as active pharmaceutical ingredients in human and veterinary medicine, in particular for the prophylaxis and / or therapy of diseases in which epithelial cells are involved.
  • diseases or inflammations or wound healing processes in the skin, respiratory organs and stomach and intestinal area such as apoplexy, angina pectoris, tumor diseases, osteolytic diseases such as osteoporosis, pathologically angiogenic diseases such as B.
  • Inflammation pulmonary fibrosis, ophthalmic diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerotic disease, psoriasis angiosis, psoriasis disease kidney failure,
  • the invention accordingly relates to peptide compounds of the formulas defined above and below and in the claims, including their physiologically acceptable salts, as medicaments, diagnostics or reagents.
  • the invention relates in particular to corresponding medicaments as inhibitors for combating diseases which are based directly or indirectly on expression of the ⁇ v ⁇ 6 integrin receptor, in particular in the case of pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis. Inflammation, infections and to influence wound healing processes.
  • the invention also relates to corresponding pharmaceutical preparations which contain at least one medicament of the formula I and, if appropriate, carriers and / or auxiliaries.
  • the invention furthermore relates to the use of the peptide compounds and / or their physiologically acceptable salts according to the claims and the description for the manufacture of a medicament for combating diseases which are based, directly or indirectly, on expression of the ⁇ v ⁇ 6 integrin receptor, in particular thus in the case of pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammation, infections and for influencing wound healing processes.
  • the pharmaceuticals according to the invention or pharmaceutical preparations containing them can be used in human or veterinary medicine.
  • Suitable carrier substances are organic or inorganic substances which are suitable for enteral (for example oral), parenteral, topical application or for application in the form of an inhalation spray and which do not react with the new compounds.
  • ren for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycene triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, petroleum jelly.
  • Tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops are used in particular for oral use, supositoses for rectal use, solutions, preferably oily or aqueous solutions, and also suspensions, emulsions or implants for parenteral use , for topical application of ointments, creams or powder.
  • the new compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injectables.
  • the specified preparations can be sterilized and / or auxiliaries such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, flavoring and / or one or more further active substances included, e.g. B. one or more vitamins.
  • auxiliaries such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, flavoring and / or one or more further active substances included, e.g. B. one or more vitamins.
  • sprays can be used which contain the active ingredient either dissolved or suspended in a propellant gas or propellant gas mixture (e.g. C0 2 or chlorofluorocarbons).
  • the active ingredient is expediently used in micronized form, it being possible for one or more additional physiologically acceptable solvents to be present, for. B. ethanol.
  • the substances according to the invention can generally be administered analogously to other known, commercially available peptides (for example described in US Pat. No. 4,472,305), preferably in doses between about 0.05 and 500 mg, in particular between 0.5 and 100 mg per dosage unit.
  • the daily dosage is preferably between about 0.01 and 20 mg / kg body weight.
  • the specific dose for each patient depends on a variety of factors, for example on the effectiveness of the particular compound used, on the age, body weight, general health, gender, on the diet, on the time and route of administration, on which Elimination rate, drug combination and severity of the disease to which the therapy applies. Parenteral administration is preferred.
  • the new compounds according to the invention can also be used as integrin ligands for the preparation of columns for affinity chromatography for the purification of integrins.
  • the complex of an avidin-derivatized carrier material, e.g. Sepharose and the new compounds are formed by methods known per se (e.g. E.A. Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology).
  • Suitable polymeric carrier materials are the polymeric solid phases known per se in peptide chemistry with preferably hydrophilic properties, for example cross-linked poly sugars such as cellulose,
  • Sepharose or Sephadex R acrylamides, polymer based on polyethylene glycol or tentacle polymers R.
  • the invention also comprises recombinant DNA sequences which contain sections which code for peptide regions which have the peptide structural motifs according to the invention.
  • DNA can be transferred to cells by particles, as described in Ch. Andree et al. Proc. 91, 12188-12192 (1994), or the transfer to cells can be increased by other means such as liposomes (Al Aronsohn and JA Hughes J. Drug Targeting, 5, 163-169 (1997)).
  • the peptides according to the invention ultimately formed by the infected cells themselves can bind directly to the ⁇ v ß 6 integrin receptor, for example of tumor cells, and block it.
  • Corresponding recombinant DNA which can be provided by known and customary techniques, can also be present, for example, in the form of virus DNA which contains sections which code for the virus coat protein.
  • virus DNA which contains sections which code for the virus coat protein.
  • Suitable viruses are, for example, adenovirus types which have been used several times as vectors for foreign genes in mammalian cells. A number of traits make them good candidates for gene therapy, such as S.J. Watkins et al. Gene Therapy 4, 1004-1012 (1997) can be found (see also J. Engelhardt et al. Hum. Gene Ther. 4, 759-769 (1993)).
  • Fibrosis transmembrane conductance regulator (CFTR) cDNA can be achieved.
  • the DNA for the sequences of this invention can also be used for cell transfections by means of retroviral or adenoviral vectors.
  • the peptides according to the invention can also be used within a liposome complex made of lipid / peptide / DNA for a transfection of cell cultures together with a liposome complex consisting of lipid / DNA (without peptide) for use in gene therapy in humans.
  • a liposome complex made of lipid / peptide / DNA for a transfection of cell cultures together with a liposome complex consisting of lipid / DNA (without peptide) for use in gene therapy in humans.
  • the production of a liposome complex from lipid / DNA peptide is described, for example, by Hart SL, et al 1998: Lipid-Mediated Enhancement of Transfection by a Non-Viral Integrin-Targeting Vector. Human Gene Therapy 9, 575-585.
  • DOTMA N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethyl- ammonium chloride
  • DOPE dioleyl phosphatidylethanolamine
  • Liposome DNA complexes for gene therapy in humans have already been described (Caplen NJ, et al 1995: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis Nature Medicine 1, 39-46).
  • the invention thus also relates to the use of appropriately modified recombinant DNA from gene-releasing systems, in particular virus DNA, for combating diseases which are based directly or indirectly on expression of ⁇ v ⁇ 6 integrin receptors, in particular in the case of pathologically angiogenic diseases,
  • Thrombosis heart attack, coronary heart disease, arteriosclerosis,
  • the HPLC analyzes (retention time Rt) were carried out in the following
  • the product was purified by RP-HPLC on Lichrosorb RP 18 (250 - 25, 7 ⁇ m, Merck KGaA) in 0.3% TFA with a gradient from 4% to 24% 2-propanol in one hour at 10 ml / min and assessment of the eluate using a UV flow photometer at 215 and 254 nm. 168 mg of product were obtained; Rt 15.5 min; FAB 973.
  • the following products were manufactured analogously:
  • the peptides according to the invention which were produced were bound to the immobilized ⁇ v ⁇ 6 receptor in solution together with fibronectin having a competitive effect and the Q value was determined as a measure of the selectivity of the binding of the peptide to be tested to ⁇ v ⁇ ⁇ .
  • the Q value is calculated from the quotient of the IC 5 o values of test peptide and a standard.
  • the linear Ac-RTDLDSLR-NH 2 (code EMD 271293) was used as the standard (lit./patent, see Pytela et al. Science 231, 1559, (1986)).
  • the binding test was carried out in detail as follows: The immobilization of soluble ⁇ v ⁇ 6 receptor on microtiter plates was carried out by diluting the protein solution in TBS ++ and then incubating overnight at 4 ° C. (100 ⁇ l / well). Unspecific binding sites were blocked by incubation (2 h, 37 ° C) with 3% (w / v) BSA in TBS ++ (200 ⁇ l / well). Excess BSA was removed by washing three times with TBSA ++.
  • Peptides were serially (1:10) diluted in TBSA ++ and incubated together with biotinylated fibronectin (2 ⁇ g / ml) with the immobilized integrin (50 ⁇ l peptide + 50 ⁇ l ligand per well; 2 h; 37 ° C). Unbound fibronectin and peptides were removed by washing three times with TBSA ++. The bound fibronectin was detected by incubation (1 h; 37 ° C) with an alkaline phosphatase-coupled anti-biotin antibody (Biorad) (1: 20000 in TBSA ++; 100 ul / well).
  • Biorad alkaline phosphatase-coupled anti-biotin antibody
  • the colohmetric 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 H 2 0; 100 ⁇ l / well). The enzyme reaction was stopped by adding 0.4 M NaOH (100 ul / well). The color intensity was determined at 405 nm in the ELISA measuring device and compared to the zero value. Wells that were not coated with receptor served as zero value. Ac-RTDLDSLR-NH 2 was used as the standard. The IC 5 o values for the peptides tested were read off from a graph and determines together with the IC50 of the standard peptide, the Q value of the peptide according to the invention.
  • Example A Injection glasses
  • a solution of 100 g of Ac-RGDLdSLR-NH 2 and 5 g of disodium hydrogenphosphate is adjusted to pH 6.5 in 3 l of double-distilled water with 2N hydrochloric acid, sterile filtered, filled into injection glasses, lyophilized and sterile sealed under sterile conditions. Each injection jar contains 5 mg of active ingredient.
  • Example B Suppositories A mixture of 20 g Ac-RGDLdSLR-NH 2 is melted with 100 g soy lecithin and 1400 g cocoa butter, poured into molds and allowed to cool. Each suppository contains 20 mg of active ingredient.
  • a solution is prepared from 1 g Ac-RGDLdSLR-NH 2 , 9.38 g NaH 2 P0 4 • 2 H 2 0, 28.48 g Na 2 HP0 4 • 12 H 2 0 and 0.1 g benzalkonium chloride in 940 ml double distilled water. It is adjusted to pH 6.8, made up to 1 I and sterilized by irradiation.
  • This solution can take the form of
  • Eye drops can be used.
  • Example D ointment
  • a mixture of 1 kg of Ac-RGDLdSLR-NH 2 , 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 tablets in a conventional manner such that each tablet contains 10 mg of active ingredient ,
  • Example F coated tablets
  • Example E tablets are pressed, which are then coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and colorant.
  • Example G capsules
  • Example H ampoules A solution of 1 kg of Ac-RGDLdSLR-NH 2 in 60 l of double-distilled water is sterile filtered, filled into ampoules, lyophilized under sterile conditions and sealed sterile. Each ampoule contains 10 mg of active ingredient.

Abstract

The invention relates to novel peptides of formula (I) Ac-Arg-X?1-Asp-X2-X3-X4-X5-X6-NH¿2 which are biologically active as ligands of the integrin αvβ6, wherein: Ac represents Acetyl, X1 represents Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala; X2 represents Leu, Ile, Nle, Val or Phe; X3 represents Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg; X4 represents Gly, Ala, Ser, β-Ala or φ-Abu; X5 represents Leu, Ile, Nle, Val or Phe, and; X6 represents Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp, whereby the aforementioned amino acids can be derivatized. The amino acid radicals are linked to one another in a peptide-like manner via the α-amino groups and the α-carboxy groups. The dextrorotatory forms and the levorotatory forms of the optically active amino acid radicals are contained as well as the salts of said peptides. Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH¿2? is excluded. The inventive peptides can also be used as effective inhibitors of the αvβ6 integrin receptor and thus for treating different diseases and pathological findings.

Description

Inhibitoren des Integrins αvßβInhibitors of the integrin α v ßβ
Die Erfindung betrifft neuartige Peptide der Formel I, welche als Liganden des Integrins αvßδ biologisch wirksam sind,The invention relates to novel peptides of the formula I which are biologically active as ligands of the integrin α v β δ ,
Ac-Arg-X1-Asp-X2-X3-X4-X5-X6-NH2 IAc-Arg-X 1 -Asp-X 2 -X 3 -X 4 -X 5 -X 6 -NH 2 I
worinwherein
Ac Acetyl,Ac Acetyl,
X1 Ser, Gly, Thr, Asp, Arg, Val, Tyr, His oder Ala,X 1 Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala,
X2 Leu, Ile, Nie, Val oder Phe,X 2 Leu, Ile, Nie, Val or Phe,
X3 Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl oder Phg,X 3 Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg,
X4 Gly, Ala, Ser, ß-Ala oder ω-Abu,X 4 Gly, Ala, Ser, ß-Ala or ω-Abu,
X5 Leu, Ile, Nie, Val oder Phe,X 5 Leu, Ile, Nie, Val or Phe,
X6 Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser oder Asp bedeuten,X 6 denotes Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp,
wobei die genannten Aminosäuren auch derivatisiert sein können, die Aminosäurereste über die α-Amino- und α-Carboxygruppen peptidartig miteinander verknüpft sind, die D- als auch die L-Formen der optisch aktiven Aminosäurereste eingeschlossen sind, sowie deren physiologisch unbedenklichen Salze, und wobei Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 ausgenommen ist.wherein the amino acids mentioned can also be derivatized, the amino acid residues are peptide-like linked via the α-amino and α-carboxy groups, the D- and the L-forms of the optically active amino acid residues are included, as well as their physiologically acceptable salts, and wherein Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 is excluded.
Der Erfindung lag die Aufgabe zugrunde, neue Verbindungen mit wertvollen Eigenschaften aufzufinden, insbesondere solche, die zur Herstellung von Arzneimitteln verwendet werden können.The invention was based on the task of finding new compounds with valuable properties, in particular those which can be used for the production of medicaments.
Es wurde gefunden, daß die erfindungsgemäßen Verbindungen und ihre Salze bei guter Verträglichkeit sehr wertvolle pharmakologische Eigenschaften besitzen.It has been found that the compounds according to the invention and their salts have very valuable pharmacological properties with good tolerability.
Die erfindungsgemäßen Peptide können als wirksame Inhibitoren des αvße Integrin-Rezeptors und somit zur Behandlung verschiedener Krankheiten und pathologischer Befunde eingesetzt werden. Andere Inhibitoren des Integrins αvß6 sind in der DE 19858857 und von S.Kraft et al. in J. Biol. Chem.274, 1979-85 (1999) beschrieben. Die erfindungsgemäßen Verbindungen sind in bezug auf die genannte Anmeldung als Auswahlerfindung zu betrachten.The peptides according to the invention can be used as effective inhibitors of the α v ß integrin receptor and thus for the treatment of various diseases and pathological findings. Other inhibitors of the integrin α v ß 6 are in DE 19858857 and by S.Kraft et al. in J. Biol. Chem. 274, 1979-85 (1999). The compounds according to the invention are to be regarded as selection inventions in relation to the application mentioned.
Integrine gehören zu der Familie von heterodimeren Klasse I - Transmembran-Rezeptoren, die in zahlreichen Zeil-Matrix- bzw. Zell-Zell- Adhäsionsvorgängen eine wichtige Rolle spielen (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47). Sie können grob in drei Klassen eingeteilt werden: die ß-i-lntegrine, die Rezeptoren für die extrazelluläre Matrix darstellen, die ß2-lntegrine, welche auf Leukozyten aktivierbar sind und während inflammatorischen Prozessen "getriggert" werden, sowie die αv - Integrine, die die Zellantwort bei Wundheilungs- und anderen pathologischen Prozessen beeinflussen (Marshall and Hart, 1996, Semin. Cancer Biol. 7, 191).Integrins belong to the family of heterodimeric class I transmembrane receptors, which play an important role in numerous cell matrix or cell-cell adhesion processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47) , They can be roughly divided into three classes: the β-integrins, which are receptors for the extracellular matrix, the β 2 integrins, which can be activated on leukocytes and are "triggered" during inflammatory processes, and the α v integrins which influence the cell response in wound healing and other pathological processes (Marshall and Hart, 1996, Semin. Cancer Biol. 7, 191).
Die Integrine αsß-i, αu ß3, aßi, αvßι, αvß3, αvßs, αvßs und αvß6 binden alle an die Arg-Gly-Asp (RGD) Peptidsequenz in natürlichen Liganden, wie z.B. Fibronektin oder Vitronektin. Lösliche RGD-haltige Peptide vermögen die Interaktion jedes dieser Integrine mit dem entsprechenden natürlichenThe integrins αsß-i, αu ß3, aßi, α v ßι, α v ß3, α v ßs, α v ßs and α v ß6 all bind to the Arg-Gly-Asp (RGD) peptide sequence in natural ligands such as fibronectin or vitronectin. Soluble peptides containing RGD are capable of the interaction of each of these integrins with the corresponding natural one
Liganden zu inhibieren. αvßβ ist ein relativ seltenes Integrin (Busk et al., 1992 J. Biol. Chem. 267(9), 5790), das bei Reperaturvorgängen in Epithelgewebe vermehrt gebildet wird und die natürlichen Matrixmoleküle Fibronectin und Tenascin bevorzugt bindet (Wang et al., 1996, Am. J. Respir. Cell Mol. Biol. 15(5), 664). Die physiologischen und pathologischen Funktionen von αvßδ sind noch nicht genau bekannt, es wird jedoch vermutet, daß dieses Integrin bei physiologischen Vorgängen und Erkrankungen (z. B. Entzündungen, Wundheilung, Tumore), bei denen epitheliale Zellen beteiligt sind, eine wichtige Rolle spielt. So wird αvßδ auf Keratinozyten in Wunden exprimiert (Haapasalmi et al., 1996, J. Invest.Inhibit ligands. α v ßβ is a relatively rare integrin (Busk et al., 1992 J. Biol. Chem. 267 (9), 5790), which is increasingly formed in epithelial tissue during repair processes and preferentially binds the natural matrix molecules fibronectin and tenascin (Wang et al ., 1996, Am. J. Respir. Cell Mol. Biol. 15 (5), 664). The physiological and pathological functions of α v ß δ are not yet exactly known, but it is believed that this integrin is important in physiological processes and diseases (e.g. inflammation, wound healing, tumors) in which epithelial cells are involved Role play. Thus, α v ß δ is expressed on keratinocytes in wounds (Haapasalmi et al., 1996, J. Invest.
Dermatol. 106(1), 42), woraus anzunehmen ist, daß neben Wundheilungs- prozessen und Entzündungen auch andere pathologische Ereignisse der Haut, wie z. B. Psoriasis, durch Agonisten oder Antagonisten des besagten Integrins beeinflußbar sind. Ferner spielt αvßβ im Atemwegsepithel eine Rolle (Weinacker et al., 1995, Am. J. Respir. Cell Mol. Biol. 12(5), 547), so daß entsprechende Agonisten / Antagonisten dieses Integrins bei Atem- wegserkrankungen, wie Bronchitis, Asthma, Lungenfibrosen und Atemwegstumoren erfolgreich eingesetzt werden könnten. Letztlich ist bekannt, daß αvß6 auch im Darmepithel eine Rolle spielt, so daß entsprechende lntegrin-Agonisten/-Antagonisten bei der Behandlung von Entzündungen, Tumoren und Wunden des Magen/Darmtraktes Verwendung finden könnten.Dermatol. 106 (1), 42), from which it can be assumed that, in addition to wound healing processes and inflammation, other pathological events of the skin, such as, for example, B. psoriasis, can be influenced by agonists or antagonists of said integrin. Furthermore, α v ßβ plays a role in the respiratory epithelium (Weinacker et al., 1995, Am. J. Respir. Cell Mol. Biol. 12 (5), 547), so that corresponding agonists / antagonists of this integrin in respiratory pathological diseases such as bronchitis, asthma, pulmonary fibrosis and respiratory tumors could be used successfully. Ultimately, it is known that α v β6 also plays a role in the intestinal epithelium, so that corresponding integrin agonists / antagonists could be used in the treatment of inflammation, tumors and wounds of the gastrointestinal tract.
Die Abhängigkeit der Entstehung von Angiogenese von der Wechselwirkung zwischen vaskulären Integrinen und extrazelluiären Matrix- proteinen ist von P.C. Brooks, R.A. Clark und D.A. Cheresh in Science 264, 569-71 (1994) beschrieben.The dependence of the development of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins has been reported by P.C. Brooks, R.A. Clark and D.A. Cheresh in Science 264, 569-71 (1994).
Es bestand somit die Aufgabe, neben den bisher bekannten natürlichen hochmolekularen Liganden und Antikörpern, die therapeutisch und diagnostisch schwer handhabbar sind, potente, spezifische bzw. selektive niedermolekulare Liganden für αvßβ, vorzugsweise Peptide, zu finden, die für die genannten therapeutischen Gebiete aber auch als Diagnostikum oder Reaqenz verwendet werden können.It was therefore the task, in addition to the previously known natural high molecular weight ligands and antibodies, which are difficult to handle therapeutically and diagnostically, to find potent, specific or selective low molecular weight ligands for α v β, preferably peptides, but for the therapeutic areas mentioned can also be used as a diagnostic or reagent.
Es wurde gefunden, daß die erfindungsgemäßen peptidischen Verbindungen und ihre Salze als lösliche Moleküle Wirkung auf Zellen ausüben, die den genannten Rezeptor tragen, oder wenn sie an Oberflächen gebunden sind, künstliche Liganden für die αvß6 - vermittelte Zellanhaftung darstellen. Vor allem wirken sie als αvß6 Integrin-Inhibitoren, wobei sie insbesondere die Wechselwirkungen des Rezeptors mit anderen Liganden hemmen, wie z. B. die Bindung von Fibronektin. Diese Wirkung kann z.B. nach der Methode nachgewiesen werden, die von J.W. Smith et al. in J. Biol. Chem. 265, 12267-12271 (1990) beschrieben wird.It has been found that the peptide compounds according to the invention and their salts act as soluble molecules on cells which carry the receptor mentioned or, if they are bound to surfaces, are artificial ligands for α v β 6 -mediated cell attachment. Above all, they act as α v ß 6 integrin inhibitors, in particular inhibiting the interactions of the receptor with other ligands, such as. B. the binding of fibronectin. This effect can be demonstrated, for example, by the method described by JW Smith et al. in J. Biol. Chem. 265, 12267-12271 (1990).
Weiter wurde gefunden, daß die neuen Substanzen bei guter Verträglichkeit sehr wertvolle pharmakologische Eigenschaften besitzen und als Arzneimittel eingesetzt werden können. Dies wird weiter unten genauer beschrieben. Die erfindungsgemäßen peptidischen Verbindungen können ferner als Diagnostika zur Detektion und Lokalisierung von pathologischen Zuständen im epithelialen System in vivo verwendet werden, wenn sie mit ent- sprechenden Markern (z.B. dem Biotinylrest ) nach dem Stand der Technik ausgestattet sind.It was also found that the new substances have very good pharmacological properties and are well tolerated and can be used as medicaments. This is described in more detail below. The peptide compounds according to the invention can also be used as diagnostics for the detection and localization of pathological conditions in the epithelial system in vivo if they are equipped with appropriate markers (for example the biotinyl residue) according to the prior art.
Die Erfindung umfaßt auch Kombinationen mit mindestens einem anderen Wirkstoff und/oder Konjugate mit anderen Wirkstoffen, wie zytotoxischen Wirkstoffen sowie Konjugate mit Radiomarkern für Röntgentherapie oder PET Diagnose aber auch Fusionsproteine mit Markerproteinen wie GFP oder Antikörpern, oder therapeutischen Proteinen wie IL-2.The invention also includes combinations with at least one other active ingredient and / or conjugates with other active ingredients, such as cytotoxic active ingredients and conjugates with radio markers for X-ray therapy or PET diagnosis, but also fusion proteins with marker proteins such as GFP or antibodies, or therapeutic proteins such as IL-2.
Einige bevorzugte Gruppen von Verbindungen können durch die folgenden Teilformeln la bis If ausgedrückt werden, die der Formel I entsprechen und worin die nicht näher bezeichneten Reste die bei der Formel I angegebene Bedeutung haben, worin jedochSome preferred groups of compounds can be expressed by the following sub-formulas Ia to If, which correspond to the formula I and in which the radicals not specified have the meaning given for the formula I, but in which
in a) X1 Ser, Gly oder Thr, bedeutet;in a) X 1 is Ser, Gly or Thr;
in b) X1 Ser, Gly oder Thr,in b) X 1 Ser, Gly or Thr,
X2 Leu, bedeutet;X 2 means Leu;
in c) X1 Ser, Gly oder Thr,in c) X 1 Ser, Gly or Thr,
X2 Leu,X 2 Leu,
X3 Asp oder D-Asp bedeutet;X 3 represents Asp or D-Asp;
in d) X1 Ser, Gly oder Thr,in d) X 1 Ser, Gly or Thr,
X2 Leu,X 2 Leu,
X3 Asp oder D-Asp,X 3 Asp or D-Asp,
X4 Gly, Ala oder Ser, bedeutet; in e) X1 Ser, Gly oder Thr,X 4 represents Gly, Ala or Ser; in e) X 1 Ser, Gly or Thr,
X2 Leu,X 2 Leu,
X3 Asp oder D-Asp,X 3 Asp or D-Asp,
X4 Gly, Ala oder Ser,X 4 Gly, Ala or Ser,
X5 Leu bedeutet;X 5 represents Leu;
in f) X1 Ser, Gly oder Thr,in f) X 1 Ser, Gly or Thr,
X2 Leu,X 2 Leu,
X3 Asp oder D-Asp,X 3 Asp or D-Asp,
X4 Gly, Ala oder Ser,X 4 Gly, Ala or Ser,
X5 Leu,X 5 Leu,
X6 Arg bedeutet; sowie deren Salze.X 6 represents Arg; as well as their salts.
Gegenstand der Erfindung sind insbesondere peptidische Verbindungen ausgewählt aus der GruppeThe invention relates in particular to peptide compounds selected from the group
Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NH2, Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NH 2 , Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH2, Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH 2 , Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NH2,Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH2, sowie deren physiologisch unbedenklichen Salze.Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH 2 , and their physiologically acceptable salts.
Die vor- und nachstehend aufgeführten Abkürzungen von Aminosäureresten stehen für die Reste folgender Aminosäuren:The abbreviations of amino acid residues listed above and below stand for the residues of the following amino acids:
Abu 4-AminobuttersäureAbu 4-aminobutyric acid
Aha 6-Aminohexansäure, 6-AminocapronsäureAha 6-aminohexanoic acid, 6-aminocaproic acid
Aib α-Amino-isobuttersäureAib α-amino isobutyric acid
Ala AlaninAla alanine
Asn AsparaginAsn asparagine
Asp AsparaginsäureAsp aspartic acid
Arg ArgininArg arginine
Bgl C-alpha-tert.-ButylglycinBgl C-alpha-tert-butylglycine
Cys CysteinCys cysteine
Dab 2,4-DiaminobuttersäureDab 2,4-diaminobutyric acid
Dap 2,3-DiaminopropionsäureDap 2,3-diaminopropionic acid
Gin GlutaminGin glutamine
Glp PyroglutaminsäureGlp pyroglutamic acid
Glu GlutaminsäureGlu glutamic acid
Gly Gly einGly Gly a
Har HomoargininHar homoarginine
His Histidin homo-Phe homo-PhenylalaninHis histidine homo-Phe homo-phenylalanine
Ile IsoleucinIle isoleucine
Leu LeucinLeu leucine
Lys LysinLys lysine
Met Methionin Nal Naphth-2-yl-alaninWith methionine Nal Naphth-2-yl alanine
Nie NorleucinNever norleucine
Orn 0 mithinOrn 0 therefore
Phe PhenylalaninPhe phenylalanine
Phg PhenylglycinPhg phenylglycine
4-Hal-Phe 4-Halogen-phenylalanin4-Hal-Phe 4-halophenylalanine
Pro ProliπPro Proliπ
Ser SerinSer Serin
Thr ThreoninThr threonine
Trp TryptophanTrp tryptophan
Tyr TyrosinTyr tyrosine
Val Valin.Val valine.
Ferner bedeuten nachstehend:Furthermore, below mean:
Ac AcetylAc Acetyl
BOC tert.-ButoxycarbonylBOC tert-butoxycarbonyl
BSA Bovine Serum AlbuminBSA Bovine Serum Albumin
CBZ oder Z BenzyloxycarbonylCBZ or Z benzyloxycarbonyl
DCCI DicyclohexylcarbodiimidDCCI dicyclohexylcarbodiimide
DMF DimethylformamidDMF dimethylformamide
EDCI N-Ethyl-N,N'-(dimethylaminopropyl)-carbodiimidEDCI N-ethyl-N, N '- (dimethylaminopropyl) carbodiimide
Et EthylEt ethyl
FCA FluoresceincarbonsäureFCA fluorescein carboxylic acid
FITC FluoresceinisothiocyanatFITC fluorescein isothiocyanate
Fmoc 9-FluorenylmethoxycarbonylFmoc 9-fluorenylmethoxycarbonyl
FTH FluoresceinthiohamstoffFTH fluoresceinthiourea
HOBt 1-HydroxybenzotriazolHOBt 1-hydroxybenzotriazole
Me MethylMe methyl
MBHA 4-Methyl-benzhydrylaminMBHA 4-methyl-benzhydrylamine
Mtr 4-Methoxy-2,3,6-trimethylphenyl-sulfonylMtr 4-methoxy-2,3,6-trimethylphenyl sulfonyl
HONSu N-HydroxysuccinimidHONSu N-hydroxysuccinimide
OBut tert.-ButylesterOBut tert-butyl ester
Oct OctanoylOct octanoyl
OMe MethylesterOMe methyl ester
OEt Ethylester Pbf 2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonylOEt ethyl ester Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonylPmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
POA PhenoxyacetylPOA phenoxyacetyl
Sal SalicyloylSal salicyloyl
TBS++ Tris buffered Saline mit 2-wertigen KationenTBS ++ Tris buffered saline with divalent cations
TBSA TBS+BSATBSA TBS + BSA
TBTU 2-(1 H-Benzotriazol-1 -yl)-1 , 1 ,3-tetramethyluronium tetrafiuoroboratTBTU 2- (1 H-benzotriazol-1-yl) -1, 1, 3-tetramethyluronium tetrafiuoroborate
TFA TrifluoressigsäureTFA trifluoroacetic acid
Trt Trityl (T phenylmethyl).Trt trityl (T phenylmethyl).
Sofern die vorstehend genannten Aminosäuren in mehreren enantiomeren Formen auftreten können, so sind vor- und nachstehend, alle diese Formen und auch ihre Gemische (z. B. die DL-Formen) eingeschlossen. Ferner können die Aminosäuren mit entsprechenden an sich bekanntenIf the above-mentioned amino acids can occur in several enantiomeric forms, all of these forms and also their mixtures (for example the DL forms) are included above and below. Furthermore, the amino acids can be used with corresponding known per se
Schutzgruppen versehen sein.Protecting groups.
In die erfindungsgemäßen Verbindungen sind auch sogenannte Prodrug- Derivate eingeschlossen, d. h. mit z. B. Alkyl- oder Acylgruppen, Zuckern oder Oligopeptiden abgewandelte Verbindungen, die im Organismus rasch zu den wirksamen erfindungsgemäßen Verbindungen gespalten werden. Hierzu gehören auch bioabbaubare Polymerderivate der erfindungsgemäßen Verbindungen, wie dies z. B. in Int. J. Pharm. 115, 61-67 (1995) beschrieben ist.So-called prodrug derivatives are also included in the compounds according to the invention, i. H. with z. B. alkyl or acyl groups, sugars or oligopeptides modified compounds that are quickly cleaved in the organism to the active compounds of the invention. This also includes biodegradable polymer derivatives of the compounds according to the invention, as described, for. B. in Int. J. Pharm. 115, 61-67 (1995).
Die genannten Aminosäuren und Aminosäurereste, wie z.B. die NH- Funktionen oder auch die C-terminale Amidfunktion können auch derivati- siert sein, wobei die N-Methyl-, N-Ethyl-, N-Propyl-, N-Benzyl- oder Cα- Methylderivate bevorzugt sind. Weiter bevorzugt sind Derivate von Asp und Glu, insbesondere die Methyl-, Ethyl, Propyl, Butyl, tert.-Butyl,The amino acids and amino acid residues mentioned, such as, for example, the NH functions or the C-terminal amide function, can also be derivatized, the N-methyl, N-ethyl, N-propyl, N-benzyl or C α - Methyl derivatives are preferred. Further preferred are derivatives of Asp and Glu, in particular the methyl, ethyl, propyl, butyl, tert-butyl,
Neopentyl- oder Benzylester der Seitenketten-carboxy-gruppen, ferner auch Derivate von Arg, das an der -NH-C(=NH)-NH2 -Gruppe mit einem Acetyl-, Benzoyl-, Methoxycarbonyi- oder Ethoxycarbonylrest substituiert sein kann. In den erfindungsgemäßen Verbindungen sind neben den Verbindungen der Formel I, die N-terminal eine Acetylgruppe tragen auch solche Verbindungen eingeschlossen, in denen Ac durch eine andere Acylfunktion ersetzt ist, wie z.B. Propionyl, Butyryl oder auch Benzoyl.Neopentyl or benzyl ester of the side chain carboxy groups, also derivatives of Arg, which may be substituted on the -NH-C (= NH) -NH 2 group with an acetyl, benzoyl, methoxycarbonyl or ethoxycarbonyl radical. In addition to the compounds of the formula I which carry an acetyl group at the N-terminal, the compounds according to the invention also include compounds in which Ac is replaced by another acyl function, such as propionyl, butyryl or also benzoyl.
Ferner sind in die erfindungsgemäßen Verbindungen auch Derivate mit eingeschlossen, welche aus den eigentlichen erfindungsgemäßen Pep- tiden und bekannten Marker-Verbindungen bestehen, die es ermöglichen, die Peptide leicht nachzuweisen. Beispiele für solche Derivate sind radioaktiv markierte, biotinylierte oder fluoreszenzmarkierte Peptide.Furthermore, the compounds according to the invention also include derivatives which consist of the actual peptides according to the invention and known marker compounds which make it possible to easily detect the peptides. Examples of such derivatives are radio-labeled, biotinylated or fluorescence-labeled peptides.
Fluoreszierender Farbstoffrest bedeutet vorzugsweise 7-Acetoxycoumarin- 3-yl, Fluorescein-5-(und/oder 6-)yl, 2',7'-Dichlorfluorescein-5-(und 6-)yl, Dihydrotetramethylrosamin-4-yl, Tetramethylrhodamin-5-(und/oder 6-)yl, 4,4-Difluor-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-ethyl oder 4,4- Difluor-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacen-3-ethyl.Fluorescent dye residue preferably means 7-acetoxycoumarin-3-yl, fluorescein-5- (and / or 6-) yl, 2 ', 7'-dichlorofluorescein-5- (and 6-) yl, dihydrotetramethylrosamin-4-yl, tetramethylrhodamine 5- (and / or 6-) yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-ethyl or 4,4-difluoro-5,7 diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl.
Geeignete funktionalisierte fluoreszierende Farbstoffreste, die als Reagenzien zur Herstellung der erfindungsgemäßen Verbindungen der Formel I dienen können, sind z. B. beschrieben in "Handbook of Fluorescent Probes and Research Chemicals, 5th Edition, 1992-1994, by R.P. Haughland, Molecular Probes, Inc.".Suitable functionalized fluorescent dye residues that can serve as reagents for the preparation of the compounds of formula I according to the invention are, for. B. described in "Handbook of Fluorescent Probes and Research Chemicals, 5th Edition, 1992-1994, by R.P. Haughland, Molecular Probes, Inc.".
Im allgemeinen sind die erfindungsgemäßen Peptide linear, sie können aber auch zyklisiert werden. Die Erfindung umfaßt nicht nur die genannten Peptide sondern auch Mischungen und Zubereitungen, welche neben diesen erfindungsgemäßen Verbindungen auch andere pharmakologische Wirkstoffe oder Adjuvantien enthalten, die die primäre pharmakologische Wirkung der erfindungsgemäßen Peptide in gewünschter Weise beinflussen können.In general, the peptides according to the invention are linear, but they can also be cyclized. The invention includes not only the peptides mentioned but also mixtures and preparations which, in addition to these compounds according to the invention, also contain other pharmacological active ingredients or adjuvants which can influence the primary pharmacological action of the peptides according to the invention in a desired manner.
Die erfindungsgemäßen Verbindungen und auch die Ausgangsstoffe zu ihrer Herstellung werden im übrigen nach an sich bekannten und häufig eingesetzten Methoden hergestellt, wie sie in der Literatur (z.B. in denThe compounds of the invention and also the starting materials for their preparation are otherwise known and common methods used, as they are in the literature (e.g. in the
Standardwerken wie Houben-Weyl, Methoden der organischen Chemie,Standard works such as Houben-Weyl, methods of organic chemistry,
Georg-Thieme-Verlag, Stuttgart) beschrieben sind, und zwar unterGeorg-Thieme-Verlag, Stuttgart) are described under
Reaktionsbedingungen, die für die genannten Umsetzungen bekannt und geeignet sind. Dabei kann man auch von an sich bekannten VariantenReaction conditions which are known and suitable for the reactions mentioned. You can also use known variants
Gebrauch machen.Make use.
Vorzugsweise können die erfindungsgemäßen Peptide mittels Fest- phasensynthese und nachfolgender Abspaltung und Reinigung hergestellt werden, wie dies z.B. von Jonczyk und Meienhofer (Peptides, Proc. 8thThe peptides according to the invention can preferably be prepared by means of solid phase synthesis and subsequent cleavage and purification, as is the case for by Jonczyk and Meienhofer (Peptides, Proc. 8th
Am. Pept. Symp., Eds. V. Hruby und D.H. Rieh, Pierce Comp. III, p. 73-77,At the. Pept. Symp., Eds. V. Hruby and D.H. Rieh, Pierce Comp. III, p. 73-77,
1983, oder Angew. Chem. 104, 1992, 375) oder gemäß Merrifield (J. Am.1983, or Angew. Chem. 104, 1992, 375) or according to Merrifield (J. Am.
Chem. Soc. 94, 1972, 3102) beschrieben wurde.Chem. Soc. 94, 1972, 3102).
Die erfindungsgemäßen Peptide können an fester Phase (manuell oder in einem Syntheseautomaten) in einer Fmoc-Strategie mit säurelabilen Seitenschutzgruppen hergestellt und mittels RP-HPLC gereinigt werden. Die Peakeinheitlichkeit kann durch RP-HPLC und die Substanzidentität mittels FAB-MS gemessen werden.The peptides according to the invention can be prepared on a solid phase (manually or in an automatic synthesizer) in an Fmoc strategy with acid-labile side-protecting groups and purified by RP-HPLC. Peak uniformity can be measured by RP-HPLC and substance identity using FAB-MS.
Im übrigen können die Peptide nach üblichen Methoden der Aminosäure- und Peptidsynthese hergestellt werden, wie dies z. B. aus Novabiochem - 1999 Catalog & Peptide Synthesis Handbook der Calbiochem-Novabio- chem GmbH, D-65796 Bad Soden, aus zahlreichen Standardwerken und publizierten Patentanmeldungen bekannt ist.In addition, the peptides can be prepared by conventional methods of amino acid and peptide synthesis, such as. B. from Novabiochem - 1999 Catalog & Peptide Synthesis Handbook from Calbiochem-Novabio-chem GmbH, D-65796 Bad Soden, is known from numerous standard works and published patent applications.
Es können schrittweise Kupplungen und Fragmentkondensationen genutzt werden. Unterschiedliche N-terminale, C-terminale und Seitenschutz- gruppen können Verwendung finden, die bevorzugt orthogonal spaltbar ausgewählt werden. Kupplungsschritte können mit unterschiedlichen Kondensationsreagenzien wie Carbodiimiden, Carbodiimidazol, solchen des Uronium-Typs wie TBTU, gemischten Anhydrid-Methoden, sowie Säurehalogenid- oder Aktivester-Methoden durchgeführt werden. Aktivierte Ester werden zweckmäßig in situ gebildet, z. B. durch Zusatz von HOBt oder N-Hydroxysuccinimid.Gradual couplings and fragment condensation can be used. Different N-terminal, C-terminal and side protection groups can be used, which are preferably selected to be orthogonally cleavable. Coupling steps can be carried out with different condensation reagents such as carbodiimides, carbodiimidazole, those of the uronium type such as TBTU, mixed anhydride methods, and acid halide or active ester methods. activated Esters are conveniently formed in situ, e.g. B. by adding HOBt or N-hydroxysuccinimide.
Eine Cyclisierung eines linearen Vorläufermoleküls mit Seitenschutz- gruppen ist ebenfalls mit solchen Kondensationsreaktionen durchführbar, wie z. B. in DE 43 10 643 oder in Houben-Weyl, I.e., Band 15/11, Seiten 1 bis 806 (1974) beschrieben.A cyclization of a linear precursor molecule with side protecting groups can also be carried out with such condensation reactions, such as. B. in DE 43 10 643 or in Houben-Weyl, I.e., Volume 15/11, pages 1 to 806 (1974).
Bei der Festphasenpeptidsynthese sind unterschiedliche Harze und Ankerfunktionen nutzbar. Harze können z.B. auf Polystyrol oderDifferent resins and anchor functions can be used in solid phase peptide synthesis. Resins can e.g. on polystyrene or
Polyacrylamid basieren, Ankerfunktionen wie Wang, o-Chlortrityl sind zur Herstellung von Peptidsäuren, Aminoxanthenoxy-Anker z.B. zur Herstellung von Paptidamiden nutzbar.Polyacrylamide based, anchor functions such as Wang, o-chlorotrityl are for the production of peptide acids, aminoxanthenoxy anchors e.g. usable for the production of paptidamides.
Biotinylierte oder fluoreszenzmarkierte Peptide / Proteine können ebenfalls nach Standardmethoden hergestellt werden (z.B. E.A. Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology; und Handbook of Fluorescent Probes and Research Chemicals, 6th Edition, 1996, by R.P. Haugland, Molecular Probes, Inc.; oder auch WO 97/14716).Biotinylated or fluorescence-labeled peptides / proteins can also be produced using standard methods (e.g. EA Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology; and Handbook of Fluorescent Probes and Research Chemicals , 6 th Edition, 1996, by Haugland RP, Molecular Probes, Inc .; or WO 97/14716).
Selbstverständlich können die erfindungsgemäßen Peptide auch durch Solvolyse, insbesondere Hydrolyse, oder durch Hydrogenolyse ihrer funktioneilen Derivate freigesetzt werden. Bevorzugte Ausgangsstoffe für die Solvolyse bzw. Hydrogenolyse sind solche, die anstelle einer oder mehrerer freier Amino- und/oder Hydroxygruppen entsprechende geschützte Amino- und/oder Hydroxygruppen enthalten, vorzugsweise solche, die anstelle eines H-Atoms, das mit einem N-Atom verbunden ist, eine Aminoschutzgruppe oder die anstelle des H-Atoms einer Hydroxy- gruppe eine Hydroxyschutzgruppe tragen. Entsprechendes gilt für Carbonsäuren, die durch Substitution ihrer -CO-OH Hydroxyfunktion mittels einer Schutzgruppe, z.B. als Ester geschützt werden können. Der Ausdruck "Aminoschutzgruppe" ist allgemein bekannt und bezieht sich auf Gruppen, die geeignet sind, eine Aminogruppe vor chemischen Umsetzungen zu schützen (zu blockieren), die aber leicht entfernbar sind, nachdem die gewünschte chemische Reaktion an anderen Stellen desOf course, the peptides according to the invention can also be released by solvolysis, in particular hydrolysis, or by hydrogenolysis of their functional derivatives. Preferred starting materials for solvolysis or hydrogenolysis are those which contain corresponding protected amino and / or hydroxyl groups instead of one or more free amino and / or hydroxyl groups, preferably those which instead of an H atom which is connected to an N atom is an amino protective group or which carry a hydroxy protective group instead of the H atom of a hydroxy group. The same applies to carboxylic acids which can be protected by substitution of their -CO-OH hydroxy function by means of a protective group, for example as an ester. The term "amino protecting group" is generally known and refers to groups which are suitable for protecting (blocking) an amino group from chemical reactions, but which are easily removable after the desired chemical reaction at other locations on the
Moleküls durchgeführt worden ist. Der Ausdruck "Hydroxyschutzgruppe" ist ebenfalls allgemein bekannt und bezieht sich auf Gruppen, die geeignet sind, eine Hydroxygruppe vor chemischen Umsetzungen zu schützen, die aber leicht entfernbar sind, nachdem die gewünschte chemische Reaktion an anderen Stellen des Moleküls durchgeführt worden ist. Das In-Freiheit-Molecule has been carried out. The term "hydroxyl protecting group" is also generally known and refers to groups which are suitable for protecting a hydroxyl group against chemical reactions, but which are easily removable after the desired chemical reaction has been carried out elsewhere in the molecule. The in freedom
Setzen der Verbindungen aus ihren funktioneilen Derivaten gelingt - je nach der benutzten Schutzgruppe - z. B. mit starken Säuren, zweckmäßig mit TFA oder Perchlorsäure, aber auch mit anderen starken anorgan- ischen Säuren wie Salzsäure oder Schwefelsäure, starken organischenDepending on the protective group used, setting the compounds from their functional derivatives succeeds, for. B. with strong acids, suitably with TFA or perchloric acid, but also with other strong inorganic acids such as hydrochloric acid or sulfuric acid, strong organic
Carbonsäuren wie Trichloressigsäure oder Sulfonsäuren wie Benzol- oder p-Toluolsulfonsäure. Hydrogenolytisch entfernbare Schutzgruppen (z. B. CBZ oder Benzyl) können z. B. durch Behandeln mit Wasserstoff in Gegenwart eines Katalysators (z. B. eines Edelmetallkatalysators wie Palladium, zweckmäßig auf einem Träger wie Kohle) abgespalten werden.Carboxylic acids such as trichloroacetic acid or sulfonic acids such as benzene or p-toluenesulfonic acid. Hydrogenolytically removable protective groups (e.g. CBZ or benzyl) can e.g. B. by treatment with hydrogen in the presence of a catalyst (z. B. a noble metal catalyst such as palladium, advantageously on a support such as coal).
Typische •Schutzgruppen für N-Termini und für seitenständige Amino- gruppen sind Z, BOC, Fmoc, solche für C-Termini oder die Asp- oder Glu- Seitenketten sind O-prim.-Alkyl (z.B. OMe oder OEt), O-tert.-Alkyl (z.B. OBut) oder OBenzyl. Für die Guanidinofunktion des Arg ist z.B. Z, BOC, N02, Mtr, Pmc oder Pbf geeignet. Alkoholische Funktionen können durch tert.-Alkylreste oder Tritylgruppen geschützt sein.Typical • protecting groups for N-termini and for pendant amino groups are Z, BOC, Fmoc, those for C-termini or the Asp or Glu side chains are O-prim.-alkyl (eg OMe or OEt), O-tert .-Alkyl (e.g. OBut) or OBenzyl. Z, BOC, N0 2 , Mtr, Pmc or Pbf are suitable for the guanidino function of Arg. Alcoholic functions can be protected by tert-alkyl radicals or trityl groups.
Die Gruppen BOC, OBut und Mtr können z. B. bevorzugt mit TFA in Di- chlormethan oder mit etwa 3 bis 5n HCI in Dioxan bei 15-30° abgespalten werden, die FMOC-Gruppe mit einer etwa 5- bis 50 %igen Lösung von Dimethylamin, Diethylamin oder Piperidin in DMF bei 15-30°. Die Tritylgruppe wird z.B. zum Schutz der Aminosäuren Histidin, Asparagin, Glutamin und Cystein eingesetzt. Die Abspaltung erfolgt, je nach gewünschtem Endprodukt, mit TFA / 10% Thiophenol, wobei die Tritylgruppe von allen genannten Aminosäuren abgespalten wird, bei Einsatz von TFA / Anisol oder TFA / Thioanisol wird nur die Tritylgruppe von His, Asn und Gin abgespalten, wogegen sie an der Cys-Seitenkette verbleibt.The groups BOC, OBut and Mtr can e.g. B. preferably with TFA in dichloromethane or with about 3 to 5N HCl in dioxane at 15-30 °, the FMOC group with an about 5 to 50% solution of dimethylamine, diethylamine or piperidine in DMF at 15 -30 °. The trityl group is used, for example, to protect the amino acids histidine, asparagine, glutamine and cysteine. Depending on the desired end product, the cleavage is carried out with TFA / 10% thiophenol, the trityl group being cleaved from all the amino acids mentioned; when using TFA / anisole or TFA / thioanisole, only the trityl group of His, Asn and Gin is cleaved, whereas they are remains on the Cys side chain.
Hydrogenolytisch entfernbare Schutzgruppen (z. B. CBZ oder Benzyl) können z. B. durch Behandeln mit Wasserstoff in Gegenwart eines Katalysators (z. B. eines Edelmetallkatalysators wie Palladium, zweckmäßig auf einem Träger wie Kohle) abgespalten werden. Als Lösungsmittel eignen sich dabei die oben angegebenen, insbesondere z. B. Alkohole wie Methanol oder Ethanol oder Amide wie DMF. Die Hydrogenolyse wird in der Regel bei Temperaturen zwischen etwa 0 und 100° und Drucken zwischen etwa 1 und 200 bar, bevorzugt bei 20-30° und 1-10 bar durchgeführt. Eine Hydrogenolyse der CBZ-Gruppe gelingt z. B. gut an 5 bis 10 %igem Pd/C in Methanol oder mit Ammomiumformiat (anstelle von Wasserstoff) an Pd/C in Methanol/DMF bei 20-30°.Hydrogenolytically removable protective groups (e.g. CBZ or benzyl) can e.g. B. by treatment with hydrogen in the presence of a catalyst (z. B. a noble metal catalyst such as palladium, advantageously on a support such as coal). Suitable solvents are the above, especially z. B. alcohols such as methanol or ethanol or amides such as DMF. The hydrogenolysis is generally carried out at temperatures between about 0 and 100 ° and pressures between about 1 and 200 bar, preferably at 20-30 ° and 1-10 bar. Hydrogenolysis of the CBZ group succeeds e.g. B. good on 5 to 10% Pd / C in methanol or with ammonium formate (instead of hydrogen) on Pd / C in methanol / DMF at 20-30 °.
Wie bereits erwähnt, umfassen die erfindungsgemäßen Peptide ihre physiologisch unbedenklichen Salze, welche ebenfalls nach Standardmethoden hergestellt werden können. So kann eine Base einer erfindungsge- mäsßen Verbindung mit einer Säure in das zugehörige Säureadditionssalz übergeführt werden, beispielsweise durch Umsetzung äquivalenter Mengen der Base und der Säure in einem inerten Lösungsmittel wie Ethanol und anschließendes Eindampfen. Für diese Umsetzung kommen insbesondere Säuren in Frage, die physiologisch unbedenkliche Salze liefern. So können anorganische Säuren verwendet werden, z.B. Schwefelsäure, Salpetersäure, Halogenwasserstoffsäuren wie Chlorwasserstoffsäure oder Bromwasserstoffsäure, Phosphorsäuren wie Orthophosphorsäure, Sul- faminsäure, ferner organische Säuren, insbesondere aliphatische, alicyc- lische, araliphatische, aromatische oder heterocyciische ein- oder mehr- basige Carbon-, Sulfon- oder Schwefelsäuren, z.B. Ameisensäure, Essig- säure, Propionsäure, Pivalinsäure, Diethylessigsäure, Malonsäure, Bernsteinsäure, Pimelinsäure, Fumarsäure, Maleinsäure, Milchsäure, Weinsäure, Äpfelsäure, Citronensäure, Gluconsäure, Ascorbinsäure, Nicotin- säure, Isonicotinsäure, Methan- oder Ethansulfonsäure, Ethandisulfon- säure, 2-Hydroxyethansulfonsäure, Benzolsulfonsäure, p-Toluolsulfon- säure, Naphthalin-mono- und -disulfonsäuren, Laurylschwefelsäure. Salze mit physiologisch nicht unbedenklichen Säuren, z.B. Pikrate, können zurAs already mentioned, the peptides according to the invention comprise their physiologically acceptable salts, which can also be prepared by standard methods. For example, a base of a compound according to the invention can be converted with an acid into the associated acid addition salt, for example by reacting equivalent amounts of the base and the acid in an inert solvent such as ethanol and subsequent evaporation. In particular, acids that provide physiologically acceptable salts are suitable for this implementation. For example, inorganic acids can be used, for example sulfuric acid, nitric acid, hydrohalic acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, and also organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or poly-based carbon atoms. , Sulfonic or sulfuric acids, e.g. formic acid, acetic Acid, propionic acid, pivalic 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, 2-dodesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, 2-sulfonic acid, 2-diacid, , p-toluenesulfonic acid, naphthalene mono- and disulfonic acids, laurylsulfuric acid. Salts with physiologically unacceptable acids, such as picrates, can be used
Isolierung und /oder Aufreinigung der erfindungsgemäßen Verbindungen verwendet werden. Andererseits kann eine Säure der erfindungsgemäßen Verbindungen durch Umsetzung mit einer Base in eines ihrer physiologisch unbedenklichen Metall- oder Ammoniumsalze übergeführt werden. Als Salze kommen dabei insbesondere die Natrium-, Kalium-, Magnesium- , Calcium- und Ammoniumsalze in Betracht, ferner substituierte Ammoniumsalze, z. B. die Dimethyl-, Diethyl- oder Diisopropylammoniumsalze, Monoethanol-, Diethanol- oder Diisopropylammoniumsalze, Cyclohexyl-, Dicyclohexyl-ammoniumsalze, Dibenzylethylendiammoniumsalze, weiterhin z. B. Salze mit Arginin oder Lysin.Isolation and / or purification of the compounds according to the invention can be used. On the other hand, an acid of the compounds according to the invention can be converted into one of their physiologically acceptable metal or ammonium salts by reaction with a base. Suitable salts here are in particular the sodium, potassium, magnesium, calcium and ammonium salts, and also substituted ammonium salts, e.g. B. the dimethyl, diethyl or diisopropylammonium salts, monoethanol, diethanol or diisopropylammonium salts, cyclohexyl, dicyclohexylammonium salts, dibenzylethylenediammonium salts, z. B. salts with arginine or lysine.
Die erfindungsgemäßen peptidischen Verbindungen können, wie bereits erwähnt, als Arzneimittelwirkstoffe in der Human- und Veterinärmedizin eingesetzt werden, insbesondere zur Prophylaxe und/oder Therapie von Erkrankungen bei denen epitheliale Zellen beteilgt sind. Besonders hervorzuheben sind hierbei Erkrankungen oder Entzündungen oder Wundhei- lungsprozesse der Haut, der Atemwegorgane und des Magen- und Darmbereichs, so zum Beispiel Apoplexie, Angina pectoris, Tumorerkrankun- gen, osteolytischen Krankheiten wie Osteoporose, pathologisch angio- genen Krankheiten wie z. B. Entzündungen, Lungenfibrose, ophthalmo- logischen Krankheiten, diabetischer Retinopathie, makularer Degeneration, Myopia, okularer Histoplasmose, rheumatischer Arthritis, Osteoarthri- tis, rubeotischem Glaukom, ulcerativer Colitis, Morbus Crohn, Atheroskler- ose, Psoriasis, Restenose nach Angioplastie, bei akutem Nierenversagen,As already mentioned, the peptide compounds according to the invention can be used as active pharmaceutical ingredients in human and veterinary medicine, in particular for the prophylaxis and / or therapy of diseases in which epithelial cells are involved. Of particular note here are diseases or inflammations or wound healing processes in the skin, respiratory organs and stomach and intestinal area, such as apoplexy, angina pectoris, tumor diseases, osteolytic diseases such as osteoporosis, pathologically angiogenic diseases such as B. Inflammation, pulmonary fibrosis, ophthalmic diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerotic disease, psoriasis angiosis, psoriasis disease kidney failure,
Nierenentzündung, mikrobiellen Infekten und Multipler Sklerose. Gegenstand der Erfindung sind demgemäß peptidische Verbindungen der oben und unten sowie in den Ansprüchen definierten Formeln einschließlich ihrer physiologisch unbedenklichen Salze als Arzneimittel, Diagnostika oder Reagenzien.Inflammation of the kidneys, microbial infections and multiple sclerosis. The invention accordingly relates to peptide compounds of the formulas defined above and below and in the claims, including their physiologically acceptable salts, as medicaments, diagnostics or reagents.
Gegenstand der Erfindung sind insbesondere entsprechende Arzneimittel als Inhibitoren zur Bekämpfung von Erkrankungen, die mittelbar oder unmittelbar auf einer Expression des αvß6 -Integrinrezeptors beruhen, insbesondere also bei pathologisch angiogenen Erkrankungen, Thrombosen, Herzinfarkt, koronaren Herzerkrankungen, Arteriosklerose, Tumoren, Osteoporose, Entzündungen, Infektionen sowie zur Beein- flussung von Wundheilungsprozessen.The invention relates in particular to corresponding medicaments as inhibitors for combating diseases which are based directly or indirectly on expression of the α v β 6 integrin receptor, in particular in the case of pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis. Inflammation, infections and to influence wound healing processes.
Gegenstand der Erfindung sind auch entsprechende pharmazeutische Zubereitungen, welche mindestens ein Arzneimittel der Formel I sowie gegebenenfalls Träger- und/oder Hilfsstoffe enthalten. Ferner ist Gegenstand der Erfindung die Verwendung der peptidischen Verbindungen und/oder ihre physiologisch unbedenklichen Salze gemäß der Ansprüche und der Beschreibung zur Herstellung eines Arzneimittels zur Bekämpfung von Erkrankungen, die mittelbar oder unmittelbar auf einer Expression des αvß6 -Integrinrezeptors beruhen, insbesondere also bei pathologisch angiogenen Erkrankungen, Thrombosen, Herzinfarkt, koronaren Herzerkrankungen, Arteriosklerose, Tumoren, Osteoporose, Entzündungen, Infektionen sowie zur Beeinflussung von Wundheilungs- prozessen. Die erfindungsgemäßen Arzneimittel bzw. sie enthaltende pharmazeutische Zubereitungen können in der Human- oder Veterinärmedizin verwendet werden. Als Trägerstoffe kommen organische oder anorganische Substanzen in Frage, die sich für die enterale (z.B. orale), parenterale, topische Applikation oder für eine Applikation in Form eines Inhalation-Sprays eignen und mit den neuen Verbindungen nicht reagie- ren, beispielsweise Wasser, pflanzliche Öle, Benzylalkohole, Alkylengly- kole, Polyethylenglykole, Glyce ntriacetat, Gelatine, Kohlehydrate wie Lactose oder Stärke, Magnesiumstearat, Talk, Vaseline. Zur oralen Anwendung dienen insbesondere Tabletten, Pillen, Dragees, Kapseln, Pui- ver, Granulate, Sirupe, Säfte oder Tropfen, zur rektalen Anwendung Sup- posito en, zur parenteralen Anwendung Lösungen, vorzugsweise ölige oder wässrige Lösungen, ferner Suspensionen, Emulsionen oder Implantate, für die topische Anwendung Salben, Cremes oder Puder. Die neuen Verbindungen können auch lyophilisiert und die erhaltenen Lyophilisate z.B. zur Herstellung von Injektionspräparaten verwendet werden. Die angegebenen Zubereitungen können sterilisiert sein und/oder Hilfsstoffe wie Gleit-, Konservierungs-, Stabilisierungs- und/oder Netzmittel, Emul- gatoren, Salze zur Beeinflussung des osmotischen Druckes, Puffersubstanzen, Färb-, Geschmacks- und /oder eine oder mehrere weitere Wirkstoffe enthalten, z. B. ein oder mehrere Vitamine. Für die Applikation als Inhalationsspray können Sprays verwendet werden, die den Wirkstoff entweder gelöst oder suspendiert in einem Treibgas oder Treibgasgemisch (z. B. C02 oder Fluorchlorkohlenwasserstoffen) enthalten. Zweckmäßig verwendet man den Wirkstoff dabei in mikronisierter Form, wobei ein oder mehrere zusätzliche physiologisch verträgliche Lösungsmittel zugegen sein können, z. B. Ethanol. Inhalationslösungen können mit Hilfe üblicher Inhalatoren verabreicht werden.The invention also relates to corresponding pharmaceutical preparations which contain at least one medicament of the formula I and, if appropriate, carriers and / or auxiliaries. The invention furthermore relates to the use of the peptide compounds and / or their physiologically acceptable salts according to the claims and the description for the manufacture of a medicament for combating diseases which are based, directly or indirectly, on expression of the α v β 6 integrin receptor, in particular thus in the case of pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammation, infections and for influencing wound healing processes. The pharmaceuticals according to the invention or pharmaceutical preparations containing them can be used in human or veterinary medicine. Suitable carrier substances are organic or inorganic substances which are suitable for enteral (for example oral), parenteral, topical application or for application in the form of an inhalation spray and which do not react with the new compounds. ren, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycene triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, petroleum jelly. Tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops are used in particular for oral use, supositoses for rectal use, solutions, preferably oily or aqueous solutions, and also suspensions, emulsions or implants for parenteral use , for topical application of ointments, creams or powder. The new compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injectables. The specified preparations can be sterilized and / or auxiliaries such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, flavoring and / or one or more further active substances included, e.g. B. one or more vitamins. For the application as an inhalation spray, sprays can be used which contain the active ingredient either dissolved or suspended in a propellant gas or propellant gas mixture (e.g. C0 2 or chlorofluorocarbons). The active ingredient is expediently used in micronized form, it being possible for one or more additional physiologically acceptable solvents to be present, for. B. ethanol. Inhalation solutions can be administered using standard inhalers.
Die erfindungsgemäßen Substanzen können in der Regel in Analogie zu anderen bekannten, im Handel befindlichen Peptiden (z.B. beschrieben in der US-A-4 472 305 ) verabreicht werden, vorzugsweise in Dosierungen zwischen etwa 0,05 und 500 mg, insbesondere zwischen 0,5 und 100 mg pro Dosierungseinheit verabreicht. Die tägliche Dosierung liegt vorzugsweise zwischen etwa 0,01 und 20 mg/kg Körpergewicht. Die spezielle Dosis für jeden Patienten hängt jedoch von den verschiedensten Faktoren ab, beispielsweise von der Wirksamkeit der eingesetzten speziellen Verbindung, vom Alter, Körpergewicht, allgemeinen Gesundheitszustand, Geschlecht, von der Kost, vom Verabreichungszeitpunkt und -weg, von der Ausscheidungsgeschwindigkeit, Arzneistoffkombination und Schwere der jeweiligen Erkrankung, welcher die Therapie gilt. Die parenterale Applikation ist bevorzugt.The substances according to the invention can generally be administered analogously to other known, commercially available peptides (for example described in US Pat. No. 4,472,305), preferably in doses between about 0.05 and 500 mg, in particular between 0.5 and 100 mg per dosage unit. The daily dosage is preferably between about 0.01 and 20 mg / kg body weight. However, the specific dose for each patient depends on a variety of factors, for example on the effectiveness of the particular compound used, on the age, body weight, general health, gender, on the diet, on the time and route of administration, on which Elimination rate, drug combination and severity of the disease to which the therapy applies. Parenteral administration is preferred.
Ferner können die neuen Verbindungen der Formel I in der analytischenFurthermore, the new compounds of formula I in the analytical
Biologie und Molekularbiologie verwendet werden. Die neuen Verbindungen der Formel I, wobei X einen über eine -CONH-, -COO-, -NH-C(=S)-NH-, -NH-C(=0)-NH- , -S02NH- oder -NHCO- Bindung verknüpften fluoreszierenden Farbstoffrest bedeutet, können als diagnostische Marker in der FACS (Fluorescence Activated Cell Sorten- Technik und Fluoreszenz-Mikroskopie verwendet werden.Biology and molecular biology are used. The new compounds of formula I, wherein X is a -CONH-, -COO-, -NH-C (= S) -NH-, -NH-C (= 0) -NH-, -S0 2 NH- or -NHCO- linked fluorescent dye residue means can be used as diagnostic markers in FACS (Fluorescence Activated Cell cultivar technique and fluorescence microscopy.
Der Einsatz von markierten Verbindungen in der Fluoreszenz-Mikroskopie ist z. B. beschrieben von Y.-L. Wang und D. L. Taylor in "Fluorescence Microscopy of Living Cells in Culture, Part A + B, Academic Press, Inc.The use of labeled compounds in fluorescence microscopy is e.g. B. described by Y.-L. Wang and D. L. Taylor in "Fluorescence Microscopy of Living Cells in Culture, Part A + B, Academic Press, Inc.
1989".1989 ".
Die neuen erfindungsgemäßen Verbindungen können auch als Integrin- iiganden zur Herstellung von Säulen für die Affinitätschromatographie zur Reindarstellung von Integrinen verwendet werden. Der Komplex aus einem Avidin-derivatisierten Trägermaterial, z.B. Sepharose und den neuen Verbindungen wird nach an sich bekannten Methoden (z.B. E.A. Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology) gebildet. Als polymere Trägermaterialien eignen sich dabei die an sich in der Peptid- chemie bekannten polymeren festen Phasen mit vorzugsweise hydrophilen Eigenschaften, beispielsweise quervernetzte Polyzucker wie Cellulose,The new compounds according to the invention can also be used as integrin ligands for the preparation of columns for affinity chromatography for the purification of integrins. The complex of an avidin-derivatized carrier material, e.g. Sepharose and the new compounds are formed by methods known per se (e.g. E.A. Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology). Suitable polymeric carrier materials are the polymeric solid phases known per se in peptide chemistry with preferably hydrophilic properties, for example cross-linked poly sugars such as cellulose,
Sepharose oder SephadexR, Acrylamide, Polymer auf Polyethylenglykol- basis oder TentakelpolymereR.Sepharose or Sephadex R , acrylamides, polymer based on polyethylene glycol or tentacle polymers R.
Die Erfindung umfaßt schließlich auch rekombinante DNA-Sequenzen, welche Abschnitte enthalten, die für Peptidbereiche codieren, die die erfindungsgemäßen peptidischen Strukturmotive aufweisen. Solche DNA kann durch Partikel auf Zellen übertragen werden, wie in Ch. Andree et al. Proc.Natl.Acad.Sci. 91 , 12188-12192 (1994) beschrieben ist, oder der Transfer auf Zellen kann durch andere Hilfsmittel, wie Liposomen, gesteigert werden (A.l. Aronsohn and J.A. Hughes J. Drug Targeting, 5, 163-169 (1997)).Finally, the invention also comprises recombinant DNA sequences which contain sections which code for peptide regions which have the peptide structural motifs according to the invention. Such DNA can be transferred to cells by particles, as described in Ch. Andree et al. Proc. 91, 12188-12192 (1994), or the transfer to cells can be increased by other means such as liposomes (Al Aronsohn and JA Hughes J. Drug Targeting, 5, 163-169 (1997)).
Der Transfer einer solchen DNA könnte demnach in Hefen, mittels Bacculo-Viren oder in Säugerzellen für die Produktion der peptidischen Substanzen dieser Erfindung benutzt werden.The transfer of such DNA could thus be used in yeast, by means of bacculoviruses or in mammalian cells for the production of the peptide substances of this invention.
Wird ein tierischer oder menschlicher Organismus mit solch einer rekombinanten DNA infiziert, dann können die durch die infizierten Zellen letztlich selbst gebildeten erfindungsgemäßen Peptide unmittelbar an den αvß6 -Integrinrezeptor, beispielsweise von Tumorzellen binden und ihn blockieren.If an animal or human organism is infected with such a recombinant DNA, the peptides according to the invention ultimately formed by the infected cells themselves can bind directly to the α v ß 6 integrin receptor, for example of tumor cells, and block it.
Entsprechende rekombinante DNA, die durch bekannte und übliche Techniken bereitgestellt werden kann, kann beispielsweise aber auch in Form von Virus-DNA vorliegen, welche Abschnitte enthält, die für das Virus-Hüllprotein codieren. Durch Infektion eines Wirtsorganismus mit derartigen rekombinanten, vorzugsweise nicht pathogenen Viren, können Wirtszellen, die das Integrin αvß6 exprimieren, bevorzugt angegriffen werden (Targetierung).Corresponding recombinant DNA, which can be provided by known and customary techniques, can also be present, for example, in the form of virus DNA which contains sections which code for the virus coat protein. By infection of a host organism with such recombinant, preferably non-pathogenic viruses, host cells which express the integrin α v ß 6 can be attacked (targeting).
Geeignete Viren sind beispielsweise Adenovirenarten, die mehrfach schon als Vektoren für fremde Gene in Säugerzellen benutzt wurden. Eine Anzahl von Eigenschaften machen sie zu guten Kandidaten für Gentherapie, wie S.J. Watkins et al. Gene Therapy 4, 1004-1012 (1997) zu entnehmen ist (siehe auch J. Engelhardt et al. Hum. Gene Ther. 4, 759- 769 (1993)) .Suitable viruses are, for example, adenovirus types which have been used several times as vectors for foreign genes in mammalian cells. A number of traits make them good candidates for gene therapy, such as S.J. Watkins et al. Gene Therapy 4, 1004-1012 (1997) can be found (see also J. Engelhardt et al. Hum. Gene Ther. 4, 759-769 (1993)).
Wie in A. Fasbender et al. J.Clin.Invest. 102, 184-193 (1998) zu finden ist, ist gemeinsames Problem bei Gentherapie durch virale und nichtvirale Vektoren die limitierte Effizienz des Gentransfers. Mit der oben beschriebenen zusätzlichen Ligandensequenz für αvß6 Integrin im Hüllprotein der Adenoviren kann eine Verbesserung des Transfers z.B. von CysticAs in A. Fasbender et al. J. Clin. 102, 184-193 (1998) is a common problem in viral and non-viral gene therapy Vectors the limited efficiency of gene transfer. With the additional ligand sequence for α v β 6 integrin described above in the coat protein of the adenoviruses, an improvement in the transfer, for example of cystic, can be achieved
Fibrosis Transmembrane Conductance Regulator (CFTR) cDNA erreicht werden.Fibrosis transmembrane conductance regulator (CFTR) cDNA can be achieved.
Ähnlich wie in der Arbeit von T. Tanaka et al. Cancer Research 58, 3362- 3369 (1998) kann statt der DNA für Angiostatin auch die DNA für die Sequenzen dieser Erfindung für Zelltransfektionen mittels retroviraler oder adenoviraler Vektoren genutzt werden.Similar to the work of T. Tanaka et al. Cancer Research 58, 3362-3369 (1998), instead of the DNA for angiostatin, the DNA for the sequences of this invention can also be used for cell transfections by means of retroviral or adenoviral vectors.
Die erfindungsgemäßen Peptide können auch innerhalb eines Liposome- komplexes aus Lipid/Peptid/DNA für eine Transfektion von Zellkulturen herstellt zusammen mit einem Liposomen-Komplex bestehend aus Lipid/ DNA (ohne Peptid) für den Einsatz in der Gentherapie am Menschen eingesetzt werden. Die Herstellung eines Liposomenkomplexes aus Lipid/DNA Peptid ist beispielsweise bei Hart S.L, et al 1998: Lipid- Mediated Enhancement of Transfection by a Non-Viral Integrin-Targeting Vector. Human Gene Therapy 9, 575-585, beschrieben. Ein Liposomenkomplex aus Lipid/Pepid/DNA ist beispielsweise aus folgenden Stammlösungen hergestellbar: 1 μg/μl Lipofectin (äquimolare Mischung aus DOTMA (= N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethyl- ammonium Chlorid ) und DOPE (Dioleyl Phosphatidylethanolamin)), 10 μg/ml Plasmid DNA und 100 μg/ml Peptid. Sowohl DNA als auch Peptid werden dazu in Zellkulturmedium gelöst. Der Liposomenkomplex wird durch Mischen der drei Komponenten in einem bestimmten Gewichtsverhältnis (Lipid: DNA: Peptid, z.B. 0,75 : 1 : 4) hergestellt. Liposomen DNA-Komplexe für eine Gentherapie am Menschen sind bereits beschrieben worden (Caplen N.J., et al 1995: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis Nature Medicine 1 , 39-46). Gegenstand der Erfindung ist somit auch die Verwendung entsprechend modifizierter rekombinater DNA von Gen-freisetzenden Systemen, insbesondere Virus-DNA, zur Bekämpfung von Krankheiten welche mittelbar oder unmittelbar auf einer Expression von αvß6 -Integrinrezeptoren beruhen, insbesondere also bei pathologisch angiogenen Erkrankungen,The peptides according to the invention can also be used within a liposome complex made of lipid / peptide / DNA for a transfection of cell cultures together with a liposome complex consisting of lipid / DNA (without peptide) for use in gene therapy in humans. The production of a liposome complex from lipid / DNA peptide is described, for example, by Hart SL, et al 1998: Lipid-Mediated Enhancement of Transfection by a Non-Viral Integrin-Targeting Vector. Human Gene Therapy 9, 575-585. A liposome complex of lipid / pepid / DNA can be prepared, for example, from the following stock solutions: 1 μg / μl lipofectin (equimolar mixture of DOTMA (= N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethyl- ammonium chloride) and DOPE (dioleyl phosphatidylethanolamine)), 10 μg / ml plasmid DNA and 100 μg / ml peptide. For this purpose, both DNA and peptide are dissolved in cell culture medium. The liposome complex is produced by mixing the three components in a specific weight ratio (lipid: DNA: peptide, for example 0.75: 1: 4). Liposome DNA complexes for gene therapy in humans have already been described (Caplen NJ, et al 1995: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis Nature Medicine 1, 39-46). The invention thus also relates to the use of appropriately modified recombinant DNA from gene-releasing systems, in particular virus DNA, for combating diseases which are based directly or indirectly on expression of α v β 6 integrin receptors, in particular in the case of pathologically angiogenic diseases,
Thrombosen, Herzinfarkt, koronaren Herzerkrankungen, Arteriosklerose,Thrombosis, heart attack, coronary heart disease, arteriosclerosis,
Tumoren, Osteoporose, Entzündungen, Infektionen sowie zur Beein- flussung von Wundheilungsprozessen.Tumors, osteoporosis, inflammation, infections and to influence wound healing processes.
Vor- und nachstehend sind alle Temperaturen in °C angegeben.All temperatures above and below are given in ° C.
Die HPLC-Analysen (Retentionszeit Rt) erfolgten in den folgendenThe HPLC analyzes (retention time Rt) were carried out in the following
Systemen:systems:
Säule 5 μm LichroSpher 60 RP-Select B (250-4), mit einem 50-minütigenColumn 5 μm LichroSpher 60 RP-Select B (250-4), with a 50 minute
Gradienten von 0 bis 80% 2-Propanol in Wasser / 0,3% Trifluoressigsäure, bei 1 ml/min Fluss und Detektion bei 215 nm.Gradients from 0 to 80% 2-propanol in water / 0.3% trifluoroacetic acid, at 1 ml / min flow and detection at 215 nm.
Massenspektrometrie (MS): El (Elektronenstoß-Ionisation) M+ Mass spectrometry (MS): El (electron impact ionization) M +
FAB (Fast Atom Bombardment) (M+H)+ FAB (Fast Atom Bombardment) (M + H) +
Beispiel 1example 1
Herstellung und Aufreinigung erfindungsgemäßer Peptide:Production and purification of peptides according to the invention:
Prinzipiell erfolgte die Herstellung und Aufreinigung mittels Fmoc-Strategie unter Protektion säurelabiler Seitenketten auf särelabilen Harzen unter Benutzung eines kommerziell erhältlichen "continuous flow" Peptid- Synthesizers entsprechend den Angaben von Haubner et al. (J. Am. Chem. Soc. 118, 1996, 17703).In principle, the production and purification was carried out by means of the Fmoc strategy while protecting acid-labile side chains on acid-labile resins using a commercially available "continuous flow" peptide synthesizer in accordance with the information from Haubner et al. (J. Am. Chem. Soc. 118, 1996, 17703).
Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2 Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH 2
2 g Amino-Xanthenyloxy-Polystyrol-Harz (Novabiochem 0.37 mmol/g) wurden nacheinander in einer Doppelkupplungstechnik 2 x mit je 0,50 g TBTU, 0,53 ml Ethyldiisopropylamin und Fmoc-Aminosäure in DMF in einem kommerziellen Synthesegerät und einer typischen Prozedur (Gerät und Handbuch Milligen 9050 PepSynthesizer™, 1987), für jeweils 30 Minuten, einem Kupplungsschritt unterworfen. Waschschritte erfolgten in DMF für 10 Minuten, Abspaltungsschritte in Piperidin/DMF (1 :4 vol) für 52 g of amino-xanthenyloxy-polystyrene resin (Novabiochem 0.37 mmol / g) were successively 2 x with 0.50 g each in a double coupling technique TBTU, 0.53 ml of ethyldiisopropylamine and Fmoc-amino acid in DMF in a commercial synthesizer and a typical procedure (device and manual Milligen 9050 PepSynthesizer ™, 1987), each for 30 minutes, subjected to a coupling step. Washing steps were carried out in DMF for 10 minutes, splitting steps in piperidine / DMF (1: 4 vol) for 5
Minuten, N-terminale Acetylierungen (Capping) wurden mit Essigsäure- anhydrid/Pyridin/DMF (2:3:15 vol) für 15 Minuten durchgeführt. Es kamen die Aminosäuren Fmoc-Arg(Pmc), danach Fmoc-Leu, danach Fmoc-Ser(But), danach Fmoc-D-Asp(OBut), danach Fmoc-Leu, danach Fmoc-Asp(OBut), danach Fmoc-Gly und schließlich Fmoc-Arg(Pmc) zumMinutes, N-terminal acetylations (capping) were carried out with acetic anhydride / pyridine / DMF (2: 3: 15 vol) for 15 minutes. There came the amino acids Fmoc-Arg (Pmc), then Fmoc-Leu, then Fmoc-Ser (But), then Fmoc-D-Asp (OBut), then Fmoc-Leu, then Fmoc-Asp (OBut), then Fmoc- Gly and finally Fmoc-Arg (Pmc) to
Einsatz. Nach Abspalten der Fmoc-Schutzgruppe vom Fmoc-Arg(Pmc)-Gly-Asp(OBut)-Leu-D-Asp(OBut)-Ser(But)-Leu-Arg(Pmc)- aminoxanthenyloxy-Polystyrolharz wurde nochmals acetyliert. Nach Waschen mit DMF und Isopropanol und folgender Trocknung am Vakuum bei Raumtemperatur wurden 2,8 g Ac-Arg(Pmc)-Gly-Asp(OBut)-Leu-D-Commitment. After the Fmoc protective group had been removed from the Fmoc-Arg (Pmc) -Gly-Asp (OBut) -Leu-D-Asp (OBut) -Ser (But) -Leu-Arg (Pmc) - aminoxanthenyloxy polystyrene resin, acetylation was carried out again. After washing with DMF and isopropanol and subsequent drying in vacuo at room temperature, 2.8 g of Ac-Arg (Pmc) -Gly-Asp (OBut) -Leu-D-
Asp(OBut)-Ser(But)-Leu-Arg(Pmc)-aminoxanthenyloxy-Polystyrolharz erhalten.Asp (OBut) -Ser (But) -Leu-Arg (Pmc) aminoxanthenyloxy polystyrene resin obtained.
Durch Behandlung dieses Peptidylharzes mit 20 ml Trifluoressigsäure/ Wasser/TIS (94:3:3 vol) für 2 Stunden bei Raumtemperatur, Filtration, Einengen im Vakuum und Verreiben mit Diethylether wurde 0,47 gTreatment of this peptidyl resin with 20 ml of trifluoroacetic acid / water / TIS (94: 3: 3 vol) for 2 hours at room temperature, filtration, concentration in vacuo and trituration with diethyl ether gave 0.47 g
Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2 (code EMD 272974) erhalten.Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH 2 (code EMD 272974).
Eine Reinigung des Produktes erfolgte per RP-HPLC auf Lichrosorb RP 18 (250 - 25, 7 μm, Merck KGaA) in 0,3 % TFA mit einem Gradienten von 4 % auf 24 % 2-Propanol in einer Stunde bei 10 ml/min und Beurteilung des Eluats mittels UV-Durchflussphotometer bei 215 und 254 nm. Es wurden 168 mg Produkt erhalten; Rt 15,5 min; FAB 973. Analog wurden die folgenden Produkte hergestellt:The product was purified by RP-HPLC on Lichrosorb RP 18 (250 - 25, 7 μm, Merck KGaA) in 0.3% TFA with a gradient from 4% to 24% 2-propanol in one hour at 10 ml / min and assessment of the eluate using a UV flow photometer at 215 and 254 nm. 168 mg of product were obtained; Rt 15.5 min; FAB 973. The following products were manufactured analogously:
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000025_0001
Nomenklatur für Aminosäuren nach Eur.J.Biochem. 138, 9-37 (1984) kleiner Buchstabe = D-AminosäureNomenclature for amino acids according to Eur.J.Biochem. 138, 9-37 (1984) small letter = D-amino acid
Beispiel 2: cvße / Fibronektin Rezeptorbindungstest:Example 2: c v esse / fibronectin receptor binding test:
Die hergestellten erfindungsgemäßen Peptide wurden in Lösung zusammen mit kompetetiv wirkenden Fibronektin an den immobiliserten αvß6 Rezeptor gebunden und der Q-Wert als Maß für die Selektivität der Bindung des zu testenden Peptids an αvßβ ermittelt. Der Q-Wert berechnet sich dabei aus dem Quotienten der IC5o-Werte von Testpeptid und einem Standard. Als Standard diente das lineare Ac-RTDLDSLR-NH2 (Code EMD 271293) (Lit./Patent vgl Pytela et al. Science 231 , 1559, (1986)). Der Bindungstest wurde im einzelnen wie folgt durchgeführt: Die Immobilisierung von löslichem αvß6 Rezeptor auf Microtiterplatten erfolgte durch Verdünnung der Proteinlösung in TBS++ und anschlies- sender Inkubation über Nacht bei 4°C (100 μl/Vertiefung). Unspezifische Bindungsstellen wurden durch Inkubation (2 h, 37°C) mit 3% (w/v) BSA in TBS++ (200 μl/Vertiefung) blockiert. Überschüssiges BSA wurde durch dreimaliges Waschen mit TBSA++ entfernt. Peptide wurden seriell (1 :10) in TBSA++ verdünnt und zusammen mit biotinyliertem Fibronektin (2 μg/ ml) mit dem immobilisierten Integrin inkubiert (50 μl Peptid + 50 μl Ligand pro Vertiefung; 2 h; 37°C). Nicht gebundenes Fibronektin und Peptide wurden durch dreimaliges Waschen mit TBSA++ entfernt. Die Detektion des gebundenen Fibronektin erfolgte durch Inkubation (1 h; 37 °C) mit einem alkalische-Phosphatase-gekoppelten anti-Biotin-Antikörper (Biorad) (1 :20000 in TBSA++; 100 μl/Vertiefung). Nach dreimaligem Waschen mit TBSA++ erfolgte die kolohmetrische Detektion durch Inkubation (10-15 min; 25°C, im Dunkeln) mit Substratlösung (5 mg Nitrophenylphosphat, 1 ml Ethanolamin, 4 ml H20; 100 μl/Vertiefung). Die Enzymreaktion wurde durch Zugabe von 0,4 M NaOH (100 μl/Vertiefung) gestoppt. Die Farbintensität wurde bei 405 nm im ELISA-Meßgerät bestimmt und gegen den Nullwert abgeglichen. Als Nullwert dienten Vertiefungen, die nicht mit Rezeptor beschichtet waren. Als Standard wurde Ac-RTDLDSLR-NH2 eingesetzt. Die IC5o-Werte für die getesteten Peptide wurden aus einer Graphik abgelesen und daraus zusammen mit dem IC50-Wert des Standardpeptids der Q-Wert des erfindungsgemäßen Peptids ermittelt.The peptides according to the invention which were produced were bound to the immobilized α v β 6 receptor in solution together with fibronectin having a competitive effect and the Q value was determined as a measure of the selectivity of the binding of the peptide to be tested to α v β β. The Q value is calculated from the quotient of the IC 5 o values of test peptide and a standard. The linear Ac-RTDLDSLR-NH 2 (code EMD 271293) was used as the standard (lit./patent, see Pytela et al. Science 231, 1559, (1986)). The binding test was carried out in detail as follows: The immobilization of soluble α v β 6 receptor on microtiter plates was carried out by diluting the protein solution in TBS ++ and then incubating overnight at 4 ° C. (100 μl / well). Unspecific binding sites were blocked by incubation (2 h, 37 ° C) with 3% (w / v) BSA in TBS ++ (200 μl / well). Excess BSA was removed by washing three times with TBSA ++. Peptides were serially (1:10) diluted in TBSA ++ and incubated together with biotinylated fibronectin (2 μg / ml) with the immobilized integrin (50 μl peptide + 50 μl ligand per well; 2 h; 37 ° C). Unbound fibronectin and peptides were removed by washing three times with TBSA ++. The bound fibronectin was detected by incubation (1 h; 37 ° C) with an alkaline phosphatase-coupled anti-biotin antibody (Biorad) (1: 20000 in TBSA ++; 100 ul / well). After washing three times with TBSA ++, the colohmetric 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 H 2 0; 100 μl / well). The enzyme reaction was stopped by adding 0.4 M NaOH (100 ul / well). The color intensity was determined at 405 nm in the ELISA measuring device and compared to the zero value. Wells that were not coated with receptor served as zero value. Ac-RTDLDSLR-NH 2 was used as the standard. The IC 5 o values for the peptides tested were read off from a graph and determines together with the IC50 of the standard peptide, the Q value of the peptide according to the invention.
Q-Wert = IC50 Testpeptid / IC50 StandardQ value = IC 50 test peptide / IC 50 standard
Q-Werte aus Versuchswiederholungen wurden gemittelt.Q values from repeated tests were averaged.
Die Ergebnisse des beschriebenen Tests sind in folgender Tabelle 1 zusammengefaßt:The results of the test described are summarized in Table 1 below:
Tabelle 1Table 1
Ergebnisse des α ßε / Fibronektin RezeptorbindungstestsResults of the α ßε / fibronectin receptor binding test
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Die nachfolgenden Beispiele betreffen pharmazeutische Zubereitungen:The following examples relate to pharmaceutical preparations:
Beispiel A: InjektionsgläserExample A: Injection glasses
Eine Lösung von 100 g Ac-RGDLdSLR-NH2 und 5 g Dinatriumhydrogen- phosphat wird in 3 I zweifach destilliertem Wasser mit 2 n Salzsäure auf pH 6,5 eingestellt, steril filtriert, in Injektionsgläser abgefüllt, unter sterilen Bedingungen lyophilisiert und steril verschlossen. Jedes Injektionsglas enthält 5 mg Wirkstoff.A solution of 100 g of Ac-RGDLdSLR-NH 2 and 5 g of disodium hydrogenphosphate is adjusted to pH 6.5 in 3 l of double-distilled water with 2N hydrochloric acid, sterile filtered, filled into injection glasses, lyophilized and sterile sealed under sterile conditions. Each injection jar contains 5 mg of active ingredient.
Beispiel B: Suppositorien Man schmilzt ein Gemisch von 20 g Ac-RGDLdSLR-NH2 mit 100 g Sojalecithin und 1400 g Kakaobutter, gießt in Formen und läßt erkalten. Jedes Suppositorium enthält 20 mg Wirkstoff.Example B: Suppositories A mixture of 20 g Ac-RGDLdSLR-NH 2 is melted with 100 g soy lecithin and 1400 g cocoa butter, poured into molds and allowed to cool. Each suppository contains 20 mg of active ingredient.
Beispiel C: LösungExample C: solution
Man bereitet eine Lösung aus 1 g Ac-RGDLdSLR-NH2, 9,38 g NaH2P04 • 2 H20, 28,48 g Na2HP04 • 12 H20 und 0,1 g Benzalkoniumchlorid in 940 ml zweifach destilliertem Wasser. Man stellt auf pH 6,8 ein, füllt auf 1 I auf und sterilisiert durch Bestrahlung. Diese Lösung kann in Form vonA solution is prepared from 1 g Ac-RGDLdSLR-NH 2 , 9.38 g NaH 2 P0 4 • 2 H 2 0, 28.48 g Na 2 HP0 4 • 12 H 2 0 and 0.1 g benzalkonium chloride in 940 ml double distilled water. It is adjusted to pH 6.8, made up to 1 I and sterilized by irradiation. This solution can take the form of
Augentropfen verwendet werden.Eye drops can be used.
Beispiel D: SalbeExample D: ointment
Man mischt 500 mg Ac-RGDLdSLR-NH2 mit 99,5 g Vaseline unter aseptischen Bedingungen.500 mg of Ac-RGDLdSLR-NH 2 are mixed with 99.5 g of petroleum jelly under aseptic conditions.
Beispiel E: TablettenExample E: tablets
Ein Gemisch von 1 kg Ac-RGDLdSLR-NH2, 4 kg Lactose, 1 ,2 kg Kartoffelstärke, 0,2 kg Talk und 0,1 kg Magnesiumstearat wird in üblicher Weise zu Tabletten verpreßt, derart, daß jede Tablette 10 mg Wirkstoff enthält.A mixture of 1 kg of Ac-RGDLdSLR-NH 2 , 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 tablets in a conventional manner such that each tablet contains 10 mg of active ingredient ,
Beispiel F: DrageesExample F: coated tablets
Analog Beispiel E werden Tabletten gepreßt, die anschließend in üblicher Weise mit einem Überzug aus Saccharose, Kartoffelstärke, Talk, Tragant und Farbstoff überzogen werden.Analogously to Example E, tablets are pressed, which are then coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and colorant.
Beispiel G: KapselnExample G: capsules
2 kg Ac-RGDLdSLR-NH2 werden in üblicher Weise in Hartgelatinekapseln gefüllt, so daß jede Kapsel 20 mg des Wirkstoffs enthält.2 kg of Ac-RGDLdSLR-NH 2 are filled into hard gelatin capsules in a conventional manner, so that each capsule contains 20 mg of the active ingredient.
Beispiel H: Ampullen Eine Lösung von 1 kg Ac-RGDLdSLR-NH2 in 60 I zweifach destilliertem Wasser wird steril filtriert, in Ampullen abgefüllt, unter sterilen Bedingungen lyophilisiert und steril verschlossen. Jede Ampulle enthält 10 mg Wirkstoff.Example H: ampoules A solution of 1 kg of Ac-RGDLdSLR-NH 2 in 60 l of double-distilled water is sterile filtered, filled into ampoules, lyophilized under sterile conditions and sealed sterile. Each ampoule contains 10 mg of active ingredient.
Beispiel I: Inhalations-SprayExample I: Inhalation spray
Man löst 14 g Ac-RGDLdSLR-NH2 in 10 I isotonischer NaCI-Lösung und füllt die Lösung in handelsübliche Sprühgefäße mit Pump-Mechanismus. Die Lösung kann in Mund oder Nase gesprüht werden. Ein Sprühstoß14 g of Ac-RGDLdSLR-NH 2 are dissolved in 10 I of isotonic NaCI solution and the solution is filled into commercially available spray vessels with a pump mechanism. The solution can be sprayed into the mouth or nose. A spray
(etwa 0,1 ml) entspricht einer Dosis von etwa 0,14 mg. (about 0.1 ml) corresponds to a dose of about 0.14 mg.

Claims

Patentansprücheclaims
1. Peptidische Verbindungen der Formel I1. Peptide compounds of the formula I.
Ac-Arg-X -Asp-X2-X3-X4-X5-X6-NH2 IAc-Arg-X -Asp-X 2 -X 3 -X 4 -X 5 -X 6 -NH 2 I
worinwherein
Ac Acetyl,Ac Acetyl,
X1 Ser, Gly, Thr, Asp, Arg, Val, Tyr, His oder Ala, X2 Leu, Ile, Nie, Val oder Phe,X 1 Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala, X 2 Leu, Ile, Nie, Val or Phe,
X3 Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl oder Phg,X 3 Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg,
X4 Gly, Ala, Ser, ß-Ala oder ω-Abu,X 4 Gly, Ala, Ser, ß-Ala or ω-Abu,
X5 Leu, Ile, Nie, Val oder Phe,X 5 Leu, Ile, Nie, Val or Phe,
X6 Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser oder AspX 6 Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp
bedeuten,mean,
wobei die genannten Aminosäuren auch derivatisiert sein können, die Aminosäurereste über die α-Amino- und α-Carboxygruppen peptidartig miteinander verknüpft sind, die D- als auch die L-Formen der optisch aktiven Aminosäurereste eingeschlossen sind, sowie deren Salze, und wobei Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 ausgenommen ist.where the amino acids mentioned can also be derivatized, the amino acid residues are linked in peptide fashion via the α-amino and α-carboxy groups, the D- and the L-forms of the optically active amino acid residues are included, as well as their salts, and where Ac- Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 is excluded.
Peptidische Verbindungen nach Anspruch 1 ausgewählt aus der GruppePeptide compounds according to claim 1 selected from the group
Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH2l Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH 2l Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2,Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NH2, Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NH 2 , Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH2, Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH 2 , Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH 2 ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH2,Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH2l Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH 2l
Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NH2,Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NH 2 ,
Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH2,Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH 2 ,
sowie deren physiologisch unbedenklichen Salze.and their physiologically acceptable salts.
5 3. Peptidische Verbindungen der Formel I gemäß Anspruch 1 und die5 3. Peptide compounds of formula I according to claim 1 and the
Verbindungen gemäß Anspruch 2 sowie deren physiologisch unbedenklichen Salze als Arzneimittel.Compounds according to claim 2 and their physiologically acceptable salts as medicaments.
Q 4. Arzneimittel nach Anspruch 3 als Inhibitor zur Bekämpfung von Q 4. Medicament according to claim 3 as an inhibitor for combating
Erkrankungen, die auf einer Expression und pathologischen Funktion von αvßβ lntegrinrezeptoren beruhen.Diseases based on expression and pathological function of α v β β integrin receptors.
5. Arzneimittel nach Anspruch 4 zur Bekämpfung von Thrombosen, 5 Herzinfarkt, koronaren Herzerkrankungen, Arteriosklerose, Tumoren, Osteoporose, Fibrösen, Entzündungen, Infektionen, Psoriasis sowie zur Beeinflussung von Wundheiiungsprozessen.5. Medicament according to claim 4 for combating thrombosis, 5 heart attack, coronary heart disease, arteriosclerosis, tumors, Osteoporosis, fibrosis, inflammation, infections, psoriasis and for influencing wound healing processes.
6. Pharmazeutische Zubereitung, enthaltend mindestens ein Arznei- mittel gemäß einem der Ansprüche 4 und 5 sowie gegebenenfalls6. Pharmaceutical preparation containing at least one medicament according to one of claims 4 and 5 and optionally
Träger- und/oder Hilfsstoffe und gegebenenfalls andere Wirkstoffe.Carriers and / or auxiliary substances and optionally other active substances.
7. Verwendung von peptidischen Verbindungen gemäß der Ansprüche 1 und 2 und/oder ihre physiologisch unbedenklichen Salze zur7. Use of peptide compounds according to claims 1 and 2 and / or their physiologically acceptable salts for
Herstellung eines Arzneimittels zur Bekämpfung von Erkrankungen, die auf einer Expression und pathologischen Funktion von αvßδ Integrinrezeptoren beruhen.Production of a medicament for combating diseases which are based on the expression and pathological function of α v β δ integrin receptors.
Verwendung nach Anspruch 7 zur Herstellung eines Arzneimittels zur Bekämpfung von Thrombosen, Herzinfarkt, koronaren Herzerkrankungen, Arteriosklerose, Tumoren, Osteoporose, Fibrösen, Entzündungen, Infektionen, Psoriasis sowie zur Beeinflussung von Wundheiiungsprozessen. Use according to claim 7 for the manufacture of a medicament for combating thromboses, myocardial infarction, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, fibrosis, inflammation, infections, psoriasis and for influencing wound healing processes.
PCT/EP2000/005404 1999-06-26 2000-06-13 INHIBITORS OF THE INTEGRIN αvβ¿6? WO2001000660A1 (en)

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CA002377224A CA2377224A1 (en) 1999-06-26 2000-06-13 Inhibitors of the integrin .alpha.v.beta.6
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EP00949177A EP1189930A1 (en) 1999-06-26 2000-06-13 INHIBITORS OF THE INTEGRIN AlPHA V BETA 6
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BR0011954-7A BR0011954A (en) 1999-06-26 2000-06-13 Integrin inhibitors alfavbeta6
KR1020017016580A KR20020015704A (en) 1999-06-26 2000-06-13 INHIBITORS OF THE INTEGRIN αVβ6
PL00352374A PL352374A1 (en) 1999-06-26 2000-06-13 Inhibitors of integrine o v beta 6
JP2001507066A JP2003503422A (en) 1999-06-26 2000-06-13 Integrin αvβ6 inhibitor
AU62630/00A AU771099B2 (en) 1999-06-26 2000-06-13 Inhibitors of the integrin alphavbeta6
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007039728A2 (en) * 2005-10-03 2007-04-12 Cancer Research Technology Ltd AVß6 PEPTIDE LIGANDS AND THEIR USES
JP2007523835A (en) * 2003-02-06 2007-08-23 メルク　パテント　ゲゼルシャフト　ミット　ベシュレンクテル　ハフトング Peptidesulfonamide
US8398975B2 (en) 2006-08-03 2013-03-19 Medimmune Limited Antibodies directed to αVβ6 and uses thereof
WO2018085415A1 (en) 2016-11-01 2018-05-11 Arrowhead Pharmaceuticals, Inc. Alpha-v beta-6 integrin ligands and uses thereof
US11597701B2 (en) 2017-11-01 2023-03-07 Arrowhead Pharmaceuticals, Inc. Integrin ligands and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6972296B2 (en) 1999-05-07 2005-12-06 Encysive Pharmaceuticals Inc. Carboxylic acid derivatives that inhibit the binding of integrins to their receptors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020708A1 (en) * 1991-05-14 1992-11-26 Board Of Regents For The University Of Oklahoma Peptide inhibitors of inflammation
WO1999007405A1 (en) * 1997-08-08 1999-02-18 The Regents Of The University Of California TREATMENT OF ACUTE LUNG INJURY AND FIBROSIS WITH ANTAGONISTS OF αvβ6
WO2000037487A1 (en) * 1998-12-19 2000-06-29 Merck Patent Gmbh αvβ6 INTEGRIN INHIBITORS

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020708A1 (en) * 1991-05-14 1992-11-26 Board Of Regents For The University Of Oklahoma Peptide inhibitors of inflammation
WO1999007405A1 (en) * 1997-08-08 1999-02-18 The Regents Of The University Of California TREATMENT OF ACUTE LUNG INJURY AND FIBROSIS WITH ANTAGONISTS OF αvβ6
WO2000037487A1 (en) * 1998-12-19 2000-06-29 Merck Patent Gmbh αvβ6 INTEGRIN INHIBITORS

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KRAFT E A: "Definition of an unexpected ligand recognition motif for alpha3ss6 integrin", JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 274, no. 4, 22 January 1999 (1999-01-22), pages 1979 - 1985, XP002136307, ISSN: 0021-9258 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007523835A (en) * 2003-02-06 2007-08-23 メルク　パテント　ゲゼルシャフト　ミット　ベシュレンクテル　ハフトング Peptidesulfonamide
WO2007039728A2 (en) * 2005-10-03 2007-04-12 Cancer Research Technology Ltd AVß6 PEPTIDE LIGANDS AND THEIR USES
WO2007039728A3 (en) * 2005-10-03 2007-09-20 Cancer Rec Tech Ltd AVß6 PEPTIDE LIGANDS AND THEIR USES
US8398975B2 (en) 2006-08-03 2013-03-19 Medimmune Limited Antibodies directed to αVβ6 and uses thereof
US8894998B2 (en) 2006-08-03 2014-11-25 Medimmune Limited Antibodies directed to αVβ6 and uses thereof
WO2018085415A1 (en) 2016-11-01 2018-05-11 Arrowhead Pharmaceuticals, Inc. Alpha-v beta-6 integrin ligands and uses thereof
EP3535397A4 (en) * 2016-11-01 2020-06-03 Arrowhead Pharmaceuticals, Inc. Alpha-v beta-6 integrin ligands and uses thereof
EP3981780A1 (en) * 2016-11-01 2022-04-13 Arrowhead Pharmaceuticals, Inc. Alpha-v beta-6 integrin ligands and uses thereof
US11597701B2 (en) 2017-11-01 2023-03-07 Arrowhead Pharmaceuticals, Inc. Integrin ligands and uses thereof

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