EP1140989A1 - Inhibiteurs de l'integrine alpha(v) beta(6) - Google Patents

Inhibiteurs de l'integrine alpha(v) beta(6)

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Publication number
EP1140989A1
EP1140989A1 EP99963507A EP99963507A EP1140989A1 EP 1140989 A1 EP1140989 A1 EP 1140989A1 EP 99963507 A EP99963507 A EP 99963507A EP 99963507 A EP99963507 A EP 99963507A EP 1140989 A1 EP1140989 A1 EP 1140989A1
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EP
European Patent Office
Prior art keywords
arg
leu
asp
peptide
thr
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP99963507A
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German (de)
English (en)
Inventor
Beate Diefenbach
Alfred Jonczyk
Sabine Kraft
Ray Mehta
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Merck Patent GmbH
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Merck Patent GmbH
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Publication date
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Publication of EP1140989A1 publication Critical patent/EP1140989A1/fr
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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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic 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
    • 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
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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 describes novel peptides which are biologically active as ligands of the integrin ⁇ v ⁇ ⁇ . These peptides all have a common structural motif, namely - Asp Leu Xaa Xaa Leu - or in a preferred form - Arg Xaa Asp Leu Xaa Xaa Leu Arg - where Xaa stands for any amino acid residue.
  • the peptides according to the invention can be used as effective inhibitors of the ⁇ v ⁇ ⁇ integrin receptor and thus for the treatment of various diseases and pathological findings.
  • Integrins belong to the family of heterodimeric class I transmembrane receptors, which play an important role in numerous cell-matrix or cell-line adhesion processes (Tuckwell et al., 1996, Symp. Soc Ex. Biol. 47). They can be roughly divided into three classes: the ßi integrins, which are receptors for the extracellular matrix, the ß 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 ⁇ sßi, ⁇ u D ß3, ⁇ sßi, ⁇ v ß ⁇ , ⁇ v ß3 and ⁇ v ß6 all bind to the Arg-Gly-Asp (RGD) peptide sequence, for example in the natural ligand fibronectin. Soluble peptides containing RGD are able to inhibit the interaction of each of these integrins with fibronectin.
  • ⁇ v ß ⁇ is a relatively rare integrin (Busk et al., 1992 J. Biol. Chem.
  • ⁇ v ß 6 is expressed on keratinocytes in wounds (Haapasalmi et al., 1996, J. Invest. Dermatol.
  • ⁇ 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 diseases, such as
  • ⁇ v ß ⁇ also plays a role in the intestinal epithelium, so that corresponding Integhn agonists / antagonists could be used in the treatment of inflammation, tumors and wounds of the gastrointestinal tract.
  • the peptide compounds of the formulas set out below and their salts, as soluble molecules have an effect 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 ⁇ 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 can be used in vivo if they are equipped with appropriate markers (e.g. the biotinyl residue) according to the prior art.
  • the invention also includes conjugates with other active substances, such as cytotoxic active substances 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.
  • the invention thus relates to peptide compounds of the formula I.
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 each independently represent an amino acid residue, the amino acids being selected independently of one another from a group consisting of Ala, Asn, Asp, Arg, Cys, Gin, Glu, Gly , Phe, His, He, Leu, Lys, Met, Nie, homo-Phe, Phg, Pro, Ser, Thr, Trp, Tyr or Val, and the amino acids mentioned can also be derivatized,
  • W 2 selected from the group OH, OR, NHR, NR 2 , NH 2 , W1 H or Acyirest
  • n, m each independently of one another a number from 0 to 15.
  • the radicals X 1 or X 6 can each independently be the same or different.
  • those amino acids or amino acid residues are also included which are derived from the natural amino acids and are homologs or isomers thereof. The amino acid residues are usually linked to one another via their alpha-amino and alpha-carboxy groups (peptide bond).
  • the invention furthermore preferably relates to those peptide compounds in which X 2 represents an amino acid residue which has been selected from the group Thr, Ser, Asp and glycine, furthermore those peptide compounds in which X 3 is an amino acid residue which has been selected from the Asp, Glu, Arg, Lys, His and Tyr, and finally those peptide compounds in which X 4 is an amino acid residue selected from the group Ser, Tyr, Thr, Gly and Val.
  • Particularly preferred peptide compounds according to the invention are those of formula V.
  • amino acids can occur in several enantiomeric forms, then above and below, for. B. as a component of the compounds of formulas I-VI, including all these forms and also their mixtures. Furthermore, the amino acids, e.g. B. as part of compounds of the formulas I-VI, provided with corresponding protective groups known per se.
  • the compounds of the formula In-VI can have one or more chiral centers and therefore exist in various stereoisomeric forms.
  • the formulas given include all of these forms, especially the D and L forms, both in enantiomeric and in racemic mixtures.
  • the formulas I and II mentioned above and below also include the corresponding salts, in particular the corresponding physiologically acceptable salts, according to the invention.
  • So-called prodrug de vate are also included in the compounds according to the invention, ie with z.
  • 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 biotinylated or fluorescence-labeled peptides.
  • the peptides according to the invention are linear, but they can also be cyclized.
  • the invention comprises not only the peptides of the formulas I to VI mentioned but also mixtures and preparations which, in addition to these compounds according to the invention, also contain other pharmacological active substances or adjuvants which can influence the primary pharmacological action of the peptides according to the invention in a desired manner.
  • the compounds according to the invention and also the starting materials for their preparation are otherwise prepared by methods which are known and are used frequently, as described in the literature (for example in the standard works such as Houben-Weyl, Methods of Organic Chemistry, Georg-Thieme-Verlag, Stuttgart , under reaction conditions which are known and suitable for the reactions mentioned, and it is also possible to use variants known per se.
  • the peptides according to the invention can preferably be prepared using
  • Solid phase synthesis and subsequent cleavage and purification can be produced, as described, for example, by Jonczyk and Meienhofer (Peptides, Proc. 8th Am. Pept. Symp., Eds. V. Hruby and DH Rieh, Pierce Comp. III, p. 73-77, 1983 , or Angew. Chem. 104, 1992, 375) or according to Merrifield (J. Am. Chem. Soc. 94, 1972, 3102). Otherwise, they can be prepared by conventional methods of amino acid and peptide synthesis, such as. B.
  • 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 of the formulas I-VI according to the invention can of course 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 by substituting their -CO-OH hydroxy function by means of a protective group, e.g. can be protected as esters.
  • 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 has been carried out at other locations in the molecule.
  • 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. Depending on the protective group used, the connections from their functional derivatives can be set free.
  • 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). The procedures are generally known and will not be described further here.
  • the peptides according to the invention comprise their physiologically acceptable salts, which can also be prepared by standard methods.
  • a base of the formula I can be converted into the associated acid addition salt using an acid, 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.
  • So 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, furthermore organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or polycarbonate, sulfonic or Sulfuric acids, e.g.
  • acetic acid formic acid, acetic acid, propionic acid, pivalic acid, diethyl acetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotanesulfonic acid, methane or ethanoisulfonic acid, methane or ethane acid Benzenesulfonic acid, p-toluenesulfonic acid, naphthalene mono- and disulfonic acids, laurylsulfuric acid.
  • Salts with physiologically unacceptable acids can be used for the isolation and / or purification of the compounds according to the invention.
  • an acid of formula I can be converted into one of its 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 diisopropyl ammonium salts, monoethanol, Diethanol or diisopropylammonium salts, cyclohexyl, dicyclohexylammonium salts, dibenzylethylenediammonium salts, further 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.
  • pulmonary fibrosis ophthalmic diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerosis, psoriasis, renal inflammation after angioplasty after angioplasty.
  • 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 the control of disorders which are based indirectly or directly on expression of the v ß ⁇ integrin receptor, thus in particular in pathological angiogenic disorders, thromboses, cardiac infarction, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammations , Infections and to influence wound healing processes.
  • Corresponding pharmaceutical preparations which contain at least one medicament of the formulas I to VI and, if appropriate, carriers and / or auxiliaries are also an object.
  • 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 production of a medicament for combating diseases which are based directly or indirectly on expression of the ⁇ v ⁇ delta integrin receptor, in particular, therefore pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammation, infections and for influencing wound healing processes.
  • the medicaments according to the invention or pharmaceutical preparations containing them can be used in human or veterinary medicine.
  • Suitable carriers 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 do not react with the new compounds, for example water, vegetable oils, benzyl alcohols , Alkyl glycols, polyethylene glycols, glycerin acetate, 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, suppositories for rectal use, solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants for which topical application 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 contain auxiliary substances such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, flavoring and / or / several other active substances, e.g. . B. one or more vitamins.
  • auxiliary substances such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, flavoring and / or / several other active substances, 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 substance is expediently used in micronized form, with one or more additional physiologically compatible solvents being present can be, e.g. B
  • 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, sex, on the diet, on the time and route of administration, on the rate of elimination, combination of drugs and severity the respective disease to which the therapy applies. Parenteral administration is preferred.
  • the invention also includes recombinant DNA sequences which contain sections which code for peptide regions which have the peptide structural motifs of the formulas I to VI according to the invention.
  • Such DNA can be transferred to cells by particles, as described in Ch. Andree et al. Proc.Natl.Acad. Be. 91, 12188-12192 (1994), or the transfer to cells can be increased by other means such as liposomes (A.I. Aronsohn and J.A. 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 from 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.
  • 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.
  • the DNA for the sequences of this invention can also be used for cell transfections by means of retroviral or adenoviral vectors.
  • 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 preparation of a liposome complex from 0 lipid / DNA / peptide is for example in
  • a lipid / peptide / DNA liposome complex is, for example, from the following
  • DOTMA N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride
  • DOPE dioleyl phosphatidylethanolamine
  • DNA and peptide are dissolved in cell culture medium.
  • the liposome complex is prepared by mixing the three components in a specific weight ratio (lipid: DNA: peptide, e.g. 0.75: 1: 4).
  • 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 ⁇ -integrin receptors, in particular in the case of pathologically angiogenic diseases, thromboses , Heart attack, coronary heart disease, arteriosclerosis, tumors, osteoporosis, inflammation, infections and to influence wound healing processes.
  • 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 of formula I are formed according to methods known per se (for example 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.
  • Suitable polymeric carrier materials are the polymeric solid phases known per se in peptide chemistry with preferably hydrophilic properties, for example crosslinked
  • Synthesizer used according to the manufacturer's instructions (Milligen). The basic steps are washing - splitting off the Fmoc protective group - washing - coupling with the next Fmoc amino acid - capping (acetylation) - washing. If an N-terminal acylation is desired after the last amino acid coupling, this takes place after the last Fmoc protective group has been split off with the corresponding activated acyl radical, e.g. the acetic anhydride.
  • the product was cleaned by RP-HPLC on Lichrosorb RP18 (250-25, 7 ⁇ m, Merck KGaA) in 0.3% TFA with a gradient from 4% to 24% 2-propanol in 2 h at 8 ml / min and assessment by means of UV flow photometer at 215 nm.
  • the fractions containing product were freeze-dried.
  • the product obtained met the expectations according to FAB-MS (Fast Atom Bombardment Mass Spectroscopy): C 4 H 73 N 15 0 15 M 1015.5 g / mol; (M + H) + is 1016.
  • the purified product Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 has in the analytical HPLC on SuperSpher RP18e (250-4, Merck KGaA) with a gradient from 0-99% A (0.08 m phosphate pH 3.5, 15% acetonitrile) to B (0.03 m phosphate pH 3.5, 70% acetonitrile) in 50 min at 1 ml / min and detection at 215 nm, a retention time of 7.22 min.
  • System A 0.3% trifluoroacetic acid with a gradient of 0 - 80% 2-propanol in 50 min on LichroSpher 60 RP-Select B ® (250-4) (Merck KGaA, Darmstadt, Germany), at 1 ml / min, and detection at 215 nm system.
  • B 0.1% trifluoroacetic acid with a gradient 30-70% acetonitrile in 50 min to Superspher 100 RP18e ® (250-4) (Merck KGaA, Darmstadt, Germany), at 1 ml / min and detection at 215 nm.
  • RGD peptides such as GRGDSPK, cyclo- (RGDfV) and the linear peptide DLYYLMDL served as comparison compounds.
  • transmembrane truncated ⁇ v ⁇ 6 from recombinant baculovirus cells.
  • an ⁇ v transfer vector was prepared and transmembrane shortened ( ⁇ TM) ⁇ v was cut out of the plasmid ⁇ v ⁇ TM (pBAc9) using the restriction enzymes EcoRI and Xbal (Mehta et al., Ref. See above) and by means of "blunt-end" ligation into the BamHI interface of pAcUW31
  • Transmembrane-shortened ⁇ 6 cDNA was cut out from the plasmid pCDNAneoß 6 (Weinacker at al., Ref. See above) using the restriction enzymes EcoRI and Xbal and likewise by means of "blunt-end" ligation into the BamHI interface of pAcUW31 "downstream" of the polyhedrin promoter cloned in.
  • the truncated ⁇ v and ß 6 containing tandem vectors were used to obtain recombinant baculovirus (Mehta et al., Ref. See above).
  • the recombinant baculoviruses were used to infect "high five" insect cells.
  • the soluble receptor was obtained after 48-71 hours of cultivation by passing the supernatant from the cell culture over affinity columns of the type mentioned above and eluting at pH 3.1. All process steps were carried out at room temperature and in the absence of any detergents. The peak fractions were neutralized, concentrated and dialyzed at 4 ° C and finally stored at -80 ° C.
  • the recombinant soluble human receptor thus obtained is biologically active and retains its ligand specificity.
  • Example 4 a v ⁇ / fibronectin receptor binding test:
  • the peptides according to the invention prepared were bound to the immobilized ⁇ v ⁇ ⁇ 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 ⁇ 6 .
  • the Q value is calculated from the quotient of the IC 50 values of the test peptide and a standard.
  • the linear hepta-RGD peptide GRGDSPK 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 soluble ⁇ v ß 6 receptor was immobilized on microtiter plates 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 ⁇ l / well).
  • Biorad alkaline phosphatase-coupled anti-biotin antibody
  • the colorimetric detection was carried out by incubation (10-15 min; 25 ° C., in the dark) with substrate solution (5 mg nitrophenyl phosphate, 1 ml ethanolamine, 4 ml H 0; 100 ⁇ l / well). The enzyme reaction was stopped by adding 0.4 M NaOH (100 ⁇ l ⁇ / 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 the zero value. GRGDSPK was used as the standard.
  • Q values less than 1 mean that they have a relatively better binding to the receptor than the standard peptide, which in absolute terms already has a good binding in competition with the natural ligand fibronectin.
  • integrin ligand binding tests were carried out with different integrins (eg ⁇ v ß 3 , ⁇ v ßs) and their corresponding ligands (eg vitronectin, fibrinogen) for comparison purposes.
  • integrins eg ⁇ v ß 3 , ⁇ v ßs
  • ligands eg vitronectin, fibrinogen
  • Solution HEPES 140 mM NaCl, 1 mM MgCl 2 in cancer, 2 mM CaCl 2, 6 mM KCI, 10 mM HEPES, 10 mM D-glucose; pH 9, is mixed lipid and DNA in the weight ratio 5: 1 (DNA lipid) 0).
  • the single dose is 30 ⁇ g DNA / 200 ⁇ l.
  • 200 ⁇ l of this lipid-DNA complex are applied to the nasal epithelium using a pump sprayer. This is repeated 10 times at 15 min intervals.
  • the total dose of DNA is 300 ⁇ g.
  • Example A Injection glasses
  • a solution of 100 g of an active ingredient of the formula I and 5 g of disodium hydrogenphosphate are adjusted to pH 6.5 in 3 l of double-distilled water with 2N hydrochloric acid, sterile filtered, filled into injection glasses, lyophilized under sterile conditions and sealed sterile. Each injection jar contains 5 mg of active ingredient.
  • a mixture of 20 g of an active ingredient of the formula I is melted with 100 g of soy lecithin and 1400 g of cocoa butter, poured into molds and allowed to cool. Each suppository contains 20 mg of active ingredient.
  • a solution is prepared from 1 g of an active ingredient of the formula I, 9.38 g of NaH2P ⁇ 4 • 2 H2O, 28.48 g of Na2HP04 • 12 H2O and 0.1 g of benzalkonium chloride in 940 ml of double-distilled water. It is adjusted to pH 6.8, made up to 1 I and sterilized by irradiation. This solution can be used in the form of eye drops.
  • Example D ointment
  • 500 mg of an active ingredient of the formula I are mixed with 99.5 g of petroleum jelly under aseptic conditions.
  • a mixture of 1 kg of active ingredient of the formula I, 4 kg of lactose, 1, 2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is compressed 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
  • each capsule contains 20 mg of the active ingredient.
  • a solution of 1 kg of active ingredient of the formula I 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.

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Abstract

L'invention concerne de nouveaux peptides biologiquement efficaces comme ligands de l'intégrine αvβ6. Ces peptides présentent tous un motif commun, à savoir - Asp Leu Xaa Xaa Leu - ou, sous une forme préférée, - Arg Xaa Asp Leu Xaa Xaa Arg -, où Xaa désigne un reste aminoacide quelconque. Les peptides selon l'invention peuvent être utilisés comme inhibiteurs efficaces du récepteur de l'intégrine αvβ6 et, par conséquent, pour le traitement de différentes maladies et de différents états pathologiques.
EP99963507A 1998-12-19 1999-12-11 Inhibiteurs de l'integrine alpha(v) beta(6) Withdrawn EP1140989A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19858857 1998-12-19
DE19858857 1998-12-19
PCT/EP1999/009842 WO2000037487A1 (fr) 1998-12-19 1999-12-11 INHIBITEURS DE L'INTEGRINE $G(a)v$G(b)¿6?

Publications (1)

Publication Number Publication Date
EP1140989A1 true EP1140989A1 (fr) 2001-10-10

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP99963507A Withdrawn EP1140989A1 (fr) 1998-12-19 1999-12-11 Inhibiteurs de l'integrine alpha(v) beta(6)

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Country Link
EP (1) EP1140989A1 (fr)
JP (1) JP2002533064A (fr)
CN (1) CN1335853A (fr)
AR (1) AR022395A1 (fr)
BR (1) BR9916323A (fr)
CA (1) CA2355874A1 (fr)
CZ (1) CZ20012212A3 (fr)
NO (1) NO20013013L (fr)
WO (1) WO2000037487A1 (fr)
ZA (1) ZA200105929B (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19929410A1 (de) * 1999-06-26 2000-12-28 Merck Patent Gmbh Inhibitoren des Integrins avß6
DE19933173A1 (de) * 1999-07-15 2001-01-18 Merck Patent Gmbh Cyclische Peptidderivate als Inhibitoren des Integrins alpha¶v¶beta¶6¶
EA011853B1 (ru) 2002-03-13 2009-06-30 Байоджен Айдек Ма Инк. АНТИТЕЛА ПРОТИВ αβ
MXPA05008313A (es) * 2003-02-06 2005-09-20 Merck Patent Gmbh Sulfonamidas peptidicas.
CN102875681A (zh) 2005-07-08 2013-01-16 拜奥根Idec马萨诸塞公司 抗-αvβ6抗体及其用途
GB0520068D0 (en) * 2005-10-03 2005-11-09 Cancer Res Technology av peptide ligand
WO2008008315A2 (fr) 2006-07-10 2008-01-17 Biogen Idec Ma Inc. COMPOSITIONS ET MÉTHODES VISANT À INHIBER LA CROISSANCE DE CANCERS À DÉFICIENCE EN smad4
MX2009001293A (es) 2006-08-03 2009-02-11 Astrazeneca Ab Anticuerpos dirigidos a (v(6 y usos de los mismos.
JP2010506944A (ja) * 2006-10-19 2010-03-04 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア インテグリンαＶβ６のアンタゴニストを用いる慢性喘息の処置および予防
WO2009093251A2 (fr) * 2008-01-24 2009-07-30 Gavish-Galilee Bio Applications Ltd Vaccin contenant un réovirus basé sur une séquence de protéine sigma c
KR101510941B1 (ko) * 2012-07-03 2015-04-10 일양약품주식회사 신규한 펩타이드 및 이의 용도
US10035860B2 (en) 2013-03-15 2018-07-31 Biogen Ma Inc. Anti-alpha V beta 6 antibodies and uses thereof
WO2014144466A1 (fr) 2013-03-15 2014-09-18 Biogen Idec Ma Inc. Anticorps anti-alpha ν bêta 6 et leurs utilisations
MA52117A (fr) 2017-02-28 2022-04-06 Morphic Therapeutic Inc Inhibiteurs de l'intégrine (alpha-v) (bêta-6)
EP4159727A1 (fr) 2017-02-28 2023-04-05 Morphic Therapeutic, Inc. Inhibiteurs de l'intégrine (alpha-v)(bêta-6)
PE20211640A1 (es) 2018-08-29 2021-08-24 Morphic Therapeutic Inc INHIBICION DE LA INTEGRINA xvß6

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
NZ219515A (en) * 1987-02-10 1989-09-27 Wellcome Found Fusion proteins comprising influenza virus ha and a nonnatural antigenic epitope

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0037487A1 *

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ZA200105929B (en) 2003-03-18
CN1335853A (zh) 2002-02-13
AR022395A1 (es) 2002-09-04
NO20013013D0 (no) 2001-06-18
CA2355874A1 (fr) 2000-06-29
BR9916323A (pt) 2001-10-30
NO20013013L (no) 2001-06-18
CZ20012212A3 (cs) 2001-09-12
WO2000037487A1 (fr) 2000-06-29
WO2000037487A8 (fr) 2001-03-29
JP2002533064A (ja) 2002-10-08

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