+

WO1997005257A1 - Inhibiteurs peptidiques, ou analogues, de l'activite du recepteur a l'urokinase - Google Patents

Inhibiteurs peptidiques, ou analogues, de l'activite du recepteur a l'urokinase Download PDF

Info

Publication number
WO1997005257A1
WO1997005257A1 PCT/US1996/012044 US9612044W WO9705257A1 WO 1997005257 A1 WO1997005257 A1 WO 1997005257A1 US 9612044 W US9612044 W US 9612044W WO 9705257 A1 WO9705257 A1 WO 9705257A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
peptide
group
ofthe
acid
Prior art date
Application number
PCT/US1996/012044
Other languages
English (en)
Inventor
Steven Rosenberg
Kerry L. Spear
Robert Valerio
Andrew Bray
Original Assignee
Chiron Corporation
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 Chiron Corporation filed Critical Chiron Corporation
Priority to AU65059/96A priority Critical patent/AU6505996A/en
Priority to EP96924666A priority patent/EP0842281A1/fr
Priority to US08/800,213 priority patent/US6030940A/en
Publication of WO1997005257A1 publication Critical patent/WO1997005257A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the fields of cellular biology and protein expression. More particularly, the invention relates to peptide and peptide analog ligands ofthe urokinase plasminogen activator receptor, and methods for preparing the same.
  • Urokinase-type plasminogen activator is a multidomain serine protease, having a catalytic "B" chain (amino acids 144-411), and an amino-terminal fragment ("ATF", aa 1-143) consisting of a growth factor-like domain (4-43) and a kringle (aa 47-135).
  • the uPA kringle appears to bind heparin, but not fibrin, lysine, or aminohexanoic acid.
  • the growth factor-like domain bears some similarity to the structure of epidermal growth factor (EGF), and is thus also referred to as an "EGF-like" domain.
  • the single chain pro-uPA is activated by plasmin, cleaving the chain into the two chain active form, which is linked together by a disulfide bond.
  • uPA binds to its specific cell surface receptor (uPAR).
  • the binding inter ⁇ action is apparently mediated by the EGF-like domain (S.A. Rabbani et al, J Biol Chem (1992) 267: 14151-56).
  • Cleavage of pro-uPA into active uPA is accelerated when pro-uPA and plasminogen -ire receptor-bound.
  • plasmin activates pro-uPA, which in turn activates more plasmin by cleaving plasminogen.
  • This positive feedback cycle is apparently limited to the receptor-based proteolysis on the cell surface, since a large excess of protease inhibitors is found in plasma, including a 2 antiplasmin, PAI-1 and PAI-2.
  • Plasmin can activate or degrade extracellular proteins such as fibrinogen, fibronectin, and zymogens. Plasminogen activators thus can regulate extracellular proteol ⁇ ysis, fibrin clot lysis, tissue remodeling, developmental cell migration, inflammation, and met- astasis. Accordingly, there is great interest in developing uPA inhibitors and uPA receptor antagonists.
  • E. Appella et al., J Biol Chem (1987) 262:4437-40 determined that receptor binding activity is localized in the EGF-like domain, and that residues 12-32 appear to be critical for binding.
  • the critical domain alone (uPA ⁇ .32) bound uPAR with an affinity of 40 nM (about 100 fold less than intact ATF).
  • One aspect ofthe invention is the set of polypeptides disclosed herein, and analogs thereof, which bind to the urokinase plasminogen activator receptor and inhibit the receptor binding activity of urokinase-type plasminogen activator.
  • Another aspect ofthe invention is a method for treating a urokinase-mod- ulated disorder, such as cancer and metastasis, by administering an effective amount of a peptide ofthe invention or an analog thereof.
  • Another aspect ofthe invention is a composition suitable for treating a urokinase-modulated disorder, comprising an effective amount of a peptide ofthe invention or an analog thereof in combination with a pharmaceutically acceptable excipient.
  • huPA refers specifically to human urokinase-type plasminogen activator.
  • the "EGF-like domain” is that portion ofthe huPA molecule responsible for mediating huPA binding to its receptor (uPAR).
  • the EGF-like domain sometimes called the growth factor- like domain (“GFD"), is located within the first 48 residues of huPA.
  • the critical residues essential for binding activity
  • “Peptides ofthe invention” and “huPAR antagonist peptides” have one ofthe following sequences:
  • LNFGCYLWCT SEQ ID NO:3
  • LNFGQYLnAYT SEQ ID NO:4
  • LNFGdCYLWCT SEQ ID NO:8
  • LCFdSCYLWYT SEQ ID NO:9
  • LNFdSCYLWCT (SEQ ID NO: 11), where dS denotes D-Ser, dC denotes D-Cys, and nA denotes 1 -naphthylalanine.
  • active analog refers to a polypeptide differing from the sequence of one ofthe peptides ofthe invention, or an active portion thereof by 1-3 amino acids, but which still exhibits a K ⁇ j ⁇ 250 nM with huPAR.
  • the differences are preferably conservative amino acid substitutions, in which an amino acid is replaced with another amino acid or amino acid analog of similar character.
  • Conservative analog substitutions include substitution of D-isomers for L-isomers, phenylglycine for phenylalanine, 1 -naphthylalanine for tryptophan, and the like.
  • Nonconservative changes are generally sub ⁇ stitutions of one ofthe above amino acids with an amino acid from a different group (e.g., substituting Asn for Glu), or substituting Cys, Met, His, or Pro for any ofthe above amino acids.
  • fusion protein refers to a protein ofthe form:
  • X ⁇ -(peptide) n -X 2 in which at least one of Xi and X 2 is a protein or polypeptide, (peptide) is a peptide ofthe invention or an active analog thereof, and n is an integer from 1 to 100.
  • Xi and X 2 may be the same or different, and may be portions ofthe same protein (e.g., the peptide may be inserted at an internal position within the primary sequence of another protein).
  • Xi and X 2 may be selected to improve expression ofthe peptide/fiision protein, to enhance purification, and/or to provide a biological activity.
  • the peptides ofthe invention may be the same or different, and may be separated by peptide spacers (e.g., if it is desired to prepare a fusion protein capable of crosslinking huPAR on the cell surface). Alternatively, the peptides may be separated by proteolytic cleavage sites, to facilitate cleavage ofthe fusion protein into individual active peptides.
  • conventional amino acid refers to the amino acids alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glu ⁇ tamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine
  • nonconventional amino acid refers to amino acids other than conventional amino acids.
  • Presently preferred nonconventional amino acids are:
  • Nle L-norleucine
  • Aabu ⁇ -aminobutyric acid
  • Hphe L-homophenylalanine
  • Nva L-norvaline
  • Dglu D-glutamic acid
  • Dphe D-phenylalanine
  • Dlys D-lysine
  • Dleu D-leucine
  • Dpro D-proline
  • Dgln D-glutamine
  • Dthr D-threonine
  • Dval D-valine
  • Dtrp D-tryptophan
  • Dtyr D-tyrosine
  • Etg L-ethylglycine
  • Tbug L-t-butylglycine
  • Pen penicillamine
  • Anap I-naphthylalanine
  • Chexa cyclohexylalanine
  • Cpen cyclopentylalanine
  • Cpro aminocyclopropane carboxylate
  • Norb aminonorbornylcarboxylate
  • Mphe L- ⁇ -methylphenylalanine
  • Mhis L- ⁇ -methylhistidine
  • Mile L- ⁇ -methylisoleucine
  • Mlys L- ⁇ -methyllysine
  • Mleu L- ⁇ -methylleucine
  • Mmet L- ⁇ -methylmethionine
  • Mval L- ⁇ -methylvaline
  • Mt L- ⁇ -methyltryptophan
  • Mtyr L- ⁇ -methyltyrosine
  • Morn L- ⁇ -methylornithine
  • Mnle L- ⁇ -methylnorleucine
  • Maabu ⁇ -amino- ⁇ -methylbutyric acid
  • Mnva L- ⁇ -methylnorvaline
  • Mhphe L- ⁇ -methylhomophenylalanine
  • Metg L- ⁇ -methylethylglycine
  • Mgabu ⁇ -methyl- ⁇ -aminobutyric acid
  • Maib ⁇ -methylaminoisobutyric acid
  • Mtbug L- ⁇ -methyl-t-butylglycine
  • Mpen ⁇ -methylpenicillamine
  • Manap ⁇ -methyl- ⁇ -naphthylalanine
  • Mchexa ⁇ -methylcyclohexylalanine
  • Mcpen ⁇ -methylcyclopentylalanine
  • Dmala D- ⁇ -methylalanine
  • Dmorn D- ⁇ -methylornithine
  • Dmcys D- ⁇ -methylcysteine
  • Dmasp D- ⁇ -methylaspartic acid
  • Dmglu D- ⁇ -methylglutamic acid
  • Dmphe D- ⁇ -methylphenylalanine
  • Dmhis D- ⁇ -methylhistidine
  • Dmile D- ⁇ -methylisoleucine
  • Dmlys D- ⁇ -methyllysine
  • Dmleu D- ⁇ -methylleucine
  • Dmmet D- ⁇ -methylmethionine
  • Dmasn D- ⁇ -methylasparagine
  • Dmpro D- ⁇ -methylproline
  • Dmgln D- ⁇ -methylglutamine
  • Dmarg D- ⁇ -methylarginine
  • Dmser D- ⁇ -methylserine
  • Dmthr D- ⁇ -methylthreonine
  • Dmval D- ⁇ -methylvaline
  • Dmtrp D- ⁇ -methyltryptophan
  • Dmtyr D- ⁇ -methyltyrosine
  • Nmala L-N-methylalanine
  • Nmcys L-N-methylcysteine
  • Nmasp L-N-methylaspartic acid
  • Nmglu L-N-methylglutamic acid
  • Nmphe L-N-methylphenylalanine
  • Nmhis L-N-methylhistidine
  • Nmile L-N-methylisoleucine
  • Nmlys L-N-methyllysine
  • Nmleu L-N-methylleucine
  • Nmmet L-N-methylmethionine
  • Nmasn L-N-methylasparagine
  • Nmchexa N-methylcyclohexylalanine
  • Dnmala D-N-methylalanine
  • Dnmorn D-N-methylornithine
  • Dnmcys D-N-methylcysteine
  • Dnmasp D-N-methylaspartic acid
  • Dnmglu D-N-methylglutamic acid
  • Dnmphe D-N-methylphenylalanine
  • Dnmhis D-N-methylhistidine
  • Dnmile D-N-methylisoleucine
  • Dnmlys D-N-methyllysine
  • Dnmleu D-N-methylleucine
  • Nhhis N-(imidazolylethyl)glycine
  • Nile N-( 1 -methylpropyl)glycine
  • Nlys N-(4-aminobutyl)glycine
  • Nleu N-(2-methylpropyl)glycine
  • Nmet N-(2-methylthioethyl)glycine
  • Nhser N-(hydroxyethyl)glycine
  • Nasn N-(carbamylmethyl)glycine
  • Ngln N-(2-carbamylethyl)glycine
  • Nval N-( 1 -methylethyl)glycine
  • Narg N-(3 -guanidinopropyl)glycine
  • Nhtrp N-(3-indolylethyl)glycine
  • Nhtyr N-(p-hydroxyphenethyl)glycine
  • Nthr N-(l -hydroxyethyl)glycine
  • Ncys N-(thiomethyl)glycine
  • Norn N-(3-aminopropyl)glycine
  • Ncpro N-cyclopropylglycine
  • Ncbut N-cyclobutyglycine
  • Nchex N-cyclohexylglycine
  • Nchep N-cycloheptylglycine
  • Ncoct N-cyclooctylglycine
  • Ncdec N-cyclodecylglycine
  • Ncund N-cycloundecylglycine
  • Ncdod N-cyclododecylglycine
  • Nbh N-(2,2-diphenylethyl)glycine
  • Nbhe N-(3,3-diphenylpropyl)glycine
  • Nnbhm N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine
  • Nnbhe N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine
  • Nbmc 1 -carboxy- l-(2,2-diphenylethylamino)cyclopropane.
  • expression vector refers to an oligonucleotide which encodes the huPAR antagonist polypeptide ofthe invention and provides the sequences necessary for its expression in the selected host cell.
  • Expression vectors will generally include a transcrip ⁇ tional promoter and terminator, or will provide for inco ⁇ oration adjacent to an endogenous promoter.
  • Expression vectors will usually be plasmids, further comprising an origin of replication and one or more selectable markers.
  • expression vectors may alternatively be viral recombinants designed to infect the host, or integrating vectors designed to integrate at a preferred site within the host's genome.
  • Expression vectors may further comprise an oligonucleotide encoding a signal leader polypeptide.
  • the huPAR antagonist When “operatively connected”, the huPAR antagonist is expressed downstream and in frame with the signal leader, which then provides for secretion ofthe huPAR antagonist polypeptide by the host to the extracellular medium.
  • the presently preferred signal leader is the Saccharomyces cerevisiae a-factor leader (particularly when modified to delete extraneous Glu-Ala sequences).
  • transcriptional promoter refers to an oligonucleotide sequence which provides for regulation ofthe DNA -> mRNA transcription process, typically based on temperature, or the presence or absence of metabolites, inhibitors, or inducers.
  • Transcrip- tional promoters may be regulated (inducible/repressible) or constitutive.
  • Yeast glycolytic enzyme promoters are capable of driving the transcription and expression of heterologous proteins to high levels, and are particularly preferred.
  • the presently preferred promoter is the hybrid ADH2/GAP promoter described in Tekamp-Olson et al, US 4,876,197 (inco ⁇ orated herein by reference), comprising the S.
  • host refers to a yeast cell suitable for expressing heterologous polypeptides.
  • suitable genera such as Saccharomyces, Schizosac ⁇ charomyces, Kluveromyces, Pichia, Hansenula, and the like.
  • yeast of the Saccharomyces genus particularly Saccharomyces cerevisiae.
  • huPA-mediated disorder refers to a disease state or malady which is caused or exacerbated by a biological activity of huPA.
  • the primary biological activity exhibited is plasminogen activation.
  • Disorders mediated by plasminogen activation include, without limitation, inappropriate angiogenesis (e.g. , diabetic retinopathy, corneal angiogenesis, Kaposi's sarcoma, and the like), metastasis and invasion by tumor cells, and chronic inflammation (e.g., rheumatoid arthritis, emphysema, and the like).
  • Fucosylated ATF or EGF-like domain are also mitogenic for tumor cells, which sometimes self-activate in an autocrine mechanism. Accordingly, the huPAR antagonist ofthe invention is effective in inhibiting the proliferation of huPA-activated tumor cells.
  • the term "effective amount" refers to an amount of huPAR antagonist polypep ⁇ tide sufficient to exhibit a detectable therapeutic effect.
  • the therapeutic effect may include, for example, without limitation, inhibiting the growth of undesired tissue or malignant cells, inhibiting inappropriate angiogenesis, limiting tissue damage caused by chronic inflammation, and the like.
  • the precise effective amount for a subject will depend upon the subject's size and health, the nature and severity ofthe condition to be treated, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation based on the information provided herein.
  • pharmaceutically acceptable refers to compounds and compositions which may be administered to mammals without undue toxicity.
  • exemplary pharmaceutically acceptable salts include mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, prop ⁇ ionates, malonates, benzoates, and the like.
  • the peptides ofthe invention may be synthesized by standard chemical methods, such as solid phase peptide synthesis.
  • the peptides composed of naturally-occurring amino acids may be expressed in an appropriate host, preferably as part of a fusion protein.
  • Peptides prepared as part of a fusion protein are preferably expressed in a host cell.
  • Presently preferred hosts are yeasts, particularly Saccharomyces, Schizosac ⁇ charomyces, Kluveromyces, Pichia, Hansenula, and the like, especially S. cerevisiae.
  • Strains MB2-1 and ABI 10 are presently preferred, as are strains JSC302 and JSC308 (for fusion protein constructs).
  • the expression vector is constructed according to known methods, and typically comprises a plasmid functional in the selected host.
  • the oligonucleotide encoding the peptide or fusion protein will generally be synthesized chemically, or cloned from a suitable source (e.g., from a bacteriophage library).
  • Stable plasmids generally require an origin of replication (such as the yeast 2 ⁇ ori), and one or more selectable markers (such as antibiotic resistance) which can be used to screen for transformants and force retention ofthe plasmid.
  • the vector should provide a promoter which is functional in the selected host cell, prefer ⁇ ably a promoter derived from yeast glycolytic enzyme promoters such as GAPDH, GAL, and ADH2.
  • the presently preferred promoter is a hybrid ADH2/GAP promoter comprising the S. cerevisiae glyceraldehyde-3 -phosphate dehydrogenase promoter in combination with the S. cerevisiae alcohol dehydrogenase II upstream activation site.
  • the expression vector should ideally provide a signal leader sequence between the promoter and the huPAR antagonist polypeptide sequence.
  • the signal leader sequence provides for translocation ofthe huPAR antagonist polypeptide through the endoplasmic reticulum and export from the cell into the extracellular medium, where it may be easily harvested.
  • signal leader sequences There are a number of signal leader sequences known that are functional in yeast. Presently preferred are the yeast ⁇ -factor leader (see U.S. 4,751,180, inco ⁇ orated herein by reference).
  • the vector may provide for integration into the host genome, as is described by Shuster, PCT WO92/01800, inco ⁇ orated herein by reference
  • Transformations into yeast can be carried out according to the method of A Hinnen et al, Proc Natl Acad Sci USA (1978) 75 1929-33, or H Ito et al, J Bacteriol (1983) 153 163-68
  • the vector integrates into the yeast genome at one or more sites homologous to its targeting sequence It is presently pre ⁇ ferred to linearize the vector by cleaving it within the targeting sequence using a restriction endonuclease, as this procedure increases the efficiency of integration
  • the number of integrated sequences may be increased by classical genetic techniques As the individual cell clones can carry integrated vectors at different locations, a genetic cross between two appropriate strains followed by sporulation and recovery of segregants can result in a new yeast strain having the integrated sequences of both original parent strains Continued cycles of this method with other integratively transformed strains can be used to further increase the copies of integrated plasmids in a yeast host strain
  • Fusion proteins may be prepared by the methods described above One may improve expression of peptides by expressing them as fusion proteins with a well-expressed protein leader or fusion partner
  • a well-expressed protein leader or fusion partner For example, Cu Zn superoxide dismutase (SOD) is highly- expressed in yeast when used as a fusion protein leader. See Cousens et al. , US 4,751 , 180, inco ⁇ orated herein by reference in full.
  • Other suitable fusion partners include ⁇ - galactosidase, human IgG antibody fragments (e.g., Fc fragments), ubiquitin, and the like.
  • secretion leaders such as the yeast ⁇ -factor leader or bacterial Omp A leader
  • secretion leaders such as the yeast ⁇ -factor leader or bacterial Omp A leader
  • fusion partners which will impart a biological activity to the fusion protein.
  • a cytotoxic protein such as ricin
  • huPAR antagonist polypeptides, active portions, active analogs, active peptoid analogs, and fusion proteins may be assayed for activity by methods known in the art. For example, the methods provided in the examples below, and in RJ. Goodson et al, Proc Natl Acad Sci USA (1994) 9J_:7129-33 are suitable.
  • active analogs may be screened conveniently and efficiently by following the method of H.M. Geysen et al, US 4,708,871.
  • Geysen described a method for synthesizing a set of over ⁇ lapping oligopeptides derived from any selected protein (e.g., aa aa 7 , aa 2 -aa 8 , aa 3 -aas > , etc.) bound to a solid phase array of pins, with a unique oligopeptide on each pin.
  • the pins are arranged to match the format of a 96-well microtiter plate, permitting one to assay all pins simultaneously, e.g., for binding to a labeled ligand.
  • huPAR antagonists are administered orally, topically, or by parenteral means, including subcutaneous and intramuscular injection, implantation of sustained release depots, intravenous injection, intranasal administration, and the like.
  • parenteral means including subcutaneous and intramuscular injection, implantation of sustained release depots, intravenous injection, intranasal administration, and the like.
  • huPAR antagonists may be administered as a pharmaceutical composition comprising a huPAR antagonist in com ⁇ bination with a pharmaceutically acceptable excipient.
  • compositions may be aqueous solutions, emulsions, creams, ointments, suspensions, gels, liposomal suspensions, and the like.
  • Suitable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol®, vegetable oils, and the like.
  • PEG polyethylene glycol
  • phosphate acetate
  • gelatin collagen
  • Carbopol® vegetable oils
  • One may additionally include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, for example, BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like.
  • Cream or ointment bases useful in formulation include lanolin, Silvadene® (Marion), Aquaphor® (Duke Laboratories), and the like.
  • Other topical formulations include aerosols, bandages, and other wound dressings.
  • Other devices include indwelling catheters and devices such as the Alzet® minipump.
  • Ophthalmic preparations may be formulated using commercially available vehicles such as Sorbi-care® (Allergan), Neodecadron® (Merck, Sha ⁇ & Dohme), Lacrilube®, and the like, or may employ topical preparations such as that described in US 5,124,155, inco ⁇ orated herein by reference. Further, one may provide a huPAR antagonist in solid form, especially as a lyophilized powder. Lyophilized formulations typically contain stabilizing and bulking agents, for example human serum albumin, sucrose, mannitol, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co.).
  • huPAR antagonist required to treat any particular disorder will of course vary depending upon the nature and severity ofthe disorder, the age and condition of the subject, and other factors readily determined by one of ordinary skill in the art.
  • the appropriate dosage may be determined by one of ordinary skill by following the methods set forth below in the examples.
  • about 0.010 mg/Kg to about 500 mg/Kg huPAR antagonist administered i.v. or subcutaneously is effective for inhibiting tissue damage due to chronic inflammation.
  • huPAR antagonist may be administered locally in a gel or matrix at a concentration of about 0.01 mg/Kg to about 50 mg/Kg.
  • Chiron Mimotopes (Melbourne, Australia):
  • LNFGCYLWCT SEQ ID NO:3
  • LNFGQYLnAYT SEQ ID NO:4
  • LNFGdCYLWCT SEQ ID NO:8
  • LCFdSCYLWYT SEQ ID NO:9
  • LNFdSCYLWCT (SEQ ED NO: 11), where dS denotes D-Ser, dC denotes D-Cys, and nA denotes 1 -naphthylalanine. All peptides were purified by preparative reverse phase HPLC and concentrations determined by amino acid analysis.
  • Example 2 Purified soluble uPAR was biotinylated with NHS-biotin (Molecular Probes) and immobilized at 0.3 Tg/mL in phosphate-buffered saline (PBS)/0.1% BSA on streptavidin- coated Immulon-2 96-well Removawell plates.
  • Human uPA N-terminal fragment (ATF; from M. Shuman, University of California, San Francisco) was iodinated by the Iodo-Gen method (Pierce). Uninco ⁇ orated 125 I was separated from labeled protein by Sephadex G-25 chromatography. The specific activity ofthe labeled protein was between 5 x 10 5 and 1 x 10° dpm/mol.
  • the iodinated tracer (100-500 pM) was incubated with the peptides prepared in Example 1 above in triplicate for 2h at room temperature in PBS/0.1% BSA in a total volume of 200 TL. The plates were washed with PBS/0.1% BSA 3X, and remaining bound radioactivity was measured on an LKB 1277 Gammamaster. Scatchard analysis was performed by using the LIGAND program (Biosoft, Milltown, NJ). The results were as follows:
  • Example 3 (Formulation of huPA Antagonists) huPA antagonist formulations suitable for use in chemotherapy are prepared as follows: A) Injectable Formulation:
  • This formulation is prepared following the procedure set forth in US 4,816,440, inco ⁇ orated herein by reference.
  • the formulation is administered by parenteral injection at the site to be treated.
  • the formulation is also generally suitable for administration as eyedrops directly to the conjunctiva, or by intranasal administration as an aerosol.
  • a concentrated formulation e.g., reducing the phosphate buffered saline to 2 mL
  • a concentrated formulation e.g., reducing the phosphate buffered saline to 2 mL
  • the minipump implanted at the site to be treated may be used.
  • LNFGQYLnAYT (SEQ ID NO:4) 1.0 mg fibronectin 69.0 mg albumin 37.5 mg water qs 3.0 mL HCl (0.01 M) qs pH 4 0
  • This dosage form is prepared following the procedure set forth in US 5,124,155, inco ⁇ orated herein by reference.
  • the fibronectin and albumin are dissolved in water to form a 3.0 mL solution, and HCl added to a pH of 4.0, causing the fibronectin to flocculate.
  • the flocculent is filtered, and combined with the peptide.
  • the mixture is then placed in a contact lens mold, and the mold closed for 30 min to form a corneal "shield" in the shape of a contact lens.
  • the shield releases peptide over a period of time, and is useful for preventing angiogenesis of corneal tissue following ophthalmic surgery.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 510 6012706 (B) TELEFAX: 510 655 3542
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Des antagonistes efficaces du récepteur de l'activateur du plasminogène, de type urokinase, possèdent des séquences choisies dans le groupe LNFGQYLWYT, LCFGCYLWYT, LNFGCYLWCT, LNFGQYLnAYT, LNFdSQYLWYT, LCFGCYLWY, LNFdSQYLnAYT, LNFGdCYLWCT, LCFdSCYLWYT, LCFdSCYLnAYT, LNFdSCYLWCT, ou leurs analogues actifs ou portions actives.
PCT/US1996/012044 1995-07-31 1996-07-19 Inhibiteurs peptidiques, ou analogues, de l'activite du recepteur a l'urokinase WO1997005257A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU65059/96A AU6505996A (en) 1995-07-31 1996-07-19 Peptide analog inhibitors of urokinase receptor activity
EP96924666A EP0842281A1 (fr) 1995-07-31 1996-07-19 Inhibiteurs peptidiques, ou analogues, de l'activite du recepteur a l'urokinase
US08/800,213 US6030940A (en) 1995-07-31 1997-02-12 Peptide analog inhibitors of urokinase receptor activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US50920895A 1995-07-31 1995-07-31
US08/509,208 1995-07-31

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US08/800,213 Continuation-In-Part US6030940A (en) 1995-07-31 1997-02-12 Peptide analog inhibitors of urokinase receptor activity

Publications (1)

Publication Number Publication Date
WO1997005257A1 true WO1997005257A1 (fr) 1997-02-13

Family

ID=24025726

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/012044 WO1997005257A1 (fr) 1995-07-31 1996-07-19 Inhibiteurs peptidiques, ou analogues, de l'activite du recepteur a l'urokinase

Country Status (3)

Country Link
EP (1) EP0842281A1 (fr)
AU (1) AU6505996A (fr)
WO (1) WO1997005257A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999005263A1 (fr) * 1997-07-25 1999-02-04 Angstrom Pharmaceuticals, Inc. Fragments d'urokinase diriges contre l'invasion et l'angiogenese et leur utilisation
WO2000001802A3 (fr) * 1998-07-01 2000-04-27 Cancerforskningsfonden Af 1989 Antagonistes peptidiques du recepteur humain de l'urokinase et leur procede de selection
US6113897A (en) * 1989-04-07 2000-09-05 Cancerforskiningsfonden Af 1989 Antibodies and their use
US6649597B1 (en) 2000-06-22 2003-11-18 The University Of Iowa Research Foundation Targeting vector to the urokinase plasminogen activator receptor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022464A1 (fr) * 1993-04-02 1994-10-13 The General Hospital Corporation Fragments de l'activateur du plasminogene urokinase
WO1994028014A2 (fr) * 1993-05-28 1994-12-08 Chiron Corporation Peptides inhibiteurs de l'activite des recepteurs d'urokinase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022464A1 (fr) * 1993-04-02 1994-10-13 The General Hospital Corporation Fragments de l'activateur du plasminogene urokinase
WO1994028014A2 (fr) * 1993-05-28 1994-12-08 Chiron Corporation Peptides inhibiteurs de l'activite des recepteurs d'urokinase

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANNUAL MEETING OF THE ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY, FORT LAUDERDALE, FLORIDA, USA, MAY 14-19, 1995. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 36 (4). 1995. S30 *
DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; CHUNG S K ET AL: "Inhibition of basic fibroblast growth factor-induced corneal angiogenesis by a urokinase plasminogen activator receptor antagonist.", XP002020944 *
MIN H Y ET AL: "Inhibition of primary tumor growth in syngeneic mice by murine urokinase receptor antagonists.", KEYSTONE SYMPOSIUM ON CANCER CELL INVASION AND MOTILITY, TAMARRON, COLORADO, USA, FEBRUARY 5-11, 1995. JOURNAL OF CELLULAR BIOCHEMISTRY SUPPLEMENT 0 (19B). 1995. 27, XP002020943 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6113897A (en) * 1989-04-07 2000-09-05 Cancerforskiningsfonden Af 1989 Antibodies and their use
WO1999005263A1 (fr) * 1997-07-25 1999-02-04 Angstrom Pharmaceuticals, Inc. Fragments d'urokinase diriges contre l'invasion et l'angiogenese et leur utilisation
US5994309A (en) * 1997-07-25 1999-11-30 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions and methods
US6696416B1 (en) 1997-07-25 2004-02-24 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US7807621B2 (en) 1997-07-25 2010-10-05 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US8110543B2 (en) 1997-07-25 2012-02-07 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
WO2000001802A3 (fr) * 1998-07-01 2000-04-27 Cancerforskningsfonden Af 1989 Antagonistes peptidiques du recepteur humain de l'urokinase et leur procede de selection
US7026282B1 (en) 1998-07-01 2006-04-11 Cancerforskningsfonden AF 1989 (Fonden Til Fremme AF Exsperimentel Cancerforskning) Peptide antagonists of the human urokinase receptor and method for selecting them
US6649597B1 (en) 2000-06-22 2003-11-18 The University Of Iowa Research Foundation Targeting vector to the urokinase plasminogen activator receptor
US7132405B2 (en) 2000-06-22 2006-11-07 The University Of Iowa Research Foundation Targeting vector to the urokinase plasminogen activator receptor
US7786088B2 (en) 2000-06-22 2010-08-31 University Of Iowa Research Foundation Targeting vector to the urokinase plasminogen activator receptor

Also Published As

Publication number Publication date
EP0842281A1 (fr) 1998-05-20
AU6505996A (en) 1997-02-26

Similar Documents

Publication Publication Date Title
US5679782A (en) Oligonucleotides encloding peptide inhibitors of urokinase receptor activity
AU671611B2 (en) Human kunitz-type protease inhibitor variants
JPH10507071A (ja) 組織因子活性インヒビターの産生
EP1871798B1 (fr) Molecules et procedes d'utilisation correspondants pour le traitement d'affections associees a mcp-1/ccr2
EP0802983B1 (fr) Expression d'inhibiteurs de l'activateur de l'urokinase-plasminogene
EP1896496B1 (fr) Inhibiteurs de proteine kinase c pour la prevention de la resistance a l'insuline et du diabete de type 2
WO1992007935A1 (fr) Proteines de fusion ciblees par clycosaminoglycane, leurs conception, construction et compositions
WO1994003483A1 (fr) Composes se liant aux recepteurs de l'endotheline
US6030940A (en) Peptide analog inhibitors of urokinase receptor activity
WO1997005257A1 (fr) Inhibiteurs peptidiques, ou analogues, de l'activite du recepteur a l'urokinase
US8691760B2 (en) Peptides, pharmaceutical compositions comprising same and uses thereof
AU710221B2 (en) Therapeutic compositions
US6849601B1 (en) Peptides
US20090215669A1 (en) Compositions and methods for treatment of diabetes
WO1994010318A1 (fr) Derives de plasminogene capables d'etre actives par la thrombine
US20100129344A1 (en) Medical uses and therapies based upon the action of azurocidin on igfbp-1
AU767850B2 (en) Novel peptides
KR20000022350A (ko) 세린 프로티아제 억제제
WO2003066089A1 (fr) Ligands de la megsine

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 08800213

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1996924666

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996924666

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1996924666

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载