[go: up one dir, main page]

WO2001025410A2 - SONDES DE DIAGNOSTIC ET AGENTS THERAPEUTIQUES DESTINES A CIBLER uPA ET uPAR - Google Patents

SONDES DE DIAGNOSTIC ET AGENTS THERAPEUTIQUES DESTINES A CIBLER uPA ET uPAR Download PDF

Info

Publication number
WO2001025410A2
WO2001025410A2 PCT/US2000/026502 US0026502W WO0125410A2 WO 2001025410 A2 WO2001025410 A2 WO 2001025410A2 US 0026502 W US0026502 W US 0026502W WO 0125410 A2 WO0125410 A2 WO 0125410A2
Authority
WO
WIPO (PCT)
Prior art keywords
upa
peptide
upar
label
xaa
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.)
Ceased
Application number
PCT/US2000/026502
Other languages
English (en)
Other versions
WO2001025410A3 (fr
Inventor
Andrew P. Mazar
Terence R. Jones
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Angstrom Pharmaceuticals Inc
Original Assignee
Angstrom Pharmaceuticals Inc
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 Angstrom Pharmaceuticals Inc filed Critical Angstrom Pharmaceuticals Inc
Priority to CA002406882A priority Critical patent/CA2406882A1/fr
Priority to EP00970496A priority patent/EP1218496A2/fr
Priority to JP2001528564A priority patent/JP2003528035A/ja
Priority to AU79868/00A priority patent/AU780730B2/en
Publication of WO2001025410A2 publication Critical patent/WO2001025410A2/fr
Publication of WO2001025410A3 publication Critical patent/WO2001025410A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • 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
    • 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)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • 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 present invention relates to conjugates of the urokinase plasminogen activator (uPA) and its fragments that bind to the cell surface receptor uPA (uPAR), the peptides and constructs labeled to deliver a diagnostic probe or a therapeutic agent to the surfaces of cells expressing uPAR.
  • the proteins and peptides of the invention are capable of carrying a suitable detectable or imageable label that will allow qualitative detection and also quantitation of uPAR levels in vitro and in vivo.
  • Such labeled peptide compositions are therefore useful as diagnostic, prognostic and imaging tools in all diseases and conditions where this receptor plays a pathological or otherwise undesirable role.
  • cDNA probes that detect uPA and uPAR-expressing cells may also be conjugated with similar diagnostic labels and used in in situ hybridization assays. Furthermore, by targeting therapeutic agents that "label" the peptide to uPAR, it is possible to achieve a number of biological effects that include cell death, the inhibition of cell movement and migration and the inhibition of angiogenesis.
  • Urokinase-type plasminogen activator has been identified as the initiator of several cascades related to tumor progression, invasion and angiogenesis.
  • the uPA system is strongly linked to pathological processes, such as cell invasion and metastasis in cancer (Dan ⁇ et al., Adv. Cancer Res., 44 :139-266 (1985)).
  • uPA pro- urokinase
  • scuPA single-chain uPA
  • uPAR pro- urokinase
  • This binding event is a prerequisite for the efficient activation of scuPA to two- chain uPA (tcuPA) in a cellular milieu (Ellis et al, J. Biol. Chem., 264:2185-88 (1989)).
  • Pro-uPA is activated by a single proteolytic cleavage between amino acid 158 (Lys) and 159 (He) to activate the proenzyme. Cleavage results in the formation of the two-chain active uPA (tcuPA).
  • uPA is a three-domain protein comprising (1) an N-terminal "growth factor domain" (GFD), (2) a kringle domain, and (3) a C-terminal serine protease domain.
  • GFD N-terminal "growth factor domain”
  • uPAR the receptor for pro-uPA, is also a multi-domain protein anchored by a glycosyl- phosphatidylinositol anchor to the outer leaf of the cell membrane (Behrendt et al, Biol. Chem. Hoppe-Seyler, 376:269-279 (1995)).
  • amino acid sequence of the N-terminus of human pro-uPA is Ser Asn Gl Leu His Gin Val Pro Ser Asn Cys Asp Cys Leu Asn Gly 1 10
  • pro-uPA SEQ ID NO:l
  • Figure 1 The structure of pro-uPA, SEQ ID NO:l, is shown in Figure 1.
  • uPA tissue plasminogen activator
  • streptokinase for thromboembolism
  • uPAR is not normally expressed at detectable levels on quiescent cells and must therefore be upregulated before it can initiate the activities of the uPA system.
  • uPAR expression is stimulated in vitro by differentiating agents such as phorbol esters (Lund et al., J. Biol. Chem.
  • uPAR appears to be up-regulated in vivo in most human carcinomas examined to date, specifically, in the tumor cells themselves, in tumor-associated endothelial cells undergoing angiogenesis and in macrophages (Pyke et al. , Cancer Res.53:1911-15 (1993) which may participate in the induction of tumor angiogenesis (Lewis et al., J. Leukoc. Biol. 57:747-751 (1995)).
  • uPAR expression in cancer patients is present in advanced disease and has been correlated with a poor prognosis in numerous human carcinomas (Hofmann et al, Cancer 75:487-92 (1996); Heiss et al., Nature Med. 7:1035-39 (1995).
  • uPAR is not expressed uniformly throughout a tumor but tends to be associated with the invasive margin and is considered to represent a phenotypic marker of metastasis in human gastric cancer.
  • uPAR expression is up-regulated only in pathological states involving extracellular matrix remodeling and cell motility such as cancer makes it an attractive marker for diagnosis as well as a selective target for therapy.
  • the present inventors have discovered that relatively large uPA peptides that are cleared rapidly from the body are useful as diagnostic agents (as well as in therapy).
  • uPA high molecular weight uPA (residues 1-411); single- chain uPA (scuPA) described above; tcuPA, DFP-uPA (tcuPA inactivated with the suicide inhibitor diisopropyl fluorophosphate), the N-terminal fragment often designated ATF, which is a peptide of 135 or 143 amino acids having the sequence of 1-135 or 1-143 of native uPA; the GFD which corresponds to residues 4-43.
  • (c) includes residues 13-30 of the uPAR-binding site of uPA.
  • (d) competes with labeled DFP-uPA (preferably [ 125 I] labeled) for binding to a cell or molecule that has a binding site for uPA, and has an IC 50 value of about 10 nM or less ⁇ that is, the protein or peptide causes 50% inhibition of the binding of the labeled ligand at concentrations of 10 nM or lower).
  • (e) is not a fusion protein wherein the uPA peptide is fused to another (non-uPA) protein or peptide.
  • Preferred detectable labels include a radionuclides, PET-imageable agents, MRI- imageable agents, fluorescers, fluorogens, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer.
  • a label permits detection or quantitation of the uPAR level in a tissue sample and can be used, therefore, as a diagnostic and a prognostic tool in all diseases where enhanced expression of the receptor plays a pathological or otherwise undesirable role, including those described herein.
  • a most preferred radionuclide is selected from the group consisting of 3 H, 14 C, 35 S, "Tc, ,2 T, 12 T, I3 T, In, 97 Ru, 67 Ga, 68 Ga, 72 As, 89 Zr and 201 T1.
  • the fluorescer or fluorogen is preferably fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o -phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green or Texas Red.
  • a protein or peptide probe of this invention e.g., labeled tcuPA
  • the present invention provides agents with increased "contrast" for detecting tumors or other sites of cells that express uP AR.
  • the present invention provides the first specifically-targeted contrast agent for magnetic resonance imaging (MRI).
  • the specificity is a result of the enhanced expression of uPAR in the target tissue, generally tumor tissue.
  • Some of the peptides that bind uPAR and, as a result undergo activation, may be quenched by the naturally occurring plasminogen activator inhibitor- 1 (PAI-1) and are taken up by a uPAR- dependent mechanism as uPA peptide-PAI-1 complexes.
  • PAI-1 plasminogen activator inhibitor- 1
  • a diagnostic label is bound to the protein or peptide through one or more diethylenetriaminepentaacetic acid (DTP A) residues that are coupled to the protein or peptide.
  • DTP A diethylenetriaminepentaacetic acid
  • the label is bound through one DTPA residue.
  • a most preferred diagnostic peptides is scuPA or ATF 1- 135 coupled to one (or more) DTPA residues, to which are bound gadolinium atoms.
  • Preferred diagnostic methods comprise MRI using these labeled peptides.
  • the protein or peptide carries a suitable therapeutic agent
  • label also referred to herein as a "therapeutic moiety.”
  • a therapeutic moiety is an atom, a molecule, a compound or any chemical component added to the peptide that renders it active in treating a disease or condition associated with enhanced expression of uPAR.
  • the peptides of the present invention are prepared by conventional means, either recombinant or by proteolysis of uPA as described in Mazar et al. , (Fibrinolysis 6
  • the therapeutically active moiety may be bound directly or indirectly to the peptide.
  • the therapeutic composition may be one in which the therapeutic moiety is bound to the active site of the uPA protein or peptide; such compositions are made from compounds described below.
  • the therapeutically labeled protein or peptide is administered as pharmaceutical composition which comprises a pharmaceutically acceptable carrier or excipient, and is preferably in a form suitable for injection.
  • the therapeutically active moiety is preferably a radionuclide.
  • radionuclides include 47 Sc, 67 Cu, 90 Y, 109 Pd, 125 I, 13 T, ,86 Re, ,88 Re, ,99 Au, 211 At, 2,2 Pb and 217 Bi.
  • the present invention also provides a uPA active site-targeting compound that covalently modifies the active site of tcuPA or a fragment or subunit thereof, which fragment or subunit retains (i) the uPA enzymatic endosite and (ii) a uPAR-binding epitope.
  • This compound may include (a) a detectable label; (b) a therapeutic moiety; or (c) a chelator that is optionally bound to a detectable label or a therapeutic moiety. The compound localizes said chelator, detectable label or therapeutic moiety to the uPA active site.
  • the above compound may be an affinity label or a uP A-activated irreversible inhibitor.
  • Preferred affinity labels are alkylating groups, such as chloromethylketone (CMK).
  • the expression (Lys, Arg) means a single amino acid that is either Lys or Arg.
  • (Xaa) 2 . 6 is Glu-Gly, resulting in compounds of the formula: (Chelator (empty) )-Glu-Gly-Arg-CMK; or
  • a molecule comprising uPA, tcuPA or a fragment or subunit thereof, which fragment or subunit retains (i) the uPA enzymatic endosite and (ii) a uPAR-binding epitope, and which uPA, tcuPA, fragment or subunit is bonded covalently to the compound described above.
  • This molecule would have the general formula: (Chelator (empty) )-(Xaa) 2 . 6 -(Lys,Arg)-(alkylating group)-uPA; or (Label-Chelator)-(Xaa) 2 . 6 -(Lys,Arg)-(alkylating group)-uPA.
  • a uPA active site-targeting peptide compound is one that binds to the endosite and one or more exosites of tcuPA or of a tcuPA fragment or subunit (which fragment or subunit retains the uPA (i) enzymatic endosite and (ii) a uPAR-binding epitope).
  • the peptide compound which covalently modifies the endosite, preferably includes either (a) detectable label, (b) a therapeutic moiety, or (c) a chelator that is optionally bound to a detectable label or to a therapeutic moiety. This peptide compound localizes said chelator, detectable label or therapeutic moiety to the uPA active site.
  • the above peptide compound of claim preferably has a structure defined by one of the following four general formulas
  • the (Label) is a detectable label
  • Xaa is any amino acid
  • Peptide Z is any peptide that binds to a surface exosite of uPA.
  • a molecule comprising uPA, tcuPA or a fragment or subunit of tcuPA bonded at the uPA endosite and at one or more exosites to the above peptide compound.
  • the protein or peptide is preferably scuPA or said N- terminal fragment of uPA residues 1-135 and the detectable label is preferably gadolinium.
  • the invention includes methods of using the above classes of molecules, specifically, in a method for detecting the presence of uPAR (i) on the surface of a cell, (ii) in a tissue, (iii) in an organ or (iv) in a biological sample, which cell, tissue, organ or sample is suspected of expressing uPAR due to a pathological state.
  • the method comprising the steps of (a) contacting the cell, tissue, organ or sample with any of the diagnostic and detectably labeled compositions and molecules described above; and (b) detecting the presence of the label associated with the cell, tissue, organ or sample.
  • both the contacting and the detecting may be conducted in vitro.
  • the contacting is in vivo and the detecting is in vitro.
  • the contacting and the detecting are in vivo.
  • a nonlimiting group of diseases or conditions treatable by therapeutically labeled peptides of this invention include primary growth of solid tumors or leukemias and lymphomas, metastasis, invasion and/or growth of tumor metastases.
  • the present invention also provides a method for inhibiting cell migration, cell invasion, cell proliferation or angiogenesis, or for inducing apoptosis, comprising contacting cells associated with undesired cell migration, invasion, proliferation or angiogenesis with an effective amount of the above therapeutically labeled composition.
  • the method is used to inhibit the invasiveness of tumor cells.
  • Diseases or conditions treatable by the present composition include primary growth of a solid tumor, leukemia or lymphoma; tumor invasion, metastasis or growth of tumor metastases; benign hyperplasia; atherosclerosis; myocardial angiogenesis; post- balloon angioplasty vascular restenosis; neointima formation following vascular trauma; vascular graft restenosis; coronary collateral formation; deep venous thrombosis; ischemic limb angiogenesis; telangiectasia; pyogenic granuloma; corneal disease; rubeosis; neovascular glaucoma; diabetic and other retinopathy; retrolental fibroplasia; diabetic neovascularization; macular degeneration; endometriosis; arthritis; fibrosis associated with a chronic inflammatory condition, traumatic spinal cord injury including ischemia, scarring or fibrosis; lung fibrosis, chemotherapy-induced fibrosis; wound healing with scarring and
  • FISH probe fluorescently labeled cDNA probe
  • Figure 1 is a schematic representation of the pro-uPA molecule (SEQ ID NO:l].
  • the N-terminal growth factor domain (ATF) of human uPA is residues 1-135.
  • uPA-uPAR interactions may exhibit varying degrees of species specificity.
  • a peptide having the amino acid sequence (or a substitution variant thereof) of the species of the animal being targeted is always preferred.
  • the uPAR-targeting peptide compounds of the invention are readily tested for their binding to uPAR, preferably by measuring their ability to inhibit the binding of [ 125 I]DFP-uPA to uPAR in a competitive ligand-binding assay.
  • the assay may employ whole cells that express uPAR, for example cell lines such as RKO or HeLa.
  • a preferred assay is conducted as follows. Cells (about 5 x 10 4 /well) are plated in medium (e.g., MEM with Earle's salts/10% FBS + antibiotics) in 24-well plates, then incubated in a humid 5% CO 2 atmosphere until the cells reach 70% confluence.
  • Catalytically inactivated high molecular weight uPA (DFP-uPA) is radioiodinated using Iodo-gen ® (Pierce) to a specific activity of about 250,000 cpm mg.
  • the cell-containing plates are then chilled on ice and the cells are washed twice (5 minutes each) with cold PBS/ 0.05% Tween-80.
  • Test compounds are serially diluted in cold PBS/ 0.1 % BSA/ 0.01% Tween-80 and added to each well to a final volume of 0.3mL 10 minutes prior to the addition of the [ 125 I]DFP-uPA. Each well then receives 9500 cpm of [ 125 I]DFP-uPA at a final concentration of 0.2 nM).
  • the plates are then incubated at 4°C for 2 hrs, after which time the cells are washed 3x (5 minutes each) with cold PBS/ 0.05% Tween-80.
  • NaOH (IN) is added to each well in 0.5 mL to lyse the cells, and the plate is incubated for 5 minutes at room temperature or until all the cells in each well are lysed as determined by microscopic examination.
  • the contents of each well are then aspirated and the total counts in each well determined using a gamma counter. Each compound is tested in triplicate and the results are expressed as a percentage of the total radioactivity measured in wells containing [ 125 I]DFP-uPA alone, which is taken to represent maximum (100 %) binding.
  • the inhibition of binding of [ 125 I]DFP-uPA to uPAR is usually dose-related, such that the concentration of the test compound necessary to produce a 50% inhibition of binding (the IC 50 value), which is expected to fall in the linear part of the curve, is easily determined.
  • the compounds of the invention have IC 50 values of less than about 10 "7 M.
  • the compounds of the invention have IC 50 values of less than about 10 "8 M and, even more preferably, less than about 10 "9 M.
  • Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma) and Matrigel® (1 x 10 6 cells in 0.2mL) s.c. in the right flank of the animals.
  • the tumors are staged to 100 mm 3 and then imaging with a test composition is initiated (50 ⁇ g/animal i.p.). Animals are subjected to the appropriate scanning or imaging method when tumors are expected to be at a range of volumes.
  • B. Xenograft Model of Metastasis The compounds of this invention can also diagnose the presence of late metastasis using an experimental metastasis model (Crowley, C.W. et al, Proc. Natl.
  • Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis.
  • PC-3 Human prostatic carcinoma cells
  • PC-3 Human prostatic carcinoma cells
  • metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer.
  • a compound of the invention (50 ⁇ g/mL) is given i.v. on day 14 and the microscopic metastases are imaged using the appropriate detection method.
  • Tissues may also be harvested from these mice after euthanasia followed by the detection of cells expressing uPA and uPAR using the cDNA probes.
  • a compound to be useful in accordance with this invention should demonstrate an ability to detect the presence of a tumor mass (primary or metastatic) as small as about 5 mm in a mouse model.
  • the uPA peptides can be detectably labeled and used, for example, to detect a peptide binding site or receptor (such as uPAR) on the surface or in the interior of a cell.
  • a peptide binding site or receptor such as uPAR
  • the fate of the peptide during and after binding can be followed in vitro or in vivo by using the appropriate method to detect the label.
  • the labeled peptide may be utilized in vivo for diagnosis and prognosis, for example to image occult metastatic foci or for other types of in situ evaluations.
  • U.S. patents disclose methods and compositions for complexing metals to larger molecules, including description of useful chelating agents.
  • the metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules.
  • These documents include: US 5,627,286 (Heteroatom-bearing ligands and metal complexes thereof); US 5,618,513 (Method for preparing radiolabeled peptides); US 5,567,408 (YIGSR peptide radiopharmaceutical applications); US 5,443,816 (Peptide-metal ion pharmaceutical preparation and method); US 5,561,220 (Technetium- 99m labeled peptides for imaging inflammation).
  • Suitable detectable labels include radioactive, fluorescent, fluorogenic, chromogenic, or other chemical labels.
  • Useful radiolabels, which are detected simply by gamma counter, scintillation counter or autoradiography include 3 H, 125 I, ,31 1, 35 S and 14 C.
  • !31 I is a useful therapeutic isotope (see below).
  • Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the fluorophore such as the dansyl group, must be excited by light of a particular wavelength to fluoresce. See, for example, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR., 1996).
  • the long wavelength rhodamines which are basically Rhodamine GreenTM derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known.
  • This group includes the tetramethylrhodamines, X-rhodamines and Texas RedTM derivatives.
  • Other preferred fluorophores for derivatizing the peptide according to this invention are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives. Also included as labels are two related inorganic materials that have recently been described: semiconductor nanocrystals, comprising, for example, cadmium sulfate (Bruchez, M.
  • quantum dots e.g., zinc-sulfide-capped cadmium selenide (Chan, W.C.W. et al, Science 281:2016-2018 (1998)).
  • the amino group of a uPA peptide is allowed to react with reagents that yield fluorescent products, for example, fluorescamine, dialdehydes such as o -phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3- dicarboxylate.
  • reagents that yield fluorescent products for example, fluorescamine, dialdehydes such as o -phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3- dicarboxylate.
  • 7-nitrobenz-2-oxa-l,3-diazole (NBD) derivatives are useful to modify amines to yield fluorescent products.
  • the peptides can also be labeled for detection using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the peptide using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA is available as the anhydride, which can readily modify the NH 2 -containing uPAR-binding peptides of this invention.
  • radionuclides may be bound to the peptide either directly or indirectly using a chelating agent such as DTPA and EDTA which is chemically conjugated, coupled or bound (which terms are used interchangeably) to the peptide.
  • a chelating agent such as DTPA and EDTA which is chemically conjugated, coupled or bound (which terms are used interchangeably) to the peptide.
  • the chemistry of chelation is well known in the art, and a varying range of molar ratios of DTPA:peptide may be used to produce the DTPA-coupled peptide, and thereby, the diagnostically labeled peptide.
  • the key limiting factor on the chemistry of coupling is that the uPA fragment must maintain its ability to bind uPAR after labeling.
  • radionuclide having diagnostic or therapeutic value can be used as the radiolabel in the present invention.
  • the radionuclide is a ⁇ - emitting or beta -emitting radionuclide, for example, one selected from the lanthanide or actinide series of the elements.
  • Positron-emitting radionuclides e.g. 68 Ga or 64 Cu, may also be used.
  • Suitable gamma -emitting radionuclides include those which are useful in diagnostic imaging applications.
  • the gamma -emitting radionuclides preferably have a half-life of from 1 hour to 40 days, preferably from 12 hours to 3 days.
  • suitable gamma -emitting radionuclides include 67 Ga, u Tn, 99m Tc, 169 Yb and ,86 Re. Most preferably, the radionuclide is 99 ⁇ c.
  • radionuclides examples include 67 Cu, 67 Ga, 68 Ga, 72 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, ⁇ Tn, 123 1, 125 1, 13 T, 169 Yb, 186 Re, and 201 T1.
  • gamma-emitting radionuclides such as 67 Ga, U I In, 99m Tc, 169 Yb and 186 Re, most preferably 99m Tc.
  • certain proteins such as transferrin and human serum albumin, have been labeled with 68 Ga
  • a number of metals (not radioisotopes) useful for MRI include gadolinium, manganese, copper, iron, gold and europium. Gadolinium is most preferred.
  • the amount of labeled peptide needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
  • the peptides can also be made detectable by coupling them to a phosphorescent or a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged peptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescers are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the peptides. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • colorimetric detection is used, based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients.
  • In situ detection of the labeled peptide may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label.
  • histological methods such as staining procedures
  • diagnostically labeled means that the peptide has attached to it a diagnostically detectable label.
  • labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include radioactive isotopes, paramagnetic isotopes, and compounds which can be imaged by positron emission tomography (PET) and by MRI.
  • PET positron emission tomography
  • MRI positron emission tomography
  • the type of detection instrument available is a major factor in selecting a radionuclide.
  • the radionuclide chosen must have a type of decay which is detectable by a particular instrument.
  • any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention.
  • Another factor in selecting a radionuclide for in vivo diagnosis is that its half- life be long enough so that the label is still detectable at the time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized.
  • a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras.
  • In vivo imaging may be used to detect occult metastases which are not observable by other methods.
  • the expression of uPAR correlates with progression of diseases in cancer patients such that patients with late stage cancer have higher levels of uPAR in both their primary tumors and metastases.
  • uPAR-targeted imaging could be used to stage tumors non-invasively or to detect other diseases which are associated with the presence of increased levels of uPAR (for example, restenosis that occurs following angioplasty).
  • the compositions of the present invention may be used in diagnostic, prognostic or research procedures in conjunction with any appropriate cell, tissue, organ or biological sample of the desired animal species.
  • biological sample any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus and the like. Also included within the meaning of this term is a organ or tissue extract and a culture fluid in which any cells or tissue preparation from the subject has been incubated.
  • the labeled peptide compounds of the invention may be incorporated into convenient dosage forms
  • the compounds of the invention are administered systemically, e.g. , by injection.
  • injection may be by any known route, preferably intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.
  • injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • sterile injectable liquid e.g., a solution
  • an ampoule or an aqueous or nonaqueous liquid suspension.
  • the present invention may be used in the diagnosis of any of a number of animal genera and species, and are equally applicable in the practice of human or veterinary medicine.
  • the compositions can be used with domestic and commercial animals, including birds and more preferably mammals, as well as humans.
  • the peptides in a form suitable for topic application are sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compounds of the invention.
  • An alternative diagnostic approach utilizes cDNA probes that are complementary to and thereby detect uPA and uPAR-expressing cells by in situ hybridization with mRNA in these cells.
  • This cDNA is preferably fluorescently labeled.
  • This method and the preparations useful therein are described below.
  • Active Site-Specific Modification of uPA and other Serine Proteases The present invention is directed to an active site-specific reagent that covalently modifies the active site ("endosite") of uPA or any serine protease and localizes a detectable label, preferably an imageable label, or a therapeutic moiety such as a radioisotope, toxin or drug conjugate, to the endosite.
  • the uPA must be tcuPA or a similar molecule that retains the enzymatic endosite of uPA and its uPAR-binding epitope.
  • a reagent is preferably an active site-directed affinity label or an enayme-activated irreversible inhibitor of uP A activity ("suicide substrate").
  • the reagent is attached directly (i.e., without the mediation of a chelating agent) to a detectable label or therapeutic moiety.
  • the reagent includes as part of its structure a chelating agent, as described above, that is either loaded with the label (or therapeutic moiety) or to which the label (or therapeutic moiety) may be added after the chelator has been delivered to the enzyme's endosite.
  • the endosite-specific reagent is preferably a modified peptide, but may also be a non-peptidic molecule, as described below.
  • any active site- directed affinity label or suicide substrate of any serine protease can be used to localize to the endosite of that protease a chelating agent or a chelate that includes a detectable (preferably imageable) label or therapeutic moiety.
  • a typical example of such a reagent is an endosite-specific affinity label comprising a tripeptide and any alkylating moiety.
  • the reagent Upon being localized to the active site of a serine protease, the reagent reacts with the active site histidine.
  • a preferred alkylating moiety is a halomethylketone, more preferably chloromethylketone ("CMK").
  • CCMK chloromethylketone
  • a preferred endosite-specific tripeptide for uPA is Glu-Gly- Arg.
  • a preferred reagent is Glu-Gly- Arg-CMK to which is bonded a label, a therapeutic moiety or a chelating agent optionally loaded with a label or therapeutic moiety.
  • This reagent molecule targets the His residue within the serine protease catalytic triad of uPA and covalently bonds to this His residue.
  • peptides that may be longer than a tripeptide are also useful - they may have even higher affinities or be more reactive than Glu-Gly- Arg for the protease endosite and could also access exosites, thereby resulting in increased binding affinity and specificity.
  • Bock reported the incorporation of spectroscopic probes into active sites of certain serine proteases by inactivating the enzyme with a thioester derivative of a peptide CHK.
  • the thiol group that is generated can serve as a unique suite for subsequent labeling with thiol-reactive probes, exemplified by 5-(iodoacetamido)fluorescein (Bock, P.E., Biochemistry 27:6633-6639 (1999), incorporated by reference in its entirety).
  • the present approach may be extended along these lines for incorporating the various types of detectable labels or therapeutic moieties not contemplated by Bock for the uses set forth herein.
  • the peptide portion of the reagent may have a basic residue (Lys or Arg) at the " SI" site.
  • the peptide part of the molecule may have the following structure (where Xaa symbolizes any amino acid residue): (Xaa) 2.6 -(Lys,Arg), wherein the (Lys, Arg) is intended to be either Lys or Arg.
  • a preferred reagent would be (Xaa) 2 . 6 -(Lys,Arg)-CMK.
  • non-peptide affinity labels or suicide substrates that inactivate a serine protease active site could also be useful in localizing to the enzyme active site (1) a detectable label or therapeutic moiety; or (2) a chelate that contains, or a chelator that has the potential to contain, a detectable label or therapeutic moiety.
  • These non-peptide reagents are not limited to those that modify the catalytic triad His residue, as compounds are known which can alkylate the active site Ser (e.g., diisopropyl fluorophosphate, DFP; and phenylmethylsulfonyl fluoride, PMSF).
  • DFP diisopropyl fluorophosphate
  • PMSF phenylmethylsulfonyl fluoride
  • a reagent containing a chelating group must be capable of modifying a thiol or amine group.
  • an amine-reactive reagent because the N-terminal amine has a pK a of about 7.5 whereas the epsilon amino group of Lys has a pK a of about 9.5, these two types of amino group will have quite different reactivities within the pH range 7.5 - 9.5.
  • a reagent delivering the chelating group can be introduced at the N-terminus of a peptidic suicide substrate in a selective manner.
  • the chelating group for an imageable label may be part of the reagent, for example, (Chelator)-(Xaa) 2 . 6 -(Lys,Arg) -CMK. It is also possible to introduce a free thiol at the N-terminus of a peptidic suicide inhibitor by introducing a Cys or HomoCys residue as one of the Xaa amino acids or by chemically modifying the N-terminus of the peptide suicide substrate. The chelator may then be part of the reagent, as described above, or it may be introduced into the (Xaa) 2 . 6 -(Lys,Arg)-CMK moiety after this peptide reagent has bound to the uPA active site. A more generic characterization of this preferred type of endosite-specific reagent is
  • the reagent may incorporate a detectable label, a therapeutic moiety, an " empty” chelator not carrying an imageable label or therapeutic moiety (but capable of accepting one), or a chelator that does carry an imageable label or therapeutic moiety.
  • the chelator, label, etc. is not necessarily incorporated into the reagent a priori; rather, the reagent would be derivatized subsequently to carry the label or moiety.
  • the structures may be characterized as follows (where " label” means detectable label):
  • Serine protease active sites can be fairly restrictive and may only accommodate a few amino acid residues or substrates of limited size (Nozawa et al, J. Biochem (1982) 7:1837-1843).
  • the endosite-specific peptide described above does not have aper se limitation on its length (although, as indicated, peptides of 3-7 residues are preferred).
  • Another embodiment is a peptide that binds to the uPA endosite and one or more exosites.
  • An example is an endosite-binding tripeptide which further comprises an additional peptide "tail" that binds to a surface exosite of the protein.
  • a well-known example of such a construct is hirulog, a thrombin inhibitor.
  • Hirulog combines the active site specificity of a thrombin-specific tripeptide and includes more peptide chain that binds to the surface of thrombin, yielding a high-affinity inhibitor of thrombin activity (Skordalakes et al. Biochemistry 13: 14420-7).
  • a range of preferred embodiments include: (Label)-(Peptide Z)-(Xaa) 2 . 6 -(Lys,Arg)-(alkylating group); (Therapeutic Moiety)-(Peptide Z)-(Xaa) 2 . 6 -(Lys,Arg)-(alkylating group),
  • a longer peptide (of between about 4 and 30 residues) that mimics the recognition region of PAI-1 is also useful as a site-specific reagent to produce a uPAR-directed imaging protein/ peptide as described above.
  • a general class of non- peptidic agents may be used to localize a chelator (and thereby, a detectable label) to the active site of uPA or of any serine protease.
  • a chelator and thereby, a detectable label
  • Such structures include, DFP and PMSF, etc.
  • a member of this group of compounds should have the following properties:
  • reagents are selective for the active site by virtue of the fact that they are enzyme activated irreversible inhibitors and will only form covalent adducts when catalyzed by the target enzyme.
  • the methods of this invention may be used to image tumor foci in a subject.
  • a vertebrate subject preferably a mammal, more preferably a human, is administered an amount of the compound effective to image a tumor focus.
  • the compound or pharmaceutically acceptable salt thereof is preferably administered in the form of a diagnostic composition as described above.
  • an effective amount means an amount sufficient to be detected using the appropriate detection system e.g., MRI detector, gamma camera, etc.
  • the minimum detectable amount will depend on the ratio of labeled peptide specifically bound to a tumor (signal) to the amount of labeled peptide either bound non-specifically or found free in plasma or in extracellular fluid.
  • the advantage of the peptides of this invention is that they are cleared rapidly from the plasma and do not accumulate in most tissues, in contrast to antibody-based contrast agents. Thus, the signal-to-noise ratio is increased and only the specifically tumor- bound peptide is detected or quantitated. This allows for accurate localization of the signal and the detection of small tumor lesions which can only be detected in the absence of high background signals.
  • the amount of the diagnostic composition to be administered depends on the precise peptide selected, the disease or condition, the route of administration, and the judgment of the skilled imaging professional.
  • a preferred dose for imaging by MRI, SPECT, etc. is an amount of up to about 100 milligrams of labeled peptide compound per kilogram of body weight.
  • a typical single dosage of the labeled peptide is between about 1 ng and about 100 mg/kg body weight.
  • An alternative diagnostic approach utilizes cDNA probes that are complementary to and thereby detect uPA- and uPAR-expressing cells by in situ hybridization with mRNA in these cells.
  • the present invention provides methods for localizing uPAR or uPA mRNA in cells using fluorescent in situ hybridization (FISH) with labeled cDNA probes that correspond to part or all of the uPA (or uPAR) coding sequence.
  • FISH fluorescent in situ hybridization
  • the basic principle of FISH is that DNA or RNA in the prepared specimens are hybridized with the probe nucleic acid of this invention that is labeled non-isotopically with, for example, a fluorescent dye, biotin or digoxigenin.
  • the hybridized signals are then detected by fluorimetric or by enzymatic methods, for example, by using a fluorescence or light microscope. The detected signal and image can be recorded on light sensitive film.
  • FISH Fluorescence Infrared spectroscopy
  • CCD charge-coupled device
  • FISH offers increased sensitivity. In additional to offering positional information, FISH allows better observation of cell or tissue morphology. Because of the nonradioactive approach, FISH has become widely used for localization of specific DNA or mRNA in a specific cell or tissue type.
  • patents and scientific articles that describe various in situ hybridization techniques and applications are: 5,912,165; 5,906,919; 5,885,531 ; 5,880,473; 5,871,932; 5,856,097; 5,837,443 ; 5,817,462; 5,784,162; 5,783,387 ; 5,750,340; 5,759,781; 5,707,797; 5,677,130; 5,665,540; 5,571,673; 5,565,322; 5,545,524 ; 5,538,869; and 5,501,954, 5,225,326, 4,888,278.
  • the compounds that may be employed in the pharmaceutical compositions of the invention include all of those compounds described above, as well as the pharmaceutically acceptable salts of these compounds.
  • the compounds of the invention possess the ability to inhibit invasiveness or angiogenesis, properties that are exploited in the treatment of cancer, in particular metastatic cancer.
  • a composition of this invention may be active per se, or may act as a "pro-drug" that is converted in vivo to the active form.
  • the compounds of the invention, as well as the pharmaceutically acceptable salts thereof may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets or injectable preparations. Solid or liquid pharmaceutically acceptable carriers may be employed.
  • the compounds of the invention are administered systemically, e.g., by injection.
  • injection may be by any known route, preferably intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.
  • Injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • a liquid carrier When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • sterile injectable liquid e.g., a solution
  • an ampoule or an aqueous or nonaqueous liquid suspension.
  • the pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for oral, parenteral, topical, transdermal, intravaginal, intrapenile, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration.
  • the pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
  • the present invention may be used in the diagnosis or treatment of any of a number of animal genera and species, and are equally applicable in the practice of human or veterinary medicine.
  • the pharmaceutical compositions can therefore be used to treat domestic and commercial animals, including birds and more preferably mammals, as well as humans.
  • the pharmaceutical composition may be administered topically or transdermally, e.g., as an ointment, cream or gel; orally; rectally; e.g., as a suppository, parenterally, by injection or continuously by infusion; intravaginally; intrapenilely; intranasally; intrabronchially; intracranially, intraaurally; or intraocularly.
  • sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compounds of the invention.
  • an effective amount of the compound for the preferred topical applications, especially for humans, it is preferred to administer an effective amount of the compound to an affected area, e.g., skin surface, mucous membrane, eyes, etc.
  • an affected area e.g., skin surface, mucous membrane, eyes, etc.
  • This amount will generally range from about 0.001 mg to about 1 g per application, depending upon the area to be treated, the severity of the symptoms, and the nature of the topical vehicle employed.
  • compositions of the invention may comprise, in addition to the labeled peptide, one or more additional anti-tumor agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim or trimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes or enzyme inhibitors, e.g., asparaginase, topoisomerase inhibitors such as etoposide; or biological response modifiers, e.g., interferons or interleukins.
  • mitotic inhibitors e.g., vinblastine
  • alkylating agents e.g., cyclophosphamide
  • folate inhibitors e.g., methot
  • compositions comprising any known cancer therapeutic in combination with the labeled peptides disclosed herein are within the scope of this invention.
  • the pharmaceutical composition may also comprise one or more other medicaments to treat additional symptoms for which the target patients are at risk, for example, anti-infectives including antibacterial, anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.
  • anti-infectives including antibacterial, anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.
  • the peptides describe herein are "therapeutically conjugated” or “therapeutically labeled” (terms which are intended to be interchangeable) and used to deliver a therapeutic agent to the site to which the compounds home and bind, such as sites of tumor metastasis or foci of infection/inflammation, restenosis or fibrosis.
  • therapeutically conjugated means that the modified peptide is conjugated to another therapeutic agent that is directed either to the underlying cause or to a "component" of tumor invasion, angiogenesis, inflammation or other pathology.
  • Examples of useful therapeutic radioisotopes include 47 Sc, 67 Cu, 90 Y, 109 Pd, 12 T, 131 I, ,86 Re, ,88 Re, ,99 Au, 2n At, 2,2 Pb and 217 Bi. These atoms can be conjugated to the peptide directly, indirectly as part of a chelate, or, in the case of iodine, indirectly as part of an iodinated Bolton-Hunter group.
  • Preferred doses of the radionuclide conjugates are a function of the specific radioactivity to be delivered to the target site which varies with tumor type, tumor location and vascularization, kinetics and biodistribution of the peptide carrier, energy of radioactive emission by the nuclide, etc.
  • Those skilled in the art of radiotherapy can readily adjust the dose of the labeled peptide in conjunction with the dose of the particular nuclide to effect the desired therapeutic benefit without undue experimentation.
  • Another therapeutic approach included here is the use of boron neutron capture therapy (NCT), where a boronated peptide is delivered to a desired target site, such as a tumor, most preferably an intracranial tumor (Barth, R.F., Cancer Invest.
  • NCT boron neutron capture therapy
  • the stable isotope 10 B is irradiated with low energy ( ⁇ 0.025 eV) thermal neutrons, and the resulting nuclear capture yields ⁇ particles and 7 Li nuclei which have high linear energy transfer and respective path lengths of about 9 and 5 ⁇ m.
  • This method is predicated on 10 B accumulation in the tumor with lower levels in blood, endothelial cells and normal tissue (e.g., brain).
  • Such delivery has been accomplished using epidermal growth factor (Yang. W. et al, Cancer Res 57:4333-4339 (1997). Because of the selective expression of uPAR in tumors, the peptides of the present invention are excellent delivery vehicles for this therapeutic moiety.
  • gadolinium specifically 157 Gd appears to be particularly advantageous for use in NCT with the present peptides. It has recently been reported (Tokumitsu, H. et al, Chem Pharm Bull 47:838-842 (1999), incorporated by reference in its entirety) that: 157 Gd has the highest thermal neutron capture cross section (255,000 barns) among naturally occurring isotopes, 66 times larger than that of 10 B; Gd neutron capture reaction releases the long range (>100 ⁇ m) prompt ⁇ -rays, internal conversion electrons, X-rays and Auger electrons. Thus, Gd-NCT may increase the chance for photons to hit tumor cells and for electrons to damage these cell locally and intensively.
  • Gd has long been used as a MRI imaging diagnostic agent. It will be possible to integrate Gd-NCT with MRI diagnosis by using the Gd-loaded dosage forms of the present peptides.
  • a preferred form of Gd for labeling the peptides of this invention for use in Gd-NCT is gadopentetic acid (Gd-DTPA).
  • Gd-DTPA gadopentetic acid
  • Other therapeutic agents which can be coupled to the peptide compounds according to the method of the invention are drugs, prodrugs, enzymes for activating pro-drugs, photosensitizing agents, gene therapeutics, antisense vectors, viral vectors, lectins and other toxins.
  • the therapeutic dosage administered is an amount that is therapeutically effective, as is known to or readily ascertainable by those skilled in the art.
  • the dose is also dependent upon the age, health, and weight of the recipient, kind of concurrent treatment(s), if any, the frequency of treatment, and the nature of the effect desired, such as, for example, anti-inflammatory effects or anti-bacterial effect.
  • Lectins are proteins, commonly derived from plants, that bind to carbohydrates.
  • cytotoxic substances are protein toxins of bacterial and plant origin (Frankel, A.E. et al, Ann. Rev. Med. 57:125-142 (1986)). These molecules binding the cell surface and inhibit cellular protein synthesis.
  • the most commonly used plant toxins are ricin and abrin; the most commonly used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin A.
  • ricin and abrin the binding and toxic functions are contained in two separate protein subunits, the A and B chains.
  • the ricin B chain binds to the cell surface carbohydrates and promotes the uptake of the A chain into the cell.
  • the ricin A chain inhibits protein synthesis by inactivating the 60S subunit of the eukaryotic ribosome Endo, Y. et al, J. Biol. Chem. 262: 5908-5912 (1987)).
  • Other plant derived toxins which are single chain ribosomal inhibitory proteins, include pokeweed antiviral protein, wheat germ protein, gelonin, dianthins, momorcharins, trichosanthin, and many others (Strip, F. et al, FEBS Lett. 795:1-8 (1986)).
  • Diphtheria toxin and Pseudomonas exotoxin A are also single chain proteins, and their binding and toxicity functions reside in separate domains of the same protein chain with full toxin activity requiring proteolytic cleavage between the two domains.
  • Pseudomonas exotoxin A has the same catalytic activity as diphtheria toxin.
  • Ricin has been used therapeutically by binding its toxic ⁇ -chain, to targeting molecules such as antibodies to enable site-specific delivery of the toxic effect.
  • Bacterial toxins have also been used as anti-tumor conjugates.
  • a toxic peptide chain or domain is conjugated to a compound of this invention and delivered in a site-specific manner to a target site where the toxic activity is desired, such as a metastatic focus.
  • Conjugation of toxins to protein such as antibodies or other ligands are known in the art (Olsnes, S. et al, Immunol. Today 70:291-295 (1989); Vitetta, E.S. et al, Ann. Rev. Immunol. 5:197-212 (1985)).
  • Cytotoxic drugs that interfere with critical cellular processes including DNA, RNA, and protein synthesis have been conjugated to antibodies and subsequently used for in vivo therapy.
  • Such drugs including, but not limited to, daunorubicin, doxorubicin, methotrexate, and mitomycin C are also coupled to the compounds of this invention and used therapeutically in this form.
  • photosensitizers may be coupled to the compounds of the invention for delivery directly to a tumor.
  • the methods of this invention may be used to inhibit tumor growth and invasion in a subject or to suppress angiogenesis induced by tumors by inhibiting endothelial cell growth and migration.
  • the methods result in inhibition of tumor metastasis.
  • a vertebrate subject preferably a mammal, more preferably a human, is administered an amount of the compound effective to inhibit tumor growth, invasion or angiogenesis.
  • the compound or pharmaceutically acceptable salt thereof is preferably administered in the form of a pharmaceutical composition as described above.
  • Doses of the compounds preferably include pharmaceutical dosage units comprising an effective amount of the peptide.
  • an effective amount is meant an amount sufficient to achieve a steady state concentration in vivo which results in a measurable reduction in any relevant parameter of disease and may include growth of primary or metastatic tumor, any accepted index of inflammatory reactivity, or a measurable prolongation of disease-free interval or of survival.
  • a reduction in tumor growth in 20 % of patients is considered efficacious (Frei III, E., The Cancer Journal 3:127-136 (1997)).
  • an effect of this magnitude is not considered to be a minimal requirement for the dose to be effective in accordance with this invention.
  • an effective dose is preferably 10-fold and more preferably 100-fold higher than the 50% effective dose (ED 50 ) of the compound in an in vivo assay as described herein.
  • the amount of active compound to be administered depends on the precise peptide or derivative selected, the disease or condition, the route of administration, the health and weight of the recipient, the existence of other concurrent treatment, if any, the frequency of treatment, the nature of the effect desired, for example, inhibition of tumor metastasis, and the judgment of the skilled practitioner.
  • a preferred dose for treating a subject, preferably mammalian, more preferably human, with a tumor is an amount of up to about 100 milligrams of active compound per kilogram of body weight.
  • a typical single dosage of the peptide is between about 1 ng and about lOOmg/kg body weight.
  • dosages in the range of about 0.01-20% concentration (by weight) of the compound, preferably 1-5%, are suggested.
  • a total daily dosage in the range of about 0.1 milligrams to about 7 grams is preferred for intravenous administration.
  • An effective amount or dose of the peptide for inhibiting invasion in vitro is in the range of about 1 picogram to about 0.5 nanograms per cell. Effective doses and optimal dose ranges may be determined in vitro using the methods described herein.
  • compositions and treatment methods are useful for inhibiting cell migration and invasion or cell proliferation in a subject having any disease or condition associated with undesired cell invasion, proliferation, angiogenesis or metastasis.
  • diseases or conditions may include primary growth of solid tumors or leukemias and lymphomas, metastasis, invasion and/or growth of tumor metastases, benign hyperplasias, atherosclerosis, myocardial angiogenesis, angiofibroma, arteriovenous malformations, post-balloon angioplasty vascular restenosis, neointima formation following vascular trauma, vascular graft restenosis, coronary collateral formation, deep venous thrombosis, ischemic limb angiogenesis, telangiectasia, pyogenic granuloma, corneal diseases, rubeosis, neovascular glaucoma, diabetic and other retinopathy, retrolental fibroplasia, diabetic neovascularization, macular degeneration,
  • compositions of the present invention are administered as soon as possible after traumatic spinal cord injury and for several days up to about two weeks thereafter to inhibit the angiogenesis and gliosis that would sterically prevent reestablishment of neuronal connectivity.
  • the treatment reduces the area of damage at the site of spinal cord injury and facilitates regeneration of neuronal function and thereby prevents paralysis.
  • the compounds of the invention are expected also to protect axons from Wallerian degeneration, reverse aminobutyrate-mediated depolarization (occurring in traumatized neurons), and improve recovery of neuronal conductivity of isolated central nervous system cells and tissue in culture.
  • the compounds of the invention are tested for their binding to uPAR by measuring their ability to inhibit the binding of [ 125 I]DFP-uPA (catalytically inactivated high molecular weight uPA) to uPAR expressed by HeLa (human cervical carcinoma) cells.
  • Cells (about 5 x 10 4 /well) are plated (in MEM with Earle's salts/10% FBS + antibiotics) in 24-well plates, then incubated in a humid 5% CO 2 atmosphere until the cells reach 70% confluence.
  • Catalytically inactivated high molecular weight uPA (DFP- uPA) is radioiodinated using Iodo-gen ® (Pierce) to a specific activity of about 250,000 cpm/mg.
  • test compounds are serially diluted in cold PBS/ 0.1 % BSA/ 0.01% Tween-80 and added to each well to a final volume of 0.3mL 10 minutes prior to the addition of the [ 125 I]DFP-uPA. Each well then receives 9500 cpm of [ 125 I]DFP-uPA at a final concentration of 0.2 nM.
  • the plates are incubated at 4°C for 2 hrs, after which time the cells are washed 3x (5 minutes each) with cold PBS/ 0.05% Tween-80.
  • the peptides of this invention inhibit the binding of [ 125 I]DFP-uPA with an IC 50 of ranging between about 0.1 nM to 2 ⁇ M. Modification of the peptides with diagnostic of therapeutic labels does not significantly affect their measured affinity constants.
  • EXAMPLE II Oregon Green-labeled-Peptide Targeting to Tumor Cells The ability of Oregon Green-labeled-peptides to localize to tumor cells is assessed by fluorescence microscopy. Tumor cells are cultured for 24 hours on coverslips (in a 6-well plate). Oregon Green-labeled peptides (1 ⁇ M) are added to each well and allowed to incubate in the presence of the cells. The coverslips are removed from the wells and washed twice (5 minutes per wash) with PBS in a second 6-well plate. The coverslips are mounted onto slides and the fluorescence observed using a fluorescent microscope. Digitized images are captured using a video camera and NIH image. Binding of Oregon Green-labeled-peptides is observed on tumor cells but is generally absent or very weak on normal cells.
  • MDA-MB-231 human breast cancer cells (1 x 10 6 ) are inoculated s.c. into the flank of female Balb/c (nu/nu) mice. Tumors are allowed to grow for varying lengths of time. Labeled uPAR targeting peptides (50 ⁇ g/injection) are injected i.p. and the ability of these peptides to localize to and detect the tumors is assessed at various times post- injection using the appropriate imaging technique.
  • the peptides described above are also tested in vivo in a model of human tumor metastasis in nude mice.
  • PC-3 cells are inoculated into nude mice i.v. at doses of 1 x 10 ⁇ cells per mouse. These mice are administered the imaging agent as above.
  • the localization of the labeled peptide is imaged using the appropriate detection technique.
  • PC-3 cells are transfected with the gene encoding the enzyme chloramphenicol acetyl-transferase (CAT).
  • CAT chloramphenicol acetyl-transferase
  • the animals are euthanized and the tumor marker probe CAT is assayed in regional lymph nodes, femurs, lungs, and brain. The localization of the labeled peptide is compared to the actual presence and amount of tumor in a given tissue or site.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une protéine ou un peptide destinés à cibler uPAR, qui est marqué(e) pour le diagnostic ou la thérapie et qui est utilisé(e) dans des procédés de diagnostic de thérapie. La protéine marquée ou le peptide marqué présente, de préférence, les propriétés suivantes: il/elle comprend au moins 38 restes d'acide aminé, dont les restes 13-30 du site de liaison à uPAR de uPA; il/elle est en compétition avec DFP-uPa marqué pour se lier à une cellule ou une molécule qui présente un site de liaison à uPA, et possède une valeur IC50 d'environ 10nM ou moins; et il n'est pas une protéine hybride dans laquelle le peptide uPA est fusionné avec une autre protéine ou un autre peptide non-uPA. Les molécules préférées sont uPA, scuPA, tcuPA, un fragment N-terminal de uPA, correspondant aux restes 1-135, un fragment N-terminal de uPA, correspondant aux restes 1-143, un fragment N-terminal de uPA, des restes 1-43 ; ou un fragment N-terminal de uPA, correspondant aux restes 4-43. Les marqueurs qui peuvent être détectés comprennent un radionucléide, un agent imageable par TEP, un agent imageable IRM, un agent fluorescent, un fluorogène, un chromophore, un chromogène, un agent phosphorescent, un chimiluminescent ou un bioluminescent. Les procédés selon la présente invention sont utilisés afin d'inhiber la migration cellulaire, l'invasion cellulaire, la prolifération cellulaire ou l'angiogenèse ou afin de produire l'apoptose, de préférence pour traiter un patient ayant une maladie ou un état pathologique liés à une migration cellulaire, une invasion cellulaire, une prolifération cellulaire ou une angiogenèse non désirées.
PCT/US2000/026502 1999-10-01 2000-09-27 SONDES DE DIAGNOSTIC ET AGENTS THERAPEUTIQUES DESTINES A CIBLER uPA ET uPAR Ceased WO2001025410A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002406882A CA2406882A1 (fr) 1999-10-01 2000-09-27 Sondes de diagnostic et agents therapeutiques destines a cibler upa et upar
EP00970496A EP1218496A2 (fr) 1999-10-01 2000-09-27 SONDES DE DIAGNOSTIC ET AGENTS THERAPEUTIQUES DESTINES A CIBLER uPA ET uPAR
JP2001528564A JP2003528035A (ja) 1999-10-01 2000-09-27 ｕＰＡおよびｕＰＡＲを標的化する診断プロ−ブおよび治療薬
AU79868/00A AU780730B2 (en) 1999-10-01 2000-09-27 Diagnostic probes and therapeutics targeting uPA and uPAR

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15701299P 1999-10-01 1999-10-01
US60/157,012 1999-10-01

Publications (2)

Publication Number Publication Date
WO2001025410A2 true WO2001025410A2 (fr) 2001-04-12
WO2001025410A3 WO2001025410A3 (fr) 2002-01-10

Family

ID=22562008

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/026502 Ceased WO2001025410A2 (fr) 1999-10-01 2000-09-27 SONDES DE DIAGNOSTIC ET AGENTS THERAPEUTIQUES DESTINES A CIBLER uPA ET uPAR

Country Status (5)

Country Link
EP (1) EP1218496A2 (fr)
JP (1) JP2003528035A (fr)
AU (1) AU780730B2 (fr)
CA (1) CA2406882A1 (fr)
WO (1) WO2001025410A2 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099780A1 (fr) * 2003-05-06 2004-11-18 Rigshospitalet Dosages immunologiques pour la detection de formes de recepteur d'urokinase
US6987270B2 (en) 2003-05-07 2006-01-17 General Electric Company Method to account for event losses due to positron range in positron emission tomography and assay of positron-emitting isotopes
WO2006036071A3 (fr) * 2004-09-29 2007-02-15 Ge Healthcare As Agents de contraste ciblant le recepteur upar
US7205392B2 (en) 2001-02-05 2007-04-17 Innoventus Project Ab Histidine-rich glycoprotein
WO2007044892A3 (fr) * 2005-10-10 2008-01-10 American Diagnostica Inc Conjugues de medicaments contenant une molecule de liaison upar et leurs utilisations
WO2007075565A3 (fr) * 2005-12-16 2008-06-05 Catherine M Shachaf Systeme diagnostic pour la detection et le diagnostic du cancer de la peau
US7927581B2 (en) 2001-07-30 2011-04-19 Factor 1A, LLC Peptide-based multimeric targeted contrast agents
WO2013118165A1 (fr) * 2012-02-07 2013-08-15 Jcr Pharmaceuticals Co., Ltd. Agents pharmaceutiques liés par fusion à l'atf pour une biodisponibilité améliorée
US9884131B2 (en) 2012-12-03 2018-02-06 Curasight Aps Positron emitting radionuclide labeled peptides for human uPAR PET imaging
WO2019067740A1 (fr) * 2017-09-27 2019-04-04 Emory University Protéines de fusion comprenant une toxine et un marqueur de cancer, nanoparticules et utilisations associées
US10994032B2 (en) 2012-05-08 2021-05-04 Trt Innovations Aps 177-Lu labeled peptide for site-specific uPAR-targeting
EP4610269A1 (fr) 2024-02-29 2025-09-03 3B Pharmaceuticals GmbH Ligands du recepteur de surface de l'activateur du plasminogene de l'urokinase (upar) utilises a des fins diagnostiques ou therapeutiques

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111462491B (zh) * 2019-12-10 2022-03-01 北京航空航天大学 一种基于匝道控制的高速公路合流区交通冲突预警方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1013285A3 (fr) * 1989-04-07 2004-05-19 Cancerforskningsfonden af 1989 (fonden til fremme af eksperimentel cancerforskning) Récepteur de l'activateur du plasminogène du type urokinase
EP0890638A4 (fr) * 1996-01-08 2001-08-22 Nissin Food Products Ltd Inhibiteur de metastases cancereuses
US5942492A (en) * 1996-11-12 1999-08-24 Angstrom Pharmaceuticals, Inc. Cyclic peptides that bind to urokinase-type plasminogen activator receptor
US6077508A (en) * 1998-03-23 2000-06-20 American Diagnostica Inc. Urokinase plasminogen activator receptor as a target for diagnosis of metastases

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7563765B2 (en) 2001-02-05 2009-07-21 Innoventus Project Ab Histidine-rich glycoprotein
US7205392B2 (en) 2001-02-05 2007-04-17 Innoventus Project Ab Histidine-rich glycoprotein
US7927581B2 (en) 2001-07-30 2011-04-19 Factor 1A, LLC Peptide-based multimeric targeted contrast agents
WO2004099780A1 (fr) * 2003-05-06 2004-11-18 Rigshospitalet Dosages immunologiques pour la detection de formes de recepteur d'urokinase
US6987270B2 (en) 2003-05-07 2006-01-17 General Electric Company Method to account for event losses due to positron range in positron emission tomography and assay of positron-emitting isotopes
US8568689B1 (en) 2004-09-29 2013-10-29 Ge Healthcare As uPAR-targeting contrast agents
RU2394837C2 (ru) * 2004-09-29 2010-07-20 Джи-И Хелткер АС Контрастный агент, нацеленный на рецептор урокиназного активатора плазминогена
WO2006036071A3 (fr) * 2004-09-29 2007-02-15 Ge Healthcare As Agents de contraste ciblant le recepteur upar
AU2005287934B2 (en) * 2004-09-29 2009-03-26 Ge Healthcare As Urokinase plasminogen activator receptor targeted contrast agent
WO2007044892A3 (fr) * 2005-10-10 2008-01-10 American Diagnostica Inc Conjugues de medicaments contenant une molecule de liaison upar et leurs utilisations
US10531824B2 (en) 2005-12-16 2020-01-14 Orlucent, Inc. Diagnostic system for the detection of skin cancer
WO2007075565A3 (fr) * 2005-12-16 2008-06-05 Catherine M Shachaf Systeme diagnostic pour la detection et le diagnostic du cancer de la peau
AU2006331910B2 (en) * 2005-12-16 2012-11-29 Amit Shachaf Diagnostic system for the detection and diagnosis of skin cancer
US8642009B2 (en) 2005-12-16 2014-02-04 Catherine M. Shachaf Diagnostic system for the detection of skin cancer
WO2013118165A1 (fr) * 2012-02-07 2013-08-15 Jcr Pharmaceuticals Co., Ltd. Agents pharmaceutiques liés par fusion à l'atf pour une biodisponibilité améliorée
US10994032B2 (en) 2012-05-08 2021-05-04 Trt Innovations Aps 177-Lu labeled peptide for site-specific uPAR-targeting
EP3590542A1 (fr) 2012-12-03 2020-01-08 Curasight APS Peptides étiquetés de radionucléides par émission de positrons pour pet scan
US9884131B2 (en) 2012-12-03 2018-02-06 Curasight Aps Positron emitting radionuclide labeled peptides for human uPAR PET imaging
US11311637B2 (en) 2012-12-03 2022-04-26 Curasight A/S Positron emitting radionuclide labeled peptides for human uPAR PET imaging
US12409240B2 (en) 2012-12-03 2025-09-09 Curasight A/S Positron emitting radionuclide labeled peptides for human uPAR PET imaging
WO2019067740A1 (fr) * 2017-09-27 2019-04-04 Emory University Protéines de fusion comprenant une toxine et un marqueur de cancer, nanoparticules et utilisations associées
US11633363B2 (en) 2017-09-27 2023-04-25 Emory University Fusion proteins having a toxin and cancer marker, nanoparticles, and uses related thereto
US12458603B2 (en) 2017-09-27 2025-11-04 Emory University Fusion proteins having a toxin and cancer marker, nanoparticles, and uses related thereto
EP4610269A1 (fr) 2024-02-29 2025-09-03 3B Pharmaceuticals GmbH Ligands du recepteur de surface de l'activateur du plasminogene de l'urokinase (upar) utilises a des fins diagnostiques ou therapeutiques
WO2025180699A1 (fr) 2024-02-29 2025-09-04 3B Pharmaceuticals Gmbh Ligands du récepteur activateur du plasminogène de type urokinase (upar) pour une utilisation diagnostique ou thérapeutique

Also Published As

Publication number Publication date
WO2001025410A3 (fr) 2002-01-10
AU780730B2 (en) 2005-04-14
CA2406882A1 (fr) 2001-04-12
EP1218496A2 (fr) 2002-07-03
AU7986800A (en) 2001-05-10
JP2003528035A (ja) 2003-09-24

Similar Documents

Publication Publication Date Title
US6277818B1 (en) Cyclic peptide ligands that target urokinase plasminogen activator receptor
AU780730B2 (en) Diagnostic probes and therapeutics targeting uPA and uPAR
JP6181265B2 (ja) クロロトキシンポリペプチドおよびコンジュゲートならびにその使用
JP4137997B2 (ja) クニッツドメインから誘導されたヒトプラスミンの阻害剤
US7807621B2 (en) Anti-invasive and anti-angiogenic compositions
AU681920B2 (en) Compositions that specifically bind to colorectal cancer cells and methods of using the same
US7960163B2 (en) Matriptase, a serine protease and its applications
JPH05304957A (ja) 酵素誘導体、それらの製造法およびそれらを含有する医薬組成物
RS50426B (sr) Tenascin -c ligandi nukleinske kiseline
JP2567536B2 (ja) Ｐａｉー２の変異体
US6896870B1 (en) Diagnostic probes and therapeutics targeting uPA and uPAR
US20040204348A1 (en) Cyclic peptide ligands that target urokinase plasminogen activator receptor
US20050232924A1 (en) Antibodies and/or conjugates thereof which bind to the amino terminal fragment of urokinase, compositions and uses thereof
WO2002069885A2 (fr) Compositions anti-invasives et anti-angiogeniques et methodes de traitement de tumeurs cerebrales et d'autres pathologies
WILL et al. Expression of urokinase-type plasminogen-activator (uPA) and Its receptor (uPAR) in human ovarian-cancer cells and in-vitro invasion capacity
KR0135980B1 (ko) 플라스미노겐 액티베이터 및 혈전용해제
JPH0377900A (ja) 血漿因子、その精製方法およびそれを有効成分として含有する血栓溶解剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2001 528564

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 79868/00

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2000970496

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2406882

Country of ref document: CA

WWP Wipo information: published in national office

Ref document number: 2000970496

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2000970496

Country of ref document: EP