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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • 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
  • C12N9/6489—Metalloendopeptidases (3.4.24)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
  • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
  • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
  • G01N2333/96486—Metalloendopeptidases (3.4.24)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to a novel protein, its fragments and mutants and to its use in detecting and testing drugs for ailments, including osteoarthritis and others characterized by up regulation of metalloproteases.
• a number of enzymes effect the breakdown of structural proteins and are structurally related metalloproteases. These include human skin fibroblast collagenase, human skin fibroblast gelatinase, human sputum collagenase and gelatinase, and human stromelysin. These are zinc-containing metalloprotease enzymes, as are the angiotensin- converting enzymes and the enkephalinases. Collagenase, stromelysin and related enzymes are important in mediating the symptomatology of a number of diseases, including rheumatoid arthritis (Mullins, D.
• Inhibitors of metalloproteases are useful in treating diseases caused, at least in part, by breakdown of structural proteins.
• a variety of inhibitors have been prepared, but there is a continuing need for metalloprotease inhibitor screens to design drugs for treating such diseases.
• Metalloproteases are a broad class of proteins which have widely varied functions. Disintegrins are zinc metalloproteases, abundant in snake venom. Alternate cloning strategies could be used. Mammalian disintegrins are a family of proteins with about 18 known subgroups. They act as cell adhesion disrupters and are also known to be active in reproduction (for example, in fertilization of the egg by the sperm, including fusion thereof, and in sperm maturation).
• Genbank a repository for gene sequences, provides several sequences of metalloproteases, including some said to encode fragments of disintegrins.
• GenBank accession # Z48444 dated February 25, 1994 discloses 2407 bases of a rat gene said to be a rat disintegrin metalloprotease gene
• GenBank accession # Z48579 dated March 2, 1995 discloses 1824 bases of a partial sequence of a gene said to be a human disintegrin metalloprotease gene
• GenBank accession # Z21961 dated October 25, 1994 discloses 2397 bases of a partial sequence of a gene said to be a bovine zinc metalloprotease gene.
• metalloprotease mediated diseases such as cancer, arthropothies (including ankylosing spondoiytis, rheumatiod arthritis, gouty arthritis (gout), inflammatory arthritis, Lyme disease and osteoarthrtis).
• This invention provides a method for identifying compounds capable of binding to the disintegrin protein, and determining the amount and affinity of a compound capable of binding to the disintegrin protein in a sample.
• This invention also provides a host cell comprising a recombinant expression vector to the disintegrin protein and a recombinant expression vector encoding to the disintegrin protein and the human disintegrin metalloprotease protein, fragment or mutant thereof, useful for these purposes.
• This invention also provides an in vivo or in vitro method for screening for osteoarthrtis and other metalloprotease based diseases, such as cancer, capable of manufacture and use in a kit form.
• protein As used herein, the terms “protein, " "protease,” and “metalloprotease” refer to a disintegrin. Preferably this is a human disintegrin as described below.
• antibody refers to an antibody to a disintegrin, or fragment thereof. These many be monoclonal or polyclonal, and can be from any of several sources. The invention also contemplates fragments of these antibodies made by any method in the protein or peptide art.
• disease screen refers to a screen for a disease or disease state.
• a disease state is the physiological or cellular or biochemical manifestation of the disease.
• this screen is used on body tissues or fluids of an animal or cell culture, using standard techniques, such as ELISA. It also contemplates "mapping" of disease in a whole body, such as by labeled antibody as described above given systemically: regardless of the detection method, preferable such detection methods include fluorescence, X-ray (including CAT scan), NMR (Including MRI), and the like.
• the term "compound screen” is related to the methods and screens related to finding compounds, determining their affinity for the protease, or designing or selecting compounds based on the screen. In another embodiment, it contemplates the use of the three dimensional structure for drug design, preferable “rational drug design”, as understood by the art. It may be preferred that the protease is in "essentially pure form", which refers to a protein reasonably free of other impurities, so as to make it useful for experiments or characterization. Use of this screening method assists the skilled artisan in finding novel structures, whether made by the chemist or by nature, which bind to and preferably inhibit the protease. These “inhibitors” may be useful in regulating or modulating the activity of the protease, and may be used to thus modulate the biological cascade that they function in. This approach affords new pharmaceutically useful compounds.
• disintegrin refers to a disintegrin, a fragment thereof, a mutant thereof or a homologue which still retains its function. This term contemplates aggracanase, and other proteases which are involved in or modulate tissue remodelling. This contemplates disintegrins from differing species, and those prepared by recombinant methods, in vitro methods, or standard peptide synthesis. Preferably the protein is a human disintegrin or mutant thereof. For the purposes of defining the mutants of the protein the preferred "native" protein is described in GenBank accession # Z48579, incorporated herein by reference and referred to in the sequence below.
• SEQ ID NO 1 describes a fragment of that DNA sequence and its transcript and SEQ ID NO 2 describes the coding region of the gene and its transcript.
• Homologue disintegrins include whole proteins with at least 90% homology as understood by the art, or fragments thereof.
• a rat protein which is 95% homologous to that of SEQ ID NO. 1 or SEQ ID NO. 2 based on the peptide sequence derived from the DNA or cDNA sequence, and a bovine protein (similarly derived) being 97-98% homologous, are both considered homologues.
• homologous cDNAs cloned from other organisms give rise to homologous proteins.
• proteins may be considered homologues based on the amino acid sequence alone. Practical limitations of amino acid sequencing would allow one to determine that a protein is homologous to another using for example comparison of the first 50 amino acids of the protein. Hence 90% homology in would allow for 5 differing amino acids in the chain of the first 50 amino acids of the homologous protein.
• the inhibition of disintegrin activity may be a predictor of efficacy in the treatment of osteoarthritis, and other diseases involving degeneration of articular cartilage.
• the metalloprotease is up regulated during osteoarthritis in tissues.
• a human disintegrin is up-regulated in human chondrocytes during osteoarthritic conditions. Inhibition of signal transduction mechanism is efficacious in disrupting the cascade of events in osteoarthritis and other diseases involving cartilage degeneration.
• up-regulation is a cause of the onset of arthritis, then interfering with the activity of this gene may be useful in treating osteoarthritis.
• the protease of the invention can be used to find inhibitors of the protease. Hence it is useful as a screening tool or for rational drug design. Without being bound by theory, the protease may modulate cellular remodeling and in fact may enhance extracellular matrix remodeling and thus enhance tissue breakdown.. Hence inhibition of disintegrin provides a therapeutic route for treatment of diseases characterized by these processes.
• a drug compound in screening, can be used to determine both the quality and quantity of inhibition. As a result such screening provides information for selection of actives, preferably small molecule actives, which are useful in treating these diseases.
• Metalloproteases active at a particularly undesired location can be targeted by conjugating a metalloprotease inhibitor to a to an antibody or fragment thereof. Conjugation methods are known in the art.
• the antibody of the invention can also be conjugated to solid supports. These conjugates can be used as affinity reagents for the purification of a desired metalloprotease, preferably a disintegrin.
• the antibody of the invention is directly conjugated to a label. As the antibody binds to the metalloprotease, the label can be used to detect the presence of relatively high levels of metalloprotease in vivo or ⁇ Q vitro cell culture.
• metalloprotease inhibiting compounds can be conjugated to antibodies. Typical conjugation methods are known in the art. These antibodies are then useful both in therapy and in monitoring the dosage of the inhibitors.
• targeting ligand which specifically reacts with a marker for the intended target tissue
• Methods for coupling the invention compound to the targeting ligand are well known and are similar to those described below for coupling to carrier.
• the conjugates are formulated and administered as described above.
• Antibodies may be made by several methods, for example, the protein may be injected into suitable (e.g., mammalian) subjects including mice, rabbits, and the like. Preferred protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum. The titers of the immune serum can readily be measured using immunoassay procedures, now standard in the art.
• the antisera obtained can be used directly or monoclonal antibodies may be obtained by harvesting the peripheral blood lymphocytes or the spleen of the immunized animal and immortalizing the antibody-producing cells, followed by identifying the suitable antibody producers using standard immunoassay techniques.
• Poh/cional or monoclonal preparations are useful in monitoring therapy or prophylaxis regimens involving the compounds of the invention.
• Suitable samples such as those derived from blood, serum, urine, or saliva can be tested for the presence of the protein at various times during the treatment protocol using standard immunoassay techniques which employ the antibody preparations of the invention.
• These antibodies can also be coupled to labels such as scintigraphic labels, e.g., technetium 99 or 1-131, using standard coupling methods.
• the labeled compounds are administered to subjects to determine the locations of excess amounts of one or more metalloproteases in vivo.
• a labelled antibody to the protein would operate a a screening tool for such enhanced expression, indicating the disease.
• Antibodies are advantageously coupled to other compounds or materials using known methods. For example, materials having a carboxyl functionality, the carboxyl residue can be reduced to an aldehyde and coupled to carrier through reaction with side chain amino groups, optionally followed by reduction of imino linkage formed. The carboxyl residue can also be reacted with side chain amino groups using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents. Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, 111.
• this gene may have a restricted tissue distribution and its expression is up regulated by potential osteoarthritis mediators.
• Enhanced expression of this gene (and hence its protein) for example, in articular chondrocytes provides a marker to monitor the development, including the earliest, asymptomatic stages, and the progression of osteoarthritis.
• an antibody raised to the protein would operate a a screening tool for such enhanced expression, indicating the disease.
• antibodies when used in a disease screen, antibodies can be conjugated to chromophore or fluorophore containing materials, or can be conjugated to enzymes which produce chromophores or fluorophores in certain conditions. These conjugating materials and methods are well known in the art. When used in this manner detection of the protein by immunoassay is straightforward to the skilled artisan. Body fluids, for example can be screened in this manner for calibration, and detection of distribution of metalloproteases, or increased levels of these proteases.
• the invention is a useful diagnostic and/or clinical marker for metalloprotease mediated diseases, such as osteoarthritis or other articular cartilage degenerative diseases.
• metalloprotease mediated diseases such as osteoarthritis or other articular cartilage degenerative diseases.
• disease When disease is detected, it may be treated before the onset of symptom or debilitation.
• antibodies can be used to target diseased tissue, for detection or treatment as described above.
• E. coli CJ236 and JM101 are known strains
• pUBl 10 is a known plasmid
• Kunkel method mutagenesis is also well known in the art.
• Variants may be made by expression systems and by various methods in various hosts, these methods are within the scope of the practice of the skilled artisan in molecular biology, biochemistry or other arts related to biotechnology.
• RNA was isolated from unstimulated and interleukin-1 stimulated cultures of normal human articular chondrocytes. The RNA was reverse transcribed into cDNA. The cDNA was subjected to a modified differential display procedure using a series of random primers.
• PCR samples generated from both stimulated and unstimulated chondrocytes were electrophoreses in adjacent lanes on polyacrylamide gels.
• the differentially expressed band was excised from the gel, cloned, and sequenced.
• the differential expression of the gene was confirmed by RNase protection and nuclear run on experiments.
• a novel partial human cDNA coding the protein is cloned from primary cultures of interleukin-1 stimulated human articular (femoral head) chondrocytes, using known methods.
• the cloned DNA of example 2 was placed in pUBl 10 using known methods.
• This plasmid is used to transform E. coli and provides a template for site-directed mutagenesis to create new mutants.
• Kunkel method mutagenesis performed altering GLN 1 ALA.
• Example 4 [ 125 I] disintegrin antibody is prepared using IODOBEADS (Pierce, Rockford, IL; immobilized chloramine-T on nonporous polystyrene beads). Lyophilized antibody (2 ⁇ g) is taken up in 50 ⁇ l of 10 mM acetic acid and added to 450 ⁇ l of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) on ice. To the tube is added 500 ⁇ Curie of 125 I (Amersham, Arlington Heights, IL) (2200Ci/mmol) in 5 ⁇ l, and one IODOBEAD. The reaction is incubated on ice for 10 min with occasional shaking. The reaction is then terminated by removal of the reaction from the IODOBEAD. To remove unreacted ⁇ H, the mixture is applied to a PD-10 gel filtration column.
• Example 5 A fluorogenic peptide (Bachem, Guelph Mills, King of Prussia, Pa) is mixed with the disintegrin and change in the fluorecence is evaluated at 2 min, as a control. Then the fluorogenic peptide is mixed with the disintegrin in the presence of the compound in evaluation in a separate run, with evaluation at 2 minutes. Data are evaluated using standard methodology to provide relative binding of the evaluated compound.
• Example 6 0.5ml of synovial fluid from the left knee of a patient is withdrawn and tested for elevated levels disintegrin by ELISA. The results indicate higher than normal disintegrin level.
• the patient is prescribed a prophylactic dose of a disintegrin inhibitor, and is administered an injection of same in the left knee before leaving the clinician's office.
• a sample of mouse derived articular cartilage is grown in a 1 micromolar solution of a small molecular weight disintegrin inhibitor. The experiment is controlled and compared to an identical culture grown with no inhibitor.
• the assay of the culture after 7 days shows that the inhibited culture has less tissue breakdown and less proteoglycan present in the serum of the culture. The result is consistent with the inhibited aggrecanase activity. Inhibition of aggrecanase would inhibit tissue breakdown and reduce the release of proteoglycan.
• Cells known to contain disintegrin are treated with a serine protease. Proteins released from the cell are measured by standard methods. Specifically the metalloprotease activity is monitored via literature methods. The amount of metalloprotease released is correlated to the amount of serine protease used to treat the cells.
• Increaeses, versus control, in src tyrosine kinase activity are measured by Western blot analysis of intracellular proteins using monoclonal antibodies specific for phosphotyrosine following cleavage and release of the disintegrin metalloprotease.
• Controls are cells that have not been treated with serine protease.
• src tyrosine kinase activity in the cell (or is it cell culture) is measured by literature meathods. Release of the metalloprotease domain of the disintegrin is also monitored via literature methods. There is a direct correlation between release of the metalloprotease domain and increases in intracellular src tyrosine kinase activity. This result is consistent with stimulation of disintegrin-mediated cell signalling by stimulation of the src tyrosine kinase cascade.
• Integrin binding is measured with a peptide containing the sequence RGD, using the protocol of Example 8. Integrin binding is measured via competitive assay, using cellular changes in shape visible via microscopy. The peptide inhibits the cellular changes as in Example 8.
• the RGD peptide inhibits cellular changes in chondrocytes.
• the osteoarthritis phenotype characterized by increased matrix synthesis and accelerated matrix metalloprotease activity does not occur.
• Other readily assayable cellular changes can be used to moniter this result, including gene expression, changes in mitotic activity, and the like.
• Example 10 A small molecular weight metalloprotease inhibitor is used to treat a tissue culture according to the method of Example 7. The release of TNF- ⁇ from the cell membrane is measured by literature methods. The inhibitor of Example 7 also decreases the amount of TNF- ⁇ secreted from the cell membrane.
• NAME HAKE, RICHARD A.
• MOLECULE TYPE DNA (genomic)
• TTAAGTCATC ATCTCCAAAC TAAACCCTCA CAAGTAACAG TTGAAGAAAA AATGGCAAGA 1604
• MOLECULE TYPE DNA (genomic)
• GCT GAA AAA AAT ACT TGT CAG CTT TAT ATT CAG ACT GAT CAT TTG TTC 865 Ala Glu Lys Asn Thr Cys Gin Leu Tyr He Gin Thr Asp His Leu Phe 760 765 770
• GAG CTC TAT GAA AAC ATT GCT GAA TGG ATT GTG GCT CAT TGG TGG GCA 2209 Glu Leu Tyr Glu Asn He Ala Glu Trp He Val Ala His Trp Trp Ala 1210 1215 1220

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• 1997
  • 1997-02-28 KR KR1019980706866A patent/KR19990087444A/ko not_active Ceased
  • 1997-02-28 WO PCT/US1997/003217 patent/WO1997031931A1/en not_active Ceased
  • 1997-02-28 EP EP97908799A patent/EP0888375A4/de not_active Withdrawn
  • 1997-02-28 NZ NZ331844A patent/NZ331844A/en unknown
  • 1997-02-28 CA CA002247827A patent/CA2247827A1/en not_active Abandoned
  • 1997-02-28 AU AU20617/97A patent/AU2061797A/en not_active Abandoned
  • 1997-02-28 JP JP9531177A patent/JPH11506023A/ja active Pending
• 1998
  • 1998-08-28 NO NO983984A patent/NO983984L/no not_active Application Discontinuation

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