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WO2006037513A1 - Agents diagnostiques et agents therapeutiques utilises dans le cadre de maladies associees a la metalloproteinase 9 matricielle (mmp9) - Google Patents

Agents diagnostiques et agents therapeutiques utilises dans le cadre de maladies associees a la metalloproteinase 9 matricielle (mmp9) Download PDF

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WO2006037513A1
WO2006037513A1 PCT/EP2005/010405 EP2005010405W WO2006037513A1 WO 2006037513 A1 WO2006037513 A1 WO 2006037513A1 EP 2005010405 W EP2005010405 W EP 2005010405W WO 2006037513 A1 WO2006037513 A1 WO 2006037513A1
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diseases
mmp9
infections
polypeptide
cancer
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PCT/EP2005/010405
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English (en)
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Stefan Golz
Ulf Brüggemeier
Andreas Geerts
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Bayer Healthcare Ag
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Publication of WO2006037513A1 publication Critical patent/WO2006037513A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human MMP9 and its regulation for the treatment of infections, cardiovacular diseases, dermatological diseases, endocrinological diseases, metabolic diseases, cancer, inflammation, gastroenterological diseases, hematological diseases, respiratory diseases, muscle skeleton diseases, neurological diseases, urological diseases, reproduction diseases in mammals.
  • MMP9 is a member of the group of protease enzymes [[Nagase et al. (1992)], US 6607879, EP 1394274].
  • proteases were recognized very early in the history of biochemistry. In the nineteenth century, one primary focus of research was on digestive proteases, like pepsin and trypsin. Proteases belong systematically to the C-N Hydrolases. More specifically, proteases catalyze the hyprolytic cleavage of a peptide bond and are therefore called peptidases as well.
  • Proteases can be classified according to several criteria, e.g. by localisation. Digestive proteases are located in the gastro-intestinal tract. These proteases are responsible for the digestion of food proteins.
  • Peptidases located extracellularly in the blood or other extracellular compartments of the body play often regulatory roles in processes like for example blood clotting, fibrinolysis, or activation of complement constituents.
  • Proteases are most commonly classified according to their mechanism of action, or to specific active groups that are present in the active center. The following groups can be distinguished: 1. Serin-peptidases, 2. cystein-peptidases, 3. aspartyl- or acidic-peptidases, 4. metallo-peptidases, or 5. peptidases with yet unclear reaction mechanism.
  • Serine proteases exhibit a serine in the catalytic site which forms a covalent ester intermediate during the catalytic reaction pathway by a nucleophilic attack on the carboxy carbon of the peptide bond.
  • a catalytic triad comprised of an aspartate, a histidine and the above mentioned serine is found. This triad functions in the reaction mechanism as a charge relay system.
  • serine protease belongs, for example, the digestive en2ymes trypsin and chymotrypsin, components of the complement cascade, enzymes involved in the blood clotting cascade, as well as enzymes that function in degradation, rebuilding and maintenace of constituents of the extracellular matrix.
  • serine protease family is the broad range of substrate specificity.
  • trypase subgroup cleave after arginine or lysine, chymases after phenylalanine or leucine, aspases after aspartate, metases after methionine and serases after serine.
  • thioester intermediate is formed by a nucleophilic attack of the cysteine on the caboxy carbon of the peptide bond.
  • a catalytic triad comprised of the cysteine, a histidine and an asparagine is found which functions as a charge relay system to facilitate the formation of the thioester intermediate.
  • cysteine proteases include lysosomal cathepsines, and cytosolic calpains.
  • the catalytic site of aspartyl proteases is composed of two aspartate residues. At the pH optimum of aspartyl proteases (2-3) one of the aspartyl carboxy groups is ionized and the other is neutral, which is important for the catalytic reaction to occur.
  • aspartyl proteases are gastric pepsins A and C, chymosin, as well as mammalian renin.
  • Metallo-peptidases are proteases, whose proteolytic activity depends on the presence of divalent cations in the active center. Examples of members of this class are carboxypeptidase A, which represents a pancreatic digestive enzyme, the Angiotension Converting Enzymes (ACE), which are responsible for the conversion of " angiotensin I to angiotensin ⁇ , or the Extracellular Matrix Metalloprotienases.
  • carboxypeptidase A which represents a pancreatic digestive enzyme
  • ACE Angiotension Converting Enzymes
  • proteases play a central role in several important cellular and intracellular processes.
  • the value as pharmaceutical targets has been proven for several proteases.
  • the protease encoded by the HTV genome is used as a target for drugs for the treatment of HTV infections, the proteasom complex has been discovered as an anti ⁇ cancer target, or Cys-proteases have been implemented as drug targets for inflammatory disorders.
  • Selective inhibitors have been developed as therapeutic agents for diseases such as HTV.
  • the identification of further disease implications of protease species and their splice variants may lead to the development of specific inhibitors or modulators, or suggest new utilities for known compounds affecting proteases.
  • This diseases may include, but are not limited to, infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HIV viruses, cancers, allergies including asthma, cardiovascular diseases including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, hematological diseases, genito-urinary diseases including urinary incontinence and benign prostate hyperplasia, osteoporosis, peripheral and central nervous system disorders including pain, Alzheimer's disease and Parkinson's disease, respiratory diseases, metabolic diseases, inflammatory diseases, gastroenterological diseases, diseases of the endocrine system, dermatological diseases, diseases of muscles or the sceleton, immunological diseases, developmental diseases or diseases of the reproductive system.
  • TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.
  • CT threshold cycle
  • Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action.
  • the TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue.
  • Expression profiling has been used in identifying genes, which are up- or downregulated in a variety of diseases.
  • An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drug treatment or in patients versus healthy individuals.
  • the premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets.
  • physiological or environmental stimuli e.g., drugs
  • the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.
  • the nucleotide sequence of MMP9 is accessible in the databases by the accession number NM_004994 and is given in SEQ ID NO:1.
  • the 72- and 92-kD type IV collagenases are members of a group of secreted zinc metalloproteases which, in mammals, degrade the collagens of the extracellular matrix. Other members of this group include interstitial collagenase and stromelysin.
  • CLG4A is secreted from normal skin fibroblasts
  • 92-kD collagenase (CLG4B) is produced by normal alveolar macrophages and granulocytes.
  • the 92-kD type IV collagenase is also known as 92-kD gelatinase, type V collagenase, or matrix metalloproteinase 9 (MMP9); see the glossary of matrix metalloproteinases provided by Nagase et al. [Nagase et al. (1992)].
  • MMP9 is published in patents US 6607879 and EP1394274.
  • the invention relates to novel disease associations of MMP9 polypeptides and polynucleotides.
  • the invention also relates to novel methods of screening for therapeutic agents for the treatment of infections, cardiovacular diseases, dermatological diseases, endocrinological diseases, metabolic diseases, cancer, inflammation, gastroenterological diseases, hematological diseases, respiratory diseases, muscle skeleton diseases, neurological diseases, urological diseases, reproduction diseases in a mammal.
  • the invention also relates to pharmaceutical compositions for the treatment of infections, cardiovacular diseases, dermatological diseases, endocrinological diseases, metabolic diseases, cancer, inflammation, gastroenterological diseases, hematological diseases, respiratory diseases, muscle skeleton diseases, neurological diseases, urological diseases, reproduction diseases in a mammal comprising a MMP9 polypeptide, a MMP9 polynucleotide, or regulators of MMP9 or modulators of MMP9 activity.
  • the invention further comprises methods of diagnosing infections, cardiovacular diseases, dermatological diseases, endocrinological diseases, metabolic diseases, cancer, inflammation, gastroenterological diseases, hematological diseases, respiratory diseases, muscle skeleton diseases, neurological diseases, urological diseases, reproduction diseases in a mammal.
  • Fig. 1 shows the nucleotide sequence of a MMP9 polynucleotide (SEQ ID NO:1).
  • Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:3).
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ ID NO:5).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • MMP9 polypeptide refers to those forms, fragments, or domains of a MMP9 polypeptide which retain the biological and/or antigenic activity of a MMP9 polypeptide.
  • Derivative refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • “Inhibitor” is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • domestic e.g., cats, dogs, etc.
  • agricultural e.g., cows, horses, sheep, etc.
  • test species e.g., mouse, rat, rabbit, etc.
  • MMP9 polynucleotide within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ E) NO: 1 ,
  • nucleic acid molecules having the sequence of SEQ ID NO: 1 (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1 ,
  • nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii);
  • nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code;
  • MMP9 polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii);
  • nucleotide sequences encoding a MMP9 have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of MMP9, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding a MMP9 disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • nucleotide sequences which encode a MMP9, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring MMP9 polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding MMP9 polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a MMP9 polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to MMP9 polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for MMP9-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding MMP9. Such probes may also be used for the detection of similar protease encoding sequences and should preferably show at least 40% nucleotide identity to MMP9 polynucleotides.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ ID
  • Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such MMP9 encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity. Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human- derived probes. Stringent conditions, within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7 % (w/v) SDS.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes MMP9. Such probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both. Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of MMP9 in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs. Rules for designing polymerase chain reaction (PCR) primers are now established, as reviewed by PCR Protocols.
  • the labels used for labeling the probes or primers of the current invention and which can provide the signal corresponding to the quantity of amplification product can take a variety of forms. As indicated above with regard to the 5' fluorogenic nuclease method, a fluorescent signal is one signal which can be measured. However, measurements may also be made, for example, by monitoring radioactivity, colorimetry, absorption, magnetic parameters, or enzymatic activity.
  • labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).
  • Fusion proteins are useful for generating antibodies against MMP9 polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of MMP9 polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • PCR-based methods can be used to extend nucleic acid sequences encoding human MMP9, for example to detect upstream sequences of MMP9 gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 0 C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5 1 regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • MMP9 can be obtained, for example, by purification from human cells, by expression of MMP9 polynucleotides, or by direct chemical synthesis.
  • MMP9 can be purified from any human cell which expresses the enzyme, including those which have been transfected with expression constructs which express MMP9.
  • a purified MMP9 is separated from other compounds which normally associate with MMP9 in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • MMP9 polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding MMP9 and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding MMP9. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors ⁇ e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJoIIa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding MMP9, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected. For example, when a large quantity of MMP9 is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding MMP9 can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • BLUESCRIPT a sequence encoding MMP9 can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • pBST vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purif ⁇ ed from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • HACs Human artificial chromosomes
  • HACs also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6M to 1OM are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding MMP9. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding MMP9, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • One such expression vector provides for expression of a fusion protein containing MMP9 and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography) Maddox, (1983)], while the enterokinase cleavage site provides a means for purifying MMP9 from the fusion protein [Porath, (1992)].
  • nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter MMP9 polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site- directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • an antibody which specifically binds to MMP9 provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to MMP9 do not detect other proteins in immunochemical assays and can immunoprecipitate MMP9 from solution.
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human anti ⁇ bodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to MMP9 can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • Candidate or test compounds or agents which bind to MMP9 and/or have a stimulatory or inhibitory effect on the activity or the expression of MMP9 are identified either in assays that employ cells which express MMP9 (cell-based assays) or in assays with isolated MMP9 (cell-free assays).
  • the various assays can employ a variety of variants of MMP9 (e.g., full-length MMP9, a biologically active fragment of MMP9, or a fusion protein which includes all or a portion of MMP9).
  • MMP9 can be derived from any suitable mammalian species (e.g., human MMP9, rat MMP9 or murine MMP9).
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of MMP9.
  • Such assays can employ full-length MMP9, a biologically active fragment of MMP9, or a fusion protein which includes all or a portion of MMP9.
  • the test compound can be obtained by any suitable means, e.g., from conventional compound libraries. Determining the ability of the test compound to modulate the activity of MMP9 can be accomplished, for example, by determining the ability of MMP9 to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which MMP9 binds or interacts with in nature.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto gluta ⁇ thione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non- adsorbed target protein or MMP9, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto gluta ⁇ thione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates,
  • antibodies which specifically bind to MMP9, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of MMP9 can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • MMP9 is expressed in various human tissues.
  • CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • Drug rashes are side effects of medications, mainly allergic reactions to medications.
  • Sweating disorders also belong to skin disorders.
  • Skin growths which are abnormal accumulations of different types of cells, may be present at birth or develop later.
  • Noncancerous (benign) growth and cancerous (malignant) growth types are distinguished.
  • Angiomas are collections of abnormally dense blood or lymph vessels that are usually located in and below the skin and that cause red or purple discolorations.
  • Pyogenic granulomas are scarlet, brown, or blue-black slightly raised areas caused by increased growth of capillaries (the smallest blood vessels) and swelling of the surrounding tissue.
  • Seborrheic keratoses sometimes called seborrheic warts
  • Examples of disorders of muscles, bursas, and tendons are spasmodic torticollis, fibromyalgia syndromes (myofascial pain syndromes, fibromyositis), bursitis, tendinitis and tenosynovitis.
  • Bacilli are a type of bacteria classified according to their distinctive rod-like shape. Bacteria are either spherical (coccal), rod-like (bacillary), or spiral/helical (spirochetal) in shape. Gram-positive or gram-negative bacilli are distinguished. Examples of gram-positive bacillary infections are are erysipelothricosis (caused by Erysipelothrix rhusiopathiae), listeriosis (caused by Listeria monocytogenes), and anthrax (caused by Bacillus anthracis). Within anthrax, pulmonary anthrax, gastrointestinal anthrax and anthrax skin sores can be distinguished.
  • rhinoviruses Influenza viruses or respiratory syncytial viruses
  • Influenza (caused by influenza A or influenza B virus)
  • Herpesvirus Infections Herpes simplex, herpes zoster, Epstein-Barr virus, cytomegalovirus, herpesvirus 6, human herpesvirus 7, or herpesvirus 8 (cause of Kaposi's sarcoma in people with AIDS), central nervous system viral infections (e.g.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.
  • the nucleotide sequences encoding MMP9 may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding MMP9 may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • Another objects of the invention are methods of the above, wherein the compound is coupled to a detectable label.
  • Other objects of the invention are methods of the above, wherein the test compound displaces a ligand which is first bound to the polypeptide.
  • antibodies are raised against MMP9, specifically monoclonal antibodies.
  • the monoclonal antibodies are screened to identify those which inhibit the binding of labeled MMP9. These monoclonal antibodies are then used therapeutically.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.

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Abstract

L'invention a trait à la MMP9 humaine qui est associée aux infections, aux troubles cardio-vasculaires, aux troubles dermatologiques, aux maladies endocriniennes, aux troubles du métabolisme, au cancer, à l'inflammation, aux troubles gastro-intestinaux, aux troubles hématologiques, aux troubles respiratoires, aux troubles squeletto-musculaires, aux troubles neurologiques, aux troubles urologiques, aux troubles de la reproduction. L'invention a également pour objet des essais permettant d'identifier des composés utiles pour traiter et prévenir des infections, des troubles cardio-vasculaires, des troubles dermatologiques, des maladies endocriniennes, des troubles du métabolisme, le cancer, l'inflammation, des troubles gastro-intestinaux, des troubles hématologiques, des troubles respiratoires, des troubles squeletto-musculaires, des troubles neurologiques, des troubles urologiques, des troubles de la reproduction. L'invention concerne aussi des composés qui se lient et/ou activent ou inhibent l'activité de MMP9 ainsi que des compositions pharmaceutiques de ces composés.
PCT/EP2005/010405 2004-10-09 2005-09-27 Agents diagnostiques et agents therapeutiques utilises dans le cadre de maladies associees a la metalloproteinase 9 matricielle (mmp9) WO2006037513A1 (fr)

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RU2474433C2 (ru) * 2007-08-15 2013-02-10 ЙЕДА РЕСЕАРЧ ЭНД ДЕВЕЛОПМЕНТ КО. ЛТД, эт ве Вайзманн Институте оф Сайнсе Регуляторы ммр-9 и их применение
US8377443B2 (en) 2010-08-27 2013-02-19 Gilead Biologics, Inc. Antibodies to matrix metalloproteinase 9
WO2013153138A1 (fr) * 2012-04-11 2013-10-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Aptamère de métalloprotéinase matricielle 9 (mmp-9) et ses utilisations
US9205122B2 (en) 2009-10-22 2015-12-08 Maresins Pharma, Inc. Herbal composition comprising ginger and goldenrod for the treatment of cold and flu
US9732156B2 (en) 2012-02-29 2017-08-15 Gilead Biologics, Inc. Methods of treating rheumatoid arthritis using antibodies to matrix metalloproteinase 9
WO2018109222A1 (fr) * 2016-12-16 2018-06-21 Universite de Bordeaux Inhibiteurs de mmp9 et leurs utilisations dans la prévention ou le traitement d'un trouble de la dépigmentation
WO2025076280A1 (fr) 2023-10-05 2025-04-10 Ashibio, Inc. Méthodes et compositions de traitement de troubles médiés par la mmp-9

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2474433C2 (ru) * 2007-08-15 2013-02-10 ЙЕДА РЕСЕАРЧ ЭНД ДЕВЕЛОПМЕНТ КО. ЛТД, эт ве Вайзманн Институте оф Сайнсе Регуляторы ммр-9 и их применение
US8999332B2 (en) 2007-08-15 2015-04-07 Yeda Research And Development Co. Ltd. Regulators of MMP-9 and uses thereof
US9205122B2 (en) 2009-10-22 2015-12-08 Maresins Pharma, Inc. Herbal composition comprising ginger and goldenrod for the treatment of cold and flu
US8377443B2 (en) 2010-08-27 2013-02-19 Gilead Biologics, Inc. Antibodies to matrix metalloproteinase 9
US8501916B2 (en) 2010-08-27 2013-08-06 Gilead Biologics, Inc. Antibodies to matrix metalloproteinase 9
US9120863B2 (en) 2010-08-27 2015-09-01 Gilead Sciences, Inc. Nucleic acids encoding antibodies to matrix metalloproteinase 9
US9260532B2 (en) 2010-08-27 2016-02-16 Gilead Biologics, Inc. Antibodies to matrix metalloproteinase 9
US9732156B2 (en) 2012-02-29 2017-08-15 Gilead Biologics, Inc. Methods of treating rheumatoid arthritis using antibodies to matrix metalloproteinase 9
WO2013153138A1 (fr) * 2012-04-11 2013-10-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Aptamère de métalloprotéinase matricielle 9 (mmp-9) et ses utilisations
WO2018109222A1 (fr) * 2016-12-16 2018-06-21 Universite de Bordeaux Inhibiteurs de mmp9 et leurs utilisations dans la prévention ou le traitement d'un trouble de la dépigmentation
CN110312737A (zh) * 2016-12-16 2019-10-08 波尔多大学 Mmp9抑制剂及其在预防或治疗色素脱失病症中的用途
US10894837B2 (en) 2016-12-16 2021-01-19 Université De Bordeaux MMP9 inhibitors and uses thereof in the prevention or treatment of a depigmenting disorder
WO2025076280A1 (fr) 2023-10-05 2025-04-10 Ashibio, Inc. Méthodes et compositions de traitement de troubles médiés par la mmp-9

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