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  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Figures 2 A, 2B and 2C similarly shows the amino acid sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of the novel HPTP-2 of the present invention.
• Figures 3A, 3B and 3C shows the northern analysis for the consensus nucleotide sequence
• a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
• An “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring HPTP.
• alleles encoding HPTP include alleles encoding HPTP.
• an "allele” or “allelic sequence” is an alternative form of the nucleic acid sequence encoding HPTP. Alleles result from a mutation, ie, a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of HPTP expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
• pGEX vectors may also 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 purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
• Proteins made in such systems are 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.
• Specific initiation signals may also be required for efficient translation of a sequence encoding HPTP. These signals include the ATG initiation codon and adjacent sequences. In cases where the sequence encoding HPTP, its initiation codon and upstream sequences are inserted into the most appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic.
• Enhancers appropriate to the cell system in use (Scharf D et al (1994) Results Probl Cell Differ 20:125-62; Bittner et al (1987) Methods in Enzymol 153:516-544).
• marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
• sequence encoding HPTP is inserted within a marker gene sequence
• recombinant cells containing the sequence encoding HPTP can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with the sequence encoding HPTP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem sequence as well.
• nucleotide and polypeptide sequences disclosed herein is based in part on the chemical and structural homology among the HPTP and the known PTPs. Because of the widespread roles of PTKs and their respective PTPs in cell growth and regulation processes in various cells and tissues, altered HPTP expression may be implicated in a variety of disorders and diseases.
• HPTP-1 or HPTP-2 may also be used for the treatment or prevention of cancer or inflammatory disease by increasing HPTP activity through the use of gene therapy or administration of HPTP or agonists of HPTP.
• HPTP activity may be decreased by the use of antisense molecules to HPTP, antibodies to HPTP, or antagonists of HPTP.
• sequences for HPTP will provide the basis for screening for agonists, antagonists or inhibitors that modulate the activity or products of HPTP.
• HPTP-specific antibodies are useful for the diagnosis and treatment of conditions and diseases associated with expression of HPTP.
• Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
• Neutralizing antibodies, ie, those which inhibit dimer formation, are especially preferred for diagnostics and therapeutics.
• various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with HPTP or any portion, fragment or oligopeptide which retains immunogenic properties.
• various adjuvants may be used to increase immunological response.
• adjuvants include but are not limited to Freund's,
• a polynucleotide sequence encoding HPTP or any part thereof may be used for diagnostic and/or therapeutic purposes.
• the sequence encoding HPTP of this invention may be used to detect and quantitate gene expression in biopsied tissues in which HPTP may be expressed in response to oncogenes.
• the diagnostic assay is useful to distinguish between absence, presence, and excess expression of HPTP and to monitor regulation of HPTP levels during therapeutic intervention.
• Included in the scope of the invention are ohgonucleotide sequences, antisense RNA and DNA molecules, and peptide nucleic acids, (PNA).
• RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
• Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
• a normal or standard profile for HPTP expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with HPTP, or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained for normal subjects with a dilution series of HPTP run in the same experiment where a known amount of substantially purified HPTP is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients affected by HPTP-associated diseases. Deviation between standard and subject values establishes the presence of disease.
• Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• a definitive diagnosis of this type may allow health professionals to begin aggressive treatment and prevent further worsening of the condition.
• 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 such as the triplet genetic code, specific base pair interactions, and the like.
• antisense molecules DNA, RNA or PNA, to the control regions of the sequence encoding HPTP, ie, the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence, are preferred.
• the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
• the determination of an effective dose is well within the capability of those skilled in the art.
• the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, eg, tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions 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 formulation.
• HPTP can be used to screen for therapeutic molecules which would ameliorate the adverse effects of inflammatory cells in autoimmune diseases.
• the STOMTUT01 cDNA library was constructed from gastric adenocarcinoma.
• the donor was a 52 year old Caucasian male who had undergone a total gastrectomy following diagnoses of an invasive grade 4 adenocarcinoma involving the gastroesophageal junction which was accompanied by symptoms of abdominal pain and abnormal loss of weight, (specimen #0092B; Mayo Clinic, Rochester MN).
• tumor cells were identified in the muscularis intestinal and invaded surrounding perigastric adipose tissue.
• metastases were detected in 6 of 18 epigastric lymph nodes biopsied. Surgical removal was extended to include parts of the duodenum and esophagus and the spleen.
• Prior to surgery the patient was being treated with Priloseq® (Omeprazole) to inhibit gastric acid secretion.
• the basis of the search is the product score which is defined as:
• nucleic acid sequences of SEQ ID Nos:2 and 4 are used to design oligo- nucleotide primers for extending a partial nucleotide sequence to full length or for obtaining 5 'sequence from genomic libraries.
• One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF).
• Primers allow the extension of the know sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest (US Patent Application 08/487,112, filed June 7, 1995, specifically incorporated by reference).
• the original, selected cDNA libraries, or a human genomic library are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
• Step 3 68° C for 6 min
• a 5-10 ml aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were selected and cut out of the gel. Further purification involves using a commercial gel extraction method such as QIAQuickTM (QIAGEN Ine). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.
• QIAQuickTM QIAGEN Ine
• Step l 94° C for 60 sec
• Step 2 94° C for 20 sec
• a portion containing 10 7 counts per minute of each of the sense and antisense oligonucleotides is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPont NEN ® ).
• an effective antisense ohgonucleotide includes any 15-20 nucleotides spanning the region which translates into the signal or early coding sequence of the polypeptide as shown in Figures 1A, IB, lC, 2A, 2B or 2C.
• Induction of an isolated, transfected bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first seven residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length HPTP.
• the signal sequence directs the secretion of HPTP into the bacterial growth media which can be used directly in the following assay for activity.
• HPTP activity is measured by the hydrolysis of P-nitrophenyl phosphate (PNPP). HPTP is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1 % b- mercaptoethanol at 37° C for 60 min. The reaction is stopped by the addition of 6 ml of 10 N NaOH and the increase in light absorbance at 410 nm of the hydrolyzed PNPP is measured using a spectrophotometer (Diamond et al, supra).
• Naturally occurring or recombinant HPTP is substantially purified by immunoaffinity chromatography using antibodies specific for HPTP.
• An immunoaffinity column is constructed by covalently coupling HPTP antibody to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

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• 1996
  • 1996-10-03 US US08/725,532 patent/US6020179A/en not_active Expired - Fee Related
• 1997
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