Classifications

• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • 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
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
• • 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
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4709—Amyloid plaque core protein
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• the method employs a human host cell transfected with an expression vector containing DNA encoding PS-1.
• the presence of the cleavage products may be detected by assaying the protein content of the host cell by using polyclonal or monoclonal antibodies raised against fragments of PS-1, such as the cleavage products, and specific to the 18kDa species over the 28kDa species and vice versa.
• Any suitable configuration of immunoassay may be employed, for example a Western blot assay or an ELISA.
• Monoclonal and polyclonal antibodies raised against fragments of PS-1 and specific to the 18kDa species over the 28kDa species and vice versa are themselves novel and form a further aspect of the invention.
• the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
• a process for producing polypeptides of the invention by recombinant techniques by expresssing a polynucleotide encoding said polypeptide in a host and recovering the expressed product.
• the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
• Suitable expression vectors include chromosomal, nonchromosomal and synthetic
• the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
• the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
• the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
• promoter an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
• promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
• regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell.
• Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
• Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
• mammalian cell culture systems can also be employed to express recombinant protein.
• mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
• Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
• the method of recovery of expressed polypeptide depends on the expression system and host selected. If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. Where the polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product. Polypeptide expressed in bacterial hosts such as E. coli may require isolation from inclusion bodies and refolding. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
• the polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
• HPLC high performance liquid chromatography
• a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
• a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT' boxes.
• a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
• Antibodies generated against polypeptides can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptide.
• the invention further provides a method of treatment or prophylaxis of Alzheimer's disease, which method comprises administering to a patient an effective amount of a compound of the invention, in particular a compound which is a modulator of the cleavage of PS-1 into 18kDa and 28kDa species
• the compounds of the invention are formulated in accordance with standard pharmaceutical practice.
• the present invention therefore also provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.
• a composition in the form of a capsule can be prepared using routine encapsulation procedures.
• pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
• composition is in unit dose form such as a tablet or capsule.
• the daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.
• an oral dose of between 1 mg and 500 mg preferably between 1 mg and 250 mg
• an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.
• the compounds will be administered for a period of continuous therapy.
• the present invention provides in a still further aspect a diagnostic method for prognosing Alzheimer's disease in a patient comprising isolating a sample of tissue from the patient, and assaying said sample for cleavage of PS-1 into 18kDa and 28kDa species.
• the presence of the cleavage products may be detected by assaying protein content of the sample by using polyclonal or monoclonal antibodies prepared against the cleavage products as described above for the method of screening compounds.
• PS-1 is proteolytically cleaved within both transfected and untransfected cells resulting in: an N-terminal fragment of 28kDa that can be immunodetected by antibodies raised to the N-terminal region of presenilin- 1 but not detected by antibodies raised to the C-terminus: and a C-terminal fragment of 18kDa that can be immunodetected with antibodies raised to the C-terminal region of presenilin- 1 but not detected with antibodies raised to the N-terminus.
• the cells are lysed in RIPA buffer consisting of 150mM sodium chloride, 1% v/v NP40, 0.5% w/v sodium deoxycholic acid, 0.1% w/v sodium dodecyl sulphate in 50mM Tris HCI pH 8.0 and assayed in a 96- well immunoassay format which utilizes specific antibodies in a sandwich assay.
• One anti-presenilin-1 antibody serves as the capture reagent for the C-terminal fragment within cells
• the second anti-presenilin-1 specific antibody (which recognises the N-terminal neoepitope generated by cleavage) serves as a component of the complex used for detection.
• a rabbit IgG specific third antibody which has been conjugated with dextran and biotinylated is detected by time-resolved fluorescence following further incubations with a streptavidin-europium complex (Delfia; EG&G Berthold and Wallac) followed by enhancer solution.
• a positive result is one in which there is change in fluorescence relative to the control group, indicating a modulation of the cleavage of as a result of the effects of the test compound.
• the results are expressed as the percentage inhibition of the signal obtained in wells conditioned with medium which contained solvent alone.

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• 1996
  • 1996-04-26 GB GBGB9608657.4A patent/GB9608657D0/en active Pending
• 1997
  • 1997-04-22 WO PCT/EP1997/002050 patent/WO1997041443A2/en not_active Application Discontinuation
  • 1997-04-22 EP EP97920740A patent/EP0900384A2/de not_active Withdrawn
  • 1997-04-22 JP JP09538552A patent/JP2000511408A/ja active Pending

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