JP2000511408A - New treatment - Google Patents

Abstract

  (57) [Summary] A method for screening compounds to identify compounds that inhibit or otherwise modulate presenilin-1 (PS-1) cleavage, comprising determining the extent of cleavage of PS-1 into 18 kDa and 28 kDa species by a test compound. , A method of measuring relative to the extent of cleavage in the absence of, the compound identified thereby and the use of the compound in therapy.

Description

DETAILED DESCRIPTION OF THE INVENTION Novel Therapies The present invention relates to assays used to identify compounds that may be useful in therapy. The invention also relates to the use of polypeptide cleavage modulators in therapy, and the use of polypeptide cleavage products in the diagnosis of Alzheimer's disease. Alzheimer's disease (AD) is a progressive degenerative disease of the central nervous system characterized clinically by dementia and neuropathologically by the presence of numerous senile plaques and neurofibrillary adhesions. Typically, AD is a late onset disease of the elderly. However, a small number of families have been reported that inherit the premature disease as an autosomal dominant trait with age-dependent penetrance. Most commonly, the age of onset of the disease is 60 years or less. The involvement of genetic factors in both early and late AD development has been shown. Mutations in at least four different loci are associated with genetic susceptibility to AD. The e4 allele of the apolipoprotein E (ApoE) gene on chromosome 19 is associated with late-onset AD (Strittmatter et al., Proc. Natl. Acad. Sci. USA 1993, 90: 1997-1981; Saunders et al., Neurology). 1993, 43: 1467-1472); Corder et al., Science 1993, 261: 921-923). Mutations in the β-amyloid precursor protein (βAPP) gene on chromosome 21 have been found in some families with early-onset AD (Goate et al., Nature 1991, 349: 704-706; Chartier-Harlin). Et al., Nature 1991, 353: 844-846; Murrell et al., Science 1991, 254: 97-99; Karlinsky et al., Neurology 1992, 42: 1445-1453). Recently, a novel AD locus in a gene called STM2 was identified on chromosome 1 (lq31-41) from gene linkage analysis of the "Volga German" kindred (Ephrat Levy-Lahad). Et al., Science 1995, 269: 970-973; ibid. 973-977). The STM2 gene has remarkable similarities to the AD-related gene, S182. Gene linkage studies on chromosome 14q24.3 have mapped the fourth locus (AD3) and may be responsible for the 70% abnormality of the early-onset autosomal dominant trait AD. (Sche11enberg et al., Science 1992, 258: 668-670; George-Hyslop et al., Nature Genet. 1992, 2: 330-334; Van Boreckhoven et al., Nature Genet. 1992, 2: 335-339). The AD3 locus is associated with the most progressive form of the disease (onset between the ages of 30 and 60), and mutations at this locus are a biologically fundamental process leading to AD. It is suggested that there is. Recently, a novel gene, S182, with five missense mutations in seven families that segregates the early-onset dominant trait AD at the AD3 locus has been cloned and described in Sherrigton et al., Nature 1995, 375: 754-760. Was done. Nucleotide sequence analysis of the S182 transcript showed heterozygous nucleotide substitutions in reverse transcriptase polymerase chain reaction products from six large family members. The putative open reading frame (ORF) of S182 encodes a protein predicted to be a 7-transmembrane protein and transmembrane (TM) helix II (L146M), VI (E246A) ), And VII (Y410C), and in the loop between TMII-TMIII (R163H) and TMVI-TMVII (V286L). In addition, a polymorphism in intron 9 of a gene that does not affect the coding region of the S182 gene (hereinafter referred to as the presenilin-1) gene has been shown to be a risk factor for late sporadic Alzheimer's disease. (M. Wragg et al., The Lancet, 347: 509-512 (1996)). The present invention is based on the finding that presenilin-1 is predominantly cleaved intracellularly between methionine 298 and alanine 299 by cells: detectable by antibodies raised against the N-terminal region of presenilin-1, A 28 kDa N-terminal fragment undetectable by raised antibodies; and an 18 kDa immunodetectable by antibodies raised to the C-terminal region of presenilin-1 but not detected by antibodies raised to the N-terminus. (D. Selkoe, Neurobiology of Aging Supplement Volume 17, number 4S Abstract 146, pS37 1996). Cleavage of presenilin-1 is an important factor in the pathology of Alzheimer's disease, since mutations in presenilin-1 cause premature AD and intronic polymorphisms are associated with an increased risk of developing late-onset sporadic Alzheimer's disease. An important event in that role. Accordingly, the present invention is a method of screening compounds to identify compounds that inhibit or otherwise modulate the cleavage of PS-1, comprising the steps of presenilin-1 (PS-1) cleavage to 18 kDa and 28 kDa species. A method is provided that comprises measuring the extent relative to the extent of cleavage in the absence of the test compound. The present invention also relates to the compounds identified thereby. In a preferred embodiment, the method uses human host cells transfected with an expression vector containing DNA encoding PS-1. The presence of the cleavage product was determined for a fragment of PS-1 such as the cleavage product, and the protein content of the host cell was determined using polyclonal or monoclonal antibodies specific for the 18 kDa species over the 28 kDa species and vice versa. It can be detected by performing an assay. Any suitable configuration of an immunoassay may be used, for example, a Western blot assay or an ELISA. Monoclonal and polyclonal antibodies raised against fragments of PS-1 and specific for 18 kDa species over 28 kDa species, and vice versa, are novel per se and form a further aspect of the invention. In particular, the present invention provides antibodies raised against polypeptides C RSLGHPEPSNLSGRPQGNSR and CRDSHLGPHRSTPESR. The present invention also provides isolated forms of the novel 18 kDa and 28 kDa cleavage products, specific antibodies thereto, and polynucleotides (DNA or RNA) encoding the polypeptide cleavage products. Cleavage sites could be determined by conventional N-terminal sequencing of the C-terminal 18 kDa species and the majority was found between methionine 298 and alanine 299 (D. Selkoe, Neurobiology of Aging Supplement Volume 17). , Number 4S Abstract 146, pS37, 1996). Based on knowledge of the cleavage site, short polypeptides (eg, amino acids) across the cleavage site that can be used in the methods of the invention in place of full-length PS-1 and can analyze and measure the enzymatic activity involved in PS-1 cleavage 3 to 10 lengths). Also, knowledge of the cleavage site allows the polypeptide to be used to generate specific antibodies in accordance with the present invention, and is useful in the methods of the present invention for the C-terminal and 18 kDa species of the 28 kDa species of PS-1. Short polypeptides corresponding to the N-terminus (eg, 3 to 20 amino acids in length) can also be designed. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material has been removed from its original environment (eg, the natural environment if it is a natural product). For example, a natural polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is isolated when separated from some or all of the coexisting materials in the natural system. Have been. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, since such a vector or composition is not part of the natural environment. Separated. Preferably, the polypeptide is in a purified form. Purified form means at least 80%, more preferably 90%, even more preferably 95% and most preferably 99% pure relative to other protein contaminants. The invention also relates to vectors containing the polynucleotides of the invention, host cells genetically engineered with the vectors of the invention, and the production of the polypeptides of the invention by recombinant techniques. Therefore, according to a further aspect of the present invention, there is provided a method for producing a polypeptide of the present invention by recombinant techniques in which a polynucleotide encoding the polypeptide is expressed in a host, and the expressed product is recovered. Alternatively, the polypeptides of the invention can be produced synthetically by conventional methods for peptide synthesis. The host cell is genetically engineered (transducted or transformed or transfected) with a vector which may be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, cosmid, phage or the like. The engineered host cells can be cultured in conventional nutrient media that have been modified to activate the promoter, select for transformants, or amplify the gene. Culture conditions, such as temperature, pH, etc., are those previously used in the host cell selected for expression, and will be apparent to those skilled in the art. Suitable expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, such as derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmid; plasmid and phage DNA, vaccinia, adenovirus, fowlpox virus and pseudotype. Vectors derived from combinations of viral DNA such as rabies are included. However, any other vector may be used as long as it is replicable and viable in the host cell. The appropriate DNA sequence may be inserted into the vector by various methods. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art. Such and other methods are deemed to be within the purview of those skilled in the art. The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to regulate mRNA synthesis. Representative examples of such promoters include the LTR or SV40 promoter, E. coli rac or trp, phage lambda P _L Promoters and other promoters known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses are included. Expression vectors also contain ribosome binding sites for translation initiation and transcription termination. Vectors may also contain appropriate sequences for amplifying expression. In addition, the expression vector contains one or more selectable marker genes to select for transformed host cells, such as dihydrofolate reductase or neomycin resistance or e. Coli tetracycline or ampicillin resistance for eukaryotic cell culture. It is preferable to provide phenotypic characteristics for The gene can be under the control of a promoter, a ribosome binding site (for bacterial expression) and, if desired, an operator (collectively referred to herein as "regulatory" elements), and the DNA sequence encoding the desired protein is Vectors containing this expression construct can be transcribed into RNA in transformed host cells. The coding sequence may or may not contain a signal peptide or leader sequence. For example, the protein sequences of the present invention can be expressed using the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. The leader sequence can be removed by the bacterial host during post-translational processing. The promoter region can be selected from the desired gene using a CAT (chloramphenicol transferase) vector or another vector having a selection marker. Two suitable vectors are PKK232-8 and PCM7. Specially named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P _R , P _L And trp. Eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is within the skill of the art. It is desirable to add, in addition to regulatory sequences, control sequences that can control the expression of the protein sequence as the host cell grows. Regulatory sequences are known to those of skill in the art and include those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Another type of control element, such as an enhancer sequence, may also be present in the vector. A particular coding sequence is placed in a vector having appropriate control sequences, and the position and orientation of the coding sequence are adjusted relative to the regulatory sequences. The expression vector is constructed such that is transcribed “under control” of the regulatory sequence (ie, the RNA polymerase that binds to the DNA molecule at the regulatory sequence transcribes the coding sequence). Modifications of the coding sequence are desirable to achieve this goal. For example, it may be necessary to modify the sequence so that it can bind to the regulatory sequence in the proper orientation, ie, maintain the reading frame. Regulatory and other control sequences may be ligated to the coding sequence prior to insertion into a vector such as the cloning vector described above. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the regulatory sequences and appropriate restriction sites. Codon usage may be changed to be compatible with the chosen host cell in order to make modifications to the coding sequence and enhance expression. In general, recombinant expression vectors provide an origin of replication and selectable markers that allow transformation of the host cell, such as the ampicillin resistance gene of E. coli and the S. cerebisiae TRP1 gene, and downstream structural sequences. Contains the promoter from the indicated high-expressed gene. The heterologous structural sequence is assembled in an appropriate state using translation initiation and termination sequences, preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic or extracellular medium. If desired, the heterologous sequence can encode a fusion protein containing an N-terminal identification peptide that confers desired properties, such as stabilization of the expressed recombinant product or ease of purification. A suitable host cell can be transformed with a vector containing the appropriate DNA sequence as described herein, and the appropriate promoter or regulatory sequence, to allow the host to express the protein. Examples of recombinant DNA vectors for cloning and host cells which can be transformed include bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (Gram negative bacteria), pGV 1106 (Gram negative bacteria), pLAFR1 (Gram negative bacteria), pME290 (Gram negative bacteria other than E. coli), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Strepto) Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), baculovirus insect cell line, YCp19 (Saccharomyces) and the like. The outline is "DNA Cloning": Vols I & II, Glover et al., Ed. IRL Press Oxford (1985) (1987); See Maniatis et al., "Molecular Cloning" Cold Spring Harbor Laboratory (1982). In some cases, it may be desirable to add a sequence that causes secretion of the polypeptide from the host organism, followed by cleaving the secretion signal. Yeast expression vectors are also known in the art. See, for example, U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; and also see European Patent Applications 103409; 100561; 96491. PSV2neo (described in J. Mol. Appl. Genet. 1: 327-341) that induces expression in mammalian cells using the SV40 late promoter or pCDNA1 (Mol. Cell) that induces expression using the CMV promoter. Biol. 7: 4125-4129). Both of these latter two vectors can be used for transient or stable (using G418 resistance) expression in mammalian cells. Insect cell expression systems, such as Drosophila, are also useful, see, for example, PCT applications WO90 / 06358 and WO92 / 06212 and EP290261-B1. Transcription of DNA by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to enhance its transcription. Examples include the SV40 enhancer 100 to 270 base pairs behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer. The host cell containing the vector can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or can be a prokaryotic cell, such as a bacterial cell. Representative examples of suitable hosts include: prokaryotes, for example, bacterial cells such as E. coli, Streptomyces, Salmonel la thyphimurium, and eukaryotes, for example, fungal cells such as yeast, Drosophila and Insect cells such as Spodoptera frugiperda, mammalian cells such as CHO, COS or Bowes melanoma, and plant cells. The selection of an appropriate host is deemed to be within the skill of the art in light of the teachings herein. The construct can be introduced into host cells by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). After transforming the appropriate host strain and allowing the host strain to grow to an appropriate cell density, the selected promoter is induced by appropriate means (eg, temperature shift or chemical induction) and the cells are cultured for an additional period. Typically, cells are harvested by centrifugation, triturated by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used for protein expression can be disrupted by conventional methods, including freeze-thaw cycles, sonication, mechanical trituration or the use of cell lysing agents, and such methods are well known to those skilled in the art. A variety of mammalian cell lines can be used to express the recombinant protein. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts (described in Gluzman, Cell, 23: 175 (1981)) and other cell lines capable of expressing compatible vectors, such as C127. , 3T3, CHO, HeLa and BHK cell lines and the like. Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, and any desired ribosome binding, polyadenylation, splice donor and acceptor sites, transcription termination sequences and 5 'flanking non-transcribed sequences. DNA sequences derived from the SV40 price and polyadenylation sites may be used to provide the desired non-transcribed genetic element. Further, a transgenic animal other than human may be used as a host. The method of recovering the expressed polypeptide depends on the chosen expression system and host. If the expression system secretes the polypeptide into the growth medium, the polypeptide can be purified directly from the medium. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from cell membrane fractions. If the polypeptide is localized on the cell surface, whole cells or isolated membranes can be used as a source for the desired gene product. Polypeptides expressed in bacterial hosts such as E. coli need to be isolated from inclusion bodies and regenerated. Selection of appropriate growth conditions and recovery methods are within the skill of the art. From recombinant cell culture 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. The polypeptide can be recovered and purified. If necessary, the configuration of the mature protein can be made complete using a protein regeneration step. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step. The polypeptide may or may not be glycosylated, depending on the host used in the recombinant production process. The polypeptide may also contain an initial methionine amino acid residue. By “recombinant” polypeptide is meant a polypeptide produced by recombinant DNA technology, ie, from a cell transformed with an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides are polypeptides produced by chemical synthesis. "Replicons" function as autonomous units of DNA replication in vivo; that is, genetic elements (eg, plasmids, chromosomes, viruses) that are capable of replicating under their own control. A "vector" is a replicon, such as a plasmid, phage, or cosmid, that another DNA segment carries so that the attached segment can replicate. "Double-stranded DNA molecule" means a polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine base) in both a relaxed and a supercoiled double-stranded helix. This term refers only to the primary and secondary structure of the molecule and does not limit any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequences can be described herein according to conventional methods that show only sequences in the 5 'to 3' direction along the sense strand of the DNA. A DNA "coding sequence" of a particular protein, or a "nucleotide sequence" that encodes a particular protein, is a DNA sequence that is transcribed and translated into a polypeptide under the control of appropriate regulatory sequences. 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 are a transcription initiation site (conveniently indicated by mapping nuclease S1) and a protein binding domain (consensus sequence) that contributes to RNA polymerase binding. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. DNA "regulatory sequence" refers generically to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, and generally provides for the expression of coding sequences (ie, transcription and translation) in host cells. When RNA polymerase binds to a promoter sequence, transcribes the coding sequence into mRNA, and then translates it into a polypeptide encoded by the coding sequence, the regulatory sequence “directs expression” of the coding sequence in the cell. A "host cell" is a cell that has been transformed or transfected, or is capable of being transformed or transfected by an exogenous DNA sequence. A cell has been "transformed" by exogenous DNA when such DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes and yeast, for example, exogenous DNA can be maintained on episomal elements, such as plasmids. With respect to eukaryotic cells, stably transformed or transfected cells are cells in which exogenous DNA has become integrated into a chromosome and is inherited by daughter cells via chromosomal replication. This stability is indicated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of daughter cells containing exogenous DNA. A "clone" is a population of cells derived from a single cell or a common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. Two DNA or polypeptide sequences are "substantially homologous" or "substantially identical" when they have at least about 85% (preferably at least about 90%, and preferably at least about 85%, nucleotides or amino acids over a given length of the molecule. Most preferably at least about 95%) and encompasses allelic variation. Also, as used herein, substantially homologous refers to a sequence that shows identity to a particular DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified in Southern hybridization experiments, for example, under stringent conditions as defined in the particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, "Current Protocols in Mol. Biol." Vol. I & II, Wiley Interscience, Ausbel et al. (Eds.) (1992). Substantially identical protein sequences can be identified by proteolytic digestion, gel electrophoresis and microsequencing. The term "functionally equivalent" means that the amino acid sequence of the protein of interest is a sequence that exhibits the same activity as that of the original protein. 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 can be produced using the polypeptide or cells expressing it as an immunogen. These antibodies can be, for example, polyclonal or monoclonal antibodies. The invention also includes chimeric, single chain and humanized antibodies and Fab fragments or the products of a Fab expression library. Such antibodies and fragments can be produced using various methods known in the art. The polypeptide can be injected directly into the animal, or the polypeptide can be administered, preferably to a non-human animal, to obtain antibodies raised against the polypeptide. The antibody thus obtained is then bound to the polypeptide itself. In this way, it is even possible to generate antibodies that bind to the fully native polypeptide using sequences encoding only fragments of the polypeptide. Monoclonal antibodies can be produced using any technique that provides antibodies produced by continuous cell line cultures. Examples include hybridoma technology (Kohler and Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72), and human monoclonal antibodies. EBV hybridoma technology for production (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. 77-96) is included. Techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be applied to produce single-chain antibodies to immunogenic polypeptides. The present invention also provides compounds that are modulators of the cleavage of PS-1 into 18 kDa and 28 kDa species and their use to modulate the function of PS-1. The present invention further provides the use of a compound according to the present invention in the manufacture of a medicament for modulating the function of PS-1. The present invention further provides a method of treating or preventing Alzheimer's disease, comprising administering to a patient an effective amount of a compound of the present invention, particularly a compound that is a modulator of the cleavage of PS-1 into 18 kDa and 28 kDa species. I will provide a. For use in therapy, the compounds of the invention are formulated according to standard pharmaceutical methods. Accordingly, the present invention also provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. Compounds that are active when administered orally can be formulated in solutions, for example, syrups, suspensions or emulsions, tablets, capzels and lozenges. Generally, the liquid formulation will be a compound in a suitable liquid carrier, such as ethanol, glycerin, a non-aqueous solvent such as polyethylene glycol, oil or water, or a pharmaceutically acceptable agent, together with a suspending agent, preservative, flavoring or coloring agent. A salt suspension or solution. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier (s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using conventional capsule methods. For example, pellets containing the active ingredient can be prepared using a standard carrier, and then filled into hard gelatin capsules; alternatively, dispersed in a suitable pharmaceutical carrier such as an aqueous gum, cellulose, silicate or oil An emulsion or suspension can be prepared and the dispersion or suspension can then be filled into soft gelatin capsules. A typical parenteral composition is a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, such as polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil. Consists of a suspension. Alternatively, the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration. A typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is active when administered in a suppository, comprising a glycol polymer, gelatin or cocoa butter or other low melting plant. Together with binders and / or lubricants, such as sexual or synthetic waxes or fats. Preferably, the composition is in unit dosage form, such as a tablet or capsule. Each dosage unit for oral administration preferably contains 1 to 250 mg of the inhibitor according to the invention (preferably 0.1 mg to 25 mg for parenteral administration). The daily dosage regimen for adult patients is calculated, for example, as the free base of the compound of formula (I) or a pharmaceutically acceptable salt thereof, and the oral dose is 1 mg to 500 mg, preferably 1 mg to 250 mg, or intravenous. The subcutaneous or intramuscular dose can be 0.1 mg to 100 mg, preferably 0.1 mg to 25 mg, and the compound is administered 1 to 4 times a day. Suitably, the compound will be administered continuously during the treatment period. In a further aspect, the present invention is a diagnostic method for predicting Alzheimer's disease in a patient, comprising isolating a tissue sample from the patient and assaying the sample for cleavage of PS-1 into 18 kDa and 28 kDa species. I will provide a. As described above for compound screening methods, the presence of a cleavage product can be detected by assaying the protein content of the sample using a polyclonal or monoclonal antibody raised against the cleavage product. The present invention is further described with reference to the following examples; however, it should be understood that the present invention is not limited to such examples. All parts or amounts are by weight unless otherwise specified. EXAMPLES Example 1 Demonstration of Cleavage of Presenilin-1 in Cells Peptide CRSLGH PEPLSNGRPQGNSR containing amino acids 42-60 in the N-terminal region of PS-1 and amino acids 344-358 in the C-terminal region of PS-1 A rabbit polyclonal antibody was raised against the peptide CRDSHLGPHRRSTPESR (R. Sherrington et al., Nature, 375: 754-760, 1995). These antibodies were: Western immunoblot analysis of cell lysates of cells transfected with the cDNA of PS-1 showed that the protein band in the transfected cells had a stronger immunoreactivity compared to the vector control cells. It is shown that the immunoreactivity for the protein band competes with the peptide used to generate the antibody. In the serum collected from the rabbit before the peptide inoculation, the serum after the peptide inoculation was It is shown that the protein band detected with the protein cannot be detected. The neuroblastoma cell line SHSY5Y was transfected with an expression vector containing the cDNA for the PS-1 gene to establish a stably transfected cell line. In transfected cells, and also in vector control cells, the PS-1 protein appears as a 43-46 kDa protein detectable with antibodies raised against both the N-terminal and C-terminal regions of the protein. Full length PS-1 protein is more easily detectable in cells transfected with PS-1 than vector control cells. PS-1 is proteolytically cleaved in both transfected and untransfected cells: detectable immunologically by antibodies raised against the N-terminal region of presenilin-1, but not at the C-terminus. A 28 kDa N-terminal fragment that is not detectable with antibodies raised against it: and an 18 kDa N-terminal fragment that can be immunologically detected with antibodies raised against the C-terminal region of presenilin-1 but not detected with antibodies raised against the N-terminus Generate a C-terminal fragment. Example 2 Assay for Modulator of Presenilin-1 Cleavage The antibodies described above can be used to measure PS-1 cleavage in cells and the effect of foreign substances that can affect the extent of PS-1 cleavage. This includes substances that inhibit or otherwise modulate the activity of the enzyme that cleaves the PS-1 protein, such substances being useful in treating Alzheimer's disease. The ability of inhibitors of presenilin-1 cleavage in human neuroblastoma cells transfected with an expression vector containing the cDNA of the presenilin-1 (PS-1) gene was tested using the following methods: pcDNA3 or pCEP4. Adherent SHSY5Y human neuroblastoma cells transfected with either PS-1 cDNA of the expression vector are grown in microtiter plates. Growth medium consisting of 88% (v / v) DMEM / NUT MIX F-12, 10% (v / v) fetal calf serum, 50 μg / ml penicillin / streptomycin, 400 μg / ml G-418 geneticin, and 2 mM glutamine (pH 7). Grow cells to 80% confluence in 4). Test compounds are dissolved in dimethylsulfoxide at a stock concentration of 2 mM and then diluted 1: 200 in growth medium. After removing the growth medium, 200 μl of the diluted compound is added to the adherent cells in three wells. Prepare three appropriate control cultures containing the diluted solvent. 95% air / 5% CO in a humidified incubator _Two And incubate at 37 ° C for 24 hours. The cells are washed twice with phosphate buffered saline (pH 7.4). Cells were immersed in a RIPA buffer consisting of 150 mM sodium chloride, 1% v / v NP40, 0.5% v / v sodium deoxycholate, 0.1% v / v sodium dodecyl sulfate in 50 mM Tris HCl (pH 8.0). Lyse and assay in a 96-well immunoassay format using specific antibodies in a sandwich assay. The first anti-presenilin-1 antibody serves as a capture agent for intracellular C-terminal fragments to detect a second anti-presenilin-1 specific antibody, which recognizes the N-terminal neo-epitope generated by cleavage. It serves as a component of the complex used. A third rabbit IgG-specific antibody bound to dextran and biotinylated is detected by fluorescence over time and then further incubated with streptavidin-europium complex (Delfia; EG & GBerthold and Wallac), followed by an enhancer solution. A positive result is a change in fluorescence relative to the control group, indicating that the effect of the test compound has modulated the cleavage. Results are expressed as% inhibition of signal obtained in wells conditioned with medium containing only solvent. Example 3 Diagnostic methods The above-mentioned antibodies are used for diagnosis as a means for predicting Alzheimer's disease and as a means for evaluating the effect of substances administered to modulate the cleavage of presenilin as a treatment for Alzheimer's disease. Thus, the extent of cleavage of PS-1 into 18 kDa and 28 kDa species in patient tissues, eg, blood, can be assayed. As described above for compound screening methods, the presence of the cleavage product may be detected by assaying the immune activity of the sample using polyclonal or monoclonal antibodies raised against the cleavage product.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 C07K 14/47 16/18 16/18 C12N 5/10 C12P 21/02 C C12P 21/02 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/53 D 33/53 C12N 5/00 B (72) Inventor Gray, Carroll 19.5 CM, UK Adablue, Essex, Harlow, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals (72) Inventor Davis, John Bee United Kingdom, CM 19.5 Adablue, Essex, Harlow, New Frontiers Science Park・ South, Smithkline Beecham Pharmaceuticals (72) Inventor Ward, Robin Buoy United Kingdom, CM 19.5 Adabrew, Essex, Harlow, New Frontiers Science Park South, SmithKline Beecham Far Matthewicals

Claims (1)

[Claims]   1. Inhibits or otherwise modulates presenilin-1 (PS-1) cleavage A method for screening a compound for identifying a compound, comprising: The extent of cleavage to 18 kDa and 28 kDa species was determined in the absence of the test compound. A method comprising measuring relative to the degree of cutting.   2. Transfected with an expression vector containing DNA encoding PS-1 The method according to claim 1, wherein a human host cell obtained is used.   3. Generated for PS-1 fragment, 18kDa than 28kDa species Polyclonal or monoclonal antibodies specific for the species or vice versa The presence of cleavage products by assaying the protein content of the host cell using The method according to claim 1 or 2, wherein the detection is performed.   4. 4. The method according to claim 3, which is a western blot assay or an ELISA. the method of.   5. Isolated form of PS-1 18 kDa or 28 kDa cleavage product.   6. Corresponds to the C-terminus of the 28 kDa species or the N-terminus of the 18 kDa species of PS-1 A polypeptide 3 to 20 amino acids in length.   7. Generated on PS-1 fragment, 18 kDa over 28 kDa species Antibodies that are species specific or vice versa.   8. The method according to claim 7, which is prepared for the polypeptide according to claim 5 or 6. The listed antibodies.   9. The polypeptide CRSLGHPELSNGRPQGNSR or CRDS 8. The antibody of claim 7, generated against HLGPPHRSTPESR.   10. The antibody according to claim 3 or 4, wherein the antibody is as defined in claims 7 to 9. The method described.   11. Polypeptides of 3 to 10 amino acids spanning the PS-1 cleavage site .   12. The method according to claim 1, wherein the polypeptide according to claim 11 is used.   13. A polynucleotide encoding the polypeptide according to claim 5, 6 or 11. Otide (DNA or RNA).   14． A vector comprising the polynucleotide according to claim 13.   15. A host cell transformed with the vector of claim 14.   16. Expressing a polynucleotide encoding the polypeptide in a host, The poiy according to claim 5, 6 or 11 by a recombinant technique for recovering the expressed product. A method for producing a peptide.   17. Triggered by PS-1 cDNA of pcDNA3 or pCEP4 expression vector. Transfected SHSY5Y human neuroblastoma cells.   18. A compound identified by the method according to claim 1.   19. Modulator of the cleavage of PS-1 into 18 kDa and 28 kDa species Certain compounds.   20. A compound according to claim 18 or 19 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:   21. 20. The manufacture of a medicament that modulates the function of PS-1. Use of the compound described in 1.   22. 21. A method for treating or preventing Alzheimer's disease, wherein the method comprises the steps of: A method comprising administering to a patient an effective amount of the compound of claim 9.   23. A diagnostic method for predicting Alzheimer's disease in a patient, the method comprising: Tissue samples from the donor and cutting PS-1 into 18 kDa and 28 kDa species. A method comprising assaying said sample for cleavage.