WO2006011485A1 - Protéine inhibant l'agglomération de peptides amyloïdes et l'effet de celle-ci - Google Patents

Protéine inhibant l'agglomération de peptides amyloïdes et l'effet de celle-ci Download PDF

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WO2006011485A1
WO2006011485A1 PCT/JP2005/013658 JP2005013658W WO2006011485A1 WO 2006011485 A1 WO2006011485 A1 WO 2006011485A1 JP 2005013658 W JP2005013658 W JP 2005013658W WO 2006011485 A1 WO2006011485 A1 WO 2006011485A1
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pgds
human
alanine
seq
mouse
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Japanese (ja)
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Yoshihiro Urade
Masako Taniike
Kosuke Aritake
Takahisa Kanekiyo
Yuji Goto
Tadato Ban
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Osaka Bioscience Institute
Osaka University
Kansai Technology Licensing Organization Co., Ltd.
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Publication of WO2006011485A1 publication Critical patent/WO2006011485A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2006—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/52—Isomerases (5)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to an agent that suppresses or improves the symptoms of Arno and Imah's disease. More specifically, the present invention relates to a drug that suppresses and improves the symptoms of Alzheimer's disease by inhibiting aggregation of amyloid peptide and interacting with a glycolipid component present on the surface of brain cell membrane.
  • a peptide forms a sheet structure in the brain parenchyma and aggregates and deposits, and is thought to be caused by the binding with lipid components present on the brain cell surface. It causes plaque formation and neurogenic changes in euron.
  • a j8 vaccine ⁇ 3 ⁇ 4 ⁇
  • Beta-sheet breaker peptides inhibit nbnllogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy. Nat Med. 1998 4: 822-6.). However, both are non-physiological substances and may have side effects.
  • Non-Patent Document 1 Neuron. 2003 38: 547-554
  • Non-Patent Document 2 Nature. 2000 408: 979-982
  • Non-Patent Document 3 Nat Med. 1998 4: 822-826
  • the present invention suppresses the aggregation of amyloid peptide and is a sugar present on the surface of brain cell membrane
  • the purpose is to provide a method for suppressing and improving Alzheimer's disease symptoms by interacting with lipid components.
  • L-PGDS Lipocalin-type prostaglandin D synthase
  • L-PGDS Lipocalin-type prostaglandin D synthase
  • the present invention is a drug used for the suppression and improvement of the symptoms of Arno and Imah's disease
  • L—PGDS Lipocalin-type prostaglandin D synthase
  • L—PGDS variants in which at least one cysteine residue of L—PGDS is replaced by another amino acid residue, eg alanine or serine;
  • a gist is a drug containing a compound selected from the group consisting of as active ingredients.
  • Substances that enhance the expression of LPGDS protein in vivo, particularly in the brain, include estrogen, darcocorticoid, interleukin-1 j8, and thyroid hormone.
  • the invention's effect include estrogen, darcocorticoid, interleukin-1 j8, and thyroid hormone.
  • the suppression of amyloid peptide aggregation and the interaction with a glycolipid component present on the surface of brain cell membrane can suppress or improve the symptoms of Alzheimer's disease. You can do good.
  • FIG. 1 is a micrograph of a micrograph in which binding of L PGDS to senile plaques in an autopsy brain of an Arno-Ima disease patient is detected by immunohistochemical staining. * Indicates amyloid 'plaque, and arrow indicates LPGDS expressed in oligodendrocytes.
  • FIG. 2 Alzheimer's disease model mouse Tg2576 amyloid 'plaque located in L
  • FIG. 3 is a graph showing the results of examining the concentration-dependent binding of human L-PGDS (Ala2.3.4) to human amyloid 'beta 1-40 peptide by surface plasmon resonance.
  • 4 is a graph showing the results of investigating the dose-dependent inhibitory effect of 4 by ThT Atsay.
  • FIG. 7 A graph showing the results obtained by examining the inhibitory effect of Ala2.3.4 on A ⁇ 1-40 peptide whose aggregation is promoted in the presence of Gandarioside GM-1 and ribosome by ThT Atsei.
  • FIG. 8 A graph showing the results of examining the dose-dependent inhibitory effect of trace on the seed-dependent aggregation of ⁇
  • ThT Atsey showed the antagonism of two mouse anti-human LPGDS monoclonal antibodies against the inhibitory effect of Ala2.3.4 peptide on seed-dependent aggregation of peptides. It is a graph to show.
  • FIG. 10 is a graph showing the results of examining the binding ability of several mouse L PGDS mutants to A
  • FIG. 5 is a photocopy showing the results of detecting the human A1-42 peptide remaining in the brain of a knockout mouse in a ligamentous and woven manner.
  • 81-42 peptide labeled with piotin was used.
  • L PGDS is the active ingredient.
  • L PGDS is derived from mammals, from human (SEQ ID NO: 1, amino acids 1 to 21 are signal peptides) and from mouse (SEQ ID NO: 2, amino acids 1 to 24 are signal peptides) Those derived from humans are preferred.
  • the second embodiment of the drug used for suppressing and improving the symptoms of the Arno-i-maima disease of the present invention comprises an L-PGDS mutant as an active ingredient.
  • the L PGDS mutant in the present invention is a substitution mutant of L PGDS, wherein at least one of the three cysteine residues is replaced with another amino acid residue, preferably glycine, alanine, serine, norin, histidine, treasure.
  • a relatively small variant of L-PGDS including substitutions with amino acids such as nin, asparagine, aspartate, etc.
  • Proteins of human L—PGDS (SEQ ID NO: 1) with the cysteines at positions 65 and 167 substituted with alanine or serine (hereinafter, may be abbreviated as “Alal. 3” and “Serl. 3”, respectively);
  • substitution mutants those in which one to several amino acid residues are further substituted with other amino acid residues can also achieve the object of the present invention.
  • Mammal-derived L PGDS is a force gene that can also obtain the tissue power of mammals. It is convenient to manufacture by recombinant technology.
  • the amino acid sequence of human or mouse-derived lipocalin-type prostaglandin D synthase (L-PGDS) is known.
  • the amino acid sequence of human-derived L—PGDS is shown in SEQ ID NO: 1 (Hoffinann A et al., J. Neurochem. 1993 Aug 61 (2) 45-46) (amino acid residues 1 to 21 of SEQ ID NO: 1) Is a signal peptide).
  • the amino acid sequence of L PGDS derived from mouse is shown in SEQ ID NO: 2 (Hoffinann A et al.,
  • the amino acid residues 1 to 24 of SEQ ID NO: 1 are signal peptides.
  • the nucleotide sequence of cDNA encoding L—PGDS is also known.
  • the nucleotide sequence of cDNA encoding human L-PGDS is shown in SEQ ID NO: 3 (nucleotide residues 1 to 63 of SEQ ID NO: 3 encode a signal peptide).
  • the nucleotide sequence of cDNA encoding mouse-derived LPG DS is shown in SEQ ID NO: 4 (nucleotide residues 1 to 72 of SEQ ID NO: 4 encode a signal peptide). Therefore, an expression vector that expresses recombinant L-PGDS can be constructed in an appropriate host system. Recombinant LPGDS can be produced by transforming host cells with the obtained expression vector and culturing the transformant under conditions suitable for expression of DNA encoding LPGDS.
  • An expression vector containing DNA encoding L-PGDS of the present invention can be constructed by methods known to those skilled in the art.
  • a vector suitable for L-PGDS expression would have a promoter for transcription initiation immediately upstream of the DNA insertion site.
  • Appropriate promoters are also known in the art and can be selected according to their functional properties in the host cell.
  • the promoter of the SV40 virus early gene, the peptide chain extension factor EF-1a promoter, the metamouthoneine gene promoter, the 13-cutin promoter, the CMV vinores promoter, etc. can be expressed in animal cell systems, Merase promoters and beta-galactosidase gene promoters can be used for expression in bacteria and E. coli. It is desirable that there is a transcription termination signal downstream of the insertion site of human hematopoietic PGD synthase DNA.
  • a selectable marker such as a drug resistance marker is present in the vector.
  • an expression vector containing a polynucleotide encoding L-PGDS Transformation may be performed simultaneously using a plasmid encoding drug resistance such as antibiotics separate from Kuta.
  • DNA encoding L-PGDS is inserted into an appropriate vector.
  • a suitable vector is selected from those known in the art in consideration of the promoter, transcription termination signal, selection marker and other conditions.
  • a DNA vector that can be used for the purpose of expressing this cDNA by inserting LPGDS cDNA and introducing it into cultured cells for example, pKCR, pEF-BOS, CDM8, pCEV4 In E. coli, such as papillomavirus DNA, pGEMEX, pUC, etc. can be mentioned.
  • the cells that can be used for the expression of LPGDS may be any cells that can replicate and can express DNA encoding LPGDS.
  • prokaryotic microorganisms such as E. coli
  • eukaryotic microorganisms such as S. cerevisiae
  • mammalian cells are used.
  • Tissue culture cells include avian or mammalian cells such as murine, rat and monkey cells. Selection and use methods of suitable host cell-vector systems are known to those skilled in the art, and a system suitable for the expression of cDNA encoding their internal L-PGDS can be arbitrarily selected.
  • a desired protein can be obtained by culturing the transformed cells according to a conventional method.
  • the medium used for the culture can be appropriately selected depending on the properties of the host. For example, when the host is E. coli, LB medium or TB medium is used. When the host is a mammalian cell, RPMI 1640 medium. Etc. can be used as appropriate.
  • the isolation and purification of the protein used in the present invention by the culture force obtained by this culture can be carried out by conventional methods.
  • the culture can be obtained by using various physical and chemical properties of the protein.
  • This processing operation can be performed. Specifically, treatment with a protein precipitating agent, ultrafiltration, high performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography and the like can be used alone or in combination.
  • the obtained protein solution can be powdered by lyophilization if necessary. In lyophilization, stabilizers such as sorbitol, mannitol, dextrose, maltose, and glycerol can be added.
  • a site-directed mutagenesis method is used to produce a mutant of LPGDS.
  • This method is well known, and kits for implementation (for example, Quick Change (registered trademark) Site-Directed Mutagenesis Kit manufactured by STRATAGENE) are commercially available.
  • a primer pair of about 30 to 40 bases containing mismaticodone at the target site was synthesized, and a wild-type plasmid was transformed into a saddle type using these primers.
  • the reaction mixture contains both the plasmid and the synthesized mutant plasmid. Treat the plasmid with Dpnl endonuclease, purify the mutant plasmid by decomposing and removing the plasmid.
  • Lactoglobulin is a protein that is contained in milk and belongs to the lipocalin family.
  • a substance that enhances the expression of PGDS is used as an active ingredient.
  • estrogen As a substance that enhances the expression of L PGDS, estrogen (Otsuki M et al. Specific regulation of lipocalin—type prostaglandin D synthase in mouse heart by estrogen receptor beta. Mol Endocrinol. 2003 Sep; 17 (9): 1844-55 Epub 2003 Jun 26.), Darciacorticoid (Garcia- Fernand ez LF et al. Dexamethasone induces lipocalin— type prostaglandin D synthase gene e xpression in mouse neuronal cells. J Neurochem. 2000 Aug; 75 (2): 460-70.
  • the active ingredient of the drug of the present invention is a protein
  • the protein is mixed with a carrier known per se It can be diluted and formulated as a liquid, for example.
  • the liquid preparation can be prepared using, for example, purified water, physiological saline, alcohols such as ethanol “propylene glycol” glycerin ”polyethylene glycol, and a solvent such as triacetin.
  • the liquid thus prepared can be used by diluting, for example, a lactate Ringer's solution, a maintenance solution comprising an infusion electrolyte solution, a postoperative recovery solution, a dehydration replenisher, a drip physiological saline solution, or the like.
  • buffering agents for pH adjustment for example, phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer, etc.
  • isotonic Agents eg sorbitol, glycerin, polyethylene glycol, propylene glycol, glucose, sodium chloride, etc.
  • Such preparations may further include pharmaceutically acceptable salts, sodium benzoyl alcohol, paraoxybenzoates, etc.
  • Benzyl alcohol «lachlormethaxinol, chlorcresol, phenethyl alcohol, sorbic acid or salts thereof, suitable antiseptic disinfectants such as thimerosal, chlorobutanol, humectants such as talc, emulsifiers such as polyethylene glycol monooleate, Auxiliary agents such as dispersants, stabilizers such as sulfites, bisulfites, metabisulfites It may be added. It is also preferable to administer as a suspending agent using gum arabic, kaolin, methylcellulose, sodium carboxymethylcellulose, etc. as a suspending agent.
  • Non-oral administration is preferred as the route of administration, for example, including intravenous administration, transcerebral spinal fluid administration, transarterial local administration by catheter, or surgical local administration.
  • the dosage of the active ingredient of the present invention is 0.01 to 20.0 mgZkgZ days, preferably 0.5 to 5 mgZkgZ days.
  • the active ingredient of the drug of the present invention is a low molecular compound such as estrogen or darcocorticoid
  • it can be mixed with a pharmaceutically acceptable carrier to produce the drug.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration. As a route of administration, parenteral administration and oral administration! /, Or deviation can be used. Powders, pills, capsules and tablets for oral administration are excipients such as ratatoses, glucose, sucrose, and maltol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc.
  • binders such as polybulal alcohol, hydroxypropylcellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin Can be manufactured.
  • the dose of the active ingredient of the present invention is 0.001 to 10 mgZkgZ day, preferably 0.01 to LmgZkgZ day.
  • the present invention uses LPGDS or a mutant gene thereof for gene therapy, and locally expresses LPGDS or a mutant in the brain, thereby producing ⁇
  • the gene of the present invention is incorporated into a vector and introduced into the brain.
  • Vectors used for gene therapy include retrovirus vectors, any virus vectors including adenovirus vectors, and ribosome vectors. More specifically, a viral vector is a gene in which a pathogenic gene is cut out using a part of the virus shell and a gene, and an L PG DS gene modified to be expressed in brain membrane or oligodendrocyte is inserted instead. is there.
  • a ribosome vector is a microsphere composed mainly of artificial lipid bilayers, which incorporates the L-PGDS gene modified so that it is expressed in brain membranes or oligodendrocytes (lipofection method).
  • a ribosome called phosphatidylserine is usually used. Instead of this negatively charged phospholipid, a cation called DOTMA (N— [1,1- (2,3 dioleoxy) pill] —N, N, N trimethylammo-um chloride) that makes it easier to produce more stable ribosomes Lipids are now on sale.
  • DOTMA N— [1,1- (2,3 dioleoxy) pill] —N, N, N trimethylammo-um chloride
  • the ribosome When positively charged, the ribosome is negatively charged, adsorbs the cells to the surface, and fuses with the cell membrane to introduce DNA into the cells.
  • This vector is administered by intravenous injection, arterial injection, intramuscular injection, intrathecal administration, or local administration.
  • the human L—PGDS gene from Ala21 to C-terminus was obtained by PCR amplification from the cDNA library, the signal peptide was deleted, and the expression vector pGEX—2T (Amersham) from the 67th to the 3 ′ end of the DNA sequence of SEQ ID NO: 3 was obtained. ⁇ Harumacia ⁇ Biotech) Then, Escherichia coli DH5a was transformed with the expression vector.
  • pGEX-2T is an expression vector for inserting the target gene into the downstream of the dartathione-S-tonsphrase (GST) gene and expressing the target protein as a GST fusion protein. Subsequently, the vector was transformed into Escherichia coli BL21 strain.
  • IPTG isopropyl 1-1-thio-13-D-galactoside
  • E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation, and then retained on a dartathione sepharose 4B column. The fusion protein retained on the column was extinguished with thrombin, and the L-PGDS fraction was eluted. Subsequently, it was purified by gel filtration column chromatography to obtain a human-derived lipocalin-type prostaglandin D synthase.
  • Example 1 In Example 1, except that human-derived PGDS-encoding cDNA was replaced with mouse-derived L PGDS-encoding cDNA (up to the 3rd end at the 70th force of the DNA sequence of SEQ ID NO: 4). Similarly, a mouse-derived lipocalin-type prostaglandin D synthase was obtained.
  • Cys65 and 167Ala expression vectors were prepared using the QuickChange Site-Directed Mutagenesis Kit.
  • E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation, and then retained on a dartathione sepharose 4B column. The fusion protein retained on the column was extinguished with thrombin, and the L-PGDS fraction was eluted. Subsequently, purification was performed by gel filtration column chromatography to obtain a human PGDS mutant (Alal. 3) in which 65 and 167 cysteines of human L-PGDS were substituted with alanine.
  • the Cys65Ser expression vector was prepared using the QuickChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE. Further, a pair of synthetic oligomers containing the mismatched codon at the target site using the obtained expression vector as a saddle type:
  • a Cyso5, 16 / 3 ⁇ 4 ⁇ expression vector was prepared using the Quick; hange; te-Directed Mutagenesis Kit.
  • An expression vector in which Cys65 and 167 were substituted with serine was transformed into Escherichia coli BL21 strain.
  • the Escherichia coli strain transformed with the expression plasmid was cultured in LB medium (containing 50 gZmL ampicillin) 37. After culturing in C, add IPTG (Isopropyl-1-thio- ⁇ -D-galatatoside) to a final concentration of 0.4 to 0.6 mM and add GST and L-PGDS fusion protein. Expression was induced and further cultured for 4-6 hours.
  • E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation, and then retained on a dartathione sepharose 4B column. The fusion protein retained on the column was extinguished with thrombin, and the L-PGDS fraction was eluted. Subsequently, it was purified by gel filtration column chromatography to obtain a mutant (Serl. 3) in which the 65th and 167th human L-PGDS were substituted with serine.
  • Example 4 except that the design of the synthetic oligomer was changed, a mutant (Ala 2.3.4) in which the 89, 167 and 186th cysteines of human L-PGDS were replaced with alanine was the same as in Example 3. Manufactured.
  • Example 6 a mutant (Ala2.3) in which murine L-PGDS 89 and 186th cysteines were substituted with alanine was produced in the same manner as in Example 6 except that the design of the synthetic oligomer was changed.
  • Example 6 as a synthetic oligomer
  • Example 6 In the same way as in Example 6, the following is a mutant (A1 a2. 3) in which mouse L-PGDS 89 and 186th systemine and 54th tributophane are replaced with alanine. Manufactured.
  • Example 6 as a synthetic oligomer
  • Mouse L A variant made of PGDS 89 and 186th cysteine and 111th histidine in alanine.
  • Example 6 as a synthetic oligomer
  • mutants were prepared by substituting the 89th and 186th systems of mouse L-PGDS and the 111th tributophane with alanine.
  • Example 6 except that the design of the synthetic oligomer was changed, in the same manner as in Example 6, a mutant was prepared in which mouse L-PGDS 89 and 186th cysteine were replaced with alanine and 43th tryptophan was replaced with riguranine. did.
  • Alzheimer's disease autopsy brain and Alzheimer's disease model mouse Tg2576 (Hsia 0 K et al., Orrelative memory deficits, Abeta elevation, ana amyloid plaques in tran sgenic mice. Science. 274: 99-102.1996; Hsiao KK et al. Age- related CNS disorder and early death in transgenic FVB / N mice overexpressing Alzheimer amyloid precur sor proteins. Neuron.l5: 1203— 18. 1995) (See Fig. 1 and Fig. 2) L-PGDS was identified to bind to the amyloid 'plaque site in both the autopsy brain of Alzheimer's disease patients and the Arno-Ima disease model mouse. [0036]
  • N D Below detection limit (> 1 ⁇ )
  • ThT is a fluorescent substance that specifically recognizes the cross-beta sheet structure, and it is known that the fluorescence intensity of ThT increases with the growth of amyloid fibrils in vitro. Therefore, a mixed solution of phosphate buffer (50 mM, pH 7.5) and NaCl (lOOmM) ⁇ A j8 1-40 (50 M) was prepared and kept at 37 ° C. At that time, a group in which the concentrations of L-PGDS analogues were adjusted and mixed was also set.
  • the aggregation reaction of human amyloid beta is promoted by adding fibrotic human amyloid beta as a seed.
  • 8 1 -40 peptide was analyzed by laser fluorescence microscopy and atomic force microscopy. (See Figures 4 and 6).
  • Aggregation reaction of human amyloid beta can also be promoted by adding gandarioside GM1 ribosome as a seed.
  • ThT assay the mutant of human L-PGDS (Ala2.3.4) also showed strong aggregation inhibitory action against GM1 liposome-dependent aggregation of A j81-40 peptide (see Fig. 7).
  • mutant mouse L-PGDS (Examples 3-11) prepared by recombinant technology
  • the surface plasmon resonance method (Biacore) described above was used to detect mutant L-PGDS for human amyloid 'beta 140 peptide.
  • the binding ability was analyzed (see Fig. 10). Dissociation constant As for Cys 89 ′ 185 Ala and His m Ala, the binding ability was 13.2 nM, for Cys 89, 185 Ala and Trp 54 Ala 4.
  • OnM Cys 89 ′ 185 Ala was 3.5 nM.
  • a positive relationship was observed between the enzyme activity of mutant L-PGDS and affinity for human amyloid 'beta 1-40 peptide (Table 2).
  • FIG. 11 is a photomicrograph of a brain section taken 1 hour after 50 ⁇ 1 intraventricular administration of 100 ⁇ piotin-labeled human amyloid beta 1-42 to wild-type mice and L-PGDS knockout mice, respectively.
  • L PGDS knockout mice Compared to wild-type mice (left), L PGDS knockout mice (right) showed aggregation of amyloid beta in the ventricle (upper center), and more intense deposition in the brain parenchyma (lower).
  • the color intensity shows that the clearance of amyloid beta, which is significantly 4.82 times stronger in L-PGDS knockout mice than in wild-type mice, is attenuated.

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Abstract

L'invention cherche à réguler et à améliorer les symptômes de la maladie d'Alzheimer. On administre un médicament contenant comme ingrédients actifs : 1) de la prostaglandine D synthétase de type lipocaline (L-PGDS) ; 2) une L-PGDS mutante ayant une substitution d'au moins un résidu de cystéine dans la L-PGDS par un autre résidu d'acide aminé ; et un polypeptide sélectionné dans le groupe constitué de : 3) la β-lactoglobuline ; et 4) une substance augmentant l'expression de la protéine L-PGDS sélectionnée dans le groupe constitué d'un estrogène, d'un glucocorticoïde, de l'interleukine-1β et d'une hormone thyroïdienne. Ce polypeptide se lie au peptide β-amyloïde et régule l'interaction entre les composants lipides présents sur la surface de la membrane des cellules du cerveau et le peptide β-amyloïde, inhibant de cette manière l'agglomération.
PCT/JP2005/013658 2004-07-27 2005-07-26 Protéine inhibant l'agglomération de peptides amyloïdes et l'effet de celle-ci WO2006011485A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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JP2007320934A (ja) * 2006-06-05 2007-12-13 Japan Health Science Foundation アミロイドβオリゴマー並びにその製造方法及び使用方法
EP3733205A4 (fr) * 2017-12-28 2021-12-08 Hyogo College Of Medicine Agent d'accélération de la production de prostaglandine d2 synthase de type lipocaline

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CN102770764B (zh) 2009-12-07 2016-04-20 皮里斯股份公司 对给定靶标具有亲和力的人脂质运载蛋白2（Lcn2，hNGAL）的突变蛋白
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BR112016027024A2 (pt) 2014-05-22 2017-10-31 Pieris Pharmaceuticals Gmbh polipeptídeos de ligação específica e seus usos
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