WO2003031615A1 - Inhibiteur de l'acide lysophosphatidique synthase - Google Patents

Inhibiteur de l'acide lysophosphatidique synthase Download PDF

Info

Publication number
WO2003031615A1
WO2003031615A1 PCT/JP2002/010342 JP0210342W WO03031615A1 WO 2003031615 A1 WO2003031615 A1 WO 2003031615A1 JP 0210342 W JP0210342 W JP 0210342W WO 03031615 A1 WO03031615 A1 WO 03031615A1
Authority
WO
WIPO (PCT)
Prior art keywords
lpld
lysophosphatidic acid
antibody
activity
acid synthase
Prior art date
Application number
PCT/JP2002/010342
Other languages
English (en)
Japanese (ja)
Inventor
Akira Tokumura
Eiji Majima
Original Assignee
Apro Life Science Institute, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Apro Life Science Institute, Inc. filed Critical Apro Life Science Institute, Inc.
Priority to JP2003534585A priority Critical patent/JPWO2003031615A1/ja
Publication of WO2003031615A1 publication Critical patent/WO2003031615A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • the present invention relates to a lysophosphatidic acid synthase (hereinafter referred to as LPLD)
  • LPLD inhibitors Substances that inhibit phosphatidic acid-producing ability (hereinafter referred to as LPLD inhibitors), screening methods for LPLD inhibitors, and lysophosphatidic acid
  • the present invention relates to an agent for preventing or treating a disease caused by the physiological activity of LPA (hereinafter referred to as LPA), a method for diagnosing the disease and the like.
  • LPA LPA
  • lipid mediators such as arachidonic acid derivatives, platelet activating factor, LPA, sphingosine-1_phosphate, and anandamide are produced.
  • LPA one of the lipid medias, has been found to have various physiological activities (Tokumura, A. et. Al., Prog. Lipid Res. 34, P151-184). (1995), Moolenaar,. H. et al. Curr. Opinion Cell Biol. 9, pl68-173 (1997)).
  • LPA is the first intermediate product of the de novo biosynthesis system of glycerol phospholipids, and the acyl group of acyl-CoA is placed at position 1 of G3P (sn-glycerol-3-phospate) by the acyltransferase. Made with additions. This reaction is the rate-limiting reaction of the phospholipid biosynthesis system, and the reaction that transfers the acyl group to the next 2-position is faster.
  • LPA phosphatidic acid
  • PA phosphatidic acid
  • the present invention relates to a method for diagnosing a disease caused by the physiological activity of LPA, a substance useful for reducing symptoms or preventing or treating a disease caused by the physiological activity of LPA, a method for screening the substance, and a medicament containing the substance
  • the purpose is to provide.
  • the present inventors have conducted intensive studies to elucidate the biosynthetic pathway of LPA, and succeeded in isolating and purifying LP LD.
  • isolating and purifying LPLD it becomes possible to produce a substance that inhibits the ability of LPLD to produce LPA.
  • Such LPLD inhibitor can suppress the expression of undesired physiological activities by LPA as described above, for example, promotion of invasion of cancer cells, promotion of cancer cell proliferation, and promotion of mobility by LPA. .
  • the LPLD inhibitor can be used as a pharmaceutically active ingredient such as an anticancer agent.
  • a medicament containing LPLD can be used as a preventive or therapeutic agent for the above-mentioned symptoms or diseases.
  • the amino acid sequence of the isolated and purified LP LD was determined and found to be Autotaxin (Mary L. Stracke, et. Al., J. Biol. Chem. Vol.267, No., pp2524-2529 (1992)). It was found to have the same structure as a known cell motility promoting protein. However, it is not known at all that Autotaxin is an LPA biosynthetic enzyme. After obtaining such new findings, the present inventors have further studied and completed the present invention.
  • Lysophosphatidic acid synthase lysophosphatidic acid synthase characterized by reacting lysophospholipid with lysophosphatidic acid synthase in the presence of a test compound, and measuring the lysophosphatidic acid synthase-producing ability of lysophosphatidic acid synthase.
  • (6) a medicament comprising the substance according to (1), (2) or (4),
  • the medicament according to the above (6) which is a therapeutic or preventive drug for cancer, male reproductive system disease, female reproductive system disease or arteriosclerosis.
  • Cancer, male reproductive system disease, female reproductive system disease, arteriosclerosis or preeclampsia characterized by measuring the activity of lysophosphatidic acid synthase or using an antibody of lysophosphatidic acid synthase Diagnostic method, (9) A kit for diagnosing cancer, male reproductive system disease, female reproductive system disease, atherosclerosis or preeclampsia, which comprises an antibody of (a) lysophospholipid or (b) lysophosphatidic acid synthase ,
  • a lysophosphatidic acid production inhibitor of lysophosphatidic acid synthase which comprises a nucleotide or an analog thereof
  • FIG. 1 shows a comparison between the determined amino acid sequence of LPLD and the amino acid sequence of human autotaxin.
  • the amino acid sequence of human autotaxin (Kawagoe H. et al, Genomics 30, 380-384 (1995)) is represented by one letter code.
  • Four peptide fragments corresponding to the sequence of human autotaxin obtained by tandem mass analysis of purified human plasma LP LD are indicated by double underlining.
  • the two N-terminal sequences of the purified LPLD determined from the analysis of the protein sequencer are indicated by single underlines.
  • a region shaded with the same sequence indicates a peptide sequence synthesized for preparing an anti-LPLD antibody.
  • FIG. 2 shows the results of Western blot of LP LD protein using an anti-LP LD antibody.
  • the purified human plasma LPLD fraction was separated on a 7.5% gel by SDS-PAGE in the absence of dithiothreitol, and then transferred to a PVDF membrane.
  • a band could be detected at a position of about 110 kDa. The position of this band is consistent with the molecular weight of the purified LPLD protein.
  • Figure 3 shows the dissolution of LP LD protein by anion exchange chromatography. The outgoing curve is shown.
  • LPLD activity was measured after a 1 hour incubation using 14: 0-LPC at a final concentration of 0.15 mM as substrate.
  • FIG. 4 shows the results of SDS-PAGE of the purified LPLD protein.
  • FIG. 5 shows the substrate specificity of human plasma LPLD.
  • A Time-dependent changes in LPLD activity and nucleotide phosphodiesterase (PDE) activity in purified plasma LPLD were measured in the presence or absence of 30 M Co 2+ .
  • PDE nucleotide phosphodiesterase
  • the present invention provides a substance that inhibits the LPA-producing ability of LPLD, that is, an LPLD inhibitor.
  • LPA includes a compound having a fatty acid at the 1- or 2-position of the glycerol skeleton and a structure in which a phosphate group is bonded at the 3-position, but the phosphate at the 3-position is cyclic. Include cyclic phosphatidic acid that also binds to position 2 Can be.
  • Examples of the compound having a fatty acid at position 1 of the glycerol skeleton include 1-acyl lysophosphatidic acid, 1-alkyl lysophosphatidic acid, 1-alkenyl lysophosphatidic acid, and the like.
  • Examples of the compound having a fatty acid at the 2-position include 2-acyl lysophosphatidic acid.
  • Fatty acids may be straight-chain or branched, and may be saturated or unsaturated.
  • the fatty acid preferably has about 12 to 24 carbon atoms.
  • the LPLD inhibitor according to the present invention among the above LPAs, those which inhibit the production of lysophosphatidic acid having an unsaturated fatty acid having about 12 to 24 carbon atoms are preferable, and those having 12 to 24 carbon atoms are preferable. Those that inhibit the production of 1-acyl lysophosphatidic acid having a degree of linear unsaturated fatty acids are more preferred.
  • the LPLD inhibitor according to the present invention may be any substance, and can be easily screened by the following method.
  • nucleotides or analogs thereof include nucleotides or analogs thereof.
  • the nucleotide is not particularly limited, and examples thereof include substances in which the sugar moiety of the nucleoside is a phosphate ester.
  • nucleoside residues in nucleotides include adenosine residues, guanosine residues, cytidine residues, liponucleoside residues such as lysine residues or lipothymidine residues; deoxyadenosine residues, deoxyguanosine residues, and deoxycytidine.
  • deoxyperidine residues such as deoxyperidine residues or deoxythymidine residues
  • dideoxyadenosine residues dideoxyguanosine residues
  • dideoxycytidine residues dideoxyperidine residues or didedeoxydysine residues
  • dideoxyliponucleoside residues such as oxythymidine residues.
  • the nucleotides include nucleoside-5'-phosphate, nucleoside-15'-diphosphate, nucleoside-15'-triphosphate, nucleoside 3'-monophosphate or liponucleoside-2 '-— Phosphoric acid and the like.
  • the nucleotide analog is not particularly limited and may be a known analog. Good.
  • dNTP N represents A, T, G or C.
  • [1-thio] triphosphate (or monothiotriphosphate) analog 7-Deaza-dGTP, 7-Deaz a-dATP, d ITP, hydroxymethyl dUTP, 2Me—AMP, 2Me_ADP, 2Me_ATP, lMe—GMP, 1Me-GDP, lMe-GTP, 5Me—CMP, 5Me—CDP, 5Me-CTP, 5MeO—CMP, 5MeO— CDP, 5 MeO—CTP or p—nitofene 5′—TMP.
  • a nucleotide or an analog thereof is ATP or p-nitrophen-5u-TMP as a particularly preferred example.
  • another preferred example of the LPLD inhibitor according to the present invention includes an antibody against LPLD or a partial peptide of LPLD (hereinafter, referred to as LPLD antibody).
  • the LPLD antibody may be, for example, a polyclonal antibody or a monoclonal antibody.
  • Such an LPLD antibody can be produced according to a known antibody production method using LPLD or a partial peptide of LPLD as an antigen.
  • the LPLD antibody is described in, for example, "Basic Experimental Methods for Proteins and Enzymes, Revised 2nd Edition (Takeo Horio-Edited by Nanedo, 1994)” or “Method in Enzymology vol.182 published by ACADEMIC PRESS, INC. 1990 "and the like.
  • a monoclonal antibody (hereinafter, referred to as an LPLD monoclonal antibody) can be produced, for example, according to the following method.
  • LPLD or a partial peptide of LPLD is administered to a warm-blooded animal together with a carrier and a diluent.
  • complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability at the time of antigen administration to the animal.
  • the above administration is usually once every 2 to 6 weeks, about 2 to 10 times in total Done once.
  • Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens. Mouse and rat mosquito S are preferably used.
  • a warm-blooded animal immunized with the antigen for example, an individual with a recognized antibody titer from a mouse is selected, and about 2 to 5 days after the final immunization, the spleen or lymph node is collected and collected.
  • a monoclonal antibody-producing hybridoma By hybridizing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared.
  • the measurement of the antibody titer in the antiserum may be performed by a known method.
  • the reaction can be performed by reacting labeled LPD or a partial peptide of LPLD with antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Köhler and Milstein (Nature, 256, 495 (1975)).
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • PEG polyethylene glycol
  • myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, and the like, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is about 10 to 80%.
  • Cell fusion can be carried out efficiently by incubating at about 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes.
  • a hybridoma culture supernatant to a solid phase such as a microplate on which an antigen is directly or adsorbed together with a carrier, Then, an anti-immunoglobulin antibody or protein A labeled with a radioactive substance or an enzyme is added, and the LPLD monoclonal antibody bound to the solid phase is detected.
  • the LPLD monoclonal antibody can be selected according to a known method or a method analogous thereto. It is usually performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminobuterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow hybridomas. For example :! RPMI 1640 medium containing about 20%, preferably about 10-20% fetal bovine serum, G1T medium containing about 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or A serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the culture temperature is usually about 20 to 40, preferably about 37 ° C.
  • the culture time is usually about 5 days to 3 weeks, preferably about 1 week to 2 weeks.
  • the cultivation is usually performed under about 5% carbon dioxide.
  • the LPLD monoclonal antibody is separated and purified according to the immunoglobulin separation and purification method.
  • Known methods for separating and purifying immunoglobulins include, for example, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption-desorption method using ion exchanger (for example, DEAE), ultracentrifugation method, gel Examples of the method include a filtration method and a specific purification method in which an antigen-bound solid phase is collected and the bond is dissociated to obtain an antibody.
  • a polyclonal antibody (hereinafter referred to as an LPLD polyclonal antibody) can be prepared by the following method. That is, the above-mentioned antigen (LPLD or LPLD partial peptide) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. From the LPLD of the present invention or a partial peptide of LPLD of the present invention. The antibody can be produced by separating and purifying the antibody.
  • any antibody can be cross-linked at any ratio as long as the antibody can be efficiently produced.
  • a method in which a carrier protein such as serum serum albumin, thyroglobulin, and hemocyanin is coupled in a weight ratio of about 0.1 to 20 and preferably about 1 to 5 with respect to 1 hapten. Is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • condensing agent there can be used, for example, daltaraldehyde dicarbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like.
  • the above-described complex of the antigen and the carrier protein or the antigen itself is administered to a site capable of producing an antibody against a warm blood animal, itself or together with a carrier or a diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the LPLD polyclonal antibody can be collected from the blood or ascites of a warm-blooded animal immunized by the above method, preferably from the blood.
  • the measurement of the LPLD polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above.
  • the separation and purification of the polyclonal antibody can be performed according to the method for separating and purifying immunoglobulin in the same manner as the above-described separation and purification of the monoclonal antibody.
  • the polyclonal antibody can be prepared by a method such as the PVP method, the Darbaru aldehyde method, and the MBS method.
  • LPLD as an antigen used in the production of LPLD antibodies can be isolated and purified from human plasma as described in the Examples below. LPLD may be isolated and purified not only from human plasma but also from extracts or cultures of various organisms. Further, LPLD can be obtained by a method for expressing a protein by genetic recombination. LPLD obtained by genetic recombination techniques may have mutations such as substitutions, deletions or additions in its amino acid sequence.
  • the number of mutation sites is one or several, and preferably about 1 to 4.
  • a method for producing LPLD by a genetic recombination technique may be in accordance with a conventional method since it is well established in the technical field. Specifically, as the above method, first, DNA encoding LPLD is inserted into a known expression vector, a host cell is transformed with the expression vector, and the transformant is cultured. And a method of extracting and purifying LPLD from a culture.
  • DNA encoding LPLD can be easily obtained from known nucleotide sequence information.
  • the nucleotide sequence information of the DNA encoding LPLD can be found in Hiroyuki Kawagoe, et.al., Submitted (04- FEB-1995) to the DDBJ / EMBL / GenBank databases. Or Hiroyuki Kawagoe, et.al., Genomics 30 (2) , p380-384 (1995) as a cDNA for phosphodiesterase Ia from human (Homo sapiens).
  • the LPLD-encoding DNA does not need to be exactly the same as the known nucleotide sequence information, but may have any homology.
  • the DNA that encodes LPLD is preferably a nucleotide sequence that hybridizes with a nucleotide sequence of known nucleotide sequence information under highly stringent conditions, and is about 70% or more, preferably about 80% or more, More preferably, DNA containing a nucleotide sequence having a homology of about 90% or more, and most preferably about 95% or more, is more preferable.
  • the high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° (:, preferably, about 60 to 70 ° C.). The conditions at 65 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • the DNA encoding LPLD can be obtained by, for example, chemical synthesis using known methods from the above-mentioned known base sequence information.
  • Examples of the chemical synthesis method include a method of chemically synthesizing with a DNA synthesizer such as a DNA synthesizer model 392 (manufactured by Perkin-Elma Inc.) using a phosphoramidite method.
  • a primer was prepared based on the base sequences at the 5 ′ end and 3 ′ end of the base sequence, and cDNA synthesized from mRNA contained in tissues or cells of various organisms or cDNA selected from a cDNA library was prepared.
  • the above DNA can also be obtained by amplifying the DNA using the PCR method (PCR Protocols, Academic Press (1990)) with type III. Furthermore, based on the known nucleotide sequence information, a DNA or polynucleotide obtained by chemically synthesizing the full length or a part of the DNA or polynucleotide is used as a probe to synthesize a cDNA or cDNA library synthesized from mRNA contained in tissues or cells of various organisms. The above DNA can also be obtained by performing colony hybridization or plaque hybridization (molecular cloning, second edition) on the DNA.
  • the amino acid sequence information of LPLD was isolated from human (Homo sapiens) by Lee, HY et. Al., Biochem. Biophys. Res. Coramun., 218 (3), p714-719 (1996). Described as autotaxin.
  • the amino acid sequence information can be obtained from Hiroyuki Kawagoe, et.al., Submitted (04-FEB-1995) to the thigh J / EMBL / GenBank databases.
  • a method known per se may be used. Specifically, for example, a method of amplifying a target DNA from the cDNA library or the like by PCR using a synthetic DNA primer having a partial base sequence of DNA encoding a known amino acid sequence, Or a method in which DNA incorporated in an appropriate vector is hybridized with a DNA fragment (probe) labeled with a DNA fragment or a synthetic DNA encoding a part or all of the known amino acid sequence described above. And the like.
  • the DNA encoding the LPLD described above is inserted into a known expression vector.
  • the expression vector examples include plasmids derived from Escherichia coli, Bacillus subtilis or yeast, bacteriophages such as ⁇ phage, and animal viruses such as retrovirus, vaccinia virus, and baculovirus.
  • the expression vector those having a promoter and optionally containing an enhancer, a splicing signal, a polyaddition signal, a selection marker, an SV40 replication origin, and the like can be used.
  • a host cell is transformed with the expression vector.
  • host cells for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • the transformed host cells are then cultured, and LPLD is obtained from the culture. Transformation methods and conditions for culturing host cells vary depending on the type of host cells. For example, Molecular Cloning 2nd Edition
  • the partial peptide of LPLD as an antigen used in the production of an antibody may be any part of LPLD obtained as described above. Reduction conditions SDS-PAGE of LP LD shows two protein bands of about 75 kDa and about 30 kDa. Therefore, the LPLD of about II OkDa is constituted by the cross-linking of these two peptides via a disulfide bridge. Therefore, in the present invention, the antibody according to the present invention can be prepared by using the two peptides of about 75 kDa or about 30 kDa as antigens. Further, mutations such as substitution, deletion, and addition may occur in the amino acid sequence. In that case, the number of mutation sites is preferably one or several, and preferably about 1 to 4. LPLD partial peptides can be chemically synthesized.
  • the LPLD inhibitor according to the present invention may be an antibody against a precursor of LPLD. That is, the antigen used in the production of the above antibody may be a precursor of LPLD.
  • LPLD antibody also includes an antibody against a precursor of LPLD.
  • LPLD is generated by various proteolytic breaks in precursor proteins with putative transmembrane domains. One of the proteolytic cleavage sites is located between arginine 35 and alanine 36, and the N-terminal portion containing the transmembrane domain is cleaved off. Using the LPLD antibody produced as described above, the amount of LPLD in the test substance can be measured.
  • Measuring the amount of LPLD enables diagnosis of a condition or disease caused by the physiological activity of LPA. That is, if the amount of LPLD is significantly higher than usual, it is highly possible that a symptom or disease caused by the physiological activity of LPA has developed, leading to early detection of the symptom or disease. Symptoms or diseases caused by the physiological activity of LPA will be described later. Measuring the amount of LPLD also allows for the diagnosis of a condition or disease caused by a higher or lower than normal LPLD in vivo. For example, in preeclampsia, the biomass of LPLD (LPA-SE) Significantly less than usual. Therefore, the amount of LP LD (L PA-SE) in the test substance collected from the living body is measured, and if the amount is significantly lower than usual, it can be diagnosed that the possibility of preeclampsia is high.
  • L PA-SE the biomass of LPLD
  • the present invention provides (a) a symptom or disease caused by the physiological activity of LPA, or (b) a symptom or disease caused by the fact that the amount of LPLD in the living body is higher than normal or lower than normal.
  • a kit for diagnosing the above-mentioned condition or disease, which comprises the LPLD antibody which comprises the LPLD antibody.
  • the method for measuring LP LD using the LP LD antibody used in the diagnostic method and the diagnostic kit according to the present invention includes an antigen in a test substance, that is, an antibody, an antigen or an antibody-antigen complex corresponding to the amount of LP LD.
  • Any method can be used as long as it is a method for detecting the amount of the compound by chemical or physical means and calculating the amount from a standard curve prepared using a standard solution containing a known amount of the antigen.
  • nephelometry, a competitive method, an immunometric method, and a sandwich method are preferably used. Among them, it is particularly preferable to use the sandwich method described below in terms of sensitivity and specificity.
  • the antibody used may be the antibody molecule itself, or the F (ab ') 2, Fab' or Fab fraction of the antibody molecule.
  • the LPLD antibody used here is LPLD isolated or produced from the same species as the species from which the test substance to be measured was collected, or LPLD produced as described above using antigens as partial antigens. Antibodies are preferred.
  • Examples of the labeling agent used in the above measurement method using a labeling substance include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance.
  • Examples of the radioisotope include 125 I, 3 H, and 14 C.
  • the enzyme is preferably a stable enzyme having a large specific activity, and examples thereof include ⁇ -galactosidase,] 3-darcosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase.
  • Examples of the fluorescent substance include fluorescamine and fluorescein isothiosinate. Luminols, luminol derivatives, Luciferin, lucigenin and the like.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test substance is reacted with the insolubilized LPLD antibody (primary reaction), and further reacted with the labeled LPLD antibody (secondary reaction), and then the activity of the labeling agent on the insolubilized carrier is measured.
  • LPLD in the test substance can be determined.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the method for diagnosing (a) a symptom or disease caused by the physiological activity of LPA, or (b) a symptom or disease caused by the fact that the amount of L PLD in the living body is higher or lower than usual. Can also be performed by measuring the activity of LPLD.
  • the activity of LPLD can be measured, for example, by measuring the LPA-producing ability of LPLD.
  • a known method may be used as a method for measuring the LPA-producing ability of LPLD.
  • it can be measured by using lysophospholipid as a substrate and measuring the amount of a reaction product formed by the decomposition of the substrate by the catalytic action of LPLD.
  • a method in which lysophosphatidylcholine is reacted with LPLD, and the produced choline or LPA is measured by a known method to measure the LPA-producing ability of LPLD is suitably used.
  • LPA can be measured by using a radiolabel, a colored substance or a fluorescent substance.
  • Labeled LPA generated from zophosphatidylcholine can be quantified by radioactivity measurement, colorimetry or fluorescence measurement.
  • the radioisotope colored substance or fluorescent substance used as the label
  • the above-mentioned known substances may be used.
  • the method of measuring choline include a method of reacting choline with choline oxidase and measuring the amount of generated hydrogen peroxide with peroxidase.
  • a test substance such as blood, collected from a living body is diluted with a 2 OmM Tris-HCl buffer (pH 8.0) or the like, if desired.
  • the lysophospholipid solution and the test substance solution are incubated at a temperature of about 37 ° C. for about 1 hour.
  • a peroxidase such as HPPA (3- (4-hydroxyphenyl) propionic acid) or HRP (horseradish peroxidase), which is a fluorescent substance, and a choline oxidase are mixed in the above mixture. Incubate this solution at about 37 ° C for about 15 minutes.
  • the fluorescence intensity of the fluorescent substance produced by the above reaction excitation wavelength: 320 nm, emission wavelength: 404 nm
  • the activity of LPLD in the test substance can be measured.
  • the present invention provides a diagnostic kit for the above-mentioned condition or disease, which comprises a lysophospholipid.
  • a lysophospholipid labeled with a radioisotope, a colored substance or a fluorescent substance is used as the lysophospholipid.
  • the activity of LPLD in the test substance decomposes the labeled lysophospholipid as a substrate, and the activity of LPLD is measured by measuring the radioactivity, colorimetry or fluorescence of the resulting labeled LPA. Can be.
  • lysophospholipid is lysophosphatidylcholine
  • another preferred embodiment is that choline oxidase and peroxidase are further combined.
  • a lysophospholipid is preferably used.
  • the present invention also provides a method of screening for an LPLD inhibitor.
  • the screening method is not particularly limited as long as it is a method capable of measuring the LPA production ability of LPLD.
  • the screening method includes, for example, reacting lysophospholipid with LPLD in the presence of a test compound, measuring LPA-producing ability of LPLD, and screening for a substance that reduces LPA-producing ability. Method.
  • the test compound used in the screening method according to the present invention includes, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • the test compound may be a novel compound or a known compound.
  • the test compound may be a single substance or a mixture.
  • the lysophospholipid used in the above-mentioned screening method is not particularly limited, and examples thereof include lysophosphatidylcholine.
  • a known method may be used as a method for measuring the productivity, and it is particularly preferable to use the method described above.
  • the LPLD used here those isolated and purified from extracts or cultures of various organisms may be used.
  • the present invention also provides a screening kit suitably used in the above-mentioned screening method, comprising a lysophospholipid and LPLD.
  • a screening kit suitably used in the above-mentioned screening method, comprising a lysophospholipid and LPLD.
  • LPLD is further contained in the diagnostic kit containing lysophospholipid described above.
  • the present invention provides a medicine containing an LPLD inhibitor.
  • the LPLD inhibitor may be administered as it is, or the LPLD inhibitor may be administered as a solution or suspension, and the solution or suspension may be administered as a granular or powdery dried product.
  • a pharmaceutical composition can be produced according to a method which is well known or commonly used in the field of pharmaceuticals.
  • the medicament according to the present invention may have any dosage form.
  • formulations suitable for oral administration include, for example, tablets, granules, fine granules, powders, syrups, solutions, capsules or suspensions.
  • Formulations suitable for parenteral administration include, for example, injections, infusions, inhalants, sprays, suppositories, transdermal absorption agents, transmucosal absorption agents and the like.
  • excipients such as lactose, glucose, sucrose and mannitol
  • disintegrants such as starch and sodium alginate
  • Lubricating agents such as magnesium stearate and talc
  • binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin
  • surfactants such as fatty acid esters
  • plasticizers such as glycerin; it can.
  • a physiologically acceptable carrier is selected from the group consisting of an LPLD inhibitor as an active ingredient, a diluent, a fragrance, a preservative, an excipient, a binder, a stabilizer and the like. Dissolve or suspend the above pharmaceutical additives Thus, a formulation suitable for parenteral administration can be prepared.
  • physiologically acceptable carrier examples include, when the preparation is an injection, a salt solution such as physiological saline, a glucose solution, a mixture of a salt solution and a glucose solution, and other D-sorbitol, D-mannitol, and the like.
  • Aqueous medium Such aqueous media may contain suitable solubilizing agents, for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), non-ionic surfactants (eg, Polysorbate 80 TM). , HCO-50, etc.) may be added.
  • an oily liquid such as sesame oil and soybean oil can be used as sesame oil and soybean oil can be used.
  • Benzyl benzoate, benzyl alcohol and the like may be added as a solubilizing agent to such an oily liquid.
  • the preparation is a suppository for enteral administration, for example, cocoa butter, hydrogenated fat, hydrogenated carboxylic acid and the like are mentioned as the carrier.
  • additives for parenteral administration include, for example, buffering agents such as phosphate buffer and sodium acetate buffer; for example, Shiridani benzalkonium.
  • soothing agents such as proforce hydrochloride; stabilizers such as human serum albumin and polyethylene glycol; and preservatives such as benzyl alcohol and phenol.
  • the medicament according to the present invention is safe and has low toxicity, it can be used, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgs, pigs, pigs, dogs, cats, dogs, Monkeys, etc.).
  • warm-blooded animals eg, rats, mice, guinea pigs, egrets, birds, higgs, pigs, pigs, dogs, cats, dogs, Monkeys, etc.
  • the dose and frequency of administration of the medicament of the present invention are not particularly limited, and are appropriately selected according to various conditions such as the type of the disease state to be treated, the administration route, the age and weight of the patient, the symptoms, and the severity of the disease. It is possible to For example, in the case of systemic administration by intravenous injection, it is preferable to administer about 0.01 to 1 mg / kg, especially about 0.1 mg / kg per adult day, and in the case of oral administration, about 0.1-1 OmgZkg, especially about lmgZkg However, the dosage is not limited to this particular example.
  • the medicament according to the present invention can be used for preventing or treating a symptom or disease caused by the physiological activity of LPA.
  • Symptoms resulting from the physiological activity of LPA include, specifically, promotion of cancer cell invasion, promotion of cancer cell proliferation, and promotion of mobility. That is, the medicament according to the present invention can be used as a prophylactic or therapeutic agent for cancer by inhibiting invasion of cancer cells, cancer cell proliferation and migration, and the like.
  • the cancer to which the medicament according to the present invention can be applied is not particularly limited.
  • the medicament according to the present invention is preferably used for the prevention or treatment of gynecological cancer such as prostate cancer, ovarian cancer, uterine cancer (endometrial cancer, cervical cancer) or peritoneal
  • Specific examples of the symptoms caused by the physiological activity of LPA include male reproductive system diseases. That is, the medicament according to the present invention can be used as an agent for preventing or treating male reproductive system diseases.
  • the male reproductive system disease include prostate hypertrophy, prostate disease such as prostate cancer or prostatitis, and the like.
  • Specific examples of the symptoms caused by the physiological activity of LPA include female reproductive system diseases. That is, the medicament according to the present invention can be used as a preventive or therapeutic agent for female reproductive system diseases.
  • Examples of the female reproductive system disease include not only the above-mentioned gynecological cancer but also endometriosis.
  • the medicament according to the invention can also be used.
  • the medicament according to the present invention can also be used as an agent for preventing or treating arteriosclerosis, particularly atherosclerosis.
  • LPA induces the attachment of mononuclear cells to vascular endothelial cells and promotes the proliferation and dedifferentiation of smooth muscle cells.
  • LPA inhibitor contained in the medicament of the present invention suppresses LPA production, This is because the action is suppressed. This is suggested by an increase in serum LPLD activity in the egret fed a high cholesterol diet.
  • the present invention can also provide a preventive or therapeutic agent for a symptom or disease caused by an in vivo amount of LPLD that is higher than normal or lower than normal, which comprises LPLD. .
  • LPLD in vivo amount of LPLD that is higher than normal or lower than normal
  • the biomass of LP LD is significantly lower than normal. Therefore, a medicament containing LPLD is useful as an agent for preventing or treating preeclampsia.
  • a medicament containing an LPLD inhibitor can be used as a prophylactic / therapeutic agent for premature labor.
  • LPLD activity decreases after childbirth as described above, it can be used as a prophylactic / therapeutic agent for various symptoms caused by the fact that LPLD activity does not decrease after childbirth.
  • LPLD increases during pregnancy, It can be used as a preventive or remedy for various symptoms associated with non-increased LD, for example, preeclampsia.
  • a drug containing LPLD can be used as a drug for promoting childbirth or as an agent for promoting labor.
  • LPLD was purified from human plasma as follows.
  • ammonium sulfate 153.7 g (30% saturated ammonium sulfate) of ammonium sulfate (hereinafter referred to as ammonium sulfate) was added to 937 ml of human plasma, left to stand on ice for 1 hour, and centrifuged at 8,000 ⁇ for 40 minutes at 4 ° C. Then, the supernatant was taken, 172 g of ammonium sulfate (60% saturated ammonium sulfate) was added to the supernatant (950 ml), the mixture was allowed to stand in ice for 1 hour, and then centrifuged at 8,000 rpr at 4 ° C for 40 minutes.
  • the LPLD activity of each fraction was measured as follows. First, the final concentration was added to saline containing 0.25 wt% BSA. 100: 1 of each fraction was mixed with 14: 1 LPC (lysophosphatidylcholine) 501 dissolved at 0.15 mM and incubated at 37 ° C for 1 hour (Primary Atsushi). At this time, choline is produced from LPC according to the amount of LPLD contained in each fraction.
  • LPC lysophosphatidylcholine
  • Fractions showing LPDL activity in this activity measurement were collected.
  • the total liquid volume of the collected fractions was 320 ml.
  • Buffer; A 10 mM sodium phosphate (pH 6.8)
  • the LP LD activity of each fraction was measured in the same manner as described above. Fractions showing LPLD activity in this activity measurement were collected. The total volume of the collected fractions was 194 ml.
  • Fractions showing LPDL activity in this activity measurement were collected. The total volume of the collected fractions was 120 ml.
  • Fractions showing LPDL activity in this activity measurement were collected.
  • the total liquid volume of the collected fractions was 180 ml.
  • Fractions showing LPDL activity in this activity measurement were collected.
  • the total liquid volume of the collected fractions was 24 ml.
  • the solution recovered as above was concentrated by ultrafiltration, and then subjected to gel filtration chromatography under the following conditions.
  • LPLD LPLD was purified from human plasma. The purified LPLD showed a molecular weight of about 110,000 by polyacrylamide electrophoresis.
  • LPLD antibody was performed according to the method described in “Basic Experimental Methods for Proteins and Enzymes, Revised 2nd Edition (Takeo Horio, published by Nanedo, 1994)”, pp. 494-498. Make it.
  • the human plasma LPLD fraction purified in Example 1 above was subjected to polyacrylamide electrophoresis (SDS-PAGE) on a 7.5% gel in the absence of dithiothreitol. A 110 kDa band was excised from this gel and treated with trypsin.
  • the protein after this treatment was directly connected to a tandem mass spectrometer (Q-Tof2) equipped with a nanoelectrospray ionization source, and a nanoscale high-performance liquid chromatograph connected to a C18 column (0.1X50 mm). The cation was analyzed by lithography, and the cation tandem mass spectrum was measured. The tandem mass spectrum was searched overnight using the Mascot Search Program.
  • PVDF The membrane was electrically (200 mA) transferred. After the membrane was stained with Coomassie brilliant blue, a band of 110 kDa was cut out, and the N-terminal amino acid sequence of the purified LP LD protein was analyzed using a protein sequencer. As a result, two types of N-terminal sequences corresponding to the partial sequences of human autoxin and phosphodiesterase I were determined. Figure 1 shows the results.
  • LPLD mocyanin
  • An equal volume of 1 mg was mixed with Freund's complete adjuvant and dispersed and administered subcutaneously to the back and thigh muscles of the egret.
  • 1 mg of KLH-peptide was mixed with an equal amount of incomplete adjuvant in Freund, and dispersed and administered subcutaneously to the back and thigh muscles of egrets for a total of 5 immunizations .
  • ammonium sulfate a saturated solution of ammonium sulfate (hereinafter referred to as ammonium sulfate) is added to 50 ml of serum, and the mixture is allowed to stand at 2 to 10 ° C. After the centrifugation, the mixture was centrifuged at 3000 rpm at 4 ° C for 45 minutes. The precipitate after centrifugation was dissolved by adding 15 ml of distilled water to the precipitate, and the obtained solution was placed in a dialysis membrane and dialyzed against a 20 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride (hereinafter referred to as PBS). (During the process, liquid exchange 3 times). After dialysis, the solution was filtered through a 0.45 m filter. Step 3 Protein A Affiliation Chromatography>
  • the solution after dialysis was applied and washed with the same buffer until no protein was eluted in the flow-through fraction. Thereafter, the antibody was eluted with a 100 mM sodium citrate solution (pH 3.0). Immediately after collecting this elute, a 1 M Tris solution (pH 9.0) was added to neutralize the antibody.
  • the eluted antibody was put into a dialysis membrane, and dialyzed against PBS (during the process, three liquid exchanges). After the analysis, the solution was filtered through a 0.45 m filter.
  • the LPLD protein purified according to Example 1 above was subjected to SDS-PAGE using a 7.5% gel, and this was electrically (200 mA) transferred to a PVDF membrane.
  • the membrane after the transfer was blocked with Proc Ace (Dainippon Pharmaceutical) at 4 ° C overnight.
  • a PVDF membrane was reacted at room temperature for 2 hours with a solution obtained by diluting the above purified antibody 1000 times with 10% PROC ACE. After washing 3 times for 5 minutes with 10% Block Ace containing 0.1% Tween 20, the horseradish peroxidase-labeled goat anti- ⁇ sagiminoglobulin G antibody was diluted 10000 times with 10% Block Ace.
  • the PVDF membrane was reacted with the solution at room temperature for 1 hour.
  • LPLD was purified from human plasma as follows.
  • Step 1 ammonium sulfate fractionation and dialysis First, 180.95 g (30% saturated ammonium sulfate) of ammonium sulfate (hereinafter referred to as ammonium sulfate) was added to 1150 ml of human plasma, left to stand on ice for 1 hour, and centrifuged at 8000 rpm for 40 minutes at 4 ° C. . Next, 204.05 g of ammonium sulfate (60% saturated ammonium sulfate) was added to the supernatant, left still in ice for 1 hour, and centrifuged at 8,000 rpm for 40 minutes at 4 ° C.
  • the obtained dialyzed solution was subjected to anion exchange chromatography under the following conditions.
  • the LPLD activity of each fraction was measured as follows. First, 14: 0 LPC (lysophosphatidylcholine) 501 dissolved in a physiological saline solution containing 0.25% by weight BSA ( ⁇ -plasma albumin) to a final concentration of 0.15 mM was prepared. Fractions 10 1 and 100 1 were mixed with 20 mM Tris-HCl buffer (pH 8.0) and incubated at 37 ° C. for 1 hour. At this time, L contained in each fraction
  • Choline is produced from LPC depending on the amount of PLD. Take 100 1 from this mixture and add 0.2 ml of 7.5 mM HPPA (3- (4-hydroxyphenyl) propionic acid) and 2.6 ml of 0.1 M Tris-HCl buffer (pH 8.5) , 2.0 U / ml HRP (horseradish peroxida Ze) 0.1 ml was added, and 300 U / ml choline oxidase 101 was further added and mixed well, and the solution was incubated at 37 ° C for 15 minutes. The choline produced at this time reacts with choline oxidase, generating hydrogen peroxide.
  • HPPA 3- (4-hydroxyphenyl) propionic acid
  • the generated hydrogen peroxide is catalyzed by HRP to form HPPA that oxidizes and emits fluorescence.
  • Choline can be quantified by measuring the fluorescence intensity of the finally generated fluorescent substance (excitation wavelength: 320 nm, fluorescence wavelength: 404 nm).
  • Step 4 Heparin affinity chromatography> The liquid collected as described above was subjected to heparin affinity chromatography under the following conditions.
  • the liquid recovered as described above was further subjected to anion exchange chromatography under the following conditions.
  • Table 1 shows the total amount of protein in the solution recovered in each step, the total PLD activity, and the yield and purification rate of the LPLD protein obtained therefrom.
  • the unit in the total activity indicates the amount (nmol) of choline produced per hour from LPC according to the amount of LPLD contained in each liquid. “-” Indicates the item whose value could not be measured.
  • the substrate specificity of LPLD purified from human plasma was examined by the following three methods. The results are shown as the average of three measurements.
  • Tomirisutiru (Myristoyl) (14: 0) -LPC is the best substrate among saturated fatty Ashiru LPC, unsaturated C 18 - fatty Ashiru LPC was higher substrate specificity than saturated fats Ashiru LPC.
  • LPLD hydrolyzes acetylated 2-arachidonyl (20: 4) — LPC to minimize acetyl transfer while measuring LPLD activity, but at a rate similar to that of acetylated 1-20: 4_LPC It was higher than the speed (Fig. 5C).
  • LPLD more efficiently supplies the preferred LPA to the endothelial differentiation genes (Edg) 7 LPA receptor.
  • Edg 7 is more responsive to 2-unsaturated acyls than to the mono- or unsaturated acyls of LPA.
  • LPLD hydrolyzes phosphatidylcholine (PCs) into two saturated medium-chain acetyl groups rather than two saturated short or long-chain acetyl groups ( Figure 5C).
  • Co 2+ increased the activity of all related analogs except acyl LPC and 1-linoleoyl (18: 2) _LPC.
  • the optimal chain length of the saturated acyl LPC was changed from 14 to 12, and the optimal chain length of the PC was changed from 10 to 8, respectively.
  • the optimal number of cis double bonds in unsaturated lacyl LPA changed from 2 to 1.
  • the present invention provides an LPLD inhibitor based on the successful isolation and purification of an enzyme (LPLD) involved in the biosynthesis of LPA, which has not been elucidated until now.
  • LPLD inhibitors can be used for preventing or treating symptoms or diseases caused by the physiological activity of LPA.
  • the medicament according to the present invention is useful for preventing or treating cancer, male reproductive disease, female reproductive disease, and arteriosclerosis.
  • the present invention also provides a screening method for a LPLD inhibitor and a screening kit. This makes it possible to easily select a substance that can be used as a preventive or therapeutic agent for the above-mentioned symptoms or diseases.
  • the diagnostic method or the diagnostic agent according to the present invention By using the diagnostic method or the diagnostic agent according to the present invention, (a) the symptom or disease caused by the physiological activity of LPA, or (b) the amount of LPLD in the living body is higher than normal or lower than normal Early detection or treatment of symptoms or diseases caused by the disease.
  • the diagnostic method or diagnostic agent according to the present invention is preferably used particularly for diagnosing cancer, male reproductive system disease, female reproductive system disease, arteriosclerosis, or preeclampsia.
  • the present invention further provides a medicament comprising LPLD.
  • a medicament comprising LPLD.
  • Such a medicament can be used as a preventive or remedy for a symptom or disease caused by a lower than normal amount of LPLD such as toxemia of pregnancy, or for promoting childbirth or promoting labor.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Gynecology & Obstetrics (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Pregnancy & Childbirth (AREA)
  • Immunology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Cette invention se rapporte à une substance qui inhibe la capacité d'une acide lysophosphatidique synthase à produire de l'acide lysophosphatidique. Cette substance est utile pour diagnostiquer les maladies attribuables à l'activité physiologique d'un acide lysophosphatidique, pour atténuer les symptômes attribuables à l'activité physiologique d'un acide lysophosphatidique ou pour prévenir ou traiter ces maladies.
PCT/JP2002/010342 2001-10-04 2002-10-03 Inhibiteur de l'acide lysophosphatidique synthase WO2003031615A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2003534585A JPWO2003031615A1 (ja) 2001-10-04 2002-10-03 リゾホスファチジン酸合成酵素阻害物質

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2001309181 2001-10-04
JP2001-309181 2001-10-04
JP2002241043 2002-08-21
JP2002-241043 2002-08-21

Publications (1)

Publication Number Publication Date
WO2003031615A1 true WO2003031615A1 (fr) 2003-04-17

Family

ID=26623726

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/010342 WO2003031615A1 (fr) 2001-10-04 2002-10-03 Inhibiteur de l'acide lysophosphatidique synthase

Country Status (2)

Country Link
JP (1) JPWO2003031615A1 (fr)
WO (1) WO2003031615A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005009469A1 (fr) * 2003-07-28 2005-02-03 Sumitomo Pharmaceuticals Co., Ltd. Nouveau medicament de regulation de la glycemie et procede de criblage associe
JP2009162616A (ja) * 2008-01-07 2009-07-23 Univ Of Tokyo オートタキシン測定による妊娠および妊娠高血圧症候群の検査方法および検査薬
JP2013127482A (ja) * 2013-03-08 2013-06-27 Univ Of Tokyo オートタキシン測定による妊娠および妊娠高血圧症候群の検査方法および検査薬
JP2014197024A (ja) * 2014-07-07 2014-10-16 国立大学法人 東京大学 オートタキシン測定による妊娠および妊娠高血圧症候群の検査方法および検査薬
JP2017026642A (ja) * 2016-11-09 2017-02-02 ニプロ株式会社 生体試料のためのリチウムイオン濃度測定キットおよびそれを用いた生体試料中のリチウムイオン濃度の測定方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993014202A1 (fr) * 1992-01-17 1993-07-22 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Autotaxine: proteine stimulant la motilite, utilisee pour le diagnostic et la therapie du cancer
WO2000068386A1 (fr) * 1999-05-07 2000-11-16 Zymogenetics, Inc. Variantes de l'autotaxine et leurs utilisations dans le traitement des troubles du metabolisme
JP2002017398A (ja) * 2000-07-07 2002-01-22 Azwell Inc リゾリン脂質の測定方法
WO2002053569A1 (fr) * 2000-12-28 2002-07-11 Azwell Inc. Procede pour doser une activite de lysophospholipase d

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993014202A1 (fr) * 1992-01-17 1993-07-22 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Autotaxine: proteine stimulant la motilite, utilisee pour le diagnostic et la therapie du cancer
WO2000068386A1 (fr) * 1999-05-07 2000-11-16 Zymogenetics, Inc. Variantes de l'autotaxine et leurs utilisations dans le traitement des troubles du metabolisme
JP2002017398A (ja) * 2000-07-07 2002-01-22 Azwell Inc リゾリン脂質の測定方法
WO2002053569A1 (fr) * 2000-12-28 2002-07-11 Azwell Inc. Procede pour doser une activite de lysophospholipase d

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
TOKUMURA A. ET AL.: "Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma", LIPIDS, vol. 33, no. 10, 1998, pages 1009 - 1015, XP002966818 *
TOKUMURA A. ET AL.: "Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients", BIOL. REPROD., vol. 61, no. 1, 1999, pages 195 - 199, XP002966819 *
WYKLE R.L. ET AL.: "lysophospholipase D", METHODS ENZYMOL., vol. 197, 1991, pages 583 - 590, XP002966817 *
WYKLE R.L. ET AL.: "Specificity of lysophospholipase D", BIOCHIM. BIOPHYS. ACTA, vol. 619, 1980, pages 58 - 67, XP002966816 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005009469A1 (fr) * 2003-07-28 2005-02-03 Sumitomo Pharmaceuticals Co., Ltd. Nouveau medicament de regulation de la glycemie et procede de criblage associe
JP2009162616A (ja) * 2008-01-07 2009-07-23 Univ Of Tokyo オートタキシン測定による妊娠および妊娠高血圧症候群の検査方法および検査薬
JP2013127482A (ja) * 2013-03-08 2013-06-27 Univ Of Tokyo オートタキシン測定による妊娠および妊娠高血圧症候群の検査方法および検査薬
JP2014197024A (ja) * 2014-07-07 2014-10-16 国立大学法人 東京大学 オートタキシン測定による妊娠および妊娠高血圧症候群の検査方法および検査薬
JP2017026642A (ja) * 2016-11-09 2017-02-02 ニプロ株式会社 生体試料のためのリチウムイオン濃度測定キットおよびそれを用いた生体試料中のリチウムイオン濃度の測定方法

Also Published As

Publication number Publication date
JPWO2003031615A1 (ja) 2005-01-27

Similar Documents

Publication Publication Date Title
US8383086B2 (en) Nicotinamide riboside kinase compositions and methods for using the same
AU2006238858B2 (en) Nicotinamide riboside kinase compositions and methods for using the same
WO2006073052A1 (fr) Nouvel inhibiteur d'angiogenèse
JP2005245469A (ja) 腫瘍サプレッサ遺伝子、並びに癌の検出、腫瘍進行のモニタおよび癌処置のための方法
US20090205056A1 (en) Method of screening for modulators of map3k11 in liver cancer cells
Khoo et al. Characterization and localization of CD38 in the vertebrate eye
JP2003521903A (ja) Ｃｄ８４様ポリペプチドおよびポリヌクレオチドに関する方法と材料
JP2015063524A (ja) 膵構造から誘導される糖ペプチド、抗体ならびに診断および治療におけるそれらの応用
WO2003031615A1 (fr) Inhibiteur de l'acide lysophosphatidique synthase
US6187559B1 (en) Phospholipase D gene
CA2398227A1 (fr) 17 proteines secretees humaines
US20070185048A1 (en) Protein Disulfide Isomerase and ABC Transporter Homologous Proteins Involved in the Regulation of Energy Homeostasis
JPH11505412A (ja) ホスホリパーゼｃホモログ
CA2391594A1 (fr) Anticorps, polypeptides et polynucleotides contenant un domaine dit en feuille de trefle
JP2002520040A (ja) 新規ｃｄ３９様ポリペプチドに関する方法および材料
JP2003047479A (ja) ビタミンｄ３水酸化酵素
Kim et al. Functional analysis of duplicated genes and N-terminal splice variant of phospholipase C-δ1 in Paralichthys olivaceus
WO2000034486A1 (fr) Phospholipase a2 secretoire humaine
WO2001021205A1 (fr) Inhibiteur et potentialisateur du facteur de couplage cf6 et utilisation correspondante
CA2420192A1 (fr) 21 proteines humaines secretees
CN115916807A (zh) 靶向棕榈酰化/去棕榈酰化循环以治疗炎性疾病
JP2004329040A (ja) ポリセラーゼ−ｉ及びその利用
JP2003246749A (ja) 好中球接着誘導剤
JP2003521251A (ja) Ｃｕｂドメイン・ポリペプチドおよびポリヌクレオチドに関する方法と材料
Aberger et al. Herbsttagung der Gesellschaft für Biologische Chemie

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AU AZ BA BB BR BY CA CN CO CR CU DM DZ EC GD GE HU ID IL IN IS JP KG KR KZ LC LK LR LV MA MD MG MK MN MX NO NZ PH PL RO RU SG SI TJ TM TN TT UA UZ VC VN YU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2003534585

Country of ref document: JP

122 Ep: pct application non-entry in european phase