WO2003031615A1 - Lysophosphatidic acid synthase inhibitor - Google Patents

Abstract

A substance which inhibits the lysophosphatidic-acid-producing ability of a lysophosphatidic acid synthase. This substance is useful for diagnosing diseases attributable to the physiological activity of a lysophosphatidic acid, for mitigating symptoms attributable to the physiological activity of a lysophosphatidic acid, or for the prevention of or treatments for the diseases.

Description

Technical field

 The present invention relates to a lysophosphatidic acid synthase (hereinafter referred to as LPLD)

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.

Background art

When stimulus is applied to the cell membrane, phospholipases are activated, and the constituent phospholipids of the cell membrane and the like are degraded, and lipid mediators such as arachidonic acid derivatives, platelet activating factor, LPA, sphingosine-1_phosphate, and anandamide are produced. Made in the body. In recent years, 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)). The physiological activities of LPA include, for example, promotion of cell proliferation, suppression of cell differentiation, promotion of invasion of cancer cells, promotion of mobility, suppression of cell adhesion, suppression of apoptosis, and the like. May cause illness. Conventionally, the following considerations have been made regarding the biosynthetic pathway of LPA. 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. Therefore, the union Since LPA is immediately converted to phosphatidic acid (PA) in the adult system, this pathway does not normally accumulate in vivo. LPA, which acts as a physiologically active substance in vivo, is considered to be a product of the phospholipid degradation system (Kazuhiko Article, Cell Engineering Vol. 17, No. 5 (1998)) o LPA as a mediator between cells Considering the function of, the pathway to produce LPA on the cell surface or extracellular fluid is a suitable purpose, and Tokumura, one of the present inventors, has the activity of producing LPA from lysophospholipid in body fluid. (Tokumura, A. et. Al., Prog. Lipid Res. 34, pl51-184 (1995)), but the substance could not be elucidated. That is, it cannot be said that the details of the LPA biosynthetic pathway by phospholipid degradation have been sufficiently elucidated, and the enzymes involved in the LPA biosynthesis have not yet been identified. Disclosure of the invention

 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. By 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. . As a result, the LPLD inhibitor can be used as a pharmaceutically active ingredient such as an anticancer agent.

On the other hand, it is also possible that symptoms such as pregnancy toxemia may appear due to the lower than normal amount of LPLD in the body. Therefore, 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.

 That is, the present invention

 (1) a substance that inhibits the ability of lysophosphatidic acid synthase to produce lysophosphatidic acid,

 (2) the lysophosphatidic acid synthase antibody

The substance described in (1),

 (3) 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. A method for screening a substance that inhibits the ability to produce thidic acid,

 (4) a substance that inhibits the lysophosphatidic acid-producing ability of lysophosphatidic acid synthase selected by the screening method according to (3),

 (5) A kit for screening a substance that inhibits the lysophosphatidic acid-producing ability of lysophosphatidic acid synthase, which comprises lysophospholipid and lysophosphatidic acid synthase,

 (6) a medicament comprising the substance according to (1), (2) or (4),

 (7) 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.

(8) 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 ,

 (10) a medicament comprising lysophosphatidic acid synthase,

(11) the medicament according to the above (10), which is a therapeutic or preventive agent for toxemia of pregnancy;

 (12) a lysophosphatidic acid production inhibitor of lysophosphatidic acid synthase, which comprises a nucleotide or an analog thereof,

About. BRIEF DESCRIPTION OF THE FIGURES

 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. When this membrane was treated with an anti-LPLD antibody and colored, 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.

 The elution curve of 280 ηπι in chromatography using a TSK gel Pio Assist Q column and the LP LD activity of each eluted fraction are 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.

 Using fractions 46 to 51 of anion exchange chromatography shown in Fig. 3 in the absence of dithiothreitol, using 7.5% gel (A;), or using 12.5% gel in the presence of 0.5% dithiothreitol (B) SDS-PAGE was performed under the conditions and silver staining was performed. Arrows indicate the UOkDa (A), 110, 77, and 30 kDa (B) LPLD protein bands. 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⁺ .

 B: The effect of ATP and p-nitrophenyl 5'-thymidine phosphate (pNP-TMP) on LPLD activity was examined using a 3.3-fold diluted solution of purified plasma LPLD.

C; substrate specificity of the purified LPLD, final concentration 0.15 mM various choline lipids and - glycerin port - 3- for phosphocholine (GPC), walk presence 30 M Co ^M was examined in the absence condition. The values on the graph are expressed as relative activities (%) with the activity of 18: 2-LPC in the absence of Co ²⁺ as 100%. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention provides a substance that inhibits the LPA-producing ability of LPLD, that is, an LPLD inhibitor.

Here, 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. As 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.

Among them, preferred examples of the LPLD inhibitor according to the present invention 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. Examples of 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. Residues, deoxyperidine residues such as deoxyperidine residues or deoxythymidine residues; dideoxyadenosine residues, dideoxyguanosine residues, dideoxycytidine residues, dideoxyperidine residues or didedeoxydysine residues. And dideoxyliponucleoside residues such as oxythymidine residues. Specifically, 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. In the present invention, a nucleotide or an analog thereof is ATP or p-nitrophen-5u-TMP as a particularly preferred example. Further, 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. Specifically, 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.

 More specifically, among the LPLD antibodies according to the present invention, a monoclonal antibody (hereinafter, referred to as an LPLD monoclonal antibody) can be produced, for example, according to the following method.

First, LPLD or a partial peptide of LPLD is administered to a warm-blooded animal together with a carrier and a diluent. At this time, 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.

 When producing monoclonal antibody-producing cells, 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. 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. For example, 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)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of 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.

Various methods can be used for screening of LPLD antibody-producing hybridomas. (A) For example, adding 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. (B) Adsorption of anti-immunoglobulin antibody or protein A Add the hybridoma culture supernatant to the solid phase For example, a method of adding LP LD or a partial peptide of LP LD labeled with a substance or enzyme to detect LP LD monoclonal antibody bound to a microplate can be used.

 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.

 Next, 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.

For example, among the LPLD antibodies according to the present invention, 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.

 Each step is described in detail below. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio between the carrier and the hapten depend on the hapten immunized by cross-linking the carrier. On the other hand, any antibody can be cross-linked at any ratio as long as the antibody can be efficiently produced. For example, 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. In addition, various condensing agents can be used for force coupling between the hapten and the carrier. Above all, as the 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. In addition, when the antigen is a partial peptide of LPLD, 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. In this case, it is preferable that 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). At this time, the LPLD-encoding DNA does not need to be exactly the same as the known nucleotide sequence information, but may have any homology. Specifically, 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. In addition, 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.

In addition, the above DNA can be easily obtained from known amino acid sequence information. 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. In addition, the amino acid sequence information can be obtained from Hiroyuki Kawagoe, et.al., Submitted (04-FEB-1995) to the thigh J / EMBL / GenBank databases. Or Hiroyuki Kawagoe, et.al., Genomics 30 (2), It is described as a phosphodiesterase I derived from human (Homo sapiens) in p380-384 (1995). Further, the amino acid sequence information is described in Murata, J et.al., J. Biol. Chem. (1994) 269: p30479-30484 or Stracke, ML et. Al., J. Biol. Chem. (1992) 267: p2524-2529, autotaxin derived from human (Homo sapiens) It is described as a precursor.

 As a method for obtaining the above DNA from known amino acid sequence information, 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. Examples of the expression vector include plasmids derived from Escherichia coli, Bacillus subtilis or yeast, bacteriophages such as λ phage, and animal viruses such as retrovirus, vaccinia virus, and baculovirus. As 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.

 Next, a host cell is transformed with the expression vector. As 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

(Molecular Cloning, 2nd ed.) It can be carried out according to known conditions described in Cold Spring Harbor Laboratory (1989) and the like. 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. Hereinafter, "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.

 As described above, 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. And a kit for diagnosing the above-mentioned condition or disease, 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. For example, 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. In the above measurement method, the antibody used may be the antibody molecule itself, or the F (ab ') 2, Fab' or Fab fraction of the antibody molecule. In addition, 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 β-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. Furthermore, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent. For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

 A detailed measurement method when the sandwich method is used as the above measurement method will be described below. First, 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. As a result, LPLD in the test substance can be determined. Also, in the immunoassay by the sandwich method, 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.

 In addition, 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. As a method for measuring the LPA-producing ability of LPLD, a known method may be used. For example, 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. More specifically, 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. At this time, as the radioisotope, colored substance or fluorescent substance used as the label, the above-mentioned known substances may be used. Examples of 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 preferred embodiment of the method for measuring the LPLD activity is described below. First, 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. Then, 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. By measuring 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.

 Further, the present invention provides a diagnostic kit for the above-mentioned condition or disease, which comprises a lysophospholipid.

 In a preferred embodiment of the diagnostic kit, a lysophospholipid labeled with a radioisotope, a colored substance or a fluorescent substance is used as the lysophospholipid. In this case, 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.

When the lysophospholipid is lysophosphatidylcholine, another preferred embodiment is that choline oxidase and peroxidase are further combined. In the diagnostic kit, a lysophospholipid is preferably used.

It is preferred that rucholine is dissolved in a physiological saline solution containing about 0.25% by weight of BSA to a final concentration of about 0.15 mM. 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. Specifically, 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.

 In the screening method according to the present invention,? And 0? A known method may be used as a method for measuring the productivity, and it is particularly preferable to use the method described above. As 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. As an embodiment of the screening kit, there is an embodiment in which LPLD is further contained in the diagnostic kit containing lysophospholipid described above. The present invention provides a medicine containing an LPLD inhibitor. Here, it is preferable to use an LPLD antibody as the LPLD inhibitor, as described above, using LPLD isolated from the same species as the species from which the test substance to be measured was collected or a partial peptide thereof as an antigen, as described above. It is more preferable to use the LPLD antibody prepared in (1). It is also preferable to use nucleotides or analogs thereof as the LPLD inhibitor.

 As the medicament, 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. However, in general, it is desirable to administer it in the form of a pharmaceutical composition containing an active ingredient LPLD inhibitor and one or more pharmaceutical additives. Such 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. For example, 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.

 In the medicament according to the present invention, for the production of solid preparations such as capsules, tablets, powders or granules, for example, 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.

In the medicament according to the present invention, 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.

Examples of the physiologically acceptable carrier 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 ^™). , HCO-50, etc.) may be added. In addition, as a carrier of the injection, for example, an oily liquid such 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. When 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.

 Specific examples of additives for parenteral administration that are suitable for parenteral administration include, for example, buffering agents such as phosphate buffer and sodium acetate buffer; for example, Shiridani benzalkonium. And soothing agents such as proforce hydrochloride; stabilizers such as human serum albumin and polyethylene glycol; and preservatives such as benzyl alcohol and phenol.

 Since 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.).

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. For example, colorectal cancer, brain tumor, head cancer, breast cancer, lung cancer, esophagus cancer, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, 、 cancer, Teng Island Cell carcinoma, Choriocarcinoma, Colon cancer, Renal cell carcinoma, Adrenal cortex cancer, Bladder cancer, Testicular cancer, Prostate cancer, Testicular tumor, Ovarian cancer, Uterine cancer, Peritoneal cancer, Choriocarcinoma, Thyroid cancer, Malignant carcinoid tumor, Skin cancer, malignant melanoma, osteosarcoma, soft tissue sarcoma, neuroblastoma, Wilms tumor, retinoblastoma, melanoma, squamous cell carcinoma and the like. In particular, 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 cancer.

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. Examples of 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. In addition, to improve the condition in which the normal function of the male reproductive system or the female reproductive system is lost but not to the extent described above, or to prevent the condition from occurring, The medicament according to the invention can also be used. Furthermore, 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.However, when 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.

 In addition, 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. . Further, it is also possible to provide a method for preventing or treating the above-mentioned symptoms or diseases characterized by administering the above-mentioned medicine. For example, in preeclampsia, 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.

Some fractions with nucleotide PDE activity are separated by electrophoresis of human plasma. Some found that the Km value for pNT-TMP (3 OmM) was much higher than for others and increased during pregnancy. 16: 0—Measurement using LPC (lysophosphatidylcholine) showed that plasma LPLD activity increased during human gestation and decreased to the level of non-pregnant women after delivery. In addition, LPLD activity in sera obtained from patients with imminent preterm birth during the third trimester (first trimester was 3 months) was significantly higher than that of normal pregnant women. Thus, LPA-mediated products of LPA play a physiological role in childbirth. Therefore, for example, a medicament containing an LPLD inhibitor can be used as a prophylactic / therapeutic agent for premature labor. In addition, since 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. On the other hand, as mentioned above, LPLD increases during pregnancy, It can be used as a preventive or remedy for various symptoms associated with non-increased LD, for example, preeclampsia. In addition, a drug containing LPLD can be used as a drug for promoting childbirth or as an agent for promoting labor. Example

 (Example 1 Purification of LPLD)

 LPLD was purified from human plasma as follows.

 First, 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 8,000ΠΜ 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. Then, 20 ml of 20 mM Tris-HCl buffer (H 8.0) was added to the precipitate (ammonium sulfate fraction) to dissolve the precipitate. 100 ml of the obtained ammonium sulfate fraction solution was put into a dialysis membrane, and the 20 mM Tris-HCl buffer solution (pH 8.0) Dialysis was performed for a total of 26 hours at 8 L (on the way, once liquid exchange). The volume after dialysis was 194 ml. The obtained dialyzed solution was subjected to anion exchange chromatography under the following conditions.

 Conditions for anion exchange mouth chromatography

Sample volume; 10ml (20 repetitions)

Column; HiPrep 16/10 Q XL (mersham harmacia bio tech) S¾)

Buffer solution: A = 20mM Tris-HCl buffer (pH8.0), B = A + 1M sodium chloride

Elution solvent gradient; 0 → 100% B 40 min (100 ml)

Flow rate: 2.5 ml / min

Fractionation: 2.5 ml / test tube

After the Yin exchange chromatography, 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. Take IOOL from this mixture, 0.2 ml of 7.5 ΙΠΜ (3- (4-hydroxyphenyl) propionic acid), 2.6 ml of 0.1 M Tris-HCl buffer (pH 8.5), 2.6 ml of 2.0 U / ml HRP (Western rust) Peroxidase (0.1 ml) was added, 300 U / ml cholinexase IOL 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. The generated hydrogen peroxide forms HPPA which oxidizes and emits fluorescence using HRP as a catalyst. Choline can be quantified by measuring the fluorescence intensity of the finally generated fluorescent substance (excitation wavelength: 320 nm, emission wavelength: 404 nm).

 Fractions showing LPDL activity in this activity measurement were collected. The total liquid volume of the collected fractions was 320 ml.

 The liquid collected as described above was subjected to hydroxyapatite chromatography under the following conditions.

 Hydroxyapatite chromatography conditions

Sample volume: 20ml (16 repetitions)

Column; Hydroxyapatite Type I Columns 5 ml (Bio-Rad)

Buffer; A = 10 mM sodium phosphate (pH 6.8),

B = 500 mM sodium phosphate (pH 6.8)

Elution solvent gradient; 0 → 50% B 50 min (100 ml), 50 → 100% B 5 min (10 ml) Flow rate: 2 ml / min

Fractionation: 2 ml / test tube

After the hydroxyapatite chromatography, 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.

 The liquid collected as described above was subjected to heparin-finished mouth chromatography under the following conditions.

 Heparin affinity chromatography conditions

Sample volume; 10ml (20 repetitions)

Column; 5 ml of HiTrap Heparin HP (from Aarasham Pharmacia Biotech)

Buffer solution: A = 50 mM Tris-HCl buffer (pH 8.0), B = A + 1 M sodium chloride Eluent gradient: 15% B 5 minutes (10 ml), 25% B 5 minutes (10 ml), 100% B 5 minutes (10 ml) (gradual)

Flow rate: 2 ml / min

Fractionation: 2 ml / test tube

 After the heparin fin take mouth chromatography, the LPLD activity of each fraction was measured in the same manner as described above.

 Fractions showing LPDL activity in this activity measurement were collected. The total volume of the collected fractions was 120 ml.

 The solution recovered as described above was subjected to concanavalin A affinity chromatography under the following conditions.

 Concanavalin A affinity chromatography conditions

Sample volume; 20ml (6 repetitions)

Column; Con Canaparin A Sepharose (manufactured by Amersham pharmaciabio tech) I HR 16/10 15 ml buffer; A = 20 mM Tris-HCl buffer + 0.5 M NaCl sodium chloride (pH7) . Four),

 B = Α + 0 · 5Μmethyl-α—D—mannopyranoside

Elution solvent gradient; 100% Β 33 minutes (50 ml) Flow rate: 1.5 ml / min

Fractionation; 1.5 ml / test tube

 After the concanavalin A affinity chromatography, the LPD activity of each fraction was measured in the same manner as described above.

 Fractions showing LPDL activity in this activity measurement were collected. The total liquid volume of the collected fractions was 180 ml.

 The liquid collected as described above was applied to anion exchange chromatography under the following conditions.

 Conditions for anion exchange chromatography

Sample volume; 15ml (12 repetitions)

Column; Resource Q (RESOURCE Q) 6 ml (Amersham Pharmacia Biotech

(amersham pharmac i a b i otech)

Buffer solution: A = 20mM Tris-HCl buffer (pH 8.0), B = A + 1M sodium chloride

Elution solvent gradient; 0 → 50% B 50 min (50 ml), 50 → 100% B 5 min (5 ml), 100% B 5 min (5 ml)

Flow rate: 2 ml / min

Fractionation: 2 ml / test tube

 After the anion exchange chromatography, the LPLP activity of each fraction was measured in the same manner as described above.

 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.

 Conditions for gel filtration mouth chromatography

Sample volume: 0.5ml (10 repetitions)

Column; Superdex 200 HR 10/30 Buffer: 50 mM Tris-HCl buffer (pH 8.0) + 0.2 M sodium chloride

Flow rate: 0.5 ml / min

Fractionation: 0.5 ml / test tube

 After the gel filtration chromatography, the LPLD 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 12 ml. As described above, LPLD was purified from human plasma. The purified LPLD showed a molecular weight of about 110,000 by polyacrylamide electrophoresis.

(Example 2 Preparation of LPLD antibody)

 Using the LPLD obtained in Example 1, production of 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.

(Example 3 Identification of amino acid sequence of LPLD)

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. As a result, four amino acid sequence fragments were identified as human autotaxin (Lee, HY et al, Biochem. Biophys. Res. Commun. 218, 714-719 (1996)) Human phosphodiesterase I (Phosphodiesterase I) with different amino acid residues (73Asp → His, 100Ala → Gly, 644Ser → Arg, 703Val → Ala, 769Tyr → His)

(Kawagoe H. et al, Genomics 30, 380-384 (1995)).

 Furthermore, the gel after SDS-PAGE was treated with polypinylidene difluoride in the same manner as above.

(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.

(Example 4 Preparation of anti-LP LD antibody)

 <Step 1 Immunization>

 In the amino acid sequence of LPLD (see Fig. 1), peptides were synthesized at positions 309 to 327, and 6.3 mg of this LPLD synthetic peptide was coupled to 13.4 mg of mocyanin (KLH) to keyhole limpet. 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. Two, four, six and eight weeks later, 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 .

 <Step 2 ammonium sulfate fractionation>

Whole blood is collected from the heron immunized as described above, and 34 ml of 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>

 After equilibrating the 40 ml Protein A column with PBS, 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.

 Step 4 Western Plot>

 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- ゥ 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. Washing with PBS for 5 minutes was repeated three times. Thereafter, the color was developed using a solution prepared by dissolving .3-amino-9-ethylcarbazole in 6 ml of dimethyl sulfoxide and 50 ml of PBS to which 30% hydrogen peroxide solution 301 was added. As shown in FIG. 2, it was found that the purified antibody specifically reacted with the 110 kDa protein. In addition, this molecular weight was consistent with the molecular weight of the LPLD protein purified in Example 1 above, and it was confirmed that this antibody correctly recognized the LPLD protein.

Example 5 Purification of Human Plasma LPLD

 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. To this precipitate (ammonium sulfate fraction) was added 20 mM Tris-HCl buffer (PH 8.0) to dissolve the precipitate, and 144 ml of the resulting ammonium sulfate fraction solution was placed in a dialysis membrane, and the 20 mM Tris-HCl buffer (pH 8.0) Dialysis was performed at 20 liters (on the way, once liquid exchange) for a total of 38 hours. The liquid volume after dialysis was 234.6 ml.

 <Step 2 Yin Exchange Mouth ①>

 The obtained dialyzed solution was subjected to anion exchange chromatography under the following conditions.

 One condition of Yin exchange exchange mouth chromatography

Sample volume: 15 ml (11 repetitions)

Column: HiPrep 16/10 Q XL (Amersham Pharmacia Biotech) nersliam harmac ia bio tecn

Buffer; A = 20 mM Tris-HCl buffer (pH 8.0), B = A + 1 M NaCl salt elution gradient; 0 → 100% B 40 min (100 ml)

Flow rate: 2.5 ml / min

Fractionation; 2.5 ml / test tube

 After anion exchange chromatography, 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 (ゥ -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. 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).

 Fractions showing LPLD activity in this activity measurement were collected. The total volume of the collected fractions was 220 ml.

 <Step 3 hydroxyapatite chromatography>

 The solution collected as described above was subjected to hide-port xyapatite chromatography under the following conditions.

 Hydroxyapatite chromatography conditions

Sample volume: 20 ml (10 repetitions)

Column: Bio-scale Ceramic Hydroxyapatite Type I Columns; 5 ml (manufactured by Bio-Rad)

Buffer: A = 10 mM sodium phosphate (pH 6.8), B = 500 mM sodium phosphate (pH 6.8)

Elution solvent gradient; 0 → 50% B 50 min (75 ml), 50 → 100% B 5 min (7.5 ml) Flow rate: 1.5 ml / min

Fractionation; 1.5ml / test tube

 After the hydroxyapatite mouth chromatography, the LPLD 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 88 ml.

Step 4 Heparin affinity chromatography> The liquid collected as described above was subjected to heparin affinity chromatography under the following conditions.

 Heparin affinity chromatography conditions

Sample volume: 15 ml (6 repetitions)

Column: HiTrap Heparin HP 5 ml (Amersham Pharmacia biotech)

Buffer: A = 50 mM Tris-HCl buffer (pH 8.0), B = AI 1 M NaCl sodium salt Elution solvent gradient: 15% B 5 min (10 ml), 25% B 5 min (10 ml), 100% B 5 min (10 ml) (gradual)

Flow rate: 2 ml / min

Fractionation: 2 ml / test tube

 After heparin affinity chromatography, the LP LD activity of each fraction was measured in the same manner as described above.

 Fractions showing L PLD activity in this activity measurement were collected. The total volume of the collected fractions was 55 ml.

 Step 5 Concanavalin A affinity chromatography>

 The solution collected as described above was subjected to concanapalin A affinity chromatography under the following conditions.

 Concanavalin A affinity chromatography conditions

Sample volume; 22 ml (2 repetitions)

Column: Con Canavalin A Sepharose (Amersham Pharmacia biotech) / HR 16/10 15 ml buffer; A = 20 mM Tris-HCl buffer + 0.5 M sodium chloride (pH 7.4), B = A + 0.5 M methyl-α-D-mannopyranoside

Elution solvent gradient; 100% B 33 min (50 ml)

Flow rate: 1.5 ml / min Fractionation: 1.5 ml / test tube

 After concanapalin A affinity chromatography, LP LD activity of each fraction was measured in the same manner as described above.

 Fractions showing L PLD activity in this activity measurement were collected. The total volume of the collected fractions was 121.6 ml.

 <Step 6 hydrophobic mouth chromatography>

 The liquid recovered as described above was subjected to hydrophobic chromatography under the following conditions.

 Conditions for hydrophobic mouth chromatography

Sample volume; 3 ml (5 repetitions)

Column; TSK Gel Phenyl 5PW (7.5X75 mm) (Tosoichi Co., Ltd.)

Buffer solution; A = 20 mM Tris-HCl buffer (pH 8.0), B = A + 1 M ammonium sulfate Elution solvent gradient; 100 → 0% B 50 min (50 ml)

Flow rate: 1 ml / min.

Fractionation: 1 ml / test tube

 After the hydrophobic chromatography, the LPLD activity of each fraction was measured in the same manner as described above.

 Fractions showing L PLD activity in this activity measurement were collected. The total volume of the collected fractions was 78.1 ml.

 <Process 7 Yin exchange exchange

 The liquid recovered as described above was further subjected to anion exchange chromatography under the following conditions.

 Conditions for anion exchange chromatography

Sample volume; 50 ml

Column: TSKgel Bio-Assist Q (4.6X50 awake) (Tokyo Saw Corporation)

Buffer: A = 20 mM Tris-HCl buffer (H 8.0) + 20% glycerol, B = A + 1 M sodium chloride

Elution solvent gradient; 0 → 50% B 50 min (25 ml), 50 → 100¾ B 5 min (2.5 ml) Flow rate: 0.5 ml / min

Fractionation: 0.5 ml / test tube

 After the Yin exchange chromatography, the LPLD activity of each fraction was measured in the same manner as described above. Figure 3 shows the results. Fractions 46 to 51 were subjected to 7.5% SDS-PAGE, and the obtained gel was stained with silver. The results are shown in FIG. The arrow is the 110 kDa band with LPLD activity. In addition, 0.5% dithiothreitol was added to the same fraction, and the mixture was heated at 95 ° C for 5 minutes, separated by 12.5¾ SDS-PAGE, and the obtained gel was stained with silver. The results are shown in Figure 4B. Under reducing conditions, the L PLD 110 kDa band was split into two bands (arrows), 77 kDa and 30 kDa. In this analysis, there was a 110 kDa band that did not decompose even under reducing conditions, indicating that some molecular species that were not cleaved inside the protein sequence were also present in this sample.

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. Table 1

(Example 6)

 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.

(A) The time course of the carohydrate degradation of palmitoyl (16: 0) -LPC and p-nitrophenyl 5'-thymidine phosphate (hereinafter referred to as ρΝΡ-ΤΜΡ) by purified plasma LP LD was measured as follows. did. That is, a purified plasma L PLD sample (0.03 ml) was diluted with 2 OmM Tris-HCl buffer (ρΗ8.0) to 0.1 mL, and together with 0.05 mL of 0.45 mM 16: 0-LPC, 30 mL 37 ° (1-24 hours at 3) in the presence and absence of Co ²⁺ [I did it. Choline produced in 0.1 ml of the Atssey mixture was measured in exactly the same manner as described in Example 1, and the LPLD activity was measured. In addition, a purified plasma LPLD sample (0.1 mL) was incubated with 0.05 ml of 0.45 mM pNP-TMP for 1 to 24 hours at 37 ° C in the presence and absence of 30 M Co2 ^+. did. The Atsey mixture (0.1 mL) was mixed with 1.0 mL of 0.1 N NaOH, the absorbance at 400 nm was measured, and the activity of nucleotide phosphodiesterase (PDE) was measured.

The results of the test are shown in FIG. 5A. LPLD activity at 24 hours was lower than PDE activity, but the Km value of LPLD activity for 16: 0-LPC calculated from the Lineweaver-Burk plot was 0.26 ± 0.5 mM, and pNP-TMP The Km value of the PDE activity for E. coli was much lower than 5.5 mM in 0.5 mM. Co ²⁺ increased not only LPLD activity but also PDE activity.

(B) The effects of ATP and pNP-TMP on LPLD activity were measured in 3.3-fold solutions of purified plasma LPLD. A mixture of ATP or pNP—TMP at the concentration shown in FIG. 5B with the diluted solution of plasma L PLD (0.1 ml) was mixed with 0.45 mM 16: 0—in the absence of Co ^{2 +.} Incubated with 0.05 ml of LPC for 24 hours. The results of such a test are shown in Figure 5B. As is clear from FIG. 5B, ATP and pNP-TMP suppressed LPLD activity in a concentration-dependent manner.

 From the results of (A) and (B) above, it was found that the purified plasma LPLD had PDE activity, and that the active sites of LPC (lysophosphatidylcholine), ATP and pNP-TMP overlapped. Was.

(C) the substrate specificity of the purified LPLD, as described above, 30 in the presence and absence of Co ^{2 +} a M, final concentration 0. 15 mM of various choline lipids shown in FIG. 5 C It was used for measurement. Such substrate specificity, in the absence of Co ²⁺ Specific activity (%) when compared to the substrate specificity of LPLD for 18: 2-LPC. The results of such a test are shown in Figure 5C.

 As a result of the above test, the substrate specificity of 1 ^? 〇 (Lysophosphatidylcholine)> alkyl LPC> alkenyl LPC was higher in the order, and no specificity for _s_n-glycerol 3-phosphocholine (GPC) was observed. This suggested that the activity required hydrophobic interaction between the fatty acid moiety and the amino acid residue in the active site.

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). Thus, 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. In addition, 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. Furthermore, the optimal number of cis double bonds in unsaturated lacyl LPA changed from 2 to 1. This CO ²⁺ -induced change in the substrate specificity of the purified PLD is due to the fact that the CO ²⁺ ion plays an important role in the two-step catalysis of au 101 ax in It suggests that it is exchanged for the bound metal ion. Industrial applicability

 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. Such LPLD inhibitors can be used for preventing or treating symptoms or diseases caused by the physiological activity of LPA. More specifically, 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.

 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. 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.

Claims

Scope of Claim 1. A substance that inhibits the lysophosphatidic acid-producing ability of lysophosphatidic acid synthase. .

2. The substance according to claim 1, which is an antibody for lysophosphatidic acid synthase.

3. The reaction of lysophospholipid with lysophosphatidic acid synthase in the presence of the test compound to measure the lysophosphatidic acid-producing ability of lysophosphatidic acid synthase, characterized in that the lysophosphatidic acid synthase lysophosphatidic acid is measured. A method for screening a substance that inhibits production ability.

4. A substance that inhibits the lysophosphatidic acid-producing ability of lysophosphatidic acid synthase selected by the screening method according to claim 3.

5. A kit for screening a substance that inhibits the lysophosphatidic acid-producing ability of lysophosphatidic acid synthase, which comprises lysophospholipid and lysophosphatidic acid synthase.

6. A medicament comprising the substance according to claim 1, 2 or 4.

7. The medicament according to claim 6, which is a therapeutic or preventive agent for cancer, male reproductive system disease, female reproductive system disease or arteriosclerosis.

8. Measure the activity of lysophosphatidic acid synthase or 'A method for diagnosing cancer, male reproductive system disease, female reproductive system disease, arteriosclerosis or toxemia of pregnancy, characterized by using an acid synthase antibody.

9. A diagnostic kit for cancer, male reproductive system disease, female reproductive system disease, arteriosclerosis or preeclampsia, which comprises an antibody of (a) lysophospholipid or (b) lysophosphatidic acid synthase .

10. A pharmaceutical comprising lysophosphatidic acid synthase.

1 1. Claims characterized in that it is a drug for the treatment or prevention of toxemia of pregnancy

Item 10. The medicament according to Item 10.

12. An inhibitor of lysophosphatidic acid-producing ability of lysophosphatidic acid synthase, which comprises a nucleotide or an analog thereof.