JP2004161779A - Composition for suppressing smooth muscle proliferation, and compositions for preventing and treating arteriosclerosis and restenosis after revascularization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a treating and diagnostic technique using a gene associated with obesity or its expression by elucidating the obesity-related gene and its expression each useful for etiology elucidation and treatment technique of several diseases associated with obesity, especially of arteriosclerosis such as stenocardia and myocardial infarction. <P>SOLUTION: The subject composition for suppressing smooth muscle proliferation, the subject composition for preventing and treating arteriosclerosis and the subject composition for preventing and treating restenosis after revascularization contain each a pharmaceutically effective amount of one selected from adipose tissue specific secretor factor apM1 and pharmaceutically permissible salts thereof together with a pharmaceutically permissible carrier. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention provides a composition for inhibiting smooth muscle proliferation, a composition for preventing and treating arteriosclerosis, and a composition for preventing and treating restenosis after vascular reconstruction, which comprises an adipose tissue-specific secretion factor apM1 (Adipose Most Abundant Gene Transcript 1) as an active ingredient. Composition.

  It is well known in modern society that obesity, which is an excessive accumulation of fat, is involved in the development of diabetes, hyperlipidemia, hypertension, and atherosclerotic diseases such as angina and myocardial infarction. Various genetic and environmental factors are involved in the cause of obesity.

  Recently, a number of obesity genes, including leptin, have been isolated from animal models. While the isolated genes are thought to be involved in the establishment of obesity in humans, environmental factors such as modern humans' excessive food intake and lack of exercise also contribute to diabetes and atherosclerosis diseases via fat accumulation. It is considered another important factor.

  In addition to searching for obesity genes, an approach to elucidate the genes that are induced in adipose tissue in hypertrophic conditions and how they affect individuals will elucidate the etiology and treatment of the above diseases It is considered that the establishment of technology has a very important meaning.

  The present inventors elucidated the etiology of obesity-related diseases, in particular, the etiology of arteriosclerosis such as angina pectoris and myocardial infarction, and elucidated obesity-related genes and their expression products useful for establishing therapeutic techniques. As a result of intensive studies with the aim of establishing treatment and diagnosis techniques for various diseases using such as the above, the accumulation of visceral fat in the abdominal cavity among fats first showed abnormal glucose tolerance, hyperlipidemia, hypertension It is clarified that it is closely connected with the etc. In addition, a large-scale sequence analysis of adipose tissue-expressed genes revealed that many secretory protein genes are expressed in adipose tissue, and that various physiologically active substance genes are particularly expressed in visceral fat. Furthermore, in addition to these known genes, a new adipose tissue-specific collagen-like protein apM1 gene was successfully cloned (see Non-Patent Document 1).

This apM1 gene encodes a secretory protein (apM1) consisting of 244 amino acids, has a collagen-like motif (GXY) of 66 amino acids, and has homology with C1q of the complement system and collagens X and VII. However, the physiological functions of the gene and its expression product apM1 were unknown.
Biochem. Biophys. Res.Commun., 221, 286-289 (1996)

  The present invention elucidates the etiology of various diseases related to obesity, in particular, arteriosclerosis such as angina pectoris and myocardial infarction, and elucidates obesity-related genes and their expression products useful for establishing therapeutic techniques. Its purpose is to establish treatment and diagnosis techniques for various diseases by utilizing it.

  The present inventors, in subsequent studies, expression of the apM1 gene by genetic engineering techniques, production of an antibody against the expressed product apM1, establishment of a measurement system for apM1 using the antibody, apM1 blood concentration by the measurement In a series of studies on the relationship between liposome and body fat distribution or various diseases, the new fact that apM1 has an inhibitory effect on smooth muscle proliferation and that its blood concentration reflects arteriosclerosis well. I found it.

  In addition, the present inventors have found that apM1 is effective in preventing and treating restenosis after revascularization such as stent percutaneous transluminal coronary vasodilation (PTCA), and thus, for example, angina pectoris and myocardial infarction etc. It has been found that it is also effective for prevention and treatment of arteriosclerosis with vascular disorders. The present invention has been completed based on such findings.

  According to the present invention, there is provided a smooth muscle growth inhibitory composition comprising at least one pharmaceutically effective amount selected from apM1 and a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. .

  Further, according to the present invention, an atherosclerosis preventive and therapeutic composition containing at least one pharmaceutically effective amount selected from apM1 and a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier is provided. Provided.

  Furthermore, according to the present invention, prevention of restenosis after revascularization surgery containing at least one pharmaceutically effective amount selected from apM1 and a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. And a therapeutic composition.

  The smooth muscle growth inhibitory composition according to the present invention, through its smooth muscle growth inhibitory action, for example, angina, myocardial infarction including thrombosis, prevention and treatment of arteriosclerosis with vascular disorders such as cerebral infarction, It is effective in preventing the progress of such arteriosclerosis. In fact, apM1 as an active ingredient of the composition of the present invention is a cell adhesion molecule VCAM-1 (Vascular Cell Adhesion Molecule-1), ELAM (Endothelial Leukocyte Adhesion Molecule), ICAM-1 ( It can exert an action of suppressing the expression of Intercelular Adhesion Molecule-1) and the like. From this, the composition of the present invention works to suppress the onset of various arteriosclerosis.

  Accordingly, the present invention also provides a pharmaceutical composition that suppresses the expression of adhesion molecules in vascular endothelial cells.

  Due to the fact that apM1 suppresses the expression of the cell adhesion molecule, the composition of the present invention is known to be a disease associated with an increase in the expression level of VCAM-1, for example, a type I allergy associated with eosinophil infiltration. It is also applicable to the prevention and treatment of bronchial asthma and the like associated with reactions.

  Further, since ICAM-1 and ELAM are also known as adhesion molecules related to inflammation, the composition of the present invention containing apM1 as an active ingredient can be used as an anti-inflammatory agent, for example, by suppressing the expression of such adhesion molecules. Therefore, it can be applied as a therapeutic agent for rheumatoid arthritis.

  Furthermore, the composition of the present invention is also effective for preventing or treating restenosis after revascularization such as stent percutaneous transluminal coronary vasodilation (stent PTCA). That is, after vascular reconstruction for coronary artery stenosis in angina pectoris and myocardial infarction, adhesion molecules to vascular endothelial cells are expressed due to ischemia reperfusion and vascular endothelial cell damage, and smooth muscle cell proliferation occurs, resulting in restenosis. Develops. The composition of the present invention is also effective for preventing ischemic restenosis after vascular reconstruction by suppressing the expression of this adhesion molecule and the proliferation of smooth muscle cells.

  According to the present invention, there is also provided a method for diagnosing arteriosclerosis. This diagnostic method utilizes a new index of smooth muscle proliferation ability (smooth muscle cell DNA synthesis ability), which measures and quantifies the amount of apM1 in a sample using a specific antibody against the apM1. It is implemented by doing.

  Hereinafter, the production of apM1 as an active ingredient in the composition of the present invention, the preparation of the composition of the present invention using this as an active ingredient, the production of an antibody against apM1, and the measurement of apM1 will be sequentially described.

In this specification, amino acids, peptides, base sequences, notation by nucleic acids and the like are represented by abbreviations of IUPAC-IUB (IUPAC-IUB Communication on Biological Nomenclature, Eur.J. Biochem., 138 , 9 (1984)), The guidelines shall be in accordance with “Guidelines for Preparing Specifications Containing Base Sequence or Amino Acid Sequence” (July 2002, edited by the Patent Office) and common symbols in the relevant field.

ApM1 can be produced by, for example, a conventional genetic engineering technique (for example, Science, 224 , 1431 (1984); Biochem. Biophys. Res. Comm., 130 , 692 (1985); Proc. Natl. Acad. Sci., USA., 80 , 5990 (1983), etc.]. In the above, as the apM1 gene, a gene previously established by the present inventors can be used (see Non-Patent Document 1).

  The apM1 can also be produced by a general chemical synthesis method according to the amino acid sequence information encoded by the above gene.

  More specifically, production of apM1 employing a genetic engineering technique involves preparing a recombinant DNA capable of expressing a gene encoding a desired protein in a host cell, introducing the recombinant DNA into a host cell, transforming the DNA, This is performed by culturing the transformant.

Here, both eukaryotes and prokaryotes can be used as host cells. The eukaryotic cells include cells of vertebrates, eukaryotic microorganisms and the like. As vertebrate cells, for example, COS cells which are monkey cells (Cell, 23 , 175 (1981)) and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sci., USA., 77 , 4216 (1980)) and the like are often used, but are not limited thereto.

As a vertebrate expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like which are usually located upstream of a gene to be expressed can be used. It may also have a replication origin if desired. Examples of the expression vector include, for example, pSV2dhfr (Mol. Cell. Biol., 1 , 854 (1981)) having an early promoter of SV40.

As eukaryotic microorganisms, yeasts are commonly used, and among them, yeast belonging to the genus Saccharomyces can be advantageously used. As an expression vector for eukaryotic microorganisms such as yeast, for example, pAM82 (Proc. Natl. Acad. Sci., USA., 80 , 1 (1983)) having a promoter for the acid phosphatase gene can be used.

  Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. When these are used as a host, for example, a plasmid vector capable of replicating in a host bacterium is used, and a promoter and an SD (Shine and Dalgarno) base sequence upstream of the gene so that the desired gene can be expressed in the vector, It is preferable to use an expression plasmid to which an initiation codon (eg, ATG) necessary for initiation of protein synthesis has been added. As Escherichia coli as the host, Escherichia coli (Escherichia coli) strain K12 and the like are often used, and as the vector, pBR322 and its improved vector are generally used, but are not limited thereto, and various known strains and vectors are not limited thereto. Also available. As the promoter, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, PL / PR promoter and the like can be used.

  A method for introducing the desired recombinant DNA thus obtained into a host cell and a method for transforming with the same can be in accordance with a general method.

  The resulting transformant can be cultured according to a conventional method, and the culture results in expression, production, accumulation or secretion of the target recombinant protein inside, outside, or on the cell membrane of the transformant. As the medium used for the culture, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cells.

The apM1 obtained as described above may be subjected to various separation operations utilizing its physical properties, chemical properties, and the like, if desired (“Biochemical Data Book II”, pp. 1175-1259, 1st edition, 1st printing, 1980 Sep. 23, 2016, published by Tokyo Kagaku Dojin; Biochemistry, 25 (25), 8274 (1986); see Eur. J. Biochem., 163 , 313 (1987)). More specifically, for example, usual reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography It can be separated and purified by various liquid chromatography such as chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, a combination thereof and the like.

  The apM1 can also be produced by a general chemical synthesis method according to the amino acid sequence information. The method includes a conventional liquid phase method and a solid phase peptide synthesis method. More specifically, such a peptide synthesis method is a so-called stepwise elongation method in which each amino acid is sequentially bonded one by one to extend the chain based on amino acid sequence information, and a fragment consisting of several amino acids is previously synthesized. And then a fragment condensation method in which each fragment is subjected to a coupling reaction.

  The condensation reaction employed in the above-mentioned peptide synthesis method can also follow a conventional method. Examples of the method include azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide). , N-hydroxy-5-norbornene-2,3-dicarboximide) method, Woodward method and the like.

  The solvent that can be used for each of these methods can also be appropriately selected from well-known general ones that are used in this kind of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.

  In the above peptide synthesis reaction, carboxyl groups in amino acids or peptides not involved in the reaction are generally esterified to lower alkyl esters such as methyl ester, ethyl ester and tertiary butyl ester such as benzyl ester and p- It can be protected as an aralkyl ester such as methoxybenzyl ester and p-nitrobenzyl ester.

  Further, an amino acid having a functional group in a side chain, for example, a hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, and the like, but such protection is not necessarily performed. There is no.

  Further, for example, a guanidino group of an arginine residue may be a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group, etc. Can be protected by various protecting groups.

  Amino acids having the above protecting groups, peptides and deprotection reaction of these protecting groups in the finally obtained protein can also be performed by a commonly used method such as a catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, hydrogen bromide, It can be carried out according to a method using hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.

  The apM1 thus obtained can be appropriately purified according to the various methods described above, for example, ion exchange resins, partition chromatography, gel chromatography, a method commonly used in the field of peptide chemistry such as countercurrent distribution, and the like. .

  The composition according to the present invention contains apM1 or a pharmaceutically acceptable salt thereof as an active ingredient. Such salts include, for example, alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, barium, ammonium and the like, alkaline earth metal salts, ammonium salts and the like. These salts are prepared according to methods well known in the art. Further, the above salts also include acid addition salts obtained by reacting apM1 with an appropriate organic or inorganic acid according to a conventional method. Representative acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate , Benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate, ascorbine Acid salts, benzenesulfonates and napsylates.

  The composition according to the present invention is generally prepared and put into practical use in the form of a pharmaceutical preparation containing a pharmaceutically effective amount of the above active ingredient together with a suitable pharmaceutical carrier.

  Carriers that can be used in the pharmaceutical preparations include diluents such as fillers, extenders, binders, humectants, disintegrants, surfactants, lubricants, and the like, which are usually used depending on the use form of the preparation. Can be exemplified by excipients and the like. These can be appropriately selected and used depending on the dosage unit form of the obtained preparation.

  As the dosage unit form of the pharmaceutical preparation, various forms can be selected according to the purpose of treatment. Representative examples include solid dosage forms such as tablets, pills, powders, powders, granules, capsules and the like, and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. These are classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments and the like according to the administration route, and can be prepared, molded or prepared according to the usual methods. . To the above-mentioned pharmaceutical preparation, various components that can be used in ordinary pharmaceutical preparations, for example, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants and the like can be appropriately added. .

  Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. These can be used alone or in combination with a surfactant or the like. In particular, this combination may improve the stability of the active ingredient in some cases.

  The L-amino acid is not particularly limited, and includes, for example, glycine, cysteine, glutamic acid and the like.

  There is no particular limitation on the saccharides, for example, glucose, mannose, galactose, monosaccharides such as fructose, sugar alcohols such as mannitol, inositol, xylitol, sucrose, maltose, disaccharides such as lactose, dextran, hydroxypropyl starch, chondroitin. Polysaccharides such as sulfuric acid and hyaluronic acid and derivatives thereof can be used.

  There is no particular limitation on the surfactant, and both ionic and nonionic surfactants can be used. This includes, for example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like.

  There is no particular limitation on the cellulose derivative, and examples thereof include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose.

  The amount of the saccharide to be added is appropriately selected from the range of about 0.0001 mg or more, preferably about 0.01 to 10 mg per 1 μg of the active ingredient. The amount of the surfactant to be added is appropriately selected from the range of about 0.00001 mg or more, preferably about 0.0001 to 0.01 mg per μg of the active ingredient. The amount of human serum albumin to be added is appropriately selected from the range of about 0.0001 mg or more, preferably about 0.001 to 0.1 mg per μg of the active ingredient. The amino acid is suitably selected from the range of about 0.001 to 10 mg per μg of the active ingredient. The amount of the cellulose derivative to be added is appropriately selected from the range of about 0.00001 mg or more, preferably about 0.001 to 0.1 mg per 1 μg of the active ingredient.

  The amount of the active ingredient contained in the pharmaceutical preparation of the present invention can be appropriately selected from a wide range, and is usually selected from the range of about 0.00001 to 70% by weight, preferably about 0.0001 to 5% by weight.

  Furthermore, as a buffering agent that can be appropriately added to the pharmaceutical preparation, boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and / or a salt corresponding thereto (for example, a sodium salt thereof, Alkali metal salts such as potassium salts, calcium salts, and magnesium salts and alkaline earth metal salts). Examples of the tonicity agent include sodium chloride, potassium chloride, sugars, glycerin and the like. Examples of the chelating agent include sodium edetate and citric acid.

  In the pharmaceutical preparation of the present invention, in addition to the liquid preparation, the liquid preparation is freeze-dried to a state where it can be stored, and then dissolved in a buffer solution containing water, fresh saline, or the like at the time of use to prepare an appropriate concentration. The solubilizer used before use is also included.

  When the pharmaceutical preparation of the present invention is formed into a tablet, the carrier may be, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, or the like. Binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone; sodium carboxymethylcellulose, calcium carboxymethylcellulose, and low-substituted hydroxypropylcellulose Disintegrators such as, starch, dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate; polyoxyethylene sorbitan fatty acid esters, lauryl sulfate Surfactants such as sodium and stearic acid monoglyceride; disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; absorption promoters such as quaternary ammonium bases and sodium lauryl sulfate; humectants such as glycerin and starch; Adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder, and polyethylene glycol can be used.

  Further, the tablet can be a tablet coated with a usual coating, if necessary, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, or a double or multilayer tablet.

  When formed into pill form, as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol and the like; laminaran And disintegrating agents such as agar.

  Capsules are prepared by mixing the active ingredient with the various carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules, and the like, according to a conventional method.

  Solutions for oral administration include conventional inert diluents, such as pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs and the like, including water. The liquid may further contain auxiliaries such as a wetting agent, an emulsion and a suspending agent, which are prepared according to a conventional method.

  When preparing a liquid preparation for parenteral administration, for example, a sterile aqueous to non-aqueous solution, emulsion, suspension or the like, as a diluent, for example, water, ethyl alcohol, propylene glycol, polyethylene glycol, ethoxylated isostearyl alcohol, polyoxylated iso Vegetable oils such as stearyl alcohol, polyoxyethylene sorbitan fatty acid esters and olive oil can be used. The liquid may also contain injectable organic esters such as ethyl oleate. These may further contain ordinary solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives, dispersants and the like.

  Sterilization of the pharmaceutical preparation can be carried out by, for example, a filtration operation of passing through a bacteria-retaining filter, blending of a bactericide, irradiation treatment and heat treatment. Also, the pharmaceutical preparation can be prepared in the form of a sterile solid composition which can be dissolved in sterile water or a suitable sterilizable medium immediately before use, and sterilized immediately before use.

  For shaping in the form of suppositories or preparations for vaginal administration, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glyceride and the like can be used as carriers.

  Pastes, creams, gels and other ointments may be used as diluents, for example, vegetable oils such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol, polyethylene glycol, silicone, bentonite and olive oil. .

  Preparations for nasal or sublingual administration can be prepared according to a conventional method using well-known standard excipients.

  The pharmaceutical preparation of the present invention may contain a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.

  The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient age, gender and other conditions, degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are administered intravenously, alone or as a mixture with ordinary rehydration solutions such as glucose and amino acids. Intramuscularly, intradermally, subcutaneously or intraperitoneally, suppositories are administered rectally, vaginal is administered intravaginally, nasal is administered intranasally, sublingual is administered orally, Ointments are topically administered transdermally.

  The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and may be appropriately selected from a wide range according to the desired therapeutic effect, administration method, treatment period, patient age, gender and other conditions. Selected. Usually, it is preferable to determine the amount such that the concentration of the active ingredient in the blood is about 1 to 200 μg / mL, more preferably about 10 to 20 μg / mL. The formulation can be administered once or several times a day.

  Hereinafter, the production of the specific antibody to apM1 will be described in detail.The specific antibody to apM1 is an antiserum (polyclonal antibody) and monoclonal antibody using a complex protein containing apM1, a fragment thereof or the like as a hapten as an immunizing antigen. It can be produced as an antibody.

  The method for producing these antibodies is well understood by those skilled in the art, and the method for producing the antibody of the present invention can also follow these conventional methods (for example, the Seikagaku Laboratory Lecture “Immunobiochemical Research Method”, The Japanese Biochemical Society). (1986)).

  More specifically, a monoclonal antibody against apM1 produces, for example, a fusion cell (hybridoma) of a mammalian plasma cell (immune cell) immunized with the above immunizing antigen and a mammalian plasmacytoma cell, and recognizes apM1 therefrom. A clone producing the desired antibody is selected and cultured by culturing the clone.

  The apM1 as the immunizing antigen used in the above is not particularly limited, and is a recombinant apM1 produced according to a known gene recombination technique, a peptide having a partial amino acid sequence thereof, or the like and other carriers. Any of composites may be used. The apM1 is known as an adipose tissue-specific secretion factor, and a protein having the same activity or action as this, for example, GBP28 (Gelatin-binding protein of 28 kDa) is also used as the above-mentioned immunizing antigen. can do.

  The mammal to be immunized with the immunizing antigen in the above method is not particularly limited, and is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion. Generally, mice, rats, rabbits, and the like are used. Is advantageously used.

  Immunization is performed by a general method, for example, by administering the above immunizing antigen to a mammal by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like. Preferably, the immunizing antigen is administered to the test animal several times every 2 to 14 days, optionally in combination with a normal adjuvant, such that the total dose is about 100 to 500 μg / mouse. . As an immunizing antigen, it is preferable to use spleen cells extracted about 3 days after the final administration.

Furthermore, various mammalian plasmacytoma cells as the other parent cells fused with the above immunocytes include already known various cells, for example, p3 (p3 / x63-Ag8) [Nature, 256 , 495-497 (1975)), p3-U1 (Current Topics in Microbiology and Immunology, 81 , 1-7 (1978)), NS-1 (Eur.J. Immunol., 6 , 511-519 (1976)), MPC-11 (Cell, 8 , 405-415 (1976)), SP2 / 0 (Nature, 276 , 269-270 (1978)), FO (J. Immunol.Meth., 35 , 1-21 (1980)), × 63.6 .5.3. [J. Immunol., 123 , 1548-1550 (1979)], S194 [J. Exp. Med., 148 , 313-323 (1978)] and the like, and R210 in rats [Nature, 277 , 131- 133 (1979)].

  The fusion reaction between the above immune cells and plasmacytoma cells can be performed according to a known method, for example, the method of Milstein et al. [Method in Enzymology, Vol. 73, pp. 3 (1981)]. More specifically, the above fusion reaction can be carried out in a usual medium in the presence of a usual fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like. An auxiliary agent such as dimethyl sulfoxide can be added to the medium as needed to further enhance the fusion efficiency.

  The ratio of immune cells to plasmacytoma cells used is not different from the usual method, and for example, it is common to use about 1 to 10 times of immune cells to plasmacytoma cells. Examples of the culture medium for the fusion reaction include various culture mediums usually used for the growth of plasmacytoma cells, such as RPMI-1640 medium, MEM medium, and other culture mediums generally used for such cell culture. Usually, it is better to remove the serum supplement such as fetal calf serum (FCS) from the medium.

  In the fusion, a predetermined amount of the immune cells and the plasmacytoma cells are mixed well in the medium, and a PEG solution pre-warmed to about 37 ° C., for example, a medium having an average molecular weight of about 1000 to 6000, is added to a normal medium. This is done by adding and mixing at a concentration of 30-60 w / v%. Thereafter, a desired hybridoma is formed by successively adding an appropriate medium, centrifuging, and removing the supernatant.

  The desired hybridoma obtained is separated by culturing it in a usual selection medium, for example, a HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT medium may be performed for a time sufficient to kill cells other than the target hybridoma (unfused cells and the like), usually several days to several weeks. The hybridoma thus obtained is subjected to a search for a target antibody and a single cloning by a usual limiting dilution method.

The target antibody-producing strain can be searched, for example, by an ELISA method (Engvall, E., Meth.Enzymol., 70 , 419-439 (1980)), a plaque method, a spot method, an agglutination method, an Ouchterlony method, a radioimmunoassay (RIA) and other methods commonly used for antibody detection ("Hybridoma method and monoclonal antibody", published by R & D Planning, pp. 30-53, March 5, 1982). For this search, the immunizing antigen can be used.

  The thus obtained hybridoma producing a desired monoclonal antibody recognizing apM1 can be subcultured in a usual medium, and can be stored in liquid nitrogen for a long time.

  The desired antibody can be collected from the hybridoma by a method of culturing the hybridoma according to a conventional method to obtain a culture supernatant thereof or a method of administering the hybridoma to a mammal compatible with the hybridoma and growing the same to obtain ascites. Etc. are adopted. The former method is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies.

  The antibody obtained as described above can be further purified by ordinary means such as salting out, gel filtration, affinity chromatography and the like. Thus, a desired apM1 monoclonal antibody having specific reactivity to apM1 is obtained.

  The present invention also provides a technique for measuring apM1 in a sample and a method for diagnosing arteriosclerosis using the technique.

  The diagnosis of arteriosclerosis is performed by measuring the amount of apM1 in a sample obtained from the blood or urine of a patient by a liquid phase or solid phase immunoassay using an antibody against apM1, and measuring the measured value of an arteriosclerosis patient. The comparison is performed by comparing the same value with that of a normal person, that is, by determining whether the amount of apM1 in the sample is higher or lower than that of a healthy person.

  Here, as the immunoassay method, an ELISA method using a sandwich method is preferable.

  The principle of this method is based on the enzyme antibody method. That is, according to this method, for example, first, an anti-human apM1 monoclonal antibody (first antibody) is immobilized on a 96-well plate, and further blocking is performed to prevent non-specific adsorption (immobilization reaction). Next, a human apM1 standard solution or specimen is added to the monoclonal antibody-immobilized plate and reacted (first reaction). After washing the plate, an anti-human apM1 antibody (second antibody) is added and reacted (second reaction). The plate is washed, and an HRP-labeled anti-rabbit IgG antibody (third antibody) is added and reacted (third reaction). After washing the plate, an enzyme reaction is performed by adding a substrate (fourth reaction), and the activity is read as an absorbance at a wavelength of 492 nm.

  In the above, a polyclonal antibody can be used as the first antibody, and a monoclonal antibody can be used as the second antibody.

  According to the above method, the higher the apM1 concentration in the standard solution or specimen used, the stronger enzyme activity (absorbance) is observed. By plotting the absorbance of the above standard solution to prepare a standard curve (calibration curve), and comparing the absorbance of the specimen with the calibration curve, it is possible to easily read apM1 in the specimen, and thus, the specimen is atherosclerotic. A diagnosis can be made as to whether or not the patient has the disease.

  Furthermore, the diagnosis of arteriosclerosis according to the present invention can be easily performed by using a specific kit, and the present invention also provides such a kit.

  Hereinafter, the kit of the present invention and a method for detecting the amount of apM1 in a sample using the kit will be described in detail.

  In the assay method using the kit of the present invention, urine or blood (particularly fasting serum or plasma) is preferable as a specimen, and the specimen can be prepared according to a conventional method after collection and blood collection from a subject.

  The kit according to the present invention contains, as an essential component, a monoclonal antibody or a polyclonal antibody against apM1 (anti-apM1 antibody), and a combination thereof with a polyclonal antibody or a monoclonal antibody against apM1, or a combination of the above monoclonal antibodies. Is preferred.

  The monoclonal antibody is preferably used in the form of a solid-phased plate that is preferably bound to a carrier on a previously immobilized plate. In addition, the gel can be used as it is in the form of an affinity gel, and the gel can be shaken and centrifuged, and prepared in an appropriate container and test tube capable of extracting the affinity gel non-bound fraction. You can also.

  It is also preferable to equilibrate the immobilized plate or gel in advance with an appropriate buffer, for example, a buffer such as 0.01 M Tris-HCl buffer (pH 7.4) +0.15 M NaCl. Further, the immobilized plate or gel may contain a usual preservative such as sodium azide.

  More preferably, a labeled anti-human apM1 polyclonal antibody is provided as a second antibody for ELISA as a further kit component of the invention. The kit of the present invention may contain a stabilizer such as saccharose or bovine serum protein and / or a preservative, if necessary. This preservative is selected from those which do not affect the experimental values when using the kit, and representative examples include, for example, diluted sodium azide and the like.

  The kit of the present invention may contain glycerin, water-soluble or water-miscible, alcohols, glycols, glycoethers, etc., and ethanol and diethyl ether for delipidation, methanol and diethyl ether, chloroform. And a mixed organic solvent such as methanol.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Production of recombinant apM1
(1) expression of apM1 in E. coli
1) apM1 PCR
The nucleotide sequence of the apM1 gene and the amino acid sequence encoded by the gene sequence are registered in a gene bank as accession number D45371. The coding region (CDS) is shown in SEQ ID NOs: 27 to 761. Its deduced amino acid sequence is as shown in SEQ ID NO: 1. In the sequence, positions 1 to 14 are signal peptides, and positions 15 to 244 are mature apM1.

  The apM1 gene was amplified by a PCR method using a plasmid provided by Dr. Funabashi, Osaka University as a template. FIG. 1 shows a restriction map of a plasmid containing the nucleotide sequence of the apM1 gene.

The PCR primer was designed to amplify the 693 bp of No. 69 to 761 in the base sequence of the apM1 gene, to add an NdeI site at the 5 ′ end and a BamHI site at the 3 ′ end, and to use an automatic DNA synthesizer. Manufactured. The sequences of the PCR primers are as shown in SEQ ID NOs: 3 (forward) and 4 (reverse).
2) Subcloning of apM1 gene The PCR product obtained in the above 1) was subcloned into pT7 Blue T-Vector (Novagen), and it was confirmed that the nucleotide sequence (pT7-apM1) had no mutation.
3) Construction of Expression Vector The expression vector pET3c (Novagen) was digested with NdeI and BamHI to obtain a fragment of about 4600 bp. Further, the pT7-apM1 obtained in 1) above was digested with NdeI and BamHI to obtain a fragment of about 700 bp. These fragments were ligated, and the resulting expression vector was designated as pET3c-apM1.
4) Expression in Escherichia coli BL21 (DE3) pLysS which is host Escherichia coli was transformed with pET3c-apM1 obtained in 3), and 2xT.Y.Amp. (Tryptone 16 g, yeast extract 10 g and NaCl 5 g) Cultured. When the cells entered the logarithmic growth phase, IPTG (isopropyl β-D-thiogalactopyranoside) was added to induce the production of recombinant apM1. E. coli before and after induction with IPTG and the inclusion body (insoluble fraction of E. coli) after IPTG induction were sampled, and the expression of apM1 was confirmed by SDS-PAGE and Western blotting.
5) Results and discussion In the expression product expressed in E. coli as described above, in the amino acid sequence of apM1, 230 amino acids from position 15 Gly to position 244 Asn excluding the signal sequence were added with Met derived from the start codon at the N-terminus. ing. Its amino acid sequence is as shown in SEQ ID NO: 2.

  When the E. coli obtained by the above method was analyzed by SDS-PAGE, a band of about 30 kD was confirmed in the E. coli and the inclusion body after IPTG induction.

  Next, when Western blotting was performed with two types of antibodies (polyclonal antibody (synthetic peptide)), both antibodies reacted with the band of about 30 kD, and no reaction was observed with the host E. coli.

  Also, when the band of about 30 kD was cut out and the sequence of the N-terminal 10 amino acids was confirmed, the sequence was the same as the expected sequence, and a minor component in which the N-terminal Met was off was also confirmed.

From the above results, it was revealed that recombinant apM1 was expressed as a protein of about 30 kD. Most of the expressed recombinant apM1 was accumulated in the cells as an inclusion body.
(2) Purification of recombinant apM1 from Escherichia coli Purification of recombinant apM1 from Escherichia coli was performed in the following five steps.
1) Culture of Escherichia coli BL21 (DE3) pLysS (manufactured by Novagen) transformed with the expression vector pET3c-apM1 was used as 2xT.Y.Amp.Cm. (trypton 16 g, yeast extract 10 g, chloramphenicol 25 μg / mL) And 5 g of NaCl) (37 ° C., shaking culture), and on the next day, the culture was diluted with a 100-fold amount of 2 × TY Amp. And further cultured. When the OD550 of the culture solution reached 0.3 to 0.5 after culturing for 2 to 3 hours, IPTG at a final concentration of 0.4 mM was added to induce production of recombinant apM1. About 3 to 5 hours after the addition of IPTG, the culture was centrifuged (5000 rpm, 20 minutes, 4 ° C.), and the resulting E. coli precipitate was cryopreserved.
2) Preparation of an inclusion body from E. coli The precipitate of E. coli was suspended in 50 mM Tris-HCl (pH 8.0), treated with lysozyme at 37 ° C for 1 hour, and then subjected to a final concentration of 0.2% Triton X-100 (Triton X-100). , Manufactured by Katayama Chemical Co., Ltd.). The solution was sonicated (BRANSON SONIFIER, output control 5, 30 sec.) And centrifuged (12000 rpm, 30 minutes, 4 ° C.) to collect a precipitate. The precipitate was suspended in 25 mL of 50 mM Tris-HCl (pH 8.0) to which 0.2% TritonX-100 was added, and subjected to ultrasonic treatment (under the same conditions as above).

The obtained solution was centrifuged, and the precipitate was washed again by the same operation, and the obtained precipitate was used as an inclusion body.
3) Refolding of the inclusion body The inclusion body was solubilized in a small amount of 7 M guanidine hydrochloride, 100 mM Tris-HCl (pH 8.0), 1% 2ME. The solution was diluted dropwise with 200 volumes of 2M Urea, 20 mM Tris-HCl (pH 8.0) and left at 4 ° C. for 3 nights.
4) Concentration of refolding solution The refolded solution was centrifuged (9000 rpm, 30 minutes, 4 ° C), and the obtained supernatant was concentrated about 100-fold by ultrafiltration using Amicon YM-10 membrane. The concentrate was dialyzed against 20 mM Tris-HCl (pH 8.0) and filtered with a 0.45 μm filter.
5) Anion exchange HPLC using DEAE-5PW
The sample obtained in 4) above was separated and purified by anion exchange high performance liquid chromatography (HPLC) using DEAE-5PW (manufactured by Tosoh Corporation). The starting buffer was 20 mM Tris-HCl (pH 7.2), elution was performed with a gradient of 1 M NaCl (0 → 1 M NaCl / 60 mL) and monitored by absorption at 280 nm. Fractions were 1 mL each, and each fraction was analyzed by SDS-PAGE.
6) Results and discussion Since recombinant apM1 was expressed as an inclusion body in Escherichia coli, the inclusion body was solubilized and refolded during purification. As a result, the recombinant apM1 was solubilized and separated on an anion exchange column. When the peak fraction (fraction Nos. 30-37) was analyzed by SDS-PAGE, a band of about 30 kD was observed. At this time, a thin smear band was confirmed in the background, but most of the protein was considered to be recombinant apM1, so this approximately 30 kD band (recombinant apM1) was used for subsequent immunization of rabbits and mice. Used as an antigen.
(3) Preparation of polyclonal and monoclonal antibodies against apM1
1) Preparation of polyclonal antibody Recombinant apM1 100 μg / body was mixed with an equal amount of complete adjuvant and immunized 5 rabbits 8 times every 2 weeks to obtain anti-apM1 polyclonal antibody (recognition number: OCT9101 to OCT9105). Obtained.
2) Preparation of monoclonal antibody Recombinant apM1 was mixed with 20 μg / body of an equal volume of a complete adjuvant, and the mice were immunized three times every two weeks, and finally immunized three days before cell fusion without an adjuvant. Cell fusion between mouse spleen lymphocytes and myeloma cells was performed by the PEG method, and hybridomas were selected in HAT medium.

  Screening of the apM1 antibody producing strain was performed by ELISA using an immunoplate coated with an antigen (recombinant apM1), and hybridomas were cloned by the limiting dilution method.

  Thus, 11 apM1 antibody-producing hybridoma strains named KOCO9101-KOCO9111 were obtained. One of them, the hybridoma (indication by the trustee for identification: KOCO9108), was placed on June 8, 1998 (Hara Deposit Date), 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, Central No. 6 It has been deposited with the National Institute of Advanced Industrial Science and Technology, the Patent Organism Depositary, and the request for transfer from the original deposit to the deposit under the Budapest Treaty was received on October 7, 1998. Its accession number is FERM BP-6542.

The single clone hybridoma was intraperitoneally administered to a pristane-treated mouse to obtain ascites (recognition number: ANOC9101 to 9111).
3) Purification of Antibody The obtained rabbit antiserum (polyclonal antibody) and mouse ascites (monoclonal antibody) were purified using a protein A column.
4) Expression of apM1 in animal cells
The apM1 cDNA was excised with EcoRI and inserted into the EcoRI site of the expression vector pCIneo (Promega). This pCIneo-apM1 was transfected into COS-1 cells (ATCC CRL1650) using LlipofectAMINE from GIBCO BRL, and the culture supernatant and cells after 72 hours were collected.
5) Western blotting of apM1 First, adipose tissue extract, COS-1 cells, COS-1 cell culture supernatant, healthy human plasma, and recombinant apM1 were subjected to SDS-PAGE with 2ME (+) and applied to a nitrocellulose membrane. Transferred.

  This membrane was reacted with an apM1 monoclonal antibody (ANOC9104) and then reacted with an HRP-labeled antibody, and then detection was performed using ECL (Amersham western blotting detection reagent).

  As a result, a band of about 35 kD was confirmed in the adipose tissue extract, pCIneo-apM1 / COS-1 cells and healthy human plasma, but could be confirmed in pCIneo / COS-1 cells and pCIneo / COS-1 cell culture supernatant. Did not.

  In the pCIneo-apM1 / COS-1 cell culture supernatant, a band of 35 kD was confirmed although it was very thin and hard to see.

Measurement of apM1 in sample
1) Western blotting of apM1 Healthy human plasma was diluted 10-fold with PBS, and 5 μl thereof was subjected to SDS-PAGE with 2ME (+) and (−), and transferred to a nitrocellulose filter. After blocking the filter, it was reacted with an anti-apM1 polyclonal antibody (OCT9101 to 9105) diluted to 1000-fold or an anti-apM1 monoclonal antibody (ANOC9101 to 9111) prepared at 5 μg / mL, and then HRP-labeled anti-rabbit IgG antibody or HRP-labeled anti-apM1 antibody. It was reacted with mouse IgG antibody and detected by ECL.

As a result, an apM1 band of about 35 kD in the case of 2ME (+) and about 70 kD in the case of 2ME (-) was confirmed for all the polyclonal antibodies. In the case of the monoclonal antibody, ANOC9104 and ANOC9108 reacted strongly with the apM1 band. From this, ANOC9104 and ANOC9108 were considered to be suitable monoclonal antibodies for use in ELISA.
2) Preparation of apM1 ELISA
The apM1 monoclonal antibody (ANOC9108) was coated on the immunoplate, and after blocking each well, apM1 standard and sample were added and incubated. Each well of the plate was washed, diluted apM1 polyclonal antibody (OCT9104) was added and incubated. Each well of the plate was washed, and diluted HRP-labeled anti-rabbit IgG antibody was added and incubated. Each well of the plate was washed, the well was developed using OPD, and the absorbance at 492 nm was measured. As the standard apM1, a recombinant apM1 expressed in Escherichia coli was purified and its protein content was quantified by a protein assay using BSA as a standard.

As a result, the detection range of the combination of ANOC9108 and OCT9104 was 0.1 ng / mL to 20 ng / mL.
3) Gel filtration and western blotting of normal human plasma When normal human plasma was measured by the apM1 ELISA described above, the concentration that could be obtained from the result of western blotting was not obtained. Therefore, normal human plasma was subjected to gel filtration using Superose12 (Pharmacia), and each fraction was subjected to SDS-PAGE and analyzed by Western blotting using ANOC9104. In addition, the molecular weight marker was subjected to gel filtration under the same conditions, and compared with the position where apM1 was eluted.

As a result, apM1 was broadly eluted in a fraction having a molecular weight of 290 kD or more. This suggests that apM1 in blood associates with other plasma components to form a large molecule of 290 kD or more, which masks the antibody recognition site. Therefore, it was considered that the antibody would be able to react if the serum was treated with a buffer containing SDS, and the following examination was performed.
4) Treatment of plasma sample Plasma was boiled with a buffer containing SDS, and the treatment conditions (boiling time, mixing ratio with SDS buffer) were examined. For boiling time, normal human plasma diluted 10-fold with SDS buffer was boiled for 10 seconds, 30 seconds, 1 minute, 3 minutes, 5 minutes, and 10 minutes, respectively, and diluted 5000 times from the plasma stock solution to apM1. It was measured. Regarding the mixing ratio with the SDS buffer, normal human plasma was diluted to 2 times, 3 times, 5 times, 10 times, and 20 times the volume of SDS buffer, respectively, boiled for 5 minutes, and finally diluted 10,000 times to apM1 Was measured.

As a result, apM1 could be detected at a level consistent with the result of Western blotting of normal plasma. Examination of the treatment conditions at that time revealed that it was appropriate to dilute the plasma 10-fold with SDS buffer and boil it for 5 minutes.
5) Dilution of plasma sample
Since the detection range of apM1 ELISA is 70 pg / mL to 20 ng / mL and the blood concentration of apM1 is in the order of μg, serum (plasma) must be diluted and measured, so determine the appropriate dilution factor. For this purpose, SDS-treated plasma was serially diluted and apM1 was measured. That is, normal human plasma was subjected to SDS treatment and serially diluted from final 200-fold to 100,000-fold to measure apM1.

As a result, it was found that apM1 was detected in proportion to the dilution ratio within the measurement range of ELISA, and it was found that it was appropriate to measure at about 5000-fold final from the absorbance and the like.
6) Examination of blood sampling conditions Blood from normal subjects was collected under serum, heparin, and EDTA sampling conditions, apM1 was measured, and the measured values of apM1 due to differences in blood sampling conditions were examined. That is, blood was collected from 10 normal persons, and serum (plasma) was subjected to SDS treatment and diluted to a final 5000-fold, and apM1 was measured.

As a result, the measured values of apM1 in the blood of the 10 normal subjects tested showed little difference due to the blood sampling method.
7) Examination of sample storage conditions
The storage conditions of the apM1 measurement specimen were examined for the plasma and the sample after the SDS treatment. The plasma was stored at 4 ° C., room temperature and 37 ° C. for 1 day, 2 days, 3 days, and 6 days.The effect of freezing and thawing of the plasma was once, twice, and four times. After repeated freeze-thawing eight times, each was subjected to SDS treatment, diluted to a final 5000-fold, and apM1 was measured. With respect to the sample after the SDS treatment, the plasma was diluted 10-fold with the SDS buffer, boiled for 5 minutes, and the same experiment was performed on the solution diluted 10-fold.

As a result, under the above conditions, the measured value of apM1 was hardly affected. Similarly, freeze-thaw was performed on plasma and samples after SDS treatment, but had almost no effect.
8) Examination of diurnal and diurnal variations Diurnal and diurnal variations in the measurement of apM1 in serum (plasma) of normal subjects were examined. Regarding the daily variation, the same sample was measured eight times to determine the CV value. Regarding the daily fluctuation, the same sample was repeatedly measured four times on different days to obtain a CV value.

As a result, the CV value was within 5% for diurnal variation, and the CV value was within 10% for diurnal variation.
9) Examination of specificity ApM1 ELISA was performed on sera of pigs, cows, horses, goats, rats and mice to examine the specificity of the measurement system. The serum of each animal was treated with SDS buffer, and serially diluted from 100-fold to 8100-fold to measure apM1.

  As a result, about 10% of cross was observed in cattle and horses, but was hardly observed in other animals.

Smooth muscle proliferation inhibitory effect of apM1 Human arterial smooth muscle cells (Clontech) were seeded on a plastic plate at a concentration of 1 × 10 ⁴ / cm ² , and 10% FBS (Gibco), 100 IU / mL penicillin And 100 μg / mL streptomycin in DMEM (Gibco) overnight, and cultured at 37 ° C. in 5% CO ₂ and 95% air.

DNA synthesis by human arterial smooth muscle cells was measured by [methyl ³ H] -thymidine incorporation (repeated four times). That is, cells seeded in a 96-well plate were cultured in DMEM supplemented with 2% FBS at 10 μg / mL apM1 and / or 10 ng / mL HB-EGF for control (recombinant human heparin-binding EGF-like growth factor, R & D (Manufactured by Systems Inc.) for 24 hours. Then [methyl ³ H] -thymidine was added at 1 μCi / well for 5 hours. The treated cells were trypsinized, removed on a glass fiber filter using an automatic cell harvester, and then the amount of [methyl ³ H] -thymidine incorporation was directly measured with a β-counter.

  The results are shown in Figure 3. In the figure, the relative DNA synthesis ratio is plotted on the vertical axis, and the control (control, when no reagent is used), the case of adding apM1 (the present invention), the case of adding HB-EGF (control), and the case of apM1 + HB- Each result in the case of adding EGF (the present invention) is shown by a bar graph.

  From the figure, it is clear that apM1 has an effect of suppressing DNA synthesis (smooth muscle proliferation) of human smooth muscle cells. In other words, the addition of apM1 significantly (p <0.001) suppressed the DNA synthesis of the control, and even in the case of DNA synthesis promoted by the addition of HB-EGF, it was significantly (p <0.001). 0.001).

  This indicates that apM1 is effective as a composition for suppressing smooth muscle growth.

Measurement of blood apM1 level in patients with coronary artery disease Patients with stenosis of 75% or more by coronary angiography (designated as CAD (+)) 24 men and 10 women and patients with only stenosis up to 75% ( The blood was collected from 66 males and 39 females, and the apM1 level in plasma was measured according to the method of the present invention described in Example 2.

At the same time, CT was taken at the level of the navel of each patient, and the abdominal fat area (VFA, Visceral Fat Area, cm ² ) of each patient was calculated from the CT.

  With the plasma apM1 level (μg / mL) as the vertical axis and VFA as the horizontal axis, the relationship between the above measured values and the calculated values was evaluated by a t-test that does not correspond to CAD (+) and values other than CAD (+). did. The result is shown in FIG.

  According to the figure, the apM1 level in the patient group with coronary artery disease (CAD (+)) was low regardless of the size of the intraperitoneal fat area (VFA). It proved to be effective.

  In addition, apM1 was important as an index of the onset and progress of arteriosclerosis, suggesting its usefulness in the application to treatment.

apM1 suppresses the development of arteriosclerosis
Human aortic vascular endothelial cells (HAEC: purchased from Clonetics) seeded on a 96-well plate were cultured in a vascular endothelial cell medium (37 ° C. under 5% CO ₂ ). Using Becton Dickinson's “Biocoat”, the cells were cultured until they became confluent, and then the culture medium was TCM199 (tissue culture medium: Osaka University Microbial Research, Nacalai Tesque) + 0.5% FCS (fetal bovine serum) : Japan Biomaterials) + 3% BSA (bovine serum albumin: Sigma).

  Then, 1, 5, 10, 25 and 50 μg / mL of the recombinant apM1 obtained in Example 1 were added thereto, and the mixture was further cultured for 18 hours. Thereafter, human recombinant TNF-α (Tumor Necrosis Factor, manufactured by R & D) was added. -α: 10 U / mL) and cultured for 6 hours (experimental group with apM1 added).

  As a control group, a group without addition of the recombinant apM1 (TNF-α stimulation only) was provided.

The adhesion molecule protein expression level of VCAM-1 (Vascular Cell Adhesion Molecule-1), ELAM (Endothelial Leukocyte Adhesion Molecule) and ICAM-1 (Intercelular Adhesion Molecule-1) expressed on the HAEC surface by stimulation with the above TNF-α is apM1. Was examined according to the cell-ELISA method (Takami, S., et al., Circulation, 97 (8), 721-728 (1998)). In addition, 6.5B5 of DAKO was used as the ICAM-1 antibody.

  An apM1-added group and a non-added group were compared, and a significant difference test was performed by a Student test.

  The results for VCAM-1 are shown in FIG.

  In FIG. 5, the abscissa represents the amount of apM1 added (μg / mL, − represents no addition), and the ordinate represents the adhesion molecule protein (VCAM-1) expressed by HAEC on the cell surface when TNF-α was not stimulated. 2) shows the relative value of the expression level of the same adhesion molecule protein in each experimental group and control group, using the expression level as a reference (1).

  FIG. 5 shows the following. That is, apM1 significantly suppressed the expression of the adhesion molecule VCAM-1 increased by TNF-α stimulation in HAEC in a concentration-dependent manner from a concentration of 1 μg / mL (p <0.05).

  It was also confirmed that apM1 similarly suppressed the expression of ELAMI and CAM-1, which are other major adhesion molecules.

It has been reported that damage to vascular endothelial cells and concomitant monocyte adhesion is important for the development of arterial effects (Ross, R., Nature, 362 (6423), 801-804 (1993)). The fact that apM1 suppressed the expression of adhesion molecules (VCAM-1, ELAM, ICAM-1, etc.) that regulates the onset process of this arterial effect means that the apM1 acts suppressively on the development of arteriosclerosis. From this, it became clear that apM1 was effective as a preventive agent for arteriosclerosis.

  According to the present invention, a composition for suppressing smooth muscle growth and a composition for suppressing expression of adhesion molecules in vascular endothelial cells, which contain apM1 as an active ingredient, are useful in the pharmaceutical field. Further, according to the present invention, a technique for measuring apM1 using an antibody specific to apM1 is provided, whereby a new diagnostic method for arteriosclerosis can be established.

1 shows a restriction map of a plasmid containing the nucleotide sequence of apM1 gene. 3 shows a standard curve prepared using recombinant apM1. 9 is a graph showing the smooth muscle inhibitory effect of apM1 measured according to Example 3. 9 is a graph showing blood apM1 levels of patients with coronary artery disease and healthy subjects measured according to Example 4. 11 is a graph showing that apM1 suppresses the expression level of an adhesion molecule on a cell surface measured according to Example 5 in a concentration-dependent manner.

Claims (3)

A smooth muscle growth inhibitory composition comprising at least one pharmaceutically effective amount selected from adipose tissue-specific secretion factor apM1 and a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. A composition for preventing and treating arteriosclerosis, comprising at least one pharmaceutically effective amount selected from adipose tissue-specific secretory factor apM1 and a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. Prevention and treatment of restenosis after revascularization including at least one pharmaceutically effective amount selected from adipose tissue-specific secretion factor apM1 and a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier Composition.

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