WO2003062416A1

WO2003062416A1 - Method of screening preventive and remedy for arteriosclerosis

Info

Publication number: WO2003062416A1
Application number: PCT/JP2003/000407
Authority: WO
Inventors: Hiromitsu Fuse; Yoshio Taniyama; Tomoko Satomi
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2002-01-21
Filing date: 2003-01-20
Publication date: 2003-07-31

Abstract

A method of screening a compound or its salt inhibiting changes in the expression of an arteriosclerosis-associated gene due to a modified LDL, a low-molecular compound or a protein contained in the modified, characterized by using the arteriosclerosis-associated gene; a kit for the screening; a compound inhibiting changes in the expression of the arteriosclerosis-associated gene which is obtained with the use of the screening method or the screening kit; preventives and remedies containing the above compound or its salt; etc.

Description

Description Atherosclerosis prevention · Screening method of therapeutic drug

The present invention relates to a method for searching for a compound useful as an arteriosclerosis inhibitor or a regression drug, and more specifically, the main cause of ischemic heart disease and cerebral infarction such as angina pectoris and myocardial infarction, which are the leading causes of death. The present invention relates to a method for screening a compound or a salt thereof for preventing or treating arteriosclerosis, a compound or a salt thereof obtained by using the screening method, and a use of the compound. Background art

The presence of atherosclerosis is important as the basis for ischemic heart disease and cerebrovascular disease, which are the leading causes of death, and are the causes of ischemic organ disease. The pathomorphological characteristics of atherosclerotic lesions are fat streaks, which are mainly composed of macrophages (foamed cells) accumulating cholesterol ester in the subendothelium, and are more advanced and smooth. This is a fibrous plaque with infiltration of muscle cells, macrophages, T cells, etc., cell necrosis, and lipid accumulation. The site of lipid accumulation shows structural weakness, hemodynamic forces trigger plaque rapture, and thrombus rapidly forms due to the reaction between tissue factor and blood coagulation factor. The breakdown of the plaque in the coronary arteries and thrombotic occlusion is closely related to the onset of so-called acute coronary syndrome such as acute myocardial infarction, unstable angina, and sudden cardiac death. (Fuster V et al.,. Engl. J. Med., 326, PP242-250, 1992).

The most important risk factor for developing acute coronary syndrome is serum cholesterol, especially low-density lipoprotein-cholesterol (LDL-C), according to several large epidemiological studies (4S study, CARE study, etc.). 0-(: (^ Evidence that serum cholesterol-lowering therapy with statins, a reductase inhibitor, reduced the incidence of heart disease events, the evidence suggests that LDL is a valid risk factor. Prove Things.

Cell biology studies revealed that macrophage cells take up oxidized LDL, which is a major lipid carrier in blood, modified by a receptor called the scavenger receptor family 1 '. This receptor does not receive negative feedback from intracellular cholesterol levels, and macrophages take up excess oxidized LDL via this receptor to form foam cells.

Hypercholesterolemic patients show hyper-LDLemia, and the oxidizability of LDL has been reported.In recent years, a quantitative system for oxidized LDL in blood using an antibody specific for oxidized LDL has been developed. Increased blood levels in patients at high risk for heart disease, and increased numbers of oxidized LDL-positive macrophage cells at lesion sites (EhardS et al, Circulation, 103, ppl955-1960, 2001) It has been strongly suggested that atherosclerotic lesions are formed at the biological level by the mechanism of foaming of macrophages by oxidized LDL.

Oxidized LDL not only foams macrophage cells, but also has an atherosclerosis-inducing effect on vascular endothelial cells, smooth muscle cells, and macrophage cells. In other words, in vascular endothelial cells, the promotion of infiltration of monocytes into the subendothelium by inducing the expression of adhesion molecules such as ICAM-1 and VCAM-1 (Khan BV et al., J Clin Invest, 95, ppl262-1270, 1995), in smooth muscle cells, promotion of intimal hyperplasia by migration and proliferation (Seinori Mori, Mebio, Vol.16 No.10, pp40-45, 1999), and in macrophage cells, TNF- Inhibition of endothelial cells and smooth muscle cells by the induction of expression of inflammatory site-like proteins such as IL-1 | 8, followed by an atherosclerosis-inducing response of those cells (Chiba et al., Mebio, Vol. 16 No. .10, pp46-50, 1999).

Glycated LDL is one of the late-stage reaction products (AGEs) and is an in-vivo substance that is increased in human circulating blood in patients with hyperlipidemia and diabetes (Akanji AO et al., Clin. Chim. Acta., 317 Pp. 176, 2002). Regarding the relationship between AGE and arteriosclerosis, there has been a report that arteriosclerotic lesion formation was suppressed by administering the extracellular AGE-binding region of the AGE receptor (RAGE) to ApoE-deficient mice (Park L. et al. Nat. Med., 4, PP1025-103K 1998). There are several possible causes of AGE inducing atherogenesis, including activation of NF-KB, p21ras, and MAP kinase in human endothelial cells. And CDC42 / rac have been reported to be activated (Schmidt AM et al., Trends Endocrinol Methabo, 11, p368-375, 2000), and a similar signaling pathway is activated in glycated LDL Have been reported (MariaF. B. et al., Diabetes, 51, 3311-3317, 2002). In other words, glycated LDL is thought to activate NF-κB etc. using its receptor (RAGE) as a transmitter, and induce an inflammatory response. In addition, since AGE accumulation is observed in the subendothelial extracellular matrix, it is considered that AGEs form crosslinks with LDL that has penetrated into the subendothelium and deposit (BrowenleeM et al., Diabetes. 34, pp938-941, 1985). In addition, AGEs have been reported to produce free radicals in macrophage (ΜΦ) cells (Schmidt AM., Et al., Diabetes, 45, pp77-88, 1996), suggesting that the deposited saccharified LDL undergoes oxidative transformation by Μφ cells and scavenger receptors. It is expected that it will be taken in via the evening and work to promote arteriosclerosis formation. '' Disclosure of the Invention

Drugs that suppress or inhibit the physiologic effects of oxidized LDL disrupt the series of cellular injury reactions associated with atherosclerotic lesion formation, resulting in acute coronary artery disease suppression and stabilization of lesions. It is considered to be effective as a novel therapeutic agent for arteriosclerosis that can be expected to prevent the onset of syndrome and prevent recurrence.

The present inventors have made intensive efforts to solve the above problems, and found that among the genes whose expression fluctuates in oxidized LDL, genes related to the onset of arteriosclerosis (arteriosclerosis-related genes), for example, LLPL is expressed by oxidized LDL LLPL-knockout mice are arteriosclerotic lesions due to the progression of arterial sclerosis lesions in genetically modified mice that cause hyperlipidemia, such as double knockout mice with apolipoprotein Ε (ΑροΕ) -knockout mice It was found to work defensively against formation (Japanese Patent Application No. 2001-152520, Japanese Patent Application No. 2001-311971, Japanese Patent Application No. 2002-145876 and PCT / JP 02/04876).

Based on these findings, the present inventors have found that a compound that suppresses LLPL expression reduction by oxidized LDL may inhibit atherosclerosis formation and, consequently, a compound that inhibits arteriosclerosis by modified LDL such as oxidized LDL or glycated LDL. Suppress fluctuations in hardening-related genes It was thought that the compound that could control might be a drug for preventing and treating arteriosclerosis, and as a result of further studies, the present invention was completed.

That is, the present invention

(1) a method for screening a compound or a salt thereof, which suppresses the fluctuation of the expression of the atherosclerosis-related gene by modified LDL or a low-molecular compound or protein contained in the modified LDL, characterized by using an atherosclerosis-related gene;

(2) Expression of the arteriosclerosis-related gene by modified LDL or a low-molecular compound or protein contained in the modified LDL, which is characterized by using cells, tissues or non-human mammals capable of expressing the arteriosclerosis-related gene. A method for screening a compound or a salt thereof,

(3) The screening method according to the above (1) or (2), wherein the expression fluctuation is decreased expression,

(4) The screening method according to any of (1) to (3) above, wherein the atherosclerosis-related gene is a lecithin: cholesterol acyltransferase-like lysophospholipase gene.

(5) The screening method according to any one of (1) to (4), wherein the modified LDL is an oxidized LDL or a glycated LDL.

(6) The screening method according to any one of the above (1) to (4), wherein the modified LDL is an oxidized LDL,

(6a) The screening method according to the above (1) to (4), wherein the modified LDL is a saccharified LDL,

(6b) the screening method according to (6a), wherein the saccharified LDL is LDL saccharified by contact with glucose;

(7) The screening method according to (6), wherein the oxidized LDL is LDL oxidized with a divalent metal ion.

(8) The screening method according to the above (7), wherein the divalent metal ion is copper,

(9) The screening method according to (6), wherein the oxidized LDL is LDL oxidized by contact with a cell derived from a mammal.

(10) Oxidation LDL contacts with divalent metal ions and mammalian cells The screening method according to the above (6), which is LDL oxidized by

(11) The screening method according to the above (6), wherein the oxidized LDL is LDL oxidized by contact with a mammalian oxidase.

(12) The screening method according to (11), wherein the oxidase is lipoxygenase,

(13) The screening method according to the above (2), wherein the cell capable of expressing an arteriosclerosis-related gene is a cell derived from a mammal.

(14) Monocytes derived from peripheral blood derived from mammals, abdominal macrophages derived from mammals, alveolar macrophages derived from mammals, aortic macrophages derived from mammals, aortic macrophages derived from mammals Smooth muscle cells from mammals, endothelial cells from mammals, fibroblasts from mammals, hepatocytes from mammals, dendritic cell lines from mammals, THP-1 (human), U937 (human) , Hep G2 (human), J774A.1 (mouse), L-1 (mouse), RAW2 64.7 (mouse), WEHI (mouse) or P388D1 (mouse). ) Described screening method,

(15) The screening method according to (1) to (14), wherein the low molecular compound contained in the oxidized LDL is at least one selected from oxidized cholesterols, lysophospholipids, ceramides and sphingomyelins.

(16) The screening method according to the above (15), wherein the oxidized cholesterol is 25-hydroxycholesterol or 7-ketocholesterol,

(17) In the presence and absence of a test compound, a cell, tissue or non-human mammal capable of expressing an atherosclerosis-related gene and a modified LDL or a low molecular weight compound or protein contained in the modified LDL And measuring the amount of mRNA encoding an arteriosclerosis-related protein in each case using the atherosclerosis-related gene DNA or its complementary DNA or a partial DNA thereof. 1) to the screening method according to (16),

(18) Contacting cells, tissues or non-human mammals capable of expressing atherosclerosis-related gene with modified LDL or a low molecular weight compound or protein contained in modified LDL in the presence and absence of test compound And in each case The screening method according to any one of (1) to (16), wherein the amount of the intracellular atherosclerosis-related protein or secreted atherosclerosis-related protein is measured.

(19) the screening method according to the above (18), wherein the amount of intracellular atherosclerosis-related protein or secreted arteriosclerosis-related protein is measured by an immunoassay, (20) ability to express atherosclerosis-related gene Contacting cells, tissues or non-human mammals with the modified LDL or low molecular weight compounds or proteins contained in the modified LDL in the presence and absence of the test compound, and in each case atherosclerosis-related protein The screening method according to any one of the above (1) to (16), wherein the activity of

(21) Cells and tissues or non-human mammals transformed with DNA linked to the atherosclerosis-related promoter or enhancer DNA with the repo overnight gene and modified LDL or low LDL contained in modified LDL A molecular compound or a protein is cultured in the presence and absence of a test compound, and a modified LDL or a modified LDL characterized by detecting expression of the reporter gene in each case. A method of screening a compound or a salt thereof, which suppresses a change in the expression of the arteriosclerosis-related gene due to the contained low molecular compound or protein,

(22) Screening for a compound or a salt thereof that suppresses a change in the expression of the atherosclerosis-related gene by a low-molecular compound or protein contained in the modified or modified LDL, characterized by containing an atherosclerosis-related gene. (23) a cell, tissue or non-human mammal capable of expressing an arteriosclerosis-related gene, characterized by a low molecular compound or protein contained in modified DL or modified LDL, A kit for screening a compound or a salt thereof that suppresses fluctuations in the expression of a curing-related gene,

(24) Modified LDL or a low-molecular-weight compound or protein contained in modified LDL, comprising cells transformed with a DNA in which a promoter or enhancer of an atherosclerosis-related gene is linked to a reporter gene. A screening kit for a compound or a salt thereof that suppresses the fluctuation of the expression of the atherosclerosis-related gene by

(25) The screening method according to any one of (1) to (21) above or A compound or a salt thereof, which suppresses a change in expression of the arteriosclerosis-related gene, obtained by using the screening kit according to any one of (22) to (24);

(2 6) A prophylactic-therapeutic agent for arteriosclerosis comprising the compound according to (25) or a salt thereof,

(27) A method for preventing or treating arteriosclerosis, which comprises administering to a mammal an effective amount of the compound or a salt thereof according to (25).

(28) Use of the compound or salt thereof according to the above (25) for producing an agent for preventing or treating arteriosclerosis is provided. BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, an arteriosclerosis-related gene (Atherosclerosis-related gene: AR gene) refers to a gene associated with the onset of arteriosclerosis. For example, adhesion molecules required for monocytes such as ICAM-KVCAM-1 to invade subendothelium (Kume N et al., J Clin Invest, 90, ppll38-1144, 1992), HB-EGF (Nishi et. al., Circ Res, 80, pp668-644, 1997), PDGF (Ochi et al., Circ Res, 77, pp530-535, 1995), M-CSF and GM-CSF (Rajavashisth TB et al., Nature, 344, pp254-257, 1990), growth factors such as MCP-1 (Yla- Herttuala et al., Proc Natl Acad Sci USA, 88, PP5252-5256, 1991), IL-l jS, TNF- Inflammatory sites such as alpha (Tipping PG et al., Am J Pathol, 142, ppl721-1728, 1993), Matrix meta-oral proteases (MMPs) that degrade intercellular matrix (Galis ZS et al., Ann NY Acad Sci, 748, PP501-507, 1995), Scavenger receptor A-1 (SRA-1) (Kodama T et al., Nature, 343, pp531-535, 1990), CD-36 (Nicholson AC et al.) , Arterioscler Thromb Vase Biol, 15, pp269-275, 1995), L0X-1 (Hoshika a H et al., Biochem Biophys Res Commun, 245, pp8 41-846, 1998). Further, the AR gene is not limited to the above-mentioned gene products, but is (1) a gene known in the literature and patent information; AR genes such as LLPL (Taniyama et al .; Biochemical and Biophysical Research) Communications (Biochem. Biophys. Res. Co mun.), 257: 50-56 (1999)); ) Future New AR genes that are found also fall under this category.

The amino acid sequence of mouse LLPL is shown in SEQ ID NO: 1, and its DNA base sequence is shown in SEQ ID NO: 2. The amino acid sequence of human LLP L is shown in SEQ ID NO: 3, and its DNA base sequence is shown in SEQ ID NO: 4.

More preferably, the AR gene is a gene that has been shown to be involved in atherosclerotic lesion formation in genetically modified mice such as transgenic mice and knockout mice, or that is involved in cell biology-proven lesion formation. Genes involved in the function of atherosclerotic lesion formation in endothelial cells, smooth muscle cells or macrophage cells are used.

In addition, the AR gene used in the present invention is a gene that undergoes a variation in expression due to the modified LDL or a low molecular compound or protein contained in the modified LDL. Expression fluctuation includes expression increase and decrease, and in the present invention, a gene whose expression is reduced by a low molecular compound or protein contained in the modified LDL or the modified LDL is preferably used. As the gene whose expression is reduced by the modified LDL or a low-molecular compound or protein contained in the modified LDL, the aforementioned LLPL or the like is used.

In the present specification, the modified LDL refers to oxidized LDL, saccharified LDL, and the like (preferably, oxidized LDL, and the like).

(1) LDL and divalent metals (eg, copper, magnesium, potassium, iron) obtained from peripheral blood of mammals (eg, humans, mice, rats, pigs, horses, sheep, monkeys, etc.) And preferably copper)

(2) contacting the LDL with mammalian-derived cells (eg, culturing),

(3) simultaneously reacting the LDL with the divalent metal and a mammal-derived cell, or

(4) LDL and the like prepared by contacting the LDL with a mammalian oxidase (eg, lipoxygenase).

Saccharified LDL is, for example, '

(1) LDL, which can be obtained from peripheral blood of mammals (eg, human, mouse, rat, pig, pigeon, hidge, monkey, etc.), and glucose are contacted and prepared. Indicates LDL and so on.

In the present specification, the low-molecular compound contained in the modified LDL refers to a low-molecular compound contained in oxidized LDL, a low-molecular compound contained in saccharified LDL, and the like (preferably, a low-molecular compound contained in oxidized LDL). The low-molecular compound contained in the oxidized LDL refers to oxidized cholesterol, lysophospholipids, ceramides, and synthetic analogs thereof. As the oxidized cholesterol, for example, 25-hydroxycholesterol, 22-hydroxycholesterol, 7-ketocholesterol and the like are used, and in addition to the above, hydroxy-, keto- or epoxy-form cholesterol and the like are used. The lysophospholipids include, for example, 1-acyl-sn-glycerol-3-phosphocholine or 1-acyl-sn-glycerol-3-phosphoserine, 2-acyl-sn-glycerol-3-phosphocholine or 2-acyl _sn-Glyceto-3-phosphoserine is used. As ceramides, for example, N-acylsphingosine, ceramide-1-phosphocholine and the like are used.

The low molecular compound contained in saccharified LDL refers to saccharified phospholipids and the like. Examples of the glycated phospholipids include glycated phosphatidylethanolamine, glycated phosphatidylserine, and glycated phosphatidylcholine.

In the present specification, the protein contained in the modified LDL refers to a protein contained in the oxidized LDL, a protein contained in the saccharified LDL, and the like (preferably, a protein contained in the oxidized LDL), and is contained in the oxidized LDL. The protein refers to oxidatively modified apolipoproteins and the like. As oxidized apolipoproteins, for example, oxidized apolipoprotein B and its peptide fragments generated by oxidation are used.

The protein contained in glycated LDL refers to glycated modified apolipoproteins and the like. As the glycated apolipoproteins, for example, glycated apolipoproteins AI—A—] !, B, C-I, and E are used.

The screening method of the present invention is a method for screening a compound or a salt thereof having an activity of controlling the expression of an AR gene, which is characterized by using an AR gene. Characterized by using cells, tissues or non-human mammals having the modified LDL or low LDL contained in the modified LDL. This is a method for screening for a compound or a salt thereof that suppresses fluctuations in the expression of the AR gene by a molecular compound or protein.

More specifically, the screening method of the present invention comprises:

(1) Contacting a cell, tissue or non-human mammal capable of expressing the AR gene with a low-molecular compound or protein contained in the modified LDL or modified LDL in the presence and absence of the test compound, Using the AR gene DNA or its complementary DNA or its partial DNA, the amount of mRNA (hereinafter, sometimes abbreviated as AR mRNA) encoding the AR protein in each case is measured. Screening method for a compound or a salt thereof that suppresses the fluctuation of the expression of the AR gene (hereinafter, abbreviated as screening method A);

(2) Contact a cell, tissue or non-human mammal capable of expressing the AR gene with a low molecular compound or protein contained in the modified LDL or modified LDL in the presence and absence of the test compound, respectively. A method for screening a compound or a salt thereof that suppresses fluctuations in the expression of the AR gene, characterized by measuring the amount of intracellular AR protein or secreted AR protein (extracellular AR protein) in the case of Abbreviated as screening method B),

(3) Contacting a cell, tissue or non-human mammal capable of expressing the AR gene with a low molecular compound or protein contained in the modified LDL or modified LDL in the presence and absence of the test compound, respectively A method for screening a compound or a salt thereof that suppresses fluctuations in the expression of the AR gene (hereinafter, abbreviated as screening method C), which comprises measuring the activity of the AR protein in the case of

(4) Transforming cells, tissues or non-human mammals transformed with DNA in which the promoter or enhancer of the AR gene is linked to a reporter gene and the modified LDL or a low-molecular compound or protein contained in the modified LDL. Culturing in the presence and absence of the test compound, and detecting the expression or activity of the reporter gene in each case. A screening method for a compound or a salt thereof that suppresses the fluctuation of AR gene expression (hereinafter abbreviated as screening method D) To do).

Hereinafter, the screening method of the present invention will be described in detail.

The screening method A of the present invention specifically includes (i) modifying a cell, tissue or non-human mammal having the ability to express the AR gene by modifying LDL and / or a low-molecular compound or protein contained therein. The amount of A RmRNA when cultured in the presence of a bacterium, and (ii) the modification of cells, tissues or non-human mammals capable of expressing the AR gene. Or comparing the amount of ARmRNA when cultured in the presence of the protein and the test compound with the modified LDL and / or the low-molecular-weight compound or protein contained in the AR gene. This is a method for screening a compound or a salt thereof that suppresses the fluctuation of expression.

Cells having the ability to express the AR gene may be, for example, any cells of mammals including humans, plants, and microorganisms, and cells derived from mammals are particularly preferable. Examples of cells derived from mammals include, for example, monocyte macrophages derived from mammals (humans, monkeys, monkeys, monkeys, rabbits, rats, mice, hamsters, etc.), peritoneal macrophages, alveolar macrophages, aortic macrophages , Smooth muscle cells, endothelial cells, fibroblasts, hepatocytes, dendritic cell lines, THP-1 (human), U937 (human), Hep G2 (human), J774 (mouse) , L-1 (mouse), RAW2 64.7 (mouse), WEHI (mouse), and P388D1 (mouse).

The cells having the ability to express the AR gene are cultured in the same manner as in a known animal cell culture method. Examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum (Science, 122, 501 (1952)), DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, Vol. 199, May 19 (1 967)], 199 medium [Proc. Zing-ob-the-Society 'For' the 'Biological Medicine (Proceeding of the Society for the Biological Medicine), Vol. 73, 1 (1950)]. Preferably, the pH is about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and if necessary, aeration and stirring may be added.

The tissue capable of expressing the AR gene may be, for example, any tissue of a mammal including humans or a plant, and particularly preferably a tissue derived from a mammal. Examples of tissues derived from mammals include brain, heart, lung, kidney, liver, kidney, spleen, testis, ovary, and intestine.

As a non-human mammal capable of expressing the AR gene, for example, a mouse, a rat, a pig, a pig, a sheep, a monkey, and the like are used.

In order to compare the expression level of mRNA by the hybridization method, a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press) , 1989).

Specifically, the amount of AR mRNA is measured by contacting RNA extracted from cells with a DNA encoding the AR gene (AR gene DNA) or its complementary DNA or a partial DNA thereof according to a known method. It is performed by measuring the amount of mRNA bound to DNA or its complementary DNA. By labeling the complementary DNA of the AR gene DNA or its partial DNA with, for example, a radioisotope or a dye, the amount of AR mRNA bound to the complementary DNA of the AR gene DNA can be easily measured. As the radioisotope, for example, ³² P, ³ H and the like are used, and as the dye, for example, fluorescein, FAM (manufactured by PE Biosystems), J〇E (manufactured by PE Biosystems), TAMRA (PE Biosystems), ROX (PE Biosystems), Cy5 (Amersham), Cy3 (Amersham) and the like are used.

In addition, the amount of AR mRNA is converted into cDNA by reverse transcriptase from RNA extracted from cells and then amplified by PCR using the DNA encoding the AR gene or its complementary DNA or its partial DNA as a primer. This can be done by measuring the amount of cDNA.

As a complementary DNA of the AR gene DNA used for measuring the amount of AR mRNA, there is a DNA (lower strand) having a sequence complementary to the AR gene DNA (upper strand). The partial DNA of the AR gene DNA is composed of, for example, about 10 to 1,000, preferably about 10 to 300, and more preferably about 10 to 30 bases in the base sequence of the AR gene DNA. Base sequence. The partial DNA of the complementary DNA of the AR gene DNA includes, for example, a DNA having a sequence complementary to the partial DNA of the above-described AR gene DNA. That is, for example, in the base sequence of the AR gene DNA, a sequence complementary to a base sequence composed of about 10 to 1000 consecutive bases, preferably about 10 to 300 bases, and more preferably about 10 to 30 bases And a DNA having:

(1) A test compound that suppresses an increase in the amount of A RmRNA caused by modified LDL and / or a low-molecular compound or protein contained therein or (2) ARmRN A by a modified LDL and / or a low-molecular compound or protein contained therein A test compound that suppresses the decrease in the amount of ARmRNA, preferably a modified LDL and / or a low-molecular compound or protein contained in the test compound, that inhibits atherosclerosis progression or atherosclerosis regression It can be selected as a compound having an effect and used as an agent for preventing and treating arteriosclerosis.

When the AR protein is LLP L, a modified LDL and / or a test compound that suppresses the reduction of LL PLmRNA by the low-molecular-weight compound or protein contained in it has an atherosclerosis progression inhibiting or atherosclerotic regression effect It can be selected as a compound and used as a drug for preventing and treating arteriosclerosis.

In the screening method A of the present invention, examples of the test compound include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. The compound may be a novel compound or a known compound.

Specifically, the screening method B of the present invention comprises: (i) modifying a cell, a tissue or a non-human mammal capable of expressing an AR gene with modified LDL and / or a low-molecular compound or protein contained therein; The amount of intracellular or extracellular AR protein when cultured in the presence, and (ii) modifying LDL and / or contained cells, tissues or non-human mammals capable of expressing the AR gene Cells cultured in the presence of small molecules or proteins and test compounds A method for screening a compound or a salt thereof, which suppresses fluctuations in AR gene expression caused by a modified LDL and / or a low-molecular compound or protein contained therein, characterized by comparing the amount of the intracellular or extracellular AR protein. is there.

The amount of the AR protein can be measured, for example, using an antibody against the AR protein or the like. The measurement method is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen (for example, the amount of the protein) in the test solution is detected by chemical or physical means. However, any measurement method may be used as long as it is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, ^[125 I], ^[13 'I], ^[3 H], ^[14 C] and the like are used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, galactosidase, i3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing a protein or enzyme 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.

In the sandwich method, the test solution is reacted with the insolubilized antibody (primary reaction), and further reacted with the labeled antibody (secondary reaction). Then, the activity of the labeling agent on the insolubilized carrier is measured. The amount of the protein of the present invention in the test solution can be determined. Wear. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the method of insolubilization can be in accordance with those described above.

In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

In the method for measuring a protein or the like by the sandwich method, an antibody used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site such as protein. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the 'C end of the protein, the antibody used in the primary reaction is preferably C An antibody that recognizes other than the end, such as the N-end, is used. It can be used for a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry method. In the competitive method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen, (F) and the labeled antigen (B) bound to the antibody are separated. (BZF separation) Measure the amount of either B or F to quantify the amount of antigen in the test solution. In this reaction method, a soluble antibody is used as the antibody, BZF separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the above antibody, or a solid phase antibody is used as the first antibody. A solid-phase method using a soluble antibody and a solid-phased antibody as the second antibody is used.

In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.

In nephelometry, the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test wave is small and only a small amount of sediment is obtained, laser-nephrometry utilizing laser-scattering is preferably used. In applying these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the protein etc. of the present invention by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, it is possible to refer to review articles, compendiums, etc. [For example, Hiroshi Irie, "Radio Nonotsusei" (Kodansha, published in 1974), Hiroshi Irie "Radio Imunoatsusei" (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Publishing, published in 1978), Eiishi Ishikawa, et al. "Enzyme Immunoassay" (Ed.) Second edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme immunoassay" (3rd edition) (Medical Publishing, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (I thigh leakage chemical 'Techniques (Part A)), ibid.Vol. 73 (Immunochemical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part A) Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods), and Ibid., Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press).

As described above, AR proteins and the like can be quantified with high sensitivity.

(1) a test compound that suppresses an increase in the amount of intracellular or extracellular AR protein due to modified LDL and / or a low molecular compound or a protein contained therein; or (2) a modified LDL and / or a low molecular compound contained therein. A test compound that suppresses a decrease in the amount of an intracellular or extracellular AR protein caused by a protein, preferably a modified LDL and / or the amount of an intracellular or extracellular AR protein caused by a protein contained in a small molecule compound or protein. A test compound that suppresses the decrease in atherosclerosis can be selected as a compound having an atherosclerosis progression-inhibiting or atherosclerotic regression effect, and can be used as an arteriosclerosis-preventing or treating drug.

When the AR protein is LLPL, a modified LDL and / or a test compound that suppresses the decrease in the amount of intracellular or extracellular LLP caused by the low-molecular compound or protein contained in the modified LDL is used to inhibit the progression of atherosclerosis or to prevent Has hardening regression It can be used as a compound to prevent and treat arteriosclerosis. As the test compound, the same compounds as described in [00 11] are used.

Cell culture can be performed in the same manner as known animal cell culture.

Specifically, the screening method C of the present invention comprises the steps of (i) modifying a cell, tissue or non-human mammal capable of expressing an AR gene with modified LDL and / or a low-molecular compound or protein contained therein. The activity of the AR protein when cultured in the presence of (i) modified cells, tissues or non-human mammals that have the ability to express the AR gene, modified LDL and / or low molecular weight compounds or The ability to compare the activity of AR proteins when cultured in the presence of the protein and the test compound is characterized by the variation in the expression of the AR gene by the modified LDL and / or low molecular compounds or proteins contained therein. This is a method for screening a compound to be suppressed or a salt thereof.

The activity of AR protein varies depending on the type of AR protein. For example, when the AR gene is LLPL, phospholipid: cholesterol acyltransferase activity, phospholipase activity, lysophospholipase activity, lysopid: ceramide acyl Examples thereof include transferase activity, PAF hydrolysis activity, PAF transesterification activity, fatty acid ester hydrolysis activity, oxidized phospholipid hydrolysis activity, and cholesterol oxide esterification activity. These activities can be measured according to a method known per se.

(1) A test compound that suppresses the increase in AR protein activity due to modified LDL and / or low-molecular-weight compounds or proteins contained therein, or (2) A test compound that modifies AR protein by modified LDL and / or low-molecular-weight compounds or proteins contained therein. A test compound that suppresses a decrease in activity is preferably a modified LDL and / or a test compound that suppresses a decrease in the activity of AR protein by a low-molecular compound or protein contained in the test compound. It can be selected as a compound and used as a drug for preventing and treating arteriosclerosis.

If the AR protein is LLPL, decrease the activity of the AR protein by the modified LDL and / or the low molecular weight compounds or proteins contained in it. The test compound to be suppressed can be selected as a compound having an atherosclerosis progression inhibiting or atherosclerotic regression effect, and can be used as an agent for preventing and treating arteriosclerosis.

As the test compound, the same compounds as described in [00 11] are used.

Cell culture can be performed in the same manner as known animal cell culture.

The screening method D of the present invention specifically relates to a cell (for example, chondrocyte, fibroblast) transformed with a DNA in which a promoter or an enhancer region of an AR gene is linked upstream of an appropriate reporter gene. Cells (eg, mouse fibroblast cell line C3H10T1 / 2), myoblasts (eg, mouse myoblast cell line C2C12), etc. are cultured in the presence of the test compound, and replaced with AR gene expression. Detecting the expression (eg, expression level or activity) of a repo overnight gene using a modified LDL and / or low-molecular-weight compounds or proteins contained in it Screening method of compound or its salt to suppress, and finally compound which suppresses fluctuation of expression of AR gene by modified LDL and / or low molecular weight compound or protein contained therein The other is a screening method of the salt.

The promoter or enhancer region of the AR gene may be a known one, or may be one cloned from a genomic DNA.

As the reporter gene, for example, j3-galactosidase gene (1 a c Z), chloramphenicol acetyltransferase gene (cat), luciferase gene, soluble alkaline phosphatase gene and the like are used.

By measuring the expression level or activity of a reporter gene product (eg, mRNA, protein) using known methods, the reduced LDL and / or the low molecular weight compound or protein contained in A test compound that increases the expression level or activity of the target gene product is selected as a compound that has the effect of controlling (particularly promoting) the activity of the promoter or enhancer of the AR gene, that is, a compound that promotes the expression of the AR gene it can.

Conversely, a modified LDL and / or a test compound that decreases the amount or activity of a reporter gene product increased by a protein or a low molecular weight compound contained in it modulates the activity of the AR gene promoter or enhancer ( In particular, select compounds that have an inhibitory action, that is, compounds that suppress AR gene expression. Can be

When the AR protein is LLPL, a modified LDL and / or a test compound that suppresses a decrease in the expression level or activity of a reporter gene product due to the low-molecular-weight compound or protein contained in the modified LDL is used as a promoter for the AR gene. Compounds that promote the activity of enhancers, i.e., compounds that promote the expression of the AR gene and inhibit the progression of atherosclerosis or have the effect of atherosclerosis regression, can be used as drugs for preventing and treating arteriosclerosis .

As the test compound, those similar to those described in [001] are used.

Cell culture can be performed in the same manner as known animal cell culture.

In addition, instead of the repo overnight gene, a drug resistance gene such as a neomycin resistance gene or a hygromycin resistance gene can be introduced, and the screening method D can be performed using the number of surviving cells as an index.

The compound selected by the screening method of the present invention, the compound or a salt thereof (preferably a pharmaceutically acceptable salt) which suppresses the fluctuation of the expression of the AR gene due to modified LDL, etc., may be a mammal (human, monkey, bush, It has a defoaming-promoting action, an arteriosclerosis-inhibiting action, an arteriosclerosis-regression action, etc. against egrets, rats, mice, and Hamus Yuichi. It can be used as a medicament such as an inhibitor or an arteriosclerosis regression agent.

Furthermore, since these compounds or salts thereof have a lipid-rich plaque regression effect, acute myocardial infarction in mammals, acute coronary syndromes such as unstable angina, peripheral arterial occlusion, and percutaneous coronary angioplasty It is also useful as a preventive and remedy for restenosis after surgery (PTCA).

The pharmaceutical composition of the present invention may be prepared using known excipients and carriers, if necessary, for example, as a solid or liquid pharmaceutical composition for oral administration or parenteral administration, according to a formulation technique known per se, It can be administered to mammals. The dose can be appropriately selected depending on the selected individual compound.

More specifically, the selected compound or a salt thereof can be used orally or parenterally by injection, infusion, inhalation, rectal injection or topical administration, as it is or in a pharmaceutical composition. Formulations (eg powders, granules, tablets, pills, Capsules, injections, syrups, emulsions, elixirs, suspensions, solutions, etc.). That is, the compound or a salt thereof can be used alone or as a mixture with a pharmaceutically acceptable carrier (adjuvant, excipient, excipient, Z or diluent, etc.).

These pharmaceutical compositions can be formulated according to ordinary methods. Such preparations can be usually produced by mixing and kneading the active ingredient with additives such as excipients, diluents and carriers. As used herein, parenteral includes subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, infusion, and the like.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be prepared according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as an aqueous solution. Permissible vehicles or solvents that can be used include water, Ringer's solution, isotonic saline and the like. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any volatile oil or fatty acid can be used, including natural or synthetic or semi-synthetic fatty oils or acids, and natural or synthetic or semi-synthetic mono-, di- or triglycerides.

Suppositories for rectal administration consist of the drug and suitable nonirritating excipients, such as cocoa balm and polyethylene glycols, which are solid at room temperature but liquid at intestinal tract and melt in the rectum, It can be manufactured by mixing with a substance that releases a drug.

In addition, a suitable base (eg, butyric acid polymer, glycolic acid polymer, butyric acid-glycolic acid copolymer, mixture of butyric acid polymer and glycolic acid polymer, polyglycerol fatty acid ester, etc.) It is also effective to form a sustained-release preparation in combination with the above. Examples of solid dosage forms for oral administration include those described above such as powders, granules, tablets, pills, and capsules. Formulations in such a dosage form include the active ingredient compound and at least one additive such as sucrose, lactose (lactose), cellulosic sugar, mannitol (D-mannitol), maltitol, dextran, dentine. Puns (eg, corn starch), microcrystalline cellulose, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic Can be produced by mixing and / or kneading with a polymer or glyceride. Such dosage forms may also comprise, as usual, further additives, such as inert diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid Antioxidants such as heart tocopherol, cysteine, disintegrants (eg, croscarmellose sodium), binders (eg, hydroxypropylcellulose), thickeners, buffering agents, sweeteners, flavoring agents Agents, perfume agents and the like. Tablets and pills can also be prepared by enteric coating. Liquid preparations for oral administration include pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, solutions and the like, which are inert diluents commonly used in the art, such as water and water. It may contain additives. These oral liquid preparations can be manufactured according to a conventional method such as mixing the active ingredient compound with an inert diluent and, if necessary, other additives. For oral administration, the amount of the active ingredient is usually about 0.01 to 99 W%, preferably about 0.1 to 90%, usually about 0.5 to 50 W%, depending on the dosage form. It is good to mix.

Dosage for a particular patient will depend on age, weight, general health, gender, diet, time of administration, mode of administration, rate of excretion, combination of drugs, and the severity of the condition the patient is treating at the time. , Depending on these and other factors. The daily dose of these pharmaceutical compositions varies depending on the condition and weight of the patient, the type of compound, the administration route, and the like. For example, when used as an arteriosclerosis inhibitor, an adult (with a body weight of about 60 kg) 1 The daily dose is about l-500 mg, preferably about 10-20 mg as an active ingredient in the case of an oral preparation, and about 0.1-200 mg in the case of a parenteral preparation as an active ingredient. It is 10 O mg, preferably about 1-5 O mg, usually about 1-2 O mg.

In the treatment of these diseases, the compounds or salts thereof selected by the screening method of the present invention may be used alone for treatment, or may be other lipid-lowering drugs or cholesterol-lowering drugs, cardioprotective drugs, Drugs for coronary artery disease, drugs for diabetes, drugs for hypothyroidism, drugs for nephrotic syndrome or treatment for chronic renal failure It may be used together with other pharmaceutical ingredients including drugs. In this case, these compounds are preferably administered as an oral preparation, and if necessary, may be administered as a rectal preparation in the form of suppositories. Possible combination components in this case include, for example, fibrates (eg, clofibrate, benzafibrate, gemfiprodil, etc.); nicotinic acid, derivatives and analogs thereof (eg, acipimox and propcol); bile acids Binding resin [eg, cholestyramine, colestipol, etc.]; Compounds that suppress cholesterol absorption [eg, sitosterol, neomycin, etc.]; Compounds that inhibit cholesterol biosynthesis [eg, oral bath, simbas, pravastin HMG—CoA reductase inhibitors]; squalene synthase inhibitors [eg, benzoxases described in EP 0 670 720, WO 97/120 24, etc.] Pin derivative etc.] squalene epoxidase inhibitors [eg, NB-598 and related compounds etc.].

Yet another possible combination component is an oxoxide squalene-lanosterol cyclase, such as a decalin derivative, an azadecalin derivative, an indane derivative, and the like. Antidiabetic drugs: Actos, Rosiglidason, Kinedak, Benfil, Hyumalin, Ouidalcon, Dalimicron, Daoneil, Noporin, Monoid, Insulinins, Darcoby, Jimerin, Rustinone, Basilcon, Demerin S, Isgilin;

Drugs for the treatment of hypothyroidism: dry thyroid (thyreoid), repothyroxine sodium (thyrazin S), liothyronidine sodium (thyronine, thyromin); drugs for nephrotic syndrome: prednisolone (prednin), prednisolone sodium succinate (Prednine), methylprednisolone sodium succinate (sol-medrol), betamethasone (lindelone);

Anticoagulant: dipyridamole (versantin), dilazep hydrochloride (comerian), tylopidine, clovidogrel, Xa inhibitor;

Drugs for chronic renal failure: diuretics [eg, furosemide (Lasix), bumetanide (Lunetron), azosemide (Diart)], antihypertensives (eg, ACE inhibitors, (enalapril maleate (Lenibeis))) and When administered in combination with Ca antagonists (manidipine), sperm receptor blockers, AII antagonists (candesartan), etc. Preferably it can be used for oral administration.

Furthermore, the compounds or salts thereof selected by the screening method of the present invention have lipid, litchi, and plaque regression, and are therefore useful as agents for preventing and treating thrombus formation. They may be used alone or in combination with the following known therapeutic agents, preferably by oral administration.

Antithrombotic drugs: anticoagulants [eg, heparin sodium, heparin calcium, perhalin calcium (ヮ -farin), Xa inhibitors], thrombolytics

[E.g., tPA, perokinase], antiplatelet drugs [e.g., aspirin, sulfinpyrazolo (anturane), dipyridamole (persantin), ticlopidine (panaludine), siloxenzozol (pletar), GPIIb / IIIa antagonist ( Leopro)];; Coronary vasodilators: difludipine, diltiazem, nicoladil, nitric acid; cardioprotective drugs: oral drugs for cardiac AT P-K, endothelin antagonists, perotinsin antagonists. In this specification, when bases and amino acids are indicated by abbreviations, I UPAC—

It is based on the abbreviation by IUB Commision on Biochemical Nomenclature or the abbreviation commonly used in the relevant field. When amino acids may have optical isomers, L-form shall be indicated unless otherwise specified.

DNA deoxylipo nucleic acid

c DNA complementary deoxylipo nucleic acid

A adenine

T thymine

G Guanin

C Sitshin

RNA liponucleic acid

mRNA messenger liponucleic acid

G 1 y: glycine

A la: Alanine

V a 1: Valine

Leu: Leucine

lie: Isoloy, ,, Ser: Serino

丄 ΐΊ Γ: Threon

C y s •, '"■ ノ,,

: Suaino

Me t: Mena Saino

G 1 u: Cretanic acid

As p: Ashafquinoic acid

L ys _β ,,,

A r g: Arginine

H i s: Histidine

Phe: Hueni J Lealanine

Ty r: Tyrosine

T r p: Tribute fan

Pro: Proline

A s n: Asparagine

G l n: Glutamine

The sequence numbers in the sequence listing in the present specification indicate the following sequences.

[SEQ ID NO: 1]

2 shows the amino acid sequence of mouse-derived LLPL.

[SEQ ID NO: 2]

_C indicates the nucleotide sequence of DNA encoding mouse-derived LLP L [SEQ ID NO: 3]

2 shows the amino acid sequence of human-derived LLPL.

[SEQ ID NO: 4]

1 shows the nucleotide sequence of DNA encoding human-derived LLP L. [SEQ ID NO: 5]

The mouse primers used in Example 1 are shown.

[SEQ ID NO: 6]

1 shows the mouse primer used in Example 1.

[SEQ ID NO: 7] 1 shows a mouse probe used in Example 1.

[SEQ ID NO: 8]

The mouse primer used in Example 5 is shown.

[SEQ ID NO: 9]

The mouse primers used in Example 5 are shown.

[SEQ ID NO: 10]

13 shows a mouse probe used in Example 5. Example

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

Example 1 Confirmation of L L P L expression decrease by oxidized L D L

According to the method of Hakamada et al. (Experimental Medicine Vol. 14, No. 12, Circulation Research Protocol, pp. 49-52 (1996)), peritoneal MAC and oral phage were obtained from C57BL / 6J mice after thioglycolate stimulation. Was prepared. Following the method of Yamamura et al. (Shinsei Kagaku Kenkyusho 4, pp.187-206 (1993)), the LDL was adjusted from ゥ sagi serum, and the method of Hakamada et al. Research Strategies, pp. 36-41

(1996)), oxidized LDL was prepared. Peritoneal macrophage cells were cultured in DMEM-25 mM HEPES (pH 7) medium containing oxidized LDL for 24 hours. After preparing total RNA from cells cultured for a predetermined time using RNeasy Mini Kit (QIAGEN), cDNA was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystemsth). Using the prepared cMA as type III, the oligomers of SEQ ID NO: 5 and SEQ ID NO: 6 as primers, and the oligomer of SEQ ID NO: 7 as probe (5, -FAM-TAATGATGCTGGCAGACCTGACGCTC-Tamra-3,) One copy of LLPL was determined by the TaqMan PCR method. Also, using TaqMan Rodent GAPDH Control Reagents (VIC Probe) (Applied Biosystems), the number of GAPDH copies was determined by the TaqMan PCR method, the LLPL copy number was corrected as the GAPDH ratio, and comparison with vehicle addition was performed. Was. Note that PBS was used as a vehicle. Table 1 shows the results.

table 1

* 1, no significant difference (Williams test result) (n = 3)

, Significant difference (Williams test result) (n = 3)

* 3, Daricelaldehyde-3-phosphate dehydrogenase As shown in Table 1, it was revealed that the expression level of LLPL did not fluctuate with LDL, but that oxidized LDL showed a dose-dependent decrease. This indicates that LLPL is a gene whose expression fluctuates specifically in oxidized LDL.

Example 2 Confirmation of L LPL expression decrease by cholesterol oxide (25-hydroxycholesterol)

Mouse peritoneal macrophage cells prepared in the same manner as in Example 1 were cultured for up to 24 hours using a DMEM-25 mM HEPES (pH 7) medium containing a test compound. Same as Example 1! After preparing tall RNA and cDNA, the expression level of LLPLL was quantified by TaqMan PCR method, corrected for GAPDH expression level, and compared with vehicle addition. The vehicle used was ethanol. Table 2 shows the results.

Table 2

* 1, no significant difference (Williams test result) (n = 3)

, Significant difference (Williams test result) (π = 3)

* 3, Daricelaldehyde-3-phosphate dehydrogenase As shown in Table 2, it was revealed that LLPL expression was reduced in a dose-dependent manner by 25-hydroxycholesterol, a typical oxidized cholesterol contained in oxidized LDL.

Example 3 Confirmation under LLP L expression level by lysophospholipid (L-α-palmitoyl lysophosphatidylcholine)

Mouse peritoneal macrophage cells prepared in the same manner as in Example 1 were cultured for up to 24 hours in a DMEM-25 mM HEPES (pH7) medium containing a test compound. After preparing total RNA and cDNA in the same manner as in Example 1, the expression level of LLPL was quantified by the TaqMan PCR method, corrected based on the expression level of the GAP dish, and compared with vehicle addition. The vehicle used was ethanol. Table 3 shows the results.

Table 3

* 1, L-

Lucolin

* 2, Daricelaldehyde-3-phosphate dehydrogenase As shown in Table 3, it is clear that L-a-palmitoyl lysophosphatidelcholine, a typical lysophospholipid contained in oxidized LDL, reduces LLPL expression. It became.

Example 4 Confirmation of L L P L protein decrease by oxidized L D L

Mouse peritoneal macrophage cells prepared in the same manner as in Example 1 were cultured for 24 hours in a DMEM-25 mM HEPE S (H7) medium containing a test substance. Furthermore, the medium was replaced with a DMEM-25 mM HEPE S (pH 7) medium containing a new test substance, and the cells were further cultured for 72 hours. i) Add SDS to the culture supernatant from which cells, etc., have been removed by centrifugation to a final concentration of 0.11%. did. ii) The cells were lysed with a lysis buffer (2 OmMT ris-15 OmM NaC 1-0.1% SDS-0.1% NaN3, pH 7.4), and then lysed. C, treated for 2 minutes was used as a sedimentation-free sample. Immunoprecipitation and Wesseven analysis were performed according to standard methods. The PCL02 polyclonal antibody was used for immunoprecipitation of LLPL, and the mlG antibody was used for detection of LLPL protein. For the quantification of LLPL, the LLPL band developed with alkaline phosphatase was analyzed densitometrically, quantified and examined. Table 4 shows the results.

Table 4

As shown in Table 4, it was revealed that the amount of intracellular LLPL and secreted LLPL protein was reduced to less than one-half in an oxidized LDL-specific manner.

Example 5 Confirmation of LLPL expression decrease by oxidized LDL in human aortic endothelial cells ao

Normal human aortic vascular endothelial cells provided by Clonetics were used. The oxidized LDL was prepared in the same manner as in Example 1. Normal human aortic endothelial cells were cultured for 24 hours using EBM-2 medium (Clonetics) supplemented with EGM-2 SingleQuots containing oxidized LDL. Using the RNeasy Mini Kit (QIAGEN) from the cells cultured for a predetermined period of time! ^ After preparing total RNA, cDNA was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystems II). Using the prepared cDNA as type I, the oligomers of SEQ ID NO: 8 and SEQ ID NO: 9 as primers, and the oligomer of SEQ ID NO: 10 as a probe (5, -FAM-TGGAGTTCCTGGACCCCAGCAAAA-Tamra-3,) The copy number of LLPL was determined by the TaqMan PCR method. In addition, the copy number of GAPDH was determined by TaqMan PCR using TaqMan GAPDH Control Reagents (Applied Biosystems), The GAPDH ratio was corrected for the number of LLP L copies, and compared with vehicle addition. Note that vehicle: PBS was used. Table 5 shows the results

Table 5

* 1, significant difference (Dannet test result) (n = 3)

* 2, Glyceraldehyde-3-phosphate dehydrogenase As shown in Table 5, it was revealed that the LLPL expression level was reduced by oxidized LDL. This indicates that LLPL is a gene whose expression is fluctuated by oxidized LDL even in human aortic endothelial cells.

Example 6 Confirmation of decreased LLPL expression by glycated LDL in mouse peritoneal macrophage cells

In the same manner as in Example 1, peritoneal macrophages are prepared from C57BL / 6J mice. LDL was adjusted according to the method of Yamamura et al. (Shinsei Kagaku Kenkyusho 4, pp.187-206 (1993)), and then adjusted to Hagida's method (Cell Engineering Separate Volume Medical Experiment Manual Series 2, Arteriosclerosis + High Glycated LDL is prepared according to the Lipidemia Research Strategy, pp. 36-41 (1996)). Glycated LDL-containing DMEM— Total RNA prepared from peritoneal macrophage cells cultured in 25 mM HEPES (pH 7) ± field for 24 hours using RNeasyMini KiKQIAGEN (TaqMan Reverse Transcription Reagents)

Prepare cDNA using (Applied Biosystems). Using the prepared cDNA as type III, determine the number of copies of LLPL by TaqMan PCR. In addition, the copy number of GAPDH is determined by TaqMan PCR method using TaqMan Rodent GAPDH Control Reagents (VIC Probe) (Applied Biosystems), and the LLPL copy number is corrected as the GAPDH ratio, and comparison with vehicle is performed. Note that PBS is used as the vehicle.

By using such a technique, it was confirmed that LLPL expression was reduced by glycated LDL. Do.

Formulation Example 1 Capsule

(1) Compound A 10 mg

(2) Lactose 9 Omg

(3) Microcrystalline cellulose 7 Omg

(4) Magnesium stearate 10 mg

1 capsule 18 Omg

After mixing the total amount of each of (1), (2) and (3) and half the amount of (4), the mixture was granulated. The remaining (4) was added thereto, and the whole was encapsulated in a gelatin capsule to obtain a capsule.

Formulation Example 2 Tablet

(1) Compound A 1 Omg

(2) Lactose 35 mg

(3) Corn starch 150 mg

(4) Microcrystalline cellulose 3 Omg

(5) Magnesium stearate 5 mg

1 tablet 23 Omg

The 2Z3 amount of all j (4) and (2) of (5) in (1), (2) and (3) were mixed and granulated. The remaining (4) and (5) were added to the granules and pressed into tablets.

Formulation Example 3 Injection

(1) Compound A 1 Omg

(2) Inosit l O Omg

(3) Benzyl alcohol 2 Omg

1 ampoule 13 Omg

(1), (2) and (3) were dissolved in distilled water for injection to a total volume of 2 ml and sealed in ampules. All steps were performed under aseptic conditions.

Compound A in Formulation Examples 1 to 3 above refers to a compound selected by the screening method of the present invention or a salt thereof. Industrial applicability

Efficient use of the modified LDL of the present invention and a method of screening for a compound for suppressing atherosclerosis-related gene expression caused by a low-molecular compound or protein contained therein and a salt thereof for preventing or treating atherosclerosis. You can choose.

Claims

The scope of the claims

I. A method for screening a compound or a salt thereof that suppresses a change in expression of the atherosclerosis-related gene by a low-molecular compound or protein contained in the modified LDL or the modified LDL, which comprises using an atherosclerosis-related gene.

2. Modified LDL or a low molecular weight compound or protein contained in the modified LDL, which is characterized by using cells, tissues or non-human mammals capable of expressing the arteriosclerosis-related gene. A method for screening a compound or a salt thereof that suppresses fluctuations in the expression of E. coli.

3. The screening method according to claim 1, wherein the variation in expression is a decrease in expression.

4. The screening method according to claim 1, wherein the atherosclerosis-related gene is a lecithin: cholesterol acyltransferase-like lysophospholipase gene.

5. The screening method according to claim 1, wherein the modified LDL is oxidized LDL or saccharified LDL.

6. The screening method according to claim 1, wherein the modified LDL is an oxidized LDL.

7. The screening method according to claim 6, wherein the oxidized LDL is LDL oxidized with a divalent metal ion.

8. The screening method according to claim 7, wherein the divalent metal ion is copper.

9. The screening method according to claim 6, wherein the oxidized LDL is LDL oxidized by contact with a cell derived from a mammal.

10. The screening method according to claim 6, wherein the oxidized LDL is LDL oxidized by contact with a divalent metal ion and a cell derived from a mammal.

I I. The screening method according to claim 6, wherein the oxidized LDL is LDL oxidized by contact with a mammalian oxidase.

12. The screening method according to claim 11, wherein the oxidase is lipoxygenase.

13. The screening method according to claim 2, wherein the cell capable of expressing an atherosclerosis-related gene is a cell derived from a mammal.

14. Cells having the ability to express atherosclerosis-related genes are monocyte macrophages derived from peripheral blood derived from mammals, peritoneal macrophages derived from mammals, alveolar macrophages derived from mammals, aortic macrophages derived from mammals, mammals Animal origin Smooth muscle cells, mammalian endothelial cells, mammalian fibroblasts, mammalian hepatocytes, mammalian dendritic cell lines, THP-1 (human), U937 (human), Hep G2 (human), J774 A.1 (mouse), L1-1 (mouse), RAW 264.7 (mouse), WEHI (mouse) or P388D1 (mouse). Screening method.

15. The screening method according to claim 1, wherein the low molecular compound contained in the oxidized LDL is at least one selected from oxidized cholesterols, lysoline lipids, ceramides, and sphingomyelins.

16. The screening method according to claim 15, wherein the oxidized cholesterol is 25-hydroxycholesterol or 7-ketocholesterol.

17. The screening method according to claim 1, wherein the modified LDL is glycated LDL.

18. The screening method according to claim 17, wherein the saccharified LDL is LDL saccharified by contact with glucose.

19. Contacting a cell, tissue or non-human mammal capable of expressing an atherosclerosis-related gene with modified LDL or a low-molecular compound or protein contained in modified LDL in the presence and absence of a test compound, The method according to any one of claims 1 to 18, wherein the amount of mRNA encoding atherosclerosis-related protein in each case is measured using atherosclerosis-related gene DNA or its complementary DNA or its partial DNA. Screening method.

20. Contacting cells, tissues or non-human mammals capable of expressing atherosclerosis-related genes with modified LDL or low molecular weight compounds or proteins contained in modified LDL in the presence and absence of test compound 19. The screening method according to claim 1, wherein the amount of intracellular atherosclerosis-related protein or secreted atherosclerosis-related protein in each case is measured.

2 1. The screening method according to claim 2, wherein the amount of intracellular atherosclerosis-related protein or secreted atherosclerosis-related protein is measured by immunoassay.

22. Contacting cells, tissues or non-human mammals capable of expressing atherosclerosis-related gene with modified LDL or low molecular weight compounds or proteins contained in modified LDL in the presence and absence of test compound , Dynamics in each case The screening method according to any one of claims 1 to 18, wherein the activity of a pulse-sclerosis-related protein is measured.

2 3. Cells, tissues or non-human mammals transformed with DNA in which the promoter or enhancer of the atherosclerosis-related gene is linked to the repo overnight gene and modified LDL or a low molecular weight compound contained in modified LDL or A modified LDL or a low LDL contained in the modified LDL, which is characterized by detecting the expression of the repo overnight gene in each case by culturing the protein with the evening protein in the presence and absence of the test compound. A method for screening a compound or a salt thereof, which suppresses a change in expression of the arteriosclerosis-related gene due to a molecular compound or protein. ,

24. A kit for screening a compound or a salt thereof that suppresses a change in the expression of the atherosclerosis-related gene caused by the modified LDL or a low-molecular compound or protein contained in the modified LDL, characterized by containing the atherosclerosis-related gene. .

25. The atherosclerosis-related gene by modified LDL or a low molecular compound or protein contained in modified LDL, characterized by containing cells, tissues or non-human mammals capable of expressing atherosclerosis-related gene A kit for screening for a compound or a salt thereof that suppresses fluctuations in expression of a compound.

26. Modified LDL or a low-molecular-weight compound contained in modified LDL, which contains cells transformed with a DNA in which a promoter or enhancer of atherosclerosis-related gene is linked to a repo allele gene. A screening kit for a compound or a salt thereof that suppresses fluctuations in the expression of the atherosclerosis-related gene due to protein.

27. Suppresses fluctuations in the expression of the arterial stiffness-related gene obtained using the screening method according to any one of claims 1 to 23 or the screening kit according to any one of claims 24 to 26 Or a salt thereof.

28. An agent for preventing or treating arteriosclerosis comprising the compound according to claim 27 or a salt thereof.

29. A method for preventing and treating arteriosclerosis, which comprises administering to a mammal an effective amount of the compound according to claim 27 or a salt thereof.

30. Use of the compound or a salt thereof according to claim 27 for producing an agent for preventing or treating arteriosclerosis.