WO2003062416A1 - Procede de criblage d'agents preventifs et de remedes pour l'arteriosclerose - Google Patents

Procede de criblage d'agents preventifs et de remedes pour l'arteriosclerose Download PDF

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Publication number
WO2003062416A1
WO2003062416A1 PCT/JP2003/000407 JP0300407W WO03062416A1 WO 2003062416 A1 WO2003062416 A1 WO 2003062416A1 JP 0300407 W JP0300407 W JP 0300407W WO 03062416 A1 WO03062416 A1 WO 03062416A1
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Prior art keywords
ldl
compound
atherosclerosis
protein
screening method
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PCT/JP2003/000407
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English (en)
Japanese (ja)
Inventor
Hiromitsu Fuse
Yoshio Taniyama
Tomoko Satomi
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Takeda Chemical Industries, Ltd.
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Publication of WO2003062416A1 publication Critical patent/WO2003062416A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 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.
  • 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.
  • LDL-C low-density lipoprotein-cholesterol
  • Hypercholesterolemic patients show hyper-LDLemia, and the oxidizability of LDL has been reported.
  • 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.
  • 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
  • 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).
  • AGE late-stage reaction products
  • 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).
  • AGE inducing atherogenesis 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- ⁇ B etc. using its receptor (RAGE) as a transmitter, and induce an inflammatory response.
  • RAGE receptor
  • 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.
  • LLPL arteriosclerotic lesions due to the progression of arterial sclerosis lesions in genetically modified mice that cause hyperlipidemia, such as double knockout mice with apolipoprotein ⁇ ( ⁇ ) -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).
  • 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.
  • 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,
  • (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.
  • 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 prophylactic-therapeutic agent for arteriosclerosis comprising the compound according to (25) or a salt thereof,
  • an arteriosclerosis-related gene refers to a gene associated with the onset of arteriosclerosis.
  • 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.
  • MCP-1 Yla- Herttuala et al., Proc Natl Acad Sci USA, 88, PP5252-5256, 1991
  • IL-l jS TNF- Inflammatory sites such as alpha
  • MMPs Matrix meta-oral proteases
  • 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.
  • 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.
  • 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.
  • the modified LDL refers to oxidized LDL, saccharified LDL, and the like (preferably, oxidized LDL, and the like).
  • LDL and divalent metals obtained from peripheral blood of mammals (eg, humans, mice, rats, pigs, horses, sheep, monkeys, etc.) And preferably copper)
  • LDL and the like prepared by contacting the LDL with a mammalian oxidase (eg, lipoxygenase).
  • a mammalian oxidase eg, lipoxygenase
  • Saccharified LDL is, for example, '
  • 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.
  • 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.
  • 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.
  • the glycated phospholipids include glycated phosphatidylethanolamine, glycated phosphatidylserine, and glycated phosphatidylcholine.
  • 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.
  • 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.
  • 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.
  • the screening method of the present invention comprises:
  • screening method C 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
  • 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.
  • 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.
  • 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)].
  • 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.
  • tissue derived from mammals include brain, heart, lung, kidney, liver, kidney, spleen, testis, ovary, and intestine.
  • 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.
  • 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.
  • the complementary DNA of the AR gene DNA or its partial 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.
  • radioisotope for example, 32 P, 3 H and the like
  • dye for example, fluorescein, FAM (manufactured by PE Biosystems), J ⁇ E (manufactured by PE Biosystems), TAMRA (PE Biosystems), ROX (PE Biosystems), Cy5 (Amersham), Cy3 (Amersham) and the like are used.
  • 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.
  • 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.
  • 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.
  • test compound examples 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the above enzyme a stable enzyme having a large specific activity is preferable.
  • galactosidase, i3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.
  • 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.
  • 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.
  • 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.
  • 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.
  • AR proteins and the like can be quantified with high sensitivity.
  • 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.
  • 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.
  • 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 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 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.
  • AR protein varies depending on the type of AR protein.
  • phospholipid cholesterol acyltransferase activity, phospholipase activity, lysophospholipase activity, lysopid: ceramide acyl
  • 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.
  • 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.
  • 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.
  • 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.
  • a cell for example, chondrocyte, fibroblast
  • 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.
  • the promoter or enhancer region of the AR gene may be a known one, or may be one cloned from a genomic DNA.
  • 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.
  • 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.
  • 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
  • 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 .
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • parenteral includes subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, infusion, and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.].
  • fibrates eg, clofibrate, benza
  • 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;
  • 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 ( ⁇ -farin), Xa inhibitors], thrombolytics
  • 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.
  • bases and amino acids are indicated by abbreviations, I UPAC—
  • a la Alanine
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • C indicates the nucleotide sequence of DNA encoding mouse-derived LLP L [SEQ ID NO: 3]
  • mice were used in Example 1 are shown.
  • Example 1 shows the mouse primer used in Example 1.
  • [SEQ ID NO: 7] 1 shows a mouse probe used in Example 1.
  • the mouse primer used in Example 5 is shown.
  • Example 13 shows a mouse probe used in Example 5.
  • oxidized LDL was prepared.
  • Peritoneal macrophage cells were cultured in DMEM-25 mM HEPES (pH 7) medium containing oxidized LDL for 24 hours.
  • cDNA was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystemsth).
  • 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.
  • 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.
  • 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.
  • a DMEM-25 mM HEPES (pH7) medium containing a test compound.
  • 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.
  • 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.
  • H7 DMEM-25 mM HEPE S
  • pH 7 DMEM-25 mM HEPE S
  • 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.
  • 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.
  • EBM-2 medium Clonetics
  • EGM-2 SingleQuots containing oxidized LDL Using the RNeasy Mini Kit (QIAGEN) from the cells cultured for a predetermined period of time! ⁇
  • cDNA was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystems II).
  • 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.
  • 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
  • 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)
  • 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.

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Abstract

L'invention concerne un procédé de criblage d'un composé ou du sel de celui-ci, lequel composé inhibe des changements dans l'expression d'un gène associé à l'artériosclérose à cause d'une lipoprotéine de faible densité modifiée ou d'un composé de faible poids moléculaire ou d'une protéine contenus dans la lipoprotéine de faible densité modifiée. Ce procédé se caractérise par l'utilisation de ce gène associé à l'artériosclérose. L'invention concerne également un kit de criblage, un composé inhibant des changements dans l'expression du gène associé à l'artériosclérose obtenu à l'aide du procédé de criblage ou du kit de criblage, des agents préventifs et des médicaments contenant le composé susmentionné ou le sel de celui-ci, etc.
PCT/JP2003/000407 2002-01-21 2003-01-20 Procede de criblage d'agents preventifs et de remedes pour l'arteriosclerose WO2003062416A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017434A2 (fr) * 1995-11-09 1997-05-15 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Utilisation de la lecithine-cholesterol acyltransferase (lcat) dans le traitement de l'atherosclerose
WO1998046767A1 (fr) * 1997-04-11 1998-10-22 Takeda Chemical Industries, Ltd. Proteines presentant une activite de lecithine-cholesterol semblable a acyltransferase, leur preparation et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017434A2 (fr) * 1995-11-09 1997-05-15 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Utilisation de la lecithine-cholesterol acyltransferase (lcat) dans le traitement de l'atherosclerose
WO1998046767A1 (fr) * 1997-04-11 1998-10-22 Takeda Chemical Industries, Ltd. Proteines presentant une activite de lecithine-cholesterol semblable a acyltransferase, leur preparation et leur utilisation

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Title
LIU M. ET AL.: "Impaired function of lecithin: cholesterol acyltransferase in atherosclerosis-susceptible white carneau pigeons: possiblke effects on metabolism of oxidized phospholipids", J. LIPID RES., vol. 39, no. 2, 1998, pages 245 - 254, XP002964712 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi

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