JP2002090364A - Searching method for arteriosclerosis depressor and regressor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find and efficient searching method for a drug affecting a cholesteric ester cycle so as to realize a searching system for efficiently searching a defoaming drug or an arteriosclerosis regressor and an arteriosclerosis treatment drug searching system different from a known mechanism. SOLUTION: From a cell cultivated in the presence of a substance to be tested and a cell cultivated in the absence of the substance to be tested, free cholesterol is selectively extracted by using a hydrous alcohol, and then, a cholesteric ester amount in the cell cultivated in the presence of the substance to be tested is compared with that in the cell cultivated in the absence of the substance to be tested. In this way, influence of the substance to be tested on cholesteric ester metabolism regulation inside the cell is evaluated. Especially, a screening method for a compound having an intracellular cholesteric ester metabolism regulating property can be realized for providing a defoaming promoting drug and arteriosclerosis depressor or regressor using the screened compound as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for searching for a compound useful as an arteriosclerosis inhibitor and a regression drug. The present invention relates to a search method for efficiently evaluating and screening drugs for preventing or treating arteriosclerosis, which is a major cause of infarction.

[0002]

2. Description of the Related Art Atherosclerosis has been studied from the pathological morphological aspect using experimental atherosclerosis and human autopsy specimens. The initial changes in atherosclerosis seen macroscopically are:
It is a spot-like or linear fat deposit called fatty streak. Histologically, this change has been found to be an accumulation of cells mainly composed of macrophages (foamed cells) that have accumulated cholesterol esters. The fatty streak eventually becomes a fibrous plaque and protrudes from the blood vessel wall, and histologically, infiltration of smooth muscle cells, macrophages, T cells, etc., cell necrosis, and lipid accumulation are observed. When the lesion further progresses, calcification, thrombus adhesion, narrowing of the blood vessel lumen, bruising of the hard spot causes thrombotic occlusion, and this leads to acute myocardial infarction, unstable angina, sudden ischemic death, etc. So-called acute corona syndrome
ry syndrome) has been shown to be closely related (Fuster V et al., N. Engl. J. Med., 3).
26 242-250, 1992). In addition, previous studies have shown that vulnerable plaques have a thin fibrous cap, are rich in lipid components such as cholesterol esters, and are unstable (Erling F et al., Circula
, 92, 657-671, 1995), removing cholesterol esters, especially cholesteryl esters, from the lesions, ie, defoaming, stabilizes unstable plaques and prevents their breakdown. It is expected to bring about stabilization and regression of atherosclerotic lesions, and to greatly reduce the occurrence and recurrence of acute coronary syndrome based on atherosclerosis. As an intracellular cholesterol metabolic system involved in foaming and defoaming of cells, a metabolic cycle involved in the interconversion of free cholesterol and cholesteryl ester, that is, the cholesteryl ester cycle (Brow)
n MS et al., J. Biol. Chem., 255, 9344-9352 (198
0)) is known, and it has been reported that the turnover rate of the cholesteryl ester cycle and its equilibrium state affect the foaming or defoaming activity of cells (Ha
kamata H. et al., Arerioscr. Thromb., 14, 1860-186
5 (1994)). The intracellular cholesteryl ester cycle
ACA mainly contributing to esterification of free cholesterol
It is said that it largely depends on the activity of T (Acyl-coenzyme A: cholestrol acyltranferase) and neutral cholesteryl ester hydrolase, which contributes to the degradation of cholesteryl ester, but there are many unclear points on the regulatory mechanism thereof. Therefore, agents that regulate the cholesteryl ester cycle or that affect the production and degradation of cholesteryl ester have the potential to promote defoaming of cells or regression of atherosclerotic lesions by a previously unknown mechanism. However, it is considered to be effective as a novel therapeutic agent for arteriosclerosis. On the other hand, conventionally, the quantification of the amount of intracellular cholesteryl ester has been carried out by mixing the total cell lipid with an organic solvent mixture (for example,
Extraction with chloroform / methanol mixture, hexane / isopropanol mixture, etc.) and then separation and quantification of cholesteryl ester by enzymatic method using cholesterol oxidase, or fractionation and quantification by TLC or liquid chromatography, etc. However, these methods are complicated, and it is difficult to measure multiple samples with high sensitivity. Therefore, it has been difficult to efficiently search for and evaluate drugs that affect the cholesteryl ester cycle.

[0003]

In view of the above circumstances, the present invention has found a method for efficiently searching for and evaluating drugs that affect the cholesteryl ester cycle, and (1) efficiently defoaming drugs or atherosclerotic lesions. The purpose of the present invention is to establish a search system capable of searching for regression drugs and (2) to establish an arteriosclerosis therapeutic drug search system different from conventionally known mechanisms.

[0004]

Means for Solving the Problems As a result of the present inventors' intensive efforts to solve the above problems, it has been found that hydroalcohols and / or cyclodextrins selectively extract and remove intracellular free cholesterol. Heading,
Utilizing this, a new intracellular cholesteryl ester metabolism regulation evaluation system was established, and intracellular cholesteryl ester cycle activity or cholesteryl ester generation activity,
Cholesteryl ester decomposition activity can be measured easily and with high sensitivity. Using this method, we succeeded in constructing a search system for intracellular cholesteryl ester cycle modulators, cholesteryl ester formation inhibitors and cholesteryl ester degradation promoters. Alcohols have been used to extract total lipids in cells and tissues, but hydroalcohols have been used for the selective extraction and removal of free cholesterol in order to directly quantify intracellular cholesteryl ester metabolic activity. No examples have been known so far. On the other hand, examples of the use of cyclodextrins for measuring a reaction system or an enzyme system involved in cholesterol metabolism include AC in cell extracts.
AT and neutral cholesterol esterase (Lisa M et a
l., Lipids, 31, 323-329 (1996)), 7α-hydroxylase (Souidi M et al., Clin. Chim. Acta, 269, 201-).
217 (1998)) and an example of measuring cholesterol export from cells (Kilsdonk EP et al., J. Biol.
Chem., 270, 17250-17256 (1995)), basic data on extraction of intracellular free cholesterol (J. Pharmac.
eutical Science, 81, 521-523 (1992)), but no examples using cyclodextrin for directly evaluating the regulation of intracellular cholesteryl ester metabolism are known.

[0005] The present invention has been completed based on the above findings, and (1) intracellular free cholesterol characterized by selectively extracting or removing free cholesterol from cells using hydroalcoholic alcohol. (2) The extraction or removal method according to the above (1), wherein the alcohol is a polar aliphatic monohydric alcohol having 1 to 4 carbon atoms, and (3) the above (1), wherein the alcohol is methanol. Extraction or removal method, (4) the extraction or removal method according to the above (1), wherein the water content of the water-containing alcohol is 5 to 60 (v / v)%, (5) the method using the water-containing alcohol and cyclodextrins ( (1) the extraction or removal method described in (1), (6) the extraction or removal method described in (1) above, wherein the cells are cells derived from a mammal, or (7) a monocyte cell derived from peripheral blood derived from a mammal. Clophages, mammalian-derived peritoneal macrophages, mammalian-derived alveolar macrophages, mammalian-derived aortic macrophages, mammalian-derived smooth muscle cells, mammalian-derived fibroblasts, mammalian-derived hepatocytes, mammalian-derived Dendritic cell line, TH
P-1 (human), U937 (human), HepG2 (human), J774 (mouse), L-1 (mouse), RAW
264.7 (mouse), WEHI (mouse) or P3
(8) the extraction or removal method according to the above (1), which is 88D1 (mouse); (8) selective extraction and removal of free cholesterol from cells using hydroalcohol or (and) cyclodextrins; (9) A method for measuring the amount of intracellular cholesteryl ester, which comprises measuring the amount of cholesteryl ester, (9) an enzymatic method using cholesterol oxidase, a TLC method, The method according to the above (8), wherein the amount of cholesteryl ester in the total lipid is determined by liquid chromatography, (10)
The method according to the above (9), wherein the solvent is chloroform-methanol or hexane-isopropanol, (11)
The method according to the above (8), wherein intracellular cholesterol is labeled, free cholesterol is selectively extracted and removed from the cell, and then the amount of the intracellular label is measured. A method for evaluating the intracellular cholesteryl ester metabolism regulating activity of a test substance, which comprises an operation of selectively extracting free cholesterol,
(13) (e) loading cells with cholesterol or cholesteryl ester, or (ii) generating cholesteryl ester in the cells to foam the cells, and the evaluation method according to the above (12). (13) wherein the loading of cholesterol or cholesteryl ester is carried out by adding (i) an alcohol solution of cholesterol or cholesteryl ester or (ii) a complex of cholesterol or cholesteryl ester and lipoprotein. (15) Lipoprotein is LDL, acetyl LDL, oxidized LDL, glycated LDL
Or the evaluation method according to the above (14), which is β-VLDL,
(16) The production of cholesteryl ester in cells is
(17) The evaluation method according to the above (13), which is performed by adding an oxidized sterol, a lipoprotein, a cholesterol-containing liposome or a cytokine, (17) the above (16), wherein the oxidized sterol is 25-hydroxycholesterol or 7-ketocholesterol. ) Described evaluation method,
(18) Lipoprotein is LDL, acetyl LDL, oxidized LD
(16) which is L, glycated LDL or β-VLDL
(19) The evaluation method according to the above (16), wherein the cytokine is interferon γ, and (20) the evaluation according to the above (12), wherein the alcohol is a polar aliphatic monohydric alcohol having 1 to 4 carbon atoms. Method (21) The evaluation method according to the above (12), wherein the alcohol is methanol, (2)
2) The water content of the water-containing alcohol is 5 to 60 (v / v)%.
(23) the evaluation method according to (12), wherein the cell is a mammal-derived cell;
(24) cells whose cells are peripheral blood-derived monocyte macrophages derived from mammals; peritoneal macrophages derived from mammals; alveolar macrophages derived from mammals; aortic macrophages derived from mammals; smooth muscle cells derived from mammals; Fibroblasts, mammalian hepatocytes, mammalian dendritic cell lines, THP-1 (human), U937 (human), HepG2 (human), J774 (mouse), L-
1 (mouse), RAW 264.7 (mouse), WEHI
(25) an evaluation method according to the above (12), which is (mouse) or P388D1 (mouse); (25) free from cells cultured in the presence of the test substance and cells cultured in the absence of the test substance; After selectively extracting cholesterol using hydroalcoholic alcohol, the intracellular cholesteryl ester content of both cells cultured in the presence and absence of the test substance is measured and compared, and the evaluation method according to (12) above is used. (26) The evaluation method according to any one of the above (12) to (25), wherein the regulation of intracellular cholesteryl ester metabolism is a regulation of intracellular cholesteryl ester forming activity.
7) The above (12), wherein the regulation of intracellular cholesteryl ester metabolism is a regulation of intracellular cholesteryl ester degrading activity.
(28) The evaluation method according to any one of (12) to (25), which is a screening method for a compound having an activity of regulating cholesteryl ester metabolism in a cell, (29) The evaluation according to (28) above, which is a method for screening a compound having an activity of inhibiting cholesteryl ester production in a cell, and the evaluation method according to the above (28), which is a method for screening a compound having an activity to promote the degradation of cholesteryl ester in a cell. Law, (3
1) The evaluation method according to the above (28), which is a screening method for a compound having a defoaming action, (32) the above (2)
8) A compound having an intracellular cholesteryl ester metabolism regulating activity or a salt thereof, which is selected by the method described in 8).
A defoaming accelerator comprising, as an active ingredient, a compound having intracellular cholesteryl ester metabolism regulating activity or a pharmaceutically acceptable salt thereof selected by the method according to the above (28),
(34) an arteriosclerosis-inhibiting or regressing agent comprising as an active ingredient a compound having intracellular cholesteryl ester metabolism regulating activity or a pharmaceutically acceptable salt thereof selected by the method according to (28); (36) a method for promoting defoaming, which comprises administering to a mammal an effective amount of a compound having a cholesteryl ester metabolism regulating activity in a cell selected by the method described in the above (28) or a salt thereof, A method for preventing or treating arteriosclerosis, which comprises administering to a mammal an effective amount of a compound having an intracellular cholesteryl ester metabolism regulating activity or a salt thereof selected by the method described in (28) above, 37)
Use of a compound having an intracellular cholesteryl ester metabolism regulating activity or a salt thereof selected by the method described in (28) for producing a defoaming accelerator, (38) Production of a prophylactic / therapeutic agent for arteriosclerosis Use of a compound or a salt thereof having an intracellular cholesteryl ester metabolism regulating activity selected by the method according to the above (28), and
(39) The use of hydroalcohol for selectively extracting or removing free cholesterol from cells is provided.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The hydroalcohol used in the present invention may be any one containing water and alcohols. As the alcohol, any polar alcohol may be used, but a polar aliphatic monohydric alcohol having 1 to 4 carbon atoms, for example, alcohols such as methanol, ethanol, isopropanol and butanol are preferable, and two or more alcohols may be used alone. You may use together. The water content of the hydrous alcohol is, for example, 5% to 60% (v /
v), preferably 10% to 30% (v / v). The hydroalcoholic used in the present invention may contain components other than water and alcohols as long as it does not adversely affect the selective extraction and removal of free cholesterol from cells. As the cyclodextrin used in the present invention, for example, α, β or γ cyclodextrin or a derivative thereof can be used.Specifically, hydroxypropyl α-cyclodextrin, hydroxypropyl β-cyclodextrin, Hydroxypropyl derivatives such as hydroxypropyl γ-cyclodextrin, dihydroxypropyl β-cyclodextrin, methyl β cyclodextrin, maltosyl β-cyclodextrin, methyl derivatives or saccharified derivatives are preferred, and they may be used alone or in combination of two or more. . The cyclodextrin is usually used in the form of an aqueous cyclodextrin solution, and the cyclodextrin concentration is, for example, about 0.1% (w / v) or more, preferably about 0.1% to about 5% (w / v). ), More preferably from about 0.5% to about 5% (w / v).

[0007] The method of extracting or removing intracellular free cholesterol of the present invention is characterized by selectively extracting and removing free cholesterol from cells using aqueous alcohol. The method for extracting or removing intracellular free cholesterol of the present invention can also be carried out using hydroalcohol and cyclodextrins. That is, the free cholesterol may be selectively extracted or removed from the cells by simultaneously or sequentially using the hydroalcohol and the cyclodextrin. The proportion of the hydroalcoholic alcohol to the cyclodextrin is not particularly limited as long as it is within a range in which the cyclodextrin can be dissolved, and when both are used simultaneously, the cyclodextrin-containing hydroalcohol may be used. More specifically, the method for extracting or removing intracellular free cholesterol of the present invention is carried out by extracting target cells using hydroalcohol, or using hydroalcohol and cyclodextrins. The target cells are not particularly limited, and may be any cells of mammals including humans, plants, and microorganisms, and cells derived from mammals are particularly preferable. Examples of mammal-derived cells include, for example, mammal-derived (human, monkey, pig, rabbit, rat, mouse, hamster, etc.) peripheral blood-derived monocyte macrophages, peritoneal macrophages, alveolar macrophages, aortic macrophages,
Smooth muscle cells, fibroblasts, hepatocytes, dendritic cell lines, etc.
THP-1 (human), U937 (human), HepG2
(Human), J774 (mouse), L-1 (mouse), R
AW264.7 (mouse), WEHI (mouse), P3
Cell lines such as 88D1 (mouse). Further, the extraction method is not particularly limited, and a method known per se, for example, Hara Aand Radin NS, Anal.Biochem. 90,
420-426 (1978) can be employed, and the extraction conditions are not particularly limited.
It is appropriate to perform the extraction for about a minute to 8 hours.

The method for measuring the amount of intracellular cholesteryl ester according to the present invention comprises the step of selectively extracting and removing free cholesterol from the inside of cells using the above-mentioned hydroalcohol or cyclodextrins, and then determining the amount of intracellular cholesteryl ester. It is done by doing. Furthermore, the method for measuring the amount of intracellular cholesteryl ester of the present invention can also be performed using hydroalcohol and cyclodextrins. The method for determining the amount of cholesteryl ester is not particularly limited.For example, from cells from which free cholesterol has been removed, total lipids are extracted with an appropriate solvent, for example, chloroform-methanol, hexane-isopropanol, etc., for example, cholesterol oxidase is extracted. The amount of cholesteryl ester in the total lipid can be quantified by a method known per se, such as an enzymatic method used, a TLC method, or liquid chromatography. Cholesteryl ester can be quantitatively determined by labeling intracellular cholesterols and selectively extracting and removing free cholesterol from cells as described in the following description or examples, and then the amount of intracellular labeling (eg, radioactivity) )
Is preferably measured.

[0009] The method for evaluating the activity of a test substance in regulating the intracellular cholesteryl ester metabolism of the test substance according to the present invention is characterized in that it comprises an operation of selectively extracting free cholesterol from the inside of cells using hydroalcohol. Furthermore, the method for evaluating the intracellular cholesteryl ester metabolism regulating activity of the test substance of the present invention can also be performed using hydroalcohol and cyclodextrins. Here, “regulation of cholesteryl ester metabolism” means regulation of cholesteryl ester production activity and degradation activity (for example, promotion of production activity, inhibition and promotion of degradation activity, inhibition), defoaming, etc.
The “evaluation method” includes a simple evaluation method of the activity of a test substance as well as a screening method of an active substance. In the evaluation method of the present invention, the cells are cultured in the presence of the test substance and in the absence of the test substance (control), and after the free cholesterol is selectively extracted from the cells by the method described above, After extraction, the amount of intracellular cholesteryl ester in each of the two cells cultured in the presence and absence of the test substance is preferably measured and compared, but not limited thereto. It may be measured by a method known per se, and the metabolic regulation activity of the test substance may be evaluated by comparison.

[0010] In the evaluation method of the present invention, intracellular cholesterol and cholesteryl ester may be labeled (for example, radioactive or fluorescent), and cells in the extraction step may be labeled with a suitable fixing agent (for example, formaldehyde). Aldehydes such as and paraformaldehyde,
Alcohols such as ethanol and methanol, and organic solvents such as acetone). The evaluation method of the present invention has a main purpose of evaluating and screening a substance useful as a defoaming accelerator, an arteriosclerosis inhibitor or an arteriosclerosis regression agent, and has an activity to inhibit intracellular cholesteryl ester production. Compounds, compounds having cholesteryl ester degradation promoting activity in cells, compounds having a defoaming effect, etc., are screened to load cholesterol or cholesteryl ester in cells to be used, or cholesteryl esters are generated in cells to generate cells. Foaming may be performed so that the activity of the test substance appears more remarkably. The loading of cells with cholesterol or cholesteryl ester can be performed as an alcohol solution or as a lipoprotein (eg, LDL or acetyl LD).
L, oxidized LDL, glycated LDL, β-VLDL, etc.), and cholesteryl esters can be produced in cells by oxidized sterols (eg, 25-hydroxycholesterol, 7-keto). Cholesterol, etc. or lipoproteins (eg, LDL, acetyl LDL, oxidized LDL, glycated LDL, β-VLDL)
), Cholesterol-containing liposomes, cytokines such as interferon γ, and the like.

[0011] Cell lines that can be used for such loading and generation include, for example, cells that can take up cholesterol or lipoprotein and accumulate cholesterol esters in cells, such as those derived from mammals (human, monkey, pig,
Rabbit, rat, mouse, hamster, etc. peripheral blood-derived monocyte macrophages, peritoneal macrophages, alveolar macrophages, aortic macrophages, smooth muscle cells, fibroblasts, hepatocytes, dendritic cell lines, etc., and THP-1 (human ), U937 (human), HepG2 (human), J77
4 (mouse), L-1 (mouse), RAW 264.7
(Mouse), WEHI (mouse), and cell lines such as P388D1 (mouse). These cells can be cultured in a medium and culture conditions suitable for the cells used. Although not particularly limited, usually, the difference between the amount of cholesteryl ester in cells of cells cultured in the presence of the test substance and the amount of cholesteryl ester in cells of control cells cultured in the absence of the test substance. However, when it is 10% or more, preferably 20% or more, it can be determined that the test substance has cholesteryl ester metabolism regulating activity. Thus,
According to the evaluation method of the present invention, the influence on the cholesteryl ester cycle or cholesteryl ester formation and decomposition of a test substance can be directly evaluated, and cholesteryl ester formation inhibitory activity, cholesteryl ester decomposition accelerating activity, defoaming effect, etc. Can be efficiently selected.

The compound having the activity of regulating intracellular cholesteryl ester metabolism, the compound having a defoaming effect or a salt thereof (preferably a pharmaceutically acceptable salt) selected from the mammals (human, monkey, , Pigs, rabbits, rats, mice, hamsters, etc.) as defoaming accelerators, arteriosclerosis inhibitors, and arteriosclerosis regression agents. Further, since these compounds or salts thereof have a lipid-rich plaque regression effect, acute myocardial infarction in mammals, acute coronary syndrome such as unstable angina, peripheral arterial occlusion, and percutaneous coronary angioplasty (PT
It is also useful as a prophylactic / therapeutic agent for restenosis after CA).
The defoaming accelerator of the present invention, a pharmaceutical composition such as an arteriosclerosis inhibitor or an arteriosclerosis regression agent, according to a formulation technique known per se, if necessary, using a known excipient or carrier, for example,
A solid or liquid pharmaceutical composition for oral administration or parenteral administration can be administered to mammals. The dose can be appropriately selected according to 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, and as it is or in a pharmaceutical composition. Formulations (eg,
Powder, granules, tablets, pills, capsules, injections, syrups, emulsions, elixirs, suspensions, solutions and the like). That is, the compound or a salt thereof can be used alone or as a mixture with a pharmaceutically acceptable carrier (adjuvant, excipient, excipient, and / or diluent).

[0013] These pharmaceutical compositions can be formulated according to ordinary methods. Such preparations can be usually produced by mixing / 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. Examples of acceptable vehicles or solvents that can be used include water, Ringer's solution, isotonic saline and the like. In addition, sterile, fixed oils may be conventionally employed as a solvent or suspending medium. For this purpose, any non-volatile oil and fatty acid can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids, and natural or synthetic or semi-synthetic mono- or di- or triglycerides. Suppositories for rectal administration include the drug and suitable nonirritating excipients, such as cocoa butter and polyethylene glycols, which are solid at room temperature but liquid at intestinal temperature and melt in the rectum, Can be manufactured by mixing with a substance that emits. Also, a suitable base (eg, butyric acid polymer, glycolic acid polymer, butyric acid-glycolic acid copolymer, a mixture of butyric acid polymer and glycolic acid polymer, polyglycerol fatty acid ester, etc.) It is also effective to make a combination sustained release preparation.

The solid dosage forms for oral administration include those described above such as powders, granules, tablets, pills and capsules. Such dosage form preparations contain the active ingredient compound and at least one additive, such as sucrose,
Lactose (lactose), cellulose sugar, mannitol (D
Mannitol), maltitol, dextran, starches (eg, corn starch), microcrystalline cellulose, agar, alginates, chitins, chitosans, pectins, tragacanth gums, gum arabic, gelatins,
It can be produced by mixing and / or kneading collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. 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 α-tocopherol and cysteine, disintegrants (eg, croscarmellose sodium), binders (eg, hydroxypropylcellulose),
Thickeners, buffering agents, sweeteners, flavoring agents, perfumes and the like can be mentioned. Tablets and pills can also be prepared with 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 optionally It may contain additives. These oral liquid preparations can be manufactured according to a conventional method such as mixing an active ingredient compound, an inert diluent, and other additives as necessary. For oral administration, it depends on the dosage form, but it is preferable to add usually about 0.01 to 99 W%, preferably about 0.1 to 90 W%, usually about 0.5 to 50 W% of the active ingredient compound. The dosage for one particular patient is
Age, weight, general health, gender, diet, administration time,
It will be determined according to these or other factors depending on the method of administration, rate of excretion, combination of drugs, and the degree of the condition of the patient being treated at that time. 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 weight of about 60 kg) per day Is about 1-500 mg, preferably about 10-200 mg, as an active ingredient in the case of an oral preparation, and about 0.1-100 m as an active ingredient in the case of a parenteral preparation.
g, preferably about 1 to 50 mg, usually about 1 to 20 mg.

In the treatment of these diseases, the selected compound or salt thereof may be used alone for treatment, or may comprise other lipid-lowering or cholesterol-lowering drugs, cardioprotectants, drugs for treating coronary artery disease, It may be used together with other pharmaceutical ingredients including a therapeutic agent for diabetes, a therapeutic agent for hypothyroidism, a therapeutic agent for nephrotic syndrome or a therapeutic agent for chronic renal failure, in which case these compounds are preferably administered as an oral preparation, If necessary, it may be administered in the form of a suppository as a rectal preparation. Possible combination components in this case include, for example, fibrates (eg, clofibrate, benzafibrate, gemfiprodil, etc.); nicotinic acid, derivatives and analogs thereof (eg, acipimox and probucol); bile acid binding resin [Examples, cholestyramine, colestipol, etc.]; compounds that suppress cholesterol absorption [eg, sitosterol, neomycin, etc.]; compounds that inhibit cholesterol biosynthesis [eg, HMG-CoA reductase inhibitors such as lovastatin, simvastatin, pravastatin] Squalene synthase inhibitors [eg, EP 0 567 006, WO 97
Benzoxazepine derivatives and the like described in Japanese Patent Application Laid-Open No. 10224] etc.] squalene epoxidase inhibitors [eg, NB-59
8 and related compounds]. Yet another possible combination component is an oxide squalene-lanosterol cyclase, such as a decalin derivative, an azadecalin derivative and an indane derivative. Antidiabetic drugs: Actos, Rosiglidason, Kinedak, Benfil, Humulin, Oygurcon, Glymicron, Daoneil, Novoline, Monotard, Insulins, Glucobay, Dimeline, Rustinone, Basilcon, Deamelin S, Isgilin; Hypothyroidism drugs: Dry thyroid (thyleoid),
Levothyroxine sodium (Tyrazine S), liothyronidine sodium (thyronine, thyromin); therapeutic agent for nephrotic syndrome: prednisolone (prednin), prednisolone sodium succinate (prednin), methylprednisolone sodium succinate (sol medrol), betamethasone (linderone) Anticoagulants: dipyridamole (versantin), dilazep hydrochloride (comerian), tilopidine, clovidogrel, Xa inhibitor; therapeutic agent for chronic renal failure: diuretics [eg, furosemide (Lasix), bumetanide (Lunetron), azosemide (Diaart) )], In combination with antihypertensive drugs (eg, ACE inhibitors, (enalapril maleate (Renibase)) and Ca antagonists (manidipine), α receptor blockers, AII antagonists (candesartan), etc. When administered, preferably it is used in oral administration.

Further, the selected compound or a salt thereof has a lipid-rich plaque regression action,
It is also useful as an agent for preventing or treating thrombus formation. They may be used alone or in combination with the following known therapeutic agents, preferably by oral administration. Antithrombotic drug: Anticoagulant (eg, heparin sodium, heparin calcium, warfarin calcium (warfarin), Xa inhibitor), thrombolytic drug (eg,
tPA, urokinase], antiplatelet drugs [eg, aspirin, sulfinpyrazolo (anturane), dipyridamole (persantin), ticlopidine (panaldine),
Cilostazol (pletal), GPIIb / IIIa antagonist (leopro)]; coronary vasodilators: nifedipine, diltiazem, nicorazil, meth nitrate; cardioprotective agents: oral drugs for cardiac ATP-K, endothelin antagonists, urotensin antagonists.

[0017]

The present invention will be described in more detail with reference to the following Examples and Preparation Examples, but the present invention is not limited thereto. Example 1 Selective removal of intracellular free cholesterol with hydrous alcohol According to the method of Hakamada et al. (Experimental Medicine Separate Volume vol.14, No. 12, Circulation Research Protocol, pp49-52 (1996)), C57BL6J after thioglycolate stimulation. Peritoneal macrophages were prepared from mice, and the method of Ishii et al. (Ishii I et al., Arte
riboscler. Thromb., 12, 1139-1145, 1992), β-ultra low density lipoprotein (β-VLDL, 1
50 μg cholesterol / ml) and 0.2 mM oleic acid / 0.2% bovine serum albumin complex, [1,
2,6,7- ³ H] -cholesterol (NEN, 37K
Bq / ml) containing RPMI 1640-25 mM HE
By culturing for 24 hours using a PES (pH 7) medium, foamed cells having cholesteryl esters accumulated in the cells were obtained. The foamed cells are washed with 80% methanol water for 10 minutes.
After extraction for 5 minutes, hexane: isopropanol (3:
Total lipids were extracted using 2). Part of the extract was TLC
Method (Merck silica gel plate No. 5582, developing solvent: petroleum ether / diethyl ether / acetic acid = 9:
1: 0.1), the free cholesterol and cholesteryl ester fractions were cut out, a liquid scintillator was added, and the radioactivity was measured. Table 1 shows the results.

Example 2 Selective Removal of Intracellular Free Cholesterol by Cyclodextrin The foamed mouse macrophages prepared in Example 1 were fixed with 10% formalin at room temperature for 10 minutes, and 2%
4 with aqueous solution of methyl β cyclodextrin (Sigma)
After extraction for 2 hours (2 hours), total lipids were further extracted with hexane: isopropanol (3: 2). A part of the extract was subjected to TLC method (Merck silica gel plate N
o. 5582, developing solvent: petroleum ether / diethyl ether / acetic acid = 9: 1: 0.1), cut off the free cholesterol and cholesteryl ester fractions,
After adding the liquid scintillator, its radioactivity was measured. Table 1 shows the results.

[0019]

[Table 1]

In Table 1, the control is the measured value of free cholesterol and cholesteryl ester of cells not extracted with aqueous methanol or methyl β-cyclodextrin. As shown in Table 1, the amount of free cholesterol in cells subjected to extraction with aqueous methanol or methyl β-cyclodextrin was reduced to 4 to 5% of that of the control, and the percentage of total cholesterol was extremely low at 3 to 5%. Met.
Therefore, since most of the cholesterol fraction remaining in the cells subjected to aqueous methanol or methyl β cyclodextrin extraction is cholesteryl ester, the amount of intracellular cholesteryl ester can be easily determined by measuring the amount of residual radioactivity in the cells. It is possible to evaluate. In other words, this method has made it possible to efficiently search for compounds that affect intracellular cholesteryl ester cycle or cholesteryl ester formation and cholesteryl ester degradation.

Example 3 Measurement of Intracellular Cholesteryl Ester Cycle Modulating Activity This example shows an example of a system for measuring the activity of an intracellular cholesteryl ester cycle modulator. Dibutyryl cyclic AMP (hereinafter sometimes abbreviated as dbc-AMP), which is well known as a compound that affects the cholesteryl ester cycle of cells. Sakr SW et al., B
iochemica et Biophysica Acta 1438, 85-98, 1999; Be
rnardDW et al., J. Biol. Chem., 266, 710-716, 1991)
Was used as a test compound to carry out this measurement system. RPMI1640-HEPES containing the test compound (pH
7) In the medium, the foamed mouse macrophages prepared in Example 1 were cultured for 24 hours. After extracting the cells with 80% methanol water for 10 minutes, 0.1N NaOH / 0.1% S
The mixture was solubilized with a DS solution, a liquid scintillator was added to a part thereof, and the radioactivity was measured. The radioactivity value was corrected with the cell protein amount measured using the Pierce BCA assay kit, and the radioactivity of the control group (without addition of the test compound) was taken as 100%, and the intracellular cholesteryl ester content ( %) Was calculated. Table 2 shows the results.

[0022]

[Table 2] As shown in Table 2, it was revealed that dibutyryl cyclic AMP exerts a cholesteryl ester cycle controlling action based on a strong cholesteryl ester decomposing activity.

Example 4 Confirmation of Defoaming-Promoting Activity of Dibutyryl Cyclic AMP The defoaming activity of the test compound was determined by comparing the foamed mouse macrophages prepared in Example 1 with RPMI containing the test compound.
After treatment with a 1640-25 mM HEPES (pH 7) medium for 24 hours, 20% fetal bovine serum (JRH) was added, and the cells were cultured for another 24 hours, followed by measurement of radioactivity released into the medium. The defoaming promoting activity value of the compound-treated group was calculated by the ratio (%) of the radioactivity amount corrected by the blank value to that of the control group. As the blank value, the radioactivity when no test compound and 20% fetal bovine serum were added was used. The defoaming promoting activity values of the control group and the compound-treated group were 0.1 N NaOH / 0.1%
After solubilizing the cells with the SDS solution, correction was performed using the cell protein amount measured using a Pierce BCA assay kit. Table 3 shows the results.

Example 5 Confirmation of Effect of Dibutyryl Cyclic AMP on Intracellular Cholesteryl Ester Content Mouse peritoneal macrophages prepared in Example 1 were subjected to β-V
RPMI with LDL (150 μg / ml) and 0.2 mM oleic acid / 0.2% bovine serum albumin complex
By culturing for 24 hours using a 1640-25 mM HEPES (pH 7) medium, foamed cells having cholesteryl esters accumulated in the cells were obtained. This foamed macrophage was converted to RPMI 1640-25 mMH containing the test compound.
After treatment with an EPES (pH 7) medium for 24 hours, total lipids were extracted using hexane: isopropanol (3: 2). After dissolving the extracted lipid with isopropanol, the total cholesterol and free cholesterol levels were measured using an enzymatic method (Wako, E-Test Wako), and the cholesteryl ester level was calculated from the difference. The amount of cell protein in the control group and the compound-treated group was 0.1 N NaOH.
/0.1% SDS solution, and measured using a Pierce BCA assay kit. The cholesteryl ester content in the compound-treated group was determined by setting the intracellular cholesteryl ester content (μg / mg cell protein) in the control group to 100%. (%) Was calculated. Table 4 shows the results.

[0025]

[Table 3]

[0026]

[Table 4]

About dibutyryl cyclic AMP,
Example 4 tests the effect of transporting cholesterol from foamed macrophages, and Example 5 tests the effect on intracellular cholesteryl ester content using a conventional measurement method. As a result, dibutyryl cyclic AM
It is clear that P exhibits a cholesterol export effect (Table 3) and a cholesteryl ester content lowering effect (Table 4). Since foamed cells are a major component of atherosclerotic lesions, the compounds found in the search system of the present invention are useful as novel therapeutic agents for atherosclerosis that suppress the formation and regression of atherosclerotic lesions.

Example 6 2- [6-chloro-4- (2-methylphenyl) -2-
Oxo-2H-chromen-3-yl] -N- (2,6-
Mouse peritoneal macrophages were prepared by the method described in the intracellular cholesteryl ester synthesis inhibition Example 1 dimethoxyphenyl) acetamide, 1% fetal bovine serum and [1,2,6,7- ³
H] -Cholesterol (NEN, 37 Kbq / ml)
Containing RPMI 1640-25 mM HEPES (pH
7) The cells were cultured in the medium for 24 hours. Macrophages labeled with ³ H-cholesterol were converted to 2- [6-chloro-
4- (2-methylphenyl) -2-oxo-2H-chromen-3-yl] -N- (2,6-dimethoxyphenyl) acetamide (hereinafter referred to as compound A), 25-
Hydroxycholesterol (Sigma, 5 μg /
ml) and RPMI 1640-25 mM HEP containing 0.2 mM oleic acid / 0.2% bovine serum albumin
After culturing in an ES (pH 7) medium for 24 hours, the content of the synthesized intracellular cholesteryl ester was determined by TLC fractionation or aqueous methanol extraction (hereinafter sometimes referred to as methanol method). Measurement using the TLC fractionation method
After extracting intracellular total lipids with a hexane: isopropanol (3: 2, v / v) mixture, TLC fractionation was performed using the conditions described in Example 1 to quantify the amount of intracellular cholesteryl ester. . The measurement using the aqueous methanol extraction method was performed by treating cells with 80% methanol for 10 minutes, solubilizing the cells with a 0.1N NaOH / 0.1% SDS solution, and measuring the radioactivity. . Each radioactivity value is 0.1N NaOH / 0.1%
Protein amount of cells solubilized with SDS solution (Pierce BCA
After correction with an assay kit), the intracellular cholesteryl ester content (%) of the compound A-treated group was calculated with the radioactivity (dpm / mg cell protein) of the control group without the test compound as 100%. Table 5 shows the results.

[0029]

[Table 5] Cholesteryl ester synthesis inhibitory action n = 3

Example 7 Intracellular Cholesteryl Ester Decomposition-Promoting Effect of Compound A Mouse peritoneal macrophage-derived foamed cells prepared by the method described in Example 1 were isolated from RPMI164 containing Compound A.
The cells were cultured in a 0-25 mM HEPES (pH 7) medium for 48 hours, and the decrease in the cholesteryl ester content in cells was determined by TLC.
Quantification was performed by the fractionation method or the aqueous methanol extraction method. T
In the measurement using the LC fractionation method, after extracting intracellular total lipids with a hexane: isopropanol (3: 2, v / v) mixture, TLC fractionation was performed using the conditions described in Example 1,
The determination was performed by quantifying the amount of intracellular cholesteryl ester. In the measurement using the aqueous methanol extraction method, the cells were treated with 80% methanol for 10 minutes, and then 0.1N Na was used.
The measurement was performed by solubilizing the cells with an OH / 0.1% SDS solution and measuring the radioactivity. Each radioactivity value is
After correcting for the protein amount of the cells solubilized with a 0.1 N NaOH / 0.1% SDS solution (Pierce BCA assay kit), the radioactivity (dpm / dpm /
(mg cell protein) as 100%, the intracellular cholesteryl ester content (%) of the compound A treatment group was calculated. Table 6 shows the results.

[0031]

[Table 6] Cholesteryl ester decomposition promoting action n = 3

When the effect of Compound A on cholesteryl ester synthesizing activity and cholesteryl ester degrading activity in cells was tested (Tables 5 and 6), it was found that Compound A has strong cholesteryl ester synthesis inhibitory activity and cholesteryl ester degradation promoting activity. I found Furthermore, the results of the conventional TLC fractionation method and the method of the present invention (hydrous alcohol extraction method) are well correlated,
The utility of the method of the invention has been proven.

Example 8 The defoaming promoting effect of Compound A was measured using the method described in Example 4. Table 7 shows the results.

[0034]

[Table 7] Defoaming promoting action n = 3

Example 9 Effect of lowering the cholesteryl ester content in cells of compound A The effect of lowering the cholesteryl ester content of cells of compound A was measured using the method described in Example 5. Table 8 shows the results.

[0036]

Table 8: Intracellular cholesteryl ester content lowering effect n = 3

When the effect of Compound A on the cholesterol export action from foamed macrophages and the intracellular cholesteryl ester content was tested, Compound A exhibited a strong cholesterol export action (Table 7) and a cholesteryl ester content lowering action (Table 8). To show that Since foamed cells are a main component of atherosclerotic lesions, Compound A is useful as a novel therapeutic agent for atherosclerosis, which suppresses formation and regression of atherosclerotic lesions.

Formulation Example 1 Capsule (1) Compound A 10 mg (2) Lactose 90 mg (3) Microcrystalline cellulose 70 mg (4) Magnesium stearate 10 mg 1 capsule 180 mg (1), (2) and (3) After mixing 1/2 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 Tablets (1) Compound A 10 mg (2) Lactose 35 mg (3) Corn starch 150 mg (4) Microcrystalline cellulose 30 mg (5) Magnesium stearate 5 mg One tablet 230 mg (1), (2) and (3) ), 2/3 of (4)
After mixing the amount and 1/2 of (5), the mixture was granulated. The remaining (4) and (5) were added to the granules and pressed into tablets

Formulation Example 3 Injection (1) Compound A 10 mg (2) Inosit 100 mg (3) Benzyl alcohol 20 mg 1 ampule 130 mg (1), (2) and (3) are distilled for injection so that the total amount is 2 ml. Dissolved in water and sealed in ampoules. All steps were performed under aseptic conditions.

[0041]

According to the method of the present invention for extracting or removing free intracellular cholesterol, the method for evaluating the activity of regulating cholesteryl ester metabolism in cells, etc., selecting a defoaming accelerator, an arteriosclerosis inhibitor or an arteriosclerosis regression agent. Can be.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/90 G01N 30/90 33/15 33/15 Z 33/48 33/48 A 33/92 33 / 92 A // A61K 31/352 A61K 31/352 C07D 311/18 C07D 311/18 (72) Inventor Shohei Nakamura 6-408 Kitaimaichi, Kashiba City, Nara Prefecture F term (reference) 2G045 BA13 BA20 BB20 CA17 CB01 DA20 DA30 DA62 DA63 DA64 DA65 DA69 DA80 FB06 FB08 4C062 EE18 4C084 AA16 MA17 MA21 MA22 MA23 MA35 MA37 MA41 MA43 MA52 MA55 MA60 MA66 ZA361 ZA362 ZA451 ZA452 ZC331 ZC332 ZC801 ZC802 4C086 BA08 MA17 MA21 MA22 MA43 MA33 MA35 MA37 MA52 MA33 MA33

Claims (39)

[Claims] 1. A method for extracting or removing intracellular free cholesterol, comprising selectively extracting or removing free cholesterol from cells using a hydroalcoholic alcohol. 2. The method according to claim 1, wherein the alcohol is a polar aliphatic monohydric alcohol having 1 to 4 carbon atoms. 3. The method according to claim 1, wherein the alcohol is methanol.
The described extraction or removal method. 4. The water-containing alcohol having a water content of 5 to 60 (v
/ V)%. 5. The method according to claim 1, wherein a hydroalcoholic alcohol and a cyclodextrin are used. 6. The method according to claim 1, wherein the cell is a mammal-derived cell. 7. A method wherein the cell is a monocyte-derived monocyte macrophage derived from a mammal, a peritoneal macrophage derived from a mammal, an alveolar macrophage derived from a mammal, an aortic macrophage derived from a mammal, a smooth muscle cell derived from a mammal, or a mammal. Derived fibroblasts, mammalian-derived hepatocytes, mammalian-derived dendritic cell lines, THP-1 (human), U937 (human), HepG2 (human), J774 (mouse), L-
1 (mouse), RAW 264.7 (mouse), WEHI
The method according to claim 1, wherein the method is (mouse) or P388D1 (mouse). 8. An intracellular cholesteryl ester which is characterized by selectively extracting and removing free cholesterol from a cell using a hydroalcohol and / or cyclodextrin, and then measuring the amount of cholesteryl ester in the cell. How to measure quantity. 9. The method according to claim 8, wherein the total lipid is extracted from the cells from which free cholesterol has been extracted and removed with a solvent, and the amount of cholesteryl ester in the total lipid is determined by an enzymatic method using cholesterol oxidase, TLC method or liquid chromatography. the method of. 10. The method according to claim 9, wherein the solvent is chloroform-methanol or hexane-isopropanol. 11. The method according to claim 8, wherein intracellular cholesterol is labeled, free cholesterol is selectively extracted and removed from inside the cell, and then the amount of intracellular labeling is measured. 12. A method for evaluating the intracellular cholesteryl ester metabolism regulating activity of a test substance, which comprises an operation of selectively extracting free cholesterol from cells using a hydroalcoholic alcohol. 13. The evaluation method according to claim 12, wherein (i) the cells are loaded with cholesterol or cholesteryl ester, or (ii) cholesteryl esters are formed in the cells to foam the cells. 14. The method of claim 11, wherein the loading of the cells with cholesterol or cholesteryl ester is carried out by adding (i) an alcohol solution of cholesterol or cholesteryl ester or (ii) a complex of cholesterol or cholesteryl ester and lipoprotein. 13
The described evaluation method. 15. The lipoprotein is LDL, acetyl LDL,
The evaluation method according to claim 14, which is oxidized LDL, glycated LDL, or β-VLDL. 16. The evaluation method according to claim 13, wherein the production of cholesteryl ester in cells is performed by adding oxidized sterols, lipoproteins, cholesterol-containing liposomes, or cytokines. 17. The method according to claim 16, wherein the oxidized sterol is 25-hydroxycholesterol or 7-ketocholesterol. 18. The lipoprotein is LDL, acetyl LDL,
The evaluation method according to claim 16, which is oxidized LDL, glycated LDL, or β-VLDL. 19. The method according to claim 19, wherein the cytokines are interferon γ.
17. The evaluation method according to claim 16, wherein 20. The method according to claim 12, wherein the alcohol is a polar aliphatic monohydric alcohol having 1 to 4 carbon atoms. 21. The method according to claim 12, wherein the alcohol is methanol. 22. The water-containing alcohol having a water content of 5 to 60.
The evaluation method according to claim 12, which is (v / v)%. 23. The method according to claim 12, wherein the cell is a mammal-derived cell. 24. A monocyte-derived macrophage derived from peripheral blood derived from a mammal, a peritoneal macrophage derived from a mammal,
Alveolar macrophages derived from mammals, aortic macrophages derived from mammals, smooth muscle cells derived from mammals, fibroblasts derived from mammals, hepatocytes derived from mammals, dendritic cell lines derived from mammals, THP-1 (Human), U937
(Human), HepG2 (human), J774 (mouse),
L-1 (mouse), RAW 264.7 (mouse), WE
The evaluation method according to claim 12, which is HI (mouse) or P388D1 (mouse). 25. A method for selectively extracting free cholesterol from a cell cultured in the presence of a test substance and a cell cultured in the absence of the test substance using aqueous alcohol, The evaluation method according to claim 12, wherein the amounts of intracellular cholesteryl esters of both cells cultured in the presence and absence thereof are measured and compared. 26. The evaluation method according to claim 12, wherein the regulation of intracellular cholesteryl ester metabolism is a regulation of intracellular cholesteryl ester forming activity. 27. The evaluation method according to claim 12, wherein the regulation of intracellular cholesteryl ester metabolism is a regulation of intracellular cholesteryl ester degrading activity. 28. A method for screening a compound having an activity of regulating intracellular cholesteryl ester metabolism.
The evaluation method according to any one of 2 to 25. 29. A method for screening a compound having an activity of inhibiting cholesteryl ester production in a cell.
8. Evaluation method according to 8. 30. A method for screening a compound having an activity of promoting the degradation of cholesteryl ester in a cell.
8. Evaluation method according to 8. 31. The evaluation method according to claim 28, which is a method for screening a compound having a defoaming action. 32. A compound having a cholesteryl ester metabolism regulating activity in a cell selected by the method according to claim 28 or a salt thereof. 33. A defoaming agent comprising, as an active ingredient, a compound having intracellular cholesteryl ester metabolism regulating activity selected by the method according to claim 28 or a pharmaceutically acceptable salt thereof. 34. An arteriosclerosis inhibitor or regression agent comprising a compound having an intracellular cholesteryl ester metabolism regulating activity selected by the method according to claim 28 or a pharmaceutically acceptable salt thereof as an active ingredient. 35. A method for promoting defoaming, comprising administering to a mammal an effective amount of a compound having a cholesteryl ester metabolism regulating activity in a cell selected by the method according to claim 28 or a salt thereof. . 36. A method for preventing arteriosclerosis, comprising administering to a mammal an effective amount of a compound having a cholesteryl ester metabolism regulating activity in a cell selected by the method according to claim 28 or a salt thereof. Method of treatment. 37. Use of a compound having a cholesteryl ester metabolism regulating activity in a cell selected by the method according to claim 28 or a salt thereof for producing a defoaming promoting agent. 38. Use of a compound having a cholesteryl ester metabolism regulating activity in a cell selected by the method according to claim 28 or a salt thereof for producing a prophylactic / therapeutic agent for arteriosclerosis. 39. Use of hydroalcohol for selectively extracting or removing free cholesterol from inside a cell.