JPH09194366A - Inhibitor of biosynthesis of triglyceride - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inhibitor of biosynthesis of a triglyceride, comprising aureothin as an active ingredient and useful for reducing the triglyceride in blood in hypertriglyceridemia and further as a preventing agent for arteriosclerosis. SOLUTION: This medicinal composition includes the inhibitor of biosynthesis of a triglyceride, an inhibitor of acetyl-CoA carboxylases, an inhibitor of the fatty acid synthase group and a lowering agent for the triglyceride in blood comprising aureothin represented by the formula, etc., as active ingredients. For example, the aureothin is found in Streptomyces thioluteus and the daily dose thereof for an adult is preferably 1μg to 1mg.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a triglyceride biosynthesis inhibitor, an acetyl CoA carboxylase inhibitor, a fatty acid synthase group inhibitor, and a blood triglyceride lowering agent containing aureosine as an active ingredient.

[0002]

2. Description of the Related Art Generally, cholesterol and triglyceride (neutral fat) in blood do not dissolve in blood by themselves, so they exist as lipoproteins by binding to apolipoprotein. Triglyceride is composed of 6 kinds of enzymes and 1 kind of enzyme group (acetyl CoA carboxylase, fatty acid synthetase group, fatty acid acyl CoA synthetase, mainly in the liver in the body, starting from acetyl CoA produced from sugar as a raw material. Glycerophosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase,
It is biosynthesized by diacylglycerol acyltransferase) and is secreted into the blood from the liver as a lipoprotein.

A condition in which blood cholesterol and / or triglyceride is higher than a standard value is called hyperlipidemia.
The condition called hyperlipidemia is fredrickson classification (WHO
According to the classification), it is classified into 6 types by lipoprotein in blood. Of these, types I, IV, and V have increased triglycerides only, type IIa has increased cholesterol, and types IIb and III have both increased (general clinical practice, 43 , 871 (199).
4)). For this reason, existing hyperlipidemic drugs (those that lower cholesterol alone or those that lower both cholesterol and triglyceride) cannot necessarily adequately address all hyperlipidemias. In particular, type IV accounts for 40 to 50% of male hyperlipidemia patients (clinical and research, 69 , 318 (1992)). In addition, in the case of secondary onset with diabetes, etc., most of them are type IV (general clinical practice, 43 , 878 (1994)).

So-called hypertriglyceridemia is a pathological condition in which the amount of triglyceride in blood is increasing, but for the past several years, it has been noted as a risk factor for arteriosclerosis and ischemic heart disease among clinicians and pharmaceutical manufacturers. It started to come.

In the field of hyperlipidemia including hypertriglyceridemia, only cholesterol, which is considered to be directly related to arteriosclerosis, has been focused so far, so there are still few drugs for lowering triglyceride, For the treatment of triglyceridemia, only clofibrate antilipemic agents and nicotinic acid preparations that exist as antilipemic agents are used. It is also a high dose when used, and therefore some side effects (J. Lipid, 5 , 64-
72 (1994)) is also a concern. Furthermore, the firm mechanism of action of these drugs has not been clarified. For these reasons, a new type of drug that has a triglyceride lowering action at a low dose, has no side effects, and has a clear mechanism of action is desired. Hypertriglyceridemia occurs due to various causes such as when there is a genetic background or when it develops secondarily from diabetes as described above (general clinical practice, 43 , 878 (1994)). A. Enhancement of triglyceride synthesis (secretion) in the liver B. It is considered that the degradation of synthesized triglyceride (present in blood as lipoprotein) by lipoprotein lipase (LPL) is delayed (clinical and research, 69, 340 (1992)). Particularly, in hypertriglyceridemia associated with diabetes, non-insulin-dependent diabetes mellitus (N
IDDM), the above A. In the case of insulin-dependent diabetes mellitus (IDDM), the above B. It is said that the cause is (clinical and research, 69 , 379 (199).
2)). Therefore, the mechanism of action of therapeutic agents for hypertriglyceridemia is triglyceride synthesis (secretion) in the liver.
And / or promote the degradation of synthesized triglycerides (present in the blood as lipoproteins) by lipoprotein lipase (LPL).

Aureosin is a known substance (Eur. J. Biochem., 219 , 691 (1994)).
But this is triglyceride synthesis inhibitory action, acetyl Co
It has not been known so far that it has an A-carboxylase inhibitory action, a fatty acid synthase group inhibitory action, or a blood triglyceride lowering action.

[0007]

The problems to be solved by the present invention are as follows: Drugs that inhibit triglyceride biosynthesis, drugs that inhibit acetyl CoA carboxylase, drugs that inhibit fatty acid synthases, and further triglycerides in blood. To obtain a drug having the action of decreasing

[0008]

[Means for Solving the Problems] In order to solve the above problems, the present inventors focus on inhibiting the triglyceride synthesis in the liver among the action mechanisms of the above-mentioned therapeutic agents for hypertriglyceridemia. As a result of intensive research, they found a substance having the desired activity.

That is, the present invention has the following formula

[0010]

Embedded image

A triglyceride biosynthesis inhibitor containing aureothin represented by the formula (Aureothin) as an active ingredient. The present invention is also an acetyl CoA carboxylase inhibitor containing aureosin represented by the above formula as an active ingredient. The present invention is also a fatty acid synthase group inhibitor containing aureosin represented by the above formula as an active ingredient. The present invention is further a blood triglyceride lowering agent containing aureosin represented by the above formula as an active ingredient. That is, the present invention is a pharmaceutical composition containing aureosin as an active ingredient.

[0012] Here, aureosin is a known substance, and found by Maeda et al. In Streptomyces thioluteus (K. Maeda, J. Antibiotics, 6A, 137 (1
953), F. Washizu, ibid., 7A, 60 (195).
4)), and the structure was determined by Hirata et al. (Y. Hirata, H. Nkada and S. Yamada, Nippon Kagaku Za.
sshi, 79, 390 (1958)).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The drug of the present invention is administered orally or parenterally, such as rectal, subcutaneous, intramuscular, intravenous, and transdermal, for the above-mentioned purposes. Intravenous administration is preferred.

For oral administration, solid or liquid preparations can be prepared. Examples of solid preparations include tablets, pills, powders and granules. In such solid formulations, the active substance is a pharmaceutically acceptable carrier,
For example, it is mixed with sodium bicarbonate, calcium carbonate, potato starch, sucrose, mannitol, carboxymethyl cellulose and the like. The formulation operation is performed by a conventional method, but additives other than the above-mentioned carrier for formulation, for example, lubricants such as calcium stearate and magnesium stearate may be contained. In addition, for example, in the solid preparation as described above, for example, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, polyvinyl alcohol phthalate, styrene maleic anhydride copolymer, or methacrylic acid, an enteric substance such as methyl methacrylate copolymer. A solution or an aqueous solution of an organic solvent may be sprayed to form an enteric-coated preparation by applying an enteric coating. Solid preparations such as powders and granules can be wrapped in enteric-coated capsules.

Liquid preparations for oral administration include, for example, emulsions, solutions, suspensions, syrups or elixirs. These formulations contain a commonly used pharmaceutically acceptable carrier such as water or liquid paraffin. Oily bases such as coconut oil, fractionated coconut oil, soybean oil and corn oil can be used as carriers. The pharmaceutically acceptable carrier includes, if necessary, auxiliary agents, fragrances, stabilizers or preservatives which are usually used. Liquid formulations may also be administered in capsules made of an absorbable material such as gelatin.
Solid formulations for rectal administration include suppositories containing the active ingredient and produced by a method known per se.

Formulations for parenteral administration are administered as sterile aqueous or non-aqueous liquids, suspensions or emulsions. Non-aqueous solutions or suspensions are pharmaceutically acceptable carriers, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil or soybean oil, and injectable organic esters such as ethyl oleate. Such formulations may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents and stabilizing agents. These solutions, suspensions and emulsions can be sterilized by appropriate treatments such as filtration through a bacteria-retaining filter, heating, blending of a bactericide or irradiation of ultraviolet rays. Alternatively, a sterile solid preparation may be produced and dissolved in sterile water or a sterile solvent for injection immediately before use. Further, vegetable oils such as soybean oil and phospholipids such as lecithin, water is added to a uniform solution of the active ingredient, and, for example, a pressure emulsion homogenizer, a fat emulsion homogenized by a homogenizer such as an ultrasonic homogenizer, and the like are also injectable. Can be used. Examples of the dosage form for transdermal administration include ointments and creams. These are manufactured by a usual method.

When the active ingredient of the present invention is used as a therapeutic agent for hypertriglyceridemia or a preventive agent for arteriosclerosis, it varies depending on the degree of disease state, age, sex, body weight, administration route, etc. of the patient. , Usually 1 μg per adult per day
~ 1 mg can be administered. Such dose is 1
It may be administered once to several times a day, for example, in 2 to 6 divided doses.

[0018]

【Example】

[Example 1] Preparation of cells Human hepatoma cells HepG2 were seeded on a 24-well plate (Costar No. 3524) at 1 x 10 ⁵ cells / well, and 1 ml of a medium (eRDF medium containing 10% bovine serum (Far Eastern Chemical Co., Ltd. )) At 37 ° C. in the presence of 5% CO ₂
Cultured for days. After aspirating the supernatant medium, add 1 ml
The cells were washed with PBS (-) buffer solution (Takara Shuzo Co., Ltd.) and aspirated. After repeating this operation twice, 1 ml of medium (eRDF medium) was newly added and the medium was replaced.

Preparation of sample Aureosin (m.
w. 397.4) is dimethyl sulfoxide (hereinafter DMS).
It is dissolved in the solution) to prepare a 1 mg / ml sample stock solution. The diluent was obtained by diluting the sample solution with DMSO.

Production of triglyceride To the cells prepared as described above, 5 μl of these sample stock solutions or their diluted solutions were added per well and ¹⁴ C-
Acetic acid solution (Amersham Code No. CFA13 in PBS
(-) Diluted 4.3 times with buffer) was added in an amount of 10 µl, and the mixture was cultured at 37 ° C in the presence of 5% CO ₂ for 1 day.

After the quantitative culturing of triglyceride was completed, the culture solution was removed, 1 ml of the extract solution (n-hexane: isopropyl alcohol = 2: 1) was added, and the mixture was allowed to stand at room temperature for 1 hour to extract lipid components in the cells. . After the treatment, the obtained extract containing the lipid component was air dried. further,
The obtained residual substance was redissolved in 20 μl of a developing solution (n-hexane: ethyl acetate = 9: 1), plotted on a thin layer chromatography plate (Tokyo Kasei S319), and diluted in the developing solution shown above. Layer chromatography was performed. After air-drying, the lipid component was developed in iodine vapor, the portion corresponding to triglyceride was cut off, and the amount of biosynthesized triglyceride was measured with a liquid scintillation counter. The results are shown in Table 1. This shows that aureosine has a triglyceride biosynthesis inhibitory activity.

[0022]

[Table 1]

[Example 2] Preparation of rat liver homogenate Report by T. Tanabe et al. (Methods In Enzymology)
, 71 , 5 (1981)), a rat liver homogenate was prepared. Male Wistar rats, 9 weeks old, were anesthetized with Nembutal, and the blood was removed, and the liver was extracted. 2 of the weight of the liver
Double the volume of buffer (100 mM potassium phosphate (pH 7.
4) 4 mM EDTA, 10 mM 2-mercaptoethanol, 0.2 mM PMSF, 250 mM sucrose)
Was added and crushed with a homogenizer (Polytron).
The disrupted liquid is separated by ultracentrifugation (100,000 xg),
The supernatant was obtained. The resulting supernatant is desalted column PD-10
(Pharmacia) for desalting to obtain rat liver homogenate.

Sample Preparation Aureosin was dissolved in DMSO to prepare a 1 mg / ml stock solution. The diluent was obtained by diluting with DMSO.

Measurement of acetyl-CoA carboxylase activity
Report constant Tanabe (T.Tanabe) et (Methods an In Enzymology
, 71 , 5 (1981)), and the acetyl CoA carboxylase activity was measured. Reaction solution A (53 mM
Tris.HCl (pH 7.5), 10.6 mM potassium citrate, 10.6 mM magnesium chloride, 0.8
mg / ml bovine serum albumin, 0.53 mM potassium phosphoenolpyruvate, 3.9 mM glutathione,
0.132mM Acetyl CoA (Wako Pure Chemical Industries), 6.3μ
g / ml lactate dehydrogenase (Oriental yeast), pyruvate kinase 15.5 μg / ml (Wako Pure Chemical Industries, Ltd.) 0.132 mM NADH (Oriental yeast) 760 μl, 100 μl of the liver homogenate prepared as described above and 5 μl of sample Was added. 37 ° C
After incubating for 10 minutes at room temperature, add 150 mM ATP to
After addition of 0 μl, the mixture was allowed to stand at room temperature for 1 minute, and 20 μl of 1M potassium hydrogen carbonate was added to start the reaction. The amount of change in absorbance at 340 nm was measured. 1 unit is 1 μmol N
The amount of ADH oxidized was 1 minute. Table 2 shows the results. This results in rat acetyl Co for aureosine.
It can be seen that it has an activity of inhibiting A carboxylase.

[0026]

[Table 2]

[Example 3] Measurement of activity of fatty acid synthase group Report by Kumar et al. (Methods In Enzymology, 71 , 86)
(1981)), the activity of fatty acid synthase was measured. Reaction solution B (114 mM potassium phosphate (pH 7.
0), 0.114 mM EDTA, 1.14 mM DT
T, 0.057 mM acetyl CoA (Wako Pure Chemical Industries), 0.
114 mM NADPH (Oriental yeast)) 875
100 μl of the liver homogenate prepared in Example 2 and 10 μl of the sample were added to μl. After incubating at 37 ° C for 3 minutes, 20 mM 10 mM malonyl CoA (Sigma) was added.
1 was added to start the reaction. The amount of change in absorbance at 340 nm was measured. 1 unit contains 1 nmol of NADPH
The amount was oxidized per minute. The results are shown in Table 3. This shows that aureosin has an activity of inhibiting the fatty acid synthase group.

[0028]

[Table 3]

Example 4 A tablet having the following composition was produced.

Active ingredient 200 μg Lactose 180 mg Potato starch 50 mg Polyvinylpyrrolidone 10 mg Magnesium stearate 5 mg Mix the active ingredient, lactose and potato starch,
This was evenly moistened with a 20% solution of polyvinylpyrrolidone in ethanol. This was passed through a 20 mm mesh sieve, dried at 45 ° C., and passed through a 15 mm mesh again. The granules thus obtained were mixed with magnesium stearate and compressed into tablets.

[Example 5] Hard gelatin capsules were prepared in which one capsule contained the following composition. Active ingredient 100 μg Microcrystalline cellulose 195 mg Amorphous silicic acid 5 mg Active ingredient, microcrystalline cellulose and unpressed amorphous silicic acid were thoroughly mixed and packed into hard gelatin capsules.

Example 6 The active ingredient was dissolved in fractionated coconut oil. Further, a soft capsule preparation was prepared by a conventional method using a soft capsule manufacturing machine so that 100 μg of the active ingredient was contained in one capsule by heating and dissolving it in a skin ingredient according to the following formulation. Skin preparation Gelatin 10 parts by weight Glycerin 5 parts by weight Sorbic acid 0.08 parts by weight Purified water 14 parts by weight

[0033]

The triglyceride biosynthesis inhibitor of the present invention,
Acetyl-CoA carboxylase inhibitor, fatty acid synthase inhibitor, and blood triglyceride lowering agent are useful for the purpose of lowering blood triglyceride in pathological conditions in which blood triglyceride is higher than the standard value, that is, hypertriglyceridemia. It is useful as a therapeutic agent for hypertriglyceridemia and as a preventive agent for arteriosclerosis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display area // (C07D 407/04 307: 28 309: 38) C07M 7:00 (72) Inventor Kazuo Kitai 4-3-2 Asahigaoka, Hino-shi, Tokyo Teijin Limited Tokyo Research Center (72) Inventor Keinori Sugimoto 4--3-2 Asahigaoka, Hino City Tokyo Teijin Limited (72) Inventor Takeuchi Takahiro Tokyo 3-2 Asahigaoka, Hino City, Tokyo Teijin Ltd. Tokyo Research Center (72) Inventor Tsutomu Kadota 4-32 Asahigaoka, Hino City, Tokyo Teijin Ltd. Tokyo Research Center

Claims (5)

[Claims] 1. A pharmaceutical composition containing Aureothin as an active ingredient. 2. A triglyceride biosynthesis inhibitor containing Aureothin as an active ingredient. 3. An acetyl-CoA carboxylase inhibitor containing Aureothin as an active ingredient. 4. A fatty acid synthase group inhibitor containing aureothin as an active ingredient. 5. A blood triglyceride lowering agent containing Aureothin as an active ingredient.