JPH0881388A - Agent for preventing arteriosclerosis - Google Patents

Abstract

PURPOSE: To obtain an arteriosclerosis-preventing agent containing κ-casein as an active ingredient, inhibiting the intake of a denatured low density lipoprotein into macrophage to prevent the deposition of choloresterol on the walls of blood vessels, and useful for preventing the arteriosclerosis or its progress. CONSTITUTION: The arteriosclerosis-preventing agent contains κ-casein and/or its hydrolysate as an active ingredient. The hydrolysate of the κ-casein is obtained e.g. by hydrolyzing the κ-casein or a protease, but the hydrolysis is preferably stopped when the fractions having mol.wts. of >=1000 dalton occupy >=30wt.% of the whole hydrolysate. The simultaneous use of a casein (e.g. α3 -casein, β-casein) except the casein fraction is not preferable because of inhibiting the action. The active ingredient is administered at a dose of 5-100mg/kg body/day, preferably 10-200mg/kg body/day, in the oral administration, and at a dose of 0.5-100mg/kg body weight/day, preferably 1-20mg/kg body weight/day, in parenteral administration.

Description

Detailed Description of the Invention

[0001]

This invention relates to an arteriosclerosis preventive agent. More specifically, the present invention relates to denatured low-density lipoprotein (hereinafter, low-density lipoprotein LD
The present invention relates to an arteriosclerosis-preventing agent that prevents cholesterol deposition on the blood vessel wall and prevents arteriosclerosis or its progression by inhibiting the uptake of (described as L) into macrophages.

[0002]

2. Description of the Related Art In arteriosclerosis such as ischemic heart disease and cerebral infarction, cholesterol ester is accumulated in the arterial wall to form a fat layer (fatty streak), and this fat layer further forms atherosclerotic plaque. It is said that the blood vessels are narrowed or blocked. This accumulation of cholesterol ester causes the foam cells (foa
m cell), but since many macrophage-derived foam cells are found in early arteriosclerotic lesions,
It has become clear that macrophages play an important role in the development of arteriosclerosis [American Journal of Pathology, Vol. 103, page 181, 1981].

There are few LDL receptors that recognize and take up normal LDL in macrophages, and scavenger receptors that specifically recognize and take in modified LDL chemically modified in vivo have been developed. Macrophages accumulate cholesterol ester and foam by overdosing modified LDL via scavenger receptors [Proceedings of the National Academy of Sciences of the United States. Proceedings of America
the National Academy of Sciences of the United Sta
tes of America), Vol. 76, pp. 333, 197
9 years]. Denatured LDL recognized by scavenger receptors include acetylated LDL, maleylated LDL, and oxidized LDL.
Examples of such modified LDL have an increased negative charge, such as DL, but in the actual living body, oxidation of LDL by free radicals is considered to be the main pathway of modified LDL production [New England Journal of Medicine ( N
ew England Journal of Medicine), 320, 9
15, page 1989].

On the other hand, high density lipoprotein (hereinafter HDL)
Is described as having a pumping action of cholesterol from foam cells once formed,
It has been suggested that this mechanism is involved in the inhibitory effect of HDL on atherosclerosis [Journal of Lipids].
(Journal of Lipid Research), Vol. 21, p. 391, 1980].

Since arteriosclerosis is caused by such a mechanism, the following measures have hitherto been adopted to prevent arteriosclerosis or prevent its progress [The Lipid] , Volume 2, Pages 494, 199
1 year]. a) lowers LDL cholesterol b) suppresses LDL denaturation (particularly oxidation) c) suppresses foaming of macrophages d) promotes pumping out cholesterol from foam cells Regarding the above a), cholesterol in diet Reduction of
BACKGROUND ART Diet therapy by ingesting dietary fiber and polyunsaturated fatty acids, etc., drug therapy by cholesterol synthesis inhibitors such as compactin, cholesterol excretion promoters such as ion exchange resins, etc. are widely practiced.

Regarding the above-mentioned b), it has become clear that drugs such as probucol have an inhibitory action on LDL oxidation. Further, with respect to the above-mentioned d), in order to increase HDL having its action, dietary therapy and drug therapy similar to those of the above-mentioned a) are carried out. In addition to HDL, thyroglobulin and casein have been found to have the effect of pumping cholesterol from foam cells [Journal of Lipid Research]
, Volume 21, page 391, 1980], but has not been put to practical use. The casein described in this document is a whole casein fraction, and it is not known whether κ-casein has an action of pumping out cholesterol in foam cells.

Further, regarding the above-mentioned c), acyl Co
It is known that an inhibitor of an enzyme involved in foam formation, such as A: cholesterol acyltransferase, present in macrophages has this action, but inhibits incorporation of denatured LDL into macrophages to suppress foam formation. Almost no studies have been conducted on prevention of arteriosclerosis or prevention of progression thereof, and it is quite known that κ-casein and its hydrolyzate have an action of inhibiting the uptake of modified LDL into macrophages. Not not.

On the other hand, κ-casein is a glycoprotein contained in the casein fraction of milk and having a molecular weight of about 19000 daltons. Milk accounts for about 15% of the total casein fraction, and human milk accounts for about 30% of the total casein fraction. κ-Casein plays an important physicochemical role in the formation of casein micelles and the formation of curd, but its specific physiological effect is not known. It has not been known at all that κ-casein inhibits the uptake of denatured LDL into macrophages, and there is no literature.

Further, regarding the hydrolyzate of κ-casein, the glycomacropeptide obtained from κ-casein by the action of rennet is added to the bifidobacteria growth action, gastric acid secretion inhibitory action, and pathogenic bacterium adhesion prevention action (JP-A-3- 22
No. 0130), and infection-preventing action (JP-A-63-284)
133), anti-plaque action and anti-cariogenic action (Japanese Patent Laid-Open No. 63-233911), and other latent peptides have opioid antagonist action and platelet aggregation inhibitory action [Milk Encyclopedia, page 113, Asakura]. Bookstore, 1
1992], etc., but it has never been known to inhibit the uptake of denatured LDL into macrophages.

[0010]

The inventors of the present invention have previously found that lactoferrin and / or a hydrolyzate of lactoferrin have an arteriosclerosis-preventing action, and have already filed a patent application (Japanese Patent Application No. Hei 10 (1999) -135242). No. 5-23055.
Hereinafter referred to as the prior application). The inventors of the present invention further conducted a search for a substance having an arteriosclerosis-preventing action after filing an earlier application, and as a result, kappa-casein and / or a hydrolyzate of kappa-casein were incorporated into modified LDL macrophages. It was found that the activity of the macrophage was suppressed by suppressing the foaming of macrophages, and that the activity was remarkably increased by purifying and using κ-casein from the total casein fraction as compared with the total casein fraction.

Based on the above new findings, the present invention provides a novel method for preventing arteriosclerosis different from the conventional method for preventing arteriosclerosis or a method for preventing the progression thereof, or a drug useful for preventing the progression thereof. The purpose is to do.

[0012]

In order to solve the above problems, the present invention provides an arteriosclerosis inhibitor containing κ-casein as an active ingredient. The present invention also provides an arteriosclerosis inhibitor containing a hydrolyzate of κ-casein as an active ingredient.

The present invention further relates to κ-casein and κ.
-An anti-arteriosclerosis agent containing a hydrolyzate of casein as an active ingredient is also provided. Hereinafter, the present invention will be described in detail. The κ-casein used in the arteriosclerosis preventive agent of the present invention is commercially available κ-casein, isolated from milk or the like of humans, cattle, sheep, goats and the like by a conventional method (for example, urea sulfate method), or a gene set. It may be produced by a replacement technique or the like.

The κ-casein hydrolyzate used in the agent for preventing arteriosclerosis of the present invention can be produced, for example, by hydrolyzing the κ-casein with an acid or a protease by a known method. However, the effect is reduced by lowering the molecular weight, so there is no particular limitation.
It is desirable to stop the hydrolysis at a ratio of (weight, the same applies hereinafter unless otherwise specified) or more.

In the present invention, kappa-casein or a hydrolyzate of kappa-casein may be used alone or in combination. However, as will be described later, combination with other casein contained in the casein fraction, for example, α _s -casein or β-casein, suppresses the action and is not desirable. The arteriosclerosis inhibitor of the present invention is
It can be orally administered to the living body in the form of ordinary foods and drugs. Also, parenterally intravenously, intraarterially, intramuscularly,
It can be administered subcutaneously, intraperitoneally, or the like, and can be administered as an injection by aseptically dissolving it in a solution such as physiological saline. When administered parenterally, the use of κ-casein derived from a different species from the target animal causes a problem with its antigenicity, so it is preferable to use κ-casein derived from the same species or a hydrolyzate thereof. .

The dose of κ-casein or the hydrolyzate of κ-casein varies depending on the kind, age and symptoms of the animal, but usually 5 mg to 1000 m when administered orally.
g / kg body weight / day, preferably 10 mg to 200 mg /
kg body weight / day, usually 0.5 m for parenteral administration
g-100 mg / kg body weight / day, preferably 1 mg-2
0 mg / kg body weight / day.

The acute toxicity of κ-casein or the hydrolyzate of κ-casein has an LD ₅₀ of 4000 mg / kg body weight or more, and the toxicity is extremely low, as is clear from Test Example 3 described later. Next, the present invention will be described in more detail with reference to test examples. Test Example 1 This test affects the specific binding of denatured LDL to macrophage scavenger receptors.
-Done to investigate the effect of casein. 1) Preparation of sample From normal human serum, a conventional method (edited by the Japanese Biochemical Society, “Sequel Biochemistry Experiment Course 3”, page 599, Tokyo Kagaku Dojin,
1986), and acetylated LDL was prepared from the LDL by a conventional method (edited by the Society of Biochemistry, Japan, "Sequel Biochemistry Experimental Course 3", page 672, Tokyo Kagaku Dojin, 1986).

The resulting acetylated LDL was radioiodinated.
Sodium (Na¹²⁵I) is used for the common law (Japan
Biochemical Society ed., "Sequel to Biochemistry Experiment Course 3", page 667
J., Tokyo Kagaku Dojin, 1986).
Sex 400 cpm / ng of protein ¹²⁵I-acetylated LD
L was prepared. 2) Test method Male Wistar rats (purchased from Japan SLC, Inc. Weight 2
Peritoneal macrophages are collected by a standard method from around 00g)
1 x 10⁶100 μl Dal for each macrophage
Becko modified Eagle medium (3% bovine serum albumin,
100 U / ml penicillin, 100 μg / ml strike
Contains leptomycin. Hereinafter, this medium is referred to as medium A
1 μg of¹²⁵I-acetylated LDL
And various concentrations of acetylated LDL or standard [journal
Journal of Dairy Sc
ience), vol. 46, p. 1183, 1963].
Add more purified bovine κ-casein and add at 4 ℃ for 1 hour
Incubated for a period of time. Centrifuge and 1% bovine serum al
Phosphate buffer containing bumine (hereinafter phosphate buffer is
Washing with BS) is repeated 3 times,
Radioactivity bound to the γ-counter (aloca
(Manufactured by the company). 3) Test result The result of this test is shown in FIG. Figure 1
¹²⁵Binding of I-acetylated LDL to macrophages
It shows the relationship with the sample concentration, the vertical axis and the horizontal axis are respectively
Against control¹²⁵Macro fur of I-acetylated LDL
The percentage of binding to di and the sample concentration are shown. ○ in the figure
And ● are acetylated LDL and bovine κ-Ca respectively.
Indicates zein.

As is clear from FIG. 1, acetylated LD
The addition of L inhibits the binding of ¹²⁵ I-acetylated LDL to macrophages, the part of which is specific binding of acetylated LDL to the scavenger receptor of macrophages. K-casein inhibited this specific binding in a concentration-dependent manner, and almost completely at high concentrations. That is, κ-casein is a modified LD
It was clearly observed that L inhibits the specific binding of scavenger receptors of macrophages.

Similar results were obtained with κ-casein and κ-casein hydrolysates derived from other animals. (Test Example 2) This test was conducted to examine the effect of various caseins on the foam formation of macrophages. 1) Sample Bovine α-casein (manufactured by Sigma; α _s -casein content: about 85%) Bovine β-casein (manufactured by Sigma) Bovine κ-casein (manufactured by Sigma) Biopulase (Nagase raw) Bovine κ-casein hydrolyzate hydrolyzed by Kagaku Kogyo Co., Ltd. for 10 minutes, the same α-casein, β-casein and κ-casein as described above were mixed at a ratio of 50:35:15 (the ratio of milk to casein). Mixture mixed in cow whole casein fraction [from milk to the usual method (Tomoyoshi Tsugo,
"Chemistry of milk", page 7, Earth Co., 1975)] 2) Test method Rat peritoneal macrophages of 3 × 10 ⁶ cells / ml suspended in medium A (prepared by the same method as Test Example 1) 1) was seeded on a commercially available 12-well plate and incubated at 37 ° C. for 2 hours to adhere to the bottom surface of the plate. After washing the macrophages three times with PBS, medium A containing 100 μg / ml acetylated LDL and various concentrations of samples
The medium was replaced with 1 ml, and the cells were cultured at 37 ° C for 16 hours. 0.2%
Of bovine serum albumin with and without PB
After thorough washing with S, intracellular lipids were extracted and total cholesterol and free cholesterol were analyzed by the enzymatic method [Journal of Lipid Resear
ch), Vol. 19, p. 514, 1978], and the amount of cholesterol ester was calculated from the difference. 3) Test results The results of this test are shown in FIG. Figure 2
The relationship between the cholesterol ester accumulated in macrophages and the sample concentration is shown, and the vertical axis and the horizontal axis respectively show the percentage of cholesterol ester accumulated in the macrophage relative to the control and the sample concentration.

As is apparent from FIG. 2, α-casein,
Compared to the action of β-casein, κ-casein significantly suppressed the accumulation of cholesterol ester in macrophages. This effect was also observed in the hydrolyzate of κ-casein. That is, it was revealed that κ-casein and its hydrolyzate specifically suppress the foaming of macrophages by denatured LDL.

On the other hand, a total casein fraction and a mixture of α-casein, β-casein and κ-casein were also found to have an action of suppressing the accumulation of cholesterol ester in macrophages. It was extremely weak as compared with the case where the hydrolyzate was used alone. That is, the action of suppressing foaming of macrophages by denatured LDL was weaker than the action expected from the ratio of κ-casein contained therein (about 15%), and κ-casein was purified from the whole casein fraction. It was revealed that its activity remarkably increased by using it. It is known that κ-casein associates with α _s -casein and β-casein to form casein micelles (Tomokichi Tsugo, “Milk Chemistry”, p. 20, Earth Company, 1975). It is speculated that this association suppresses the action of κ-casein.

As a modified LDL for foaming macrophages, instead of acetylated LDL, a conventional method [Journal of Clinical Investigation (J
ournal of Clinical Investigation), Vol. 81, Vol. 7
A similar test was conducted using oxidized LDL prepared according to page 20, 1988], and almost the same result was obtained. Test Example 3 This test was conducted to examine the acute toxicity of κ-casein and κ-casein hydrolysates. 1) Test animals Eight groups (1 group) of 6-week-old CD (SD) rats of both sexes (purchased from Charles River Japan) were randomly selected.
It was divided into 5 groups). 2) Test method Bovine κ-casein (the same as in Test Example 1) and bovine κ-casein (the same as in Test Example 1) were hydrolyzed with biopulase (Nagase Seikagaku Corp.) for 10 minutes to prepare. Dissolve the hydrolyzate in water for injection (Otsuka Pharmaceutical Co., Ltd.),
1000, 2000 and 4000 mg / kg body weight
A single oral gavage was administered using a needle with a metal ball at a ratio of 1 to test acute toxicity. 3) Test results The results of this test are shown in Table 1. As is clear from Table 1, no death occurred in any of the cases where bovine κ-casein and bovine κ-casein hydrolyzate were administered. Therefore, the LD ₅₀ of bovine κ-casein and bovine κ-casein hydrolyzate were both 4000 mg / kg or more. It should be noted that, when the tests were carried out by changing the types of κ-casein and κ-casein hydrolyzate, almost the same results were obtained.

[0024]

[Table 1]

Next, the present invention will be described in more detail and concretely by showing examples, but the present invention is not limited to the following examples.

[0026]

【Example】

Example 1 A tablet antiarteriosclerosis agent having the following composition per tablet was prepared. Bovine κ-casein hydrolyzate 20.0 (mg) [Prepared by hydrolyzing bovine κ-casein (the same as in Test Example 1) with biopulase (Nagase Seikagaku Corporation) for 10 minutes] Lactose monohydrate Material (manufactured by Wako Pure Chemical Industries, Ltd.) 30.0 Corn starch (manufactured by Wako Pure Chemical Industries, Ltd.) 19.8 Crystalline cellulose (manufactured by Asahi Kasei Corporation) 28.0 Magnesium silicate pentahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 2.0 Magnesium stearate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 Bovine kappa-casein hydrolyzate, lactose monohydrate, corn starch and crystalline cellulose are uniformly kneaded while appropriately adding sterile purified water to the mixture. Then, the mixture was dried at 50 ° C. for 3 hours, magnesium silicate pentahydrate and magnesium stearate were added to and mixed with the obtained dried product, and the mixture was tableted by a tableting machine by a conventional method. Example 2 1 ml of water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added with a known method [Journal of Bioche
mistry), vol. 90, page 1005, 1981)
Dissolved at a ratio of 10 mg of human κ-casein purified by 4. Adjust to 10 liters, sterilize by filtration, and use 10 ml by the conventional method.
Each was filled in a vial bottle and freeze-dried to prepare a freeze-dried arteriosclerosis inhibitor for injection. Example 3 Purified bovine κ-casein (the same as in Test Example 1) 5
0 part (weight; the same applies hereinafter), 30 parts of a hydrolyzate obtained by hydrolyzing this bovine κ-casein with rennin (manufactured by Sigma) for 30 minutes, 50 parts of corn starch (manufactured by Matsutani Chemical Co., Ltd.), sucrose 50 parts (manufactured by Toyo Seito Co., Ltd.) and L-
20 parts of sodium ascorbate (manufactured by Nippon Roche) was thoroughly mixed, and 2 g of the mixture was packed in a bag to prepare a powdery arteriosclerosis inhibitor for oral ingestion.

[0027]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention provides an arteriosclerosis inhibitor that suppresses macrophage foaming by specifically inhibiting the uptake of denatured LDL into macrophages. This makes it possible to effectively prevent arteriosclerosis or prevent its progression.

[Brief description of drawings]

FIG. 1 shows the relationship between binding of ¹²⁵ I-acetylated LDL to macrophages and sample concentration.

FIG. 2 shows the relationship between cholesterol ester accumulated in macrophages and sample concentration.

Claims (3)

[Claims] 1. An anti-arteriosclerosis agent comprising κ-casein as an active ingredient. 2. An anti-arteriosclerosis agent containing a hydrolyzate of κ-casein as an active ingredient. 3. An anti-arteriosclerosis agent containing κ-casein and a hydrolyzate of κ-casein as an active ingredient.