JPH06234655A - Modified low density lipoprotein binder - Google Patents

Abstract

PURPOSE:To obtain the modified LDL binder not only specifically binding to a modified LDL but also inhibiting the intake of the modified LDL into macrophage. CONSTITUTION:This modified low density lipoprotein binder contains lactoferrin and/or the hydrolysate of the lactoferrin as active ingredients. Since this binder specifically binds to modified LDL, it can selectively remove the modified LDL without substantially deteriorating other ingredients in blood, and since specifically binding to the modified LDL and inhibiting the intake of the modified LDL into macrophage, the modified LDL binder can inhibit the foaming of the macrophage. Thereby, the crisis of arterial sclerosis or its progress can effectively prevented.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to denatured low density lipoprotein binders. More specifically, the present invention relates to a denatured low-density lipoprotein binding agent useful as a preventive agent for arteriosclerosis or a preventive agent thereof.

[0002]

2. Description of the Related Art In arteriosclerosis such as ischemic heart disease and cerebral infarction, cholesterol ester accumulates on the arterial wall to form a fatty streak, and atherosclerotic plaques are formed. It is believed to be caused by stenosis and occlusion. The accumulation of this cholesterol ester is due to the aggregation of foam cells containing a large amount of cholesterol ester, but since macrophage-derived foam cells are often found in the early arteriosclerotic lesions, macrophages show arteriosclerosis. It has become clear that it plays an important role in the onset of disease [American Journal of Pathology, Vol. 103,
Pp. 181, 1981].

[0003] There are few LDL receptors that recognize and take up normal low-density lipoprotein (hereinafter sometimes referred to as LDL) in macrophages, and specific denatured LDL chemically modified in vivo is specific. The scavenger receptor that has been recognized and taken up is developed, and macrophage accumulates cholesterol ester and foams by excessively taking up denatured LDL via the scavenger receptor [Proceedings of the National. Academy of Sciences of the United States of America (Proceedings o
f the National Academy of Sciences of the United St
ates of America), 76, 333, 19
1979]. Denatured LDL recognized by scavenger receptors
Examples of the modified LDL include acetylated LDL, maleylated LDL, oxidized LDL, etc., which have particularly increased negative charges.
Is believed to be the major pathway for the generation of denatured LDL [New England Journal of Medicine, Volume 320,
P. 915, 1989].

Due to such a mechanism of onset of arteriosclerosis, the following measures have been conventionally adopted to prevent arteriosclerosis or prevent its progress [The Lipi
d), Volume 2, p. 494, 1991]. a) lowering LDL cholesterol b) suppressing LDL denaturation (particularly oxidation) c) suppressing macrophage foaming Regarding the above a), reduction of dietary cholesterol,
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 b), it has become clear that drugs such as probucol have an inhibitory effect on LDL oxidation. Further, regarding the above-mentioned c), it is known that an inhibitor of acyl CoA: cholesterol acyltransferase of macrophages has this action.

[0005]

However, the modified LDL produced in the intermediate step between the above b) and c).
Almost no studies have been conducted on the prevention of arteriosclerosis or its progression by inhibiting the uptake of macrophages into macrophages, let alone the effect of inhibiting the uptake of denatured LDL into macrophages by lactoferrin and its hydrolysates. It was unknown at all.

[0006] Lactoferrin is a basic iron-binding protein contained in milk, tears, exocrine fluid such as saliva, blood and the like. Although many studies have been carried out on the physiological action of lactoferrin, it is difficult to say that it has been fully elucidated because of its wide variety of physiological actions. It was its antibacterial activity that was pointed out from the beginning when the presence of lactoferrin in milk was confirmed [Biochimica et Biophica.
ysica Acta), Volume 45, Page 413, 1960.
Year] against E. coli, staphylococci and enterococci.
It has been reported to have antibacterial activity at a concentration of 5 to 30 mg [Journ of Daily Science (Journ
al of Dairy Science), Volume 67, Page 606, 1
984]. Other physiological effects of lactoferrin in milk include iron absorption promoting action, cell growth promoting action, and the like [Monthly Food Chemical, Vol. 7, Vol.
Issue, p. 61, 1991]. Lactoferrin is also produced by neutrophils and is also present in body fluids such as blood, and it has been pointed out that it is also involved in the suppression of inflammation [American Journal of Pathology (Ame
rican Journal of Pathology), 99, 413, 1980].

However, lactoferrin is a modified LD
It has the property of binding specifically to L, and also denatured LDL
It has not been known at all until now that the uptake of saccharin into macrophages is inhibited, and there is no literature. Further, the antibacterial action of lactoferrin hydrolysates has also been reported [Journal of Dairy Science, Vol. 74, 413.
7, p. 1991], having the property of binding specifically to denatured LDL, and inhibiting the uptake of denatured LDL into macrophages has never been known.

The present invention has been made in view of the above circumstances, and is completely different from the conventional method for preventing arteriosclerosis or the method for preventing the progress thereof, and is useful for preventing or preventing the progression of arteriosclerosis. It is an object of the present invention to provide a modified LDL-binding agent that not only specifically binds to modified LDL but also inhibits uptake of modified LDL into macrophages as a drug.

[0009]

The inventors of the present invention have
As a result of intensive studies to solve the above problems, it was found that lactoferrin and its hydrolyzate have the property of specifically binding to modified LDL and further inhibit the uptake of modified LDL into macrophages. It started and completed this invention.

That is, the present invention provides a modified low-density lipoprotein binder containing lactoferrin and / or a hydrolyzate of lactoferrin as an active ingredient. Hereinafter, the present invention will be described in detail. The lactoferrin used in the modified LDL-binding agent of the present invention is obtained, for example, by commercially available lactoferrin or by isolating it from a secretory fluid such as milk of humans, cattle, sheep, goats, etc. by a conventional method (for example, ion exchange chromatography). Or produced by genetic recombination technology. Further, apolactoferrin deironed with hydrochloric acid, citric acid, or the like, metal saturated lactoferrin saturated with metals such as iron, copper, zinc, manganese, or the like, and an arbitrary mixture thereof may be used.

The lactoferrin hydrolyzate used in the modified LDL binder of the present invention is produced, for example, by hydrolyzing the above-mentioned lactoferrin with an acid or a protease. However, since the action is weakened by lowering the molecular weight, there is no particular limitation, but it is desirable that the decomposition is carried out to the extent that the molecular weight is not extremely lowered. In the present invention, lactoferrin or a hydrolyzate of lactoferrin may be used alone, or both may be used in combination.

The modified LDL binder of the present invention is fixed to a carrier by a known method and aseptically sealed in a container,
A filter that allows only fluid to pass through both ends of the container can be applied as a dialysis device that selectively removes denatured LDL from body fluid. The modified L of the present invention
The DL binding agent can also be aseptically dissolved in a solution such as physiological saline and administered to a living body. In this case, it can be used as a drug that specifically binds to denatured LDL in body fluid and inhibits uptake of denatured LDL into macrophages. Since sexuality is a problem, it is desirable to use lactoferrin derived from the same species or a hydrolyzate thereof.

The dose of lactoferrin and / or lactoferrin hydrolyzate may vary depending on the type and age of the animal, but it is usually 0.2 mg to 80 mg / kg body weight / kg regardless of whether arteriosclerosis develops. Day,
Desirably, the dose is 0.4 mg to 10 mg / kg body weight / day. The acute toxicity of lactoferrin and the hydrolyzate of lactoferrin is, as is clear from Test Example 7 described later,
LD50 is 4000 mg / kg or more, and toxicity is extremely low.

Next, the function and effect of the present invention will be described in detail with reference to test examples. (Test Example 1) This test was conducted to examine whether or not bovine lactoferrin has a property of binding to acetylated LDL. 1) Sample From normal human serum, a conventional method (edited by the Biochemical Society of Japan, "Lecture on Biochemistry Experiment 3", page 599, Tokyo Kagaku Dojin, 198)
Aqueous solution containing 5 μg of LDL prepared in 6 years) From the above LDL, the conventional method (edited by the Biochemical Society of Japan, “Lecture on Biochemistry Experiment 3”, page 672, Tokyo Kagaku Dojin, 19
1986) and an aqueous solution containing 5 μg of acetylated LDL. 5 μg of acetylated LDL and bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., the same applies hereinafter). Aqueous solution containing 5 μg 5 μg of acetylated LDL and 5 μg of bovine lactoferrin
Aqueous solution containing 5 μg of acetylated LDL and 50 μ of bovine lactoferrin
However, the weight of the lipoprotein represents the weight of the protein component (hereinafter the same). 2) Test method Each sample was subjected to agarose gel (Universal Gel 8 manufactured by Ciba Corning) using 0.05 M barbital buffer, and the protein was stained with Coomassie Brilliant Blue. 3) Test result The result of this test is as shown in FIG. FIG. 1 is a trace diagram of an electrophoretic photograph. LDL (sample) is negatively charged by acetylation and moves to the positive electrode side (sample). However, when acetylated LDL coexisted with bovine lactoferrin, it bound to bovine lactoferrin to form a single band, and moved to the neutral side depending on the concentration of bovine lactoferrin (sample ~). That is, it was revealed that bovine lactoferrin has an action of binding to acetylated LDL and neutralizing its negative charge. Similar results were obtained for the lactoferrin hydrolyzate. (Test Example 2) In this test, bovine lactoferrin was oxidized to L
It was carried out to examine whether or not it has the property of binding to DL. 1) Sample Aqueous solution containing 5 μg of LDL same as in Test Example 1 From the above sample, the conventional method [Journal of Clinical Inv
estigation), Vol. 81, p. 720, 1988
Aqueous solution containing 5 μg of oxidized LDL prepared in the above] Aqueous solution containing 5 μg of acetylated LDL same as in Test Example 1 Aqueous solution containing 5 μg of oxidized LDL and 0.5 μg of bovine lactoferrin Aqueous solution containing 5 μg of oxidized LDL and 5 μg of bovine lactoferrin Aqueous solution 2) Test method The same method as in Test Example 1 was used. 3) Test result The result of this test is as shown in FIG. FIG. 2 is a trace diagram of an electrophoretic photograph, in which the symbols (1) to (3) represent the samples described above. When oxidized LDL was allowed to coexist with bovine lactoferrin, it bound bovine lactoferrin to form a single band, and migrated to the neutral side depending on the concentration of bovine lactoferrin (sample-). That is, bovine lactoferrin is not only acetylated LDL, but oxidized LDL that plays a major role in the development of arteriosclerosis in vivo.
It has been revealed that they also have a function of binding with and neutralizing the negative charge. Similar results were obtained for the lactoferrin hydrolyzate. (Test Example 3) In this test, bovine lactoferrin was denatured L
It was performed to examine whether the property of binding to DL is specific to denatured LDL. 1) Sample Aqueous solution containing 5 μg of LDL and 5 μg of bovine lactoferrin identical to those in Test Example 1 From normal human serum, a conventional method (edited by the Biochemical Society of Japan, “Zokusei Chemistry Experimental Course 3”, page 599, Tokyo Kagaku Dojin, 198).
High density lipoprotein (hereinafter referred to as H
An aqueous solution containing 5 μg (described as DL) and 5 μg of bovine lactoferrin 2) Test method The same method as in Test Example 1 was used. 3) Test result The result of this test is as shown in FIG. FIG. 3 is a trace diagram of an electrophoretic photograph, where and represent the above-mentioned samples, respectively. Ordinary LDL and HDL were detected as bands separate from bovine lactoferrin even when coexisting with bovine lactoferrin (sample and). That is, it was revealed that bovine lactoferrin does not bind to ordinary lipoproteins, but only specifically to denatured LDL. Similar results were obtained for the lactoferrin hydrolyzate. (Test Example 4) In this test, lactoferrin was denatured LDL.
It was performed to examine whether the property of binding with is specific to lactoferrin. 1) Sample Aqueous solution containing 5 μg of the same LDL as in Test Example 1 Aqueous solution containing 5 μg of the same acetylated LDL as in Test Example 1 Aqueous solution containing 5 μg of human lactoferrin (manufactured by Calbiochem) 5 μg of acetylated LDL and 5 μg of human lactoferrin same as in Test Example 1 Aqueous solution containing 5 μg of Uciapotransferrin (manufactured by Kanto Kagaku) Aqueous solution containing 5 μg of acetylated LDL and 5 μg of Uciapotransferrin Aqueous solution containing 5 μg of bovine iron transferrin (manufactured by Kanto Kagaku) Same as Test Example 1 Aqueous solution containing 5 µg of acetylated LDL and 5 µg of bovine iron transferrin 2) Test method The same method as in Test Example 1 was used. 3) Test result The result of this test is as shown in FIG. FIG. 4 is an electrophoretic photographic trace diagram, in which ˜ represents the samples. Like bovine lactoferrin, human lactoferrin bound to denatured LDL to form a single band, neutralizing the negative charge of denatured LDL (sample). However, transferrin, which is an iron-binding protein homologous to lactoferrin, coexists with denatured LDL,
Detected as a band separate from denatured LDL, and denatured L
It had no effect on the DL charge. That is, it was revealed that the property of binding to denatured LDL is specific to lactoferrin. Similar results were obtained for lactoferrin and transferrin hydrolysates. (Test Example 5) In this test, lactoferrin was denatured LDL
It was carried out to examine the effect of the property of specifically binding to LDL on the specific binding of denatured LDL to the macrophage scavenger receptor. 1) Test method Acetylated LDL was treated with radioactive sodium iodide (Na 1
25I) and labeled by the conventional method [Zokusei Kagaku Kenkyu Koza 3 (Tokyo Kagaku Dojin), page 667, 1986],
125 I-acetylated LDL of radioactive 400 cpm / ng protein was prepared. Peritoneal macrophages were collected from male Wistar rats (manufactured by SLC Japan, about 200 g) by a conventional method, and 1 × 10 6 macrophages were added to 100 μl of Dulbecco's modified Eagle medium (3% bovine serum albumin, 100 U / ml penicillin, 10
Suspended in 0 μg / ml streptomycin; hereinafter referred to as medium A), and suspended in 1 μg of 125 I-acetylated LDL and various concentrations of acetylated LDL or bovine lactoferrin (all the same as in Test Example 1).
Was added and incubated at 4 ° C. for 1 hour. Centrifugation and washing with PBS (containing 1% bovine serum albumin) were repeated 3 times, and the radioactivity bound to macrophages was measured by a γ-counter. 2) Test results The test results are shown in Fig. 5. FIG. 5 shows 125
The relationship between the binding of I-acetylated LDL to macrophages and the sample concentration is shown, and the vertical axis shows 125 relative to the control.
The percentage of binding of I-acetylated LDL to macrophages is shown, and the horizontal axis shows the sample concentration. In the figure, ○ and ● represent acetylated LDL and bovine lactoferrin, respectively.

125 by addition of acetylated LDL
Although the binding of I-acetylated LDL to macrophages is inhibited, this inhibited portion is the specific binding of acetylated LDL to the macrophage scavenger receptor. Lactoferrin inhibited this specific binding in a concentration-dependent manner, and almost completely at high concentrations. That is, it was revealed that lactoferrin specifically binds to the denatured LDL to eliminate the property of the denatured LDL as a ligand of the macrophage scavenger receptor. Similar results were obtained for the lactoferrin hydrolyzate. (Test Example 6) This test was conducted to examine the effect of lactoferrin on the foaming of macrophages. 1) Sample Bovine lactoferrin same as in Test Example 1 Bovine lactoferrin hydrolyzate obtained by hydrolyzing the above bovine lactoferrin with biopulase (manufactured by Nagase Seikagaku Corp.) for 10 minutes Same as Test Example 4 Usiapotransferrin Same as Test Example 4 Bovine iron transferrin of 2) Test method 3 x 106 cells / ml of rat peritoneal macrophages suspended in medium A were seeded in 1 ml each on a commercially available 12-well plate and cultured at 37 ° C for 2 hours to attach the macrophages to the bottom surface. Let After washing 3 times with PBS, 100 μg / m
The medium was replaced with 1 ml of medium A containing 1 acetylated LDL and samples of various concentrations, and the cells were cultured at 37 ° C. for 16 hours. 0.
The cells were thoroughly washed with PBS containing 2% bovine serum albumin and PBS containing no bovine serum albumin, intracellular lipids were extracted, and total cholesterol and free cholesterol were analyzed by the enzymatic method [Journal of Lipid Research (Journal of Lipid Research).
search), Vol. 19, p. 514, 1978], and the difference is defined as cholesterol ester. 3) Test results The test results are shown in Fig. 6. FIG. 6 shows the relationship between cholesterol ester accumulated in macrophages and the sample concentration. The vertical axis represents the percentage of cholesterol ester accumulated in macrophages relative to the control, and the horizontal axis represents the sample concentration. Transferrin did not affect the accumulation of cholesterol ester in macrophages, whereas lactoferrin significantly suppressed it. This effect was also observed in the hydrolyzate of lactoferrin. That is, it was revealed that lactoferrin and its hydrolyzate suppressed foaming of macrophages due to modified LDL. Test Example 7 This test was conducted to examine the acute toxicity of lactoferrin and lactoferrin hydrolyzate. 1) Test animal Six groups of 6-week-old CD (SD) rats of both sexes (manufactured by Charles River Japan) were randomly assigned to 8 groups (5 groups per group).
). 2) Test method Bovine lactoferrin and a hydrolyzate of bovine lactoferrin obtained by hydrolyzing it with bioprase for 10 minutes were dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) to give 100 per 1 kg of body weight.
A single oral gavage at a rate of 0, 2000 or 4000 mg was performed using a needle with a metal ball, and acute toxicity was tested. 3) Test results The results of this test are shown in Table 1. As is clear from Table 1, no death was observed in any of the cases where bovine lactoferrin and bovine lactoferrin hydrolyzate were administered. Therefore, the LD50 of bovine lactoferrin and bovine lactoferrin hydrolyzate were both 4000
It was more than mg / kg. It should be noted that, although the lactoferrin hydrolyzate was changed and tested, similar results were obtained.

[0016]

[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.

[0018]

【Example】

Example 1 Porous cellulose gel (manufactured by Chisso Co., CK gel A-3)
To 100 ml, 40 g of 20% sodium hydroxide, 120 g of heptane, and 10 drops of nonionic surfactant (Tao 20 manufactured by Kao Atlas Co., Ltd.) were added, and the mixture was stirred at 40 ° C. for 2 hours, after which 50 g of epichlorohydrin was added and stirred for 2 hours. Then, the gel was washed with water and filtered to obtain a cellulose gel having an epoxy group introduced therein. In addition, a method known from human milk [Blood Cells, Volume 15, 37]
1 page, 1989], 1 g of human lactoferrin purified in 200 ml of purified water was added and a solution adjusted to pH 10 was added, and the mixture was shaken at room temperature for 24 hours, washed with water and filtered, and a human lactoferrin-immobilized cellulose gel was obtained. 100 ml was obtained. This was aseptically sealed in a container that was a filter that allowed only fluid to pass through both ends, to prepare a modified LDL binder for hemodialysis. Example 2 A modified LDL binder for tablets having the following composition per tablet was prepared.

Bovine lactoferrin hydrolyzate 20.0 (mg) [Bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) hydrolyzed with biopulase (manufactured by Nagase Seikagaku Corporation) for 10 minutes] Lactose monohydrate (Wako Pure Chemical Industries, Ltd.) 30.0 Corn starch (manufactured by Wako Pure Chemical Industries) 19.8 Crystalline cellulose (manufactured by Asahi Kasei Corporation) 28.0 Magnesium silicate pentahydrate (manufactured by Wako Pure Chemical Industries) 2.0 Stearic acid Magnesium (manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 A mixture of bovine lactoferrin hydrolyzate, lactose monohydrate, corn starch and crystalline cellulose is uniformly kneaded while appropriately adding sterilized purified water, and the mixture is kneaded at 50 ° C. for 3 hours. After drying, magnesium silicate pentahydrate and magnesium stearate are added to the obtained dried product and mixed, and tableted by a tableting machine by a conventional method. It was. Example 3 In 1 ml of water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), 10 mg of human lactoferrin purified by the same method as in Example 1 and 49.5 mg of D-mannite (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved, The pH was adjusted to about 7 with an aqueous solution of phosphate buffer powder (manufactured by Wako Pure Chemical Industries, Ltd.), sterilized by filtration, 10 ml each was filled in vials by a conventional method, freeze-dried, and freeze-dried modified LDL for injection. A binder was prepared.

[0020]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, the modified LDL can be selectively removed with specific binding to the modified LDL without substantially impairing other components in the body fluid. Provided is a novel modified LDL binding agent capable of inhibiting the uptake of modified LDL into macrophages and suppressing macrophage foaming. This makes it possible to effectively prevent the onset and progression of arteriosclerosis.

[Brief description of drawings]

FIG. 1 is a trace diagram of an electrophoretic photograph.

FIG. 2 is a trace diagram of an electrophoretic photograph.

FIG. 3 is a trace diagram of an electrophoretic photograph.

FIG. 4 is a trace diagram of an electrophoretic photograph.

FIG. 5 shows the relationship between binding of 125 I-acetylated LDL to macrophages and sample concentration.

FIG. 6 shows the relationship between cholesterol ester accumulated in macrophages and sample concentration.

Front page continuation (72) Inventor Kozo Kawase 761-1 Shiraho, Urawa City, Saitama Prefecture (72) Inventor Mitsunori Takase 138-10 Minamichumaru, Omiya City, Saitama Prefecture (72) Inventor Mikio Kajikawa Asahi Ward, Yokohama City, Kanagawa Prefecture Town 2672-41

Claims (1)

[Claims] 1. A modified low-density lipoprotein binder comprising lactoferrin and / or a hydrolyzate of lactoferrin as an active ingredient.