JPH05186367A - Therapeutic agent for hyperlipemia - Google Patents

Abstract

PURPOSE:To provide the subject therapeutic agent having such effect as to reduce blood cholesterol efficiently in the body, containing, as active ingredient, interleukin-6. CONSTITUTION:The objective therapeutic agent containing, as active ingredient, interleukin-6 produced by cultured human cells. As necessary, this agent is formulated with additive (s) like human serum albumin, gelatin, mannitol, sorbitol, sorbitol, or lactose into a pharmaceutical preparation. Besides injections, this agent can be made into a pharmaceutical preparation suitable for transmucosal administration such as peroral, pernasal, transpulmonary, or transintestinal administration. Its dose is 0.0001-300mug/kg body weight/day. The interleukin-6, because of its presence in the body, is more compatible with the body than synthetic medicines.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel therapeutic drug for hyperlipidemia.

[0002]

2. Description of the Related Art Recently, HMG has been used as a therapeutic drug for hyperlipidemia.
-A CoA reductase inhibitor has been developed as a synthetic drug that inhibits cholesterol synthesis in vivo. But,
The therapeutic drug for hyperlipidemia is artificially synthesized,
It may artificially inhibit the enzymatic reaction in the body. Originally, it is more desirable to apply a substance that exists in the living body and has a desired function as a medicine because it is more desirable in terms of not disturbing the rhythm of the living body. A useful therapeutic agent has not yet been developed.

On the other hand, interleukin 6 (hereinafter IL-
Abbreviated as 6) is interferon β ₂ (Zilberstein, A.
et.at., EMBO J. 5, 2529-2537 , 1986), B cell differentiation factor (BSF-2): (Hirano, T. et.al., Nat.
ure, 324, 73-76 , 1986), 26-KDa protein (Hage
man, G.et.al., Eur.J.Biochem., 159 , 625-632, 1986),
Hybridoma / Plasmacytoma Growth Factor (VanDam
me, J.et.al., J.Exp.Med., 165 , 914-919,1987), hepatocyte stimulating factor (HSF): (Andus, T.et.al., FEBS Le
tt., 221, 18-22,1987; Gauldie, J.et.al., Proc.Natl.Aca
d.Sci.USA, 84 , 7251-7255, 1987), etc., which have been studied separately, have been found to be the same molecule.
It has been proposed to call it, and its name is well established. As described above, many physiological activities have been reported for IL-6, but a clear effect on lipid metabolism has not yet been shown. Slightly, Grunfeld, C.et.al., Cancer Res., 5
0, the 4233-4238 (1990), were administered IL-6 in mice is but transient fatty acid synthesis in hepatocytes is increased, liver cholesterol - only Le synthesis is described as not fluctuate Is.

[0004]

The problem to be solved by the present invention is to provide a physiologically active substance which originally has an action in vivo as a medicine. Among other things, the object of the present invention is to provide an in-vivo physiologically active substance having an action of efficiently reducing blood cholesterol in the body.

[0005]

The above object can be achieved by the present invention described below. That is, the present invention relates to a therapeutic drug for hyperlipidemia containing interleukin 6 as an active ingredient. The present invention finds that IL-6 efficiently reduces blood cholesterol, and shows that IL-6 is useful as a novel therapeutic drug for hyperlipidemia that has not been obtained hitherto. ..

The IL-6 used in the present invention is not particularly limited, and IL-6 obtained by a known method is preferably used. For example, those obtained by culturing IL-6 producing cells, or recombinant type I obtained by the gene recombination method.
L-6 is also acceptable. More preferably, those obtained by culturing IL-6-producing human cells are used.

IL-6 obtained from cultured human cells is preferable because it is possible to avoid contamination of impurities derived from species other than human, and the obtained IL-6 is close to IL-6 which originally works in vivo. .. That is, the structure containing a sugar chain and fine modification is similar to that of IL-6 in a human body, and thus antibody production can be relatively eliminated when administered as a drug to humans, which is preferable. Therefore, IL-6 produced by human cultured cells can be expected to be more effective in vivo.

IL-6 produced by the cultured human cells of the present invention
Means IL-6 obtained by culturing cells of human origin, and more specifically, normal cells, that is, adherent humans that have not become cancerous (extremely transformed) or are not derived from cancer cells. IL-6 obtained by culturing cells is preferred. IL-6 obtained under these conditions usually has a sugar chain-added structure. As normal human cells not derived from cancer cells, particularly fibroblasts,
Endothelial cells, stromal cells, etc. are considered to be one of the sources of IL-6 in vivo, and some of them are particularly preferably used because they can be cultured in a form close to normal cells, but are not particularly limited to these. It is not something that will be done.

Among the normal human cells preferably used in the present invention, particularly suitable cells are adherent and can be cultured under general cell culture conditions. General culture flask,
A roller bottle, a culture method using microcarriers (fine particles), and the like are preferably used, but are not limited thereto. Thus, almost pure IL-6 can be obtained from the culture broth obtained by culturing human cells by a conventional purification method. Examples of these culturing and purifying methods are shown in Examples, but are not limited to these.

On the other hand, recombinant IL-6 can be produced by a known method. As an example, an example in which Escherichia coli is used as a host is shown as an example, but other than this, it can be produced by using widely known gene manipulation methods. For example, it can also be prepared by introducing the IL-6 gene into animal cells such as hamster cells, mouse cells, monkey cells, etc. or yeast by linking it downstream of a promoter or the like that functions in the host.

The composition of the present invention contains human IL-6 produced by the above-mentioned method as a main component. As the other components, general pharmaceutical additives are selected. Of course, the object of the present invention can be achieved without additives. Additives are generally added primarily for stabilization. Such a pharmaceutical additive is selected from the proteins and / or saccharides described in the local law that can be used as a pharmaceutical additive. Particularly suitably selected from human serum albumin (HSA), gelatin, mannitol, sorbitol, lactose and the like, but not limited to these.

In order to specifically achieve the blood cholesterol-reducing activity which is the object of the present invention, the composition containing IL-6 thus obtained is administered to a living body. The administration method is not particularly limited, but an appropriate one is selected from general injections such as intravenous injection, subcutaneous injection, intramuscular injection, and intravenous drip infusion. In some cases, transmucosal administration methods such as oral, nasal, pulmonary, and enteral administrations are also suitably performed.

The present invention also relates to human IL-6 and other drugs,
It also includes simultaneous or sequential combined administration with a biological agent, a synthetic pharmaceutical agent and the like. Other agents include IL-
6, which has blood cholesterol lowering activity independently of 6,
Alternatively, it is selected from those which enhance or assist the activity of IL-6.

The effective dose of IL-6 is selected in the range of 0.0001 to 300 μg per 1 kg of body weight per day. Suitably 0.001-1 per kg body weight
It is selected in the range of 0 μg. The above-mentioned dose varies depending on the symptoms and is not limited to these values.
The number of administrations is usually selected in the range of 1 to 2 times a day, or once in 2 to several days, but is not limited thereto.

[0015]

EXAMPLES The present invention will now be described in more detail and more specifically by way of examples, which, of course, are not intended to limit the present invention. The IL-6 activity was evaluated by the following method.

Evaluation method of biological activity: Cell line 7TD1 (IL
-6-dependent hybridoma cells (J. van Snick et al., E
uropean J. Immunol., 18, 193-197 (1988)),
By adding an appropriate amount of IL-6 to it, 7TD
1 cell proliferation was measured by the MTT method, and a standard IL-
The bioactivity of IL-6 was assessed by comparison with the proliferative activity for 6 serially diluted samples. As standard IL-6, human IL-6ELIS shown below by Toray Industries, Inc.
The same as the one attached to the A kit was used.

ELISA (enzyme-linked immunosorbent assay) method: anti-IL
-6 antibody (N.Ida et al., Biochem.Biophys.Res.Commun.,
165,728-734, (1989)). IL-6 was evaluated using a human IL-6 ELISA kit manufactured by Toray Industries, Inc., sold by Toray Fuji Bionics.

Example 1 Preparation of E. coli-derived IL-6: Known literature (T. Hirano et al.,
Nature, vol. 324, 73 (1986)), which has an IL-6 cDNA having the same gene sequence as the backbone I
The L-6 expression vector was prepared by the following method.

Thyroid cancer-derived cell line NIM-1 cells (Kaoru Toyama, Journal of Japanese Society of Hematology, 53, 805 (199
0)) is cultivated, and the following two chemically synthesized DNA oligomers CCGATCCGATGCCATAGCCCCCAGGGA are used as a substrate with a cDNA mixture synthesized by reverse transcriptase from mRNA prepared by a usual method.
(Sequence ID No. 1 in the Sequence Listing) and GCCACGGATCCCCATATTTGCCGAAG
A PCR reaction was carried out using (SEQ ID NO: 2 in the sequence listing) as a primer. After the obtained amplified DNA was digested with restriction enzymes ClaI and BamHI, the obtained DNA fragment was used for Escherichia coli expression vector-pKM6.
(Tanaka et al., J. Interferon Res., 6, 429-35 (198
6)) is inserted between the ClaI site and the BglII site,
The expressed IL-6 vector-pKMIL-6 was obtained. This pK
MIL-6 was introduced into Escherichia coli HB101 to obtain a recombinant. This recombinant was cultured as described below to prepare Escherichia coli recombinant IL-6.

Escherichia coli HB101 / pKMIL-6 containing a human interleukin-6 expression plasmid
Cultured using an L jar. 30 L of growth medium (1 potassium phosphate 0.3%, disodium phosphate 0.6
%, Sodium chloride 0.5%, ammonium chloride 0.1
%, 0.5% glucose, 0.5% casamino acid, magnesium sulfate 1 mM, ferrous 3μM sulfate, vitamin B ₁ 6
μg / ml, ampicillin 50 μg / ml) was placed in a 30 L jar to inoculate the above recombinant. Jar
The operation was performed under the conditions of a stirring number of 300 rpm, an air flow rate of 1 VVM, and 25 ° C. Indole acrylic acid, which is an inducer of tryptophan operon, was added, and the mixture was cultured for 60 hours while adding glucose and casamino acid. Cultured cells are 10,000
It was collected by centrifugation at 0 × g for 20 minutes. The cells are
About 895 g was obtained. Collected cells with 1 mM EDTA, 1
The suspension was suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 00 mM NaCl so that the OD550 nm was 20.
The cells were disrupted with Manton-Gorrin and centrifuged to recover the disrupted extract. The amount of protein in the extract is 235
g, interleukin-6 was 495 mg. Here, the amount of IL-6 was measured by the above-mentioned ELISA method (hereinafter the same).

The extract was adsorbed on 5.5 L of a silica column and eluted with an acidic solution. 462 mg of IL-6 was recovered. Ammonium sulfate was added to the eluate at a final concentration of 1.33 M, and the insoluble impurities were removed by centrifugation. Next, it was adsorbed on 200 ml of a butyl column (Butyl Toyopearl Tosoh Corporation) and eluted with a low salt neutral solution. SDS-PAGE
By the purity assay method, 237 mg of IL-6 having a purity of 84% was obtained. The eluted IL-6 was directly used for the heparin column (AF
Heparint Yopearl (manufactured by Tosoh Corporation) 80 ml was adsorbed. Elute with neutral salt buffer. IL- with a purity of 91%
114 mg of 6 was obtained. The eluate was purified again with 200 ml of a butyl column (Butyl Toyopearl Tosoh Corporation) to obtain 66 mg of IL-6. IL-6 prepared above
Was 95% or more by reverse phase HPLC method. Above I
L-6 is an IL having activity according to the evaluation method described in Example 1.
It was confirmed to be -6.

Example 2 Preparation of human cell-derived IL-6: IL-6 of the present invention was prepared by the following method as an example. Human fibroblasts were bead-cultured in a 2 L glass culture tank in an Eagle MEM medium containing 1 L of 5% NCS so that the cell number was 10 ⁶ / ml (beads: "Cytodex 1", ( Pharmacia), 37 ° C). Then, the medium was 1 L of serum-free Eagle MEM medium containing a small amount of carboxymethyl cellulose.
And 100,000 units / L of human natural interferon β was added as priming. The next day, poly I: poly C 50 mg / L and cycloheximide 10 mg / L were further added. 4 hours later, actinomycin D 4 mg / L
Then, after 1 hour, the medium was replaced with an Eagle MEM medium containing a small amount of methylcellulose as a production medium, and superinduction treatment was performed. Then 2
The culture was continued for a day (37 ° C.).

After stirring was stopped and the microcarriers were allowed to settle, the supernatant and the washing solution with the production medium were filtered, and 1 L was transferred to another container equipped with a stirring device. Sterilized "Blu-Sepharose CL-6BFF" (Pharmacia) was added to this production solution, and batch adsorption was performed while stirring at 15 ° C for 4 days. After the stirring was stopped, the bull-carrier was allowed to settle and the supernatant was transferred to another container. The silica carrier was sterilized in a sodium phosphate buffer solution under high pressure steam (121 ° C, 30 minutes) and then 4 m in length.
Each column was packed in two columns and connected in series. To this, the flow-through of the blue-carrier was passed at a flow rate of 20 ml / hr. After total flow, the two columns were purified separately.
After each flowing 25 ml of sodium phosphate buffer,
Fraction 1 containing interleukin-6 by flowing 20 mM hydrochloric acid
0 ml was collected. Ammonium sulfate was further added to the hydrochloric acid recovery solution so that the concentration was 1.33 M, and the mixture was gently stirred overnight at 4 ° C. The precipitate was removed by centrifugation at 3000 rpm for 30 minutes (4 ° C).

The separated supernatant is subjected to hydrophobic chromatography.
Butyl toyopar 650M "1 ml (Tosoh Corp.), which is a carrier, was passed through a column packed for adsorption. This column was adsorbed. 20mM hydrochloric acid containing 1.33M ammonium sulfate and 50mM phosphoric acid containing 1.33M ammonium sulfate After washing with a sodium buffer solution, it was recovered with a 50 mM sodium phosphate buffer solution, followed by reverse-phase chromatography ODS column (C ₁₈ ) (YMC-Pac).
k ODS A-312 S-5 120A, YMC
High Performance Liquid Chromatography (Shimadzu LC)
-4A), a human natural interleukin was gradient-eluted with water containing 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid.
Six peaks were collected. The human natural interleukin-6 thus obtained was subjected to gel filtration with "Sephadex G-25" (Pharmacia) using 5 mM formic acid as a solvent to obtain an interleukin-6 solution containing no acetonitrile.

The purity of the IL-6 prepared above was determined by the reversed phase HP.
It was 95% or more by LC method. It was confirmed that the above IL-6 was active IL-6 by the above-mentioned evaluation method.

Example 3 Measurement of blood cholesterol level by subcutaneous injection of rabbit: Using the IL-6 obtained in Example 2 as a test sample, rabbit NZ
W type, male, 17 weeks old, weight 2.8-3.3 Kg (Japan S
(Purchased from LC). The dosage form is purified IL-6
(The stock solution is neutral phosphate buffer) diluted with physiological saline is used. 10 μg / Kg is subcutaneously applied to the back by 1
It was administered once daily for 14 days.

The blood cholesterol level of the test rabbit was measured by the following protocol. Blood sampling is 2, 6 after the start of administration.
On the 10th and 15th days, the ear vein was wiped with alcohol cotton to inflate the blood vessel, and the blood vessel was incised with a lancet and then collected into a Benoject blood collection tube (Terumo). Leave whole blood at room temperature for 30 minutes,
Serum was obtained by centrifugation at 3000 rpm at 4 ° C for 15 minutes.

Using the above serum, Fuji Dry Chem 550
Serum biochemical marker 1 at 0V (Fujifilm)
The measurement was performed on 5 birds in each group. As a result, the total cholesterol amount decreased daily, and on day 15, it decreased to about half of the value before the start of administration.

The total cholesterol value for each measurement day was as follows (unit: mg / ml). The measurement on the 0th day is the first
Immediately before repeated administration. Day 0 64.3 ± 8.9 Day 2 58.8 ± 7.0 Day 6 44.8 ± 6.7 Day 10 28.8 ± 8.1 Day 15 24.0 ± 3.9

Example 4 Measurement of Blood Cholesterol Level by Intravenous Injection in Rabbit: The same experiment as in Example 3 was carried out using the test sample obtained in Example 1. The method of administration is intravenous injection, and the dose is 10 μg /
It was set to kg. As a result, a decrease in the total cholesterol level was observed daily.

The total cholesterol values for each measurement day were as follows. (Unit: mg / ml) Day 0 62.4 ± 7.8 Day 2 59.1 ± 8.4 Day 6 49.5 ± 6.0 Day 10 35.9 ± 7.3 Day 15 30 0.7 ± 6.1

Example 5 Measurement of blood cholesterol level by subcutaneous release of rabbits:
Using the test sample obtained in Example 1, an Alza osmotic pump (model: 2ML1, Muromachi Kikai) was implanted under the skin on the back subcutaneously, and continuous administration was performed at a low dose of 1 μg / kg / day for 7 days. The experiment was done. As a result, a decrease in total cholesterol was observed.

The total cholesterol values for each measurement day were as follows. (Unit: mg / ml) Day 0 39.0 ± 6.5 Day 4 45.3 ± 11.7 Day 8 30.0 ± 3.1 Day 10 31.0 ± 1.5

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, a therapeutic drug for hyperlipidemia having an activity of effectively reducing blood cholesterol in vivo can be obtained. Further, since IL-6 exists in the living body, an excellent therapeutic drug for hyperlipidemia that is more harmonized with the living body than synthetic drugs can be expected.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 25 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CCGATCGATG CCAGTACCCC CAGGA 25

SEQ ID NO: 2 Sequence Length: 25 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GCCACGGATC CTACATTTGC CGAAG 25

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Masanobu Naruto 1111 Tehiro, Kamakura City, Kanagawa Prefecture

Claims (2)

[Claims] 1. A therapeutic agent for hyperlipidemia containing interleukin 6 as an active ingredient. 2. The therapeutic drug for hyperlipidemia according to claim 1, wherein interleukin 6 is produced by cultured human cells.