JP2001233784A - Interleukin-18 production promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine that can efficiently promote the production of interleukin-18 that is clinically useful with high safety. SOLUTION: This is a medicine that promotes the production of interleukin-18 and comprises a mixture of one kind or two or more kinds selected from the group consisting of macrophage colony stimulating factors and their pharmaceutically acceptable salts as active ingredients.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an interleukin-1.
8 It relates to a production promoter. More specifically, the present invention relates to a drug that promotes the production of factors that suppress the onset and progression of diabetes in humans and mammals, and the production of factors that suppress bone metastasis of cancer cells. In the present specification, percentages are by weight unless otherwise specified.

[0002]

2. Description of the Related Art In general, proliferation and differentiation of hematopoietic cells require specific growth and differentiation factors, and various mature blood cells such as erythrocytes, granulocytes, macrophages, eosinophils, platelets, A number of differentiation and growth factors are involved before becoming lymphocytes. On the other hand, living organisms respond to infections such as bacteria and viruses, tumors, cell damage, and the like by immune responses. The immune responses are regulated by direct or indirect interactions between immunocompetent cells. I have. 2. Description of the Related Art In recent years, biological response modifiers that have an effect of improving the defense against foreign substances by acting on immune functions have attracted attention from the perspective of cancer treatment. Substances collectively referred to as cytokines such as interleukins, colony stimulating factors, TNF (tumor necrosis factor), and interferons have been successively shown to be involved in these differentiation, proliferation and immune functions. Also, a method of producing a cytokine by a genetic engineering technique and using it as a drug has been studied [Science, Vol. 235, No. 1504-1].
508, 1987, and the same magazine, vol. 378, No. 6552, pp. 88-91, 1994].

[0003] At present, 18 types of cytokines belonging to interleukin have been identified, and among them, interleukin-18 (hereinafter sometimes abbreviated as "IL-18") is a signal transmitter in the immune system. It is a type of cytokine that is a substance. At the beginning of the discovery, the present cytokine was disclosed in Japanese Patent Application Laid-Open Nos.
191098, JP-A-9-289896,
And [Nature, Vol. 378, No. 6552
No., pp. 88-91, 1994], was expressed as an interferon-γ inducer,
Later it was called IL-18 [Journal of Immunology,
Vol. 156, pp. 4274-4279, 1996
Year].

[0004] IL-18 is structurally similar in function to interleukin-12, a protein of the interleukin-1 family. IL-18 is an interferon from T cells and natural killer cells.
Strongly induces γ production, but also induces granulocyte / macrophage colony stimulating factor, tumor necrosis factor, interleukin-4, interleukin-5, interleukin-8, interleukin-10, and interleukin-13 And also has the property of enhancing the cytotoxicity of natural killer cells and the property of inducing the production of natural killer cells [Macrophage Journal, Vol. 6, No. No. 3, pages 5-6, 1999, and Kohei Miyazono and Kazuo Sumura, "Revised Versions of Cytokines and Growth Factors", No. 46
47 pages, Yodosha, 1998].

[0005] Experiments using animal models have strongly suggested that IL-18 is involved in the pathogenesis of diseases. That is, in NOD (nonobese diabetic) mice, which are models of insulin-dependent diabetes, expression of IL-18 is observed in parallel with the activity of the disease state, but the administration of IL-18 suppresses the development of the disease. [Journal of Immunology, vol. 163, no.
1236, 1999].

Further, it has been reported that IL-18 has an inhibitory effect on bone metastasis of human lung cancer cells (MDA-231) [Anticancer Research (Antica)
ncerResearch), Vol. 19, pp. 4131-4138, 1999]. It is also disclosed that IL-18 has an osteoclast formation inhibitory effect (JP-A-10-2).
36974). On the other hand, macrophage colony stimulating factor is a factor that acts on bone marrow cells to promote differentiation and proliferation into granulocytes and macrophages. Macrophage colony stimulating factor that promotes differentiation and proliferation of monocytes and macrophages.・ Granulocyte colony stimulating factor that promotes proliferation, and is classified as a granulocyte macrophage colony stimulating factor that promotes the differentiation and proliferation of granulocytes and macrophages. Pages 46-47, Yodosha, 1
998).

[0007] Macrophage colony stimulating factor (Macrop
hage Colony Stimulating Factor. Hereinafter, it may be abbreviated as M-CSF. ) Is a homodimeric glycoprotein with a molecular weight of 84,000 daltons discovered in human urine by Takaku et al. In 1978, and acts on monocyte precursor cells in the bone marrow of mammals to produce monocytes and macrophages. It promotes the differentiation and proliferation of cells and acts on mature macrophages to promote granulocyte colony stimulating factor (Granulocyte Colony Stimulati).
ng Factor), and is known to promote an increase in the number of granulocytes in peripheral blood, which has been reduced by cancer chemotherapy and the like [International Conference on Mucosal Immunology, page 109, 1990]
Year]. In vivo, macrophage colony-stimulating factor is involved in the formation of osteoclasts and the expression of their functions, and has extremely important functions such as bone formation and provision of a bone marrow cavity, which is a site of hematopoiesis. Furthermore, since it also has an activity of enhancing functions such as lipid metabolism, its use as a blood cholesterol lowering agent is also being studied (Toshio Suda, “Fate of blood stem cells”, page 64, Yodosha, 1992).

The use of macrophage colony stimulating factor is effective for bone marrow transplantation in hematopoietic diseases (Japanese Patent Application Laid-Open No. 62-27010) and to increase the antitumor effect of a cancer immunotherapy agent. Kaihei 1-19322
No. 7), effective in preventing thrombocytopenia (JP-A-1-207244), effective in enhancing the function of transplanted bone marrow (Japanese Patent Publication No. 6-10137), effective in treating malignant tumors (JP-A-2-225418), it is effective as an adjuvant for platinum complex antineoplastic agents (JP-A-2-264729), and it is effective for the treatment of hyperlipidemia. JP-A-2-258728, JP-A-2-264728 and JP-A-3-21
No. 25), being effective for the treatment of myelodysplastic syndrome (JP-A-3-17021), being effective for the prevention and treatment of nephritis (JP-A-8-104648), Effective for treatment (Japanese Unexamined Patent Publication No.
178334) and the like. However, these macrophage colony stimulating factors are
It is not known to have a -18 production promoting effect and is not described in the literature.

[0009]

DISCLOSURE OF THE INVENTION In view of the above-mentioned prior art, the present inventors have intensively studied a substance that promotes the production of a factor that suppresses the onset and progression of diabetes and a factor that suppresses bone metastasis of cancer cells. As a result, the present inventors have found that macrophage colony stimulating factors have the effect, and completed the present invention. An object of the present invention is to provide a factor that suppresses the onset and progression of diabetes and a production promoter of a factor that suppresses bone metastasis of cancer cells.

[0010]

DISCLOSURE OF THE INVENTION The present invention for solving the above-mentioned problems is an agent for promoting the production of interleukin-18, comprising macrophage colony-stimulating factors and pharmaceutically acceptable salts thereof. An interleukin-18 production promoter containing, as an active ingredient, one or more mixtures selected from the group consisting of: pharmaceutically acceptable salts of macrophage colony stimulating factors,
Interleukin-18, a macrophage colony stimulating factor that is an acid addition salt, metal complex, or carboxylate
In some embodiments, it is a production promoter.

[0011]

Next, the present invention will be described in detail. The active ingredient for promoting the production of a factor having an action of suppressing the onset and progression of diabetes and an action of suppressing bone metastasis of cancer cells in the present invention is a macrophage colony stimulating factor, and a pharmaceutically acceptable factor of the factor. One or a mixture of two or more selected from the group consisting of the following salts: The macrophage colony stimulating factor used in the present invention is a macrophage colony stimulating factor or a pharmacologically acceptable derivative of the macrophage colony stimulating factor, and particularly preferably the macrophage colony stimulating factor itself. The method for producing the macrophage colony stimulating factor is not particularly limited. For example, it can be isolated from human urine by a physicochemical method, or can be produced by genetic engineering. .

An example of a specific manufacturing method is as follows. JP-A-63-198700 and JP-A-63-250400 describe methods for chemically isolating human urine.
Gazette, Japanese Unexamined Patent Publication No. Sho 64-22899,
Japanese Patent Application Laid-Open No. 501607/501 and a method disclosed in Japanese Patent Application Laid-Open No. 1-502397 are known as methods for producing by genetic engineering. The macrophage colony stimulating factor produced by the above method acts on mammalian bone marrow cells in soft agar to promote the formation of colonies consisting of monocytes and macrophage cells (hereinafter referred to as colony stimulating activity). There is).

As the macrophage colony stimulating factor used in the present invention, any factor having the same colony stimulating activity can be used, and the type thereof is not particularly limited. More specifically, Table 1-5
Macrophage colony stimulating factor produced by the production method of JP-A-02397 is particularly desirable, a peptide having the amino acid sequence of the macrophage colony stimulating factor, a peptide in which a part of the amino acid sequence of the macrophage colony stimulating factor has been omitted or substituted, the macrophage Peptides having other amino acids inserted or added to a part of the amino acid sequence of the colony stimulating factor can be exemplified, but are not limited thereto, and all those having the aforementioned colony stimulating activity are used in the present invention. be able to.

If necessary, a buffer containing human serum albumin, saccharides, etc. as a stabilizer is added to the macrophage colony stimulating factor obtained by the chemical method or the genetic engineering method as described above to dissolve the macrophage colony stimulating factor. Hereinafter, it is possible to produce an IL-18 production promoter by sterile filtration and freeze-drying by a conventional method.
In addition, pharmacologically acceptable components can be added in addition to human serum albumin and saccharides.

In the present invention, the active ingredient for promoting the production of IL-18 is one or more selected from the group consisting of the macrophage colony stimulating factors and pharmaceutically acceptable salts of the factors. And pharmaceutically acceptable salts of the factors include non-toxic salts such as acid addition salts and metal complexes, and carboxylate salts. More specifically, the acid addition salt is a hydrochloride,
Sulfates, phosphates, oxalates, acetates, citrates,
Ascorbate, tartrate, and the like; metal complexes such as zinc, iron, calcium, magnesium, and aluminum complexes; and carboxylate salts such as sodium and potassium alkali metal salts, calcium salts, and magnesium salts. Examples thereof include alkaline earth metal salts and ammonium salts. These salts can be produced by a conventional method.

The IL-18 production promoter of the present invention is prepared by sterilizing the macrophage colony stimulating factor and / or a pharmaceutically acceptable salt of the macrophage colony stimulating factor produced as described above, for example, by a conventional method. It can be dissolved in physiological saline for injection, distilled water for injection, etc. to produce a preparation. The IL-18 production promoter of the present invention is, for example, dissolved in physiological saline for injection, distilled water for injection, or the like, and in an appropriate form such as infusion, intravenous injection, subcutaneous injection, intraperitoneal administration, age, symptoms, etc. The effective amount can be appropriately administered according to the above.

The IL-18 production promoter of the present invention contains macrophage colony stimulating factor, which is a natural product naturally contained in human body fluids, as an active ingredient, and therefore has the advantage that side effects are extremely slight. There is. From the results of animal studies, the LD ₅₀ for these the macrophage colony stimulating factor, in the intravenous administration 100 mg / kg body weight or more, the oral administration 300 mg / kg body weight or more, subcutaneously at 200 mg / kg body weight or higher. IL- of the present invention
The effective dose of the 18 production enhancer is about 500 μg / day / kg body weight, calculated from the animal test results described below.

Next, the operation and effect of the present invention will be specifically described with reference to test examples. Test Example 1 This test was performed to quantify the amount of IL-18 induced in the spleen of mice to which macrophage colony stimulating factor was administered.

(1) Preparation of samples 1) Treatment of test animals Sixty-five week-old male C57BL / 6 mice (purchased from Charles River Japan) were acclimated for 1 week by a conventional method, and then randomly 10 mice per group , And each group was treated as follows. Placebo group 200 μl / mouse of physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) was administered from the tail vein. Drug administration group Human M-CSF (manufactured by Morinaga Milk Products) 500 μg / kg of body weight
Once (1 day; 1 dose group), 3 times (3 days; 3 dose groups), 5 times (5 days; 5 dose groups), 7 times (7 days; 7 dose groups), It was administered 9 times (9 days, 9 doses) via the tail vein, and 3 hours after the final administration, the spleen was removed.

2) Treatment of spleen The removed spleen was put into a hand homogenizer, and an organ 10
1 ml of a homogenizing solution per 0 mg [9.1 mM disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.7
Phosphate buffer pH 7.4 containing mM mM sodium dihydrogen phosphate (Wako Pure Chemical Industries, Ltd.) and 150 mM sodium chloride (Wako Pure Chemical Industries, Ltd.), and a final concentration of 1% NP40 (Sigma), and Final concentration 0.5% sodium deoxycholate (manufactured by Wako Pure Chemical Industries), final concentration 0.1% sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries), and final concentration 100 μg / ml (p-amidinophenyl) A solution containing methanesulfonyl fluoride hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.)]. Thereafter, the entire amount was transferred to a microtube and placed at 4 ° C., 15,000.
After centrifugation at 0 rotation for 10 minutes, the supernatant was recovered, and further centrifuged under the same conditions to recover the supernatant, thereby preparing a sample.

(2) Test Method 100 μg of each obtained sample was subjected to SDS electrophoresis using sodium dodecyl sulfate (hereinafter abbreviated as SDS) polyacrylamide electrophoresis gel (manufactured by Tefco, concentration: 16%). . After completion of the electrophoresis, the protein separated in the gel is subjected to a polyvinylidene difluoride membrane filter (manufactured by Amersham Pharmacia Biotech. Hy).
bond-P. Catalog number, RPN2020F. ), 5% skim milk (Diff Laboratories)
At room temperature using a solution containing 10 mM Tris-HCl buffer pH 7.4 (manufactured by Wako Pure Chemical Industries), 150 mM sodium chloride (manufactured by Wako Pure Chemical Industries), and 0.1% Tween 20 (manufactured by Sigma). For 2 hours. Subsequently, a solution containing 10 mM Tris-HCl buffer (pH 7.4) containing 0.5% skim milk, 150 mM sodium chloride, 0.1% Tween 20, and 0.2% sodium azide (manufactured by Nacalai Tesque, Inc.) (hereinafter referred to as antibody (Abbreviated as diluent solution)), and reacted at 4 ° C. overnight as a primary antibody with a solution of an anti-mouse IL-18 goat antibody (manufactured by R & D Systems) diluted to a final concentration of 0.1 μg / ml.

After completion of the reaction, 10 mM Tris-HCl buffer pH 7.4, 150 mM sodium chloride, and 0.1%
After washing with Tween 20 (hereinafter abbreviated as washing solution),
Further, a horseradish peroxidase-labeled anti-goat IgG-rabbit antibody (manufactured by ICN Corporation) diluted 10,000 times with an antibody diluting solution was reacted as a secondary antibody at room temperature for 1 hour. After the reaction, the membrane filter was washed with a washing solution, immersed in a reaction solution for detection of ECL-Plus western blotting (manufactured by Amersham Pharmacia Biotech) for 5 minutes, exposed to Hyperfilm ECL (manufactured by Amersham Pharmacia Biotech), and exposed to a band. Was quantified with a densitometer, and the average value of each group was calculated and tested.

(3) Test Results The results of this test are as shown in Table 1. Table 1 shows IL-18 detected by Western blotting.
The result of quantifying the band of No. with a dentocytometer is shown. As is clear from Table 1, the placebo group, human MC
SF single dose group, human M-CSF three dose group, human M-
IL-in the spleen extracted from the CSF 5 times administration group, human M-CSF 7 times administration group, and human M-CSF 9 times administration group
18 were expressed at 64 ng, 85 ng, and 302, respectively.
ng, 753 ng, 1291 ng, and 1075 ng, and the production amount of IL-18 in the group to which human M-CSF was administered 7 times showed the maximum value. Therefore, it was found that it was necessary to administer human M-CSF at least seven times. In addition, MC
Tests were performed with different types of SFs, and almost the same results were obtained.

[0024]

[Table 1]

Test Example 2 This test was performed to test the acute toxicity of M-CSF, which is the main component of the drug of the present invention. (1) Preparation of sample Human M-CSF (manufactured by Morinaga Milk Industry Co., Ltd.) was dissolved in physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and aseptically filtered to prepare a sample solution.

(2) Test method The acute toxicity test method is described in [Basic and Clinical, Vol. 22, No. 7,
1649-1659, 1988] using 8-week-old mice (intravenous, subcutaneous, oral).

(3) Test Results No death occurred in any of the administration groups, and no change was found in histopathology. L of the human M-CSF
D ₅₀ is greater than 100 mg / kg body weight intravenously, greater than 300 mg / kg body weight for oral administration and less than 20 mg for subcutaneous administration.
It was 0 mg / kg body weight or more. Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0028]

Example 1 An injection having the following composition was produced by a conventional method. Human M-CSF (manufactured by Morinaga Milk Industry Co., Ltd.) 0.5 (%) Sodium chloride (manufactured by Wako Pure Chemical Industries) 0.9 Distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) 98.6

Example 2 An injection having the following composition was produced by a conventional method. Human M-CSF (Morinaga Dairy) 0.5 (%) Actinomycin D (Sigma) 0.005 Sodium chloride (Wako Pure Chemical Industries) 0.9 Distilled water for injection (Otsuka Pharmaceutical) 98.595

Example 3 Tablets having the following composition per tablet were produced by a conventional method. Human M-CSF (Morinaga Dairy) 1.0 (mg) Actinomycin D (Sigma) 0.02 Lactose (Wako Pure Chemical Industries) 162.98 Crystalline cellulose (Wako Pure Chemical Industries) 30 5.0 Polyvinylpyrrolidone (manufactured by Sigma) 5.0

[0031]

As described in detail above, the present invention relates to a novel IL-18 production promoter, and the effects of the present invention are as follows. 1) It is possible to surely promote the production of IL-18 in humans and mammals. 2) M-CSFs have already been approved as pharmaceuticals, their safety has been confirmed, and no side effects due to administration of the preparation of the present invention are observed. 3) The IL-18 production promoter of the present invention has an effect of suppressing the onset and progress of diabetes and an effect of suppressing bone metastasis of cancer cells.

Continued on the front page (72) Inventor Tetsuya Kuhara 5-1-1, Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Co., Ltd.Biological Science Research Institute (72) Inventor Taketo Ito 5-83, Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry F-term (reference) in Biological Science Research Institute Inc. 4C084 AA02 BA44 CA39 DA19 MA01 MA17 MA35 MA66 NA07 NA14 ZB262 ZC352 ZC412

Claims (2)

[Claims] 1. An agent for promoting the production of interleukin-18, which comprises macrophage colony-stimulating factors,
And an interleukin-18 production promoter comprising as an active ingredient one or a mixture of two or more selected from the group consisting of pharmaceutically acceptable salts of the factors. 2. The interleukin- according to claim 1, wherein the pharmaceutically acceptable salt of the macrophage colony stimulating factor is an acid addition salt, a metal complex, or a carboxylate.
18 Production promoter.