JP2002179579A - Method for biologically utilizing apoptosis-inducing polysaccharides - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for utilizing polysaccharides which are useful for inducing cell apoptosis and formed by microorganisms. SOLUTION: This method comprises utilizing a compound expressed by formula (I) or its salt as an apoptosis-inducing agent, wherein the compound comprises a polysaccharide obtained by subjecting Pseudomonas culture to separation and refinement. The polysaccharide is expected to be useful as a medicament, for example, as a remedy for malignant tumors, autoimmune diseases, and inflammations.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention is useful in the field of medicine, and more specifically to a method of using a polysaccharide as an apoptosis inducer closely related to cell metabolism and the like.

[0002]

2. Description of the Related Art The death of cells constituting an organism is roughly classified into apoptosis and necrosis. Necrosis is cell death caused by aggravation of the environment or physical damage to cells, whereas apoptosis, by contrast, is actively regulated cell death [M. Sasada : Blood Frontier, 10, 1367-1372 (2000)].

In recent years, cell death due to this apoptosis has
It attracts attention in basic research fields such as biology and medicine, and in industrial fields such as pharmaceutical manufacturing. The reason is that apoptosis is a physiological phenomenon such as morphogenesis during embryonic development, normal cell rotation, immune system construction and maintenance, hormone-dependent cell death control, and pathological phenomena such as radiation and It is being clarified that it is extremely widespread and important, such as cell death due to drugs and cell death due to viral infection, as well as a deep relationship with various diseases. With the development of various anticancer drugs that cause cancer cell destruction by apoptosis, and the recent progress in genetic research, what kind of genes control apoptosis itself and how is the information leading to apoptosis? The accumulation of knowledge on whether or not it is transmitted has led to basic interests in cell biology, etc. [T. Murate: Blood Frontier, 1
0, 1373-1381 (2000) and Yuo: Blood Frontier,
10, 1383-1395 (2000)].

The above findings suggest that by inducing apoptosis, cells whose presence is undesirable in living organisms, such as autoreactive lymphocytes of patients with autoimmune diseases, lymphocytes sensitized to allergens of allergic patients, This indicates that cancer cells and the like can be eliminated, and for that purpose, a role of an apoptosis-inducing agent is expected. In addition, it is considered that by inducing apoptosis of bacteria and viruses from the outside, infection treatment from bacteria can be promoted, or metabolism of the skin can be promoted by inducing apoptosis of epidermal cells of the skin. Therefore, there has been a movement to develop factors and drugs that control apoptosis and explore the possibility of applying them to the treatment of these diseases. However, the number is still small and it is desired to provide new compounds.

Among them, one related to polysaccharides is that heparin strongly induces apoptosis of human lymphoblasts [E. Erduran et al .: American
Journal of Hematology, 61, 90-93 (1999)], and sulfated polysaccharides produced by marine microalgae
It is known that K562 has an apoptosis-inducing action [K. Sogawa et al .: Journal of Marine Bio
technology, 6, 241-243 (1998)].

According to morphological observation using an electron microscope, cell death due to apoptosis was observed by apoptosis, including chromosome aggregation, fragmentation of the cell nucleus, loss of microvilli on the cell surface, and cytoplasmic condensation. It has been shown that cells are rapidly phagocytosed and processed by macrophages and the like. It is also well known that apoptosis accompanied by biochemical characteristics such as fragmentation of chromosomal DNA is often observed.

On the other hand, the sulfated polysaccharide represented by the structural formula (I) is a compound separated and purified from a culture solution of a marine microorganism, and is known to exhibit weak antiviral activity against a certain major virus. Mazda (M.Matsuda et al .: Journal of the Fisheries Society of Japan, 59, 535-538 (1993) and Mazda (M.Matsuda)
Et al .: Marine Biotechnology, 1, 68-73 (1999)]. However, it is not known that it has a biological activity, particularly an apoptosis-inducing effect, and it is not described in the literature.

[0008]

An object of the present invention is to develop and provide a polysaccharide separated and purified from a culture of a marine microorganism as an apoptosis inducer.

[0009]

Means for Solving the Problems The present inventors have searched for various compounds, and as a result, have found that polysaccharides produced by marine microorganisms have an action of inducing apoptosis, and completed the present invention.

The polysaccharide used in the present invention is the same as the sulfated polysaccharide described in the literature [M. Matsuda et al .: 1993, supra, and optionally a pharmaceutically and pharmaceutically acceptable salt thereof. .

That is, the present invention relates to the use of a polysaccharide represented by the above structural formula (I) or a physiologically acceptable salt thereof as an apoptosis inducer.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION As a microorganism capable of producing a polysaccharide used in the present invention, marine Pseudomonas
(Pseudomonas) microorganisms, and more specifically, Pseudomonas sp. WAK-1. This strain is stored in the Matsuda Laboratory, Department of Bioresources and Food Chemistry, Faculty of Agriculture, Kagawa University [see M. Matsuda et al .: Journal of the Fisheries Society of Japan, 58, 1735-1741 (1992)].

The polysaccharide producing strain Pseudomonas
The polysaccharide represented by the above structural formula (I) based on Pseudomonas sp. WAK-1 is hereinafter referred to as WAK-APO.

A culture containing WAK-APO can be obtained by inoculating a marine Pseudomonas strain into a nutrient-containing medium and growing it. The medium is preferably a liquid medium containing nutrients such as a carbon source and a nitrogen source which can be assimilated by the microorganism and various inorganic salts necessary for growth. Specifically, for example, as the carbon source, glucose, fructose, sucrose and the like can be mentioned, and these are used alone or as a mixture. Nitrogen sources include meat extract, yeast extract,
Polypeptone, other organic or inorganic substances, and the like are used, and they are used alone or as a mixture. As the inorganic salt, calcium carbonate, sodium chloride, various phosphates and the like can be used. In addition, if necessary,
Trace amounts of heavy metal salts such as iron, manganese, zinc, and cobalt can also be added. Further, a medium prepared with seawater is preferable, but artificial seawater or a 2 to 3% by weight saline solution may be used.

The culturing method may be the same as the method for producing a metabolite of a general microorganism, and may be either solid culture or liquid culture, but liquid culture is more preferable. In the case of liquid culture, any of stirring culture, shaking culture, aeration culture, etc. may be performed, but if cultured under substantially shaking conditions, the sulfated polysaccharide used in the present invention is selected in the culture. (See Japanese Patent Application No. 2000-265079). If foaming is severe,
As an antifoaming agent, for example, vegetable oils such as soybean oil, higher alcohols such as octadecanol, various silicon compounds and the like may be appropriately added.

The cultivation usually has a pH of 6.0 to 8.0, preferably 6.5.
In the range of ~ 7.5, the temperature is 15 ~ 35 ° C, preferably 25 ~ 30 ° C
Is appropriate. The cultivation time is selected to be a period in which the production of the polysaccharide used in the present invention is maximized, but is usually 48 to 144 hours, preferably 72 to 96 hours.

The thus obtained culture contains the polysaccharide used in the present invention. In collecting the polysaccharide, WAK-APO is extracellular, so after removing cells and other solid components in the culture in advance, ordinary separation means, for example, solvent precipitation, ion exchange resin, etc. Separation and purification can be carried out by a method generally used for recovering polysaccharides from impurities, such as a method or adsorption or partition chromatography, gel filtration, dialysis, and freeze-drying, alone or in an appropriate combination.

As an example, a solvent such as ethanol is added to a solution obtained by removing the solid content to precipitate the polysaccharide. The obtained polysaccharide is dissolved in water, and a quaternary ammonium salt such as cetyltrimethylammonium bromide solution is added thereto to precipitate the polysaccharide as a complex with cetyltrimethylammonium bromide. The polysaccharide can be obtained by dissolving the precipitate in water containing sodium chloride, precipitating with ethanol, dissolving the precipitate in water, dialysis and freeze-drying.

The polysaccharide thus obtained is analyzed by DEAE-cellulose ion-exchange column chromatography, homogeneity analysis by electrophoresis, sugar composition analysis, sulfate group analysis, nuclear magnetic resonance analysis, and the like. [Mazda (Matsuda) et al.,
(1993)].

Using the obtained polysaccharide as a test sample, an apoptosis induction test is performed by using human myeloid leukemia cells U937 by a cell morphological change and DNA fragmentation analysis.

[0021]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Reference Examples and Test Examples, but the present invention is not limited to Examples. Reference example Reference [M.Matsuda et al .: 1
992], a medium prepared from seawater having a composition of peptone 0.5% and yeast extract 0.1% was sterilized in an autoclave at 121 ° C. for 20 minutes. Pseudomonas sp. WAK-1 (Pseudomonas sp. (10 mL), and cultured with shaking at 28 ° C. for 72 hours. Then, the preculture solution was inoculated into the above sterilized medium (200 mL) containing 3% sucrose in a 500 mL Erlenmeyer flask, and cultured at 28 ° C. for 72 hours with shaking. After the culture, the culture-finished solution was centrifuged to remove the cells, and twice the amount of ethanol was added to the supernatant to obtain a white precipitate. The precipitate was collected, dissolved in water (200 mL), and a 5% cetyltrimethylammonium bromide aqueous solution was gradually added until no new precipitate was formed, to precipitate a polysaccharide as a complex with cetyltrimethylammonium bromide. . After washing the complex with water to remove excess cetyltrimethylammonium bromide, the complex was dissolved in a 4M aqueous sodium chloride solution (200 ml). To this solution, twice the amount of ethanol was added to precipitate the polysaccharide. The obtained precipitate was dissolved in water, dialyzed against water, and lyophilized to obtain an acidic polysaccharide (about 0.1 g). This polysaccharide (102 mg) was then purified to further purify the polysaccharide.
A DEAE-cellulose column (2.3
× 22.5 cm). In 0.01 M phosphate buffer (pH 7.0)
After removing the fraction eluted with 0.6M sodium chloride, 0.8M
Fractions eluted with sodium chloride were collected, dialyzed, and lyophilized to give the polysaccharide (53 mg). The polysaccharide obtained in this manner was checked for homogeneity using cellulose acetate membrane electrophoresis, and analyzed for sugar composition, sulfate group content, and nuclear magnetic resonance.
(I) was confirmed to be a polysaccharide represented by
(M. Matsuda) et al. (1993)].

Next, the following test was conducted to confirm the effects of the present invention on the WAK-APO obtained above. When detecting the apoptosis-inducing action, it is necessary to clarify the difference from necrosis, which is cell necrosis. As described above, the cells in which apoptosis is induced first shrink, the chromatin is condensed, and the condensed nuclei are fragmented. In addition, the microvilli on the cell surface disappear and become smooth, and the cell surface molecular markers responsible for the self-recognition mechanism are gradually lost. In addition, large and small protrusions appear on the cell surface and eventually fragment into apoptotic bodies. These apoptotic bodies are eventually eliminated in vivo by phagocytic cells such as macrophages.

Therefore, paying attention to these phenomena, WAK-APO was added to human myeloid leukemia cells U937, and after the reaction was completed, a plurality of methods described below were used to detect whether apoptosis was induced. Experiments.

Test Example The apoptosis-inducing effect of WAK-APO obtained in the above Reference Example on human cultured cells was tested in vitro. Human monocyte leukemia cells U937 were used as cultured cells. The medium used was RPMI containing 10% FBS. Preculture the cells, adjust the cells in the log phase to 3 × 105 cells / mL with a medium, and dispense 1 mL each into a 24-well culture plate,
The final concentration of 10 μL of the sample solution was 10, 1, respectively.
0.1 and 0.01 μg / mL were added and mixed. The cells were cultured at 37 ° C. and 5% CO 2 concentration for 24 hours. After culturing for 24 hours, the morphological changes of the cells in each well were photographed and observed, and DNA fragmentation analysis was performed by agarose electrophoresis.

In the observation of the morphological change, those with no morphological change (-) and those with morphological change observed in many cells were identified.
(+), Cells that seemed dead were judged as (++), and DNA fragmentation analysis showed no DNA ladder (-), while DNA degradation was observed. (+) And DNA
The one with a clear ladder was judged as (++). Table 1 shows the test results.

[0026]

[Table 1]

As shown in Table 1, no morphological change or DNA fragmentation was observed in the control, but WAK-A
In U937 cells treated with PO, cell morphological changes characteristic of apoptosis were observed at concentrations of 0.1 and 1 μL / mL. 0.1
A ladder-like electrophoresis image composed of DNA fragmented at a concentration of 1 μL / mL was observed, and DNA fragmentation characteristic of apoptosis was observed.

As shown by the above test results, it was confirmed that WAK-APO produced in this Reference Example had an apoptosis-inducing effect.

[0029]

The polysaccharide WAK-APO described in the present invention comprises:
Since it exhibits an apoptosis-inducing effect on cultured human cells, it can control the cell cycle, and can be expected to be used as a drug, for example, a therapeutic drug for malignant tumors, autoimmune diseases, inflammation, and the like.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C08B 37/00 C08B 37/00 H

Claims (1)

[Claims] [1] A method of using a compound represented by the structural formula (I) or a salt thereof as an apoptosis-inducing agent as an active ingredient. Embedded image