JPH09227392A - Antitumor agent and production thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce an antitumor agent, comprising a microbial cell of a microorganism belonging to the genus Enterococcus or a treated microbial cell prepared by carrying out the enzymic treatment of the microbial cell and heat treatment thereof as an active ingredient and having high immunopotentiating effects without any adverse effects. SOLUTION: This antitumor agent comprises a microbial cell of a microorganism belonging to the genus Enterococcus or a treated microbial cell prepared by carrying out the enzymic treatment of the microbial cell with a lytic enzyme, etc., and heat treatment thereof as an active ingredient. For example, Enterococcus faecalis NF-1011 (FERM P-12564) is cited as the microorganism belonging to the genus Enterococcus. A lysozyme is preferred as the enzyme used for the enzymic treatment. Treating conditions with the enzyme are preferably 10-3000μg/ml final concentration of the lysozyme, 37 deg.C temperature and 1-3hr treating time. For example, the heat treatment is conducted by treating the microbial cell treated with the enzyme at 110 deg.C for 10min in an autoclave. The objective antitumor agent can usually be administered in a daily dose of 0.002-0.1g/kg body weight active ingredient for an adult in one or several divided portions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent and a method for producing the same.

[0002]

2. Description of the Related Art Most of the antitumor agents currently in main use have a function of acting as a blocking agent during cell growth. An alkylating agent and a platinum agent having a DNA synthesis inhibitory action, an antimetabolite that is taken in during the metabolism of cells and inhibits the action of enzymes, and an antitumor action that acts on metabolic enzymes and nucleic acids derived from natural products Examples include cancer antibiotics and plant alkaloids.
Most of these drugs act specifically on cells that are actively undergoing cell division, so they can be applied to normal cells as well as bone marrow cells and intestinal epithelial cells, which have a relatively rapid cell division cycle, as well as cancer cells. The drug acts. Therefore, serious side effects such as a decrease in immunity due to a decrease in white blood cell function and gastrointestinal disorders are observed.

It is known that immune cells such as lymphocytes have an action of recognizing and injuring tumor cells. BRM (Biological Response Mod)
ifiers) therapy, which activates immune cells in the body, especially effector cells such as T cells, by administering a substance derived from bacteria, polysaccharides, etc., and increases the rate of damage to tumor cells. Until now, Lentinan, OK-432, P
Immunostimulants such as SK and cytokines such as interferon and interleukin are used. However, these drugs also have side effects such as rash, drug hypersensitivity, and fever, and in particular, injection preparations tend to have side effects.

[0004]

The chemotherapeutic agents currently used as antitumor agents always have side effects due to the mechanism of action. On the other hand, BRM preparations are used for the purpose of recovering the immunocompetence that is reduced by the onset of cancer and suppressing the growth rate of tumors. Therefore, there is a need for a drug that has no side effects, enhances immune system in the body and suppresses tumor growth.

[0005]

[Means for Solving the Problems] Enterococcus, which is a kind of lactic acid bacterium, is known to have a function of a BRM preparation, that is, an antitumor effect by enhancing immunity (Ohashi et al., Pharmaceutical Journal, 113. 396-399 (1
993)). As a result of various investigations by the present inventors in order to obtain cells having a stronger antitumor effect or a treated product thereof, the treated cells obtained by treating lactic acid bacteria cells with an enzyme are It was found that the antitumor effect was much stronger than that of the treated cells, and the present invention was completed.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is characterized in that a lactic acid bacterium belonging to the genus Enterococcus is treated with an enzyme and further heat-treated to obtain a treated product of the bacterium. The enzyme used in the present invention includes lysozyme, chitinase, N-
It is possible to use any enzyme as long as it is a lytic enzyme such as acetylglucosaminidase or a converting or degrading enzyme for the cell surface structure or antibiotics, etc., which enhances the antitumor effect of the cell by enzymatic treatment. However, it is lysozyme that can get particularly effective results. Further exemplifying the conditions for cell treatment, treatment with a final lysozyme concentration of 10 to 3000 μg / ml at 37 ° C. for 1 to 3 hours is preferable.

To prepare the bacterial cells or processed products thereof, additives such as carriers such as starch, lactose, soybean protein, excipients, binders, disintegrating agents, lubricants, stabilizers, and flavoring agents are added. It is formulated into tablets and granules by a well-known method.

The amount used varies depending on symptoms, age, etc.
As an active ingredient, 0.002 to 0.1 g / kg body weight per day can be usually administered to an adult once or several times a day.

[0009]

EXAMPLES Examples will be shown below, but the present invention is not limited to the description of these Examples.

Embodiment 1 FIG. (Cultivation of lactic acid bacteria) Enterococcus faecali
s) NF-1011 (Microtechnology Research Institute, No. 12564) was inoculated into a Rogosa liquid medium having the composition shown below (the number of bacteria: 10
⁶ cells / ml), cultivated at 37 ℃ for 10 to 16 hours, and the viable cell count is about 1
A culture solution of ⁰⁹ cells / ml was obtained. The obtained culture solution is 12,000 rpm
The cells were collected by centrifuging at 20 ° C. for 20 minutes and washed twice with distilled water to obtain cells.

The composition of Rogosa liquid medium is shown below. Trypticase 10 g Yeast extract 5 g Tryptose 3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml Distilled water 1000 ml (adjusted to pH 7.0, 121 ° C. 15 minutes heat-sterilized in) (1) _{saline: MgSO 4 · 7H 2 O 11.5g} FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4g distilled 100 ml of water

Embodiment 2 FIG. (Enzyme treatment and heat treatment) Bacteria obtained in Example 1: Sterile distilled water = 1:10 (v /
It was resuspended so that the ratio of v) was obtained. To this, filter-sterilized egg white lysozyme for food addition (manufactured by Taiyo Kagaku Co., Ltd.) was added to a final concentration of 100 μg / ml, and 37
Incubated at 2 ° C. for 2 hours. Next, 1
Heat treatment by autoclave at 10 ℃ for 10 minutes,
After the bacterial solution was cooled, it was dried with a freeze dryer to obtain a powder sample.

Embodiment 3 FIG. (Heat Treatment) Bacteria obtained in Example 1: Sterile distilled water = 1: 10 (v /
Suspended at the ratio of v). This at 110 ℃ 10
After heat treatment by autoclave for 1 minute, 110 ℃
The egg white lysozyme used in Example 2, which had been inactivated by heating for 10 minutes, was added in an amount corresponding to a final concentration of 100 μg / ml. Next, freeze-drying was performed to obtain a powder sample.

Embodiment 4 FIG. (Anti-tumor effect) C3H / He N strain mice (male, 7 weeks old) purchased from Japan SLC Co., Ltd. were measured according to body weight according to the preparation administration group (Group A) obtained in Example 2 and the standard obtained in Example 3. The product was divided into 3 groups (10 animals each), a product administration group (B group) and a control group (C group).

MM4 intraperitoneally in C3H / He N mouse
6 breast cancers were transplanted, and one week later, cancer cells were collected and CaCl
₂ and suspended in phosphate buffered physiological saline (PBS (-)) that does not contain MgCl ₂ .6H ₂ O, and the cells were washed by repeating centrifugation at 700 rpm for 5 minutes 3 times, and then PBS.
The cell concentration was adjusted with (-). This cell suspension was transplanted into the abdominal skin of the mice of all the above groups so that the number of viable cells was 1 × 10 ⁶ cells / mouse.

Each of the preparations obtained in Examples 2 and 3 was mixed with powdered feed (CE-2: CLEA Japan, Inc.) in an amount of 5% to prepare a mixed feed. From the day after the transplantation of MM46 breast cancer cells, each mouse group (group A and group B)
I was allowed free intake every day. Group C received a powdered feed containing no preparation.

After the cancer transplantation, the size of the formed tumor was measured. From the 4th day after cancer transplantation, the major axis (a) and the minor axis (b) were measured using calipers every 3 days, and the size (diameter) of the tumor was determined by the following formula. The results are shown in FIG. Tumor diameter (mm) = (axb) ^1/2

Compared with the C group, the A group and the B group had a significantly smaller tumor diameter 18 days after the cancer transplantation (risk rate 1% to 5%).
This effect persisted until 38 days after cancer transplantation. In addition, when group A and group B were compared, the tumor diameter of group A tended to become smaller than 25 days after cancer transplantation.

41 days after the cancer transplantation, the tumor parts of all the mice were excised and their weights were measured. The results are shown in FIG. In contrast to the C group, the B group showed a significant weight suppression with a risk rate of 5%. On the other hand, group A also showed a significant reduction in tumor weight at a risk rate of 1%,
The inhibitory effect was higher than that of group B.

[0020]

EFFECTS OF THE INVENTION The enzyme-treated lactic acid bacterium obtained in the present invention has no side effects and has a high immunostimulatory effect, so that it suppresses tumor growth more efficiently. It is also possible to use this treated product of bacterial cells in combination with an existing anticancer agent.

[Brief description of drawings]

FIG. 1 is a diagram showing changes in tumor diameter of MM46 breast cancer.

FIG. 2 is a diagram showing the average tumor weight per mouse.

Claims (4)

[Claims] 1. An antitumor agent which is a microbial cell of a microorganism belonging to the genus Enterococcus as an active ingredient, or a microbial cell that has been treated with an enzyme that acts on the microbial cell such as a lytic enzyme and by heat treatment. 2. The antitumor agent according to claim 1, wherein the microorganism belonging to the genus Enterococcus is Enterococcus faecalis. 3. A method for producing an antitumor agent, which comprises subjecting a bacterial cell of a microorganism belonging to the genus Enterococcus to an enzymatic treatment such as a lytic enzyme which acts on the bacterial cell and a heat treatment. 4. The method for producing an antitumor agent according to claim 3, wherein the microorganism belonging to the genus Enterococcus is Enterococcus faecalis.