JPS6011686B2 - Novel anti-tumor agent - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel antitumor agent and a method for producing the same.

In recent years, it has been reported that various bacterial cells are effective against malignant tumors, especially BCG, which is a viable form of Mycobacterium bovis, and killed bacteria of the Corynebacterium genus, such as Corynebacterium pervum. , as well as group A hemolytic streptococci treated with penicillin, are attracting attention.

All of these are based on injecting the bacterial cells themselves, so
Side effects such as fever, nausea, war pain, and induration and pain at the injection site have become problems after injection. In the case of live bacteria, a thick infestation often occurs at the injection site, and patients with malignant tumors generally have significantly reduced body defenses, so there is a risk of infection even when injected with low pathogenic bacteria. . The present inventors discovered that live bacteria of the genus Bifidobacterium, which is a non-pathogenic anaerobic bacterium resident in the armpits, have a remarkable anti-tumor effect, and as a result of further investigation, the dead bacteria It was confirmed that the bacteria also have strong anti-tumor effects similar to live bacteria.

The present invention is based on this knowledge, and is an antitumor agent containing live or killed bacteria of the genus Bifidobacterium as an active ingredient.

Furthermore, the present invention provides an antitumor drug containing live or dead bacteria of the genus Bifidobacterium as an active ingredient, which comprises culturing the bacteria of the genus Bifidobacterium and separating the bacteria from the culture solution. This is the manufacturing method of the drug.

Bacteria of the genus Bifidobacterium are known, and the microbiological properties of these strains are described in detail in "Purges of Determinative Bacteriology", 8th edition, pages 669-67 (1974). There is.

For example, the following bacterial strains can be used in the present invention. Bifidobacterium infantis SI strain 2 (National Institute of Technology Deposit Accession No. 3948), Pifidobacterium animalis BiMO-Bif strain 11 (Story No. 394), Bifidobacterium adretusen Theis aEI9 umbrella strain (
Pifidobacterium pifidum aE strain 319 (Public No. 3951).

The bacteria to be used are cultured under anaerobic conditions, for example, by replacing the air with carbon dioxide gas, nitrogen gas, or the like.

The medium must contain a large amount of reducing substances; for example, a medium containing liver extract, thioglycolic acid, L-cysteine, ascorbic acid, sodium sulfide, and other compounds is used. Bacteria of the genus Bifidobacterium require special nutritional factors for their growth, and these are called "bifidus factors".
It is called. Examples of the hyphidase factor include penzoyl-D-glucosamine and 4'-phosphopantetheine-S-sulfonic acid. The anti-tumor agent of the present invention can be produced by separating bacterial cells from a culture solution.

The bacterial cells are separated from the culture solution as it is or after killing the bacteria by a conventional method such as centrifugation. Killing of bacteria can be carried out by appropriate means, such as leaving the culture solution in the air, heating, formaldehyde treatment, phenol treatment, etc., before or after separating the bacterial cells. The live or dead cells thus obtained can be dried by a conventional method, such as by freeze-drying, and stored as a powder or as a suspension in physiological saline, for example. The antitumor topical agent of the present invention is generally used in the form of an injection. The dosage is generally 0.1 to 10 9/k per adult as the amount of dry bacterial cells.
It is 9.

The anti-tumor agents of the invention may contain customary auxiliaries or additives.

It was unexpected that Pifidobacterium bacteria had an antitumor effect.

As shown in the experimental example, Bifidobacterium bacteria are
The live bacteria and dead bacteria are Meth, which is a transplant system of methylcholanthrene-induced macropupil derived from inbred mouse BALB/c.
- Since it is effective against A, it is considered to be effective against spontaneous cancer as well. All types of Bifidobacterium bacteria used in the present invention are non-pathogenic, and killed bacteria can also be used, so there is no risk of causing infection and allergic diseases caused by pathogenic bacteria. It is excellent in that there is no need to take this into account. The results of an experiment regarding the anti-tumor effect of the anti-tumor agent of the present invention are shown below.

The experimental animals used were inbred BALB/c male mice (8
Each group consisted of 7 to 10 animals (~9 weeks old, body weight 20±2 days), and Hitomito transplanted Meth-A (methylcholanthrene-induced sarcoma) into the animals. The control substance was phosphate buffered saline (PH7.0) (hereinafter referred to as P plaque), and the comparison substance was OK-4, a known antitumor mimetic.
32 (group A hemolytic streptococcus preparation) was used. Experimental example Meth-A seed water was added subcutaneously to the abdomen of mouse A to a cell count of 5 x 1.
The cells were transplanted into mice.

In the first group, a suspension of live bacteria of Bifidobacterium infantis SI Genmori obtained in Example 1 (described later) (bacteria count: approximately 1 o/ground) was added to the tumor 2 days after transplantation, 4 Days later, 6 days later, and 8 days later, each tumor was injected once a day in an amount of 0.1 μm. The second group included P as a control substance.
The first group received plaque at an amount of 0.1'/mouse, and the third group received OK-432 as a comparison substance at an amount of Madarahata/mouse.
injections at the same intervals as the groups. The results are shown in Table 1. Table 1 As is clear from the results, all of the mice in the control and comparison groups died of the tumor within 50 days, while the group treated with live Bifidobacterium infantis SI2 beads died in 50% of the mice. of mice healed and survived within 50 days.

Furthermore, in the case of the present invention, the average survival period of mice that died at the tumor site was also significantly extended compared to the control group. Experimental Example B An experiment was conducted in the same manner as in Experimental Example A, and Meth-A was transplanted into the abdomen of a mouse at a cell count of 2.5×1 p/mouse.

Live bacteria of Bifidobacterium infantes SI Genju obtained in Example 1, killed bacteria of the same strain obtained in Example 2, and Bifidobacterium adolescentis obtained in Example 4. Each suspension of live bacteria of the aEI9 umbrella strain (bacterial count: about 1 0/' in each case) was injected into the tumor in the same machine as in Experimental Example A. Figure 1 shows the results of observing changes in the average diameter of the tumor moat by palpation from 10 to 27 days after transplantation.

In addition, in this experiment, the number of survivors after 6 weeks of pupil field transplantation was the second largest.
It was as shown in the table. Table 2 As is clear from the results shown in Figures 1 and 2, live and dead Bifidobacterium infantis SI2 bacteria have excellent anti-tumor effects, and are effective against mice with regression of the lumbar spine. There were many of them.

That is, Bifidobacterium infantis SI
The dead bacteria of Kusu showed the same effect as the live bacteria. The effect of Pifidobacterium adressenteis aEI9 umbrella strain is O
It was at the same level as K-432. Experimental Example C An experiment was conducted in the same manner as in Experimental Example A, and Meth-A was harvested from the mouse and transplanted into the mouse at a cell count of 2.5×1 p/mouse.

Bifidobacterium infantis SI 2 beads, Bifidobacterium animalis Bi and other ○-Bif-1 strains, Bifidobacterium addresscentis aEI9 umbrella strain and Each live bacterial suspension of Fidobacterium bifidum aE31 Kusu was injected intraperitoneally in the same manner as in Experimental Example A.
Changes in the diameter of the tumor were observed by palpation from 9 days to 30 days after seed wound transplantation. The results are shown in FIG. Sogeshi,
The anti-tumor effect in Bifidobacterium infantis SI Kusu and Bifidobacterium adrecentis aEI9 strain was destroyed, while Bifidobacterium animalis BifMO-Bif-1 strain and Bifidobacterium Um bifidoum aE319 *Ma, O
It showed almost the same effect as K-432. Experimental Example ○ A suspension of live and dead Bifidobacterium infantis SI Genju bacteria obtained in Examples 1 and 2 was applied to male and female BALB/c mice (10 each group) with an average body weight of 22 mm. Table 3 shows the results of measuring the acute toxicity value (LD50).

The LD50 value is expressed as the dry amount of bacterial cells, and 9 of 1 corresponds to approximately 1 and 0 of bacteria. Table 3 From the results, it is clear that the antitumor agent of the present invention has extremely low toxicity.

Example 1 One platinum loopful of overnight cultured inoculum of Pifidobacterium infantis strain SI2 was inoculated onto 50 sheets of a medium having the following composition, and the mixture was placed in an anaerobic jar replaced with carbon dioxide gas and nitrogen gas (volume ratio 1:9). Then, culture at 370° C. for 2 hours.

Medium composition: tomato juice infusion 4
00 Neobeptone (Difco) 15 Multi-liver extract 75 Secreted glucose 20 Soluble starch 0.5 Sodium chloride 5 L Cysteine/HC1/Day 20 0.2 Add distilled water to make a total volume of 525' PH = 6.8 to 7.0 The tomato juice infusion solution is a liquid obtained by adding 1 volume part of distilled water to 1 volume part of tomato juice liquid, flooding it at 100°C for 1 hour, adjusting the pH to 7.0, and filtering it. .

The liver extract is obtained by heating the liver end with 170 ml of distilled water for 1 hour at 50-60 pm, then heating it for several minutes at 100 pm, and then heating it for a few minutes. Immediately after culturing, cool on ice and centrifuge at 5°C for 18 minutes at 5000x.
The supernatant is removed, and the precipitated bacterial cells are washed twice by centrifugation using P plaques.

The obtained washed bacterial cells are suspended in 1/5 volume of PBS of the medium to obtain a viable bacterial suspension. Example 2 A culture solution of Bifidobacterium infantis strain SI2 cultured in the same manner as in Example 1 is left as is for 37q01 week while introducing sterile air.

A part of it was inoculated into a new medium and a culture test was conducted under anaerobic conditions. The result was negative and the bacteria were killed. This bacteria solution was collected from a distance in the same manner as in Example 1, washed with PBS, and then used as a suspension of killed bacteria in P mother.

Example 3 Bifidobacterium animalis strain BifMO-Bjf-1 is cultured in the same manner as in Example 1, and treated in the same manner as described above to obtain a viable bacterial suspension.

Example 4 When Bifidobacterium adrecentheis aEI9 umbrella strain was cultured in the same manner as in Example 1 and treated in the same manner,
A viable bacterial suspension is obtained.

Example 5 Bifidobacterium IJum aE31 kusu is cultured and treated in the same manner as in Example 1 to obtain a viable bacterial suspension.

When the culture solutions of Examples 3 to 5 are left in sterile air in the same manner as in Example 2, dead cells of each of them are obtained.

[Brief explanation of the drawing]

FIGS. 1 and 2 are graphs showing the effects of the anti-tumor agent of the present invention. Figure 1 Figure 2

Claims (1)

[Scope of Claims] 1. An antitumor agent containing live or dead bacteria of the genus Bifidobacterium as an active ingredient. 2. A method for producing an antitumor agent containing live or dead bacteria of the genus Bifidobacterium as an active ingredient, which comprises culturing the bacteria of the genus Bifidobacterium and separating the bacteria from the culture solution. 3. The method according to claim 2, characterized in that the bacterial cells are killed before or after isolation.