JPS6153219A - Antitumor agent - Google Patents

Abstract

PURPOSE:An antitumor agent containing the bacterial cell or endospore of butyric bacteria MII 588 strain as an active component. CONSTITUTION:An antitumor agent effective not only to nodular tumor of sarcoma 180 but also to streptolytic preparation and other same kind of tumor such as mouse leukemia L/210, etc., by using a butyric bacteria [Clostridium butyricum] MII 588 strain (FERM-P No.7765), as an active component. The agent exhibits especially remarkable effect by the combined use with a synthetic carcinostatic agent such as cyclophosphamide, etc. The agent can be prepared by culturing the strain in a proper medium (e.g. shown in the table I and table II) at a specific temperature for a specific time, collecting the bacterial cells from the culture liquid by centrifugal separation, dispersing the cells in distilled water, stirring, washing by using a centrifugal separator, freeze-drying, and finally pulverizing the product. It is administered by intravenous injection or peritoneal infusion at a dose of 0.5-2.5g/day in terms of dry bacterial cell.

Description

[Detailed description of the invention] Clostridium butyrjcum
) is a known anti-tumor agent containing bacterial cells or endospores as an active ingredient.

In recent years, drugs used to treat malignant tumors, such as immunostimulants and interferons, have been linked to cancer treatment by modifying the host's biological response to cancer.

So-called biological response modifiers (Biological
Resp-onse Modifier,, BRM
) is being actively researched.

These are generally different from traditional cytotoxic substances. It is attractive as a cancer therapeutic because it has few side effects on the host.

Among these, those that use bacterial cells or bacterial cell components as immunostimulants include hemolytic streptococcal preparations (Oka
moto. H. et al. :Studie
s on the anti-csncer a
nd 5treptolysin S-forming
abilities of hemolytle 8t
reptocococci, Jpn. J. Mic
robioL.

△ヱ． 3ko3-33bu. /tat7), Mycobacterium tuberculosis.

R. O. G. (Mathe, Go et a
l. : Active immunothera-p
y for acute i:ymphoblasti
c leukemia, Lancetsl, Ata7
-A Tata. 19Ata: and &zurua, 1. e
tal. : Biologically actlv
e components frommycabact
erial call walls. Mu Isolati
on and composition of cell
wall skeleton and comp-o
nent PJ, J. Natl. Cancer
Inst. , ! 2. Ta! 110/, /ta74A
reference) and Nocardia cells and cell components (in zurr)
, a, 1. et al. : Antitumor
ofcell wall skeletons
d peptide glycol pids of my
cobacterium and related m
icroorganisms.

Gann, l! , 41 ta J-! Oj, / 974A).

Oorynebacterium pary+um (
Israel + L. : Oo'ryn-ebac
terium paryu+n * Plenum P
res: New 'York.

/T7tA), Llsterla monoc
ytogenesis(Kamisango*K,
et al.: Immunomodulatlon
bymicrobial products
and related synthetic
mpounds * ExcerptaΔ(edica
, JRI/, 1PI/), and tumor necrosis factor (Oarswell, E, Pt, et al.
Al: ? hn endotoxin-indu
c-ed serum factor that
causes necrosis oftum
ors, Proc, Na11. Acad, 3
ci, , U, LA, , , 72 mJttt
-3470, /97) have been in the spotlight, and more recently, Lactopacillas (L.
There have been reports of anti-tumor effects on a number of bacterial strains, including S. actobacillus. However, so far no obligate anaerobes, especially Clostridium, have been observed. On the other hand, butyric acid bacteria that live in the intestine are known to produce an effective factor for anti-septic bacteria in their culture solution, and have long been used as a measuring preparation with intestinal regulating effects.

The present inventors found that butyrate vM (Clostridium butericum) MIIzrr strain derived from the human intestinal tract was a trophozoite.

Regardless of endospores, whether in live or dead form, Sarco7
In addition to 110 nodular tumors, streptococcal agents and other conventional immunostimulants are ineffective in the development of mouse leukemia L/L.
It was found that this drug is effective against syngeneic tumors such as 210, and is especially effective when used in combination with synthetic anticancer agents such as cyclophospha2.

Butyric acid bacteria [Clostridium butyricum (Clos-t)]
ridium butyrlcum)) M II!
r 1 share is Showa! On March 3, 2015, the Agency of Industrial Science and Technology entrusted the micro-machine to the Institute of Materials Science and Technology, and its accession number was No. 7.
No. 76J- (FEBMP-77at).

The main morphological and biochemical properties of Butyric acid bacterium MIIzrr were stored as a standard strain at the Institute of Applied Microbiology, the University of Tokyo.
A comparison with IM MI900/ is as shown in Table 1 and Table 1.

Table 1 Dairy RGMrIjrt and C1ostrid sum
Ratio with butyricamIAM /ri00/
! morphological properties) An λ butyric acid-MBrrr and (31o
stridium butyrlcumI人M/ri00
Comparison with (biochemical properties) MIIrrr bacterium is a Durham-positive bacillus that forms endophytic spores and is an obligate anaerobe, so it is based on Bergey's classification book (Bergey's Man
ual of DeterminativeBacte
riology, 1st edition, Williams & W.
According to Ilkins 0Oe11ri7hiro), it is included in either the genus Clostridium or the genus Desulfotomaculum. However, since this bacterium does not reduce sulfate in a lactate-sulfate medium, there is no question that it is included in the genus Clostridium. Furthermore, as shown in the table, the morphological and biochemical properties are identical to the standard strain IAM/ri00/ of the butyric acid bacterium Clostridium butyricum. However, the IAM/ri00/ strain differed from the description in Purgy's taxonomy in that it lacked the ability to ferment xylose and lysase, and also differed from the MIIrrr strain. The glycerol fermentation ability was also different, but this is not a problem since it is originally stated that it varies depending on the strain.

When producing the antitumor agent f:1B of the present invention, butyric acid bacteria Ml[l
RR can be cultured using methods commonly used to culture obligate anaerobes. Generally, in order to obtain vegetative bacterial cells, a medium with low spore formation, such as P.
YG medium is recommended, and its composition is as shown below.

PYG medium medium layer course 20f peptone 20? Yeast extract 10f Distilled water 1000 at l) H7.0 Most of the bacteria can be obtained as vegetative forms by culturing in this medium at 37°C for 1.21 Jt.

O8 medium is used to produce secondary endospores,
Its composition is as shown below.

O8 medium: Cane starch Otaamino solution* 20ri0a003 7. j? distilled water
1000m1 00O7,0 *Amino liquid soy sauce squeezer (flavor liquid: manufactured by Ajinomoto Co., Inc.) This medium makes 3
If cultured at 7°C for 72 hours, more than 10 of the bacteria will become endospores.

In producing the antitumor agent of the present invention, after culturing in the above-mentioned medium at a predetermined temperature for 1 hour, the culture solution is centrifuged to collect bacteria. The collected cells are suspended in distilled water, stirred, centrifuged, washed two or three times, and then freeze-dried to powder.

There is no difference in biological activities such as antitumor activity and toxicity of Bacillus butyric acid bacteria MIIJR♂ whether it is a lyophilized live cell or a dead cell that has been heat-treated at 100° C. for 73 minutes.

Powdered bacterial cells and endospores can usually be used as is as an antitumor agent, but if necessary, a preservability improver or other drugs may be added to form a formulation. No special conditions are required for storage after formulation, as long as it is kept isolated from the outside air, such as by encapsulating it in an ampoule or capsule. When used, it is suspended in physiological saline and administered intravenously or intraperitoneally. The dosage is 0 per day for adults as dry bacterial cells.
,! ~2Jf.

Next, test examples of the antitumor effect and immunostimulatory effect of the antitumor agent of the present invention will be shown.

l Antitumor effect test (1) One week old IOR male mouse/group of 10 mice was inoculated with f(t)/×10 sarcoma iro cells into the golden flank, and 21 hours later. Heat-killed cells of butyric acid bacteria 0.2η/mouse/ny/mouse were heated to 0.2η/mouse. Suspended in physiological saline and injected intravenously once a day for J days. Three weeks later, the mice were sacrificed and the tumors were removed.
The weight was measured and the growth inhibition rate relative to the control was calculated. The results are shown in Table 3.

Table 3. The effect of M'firrr dead cells and dead endospores on Sarcoma/♂O, that is, the administration of dead cells/my/mice showed extremely significant tumor suppression of J''t, 7chi.

Considerable effects were seen even at 0.2 my/mouse.

A similar suppressive effect was obtained with dead endospores, but it seemed to be less effective than with dead bacterial bodies.

(7 mice/group 1Q of male BDF1 mice were inoculated intraperitoneally with 6 L/-210 leukemia cells, and 2≠hours later, butyric acid@Mf
LJRR Killed Bacterial Bacterial L Killed Bacterial Bacterial Body No. 1 Suspended in physiological saline and injected into the peritoneal cavity! We investigated the life-prolonging effects of administering the drug 7 times a day for 7 days. The result is shown in the table 1° table ≠ L/21
As is clear from the results in Table 1, killed bacteria alone are effective according to the standards of the National Cancer Institute, but especially with low concentrations of cyclophosphamide. It is clear that when used in combination, extremely excellent antitumor effects are exhibited.

Demonstration of immunostimulatory effect It is not difficult to imagine that the anti-inflammatory effect described above is due to the host-mediated anti-inflammatory effect due to immunostimulatory effect.
In order to detect these effector cells, an experiment was conducted using a cell sorter (cell analyzer) O8-SO (manufactured by Showa Denko). Intraperitoneal administration of dead bacteria/■/mouse/day/day to Ba1b/e male mice of ≠ weeks of age After 1 day, Asiflo 0M1m intraperitoneal macrophages were infected with the above by indirect fluorescent antibody method. Measured using a cell sorter. As a result, a significant increase in the number of effector cells was observed upon administration of butyric acid bacteria cells.

As described above, it has been revealed that the anti-cancer agent according to the present invention not only has an excellent anti-tumor effect, but also that its effect is significantly amplified when used in combination with other anti-cancer agents. Butyric acid bacteria have many advantages, such as being a non-pathogenic bacterium, being effective with heat-killed bacterial bodies or dead endospores, having fewer restrictions on use, and being able to be produced at low cost. It's a thing.

All examples of the present invention are shown below.

Example/ Butyric acid bacteria M 17 j r♂ (Feikoken Bacteria No. 77tj)
Add 2μ of the same medium to 1 liter of PYG medium as in the test example above.
The inoculum was inoculated so that the amount of the inoculum was 10% of the culture after pre-cultivation, and cultured at 37° C. for 2 hours. At this point, the number of viable bacteria is J
X/0/d! reached. After culturing, the bacterial cells were separated from the culture solution using a cooling continuous centrifuge and washed with distilled water to obtain 32 viable bacterial cells (100 kg as dried bacterial cells). All the obtained bacterial cells were turbid in the collected water for 4 hours and heated to 100°C. The mixture was sterilized by heating for a minute, dispensed into 10 mt ampoules, and after freeze-drying, the ampoules were melt-sealed.

Stored at 1°C. When the anti-tumor effect was tested using this dead bacteria powder according to Test Example/(1),
The tumor growth inhibition rate was high.

Example: Cobutyric acid bacteria MIIrr♂@ was added to O8 medium z in the same manner as in Example.
t'tc was inoculated and cultured at 37°C for 72 hours.

Endospores were collected using a refrigerated continuous centrifuge and washed with distilled water to obtain wet bacterial cells/2Jf. This was dried with hot air at 20°C to obtain powder J'2. The obtained dried bacterial cells were divided into 100 μl portions into vials and stored at 20° C. for 2 months.

These preserved endospores were suspended in physiological saline and the effects of the cyclophosphants on mouse L/λ10 leukemia were measured in accordance with Test Example/(2). When used in combination with Famido, a 220% life extension effect was observed.

Procedural amendment (voluntary) November 25, 1985

Claims (1)

[Claims] Butyric acid bacteria [Clostridium butyricum (Clos-t)]
ridium butyricum)] An antitumor agent containing bacterial cells or endospores of MII588 strain as an active ingredient.