JP2001097869A - New antitumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antitumor gent containing a microorganism or the product of an intracellular enzyme of the microorganism having the antitumor activity effective against various kinds of tumors as active ingredient. SOLUTION: This antitumor agent contains a microorganism belonging to acetic acid bacteria or the product of its intracellular enzyme as active ingredient. The acetic acid bacteria are preferably a microorganism belonging to the genus Gluconobacter, the genus Acetobacter or intermediate strain groups of these genera.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an acetic acid bacterium (Acetic A).
The present invention relates to a novel antitumor agent comprising, as an active ingredient, a microorganism belonging to Cid Bacteria) or an enzyme product thereof.

[0002]

2. Description of the Related Art In the field of chemotherapy for cancer patients, metabolites derived from many microorganisms, such as bleomycin and adriamycin, have been found as substances having an antitumor effect and are used clinically. However, the effect is not always constant for a wide variety of tumors,
There is a need for the development of better antitumor agents. On this occasion,
The present inventors have found that acetic acid bacteria produce substances having high antitumor activity.

Although it has been reported that certain acetic acid bacteria produce antibiotics, it has not been reported that microorganisms belonging to acetic acid bacteria produce substances having antitumor activity. In addition, there are few reports on the production of antibiotics, and a slight amount of Gluconobacter sp. W-315 (later related strain Frateu)
ria (pseudoacetic acid bacterium) sp. w-315 produced polyene antibiotic Enacyloxin (J. Antibiotics, Chapter 45)
Vol. 4, No. 4, pp. 470-475, 1992), Gluconobacter oxyd
ans CCRC10383 strain produced Cephalexin (Proc. Natl. Sc
i. Council, ROC, Vol. 16, No. 4, pp. 184-187, 1992
Gluconobacter oxydans subsp F-
F-2702 substance (Jikeikai Med.
J., Vol. 37, pp. 335-342, 1990).

The present inventors have developed Gluconobact. Subsp. F-2702, a kind of acetic acid bacterium, isolated from a soil sample previously collected from Hirabari, Nagoya City.
er oxydans subsp F-2702)
(F-2702 strain) has been found to accumulate F-2702 substances having strong antifungal activity when cultured under aerobic conditions (Jikeikai Med. J., Vol. 37, pp. 325-333,
1990, Japanese Patent Publication No. 4-7357). In addition, F-2
Strain 702 was sent to the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology (currently, the Institute of Biotechnology and Industrial Technology) by Microbiological Laboratories No. 7449 (FE
RM P-7449) and can be used effectively.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antitumor agent comprising a microorganism having an antitumor activity having an effect on a variety of tumors or an enzyme product of the cells thereof as an active ingredient. It is to be.

[0006]

As a result of various studies on metabolites of microorganisms, the present inventors have found that microorganisms belonging to acetic acid bacteria or substances produced by the intracellular enzymes thereof, especially F-2,
The present inventors have found that 702 substances show high antitumor activity and completed the present invention. That is, the present invention provides an antitumor agent comprising, as an active ingredient, a microorganism belonging to acetic acid bacteria or an intracellular enzyme product thereof.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The acetic acid bacteria taxonomically include two genera of the genus Gluconobacter and the genus Acetobacter.
Manual of Determinative Bacteriology Eighth Edition: Bergey's Manual of Determinative Ba
cteriolgy 8th Edition, 1974). It is also known that there is an intermediate strain having an intermediate property between both bacteria (J. Gen. Appl. Microbiol., Vol. 10, pp. 95-125, 19).
64, Int. J. Syst. Bacteriol., Vol. 30, pp. 547-556,
1980). For example, according to a recent method for classifying the base sequence of 16S ribosomal RNA, the genus Gluconoacetobacter has been proposed as an intermediate genus of the genus Gluconobacter and the genus Acetobacter (Biosci.
Biotechnol. Biochem., Vol. 61, No. 8, pp. 1244-1251,
1997).

The microorganism belonging to the acetic acid bacterium in the present invention refers to genus Gluconobacter, Acetobacter, and their intermediate strains, which are systematically related.
In addition, mutants treated with a mutagen such as ethyl methanesulfonate, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine are also included. Substances having an antitumor effect produced by these cells are an object of the present invention. Of these, microorganisms belonging to the genus Gluconobacter are preferred, and in particular, Gluconobacter oxydans.
subsp ・ F-2702 (Gluconobacter oxydans subsp F-270
2) Bacteria are preferred (see Japanese Patent Publication No. 4-7357).

A substance having antitumor activity can be isolated by culturing an acetic acid bacterium, for example, as described in Japanese Patent Publication No. 4-7357. In summary, bacteria are identified as glucose, peptone, yeast extract, malt extract,
It can be cultured in a medium using a nutrient source such as a granulation extract, and the medium may further contain an amino acid. An appropriate substance that promotes the growth of bacteria, such as vitamins, may be added to the medium. The cultivation method is the same as that for general aerobic bacteria.However, for a relatively small laboratory scale, it is better to increase the contact surface between air and the culture solution as much as possible by static liquid cultivation. Culture is not suitable. The pH suitable for the culture is pH 6.0 to 8.0, and the temperature is 27 ° C to 37 ° C.
The culture is usually performed at pH 6.8 and at a temperature of 34 ° C. The culturing days are usually 4 to 10 days.

In order to extract and purify an intracellular enzyme product having antitumor activity from a culture supernatant, a commonly used extraction method is used.
Purification means can be used.For example, an extraction method using an organic solvent such as ethyl acetate, chloroform, and diethyl ether, and a transsolution using a solution such as aqueous sodium bicarbonate or a phosphate buffer are repeated, and further column chromatography is performed. Use in combination.

The intracellular enzyme product of the acetic acid bacterium thus extracted and purified is not particularly limited as long as it has antitumor activity.
Substances are particularly preferred. F-2702 material has the following physicochemical and biological properties. Elemental analysis (%): 64.88% carbon, 5.52% hydrogen (excluding nitrogen, sulfur, phosphorus, and halogen) Molecular weight (mass spectrometry): Approx. 300 Melting point: 119 ° C or higher (decomposition) Specific rotation ([α] 20D): + 1.4035 ° (C = 0.713, methanol) Ultraviolet absorption spectrum (λmax, methanol): 315 nm, 23
5nm, 210nm Infrared absorption spectrum (main absorption value): 3400, 3100-280
0, 2700-2600, 1700, 1620, 1590, 1520, 1460, 1390,
1330, 1280, 1140, 1090, 1050, 990, 960, 940, 870,
820, 700cm-1 Solubility: Easily soluble in DMSO, methanol and ethanol. Soluble in ethyl acetate and diethyl ether. Insoluble in benzene and n-hexane. Soluble in water. Color reaction: yellow to orange with concentrated sulfuric acid, concentrated hydrochloric acid or 70% perchloric acid. Positive-ferric chloride reaction, 2,4-dinitrophenylhydrazine reaction, Ehrlich's diazo reaction. False positive-Ehrlich reaction Appearance: yellow-orange Antibacterial spectrum: shows growth inhibitory activity against molds and yeasts.

The antitumor agent of the present invention containing a microorganism belonging to the acetic acid bacterium or its intracellular enzyme product as an active ingredient has antitumor activity against various tumors, for example, liver cancer, pharyngeal cancer, leukemia. , Lymphoma and the like.

When the antitumor agent of the present invention is used for treatment,
Various dosage forms can be used depending on the patient's condition and usage, for example, internal preparations such as powders, fine granules, granules, capsules and tablets, external preparations such as ointments and cataplasms, and injections Alternatively, suppositories and the like can be mentioned. The acetic acid bacterium or its intracellular enzyme product is appropriately mixed with physiologically acceptable excipients, disintegrants, binders, lubricants, preservatives, additives such as dissolving agents, and using a well-known method. Thus, it can be prepared into a dosage form as described above.

The dose of the antitumor agent of the present invention when it is used for actual treatment is appropriately determined in consideration of the age, weight, sex, symptoms, etc. of the target patient. For example, F-2702
When the substance is administered orally, usually 0.00
In the case of parenteral administration in the range of 1 to 10 mg / kg, it is usually used in the range of 0.01 to 1 mg / kg per day for adults. When the antitumor agent of the present invention is used for antitumor purposes, no apparent toxicity is observed.

[0015]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Extraction and Purification of F-2702 Substance Using a Rubin flask, containing 1% glucose, 0.5% malt extract, 0.5% yeast extract, 0.5% meat extract and 1% peptone. Strain F-2702 in medium at 34 ° C
For 4 days under aerobic conditions. 10 L of the culture solution was separated into bacterial cells and culture supernatant by centrifugation. The pH of the supernatant was adjusted to pH 3.0 with HCl and extracted with ethyl acetate. The solvent layer (6 L) was concentrated under vacuum at 30 ° C.
The solution was transferred into a 1 M phosphate buffer (pH 7.8). The pH of the aqueous layer (200 ml) was adjusted to pH 3.0 with HCl,
Extracted with CHCl ₃ . The solvent layer was again dissolved in 0.1 M phosphate buffer (pH 7.8), and the aqueous layer (200 ml)
Was adjusted to pH 3.0 with HCl, and extracted with diethyl ether. The solvent layer (200 ml) was dried over anhydrous sodium sulfate and concentrated at 30 ° C. under vacuum. The obtained crude product was subjected to Sephadex LH-20 (Pharmacia) column (2.5 × 45) using methanol as an eluent.
0 cm) by gel filtration.
Sephadex LH-20 column using ₂ Cl ₂ (2.
Purification by gel filtration (5 × 90 cm) isolated the active fraction. The activity was examined by cytotoxicity to U937 cells.

Example 2 Cytotoxicity test Using various types of tumor cells, the cytotoxicity of F-2702 was investigated. The following cells were subjected to the test. All cells were purchased from the American Type Culture Collection (ATCC). Suspension cells: U937 (human malignant tissue lymphoma cells) P388D1 (mouse lymphoma cells) THP-1 (human monocytic leukemia cells) Adherent cells: NIH-3T3 (mouse fibroblast cells) Hep G2 (human hepatoma cells) KB (human nasopharyngeal carcinoma cells) Each cell was seeded at 1 × 10 ⁴ cells in a 96-well plate, and further freeze-dried by a conventional method.
The two substances were added to cells at a final concentration of 160 μg / mL so as to be serially diluted 2-fold, and cultured at 37 ° C. for 3 days in a carbon dioxide incubator. Three days later, Cell Counting
The survival rate of each cell was examined using Kit (Dojindo).
The results are shown in FIGS. From these results, F-
It was confirmed that 2702 substances had cytotoxicity against various tumor cells. This cytotoxicity acts more strongly on suspension cells than on adherent cells, and U937, P388D
1. The growth of THP-1 could be almost suppressed at a concentration of 20 μg / mL. Furthermore, it was shown that this cytotoxicity is exerted in a relatively short time.

Example 3 Apoptosis Assay Apoptosis is a general term for cell death characterized by DNA fragmentation, chromatin condensation, formation of apoptotic bodies, and the like. In the process of development, apoptosis is also considered to be a cell suicide mechanism, as cells differentiate into various tissues and a certain number of cells that have completed their mission die by apoptosis. In recent years, induction of apoptosis of cancer cells by anticancer drugs and radiation has been widely used for determining the effects of anticancer drugs and for investigating and studying drug resistance (Int. J. Oncol., 1, 639-648, 1992). . DNA fragmentation of nucleosome units due to apoptosis can be characterized using Lagarose gel electrophoresis with the characteristic La
It can be confirmed by detecting the der pattern (Analysis of cell death by flowcytometry.
In: Cell Growth and Apoptosis.Apractical approac
h (Studzinski GP.ed), IRL press, Oxford, 143-167
P., 1995). Therefore, using HL-60 cells (promyelocytic leukemia cells), the presence or absence of induction of apoptosis by the F-2702 substance was examined.

HL-60 cells were purchased from the American Type Culture Collection. This HL-60
The cells were seeded at 1 × 10 ⁶ cells in a 35 mm diameter petri dish, and the F-2702 substance was added to the cells at a concentration of 20 μg / mL. Staurosporine at 1 μg / mL was used as a control substance. After culturing for 5 hours, the DNA was recovered using an Apollader kit (Takara Shuzo) according to the attached instructions, and the DNA was collected and subjected to 2% agarose gel electrophoresis to examine the fragmentation of the DNA. As a result, F-2702
For both the substance and staurosporine, a Ladder pattern indicating DNA fragmentation of nucleosome units was detected, and induction of apoptosis was confirmed (FIG. 3).

[0020]

Industrial Applicability According to the present invention, it has been found that a microorganism belonging to acetic acid bacterium or an intracellular enzyme product thereof has excellent antitumor activity, and an antitumor agent which can be used for a wide variety of tumors is provided. Is done.

[Brief description of the drawings]

FIG. 1 shows the effect of F-2702 product on tumor cells.

FIG. 2 shows the effect of F-2702 product on tumor cells.

FIG. 3 shows the apoptosis-inducing effect of F-2702 product on HL-60 cells.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 BA03 BA04 BB08 BB15 BC02 BE09 BE10 BE11 BE14 BE19 BG02 BG03 BH01 BH02 BH06 BH10 CA02 DA02 DA05 4C087 AA01 AA02 BC45 CA09 CA10 CA11 CA22 CA25 CA37 MA01 NA14 ZB21 ZB ZB26

Claims (4)

[Claims] An antitumor agent comprising a microorganism belonging to acetic acid bacterium (Acetic Acid Bacteria) or an intracellular enzyme product thereof as an active ingredient. 2. The method according to claim 1, wherein the acetic acid bacterium is Gluconob.
The antitumor agent according to claim 1, which is a microorganism belonging to the genus acter), the genus Acetobacter, or an intermediate strain thereof. 3. The microorganism belonging to the genus Gluconobacter is Gluconobacter oxydans subsp. F-2702.
The antitumor agent according to claim 1 or 2, which is a (Gluconobacter oxydans subsp F-2702) bacterium. 4. The antitumor according to any one of claims 1 to 3, wherein the produced substance is an F-2702 substance having the following physicochemical properties and biological properties, and a pharmaceutically acceptable salt thereof. Agent. Elemental analysis (%): 64.88% carbon, 5.52% hydrogen (excluding nitrogen, sulfur, phosphorus, and halogen) Molecular weight (mass spectrometry): Approx. 300 Melting point: 119 ° C or higher (decomposition) Specific rotation ([α] 20D): + 1.4035 ° (C = 0.713, methanol) Ultraviolet absorption spectrum (λmax, methanol): 315 nm, 23
5nm, 210nm Infrared absorption spectrum (main absorption value): 3400, 3100-280
0, 2700-2600, 1700, 1620, 1590, 1520, 1460, 1390,
1330, 1280, 1140, 1090, 1050, 990, 960, 940, 870,
820, 700cm-1 Solubility: Easily soluble in DMSO, methanol and ethanol. Soluble in ethyl acetate and diethyl ether. Insoluble in benzene and n-hexane. Soluble in water. Color reaction: yellow to orange with concentrated sulfuric acid, concentrated hydrochloric acid or 70% perchloric acid. Positive-ferric chloride reaction, 2,4-dinitrophenylhydrazine reaction, Ehrlich's diazo reaction. False positive-Ehrlich reaction Appearance: yellow-orange Antibacterial spectrum: shows growth inhibitory activity against molds and yeasts.