JP2003261453A - Antitumor agent and radiation-protecting agent consisting of e. faecalis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop an effective antitumor agent and a radiation-protecting agent. <P>SOLUTION: The antitumor agent and the radiation-protecting agent are obtained by using a water soluble extract of heat-killed microbial cells of a new lactobacillus, Enterococcus faecalis 2001 strain. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the development of antitumor agents and radioprotective agents.

[0002]

2. Description of the Related Art At present, various treatment methods have been developed for cancer, which is the leading cause of death in Japan, such as surgical therapy or therapy with a chemical anti-cancer agent. However, all the therapies have serious side effects, and at present, the results have not been sufficiently achieved.

Radiation is also used to treat cancer, but its side effects pose a problem. Irradiation of the maternal body during the most susceptible fetal organogenesis period has effects on embryonic death, external malformations, and fetal weight. Therefore, if Enterococcus Faecalis can be administered to reduce these effects , The radioprotective effect of normal cells is clear. The development of radioprotective agents without side effects is awaited. Conventionally, chemical agents have been used as radioprotective agents, but their effects are not terribly strong, side effects are strong, and effective agents have not been found.

[0004]

The problem is to develop an effective antitumor agent and radioprotective agent.

[0005]

[Means for Solving the Problems] The inventors of the present invention have made diligent efforts to solve the above problems, and as a result, a novel lactic acid bacterium Entero
It was found that the problem can be solved by using a water-soluble extract of heat-killed cells of coccus Faecalis (hereinafter referred to as EF) 2001 strain.

That is, the present invention is (1) Enterococcus Faecalis EF-2001.
An antitumor agent containing a water-soluble extract of heat-killed strains of a strain as an active ingredient, (2) Enterococcus
(3) Heat-killed cells of Enterococcus Faecalis , which contains a water-soluble extract of heat-killed cells of Fecalis ( Enterococcus Faecalis ) EF-2001 as an active ingredient. radioprotective agent characterized by containing a water-soluble extract as an active ingredient, (4) Enterococcus faecalis (Enterococcus faecalis) is Enterococcus faecalis (Enterococcus faecalis) EF-2001 strain (3) radiation according protectant, (5) Enterococcus faecalis (Enterococcus faecalis) tumor growth inhibition method comprising administering an EF-2001 strain soluble extract of heat-killed cells of, (6) Enterococcus faecalis (Enterococcus faecalis) (7) Enterococcus, which comprises administering a water-soluble extract of heat-killed cells of Faecalis (Enterococcus Faecali
s ) is Enterococcus Faculis ( Enterococcus Fa
ecalis ) EF-2001 strain (6). EF-2001 is an enterococcal Enterococcus Faecalis -2001 strain classified from the stool of a normal person and has the following properties.

It is a Gram-positive coccus. Shape of colony (trypto soya agar medium, cultured for 24 hours): diameter 1.0 mm, smooth, regular circle, white colony morphology: sphere to oval (1.0 × 1.5 μm) Liquid medium is well linked. No spore formation. Facultative anaerobic. Fermenting glucose to produce lactic acid (final pH 4.3). No gas production. Catalase negative. Proliferate at 10-45 ° C (optimal 3
7 ° C). Proliferated to pH 9.6, 6.5% NaCl, 40% bile.
0.04% Potassium tellurium potassium positive. 0.01% tetrazolium positive. 0.1% methylene blue milk
Positive. Hydrolyze arginine.

Amygdalin, cellobiose, fructose, galactose, glucose, glycerol, lactose, maltose, mannose, mannitol, ribose, salicin, sucrose, melezitose, and sorbitol are fermented to produce an acid. Resistant to 60 ° C for 30 minutes. Digests casein and gelatin. Decarboxylate tyrosine to tyramine. Lancefield antigen group D. GC% 35.0 ±
The lactic acid bacterium Enterococc is a new bacterium due to its 1.0% or higher property
It turned out to be us Faecalis .

[0009] The heat-killed cells of this EF-2001 (hereinafter referred to as EFH-201
Say. ) Was first simply fractionated into a water-soluble fraction and an insoluble fraction, and the antitumor activity of the water-soluble fraction against transplanted cancer in mice was examined. The method of fractionating into the water-soluble fraction and the insoluble fraction is not particularly limited, but the following method is available, for example.

(1) Heat-killed cells prepared by any method are suspended in water at about 20 to 26 ° C. to elute soluble components. Then, the cells are centrifuged to remove the cells, and the supernatant is concentrated if necessary and dried to obtain a powdery water-soluble extract. (2) Viable cells or heat-killed cells are crushed by a method such as ultrasonication and then suspended in water at about 20 to 26 ° C to elute soluble components. Then, the cells are centrifuged to remove the disrupted cells, and the supernatant is concentrated if necessary and dried to obtain a powdery water-soluble extract.

The method (2) is particularly effective because it is particularly excellent in the extraction efficiency of the active ingredient. Regarding the Enterococcus Faecalis EF-2001 strain, a request was made to the Director of the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, but it was not accepted as a rejection. Therefore, in order to meet the demand for sale, Enterococcus F.
The aecalis ) EF-2001 strain is stored at the BRM Research Laboratories of Nippon Belm Co., Ltd.

Next, focusing on the antioxidant action and systemic immune system activating action of EF-2001, EF-200 was kept in the living body of mice for a certain period of time.
The protective effect of EF-2001 against radiation at the individual level was observed by irradiating pregnant mice during the period of organogenesis with the administration of 1. As a result, radioprotective effects were revealed in embryo death, malformation and fetal weight. The above-mentioned antitumor agent and radioprotective agent can be prepared into a preparation by a usual preparation method, and a common carrier, excipient, binder,
Tablets and granules can be prepared using an auxiliary agent such as a disintegrant.

The dose of the water-soluble extract of EFH-201 is
Although it varies depending on the activity of the extract, the symptoms of the patient, the age, etc., the antitumor agent is usually 100 mg / day / kg body weight for adults, and the radioprotective agent is 60 mg / day / kg body weight for adults. It is preferable to administer a degree. Furthermore, for antitumor agents, EFH-201
It is also possible to administer the water-soluble extract of (1) in food in an amount comparable to the above dose. Examples of foods include health supplements and general foods such as udon, bread, yogurt, tofu, and confectionery.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION (Example 1) Examination of antitumor effect Experimental animals ICR male mice, BALB / c male mice and dd
Y male mice (4 weeks old) and Swiss-Webstar male and female mice (8 weeks old) were purchased from Japan SLC Co., Ltd. and used for an experiment after preliminary breeding for 1 week or more. Swiss-Web
Male and female star mice were mated at the age of maturity, and then only one female mouse was bred until delivery. Throughout the preliminary breeding and experimental period, temperature 23 ± 1 ° C, humidity 55 ± 10
%, Lighting for 12 hours, and ventilation conditions were 10 times / hr.

Extraction of Water-Soluble Fraction Derived from EF-2001 Heated Dead Cells (EFH-201) To 20 g of EFH-201 was added 200 ml of distilled water, and the mixture was stirred at room temperature for 2 hours. After that, centrifuge (5000 rp
The water-soluble fraction obtained by collecting, concentrating and freeze-drying the collected supernatant was used as an EFH-201 water-soluble extract in the experiment.

Effect of increasing the ratio of lymphocytes to polymorphonuclear leukocytes (L / P activity) by EFH-201 water-soluble extract According to the method of Hand et al., Swiss-Web in which immunity is immature.
Litter newborns (within 6-12 hours after birth) of star female mice were divided into two groups, and 200 g of the EFH-201 water-soluble extract (dissolved in physiological saline) was used for each of the pups in the sample group.
The ss-Webstar mouse was intraperitoneally administered. Saline (0.05 ml) was similarly administered to the newborn Swiss-Webstar mice in the control group. Then, blood was collected from the tail vein of the mouse before administration and on the 6th, 10th, and 14th days after administration to prepare a blood smear.
The L / P ratio was calculated by counting the total number of lymphocytes (L) and polymorphonuclear leukocytes (P) in the sample under (400 times) 100.

Antitumor activity of water-soluble extract of EFH-201 Sarcoma was subcutaneously placed on the right lower abdomen of ICR male mice (5 weeks old).
-180 (1.7 × 10 ⁶ cells / mouse) was transplanted. Also, 6
Meth-A fibrosarcoma cells were subcutaneously injected into the right and left lower abdomen of 1-week-old BALB / c male mice at 1 × 10 each.
⁶ and 2 × 10 ⁵ were transplanted (Double-grafted tumor
or system method).

EFH-201 water-soluble extract dissolved in physiological saline on the 3rd, 4th and 5th day after transplantation of both types of tumor cells 1 mg /
0.1 ml was administered 3 times into the right lower abdominal tumor. Saline was similarly administered to the control group. The size of the enlarged and shaped tumor is shown by the product of the major axis and the minor axis (area mm ² ).
Measurements were taken from day 2 to day 25 every other day. Then, the tumor was excised on the 25th or 26th and the weight thereof was measured. In addition,
8-10 mice were used per group.

Statistical Treatment The data obtained in each animal experimental group are shown as mean ± standard deviation, and the statistical test between the control group and the test substance administration group was St.
udent t-Tested, p <0.05 (risk factor:
5% or less) was considered to be significantly different.

Results 1. Swiss-Webs with EFH-201 water soluble extract
Increase in L / P in blood of tar mouse In neonatal Swiss-Webstar mouse intraperitoneal administration group (EFH-201 water-soluble extract), 6,10,14 after administration
The increase of lymphocytes was remarkable as compared with polymorphonuclear leukocytes in the blood with the passage of time and time. Therefore, the lymphocyte-to-polymorphonuclear leukocyte (L / P) ratio also showed a gradual increase in the control group, whereas it showed a sharp increase in the water-soluble extract-administered group, compared with the control group. L / P was significantly high (p <0.05) at any time point, and the significant difference among the experimental groups on day 14 was significant (p <0.01). Figure 1
Shown in.

2. Antitumor Activity of EFH-201 Water-Soluble Extract The effect of EFH-201 water-soluble extract on the growth inhibition of Sarcoma-180 cells transplanted subcutaneously in the abdomen of ICR mice was examined, and as shown in FIG. The tumor size (mm ² ) in the EFH-201 water-soluble extract group was significantly smaller than that in the group from the ^second administration (4 days after tumor implantation) throughout the measurement period, showing a significant tumor growth inhibitory effect. It was observed. After measuring the tumor size on the 26th day, the tumor was excised, and the measured value of the weight is shown in Table 1. The tumor weight of the EFH-201 water-soluble extract-administered group was obviously smaller than that of the control group, and the tumor growth inhibition rate was 70.6%.

[0022]

[Table 1]

Next, EFH-20 which affects the growth inhibition (Double-grafted tumor system method) of Meth-A fibrosarcoma cells transplanted subcutaneously on the right and left abdomen of BALB / c mice.
1 The results of examining the effect of the water-soluble extract are shown in FIG. The tumor (A) on the right side of the abdomen tended to be suppressed in the extract-administered group from the 13th day after transplantation as compared with the control group, and the tumor size was obviously small from the 15th day. On the other hand, there was no statistically significant difference in the size of the tumor on the left side (B) between the extract-administered group and the control group, but the growth of the tumor in the extract-administered group tended to be suppressed. there were.

On the 25th day, the tumor on the right side of the abdomen was removed. The tumor weight was 1.46 ± 0.25 g in the control group, and EFH-201
In the water-soluble extract-administered group, it was 0.64 ± 0.21 g, which was clearly small. Also, Meth with EFH-201 water soluble extract
-A tumor inhibition rate was calculated to be 56.4%, 1 in 10
The tumor completely regressed in one mouse. In the control group, no BALB / c mouse in which the tumor completely regressed was found. On the other hand, with respect to the left-sided tumor, the tumor size was not significantly different between the extract group and the control group, and therefore the tumor was not excised.

Conclusion Neonatal Swiss-Webs of water-soluble extract of EFH-201
The intraperitoneal administration to the tar mouse was carried out with the L
/ P was increased. This is a phenomenon that the increase of lymphocytes was more remarkable than that of polymorphonuclear leukocytes centered on neutrophils, and a component having an action of stimulating the division and proliferation mechanism of lymphocytes was contained in the extract. Is considered to exist.

Further, the EFH-201 water-soluble extract against the tumor cell Sarcoma-180 cells transplanted subcutaneously into the abdomen of ICR mice showed a growth inhibitory effect. Meth-inoculated subcutaneously into the abdomen
EFH-201 water-soluble extract administered into A fibrosarcoma cell tumor clearly suppressed the proliferation of the tumor cells.

(Example 2) Examination of radiation protection effect Experimental animal At Charles River Japan, closed colony ICR (Crj: CD-01 (Swiss Hauchka)) mice were used. After the purchase, in order to acclimate to the breeding conditions of the present invention, preliminary breeding was carried out for at least one week, and the experiment was started. As the age of mice, 9 to 13 weeks of female mice and 9 to 20 weeks of male mice were used. In order to make a Female mouse pregnant, put 1 or 2 Female mice in the gauge of Male mouse, AM6: 00 ~ AM9: 00
For 3 hours with male mice.

Radiation conditions The radiation irradiation device and irradiation conditions used were a Philips radiation irradiation device (225 kV). The dose rate was 0.35 / min., And the irradiation dose was 2 Gy to the whole body of the maternal mouse 8 days after pregnancy. Experimental group Experimental group, Control group, EFH-201 administration only group (hereinafter, Sham
Control group), 2 Gy radiation alone irradiation group, EFH-201 was administered, and 2 Gy radiation irradiation group were classified into 4 groups in total. The number of mice used in each group is 20 or more at the individual level.

Method of Observing Effects 1) Observation of external surface malformation: Female mice were bred, maternal mice confirmed to be pregnant were irradiated with radiation on the 8th day of pregnancy, and mother mice were cervical dislocation on the 18th day of pregnancy. Killed by
The fetus was removed from the maternal mouse. Then, the effects on pre-implantation mortality rate, embryo mortality rate (implantation scar, placenta remnant, resorption embryo, macerated fetus), fetal mortality rate, external malformation rate and fetal weight were observed.

The fetal weight is measured by a Sartorius top plate scale (Ma
x20). 2) Statistical processing of data: In statistical processing, first,
Since the fetal weight follows a normal distribution, t-test was performed. As for the statistical processing of individual-level data, non-parametric 1: 1 Wilcoxon test was performed for pre-implantation mortality rate, embryo mortality rate, external surface malformation rate, skeletal malformation rate, and the like.

Results 1) Pre-implantation mortality rate: Table 2 shows the implantation rate. The landing rate is
The 2Gy radiation-irradiated group and the EFH-201-administered 2Gy-irradiated group were slightly higher than the Control group and the Sham Control group, but no statistically significant difference was observed. Also, 2
No significant difference was observed between the Gy radiation-only irradiation group and the EFH-201-administered 2 Gy radiation irradiation group.

[0032]

[Table 2]

2) Embryonic mortality: The results of embryonic mortality are shown in FIG. The embryo mortality is higher than that of the Control group and the Sham Control group.
A significant difference was observed between the 2 Gy radiation alone irradiation group and the 2 Gy radiation irradiation group to which EFH-201 was administered (P <0.001). Furthermore,
As can be seen from Table 1, 62.3 in the 2 Gy single irradiation group.
However, in the 2Gy irradiation group administered with EFH-201, it was 47.5%. Therefore, the 2Gy irradiation group administered with EFH-201 was more than the 2Gy irradiation alone irradiation group, The embryo mortality is clearly suppressed.

3) Fetal mortality: The results of fetal mortality are shown in FIG.
Shown in. There was no significant difference in all treatments compared to the Control and Sham Control groups. 4) Outer surface malformation incidence: The results of the outer surface malformation incidence are shown in FIG. Compared to the Control group and the Sham Control group, a significant difference was observed between the 2Gy radiation alone irradiation group and the 2Gy radiation irradiation group administered with EFH-201 (P <0.001).

5) Types of malformations: The major external malformations that occurred were exencephalopathy (Fig. 7), abnormal tail (Fig. 8), cleft palate (Fig. 9) and the like. The most prominent external surface malformation was exencephalopathy in both the 2Gy irradiation alone group and the EFH-201-administered 2Gy irradiation group, followed by a tail abnormality.

6) Fetal weight: The results of fetal weight are shown in FIG. FIG. 10 is a graph in which data is statistically processed by the T-test test and plotted. Control group and Sham Cont
Compared to the rol group, the fetus body weight was decreased in the 2 Gy radiation-only irradiation group (P <0.05). But with the Control group
No reduction in fetal weight was observed in the 2 Gy irradiation group to which EFH-201 was administered as compared to the Sham Control group. Further, as shown in Table 2, the body weight of the fetus in the 2Gy irradiation group to which EFH-201 was administered was obviously higher than that in the 2Gy irradiation alone group.

Conclusion From the above results, it is considered that the cells of embryos and fetuses damaged by radiation had their immunity enhanced by EFH-201 and the repair ability of the cells was enhanced, so that a protective effect against radiation was exhibited. The above Example 2 was carried out on EFH-201, that is, the heat-killed bacterial cell powder of the EF-2001 strain, but other Enterococcus faecalis (accession number)
Similar effects can be obtained with ATCC10100, 14428, 19434).

[0038]

Effect of the Invention] Enterococcus faecalis, especially Enterococcus faecalis (Enterococcus Faecali
s ) By using a water-soluble extract of heat-killed cells of the EF-2001 strain, radiation can be protected safely and simply at the same time as the antitumor effect.

[Brief description of drawings]

FIG. 1 Swiss-We with EFH-201 water-soluble extract
The figure which shows the lymphocyte-to-polymorphonuclear leukocyte ratio increasing action in the peripheral blood of bstar mice.

FIG. 2 shows the antitumor effect of EFH-201 water-soluble extract against Sarcoma-180 solid tumor transplanted into ICR mice.

FIG. 3 shows the antitumor effect of EFH-201 water-soluble extract against Meth-A fibrosarcoma in BALB / c mice.

FIG. 4 is a diagram showing embryo mortality upon irradiation of mating ICR mice.

FIG. 5 is a view showing the fetal mortality rate of a hybridized ICR mouse to irradiation.

FIG. 6 is a graph showing the incidence of malformation in irradiated ICR mice upon irradiation.

FIG. 7 shows normal mice and exencephalopathy.

FIG. 8 is a diagram showing an abnormality of the tail.

FIG. 9 shows cleft palate.

FIG. 10 shows the fetal weight of a fetus in response to irradiation of a mating ICR mouse.

Claims (7)

[Claims] 1. Enterococcus faecalis ( Entero
coccus Faecalis ) An antitumor agent comprising a water-soluble extract of heat-killed cells of EF-2001 strain as an active ingredient. 2. Enterococcus faecalis ( Entero
coccus Faecalis ) EF-2001 strain containing a water-soluble extract of heat-killed cells as an active ingredient. 3. Enterococcus faecalis ( Entero
Coccus Faecalis ) is a radioprotective agent containing a water-soluble extract of heat-killed cells as an active ingredient. 4. Enterococcus faecalis ( Entero
coccus Faecalis ) is Enterococcus faecalis ( E
nterococcus Faecalis ) EF-2001 strain.
The described radiation protector. 5. Enterococcus faecalis ( Entero
coccus Faecalis ) A method for suppressing tumor growth, which comprises administering a water-soluble extract of heat-killed cells of EF-2001 strain. 6. Enterococcus faecalis ( Entero
Coccus Faecalis ) is a heat-killed cell body, and a water-soluble extract is administered to the radiation protection method. 7. Enterococcus faecalis ( Entero
coccus Faecalis ) is Enterococcus faecalis ( E
nterococcus Faecalis ) EF-2001 strain.
Radiation protection method described.