JPH0258501A - Lipopolysaccharide and use thereof - Google Patents

Abstract

PURPOSE:To obtain a lipopolysaccharide, useful as interferon inducers, tumor necrotic factor inducers and carcinostatic agents and contained in Gram-negative bacterial cells with low toxicity to mammals without containing 2-keto-3- deoxyoctonates. CONSTITUTION:The objective lipopolysaccharide with low toxicity to mammals without containing 2-keto-deoxyoctonates. The above-mentioned lipopolysaccharide is obtained by culturing Gram-negative methanol assimilating bacteria, such as Pseudomonas extroquens NCIB9399.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a novel low-toxicity ribopolysaccharide (140 pO1yS
nCcharide (hereinafter referred to as LPS), as well as interferon-inducing agents and tumor necrosis factor (Tumor
Necrosis Facf (hereinafter referred to as TNF) inducer and anticancer agent. More specifically, the low-toxicity L P contained in G11-negative bacterial cells
Regarding S. When used in combination with immunostimulants such as lentinan and cyclophosphamite, LPS not only exhibits even higher anticancer effects, but also has the ability to induce interferon and TNF, making it useful for the treatment of cancer and viral infections. used as an agent.

[Prior Art] In recent years, as a type of chemotherapy for cancer, there has been interest in immunotherapy, which treats cancer by strengthening the body's inherent immune capacity to recognize cancer as a foreign substance and eliminate it.

Examples of drugs that have such functions include Krestin, which is extracted with hot water from the mycelium of Corsiella versicolor, lentinan, which has an immunostimulating function and which is extracted with hot water from Shiitake mushrooms, bisibanil, which is an anti-malignant tumor streptococcus preparation, and Mycobacterium tuberculosis. Immune stimulants such as BCG, which is a live bacterial vaccine, are known and are already in clinical use.

However, these immunostimulants alone are not practically sufficient, and in order to increase this antitumor effect, studies have been made on the effects of combination therapy in combination with conventional treatments. For example, MM4611 was administered subcutaneously to the inguinal region of C3)1/He mice using a combination of lentinan and E. coli LPs.
! The cells were transplanted and administered once every 12 days after transplantation.
Good results have been obtained, with 0% of mice being completely cured (Japanese Patent Application Laid-open No. 46818/1983).

In addition, poly! -PolyC, TC13ASB mother mannan, etc. have been clarified (S, Abe et al.
al,:GannJ,'3.91-96;Febru
ary, 1982〉.

- In parallel, lymphokines, which are endogenous substances between immunocompetent cells, include interferon,
and i' NF have been attracting attention.

In other words, interferon is a glycoprotein that gives the living body the ability to protect against virus infection, and is a substance produced by the living body when stimulated by virus particles, bacterial cells, or interferon inducers, and its effects are not limited to antiviral effects. It has come to be known that it exhibits antimicrobial activity, anti-cell proliferation activity, and phagocytic ability-enhancing effect on phagocytes. Furthermore, it has been reported that it exhibits anti-tumor activity against some tumors, and has attracted attention as a therapeutic agent for viral infections and anti-tumours, and clinical applications have already begun. Also, 'r N F is 1975 I T' L,
It is a glycoprotein with a molecular weight of 40,000 discovered by J. J., 01d, etc. Animals such as mice and rabbits are first stimulated with BCG, Corynebacterium uno, Barbum, Zymosan, etc., and 10 to 14 days later, secondary stimulation with PS is performed. It is well known that this causes the blood to become fertile. When the blood serum obtained in this way is applied to tumor-bearing animals, it causes significant hemorrhagic necrosis, which has led to expectations for its clinical application. However, in terms of therapeutic methods using these as therapeutic agents, interferon is produced by applying a virus to human diploid cells that can be cultured and released into the culture medium, or interferon produced by microorganisms using recombinant DNA. In the case of TNF, it is purified from serum collected after primary and secondary stimulation of rabbits, etc., or from culture fluid obtained by culturing appropriate animal cells. TN
These methods involve directly administering F to the patient, and both methods involve directly administering the substance produced by external means to the patient. In addition, there was an idea to treat viral infections and cancer by injecting PS and making interferon and TNF endogenous, but in this case too, conventional LPS
Because it is highly toxic, Il! It could not be used on the floor.

[Problems to be solved by the invention] Conventional LPS is terrible! These are obtained from bacteria and Salmonella enterica, and because they are highly toxic to mammals, they are often used in conjunction with cancer treatment, interferon, or 'rNl.
It has not been possible to use it clinically as an inducer.

[Means and actions for solving the problem] The inventors of wood
We have conducted extensive research to develop cancer combination therapy agents, interferon inducers, and TNF inducers that have low toxicity to mammals and can be applied clinically. There is a group of bacteria that have PS, and from these bacteria, PS has low toxicity and exerts a strong anticancer effect when used in combination with other immunostimulants, and also induces interferon and TNF, respectively. The present invention was achieved based on the discovery that the present invention has the ability to

That is, the present invention is characterized in that it does not contain 2-keto-3-deoxyoctonate and has low toxicity to mammals. The present invention relates to a novel ribopolysaccharide derived from J bacterial cells, as well as an interferon inducer, a tumor necrosis factor inducer, and an antitumor agent, which are characterized by containing this novel ribopolysaccharide as an active ingredient.

The bacteria containing the novel low-toxicity LPS of the present invention are not particularly limited as long as they are Gram-negative methanol-assimilating bacteria, but representative strains are exemplified as follows. That is, (MG-1009): Pseudomonas extroque
ns) NCIB9399 [J, Gen, Appl, M
icrobiol, 25.343 (1979) Reference ('IG-1021) Nibrotaminobacter ruber (Prota bacterium nobacter ruber)
NCl82879 [J, Gen, Appl, Mic
robtol, 25. , 343 (1979)] (Lu G-1023): Pseudomonas sp.
eudononas sp,) NCIB9686 [
8iochem, J., 92,609 (1964)
Reference] (MG-1039): Methylomonas methanolica
) NRRl, B-5458 [Proc, 4th I
Fs: Ferment, Technol'roday
, p497 (1972)] ('IG-1090
): Pseudomonas methylotropha (1'Seudo
monas mct, hylotropha) NC1
[051/I[l'roc, Int, symp, Mi
crobiol, Growthon C3-Com
pd, p, 23 (1975), Special Publication No. 55-1038
(MG-1091) : Pseudomonas methylotropha (f'seudomonas methylot
ropha) NCIBIO592 [I'roc, In
t, Symp, Microbiol, Growth
C1-Compd, p, 23 (1975), see Special Publication No. 55-1038] (MG-1105): Microcyclus aquaticus
ATCC25396 [J, Gen, 8pp1. Micro
obio1. ．． 25. 343 (+979) reference and (MG-1284): 1'aracoccus denitrificans
ns) IFO] 3301 [lnt, J, 5yst, Ba
cteriol, +9,375 (1969) n,! j
Examples include:

Note that (MG-) in the strain name above indicates the number of the strain owned by the inventors.

(+)1. , Preparation of PS Highly toxic LPS can be obtained from these low-toxicity LPS-producing bacteria, for example, in a manner similar to that of N.

■Culture of low-toxicity t, ps-producing bacteria The cultivation method of methanol-assimilating bacteria, which is a low-toxicity [, PS-producing bacteria, may be any known culture method of methanol-assimilating bacteria, and there are no particular restrictions. However, for practical purposes, for example, Japanese Patent Application Laid-Open No. 11790/1983. Japanese Patent Publication No. 54-129182,
It is cultured by the method disclosed in Japanese Patent Publication No. 53-6277, Japanese Patent Publication No. 56-3515, etc. That is, culture is performed using a medium having the composition shown below.

(NH4)2504 3 gKl12PO4
] , 4 3 Ns2PO42,1g '1.5 (+4・711,20 0.2 :C
aCl2-2820 30 mgFcC(,l
lr, (17・X1120 30 m3M, C1
2.4820 5 mgZ, 5 (14.7)
120 5 mg.

CuSO4・51120 0.5 m3 Vitamin mixture 1 ml Methanol 10 people Pure water l r+87.0 In addition, the N monthly income of the vitamin mixture in the above medium is:
Distilled water or pure water 11 contains 20 μg of biotin, 4 m3 of pantodenic calcium lx, 207 t2 of confectic acid, 20 m8 of inositol, 4 mz of nicotinic acid, 4 B of pyridoxine salt, 4+B of thiamine jh acid, and FAIL 2”S of para-aminobenzoin. It contains riboflavin 2B.

The culture medium to be used is preferably one that has methanol as its main carbon source as mentioned above, but in the case of bacteria that can assimilate carbohydrates other than methanol, such as glucose, Any available carbon source can also be used. When using methanol as an carbonation source, the methanol concentration in the culture method is preferably 6 weight I or less, and from the viewpoint of good bacterial growth, preferably 2 weight MZ or less. As a nitrogen source, for example, ammonium ,salt,
Nitrogen-free compounds such as nitrates or organic nitrogen-containing substances such as urine, corn stew liquor, casein, peptone, etc. may be used. Other inorganic salt compounds include, for example, calcilano, salt, magnesium salt, potash J1 salt, natriuno, salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt, boron compound, iodine Compounds etc. are used.

The culture conditions are, for example, a temperature of about 20 to 40°C, preferably about 25 to 37°C, and p)I of about 5 to 9, preferably about 6 to 8. Culture is carried out aerobically under these conditions. Further, there is no particular restriction on the concentration of dissolved oxygen in the culture solution, but it is usually preferably about 0.5 to 20 ppm. For this purpose, measures such as adjusting the amount of ventilation, stirring, and increasing the pressure inside the culture tank are adopted. Furthermore, the culture method may be either batch culture or continuous VC culture.

Bacterial cells are separated from the culture medium B obtained by culturing in this manner, for example, by a conventional bacterial cell separation means such as a centrifuge and filtration. The isolated wet bacterial cells are stored at a low temperature, for example, 4''CN degrees, until they are used to prepare dry Qt, PS by a method such as freeze drying or spray drying.

■ Extraction and purification of LPS In the present invention, to extract LPS from the facepiece, a normal method for extracting LPS from four bacteria can be applied.
For example, the water-phenol method and the trichloroacetic acid method are preferred. As a water-phenol method, for example, Westp
hal et al [Z, Naturforsch7b
, +48 (1952)].

After stirring, it is cooled to room temperature, centrifuged, the upper layer of water is collected, dialyzed, and freeze-dried to obtain crude L F.
' Get S.

Further, as the trichloroacetic acid method, there is, for example, the method described in "Bacterial Endotoxin Murasaki", edited by Tada Kima et al., published by Kodansha. That is, the bacterial cells were suspended in water and then cooled to 4°C.
Add α and shake to extract. After centrifugation, add cold ethanol to the upper layer, collect the resulting precipitate, dialyze the aqueous solution dissolved in water, and freeze-dry.

The crude LPS obtained by these methods can be processed by fermentation method using ribonuclease, gel [one-pass method,
Alternatively, it is further purified by Ifi centrifugation method or the like.

(2)1. , Physical properties of F'S Representative low toxicity LPS producing bacterium MG1023 of the present invention
ps obtained from the strain as described above has the following physical properties.

For reference, E. coli 0127881. P.S.
(manufactured by Difco) is also shown.

■Chemical composition Total vJ amount (measured by phenol-sulfuric acid method), protein amount (1
,, measured by o wry method), amount of hexosamine (
Measured by Erson-Morgan method)
, total phosphorus content (measured by Chen method) and lipid content (L
Table 1 shows r'S and 6M1 (measured by hydrolyzing C1 at 100° C. for 2/1 hour, extracting with chloroform 1, weighing α), respectively.

Table - Chemical composition of ILPS ■ Constituent monosaccharide LPS was hydrolyzed with sulfuric acid to release neutral sugars, which were then subjected to thin layer chromatography using an Avicel cellulose plate (the intermediary solvent was 90% phenol/1% aqueous ammonia =
The tunic sugars detected in 4/1) include rhamnose, ribose, glucose, galactose, and other R
The sugar f=0.82 and Rf"0.64.

However, the structure of the latter two was unknown.

Furthermore, Table 2 shows the content of the eastern saccharides determined by high performance liquid chromatography.

Table 2 Constituent sugars and content of LPS ■ 2-keto 3-deoxyoctonate (hereinafter referred to as rKI
)O") KDO has been detected in Escherichia coli LPS' and the like as a specific sugar that participates in the binding of polysaccharides constituting LPS and libido Δ. KDO can be analyzed qualitatively and quantitatively by the periodic acid-thiobarbituric acid method, and the coloring solution is 5
It has maximum absorption (λ□,) at 50°. However, the maximum absorption wavelength of the low toxicity LPS coloring liquid of the present invention is 535 nm.
m, and low toxicity LPS does not contain Kl)O in its molecule.

■Nari Yoko, fatty acid I, PS as a 3% HCl-methanol solution, 1
Table 3 shows the fatty acids detected by gas chromatography after heating at 05°C for 24 hours to form methyl esters.

(Unit is percentage of dry weight of LPS) Table 3L
I) Constituent fatty acids of S ■ Molecular weight The molecular weights determined by the gel filtration method are two types: one of 300,000 Daltons and one larger than 300,000 Daltons.

(3) The toxicity of LPS1, the toxicity of PS using horseshoe crab blood cell components1
One Last G Experience (Bull, Johns l1 opkins
1losp, 98325 (1956) Levin,
J Ban3. It can be determined by acute toxicity tests using F, [+, ) or mice.

In the Limulus test, LPS solutions of various concentrations were added to a vial containing horseshoe crab blood cell components and allowed to stand at 37°C for 1 hour.
This is a test method in which the vial is then slowly inverted 180 degrees to observe the collapse of the gel, and the toxicity is determined at an LPS concentration of 1 degree, at which the gel hardens and does not collapse even if it is tipped over. The solidification strength of this gel is proportional to the toxicity of PS, and the more toxic, the smaller the amount of LPS
The gel will solidify.

While E. coli LPS solidifies as a gel at an extremely low concentration of 10 to 5 μg/ml, the LPS of the present invention has the lowest concentration at which it solidifies at ]0-2μ8/m, which is the least toxic. did not solidify even at 10-'μ87m1, and
It is an LPS that is more than 100 times less toxic than J bacteria 1°PS.

In addition, the toxicity can also be determined by an acute toxicity test using mice, but this method can also be used to
The toxicity of S is 1150 to I of the toxicity of E. coli 1-PS.
/120 or less, and its toxicity is extremely low.

(/1) Method for inducing interferon The low toxicity r, ps of the present invention can be used as an oral or parenteral drug at any concentration through intensive formulation.

That is, for example, in order to induce interferon using the low toxicity LPS preparation of the present invention, the amount of LPS should be 0.
03-30mg/kg body weight, preferably 0.03-3
It is administered orally or parenterally, preferably intravenously, at a rate of mg/kg body weight.

(5) Method for inducing TNF In order to induce TNF using the low toxicity LPSfiA agent of the present invention, Corynebacterium barbum 1 to 200 m
g/kg body weight or zymosan 1-100mg/
l (8 body weight administered orally or parenterally, 1
After 0 to 14 days, the r-ps formulation of the present invention is
Orally or non-1Y at a rate of 0.03-301T18/kg1 preferably 0.3-3B/8 as ps nails,
Administer orally, preferably intravenously.

(6) Tumor suppression method When the low toxicity LPS preparation of the present invention is used as an antitumor agent in combination with an immunostimulant, for example, when lentinan is used as the immunostimulant, lentinan is subcutaneously administered. Injection: 0.01-30mg/kg per day
:l< means 0.05~10m3/kg body weight. −
On the other hand, the low toxicity 1, PS of the present invention is 0.0% per day by subcutaneous injection.
0001~Imp/kg body weight preferably 0.001~O
，! +n3/1 Famous Weight Throwing Example [Example] The present invention will be further explained by the following example.

Example 1 Preparation of T, PS from MG-1023 strain 1'seudomonas sp, NclB9686 (
MG-1023) in the above culture + 1 t s h h, per 11 culture fluids (N) 14) 2s0438, K1121'
041.43. Na2PO42,1g-MeSO4
”7112(] 0.28, (:aCI2・2t12Q
30m3, F, C611507・XlI20 30
m3, M, Cl2-/111205m2B, 7. ．． 5
O4-7H205mg, C,5O4-511, (10,
5B, 1ml of vitamin mixture tα, and methanol rag were cultured in a medium containing 1ml of methanol rag, and approximately 1803 dry-light bacterial cells were collected from 201 of the welding solution. )Ta. 100g of this dried bacterial cell at 65-75℃
The mixture was dispersed twice in 3,500 ml of warm water and kept at the same temperature.

An equal amount of 90% phenol water-soluble 7α heated to 65 to 75° C. was added to this cell suspension and maintained at the same temperature. This mixture was shaken vigorously for 10 minutes. After shaking, transfer the mixture to a stainless steel container and heat at 65-75°C.
It was heated again. This solution was then allowed to rise again for 10 minutes. Repeat this operation three times for 10 minutes each,
The protein bound to r-ps was sufficiently denatured and released, and at the same time, the target LPS was eluted into the mixture. The mixed solution that has been shaken and stirred is cooled to 4°C, then centrifuged (80QOXg, 20 minutes) to remove ice fields and ice.
It was separated into three layers: a layer and a phenol layer. The upper aqueous layer containing r and ps was gently distilled. Boil the remaining liquid again at 65~
The mixture was heated to 75°C, an equal amount of water at 65 to 75°C was added, and the above extraction operation was repeated, followed by centrifugation, and the aqueous layer was collected.

The aqueous layers collected at the first and second times were mixed, transferred to a dialysis machine, and dialyzed against running water for 2 days and nights. The obtained dialysis fluid was
It was concentrated to about 200 ml under reduced pressure at 40°C.

Since the concentrated solution contained nucleic acids as impurities as it was, it was treated to remove nucleic acids using an enzymatic method (ribonuclease). That is, a predetermined amount of the above-mentioned concentrated solution is added with diluted tris-hydrochloric acid. + I solution p] (8 was added to adjust the moisture level to 0.051'l. To this, Ribonuc 1 Noase Δ (mixed with SIG) dissolved at a ratio of 1 part in 1 ml of the above-mentioned mild liquid was added (after concentration). (in the case of 200 ml, approximately 40 ml) was added to make the reaction solution, and this was
Incubated for 2-33 hours at <RTIgt;C. Next, this reaction solution was heated to 65 to 75°C, and an equal amount of a 90% phenol aqueous solution heated to the same temperature was added to the reaction solution, followed by shaking. This operation stopped the enzymatic reaction and at the same time denatured the fermented protein and transferred it to the phenol layer. After cooling the shaking liquid to 4°C, it was centrifuged to collect the aqueous layer.

After dialysis of this aqueous layer to remove phenol and low-molecular nucleic acids, the dialyzed solution was frozen and dried for 1 ri to yield approximately 4 g of purified L.
Got PS.

Actual Ft Example 2 Preparation of LPS of h+c-+o3q strain '1ethylomonas met, hanolic
a NRRI, B-5458 (MG-1039) was cultured in the same manner as in Example to obtain a dried facepiece. Using 8 g of this dried bacterial cell, crude LPS1.6 was prepared in the same manner as in Example 1.
I got g. This crude solution was made into a 1-3% aqueous solution and centrifuged at 1,105,000x for 20 hours to obtain a pale yellow agar-like precipitate. This precipitate was freeze-dried and purified 1. PS+, oi3 was obtained.

Example 3 Reno-Rath test of LPS Physiological saline solutions of LPS obtained from various specimens were prepared and sequentially diluted 10-fold to obtain 0-1 to 3
A solution of O-5z/ml was prepared. Add 0.2 ml of each of these to the vial containing horseshoe crab blood cell components of the Limulus kit (manufactured by Wako Pure Chemical Industries, Ltd.), let stand at 37°C for 1 hour, and then gently invert the vial on a flat plate to allow gelation. The presence or absence was observed.

The results are shown in Table-4.

While E. coli LPS caused gelation at a concentration of 10-5 μg/ml, the gelation concentration of the LPS of the present invention was 10-10-2 μg/ml or higher.

Furthermore, the LPS of the present invention does not gel at all.
There is also S, and its toxicity is higher than that of E. coli LPS.
It was less than 1000.

Table-/I 1. PS limulus test (It becomes ten-nigel.

Example/l Acute toxicity test of LPS An acute toxicity test was conducted using PS as a control, and its LD5. The strength of toxicity was compared based on the values. That is, L
Dissolve PS in physiological saline to prepare SPF at 6-7 weeks of age.
ddy was administered intraperitoneally and into the solid vein of male mice (- group 4 to 8 mice), and LD was determined based on the course of death after administration.
I found the value. These results are shown in Table-5 and Table-6.

From Table-5, large fli [LPs are LD 5o14-31
However, with the LPS of the present invention, no fatal cases were observed even at the highest ly level tested here, 1690 mz/3, and the Lr) So value was 2 or more at 1690 mg/2, and E. coli Compared to the toxicity of LPS, at most 11
The toxicity was extremely low, ranging from 50 times to 1/120 times.

Table 51, I's 2, Sexual toxicity test (intraperitoneal injection) Also, from Table 6, the LD50 value of E. coli LPS is 7.9
~15.7mg/kg, whereas 嘱-1039
With LPS, no deaths were observed even at the highest dose tested, 62.9 mg/J. , Dl, value is 62.9
m8/J or more, which is more than 10 times the value of LPS of 115 bacteria. Considering the results of the Limulus test as well, LPS of MG-1039 is substantially non-toxic.

Table-61, Acute maternal test of Ps (tail vein stage) Example 5
Induction of interferon Interferon was induced using L1) S obtained by the water-phenol method from various low-toxic PS-producing bacteria of the present invention. In other words, ■Mouse (d
d y, male, 7 weeks old, weight: 30 ~; U58) From the tail vein, IO μg and 1001 LPS were obtained, respectively.
0.1 ml of L I) S dissolved in physiological saline was administered to each mouse, and 2 hours later, 0.1 ml of L I) S was administered from the heart.
3 - 0.7ml of blood was collected, left at 37℃ for 30 minutes, centrifuged at 3000 rpm for 10 minutes, and the supernatant was taken.
Interferon activity was measured.

■Interferon activity was measured at 50% using L-929 cells and bovine vesicular stomatitis virus (hereinafter referred to as ■S■).
Measured by plaque reduction method (enhanced version of interferon p
l+ Shigeyasu Kobayashi, Kodansha Scientific, Modern Medicine 29 p6601974 Masahiko Iizuka).

That is, 2xl O'cel in 966 microplates.
-929 cells were inoculated at a volume of 110.1 ml/well and cultured at -8% to form a Molyer layer. Then,
Remove the culture medium in the wells and add fetal bovine serum (FCS) 1
% Minimum Essential Medium (MEM Medium)
After washing with
well) and cultured for 2 hours in a carbon dioxide gas incubator at 37°C.

After 2 hours, unadsorbed virus solution was removed using an aspirator, and 100 μm of 0.5% methylcellulose solution was added.
It was cultured for 2 days at 37°C in a carbon dioxide gas culture device. VS■ Infection Two days after infection, the cells were removed using a methyl cellulose filter, and 1% crystal violet was added to each well to fix and stain the cells. After 2 hours, this was washed with water, air-dried, and the number of plaques was counted using a stereomicroscope. Taking the dilution factor of the serum on the horizontal axis and the number of plaques on the vertical axis, taking the control plaque number as 100, find the dilution factor of the serum that will give a 50% plaque number, and prepare it in advance in the next step. The interferon titer was calculated from the standard calibration curve.

■The standard calibration curve is an interferon sample (International m rank IU)
Interferon dilution standards with three concentrations were prepared by appropriately diluting the interferon (with activity indicated), and the dilution factor at which each diluted product would increase 50% plaque formation was determined. A standard calibration curve was created by plotting the dilution factor giving 50% plaque formation on the vertical axis and the interferon titer on the horizontal axis, and plotting the dilution factor giving 50% plaque formation inhibition given each interferon titer.

Table 7 shows the interferon-inducing activity of LPS obtained from each bacteria. In the projection area of IOttg/mouse, it showed an activity equal to or higher than that of the control E. coli LPS. Since the LPS of the present invention has low toxicity, it was possible to increase the tail throw. That is, 1. from E. coli. Regarding the administration of PS,]0713/mice showed interferon-inducing activity, but l007L5/mice showed toxicity symptoms such as piloerection, sedation, ptosis, and staggering gait, so measurements were not carried out. I didn't do it. On the other hand, for the L P S throw of the present invention, lO
A high interferon-inducing activity was shown at μg/mouse, but even at 100 μg/mouse, no toxic symptoms such as weakness were observed, and interferon could still be induced. Note that 1 of the present invention
゜For PS injection, the interferon inducing activity was 101. 11 for t3/mouse! 10 μg of LPS from IQM was inferior to that of I-PS from M, but increased in the case of 100 μg/mouse.
/ In some cases, it was nearly twice as high as in the case of mice.

Table 7 Interferon-inducing activity Example 6 Induction of TNF TNF' was induced using LPS obtained in the present invention.

Corynebacterium vulva, III+8 was intraperitoneally administered to 5-week-old ddY female mice (body hair 25-308).
After 10 days of rearing with 2'y- 10 ) t B/mouse, 100 μs/mouse and 1 as LPS, respectively.
0.1 ml of a physiological saline solution of PS was administered through the tail vein at a concentration of 0.000 μg/mouse. Throw ty-
After 2 hours, 0.3 to 0.7 ml of blood was collected from the heart, left at 37°C for 30 minutes, centrifuged at 3000 rpm for 30 minutes, the supernatant was collected, and the TNF activity of the supernatant was determined as follows. It was measured.

That is, 8 x 104 cells in a 96-well microplate.
L-929 cells were inoculated at f10.1 ml/well and left to stand for 3 hours. Then, 50μ of diluted serum
m was added and cultured overnight. After culturing, the cells were stained with 1% crystal violet (ethanol solution), dried at room temperature, and then stained with 0.5% sodium dodecyl sulfate solution at 100 μm/min.
Add to the well and collect after 3 hours of elution, and measure the absorbance of the collected solution as λ=
Measured at 590r+m.

After culturing L-929 cells without adding serum, the cells were treated in the same manner as described above, and the dilution ratio of the serum that gave an absorbance of 1/2 of the absorbance of the control was determined as the TNF-inducing activity.

Table 8 shows the TNF-inducing activity of each 1=PS. As a control, the LPS-induced activity of E. coli 012788 is also shown.

Table 8 LPS showed high TNF-inducing activity, but 100
)i3/mouse and 1ooOB3/mouse showed obvious toxicity symptoms such as piloerection, ptosis, sedation, and staggering gait as described above, so measurements were not performed. On the other hand, for the administration of LPS from E. coli of the present invention, 10 μgl was used] 00
Since the dose of 0 μg/mouse is not suitable for practical use, we measured the activity of MG-1284 on a wipe, but even with such a high dose, there were no cases. No symptoms observed, still 1'N
F can be induced.

Example 7 Anticancer effect The anticancer activity of LPS obtained in Example 1 was investigated in the case of LPS alone and when used in combination with lentinan. For comparison, E. coli 0127 f181. ,
A similar test was conducted for PS.

That is, 3XI06 Ehrlich cancer cells were transplanted into the right inguinal region of a 6-week-old ddY male mouse, and after raising it for 12 days,... , PS was dissolved in physiological saline and administered intraperitoneally. 1 on the 238th day after administration! ! Describe the situation,
The extent was compared with the control group, and the tumor i11 correct rate was calculated and the number of complete disappearance of tumors was estimated at 1:1. These results are shown in Table-9 and Table-10, respectively.

MG-1023 (7) L P S The tumor inhibition rate when administered with I52 injected with lentinan and Escherichia coli r,
, ps, but MG-1023 glue,
When PS is used in combination with lentinan, it is a big problem! Bacterium 1 showed a remarkable tumor inhibiting effect similar to the case when PS and lentinan were used together. This time, we compared the effects of two administrations, but if we consider it from a practical perspective, it is common to use multiple administrations, and in such cases, 1. ])S is advantageous over E. coli Ll)S in that it is non-reactive and can be used repeatedly.

Table-9 Effect of combined use of lentinan and colonic mLPs” Standard deviation specimen 1 Physiological saline 2. Lentinan 6.25mg/ky.

3 Large a = Bacteria r, PS 0.5m8/kg4 Lentinan 6.25mg/8 + Escherichia coli T, PS5
〃 + 〃 6 〃 + 〃 7 tt--4-tto, 5mg/kg O805...z/3 0.005m3/5 0.0005+n (: Table 10 Effects of combined use of lentinan and MG-+023 LPS [Effects of the Invention] 1. PS of the present invention is superior to conventional 1 in that it induces interferon and TNF at low doses, and also exhibits effects against cancer when used in combination with immunostimulants. Although it is the same as PS, conventional LPS has extremely low toxicity.
This is significantly different from the previous method, and makes it possible to use it in actual clinical settings.

9 Standard deviation; 0 1 animal died during the test Specimen 1 Physiological saline 2 Lentinan 6.25 mg/kz 8MG-1023LPS 1. Omz/ni39 Lentinan 6.25n+3/kg+ MG-10231, PS
310 // to 1.0m3/
+ tt O, Img/kg
11 // +t
t(), 01m3ha<8.

Claims (3)

[Claims] (1) Ribopolysaccharide derived from bacteria that does not contain 2-keto-3-deoxyoctonate and has low toxicity to mammals (2) An interferon inducer that does not contain 2-keto-3-deoxyoctonate and contains ribopolysaccharide derived from bacteria, which has low toxicity to mammals, as an active ingredient. (3) A tumor necrosis factor inducer (4) characterized in that it does not contain 2-keto-3-deoxyoctonate and contains ribopolysaccharide derived from bacteria, which has low toxicity to mammals, as an active ingredient. An antitumor agent that does not contain 2-keto-3-deoxyoctonate and contains ribopolysaccharide derived from bacteria that has low toxicity to mammals as an active ingredient.