JPS63196521A - Tumor cell disorder factor inducer - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Za! The present invention relates to a tumor cytotoxic factor inducer.

Conventional technology Neutral protease, complement, arginase, active enzyme group,
The l! l! Among tumor cytotoxic factors, the effects of TNF are known to closely match the spectrum of tumor cytotoxicity of macrophages;
It is considered to be the most important effector of macrophage antitumor activity.

TNF released from macrophages
In addition to -α, TNF-β released from lymphocytes
are known to cause necrosis of cancer when administered to tumor-bearing animals, and inhibit the proliferation or cause death of certain cancer cells in vitro. However, in order to cause macrophages to produce and release tumor cytotoxic factors such as TNF, Carsw
(1975), macrophages were first treated with BCG (Bacillus Cal).
Guerin), and then treated with an immunostimulant such as
It is necessary to administer an inducing agent such as endotoxin.

Other tumor cytotoxic factor inducers include Lipi
dA and its derivatives are known.

However, the substances known to have the above-mentioned toxic factor-inducing effect have strong toxicity and side effects, and it has been difficult to use them as drugs for inducing tumor cytotoxic factors.

Problems to be Solved by the Invention An object of the present invention is to provide a tumor cytotoxic factor inducer comprising a highly safe substance that is not toxic as shown by endotoxin or Lipid A.

Means for Solving the Problems In the course of extensive research on treated products of Lactobacillus, the present inventors discovered that a polysaccharide-peptidoglycan complex obtained by treating Lactobacillus with N-7 cetylmuramidase was excellent. I learned that it exhibits a tumor cytotoxic factor-inducing effect.

The present invention is based on the above-mentioned novel findings, and provides a tumor cytotoxic factor inducer containing a polysaccharide-peptidoglycan complex derived from the cell wall of Lactobacillus as an active ingredient.

The surface layer of the cell wall of Lactobacillus bacteria has a layer mainly composed of peptidoglycan, and further outside of this layer there are teichoic acids, polysaccharides, and the like. Peptidoglycan is 4 or 5
The basic unit is a trisaccharide unit in which N-7 cetylmuramic acid and N-7 cetylglufusamine, which are substituted with a peptide value of 7 amino acids, is linked, and this is a large It is a molecularized product, and in the cell wall, it is bound to the above-mentioned teichoic acid and polysaccharide via phosphoric acid. When treated with Lactobacillus N-7 cetylmuramidase, the β-1,4 bond between N-7 cetylmuramic acid and N-7 cetylglucosamine is cleaved, leaving the bond between peptidoglycan and polysaccharide intact. The above-mentioned basic unit and the basic units bonded to each other through the peptide chain portion are released. The typical ones are shown below.

R-L-A 1a-D-G 1u-L-Lys-D-A
laRL-Ala D-Glu-L-Lys-D-
Ala-Asp R-L-A 1a-D-G 1u-L-Lys-D-A
la-Asp R-L-A 1a-D-G l u-L-Lys-D-
A lm However, the abbreviations in the formula each mean the following units.

R: Polysaccharide HO-P=0 ■ H-C-CH.

■ C=0 L-Ala: L-alanine D-Glu: D-glutamine L-Lys: L-lysine D-Asp: D-7 Sparagine A mixture of these complex compounds induces the tumor cytotoxic factor of the present invention. It is a polysaccharide-peptidoglycan complex that constitutes the drug.

Although the sugar composition and amino acid sequence of the polysaccharide-peptidoglycan complex differs slightly depending on the type of raw material Lactobacillus, all of the materials obtained from the Lactobacillus described below have the above amino acid sequence, and the tumor cytotoxicity of the present invention It is preferred as a polysaccharide-peptidoglycan complex constituting the factor inducer.

L, casei (L, casei), L, buhineri (
L. buchneri), L. euglete 4 (L.
, jugurti), L, salmon (L, 5ake), L
, lactis (L, 1actis), L. coryneformis (L.

coryneformis), L, 7 Sidophilus (L
, acidophilus), L. pastori 7nus (
L. pastoriinus), L. carpathus (L.
curvatus), L, Saliparius (L, 5a
livarius), L.

xylosus, L. del-brueckii, L. leichmannii, L. bulgalicus, L. helveticus.

helvetius), L, plebis (L, bre
vis), (however, L is Lactobacillus LacLo
bacillus (same in the following sentences).

Details of the method for producing the polysaccharide/peptidoglycan complex, which is the active ingredient of the tumor cytotoxic factor inducer of the present invention, from Lactobacillus are as follows.

The lactic acid bacterium used as the raw material may be cultured by a conventional method using an appropriate medium that can be used for culturing lactic acid bacterium, and there is no need to use a special culture method. The cells are preferably heated to kill them, suspended in water, and treated with N-acetylmuramidase.

When treated at about 5-6 9H and a temperature of about 37-50°C for about 12-24 hours, the cell wall is dissolved. After centrifuging the reaction mixture and enzymatically treating the supernatant again, decompose the nucleic acids with a nuclease, and then add trypsin to decompose the proteins.Then, dialyze against distilled water, and freeze-dry the dialyzed solution. For example, the desired polysaccharide-peptidoglycan complex can be obtained.

The polysaccharide-peptidoglycan complex obtained as described above can be used as it is or if necessary, further purified.
Structure of the tumor cytotoxic factor inducer of the present invention! can be an ingredient.

Polysaccharide-peptidoglycan complexes are water-soluble and stable substances. In other words, the activity of the freeze-dried powder does not decrease for more than a year when stored at room temperature, and the activity of the lyophilized powder does not decrease for more than 6 months when stored frozen at -20°C, and the activity does not decrease after freezing and thawing. Physiological saline solution, which does not lose its activity even after repeating 3 times, has an additional 100
No activity decrease occurs even when heated at ℃ for 10 minutes.

Therefore, when producing a tumor cytotoxic factor inducer from a polysaccharide-peptidoglycan complex, it can be formulated into an injection, tablet, powder, etc. by any method and used for intravenous injection, oral administration, etc. can.

The standard dosage of the tumor cytotoxic factor inducing agent according to the present invention is about 0.8 to 80 infusions of polysaccharide-peptidoglycan complex per 8 parts of body weight.

The toxicity of the polysaccharide-peptidoglycan complex is determined by the following LD,
. As is clear from the values (values for BALB/c male mice of 7 weeks old and weighing 25 g), it is not observed at all.

For oral administration For intravenous administration of 2000 mg/kg or more For intraperitoneal administration of 800 mg/kg or more
800 mg/kg or more Effects of the Invention The tumor cytotoxic factor inducer of the present invention has no toxicity or side effects, and is water-soluble, so it can be easily formulated into injections or any other dosage form. *In addition, since it is obtained from Lactobacillus artefacts, raw materials are easy to obtain and manufacturing is also easy.

Taking advantage of these features, the tumor cytotoxic factor inducer of the present invention can be used in living organisms by various administration routes, and can induce effective damaging activity against tumor cells.

Furthermore, it can be widely used for many purposes, such as being used as an inducing agent in the mass production of tumor cytotoxic factors.

Below, the present invention will be explained with reference to Examples. In the Examples, the A cancer cytotoxic factor-inducing effect was confirmed by the following method.

Test method: 0.5a+g of killed Lactobacillus casei cells were administered intraperitoneally to 8- to 9-week-old BALB/c mice, and peritoneally exudated cells were collected 5 days later. Density gradient method (Gutierr)
eye et al.: J, Immuno, Methods
, 29, 57.

(1979) to obtain peritoneal macrophages with a purity of 99%.

This was suspended in RPMl-1640 medium supplemented with 10% calf fat serum (manufactured by Sigma) at a density of 2 x 10 cells/1, and the resulting suspension was poured into a 35-ring culture petri dish. Dispense. A specimen (dissolved in RPMI-1640 medium) is added thereto and cultured for 18 hours. after that,
The culture supernatant was centrifuged for 10 minutes, and further added with RPMI-164.
Dialyze for 48 hours against 0 medium.

Separately, RPMI-164 supplemented with 10% calf fat serum.
Suspend L929 cells in 0 medium (IXIO@/a+I
), the obtained suspension is dispensed into 96-well microplates in 10011 portions and cultured for 2 hours in 5% carbon dioxide gas.

Thereafter, a dialyzed solution of the dialyzed culture supernatant was diluted with RPMI-1640 medium supplemented with calf fetus and added to each microplate, followed by culturing at 37°C in 5% carbon dioxide gas for 72 hours. Next, the culture supernatant is discarded, and Hank's solution is added to each well for washing. After removing Hank's solution, 20
Add 0 μm ethanol to each well and leave it at room temperature for 5 minutes, then remove the ethanol. Then, add 10 μm to each well to stain the ethanol-fixed L929 cells.
% Giemsa solution, left at room temperature for 15 minutes, washed with tap water, and dried at 50°C. Then 10 in each well
A 50% ethanol solution containing 0 μl of 0.05N-HCI was added to extract the dye that was staining the cells.
The absorbance of and 49on- is measured, and the cytotoxicity of the tumor cytotoxic factor induced by the sample to L929 cells is calculated using the following formula.

Cytotoxicity (%) = difference in absorbance between 620 nm and 490 nm when sample is added and 490 nm Example 1 Lactobacillus casei YIT9018 (Huikokenjo) No. 665) was inoculated into Rogosa medium and incubated at 37℃ for 24 hours.
After culturing for an hour, the bacteria were collected by centrifugation and washed four times with distilled water.

The washed bacterial cells were heated to 100° C. for 20 minutes and then freeze-dried.

1 g of soybean powder obtained above was added to 200 ml of 5-M tris-malate buffer (pH 5, 8, 21 M MgCl
□), 10 tg of N-7 cetyl muramidase SG (Seikagaku Corporation) was added, and the mixture was reacted at 37°C for 16 hours. After that, the reaction mixture was heated with 5000XQ for 2 hours.
The precipitate containing the cytoplasmic part was removed by centrifugation for 0 minutes, and the supernatant was further reacted for 18 hours. Then, 10 mg was added to the reaction solution.
DNase and 10g of RNase (both products from Sigma) were added and reacted at 37°C for 12 hours to decompose the mixed nucleic acids, and then 10g of trypsin (product from Sigma) was added. The mixed proteins were decomposed by reacting at 37°C for 16 hours. The resulting reaction mixture was dialyzed against a large amount of distilled water at 4°C, and the dialyzed solution was then freeze-dried to obtain about 400 mg of a polysaccharide-peptidoglycan complex.

Its infrared absorption spectrum is shown in FIG.

As a result of investigating the tumor cytotoxic factor-inducing effect of the above polysaccharide-peptidoglycan complex, as shown in Table 1,
The polysaccharide-peptidoglycan complex induced tumor cytotoxic factor release from peritoneal macrophages at concentrations of 1011 g/+ml and above, and released the most tumor cytotoxic factors at a concentration of 5031 g/+*l.

Table 1 -4.7±4.1 100 83.6±0.6 50 93.1±0.3 10 70.6±0.9 Example 2 In the same manner as in Example 1, various Lactobacillus bacteria were killed. Polysaccharide-peptidoglycan complexes are produced from bacterial cells, and about them,
The tumor cytotoxic factor-inducing effect was investigated. Table 2 shows the results.
In the case of the control example shown in Figure 2, in which the polysaccharide peptidoglycan complex was not added, the cytotoxicity was 4.7±2.1.

L, casei YIT9018 67.7±
1. IL, casei YITO12372, 8±1.4L
, Kazei YITOO10575, 6±2.3L, Nigurti YITOO8573, 4±2. OL, Helveticus YITOO8381,2±1.7L, Salivarius YITO10476,0±2.5

[Brief explanation of the drawing]

FIG. 1 is an infrared spectrum diagram of the polysaccharide/peptidoglycan complex according to Example 1.

Claims (1)

[Claims] A tumor cytotoxic factor inducer containing a polysaccharide-peptidoglycan complex derived from the cell wall of Lactobacillus as an active ingredient.