JPH0699314B2 - Macrophage activator - Google Patents

Description

TECHNICAL FIELD The present invention relates to a macrophage activator.

Conventional technology Patients with serious underlying diseases such as cancer and severe burns and elderly patients are hospitalized with attenuated bacteria (so-called opportunistic infection).
Is likely to occur, and the establishment of a protective measure is desired. Conventionally, treatment of opportunistic infections is mainly carried out with antibiotics, but as is well known, the use of antibiotics has a problem that the bacteria acquire resistance. Therefore, it is desirable to improve the infection defense ability of the living body without relying only on antibiotics, and effective means for that purpose are being searched for.

Lactobacillus cells are one of the substances that have been confirmed to have an effect of improving biological defense ability in experiments using mice. The action is
Cells capable of processing a large number of foreign substances (senescent red blood cells, bacteria, viruses, fungi, protozoa, etc.)-Macrophages-
To improve the defense against infection, etc.
It is believed to produce favorable results. However, it is not known what component of Lactobacillus is involved in macrophage activation. Therefore, there is no choice but to use the bacterium itself that intends to utilize lactobacillus for macrophage activation, and to use it in a dosage form that is desired to be water-soluble, such as an injectable drug, that does not contain substances unrelated to drug efficacy as much as possible. Of course, I could not do it, but I could not expect a sufficient effect even if I took it internally.

Problems to be Solved by the Invention The object of the present invention is to solve the above-mentioned problems in the case of utilizing lactobacillus for the activation of macrophages. It is to find a substance as close as possible and to provide a macrophage activator using it.

Means for Solving the Problems As a result of extensive research on a treated product of lactobacillus, the above-mentioned macrophage activating action exhibited by lactobacillus, for example, Lactobacillus casei, is due to the fact that the lactobacillus exhibiting the activating action is treated with N-acetyl lamidodes. It was found to be found in the polysaccharide-peptidoglycan complex obtained by treating with.

The present invention is based on the above new findings, L-alanine,
Macrophage activation using a polysaccharide-peptidoglycan complex derived from lactobacillus cell wall, which has a peptide chain containing D-glutamine and L-lysine as essential constituent amino acids and a sugar chain containing glucose and rhamnose as essential constituent sugars The agent is provided.

At the surface of the cell wall of lactobacillus, there is a layer mainly consisting of peptidoglycan, and on the outside thereof, teichoic acid and polysaccharides are present. 4 or 5 peptidoglycan
The basic unit is a disaccharide unit linked to N-acetylmuramic acid and N-acetylglucosamine, which is substituted with a peptide chain consisting of individual amino acids.This is a macromolecule that is crosslinked by other amino acids and peptides. In the cell wall, they are bound to the above-mentioned teichoic acid and polysaccharide via phosphate. When Lactobacillus is treated with N-acetyl ramidose, the β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine is cleaved, leaving the bond between the peptidoglycan and the polysaccharide as described above. The basic unit and those in which the basic units are linked to each other at the peptide chain portion are released.
The representative ones are shown below.

However, the abbreviations in the formulas mean the following units, respectively.

L-Ala: L-alanine D-Glu: D glutamine L-Lys: L-lysine D-Asp: D-asparagine The “polysaccharide” in R above is glucose and rhamnose as essential constituent sugars, and galactose It may be included as a constituent sugar.

The mixture of these complex compounds is the polysaccharide-peptidoglycan complex that constitutes the macrophage activator of the present invention.

The sugar composition and amino acid sequence in the polysaccharide-peptidoglycan complex differ slightly depending on the type of the starting lactobacillus, but all of the following lactobacilli are of the above sugar composition and amino acid sequence, and the macrophage of the present invention Preferred as a polysaccharide-peptidoglycan complex that constitutes the activator. L. casei, L. buchneri, L. jugurt
i), L.sake, L.lactis, L.
L. coryneformis, L. acidophilus, L. pastorian
us), L. curvatus, L. salivarius, L. xylosus, L. del-brueckii, L. reichmanni.
leichmannii), L. bulgalicus, L.
L. helveticus, L. brevis
is). (However, L. is an abbreviation for Lactobacillus. The same applies in the following sentences).

The details of the method for producing a polysaccharide-peptidoglycan complex, which is an active ingredient of the macrophage activator of the present invention, from lactobacilli are as follows.

The lactic acid bacillus used as a raw material may be one that has been cultured by an ordinary method using an appropriate medium that can be used for culturing the lactic acid bacterium, and does not need to be a special culture method. After culturing, the cells are washed by a conventional method, preferably heated to make dead cells, suspended in water, and treated with N-acetylmuramidice. pH
When the cells are treated at about 5 to 6 and a temperature of about 37 to 50 ° C for about 12 to 24 hours, the cell wall is dissolved. After centrifuging the reaction mixture and treating the supernatant with an enzyme again, the nucleic acid is decomposed with a nucleolytic enzyme,
Further trypsin is added to decompose the protein. Then, the product is dialyzed against distilled water and the dialyzed solution is freeze-dried to obtain the desired polysaccharide-peptidoglycan complex.

The polysaccharide-peptidoglycan complex obtained as described above may be purified as it is or may be further purified, if necessary.
It can be a constituent of the macrophage activator of the present invention. The polysaccharide-peptidoglycan complex is a water-soluble and highly stable substance. That is, the activity of freeze-dried powder does not decrease for more than a year when stored at room temperature, and the activity of physiological saline solution does not decrease for more than 6 months when stored frozen at -20 ° C, and freeze-thaw does not occur. Activity is not diminished even after repeating three times. Physiological saline solution does not lose its activity even when heated at 100 ° C for 10 minutes.
Therefore, when a macrophage activator is produced from a polysaccharide-peptidoglycan complex, it can be formulated into an injection, tablet, powder or the like by any method and used in the form of intravenous injection or oral administration.

The standard dose of the macrophage activator according to the present invention is about 0.8-80 mg as polysaccharide-peptidoglycan complex per kg body weight.

The toxicity of the polysaccharide-peptidoglycan complex is shown by the following LD ₅₀ value (value for 7-week-old BALB / c male mice weighing about 25 g).
As you can see, it is not recognized at all.

Oral administration 2000 mg / kg or more Intravenous administration 800 mg / kg or more Intraperitoneal administration 800 mg / kg or more Advantageous effects of the invention The macrophage activator of the present invention is nontoxic, There is no problem of obtaining resistance,
Effective against a wide range of infectious diseases because it is effective against both Gram-positive and Gram-negative bacteria, and is expected to be effective against diseases other than infectious diseases because it activates macrophages. Therefore, it has many features such as easy preparation of injections and other arbitrary dosage forms.

EXAMPLES Hereinafter, the present invention will be described with reference to examples. Example 2
In each of the following examples, the bacterial infection protective effect of the polysaccharide-peptidoglycan complex was confirmed by the following method.

Test method: Seven-week-old BALB / c mice were used as test animals, one group consisting of 5 mice. These mice were inoculated intraperitoneally with 10 ⁶ Listeria monocytogenes EDG strains or Salmonella tybhimurium LT-2 strains, and the number of viable bacteria inoculated intraperitoneally at 24 hours was measured, or Mice inoculated intraperitoneally with an amount of fungus are observed until 14 days after inoculation. In this case, the experimental group was intraperitoneally inoculated with a physiological saline solution of a polysaccharide-peptidoglycan complex 3 to 10 days before inoculation of the infectious bacteria. The control group does not receive this pretreatment. The protective effect against infection was determined by the survival rate of infected mice or the growth inhibition rate of infected bacteria (according to the following formula.

Growth inhibition rate (%) = 100 × (1-X / Y) where X: average value of viable cell count in the experimental group Y: average value of viable cell count in the untreated control group Production Example 1 L. casei YIT9018 (Microtechnology Research Institute, No. 665) was inoculated into Rogosa's medium and cultured at 37 ° C. for 2 hours, then the cells were collected by centrifugation and washed 4 times with distilled water. 10 washed cells
After heating at 0 ° C. for 20 minutes, it was freeze-dried.

1 g of the bacterial powder obtained above was suspended in 200 ml of 5 mM Tris-malate buffer (pH 5.8, containing ₂ mM MgCl ₂ ) to give 10
N-acetylmuramidice SG (Seikagaku Kogyo product) (mg) was added, and the mixture was reacted at 37 ° C for 16 hours. Then, add 50 to the reaction mixture.
The precipitate containing the cytoplasmic portion was removed by centrifugation at 00 × g for 20 minutes, and the supernatant was further reacted for 18 hours. Then 10 in the reaction mixture
DNase of 10 mg and RNase of 10 mg (both are products of Sigma) are added, and the mixed nucleic acid is decomposed by reacting at 37 ° C for 12 hours, and then 10 mg of trypsin (product of Sigma) is added and the mixture is incubated at 37 ° C for 16 hours. By reacting, the mixed proteins were decomposed. The obtained reaction mixture was dialyzed against a large amount of distilled water at 4 ° C., and then the dialyzed solution was freeze-dried to obtain about 400 mg of a polysaccharide-peptidoglycan complex.
The infrared absorption spectrum is shown in FIG.

Production Example 2 A polysaccharide-peptidoglycan complex was produced in the same manner as in Production Example 1 except that L. eugleti YIT0085 strain was used as the lactobacillus.

Production Example 3 The polysaccharide-peptidoglycan complex produced in Production Example 1 was fractionated by gel filtration using Sephacryl S-300 (Pharmacia). As a result of investigating the eluate with a 50 mM (NH ₄ ) ₂ CO ₃ solution, the polysaccharide-peptidoglycan complex has a molecular weight of about 100,000 or more (hereinafter PS-PG ・ A) and a molecular weight of about 40,000 or less ( (Hereinafter referred to as PS-PG / B)).

Further, the polysaccharide-peptidoglycan complex according to Production Example 2 was fractionated in the same manner as above by gel filtration using Sephacryl S-200 (Pharmacia) to obtain a fraction having a molecular weight of about 50,000 (hereinafter PS- PG ・ C) and the molecular weight is about 2.5
Ten thousand fractions (hereinafter referred to as PS-PG / D) were obtained.

Each of the above fractions had a sugar composition as shown in Table 1.

Example 1 The polysaccharide-peptidoglycan complex according to Production Example 1 was dissolved in sterile physiological saline to a concentration of 5 mg / ml, and 0.1 ml of the resulting solution was added to a 7-week-old mouse (BALB / c mouse, body weight). About 25 g, 4 animals per group) was intraperitoneally injected. 1-10 after administration
The mice in each group were sequentially bled and sacrificed during the day, and the abdominal cavity was immediately washed by injecting 5 ml of Hanks buffer (pH 7.0) into the abdominal cavity. Next, the intraperitoneal lavage fluid was taken out with a syringe, and the number of white blood cells contained therein was measured with a hemocytometer. On the other hand, peritoneal exudate cells contained in the peritoneal lavage fluid were smeared on a slide glass, dried, fixed with methanol, stained with Giemsa solution and then observed under a microscope to determine the macrophage content in all cells. .

The number of macrophages (average value) obtained by multiplying the number of white blood cells measured by the above method by the macrophage content rate
Was as shown in Table 1. The number of macrophages similarly measured in the untreated control group was 6.35 × 10 ⁵ , and it was confirmed that administration of the polysaccharide-peptidoglycan complex, that is, the macrophage activator of the present invention markedly increased the number of macrophages.

Example 2 With respect to the polysaccharide-peptidoglycan complex according to Production Example 1, the protective effect against Listeria monocytogenes infection was examined. The dose of the polysaccharide-peptidoglycan complex to the experimental group mice was 20 mg / kg.

As a result, all the mice in the untreated control group died by the Listeria monocytogenes infection by the 4th day, whereas all the mice in the experimental group survived by the 14th day. In addition, the intraperitoneal bacterial growth 24 hours after the bacterial inoculation was also significantly suppressed as shown in Table 3.

Example 3 With regard to the polysaccharide-peptidoglycan complex according to Production Example 1, the protective effect against Salmonella typhimurium infection was examined. Infection 5
When 20 mg / kg of the polysaccharide-peptidoglycan complex was administered the day before, the growth inhibition rate of the bacteria was 96.3%.

Example 4 With regard to the polysaccharide-peptidoglycan complex according to Production Example 1, the protective effect against Listeria monocytogenes infection was examined. In this case, the polysaccharide-peptidoglycan complex was administered at 0.8 to 80 mg / kg 5 days before infection. The results are shown in Table 4.

Example 5 Polysaccharide-peptidoglycan complex according to Production Examples 1 and 2, and L. casei YI in the same manner as in Production Example 1.
T0123 strain, L. casei YIT0105 strain, L. salivarius YIT0104
, L. helveticus YIT0083 strain, etc.
The peptidoglycan complex was examined for its protective effect against Listeria monocytogenes. However, the polysaccharide-peptidoglycan complex was administered at 20 mg / kg 5 days before bacterial infection. The results are shown in Table 5.

Example 6 With respect to the polysaccharide-peptidoglycan complex fractions according to Production Example 3, that is, PS-PG · A to D, the protective effect against infection with Listeria monocytogenes was examined. However, the polysaccharide-peptidoglycan complex was administered at 20 mg / kg 5 days before bacterial infection.

The results are shown in Table 6.

[Brief description of drawings]

FIG. 1 is an infrared spectrum diagram of a polysaccharide-peptidoglycan complex according to Production Example 1.

 ─────────────────────────────────────────────────── ─── Continuation of front page (56) References Infection and Immunity, vol. 24, No. 2, PP 308-312 (1979) Infection and Immunity, vol, 41, No. 2, PP 462-469 (1983)

Claims (1)

[Claims] 1. Lactobacillus cell wall-derived polysaccharide-peptidoglycan complex having a peptide chain containing L-alanine, D-glutamine and L-lysine as essential constituent amino acids and a sugar chain containing glucose and rhamnose as essential constituent sugars. A macrophage activator containing the body as an active ingredient.