JPH06219956A - Immunostimulant - Google Patents

Abstract

(57) [Summary] [Objective] To provide a novel immunostimulant having a cytokine-inducing ability. [Structure] An immunostimulant which can be obtained from a culture supernatant of Streptoverticillium hachijowens, exhibits a cytokine-inducing ability, and contains a substance having the following physicochemical properties as an active ingredient. a) Appearance: White to pale yellow powder b) Elemental analysis: C35.5-39.3%; H5.3-6.0%;
N 11.3 to 12.6%; O 21.2 to 26.8% c) Molecular weight of organic substance: 30,000 to 100,000 d) Red external absorption spectrum: Clear peaks are observed in the following ranges. 3300 ~ 3500, 1650 ~ 1660, 1538
~ 1540, 1227 ~ 1229, 1064 ~ 1065cm ^-1 e) Sugar content: 70 ~ 150μg / mg f) Protein content: 70 ~ 230μg / mg g) Sugar / protein content ratio: 0.9 ~ 1.2 (weight ratio)

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel immunostimulant, particularly to an immunostimulant which is expected to be effective as an antitumor agent, a therapeutic agent for immunodeficiency and the like.

[0002]

2. Description of the Related Art It has been conventionally known that various antitumor antibiotics are produced by microorganisms. Some of them have been put to practical use as anticancer agents. In particular, mitomycin C, bleomycin, adriamycin, neocarzinostatin and the like are known as anticancer agents derived from actinomycetes and are applied to therapy. However, many anti-tumor antibiotics cause myelosuppression, gastrointestinal disorders,
With side effects such as cardiac dysfunction and hair loss, it is a great burden for cancer patients. Further, in many cancer patients, the immunocompetence is lowered, the progression of cancer is promoted, the resistance to various bacteria is diminished, and serious infections are likely to occur. Against this background, the concept of quality of life has recently been considered as a treatment method for cancer from the clinical side, and in the field of anti-cancer agents as well, there are fewer side effects and the immunopotency of cancer patients has been improved. Increasingly trying to treat cancer is becoming more popular.

Streptoverticillium hachij which is one of the actinomycetes
The presence of a polyene antibiotic, tricomycin, which is effective against vaginal trichomoniasis, is known as an active ingredient derived from oense) (synonymous with Streptomyces hachijoensis). Further, one of the present inventors, Soeda, obtained an antitumor substance having a molecular weight of 1200 from Streptoverticillium hachijowens H-2609 strain (IFO 12782) (ATCC 19769) (Japanese Patent Publication No. 49-42560).
No. 14, Defense Hygiene Vol. 14, No. 1, Page 1, 1967, Japanese Examined Patent Publication No. 52-50278). Also, Tsukuni et al. Obtained an antitumor substance mainly composed of a protein having a molecular weight of 15,000 to 35,000 using the same bacterium (Japanese Patent Publication No. 56-9517). Kono obtained an antitumor agent mainly composed of a protein having a molecular weight of 20,000 to 60,000 using the same bacterium (JP-A-2-53729). In addition, substances that induce cytokines such as interleukins and tumor necrosis factors are useful for the treatment of cancer, immunodeficiency, infectious diseases, etc. (Japanese Patent Publication No. 4-69610, Japanese Patent Publication No. 4-5005,
JP-A-2-258723, JP-A-62-19533, etc.).

[0004]

In the field of cancer chemotherapy, various antitumor agents are required in response to various cancer types and symptoms. An object of the present invention is to provide an immunostimulant that is expected to be useful as an antitumor agent or a therapeutic agent for immunodeficiency.

[0005]

Means for Solving the Problems The present invention can be obtained from a culture supernatant of Streptoverticillium hachijowens and exhibits a cytokine-inducing ability and contains a substance having the following physicochemical properties as an active ingredient. Immunostimulant. a) Appearance: White to pale yellow powder b) Elemental analysis: C35.5 to 39.3%; H5.3 to 6.0%;
N 11.3 to 12.6%; O 21.2 to 26.8% c) Molecular weight of organic substance: 30,000 to 100,000 d) Red external absorption spectrum: Clear peaks are observed in the following ranges. 3300 ~ 3500, 1650 ~ 1660, 1538
~ 1540, 1227 ~ 1229, 1064 ~ 1065cm ^-1 e) Sugar content: 70 ~ 150μg / mg f) Protein content: 70 ~ 230μg / mg g) Sugar / protein content ratio: 0.9 ~ 1.2 (weight ratio) Above The components other than C, H, N, and O measured by elemental analysis in step S1 are inorganic substances, and the inorganic portion is Na, phosphate ion,
It is considered to be composed of ash and water. In the above, elemental analysis is thermal conductivity detection method (Heraeus CHN-0-Rapid is used), molecular weight is gel filtration method, infrared absorption spectrum is KB
r Tablet method, sugar content was phenol-sulfuric acid method, and protein content was Bio-Rad's Protein Assay Kid (same in Example 1).

The active ingredient of the immunostimulant of the present invention can be obtained from the culture supernatant of Streptoverticillium hachijowens. The mycological properties of Streptoverticillium hachijowens are "Trichomycin, a new antibiotic with anti-tricomonas and anti-cancer action".
(Hosoya et al., Journal of Antibiotics No. 1
(Vol. 10, p. 564, 1952), the H-2609 strain of Streptoverticillium hachijowens is described and also "Studies on the Antibiotics Substance Producing Strains H2075, H-2609 and. H-303
0 "(The Journal of Antibiotics
Series A, Volume 7, Issue 1, Page 10, 1954), H. of Streptoverticillium hachijowens
-2609 and H-2075 strains are mentioned. In addition, the H-2609 strain is IFO 12782 (ATCC 19769).
Has been deposited as. In the present invention, H-2609
Strains, H-2075 strains, H-3030 strains can be used as specific strains, but not limited to these, as long as they belong to Streptoverticillium hachijowens and have the ability to produce the above active ingredients, other The wild strains of No. 1 and No. 2 can also be used, and the mutant strains obtained by mutagenizing these microorganisms by a conventional method (for example, ultraviolet irradiation, mutagenizing treatment, genetic engineering treatment) can also be used.

Cultivation of the radiant bacterium Streptoverticillium hachijowens can be carried out by the following method. As the nutrient source, known ones conventionally used for culturing radiation bacteria can be used. For example, glucose, molasses, starch, dextrin, sucrose, starch syrup, animal and vegetable oils and the like can be used as the carbon source. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions, if necessary. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of active ingredients can be added appropriately. As a culturing method, a culturing method under aerobic conditions is suitable, and a suitable temperature for culturing is 27 to 30 ° C, but in most cases, culturing is performed at around 28 ° C. The pH range suitable for culturing is 7.0 to 9.0. By culturing under the above conditions for 3 to 7 days, a highly active culture solution can be obtained.

Separation of the active ingredient from the culture solution is carried out as follows. First, the bacterial cells are removed from the culture solution, an electrolyte inorganic salt such as zinc chloride is added to the bacterial cell removal solution to salt out, the resulting precipitate is collected, and an aqueous solution of an alkali phosphate such as dibasic sodium phosphate is added to this. Extraction is performed, and a hydrophilic organic solvent such as methanol is added to the extract to collect a precipitated fraction, which is dissolved in water to remove insoluble matter to obtain a solution. This product can be used as it is in the next step, but if necessary, it may be subjected to the next step as a dried product by freeze-drying or the like. The process up to the acquisition of this solution or freeze-dried product is
As described in No. 49-42560, more specifically, it is carried out as follows, for example. That is, the cells were removed from the culture solution, and 50% (w / v) of an aqueous solution of an electrolyte inorganic salt such as zinc chloride or ammonium sulfate was added to the supernatant to a final concentration of about 2.5% for salting out, The resulting precipitate is collected and
About 55% (w / v) of the aqueous solution of sodium phosphate such as dibasic sodium phosphate was added to the supernatant at a concentration of about 55% (w / v) for extraction. , A lower alcohol such as propanol or butanol, or a ketone solvent such as acetone or isobutyl ketone is added to collect a fraction to be precipitated. The collected precipitate is dissolved in distilled water, filtered with an appropriate filter medium to remove insoluble matter, and the filtrate is concentrated, dialyzed and lyophilized as necessary (primary separated product). This primary isolate contains both immunostimulatory and cytocidal actives.

Next, this primary isolate is subjected to gel permeation chromatography (GPC) to obtain a fraction having a molecular weight of 30,000 to 100,000. In this gel permeation chromatography, for example, G3000PW (manufactured by Tosoh Corporation) is used as a column, and 20 mM phosphate buffer solution (pH 7.
2) It can be performed using +0.2 M NaCl. The eluate is concentrated, and if necessary, dialyzed and lyophilized to obtain the active ingredient of the immunostimulant of the present invention. In the above concentration, for example, exclusion molecular weight 3
This can be done by ultrafiltration using a membrane of any kind. The active ingredient of the immunostimulant of the present invention can be obtained as described above and has the above-mentioned physical properties. The active ingredient usually contains, for example, 10% or less of an inorganic substance depending on the degree of removal of the inorganic substance by dialysis, ultrafiltration or the like. Therefore,
When the inorganic substances are completely removed, the above physical properties may be slightly different, but the use of such an active ingredient naturally falls within the range of the immunostimulant of the present invention. This active ingredient has no cytocidal activity, but has the ability to induce cytokines. Specifically, it has the ability to induce TNF-α as one of tumor necrosis factor (TNF) and the ability to induce IL-1 as one of interleukins (IL).
It should be noted that, compared with the primary isolate, this active ingredient shows a marked difference in that it is substantially free of cell-killing components, and that TNF-α-inducing ability and IL-1 inducing ability are much higher. .

Immunotherapy of cancer is a method of curing cancer by increasing the resistance of the tumor-bearing host to cancer, and the activation of immunocompetent cells is the basis of the therapy. Since this active ingredient activates macrophages and spleen cells, which are immunocompetent cells, and damages tumor cells, it can be greatly expected as an immunotherapeutic agent for cancer. Unlike chemotherapeutic agents, immunotherapeutic agents are unlikely to show side effects or toxicity such as blood disorders, digestive organ disorders, lung disorders, cardiotoxicity, nervous system disorders, skin disorders, secondary carcinogenesis, etc. It has the advantage that it can be used with peace of mind without causing other hematopoietic disorders or hepatorenal dysfunction.

Regarding the application of the present active ingredient to therapy, it is preferable to use it as an injection from the results of animal experiments. Particularly, subcutaneous injection stimulates the reticuloendothelial system in vivo to enhance the immune system. Good to do. Also, it can be used in the form of oral preparations, suppositories, etc. by selecting the formulation method. The injection may be subcutaneous, intramuscular, or intravenous injection, and a dosage form such as a suspension, a solution, or a powder that is dissolved at the time of use is used. Moreover, it is not necessary to include a local anesthetic in the injection. The dosage form as an injection usually includes a sterile water solution. The above-mentioned preparations also include buffers pH adjusters (sodium hydrogen phosphate, citric acid, etc.), isotonicity agents (sodium chloride, glucose, etc.), preservatives (methyl paraoxybenzoate, propyl p-hydroxybenzoate, etc.) And other pharmaceutical adjuvants other than water can be contained. The formulation can be sterilized by filtration through a bacteria-retaining filter, incorporation of a bactericide into the composition, irradiation or heating of the composition. The preparation can also be produced as a sterilized solid composition and dissolved in sterilized hydrogen before use. The dose of the active ingredient of the present invention is appropriately selected depending on the symptoms of the patient, but in general, it is preferable to administer 1 to 100 mg / kg to an adult in several divided doses a day. It is preferably administered intramuscularly, intravenously or by injection into the affected area.

[0012]

EXAMPLES Next, the present invention will be specifically described by way of examples. Example 1 1) Production of the active ingredient according to claim 1 Streptoverticillium hachijowens H-2609
2.0 ml of the preserved strain of the strain was poured into 50 ml of Sabouraud medium agar medium having the following composition in Spitz Kolben and statically cultured at 28 ° C. for 8 days. The emerged colonies (for 2 Spitskolbens) were inoculated by platinum loop into 100 ml of each liquid medium of the following composition in 6 500 ml Sakaguchi flasks, and cultured with shaking at 28 ° C. and 150 rpm for 3 days. The whole amount of the culture (about 600 ml) was added to 16 L of liquid medium having the same composition as above in a 30 L fermentor, and aerobically stirred culture was performed at 28 ° C., 245 rpm, and aeration rate of 16 L / min for 3 days. Sabouraud agar medium and preparation Glucose 30g, peptone 10g and agar 20g were added to pure water 1000
Dissolve in ml. No pH adjustment. Liquid medium and preparation (1) Polypeptone 5g, Proteose peptone (Difco) 10
g, glucose 5g, sodium chloride 7.5g and meat extract 3g
Dissolved in 1000 ml of pure water. (2) Dissolve 20 g of potato starch in 200 ml of pure water. The liquid (1) is heated to 80 ° C., and the liquid (2) is divided into three portions and added and dissolved with stirring.

After the completion of the culture, the culture was sterilized by centrifugation and the supernatant was suction filtered. 50% ZnCl ₂ 0.6 L in the filtrate (12 L)
Was added, and the mixture was stirred for 1 minute, allowed to stand for 9 minutes, and then suction filtered in the same manner as above. Dissolve the precipitate in 10% Na ₂ HPO ₄ 6.6 L,
The mixture was stirred for 1 hour, allowed to stand at 4 ° C. overnight, and suction filtered in the same manner as above. 26.4 L of methanol (4 ° C.) was added to 6.6 L of the filtrate, and the mixture was stirred for 5 minutes and suction filtered in the same manner as above. The obtained precipitate was dissolved in 7.92 L of water and suction filtered in the same manner as above. The filtrate (7.9 L) was freeze-dried to obtain 226.7 g of a primary isolate.

92 g of the primary isolate was added to 22 mM phosphate buffer (pH 7.2)
G3000PW dissolved in 920ml and filled with the same buffer
Pour onto φ21.5 × 60 cm column, 20 mM containing 0.2 M NaCl
Gel permeation chromatography was carried out by passing through a phosphate buffer (pH 7.2) at a passing rate of 2.5 ml / min. Eliminate 2800 ml of eluate after the first 550 ml of eluate Molecular weight 3
It was subjected to ultrafiltration (pressure 10 Kg / cm ² ) using a membrane of 40,000 for 40 minutes. The inner solution is freeze-dried, the freeze-dried product is dissolved in water, dialyzed against water, and the inner solution is freeze-dried to obtain a freeze-dried product (the active ingredient of the immunostimulant of the present invention, hereinafter referred to as "the present product"). Say) 10
0 mg was obtained. The appearance of this substance is white to pale yellow powder, and the physical properties are as follows. Regarding the physical properties, it is considered that the components other than C, H, N, and O measured by elemental analysis are inorganic substances, and the inorganic portion is composed of Na, phosphate ions, ash, water, and the like. Elemental analysis: C39.3; H6.0; N11.3; O21.2 Molecular weight of organic matter: 30,000 to 100,000 Red external absorption spectrum: Fig. 1 Ultraviolet absorption spectrum (measured in aqueous solution): Fig. 2 Sugar content: 79 μg / mg Protein content: 71 μg / mg Sugar / protein content ratio: 1.11

2) Measurement of TFN-α Inducing Ability (Induction Method) 0.5 ml of mineral oil was intraperitoneally administered to 7-week-old male Balb / c mice. 6 days later, 10 ml of cold Hanks liquid
Peritoneal exudate cells (PEC) were washed out with 5 ml of tris ammonium chloride solution and the red blood cells were removed by centrifugation. DMEM containing 10% fetal bovine serum (FBS) in the supernatant
10 ml of a medium (hereinafter simply referred to as FBS-containing DMEM medium) was added and centrifuged to precipitate macrophages. The operation of washing the macrophages by adding 10 ml of FBS-containing DMEM medium to the precipitate and centrifuging was repeated twice to collect macrophages. This is DME containing FBS
The cells were suspended in M medium at 4.0 × 10 ⁶ / ml, dispensed to a 96-well microplate in an amount of 100 μl, and cultivated at 37 ° C. for 1 to 2 hours. Non-adherent cells were removed by removing the culture medium by pipetting, and FBS-containing DMEM medium was added to 200 μl / well. Then, 22 μl each of the aqueous solution of the present invention obtained in 1) above at various concentrations was added to each well and incubated at room temperature for 40 hours. Culture broth at 4 ℃, 220 xg
After centrifugation for 10 minutes, the supernatant was obtained.

(Measuring method) (TNF-α / Zenzyme Co., Ltd.)
100 μl of each supernatant was separately added to a 96-well microplate on which anti-mouse TNF monoclonal antibody was immobilized, and incubated at room temperature for 120 minutes. After removing the liquid by pipetting, 100 μl of an aqueous solution of donkey anti-mouse TNF polyclonal antibody was added to each well and incubated at room temperature for 90 minutes. After removing the liquid by pipetting, 100 μl of an aqueous solution of peroxidase-conjugated goat anti-donkey Ig antibody was added to each well and incubated at room temperature for 60 minutes. After pipetting off the liquid, ortho-phenylenediamine aqueous solution 100 μ
l, add and react for 4-6 minutes, then 100 μl of 2N sulfuric acid
Stop the reaction by adding and the optical density (absorbance) at 490 nm
Was measured. As a result, the amount of TNF-α induction of the product of the present invention was 3200 pg / ml at a concentration during induction of 7.5 μg / ml. Incidentally, as a result of the same test in place of the product of the present invention, 0 in the primary separated product.
It was 1600 pg / ml at 5 mg / ml, which clearly shows that the specific activity of the product of the present invention was significantly improved.

3) Measurement of IL-1α inducing ability (induction method) 100
A supernatant was obtained by performing the same operation as the induction method in the measurement of TFN-α inducing ability except that the solution was dispensed to μl. (Measurement method) (by Zenzyme IL-1α / ELISA method) 100 μl of each supernatant was separately added to a 96-well microplate on which anti-mouse IL-1 monoclonal antibody was immobilized,
Incubated at 37 ° C for 40 minutes. After removing the liquid by pipetting, 100 μl of an aqueous solution of biotin-conjugated anti-mouse IL-1 polyclonal antibody was added to each well, and the mixture was incubated at 37 ° C.
Incubated for 40 minutes. After removing the liquid by pipetting, 100 μl of an aqueous solution of peroxidase-conjugated avidin was added to each well and incubated at 37 ° C. for 25 minutes. After removing the liquid by pipetting, 100 μl of a mixture of hydrogen peroxide solution and tetramethylbenzidine solution was added.
After ˜5 minutes, 100 μl of IM sulfuric acid was added to stop the reaction.
Then, the optical density (absorbance) was measured at 450 nm. As a result, the amount of IL-1α induction of the product of the present invention was 60 μg / ml at the time of induction.
Was 250 pg / ml. As a result of the same test in place of the product of the present invention, the primary isolate was 2 mg / ml and 350 pg / ml,
From this, it is clear that the product of the present invention has a significantly improved specific activity.

[0018]

The active ingredient of the immunostimulant according to the present invention has an action of inducing the production of cytokines such as TFN-α and IL-1 in cells such as macrophages, and the immunostimulant and the immunostimulant antitumor agent, Expected to be useful as a therapeutic agent for immunodeficiency.

[Brief description of drawings]

1 shows the infrared absorption spectrum (KBr tablet method) of the active ingredient of the immunostimulant of the present invention obtained in Example 1. FIG.

FIG. 2 shows the ultraviolet absorption spectrum (measured in an aqueous solution) of the active ingredient of the immunostimulant of the present invention obtained in Example 1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1:01) (72) Inventor Hiroshi Kawashima 1-7-4- Sakaigawa, Tobata-ku, Kitakyushu, Fukuoka 202 (72) Inventor Shigeru Okamoto 2-4-6, Nakai, Kokurakita-ku, Kitakyushu-shi, Fukuoka (72) Inventor Hirokatsu Sekiya 4-chome, Nakai-ku, Kitakyushu, Kitakyushu, Fukuoka

Claims (1)

[Claims] 1. An immunostimulant, which can be obtained from a culture supernatant of Streptoverticillium hachijowens, exhibits a cytokine-inducing ability, and contains a substance having the following physicochemical properties as an active ingredient. a) Appearance: White to pale yellow powder b) Elemental analysis: C35.5 to 39.3%; H5.3 to 6.0%;
N 11.3 to 12.6%; O 21.2 to 26.8% c) Molecular weight of organic substance: 30,000 to 100,000 d) Red external absorption spectrum: Clear peaks are observed in the following ranges. 3300 ~ 3500, 1650 ~ 1660, 1538
~ 1540, 1227 ~ 1229, 1064 ~ 1065cm ^-1 e) Sugar content: 70 ~ 150μg / mg f) Protein content: 70 ~ 230μg / mg g) Sugar / protein content ratio: 0.9 ~ 1.2 (weight ratio)