JPS6230198B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antitumor active substance obtained from the cells of a microorganism belonging to the genus Propionibacterium and a preparation containing the active substance as an active ingredient. It has been known for a long time that bacteria belonging to the genus Propionibacterium have physiological activities such as antitumor and adjuvant effects, and some of them are also used clinically. However, the cells of this microorganism have various side effects, and in order to alleviate these side effects, attempts have been made mainly to separate the active substance from the cell wall. Examples of these include cell wall skeletons and polymeric mucopeptides prepared from cell walls, but it is said that those with high antitumor activity also have strong toxicity, and those with low toxicity do not necessarily have high antitumor activity. It has its drawbacks. Next, as a result of various studies on antitumor active substances obtained from Mycobacterium tuberculosis, the present inventors learned that although the cell wall exhibits high antitumor activity, its toxicity is the same as that of the raw material, live bacteria. We investigated a method for obtaining a substance with antitumor activity equivalent to or higher than that of bacterial cells and less toxic from a fraction obtained by removing as much of the bacteria as possible, and found that the desired substance could be obtained by a simple method. Therefore, a patent [Japanese Patent Application No. 53-26519 (see Japanese Patent Application Laid-open No. 54-140710)]
has been applied for. Furthermore, in order to improve the above-mentioned drawbacks of antitumor agents made from Propionibacterium bacteria and bacterial cell extracts, the present inventors have investigated various preparation methods for active substances based on their experience with Mycobacterium tuberculosis. As a result, we confirmed that active substances with high antitumor activity and low toxicity can be obtained from bacteria belonging to the genus Propionibacterium using the same method used for Mycobacterium tuberculosis. Completed the invention. The strains belonging to the genus Propionibacterium used in the present invention include the known Propionibacterium strains described in Versey's Manual of Determinative Bacteriology, 8th edition, pages 634 to 641.・Propionibacteruim acnes, Propionibacterium granulosum
Propionibacterium avidum, Propionibacterium avidum, etc. have high antitumor activity as whole bacteria.
A strain with low pathogenicity is preferred. For cultivating the above-mentioned bacterial strain in the present invention, a conventional anaerobic culture method can be used. For example, nitrogen sources include meat extract, peptone, phyton, yeast extract, etc., carbon sources include glucose, reducing agents include cysteine hydrochloride, sodium thioglycolate, potassium phosphate, magnesium sulfate, etc.
It is preferable to use a medium supplemented with sodium chloride or the like. The culture is preferably left at 30 to 40°C for 2 to 15 days. The obtained bacterial cells can be collected by centrifugation, washed with physiological saline, etc., and then subjected to the next operation as is, or, if necessary, sterilized with phenol or mild heat, and then subjected to the next operation. You may do so. After mixing the obtained bacterial cells with water, preferably an appropriate buffer solution, the cells are crushed using a bacterial cell crusher, such as a Dyno-mill or French.
A suspension of crushed bacterial cells is obtained by crushing the bacterial cells using a press or the like. Suitable buffers include, for example, phosphate, borate, acetate, citrate,
There are tartrates, succinates, trishydroxymethylaminomethane, etc., and the concentration used is 0.001
~1M, preferably 0.01~0.1M is appropriate. The obtained suspension of crushed bacterial cells can be filtered or centrifuged to obtain a cell-free extract suitable for the raw material. The cell-free extract in the present invention refers to unbroken bacterial cells obtained by filtration or centrifugation from a suspension of crushed bacterial cells.
This is a fraction from which cell wall fractions, etc. are removed as much as possible, and are removed during this separation procedure so that the final substance of the present invention does not contain more than 20% cell wall fractions. is preferable. The flocculant used for the purpose of precipitating the substance of the present invention from the cell-free extract can be selected from a fairly wide range. For example, polyvalent metal salts such as aluminum sulfate, calcium chloride, magnesium chloride, ferric chloride, manganese chloride, etc., water-soluble basic antibiotics such as streptomycin, kanamycin, or salts thereof are suitable. The amount of flocculant used is selected depending on the type of flocculant, but
For example, polyvalent metal salts: 0.1-10% (W/V), preferably 0.1-3% (W/V) Water-soluble basic antibiotics: 0.1-10% (W/V)
V) It is appropriate to use preferably 0.1 to 1% (W/V) relative to the cell-free extract. These flocculants are added to the cell-free extract to precipitate the substance of the present invention. For example, if a predetermined amount of the flocculant is required, it is added to the extract as an aqueous solution and mixed. A precipitate forms over time. This precipitate is separated from the mother liquor by a method such as filtration or centrifugation and collected. Furthermore, it is preferable to leave the mixture to stand after adding the flocculant, from the viewpoint of growth of the precipitate and improvement of the sedimentation rate of the precipitate. A few hours of standing time is sufficient. The substance of the present invention separated from the mother liquor by a method such as filtration or centrifugation may be used as is, but if possible, it is better to remove the flocculant contained in the precipitate.
To do this, for example, operate as follows. The separated substance of the present invention is suspended in water or an appropriate buffer. Next, the flocculant is removed by means such as dialysis, gel filtration, ultrafiltration, etc., and then freeze-dried to obtain the purified substance of the present invention. Suitable buffers that can be used in this purification procedure include, for example, phosphate, borate, acetate, citrate, tartrate, succinate, trishydroxymethylaminomethane, etc., and the concentration used is
0.001-2M, preferably 0.01-0.2M is suitable. The pH of the buffer solution is preferably in the range of 4 to 10. PH is 4
If the amount is less than 1, the dispersibility of the substance of the present invention decreases and the removal of the flocculant becomes insufficient. On the other hand, if the pH exceeds 10, the substance of the present invention may be decomposed, which is not preferable. In addition, when a water-soluble basic antibiotic such as streptomycin is used as a coagulant, streptomycin can be easily removed by using a salted buffer solution containing, for example, 0.1 to 0.5 M of common salt to increase the ionic strength. You may add 0.5M or more of common salt, but even if you add 0.5M or more, the streptomycin removal effect will not improve any further. As the drying method, it is preferable to use a freeze-drying method in order to avoid deterioration of the substance of the present invention. In addition, when preparing the substance of the present invention from live bacteria,
All operations are preferably carried out at 0 to 10°C to prevent denaturation due to heat or enzymes and to facilitate separation operations. The substance of the present invention has a complex composition mainly consisting of carbohydrates, proteins, lipids, and nucleic acids. There is no major difference in the composition of the substances of the present invention obtained by the flocculants used in the present invention, and they all have high antitumor activity, little toxicity and side effects,
It can be used as an active ingredient of antitumor agents.
Furthermore, the method for producing it is extremely simple, the yield is high, and it is an extremely useful substance. The substance of the present invention is mainly administered to animals as an injection alone or together with an antigenic substance, but the substance of the present invention has the property of being emulsifiable in either water or oil. For example, the substance of the present invention may be emulsified in physiological saline, or this suspension may be further suspended in vegetable oil or mineral oil (water-in-oil type), or the substance of the present invention may be directly applied to vegetable oil or mineral oil. After being suspended in oil, this suspension is further suspended in physiological saline to be used as a suspension (oil-in-water type). The dose of the substance of the present invention is appropriately selected depending on the animal species, administration route, and administration schedule, but it is 1 to 100 mg/Kg for intraperitoneal administration to mice, 2 to 250 mg/Kg for subcutaneous administration, and peritoneal administration for guinea pigs. 1-100mg/Kg for intradermal administration, 0.05-100 for subcutaneous administration
mg/Kg. The administration site of the substance of the present invention may be either within the tumor or at a site remote from the tumor. The amount of the substance of the present invention to be administered to various tumor types is the same as or a fraction of that of the raw material live bacteria, and exhibits the same antitumor effect as live bacteria. Furthermore, the substance of the present invention exhibits strong antitumor effects against both solid cancer and ascites cancer. For example, cancer types for which the substance of the present invention is effective include Ehrlichi's ascites tumor, solid tumor, and Sarcoma 180.
Ascites tumor, solid tumor, melanoma, etc. Since the substance of the present invention is prepared from a cell-free extract, the infectivity from the substance of the present invention is originally low;
By using an anti-inflammatory substance as a coagulant, the risk of infection can be completely eliminated. The following is an example of the method for producing the substance of the present invention and the formulation method when using the substance of the present invention as an antitumor agent.
Further, the usefulness of the material of the present invention will be explained using test examples. Example 1 Propionibacterium avidum (Propionibacteruim avidum) ATCC25577 was prepared in a medium with the following composition: Heart Infusion Medium (manufactured by Eiken Chemical) 25 g Glucose 5 g Sodium thioglycolate 0.5 g Water was added to make a total volume of 700 ml to 1000 ml. The bacteria were inoculated into a Rubin with a rubber stopper, and the air in the Rubin was replaced with nitrogen, followed by static culture at 37°C for 5 days. After the culture was completed, phenol was added to the culture at a final concentration of 5% and left at room temperature for 2 days. Bacterial cells were collected from the resulting culture by centrifugation and washed three times with physiological saline to obtain wet bacterial cells. To 190g of this wet bacterial body, 10mM phosphate buffer) (PH
7.0) After adding and suspending 1, crush the bacterial cells while cooling on ice using a Dyno-Mill, and centrifuge the resulting crushed bacterial cells under cooling (10,000 x g, 20 minutes). Then, unbroken bacterial bodies and cell wall fractions were removed to obtain 870 ml of a cell-free extract. Add streptomycin sulfate to 200ml of this extract.
After adding 0.6 g and stirring, the mixture was allowed to stand overnight at 0 to 4°C. The generated precipitate was centrifuged under cooling (10,000×
g, 20 minutes) and then diluted with 50 ml of salted buffer, which is 10 mM phosphate buffer (PH7.0) with 0.5 M of sodium chloride added.
This suspension was packed into a cellophane dialysis tube, and dialyzed overnight at 0 to 4°C using the above-mentioned salted buffer 1 as the external dialysis fluid.Then, the external dialysis fluid was replaced with distilled water and dialyzed overnight. The obtained dialysis fluid was freeze-dried to obtain 870m of the active substance of the present invention. This active substance will hereinafter be referred to as NP-1. Example 2 Manganese chloride 2 was added to 200 ml of the extract obtained in Example 1.
After stirring well, the mixture was allowed to stand overnight at 0 to 4°C. The generated precipitate was centrifuged under cooling (10,000×
g, 20 minutes), suspend it in 50 ml of 10 mM phosphate buffer (PH7.0), pack this suspension into a cellophane dialysis tube, use the above buffer 1 as the external dialysis solution, Dialysis was performed overnight at 0 to 4°C, and the dialyzed fluid was further dialyzed overnight with distilled water, and the resulting dialyzed fluid was freeze-dried to obtain 790m of the active substance of the present invention. This active substance will hereinafter be referred to as NP-2. Example 3 Propionibacterium granulosum ATCC25564
was inoculated into the same medium used in Example 1 and cultured. After the cultivation was completed, the resulting culture was sterilized in the same manner as in Example 1. 160 g of the obtained wet bacterial cells were treated in the same manner as in Example 1 to obtain 3.36 g of the active substance of the present invention. This active substance will hereinafter be referred to as NP-3. Example 4 Preparation of the substance of the present invention Add 0.5 ml of physiological saline to 10 mg of N-P-1, thoroughly grind and suspend in a mortar, then add 20 ml of physiological saline and mix well to prepare N-P-1. A suspension of one substance in physiological saline was obtained. The substance of the present invention was also suspended in physiological saline in the form of an emulsion. Example 5 Preparation of the substance of the present invention 2.5 ml of physiological saline was added to 25 mg of NP-1,
After thoroughly grinding and suspending in a mortar, add 15% Arlacel A.
2.5 ml of liquid paraffin containing Arlacel A (manufactured by Sanko Pure Chemical Industries, Ltd.) was added and homogenized to obtain a water-in-oil formulation of the substance of the present invention. Test Example 1 Composition of the substance of the present invention The composition of the substance of the present invention is shown in Table 1. The compositional analysis also serves as a color reaction test and is carried out by the following method. (1) Carbohydrate phenol sulfuric acid method (M. Dubois, K.
A.Gilles, JKHamilton, PARebers,
and F. Smith, analytical chemistry
28 , 350, 1956) (2) Protein Lowry method (CDStauffer,
Analytical Biochemistry 69 , 646,
(1975) (3) Lipid Bligh-Dyer method (EGBligh)
and W.J.Dyer, Can.J.Biochem.Physiol.
, 37 , 911, 1959) (4) Nucleic acids Schmitt-Tannhauser, Schneider method (WCSchneider, J.Biol.Chem.
, 164 , 747, 1946) and the diphenylamine method (K.Burton, Biochem.J.,
62 315, 1956) and the Orcinol method (W.
Mejbaum, Z. Physiol, Chem 258 ,
117, 1939).

[Table] Test Example 2 Antitumor effect on murine Sarcoma solid tumor 2 × 10 ⁶ Sarcoma tumor cells/mouse were intradermally inoculated into 6 ICR female mice per group, and the tumor diameter was approximately After reaching 5 mm, 0.1 ml of the antitumor agent prepared by the method of Example 4 was administered twice a week for a total of 6
It was administered intratumorally. The number of cured animals 6 weeks after tumor inoculation is shown in Table 2. For comparison, Propionibacterium avidum was used instead of the substance of the present invention.
avidum) ATCC25577 Phenol-treated bacterial cells (Pa
A formulation using the same amount of WC) and a formulation with a similar composition without the active substance were used as a control.

[Table] All of the active substances of the present invention inhibited the growth of the same type of tumor and led to cure. Test Example 3 Antitumor effect on mouse melanoma solid tumor. Melanoma B-16 tumor cells 1x were subcutaneously injected into 6 C57BL/6J female mice per group.
After the tumor diameter reached approximately ⁵ mm, 0.1 ml of the antitumor agent prepared according to the method of Example 5 was administered into the tumor twice a week for a total of 6 times. The number of cured animals 6 weeks after tumor inoculation is shown in Table 3. For comparison, Propionibacterium avidum was used instead of the substance of the present invention.
avidum) ATCC25577 Phenol-treated bacterial cells (Pa
A formulation using the same amount of WC) and a formulation with a similar composition containing no active substance were used as a control example.

[Table] All of the active substances of the present invention inhibited the growth of syngeneic tumors and led to cure. Test Example 4 Acute Toxicity The substance of the present invention suspended in physiological saline was intraperitoneally administered to 5-week-old DDY male mice (6 mice per group), and the LD50
The values (50% lethal dose, mg/kg body weight) were determined and shown in Table 4.

[Table] The toxicity of the active substances of the present invention is extremely low.

Claims (1)

[Claims] 1. A bacterial cell belonging to the genus Propionibacterium,
An antitumor active substance obtained as a precipitate by adding a flocculant to the liquid phase of a cell-free extract obtained by disruption in a buffer solution with a pH not exceeding 10, and having the following properties: . (1) Composition Carbohydrates (phenol sulfate method) 12-16% Proteins (Lowry method) 38-46% Lipids (Blei-Dyer method) 20-26% Nucleic acids (diphenylamine method and orcinol method) 8-16% (2) Color reaction carbohydrate (phenol sulfuric acid method) Positive protein (Lowry method) Positive nucleic acid (diphenylamine method and orcinol method) All positive 2 Confirm that the flocculant is a water-soluble basic antibiotic or its salt. An antitumor active substance according to claim 1, characterized in: 3. The antitumor active substance according to claim 2, wherein the flocculant is streptomycin sulfate. 4. The antitumor active substance according to claim 1, wherein the flocculant is a polyvalent metal salt. 5. Bacteria belonging to the genus Propionibacterium,
The active ingredient is a precipitate obtained by adding a flocculant to the liquid phase of a cell-free extract obtained by disruption in a buffer solution with a pH not exceeding 10, and having the following properties: An antitumor agent characterized by: (1) Composition Carbohydrates (phenol sulfate method) 12-16% Proteins (Lowry method) 38-46% Lipids (Blei-Dyer method) 20-26% Nucleic acids (diphenylamine method and orcinol method) 8-16% (2) Color reaction carbohydrate (phenol sulfuric acid method) Positive protein (Lowry method) Positive nucleic acid (diphenylamine method and orcinol method) All positive