JPS5973525A - Novel rhamnose homopolymer and antitumor agent containing said compound as active component - Google Patents

Abstract

NEW MATERIAL:The rhamnose homopolymer (composed of beta1 2, 2 3 bonds and having a molecular weight of about 16,000-27,000). USE:An antitumor agent. PROCESS:A bacterial strain belonging to Clostridium genus (e.g. Clostridium saccharoperbutylacetonicum ATCC 27022) is cultured in a medium containing glucose, sucrose, etc. as a carbon source. After proliferation of the bacterial strain, the medium is centrifuged at 6,000-12,000r.p.m, wased with cold water, and centrifuged again to remove the foreign materials and recover the bacterial cells. The cells are extracted wtih water, and the extract is added with a solvent such as acetone to obtain a precipitate. The precipitate is dissolved in pure water, dialyzed with pure water, concentrated under reduced pressure, freeze-dried, and treated by column chromatography, etc. to obtain the objective rhamnose homopolymer. It is administered to a man as an injection for intravenous, intra-arterial or local use, or as an oral drug such as capsule.

Description

[Detailed description of the invention]

The present invention relates to a novel rhamnose homopolymer and an antitumor agent containing the same as an active ingredient. Kiri and Watanabe regarding the composition of bacterial cell walls, especially those of Clostridium bacteria. Various people have reported that the PG layer (peptidegly-ca
n) and ps layer (polysaccharide) are 4
:1 ratio. (PS: Watanabe et al. 50th Anniversary of the Japanese Society of Agricultural Chemistry (Abstract 2-6 October 1974) (Journal of the Japan Stratification Society 48N-162 (1974)) PG: Miscellaneous et al. Same as the above conference lecture (Summary) 2 of 5 1974 1
(Journal of the Japan Stratification Society 48N-162 (1974)) was published and is well known. On the other hand, regarding the antitumor properties of bacterial cell walls, Mycobacterium tuberculosis (Yamamura et al. Japan Clinical 32542
(1974) ) and lactic acid bacteria (1,G, Bogdano
v et al FBBS 1etters 57259 (19
75) ') have been reported to have antitumor properties, and the present inventors have obtained cell wall segments obtained by a conventional method by sonicating Clostridium cells and adjusting centrifugal force, and insoluble cells produced by a specific Clostridium species. Too many! Class C-45
He discovered that it had remarkable antitumor properties against ascites cancer in mice, completed an invention based on this discovery, and applied for a patent. (
JP-A-54-76817 and JP-A-54-76896
No.) The present inventors subsequently conducted further research and found that the slightly acidic aqueous solution extraction segment or the alkaline aqueous solution extraction segment of bacteria belonging to the genus Clostridium or Brevibacterium was better than the cell wall segment and insoluble polysaccharide of the previously filed invention. He discovered that this substance also exhibits strong antitumor properties, completed an invention based on this, and applied for a patent. (Special application 1972-2497
No. 9, special application 1982-. 31535) The present inventors continued their research on the antitumor active substances contained in the cells of the genus Clostridium and Brevibacterium, and found that they It was discovered that the extracted fraction also showed strong antitumor properties, and based on this, he completed an invention and applied for a patent. (Special application No. 57-34396
No.) The present inventors continued their research to elucidate the antitumor components of bacterial cells belonging to the genus Cross 1 to Rhydium that were extracted with water, and found that among the same components, the antitumor components that are strong in the class M category They discovered that this is a novel rhamnose homopolymer and completed the present invention. That is, the present invention is a rhamnose homopolymer having a molecular weight of about 16,000 to 27,000 and consisting of βI-2.1-3 bonds, and an antitumor agent containing this as an active ingredient. The present invention is directed to bacterial strains belonging to the genus Clostridium, such as Clostridium satucaloperbutylacetonicum (Clostridium satucaloperbutylacetonicum).
Stridium 5accha-roperbu
tylacetonicum) ATCC27022 by a conventional method. The composition of the medium in this case is glucose, sucrose, starch, and maltose as the carbon source, which is the usual medium composition. Starch saccharification liquid, cane molasses, sugar beet molasses, etc. are used, and nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, ammonium acetate, nitrate salts, chloride, soybean meal, cottonseed meal, peptone, and rice bran. Organic nitrogen sources such as , corn steep liquor and yeast extract, minerals such as phosphates and magnesium salts, and lime carbonate added to prevent acidification of the medium are used. Among these media, a medium and culture conditions suitable for the strain used may be appropriately selected and used. Any known method can be used to recover bacterial cells from these growth media. Usually, it is spun down by centrifugation at 6000 to 1200 o r, p, m,
Contaminants are removed and collected by washing with cold water and repeated centrifugation. The cells of the bacteria belonging to the genus Clostridium thus obtained are extracted with water. In this case, although the extract does not change depending on the temperature, it is more convenient to perform the extraction under heating in terms of the amount of extraction, extraction time, etc. The extraction time ranges from about 30 minutes to one day with stirring. The aqueous extract extracted as described above is further subjected to solvent precipitation. To perform solvent precipitation on the aqueous extract, a water-miscible solvent such as acetone or alcohol, preferably a lower alcohol such as ethanol, is added to the aqueous extract to form a precipitate, which is collected. This precipitate is dissolved in pure water, dialyzed against pure water, the dialyzed solution is concentrated under reduced pressure, freeze-dried, and fractionated using column chromatography, Sephadex, etc. Among them, both polysaccharides with strong antitumor activity are collected, and con
Rhamnose pomopolymer was obtained by A-cellophalose affinity chromatography. This polysaccharide has a molecular weight of approximately 16.000 to 27.000 as determined by gel filtration using column chromatography, and gas chromatography analysis shows that the constituent sugars are approximately 100% rhamnose, optical rotation plus, and methylation analysis using Hakomori method indicates β1.
-2,1→3 bond. This substance is a new substance that has not been described in any literature. The substance of the present invention has strong antitumor properties and can be used as an active ingredient as an antitumor agent. 1 shows test results of antitumor properties of the substance of the present invention. Test method: As test animals, mice with a weight of 20 to 25 g were selected from 5 withdrawn mice of ddY♀, and one week after inheriting Sarcoma-180, the mice were incubated with PBS'→ so that the cell count was 106. Dilute and implant intraperitoneally. The sample is 1w/0.2m 7! After suspending in PBS' → 115°C. Autoclave for 5 minutes and administer 0.2 mj2 intraperitoneally 10 times. Administration was 2 days before Sarcoma-180 implantation.
times (-6, -3), 8 times after transplantation (2, 3, 4 degrees 5.6
, 7, 8.9) Administer. PBS as control

Administer only [-]. Observe the survival number over 100 days in test mice 10w4. The antitumor properties of the substances obtained in the following examples were tested by the method described above, and the results are shown in Table 1 below. As is clear from the above results, the substance of the present invention exhibits extremely strong antitumor properties. When administering the substance of the present invention, it is dissolved in distilled water for injection or physiological saline. In that case, the administration method for animals is intraperitoneal injection. Subcutaneous injections and water-soluble substances are administered as injections such as intravascular injection into veins or arteries and local administration, and for humans, injections such as intravascular injection into veins or arteries or local administration, or capsules etc. It is administered as an oral drug, and the dosage is determined by taking into account the results of animal studies and various circumstances, and the total dosage is administered continuously or intermittently within a range that does not exceed a certain amount. However, the dosage depends on the method of administration, the circumstances of the patient or treated animal, such as age, body weight, sex, susceptibility to diet, administration time,
Of course, the administration may be changed as appropriate depending on the concomitant drugs, the patient, or the severity of the disease. Next, an example of manufacturing the substance of the present invention will be given. Example 1 Clostridium satucaloperbutylacetonicum A
250 mj! After culturing 1 volume of TYA medium in a fermentation bottle at 30°C and initial pH 6.5 for 15 hours, 50m7 of this culture solution was added. 500r+l in 11 fermentation bottles! 〒Inoculated into a dispensed YA medium, cultured for 15 hours in the same way, and inoculated 300 mL of this culture into a 51 flat-bottomed flask containing TYA medium dispensed at 37°C, cultured in the same way for 15 hours, and cultured for 15 hours. Perform culture. For the main culture, put TYA medium 41 in each of five 5Il flat-bottomed flasks, and add the above preculture solution to 500 m
# of seeds and static culture with cotton plugs at 30°C. After about 60 hours, 12 g/j of butanol! After production, the culture solution was combined at 12.00 Or. pom. The cells were suspended in cold water at 6.00 Or. p,m
．． . Cells are washed by repeating freezing centrifugation (4°C) for 10 minutes three times. The cells are suspended in 750 mJ of cold water, 1.5 m/l of formalin is added and stirred, and then extracted with stirring in a 70°C constant temperature bath for 30 minutes. This extract was heated to 12,00 Orr.
p. m. , Centrifuge for 30 minutes, collect the supernatant,
Add alcohol to a final concentration of 85%, stir, and set aside overnight. The resulting fibrous material was heated to 6.0OOr.
．． p. m. , collected by centrifugation for 10 minutes, approximately 300
Dissolve in mJ of water, perform running water dialysis with tap water for 2 days and pure water for 1 day, and freeze-dry. This was suspended in pure water to obtain a water-soluble fraction.
H 7.0) 30mj! I) EAE-Sephadex A-50 (column size φ2 cm x 35 cm) which had been equilibrated with the above-mentioned Nohuffer in advance. Flow the above-mentioned buffer over 500ml and collect the flow-through fraction. This passing fraction is about 5QmI! Sephadex G-50 (column size φ2.7cm x 90c+n
), color was developed using the Anthrone sulfuric acid method, and polysaccharide fraction F-1 (both fractions from the 35th to the 60th fraction) having an absorption at 620 nm was obtained. F-1 fraction is rhamnose (90.3%)
, galactose (3.5%) and glucose (6.2%
) and has an average molecular weight of about 19,000. F-1 had a weight of 26 μm as a freeze-dried sample. This F-IIi minute 80
(2) was dissolved in 10 ml of 0.15M PBS (phosphate bath saline) (pH 6.8) and placed on ConA-Sepharose (column size φ2, 5 cm x 25 cm) equilibrated with the above-mentioned buffer. Elute with the above-mentioned buffer, and collect the eluate in 5 mp aliquots. Both parts of the color are developed using the phenol-sulfuric acid method and have absorption at 490 nm (both parts from the 10th to the 25th part).
Collect and freeze-dry this to make 50tnj of pure water! Sephadex G-75 (column size φ'l, 7
C+n x 90 cm> to desalt, elute with pure water, and collect 5 ml of the eluate. Both parts are colored with phenol sulfuric acid and absorb at 490 nm (from the 30th line to the 8th line).
0) were collected and freeze-dried to obtain a homopolymer 64 of rhamnose, the substance of the present invention. As a constituent monosaccharide analysis of the substance of the present invention, the substance of the present invention was
M was hydrolyzed with TFA (refluoroacetic acid) to obtain a TMS (trimethyldisilazane) derivative by the method of Sweely et al., and quantification was performed by heating gas chromatography using a 3% 5E-52 column. However, except for traces of glucose, everything was rhamnose. When the optical rotation of the substance of the present invention was measured, it showed a positive value. The infrared absorption curve of this substance [liquid paraffin (
(manufactured by Merck & Co.)] was observed to be absorbed by Kaiser 840. Based on this, it is determined that this substance is β type. The substance of the present invention is dex1-lan T-40 with a molecular weight of 40.0.
00. Dextran T-10 molecular weight 10,000 was used as a standard substance, Sephadex G-75 (column size φ
The molecular weight was roughly estimated to be 16,000 to 27,000 from the elution position by gel filtration (2.7 cm x 9Q cm). For sugar chain structure analysis of the substance of the present invention, the substance of the present invention was completely methylated by Hakomori's method, and TF of formic acid (ConC) 2M was used.
After hydrolyzing with A, reducing with sodium borohydride and acetylating with a 1:1 mixture of pyridine and acetic anhydride, the partially methylated sugar alcohol acetylated mixture obtained was mixed with 3% E.
It was subjected to gas chromatography using CN55M and Schiller capillary columns, and the detected peaks were compared with the peaks of standard partially methylated sugar alcohol acetate to identify each partially methylated sugar component. The predicted sugar chain bonds of the partially methylated sugar alcohol obtained by gas chromatography are shown in Table 2 below. 1 Table 2 2 As described above, the substance of the present invention is a new rhamnose homopolymer and has extremely strong anti-JII! It has tumorigenic properties and is useful as an antitumor agent. Patent Applicant Sanraku Ocean Co., Ltd. Agent Riki Arai (Two Idiots)

Claims (1)

[Claims] 1. Molecular weight consisting of β1-2.2-3 bonds of about 16.0
00-27,000 rhamnose homopolymer. 2. Molecular weight approximately 1 consisting of β1→2,1→3￥It3 combination
An antitumor agent containing a rhamnose homopolymer of 6,000 to 27,000 as an active ingredient.