JPS61271224A - Antitumor active substance - Google Patents

Abstract

PURPOSE:The titled substance obtained by treating microbial cells of Streptococcus faecium with hot water, and salting out the resultant extract. CONSTITUTION:An antitumor active substance obtained by cultivating a strain, e.g. Streptococcus faecium R 672 strain; FERM-P No.8167) belonging to Streptococcus faecium, and collecting the microbial cells from the culture by the centifugation, washing the micobial cells with physiological saline solution, etc., adding an adequate amount of purified water thereto, heating the resultant mixture, and extracting the substance with hot water. The amount of the purified water to be used for extraction with the hot water is 5-50 times, preferably 8-15 times based on the wet weight of the microbial cells. The extraction temperature is 4-100 deg.C. A salt, e.g. ammonium sulfate, is used to salt out the extract, and deposited precipitate is separated by filtration or centrifugation, etc., and the resultant residual salt is removed by dialysis, gel filtration, etc. The resultant precipitate is then freeze-dried to give the aimed antitumor active substance. The preferred dosage form is injection and transfusion solution, and the dose thereof is 1-2,000mg/day.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to antitumor active substances, and more particularly to Streptococcus faecium (Streptococcus faecium).
The present invention relates to a low toxicity and highly active anti-tumor active substance obtained from C. cus faecium.

Prior Art It is widely known clinically and empirically that certain bacteria belonging to the genus Streptococcus have antitumor activity, and in particular that streptococci (hemolytic streptococci) with β-hemolytic properties exhibit anticancer effects. It is being Today, various types of antitumor preparations or anticancer drugs are used, but there are problems such as strong side effects, insufficient treatment, low activity, or instability. Furthermore, the development of effective antitumor active substances is desired.

Under these circumstances, it has been proposed to utilize the antitumor activity of the above-mentioned bacteria of the genus Streptococcus.

First, attempts were made to use the bacterial cells themselves, but since these streptococci are the pathogens of erysipelas and various other infectious diseases, using the bacterial components for treatment often resulted in side effects such as fever. . Furthermore, most of the bacteria that have been known to have antitumor activity are highly toxic and their activity is unstable, making them unsuitable for use as bacterial cells themselves. Attempts have also been made to isolate active ingredients associated with antitumor activity from the cells of these bacteria. However, conventional separation operations are not only extremely complicated, but also the activity of the obtained substances is not high compared to that of the starting bacterial cells.

Therefore, there has been a desire for the emergence of bacterial strains with high antitumor activity and low toxicity, as well as the development of a method for simply and efficiently separating active substances from them.

In view of the above-mentioned circumstances, the inventors of the present invention have conducted research with the aim of discovering more useful bacterial strains and developing efficient isolation methods for active substances. Streptococcus faecium, which had not been previously studied, shows high antitumor activity and low toxicity compared to previous bacteria, and the hot water extract of its cells has a large amount of antitumor activity, and it is also possible to use it in an appropriate manner. They discovered that the salt can be efficiently precipitated at a certain concentration of salt, and completed the present invention.

That is, the present invention provides an antitumor active substance obtained by treating bacterial cells of Streptococcus faecium with hot water and salting out the extract.

Examples of the Streptococcus faecium strain used in the present invention include Streptococcus faecium R672 strain.

This strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (Accession number: Microbiological Research Institute No. 8167 (Accession Sunday 60
3/28)). In addition, Streptococcus faecium other than the above-mentioned strains can be used in the same way, and these include, for example, S. faecium, Streptococcus faecium IF012256, IFO
Includes I236/7, etc.

In order to obtain the antitumor active substance of the present invention, the above-mentioned bacterial strain is cultured by a conventional method, and the bacterial cells are collected from the culture by centrifugation. Next, after washing the bacterial cells with physiological saline or the like, an appropriate amount of purified water is added and heated to perform hot water extraction. The amount of purified water used for hot water extraction is approximately 5 to 50% of the wet weight of the bacterial cells.
twice, preferably 8 to 15 times. Also, the extraction temperature is 4
-100°C (preferably 60-80°C), and is usually carried out for 10-120 minutes with stirring. When hot water extraction is carried out under the above conditions, the active substances of the bacterial cells are efficiently extracted.

Various salts for salting out are used for the purpose of precipitating the active substance from the extract. Examples of these salts include ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate, and sodium chloride.

Among these, ammonium sulfate is preferred. These salts are added to the extract in a predetermined saturated solution, for example, a 70% saturated ammonium sulfate solution, preferably a 20% to 40% saturated ammonium sulfate solution, and are stirred under water cooling to form a precipitate. When salting out with ammonia sulfate at the above concentration, the antitumor active substance of the present invention can be easily and efficiently obtained without impairing the activity of the active substance and without accompanying a large amount of unnecessary substances. .

It should be noted that those skilled in the art will easily understand that the salt concentration can vary depending on the type of salt used.

Next, the precipitate is separated according to standard techniques such as filtration or centrifugation. Subsequently, residual salts are removed by dialysis, gel filtration, ultrafiltration, etc., and then freeze-dried to obtain the desired antitumor active substance.

The substance of the present invention thus obtained was a substance with a complex composition mainly consisting of carbohydrates, proteins, lipids, nucleic acids, and riboteichoic acid.

The substance of the present invention showed significantly high antitumor activity in animal experiments. Therefore, this substance can be formulated into a desired drug layer using an appropriate carrier or excipient, or used in combination with other drugs to form a pharmaceutical composition, which can be used clinically to treat various tumors. Preferred dosage forms are injections and infusions.

The dosage in this case can be 1 to 2000 mg/day. Moreover, its acute toxicity is LD5. >750m
g/kg (i.p. to mice).

) administration).

The present invention will be explained in more detail with reference to Examples below.

Example 1 Streptococcus faecium R672 at 200 mQ
Inoculate six Erlenmeyer flasks containing trypto soya broth and culture overnight at 37°C.

This culture is further inoculated into 6 Erlenmeyer flasks containing 3Q trypto soya broth medium and cultured at 37°C for 24 hours. 29 by centrifuging the resulting culture.
g of wet bacterial cells are obtained. A 10-fold amount of purified water is added to the bacterial cells, stirred at 80°C for 30 minutes, and then centrifuged to obtain a hot water extract. Ammonium sulfate is added to this extract under water cooling to give a saturation of 40% (approximately 20% by weight), and the mixture is stirred.

The generated precipitate was collected by centrifugation under cooling, and this was poured into purified water 5
Suspend at 0 mQ, pack into a dialysis tube (Spectrabore 3), and dialyze against purified water 2012 overnight. After removing the remaining ammonium sulfate by changing the external dialysis solution twice, the dialysis solution was freeze-dried to obtain 397 mg of the active substance of the present invention.

Example 2 Hot water extract of bacterial cells obtained by the same method as Example 1 470x
Q is packed in a dialysis tube and dialyzed against purified water to remove low-molecular substances, and then ammonium sulfate is added to the dialysate solution to achieve 20% saturation under water cooling and stirred. The precipitate ③ formed is collected by centrifugation under cooling. Further, under water cooling, ammonium sulfate was added to the supernatant to make it 40% saturated, and the mixture was stirred.

The formed precipitate (■) is collected by centrifugation under cooling. Each precipitate was dialyzed in the same manner as in Example 1, and the dialyzed solution was freeze-dried to obtain 78 active substances (35119).

The antitumor activity of the active substances obtained in the above examples was demonstrated in animal experiments shown in the following test examples.

Test Example 1 A mouse-derived colon cancer Colon26 tumor is subcutaneously implanted into a BALB/C mouse (4 weeks old) weighing around 209 kg.

Five days after transplantation, animals with tumors of uniform size are assigned to 5 groups of 5 animals per group. The administration group receives 200 m9/kg of the active substance of the invention intraperitoneally. On the other hand, the same amount of physiological saline is administered to the control group (control value).

Long axis (a) and short axis (b) of tumor every day until 3rd day after administration.
is measured externally with a caliper, the tumor volume is calculated using the formula 1/2ab', and the growth inhibition rate A relative to the control is determined using the formula shown below. Further, 4 days after administration, the mice are sacrificed, the tumor is excised, its weight is measured, and the growth inhibition rate B is determined in the same manner. The results are shown in Table 1.

Growth inhibition rate A (1 to 3 days after administration) Growth inhibition rate B (4 days after administration)! Growth inhibition rate samples against Lf Colon26 were all administered at 300 m9/kg intraperitoneally.

Sample 1: S, faecium R672 bacterial cell hot water extract Sample 2: 40% saturated ammonium sulfate precipitate according to Example 1 Sample 3: 20% saturated ammonium sulfate precipitate according to Example 2 Sample 4: 20% to 40 according to Example 2 % Saturated ammonium sulfate precipitate *Significant difference at risk rate of 5% or less Patent applicant Rohto Pharmaceutical Co., Ltd. Agent Patent attorney Said Ao (one other person) procedural amendment June 29, 1985 1゜ incident Indication: 1985 Patent Application No. 1129162, Name of the invention: Anti-tumor active substance 3, Relationship with the person making the amendment Patent applicant address: 1-8-1 Tatsunishi, Ikuno-ku, Osaka-shi, Osaka Name: Rohto I Medicine Co., Ltd. 4, agent address: 7, Kokugi Building, 2-10 Honmachi, Higashi-ku, Osaka, Osaka Prefecture.
Contents of the amendment The following points in the description will be amended.

■) Page 5, line 7, “For example 70%” should be changed to “For example 10%~”
80%” was corrected.

2) Delete "(approximately 20% by weight)" on page 7, lines 8-9.

3) On page 7, line 13, “furthermore” was amended to “further.”

4) Page 9, line 5 rA (1 to 3 days after administration)” to “A(
1 to 3 days after administration)".

5) On page 9, line 8, "after administration" was corrected to "administration."

6) 2nd line directly below 9th r300mg/KgJ [200m
g/K gJ and correction.

that's all

Claims (1)

[Claims] 1. An antitumor active substance obtained by treating Streptococcus faecium cells with hot water and salting out the extract. 2. The substance according to item 1, wherein the temperature of the hot water treatment is 50 to 100°C. 3. The substance according to item 1, which is salted out from the extract with ammonium sulfate at a final concentration of about 20% by weight.