NL8105281A - POLYSACCHARIDE MPS-80 HIGH MOULCULAR WEIGHT, METHOD FOR PREPARING IT AND THERAPEUTIC USE. - Google Patents

Description

* φ -1- 22265 / Vk / mb · * "· ** *

Short designation: High molecular weight polysaccharide MPS-80, method for its preparation and therapeutic use.

The invention relates to a new polysaccharide MPS-80. The invention further relates to a method for the preparation thereof and for the therapeutic use, in particular as an anti-tumor agent.

More particularly, the invention relates to a polysaccharide MPS-80 with a high viscosity and an anti-tumor action.

High molecular weight polysaccharide MPS-80 according to the invention can be obtained by a culture for high molecular polysaccharide MPS-80 producing bacteria belonging to the group consisting of genera Lactobacillus and Streptococcus. As examples for the bacteria capable of producing high molecular weight polysaccharide MPS-80 may be mentioned Lactobacillus Jugurti no. 851 "FERM BP-66 (FERM-P no. 5851)" * and Streptococcus thermophilus no. 127 "FERM BP-65 (FERM-P no. 5850), f deposited with Fermentation 20 Research Institute, Agency of Industrial Science and Technology. These bacteria can be used successfully.

The present invention therefore relates to high molecular weight polysaccharide MPS-80 and to a process for its preparation. High molecular weight polysaccharide MPS-80 can be obtained by culturing bacteria capable of producing high molecular weight polysaccharide MPS-80 and separating them from the liquid fraction and the bacteria-containing fraction of the culture mixture.

As culture media, any culture medium with an unlimited composition can be used. Examples of this are a preferred lactic acid bacteria-containing culture medium containing skim milk or whey.

With regard to the culture conditions, it can be stated that good results can be obtained, for example, by a static culture at a temperature of 20-45 ° C. The culture can be accomplished under conditions that allow the bacteria producing the high molecular weight polysaccharide MPS-80 to grow, whereby the high molecular weight polysaccharide MPS-80 is grown. · Ί. $ -2-22265 / Vk / rab.

The culture mixture of the bacteria containing the high molecular weight. producing polysaccharide MPS-80 is separated by centrifugation in a liquid fraction and a bacteria-containing fraction. From these 5 fractions the high molecular polysaccharide MPS-80 is collected which has an anti-tumor effect. The high molecular polysaccharide MPS-80 can be collected from the liquid fraction by a well known method such as gel filtration, chromatographic ion exchange, salting out, solvent fractionation, dialysis and the like. These operations can be used alone, or suitably combined. The high molecular polysaccharide MPS-80 can be collected from the bacteria-containing fraction, for example, by extracting it with water and then treating the extract in the same manner as above to collect it from the liquid fraction.

In the method of the invention, generally, the bacteria producing the high molecular polysaccharide MPS-80 are grown in a whey-containing medium and the culture mixture is centrifuged to obtain a separated top liquid layer. Then an organic solvent is added to this top liquid layer to form a precipitate.

On the other hand, the bacteria-containing fraction obtained by the centrifugation is subjected to the extraction with water and then the extract is subjected to the centrifugation to obtain an extracted supernatant.

An organic solvent is added to the liquid above to obtain a precipitate.

Both precipitates obtained as indicated above are dissolved in water and an organic solvent is added thereto to effect reprecipitation. The precipitates are dissolved in a suitable buffer solution and supplied to an ion exchanger which is sufficiently buffered with the same buffer solution as indicated above. Then the same buffer solution is passed through an ion exchanger. A liquid with the high molecular polysaccharide that is not adsorbed on the ion exchanger is recovered and dialyzed using ion-exchanged water. The dialyzed solution is lyophilized to obtain purified high molecular polysaccharide MPS-80.

The thus obtained high molecular polysaccharide MPS-80 8105281 • *.

f Λ -3- 22265 / Vk / mb has an excellent anti-tumor effect and is suitable as an anti-tumor agent, while its high viscosity makes it suitable as a thickener.

Physicochemical properties of the high molecular weight polysaccharide 5 MPS-80 of the invention are: 1) elemental analysis: 42.2 $ C, 6.9 $ H, and 50.4 $ 0.

2) Molecular weight: a) Molecular weight determined by ultrafiltration method: The results of the ultrafiltration by Sepharose 2B are indicated at 10 in Fig. 1. This polysaccharide is fractionated near the open volume under conditions in a 2.5 x column 40.5 cm, 5 g fraction, 2.5 mg (1 ml) sample which is applied and 0.05 M phosphate buffer solution (pH = 6.0) are used as developer.

b) molecular weight determined by ultracentrifugation: A sample was dissolved in 0.2 M phosphate buffer solution (pH = 7.3) and the resulting solution with a concentration of 0.1 $ was subjected to ultracentrifugation (set rotation speed: 51,200 revolutions per minute) to obtain a sedimentation constant of 7.90S (S: Svedberg unit). (The sedimentation pattern was single).

3) Melting point (decomposition point): this polysaccharide begins to decolorize at about 262 ° C and turns black at 263-264 ° C.

18 4) Specific rotational power: [<x] Q = +33.2 (concentration = 0.5 $).

5) Ultraviolet absorption spectrum: see fig. 2.

25 6) Infrared absorption spectrum: see fig. 3.

7) Solubility in Solvents: Soluble in water, but practically insoluble in methanol, ethanol, acetone and ether.

8) Color reactions: a) Molonic reaction: + '30 b) anthron reaction: + c) cysteine sulfuric acid reaction: + d) aniline hydrochloric acid reaction: - e) carbazole sulfuric acid reaction: - f) Elson-Morgan reaction: - 35 g) Biuret reaction: - 9) Basicity, acidity or neutral: 0.1-0.5% aqueous solution of this polysaccharide has a neutral pH.

8105281-4-22265 / Vk / rab 10) Color: lyophilized, polysaccharide is obtained in the form of white fibers.

11) Saccharide is part of the polysaccharide: Saccharide being part of the polysaccharide was examined by gas-liquid chromatographic analysis using 5% SE-52 (2m column) which was performed as follows: the column was heated to 150 ° C-230 ° C (3 ° C / minute). The sample was hydrolyzed with 2N-H 2 SO 4 in boiling water for 4 hours, then neutralized with barium carbonate and subjected to filtration. The filtrate was demineralized with Amberlite IRA-410 and Amberlite IR-120B, evaporated to dryness and subjected to TMS treatment followed by GLC. As a result, glucose and galactose was observed as the saccharide present in the polysaccharide.

12) Amount of saccharide as part of the polysaccharide: the sample was hydrolyzed with 2N-H 2 SO 4 in boiling water for 4 hours, neutralized with barium carbonate and subjected to filtration. The filtrate was examined to determine how much saccharide was part of the polysaccharide. The glucose to galactose ratio was 2.2-1.9 to 1.

13) C-NMR spectrum (in D20 with TMS as standard) (ppm): the results are shown in Fig. 4.

14) Decomposition with Enzymes: This polysaccharide was dissolved in 0.05 M acetate buffer solution and subjected to reaction with various enzymes. The enzyme decomposition was evaluated based on the increased amount of reduced sugar by the Somogyi-Nelson method. The enzymes used and the reaction conditions were: (the enzymes a) -e are products of Boehringer).

a) öd-amylase, pH = 5.9, temperature is 37 ° C, duration is 4 hours, 30 b) ^ - amylase, pH s 4.8, temperature is 30 ° C, duration is 4 hours, o) / 3-galactosidase, pH 4.8, temperature is 30 ° C, time is 4 hours, d) amyloglueosidase, pH = 4.8, temperature is 30 ° C, time is 4 hours, e) <X. galactosidase, pH = 4.8, temperature is 30 ° C, duration is 4 hours.

No increase was observed in the amount of 8105281-52265 / Vk / mb reduced sugar under the above conditions a) -e).

15) LD * Female mice (ddY 5 w), du with an average body weight of 21.3 g, each group consisting of 7 mice, were used for this experiment. A solution of a sample in physiological saline was administered intraperitoneally in varying amounts to the mice once and these were observed for 10 days to determine the LD1 value. As a result, it was found that the LD_ value was not less than 200 mg / kg

DU

body weight.

The invention will be further elucidated by means of the following examples.

Example I

Lactobacillus Jugurti no. 851 "FERM BP-66 (FERM-P no. 5051)" * was seeded in 10 liters of whey medium (10 wt? Whey powder plus 0.5 wt.? 15 brewer's yeast extract) and the static culture was maintained for 24 hours was effected at a temperature of 37 ° C. After the end of the culture, the culture mixture was centrifuged (10,000 rpm for 15 minutes) to obtain 9.2 liters of the culture supernatant. Ethyl alcohol (99.5?) was added of the supernatant in such an amount that the final concentration thereof would be 35 vol .. A precipitate formed in this operation was centrifuged (10,000 rpm for 5 minutes) to obtain 1.2 g of precipitate.

Water which was ion-exchanged was added to the precipitate to obtain a solution. Insoluble material was removed from the solution by centrifugation (10,000 rpm for 15 minutes). This treatment was repeated a total of three times to obtain 800 mg of crude purified product.

The crude purified product obtained as indicated above was dissolved in 0.05 M phosphate buffer solution (pH 6.0) and the solution was fed to a column filled with diethylaminoethyl cellulose (DEAE cellulose) buffered sufficiently with the same buffer solution . The buffer solution was passed through the column and then the solution containing the substance not adsorbed on DEAE cellulose was collected. The solution with the non-adsorbed substance was lyophilized, dissolved in water that had been ion-exchanged, and dialyzed through a dialysis tube using water that had been ion-exchanged at 10 ° C for four days. After finishing dialysis 8105281-62265 / Vk / mb, the solution thus obtained was lyophilized, from which 500 mg of lyophilized sample of high molecular weight polysaccharide MPS-80 was obtained.

Example II

To 10 liters of a 10 wt.% Whey powder solution, 0.5 wt. 56 brewer's yeast extract was added to obtain a culture medium.

Streptococcus thermophilus No. 127, "FERM BP-65 (FERM-P No. 5850)" was seeded in this medium and the static culture was effected for 20 hours at a temperature of 37 ° C. After the end of the culture, the culture mixture was centrifuged (10,000 revolutions per minute, for 15 minutes) to obtain 9 liters of supernatant culture liquid. Ethyl alcohol (99.550) was added to the supernatant in such an amount that its final concentration would be 50 vol.%. By centrifugation (10,000 rpm, 5 minutes), 1.7 g of precipitate obtained.

Ion-exchanged mater was added to the precipitate to obtain a solution. An insoluble material was removed from the solution by centrifugation (10,000 rpm, for 15 minutes).

• This treatment was repeated a total of three times to obtain d, 2. g roughly purified product.

The crude purified product obtained as indicated above was dissolved in 0.05 M phosphate buffer solution (pH = 6.0) and the solution was fed to a column filled with diethylaminoethyl cellulose (DEAE cellulose) which had been sufficiently buffered with the same buffer solution.

The buffer solution was passed through the column and then the solution containing a substance that had not been adsorbed on DEAE cellulose was collected. The unadsorbed substance solution was freeze-dried, dissolved in water that had been ion-exchanged and dialyzed by a dialysis tube using water that had been ion-exchanged at 10 ° C for four days.

After dialysis had ended, the solution thus treated was freeze-dried to give 800 mg of a freeze-dried sample of high molecular weight polysaccharide MPS-80.

After column chromatography with Sepharose 2B and ultracentrifugal analysis, it was shown that the above two 8105281 m -7-22265 / Vk / mb samples were the same single substance. When the sample was dissolved in water, a colorless clear solution was obtained.

The invention is further elucidated by means of the following experiments and with reference to the drawing in which: Fig. 1 shows the molecular weight distribution of the high molecular polysaccharide MPS-80 obtained by ultrafiltration, where a is the high molecular polysaccharide MPS -80 represents and b, fBlue Dextran ", FIG. 2 represents the UV absorption spectrum of high molecular weight polysaccharide MPS-80 and FIG. 3 represents its infrared absorption spectra, and FIG. 4 shows the C-NMR- spectrum of this.

Experiment 1

Influence on Ehrlich carcinoma ascites.

For this purpose, 8-week-old female ddi mice were used.

Each group consisted of 7 mice. Ehrlich ascites tumor cells in the amount of 1 x 10, which had been cultured in ddY mice for one week, were seeded intraperitoneally. From the following day, a physiological saline solution was administered to a comparison group and a solution of the high molecular polysaccharide MPS-80 obtained according to Example I in physiological saline was administered to the test group, which administration was administered intraperitoneally for Was run continuously for 9 days. The dosages were 15 mg / kg / day and 60 mg / kg / day.

The influence of the high molecular polysaccharide MPS-80 on Ehrlich carcinoma ascites was assessed depending on the extent of the extension of life compared to the blank group. The results obtained are summarized in Table A.

TABLE A ~ 30 mean rate of number of animals dose survival length of those still alive / ke-Zdao-l time the lifespans after 60 days img / icg / dagj (days) __ (J) __ blank 28 100 0/7 35 15 28 100 3/7 60> 60> 214 5/7 8105281 - * φ -8- 22265 / Vk / mb

The results of Table A show that the high molecular polysaccharide MPS-80 had strong anti-tumor activity against Ehrlich carcinoma ascites.

Experiment 2 5 Influence on Ehrlich carcinoma ascites.

Female ddY mice 8 weeks old were used in this experiment. Each group consisted of 7 mice. Ehrlich ascites tumor cells, which had been sequentially cultured using ddY mice for one week, were seeded intraperitoneally in an amount of 1 x 10 4. On this same day, a blank group was administered intraperitoneally with a physiological saline solution and a solution of the high molecular polysaccharide MPS-80 obtained according to Example II, in a physiological saline solution to the test group. Administration was continued for three days. The 15 doses were 12.5 mg / kg / day, 25.0 mg / kg / day and 50 mg / kg / day.

The results obtained are summarized in Table B, TABLE B.

mean extent of number of animals dose survival length of those who were still alive - - (me / kc / dac) time of life after 60 days 20 mg / kg / day y (days) __ (%) __ blank 29 100 0 / 7 12.5 33 114 1/7 25.0 39 134 3/7 25 50.0 29 124 2/7

The results of Table B show that the high molecular weight polysaccharide MPS-80 had an effect on some 30 animals still alive after 60 days, although no notable influence was observed on the extent of the life extension.

Experiment 3

The influence on subcutaneously implanted Ehrlich carcinoma.

Female ddY mice 8 weeks old were used for this. Each group consisted of 8 mice. Ehrlich ascites tumor cells that had been cultured using ddY mice for one week were implanted subcutaneously into the mice in an amount of 1 x 10 4. From the next day, a physiological saline 8105281 m -9-22265 / Vk / mb solution was administered to a comparative group and a solution of the high molecular polysaccharide MPS-80 obtained as indicated in Example X was administered in a physiological saline administered intraperitoneally to the test group for 8 days continuously. The dosages were 12.5 mg / kg / day and 25.0 mg / kg / day.

The influence of the high molecular polysaccharide MPS-80 on subcutaneously implanted Ehrlich carcinoma was assessed based on the mice in which the solid tumor completely deteriorated. The results obtained are summarized in Table C.

10 TABLE C

number of mice whose solid tumor dose has decreased 50 days after the (mg / kg / day) transplant 15 blank 0/8 12.5 3/8 25.0 4/8 20

It is clear from the results of Table C that the high molecular polysaccharide MPS-80 had notable effects on the subcutaneously implanted Ehrlich carcinoma.

Experiment 4 25 Influence on ascites Sarcoma-180.

Female ddY mice 8 weeks old were used. Each group consisted of 5 mice. Sarcoma 180 ascites tumor cells which had been cultured using ddY mice for one week were seeded intraperitoneally in an amount of 1 x 10.

From the next day, a physiological saline solution was administered to a comparative group and a solution of high molecular polysaccharide MPS-80 as obtained according to Example I, in a physiological saline solution, was administered intraperitoneally continuously for 9 days to the test group. The dosages were 12.5 mg / kg / day, 25.0 mg / kg / day and 50 mg / kg / day. The influence of the high molecular polysaccharide MPS-80 on ascites Sarcoma-180 was assessed by the extent of the extension of life compared to the blank group. The results obtained are summarized in Table D.

8105281 -TO- 22265 / Vk / mb. λ

TABLE D

average degree of number of animals dose survival length of those still alive /. »Lifetime after 60 days (mg / tcg / day) jdays) (t) __ 5 bianco 23 100 0/5 12.5 24 104 2/5 25.0> 60> 261 3/5 50.0 44 191 2/5 10 ____

The results of Table D show that the high molecular polysaccharide MPS-80 has a strong anti-tumor effect on ascites Sarcoma-180.

15 Experiment 5

The influence on lymphocytic leukemia P-388.

Male CDF1 mice 6 weeks old were used for this. Each group consisted of 8 mice. P-388 cells were cultured using DBA mice for a week and 3 were transplanted in an amount of 5 x 10 intraperitoneally.

The influences of these were investigated using a combination of Mitomycin C with high molecular polysaccharide MPS-80 obtained in Example I.

On the day after transplantation, the intraperitoneal administration of a physiological saline was started to the blank group.

Administration was continued for 10 days. Mitomycin C was administered once in an amount of 1 mg / kg to a group to which only Mitomycin C was administered. The group to be investigated was administered both Mitomycin C and high molecular polysaccharide MPS-80, 30 Mitomycin C in an amount of 1 mg / kg being administered only once the day after transplantation, and then high molecular polysaccharide MPS-80 was administered administered intraperitoneally continuously for 9 days from the day after Mitomycin C administration. The high molecular polysaccharide MPS-80 dose was 12.5 mg / kg / day, 25.0 rag / kg / day, 50.0 mg / kg / day, and 77.5 mg / kg / day.

The influence obtained using high molecular polysaccharide MPS-80 in combination with Mitomycin C was assessed depending on the extent of extension of life and the number of animals that were still alive 8105281-11-22 65 / Vk / mb after 70 days.

The results obtained are summarized in Table E.

TABLE E

mean extent of number of animals 5 doserine (ms / ks / dae) survival length of the still alive dose ung / kg / dayyear life after 70 days __ (days) __ (%) __ blank 1410 100 0 / 8

Mitomyein C (1 mg) i8.0 129 0/8 10 * 1 mg M-C 1 + 12.5 mg MPS ^ 21.0 150 0/8 1 mg M-C + 25.0 mg MPS 19.5 139 0 / 8 1 mgM-C + 50.0 mg MPS 20.5 146 0/8 15 1 mgM-C +77.5 mg MPS 21.5 154 2/8 »1)

Comments: M-C = Mitomycin-C

MPS = high molecular weight polysaccharide MPS-80.

From the results of Table E it is clear that the extent of the extension of the life span and the number of animals still alive after 70 days reflects the influence obtained by the combination of the high molecular polysaccharide MPS-80 with Mitomyein C.

According to the invention, a new substance is obtained in the form of high molecular weight polysaccharide MPS-80 by culturing bacteria producing high molecular weight polysaccharide MPS-80 belonging to genera Lactobacillus or Streptococcus. This new high molecular weight polysaccharide MPS-80 has excellent anti-tumor activity and based on this it is expected that this substance can be used as an anti-tumor agent.

- CONCLUSIONS - 8105281

Claims (16)

2. Molecular weight: a) Molecular weight determined by ultrafiltration method: the results of the ultrafiltration by Sepharose 2B are shown in Fig. 1. This polysaccharide is fractionated near the open volume under conditions in a 2.5 x 40.5 cm column .5 g fraction, 2.5 mg (1 ml) sample which is applied and 0.05 M phosphate buffer solution (pH = 6.0) is used as developer. b) molecular weight determined by ultracentrifugation: A sample was dissolved in 0.2 M phosphate buffer solution (pH = 7.3) and the resulting solution at a concentration of 0.1 $ was subjected to ultracentrifugation (set rotation speed; 51,200 revolutions per minute ) to obtain a sedimentation constant of 7.98S (S: Svedberg unit). (The sedimentation pattern was single). 3. Melting point (decomposition point): this polysaccharide begins to decolorize at about 262 ° C and turns black at 263-264 ° C. 4. Specific rotational power {oJ} +33.2 (concentration = 0.5 $). 5. Ultraviolet absorption spectrum: see fig. 2. 6. Infrared absorption spectrum: see fig. 3 · Solubility in solvents: soluble in water, but practically insoluble in methanol, ethanol, acetone and ether. 8. Color reactions: a) Molecular reaction: + b) anthron reaction: + c) cysteine sulfuric acid reaction: + d) aniline hydrochloric acid reaction: - e) carbazole sulfuric acid reaction: - f) Elson-Morgan reaction: - g) Biuret reaction : - 9. Basicity, Acidity or Neutral: 0.1-0.5% aqueous solution of this polysaccharide has a neutral pH. 8105281 "τ -13- 22265 / Vk / mb 10. Color: Lyophilized polysaccharide is obtained in the form of white fibers. 11. Saccharide forming part of the polysaccharide: Saccharide forming part of the polysaccharide was examined by gas-liquid chromatographic analysis using 5% SE-52 (2m column) which was performed as follows: The sample was hydrolyzed with ZN-HgSO4. , in boiling water for 4 hours, then neutralized with barium carbonate and subjected to filtration. The filtrate was demineralized with Amberlite IRA-410 and Amberlite IR-120B, evaporated to dryness and subjected to TMS treatment followed by GLC. As a result, glucose and galactose was observed as the saccharide present in the polysaccharide. 12. Amount of saccharide as constituent of the polysaccharide: The sample was hydrolysed with ZN-SO 2 in boiling water for 4 hours, neutralized with barium carbonate and subjected to filtration. The filtrate was examined enzymatically to determine how much saccharide was part of the polysaccharide. The glucose to galactose ratio was 2.2-1.9 to 1. 13. C-NMR spectrum (in D20 with TMS as standard) (ppm): The results are shown in Figure 4. 14. Decomposition with Enzymes: This polysaccharide was dissolved in 0.05 M acetate buffer solution and subjected to reaction with various enzymes. The enzymes decomposition was evaluated based on the increased amount of reduced sugar according to the Somogyi-Nelson method. The enzymes used and the reaction conditions were: (enzymes a) -e are products of Boehringer). a) -amylase, pH = 5.9, temperature is 37 ° C, duration is 4 hours, b) β-amylase, pH = 4.8, temperature is 30 ° C, duration is 30. hours, c) β-galactosidase, pH = 4.8, temperature is 30 ° C, time is 4 hours, d) amyloglucosidase, pH = 4.8, temperature is 30 ° C, time is 4 hours, 35 e) oC-galactosidase, pH = 4.8, temperature is 30 ° C, duration is 4 hours. No increase in the amount of reduced sugar was observed under the above conditions a) -e). 8105281 • »_ ε -14- 22265 / Uk / rab 15. Female sex LDgQ-houses (ddY 5 w), with an average body weight of 21.3 g, each group consisting of 7 mice, were used for this experiment. A solution of a sample in physiological saline was administered intraperitoneally 1 time in varying amounts to the mice and these were observed for 10 days to determine the LD ^ Q value. As a result, the LD10 value was found to be not less than 200 mg / kg body weight. 2. Process for preparing high molecular polysaccharide MPS-80, characterized in that high molecular polysaccharide MPS-80 is prepared by culturing bacteria producing high molecular polysaccharide MPS-80 belonging to the group consisting of genera Lactobacillus and Streptococcus and collecting high molecular polysaccharide MPS-80 from the culture mixture. 15 3 · Anti-tumor agent, characterized in that it contains high molecular polysaccharide MPS-80 as active substance. The method according to claim 2, characterized in that bacteria belonging to the group consisting of Lactobacillus Jugurti no. 851 "FERM BP-66 (FERM-P no. 5851)" and 20 Streptococcus thermophilus no. 127 "FERM BP are used. -65 (FERM-P no. 5850). Eindhoven, November 1981 8105281