DE2615099A1 - PROCESS FOR MANUFACTURING A NEW ANTICARCINOMA SUBSTANCE - Google Patents

Description

No. 8-1-108, 2-chome, Ebisu-Nishi
Shibuya-ku

Tokyo (Japan)

Process for the production of a new anti-cancer substance

The invention relates to improving a method for producing a new anti-carcinoma substance from the supernatant obtained by removing the mycelium from a culture solution obtained from the Cultivation of the Hachijoensis Streptomycin, which is a streptomycin obtained in 1951 by Hosoya, Komatsu, Soeda, Yamaguchi, and Sonoda was discovered.

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26th

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The microbiological properties of Streptomyces hachijoensis are in Hosoya, Komatsu, Soeda, Yamaguchi and Sonoda, "Trichomycin, New Antibiotic Substance Having Trichimonacidal and Anti-Fungal Activities" (Journal of Antibiotics, Volume 10, Page 564 (1952)) with regard to the Hachijoensis Streptomycin culture strain H-2609 and in Yamaguchi "Studies of the Antibiotic Substance Producing Strains H-2075, H-2609 (Hachijoensis Streptomycin No. Sp.) And H-3030 (The Journal of Antibiotics Series a, Volume VII, No. 1, pages 10-14 (1954) with a view to to the culture strains H-2609 and H-2075 of Streptomyces Hachijoensis.

Methods for producing medicaments using Streptomyces hachijoensis are known. in the In particular, a method can be mentioned here in which the mycelium of the strain H-2609 of Streptomyces Hachijoensis is extracted by aqueous acetone or aqueous methanol and a method in which a culture solution which contains mycelium of the weakly acidic culture strain, whereupon a small amount of activated Clay was added to the culture solution, the mixture was stirred, and the substance absorbed on the activated clay with aqueous Acetone and / or aqueous methanol is extracted. Trichomycin, which has antitrichomonal, antifungal and anti-fermentation activities is produced by this process.

Recently, a method for producing a new anti-carcinoma substance has been developed in which the same Strain as used in the above methods for making trichomycin, but the end product, its Use and the extraction stage for the effective component completely different from these processes for producing

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ORIGINAL INSPECTED

261bÜ9G

Position of trichomycins are.

This new anti-cancer substance is made after a procedure obtained in which the mycelium is removed from a culture solution of Streptomyces hachijoensis to produce a Precipitation, a zinc chloride solution was added to the supernatant, the precipitate was collected, to adjust the pH value to 7.0 to 9 »4 and thus for the extraction of the precipitate sodium hydrogen phosphate solution to the collected Precipitate added, at least one of the three solvents methanol, ethanol or acetone to the extract added to initiate the precipitation, then the resulting precipitate dissolved in distilled water and the solution is finally filtered off sterile.

The aim of the invention is to obtain a new anti-carcinoma substance, which is improved over the substance discussed above, as well as a method for producing a such new anti-carcinoma substance, according to which mycelium was removed from a culture solution of Streptomyces hachijoensis an organic solvent is added to the supernatant, the resulting precipitate is collected, water, saline solution, Netrium hydrogen phosphate or glucose was added to the precipitate and the solution was then filtered aseptic will.

The substance obtained by the process according to the invention has a significantly higher activity than the preparation which is obtained by the procedure described above.

When administering anti-cancer substances that are already known arise hypoleucocytosis or the like as undesirable side effects. Therefore it is not to-

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ORIGINAL INSPECTED

casual, these anti-cancer addictions over a long time For a period of time and usually the administration is interrupted »The anti-cancer substance after the invention, however, does not cause hypoleucocytosis and other disorders of hemopoietic organs, no liver dysfunction, Kidney dysfunction, pulmonary fibrosis, or the like, even if they have been for a long time administered away.

The physico-chemical properties of the new anti-cancer substance according to the invention are listed below:

1. Appearance:

white to light yellow powder

2. Solubility:

Easily soluble in water, insoluble in methanol, ethanol, chloroform, ether, pyridine, ethylene chloride, dioxane and Acetic acid.

3. Optical rotation:
+ 17.1 ° in water

4. Color reactions:

reacts positively to Fehling's reaction, silver mirror reaction and the Molisch reaction, and negative for the ninhydrin reaction and with xantoproteinic acid.

5. Values of the elemental analysis:

C: 33.8 %
H: 6.33 %

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ORIGINAL INSPECTED

-r- - 5-

O: 59.86 %
Ash content: 15.2%

6. Molecular weight:

The molecular weight without ash is 1200 (measured by the vapor pressure difference method using a Hitchi 115 molecular weight measuring instrument)

7. Molecular formula:

From the elemental analysis values given above and the result of the molecular weight measurement, it is assumed as given that the substance without the ash content can be expressed by the following formula:

^C 34 ^H 76 ° 45

8. Ultraviolet absorption:

A maximum absorption is observed at 277 m / in the case of an aqueous solution.

9. Infrared absorption:

Clear maxima occur at 1030, 1655, 2920 and 3450 cm "* ¹

The anti-cancer test was made with the new anti-cancer Substance carried out according to the invention. It was used in the experiment of Ehrlich's ascites tumor, whereby 0.1 ml of a solution containing 10 ^ / ml of Ehrlich's mycelium Ascites tumor, mice from the DD-S series were injected. The new one came within 3 hours Substance injected once intraperineally. (The dose was 6.25, 12.5, 25.50 or 100 g). The result was the finding that tumor can be prevented in the mice

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if they had an injection of the new substance at a dose of 12, 5 or more.

Based on the Invitro test developed by the inventor of the present invention, it was found that the substance exhibited high anti-carcinogenic activity when subjected to a deactivation test by color reduction using 2, 6-dichlophenol-indophenol. Also with the catalytic reaction test (a modification of the Niike method) showed the new substance against Carcinomas have high anti-carcinogenic activity.

1 to 2 mg of the new anticarcinoma substance were in 0.5 to 1.0 ml of a physiological saline solution dissolved and injected subcutaneously once a day into patients who had received a beneficial gastric resection and no undesirable side effects were found.

The invention is described in more detail below with reference to the examples in connection with the drawings:

The drawings show in

Fig. 1 is an ultraviolet absorption spectrum of the new Anti-cancer substance

and in

Fig. 2 shows an infrared absorption spectrum of the substance according to the invention.

example 1

(a) The strain H-2609 of Streoptomyces Hachijoensis was in

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2615039

a culture solution of 5 1 applied, the 0.5 % polypeptones, 1.0% proteose peptone (brand name of the Difco & Co. product), 0.3 % meat extract (brand name of the Difco & Co. product), 0 , 75 % sodium chloride, 0.5 % glucose and 2.0% starch. The shaken culture is kept at 27 to 30 ° C for 4 to 5 days. Mycelium is removed from the culture solution by centrifugation and the supernatant is collected.

(b) Cooled methanol is added to the supernatant in four times the amount thereof, the resulting precipitate collected and a dilute sodium hydrogen phosphate solution added to the precipitation in order to adjust the pH to a weakly alkaline value between 7.0 and 9.0 and effect the extraction. The resulting extract is filtered off germ-free and freeze-dried to remove the obtain new anti-cancer substance.

Instead of methanol, ethanol or acetone can also be used in the experiment. Instead of sodium hydrogen phosphate solution Saline, distilled water, or glucose can also be used in the experiment.

Example 2

A saturated ammonium sulfate solution is added to the supernatant obtained in (a) of Example 1 to a saturation of 1/3, after which the supernatant under reduced pressure at a temperature of less than 30 ^° C. to a volume of the supernatant of 1/5 to was concentrated to 1/10 of the original amount. The precipitate formed was removed. A saturated ammonium sulfate solution was added until the supernatant became saturated

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of 2/3. The precipitate was collected and it became distilled water (or saline solution) was added to the precipitate to dissolve it therein. The solution was through a cell glass tube (or a beef intestinal membrane) dialyzed. After dialysis, the dialysate is filtered germ-free and freeze-dried so that the "intended." obtain new anti-cancer substance.

Example 3

A 50% strength zinc chloride solution is added to the supernatant obtained in (a) of Example 1 and the resulting precipitate is collected. A 10% sodium hydrogen phosphate solution is added to the precipitate in order to adjust the pH to a weakly alkaline value between 7.0 and 9.0 and to effect the extraction. A saturated ammonium sulfate solution is added to the extract up to a saturation of 1/3 and the precipitate that has formed is removed. A saturated ammonium sulfate solution is added to the supernatant until the solution has a saturation of 2/3. The precipitate formed is collected and distilled water (or saline solution) is added to dissolve it therein. The solution is dialyzed through a cellophane tube (or beef intestinal membrane). After dialysis, the dialysate is filtered sterile and freeze-dried in order to obtain the intended anti-carcinoma substance.

Example 4

A 50 % zinc chloride solution was added to the supernatant obtained in (a) of Example 1, and the resulting precipitate was collected. A 10 % sodium

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hydrogen phosphate solution was added to the precipitate in order to adjust the pH to a weakly alkaline value between 7.0 and 9.0 and to effect the extraction. A saturated ammonium sulfate solution was added to the extract to a saturation of 1/3 and the precipitate formed was removed. A saturated ammonium sulfate solution was added to the supernatant until the solution had a saturation of 2/3. The formed precipitate was collected and distilled water (or saline solution) to dissolve it therein. The solution was dialyzed through a cellophane tube (or bovine intestinal membrane). After dialysis, the Bialysat at a temperature of less than 30 ⁰ C under reduced pressure to 1/5 of its original volume was concentrated to 1/10. Four times the amount of cold methanol is added to the concentrate. The precipitate formed is collected and dissolved in distilled water (or saline solution). The solution is filtered aseptically and freeze-dried so as to obtain the intended anti-carcinoma substance.

In each of Examples 1 to 4 above, the yield of the novel anticarcinoma substance was about 500 mg.

Since the new anti-cancer substance has gone through the sterilization phase and is in powder form, it can administered to the body in the form of a solution in distilled water, physiological saline or glucose solution will.

When administering the new anti-carcinoma substance according to the invention to patients with gastric cancer, esophageal cancer, Throat cancer, lung cancer, ovarian cancer and uterine cancer, especially in patients with gastric cancer who received a salutary gastric resection and in

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administration of the new Antikarzinoii substance to patients, where surgery was impossible became relatively satisfactory Results achieved.

Claims: - 11 -

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Claims (4)

AA Claims (T) Process for the production of a new anti-carcinoma substance, characterized in that Mycelium removed from a culture solution of Streptomyces hachijoensis, an organic solvent from the supernatant added to induce precipitation, the precipitate collected, the collected precipitate with water, Saline, sodium hydrogen phosphate or glucose is extracted, and then the extract is filtered aseptic. 2. A method for producing a new anti-cancer substance, characterized in that Mycelium removed from a culture solution of Streptomyces Hachijoensis, the supernatant concentrated under reduced pressure, the concentrate is fractionated by ammonium sulfate, collected the precipitate formed during fractionation by the ammonium sulphate, the collected precipitate in water or dissolved in a saline solution and this solution is subjected to dialysis. 3. A method for the preparation of a new anti-carcinoma substance, characterized in that Mycelium from a culture solution of Streptomyces hachijoensis removed, a zinc chloride solution was added to the supernatant to obtain a precipitate, the precipitate collected, sodium hydrogen phosphate solution of the collected precipitate to adjust the pH to 7.0 to 9.0 and thus added to extract the precipitate, the extract subjected to fractionation by ammonium sulfate, the precipitate formed during fractionation by the ammonium sulfate is collected, the collected precipitate 609R4W1177 - 12 - - ftf - * 4Ά dissolved in water or a saline solution, this solution is subjected to dialysis and the dialysate is filtered sterile will. 4. A process for the preparation of a new anti-carcinoma substance, characterized in that Mycelium removed from a culture solution, a zinc chloride solution to the resulting supernatant to obtain added to a precipitate, the precipitate collected Sodium hydrogen phosphate solution of the collected precipitation to adjust the pH to 7.0 to 9 »0 and added to extract the precipitate, the extract subjected to fractionation by ammonium sulfate, then Dialysis and concentration under reduced pressure, an organic solvent with resulting concentrate to obtain a precipitate added, the precipitate dissolved in water or a saline solution and then the solution is filtered sterile. 609844/1177