DE1211749B - Process for obtaining lysis products from malignant tissues - Google Patents

Description

Process for obtaining lysis products from malignant tissues Es it is known that in the settlement of non-pathogenic, anaerobically growing microorganisms signs of lysis occur in malignant tissues.

J. R. MÖse and G. MÖse describe such phenomena in the Exposure to an Amylobacter strain from Ehrlich tumors (Journal of Cancer Research, 63 [1959], pp. 63 to 74, and 63 [19601, p. 447; German patent specification 1 118 403).

For the extraction of lysates from malignant tissues is very effective disadvantageous from that the vegetative forms of the microorganisms used, the cause the lysis processes alone. soon after a short growing period Spore pass over, whereby the lysis processes in one for the recovery of good Yields of lysis products are terminated at an early stage. For this reason the lysis phenomena described above could not yet be economically evaluated Find.

There has now been a method which avoids the disadvantage mentioned Extraction of lysis products found from malignant tissues, which is characterized is that on such malignant tissue non-pathogenic anaerobic microorganisms in the presence from physiologically compatible heavy metal salts or complexes can act and the lysis products obtained thereby isolated.

Carcinomas, sarcomas or leukomas, for example, are considered to be malignant tissue Consider and as non-pathogenic anaerobic microorganisms those of the genus Clostridium, preferably Clostridium butyricum, acetobutylicum, tyrobutyricum, tertium, rockenium or pectinovorum. Heavy metal salts or complexes are preferably used iron, copper, manganese, cobalt or zinc.

The isolation of the process products, d. H. of the lysis products in a manner known per se, for. B. by so-called puncture or other mechanical Separation from the cell structure.

The lysis products obtained in this way are distinguished by a twofold Effect off; on the one hand they have an antihypertensive effect, on the other hand they cause a Increase in serum bactericide. They can therefore be used as medicines. The superiority of the products of the process over lysates, which according to the state of in the art is shown, for example, that the former are effective against serum bactericidal increase considerably more. So the comparative investigation of the after Example 1 obtained lysate sterilized with penicillin in the serum bactericide test the quotient 0.3, while that mentioned in the last paragraph of Example 1, without Shared use of the iron dextran complex obtained a bactericidal index in the aforementioned test of 1.0. The indices were determined on mice 21 hours after the subcutaneous route Administration of the lysates.

The advantages of the method according to the invention still exist in that even with numerous tribes more apathogenic. anaerobically growing microorganisms, with which, without the addition of the above-mentioned salts or complexes of heavy metals, preferably iron. lysis products only give rise to such products to a small extent let win in good yield. It is also possible with the present method. use malignant tissue of various ages to obtain lysis products, while so far only limited ages of malignant tissues after exposure to anaerobic microorganisms tended to lyse.

In addition, it is essential for the claimed method Addition of salts or complexes of heavy metals. preferably of iron, achieved that for the carrier of malignant tissue, so z. B. rats, mice, guinea pigs, rabbits or dogs, toxic side effects in the formation of the lysis products and a any resulting reduction in yield can be avoided.

Suitable for carrying out the method according to the invention malignant tissue, for example carcinoma, sarcoma. Leukomas in vivo, or tissue cultures malignant strains such as I-IeLa. Hep. 1 or Hep. 3.

As microorganisms, anaerobic, apathogenic strains z. B. off of the genus Clostridium used, preferably Clostridium butyricum. acetobutylicum, tyrobutyricum, tertium, rockenium or pectinovorum.

When using malignant tissues in vivo, the spores are e.g. B. administered parenterally to one of the clostridial strains mentioned. The heavy metal salts respectively. Complexes are given orally or, preferably, parenterally.

As heavy metal compounds are such. 13. the physiological compatible salts or complexes of iron, copper, manganese, cobalt or zinc. Preferably iron compounds such as ferrous chloride, ferrous sulfate, ferrogluconate, ferrous tartrate, Ferrohydroxyd or iron dextran complexes used.

In the same way, cobalt gluconate, cobalt dextran complexes or Manganese citrate can be used.

The heavy metal salts or complexes mentioned are in physiological tolerated doses. Occurs in the course of 1 to 4 days after application a lysis of the malignant tissue, which is recognizable by the fluctuation of the previous coarse or solid tumor.

The resulting lysate is obtained by puncture. The one in the lysate contained vegetative forms of applied anaerobic microorganisms can be killed by adding antibiotics, e.g. penicillin or tetracycline.

Example 1 Mice with induced methylcholanthrene sarcoma, whose Diameter is between 6 and 40 mm for a weight of 1 to 3 g parenterally with 0.5 ml of an aqueous, about 100 million spores of the Clostridium butyricum-containing suspension treated. Simultaneously, or 24 hours after Starting with the application of spores, an iron dextran complex is added in the dosage of 7.5 mg given per day for 3 to 4 days. From the outside based on the tactile findings recognizable lysis determines the point in time at which the lysate is obtained by puncture.

With an initial tumor weight of 3 g, 2.8 to 3 g of lysate are obtained. The lysis is therefore 95%.

The lysate is sterilized by adding 10 units of penicillin.

In the comparative experiment without administration of iron dextran complex showed of ten mice only four mice a lysis of an average of 17010, six mice showed no lysis.

Example 2 rats having a lymphoid leukoma with a diameter between 8 and 60 mm and a tumor weight of 1 to 10 g, each is 0.5 ml of an aqueous spore suspension of Clostridium tyrobutyricum containing about 100 million spores administered parenterally. At the same time, or 24 hours later starting, a cobalt dextran complex is used in the dosage of 7.5 y per day Given 4 days. The onset of lysis can be seen macroscopically. When completed The lysate is lysed by puncture.

The yield of lysate is 900/0. By adding 10 units Penicillin is used to sterilize the lysate.

Example 3 The transplantation of Ehrlich's carcinoma is as follows performed; Subcutaneous tumors are carefully dissected and placed in an embryo crusher (Bergmann model) crushed. The tumor mass becomes 1:10 with physiological saline solution diluted. 0.1 ml of this suspension is applied subcutaneously to the neck of Swiss Mice inoculated. The well-localized tumors reach an approximate one after 8 to 10 days Diameter of 12 mm. Twenty animals that carry such tumors will die in the spore doses given in Table 1, each in 0.2 ml of physiological saline solution injected intravenously.

Treatment with iron dextran is started 24 hours after the spore application intramuscularly on ten animals given daily doses of 7.5 mg Fe have been pretreated.

The evaluation takes place on the 8th day after the spore injection. The increase the effectiveness of the combination treatment can be seen from the table below.

Table 1 Tumor size average Spore dose treatment number of diameter number of animals of lysis tumor weight Clostridium butyricum with a dose of iron dextran lyses mm% g 108 / 0.2 ml. v. none 10 to 14 10 10 41 1.69 108 / 0.2 ml. v. 4.7.5 mg Fe daily dose 10 to 14 10 10 90 0.49 24 hours after spores Control 10 to 14 10 0 0 1.27 Example 4 The induced methylcholanthrene fibrosarcoma develops in the course of 8 to 12 weeks after subcutaneous injection of 0.5 mg methylcholanthrene (Roche) in 0.5% cellulose ester suspension in Tylose (MH 300 p Kalle AG.) [Test animals: Swiss Mice].

The treatment and evaluation are carried out as in Example 3. Table 2 shows the clear effect of the combination treatment.

Table 2 Tumor size average Spore dose treatment number of diameter number of animals of lysis tumor weight Clostridium butyricum with a dose of iron dextran lyses mm% g 108 / 0.2 ml. v. none 13 to 20 10 4 7 4.17 100 / 0.2 ml. v. 3 7.5 mg Fe daily dose 14 to 18 10 10 66 1.76 24 hours after spores Control 13 to 20 10 0 0 4.99 Example 5 The transplantation of Ehrlich's carcinomas is carried out analogously to Example 4. The corresponding treatment takes place at an earlier point in time, namely when the tumors have an average diameter of 4 or 8 mm (test animals: Swiss Mice). The evaluation was made on the 12th day after the administration of the spores.

Table 3 Spore Dose Treatment Tumor Number of Circumference Average Clostridium butyricum with a dose of iron dextran lyses mm% g 108 / 0.2 ml iv none 4 10 0 0 0.64 108 / 0.2 ml iv 5. 7.5 mg Fe daily dose 4 10 2 20 0.47 24 hours after spores Control 4 10 0 0 0.59 108 / 0.2 ml iv none 8 10 1 6 1.86 108 / 0.2 ml 5 5. 7.5 mg Fe daily dose 8 9 3 20.2 0.93 24 hours after spores Control 8 10 0 0 2.07 Example 6 Solid Ehrlich carcinomas are treated as in Example 3, but with the difference that vanadyl sulfate is used instead of iron dextran. The success of the combination treatment can be seen from Table 4 (evaluation on the 10th day after the spore injection).

Table 4 Tumor size average Spore dose treatment number of diameter number of animals of lysis tumor weight Clostridium butyricum with vanadyl sulfate lyses mm% g 108 / 0.2 ml iv 5. 10 mg daily dose 10 to 12 5 5 72 0.59 24 hours after spores 108 / 0.2 ml iv none 9 to 12 10 10 33 2.35 Example 7 If, instead of vanadyl sulfate, manganese (II) chloride is used for the combination treatment and the procedure is otherwise as in Example 6, the result shown in Table 5 below is obtained.

Table 5 Tumor circumference mean Spore dose treatment number of diameter number of animals of lysis tumor weight Clostridium butyricum with manganese (II) chloride lyses mm 108 / 0.2 ml iv 5 10 mg daily dose 10 to 13 9 9 @ | 77 0.72 24 hours after spores 108 / 0.2 ml iv none 11 to 16 5 5 52 2.27 Example 8 The treatment of solid Ehrlich carcinomas is carried out analogously to Example 3, but with the difference that the cobalt salt of sebacic acid is used instead of iron dextran. The success of the combination treatment is shown in Table 6 (evaluation on the 10th day after the spore injection).

Table 6 Treatment tumor size average Spore dose number of with cobalt salt of sebacin diameter number of animals of lysis tumor weight Clostridium butyricum lyses acid mm% g 108 / 0.2 ml iv 5. 2 mg daily dose 8 to 14 9 8 56 1.26 24 hours after spores 108 / 0.2 ml iv none 11 to 16 5 5 52 2.27 Example 9 Solid Ehrlich carcinomas are treated as in Example 3, with the difference that, instead of iron dextran, basic bismuth (II) nitrate is used. The success of the combination treatment is shown in Table 7 (evaluation on the 10th day after the spore injection).

Table 7 Treatment tumor size average Spore dose number of with basic bismuth (II) - by the number of animals the lyses tumor weight Clostridium butyricum lyses nitrate mm% g 108 / 0.2 ml 5. 5 mg daily dose 11 5 5 54 0.98 24 hours after spores 108 / 0.2 ml iv none 8 to 12 10 10 33 2.35 Example 10 The treatment of solid Ehrlich carcinomas is carried out analogously to Example 3, but with the difference that zirconyl chloride is used instead of iron dextran. The success of the combination treatment is shown in Table 8 (evaluation on the 10th day after the spore injection).

Table 8 Tumor circumference mean Spore dose treatment number of diameter number of animals of lysis tumor weight Clostridium butyricum with zirconyl chloride lyses mm% g 108 / 0.2 ml 5th lmg daily dose 8 to 11 5 5 44 1.67 24 hours after spores 108 / 0.2 ml iv none 8 to 12 10 10 33 2.35 Example 11 The treatment of solid Ehrlich carcinomas is carried out analogously to Example 3, but with the difference that manganese (II) sulfate is used instead of iron dextran. The success of the combination treatment is shown in Table 9 (evaluation on the 10th day after the spore injection).

Table 9 Tumor circumference mean Spore dose treatment number of diameter number of animals of lysis tumor weight Clostridium butyricum with manganese (II) lyses mm% g 108 / 0.2 ml iv 5. 10 mg daily dose 8 to 12 10 9 55 0.59 24 hours after spores 108 / 0.2 ml iv none 8 to 12 10 10 33 2.35 Example 12 Instead of Ehrlich's carcinomas, Harding-Passey melanomas are used. Transplantation of the tumors and treatment are carried out analogously to Example 3.

The results are shown in Table 10. The spore cans will Injected intravenously as soon as the tumors reach a diameter of 11 to 17 mm to have.

Table 10 Scope average Spore dose treatment number of Number of animals in the lysis tumor weight Clostridium butyricum with iron dextran lyses 1 010 1 g 108 / 0.2 ml iv 5 5. 7.5 mg Fe daily dose 5 5 86 0.39 24 hours after spores 108 / 0.2 ml IV none 5 4 32 3.33 Example 13 Instead of Ehrlich's carcinoma, lymph sarcoma 4946 is used. The tumors are transplanted and treated as in Example 3. The results are shown in Table 11. The spore doses are injected intravenously as soon as the tumors have reached a diameter of 11 to 17 mm.

Table 11 Scope average Spore dose treatment number of Number of animals in the lysis tumor weight Clostridium butyricum with iron dextran lyses % g 108 / 0.2 ml. v. 5 7.5 5.7.5 mg Fe daily dose 10 10 82 0.43 24 hours after spores 108 / 0.2 ml iv | none | 10 | 0 | 0 | 1.46 Example 14 Instead of Ehrlich's carcinomas, the transplanted methylch. Sarcoma 2677 inserted. The tumors are transplanted and treated as in Example 3. The results are shown in Table 12. The spore doses are injected intravenously as soon as the tumors have reached a diameter of 11 to 17 mm.

Table 12 Scope average Spore dose treatment number of Number of animals in the lysis tumor weight Clostridium butyricum with iron dextran lyses % g 108 / 0.2 ml iv 5 5. 7.5 mg Fe daily dose 10 9 63 0.64 24 hours after spores 108 / 0.2 ml iv none 10 5 25 2.06 Example 15 The tumors used are spontaneous tumors from mice. The tumors were grown in female mice. Most of the cases are adenocarcinomas of the mammary glands. These tumors are used as soon as they reach a diameter of 10 to 17 mm.

The treatment is carried out as in Example 3.

Table 13 Scope average Spore dose treatment number of Number of animals in the lysis tumor weight Clostridium butyricum with iron dextran lyses % g 108 / 0.2 ml iv 5 5. 7.5 mg Fe daily dose 9 8 55 1.56 24 hours after spores 108 / 0.2 ml iv none 9 4 17 2.31 Example 16 Instead of Clostridium butyricum spores, Clostridium tyrobutyricum spores were used for the treatment. The increase in effectiveness caused by the additional use of iron dextran can be seen from Table 14. The test conditions are the same as in Example 15.

Table 14 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium tyrobutyricum with iron dextran lyses mm% g 108 / 0.2 ml iv 5 5. 7.5 mg Fe daily dose 10 to 14 5 74 0.82 24 hours after spores 108 / 0.2 ml iv none 9 to 13 5 38 1.73 Example 17 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium acetobutylicum spores are used. The increase in effectiveness caused by the additional use of iron dextran can be seen from Table 15.

Table 15 Spore dose Tumor circumference Average Treatment number of Clostridium diameter of the lyses tumor weight with iron dextran lyses acetobutylicum mm% g 108 / 0.2 ml iv 5 5: 7.5 mg Fe daily dose 10 to 14 4 64 0.94 24 hours after spores 108 / 0.2 ml iv none 10 to 14 4 36 3.38 Example 18 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium rockineum spores are used. The increase in effectiveness achieved through the combined use of iron dextran can be seen from Table 16.

Table 16 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium felsineum with iron dextran lyses mm% g 108 / 0.2 ml iv 5. 7.5 mg Fe daily dose 9 to 11 5 57 1.01 24 hours after spores 108 / 0.2 ml iv none 10 to 12 4 32 1.85 Example 19 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium sporogenes spores are used. The increase in effectiveness achieved by the combined use of iron dextran can be seen from Table 17.

Table 17 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium sporogenges with iron dextran lyses mm% g 108 / 0.2 ml iv 5. 7.5 mg Fe daily dose 12 to 14 5 64 0.88 24 hours after spores 108 / 0.2 ml iv none 11 to 15 5 30 1.78 Example 20 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium cochlearum spores are used. The increase in effectiveness achieved through the combined use of iron dextran can be seen from Table 18.

Table 18 Spore Dose Treatment Tumor Number of Circumference Average diameter of the lyses tumor weight Clostridium cochlearum with iron dextran lyses mm% g 108 / 0.2 ml iv 5. 7.5 mg Fe daily dose 6 to 10 5 86 0.35 24 hours after spores 108 / 0.2 ml iv none 8 to 10 5 42 1.85 Example 21 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium pasteurianum spores are used. The increase in effectiveness achieved by the combined use of iron dextran can be seen from Table 19.

Table 19 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium pasteurianum with iron dextran lysis mm% g 108 / 0.2 ml iv 5 5. 7.5 mg Fe daily dose 12 to 14 5 52 1.63 24 hours after spores 108 / 0.2 ml iv none 11 to 13 4 28 2.51 Example 22 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium roseum spores are used. The increase in effectiveness achieved through the combined use of iron dextran can be seen from Table 20.

Table 20 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium roseum with iron dextran lyses mm% g 5. 7.5 mg Fe daily dose 8 to 10 5 72 0.66 108 / 0.2 ml iv 24 hours after spores 108 / 0.2 ml iv none 8 to 10 5 42 1.71 Example 23 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium paraputrificum spores are used. The increase in effectiveness achieved by the combined use of iron dextron can be seen from Table 21.

Table 21 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium paraputrificum with iron dextran lyses mm% g 108 / 0.2 ml iv 5. 7.5 Fe daily dose 8 to 12 5 70 0.97 24 hours after spores 108 / 0.2 ml iv none 10 to 12 5 44 1.81 Example 24 The test conditions are the same as in Example 15, with the difference that Clostridium tertium spores are used instead of Clostridium butyricum spores. The increase in effectiveness achieved by the combined use of iron dextron can be seen from Table 22.

Table 22 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium tertium with iron dextran lyses mm% g 108 / 0.2 ml iv 5. 7.5 mg Fe daily dose 8 to 12 5 60 0.73 24 hours after spores 108 / 0.2 ml IV none 8 to 12 4 44 2.97 Example 25 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium multifermentan spores are used. The increase in effectiveness achieved by the combined use of iron dextron can be seen from Table 23. Table 23 Spore dose Tumor circumference Average Treatment number of Clostridium diameter of the lyses tumor weight multifermentan with bisendextran mm 0/0 g 108 / 0.2 ml iv 5. 7.5 mg Fe daily dose 8 to 11 5 64 0.57 24 hours after spores 108 / 0.2 ml iv none 6 to 10 3 16 1.65 Example 26 The test conditions are the same as in Example 15, with the difference that instead of Clostridium butyricum spores, Clostridium pectinovorum spores are used. The increase in effectiveness achieved by the combined use of iron dextron can be seen from Table 24.

Table 24 Tumor circumference mean Spore dose treatment number of diameter of the lyses tumor weight Clostridium pectinovorum with iron dextran lysis mm% g 108 / 0.2 ml iv 5 5. 7.5 mg Fe daily dose 8 to 13 5 66 0.65 24 hours after spores 108 / 0.2 ml iv none 6 to 12 4 20 1.59 Example 27 Tumor material is removed from freshly killed rats with well-developed Yoshida sarcoma under sterile conditions and cut into slices approximately 2 to 4 mm thick and 20 to 30 mm in size. These slices are placed in a mixture of brain liverheart medium (Baltimore Biological Lab / USA.) And trypticase soy broth (Difco Lab) in a ratio of 1: 1. 1 to 50 million spores of Clostridium butyricum and 3.75 mg of iron dextranate per milliliter of nutrient medium are then added to the medium. This is followed by incubation at 370 ° C. The tumor material is completely lysed over the course of 3 to 4 days. The lysate is freed from insoluble components by filtration and sterilized by adding penicillin.

Claims (4)

Claims: 1. Process for the production of lysis products from malignant tissues, thereby denotes that such malignant tissue is apathogenic Anaerobic microorganisms in the presence of physiologically compatible heavy metal salts or complexes can act and the lysis products obtained thereby isolated. 2. The method according to claim 1, characterized in that as malignant tissue carcinomas, sarcomas or leukomas are used. 3. The method according to claim 1, characterized in that as non-pathogenic anaerobic microorganisms of the genus Clostridium are used, preferably Clostridium butyricum, acetobutylicum, tyrobutyricum, tertium, rockenium or pectinovorum. 4. The method according to claim 1, characterized in that as Heavy metal salts or complexes those of iron, copper, manganese, cobalt or zinc used. Documents considered: German Auslegeschrift No. 1 118 403.