SK277936B6 - Method of production of preparations from cell walls or whole cells - Google Patents

Abstract

Method of production of preparations from cell walls or whole cells from Propionibacterium granulosum, Propionibacterium avidum and/or Propionibacterium acnes for use in radiotherapy and chemotherapy of tumors. Liquid culture of mentioned propionibacterium is treated by GasPak method by anaerobic conditions at temperature 37 degrees of Celsius, in this way gained growth of mass is grafted with dense flowed mass of propionibacterium, after incubation during 72 hours at 37 degrees of Celsius are cells separated by with centrifuge by cooling, the sediment is washed with distilled water and then is disintegrated by milling with glass beads, glass beads are separated from homogenisate by filtration on sintered glass by using of phosphate buffers with pH 7,2 value, milk suspension containing cell walls and cytoplasms is put through centrifuge sediment is taken into phosphate buffer by pH 7,2 value, autolytic enzymes are inactivated by boiling, cell walls containing proteine are cleared by incubation with trypsine suspension and toluene, cell walls are separated cleaned with trypsine digestion, washed with distilated water and then they are dried by frozen sublimation.

Description

A method of making cell wall or whole cell formulations of Propionibacterium granulosum, Propionibacterium avidum and / or Propionibacterium acnes for use in radiotherapy and tumor chemotherapy. The liquid culture of the respective propionibacterium is treated with the GasPak method under anaerobic conditions at 37 ° C, the meat growth thus obtained is inoculated with a dense propionibacterium washout, after incubation for 72 hours at 37 ° C, the cells are separated by centrifugation under cooling, washed with distilled water. it is then disintegrated by milling with glass beads, the glass beads are separated from the homogenate by filtration on a glass frit using phosphate buffers at pH 7.2, the milk-white suspension containing cell walls and centrifuged, the sediment is taken up in phosphate buffer at pH 7.2, autolytic enzymes are inactivated by boiling, cell walls containing protein are further purified by incubation with trypsin suspension and toluene, centrifuged cell walls purified by trypsin digestion, washed with distilled water and then freeze-dried.

Technical field

The invention relates to propionibacteria, in particular Propionibacterium granulosum, Propionibacterium avidum and / or Propionibacterium acnes for the production of cell preparations or whole cell preparations, which are useful in the treatment of tumors, as well as in radio or tumor chemotherapy.

BACKGROUND OF THE INVENTION

Preparations of anaerobic Coryneforms are known, for example C-Parvu, CN 6134, which are described as anti-tumor drugs. However, it has been shown that these preparations lead to undesirable side effects and complications in systemic or local therapy, so the literature recommends avoiding doses higher than 7.5 mg / m ^{2 of} surface area. This amount corresponds to approximately 20 mg per patient.

SUMMARY OF THE INVENTION

Preparations from whole cell deaths or cell wall propionibacteria have been found to be extremely effective in the chemotherapy or radiotherapy of tumors. These preparations are particularly suitable for supportive therapy. Efficacy is significant not only for topical use.

Experiments have shown that P. acnes, P. granulosum and P. avidum after intrapcritoneal injection to mice at 1.5 mg / mouse significantly prolonged the survival time of lethal (0 ^ 2193 C / kg) irradiated mice, with P. granulosum being the most effective . The preparations of the invention have been found to be suitable for stimulating CFU-S proliferation, i. to stimulate the travel of hematopoietic colony-forming units from the bone marrow to enter the cell cycle and to travel to the peripheral blood. This finding implies in clinical practice that the compositions of the invention are valuable as adjunctive therapies in radiotherapy of tumors, since they significantly increase the patient's tolerance to radiation. In addition, experiments have found that the compositions of the invention are effective against sarcoma 180 in mice and result in regression of more than 70% of sarcoma 180 tumors when administered intratumorally.

It has also been found that the formulations of the invention, when administered systemically to patients with primary or secondary lung tumors as adjunctive therapy in chemotherapy, help to prevent the major complications of cancer chemotherapy, namely the risk of infection. Accordingly, the compositions of the invention can be used as important antibacterially effective adjunctive therapies in cancer chemotherapy.

However, these results, which are exemplified in the following data of several in vivo tests, must be assessed as unexpected in any respect.

Cell wall preparations have proved to be particularly useful, since the risk of undesirable side effects is particularly low with this type of preparation. To date, cell wall preparations have not been clinically tested.

Accordingly, the present invention provides a process for the manufacture of cell wall or whole cell formulations of Propionibacterium granulosum, Propionibacterium avidum, and / or Propionibacterium acnes for use in radiotherapy and tumor chemotherapy, which comprises treating a liquid culture of a propionibacterium with the GasPak method. , in an airtight enclosure under anaerobic conditions formed with borohydride, sodium bicarbonate, citric acid and water at 37 ° C, the mass growth thus obtained is inoculated with a leach of propionibacterium, after incubation for 72 hours at 37 ° C, the cells are separated by centrifugation with cooling, the sediment is washed with distilled water and, if necessary, disintegrated then by grinding with glass beads, preferably with a diameter in the range of 0.17 to

0.18 mm, preferably using twice the volume of these glass beads, after microscopic examination for the absence of whole cells, the glass beads are separated from the homogenate by filtration on a glass frit using phosphate buffers at pH Ί2, the milky suspension containing cell walls and cytoplasms centrifuged , the sediment is taken up in phosphate buffer at pH Ί2, autolytic enzymes are inactivated by boiling, cell walls containing protein are further purified by incubation with trypsin suspension and toluene, centrifuged cell walls purified by trypsin digestion, washed with distilled water and then freeze dried .

According to a preferred embodiment of the invention, Propionibacterium granulosum and Propionibacterium avidum, in particular Propionibacterium granulosum strain KP 45 and Propionibacterium avidum strain KP 40 are used.

Injectable suspensions are preferably prepared in phosphate buffered saline. A particularly preferred concentration is 0.5 to 50 mg of active material per ml of solution. The preferred concentration is 2 to 12 mg / ml solution, especially 5 to 7.5 mg / ml solution. Especially suitable are intravenous infusion solutions which contain 15 to 30 mg of the preparation in 100 ml of solution. Such dosages have proven to be particularly advantageous for application in the treatment of primary and secondary neoplastic lung diseases, the first injection being given at least 10 days prior to chemotherapeutic treatment.

As the phosphate buffer, it is expedient to use a NaHPO./KH3PO4 phosphate buffer which conveniently has a pH of about 7.2.

The invention is not limited to the use of any special strain of propionibacteria. Various strains of propionibacteria are contemplated, for example, in FEMS Microbiology Letters, Volume 2, no. 1, July 1977, pages 5-9.

P. granulosum KP 45 and P. avidum KP 40 strains are particularly preferred. They were deposited in the German collection of microorganisms DSM, Gesellschaft für Biotechnologische Forschung mbH, Grisebachstrasse 8, D-3400 Gotttingen, NSE, under the deposit number DSM 1773 for P. granulosum KP 45 and 1772 for P. avidum KP 40, dated 11.3.1980. Both strains can be obtained from DSM in accordance with the declaration of release to be filed by the depositor with DSM and the German Patent Office (i.e. Form P2750).

P. granulosum, P. acnes and P. avidum can be isolated and grown from acne efflorescence streaks (Acne vulgaris, Acne papulopustulosa and Acnes conclobata). From a taxonomic standpoint, compare Hautartzt 30,242-267 (1979).

Other propionibacterial strains for use in the invention are described, for example, in Applied Microbiology, February 1973, pages 222-229, American Society for Microbiology, Vol. 25, No. 2.

Characterization of P. avidum propionibacteria strain KP 40 a

P. granulosum KP 45

SK 277936 Β6

Kati č. source Kind of Blocheeic reactions (Lit. i) sucrose Nal- Sortose bit Man- Sali- Ino-Eaculin · hydrolysis thread tin The 45 Selection of wound infection P. granulosu · 4 4 - - - - The 40 Taken with a sweat clinically healthy P. avidua 4 + - + + - 44-

KmA no. source Kind of Biochemical reactions (Lit. 1) indole Hitrate Gelatin Dextrin Melicitosis K 45 Selection of wound infection P. granulou · - - - 4 4 K 40 Taken from clinically healthy lapses P. avidu * - - 4 4 4

1 iKaeô jó. Serology (Lit t 2) Phag lysotype (Lit.3) source Kind of P.acnes KB P.granulocun P. 95 D34 56 avidun 0575 31 Kôl..KÔ13 • K 45 selection of infections. wounds P. granuloeua - + - - NT K 4012Created from (clinically and healthy) I i P. evidua - - - 4 - - NT

NT = untypable

1. G. Pulverer and H. L. Ko: Farmentative and Serological Studies on Propionibacterium acnes and Appl. Microb. 25: 222-229.1973

2. U. Höffler, H. L. Ko and G. Pulverer: Serotyping of Propionibacterium acnes and related microbial species. Fems Microbiology Letters, Vol. 2, 5 to 9,1977

3. E. C. Jong, H. L. Ko, and G. Pulverer Studies on Bacteriophages of P. acnes Med. Microbiol. Immunol. 161, 263-271, 1975

Der Hautarzt 30,242 to 247,179

DETAILED DESCRIPTION OF THE INVENTION

Differentiation of different types of propionibacteria from the acoresulence of Acne vulgaris.

The following examples serve to explain the preparation of the preparations of the invention.

Example 1

General procedure for obtaining cell walls.

The liquid culture of the respective propionibacterium was grown in a 1 liter volume (Erlenmeyer flask) at 37 ° C, under anaerobic conditions (GasPak method, BBL).

For this large scale cultivation, medium (A-broth ⁺ or tryptic soybean, Difco) with a dense suspension of propionibacteria was inoculated. The inoculum suspension was prepared by triturating a three-day Aagar propionibacterium culture in 10 ml of broth. After a culture time of 72 hours at 37 ° C, the cells were separated by centrifugation at 10,000 g (20 min) in a liquid Sorval R2 centrifuge. The sediment was washed three times with distilled water and then mixed with twice the volume of glass beads (diameter 0.17 to 0.18 mm) and disintegrated in a 10 cell mill for 1 to 1.5 hours. When the preparation was subjected to microscopic examination (Grampreparat) and no whole cells were present, the glass beads were separated on the G1 tritium using phosphate buffer (pH 7.2) from the homogenate.

The milk suspension (containing cell walls and cytoplasm) was centrifuged for 20 minutes at 40,000 g and the sediment was taken up in phosphate buffer (pH 7.2). Autolytic enzymes were inactivated by boiling in a water bath for 10 minutes.

These still protein-containing cell walls were further purified by incubation at 37 ° C for 24 hours with trypsin (Merck, 0.5 mg / ml suspension) and 1 ml of toluene per 100 ml suspension (to prevent bacterial growth).

The tryptic digestion of the purified cell wall was then centrifuged at 40,000 g (for 30 minutes) and washed three times with distilled water and then lyophilized.

+) A- Broth:

Bacto Casitone 12 g Bacto yeast extract 12 g KH2PO4 4 g MgSO4.7H2O 1 g glucose 4 g distilled water up to 1000 ml PH for A-agar + 28 g Bacto agar 72

++) Phosphate buffer:

1/15 MNajHPOUHjOa

Dissolve 1/15 M of KH3PCU in 1000 ml of distilled water, mix 612 g of sodium hydrogen phosphate solution with 388 g of potassium dihydrogen phosphate solution and adjust the pH to 7.2.

+++) Cell gas: Vibrogen - cell gas vi 3, Edmund Btthler, 7400 TUbingen.

Example 2

Preparation of suspensions of P. acnes (strain ATCO 6919), p. avidum (strain 0575, KP 40) and P. granulosum (strain KP 45)

According to the general procedure of Example 1, lyophilized propionibacteria of this kind are suspended in a sodium chloride solution with a phosphate buffer of the above-mentioned species at a concentration of 5.0 and 7.5 mg of Ani, respectively. The solution obtained is particularly suitable for intraperitoneal injection.

Example 3

Using the procedure of Example 2, an intraperitoneal injectable suspension of P. granulosum strain 45, 95-K, P. acnes strain ATCC 6919 and P. avidum strain 0575, KP 40 is prepared from lyophilized material at a concentration of 5 or 7.5 mg / ml. ml of cell material.

Example 4

Preparation of injectable suspensions for additional treatment in chemotherapy.

P. granulosum strain KP 45 was prepared according to the general procedure of Example 1. A suspension of 30 mg of cell material in 100 ml of a diffusion solution was prepared for intravenous administration.

The preparations of the invention may contain conventional examples. The preparations may be provided in ampoules or may be filled into sterile vials with a puncturable cap. Assembling with separate storage of components for preparation at the time of application is also possible.

In vivo tests

Haemopoiesis in mice treated with propionibacteria after lethal irradiation.

Lethally irradiated mice of 0.2193 C / kg were injected intraperitoneally with three strains of propionibacteria (P. acnes, P. granulosum and P. avidum) at a dose of 1.5 mg / mouse each. It was found that P. granulosum prolonged the survival time of irradiated mice the most.

Adult, 8-week-old male Surss mice were used. Animals were fed a standard diet, water was available ad libitum. Water and feed were sterilized. The water contained oxytetracycline (1 g per 1000 ml).

Irradiation: Mice were irradiated in a rotating methacrylate cage (10 mice in a cage) at a distance of 50 cm from a Med 250 Medico unit having a power of 200 kV and equipped with a 0.5 mm Cu-filter, dose

0, 1677 C / kg or 0.2193 C / kg, respectively. An irradiation dose of about 0.01935 C / kg per minute was determined by a CT-1 thermoluminescent R-dosimeter.

P. acnes strain ATCC 6919, P. avidum strain 0575, KP 40 and P. granulosum strain KP 45 were used for the experiments in the form of killed whole cells and in the form of cell walls. Lyophilized propionibacteria were suspended in 7.5 mg / ml sodium chloride phosphate buffer solution and the 02 ml suspension was injected intraperitoneally (1.5 mg per mouse).

Examination of exogenous spleen colonies

At 3, 2 and 1 days prior to bone marrow transplantation, 1.5 mg mice were injected

P. granulosum. Bone marrow cell suspension was prepared so that both femurs were washed with sterile Parker medium. Cells were counted in a hemocytometer. 5 x 10 ^s cells capable of reproduction in 0.2 ml of Parker medium were injected per mouse into the tail vein which was four hours prior bone marrow irradiated 0.2193 C / kg. Control mice were injected with 02 ml of medium or 5 x 10 'bone marrow cells of normal mice (not treated with P. granulosum injection). Another group of mouse donors received P. granulosum first, then the animals were anesthetized with ether and blood was collected from the retrobulb plexus. Blood cells were counted in a hemocytometer and aliquots of blood with 5 x 10 ⁵ nuclear cells were injected into the tail vein of mice irradiated with 0.2193 C / kg. Control mice received the same amount of blood that was collected from normal mice (untreated P. granulosum). All mice were sacrificed and weighed 9 days after blood transfusion or bone marrow transplantation, the spleen was removed, weighed again and the number of colonies in the spleen fixed in chloroform: ethanol (1: 3) was determined.

Examination of endogenous spleen colonies.

3.2, or 1 or 0 days prior to irradiation of 0.1677 C / kg, mice were injected intraperitoneally with P. granulosum. Control mice received equal volumes of phosphate buffered saline. Nine days after irradiation, animals were sacrificed, spleens removed, weighed, and colonies counted as in the previous exogenous colony evaluation test.

Survival test.

Mice were divided into 7 groups, with 15 mice each irradiated with 0/2193 C / kg X-rays in each group. At 4 hours post-irradiation, the animals were treated with cell walls of propionibacteria or whole cells. Control mice received equal volumes of phosphate buffered saline. On the 10th day after irradiation, the number of surviving animals was determined in each group.

Student's test was used for statistical analysis.

In the appended FIG. 1 is a graphical effect of the test propionibacteria on the survival time of lethally irradiated mice. All three strains of propionibacteria resulted in a significant increase in survival time compared to control animals. P. granulosum has proven to be more effective than P. acnes and P. avi4 dum in the form of whole cell preparations and in cell wall preparations. (strain KP 45).

Effect of P. granulosum on endogenous spleen colonies

Table 1 Number of endogenous spleen colonies in mice treated with Propionibacterium granulosum (strain 45) (mean + standard deviation)

Relative Number of endogenous spleen weight spleens

Control (650 R) 1.B2. + -. 0.45 1.24 to 0.67 P. granulosum - 3 days before irradiation 1.66 ± 0.24 10.60 1 4.45 P. granulosum - 2 days before irradiation 1.73 i 0.21 8.17 to 3.49 P. granulosum - 1 days before irradiation 2.18 and 0.42 11.20 t 5.30 P- granulosum - 4 hours after irradiation 2.32 to 0.38 14.10 ± 4.70

^x - p <0.01

However, the relative spleen weight of irradiated mice increased - The number of exogenous spleen colonies was significantly only when P. granulosum was applied increased after treatment.

1 day before or 4 hours after irradiation 0.1677 C / kg.

Influence of P. granulosum on formation of exogenous spleen colonies (strain KP 45)

Table 2 Number of exogenous colonies after bone marrow transplantation in mice treated with P. granulosum (strain KP 45) (mean ± standard deviation)

transfusions Relative weight of spleen Number of exogenous spleen colonies CFU-S for 10 ⁶ bone marrow nuclear cells Control (0.5 ml Parker medium) 0.99 ± 0.19 1.12 í 0.32 11.20 ± 3.2 Bone marrow of normal mice 1.52 i 0.31 20.40 ± 2.14 204.00 21.4 Bone marrow of mice treated for 3 days 1.51 and 0.49 10.30 t 1.87 ^{3 c} 103.00 + 18.7 before transfusion of P. granulosum Bone marrow of mice treated for 2 days 1.75 * 0.44 11.60 ± 4.4 ^X 116.00 ± 44.2 ^X before transfusion of P. granulosum Bone marrow mice treated 1 day prior to transfusion 1.69 + 0.27 10.70 ± 4.3 ^{X X} 107.00 + 43.3 ^X P. granulosum

* - p <o, oi

SK 277936 Β6

No differences in the relative weight of the spleen were found in normal bone marrow transplanted mice and in mice treated with bone marrow donor mice treated 3.2 or 1 day prior to transplantation. This treatment resulted in a significant loss of exogenous spleen colonies in animals irradiated with 0.2193 C4g as compared to animals given untreated bone marrow donor mice. This Table 3 Number of exogenous spleen colonies after (mean + standard deviation) shows the decrease in the number of CFU-S in the bone marrow of mice treated with P. granulosum. Otherwise, blood injections of P. granulosum-treated mice resulted in a significant increase in the spleen spleen weight and spleen colony counts in the lethal-irradiated test animals as compared to the blood injections of untreated donor animals as shown in Table 3 below.

transfusion of mice treated with P. granulosum (strain KP 45)

Relative mass

LeukoI Cytosis in Transfusion Transfusion. spleen blood

Number of exogenous spleen colonies

CFU-S for 10 ⁶ bone marrow nuclear cells

Blood of nornalnych nici i

: Mouse Blood ^! 3 days prior to transfusion with P.granulosum

Blood of mice treated 2 days before transfusion with P.granulosum

6480

9960

10120

1:41

2.62

3:45

00:23

0.88 ^x

1.81 ^x

3.80 ± 2.2

9.16 ± 1.4 ^X

10.2 i 2.6 *

1.17

00:28

1.84

1.2

0.31 ^x

O.34 ^x

Blood was cleared the day before P.granulosus transfusion

9650

3.23

1.48 ^x

7.40 tonnes 1.8 ^XX

1:54

0.23 ^{xx x} - p <0.01

XX

- p <0.05

Blood was taken from donors - mice that received P. granulosum injection two or three days before the blood transfusion, to stimulate spleen colony formation. However, blood transfusion leukocytosis was elevated after treatment with P. granulosum 3, 2, or 1 day prior to blood collection.

In the experiments carried out, P. granulosum 15 proved to be the most effective agent in lethal-irradiated rats. The lethal effect of the lethal dose of X-rays is the result of inhibition of the proliferative ability of haemopoietic stem cells and damage to internal organ epithelial cells and the consequent development of general saprophytic bacterial infections. In any case, the previous experiment shows that mice treated with propionibacteria do not show signs of diarrhea and / or gastrointestinal bleeding. The protective effect of propionibacteria can also be attributed to the healing of the hematopoietic bone marrow stem cells after irradiation.

The action of P. granulosum, P. acnes and P. avidum in experimental mouse sarcoma tumor 180 in mice. 30

All three propionibacteria mentioned above were administered intraperitoneally or intratumorally in multiple doses of 1 mg / mouse each, and were shown to be effective in retarding growth and stimulating sarcoma regression 180 in CFW-mice. In addition, survival of mice with sarcoma 180 was prolonged after application of the propionibacteria.

Used propionibacteria.

Propionibacterium granulosum strain KP 45 (isolated from an infected wound at the Institute of Hygiene of the University of Cologne) was used in the experiments. Propionibacterium acnes strain 6919 (ATCC) Propionibacterium avidum strain 0575 (CC Cummins, Anaerobe Labs., Virginia Polytechnic Inst. And State University, Blackburg), KP 40. Said propionibacteria were lyophilized and suspended in 5 mg sodium chloride phosphate buffer solution / ml 02 ml of suspension was administered intraperitoneally or intratumorally to tumor-bearing mice.

Mice with tumors.

Adult male CFW mice were used in this tumor system. Tumors were induced as described in Radio Sc.12 (1978), pages 185-189. For this purpose, sarcoma type 180 tumors were from donors 6.

- mice excised, tumor tissue killed, trypsinized (0.25% trypsin, 15 minutes at 37 ° C) and filtered. The number of cells was counted and set in 1 ml to 10 * cells capable of propagation. 0.1 ml of suspension was given by subcutaneous, intrascapular injection. This procedure led to the development of detectable tumors after about 4 to 5 days. Rapid tumor growth occurs between days 4 and 14 after transplantation, with a lethal effect between days 20 and 28.

In total, 225 male CFW mice were used in the experiments. In the survival assay, 105 mice were divided into 7 groups (15 mice per group) and animals of experimental groups (I to VI) were treated on days 0.4, 8, 12 and 16 after tumor cell implantation by injection of propionibacteria. P. granulosum, P. avidum or P. acnes were injected intrapertoneally (3 groups) or intratumorally (3 groups) at a dose of 1 mg / mouse. Control mice received intraperitoneal or intratumoral injections of PBS. The number of surviving animals was recorded on the 16th and 20 th day, respectively, after the implantation of the tumor cells, and every other day to 44 th day after the implantation. The remaining 120 animals were divided into 85 groups and mice of experimental groups (I-VI) were treated with propionibacterial injection as the first and control mice (groups VII and VRI) were treated with PBS. On day 20 after tumor cell implantation, all mice were sacrificed and tumors excised and weighed. The arithmetic mean and standard deviations were determined for each group and all tumors of the experimental groups that were more than two standard deviations below the mean control value were designated as regressive. The results were analyzed statistically by Student's t15 assay (tumor mass) or by Chi-square analysis with Yates correction (tumor regression and mouse survival).

The results obtained are shown in Table 4 below.

Table 4 Survival of CFW-sarcoma 180 mice treated with propionibacteria (1 mg / mouse) 0.4, 8, 12, and 16 days after tumor cell implantation

Days after implantation of recruitment cells

19 20 21 22 23 24 25 26 27 28 29 30

inspection 15 13 10 7 3 0 p. granulosum i ntra per i tonal 15 15 15 15 15 12 10 9 9 7 6 5 3 3 P. acnes intraperitoneally 15 15 15 15 15 14 10 8 8 6 5 5 2 1 P. avidum intraperitoneally 15 15 15 14 14 13 11 10 8 5 4 4 0 P. granulosum intratumorally 15 15 15 15 15 15 13 12 11 9 7 7 6 5 P. acnes intratumorally 15 15 15 15 15 14 12 11 9 7 7 7 5 2 P. avidum intratumorally 15 15 15 15 15 15 14 13 10 8 8 6 4 3

The following Table 5 shows the effect of propionibac20 thorium on growth and sarcoma 180 in CFW mice.

Table 5 Tumor weight and number of regression tumors in sarcoma 180 mice treated with propionibacteria (1 mg / mouse) at 0, 4, 8, 12 and 16 days after tumor cell implantation

Intraperitoneal injection Weight. Number Percent

tumor (G) regression tumors regression inspection 2631 * 321 0/15 0 Propionibacterium granulosum 642 t 8/15 53.3 Propionibacterium avidum 1032 ± 381 6/15 40.0 Propionibacterium 1126 ± 412 5/15 * 33.3

acnes

! Intratumoral injection Tumor mass (G) Number of regression tumors Percentage of regression I Control 2631 ± 321 0/15 0 | Propionibacterium 1granulosum 538 and 212 11/15 73.3 Propionibacterium avidum 842 ± 246 10/15 66.6 Propionibacterium acnes 731 t 9/15 60.0

When administered intraperitoneally at a single dose of 1 mg / mouse, all three of the propionibacterial strains tested showed significant splenic enlargement at day 4 after injection, with a further increase in spleen mass at day 6-14. This enlargement of the spleen mirrors the stimulation of the reticuloendothelial system and proceeds in parallel with the antitumor activity of these immunostimulators.

P. granulosum resulted in regression of more than 70% of examined sarcoma 180 tumors after intratumoral administration.

Cytostatic activity in vivo in mouse tumor cells after Propionibacterium granulosum.

In another study, the cytostatic action of Propionibacterium granulosum strain KP 45 was investigated in sarcoma L-1 and BALB / c - mice (lung). Here, too, there was significant efficacy compared to untreated animals.

4. Antibacterial activity as adjunctive therapy in chemotherapy of primary and secondary lung tumors.

patients with primary or secondary metastatic lung tumors were selected for these trials. 10 patients received intravenous infusions of 30 mg P. granulosum in 100 ml intravenous infusion solution. The other 20 patients served as controls.

All patients were treated with chemotherapy to synchronize the proliferation of neoplastic cells. Each cycle lasted 3 days: day 1 vincristine, 1.5 mg intravenously at 8 am and 8 pm, day 2 methotrexate, 25 mg intramuscular at 8 pm, day 3 methotrexate 25 mg intramuscular at 8 am followed by methotrexate intravenously at 25 mg at 2 pm and infusion of 30 mg / kg cyclophosphamide at the same time.

This chemotherapy treatment was repeated three times every 21 days.

Overall chemotherapy consisted of the three cycles mentioned above, starting on day 1, 22 and 43 of observation.

On day 3, 10, 31, and 52 days of observation, P. granulosum was administered at a dose of 30 mg / 100 ml intravenous infusion solution over 15 minutes (the last day of the first chemotherapy cycle and then 1 week after the end of each cycle).

Five cases of bacterial infections (three pneumonia, one sepsis and 1 case of angina tonsillaris) occurred during the 60-day observation period in patients receiving chemotherapy alone, while no symptoms of bacterial infections occurred in 10 patients receiving chemotherapy and intravenous P. granulosum infusions. This difference can be described as statistically significant.

Patient tolerance to intravenous infusions of P. granulosum strain KP 45 was good. During the course of the 30 mg P. granulosum infusion during the course of the infusion, feelings of cold and then fever of 39 ° C (2 to 12 hours after infusion) occurred, but the following day no side effects were detected. Repeated infusions of P. granulosum did not lead to the development of delayed hypersensitivity 10 over a 60-day observation period. This concludes that an intravenous infusion of 30 to 240 mg of P. granulosum strain KP 45 can be performed in patients without the risk of serious side effects or complications.

5. General instructions for topical application (gastric and intestinal cancer).

mg of the lyophilized product is suspended in 1 ml of 1% xylocaine in sodium chloride solution and administered 20 injections intratumorally through the skin (1 mm diameter) using an 80 cm long, rigid polyethylene guide and a tightly ordered intramuscular needle (size 18) without pineal gland. The guide is guided through the biopsy channel into the endoscope (gastrofibroscope or wheel 25 of the nose), wherein the non-plastic tumor under visual control is injected and the needle is inserted into the tumor tissue 0.5-1 cm. The product is injected and rinsed with 1-2 ml of 1% xylocaine in sodium chloride solution.

Intratumoral injections (10 mg of product each) are administered, for example, on the first three days, then on the 10th and 17th day of observation.

The attached graph shows the values (survival percentage and number of surviving experimental animals, depending on the number of days after irradiation of the experimental animals) corresponding to the effect of the propionibacteria used, after irradiation of mice with a lethal dose of 0.2193 C / kg.

In the attached graph, the individual lines correspond to the following propionibacteria and preparations thereof:

line A (marked with open squares) P. granulosum (whole cells), line B (marked with solid squares) P. granulosum (cell walls), line C (marked with open circles) P. acnes (whole 45 cells), line D (marked with open circles) P. acnes (cell walls) Line E (marked with open triangles P. avidum (whole cells) Line F (marked with solid triangles) P. avidum

SK 277936 G6 (cell walls), line G (indicated by circles with a dot inside) untreated controls.

Claims (8)

A process for the manufacture of cell wall or whole cell preparations from Propionibacterium granulosum, Propionibacterium avidum and / or Propionibacterium acnes for use in radiotherapy and tumor chemotherapy, characterized in that the liquid culture of the respective propionibacterium is treated by the GasPak method. in an airtight enclosure under anaerobic conditions formed with borohydride, sodium bicarbonate, citric acid and water at 37 ° C, the mass growth thus obtained is inoculated with a dense flush of propionibacterium after incubation for 72 hours at 37 ° C the cells are separated by centrifugation under cooling, the sediment is washed with distilled water and, if necessary, then disintegrated by milling with glass beads, preferably having a diameter in the range of 0.17 to 018 mm, preferably using 20 times the volume of these glass beads, after microscopic inspection to the absence of the whole cells, the glass beads are separated from the homogenate by filtration on a glass frit using phosphate buffers at pH 7.2, the milk suspension containing cell walls 25 and the cytoplasms centrifuged, the sediment is taken up in phosphate buffer at pH 7.2, autolytic enzymes are inactivated by boiling, the cell walls containing the protein are further purified by incubation with a thypsine suspension and toluene, centrifuged cell walls purified by trypsin digestion, washed with distilled water and then freeze-freed. Method according to claim 1, characterized in that the obtained cell walls are incubated at 37 ° C for 24 hours. 35 Method according to claim 1, characterized in that thiypsine is used in the suspension of cell wall and cytoplasm in a suspension of 0.5 mg / ml and the addition of toluene to the leach is 1 ml per 100 ml. The method according to claim 1, characterized in that the starting propionibacterium which is cultivated and subjected to processing is preferably Propionibacterium granulosum strain 45. Method according to claim 1, characterized in that the cultivated and treated propionibacterium is also preferably a strain of Propionibacterium avidum KP 40. Method according to claim 1, characterized in that, as the medium for the cultivation of the mass of the propionibacterium used, a broth of the following composition is used. Bacto Casitone 12g yeast extract Bacto 12g MgSO4.7H2O 1g glucose 4g distilled water to 1000 ml pH12 for A-agar + 28 g Bacto agar. Method according to claim 1, characterized in that the phosphate buffer, by which the glass beads are separated from the homogenate after the cell disintegration and into which the sediment is removed, is prepared in advance by 1/15 M NajHPC> 4.2H2O and 1/15 M KH2PO4 are dissolved in 1000 ml of distilled water each, 612 g of sodium hydrogen phosphate solution are mixed with 388 g of potassium dihydrogen phosphate solution, the pH being adjusted to 12- The process according to claim 1, characterized in that the lyophilized propionibacteria are suspended in a 5.0 or 7.5 mg / ml sodium chloride phosphate solution.