FI75094B - FOERFARANDE FOER FRAMSTAELLNING AV PROPIONIBACTERIUM GRANULOSUM, PROPIONIBACTERIUM AVIDUM OCH / ELLER PROPIONIBACTERIUM ACNES CELLVAEGGS- ELLER HELCELLSPRODUKTER. - Google Patents

Description

ί 75094

Method for the preparation of a cell wall or whole cell preparation of Propionibacterium granulosum, Propionibacterium avidum and / or Propionibacterium acnes

The invention relates to a process for the preparation of a cell wall or whole cell preparation of Propionibacterium granulosum, Propionibacterium avidum and / or Propionibacterium acnes for use in the radiotherapy or chemotherapy of tumors.

Preparations of anaerobic coryneforms, for example C-Parvum, CN 6134, which have been described as antitumor agents, are already known. However, it has been shown that in systemic or topical therapy, these preparations can lead to undesirable side effects and complications, so it is recommended in the literature to avoid doses higher than 7.5 mg / m2. This amount corresponds to approximately 20 mg per patient.

According to the invention, it has been found that preparations based on killed whole cells or cell walls of propionic acid bacteria are very effective in the chemotherapy or radiotherapy of tumors. These preparations are particularly suitable for supportive therapy. When administered topically, the efficacy is particularly high.

The method according to the invention is characterized in that the liquid culture of the propionic bacterium in question is treated according to the Gas-Pak method under anaerobic conditions, after incubation the cells are separated by cold centrifugation and the sediment is washed with distilled water and possibly then crushed with glass beads no longer present, the glass beads are separated from the homogenate by glass sinter using phosphate buffer pH 7.2, the milky suspension containing cell walls and cytoplasm is separated by centrifugation, the sediment is taken up in phosphate buffer pH 7.2, the autolytic enzymes are inactivated by inactivation, purified by incubation with trypsin suspension and toluene, trypsin-thawed cell walls are separated by centrifugation, washed with distilled water and lyophilized.

Thus, it has been shown experimentally that P. acnes, P. granulosum, and P. avidum significantly prolong the lifespan of lethal (850 R) irradiated mice when injected intraperitoneally into mice at 1.5 mg per mouse. P. granulosum is most effective in this case. It was found that the preparation according to the invention is suitable for stimulating CFU-S profiling, i. it stimulates the transport of hematopoietic colony-forming units from the bone marrow to the cell cycle and to the peripheral blood. In clinical practice, this finding means that the formulations of the invention are valuable as adjunctive therapy in radiotherapy of tumors because they substantially improve the radiation tolerance of patients.

In addition, studies have shown that the preparations obtained by the invention are effective against murine Sarcoma 180 in mice and regress more than 70% of Sarcoma 180 tumors when applied intratumorally.

It was also found that the formulations of the invention, when administered systemically as adjunctive chemotherapy to patients with primary or secondary lung tumors, help to avoid one of the 750,794 skin complications of tumor chemotherapy, namely the risk of infection. Thus, it has been found that the formulations of the invention can be used in cancer chemotherapy as excellent, antibacterially effective adjuvants.

These results, which have been proven below by some in vivo experiments, must be considered unexpected in all respects.

The cell wall preparations according to the invention have proved to be particularly useful, since preparations of this type have a particularly low risk of adverse side effects. According to the state of the art, cell wall preparations have not yet been clinically studied.

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

It is recommended to prepare injectable suspensions in phosphate-buffered saline. In this case, it is particularly expedient for the concentration of active substance to be 0.5 to 50 mg per ml of solution. A concentration of 2 to 12 mg / ml of solution, especially 5 to 7.5 mg / ml of solution, is preferred.

Particularly suitable are intravenous infusion solutions containing 15 to 30 mg of the preparation in 100 ml of solution. Such a dose level has proven to be particularly advantageous in the treatment of primary and secondary pulmonary plasmas, the first injection being at least 10 days before the chemotherapeutic treatment.

4,75094

As the phosphate buffer, Na 2 HPO 4 / KH 2 PO 4 phosphate buffer, suitably having a pH of about 7.2, is suitably used.

The method is not limited to any particular propionic acid bacterial strain. Various propionic acid bacterial strains are possible, for example the propionic acid bacteria described in FEMS Microbiology Letter, Volume 2, No. 1, July 1977, pages 5-9.

The strains P.granulosum KP 45 and P.avidum KP 40 are considered to be particularly good. (P.granulosum KP 45) and 1772 (P. avidum KP 40), dated 11.03.1980. Both positions are available from the DSM collection in accordance with the assignment agreement between the DSM collection and the Patent Office of the Federal Republic of Germany (eg form P 2750).

The bacteria P.granulosum, p.acnes and P.avidum can usually be isolated and cultured from Acne Effluoresensensives (Acne vulgaris, Acne papulopustulosa and Acnes conclobata). in terms of taxonomy cf. Hautarzt 30, 242-267 (1979).

Other strains of propionic acid bacteria which may be used in the invention are described, for example, in Applied Microbiology, February 1973, pages 222-229, American Society for Microbiology, Volume 25, No. 2.

5 75094

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The following examples illustrate the preparation of the preparations according to the invention.

Example 1

General method for recovering cell walls

Liquid culture of this propionic acid bacterium was performed in one liter (Erlenmeyer flask) at 37 ° C under anaerobic conditions (Gas-Pak method, BBL).

For pulp growth, the medium (A broth + or Triptic soybean broth, Difco) was inoculated with a thick slurry of propionic acid bacteria. The inoculum slurry was prepared by grinding a 3-day-old A agar culture of propionic acid into 10 ml of nutrient broth. After 72 hours of incubation at 37 ° C, the liquid was separated from the cells by centrifugation in a Sorval R2 cold centrifuge at 10,000 g (20 minutes). The sediment was washed three times with distilled water and then mixed with twice the volume of glass beads (0 0.17 to 0.18 mm) and crushed in a cell mill for +++ 1-1 1/2 hours.

After microscopically checking (gram preparation) that no more whole cells were present, the glass beads were separated from the homogenate by G-1 sinter using phosphate buffer ++ (pH 7.2).

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

These still protein-containing cell walls were purified by incubation for 24 hours at 37 ° C with trypsin (Merck, 0.5 mg / ml suspension) and toluene slurry (1 ml 100 none) (to prevent bacterial growth).

8 75094

Trypsin-thawed cell walls were then centrifuged at 40,000 g (30 minutes) and washed three times with distilled water, followed by freeze-drying.

+) A-1iemi:

Bacto Casitone 12 g

Yeast extract of Bacto 12 g KH2PO4 4 g

MgSO 4 * 7H 2 O 1 g

Glucose 4 g

Dist. water 1000 ml, pH 7.2 A-Agar + 28 g Bacto Agar.

++) Phosphate buffer: 1/15 M Ν32ΗΡ04 * 2Η20 and 1.15 M KH2PO4 were each dissolved in 100 ml of distilled water; 612 g of the secondary sodium phosphate solution were mixed with 388 g of the primary potassium phosphate solution and the pH was adjusted to 7.2.

+++) Cell mill: Vibrogen screen mill vi 3, Edmund Biihler, 7400 Tiibingen.

Example 2 Preparation of suspensions of P.acnes (strain ATCC 6919) .P.avidum (strain 0575. KP 40) and P.qranulosum (strain KP 45)

By the general method of Example 1, lyophilized propionic acid bacteria of said nature are suspended at a concentration of 5.0 or 7.5 mg / ml in the above-mentioned phosphate-buffered saline solution. The resulting suspension is particularly suitable for intraperitoneal injection.

9 75094

Example 3 Using the procedure of Example 2, suspensions of P. granulosum (strain KP 45, 95 K), P. acnes (strain ATCC 6919) and P. avidum (strain 0575, KP 40) can be prepared from lyophilized material and injected intraperitoneally. and at a concentration of 5 or 7.5 mg / ml of cellular material.

Example 4

Preparation of Injectable Suspensions for Chemotherapy Supporting Treatment P. granulosum, strain KP 45, is treated by the general method of Example 1. For intravenous administration, a suspension is prepared containing 30 mg of cellular material in 100 ml of solution for infusion.

The preparations according to the invention may contain conventional additives. The preparations may be in ampoule form or may be filled into sterile bottles fitted with a piercing cap. It may also be possible to store the ingredients separately, in which case they are manufactured at the time of use.

In vivo studies 1. Hemopoiesis in propionic acid-treated mice after lethal irradiation

Three strains of propionic acid bacteria (P.acnes, P.granulosum and P.avidum) were injected intraperitoneally into lethal irradiated mice (850 R) at a dose of 1.5 mg per mouse each. In this case, it turned out that P.granulosum prolonged the lifespan of the most irradiated mice.

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Adult, 8-week-old male Suiss mice were used in the experiment. The animals were fed a standard diet.

Water was available ad libidum. Water and feed were sterilized. The water contained oxytetracycline (1 g / 1000 ml).

Irradiation: Mice were irradiated at a dose of 650 or 850 R in a rotating methacrylate cage (10 mice per cage) with a THX 250 Medicor unit operating at 200 kV and equipped with a 0.5 mm Cu filter 50 cm away. The CT-1 Thermoluminescence R dosimeter was used to determine the amount of radiation at about 75 R / min.

P.acnes strain ATCC 6919, P.avidum strain 0575, KP 40 and P.granulosum strain KP 45 were used as killed whole cells and cell walls. Lyophilized propionic acid bacteria were suspended in phosphate-buffered saline at a concentration of 7.5 mg / ml, and the suspension was injected intraperitoneally in 0.2 ml (1.5 mg / mouse).

Examination of exogenous anthrax colonies

Donor mice were injected with 1.5 mg of P. qranulosum bacteria, 3, 2, or 1 day before bone marrow transplantation. The bone marrow-cell suspension was prepared by rinsing both femurs with sterile Parker's medium. Cells were counted on a hemocytometer. Each mouse irradiated with 850 R at the dose of 850 R 4 hours before bone marrow inoculation was injected into the tail vein with 0.2 x 10 5 proliferating cells in 0.2 ml of Parker's medium. Control mice were injected with 0.2 ml of medium or 5 x 10 6 bone marrow cells from normal mice (not treated with P. granulosum bacteria). Another group of donor mice were given P. granulosum bacteria as described above, after which the animals were anesthetized with ether and blood was drained from the retrobulbar plexus. Blood cells were counted with a hemocytometer, and aliquots of blood containing 11,75094 5 x 10 7 nucleated cells were indicated in the tail vein of mice irradiated with 850 R. The control mouse broth was given an equal volume of blood taken from normal mice (which had not been treated with P. qranulosum bacteria). Nine days after blood or bone marrow inoculation, all mice were sacrificed and weighed, the spleen was removed, reweighed, and the number of colonies per spleen after chloroform / ethanol (1: 3) fixation was determined.

Investigation of endogenous colonies

Mice were injected intraperitoneally with P. qranulosum bacteria for 3, 2, or 1, or 0 days before being irradiated with a 650 R dose. Control mice were given equal volumes of PBS (phosphate buffered saline). Nine days after irradiation, the animals were sacrificed, the spleens removed, weighed, and colonies counted as in the previous test used to determine exogenous splenic colonies.

Surviving, jääntitesti

The mice were divided into 7 groups of 15 mice in each group, and the mice were irradiated with X-rays at 850 R dosage. Four hours after irradiation, the animals were injected with the cell walls or whole cells of propionic acid bacteria. Control mice were given equal volumes of PBS. The number of surviving animals in each group was counted 10 days after irradiation and beyond.

Student's t-Test was used for statistical analysis.

The accompanying Figure 1 shows the effect of graphically studied propionic acid bacteria on the survival of lethal irradiated mice. All three propionic acid bacterial strains significantly prolonged survival compared to comparator animals. P.qranulosum proved to be more effective than P.acnes and P.avidum as a whole cell preparation and a cell wall preparation.

Effect of P.qranulosum on endoenic colonies (strain KP 45)

TABLE I

Number of endogenous splenic colonies in mice treated with P.qranulosum propionic acid bacteria (strain KP 45) (mean + standard deviation)

Spleen relative number of eridogenic colonies

Comparisons / 650 R / 1.82 + 0.45 1.24 + 0.67 P.granulosum- 3 days before irradiation 1.66 _ + 0.24 10.6 + 4.45 P.granulosum - 2 days before irradiation 1.73 + 0 »21 8.17 + 3.49x P.granulosum - 1 day before irradiation 2.18 + 0.42 11.2 + 5.301 ''“ “'" "' '*" "T" "- - - ----- - - - - “Γ r - 1 1-1 _1" P.granulosum - 4 hours after irradiation 2.32 + 0.38 14.1 + 4.70x - p <0, 01 The relative spleen weight of irradiated mice decreased only when P. granulosum was administered 1 day before 650 R irradiation or 4 hours after irradiation 13 75094 However, the number of exogenous spleen colonies increased significantly with all treatments.

Effect of P.qranulosum1 on the formation of exogenous splenic colonies (strain KP 45) 14 75094 ro 3 I · —f

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No differences were observed between the relative spleen weights of mice inoculated with normal bone marrow and mice inoculated with P.granulosum 2, 3, or 1 day before inoculation of donated animals. This treatment significantly reduced the number of exogenous splenic colonies in 830 R-treated animals compared to animals given bone marrow from untreated donor animals. This finding indicates a decrease in the number of CFU-S in the bone marrow of P. granulosum-treated mice. On the other hand, injection of blood from mice treated with P. granulosum into lethal irradiated experimental animals significantly increased the relative weights and number of spleen colonies compared to injection of blood from untreated donor animals, as shown in Table III below: m 75094

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Colon formation was most effectively stimulated by blood taken from donor mice injected with P.granulosum 2 or 3 days before transfusion. However, the number of leukocytes in the inoculated blood increased after treatment with P. granulosum 3, 2, or 1 day before blood collection.

In previous experiments, P. granulosum proved to be the most effective agent in lethal irradiated rats. The lethal effect of a lethal dose of X-rays is due to inhibition of the ability of hematopoietic stem cells to proliferate and damage to visceral epithelial cells, which in turn results in the development of a general infection caused by saprophytic bacteria. Based on the previous experiment, mice treated with propionic acid bacteria show no symptoms of diarrhea and / or gastrointestinal bleeding. The protective effect of propionic acid bacteria can thus be attributed to the better recovery of bone marrow hematopoietic stem cells after irradiation.

2. Efficacy of P.granulosum, P.acnes and P ♦ avidum bacteria against murine experimental Murine Sarcoma 180 tumor

All three of said propionic acid bacteria were applied intraperitoneally or intratumorally in several doses per mg / mouse. In CFW mice, they were shown to slow the growth of Sarcoma 180 and stimulate its regression. In addition, application of propionic acid bacteria extended the lifespan of Sarcoma 180 mice.

Used propionic acid bacteria

Propionic acid bacterial strains granulosum strain KP 45 (isolated from skin infection at the Institute of Hygiene of the University of Cologne) were used in the experiments, propionic acid bacterial strain acnes strain 6919 (ATCC) propionic acid bacterial strain is 750 9 4 avidum strain 0575 (CS Cummins, Virginia Cummins, Anaerobe KP 40. Said propionic acid bacteria were lyophilized and suspended in phosphate buffered saline at a concentration of 5 mg / ml. Tumor mice were given 0.2 ml of the suspension intraperitoneally or intratumorally.

Tumor Mice Adult CFW male mice were used in this tumor system. Tumors were induced in Radio Sci. 12 (1978), pages 185-189.

To this end, Sarcoma 180 tumors from donor mice were dissected, the tumor tissue was minced, trypsinized (0.25% trypsin, 15 minutes at 37 ° C) and filtered. The number of cells was counted and the cell concentration per 10 ml of saline was adjusted to 10 proliferating cells. The suspension was injected 0.1 ml subcutaneously and intrascapularly. By doing so, known tumors developed within about 4-5 days. Rapid tumor growth occurred after transplantation between days 4 and 14, and a lethal effect developed between days 20 and 28.

A total of 225 CFW male mice were used in the experiments. In the survival experiment, 105 mice were divided into seven groups (15 mice per group) and animals in experimental groups (I to VI) were injected with propionic acid bacteria after inoculation of tumor cells on days 0, 4, 8, 12, and 16. P.granulosum, P.avidum, or P.acnes injected intraperitoneally (3 groups) or intratumorally (3 groups) at a dose of 1 mg / mouse. Control mice were injected intraperitoneally or intratumorally with PBS. The number of surviving animals was counted on the 16th or 20th day after tumor cell inoculation and every other day thereafter 19,75094 until day 44 of inoculation. The remaining 120 mice were divided into eight groups, and mice in experimental groups (I to VI) were injected with propionic acid bacteria as above, and control mice (groups VII and VIII) were injected with PBS. On day 20 after inoculation of tumor cells, all mice were sacrificed, tumors were excised and weighed. An arithmetic mean and standard deviation were determined for each group, and all tumors in the experimental groups that were more than two standard deviations below the control mean were counted as regressive. Results were statistically analyzed by Student's t-Test (tumor mass) or Yates' Chi-Quadrat analysis (regression of tumor and mouse survival).

The results obtained are summarized in the following Table IV.

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The following Table V shows the effect of propionic acid bacteria on the growth and regression of Sarcoma 180 in CFW mice.

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23 75094

When all three propionic acid bacterial strains tested were administered intraperitoneally at a single dose of 1 mg / mouse, the spleen increased significantly on day 4 after injection and the spleen mass continued to increase on days 6-14.

This increase in spleen size illustrates the re-stimulation of the reticuloendothelial system and runs in parallel with the antitumor activity of these immunostimulators.

P.qranulosum regressed more than 70% of the Sarcoma 180 tumors studied when administered intratumorally.

3. Cytostatic potency of propionic acid bacterium P.qranulosum in vivo in Murine tumor cells

A further study investigated the cytostatic effect of the propionic acid bacterium P.granulosum strain KP 45 on Sarcoma L-1 (lung) of BALB / c mice. Again, the efficacy was significant compared to untreated animals.

4. Antibacterial Effect on Chemotherapy Supporting Primary or Secondary Lung Tumors 30 Patients with primary or secondary metastatic lung tumors were selected for this test. 10 patients received an intravenous infusion of 30 mg P.qranulosum in 100 ml of intravenous infusion solution. The other 20 patients served as a control.

All patients were treated chemotherapeutically to synchronize neoplastic cell proliferation. Each treatment lasted for three days: Day 1: Vincristine 1.5 mg intravenously at 8 am and 8 pm, Day 2: Methotrexate 25 mg intramuscularly at 8 pm, Day 3: Methotrexate 25 mg intramuscularly at 8 am, followed by intravenous methotrexate 25 mg at 2 pm and at the same time 30 mg / kg cyclophosphamide by infusion.

24 75094 This chemotherapeutic treatment was repeated three times every 21 days.

The entire chemotherapy comprised the three cycles mentioned above, which began on days 1. 22 and 43 of follow-up.

During observation days 3, 10, 31, and 52, P. granulosum was administered at a dose of 30 mg / 100 ml as an intravenous infusion solution over 15 minutes (on the last day of the first chemotherapeutic period and one day after the end of each cycle).

In five patients treated with chemotherapy alone, bacterial infections (three cases of pneumonia, one case of sepsis and one case of Anqina Tonsillaris) occurred during the 60-day follow-up period, whereas bacterial infections did not show any symptoms in the 10 patients treated with chemotherapy and infusion. P.qranulosumia. This difference must be considered statistically significant.

Patients tolerated intravenous infusions of P. granulosum strain KP 45 well. During the infusion of 30 mg P.granulosum, a feeling of cold occurred twice and finally a fever of 39 ° (2 to 12 hours after the infusion), but the following day these side effects could no longer be observed. A new infusion of P. qranulosum did not result in the development of delayed hypersensitivity during the 60-day observation period. From this it must be concluded that P.qranulosum strain KP 45 can be infused intravenously at 30 to 240 mg without the risk of serious side effects or complications in patients.

25 7 5 0 9 4 5. General instructions for topical administration (gastrointestinal tract) 10 mg of lyophilized product are suspended in 1 ml of 1% xylocaine in saline and injected intratumorally through the skin (diameter 1 mm), using an 80 cm long, rigid polyethylene tube fitted with a fixed intramuscular needle (18 Gauge) without an epiphysis. The tubes are inserted through a bioptic channel into an endoscope (Gastro-fiber or colonoscope), whereupon the visually observed neoplastic tumor is functionalized and the needle is inserted into 0.5 to 1 cm of tumor tissue. The product is injected through a tube and rinsed with 1-2 ml of 1% xylocaine in saline.

Intratumoral injection (10 mg of product each) is performed, for example, on the first three days of monitoring, then on days 10 and 17.

Claims (11)

A method of producing single-cell wall or whole-cell wall preparations of Propionbacterium granulosum, Propionbacterium avidum and / or Propionbacterium acne cells for use in radio or chemotherapy of tumors, characterized in that a liquid culture of said sample peony bacteria are treated according to the Gas-Pak method under anaerobic conditions, after incubation the cells are separated by cold slurry and the sediment is washed with distilled water and possibly then crushed with glass beads in a cell mill, when the microscopic control shows that whole cells no longer exist, the glass beads is separated from the homogenate by an ice cream using a pH 7.2 phosphate buffer, the cell walls and cytoplasm containing milky suspension are centrifuged and the sediment is taken up in phosphate buffer with pH 7.2, autolytic enzymes inactivated by boiling, the recovered proteinaceous cell wall is purified by incubation with tr ypsin suspension and toluene, the cell walls purified by trypsin containment are separated by centrifugation, washed with distilled water and lyophilized. Process according to claim 1, characterized in that the Gas-Pak method is carried out at a temperature of 37 ° C. Method according to claim 1, characterized in that the incubation is carried out for a period of 72 hours and at a temperature of 37 ° C. Method according to claim 1, characterized in that the crushing of the cells in the cell mill is carried out by using the double volume of glass beads for 1-2 hours. Process according to claim 3, characterized in that glass beads with a diameter of 0.17 - 0.18 mm are used.