CZ278484B6 - Process for preparing preparations from cell walls or the whole cells - Google Patents

Abstract

The use of Propionibacterium granulosum, P.avidum and/or P.acnes for prodn. of cell-wall or whole-cell prepns. in the radio- or chemotheraphy of tumours is claimed. Pref. strains are P. granulosum KP45 (DSM 1773) and P.avidum KP40 (DSM 1772). Prepns. for i.p., i.v., or intratumoral admin. pref. contain 0.5-50 (esp. 5-7.5) mg/ml. of killed cells or cell walls suspended in phosphate-buffered saline (pH 7.2). The prepns. increase tolerance to radiation, promote tumour regression and increase resistance to bacterial infection.

Description

The invention relates to the use of propionibacteria. In particular, it relates to the use of 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.

Preparations of anaerobic Corynefors, for example C-Parvum, CN 6134, are known which are described as anti-tumor drugs. However, these preparations have been shown to result in 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 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 propionibacteria are extremely effective in the chemotherapy or radiotherapy of tumors. These preparations are particularly suitable for supportive therapy. Efficacy is particularly important when used locally.

Experiments have shown that P. acnes, P. granulosum and P. avidum after intraperitoneal 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 formulations of the invention have been found to be useful for stimulating CFU-S proliferation, i.e., to stimulate the hematopoietic colony forming units from the bone marrow to enter the cell cycle and to the peripheral blood. This finding means in clinical practice that the preparations of the invention are valuable as an adjunct therapy in radiotherapy of tumors, as they significantly increase the patient's radiation tolerance. In addition, experiments have found that the preparations of the invention are effective against sarcoma 180 in mice and result in regression of more than 70% of sarcoma type 180 tumors when administered intratumorally.

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

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

Cell wall preparations have proved to be particularly useful, since the risk of undesirable side effects is particularly low in this type of preparation. According to the prior art, 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, Propionicbacterium avidum and / or Propionibacterium acnes for use in radiotherapy and tumor chemotherapy, which comprises treating a liquid culture of the propionic bacterium in question. GasPak, sealed

Under anaerobic conditions formed with borohydride, bicarbonate. Sodium, citric acid and water at 37 ° C, the mass growth thus obtained is inoculated with a dense propionibacterium wash, 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 it is then disintegrated by milling with glass beads, preferably with a diameter in the range

0.17 mm 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 of pH 7.2, a milk suspension containing cell walls and cytoplasms are centrifuged, 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 distilled water and then freeze-dried.

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

Injectable suspensions are preferably prepared in phosphate buffered saline. A particularly suitable 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. Intravenous infusion solutions containing 15 to 30 mg of the preparation in 100 ml of solution are particularly suitable. Such dosages have proven to be particularly advantageous for administration in the treatment of primary and secondary neoplastic lung diseases, wherein the first injection should be given at least 10 days prior to chemotherapeutic treatment.

Suitably, Na ₂ HPO ₄ / KH ₂ PO ₄ -phosphate buffer, which has a pH of about 7.2, is preferably used as the phosphate buffer.

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 to 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 Göttingen, Germany, under deposit number DSM 1773 for

P. granulosum KP 45 and 1772 for P. avidum KP 40 dated March 11, 1980. Both strains can be obtained from DSM in accordance with the declaration of release to be filed by the depositor with DSM and German Pat. (form P 2750).

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

Other propionibacterial strains are contemplated

-27878484 B6 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.

Table - Characterization of P. avidum propionibacteria strain KP 40 and P. granulosum KP 45

Tribe Origin no. Species Sucrose Biochemical reactions (Lit. 1) Mal- Sor- Mannit Salin- Ino Esculin- Indol tosis bit activity hydrolysis Nitrate Želá- Dex- .Melicitosis tina trin K 45 Choose from P.granu- wound infection losům + + + + K 40 Taken from a clinically healthy release P.avidum + + - + + - ++ + + + Table - continuation Strain Origin Second Seriology (Lit. 2) Phag lysotype C. P. acnes P. granulosum P. avidum (Lit. 3) KB 95 D34 58 0575 31 Kol ... ... K613

KP 45 Selection of in- P. granulated lottery tickets - + - - NT wounds

KP 40 Taken from pre- P. avidum - - - + - - NT is clinically healthy

NT = untypable

1) G. Pulverer and H. L. Ko

Propionibacterium acnes und

App. Microb. 25: 222-229, 1973

2) U. Hoffler, H. L. Ko and G. Pulverer

Serotyping of Propionibacterium acnes and related microbial species. Fems Microbiology Letters, Vol. 2 to 9, 1977

3) E.C. Jong, H.L.K., and G. Pulverer

Bacteriophages of P. acnes Med. Microbiol. Immunol. 161, 263-271, 1975

Der Hautarzt 1979, 30, 242-247

Differentiation of different types of propionibacteria from acne vulgaris efflorescence.

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

Example 1

General procedure for obtaining cell walls

Liquid culture relevant. propinibacteria were grown in a 1 liter volume (in an Erlenmeyer flask) at 37 ° C, under anaerobic conditions (Gas-Pak method, BBL).

Medium (A-broth ⁺ or tryptic soybean, Difco) with a dense suspension of propionibacteria was inoculated for this large-scale cultivation. The inoculum suspension was prepared by spreading a three-day propionibacterial A-agar 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 refrigerated Sorval R2 centrifuge from the liquid. The sediment was washed three times with distilled water and then mixed with twice the volume of glass beads (φ 0.17 to 0.18 mm) and disintegrated in a cell mill ⁺⁺⁺ for 1 to 1.5 hours. When the preparation was subjected to microscopic examination (Gram preparation) and no whole cells were present, the glass beads were separated on a Gl frit 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 (Gauge, 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 12g

Bacto yeast extract 12g

KH ₂ PO ₄ 4g

MgSO ₄ .7H ₂ O 1g glucose 4g distilled water ad 1000 ml, pH 7.2 for A-agar + 28 g Bacto agar ++) Phosphate buffer:

1/15 M Na ₂ HPO ₄ .2H ₂ O a

Dissolve 1/15 M KH ₂ PO ₄ in 1 000 ml of distilled water, mix 612 g of sodium phosphate solution with 388 g

The pH of the potassium phosphate solution was adjusted to 7.2.

+++) Cell mill:

The vibrogen cell mill knows 3,

Edmund Bohler, 7400 Tübingen.

Example 2

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

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

Example 3

Using the procedure of Example 2, intraperitoneally injectable suspensions of P. granulosum strain 45, 95-K, P. acnes strain ATCC 6919 and P. avidum strain 0575, KP 40 are prepared from lyophilized material at a concentration of 5 and 7.5 mg, respectively. ml of cell material.

Example 4

Preparation of injected suspensions for supplementary chemotherapy therapy

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 infusion 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, sterile vials. Ready-made clothing with separate storage of components for preparation at the time of application is also possible.

In vivo tests

Hemopoiesis in mice treated with propionibacteria after lethal irradiation

Lethally irradiated mice 0.2193 C / kg were administered intraperitoneally injected three strains propionibc ^'erií (P. acnes, P. granulosum and P. avidum), each at 1.5 mg / mouse. It was found that P. granulosum prolonged the survival 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).

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Irradiation: Mice were irradiated in a rotating methacrylate cage (10 mice per cage) at a distance of 50 cm from the THX 250 Medicorunit, which had a power input of 200 kV and was fitted with a 0.5 mm Cu-filter at a dose of 650 or 0.2193 C / kg. . An irradiation dose of about 0.1677 C / kg per minute was determined by CT-1 thermoluminescence 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 whole cell death and cell walls. Lyophilized propionibacteria were suspended in 7.5 mg / ml sodium chloride phosphate buffer solution and 0.2 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 P. granulosum was injected into the donor mice. 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 ⁵ cells capable of multiplication in 0.2 ml Parker medium were injected into each tail vein, which was irradiated with 0.2193 C / kg 4 hours prior to bone marrow transplantation. Control mice were injected with 0.2 ml of medium or 5 x 10 6 cells of bone marrow cells of normal mice (not treated with P. granulosum injections). Another group of mouse donors was treated with P. granulosum as before, then the animals were anesthetized with ether and blood was collected from the retrobulbar 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 amounts of blood that were collected from normal mice (untreated P. granulosum). All mice were sacrificed and weighed 9 days after blood or bone marrow transfusion, the spleen was removed, weighed again and the spleen colony count was fixed in chloroform: ethanol (1: 3).

Examination of endogenous spleen colonies

Mice were injected intraperitoneally with P. granulosum 3, 2, or 1 or 0 days prior to irradiation of 0.1677 C / kg. Control mice received equal volumes of PBS (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 seven groups, each containing 15 mice irradiated with 0.2193 C / kg X-rays. 4 hours after irradiation, animals were treated with cell walls of propionibacteria or whole cells. Control mice received equal volumes of PBS. On the 10th day after irradiation, the number of surviving animals was determined in each group.

Student's t-test was used for statistical analysis

In the attached Fig. 1, the effect of the test subjects is shown graphically

-6US 278484 B6 for survival of lethally irradiated mice. All three strains of propionibacteria resulted in a marked increase in survival compared to control animals. P. granulosum has proven to be more effective than P. acnes and P. avidum in the form of whole cell preparations and cell wall preparations.

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

Table I

Number of endogenous spleen colonies in mice treated with Propionibacterium granulosum (strain KP 45) (Mean ± standard deviation)

* Relative weight of spleen Number of endogenous spleen colonies Control (650 R) 1.82 ± 0. 45 1.24 ± 0.67 P. granulosum - 3 days before irradiation 1.66 ± 0.24 10.6 ± 4.45 ^x P. granulosum - 2 days before irradiation 1.73 ± 0.21 8.17 ± 3.49 ^x

P. granulosum - 1 day

before radiation 2.18 ± 0.42 11.2 ± 5.30 ^x P. granulosum - 4 hrs after irradiation 2.32 ± 0.38 14.1 ± 4.70 ^{x x} - p < 0.01 ......— - ------- -

The relative spleen weight of irradiated mice increased only when P. granulosum was applied 1 day before or 4 hours after irradiation of 0.1677 C / kg. However, the number of exogenous spleen colonies increased significantly after all treatments.

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

Table II

Number of exogenous colonies after bone marrow transfusion of mice treated with P. granulosum (strain KP 45) (mean + standard deviation)

Transfusion Relative weight of spleen Number of exogenous spleen colonies CFU-S for 16® bone marrow nuclear cells Control (0.5 ml Parker medium) 0.99 + 0.19 1.12 + 0.32 11,2 i 3,2 Bone marrow of normal mice 1.52 and 0.31 20.4 + 2.14 204.0 + 21.4 Bone marrow of mice treated for 3 days before transfusion of P. granulosum 1.51 + 0.49 ± 10.3 l 87 ^x 103.0 ± 18.7 ^X

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Continuation of Table II

Bone marrow of mice treated 2 days before P. grululosum transfusion: 1.75 + 0.44 11.6 + 4.4 ^X 116.0 + 44.2 ^X

Bone marrow of mice treated 1 day before P. grululosum transfusion 1.69 + 0.27 10.7 + 4.3 ^{X of} 107.0 + 43.3 ^X x - p <0.01

No differences in relative spleen weights were found in normal bone marrow transplanted mice and donor-treated bone marrow mice treated 2, 3, or 1 day prior to transplantation. This treatment resulted in a significant loss of exogenous spleen colonies in animals irradiated with 0.2193 C / kg compared to animals given bone marrow of untreated donor-mice. This finding shows a decrease in the number of CFU-S in the bone marrow of mice treated with P. grululosum. Otherwise, blood injections of P. gránulosum-treated mice resulted in a significant increase in the relative spleen weight and spleen colony counts in the lethal-irradiated experimental animals compared to the blood injections of untreated donor animals as shown in Table III below:

Table III

Number of exogenous spleen colonies after blood transfusion of mice treated with P. grululosum (strain KP 45)

(Mean values + standard deviation) Transfusion Leukocytosis u transfused blood Relative weight of spleen Number of exogenous spleen colonies CFU-S for 10® bone marrow nuclear cells Blood of normal mice 6480 1.41 + 0.23 3.8 + 2.2 - 1.17 + 0.28 Blood of mice treated for 3 days before transfusion of P. grululosum 9960 2.62 + 0.88 ^x 9.16 + 1.4 ^X 1.84 + 0.31 ^x Blood of mice treated for 2 days prior to transfection of p. grululosum 10120 3.45 + 1.81 ^x 10.2 + 2.6 ^X 2.01 + 0.34 ^x Blood of mice treated for 1 day before transfusion of P. grululosum 9650 3.23 + 1.48 ^x 7.4 + 1.8 1.54 + 0.23

x - p <0.01 xx - p <0.05

In stimulating spleen colony formation, blood collected from donor-mice given P. gránulosum injection two or three days prior to blood transplantation was the most effective. However, leukocytosis in the transplanted blood was increased after treatment with P. grululosum 3, 2 or 1 day prior to blood collection.

In the experiments conducted, P. gránulosum proved to be the most effective agent in lethally 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 the epithelial cells of internal organs with the consequent development of general saprophytic bacterial infections. In any case, the foregoing experiment shows that mice treated with propionibacteria do not develop symptoms of diarrhea and / or gastrointestinal bleeding. The protective effect of propionibacteria can therefore be attributed to the healing of hematopoietic bone marrow stem cells after irradiation.

2. Treatment of P. granulosum, P. acnes and P. avidum in experimental mouse tumor sarcoma 180 in mice

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

Propionibacteria used

Propionibacterium granulosum KP 45 (isolated from 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 sodium chloride phosphate buffer at a concentration of 5 mg. / ml 0.2 ml of suspension was administered intraperitoneally or intratumorally to tumor-bearing mice.

Mice and tumors

Adult male CFW mice were used in this tumor system. Tumors were induced as described in Radio Sci. 12 (1978), pages 185-189. For this purpose, sarcoma 180 tumors were excised from donor-mice, the tumor tissue was pulverized, trypsinized (0.25% trypsin, 15 minutes at 37 ° C) and filtered. The number of cells was counted and set in 1 ml to 10 ⁸ cells capable of multiplication. 0.1 ml of suspension was given by subcutaneous, intrascapular injection. This procedure led to the development of appreciable tumors after about 4 to 5 days. Rapid tumor growth occurred between day 4 and day 14 after transplantation, with a lethal effect between day 20 and day 28.

In total, 225 male CFW mice were used in the experiments. In the survival assay, 105 mice were divided into seven 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 propionibacterial injection. P. granulosum, P. avidum or P. acnes was injected intraperitoneally (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 days 1 and 20, respectively, after tumor cell implantation, and then every other day to day 44 after implantation. The remaining 120 animals were divided into 8 groups and mice of experimental groups (I to VI) were treated with propionibacteria as before and control mice (groups VII and VIII) 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 vessels

The experimental groups that were more than two standard deviations after the mean control value were designated as regressive. The results were analyzed statistically by Student's t-test (tumor mass) or by Chi-square analysis with Yates correction (tumor regression and mouse survival).

The results obtained are shown in Table VI below.

Table IV

Survival of CFW 180 sarcoma mice treated with propionibacteria (1 mg / mouse) 0, 4, 8, 12 and 16 days after tumor cell implantation

Days after tumor cell implantation

16 20 May 22nd 24 26 28 30 32 34 36 38 40 42 44 Control 15 Dec 13 10 7 3 0 P. granulosum intraperitoneally 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 12 10 9 9 7 6 5. 3 3 P. acnes intraperitoneally 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 14 12 8 8 7 6 5 2 1 P. avidum intraperitoneally 15 Dec 15 Dec 15 Dec 14 14 13 11 10 8 5 4 4 0 P. granulosum intratumorally 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 13 12 11 9 7 7 6 3 P. acnes intratumorally 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 14 12 11 9 7 7 7 5 2 P. avidum intratumorally 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 15 Dec 14 13 '10 8 8 6 4 3

The following Table V shows the effect of propionibacteria on sarcoma growth and regression in CFW-mice.

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Table V

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

Intraperitoneal injection _ Intratumoral injection

Weight of tumors (G) Number of regression tumors Percentage of regression Weight of tumors (G) Number of regression tumors Percentage of regression Control 2631 t 0/15 0 2631 + 321 • 0/15 0 Propionibacterium granulosum 842 + 312 8/15 53.3 538 + 212 11/15 73.3 Propionibacterium avidum 1032 i. 381 6/15 40.0 842 + 246 10/15 66.6 Propionibacterium acnes 1126 and 412 5/15 33.3 731 and 218 9/15 60.0

With a single dose of 1 mg / mouse intraperitoneally, all three strains of propionibacteria 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 tumors 180 following intratumoral administration.

Cytostatic activity in vivo in mouse tumor cells after Propionibacterium granulosum

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

4. Antibacterial activity as adjunctive therapy in the chemotherapy of primary and secondary lung tumors of patients with primary or secondary metastatic lung tumors was selected for these trials. 10 patients received an intravenous infusion 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 run lasted 3 days:

Day 1 vincristine, 1.5 mg intravenously at 8 am and 8 pm,

Day 2 methotrexate, 25 mg intramuscularly at 20 hours, Day 3 methotrexate 25 mg intramuscularly at 8 hours, followed by methotrexate intravenously at 25 mg at 14 hours and infusion 30mg / kg

-11EN 278484 B6 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 one 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 infusions of P. granulosum. This difference can be described as statistically significant.

Patients' tolerability to intravenous infusions of P. granulosum strain KP 45 was good. During the infusion of 30 mg P. granulosum, although cold sensations occurred and then fever up to 39 ° C (2 to 12 hours after infusion), these side effects were no longer detectable the next day. Repeated infusions of P. granulosum did not lead to the development of delayed hypersensitivity during the 60-day observation. It is concluded that intravenous infusion of 30 to 240 mg of P. granulosum strain KP 45 can be performed in patients without risk of serious side effects or complications.

5. General instructions for topical application (gastric and intestinal cancer) mg of lyophilized product is suspended in 1 ml of 1% xylocaine in sodium chloride solution and injected intratumorally through the skin (1 mm diameter) using an 80 cm long, rigid polyethylene duct. with a fixed intramuscular needle (size 18) without pineal gland. The line is guided through the biopsy channel into an endoscope (gastrofiberoscope or colonoscope), wherein the neoplastic tumor that is under visual control is injected and the needle is inserted into the tumor tissue of 0.5 to 1 cm. The product is administered by injection and is rinsed with 1-2 ml of 1% xylocaine in sodium chloride solution.

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

The attached graph shows the values / percent survival and the number of surviving test animals, depending on the number of days after irradiation of the test 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 /

-12EN 278484 B6 line C / marked with open circles / P. acnes / whole cells / line D / marked with full circles / P. acnes / cell walls / line E / marked with open triangles / P. avidum / whole cells / line F / marked with solid triangles / P. avidum / cell wall /, line G / marked with dot circles inside / untreated control.

PATENT CLAIMS

Claims (8)

A method for the manufacture of cell wall or whole cell formulations from Propionibacterium granulosum, Propionibacterium avidum, and / or Propionibacterium acnes for use in radiotherapy and tumor chemotherapy, characterized in that the liquid culture of the propionibacterium in question is treated by a GasPak method in an airtight confined space. under anaerobic conditions formed with borohydride, sodium bicarbonate, citric acid and water at 37 ° C, the mass growth thus obtained is inoculated with a thick wash of propionibacterium, after incubation for 72 hours at 37 ° C the cells are separated by centrifugation under cooling, sediment washed with distilled water and, if necessary, disintegrated by milling with glass beads, preferably having 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 of cells, the glass beads are separated from the homogenate by filtration on a glass frit using phosphate buffers at pH 7.2, the milky suspension containing cell walls and the cytoplasm 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 trypsin suspension and toluene, centrifuged cell walls purified by trypsin digestion, washed with distilled water and then freeze-dried. Method according to claim 1, characterized in that the obtained cell walls are incubated at 37 ° C for 24 hours. Method according to claim 1, characterized in that trypsin is used in the suspension of cell walls and cytoplasm in 0.5 mg / ml suspension and the addition of toluene to the washout is 1 ml per 100 ml. 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 cultured and treated propionibacterium is also preferably a strain of Propionibacterium avidum kp 40. -13GB 278484 B6 The method according to claim 1, characterized in that an α-broth of the following composition is used as the medium for culturing the mass of the propionibacterium: Bacto Casitone 12 g Bacto yeast extract 4 g MgS0 ₄ . 7 H ₂ O 1 g glucose 4 g Distilled water up to 1000 ml PH for α-agar + 28 g Bacto agar. 7.2 The method according to claim 1, characterized by a buffer by which the glass cells are separated from the homogenate and the mentholes, prepared by pre-treating the phosphate beads after disintegration with which the gray / 15 M is removed. Na ₂ HPO ₄ . 2 H ₂ 0 a 1/15 M KH ₂ PO ₄ always dissolves in 1000 ml of distilled water; and 612 g of sodium hydrogen phosphate solution are mixed 388 g of potassium dihydrogen phosphate solution, the pH being adjusted to 7.2. Process according to claim 1, characterized in that the lyophilized propionibacteria are suspended in a phosphate buffered saline solution at a concentration of 5.0 and 7.5 mg / ml, respectively, in the final step of the process.