IE51203B1 - Cell-wall and whole-cell preparations for the treatment of tumours - Google Patents

Description

The present invention is concerned with cell-wall preparations and whole-cell preparations which are useful for the treatment of tumours, as well as in the radiotherapy and chemotherapy of tumours, which prepar5 ations have been obtained by the use of certain strains of Propionibacteria.

Preparations of anaerobic Co ryneforms, for example C. parvum CN 6134, are known and have been described as antitumour agents. However, it has been found that in systematic or local therapy, these preparations give rise to undesirable side effects and complications so that it is recommended in the liter2 ature that doses of more than 7.5 mg/m of surface area are to be avoided, this amount corresponding approximately to 20 mg. per patient.

We have now found that preparations based on killed whole cells or cell walls of certain strains of Propionibacteria are extraordinarily effective in the chemotherapy and radiotherapy of tumours. These preparations are particularly suitable for supportive therapy and their effect is especially great in the case of local administration.

We have found experimentally that certain strains of P. qranuloBum and P. avidum. when injected intra25 peritoneally into mice at a dose of 1.5 mg. per mouse, significantly prolong the survival period of mice which have been subjected to lethal radiation (650 R), -3a strain of P. qranulosum being the roost effective.

We have found that the preparations according to the present invention stimulate CFU-S proliferation, i.e. stimulate the migration of haemopoietic colony-forming units from the bone marrow to enter into the cell cycle and to migrate to the peripheral blood. In clinical practice, this finding means that the preparations according to the present invention are valuable for use in supportive therapy in the radiotherapy of tumours since they substantially increase the tolerance of the patients to radiation.

In addition, we have found that the preparations according to the present invention are effective against murine sarcoma 160 in mice and lead to a regression of more than 70% of the sarcoma 180 tumours when administered intratumorally.

We have also found that the preparations according to the present invention, when administered systemically to patients with primary or secondary lung tumours as a supplementary treatment with chemotherapy, help to avoid one of the chief complications in the chemotherapy of tumours, namely, the danger of infections. Thus, it appears that the preparations according to the present invention may be used as excellent supplementary agents, with an antibacterial effect, in the chemotherapy of cancer. -4The above-mentioned results, which are demonstrated hereinafter by in vivo experimental data, are completely unexpected.

The cell-wall preparations according to the present invention have proved to be especially useful since, with this type of preparations, the danger of undesirable side-effects is particularly low. Cellwall preparations have not been previously investigated clinically.

Thus, the present invention provides cell-wall preparations and killed whole-cell preparations for use in the radiotherapy and chemotherapy of tumours, which preparations have been obtained from Propionibacterium granulosum Strain KP 45 and Propionibacterium aviduro Strain KP 40.

It is preferable to prepare injectable suspensions in phosphate-buffered sodium chloride solution, a concentration of 0.5 to 50 mg. of active material per ml. of solution being particularly useful, a concentration of 2 to 12 mg/ml of solution and particularly of 5 to 7.5 mg/ml of solution being preferred.

Intravenous infusion solutions containing 15 to 30 mg. of the preparation in 100 ml. of solution are especially preferred. Such a dosage quantity has proved to be especially advantageous for administration in the treatment of primary and secondary lung neoplasms in which a first injection should take place at -5least 10 days before chemotherapeutic treatment.

It is preferable to use a disodium hydrogen phosphate/monopotassium dihydrogen phosphate buffer with a pH of about 7.2.

The strains P. qranulosum KP 45 and P. avidum KP 40 have been deposited with the DSM (Deutsche 5anmlung von Mikroorganismen, Gesellschaft fur Biotechnologische Forschung mbH (German Collection of Microorganisms, Society for Biotechnological Research, Lts.), Grisebachstrasse 8, D-3400 Gottingen, West Germany), under DSM deposit numbers 1773 for P. qranulosum KP 45 and 1772 for avidum KP 40, on the 11th March, 1980. Both strains are available from the DSM in conformity with the release document (at present Form 2750) which is to be presented to the DSM and the German Patent Office.

P. qranulosum and P. avidum can also be isolated and grown from smears of acne efflorescences (acne vulgaris, acne papulopustulosa and acne conglobata), the taxonomy of which is given, for example, in Der Hautarzt, 30, 242-267/1979.

The following Examples are given for the purpose of illustrating the present invention:51203 -6Example 1.

General procedure for obtaining cell walls.

The particular Propionibacterium was cultured in a 1 litre Erlenmeyer flask at 37°C. under anaerobic 5 conditions (Gas-Pak Process, BBL).

For this mass culture, the medium (A-bouillon* or Triptic soy broth, Difco) was inoculated with a thick suspension of Propionibacterium. The suspension used for the inoculation was prepared by triturating a three-day A agar culture of Propionibacterium in 10 ml of bouillon. After an incubation period of 72 hours at 37°C., the cells were separated from the liquid by centrifuging at 10,000 g (20 min.) in a Sorval R2 cooled centrifuge. The sediment was washed three times with distilled water and then mixed with double the 51293 -7volume of glass beads (diameter 0.17 - 0.18 mm.) and ground in a cell mill *** for 1 to 1.5 hours. After carrying out a microscopic examination (Gram’s preparation) to ascertain that whole cells were no longer present, the glass beads were separated from the homogenisate by using a phosphate buffer ** (pH 7.2) and a G-l frit.

The milky 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). The autolytic enzymes were inactivated by boiling for 10 minutea on a water-bath.

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

The cell walla which had been purified by digestion with trypsin were finally centrifuged off (30 minutea), washed 3 times with distilled water and then freezedried.

* A-boulllon Bacto casitone 12 g.

Bacto yeast extract 12 g.

KH2P04 4 g.

MgSO4-7H20 1 g. glucose 4 g.

-Bdistilled water to make up 1000 ml., pH 7.2 for A agar + 28 g. of Bacto agar. ** phosphate buffer 1/15 M Na2HP04-2H20 and 1/15 M KH2P04 were each dissolved in 1000 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 Zellmuhle vi 3, Edmund Buhler, 7400 Tubingen, Germany.

Example 2.

Preparation of suspensions of P. avidum (Strain 0575, KP 40) and P. qranulosum (Strain KP 45).

According to the general procedure of Example 1, lyophilised Propionibacteria of the types mentioned are suspended in the phosrphate-buffered salt solution described above in a concentration of 5.0 or 7.5 mg/ml The suspension obtained is especially useful for intraperitoneal injection.

Example 3.

Using the procedure of Example 2, suspensions which can be injected intraperitoneally are prepared from lyophilised P. qranulosum, Strain KP 45, 95 K and P. avidum. Strain 0575, KP 40, with a concentration of 5 or 7.5 mg/ml of cell material. -9Examole 4.

Preparation of injectable suspensions for supplementary treatment in chemotherapy.

P. qranulosum. Strain KP 45, is prepared according 5 to the general procedure of Example 1. A suspension is made of 30 mg. of cell material in 100 ml. of infusion solution for intravenous administration.

The preparations according to the present invention may contain conventional adjuvants. The preparations may i0 be in ampoule form or they may be in sterile bottles with seals which can be pierced. Formulations with separate storage of the individual components, which are mixed together when they are to be administered, can also be made.

The following Table gives characterisation details of the above-mentioned P, avidum Strain KP 40 and P. qranulosum Strain KP 45: in CL ΙΌ X *4 Li ρ Φ 3 to « U 0 Π3 «rt Α 3 •rt C C ro 0 Ll •W CP D- 0 • Li CL CL V Φ c X IQ to O to 0 CL c X 0 •rt c 4J «rt 10 10 n u •rt to Ll to Φ to ε υ P ?o *0 L •rt iO > X Q ue CL 4J υ (0 φ ζ •-Η IQ υ -rt ε φ Α U Ο •rt η 1 1 Φ «—1 -rt 0) Φ N 0 χ -η to + + 1 C X -H φ Li + + Q to 1 a- ne Φ -rt 1 + O to 1 Φ Nit rat I 1 1 Ό Φ C rt HI 0 1 1 1 ό ID 3 Li rt □ c *D n 1 + η -π > > ΗΗίΗ + I (0 rt 0 0 C «Ρ •rt rt1 1 1 •rt c |Q U 1 + CO 1 c rt C 0 <0 to r-H 1 + 1 L< 0 0 +J CO -rt I1 1 *J φ rt O IQ 0 + + Σ 1 X Φ Sacc aros + + o 1 0] 11 Φ c rtl rt IQ Ρ > 0 Li Φ 0 CO e 0. • 0 •.3 CO CLlrrt 0.ΙΌ E E > 0 c « rt c Li 0 Li C to Uj *c rt 'Ll to to « >4J O' c to 4J ou.ee rt Li P υ C P rt 4J Φ Li 10 p φ Φ Q, IQ C Ή -rt o Φ 3 Uj X Φ Q) -rt 10 4J ε C C Li Li rt W <0 co tO to α « o x a c rt m o 10 0 z to CL CL to tf X Type of phago cytolyais ' Koi.....Koi 3 £ £ Ci & 0 H 0 •rt k 0 « E 0 Ό •rt > Λ a rj ΓΛ 1 1 m c· m O 1 1 co in 1 + P.granulosum 0· m D 1 1 in O' + 1 0 0 υ n 0 X cu 1 1 Species 1 3 c 0 kJ 0 fa ε η ii Origin 1 c i a ι 0 Φ -I H U Uj « >4J <0 JJ 0.^ o u x c k Ό 0 C « —< -u Φ β £ e φ 4) e -H e c -η 4) 0 □ Uj X 0 +> 41 -H « *) E k p C « Μ 3 Url ># 5 wiuS-w £j*j ae υ-ίχ a Strain No. in o 0· 0* cu α X X ta A Φ 0 c «rJ Ό u AJ 0 0 c A 0 0 E ku rj □ σ e 0 rt C Uj kJ Ij 3 • 0 kJ »rt Ό •0 AJ 0 Q £ 0 0 Σ 0 N A C C 0 » •rt 0 0 0 0 c Ρ N £ 0 0 0 c U C •rt • 0 u Q.UJ N 0 0 •rt 0 0 E kj ό 0 0 3 • cu kJ υ •rl fa • 0 £ kJ c O' 3 0 0 U4 Oh H a AJ 0 O' Uj 0 υ 0 iH M-l φ 0 0 0i\W kJ A 0 •rl Γ* 1 0 rt σ TJ 0· ffl rJ c · 0 3 f\ ·Η Uj Of* A A 1 M UJ •rt r* a V5 cj 0 M ao' 0 0· σ 0 H •rt r4 (\ rJ 0 u\ kJ 0 3 •rt fa O' 0 u - > U 1 A •rt O 1 0 uj tn U O'cnl 0) σ 0 0 0 c ι—i ffl • u 3 0 AJ < > £ £ k· 0 •rl » 0 0 Li » 0 aw ω 0 3 £ £ k 0 AJ 0 υ AJ 0 0 Ό 3 < 0 AJ •H c 0 0 kJ AJ *0 «) S-rl £ 0 0 3 0 0 W -4 AJ · 0 kJ 0 μ to m > 0 CLUJ > , c· ♦+4 O & ·· b » o> A £ 0 kJ o ·· -4 0 *0 0 0 0 H kJ\ 4-> £ ·Η t4 Lj 0 0H C 0 U 0 « -rt k c· 0 0 $ > A 0 cj e .· A a H 0 > 1 c m * 0 3 M -ι m φ > 0 fa 0 3 Ό fa ο» A « fa CJ Η *rt •rt • ε \ C kJ o • * • I O' 0 0 0 Oh| 0 Ci ’rt AJ *D E Φ X (M 0. 0 £ 0 *0H 1 0 0 0 fa C • rs k Λ 0 - «j CJ CU •rt 0 • • • cj c X · OH Ό X kJ 0 0 X 0 Φ • 0 •»J • 0 c A Otnlc a J -rt • 3 £ C rsilu 0 • 0 J E 4J 0 •rl w X 0 * E • «-4 Cu δ . X H 0 k β Ό - 0 Λ 0 0 0 0 kJ • · u kJ -rt 3 0 H 0Ή AJ 0 υ A 0 rJ 0 C 0 o > •rt v •rt UJ -rl 0 -rt c rJ Σ 0 kJ uj A bXl c 3 kJ 0 0 0 p 0 CU . φ > X k • Lj υ D.4J 0 υ υ • Q, C Uj • -rt • «rt M OCO 0 3> E u ε fa —»» · Z —1 Cj n -12In vivo Investigations. 1. Haemopoiesis in mice treated with Propionibaeteria after lethal radiation.

Two strains of Propionibaeteria (P. qranulosum 5 and P. avidum) were injected intraperitoneally into mice which had been subjected to lethal radiation (850 R), in a dosage, in each case, of 1.5 mg. per mouse. It was found that P. qranulosum prolonged the most the survival period of the mice which had been subjected to radiation.

Adult, 8 week-old male Swiss mice were used.

The animals were fed a standard diet. Water was freely available. The water and food were sterilised. The water contained oxytetracycline (1 g. per 1000 ml.).

Radiation.

The mice were subjected to radiation of 650 or 850 R in a rotating metacrylate cage (10 mice per cage), at a distance of 50 cm from a THX 250 Medicor unit, which was operated at 200 kV and provided with a 0.5 mm Cu filter. The radiation rate of about 75 R per minute was determined with a CT-1 thermoluminescence R-dosimeter.

P. avidum. Strain 0575, KP 40 and P. qranulosum. Strain KP 45, were used for the experiments in the form of killed whole cells and in the form of cell walls. The lyophilised Propionibacteria were suspended in a phosphate-buffered salt solution in a concentration of 7.5 mg/ml and 0.2 ml. of -13the suspension was injected intraperitoneally (1.5 mg. per mouse).

Investigation of exogenous spleen colonies. 3, 2 or 1 day before the transplant of bone marrow, donor mice were injected with 1.5 mg. of P. qranulosum. The bone marrow cell suspension was prepared by washing both thigh bones with sterile Parker medium. The cells were counted in a haemocytometer. 5 χ 103 cells which were capable of reproduction in 0.2 ml. of Parker medium were injected into the tail vein of each mouse which had been subjected to radiation of 350 R 4 hours before the bone marrow transplant. 0.? ml. of medium or 5 x 103 bone marrow cells from normal mice (not treated with P. qranulosum) were injected into the control mice. P. qranulosum was administered, as before, to another group of donor mice; the animals were then anaesthetised with diethyl ether and blood was removed from the retrobulbar plexus. The blood cells were counted in a haemocytometer and aliquot portions of blood with 5 χ 105 nucleated cells were injected into the tail veins of mice which had been subjected to radiation of 850 R. The control mice received equal volumes of blood which had been taken from normal mice (not treated with P. qranulosum).

All the mice were killed and weighed 9 days after the transplant of blood or bone marrow, the spleen was removed, weighing was carried out again and the -14number of colonies per spleen was determined after fixing in chloroform : ethanol {1 : 3 v/v). Investigation of endogenous spleen colonies. 3, 2, 1 or 0 days before being subjected to 5 radiation with 650 R, mice were injected intraperitoneally with P. granulosum. The control mice received the same volumes of PBS (phosphate-buffered salt solution). 9 days after being subjected to radiation, the animals were killed, the spleens were removed, weighed and the colonies were counted, as in the above test to determine the exogenous spleen colonies.

Survival test.

Mice were divided into 7 groups, with 15 mice per group in each case, and were subjected to X-ray radiation of 850 R. 4 hours after being subjected to radiation, cell walls or whole cells of Propionibacteria were injected into the animals. The control mice received the same volumes of PBS. From the 10th day after radiation, the number of surviving animals in each group was counted.

The Student t-test was used for the statistical analysis.

The effect of the Propionibacteria which were investigated on the survival rate of the mice which had been subjected to lethal radiation is given in graphic form in the Figure of the accompanying drawing. In comparison with the control animals, both strains -15of Propionibacterja led to a significant prolongation of the survival span. P. qranulosum. in the form of a whole cell preparation and in the form of a cellwall preparation, proved to be more effective than P. avidurn.

The influence of P. qranulosum Strain KP 45 on endogenous spleen colonies.

TABLE I The number of endogenous spleen colonies in mice 10 treated with Propionibacterium qranulosum (Strain KP 45) (average value + 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 radiation 1.66 + 0.24 10.6 + 4.45 x P. qranulosum 2 days before radiation 1.73 + 0.21 8.17 + 3.49 x P. qranulosum T day before radiation 2.18 + 0.42 11.2 + 5.30 x P. qranulosum 4 hours after radiation 2.32 + 0.38 14.1 + 4.70 X x = p £ 0.01 -16The relative weight of the spleens of the mice which were subjected to radiation only increased if p. qranulosum was administered 1 day before or 4 hours a after radiation with 650 R. However, the number of 5 exogenous spleen colonies in all treatments increased significantly.

The effect of P, qranulosum Strain KP 45 on the formation of exogenous spleen colonies.

No differences were established in the relative 10 weight of the spleen between mice which had received normal bone-marrow transplants and mice which had received bone marrow from donor animals which had been treated with P. qranulosum 2, 3 or 1 days before the transplant. This treatment led to a significant decrease in the number of exogenous spleen colonies in animals which had been subjected to radiation of 850 R, in comparison with animals to whom the bone marrow of untreated donor animals had been given. This finding shows a decrease in the number of CFU-S in the bone marrow of mice which were treated with P. qranulosum.

On the other hand, an injection of blood from mice which had been treated with P. qranulosum in experimental animals which had been subjected to lethal radiation led to a significant increase in the values of the relative spleen weights and the number of spleen colonies, in comparison with the injection of blood from untreated donor animals, as can be seen from the following Table III:51203 -1'Number of exogenous colonies after transfusion of bone marrow from mice treated with P. granulosum (Strain KP 45) CFU-S per 10θ nucleated bone marrow cells CN • m + 1 CN »-4 204.0 + 21.4 κ GO •Η + 1 o • ro O ri X rsj 41 O Φ 107.0 + 43.3 x Number of exogenous 1 spleen colonies 1 1 CM fO o + 1 CN r-4 rH 20.4 + 2.14 X r* cc »-4 + 1 ro C r4 11.6 + 4.4 X 1 1_ X Π τί 41 r* d rH Relative weight of spleen 0.99 + 0.19 r—4 co o + 1 CN Λ e-ί 1.51 + 0.49 Ti TT d +t in r-1 1.69 + 0.27 Transfusion Control (0.5 ml of Parker medium) Bone marrow from normal mice Bone marrow from mice treated with P. granulosum 3 days before the transfusion Bone marrow from mice treated with P. qranulosum 2 days before, the transfusion -1 Bone marrow from mice treated with P. granulosum 1 day before the transfusion p 0.01 -18Number of exoqenous spleen colonies after transfusion of blood from mice treated with P. granulosum (Strain KP 45) \O O Φ c 0 tn CO X rt X X X m rrt X rt oj m n OJ • • • ΜΌ Φ O o o O W Φ o + 1 41 + 1 41 ro ? ω 0) 0 r* 0- rt 1 rt P C o in D ϋ P • • • • Eh 3 <0 rt rt CM rt U C E 1 0 c X Φ X X φ Φ 5ί X X rt Ol • sO X 44 cl tn • rt • • 0 tn φ ΓΜ Oi rt •rt 41 P 0 C + 1 41 41 φ 3 0 sO *— Λ 0 -1 GC .«rt Oi 0 F C 0 • • • • c 3 Φ u m 0) O Γ 0 z σ •rt ri ri ro £ •rt σ X X X > •rt 0 m co rt a? Φ Φ c Ol cc CO Ό ? Φ • • • • Φ o o rt rt Ό Φ rt Li > a + 1 41 41 41 <0 0 Ό 4> Oi in m C 0 44 Φ Ol (0 rt 0 • 1 • • ri Φ Ol m tn CC 41 £ ri Ό tn •Η 0 a) ϊ 0 3 rt tn Λ 0 •rt > tn Ό 0 Φ o O O O w ri 0 co >0 Oi in O' > 3 Ό· cn rt sT <0 υ 44 so cn o σ» P 0 0 ·—f φ υ C > 3 ro < Φ P U ri 0 0 > >. Ό to Ό Φ Ό 0 Φ ΦΌ C C ΦΌ C u ri 0 4» 0 ri ϋ •rt rc fn -rt 0 Ol Ή 0 rt •rt E Φ 0 Φ 0 Φ 0 w ε 3 l ε 3 ε 3 X 3 44 4» 3 44 4» 3 4-) C (0 0 0 0 0 0 0 0 E Φ 0 P Φ O C Φ 0 C •rt P U rt 0 U rt 0 U rt 0 tn 0 •rt 3 P -rt 3 P •rt 3 P 3 c ε c •P ε c ri ε e ri 44 <0 0 0 tn ε E Φ ε n Φ E u Φ c 0 ο ϋ £ O 3 £ O 0 £ ro P P ri P ri P 4) P 44 44 · 44 · · E- 0.1 φ 0*1 Φ ο* φ Ό *0 P *0 P *O P 0 OX 0 0 £ 0 ο a 0 0 0 ri 44 0 4> Ή Ό ri ri φ Φ Φ ca β 3 Λ ffl 3 Λ m 3 X o a o V a 0.05 ex I -19Blood taken from donor mice which had been injected with P. granulosum two or three days before the blood transplant was most effective in stimulating the formation of spleen colonies. However, leucocytosis in transplanted blood was increased after treatment with P. granulosum 3, 2 or 1 day before the blood was taken.

In the above experiments, P. granulosum proved to be the most effective agent in rats which had been subjected to lethal radiation. The fatal effect of the lethal dose of x-rays is the result of inhibiting the proliferative ability of the haemopoietic parent cells and of the damage to the epithelial cells of the internal organs, with subsequent development of generalised infections by saprophytic bacteria. It is, of course, evident from the above experiment that mice treated with Propionibacteria do not exhibit any symptoms of diarrhoea and/or haemorrhages of the digestive tract. Therefore, the protective effect of the Propionibacteria can be ascribed to the increased recovery of the haemopoietic parent cells of the bone marrow after the radiation. 2. The effectiveness of P. granulosum and P. avidum in experimental murine sarcoma 180-tumour in mice.

Both previously mentioned Propionibacteria were administered intraperitoneally or intratumorally in several doses of 1 mg. per mouse in each case and -20proved to be effective in retarding the growth and stimulating the regression of sarcoma 180 in CFW-mice. In addition, the use of Propionibacterja results in the prolongation of the survival of mice with sarcoma 180.

The Propionibacterja used.

Propionibacterium qranulosum Strain KP 45 (isolated from a wound infection in the Hygiene Institute of the University of Cologne) and Propioni10 bacterium avidum Strain 0575 (C.S. Cummins, Anaerobe Labs, Virginia Polytechnic Inst, and State University, Blacksburg), KP 40 were used for the experiments. The Propionibacterja mentioned were lyophilised and suspended in a phosphate-buffered salt solution in a concentration of 5 mg/ml. 0.2 ml. of the suspension was administered intraperitoneally or intratumorally to mice which had tumours.

The mice havinq tumours.

In the case of this tumour system, adult male CFW mice were used. The tumours were induced as described in Radio Sci., 12, 185-189/1978. For this purpose, sarcoma 180 tumours from donor mice were dissected, tumour tissue was minced, trypsinised (0.25% trypsin. 15 minutes at 37°C.) and filtered. β The number of cells was counted and adjusted to 10 cells capable of reproduction per 1 ml. of salt solution. 0.1 ml. of the suspension was injected -21subcutaneously and intrascapularly. This procedure led to the development of palpable tumours after about 4 to 5 days. Rapid growth of the tumours took place between the 4th and the 14th day after the transplant, with the lethal effect appearing between the 20th and 28th day.

A total of 225 male CFW-mice were used in the investigations. In the survival tests, 105 mice were divided up into seven groups (15 mice in each group) and the animals of the experimental groups (I to VI) were injected with Propionibacteria on days 0, 4, 8, and 16 after the inplantation of the tumour cells.

P. qranulosum or P. avidum was injected intraperitoneally (3 groups) or intratumorally (3 groups) at a dosage of 1 mg/mouse. The control mice received intraperitoneal or intratumoral injections of PBS.

The number of surviving animals was recorded on the 16th or 20th day after inplantation of the tumour cells and then every second day up to the 44th day after inplantation. The remaining 120 animals were divided up into 8 groups. Mice of experimental groups (I to VI) were injected with Propionibacteria as before and the control mice (groups VII and VIII) were injected with PBS. On the 20th day after the inplantation of the tumour cells, all the mice were sacrificed and the tumours were dissected and weighed. The arithmetical average and the standard deviations were determined for -22each group. All the tumours of the experimental groups which were more than two standard deviations below the average value for the control were recorded as regressive. The results were analysed statistically by means of the Student t-test (tumour mass) or the Chi-Square-Analysis with Yates Correction (regression of the tumours and survival of the mice). The results obtained are summarised in the following Table IV :51203 -23JC 4J <0 '2H Ό 4J C C Π3 4) IS tf «I GJ »H P CD H 4» φ • O • 3 OOO co g η n 3 >+J Μ β ε Ό Ή Ο Ο U β k Ο β 10 ο Ο — ·γΙ 0) ·Ρ οια η β α ·ρ -π ο β ► Ε « «\Η ί ο * « e UH Η — Φ £Uk U Φ , 4J 41 <μ υ <μ Ο tf tf Η »Η Λ C > ο •Η ·Η fc& Ο Ρ co α 5Τ ci ci m CM m o vo Mt « ο in Mt N ID ••Ί Ό· υ Ρ η vo r- CO 3 ci £ φ c· m σι CO ο £ 41 Ch co H o tw C1 H rd 0 C ο Ν Ot o CM Cl •Η η rd rd rd «0 41 C ο O rd Cl Μί <0 η rd rd rd rd Η % *Η co O Cl m m in ci rd rd rd rd Ρ V 41 (μ νθ ci CM V in m « ext rd rd rd rd & c^ m di in in Ό CJ rd rd rd rd Cf o in tn in in CM rd rd rd rd rd o o in m in tn Ct rd rd rd rd rd vo m in in m m rd rd H H rd rd rd rd tf « GJ 0) I β c rd rd 0 0 10 <0 6 41 £ P P 3 Ή •H •p Q 0 « P P io b E 0 Φ φ o 3 3 rd Ot e & rd 41 6 +* Π 0 3 3 « 3 io c P tt P β k <0 k o <0 41 •ri 4l fl 41 ♦rl 41 ρ k β > c k β > c 41 σ -w fl td σ -κ tf ·Η β 0 υ Μ 0.Ί -24The following Table V below shows the influence of Propionibacteria on the growth and regression of sarcoma 180 in CFW mice :512 0 3 *0 4 CJ c ΦΗ c o k A n Φ k ε « k SJ A Q υ intratumoral injection percentage of | regression o 73.3 66.6 number of regressed tumours 0/15 11/15 10/15 tumour mass (g) 2631 + 321 ci rH ci + 1 CO m m vO «r Ci +1 ci co Intraperitoneal injection ω c 0» 0 «S -k jj 0 c a ΦΉ Φ U 0 k k 0» Φ Φ a n o 53.3 40.0 number of regressed tumours 0/15 8/15 tn rH to tumour mass (g) rH CJ tn +1 rH tn κθ Ci 842 + 312 1 1032 + 381 Control § Ή k Φ 4J ί § •Η <0 e o 0 -1 •H 3 0 c O «3 b b 0« 01 Prooionibacterium | avidum -26With the intraperitoneal administration of a single dose of 1 mg/mouse, there was a significant enlargement of the spleen on the 4th day after injection, with further enlargement of the spleen mass on days 6 to 14, with both strains of Propionibacteria tested. This enlargement of the spleen reflects the stimulation of the reticuloendothelial system and runs parallel to the antitumour activity of these immunostimulators. £. qranulosum led to a regression of more than 70% in the sarcoma 180 tumours which were investigated when administered intratumorally. 3. In vivo cytostatic effectiveness in murine tumour cells by Propionibacterium qranulosum.

In a further investigation, the cytostatic effect of Propionibacterium qranulosum Strain KP 45 on sarcoma L-l in BALB/c mice (lung) was investigated. Here, too, a significant effectiveness was observed in comparison with untreated animals. 4. Antibacterial effect as a supplementary treatment in the chemotherapy of primary or secondary lung tumours.

For this test, 30 patients with primary or secondary metastatic lung tumours were selected. 10 of the patients received intravenous infusions of 30 mg. of P. qranulosum in 100 ml. of intravenous infusion solution, the other 20 patients serving as a control. -27All the patients were treated chemotherapeutically to synchronise the proliferation of neoplastic cells.

Each course lasted 3 days: 1st day: 1.5 mg. of vincristine intravenously at 8 a.m. and at 8 p.m., 2nd day 25 mg. of methotrexate intramuscularly at 8 p.m.. 3rd day: 25 mg. of methotrexate intramuscularly at 8 a.m., followed by 25 mg. of methotrexate intravenously at 2 p.m. and an infusion of 30 mg/kg of cyclophosphamide at the same time.

This chemotherapeutic treatment was repeated three times every 21 days.

The total chemotherapy consisted of the above three cycles which began in each case on the 1st, 22nd and 43rd day of observation.

During the 3rd, 10th, 31st and 52nd day of observation, P. qranulosum was administered at a dose of 30 mg/100 ml. of intravenous infusion solution in the course of 15 minutes (on the last day of the first chemotherapeutic cycle and then 1 week after the end of each cycle).

In the case of patients who were only treated therapeutically, five cases of bacterial infection appeared (three pneumonias, one sepsis and one case of angina tonsillaris) during a 60-day period of observation, while in the 10 patients treated with chemotherapy and intravenous infusions of P. qranulosum, no symptoms of bacterial infection appeared. This difference is statistically significant. -28The tolerance of the patients to intravenous infusions of P. qranulosum Strain KP 45 was good.

During the infusion, in an amount of 30 mg. of P. qranulosum, chills and then fever up to 39°C. did, indeed, appear; on the following day, however, these side-effects were no longer observed. Repeated infusions of P. qranulosum did not lead to the development of a ” Z delayed hypersensitivity during the 60-day observation. From this, it is concluded that the intravenous infusion of 30 to 240 mg. of P. qranulosum Strain KP 45 can be carried out without the risk of serious side effects or complications in patients.

. General directions for local administration (cancer of the stomach and intestine) mg. of lyophilised product are suspended in ml. of 1% xylocaine in salt solution and injected intratumorally through the skin (diameter 1 mm.), using an 80 cm. long, stiff polyethylene tube with a firmly attached intramuscular needle (18 gauge) without an epiphysis. The tube is introduced by means of a biopsy cannula in an endoscope (gastrofiberscope or colonoscope) and the neoplastic tumour, which is under visual control, is punctured and the needle is introduced to a depth of 0.5 to 1 cm. into the tumour tissue. The product is 25 injected through the tube and washing is carried out with 1 to 2 ml. of 1% xylocaine in saline. -29The intratumoral injections (10 mg. of product in each case) are administered, for example, during the first three days and then on the 10th and on the 17th day of observation.

Claims (5)

1. CLAIMS:1. A cell-wall preparation and a killed whole-cell preparation for use in the radiotherapy and chemotherapy of tumours, which preparation has been 5 obtained from Propionibacterium qranulosum Strain KP 45 and/or from Propionibacterium avidum Strain KP 40.

2. A preparation according to claim 1, in the form of an injectable suspension in a phosphate-buffered 10 salt solution.

3. A preparation according to claim 2, in the form of a suspension of whole cells or cell walls in phosphate-buffered salt solution with a concentration of 0.5 to 50 mg. of active material per ml. of 15 solution.

4. A preparation according to claim 3, in the form of a suspension of whole cells or cell walls in phosphate-buffered salt solution with a concentration of 2 to 12 mg. of active material per ml. of solution. 20 5. A preparation according to claim 4, in the form of a suspension of whole cells or cell walls in phosphate-buffered salt solution with a concentration of 5 to 7.5 mg. of active material per ml. of solution. 25 6. A preparation according to claim 1, in the form of an intravenous infusion solution containing 15 to 30 mg. of active material per 100 ml. of solution. -317. A preparation according to any of claims 2 to 5, wherein the phosphate-buffered salt solution contains disodium hydrogen phosphate and monopotassium dihydrogen phosphate.

5. 8. Preparations according to claim 1, substantially as hereinbefore described and exemplified.