DE2527636A1 - METHOD FOR PRODUCING MYCOBACTERIAL FRACTIONS - Google Patents

Description

Agence Nationale de Valorisation de la Recherche (ANVAR), 13, Rue Madeleine Michelis, 92522 Neuilly sur Seine,

France

Process for the preparation of mycobacterial fractions

The invention relates to new mycobacterial fractions, which have been isolated from different species of the genus Mycobacteria. The invention also relates to a manufacturing method for such factions.

The mycobacterial fractions according to the invention have valuable properties for protecting warm-blooded animals against Tumor and viral agents. They also act as adjuvants in the biological response to antigens.

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Whole mycobacterial cells, e.g. from Bacillus Calmette-Guerin (BCG), are currently used clinically to treat cancer. They prevent and cause tumor formation even a regression of various experimental tumors in animals. It has also been shown that BCG had a modest survival increase from having Columbia SK virus infected mice. In addition, mycobacteria have an immune response that occurs in the cell medium and that they induce the production of circulating antibodies to a wide variety increase of antigens to which test animals have been exposed. They are particularly suitable for this purpose, though they are used in an oily emulsion known as Freund's complete adjuvant. All of these valuable Activities are obtained using whole, unpurified mycobacterial cells, creating a large A variety of biological responses can be caused, some of which are so toxic that use is restricted in clinical therapy.

Live BCG is currently used as an immunopotentiating agent in the therapy of human tumors. at however, the use of such live mycobacteria can result in various complications, one of which the most common ulcerations at the site of BCG inoculation are 2 to 3 months to heal are. Fever and dizzy spells are often soon after multiple inoculations of sensitized Patient observed. Systemic BCG infections with prolonged Fever, hepatosplenomegaly, liver function test abnormalities, and other systemic infections have been observed in individuals who have been inoculated with BCG. It seems likely that

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the risk of such infections with BCG therapy is greater in patients whose immune responses are subdued.

It was found that non-living mycobacterial preparations can be used as tumor growth inhibitors in place of live mycobacteria.

The aim of the invention is to obtain new, purified mycobacterial cell fractions to provide that are able to warm-blooded animals a higher protection against both tumors as well as to confer viral infections than the use of whole cell mycobacteria preparations. In contrast to whole mycobacterial cells, which have a low antiviral activity when injected intravenously and no activity when injected intraperitoneally will have, induce the new fractions according to the invention a very strong protection against a viral Infection if injected by any of these routes. You are also able to control the intensity of the Boost immune response to a wide variety of antigens.

In the process according to the invention, the procedure for preparing the protective agent according to the invention is that one a suspension of broken or shattered mycobacterial cells in an emulsion of a subjecting aqueous solution and an immiscible organic solvent to a violent disintegration treatment, the solvent being of the type which, when appropriately mixed with water, form an emulsion therewith can form, the separation of the cell debris from this medium on the one hand and from the water and from the solution

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by means of the emulsion to separate phases on the other hand and that one of the solvent, the solid material. rial freed, which is either suspended in it or an intermediate phase system between the water and solvent phase or both, the solid material containing the protective agent.

Forms in an advantageous embodiment of the invention the starting suspension from a suspension of whole cells in a mixture of water and a Immiscible organic solvents are suspended, then the suspension of a disintegration, for example by mechanical shattering or violent agitation or agitation.

The separation of the cell debris on the one hand and the solvent phases on the other hand it is advantageously carried out by centrifugation.

In a preferred embodiment of the method of removing the solid material from the organic solvent, in which it is suspended, you can first remove the aqueous phase, then the solid material that is suspended in the organic phase, or recover the solid interphase material, this material in water or an aqueous solution, for example in an aqueous physiological emulsion, and redisperse the resulting Remove the remaining organic solvent from the product.

The solid of the suspension contains the active ingredient according to the invention. It can be separated and dried, in particular freeze-dried or lyophilized. It

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Freeze-drying or lyophilization of the entire suspensions can also be carried out.

According to an advantageous method of manufacture of the protective agent according to the invention, the myco- "bacterial cells are treated in the following way:

1. The mycobacterial cells are submerged with water Mixed to form an aqueous suspension thereof.

2. The resulting suspension is then mixed with a suitable water-immiscible organic solvent which is capable of, by appropriate mixing, in the presence if necessary of an emulsifying agent to form an emulsion.

3. The cells of the resulting emulsion are agitated by vigorous mechanical agitation or agitation or circulation broken up by a cell disruptor such as a Manton-Gaulin press.

4. The aqueous part and the cell debris are removed by appropriate measures, for example centrifugation, separated from the non-aqueous portion of the emulsion.

5. The solid material suspended in the organic phase is recovered.

6. The solid obtained in this way is dispersed in water and the resulting suspension is freed from the remaining organic solvent.

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7. The aqueous suspension is dried, whereby the active ingredient according to the invention is obtained.

The water-immiscible organic solvent that makes up the combination used with the above aqueous suspension of mycobacterial cells is advantageous a solvent for lipids. It is advantageously selected from the group of saturated aliphatic and cyclic hydrocarbons, in particular having 5 to 12 carbon atoms. Examples of such solvents are Hexane, octane, petroleum ether, cyclohexane. Mixtures of such saturated hydrocarbons can also be used will.

Advantageous water / solvent ratios are between 75 and 40, preferably 65 and 55 vol. -96 of water and 25 and 60, preferably 35 and 45 vol .-? 6 organic solvents. Naturally, such relationships can be left without going outside the scope of the invention got.

Suitable emulsifying agents which can be used to obtain the desired emulsion can be any be non-ionic detergent compounds, such as polyethylene oxide sorbitol monooleate (Tween 80), polyoxyethylene lauryl ether (Brij 30) and octylphenoxypolyethoxyethanol (Triton X-100).

If necessary, the product obtained can be subjected to further cleaning treatments, e.g. a Differential solubilization in various organic solvents, which is either solubilized or non-solubilized fraction is obtained, which the active

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contains substance. For example, after an initial extraction with hot acetone or hot ethanol (which 55 to 65% of the inert material is removed) Chloroform / methanol extract with high antiviral and high antitumor activity can be obtained. This chloroform / methanol extract can be further fractionated by either

(1) ether solubilization followed by methanol precipitation, or

(2) chromatography on a silica column using ether, ether ethyl acetate or Ether / methanol mixtures for development

can happen. The latter stage gave a fraction with high antiviral and high splenomegalic activity.

The invention is illustrated in the examples. example 1

37 g of dried phenolic cells from M. kansasaii ATCC 21982 were suspended in 1.5 l of physiological saline solution and the mixture was emulsified with 1.1 l of hexane containing 15 ml of Tween-80 (polyethylene oxide sorbitol monooleate) by vigorous mechanical stirring for 2 hours . The emulsion was then centrifuged at 5000 g for 1 hour at 20 ^° C., which resulted in a separation into a lower aqueous layer, a solid intermediate phase layer and an upper hexane layer. The aqueous layer was carefully suctioned off. The interphase material was resuspended in the hexane layer by mechanical agitation

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IMD, the suspension was 1 h at 20 ⁰ C and 10000 g centrifuged again. The resulting clear hexane layer was decanted and the white gelatinous layer was separated from the packed cell debris. The white gelatinous material was suspended in water and the resulting suspension was freeze-dried to give 160 mg of an amorphous white powder, hereinafter referred to as M. kansasii IPM (interphase material).

Example 2

280 g (wet weight) of cells from M. phlei ATCC 11758 were obtained suspended in 2.8 1 physiological saline solution and the suspension was mixed with 2.1 1 hexane and 28 ml Tween-80 mixed. The mixture was emulsified by vigorous mechanical agitation for 3 hours. The phases were centrifuged separated to give an intermediate phase material isolated as in Example 1. It was 720 mg of a white amorphous material, hereinafter referred to as M. phlei IPM.

Example 3

150 g (wet weight) of phenolized cells from the M. tuberculosis (var. Bovis) strain BCG became physiological in 1.5 liters Suspended saline solution and the suspension was mixed with 1.2 l of hexane and 15 ml of Tween-80. After emulsification, Centrifugation and drying as in Example 1 gave 450 mg of a white amorphous product as the freeze-dried product Obtained substance hereinafter referred to as BCG-IPM.

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Example 4

200 g of wet, freshly obtained cells of Mycobacterium smegmatis (ATCC 21732) were suspended in 2.1% of a 0.9% aqueous saline solution and the mixture was dispersed in a Waring mixer for about 10 minutes at room temperature. 1.2 l of hexane and 24 ml of Tween-80 were added to the smooth suspension with mechanical agitation or stirring. The resulting emulsion was cooled by a pressure cell for continuous flow (Manton-Gaulin) at a pressure of 633 passed a Abstromtemperatur of at most 30 ⁰ C kg / cm. The resulting mixture was recycled two more times through the press with cooling. The homogenate was then centrifuged for 90 min at 5000 g and 25 ° C., whereby a multi-phase system was obtained which consisted of a lower cell debris layer, a lower opaque aqueous layer and an upper gelatinous hexane layer.

The aqueous layer was carefully aspirated from the centrifuge tube and discarded.

The viscous gelatinous hexane layer was then carefully decanted into a suitable evaporator, where it was mixed with 500 ml of water. The entire mixture was then heated ^{to about 38 °} C. under reduced pressure to remove the hexane.

The remaining milky aqueous residue was then freeze-dried, yielding 29.0 g of a fluffy, slightly colored amorphous solid, hereinafter referred to as M. smegmatis IPM.

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The product IPM isolated as above is a white to amber-colored fluffy solid, which in neutral is insoluble in aqueous systems and only partially soluble in organic solvents. IPM can go through the following elemental analysis:

C: 51 to 57%

H: 7.5 to 8.7%

N: 5.0 to 6.5%

P: 0.5 to 1.2%

and by the following composition:

Neutral carbohydrates 21 to 23%

Amino sugar 0.8 to 1.8%

Amino acids 24 to 28%

Lipids (free) 40 to 43%

be characterized. These values indicate that IPM is part of the wall of the mycobacterial cell.

Based on the examination with the electron microscope and the ultracentrifuge, IPM appears to be a heterogeneous material to be of varying particle size. It can be in an aqueous saline solution, consisting of a carrier 10% polyethylene glycol 400 in saline or in water containing 0.02% Tween-80, with formation of stable milky dispersions suitable for injection.

These IPM preparations are for the protection of warm-blooded animals highly active against a large number of experimental tumors or viral infections and they also increase the immune response

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response to antigens (auxiliary substance reaction). If they are rabbits injected with doses up to 100 µg / kg then are they don't fumed.

Antitumor activity

It has been found that the new products obtained by the above methods have significant protective activity against a variety of experimental tumors including leukemia L-1210, Ehrlich ascites carcinoma and one have syngeneic lymphoid leukemia. The test methods used and test results obtained are described below.

Leukemia L-1210:

The test materials were groups of 10 B6DF1 mice in Single doses of 100 μg / mouse by intraperitoneal injection of the test material in a Hanks-BSS solution. In each experiment, two control groups of each 10 mice used. The control mice received either the diluent alone (negative controls) or the Pasteur strain BCG at 100 µg / mouse intraperitoneally (Positive controls). 8 days after administration of test material, BCG or diluent alone all animals were implanted with L-1210 leukemia cells, freshly collected from ascite-bearing B6DF1 donor mice. Each test mouse received intraperitoneally 500 cells and all animals were observed until death. Mice that lived 35 days after tumor implantation became regarded as long-term surviving mice as the control animals treated with the diluent were invariable Died 14 days after implantation. With everyone

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In the group, the mortality was recorded and plotted on a coordinate system (in%) against the time expressed in days (T). The mean time to death (Y ₅₀ ) ¹ ^ the standard deviation were determined graphically and the probability values were calculated according to the Student's t-test. In this system, a delay in the mean time to death of 2 days or more has been found to be highly significant (p = 0.001) and materials that give a delay of this magnitude are said to be very active.

Figures 1 and 2 describe the delay in leukemia mortality after treatment with IPM of M. smegmatis or M. phlei (curve no. 1) pr, with whole BCG cells (Curve No. 2). The mortality response of the untreated control animals is given for comparison purposes (Curve No. 3).

Ehrlich Ascites Carcinoma:

Pasteur strain BCG (phenol killed cells) or IPM obtained from M. smegmatis or M. kansasii became groups of 6-week-old hybrid female mice (C57BLxAKR) administered in various dosages. All preparations were suspended in normal saline and injected intraperitoneally. After 14 days, all of the mice became intraperitoneal implanted with 10 Ehrlich ascites carcinoma cells fresh from the ascites fluid from donor mice had been won.

From the values in Table I, which are typical results

if there are several results, it becomes clear

that the mean survival time of the untreated control

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animals was 18 days, after 30 days there were no more surviving animals. In contrast, mice were those treated with 100 µg BCG effectively protected, however less than those with IPM preparations which were active even at smaller doses.

Table I.

Ehrlich's ascites carcinoma. Comparison between BCG and IPM, extracted from M. smegmatis or M. kansasii

Treatment * dose of mycobacterial (ug) preparation '

mean survival after lifetime ** (days) 60 days ***

Untreated control animals

IPM M. smegmatis

IPM M. kansasii

100 10 30

100 30

100

'18> 60> 60> 60> 60> 60> 60

0/10

7/9 7/10 6/8 10/10 6/10 8/10

* All mycobacterial immunostimulants were given i.p.

14 days before inoculation of the tumor cells on the i.p.

¥ eg injected.
** Mean survival time: The day on which 50% of the mice

were dead.
*** Survivors / total animals.

The Pasteur strain BCG and IPM obtained from M. phlei were compared with each other in terms of protective activity. Groups of 10 female hybrid mice B6DF1 (Jax)

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a single intraperitoneal injection of the test material was given 8 days prior to tumor implantation. On day 0, all mice received an intraperitoneal implantation of 1Cr Ehrlich ascites cells (Gresser strain), freshly collected from donors of the DBA / 2 strain. The mortality response was recorded and all of the mice living on the 85th day were sacrificed and autopsied.

Untreated control animals had a mean time to Death of 21 days with all mice dead on day 27. In the IM treated group, all animals survived the 85 day observation period and they remained in good health. BCG treatment resulted in 9/10 long-term survivors. All surviving mice were free of detectable malignancies at autopsy.

Syngeneic lymphoid leukemia:

The lymphoid leukemia used in the following experiments appeared spontaneously in a female hybrid naaus (057BLxAKk) in the laboratory. This was about a syngeneic one. Tuaor going through a row of passages maintained in this hybrid stamra and the self

7 after an inoculation of 5 x 10 cells not into one Parent strain is transplantable. As a test inoculum was used a suspension of viable cells obtained from the spleen of donor mice 14 days after intraperitoneally laplantierung νοϊΐ 10 cells / mouse was obtained was. Previous research had shown that one Inoculate all mice as low as 10 viable cells an average time to death of 24 days. There is also a pronounced reverse relationship

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between the number of implanted tumor cells and the average survival time.

The test materials were administered intraperitoneally 8 days before the inoculation of 2 × 10 leukemia cells on the same Injected away. The test results indicated that injection of these mycobacterial fractions against the mice effectively protected syngeneic leukemia. In Table II the activity of BCG and IFM extracted from M. smegmatis or M. kansasii, given in comparison. It can be seen that both IPM preparations in this severe tumor system are more active than BCG.

Table II

Syngeneic lymphoid leukemia. Comparison between BCG and IPM extracted from M. smegmatis or M. kansasii.

Treatment * dose mean survival naclv

mycobacterial (/ ig) lifetime ** (days) 60 days *** Preparation '

Controls (saline solution

0/10 0/10 1/10 1/10 1/10 3/10 4/10 1/10 3/10 2/10

All mycobacterial preparations were i.p. 8 days before inoculating the tumor cells by the i.p. route of administration injected.

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sung 10 24 BCG in saline 30th 31 100 30th 10
30th 31 IPM M. smegmatis in
Saline solution 100 28
37 10
30th 35 IPM M. kansasii in
Saline solution 100 29
37 39

** Mean survival time: the day on which $ 5C of the mice were dead.
*** Survivors / total animals.

In another experiment, two different batches of IHi extracted from M. smegmatis were suspended in a vehicle, the one from 1Obigem aqueous polyethylene glycol 400, mixed with 0.02% aqueous Tween-80 (this vehicle is hereinafter referred to as PEG). The IPM in PEG was compared for activity with BCG suspended in saline. They were control groups intended for both diluents. Under these conditions, too, it was found that the IPM preparations were more active were as BCG as can be seen from Table III.

Table III

Syngeneic lymphoid leukemia. Comparison between BCG and two different approaches of extracted from M .. smegmatis IPM.

Treatment*
mycobacterial
preparation dose
(/ ig) medium over
lifetime **
(Days) Survive after
60 days *** Controls (saline solution
sung) 24 1/38 Controls (PEG) 23 0/28 BCG in saline 10 23 0/10 30th 32 3/20 100 30th 5/20 IPI-I M. smegmatis in PEG
(Approach 1) 10
30th 29
36 2/10
4/10 100 40 5/10 IPM M. smegmatis in PEG
(Approach 2) 10
30th 27
32 1/9
1/10 100 47 5/10

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* All mycobacterial immunostiraulants were administered i.p. 8 days before inoculating the tumor cells on the i.p. Injected away.

** Mean survival time: the day on which 50% of the mice were dead.

* · * ■ * Survivors / total animals.

The activity of IPM appears to be increased by the administration of a repeated dose, which is in contrast to the response obtained with BCG. In the following experiments all treated mice received 100 iig BCG or IPM 8 days prior to implantation of the Leukämieζeilen 24 h after tumor implantation, the mice received a second injection of the test materials with doses of 10, 30 or 100 u g / mouse. As can be seen from Table IV, the activity of IPM appears to be enhanced under these conditions, which is not the case with BCG.

Table IV

Syngeneic lymphoid leukemia. Comparison between repeated injections of BCG and of IPM.

Treatment doses * mean survival

mycobacterial second injection lifetime ** after Preparation D + 1 (days) days ***

Controls (saline controls (PEG) BCG in saline

IPM in PEG

ns 24 0/10 Il 22nd 0/10 10 H 28 1/10 30th Il 32 2/10 100 Il 30th 1/10 10 Il > 50 5/9 30th > 50 7/10 100 > 50 6/10

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* All treated mice received 100 µl BCG or IPM 8 days before implantation of the tumor cells and 9 days before a second injection of the same material with different dosages.

** Mean survival time: the day on which 50% of the mice were dead.

*** Survivors / total animals.

The antitumor activity of IPM is increased when using this immunostimulant administered with irradiated leukemia cells. In the example below, all were treated Mice 30 or 100 µg BCG or from IPM 8 days before implantation of viable leukemia cells. Half of these groups received the mycobacterial preparations mixed with 10 irradiated leukemia parts. There were two control groups used: a) untreated mice and b) immunized control animals, which the irradiated cells without mycobacterial Preparations received.

As can be seen from Table V, the administration of lethally irradiated syngeneic tumor cells appeared to be combined with either BCG or IPM compared to treatment with either of these materials alone To confer degree of protection. The increased protection in this experiment was after combination with IPM more evident than in the case with BCG. Although the mechanism of action is still unclear, such results suggest clearly suggests a mutual enhancement of antitumor activity when tumor cells are killed and IFM administered in combination.

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Table V

Syngeneic lymphoid leukemia. Comparison between alone IPM and BCG injected or administered with irradiated leukemia cells.

Treatment ⁵

Dose of mycobacterial
Preparation (ug)

medium survival ^, Survival after 50 days *** days ** '(days)

Controls, non-immunized

Controls, immunized ** BCG, unimmunized BCG, non-immunized BCG, immunized BCG, immunized IPM, non-immunized IPM, non-immunized IPM, immunized IPM, immunized

- 23 0/20 - 23 2/10 30th 29 1/10 100 31 2/10 30th 34 4/10 100 34 3/9 30th 30th 2/10 100 40 5/10 30th 38 4/10 100 > 50 8/10

* All injections were made by the i.p. route 8 days before the inoculation of viable tumor cells on the

same way.
** immunized = 10 irradiated leukemia cells (with or

without immunostimulants)
*** mean survival time: the day on which 50% of the mice

were dead. **** survivors / total animals.

Antiviral activity

The substances were tested for their protective effect against you
pathogenic virus examined as follows. The test materials

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were suspended in PEG by sonication. The dose of the test material ranged from 10 to 100 »g / kg, groups of 30 mice (10 g body weight, Charles River CD-1 strain) were inoculated either intraperitoneally or intravenously with the desired dose of the material and held for 14 days * At this point (day 0) , 20 mice per group were given a lethal virus infection and the remaining 10 animals were sacrificed to measure the weight of the spleen. The virus load was 20 LD ₁ -Q (mean lethal dose) of Columbia SK virus injected intraperitoneally. The deaths were recorded daily and the trial was terminated on day 12. The mean survival time was calculated taking into account both the dead and surviving mice. Deaths from infection with Columbia SK occurred 3 or more days after administration, so deaths that occurred prior to day 3 were considered non-specific and excluded from the calculations. The mean survival time of the untreated control animals was 4 to 5 days. The increase in the mean survival time was expressed as a percentage increase over the untreated control animals. The spleen weights were measured 14 days after injection of the test material. The group mean spleen weights were compared with those of control mice administered with the diluent only. The ratio was expressed as a percentage change.

Table VI gives the results of typical test runs using the dose response of IPM was assessed on the basis of both the increase in spleen weights and the increase in mean survival time.

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mg / kg
ip Spleen weight
mg increase 186 Resistance to
Viral infection
Overall to-middle
then survive
bens2eit 4.13 the
increase
* 85 107 30/30 10.00 «Ta Table VI 100 243 79 6/20 7.90 142 32 176 13/20 6.45 91 10 152 17/20 56 Dose response from IFM by M. smegmatis carrier
control M. smegmatis IFM F-3798

A time study showed that significant resistance to Columbia SK infection could be obtained when the IFM was administered as early as 28 days prior to virus challenge (Table VII).

The results given in Table VIII show that IFM was effective on both M. smegmatis and M. phlei, when injected either intraperitoneally or intravenously. However, BCG cells were intact when administered intraperitoneally Way ineffective. M. kansasii IPM was tested IV route only.

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α co as

Table VII Spleen weight
mg gain, 5 - » Mice 4.53 - Time study by IPM "at 113 24
70 5.20
4.45 15th
-2 Day after the
injection 140
192 79 9.20 103 control 202 205 9.75 11-5 * M. smegmatis IPM
F-4073, 100 mg / kg IP 1
3 345 47 9.40 108 ^ 7th 166 Resistance to the viral infection
% Deaths / mean overgrowth, %
Total lifetime -I4 29/30 28 18/20
19/20 8/20 8/20 8/20

Table VIII Biological activity after ip or iv injection

IPM

Way spleen weight resistance to Col-SK exposure

mg increase, % deaths / total mean over-increase, %

lifetime

cn CJ to oo OO

control

M. tuberculosis (var. Bovis) whole cells M. bais BCG 6/11 Pasteur Institute

smeginatis F-4073 100 mg / kg

, phlei IPM F-4135 100 mg / kg

M. kansasii IPM F-4415 100 mg / kg

- 106 - 29/30 4.93 51 IV 214 102 15/20 7.45 2 IP 142 34 20/20 5.05 101 IV 241 ' 127 10/20 9.90 85 IP 234 121 11/20 9.10 96 IV 330 211 9/20 9.65 107 IP 248 134 7/20 io.20 110 IV 9/20 10.30

Resource activity

The adjuvant activity of IPM on the titre of serum antibodies was determined in guinea pigs using ovalbumin as the test antigen. The antibody titers were measured by quantitative precipitation and passive hemagglutination. 1 mg of soluble antigen (ovalbumin) was added given to 50 µg IPM. The mixture was combined with the incomplete Freund's Vision Aid (FIA), thereby forming a water-in-oil emulsion. 0.1 ml this emulsion was injected into the hind paw of Hartley male guinea pigs. The control animals received Injections of ovalbumin only in FIA or injections from the Complete Friends' Vision Aid (FCA), consisting of FIA and M. butyricum cells. The antibody titers versus ovalbumin were measured 21 days after injection. The titers were determined by quantitative precipitation according to the method of Gierer and Schramm (Zeit, für Naturforsch., 1956, 116: 138) or by passive haemagglutination of erythrocytes coated with ovalbumin, according to Stavitsky (J; Immunol., 1954, 72/360 to 368) certainly. The results are shown in Table IX.

The figures given correspond to the mean of groups of 6 guinea pigs. The results are as micrograins Antigen-antibody complex per ml of serum (in the case of quantitative precipitation) and as the reciprocal of the serum dilution given for the hemagglutination values.

The delayed hypersensitivity was achieved by intradermal injection of 0.1 ml of saline solution, which contains either 5 wg ovalbumin, 100 I.U. old tuberculin or IPM at doses of 10 or 50 µg.

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As can be seen from Table IX, the interphase material yielded an elevated antibody titer comparable to that of the full one Friend's seeing aid is received.

Administration of IPM in Freund's incomplete device induced a greatly delayed hypersensitization response to ovalbumin and to IFM itself. The answer to tuberculin was however, weaker than that observed in control animals which see with full friend Aids had been sensitized.

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Table IX

Resource activity

Treatment * passive hemagglu- quantitative delayed © hypersensitivity to

tination precipitation ovalbumin tuberculin IPM

5 ^ g 100 I.U. 50 ug 10 ug

FIA 1/500 <200 0/6 0/6 0/6 '0/6

^ FCA 1/1200 2470 6/6 pos. 6/6 pos. 6/6 pos. 6/6 pos.

° FIA + IFM 50 ug 1/1000 2730 6/6 pos. 2/6 pos. 6/6 pos. 6/6 pos. *

£ ' V6 neg.

OO gp

CO. '*

> *. * male guinea pigs weighing 300 g were placed in both hind paws on day 0.

_{€ O} 0.1 ml injected a water-in-oil emulsion containing 0.5 mg of ovalbumin.
- * The ovalbumin skin test was done on day 18.
The animals were sacrificed on day 21.

cn ro -j an co cn

Comparative induction of hyperreactivity to endotoxin

It is known that BCG-treated mice become highly susceptible to endotoxin lethality. The resistance versus endotoxin was measured by intravenous injection of 300 µg IPM or BCG (phenol-killed). The mice were administered 14 days prior to exposure to 1, 5, 25, 125 or 500 µg of endotoxin the same way. The IPM was suspended in PEG, while the BCG was either in saline or PEG was suspended. A fourth group of animals received PEG only.

The endotoxin used was produced from Salmonella enteritidis by the phenol / water process. The mean lethal dose (LD ₅₀ ) was determined according to the method of Reed and Muench. As can be seen from Table X, which contains the results of a typical experiment, 300 µg BCG sensitized the mice to the endotoxin. The LD ^ Q values of less than 2 µg are in sharp contrast to the LD ^ Q values of 290 µg in non-sensitized control animals. However, 300 µg of IPM made the mice less sensitive to the endotoxin than BCG, the LD ₁ -Q being 35 η g. This shows that although IPM has more antitumor activity than BCG, it is less sensitizing to endotoxin.

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Table X Hyperreactivity to Endotoxin

Treatment, day 14 doses of endotoxin, µg

1 5 25 125 500 ^DO

Controls (EEG) 0/8 * 7/8 = 290 u;

BCG in saline,

300 µg IV 4/8 6/8 8/8 8/8

BCG in PEG 300 µg i.v. 6/8 6/8 8/8 8/8 <2 "

IPM in PEG 300 µg i.v. 1/8 2/8 4/8 4/8 35 "

* Deaths / total

The agents according to the invention are primarily intended for use as injectable suspensions. Suitable Suspensions are obtained by placing the mycobacterial fractions in a carrier made of polyethylene glycol, Tween-80 and suspended in water.

The agents according to the invention can use the BCG in BCG therapy replace and especially as immunopotentiating agents in the therapy of tumors and viral infections Warm-blooded animals are used. They can also be used to oppose the intensity of the immune response a wide variety of antigens. Although the agents according to the invention have a strong BCG-like therapeutic If they are active, they are tolerated much better by the patients.

Injections four times a week of 0.15 mg of the agent (two injections in the thigh and two injections in the Outside of the arm in the pit above the shoulder spine) were taken from human patients for periods of 3 to Well tolerated for 6 months.

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Claims (21)

Claims Process for the preparation of mycobacterial fractions with antitumoral, antiviral and auxiliary activity, characterized in that that a suspension of broken mycobacterial cells in an emulsion from an aqueous solution and subjecting an immiscible organic solvent to vigorous disintegration treatment, wherein such a solvent is used which can form an emulsion when mixed with water, of this Medium separates the cell debris and separates the water and the solvent of the emulsion into separate phases, and that the solid material, which is either suspendjart is present or forms an interphase system between the water and solvent phases or both, of the solvent freed, the solid material containing the protective agent. 2. The method according to claim 1, characterized in that the starting suspension is made a suspension of whole cells formed in a mixture of water and an immiscible organic Solvent are suspended, and then the suspension is broken apart, for example a mechanical Break apart and subject to violent agitation or agitation. 3. The method according to claim 1 or 2, characterized in g e that one can remove the solid suspended material from the organic solvent carried out in such a way that you first the aqueous phase -30- 509882/0910 removed, then the solid material suspended in the organic phase or the solid interphase material wins this material in water or an aqueous solution, e.g. an aqueous physiological emulsion, redispersed and that the resulting suspension of the remaining organic solvent freed. 4. The method according to claim 1, characterized in that there is an aqueous suspension of mycobacterial cells form the resulting suspension with a water-immiscible organic solvent and an emulsifying agent combined which breaks apart or shatters cells in the emulsion thus formed, separating the aqueous part and the cell debris from the non-aqueous part, the solid material from the non-aqueous part wins, the solid material is suspended in water and that the aqueous suspension is dried, whereby fractions are obtained which are characterized by their antitumoral, antiviral and adjuvant activity are. 5. The method according to claim 4, characterized in that the cells are broken apart by means of a violent mechanical agitation or stirring or circulation through a Zeildisintegrator. 6. The method according to claim 1, characterized in that a polyethylene oxide sorbitol monooleate is used as the emulsifying agent used. 7. The method according to any one of claims 1 to 6, characterized in _f wherein the organic solvent used · a solvent for lipids. -31- 509882/0910 8. The method according to any one of claims 1 to 6, characterized characterized in that the solvent used is saturated aliphatic and / or cyclic hydrocarbons, especially with 5 to 12 carbon atoms. 9. The method according to claim 8, characterized in that the water-immiscible organic Solvent hexane used. 10. The method according to any one of claims 7 to 9, characterized in that there is water / solvent ratios uses from 75 to 40% by volume of water and 25 to 60% by volume of organic solvent. 11. The method according to any one of claims 7 to 9, characterized characterized in that one has water / solvent ratios uses from 65 to 55% by volume of water and 35 to 45% by volume of organic solvent. 12. The method according to any one of claims 1 to 11, characterized in that one mycobacterial cells of the species Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium phlei and / or Mycobacterium smegmatis used. 13. The method according to claim 4, characterized in that the mycobacterial cells suspended in an aqueous saline solution. 14. The method according to claim 5, characterized in that the cells in the manner -32- 509882/0910 breaks by passing the emulsion through a pressure cell with continuous flow. 15. The method according to claim 4, characterized in that the non-aqueous part is a solid Contains intermediate phase layer and an upper organic layer. 16. The method according to any one of claims 1 to 15, characterized in that the solid obtained, containing the active ingredient, dries, freeze-dried or lyophilized. 17. The method according to any one of claims 1 to 16, characterized in that the agent continues by differential solubilization in organic solvents and that one either solubilized or non-solubilized fraction which wins the Contains active ingredient. 18. Mycobacterial fractions with anti-tumor, anti-viral and auxiliary activity, characterized in that it consists of aqueous suspensions of mycobacterial cells have been produced by the method of any one of claims 1 to 17. 19. Mycobacterial fractions according to claim 18, characterized marked that they. from aqueous suspensions of mycobacterial cells of the species Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium kansasii and / or Mycobacterium phlei by emulsifying the Suspension, breaking apart or shattering of the -33- 509882/0910 len, separation of the solid, suspended in the organic phase Material, dispersing the solid material in water to remove any residual organic material Substances and drying of the aqueous suspension have been obtained, with the desired fraction in the form of a white until amorphous amorphous solid is obtained. 20. Mycobacterial cell wall fractions in the form of a white to amber-colored fluffy solid which is insoluble in neutral aqueous systems and only partially soluble in organic solvents, characterized by the following elemental analysis : C: 51 to 57%
Hi 7.5 to 8.7%
N: 5.8 to 6.5%
P: 0.5 to 1.2% and the following composition: Neutral carbohydrates 21 to 23% Amino sugar 0.8 to 1.8% Amino acids 24 to 28% Lipids (free) 40 to 43%. 21. Pharmaceutical agent, in particular for the therapy of tumors and viral infections in warm-blooded animals, thereby characterized in that it is used as an active ingredient together with a pharmaceutical carrier at least contains one of the mycobacterial fractions according to any one of claims 18 to 20. 509882/0910