DE2713271A1 - MITOGENIC AGENTS DETAILED FROM BACTERIAL PEPTIDOGLYCANS - Google Patents

Description

Patent attorneys Dipu-Ing. H.Weickmann, Dipu-Phys. Dr. K. Fincke

Dipl.-Ing. FAWeickmann, Dipl.-Chem. B. Huber

8 MUNICH 86, DEN

PO Box 860 820

MDHLSTRASSE 22, CALL NUMBER 98 39 21/22 Case: PL-OO96- 77-B HtM / cb AGENCE NATIONALE DE VALORIZATION DE LA RECHERCHE

(ANVAR)

13, rue Madeleine Michelis 92522 Neuilly Sur Seine / France

Mitogenic agents, particularly those produced by bacterial peptides

glycans originate.

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The present invention relates to agents having mitogenic properties. Because of these properties, they make biological reagents for research and diagnosis of If these agents are administered to humans or animals, they can cause non-specific stimulation of Lymphocytes cause B, resulting in increased formation manifested by antibodies that act against a broad category of antigens. They also favor development of stem cells so that they can be used for the curative treatment of diseases that result in a deficiency in immunocompetent and haematopoietic cells.

It is known that certain fractions that have such mitogenic properties, starting from whole cells, obtained from Nocardia cultures. In particular, they are the subject of the German patent application (French patent application No. 74 145OO of

April 25, 1974) of the applicant, in which a process for the production of such mitogenic fractions is indicated. You can use these fractions in particular starting from a first aqueous Fraction obtained, which in turn has been separated from a suspension containing the products of the digestion of Nocardia cells, which has previously been freed from lipids, in the presence of a muramidase, in particular lysozyme, in a contains aqueous buffer solution. Fractions are obtained which are even more enriched in the mitogenic agent, in particular by separating the solubilized peptidoglycan fractions contained in the first aqueous fraction, which by Fragmentation of the glycan chains of the peptidoglycan by at least partial hydrolysis of the 1,4-ß-bonds between alternating N-acetylglucosaminyl groups and N-acylmuramyl groups were formed in the glycan chains. These peptidoglycan fragments, their valuable adjuvant properties and / or unspecific

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specific immunostimulatory properties are described, however, have little or no mitogenic effect.

The last-mentioned separation can be carried out by filtration through a molecular sieve, for example gels of the type that are commercially available under called "Sephadex", or can be obtained by a modified procedure set forth therein consists in resuspending the aforementioned first fraction in freeze-dried form in concentrated acetic acid and in particular by centrifugation the undissolved fraction which exerts the mitogenic effect is recovered while in the liquid phase contains the peptidoglycan fragments with adjuvant properties but remain in dissolved form without mitogenic effects.

The present invention now provides new mitogenic agents, particularly water-soluble agents that differ from those already known distinguish whether they are directly derivatives of the peptidoglycans of the bacterial cell walls now come from Nocardiae or other types of bacteria. They develop a mitogenic effect both in vitro and in vivo. In the latter case, this mitogenic effect is not only manifested in mice given these agents but also on other animal species. They are thus suitable for the production of preparations with the intention can be administered to exploit the mitogenic effect of their active ingredients, so that these preparations on the basis of this Fact represent medicinal products.

The invention therefore also relates to pharmaceutical preparations which contain these water-soluble mitogenic agents which are derived from the peptidoglycans mentioned, and which together with physiologically compatible pharmaceutical binding agents

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agents, carrier materials and / or auxiliaries in the form of isotonic, sterile or sterilizable and injectable aqueous solutions, these preparations being free of oil are.

In general, the invention relates to the use of the mitogenic Means that is formed from a polymer which has only a low content, in particular a content of less than 10 percent by weight, based on the weight of the polymer, of neutral sugars or completely free thereof which agent has a polysaccharide skeleton derived from monomeric units each of which is N-acetylglucosaminyl-N-acylmuramyl disaccharide units contain acetyl groups or glycolyl groups (glycoloyl groups) as acyl residues these units are present in such a number that the mitogenic effect of the agent manifests itself, in particular its ability to stimulate the lymphocytes B of mice or rabbits.

In particular, the agent is a bacterial peptidoglycan, the glycan skeleton of which is essentially intact or at least a sufficiently large number of disaccharide units includes that the state defined above is reached.

In particular, you can use the agent starting from bacterial cell walls, which one previously - especially in the case of Nocardiae, Mycobacteria and Corynebacteria - of proteins and lipids has freed, obtained by acid hydrolysis of these cell walls, which one under mild conditions, especially under action a dilute hydrochloric acid solution causes to limit the effect of hydrolysis to solubilizing the bulk of their arabinogalactans or, in the case of bacteria other than those mentioned above, their

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glycans various polysaccharides, and, in the case of the above mentioned species, if not the cleavage of their mycolic acids (acides mycoliques), at least the loosening of the bond the latter to the glycan skeleton so that it can be treated with chloroform or a chloroform-methanol mixture in the course of a re-treatment to remove the lipids with the aid of solvents, which is always desired, can be extracted can.

The separation of the majority of the neutral sugars that are present in the bacterial cell walls, their mitogenic effect already was mentioned, and the loosening of the bonds over which the mycolinic acids or possibly corresponding components bound to the glycan chains, if not their complete cleavage, have the consequence that the mitogenic effect of the products obtained, compared to the effect of the cell walls used as starting material, maintained or increased will.

The present invention is based on the finding that the mitogenic effect of these products appears to be linked to the glycan chain itself, provided that this has a sufficiently large number of disaccharide units of the kind defined above having.

The invention therefore also relates in particular to mitogenic agents which consist of glycan chains which, if appropriate, also have peptide substituents which, however, are in turn free of inter-peptide bonds to an extent, that the water solubility of these agents is ensured, which can go so far that the glycan chains are free of any Substituents of peptide nature.

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It should be remembered that it has been shown that the walls of all bacteria examined so far are a peptidoglycan contain, which has a glycan skeleton, which is built up from a chain of monomeric units of disaccharide units, which can be represented by the following general formula

CH ₂ OH

CH ₀ OH
ι 2.

^^ \) H ψ

.0.

\)H ψ

H NHAc

HAc

CH ₃ -CH-COOH

these glycan chains being substituted by peptide units which have acid groups of their N-acetylmuramyl units (or their N-glycolylmuramyl units in the case of certain types of bacteria) are bound to these glycan chains, these peptide units being crosslinked with one another. In certain cases is part of the N-acetylglucosamine groups of these disaccharide units partially deacetylated.

It could be shown that the peptide units mentioned generally comprise sequences, the first groups of which are the most frequent the following are: L-Ala-> D-Glu-hneso-DAP, where the bond of these peptide units to the glycan generally takes place via the L-alanyl group. In certain bacteria the mesoc £, £ -diaminopimelic acid by another amino acid, for example lysine, which in turn can optionally be substituted. Especially with regard to the functional Groups of amino acids that are involved in building the said

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Sequence are not involved, certain variations are possible so that these are either in free form, substituted or be involved in the formation of interpeptide bonds. It is, however, to explain the present Invention need not go into the details of these variations.

The break of the interpeptide bonds between the glycan chains, whereby the product obtained becomes water-soluble, can be achieved with the help of endopeptidases, for example the bacteriolytic endopeptidases of Streptomyces albus G, the products obtained being made up of water-soluble glycan chains which still have peptide chains, in particular up to contain up to 9 or 10 amino acids, which, however, are no longer linked to one another. The proportion of these peptide chains can be further reduced or they can be eliminated entirely by using N-acetylmuramyl-L-Ala-amidases, for example the enzyme known as "Amidase from Myxobacter AL ₁ ". These peptidases and other enzymes which lead to the same result are described, for example, in the article by Jean-Marie Ghuysen entitled "Use of Bacteriolytic Enzymes in the Determination of Wall Structure and Their Role in Cell Metabolism" (Bacteriological Reviews, Dec. 1968, Vol. 32, No. 4, Pt. 2, pages 425 to 464).

It should be noted that the short peptide chains that may be are bound to the glycan chains, the mitogenic effect of the water-soluble products obtained in this way, apparently not participating.

Possess the water-soluble mitogenic agents of the present invention a characteristic structure, the essential part of which is through

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the following general formula

QIc N Ac - Mur N X - GIc N Ac - Mur N Χι ι peptide peptide

η 2

can be played back in the

the "peptide" groups stand for the peptide chains mentioned (which may also be missing), the abbreviations "GIc N Ac "and" Mur N X "for N-acetylglucosaminyl groups and N-acylmuramyl groups, respectively where "X" stands for an acetyl group (Ac) or a glycolyl group (Glycoloylgruppe) (Glyc) and "n" has such a large value that said mitogenic effect can manifest itself.

The above definition also includes water-soluble ones according to the invention mitogenic agents whose N-acetylglucosamine groups are optionally partially deacetylated. This also applies to all preferred groups of mitogens according to the invention Funds, of which we will speak in the following.

"N" preferably has a value of at least 6 or more, this value being in particular between 10 and 45.

Preferred water-soluble mitogenic agents according to the invention are Means having a structure, the essential part of which either by the following general formula

31c N Ac - Mur N Glyc GIc N Ac - Mur N Glyc

L-AIa L-AIa

D-isoGln D-isoGln

I I

Y Y

or by the following general formula

η 2

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SIc N Ac - Mur N Glyc

L-Al a D-isoGln

GIc N Ac - Mur N Glyc-L-Äla

Y
D-Al a

D-isoGln

Y
D-AIa

η 2

can be reproduced, with those given in these formulas Abbreviations have the meanings defined above, while “AIa” for the alanyl group, “isoGln” for the isoglutamine group and "Y" for a meso <X, £ -diaminopimelic acid group which may optionally be in the amide form.

The mitogenic agents corresponding to the above formula can in particular, starting from cell walls of Nocardiae that have been freed from proteins and lipids, such as N. rubra or N. opaca these cell walls are previously subjected to acid hydrolysis under the action of a 0.1 N hydrochloric acid solution, followed by a renewed removal of the lipids.

Other characteristic mitogenic agents have a structure its essential part by the following general formula

lc N Ac - Mur N Ac

GIc N Ac - Mur N Ac-L-AIa

Z
W.

η 2

or the following general formula

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lc N Ac - Mur N Ac

GIc N Ac - Mur N Ac-L-AIa

W.
D-AIa

can be reproduced, the abbreviations of the above general formulas following the definitions given above and "Z" for a D-glutamic acid group or a D-isoglutamine group and "W" for a meso-OC, £ -diaminopimelic acid group (meso-DAP group), which may optionally be in the amide form, or a lysyl group, which in turn may optionally can be substituted by a short peptide chain.

Such mitogenic agents are obtained, for example, starting from B. cereus, B. subtilis, M. roseus and Staphylococcus albus.

Another family of preferred mitogens according to the invention Agents includes that group of compounds whose structure as an essential component units of the following general formula

lc N Ac - Mur N X

GIc N Ac - Mur N X

contains, whereby the abbreviations mentioned are those given above Have meanings so that these products are built up from glycan chains, some of which have carboxyl groups are completely freed from peptide chains.

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Further embodiments, objects and advantages of the invention result from the further description of examples of mitogenic agents with glycan chains and their manufacturing processes.

First, the conditions are described under which the cell walls of different bacteria are obtained, from which one can extract the mitogenic agents under conditions which are explained below.

a) Cultivation of Nocardia rubra, ATCC 14 898 and Nocardia opaca, ATCC 21953 or the strain of the Institut Pasteur (Paris).

The strains mentioned above are grown in a fermenter with a capacity of 20 l to 14 l of a medium that one has inoculated with 500 ml of a preculture that has been grown on the same medium.

The medium used for the cultivation of Nocardia rubra consists of 2.5 % heart infusion broth (Heart Infusion Broth, Difco), 10 ml / 1 glycerine and 0.25 g / l Na ₂ HPO ₄ . 12 H ₃ O, the medium being adjusted to a pH of 7.4 to 7.6.

The medium for growing Nocardia opaca consists of 0.2% yeast extract (Difco), 0.4% meat extract (Difco), 2% bactopeptone (Difco) and 0.5% sodium chloride and is adjusted to a pH of 7.2 set.

In both cases, the microorganisms are grown under the same conditions, apart from the temperatures of the Cultures (which are 25 ° C in the case of N. rubra and 30 ° C in the case of N. opaca). The microorganisms are cultivated under Stir by turning the spiral of the fermenter with a rotary

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number of 250 min and by aerating the medium with 2 air / min.

The incubation is interrupted after 3 to 4 days. In the case of N. rubra, the culture is brick-red in color.

The cultures are then recovered by centrifugation, washed with distilled water and stored until used at -20 ° C.

To obtain the cell walls of these cells, they are suspended the cells in 5 times their weight in distilled water in the container of a homogenizer or a "mixer" and passes it three times through a pressure grinding device (commercially available under the name Manton-Gaulin is known) using a pressure

2
of 750 kg / cm. After the first pass, DNase is used

The homogeneous material finally obtained is diluted with 3 times the volume of distilled water and centrifuged three times for 15 minutes at 8OO g in a refrigerated centrifuge, with each of these centrifuging operations centrifuge residues formed containing the unbroken cells. The finally The liquid phase obtained is then centrifuged at 27500 g for 50 minutes. A centrifuge residue is obtained, which consists of the raw cell walls of the cells in question.

These cell walls are then subjected to the action of proteolytic enzymes, in particular the action of trypsin and chymotrypsin overnight at 22 ° C., these enzymes being used in an amount that is 0.1 % based on

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is the weight of the raw cell walls, where in a 0.066 m phosphate buffer with a pH of 7.8 works, to which one has previously added a few drops of toluene in order to avoid any contamination. The cell walls are then separated by centrifugation and then initially by resuspending and centrifuging washed three times in the phosphate buffer and then three times in water, to form the protein-stripped cell walls.

These protein-stripped cell walls are then removed freed from the lipids at room temperature by treating them one after the other with acetone and then with an ethyl ether-ether mixture (1 vol. Alcohol per vol. Ether) and finally with a mixture of 2 vol. Chloroform and 1 vol. Methanol extracted. Finally, they are resuspended in acetone and dried.

b) Preparation of the cell walls of Micrococcus roseus

The cells of M. roseus (strain no. 5693 of the Pasteur Institute, Paris) are placed in 2 1-Erlenmeyer flasks containing 800 ml of a "Nutrient broth" (Difco) contained as a culture medium, on a vibrating table (which is called "Biolafitte" is known) at 25 C, whereupon the cultured cells after 2/3 of the exponential growth phase wins.

The bacteria are broken up by stirring in the presence of small glass beads. For a typical measure In this way, 30 g (fresh weight) of the bacteria are suspended in 150 ml of distilled water and 150 ml of glass beads are added with a diameter of 0.17 to 0.18 mm and brings the mixture in a 400 ml bowl of a homogenizer (Omni-Mixer, Sorvall) made by immersion in a

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Bath is cooled with ice-cold water. The mixer is started at full speed for 30 minutes. After decanting off the glass beads, store the supernatant liquid and add the supernatant liquid obtained by washing the beads twice with 100 ml of distilled water. The combined supernatants are then centrifuged three times for 10 minutes at 800 g in a refrigerated centrifuge to remove the fragments of the glass beads and the unbroken bacteria. The supernatant liquid obtained in this way is then centrifuged for 1 hour at 27500 g in a refrigerated centrifuge. The centrifuge residue obtained in this way, which consists of the cell walls, is then suspended using a Potter rubbing device with a tetrafluoroethylene flask (Teflon) in 150 ml of a 0.066 m phosphate buffer with a pH of 7.8 containing 3O mg of trypsin and 30 mg of chymotrypsin is added, after which it is incubated overnight at room temperature in the presence of a few drops of toluene, which are intended to prevent contamination. After centrifugation for 1 hour at 27500 g, the centrifuge residue comprising the cell walls from which proteins have been removed is washed by resuspending with the aid of the Potter rubbing device with tetrafluoroethylene flask and centrifuging for 1 hour at 27500 g at +4 ⁰ C, with three times Wash 150 ml of the 0.066 M phosphate buffer with a pH of 7.8 and three times with 150 ml of distilled water. The cell walls are then freeze-dried.

The cell walls obtained in this way (cleaned cell walls) consist of a peptidoglycan and one or more other polymers that consist of neutral sugars because the only amino acids and amino sugars that can be found in the cell walls are from peptidoglycan.

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In particular from the publications of:

(1) J.F. Petit, E. Munoz and J.M. Ghuysen, Biochemistry, ^, 2764-2776 (1966)

(2) E. Munoz, J.M. Ghuysen, M. Leyh-Bouille, J.F. Petit, H. Heymann, E. Bricas and P. Lefrancier, Biochemistry, 5, 3748-3764 (1966)

it is known that the peptidoglycan of M. roseus consists of repeating N-acetylglucosaminyl-ß-1,4-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysyl-D-alanine units in which the £ -amino group of lysine is substituted by a peptide (L-AIa) ₃ -L-Thr); wherein the interpeptide bonds consist in a bond between the terminal D-alanine of a tetrapeptide and the terminal L-alanine of the peptide (L-AIa) ₃ -L-Thr which substitutes the lysine of another tetrapeptide.

c) Obtaining the walls of B. cereus

Bacillus cereus T cells are cultured on the medium known under the name "Antibiotic Medium 3" (Difco) is, at 37 ° C in 2 1 Erlenmeyer flasks containing 800 ml of the medium and which are moved on a stirring table (Biolafitte). After 2/3 of the exponential growth phase the culture is obtained by centrifugation. Then follow the procedure already described for M. roseus the bacteria were broken open, their cell walls recovered, washed and freed from proteins and finally washed again and isolated.

d) Obtaining the cell walls of B. subtilis (NCTC 3610)

The strain is grown on a nutrient broth (Nutrient

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broth, Difco) as a medium, which has a concentration that is 1.5 times greater than that of the manufacturer recommended, using conditions similar to those described for M. roseus.

The cells are recovered when the optical density of the medium at 600 nm is 1.1 (Duospac spectrophotometer, Jobin et Yvon).

The culture is centrifuged off and the centrifuge residue is suspended again in 20 times the amount by weight of water, whereupon the suspension obtained is twice in succession by a press (French), which is operated at a pressure of 1200 kg /

2
cm is operated, with the medium between the

DNase is added to both passages.

The suspension obtained, after breaking up these last bacteria, is then treated as it is for M. roseus is described, but the centrifuge residue obtained after centrifugation at 27500 g suspended in an isotonic sodium chloride solution and brought to the boil prior to treatment with the proteolytic enzymes is heated to destroy the autolytic enzymes present in the cell walls.

example 1

Obtaining cell walls, proteins and possibly Lipids are freed and which are practically freed from their polysaccharides by acid hydrolysis, those of peptidoglycan are different.

The products in question are hereinafter referred to as "acid-treated cell walls".

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These "acid-treated cell walls" are obtained from the lipid and protein depleted cell walls described above using the following conditions:

a) "Acid-treated cell walls" from N. opaca and N. rubra

One 4OO suspended the corresponding cell walls in 4O mL of 0.1 mg n-hydrochloric acid, and preserves them for 12 hours at 60 ⁰ C in this medium. Then they are washed three times with water by suspending them in water and then centrifuging them. They are then subjected again to a treatment to remove the lipids with chloroform at room temperature. It is filtered and the cell walls are recovered, which are then washed with acetone and dried. The analysis shows that these cell walls have practically lost all of their arabinogalactans and that the residues of the nocardomycolic acids can be extracted with chloroform.

b) "Acid-treated cell walls" of the other bacteria examined

1 g of the cleaned cell walls is suspended in 100 ml of 0.1 n-hydrochloric acid and keep them in this medium for 12 hours at 60 ° C. The cell walls are subsequently washed by centrifuging five times and resuspending in water.

They are then freeze-dried or dried in acetone. In this way, the corresponding "acid-treated cell walls" are obtained, the main component of which is peptidoglycan that is essentially free of neutral sugars and a substantial proportion of all others Ingredients other than peptidoglycan.

See the following section dealing with pharmacological

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Properties concerned are obtained differently
Products are designated as follows:

(1) Peptidoglycan (acid) from N. rubra

(2) peptidoglycan (acid) from N. opaca

(3) Peptidoglycan (acid) from M. roseus

(4) Peptidoglycan (acid) from B. cereus

(5) peptidoglycan (acid) from B. subtilis.

The effect of acid hydrolysis on the cell walls results
can be clearly seen from the following Table I, in which the results of the analysis of the crude cell walls of N. rubra on the one hand and the cell walls of N. rubra after acid hydrolysis on the other hand are given. The corresponding lipid levels have not been determined.

The differences in the levels of neutral sugars in the "peptidoglycan (acid) of N. rubra" greatly diminished are, and with respect to the corresponding increases in the content of amino acids and in particular of amino sugars are significant.

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TABLE I.

Composition of the cell walls of N. rubra before removal of proteins and lipids or after the acid hydrolysis and the renewed removal of the lipids

neutral sugar % nmoles / mg Together
settlement Amino sugar % nmoles / mg Together
settlement amino acids % nmoles / mg DAP nmoles / mg Raw cell walls
Acid Treated
Cell walls 28.2
5.58 1516
300 GaI, era,
Man, GIc
GaI, era,
Man 12.8
38.4 5OO
1500 Mur, Glc,
NH "
Mur, GIc
NH ₂ 31.91
38.5 2659.9
2967.50 330.70
766.57

NJ CJl

Example 2

Preparation of water-soluble mitogenic glycans from certain compounds of the above-mentioned compounds 1 to 5 under the action of the peptidases from S. albus G.

The above-mentioned bacteriolytic enzymes are used, which are described by JM Ghuysen et Coil, in the article "An improved technique for the preparation of Streptomyces peptidases and N-Acetylmuramyl-L-alanine-amidase active on bacterial wall peptidoglycans" (Biochemistry, 8, (1969) 213 to 222), until crude peptidases are obtained.

Then 6 mg of the named "peptidoglycans (acid)" are incubated for 6 hours at 37 ° C. together with 250 μl of the named Peptidases (which, under the conditions described in the above-mentioned publication, 40 ml of the filtrate of the culture obtained corresponds) in 1 ml of a 0.005 M veronal buffer with a pH of 9.0. The enzyme is then neutralized to a pH of 7.0 inactivated by heating the medium to 100 ° C for 10 minutes.

The medium is then centrifuged and the solution containing the water-soluble mitogenic agent is recovered.

If this process is applied to the corresponding "paptidoglycans (acid)" under the above-mentioned conditions, one obtains one aqueous solutions of mitogenic agents, which are referred to below with the following names:

(6) N. rubra (S. albus G)

(7) B. cereus (S. albus G)

(8) M. roseus (S. albus G)

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The results of the analysis of the product N. rubra (S. albus G) are given in Table II below. The analytical values of peptidoglycan fragments are also for comparison purposes obtained by solubilizing the same "acid-treated peptidoglycan" which material one has formed from the same strain of N. rubra with lysozyme.

This latter product is obtained in particular by suspending it of 50 mg of the "peptidoglycan (acid) from N. rubra" in 5 ml of a 0.05 m ammonium acetate solution with a pH value of 6.3 and by incubating the suspension for 16 hours at 37 ° C in the presence of 1 mg of lysozyme and a few drops Toluene. The mixture obtained after incubation is then centrifuged, whereupon the centrifuge residue is removed rejects. The supernatant fluid is recovered and contains the product identified in Table II below as "N. rubra (Lysozyme) ".

TABLE II

Comparative analysis of the products obtained by solubilizing "peptidoglycan (acid) of N. rubra" under the action the peptidase obtained from S. albus G or from lysozyme.

Factions Percentage of the
Weight of cell
walls in Lö
sung have gone Percentage atz "of
meso-DAP, the
a free amino
group has Color index at
the morgan
Elson reaction («) N. rubra
(S. albus G)
I. rubra
(Lysozyme) 80 - 85 t
90 Z 85 - 90 t
50 t 0.15
0.84

(*) expressed as disaccharide.

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It is noted that the two enzymes solubilized an amount by weight approximately equal to the weight of the treated Cell walls. On the other hand, there are considerable differences in the content of the soluble solids obtained Fractions of meso-DAP, which has a free amino group. The same is also true of the color indices that you are looking for determined using the Morgan-Elson method, this index being representative of the proportion of free disaccharides.

The increased percentage of meso-DAP groups that are free Have amino group, which is found in the fraction "N. rubra (S. albus G)", shows that the peptidase in the range of Interpeptide bonds worked well.

In contrast, in the case of the "N. rubra (lysozyme)" fraction, it can be seen that a large proportion (50%) of the meso-DAP groups still participates in interpeptide bonds.

Furthermore, the color ratio determined by the Morgan-Elson method shows that as the amount of disaccharide based on the total amount to Osamin, divided by 2 for the fraction "N. rubra (S. albus G)" that the glycan chains by the Enzyme preparation are only slightly or not at all attacked. In contrast, the high color index that one has at of the "N. rubra (lysozyme)" fraction found the presence of a large proportion of free disaccharides, which the The fact confirms that the lysozyme has the glycan chains of the starting material peptidoglycan in several places has split. This fraction "N. rubra (lysozyme)" is also found in the investigations described below with respect to of mitogenicity as inactive.

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Example 3

Preparation of water-soluble mitogenic glycans from certain compounds of the above-mentioned compounds 1 to 5 under the action of the amidase of Myxobacter AL ..

The enzyme is prepared according to the method described by J.C. Ensign and R.S. Wolfe (J. Bacteriol. 91, (1966) 524-534), where instead of filtration through Sephadex GlOO, filtration via Sephadex G25.

In the following, the procedure is described as an example, which is applied to the cell walls of N. rubra, it being understood that that equivalent fractions are obtained if the other above-mentioned peptidoglycans are treated under similar conditions.

400 mg of the cell walls of "acid-treated Nocardia rubra" are suspended in 60 ml of a 0.02 m veronal buffer and the suspension is mixed with a dissolving preparation of the amidase from Myxobacter AL ₁ in such an amount that the cell walls are lysed in the course of 2 Hours at 37 ° C is complete. It is then incubated for 20 hours in order to achieve the most complete enzyme action possible.

The dissolving amidase of the Myxobacter AL ₁ dissolves the peptidoglycan by cleaving the amide bonds between the carboxyl groups and the muramic acid residues of the glycan chains and the amino group of the L-alanine of the peptide chains.

The active product obtained is hereinafter referred to as follows:

(9) N. rubra (Myxobacter)

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After centrifugation, which serves to remove a small amount of residual turbidity, the lysate is concentrated in a rotary evaporator and applies it to a column loaded with Sephadex G25, which has been equilibrated with 0.1 η-acetic acid, whereupon one elutes with the same eluent.

The effluent from the column is analyzed after hydrolysis, with a first peak showing all the amino sugars of the lysate and some Contains amino acids, while the following peaks only contain amino acids.

Only the first peak is active. It owes its effect to the glycan chains that were formed by the action of the enzyme.

The product contained in this peak is hereinafter referred to as

(10) Designated N. rubra (Myxobacter-Sephadex G25).

Under similar conditions, starting from the corresponding one obtains "Acid-treated peptidoglycan" means the water-soluble mitogenic product, hereinafter referred to as

(11) M. roseus (Myxobacter) is called.

Pharmacological properties of the fractions obtained.

The pharmacological properties of the mitogenic according to the invention Means manifest themselves among other things in their ability in a non-specific way to the cells of the bone marrow lymphocytes B to stimulate. This effect is due to the ability of the spleen lymphocytes (of mice and rabbits) to produce more tritiated thymidine to absorb (by incorporation) than the cells of the control lymphocytes B, detected.

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1. Cultivation of the lymphocytes

The lymphocytes are separated from the spleen of mice or rabbits using the technique of C. Bona et Coll. (Eur. J. Immun. 2, (1972) 434). Various strains of mice are used, in particular mice of the AKR strain 2-3 months of age and thymus-deficient mice of the Nude strain of 4-8 weeks old, bred in the CNRS laboratory in Orleans, which belong to a wild line, the are not related to a naked mutation and are from the Institute of Animal Genetics, Edinburgh.

Spleen cells from rabbits of the Bouscat strain, aged 4 to 6 weeks, are also used; Garches to be bred.

1.5 χ 10 spleen lymphocytes from mice are cultured during 48 hours at 37 ° C in 1 ml of the medium known as RPMI-1640 (Eurobio), which is mixed with 5% of the serum of calf fetuses (flow labs).

In the same way, 2.5 × 10 rabbit spleen lymphocytes are cultivated for 72 hours at 37 ° C. in a medium known as "Eagle medium" to which 10 % autologous serum has been added and which is heated has inactivated at 56 ° C for 30 minutes.

2. Incorporation of tritiated thymidine

16 hours before the cells are obtained, IyU Ci H-thymidine (1 Ci / mmol, Saclay, France) is added to each culture. After At the end of the incubation, an ICO times larger amount of non-radioactive thymidine is added. After centrifugation during

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10 minutes at 450 g, the supernatant liquids are separated off, the centrifuge residue is precipitated with trichloroacetic acid and suspend it again in the usual way before determining the activity with the aid of a scintillation counter.

As can be seen from the following Table III, the examined, Compounds according to the invention are all mitogenic Effect resulting in an increase in the incorporation of tritiated thymidine by the splenic lymphocytes of the test animals manifested. The different fractions are listed in the left column of this table. In the columns that correspond to the animals from which the examined lymphocytes originated, the values of the stimulation index are given, observed at the doses of the compounds tested (indicated in brackets next to the corresponding values of the stimulation indices), remembering that that the stimulation indices represent the ratios of the radioactivity of the stimulated cells to the control cells.

It can be seen that an increase in the determined stimulation index is observed in practically all cases. the Agents derived from Nocardiae cells are particularly effective on mouse lymphocytes. You are too effective against rabbits. By applying the same test, the mitogenic effects of the "acid-treated Peptidoglycans "from Nocardia examined against lymphocytes derived from samples of circulating human blood. The The results obtained in these various investigations readily reveal the unspecific effect of the invention recognize mitogenic agents.

709840/09

TABLE III

Art . of the product Mitogenicity (100 / ug) (Stimulation index) NUDE Rabbits (l00 _{/ U} g) Mouse AKR (100 _{/ U} g) mouse (100 _{/ ϋ3} ) 1.94 (100 _{/ U} g) (D Peptidoglycan (acid)
by N. rubra 7.90 11.34 (10O ₇ Ug) 10.73 (10O ₇ Ug) (2) Peptidoglycan (acid)
by N. opaca 8.17 14.07 (0.1 l / ug) 3.52 (10 / Ug) (3) Peptidoglycan (acid)
by M. roseus 1.20 (10O ₇ Ug) 16.95 (lOO ^ g) (4) Peptidoglycan (acid)
from B. cereus (100 _{/ U} g) 4.1 (100 _{/ U} g) 1.78 (1 / ug) (5) Peptidoglycan (acid)
from B. subtilis (100 / ug) 3.94 (100, Ug) 2.23 (1 / ug) (6) N. rubra (S. albus G) 9.6 7.61 4.19 (10O ₇ Ug) (7) B. osreus (S. albus G) 3.24 (100 _{/ U} g) (100 _{/ U} g) 1.76 (8th) M. roseus (S. albus G) (lOO ^ ug) 2.86 (9) N. rubra (Myxobacter) 2.88 (100 / Ug) (ΙΟ) N. rubra (Myxobacter-
Sephadex G25) 3.53 (ID M. roseus (Myxobacter) 4.3

Because of their biological effects, those according to the invention are Means suitable for various purposes, in particular to stimulate cells of lymphoid organs (or lymphoid systems), and both in vitro and in vivo. Some of these uses are given below by way of example :

a) As biological reagents for research use they have of very great interest as it stimulates lymphocytes B in various animal species including monkeys and also People, effect;

709840/094 *

b) as a biological reagent for medical purposes, they enable diagnosis starting from cells of lymphoid organs detect the immune deficiency in the cells producing the antibodies;

c) for preventive treatment: after the injection of the product (for which one preferably uses water-soluble agents) in animals one observes an unspecific overall stimulation, which causes an increase in antibodies against various antigens;

d) The particularly water-soluble mitogenic agents can be used for the healing treatment of deficiency diseases of the bone marrow which are caused by accidents (for example due to radiation) or by idiopathic causes (for example myelosclerosis) can be caused. They are preferably used together with liquid, injectable sterile carrier materials, such as an isotonic saline solution or an isotonic glucose solution, administered by injection.

709840/0944

Claims (1)

Claims 1. Use of the mitogenic agent made of a polymer is formed which has only a low content, in particular a content of less than 10 percent by weight Has neutral sugars or is completely free thereof, which agent has a polysaccharide skeleton that of monomeric Units derived are each N-acetylglucosaminyl-N-acylmuramyl disaccharide units contain, which have acetyl groups or glycolyl groups as acyl radicals and their N-acetylglucosamine group is optionally partially deacetylated, these units being present in such a number are that the mitogenic effect of the agent manifests itself, in particular its ability to stimulate lymphocytes B of mice and rabbits, to stimulate cells of the lymphoid organs, especially lymphocytes B. 2. Use according to claim 1, characterized in that a bacterial peptidoglycan is used as the mitogenic agent, the glycan skeleton of which is essentially intact. 3. Use according to claim 2, characterized in that one uses as a mitogenic agent an agent that is based on bacterial cell walls, which one previously from proteins and possibly has freed from lipids, obtained by acid hydrolysis, which is obtained under mild conditions, especially under action a dilute hydrochloric acid solution to limit the effect of hydrolysis on solubilizing the Main amount of arabinogalactans in the case of mycobacteria, corynebacteria and nocardiae or those which may be present, polysaccharides other than peptidoglycan in the case of other bacterial species. and, in the case of the above-mentioned species, 709840 / 09U ORIGINAL INSPECTED on, if not the cleavage of their mycolinic acids, at least the loosening of the binding of the latter to the glycan skeleton, so that they can be extracted with chloroform or a chloroform-methanol mixture, the obtained in this way Products preferably a complementary treatment with solvents such as those mentioned above to remove the lipids have been subjected to. 4. Use according to claims 1 or 2, characterized in that that the mitogenic agent is made up of glycan chains which may still have peptide substituents, which, however, have at least such an extent of interpeptide bonds are free that the water solubility of the agent is ensured. 5. Use according to claim 4, characterized in that the water-soluble mitogenic agent has a structure whose essential part is represented by the following general formula ; ic N Ac - Mur N X - GIc N Ac - Mur N X- I I Peptide peptide η 2 can be played back in the the "peptide" groups stand for the peptide chains mentioned (which may be missing), "GIc N Ac" stands for N-acetylglucosaminyl groups (certain of which may be partially deacetylated), "Mur N X" stands for N-acylmuramyl groups, with "X" an acetyl group or a glycolyl group, and "n" has a value of at least 6, in particular a value between 10 and 45 owns. 709840/0944 6. Use according to claim 4, characterized in that the glycan chains of said water-soluble mitogenic agent Carry substituent groups that are formed by short peptide chains that have at most 10 amino acids and on N-acylmuramyl groups are bound, which are present between the glycan chains. 7. Use according to claim 6, characterized in that the water-soluble mitogenic agent has a structure whose essential part by the following general formula GIc N Ac - Mur N Glyc L-AIa D-isoGln GlC N AC - Mur N Glyc
L-AIa D-isoGln η 2 or the following general formula N Ac - Mur N Glyc L-AIa D-isoGln Y D-AIa GIc N Ac - Mur N Glyc
L-AIa D-isoGln Y
D-AIa η 2 can be reproduced, where "GIc N Ac", "Mur N Glyc" and "n" have the meanings given in claim 5 and "AIa" stands for an alanyl group,
"isoGln" stands for an isoglutamine group and "Y" stands for a meso-Ot, ε-diaminopimelic acid group which may be present in the amide form. 8. Use according to claim 6, characterized in that the water-soluble mitogenic agent has a structure whose essential part by the following general formula 709840 / 09U GIc N Ac - Mur N Ac L-AIa Z W • V. GIc N Ac - Mur N Ac
L-AIa
Z
W. η 2 or the following general formula GIc N Ac - Mur N Ac L-AIa D-AIa GIc N Ac - Mur N Ac L-AIa D-AIa η 2 can be reproduced in which "GIc N Ac", "Mur N Ac" and "n" have the meanings given in claim 5, and
"Z" for a D-glutamic acid or a D-isoglutamine group and "W" for a meso-Ct, £ diaminopimelic acid group which may be present in the amide form (meso-DAP group) or a lysyl group, which may be substituted by a short peptide chain can be, stand · »- 9. Use according to claim 6, characterized in that the water-soluble mitogenic agent has a structure whose essential part by the following general formula lc N Ac - Mur N X GIc N Ac - Mur N X-J can be reproduced in the "GIc N Ac", "Mur N X" and "n" have the meanings given in claim 5, so that the 709840 / 09U Products are made up of glycan chains, their carboxyl groups are partially or completely free of peptide chains. 10. Use of a water-soluble mitogenic agent which is obtainable by a process which consists in that an insoluble peptidoglycan - which is derived from bacterial cell walls essentially freed from lipids and proteins, which has previously preferably undergone mild acid hydrolysis, in particular under the action a dilute hydrochloric acid solution, have been subjected to the separation of the majority of the neutral sugars and at least a loosening of the bonds between the peptidoglycan and the mycolic acids or other polysaccharides so that they can be removed if necessary by extraction with chloroform, - the dissolving effect of a Exposes peptidase enzyme, such as a bacteriolytic endopeptidase, such as that of Streptomyces albus G or the amidase of Myxobacter AL ₁ , for stimulating cells of lymphoid organs, in particular of lymphocytes B. 11. Use according to claim 1, characterized in that the mitogenic agent used is the agent which is obtained when either the method of claim 3 or the method of claim 10 is applied to cell walls of Nocardiae such as N. opaca, N. rubra or N. corallina. 12. Use according to claim 1, characterized in that the mitogenic agent used is that which is obtained using either the method of claim 3 or the method of claim IO on bacterial cell walls such as those of B. cereus, B. subtilis, M. roseus or Staphylococcus albus. 709840/0944 Λ.3j Pharmaceutical preparation with drug effect, a characterized in that it is a mitogenic agent as an active ingredient, as defined in one of claims 1 to 12, and contains pharmaceutical carrier materials, binders and / or auxiliaries. 14. Preparation according to claim 13, characterized in that it is free from oil. 15. Preparation according to claim 14, characterized in that it is in the form of an isotonic, sterilized or sterilizable and injectable aqueous solution. 16. Laboratory reagent for in vitro stimulation of Cells of lymphoid organs, especially lymphocytes B, characterized in that it contains a mitogenic agent as the mitogenic active ingredient, as described in claims 1 to 12 is defined. 709840 / 09U