GB1580753A

GB1580753A - Pharmaceutical compositions containing water-soluble mitogenic agents

Info

Publication number: GB1580753A
Application number: GB12684/77A
Authority: GB
Original assignee: Agence National de Valorisation de la Recherche ANVAR
Current assignee: Bpifrance Financement SA
Priority date: 1976-03-26
Filing date: 1977-03-25
Publication date: 1980-12-03
Also published as: JPS52145511A; DE2713271A1; FR2345158B1; BE852924A; FR2345158A1

Description

(54) IMPROVEMENTS IN AND RELATING TO PHARMACEUTICAL COMPOSITIONS CONTAINING WATER-SOLUBLE MITOGENIC AGENTS (71) We, AGENCE NATIONAL DE VALORISATION DE LA RECHER CHE (ANVAR), a French body corporate, of 13, rue Madeleine Michelis, 92522, Neuilly Sur-Seine, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The invention relates to pharmaceutical compositions containing water-soluble agents having mitogenic properties. These properties make them biological reagents of great value for research and diagnosis. These agents, when they are administered to men or animals, can induce a non-specific stimulation of lymphocytes B which is shown by an increased production of antibodies directed against large categories of anigens. Further, they encourage the development of the stem cells, so that their use may be considered in the curative treatment of diseases resulting from deficiencies of immunocompetent and hematopoietic cells.

It is known that certain fractions having such mitogenic properties have already been obtained from whole cells obtained from Nocardia cultures. Our U.K. Patent No.

1,509,222 describes and claims a process for obtaining such mitogenic fractions. These fractions can be obtained in particular from a first aqueous fraction, itself separated from a suspension containing the products of digestion of previously delipidated Nocardia cells, in the presence of a muramidase, more particularly of lysozyme, in an aqueous buffered solution. Fractions are obtained which are still more enriched in mitogenic agent, especially by separation of the peptidoglycan fractions dissolved in the first aqueous fraction, and resulting from the fragmentation of the glycan chains of the peptidoglycan, owing to the at least partial hydrolysis of the 1,4--P linkages between the alternate N-acetylglucosaminyl and N-acylmuramyl groups in its glycan chains. These fragments of peptidoglycano, of which the valuable non-specific adjuvant and/or immunostimulant properties have been described in another connection, are however revealed as only having a weak or even no mitogenic activity.

The last separation mentioned may be effected by filtration on a molecular sieve, for example on gels of the type of those known commercially by the name SEPHADEX, or according to an alternative process, by suspension of a lyophilizate of the aforesaid first fraction in concentrated acetic acid and by recovery, especially by centrifuging, of the undissolved fraction, which contains the mitogenic activity, while the liquid phase retains the adjuvant but not mitogenic peptidoglycan fragments in the dissolved state. ("SEPHA DEX" is a Trade Mark).

The present invention provides mitogenic agents, notably water-soluble ones, quite distinct from those mentioned above, in that they are directly derived from the peptidoglycans of the bacterial walls, whether they come from Nocardiae or other bacterial species. They have a mitogenic activity in vitro and in vivo as well. In the latter case the mitogenic activity shows up not only in mice, but also in other species of animals. They are therefore suitable for the manufacture of administrable compositions in order to put into practice their mitogenic activities.

According to the present invention there is provided an oil-free pharmaceutical composition containing a mitogenic agent in association with a pharmaceutical carrier, the agent being formed from a water-soluble polymer which may have a content of neutral sugars of less than 10% by weight and comprising glycan chains including a polysaccharide skeleton derived from monomeric groups, con taining respectively N-acetyl-glucosaminyl-Nacylmuramyl disaccharide units, in which the acyl group consists of an acetyl or glycolyl group, the N-acetylglucosamine moiety being optionally partly deacetylated and the glycan chains optionally carrying also peptide substituents, which substituents are sufficiently free from interpeptide linkages to ensure the water-solubility of the mitogenic agent.

In order for the mitogenic agent to mani- fest its mitogenic activity, which may be tested by its aptitude to stimulate the B lymphocytes of mice or rabbits, there must be sufficient disaccharide units.

The mitogenic agent may be obtained for example from essentially delipidated and deproteinized bacterial walls - especially of Nocardiae, Mycobacteria and Corynebacteria - by acid hydrolysis of these walls, under sufficiently mild conditions, particularly by means of a dilute solution of hydrochloric acid, in order to limit the effects of the hydrolysis to the solubilization of the major part of their arabinogalactans or, in the case of bacteria other than those above-mentioned, of their polysaccharide fractions, if any, other than those of their peptidoglycans, and in the case of the above-mentioned species, to the separation of their mycolic acids, and at least the weakening of the linkages which connect these latter to the glycan skeleton, in order to make them extractable, for example by chloroform or by a chloroform-methanol mixture, in the course of a subsequent delipidation treatment by solvents which is then still desirable.

The separation of the major part of the neutral sugars contained in the bacterial walls, the mitogenic activity of which had already been mentioned, and the weakening of the linkages connecting the mycolic acids, or possible corresponding constituents, to the glycan chains, if not their complete separation, have the effect of preserving or enhancing the mitogenic activity of the products obtained, compared with that of the initial walls.

The invention follows from the discovery that the mitogenic activity of these products appears to be bound to the glycan chain itself, however subject to its comprising a sufficient number of disaccharide units as defined above.

It will be recalled that it has been shown that the walls of all the bacteria studied up to the present contain a peptidoglycan having a glycan skeleton formed from a chain of monomer units formed of disaccharide units, which may be represented by the following formula:

these glycan chains being substituted by peptide units fixed to the glycan chains through the acid groups carried by their N-acetylmuramyl units (or N-glycolylmuramyl units for certain bacterial species), these peptide units being crosslinked between them. In some cases part of the N-acetylglucosamine groups of these disaccharide units are partially deacetylated.

It has been shown that the aforesaid peptide units comprise in general sequences of which the first members are most often L-AlaD-GlumesDAP, the fixation of these peptide units on the glycan being generally effected through the Galanyl group. In some bacteria, the meso a-e-diaminopimelic acid is replaced by another amino-acid, for example lysine, itself possibly substituted. There may exist certain variations concerning especially the functional groups of the amino-acids which do not participate in the constitution of the aboveindicated sequence, which may be either free or substituted or engaged in interpeptide linkages. It is not necessary, however, for the requirements of the description of this invention, to enter into the details of these variations.

The rupture of the interpeptide linkages between the glycan chains, which makes the product obtained water-soluble, may be effected for example by endopeptidases, such as for example the bacteriolytic endopep tidases of Streptomyces albus G, the products obtained being then formed from watersoluble glycan chains also -carrying peptide chains which may for example comprise up to 9 or 10 amino-acids, these being hewever no longer cross-linked between them. The proportion of these peptide chains can also be reduced, or even suppressed if one has recourse to N - acetylmuramyl - L - Ala - amidases, for example that denoted by the expression "amidase of Myxobacter Awl,". These peptidases, like other enzymes which enable the same result to be obtained, are, for example, described in the article entitled "Use of Bac teriolytic Enzymes in the Determination of Wall Structure and Their Role in Cell Metabolism" by Jean-Marie Ghuysen, published in "Bacteriological Reviews, Dec. 1968, p.

425--464, vol. 32, No. 4, Pt. 2".

It is to be noted that the short peptide chains which may be fixed on the glycan chains do not appear to participate in the mitogenic action of the water-soluble products which are thus obtained.

The water-soluble mitogenic agents according to the invention have a characteristic structure of which the essential part may be represented by the general formula:

Gic N X - Gle N Ac -Et N X-lf pSWe peptSe J 2n in which the "peptide" groups denote the aforesaid peptide chains (which may possibly be absent), the abbreviations "Glc N Ac" and "Mur N X" denote the N-acetylglucos aminyl groups and the N-acylmuramyl groups respectively, with "X" being an acetyl (Ac) group or a glycolyl (Glyc) group and "n" has a value large enough for the aforesaid mitogenic activity to be able to be manifested.

Also included in the definition which has just been given are the water-soluble mito genic agents according to the invention, in which the N-acetylglucosamine groups may partly be deacetylated. This also applies to all the preferred families of mitogenic agents according to the invention which will be defined later.

Preferably "not' is at least equal to or superior to 6, especially from 10 to 45.

Among the preferred water-soluble mitogenic agents according to the invention may be mentioned those which have a structure of which the essential part may be represented either by the general formula:

or by the general formula:

in which in addition to the abbreviations already defined, "Ala" means an alanyl group, "isoGln" is an isoglutamine group and "Y" is a meso-a-6-diaminopimelic acid group, possibly containing an amido group.

The mitogenic agents corresponding to the above formula may more particularly be obtained from deproteinised and delipidated walls of Nocardiae, such as N.rubra or N.opaca, these walls having been previously subjected to acid hydrolysis with a solution of 0.1 N HCI, followed by further delipidation.

Other characteristic mitogenic agents have a structure of which the essential part may be represented, either by the general formula:

or by the general formula:

in which in addition to the abbreviations already defined, "Z" is a D-glutamic acid or D-isoglutamine group and "W" is a meso DAP group, possibly containing an amido group, or a lysyl group, possibly itself substituted by a short peptide chain.

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

Another family of preferred mitogenic agents according to the invention is that of which the essential part may be represented by the general formula:

in which the abbreviations have the meaning already given, these products being in consequence constituted of glycan chains of which the carboxyl groups are partly or wholly free from any peptide chains.

Other characteristics of the invention will appear in the course of the description of examples of mitogenic agents containing glycan chains and their processes of manufacture.

First of all the conditions in which the walls of the different bacteria may be obtained will be described. The conditions under which the mitogenic agents have been obtained from said walls will be subsequently described.

(a) Culture of Nocardia rubra, ATCC 14,898 and Nocardia opaca, ATCC 21,953 (strains from the Pasteur Institute of Paris).

The strains indicated above were cultivated in a fermenter of 20 litres, on 14 litres of a medium inoculated with 500 ml of a precul- ture effected on the same medium.

The medium used for the culture of Nocardia rubra was composed of 2.5% of "Heart Infusion Broth" (Difco), 10 ml/litre of glycerol and 0.25 g/litre of Na2HPO4, 12 H2O, the pH having been adjusted to 7.4-7.6.

("Difco" is a Trade Mark).

The culture medium of Nocardia opaca was composed of 0.2% of yeast extract (Difco), 0.4% of meat extract (Difco), 2% of bactopeptone (Difco) and 0.5% of NaCI, the pH having been adjusted to 7.2.

In both cases the cultures were carried out under the same conditions, except with respect to the temperatures of culture (25 C for N. rubra and 30"C for N. opaca). They were carried out under stirring, by rotation of the propeller of the fermenter at 250 revolutions per minute, and with aeration of the medium, at the rate of 2 litres of air per minute.

The cultures were interrupted at the end of 3 to 4 hours. In the case of N. rubra, the culture then had a brick red colour.

The cultures were then collected by centrifuging, washed with distilled water and stored at -200,C until their use.

In order to obtain the walls of these cells, the latter were suspended in five times their weight of distilled water in the container of a homogeniser or "mixer" and subjected to three passages through a pressure grinder known by the trademark MANTON-GAU- LIN, under a pressure of 750 kg per square centimetre. DNase was added after the first passage.

The homogenised product finally obtained was diluted with three times its volume of distilled water and centrifuged three times for 15 minutes, at 800 g in a cooled centrifuge, the deposits obtained at the end of each of these operations, and containing the unbroken cells, being then eliminated. The liquid phase finally obtained was then subjected to centrifuging at 27,500 g for 50 minutes. The deposit made up of the crude cell walls was collected.

The latter was then subjected to the action of proteolytic enzymes, especially for one night at 220C with trypsin and chymotrypsin, with amounts of each of these enzymes of 0.1% of the weight of the crude cell walls, within a 0.066 M phosphate buffer, at pH 7.8, to which were previously added a few drops of toluene in order to prevent any contamination. The walls were separated by centrifuging, then subjected to washings each of which comprised taking them up and suspending them in the phosphate buffer, for the first three times, then in water, for the next three times, and then centrifuging again, whereby deproteinised walls were finally obtained.

These deproteinised walls were then delipidated at ordinary temperature by successive extractions with acetone, then with an ethyl alcohol-ether mixture (1 volume of alcohol to 1 volume of ether), and finally with a mixture of 2 volumes of chloroform to 1 volume of methanol. They were finally put back in suspension in acetone and dried.

(b) Preparation of the walls of Micrococcus roseus.

The cells of M. roseus (strain No. 5693 of the Pasteur Institute of Paris) were cultivated on the culture medium "Nutrient broth" (Difco) in 2 litres flasks containing 800 ml of medium placed on an agitation table known by the name of "BIOLAFITTE", at 250C, and were collected at i of the exponential phase of growth.

The bacteria were crushed by agitation in the presence of small glass balls: in a typical experiment, 30 g of freshly weighed bacteria were suspended in 150 ml of distilled water; 150 ml of small glass balls 0.17--0.18 mm in diameter were added and the mixture was placed in the bowl of 400 ml of a homogeniser of the "Omni-mixer" type (SORVALL) cooled by immersion in a bath of iced water.

The mixer was run at full speed for 30 minutes. After settling of the glass balls, the supernatant was recovered. The supernatants obtained after two successive washings of these balls each time with 100 ml of distilled water were pooled with the first supernatant and the pooled liquid was centrifuged three times for 10 minutes at 800 g in a cooled centrifuge in order to remove the glass ball debris and uncrushed bacteria. The resulting supernatant was centrifuged for one hour at 27,500 g in a cooled centrifuge: the deposit constituted of walls was suspended in a POTTER grinder with a tetrafluoroethylene piston in 150 ml of phosphate 0.066 M buffer, pH 7.8, containing 30 mg of trypsin and 30 mg of chymotrypsin, then incubated for one night at ambient temperature in the presence of a few drops of toluene to prevent contamination. After centrifuging for one hour at 27,500 g, the deposit of deproteinised walls was washed by resuspending it within the POTTER grinder, three times within 150 ml of 0.066 M phosphate buffer, pH 7.8, and three times within 150 ml of distilled water with intermediate centrifuging for an hour at 27,500 g at +40C. The walls were then lyophilised.

The walls thus obtained (purified walls) were constituted by a peptidoglycan and one or more other polymers made from neutral sugars, since the only amino-acids and aminosugars detected in the wall were those of the peptidoglycan.

From publications made respectively by (1) J. F. Petit, E. Munoz and J. M. Ghuysen, Biochemistry, 5, 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 is made up of repetitive units of N-acetylglycosaminyl - p - 1,4 - N - acetylmuramyl L - alanyl - D - isoglutaminyl - L - lysyl D-alanine, in which the e-aminated group of the lysine is substituted by a peptide (L-Ala) 3-L-Thr; the interpeptide linkages are made by linkages between the terminal D-Ala of a tetrapeptide and the terminal L-Ala of the peptide (L-Ala) 3-L-Thr substituting the lysine of another tetrapeptide.

(c) Obtaining the walls of B. cereus.

Cells of Bacillus cereus T have been cultivated on the medium known by the name of "Antibiotic Medium 3" (Difco) at 370C, in 2 litres flasks containing 800 ml of medium and agitated on a Biolafitte agitation table.

The culture has been collected by centrifuging, at two-thirds of the exponential phase of growth.

The bacteria were then crushed or broken, the walls recovered, washed and deproteinised, finally washed again and collected according to the process already described for Al. roseus.

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

The strain was cultivated at 37 C on the medium "Nutrient broth" (Difco) at a concentration 1.5 times greater than that recommended by the manufacturer, under conditions similar in other respects to those described for M. roseus.

The cells were collected when the optical density of the medium, at 600 nm, was 1.1 (sDectrophotometer DUOSPAC, JOBIN and YVON) .

The culture was centrifuged and the deposit was put in suspension in twenty times its weight of water; the suspension obtained was subjected to two successive passages through a FRENCH press at 1200 kg per square centimetre, the DNase being added to the medium between the two passages. In the French press a suspension of cells to be ruptured is passed through a needle under high pressure.

The suspension obtained was then treated like that obtained from Al. roscos. after nlp- ture of these latter bacteria, the deposit of walls obtained after centrifuging of 27 ,5()0 g being, however, before the treatment with the proteolytic enzyme%., suspended in a solu tion of isotonic sodium chloride and brought to the boil to destroy the autolytic enzymes contained in these walls.

Preparation of precursors.

Obtaining the deproteinised walls, possibly delipidated, and practically freed from their polysaccharides other than those of the pep tidoglycan, by acid hydrolysis.

The products in question will be subsequently denoted by the expression "acidtreated walls".

These "acid-treated walls" were obtained in the following conditions from the delipidated and deproteinised walls described above.

(a) "Acid-treated walls" of N. opaca and N. ruhra.

400 mg of the corresponding walls were suspended in 40 ml of 0.1 N hydrochloric acid and maintained in this medium for 12 hours at 60"C. They were then washed three times with water, by putting in suspension therein, followed by centrifuging. They were then subjected to a further delipidation treatment with chloroform at the ambient temperature. The product was filtered and the walls were recovered, washed with acetone and dried. Analysis showed that these walls had practically lost all their arabinogalactans and that the residues of the nocardomycolic acids were extractable with chloroform.

(b) "Acid-treated walls" of the other bacteria studied.

1 g of the purified walls was suspended in 100 ml of 0.1 N hydrochloric acid and mainrained in this medium for 12 hours at 600 C.

The walls were then washed five times, each washing comprising suspending the walls in water and recovering them bv centrifuging.

They were then lyophilised or dried with acetone. The corresponding "acid-treated walls" were thus obtained, of which the major constituent was peptidoglycan, approximately free from neutral sugars and a considerable part of all other constituents foreign to the peptidoglycan.

In the account of the pharmacological properties, reference will be made to the differenr products obtained under the following references: (1) (acid) peptidoglycan of N. rubra; (2) (acid) peptidoglycan of N. opaca; (3) (acid) peptidoglycan of N roseus, (4) (acid) peptidoglycan of B. ,.r(uS (5) (acid) peptidoglycan of B. subtilis.

The effect of acid hydrolysis on the walls appears clearly on examination of the Table I below, which provides the results of analysis of the walls of N. ruba, in their crude state and after acid hydrolysis respectively. Their respective lipid contents have not been determined.

The differences were significant at the level of the proportions of neutral sugars, very reduced in the "(acid) peptidoglycan of N. rubra", and of the resulting increases of the contents of amino-acids and, more still, of amino-sugars.

TABLE I Composition of the walls of N. rubra before deproteinisation and delipidation and after acid hydrolysis and redelipidation respectively

Neutral sugars Amino-sugars Amino-acids com- com- DAP nmol/ posi- nmol/ posi- nmol/ nmol/ % mg tion % mg tion % mg mg Crude cellular walls 28.2 1516 Gal, 12.8 500 Mur, 31.91 2659.9330.70 Ara, Glc, Glc, NH, Man Acid-treated cellular 5.58 300 walls Ara, Glc, Man NH2 Example I.

Obtaining water-soluble mitogenic glycans from some of the compounds 1 to 5 above, by the action of the peptidases of S. albus G.

Recourse was had to the above-indicated bacteriolytic enzymes, purified according to the technique described by J. M. Ghuysen et al., in the article entitled "An improved technique for the preparation of Streptomyces peptidases and N - acetylmuramyl - b alanine amidase active on bacterial wall peptidoglycans" [Biochemistry, 8, 213-222, (.1969)] for obtaining crude peptidases.

6 mg of the said "(acid) peptidoglycans" were incubated for 6 hours at 370C, with 250 microlitres of the said peptidase (corresponding to 40 ml of the filtrate from the culture obtained under the conditions described in the above-mentioned publication), in one volume of 1 ml of a 0.005 M veronal buffer at pH 9.0. The enzyme was then inactivated, after neutralisation, to pH 7.0, and heating of the medium at 100"C for 10 minutes.

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

When the process was applied in the aboveindicated conditions, there were obtained, from the said corresponding "(acid) peptido glycans", the aqueous solutions of the mito genic agents to which reference will be made subsequently under the following expressions: (6) N. rubra (S. albus G); (7) B. cereus (S. albus G); (8) N. rosecus (S. albus G); In Table II below, the results of the analysis of the product N. rubra (S. albus G) have been indicated. By way of comparison, there has also been shown the analytical characteristics of the fragments of peptidoglycan obtained by solubilization by lysozyme of the same "acid-treated peptidoglycan", obtained from the same strain of N. rubra.

This last product was obtained by suspending 50 mg of the "(acid) peptidoglycan of N. rubra" in 5 ml of a 0.05 M solution of ammonium acetate, at pH 6.3, incubating the suspension for 16 hours at 37"C, in the presence of 1 mg of lysozyme, and a few drops of toluene. The incubation mixture was then centrifuged and the deposit was eliminated. The supernatant, which was collected, contained the product denoted by the expression "N. rubra (lysozyme)" in the Table H below.

TABLE 11 Compared analyses of the products resulting from the solubilization of the "(acid) peptidoglycane of N. rubra" under the actions of the peptidase of S. albus G and of the lysozyme respectively

% of the weight Index of colouration of cellular walls % of meso-DAP in the passed into the having a free MORGAN-ELSON (*) Fractions solution amino group reaction N. rubra 80-85 % 85-90 % 0.15 (S. albus G) N. rubra 90 % 50 % 0.84 (ly sozyme) (*) expressed as disaccharide.

It was found that the two enzymes caused the solubilization of similar weight proportions of the treated cell walls. On the other hand, important variations were observed at the level of the contents of each of the soluble fractions obtained in meso-DAP having a free amino group. The same applied to the colouration indices measured by the MOR GAN-ELSON method, which indices are representative of the proportions of free disaccharides.

The high percentage of meso-DAP groups having a free amino group which was found in the "fraction N. rubra (S. albus G)", testifies that the peptidase did act essentially at the level of the interpeptide linkages.

On the contrary, in the case of the fraction "N. rubra (lysozyme)", it was found that a large part (50%) of the meso-DAP groups were always still engaged in the interpeptide linkages.

In the same way, the ratio of the colouration obtained by the method of MOR GAN-ELSON, expressed as disaccharide to the total quantity of osamine, divided by 2, and measured by the method of ELSON-MORGAN, for "N. rubra (S. albus G)", shows that the glycan chains had only been little or not affected by the enzyme preparation. On the contrary, the high index of colouration which was found in the fraction "N. rubra (lysozyme)" demonstrated the presence of a large proportion of free disaccharides, which confirmed that the lysozyme had broken the glycan chains of the initial peptidoglycan in numerous points. This fraction "N. rubra (lysozyme)" was shown to be equally inactive in the tests of mitogenicity which will be described later.

Example II.

Obtaining water-soluble mitogenic glycans from some of the compounds 1 to 5 above, by the action of the amidase of Myxo bacter AL1.

The enzyme was prepared according to the technique described by J. C. ENSIGN and R. S. WOLFE, J. Bacteriol. 91, 524-534 (1966), the filtration on SEPHADEX G100 being replaced by a filtration on SEPHADEX G25.

Hereinafter there is described, by way of example, the process applied to the walls of N. rubra, it being understood that the equivalent fractions have been obtained under similar conditions, from on a SEPHADEX G25 column equilibrated with 0.1 N acetic acid; the elution was then carried out with the same solution.

The effluent of the column was analysed after hydrolysis: a first peak contained all the amino-sugars of the lysate and some aminoacids; the following peaks only contained amino-acids.

Only the first peak was active. Its activity was attributed tn the glvc'n chains obtained by the action of the enzyme.

The product contained in this peak was denoted further by the expression: (10) N. rubra (Myxobacter-SEPHADEX G25) Under similar conditions there was obtained from the corresponding "acid-treated peptidoglycan" the water-soluble mitogenic product denoted hereafter as follows: (11) M. roseus (Myxobacter) PHARMACOLOGICAL PROPERTIES OF THE FRACTIONS OBTAINED.

The mitogenic agents according to the invention possess among others the capacity of stimulating in a non-specific way the B lymphocytes cells derived from bone marrow.

This activity was demonstrated by the aptitude of splenic lymphocytes (from mouse and rabbit) to absorb (by incorporation) more tritium-containing thymidine than the B lymphocyte cells of the controls.

1) Culture of the lymphocytes.

Lymphocytes were separated from the spleens of mice or rabbits, by having recourse to the technique of C. BONA et al, Eur. J.

Imm., 2, 434, 1972. Various types of mice have been used, notably from 2 to 3 months old AKR mice and from four to eight weeks old mice, of the mouse athymic NUDE species, reared at the C.N.R.S. laboratory of Orleans, belonging to a wild non-inbred progeny for nude mutation, and coming from "The Institute of Animal Genetics" of Edinburgh.

Recourse has also been had to splenic cells of from four to six weeks old BOUSCAT rabbits, coming from the breeding of the PAS TEUR INSTITUTE of Garches.

1.5 x 106 splenic lymphocytes of mice were cultivated for 48 hours, at 370C, in 1 ml of the medium known as the RPMT-1640 (Eurobio), to which 5''/, of foetal calf serum 'Flow-labs) had been added.

In the same way 2.5 x 10" splenic lymphocytes of the rabbit were cultivated for 72 hours, at 37"C, in 1 ml of the medium known as the "Eagle medium", to which had been added 10% of autologous serum inactivated by heating for 30 minutes at 560C.

2) Incorporation of tritium-containing thymidine.

1 ssCi. of 3H-thymidine (lCi/mMole, Saclay, France) was added to each culture 16 hours before the cells were harvested. At the end of the incubation, an amount 100 times greater of non-radioactive thymidine was added. After centrifuging at 450 g for 10 minutes, the supernatant was removed, the deposit was precipitated with trichloracetic acid and resuspended according to the con ventional techniques, before being measured on the scintillation counter.

As may be found by examination of Table III which follows, the agents according to the invention which have been tested all have a mitogenic action which shows itself by the increase of the incorporation of tritium-con taining thymidine by the splenic lymphocytes of the animals studied. In the left-hand columns the various fractions are identified.

There has been indicated in the columns corresponding to the animals which provided the lymphocytes tested, the observed values of the stimulation index of the agents tested, at the doses indicated between brackets at the side of the corresponding values of the stimu lation index. The stimulation index is the ratio of the radio-activity of the stimulated cells to those of control cells.

As may be found, in practically all cases an increase of the measured stimulation index was observed. The agents derived from cells of Nocardiae are particularly active with regard to the lymphocytes of mice. They are equally so with regard to the rabbit. In the same manner, it has been verified when resort ing to the same test, that the "acid-treated peptidoglycans" of Nocardia were active on lymphocytes of circulating blood of human origin. Accordingly these tests establish the non-specific character of the mitogenic agents according to the invention.

TABLE Ill

Mitogenicity (stimulation index) Nature of the product mice AKR mice NUDE rabbit (l) (acid) peptidoglycan of 7 9() (100 g) 11.34 (lOOMg) 194 (100 g) N rubra (2) (acid) peptidoglycan of 8. 17 (100 g) 14.07 (InoMg) 10.73 (100 g) N. opaca (3) (acid) peptidoglycan of 1.20 (0.1 g) 3.5l (100 g) N roseus (4) (acid) 4.1 (100 g) 16.95 (1o g) peptidoglycan of B. cereus (5) (acid) peptidoglycan of 3.94 (100 g) 1.78 (100 g) B. subtilis (6) N. rubra (S. albus G) 9.6 (100 g) 7.61 (10011g) 2.23 (1 g) (7) B. cereus (S. albus G) 3.24 (100 g) 4.19 (all) (8) N. roseus (S. aibus G) 2.86 (100 g) 1.76 (100s1g) (9) N. nlbra (Myxobacter) 288 (100 g) (10) N rahra (myxobacter- 3.53 (100 g) SEPHADEX G 25) (11) M.. roseus (Myxobacter) 43 (100 g) The biological effects of the agents according to the invention make them suitable for various applications, of which some are indicated hereafter by way of examples: (a) biological reagent for research of great interest which permits the stimulation of the B lymphocytes in several animal species, the monkey and even man; (b) medical biological reagent, for diagnosis, from the cells of the lymph organ cells, of immunity deficiencies relating to the cells which produce antibodies; (c) preventive treatment: after the injection of the product (preferably chosen from among the water-soluble agents) in the animal, a global and non-specific stimu lation is observed which produces an increase of the antibodies directed against large categories of antigens; (d) use in the curative treatment of the defi ciency diseases of bone marrow having accidental causes (for example irradia tion) or idiopathic causes (for example mvelo-sclerosis) and for the stimulation of stem cells. They are then administered preferably by injection in association with injectable sterile liquid vehicles, such as a saline or glucose isotonic solution.

The water-soluble mitogenic agents are valuable, particularly in that it is possible to use them in the form of oil-free compositions.

WHAT WE CLAIM IS: 1. An oil-free pharmaceutical composition containing a mitogenic agent in association with a pharmaceutical carrier, the agent being formed from a water-soluble polymer which may have a content of neutral sugars of less than 10% by weight and comprising glycan chains including a polysaccharide skeleton derived from monomeric groups, containing respectively N - acetyl - glucosaminyl - Nacylmuramyl disaccharide units, in which the acyl group consists of an acetyl or glycolyl group, the N-acetylglucosamine moiety being optionally partly deacetylated and the glycan chains optionally carrying also peptide substituents, which substituents are sufficiently free from interpeptide linkages to ensure the water-solubility of the mitogenic agent.

2. A composition as claimed in claim 1 in which the mitogenic agent has the structure of which the essential part is represented by the general formula:

G[e N Ac Mur N X-- Gle NAc-ErN X hide pcik 2 wherein "peptide" represents a peptide chain, "Glc N Ac" represents an N-acetylglucosaminyl group, "Mur N X" represents an Nacylmuramyl group with "X" being an acetyl or glycolyl group and "n" is at least 6.

3. A composition as claimed in claim 2 in which each peptide chain comprises at most ten amino acid residues.

4. A composition as claimed in claim 3 in which the essential part of the structure of the water-soluble mitogenic agent is represented by the general formula:

or by the general formula:

in which in addition to the abbreviations already defined, "Ala" represents an alanyl group, "isoGln" represents an isoglutamine group and "Y" represents a meso-la--di- aminopimelic acid group, optionally containing an amido group and in which the Nacetyl-glucosaminyl groups are optionally partly deacetylated.

5. A composition as claimed in claim 3 in which the essential part of the structure of the water-soluble mitogenic agent is represented by the general formula:

or by the general formula:

in which in addition to the abbreviations already defined, "Z" represents a D-glutamic acid or D-isoglutamine group and "W" represents a meso-s-diaminopimelic acid group optionally containing an amide group or a lysyl group and in which the N-acetylglucosaminyl groups are optionally partly deacetylated.

6. A composition as claimed in claim 1 in which the mitogenic agent has a structure of which the essential part is represented by the general formula:

wherein CTle N Ac represents an N-acetylglucosaminyl group Mur N X represents an N-acylmuramyl group with X being an acetyl or glycolyl group and n is at least 6 and in which the N-acetylglucosaminyl groups are optionally partly deacetylated.

7. A composition as claimed in any of claims 2 to 6 in which n is 10 to 45.

8. A composition as claimed in any of claims 1 to 7 in which some of the N-acetylglucosaminyl groups are partly deacetylated.

9. A composition as claimed in any of claims 1 to 8 in which the water-soluble mitogenic agent is obtained by a process which comprises subjecting an insoluble peptidoglycan derived from essentially delipidated

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. preferably by injection in association with injectable sterile liquid vehicles, such as a saline or glucose isotonic solution. The water-soluble mitogenic agents are valuable, particularly in that it is possible to use them in the form of oil-free compositions. WHAT WE CLAIM IS:

1. An oil-free pharmaceutical composition containing a mitogenic agent in association with a pharmaceutical carrier, the agent being formed from a water-soluble polymer which may have a content of neutral sugars of less than 10% by weight and comprising glycan chains including a polysaccharide skeleton derived from monomeric groups, containing respectively N - acetyl - glucosaminyl - Nacylmuramyl disaccharide units, in which the acyl group consists of an acetyl or glycolyl group, the N-acetylglucosamine moiety being optionally partly deacetylated and the glycan chains optionally carrying also peptide substituents, which substituents are sufficiently free from interpeptide linkages to ensure the water-solubility of the mitogenic agent.

or by the general formula:

7. A composition as claimed in any of claims 2 to 6 in which n is 10 to 45.

and deproteinised bacterial walls to the lytic action of a peptidase enzyme.

10. A composition as claimed in claim 9 in which prior to the lytic action, the bacterial walls have been subjected to acid hydrolysis under sufficiently mild conditions to effect separation of at least 90% of their neutral sugars and to at least weaken the linkages between the glycan chains and the mycolic acids or other polysaccharides.

11. A composition as claimed in any of claims 1 to 10 wherein the bacterial walls are those of Nocardiae.

12. A composition as claimed in any of claims 1 to 10 wherein the bacterial walls are those of B. cereus, B. subtilis, M. roseus or Staphylococcus albus.

13. A composition as claimed in any of claims 1 to 12 in the form of an isotonic, sterilized or sterilizable, injectable aqueous solution.

14. A composition as claimed in any of claims 1 to 13 in which the number of the N - acetylglucosaminyl - N - acylmuramyl disaccharide units is sufficient to stimulate the Iymphocvtes B of mice or rabbits.

15. Oil-free, sterile pharmaceutical compositions substantially as herein described.