CA1060796A - Oil-free adjuvant composition containing muramyl dipeptide - Google Patents

Abstract

A B S T R A C T

The invention relates to an oil-free adjuvant compo-sition, containing N-acetyl-muramyl-L-alanyl-D-glutamic acid as an active principle, associated to a pharmaceutically acceptable vehicle. This composition can act as an immuno-logical adjuvant to stimulate a host's immunitary response to different sorts of antigens.

Description

106079~
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The invention relates to oil-free drug compounds, acting as immunological adjuvants to stimulate a host's immunitary response to different sorts of antigens. More particularly, the invention concerns drug compounds of said type, capable of reinforcing and enhancing the action of weak mmunogens.
More particularly, the invention relates to adjuvant -drug compounds, likely to be used for immunizing man and warm blood animals against bacteria, viral and parasitic infections and also against various tissue antigens having a normal or pathological origin, among others tumors.
Adjuvant agents have already been described in Canadian pending application S.N. 155,915 filed Nov. 8, 1972, -now Can. Pat. 1,013,691, July 12, 1977 which are obtained more particularly by a process consisting in cultivating a strain of Mycobacteria, Nocardia cells or related micro-organisms, collecting the cells of the cultivated strains, causing their disruption and recovering the disrupted cell-walls (for instance by differential centrifugation) and then separating and eliminating the waxes, free lipids, proteins and nucleic acids, causing the digestion of the delipidated substance originating from cell-walls and having previously been sus-pended in an aqueous medium, by a murolytic enzyme, such as lysozyme, eliminating the solid residue and collecting the aqueous fraction which contains aforesaid soluble agents, further purification of which is obtained, i.e. by filtration of said aqueous fraction through a molecular sieve of the SEPHADEX~type (polydextran gel) or alike.
As already indicated in this patent, the active part of these adiuvant agents consisted essentially of an oligomer, the monomer unit of which fairly corresponds to that of the ~ ~ ' - ' - - , cell-walls polymers of Mycobacteria or Nocardia cells from which they originate.
As has been demonstrated in this patent, the hydro-soluble adjuvant agents of the type referred to, show a noteworthy adjuvant activity when administered in the form of an aqueous-oily emulsion to test animals. They have also proved to show a certain adjuvant activity when administered in the form of an aqueous solution (test of N.K. Jerne et al.
described in "Cell bound antibodies" ed. B. Ames and 10 H. Koprovski Wistar Institute Press, Philadelphia, 1963).
However, Jerne's test concerns but locally formed antibodies;
it is not representative of a non specific immunostimulant activity tending to increase the rate of circulating anti-bodies; still these adjuvant agents contained in an aqueous solution do not result in an increase in vivo of the rate of circulating antibodies (present in the serum).
Under such conditions, it appears that the adjuvant activity of aqueous solutions containing the agents described in the above mentioned application could, at best, have but a very reduced therapeutical interest; subsequent studies of these agents in view of their therapeutical applications have thus been carried out, using an aqueous-oily emulsion. Par-ticularly, the same applied as concerns different products ' -of ever-decreasingly low molecular masses, which have been extracted from above adjuvants or derivate therefrom.
As disclosed in the French patent No. 73337806, the adjuvant activity of thus obtained preparations when adminis-tered within an aqueous-oily emulsion, had been attributed to the presence in these preparations of soluble fragments of the peptidoglycans contained in the cell walls of bacteria treated by the above process. More particularly, the above appli-. .

`` . cation described hydrosoluble adjuvant agents consisting of ` peptidoglycan fragments formed of polysaccharides-polypeptides of low molecular weight, particularly of oligosaccharides-oligopeptides, the polysaccharide part of which consisted in N-acetyl-glucosamine units, on the one hand, and N-acyl-muramiC acid more particularly N-glycolyl-muramic acid, on the other hand.
AS has been ascertained in Patent No. 74 22909, the N-acetyl-glucosamine portion was not necessary to the adjuvant action of agents containing peptidoglycane soluble fragments.
Particularly, this application describes a synthetic hydro-soluble adjuvant agent consisting of N-acetyl-muramic acid carrying a dipeptide group, the latter being preferably formed of L-alanine, directly linked to the N-acetyl-muramic acid, and of D-glutamic acid, the latter being preferably the amide derivative (on the ~-carboxyl group). The product considered - was thus, in the latter case, the so-called "N-acetyl-muramyl-L-alanyl-D-isoglutamine" (Mur-NAc-L-Ala-D-Glu-~-NH2) of formula:

I H, OH
~\~ ' , HO ~ ~

¦ NHAc CH3 (IH2)2 COOH

: This product is also identified as follows: 2-(2-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-~-isoglutamine.

Like the adjuvant agents formed of more important ;
molecules, the constitution of which has been recalled here-.- above, the Mur-NAc-L-Ala-D-Glu-~-NH2 has exhibited an excel-lent adjuvant activity when administered to test animals in the form of an aqueous-oily emulsion.
However, the necessity of resorting to aqueous-oily emulsions in order to bring out the efficient adjuvant action of agents of the type referred to, entails many drawbacks.
Most of the oils used, up to the present, for the preparation of such emulsions, are formed of paraffin or analogous oils, which cannot be metabolized. Moreover, the emulsifying agents needed for stabilizing said emulsions, for instance those known under the designation "ARLACEL~"do not exhibit the desirable inocuity. All aforesaid drawbacks have thus con-siderably reduced the use of the hydrosoluble adjuvant agents .~ described hereabove in therapy, particularly human therapy.
In order to overcome, at least partly, such drawbacks, it has been proposed to prepare aqueous-oily emulsions, using vegetal oils. Various experiences have been ; tempted that way, but most authors have stumbled against the problem of stabilizing these emulsions.
The invention described in French patent No.
75 04003, has substantially improved the situation. It -- describes a stabilized water-vegetal oil emulsion, usable as a vehicle for administering agents of the type referred to, especially the above mentioned N-acetyl-muramyl-L-alanyl-D-isoglutamine. Nevertheless, this invention, though achieving an important improvement, does not remove the need for a water-oil emulsion.
The invention derives also from the essential discovery that, against any expectation, the substance consti-- tuted by N-acetyl-muramyl-L-alanyl-D-glutamic acid, has a ~ ~ - 4 -.
. noteworthy non specific adjuvant activity, when administered .
~; to an host under the form of an oil-free aqueous sol ution.

- N-acetyl-muramyl-L-alanyl-D-glutamic acid, which may be designated by the abbreviated form:

Mur-NAc-L-Ala-D-Glu-OH
corresponds to the following formula:

.~ CH OH

-.`., V \l ' OH ~

/ NHAc H3C - CH - ICl - NH - CH - CO - NH - CH - COOH

:. O CH3 (IH2)2 ;......................................................... COOH
,'5 ':~, To identify said substance, one may have recourse to official nomenclature. It iS then called: 2-(2-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid.

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.~ Taking into account itS chemical relationship with ~eS previouS adjuvants, constituted by bacterial extracts, it was quite unforeseeable that N-acetyl-muramyl-L-alanyl-D-glutamic acid should be able of exerting such an action, more especially , . .
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-10f~0796 of enhancing the production of circulating antibodies in vivowithout having recourse, in the administration medium, to an oil, either of mineral or vegetal origin. This discovery of the adjuvant properties of oil-free compositions - especially aqueous solutions - represents an essential advance in the field of therapeutical uses, especially for men, of drugs having a non specific adjuvant activity with respect to immunity. As a matter of fact, N-acetyl-muramyl-L-alanyl-D-glutamic acid which is not only soluble but has also been discovered to be active in aqueous solution, can exert its activity in physiological serums, which was not the case for the previously known non specific adjuvants of immunity.
The non specific adjuvant activity of said substance in oil-free aqueous solution is all the more noteworthy as the same substance, when administered within an aqueous-oily emulsion, presents but a much lower activity. This reversal --of the intensities of the properties of adjuvant activity according to the nature of the medium used was, of course, quite unexpected, considering what was already known of previous adjuvants, the nature of which has been recalled hereabove.
It has been moreover observed that, contrarily to the previously described hydrosoluble adjuvant agents, N-acetyl-muramyl-L-alanyl-D-glutamic acid induced no sensiti-zation of the host, either against N-acetyl-muramyl-L-alanyl-D-glutamic acid itself, or against various previously cited adjuvants, prepared from mycobacteria or analogous. This sensitization, induced by the previously described hydro-soluble adjuvants, appears in the form of hypersensitivity reactions of tuberculinic type on subjects having previously been put into contact with the adjuvants themselves or with : -` 10~()796 mycobacterial germs or extracts.
Thus, N-acetyl-muramyl-L-alanyl-D-glutamic acid, does not constitute a sensitizer; in other words, it has no immuno-genic properties, particularly, it does not bring about any reaction on subjects, either sensitive or sensitized, to tuber-culinic bacilla. Therefore, it has not an antigenic structure of the same type as that of previous adjuYants. N-acetyl-muramyl-L-alanyl-D-glutamic acid is therefore, in that respect, perfectly harmless for subjects which have previously been - 10 contacted, either with the product itself or with the above mentioned previous adjuvants, or with mycobacteria or analo-gou s .
~ Thus, the invention relates to oil-free compositions - containing N-acetyl-muramyl-L-alanyl-D-glutamic acid, associ-ated with a pharmaceutically acceptable vehicle. By oil-free compositions is more particularly meant compositions free of h an oil which is liquid at ambient temperature, say at 25C.
' The invention relates particularly to oil-free ; solutions containing N-acetyl-muramyl-L-alanyl-D-glutamic acid, - 20 more especially sterile or sterilizable solutions, injectable or able to be used, more especially extemporaneously, for the preparation of injectable solutions. It particularly concerns physiological aqueous solutions, more especially isotonic ones, - such as saline or glucosed solutions, of one of these com-pounds. It is to be understood that these examples infer no limitation as to the definition of physiologically acceptable products which may be used to constitute isotonic injectable ; solutions.
However, the invention concerns also N-acetyl-muramyl-L-alanyl-D-glutamic acid solutions in distilled water or in water totally free from products such as mineral salts : ~060796 ` which, upon evaporation, leave a solid residue, such solutions being able to be used subsequently for the preparation of vaccines.
The invention also relates to compositions or solutions of the above indicated type in which N-acetyl-muramyl-L-alanyl-D-glutamic acid are associated to a vacci-nating antigen, particularly to a weak immunogen, in order to reinforce the host's organism capacity to produce antibodies against this antigen.
The invention relates also to N-acetyl-muramyl-L-alanyl-D-glutamic acid as a lyophilised product this lyophili-sate possibly containing also a vaccinating antigen mixed with N-acetyl-muramyl-L-alanyl-D-glutamic acid.
The invention relates also to oil-free pharmaceutical ,3 compositions containing the adjuvant agent, more especially injectable aqueous vaccinating compositions containing, in ` addition to the adjuvant, an active principle of vaccine, these pharmaceutical compositions being able to be adminis~ered by - sub-cutaneous, intradermic or intramuscular injections, or still by scarification.
It also relates to pharmaceutical compositions which may be administered by other routes, i.e. orally or rectally, or under the form of aerosols intended for entering into contact with mucous membranes, more especially, ocular, nasal or vaginal membranes.
Consequently, it concerns also pharmaceutical compo-sitions in which N-acetyl-muramyl-L-alanyl-D-glutamic acid are associated to solid or liquid pharmaceutically acceptable vehi-cles, suitable for the constitution of forms intended for oral, ocular or nasal administration or with excipients, such as glycerides or analogous, suitable for the constitution of forms 106~)796 to be administered rectally, or still with gelatinous excipi-ents, for vaginal administration. It also concerns liquid isotonic compositions containing N-acetyl-muramyl-L-alanyl-D-glutamic acid adapted for administration on ocular or nasal mucous membranes. It finally relates to compositions consti- -tuted by liquefied, pharmaceutically acceptable gases, of the "propellent" type in which the N-acetyl-muramyl-L-alanyl-D-glutamic acid are dissolved or kept in suspension, the ;~ expansion of which causes the dispersion of an aerosol.
The invention also relates to a pharmaceutical presentation containing one or several unit doses of N-acetyl-muramyl-L-alanyl-D-glutamic acid, each dose from about 10 to about 100 mg, preferably 30 mg under lyophilised form, on the - one hand, and a corresponding number of ampullae, each con- taining 1 ml of isotonic solute, for instance chlorinated solute, for the extemporaneous preparation of a dosed solution, ~-more especially for administration by injection, on the other hand.
In the pharmaceutical presentations for adminis-tration through oral route, the unit dose of N-acetyl-muramyl-L-alanyl-D-glutamic acid is comprised between about 10 and about 200 mg.
Other characteristics of the invention will still appear in the course of the following description of tests evidencing the pharmacological properties of oil-free aqueous solutions containing N-acetyl-muramyl-L-alanyl-D-glutamic acid and of an example of synthesis of N-acetyl-muramyl-L-alanyl-D-glutamic acid.
- It is to be noted that, in the present case, the word oil means fatty substances or analogous in the liquid form at ambient temperatures.

~; 1060796 I - Mur-NAc-L-Ala-D-Glu-(OH) synthesis ` This compound, the official name is 2-(2-acetamido-

2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid, is prepared stepwise. First, the peptidic moiety is synthesized, then this moiety is bounded to the muramyl moiety; finally, the functions blocked during the synthesis are freed.
BOC-L-alanyl-D-glutamic benzyldiester (I) , 2,3 9 (8 m moles) of t.butyloxy carbonyl-L-alanin succinimid ester, the amino function of which is protected by the t.butyloxy carbonyl group (BOC-L-Ala-OSu), are added with stirring to a solution in dimethylformamid of 4,5 9 (9 m moles) D-glutamic dibenzylester p.toluen sulfonate and of 1 ml ;~ (9 m moles) N-methylmorpholine. The mixture remains at room temperature during 12 hours, then evaporated to dryness. The ; dry compound is dissolved in 50 ml acetic ethyl ester and washed successively with a 10% citric acid solution; water; a ~`
; lN sodium bicarbonate solution; water. The acetic ethyl ester part is dried with MgS04, filtered and concentrated. The compound is crystallized out of a mixture of ethyl acetate-hexane, 2,50 9 (67,5%) of the desired product are obtained.

~D5 ~ + 7 3 The analysis by element is C27H3407N2 (498,5) C % H % N %
theoretical : 65 6.9 5.6 found : 64.85 7.0 5.5 2-(benzyl-2-acetamido-4,6 benzylidene-2-deoxy-3-0-D-gluco-pyranosyl)-D-propionyl-(O-benzyl)-L-alanyl-D-glutamic benzyl-diester (II)500 mg (1 m mole) of compound (I) are treated with a lN hydrochloric solution in glacial acetic acid. Thirty g ' ~ ' , , ' ~

"` 1060796 '~-, ...
minutes later, the mixture is evaporated to dryness. The oil ` obtained is dissolved in a mixture of acetonitrile-dimethyl- -formamid (2/1 v.v.). The mixture is cooled to 0C and 0,141 ml (1 m mole) triethylamin is added. This solution is poured with stirring in a dispersion at 0C containing 472 mg (1 m mole) of benzyl-2-acetamido-4,6-0-benzylidene-3-0-(D-l carboxyethyl)-2-deoxy-~-D-glucopyranoside and 0,141 ml (1 m mole) of triethylamine in 25 ml of the mixture acetonitrile-dimethylformamid (2/1 v.v.).
The mixture remains 12 hours at room temperature, then is concentrated and the residue is poured in a 10% acetic acid solution. The precipitate is filtered, thoroughly washed with water and dried. 800 mg (94%) of the desired product are --obtained. `~
M P = 198-199 C ~ :
~2D5 - 4,92 (dimethylformamide) When the product is crystallized out of ethanol, the ~-melting point is 220C.
The analysis by element is ;
C47H53012N3 (851,96) C % H % N %
theoretical :66.26 6.27 4.93 found : 66.34 6.45 4.92 2-(2-acetamido-2-deoxy-3-0-D-gl ~-iopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid (III) 700 mg (0,8 m mole) of compound II are treated 1 hour by 40 ml of a 60% acetic acid solution in a boiling water-bath. The mixture is evaporated to dryness (and desic-cated with MgS04). The residue is added to a mixture of chloroform-methanol (3,3 v.v.) and passed through a silica column (35 g) previously contacted with the same solvent mixture. The eluated fractions containing the product are -:

;

recovered evaporated to dryness (their homogeneity is tested by thin layer chromatography on silica gel with the same mixture of solvents). 185 mg (30%) of intermediate compound are obtained.
76 mg of this intermediate are dissolved in 15 ml of glacial acetic acid and their hydrogenated with 5% palladium on coal as catalyst. The mixture is filtered, evaporated to dryness and precipitated out of an ether-methanol-acetone - solvent mixture. 45 mg (92%) of the desired product are obtained.
M P = 150-155C
~2D5 - + 33 (in glacial acetic acid) The analysis by element is Cl9H3112N3 1H2 (511,48) C % N % H %
theoretical : 44.6 6.5 8.2 found : 44.7 6.4 8.1 IV - PHARMACOLOGY (Mur-NAc-L-Ala-D-Glu-(OH)) 1) Toxicity The compound according to the invention has been compared with the lipopolysaccharide (LPS) prepared by the phenol-water procedure from S.ente~iditis with regard to their lethal dose (LD). The LPS is known to exhibit an adjuvant activity when administered in water solution, but cannot be used as adjuvant product due to its high toxicity.
The method used to carry out these experiments is ~-described by Chedid and al. in Ann. N.Y. Acad. Sci. 133 : 712, 1966. The tests are made on two month old Swiss common stock mice. The assayed products are administered by intravenous injection.
The LD50 of the LPS is found to be about 300 ~9 for normal mice. With the very susceptible adrenalectomized mice, , '' - 1 1 -' ;'' ' ~ - . ~ - .'' . .
.. ~ .

the LD50 is lowered to 0,02 ~9.
300 ~9 of Mur-NAc-L-Ala-D-Glu-(OH) were administered to adrenalectomized mice. All mice survived this injection.
The product according to the invention is then innocuous at the doses at which it exhibits a good adjuvanti-city (these doses are determined in the following tests).
2) Adjuvanticity in aqueous solution In these experiments, the antibody levels in mice are determined following the injections of various compo-;10 sitions. The injected compositions contain bovine serum -albumin (BSA) as antigen and the adjuvant compound according to the invention. Other mice received BSA and the WSA adju--vant compound prepared from M.smegmatis according to Adam and al. method (Infec. Immun., 7, 855-861, 1973). The compo-sitions are injected in solution in saline. The injected dose of BSA is 0,5 mg per mouse, the recall dose (30 days after the first injection) contains 0,1 mg of BSA. Each tested composition is administered to eight mice. At various tim-e intervals following the injection, the mice are bled to check the antibody level.
.The antibody estimation is made either by passive hemagglutlnation (PHA) using formalinized sheep red blood `cells coated with the tested antigen, or according to method taught by A.A. Hirata and M.W. Brandiss (J. Immunol., 100, 641-648, 1968), or by the so-called antigen binding capacity method described by P. Minden and S. Farr (Handbook of Exp.
Immunol., p. 463, Weir D.M. ed. Blackwell Scientific Pub., Oxford and Edinburgh, 1967).
,;`Results of these experiments are given in Table A. -As can be seen, the Mur-NAc-L-Ala-D-Glu-(OH) administered in saline induces an enhancement of the antibody titer, whereas ~ 060796 the WSA is practically inactive.
Table A

Treatment Day 14 Day 28 Day 34 Day 36 dose/mouse PHA ABC PHA ABC PHA ABC PHA ABC

Control - BSA < 3 <20 < 3 <20 3 <20 3 <20 BSA ~ WSA 300 ~9 < 3 <20 < 3 <20 6 <20 6 65 BSA + Mur-MAc-L-Ala- 3 <20 50 80 400 320 2,130 630 D-Glu-~OH~ 100 ~9 _

3) Adjuvanticity with a water in oil emulsion (comparative assay) As previously, the adjuvanticity is estimated from the increase of the antibody level following an antigen injection. In these experiments, ovalbumin is used as antigen. Assays are carried out on Hartley's guinea pigs weighing 350 9. The animals are injected with 0,1 ml in each posterior footpad. Each dose contains 1 mg ovalbumin and either the Freund's complete adjuvant (FCA) or the Freund's incomplete adjuvant (FIA~ or this one with the compound of the invention (50 ~9). The antibody titer is estimated 21 days after the injection, by passive hemagglutination as indicated above, or by precipitation according to Folin's method.
A skin test for delayed hypersensitivity is made 18 days after the injection on the same animals. They received 5 ~9 of ovalbumin by intradermal injection. 48 hours later, the diameter of reaction is measured (in mm).
It can be seen from the results given in Table B
that the Mur-NAc-L-Ala-D-Glu-(OH), under the condition of this experiment, does not induce an increase of the antibody titer when injected with the oil constituted by the Freund's incom-plete adjuvant. The skin test reaction is light compared with the one obtained with the Freund's complete adjuvant (FCA).
Table B

Treatment Skin test Antibody a. ovalbumin dose/guinea-pigOvalbumin 5 ~ugPrecipitinAgglutinin , FIA (controls) ~0,0,0,0,0,0 500 1,000 ovalbumin FCA + ovalbumin10,8,10,12,113,200 2,400 FIA ~ Mur-NAc-L-Ala-D-Glu-(OH) 50 ,ug t0,0,5,6 500 900 ovalbumin . 4) Induced hypersensitivity against various mycobacteria ; antigens especially tuberculin The capacity of Mur-NAc-L-Ala-D-Glu-(OH) to induce ~: hypersensitivity reaction against various compounds injected with Freund's incomplete adjuvant (FIA) has been studied. For comparison, simultaneous tests were carried out with Freund's complete adjuvant (FCA).
The estimation is made as previously described.
According to the results given in table C, no hyper-20 sensitivity reaction is observed against FIA ~ Mur-NAc-L-Ala-, D-Glu-(OH). Thus the Mur-NAc-L-Ala-D-Glu-(OH) is not immuno-, genic.
Table C
.
Skin test Treatment l dose/guinea- Purified Old Mur-NAc-L
Pi9 tuberculin tuberculin WSA Ala-D-Glu-,- 50 IU 50 IU 5 ~9 (OH) 5 1l9 FCA 10,7,10,7,10,714,10,13,12,13,5 6,7,0,6,3,5 0,0,0,0,0,0 30 FIA~Mur-NAc-¦ L-Ala-D-Glu- 0,0,0,0,0,0 0,0,0,0,0,0 0,0,0,0,0,0 0,0,0,0,0,0 ,~ .

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V - PHARMACEUTICAL FORM
This form is to be used for the extemporaneous preparation of dosed injectable adjuvant solution and contains:
- unit doses of 30 mg lyophilized 2-(2-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid, - for each of the above doses an ampoule containing 1 ml of saline (isotonic solution of NaCl).
Thus, oil-free adjuvant compositions are obtained which are deprived of toxicity or noxious secondary effects and, therefore, particularly proper for therapeutical appli-cations.
Adjuvant compositions are obtained, which may be used to increase the efficiency of vaccines of bacterial or viral origin, more especially if they are light immunogens.
More especially, they may be used to increase the host's immunity (either human or animal) against infections of bacterial or viral origin, tumor antigens, protozoa antige-ns, ~-esw. They are also useful for making serums containing active antibodies against said antigens.

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Claims (14)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. Oil-free adjuvant composition, containing N-acetyl-muramyl-L-alanyl-D-glutamic acid as an active principle, associated to a pharmaceutically acceptable vehicle.

2. Composition according to Claim 1, characterized in that it is constituted by an aqueous solution of above compound.

3. Solution according to Claim 2, characterized in that the aqueous solvent is constituted by distilled water or by water after evaporation of which no mineral residue is left.

4. Solution according to Claim 2, characterized in that the aqueous solvent is constituted by an isotonic solution.

5. Solution according to any of Claims 2, 3 and 4, characterized in that it is sterile.

6. Solution according to Claim 4, alone or in combi-nation with Claim 5, characterized in that it is an injectable solution.

7. Solution according to any of Claims 2, 3 and 4, characterized in that it contains, in addition, a vaccinating antigen.

8. Composition according to Claim 1, characterized in that it contains, in addition to N-acetyl-muramyl-L-alanyl-D-glutamic acid, a pharmaceutically acceptable vehicle permitting oral administration of said composition, more especially a solid vehicle.

9. Composition according to Claim 1, characterized in that it contains, in addition to N-acetyl-muramyl-L-alanyl-D-glutamic acid, a vehicle of the type of those adapted to rectal administration of such composition.

10. Composition according to Claim 1, characterized in that it contains, in addition to N-acetyl-muramyl-L-alanyl-D-glutamic acid, liquefied gases of the "propellent" type, in which its active principles are dissolved or maintained in suspension, and the expansion of which causes the dispersion of an aerosol.

11. Composition according to Claim 1, characterized in that it contains, in addition to N-acetyl-muramyl-L-alanyl-D-glutamic acid, a vehicle of the type of those adapted for vaginal administration of such a composition, more especially those consisting in gelatinous excipients.

12. Composition according to Claim 1, characterized in that it contains, in addition to N-acetyl-muramyl-L-alanyl-D-glutamic acid, an aqueous isotonic solution adapted for nasal, ocular or vaginal local administration.

13. Composition according to Claim 8, 9 or 10, wherein there is added a vaccinating agent.

14. Composition according to Claim 11 or 12, wherein there is added a vaccinating agent.