WO1993025236A1 - Humoral immunity and cell mediation adjuvant composition inducing no response to auto-antigenic determinants - Google Patents

Abstract

Process for the preparation of a pharmaceutic composition having a selective immunity adjuvant action bringing into play a humoral response or a cellular mediation response, preferably both at once, directed against given antigens, in the absence of any induction of auto-immune disease linked to a response to auto-antigenic determinants. The adjuvant principle of said pharmaceutic composition consists of MDP-DD-GDP of formula (I).

Description

ADJUVANT COMPOSITION OF HUMOR AND CELL MEDIATION IMMUNITY WITHOUT RESULTING IN RESPECT OF SELF-ANTIGENIC DETERMINANTS

Freund's Complete Adjuvant (ACF) is the strongest known immuno-adjuvant capable of increasing specific humoral and cell-mediated immune responses to an antigen. It is composed of mycobacteria administered in a water in mineral oil emulsion, the use of this adjuvant in human or veterinary clinic is excluded, because it induces significant side reactions, in particular induction of polyarthritis and sensitization to tuberculin. It is already known that we can reproduce the adjuvant effects without the side effects of mycobacteria by replacing them with a small molecule of 500 daltons, N-acetyl-muramyl-L-alanyl-D-isoglutamine, often abbreviated as MurNac- L-Ala-D-isoGln, also known as "Muramyl-dipeptide" or "MDP". This molecule consists of a structural element of the mycobacterial wall. Many analogs of MDP also exhibit adjuvant activities like MDP, they can be produced by chemical synthesis. These analogues, often called "Muramyl-peptides" are generally derived from MDP by one or more of the following modifications:

- replacement of the L-alanyl residue of the peptide chain by another L-aminoacyl residue or by the glycyl residue, substitutions on at least one of the carboxyl groups of the D-glutamyl residue, substitutions at positions 1, 4 and 6 of the muramyl group. These substitutions are sometimes accompanied by modulation of the adjuvant properties of MDP. For example, the lipophilic derivatives of the Muramyl peptide are often stronger adjuvants than the hydrophilic derivatives.

Other types of modifications to the MDP molecule can, however, alter the spectrum of its biological properties to a greater extent. For example — and with the notable exception of N-acetyl-muramyl-L- (ethyl) alanyl-D-isoglutamine— substitutions of the L-alanyl residue of the peptide chain are most often accompanied by the loss of adjuvant properties of the corresponding modified muramyl peptide. The replacement in MDP of the L-alanyl residue by a D-alanyl residue to obtain "MDP-DD" even results in the appearance of contrary properties. This molecule is then endowed with anti-adjuvant or immuno-suppressive properties.

But to return to muramyl peptides endowed with adjuvant properties, it is also known that their administration with an antigen in a water-in-mineral oil emulsion makes it possible to obtain very strong specific humoral and cell-mediated responses. This activity is especially sought after in the case of certain prophylactic or therapeutic vaccines intended to fight against especially serious diseases. Among these diseases, mention may be made of viral diseases such as AIDS, and various types of cancer, the specific antigens of which can be determined for the tumor. In all the cases cited, it is necessary to induce high levels of humoral and also cell-mediated response. These response levels could be obtained by using Freund's Complete Adjuvant or rather lipophilic derivatives of MDP, in water-in-oil emulsion in order to avoid the side effects due to the adjuvant. complete by Freund. But here is added a risk which is linked to the composition of the vaccine preparations. Indeed, these can contain auto-antigens, that is to say structures capable of causing an autoimmune disease. Thus: a) the AIDS virus used in certain vaccines has been cultured on human cells or cell lines derived from human cells. Even after purification there is a potential risk of the presence of autoantigens in the vaccine preparation. ) certain "cancer vaccines", which are already the subject of clinical trials, contain structures comprising both tumor-specific antigenic determinants and antigenic determinants of the host's natural tissue.

However, it is in the case of these particularly serious pathologies that the clinician would like to be able to induce strong humoral and cell-mediated responses in the host, but against the only specific antigenic determinants of pathogens are the cause (case of AIDS) or tumor antigenic determinants. Such responses are specially sought in the case of certain prophylactic or therapeutic vaccines intended to combat especially serious diseases, including viral diseases such as AIDS, and different types of cancer, when it is possible to identify the specific antigens of the tumor. But in these examples we see that if we induce a very strong immune response vis-à-vis structures capable of evoking a protective response, we risk also inducing an autoimmune reaction linked to a reaction towards -vis host structures present in the administered composition containing the vaccine. This is what we actually observe in experimental models where autoantigens are administered with an adjuvant of the "Freund Complete Adjuvant" type, or an incomplete Freund adjuvant (AIF) containing MurNac-L-Ala-D-isoGln 1,2-dipalmitoyl-sn-glycerol or " MDP-GDP ".

This is why it is quite unexpected that a molecule of the family of lipophilic Muramyl-peptides has been shown to have unique properties. Indeed N-acetyl-muramyl-D-alanyl- D- (7- [Sn dipalmytoyl] glycérόl) isoglutamine (Mur- Nac-D-Ala-DIsoGln 1,2-dipalmrtoyl-sn-glycerol or MDP- DD-GDP ) is as strongly adjuvant as the other lipophilic Muramyl-peptides, in particular when it is administered in the form of a water-in-oil dispersion and when its action is exerted against antigenic determinants foreign to the host. On the other hand, even when administered in a water-in-oil emulsion, it does not induce autoimmune disease linked to a response towards autoantigenic determinants.

MDP-DD-GDP, of formula:

est une molécule en soi connue. Il est fait plus spécifiquement référence au brevet européen n°85.400921.4/0165123 dans lequel est décrit un procédé pour sa préparation. Ce brevet était plus particulièrement relatif à une classe de molécules, parmi lesquelles le MDP-DD-GDP, choisies en raison de leur action d'activation des macrophages.

is a molecule known per se. More specifically, reference is made to European Patent No. 85.400921.4 / 0165123 in which a process for its preparation is described. This patent related more particularly to a class of molecules, among which MDP-DD-GDP, chosen because of their macrophage activation action.

The invention therefore relates more particularly to a pharmaceutical composition using MDP-DD-GDP for its application as an adjuvant capable of inducing a selective response vis-à-vis antigens foreign to the organism of the host (heteroantigens) in the absence of any induction of autoimmune disease linked to a response to autoantigenic determinants. Although this action may manifest itself during administration in the form of a single-phase aqueous solution, it is preferable to use it in the form of a two-phase, injectable dispersion, based on an aqueous solution. and a pharmaceutically acceptable hydrophobic component. This constituent is often lipid in nature. But more generally the pharmaceutically acceptable hydrophobic constituent intervening or having to intervene in the constitution of the above-mentioned two-phase dispersion is chosen in such a way that its presence within the dispersion containing the MDP-DD-GDP induces a reinforcement of the humoral response or of the cell-mediated response, or preferably both, in the recipient host vis-à-vis the vaccinating antigen administered to the host in the presence of MDP-DD-GDP.

This hydrophobic constituent will most often consist of a mineral oil, for example paraffin oil, or vegetable oil. Water-in-vegetable oil emulsions which can be used for the constitution of adjuvant compositions are described, for example in French patent n ° 7504003. The hydrophobic compound can also be the basis of lipids capable of forming liposomes. Reference is again made to European patent No. 0165123 already identified above, with regard to the constitution of such liposomes. It can also be formed from a hydrophobic detergent. The hydrophobic compound can also be based on polyoxylene-polyoxypropylene polymers, such as those known under the trademark PLURONIC and / or polyoxyethylene esters of sorbitan, in particular those marketed under the trademark TWEEN 20. The hydrophobic compound can also be formed by microspheres, having sizes normally less than 7 microns.

The invention is of particular interest when it is applied to the induction of a vaccinating and more generally immunogenic response with regard to an antigen which is not devoid of antigenic determinants characteristic of the self or capable of crossing immunologically with these antigenic determinants. These can either belong to the vaccinating antigen, or be associated with it under conditions which prevent a complete prior purification of the vaccinating antigen. As we have seen, this situation can arise each time that the vaccinating antigen has been produced, for example in human or animal cells (for example inactivated viruses or fractions of virus cultivated in these cells).

The invention finds a particularly advantageous application in the constitution - or for the production - of vaccinating pharmaceutical compositions containing an inactivated HIV virus, or an anti-tumor antigen or also certain parasitic antigens capable of giving rise to cross-reactions with certain host structures.

It goes without saying that whenever a preparation of the vaccinating antigen involves a risk of the presence of an auto-antigenic determinant or crossing with the determinants of the self at the end of this preparation, it should be considered as "vaccinating principle not completely devoid of auto-antigenic constituents ".

In particular, the invention finds an advantageous application in combination with any virus or fraction of virus obtained by culture in human or mammalian cells, or also with any antigen whose conditions of production introduce a risk of contamination by structural elements. belonging to human or mammalian cells, in particular cells of the white line.

It is understood that the pharmaceutical composition as it has just been defined can be prepared extemporaneously. Furthermore, if the antigen is often administered at the same time as the other constituents of the pharmaceutical composition, the invention cannot be limited to this specific case. The administration of MDP-DD-GDP in the form of a two-phase dispersion on the one hand, and that of the vaccinating antigen on the other hand, can also be done in a non-simultaneous manner, the only condition being that the time intervals which flow between these separate administrations are such that the munological adjuvant effect of the preparation containing MDP-DD-GDP against the vaccinating antigen can be manifested.

In other words, the term "pharmaceutical composition" as used above covers both compositions formed from the various named constituents and found in a premixing stage, that all of these different constituents taken in isolation but combined at the time (s) of their respective administration (s) to patients (humans or animals) under conditions allowing obtaining the above-mentioned results.

The relative proportions in the emulsion of the aqueous solution and of the hydrophobic component are chosen such that an emulsion which is sufficiently stable, in particular at the time of administration, is capable of being formed between the aqueous solution and the hydrophobic component. In particular, when the hydrophobic constituent consists of a mineral oil, the proportion of the latter will preferably be from 50% to 90% (volume / volume) relative to the entire mixture.

It goes without saying that these intervals are given only as examples. They are in no way limiting.

The invention will be further illustrated by the following examples. As we can see, the tests used are very severe, since the animals receiving the preparation capable of inducing autoimmunization are paralyzed and die.

Example 1:

Absence of induction of an autoimmune disease (allergic encephalomyelitis) in guinea pigs after administration of the basic myelin protein with MDP-DD-GDP in water-in-oil emulsion.

The basic protein of myelin is an auto¬ antigen known for its capacity to induce an immunological reaction vis-à-vis the myelin present in the spinal cord and the brain. The animals are Hartley male guinea pigs. They receive in the pads of the hind legs, 50 μg of protein basic. The negative controls receive the antigen in an emulsion of Freund's Incomplete Adjuvant (oil only) and the positive controls in an emulsion of Complete Adjuvant (oil + mycobacteria). The experimental batches receive the incomplete adjuvant added with 50 μg MDP-GDP (Mur-Nac-L-Ala-D-isoGln- GDP) or MDP-Thr-GDP (Mur-Nac-L-Thr-D-isoGln- GDP) or MDP-DD-GDP.

The animals are weighed weekly, clinical signs appear after 10 days and the animals die between the 12th and 19th days. After 3 weeks, cuti-reactions are practiced on healthy animals and on animals that have survived the disease. Blood is also taken by cardiac puncture to test for antibodies by ELISA.

Table 1 gives indications on the number of animals of each group which was affected by clinical signs characteristic of allergic encephalomyelitis: weight loss, paralysis of the hind legs then generalized, incontinence. the number of dead animals per group, the anti-myelin basic protein antibody titers, and the presence of a positive cuti-reaction (when tested) in the survivors. NT stands for "untested".

It can be seen from table 1 that only the negative control batch and the batch which received MDP-DD-GDP as an adjuvant are immune to the disease. However, the animals which received MDP-DD-GDP synthesized antibodies and had positive skin reactions, when a cuti-reaction was carried out with the basic protein.

On table 2, we see the evolution of the weight. The average of the weights is given for each batch.

TABLE 2

Week Week Week Week

Immunization 1 2 3 4

Basic protein + AIF 510 537 620 695

Basic protein + ACF 500 560 425 450

Basic protein + AIF + MDP-GDP 480 550 546 480

Basic protein + AIF + MDP-L-Thr-GDP 480 549 466 *

Basic protein + AIF + MDP-DD-GDP 490 568 638 685

: 2 survivors: 3 survivors It can be seen from the examination of this table that the injection of the basic protein is not accompanied by a loss of weight, on the contrary, of the animals treated, when these have not received in addition to the protein basic, that the AIF, or both the AIF and the MDP-DD-GDP, while there is a loss of weight (insofar as the animals survive) in those of animals which have received in addition to the basic protein, either from ACF, or from AIF and MDP-GDP, or again from AIF and MDP-Thr-GDP.

Example 2;

Absence of toxic synergy between MDP-DD-GDP and endotoxins;

25 g female Swiss mice receive physiological water via the venous route, either alone (negative control) or containing lipophilic derivatives of MDP at a dose of 300 μg. Two hours later they still receive 25 μg of endotoxin (lipopolysaccharide from S. Enderitidis) by vein. It is seen from Table 3 that MDP-GDP sensitized mice to the administration of endotoxin unlike MDP-DD-GDP and MDP-Thr-GDP.

Exemple 3:

Example 3:

The lipophilic derivatives were tested (according to the method described in the European Pharmacopoeia) for their pyrogenic activity and the MDP-DD-GDP was found to be 10 times less pyrogenic than the MDP-GDP.

It goes without saying that MDP-DD-GDP can be replaced by analogous structures in which the first amino acid of the peptide chain has a D configuration (in other words a muramyl peptide type DD, preferably a product of this type having a chain lipophilic). Thus form part of the invention all the pharmaceutical compositions which have been defined above, but in which the MDP-DD-GDP would be replaced by another adjuvant muramyl-peptide of the DD type, since in experimental protocols making the object of the tests which have been illustrated in examples 1 and 2, this other muramyl-peptide would induce results similar to those obtained with MDP-DD-GDP in test animals, namely absence of induction of a allergic encephalomyelitis in guinea pigs after administration of the basic protein myelin and this other muramyl-peptide type DD, and absence of toxic synergy in guinea pigs between this latter product and bacterial endotoxins, in particular of S. enteritidiε.

Among the muramyl-peptides of type DD capable of being used in the pharmaceutical compositions according to the invention, mention will be made of those which would not induce autoimmune manifestations in the above-mentioned experimental protocols and which would correspond to the following general formula :

dans laquelle les substituants R_1# R₂, X, Y et B ont les significations suivantes :

in which the substituents R _{1 #} R ₂ , X, Y and B have the following meanings:

R ₁ : is H or CH3.

R ₂ : is a hydrogen atom, an NH2 group, or a group W consisting of a hydrocarbon chain comprising from 1 to 4 carbon atoms.

X: is an aminoacyl residue of configuration D comprising: alanyl, valyl, isoleucyl, leucyl, seryl, threonyl, prolyl, methionyl.

Y: has the same meaning as R ₂ or is a hydrocarbon chain of linear or branched structure comprising from 4 to 90 carbon atoms, or also a group -CH ₂ -CHO (R ₃ ) -CH20 (R ₄ ), with R ₃ and R ^ being hydrocarbon chains, identical or different, comprising from 8 to 20 carbon atoms.

A, B and C: are, with regard to each of them, independently of each other, either a direct bond, or an oxygen atom, or a structure -NH-CHCR ^ -CO-, corresponding to all aminoacyl of configuration L, with R5 being an aminoacyl residue characteristic of one of the 20 natural amino acids. Z: is a hydrogen atom or else is a hydrocarbon acyl group of linear or branched structure comprising from 1 to 90 carbon atoms, or else a CO-CHO- (R ₃ ) -CH ₂ 0 (R ₄ ) group, with R ₃ and R ₄ being hydrocarbon chains, identical or different, comprising from 8 to 20 carbon atoms.

As examples of compounds which can be used advantageously, mention will be made of: MurNac-D-Thr-D-IsoGln 1,2-dipalmitoyl-sn-glycerol

For methods of preparing these products, reference may be made, for example, to European patent No. 0165123 or to European patent No. 0004512.

As a variant, the invention relates to a process for the preparation of a pharmaceutical composition capable of preventing the induction of an autoimmune disease linked to a response towards autoantigenic determinants, when this composition contains a principle active capable of exerting a selective adjuvant action of the immunity implementing a humoral response or a response mediated by cell, preferably both at the same time, against determined antigens, characterized in that the adjuvant principle of this pharmaceutical composition consists of a muramyl-peptide of formula I or II.

The invention therefore also relates to compositions of the genus in question, containing an adjuvant active principle which is not free from auto-antigenic determinants, associated with a muramyl-peptide of formula I or of general formula II.

Under the influence of these particular adjuvants, there is no induction of an autoimmune response even if the vaccine preparation contains autoantigens. In other words the MDP-DD-GDP or the compounds of general formula II appear adjuvant-active vis-à-vis hetero-antigens and adjuvant-inactive (or passive) vis-à-vis auto¬ antigens.

Therefore the pharmaceutical composition which is the subject of the patent makes it possible not only to obtain an immune response directed only against antigens distinct from the self but to prevent the triggering of an autoimmune disease linked to the autoantigenic determinants (or elements of the self) possibly present in the vaccinating pharmaceutical composition.

By "composition according to the invention" is meant both a composition containing simultaneously or in a mixture the heterogeneous against which protection is sought, if necessary not devoid of auto-antigens, on the one hand, and the compound of formula I or of general formula II, on the other hand, that any form of association intended to allow the administrations of the two principles at distinct times, the time interval separating these distinct moments being however sufficiently short so that these two principles are still able to interact with each other in vivo as described.

Claims

1. Process for the preparation of a pharmaceutical composition exerting a selective adjuvant action of immunity using a humoral response or a cell-mediated response, preferably both at the same time, against specific antigens, in particular the absence of any induction of autoimmune disease linked to a response towards autoantigenic determinants, characterized in that the adjuvant principle of this pharmaceutical composition consists of a muramyl-peptide of type DD, of formula :

in which the substituents R _{1 f} R ₂ , X, Y and B have the following meanings: _n : is H or CH3. R ₂ : is a hydrogen atom, an NH2 group, or a group consisting of a hydrocarbon chain comprising from 1 to 4 carbon atoms. X: is an aminoacyl residue of configuration D comprising: alanyl, valyl, isoleucyl, leucyl, seryl, threonyl, prolyl, methionyl. Y: has the same meaning as R ₂ or is a hydrocarbon chain of linear or branched structure comprising from 4 to 90 carbon atoms, or else a group -CH ₂ -CHO (R ₃ ) -CH20 (R, with R ₃ and R ₄ being identical or different hydrocarbon chains comprising from 8 to 20 carbon atoms. A, B and C: are, with regard to each of them, independently of each other, either a direct bond, or an oxygen atom, or a structure -NH-CH (R ₅ ) -CO-, corresponding to any aminoacyl of configuration L, with R5 being an aminoacyl residue characteristic of one of the 20 natural amino acids. Z: is a hydrogen atom or else is a hydrocarbon acyl group of linear or branched structure comprising from 1 to 90 carbon atoms, or else a CO-CHO- (R ₃ ) -CH ₂ 0 (R ₄ ) group, with R ₃ and R ₄ being hydrocarbon chains, identical or different, comprising from 8 to 20 carbon atoms. 2. Method according to claim 1, characterized in that the adjuvant principle consists of MDP-DD-GDP. 3. Process for the preparation of a pharmaceutical composition capable of preventing the induction of an autoimmune disease linked to a response towards auto-antigenic determinants, when this composition contains an active principle capable of exerting selective adjuvant action of immunity using a humoral response or a cell-mediated response, preferably both at the same time, against specific antigens, this principle active being, where appropriate, not devoid of elements capable of immunologically crossing with elements of the self of the host for which this composition is intended, this method being characterized in that the adjuvant principle of this pharmaceutical composition consists of a muramyl peptide as defined in claim 1 or 2. 4. Method according to any one of claims 1 to 3, applied to the preparation of a vaccine composition against a pathogenic agent to which the above antigens correspond, this composition then containing these antigens. 5. Method according to any one of claims 1 to, for the production of vaccine compositions against a pathogenic agent not devoid of antigenic determinants characteristic of the self or of antigenic determinants capable of crossing immunologically with structures of the self. 6. Method according to claim 5, characterized in that the antigen is a virus or a fraction of virus obtained by culture in human or mammalian cells. 7. Method according to claim 5, characterized in that the antigen is an antigen whose conditions of production introduce a risk of contamination by structural elements belonging to human or mammalian cells, in particular cells of the line white. 8. Method according to any one of claims 1 to 7, characterized in that the antigen is an attenuated HIV virus or a vaccine antigen of this virus. 9. Method according to any one of claims 1 to 7, characterized in that the antigen is an antitumor antigen. 10. Method according to any one of claims 1 to 7, characterized in that the antigen is a parasite antigen capable of giving rise to cross-immunological reactions with certain structures of the host. 11. Pharmaceutical composition using a muramyl-peptide of type DD, within a two-phase, injectable dispersion, based on an aqueous solution and a pharmaceutically acceptable hydrophobic constituent, this muramylpeptide of type DD having the formula:

R, C3-X- »H-Ç rH-CD-Λ-1? _S .H l . CH _z -co - & - γ in which the substituents R ,, R ₂ , X, Y and B have the following meanings: R,: is H or CH3. R ₂ : is a hydrogen atom, an NH2 group, or a group W consisting of a hydrocarbon chain comprising from 1 to 4 carbon atoms. X: is an aminoacyl residue of configuration D comprising: alanyl, valyl, isoleucyl, leucyl, seryl, threonyl, prolyl, methionyl. Y: has the same meaning as R ₂ or is a hydrocarbon chain of linear or branched structure comprising from 4 to 90 carbon atoms, or also a group -CH ₂ -CHO (R ₃ ) -CH20 (R ₄ ), with R ₃ and, R ₄ being hydrocarbon chains, identical or different, comprising from 8 to 20 carbon atoms. , B and C: are, with regard to each of them, independently of each other, either a direct bond, either an oxygen atom, or a structure -NH-CH (R ₅ ) -CO-, corresponding to any aminoacyl of configuration L, with R5 being an aminoacyl residue characteristic of one of the 20 natural amino acids. Z: is a hydrogen atom or else is a hydrocarbon acyl group of linear or branched structure comprising from 1 to 90 carbon atoms, or else a CO-CHO- (R ₃ ) -CH ₂ 0 (R_,) group, with R ₃ and R ₄ being hydrocarbon chains, identical or different, comprising from 8 to 20 carbon atoms. 12. Composition according to claim 11, characterized in that the muramyl-peptide of type DD, is MDP-DD-GDP. 13. Composition according to claim 11 or 12, characterized in that the muramyl-peptide of type DD is there in association with a vaccinating antigen. 14. Composition according to any one of claims 11 to 13, characterized in that the hydrophobic constituent consists of a mineral oil, for example paraffin oil, or vegetable oil. 15. Composition according to any one of claims 11 to 13, characterized in that the hydrophobic constituent is based on lipids capable of forming liposomes. 16. Composition according to any one of claims 11 to 13, characterized in that the hydrophobic compound is based on polyoxylene-polyoxypropylene polymers, such as those known under the trademark PLURONIC and / or polyoxyethylene esters of sorbitan, in particular of those marketed under the TWEEN 20 brand. 17. Composition according to claim 13, if appropriate in combination with any one of claims 14 to 16, characterized in that the antigen is an antigen not devoid of antigenic determinants characteristic of itself or an antigen capable of crossing immunologically with these antigenic determinants of the self. 18. Composition according to any one of claims 11 to 16, characterized in that the pharmaceutically acceptable hydrophobic constituent is chosen such that its presence within the dispersion containing the muramyl-peptide of type DD induces in the recipient host a strengthening of the humoral response or of the cell-mediated response, or preferably both. 19. Composition according to any one of claims 11 to 16, characterized in that the antigen is an attenuated HIV virus or a vaccine antigen of this virus. 20. Composition according to any one of claims 11 to 16, characterized in that the antigen is an antitumor antigen. 21. Composition according to any one of claims 11 to 16, characterized in that the antigen is a parasite antigen capable of giving rise to crossed immunological reactions with certain structures of the host. 22. Composition according to any one of claims 11 to 16, characterized in that the antigen is a virus or a fraction of virus obtained by culture in human or mammalian cells. 23. Composition according to any one of claims 11 to 16, characterized in that the antigen is an antigen whose conditions of production introduce a risk of contamination by structural elements belonging to human or mammalian cells, in particularly cells of the white line.