EP0741573A1

EP0741573A1 - Use of diesters of muramyl peptides in oral form as immunostimulating agents

Info

Publication number: EP0741573A1
Application number: EP95907694A
Authority: EP
Inventors: Louis Chedid; Françoise Audibert; Pierre Lefrancier
Original assignee: Vacsyn SA
Current assignee: Vacsyn SA
Priority date: 1994-01-25
Filing date: 1995-01-24
Publication date: 1996-11-13
Also published as: AU1580995A; WO1995019777A2; WO1995019777A3; CA2181899A1; JPH09509409A; FR2715305B1; FR2715305A1

Abstract

The invention relates to an externally active immunostimulating pharmaceutical composition characterized in that it contains a diester having the general formula (I) wherein R is CH3, X is L-Ala or L-Thr, R1 is a hydrocarbonaceous group having from 1 to 4 carbon atoms, R2 is a hydrocarbonaceous group having 1 or 2 carbon atoms, in an externally administered formulation compatible with an administration of active principle comprised between 0.1 mg and 5 mg par kg of weight to human beings or animals.

Description

DIESTERS OF MURAMYL PEPTIDES IN AN ORAL FORM

The present invention relates to an immunostimulatory pharmaceutical composition, active by external administration, in particular by oral, nasal, transcutaneous, aerosol route; immunoεtimulant this effect can be exploited either to an activity of ¹ immunoadjuvant in combination with a vaccine, either prophylactic or therapeutic treatment of an infection optionally in combination with other suitable treatment type ¹ infection.

Mura yl peptides and their ester derivatives are a family of molecules which have been described in numerous publications and numerous patents since the first description of N-acetyl-Muramyl-L-Alanyl-Disoglutamine (MDP for muramyl dipeptide).

Briefly, they are capable of exercising a wide variety of biological activities including - immuno-adjuvant activity, anti-infectious activity, anti-tumor activity, anti-allergic activity and regulation of cytoin secretion. However, a certain number of these muramyl peptides, while they may exhibit quite promising biological activities, often have side effects or drawbacks which prevent their use as a medicament, in particular toxic effects or difficulty in formulation. due to their low solubility in aqueous solutions.

They also exhibit great variability in their spectrum of action.

The only immunoadjuvant used in human clinics is currently aluminum hydroxide, but this compound is only active systemically, i.e. it must be injected parenterally and is not active after oral or transcutaneous administration. In addition, it produces undesirable effects such as the induction of antibodies of the IgE type which create a risk of allergy. In addition, it primarily produces a humoral response and does not promote the production of secretory immunoglobulins.

The search, therefore, for an immunosti ulant which has at the same time advantages of effectiveness, formulation, and toxicity remains a current problem which had never been solved by any of the derivatives. CDM; in particular, an active immunomodulator after administration by the external route, in particular by the oral or percutaneous route or aerosol, can make it possible to envisage a wider and more flexible use of this immunostimulant.

The present invention has made it possible to select from the many derivatives and analogues of MDP diesters of general formula I:

in which :

- R is CH3,

- X is L-Ala or L-Thr,

- RI is a hydrocarbon group having from 1 to 4 carbon atoms,

- R2 is a hydrocarbon group having 1 to 2 carbon atoms.

A particular compound corresponding to this formula is the muradimetide of formula II:

Nac-Mur-L-Ala-D-Gl [OMe] -OMe which muradi etide is soluble in water, has a low toxicity and can thus be incorporated as active principle in an immunostimulatory pharmaceutical composition which is active externally, in particular by oral, percutaneous or aerosol route, and this in a formulation compatible with an administration of active ingredient between 0.1 mg and 5 mg per kg of weight.

Muradimetide is a synthetic molecule, a synthesis of which has been described in French patent FR760680.

The invention therefore consists, among all of the derivatives of muramyl dipeptide, in selecting a compound corresponding to the above formula from derivatives which had already been described previously, said derivatives having both remarkable immunostimulatory qualities which make it possible to use them. as vaccine adjuvants, as an antibacterial or antiviral substance, and whose low toxicity also allows their administration by the external route, in particular oral, nasal or aerosol or percutaneous and not having the side effects observed for CDP or other derivatives, in particular mono-esters.

The combination of all these characteristics makes it possible to select the derivatives of general formula

I and, more particularly, the muradimetide of formula

II as active principle of an immunostimulatory pharmaceutical composition which can be administered externally, in particular by oral, percutaneous or aerosol route, active in a formulation compatible with administration of between 0.1 mg and 5 mg / kg of weight in humans or the animal. The preferred doses are from 0.5 mg to 3 mg / kg.

An advantageous property of the composition according to the invention is that it is an immunoadjuvant usable as an accompaniment to an effect induced by vaccination. More particularly, the pharmaceutical composition used as an adjuvant can be administered before, during or after a vaccination regardless of the mode of administration of the latter and by this immunoadjuvant effect lead to increased synthesis of protective antibodies and allow, the if necessary, the saving of one or two booster calls necessary in the context of conventional vaccination. The adjuvant activity is also obtained if the first administration of the molecule is carried out at the time of the recall. In all cases, both an increase in the antibodies and a change in the isotypic profile are obtained in favor of secretory immunity.

The immunostimulatory pharmaceutical composition of the invention can also be used to prevent or treat viral, bacterial, parasitic or other infections.

Muradimetide in particular has the quite remarkable capacity among all of the derivatives of MDP to be active in the protection of the subjects treated against an infection when it is administered externally after said infection, a particularly interesting example of external route being the oral route (per os).

The diesters of formula 1 can be used for the manufacture of an immunostimulating pharmaceutical composition which can be administered externally, and if the oral route is particularly advantageous, other routes such as the mucosa route, the percutaneous route, or the aerosol can also be used.

Another quite remarkable feature of muradimetide and the other compounds included in the formula I above in the pharmaceutical composition of the invention is their capacity to exhibit a synergy in the protective effect with regard to an infection when it is administered jointly or in parallel with another antiviral or anti¬ infectious compound . For example, when the composition of the invention is used in combination with AZT in the treatment of HIV infections, protection is observed when AZT is administered at a suboptimal dose of 0.01 M AZT, a dose which does not confer no protection against infection when administered alone, this use may be simultaneous, separate or spread over time.

The following examples will show in a nonlimiting and more demonstrative way, how the combination of the qualities of the muradimetide, chosen by way of example, in the compounds of formula I make it a candidate of choice in the manufacture of an immunostimulatory composition which can be administered by the external, and especially orally.

In these examples, the figures have the following meanings:

FIG. 1 represents the adjuvant activity of muradimetide (MDM) administered orally on the antibody response of the mouse to a hepatitis B vaccine. Two groups of 8 female Swiss mice aged 7 to 8 weeks receive subcutaneously on day 1 and on day 30, 1 μg of antigens (HBS Ag) absorbed on aluminum (135 μg). An experimental group simultaneously receives 1 mg of muradimetide orally.

FIG. 2 represents the protective activity of muradimetide against infection by the EMC virus, that is to say the survival rate at 60 days expressed as a percentage of Swiss mice treated with muradimetide systemically after infection with EMCV.

- Figure 3 represents the dose-response activity of muradimetide administered in the protection of mice against EMCV infection.

- Figure 4 shows the effect of MDP or muradimetide (MDM) in combination or not with antiretroviral treatment with AZT on the production of HIV antigens in cultures of U937 cells infected with the HIV virus.

- EXAMPLE I: General pharmacology:

1) Study of pyrogenicity:

Table 1 is a comparison of the pyrogenic effect in rabbits of muradimetide and of MDP administered by the venous route (i.v.). The pyrogenicity tests were carried out following the rules of the European Pharmacopoeia.

These data show that muradimetide is not pyrogenic when administered to rabbits at a dose of 3 mg / kg, while the minimum pyrogenic dose of MDP is close to 0.33 mg / kg. Indeed, when 6 rabbits per group are used, the pharmacopoeia says a pyrogen-free product a) at the dose administered when the sum of the maximum temperature rises observed in these rabbits during the duration of the test is less than 3 '. b) pyrogenic when this sum is greater than 4 ° and doubtful therefore that the test must be repeated on a larger number of animals when the sum is greater than 3 ^e . Table 1

Muradimetide MDP

Dosages Delta T Rabbit Delta T Rabbit n ° n °

0.33mg / kg 1 0.10 1 0.10 2 0.30 2 0.20 3 0.40 3 0.30 4 0.20 4 1.20 5 0.05 5 0.60 6 0.00 6 0.65

1 ° .05 3 ° .05

1 mg / kg 1 0.75 1 0.60 2 0.50 2 1.20 3 0.80 3 0.50 4 0.40 4 0.70 5 0.10 5 0.40 6 0.10 6 0 , 80

2 ° .65 4 ° .20

3 mg / kg 1 1.10 1 1.50 2 0.55 2 0.30 3 0.20 3 0.45 4 0.15 4 0.50 5 0.70 5 0.80 6 0.20 6 0 , 50

2 ° .90 4 ° .05

2) Measurement of leukopenia in rabbits: Most derivatives and analogues of MDP induce leukopenia when administered to rabbits. On the contrary, muradimetide does not modify the blood count as indicated in table 2 below: Table 2

Leukocyte treatment / mm3

100 μg / kg / i.v. T.O T + 3 hours

Muradimetide 7850 7700 CDM 7600 2000

3) Absence of slow sleep induction: Muradimetide and MDP were administered to rabbits either I.V. or by the intracerebroventricular route (I.C.V.) or orally (P.O.). The minimum somnogenic doses capable of inducing REM sleep are shown in the following table. These results show that the minimum somnogenic dose of muradimetide is 10 times higher than that of MDP parenterally and that it is not active by the oral route.

Table 3

Absence of somnogenic activity of orally administered muradimetide

Administration Minimum somnogenic doses / kg

Muradimetide MDP

IV 1 mg 100 μg

I.C.V. 1 μg 0.025 μg

PO negative 10 mg 1 mg - EXAMPLE II: Adjuvant activity of muradimetide in oral administration:

Muradimetide is administered orally simultaneously with parenteral vaccination with an antigen.

1) Response to human serum albumin (BSA):

The adjuvant activity of muradimetide, that is to say its capacity to increase the antibody response to the antigen (BSA) has been compared with the activity of several other muramyl peptides after administration to mice either orally, either subcutaneously.

BSA is administered as an antigen, always by the subcutaneous route. The results are shown in the following table 4:

Table 4

-

Administration

Muramyl peptides Short name of adjuvant

Per os S.C.

Witnesses (without adjuvant) 30 30

Nac-Mur-L-Ala-D-iGln MDP 1100 700

Nac-Mur-L-Ser-D-iGln MDP serine 450 1300

Nac-Mur-N-Methyl-L-Ala-D-iGln 100 450

Nac-Mur-L-Ala-D-Gln-OMe Mura etide 100 500

Nac-Mur-L-Ala-D-Gln-OnBu Murabutide 100 700

Nac-Mur-L-Ala-D-Glu MDPA 200 1000

Nac-Mur-L-Ala-D-Glu * (OMe) OMe Muradimetide 1100 1350

Nac-Mur-L-Ala-D-Glu (OMe) OnBu 200 500

Nac-Mur-L-Ala-D-Glu-OMe 200 450

Nac-Mur-L-Ala-D-Glu-OnBu 80 550

* D-Glu can be substituted on the carboxyl functions in or 7. The substitutions made in ₇ are indicated in brackets. BSA is given subcutaneously at a rate of 0.5 mg on day 0 and on day 30. MDP and its derivatives or analogues are given only on day 0, either at a rate of 1 mg per os, or of 0, 1 mg mixed with the antigen subcutaneously.

Antibodies are measured by passive hemaglutination on day 36.

We see in this table that three of the most effective compounds are MDP, MDPA and muradimetide. Since serine MDP is highly pyrogenic, it cannot be used therapeutically.

The more detailed results comparing these three compounds are given in the following table 5:

Table 5

Comparison between the adjuvant activity of MDP and muradimetide given subcutaneously or orally

Mouse Antibody Response Adjuvant (Title Range)

Witnesses (without 150 30 (<3-400) adjuvant)

MDP S.C. 150 700 (50-3200) MDP P.O. 50 1100 (50-3200)

Muradimetide S.C. 32 1350 (100-3200) Muradimetide P.O. 16 1100 (50-3200)

MDPA S.C. 32 1000 (200-3200) MDPA P.O. 16 200 (12-800)

The experimental conditions are the same as those described in Table 4 above.

These data show that only two molecules, MDP and muradimetide, are so active significant when given orally simultaneously with the parenterally administered antigen.

Interestingly, MDPA, of which muradimetide is the methyl diester derivative, is active when administered systemically, but weakly active when administered orally indicating that the biological activity of muradimetide administered orally cannot be attributed to the formation of MDPA resulting from hydrolysis of its methyl ester groups.

Given the toxicity of MDP as it is known and recalled above, it is clear that, among all the derivatives and analogues of MDP, only muradimetide has the combination of the qualities of non-toxicity and of activity adjuvant when administered orally.

2) Response to a hepatitis B vaccine: _

The antigen used is a recombinant hepatitis B virus (HBS) surface antigen obtained by genetic recombination and administered, absorbed on aluminum hydroxide.

It was injected subcutaneously into Swiss mice on day 1 and day 21. Muradimetide was given orally at the same time as the vaccine.

The results obtained are shown in Figure 1; in this figure, Swiss mice (8 per group) receive subcutaneously 1 μg of HBS Ag (Pasteur Mérieux Sera and Vaccines) and muradimetide orally (1 mg). A reminder is given on the thirtieth day.

FIG. 1 clearly indicates that this oral administration has considerably increased the response to the vaccine and allows a much earlier production of antibodies against the vaccinating antigen. 3) Response to a synthetic peptide of Escherichia coli administered orally:

The peptide represents the copy of a vaccine structure of a surface antigen of Escherichia coli.

This antigen is known to be a potentially protective antigen in humans.

The peptide was encapsulated in poly (DL-lactide coglycolide) microspheres and given orally to female Swiss mice either alone, or with muradimetide administered either subcutaneously or orally. The antibody response in the serum was evaluated by ELISA test either against the peptide or against the natural structure of the protein.

Table 6 below represents the recognition of the natural protein by the antibodies obtained after oral administration of the synthetic peptide encapsulated in the microspheres.

Table 6

Antibody Response Groups

Peptide Protein synthé¬ natural tick

E. coli peptide P.O. 300 200

E. coli peptide P.O. 1100 1200

+ muradimetide S.C.

E. coli peptide + muradimetide P.O. 4000 2000

Swiss mice received 10 μg of the synthetic peptide orally on days 1 and 30. The peptide was incorporated into the microspheres and the muradimetide in saline was administered either by subcutaneous (100 μg), or orally at a rate of 1 mg per mouse.

These data show that muradimetide, particularly when given orally, has made it possible to obtain antibodies in the serum recognizing the synthetic antigen and its natural structure.

4) Muradimetide is administered orally with secondary immunization by an antigen administered parenterally:

The influence of oral administration of muramyl peptide at the time of a secondary injection of immunizing antigen was evaluated by the production of isotype of antibodies produced in serum and by the presence of secretory IgA (sought in the intestinal fluid).

A protective Shistosoma mansoni antigen (SMAg) was administered subcutaneously to Swiss mice (a group of 8) on day 1 and day 21; this antigen is absorbed on aluminum hydroxide.

For secondary immunization, mice receive 1 mg of muradimetide orally. The results are given in the following table:

Table 7

Influence of orally administered muradimetide at booster time on the humoral response to SMAg

These results demonstrate that the muramyl dipeptide administered during secondary immunization can increase and modulate the response to an antigen administered parenterally.

Administration of muradimetide increased the specific synthesis of secretory IgA. Surprisingly, it considerably reduced the production of IgE in the serum, confirming in this that the oral administration of muradimetide can modulate the isotypic profile of the antibody response.

4) Muradimetide and the antigen are administered orally; muradimetide is only administered during secondary immunization.

RESPONSE TO THE CHOLERIC TOXIN β CHAIN

The influence of muradimetide administered orally at the same time as secondary immunization has also been studied in the antibody response to an active immunogen by the oral route: the cholera toxin β chain (βCT). Experiments were carried out in a group of 8 female Swiss mice receiving each of them orally 10 μg of βC, two times three weeks apart.

Antibodies are measured by the ELISA technique in serum and in vaginal lavage on day 28.

The results are expressed in Table 8 below.

Table 8

Adjuvant activity of CDM associated with oral vaccine

ANTI-3CT ADJUVANT

In the In vaginal fluid serum

IgG IgA IgG IgA

Control (without adjuvant) 1800 900 20 5

Muradimetide p.o. 1 mg 4600 2700 35 85 at day 25

This experience shows that muradimetide administered orally at the same time as an immunization booster with an oral vaccine can exercise its adjuvant power both at the level of systemic immunity and at the level of mucosal immunity measured by amount of secretory IgA. EXAMPLE III Anti-Infectious Activity of Muradimetide Administered Orally:

1) Protection against bacteria: the: Infection with a mycobacterium

Many strains of mycobacteria, in particular among mycobacteria tuberculosis, are resistant to antibiotics. Tuberculosis has once again become a major health problem and of course particularly in immunosuppressed individuals. A drug administered orally and increasing the defenses against this type of disease is therefore of considerable importance not only in the field of tuberculosis but also in that of opportunistic infections linked to many strains of mycobacteria.

Muradimetide has been shown to protect mice from Mycobacterium Fortuitum infection. This effect is obtained in particular by animals treated with AZT which has the known side effects of causing immunosuppression and consequently of increasing the sensitivity to bacterial or viral aggressions.

One model used is Mycobacterium Fortuitum infection in mice. The animals are 8 week old female C57B1 / 6 mice. They are divided into 3 groups of 8 animals: a) a group of controls receives AZT per os for 3 weeks at a rate of 3 mg per mouse, three times per week, b) a second group of controls is not treated, c) the experimental group receives Muradimetide at a dose of 1 mg at the same time as AZT.

The animals of the three groups are tested the day after the last administration of AZT alone or in combination. They receive an injection by vein of 2 x 10 ⁷ infecting units of M. Fortuitum. The animals are followed for 20 days.

The cumulative results of two experiments carried out under the same experimental conditions are given in Table 9 below:

Table 9

Survival to the day

1 5 10 15 20

Untreated witness 16 4 1 0 0

Witness - AZT 16 6 2 2 2

AZT + Muradimetide group 16 16 14 14 14

It is clear that Muradimetide has demonstrated very strong anti-mycobacterial activity.

lb: Infection with Klebsiella pneumoniae Muradimetide is administered either parenterally or orally before or after a lethal challenge with Klebsiella pneumoniae. This administration can, as shown in Table 10 below, protect the treated mice from death; this protection can be obtained both in adults and in newborns.

Table 10

Antibacterial activity of muradimetide administered orally and systemically compared to the parent molecule MDPA

Treatment% protection (number of animals)

Day 1 (before challenge)

53 (16)

2 mg p.o. 50 (24) 100 μg ε.c * 75 (37)

56 (24)

2 mg p.o. 25 (16)

Hour + 1 (after challenge)

69 (16) 6.9 (16)

The animals are 6/7 week old Swiss female mice, except for the group marked with an asterisk which consists of newborn mice.

These results demonstrate that muradimetide is endowed with non-specific immunostimulatory activity.

Surprisingly, it shows itself as one of the derivatives or analogues of MDP which is particularly effective, when it is administered orally and also both before and after the challenge with a lethal dose of bacteria.

On the contrary, its parent molecule, MDPA, is also active as muradimetide when administered systemically, but, on the other hand, does not show significant activity when it is administered orally (only 25% protection, compared to 50% for muradimetide).

2) Protection against viruses:

Muradimetide can protect hosts against viral infections of various ethologies as will be shown below in which the immunostimulant has been shown to be active in the same way after systemic administration or oral administration. a) Influenza virus:

The APR / 8 influenza virus is capable of inducing lethal pneumonia in adult mice.

Muradimetide administered orally 24 hours after the infectious challenge given orally was able to protect 30% of the animals observed for two weeks. b) 1 • encephalomyocarditis virus (iEMCV): EMCV induces in mice a severe encephalomyocarditis leading to death after 5/6 days under our experimental conditions (100 LD50 given intraperitoneally). The protective effect of muradimetide was evaluated in this model after intraperitoneal administration. b.l. Prophylactic effect:

Muradimetide is administered at a dose of 2 or 20 mg / kg depending on the experiments in mice 24 h before infection with EMCV at a dose of 100 LD50. The animals are observed for 60 days.

The comparative results obtained with different derivatives of MDP in the following table 11: Table 11 Prophylactic effect Antiviral activity of hydrophilic derivatives of the family of muramyl peptides administered to the mouse before the injection of 100 LD50 of the EMCV virus

Muramyl peptides% survival (30 μg administered 24 hours before the virus)

0 0

CDM 0

CDM OC ₆ H ₉ 10

Muradimetide 30

Murametide 10

It is evident from reading this table that muradimetide alone, without subsequent interferon injection, makes it possible to obtain a survival percentage of 30% of the mice.

To better highlight the remarkable activity of muradimetide in the EMCV model, dose-response experiments were undertaken using muradimetide or other derivatives of muramyl peptides administered 24 h before viral infection, the results are presented in the following table 12:

Table 12 Effective dose 50 of the hydrophilic derivatives of the family of muramyl peptides administered to the mouse before the injection of 100 LD50 of the EMCV virus

Muramyl peptides DE50

CDM> 600 μg

MDPA (OCH ₃ ) OC ₄ H ₉ 600 μg

Muradimetide 150 μg

Murametide 300 μg

ED50: dose of the test compound making it possible to ensure the survival of half of the animals.

We see in Table 12 that a dose of 150 μg of muradimetide is sufficient to obtain 50% survival.

The anti-viral activity of muradimetide is illustrated by FIG. 2 which shows that at a dose of 300 μg muradimetide protects more than 60% of the mice against death by viral infection, the MDP only protects it from 20% of the animals .

FIG. 3 represents the dose response activity of muradimetide in the protection of mice against infection with EMCV.

Swiss mice (group of 10) were pretreated with muradimetide before infection with 100 LD50 of EMCV infectious units and the mice were observed for 60 days.

We can see in this figure that muradimetide alone protects 80% of animals at the highest dose of 600 μg / mouse equivalent to 30 mg / kg.

Muradimetide has also been shown to be active by oral administration unlike other muramyl peptide derivatives. The administration of muradimetide by the oral route 1 h after the infectious challenge is capable of increasing the activity of IFN / β administered at the same time by the intraperitoneal route, as shown in Table 13 below:

Table 13

Treatment Survival rate

1 hour after the final challenge at 60 days

PBS (control) 0/20

Muradimetide (150 mg / kg) p.o. 1/20 IFN-Q - // 3 25,000 IU i.p. 6/20 Combined treatment 10/20 MDM p.o. + IFN i.p.

c) Herpes Si plex type II virus (HSV II): HSV II induced in mice - severe necrosis in the liver; muradimetide at a dose of 300 μg administered after an infectious challenge with 2.10 ⁴ IU of HSV II is capable of inhibiting the development of necrosis on the fifth day, the period which represents the peak of deterioration in the liver.

The data represented in the following table 14 were obtained using groups of 10 female Swiss mice:

Table 14 Activity of muradimetide in the treatment of HSV II infection

Treatment Number of necroses% at + 1 hour per liver of mean inhibition ± SD

None 94 ± 4 0

CDM 63 ± 7 33

Muradimetide 35 ± 4 63

In these experiments, muradimetide is able to interfere with the necrotic activity of the virus disseminated in the liver. d) Human immunodeficiency virus: The effect of combining muramyl peptides with suboptimal doses of anti-retroviral drugs such as 2 ', 3' -dioxycytidine (ddC), azidothymidine (AZT) and others inhibitory agents, was investigated in a model of HIV infection of cells of human origin U937 monoplastoid Con positive.

The experimental protocol used to study the effect of different treatments on viral infection in these cells is as follows:

Uninfected U937 cell lines are incubated for 2 h with a pool of virus in the presence of 2 g of polybrene. After infection, the cells are centrifuged and the cultures are. then seeded in RPMI medium supplemented with fetal calf serum. Different amounts of muramyl peptide derivatives and anti-retroviral agents are dissolved in the culture as shown below. The cells are then counted by microscopic observation after staining with trypan blue. Association of muramyl peptide derivatives with AZT:

The measurement of the HIV antigen present in infected cell cultures treated with MDP or muradimetide alone or in combination with AZT makes it possible to show the inhibitory activity of these molecules.

FIG. 4 representing the effect of treatments for AZT, MDP or muradimetide (MDM) administered alone or in combination.

The inhibitory activity of suboptimal doses of 0.01 M AZT is manifested on day 10, but not on day 3 following HIV infection.

A significant synergistic effect is obtained using 10 μg of MDP or muradimetide in combination with the 0.01 M suboptimal dose of AZT as shown on the antigen / HIV test.

The combination of muramyl peptides. _. and AZT also shows synergistic activity on day 10, a period of time for which the muramyl peptides alone are ineffective.

Effect of Muradimetide on "MAIDS" mice

There is an experimental viral model in mice that reproduces certain signs of AIDS. This model is called MAIDS for "Murine Acquired Immuno Deficiency Syndrome". This model is considered to represent a definite value in the search for drugs to prevent or treat symptoms linked to HIV infection.

The murine leukemia LP-BM5 virus is a defective retrovirus which is capable of infecting in particular the C57B1 / 6 mice. It has been described by Mosier DE et al in J. Exp Med. (1985) 161 766: 784 and in J. Exp. Med (1987) 165: 1737-1742. Animals affected by the retrovirus have lymphadenopathy, an enlarged spleen (equal spleno), elevations in serum IgG and a decrease in mature T helper T cells accompanied by suppression of T cell responses to mitogens and an inability to respond to antigenic stimuli.

The present experience shows that treatment with Muradimetide in such mice infected with the retrovirus for 7 days has made it possible to correct the symptoms of the disease.

The animals are C57B1 / 6 female mice from 7 to 8 older, free of mouse specific pathogens. Groups of 10 animals are infected peritoneally with infectious supernatants containing the retrovirus LP-BM5.

7 days after infection, the experimental group receives Muradimetide, either administered orally or subcutaneously, at a dose of 100 μg per day for 21 days.

It is observed at the end of the treatment that Muradimetide has made it possible to inhibit the abnormal development of the spleen and that the spleen cells of the treated animals respond to mitogens unlike the cells taken from untreated animals. This has been observed for the response to LPS and Con-A (specific for T cells). During the duration of the treatment, there is no longer any difference between the 2 groups. Finally, treatment with Muradimetide helps maintain a normal level of T helper cells.

Claims

1. Immunostimulatory pharmaceutical composition active externally, characterized in that it contains a diester of general formula I:

in which :

- R is CH3,

- X is L-Ala or L-Thr,

- RI is a hydrocarbon group having from 1 to 4 carbon atoms,

- R2 is a hydrocarbon group having 1 to 2 carbon atoms, in an external administration formulation compatible with an administration of active principle of between 0.1 mg and 5 mg per kg of weight in humans or animals.

2. Immunostimulatory pharmaceutical composition according to claim 1, characterized in that the compound is the muradimetide of formula II:

Nac-Mur-L-Ala-D-Glu [OMe] -OMe

3. Pharmaceutical composition according to claims 1 and 2, characterized in that the external route is the oral route.

4. Pharmaceutical composition according to claims 1 and 2, characterized in that the external route is the aerosol route.

5. Pharmaceutical composition according to claims 1 and 2, characterized in that the external route is the percutaneous route.

6. Composition according to one of claims 1 to 5, characterized in that it is an immunoadjuvant used in accompaniment of an effect induced by a vaccination.

7. Composition according to claim 6, characterized in that it can be administered externally before, during or after a vaccination whatever the mode of administration of said vaccination.

8. Composition according to one of claims 1 to 5, characterized in that it is used to treat or prevent the effects of pathogenic infection, in particular bacterial, viral or parasitic infection.

9. Composition according to claim 8, characterized in that the bacterial infection capable of being treated is a mycobacterial infection

10. Composition according to one of claims 1 to 5, characterized in that it is used in combination with an antiviral agent, or an anti-retroviral agent, the composition and the antiviral agent being used simultaneously, separately or spread out over time.

11. Composition according to claim 9, characterized in that it is used in combination with a nucleoside analog, in particular AZT (azidothymidine) or, 2 ′, 3-dideoxycytidine, for simultaneous use separated or spread out in the tempε.

12. Use of a die of formula:

for the manufacture of an immunostimulatory pharmaceutical composition which can be administered externally in a formulation compatible with administration of active ingredient between 0.1 and 5 mg per kg of weight in humans or animals.

13. Use according to claim 12 characterized in that the diester is the muradimetide of formula II:

Nac-Mur-L-Ala-D-Glu [OMe] -OMe.

14. Use according to one of claims 12 or 13 characterized in that the formulation of the diester is compatible with an administration of active ingredient of between 0.5 and 3 mg / kg of weight in humans or animals.

15. Use according to one of claims 12, 13 or 14 characterized in that the external route is the oral route, the mucosa route, the percutaneous route or the aerosol route.

16. Use of muradimetide according to claims 12 to 15, for the preparation of an immunoadjuvant of vaccine.

17. Use according to claims 12 to 15 for the preparation of a medicament intended to be used simultaneously in a separate or spread out manner with an anti-infectious agent.

18. Use according to claim 17, characterized in that the anti-infectious agent is an antiviral agent, in particular a nucleotide analog.

19. Use according to claims 12 to 15, characterized in that the pharmaceutical composition makes it possible to treat bacterial infections, in particular mycobacterial infections.