Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants

Definitions

• the present invention relates to an immunostimulatory pharmaceutical composition, active by external administration, in particular by oral, nasal, transcutaneous, aerosol route; immuno ⁇ timulant 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).
• 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.
• 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:
• - 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.
• 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.
• 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.
• 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.
• composition according to the invention is an immunoadjuvant usable as an accompaniment to an effect induced by vaccination.
• 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.
• 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 .
• AZT AZT
• a dose which does not confer no protection against infection when administered alone, this use may be simultaneous, separate or spread over time.
• FIG. 1 represents the adjuvant activity of muradimetide (MDM) administered orally on the antibody response of the mouse to a hepatitis B vaccine.
• MDM muradimetide
• 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.
• FIG. 3 represents the dose-response activity of muradimetide administered in the protection of mice against EMCV infection.
• FIG. 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.
• MDM muradimetide
• 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.
• 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.
• Muradimetide is administered orally simultaneously with parenteral vaccination with an antigen.
• 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.
• 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.
• MDPA of which muradimetide is the methyl diester derivative
• muradimetide is the methyl diester derivative
• the antigen used is a recombinant hepatitis B virus (HBS) surface antigen obtained by genetic recombination and administered, absorbed on aluminum hydroxide.
• HBS hepatitis B virus
• 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.
• 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 represents the recognition of the natural protein by the antibodies obtained after oral administration of the synthetic peptide encapsulated in the microspheres.
• 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).
• SMAg Shistosoma mansoni antigen
• mice receive 1 mg of muradimetide orally.
• the results are given in the following table:
• 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.
• Muradimetide and the antigen are administered orally; muradimetide is only administered during secondary immunization.
• Antibodies are measured by the ELISA technique in serum and in vaginal lavage on day 28.
• 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.
• mice 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 animals are 6/7 week old Swiss female mice, except for the group marked with an asterisk which consists of newborn mice.
• MDPA its parent molecule
• 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.
• the APR / 8 influenza virus is capable of inducing lethal pneumonia in adult mice.
• iEMCV encephalomyocarditis virus
• 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.
• Muramyl peptides% survival (30 ⁇ g administered 24 hours before the virus)
• ED50 dose of the test compound making it possible to ensure the survival of half of the animals.
• FIG. 2 shows that at a dose of 300 ⁇ g 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.
• 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 / ⁇ administered at the same time by the intraperitoneal route, as shown in Table 13 below:
• 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.
• HSV II Herpes Si plex type II virus: HSV II induced in mice - severe necrosis in the liver; muradimetide at a dose of 300 ⁇ g 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.
• 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:
• 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 ⁇ g 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.
• MAIDS Murine Acquired Immuno Deficiency Syndrome
• 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 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.
• the experimental group receives Muradimetide, either administered orally or subcutaneously, at a dose of 100 ⁇ g per day for 21 days.
• 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.

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• 1994
  • 1994-01-25 FR FR9400786A patent/FR2715305B1/fr not_active Expired - Fee Related
• 1995
  • 1995-01-24 EP EP95907694A patent/EP0741573A1/de not_active Withdrawn
  • 1995-01-24 AU AU15809/95A patent/AU1580995A/en not_active Abandoned
  • 1995-01-24 JP JP7519390A patent/JPH09509409A/ja active Pending
  • 1995-01-24 CA CA002181899A patent/CA2181899A1/fr not_active Abandoned
  • 1995-01-24 WO PCT/FR1995/000077 patent/WO1995019777A2/fr not_active Application Discontinuation

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