Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1277—Processes for preparing; Proliposomes
  • A61K9/1278—Post-loading, e.g. by ion or pH gradient
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2013—IL-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • 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
  • A61P35/00—Antineoplastic agents
• • 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

Definitions

• the present invention relates to an immunostimulatory composition further having properties for amplifying the protective power of vaccinating antigens, and in particular of the rabies vaccine for human or veterinary use.
• Interleukin-2 is a lymphokine that plays a key role in the immune response.
• T cell growth factor IL-2 is produced by lymphoid cells (mainly T cells) stimulated by antigens or mitogens.
• IL-2 has a wide variety of immunoregulatory properties. It promotes the proliferation and / or differentiation of cells which express a specific IL-2 receptor, in particular T cells, including CTL (cytotoxic T lymphocytes), natural killer cells (NK) and B cells. Due to its immunostimulatory action, IL-2 is now used in immunotherapy of human patients, such use is made all the easier since we now have recombinant human IL-2.
• vaccines which consist of inactivated particles, which do not exhibit the usual undesirable local and general side reactions, due to the fact that they consist of very immunogenic "immunosomes", which are formed by antigens attached to the membrane of a preformed liposome, and which are more immunogenic than the viral antigen subunits used for the preparation of the immunosome.
• the liposomes, on the membrane of which the antigens are anchored, are, in fact, transport structures of fundamental interest: these structures consist of a double layer of lipids, in general phospholipids such as lecithins, in alternation with water or an aqueous solution, closed to form a "droplet", to which one can add, by inclusion and / or anchoring, various constituents - drugs, enzymes in particular - which one seeks to make convey by the blood to target organs, without these constituents being degraded on their journey.
• IJ FIDLER see his article "Activation of macrophages by liposomes containing both lymphokines and muramyl dipeptide for treatment of heterogeneous metastases", published in Dev. Target-Oriented Anticancer Drugs, 1983, 219-226, applied the properties of liposomes for the treatment of metastases from solid tumors.
• immunosomes liposomes carrying on their membrane the purified glycoprotein which constitutes one of the forms of the rabies antigen
• immunosomes are capable of inducing a specific IL-2 response whereas an equivalent quantity of the purified glycoprotein used alone does not determine production of IL-2.
• VNAb antibody neutralizing the virus
• CTL cytotoxic T lymphocytes
• ADCC antibody-dependent cytotoxic cells
• IFN Interferon
• WIKTOR et al. PROC. NAT. ACAD. SCI. USA, 1977, 74, 334-338 and DEVELOP. BIOL STANDARD, 1978, 40, 255-264 have suggested that cytotoxic T cells play a role important role in the elimination of the rabies virus. From this and taking into account that the in vitro proliferation of CTLs depends on the presence of IL-2, the inventors have been able to establish, surprisingly, that IL-2 of exogenous origin is capable of ensuring , alone or associated with antigens, an immunoprotective or immunotherapeutic action, when it is associated with liposomes. They were also able to surprisingly establish:
• IL-2 of exogenous origin associated with liposomes and with an antigen, in particular with the purified glycoprotein of the rabies virus, induces an amplification of the protective power of the atigen
• the subject of the present invention is a composition having immunostimulatory properties, characterized in that it comprises a lipid support, interleukin-2 (IL-2) or another lymphokine exerting an action similar to that of IL- 2, as well as optionally an agent having immunizing properties.
• IL-2 interleukin-2
• said composition comprises in combination, preformed liposomes and interleukin-2 (IL-2) or another lymphokine which exerts an action similar to that of IL-2, which composition exhibits properties for amplifying the activity of IL-2.
• IL-2 interleukin-2
• the composition according to the invention comprises, in combination with preformed liposomes, ⁇ nterleukme-2 and an immunogenic agent, in particular an antigen, which composition has properties for amplifying the activity of IL-2.
• the antigen is chosen so that it is capable of inducing vaccination.
• the liposomes are unilamellar and constituted by a single outer layer of a bilayer interfacial phase surrounding an aqueous polar phase, the interfacial layer containing at least one phospholipid chosen from the group which includes in particular phosphatidylcholine and cholesterol.
• the process for preparing the compositions in accordance with the invention comprises mixing the lipid support, in particular liposomes preformed with IL-2 and optionally with an appropriate immunogenic agent.
• the present invention also relates to pharmaceutical compositions containing an immunostimulatory composition according to the invention.
• compositions according to the invention can be administered alone or in combination with inert carriers which are acceptable from the pharmaceutical point of view.
• compositions according to the invention can be used, in humans or animals, in the prevention and treatment of viral infections, in particular herpes HSV2, as an adjuvant for prophylactic or therapeutic vaccination, in particular anti-rabies, as an immunostimulant, in particular in the treatment of cancers, etc ...
• IL-2 which is hydrophobic in- would interact with liposomes (adsorption for example, as is the case with albumin and interferon alpha) and would then be better recognized by the T cell receptor whose proliferation would be amplified; or, the simultaneous solicitation of several receptors of the same cell by the IL-2 absorbed on the surface of the liposomes would be accompanied by a greater proliferation of the cells.
• compositions according to the invention will be better understood using the additional description which follows, which refers to examples of preparations of the compositions according to the invention, reports of experiments relating to the potentiation of the substance associated with liposomes. , compositions according to the invention.
• liposomes vesicles consisting of two monomolecular layers of phospholipids
• OGP dialysable detergent
• PC phosphatidylcholine
• Chol cholesterol
• Example 2 Preparation of a liposome-IL-2 mixture.
• Example 3 0.3 ⁇ M of liposomes prepared according to Example 1 are mixed with 0.5 IU / ml of IL-2.
• Example 4 The procedure is as described in Example 2, using 2.5 IU / ml of IL-2.
• Example 4 The procedure is as described in Example 2, using 2.5 IU / ml of IL-2.
• Example 6 Preparation of a liposome-IL-2-antigen mixture.
• Example 3 The procedure is as described in Example 3 and, as antigen, envelope glycoprotein of the rabies virus is added.
• Example 7 Effect of different antigens and liposomes on the activity of IL-2 and the proliferation of CTLL (Cytotoxic-T Lymphocytes line).
• This table shows two series of experiments; in the first, the same amount of IL-2 was mixed with different antigens and then titrated for its ability to induce cell proliferation; in the second, IL-2 was previously mixed with liposomes (0.3 ⁇ M lipids), then with antigens.
• the amplification factor is expressed by the ratio of the cpm measured with the IL-2-product mixture to the cpm measured with the same amount of IL-2 but without antigen or liposomes.
• - CTLL cells correspond to T lymphocytes of mice which have been transformed and therefore can be maintained in culture by successive passages. These are cells whose survival and development depend on the presence of interleukme-2 (5-10 International Units per ml -Ul / ml-). They are grown in suspension in the presence of 10% fetal bovine serum and 10% CO 2 (complete RPMI-CTLL-1 medium). The CTLL cells used to assay IL-2 are therefore completely IL-2-dependent. In particular, two T-cytotoxic cell lines (CTLLD and CTLLc) were tested.
• the amplification of proliferation is due to a potentiation of IL-2 by the liposomes (we can think of a simultaneous solicitation of several IL-2 receptors by the mixture IL-2-liposomes) rather than an interaction between the liposome and the antigen or cell.
• the virus induces a greater proliferation of CTLL when it is added to a mixture of IL-2 and liposomes (amplification factor 3.7 to 4.5) than when it is mixed with liposomes then added to IL-2 (amplification factor 1.3 to 1.82).
• the liposome mixed with IL-2 enhances cell proliferation. This amplification is illustrated in FIG.
• This amplification is greater with the CTLLc line. It is dependent on the quantity of liposomes (expressed in nanomoles of lipids) and on the quantity of IL-2 whose activity is susceptible of being amplified. The best amplification was obtained with the CTLLc line for 0.046 to 0.187 units of IL-2 and 20 nanomole ⁇ of lipids (amplification by a factor equal to or greater than 2). The high liposome concentrations no longer amplify.
• the lipo ⁇ ome ⁇ alone do not amplify the proliferation of CTLL cells, as shown in FIG. 2, which includes on the abscissa the concentration of liposomes expressed in nanomoles of lipid per 20,000 cells and on the ordinate the percentage of proliferation, taking as 100 %, the proliferation results obtained after treatment with the medium alone.
• the curve corresponds to the CTLLc line, the curve corresponds to the lineage
• Liposomes 1/10 FA * 1/20 FA * ⁇ M lipids
• liposomes to IL-2 in its "associated" form, amplifies the activity of the latter by a factor of 1.6-1.7.
• Example 8 Combination of recombinant human interleukin-2 with rabies treatment.
• IL-2 stimulates the proliferation of cytotoxic T lymphocytes which are mainly responsible for rabies protection.
• this product could be used in humans if it was found to be non-toxic and effective in animals;
• Table III above shows that, in the context of the control of vaccines after exposure to wild virus, hamsters have been treated with IMS or with the inactivated virus in combination or not with recombinant human IL-2; in the second experiment, liposomes were either mixed with IL-2 or injected separately two hours after IL-2.
• Example 9 Potentiation of protective power test (pre-exposure) by the association of IL-2 with vaccination.
• IL-2 Given the place of IL-2 in the immune response and the fact that wild rabies specifically rewards production, recombinant human IL-2 has been associated with vaccinations. In addition, it has been demonstrated a phenomenon of amplification of the proliferation of T cells in the presence of liposomes and IL-2 (Example 7); a mixture of liposomes and purified virus was also used to verify whether the liposomes amplified the action of exogenous IL-2, or even perhaps endogenous IL-2.
• IL-2 is capable of amplifying the protective power of IMS. Indeed, while only 30% of the animals are protected when injecting IMS (at a dilution lower than the effective dose 50%), all of the animals are protected by adding IL-2 for amounts varying from 10 at 100 IU / ml. In addition, it appears that a late injection (14 days) is not as effective.
• the level of mterIeukin-2 at the time of the recall (or in the vicinity) plays an important role in the subsequent protection against rabies.
• This level of IL-2 is amplified by the presence of liposomes or immunosomes.
• mice were vaccinated on days 0 and 7 and then tested on day 21. Some of them received one or more injections of human IL-2 recombinant. Protection is expressed by the ratio of the number of surviving animals to the number of animals tested. In addition, for some of them, the level of neutralizing antibody (AcN) and the level of IL-2 were determined on the day of the test after simple stimulation with the culture medium in the absence of antigen.
• AcN neutralizing antibody

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• 1987
  • 1987-12-21 FR FR8717799A patent/FR2624741B1/fr not_active Expired - Lifetime
• 1988
  • 1988-12-20 PT PT89271A patent/PT89271A/pt not_active Application Discontinuation
  • 1988-12-21 JP JP1500768A patent/JPH05504756A/ja active Pending
  • 1988-12-21 WO PCT/FR1988/000630 patent/WO1989005631A1/fr not_active Application Discontinuation
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